Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

Official Journal L 142 , 31/05/2008 P. 0001 - 0739

Council Regulation (EC) No 440/2008

of 30 May 2008

laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

(Text with EEA relevance)

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Community,

Having regard to Regulation (EC) No 1907/2006 of 18 December 2006 of the European Parliament and of the Council concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC [1], and in particular Article 13(3)thereof,

Whereas:

(1) Pursuant to Regulation (EC) No 1907/2006, test methods are to be adopted at Community level for the purposes of tests on substances where such tests are required to generate information on intrinsic properties of substances.

(2) Council Directive 67/548/EEC of 27 June 1967 on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances [2], laid down, in Annex V, methods for the determination of the physico-chemical properties, toxicity and ecotoxicity of substances and preparations. Annex V to Directive 67/548/EEC has been deleted by Directive 2006/121/EC of the European Parliament and of the Council with effect from 1 June 2008.

(3) The test methods contained in Annex V to Directive 67/548/EEC should be incorporated into this Regulation.

(4) This Regulation does not exclude the use of other test methods, provided that their use is in accordance with Article 13(3) of Regulation 1907/2006.

(5) The principles of replacement, reduction and refinement of the use of animals in procedures should be fully taken into account in the design of the test methods, in particular when appropriate validated methods become available to replace, reduce or refine animal testing.

(6) The provisions of this Regulation are in accordance with the opinion of the Committee established under Article 133 of Regulation (EC) No 1907/2006,

HAS ADOPTED THIS REGULATION:

Article 1

The test methods to be applied for the purposes of Regulation 1907/2006/EC are set out in the Annex to this Regulation.

Article 2

The Commission shall review, where appropriate, the test methods contained in this Regulation with a view to replacing, reducing or refining testing on vertebrate animals.

Article 3

All references to Annex V to Directive 67/548/EEC shall be construed as references to this Regulation.

Article 4

This Regulation shall enter into force on the day following its publication in the Official Journal of the European Union.

It shall apply from 1 June 2008.

Done at Brussels, 30 May 2008.

For the Commission

Stavros Dimas

Member of the Commission

[1] OJ L 396, 30.12.2006, p. 1, as corrected by OJ L 136, 29.5.2007, p. 3.

[2] OJ 196, 16.8.1967, p. 1. Directive as last amended by Directive 2006/121/CE of the European Parliament and of the Council (OJ L 396, 30.12.2006, p. 850, as corrected by OJ L 136, 29.5.2007, p. 281).

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ANNEX

PART A: METHODS FOR THE DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES

TABLE OF CONTENTS

A.1. MELTING/FREEZING TEMPERATURE

A.2. BOILING TEMPERATURE

A.3. RELATIVE DENSITY

A.4. VAPOUR PRESSURE

A.5. SURFACE TENSION

A.6. WATER SOLUBILITY

A.8. PARTITION COEFFICIENT

A.9. FLASH-POINT

A.10. FLAMMABILITY (SOLIDS)

A.11. FLAMMABILITY (GASES)

A.12. FLAMMABILITY (CONTACT WITH WATER)

A.13. PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS

A.14. EXPLOSIVE PROPERTIES

A.15. AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)

A.16. RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS

A.17. OXIDISING PROPERTIES (SOLIDS)

A.18. NUMBER — AVERAGE MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS

A.19. LOW MOLECULAR WEIGHT CONTENT OF POLYMERS

A.20. SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN WATER

A.21. OXIDISING PROPERTIES (LIQUIDS)

A.1. MELTING/FREEZING TEMPERATURE

1. METHOD

The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.1. INTRODUCTION

The methods and devices described are to be applied for the determination of the melting temperature of substances, without any restriction with respect to their degree of purity.

The selection of the method is dependent on the nature of the substance to be tested. In consequence the limiting factor will be according to, whether or not the substance can be pulverised easily, with difficulty, or not at all.

For some substances, the determination of the freezing or solidification temperature is more appropriate and the standards for these determinations have also been included in this method.

Where, due to the particular properties of the substance, none of the above parameters can be conveniently measured, a pour point may be appropriate.

1.2. DEFINITIONS AND UNITS

The melting temperature is defined as the temperature at which the phase transition from solid to liquid state occurs at atmospheric pressure and this temperature ideally corresponds to the freezing temperature.

As the phase transition of many substances takes place over a temperature range, it is often described as the melting range.

Conversion of units (K to oC)

t = T - 273,15

t : Celsius temperature, degree Celsius (oC)

T : thermodynamic temperature, kelvin (K)

1.3. REFERENCE SUBSTANCES

Reference substances do not need to be employed in all cases when investigating a new substance. They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

Some calibration substances are listed in the references (4).

1.4. PRINCIPLE OF THE TEST METHOD

The temperature (temperature range) of the phase transition from the solid to the liquid state or from the liquid to the solid state is determined. In practice while heating/cooling a sample of the test substance at atmospheric pressure the temperatures of the initial melting/freezing and the final stage of melting/freezing are determined. Five types of methods are described, namely capillary method, hot stage methods, freezing temperature determinations, methods of thermal analysis, and determination of the pour point (as developed for petroleum oils).

In certain cases, it may be convenient to measure the freezing temperature in place of the melting temperature.

1.4.1. Capillary method

1.4.1.1. Melting temperature devices with liquid bath

A small amount of the finely ground substance is placed in a capillary tube and packed tightly. The tube is heated, together with a thermometer, and the temperature rise is adjusted to less than about 1 K/min during the actual melting. The initial and final melting temperatures are determined.

1.4.1.2. Melting temperature devices with metal block

As described under 1.4.1.1, except that the capillary tube and the thermometer are situated in a heated metal block, and can be observed through holes in the block.

1.4.1.3. Photocell detection

The sample in the capillary tube is heated automatically in a metal cylinder. A beam of light is directed through the substance, by way of a hole in the cylinder, to a precisely calibrated photocell. The optical properties of most substances change from opaque to transparent when they are melting. The intensity of light reaching the photocell increases and sends a stop signal to the digital indicator reading out the temperature of a platinum resistance thermometer located in the heating chamber. This method is not suitable for some highly coloured substances.

1.4.2. Hot stages

1.4.2.1. Kofler hot bar

The Kofler hot bar uses two pieces of metal of different thermal conductivity, heated electrically, with the bar designed so that the temperature gradient is almost linear along its length. The temperature of the hot bar can range from 283 to 573 K with a special temperature-reading device including a runner with a pointer and tab designed for the specific bar. In order to determine a melting temperature, the substance is laid, in a thin layer, directly on the surface of the hot bar. In a few seconds a sharp dividing line between the fluid and solid phase develops. The temperature at the dividing line is read by adjusting the pointer to rest at the line.

1.4.2.2. Melt microscope

Several microscope hot stages are in use for the determination of melting temperatures with very small quantities of material. In most of the hot stages the temperature is measured with a sensitive thermocouple but sometimes mercury thermometers are used. A typical microscope hot stage melting temperature apparatus has a heating chamber which contains a metal plate upon which the sample is placed on a slide. The centre of the metal plate contains a hole permitting the entrance of light from the illuminating mirror of the microscope. When in use, the chamber is closed by a glass plate to exclude air from the sample area.

The heating of the sample is regulated by a rheostat. For very precise measurements on optically anisotropic substances, polarised light may be used.

1.4.2.3. Meniscus method

This method is specifically used for polyamides.

The temperature at which the displacement of a meniscus of silicone oil, enclosed between a hot stage and a cover-glass supported by the polyamide test specimen, is determined visually.

1.4.3. Method to determine the freezing temperature

The sample is placed in a special test tube and placed in an apparatus for the determination of the freezing temperature. The sample is stirred gently and continuously during cooling and the temperature is measured at suitable intervals. As soon as the temperature remains constant for a few readings this temperature (corrected for thermometer error) is recorded as the freezing temperature.

Supercooling must be avoided by maintaining equilibrium between the solid and the liquid phases.

1.4.4. Thermal analysis

1.4.4.1 Differential thermal analysis (DTA)

This technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the temperature record.

1.4.4.2 Differential scanning calorimetry (DSC)

This technique records the difference in energy inputs into a substance and a reference material, as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic (melting) or exothermic (freezing) departure from the base line of the heat flow record.

1.4.5. Pour point

This method was developed for use with petroleum oils and is suitable for use with oily substances with low melting temperatures.

After preliminary heating, the sample is cooled at a specific rate and examined at intervals of 3 K for flow characteristics. The lowest temperature at which movement of the substance is observed is recorded as the pour point.

1.5. QUALITY CRITERIA

The applicability and accuracy of the different methods used for the determination of the melting temperature/melting range are listed in the following table:

TABLE: APPLICABILITY OF THE METHODS

A. Capillary methods

Melting temperature devices with liquid bath | yes | only to a few | 273 to 573 K | ±0,3 K | JIS K 0064 |

Melting temperature with metal block | yes | only to a few | 293 to >573 K | ±0,5 K | ISO 1218 (E) |

Photocell detection | yes | several with appliance devices | 253 to 573 K | ±0,5 K | |

B. Hot stages and freezing methods

Kofler hot bar | yes | no | 283 to > 573 K | ± 1K | ANSI/ASTM D 3451-76 |

Melt microscope | yes | only to a few | 273 to > 573 K | ±0,5 K | DIN 53736 |

Meniscus method | no | specifically for polyamides | 293 to > 573 K | ±0,5 K | ISO 1218 (E) |

Freezing temperature | yes | yes | 223 to 573 K | ±0,5 K | e.g. BS 4695 |

C. Thermal analysis

Differential thermal analysis | yes | yes | 173 to 1273 K | up to 600 K ±0,5 K up to 1273 K ±2,0 K | ASTM E 537-76 |

Differential scanning calorimetry | yes | yes | 173 to 1273 K | up to 600 K ±0,5 K up to 1273 K ±2,0 K | ASTM E 537-76 |

D. Pour point

1.6. DESCRIPTION OF THE METHODS

The procedures of nearly all the test methods have been described in international and national standards (see Appendix 1).

1.6.1. Methods with capillary tube

When subjected to a slow temperature rise, finely pulverised substances usually show the stages of melting shown in figure 1.

Figure 1

Stage A

Stage B

Stage C

Stage D

Stage E

Stage A

(beginning of melting): fine droplets adhere uniformly to the inside wall of the capillary tube

Stage B

a clearance appears between the sample and the inside wall due to shrinkage of the melt

Stage C

the shrunken sample begins to collapse downwards and liquefies

Stage D

a complete meniscus is formed at the surface but an appreciate amount of the sample remains solid

Stage E

(final stage melting): there are no solid particles

+++++ TIFF +++++

During the determination of the melting temperature, the temperatures are recorded at the beginning of the melting and at the final stage.

1.6.1.1. Melting temperature devices with liquid bath apparatus

Figure 2 shows a type of standardised melting temperature apparatus made of glass (JIS K 0064); all specifications are in millimeters.

Figure 2

A: Measurement vessel

B: Stopper

C: Vent

D: Thermometer

E: Auxiliary thermometer

F: Bath liquid

G: Capillary tube made of glass, 80 to 100 mm in length, 1,0 ± 0,2 mm inner diameter, 0,2 to 0,3 mm wall thickness

H: Side tube

+++++ TIFF +++++

Bath liquid:

A suitable liquid should be chosen. The choice of the liquid depends upon the melting temperature to be determined, e.g. liquid paraffin for melting temperatures no higher than 473 K, silicone oil for melting temperatures no higher than 573 K.

For melting temperatures above 523 K, a mixture consisting of three parts sulphuric acid and two parts potassium sulphate (in mass ratio) can be used. Suitable precautions should be taken if a mixture such as this is used.

Thermometer:

Only those thermometers should be used which fulfil the requirements of the following or equivalent standards:

ASTM E 1-71, DIN 12770, JIS K 8001.

Procedure:

The dry substance is finely pulverised in a mortar and is put into the capillary tube, fused at one end, so that the filling level is approximately 3 mm after being tightly packed. To obtain a uniform packed sample, the capillary tube should be dropped from a height of approximately 700 mm through a glass tube vertically onto a watch glass.

The filled capillary tube is placed in the bath so that the middle part of the mercury bulb of the thermometer touches the capillary tube at the part where the sample is located. Usually the capillary tube is introduced into the apparatus about 10 K below the melting temperature.

The bath liquid is heated so that the temperature rise is approximately 3 K/min. The liquid should be stirred. At about 10 K below the expected melting temperature the rate of temperature rise is adjusted to a maximum of 1 K/min.

Calculation:

The calculation of the melting temperature is as follows:

T = TD + 0,00016 (TD - TE) n

where:

T = corrected melting temperature in K

TD = temperature reading of thermometer D in K

TE = temperature reading of thermometer E in K

n = number of graduations of mercury thread on thermometer D at emergent stem.

1.6.1.2. Melting temperature devices with metal block

Apparatus:

This consists of:

- a cylindrical metal block, the upper part of which is hollow and forms a chamber (see figure 3),

- a metal plug, with two or more holes, allowing tubes to be mounted into the metal block,

- a heating system, for the metal block, provided for example by an electrical resistance enclosed in the block,

- a rheostat for regulation of power input, if electric heating is used,

- four windows of heat-resistant glass on the lateral walls of the chamber, diametrically disposed at right-angles to each other. In front of one of these windows is mounted an eye-piece for observing the capillary tube. The other three windows are used for illuminating the inside of the enclosure by means of lamps,

- a capillary tube of heat-resistant glass closed at one end (see 1.6.1.1).

Thermometer:

See standards mentioned in 1.6.1.1. Thermoelectrical measuring devices with comparable accuracy are also applicable.

Figure 3

Thermometer

Capillary tube

Metal plug

Eye-piece

Lamp

Electrical resistance

Metal heating block

+++++ TIFF +++++

1.6.1.3. Photocell detection

Apparatus and procedure:

The apparatus consists of a metal chamber with automated heating system. Three capillary are filled accordingly to 1.6.1.1 and placed in the oven.

Several linear increases of temperature are available for calibrating the apparatus and the suitable temperature rise is electrically adjusted at a pre-selected constant and linear rate. recorders show the actual oven temperature and the temperature of the substance in the capillary tubes.

1.6.2. Hot stages

1.6.2.1. Kofler hot bar

See Appendix.

1.6.2.2. Melt microscope

See Appendix.

1.6.2.3. Meniscus method (polyamides)

See Appendix.

The heating rate through the melting temperature should be less than 1 K/min.

1.6.3. Methods for the determination of the freezing temperature

See Appendix.

1.6.4. Thermal analysis

1.6.4.1. Differential thermal analysis

See Appendix.

1.6.4.2. Differential scanning calorimetry

See Appendix.

1.6.5. Determination of the pour point

See Appendix.

2. DATA

A thermometer correction is necessary in some cases.

3. REPORTING

The test report shall, if possible, include the following information:

- method used,

- precise specification of the substance (identity and impurities) and preliminary purification step, if any,

- an estimate of the accuracy.

The mean of at least two measurements which are in the range of the estimated accuracy (see tables) is reported as the melting temperature.

If the difference between the temperature at the beginning and at the final stage of melting is within the limits of the accuracy of the method, the temperature at the final stage of melting is taken as the melting temperature; otherwise the two temperatures are reported.

If the substance decomposes or sublimes before the melting temperature is reached, the temperature at which the effect is observed shall be reported.

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

4. REFERENCES

(1) OECD, Paris, 1981, Test Guideline 102, Decision of the Council C(81) 30 final.

(2) IUPAC, B. Le Neindre, B. Vodar, eds. Experimental thermodynamics, Butterworths, London 1975, vol. II, p. 803-834.

(3) R. Weissberger ed.: Technique of organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter VII.

(4) IUPAC, Physicochemical measurements: Catalogue of reference materials from national laboratories, Pure and applied chemistry, 1976, vol. 48, p. 505-515.

Appendix

For additional technical details, the following standards may be consulted for example.

1. Capillary methods

1.1. Melting temperature devices with liquid bath

ASTM E 324-69 | Standard test method for relative initial and final melting points and the melting range of organic chemicals |

BS 4634 | Method for the determination of melting point and/or melting range |

DIN 53181 | Bestimmung des Schmelzintervalles von Harzen nach Kapillarverfarehn |

JIS K 00-64 | Testing methods for melting point of chemical products |

1.2. Melting temperature devices with metal block

DIN 53736 | Visuelle Bestimmung der Schmelztemperatur von teilkristallinen Kunststoffen |

ISO 1218 (E) | Plastics — polyamides — determination of "melting point" |

2. Hot stages

2.1. Kofler hot bar

ANSI/ASTM D 3451-76 | Standard recommended practices for testing polymeric powder coatings |

2.2. Melt microscope

DIN 53736 | Visuelle Bestimmung der Schmelztemperatur von teilkristallinen Kunststoffen |

2.3. Meniscus method (polyamides)

ISO 1218 (E) | Plastics — polyamides — determination of "melting point" |

ANSI/ASTM D 2133-66 | Standard specification for acetal resin injection moulding and extrusion materials |

NF T 51-050 | Résines de polyamides. Détermination du "point de fusion" méthode du ménisque |

3. Methods for the determination of the freezing temperature

BS 4633 | Method for the determination of crystallising point |

BS 4695 | Method for Determination of Melting Point of petroleum wax (Cooling Curve) |

DIN 51421 | Bestimmung des Gefrierpunktes von Flugkraftstoffen, Ottokraftstoffen und Motorenbenzolen |

ISO 2207 | Cires de pétrole: détermination de la température de figeage |

DIN 53175 | Bestimmung des Erstarrungspunktes von Fettsäuren |

NF T 60-114 | Point de fusion des paraffines |

NF T 20-051 | Méthode de détermination du point de cristallisation (point de congélation) |

ISO 1392 | Method for the determination of the freezing point |

4. Thermal analysis

4.1. Differential thermal analysis

ASTM E 537-76 | Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis |

ASTM E 473-85 | Standard definitions of terms relating to thermal analysis |

ASTM E 472-86 | Standard practice for reporting thermoanalytical data |

DIN 51005 | Thermische Analyse, Begriffe |

4.2. Differential scanning calorimetry

ASTM E 537-76 | Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis |

ASTM E 473-85 | Standard definitions of terms relating to thermal analysis |

ASTM E 472-86 | Standard practice for reporting thermoanalytical data |

DIN 51005 | Thermische Analyse, Begriffe |

5. Determination of the pour point

NBN 52014 | Echantillonnage et analyse des produits du pétrole: Point de trouble et point d'écoulement limite — Monsterneming en ontleding van aardolieproducten: Troebelingspunt en vloeipunt |

ASTM D 97-66 | Standard test method for pour point of petroleum oils |

ISO 3016 | Petroleum oils — Determination of pour point |

A.2. BOILING TEMPERATURE

1. METHOD

The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.1. INTRODUCTION

The methods and devices described here can be applied to liquid and low melting substances, provided that these do not undergo chemical reaction below the boiling temperature (for example: auto-oxidation, rearrangement, degradation, etc.). The methods can be applied to pure and to impure liquid substances.

Emphasis is put on the methods using photocell detection and thermal analysis, because these methods allow the determination of melting as well as boiling temperatures. Moreover, measurements can be performed automatically.

The "dynamic method" has the advantage that it can also be applied to the determination of the vapour pressure and it is not necessary to correct the boiling temperature to the normal pressure (101,325 kPa) because the normal pressure can be adjusted during the measurement by a manostat.

Remarks:

The influence of impurities on the determination of the boiling temperature depends greatly upon the nature of the impurity. When there are volatile impurities in the sample, which could affect the results, the substance may be purified.

1.2. DEFINITIONS AND UNITS

The normal boiling temperature is defined as the temperature at which the vapour pressure of a liquid is 101,325 kPa.

If the boiling temperature is not measured at normal atmospheric pressure, the temperature dependence of the vapour pressure can be described by the Clausius-Clapeyron equation:

log p =

ΔH

+const.

where:

p = the vapour pressure of the substance in pascals

Δ Hv = its heat of vaporisation in J mol-1

R = the universal molar gas constant = 8,314 J mol-1 K-1

T = thermodynamic temperature in K

The boiling temperature is stated with regard to the ambient pressure during the measurement.

Conversions

Pressure (units: kPa)

100 kPa =

1 bar = 0,1 MPa

("bar" is still permissible but not recommended)

133 Pa =

1 mm Hg = 1 Torr

(the units "mm Hg" and "Torr" are not permitted)

1 atm =

standard atmosphere = 101325 Pa

(the unit "atm" is not permitted)

Temperature (units: K)

t = T - 273,15

t : Celsius temperature, degree Celsius (oC)

T : thermodynamic temperature, kelvin (K)

1.3. REFERENCE SUBSTANCES

Some calibration substances can be found in the methods listed in the Appendix.

1.4. PRINCIPLE OF THE TEST METHOD

Five methods for the determination of the boiling temperature (boiling range) are based on the measurement of the boiling temperature, two others are based on thermal analysis.

1.4.1. Determination by use of the ebulliometer

Ebulliometers were originally developed for the determination of the molecular weight by boiling temperature elevation, but they are also suited for exact boiling temperature measurements. A very simple apparatus is described in ASTM D 1120-72 (see Appendix). The liquid is heated in this apparatus under equilibrium conditions at atmospheric pressure until it is boiling.

1.4.2. Dynamic method

This method involves the measurement of the vapour recondensation temperature by means of an appropriate thermometer in the reflux while boiling. The pressure can be varied in this method.

1.4.3. Distillation method for boiling temperature

This method involves distillation of the liquid and measurement of the vapour recondensation temperature and determination of the amount of distillate.

1.4.4. Method according to Siwoloboff

A sample is heated in a sample tube, which is immersed in a liquid in a heat-bath. A fused capillary, containing an air bubble in the lower part, is dipped in the sample tube.

1.4.5. Photocell detection

Following the principle according to Siwoloboff, automatic photo-electrical measurement is made using rising bubbles.

1.4.6. Differential thermal analysis

This technique records the difference in temperatures between the substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the temperature record.

1.4.7. Differential scanning calorimetry

This technique records the difference in energy inputs into a substance and a reference material as a function of temperature, while the substance and reference material are subjected to the same controlled temperature programme. This energy is the energy necessary to establish zero temperature difference between the substance and the reference material. When the sample undergoes a transition involving a change of enthalpy, that change is indicated by an endothermic departure (boiling) from the base line of the heat flow record.

1.5. QUALITY CRITERIA

The applicability and accuracy of the different methods used for the determination of the boiling temperature/boiling range are listed in table 1.

Table 1:

Comparison of the methods

Method of measurement | Estimated accuracy | Existing standard |

Ebulliometer | ±1,4 K (up to 373 K) [5] [6] ±2,5 K (up to 600 K) [5] [6] | ASTM D 1120-72 [5] |

Dynamic method | ±0,5 K (up to 600 K) [6] | |

Distillation process (boiling range) | ±0,5 K (up to 600 K) | ISO/R 918, DIN 53171, BS 4591/71 |

According to Siwoloboff | ± 2 K (up to 600 K) [6] | |

Photocell detection | ±0,3 K (up to 373 K) [6] | |

Differential thermal calorimetry | ±0,5 K (up to 600 K) ±2,0 K (up to 1273 K) | ASTM E 537-76 |

Differential scanning calorimetry | ±0,5 K (up to 600 K) ±2,0 K (up to 1273 K) | ASTM E 537-76 |

1.6. DESCRIPTION OF THE METHODS

The procedures of some test methods have been described in international and national standards (see Appendix).

1.6.1. Ebulliometer

See Appendix.

1.6.2. Dynamic method

See test method A.4 for the determination of the vapour pressure.

The boiling temperature observed with an applied pressure of 101,325 kPa is recorded.

1.6.3. Distillation process (boiling range)

See Appendix.

1.6.4. Method according to Siwoloboff

The sample is heated in a melting temperature apparatus in a sample tube, with a diameter of approximately 5 mm (figure 1).

Figure 1 shows a type of standardised melting and boiling temperature apparatus (JIS K 0064) (made of glass, all specifications in millimetres).

Figure 1

A: Measuring vessel

B: Stopper

C: Vent

D: Thermometer

E: Auxiliary thermometer

F: Bath liquid

G: Sample tube, maximum 5 mm outer diameter; containing a capillary tube, approximately 100 mm long, approximately 1 mm long inner diameter and approximately 0,2 to 0,3 mm wall-thickness

H: Side tube

+++++ TIFF +++++

A capillary tube (boiling capillary) which is fused about 1 cm above the lower end is placed in the sample tube. The level to which the test substance is added is such that the fused section of the capillary is below the surface of the liquid. The sample tube containing the boiling capillary is fastened either to the thermometer with a rubber band or is fixed with a support from the side (see figure 2).

Figure 2 Principle according to Siwoloboff | Figure 3 Modified principle |

+++++ TIFF +++++

| |

The bath liquid is chosen according to boiling temperature. At temperatures up to 573 K, silicone oil can be used. Liquid paraffin may only be used up to 473 K. The heating of the bath liquid should be adjusted to a temperature rise of 3 K/min at first. The bath liquid must be stirred. At about 10 K below the expected boiling temperature, the heating is reduced so that the rate of temperature rise is less than 1 K/min. Upon approach of the boiling temperature, bubbles begin to emerge rapidly from the boiling capillary.

The boiling temperature is that temperature when, on momentary cooling, the string of bubbles stops and fluid suddenly starts rising in the capillary. The corresponding thermometer reading is the boiling temperature of the substance.

In the modified principle (figure 3) the boiling temperature is determined in a melting temperature capillary. It is stretched to a fine point about 2 cm in length (a) and a small amount of the sample is sucked up. The open end of the fine capillary is closed by melting, so that a small air bubble is located at the end. While heating in the melting temperature apparatus (b), the air bubble expands. The boiling temperature corresponds to the temperature at which the substance plug reaches the level of the surface of the bath liquid (c).

1.6.5. Photocell detection

The sample is heated in a capillary tube inside a heated metal block.

A light beam is directed, via suitable holes in the block, through the substance onto a precisely calibrated photocell.

During the increase of the sample temperature, single air bubbles emerge from the boiling capillary. When the boiling temperature is reached the number of bubbles increases greatly. This causes a change in the intensity of light, recorded by a photocell, and gives a stop signal to the indicator reading out the temperature of a platinum resistance thermometer located in the block.

This method is especially useful because it allows determinations below room temperature down to 253,15 K (– 20 oC) without any changes in the apparatus. The instrument merely has to be placed in a cooling bath.

1.6.6. Thermal analysis

1.6.6.1. Differential thermal analysis

See Appendix.

1.6.6.2. Differential scanning calorimetry

See Appendix.

2. DATA

At small deviations from the normal pressure (max. ± 5 kPa) the boiling temperatures are normalised to Tn by means of the following number-value equation by Sidney Young:

Tn = T + (fT × Δp)

where:

Δp = (101,325 - p) [note sign]

P = pressure measurement in kPa

fT = rate of change of boiling temperature with pressure in K/kPa

T = measured boiling temperature in K

Tn = boiling temperature corrected to normal pressure in K

The temperature-correction factors, fT, and equations for their approximation are included in the international and national standards mentioned above for many substances.

For example, the DIN 53171 method mentions the following rough corrections for solvents included in paints:

Table 2:

Temperature — corrections factors fT

Temperature T (K) | Correction factor fT (K/kPa) |

323,15 | 0,26 |

348,15 | 0,28 |

373,15 | 0,31 |

398,15 | 0,33 |

423,15 | 0,35 |

448,15 | 0,37 |

473,15 | 0,39 |

498,15 | 0,41 |

523,15 | 0,4 |

548,15 | 0,45 |

573,15 | 0,47 |

3. REPORTING

The test report shall, if possible, include the following information:

- method used,

- precise specification of the substance (identity and impurities) and preliminary purification step, if any,

- an estimate of the accuracy.

The mean of at least two measurements which are in the range of the estimated accuracy (see table 1) is reported as the boiling temperature.

The measured boiling temperatures and their mean shall be stated and the pressure(s) at which the measurements were made shall be reported in kPa. The pressure should preferably be close to normal atmospheric pressure.

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

4. REFERENCES

(1) OECD, Paris, 1981, Test Guideline 103, Decision of the Council C (81) 30 final.

(2) IUPAC, B. Le Neindre, B. Vodar, editions. Experimental thermodynamics, Butterworths, London, 1975, vol. II.

(3) R. Weissberger edition: Technique of organic chemistry, Physical methods of organic chemistry, Third Edition, Interscience Publications, New York, 1959, vol. I, Part I, Chapter VIII.

Appendix

For additional technical details, the following standards may be consulted for example.

1. Ebulliometer

1.1. Melting temperature devices with liquid bath

ASTM D 1120-72 | Standard test method for boiling point of engine anti-freezes |

2. Distillation process (boiling range)

ISO/R 918 | Test Method for Distillation (Distillation Yield and Distillation Range) |

BS 4349/68 | Method for determination of distillation of petroleum products |

BS 4591/71 | Method for the determination of distillation characteristics |

DIN 53171 | Losungsmittel für Anstrichstoffe, Bestimmung des Siedeverlaufes |

NF T 20-608 | Distillation: détermination du rendement et de l'intervalle de distillation |

3. Differential thermal analysis and differential scanning calorimetry

ASTM E 537-76 | Standard method for assessing the thermal stability of chemicals by methods of differential thermal analysis |

ASTM E 473-85 | Standard definitions of terms relating to thermal analysis |

ASTM E 472-86 | Standard practice for reporting thermoanalytical data |

DIN 51005 | Thermische Analyse, Begriffe |

A.3. RELATIVE DENSITY

1. METHOD

The methods described are based on the OECD Test Guideline (1). The fundamental principles are given in reference (2).

1.1. INTRODUCTION

The methods for determining relative density described are applicable to solid and to liquid substances, without any restriction in respect to their degree of purity. The various methods to be used are listed in table 1.

1.2. DEFINITIONS AND UNITS

The relative density D204 of solids or liquids is the ratio between the mass of a volume of substance to be examined, determined at 20 oC, and the mass of the same volume of water, determined at 4 oC. The relative density has no dimension.

The density, ρ, of a substance is the quotient of the mass, m, and its volume, v.

The density, ρ, is given, in SI units, in kg/m3.

1.3. REFERENCE SUBSTANCES (1) (3)

1.4. PRINCIPLE OF THE METHODS

Four classes of methods are used.

1.4.1. Buoyancy methods

1.4.1.1. Hydrometer (for liquid substances)

Sufficiently accurate and quick determinations of density may be obtained by the floating hydrometers, which allow the density of a liquid to be deduced from the depth of immersion by reading a graduated scale.

1.4.1.2. Hydrostatic balance (for liquid and solid substances)

The difference between the weight of a test sample measured in air and in a suitable liquid (e.g. water) can be employed to determine its density.

For solids, the measured density is only representative of the particular sample employed. For the determination of density of liquids, a body of known volume, v, is weighed first in air and then in the liquid.

1.4.1.3. Immersed body method (for liquid substances) (4)

In this method, the density of a liquid is determined from the difference between the results of weighing the liquid before and after immersing a body of known volume in the test liquid.

1.4.2. Pycnometer methods

For solids or liquids, pycnometers of various shapes and with known volumes may be employed. The density is calculated from the difference in weight between the full and empty pycnometer and its known volume.

1.4.3. Air comparison pycnometer (for solids)

The density of a solid in any form can be measured at room temperature with the gas comparison pycnometer. The volume of a substance is measured in air or in an inert gas in a cylinder of variable calibrated volume. For the calculation of density one mass measurement is taken after concluding the volume measurement.

1.4.4. Oscillating densitimeter (5) (6) (7)

The density of a liquid can be measured by an oscillating densitimeter. A mechanical oscillator constructed in the form of a U-tube is vibrated at the resonance frequency of the oscillator which depends on its mass. Introducing a sample changes the resonance frequency of the oscillator. The apparatus has to be calibrated by two liquid substances of known densities. These substances should preferably be chosen such that their densities span the range to be measured.

1.5. QUALITY CRITERIA

The applicability of the different methods used for the determination of the relative density is listed in the table.

1.6. DESCRIPTION OF THE METHODS

The standards given as examples, which are to be consulted for additional technical details, are attached in the Appendix.

The tests have to be run at 20 oC, and at least two measurements performed.

2. DATA

See standards.

3. REPORTING

The test report shall, if possible, include the following information:

- method used,

- precise specification of the substance (identity and impurities) and preliminary purification step, if any.

204, shall be reported as defined in 1.2, along with the physical state of the measured substance.

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

Table:

Applicability of methods

Method of measurement | Density | Maximum possible dynamic viscosity | Existing Standards |

solid | liquid |

1.4.1.1.Hydrometer | | yes | 5 Pa s | ISO 387, ISO 649-2, NF T 20-050 |

(b)liquids | | yes | 5 Pa s | ISO 901 and 758 |

1.4.1.3.Immersed body method | | yes | 20 Pa s | DIN 53217 |

1.4.2.Pycnometer | | | | ISO 3507 |

(b)liquids | | yes | 500 Pa s | ISO 758 |

1.4.4.Oscillating densitimer | | yes | 5 Pa s | |

4. REFERENCES

(1) OECD, Paris, 1981, Test Guideline 109, Decision of the Council C(81) 30 final.

(2) R. Weissberger ed., Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Chapter IV, Interscience Publ., New York, 1959, vol. I, Part 1.

(3) IUPAC, Recommended reference materials for realization of physico-chemical properties, Pure and applied chemistry, 1976, vol. 48, p. 508.

(4) Wagenbreth, H., Die Tauchkugel zur Bestimmung der Dichte von Flüssigkeiten, Technisches Messen tm, 1979, vol. II, p. 427-430.

(5) Leopold, H., Die digitale Messung von Flüssigkeiten, Elektronik, 1970, vol. 19, p. 297-302.

(6) Baumgarten, D., Füllmengenkontrolle bei vorgepackten Erzeugnissen -Verfahren zur Dichtebestimmung bei flüssigen Produkten und ihre praktische Anwendung, Die Pharmazeutische Industrie, 1975, vol. 37, p. 717-726.

(7) Riemann, J., Der Einsatz der digitalen Dichtemessung im Brauereilaboratorium, Brauwissenschaft, 1976, vol. 9, p. 253-255.

Appendix

For additional technical details, the following standards may be consulted for example.

1. Buoyancy methods

1.1. Hydrometer

DIN 12790, ISO 387 | Hydrometer; general instructions |

DIN 12791 | Part I: Density hydrometers; construction, adjustment and use Part II: Density hydrometers; standardised sizes, designation Part III: Use and test |

ISO 649-2 | Laboratory glassware: Density hydrometers for general purpose |

NF T 20-050 | Chemical products for industrial use — Determination of density of liquids — Areometric method |

DIN 12793 | Laboratory glassware: range find hydrometers |

1.2. Hydrostatic balance

For solid substances

ISO 1183 | Method A: Methods for determining the density and relative density of plastics excluding cellular plastics |

NF T 20-049 | Chemical products for industrial use — Determination of the density of solids other than powders and cellular products — Hydrostatic balance method |

ASTM-D-792 | Specific gravity and density of plastics by displacement |

DIN 53479 | Testing of plastics and elastomers; determination of density |

For liquid substances

ISO 901 | ISO 758 |

DIN 51757 | Testing of mineral oils and related materials; determination of density |

ASTM D 941-55, ASTM D 1296-67 and ASTM D 1481-62 |

ASTM D 1298 | Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer method |

BS 4714 | Density, specific gravity or API gravity of crude petroleum and liquid petroleum products by hydrometer method |

1.3. Immersed body method

DIN 53217 | Testing of paints, varnishes and similar coating materials; determination of density; immersed body method |

2. Pycnometer methods

2.1. For liquid substances

ISO 3507 | Pycnometers |

ISO 758 | Liquid chemical products; determination of density at 20 oC |

DIN 12797 | Gay-Lussac pycnometer (for non-volatile liquids which are not too viscous) |

DIN 12798 | Lipkin pycnometer (for liquids with a kinematic viscosity of less than 100 10-6 m2 s-1 at 15 oC) |

DIN 12800 | Sprengel pycnometer (for liquids as DIN 12798) |

DIN 12801 | Reischauer pycnometer (for liquids with a kinematic viscosity of less than 100. 10-6 m2 s-1 at 20 oC, applicable in particular also to hydrocarbons and aqueous solutions as well as to liquids with higher vapour pressure, approximately 1 bar at 90 oC) |

DIN 12806 | Hubbard pycnometer (for viscous liquids of all types which do not have too high a vapour pressure, in particular also for paints, varnishes and bitumen) |

DIN 12807 | Bingham pycnometer (for liquids, as in DIN 12801) |

DIN 12808 | Jaulmes pycnometer (in particular for ethanol — water mixture) |

DIN 12809 | Pycnometer with ground-in thermometer and capillary side tube (for liquids which are not too viscous) |

DIN 53217 | Testing of paints, varnishes and similar products; determination of density by pycnometer |

DIN 51757 | Point 7: Testing of mineral oils and related materials; determination of density |

ASTM D 297 | Section 15: Rubber products — chemical analysis |

ASTM D 2111 | Method C: Halogenated organic compounds |

BS 4699 | Method for determination of specific gravity and density of petroleum products (graduated bicapillary pycnometer method) |

BS 5903 | Method for determination of relative density and density of petroleum products by the capillary — stoppered pycnometer method |

NF T 20-053 | Chemical products for industrial use — Determination of density of solids in powder and liquids — Pyknometric method |

2.2. For solid substances

ISO 1183 | Method B: Methods for determining the density and relative density of plastics excluding cellular plastics |

NF T 20-053 | Chemical products for industrial use — Determination of density of solids in powder and liquids — Pyknometric method |

DIN 19683 | Determination of the density of soils |

3. Air comparison pycnometer

DIN 55990 | Part 3: Prüfung von Anstrichstoffen und ähnlichen Beschichtungsstoffen; Pulverlack; Bestimmung der Dichte |

DIN 53243 | Anstrichstoffe; chlorhaltige Polymere; Prüfung |

A.4. VAPOUR PRESSURE

1. METHOD

The majority of the methods described are based on the OECD Test Guideline (1). The fundamental principles are given in references (2) and (3).

1.1. INTRODUCTION

It is useful to have preliminary information on the structure, the melting temperature and the boiling temperature of the substance to perform this test.

There is no single measurement procedure applicable to the entire range of vapour pressures. Therefore, several methods are recommended to be used for the measurement of vapour pressure from < 10-4 to 105 Pa.

Impurities will usually affect the vapour pressure, and to an extent which depends greatly upon the kind of impurity.

Where there are volatile impurities in the sample, which could affect the result, the substance may be purified. It may also be appropriate to quote the vapour pressure for the technical material.

Some methods described here use apparatus with metallic parts; this should be considered when testing corrosive substances.

1.2. DEFINITIONS AND UNITS

The vapour pressure of a substance is defined as the saturation pressure above a solid or liquid substance. At the thermodynamic equilibrium, the vapour pressure of a pure substance is a function of temperature only.

The SI unit of pressure which should be used is the pascal (Pa).

Units which have been employed historically, together with their conversion factors, are:

1 Torr (≡ 1 mm Hg) | = 1,333 × 102 Pa |

1 atmosphere | = 1,013 × 105 Pa |

1 bar | = 105 Pa |

The SI unit of temperature is the kelvin (K).

The universal molar gas constant R is 8,314 J mol-1 K-1

The temperature dependence of the vapour pressure is described by the Clausius-Clapeyron equation:

log p =

ΔH

+const.

where:

p = the vapour pressure of the substance in pascals

ΔHv = its heat of vaporisation in Jmol-1

R = the universal molar gas constant in Jmol-1 K-1

T = thermodynamic temperature in K

1.3. REFERENCE SUBSTANCES

1.4. PRINCIPLE OF THE TEST METHODS

For determining the vapour pressure, seven methods are proposed which can be applied in different vapour pressure ranges. For each method, the vapour pressure is determined at various temperatures. In a limited temperature range, the logarithm of the vapour pressure of a pure substance is a linear function of the inverse of the temperature.

1.4.1. Dynamic method

In the dynamic method, the boiling temperature which pertains to a specified pressure is measured.

Recommended range:

103 up to 105 Pa.

This method has also been recommended for the determination of normal boiling temperature and is useful for that purpose up to 600 K.

1.4.2. Static method

In the static process, at thermodynamic equilibrium, the vapour pressure established in a closed system is determined at a specified temperature. This method is suitable for one component and multicomponent solids and liquids.

Recommended range:

10 up to 105 Pa.

This method can also be used in the range 1 to 10 Pa, providing care is taken.

1.4.3. Isoteniscope

This standardised method is also a static method but is usually not suitable for multicomponent systems. Additional information is available in ASTM method D-2879-86.

Recommended range:

from 100 up to 105 Pa.

1.4.4. Effusion method: Vapour pressure balance

The quantity of substance leaving a cell per unit time through an aperture of known size is determined under vacuum conditions such that return of substance into the cell is negligible (e.g. by measurement of the pulse generated on a sensitive balance by a vapour jet or by measuring the weight loss).

Recommended range:

10-3 to 1 Pa.

1.4.5. Effusion method: By loss of weight or by trapping vaporisate

The method is based on the estimation of the mass of test substance flowing out per unit of time of a Knudsen cell (4) in the form of vapour, through a micro-orifice under ultra-vacuum conditions. The mass of effused vapour can be obtained either by determining the loss of mass of the cell or by condensing the vapour at low temperature and determining the amount of volatilised substance using chromatographic analysis. The vapour pressure is calculated by applying the Hertz-Knudsen relation.

Recommended range:

10-3 to 1 Pa.

1.4.6. Gas saturation method

A stream of inert carrier gas is passed over the substance in such a way that it becomes saturated with its vapour. The amount of material transported by a known amount of carrier gas is measurable either by collection in a suitable trap or by an intrain analytical technique. This is then used to calculate the vapour pressure at a given temperature.

Recommended range:

10-4 to 1 Pa.

This method can also be used in the range 1 to 10 Pa providing care is taken.

1.4.7. Spinning rotor

In the spinning rotor gauge, the actual measuring element is a small steel ball which is suspended in a magnetic field and rotates with high speed. The gas pressure is deduced from the pressure-dependent slow-down of the steel ball.

Recommended range:

10-4 to 0,5 Pa.

1.5. QUALITY CRITERIA

The various methods of determining the vapour pressure are compared as to application, repeatability, reproducibility, measuring range, existing standard. This is done in the following table.

Table:

Quality criteria

solid | liquid |

| | | 1 to 5 % | 1 to 5 % | 2 × 103 Pa to 105 Pa | — |

1.4.2.Static method | yes | yes | 5 to 10 % | 5 to 10 % | 10 Pa to 105 Pa [8] | NFT 20-048 (5) |

1.4.3.Isoteniscope | yes | yes | 5 to 10 % | 5 to 10 % | 102 Pa to 105 Pa | ASTM-D 2879-86 |

1.4.4.Effusion method Vap.Pres.balance | yes | yes | 5 to 20 % | 5 to 50 % | 10-3 Pa to 1 Pa | NFT 20-047(6) |

1.4.5.Effusion method weigt loss | yes | yes | 10 to 30 % | — | 10-3 Pa to 1 Pa | — |

1.4.6.Gas saturation method | yes | yes | 10 to 30 % | Up to 50 % | 10-4 Pa to 1 Pa [8] | — |

1.4.7.Spinning rotor method | yes | yes | 10 to 20 % | — | 10-4 Pa to 0,5 Pa | — |

1.6. DESCRIPTION OF THE METHODS

1.6.1. Dynamic measurement

1.6.1.1. Apparatus

The measuring apparatus typically consists of a boiling vessel with attached cooler made of glass or metal (figure 1), equipment for measuring the temperature, and equipment for regulating and measuring the pressure. A typical measuring apparatus shown in the drawing is made from heat-resistant glass and is composed of five parts:

The large, partially double-walled tube consists of a ground jacket joint, a cooler, a cooling vessel and an inlet.

The glass cylinder, with a Cottrell "pump", is mounted in the boiling section of the tube and has a rough surface of crushed glass to avoid "bumping" in the boiling process.

The temperature is measured with a suitable temperature sensor (e.g. resistance thermometer, jacket thermocouple) immersed in the apparatus to the point of measurement (No 5, figure 1) through a suitable inlet (e.g. male ground joint).

The necessary connections are made to the pressure regulation and measuring equipment.

The bulb, which acts as a buffer volume, is connected with the measuring apparatus by means of a capillary tube.

The boiling vessel is heated by a heating element (e.g. cartridge heater) inserted into the glass apparatus from below. The heating current required is set and regulated via a thermocouple.

The necessary vacuum of between 102 Pa and approximately 105 Pa is produced with a vacuum pump.

A suitable valve is used to meter air or nitrogen for pressure regulation (measuring range approximately 102 to 105 Pa) and ventilation.

Pressure is measured with a manometer.

1.6.1.2. Measurement procedure

The vapour pressure is measured by determining the boiling temperature of the sample at various specified pressures between roughly 103 and 105 Pa. A steady temperature under constant pressure indicates that the boiling temperature has been reached. Frothing substances cannot be measured using this method.

The substance is placed in the clean, dry sample vessel. Problems may be encountered with non-powder solids but these can sometimes be solved by heating the cooling jacket. Once the vessel has been filled the apparatus is sealed at the flange and the substance degassed. The lowest desired pressure is then set and the heating is switched on. At the same time, the temperature sensor is connected to a recorder.

Equilibrium is reached when a constant boiling temperature is recorded at constant pressure. Particular care must be taken to avoid bumping during boiling. In addition, complete condensation must occur on the cooler. When determining the vapour pressure of low melting solids, care should be taken to avoid the condenser blocking.

After recording this equilibrium point, a higher pressure is set. The process is continued in this manner until 105 Pa has been reached (approximately 5 to 10 measuring points in all). As a check, equilibrium points must be repeated at decreasing pressures.

1.6.2. Static measurement

1.6.2.1. Apparatus

The apparatus comprises a container for the sample, a heating and cooling system to regulate the temperature of the sample and measure the temperature. The apparatus also includes instruments to set and measure the pressure. Figures 2a and 2b illustrate the basic principles involved.

The sample chamber (figure 2a) is bounded on one side by a suitable high-vacuum valve. A U-tube containing a suitable manometer fluid is attached to the other side. One end of the U-tube branches off to the vacuum pump, the nitrogen cylinder or ventilation valve, and a manometer.

A pressure gauge with a pressure indicator can be used instead of a U-tube (figure 2b).

In order to regulate the temperature of the sample, the sample vessel together with valve and U-tube or pressure gauge is placed in a bath which is kept at a constant temperature of ±0,2 K. The temperature measurements are taken on the outside wall of the vessel containing the sample or in the vessel itself.

A vacuum pump with an upstream cooling trap is used to evacuate the apparatus.

In method 2a the vapour pressure of the substance is measured indirectly using a zero indicator. This takes into account the fact that the density of the fluid in the U-tube alters if the temperature changes greatly.

The following fluids are suitable for use as zero indicators for the U-tube, depending on the pressure range and the chemical behaviour of the test substance: silicone fluids, phthalates. The test substance must not dissolve noticeably in or react with the U-tube fluid.

For the manometer, mercury can be used in the range of normal air pressure to 102 Pa, while silicone fluids and phthalates are suitable for use below 102 Pa down to 10 Pa. Heatable membrane capacity manometers can even be used at below 10-1 Pa. There are also other pressure gauges which can be used below 102 Pa.

1.6.2.2. Measurement procedure

Before measuring, all components of the apparatus shown in figure 2 must be cleaned and dried thoroughly.

For method 2a, fill the U-tube with the chosen liquid, which must be degassed at an elevated temperature before readings are taken.

The test substance is placed in the apparatus, which is then closed and the temperature is reduced sufficiently for degassing. The temperature must be low enough to ensure that the air is sucked out, but — in the case of multiple component system — it must not alter the composition of the material. If required, equilibrium can be established more quickly by stirring.

The sample can be supercooled with e.g. liquid nitrogen (taking care to avoid condensation of air or pump fluid) or a mixture of ethanol and dry ice. For low-temperature measurements use a temperature-regulated bath connected to an ultra-cryomat.

With the valve over the sample vessel open, suction is applied for several minutes to remove the air. The valve is then closed and the temperature of the sample reduced to the lowest level desired. If necessary, the degassing operation must be repeated several times.

When the sample is heated the vapour pressure increases. This alters the equilibrium of the fluid in the U-tube. To compensate for this, nitrogen or air is admitted to the apparatus via a valve until the pressure indicator fluid is at zero again. The pressure required for this can be read off a precision manometer at room temperature. This pressure corresponds to the vapour pressure of the substance at that particular measuring temperature.

Method 2b is similar but the vapour pressure is read off directly.

The temperature-dependence of vapour pressure is determined at suitably small intervals (approximately 5 to 10 measuring points in all) up to the desired maximum. Low-temperature readings must be repeated as a check.

If the values obtained from the repeated readings do not coincide with the curve obtained for increasing temperature, this may be due to one of the following:

1. the sample still contains air (e.g. high-viscosity materials) or low-boiling substances, which is/are released during heating and can be removed by suction following further supercooling;

2. the cooling temperature is not low enough. In this case liquid nitrogen is used as the cooling agent.

If either l or 2 is the case, the measurements must be repeated;

3. the substance undergoes a chemical reaction in the temperature range investigated (e.g. decomposition, polymerisation).

1.6.3. Isoteniscope

A complete description of this method can be found in reference 7. The principle of the measuring device is shown in figure 3. Similarly to the static method described in 1.6.2, the isoteniscope is appropriate for the investigation of solids or liquids.

In the case of liquids, the substance itself serves as the fluid in the auxiliary manometer. A quantity of the liquid, sufficient to fill the bulb and the short leg of the manometer section, is put in the isoteniscope. The isoteniscope is attached to a vacuum system and evacuated, then filled by nitrogen. The evacuation and purge of the system is repeated twice to remove residual oxygen. The filled isoteniscope is placed in an horizontal position so that the sample spreads out into a thin layer in the sample bulb and manometer section (U-part). The pressure of the system is reduced to 133 Pa and the sample gently warmed until it just boils (removal of dissolved fixed gases). The isoteniscope is then placed so that the sample returns to the bulb and short leg of the manometer, so that both are entirely filled with liquid. The pressure is maintained as for degassing; the drawn-out tip of the sample bulb is heated with a small flame until sample vapour released expands sufficiently to displace part of the sample from the upper part of the bulb and manometer arm into the manometer section of the isoteniscope, creating a vapour-filled, nitrogen-free space.

The isoteniscope is then placed in a constant temperature bath, and the pressure of nitrogen is adjusted until its pressure equals that of the sample. Pressure balance is indicated by the manometer section of the isoteniscope. At the equilibrium, the vapour pressure of nitrogen equals the vapour pressure of the substance.

In the case of solids, depending on the pressure and temperature range, the manometer liquids listed in 1.6.2.1 are used. The degassed manometer liquid is filled into a bulge on the long arm of the isoteniscope. Then the solid to be investigated is placed in the bulb and is degassed at elevated temperature. After that the isoteniscope is inclined so that the manometer liquid can flow into the U-tube. The measurement of vapour pressure as a function of temperature is done according to 1.6.2.

1.6.4. Effusion method: vapour pressure balance

1.6.4.1. Apparatus

Various versions of the apparatus are described in the literature (1). The apparatus described here illustrates the general principle involved (figure 4). Figure 4 shows the main components of the apparatus, comprising a high-vacuum stainless steel or glass container, equipment to produce and measure a vacuum and built-in components to measure the vapour pressure on a balance. The following built-in components are included in the apparatus:

- an evaporator furnace with flange and rotary inlet. The evaporator furnace is a cylindrical vessel, made of e.g. copper or a chemically resistant alloy with good thermal conductivity. A glass vessel with a copper wall can also be used. The furnace has a diameter of approximately 3 to 5 cm and is 2 to 5 cm high. There are between one and three openings of different sizes for the vapour stream. The furnace is heated either by a heating spiral around the outside. To prevent heat being dissipated to the base plate, the heater is attached to the base plate by a metal with low thermal conductivity (nickel-silver or chromium-nickel steel), e.g. a nickel-silver pipe attached to a rotary inlet if using a furnace with several openings. This arrangement has the advantage of allowing the introduction of a copper bar. This allows cooling from the outside using a cooling bath,

- if the copper furnace lid has three openings of different diameters at 90o to each other, various vapour pressure ranges within the overall measuring range can be covered (openings between approximately 0,30 and 4,50 mm diameter). Large openings are used for low vapour pressure and vice versa. By rotating the furnace the desired opening or an intermediate position in the vapour stream (furnace opening — shield — balance pan) can be set and the stream of molecules is released or deflected through the furnace opening onto the scale pan. In order to measure the temperature of the substance, a thermocouple or resistance thermometer is placed at a suitable point,

- above the shield is a balance pan belonging to a highly sensitive microbalance (see below). The balance pan is approximately 30 mm in diameter. Gold-plated aluminium is a suitable material,

- the balance pan is surrounded by a cylindrical brass or copper refrigeration box. Depending on the type of balance, it has openings for the balance beam and a shield opening for the stream of molecules and should guarantee complete condensation of the vapour on the balance pan. Heat dissipation to the outside is ensured e.g. by a copper bar connected to the refrigeration box. The bar is routed through the base plate and thermally insulated from it, e.g. with a chromium-nickel steel tube. The bar is immersed in a Dewar flask containing liquid nitrogen under the base plate or liquid nitrogen is circulated through the bar. The refrigeration box is thus kept at approximately - 120 oC. The balance pan is cooled exclusively by radiation and is satisfactory for the pressure range under investigation (cooling approximately 1 hour before the start of measurement),

- the balance is positioned above the refrigeration box. Suitable balances are e.g. a highly sensitive 2-arm electronic microbalance (8) or a highly sensitive moving coil instrument (see OECD Test Guideline 104, Edition 12.05.81),

- the base plate also incorporates electrical connections for thermocouples (or resistance thermometers) and heating coils,

- a vacuum is produced in the vessel using a partial vacuum pump or high-vacuum pump (required vacuum approximately 1 to 2 × 10-3 Pa, obtained after 2 h pumping). The pressure is regulated with a suitable ionisation manometer.

1.6.4.2. Measurement procedure

The vessel is filled with the test substance and the lid is closed. The shield and refrigeration box are slid across the furnace. The apparatus is closed and the vacuum pumps are switched on. The final pressure before starting to take measurements should be approximately 10-4 Pa. Cooling of the refrigeration box starts at 10-2 Pa.

Once the required vacuum has been obtained, start the calibration series at the lowest temperature required. The corresponding opening in the lid is set, the vapour stream passes through the shield directly above the opening and strikes the cooled balance pan. The balance pan must be big enough to ensure that the entire stream guided through the shield strikes it. The momentum of the vapour stream acts as a force against the balance pan and the molecules condense on its cool surface.

The momentum and simultaneous condensation produce a signal on the recorder. Evaluation of the signals provides two pieces of information:

1. in the apparatus described here the vapour pressure is determined directly from the momentum on the balance pan (it is not necessary to know the molecular weight for this (2)). Geometrical factors such as the furnace opening and the angle of the molecular stream must be taken into account when evaluating the readings;

2. the mass of the condensate can be measured at the same time and the rate of evaporation can be calculated from this. The vapour pressure can also be calculated from the rate of evaporation and molecular weight using the Hertz equation (2).

p = G

2 πRT×10

where

G = evaporation rate (kg s-1 m-2)

M = molar mass (g mol-1)

T = temperature (K)

R = universal molar gas constant (Jmol-1 K-1)

p = vapour pressure (Pa)

After the necessary vacuum is reached, the series of measurements is commenced at the lowest desired measuring temperature.

For further measurements, the temperature is increased by small intervals until the maximum desired temperature value is reached. The sample is then cooled again and a second curve of the vapour pressure may be recorded. If the second run fails to confirm the results of the first run, then it is possible that the substance may be decomposing in the temperature range being measured.

1.6.5. Effusion method — by loss of weight

1.6.5.1. Apparatus

The effusion apparatus consists of the following basic parts:

- a tank that can be thermostated and evacuated and in which the effusion cells are located,

- a high vacuum pump (e.g. diffusion pump or turbomolecular pump) with vacuum gauge,

- a trap, using liquefied nitrogen or dry ice.

An electrically heated, aluminium vacuum tank with four stainless steel effusion cells is shown in figure 5 for example. The stainless steel foil of about 0,3 mm thickness has an effusion orifice of 0,2 to 1,0 mm diameter and is attached to the effusion cell by a threaded lid.

1.6.5.2. Measurement procedure

The reference and test substances are filled into each effusion cell, the metal diaphragm with the orifice is secured by the threaded lid, and each cell is weighed to within an accuracy of 0,1 mg. The cell is placed in the thermostated apparatus, which is then evacuated to below one tenth of the expected pressure. At defined intervals of time ranging from 5 to 30 hours, air is admitted into the apparatus, and the loss in mass of the effusion cell is determined by reweighing.

In order to ensure that the results are not influenced by volatile impurities, the cell is reweighed at defined time intervals to check that the evaporation rate is constant over at least two such intervals of time.

The vapour pressure p in the effusion cell is given by:

p =

where

p = vapour pressure (Pa)

m = mass of the substance leaving the cell during time t (kg)

t = time (s)

A = area of the hole (m2)

K = correction factor

R = universal gas constant (Jmol-1 K-1)

T = temperature (K)

M = molecular mass (kg mol-1)

The correction factor K depends on the ratio of length to radius of the cylindrical orifice:

ratio | 0,1 | 0,2 | 0,6 | 1,0 | 2,0 |

K | 0,952 | 0,909 | 0,771 | 0,672 | 0,514 |

The above equation may be written:

p = E

E =

1KA2πR and is the effusion cell constant.

This effusion cell constant E may be determined with reference substances (2,9), using the following equation:

E =

where:

p(r) = vapour pressure of the reference substance (Pa)

M(r) = molecular mass of the reference substance (kg × mol-1)

1.6.6. Gas saturation method

1.6.6.1. Apparatus

A typical apparatus used to perform this test comprises a number of components given in figure 6a and described below (1).

Inert gas:

The carrier gas must not react chemically with the test substance. Nitrogen is usually sufficient for this purpose but occasionally other gases may be required (10). The gas employed must be dry (see figure 6a, key 4: relative humidity sensor).

Flow control:

A suitable gas-control system is required to ensure a constant and selected flow through the saturator column.

Traps to collect vapour:

These are dependent on the particular sample characteristics and the chosen method of analysis. The vapour should be trapped quantitatively and in a form which permits subsequent analysis. For some test substances, traps containing liquids such as hexane or ethylene glycol will be suitable. For others, solid absorbents may be applicable.

As an alternative to vapour trapping and subsequent analysis, in-train analytical techniques, like chromatography, may be used to determine quantitatively the amount of material transported by a known amount of carrier gas. Furthermore, the loss of mass of the sample can be measured.

Heat exchanger:

For measurements at different temperatures it may be necessary to include a heat-exchanger in the assembly.

Saturator column:

The test substance is deposited from a solution onto a suitable inert support. The coated support is packed into the saturator column, the dimensions of which and the flow rate should be such that complete saturation of the carrier gas is ensured. The saturator column must be thermostated. For measurements above room temperature, the region between the saturator column and the traps should be heated to prevent condensation of the test substance.

In order to lower the mass transport occurring by diffusion, a capillary may be placed after the saturator column (figure 6b).

1.6.6.2. Measurement procedure

Preparation of the saturator column:

A solution of the test substance in a highly volatile solvent is added to a suitable amount of support. Sufficient test substance should be added to maintain saturation for the duration of the test. The solvent is totally evaporated in air or in a rotary evaporator, and the thoroughly mixed material is added to the saturator column. After thermostating the sample, dry nitrogen is passed through the apparatus.

Measurement:

The traps or in-train detector are connected to the column effluent line and the time recorded. The flow rate is checked at the beginning and at regular intervals during the experiment, using a bubble meter (or continuously with a mass flow-meter).

The pressure at the outlet to the saturator must be measured. This may be done either:

(a) by including a pressure gauge between the saturator and traps (this may not be satisfactory because this increases the dead space and the adsorptive surface); or

(b) by determining the pressure drops across the particular trapping system used as a function of flow rate in a separate experiment (this may be not very satisfactory for liquid traps).

The time required for collecting the quantity of test substance that is necessary for the different methods of analysis is determined in preliminary runs or by estimates. As an alternative to collecting the substance for further analysis, in-train quantitative analytical technique may be used (e.g. chromatography). Before calculating the vapour pressure at a given temperature, preliminary runs are to be carried out to determine the maximum flow rate that will completely saturate the carrier gas with substance vapour. This is guaranteed if the carrier gas is passed through the saturator sufficiently slowly so that a lower rate gives no greater calculated vapour pressure.

The specific analytical method will be determined by the nature of the substance being tested (e.g. gas chromatography or gravimetry).

The quantity of substance transported by a known volume of carrier gas is determined.

1.6.6.3. Calculation of vapour pressure

Vapour pressure is calculated from the vapour density, W/V, through the equation:

p =

where:

p = vapour pressure (Pa)

W = mass of evaporated test substance (g)

V = volume of saturated gas (m3)

R = universal molar gas constant (Jmol-1 K-1)

T = temperature (K)

M = molar mass of test substance (g mol-1)

Measured volumes must be corrected for pressure and temperature differences between the flow meter and the thermostated saturator. If the flow meter is located downstream from the vapour trap, corrections may be necessary to account for any vaporised trap ingredients (1).

1.6.7. Spinning rotor (8, 11, 13)

1.6.7.1. Apparatus

The spinning rotor technique can be carried out using a spinning rotor viscosity gauge as shown in figure 8. A schematic drawing of the experimental set-up is shown in figure 7.

The measuring apparatus typically consists of a spinning rotor measuring head, placed in a thermostated enclosure (regulated within 0,1 oC). The sample container is placed in a thermostated enclosure (regulated within 0,01 oC), and all other parts of the set-up are kept at a higher temperature to prevent condensation. A high-vacuum pump device is connected to the system by means of high-vacuum valves.

The spinning rotor measuring head consists of a steel ball (4 to 5 mm diameter) in a tube. The ball is suspended and stabilised in a magnetic field, generally using a combination of permanent magnets and control coils.

The ball is made to spin by rotating fields produced by coils. Pick-up coils, measuring the always present low lateral magnetisation of the ball, allow its spinning rate to be measured.

1.6.7.2. Measurement procedure

When the ball has reached a given rotational speed v(o) (usually about 400 revolutions per second), further energising is stopped and deceleration takes place, due to gas friction.

The drop of rotational speed is measured as a function of time. As the friction caused by the magnetic suspension is negligible as compared with the gas friction, the gas pressure p is given by:

p =

rρ

× ln

where:

__NEWLINE__c— = average speed of the gas molecules

r = radius of the ball

ρ = mass density of the ball

σ = coefficient of tangential momentum transfer (ε = 1 for an ideal spherical surface of the ball)

t = time

v(t) = rotational speed after time t

v(o) = initial rotational speed

This equation may also be written:

p =

rρ

- t

× t

where tn, tn-1 are the times required for a given number N of revolutions. These time intervals tn and tn-1 succeed one another, and tn > t n-1.

— is given by:

where:

T = temperature

R = universal molar gas constant

M = molar mass

2. DATA

The vapour pressure from any of the preceding methods should be determined for at least two temperatures. Three or more are preferred in the range 0 to 50 oC, in order to check the linearity of the vapour pressure curve.

3. REPORTING

The test report shall, if possible, include the following information:

- method used,

- precise specification of the substance (identity and impurities) and preliminary purification step, if any,

- at least two vapour pressure and temperature values, preferably in the range 0 to 50 oC,

- all of the raw data,

- a log p versus 1/T curve,

- an estimate of the vapour pressure at 20 or 25 oC.

If a transition (change of state, decomposition) is observed, the following information should be noted:

- nature of the change,

- temperature at which the change occurs at atmospheric pressure,

- vapour pressure at 10 and 20 oC below the transition temperature and 10 and 20 oC above this temperature (unless the transition is from solid to gas).

All information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

4. REFERENCES

(1) OECD, Paris, 1981, Test Guideline 104, Decision of the Council C(81) 30 final.

(2) Ambrose, D. in B. Le Neindre, B. Vodar, (Eds.): Experimental Thermodynamics, Butterworths, London, 1975, vol. II.

(3) R. Weissberger ed.: Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed. Chapter IX, Interscience Publ., New York, 1959, vol. I, Part I.

(4) Knudsen, M. Ann. Phys. Lpz., 1909, vol. 29, 1979; 1911, vol. 34, p. 593.

(5) NF T 20-048 AFNOR (September 85) Chemical products for industrial use — Determination of vapour pressure of solids and liquids within range from 10-1 to 105 Pa — Static method.

(6) NF T 20-047 AFNOR (September 85) Chemical products for industrial use — Determination of vapour pressure of solids and liquids within range from 10-3 to 1 Pa — Vapour pressure balance method.

(7) ASTM D 2879-86, Standard test method for vapour pressure-temperature relationship and initial decomposition temperature of liquids by isoteniscope.

(8) G. Messer, P. Rohl, G. Grosse and W. Jitschin. J. Vac. Sci. Technol.(A), 1987, 'Vol. 5 (4), p. 2440.

(9) Ambrose, D.; Lawrenson, I.J.; Sprake, C.H.S. J. Chem. Thermodynamics 1975, vol. 7, p. 1173.

(10) B.F. Rordorf. Thermochimica Acta, 1985, vol. 85, p. 435.

(11) G. Comsa, J.K. Fremerey and B. Lindenau. J. Vac. Sci. Technol., 1980, vol. 17 (2), p. 642.

(12) G. Reich. J. Vac. Sci. Technol., 1982, vol. 20 (4), p. 1148.

(13) J.K. Fremerey. J. Vac. Sci. Technol.(A), 1985, vol. 3 (3), p. 1715.

Appendix 1

Estimation method

INTRODUCTION

Calculated values of the vapour pressure can be used:

- for deciding which of the experimental methods is appropriate,

- for providing an estimate or limit value in cases where the experimental method cannot be applied due to technical reasons (including where the vapour pressure is very low),

- to help identify those cases where omitting experimental measurement is justified because the vapour pressure is likely to be < 10-5 Pa at ambient temperature.

ESTIMATION METHOD

The vapour pressure of liquids and solids can be estimated by use of the modified Watson Correlation (a). The only experimental data required is the normal boiling point. The method is applicable over the pressure range from 105 Pa to 10-5 Pa.

Detailed information on the method is given in "Handbook of Chemical Property Estimation Methods" (b).

CALCULATION PROCEDURE

According to (b) the vapour pressure is calculated as follows:

ln P

≈

3-2

where:

T = temperature of interest

Tb = normal boiling point

Pvp = vapour pressure at temperature T

ΔHvb = heat of vaporisation

ΔZb = compressibility factor (estimated at 0,97)

m = empirical factor depending on the physical state at the temperature of interest

further:

where KF is an empirical factor considering the polarity of the substance. For several compound types, KF factors are listed in reference (b).

Quite often, data are available in which a boiling point at reduced pressure is given. In such a case, according to (b), the vapour pressure is calculated as follows:

ln P

≈ ln P

Δ Hv

Δ Z

3-2

where T1 is the boiling point at the reduced pressure P1.

REPORT

When using the estimation method, the report shall include a comprehensive documentation of the calculation.

LITERATURE

(a) K.M. Watson, Ind. Eng. Chem., 1943, vol. 35, p. 398.

(b) W.J. Lyman, W.F. Reehl, D.H. Rosenblatt. Handbook of Chemical Property Estimation Methods, Mc Graw-Hill, 1982.

Appendix 2

Figure 1

Apparatus for determining the vapour pressure curve according to the dynamic method

1 = Thermocouple

2 = Vacuum buffer volume

3 = Pressure gauge

4 = Vacuum

5 = Measuring point

6 = Heating element circa 150 W

+++++ TIFF +++++

Figure 2a

Apparatus for determining the vapour pressure curve according to the static method (using a U-tube manometer)

1. Test substance

2. Vapour phase

3. High vacuum valve

4. U-tube (auxiliary manometer)

5. Manometer

6. Temperature bath

7. Temperature measuring device

8. To vacuum pump

9. Ventilation

+++++ TIFF +++++

Figure 2b

Apparatus for determining the vapour pressure curve according to the static method (using a pressure indicator)

1. Test substance

2. Vapour phase

3. High vacuum valve

4. Pressure gauge

5. Pressure indicator

6. Temperature bath

7. Temperature measuring device

+++++ TIFF +++++

Figure 3

Isoteniscope (see reference 7)

1. To pressure control and measurement system

2. 8 mm OD tube

3. Dry nitrogen in pressure system

4. Sample vapour

5. Small tip

6. Liquid sample

+++++ TIFF +++++

Figure 4

Apparatus for determining the vapour pressure curve according to the vapour pressure balance method

1. Test substance

2. Vapour phase with vapour stream

3. Evaporation furnace with rotary inlet

3a. Furnace lid with opening

4. Furnace heating (refrigeration)

5. Measurement of temperature of sample

6. Refrigeration box

7. Shield

8. Refrigeration bar for refrigeration box

9. Balance pan

10. Microbalance

11. To recorder

12. To high-vacuum pump

+++++ TIFF +++++

Figure 5

Example of apparatus for evaporation at low pressure by effusion methode, with an effusion cell volume of 8 cm3

1 Connection to vacuum

2 Wells for platinum resistance thermometer or temperature measurement and control (2)

3 Lid for vacuum tank

4 O-ring

5 Aluminium vacuum tank

6 Device for installing and removing the effusion cells

7 Threaded lid

8 Butterfly nuts (6)

9 Bolts (6)

10 Stainless steel effusion cells

11 Heater cartridges (6)

+++++ TIFF +++++

Figure 6a

An example of a flow system for the determination of vapour pressure by the gas saturation method

1 = Flow regulator

2 = Heat exchanger

3 = Needle valves

4 = Relative humidity sensor

5 = Saturation columns

6 = PTFE joints

7 = Flow meter

8 = Trap (absorber)

9 = Oil trap

10 = Fritted bubbler

Vent

Manifold

+++++ TIFF +++++

Figure 6b

An example of system for the determination of vapour pressure by the gas saturation method, with a capillary placed after the saturation chamber

1. Thermal mass flowmeter

2. Manometer

3. Temperature-controlled chamber

4. Thermostating coil for carrier gas

5. Thermometer (Pt 100)

6. Gas saturation chamber

7. Capillary

8. Absorption vessels

9. Gas meter

10. Cold trap

+++++ TIFF +++++

Figure 7

Example of the experimental set-up for spinning rotor method

Vapour pressure apparatus

A. spinning rotor sensor head;

B. sample cell;

C. thermostat;

D. vacuum line (turbo pump);

E. air thermostat.

+++++ TIFF +++++

Figure 8

Example of spinning rotor measuring head

1. Ball;

2. Evacuated tubular extension of 6;

3. Permanent magnets (2);

4. Coils (2) for vertical stabilization;

5. Driving coils (4);

6. Connection flange.

+++++ TIFF +++++

A.5. SURFACE TENSION

1. METHOD

The methods described are based on the OECD Test Guideline (1). The fundamental principles are given in reference (2).

1.1. INTRODUCTION

The described methods are to be applied to the measurement of the surface tension of aqueous solutions.

It is useful to have preliminary information on the water solubility, the structure, the hydrolysis properties and the critical concentration for micelles formation of the substance before performing these tests.

The following methods are applicable to most chemical substances, without any restriction in respect to their degree of purity.

The measurement of the surface tension by the ring tensiometer method is restricted to aqueous solutions with a dynamic viscosity of less than approximately 200 mPa s.

1.2. DEFINITIONS AND UNITS

The free surface enthalpy per unit of surface area is referred to as surface tension.

The surface tension is given as:

N/m (SI unit) or

mN/m (SI sub-unit)

1 N/m = 103 dynes/cm

1 mN/m = 1 dyne/cm in the obsolete cgs system

1.3. REFERENCE SUBSTANCES

Reference substances which cover a wide range of surface tensions are given in references 1 and 3.

1.4. PRINCIPLE OF THE METHODS

The methods are based on the measurement of the maximum force which is necessary to exert vertically, on a stirrup or a ring in contact with the surface of the liquid being examined placed in a measuring cup, in order to separate it from this surface, or on a plate, with an edge in contact with the surface, in order to draw up the film that has formed.

Substances which are soluble in water at least at a concentration of 1 mg/l are tested in aqueous solution at a single concentration.

1.5. QUALITY CRITERIA

These methods are capable of greater precision than is likely to be required for environmental assessment.

1.6. DESCRIPTION OF THE METHODS

A solution of the substance is prepared in distilled water. The concentration of this solution should be 90 % of the saturation solubility of the substance in water; when this concentration exceeds 1 g/l, a concentration of 1 g/l is used for testing. Substances with water solubility lower than 1 mg/l need not be tested.

1.6.1. Plate method

See ISO 304 and NF T 73-060 (Surface active agents — determination of surface tension by drawing up liquid films).

1.6.2. Stirrup method

See ISO 304 and NF T 73-060 (Surface active agents — determination of surface tension by drawing up liquid films).

1.6.3. Ring method

See ISO 304 and NF T 73-060 (Surface active agents — determination of surface tension by drawing up liquid films).

1.6.4. OECD harmonised ring method

1.6.4.1. Apparatus

Commercially available tensiometers are adequate for this measurement. They consist of the following elements:

- mobile sample table,

- force measuring system,

- measuring body (ring),

- measurement vessel.

1.6.4.1.1. Mobile sample table

The mobile sample table is used as a support for the temperature-controlled measurement vessel holding the liquid to be tested. Together with the force measuring system, it is mounted on a stand.

1.6.4.1.2. Force measuring system

The force measuring system (see figure) is located above the sample table. The error of the force measurement shall not exceed ± 10-6 N, corresponding to an error limit of ±0,1 mg in a mass measurement. In most cases, the measuring scale of commercially available tensiometers is calibrated in mN/m so that the surface tension can be read directly in mN/m with an accuracy of 0,1 mN/m.

1.6.4.1.3. Measuring body (ring)

The ring is usually made of a platinum-iridium wire of about 0,4 mm thickness and a mean circumference of 60 mm. The wire ring is suspended horizontally from a metal pin and a wire mounting bracket to establish the connection to the force measuring system (see figure).

Figure

Measuring body

(All dimensions expressed in millimetres)

Mounting bracket

Ring

+++++ TIFF +++++

1.6.4.1.4. Measurement vessel

The measurement vessel holding the test solution to be measured shall be a temperature-controlled glass vessel. It shall be designed so that during the measurement the temperature of the test solution liquid and the gas phase above its surface remains constant and that the sample cannot evaporate. Cylindrical glass vessels having an inside diameter of not less than 45 mm are acceptable.

1.6.4.2. Preparation of the apparatus

1.6.4.2.1. Cleaning

Glass vessels shall be cleaned carefully. If necessary they shall be washed with hot chromo-sulphuric acid and subsequently with syrupy phosphoric acid (83 to 98 % by weight of H3PO4), thoroughly rinsed in tap water and finally washed with double-distilled water until a neutral reaction is obtained and subsequently dried or rinsed with part of the sample liquid to be measured.

The ring shall first be rinsed thoroughly in water to remove any substances which are soluble in water, briefly immersed in chromo-sulphuric acid, washed in double-distilled water until a neutral reaction is obtained and finally heated briefly above a methanol flame.

Note:

Contamination by substances which are not dissolved or destroyed by chromo-sulphuric acid or phosphoric acid, such as silicones, shall be removed by means of a suitable organic solvent.

1.6.4.2.2. Calibration of the apparatus

The validation of the apparatus consists of verifying the zero point and adjusting it so that the indication of the instrument allows reliable determination in mN/m.

Mounting:

The apparatus shall be levelled, for instance by means of a spirit level on the tensiometer base, by adjusting the levelling screws in the base.

Zero point adjustment:

After mounting the ring on the apparatus and prior to immersion in the liquid, the tensiometer indication shall be adjusted to zero and the ring checked for parallelism to the liquid surface. For this purpose, the liquid surface can be used as a mirror.

Calibrations:

The actual test calibration can be accomplished by means of either of two procedures:

(a) Using a mass: procedure using riders of known mass between 0,1 and 1,0 g placed on the ring. The calibration factor, Φa by which all the instrument readings must be multiplied, shall be determined according to equation (1).

Φa=σrσa | |

where:

mg2b (mN/m)

m = mass of the rider (g)

g = gravity acceleration (981 cm s-2 at sea level)

b = mean circumference of the ring (cm)

σa = reading of the tensiometer after placing the rider on the ring (mN/m).

(b) Using water: procedure using pure water whose surface tension at, for instance, 23 oC is equal to 72,3 mN/m. This procedure is accomplished faster than the weight calibration but there is always the danger that the surface tension of the water is falsified by traces of contamination by surfactants.

The calibration factor, Φb by which all the instrument readings shall be multiplied, shall be determined in accordance with the equation (2):

Φb=σoσg | |

where:

σo = value cited in the literature for the surface tension of water (mN/m)

σg = measured value of the surface tension of the water (mN/m) both at the same temperature.

1.6.4.3. Preparation of samples

Aqueous solutions shall be prepared of the substances to be tested, using the required concentrations in water, and shall not contain any non-dissolved substances.

The solution must be maintained at a constant temperature (±0,5 oC). Since the surface tension of a solution in the measurement vessel alters over a period of time, several measurements shall be made at various times and a curve plotted showing surface tension as a function of time. When no further change occurs, a state of equilibrium has been reached.

Dust and gaseous contamination by other substances interfere with the measurement. The work shall therefore be carried out under a protective cover.

1.6.5. Test conditions

The measurement shall be made at approximately 20 oC and shall be controlled to within ±0,5 oC.

1.6.6. Performance of test

The solutions to be measured shall be transferred to the carefully cleaned measurement vessel, taking care to avoid foaming, and subsequently the measurement vessel shall be placed onto the table of the test apparatus. The table-top with measurement vessel shall be raised until the ring is immersed below the surface of the solution to be measured. Subsequently, the table-top shall be lowered gradually and evenly (at a rate of approximately 0,5 cm/min) to detach the ring from the surface until the maximum force has been reached. The liquid layer attached to the ring must not separate from the ring. After completing the measurements, the ring shall be immersed below the surface again and the measurements repeated until a constant surface tension value is reached. The time from transferring the solution to the measurement vessel shall be recorded for each determination. Readings shall be taken at the maximum force required to detach the ring from the liquid surface.

2. DATA

In order to calculate the surface tension, the value read in mN/m on the apparatus shall be first multiplied by the calibration factor Φa or Φb (depending on the calibration procedure used). This will yield a value which applies only approximately and therefore requires correction.

Harkins and Jordan (4) have empirically determined correction factors for surface-tension values measured by the ring method which are dependent on ring dimensions, the density of the liquid and its surface tension.

Since it is laborious to determine the correction factor for each individual measurement from the Harkins and Jordan tables, in order to calculate the surface tension for aqueous solutions the simplified procedure of reading the corrected surface-tension values directly from the table may be used. (Interpolation shall be used for readings ranging between the tabular values.)

Table:

Correction of the measured surface tension

Only for aqueous solutions, ρ = 1 g/cm3

r | = 9,55 mm (average ring radius) |

r | = 0,185 mm (ring wire radius) |

Experimental Value (mN/m) | Corrected Value (mN/m) |

Weight calibration (see 1.6.4.2.2(a)) | Water calibration (see 1.6.4.2.2(b)) |

20 | 16,9 | 18,1 |

22 | 18,7 | 20,1 |

24 | 20,6 | 22,1 |

26 | 22,4 | 24,1 |

28 | 24,3 | 26,1 |

30 | 26,2 | 28,1 |

32 | 28,1 | 30,1 |

34 | 29,9 | 32,1 |

36 | 31,8 | 34,1 |

38 | 33,7 | 36,1 |

40 | 35,6 | 38,2 |

42 | 37,6 | 40,3 |

44 | 39,5 | 42,3 |

46 | 41,4 | 44,4 |

48 | 43,4 | 46,5 |

50 | 45,3 | 48,6 |

52 | 47,3 | 50,7 |

54 | 49,3 | 52,8 |

56 | 51,2 | 54,9 |

58 | 53,2 | 57,0 |

60 | 55,2 | 59,1 |

62 | 57,2 | 61,3 |

64 | 59,2 | 63,4 |

66 | 61,2 | 65,5 |

68 | 63,2 | 67,7 |

70 | 65,2 | 69,9 |

72 | 67,2 | 72,0 |

74 | 69,2 | — |

76 | 71,2 | — |

78 | 73,2 | — |

This table has been compiled on the basis of the Harkins-Jordan correction. It is similar to that in the DIN Standard (DIN 53914) for water and aqueous solutions (density ρ = 1 g/cm3 and is for a commercially available ring having the dimensions R = 9,55 mm (mean ring radius) and r = 0,185 mm (ring wire radius). The table provides corrected values for surface-tension measurements taken after calibration with weights or calibration with water.

Alternatively, without the preceding calibration, the surface tension call can be calculated according to the following formula:

σ=

where:

F = the force measured on the dynamometer at the breakpoint of the film

R = the radius of the ring

f = the correction factor (1)

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, include the following information:

- method used,

- type of water or solution used,

- precise specification of the substance (identity and impurities),

- measurement results: surface tension (reading) stating both the individual readings and their arithmetic mean as well as the corrected mean (taking into consideration the equipment factor and the correction table),

- concentration of the solution,

- test temperature,

- age of solution used; in particular the time between preparation and measurement of the solution,

- description of time dependence of surface tension after transferring the solution to the measurement vessel,

- all information and remarks relevant for the interpretation of results have to be reported, especially with regard to impurities and physical state of the substance.

3.2. INTERPRETATION OF RESULTS

Considering that distilled water has a surface tension of 72,75 mN/m at 20 oC, substances showing a surface tension lower than 60 mN/m under the conditions of this method should be regarded as being surface-active materials.

4. REFERENCES

(1) OECD, Paris, 1981, Test Guideline 115, Decision of the Council C(81) 30 final.

(2) R. Weissberger ed.: Technique of Organic Chemistry, Physical Methods of Organic Chemistry, 3rd ed., Interscience Publ., New York, 1959, vol. I, Part I, Chapter XIV.

(3) Pure Appl. Chem., 1976, vol. 48, p. 511.

(4) Harkins, W.D., Jordan, H.F., J. Amer. Chem. Soc., 1930, vol. 52, p. 1751.

A.6. WATER SOLUBILITY

1. METHOD

The methods described are based on the OECD Test Guideline (1).

1.1. INTRODUCTION

It is useful to have preliminary information on the structural formula, the vapour pressure, the dissociation constant and the hydrolysis (as a function of pH) of the substance to perform this test.

No single method is available to cover the whole range of solubilities in water.

The two test methods described below cover the whole range of solubilities but are not applicable to volatile substances:

- one which applies to essentially pure substances with low solubilities, (< 10-2 grams per litre), and which are stable in water, referred to as the "column elution method",

- the other which applies to essentially pure substances with higher solubilities (> 10-2 grams per litre), and which are stable in water, referred to as the "flask method".

The water solubility of the test substance can be considerably affected by the presence of impurities.

1.2. DEFINITION AND UNITS

The solubility in water of a substance is specified by the saturation mass concentration of the substance in water at a given temperature. The solubility in water is specified in units of mass per volume of solution. The SI unit is kg/m3 (grams per litre may also be used).

1.3. REFERENCE SUBSTANCES

1.4. PRINCIPLE OF THE TEST METHOD

The approximate amount of the sample and the time necessary to achieve the saturation mass concentration should be determined in a simple preliminary test.

1.4.1. Column elution method

This method is based on the elution of a test substance with water from a micro-column which is charged with an inert support material, such as glass beads or sand, coated with an excess of test substance. The water solubility is determined when the mass concentration of the eluate is constant. This is shown by a concentration plateau as a function of time.

1.4.2. Flask method

In this method, the substance (solids must be pulverised) is dissolved in water at a temperature somewhat above the test temperature. When saturation is achieved the mixture is cooled and kept at the test temperature, stirring as long as necessary to reach equilibrium. Alternatively, the measurement can be performed directly at the test temperature, if it is assured by appropriate sampling that the saturation equilibrium is reached. Subsequently, the mass concentration of the substance in the aqueous solution, which must not contain any undissolved particles, is determined by a suitable analytical method.

1.5. QUALITY CRITERIA

1.5.1. Repeatability

For the column elution method, < 30 % may be obtainable; for the flask method, < 15 % should be observed.

1.5.2. Sensitivity

This depends upon the method of analysis, but mass concentration determinations down to 10-6 grams per litre can be determined.

1.6. DESCRIPTION OF THE METHOD

1.6.1. Test conditions

The test is preferably run at 20 ±0,5 oC. If a temperature dependence is suspected in the solubility (> 3 % per oC), two other temperatures at least 10 oC above and below the initially chosen temperature should also be used. In this case, the temperature control should be ±0,1 oC. The chosen temperature should be kept constant in all relevant parts of the equipment.

1.6.2. Preliminary test

To approximately 0,1 g of the sample (solid substances must be pulverised) in a glass-stoppered 10 ml graduated cylinder, increasing volumes of distilled water at room temperature are added according to the steps shown in the table below:

0,1 g soluble in "x" ml of water | 0,1 | 0,5 | 1 | 2 | 10 | 100 | > 100 |

Approximative solubility (grams per litre) | > 1000 | 1000 to 200 | 200 to 100 | 100 to 50 | 50 to 10 | 10 to 1 | < 1 |

After each addition of the indicated amount of water, the mixture is shaken vigorously for 10 minutes and is visually checked for any undissolved parts of the sample. If, after addition of 10 ml of water, the sample or parts of it remain undissolved, the experiment has to be repeated in a 100 ml measuring cylinder with larger volumes of water. At lower solubilities the time required to dissolve a substance can be considerably longer (at least 24 h should be allowed). The approximate solubility is given in the table under that volume of added water in which complete dissolution of the sample occurs. If the substance is still apparently insoluble, more than 24 h should be allowed (96 h maximum), or further dilution should be undertaken to ascertain whether the column elution or flask solubility method should be used.

1.6.3. Column elution method

1.6.3.1. Support material, solvent and eluent

The support material for the column elution method should be inert. Possible materials which can be employed are glass beads and sand. A suitable volatile solvent of analytical reagent quality should be used to apply the test substance to the support material. Water which has been double distilled in glass or quartz apparatus should be employed as the eluent.

Note:

Water directly from an organic ion exchanger must not be used.

1.6.3.2. Loading of the support

Approximately 600 mg of support material is weighed and transferred to a 50 ml round-bottom flask.

A suitable, weighed amount of test substance is dissolved in the chosen solvent. An appropriate amount of this solution is added to the support material. The solvent must be completely evaporated, e.g. in a rotary evaporator; otherwise water saturation of the support is not achieved due to partition effects on the surface of the support material.

The loading of support material may cause problems (erroneous results) if the test substance is deposited as an oil or a different crystal phase. The problem should be examined experimentally and the details reported.

The loaded support material is allowed to soak for about two hours in approximately 5 ml of water, and then the suspension is added to the microcolumn. Alternatively, dry loaded support material may be poured into the microcolumn, which has been filled with water, and then equilibrated for approximately two hours.

Test procedure:

The elution of the substance from the support material can be carried out in one of two different ways:

- recirculating pump (see figure 1),

- levelling vessel (see figure 4).

1.6.3.3. Column elution method with recirculating pump

Apparatus

A schematic arrangement of a typical system is presented in figure 1. A suitable microcolumn is shown in figure 2, although any size is acceptable, provided it meets the criteria for reproducibility and sensitivity. The column should provide for a headspace of at least five bed volumes of water and be able to hold a minimum of five samples. Alternatively, the size can be reduced if make-up solvent is employed to replace the initial five bed volumes removed with impurities.

The column should be connected to a recirculating pump capable of controlling flows of approximately 25 ml/h. The pump is connected with polytetrafluoroethylene (P.T.F.E.) and/or glass connections. The column and pump, when assembled, should have provision for sampling the effluent and equilibrating the headspace at atmospheric pressure. The column material is supported with a small (5 mm) plug of glass wool, which also serves to filter out particles. The recirculating pump can be, for example, a peristaltic pump or a membrane pump (care must be taken that no contamination and/or absorption occurs with the tube material).

Measurement procedure

The flow through the column is started. It is recommended that a flow rate of approximately 25 ml/hr be used (this corresponds to 10 bed volumes/hr for the column described). The first five bed volumes (minimum) are discarded to remove water-soluble impurities. Following this, the recirculating pump is allowed to run until equilibration is established, as defined by five successive samples whose concentrations do not differ by more than ± 30 % in a random fashion. These samples should be separated from each other by time intervals corresponding to the passage of at least 10 bed volumes of the eluent.

1.6.3.4. Column elution method with levelling vessel

Apparatus (see figures 4 and 3)

Levelling vessel: the connection to the levelling vessel is made by using a ground glass joint which is connected by PTFE tubing. It is recommended that a flow rate of approximately 25 ml/hr be used. Successive eluate fractions should be collected and analysed by the chosen method.

Measurement procedure

Those fractions from the middle eluate range where the concentrations are constant (± 30 %) in at least five consecutive fractions are used to determine the solubility in water.

In both cases (using a recirculating pump or a levelling vessel), a second run is to be performed at half the flow rate of the first. If the results of the two runs are in agreement, the test is satisfactory; if there is a higher apparent solubility with the lower flow rate, then the halving of the flow rate must continue until two successive runs give the same solubility.

In both cases (using a recirculating pump or a levelling vessel) the fractions should be checked for the presence of colloidal matter by examination for the Tyndall effect (light scattering). Presence of such particles invalidates the results, and the test should be repeated with improvements in the filtering action of the column.

The pH of each sample should be recorded. A second run should be performed at the same temperature.

1.6.4. Flask method

1.6.4.1. Apparatus

For the flask method the following material is needed:

- normal laboratory glassware and instrumentation,

- a device suitable for the agitation of solutions under controlled constant temperatures,

- a centrifuge (preferably thermostated), if required with emulsions, and

- equipment for analytical determination.

1.6.4.2. Measurement procedure

The quantity of material necessary to saturate the desired volume of water is estimated from the preliminary test. The volume of water required will depend on the analytical method and the solubility range. About five times the quantity of material determined above is weighed into each of three glass vessels fitted with glass stoppers (e.g. centrifuge tubes, flasks). The chosen volume of water is added to each vessel, and the vessels are tightly stoppered. The closed vessels are then agitated at 30 oC. (A shaking or stirring device capable of operating at constant temperature should be used, e.g. magnetic stirring in a thermostatically controlled water bath). After one day, one of the vessels is removed and re-equilibrated for 24 hours at the test temperature with occasional shaking. The contents of the vessel are then centrifuged at the test temperature, and the concentration of test substance in the clear aqueous phase is determined by a suitable analytical method. The other two flasks are treated similarly after initial equilibration at 30 oC for two and three days, respectively. If the concentration results from at least the last two vessels agree with the required reproducibility, the test is satisfactory. The whole test should be repeated, using longer equilibration times, if the results from vessels 1, 2 and 3 show a tendency to increasing values.

The measurement procedure can also be performed without pre-incubation at 30 oC. In order to estimate the rate of establishment of the saturation equilibrium, samples are taken until the stirring time no longer influences the concentration of the test solution.

The pH of each sample should be recorded.

1.6.5. Analysis

A substance-specific analytical method is preferred for these determinations, since small amounts of soluble impurities can cause large errors in the measured solubility. Examples of such methods are: gas or liquid chromatography, titration methods, photometric methods, voltammetric methods.

2. DATA

2.1. COLUMN ELUTION METHOD

The mean value from at least five consecutive samples taken from the saturation plateau should be calculated for each run, as should the standard deviation. The results should be given in units of mass per volume of solution.

The means calculated on two tests using different flows are compared and should have a repeatability of less than 30 %.

2.2. FLASK METHOD

The individual results should be given for each of the three flasks and those results deemed to be constant (repeatability of less than 15 %) should be averaged and given in units of mass per volume of solution. This may require the reconversion of mass units to volume units, using the density when the solubility is very high (> 100 grams per litre).

3. REPORTING

3.1. COLUMN ELUTION METHOD

The test report shall, if possible, include the following information:

- the results of the preliminary test,

- precise specification of the substance (identity and impurities),

- the individual concentrations, flow rates and pH of each sample,

- the means and standard deviations from at least five samples from the saturation plateau of each run,

- the average of the two successive, acceptable runs,

- the temperature of the water during the saturation process,

- the method of analysis employed,

- the nature of the support material employed,

- loading of support material,

- solvent used,

- evidence of any chemical instability of the substance during the test and the method used,

- all information relevant for the interpretation of the results, especially with regard to impurities and physical state of the substance.

3.2. FLASK METHOD

The test report shall, if possible, include the following information:

- the results of the preliminary test,

- precise specification of the substance (identity and impurities),

- the individual analytical determinations and the average where more than one value was determined for each flask,

- the pH of each sample,

- the average of the value for the different flasks which were in agreement,

- the test temperature,

- the analytical method employed,

- evidence of any chemical instability of the substance during the test and the method used,

- all information relevant for the interpretation of the results, especially with regard to impurities and physical state of the substance.

4. REFERENCES

(1) OECD, Paris, 1981, Test Guideline 105, Decision of the Council C(81) 30 final.

(2) NF T 20-045 (AFNOR) (September 85) Chemical products for industrial use — Determination of water solubility of solids and liquids with low solubility — Column elution method.

(3) NF T 20-046 (AFNOR) (September 85) Chemical products for industrial use — Determination of water solubility of solids and liquids with high solubility — Flask method.

Appendix

Figure 1

Column elution method with recirculating pump

Atmospheric equilibration

Flow meter

Microcolumn

Thermostatically controlled circulating pump

Recirculating pump

Two-way valve for sampling

+++++ TIFF +++++

Figure 2

A typical microcolumn

(All dimensions in millimetres)

(Connection for ground glass joint)

Headspace

Interior 5

Exterior 19

Plug of glass wool

Stopcock with two-way action

+++++ TIFF +++++

Figure 3

A typical microcolumn

(All dimensions in millimetres)

Connection for ground glass joint

Headspace

Interior 5

Exterior 19

Stopcock

+++++ TIFF +++++

Figure 4

Column elution method with levelling vessel

1 = Levelling vessel (e.g. 2,5 litre flask)

2 = Column (see figure 3)

3 = Fraction collector

4 = Thermostat

5 = Teflon tubing

6 = Ground glass joint

7 = Water line (between thermostat and column, inner diameter: approximately 8 mm)

+++++ TIFF +++++

A.8. PARTITION COEFFICIENT

1. METHOD

The "shake flask" method described is based on the OECD Test Guideline (1).

1.1. INTRODUCTION

It is useful to have preliminary information on structural formula, dissociation constant, water solubility, hydrolysis, n-octanol solubility and surface tension of the substance to perform this test.

Measurements should be made on ionisable substances only in their non-ionised form (free acid or free base) produced by the use of an appropriate buffer with a pH of at least one pH unit below (free acid) or above (free base) the pK.

This test method includes two separate procedures: the shake flask method and high performance liquid chromatography (HPLC). The former is applicable when the log Pow value (see below for definitions) falls within the range - 2 to 4 and the latter within the range 0 to 6. Before carrying out either of the experimental procedures a preliminary estimate of the partition coefficient should first be obtained.

The shake-flask method applies only to essentially pure substances soluble in water and n-octanol. It is not applicable to surface active materials (for which a calculated value or an estimate based on the individual n-octanol and water solubilities should be provided).

The HPLC method is not applicable to strong acids and bases, metal complexes, surface-active materials or substances which react with the eluent. For these materials, a calculated value or an estimate based on individual n-octanol and water solubilities should be provided.

The HPLC method is less sensitive to the presence of impurities in the test compound than is the shake-flask method. Nevertheless, in some cases impurities can make the interpretation of the results difficult because peak assignment becomes uncertain. For mixtures which give an unresolved band, upper and lower limits of log P should be stated.

1.2. DEFINITION AND UNITS

The partition coefficient (P) is defined as the ratio of the equilibrium concentrations (ci) of a dissolved substance in a two-phase system consisting of two largely immiscible solvents. In the case n-octanol and water:

The partition coefficient (P) therefore is the quotient of two concentrations and is usually given in the form of its logarithm to base 10 (log P).

1.3. REFERENCE SUBSTANCES

Shake-flask method

HPLC method

In order to correlate the measured HPLC data of a compound with its P value, a calibration graph of log P versus chromatographic data using at least six reference points has to be established. It is for the user to select the appropriate reference substances. Whenever possible, at least one reference compound should have a Pow above that of the test substance, and another a Pow below that of the test substance. For log P values less than 4, the calibration can be based on data obtained by the shake-flask method. For log P values greater than 4, the calibration can be based on validated literature values if these are in agreement with calculated values. For better accuracy, it is preferable to choose reference compounds which are structurally related to the test substance.

Extensive lists of values of log Pow for many groups of chemicals are available (2)(3). If data on the partition coefficients of structurally related compounds are not available, then a more general calibration, established with other reference compounds, may be used.

A list of recommended reference substances and their Pow values is given in Appendix 2.

1.4. PRINCIPLE OF THE METHOD

1.4.1. Shake-flask method

In order to determine a partition coefficient, equilibrium between all interacting components of the system must be achieved, and the concentrations of the substances dissolved in the two phases must be determined. A study of the literature on this subject indicates that several different techniques can be used to solve this problem, i.e. the thorough mixing of the two phases followed by their separation in order to determine the equilibrium concentration for the substance being examined.

1.4.2. HPLC method

HPLC is performed on analytical columns packed with a commercially available solid phase containing long hydrocarbon chains (e.g. C8, C18) chemically bound onto silica. Chemicals injected onto such a column move along it at different rates because of the different degrees of partitioning between the mobile phase and the hydrocarbon stationary phase. Mixtures of chemicals are eluted in order of their hydrophobicity, with water-soluble chemicals eluted first and oil-soluble chemicals last, in proportion to their hydrocarbon-water partition coefficient. This enables the relationship between the retention time on such a (reverse phase) column and the n-octanol/water partition coefficient to be established. The partition coefficient is deduced from the capacity factor k, given by the expression:

-t

in which, tr = retention time of the test substance, and to = average time a solvent molecule needs to pass through the column (dead-time).

Quantitative analytical methods are not required and only the determination of elution times is necessary.

1.5. QUALITY CRITERIA

1.5.1. Repeatability

Shake-flask method

In order to assure the accuracy of the partition coefficient, duplicate determinations are to be made under three different test conditions, whereby the quantity of substance specified as well as the ratio of the solvent volumes may be varied. The determined values of the partition coefficient expressed as their common logarithms should fall within a range of ±0,3 log units.

HPLC method

In order to increase the confidence in the measurement, duplicate determinations must be made. The values of log P derived from individual measurements should fall within a range of ±0,1 log units.

1.5.2. Sensitivity

Shake-flask method

The measuring range of the method is determined by the limit of detection of the analytical procedure. This should permit the assessment of values of log Pow in the range of - 2 to 4 (occasionally when conditions apply, this range may be extended to log Pow up to 5) when the concentration of the solute in either phase is not more than 0,01 mol per litre.

HPLC method

The HPLC method enables partition coefficients to be estimated in the log Pow range 0 to 6.

Normally, the partition coefficient of a compound can be estimated to within ± l log unit of the shake-flask value. Typical correlations can be found in the literature (4)(5)(6)(7)(8). Higher accuracy can usually be achieved when correlation plots are based on structurally-related reference compounds (9).

1.5.3. Specificity

Shake-flask method

The Nernst Partition Law applies only at constant temperature, pressure and pH for dilute solutions. It strictly applies to a pure substance dispersed between two pure solvents. If several different solutes occur in one or both phases at the same time, this may affect the results.

Dissociation or association of the dissolved molecules result in deviations from the Nernst Partition Law. Such deviations are indicated by the fact that the partition coefficient becomes dependent upon the concentration of the solution.

Because of the multiple equilibria involved, this test method should not be applied to ionisable compounds without applying a correction. The use of buffer solutions in place of water should be considered for such compounds; the pH of the buffer should be at least 1 pH unit from the pKa of the substance and bearing in mind the relevance of this pH for the environment.

1.6. DESCRIPTION OF THE METHOD

1.6.1. Preliminary estimate of the partition coefficient

The partition coefficient is estimated preferably by using a calculation method (see Appendix 1), or where appropriate, from the ratio of the solubilities of the test substance ill the pure solvents (10).

1.6.2. Shake-flask method

1.6.2.1. Preparation

n-Octanol: the determination of the partition coefficient should be carried out with high purity analytical grade reagent.

Water: water distilled or double distilled in glass or quartz apparatus should be employed. For ionisable compounds, buffer solutions in place of water should be used if justified.

Note:

Water taken directly from an ion exchanger should not be used.

1.6.2.1.1. Pre-saturation of the solvents

Before a partition coefficient is determined, the phases of the solvent system are mutually saturated by shaking at the temperature of the experiment. To do this, it is practical to shake two large stock bottles of high purity analytical grade n-octanol or water each with a sufficient quantity of the other solvent for 24 hours on a mechanical shaker and then to let them stand long enough to allow the phases to separate and to achieve a saturation state.

1.6.2.1.2. Preparation for the test

The entire volume of the two-phase system should nearly fill the test vessel. This will help prevent loss of material due to volatilisation. The volume ratio and quantities of substance to be used are fixed by the following:

- the preliminary assessment of the partition coefficient (see above),

- the minimum quantity of test substance required for the analytical procedure, and

- the limitation of a maximum concentration in either phase of 0,01 mol per litre.

Three tests are carried out. In the first, the calculated volume ratio of n-octanol to water is used; in the second, this ratio is divided by two; and in the third, this ratio is multiplied by two (e.g. 1:1, 1:2, 2:1).

1.6.2.1.3. Test substance

A stock solution is prepared in n-octanol pre-saturated with water. The concentration of this stock solution should be precisely determined before it is employed in the determination of the partition coefficient. This solution should be stored under conditions which ensure its stability.

1.6.2.2. Test conditions

The test temperature should be kept constant (± 1 oC) and lie in the range of 20 to 25 oC.

1.6.2.3. Measurement procedure

1.6.2.3.1. Establishment of the partition equilibrium

Duplicate test vessels containing the required, accurately measured amounts of the two solvents together with the necessary quantity of the stock solution should be prepared for each of the test conditions.

The n-octanol phases should be measured by volume. The test vessels should either be placed in a suitable shaker or shaken by hand. When using a centrifuge tube, a recommended method is to rotate the tube quickly through 180o about its transverse axis so that any trapped air rises through the two phases. Experience has shown that 50 such rotations are usually sufficient for the establishment of the partition equilibrium. To be certain, 100 rotations in five minutes are recommended.

1.6.2.3.2. Phase separation

When necessary, in order to separate the phases, centrifugation of the mixture should be carried out. This should be done in a laboratory centrifuge maintained at room temperature, or, if a non-temperature controlled centrifuge is used, the centrifuge tubes should be kept for equilibration at the test temperature for at least one hour before analysis.

1.6.2.4. Analysis

For the determination of the partition coefficient, it is necessary to determine the concentrations of the test substance in both phases. This may be done by taking an aliquot of each of the two phases from each tube for each test condition and analyzing them by the chosen procedure. The total quantity of substance present in both phases should be calculated and compared with the quantity of the substance originally introduced.

The aqueous phase should be sampled by a procedure that minimises the risk of including traces of n-octanol: a glass syringe with a removable needle can be used to sample the water phase. The syringe should initially be partially filled with air. Air should be gently expelled while inserting the needle through the n-octanol layer. An adequate volume of aqueous phase is withdrawn into the syringe. The syringe is quickly removed from the solution and the needle detached. The contents of the syringe may then be used as the aqueous sample. The concentration in the two separated phases should preferably be determined by a substance-specific method. Examples of analytical methods which may be appropriate are:

- photometric methods,

- gas chromatography,

- high-performance liquid chromatography.

1.6.3. HPLC method

1.6.3.1. Preparation

Apparatus

A liquid chromatograph, fitted with a pulse-free pump and a suitable detection device, is required. The use of an injection valve with injection loops is recommended. The presence of polar groups in the stationary phase may seriously impair the performance of the HPLC column. Therefore, stationary phases should have the minimal percentage of polar groups (11). Commercial microparticulate reverse-phase packings or ready-packed columns can be used. A guard column may be positioned between the injection system and the analytical column.

Mobile phase

HPLC grade methanol and HPLC grade water are used to prepare the eluting solvent, which is degassed before use. Isocratic elution should be employed. Methanol/water ratios with a minimum water content of 25 % should be used. Typically a 3:1 (v/v) methanol-water mixture is satisfactory for eluting compounds of log P 6 within an hour, at a flow rate of 1 ml/min. For compounds of high log P it may be necessary to shorten the elution time (and those of the reference compounds) by decreasing the polarity of the mobile phase or the column length.

Substances with very low solubility in n-octanol tend to give abnormally low log Pow values with the HPLC method; the peaks of such compounds sometimes accompany the solvent front. This is probably due to the fact that the partitioning process is too slow to reach the equilibrium in the time normally taken by an HPLC separation. Decreasing the flow rate and/or lowering the methanol/water ratio may then be effective to arrive at a reliable value.

Test and reference compounds should be soluble in the mobile phase in sufficient concentrations to allow their detection. Only in exceptional cases may additives be used with the methanol-water mixture, since additives will change the properties of the column. For chromatograms with additives it is mandatory to use a separate column of the same type. If methanol-water is not appropriate, other organic solvent-water mixtures call be used, e.g. ethanol-water or acetonitrile-water.

The pH of the eluent is critical for ionisable compounds. It should be within the operating pH range of the column, which is usually between 2 and 8. Buffering is recommended. Care must be taken to avoid salt precipitation and column deterioration which occur with some organic phase/buffer mixtures. HPLC measurements with silica-based stationary phases above pH 8 are not advisable since the use of an alkaline, mobile phase may cause rapid deterioration in the performance of the column.

Solutes

The reference compounds should be the purest available. Compounds to be used for test or calibration purposes are dissolved in the mobile phase if possible.

Test conditions

The temperature during the measurements should not vary by more than ± 2 K.

1.6.3.2. Measurement

Calculation of dead time to

The dead time to can be determined by using either a homologous series (e.g. n-alkyl methyl ketones) or unretained organic compounds (e.g. thiourea or formamide). For calculating the dead time to by using a homologous series, a set of at least seven members of a homologous series is injected and the respective retention times are determined. The raw retention times tr (nc + 1) are plotted as a function of tr(nc) and the intercept a and slope b of the regression equation:

tr(nc + 1) = a + b tr(nc)

are determined (nc = number of carbon atoms). The dead time to is then given by:

to = a/(1 - b)

Calibration graph

The next step is to construct a correlation plot of log k values versus log p for appropriate reference compounds. In practice, a set of between 5 and 10 standard reference compounds whose log p is around the expected range are injected simultaneously and the retention times are determined, preferably on a recording integrator linked to the detection system. The corresponding logarithms of the capacity factors, log k, are calculated and plotted as a function of the log p determined by the shake-flask method. The calibration is performed at regular intervals, at least once daily, so that possible changes in column performance can be allowed for.

Determination of the capacity factor of the test substance

The test substance is injected in as small a quantity of mobile phase as possible. The retention time is determined (in duplicate), permitting the calculation of the capacity factor k. From the correlation graph of the reference compounds, the partition coefficient of the test substance can be interpolated. For very low and very high partition coefficients, extrapolation is necessary. In those cases particular care has to be taken of the confidence limits of the regression line.

2. DATA

Shake-flask method

The reliability of the determined values of P can be tested by comparison of the means of the duplicate determinations with the overall mean.

3. REPORTING

The test report shall, if possible, include the following information:

- precise specification of the substance (identity and impurities),

- when the methods are not applicable (e.g. surface active material), a calculated value or an estimate based on the individual n-octanol and water solubilities should be provided,

- all information and remarks relevant for the interpretation of results, especially with regard to impurities and physical state of the substance.

For shake-flask method:

- the result of the preliminary estimation, if any,

- temperature of the determination,

- data on the analytical procedures used in determining concentrations,

- time and speed of centrifugation, if used,

- the measured concentrations in both phases for each determination (this means that a total of 12 concentrations will be reported),

- the weight of the test substance, the volume of each phase employed in each test vessel and the total calculated amount of test substance present in each phase after equilibration,

- the calculated values of the partition coefficient (P) and the mean should be reported for each set of test conditions as should the mean for all determinations. If there is a suggestion of concentration dependency of the partition coefficient, this should be noted in the report,

- the standard deviation of individual P values about their mean should be reported,

- the mean P from all determinations should also be expressed as its logarithm (base 10),

- the calculated theoretical Pow when this value has been determined or when the measured value is > 104,

- pH of water used and of the aqueous phase during the experiment,

- if buffers are used, justification for the use of buffers in place of water, composition, concentration and pH of the buffers, pH of the aqueous phase before and after the experiment.

For HPLC method:

- the result of the preliminary estimation, if any,

- test and reference substances, and their purity,

- temperature range of the determinations,

- pH at which the determinations are made,

- details of the analytical and guard column, mobile phase and means of detection,

- retention data and literature log P values for reference compounds used in calibration,

- details of fitted regression line (log k versus log P),

- average retention data and interpolated log P value for the test compound,

- description of equipment and operating conditions,

- elution profiles,

- quantities of test and references substances introduced in the column,

- dead-time and how it was measured.

4. REFERENCES

(1) OECD, Paris, 1981, Test Guideline 107, Decision of the Council C(81) 30 final.

(2) C. Hansch and A.J. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York, 1979.

(3) Log P and Parameter Database, A tool for the quantitative prediction of bioactivity (C. Hansch, chairman, A.J. Leo, dir.) — Available from Pomona College Medical Chemistry Project 1982, Pomona College, Claremont, California 91711.

(4) L. Renberg, G. Sundström and K. Sundh-Nygärd, Chemosphere, 1980, vol. 80, p. 683.

(5) H. Ellgehausen, C. D'Hondt and R. Fuerer, Pestic. Sci., 1981, vol. 12, p. 219.

(6) B. McDuffie, Chemosphere, 1981, vol. 10, p. 73.

(7) W.E. Hammers et al., J. Chromatogr., 1982, vol. 247, p. 1.

(8) J.E. Haky and A.M. Young, J. Liq. Chromat., 1984, vol. 7, p. 675.

(9) S. Fujisawa and E. Masuhara, J. Biomed. Mat. Res., 1981, vol. 15, p. 787.

(10) O. Jubermann, Verteilen und Extrahieren, in Methoden der Organischen Chemie (Houben Weyl), Allgemeine Laboratoriumpraxis (edited by E. Muller), Georg Thieme Verlag, Stuttgart, 1958, Band I/1, p. 223-339.

(11) R.F. Rekker and H.M. de Kort, Euro. J. Med. Chem., 1979, vol. 14, p. 479.

(12) A. Leo, C. Hansch and D. Elkins, Partition coefficients and their uses. Chem. Rev., 1971, vol. 71, p. 525.

(13) R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977.

(14) NF T 20-043 AFNOR (1985). Chemical products for industrial use — Determination of partition coefficient — Flask shaking method.

(15) C.V. Eadsforth and P. Moser, Chemosphere, 1983, vol. 12, p. 1459.

(16) A. Leo, C. Hansch and D. Elkins, Chem. Rev., 1971, vol. 71, p. 525.

(17) C. Hansch, A. Leo, S.H. Unger, K.H. Kim, D. Nikaitani and E.J. Lien, J. Med. Chem., 1973, vol. 16, p. 1207.

(18) W.B. Neely, D.R. Branson and G.E. Blau, Environ. Sci. Technol., 1974, vol. 8, p. 1113.

(19) D.S. Brown and E.W. Flagg, J. Environ. Qual., 1981, vol. 10, p. 382.

(20) J.K. Seydel and K.J. Schaper, Chemische Struktur und biologische Aktivität von Wirkstoffen, Verlag Chemie, Weinheim, New York, 1979.

(21) R. Franke, Theoretical Drug Design Methods, Elsevier, Amsterdam, 1984.

(22) Y.C. Martin, Quantitative Drug Design, Marcel Dekker, New York, Base1, 1978.

(23) N.S. Nirrlees, S.J. Noulton, C.T. Murphy, P.J. Taylor; J. Med. Chem., 1976, vol. 19, p. 615.

Appendix 1

Calculation/estimation methods

INTRODUCTION

A general introduction to calculation methods, data and examples are provided in the Handbook of Chemical Property Estimation Methods (a).

Calculated values of Pow can be used:

- for deciding which of the experimental methods is appropriate (shake-flask range: log Pow: - 2 to 4, HPLC range: log Pow: 0 to 6),

- for selecting the appropriate test conditions (e.g. reference substances for HPLC procedures, volume ratio n-octanol/water for shake flask method),

- as a laboratory internal check on possible experimental errors,

- for providing a Pow-estimate in cases where the experimental methods cannot be applied for technical reasons.

ESTIMATION METHOD

Preliminary estimate of the partition coefficient

The value of the partition coefficient can be estimated by the use of the solubilities of the test substance in the pure solvents: For this:

saturation c

CALCULATION METHODS

Principle of the calculation methods

All calculation methods are based on the formal fragmentation of the molecule into suitable substructures for which reliable log Pow-increments are known. The log Pow of the whole molecule is then calculated as the sum of its corresponding fragment values plus the sum of correction terms for intramolecular interactions.

Lists of fragment constants and correction terms ate available (b)(c)(d)(e);. Some are regularly updated (b).

Quality criteria

In general, the reliability of the calculation method decreases with increasing complexity of the compound under study. In the case of simple molecules with low molecular weight and one or two functional groups, a deviation of 0,1 to 0,3 log Pow units between the results of the different fragmentation methods and the measured value can be expected. In the case of more complex molecules the margin of error can be greater. This will depend on the reliability and availability of fragment constants, as well as on the ability to recognise intramolecular interactions (e.g. hydrogen bonds) and the correct use of the correction terms (less of a problem with the computer software CLOGP-3) (b). In the case of ionising compounds the correct consideration of the charge or degree of ionisation is important.

Calculation procedures

Hansch π-method

The original hydrophobic substituent constant, π, introduced by Fujira et al. (f) is defined as:

πx = log Pow (PhX) - log Pow (PhH)

where Pow (PhX) is the partition coefficient of an aromatic derivative and Pow (PhH) that of the parent compound

(e.g. πCl = log Pow (C6H5Cl) - log Pow (C6H6) = 2,84 - 2,13 = 0,71).

According to its definition the π-method is applicable predominantly for aromatic substitution. π-values for a large number of substituents have been tabulated (b)(c)(d). They are used for the calculation of log Pow for aromatic molecules or substructures.

Rekker method

According to Rekker (g) the log Pow value is calculated as follows:

log P

(interactious terms)

where fi represents the different molecular fragment constants and ai the frequency of their occurrence in the molecule under investigation. The correction terms can be expressed as an integral multiple of one single constant Cm (so-called magic constant). The fragment constants fi and Cm were determined from a list of 1054 experimental Pow values (825 compounds) using multiple regression analysis (c)(h). The determination of the interaction terms is carried out according to set rules described in the literature (e)(h)(i).

Hansch-Leo method

According to Hansch and Leo (c), the log Pow value is calculated from:

log P

where fi represents the different molecular fragment constants, Fj the correction terms and ai, bj the corresponding frequencies of occurrence. Derived from experimental Pow values, a list of atomic and group fragmental values and a list of correction terms Fj (so-called factors) were determined by trial and error. The correction terms have been ordered into several different classes (a)(c). It is relatively complicated and time consuming to take into account all the rules and correction terms. Software packages have been developed (b).

Combined method

The calculation of log Pow of complex molecules can be considerably improved, if the molecule is dissected into larger substructures for which reliable log Pow values are available, either from tables (b)(c) or from one's own measurements. Such fragments (e.g. heterocycles, anthraquinone, azobenzene) can then be combined with the Hansch π-values or with Rekker or Leo fragment constants.

Remarks

(i) The calculation methods can only be applied to partly or fully ionised compounds when it is possible to take the necessary correction factors into account;

(ii) if intramolecular hydrogen bonds can be assumed, the corresponding correction terms (approx. +0,6 to +1,0 log Pow units) have to be added (a). Indications for the presence of such bonds can be obtained from stereo models or spectroscopic data of the molecule;

(iii) If several tautomeric forms are possible, the most likely form should be used as the basis of the calculation;

(iv) the revisions of lists of fragment constants should be followed carefully.

Report

When using calculation/estimation methods, the test report shall, if possible, include the following information:

- description of the substance (mixture, impurities, etc.),

- indication of any possible intramolecular hydrogen bonding, dissociation, charge and any other unusual effects (e.g. tautomerism),

- description of the calculation method,

- identification or supply of database,

- peculiarities in the choice of fragments,

- comprehensive documentation of the calculation.

LITERATURE

(a) W.J. Lyman, W.F. Reehl and D.H. Rosenblatt (ed.), Handbook of Chemical Property Estimation Methods, McGraw-Hill, New York, 1983.

(b) Pomona College, Medicinal Chemistry Project, Claremont, California 91711, USA, Log P Database and Med. Chem. Software (Program CLOGP-3).

(c) C. Hansch, A.J. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley, New York, 1979.

(d) A. Leo, C. Hansch, D. Elkins, Chem. Rev., 1971, vol. 71, p. 525.

(e) R.F. Rekker, H.M. de Kort, Eur. J. Med. Chem. -Chill. Ther. 1979, vol. 14, p. 479.

(f) T. Fujita, J. Iwasa and C. Hansch, J. Amer. Chem. Soc., 1964, vol. 86, p. 5175.

(g) R.F. Rekker, The Hydrophobic Fragmental Constant, Pharmacochemistry Library, Elsevier, New York, 1977, vol. 1.

(h) C.V. Eadsforth, P. Moser, Chemosphere, 1983, vol. 12, p. 1459.

(i) R.A. Scherrer, ACS, American Chemical Society, Washington D.C., 1984, Symposium Series 255, p. 225.

Appendix 2

Recommended Reference Substances for the HLPC Method

No | Reference Substance | log Pow | pKa |

1 | 2-Butanone | 0,3 | |

2 | 4-Acetylpyridine | 0,5 | |

3 | Aniline | 0,9 | |

4 | Acetanilide | 1,0 | |

5 | Benzylalcohol | 1,1 | |

6 | p-Methoxyphenol | 1,3 | pKa = 10,26 |

7 | Phenoxy acetic acid | 1,4 | pKa = 3,12 |

8 | Phenol | 1,5 | pKa = 9,92 |

9 | 2,4-Dinitrophenol | 1,5 | pKa = 3,96 |

10 | Benzonitrile | 1,6 | |

11 | Phenylacetonitrile | 1,6 | |

12 | 4-Methylbenzyl alcohol | 1,6 | |

13 | Acetophenone | 1,7 | |

14 | 2-Nitrophenol | 1,8 | pKa = 7,17 |

15 | 3-Nitrobenzoic acid | 1,8 | pKa = 3,47 |

16 | 4-Chloraniline | 1,8 | pKa = 4,15 |

17 | Nitrobenzene | 1,9 | |

18 | Cinnamic alcohol | 1,9 | |

19 | Benzoic acid | 1,9 | pKa = 4,19 |

20 | p-Cresol | 1,9 | pKa = 10,17 |

21 | Cinnamic acid | 2,1 | pKa = 3,89 cis 4,44 trans |

22 | Anisole | 2,1 | |

23 | Methylbenzoate | 2,1 | |

24 | Benzene | 2,1 | |

25 | 3-Methylbenzoic acid | 2,4 | pKa = 4,27 |

26 | 4-Chlorophenol | 2,4 | pKa = 9,1 |

27 | Trichloroethylene | 2,4 | |

28 | Atrazine | 2,6 | |

29 | Ethylbenzoate | 2,6 | |

30 | 2,6-Dichlorobenzonitrile | 2,6 | |

31 | 3-Chlorobenzoic acid | 2,7 | pKa = 3,82 |

32 | Toluene | 2,7 | |

33 | 1-Naphthol | 2,7 | pKa = 9,34 |

34 | 2,3-Dichloroaniline | 2,8 | |

35 | Chlorobenzene | 2,8 | |

36 | Allyl-phenylether | 2,9 | |

37 | Bromobenzene | 3,0 | |

38 | Ethylbenzene | 3,2 | |

39 | Benzophenone | 3,2 | |

40 | 4-Phenylphenol | 3,2 | pKa = 9,54 |

41 | Thymol | 3,3 | |

42 | 1,4-Dichlorobenzene | 3,4 | |

43 | Diphenylamine | 3,4 | pKa = 0,79 |

44 | Naphthalene | 3,6 | |

45 | Phenylbenzoate | 3,6 | |

46 | Isopropylbenzene | 3,7 | |

47 | 2,4,6-Trichlorophenol | 3,7 | pKa = 6 |

48 | Biphenyl | 4,0 | |

49 | Benzylbenzoate | 4,0 | |

50 | 2,4-Dinitro-6 sec. butyophenol | 4,1 | |

51 | 1,2,4-Trichlorobenzene | 4,2 | |

52 | Dodecanoic acid | 4,2 | |

53 | Diphenylether | 4,2 | |

54 | n-Butylbenzene | 4,5 | |

55 | Phenanthrene | 4,5 | |

56 | Fluoranthene | 4,7 | |

57 | Dibenzyl | 4,8 | |

58 | 2,6-Diphenylpyridine | 4,9 | |

59 | Triphenylamine | 5,7 | |

60 | DDT | 6,2 | |

Other reference substances of low log Pow |

1 | Nicotinic acid | -0,07 | |

A.9. FLASH-POINT

1. METHOD

1.1. INTRODUCTION

It is useful to have preliminary information on the flammability of the substance before performing this test. The test procedure is applicable to liquid substances whose vapours can be ignited by ignition sources. The test methods listed in this text are only reliable for flash-point ranges which are specified in the individual methods.

The possibility of chemical reactions between the substance and the sample holder should be considered when selecting the method to be used.

1.2. DEFINITIONS AND UNITS

The flash-point is the lowest temperature, corrected to a pressure of 101,325 kPa, at which a liquid evolves vapours, under the conditions defined in the test method, in such an amount that a flammable vapour/air mixture is produced in the test vessel.

Units: oC

t = T - 273,15

(t in oC and T in K)

1.3. REFERENCE SUBSTANCES

1.4. PRINCIPLE OF THE METHOD

The substance is placed in a test vessel and heated or cooled to the test temperature according to the procedure described in the individual test method. Ignition trials are carried out in order to ascertain whether or not the sample flashed at the test temperature.

1.5. QUALITY CRITERIA

1.5.1. Repeatability

The repeatability varies according to flash-point range and the test method used; maximum 2 oC.

1.5.2. Sensitivity

The sensitivity depends on the test method used.

1.5.3. Specificity

The specificity of some test methods is limited to certain flash-point ranges and subject to substance-related data (e.g. high viscosity).

1.6. DESCRIPTION OF THE METHOD

1.6.1. Preparations

A sample of the test substance is placed in a test apparatus according to 1.6.3.1 and/or 1.6.3.2.

For safety, it is recommended that a method utilising a small sample size, circa 2 cm3, be used for energetic or toxic substances.

1.6.2. Test conditions

The apparatus should, as far as is consistent with safety, be placed in a draught-free position.

1.6.3. Performance of the test

1.6.3.1. Equilibrium method

See ISO 1516, ISO 3680, ISO 1523, ISO 3679.

1.6.3.2. Non-equilibrium method

Abel apparatus:

See BS 2000 part 170, NF M07-011, NF T66-009.

Abel-Pensky apparatus:

See EN 57, DIN 51755 part 1 (for temperatures from 5 to 65 oC), DIN 51755 part 2 (for temperatures below 5 oC), NF M07-036.

Tag apparatus:

See ASTM D 56.

Pensky-Martens apparatus:

See ISO 2719, EN 11, DIN 51758, ASTM D 93, BS 2000-34, NF M07-019.

Remarks:

When the flash-point, determined by a non-equilibrium method in 1.6.3.2, is found to be 0 ± 2 oC, 21 ± 2 oC or 55 ± 2 oC, it should be confirmed by an equilibrium method using the same apparatus.

Only the methods which can give the temperature of the flash-point may be used for a notification.

To determine the flash-point of viscous liquids (paints, gums and similar) containing solvents, only apparatus and test methods suitable for determining the flash-point of viscous liquids may be used.

See ISO 3679, ISO 3680, ISO 1523, DIN 53213 part 1.

2. DATA

3. REPORTING

The test report shall, if possible, include the following information:

- the precise specification of the substance (identification and impurities),

- the method used should be stated as well as any possible deviations,

- the results and any additional remarks relevant for the interpretation of results.

4. REFERENCES

None.

A.10. FLAMMABILITY (SOLIDS)

1. METHOD

1.1. INTRODUCTION

It is useful to have preliminary information on potentially explosive properties of the substance before performing this test.

This test should only be applied to powdery, granular or paste-like substances.

In order not to include all substances which can be ignited but only those which burn rapidly or those whose burning behaviour is in any way especially dangerous, only substances whose burning velocity exceeds a certain limiting value are considered to be highly flammable.

It can be especially dangerous if incandescence propagates through a metal powder because of the difficulties in extinguishing a fire. Metal powders should be considered highly flammable if they support spread of incandescence throughout the mass within a specified time.

1.2. DEFINITION AND UNITS

Burning time expressed in seconds.

1.3. REFERENCE SUBSTANCES

Not specified.

1.4. PRINCIPLE OF THE METHOD

The substance is formed into an unbroken strip or powder train about 250 mm long and a preliminary screening test performed to determine if, on ignition by a gas flame, propagation by burning with flame or smouldering occurs. If propagation over 200 mm of the train occurs within a specified time then a full test programme to determine the burning rate is carried out.

1.5. QUALITY CRITERIA

Not stated.

1.6. DESCRIPTION OF METHOD

1.6.1. Preliminary screening test

The substance is formed into an unbroken strip or powder train about 250 mm long by 20 mm wide by 10 mm high on a non-combustible, non-porous and low heat-conducting base plate. A hot flame from a gas burner (minimum diameter 5 mm) is applied to one end of the powder train until the powder ignites or for a maximum of two minutes (five minutes for powders of metals or metal-alloys). It should be noted whether combustion propagates along 200 mm of the train within the 4 minutes test period (or 40 minutes for metal powders). If the substance does not ignite and propagate combustion either by burning with flame or smouldering along 200 mm of the powder train within the four minutes (or 40 minutes) test period, then the substance should not be considered as highly flammable and no further testing is required. If the substance propagates burning of a 200 mm length of the powder train in less than four minutes, or less than 40 minutes for metal powders, the procedure described below (point 1.6.2. and following) should be carried out.

1.6.2. Burning rate test

1.6.2.1. Preparation

Powdery or granular substances are loosely filled into a mould 250 mm long with a triangular cross-section of inner height 10 mm and width 20 mm. On both sides of the mould in a longitudinal direction two metal plates are mounted as lateral limitations which project 2 mm beyond the upper edge of the triangular cross section (figure). The mould is then dropped three times from a height of 2 cm onto a solid surface. If necessary the mould is then filled up again. The lateral limitations are then removed and the excess substance scraped off. A non-combustible, non-porous and low heat-conducting base plate is placed on top of the mould, the apparatus inverted and the mould removed.

Paste-like substances are spread on a non-combustible, non-porous and low heat-conducting base plate in the form of a rope 250 mm in length with a cross section of about 1 cm2.

1.6.2.2. Test conditions

In the case a moisture-sensitive substance, the test should be carried out as quickly as possible after its removal from the container.

1.6.2.3. Performance of the test

Arrange the pile across the draught in a fume cupboard.

The air-speed should be sufficient to prevent fumes escaping into the laboratory and should not be varied during the test. A draught screen should be erected around the apparatus.

A hot flame from a gas burner (minimum diameter of 5 mm) is used to ignite the pile at one end. When the pile has burned a distance of 80 mm, the rate of burning over the next 100 mm is measured.

The test is performed six times, using a clean cool plate each time, unless a positive result is observed earlier.

2. DATA

The burning time from the preliminary screening test (1.6.1) and the shortest burning time in up to six tests (1.6.2.3) are relevant for evaluation.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, include the following information:

- the precise specification of the substance (identification and impurities),

- a description of the substance to be tested, its physical state including moisture content,

- results from the preliminary screening test and from the burning rate test if performed,

- all additional remarks relevant to the interpretation of results.

3.2. INTERPRETATION OF THE RESULT

Powdery, granular or paste-1ike substances are to be considered as highly flammable when the time of burning in any tests carried out according to the test procedure described in 1.6.2 is less than 45 seconds. Powders of metals or metal-alloys are considered to be highly flammable when they can be ignited and the flame or the zone of reaction spreads over the whole sample in 10 minutes or less.

4. REFERENCES

NF T 20-042 (September 85) Chemical products for industrial use. Determination of the flammability of solids.

Appendix

Figure

Mould and accessories for the preparation of the pile

(All dimensions in millimetres)

Length of the mould: 250 mm

Material: aluminium

+++++ TIFF +++++

A.11. FLAMMABILITY (GASES)

1. METHOD

1.1. INTRODUCTION

This method allows a determination of whether gases mixed with air at room temperature (circa 20 oC) and atmospheric pressure are flammable and, if so, over what range of concentrations. Mixtures of increasing concentrations of the test gas with air are exposed to an electrical spark and it is observed whether ignition occurs.

1.2. DEFINITION AND UNITS

The range of flammability is the range of concentration between the lower and the upper explosion limits. The lower and the upper explosion limits are those limits of concentration of the flammable gas in admixture with air at which propagation of a flame does not occur.

1.3. REFERENCE SUBSTANCES

Not specified.

1.4. PRINCIPLE OF THE METHOD

The concentration of gas in air is increased step by step and the mixture is exposed at each stage to an electrical spark.

1.5. QUALITY CRITERIA

Not stated.

1.6. DESCRIPTION OF THE METHOD

1.6.1. Apparatus

The test vessel is an upright glass cylinder having a minimum inner diameter of 50 mm and a minimum height of 300 mm. The ignition electrodes are separated by a distance of 3 to 5 mm and are placed 60 mm above the bottom of the cylinder. The cylinder is fitted with a pressure-release opening. The apparatus has to be shielded to restrict any explosion damage.

A standing induction spark of 0,5 sec. duration, which is generated from a high voltage transformer with an output voltage of 10 to 15 kV (maximum of power input 300 W), is used as the ignition source. An example of a suitable apparatus is described in reference (2).

1.6.2. Test conditions

The test must be performed at room temperature (circa 20 oC).

1.6.3. Performance of the test

Using proportioning pumps, a known concentration of gas in air is introduced into the glass cylinder. A spark is passed through the mixture and it is observed whether or not a flame detaches itself from the ignition source and propagates independently. The gas concentration is varied in steps of 1 % vol. until ignition occurs as described above.

If the chemical structure of the gas indicates that it would be non-flammable and the composition of the stoichiometric mixture with air can be calculated, then only mixtures in the range from 10 % less than the stoichiometric composition to 10 % greater than this composition need be tested in 1 % steps.

2. DATA

The occurrence of flame propagation is the only relevant information data for the determination of this property.

3. REPORTING

The test report shall, if possible, include the following information:

- the precise specification of the substance (identification and impurities),

- a description, with dimensions, of the apparatus used,

- the temperature at which the test was performed,

- the tested concentrations and the results obtained,

- the result of the test: non-flammable gas or highly flammable gas,

- if it is concluded that the gas is non-flammable then the concentration range over which it was tested in 1 % steps should be stated,

- all information and remarks relevant to the interpretation of results have to be reported.

4. REFERENCES

(1) NF T 20-041 (September 85) Chemical products for industrial use. Determination of the flammability of gases.

(2) W. Berthold, D. Conrad, T. Grewer, H. Grosse-Wortmann "Entwicklung einer Standard-Apparatur zur Messung von Explosionsgrenzen". Chem.-Ing.- Tech. 1984, vo1. 56, 2, 126-127. , T. Redeker und H. Schacke, p. 126-127.

A.12. FLAMMABILITY (CONTACT WITH WATER)

1. METHOD

1.1. INTRODUCTION

This test method can be used to determine whether the reaction of a substance with water or damp air leads to the development of dangerous amounts of gas or gases which may be highly flammable.

The test method can be applied to both solid and liquid substances. This method is not applicable to substances which spontaneously ignite when in contact with air.

1.2. DEFINITIONS AND UNITS

Highly flammable: substances which, in contact with water or damp air, evolve highly flammable gases in dangerous quantities at a minimum rate of 1 litre/kg per hour.

1.3. PRINCIPLE OF THE METHOD

The substance is tested according to the step by step sequence described below; if ignition occurs at any step, no further testing is necessary. If it is known that the substance does not react violently with water then proceed to step 4 (1.3.4).

1.3.1. Step 1

The test substance is placed in a trough containing distilled water at 20 oC and it is noted whether or not the evolved gas ignites.

1.3.2. Step 2

The test substance is placed on a filter paper floating on the surface of a dish containing distilled water at 20 oC and it is noted whether or not the evolved gas ignites. The filter paper is merely to keep the substance in one place to increase the chances of ignition.

1.3.3. Step 3

The test substance is made into a pile approximately 2 cm high and 3 cm diameter. A few drops of water are added to the pile and it is noted whether or not the evolved gas ignites.

1.3.4. Step 4

The test substance is mixed with distilled water at 20 oC and the rate of evolution of gas is measured over a period of seven hours, at one-hour intervals. If the rate of evolution is erratic, or is increasing, after seven hours, the measuring time should be extended to a maximum time of five days. The test may be stopped if the rate at any time exceeds 1 litre/kg per hour.

1.4. REFERENCE SUBSTANCES

Not specified.

1.5. QUALITY CR1TERIA

Not stated.

1.6. DESCRIPTION OF METHODS

1.6.1. Step 1

1.6.1.1. Test conditions

The test is performed at room temperature (circa 20 oC).

1.6.1.2. Performance of the test

A small quantity (approximately 2 mm diameter) of the test substance should be placed in a trough containing distilled water. A note should be made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.

1.6.2. Step 2

1.6.2.1. Apparatus

A filter-paper is floated flat on the surface of distilled water in any suitable vessel, e.g. a 100 mm diameter evaporating dish.

1.6.2.2. Test conditions

The test is performed at room temperature (circa 20 oC).

1.6.2.3. Performance of the test

A small quantity of the test substance (approximately 2 mm diameter) is placed onto the centre of the filter-paper. A note should be made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.

1.6.3. Step 3

1.6.3.1. Test conditions

The test is performed at room temperature (circa 20 oC).

1.6.3.2. Performance of the test

The test substance is made into a pile approximately 2 cm high and 3 cm diameter with an indentation in the top. A few drops of water are added to the hollow and a note is made of whether (i) any gas is evolved and (ii) if ignition of the gas occurs. If ignition of the gas occurs then no further testing of the substance is needed because the substance is regarded as hazardous.

1.6.4. Step 4

1.6.4.1. Apparatus

The apparatus is set up as shown in the figure.

1.6.4.2. Test conditions

Inspect the container of the test substance for any powder < 500 μm (particle size). If the powder constitutes more than 1 % w/w of the total, or if the sample is friable, then the whole of the substance should be ground to a powder before testing to allow for a reduction in particle size during storage and handling; otherwise the substance is to be tested as received. The test should be performed at room temperature (circa 20 oC) and atmospheric pressure.

1.6.4.3. Performance of the test

10 to 20 ml of water are put into the dropping funnel of the apparatus and 10 g of substance are put in the conical flask. The volume of gas evolved can be measured by any suitable means. The tap of the dropping funnel is opened to let the water into the conical flask and a stop watch is started. The gas evolution is measured each hour during a seven hour period. If, during this period, the gas evolution is erratic, or if, at the end of this period, the rate of gas evolution is increasing, then measurements should be continued for up to five days. If, at any time of measurement, the rate of gas evolution exceeds 1 litre/kg per hour, the test can be discontinued. This test should be performed in triplicate.

If the chemical identity of the gas is unknown, the gas should be analysed. When the gas contains highly flammable components and it is unknown whether the whole mixture is highly flammable, a mixture of the same composition has to be prepared and tested according to the method A.11.

2. DATA

The substance is considered hazardous if:

- spontaneous ignition takes place in any step of the test procedure,

- there is evolution of flammable gas at a rate greater than 1 litre/kg of the substance per hour.

3. REPORTING

The test report shall, if possible, include the following information:

- the precise specification of the substance (identification and impurities),

- details of any initial preparation of the test substance,

- the results of the tests (steps 1, 2, 3 and 4),

- the chemical identity of gas evolved,

- the rate of evolution of gas if step 4 (1.6.4) is performed,

- any additional remarks relevant to the interpretation of the results.

4. REFERENCES

(1) Recommendations on the transport of dangerous goods, test and criteria, 1990, United Nations, New York.

(2) NF T 20-040 (September 85) Chemical products for industrial use. Determination of the flammability of gases formed by the hydrolysis of solid and liquid products.

Appendix

Figure

Apparatus

+++++ TIFF +++++

A.13. PYROPHORIC PROPERTIES OF SOLIDS AND LIQUIDS

1. METHOD

1.1. INTRODUCTION

The test procedure is applicable to solid or liquid substances, which, in small amounts, will ignite spontaneously a short time after coming into contact with air at room temperature (circa 20 oC).

Substances which need to be exposed to air for hours or days at room temperature or at elevated temperatures before ignition occurs are not covered by this test method.

1.2. DEFINITIONS AND UNITS

Substances are considered to have pyrophoric properties if they ignite or cause charring under the conditions described in 1.6.

The auto-flammability of liquids may also need to be tested using method A.15. Auto-ignition temperature (liquids and gases).

1.3. REFERENCE SUBSTANCES

Not specified.

1.4. PRINCIPLE OF THE METHOD

The substance, whether solid or liquid, is added to an inert carrier and brought into contact with air at ambient temperature for a period of five minutes. If liquid substances do not ignite then they are absorbed onto filter paper and exposed to air at ambient temperature (circa 20 oC) for five minutes. If a solid or liquid ignites, or a liquid ignites or chars a filter paper, then the substance is considered to be pyrophoric.

1.5. QUALITY CRITERIA

Repeatability: because of the importance in relation to safety, a single positive result is sufficient for the substance to be considered pyrophoric.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Apparatus

A porcelain cup of circa 10 cm diameter is filled with diatomaceous earth to a height of about 5 mm at room temperature (circa 20 oC).

Note:

Diatomaceous earth or any other comparable inert substance which is generally obtainable shall be taken as representative of soil onto which the test substance might be spilled in the event of an accident.

Dry filter paper is required for testing liquids which do not ignite on contact with air when in contact with an inert carrier.

1.6.2. Performance of the test

(a) Powdery solids

1 to 2 cm3 of the substance to be tested is poured from circa 1 m height onto a non-combustible surface and it is observed whether the substance ignites during dropping or within five minutes of settling.

The test is performed six times unless ignition occurs;

(b) liquids

Circa 5 cm3 of the liquid to be tested is poured into the prepared porcelain cup and it is observed whether the substance ignites within five minutes.

If no ignition occurs in the six tests, perform the following tests:

A 0,5 ml test sample is delivered from a syringe to an indented filter paper and it is observed whether ignition or charring of the filter paper occurs within five minutes of the liquid being added. The test is performed three times unless ignition or charring occurs.

2. DATA

2.1. TREATMENT OF RESULTS

Testing can be discontinued as soon as a positive result occurs in any of the tests.

2.2. EVALUATION

If the substance ignites within five minutes when added to an inert carrier and exposed to air, or a liquid substance chars or ignites a filter paper within five minutes when added and exposed to air, it is considered to be pyrophoric.

3. REPORTING

The test report shall, if possible, include the following information:

- the precise specification of the substance (identification and impurities),

- the results of the tests,

- any additional remark relevant to the interpretation of the results.

4. REFERENCES

(1) NF T 20-039 (September 85) Chemical products for industrial use. Determination of the spontaneous flammability of solids and liquids.

(2) Recommendations on the Transport of Dangerous Goods, Test and criteria, 1990, United Nations, New York.

A.14. EXPLOSIVE PROPERTIES

1. METHOD

1.1. INTRODUCTION

The method provides a scheme of testing to determine whether a solid or a pasty substance presents a danger of explosion when submitted to the effect of a flame (thermal sensitivity), or to shock or friction (sensitivity to mechanical stimuli), and whether a liquid substance presents a danger of explosion when submitted to the effect of a flame or shock.

The method comprises three parts:

(a) a test of thermal sensitivity (1);

(b) a test of mechanical sensitivity with respect to shock (1);

The method yields data to assess the likelihood of initiating an explosion by means of certain common stimuli. The method is not intended to ascertain whether a substance is capable of exploding under any conditions.

The method is appropriate for determining whether a substance will present a danger of explosion (thermal and mechanical sensitivity) under the particular conditions specified in the directive. It is based on a number of types of apparatus which are widely used internationally (1) and which usually give meaningful results. It is recognised that the method is not definitive. Alternative apparatus to that specified may be used provided that it is internationally recognised and the results can be adequately correlated with those from the specified apparatus.

The tests need not be performed when available thermodynamic information (e.g. heat of formation, heat of decomposition) and/or absence of certain reactive groups (2) in the structural formula establishes beyond reasonable doubt that the substance is incapable of rapid decomposition with evolution of gases or release of heat (i.e. the material does not present any risk of explosion). A test of mechanical sensitivity with respect to friction is not required for liquids.

1.2. DEFINITIONS AND UNITS

Explosive:

Substances which may explode under the effect of flame or which are sensitive to shock or friction in the specified apparatus (or are more mechanically sensitive than 1,3-dinitrobenzene in alternative apparatus).

1.3. REFERENCE SUBSTANCES

1,3-dinitrobenzene, technical crystalline product sieved to pass 0,5 mm, for the friction and shock methods.

Perhydro-1,3,5-trinitro-1,3,5-triazine (RDX, hexogen, cyclonite — CAS 121-82-4), recrystallised from aqueous cyclohexanone, wet-sieved through a 250 μm and retained on a 150 μm sieve and dried at 103 ± 2 oC (for four hours) for the second series of friction and shock tests.

1.4. PRINCIPLE OF THE METHOD

Preliminary tests are necessary to establish safe conditions for the performance of the three tests of sensitivity.

1.4.1. Safety-in-handling tests (3)

For safety reasons, before performing the main tests, very small samples (circa 10 mg) of the substance are subjected to heating without confinement in a gas flame, to shock in any convenient form of apparatus and to friction by the use of a mallet against an anvil or any form of friction machine. The objective is to ascertain if the substance is so sensitive and explosive that the prescribed sensitivity tests, particularly that of thermal sensitivity, should be performed with special precautions so as to avoid injury to the operator.

1.4.2. Thermal sensitivity

The method involves heating the substance in a steel tube, closed by orifice plates with differing diameters of hole, to determine whether the substance is liable to explode under conditions of intense heat and defined confinement.

1.4.3. Mechanical sensitivity (shock)

The method involves subjecting the substance to the shock from a specified mass dropped from a specified height.

1.4.4. Mechanical sensitivity (friction)

The method involves subjecting solid or pasty substances to friction between standard surfaces under specified conditions of load and relative motion.

1.5. QUALITY CRITERIA

Not stated.

1.6. DESCRIPTION OF METHOD

1.6.1. Thermal sensitivity (effect of a flame)

1.6.1.1. Apparatus

The apparatus consists of a non-reusable steel tube with its re-usable closing device (figure 1), installed in a heating and protective device. Each tube is deep-drawn from sheet steel (see Appendix) and has an internal diameter of 24 mm, a length of 75 mm and wall thickness of 0,5 mm. The tubes are flanged at the open end to enable them to be closed by the orifice plate assembly. This consists of a pressure-resistant orifice plate, with a central hole, secured firmly to a tube using a two-part screw joint (nut and threaded collar). The nut and threaded collar are made from chromium-manganese steel (see Appendix) which is spark-free up to 800 oC. The orifice plates are 6 mm thick, made from heat-resistant steel (see Appendix), and are available with a range of diameters of opening.

1.6.1.2. Test conditions

Normally the substance is tested as received although in certain cases, e.g. if pressed, cast or otherwise condensed, it may be necessary to test the substance after crushing.

For solids, the mass of material to be used in each test is determined using a two-stage dry run procedure. A tared tube is filled with 9 cm3 of substance and the substance tamped with 80 N force applied to the total cross-section of the tube. For reasons of safety or in cases where the physical form of the sample can be changed by compression other filling procedures may be used; e.g. if the substance is very friction sensitive then tamping is not appropriate. If the material is compressible then more is added and tamped until the tube is filled to 55 mm from the top. The total mass used to fill the tube to the 55 mm level is determined and two further increments, each tamped with 80 N force, are added. Material is then either added with tamping, or taken out, as required, to leave the tube filled to a level 15 mm from the top. A second dry run is performed, starting with a tamped quantity of a third of the total mass found in the first dry run. Two more of these increments are added with 80 N tamping and the level of the substance in the tube adjusted to 15 mm from the top by addition or subtraction of material as required. The amount of solid determined in the second dry run is used for each trial; filling being performed in three equal amounts, each compressed to 9 cm3 by whatever force is necessary. (This may be facilitated by the use of spacing rings).

Liquids and gels are loaded into the tube to a height of 60 mm taking particular care with gels to prevent the formation of voids. The threaded collar is slipped onto the tube from below, the appropriate orifice plate is inserted and the nut tightened after applying some molybdenum disulphide based lubricant. It is essential to check that none of the substance is trapped between the flange and the plate, or in the threads.

Heating is provided by propane taken from an industrial cylinder, fitted with a pressure regulator (60 to 70 mbar), through a meter and evenly distributed (as indicated by visual observation of the flames from the burners) by a manifold to four burners. The burners are located around the test chamber as shown in figure 1. The four burners have a combined consumption of about 3,2 litres of propane per minute. Alternative fuel gases and burners may be used but the heating rate must be as specified in figure 3. For all apparatus, the heating rate must be checked periodically using tubes filled with dibutyl phthalate as indicated in figure 3.

1.6.1.3. Performance of the tests

Each test is performed until either the tube is fragmented or the tube has been heated for five minutes. A test resulting in the fragmentation of the tube into three or more pieces, which in some cases may be connected to each other by narrow strips of metal as illustrated in figure 2, is evaluated as giving an explosion. A test resulting in fewer fragments or no fragmentation is regarded as not giving an explosion.

A series of three tests with a 6,0 mm diameter orifice plate is first performed and, if no explosions are obtained, a second series of three tests is performed with a 2,0 mm diameter orifice plate. If an explosion occurs during either test series then no further tests are required.

1.6.1.4. Evaluation

The test result is considered positive if an explosion occurs in either of the above series of tests.

1.6.2. Mechanical sensitivity (shock)

1.6.2.1. Apparatus (figure 4)

The essential parts of a typical fall hammer apparatus are a cast steel block with base, anvil, column, guides, drop weights, release device and a sample holder. The steel anvil 100 mm (diameter) × 70 mm (height) is screwed to the top of a steel block 230 mm (length) × 250 mm (width) × 200 mm (height) with a cast base 450 mm (length) × 450 mm (width) × 60 mm (height). A column, made from seamless drawn steel tube, is secured in a holder screwed on to the back of the steel block. Four screws anchor the apparatus to a solid concrete block 60 × 60 × 60 cm such that the guide rails are absolutely vertical and the drop weight falls freely. 5 and 10 kg weights, made from solid steel, are available for use. The striking head of each weight is of hardened steel, HRC 60 to 63, and has a minimum diameter of 25 mm.

The sample under test is enclosed in a shock device consisting of two coaxial solid steel cylinders, one above the other, in a hollow cylindrical steel guide ring. The solid steel cylinders should be of 10 (-0,003, -0,005) mm diameter and 10 mm height and have polished surfaces, rounded edges (radius of curvature 0,5 mm) and a hardness of HRC 58 to 65. The hollow cylinder must have an external diameter of 16 mm, a polished bore of 10 (+0,005, +0,010) mm and a height of 13 mm. The shock device is assembled on an intermediate anvil (26 mm diameter and 26 mm height) made of steel and centred by a ring with perforations to allow escape of fumes.

1.6.2.2. Test conditions

The sample volume should be 40 mm3, or a volume to suit any alternative apparatus. Solid substances should be tested in the dry state and prepared as follows:

(a) powdered substances are sieved (sieve size 0,5 mm); all that has passed through the sieve is used for testing;

(b) pressed, cast or otherwise condensed substances are broken into small pieces and sieved; the sieve fraction from 0,5 to 1 mm diameter is used for testing and should be representative of the original substance.

Substances normally supplied as pastes should be tested in the dry state where possible or, in any case, following removal of the maximum possible amount of diluent. Liquid substances are tested with a 1 mm gap between the upper and lower steel cylinders.

1.6.2.3. Performance of the tests

A series of six tests are performed dropping the 10 kg mass from 0,40 m (40 J). If an explosion is obtained during the six tests at 40 J, a further series of six tests, dropping a 5 kg mass from 0,15 m (7,5 J), must be performed. In other apparatus, the sample is compared with the chosen reference substance using an established procedure (e.g. up-and-down technique etc.).

1.6.2.4. Evaluation

The test result is considered positive if an explosion (bursting into flame and/or a report is equivalent to explosion) occurs at least once in any of the tests with the specified shock apparatus or the sample is more sensitive than 1,3-dinitrobenzene or RDX in an alternative shock test.

1.6.3. Mechanical sensitivity (friction)

1.6.3.1. Apparatus (figure 5)

The friction apparatus consists of a cast steel base plate on which is mounted the friction device. This consists of a fixed porcelain peg and moving porcelain plate. The porcelain plate is held in a carriage which runs in two guides. The carriage is connected to an electric motor via a connecting rod, an eccentric cam and suitable gearing such that the porcelain plate is moved, once only, back and forth beneath the porcelain peg for a distance of 10 mm. The porcelain peg may be loaded with, for example, 120 or 360 newtons.

The flat porcelain plates are made from white technical porcelain (roughness 9 to 32 μm) and have the dimensions 25 mm (length) × 25 mm (width) × 5 mm (height). The cylindrical porcelain peg is also made of white technical porcelain and is 15 mm long, has a diameter of 10 mm and roughened spherical end surfaces with a radius of curvature of 10 mm.

1.6.3.2. Test conditions

The sample volume should be 10 mm3 or a volume to suit any alternative apparatus.

Solid substances are tested in the dry state and prepared as follows:

(a) powdered substances are sieved (sieve size 0,5 mm); all that has passed through the sieve is used for testing;

(b) pressed, cast or otherwise condensed substances are broken into small pieces and sieved; the sieve fraction < 0,5 mm diameter is used for testing.

Substances normally supplied as pastes should be tested in the dry state where possible. If the substance cannot be prepared in the dry state, the paste (following removal of the maximum possible amount of diluent) is tested as a 0,5 mm thick, 2 mm wide, 10 mm long film, prepared with a former.

1.6.3.3. Performance of the tests

The porcelain peg is brought onto the sample under test and the load applied. When carrying out the test, the sponge marks of the porcelain plate must lie transversely to the direction of the movement. Care must be taken that the peg rests on the sample, that sufficient test material lies under the peg and also that the plate moves correctly under the peg. For pasty substances, a 0,5 mm thick gauge with a 2 × 10 mm slot is used to apply the substance to the plate. The porcelain plate has to move 10 mm forwards and backwards under the porcelain peg in a time of 0,44 seconds. Each part of the surface of the plate and peg must only be used once; the two ends of each peg will serve for two trials and the two surfaces of a plate will each serve for three trials.

A series of six tests are performed with a 360 N loading. If a positive event is obtained during these six tests, a further series of six tests must be performed with a 120 N loading. In other apparatus, the sample is compared with the chosen reference substance using an established procedure (e.g. up-and-down technique, etc.).

1.6.3.4. Evaluation

The test result is considered positive if an explosion (crepitation and/or a report or bursting into flame are equivalent to explosion) occurs at least once in any of the tests with the specified friction apparatus or satisfies the equivalent criteria in an alternative friction test.

2. DATA

In principle, a substance is considered to present a danger of explosion in the sense of the directive if a positive result is obtained in the thermal, shock or friction sensitivity test.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, include the following information:

- identity, composition, purity, moisture content, etc. of the substance tested,

- the physical form of the sample and whether or not it has been crushed, broken and/or sieved,

- observations during the thermal sensitivity tests (e.g. sample mass, number of fragments, etc.),

- observations during the mechanical sensitivity tests (e.g. formation of considerable amounts of smoke or complete decomposition without a report, flames, sparks, report, crepitation, etc.),

- results of each type of test,

- if alternative apparatus has been used, scientific justification as well as evidence of correlation between results obtained with specified apparatus and those obtained with equivalent apparatus must be given,

- any useful comments such as reference to tests with similar products which might be relevant to a proper interpretation of the results,

- all additional remarks relevant for the interpretation of the results.

3.2. INTERPRETATION AND EVALUATION OF RESULTS

The test report should mention any results which are considered false, anomalous or unrepresentative. If any of the results should be discounted, an explanation and the results of any alternative or supplementary testing should be given. Unless an anomalous result can be explained, it must be accepted at face value and used to classify the substance accordingly.

4. REFERENCES

(1) Recommendations on the Transport of Dangerous Goods: Tests and criteria, 1990, United Nations, New York.

(2) Bretherick, L., Handbook of Reactive Chemical Hazards, 4th edition, Butterworths, London, ISBN 0-750-60103-5, 1990.

(3) Koenen, H., Ide, K.H. and Swart, K.H., Explosivstoffe, 1961, vol. 3, 6-13 and 30-42.

(4) NF T 20-038 (September 85) Chemical products for industrial use — Determination of explosion risk.

Appendix

Example of material specification for thermal sensitivity test (see DIN 1623)

(1) Tube: Material specification No 1.0336.505 g

(2) Orifice plate: Material specification No 1.4873

(3) Threaded collar and nut: Material specification No 1.3817

Figure 1

Thermal sensitivity test apparatus

(all dimensions in millimetres)

Fig. 1a Steel tube and accessories

(1) tube

(1a) outer flange

(2) threaded collar; low-friction thread

(3) orifice plate a = 2,0 or 6,0 mm diameter

(4) nut b = 10 mm diameter

(5) chamfered surface

(6) 2 flat for spanner size 41

Fig. 1b Heating and protective device

(7) 2 flat for spanner size 36

(8) splinter-proof box

(9) 2 supporting rods for tube

(10) assembled tube

(11) position for rear burner; the other burners are visible

(12) pilot jet

+++++ TIFF +++++

Figure 2

Thermal sensitivity test

(example of fragmentation)

No explosion

Explosion

+++++ TIFF +++++

Figure 3

Heating rate calibration for thermal sensitivity test

Temperature (°C)

Time (s)

+++++ TIFF +++++

Temperature/time curve obtained on heating dibutyl phtalate (27 cm3) in a closed (1,5 mm orifice plate) tube using a propane flow rate of 3,2 litre/minute. The temperature is measured with a 1 mm diameter stainless steel sheathed chromel/alumel thermocouple, placed centrally 43 mm below the rim of the tube. The heating rate between 135 oC and 285 oC should be between 185 and 215 K/minute.

Figure 4

Shock test apparatus

(all dimensions in millimetres)

Fig. 4a Fall-hammer, front and side, general view

(1) base, 450 x 450 x 60

(2) steel block, 230 x 250 x 200

(3) anvil, 100 diameter x 70

(4) column

(5) median cross-member

(6) 2 guides

(7) toothed rack

Fig. 4b Fall-hammer, lower part

(8) graduated scale

(9) fall-hammer (drop mass)

(10) holding and releasing device

(11) locating plate

(12) intermediate anvil (interchangeable), 26 diameter x 26

(13) locating ring with orifices

(14) impact device

+++++ TIFF +++++

Figure 4

Continued

Fig. 4c Shock device for substances in powdered or paste-like form

Fig. 4d Shock device for liquid substances

(1) steel cylinders

(2) guide ring for steel cylinders

(3) locating ring with orifices

(a) vertical section

(b) plan

(4) rubber ring

(5) liquid substance (40 mm3)

(6) space free from liquid

Fig. 4e Hammer (drop mass of 5 kg)

(1) suspension spigot

(2) height marker

(3) positioning groove

(4) cylindrical striking head

(5) rebound catch

+++++ TIFF +++++

Figure 5

Friction sensitivity apparatus

Fig. 5a Friction apparatus; elevation and plan view

(1) steel base

(2) movable carriage

(3) porcelain plate, 25 x 25 x 5 mm, held on carriage

(4) fixed porcelain peg, 10 diameter x 15 mm

(5) sample under test, approximately 10 mm3

Fig. 5b Starting position of peg on sample

(6) peg-holder

(7) loading arm

(8) counterweight

(9) switch

(10) wheel for setting carriage at starting position

(11) direction to electric drive motor

+++++ TIFF +++++

A.15. AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES)

1. METHOD

1.1. INTRODUCTION

Explosive substances and substances which ignite spontaneously in contact with air at ambient temperature should not be submitted to this test. The test procedure is applicable to gases, liquids and vapours which, in the presence of air, can be ignited by a hot surface.

The auto-ignition temperature can be considerably reduced by the presence of catalytic impurities, by the surface material or by a higher volume of the test vessel.

1.2. DEFINITIONS AND UNITS

The degree of auto-ignitability is expressed in terms of the auto-ignition temperature. The auto-ignition temperature is the lowest temperature at which the test substance will ignite when mixed with air under the conditions defined in the test method.

1.3. REFERENCE SUBSTANCES

Reference substances are cited in the standards (see 1.6.3). They should primarily serve to check the performance of the method from time to time and to allow comparison with results from other methods.

1.4. PRINCIPLE OF THE METHOD

The method determines the minimum temperature of the inner surface of an enclosure that will result in ignition of a gas, vapour or liquid injected into the enclosure.

1.5. QUALITY CRITERIA

The repeatability varies according to the range of auto-ignition temperatures and the test method used.

The sensitivity and specificity depend on the test method used.

1.6. DESCRIPTION OF THE METHOD

1.6.1. Apparatus

The apparatus is described in the method referred to in 1.6.3.

1.6.2. Test conditions

A sample of the test substance is tested according to the method referred to in 1.6.3.

1.6.3. Performance of the test

See IEC 79-4, DIN 51794, ASTM-E 659-78, BS 4056, NF T 20-037.

2. DATA

Record the test-temperature, atmospheric pressure, quantity of sample used and time-1ag until ignition occurs.

3. REPORTING

The test report shall, if possible, include the following information:

- the precise specification of the substance (identification and impurities),

- the quantity of sample used, atmospheric pressure,

- the apparatus used,

- the results of measurements (test temperatures, results concerning ignition, corresponding time-lags),

- all additional remarks relevant to the interpretation of results.

4. REFERENCES

None.

A.16. RELATIVE SELF-IGNITION TEMPERATURE FOR SOLIDS

1. METHOD

1.1. INTRODUCTION

Explosive substances and substances which ignite spontaneously in contact with air at ambient temperature should not be submitted to this test.

The purpose of this test is to provide preliminary information on the auto-flammability of solid substances at elevated temperatures.

If the heat developed either by a reaction of the substance with oxygen or by exothermic decomposition is not lost rapidly enough to the surroundings, self-heating leading to self-ignition occurs. Self-ignition therefore occurs when the rate of heat-production exceeds the rate of heat loss.

The test procedure is useful as a preliminary screening test for solid substances. In view of the complex nature of the ignition and combustion of solids, the self-ignition temperature determined according to this test method should be used for comparison purposes only.

1.2. DEFINITIONS AND UNITS

The self-ignition temperature as obtained by this method is the minimum ambient temperature expressed in oC at which a certain volume of a substance will ignite under defined conditions.

1.3. REFERENCE SUBSTANCE

None.

1.4. PRINCIPLE OF THE METHOD

A certain volume of the substance under test is placed in an oven at room temperature; the temperature/time curve relating to conditions in the centre of the sample is recorded while the temperature of the oven is increased to 400 oC, or to the melting point if lower, at a rate of 0,5 oC/min. For the purpose of this test, the temperature of the oven at which the sample temperature reaches 400 oC by self-heating is called the self-ignition temperature.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE METHOD

1.6.1. Apparatus

1.6.1.1. Oven

A temperature-programmed laboratory oven (volume about 2 litres) fitted with natural air circulation and explosion relief. In order to avoid a potential explosion risk, any decomposition gases must not be allowed to come into contact with the electric heating elements.

1.6.1.2. Wire mesh cube

A piece of stainless steel wire mesh with 0,045 mm openings should be cut according to the pattern in figure 1. The mesh should be folded and secured with wire into an open-topped cube.

1.6.1.3. Thermocouples

Suitable thermocouples.

1.6.1.4. Recorder

Any two-channel recorder calibrated from 0 to 600 oC or corresponding voltage.

1.6.2. Test conditions

Substances are tested as received.

1.6.3. Performance of the test

The cube is filled with the substance to be tested and is tapped gently, adding more of the substance until the cube is completely full. The cube is then suspended in the centre of the oven at room temperature. One thermocouple is placed at the centre of the cube and the other between the cube and the oven wall to record the oven temperature.

The temperatures of the oven and sample are continuously recorded while the temperature of the oven is increased to 400 oC, or to the melting point if lower, at a rate of 0,5 oC/min.

When the substance ignites the sample thermocouple will show a very sharp temperature rise above the oven temperature.

2. DATA

The temperature of the oven at which the sample temperature reaches 400 oC by self-heating is relevant for evaluation (see figure 2).

3. REPORTING

The test report shall, if possible, include the following information:

- a description of the substance to be tested,

- the results of measurement including the temperature/time curve,

- all additional remarks relevant for the interpretation of the results.

4. REFERENCES

NF T 20-036 (September 85) Chemical products for industrial use. Determination of the relative temperature of the spontaneous flammability of solids.

Figure 1

Pattern of 20 mm test cube

+++++ TIFF +++++

Figure 2

Typical temperature/time curve

sample temperature

oven temperature

ignition temperature

Time

+++++ TIFF +++++

A.17. OXIDISING PROPERTIES (SOLIDS)

1. METHOD

1.1. INTRODUCTION

It is useful to have preliminary information on any potentially explosive properties of the substance before performing this test.

This test is not applicable to liquids, gases, explosive or highly flammable substances, or organic peroxides.

This test need not be performed when examination of the structural formula establishes beyond reasonable doubt that the substance is incapable of reacting exothermically with a combustible material.

In order to ascertain if the test should be performed with special precautions, a preliminary test should be performed.

1.2. DEFINITION AND UNITS

Burning time: reaction time, in seconds, taken for the reaction zone to travel along a pile, following the procedure described in 1.6.

Burning rate: expressed in millimetres per second.

Maximum burning rate: the highest value of the burning rates obtained with mixtures containing 10 to 90 % by weight of oxidiser.

1.3. REFERENCE SUBSTANCE

Barium nitrate (analytical grade) is used as reference substance for the test and the preliminary test.

The reference mixture is that mixture of barium nitrate with powdered cellulose, prepared according to 1.6, which has the maximum burning rate (usually a mixture with 60 % barium nitrate by weight).

1.4. PRINCIPLE OF THE METHOD

A preliminary test is carried out in the interests of safety. No further testing is required when the preliminary test clearly indicates that the test substance has oxidising properties. When this is not the case, the substance should then be subject to the full test.

In the full test, the substance to be tested and a defined combustible substance will be mixed in various ratios. Each mixture is then formed into a pile and the pile is ignited at one end. The maximum burning rate determined is compared with the maximum burning rate of the reference mixture.

1.5. QUALITY CRITERIA

If required, any method of grinding and mixing is valid provided that the difference in the maximum rate of burning in the six separate tests differs from the arithmetic mean value by no more than 10 %.

1.6. DESCRIPTION OF THE METHOD

1.6.1. Preparation

1.6.1.1. Test substance

Reduce the test sample to a particle size < 0,125 mm using the following procedure: sieve the test substance, grind the remaining fraction, repeat the procedure until the whole test portion has passed the sieve.

Any grinding and sieving method satisfying the quality criteria may be used.

Before preparing the mixture the substance is dried at 105 oC, until constant weight is obtained. If the decomposition temperature of the substance to be tested is below 105 oC, the substance has to be dried at a suitable lower temperature.

1.6.1.2. Combustible substance

Powdered cellulose is used as a combustible substance. The cellulose should be a type used for thin-layer chromatography or column chromatography. A type with fibre-lengths of more than 85 % between 0,020 and 0,075 mm has proved to be suitable. The cellulose powder is passed through a sieve with a mesh-size of 0,125 mm. The same batch of cellulose is to be used throughout the test.

Before preparing the mixture, the powdered cellulose is dried at 105 oC until constant weight is obtained.

If wood-meal is used in the preliminary test, then prepare a soft-wood wood-meal by collecting the portion which passes through a sieve mesh of 1,6 mm, mix thoroughly, then dry at 105 oC for four hours in a layer not more than 25 mm thick. Cool and store in an air-tight container filled as full as practicable until required, preferably within 24 hours of drying.

1.6.1.3. Ignition source

A hot flame from a gas burner (minimum diameter 5 mm) should be used as the ignition source. If another ignition source is used (e.g. when testing in an inert atmosphere), the description and the justification should be reported.

1.6.2. Performance of the test

Note:

Mixtures of oxidisers with cellulose or wood-meal must be treated as potentially explosive and handled with due care.

1.6.2.1. Preliminary test

The dried substance is thoroughly mixed with the dried cellulose or wood-meal in the proportions 2 of test substance to 1 of cellulose or wood-meal by weight and the mixture is formed into a small cone-shaped pile of dimensions 3,5 cm (diameter of base) × 2,5 cm (height) by filling, without tamping, a cone-shaped former (e.g. a laboratory glass funnel with the stem plugged).

The pile is placed on a cool, non-combustible, non-porous and low heat-conducting base plate. The test should be carried out in a fume cupboard as in 1.6.2.2.

The ignition source is put in contact with the cone. The vigour and duration of the resultant reaction are observed and recorded.

The substance is to be considered as oxidising if the reaction is vigorous.

In any case where the result is open to doubt, it is then necessary to complete the full train test described below.

1.6.2.2. Train test

Prepare oxidiser cellulose-mixtures containing 10 to 90 % weight of oxidiser in 10 % increments. For borderline cases, intermediate oxidiser cellulose mixtures should be used to obtain the maximum burning rate more precisely.

The pile is formed by means of a mould. The mould is made of metal, has a length of 250 mm and a triangular cross-section with an inner height of 10 mm and an inner width of 20 mm. On both sides of the mould, in the longitudinal direction, two metal plates are mounted as lateral limitations which project 2 mm beyond the upper edge of the triangular cross-section (figure). This arrangement is loosely filled with a slight excess of mixture. After dropping the mould once from a height of 2 cm onto a solid surface, the remaining excess substance is scraped off with an obliquely positioned sheet. The lateral limitations are removed and the remaining powder is smoothed, using a roller. A non-combustible, non-porous and low heat-conducting base plate is then placed on the top of the mould, the apparatus inverted and the mould removed.

Arrange the pile across the draught in a fume cupboard.

The air-speed should be sufficient to prevent fumes escaping into the laboratory and should not be varied during the test. A draught screen should be erected around the apparatus.

Due to hygroscopicity of cellulose and of some substances to be tested, the test should be carried out as quickly as possible.

Ignite one end of the pile by touching with the flame.

Measure the time of reaction over a distance of 200 mm after the reaction zone has propagated an initial distance of 30 mm.

The test is performed with the reference substance and at least once with each one of the range of mixtures of the test substance with cellulose.

If the maximum burning rate is found to be significantly greater than that from the reference mixture, the test can be stopped; otherwise the test should be repeated five times for each of the three mixtures giving the fastest burning rate.

If the result is suspected of being a false positive, then the test should be repeated using an inert substance with a similar particle size, such as kieselguhr, in place of cellulose. Alternatively, the test substance cellulose mixture, having the fastest burning rate, should be retested in an inert atmosphere (< 2 % v/v oxygen content).

2. DATA

For safety reasons the maximum burning rate — not the mean value — shall be considered to be the characteristic oxidising property of the substance under test.

The highest value of burning rate within a run of six tests of a given mixture is relevant for evaluation.

Plot a graph of the highest value of burning rate for each mixture versus the oxidiser concentration. From the graph take the maximum burning rate.

The six measured values of burning rate within a run obtained from the mixture with the maximum burning rate must not differ from the arithmetic mean value by more than 10 %; otherwise the methods of grinding and mixing must be improved.

Compare the maximum burning rate obtained with the maximum burning rate of the reference mixture (see 1.3).

If tests are conducted in an inert atmosphere, the maximum reaction rate is compared with that from the reference mixture in an inert atmosphere.

3. REPORT

3.1. TEST REPORT

The test report shall, if possible, include the following information:

- the identity, composition, purity, moisture content etc. of the substance tested,

- any treatment of the test sample (e.g. grinding, drying),

- the ignition source used in the tests,

- the results of measurements,

- the mode of reaction (e.g. flash burning at the surface, burning through the whole mass, any information concerning the combustion products, etc.),

- all additional remarks relevant for the interpretation of results, including a description of the vigour (flaming, sparking, fuming, slow smouldering, etc.) and approximate duration produced in the preliminary safety/screening test for both test and reference substance,

- the results from tests with an inert substance, if any,

- the results from tests in an inert atmosphere, if any.

3.2. INTERPRETATION OF THE RESULT

A substance is to be considered as an oxidising substance when:

(a) in the preliminary test, there is a vigorous reaction;

(b) in the full test, the maximum burning rate of the mixtures tested is higher than or equal to the maximum burning rate of the reference mixture of cellulose and barium nitrate.

In order to avoid a false positive, the results obtained when testing the substance mixed with an inert material and/or when testing under an inert atmosphere should also be considered when interpreting the results.

4. REFERENCES

NF T 20-035 (September 85) Chemical products for industrial use. Determination of the oxidising properties of solids.

Appendix

Figure

Mould and accessories for the preparations of the pile

(All dimensions in millimetres)

+++++ TIFF +++++

A.18. NUMBER-AVERAGE MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION OF POLYMERS

1. METHOD

This Gel Permeation Chromatographic method is a replicate of the OECD TG 118 (1996). The fundamental principles and further technical information are given in reference (1).

1.1. INTRODUCTION

Since the properties of polymers are so varied, it is impossible to describe one single method setting out precisely the conditions for separation and evaluation which cover all eventualities and specificities occurring in the separation of polymers. In particular, complex polymer systems are often not amenable to gel permeation chromatography (GPC). When GPC is not practicable, the molecular weight may be determined by means of other methods (see Appendix). In such cases, full details and justification should be given for the method used.

The method described is based on DIN Standard 55672 (1). Detailed information about how to carry out the experiments and how to evaluate the data can be found in this DIN Standard. In case modifications of the experimental conditions are necessary, these changes must be justified. Other standards may be used, if fully referenced. The method described uses polystyrene samples of known polydispersity for calibration and it may have to be modified to be suitable for certain polymers, e.g. water soluble and long-chain branched polymers.

1.2. DEFINITIONS AND UNITS

The number-average molecular weight Mn and the weight average molecular weight Mw are determined using the following equations:

Mn=Σni=1HiΣni=1Hi/Mi | Mw=Σni=1Hi × MiΣni=1Hi |

where,

Hi is the level of the detector signal from the baseline for the retention volume Vi,

Mi is the molecular weight of the polymer fraction at the retention volume Vi, and

n is the number of data points.

The breadth of the molecular weight distribution, which is a measure of the dispersity of the system, is given by the ratio Mw/Mn.

1.3. REFERENCE SUBSTANCES

Since GPC is a relative method, calibration must be undertaken. Narrowly distributed, linearly constructed polystyrene standards with known average molecular weights Mn and Mw and a known molecular weight distribution are normally used for this. The calibration curve can only be used in the determination of the molecular weight of the unknown sample if the conditions for the separation of the sample and the standards have been selected in an identical manner.

A determined relationship between the molecular weight and elution volume is only valid under the specific conditions of the particular experiment. The conditions include, above all, the temperature, the solvent (or solvent mixture), the chromatography conditions and the separation column or system of columns.

The molecular weights of the sample determined in this way are relative values and are described as "polystyrene equivalent molecular weights". This means that dependent on the structural and chemical differences between the sample and the standards, the molecular weights can deviate from the absolute values to a greater or a lesser degree. If other standards are used, e.g. polyethylene glycol, polyethylene oxide, polymethyl methacrylate, polyacrylic acid, the reason should be stated.

1.4. PRINCIPLE OF THE TEST METHOD

Both the molecular weight distribution of the sample and the average molecular weights (Mn, Mw) can be determined using GPC. GPC is a special type of liquid chromatography in which the sample is separated according to the hydrodynamic volumes of the individual constituents (2).

Separation is effected as the sample passes through a column which is filled with a porous material, typically an organic gel. Small molecules can penetrate the pores whereas large molecules are excluded. The path of the large molecules is thereby shorter and these are eluted first. The medium-sized molecules penetrate some of the pores and are eluted later. The smallest molecules, with a mean hydrodynamic radius smaller than the pores of the gel, can penetrate all of the pores. These are eluted last.

In an ideal situation, the separation is governed entirely by the size of the molecular species, but in practice it is difficult to avoid at least some absorption effects interfering. Uneven column packing and dead volumes can worsen the situation (2).

Detection is effected by, e.g. refractive index or UV-absorption, and yields a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate the column by passing down polymers of known molecular weight and, ideally, of broadly similar structure e.g. various polystyrene standards. Typically a Gaussian curve results, sometimes distorted by a small tail to the low molecular weight side, the vertical axis indicating the quantity, by weight, of the various molecular weight species eluted, and the horizontal axis the log molecular weight.

1.5. QUALITY CRITERIA

The repeatability (Relative Standard Deviation: RSD) of the elution volume should be better than 0,3 %. The required repeatability of the analysis has to be ensured by correction via an internal standard if a chromatogram is evaluated time-dependently and does not correspond to the above mentioned criterion (1). The polydispersities are dependent on the molecular weights of the standards. In the case of polystyrene standards typical values are:

Mp < 2000 | Mw/Mn < 1,20 |

2000 ≤ Mp ≤ 106 | Mw/Mn < 1,05 |

Mp > 106 | Mw/Mn < 1,20 |

(Mp is the molecular weight of the standard at the peak maximum)

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparation of the standard polystyrene solutions

The polystyrene standards are dissolved by careful mixing in the chosen eluent. The recommendations of the manufacturer must be taken into account in the preparation of the solutions.

The concentrations of the standards chosen are dependent on various factors, e.g. injection volume, viscosity of the solution and sensitivity of the analytical detector. The maximum injection volume must be adapted to the length of the column, in order to avoid overloading. Typical injection volumes for analytical separations using GPC with a column of 30 cm × 7,8 mm are normally between 40 and 100 μl. Higher volumes are possible, but they should not exceed 250 μl. The optimal ratio between the injection volume and the concentration must be determined prior to the actual calibration of the column.

1.6.2. Preparation of the sample solution

In principle, the same requirements apply to the preparation of the sample solutions. The sample is dissolved in a suitable solvent, e.g. tetrahydrofuran (THF), by shaking carefully. Under no circumstances should it be dissolved using an ultrasonic bath. When necessary, the sample solution is purified via a membrane filter with a pore size of between 0,2 and 2 μm.

The presence of undissolved particles must be recorded in the final report as these may be due to high molecular weight species. An appropriate method should be used to determine the percentage by weight of the undissolved particles. The solutions should be used within 24 hours.

1.6.3. Apparatus

- solvent reservoir,

- degasser (where appropriate),

- pump,

- pulse dampener (where appropriate),

- injection system,

- chromatography columns,

- detector,

- flowmeter (where appropriate),

- data recorder-processor,

- waste vessel.

It must be ensured that the GPC system is inert with regard to the utilised solvents (e.g. by the use of steel capillaries for THF solvent).

1.6.4. Injection and solvent delivery system

A defined volume of the sample solution is loaded onto the column either using an auto-sampler or manually in a sharply defined zone. Withdrawing or depressing the plunger of the syringe too quickly, if done manually, can cause changes in the observed molecular weight distribution. The solvent-delivery system should, as far as possible, be pulsation-free ideally incorporating a pulse dampener. The flow rate is of the order of 1 ml/min.

1.6.5. Column

Depending on the sample, the polymer is characterised using either a simple column or several columns connected in sequence. A number of porous column materials with defined properties (e.g. pore size, exclusion limits) are commercially available. Selection of the separation gel or the length of the column is dependent on both the properties of the sample (hydrodynamic volumes, molecular weight distribution) and the specific conditions for separation such as solvent, temperature and flow rate (1)(2)(3).

1.6.6. Theoretical plates

The column or the combination of columns used for separation must be characterised by the number of theoretical plates. This involves, in the case of THF as elution solvent, loading a solution of ethyl benzene or other suitable non-polar solute onto a column of known length. The number of theoretical plates is given by the following equation:

N=5,54VeW1/22 | or | N=16VeW2 |

where,

N = the number of theoretical plates

Ve = the elution volume at the peak maximum

W = the baseline peak width

W1/2 = the peak width at half height

1.6.7. Separation efficiency

In addition to the number of theoretical plates, which is a quantity determining the bandwidth, a part is also played by the separation efficiency, this being determined by the steepness of the calibration curve. The separation efficiency of a column is obtained from the following relationship:

e,M

e,(10M

)

≥6,0

where,

Ve, Mx = the elution volume for polystyrene with the molecular weight Mx

Ve,(10.Mx) = the elution volume for polystyrene with a ten times greater molecular weight

The resolution of the system is commonly defined as follows:

=2×

-V

log

where,

Ve1, Ve2 = the elution volumes of the two polystyrene standards at the peak maximum

W1, W2 = the peak widths at the base-line

M1, M2 = the molecular weights at the peak maximum (should differ by a factor of 10)

The R-value for the column system should be greater than 1.7 (4).

1.6.8. Solvents

All solvents must be of high purity (for THF purity of 99,5 % is used). The solvent reservoir (if necessary in an inert gas atmosphere) must be sufficiently large for the calibration of the column and several sample analyses. The solvent must be degassed before it is transported to the column via the pump.

1.6.9. Temperature control

The temperature of the critical internal components (injection loop, columns, detector and tubing) should be constant and consistent with the choice of solvent.

1.6.10. Detector

The purpose of the detector is to record quantitatively the concentration of sample eluted from the column. In order to avoid unnecessary broadening of peaks the cuvette volume of the detector cell must be kept as small as possible. It should not be larger than 10 μl except for light scattering and viscosity detectors. Differential refractometry is usually used for detection. However, if required by the specific properties of the sample or the elution solvent, other types of detectors can be used, e.g. UV/VIS, IR, viscosity detectors, etc.

2. DATA AND REPORTING

2.1. DATA

The DIN Standard (1) should be referred to for the detailed evaluation criteria as well as for the requirements relating to the collecting and processing of data.

For each sample, two independent experiments must be carried out. They have to be analysed individually.

Mn, Mw, Mw/Mn and Mp must be provided for every measurement. It is necessary to indicate explicitly that the measured values are relative values equivalent to the molecular weights of the standard used.

After determination of the retention volumes or the retention times (possibly corrected using an internal standard), log Mp values (Mp being the peak maxima of the calibration standard) are plotted against one of those quantities. At least two calibration points are necessary per molecular weight decade, and at least five measurement points are required for the total curve, which should cover the estimated molecular weight of the sample. The low molecular weight end-point of the calibration curve is defined by n-hexyl benzene or another suitable non-polar solute. The number average and the weight-average molecular weights are generally determined by means of electronic data processing, based on the formulas of section 1.2. In case manual digitisation is used, ASTM D 3536-91 can be consulted (3).

The distribution curve must be provided in the form of a table or as figure (differential frequency or sum percentages against log M). In the graphic representation, one molecular weight decade should be normally about 4 cm in width and the peak maximum should be about 8 cm in height. In the case of integral distribution curves the difference in the ordinate between 0 and 100 % should be about 10 cm.

2.2. TEST REPORT

The test report must include the following information:

2.2.1. Test substance:

- available information about test substance (identity, additives, impurities),

- description of the treatment of the sample, observations, problems.

2.2.2. Instrumentation:

- reservoir of eluent, inert gas, degassing of the eluent, composition of the eluent, impurities,

- pump, pulse dampener, injection system,

- separation columns (manufacturer, all information about the characteristics of the columns, such as pore size, kind of separation material, etc., number, length and order of the columns used),

- number of the theoretical plates of the column (or combination), separation efficiency (resolution of the system),

- information on symmetry of the peaks,

- column temperature, kind of temperature control,

- detector (measurement principle, type, cuvette volume),

- flowmeter if used (manufacturer, measurement principle),

- system to record and process data (hardware and software).

2.2.3. Calibration of the system:

- detailed description of the method used to construct the calibration curve,

- information about quality criteria for this method (e.g. correlation coefficient, error sum of squares, etc.),

- information about all extrapolations, assumptions and approximations made during the experimental procedure and the evaluation and processing of data,

- all measurements used for constructing the calibration curve have to be documented in a table which includes the following information for each calibration point:

- name of the sample,

- manufacturer of the sample,

- characteristic values of the standards Mp, Mn, Mw, Mw/Mn, as provided by the manufacturer or derived by subsequent measurements, together with details about the method of determination,

- injection volume and injection concentration,

- Mp value used for calibration,

- elution volume or corrected retention time measured at the peak maxima,

- Mp calculated at the peak maximum,

- percentage error of the calculated Mp and the calibration value.

2.2.4. Evaluation:

- evaluation on a time basis: methods used to ensure the required reproducibility (method of correction, internal standard, etc.),

- information about whether the evaluation was effected on the basis of the elution volume or the retention time,

- information about the limits of the evaluation if a peak is not completely analysed,

- description of smoothing methods, if used,

- preparation and pre-treatment procedures of the sample,

- the presence of undissolved particles, if any,

- injection volume (μl) and injection concentration (mg/ml),

- observations indicating effects which lead to deviations from the ideal GPC profile,

- detailed description of all modifications in the testing procedures,

- details of the error ranges,

- any other information and observations relevant for the interpretation of the results.

3. REFERENCES

(1) DIN 55672(1995) Gelpermeationschromatographie (GPC) mit Tetrahydrofuran (THF) als Elutionsmittel, Teil 1.

(2) Yau, W.W., Kirkland, J.J., and Bly, D.D. eds., (1979) Modern Size Exclusion Liquid Chromatography, J. Wiley and Sons.

(3) ASTM D 3536-91, (1991). Standard Test Method for Molecular Weight Averages and Molecular Weight Distribution by Liquid Exclusion Chromatography (Gel Permeation Chromatography-GPC) American Society for Testing and Materials, Philadelphia, Pennsylvania.

(4) ASTM D 5296-92, (1992) Standard Test Method for Molecular Weight Averages and Molecular Weight Distribution of Polystyrene by High Performance Size-Exclusion Chromatography. American Society for Testing and Materials, Philadelphia, Pennsylvania.

Appendix

Examples of other methods for determination of number average molecular weight (Mn) for polymers

Gel permeation chromatography (GPC) is the preferred method for determination of Mn, especially when a set of standards are available, whose structure are comparable with the polymer structure. However, where there are practical difficulties in using GPC or there is already an expectation that the substance will fail a regulatory Mn criterion (and which needs confirming), alternative methods are available, such as:

1. Use of colligative properties

1.1. Ebullioscopy/Cryoscopy

involves measurement of boiling point elevation (ebullioscopy) or freezing point depression (cryoscopy) of a solvent, when the polymer is added. The method relies on the fact that the effect of the dissolved polymer on the boiling/freezing point of the liquid is dependent on the molecular weight of the polymer (1) (2).

Applicability, Mn < 20000.

1.2. Lowering of vapour pressure

involves the measurement of the vapour pressure of a chosen reference liquid before and after the addition of known quantities of polymer (1) (2).

Applicability, Mn < 20000 (theoretically; in practice however of limited value).

1.3 Membrane osmometry

relies on the principle of osmosis, i.e. the natural tendency of solvent molecules to pass through a semi-permeable membrane from a dilute to a concentrated solution to achieve equilibrium. In the test, the dilute solution is at zero concentration, whereas the concentrated solution contains the polymer. The effect of drawing solvent through the membrane causes a pressure differential that is dependent on the concentration and the molecular weight of the polymer (1) (3) (4).

Applicability, Mn between 20000 - 200000.

1.4 Vapour phase osmometry

involves comparison of the rate of evaporation of a pure solvent aerosol to at least three aerosols containing the polymer at different concentrations (1)(2)(4).

Applicability, Mn < 20000.

2. End-group analysis

To use this method, knowledge of both the overall structure of the polymer and the nature of the chain terminating end groups is needed (which must be distinguishable from the main skeleton by, e.g. NMR or titration/derivatisation). The determination of the molecular concentration of the end groups present on the polymer can lead to a value for the molecular weight (7) (8) (9).

Applicability, Mn up to 50000 (with decreasing reliability).

3. References

(1) Billmeyer, F.W. Jr., (1984) Textbook of Polymer Science, 3rd Edn., John Wiley, New York.

(2) Glover, C.A., (1975) Absolute Colligative Property Methods. Chapter 4. In: Polymer Molecular Weights, Part I P.E. Slade, Jr. ed., Marcel Dekker, New York.

(3) ASTM D 3750-79, (1979) Standard Practice for Determination of Number-Average Molecular Weight of Polymers by Membrane Osmometry. American Society for Testing and Materials, Philadelphia, Pennsylvania.

(4) Coll, H. (1989) Membrane Osmometry. In: Determination of Molecular Weight, A.R. Cooper ed., J. Wiley and Sons, pp. 25-52.

(5) ASTM 3592-77, (1977) Standard Recommended Practice for Determination of Molecular Weight by Vapour Pressure, American Society for Testing and Materials, Philadelphia, Pennsylvania.

(6) Morris, C.E.M., (1989) Vapour Pressure Osmometry. In: Determinationn of Molecular Weight, A.R. Cooper ed., John Wiley and Sons.

(7) Schröder, E., Müller, G., and Arndt, K-F., (1989) Polymer Characterisation, Carl Hanser Verlag, Munich.

(8) Garmon, R.G., (1975) End-Group Determinations, Chapter 3 In: Polymer Molecular Weights, Part I, P.E. Slade, Jr. ed., Marcel Dekker, New York.

(9) Amiya, S., et al. (1990) Pure and Applied Chemistry, 62, 2139-2146.

A.19. LOW MOLECULAR WEIGHT CONTENT OF POLYMERS

1. METHOD

This Gel Permeation Chromatographic method is a replicate of the OECD TG 119 (1996). The fundamental principles and further technical information are given in the references.

1.1. INTRODUCTION

1.2. DEFINITIONS AND UNITS

Low molecular weight is arbitrarily defined as a molecular weight below 1000 dalton.

The number-average molecular weight Mn and the weight average molecular weight Mw are determined using the following equations:

Mn=Σni=1HiΣni=1Hi/Mi | Mw=Σni=1Hi × MiΣni=1Hi |

where,

Hi = the level of the detector signal from the baseline for the retention volume Vi,

Mi = the molecular weight of the polymer fraction at the retention volume Vi, and n is the number of data points

The breadth of the molecular weight distribution, which is a measure of the dispersity of the system, is given by the ratio Mw/Mn.

1.3. REFERENCE SUBSTANCES

1.4. PRINCIPLE OF THE TEST METHOD

Detection is effected by e.g. refractive index or UV-absorption and yields a simple distribution curve. However, to attribute actual molecular weight values to the curve, it is necessary to calibrate the column by passing down polymers of known molecular weight and, ideally, of broadly similar structure, e.g. various polystyrene standards. Typically a Gaussian curve results, sometimes distorted by a small tail to the low molecular weight side, the vertical axis indicating the quantity, by weight, of the various molecular weight species eluted, and the horizontal axis the log molecular weight.

The low molecular weight content is derived from this curve. The calculation can only be accurate if the low molecular weight species respond equivalently on a per mass basis to the polymer as a whole.

1.5. QUALITY CRITERIA

Mp < 2000 | Mw/Mn < 1,20 |

2000< Mp< 106 | Mw/Mn < 1,05 |

Mp > 106 | Mw/Mn < 1,20 |

(Mp is the molecular weight of the standard at the peak maximum)

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparation of the standard polystyrene solutions

The polystyrene standards are dissolved by careful mixing in the chosen eluent. The recommendations of the manufacturer must be taken into account in the preparation of the solutions.

1.6.2. Preparation of the sample solution

1.6.3. Correction for content of impurities and additives

Correction of the content of species of M < 1000 for the contribution from non-polymer specific components present (e.g. impurities and/or additives) is usually necessary, unless the measured content is already < 1 %. This is achieved by direct analysis of the polymer solution or the GPC eluate.

In cases where the eluate, after passage through the column, is too dilute for a further analysis it must be concentrated. It may be necessary to evaporate the eluate to dryness and dissolve it again. Concentration of the eluate must be effected under conditions which ensure that no changes occur in the eluate. The treatment of the eluate after the GPC step is dependent on the analytical method used for the quantitative determination.

1.6.4. Apparatus

GPC apparatus comprises the following components:

- solvent reservoir,

- degasser (where appropriate),

- pump,

- pulse dampener (where appropriate),

- injection system,

- chromatography columns,

- detector,

- flowmeter (where appropriate),

- data recorder-processor,

- waste vessel.

It must be ensured that the GPC system is inert with regard to the utilised solvents (e.g. by the use of steel capillaries for THF solvent).

1.6.5. Injection and solvent delivery system

1.6.6. Column

1.6.7. Theoretical plates

N=5,54VeW1/22 | or | N=16VeW2 |

where,

N = the number of theoretical plates

Ve = the elution volume at the peak maximum

W = the baseline peak width

W1/2 = the peak width at half height

1.6.8. Separation efficiency

e,M

e,(10M

)

≥6,0

where,

Ve, Mx = the elution volume for polystyrene with the molecular weight Mx

Ve,(10.Mx) = the elution volume for polystyrene with a ten times greater molecular weight

The resolution of the system is commonly defined as follows:

=2×

-V

log

where,

Ve1, Ve2 = the elution volumes of the two polystyrene standards at the peak maximum

W1, W2 = the peak widths at the base-1ine

M1, M2 = the molecular weights at the peak maximum (should differ by a factor of 10).

The R-value for the column system should be greater than 1,7 (4).

1.6.9. Solvents

1.6.10. Temperature control

The temperature of the critical internal components (injection loop, columns, detector and tubing) should be constant and consistent with the choice of solvent.

1.6.11. Detector

2. DATA AND REPORTING

2.1. DATA

The DIN Standard (1) should be referred to for the detailed evaluation criteria as well as for the requirements relating to the collecting and processing of data.

For each sample, two independent experiments must be carried out. They have to be analysed individually. In all cases it is essential to determine also data from blanks, treated under the same conditions as the sample.

It is necessary to indicate explicitly that the measured values are relative values equivalent to the molecular weights of the standard used.

After determination of the retention volumes or the retention times (possibly corrected using an internal standard), log Mp values (Mp being the peak maxima of the calibration standard) are plotted against one of those quantities. At least two calibration points are necessary per molecular weight decade, and at least five measurement points are required for the total curve, which should cover the estimated molecular weight of the sample. The low molecular weight end-point of the calibration curve is defined by n-hexyl benzene or another suitable non-polar solute. The portion of the curve corresponding to molecular weights below 1000 is determined and corrected as necessary for impurities and additives. The elution curves are generally evaluated by means of electronic data processing. In case manual digitisation is used, ASTM D 3536-91 can be consulted (3).

If any insoluble polymer is retained on the column, its molecular weight is likely to be higher than that of the soluble fraction, and if not considered would result in an overestimation of the low molecular weight content. Guidance for correcting the low molecular weight content for insoluble polymer is provided in the Appendix.

2.2. TEST REPORT

The test report must include the following information:

2.2.1. Test substance:

- available information about test substance (identity, additives, impurities),

- description of the treatment of the sample, observations, problems.

2.2.2. Instrumentation:

- reservoir of eluent, inert gas, degassing of the eluent, composition of the eluent, impurities,

- pump, pulse dampener, injection system,

- separation columns (manufacturer, all information about the characteristics of the columns, such as pore size, kind of separation material, etc., number, length and order of the columns used),

- number of the theoretical plates of the column (or combination), separation efficiency (resolution of the system),

- information on symmetry of the peaks,

- column temperature, kind of temperature control,

- detector (measurement principle, type, cuvette volume),

- flowmeter if used (manufacturer, measurement principle),

- system to record and process data (hardware and software).

2.2.3. Calibration of the system:

- detailed description of the method used to construct the calibration curve,

- information about quality criteria for this method (e.g. correlation coefficient, error sum of squares, etc.),

- information about all extrapolations, assumptions and approximations made during the experimental procedure and the evaluation and processing of data,

- all measurements used for constructing the calibration curve have to be documented in a table which includes the following information for each calibration point:

- name of the sample,

- manufacturer of the sample,

- characteristic values of the standards Mp, Mn, Mw, Mw/Mn, as provided by the manufacturer or derived by subsequent measurements, together with details about the method of determination,

- injection volume and injection concentration,

- Mp value used for calibration,

- elution volume or corrected retention time measured at the peak maxima,

- Mp calculated at the peak maximum,

- percentage error of the calculated Mp and the calibration value.

2.2.4. Information on the low molecular weight polymer content:

- description of the methods used in the analysis and the way in which the experiments were conducted,

- information about the percentage of the low molecular weight species content (w/w) related to the total sample,

- information about impurities, additives and other non-polymer species in percentage by weight related to the total sample.

2.2.5. Evaluation:

- evaluation on a time basis: all methods to ensure the required reproducibility (method of correction, internal standard etc.),

- information about whether the evaluation was effected on the basis of the elution volume or the retention time,

- information about the limits of the evaluation if a peak is not completely analysed,

- description of smoothing methods, if used,

- preparation and pre-treatment procedures of the sample,

- the presence of undissolved particles, if any,

- injection volume (μl) and injection concentration (mg/ml),

- observations indicating effects which lead to deviations from the ideal GPC profile,

- detailed description of all modifications in the testing procedures,

- details of the error ranges,

- any other information and observations relevant for the interpretation of the results.

3. REFERENCES

(1) DIN 55672 (1995) Gelpermeationschromatographie (GPC) mit Tetrahydrofuran (THF) als Elutionsmittel, Teil 1.

(2) Yau, W.W., Kirkland, J.J., and Bly, D.D. eds. (1979) Modern Size Exclusion Liquid Chromatography, J. Wiley and Sons.

(3) ASTM D 3536-91, (1991) Standard Test method for Molecular Weight Averages and Molecular Weight Distribution by Liquid Exclusion Chromatography (Gel Permeation Chromatography-GPC). American Society for Testing and Materials, Philadelphia, Pennsylvania.

(4) ASTM D 5296-92, (1992) Standard Test method for Molecular Weight Averages and Molecular Weight Distribution of Polystyrene by High Performance Size-Exclusion Chromatography. American Society for Testing and Materials, Philadelphia, Pennsylvania.

Appendix

Guidance for correcting low molecular content for the presence of insoluble polymer

When insoluble polymer is present in a sample, it results in mass loss during the GPC analysis. The insoluble polymer is irreversibly retained on the column or sample filter while the soluble portion of the sample passes through the column. In the case where the refractive index increment (dn/dc) of the polymer can be estimated or measured, one can estimate the sample mass lost on the column. In that case, one makes a correction using an external calibration with standard materials of known concentration and dn/dc to calibrate the response of the refractometer. In the example hereafter a poly(methyl methacrylate) (pMMA) standard is used.

In the external calibration for analysis of acrylic polymers, a pMMA standard of known concentration in tetrahydrofuran, is analysed by GPC and the resulting data are used to find the refractometer constant according to the equation:

K = R/(C × V × dn/dc)

where:

K = the refractometer constant (in microvolt second/ml),

R = the response of the pMMA standard (in microvolt/second),

C = the concentration of the pMMA standard (in mg/ml),

V = the injection volume (in ml), and

dn/dc = the refractive index increment for pMMA in tetrahydrofuran (in ml/mg).

The following data are typical for a pMMA standard:

R = 2937891

C = 1,07 mg/ml

V = 0,1 ml

dn/dc = 9 × 10-5 ml/mg

The resulting K value, 3,05 × 1011 is then used to calculate the theoretical detector response if 100 % of the polymer injected had eluted through the detector.

A.20. SOLUTION/EXTRACTION BEHAVIOUR OF POLYMERS IN WATER

1. METHOD

The method described is a replicate of the revised version of OECD TG 120 (1997). Further technical information is given in reference (1).

1.1. INTRODUCTION

For certain polymers, such as emulsion polymers, initial preparatory work may be necessary before the method set out hereafter can be used. The method is not applicable to liquid polymers and to polymers that react with water under the test conditions.

When the method is not practical or not possible, the solution/extraction behaviour may be investigated by means of other methods. In such cases, full details and justification should be given for the method used.

1.2. REFERENCE SUBSTANCES

None.

1.3. PRINCIPLE OF THE TEST METHOD

The solution/extraction behaviour of polymers in an aqueous medium is determined using the flask method (see A.6 Water Solubility, Flask method) with the modifications described below.

1.4. QUALITY CRITERIA

None.

1.5. DESCRIPTION OF THE TEST METHOD

1.5.1. Equipment

The following equipment is required for the method:

- crushing device, e.g. grinder for the production of particles of known size,

- apparatus for shaking with possibility of temperature control,

- membrane filter system,

- appropriate analytical equipment,

- standardised sieves.

1.5.2. Sample preparation

A representative sample has first to be reduced to a particle size between 0,125 and 0,25 mm using appropriate sieves. Cooling may be required for the stability of the sample or for the grinding process. Materials of a rubbery nature can be crushed at liquid nitrogen temperature (1).

If the required particle size fraction is not attainable, action should be taken to reduce the particle size as much as possible, and the result reported. In the report, it is necessary to indicate the way in which the crushed sample was stored prior to the test

1.5.3. Procedure

Three samples of 10 g of the test substance are weighed into each of three vessels fitted with glass stoppers and 1000 ml of water is added to each vessel. If handling an amount of 10 g polymer proves impracticable, the next highest amount which can be handled should be used and the volume of water adjusted accordingly.

The vessels are tightly stoppered and then agitated at 20 oC. A shaking or stirring device capable of operating at constant temperature should be used. After a period of 24 hours, the content of each vessel is centrifuged or filtered and the concentration of polymer in the clear aqueous phase is determined by a suitable analytical method. If suitable analytical methods for the aqueous phase are not available, the total solubility/extractivity can be estimated from the dry weight of the filter residue or centrifuged precipitate.

It is usually necessary to differentiate quantitatively between the impurities and additives on the one hand and the low molecular weight species on the other hand. In the case of gravimetric determination, it is also important to perform a blank run using no test substance in order to account for residues arising from the experimental procedure.

The solution/extraction behaviour of polymers in water at 37 oC at pH 2 and pH 9 may be determined in the same way as described for the conduct of the experiment at 20 oC. The pH values can be achieved by the addition of either suitable buffers or appropriate acids or bases such as hydrochloric acid, acetic acid, analytical grade sodium or potassium hydroxide or NH3.

Depending on the method of analysis used, one or two tests should be performed. When sufficiently specific methods are available for direct analysis of the aqueous phase for the polymer component, one test as described above should suffice. However, when such methods are not available and determination of the solution/extraction behaviour of the polymer is limited to indirect analysis by determining only the total organic carbon content (TOC) of the aqueous extract, an additional test should be conducted. This additional test should also be done in triplicate, using ten times smaller polymer samples and the same amounts of water as those used in the first test.

1.5.4. Analysis

1.5.4.1. Test conducted with one sample size

Methods may be available for direct analysis of polymer components in the aqueous phase. Alternatively, indirect analysis of dissolved/extracted polymer components, by determining the total content of soluble parts and correcting for non polymer-specific components, could also be considered.

Analysis of the aqueous phase for the total polymeric species is possible:

either by a sufficiently sensitive method, e.g.:

- TOC using persulphate or dichromate digestion to yield CO2 followed by estimation by IR or chemical analysis,

- Atomic Absorption Spectrometry (AAS) or its Inductively Coupled Plasma (ICP) emission equivalent for silicon or metal containing polymers,

- UV absorption or spectrofluorimetry for aryl polymers,

- LC-MS for low molecular weight samples,

or by vacuum evaporation to dryness of the aqueous extract and spectroscopic (IR, UV, etc.) or AAS/ICP analysis of the residue.

If analysis of the aqueous phase as such is not practicable, the aqueous extract should be extracted with a water-immiscible organic solvent e.g. a chlorinated hydrocarbon. The solvent is then evaporated and the residue analysed as above for the notified polymer content. Any components in this residue which are identified as being impurities or additives are to be subtracted for the purpose of determining the degree of solution/extraction of the polymer itself.

When relatively large quantities of such materials are present, it may be necessary to subject the residue to e.g. HPLC or GC analysis to differentiate the impurities from the monomer and monomer-derived species present so that the true content of the latter can be determined.

In some cases, simple evaporation of the organic solvent to dryness and weighing the dry residue may be sufficient.

1.5.4.2. Test conducted with two different sample sizes

All aqueous extracts are analysed for TOC.

A gravimetric determination is performed on the undissolved/not extracted part of the sample. If, after centrifugation or filtering of the content of each vessel, polymer residues remain attached to the wall of the vessel, the vessel should be rinsed with the filtrate until the vessel is cleared from all visible residues. Following which, the filtrate is again centrifuged or filtered. The residues remaining on the filter or in the centrifuge tube are dried at 40 oC under vacuum and weighed. Drying is continued until a constant weight is reached.

2. DATA

2.1. TEST CONDUCTED WITH ONE SAMPLE SIZE

The individual results for each of the three flasks and the average values should be given and expressed in units of mass per volume of the solution (typically mg/l) or mass per mass of polymer sample (typically mg/g). Additionally, the weight loss of the sample (calculated as the weight of the solute divided by the weight of the initial sample) should also be given. The relative standard deviations (RSD) should be calculated. Individual figures should be given for the total substance (polymer + essential additives, etc.) and for the polymer only (i.e. after subtracting the contribution from such additives).

2.2. TEST CONDUCTED WITH TWO DIFFERENT SAMPLE SIZES

The individual TOC values of the aqueous extracts of the two triplicate experiments and the average value for each experiment should be given expressed as units of mass per volume of solution (typically mgC/l), as well as in units of mass per weight of the initial sample (typically mgC/g).

If there is no difference between the results at the high and the low sample/water ratios, this may indicate that all extractable components were indeed extracted. In such a case, direct analysis would normally not be necessary.

The individual weights of the residues should be given and expressed in percentage of the initial weights of the samples. Averages should be calculated per experiment. The differences between 100 and the percentages found represent the percentages of soluble and extractable material in the original sample.

3. REPORTING

3.1. TEST REPORT

The test report must include the following information:

3.1.1. Test substance:

- available information about test substance (identity, additives, impurities, content of low molecular weight species).

3.1.2. Experimental conditions:

- description of the procedures used and experimental conditions,

- description of the analytical and detection methods.

3.1.3. Results:

- results of solubility/extractivity in mg/l; individual and mean values for the extraction tests in the various solutions, broken down in polymer content and impurities, additives, etc.,

- results of solubility/extractivity in mg/g of polymer,

- TOC values of aqueous extracts, weight of the solute and calculated percentages, if measured,

- the pH of each sample,

- information about the blank values,

- where necessary, references to the chemical instability of the test substance, during both the testing process and the analytical process,

- all information which is important for the interpretation of the results.

4. REFERENCES

(1) DIN 53733 (1976) Zerkleinerung von Kunststofferzeugnissen für Prüfzwecke.

A.21. OXIDISING PROPERTIES (LIQUIDS)

1. METHOD

1.1. INTRODUCTION

This test method is designed to measure the potential for a liquid substance to increase the burning rate or burning intensity of a combustible substance, or to form a mixture with a combustible substance which spontaneously ignites, when the two are thoroughly mixed. It is based on the UN test for oxidising liquids (1) and is equivalent to it. However, as this method A.21 is primarily designed to satisfy the requirements of Regulation (EC) No 1907/2006, comparison with only one reference substance is required. Testing and comparison to additional reference substances may be necessary when the results of the test are expected to be used for other purposes. [9]

This test need not be performed when examination of the structural formula establishes beyond reasonable doubt that the substance is incapable of reacting exothermically with a combustible material.

It is useful to have preliminary information on any potential explosive properties of the substance before performing this test.

This test is not applicable to solids, gases, explosive or highly flammable substances, or organic peroxides.

This test may not need to be performed when results for the test substance in the UN test for oxidising liquids (1) are already available.

1.2. DEFINITIONS AND UNITS

Mean pressure rise time is the mean of the measured times for a mixture under test to produce a pressure rise from 690 kPa to 2070 kPa above atmospheric.

1.3. REFERENCE SUBSTANCE

65 % (w/w) aqueous nitric acid (analytical grade) is required as a reference substance. [10]

Optionally, if the experimenter foresees that the results of this test may eventually be used for other purposes [9], testing of additional reference substances may also be appropriate. [11]

1.4. PRINCIPLE OF THE TEST METHOD

The liquid to be tested is mixed in a 1 to 1 ratio, by mass, with fibrous cellulose and introduced into a pressure vessel. If during mixing or filling spontaneous ignition occurs, no further testing is necessary.

If spontaneous ignition does not occur the full test is carried out. The mixture is heated in a pressure vessel and the mean time taken for the pressure to rise from 690 kPa to 2070 kPa above atmospheric is determined. This is compared with the mean pressure rise time for the 1:1 mixture of the reference substance(s) and cellulose.

1.5. QUALITY CRITERIA

In a series of five trials on a single substance no results should differ by more than 30 % from the arithmetic mean. Results that differ by more than 30 % from the mean should be discarded, the mixing and filling procedure improved and the testing repeated.

1.6. DESCRIPTION OF THE METHOD

1.6.1. Preparation

1.6.1.1. Combustible substance

Dried, fibrous cellulose with a fibre length between 50 and 250 μm and a mean diameter of 25 μm [12], is used as the combustible material. It is dried to constant weight in a layer not more than 25 mm thick at 105 oC for four hours and kept in a desiccator, with desiccant, until cool and required for use. The water content of the dried cellulose should be less than 0,5 % by dry mass [13]. If necessary, the drying time should be prolonged to achieve this. [14] The same batch of cellulose is to be used throughout the test.

1.6.1.2. Apparatus

1.6.1.2.1. Pressure vessel

A pressure vessel is required. The vessel consists of a cylindrical steel pressure vessel 89 mm in length and 60 mm in external diameter (see figure 1). Two flats are machined on opposite sides (reducing the cross-section of the vessel to 50 mm) to facilitate holding whilst fitting up the firing plug and vent plug. The vessel, which has a bore of 20 mm diameter is internally rebated at either end to a depth of 19 mm and threaded to accept 1'' British Standard Pipe (BSP) or metric equivalent. A pressure take-off, in the form of a side arm, is screwed into the curved face of the pressure vessel 35 mm from one end and at 90o to the machined flats. The socket for this is bored to a depth of 12 mm and threaded to accept the 1/2" BSP (or metric equivalent) thread on the end of the side-arm. If necessary, an inert seal is fitted to ensure a gas-tight seal. The side-arm extends 55 mm beyond the pressure vessel body and has a bore of 6 mm. The end of the side-arm is rebated and threaded to accept a diaphragm type pressure transducer. Any pressure-measuring device may be used provided that it is not affected by the hot gases or the decomposition products and is capable of responding to rates of pressure rise of 690-2070 kPa in not more than 5 ms.

The end of the pressure vessel farthest from the side-arm is closed with a firing plug which is fitted with two electrodes, one insulated from, and the other earthed to, the plug body. The other end of the pressure vessel is closed by a bursting disk (bursting pressure approximately 2200 kPa) held in place with a retaining plug which has a 20 mm bore. If necessary, an inert seal is used with the firing plug to ensure a gas-tight fit. A support stand (figure 2) holds the assembly in the correct attitude during use. This usually comprises a mild steel base plate measuring 235 mm × 184 mm × 6 mm and a 185 mm length of square hollow section (S.H.S.) 70 mm × 70 mm × 4 mm.

A section is cut from each of two opposite sides at one end of the length of S.H.S. so that a structure having two flat sided legs surmounted by 86 mm length of intact box section results. The ends of these flat sides are cut to an angle of 60o to the horizontal and welded to the base plate. A slot measuring 22 mm wide × 46 mm deep is machined in one side of the upper end of the base section such that when the pressure vessel assembly is lowered, firing plug end first, into the box section support, the side-arm is accommodated in the slot. A piece of steel 30 mm wide and 6 mm thick is welded to the lower internal face of the box section to act as a spacer. Two 7 mm thumb screws, tapped into the opposite face, serve to hold the pressure vessel firmly in place. Two 12 mm wide strips of 6 mm thick steel, welded to the side pieces abutting the base of the box section, support the pressure vessel from beneath.

1.6.1.2.2. Ignition system

The ignition system consists of a 25 cm long Ni/Cr wire with a diameter 0,6 mm and a resistance of 3,85 ohm/m. The wire is wound, using a 5 mm diameter rod, in the shape of a coil and is attached to the firing plug electrodes. The coil should have one of the configurations shown in figure 3. The distance between the bottom of the vessel and the underside of the ignition coil should be 20 mm. If the electrodes are not adjustable, the ends of the ignition wire between the coil and the bottom of the vessel should be insulated by a ceramic sheath. The wire is heated by a constant current power supply able to deliver at least 10 A.

1.6.2. Performance of the test [15]

The apparatus, assembled complete with pressure transducer and heating system but without the bursting disk in position, is supported firing plug end down. 2,5 g of the liquid to be tested is mixed with 2,5 g of dried cellulose in a glass beaker using a glass stirring rod [16]. For safety, the mixing should be performed with a safety shield between the operator and mixture. If the mixture ignites during mixing or filling, no further testing is necessary. The mixture is added, in small portions with tapping, to the pressure vessel making sure that the mixture is packed around the ignition coil and is in good contact with it. It is important that the coil is not distorted during the packing process as this may lead to erroneous results [17]. The bursting disk is placed in position and the retaining plug is screwed in tightly. The charged vessel is transferred to the firing support stand, bursting disk uppermost, which should be located in a suitable, armoured fume cupboard or firing cell. The power supply is connected to the external terminals of the firing plug and 10 A applied. The time between the start of mixing and switching on the power should not exceed 10 minutes.

The signal produced by the pressure transducer is recorded on a suitable system which allows both evaluation and the generation of a permanent record of the time pressure profile obtained (e.g. a transient recorder coupled to a chart recorder). The mixture is heated until the bursting disk ruptures or until at least 60 s have elapsed. If the bursting disk does not rupture, the mixture should be allowed to cool before carefully dismantling the apparatus, taking precautions to allow for any pressurisation which may occur. Five trials are performed with the test substance and the reference substance(s). The time taken for the pressure to rise from 690 kPa to 2070 kPa above atmospheric is noted. The mean pressure rise time is calculated.

In some cases, substances may generate a pressure rise (too high or too low), caused by chemical reactions not characterising the oxidising properties of the substance. In these cases, it may be necessary to repeat the test with an inert substance, e.g. diatomite (kieselguhr), in place of the cellulose in order to clarify the nature of the reaction.

2. DATA

Pressure rise times for both the test substance and the reference substance(s). Pressure rise times for the tests with an inert substance, if performed.

2.1. TREATMENT OF RESULTS

The mean pressure rise times for both the test substance and the reference substances(s) are calculated.

The mean pressure rise time for the tests with an inert substance (if performed) is calculated.

Some examples of results are shown in Table 1.

Table 1

Examples of results [18]

Substance [19] | Mean pressure rise time for a 1:1 mixture with celulose (ms) |

Ammonium dichromate, saturated aqueous solution | 20800 |

Calcium nitrate, saturated aqueous solution | 6700 |

Ferric nitrate, saturated aqueous solution | 4133 |

Lithium perchlorate, saturated aqueous solution | 1686 |

Magnesium perchlorate, saturated aqueous solution | 777 |

Nickel nitrate, saturated aqueous solution | 6250 |

Nitric acid, 65 % | 4767 [20] |

Perchloric acid, 50 % | 121 [20] |

Perchloric acid, 55 % | 59 |

Potassium nitrate, 30 % aqueous solution | 26690 |

Silver nitrate, saturated aqueous solution | [21] |

Sodium chlorate, 40 % aqueous solution | 2555 [20] |

Sodium nitrate, 45 % aqueous solution | 4133 |

Inert substance | |

Water: cellulose | [21] |

3. REPORT

3.1. TEST REPORT

The test report should include the following information:

- the identity, composition, purity, etc. of the substance tested,

- the concentration of the test substance,

- the drying procedure of the cellulose used,

- the water content of the cellulose used,

- the results of the measurements,

- the results from tests with an inert substance, if any,

- the calculated mean pressure rise times,

- any deviations from this method and the reasons for them,

- all additional information or remarks relevant to the interpretation of the results.

3.2. INTERPRETATION OF THE RESULTS [22]

The test results are assessed on the basis of:

(a) whether the mixture of test substance and cellulose spontaneously ignites; and

(b) the comparison of the mean time taken for the pressure to rise from 690 kPa to 2070 kPa with that of the reference substance(s).

A liquid substance is to be considered as an oxidiser when:

(a) a 1:1 mixture, by mass, of the substance and cellulose spontaneously ignites; or

(b) a 1:1 mixture, by mass, of the substance and cellulose exhibits a mean pressure rise time less than or equal to the mean pressure rise time of a 1:1 mixture, by mass, of 65 % (w/w) aqueous nitric acid and cellulose.

In order to avoid a false positive result, if necessary, the results obtained when testing the substance with an inert material should also be considered when interpreting the results.

4. REFERENCES

(1) Recommendations on the Transport of Dangerous Goods, Manual of Tests and Criteria. 3rd revised edition. UN Publication No: ST/SG/AC.10/11/Rev. 3, 1999, page 342. Test O.2: Test for oxidising liquids.

Figure 1

Pressure vessel

(A) Pressure vessel body

(B) Bursting disk retaining plug

(D) Soft lead washer

(E) Bursting disc

(F) Side arm

(G) Pressure transducer head

(H) Washer

(J) Insulated electrode

(K) Earthed electrode

(L) Insulation

(M) Steel cone

(N) Washer distorting groove

+++++ TIFF +++++

Figure 2

Support stand

+++++ TIFF +++++

Figure 3

Ignition system

(A) Ignition coil

(B) Insulation

(D) Firing plug

+++++ TIFF +++++

Note: either of these configurations may be used.

[1] Dependent on type of instrument and on degree of purity of the substance.

[2] Dependent on type of instrument and on degree of purity of the substance

[3] Dependent on type of instrument and on degree of purity of the substance

[4] Dependent on type of instrument and on degree of purity of the substance

[5] This accuracy is only valid for the simple device as for example described in ASTM D 1120-72; it can be improved with more sophisticated ebulliometer devices.

[6] Only valid for pure substances. The use in other circumstances should be justified.

[7] Dependent of the degree of purity.

[8] These methods can also be used in the range 1 to 10 Pa providing care is taken.

[9] As, for example, in the framework of UN transport regulations.

[10] The acid should be titrated before testing to confirm its concentration.

[11] E.g.: 50 % (w/w) perchloric acid and 40 % (w/w) sodium chlorate are used in reference 1.

[12] E.g. Whatman Column Chromatographic Cellulose Powder CF 11, catalogue No 4021 050.

[13] Confirmed by, e.g. Karl-Fisher titration.

[14] Alternatively, this water content can also be achieved by, e.g. heating at 105 oC under vacuum for 24 h.

[15] Mixtures of oxidisers with cellulose must be treated as potentially explosive and handled with due care.

[16] In practice this can be achieved by preparing a 1:1 mixture of the liquid to be tested and cellulose in a greater amount than needed for the trial and transferring 5 ±0,1 g to the pressure vessel. The mixture is to be freshly prepared for each trial.

[17] In particular, contact between the adjacent turns of the coil must be avoided.

[18] See reference (1) for classification under the UN transport scheme.

[19] Saturated solutions should be prepared at 20 oC.

[20] Mean value from interlaboratory comparative trials.

[21] Maximum pressure of 2070 kPa not reached.

[22] See reference 1 for interpretation of the results under the UN transport regulations using several reference substances.

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PART B: METHODS FOR THE DETERMINATION OF TOXICITY AND OTHER HEALTH EFFECTS

TABLE OF CONTENTS

GENERAL INTRODUCTION

B.1 bis. ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE

B.1 tris. ACUTE ORAL TOXICITY — ACUTE TOXIC CLASS METHOD

B.2. ACUTE TOXICITY (INHALATION)

B.3. ACUTE TOXICITY (DERMAL)

B.4. ACUTE TOXICITY: DERMAL IRRITATION/CORROSION

B.5. ACUTE TOXICITY: EYE IRRITATION/CORROSION

B.6. SKIN SENSITISATION

B.7. REPEATED DOSE (28 DAYS) TOXICITY (ORAL)

B.8. REPEATED DOSE (28 DAYS) TOXICITY (INHALATION)

B.9. REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)

B.10. MUTAGENICITY — IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST

B.11. MUTAGENICITY — IN VIVO MAMMALIAN BONE MARROW CHROMOSOME ABERRATION TEST

B.12. MUTAGENICITY — IN VIVO MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST

B.13/14. MUTAGENICITY: REVERSE MUTATION TEST USING BACTERIA

B.15. MUTAGENICITY TESTING AND SCREENING FOR CARCINOGENICITY GENE MUTATION — SACCHAROMYCES CEREVISIAE

B.16. MITOTIC RECOMBINATION — SACCHAROMYCES CEREVISIAE

B.17. MUTAGENICITY — IN VITRO MAMMALIAN CELL GENE MUTATION TEST

B.18. DNA DAMAGE AND REPAIR — UNSCHEDULED DNA SYNTHESIS — MAMMALIAN CELLS IN VITRO

B.19. SISTER CHROMATID EXCHANGE ASSAY IN VITRO

B.20. SEX-LINKED RECESSIVE LETHAL TEST IN DROSOPHILA MELANOGASTER

B.21. IN VITRO MAMMALIAN CELL TRANSFORMATION TESTS

B.22. RODENT DOMINANT LETHAL TEST

B.23. MAMMALIAN SPERMATOGONIAL CHROMOSOME ABERRATION TEST

B.24. MOUSE SPOT TEST

B.25. MOUSE HERITABLE TRANSLOCATION

B.26. SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90 — DAY ORAL TOXICITY STUDY IN RODENTS

B.27. SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90 — DAY ORAL TOXICITY STUDY IN NON-RODENTS

B.28. SUB-CHRONIC DERMAL TOXICITY STUDY 90-DAY REPEATED DERMAL DOSE STUDY USING RODENT SPECIES

B.29. SUB-CHRONIC INHALATION TOXICITY STUDY 90-DAY REPEATED INHALATION DOSE STUDY USING RODENT SPECIES

B.30. CHRONIC TOXICITY TEST

B.31. PRENATAL DEVELOPMENTAL TOXICITY STUDY

B.32. CARCINOGENICITY TEST

B.33. COMBINED CHRONIC TOXICITY/CARCINOGENICITY TEST

B.34. ONE-GENERATION REPRODUCTION TOXICITY TEST

B.35. TWO-GENERATION REPRODUCTION TOXICITY STUDY

B.36. TOXICOKINETICS

B.37. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES FOLLOWING ACUTE EXPOSURE

B.38. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES 28 DAY REPEATED DOSE STUDY

B.39. UNSCHEDULED DNA SYNTHESIS (UDS) TEST WITH MAMMALIAN LIVER CELLS IN VIVO

B.40. IN VITRO SKIN CORROSION: TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST (TER)

B.40 BIS. IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST

B.41. IN VITRO 3T3 NRU PHOTOTOXICITY TEST

B.42. SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY

B.43. NEUROTOXICITY STUDY IN RODENTS

B.44. SKIN ABSORPTION: IN VIVO METHOD

B.45. SKIN ABSORPTION: IN VITRO METHOD

GENERAL INTRODUCTION

A. CHARACTERISATION OF THE TEST SUBSTANCE

The composition of the test substance, including major impurities, and its relevant physico-chemical properties including stability, should be known prior to the initiation of any toxicity study.

The physico-chemical properties of the test substance provide important information for the selection of the route of administration, the design of each particular study and the handling and storage of the test substance.

The development of an analytical method for qualitative and quantitative determination of the test substance (including major impurities when possible) in the dosing medium and the biological material should precede the initiation of the study.

All information relating to the identification, the physico-chemical properties, the purity, and behaviour of the test substance should be included in the test report.

B. ANIMAL CARE

Stringent control of environmental conditions and proper animal care techniques are essential in toxicity testing.

(i) Housing conditions

The environmental conditions in the experimental animal rooms or enclosures should be appropriate to the test species. For rats, mice and guinea pigs, suitable conditions are a room temperature of 22 oC ± 3 oC with a relative humidity of 30 to 70 %; for rabbits the temperature should be 20 ± 3 oC with a relative humidity of 30 to 70 %.

Some experimental techniques are particularly sensitive to temperature effects and, in these cases, details of appropriate conditions are included in the description of the test method. In all investigations of toxic effects, the temperature and humidity should be monitored, recorded, and included in the final report of the study.

Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. Details of the lighting pattern should be recorded and included in the final report of the study.

Unless otherwise specified in the method, animals may be housed individually, or be caged in small groups of the same sex; for group caging, no more than five animals should be housed per cage.

In reports of animal experiments, it is important to indicate the type of caging used and the number of animals housed in each cage both during exposure to the chemical and any subsequent observation period.

(ii) Feeding conditions

Diets should meet all the nutritional requirements of the species under test. Where test substances are administered to animals in their diet the nutritional value may be reduced by interaction between the substance and a dietary constituent. The possibility of such a reaction should be considered when interpreting the results of tests. Conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of the diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method.

Dietary contaminants which are known to influence the toxicity should not be present in interfering concentrations.

C. ALTERNATIVE TESTING

The European Union is committed to promoting the development and validation of alternative techniques which can provide the same level of information as current animal tests, but which use fewer animals, cause less suffering or avoid the use of animals completely.

Such methods, as they become available, must be considered wherever possible for hazard characterisation and consequent classification and labelling for intrinsic hazards and chemical safety assessment.

D. EVALUATION AND INTERPRETATION

When tests are evaluated and interpreted, limitations in the extent to which the results of animal and in vitro studies can be extrapolated directly to man must be considered and therefore, evidence of adverse effects in humans, where available, may be used for confirmation of testing results.

E. LITERATURE REFERENCES

Most of these methods are developed within the framework of the OECD programme for Testing Guidelines, and should be performed in conformity with the principles of Good Laboratory Practice, in order to ensure as wide as possible "mutual acceptance of data".

Additional information may be found in the references listed in the OECD guidelines and the relevant literature published elsewhere.

B.1 bis. ACUTE ORAL TOXICITY — FIXED DOSE PROCEDURE

1. METHOD

This test method is equivalent to OECD TG 420 (2001)

1.1. INTRODUCTION

Traditional methods for assessing acute toxicity use death of animals as an endpoint. In 1984, a new approach to acute toxicity testing was suggested by the British Toxicology Society based on the administration at a series of fixed dose levels (1). The approach avoided using death of animals as an endpoint, and relied instead on the observation of clear signs of toxicity at one of a series of fixed dose levels. Following UK (2) and international (3) in vivo validation studies the procedure was adopted as a testing method in 1992. Subsequently, the statistical properties of the Fixed Dose Procedure have been evaluated using mathematical models in a series of studies (4)(5)(6). Together, the in vivo and modelling studies have demonstrated that the procedure is reproducible, uses fewer animals and causes less suffering than the traditional methods and is able to rank substances in a similar manner to the other acute toxicity testing methods.

Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing (7). This guidance document also contains additional information on the conduct and interpretation of Testing Method B.1bis.

It is a principle of the method that in the main study only moderately toxic doses are used, and that administration of doses that are expected to be lethal should be avoided. Also, doses that are known to cause marked pain and distress, due to corrosive or severely irritant actions, need not be administered. Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed, and are considered in the interpretation of the test results in the same way as animals that died on test. Criteria for making the decision to kill moribund or severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject of a separate Guidance Document (8).

The method provides information on the hazardous properties and allows the substance to be ranked and classified according to the Globally Harmonised System (GHS) for the classification of chemicals which cause acute toxicity (9).

The testing laboratory should consider all available information on the test substance prior to conducting the study. Such information will include the identity and chemical structure of the substance; its physico-chemical properties; the results of any other in vitro or in vivo toxicity tests on the substance; toxicological data on structurally related substances; and the anticipated use(s) of the substance. This information is necessary to satisfy all concerned that the test is relevant for the protection of human health, and will help in the selection of an appropriate starting dose.

1.2. DEFINITIONS

Acute oral toxicity: refers to those adverse effects occurring following oral administration of a single dose of a substance or multiple doses given within 24 hours.

Delayed death: means that an animal does not die or appear moribund within 48 hours but dies later during the 14-day observation period.

Dose: is the amount of test substance administered. Dose is expressed as weight of test substance per unit weight of test animal (e.g. mg/kg).

Evident toxicity: is a general term describing clear signs of toxicity following the administration of test substance (see (3) for examples) such that at the next highest fixed dose either severe pain and enduring signs of severe distress, moribund status (criteria are presented in the Humane Endpoints Guidance Document (8)), or probable mortality in most animals can be expected.

GHS: Globally Harmonised Classification System for Chemical Substances and Mixtures. A joint activity of OECD (human health and the environment), UN Committee of Experts on Transport of Dangerous Goods (physical-chemical properties) and ILO (hazard communication) and coordinated by the Interorganisation Programme for the Sound Management of Chemicals (IOMC).

Impending death: when moribund state or death is expected prior to the next planned time of observation. Signs indicative of this state in rodents could include convulsions, lateral position, recumbence and tremor. (See the Humane Endpoint Guidance Document (8) for more details).

LD50(median lethal dose): is a statistically derived single dose of a substance that can be expected to cause death in 50 % of animals when administered by the oral route. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (mg/kg).

Limit dose: refers to a dose at an upper limitation on testing (2000 or 5000 mg/kg).

Moribund status: being in a state of dying or inability to survive, even if treated. (See the Humane Endpoint Guidance Document (8) for more details).

Predictable death: presence of clinical signs indicative of death at a known time in the future before the planned end of the experiment, for example: inability to reach water or food. (See the Humane Endpoint Guidance Document (8) for more details).

1.3. PRINCIPLE OF THE TEST METHOD

Groups of animals of a single sex are dosed in a stepwise procedure using the fixed doses of 5, 50, 300 and 2000 mg/kg (exceptionally an additional fixed dose of 5000 mg/kg may be considered, see Section 1.6.2). The initial dose level is selected on the basis of a sighting study as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality. Clinical signs and conditions associated with pain, suffering, and impending death, are described in detail in a separate OECD Guidance Document (8). Further groups of animals may be dosed at higher or lower fixed doses, depending on the presence or absence of signs of toxicity or mortality. This procedure continues until the dose causing evident toxicity or no more than one death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Selection of animal species

The preferred rodent species is the rat, although other rodent species may be used. Normally females are used (7). This is because literature surveys of conventional LD50 tests show that usually there is little difference in sensitivity between the sexes, but in those cases where differences are observed, females are generally slightly more sensitive (10). However, if knowledge of the toxicological or toxicokinetic properties of structurally related chemicals indicates that males are likely to be more sensitive then this sex should be used. When the test is conducted in males, adequate justification should be provided.

Healthy young adult animals of commonly used laboratory strains should be employed. Females should be nulliparous and non-pregnant. Each animal, at the commencement of its dosing, should be between eight and 12 weeks old and its weight should fall in an interval within ± 20 % of the mean weight of any previously dosed animals.

1.4.2. Housing and feeding conditions

The temperature of the experimental animal room should be 22 oC (± 3 oC). Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. Animals may be group-caged by dose, but the number of animals per cage must not interfere with clear observations of each animal.

1.4.3. Preparation of animals

The animals are randomly selected, marked to permit individual identification, and kept in their cages for at least five days prior to the start of dosing to allow for acclimatisation to the laboratory conditions.

1.4.4. Preparation of doses

In general test substances should be administered in a constant volume over the range of doses to be tested by varying the concentration of the dosing preparation. Where a liquid end product or mixture is to be tested however, the use of the undiluted test substance, i.e. at a constant concentration, may be more relevant to the subsequent risk assessment of that substance, and is a requirement of some regulatory authorities. In either case, the maximum dose volume for administration must not be exceeded. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the volume should not normally exceed 1ml /100 g of body weight: however in the case of aqueous solutions 2 ml/100 g body weight can be considered. With respect to the formulation of the dosing preparation, the use of an aqueous solution/suspension/emulsion is recommended wherever possible, followed in order of preference by a solution/suspension/emulsion in oil (e.g. corn oil) and then possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the vehicle should be known. Doses must be prepared shortly prior to administration unless the stability of the preparation over the period during which it will be used is known and shown to be acceptable.

1.5. PROCEDURE

1.5.1. Administration of doses

The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation canula. In the unusual circumstance that a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours.

Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld over-night; with the mouse, food but not water should be withheld for three to four hours). Following the period of fasting, the animals should be weighed and the test substance administered. After the substance has been administered, food may be withheld for a further three to four hours in rats or one to two hours in mice. Where a dose is administered in fractions over a period of time, it may be necessary to provide the animals with food and water depending on the length of the period.

1.5.2. Sighting study

The purpose of the sighting study is to allow selection of the appropriate starting dose for the main study. The test substance is administered to single animals in a sequential manner following the flowcharts in Appendix 1. The sighting study is completed when a decision on the starting dose for the main study can be made (or if a death is seen at the lowest fixed dose).

The starting dose for the sighting study is selected from the fixed dose levels of 5, 50, 300 and 2000 mg/kg as a dose expected to produce evident toxicity based, when possible, on evidence from in vivo and in vitro data from the same chemical and from structurally related chemicals. In the absence of such information, the starting dose will be 300 mg/kg.

A period of at least 24 hours will be allowed between the dosing of each animal. All animals should be observed for at least 14 days.

Exceptionally, and only when justified by specific regulatory needs, the use of an additional upper fixed dose level of 5000 mg/kg may be considered (see Appendix 3). For reasons of animal welfare concern, testing of animals in GHS Category 5 ranges (2000-5000 mg/kg is discouraged and should only be considered when there is a strong likelihood that the results of such a test have a direct relevance for protecting human or animal health or the environment.

In cases where an animal tested at the lowest fixed dose level (5 mg/kg) in the sighting study dies, the normal procedure is to terminate the study and assign the substance to GHS Category 1 (as shown in Appendix 1). However, if further confirmation of the classification is required, an optional supplementary procedure may be conducted, as follows. A second animal is dosed at 5 mg/kg. If this second animal dies, then GHS Category 1 will be confirmed and the study will be immediately terminated. If the second animal survives, then a maximum of three additional animals will be dosed at 5 mg/kg. Because there will be a high risk of mortality, these animals should be dosed in a sequential manner to protect animal welfare. The time interval between dosing each animal should be sufficient to establish that the previous animal is likely to survive. If a second death occurs, the dosing sequence will be immediately terminated and no further animals will be dosed. Because the occurrence of a second death (irrespective of the number of animals tested at the time of termination) falls into outcome A (two or more deaths), the classification rule of Appendix 2 at the 5 mg/kg fixed dose is followed (Category 1 if there are two or more deaths or Category 2 if there is no more than one death). In addition, Appendix 4 gives guidance on the classification in the EU system until the new GHS is implemented.

1.5.3. Main study

1.5.3.1. Numbers of animals and dose levels

The action to be taken following testing at the starting dose level is indicated by the flowcharts in Appendix 2. One of three actions will be required; either stop testing and assign the appropriate hazard classification class, test at a higher fixed dose or test at a lower fixed dose. However, to protect animals, a dose level that caused death in the sighting study will not be revisited in the main study (see Appendix 2). Experience has shown that the most likely outcome at the starting dose level will be that the substance can be classified and no further testing will be necessary.

A total of five animals of one sex will normally be used for each dose level investigated. The five animals will be made up of one animal from the sighting study dosed at the selected dose level together with an additional four animals (except, unusually, if a dose level used on the main study was not included in the sighting study).

The time interval between dosing at each level is determined by the onset, duration, and severity of toxic signs. Treatment of animals at the next dose should be delayed until one is confident of survival of the previously dosed animals. A period of three or four days between dosing at each dose level is recommended, if needed, to allow for the observation of delayed toxicity. The time interval may be adjusted as appropriate, e.g. in case of inconclusive response.

When the use of an upper fixed dose of 5000 mg/kg is considered, the procedure outlined in Appendix 3 should be followed (see also section 1.6.2).

1.5.3.2. Limit test

The limit test is primarily used in situations where the experimenter has information indicating that the test material is likely to be nontoxic, i.e., having toxicity only above regulatory limit doses. Information about the toxicity of the test material can be gained from knowledge about similar tested compounds or similar tested mixtures or products, taking into consideration the identity and percentage of components known to be of toxicological significance. In those situations where there is little or no information about its toxicity, or in which the test material is expected to be toxic, the main test should be performed.

Using the normal procedure, a sighting study starting dose of 2000 mg/kg (or exceptionally 5000 mg/kg) followed by dosing of a further four animals at this level serves as a limit test for this guideline.

1.6. OBSERVATIONS

Animals are observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first four hours, and daily thereafter, for a total of 14 days, except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead. However, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, time of onset and length of recovery period, and may thus be extended when considered necessary. The times at which signs of toxicity appear and disappear are important, especially if there is a tendency for toxic signs to be delayed (11). All observations are systematically recorded, with individual records being maintained for each animal.

Additional observations will be necessary if the animals continue to display signs of toxicity. Observations should include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The principles and criteria summarised in the Humane Endpoints Guidance Document should be taken into consideration (8). Animals found in a moribund condition and animals showing severe pain or enduring signs of severe distress should be humanely killed. When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.

1.6.1. Body weight

Individual weights of animals should be determined shortly before the test substance is administered and at least weekly thereafter. Weight changes should be calculated and recorded. At the end of the test surviving animals are weighed and then humanely killed.

1.6.2. Pathology

All test animals (including those that die during the test or are removed from the study for animal welfare reasons) should be subjected to gross necropsy. All gross pathological changes should be recorded for each animal. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 or more hours after the initial dosing may also be considered because it may yield useful information.

2. DATA

Individual animal data should be provided. Additionally, all data should be summarised in tabular form, showing for each test group the number of animals used, the number of animals displaying signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of individual animals, a description and the time course of toxic effects and reversibility, and necropsy findings.

3. REPORTING

3.1. TEST REPORT

The test report must include the following information, as appropriate:

Test substance:

- physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation),

- identification data, including CAS number.

Vehicle (if appropriate):

- justification for choice of vehicle, if other than water.

Test animals:

- species/strain used,

- microbiological status of the animals, when known,

- number, age and sex of animals (including, where appropriate, a rationale for use of males instead of females),

- source, housing conditions, diet, etc.

Test conditions:

- details of test substance formulation, including details of the physical form of the material administered,

- details of the administration of the test substance including dosing volumes and time of dosing,

- details of food and water quality (including diet type/source, water source),

- the rationale for the selection of the starting dose.

Results:

- tabulation of response data and dose level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity and duration of effects),

- tabulation of body weight and body weight changes,

- individual weights of animals at the day of dosing, in weekly intervals thereafter, and at time of death or sacrifice,

- date and time of death if prior to scheduled sacrifice,

- time course of onset of signs of toxicity and whether these were reversible for each animal,

- necropsy findings and histopathological findings for each animal, if available.

Discussion and interpretation of results.

Conclusions.

4. REFERENCES

(1) British Toxicology Society Working Party on Toxicity (1984) Special report: a new approach to the classification of substances and preparations on the basis of their acute toxicity. Human Toxicol., 3, p. 85-92.

(2) Van den Heuvel, M.J., Dayan, A.D. and Shillaker, R.O (1987) Evaluation of the BTS approach to the testing of substances and preparations for their acute toxicity. Human Toxicol.‚ 6, p. 279-291.

(3) Van den Heuvel, M.J., Clark, D.G., Fielder, R.J., Koundakjian, P.P., Oliver, G.J.A., Pelling, D., Tomlinson, N.J. and Walker, A.P (1990) The international validation of a fixed-dose procedure as an alternative to the classical LD50 test. Fd. Chem. Toxicol. 28, p. 469-482.

(4) Whitehead, A. and Curnow, R.N (1992) Statistical evaluation of the fixed-dose procedure. Fd. Chem. Toxicol., 30, p. 313-324.

(5) Stallard, N. and Whitehead, A (1995) Reducing numbers in the fixed-dose procedure. Human Exptl. Toxicol. 14, p. 315-323.

(6) Stallard, N., Whitehead, A and Ridgeway, P. (2002) Statistical evaluation of the revised fixed dose procedure. Hum. Exp. Toxicol., 21, p. 183-196.

(7) OECD (2001) Guidance Document on Acute Oral Toxicity Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No 24. Paris

(8) OECD (2000) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph Series on Testing and Assesment No 19.

(9) OECD (1998) Harmonised Integrated Hazard Classification for Human Health and Environmental Effects of Chemical Substances as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals in November 1998, Part 2, p. 11 [http://webnet1.oecd.org/oecd/pages/home/displaygeneral/0,3380, EN-documents-521-14-no-24-no-0,FF.html].

(10) Lipnick, R.L., Cotruvo, J.A., Hill, R.N., Bruce, R.D., Stitzel, K.A., Walker, A.P., Chu, I., Goddard, M., Segal, L., Springer, J.A. and Myers, R.C (1995) Comparison of the Up-and-Down, Conventional LD50, and Fixed-Dose Acute Toxicity Procedures. Fd. Chem. Toxicol. 33, p. 223-231.

(11) Chan P.K and A. W Hayes (1994) Chapter 16 Acute Toxicity and Eye Irritation. In: Principles and Methods of Toxicology. 3rd Edition. A.W. Hayes, Editor. Raven Press Ltd. New York, USA.

Appendix 1

FLOW CHART FOR THE SIGHTING STUDY

Starting dose: 5 mg/kg

START

1 animal 5 mg/kg

1 animal 50 mg/kg

1 animal 300 mg/kg

1 animal 2000 mg/kg

Classify GHS

Category 1*

Main study starting Dose (mg/kg):

starting dose: 50 mg/kg

START

1 animal 5 mg/kg

1 animal 50 mg/kg

1 animal 300 mg/kg

1 animal 2000 mg/kg

Classify GHS

Category 1*

Main study starting Dose (mg/kg):

Outcome

death

evident toxicity

no evident toxicity and no death

* for outcome at 5 mg/kg there is an optional supplementary procedure to confirm the GHS classification: see section 1.5.2

+++++ TIFF +++++

Starting dose: 300 mg/kg

START

1 animal 5 mg/kg

1 animal 50 mg/kg

1 animal 300 mg/kg

1 animal 2000 mg/kg

Classify GHS

Category 1*

Main study starting Dose (mg/kg):

starting dose: 2000 mg/kg

START

1 animal 5 mg/kg

1 animal 50 mg/kg

1 animal 300 mg/kg

1 animal 2000 mg/kg

Classify GHS

Category 1*

Main study starting Dose (mg/kg):

Outcome

death

evident toxicity

no evident toxicity and no death

* for outcome at 5 mg/kg there is an optional supplementary procedure to confirm the GHS classification: see section 1.5.2

+++++ TIFF +++++

Appendix 2

FLOW CHART FOR THE MAIN STUDY

Starting dose: 5 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg*

5 animals 300 mg/kg

5 animals 2000 mg/kg

Classify GHS

Category

5/Unclassified

Starting dose: 50 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg*

5 animals 2000 mg/kg

Classify GHS

Category

5/Unclassified

Outcome

≥ 2 deaths

≥ 1 with evident toxicity and / or 1 death

No evident toxicity and no death

Group size

The 5 animals in each main study group will include any animal tested at that dose level in the sighting study

* Animal welfare override

If this dose level caused death in the sighting study, then no further animals will be tested. Go directly to outcome

+++++ TIFF +++++

Starting dose: 300 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg*

Classify GHS

Category

5/Unclassified

starting dose: 2000 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg

Classify GHS

Category

5/Unclassified

Outcome

≥ 2 deaths

≥ 1 with evident toxicity and / or 1 death

No evident toxicity and no death

Group size

The 5 animals in each main study group will include any animal tested at that dose level in the sighting study

* Animal welfare override

If this dose level caused death in the sighting study, then no further animals will be tested. Go directly to outcome

+++++ TIFF +++++

Appendix 3

CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD50 VALUES EXCEEDING 2000 MG/KG WITHOUT THE NEED FOR TESTING.

Criteria for hazard Category 5 are intended to enable the identification of test substances which are of relatively low acute toxicity hazard but which, under certain circumstances may present a danger to vulnerable populations. These substances are anticipated to have an oral or dermal LD50 in the range of 2000-5000 mg/kg or equivalent doses for other routes. Test substances could be classified in the hazard category defined by: 2000 mg/kg < LD50 < 5000 mg/kg (Category 5 in the GHS) in the following cases:

(a) if directed to this category by any of the testing schemes of Appendix 2, based on mortality incidences

(b) if reliable evidence is already available that indicates the LD50 to be in the range of Category 5 values; or other animal studies or toxic effects in humans indicate a concern for human health of an acute nature;

(c) through extrapolation, estimation or measurement of data if assignment to a more hazardous class is not warranted; and

- reliable information is available indicating significant toxic effects in humans, or

- any mortality is observed when tested up to Category 4 values by the oral route, or

- where expert judgement confirms significant clinical signs of toxicity, when tested up to Category 4 values, except for diarrhoea, piloerection or an ungroomed appearance, or

- where expert judgement confirms reliable information indicating the potential for significant acute effects from the other animal studies.

TESTING AT DOSES ABOVE 2000 MG/KG

Exceptionally, and only when justified by specific regulatory needs, the use of an additional upper fixed dose level of 5000 mg/kg may be considered. Recognising the need to protect animal welfare, testing at 5000 mg/kg is discouraged and should only be considered when there is a strong likelihood that the results of such a test would have a direct relevance for protecting animal or human health (9).

Sighting study

The decision rules governing the sequential procedure presented in Appendix 1 are extended to include a 5000 mg/kg dose level. Thus, when a sighting study starting dose of 5000 mg/kg is used outcome A (death) will require a second animal to be tested at 2000 mg/kg; outcomes B and C (evident toxicity or no toxicity) will allow the selection of 5000 mg/kg as the main study starting dose. Similarly, if a starting dose other than 5000 mg/kg is used then testing will progress to 5000 mg/kg in the event of outcomes B or C at 2000 mg/kg; a subsequent 5000 mg/kg outcome A will dictate a main study starting dose of 2000 mg/kg and outcomes B and C will dictate a main study starting dose of 5000 mg/kg.

Main study

The decision rules governing the sequential procedure presented in Appendix 2 are extended to include a 5000 mg/kg dose level. Thus, when a main study starting dose of 5000 mg/kg is used, outcome A (≥ 2 deaths) will require the testing of a second group at 2000 mg/kg; outcome B (evident toxicity and/or ≤ 1 death) or C (no toxicity) will result in the substance being unclassified according to GHS. Similarly, if a starting dose other than 5000 mg/kg is used then testing will progress to 5000 mg/kg in the event of outcome C at 2000 mg/kg; a subsequent 5000 mg/kg outcome A will result in the substance being assigned to GHS Category 5 and outcomes B or C will lead to the substance being unclassified.

Appendix 4

TEST METHOD B.1 bis

Guidance on classification according to the EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS) (taken from reference (8))

Starting dose: 5 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg

Classify EU

starting dose: 50 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg *

5 animals 2000 mg/kg

Classify EU

Outcome

≥ 2 deaths

≥ 1 with evident toxicity and/or 1 death

No evident toxicity and no death

T+ = very toxic

T = toxic

H = harmful

U = unclassified

* Animal welfare override If this dose level caused death in the sighting study, then no futher animals will be tested. Go directely to outcome

Group size The 5 animals in each main study group will include any animal tested at that dose level in the study

+++++ TIFF +++++

Starting dose: 300 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg*

Classify EU

Starting dose: 2000 mg/kg

START

5 animals 5 mg/kg

5 animals 50 mg/kg

5 animals 300 mg/kg

5 animals 2000 mg/kg

Classify EU

Outcome

≥ 2 deaths

≥ 1 with evident toxicity and / or 1 death

No evident toxicity and no death

T+ = very toxic

T = toxic

H = harmful

U + unclassified

Group size

The 5 animals in each main study group will include any animal tested at that dose level in the sighting study

* Animal welfare override

If this dose level caused death in the sighting study, then no further animals will be tested. Go directly to outcome

+++++ TIFF +++++

B.1 tris. ACUTE ORAL TOXICITY — ACUTE TOXIC CLASS METHOD

1. METHOD

This test method is equivalent to OECD TG 423 (2001)

1.1. INTRODUCTION

The acute toxic class method (1) set out in this test is a stepwise procedure with the use of three animals of a single sex per step. Depending on the mortality and/or the moribund status of the animals, on average two to four steps may be necessary to allow judgement on the acute toxicity of the test substance. This procedure is reproducible, uses very few animals and is able to rank substances in a similar manner to the other acute toxicity testing methods. The acute toxic class method is based on biometric evaluations (2)(3)(4)(5) with fixed doses, adequately separated to enable a substance to be ranked for classification purposes and hazard assessment. The method as adopted in 1996 was extensively validated in vivo against LD50 data obtained from the literature, both nationally (6) and internationally (7).

Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing (8). This Guidance Document also contains additional information on the conduct and interpretation of testing method B.1tris.

Test substances, at doses that are known to cause marked pain and distress due to corrosive or severely irritant actions, need not be administered. Moribund animals, or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed, and are considered in the interpretation of the test results in the same way as animals that died on test. Criteria for making the decision to kill moribund or severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject of a separate Guidance Document (9).

The method uses pre-defined doses and the results allow a substance to be ranked and classified according to the Globally Harmonised System for the classification of chemicals which cause acute toxicity (10).

In principle, the method is not intended to allow the calculation of a precise LD50, but does allow for the determination of defined exposure ranges where lethality is expected since death of a proportion of the animals is still the major endpoint of this test. The method allows for the determination of an LD50 value only when at least two doses result in mortality higher than 0 % and lower than 100 %. The use of a selection of pre-defined doses, regardless of test substance, with classification explicitly tied to number of animals observed in different states improves the opportunity for laboratory to laboratory reporting consistency and repeatability.

The testing laboratory should consider all available information on the test substance prior to conducting the study. Such information will include the identity and chemical structure of the substance; its physico-chemical properties; the result of any other in vivo or in vitro toxicity tests on the substance; toxicological data on the structurally related substances; and the anticipated use(s) of the substance. This information is necessary to satisfy all concerned that the test is relevant for the protection of human health and will help in the selection of the most appropriate starting dose.

1.2. DEFINITIONS

Acute oral toxicity: refers to those adverse effects occurring following oral administration of a single dose of a substance or multiple doses given within 24 hours.

Delayed death: means that an animal does not die or appear moribund within 48 hours but dies later during the 14-day observation period.

Dose: is the amount of test substance administered. Dose is expressed as weight of test substance per unit weight of test animal (e.g. mg/kg).

Impending death: when moribund state or death is expected prior to the next planned time of observation. Signs indicative of this state in rodents could include convulsions, lateral position, recumbence and tremor (See the Humane Endpoint Guidance Document (9) for more details).

LD50(median lethal oral dose): is a statistically derived single dose of a substance that can be expected to cause death in 50 % of animals when administered by the oral route. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (mg/kg).

Limit dose: refers to a dose at an upper limitation on testing (2000 or 5000 mg/kg).

Moribund status: being in a state of dying or inability to survive, even if treated (See the Humane Endpoint Guidance Document (9) for more details).

Predictable death: presence of clinical signs indicative of death at a known time in the future before the planned end of the experiment; for example: inability to reach water or food. (See the Humane Endpoint Guidance Document (9) for more details).

1.3. PRINCIPLE OF THE TEST

It is the principle of the test that, based on a stepwise procedure with the use of a minimum number of animals per step, sufficient information is obtained on the acute toxicity of the test substance to enable its classification. The substance is administered orally to a group of experimental animals at one of the defined doses. The substance is tested using a stepwise procedure, each step using three animals of a single sex (normally females). Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step, i.e.;

- no further testing is needed,

- dosing of three additional animals, with the same dose,

- dosing of three additional animals at the next higher or the next lower dose level.

Details of the test procedure are described in Appendix 1. The method will enable a judgement with respect to classifying the test substance to one of a series of toxicity classes defined by fixed LD50 cut-off values.

1.4. DESCRIPTION OF THE METHOD

1.4.1. Selection of animal species

The preferred rodent species is the rat, although other rodent species may be used. Normally females are used (9). This is because literature surveys of conventional LD50 tests show that, although there is little difference in sensitivity between the sexes, in those cases where differences are observed females are generally slightly more sensitive (11). However if knowledge of the toxicological or toxicokinetic properties of structurally related chemicals indicates that males are likely to be more sensitive, then this sex should be used. When the test is conducted in males, adequate justification should be provided.

1.4.2. Housing and feeding conditions

The temperature in the experimental animal room should be 22 oC (± 3 oC). Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. Animals may be group-caged by dose, but the number of animals per cage must not interfere with clear observations of each animal.

1.4.3. Preparation of animals

The animals are randomly selected, marked to permit individual identification, and kept in their cages for at least five days prior to dosing to allow for acclimatisation to the laboratory conditions.

1.4.4. Preparation of doses

In general, test substances should be administered in a constant volume over the range of doses to be tested by varying the concentration of the dosing preparation. Where a liquid end product or mixture is to be tested however, the use of the undiluted test substance, i.e. at a constant concentration, may be more relevant to the subsequent risk assessment of that substance, and is a requirement of some regulatory authorities. In either case, the maximum dose volume for administration must not be exceeded. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. In rodents, the volume should not normally exceed 1 ml/100 g of body weight: however in the case of aqueous solutions 2 ml/100 g body weight can be considered. With respect to the formulation of the dosing preparation, the use of an aqueous solution/suspension/emulsion is recommended wherever possible, followed in order of preference by a solution/suspension/emulsion in oil (e.g. corn oil) and then possibly solution in other vehicles. For vehicles other than water the toxicological characteristics of the vehicle should be known. Doses must be prepared shortly prior to administration unless the stability of the preparation over the period during which it will be used is known and shown to be acceptable.

1.5. PROCEDURE

1.5.1. Administration of doses

Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld overnight, with the mouse, food but not water should be withheld for three or four hours). Following the period of fasting, the animals should be weighed and the test substance administered. After the substance has been administered, food may be withheld for a further three or fours hours in rats or one or two hours in mice. Where a dose is administered in fractions over a period it may be necessary to provide the animals with food and water depending on the length of the period.

1.5.2. Number of animals and dose levels

Three animals are used for each step. The dose level to be used as the starting dose is selected from one of four fixed levels, 5, 50, 300 and 2000 mg/kg body weight. The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. The flowcharts of Appendix 1 describe the procedure that should be followed for each of the starting doses. In addition, Appendix 4 gives guidance on the classification in the EU system until the new GHS is implemented.

When available information suggests that mortality is unlikely at the highest starting dose level (2000 mg/kg body weight), then a limit test should be conducted. When there is no information on a substance to be tested, for animal welfare reasons it is recommended to use the starting dose of 300 mg/kg body weight.

The time interval between treatment groups is determined by the onset, duration, and severity of toxic signs. Treatment of animals at the next dose should be delayed until one is confident of survival of the previously dosed animals.

Exceptionally, and only when justified by specific regulatory needs, the use of additional upper dose level of 5000 mg/kg body weight may be considered (see Appendix 2). For reasons of animal welfare concern, testing of animals in GHS Category 5 ranges (2000-5000 mg/kg) is discouraged and should only be considered when there is a strong likelihood that the results of such a test would have a direct relevance for protecting human or animal health or the environment.

1.5.3. Limit test

The limit test is primarily used in situations where the experimenter has information indicating that the test material is likely to be non-toxic, i.e., having toxicity only above regulatory limit doses. Information about the toxicity of the test material can be gained from knowledge about similar tested compounds or similar tested mixtures or products, taking into consideration the identity and percentage of components known to be of toxicological significance. In those situations where there is little or no information about its toxicity, or in which the test material is expected to be toxic, the main test should be performed.

A limit test at one dose level of 2000 mg/kg body weight may be carried out with six animals (three animals per step). Exceptionally a limit test at one dose level of 5000 mg/kg may be carried out with three animals (see Appendix 2). If test substance-related mortality is produced, further testing at the next lower level may need to be carried out.

1.6. OBSERVATIONS

Animals are observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first four hours, and daily thereafter, for a total of 14 days, except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead. However, the duration of observation should not be fixed rigidly. It should be determined by the toxic reactions, time of onset and length of recovery period, and may thus be extended when considered necessary. The times at which signs of toxicity appear and disappear are important, especially if there is a tendency for toxic signs to be delayed (12). All observations are systematically recorded with individual records being maintained for each animal.

Additional observations will be necessary if the animals continue to display signs of toxicity. Observations should include changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The principles and criteria summarised in the Humane Endpoints Guidance Document (9) should be taken into consideration. Animals found in a moribund condition and animals showing severe pain or enduring signs of severe distress should be humanely killed. When animals are killed for humane reasons or found dead, the time of death should be recorded as precisely as possible.

1.6.1. Body weight

Individual weights of animals should be determined shortly before the test substance is administered, and at least weekly thereafter. Weight changes should be calculated and recorded. At the end of the test surviving animals are weighed and humanely killed.

1.6.2. Pathology

All test animals (including those that die during the test or are removed from the study for animal welfare reasons) should be subjected to gross necropsy. All gross pathological changes should be recorded for each animal. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 or more hours may also be considered because it may yield useful information.

2. DATA

3. REPORTING

3.1. Test report

The test report must include the following information, as appropriate:

Test substance:

- physical nature, purity, and, where relevant, physico-chemical properties (including isomerisation),

- identification data, including CAS number.

Vehicle (if appropriate):

- justification for choice of vehicle, if other than water.

Test animals:

- species/strain used,

- microbiological status of the animals, when known,

- number, age, and sex of animals (including, where appropriate, a rationale for the use of males instead of females),

- source, housing conditions, diet, etc.

Test conditions:

- details of test substance formulation including details of the physical form of the material administered,

- details of the administration of the test substance including dosing volumes and time of dosing,

- details of food and water quality (including diet type/source, water source),

- the rationale for the selection of the starting dose.

Results:

- tabulation of response data and dose level for each animal (i.e. animals showing signs of toxicity including mortality; nature, severity, and duration of effects),

- tabulation of body weight and body weight changes,

- individual weights of animals at the day of dosing, in weekly intervals thereafter, and at the time of death or sacrifice,

- date and time of death if prior to scheduled sacrifice,

- time course of onset of signs of toxicity, and whether these were reversible for each animal,

- necropsy findings and histopathological findings for each animal, if available.

Discussion and interpretation of results.

Conclusions.

4. REFERENCES

(1) Roll R., Höfer-Bosse Th. And Kayser D (1986) New Perspectives in Acute Toxicity Testing of Chemicals. Toxicol. Lett., Suppl. 31, p. 86.

(2) Roll R., Riebschläger M., Mischke U. and Kayser D (1989) Neue Wege zur Bestimmung der akuten Toxizität von Chemikalien. Bundesgesundheitsblatt 32, p. 336-341.

(3) Diener W., Sichha L., Mischke U., Kayser D. and Schlede E (1994) The Biometric Evaluation of the Acute-Toxic-Class Method (Oral). Arch. Toxicol. 68, p. 559-610.

(4) Diener W., Mischke U., Kayser D. and Schlede E., (1995) The Biometric Evaluation of the OECD Modified Version of the Acute-Toxic-Class Method (Oral). Arch. Toxicol. 69, p. 729-734.

(5) Diener W., and Schlede E., (1999) Acute Toxicity Class Methods: Alterations to LD/LC50 Tests. ALTEX 16, p. 129-134.

(6) Schlede E., Mischke U., Roll R. and Kayser D., (1992). A National Validation Study of the Acute-Toxic- Class Method — An Alternative to the LD50 Test. Arch. Toxicol. 66, 455-470.

(7) Schlede E., Mischke U., Diener W. and Kayser D., (1994) The International Validation Study of the Acute-Toxic-Class Method (Oral). Arch. Toxicol. 69, p. 659-670.

(8) OECD, (2001) Guidance Document on Acute Oral Toxicity Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No 24. Paris.

(9) OECD, (2000) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph Series on Testing and Assessment No 19.

(10) OECD, (1998) Harmonised Integrated Hazard Classification System For Human Health And Environmental Effects Of Chemical Substances as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals in November 1998, Part 2, p. 11 [http://webnet1.oecd.org/oecd/pages/home/displaygeneral/0,3380, EN-documents-521-14-no-24-no-0,FF.html].

(11) Lipnick R. L., Cotruvo, J.A., Hill R. N., Bruce R. D., Stitzel K. A., Walker A. P., Chu I.; Goddard M., Segal L., Springer J. A. and Myers R. C. (1995) Comparison of the Up-and Down, Conventional LD50, and Fixed Dose Acute Toxicity Procedures. Fd. Chem. Toxicol 33, p. 223-231.

(12) Chan P.K. and A.W. Hayes. (1994). Chap. 16. Acute Toxicity and Eye Irritancy. Principles and Methods of Toxicology. Third Edition. A.W. Hayes, Editor. Raven Press, Ltd., New York, USA.

Appendix 1

PROCEDURE TO BE FOLLOWED FOR EACH OF THE STARTING DOSES

GENERAL REMARKS

For each starting dose, the respective testing schemes as included in this Appendix outline the procedure to be followed.

- Appendix 1 a: starting dose is 5 mg/kg bw,

- Appendix 1 b: starting dose is 50 mg/kg bw,

- Appendix 1 c: starting dose is: 300 mg/kg bw,

- Apendix 1 d: starting dose is: 2000 mg/kg bw.

Depending on the number of humanely killed or dead animals, the test procedure follows the indicated arrows.

Appendix 1A

TEST PROCEDURE WITH A STARTING DOSE OF 5 MG/KG BODY WEIGHT

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1

> 0 - 5

Category 2

> 5 - 50

Category 3

> 50 - 300

Category 4

> 300 - 2000

Category 5

> 2000 - 5000

∞

LD50 cut-off mg/kg b.w.

- per step 3 animals of a single sex (normally females) are used

- 0, 1, 2, 3: Number of moribund or dead animals at each step

- GHS: Globally Harmonised Classification System (mg/kg b.w.)

- ∞: unclassified

- Testing at 5000 mg/kg b.w.: see Appendix 2

+++++ TIFF +++++

Appendix 1B

TEST PROCEDURE WITH A STARTING DOSE OF 50 MG/KG BODY WEIGHT

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1

> 0 - 5

Category 2

> 5 - 50

Category 3

> 50 - 300

Category 4

> 300 - 2000

Category 5

> 2000 - 5000

∞

3 (at 50) at the 1st step

other

LD50 cut-off mg/kg b.w.

- per step 3 animals of a single sex (normally females) are used

- 0, 1, 2, 3: Number of moribund or dead animals at each step

- GHS: Globally Harmonised Classification System (mg/kg b.w.)

- ∞: unclassified

- Testing at 5000 mg/kg b.w.: see Appendix 2

+++++ TIFF +++++

Appendix1C

TEST PROCEDURE WITH A STARTING DOSE OF 300 MG/KG BODY WEIGHT

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1

> 0 - 5

Category 2

> 5 - 50

Category 3

> 50 - 300

Category 4

> 300 - 2000

Category 5

> 2000 - 5000

∞

3 (at 50) at the 1st step

other

3 (at 50) at the 1st step

other

LD50 cut-off mg/kg b.w.

- per step 3 animals of a single sex (normally females) are used

- 0, 1, 2, 3: Number of moribund or dead animals at each step

- GHS: Globally Harmonised Classification System (mg/kg b.w.)

- ∞: unclassified

- Testing at 5000 mg/kg b.w.: see Appendix 2

+++++ TIFF +++++

Appendix 1D

TEST PROCEDURE WITH A STARTING DOSE OF 2000 MG/KG BODY WEIGHT

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1

> 0 - 5

Category 2

> 5 - 50

Category 3

> 50 - 300

Category 4

> 300 - 2000

Category 5

> 2000 - 5000

∞

3 (at 50) at 1st step

other

3 (at 300) at 1st step

other

3 (at 2000) at 1st step

2 (at 2000) at 1st step

other

LD50 cut-off mg/kg b.w.

- per step 3 animals of a single sex (normally females) are used

- 0, 1, 2, 3: Number of moribund or dead animals at each step

- GHS: Globally Harmonised Classification System (mg/kg b.w.)

- ∞: unclassified

- Testing at 5000 mg/kg b.w.: see Appendix 2

+++++ TIFF +++++

Appendix 2

CRITERIA FOR CLASSIFICATION OF TEST SUBSTANCES WITH EXPECTED LD50 VALUES EXCEEDING 2000 MG/KG WITHOUT THE NEED FOR TESTING

Criteria for hazard Category 5 are intended to enable the identification of test substances which are of relatively low acute toxicity hazard but which, under certain circumstances may present a danger to vulnerable populations. These substances are anticipated to have an oral or dermal LD50 in the range of 2000-5000 mg/kg or equivalent doses for other routes. The test substance should be classified in the hazard category defined by: 2000 mg/kg < LD50 < 5000 mg/kg (Category 5 in the GHS) in the following cases:

(a) If directed to this category by any of the testing schemes of Appendix 1a-1d, based on mortality incidences;

(c) through extrapolation, estimation or measurement of data if assignment to a more hazardous class is not warranted; and

- reliable information is available indicating significant toxic effects in humans, or

- any mortality is observed when tested up to Category 4 values by the oral route, or

- where expert judgement confirms significant clinical signs of toxicity, when tested up to Category 4 values, except for diarrhoea, piloerection or an ungroomed appearance, or

- where expert judgement confirms reliable information indicating the potential for significant acute effects from the other animal studies.

TESTING AT DOSES ABOVE 2000 MG/KG

Recognising the need to protect animal welfare, testing of animals in Category 5 (5000 mg/kg) ranges is discouraged and should only be considered when there is a strong likelihood that results of such a test have a direct relevance for protecting human or animal health (10). No further testing should be conducted at higher dose levels.

When testing is required a dose of 5000 mg/kg, only one step (i.e. three animals) is required. If the first animal dosed dies, then dosing proceeds at 2000 mg/kg in accordance with the flowcharts in Appendix 1. If the first animal survives, two further animals are dosed. If only one of the three animals dies, the LD50 value is expected to exceed 5000 mg/kg. If both animals die, then dosing proceeds at 2000 mg/kg.

Appendix 3

TEST METHOD B.1 tris: Guidance on classififcation according to EU scheme to cover the transition period until full implementation of the Globally Harmonised Classification System (GHS) (taken from reference (8))

Start

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

GHS

Category 1

> 0 - 5

Category 2

> 5 - 50

Category 3

> 50 - 300

Category 4

> 300 - 2000

Category 5

> 2000 - 5000

∞

LD50 cut-off

mg/kg b. w.

EU/chemicals Liquid pesticides

EU solid pesticides

UN liquids

UN solids

Switzerland

US EPA crk

Japan PDSCA

Canada/WHMIS/US OSHA

US EPA pesticides

US CPSC

Canada pesticides

- per step 3 animals of a single sex (normally female) are used

- 0,1, 2, 3: Number of moribund or dead animals at each step

- ∞: unclassified

- GHS: Globally Harmonised Classification System (mg/kg b. w.)

+++++ TIFF +++++

Start

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

5 mg/kg 3 animals

50 mg/kg 3 animals

300 mg/kg 3 animals

2000 mg/kg 3 animals

GHS

Category 1 > 0 - 5

Category 2 > 5 - 50

Category 3 > 50 - 300

Category 4 > 300 - 2000

Category 5 > 2000 - 5000

(at 50)

other

LD50 cut-off

mg/kg b. w.

EU/chemicals Liquid pesticides

EU solid pesticides

UN liquids

UN solids

Switzerland

US EPA crk

Japan PDSCA

Canada/WHMIS/US OSHA

US EPA pesticides

US CPSC

Canada pesticides

- per step 3 animals of a single sex (normally female) are used

- 0,1, 2, 3: Number of moribund or dead animals at each step

- ∞: unclassified

- *: at first step

- GHS: Globally Harmonised Classification System (mg/kg b. w.)

+++++ TIFF +++++

Start

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

GHS

Category 1

> 0 - 5

Category 2

> 5 - 50

Category 3

> 50 - 300

Category 4

> 300 - 2000

Category 5

> 2000 - 5000

(at 50)

other

(at 300)

other

LD50 cut-off

mg/kg b. w.

EU/chemicals Liquid pesticides

EU solid pesticides

UN liquids

UN solids

Switzerland

US EPA crk

Japan PDSCA

Canada/WHMIS/US OSHA

US EPA pesticides

US CPSC

Canada pesticides

- per step 3 animals of a single sex (normally female) are used

- 0,1,2,3: Number of moribund or dead animals at each step

- ∞: unclassified

- *: at first step

- GHS: Globally Harmonised Classification System (mg/kg b. w.)

+++++ TIFF +++++

Start

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

5 mg/kg

3 animals

50 mg/kg

3 animals

300 mg/kg

3 animals

2000 mg/kg

3 animals

GHS

Category 1

> 0 - 5

Category 2

> 5 - 50

Category 3

> 50 - 300

Category 4

> 300 - 2000

Category 5

> 2000 - 5000

(at 50)

other

(at 300)

other

(at 2000)

other

LD50 cut-off

mg/kg b. w.

EU/chemicals Liquid pesticides

EU solid pesticides

UN liquids

UN solids

Switzerland

US EPA crk

Japan PDSCA

Canada/WHMIS/US OSHA

US EPA pesticides

US CPSC

Canada pesticides

- per step 3 animals of a single sex (normally female) are used

- 0, 1, 2, 3: Number of moribund or dead animals at each step

- ∞: unclassified

- *: at first step

- GHS: Globally Harmonised Classification System (mg/kg b. w.)

+++++ TIFF +++++

B.2. ACUTE TOXICITY (INHALATION)

1. METHOD

1.1. INTRODUCTION

It is useful to have preliminary information on the particle size distribution, the vapour pressure, the melting point, the boiling point, the flash point and explosivity (if applicable) of the substance.

See also General introduction Part B (A).

1.2. DEFINITIONS

See General introduction Part B (B).

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

Several groups of experimental animals are exposed for a defined period to the test substance in graduated concentrations, one concentration being used per group. Subsequently observations of effects and deaths are made. Animals, which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.

Animals showing severe and enduring signs of distress and pain may need to be humanely killed. Dosing test substances in a way known to cause marked pain and distress due to corrosive or severe irritating properties need not be carried out.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

The animals are kept under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test healthy young animals are randomiseds and assigned to the required number of groups. They need not be subjected to simulated exposure unless this is indicated by the type of exposure apparatus being used.

Solid test substances may need to be micronised in order to achieve particles of an appropriate size.

Where necessary a suitable vehicle may be added to the test substance to help generate an appropriate concentration of the test substance in the atmosphere and a vehicle control group should then be used. If a vehicle or other additives are used to facilitate dosing, they should be known not to produce toxic effects. Historical data can be used if appropriate.

1.6.2. Test conditions

1.6.2.1. Experimental animals

Unless there are contra-indications the rat is the preferred species. Commonly used laboratory strains should be employed. For each sex, at the start of the test the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.

1.6.2.2. Number and sex

At least 10 rodents (five female and five male) are used at each concentration level. The females should be nulliparous and non-pregnant.

Note: in acute toxicity tests with animals of a higher order than rodents, the use of smaller numbers should be considered. Doses should be carefully selected, and every effort should be made not to exceed moderately toxic doses. In such tests administration of lethal doses of the test substance should be avoided.

1.6.2.3. Exposure concentrations

These should be sufficient in number, at least three, and spaced appropriately to produce test groups with a range of toxic effects and mortality rates. The data should be sufficient to produce a concentration mortality curve and, where possible, permit an acceptable determination of an LC50.1.6.2.4. Limit test

1.6.2.4. Limit test

If an exposure of five male and five female test animals to 20 mg per litre of a gas or 5 rug per litre of an aerosol or a particulate for four hours (or where this is not possible due to the physical or chemical, including explosive, properties of the test substance, the maximum attainable concentration) produces no compound related mortality within 14 days further testing may not be considered necessary (18th ATP, dir. 93/21/EEC, Ll10/93)

1.6.2.5. Exposure time

The period of exposure should be four hours.

1.6.2.6. Equipment

The animals should be tested with inhalation equipment designed to sustain a dynamic airflow of at least 12 air changes per hour, to ensure an adequate oxygen content and an evenly distributed exposure atmosphere. Where a chamber is used its design should minimise crowding of the test animals and maximise their exposure by inhalation to the test substance. As a general rule to ensure stability of a chamber atmosphere the total "volume" of the test animals should not exceed 5 % of the volume of the test chamber. Oro-nasal, head only, or whole body individual chamber exposure may be used; the first two will help to minimise the uptake of the test substance by other routes.

1.6.2.7. Observation period

The observation period should be at least 14 days. However, the duration of observations should not be rigidly fixed. It should be determined by the toxic reactions, their rate of onset and the length of the recovery period; it may thus be extended when considered necessary. The time at which signs of toxicity appear and disappear and the time of death are important, especially if there is a tendency for deaths to be delayed.

1.6.3. Procedure

Shortly before exposure, the animals are weighed, and then exposed to the test concentration in the designated apparatus for a period of four hours, after equilibration of the chamber concentration. Time for equilibration should be short. The temperature at which the test is performed should be maintained at 22 ± 3 oC. Ideally the relative humidity should be maintained between 30 % and 70 %, but in certain instances (e.g. tests of some aerosols) this may not be practicable. Maintenance of a slight negative pressure inside the chamber (≥ 5 mm of water) will prevent leakage of the test substance into the surrounding area. Food and water should be withheld during exposure. Suitable systems for the generation and monitoring of the test atmosphere should be used. The system should ensure that stable exposure conditions are achieved as rapidly as possible. The chamber should be designed and operated in such a way that a homogeneous distribution of the test atmosphere within the chamber is maintained.

Measurements or monitoring should be made:

(a) of the rate of air flow (continuously);

(b) of the actual concentration of the test substance measured in the breathing zone at least three times during exposure (some atmospheres, e.g. aerosols at high concentrations, may need more frequent monitoring). During the exposure period the concentration should not vary by more than ± 15 % of the mean value. However in the case of some aerosols, this level of control may not be achievable and a wider range would then be acceptable. For aerosols, particle size analysis should be performed as often as necessary (at least once per test group);

During and following exposure, observations are made and recorded systematically; individual records should be maintained for each animal. Observations should be made frequently during the first day. A careful clinical examination should be made at least once each working day, other observations should be made daily with appropriate actions taken to minimise loss of animals from the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.

Observations should include changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Particular attention should be directed to observation of respiratory behaviour, tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of death should be recorded as precisely as possible. Individual weights of animals should be determined weekly after exposure, and at death.

Animals that die during the test and those surviving at the termination of the test are subjected to necropsy with particular reference to any changes in the upper and lower respiratory tract. All gross pathological changes should be recorded. Where indicated, tissues should be taken for histopathological examination.

2. DATA

Data should be summarised in tabular form showing for each test group the number of animals at the start of the test, time of death of individual animals, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings. Changes in weight must be calculated and recorded when survival exceeds one day. Animals, which are humanely killed due to compound-related distress and pain are recorded as compound-related deaths. The LC50 should be determined by a recognised method. Data evaluation should include the relationship, if any, between the animal's exposure to the test substance and the incidence and severity of all abnormalities, including behavioural and clinical abnormalities, gross lesions, body weight changes, mortality and any other toxic effects.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, include the following information:

- species, strain, source, environmental conditions, diet, etc.,

- test conditions: description of exposure apparatus, including design, type, dimensions, source of air, system for generating aerosols, method of conditioning air and the method of housing animals in a test chamber when this is used. The equipment for measuring temperature, humidity, and aerosol concentrations and particle size distribution should be described.

Exposure data

These should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and shall, if possible, include:

(a) airflow rates through the inhalation equipment;

(b) temperature and humidity of the air;

(d) nature of vehicle, if used;

(e) actual concentrations in test breathing zone;

(f) The mass median aerodynamic diameter (Mmad) and the geometric standard deviation (GSD);

(g) equilibration period;

(h) exposure period;

- tabulation of response data by sex and exposure level (i.e. number of animals that died or were killed during the test, number of animals showing signs of toxicity, number of animals exposed),

- time of death during or following exposure, reasons and criteria used for humane killing of animals,

- all observations,

- LC50 value for each sex determined at the end of the observation period (with method of calculation specified),

- 95 % confidence interval for the LC50 (where this can be provided),

- dose/mortality curve and slope (where permitted by the method of determination),

- necropsy findings,

- any histopathological findings,

- discussions of the results (particular attention should be given to the effect that humane killing of animals during the test may have on the calculated LC50 value),

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B (D).

4. REFERENCES

See General introduction Part B (E).

B.3. ACUTE TOXICITY (DERMAL)

1. METHOD

1.1. INTRODUCTION

See General introduction Part B (A).

1.2. DEFINITION

See General introduction Part B (B).

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The test substance is applied to the skin in graduated doses to several groups of experimental animals, one dose being used per group. Subsequently, observations of effects and deaths are made. Animals, which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

The animals are kept in their experimental cages under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test, healthy young adult animals are randomised and assigned to the treatment groups. Approximately 24 hours before the test, fur should be removed by clipping or shaving from the dorsal area of the trunk of the animals. When clipping or shaving the fur, care must be taken to avoid abrading the skin which could alter its permeability. Not less than 10 % of the body surface should be clear for the application of the test substance. When testing solids, which may be pulverised if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. When a vehicle is used, the influence of the vehicle on penetration of skin by the test substance should be taken into account. Liquid test substances are generally used undiluted.

1.6.2. Test conditions

1.6.2.1. Experimental animals

The adult rat or rabbit may be used. Other species may be used but their use would require justification. Commonly used laboratory strains should be employed. For each sex, at the start of the test the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.

1.6.2.2. Number and sex

At least five animals are used at each dose level. They should all be of the same sex. If females are used, they should be nulliparous and non-pregnant. Where information is available demonstrating that a sex is markedly more sensitive, animals of this sex should be dosed.

Note: in acute toxicity tests with animals of a higher order than rodents, the use of smaller numbers should be considered. Doses should be carefully selected, and every effort should be made not to exceed moderately toxic doses. In such tests, administration of lethal doses of the test substance should be avoided.

1.6.2.3. Dose levels

These should be sufficient in number, at least three, and spaced appropriately to produce test groups with a range of toxic effects and mortality rates. Any irritant or corrosive effects should be taken into account when deciding on dose levels. The data should be sufficient to produce a dose/response curve and, where possible, permit an acceptable determination of the LD50.

1.6.2.4. Limit test

A limit test at one dose level of at least 2000 mg/kg bodyweight may be carried out in a group of five male and five female animals, using the procedures described above. If compound-related mortality is produced, a full study may need to be considered.

1.6.2.5. Observation period

The observation period should be at least 14 days. However, the duration of observation should not be rigidly fixed. It should be determined by the toxic reactions, their rate of onset and the length of the recovery period; it may thus be extended when considered necessary. The time at which signs of toxicity appear and disappear, their duration and the time of death are important, especially if there is a tendency for deaths to be delayed.

1.6.3. Procedure

Animals should be caged individually. The test substance should be applied uniformly over an area, which is approximately 10 % of the total body surface area. With highly toxic substances the surface area covered may be less but as much of the area should be covered with a layer as thin and uniform as possible.

Test substances should be held in contact with the skin with a porous gauze dressing and non-irritating tape throughout a 24-hour exposure period. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance but complete immobilisation is not a recommended method.

At the end of the exposure period, residual test substance should be removed, where practicable, using water or some other appropriate method of cleansing the skin.

Observations should be recorded systematically as they are made. Individual records should be maintained for each animal. Observations should be made frequently during the first day. A careful clinical examination should be made at least once each working day, other observations should be made daily with appropriate actions taken to minimise loss of animals to the study, e.g. necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals.

Observations should include changes in fur, treated skin, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Particular attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of death must be recorded as precisely as possible. Animals that die during the test and those surviving at the termination of the test are subjected to necropsy. All gross pathological changes should be recorded. Where indicated, tissues should be taken for histopathological examination.

Assessment of toxicity in the other sex

After completion of the study in one sex, at least one group of five animals of the other sex is dosed to establish that animals of this sex are not markedly more sensitive to the test substance. The use of fewer animals may be justified in individual circumstances. Where adequate information is available to demonstrate that animals of the sex tested are markedly more sensitive, testing in animals of the other sex may be dispensed with.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, time of death of individual animals, number of animals displaying other signs of toxicity, description of toxic effects and necropsy findings. Individual weights of animals should be determined and recorded shortly before the test substance is applied, weekly thereafter, and at death; changes in weight should be calculated and recorded when survival exceeds one day. Animals, which are humanely killed due to compound-related distress and pain are recorded as compound-related deaths. The LD50 should be determined by a recognised method.

Data evaluation should include an evaluation of relationships, if any, between the animal's exposure to the test substance and the incidence and severity of all abnormalities, including behavioural and clinical abnormalities, gross lesions, body weight changes, mortality, and any other toxicological effects.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, include the following information:

- species, strain, source, environmental conditions, diet, etc.,

- test conditions (including method of skin cleansing and type of dressing: occlusive or not occlusive),

- dose levels (with vehicle, if used, and concentrations),

- sex of animals dosed,

- tabulation of response data by sex and dose level (i.e. number of animals that died or were killed during the test, number of animals showing signs of toxicity, number of animals exposed),

- time of death after dosing, reasons and criteria used for humane killing of animals,

- all observations,

- LD50 value for the sex subjected to a full study, determined at 14 days with the method of determination specified,

- 95 % confidence interval for the LD50 (where this can be provided),

- dose/mortality curve and slope where permitted by the method of determination,

- necropsy findings,

- any histopathological findings,

- results of any test on the other sex,

- discussion of results (particular attention should be given to the effect that humane killing of animals during the test may have on the calculated LD50 value),

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B (D).

4. REFERENCES

See General introduction Part B (E).

B.4. ACUTE TOXICITY: DERMAL IRRITATION/CORROSION

1. METHOD

This method is equivalent to the OECD TG 404 (2002).

1.1. INTRODUCTION

In the preparation of this updated method special attention was given to possible improvements in relation to animal welfare concerns and to the evaluation of all existing information on the test substance in order to avoid unnecessary testing in laboratory animals. This method includes the recommendation that prior to undertaking the described in vivo test for corrosion/irritation of the substance, a weight-of-the-evidence analysis be performed on the existing relevant data. Where insufficient data are available, they can be developed through application of sequential testing (1). The testing strategy includes the performance of validated and accepted in vitro tests and is provided as an Appendix to this method. In addition, where appropriate, the successive, instead of simultaneous, application of the three test patches to the animal in the initial in vivo test is recommended.

In the interest of both sound science and animal welfare, in vivo testing should not be undertaken until all available data relevant to the potential dermal corrosivity/irritation of the substance have been evaluated in a weight-of-the-evidence analysis. Such data will include evidence from existing studies in humans and/or laboratory animals, evidence of corrosivity/irritation of one or more structurally related substances or mixtures of such substances, data demonstrating strong acidity or alkalinity of the substance (2)(3), and results from validated and accepted in vitro or ex vivo tests (4)(5)(5a). This analysis should decrease the need for in vivo testing for dermal corrosivity/irritation of substances for which sufficient evidence already exists from other studies as to those two endpoints.

A preferred sequential testing strategy, which includes the performance of validated and accepted in vitro or ex vivo tests for corrosion/irritation, is included as an Appendix to this Method. The strategy was developed at, and unanimously recommended by the participants of, an OECD workshop (6), and has been adopted as the recommended testing strategy in the Globally Harmonised System for the Classification of Chemical Substances (GHS) (7). It is recommended that this testing strategy be followed prior to undertaking in vivo testing. For new substances it is the recommended a stepwise testing approach for developing scientifically sound data on the corrosivity/irritation of the substance. For existing substances with insufficient data on dermal corrosion/irritation, the strategy should be used to fill missing data gaps. The use of a different testing strategy or procedure, or a decision not to use a stepwise testing approach, should be justified.

If a determination of corrosivity or irritation cannot be made using a weight-of-the-evidence analysis, consistent with the sequential testing strategy, an in vivo test should be considered (see Appendix).

1.2. DEFINITIONS

Dermal irritation: is the production of reversible damage of the skin following the application of a test substance for up to four hours.

Dermal corrosion: is the production of irreversible damage of the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to four hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14 days, by discoloration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to evaluate questionable lesions.

1.3. PRINCIPLE OF THE TEST METHOD

The substance to be tested is applied in a single dose to the skin of an experimental animal; untreated skin areas of the test animal serve as the control. The degree of irritation/corrosion is read and scored at specified intervals and is further described in order to provide a complete evaluation of the effects. The duration of the study should be sufficient to evaluate the reversibility or irreversibility of the effects observed.

Animals showing continuing signs of severe distress and/or pain at any stage of the test should be humanely killed, and the substance assessed accordingly. Criteria for making the decision to humanely kill moribund and severely suffering animals can be found in reference (8).

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparation for the in vivo test

1.4.1.1. Selection of animal species

The albino rabbit is the preferable laboratory animal and healthy young adult rabbits are used. A rationale for using other species should be provided.

1.4.1.2. Preparation of the animals

Approximately 24 hours before the test, fur should be removed by closely clipping the dorsal area of the trunk of the animals. Care should be taken to avoid abrading the skin, and only animals with healthy, intact skin should be used.

Some strains of rabbit have dense patches of hair that are more prominent at certain times of the year. Such areas of dense hair growth should not be used as test sites.

1.4.1.3. Housing and feeding conditions

Animals should be individually housed. The temperature of the experimental animal room should be 20 oC (± 3 oC) for rabbits. Although the relative humidity should be at least 30 % and preferably not exceed 70 %, other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unrestricted supply of drinking water.

1.4.2. Test procedure

1.4.2.1. Application of the test substance

The test substance should be applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which is held in place with non-irritating tape. In cases in which direct application is not possible (e.g. liquids or some pastes), the test substance should first be applied to the gauze patch, which is then applied to the skin. The patch should be loosely held in contact with the skin by means of a suitable semi-occlusive dressing for the duration of the exposure period. If the test substance is applied to the patch, it should be attached to the skin in such a manner that there is good contact and uniform distribution of the substance on the skin. Access by the animal to the patch and ingestion or inhalation of the test substance should be prevented.

Liquid test substances are generally used undiluted. When testing solids (which may be pulverised, if considered necessary), the test substance should be moistened with the smallest amount of water (or, where necessary, of another suitable vehicle) sufficient to ensure good skin contact. When vehicles other than water are used, the potential influence of the vehicle on irritation of the skin by the test substance should be minimal, if any.

At the end of the exposure period, which is normally four hours, residual test substance should be removed, where practicable, using water or an appropriate solvent without altering the existing response or the integrity of the epidermis.

1.4.2.2. Dose level

A dose of 0,5 ml. of liquid or 0,5 g of solid or paste is applied to the test site.

1.4.2.3. Initial test (in vivo dermal irritation/corrosion test using one animal)

It is strongly recommended that the in vivo test be performed initially using one animal, especially when the substance is suspected to have corrosion potential. This is in accordance with the sequential testing strategy (see Appendix 1).

When a substance has been judged to be corrosive on the basis of a weight-of-the-evidence analysis, no further animal testing is needed. For most substances suspected of being corrosive, further in vivo testing is normally not necessary. However, in those cases where additional data are felt warranted because of insufficient evidence, limited animal testing may be carried out using the following approach: up to three tests patches are applied sequentially to the animal. The first patch is removed after three minutes. If no serious skin reaction is observed, a second patch is applied and removed after one hour. If the observations at this stage indicate that exposure can humanely be allowed to extend to four hours, a third patch is applied and removed after four hours, and the response is graded.

If a corrosive effect is observed after any of the three sequential exposures, the test is immediately terminated. If a corrosive effect is not observed after the last patch is removed, the animal is observed for 14 days, unless corrosion develops at an earlier time point.

In those cases in which the test substance is not expected to produce corrosion but may be irritating, a single patch should be applied to one animal for four hours.

1.4.2.4. Confirmatory test (in vivo dermal irritation test with additional animals)

If a corrosive effect is not observed in the initial test, the irritant or negative response should be confirmed using up to two additional animals, each with one patch, for an exposure period of four hours. If an irritant effect is observed in the initial test, the confirmatory test may be conducted in a sequential manner, or by exposing two additional animals simultaneously. In the exceptional case, in which the initial test is not conducted, two or three animals may be treated with a single patch, which is removed after four hours. When two animals are used, if both exhibit the same response, no further testing is needed. Otherwise, the third animal is also tested. Equivocal responses may need to be evaluated using additional animals.

1.4.2.5. Observation period

The duration of the observation period should be sufficient to evaluate fully the reversibility of the effects observed. However, the experiment should be terminated at any time that the animal shows continuing signs of severe pain or distress. To determine the reversibility of effects, the animals should be observed up to 14 days after removal of the patches. If reversibility is seen before 14 days, the experiment should be terminated at that time.

1.4.2.6. Clinical observations and grading of skin reactions

All animals should be examined for signs of erythema and oedema, and the responses scored at 60 minutes, and then at 24, 48 and 72 hours after patch removal. For the initial test in one animal, the test site is also examined immediately after the patch has been removed. Dermal reactions are graded and recorded according to the grades in the Table below. If there is damage to skin which cannot be identified as irritation or corrosion at 72 hours, observations may be needed until day 14 to determine the reversibility of the effects. In addition to the observation of irritation, all local toxic effects, such as defatting of the skin, and any systemic adverse effects (e.g. effects on clinical signs of toxicity and body weight), should be fully described and recorded. Histopathological examination should be considered to clarify equivocal responses.

The grading of skin responses is necessarily subjective. To promote harmonisation in grading of skin response and to assist testing laboratories and those involved in making and interpreting the observations, the personnel performing the observations need to be adequately trained in the scoring system used (see Table below). An illustrated guide for grading skin irritation and other lesions could be helpful (9).

2. DATA

2.1. PRESENTATION OF RESULTS

Study results should be summarised in tabular form in the final test report and should cover all items listed in section 3.1.

2.2. EVALUATION OF RESULTS

The dermal irritation scores should be evaluated in conjunction with the nature and severity of lesions, and their reversibility or lack of reversibility. The individual scores do not represent an absolute standard for the irritant properties of a material, as other effects of the test material are also evaluated. Instead, individual scores should be viewed as reference values, which need to be evaluated in combination with all other observations from the study.

Reversibility of dermal lesions should be considered in evaluating irritant responses. When responses such as alopecia (limited area), hyperkeratosis, hyperplasia and scaling, persist to the end of the 14-day observation period, the test substance should be considered an irritant.

3. REPORTING

3.1. TEST REPORT

The test report must include the following information:

Rationale for in vivo testing: weight-of-evidence analysis of pre-existing test data, including results from sequential testing strategy:

- description of relevant data available from prior testing,

- data derived at each stage of testing strategy,

- description of in vitro tests performed, including details of procedures, results obtained with test/reference substances,

- weight-of-the-evidence analysis for performing in vivo study.

Test substance:

- identification data (e.g. CAS number, source, purity, known impurities, lot number),

- physical nature and physicochemical properties (e.g. pH, volatility, solubility, stability),

- if mixture, composition and relative percentages of components.

Vehicle:

- identification, concentration (where appropriate), volume used,

- justification for choice of vehicle.

Test animals:

- species/strain used, rationale for using animals other than albino rabbit,

- number of animals of each sex,

- individual animal weights at start and conclusion of test,

- age at start of study,

- source of animals, housing conditions, diet, etc.

Test conditions:

- technique of patch site preparation,

- details of patch materials used and patching technique,

- details of test substance preparation, application, and removal.

Results:

- tabulation of irritation/corrosion response scores for each animal at all time points measured,

- descriptions of all lesions observed,

- narrative description of nature and degree of irritation or corrosion observed, and any histopathological findings,

- description of other adverse local (e.g. defatting of skin) and systemic effects in addition to dermal irritation or corrosion.

- Discussion of results

4. REFERENCES

(1) Barratt, M.D., Castell, J.V., Chamberlain, M., Combes, R.D., Dearden, J.C., Fentem, J.H., Gerner, I., Giuliani, A., Gray, T.J.B., Livingston, D.J., Provan, W.M., Rutten, F.A.J.J.L., Verhaar, H.J.M., Zbinden, P. (1995) The Integrated Use of Alternative Approaches for Predicting Toxic Hazard. ECVAM Workshop Report 8. ATLA 23, p. 410-429.

(2) Young, J.R., How, M.J., Walker, A.P., Worth W.M.H. (1988) Classification as Corrosive or Irritant to Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on Animals. Toxicollogy In Vitro, 2, p. 19-26.

(3) Worth, A.P., Fentem, J.H., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Liebsch, M. (1998) Evaluation of the proposed OECD Testing Strategy for skin corrosion. ATLA 26, p. 709-720.

(4) ECETOC (1990) Monograph No 15, "Skin Irritation", European Chemical Industry, Ecology and Toxicology Centre, Brussels.

(5) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsail, D.J., Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology In Vitro 12, p. 483-524.

(5a) Testing Method B.40 Skin Corrosion.

(6) OECD (1996) OECD Test Guidelines Programme: Final Report of the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held in Solna, Sweden, 22-24 January 1996 (http://www.oecd1.org/ehs/test/background.htm).

(7) OECD (1998) Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www.oecd1.org/ehs/Class/HCL6.htm).

(8) OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Environmental Health and Safety Publications. Series on Testing and Assessment No 19 (http://www.oecd1.org/ehs/test/monos.htm).

(9) EPA (1990). Atlas of Dermal Lesions, (20T-2004). United States Environmental Protection Agency, Office of Pesticides and Toxic Substances, Washington, DC, August 1990.

[Available from OECD Secretariat upon request].

Table I

GRADING OF SKIN REACTIONS

Erythema and Eschar formation

No erythema … | 0 |

Very slight erythema (barely perceptible) … | 1 |

Well defined erythema … | 2 |

Moderate to severe erythema … | 3 |

Severe erythema (beef redness) to eschar formation preventing grading of erythema … | 4 |

Maximum possible: 4

Oedema formation

No oedema … | 0 |

Very slight oedema (barely perceptible) … | 1 |

Slight oedema (edges of area well defined by definite raising) … | 2 |

Moderate oedema (raised approximately 1 mm) … | 3 |

Severe oedema (raised more than 1 mm and extending beyond area of exposure) … | 4 |

Maximum possible: 4

Histopathological examination may be carried out to clarify equivocal responses.

Appendix

A Sequential Testing Strategy for Dermal Irritation and Corrosion

GENERAL CONSIDERATIONS

In the interest of sound science and animal welfare, it is important to avoid the unnecessary use of animals and to minimise any testing that is likely to produce severe responses in animals. All information on a substance relevant to its potential skin corrosivity/irritancy should be evaluated prior to considering in vivo testing. Sufficient evidence may already exist to classify a test substance as to its dermal corrosion or irritation potential without the need to conduct testing in laboratory animals. Therefore, utilising a weight-of-the-evidence analysis and a sequential testing strategy, will minimise the need for in vivo testing, especially if the substance is likely to produce severe reactions.

It is recommended that a weight-of-the-evidence analysis be used to evaluate existing information regarding the skin irritation and corrosion of substances to determine whether additional studies, other than in vivo dermal studies, should be performed to help characterise such potential. Where further studies are needed, it is recommended that the sequential testing strategy be utilised to develop the relevant experimental data. For substances which have no testing history, the sequential testing strategy should be utilised to develop the data set needed to evaluate its dermal corrosion/irritation potential. The testing strategy described in this Appendix was developed at an OECD workshop (1) and was later affirmed and expanded in the Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, in November 1998 (2).

Although this sequential testing strategy is not an integral part of testing method B.4, it expresses the recommended approach for the determination of skin irritation/corrosion characteristics. This approach represents both best practice and an ethical benchmark for in vivo testing for skin irritation/corrosion. The testing method provides guidance for the conduct of the in vivo test and summarises the factors that should be addressed before initiating such a test. The strategy provides an approach for the evaluation of existing data on the skin irritation/corrosion properties of test substances and a tiered approach for the generation of relevant data on substances for which additional studies are needed, or for which no studies have been performed. It also recommends the performance of validated and accepted in vitro or ex vivo tests for skin corrosion/irritation under specific circumstances.

DESCRIPTION OF THE EVALUATION AND TESTING STRATEGY

Prior to undertaking tests as part of the sequential testing strategy (Figure), all available information should be evaluated to determine the need for in vivo skin testing. Although significant information might be gained from the evaluation of single parameters (e.g. extreme pH), the totality of existing information should be considered. All relevant data on the effects of the substance in question, or its analogues, should be evaluated in making a weight-of-the-evidence decision, and a rationale for the decision should be presented. Primary emphasis should be placed upon existing human and animal data on the substance, followed by the outcome of in vitro or ex vivo testing. In vivo studies of corrosive substances should be avoided whenever possible. The factors considered in the testing strategy include:

Evaluation of existing human and animal data (Step 1). Existing human data, e.g. clinical or occupational studies and case reports, and/or animal test data, e.g. from single or repeated dermal exposure toxicity studies, should be considered first, because they provide information directly related to effects on the skin. Substances with known irritancy or corrosivity, and those with clear evidence of non-corrosivity or non-irritancy, need not be tested in in vivo studies.

Analysis of structure activity relationships (SAR) (Step 2). The results of testing of structurally related substances should be considered, if available. When sufficient human and/or animal data are available on structurally related substances or mixtures of such substances to indicate their skin corrosion/irritancy potential, it can be presumed that the test substance being evaluated will produce the same responses. In those cases, the test substance may not need to be tested. Negative data from studies of structurally related substances or mixtures of such substances do not constitute sufficient evidence of non-corrosivity/non-irritancy of a substance under the sequential testing strategy. Validated and accepted SAR approaches should be used to identify both dermal corrosion and irritation potential.

Physicochemical properties and chemical reactivity (Step 3). Substances exhibiting pH extremes such as ≤ 2,0 and ≥ 11,5 may have strong local effects. If extreme pH is the basis for identifying a substance as corrosive to skin, then its acid/alkali reserve (or buffering capacity) may also be taken into consideration (3)(4). If the buffering capacity suggests that a substance may not be corrosive to the skin, then further testing should be undertaken to confirm this, preferably by the use of a validated and accepted in vitro or ex vivo test (see steps 5 and 6).

Dermal toxicity (Step 4). If a chemical has proven to be very toxic by the dermal route, an in vivo dermal irritation/corrosion study may not be practicable because the amount of test substance normally applied could exceed the very toxic dose and, consequently result in the death or severe suffering of the animals. In addition, when dermal toxicity studies utilising albino rabbits have already been performed up to the limit dose level of 2000 mg/kg body weight or higher, and no dermal irritation or corrosion has been seen, additional testing for skin irritation/corrosion may not be needed. A number of considerations should be borne in mind when evaluating acute dermal toxicity in previously performed studies. For example, reported information on dermal lesions may be incomplete. Testing and observations may have been made on a species other than the rabbit, and species may differ widely in sensitivity of their responses. Also the form of test substance applied to animals may not have been suitable for assessment of skin irritation/corrosion (e.g., dilution of substances for testing dermal toxicity (5). However, in those cases in which well-designed and conducted dermal toxicity studies have been performed in rabbits, negative findings may be considered sufficient evidence that the substance is not corrosive or irritating.

Results from in vitro or ex vivo tests (Steps 5 and 6). Substances that have demonstrated corrosive or severe irritant properties in a validated and accepted in vitro or ex vivo test (6)(7) designed for the assessment of these specific effects, need not be tested in animals. It can be presumed that such substances will produce similar severe effects in vivo.

In vivo test in rabbits (Steps 7 and 8). Should a weight-of the-evidence decision be made to conduct in vivo testing, it should begin with an initial test using one animal. If the results of this test indicate the substance to be corrosive to the skin, further testing should not be performed. If a corrosive effect is not observed in the initial test, the irritant or negative response should be confirmed using up to two additional animals for an exposure period of four hours. If an irritant effect is observed in the initial test, the confirmatory test may be conducted in a sequential manner, or by exposing the two additional animals simultaneously.

REFERENCES

(1) OECD, (1996) Test Guidelines Programme: Final Report on the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held on Solna, Sweden, 22-24 January 1996 (http://www1.oecd.org/ehs/test/background.htm).

(2) OECD, (1998) Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www1.oecd.org/ehs/Class/HCL6.htm).

(3) Worth, A.P., Fentem J.H., Balls M., Botham P.A., Curren R.D., Earl L.K., Esdaile D.J., Liebsch M., (1998). An Evaluation of the Proposed OECD Testing Strategy for Skin Corrosion. ATLA 26, p. 709-720.

(4) Young, J.R., How, M.J., Walker, A.P., Worth, W.M.H., (1988). Classification as Corrosive or Irritant to Skin of Preparations Containing Acidic or Alkaline Substances, Without Testing on Animals. Toxicology In Vitro, 2(1), p. 19-26.

(5) Patil, S.M., Patrick, E., Maibach, H.I. (1996) Animal, Human, and In Vitro Test Methods for Predicting Skin Irritation, in: Francis N. Marzulli and Howard I. Maibach (editors): Dermatotoxicology. Fifth Edition ISBN 1-56032-356-6, Chapter 31, p. 411-436.

(6) Testing Method B.40.

(7) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology In Vitro 12, p. 483-524.

Figure

TESTING AND EVALUATION STRATEGY FOR DERMAL IRRITATION/CORROSION

Activity

Finding

Conclusion

Corrosive

Apical endpoint; considered corrosive. No testing is needed.

Irritating

Apical endpoint; considered to be an irritant. No testing is needed.

Not corrosive/not irritating

Apical endpoint; considered not corrosive or irritating. No testing required.

Existing human and/or animal data showing effects on skin or mucous membranes

↓

No information available, or available information is not conclusive

↓

Perform SAR evaluation for skin corrosion/irritation

Predict severe damage to skin

Considered corrosive. No testing is needed.

Predict irritation to skin

Considered an irritant. No testing is needed.

↓

No predictions can be made, or predictions are not conclusive or negative

↓

Measure pH (consider buffering capacity, if relevant)

pH ≤ 2 or ≥ 11.5 (with high buffering capacity, if relevant)

Assume corrosivity. No testing is needed.

↓

2 < pH < 11.5, or pH ≤ 2.0 or ≥ 11.5 with low/no buffering capacity, if relevant

↓

Evaluate systemic toxicity data via dermal route (1)

Very toxic

No further testing is needed.

Not corrosive or irritating when tested to limit dose of 2000 mg/kg body weight or higher, using rabbits

Assume not corrosive or irritating. No further testing is needed.

↓

Such information is not available or is non-conclusive

↓

Perform validated and accepted in vitro or ex vivo test for skin corrosion

Corrosive response

Assume corrosivity in vivo. No further testing is needed.

↓

Substance is not corrosive

↓

Perform validated and accepted in vitro or ex vivo test for skin irritation

Irritant response

Assume irritancy in vivo. No further testing is needed.

↓

Validated in vitro or ex vivo testing methods for skin irritation are not yet available or substance is not an irritant

↓

Perform initial in vivo rabbit test using one animal

Severe damage to skin

Considered corrosive. No further testing is needed.

↓

No severe damage

↓

Perform confirmatory test using one or two additional animals

Corrosive or irritating

Considered corrosive or irritating. No further testing is needed

Not corrosive or irritating

Considered not corrosive or irritating. No further testing needed

(1) can be considered before Steps 2 and 3.

+++++ TIFF +++++

B.5. ACUTE TOXICITY: EYE IRRITATION/CORROSION

1. METHOD

This method is equivalent to the OECD TG 405 (2002)

1.1. INTRODUCTION

In the preparation of this updated method special attention was given to possible improvements through the evaluation of all existing information on the test substance in order to avoid unnecessary testing in laboratory animals and thereby address animal welfare concerns. This method includes the recommendation that prior to undertaking the described in vivo test for acute eye irritation/corrosion, a weight-of-the-evidence analysis be performed (1) on the existing relevant data. Where insufficient data are available, it is recommended that they be developed through application of sequential testing (2)(3). The testing strategy includes the performance of validated and accepted in vitro tests and is provided as an Appendix to the testing method. In addition, the use of an in vivo dermal irritation/corrosion test to predict eye corrosion prior to consideration of an in vivo eye test is recommended.

In the interest of both sound science and animal welfare, in vivo testing should not be considered until all available data relevant to the potential eye corrosivity/irritation of the substance has been evaluated in a weight-of-the-evidence analysis. Such data will include evidence from existing studies in humans and/or laboratory animals, evidence of corrosivity/irritation of one or more structurally related substances or mixtures of such substances, data demonstrating high acidity or alkalinity of the substance (4)(5), and results from validated and accepted in vitro or ex vivo tests for skin corrosion and irritation (6)(6a). The studies may have been conducted prior to, or as a result of, a weight-of-the-evidence analysis.

For certain substances, such an analysis may indicate the need for in vivo studies of the ocular corrosion/irritation potential of the substance. In all such cases, before considering the use of the in vivo eye test, preferably a study of the in vivo dermal effects of the substance should be conducted first and evaluated in accordance with testing method B.4 (7). The application of a weight-of-the-evidence analysis and the sequential testing strategy should decrease the need for in vivo testing for eye corrosivity/irritation of substances for which sufficient evidence already exists from other studies. If a determination of eye corrosion or irritation potential cannot be made using the sequential testing strategy, even after the performance of an in vivo study of dermal corrosion and irritation, an in vivo eye corrosion/irritation test may be performed.

A preferred sequential testing strategy, which includes the performance of validated in vitro or ex vivo tests for corrosion/irritation, is included in the Appendix to this testing method. The strategy was developed at, and unanimously recommended by the participants of, an OECD workshop (8), and has been adopted as the recommended testing strategy in the Globally Harmonised System for the Classification of Chemical Substances (GHS) (9). It is recommended that this testing strategy be followed prior to undertaking in vivo testing. For new substances it is the recommended stepwise testing approach for developing scientifically sound data on the corrosivity/irritation of the substance. For existing substances with insufficient data on skin and eye corrosion/irritation, the strategy should be used to fill missing data gaps. The use of a different testing strategy or procedure, or the decision not to use a stepwise testing approach, should be justified.

1.2. DEFINITIONS

Eye irritation: is the production of changes in the eye following the application of a test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.

Eye corrosion: is the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application.

1.3. PRINCIPLE OF THE TEST METHOD

The substance to be tested is applied in a single dose to one of the eyes of the experimental animal; the untreated eye serves as the control. The degree of eye irritation/corrosion is evaluated by scoring lesions of conjunctiva, cornea, and iris, at specific intervals. Other effects in the eye and adverse systemic effects are also described to provide a complete evaluation of the effects. The duration of the study should be sufficient to evaluate the reversibility or irreversibility of the effects.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparation for the in vivo test

1.4.1.1. Selection of species

The albino rabbit is the preferable laboratory animal, and healthy young adult animals are used. A rationale for using other strains or species should be provided.

1.4.1.2. Preparation of animals

Both eyes of each experimental animal provisionally selected for testing should be examined within 24 hours before testing starts. Animals showing eye irritation, ocular defects, or pre-existing corneal injury should not be used.

1.4.1.3. Housing and feeding conditions

1.4.2. Test procedure

1.4.2.1. Application of the test substance

The test substance should be placed in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids are then gently held together for about one second in order to prevent loss of the material. The other eye, which remains untreated, serves as a control.

1.4.2.2. Irrigation

The eyes of the test animals should not be washed for at least 24 hours following instillation of the test substance, except for solids (see Section 1.4.2.3.2), and in case of immediate corrosive or irritating effects. At 24 hours a washout may be used if considered appropriate.

Use of a satellite group of animals to investigate the influence of washing is not recommended unless it is scientifically justified. If a satellite group is needed, two rabbits should be used. Conditions of washing should be carefully documented, e.g. time of washing; composition and temperature of wash solution; duration, volume, and velocity of application.

1.4.2.3. Dose level

1.4.2.3.1. Testing of liquids

For testing liquids, a dose of 0,1 ml is used. Pump sprays should not be used for instilling the substance directly into the eye. The liquid spray should be expelled and collected in a container prior to instilling 0,1 ml into the eye.

1.4.2.3.2. Testing of solids

When testing solids, pastes, and particulate substances, the amount used should have a volume of 0,1 ml or a weight of not more than 100 mg. The test material should be ground to a fine dust. The volume of solid material should be measured after gently compacting it, e.g. by tapping the measuring container. If the solid test substance has not been removed from the eye of the test animal by physiological mechanisms at the first observation time point of one hour after treatment, the eye may be rinsed with saline or distilled water.

1.4.2.3.3. Testing of aerosols

It is recommended that all pump sprays and aerosols be collected prior to instillation into the eye. The one exception is for substances in pressurised aerosol containers, which cannot be collected due to vaporisation. In such cases, the eye should be held open, and the test substance administered to the eye in a simple burst of about one second, from a distance of 10 cm directly in front of the eye. This distance may vary depending on the pressure of the spray and its contents. Care should be taken not to damage the eye from the pressure of the spray. In appropriate cases, there may be a need to evaluate the potential for "mechanical" damage to the eye from the force of the spray.

An estimate of the dose from an aerosol can be made by simulating the test as follows: the substance is sprayed on to weighing paper through an opening the size of a rabbit eye placed directly before the paper. The weight increase of the paper is used to approximate the amount sprayed into the eye. For volatile substances, the dose may be estimated by weighing a receiving container before and after removal of the test material.

1.4.2.4. Initial test (in vivo eye irritation/corrosion test using one animal)

As articulated in the sequential testing strategy (see Appendix 1), it is strongly recommended that the in vivo test be performed initially using one animal.

If the results of this test indicate the substance to be corrosive or a severe irritant to the eye using the procedure described, further testing for ocular irritancy should not be performed.

1.4.2.5. Local anaesthetics

Local anaesthetics may be used on a case-by-case basis. If the weight-of-the-evidence analysis indicates that the substance has the potential to cause pain, or initial testing shows that a painful reaction will occur, a local anaesthetic may be used prior to instillation of the test substance. The type, concentration, and dose of the local anaesthetic should be carefully selected to ensure that differences in reaction to the test substance will not result from its use. The control eye should be similarly anaesthetised.

1.4.2.6. Confirmatory test (in vivo eye irritation test with additional animals)

If a corrosive effect is not observed in the initial test, the irritant or negative response should be confirmed using up to two additional animals. If a severe irritant effect is observed in the initial test indicating a possible strong (irreversible) effect in the confirmatory testing, it is recommended that the confirmatory test be conducted in a sequential manner in one animal at a time, rather than exposing the two additional animals simultaneously. If the second animal reveals corrosive or severe irritant effects, the test is not continued. Additional animals may be needed to confirm weak or moderate irritant responses.

1.4.2.7. Observation period

The duration of the observation period should be sufficient to evaluate fully the magnitude and reversibility of the effects observed. However, the experiment should be terminated at any time that the animal shows continuing signs of severe pain or distress (9). To determine reversibility of effects, the animals should be observed normally for 21 days post administration of the test substance. If reversibility is seen before 21 days, the experiment should be terminated at that time.

1.4.2.7.1. Clinical observations and grading of eye reactions

The eyes should be examined at one, 24, 48, and 72 hours after test substance application. Animals should be kept on test no longer than necessary once definitive information has been obtained. Animals showing continuing severe pain or distress should be humanely killed without delay, and the substance assessed accordingly. Animals with the following eye lesions post-instillation should be humanely killed: corneal perforation or significant corneal ulceration including staphyloma; blood in the anterior chamber of the eye; grade 4 corneal opacity which persists for 48 hours; absence of a light reflex (iridial response grade 2) which persists for 72 hours; ulceration of the conjunctival membrane; necrosis of the conjuctivae or nictitating membrane; or sloughing. This is because such lesions generally are not reversible

Animals that do not develop ocular lesions may be terminated not earlier than three days post instillation. Animals with mild to moderate lesions should be observed until the lesions clear, or for 21 days, at which time the study is terminated. Observations should be performed at seven, 14, and 21 days in order to determine the status of the lesions, and their reversibility or irreversibility.

The grades of ocular reaction (conjunctivae, cornea and iris) should be recorded at each examination (Table I). Any other lesions in the eye (e.g. pannus, staining) or adverse systemic effects should also be reported.

Examination of reactions can be facilitated by use of a binocular loupe, hand slit-lamp, biomicroscope, or other suitable device. After recording the observations at 24 hours, the eyes may be further examined with the aid of fluorescein.

The grading of ocular responses is necessarily subjective. To promote harmonisation of grading of ocular response and to assist testing laboratories and those involved in making and interpreting the observations, the personnel performing the observations need to be adequately trained in the scoring system used.

2. DATA

2.2. EVALUATION OF RESULTS

The ocular irritation scores should be evaluated in conjunction with the nature and severity of lesions, and their reversibility or lack of reversibility. The individual scores do not represent an absolute standard for the irritant properties of a material, as other effects of the test material are also evaluated. Instead, individual scores should be viewed as reference values and are only meaningful when supported by a full description and evaluation of all observations.

3. REPORTING

3.1. TEST REPORT

The test report must include the following information:

Rationale for in vivo testing: weight-of-the-evidence analysis of pre-existing test data, including results from sequential testing strategy

- description of relevant data available from prior testing,

- data derived in each step of testing strategy,

- description of in vitro tests performed, including details of procedures, results obtained with test/reference substances,

- description of in vivo dermal irritation/corrosion study performed, including results obtained,

- weight-of-the-evidence analysis for performing in vivo study.

Test substance:

- identification data (e.g. CAS number, source, purity, known impurities, lot number),

- physical nature and physicochemical properties (e.g. pH, volatility, solubility, stability, reactivity with water),

- in case of a mixture, composition and relative percentages of components,

- if local anaesthetic is used, identification, purity, type, dose, and potential interaction with test substance.

Vehicle:

- identification, concentration (where appropriate), volume used,

- justification for choice of vehicle.

Test animals:

- species/strain used, rationale for using animals other than albino rabbit,

- age of each animal at start of study,

- number of animals of each sex in test and control groups (if required),

- individual animal weights at start and conclusion of test,

- source, housing conditions, diet, etc.

Results:

- description of method used to score irritation at each observation time (e.g. hand slitlamp, biomicroscope, fluorescein),

- tabulation of irritant/corrosive response data for each animal at each observation time up to removal of each animal from the test,

- narrative description of the degree and nature of irritation or corrosion observed,

- description of any other lesions observed in the eye (e.g. vascularisation, pannus formation, adhesions, staining),

- description of non-ocular local and systemic adverse effects, and histopathological findings, if any.

Discussion of results.

3.2. INTERPRETATION OF THE RESULTS

Extrapolation of the results of eye irritation studies in laboratory animals to humans is valid only to a limited degree. In many cases the albino rabbit is more sensitive than humans to ocular irritants or corrosives.

Care should be taken in the interpretation of data to exclude irritation resulting from secondary infection.

4. REFERENCES

(2) de Silva, O., Cottin, M., Dami, N., Roguet, R., Catroux, P., Toufic, A., Sicard, C., Dossou, K.G., Gerner, I., Schlede, E., Spielmann, H., Gupta, K.C., Hill, R.N., (1997) Evaluation of Eye Irritation Potential: Statistical Analysis and Tier Testing Strategies. Food Chem. Toxicol 35, p. 159-164.

(3) Worth A.P. and Fentem J.H., (1999) A general approach for evaluating stepwise testing strategies ATLA 27, p. 161-177

(4) Young, J.R., How, M.J., Walker, A.P., Worth W.M.H., (1988) Classification as Corrosive or Irritant to Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on Animals. Toxicollogy In Vitro, 2, p. 19-26.

(5) Neun, D.J. (1993) Effects of Alkalinity on the Eye Irritation Potential of Solutions Prepared at a Single pH. J. Toxicol. Cut. Ocular Toxicol. 12, p. 227-231.

(6) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsaile, D.J., Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology In Vitro 12, p. 483-524.

(6a) Testing Method B.40 Skin Corrosion.

(7) Testing method B.4. Acute toxicity: dermal irritation/corrosion.

(8) OECD, (1996) OECD Test Guidelines Programme: Final Report of the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held in Solna, Sweden, 22-24 January 1996 (http://www.oecd.org/ehs/test/background.htm).

(9) OECD, (1998) Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998 (http://www.oecd.org/ehs/Class/HCL6.htm).

(10) OECD, (2000) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Environmental Health and Safety Publications. Series on Testing and Assessment No 19 (http://www.oecd.org/ehs/test/monos.htm).

Table I

GRADING OF OCULAR LESIONS

Cornea

Opacity: degree of density (readings should be taken from most dense area) [*]

No ulceration or opacity … | 0 |

Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible … | 1 |

Easily discernible translucent area; details of iris slightly obscured … | 2 |

Nacrous area; no details of iris visible; size of pupil barely discernible … | 3 |

Opaque cornea; iris not discernible through the opacity … | 4 |

Maximum possible: 4

Iris

Normal … | 0 |

Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is considered to be an effect) … | 1 |

Hemorrhage, gross destruction, or no reaction to light … | 2 |

Maximum possible: 2

Conjunctivae

Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris)

Normal … | 0 |

Some blood vessels hyperaemic (injected) … | 1 |

Diffuse, crimson colour; individual vessels not easily discernible … | 2 |

Diffuse beefy red … | 3 |

Maximum possible: 3

Chemosis

Swelling (refers to lids and/or nictating membranes)

Normal … | 0 |

Some swelling above normal … | 1 |

Obvious swelling, with partial eversion of lids … | 2 |

Swelling, with lids about half closed … | 3 |

Swelling, with lids more than half closed … | 4 |

Maximum possible: 4

Appendix

A Sequential Testing Strategy for Eye Irritation and Corrosion

GENERAL CONSIDERATIONS

In the interests of sound science and animal welfare, it is important to avoid the unnecessary use of animals, and to minimise testing that is likely to produce severe responses in animals. All information on a substance relevant to its potential ocular irritation/corrosivity should be evaluated prior to considering in vivo testing. Sufficient evidence may already exist to classify a test substance as to its eye irritation or corrosion potential without the need to conduct testing in laboratory animals. Therefore, utilising a weight-of-the-evidence analysis and sequential testing strategy will minimise the need for in vivo testing, especially if the substance is likely to produce severe reactions.

It is recommended that a weight-of-the-evidence analysis be used to evaluate existing information pertaining to eye irritation and corrosion of substances and to determine whether additional studies, other than in vivo eye studies, should be performed to help characterise such potential. Where further studies are needed, it is recommended that the sequential testing strategy be utilised to develop the relevant experimental data. For substances which have no testing history, the sequential testing strategy should be utilised to develop the data needed to evaluate its eye corrosion/irritation. The testing strategy described in this Appendix was developed at an OECD workshop (1). It was subsequently affirmed and expanded in the Harmonised Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, in November 1998 (2).

Although this testing strategy is not an integrated part of testing method B.5, it expresses the recommended approach for the determination of eye irritation/corrosion properties. This approach represents both best practice and an ethical benchmark for in vivo testing for eye irritation/corrosion. The testing method provides guidance for the conduct of the in vivo test and summarises the factors that should be addressed before considering such a test. The sequential testing strategy provides a weight-of-the-evidence approach for the evaluation of existing data on the eye irritation/corrosion properties of substances and a tiered approach for the generation of relevant data on substances for which additional studies are needed or for which no studies have been performed. The strategy includes the performance first of validated and accepted in vitro or ex vivo tests and then of testing method B.4 skin irritation/corrosion studies under specific circumstances (3)(4).

DESCRIPTION OF THE STEPWISE TESTING STRATEGY

Prior to undertaking tests as part of the sequential testing strategy (Figure), all available information should be evaluated to determine the need for in vivo eye testing. Although significant information might be gained from the evaluation of single parameters (e.g., extreme pH), the totality of existing information should be assessed. All relevant data on the effects of the substance in question, and its structural analogues, should be evaluated in making a weight-of-the-evidence decision, and a rationale for the decision should be presented. Primary emphasis should be placed upon existing human and animal data on the substance, followed by the outcome of in vitro or ex vivo testing. In vivo studies of corrosive substances should be avoided whenever possible. The factors considered in the testing strategy include:

Evaluation of existing human and animal data (Step 1). Existing human data, e.g. clinical and occupational studies, and case reports, and/or animal test data from ocular studies should be considered first, because they provide information directly related to effects on the eyes. Thereafter, available data from human and/or animal studies investigating dermal corrosion/irritation should be evaluated. Substances with known corrosivity or severe irritancy to the eye should not be instilled into the eyes of animals, nor should substances showing corrosive or irritant effects to the skin; such substances should be considered to be corrosive and/or irritating to the eyes as well. Substances with sufficient evidence of non-corrosivity and non-irritancy from previously performed ocular studies should also not be tested in in vivo eye studies.

Analysis of structure activity relationships (SAR) (Step 2). The results of testing of structurally related chemicals should be considered, if available. When sufficient human and/or animal data are available on structurally related substances or mixtures of such substances to indicate their eye corrrosion/irritancy potential, it can be presumed that the test substance will produce the same responses. In those cases, the substance may not need to be tested. Negative data from studies of structurally related substances or mixtures of such substances do not constitute sufficient evidence of non-corrosivity/non-irritancy of a substance under the sequential testing strategy. Validated and accepted SAR approaches should be used to identify the corrosion and irritation potential for both dermal and ocular effects.

Physicochemical properties and chemical reactivity (Step 3). Substances exhibiting pH extremes such as ≤ 2,0 or ≥ 11,5 may have strong local effects. If extreme pH is the basis for identifying a substance as corrosive or irritant to the eye, then its acid/alkaline reserve (buffering capacity) may also be taken into consideration (5)(6). If the buffering capacity suggests that a substance may not be corrosive to the eye, then further testing should be undertaken to confirm this, preferably by the use of a validated and accepted in vitro or ex vivo test (see Section step 5 and 6).

Consideration of other existing information (Step 4). All available information on systemic toxicity via the dermal route should be evaluated at this stage. The acute dermal toxicity of the test substance should also be considered. If the test substance has been shown to be very toxic by the dermal route, it may not need to be tested in the eye. Although there is not necessarily a relationship between acute dermal toxicity and eye irritation/corrosion, it can be assumed that if an agent is very toxic via the dermal route, it will also exhibit high toxicity when instilled into the eye. Such data may also be considered between Steps 2 and 3.

Results from in vitro or ex vivo tests (Steps 5 and 6). Substances that have demonstrated corrosive or severe irritant properties in an in vitro or ex vivo test (7)(8) that has been validated and accepted for the assessment specifically of eye or skin corrosivity/irritation, need not be tested in animals. It can be presumed that such substances will produce similar severe effects in vivo. If validated and accepted in vitro/ex vivo tests are not available, one should bypass Steps 5 and 6 and proceed directly to Step 7.

Assessment of in vivo dermal irritancy or corrosivity of the substance (Step 7). When insufficient evidence exists with which to perform a conclusive weight-of-the-evidence analysis of the potential eye irritation/corrosivity of a substance based upon data from the studies listed above, the in vivo skin irritation/corrosion potential should be evaluated first, using testing method B.4 (4) and its accompanying Appendix (9). If the substance is shown to produce corrosion or severe skin irritation, it should be considered to be a corrosive eye irritant unless other information supports an alternative conclusion. Thus, an in vivo eye test would not need to be performed. If the substance is not corrosive or severely irritating to the skin, an in vivo eye test should be performed.

In vivo test in rabbits (Steps 8 and 9): in vivo ocular testing should begin with an initial test using one animal. If the results of this test indicate the substance to be a severe irritant or corrosive to the eyes, further testing should not be performed. If that test does not reveal any corrosive or severe irritant effects, a confirmatory test is conducted with two additional animals.

REFERENCES

(1) OECD, (1996) OECD Test Guidelines Programme: Final Report of the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Held in Solna, Sweden, 22-24 January 1996 (http://www.oecd.org/ehs/test/background.htm).

(3) Worth, A.P. and Fentem J.H., (1999). A General Approach for Evaluating Stepwise Testing Strategies. ATLA 27, p. 161-177.

(4) Testing method B.4. Acute Toxicity: dermal irritation/corrosion.

(5) Young, J.R., How, M.J., Walker, A.P., Worth W.M.H., (1988) Classification as Corrosive or Irritant to Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on Animals. Toxicolohy In Vitro, 27, p. 19-26.

(6) Neun, D.J., (1993) Effects of Alkalinity on the Eye Irritation Potential of Solutions Prepared at a Single pH. J. Toxicol. Cut. Ocular Toxicol. 12, p. 227-231.

(7) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsail, D.J., Holzhutter, H.G. and Liebsch, M., (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology In Vitro 12, p. 483-524.

(8) Testing Method B.40 Skin Corrosion.

(9) Appendix to Testing method B.4: A Sequential Testing Strategy for Skin Irritation and Corrosion.

Figure

TESTING AND EVALUATION STRATEGY FOR EYE IRRITATION/CORROSION

Activity

Finding

Conclusion

Existing human and/or animal data showing effects on eyes

Severe damage to eyes

Apical endpoint; consider corrosive to eyes. No testing is needed.

Eye irritant

Apical endpoint; consider irritating to eyes. No testing is needed.

Not corrosive/not irritating to eyes

Apical endpoint; considered non-corrosive and non-irritating to eyes. No testing required.

Existing human and/or animal data showing corrosive effects on skin

Skin corrosive

Assume corrosivity to eyes. No testing is needed.

Existing human and/or animal data showing severe irritant effects on skin

Severe skin irritant

Assume irritating to eyes. No testing is needed.

↓

no information available, or available information is not conclusive

↓

Perform SAR for eye corrosion/irritation

Predict severe damage to eyes

Assume corrosivity to eyes. No testing is needed.

Predict irritation to eyes

Assume irritating to eyes. No testing is needed.

Perform SAR for skin corrosion

Predict skin corrosivity

Assume corrosivity to eyes. No testing is needed.

↓

No predictions can be made, or predictions are not conclusive or negative

↓

Measure pH (buffering capacity, if relevant)

pH ≤ 2 or ≥ 11.5 (with high buffering capacity, if relevant)

Assume corrosivity to eyes. No testing is needed.

↓

2< pH < 11.5, or pH ≤ 2.0 or ≥ 11.5 with low/no buffering capacity, if relevant

↓

Evaluate systemic toxicity via the dermal route

Very toxic at concentrations that would be tested in the eye

Substance would be too toxic for testing. No testing is needed.

↓

Such information is not available, or substance is not very toxic

↓

+++++ TIFF +++++

Perform validated and accepted in vitro or ex vivo test for eye corrosion

Corrosive response

Assume corrosivity to eyes. No further testing is needed.

↓

Substance is not corrosive, or validated in vitro or ex vivo testing methods for eye corrosion are not yet available

↓

Perform validated and accepted in vitro or ex vivo test for eye irritation

Irritant response

Assume irritancy to eyes. No further testing is needed.

↓

Substance is not an irritant, or validated in vitro or ex vivo testing methods for eye irritation are not yet available

↓

Experimentally assess in vivo skin irritation/corrosion potential (see Testing method B.4 including its Annex)

Corrosive or severe irritant response

Assume corrosivity to eyes. No further testing is needed.

↓

Substance is not corrosive or severely irritating to skin

↓

Perform initial in vivo rabbit eye test using one animal

Severe damage to eyes

Consider corrosive to eyes. No further testing is needed.

↓

No severe damage, or no response

↓

Perform confirmatory test using one or two additional animals

Corrosive or irritating

Consider corrosive or irritating to eyes. No further testing is needed

Not corrosive or irritating

Consider non-irritating and non-corrosive to eyes. No further testing is needed.

+++++ TIFF +++++

B.6. SKIN SENSITISATION

1. METHOD

1.1. INTRODUCTION

Remarks:

The sensitivity and ability of tests to detect potential human skin sensitisers are considered important in a classification system for toxicity relevant to public health.

There is no single test method which will adequately identify all substances with a potential for sensitising human skin and which is relevant for all substances.

Factors such as the physical characteristics of a substance, including its ability to penetrate the skin, must be considered in the selection of a test.

Two types of tests using guinea pigs have been developed: the adjuvant-type tests, in which an allergic state is potentiated by dissolving or suspending the test substance in Freunds Complete Adjuvant (FCA), and the non-adjuvant tests.

Adjuvant-type tests are likely to be more accurate in predicting a probable skin sensitising effect of a substance in humans than those methods not employing Freunds Complete Adjuvant and are thus the preferred methods.

The Guinea-Pig Maximisation Test (Gpmt) is a widely used adjuvant-type test. Although several other methods can be used to detect the potential of a substance to provoke skin sensitisation reaction, the Gpmt is considered to be the preferred adjuvant technique.

With many chemical classes, non-adjuvant tests (the preferred one being the Buehler test) are considered to be less sensitive.

In certain cases there may be good reasons for choosing the Buehler test involving topical application rather than the intradermal injection used in the Guinea-Pig Maximisation Test. Scientific justification should be given when the Buehler test is used.

The Guinea-Pig Maximisation Test (Gpmt) and the Buehler test are described in this method. Other methods may be used provided that they are well-validated and scientific justification is given.

If a positive result is seen in a recognised screening test, a test substance may be designated as a potential sensitiser, and it may not be necessary to conduct a further guinea pig test. However, if a negative result is seen in such a test, the guinea pig test must be conducted using the procedure described in this tes method.

See also General introduction Part B.

1.2. DEFINITIONS

Skin sensitisation: (allergic contact dermatitis) is an immunologically mediated cutaneous reaction to a substance. In the human, the responses may be characterised by pruritis, erythema, oedema, papules, vesicles, bullae or a combination of these. In other species the reactions may differ and only erythema and oedema may be seen.

Induction exposure: an experimental exposure of a subject to a test substance with the intention of inducing a hypersensitive state.

Induction period: a period of at least one week following an induction exposure during which a hypersensitive state may be developed.

Challenge exposure: an experimental exposure of a previously treated subject to a test substance following an induction period, to determine if the subject reacts in a hypersensitive manner.

1.3. REFERENCE SUBSTANCES

The sensitivity and reliability of the experimental technique used should be assessed every six months by use of substances, which are known to have mild-to-moderate skin sensitisation properties.

In a properly conducted test, a response of at least 30 % in an adjuvant test and at least 15 % in a non-adjuvant test should be expected for mild/moderate sensitisers.

The following substances are preferred.

CAS numbers | EINECS numbers | EINECS names | Common names |

101-86-0 | 202-983-3 | α-hexylcinnamaldehyde | α-hexylcinnamaldehyde |

149-30-4 | 205-736-8 | Benzothiazole-2-thiol (mercaptobenzothiazole) | kaptax |

94-09-7 | 202-303-5 | Benzocaine | norcaine |

There may be circumstances where, given adequate justification other control substances meeting the above criteria may be used.

1.4. PRINCIPLE OF THE TEST METHOD

The test animals are initially exposed to the test substance by intradermal injections and/or epidermal application (induction exposure). Following a rest period of 10 to 14 days (induction period), during which an immune response may develop, the animals are exposed to a challenge dose. The extent and degree of skin reaction to the challenge exposure in the test animals is compared with that demonstrated by control animals which undergo sham treatment during induction and receive the challenge exposure.

1.5. DESCRIPTION OF THE TEST METHODS

If removal of the test substance is considered necessary, this should be achieved using water or an appropriate solvent without altering the existing response or the integrity of the epidermis.

1.5.1. Guinea-Pig Maximisation Test (GPMT)

1.5.1.1. Preparations

Healthy young adult albino guinea pigs are acclimatised to the laboratory conditions for at least five days prior to the test. Before the test, animals are randomised and assigned to the treatment groups. Removal of hair is by clipping, shaving or possibly by chemical depilation, depending on the test method used. Care should be taken to avoid abrading the skin. The animals are weighed before the test commences and at the end of the test.

1.5.1.2. Test conditions

1.5.1.2.1. Test animals

Commonly used laboratory strains of albino guinea-pigs are used.

1.5.1.2.2. Number and sex

Male and/or female animals can be used. If females are used, they should be nulliparous and non-pregnant.

A minimum of 10 animals is used in the treatment group and at least five animals in the control group. When fewer than 20 test and 10 control guinea pigs have been used, and it is not possible to conclude that the test substance is a sensitiser, testing in additional animals to give a total of at least 20 test and 10 control animals is strongly recommended.

1.5.1.2.3. Dose levels

The concentration of the test substance used for each induction exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. The concentration used for the challenge exposure should be the highest non-irritant dose. The appropriate concentrations should be determined from a pilot study using two or three animals, if other information is not available. Consideration should be given to the use of FCA-treated animals for this purpose.

1.5.1.3. Procedure

1.5.1.3.1. Induction

Day 0-treated group

Three pairs of intradermal injections of 0,1 ml volume are given in the shoulder region which is cleared of hair so that one of each pair lies on each side of the midline.

Injection 1: a 1:1 mixture (v/v) FCA/water or physiological saline.

Injection 2: the test substance in an appropriate vehicle at the selected concentration.

Injection 3: the test substance at the selected concentration formulated in a 1:1 mixture (v/v) FCA/water or physiological saline.

In injection 3, water soluble substances are dissolved in the aqueous phase prior to mixing with FCA. Liposoluble or insoluble substances are suspended in FCA prior to combining with the aqueous phase. The final concentration of test substance shall be equal to that used in injection 2.

Injections 1 and 2 are given close to each other and nearest the head, while 3 is given towards the caudal part of the test area.

Day 0-control group

Three pairs of intradermal injections of 0,1 ml volume are given in the same sites as in the treated animals.

Injection 1: a 1:1 mixture (v/v) FCA/water or physiological saline.

Injection 2: the undiluted vehicle.

Injection 3: a 50 % w/v formulation of the vehicle in a 1:1 mixture (v/v) FCA/water or physiological saline.

Day 5-7-treated and control groups

Approximately 24 hours before the topical induction application, if the substance is not a skin irritant, the test area, after close-clipping and/or shaving is treated with 0,5 ml of 10 % sodium lauryl sulphate in vaseline, in order to create a local irritation.

Day 6-8-treated group

The test area is again cleared of hair. A filter paper (2 × 4 cm) is fully-loaded with test substance in a suitable vehicle and applied to the test area and held in contact by an occlusive dressing for 48 hours. The choice of the vehicle should be justified. Solids are finely pulverised and incorporated in a suitable vehicle. Liquids can be applied undiluted, if appropriate.

Day 6-8-control group

The test area is again cleared of hair. The vehicle only is applied in a similar manner to the test area and held in contact by an occlusive dressing for 48 hours.

1.5.1.3.2. Challenge

Day 20-22-treated and control groups

The flanks of treated and control animals are cleared of hair. A patch or chamber loaded with the test substance is applied to one flank of the animals and, when relevant, a patch or chamber loaded with the vehicle only may also be applied to the other flank. The patches are held in contact by an occlusive dressing for 24 hours.

1.5.1.3.3. Observation and Grading: treated and control groups

- approximately 21 hours after removing the patch the challenge area is cleaned and closely-clipped and/or shaved and depilated if necessary;

- approximately three hours later (approximately 48 hours from the start of the challenge application) the skin reaction is observed and recorded according to the grades shown in the Appendix;

- approximately 24 hours after this observation a second observation (72 hours) is made and once again recorded.

Blind reading of test and control animals is encouraged.

If it is necessary to clarify the results obtained in the first challenge, a second challenge (i.e. a rechallenge), where appropriate with a new control group, should be considered approximately one week after the first one. A rechallenge may also be performed on the original control group.

All skin reactions and any unusual findings, including systemic reactions, resulting from induction and challenge procedures should be observed and recorded according to the grading scale of Magnusson/Kligman (See Appendix). Other procedures, e.g. histopathological examination, the measurement of skin fold thickness, may be carried out to clarify doubtful reactions.

1.5.2. Buehler test

1.5.2.1. Preparations

Healthy young adult albino guinea-pigs are acclimatised to the laboratory conditions for at least five days prior to the test. Before the test, animals are randomised and assigned to the treatment groups. Removal of hair is by clipping, shaving or possibly by chemical depilation, depending on the test method used. Care should be taken to avoid abrading the skin. The animals are weighed before the test commences and at the end of the test.

1.5.2.2. Test conditions

1.5.2.2.1. Test animals

Commonly used laboratory strains of albino guinea-pigs are used.

1.5.2.2.2. Number and sex

Male and/or female animals can be used. If females are used, they should be nulliparous and non-pregnant.

A minimum of 20 animals is used in the treatment group and at least 10 animals in the control group.

1.5.2.2.3. Dose levels

The concentration of test substance used for each induction exposure should be the highest possible to produce a mild but not excessive irritation. The concentration used for the challenge exposure should be the highest non-irritating dose. If necessary, the appropriate concentration can be determined from a pilot study using two or three animals.

For water soluble test materials, it is appropriate to use water or a dilute non-irritating solution of surfactant as the vehicle. For other test materials 80 % ethanol/water is preferred for induction and acetone for challenge.

1.5.2.3. Procedure

1.5.2.3.1. Induction

Day 0-treated group

One flank is cleared of hair (closely-clipped). The test patch system should be fully loaded with test substance in a suitable vehicle (the choice of the vehicle should be justified; liquid test substances can be applied undiluted, if appropriate).

The test patch system is applied to the test area and held in contact with the skin by an occlusive patch or chamber and a suitable dressing for six hours.

The test patch system must be occlusive. A cotton pad is appropriate and can be circular or square, but should approximate 4-6 cm2. Restraint using an appropriate restrainer is preferred to assure occlusion. If wrapping is used, additional exposures may be required.

Day 0-control group

One flank is cleared of hair (closely-clipped). The vehicle only is applied in a similar manner to that used for the treated group. The test patch system is held in contact with the skin by an occlusive patch or chamber and a suitable dressing for six hours. If it can be demonstrated that a sham control group is not necessary, a naive control group may be used.

Days 6-8 and 13-15-treated and control group

The same application as on day 0 is carried out on the same test area (cleared of hair if necessary) of the same flank on day 6-8, and again on day 13-15.

1.5.2.3.2. Challenge

Day 27-29-treated and control group

The untreated flank of treated and control animals is cleared of hair (closely-clipped). An occlusive patch or chamber containing the appropriate amount of test substance is applied, at the maximum non-irritant concentration, to the posterior untreated flank of treated and control animals.

When relevant, an occlusive patch or chamber with vehicle only is also applied to the anterior untreated flank of both treated and control animals. The patches or chambers are held in contact by a suitable dressing for six hours.

1.5.2.3.3. Observation and grading

- approximately 21 hours after removing the patch the challenge area is cleared of hair,

- approximately three hours later (approximately 30 hours after application of the challenge patch) the skin reactions are observed and recorded according to the grades shown in the Appendix,

- approximately 24 hours after the 30 hour observation (approximately 54 hours after application of the challenge patch) skin reactions are again observed and recorded.

Blind reading of the test and control animals is encouraged.

All skin reactions and any unusual findings, including systemic reactions, resulting from induction and challenge procedures should be observed and recorded according to the Magnusson/Kligman grading scale (See Appendix). Other procedures, e.g. histopathological examination, the measurement of skin fold thickness, may be carried out to clarify doubtful reactions.

2. DATA (GPMT and Buehler test)

Data should be summarised in tabular form, showing for each animal the skin reactions at each observation.

3. REPORTING (GPMT and Buehler test)

If a screening assay is performed before the guinea pig test the description or reference of the test (e.g. Mouse Ear Swelling Test (MEST)), including details of the procedure, must be given together with results obtained with the test and reference substances.

Test report (GMPT and Buehler test)

The test report shall, if possible, include the following information:

Test animals:

- strain of guinea-pig used,

- number, age and sex of animals,

- source, housing conditions, diet, etc.,

- individual weights of animals at the start of the test.

Test conditions:

- technique of patch site preparation,

- details of patch materials used and patching technique,

- result of pilot study with conclusion on induction and challenge concentrations to be used in the test,

- details of test substance preparation, application and removal,

- justification for choice of vehicle,

- vehicle and test substance concentrations used for induction and challenge exposures and the total amount of substance applied for induction and challenge.

Results:

- a summary of the results of the latest sensitivity and reliability check (see 1.3) including information on substance, concentration and vehicle used,

- on each animal including grading system,

- narrative description of the nature and degree effects observed,

- any histopathological findings.

Discussion of results.

Conclusions.

4. REFERENCES

This method is analogous to OECD TG 406.

Appendix

TABLE

Magnusson/Kligman grading scale for the evaluation of challenge patch test reactions

0 = no visible change

1 = discrete or patchy erythema

2 = moderate and confluent erythema

3 = intense erythema and swelling

B.7. REPEATED DOSE (28 DAYS) TOXICITY (ORAL)

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITIONS

See General introduction Part B.

1.3. PRINCIPLE OF THE TEST METHOD

The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 28 days. During the period of administration the animals are observed closely, each day for signs of toxicity. Animals which die or are killed during the test are necropsied and at the conclusion of the test surviving animals are killed and necropsied.

This method places more emphasis on neurological effects as a specific endpoint, and the need for careful clinical observations of the animals, so as to obtain as much information as possible, is stressed. The method should identify chemicals with neurotoxic potential, which may warrant further indepth investigation of this aspect. In addition, the method may give an indication of immunological effects and reproductive organ toxicity.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparations

Healthy young adult animals are randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals are identified uniquely and kept in their cages for at least five days prior to the start of the study to allow for acclimatisation to the laboratory conditions.

The test substance is administered by gavage or via the diet or drinking water. The method of oral administration is dependent on the purpose of the study, and the physical/chemical properties of the substance.

Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined.

1.4.2. Test conditions

1.4.2.1. Test animals

The preferred rodent species is the rat, although other rodent species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. The females should be nulliparous and non-pregnant. Dosing should begin as soon as possible after weaning and, in any case, before the animals are nine weeks old.

At the commencement of the study the weight variation of animals used should be minimal and not exceed ± 20 % of the mean weight of each sex.

Where a repeated dose oral study is conducted as a preliminary to a long term-study, preferably animals from the same strain and source should be used in both studies.

1.4.2.2. Number and sex

At least 10 animals (five female and five male) should be used at each dose level. If interim kills are planned, the number should be increased by the number of animals scheduled to be killed before the completion of the study.

In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high dose level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.

1.4.2.3. Dose levels

Generally, at least three test groups and a control group should be used. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used in administering the test substance, the control group should receive the vehicle in the highest volume used.

If from assessment of other data, no effects would be expected at a dose of 1000 mg/kg bw/d, a limit test may be performed. If there are no suitable data available, a range finding study may be performed to aid the determination of the doses to be used.

Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available for the test substance or related materials. The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and no-observed-adverse effects at the lowest dose level (NOAEL). Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of 10) between dosages.

For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals' body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight.

Where a repeated dose study is used as a preliminary to a long term-study, a similar diet should be used in both studies.

1.4.2.4. Limit test

If a test at one dose level of at least 1000 mg/kg body weight/day or, for dietary or drinking water administration, an equivalent percentage in the diet or drinking water (based upon body weight determinations), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.

1.4.2.5. Observation period

The observation period should be 28 days. Animals in a satellite group scheduled for follow-up observations should be kept for at least a further 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

1.4.3. Procedure

The animals are dosed with the test substance daily seven days each week for a period of 28 days; use of a five-day per week dosing regime needs to be justified. When the test substance is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should not exceed 1 ml/100 g body weight, except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

1.4.3.1. General observations

General clinical observations should be made at least once a day, preferably at the same time(s) each day and considering the peak period of anticipated effects after dosing. The health condition of the animals should be recorded. At least twice daily, all animals are observed for morbidity and mortality. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.

Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time, each time. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) should also be recorded.

In the fourth exposure week sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli), assessment of grip strength and motor activity assessment should be conducted. Further details of the procedures that could be followed are given in the literature (see General introduction Part B).

Functional observations conducted in the fourth exposure week may be omitted when the study is conducted as a preliminary study to a subsequent subchronic (90-day) study. In that case, the functional observations should be included in this follow-up study. On the other hand, the availability of data on functional observations from the repeated dose study may enhance the ability to select dose levels for a subsequent subchronic study.

Exceptionally, functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.

1.4.3.2. Body weight and food/water consumption

All animals should be weighed at least once a week. Measurements of food and water consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly.

1.4.3.3. Haematology

The following haematological examinations should be made at the end of the test period: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count, platelet count and a measure of blood clotting time/potential.

Blood samples should be taken from a named site just prior to or as part of the procedure for killing the animals, and stored under appropriate conditions.

1.4.3.4. Clinical biochemistry

Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained of all animals just prior to or as part of the procedure for killing the animals (apart from those found moribund and/or intercurrently killed). Overnight fasting of the animals prior to blood sampling is recommended [2]. Investigations of plasma or serum shall include sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, and sorbitol dehydrogenase). Measurements of additional enzymes (of hepatic or other origin) and bile acids may provide useful information under certain circumstances.

Optionally, the following urine analysis determinations could be performed during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.

In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphate, fasting triglycerides, specific hormones, methaemoglobin and cholinesterase. These need to be identified for substances in certain classes or on a case-by-case basis.

Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect with a given substance.

If historical baseline data are inadequate, consideration should be given to determination of haematological and clinical biochemistry variables before dosing commences.

1.4.3.5. Gross necropsy

All animals in the study shall be subjected to a full, detailed gross necropsy, which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain and heart of all animals should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying.

The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), gonads, accessory sex organs (e.g. uterus, prostate), urinary bladder, lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, and a section of bone marrow (or, alternatively, a fresh mounted bone marrow aspirate). The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved.

1.4.3.6. Histopathological examination

Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups. These examinations should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group.

All gross lesions shall be examined.

When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.

2. DATA

Individual data should be provided. Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons and the time of any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion.

When possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods should be selected during the design of the study.

3. REPORTING

TEST REPORT

The test report shall, if possible, include the following information:

Test animals:

- species/strain used,

- number, age and sex of animals,

- source, housing conditions, diet, etc.,

- individual weights of animals at the start of the test in weekly intervals thereafter and at the end of the test.

Test conditions:

- justification for choice of vehicle, if other than water,

- rationale for dose level selection,

- details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation,

- details of the administration of the test substance,

- conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable,

- details of food and water quality.

Results:

- body weight/body weight changes,

- food consumption, and water consumption, if applicable,

- toxic response data by sex and dose level, including signs of toxicity,

- nature, severity and duration of clinic observations (whether reversible or not),

- sensory activity, grip strength and motor activity assessments,

- haematological tests with relevant base-line values,

- clinical biochemistry tests with relevant base-line values,

- body weight at killing and organ weight data,

- necropsy findings,

- a detailed description of all histopathological findings,

- absorption data if available,

- statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

4. REFERENCES

This method is analogous to OECD TG 407.

B.8. REPEATED DOSE (28 DAYS) TOXICITY (INHALATION)

1. METHOD

1.1. INTRODUCTION

See also General introduction Part B (A).

1.2. DEFINITION

See General introduction Part B (B).

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

Several groups of experimental animals are exposed daily for a defined period to the test substance in graduated concentrations, one concentration being used per group, for a period of 28 days. Where a vehicle is used to help generate an appropriate concentration of the test substance in the atmosphere, a vehicle control group should be used. During the period of administration the animals are observed daily to detect signs of toxicity. Animals, which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

The animals are kept under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test, healthy young animals are randomised and assigned to the required number of groups. Where necessary, a suitable vehicle may be added to the test substance to help generate an appropriate concentration of the substance in the atmosphere. If a vehicle or other additive is used to facilitate dosing, it should be known not to produce toxic effects. Historical data can be used if appropriate.

1.6.2. Test conditions

1.6.2.1. Experimental animals

Unless there are contra-indications, the rat is the preferred species. Commonly used laboratory strains of young healthy animals should be employed.

At the commencement of the study the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.

1.6.2.2. Number and sex

At least 10 animals (five female and five male) should be used for each test group. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the numbers should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high concentration level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.

1.6.2.3. Exposure concentration

At least three concentrations are required, with a control or a vehicle control (corresponding to the concentration of vehicle at the highest level) if a vehicle is used. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test-group animals. The highest concentration should result in toxic effects but no, or few, fatalities. The lowest concentration should not produce any evidence of toxicity. Where there is a usable estimation of human exposure, the lowest concentration should exceed this. Ideally, the intermediate concentration should produce minimal observable toxic effects. If more than one intermediate concentration is used the concentrations should be spaced to produce a gradation of toxic effects. In the low and intermediate groups and in the controls, the incidence of fatalities should be low to permit a meaningful evaluation of the results.

1.6.2.4. Exposure time

The duration of daily exposure should be six hours but other periods may be needed to meet specific requirements.

1.6.2.5. Equipment

The animals should be tested in inhalation equipment designed to sustain a dynamic airflow of at least 12 air changes per hour to ensure an adequate oxygen content and an evenly distributed exposure atmosphere. Where a chamber is used its design should minimise crowding of the test animals and maximise their exposure by inhalation of the test substance. As a general rule to ensure stability of a chamber atmosphere the total "volume" of the test animals should not exceed 5 % of the volume of the test chamber. Oro-nasal, head only, or individual whole body chamber exposure may be used; the first two will minimise uptake by other routes.

1.6.2.6. Observation period

The experimental animals should be observed daily for signs of toxicity during the entire treatment and recovery period. The time of death and the time at which signs of toxicity appear and disappear should be recorded.

1.6.3. Procedure

The animals are exposed to the test substance daily, five to seven days per week, for a period of 28 days. Animals in any satellite groups scheduled for follow-up observations should be kept for a further 14 days without treatment to detect recovery from, or persistence of toxic effects. The temperature at which the test is performed should be maintained at 22 ± 3 oC.

Ideally, the relative humidity should be maintained between 30 and 70 %, but in certain instances (e.g. tests of some aerosols) this may not be practicable. Maintenance of a slight negative pressure inside the chamber (≤ 5 mm of water) will prevent leakage of the test substance into the surrounding area. Food and water should be withheld during exposure.

A dynamic inhalation system with a suitable analytical concentration control system should be used. To establish suitable exposure concentrations a trial test is recommended. The airflow should be adjusted to ensure that conditions throughout the exposure chamber are homogeneous. The system should ensure that stable exposure conditions are achieved as rapidly as possible.

Measurements or monitoring should be made:

(a) of the rate of airflow (continuously),

(b) of the actual concentration of the test substance measured in the breathing zone. During the daily exposure period the concentration should not vary by more than ± 15 % of the mean value. However, in the case of some aerosols, this level of control may not be achievable and a wider range would then be acceptable. During the total duration of the study, the day-to-day concentrations should be held as constant as practicable. For aerosols, at least one particle size analysis should be performed per test group weekly,

During and following exposure observations are made and recorded systematically; individual records should be maintained for each animal. All the animals should be observed daily and signs of toxicity recorded including the time of onset, their degree and duration. Observations should include changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Measurements should be made weekly of the animals' weight. It is also recommended that food consumption is measured weekly. Regular observation of the animals is necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period, all survivors in the non-satellite treatment groups are necropsied. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.

The following examinations shall be made at the end of the test on all animals including the controls:

(i) haematology, including at least haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count and a measure of clotting potential;

(ii) clinical blood biochemistry including at least one parameter of liver and kidney function: serum alanine aminotransferase (formerly known as glutamic pyruvic transaminase), serum aspartate aminotransferase (formerly known as glutamic oxaloacetic transaminase), urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements;

Other determinations, which may be necessary for an adequate toxicological evaluation include calcium, phosphorus, chloride, sodium, potassium, fasting glucose analysis of lipids, hormones, acid/base balance, methaemoglobin and cholinesterase activity.

Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed toxic effects.

1.6.3.1. Gross necropsy

All animals in the study should be subjected to a full gross necropsy. At least the liver, kidneys, adrenals, lungs, and testes should be weighed wet as soon as possible after dissection to avoid drying. Organs and tissues (the respiratory tract, liver, kidneys, spleen, testes, adrenals, heart, and any organs showing gross lesions or changes in size) should be preserved in a suitable medium for possible future histopathological examination. The lungs should be removed intact, weighed and treated with a suitable fixative to ensure that lung structure is maintained.

1.6.3.2. Histopathological examination

In the high-concentration group and in the control(s), histological examination should be performed on preserved organs and tissues. Organs and tissues showing defects attributable to the test substance at the highest dosage level should be examined in all lower-dosage groups. Animals in any satellite groups should be examined histologically with particular emphasis on those organs and tissues identified as showing effects in the other treated groups.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test and the number of animals displaying each type of lesion.

All observed results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, include the following information:

- species, strain, source, environmental conditions, diet, etc.,

- test conditions.

Description of exposure apparatus including design, type, dimensions, source of air, system for generating aerosols, method of conditioning air, treatment of exhaust air and the method of housing animals in a test chamber when this is used. The equipment for measuring temperature, humidity and, where appropriate, stability of aerosol concentrations or particle size distribution, should be described.

Exposure data:

These should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and shall, if possible, include:

(a) airflow rates through the inhalation equipment;

(b) temperature and humidity of air;

(c) nominal concentrations (total amount of test substance fed into the inhalation equipment divided by the volume of air);

(d) nature of vehicle, if used;

(e) actual concentrations in test breathing zone;

(f) the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD);

- toxic response data by sex and concentration,

- time of death during the study or whether animals survived to termination,

- description of toxic or other effects, no-effect level,

- the time of observation of each abnormal sign and its subsequent course,

- food and body-weight data,

- haematological tests employed and results,

- clinical biochemistry tests employed and results,

- necropsy findings,

- a detailed description of all histopathological findings,

- a statistical treatment of results where possible,

- discussion of the results,

- interpretation of results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B (D).

4. REFERENCES

See General introduction Part B (E).

B.9. REPEATED DOSE (28 DAYS) TOXICITY (DERMAL)

1. METHOD

1.1. INTRODUCTION

See General introduction Part B (A).

1.2. DEFINITIONS

See General introduction Part B (B).

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The test substance is applied daily to the skin in graduated doses to several groups of experimental animals, one dose per group, for a period of 28 days. During the period of application, the animals are observed daily to detect signs of toxicity. Animals, which die during the test, are necropsied and at the conclusion of the test surviving animals are necropsied.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

The animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. Before the test, healthy young animals are randomised and assigned to the treatment and control groups. Shortly before testing, fur is clipped from the dorsal area of the trunk of the test animals. Shaving may be employed but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care must be taken to avoid abrading the skin. Not less than 10 % of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of the covering. When testing solids, which may be pulverised if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. Liquid test substances are generally used undiluted. Daily application on a five to seven-day per week basis is used.

1.6.2. Test conditions

1.6.2.1. Experimental animals

The adult rat, rabbit or guinea-pig may be used. Other species may be used but their use would require justification.

At the commencement of the study, the range of weight variation in the animals used should not exceed ± 20 % of the appropriate mean value.

1.6.2.2. Number and sex

At least 10 animals (five female and five male) with healthy skin should be used at each dose level. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the numbers should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 10 animals (five animals per sex) may be treated with the high dose level for 28 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 14 days post-treatment. A satellite group of 10 control animals (five animals per sex) is also used.

1.6.2.3. Dose levels

At least three dose levels are required with a control or a vehicle control if a vehicle is used. The exposure period should be at least six hours per day. The application of the test substance should be made at similar times each day, and adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body-weight. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. Where a vehicle is used to facilitate dosing, the vehicle control group should be dosed in the same way as the treated groups, and receive the same amount as that received by the highest dose level group. The highest dose level should result in toxic effects but produce no, or few, fatalities. The lowest dose level should not produce any evidence or toxicity. Where there is a usable estimation of human exposure, the lowest level should exceed this. Ideally, the intermediate dose level should produce minimal observable toxic effects. If more than one intermediate dose is used the dose levels should be spaced to produce a gradation of toxic effects. In the low and intermediate groups and in the controls, the incidence of fatalities should be low in order to permit a meaningful evaluation of the results.

If application of the test substance produces severe skin irritation, the concentrations should be reduced and this may result in a reduction in, or absence of, other toxic effects at the high dose level. Moreover if the skin has been badly damaged it may be necessary to terminate the study and undertake a new study at lower concentrations.

1.6.2.4. Limit test

If a preliminary study at a dose level of 1000 mg/kg, or a higher dose level related to possible human exposure where this is known, produces no toxic effects, further testing may not be considered necessary.

1.6.2.5. Observation period

The experimental animals should be observed daily for signs of toxicity. The time of death and the time at which signs of toxicity appear and disappear should be recorded.

1.6.3. Procedure

Animals should be caged individually. The animals are treated with the test substance, ideally on seven days per week, for a period of 28 days. Animals in any satellite groups scheduled for follow-up observations should be kept for a further 14 days without treatment to detect recovery from or persistence of toxic effects. Exposure time should be at least six hours per day.

The test substance should be applied uniformly over an area, which is approximately 10 % of the total body surface area. With highly toxic substances, the surface area covered may be less but as much of the area as possible should be covered with as thin and uniform a layer as possible.

During exposure the test substance is held in contact with the skin with porous gauze dressing and non-irritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance but complete immobilisation is not a recommended method. As an alternative a "collar protective device" may be used.

At the end of the exposure period, residual test substance should be removed, where practicable, using water or some other appropriate method of cleansing the skin.

All the animals should be observed daily and signs of toxicity recorded including the time of onset, their degree and duration. Observations should include changes in skin and fur, eyes and mucous membranes as well as respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Measurements should be made weekly of the animals' weight. It is also recommended that food consumption is measured weekly. Regular observation of the animals is necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period, all survivors in the non-satellite treatment groups are necropsied. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.

The following examinations shall be made at the end of the test on all animals including the controls:

(1) haematology, including at least haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential;

(2) clinical blood biochemistry including at least one parameter of liver and kidney function: serum alanine aminotransferase (formerly known as glutamic pyruvic transaminase), serum aspartate aminotransferase (formerly known as glutamic oxaloacetic transaminase), urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein;

Other determinations which may be necessary for an adequate toxicological evaluation include calcium, phosphorus, chloride, sodium, potassium, fasting glucose, analysis of lipids, hormones, acid/base balance, methaemoglobin and cholinesterase activity.

Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.

1.6.4. Gross necropsy

All animals in the study should be subjected to a full gross necropsy. At least the liver, kidneys, adrenals, and testes should be weighed wet as soon as possible after dissection, to avoid drying. Organs and tissues, i.e. normal and treated skin, liver, kidney, spleen, testes, adrenals, heart, and target organs (that is those organs showing gross lesions or changes in size) should be preserved in a suitable medium for possible future histopathological examination.

1.6.5. Histopathological examination

In the high dose group and in the control group, histological examination should be performed on the preserved organs and tissues. Organs and tissues showing defects attributable to the test substance at the highest dosage level should be examined in all lower-dosage groups. Animals in the satellite group should be examined histologically with particular emphasis on those organs and tissues identified as showing effects in the other treated groups.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test and the number of animals displaying each type of lesion.

All observed results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, include the following information:

- animal data (species, strain, source, environmental conditions, diet, etc.),

- test conditions (including the type of dressing: occlusive or not-occlusive),

- dose levels (including vehicle, if used) and concentrations,

- no-effect level, where possible,

- toxic response data by sex and dose,

- time of death during the study or whether animals survived to termination,

- toxic or other effects,

- the time of observation of each abnormal sign and its subsequent course,

- food and body-weight data,

- haematological tests employed and results,

- clinical biochemistry tests employed and results,

- necropsy findings,

- a detailed description of all histopathological findings,

- statistical treatment of results where possible,

- discussion of the results,

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B (D).

4. REFERENCES

See General introduction Part B (E).

B.10. MUTAGENICITY — IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST

1. METHOD

This method is a replicate of the OECD TG 473, In Vitro Mammalian Chromosome Aberration Test (1997).

1.1. INTRODUCTION

The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells (1)(2)(3). Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. However, this method is not designed to measure numerical aberrations and is not routinely used for that purpose. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumour-suppressor genes of somatic cells are involved in cancer induction in humans and experimental animals.

The in vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. The cells used are selected on the basis of growth ability in culture, stability of the karyotype, chromosome number, chromosome diversity and spontaneous frequency of chromosome aberrations.

Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian in vivo conditions. Care should be taken to avoid conditions which would lead to positive results which do not reflect intrinsic mutagenicity and may arise from changes in pH, osmolality or high levels of cytotoxicity (4)(5).

This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through mechanisms other than direct DNA damage.

See also General introduction Part B.

1.2. DEFINITIONS

Chromatid-type aberration: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids.

Chromosome-type aberration: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.

Endoreduplication: a process in which after an S period of DNA replication, the nucleus does not go into mitosis but starts another S period. The result is chromosomes with four, eight, 16, …chromatids.

Gap: an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatids).

Mitotic index: the ratio of cells in metaphase divided by the total number of cells observed in a population of cells; an indication of the degree of proliferation of that population.

Numerical aberration: a change in the number of chromosomes from the normal number characteristic of the cells utilised.

Polyploidy: a multiple of the haploid chromosome number (n) other than the diploid number (i.e. 3n, 4n and so on).

Structural aberration: a change in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.

1.3. PRINCIPLE OF THE TEST METHOD

Cell cultures are exposed to the test substance both with and without metabolic activation. At predetermined intervals after exposure of cell cultures to the test substance, they are treated with a metaphase-arresting substance (e.g. Colcemid® or colchicine), harvested, stained and metaphase cells are analysed microscopically for the presence of chromosome aberrations.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparations

1.4.1.1. Cells

A variety of cell lines, strains or primary cell cultures, including human cells, may be used (e.g. Chinese hamster fibroblasts, human or other mammalian peripheral blood lymphocytes).

1.4.1.2. Media and culture conditions

Appropriate culture media and incubation conditions (culture vessels, CO2 concentration, temperature and humidity) should be used in maintaining cultures. Established cell lines and strains should be checked routinely for stability in the modal chromosome number and the absence of mycoplasma contamination and should not be used if contaminated. The normal cell cycle time for the cells and culture conditions used should be known.

1.4.1.3. Preparation of cultures

Established cell lines and strains: cells are propagated from stock cultures, seeded in culture medium at a density such that the cultures will not reach confluency before the time of harvest, and incubated at 37 oC.

Lymphocytes: whole blood treated with an anti-coagulant (e.g. heparin) or separated lymphocytes obtained from healthy subjects are added to the culture medium containing a mitogen (e.g. phytohaemagglutinin) and incubated at 37 oC.

1.4.1.4. Metabolic activation

Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as: Aroclor 1254 (6)(7)(8)(9), or a mixture of phenobarbitone and ß–naphthoflavone (10)(11)(12).

The post-mitochondrial fraction is usually used at concentrations in the range from 1-10 % v/v in the final test medium. The condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases it may be appropriate to utilise more than one concentration of post-mitochondrial fraction.

A number of developments, including the construction of genetically engineered cell lines expressing specific activating enzymes, may provide the potential for endogenous activation. The choice of the cell lines used should be scientifically justified (e.g. by the relevance of the cytochrome P450 isoenzyme for the metabolism of the test substance).

1.4.1.5. Test substance/Preparation

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the cells. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.

1.4.2. Test conditions

1.4.2.1. Solvent/vehicle

The solvent/vehicle should not be suspected of chemical reaction with the test substance and should be compatible with the survival of the cells and the S9 activity. If other than well-known solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used should be free of water. Water can be removed by adding a molecular sieve.

1.4.2.2. Exposure concentrations

Among the criteria to be considered when determining the highest concentration are cytotoxicity, solubility in the test system and changes in pH or osmolality.

Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell integrity and growth, such as degree of confluency, viable cell counts, or mitotic index. It may be useful to determine cytotoxicity and solubility in a preliminary experiment.

At least three analysable concentrations should be used. Where cytotoxicity occurs, these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentrations should be separated by no more than a factor between 2 and √10. At the time of harvesting, the highest concentration should show a significant reduction in degree of confluency, cell count or mitotic index, (all greater than 50 %). The mitotic index is only an indirect measure of cytotoxic/cytostatic effects and depends on the time after treatment. However, the mitotic index is acceptable for suspension cultures in which other toxicity measurements may be cumbersome and impractical. Information on cell cycle kinetics, such as average generation time (AGT), could be used as supplementary information. AGT, however, is an overall average that does not always reveal the existence of delayed subpopulations, and even slight increases in average generation time can be associated with very substantial delay in the time of optimal yield of aberrations.

For relatively non-cytotoxic substances, the maximum test concentration should be 5 μl/ml, 5 mg/ml or 0,01 M, whichever is the lowest.

For relatively insoluble substances that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium at the end of the treatment period. In some cases (e.g. when toxicity occurs only at higher than the lowest insoluble concentration) it is advisable to test at more than one concentration with visible precipitation. It may be useful to assess solubility at the beginning and the end of the treatment, as solubility can change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring.

1.4.2.3. Negative and positive controls

Concurrent positive and negative (solvent or vehicle) controls, both with and without metabolic activation, should be included in each experiment. When metabolic activation is used, the positive control chemical should be the one that requires activation to give a mutagenic response.

Positive controls should employ a known clastogen at exposure levels expected to give a reproducible and detectable increase over background, which demonstrates the sensitivity of the test system.

Positive control concentrations should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. Examples of positive control substances include:

Metabolic Activation condition | Substance | CAS No | EINECS No |

Absence of exogenous metabolic Activation | Methyl methanesulphonate | 66-27-3 | 200-625-0 |

Ethyl methanesulphonate | 62-50-0 | 200-536-7 |

Ethyl nitrosourea | 759-73-9 | 212-072-2 |

Mitomycin C | 50-07-7 | 200-008-6 |

4-Nitroquinoline-N-oxide | 56-57-5 | 200-281-1 |

Presence of exogenous metabolic Activation | Benzo[a]pyrene | 50-32-8 | 200-028-5 |

Cyclophosphamide Cyclophosphamide monohydrate | 50-18-0 6055-19-2 | 200-015-4 |

Other appropriate positive control substances may be used. The use of chemical class-related positive control chemicals should be considered, when available.

Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment cultures, should be included for every harvest time. In addition, untreated controls should also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.

1.4.3. Procedure

1.4.3.1. Treatment with the test substance

Proliferating cells are treated with the test substance in the presence and absence of a metabolic activation system. Treatment of lymphocytes should commence at about 48 hours after mitogenic stimulation.

1.4.3.2. Duplicate cultures should normally be used at each concentration, and are strongly recommended for negative/solvent control cultures. Where minimal variation between duplicate cultures can be demonstrated (13)(14), from historical data, it may be acceptable for single cultures to be used at each concentration.

Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels (15)(16).

1.4.3.3. Culture harvest time

In the first experiment, cells should be exposed to the test substance, both with and without metabolic activation, for three to six hours, and sampled at a time equivalent to about 1,5 normal cell cycle length after the beginning of treatment (12). If this protocol gives negative results both with and without activation, an additional experiment without activation should be done, with continuous treatment until sampling at a time equivalent to about 1,5 normal cell cycle lengths. Certain chemicals may be more readily detected by treatment/sampling times longer than 1,5 cycle lengths. Negative results with metabolic activation need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided.

1.4.3.4. Chromosome preparation

Cell cultures are treated with Colcemid® or colchicine usually for one to three hours prior to harvesting. Each cell culture is harvested and processed separately for the preparation of chromosomes. Chromosome preparation involves hypotonic treatment of the cells, fixation and staining.

1.4.3.5. Analysis

All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. Since fixation procedures often result in the breakage of a proportion of metaphase cells with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to the modal number ± 2 for all cell types. At least 200 well spread metaphases should be scored per concentration and control, equally divided amongst the duplicates, if applicable. This number can be reduced when high number of aberrations is observed.

Though the purpose of the test is to detect structural chromosome aberrations, it is important to record polyploidy and endoreduplication when these events are seen.

2. DATA

2.1. TREATMENT OF RESULTS

The experimental unit is the cell, and therefore the percentage of cells with structural chromosome aberration(s) should be evaluated. Different types of structural chromosome aberrations should be listed with their numbers and frequencies for experimental and control cultures. Gaps are recorded separately and reported but generally not included in the total aberration frequency.

Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiments should also be recorded.

Individual culture data should be provided. Additionally, all data should be summarised in tabular form.

There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using modification of experimental conditions. The need to confirm negative results has been discussed in 1.4.3.3. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing and the metabolic activation conditions.

2.2. EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (3)(13). Statistical significance should not be the only determining factor for a positive response.

An increase in the number of polyploid cells may indicate that the test substance has the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. An increase in the number of cells with endoreduplicated chromosomes may indicate that the test substance has the potential to inhibit cell cycle progression (17)(18).

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this system.

Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.

Positive results from the in vitro chromosome aberration test indicate that the test substance induces structural chromosome aberrations in cultured mammalian somatic cells. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in cultured mammalian somatic cells.

3. REPORTING

TEST REPORT

The test report must include the following information:

Solvent/Vehicle:

- justification for choice of vehicle,

- solubility and stability of the test substance in solvent/vehicle, if known.

Cells:

- type and source of cells,

- karyotype features and suitability of the cell type used,

- absence of mycoplasma, if applicable,

- information on cell cycle length,

- sex of blood donors, whole blood or separated lymphocytes, mitogen used,

- number of passages, if applicable,

- methods for maintenance of cell culture, if applicable,

- modal number of chromosomes.

Test conditions:

- identity of metaphase arresting substance, its concentration and duration of cell exposure,

- rationale for selection of concentrations and number of cultures including, e.g. cytotoxicity data and solubility limitations, if available,

- composition of media, CO2 concentration if applicable,

- concentration of test substance,

- volume of vehicle and test substance added,

- incubation temperature,

- incubation time,

- duration of treatment,

- cell density at seeding, if appropriate,

- type and composition of metabolic activation system, including acceptability criteria,

- positive and negative controls,

- methods of slide preparation,

- criteria for scoring aberrations,

- number of metaphases analysed,

- methods for the measurements of toxicity,

- criteria for considering studies as positive, negative or equivocal.

Results:

- signs of toxicity, e.g. degree of confluency, cell cycle data, cell counts, mitotic index,

- signs of precipitation,

- data on pH and osmolality of the treatment medium, if determined,

- definition for aberrations, including gaps,

- number of cells with chromosome aberrations and type of chromosome aberrations given separately for each treated and control culture,

- changes in ploidy if seen,

- dose-response relationship, where possible,

- statistical analyses, if any,

- concurrent negative (solvent/vehicle) and positive control data,

- historical negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations.

Discussion of results.

Conclusions.

4. REFERENCES

(1) Evans, H. J., (1976) Cytological Methods for Detecting Chemical Mutagens. In: Chemical mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. (ed) Plenum Press, New York and London, p. 1-29.

(2) Ishidate, M. Jr. and Sofuni, T., (1985). The In Vitro Chromosomal Aberration Test Using Chinese Hamster Lung (CHL) Fibroblast Cells in Culture. In: Progress in Mutation Research, Vol. 5, Ashby, J. et al., (Eds) Elsevier Science Publishers, Amsterdam-New York-Oxford, p. 427-432.

(3) Galloway, S.M., Armstrong, M.J., Reuben, C., Colman, S., Brown, B., Cannon, C., Bloom, A.D., Nakamura, F., Ahmed, M., Duk, S., Rimpo, J., Margolin, G.H., Resnick, MA, Anderson, G. and Zeiger, E., (1978). Chromosome aberration and sister chromatic exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Environs. Molec. Mutagen 10 (suppl. 10), p. 1-175.

(4) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M.Jr., Brusick, D., Ashby, J. and Myhr, B.C., (1991) Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Res, 257, p. 147-204.

(5) Morita, T., Nagaki, T., Fukuda, I. and Okumura, K., (1992). Clastogenicity of low pH to Various Cultured Mammalian Cells. Mutation Res., 268, p. 297-305.

(6) Ames, B.N., McCann, J. and Yamasaki, E., (1975). Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test. Mutation Res., 31, p. 347-364.

(7) Maron, D.M. and Ames, B.N., (1983) Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, p. 173-215.

(8) Natarajan, A.T., Tates, A.D., van Buul, P.P.W., Meijers, M. and de Vogel, N., (1976) Cytogenetic Effects of Mutagen/Carcinogens after Activation in a Microsomal System in Vitro, I. Induction of Chromosome Aberrations and Sister Chromatid Exchange by Diethylnitrosamine (DEN) and Dimethylnitrosamine (DMN) in CHO Cells in the Presence of Rat-Liver Microsomes. Mutation Res., 37, p. 83-90.

(9) Matsuoka, A., Hayashi, M. and Ishidate, M. Jr., (1979) Chromosomal Aberration Tests on 29 Chemicals Combined with S9 Mix In Vitro. Mutation Res., 66, p. 277-290.

(10) Elliot, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M. and Wolf, R.C., (1992). Report of UK Environmental Mutagen Society Working Party. Alternatives to Aroclor 1254-induced S9 in In Vitro Genotoxicity Assays. Mutagenesis, 7, p. 175-177.

(11) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T., (1976) A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. In: de Serres, F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. (eds) In Vitro Metabolic Activation in Mutagenesis Testing, Elsevier, North-Holland, p. 85-88.

(12) Galloway, S.M., Aardema, M.J., Ishidate, M.Jr., Ivett, J.L., Kirkland, D.J., Morita, T., Mosesso, P., Sofuni, T., (1994) Report from Working Group on in In Vitro Tests for Chromosomal Aberrations. Mutation Res., 312, p. 241-261.

(13) Richardson, C., Williams, D.A., Allen, J.A., Amphlett, G., Chanter, D.O. and Phillips, B., (1989) Analysis of Data from In Vitro Cytogenetic Assays. In: Statistical Evaluation of Mutagenicity Test Data. Kirkland, D.J., (ed) Cambridge University Press, Cambridge, p. 141-154.

(14) Soper, K.A. and Galloway, S.M., (1994) Replicate Flasks are not Necessary for In Vitro Chromosome Aberration Assays in CHO Cells. Mutation Res., 312, p. 139-149.

(15) Krahn, D.F., Barsky, F.C. and McCooey, K.T., (1982) CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. In: Tice, R.R., Costa, D.L., Schaich, K.M. (eds). Genotoxic Effects of Airborne Agents. New York, Plenum, p. 91-103.

(16) Zamora, P.O., Benson, J.M., Li, A.P. and Brooks, A.L., (1983) Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis, 5, p. 795-801.

(17) Locke-Huhle, C., (1983) Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest. Mutation Res., 119, p. 403-413.

(18) Huang, Y., Change, C. and Trosko, J.E., (1983) Aphidicolin-induced endoreduplication in Chinese hamster cells. Cancer Res., 43, p. 1362-1364.

B.11. MUTAGENICITY — IN VIVO MAMMALIAN BONE MARROW CHROMOSOME ABERRATION TEST

1. METHOD

This method is a replicate of the OECD TG 475, Mammalian Bone Marrow Chromosome Aberration Test (1997).

1.1. INTRODUCTION

The mammalian in vivo chromosome aberration test is used for the detection of structural chromosome aberrations induced by the test substance to the bone marrow cells of animals, usually rodents (1)(2)(3)(4). Structural chromosome aberrations may be of two types, chromosome or chromatid. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. With the majority of chemical mutagens, induced aberrations are of the chromatid-type, but chromosome-type aberrations also occur. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumour-suppressor genes are involved in cancer in humans and experimental systems.

Rodents are routinely used in this test. Bone marrow is the target tissue in this test, since it is a highly vascularised tissue, and it contains a population of rapidly cycling cells that can be readily isolated and processed. Other species and target tissues are not the subject of this method.

This chromosome aberration test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species and among tissues. An in vivo test is also useful for further investigation of a mutagenic effect detected by in vitro test.

If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.

See also General introduction Part B.

1.2. DEFINITIONS

Chromatid-type aberration: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids.

Chromosome-type aberration: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.

Gap: an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatid(s).

Numerical aberration: a change in the number of chromosomes from the normal number characteristic of the cells utilised.

Polyploidy: a multiple of the haploid chromosome number (n) other than the diploid number (i.e. 3n, 4n and so on).

1.3. PRINCIPLE OF THE TEST METHOD

Animals are exposed to the test substance by an appropriate route of exposure and are sacrificed at appropriate times after treatment. Prior to sacrifice, animals are treated with a metaphase arresting agent (e.g. Colcemid® or colchicine). Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells are analysed for chromosome aberrations.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparations

1.4.1.1. Selection of animal species

Rats, mice and Chinese hamsters are commonly used, although any appropriate mammalian species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. At the commencement of the study, the weight variation of animals should be minimal and not exceed ± 20 % of the mean weight of each sex.

1.4.1.2. Housing and feeding conditions

General conditions referred in the General introduction to Part B are applied although the aim for humidity should be 50-60 %.

1.4.1.3. Preparation of the animals

Healthy young adult animals are randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals are identified uniquely. The animals are acclimated to the laboratory conditions for at least five days.

1.4.1.4. Preparation of doses

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted, if appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.

1.4.2. Test conditions

1.4.2.1. Solvent/Vehicle

The solvent/vehicle should not produce toxic effects at the dose levels used, and should not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle should be considered first.

1.4.2.2. Controls

Concurrent positive and negative (solvent/vehicle) controls should be included for each sex in each test. Except for treatment with the test substance, animals in the control groups should be handled in an identical manner to the animals in the treated groups.

Positive controls should produce structural aberrations in vivo at exposure levels expected to give a detectable increase over background. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. The use of chemical class related positive control chemicals may be considered, when available. Examples of positive control substances include:

Substance | CAS No | EINECS No |

Ethyl methanesulphonate | 62-50-0 | 200-536-7 |

Ethyl nitrosourea | 759-73-9 | 212-072-2 |

Mitomycin C | 50-07-7 | 200-008-6 |

Cyclophosphamide Cyclophosphamide monohydrate | 50-18-0 6055-19-2 | 200-015-4 |

Triethylenemelamine | 51-18-3 | 200-083-5 |

Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups, should be included for every sampling time, unless acceptable inter-animal variability and frequencies of cells with chromosome aberrations are available from historical control data. If single sampling is applied for negative controls, the most appropriate time is the first sampling time. In addition, untreated controls should also be used unless there are historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle.

1.5. PROCEDURE

1.5.1. Number and sex of animals

Each treated and control group must include at least five analysable animals per sex. If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences in toxicity between sexes, then testing in a single sex will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.

1.5.2. Treatment schedule

Test substances are preferably administered as a single treatment. Test substances may also be administered as a split dose, i.e. two treatments on the same day separated by no more than a few hours, to facilitate administering a large volume of material. Other dose regimens should be scientifically justified.

Samples should be taken at two separate times following treatment on one day. For rodents, the first sampling interval is 1,5 normal cell cycle length (the latter being normally 12-18 hr) following treatment. Since the time required for uptake and metabolism of the test substance as well as its effect on cell cycle kinetics can affect the optimum time for chromosome aberration detection, a later sample collection 24 hr after the first sample time is recommended. If dose regimens of more than one day are used, one sampling time at 1,5 normal cell cycle lengths after the final treatment should be used.

Prior to sacrifice, animals are injected intraperitoneally with an appropriate dose of a metaphase arresting agent (e.g. Colcemid® or colchicine). Animals are sampled at an appropriate interval thereafter. For mice this interval is approximately three to five hours; for Chinese hamsters this interval is approximately four to five hours. Cells are harvested from the bone marrow and analysed for chromosome aberrations.

1.5.3. Dose levels

If a range finding study is performed because there are no suitable data available, it should be performed in the same laboratory, using the same species, strain, sex and treatment regimen to be used in the main study (5). If there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity in the bone marrow (e.g. greater than 50 % reduction in mitotic index).

1.5.4. Limit test

If a test at one dose level of at least 2000 mg/kg body weight using a single treatment, or as two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based on data from structurally related substances, then a full study using three dose levels may not be considered necessary. For studies of a longer duration, the limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.

1.5.5. Administration of doses

The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100 g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

1.5.6. Chromosome preparation

Immediately after sacrifice, bone marrow is obtained, exposed to hypotonic solution and fixed. The cells are then spread on slides and stained.

1.5.7. Analysis

The mitotic index should be determined as a measure of cytotoxicity in at least 1000 cells per animal for all treated animals (including positive controls) and untreated negative control animals.

At least 100 cells should be analysed for each animal. This number could be reduced when high numbers of aberrations are observed. All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. Since slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to the number 2n ± 2.

2. DATA

2.1. TREATMENT OF RESULTS

Individual animal data should be presented in tabular form. The experimental unit is the animal. For each animal the number of cells scored, the number of aberrations per cell and the percentage of cells with structural chromosome aberration(s) should be evaluated. Different types of structural chromosome aberrations should be listed with their numbers and frequencies for treated and control groups. Gaps are recorded separately and reported but generally not included in the total aberration frequency. If there is no evidence for a difference in response between the sexes, the data from both sexes may be combined for statistical analysis.

2.2. EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a dose-related increase in the relative number of cells with chromosome aberrations or a clear increase in the number of cells with aberrations in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (6). Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.

An increase in polyploidy may indicate that the test substance has the potential to induce numerical chromosome aberrations. An increase in endoreduplication may indicate that the test substance has the potential to inhibit cell cycle progression (7)(8).

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.

Positive results from the in vivo chromosome aberration test indicate that a substance induces chromosome aberrations in the bone marrow of the species tested. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in the bone marrow of the species tested.

The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g. systemic toxicity) should be discussed.

3. REPORTING

3.1. TEST REPORT

The test report must include the following information:

Solvent/Vehicle:

- justification for choice of vehicle,

- solubility and stability of the test substance in solvent/vehicle, if known,

Test animals:

- species/strain used,

- number, age and sex of animals,

- source, housing conditions, diet, etc.,

- individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group,

Test conditions:

- positive and negative (vehicle/solvent) controls,

- data from range-finding study, if conducted,

- rationale for dose level selection,

- details of test substance preparation,

- details of the administration of the test substance,

- rationale for route of administration,

- methods for verifying that the test substance reached the general circulation or target tissue, if applicable,

- conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable,

- details of food and water quality,

- detailed description of treatment and sampling schedules,

- methods for measurements of toxicity,

- identity of metaphase arresting substance, its concentration and duration of treatment,

- methods of slide preparation,

- criteria for scoring aberrations,

- number of cells analysed per animal,

- criteria for considering studies as positive, negative or equivocal.

Results:

- signs of toxicity,

- mitotic index,

- type and number of aberrations, given separately for each animal,

- total number of aberrations per group with means and standard deviations,

- number of cells with aberrations per group with means and standard deviations,

- changes in ploidy, if seen,

- dose-response relationship, where possible,

- statistical analyses, if any,

- concurrent negative control data,

- historical negative control data with ranges, means and standard deviations,

- concurrent positive control data.

Discussion of the results.

Conclusions.

4. REFERENCES

(1) Adler, I.D., (1984) Cytogenetic Tests in Mammals. In: Mutagenicity Testing: a Practical Approach. S. Venitt and J.M. Parry (Eds). IRL Press, Oxford, Washington D.C., p. 275-306.

(2) Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, A.F. and Shelby, M. (1987). Mammalian In Vivo Cytogenetic Assays: Analysis of Chromosome Aberrations in Bone Marrow Cells. Mutation Res., 189, p. 157-165.

(3) Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., Bootman, J. and Henderson, L., (1990) In Vivo Cytogenetic Assays. In: D.J. Kirkland (Ed.) Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report. Part I revised. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 115-141.

(4) Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., Blakey, D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., F.B., Pacchierotti, F., Preston, R.J., Romagna, F., Shimada, H., Sutou, S. and Vannier, B., (1994) Report from the Working Group on the In Vivo Mammalian Bone Marrow Chromosomal Aberration Test. Mutation Res., 312, p. 305-312.

(5) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D.J. and Richold, M. (1992). Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose setting in In vivo Mutagenicity Assays. Mutagenesis, 7, p. 313-319.

(6) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G. and Savage, J.R.K. (1989). Statistical Analysis of In Vivo Cytogenetic Assays. In: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report Part III. Statistical Evaluation of Mutagenicity Test Data. D.J. Kirkland, (Ed.) Cambridge University Press, Cambridge. p. 184-232.

(7) Locke-Huhle, C., (1983) Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest. Mutation Res. 119, p. 403-413.

(8) Huang, Y., Change, C. and Trosko, J.E., (1983) Aphidicolin-induced endoreduplication in Chinese hamster cells. Cancer Res., 43, p. 1362-1364.

B.12. MUTAGENICITY — IN VIVO MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST

1. METHOD

This method is a replicate of the OECD TG 474, Mammalian Erythrocyte Micronucleus Test (1997).

1.1. INTRODUCTION

The mammalian in vivo micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents.

The purpose of the micronucleus test is to identify substances that cause cytogenetic damage, which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.

When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.

The bone marrow of rodents is routinely used in this test since polychromatic erythrocytes are produced in that tissue. The measurement of micronucleated immature (polychromatic) erythrocytes in peripheral blood is equally acceptable in any species in which the inability of the spleen to remove micronucleated erythrocytes has been demonstrated, or which has shown an adequate sensitivity to detect agents that cause structural or numerical chromosome aberrations. Micronuclei can be distinguished by a number of criteria. These include identification of the presence or absence of a kinetochore or centromeric DNA in the micronuclei. The frequency of micronucleated immature (polychromatic) erythrocytes is the principal endpoint. The number of mature (normochromatic) erythrocytes in the peripheral blood that contain micronuclei among a given number of mature erythrocytes can also be used as the endpoint of the assay when animals are treated continuously for four weeks or more.

This mammalian in vivo micronucleus test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species, among tissues and among genetic endpoints. An in vivo assay is also useful for further investigation of a mutagenic effect detected by an in vitro system.

If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.

See also General introduction Part B.

1.2. DEFINITIONS

Centromere (Kinetochore): region(s) of a chromosome with which spindle fibers are associated during cell division, allowing orderly movement of daughter chromosomes to the poles of the daughter cells.

Micronuclei: small nuclei, separate from and additional to the main nuclei of cells, produced during telophase of mitosis (meiosis) by lagging chromosome fragments or whole chromosomes.

Normochromatic erythrocyte: mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by stains selective for ribosomes.

Polychromatic erythrocyte: immature erythrocyte, in an intermediate stage of development, that still contains ribosomes and therefore can be distinguished from mature, normochromatic erythrocytes by stains selective for ribosomes.

1.3. PRINCIPLE OF THE TEST METHOD

Animals are exposed to the test substance by an appropriate route. If bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained (1)(2)(3)(4)(5)(6)(7). When peripheral blood is used, the blood is collected at appropriate times after treatment and smear preparations are made and stained (4)(8)(9)(10). For studies with peripheral blood, as little time as possible should elapse between the last exposure and cell harvest. Preparations are analysed for the presence of micronuclei.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparations

1.4.1.1. Selection of animal species

Mice or rats are recommended if bone marrow is used, although any appropriate mammalian species may be used. When peripheral blood is used, mice are recommended. However, any appropriate mammalian species may be used provided it is a species in which the spleen does not remove micronucleated erythrocytes or a species, which has shown an adequate sensitivity to detect agents that cause structural or numerical chromosome aberrations. Commonly used laboratory strains of young healthy animals should be employed. At the commencement of the study, the weight variation of animals should be minimal and not exceed ±20 % of the mean weight of each sex.

1.4.1.2. Housing and feeding conditions

General conditions referred in the General introduction to Part B are applied although the aim for humidity should be 50-60 %.

1.4.1.3. Preparation of the animals

Healthy young adult animals are randomly assigned to the control and treatment groups. The animals are identified uniquely. The animals are acclimated to the laboratory conditions for at least five days. Cages should be arranged in such a way that possible effects due to cage placement are minimised.

1.4.1.4. Preparation of doses

1.4.2. Test conditions

1.4.2.1. Solvent/Vehicle

The solvent/vehicle should not produce toxic effects at the dose levels used, and should not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with reference data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle should be considered first.

1.4.2.2. Controls

Positive controls should produce micronuclei in vivo at exposure levels expected to give a detectable increase over background. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. In addition, the use of chemical class-related positive control chemicals may be considered, when available. Examples of positive control substances include:

Substance | CAS No | EINECS No |

Ethyl methanesulphonate | 62-50-0 | 200-536-7 |

N-ethyl-N-nitrosourea | 759-73-9 | 212-072-2 |

Mitomycin C | 50-07-7 | 200-008-6 |

Cyclophosphamide Cyclophosphamide monohydrate | 50-18-0 6055-19-2 | 200-015-4 |

Triethylenemelamine | 51-18-3 | 200-083-5 |

Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups should be included for every sampling time, unless acceptable inter-animal variability and frequencies of cells with micronuclei are demonstrated by historical control data. If single sampling is applied for negative controls, the most appropriate time is the first sampling time. In addition, untreated controls should also be used unless there are historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle.

If peripheral blood is used, a pre-treatment sample may also be acceptable as a concurrent negative control, but only in the short peripheral blood studies (e.g. 1-3 treatment(s)) when the resulting data are in the expected range for the historical control.

1.5. PROCEDURE

1.5.1. Number and sex of animals

Each treated and control group must include at least five analysable animals per sex (11). If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences between sexes in toxicity, then testing in a single sex will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.

1.5.2. Treatment schedule

No standard treatment schedule (i.e. one, two or more treatments at 24 h intervals) can be recommended. The samples from extended dose regimens are acceptable as long as a positive effect has been demonstrated for this study or, for a negative study, as long as toxicity has been demonstrated or the limit dose has been used, and dosing continued until the time of sampling. Test substances may also be administered as a split dose, i.e., two treatments on the same day separated by no more than a few hours, to facilitate administering a large volume of material.

The test may be performed in two ways:

(a) animals are treated with the test substance once. Samples of bone marrow are taken at least twice, starting not earlier than 24 hours after treatment, but not extending beyond 48 hours after treatment with appropriate intervals between samples. The use of sampling times earlier than 24 hours after treatment should be justified. Samples of peripheral blood are taken at least twice, starting not earlier than 36 hours after treatment, with appropriate intervals following the first sample, but not extending beyond 72 hours. When a positive response is recognised at one sampling time, additional sampling is not required.

(b) if two or more daily treatments are used (e.g. two or more treatments at 24 hour intervals), samples should be collected once between 18 and 24 hours following the final treatment for the bone marrow and once between 36 and 48 hours following the final treatment for the peripheral blood (12);

Other sampling times may be used in addition, when relevant.

1.5.3. Dose levels

If a range finding study is performed because there are no suitable data available, it should be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study (13). If there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity in the bone marrow (e.g. a reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow or peripheral blood).

1.5.4. Limit test

If a test at one dose level of at least 2000 mg/kg body weight using a single treatment, or as two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. For studies of a longer duration, the limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.

1.5.5. Administration of doses

The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100 g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

1.5.6. Bone marrow/blood preparation

Bone marrow cells are usually obtained from the femurs or tibias immediately following sacrifice. Commonly, cells are removed from femurs or tibias, prepared and stained using established methods. Peripheral blood is obtained from the tail vein or other appropriate blood vessel. Blood cells are immediately stained supravitally (8)(9)(10) or smear preparations are made and then stained. The use of a DNA specific stain (e.g. acridine orange (14) or Hoechst 33258 plus pyronin-Y (15)) can eliminate some of the artifacts associated with using a non DNA specific stain. This advantage does not preclude the use of conventional stains (e.g. Giemsa). Additional systems (e.g. cellulose columns to remove nucleated cells (16)) can also be used provided that these systems have been shown to adequately work for micronucleus preparation in the laboratory.

1.5.7. Analysis

The proportion of immature among total (immature + mature) erythrocytes is determined for each animal by counting a total of at least 200 erythrocytes for bone marrow and 1000 erythrocytes for peripheral blood (17). All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. At least 2000 immature erythrocytes per animal are scored for the incidence of micronucleated immature erythrocytes. Additional information may be obtained by scoring mature erythrocytes for micronuclei. When analysing slides, the proportion of immature erythrocytes among total erythrocytes should not be less than 20 % of the control value. When animals are treated continuously for four weeks or more, at least 2000 mature erythrocytes per animal can also be scored for the incidence of micronuclei. Systems for automated analysis (image analysis and cell suspensions flow cytometry) are acceptable alternatives to manual evaluation if appropriately justified and validated.

2. DATA

2.1. TREATMENT OF RESULTS

Individual animal data should be presented in tabular form. The experimental unit is the animal. The number of immature erythrocytes scored, the number of micronucleated immature erythrocytes, and the number of immature among total erythrocytes should be listed separately for each animal analysed. When animals are treated continuously for four weeks or more, the data on mature erythrocytes should also be given if it is collected. The proportion of immature among total erythrocytes and, if considered applicable, the percentage of micronucleated erythrocytes is given for each animal. If there is no evidence for a difference in response between the sexes, the data from both sexes may be combined for statistical analysis.

2.2. EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (18)(19). Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.

Positive results in the micronucleus test indicate that the substance induces micronuclei which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce micronuclei in the immature erythrocytes of the test species.

The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g. systemic toxicity) should be discussed.

3. REPORTING

TEST REPORT

The test report should include the following information:

Solvent/Vehicle:

- justification for choice of vehicle,

- solubility and stability of the test substance in solvent/vehicle, if known.

Test animals:

- species/strain used,

- number, age and sex of animals,

- source, housing conditions, diet, etc.,

- individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group.

Test conditions:

- positive and negative (vehicle/solvent) control data,

- data from range-finding study, if conducted,

- rationale for dose level selection,

- details of test substance preparation,

- details of the administration of the test substance,

- rationale for route of administration,

- methods for verifying that the test substance reached the general circulation or target tissue, if applicable,

- conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable,

- details of food and water quality,

- detailed description of treatment and sampling schedules,

- methods of slide preparation,

- methods for measurements of toxicity,

- criteria for scoring micronucleated immature erythrocytes,

- number of cells analysed per animal,

- criteria for considering studies as positive, negative or equivocal.

Results:

- signs of toxicity,

- proportion of immature erythrocytes among total erythrocytes,

- number of micronucleated immature erythrocytes, given separately for each animal,

- mean ± standard deviation of micronucleated immature erythrocytes per group,

- dose-response relationship, where possible,

- statistical analyses and methods applied,

- concurrent and historical negative control data,

- concurrent positive control data.

- Discussion of the results.

- Conclusions.

4. REFERENCES

(1) Heddle, J.A., (1973) A Rapid In Vivo Test for Chromosomal Damage, Mutation Res., 18, p. 187-190.

(2) Schmid, W., (1975) The Micronucleus Test, Mutation Res., 31, p. 9-15.

(3) Heddle, J.A., Salamone, M.F., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J.G. and Newell, G.W. (1983). The Induction of Micronuclei as a Measure of Genotoxicity. Mutation Res., 123, p. 61-118.

(4) Mavournin, K.H., Blakey, D.H., Cimino, M.C., Salamone, M.F. and Heddle, J.A., (1990) The In Vivo Micronucleus Assay in Mammalian Bone Marrow and Peripheral Blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 239, p. 29-80.

(5) MacGregor, J.T., Schlegel, R. Choy, W.N., and Wehr, C.M., (1983) Micronuclei in Circulating Erythrocytes: A Rapid Screen for Chromosomal Damage During Routine Toxicity Testing in Mice. Iin: "Developments in Science and Practice of Toxicology", Ed. A.W. Hayes, R.C. Schnell and T.S. Miya, Elsevier, Amsterdam, p. 555-558.

(6) MacGregor, J.T., Heddle, J.A. Hite, M., Margolin, G.H., Ramel, C., Salamone, M.F., Tice, R.R., and Wild, D., (1987) Guidelines for the Conduct of Micronucleus Assays in Mammalian Bone Marrow Erytrocytes. Mutation Res., 189, p. 103-112.

(7) MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.E., (1990) The in vivo Erythrocyte Micronucleus Test: Measurement at Steady State Increases Assay Efficiency and Permits Integration with Toxicity Studies. Fund. Appl. Toxicol., 14, p. 513-522.

(8) Hayashi, M., Morita, T., Kodama, Y., Sofuni, T. and Ishidate, M. Jr., (1990) The Micronucleus Assay with Mouse Peripheral Blood Reticulocytes Using Acridine Orange-Coated Slides. Mutation Res., 245, p. 245-249.

(9) The Collaborative Study Group for the Micronucleus Test, (1992) Micronucleus Test with Mouse Peripheral Blood Erythrocytes by Acridine Orange Supravital Staining: The Summary Report of the 5th Collaborative Study by CSGMT/JEMMS. MMS. Mutation Res., p. 278, 83-98.

(10) The Collaborative Study Group for the Micronucleus Test (CSGMT/JEMMS. MMS: The Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan), (1995) Protocol recommended for the short-term mouse peripheral blood micronucleus test. Mutagenesis, 10, p. 153-159.

(11) Hayashi, M., Tice, R.R., MacGregor, J.T., Anderson, D., Blackey, D.H., Kirsch-Volders, M., Oleson, Jr. F. B., Pacchierotti, F., Romagna, F., Shimada, H., Sutou, S. and Vannier, B., (1994) In Vivo Rodent Erythrocyte Micronucleus Assay. Mutation Res., 312, p. 293-304.

(12) Higashikuni, N. and Sutou, S., (1995) An optimal, generalised sampling time of 30 ± 6 h after double dosing in the mouse peripheral blood micronucleus test. Mutagenesis, 10, p. 313-319.

(13) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D.J. and Rochold, M., (1992) Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo Mutagenicity Assays. Mutagenesis, 7, p. 313-319.

(14) Hayashi, M., Sofuni, T. and Ishidate, M. Jr., (1983) An Application of Acridine Orange Fluorescent Staining to the Micronucleus Test. Mutation Res., 120, p. 241-247.

(15) MacGregor, J.T., Wehr, C.M. and Langlois, R.G., (1983) A Simple Fluorescent Staining Procedure for Micronuclei and RNA in Erythrocytes Using Hoechst 33258 and Pyronin Y. Mutation Res., 120, p. 269-275.

(16) Romagna, F. and Staniforth, C.D., (1989) The automated bone marrow micronucleus test. Mutation Res., 213, p. 91-104.

(17) Gollapudi, B. and McFadden, L.G., (1995) Sample size for the estimation of polychromatic to normochromatic erythrocyte ratio in the bone marrow micronucleus test. Mutation Res., 347, p. 97-99.

(18) Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, D.G. and Henderson, L., (1990) In Vivo Cytogenetics Assay. In: D.J. Kirkland (Ed.) Basic Mutagenicity tests. UKEMS Recommended Procedures. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report, Part I, revised. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 115-141.

(19) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G. and Savage, J.R.K., (1989) Statistical Analysis of In Vivo Cytogenetic Assays. In: D.J. Kirkland (Ed.) Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report, Part III. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 184-232.

B.13/14. MUTAGENICITY: REVERSE MUTATION TEST USING BACTERIA

1. METHOD

This method is a replicate of the OECD TG 471, Bacterial Reverse Mutation Test (1997).

1.1. INTRODUCTION

The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs (1)(2)(3). The principle of this bacterial reverse mutation test is that it detects mutations, which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesise an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino-acid required by the parent test strain.

Point mutations are the cause of many human genetic diseases and there is substantial evidence that point mutations in oncogenes and tumour-suppressor genes of somatic cells are involved in tumour formation in humans and experimental animals. The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. Many of the test strains have several features that make them more sensitive for the detection of mutations including responsive DNA sequences at the reversion sites, increased cell permeability to large molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair processes. The specificity of the test strains can provide some useful information on the types of mutations that are induced by genotoxic agents. A very large data base of results for a wide variety of structures is available for bacterial reverse mutation tests and well-established methodologies have been developed for testing chemicals with different physico-chemical properties, including volatile compounds.

See also General introduction Part B.

1.2. DEFINITIONS

A reverse mutation test in either Salmonella typhimurium or Escherichia coli detects mutation in an amino-acid requiring strain (histidine or tryptophan, respectively) to produce a strain independent of an outside supply of amino-acid.

Base pair substitution mutagens are agents that cause a base change in DNA. In a reversion test this change may occur at the site of the original mutation, or at a second site in the bacterial genome.

Frameshift mutagens are agents that cause the addition or deletion of one or more base pairs in the DNA, thus changing the reading frame in the RNA.

1.3. INITIAL CONSIDERATIONS

The bacterial reverse mutation test utilises prokaryotic cells, which differ from mammalian cells in such factors as uptake, metabolism, chromosome structure and DNA repair processes. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. In vitro metabolic activation systems cannot mimic entirely the mammalian in vivo conditions. The test therefore does not provide direct information on the mutagenic and carcinogenic potency of a substance in mammals.

The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity. An extensive database has demonstrated that many chemicals that are positive in this test also exhibit mutagenic activity in other tests. There are examples of mutagenic agents, which are not detected by this test; reasons for these shortcomings can be ascribed to the specific nature of the endpoint detected, differences in metabolic activation, or differences in bioavailability. On the other hand, factors, which enhance the sensitivity of the bacterial reverse mutation test can lead to an overestimation of mutagenic activity.

The bacterial reverse mutation test may not be appropriate for the evaluation of certain classes of chemicals, for example highly bactericidal compounds (e.g. certain antibiotics) and those which are thought (or known) to interfere specifically with the mammalian cell replication system (e.g. some topoisomerase inhibitors and some nucleoside analogues). In such cases, mammalian mutation tests may be more appropriate.

Although many compounds that are positive in this test are mammalian carcinogens, the correlation is not absolute. It is dependent on chemical class and there are carcinogens that are not detected by this test because they act through other, non-genotoxic, mechanisms or mechanisms absent in bacterial cells.

1.4. PRINCIPLE OF THE TEST METHOD

Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium. In the preincubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after two or three days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.

Several procedures for performing the bacterial reverse mutation test have been described. Among those commonly used are the plate incorporation method (1)(2)(3)(4), the preincubation method (2)(3)(5)(6)(7)(8), the fluctuation method (9)(10), and the suspension method (11). Modifications for the testing of gases or vapours have been described (12).

The procedures described in the method pertain primarily to the plate incorporation and preincubation methods. Either of them is acceptable for conducting experiments both with and without metabolic activation. Some substances may be detected more efficiently using the preincubation method. These substances belong to chemical classes that include short chain aliphatic nitrosamines, divalent metals, aldehydes, azo-dyes and diazo compounds, pyrollizidine alkaloids, allyl compounds and nitro compounds (3). It is also recognised that certain classes of mutagens are not always detected using standard procedures such as the plate incorporation method or preincubation method. These should be regarded as "special cases" and it is strongly recommended that alternative procedures should be used for their detection. The following "special cases" could be identified (together with examples of procedures that could be used for their detection): azo-dyes and diazo compounds (3)(5)(6)(13), gases and volatile chemicals (12)(14)(15)(16) and glycosides (17)(18). A deviation from the standard procedure needs to be scientifically justified.

1.5. DESCRIPTION OF THE TEST METHOD

1.5.1. Preparations

1.5.1.1. Bacteria

Fresh cultures of bacteria should be grown up to the late exponential or early stationary phase of growth (approximately 109 cells per ml). Cultures in late stationary phase should not be used. It is essential that the cultures used in the experiment contain a high titre of viable bacteria. The titre may be demonstrated either from historical control data on growth curves, or in each assay through the determination of viable cell numbers by a plating experiment.

The recommended incubation temperature is 37 oC.

At least five strains of bacteria should be used. These should include four strains of S. typhimurium (TA 1535; TA 1537 or TA97a or TA97; TA98; and TA100) that have been shown to be reliable and reproducibly responsive between laboratories. These four S. typhimurium strains have GC base pairs at the primary reversion site and it is known that may not detect certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E. coli WP2 strains or S. typhimurium TA102 (19), which have an AT base pair at the primary reversion site. Therefore the recommended combination of strains is:

- S. typhimurium TA1535, and

- S. typhimurium TA1537 or TA97 or TA97a, and

- S. typhimurium TA98, and

- S. typhimurium TA100, and

- E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102.

In order to detect cross-linking mutagens it may be preferable to include TA102 or to add a DNA repair-proficient strain of E. coli [e.g. E. coli WP2 or E. coli WP2 (pKM101)]

Established procedures for stock culture preparation, marker verification and storage should be used. The amino-acid requirement for growth should be demonstrated for each frozen stock culture preparation (histidine for S. typhimurium strains, and tryptophan for E. coli strains). Other phenotypic characteristics should be similarly checked, namely: the presence or absence of R-factor plasmids where appropriate [i.e. ampicillin resistance in strains TA98, TA100 and TA97a or TA97, WP2 uvrA and WP2 uvrA (pKM101), and ampicillin + tetracycline resistance in strain TA102]; the presence of characteristic mutations (i.e. rfa mutation in S. typhimurium through sensitivity to crystal violet, and uvrA mutation in E. coli or uvrB mutation in S. typhimurium, through sensitivity to ultraviolet light) (2)(3). The strains should also yield spontaneous revertant colony plate counts within the frequency ranges expected from the laboratory's historical control data and preferably within the range reported in the literature.

1.5.1.2. Medium

An appropriate minimal agar (e.g. containing Vogel-Bonner minimal medium E and glucose), and an overlay agar containing histidine and biotin or tryptophan to allow for a few cell divisions, is used (1)(2)(9).

1.5.1.3. Metabolic activation

Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (1)(2) or a combination of Phenobarbitone and ß-naphthoflavone (18)(20)(21). The post-mitochondrial fraction is usually used at concentrations in the range from 5 to 30 % v/v in the S9-mix. The choice and condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases, it may be appropriate to utilise more than one concentration of post-mitochondrial fraction. For azo-dyes and diazo-compounds, using a reductive metabolic activation system may be more appropriate (6)(13).

1.5.1.4. Test substance/Preparation

Solid test substances should be dissolved or suspended in appropriate solvents or vehicles and diluted if appropriate prior to treatment of the bacteria. Liquid test substances may be added directly to the test systems and/or diluted prior to treatment. Fresh preparations should be employed unless stability data demonstrate the acceptability of storage.

The solvent/vehicle should not be suspected of chemical reaction with the test substance and should be compatible with the survival of the bacteria and the S9 activity (22). If other than well-known solvent/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle be considered first. When testing water-unstable substances, the organic solvents used should be free of water.

1.5.2. Test conditions

1.5.2.1. Test strains (see 1.5.1.1)

1.5.2.2. Exposure concentration

Amongst the criteria to be taken into consideration when determining the highest amount of the test substance to be used are the cytotoxicity and the solubility in the final treatment mixture.

It may be useful to determine toxicity and insolubility in a preliminary experiment. Cytotoxicity may be detected by a reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or the degree of survival of treated cultures. The cytotoxicity of a substance may be altered in the presence of metabolic activation systems. Insolubility should be assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.

The recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 μl/plate. For non-cytotoxic substances that are not soluble at 5 mg/plate or 5 μl/plate, one or more concentrations tested should be insoluble in the final treatment mixture. Test substances that are cytotoxic already below 5 mg/plate or 5 μl/plate should be tested up to a cytotoxic concentration. The precipitate should not interfere with the scoring.

At least five different analysable concentrations of the test substance should be used with approximately half log (i.e. √10) intervals between test points for an initial experiment. Smaller intervals may be appropriate when a concentration-response is being investigated. Testing above the concentration of 5 mg/plate or 5 μl/plate may be considered when evaluating substances containing substantial amounts of potentially mutagenic impurities.

1.5.2.3. Negative and positive controls

Concurrent strain-specific positive and negative (solvent or vehicle) controls, both with and without metabolic activation, should be included in each assay. Positive control concentrations that demonstrate the effective performance of each assay should be selected.

For assays employing a metabolic activation system, the positive control reference substance(s) should be selected on the basis of the type of bacteria strains used.

The following substances are examples of suitable positive controls for assays with metabolic activation:

CA numbers | EINECS numbers | Names |

781-43-1 | 212-308-4 | 9,10-dimethylanthracene |

57-97-6 | 200-359-5 | 7,12-dimethylbenz[a]anthracene |

50-32-8 | 200-028-5 | benzo[a]pyrene |

613-13-8 | 210-330-9 | 2-aminoanthracene |

50-18-0 | | cyclophosphamide |

6055-19-2 | 200-015-4 | cyclophosphamide monohydrate |

The following substance is a suitable positive control for the reductive metabolic activation method:

CA numbers | EINECS numbers | Names |

573-58-0 | 209-358-4 | Congo Red |

2-Aminoanthracene should not be used as the sole indicator of the efficacy of the S9-mix. If 2-aminoanthracene is used, each batch of S9 should also be characterised with a mutagen that requires metabolic activation by microsomal enzymes, e.g. benzo[a]pyrene, dimethylbenzanthracene.

The following substances are examples of strain-specific positive controls for assays performed without exogenous metabolic activation system:

CAS numbers | EINECS numbers | Names | Strain |

26628-22-8 | 247-852-1 | Sodium azide | TA 1535 and TA 100 |

607-57-8 | 210-138-5 | 2-nitrofluorene | TA 98 |

90-45-9 | 201-995-6 | 9-aminoacridine | TA 1537, TA 97 and TA 97a |

17070-45-0 | 241-129-4 | ICR 191 | TA 1537, TA 97 and TA 97a |

80-15-9 | 201-254-7 | Cumene hydroperoxide | TA 102 |

50-07-7 | 200-008-6 | Mitomycin C | WP2 uvrA and TA102 |

70-25-7 | 200-730-1 | N-ethyl-N-nitro-N-nitrosoguanidine | WP2, WP2uvrA and WP2uvrA(pKM101) |

56-57-5 | 200-281-1 | 4-nitroquinoline-1-oxide | WP2, WP2uvrA and WP2uvrA(pKM101) |

3688-53-7 | | Furylfuramide (AF2) | plasmid-containing strains |

Other appropriate positive control reference substances may be used. The use of chemical class-related positive control chemicals should be considered, when available.

Negative controls, consisting of solvent or vehicle alone, without test substance, and otherwise treated in the same way as the treatment groups, should be included. In addition, untreated controls should also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.

1.5.3. Procedure

For the plate incorporation method (1)(2)(3)(4), without metabolic activation, usually 0,05 ml or 0,1 ml of the test solutions, 0,1 ml of fresh bacterial culture (containing approximately 108 viable cells) and 0,5 ml of sterile buffer are mixed with 2,0 ml of overlay agar. For the assay with metabolic activation, usually 0,5 ml of metabolic activation mixture containing an adequate amount of post-mitochondrial fraction (in the range from 5 to 30 % v/v in the metabolic activation mixture) are mixed with the overlay agar (2,0 ml), together with the bacteria and test substance/test solution. The contents of each tube are mixed and poured over the surface of a minimal agar plate. The overlay agar is allowed to solidify before incubation.

For the preincubation method (2)(3)(5)(6), the test substance/test solution is preincubated with the test strain (containing approximately 108 viable cells) and sterile buffer or the metabolic activation system (0,5 ml) usually for 20 min. or more at 30-37 oC prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. Usually, 0,05 or 0,1 ml of test substance/test solution, 0,1 ml of bacteria, and 0,5 ml of S9-mix or sterile buffer are mixed with 2,0 ml of overlay agar. Tubes should be aerated during pre-incubation by using a shaker.

For an adequate estimate of variation, triplicate plating should be used at each dose level. The use of duplicate plating is acceptable when scientifically justified. The occasional loss of a plate does not necessarily invalidate the assay.

Gaseous or volatile substances should be tested by appropriate methods, such as in sealed vessels (12)(14)(15)(16).

1.5.4. Incubation

All plates in a given assay should be incubated at 37 oC for 48-72 hours. After the incubation period, the number of revertant colonies per plate is counted.

2. DATA

2.1. TREATMENT OF RESULTS

Data should be presented as the number of revertant colonies per plate. The number of revertant colonies on both negative (solvent control, and untreated control if used) and positive control plates should also be given. Individual plate counts, the mean number of revertant colonies per plate and the standard deviation should be presented for the test substance and positive and negative (untreated and/or solvent) controls.

There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments. Study parameters that might be modified include the concentration spacing, the method of treatment (plate-incorporation or liquid pre-incubation), and metabolic activation conditions.

2.2. EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system (23). Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (24). However, statistical significance should not be the only determining factor for a positive response.

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.

Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.

3. REPORTING

TEST REPORT

The test report must include the following information:

Solvent/Vehicle:

- justification for choice of solvent/vehicle,

- solubility and stability of the test substance in solvent/vehicle, if known.

Strains:

- strains used,

- number of cells per culture,

- strain characteristics.

Test conditions:

- amount of test substance per plate (mg/plate or μl/plate) with rationale for selection of dose and number of plates per concentration,

- media used,

- type and composition of metabolic activation system, including acceptability criteria,

- treatment procedures.

Results:

- signs of toxicity,

- signs of precipitation,

- individual plate counts,

- the mean number of revertant colonies per plate and standard deviation,

- dose-response relationship, where possible,

- statistical analyses, if any,

- concurrent negative (solvent/vehicle) and positive control data, with ranges, means and standard deviations,

- historical negative (solvent/vehicle) and positive control data with ranges, means and standard deviations.

Discussion of results.

Conclusions.

4. REFERENCES

(1) Ames, B.N., McCann, J. and Yamasaki E., (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res., 31, p. 347-364.

(2) Maron, D.M. and Ames, B.N., (1983) Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, p. 173-215.

(3) Gatehouse, D., Haworth, S., Cebula, T., Gocke, E., Kier, L., Matsushima, T., Melcion, C., Nohmi, T., Venitt, S. and Zeiger, E., (1994) Recommendations for the Performance of Bacterial Mutation Assays. Mutation Res., 312, p. 217-233.

(4) Kier, L.D., Brusick D.J., Auletta, A.E., Von Halle, E.S., Brown, M.M., Simmon, V.F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T.K. and Ray V., (1986) The Salmonella typhimurium/Mammalian Microsomal Assay: A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res., 168, p. 69-240.

(5) Yahagi, T., Degawa, M., Seino, Y.Y., Matsushima, T., Nagao, M., Sugimura, T. and Hashimoto, Y., (1975) Mutagenicity of Carcinogen Azo Dyes and their Derivatives, Cancer Letters, 1, p. 91-96.

(6) Matsushima, M., Sugimura, T., Nagao, M., Yahagi, T., Shirai, A. and Sawamura, M., (1980) Factors Modulating Mutagenicity Microbial Tests. In: Short-term Test Systems for Detecting Carcinogens. Ed. Norpoth K.H. and Garner, R.C., Springer, Berlin-Heidelberg-New York. p. 273-285.

(7) Gatehouse, D.G., Rowland, I.R., Wilcox, P., Callender, R.D. and Foster, R., (1980) Bacterial Mutation Assays. In: Basic Mutagenicity Tests: UKEMS Part 1 Revised. Ed. D.J. Kirkland, Cambridge University Press, p. 13-61.

(8) Aeschacher, H.U., Wolleb, U. and Porchet, L., (1987) Liquid Preincubation Mutagenicity Test for Foods. J. Food Safety, 8, p. 167-177.

(9) Green, M.H.L., Muriel, W.J. and Bridges, B.A., (1976) Use of a simplified fluctuation test to detect low levels of mutagens. Mutation Res., 38, p. 33-42.

(10) Hubbard, S.A., Green, M.H.L., Gatehouse, D. and J.W. Bridges (1984) The Fluctuation Test in Bacteria. In: Handbook of Mutagenicity Test Procedures. 2nd Edition. Ed. Kilbey, B.J., Legator, M., Nichols, W. and Ramel, C., Elsevier, Amsterdam-New York-Oxford, p. 141-161.

(11) Thompson, E.D. and Melampy, P.J., (1981) An Examination of the Quantitative Suspension Assay for Mutagenesis with Strains of Salmonella typhimurium. Environmental Mutagenesis, 3, p. 453-465.

(12) Araki, A., Noguchi, T., Kato, F. and T. Matsushima (1994) Improved Method for Mutagenicity Testing of Gaseous Compounds by Using a Gas Sampling Bag. Mutation Res., 307, p. 335-344.

(13) Prival, M.J., Bell, S.J., Mitchell, V.D., Reipert, M.D. and Vaughn, V.L., (1984) Mutagenicity of Benzidine and Benzidine-Congener Dyes and Selected Monoazo Dyes in a Modified Salmonella Assay. Mutation Res., 136, p. 33-47.

(14) Zeiger, E., Anderson, B.E., Haworth, S., Lawlor, T. and Mortelmans, K., (1992) Salmonella Mutagenicity Tests. V. Results from the Testing of 311 Chemicals. Environ. Mol. Mutagen., 19, p. 2-141.

(15) Simmon, V., Kauhanen, K. and Tardiff, R.G., (1977) Mutagenic Activity of Chemicals Identified in Drinking Water. In Progress in Genetic Toxicology, D. Scott, B. Bridges and F. Sobels (Eds.) Elsevier, Amsterdam, p. 249-258.

(16) Hughes, T.J., Simmons, D.M., Monteith, I.G. and Claxton, L.D., (1987) Vaporisation Technique to Measure Mutagenic Activity of Volatile Organic Chemicals in the Ames/Salmonella Assay. Environmental Mutagenesis, 9, p. 421-441.

(17) Matsushima, T., Matsumoto, A., Shirai, M., Sawamura, M., and Sugimura, T., (1979) Mutagenicity of the Naturally Occurring Carcinogen Cycasin and Synthetic Methylazoxy Methane Conjugates in Salmonella typhimurium. Cancer Res., 39, p. 3780-3782.

(18) Tamura, G., Gold, C., Ferro-Luzzi, A. and Ames, B.N., (1980) Fecalase: A Model for Activation of Dietary Glycosides to Mutagens by Intestinal Flora. Proc. Natl. Acad. Sci. USA, 77, p. 4961-4965.

(19) Wilcox, P., Naidoo, A., Wedd, D.J. and Gatehouse, D.G., (1990) Comparison of Salmonella typhimurium TA 102 with Escherichia coli WP2 Tester strains. Mutagenesis, 5, p. 285-291.

(20) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T., (1976) A Safe Substitute for Polychlorinated Biphenyls as an Inducer or Metabolic Activation Systems. In: "In vitro metabolic Activation in Mutagenesis Testing" Eds. F.J. de Serres et al. Elsevier, North Holland, p. 85-88.

(21) Elliot, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M. and Wolf, R.C., (1992) Alternatives to Aroclor 1254-induced S9 in in vitro Genotoxicity Assays. Mutagenesis, 7, p. 175-177.

(22) Maron, D., Katzenellenbogen, J. and Ames, B.N., (1981) Compatibility of Organic Solvents with the Salmonella/Microsome Test. Mutation Res., p. 88343-350.

(23) Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E. and Zeiger, E., (1987) Guide for the Salmonella typhimurium/Mammalian Microsome Tests for Bacterial Mutagenicity. Mutation Res. 189, p. 83-91.

(24) Mahon, G.A.T., Green, M.H.L., Middleton, B., Mitchell, I., Robinson, W.D. and Tweats, D.J., (1989) Analysis of Data from Microbial Colony Assays. In: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Part II. Statistical Evaluation of Mutagenicity Test Data. Ed. Kirkland, D.J., Cambridge University Press, p. 28-65.

B.15. MUTAGENICITY TESTING AND SCREENING FOR CARCINOGENICITY GENE MUTATION — SACCHAROMYCES CEREVISIAE

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITION

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

A variety of haploid and diploid strains of the yeast Saccharomyces cerevisiae can be used to measure the production of gene mutations induced by chemical agents with and without metabolic activation.

Forward mutation systems in haploid strains, such as the measurement of mutation from red, adenine-requiring mutants (ade-1, ade-2) to double adenine-requiring white mutants and selective systems such as the induction of resistance to canavnaine and cycloheximide, have been utilised.

The most extensively validated reverse mutation system involves the use of the haploid strain XV 185-14C which carries the ochre nonsense mutations ade 2-1, arg 4-17, lys 1-1 and trp 5-48, which are reversible by base substitution mutagens that induce site specific mutations or ochre suppressor mutations. XV 185-14C also carries the his 1-7 marker, a missense mutation reverted mainly by second site mutations, and the marker hom 3-10 which is reverted by frameshift mutagens.

In diploid strains of S. cerevisiae the only extensively used strain is D7 which is homozygous for ilv 1-92.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

Preparations

Solutions of test chemicals and control should be prepared just prior to testing, using an appropriate vehicle. In the case of organic compounds, which are not water soluble, not more than a 2 % solution v/v of organic solvents such as ethanol, acetone or dimethylsulphoxide (DMSO) should be used. The final concentration of the vehicle should not significantly affect cell viability and growth characertistics.

Metabolic activation

Cells should be exposed to test chemicals both in the presence and absence of an appropriate exogenous metabolic activation system.

The most commonly used system is a co-factor supplemented post-mitochondrial fraction from the livers of rodents pre-treated with enzyme inducing agents. The use of other species, tissues, post-mitochondrial fractions, or procedures may also be appropriate for metabolic activation.

Test conditions

Tester strains

The haploid strain XV 185-14C and the diploid strain D7 are the most used in gene mutation studies. Other strains may also be appropriate.

Media

Appropriate culture media are used for the determination of survival and mutant numbers.

Use of negative and positive controls

Positive, untreated and solvent controls should be performed concurrently. Appropriate positive control chemicals should be used for each specific mutational endpoint.

Exposure concentration

At least five adequately spaced concentrations of the test substance should be used. For toxic substances, the highest concentration tested should not reduce survival below 5 to 10 % . Relatively water-insoluble substances should be tested up to their limit of solubility, using appropriate procedures. For freely water-soluble non-toxic substances, the upper concentration should be determined on a case by case basis.

Incubation conditions

The plates are incubated four to seven days at 28 to 30 oC in the dark.

Spontaneous mutation frequencies

Sub-cultures should be used with spontaneous mutation frequencies within the accepted normal range.

Number of replicates

At least three replicate plates should be used per concentration for the assay of prototrophs produced by gene mutation and for cell viability. In the case of experiments using markers such as hom 3-10 with a low mutation rate, the number of plates used must be increased to provide statistically relevant data.

Procedure

Treatment of S. cerevisiae strains is usually performed in a liquid test procedure involving either stationary or growing cells. Initial experiments should be carried out on growing cells: 1-5 × 107 cells/ml are exposed to the test chemical for up to 18 hours at 28 to 37 oC with shaking; an adequate amount of metabolic activation system is added during treatment when appropriate. At the end of the treatment, cells are centrifuged, washed and seeded upon an appropriate culture medium. After incubation, plates are scored for survival and the induction of gene mutation. If the first experiment is negative, then a second experiment should be carried out using stationary phase cells. If the first experiment is positive it is confirmed in an appropriate independent experiment.

2. DATA

Data should be presented in tabular form indicating the number of colonies counted, number of mutants, survival and mutant frequency. All results should be confirmed in an independent experiment. The dara should be evaluated using appropriate statistical methods.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- strain used,

- test conditions: stationary phase or growing cells, compositions of media, incubation temperature and duration, metabolic activation system,

- treatment conditions: exposure levels, procedure and duration of treatment, treatment temperature, positive and negative controls,

- number of colonies counted, number of mutants, survival and mutant frequency, dose/response relationship if applicable, statistical evaluation of data,

- discussion of results,

- interpretation of results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.16. MITOTIC RECOMBINATION — SACCHAROMYCES CEREVISIAE

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITION

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

Mitotic recombination in Saccharomyces cerevisiae can be detected between genes (or more generally between a gene and its centromere) and within genes. The former event is called mitotic crossing-over and generates reciprocal products whereas the latter event is most frequently non-reciprocal and is called gene conversion. Crossing-over is generally assayed by the production of recessive homozygous colonies or sectors produced in a heterozygous strain, whereas gene conversion is assayed by the production of prototrophic revertants produced in an auxotrophic heteroallelic strain carrying two different defective alleles of the same gene. The most commonly used strains for the detection of mitotic gene conversion are D4 (heteroallelic at ade 2 and trp 5) D7 (heteroallelic at trp 5) BZ34 (heteroallelic at arg 4) and JDl (heteroallelic at his 4 and trp 5). Mitotic crossing-over producing red and pink homozygous sectors can be assayed in D5 or in D7 (which also measures mitotic gene conversion and reverse mutation at ilv 1-92) both strains being heteroallelic for complementing alleles of ade 2.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

Preparations

Solutions of test chemicals and control or reference compounds should be prepared just prior to testing, using an appropriate vehicle. With organic compounds that are water insoluble not more than a 2 % solution v/v of organic solvents such as ethanol, acetone or dimethylsulphoxide (DMSO) should be used. The final concentration of the vehicle should not significantly affect cell viability and growth characteristics.

Metabolic activation

Cells should be exposed to test chemicals both in the presence and absence of an appropriate exogenous metabolic activation system. The system most commonly used is a co-factor supplemented post-mitochondrial fraction from the livers of rodents pre-treated with enzyme inducing agents. The use of other species, tissues, post-mitochondrial fractions, or procedures may also be appropriate for metabolic activation.

Test conditions

Tester strains

The most frequently used strains are the diploids D4, D5, D7 and JD1. The use of other strains may be appropriate.

Media

Appropriate culture media are used for the determination of survival and the frequency of mitotic recombination.

Use of negative and positive controls

Positive, untreated and solvent controls should be performed concurrently. Appropriate positive control chemicals should be used for each specific recombination endpoint.

Exposure concentrations

At least five adequately spaced concentrations of the test substance should be used. Among the factors to be taken into consideration are cytotoxicity and solubility. The lowest concentration must have no effect on celf viability. For toxic chemicals, the highest concentration tested should not reduce survival below 5 to 10 %. Relatively water-insoluble chemicals should be tested up to the limit of solubility using appropriate procedures. For freely water-soluble non-toxic substances the upper concentration should be determined on a case by case basis.

Cells may be exposed to test chemicals in either the stationary phase or during growth for periods of up to 18 hours. However, for long treatment times cultures should be microscopically inspected for spore formation, the presence of which invalidates the test.

Incubation conditions

The plates are incubated in the dark for four to seven days at 28 to 30 oC. Plates used for the assay of red and pink homozygous sectors produced by mitotic crossing-over should be kept in a refrigerator (about 4 oC) for a further one to two days before scoring to allow for the development of the appropriate pigmented colonies.

Spontaneous mitotic recombination frequencies

Sub-cultures should be used with spontaneous mitotic recombination mutation frequencies within the accepted normal range.

Number of replicates

A minimum of three replicate plates should be used per concentration for the assay of prototrophs produced by mitotic gene conversion and for viability. In the case of the assay of recessive homozygosis produced by mitotic crossing-over, the plate number should be increased to provide an adequate number of colonies.

Procedures

Treatment of S. cerevisiae strains is usually performed in a liquid test procedure involving either stationary or growing cells. Initial experiments should be done on growing cells. 1-5 × 107 celles/ml are exposed to the test chemical for up to 18 hours at 28 to 37 oC with shaking; an adequate amount of metabolic activation system is added during treatment when appropriate.

At the end of the treatment, cells are centrifuged, washed and seeded upon appropriate culutre medium. After incubation plates are scored for survival and the induction of mitotic recombination.

If the first experiment is negative, then a second experiment should be carried out using stationary phase cells. If the first experiment is positive it is confirmed in an independent experiment.

2. DATA

Data should be presented in tabular form indicating the number of colonies counted, the number of recombinants, survival and the frequency of recombinants.

Results should be confirmed in an independent experiment.

The data should be evaluated using appropriate statistical methods.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- strain used,

- test conditions: stationary phase or growing cells, composition of media, incubation temperature and duration, metabolic activation system,

- treatment conditions: exposure concentration, procedure and duration of treatment, treatment temperature, positive and negative controls,

- number of colonies counted, number of recombinants; survival and recombination frequency, dose/response relationship if applicable, statistical evaluation of data,

- discussion of the results,

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.17. MUTAGENICITY — IN VITRO MAMMALIAN CELL GENE MUTATION TEST

1. METHOD

This method is a replicate of the OECD TG 476, In Vitro Mammalian Cell Gene Mutation Test (1997).

1.1. INTRODUCTION

The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, CHO-AS52 and V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid cells (1). In these cell lines the most commonly-used genetic endpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT), and a transgene of xanthine-guanine phosphoribosyl transferase (XPRT). The TK, HPRT and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK and XPRT may allow the detection of genetic events (e.g. large deletions) not detected at the HPRT locus on X-chromosomes (2)(3)(4)(5)(6).

In the in vitro mammalian cell gene mutation test, cultures of established cell lines or cell strains can be used. The cells used are selected on the basis of growth ability in culture and stability of the spontaneous mutation frequency.

Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian in vivo conditions. Care should be taken to avoid conditions, which would lead to results not reflecting intrinsic mutagenicity. Positive results, which do not reflect intrinsic mutagenicity may arise from changes in pH, osmolality or high levels of cytotoxicity (7).

See also General introduction Part B.

1.2. DEFINITIONS

Forward mutation: a gene mutation from the parental type to the mutant form which gives rise to an alteration or a loss of the enzymatic activity of the function of the encoded protein.

Base pair substitution mutagens: substances, which cause substitution of one or several base pairs in the DNA.

Frameshift mutagens: Substances, which cause the addition or deletion of single or multiple base pairs in the DNA molecule.

Phenotypic expression time: a period during which unaltered gene products are depleted from newly mutated cells.

Mutant frequency: the number of mutant cells observed divided by the number of viable cells.

Relative total growth: increase in cell number over time compared to a control population of cells; calculated as the product of suspension growth relative to the negative control times cloning efficiency relative to negative control.

Relative suspension growth: increase in cell number over the expression period relative to the negative control.

Viability: the cloning efficiency of the treated cells at the time of plating in selective conditions after the expression period.

Survival: the cloning efficiency of the treated cells when plated at the end of the treatment period; survival is usually expressed in relation to the survival of the control cell population.

1.3. PRINCIPLE OF THE TEST METHOD

Cells deficient in thymidine kinase (TK) due to the mutation TK+/- -> TK-/- are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not. Similarly, cells deficient in HPRT or XPRT are selected by resistance to 6-thioguanine (TG) or 8-azaguanine (AG). The properties of the test substance should be considered carefully if a base analogue or a compound related to the selective agent is tested in any of the mammalian cell gene mutation tests. For example, any suspected selective toxicity by the test substance for mutant and non-mutant cells should be investigated. Thus, performance of the selection system/agent must be confirmed when testing chemicals structurally related to the selective agent (8).

Cells in suspension or monolayer culture are exposed to the test substance, both with and without metabolic activation, for a suitable period of time and subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection (9)(10)(11)(12)(13). Cytotoxicity is usually determined by measuring the relative cloning efficiency (survival) or relative total growth of the cultures after the treatment period. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each selected locus and cell type, to allow near-optimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted. The mutant frequency is derived from the number of mutant colonies in selective medium and the number of colonies in non-selective medium.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparations

1.4.1.1. Cells

A variety of cell types are available for use in this test including subclones of L5178Y, CHO, CHO-AS52, V79 or TK6 cells. Cell types used in this test should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency. Cells should be checked for mycoplasma contamination and should not be used if contaminated.

The test should be designed to have a predetermined sensitivity and power. The number of cells, cultures and concentrations of test substance used should reflect these defined parameters (14). The minimal number of viable cells surviving treatment and used at each stage in the test should be based on the spontaneous mutation frequency. A general guide is to use a cell number, which is at least 10 times the inverse of the spontaneous mutation frequency. However, it is recommended to utilise at least 106 cells. Adequate historical data on the cell system used should be available to indicate consistent performance of the test.

1.4.1.2. Media and culture conditions

Appropriate culture media, and incubation conditions (culture vessels, temperature, CO2 concentration, and humidity) should be used. Media should be chosen according to the selective systems and cell type used in the test. It is particularly important that culture conditions should be chosen that ensure optimal growth of cells during the expression period and colony forming ability of both mutant and non-mutant cells.

1.4.1.3. Preparation of cultures

Cell are propagated from stock cultures, seeded in culture medium and incubated at 37 oC. Prior to use in this test, cultures may need to be cleansed of pre-existing mutant cells.

1.4.1.4. Metabolic activation

Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The most commonly used system is a cofactor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents treated with enzyme-inducing agents such as Aroclor 1254 (15)(16)(17)(18) or a combination of phenobarbitone and ß–naphthoflavone (19)(20).

The post-mitochondrial fraction is usually used at concentrations in the range from 1-10 % v/v in the final test medium. The choice and condition of a metabolic activation system may depend upon the class of chemical being tested. In some cases it may be appropriate to utilise more than one concentration of post-mitochondrial fraction.

1.4.1.5. Test substance/Preparation

1.4.2. Test conditions

1.4.2.1. Solvent/Vehicle

1.4.2.2. Exposure concentrations

Among the criteria to be considered when determining the highest concentration are cytotoxicity, solubility in the test system and changes in pH or osmolality.

Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell integrity and growth, such as relative cloning efficiency (survival) or relative total growth. It may be useful to determine cytotoxicity and solubility in a preliminary experiment.

At least four analysable concentrations should be used. Where there is cytotoxicity, these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean that the concentration levels should be separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10-20 % (but not less than 10 %) relative survival (relative cloning efficiency) or relative total growth. For relatively non-cytotoxic substances, the maximum test concentration should be 5 mg/ml 5 μl/ml, or 0,01 M, whichever is the lowest.

Relatively insoluble substances should be tested up to or beyond their limit of solubility under culture conditions. Evidence of insolubility should be determined in the final treatment medium to which cells are exposed. It may be useful to assess solubility at the beginning and the end of the treatment, as solubility can change during the course of exposure in the test system due to presence of cells, S9, serum, etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring.

1.4.2.3. Controls

Concurrent positive and negative (solvent or vehicle) controls, both with and without metabolic activation should be included in each experiment. When metabolic activation is used, the positive control chemical should be the one that requires activation to give a mutagenic response.

Examples of positive control substances include:

Ethyl nitrosourea | 759-73-9 | 212-072-2 |

TK (small and large colonies) | Methyl methanesulphonate | 66-27-3 | 200-625-0 |

XPRT | Ethyl methanesulphonate | 62-50-0 | 200-536-7 |

Ethyl nitrosourea | 759-73-9 | 212-072-2 |

N-Nitrosodimethylamine | 62-75-9 | 200-549-8 |

7,12-Dimethylbenzanthracene | 57-97-6 | 200-359-5 |

TK (small and large colonies) | Cyclophosphamide | 50-18-0 | 200-015-4 |

Cyclophosphamide monohydrate | 6055-19-2 | |

Benzo[a]pyrene | 50-32-8 | 200-028-5 |

3-Methylcholanthrene | 56-49-5 | 200-276-5 |

XPRT | N-Nitrosodimethylamine (for high levels of S-9) | 62-75-9 | 200-549-8 |

Benzo[a]pyrene | 50-32-8 | 200-028-5 |

Other appropriate positive control reference substances may be used, e.g. if a laboratory has a historical data base on 5-Bromo 2'-deoxyuridine (CAS n. 59-14-3, Einecs n. 200-415-9), this reference substance could be used as well. The use of chemical class-related positive control chemicals should be considered, when available.

Negative controls, consisting of solvent or vehicle alone in the treatment medium, and treated in the same way as the treatment groups, should be included. In addition, untreated controls should also be used unless there are historical control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent.

1.4.3. Procedure

1.4.3.1. Treatment with the test substance

Proliferating cells should be exposed to the test substance both with and without metabolic activation. Exposure should be for a suitable period of time (usually three to six hours is effective). Exposure time may be extended over one or more cell cycles.

Either duplicate or single treated cultures may be used at each concentration tested. When single cultures are used, the number of concentrations should be increased to ensure an adequate number of cultures for analysis (e.g. at least eight analysable concentrations). Duplicate negative (solvent) control cultures should be used.

Gaseous or volatile substances should be tested by appropriate methods, such as in sealed culture vessels (21)(22).

1.4.3.2. Measurement of survival, viability and mutant frequency

At the end of the exposure period, cells are washed and cultured to determine survival and to allow for expression of the mutant phenotype. Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period.

Each locus has a defined minimum time requirement to allow near optimal phenotypic expression of newly induced mutants (HPRT and XPRT require at least six to eight days, and TK at least two days). Cells are grown in medium with and without selective agent(s) for determination of numbers of mutants and cloning efficiency, respectively. The measurement of viability (used to calculate mutant frequency) is initiated at the end of the expression time by plating in non-selective medium.

If the test substance is positive in the L5178Y TK+/- test, colony sizing should be performed on at least one of the test cultures (the highest positive concentration) and on the negative and positive controls. If the test substance is negative in the L5178Y TK+/- test, colony sizing should be performed on the negative and positive controls. In studies using TK6TK+/-, colony sizing may also be performed.

2. DATA

2.1. TREATMENT OF RESULTS

Data should include cytotoxicity and viability determination, colony counts and mutant frequencies for the treated and control cultures. In the case of a positive response in the L5178Y TK+/- test, colonies are scored using the criteria of small and large colonies on at least one concentration of the test substance (highest positive concentration) and on the negative and positive control. The molecular and cytogenetic nature of both large and small colony mutants has been explored in detail (23)(24). In the TK+/- test, colonies are scored using the criteria of normal growth (large) and slow growth (small) colonies (25). Mutant cells that have suffered the most extensive genetic damage have prolonged doubling times and thus form small colonies. This damage typically ranges in scale from the losses of the entire gene to karyotypically visible chromosome aberrations. The induction of small colony mutants has been associated with chemicals that induce gross chromosome aberrations (26). Less seriously affected mutant cells grow at rates similar to the parental cells and form large colonies.

Survival (relative cloning efficiencies) or relative total growth should be given. Mutant frequency should be expressed as number of mutant cells per number of surviving cells.

Individual culture data should be provided. Additionally, all data should be summarised in tabular form.

There is no requirement for verification of a clear positive response. Equivocal results should be clarified by further testing preferably using modification of experimental conditions. Negative results need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification should be provided. Modification of study parameters to extend the range of conditions assessed should be considered in follow-up experiments for either equivocal or negative results. Study parameters that might be modified include the concentration spacing and the metabolic activation conditions.

2.2. EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response.

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this system.

Although most studies will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of times the experiment is repeated.

Positive results from the in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells used. A positive concentration response that is reproducible is most meaningful. Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used.

3. REPORTING

TEST REPORT

The test report must include the following information:

Solvent/Vehicle:

- justification for choice of vehicle/solvent,

- solubility and stability of the test substance in solvent/vehicle, if known,

Cells:

- type and source of cells,

- number of cell cultures,

- number of cell passages, if applicable,

- methods for maintenance of cell culture, if applicable,

- absence of mycoplasma.

Test conditions:

- rationale for selection of concentrations and number of cultures including, e.g. cytotoxicity data and solubility limitations, if available,

- composition of media, CO2 concentration,

- concentration of test substance,

- volume of vehicle and test substance added,

- incubation temperature,

- incubation time,

- duration of treatment,

- cell density during treatment,

- type and composition of metabolic activation system, including acceptability criteria,

- positive and negative controls,

- length of expression period (including number of cells seeded, and subcultures and feeding schedules, if appropriate),

- selective agents,

- criteria for considering tests as positive, negative or equivocal,

- methods used to enumerate numbers of viable and mutant cells.

- definition of colonies of which size and type are considered (including criteria for "small" and "large" colonies, as appropriate).

Results:

- signs of toxicity,

- signs of precipitation,

- data on pH and osmolality during the exposure to the test substance, if determined,

- colony size if scored for at least negative and positive controls,

- laboratory's adequacy to detect small colony mutants with the L5178Y TK+/- system, where appropriate,

- dose-response relationship, where possible,

- statistical analyses, if any,

- concurrent negative (solvent/vehicle) and positive control data,

- historical negative (solvent/vehicle) and positive control data with ranges, means and standard deviations,

- mutant frequency.

Discussion of results.

Conclusions.

4. REFERENCES

(1) Moore, M.M., DeMarini, D.M., DeSerres, F.J. and Tindall, K.R. (Eds.), (1987) Banbury Report 28: Mammalian Cell Mutagenesis, Cold Spring Harbor Laboratory, New York.

(2) Chu, E.H.Y. and Malling, H.V., (1968) Mammalian Cell Genetics. II. Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells In Vitro, Proc. Natl. Acad. Sci., USA, 61, p. 1306-1312.

(3) Liber, H.L. and Thilly, W.G., (1982) Mutation Assay at the Thymidine Kinase Locus in Diploid Human Lymphoblasts. Mutation Res., 94, p. 467-485.

(4) Moore, M.M., Harington-Brock, K., Doerr, C.L. and Dearfield, K.L., (1989) Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO HGPRT Loci. Mutagenesis, 4, p. 394-403.

(5) Aaron, C.S. and Stankowski, Jr.L.F., (1989) Comparison of the AS52/XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates. Mutation Res., 223, p. 121-128.

(6) Aaron, C.S., Bolcsfoldi, G., Glatt, H.R., Moore, M., Nishi, Y., Stankowski, Jr.L.F., Theiss, J. and Thompson, E., (1994) Mammalian Cell Gene Mutation Assays Working Group Report. Report of the International Workshop on Standardisation of Genotoxicity Test Procedures. Mutation Res., 312, p. 235-239.

(7) Scott, D., Galloway, S.M., Marshall, R.R., Ishidate, M., Brusick, D., Ashby, J. and Myhr, B.C., (1991) Genotoxicity Under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutation Res., 257, p. 147-204.

(8) Clive, D., McCuen, R., Spector, J.F.S., Piper, C. and Mavournin, K.H., (1983) Specific Gene Mutations in L5178Y Cells in Culture. A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res., 115, p. 225-251.

(9) Li, A.P., Gupta, R.S., Heflich, R.H. and Wasson, J.S., (1988) A Review and Analysis of the Chinese Hamster Ovary/Hypoxanthine Guanine Phosphoribosyl Transferase System to Determine the Mutagenicity of Chemical Agents: A Report of Phase III of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res., 196, p. 17-36.

(10) Li, A.P., Carver, J.H., Choy, W.N., Hsie, A.W., Gupta, R.S., Loveday, K.S., O'Neill, J.P., Riddle, J.C., Stankowski, L.F. Jr. and Yang, L.L., (1987) A Guide for the Performance of the Chinese Hamster Ovary Cell/Hypoxanthine-Guanine Phosphoribosyl Transferase Gene Mutation Assay. Mutation Res., 189, p. 135-141.

(11) Liber, H.L., Yandell, D.W and Little, J.B., (1989) A Comparison of Mutation Induction at the TK and HPRT Loci in Human Lymphoblastoid Cells: Quantitative Differences are Due to an Additional Class of Mutations at the Autosomal TK Locus. Mutation Res., 216, p. 9-17.

(12) Stankowski, L.F. Jr., Tindall, K.R. and Hsie, A.W., (1986) Quantitative and Molecular Analyses of Ethyl Methanosulphonate -and ICR 191- Induced Molecular Analyses of Ethyl Methanosulphonate -and ICR 191-Induced Mutation in AS52 Cells. Mutation Res., 160, p. 133-147.

(13) Turner, N.T., Batson, A.G. and Clive, D., (1984) Procedures for the L5178Y/TK+/- — TK+/- Mouse Lymphoma Cell Mutagenicity Assay. In: Kilbey, B.J. et al (eds.) Handbook of Mutagenicity Test Procedures, Elsevier Science Publishers, New York, p. 239-268.

(14) Arlett, C.F., Smith, D.M., Clarke, G.M., Green, M.H.L., Cole, J., McGregor, D.B. and Asquith, J.C., (1989) Mammalian Cell Gene Mutation Assays Based upon Colony Formation. In: Statistical Evaluation of Mutagenicity Test Data, Kirkland, D.J., Ed., Cambridge University Press, p. 66-101.

(15) Abbondandolo, A., Bonatti, S., Corti, G., Fiorio, R., Loprieno, N. and Mazzaccaro, A., (1977) Induction of 6-Thioguanine-Resistant Mutants in V79 Chinese Hamster Cells by Mouse-Liver Microsome-Activated Dimethylnitrosamine. Mutation Res., 46, p. 365-373.

(16) Ames, B.N., McCann, J. and Yamasaki, E., (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res., 31, p. 347-364.

(17) Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown M.M.M., (1979) Validation and Characterisation of the L5178Y/TK+/- Mouse Lymphoma Mutagen Assay System. Mutat. Res., 59, p. 61-108.

(18) Maron, D.M. and Ames, B.N., (1983) Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, p. 173-215.

(19) Elliott, B.M., Combes, R.D., Elcombe, C.R., Gatehouse, D.G., Gibson, G.G., Mackay, J.M. and Wolf, R.C., (1992) Alternatives to Aroclor 1254-Induced S9 in In Vitro Genotoxicity Assays. Mutagenesis, 7, p. 175-177.

(20) Matsushima, T., Sawamura, M., Hara, K. and Sugimura, T., (1976) A Safe Substitute for Polychlorinated Biphenyls as an Inducer of Metabolic Activation Systems. In: In vitro Metabolic Activation in Mutagenesis Testing, de Serres, F.J., Fouts, J.R., Bend, J.R. and Philpot, R.M. (eds), Elsevier, North-Holland, p. 85-88.

(21) Krahn, D.F., Barsky, F.C. and McCooey, K.T., (1982) CHO/HGPRT Mutation Assay: Evaluation of Gases and Volatile Liquids. In: Tice, R.R., Costa, D.L., Schaich, K.M. (eds). Genotoxic Effects of Airborne Agents. New York, Plenum, p. 91-103.

(22) Zamora, P.O., Benson, J.M., Li, A.P. and Brooks, A.L., (1983) Evaluation of an Exposure System Using Cells Grown on Collagen Gels for Detecting Highly Volatile Mutagens in the CHO/HGPRT Mutation Assay. Environmental Mutagenesis, 5, p. 795-801.

(23) Applegate, M.L., Moore, M.M., Broder, C.B., Burrell, A. and Hozier, J.C., (1990) Molecular Dissection of Mutations at the Heterozygous Thymidine Kinase Locus in Mouse Lymphoma Cells. Proc. Natl. Acad. Sci. USA, 87, p. 51-55.

(24) Moore, M.M., Clive, D., Hozier, J.C., Howard, B.E., Batson, A.G., Turner, N.T. and Sawyer, J., (1985) Analysis of Trifluorothymidine-Resistant (TFTr) Mutants of L5178Y/TK+/- Mouse Lymphoma Cells. Mutation Res., 151, p. 161-174.

(25) Yandell, D.W., Dryja, T.P. and Little, J.B., (1990) Molecular Genetic Analysis of Recessive Mutations at a Heterozygous Autosomal Locus in Human Cells. Mutation Res., 229, p. 89-102.

(26) Moore, M.M. and Doerr, C.L., (1990) Comparison of Chromosome Aberration Frequency and Small-Colony TK-Deficient Mutant Frequency in L5178Y/TK+/- - 3.7.2C Mouse Lymphoma Cells. Mutagenesis, 5, p. 609-614.

B.18. DNA DAMAGE AND REPAIR — UNSCHEDULED DNA SYNTHESIS — MAMMALIAN CELLS IN VITRO

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITION

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The Unscheduled DNA Synthesis (UDS) test measures the DNA repair synthesis after excision and removal of a stretch of DNA containing the region of damage induced by chemical and physical agents. The test is based on the incorporation of tritium labelled thymidine (3H-TdR) into the DNA of mammalian cells, which are not in the S phase of the cell cycle. The uptake of 3H-TdR may be determined by autoradiography or by liquid scintillation counting (LSC) of DNA from the treated cells. Mammalian cells in culture, unless primary rat hepatocytes are used, are treated with the test agent with and without an exogenous metabolic activation system. UDS may also be measured in in vivo systems.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

Preparations

Test chemicals and control or reference substances should be prepared in growth medium or dissolved or suspended in appropriate vehicles and then further diluted in growth medium for use in the assay. The final concentration of the vehicle should not affect cell viability.

Primary cultures of rat hepatocytes, human lymphocytes or established cell lines (e.g. human diploid fibroblasts) may be used in the assay.

Cells should be exposed to the test chemical both in the presence and absence of an appropriate metabolic activation system.

Test conditions

Number of cultures

At least two cell cultures for autoradiography and six cultures (or less if scientifically justified) for LSC UDS determinations are necessary for each experimental point.

Use of negative and positive controls

Concurrent positive and negative (untreated and/or vehicle) controls with and without metabolic activation should be included in each experiment.

Examples of positive controls for the rat hepatocyte assay include 7,12- dimethylbenzathracene (7,12- DMBA) or 2-acetylaminofluorene (2-AAF). In the case of established cell lines 4-nitroquinoline-N-oxide (4-NQO) is an example of a positive control for both the autoradiographic and LSC assays performed without metabolic activation; N-dimethylnitrosamine is an example of a positive control compound when metabolic activation systems are used.

Exposure concentrations

Multiple concentrations of the test substance over a range adequate to define the response should be used. The highest concentration should elicit some cytotoxic effects. Relatively water-insoluble compounds should be tested up to the limit of solubility. For freely water-soluble non-toxic chemicals, the upper test chemical concentration should be determined on a case-by-case basis.

Cells

Appropriate growth media, CO2 concentration, temperature and humidity should be used in maintaining cultures. Established cell lines should be periodically checked for Mycoplasma contamination.

Metabolic activation

A metabolic activation system is not used with primary hepatocyte cultures. Established cell lines and lymphocytes are exposed to test substance both in the presence and absence of an appropriate metabolic activation system.

Procedure

Preparation of cultures

Established cell lines are generated from stock cultures (e.g. by trypsinisation or by shaking off), seeded in culture vessels at appropriate density, and incubated at 37 oC.

Short-term cultures of rat hepatocytes are established by allowing freshly dissociated hepatocytes in an appropriate medium to attach themselves to the growing surface.

Human lymphocyte cultures are set up using appropriate techniques.

Treatment of the cultures with the test substance

Primary rat hepatocytes

Freshly isolated rat hepatocytes are treated with the test substance in a medium containing 3H-TdR for an appropriate length of time. At the end of the treatment period, medium should be drained off the cells, which are then rinsed fixed and dried. Slides should be dipped in autoradiographic emulsion (alternative stripping film may be used), exposed, developed, stained and counted.

Established cell lines and lymphocytes

Autoradiographic techniques: cell cultures are exposed to the test substance for appropriate durations followed by treatment with 3H-TdR. The times will be governed by the nature of the substance, the activity of metabolising systems and the type of cells. To detect the peak of UDS, 3H-TdR should be added either simultaneously with the test subsrance or within a few minutes after exposure to the test substance. The choice between these two procedures will be influenced by possible interactions between test substance and 3H-TdR. In order to discriminate between UDS and semi-conservative DNA replication, the latter can be inhibited, for example, by the use of an arginine-deficient medium, low serum content or by hydroxyurea in the culture medium.

LSC measurements of UDS: prior to treatment with test substance, entry of cells into S-phase should be blocked as described above; cells should then be exposed to test chemical as described for autoradiography. At the end of the incubation period, DNA should be extracted from the cells and the total DNA content, and the extent of 3H-TdR, incorporation determined.

It should be noted that, where human lymphocytes are used in the above techniques, the suppression of semi-conservative DNA replication is unnecessary in unstimulated cultures.

Analysis

Autoradiographic determinations

In determining UDS in cells in culture, S-phase nuclei are not counted. At least 50 cells per concentration should be counted. Slides should be coded before counting. Several widely separated random fields should be counted on each slide. The amount of 3H-TdR incorporation in the cytoplasm should be determined by counting three nucleus-sized areas in the cyptoplasm of each cell counted.

LSC determinations

An adequate number of cultures should be used at each concentration and in the controls in LSC UDS determinations.

All results should be confirmed in an independent experiment.

2. DATA

Data should be presented in tabular form.

2.1. AUTORADIOGRAPHIC DETERMINATIONS

The extent of 3H-TdR incorporation in the cytoplasm and the number of grains found over the cell nucleus should be recorded separately.

Mean, median and mode may be used to describe the distribution of the extent of 3H-TdR incorporation in the cytoplasm and the number of grains per nucleus.

2.2. LSC DETERMINATIONS

For LSC determinations, 3H-TdR incorporation should be reported as dpm/μg DNA. The mean dpm/μg DNA with standard deviation may be used to describe the distribution of incorporation.

Data should be evaluated using appropriate statistical methods.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- cells used, density and passage number at time of treatment, number of cell cultures,

- methods used for maintenance of cell cultures including medium, temperature and CO2 concentration,

- test substance, vehicle, concentrations and rationale for selection of concentrations used in the assay,

- details of metabolic activation systems,

- treatment schedule,

- positive and negative controls,

- autoradiographic technique used,

- procedures used to block entry of cells into S-phase,

- procedures used for DNA extraction and determination of total DNA content in LSC determination,

- dose/response relationship, where possible,

- statistical evaluation,

- discussion of results,

- interpretation of results.

3.2. VALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.19. SISTER CHROMATID EXCHANGE ASSAY IN VITRO

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITION

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The Sister Chromatid Exchange (SCE) assay is a short-term test for the detection of reciprocal exchanges of DNA between two sister chromatids of a duplicating chromosome. SCEs represent the interchange of DNA replication products at apparently homologous loci. The exchange process presumably involves DNA breakage and reunion, although little is known about its molecular basis. Detection of SCEs requires some means of differentially labelling sister chromatids and this can be achieved by incorporation of bromodeoxyuridine (BrdU) into chromosomal DNA for two cell cycles.

Mammalian cells in vitro are exposed to the test chemical with and without a mammalian exogenous metabolic activation system, if appropriate, and cultured for two rounds of replication in BrdU-containing medium. After treatment with a spindle inhibitor (e.g. colchicine) to accumulate cells in a metaphase-like stage of mitosis (c-metaphase), cells are harvested and chromosome preparations are made.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

- Primary cultures, (human lymphocytes) or established cell lines (e.g. Chinese hamster ovary cells) may be used in the assay. Cell lines should be checked for Mycoplasma contamination,

- appropriate culture media and incubation conditions (e.g. temperature, culture vessels, CO2 concentration and humidity) should be used,

- test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. The final concentration of a vehicle in the culture system should not significantly affect, cell viability or growth rate and effects on SCE frequency should be monitored by a solvent control,

- cells should be exposed to the test substance both in the presence and absence of an exogenous mammalian metabolic activation system. Alternatively, where cell types with intrinsic metabolic activity are used, the rate and nature of the activity should be appropriate to the chemical class being tested.

1.6.2. Test conditions

Number of cultures

At least duplicate cultures should be used for each experimental point.

Use of negative and positive controls

Positive controls, using both a direct acting compound and a compound requiring metabolic activation should be included in each experiment; a vehicle control should also be used.

The following are examples of substances which might be used as positive controls:

- direct acting compound:

- ethylmethanesulphonate,

- indirect acting compound:

- cyclophosphamide.

When appropriate, an additional positive control of the same chemical class as the chemical under test may be included.

Exposure concentrations

At least three adequately spaced concentrations of the test substance should be used. The highest concentration should give rise to a significant toxic effect but must still allow adequate cell replication to occur. Relatively water-insoluble substances should be tested up to the limit of solubility using appropriate procedures. For freely water-soluble non-toxic substances the upper test substance concentration should be determined on a case-by-case basis.

1.6.3. Procedure

Preparation of cultures

Established cell lines are generated from stock cultures (e.g. by trypsinisation or by shaking off), seeded in culture vessels at appropriate density and incubated at 37 oC. For monolayer cultures, the number of cells per culture vessel should be adjusted so that the cultures are not much more than 50 % confluent at the time of harvest. Alternatively, cells may be used in suspension culture. Human lymphocyte cultures are set up from heparinized blood, using appropriate techniques, and incubated at 37 oC.

Treatment

Cells in an exponential stage of growth are exposed to the test substance for a suitable period of time; in most cases one to two hours may be effective, but the treatment time may be extended up to two complete cell cycles in certain cases. Cells without sufficient instrinsic metabolic activity should be exposed to the test chemical in the presence and absence of an appropriate metabolic activation system. At the end of the exposure period, cells are washed free of test substance and cultured for two rounds of replication in the presence of BrdU. As an alternative procedure cells may be exposed simultaneously to the test chemical and BrdU for the complete culture time of two cell cycles.

Human lymphocyte cultures are treated while they are in a semisynchronous condition.

Cells are analysed in their second post-treatment division, ensuring that the most sensitive cell cycle stages have been exposed to the chemical. All cultures to which BrdU is added should be handled in darkness or in dim light from incandescent lamps up to the harvesting of cells in order to minimise photolysis of BrdU-containing DNA.

Harvesting of cells

Cell cultures are treated with a spindle inhibitor (e.g. colchicine) one to four hours prior to harvesting. Each culture is harvested and processed separately for the preparation of chromosomes.

Chromosome preparation and staining

Chromosome preparations are made by standard cytogenetic techniques. Staining of slides to show SCEs can be performed by several techniques, (e.g. the fluorescence plus Giemsa method).

Analysis

The number of cells analysed should be based on the spontaneous control frequency of SCE. Usually, at least 25 well-spread metaphases per culture are analysed for SCEs. Slides are coded before analysis. In human lymphocytes only metaphases containing 46 centromeres are analysed. In established cell lines only metaphases containing ± 2 centromeres of the modal number are analysed. It should be stated whether or not centromeric switch of label is, scored as an SCE. The results should be confirmed in an independent experiment.

2. DATA

Data should be presented in tabular form. The number of SCEs for each metaphase and the number of SCEs per chromosome for each metaphase should be listed separately for all treated and control cultures.

The data should be evaluated using appropriate statistical methods.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- cells used, methods of maintenance of cell culture,

- test conditions: composition of media, CO2 concentration, concentration of test substance, vehicle used, incubation temperature, treatment time, spindle inhibitor used, its concentration and the duration of treatment with it, type of mammalian activation system used, positive and negative controls,

- number of cell cultures per experimental point,

- details of the technique used for slide preparation,

- number of metaphases analysed (data given separately for each culture),

- mean number of SCE per cell and per chromosome (data given separately for each culture),

- criteria for scoring SCE,

- rationale for dose selection,

- dose-response relationship, if applicable,

- statistical evaluation,

- discussion of results,

- interpretation of results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.20. SEX-LINKED RECESSIVE LETHAL TEST IN DROSOPHILA MELANOGASTER

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITION

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLES OF THE TEST METHOD

The sex-linked recessive lethal (SLRL) test using Drosophila melanogaster detects the occurrence of mutations, both point mutations and small deletions, in the germ line of the insect. This test is a forward mutation assay capable of screening for mutations at about 800 loci on the X-chromosome; this represents about 80 % of all X-chromosal loci. The X-chromosome represents approximately one-fifth of the entire haploid genome.

Mutations in the X-chromosome of Drosophila melanogaster are phenotypically expressed in males carrying the mutant gene. When the mutation is lethal in the hemizygous condition, its presence is inferred from the absence of one class of male offspring out of the two that are normally produced by a heterozygous female. The SLRL test takes advantage of these facts by means of specially marked and arranged chromosomes.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

Preparations

Stocks

Males of a well-defined wild-type stock and females of the Muller-5 stock may be used. Other appropriately marked female stocks with multiple inverted X-chromosomes may also be used.

Test substance

Test substances should be dissolved in water. Substances, which are insoluble in water may be dissolved or suspended in appropriate vehicles (e.g. a mixture of ethanol and Tween-60 or 80), then diluted in water or saline prior to administration. Dimethylsulphoxide (Dmso) should be avoided as a vehicle.

Number of animals

The test should be designed with a predetermined sensitivity and power. The spontaneous mutant frequency observed in the appropriate control will influence strongly the number of treated chromosomes that must be analysed.

Route of administration

Exposure may be oral, by injection or by exposure to gases or vapours. Feeding of the test substance may be done in sugar solution. When necessary, substances may be dissolved in a 0,7 % NaCl solution and injected into the thorax or abdomen.

Use of negative and positive controls

Negative (vehicle) and positive controls should be included. However, if appropriate laboratory historical control data are available, no concurrent controls are needed.

Exposure levels

Three exposure levels should be used. For a preliminary assessment one exposure level of the test substance may be used, that exposure level being either the maximum tolerated concentration or that producing some indication of toxicity. For non-toxic substances exposure to the maximum practicable concentration should be used.

Procedure

Wild-type males (three to five days old) are treated with the test substance and mated individually to an excess of virgin females from the Muller-5 stock or from another appropriately marked (with multiple inverted X-chromosomes) stock. The females are replaced with fresh virgins every two to three days to cover the entire germ cell cycle. The offspring of these females are scored for lethal effects corresponding to the effects on mature sperm, mid or late-stage spermatids, early spermatids, spermatocytes and spermatogonia at the time of treatment.

Heterozygous F1 females from the above crosses are allowed to mate individually (i.e. one female per vial) with their brothers. In the F2 generation, each culture is scored for the absence of wild-type males. If a culture appears to have arisen from an F1 female carrying a lethal in the parental X-chromosome (i.e. no males with the treated chromosome are observed) daughters of that female with the same genotype should be tested to ascertain whether the lethality is repeated in the next generation.

2. DATA

Data should be tabulated to show the number of X-chromosomes tested, the number of non-fertile males and the number of lethal chromosomes at each exposure concentration and for each mating period for each male treated. Numbers of clusters of different sizes per male should be reported. These results should be confirmed in a separate experiment.

Appropriate statistical methods should be used in evaluation sex-linked recessive lethal tests. Clustering of recessive lethals originating from one male should be considered and evaluated in an appropriate statistical manner.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- stock: Drosophila stocks or strains used, age of insects, number of males treated, number of sterile males, number of F2 cultures established, number of F2 cultures without progeny, number of chromosomes carrying a lethal detected at each germ cell stage,

- criteria for establishing the size of treated groups,

- test conditions. detailed description of treatment and sampling schedule, exposure levels, toxicity data, negative (solvent) and positive controls, if appropriate,

- criteria for scoring lethal mutations,

- exposure/effect relationship where possible,

- evaluation of data,

- discussion of results,

- interpretation of results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.21. IN VITRO MAMMALIAN CELL TRANSFORMATION TESTS

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITION

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

Mammalian cell culture systems may be used to detect phenotypic changes in vitro induced by chemical substances associated with malignant transformation in vivo. Widely used cells include C3H10T1/2, 3T3, SHE, Fischer rat and the tests rely on changes in cell morphology, focus formation or changes in anchorage dependence in semi-solid agar. Less widely used systems exist which detect other physiological or morphological changes in cells following exposure to carcinogenic chemicals. None of the in vitro test endpoints has an established mechanistic link with cancer. Some of the test systems are capable of detecting tumour promotors. Cytotoxicity may be determined by measuring the effect of the test material on colony-forming abilities (cloning efficiency) or growth rates of the cultures. The measurement of cytotoxicity is to establish that exposure to the test chemical has been toxicologically relevant but cannot be used to calculate transformation: frequency in all assays since some may involve prolonged incubation and/or replating.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

Preparations

Cells

A variety of cell lines or primary cells are available depending on the transformation test being used. The investigator must ensure that the cells in the test being performed exhibit the appropriate phenotypic change after exposure to known carcinogens and that the test, in the investigator's laboratory, is of proven and documented validity and reliability.

Medium

Media and experimental conditions should be used that are appropriate to the transformation assay in use.

Test substance

Test substances may be prepared in culture media or dissolved or suspended in appropriate vehicles prior to treatment of the cells. The final concentration of the vehicle in the culture system should not affect cell viability, growth rate or transformation incidence.

Metabolic activation

Cells should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. Alternatively, when cell types are used that possess intrinsic metabolic activity, the nature of the activity should be known to be appropriate to the chemical class being tested.

Test conditions

Use of negative and positive controls

Positive controls, using both a direct-acting compound and a compound requiring metabolic activation should be included in each experiment; a negative (vehicle) control should also be used.

The following are examples of substances, which might be used as positive controls:

- Direct-acting chemicals:

- Ethylmethanesulphonate,

- β-propiolactone,

- Compounds requiring metabolic activation:

- 2-acetylaminofluorene,

- 4-dimethylaminoazobenzene,

- 7,12-dimethylbenzanthracene.

When appropriate, an additional positive control of the same chemical class as the compound under test should be included.

Exposure concentrations

Several concentrations of the test substance should be used. These concentrations should yield a concentration-related toxic effect, the highest concentration producing a low level of survival and the survival in the lowest concentration being approximately the same as that in the negative control. Relatively water-insoluble substances should be tested up to the limit of solubility using appropriate procedures. For freely water-soluble non-toxic substances the upper test substance concentration should be determined on a case-by-case basis.

Procedure

Cells should be exposed for a suitable period of time depending on the test system in use, and this may involve re-dosing accompanied by a change of medium (and if necessary, fresh metabolic activation mixture) if exposure is prolonged. Cells without sufficient intrinsic metabolic activity should be exposed to the test substance in the presence and absence of an appropriate metabolic activation system. At the end of the exposure period, cells are washed free of test substance and cultured under conditions appropriate for the appearance of the transformed phenotype being monitored and the incidence of transformation determined. All results are confirmed in an independent experiment.

2. DATA

Data should be presented in tabular form and may take a variety of forms according to the assay being used e.g. plate counts, positive plates or numbers of transformed cells. Where appropriate, survival should be expressed as a percentage of control levels and transformation frequency expressed as the number of transformants per number of survivors. Data should be evaluated using appropriate statistical methods.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- cell type used, number of cell cultures, methods for maintenance of cell cultures,

- test conditions: concentration of test substance, vehicle used, incubation time, duration and frequency of treatment, cell density during treatment, type of exogenous metabolic activation system used, positive and negative controls, specification of phenotype being monitored, selective system used (if appropriate), rational for dose selection,

- method used to enumerate viable and transformed cells,

- statistical evaluation,

- discussion of results,

- interpretation of results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.22. RODENT DOMINANT LETHAL TEST

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITION

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

Dominant lethal effects cause embryonic or foetal death. Induction of dominant lethals by exposure to a chemical substance indicates that the substance has affected germinal tissue of the test species. It is generally accepted that dominant lethals are due to chromosomal damage (structural and numerical anomalies). Embryonic death if females are treated may also be the result of toxic effects.

Generally, male animals are exposed to the test compound and mated to untreated virgin females. The various germ cell stages can be tested separately by the use of sequential mating intervals. The increase of dead implants per female in the treated group over the dead implants per female in the control group reflects the post-implantational loss. Pre-implantational loss can be estimated based on corpora lutea counts or by comparing the total implants per female in treated and control groups. The total dominant lethal effect is the sum of pre- and post-implantational loss. The calculation of the total dominant lethal effect is based on comparison of the live implants per female in the test group to the live implants per female in the control group. A reduction in the number of implants at certain intervals may be the result of cell killing (i.e. of spermatocytes and/or spermatogonia).

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

Preparations

When possible, test substances should be dissolved or suspended in isotonic saline. Chemicals insoluble in water may be dissolved or suspended in appropriate vehicles. The vehicle used should neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test chemical should be employed.

Test conditions

Route of administration

The test compound should generally be administered only once. Based on toxicological information a repeated treatment schedule can be employed. The usual routes of administration are oral intubation or intraperitoneal injection. Other routes of administration may be appropriate.

Experimental animals

Rats or mice are recommended as the test species. Healthy fully sexually mature animals are randomised and assigned to treatment and control groups.

Number and sex

An adequate number of treated males should be used, taking into account the spontaneous variation of the biological character being evaluated. The number chosen should be based on the pre-determined sensitivity of detection and power of significance. For example in a typical test, the number of males in each dose group should be sufficient to provide between 30 and 50 pregnant females per mating interval.

Use of negative and positive controls

Generally concurrent positive and negative (vehicle) controls should be included in each experiment. When acceptable positive control results are available from experiments conducted recently in the same laboratory these results can be used instead of a concurrent positive control. Positive control substances should be used at an appropriate low dose (e.g. MMS, intraperitoneally, at 10 mg/kilogram) to demonstrate the test sensitivity.

Dose levels

Normally, three dose levels should be used. The high dose should produce signs of toxicity or reduced fertility in the treated animals. In certain cases a single high dose level may be sufficient.

Limit test

Non-toxic substances should be tested at 5 g/kilogram on a single administration or at 1 g/kilogram/day on repeated administration.

Procedure

Several treatment schedules are available. Single administration of the test substance is the most widely used. Other treatment schedules may be used.

Individual males are mated sequentially to one or two untreated virgin females at appropriate intervals after treatment. Females should be left with the males for at least the duration of one oestrous cycle or until mating has occurred as determined by the presence of sperm in the vagina or by the presence of a vaginal plug.

The number of matings following treatment is governed by the treatment schedule and should ensure that all germ cell stages are sampled after treatment.

Females are sacrificed in the second half of pregnancy and uterine contents are examined to determine the number of dead and live implants. The ovaries may be examined to determine the number of corpora lutea.

2. DATA

Data should be tabulated to show the number of males, the number of pregnant females, and the number of non-pregnant females. Results of each mating, including the identity of each male and female, should be reported individually. For each female, week of mating, dose level received by the males, the frequencies of live implants and of dead implants should be recorded.

The calculation of the total dominant lethal effect is based on comparison of the live implants per female in the test group to the live implants per female in the control group. The ratio of dead to live implants from the treated group compared to the same ratio from the control group is analysed to indicate the post-implantation loss.

If the data are recorded as early and late deaths, the tables should make that clear. If pre-implantation loss is estimated, it should be reported. Pre-implantation loss can be calculated as a discrepancy between the number of corpora lutea and the number of implants or as a reduction in the average number of implants per uterus in comparison with control matings.

Data are evaluated using appropriate statistical methods.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- species, strain, age and weights of animals used, number of animals of each sex in experimental and control groups,

- test substance, vehicle, dose levels tested and rationale for dose selection, negarive and positive controls, toxicity data,

- route and treatment schedule,

- mating schedule,

- method used to determine that mating has occurred,

- time of sacrifice,

- criteria for scoring dominant lethals,

- dose/response relationship, if applicable,

- statistical evaluation,

- discussion of results,

- interpretation of results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.23. MAMMALIAN SPERMATOGONIAL CHROMOSOME ABERRATION TEST

1. METHOD

This method is a replicate of the OECD TG 483, Mammalian Spermatogonial Chromosome Aberration Test (1997).

1.1. INTRODUCTION

The purpose of the in vivo mammalian spermatogonial chromosome aberration test is to identify those substances that cause structural chromosome aberrations in mammalian spermatogonial cells (1)(2)(3)(4)(5). Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur. This method is not designed to measure numerical aberrations and is not routinely used for that purpose. Chromosome mutations and related events are the cause of many human genetic diseases.

This test measures chromosome events in spermatogonial germ cells and is, therefore, expected to be predictive of induction of inheritable mutations in germ cells.

Rodents are routinely used in this test. This in vivo cytogenetic test detects chromosome aberrations in spermatogonial mitoses. Other target cells are not the subject of this method.

To detect chromatid-type aberrations in spermatogonial cells, the first mitotic cell division following treatment should be examined before these lesions are lost in subsequent cell divisions. Additional information from treated spermatogonial stem cells can be obtained by meiotic chromosome analysis for chromosome-type aberrations at diakinesis-metaphase I when the treated cells become spermatocytes.

This in vivo test is designed to investigate whether somatic cell mutagens are also active in germ cells. In addition, the spermatogonial test is relevant to assessing mutagenicity hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes.

A number of generations of spermatogonia are present in the testis with a spectrum of sensitivity to chemical treatment. Thus, the aberrations detected represent an aggregate response of treated spermatogonial cell populations, with the more numerous differentiated spermatogonial cells predominating. Depending on their position within the testis, different generations of spermatogonia may or may not be exposed to the general circulation, because of the physical and physiological Sertoli cell barrier and the blood-testis barrier.

If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.

See also General introduction Part B.

1.2. DEFINITIONS

Chromatid-type aberration: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids.

Chromosome-type aberration: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.

Gap: an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatids.

Numerical aberration: a change in the number of chromosomes from the normal number characteristic of the animals utilised.

Polyploidy: a multiple of the haploid chromosome number (n) other than the diploid number (i.e. 3n, 4n and so on).

Structural aberration: a change in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, observed as deletions, intrachanges or interchanges.

1.3. PRINCIPLE OF THE TEST METHOD

Animals are exposed to the test substance by an appropriate route of exposure and are sacrificed at appropriate times after treatment. Prior to sacrifice, animals are treated with a metaphase-arresting substance (e.g. Colcemid® or colchicine). Chromosome preparations are then made from germ cells and stained, and metaphase cells are analysed for chromosome aberrations.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparations

1.4.1.1. Selection of animal species

Male Chinese hamsters and mice are commonly used. However, males of other appropriate mammalian species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. At the commencement of the study the weight variation of animals should be minimal and not exceed ± 20 % of the mean weight.

1.4.1.2. Housing and feeding conditions

General conditions referred in the General introduction to Part B are applied although the aim for humidity should be 50-60 %.

1.4.1.3. Preparation of the animals

Healthy young adult males are randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals are identified uniquely. The animals are acclimated to the laboratory conditions for at least five days prior to the start of the study.

1.4.1.4. Preparation of doses

1.4.2. Test conditions

1.4.2.1. Solvent/Vehicle

The solvent/vehicle should not produce toxic effects at the dose levels used and should not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported by data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle should be considered first.

1.4.2.2. Controls

Concurrent positive and negative (solvent/vehicle) controls should be included in each test. Except for treatment with the test substance, animals in the control groups should be handled in an identical manner to animals in the treated groups.

Positive controls should produce structural chromosome aberrations in vivo in spermatogonial cells when administered at exposure levels expected to give a detectable increase over background.

Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. In addition, the use of chemical class-related positive control chemicals may be considered, when available. Examples of positive control substances include:

Substance | CAS No | Einecs No |

Cyclophosphamide Cyclophosphamide monohydrate | 50-18-0 6055-19-2 | 200-015-4 |

Cyclohexylamine | 108-91-8 | 203-629-0 |

Mitomycin C | 50-07-7 | 200-008-6 |

Monomeric acrylamide | 79-06-1 | 201-173-7 |

Triethylenemelamine | 51-18-3 | 200-083-5 |

Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups, should be included for every sampling time, unless acceptable inter-animal variability and frequency of cells with chromosome aberrations are demonstrated by historical control data. In addition, untreated controls should also be used unless there are historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle.

1.5. PROCEDURE

1.5.1. Number of animals

Each treated and control group must include at least five analysable males.

1.5.2. Treatment schedule

Test substances are preferably administered once or twice (i.e. as a single treatment or as two treatments). Test substances may also be administered as a split dose, i.e. two treatments on the same day separated by no more than a few hours, to facilitate administering a large volume of material. Other dose regimens should be scientifically justified.

In the highest dose group two sampling times after treatment are used. Since cell cycle kinetics can be influenced by the test substance, one early and one late sampling time are used around 24 and 48 hours after treatment. For doses other than the highest dose, a sampling time of 24 hours or 1,5 cell cycle length after treatment should be taken, unless another sampling time is known to be more appropriate for detection of effects (6).

In addition, other sampling times may be used. For example in the case of chemicals, which may induce chromosome lagging, or may exert S-independent effects, earlier sampling times may be appropriate (1).

The appropriateness of a repeated treatment schedule needs to be identified on a case-by-case basis. Following a repeated treatment schedule the animals should then be sacrificed 24 hours (1,5 cell cycle length) after the last treatment. Additional sampling times may be used where appropriate.

Prior to sacrifice, animals are injected intraperitoneally with an appropriate dose of a metaphase arresting substance (e.g. Colcemid® or colchicine). Animals are sampled at an appropriate interval thereafter. For mice this interval is approximately three to five hours, for Chinese hamsters this interval is approximately four to five hours.

1.5.3. Dose levels

If a range finding study is performed because there are no suitable data available, it should be performed in the same laboratory, using the same species, strain and treatment regimen to be used in the main study (7). If there is toxicity, three dose levels are used for the first sampling time. These dose levels should cover a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality.

Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity in the spermatogonial cells (e.g. a reduction in the ratio of spermatogonial mitoses to first and second meiotic metaphases; this reduction should not exceed 50 %).

1.5.4. Limit test

If a test at one dose level of at least 2000 mg/kg body weight/day using a single treatment, or as two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.

1.5.5. Administration of doses

The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100 g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

1.5.6. Chromosome preparation

Immediately after sacrifice, cell suspensions are obtained from one or both testes, exposed to hypotonic solution and fixed. The cells are then spread on slides and stained.

1.5.7. Analysis

For each animal at least 100 well-spread metaphase should be analysed (i.e. a minimum of 500 metaphases per group). This number could be reduced when high numbers of aberrations are observed. All slides, including those of positive and negative controls, should be independently coded before microscopic analysis. Since fixation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, the cells scored should contain a number of centromeres equal to the number 2n ± 2.

2. DATA

2.1. TREATMENT OF RESULTS

Individual animal data should be presented in a tabular form. The experimental unit is the animal. For each individual animal the number of cells with structural chromosome aberrations and the number of chromosome aberrations per cell should be evaluated. Different types of structural chromosome aberrations should be listed with their numbers and frequencies for treated and control groups. Gaps are recorded separately and reported but generally not included in the total aberration frequency.

If mitosis as well as meiosis is observed, the ratio of spermatogonial mitoses to first and second meiotic metaphases should be determined as a measure of cytotoxicity for all treated and negative control animals in a total sample of 100 dividing cells per animal to establish a possible cytotoxic effect. If only mitosis is observed, the mitosis index should be determined in at least 1000 cells for each animal.

2.2. EVALUATION AND INTERPRETATION OF RESULTS

There are several criteria for determining a positive result, such as a dose-related increase in the relative number of cells with chromosome aberrations or a clear increase in the number of cells with aberrations in a single dose at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (8). Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.

A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.

Positive results from the in vivo spermatogonial chromosome aberration test indicate that the test substance induces structural chromosome aberrations in the germ cells of the species tested. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in the germ cells of the species tested.

The likelihood that the test substance or its metabolites reach the target tissue should be discussed.

3. REPORTING

TEST REPORT

The test report must include the following information:

Solvent/Vehicle:

- justification for choice of vehicle,

- solubility and stability of the test substance in solvent/vehicle, if known.

Test animals:

- species/strain used,

- number and age of animals,

- source, housing conditions, diet, etc.,

- individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group.

Test conditions:

- data from range finding study, if conducted,

- rationale for dose level selection,

- rationale for route of administration,

- details of test substance preparation,

- details of the administration of the test substance,

- rationale for sacrifice times,

- conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable.

- details of food and water quality,

- detailed description of treatment and sampling schedules,

- methods for measurement of toxicity,

- identity of metaphase arresting substance, its concentration and duration of treatment,

- methods of slide preparation,

- criteria for scoring aberrations,

- number of cells analysed per animal,

- criteria for considering studies as positive, negative or equivocal.

Results:

- signs of toxicity,

- mitotic index,

- ratio of spermatogonial mitoses cells to first and second meiotic metaphases,

- type and number of aberrations, given separately for each animal,

- total number of aberrations per group,

- number of cells with aberrations per group,

- dose-response relationship, if possible,

- statistical analyses, if any,

- concurrent negative control data,

- historical negative control data with ranges, means and standard deviations,

- concurrent positive control data,

- changes in ploidy, if seen.

Discussion of results.

Conclusions.

4. REFERENCES

(1) Adler, I.D., (1986) Clastogenic Potential in Mouse Spermatogonia of Chemical Mutagens Related to their Cell-Cycle Specifications. In: Genetic Toxicology of Environmental Chemicals, Part B: Genetic Effects and Applied Mutagenesis, Ramel, C., Lambert, B. and Magnusson, J. (Eds.) Liss, New York, p. 477-484.

(2) Adler, I.D., (1984) Cytogenetic tests in Mammals. In: Mutagenicity Testing: a Practical Approach. Ed. S. Venitt and J.M. Parry. IRL Press, Oxford, Washington DC, p. 275-306.

(3) Evans, E.P., Breckon, G. and Ford, C.E., (1964) An Air-Drying Method for Meiotic Preparations from Mammalian Testes. Cytogenetics and Cell Genetics, 3, p. 289-294.

(4) Richold, M., Ashby, J., Chandley, A., Gatehouse, D.G. and Henderson, L., (1990) In Vivo Cytogenetic Assays, In: D.J. Kirkland (Ed.) Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I revised. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 115-141.

(5) Yamamoto, K. and Kikuchi, Y., (1978) A New Method for Preparation of Mammalian Spermatogonial Chromosomes. Mutation Res., 52, p. 207-209.

(6) Adler, I.D., Shelby M.D., Bootman, J., Favor, J., Generoso, W., Pacchierotti, F., Shibuya, T. and Tanaka N., (1994) International Workshop on Standardisation of Genotoxicity Test Procedures. Summary Report of the Working Group on Mammalian Germ Cell Tests. Mutation Res., 312, p. 313-318.

(7) Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D.J. and Richold, M., (1992) Report of British Toxicology Society/UK Environmental Mutagen Society Working group: Dose setting in In Vivo Mutagenicity Assays. Mutagenesis, 7, p. 313-319.

(8) Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G. and Savage, J.R.K., (1989) Statistical Analysis of In Vivo Cytogenetic Assays In: D.J. Kirkland (Ed.) Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing, report, Part III. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 184-232.

B.24. MOUSE SPOT TEST

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITION

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

This is an in vivo test in mice in which developing embryos are exposed to the chemicals. The target cells in the developing embryos are melanoblasts, and the target genes are those which control the pigmentation of the coat hairs. The developing embryos are heterozygous for a number of these coat colour genes. A mutation in, or loss of (by a variety of genetic events), the dominant allele of such a gene in a melanoblast results in the expression of the recessive phenotype in its descendant cells, constituting a spot of changed colour in the coat of the resulting mouse. The number of offspring with these spots, mutations, are scored and their frequency is compared with that among offspring resulting from embryos treated with the solvent only. The mouse spot test detects presumed somatic mutations in foetal cells.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

Preparations

When possible, test substances are dissolved or suspended in isotonic saline. Chemicals insoluble in water are dissolved or suspended in appropriate vehicles. The vehicle used should neither interfere with the test chemical nor produce toxic effects. Fresh preparations of the test chemical should be used.

Experimental animals

Mice of the T strain (nonagouti, a/a; chinchilla, pink eye, cchp/cchp; brown, b/b; dilute, short ear, d se/d se; piebald spotting, s/s) are mated either with the HT strain (pallid, nonagouti, brachypody, pa a bp/pa a bp; leaden fuzzy, ln fz/ln fz; pearl pe/pe) or with C57BL (nonagouti, a/a). Other appropriate crosses such as between Nmri (nonagouti, a/a; albino, c/c) and DBA (nonagouti, a/a; brown, b/b; dilute d/d) may be used provided they produce nonagouti offspring.

Number and sex

Sufficient pregnant females are treated to provide an appropriate number of surviving offspring for each dose level used. The appropriate sample size in governed by the number of spots observed in the treated mice and the scale of the control data. A negative result is acceptable only when at least 300 offspring from females treated with the highest dose have been scored.

Use of negative and positive controls

Concurrent control data from mice treated with the vehicle only (negative controls) should be available. Historical control data from the same laboratory may be pooled to increase the sensitivity of the test provided they are homogeneous. Positive control data recently obtained in the same laboratory from treatment with a chemical known to show mutagenicity by this test should be available if no mutagenicity of the test chemical is detected.

Route of administration

The usual routes of administration are oral intubation or intraperitoneal injection of the pregnant females. Treatment by inhalation or other routes of administration are used when appropriate.

Dose levels

At least two dose levels are used including one showing signs of toxicity or reduced litter size. For non-toxic chemicals exposure to the maximum practicable dose should be used.

Procedure

A single treatment is normally given on day 8,9 or 10 of pregnancy, counting as day 1 the day on which the vaginal plug is first dbserved. These days correspond to 7,25, 8,25 and 9,25 days after conception. Successive treatments over these days may be used.

Analysis

The offspring are coded and scored for spots between three and four weeks after birth. Three classes of spots are distinguished:

(a) white spots within 5 mm of the mid-ventralline which are presumed to result from cell killing (WMVS);

(b) yellow, agouti-like, spots associated with mammae, genitalia, throat, axillary and inguinal areas and on the mid-forehead, which are presumed to result from misdifferentiation (MDS); and

All three classes are scored but only the last, RS, is of genetic relevance. Problems of distinguishing between MDS and RS may be solved by fluorescence microscopy of sample hairs.

Obvious gross morphological abnormalities of the offspring should be noted.

2. DATA

The data are presented as the total number of offspring scored and the number having one or more presumed somatic mutation spots. Treatment and negative control data are compared by appropriate methods. Data are also presented on a per-litter basis.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- the strains used in the cross,

- the number of pregnant females in the experimental and control groups,

- the average litter size in the experimental and control groups at birth and at weaning,

- the dose level(s) of the test chemical,

- the solvent used,

- the day of pregnancy of which treatment was given,

- the route of treatment,

- the total number of offspring scored, and the number with WMVS, MDS and RS in the experimental and control groups,

- gross morphological abnormalities,

- dose/response relationship of RS when possible,

- statistical evaluation,

- discussion of results,

- interpretation of results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.25. MOUSE HERITABLE TRANSLOCATION

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITION

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The mouse heritable translocation test detects structural and numerical chromosome changes in mammalian germ cells as recovered in first generation progeny. The types of chromosome changes detected are reciprocal translocations and, if female progeny are included, X-chromosome loss. Carriers of translocations and XO-females show reduced fertility which is used to select F1 progeny for cytogenetic analysis. Complete sterility is caused by certain types of translocations (X-autosome and c-t type). Translocations are cytogenetically observed in meiotic cells at diakinesis- metaphase I of male individuals, either F1 males or male offspring of F1 females. The XO-females are cytogenetically identified by the presence of only 39 chromosomes in bone marrow mitoses.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

Preparations

The test chemicals are dissolved in isotonic saline. If insoluble they are dissolved or suspended in appropriate vehicles. Freshly prepared solutions of the test compound are employed. If a vehicle is used to facilitate dosing, it must not interfere with the test compound or produce toxic effects.

Route of administration

Routes of administration are usually oral intubation or intraperitoneal injection. Other routes of administration may be appropriate.

Experimental animals

For the ease of breeding and cytological verification these experiments are performed with mice. No specific mouse strain is required. However, the average litter-size of the strain should be greater than eight and be relatively constant.

Healthy sexually mature animals are used.

Number of animals

The number of animals necessary depends upon the spontaneous translocation frequency and the minimal rate of induction required for a positive result.

The test is usually performed by analyses of male F1 progeny. At least 500 male F1 progeny should be tested per dose group. If female F1 progeny are included, 300 males and 300 females are required.

Use of negative and positive controls

Adequate control data, derived from concurrent and historic control should be available. When acceptable positive control results are available from experiments conducted recently in the same laboratory these results can be used instead of a concurrent positive control.

Dose levels

One dose level is tested, usually the highest dose associated with the production of minimal toxic effects, but without affecting reproductive behaviour or survival. To establish a dose/response relationship two additional lower doses are required. For non-toxic chemicals exposure to the maximum practicable dose should be used.

Procedure

Treatment and mating

Two treatment schedules are available. Single administration of the test substance is most widely used. Administration of the test substance on seven days per week for 35 days may also be used. The number of matings following treatment is governed by the treatment schedule and should ensure that all treated germ cell stages are sampled. At the end of the mating period females are caged individually. When females give birth, the date, litter size and sex of progeny are recorded. All male progeny are weaned and all female progeny are discarded unless they are included in the experiment.

Testing for translocation heterozygosity

One of two possible methods is used:

- fertility testing of F1 progeny and subsequent verification of possible translocation carriers by cytogenetic analysis,

- cytogenetic analysis of all male F1 progeny without prior selection by fertility testing.

(a) Fertility testing

Reduced fertility of an Fl individual can be established by litter size observation and/or analysis of uterine contents of female mates.

Criteria for determining normal and reduced fertility must be established for the mouse strain used.

Litter size observation: F1 males to be tested are caged individually with females either from the same experiment or from the colony. Cages are inspected daily beginning 18 days after mating. Litter size and sex of the F2 progeny are recorded at birth and litters are discarded thereafter. If female F1 progeny are tested the F2 progeny of small litters are kept for further testing. Female translocation carriers are verified by cytogenetic analysis of a translocation in any of their male offspring. XO-females are recognised by the change in sex ratio among their progeny from 1:1 to 1:2 males versus females. In a sequential procedure, normal Fl animals are eliminated from further testing if the first F2 litter reaches or exceeds a predetermined normal value, otherwise a second or third F2 litter is observed.

F1 animals that cannot be classified as normal after observation of up to three F2 litters are either tested further by analysis of uterine contents of female mates or directly subjected to cytogenetic analysis.

Analysis of uterine contents: the reduction in litter size of translocation carriers is due to embryonic death so that a high number of dead implants is indicative of the presence of a translocation in the animal under test. F1 males to be tested are mated to two to three females each. Conception is established by daily inspection for vaginal plugs in the morning. Females are sacrificed 14 to 16 days later and living and dead implants in their uteri are recorded.

(b) Cytogenetic analysis

Testes preparations are made by the air-drying technique. Translocation carriers are identified by the presence of multivalent configurations at diakinesis-metaphase I in primary spermatocytes. Observation of at least two cells with multivalent association constitutes the required evidence that the tested animal is a translocation carrier.

If no breeding selection has been performed all F1 males are inspected cytogenetically. A minimum of 25 diakinesis-metaphase I cells per male must be scored microscopically. Examination of mitotic metaphases, in spermatogonia or bone-marrow, is required in F1 males with small testes and meiotic breakdown before diakinesis or from F1 female XO suspects. The presence of an unusually long and/or short chromosome in each of 10 cells is evidence for a particular male sterile translocation (c-t type). Some X-autosome translocations that cause male sterility may only be identified by banding analysis of mitotic chromosomes. The presence of 39 chromosomes in all of 10 mitoses is evidence for an XO condition in a female.

2. DATA

Data are presented in tabular form.

The mean litter size and sex ratio from parental matings at birth and weaning are reported for each mating interval.

For fertility assessment of F1 animals, the mean litter size of all normal matings and the individual litter sizes of F1 translocation carriers are presented. For analysis of uterine contents, the mean number of living and dead implants of normal matings and the individual numbers of living and dead implants for each mating of F1 translocation carriers are reported.

For cytogenetic analysis of diakinesis-metaphase I, the numbers of types of multivalent configurations and the total number of cells are listed for each translocation carrier.

For sterile F1 individuals, the total number of matings and the duration of the mating period are reported. Testes weights and cytogenetic analysis details are given.

For XO females, the mean litter size, sex ratio of F1 progeny and cytogenetic analysis results are reported.

Where possible F1 translocation carriers are preselected by fertility tests, the tables have to include information on how many of these were confirmed translocation heterozygotes.

Data from negative controls and the positive control experiments are reported.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- strain of mice, age of animals, weights of treated animals,

- numbers of parental animals of each sex in experimental and control groups,

- test conditions, detailed description of treatment, dose levels, solvents, mating schedule,

- number and sex of offspring per female, number and sex of offspring raised for translocation analysis,

- time and criteria of translocation analysis,

- number and detailed description of translocation carriers, including breeding data and uterine content data, if applicable;

- cytogenetic procedures and details of microscopic analysis, preferably with pictures,

- statistical evaluation,

- discussion of results,

- interpretation of results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.26. SUB-CHRONIC ORAL TOXICITY TESTREPEATED DOSE 90 – DAY ORAL TOXICITY STUDY IN RODENTS

1. METHOD

This sub-chronic oral toxicity test method is a replicate of the OECD TG 408 (1998).

1.1. INTRODUCTION

In the assessment and evaluation of the toxic characteristics of a chemical, the determination of sub-chronic oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained from acute or repeated dose 28-day toxicity tests. The 90-day study provides information on the possible health hazards likely to arise from repeated exposure over a prolonged period of time covering post-weaning maturation and growth well into adulthood. The study will provide information on the major toxic effects, indicate target organs and the possibility of accumulation, and can provide an estimate of a no-observed-adverse-effect level of exposure which can be used in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.

The method places additional emphasis on neurological endpoints and gives an indication of immunological and reproductive effects. The need for careful clinical observations of the animals, so as to obtain as much information as possible, is also stressed. This study should allow for the identification of chemicals with the potential to cause neurotoxic, immunological or reproductive organ effects, which may warrant further in-depth investigation.

See also General introduction Part B.

1.2. DEFINITIONS

Dose: is the amount of test substance administered. Dose is expressed as weight (g, mg) or as weight of test substance per unit weight of test animal (e.g. mg/kg), or as constant dietary concentrations (ppm).

Dosage: is a general term comprising of dose, its frequency and the duration of dosing.

NOAEL: is the abbreviation for no-observed-adverse-effect level and is the highest dose level where no adverse treatment-related findings are observed.

1.3. PRINCIPLE OF THE TEST METHOD

The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 90 days. During the period of administration the animals are observed closely for signs of toxicity. Animals, which die or are killed during the test are necropsied and, at the conclusion of the test, surviving animals are also killed and necropsied.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparations of animals

1.4.2. Preparations of doses

1.4.3. Test conditions

1.4.3.1. Experimental animals

The preferred species is the rat, although other rodent species, e.g. the mouse, may be used. Commonly used laboratory strains of young healthy adult animals should be employed. The females should be nulliparous and non-pregnant. Dosing should begin as soon as possible after weaning and, in any case, before the animals are nine weeks old. At the commencement of the study the weight variation of animals used should be minimal and not exceed ± 20 % of the mean weight of each sex. Where the study is conducted as a preliminary to a long term chronic toxicity study, animals from the same strain and source should be used in both studies.

1.4.3.2. Number and sex

At least 20 animals (10 female and 10 male) should be used at each dose level. If interim kills are planned, the number should be increased by the number of animals scheduled to be killed before the completion of the study. Based on previous knowledge of the chemical or a close analogue, consideration should be given to including an additional satellite group of ten animals (five per sex) in the control and in the top dose group for observation, after the treatment period, of reversibility or persistence of any toxic effects. The duration of this post-treatment period should be fixed appropriately with regard to the effects observed.

1.4.3.3. Dose levels

At least three dose levels and a concurrent control shall be used, except where a limit test is conducted (see 1.4.3.4). Dose levels may be based on the results of repeated dose or range finding studies and should take into account any existing toxicological and toxicokinetic data available for the test substance or related materials. Unless limited by the physical-chemical nature or biological effects of the test substance, the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. A descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a no-observed-adverse-effect level (NOAEL) at the lowest dose level. Two to four-fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of about 6-10) between dosages.

The control group shall be an untreated group or a vehicle-control group if a vehicle is used in administering the test substance. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to those in the test groups. If a vehicle is used, the control group shall receive the vehicle in the highest volume used. If a test substance is administered in the diet, and causes reduced dietary intake, then a pair-fed control group may be useful in distinguishing between reductions due to palatability or toxicological alterations in the test model.

Consideration should be given to the following characteristics of the vehicle and other additives, as appropriate: effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.

1.4.3.4. Limit test

If a test at one dose level, equivalent to at least 1000 mg/kg body weight/day, using the procedures described for this study, produces no-observed-adverse-effects and if toxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.

1.5. PROCEDURE

1.5.1. Administration of doses

The animals are dosed with the test substance daily seven days each week for a period of 90 days. Any other dosing regime, e.g. five days per week, needs to be justified. When the test substance is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should not exceed 1 ml/100 g body weight, except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animal's body weight may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where a 90-day study is used as a preliminary to a long term chronic toxicity study, a similar diet should be used in both studies.

1.5.2. Observations

The observation period should be at least 90 days. Animals in a satellite group scheduled for follow-up observations should be kept for an appropriate period without treatment to detect persistence of, or recovery from toxic effects.

General clinical observations should be made at least once a day, preferably at the same time(s) each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition of the animals should be recorded. At least twice daily, usually at the beginning and end of each day, all animals are inspected for signs of morbidity and mortality.

At least once prior to the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made outside the home cage, preferably in a standard arena and at similar times on each occasion. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory. Effort should be made to ensure that variations in the observation conditions are minimal. Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) should also be recorded (1).

Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the administration of the test substance and at the termination of the study, preferably in all animals but at least in the high dose and control groups. If changes in the eyes are detected all animals should be examined.

Towards the end of the exposure period and in any case not earlier than in week 11, sensory reactivity to stimuli of different types (1) (e.g. auditory, visual and proprioceptive stimuli) (2), (3), (4), assessment of grip strength (5) and motor activity assessment (6) should be conducted. Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could also be used.

Functional observations conducted towards the end of the study may be omitted when data on functional observations are available from other studies and the daily clinical observations did not reveal any functional deficits.

Exceptionally, functional observations may also be omitted for groups that otherwise reveal signs of toxicity to an extent that would significantly interfere with the functional test performance.

1.5.2.1. Body weight and food/water consumption

All animals should be weighed at least once a week. Measurements of food consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly. Water consumption may also be considered for dietary or gavage studies during which drinking activity may be altered.

1.5.2.2. Haematology and clinical biochemistry

Blood samples should be taken from a named site and stored, if applicable, under appropriate conditions. At the end of the test period, samples are collected just prior to or as part of the procedure for killing the animals.

The following haematological examinations should be made at the end of the test period and when any interim blood samples may have been collected: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count and a measure of blood clotting time/potential.

Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained from each animal just prior to or as part of the procedure for killing the animals (apart from those found moribund and/or intercurrently killed). In a similar manner to haematological investigations, interim sampling for clinical biochemical tests may be performed. Overnight fasting of the animals prior to blood sampling is recommended [3]. Determinations in plasma or serum should include sodium, potassium, glucose, total cholesterol, urea, blood urea nitrogen, creatinine, total protein and albumin, and more than two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, and sorbitol dehydrogenase). Measurements of additional enzymes (of hepatic or other origin) and bile acids, which may provide useful information under certain circumstances, may also be included.

Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.

In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium, phosphorus, fasting triglycerides, specific hormones, methaemoglobin and cholinesterase. These need to be identified for chemicals in certain classes or on a case-by-case basis.

Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect from a given substance.

If historical baseline data are inadequate, consideration should be given as to whether haematological and clinical biochemistry variables need to be determined before dosing commences; it is generally not recommended that this data be generated before treatment (7).

1.5.2.3. Gross necropsy

All animals in the study shall be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, thymus, spleen, brain and heart of all animals (apart from those found moribund and/or intercurrently killed) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying.

The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and medulla/pons), spinal cord (at three levels: cervical, mid-thoracic and lumbar), pituitary, thyroid, parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines (including Peyer's patches), liver, pancreas, kidneys, adrenals, spleen, heart, trachea and lungs (preserved by inflation with fixative and then immersion), aorta, gonads, uterus, accessory sex organs, female mammary gland, prostate, urinary bladder, gall bladder (mouse), lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, a section of bone marrow (and/or a fresh bone marrow aspirate), skin and eyes (if changes were observed during ophthalmological examinations). The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved.

1.5.2.4. Histopathology

All gross lesions should be examined.

When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.

2. DATA AND REPORTING

2.1. DATA

When applicable, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods and the data to be analysed should be selected during the design of the study.

2.2. TEST REPORT

The test report must include the following information:

2.2.1. Test substance:

- physical nature, purity and physico-chemical properties,

- identification data,

- vehicle (if appropriate): justification for choice of vehicle, if other than water.

2.2.2. Test species:

- species and strain used,

- number, age and sex of animals,

- source, housing conditions, diet etc.,

- individual weights of animals at the start of the test.

2.2.3. Test conditions:

- rationale for dose level selection,

- details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation,

- details of the administration of the test substance,

- actual doses (mg/kg body weight/day), and conversion factor from diet/drinking water test substance concentration (ppm) to the actual dose, if applicable,

- details of food and water quality.

2.2.4. Results:

- body weight and body weight changes,

- food consumption, and water consumption, if applicable,

- toxic response data by sex and dose level, including signs of toxicity,

- nature, severity and duration of clinical observations (whether reversible or not),

- results of ophthalmological examination,

- sensory activity, grip strength and motor activity assessments (when available),

- haematological tests with relevant base-line values,

- clinical biochemistry tests with relevant base-line values,

- terminal body weight, organ weights and organ/body weight ratios,

- necropsy findings,

- a detailed description of all histopathological findings,

- absorption data if available,

- statistical treatment of results, where appropriate,

Discussion of results.

Conclusions.

3. REFERENCES

(1) IPCS (1986) Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. Environmental Health Criteria Document No 60.

(2) Tupper, D.E., Wallace, R.B., (1980) Utility of the Neurologic Examination in Rats. Acta Neurobiol. Exp., 40, p. 999-1003.

(3) Gad, S.C., (1982) A Neuromuscular Screen for Use in Industrial Toxicology. J.Toxicol. Environ. Health, 9, p. 691-704.

(4) Moser, V.C., Mc Daniel, K.M., Phillips, P.M., (1991) Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol., 108, p. 267-283.

(5) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T., (1979) A Method for the Routine Assesment of Fore- and Hind- limb grip Strength of Rats and Mice. Neurobehav. Toxivol., 1, p. 233-236.

(6) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., Tilson H.A., MacPhail R.C., (1991) Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol., 13, p. 599-609.

(7) Weingand K., Brown G., Hall R. et al., (1996) "Harmonisation of Animal Clinic Pathology Testing in Toxicity and Safety Studies", Fundam. & Appl. Toxicol., 29, p. 198-201.

B.27. SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90-DAY ORAL TOXICITY STUDY IN NON-RODENTS

1. METHOD

This sub-chronic oral toxicity test method is a replicate of the OECD TG 409 (1998).

1.1. INTRODUCTION

In the assessment and evaluation of the toxic characteristics of a chemical, the determination of sub-chronic oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained from acute or repeated dose 28-day toxicity tests. The 90-day study provides information on the possible health hazards likely to arise from repeated exposure over a period of rapid growth and into young adulthood. The study will provide information on the major toxic effects, indicate target organs and the possibility of accumulation, and can provide an estimate of a no-observed-adverse-effect level of exposure which can be used in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.

The test method allows for the identification in non-rodent species of adverse effects of chemical exposure and should only be used:

- where effects observed in other studies indicate a need for clarification/characterisation in a second, non-rodent species, or

- where toxicokinetic studies indicate that the use of a specific non-rodent species is the most relevant choice of laboratory animal, or

- where other specific reasons justify the use of a non-rodent species.

See also General introduction Part B.

1.2. DEFINITIONS

Dosage: is a general term comprising of dose, its frequency and the duration of dosing.

NOAEL: is the abbreviation for no-observed-adverse-effect level and is the highest dose level where no adverse treatment-related findings are observed.

1.3. PRINCIPLE OF THE TEST METHOD

The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 90 days. During the period of administration the animals are observed closely for signs of toxicity. Animals, which die or are killed during the test are necropsied and at the conclusion of the test surviving animals are also killed and necropsied.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Selection of animal species

The commonly used non-rodent species is the dog, which should be of a defined breed; the beagle is frequently used. Other species, e.g. swine, mini-pigs, may also be used. Primates are not recommended and their use should be justified. Young, healthy animals should be employed, and in the case of the dog, dosing should begin preferably at four to six months and not later than nine months of age. Where the study is conducted as a preliminary to a long-term chronic toxicity study, the same species/breed should be used in both studies.

1.4.2. Preparation of animals

Healthy young animals, which have been acclimated to laboratory conditions and have not been subjected to previous experimental procedures, should be used. The duration of acclimatisation will depend upon the selected test species and their source. At least five days for dogs or purpose bred swine from a resident colony and at least two weeks for these animals if from external sources are recommended. The test animals should be characterised as to species, strain, source, sex, weight and/or age. Animals should be randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. Each animal should be assigned a unique identification number.

1.4.3. Preparations of doses

The test substance may be administered in the diet or in the drinking water, by gavage or in capsules. The method of oral administration is dependent on the purpose of the study, and the physical-chemical properties of the test material.

1.5. PROCEDURE

1.5.1. Number and sex of animals

At least eight animals (four female and four male) should be used at each dose level. If interim kills are planned, the number should be increased by the number of animals scheduled to be killed before the completion of the study. The number of animals at the termination of the study must be adequate for a meaningful evaluation of toxic effects. Based on previous knowledge of the substance or a close analogue, consideration should be given to including an additional satellite group of eight animals (four per sex) in control and in top dose group for observation after the treatment period of reversibility or persistence of any toxic effects. The duration of this post-treatment period should be fixed appropriately with regard to the effects observed.

1.5.2. Dosage

At least three dose levels and a concurrent control shall be used, except where a limit test is conducted (see 1.5.3). Dose levels may be based on the results of repeated dose or range finding studies and should take into account any existing toxicological and toxicokinetic data available for the test compound or related materials. Unless limited by the physical-chemical nature or biological effects of the test substance, the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. A descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a no-observed-adverse-effect level (Noael) at the lowest dose level. Two to fourfold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of about 6–10) between dosages.

1.5.3. Limit test

1.5.4. Administration of doses

The animals are dosed with the test substance daily seven days each week for a period of 90 days. Any other dosing regime, e.g. five days per week, needs to be justified. When the test substance is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. Normally the volume should be kept as low as possible. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animal's body weight may be used; any alternative used must be specified. For a substance administered by gavage or by capsule, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where the 90 day study is used as a preliminary to a long term chronic toxicity study, a similar diet should be used in both studies.

1.5.5. Observations

General clinical observations should be made at least once a day, preferably at the same time(s) each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition of the animals should be recorded. At least twice daily, usually at the beginning and end of each day, all animals should be inspected for signs of morbidity and mortality.

At least once prior to the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made, where practical outside the home cage in a standard arena and preferably at similar times on each occasion. Effort should be made to ensure that variations in the observation conditions are minimal. Signs of toxicity should be carefully recorded, including time of onset, degree and duration. Observations should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or any bizarre behaviour should also be recorded.

Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the administration of the test substance and at the termination of the study, preferably in all animals but at least in the high dose and control groups. If treatment related changes in the eyes are detected all animals should be examined.

1.5.5.1. Body weight and food/water consumption

1.5.5.2. Haematology and clinical biochemistry

Haematology, including haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count and a measure of clotting potential such as clotting time, prothrombin time, or thromboplastin time should be investigated at the start of the study, then either at monthly intervals or midway through the test period and finally at the end of the test period.

Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained from all animals at the start, then either at monthly intervals or midway through the test and finally at the end of the test period. Test areas, which should be considered are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the test substance. Animals should be fasted for a period appropriate to the species prior to blood sampling. Suggested determinations include calcium, phosphorus, chloride, sodium, potassium, fasting glucose, alanine aminotransferase, aspartate aminotransferase, ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements.

Urinalysis determinations should be performed at least at the start, then midway and finally at the end of the study using timed urine volume collection. Urinalysis determinations include appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells. Additional parameters may be employed where necessary to extend the investigation of observed effect(s).

In addition, studies to investigate markers of general tissue damage should be considered. Other determinations, which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methaemoglobin, and cholinesterase inhibition. Additional clinical biochemistry may be employed where necessary to extend the investigation of observed effects. These need to be identified for chemicals in certain classes or on a case-by-case basis.

Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect from a given substance.

1.5.5.3. Gross necropsy

All animals in the study shall be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver with gall bladder, kidneys, adrenals, testes, epididymides, ovaries, uterus, thyroid (with parathyroids), thymus, spleen, brain and heart of all animals (apart from those found moribund and/or inter-currently killed) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying.

The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and medulla/pons), spinal cord (at three levels: cervical, mid-thoracic and lumbar), pituitary, eyes, thyroid, parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines (including Peyer's patches), liver, gall bladder, pancreas, kidneys, adrenals, spleen, heart, trachea and lungs, aorta, gonads, uterus, accessory sex organs, female mammary gland, prostate, urinary bladder, lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, a section of bone marrow (and/or a fresh bone marrow aspirate) and skin. The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved.

1.5.5.4. Histopathology

Full histopathology should be carried out on the preserved organs and tissues in at least all animals in control and high dose group. The examination should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group.

All gross lesions should be examined.

When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.

2. DATA AND REPORTING

2.1. DATA

2.2. TEST REPORT

The test report must include the following information:

2.2.1. Test substance:

- physical nature, purity and physico-chemical properties,

- identification data,

- vehicle (if appropriate): justification for choice of vehicle, if other than water.

2.2.2. Test species:

- species and strain used,

- number, age and sex of animals,

- source, housing conditions, diet etc.,

- individual weights of animals at the start of the test.

2.2.3. Test conditions:

- rationale for dose level selection,

- details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation,

- details of the administration of the test substance,

- actual doses (mg/kg body weight/day), and conversion factor from diet/drinking water test substance concentration (ppm) to the actual dose, if applicable,

- details of food and water quality.

2.2.4. Results:

- body weight/body weight changes,

- food consumption, and water consumption, if applicable,

- toxic response data by sex and dose level, including signs of toxicity,

- nature, severity and duration of clinical observations (whether reversible or not),

- ophthalmological examination,

- haematological tests with relevant base-line values,

- clinical biochemistry tests with relevant base-line values,

- terminal body weight, organ weights and organ/body weight ratios,

- necropsy findings,

- a detailed description of all histopathological findings,

- absorption data if available,

- statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

B.28. SUB-CHRONIC DERMAL TOXICITY STUDY 90-DAY REPEATED DERMAL DOSE STUDY USING RODENT SPECIES

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITIONS

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The test substance is applied daily to the skin in graduated doses to several groups of experimental animals, one dose per group for a period of 90 days. During the period of application the animals are observed daily to detect signs of toxicity. Animals, which die during the test are necropsied, and at the conclusion of the test surviving animals are necropsied.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

The animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. Before the test healthy young animals are randomised and assigned to the treated and control groups. Shortly before testing fur is clipped from the dorsal area of the trunk of the test animals. Shaving may be employed but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care must be taken to avoid abrading the skin. Not less than 10 % of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of the covering. When testing solids, which may be pulverised if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. Liquid test substances are generally used undiluted. Daily application on a five to seven-day per week basis is used.

1.6.2. Test conditions

1.6.2.1. Experimental animals

The adult rat, rabbit or guinea pig may be used. Other species may be used but their use would require justification. At the commencement of the test the range of the weight variation should be ± 20 % of the mean weight. Where a sub-chronic dermal study is conducted as a preliminary to a long-term study, the same species and strain should be used in both studies.

1.6.2.2. Number and sex

At least 20 animals (10 female and 10 male) with healthy skin should be used at each dose level. The females should be nulliparous and non-pregnant. If interim sacrifices are planned the number should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 20 animals (10 animals per sex) may be treated with the high-dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 28 days post-treatment.

1.6.2.3. Dose levels

At least three dose levels are required with a controle or a vehicle control if a vehicle is used. The exposure period should be at least six hours per day. The application of the test substance should be made at similar times each day, and the amount of substance applied adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body weight. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. Where a vehicle is used to facilitate dosing, the vehicle control group should be dosed in the same way as the treated groups, and receive the same amount as that received by the highest dose level group. The highest dose level should result in toxic effects but produce no, or few, fatalities. The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest level should exceed this. Ideally, the intermediate dose level should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose levels should be spaced to produce a gradation of toxic effects. In the low and intermediate groups, and in the controls, the incidence of fatalities should be low, in order to permit a meaningful evaluation of the results.

If application of the test substance produces severe skin irritation the concentrations should be reduced and this may result in a reduction in, or absence of, other toxic effects al: the high-dose level. If the skin has been badly damaged it may be necessary to terminate the study and undertake a new study at lower concentrations.

1.6.3. Limit test

If a preliminary study at a dose level of 1000 mg/kilograms, or a higher dose level related to possible human exposure where this is known, produces no toxic effects, further testing may not be considered necessary.

1.6.4. Observation period

The experimental animals should be observed daily for signs of toxicity. The time of death and the time at which signs of toxicity appear and disappear should be recorded.

1.6.5. Procedure

Animals should be caged individually. The animals are treated with the test substance, ideally on seven days per week, for a period of 90 days.

Animals in any satellite groups scheduled for follow-up observations should be kept for a further 28 days without treatment to detect recovery from, or persistence of, toxic effects. Exposure time should be six hours per day.

The test substance should be applied uniformly over an area, which is approximately 10 % of the total body surface area. With highly toxic substances, the surface area covered may be less but as much of the area should be covered with as thin and uniform a film as possible.

During exposure the test substance is held in contact with the skin with a porous gauze dressing and non-irritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance but complete immobilisation is not a recommended method.

At the end of the exposure period, residual test substance should be removed where practicable using water or some other appropriate method of cleansing the skin.

All the animals should be observed daily and signs of toxicity recorded, including the time of onset, their degree and duration. Cageside observations should include changes in skin and fur, eyes and mucous membranes, as well as respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Measurements should be made of food consumption weekly and the animals weighed weekly. Regular observations of the animals are necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period all survivors in the non-satellite treatment groups are necropsied. Moribund animals should be removed and necropsied when noticed.

The following examinations are customarily made on all animals including the controls:

(a) ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to exposure to the test substance and at the termination of the study, preferably in all animals but at least in the high-dose and control groups. If changes in the eyes are detected all animals should be examined.

(b) haematology, including haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential, such as clotting time, prothrombin time, thromboplastin time, or platelet count, should be investigated at the end of the test period.

(c) clinical biochemistry determination on blood should be carried out at the end of the test period. Test areas, which are considered appropriate to all studies are electrolyte balance, carbohydrate metabolism, liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. Suggested determinations are calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), sercum glutamic pyruvic transaminase [4], serum glutamic oxaloacetic transaminase [5], ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methaemoglobin and choliensterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.

(d) urinalysis is not required on a routine basis but only when there is an indication based on expected or observed toxicity.

If historical baseline data are inadequate, consideration should be given to determination of haem a to logical and clinical biochemistry parameters before dosing commences.

Gross necropsy

All animals should be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals and testes must be weighed wet as soon as possible after dissection to avoid drying. The following organs and tissues should be preserved in a suitable medium for possible future histopathological examination: all gross lesions, brain — including sections of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/parathyroid, any thymic tissue, (trachea), lungs, heart, aorta, salivary glands, liver, spleen, kidneys, adrenals, pancreas, gonads, uterus, accessory genital organs, gall bladder (if present), oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, representative lymph node, (female mammary gland), (thigh musculature), peripheral nerve, (eyes), (sternum with bone marrow), (femur — including articular surface), (spinal cord at three levels — cervical, mid-thoracic and lumbar), and (exorbital lachrymal glands). The tissues mentioned between brackets need only be examined if indicated by signs of toxicity or target organ involvement.

Histopathological examination

(a) Full histopathology should be carried out on normal and treated skin and on organs and tissues of animals in the control and high-dose groups.

(b) all gross lesions should be examined.

(d) where rats are used, lungs of animals in the low- and intermediate-dose groups should be subjected to histopathological examination for evidence of infection, since this provides a convenient assessment of the state of health of the animals. Further histopathological examination may not be required routinely on the animals in these groups, but must always be carried out in organs, which show evidence of lesions in the high-dose group.

(e) when a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the other treated groups.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion. Results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- species, strain, source, environmental conditions, diet,

- test conditions,

- dose levels (including vehicle, if used) and concentrations,

- toxic response data by sex and dose,

- no-effect level, where possible,

- time of death during the study or whether animals survived to termination,

- description of toxic or other effects,

- the time of observation of each abnormal sign and its subsequent course,

- food and bodyweight data,

- ophthalmological findings,

- haematological tests employed and all results,

- clinical biochemistry tests employed and all results (including results of any urinalysis),

- necropsy findings,

- a detailed description of all histopathological findings,

- statistical treatment of results where possible,

- discussion of the results,

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.29. SUB-CHRONIC INHALATION TOXICITY STUDY 90-DAY REPEATED INHALATION DOSE STUDY USING RODENT SPECIES

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITIONS

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

Several groups of experimental animals are exponed daily for a defined period to the test substance in graduated concentrations, one concentration being used per group, for a period of 90 days. Where a vehicle is used to help generate an appropriate concentration of the test substance in the atmosphere, a vehicle control group should be used. During the period of administration the animals are observed daily to detect signs of toxicity. Animals, which die during the test are necropsied and at the conclusion of the test surviving animals are necropsied.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

The animals are kept under the experimental housing and feeding conditions for at least five days prior to the experiment. Before the test, healthy young aimals are randomised and assigned to the treatment and control groups. Where necessary, a suitable vehicle may be added to the test substance to help generate an appropriate concentration of the substance in the atmosphere. If a vehicle or other additives are used to facilitate dosing, they should be known not to produce toxic effects. Historical data can be used if appropriate.

1.6.2. Test conditions

1.6.2.1. Experimental animals

Unless there are contra-indications, the rat is the preferred species. Commonly used laboratory strains of young healthy animals should be employed. At the commencement of the study the range of weight variation of animals used should not exceed ± 20 % of the appropriate mean value. Where a subchronic inhalation study is conducted as a preliminary to a long-term study, the same species and strain should be used in both studies.

1.6.2.2. Number and sex

At least 20 animals (10 female and 10 male) should be used for each exposure concentration. The females should be nulliparous and non-pregnant. If interim sacrifices are planned the number should be increased by the number of animals scheduled to be sacrified before the completion of the study. In addition, a satellite group of 20 animals (10 animals per sex) may be treated with the high concentration level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 28 days post treatment.

1.6.2.3. Exposure concentrations

At least three concentrations are required, with a control or a vehicle control (corresponding to the concentration of vehicle at the highest level) if a vehicle is used. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. The highest concentration should result in toxic effects but no, or few, fatalities. Where there is a usable estimation of human exposure the lowest level should exceed this. Ideally, the intermediate concentration should produce minimal observable toxic effects. If more than one intermediate concentration is used the concentrations should be spaced to produce a gradation of toxic effects. In the low and intermediate groups, and in the controls, the incidence of fatalities should be low to permit a meaningful evaluation of the results.

1.6.2.4. Exposure time

The duration of daily exposure should be six hours after equilibration of the chamber concentrations. Other durations may be used to meet specific requirements.

1.6.2.5. Equipment

The animals should be tested in inhalation equipment designed to sustain a dynamic air flow of at least 12 air changes per hour to ensure an adequate oxygen content and an evenly distributed exposure atmosphere. Where a chamber is used its design should minimise crowding of the test animals and maximise their exposure by inhalation to the test substance. As a general rule, to ensure stability of a chamber atmosphere the total volume of the test animals should not exceed 5 % of the volume of the test chamber. Oro-nasal, head only, or whole body individual chamber exposure may be used; the first two will minimise uptake by other routes.

1.6.2.6. Observation period

1.6.3. Procedure

The animals are exposed to the test substance daily, five to seven days per week, for a period of 90 days. Animals in any satellite groups scheduled for follow-up observations should be kept for a further 28 days without treatment to detect recovery from, or persistence of, toxic effects. The temperature at which the test is performed should be maintained at 22 ± 3 oC. Ideally, the relative humidity should be maintained between 30 % and 70 %, but in certain instances (e.g. tests of aerosols) this may not be practicable. Food and water should be withheld during exposure.

A dynamic inhalation system with a suitable analytical concentration control system should be used. To establish suitable exposure concentrations a trial test is recommended. The air flow should be adjusted to ensure that conditions throughout the exposure chamber are homogeneous. The system should ensure that stable exposure conditions are achieved as rapidly as possible.

Measurements or monitoring should be made of:

(a) the rare of air flow (continuously);

(b) the actual concentration of the test substance measured in the breathing zone. During the daily exposure period the concentration should not vary by more than ± 15 % of the mean value. However, in the case of dusts and aerosols, this level of control may not be achievable and a wider range would then be acceptable. During the total duration of the study, the day-to-day concentrations should be held as constant as practicable. During the development of the generating system, particle-size analysis should be performed to establish the stability of aerosol concentrations. During exposure, analysis should be conducted as often as necessary to determine the consistency of particle-size distribution;

(d) during and following exposure, observations are made and recorded systematically; individual records should be maintained for each animal. All the animals should be observed daily and signs of toxicity recorded including the time of onset, their degree and duration. Cageside observations should include: changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous systems; somatomotor activity and behaviour pattern. Measurements should be made of food consumption weekly and the animals weighed weekly. Regular observation of the animals is necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the exposure period all surviving animals are necropsied. Moribund animals should be removed and necropsied when noticed.

The following examinations are customarily made on all animals including the controls:

(a) ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the exposure to the test substance and at the termination of the study, preferably in all animals but at least in the high-dose and control groups. If changes in the eyes are detected all animals should be examined;

(c) clinical biochemistry determination on blood should be carried out at the end of the test period. Test areas, which are considered appropriate to all studies are electrolyte balance, carbohydrate metabolism, liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. Suggested determinations are calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), serum glutamic pyruvic transaminase [4], serum glutamic oxaloacetic transaminase [5], ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements. Other determinations, which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methaemoglobin and cholinesterase activity. Additional clinical biochemistry may be employed where necessary to extend the investigation of observed effects;

(d) urinalysis is not required on a routine basis but only when there is an indication based on expected or observed toxicity.

If historical baseline data are inadequate, consideration should be given to determination of haematological and clinical biochemistry parameters before dosing commences.

Gross necropsy

All animals should be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals and testes should be weighed wet as soon as possible after dissection to avoid drying. The following organs and tissues should be preserved in a suitable medium for possible future histopathological examination: all gross lesions, lungs —which should be removed intact, weighed and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure), nasopharyngeal tissues, brain —including sections of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/parathyroid, any thymic tissue, trachea, lungs, heart, aorta, salivary glands, liver, spleen, kidneys, adrenals, pancreas, gonads, uterus (accessory genital organs), (skin), gall bladder (if present), oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, representative lymph node, (female mammary gland), (thigh musculature), peripheral nerve, (eyes), sternum with bone marrow, (femur, including articular surface), and (spinal cord at three levels — cervical, mid-thoracic and lumbar). The tissues mentioned between brackets need only be examined if indicated by signs of toxicity, or target organ involvement.

Histopathological examination

(a) Full histopathology should be carried out on the respiratory tract and other organs and tissues of all animals in the control and high-dose groups.

(b) all gross lesions should be examined.

(d) lungs of animals in the low – and intermediate-dose group should also be subjected to histopathological examination; since this can provide a convenient assessment of the state of health of the animals. Further histopathological examination may not be required routinely on the animals in these groups but must always be carried out on organs, which show evidence of lesions in the high-dose group.

(e) when a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in other treated groups.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the types of lesions and the percentage of animals displaying each type of lesion. Results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- species, strain, source, environmental conditions, diet,

- test conditions:

description of exposure apparatus: including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing animals in a test chamber then this is used. The equipment for measuring temperature, humidity and, where appropriate, stability of aerosol concentrations or particle size, should be described.

Exposure data: these should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and should include:

(a) air flow rates through the inhalation equipment;

(b) temperature and humidity of air;

(c) nominal concentrations (total amount of test substance fed into the inhalation equipment divided by the volume of air);

(d) nature of vehicle, if used;

(e) actual concentrations in test breathing zone;

(f) median particle sizes (where appropriate):

- toxic response data by sex and concentration,

- no-effect level when possible,

- time of death during the study or whether animals surived to termination,

- description of toxic or other effects,

- the time of observation of each abnormal sign and its subsequent course,

- food and bodyweight data,

- ophthalmological findings,

- haematological tests employed and results,

- clinical biochemistry tests employed and results (including results of any urinalysis),

- necropsy findings,

- a detailed description of all histopathological findings,

- statistical treatment of results where appropriate,

- discussion of the results,

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.30. CHRONIC TOXICITY TEST

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITIONS

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The test substance is administered normally seven days per week, by an appropriate route, to several groups of experimental animals, one dose per group, for a major portion of their life span. During and after exposure to the test substance, the experimental animals are observed daily to detect signs of toxicity.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

1.6.2. Test conditions

1.6.2.1. Experimental animals

The preferred species is the rat.

Based upon the results of previously conducted studies other species (rodent or non-rodent) may be used. Commonly used laboratory strains of young healthy animals should be employed and dosing should begin as soon as possible after weaning.

At the commencement of the study the weight variation in the animals used should not exceed ± 20 % of the mean value. Where a sub-chronic oral study is conducted as a preliminary to a long-term study, the same species/breed and strain should be used in both studies.

1.6.2.2. Number and sex

For rodents at least 40 animals (20 female and 20 male) should be used at each dose level and concurrent control group. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed before the completion of the study.

For non-rodents a smaller number of animals, but at least four per sex per group, is acceptable.

1.6.2.3. Dose levels and frequency of exposure

At least three dose levels should be used in addition to the concurrent control group. The highest dose level should elicit definite signs of toxicity without causing excessive lethality.

The lowest dose level should not produce and evidence of toxicity.

The intermediate dose(s) should be established in a mid-range between the high and low doses.

The selection of dose levels should take into account data from preceding toxicity tests and studies.

Frequency of exposure is normally daily. If the chemical is administered in the drinking water or mixed in the diet it should be continuously available.

1.6.2.4. Controls

A concurrent control group which is identical in every respect to the treated groups, except for exposure to the test substance, should be used.

In special circumstances, such as in inhalation studies involving aerosols or the use of an emulsifier of uncharacterised biological activity in oral studies, a concurrent negative control group should also be used. The negative control group is treated in the same manner as the test groups except that the animals are not exposed to the test substance or any vehicle.

1.6.2.5. Route of administration

The two main routes of administration are oral and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the likely route of exposure in humans.

The use of the dermal route presents considerable practical problems. Chronic systemic toxicity resulting from percutaneous absorption can normally be inferred form the results of another oral test and a knowledge of the extent of percutaneous absorption derived from preceding percutaneous toxicity tests.

1.6.2.6. Oral studies

Where the test substance is absorbed from the gastrointestinal tract, and if the ingestion route is one by which humans may be exposed, the oral route of administration is preferred unless there are contra-indications. The animals may receive the test substance in the diet, dissolved in drinking water or given by capsule. Ideally, daily dosing on a seven-day per week basis should be used because dosing on a five-day per week basis may permit recovery or withdrawal toxicity in the non-dosing period and thus affect the result and subsequent evaluation. However, based primarily on practical considerations, dosing on a five-day per week basis is considered to be acceptable.

1.6.2.7. Inhalation studies

Because inhalation studies present technical problems of greater complexity than the other routes of administration, more detailed guidance on this mode of administration is given here. It should also be noted that intratracheal instillation may constitute a valid alternative in specific situations.

In both cases, the animals are usually exposed to fixed concentrations of test substance. A major difference between intermittent and continuous exposure is that with the former there is a 17 to 18 hour period in which animals may recover from the effects of each daily exposure, with an even longer recovery period during weekends.

The choice of intermittent or continuous exposure depends on the objectives of the study and on the human exposure that is to be simulated. However, certain technical difficulties must be considered. For example, the advantages of continuous exposure for simulating environmental conditions may be offset by the necessity for watering and feeding during exposure, and by the need for more complicated (and reliable) aerosol and vapour, generation and monitoring techniques.

1.6.2.8. Exposure chambers

The animals should be tested in inhalation chambers designed to sustain a dynamic flow of at least 12 air changes per hour to assure adequate oxygen content and an evenly distributed exposure atmosphere. Control and exposure chambers should be identical in construction and design to ensure exposure conditions comparable in all respects except for exposure to the test substances. Slight negative pressure inside the chamber is generally maintained to prevent leakage of the test substance into the surrounding area. The chambers should minimise the crowding of test animals. As a general rule, to ensure the stability of the chamber atmosphere, the total volume of the test animals should not exceed 5 % of the volume of the chamber.

Measurements or monitoring should be made of:

(i) air flow: the rate of air flow through the chamber should preferably be monitored continuously;

(ii) concentration: during the daily exposure period the concentration of the test substance should not vary more than ± 15 % of the mean value;

(iii) temperature and humidity: for rodents, the temperature should be maintained at 22 ± 2 oC, and the humidity within the chamber at 30 to 70 %, except when water is used to suspend the test substance in the chamber atmosphere. Preferably both should be monitored continuously;

(iv) particle size measurements: particle-size distribution should be determined in chamber atmospheres involving liquid or solid aerosols. The aerosol particles should be of respirable size for the test animal used. Samples of the chamber atmospheres should be taken in the breathing zone of the animals. The air sample should be representative of the distribution of the particles to which the animals are exposed and should account, on a gravimetric basis, for all the suspended aerosol even when much of the aerosol is not respirable. Particle size analyses should be carried out frequently during the development of the generating system to ensure the stability of the aerosol and thereafter as often as necessary during the exposures to determine adequately the consistency of the particle distribution to which the animals have been exposed.

1.6.2.9. Duration of study

The duration of the period of administration should be at least 12 months.

1.6.3. Procedure

Observations

A careful clinical examination should be made at least once each day. Additional observations should be made daily with appropriate actions taken to minimise loss of animals to the study, for example necropsy or refrigeration of those animals found dead and isolation or sacrifice of weak or moribund animals. Careful observations should be made to detect onset and progression of all toxic effects as well as to minimise loss due to disease, autolysis or cannibalism.

Clinical signs, including neurological and ocular changes as well as mortality, should be recorded for all animals. Time of onset and progression of toxic conditions, including suspected tumours, should be recorded.

Bodyweights should be recorded individually for all animals once a week during the first 13 weeks of the test period and at least once every four weeks thereafter. Food intake should be determined weekly during the first 13 weeks of the study, and then at approximately three-monthly intervals unless health status or body weight changes dictate otherwise.

Haematological examination

Haematological examination (e.g. haemoglobin content; packet cell volume, total red blood cells, total white blood cells, platelets or other measures of clotting potential) should be performed at three months, six months, and thereafter at approximately six-month intervals and at termination on blood samples collected form all non-rodents and from 10 rats/sex of all groups. If possible, samples should be from the same rats at each interval. In addition, a pre-test sample should be collected from non-rodents.

If clinical observations suggest a deterioration in the health of the animals during the study, a differential blood count of the affected animals should be performed.

A differential blood count is performed on samples from the animals in the highest dose group and the controls. Differential blood counts are performed for the next lower group(s) only if there is a major discrepancy between the highest group and the controls, or if indicated by pathological findings.

Urinalysis

Urine samples from all non-rodents and from 10 rats/sex of all groups, if possible from the same rats at the same intervals as haematological examination, should be collected for analysis. The following determinations should be made for either individual animals or on a pooled sample/sex/group for rodents:

- appearance: volume and density for individual animals,

- protein, glucose, ketones, occult blood (semi-quantitatively),

- microscopy of sediment (semi-quantitatively).

Clinical chemistry

At approximately six-monthly intervals and at termination, blood samples are drawn for clinical chemistry measurements from all non-rodents and 10 rats/sex of all groups, if possible, from the same rats at each interval. In addition, a pre-test sample should be collected from non-rodents. Plasma is prepared from these samples and the following determinations are made:

- total protein concentration,

- albumin concentration,

- liver function tests (such as alkaline phosphatase activity, glutamic pyruvic transaminase [4] activity and glutamic oxaloacetic transaminase [5] activity), gamma glutamyl transpeptldase, ornithine decarboxylase,

- carbohydrate metabolism such as fasting blood glucose,

- kidney function tests such as blood urea nitrogen.

Gross necropsy

Full gross necropsy should be performed on all animals, including those which died during the experiment or were killed having been found in a moribund condition. Prior to sacrifice, samples of blood should be collected from all animals, for differential blood counts. All grossly visible lesions, tumours or lesions suspected of being tumours should be preserved. An attempt should be made to correlate gross observations with the microscopic findings.

All organs and tissues should be preserved for histopathological examination. This usually concerns the following organs and tissues: brain [6] (medullaipons, cerebellar correx, cerebral cortex), pituitary, thyroid (including parathyroid), thymus, lungs (including trachea), heart, aorta, salivary glands, liver [6], spleen, kidneys [6], adrenals [6], oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, uterus, urinary bladder, lymph nodes, pancreas, gonads [6], accessory genital organs, female mammary gland, skin, musculature, peripheral, nerve, spinal cord (cervical, thoracic, lumbar), sternum with bone marrow and femur (including joint) and eyes. Inflation of lungs and urinary bladder with a fixative is the optimal way to preserve these tissues; inflation of the lungs in inhalation studies is essential for appropriate histopathological examination. In special studies such as inhalation studies, the entire respiratory tract should be studied, including nose, pharynx and larynx.

If other clinical examinations are carried out, the information obtained from these procedures should be available before microscopic examination, because it may give significant guidance to the pathologist.

Histopathology

All visible changes, particularly tumours and other lesions occurring in any organ should be examined microscopically. In addition, the following procedures are recommended:

(a) microscopic examination of all preserved organs and tissues with complete description of all lesions found in:

1. all animals that died or were killed during the study;

2. all of the high-dose group and controls;

(b) organs or tissues showing abnormalities caused, or possibly caused, by the test substance are also examined in the lower-dose groups;

(c) where the result of the test gives evidence of substantial reduction of the animals' normal lifespan or the induction of effects that might affect a toxic response, the next-lower dose level should be examined as described above;

(d) information on the incidence of lesions normally occurring in the strain of animals used, under the same laboratory conditions, i.e. historical control data, is indispensable for correctly assessing the significance of changes observed in treated animals.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions and the percentage of animals displaying each type of lesion. Results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

TEST REPORT

The test report shall, if possible, contain the following information:

- species, strain, source, environmental conditions, diet,

- test conditions:

Description of exposure apparatus:

Including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing animals in a test chamber when this is used. The equipment for measuring temperature, humidity and, where appropriate, stability of aerosol concentration or particle size, should be described.

Exposure data:

These should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and should include:

(a) air flow rates through the inhalation equipment;

(b) temperature and humidity of air;

(c) nominal concentrations (total amount of test substance fed into the inhalation equipment divided by the volume of air);

(d) nature of vehicle, if used;

(e) actual concentrations in test breathing zone;

(f) median particle sizes (where appropriate):

- dose levels (including vehicle, if used) and concentrations,

- toxic response data by sex and dose,

- no-effect level,

- time of death during the study or whether animals survived to termination,

- description of toxic and other effects,

- the time of observation of each abnormal sign and its subsequent course,

- food and bodyweight data,

- ophthalmological findings,

- haematological tests employed and all results,

- clinical biochemistry tests employed and all results (including results of any urinalysis),

- necropsy findings,

- a detailed description of all histopathological findings,

- statistical treatment of results where possible,

- discussion of the results,

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.31. PRENATAL DEVELOPMENTAL TOXICITY STUDY

1. METHOD

This method is a replicate of OECD TG 414 (2001).

1.1. INTRODUCTION

This method for developmental toxicity testing is designed to provide general information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism in utero; this may include assessment of maternal effects as well as death, structural abnormalities, or altered growth in the foetus. Functional deficits, although an important part of development, are not an integral part of this test method. They may be tested for in a separate study or as an adjunct to this study using the test method for developmental neurotoxicity. For information on testing for functional deficiencies and other postnatal effects the test method for the two-generation reproductive toxicity study and the developmental neurotoxicity study should be consulted as appropriate.

This test method may require specific adaptation in individual cases on the basis of specific knowledge on e.g. physicochemical or toxicological properties of the test substance. Such adaptation is acceptable, when convincing scientific evidence suggests that the adaptation will lead to a more informative test. In such a case, this scientific evidence should be carefully documented in the study report.

1.2. DEFINITIONS

Developmental toxicology: the study of adverse effects on the developing organism that may result from exposure prior to conception, during prenatal development, or postnatally to the time of sexual maturation. The major manifestations of developmental toxicity include 1) death of the organism, 2) structural abnormality, 3) altered growth, and 4) functional deficiency. Developmental toxicology was formerly often referred to as teratology.

Adverse effect: any treatment-related alteration from baseline that diminishes an organism's ability to survive, reproduce or adapt to the environment. Concerning developmental toxicology, taken in its widest sense it includes any effect which interferes with normal development of the conceptus, both before and after birth.

Altered growth: an alteration in offspring organ or body weight or size.

Alterations (anomalies): structural alterations in development that include both malformations and variations (28).

Malformation/Major abnormality: structural change considered detrimental to the animal (may also be lethal) and is usually rare.

Variation/Minor abnormality: structural change considered to have little or no detrimental effect on the animal; may be transient and may occur relatively frequently in the control population.

Conceptus: the sum of derivatives of a fertilised ovum at any stage of development from fertilisation until birth including the extra-embryonic membranes as well as the embryo or foetus.

Implantation (nidation): attachment of the blastocyst to the epithelial lining of the uterus, including its penetration through the uterine epithelium, and its embedding in the endometrium.

Embryo: the early or developing stage of any organism, especially the developing product of fertilisation of an egg after the long axis appears and until all major structures are present.

Embryotoxicity: detrimental to the normal structure, development, growth, and/or viability of an embryo.

Foetus: the unborn offspring in the post-embryonic period.

Foetotoxicity: detrimental to the normal structure, development, growth, and/or viability of a foetus.

Abortion: the premature expulsion from the uterus of the products of conception: of the embryo or of a nonviable foetus.

Resorption: a conceptus which, having implanted in the uterus, subsequently died and is being, or has been resorbed.

Early resorption: evidence of implantation without recognisable embryo/foetus

Late resorption: dead embryo or foetus with external degenerative changes

NOAEL: abbreviation for no-observed-adverse-effect level and is the highest dose or exposure level where no adverse treatment-related findings are observed.

1.3. REFERENCE SUBSTANCE

None.

1.4. PRINCIPLE OF THE TEST METHOD

Normally, the test substance is administered to pregnant animals at least from implantation to one day prior to the day of scheduled kill, which should be as close as possible to the normal day of delivery without risking loss of data resulting from early delivery. The test method is not intended to examine solely the period of organogenesis, (e.g. days 5-15 in the rodent, and days 6-18 in the rabbit) but also effects from preimplantation, when appropriate, through the entire period of gestation to the day before caesarean section. Shortly before caesarean section, the females are killed, the uterine contents are examined, and the foetuses are evaluated for externally visible anomalies and for soft tissue and skeletal changes.

1.5. DESCRIPTION OF THE TEST METHOD

1.5.1. Selection of animal species

It is recommended that testing be performed in the most relevant species, and that laboratory species and strains which are commonly used in prenatal developmental toxicity testing be employed. The preferred rodent species is the rat and the preferred non-rodent species is the rabbit. Justification should be provided if another species is used.

1.5.2. Housing and feeding conditions

The temperature in the experimental animal room should be 22 oC (± 3o) for rodents and 18 oC (± 3o) for rabbits. Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water.

Mating procedures should be carried out in cages suitable for the purpose. While individual housing of mated animals is preferred, group housing in small numbers is also acceptable.

1.5.3. Preparation of the animals

Healthy animals, which have been acclimated to laboratory conditions for at least five days and have not been subjected to previous experimental procedures, should be used. The test animals should be characterised as to species, strain, source, sex, weight and/or age. The animals of all test groups should, as nearly as practicable, be of uniform weight and age. Young adult nulliparous female animals should be used at each dose level. The females should be mated with males of the same species and strain, and the mating of siblings should be avoided. For rodents day 0 of gestation is the day on which a vaginal plug and/or sperm are observed; for rabbits day 0 is usually the day of coitus or of artificial insemination, if this technique is used. Mated females should be assigned in an unbiased manner to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. Each animal should be assigned a unique identification number. Mated females should be assigned in an unbiased manner to the control and treatment groups, and if the females are mated in batches, the animals in each batch should be evenly distributed across the groups. Similarly, females inseminated by the same male should be evenly distributed across the groups.

1.6. PROCEDURE

1.6.1. Number and sex of animals

Each test and control group should contain a sufficient number of females to result in approximately 20 female animals with implantation sites at necropsy. Groups with fewer than 16 animals with implantation sites may be inappropriate. Maternal mortality does not necessarily invalidate the study providing it does not exceed approximately 10 %.

1.6.2. Preparation of doses

If a vehicle or other additive is used to facilitate dosing, consideration should be given to the following characteristics: effects on the absorption, distribution, metabolism, and retention or excretion of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals. The vehicle should neither be developmentally toxic nor have effects on reproduction.

1.6.3. Dosage

Normally, the test substance should be administered daily from implantation (e.g. day 5 post mating) to the day prior to scheduled caesarean section. If preliminary studies, when available, do not indicate a high potential for preimplantation loss, treatment may be extended to include the entire period of gestation, from mating to the day prior to scheduled kill. It is well known that inappropriate handling or stress during pregnancy can result in prenatal loss. To guard against prenatal loss from factors which are not treatment related, unnecessary handling of pregnant animals as well as stress from outside factors such as noise should be avoided.

At least three dose levels and a concurrent control should be used. Healthy animals should be assigned in an unbiased manner to the control and treatment groups. The dose levels should be spaced to produce a gradation of toxic effects. Unless limited by the physical/chemical nature or biological properties of the test substance, the highest dose should be chosen with the aim to induce some developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering. At least one intermediate dose level should produce minimal observable toxic effects. The lowest dose level should not produce any evidence of either maternal or developmental toxicity. A descending sequence of dose levels should be selected with a view to demonstrating any dosage-related response and no-observed-adverse-effect level (NOAEL). Two- to four-fold intervals are frequently optimal for setting the descending dose levels, and the addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of 10) between dosages. Although establishment of a maternal NOAEL is the goal, studies which do not establish such a level may also be acceptable (1).

Dose levels should be selected taking into account any existing toxicity data as well as additional information on metabolism and toxicokinetics of the test substance or related materials. This information will also assist in demonstrating the adequacy of the dosing regimen.

A concurrent control group should be used. This group should be a sham-treated control group or a vehicle-control group if a vehicle is used in administering the test substance. All groups should be administered the same volume of either test substance or vehicle. Animals in the control group(s) should be handled in an identical manner to test group animals. Vehicle control groups should receive the vehicle in the highest amount used (as in the lowest treatment group).

1.6.4. Limit test

If a test at one dose level of at least 1000 mg/kg body weight/day by oral administration, using the procedures described for this study, produces no observable toxicity in either pregnant animals or their progeny and if an effect would not be expected based upon existing data (e.g. from structurally and/or metabolically related compounds), then a full study using three dose levels may not be considered necessary. Expected human exposure may indicate the need for a higher oral dose level to be used in the limit test. For other types of administration, such as inhalation or dermal application, the physico-chemical properties of the test substance often may indicate and limit the maximum attainable level of exposure (for example, dermal application should not cause severe local toxicity).

1.6.5. Administration of doses

The test substance or vehicle is usually administered orally by intubation. If another route of administration is used, the tester should provide justification and reasoning for its selection, and appropriate modifications may be necessary (2)(3)(4). The test substance should be administered at approximately the same time each day.

The dose to individual animals should normally be based on the most recent individual body weight determination. However, caution should be exercised when adjusting the dose during the last trimester of pregnancy. Existing data should be used for dose selection to prevent excess maternal toxicity. However, if excess toxicity is noted in the treated dams, those animals should be humanely killed. If several pregnant animals show signs of excess toxicity, consideration should be given to terminating that dose group. When the substance is administered by gavage, this should preferably be given as a single dose to the animals using a stomach tube or a suitable intubation canula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. The volume should not exceed 1 ml/100 g body weight, except in the case of aqueous solutions where 2 ml/100 g body weight may be used. When corn oil is used as a vehicle, the volume should not exceed 0.4 ml/100 g body weight. Variability in test volume should be minimised by adjusting the concentrations to ensure a constant volume across all dose levels.

1.6.6. Observations of the dams

Clinical observations should be made and recorded at least once a day, preferably at the same time(s) each day taking into consideration the peak period of anticipated effects after dosing. The condition of the animals should be recorded including mortality, moribundity, pertinent behavioural changes, and all signs of overt toxicity.

1.6.7. Body weight and food consumption

Animals should be weighed on day 0 of gestation or no later than day 3 of gestation if time-mated animals are supplied by an outside breeder, on the first day of dosing, at least every three days during the dosing period and on the day of scheduled kill.

Food consumption should be recorded at three-day intervals and should coincide with days of body weight determination.

1.6.8. Post-mortem examination

Females should be killed one day prior to the expected day of delivery. Females showing signs of abortion or premature delivery prior to scheduled kill should be killed and subjected to a thorough macroscopic examination.

At the time of termination or death during the study, the dam should be examined macroscopically for any structural abnormalities or pathological changes. Evaluation of the dams during caesarean section and subsequent foetal analyses should be conducted preferably without knowledge of treatment group in order to minimise bias.

1.6.9. Examination of uterine contents

Immediately after termination or as soon as possible after death, the uteri should be removed and the pregnancy status of the animals ascertained. Uteri that appear non gravid should be further examined (e.g. by ammonium sulphide staining for rodents and Salewski staining or a suitable alternative method for rabbits) to confirm the non-pregnant status (5).

Gravid uteri including the cervix should be weighed. Gravid uterine weights should not be obtained from animals found dead during the study.

The number of corpora lutea should be determined for pregnant animals.

The uterine contents should be examined for numbers of embryonic or foetal deaths and viable foetuses. The degree of resorption should be described in order to estimate the relative time of death of the conceptus (see Section 1.2).

1.6.10. Examination of foetuses

The sex and body weight of each foetus should be determined.

Each foetus should be examined for external alterations (6).

Foetuses should be examined for skeletal and soft tissue alterations (e.g. variations and malformations or anomalies) (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Categorisation of foetal alterations is preferable but not required. When categorisation is done, the criteria for defining each category should be clearly stated. Particular attention should be paid to the reproductive tract which should be examined for signs of altered development.

For rodents, approximately one-half of each litter should be prepared and examined for skeletal alterations. The remainder should be prepared and examined for soft tissue alterations, using accepted or appropriate serial sectioning methods or careful gross dissection techniques.

For non-rodents, e.g. rabbits, all foetuses should be examined for both soft tissue and skeletal alterations. The bodies of these foetuses are evaluated by careful dissection for soft tissue alterations, which may include procedures to further evaluate internal cardiac structure (25). The heads of one-half of the foetuses examined in this manner should be removed and processed for evaluation of soft tissue alterations (including eyes, brain, nasal passages and tongue), using standard serial sectioning methods (26) or an equally sensitive method. The bodies of these foetuses and the remaining intact foetuses should be processed and examined for skeletal alterations, utilising the same methods as described for rodents.

2. DATA

2.1. TREATMENT OF RESULTS

Data shall be reported individually for the dams as well as for their offspring and summarised in tabular form, showing for each test group and each generation the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons, the time of any death or humane kill, the number of pregnant females, the number of animals showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the types of embryo/foetal observations, and all relevant litter data.

Numerical results should be evaluated by an appropriate statistical method using the litter as the unit for data analysis. A generally accepted statistical method should be used; the statistical methods should be selected as part of the design of the study and should be justified. Data from animals that do not survive to the scheduled kill should also be reported. These data may be included in group means where relevant. Relevance of the data obtained from such animals, and therefore inclusion or exclusion from any group mean(s), should be justified and judged on an individual basis.

2.2. EVALUATION OF RESULTS

The findings of the Prenatal Developmental Toxicity Study should be evaluated in terms of the observed effects. The evaluation will include the following information:

- maternal and embryo/foetal test results, including the evaluation of the relationship, or lack thereof, between the exposure of the animals to the test substance and the incidence and severity of all findings,

- criteria used for categorising foetal external, soft tissue, and skeletal alterations if categorisation has been done,

- when appropriate, historical control data to enhance interpretation of study results,

- the numbers used in calculating all percentages or indices,

- adequate statistical analysis of the study findings, when appropriate, which should include sufficient information on the method of analysis, so that an independent reviewer/statistician can re-evaluate and reconstruct the analysis.

In any study which demonstrates the absence of any toxic effects, further investigations to establish absorption and bioavailability of the test substance should be considered.

2.3. INTERPRETATION OF RESULTS

A prenatal developmental toxicity study will provide information on the effects of repeated exposure to a substance during pregnancy on the dams and on the intrauterine development of their progeny. The results of the study should be interpreted in conjunction with the findings from subchronic, reproduction, toxicokinetic and other studies. Since emphasis is placed both on general toxicity in terms of maternal toxicity and on developmental toxicity endpoints, the results of the study will allow to a certain extent for the discrimination between developmental effects occurring in the absence of general toxicity and those which are only induced at levels that are also toxic to the maternal animal (27).

3. REPORTING

3.1. TEST REPORT

The test report must include the following specific information:

Test substance:

- physical nature and, where relevant, physiochemical properties,

- identification including CAS number if known/established,

- purity.

Vehicle (if appropriate):

- justification for choice of vehicle, if other than water.

Test animals:

- species and strain used,

- number and age of animals,

- source, housing conditions, diet, etc.,

- individual weights of animals at the start of the test.

Test conditions:

- rationale for dose level selection,

- details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation,

- details of the administration of the test substance,

- conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable,

- environmental conditions,

- details of food and water quality.

Results:

Maternal toxic response data by dose, including but not limited to:

- the number of animals at the start of the test, the number of animals surviving, the number pregnant, and the number aborting, number of animals delivering early,

- day of death during the study or whether animals survived to termination,

- data from animals that do not survive to the scheduled kill should be reported but not included in the inter-group statistical comparisons,

- day of observation of each abnormal clinical sign and its subsequent course,

- body weight, body weight change and gravid uterine weight, including, optionally, body weight change corrected for gravid uterine weight,

- food consumption and, if measured, water consumption,

- necropsy findings, including uterine weight,

- NOAEL values for maternal and developmental effects should be reported.

Developmental endpoints by dose for litters with implants, including:

- number of corpora lutea,

- number of implantations, number and percent of live and dead foetuses and resorptions,

- number and percent of pre- and post-implantation losses.

Developmental endpoints by dose for litters with live foetuses, including:

- number and percent of live offspring,

- sex ratio,

- foetal body weight, preferably by sex and with sexes combined,

- external, soft tissue, and skeletal malformations and other relevant alterations,

- criteria for categorisation if appropriate,

- total number and percent of foetuses and litters with any external, soft tissue, or skeletal alteration, as well as the types and incidences of individual anomalies and other relevant alterations.

Discussion of results.

Conclusions.

4. REFERENCES

(1) Kavlock R.J. et al., (1996) A Simulation Study of the Influence of Study Design on the Estimation of Benchmark Doses for Developmental Toxicity. Risk Analysis 16; p. 399-410.

(2) Kimmel, C.A. and Francis, E.Z., (1990) Proceedings of the Workshop on the Acceptability and Interpretation of Dermal Developmental Toxicity Studies. Fundamental and Applied Toxicology 14; p. 386-398.

(3) Wong, B.A., et al., (1997) Developing Specialised Inhalation Exposure Systems to Address Toxicological Problems. CIIT Activities 17; p. 1-8.

(4) US Environmental Protection Agency, (1985) Subpart E-Specific Organ/Tissue Toxicity, 40 CFR 798.4350: Inhalation Developmental Toxicity Study.

(5) Salewski, E., (1964) Faerbermethode zum Makroskopischen Nachweis von Implantations Stellen am Uterusder Ratte. Naunyn-Schmeidebergs Archiv fur Pharmakologie und Experimentelle Pathologie, 247, 367.

(6) Edwards, J.A., (1968) The external Development of the Rabbit and Rat Embryo. In Advances in Teratology. D.H.M. Woolam (ed.) Vol. 3. Academic Press, NY.

(7) Inouye, M. (1976) Differential Staining of Cartilage and Bone in Fetal Mouse Skeleton by Alcian Blue and Alizarin Red S. Congenital Anomalies 16, p. 171-173.

(8) Igarashi, E. et al., (1992) Frequency Of Spontaneous Axial Skeletal Variations Detected by the Double Staining Techniquefor Ossified and Cartilaginous Skeleton in Rat Foetuses. Congenital Anomalies 32, p. 381-391.

(9) Kimmel, C.A. et al. (1993) Skeletal Development Following Heat Exposure in the Rat. Teratology, 47 p.229-242.

(10) Marr, M.C. et al. (1988) Comparison of Single and Double Staining for Evaluation of Skeletal Development: The Effects of Ethylene Glycol (EG) in CD Rats. Teratology 37; 476.

(11) Barrow, M.V. and Taylor, W.J. (1969) A Rapid Method for Detecting Malformations in Rat Foetuses. Journal of Morphology, 127, p. 291-306.

(12) Fritz, H. (1974) Prenatal Ossification in Rabbits ss Indicative of Foetal Maturity. Teratology, 11 p. 313-320.

(13) Gibson, J.P. et al. (1966) Use of the Rabbit in Teratogenicity Studies. Toxicology and Applied Pharmacology, 9, p. 398-408.

(14) Kimmel, C.A. and Wilson, J.G. (1973) Skeletal Deviation in Rats: Malformations or Variations? Teratology, 8, p. 309-316.

(15) Marr, M.C. et al. (1992) Developmental Stages of the CD (Sprague-Dawley) Rat Skeleton after Maternal Exposure to Ethylene Glycol. Teratology, 46, p. 169-181.

(16) Monie, I.W. et al. (1965) Dissection Procedures for Rat Foetuses Permitting Alizarin Red Staining of Skeleton and Histological Study of Viscera. Supplement to Teratology Workshop Manual, p. 163-173.

(17) Spark, C. and Dawson, A.B. (1928) The Order and Time of appearance of Centers of Ossification in the Fore and Hind Limbs of the Albino Rat, with Special Reference to the Possible Influence of the Sex Factor. American Journal of Anatomy, 41, p. 411-445.

(18) Staples, R.E. and Schnell, V.L. (1964) Refinements in Rapid Clearing Technique in the KOH-Alizarin Red S Method for Fetal Bone. Stain Technology, 39, p. 61-63.

(19) Strong, R.M. (1928) The Order Time and Rate of Ossification of the Albino Rat (Mus Norvegicus Albinus) Skeleton. American Journal of Anatomy, 36, p. 313-355.

(20) Stuckhardt, J.L. and Poppe, S.M. (1984) Fresh Visceral Examination of Rat and Rabbit Foetuses Used in Teratogenicity Testing. Teratogenesis, Carcinogenesis, and Mutagenesis, 4, p. 181-188.

(21) Walker, D.G. and Wirtschafter, Z.T. (1957) The Genesis of the Rat Skeleton. Thomas, Springfield, IL.

(22) Wilson, J.G. (1965) Embryological Considerations in Teratology. In Teratology: Principles and Techniques, Wilson J.G. and Warkany J. (eds). University of Chicago, Chicago, IL, p. 251-277.

(23) Wilson, J.G. and Fraser, F.C. (eds). (1977) Handbook of Teratology, Vol. 4. Plenum, NY.

(24) Varnagy, L. (1980) Use of Recent Fetal Bone Staining Techniques in the Evaluation of Pesticide Teratogenicity. Acta Vet. Acad. Sci. Hung, 28, p. 233-239.

(25) Staples, R.E. (1974) Detection of visceral Alterations in Mammalian Foetuses. Teratology, 9, p. 37-38.

(26) Van Julsingha, E.B. and C.G. Bennett (1977) A Dissecting Procedure for the Detection of Anomalies in the Rabbit Foetal Head. In: Methods in Prenatal Toxicology Neubert, D., Merker, H.J. and Kwasigroch, T.E. (eds.). University of Chicago, Chicago, IL, p. 126-144.

(27) US Environmental Protection Agency (1991) Guidelines for Developmental Toxicity Risk Assessment. Federal Register, 56, p. 63798-63826.

(28) Wise, D.L. et al. (1997) Terminology of Developmental Abnormalities in Common Laboratory Mammals (Version 1) Teratology, 55, p. 249-292.

B.32. CARCINOGENICITY TEST

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITIONS

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The test substance is administered normally seven days per week, by an appropriate route, to several groups of experimental animals, one dose per group, for a major portion of their lifespan. During and after exposure to the test substance, the experimental animals are observed daily to detect signs of toxicity, particularly the development of tumours.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Experimental animals

1.6.2. Number and sex

For rodents at least 100 animals (50 female and 50 male) should be used at each dose level and concurrent control group. The females should be nulliparous and non-pregnant. If interim sacrifices are planned the number should be increased by the number of animals scheduled to be sacrificed before the completion of the study.

1.6.3. Dose levels and frequency of exposure

At least three dose levels should be used in addition to the concurrent control group. The highest dose level should elicit signs of minimal toxicity, such as a slight depression of bodyweight gain (less than 10 %), without substantially altering the normallifespan due to effects other than tumours.

The lowest dose level should not interfere with normal growth, development and longevity of the animal or produce any indication of toxicity. In general, this should not be lower than 10 % of the high dose.

The intermediate dose(s) should be established in a mid-range between the high and low doses.

The selection of dose levels should take into account data from preceding toxicity tests and studies.

Frequency of exposure is normally daily.

If the chemical is administered in the drinking water or mixed in the diet it should be continuously available.

1.6.4. Controls

A concurrent control group which is identical in every respect to the treated groups, except for exposure to the test substance, should be used.

In special circumstances, such as in inhalation studies involving aerosols or the use of an emulsifier of uncharacterised biological activity in oral studies, an additional control group which is not exposed to the vehicle should be used.

1.6.5. Route of administration

The three main routes of administration are oral, dermal and inhalation. The choice of the route of administration depends upon the physical and chemical characteristics of the test substance and the likely route of exposure in humans.

1.6.5.1. Oral studies

Where the test substance is absorbed from the gastro-intestinal tract, and if the ingestion route is one by which humans may be exposed, the oral route of administration is preferred, unless there are contra-indications. The animals may receive the test substance in their diet, dissolved in drinking water or given by capsule.

Ideally, daily dosing on a seven-day per week basis should be used because dosing on a five-day per week basis may permit recovery or withdrawal toxicity in the non-dosing period and thus affect the result and subsequent evaluation. However, based primarily on practical considerations, dosing on a five-day per week basis is considered to be acceptable.

1.6.5.2. Dermal studies

Cutaneous exposure by skin painting may be selected to simulate a main route of human exposure and as a model system for induction of skin lesions.

1.6.5.3. Inhalation studies

Because inhalation studies present technical problems of greater complexity than the other routes of administration, more detailed guidance on this mode of administration is given here. It should be noted that intratracheal instillation may constitute a valid alternative in specific situations.

Long-term exposures are usually patterned on projected human exposure, giving the animals either a daily exposure of six hours after equilibration of chamber concentrations, for five days a week (intermittent exposure), or, relevant to possible environmental exposure, 22 to 24 hours of exposure per day for seven days a week (continuous exposure), with about an hour for feeding the animals daily at a similar time and maintaining the chambers. In both cases, the animals are usually exposed to fixed concentrations of test substance. A major difference between intermittent and continuous exposure is that with the former there is a 17 to 18 hour period in which animals may recover from the effects of each daily exposure with an even longer recovery period during weekends.

The choice of intermittent or continuous exposure depends on the objectives of the study and on the human exposure that is to be simulated. However, certain technical difficulties must be considered. For example, the advantages of continuous exposure for simulating environmental conditions may be offset by the necessity for watering and feeding during exposure and by the need for more complicated (and reliable) aerosol and vapour generation and monitoring techniques.

1.6.6. Exposure chambers

Measurements or monitoring should be made of:

(i) air flow: the rate of air flow through the chamber should preferably be monitored continuously;

(ii) concentration: during the daily exposure period the concentration of the test substance should not vary more than ± 15 % of the mean value. During the total duration of this study, the day-to-day concentrations should be held as constant as practicable;

(iii) temperature and humidity: for rodents, the temperature should be maintained at 22 ± 2 oC and the humidity within the chamber at 30 to 70 %, except when water is used to suspend the test substance in the chamber atmosphere. Preferably both should be monitored continuously;

(iv) particle size measurements: particle size distribution should be determined in chamber atmospheres involving liquid or solid aerosols. The aerosol particles should be of respirable size for the test animal used. Samples of the chamber atmospheres should be taken in the breathing zone of the animals. The air sample should be representative of the distribution of the particles to which the animals are exposed and should account, on a gravimetric basis, for all of the suspended aerosol even when much of the aerosol is not respirable. Particle size analyses should be carried out frequently during the development of the generating system to ensure the stability of the aerosol and thereafter as often as necessary during the exposures to determine adequately the consistency of the particle distribution to which the animals have been exposed.

1.6.7. Duration of study

The duration of a carcinogenicity test comprises the major portion of the normal lifespan of the test animals. The termination of the test should be at 18 months for mice and hamsters and 24 months for rats; however, for certain strains of animals with greater longevity and/or low spontaneous tumour rate, termination should be at 24 months for mice and hamsters and at 30 months for rats. Alternatively, termination of such an extended study is acceptable when the number of survivors in the lowest dose or control group reaches 25 %. When terminating a test in which there is an apparent sex difference in response, each sex should be considered separately. Where only the high-dose group dies prematurely for obvious reasons of toxicity, this need not trigger termination providing toxic manifestations are not causing problems in the other groups. For a negative test result to be acceptable, not more than 10 % of any group may be lost from the experiment due to autolysis, cannibalism or management problems and the survival of all groups is not less than 50 % at 18 months for mice and hamsters and at 24 months for rats.

1.6.8. Procedure

1.6.8.1. Observations

Daily cageside observations should include changes in skin and fur, eyes and mucous membranes as well as respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern.

Regular observations of the animals is necessary to ensure that, as far as possible, animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. Moribund animals should be removed and necropsied when noticed.

Clinical signs and mortality should be recorded for all animals. Special attention must be paid to tumour development: the time of onset; location, dimensions, appearance and progression of each grossly visible or palpable tumour should be recorded.

Measurements should be made of food consumption (and water consumption when the test substance is administered in the drinking water) weekly during the first 13 weeks of the study and then at approximately three-month intervals unless health status or body weight changes dictate otherwise.

Bodyweights should be recorded individually for all animals once a week during the first 13 weeks of the test period and at least once every four weeks thereafter.

1.6.8.2. Clinical examinations

Haematology

If cage side observations suggest a deterioration in health of the animals during the study, a differential blood count of the affected animals should be performed.

At 12 months, 18 months, and prior to sacrifice, a blood smear is obtained from the animals. A differential blood count is performed on samples from the animals in the high-dose group and the controls. If these data, particularly those obtained prior to sacrifice, or data from the pathological examination indicate a need, differential blood counts should be performed on the next-lower group(s) as well.

Gross necropsy

Full gross necropsy should be performed on all animals, including those which died during the experiment or were sacrificed having been found in a moribund condition. All grossly visible tumours or lesions, or lesions suspected of being tumours, should be preserved.

The following organs and tissues should be preserved in suitable media for possible future histopathological examination: brain (including sections of medulla/pons, cerebellar cortex, cerebral cortex), pituitary, thyroid/parathyroid, any thymic tissue, trachea and lungs, heart, aorta, salivary glands, liver, spleen, kidneys, adrenals, pancreas, gonads, uterus, accessory genital organs, skin, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, representative lymph node, female mammary gland, thigh musculature, peripheral nerve, sternum with bone marrow, femur (including joint), spinal cord at three levels (cervical, mid-thoracic and lumbar) and eyes.

Inflation of lungs and urinary bladder with a fixative is the optimal way to preserve these tissues; inflation of the lungs in inhalation studies is essential for appropriate histopathological examination. In inhalation studies, the entire respiratory tract should be preserved, including nasal cavity, pharynx and larynx.

Histopathology

(a) Full histopatology should be carried out on the organs and tissues of all animals that died or were sacrificed during the test and all animals in the control and high-dose groups;

(b) all grossly visible tumours or lesions suspected of being tumours should be examined microscopically in all groups,

(c) if there is a significant difference in the incidence of neoplastic lesions in the high-dose and control groups, histopathology should be carried out on that particular organ or tissue in the other groups,

(d) if the survival of the high-dose group is substantially less than the control then the next-lower dose group should be examined fully,

(e) if there is evidence in the high-dose group of the induction of toxic or other effects that might affect a neoplastic response, the next-lower dose level should be examined fully.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing tumours detected during the test, the time of detection and the number of animals found to have tumours following sacrifice. Results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- species, strain, source, environmental conditions, diet,

- test conditions:

3.1.1. Description of exposure apparatus:

including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing animals in a test chamber when this is used. The equipment for measuring temperature, humidity and, where appropriate, stability of aerosol) concentration or particle size, should be described.

3.1.2. Exposure data:

these should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation) and should include:

(a) air flow rates through the inhalation equipment;

(b) temperature and humidity of air;

(c) nominal concentrations (total amount of test substance fed into the inhalation equipment divided by the volume of air);

(d) nature of vehicle, if used;

(e) actual concentrations in test breathing zone;

(f) median particle sizes (where appropriate),

- dose levels (including vehicle, if used) and concentrations,

- tumour incidence data by sex, dose and tumour type,

- time of death during the study or whether animals survived to termination,

- toxic response data by sex and dose,

- description of toxic or other effects,

- the time of observation of each abnormal sign and its subsequent course,

- food and bodyweight data,

- haematological tests employed and all results,

- necropsy findings,

- a detailed description of all histopathological findings,

- statistical treatment of results with a description of the methods used,

- discussion of the results,

- interpretation of the result.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.33. COMBINED CHRONIC TOXICITY/CARCINOGENICITY TEST

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITIONS

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The objective of a combined chronic toxicity carcinogenicity test is to determine the chronic and carcinogenic effects of a substance in a mammalian species following prolonged exposure.

To this end a carcinogenicity test is supplemented with a least one treated satellite group and a control satellite group. The dose used for the high-dose satellite group may be higher than that used for the high-dose group in the carcinogenicity test. The animals in the carcinogenicity test are examined for general toxicity as well as for carcinogenic response. The animals in the treated satellite group are examined for general toxicity.

The test substance is administered normally seven days per week, by an appropriate route, to several groups of experimental animals, one dose per group, for a major portion of their lifespan. During and after exposure to the test substance, the experimental animals are observed daily to detect signs of toxicity and the development of tumours.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Experimental animals

The preferred species is the rat. Based upon the results of previously conducted tests other species (rodent or non-rodent) may be used. Commonly used laboratory strains of young healthy animals should be employed and dosing should begin as soon as possible after weaning.

At the commencement of the test the weight variation in the animals used should not exceed ± 20 % of the mean value. Where a sub-chronic oral test is conducted as a preliminary to a long-term test, the same species and breed/strain should be used in both studies.

1.6.2. Number and sex

For rodents, at least 100 animals (50 female and 50 male) should be used at each dose level and concurrent control group. The females should be nulliparous and non-pregnant. If interim sacrifices are planned, the number should be increased by the number of animals scheduled to be sacrificed before the completion of the study.

The treated satellite group(s) for the evaluation of pathology other than tumours should contain 20 animals of each sex, while the satellite control group should contain 10 animals of each sex.

1.6.3. Dose levels and frequency of exposure

For carcinogenicity testing purposes, at least three dose levels should be used in addition to the concurrent control group. The highest dose level should elicit signs of minimal toxicity, such as a slight depression of body weight gain (less than 10 %), without substantially altering the normal lifespan due to effects other than tumours.

The intermediate dose(s) should be established in a mid-range between the high and low doses.

The selection of dose levels should take into account data from preceding toxicity tests and studies.

For chronic toxicity testing purposes, additional treated groups and a concurrent control satellite group are included in the test. The high dose for treated satellite animals should produce definite signs of toxicity.

Frequency of exposure is normally daily.

If the chemical is administered in the drinking water or mixed in the diet, it should be continuously available.

1.6.4. Controls

A concurrent group which is identical in every respect to the treated groups, except for exposure to the test substance should be used.

In special circumstances, such as in inhalation studies involving aerosols or the use of an emulsifier of uncharacterized biological activity in oral studies, an additional control group which is not exposed to the vehicle should be utilised.

1.6.5. Route of administration

1.6.5.1. Oral tests

Where the test substance is absorbed from the gastro-intestinal tract and the ingestion route is one by which humans may be exposed, the oral route of administration is preferred, unless there are contra-indications. The animals may receive the test substance in their diet, dissolved in drinking water or given by capsule.

1.6.5.2. Dermal tests

Cutaneous exposure by skin painting may be selected to simulate a main route of human exposure and as a model system for induction of skin lesions.

1.6.5.3. Inhalation tests

Because inhalation tests present technical problems of greater complexity than the other routes of administration, more detailed guidance on this mode of administration is given here. It should be noted that intratracheal instillation may constitute a valid alternative in specific situations.

Long-term exposures are usually patterned on projected human exposure, giving the animals either a daily exposure of six hours after equilibration of chamber concentrations, for five days a week (intermittent exposure), or, relevant to possible environmental exposure, 22 to 24 hours of exposure per day for seven days a week (continuous exposure), with about an hour for feeding the animals daily at a similar time and maintaining the chambers. In both cases, the animals are usually exposed to fixed concentrations of test substance. A major difference between intermittent and continuous exposure is that, with the former, there is a 17 to 18 hour period in which animals may recover from the effects of each daily exposure, with an even longer recovery period during weekends.

The choice of intermittent or continuous exposure depends on the objectives of the test and on the human exposure that is to be simulated. However, certain technical difficulties must be considered. For example, the advantages of continuous exposure for simulating environmental conditions may be offset by the necessity for watering and feeding during exposure and by the need for more complicated (and reliable) aerosol and vapour generation and monitoring techniques.

1.6.6. Exposure chambers

Measurements or monitoring should be made of:

(i) air flow: the rate of air flow through the chamber should preferably be monitored continuously;

(ii) concentration: during the daily exposure period the concentration should not vary more than ± 15 % of the mean value. During the total duration of this study, the day-to-day concentrations should be held as constant as practicable;

1.6.7. Duration of test

The duration of the carcinogenicity part of the test comprises the major portion of the normal life span of the test animals. The termination of the test should be at 18 months for mice and hamsters and 24 months for rats; however, for certain strains of animals with greater longevity and/or low spontaneous tumour rate, termination should be at 24 months for mice and hamsters and at 30 months for rats. Alternatively, termination of such an extended test is acceptable when the number of survivors in the lowest dose or control group reaches 25 %. When terminating a test in which there is an apparent sex difference in response, each sex should be considered separately. Where only the high-dose group dies prematurely for obvious reasons of toxicity, this need not trigger termination providing toxic manifestations are not causing problems in the other groups. For a negative test result to be acceptable not more than 10 % of any group may be lost from the experiment due to autolysis, cannibalism or management problems, and the survival of all groups is not less than 50 at 18 months for mice and hamsters and at 24 months for rats.

The satellite groups of 20 dosed animals per sex and 10 associated control animals per sex used for chronic toxicity testing should be retained in the test for at least 12 months. These animals should be scheduled for sacrifice for an examination of test-substance-related pathology uncomplicated by gerontological changes.

1.6.8. Procedure

1.6.8.1. Observations

Daily cageside observations should be made and should include changes in skin and fur, eyes and mucous membranes as well as respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern.

Clinical examination should be performed at appropriate intervals on animals in the treated satellite group(s).

Regular observations of the animals is necessary to ensure, as far as possible, that animals are not lost from the test due to causes such as cannibalism, autolysis of tissues or misplacment. Moribund animals should be removed and necropsied when noticed.

Clinical signs, including neurological and ocular changes as well as mortality should be recorded for all animals. Special attention must be paid to tumour development: the time of onset, location, dimensions, appearance and progression of each grossly visible or palpable tumour should be recorded; the time of onset and progression of toxic conditions should be recorded.

Bodyweights should be recorded individually for all animals once a week during the first 13 weeks of the test period and at least once every four weeks thereafter.

1.6.8.2. Clinical examinations

Haematology

Haematological examination (e.g. haemoglobin content, packed cell volume, total red blood cells, total white blood cells, platelets, or other measures of clotting potential) should be performed at three months, six months and at approximately six-month intervals thereafter, and at termination on blood samples collected from 10 rats/sex of all groups. If possible, samples should be from the same rats at each interval.

If cageside observations suggest a deterioration in the health of the animals during the study, a differential blood count of the affected animals should be performed. A differential blood count is performed on samples of those animals in the highest dose group and the controls. Differential blood counts are performed for the next lower group(s) only if there is a major discrepancy between the highest group and the controls, or if indicated by pathological findings.

Urinalysis

Urine samples from 10 rats/sex of all groups, if possible from the same rats at the same intervals as haematological examination, should be collected for analysis. The following determinations should be made from either individual animals or on a pooled sample/sex/group of rodents:

- appearance: volume and density for individual animals,

- protein, glucose, ketones, occult blood (semi-quantitatively),

- microscopy of sediment (semi-quantitatively).

Clinical chemistry

At approximately six-monthly intervals, and at termination, blood samples are drawn for clinical chemistry measurements from all non-rodents and 10 rats/sex of all groups, if possible, from the same rats at each interval. In addition, a pre-test sample should be collected from non-rodents. Plasma is prepared from these samples and the following determinations are made:

- total protein concentration,

- albumin concentration,

- carbohydrate metabolism such as fasting blood glucose,

- kidney function tests such as blood urea nitrogen.

Gross necropsy

Full gross necropsy should be performed in all animals, including those which died during the experiment or were sacrificed having been found in a moribund condition. Prior to sacrifice, samples of blood should be collected from all animals for differential blood counts. All grossly visible tumours or lesions suspected of being tumours should be preserved. An attempt should be made to correlate gross observations with the microsopic findings.

All organs and tissues should be preserved for histopathological examination. This usually concerns the following organs and tissues: brain [7] (medulla/pons, cerebellar cortex, cerebral cortex); pituitary, thyroid (including parathyroid), thymus, lungs (including trachea), heart, aorta, salivary glands, liver [7], spleen, kidneys [7], adrenals [7], oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, lymph nodes, pancreas, gonads [7], accessory genital organs; female mammary gland, skin, musculature, peripheral nerve, spinal cord (cervical, thoracic, lumbar), sternum with bone marrow and femur (including joint) and eyes.

Although inflation of lungs and urinary bladder with a fixative is the optimal way to preserve these tissues, inflation of the lungs in inhalation studies is a necessary requirement for appropriate histopathological examination. In special studies such as inhalation studies, the entire respiratory tract should be studied, including nose, pharynx and larynx.

If other clinical examinations are carried out, the information obtained from these procedures should be available before microsopic examination, because it may give significant guidance to the pathologist.

Histopathology

For the chronic toxicity testing portion:

Detailed examination should be made of all preserved organs of all animals of the satellite high-dose and control groups. Where test-substance-related pathology is found in the high-dose satellite group, target organs of all other animals in any other treated satellite group should be subjected to full and detailed histological examination as well as those of the heated groups in the carcinogenicity testing portion of the study at its termination.

For the carcinogenicity testing portion:

(a) full histopathology should be carried out on the organs and tissues of all animals that died or were sacrificed during the test, and of all animals in the control and high-dose groups;

(b) all grossly visible tumours or lesions suspected of being tumours in all groups occurring in any organ should be examined microscopically;

(d) if the survival of the high-dose group is substantially less than the control then the next-lower dose group should be examined fully;

(e) If there is evidence in the high-dose group of the induction of toxic or other effects that might affect a neoplastic response, the next-lower dose level should be examined fully.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing tumours or toxic effects detected during the test, the time of detection and the number of animals found to have tumours following sacrifice. Results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

3.1. TEST REPORT

The test report shall, if possible, contain the following information:

- species, strain source, environmental conditions, diet,

- test conditions:

3.1.1. Description of exposure apparatus:

including design, type, dimensions, source of air, system for generating particulates and aerosols, method of conditioning air, treatment of exhaust air and the method of housing animals in a test chamber when this is used. The equipment for measuring temperature, humidity and, where appropriate, stability of aerosol concentration or particle size, should be described.

3.1.2. Exposure data:

these should be tabulated and presented with mean values and a measure of variability (e.g. standard deviation), and should include:

(a) air flow rates through the inhalation equipment;

(b) temperature and humidity of air;

(c) nominal concentrations (total amount of test substance fed into the inhalation equipment divided by the volume of air);

(d) nature of vehicle, if used;

(e) actual concentrations in test breathing zone;

(f) median particle sizes (where appropriate):

- dose levels (including vehicle, if used) and concentrations,

- tumour incidence data by sex, dose and tumour type,

- time of death during the study or whether animals survived to termination, including satellite group,

- toxic response data by sex and dose,

- description of toxic or other effects,

- the time of observation of each abnormal sign and its subsequent course,

- ophthalmological findings,

- food and bodyweight data,

- haematological tests employed and all results,

- clinical biochemistry test employed and all results (including any urinalysis),

- necropsy findings,

- a detailed description of all histopathological findings,

- statistical treatment of results with a description of the methods used,

- discussion of the results,

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.34. ONE-GENERATION REPRODUCTION TOXICITY TEST

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITIONS

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The test substance is administered in graduated doses to several groups of males and females. Males should be dosed during growth and for at least one complete spermatogenic cycle (approximately 56 days in the mouse and 70 days in the rat) in order to elicit any adverse effects on spermatogenesis by the test substance.

Females of the parental (P) generation should be dosed for at least two complete oestrous cycles in order to e adverse effects on oestrus by the test substance. The animals are then mated. The test substance is administered to both sexes during the mating period and thereafter only to females during pregnancy and for the duration of the nursing period. For administration by inhalation the method will require modification.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

Before the test, healthy young adult animals are randomised and assigned to the treated and control groups. The animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. It is recommended that the test substance be administered in the diet or drinking water. Other routes of administration are also acceptable. All animals should be dosed by the same method during the appropriate experimental period. If a vehicle or other additives are used to facilitate dosing, they should be known not to produce toxic effects. Dosing should be on a seven-day per week basis.

1.6.2. Experimental animals

Selection of species

The rat or mouse are the preferred species. Healthy animals, not subjected to previous experimental procedures, should be used. Strains with low fecundity should not be used. The test animals should be characterized as to species, strain, sex, weight and/or age.

For an adequate assessment of fertility, both males and females should be studied. All test and control animals should be weaned before dosing begins.

Number and sex

Each treated and control group should contain a sufficient number of animals to yield about 20 pregnant females at or near term.

The objective is to produce enough pregnancies and offspring to assure a meaningful evaluation of the potential of the substance to affect fertility, pregnancy and maternal behaviour in P generation animals and suckling, growth and development of the F1 offspring from conception to weaning.

1.6.3. Test conditions

Food and water should be provided ad libitum. Near parturition, pregnant females should be caged separately in delivery or maternity cages and may be provided with nesting materials.

1.6.3.1. Dose levels

At least three treated groups and a control group should be used. If a vehicle is used in administering the test substance, the control group should receive the vehicle in the highest volume used. If a test substance causes reduced dietary intake or utilisation, then the use of a paired fed control group may be considered necessary. Ideally, unless limited by the physical/chemical nature or biological effects of the test substance, the highest dose level should induce toxicity but not mortality in the parental (P) animals. The intermediate dose(s) should induce minimal toxic effects attributable to the test substance, and the low dose should not induce any observable adverse effects on the parents or offspring. When administered by gavage or capsule the dosage given to each animal should be based on the individual animal's body weight and adjusted weekly for changes in body weight. For females during pregnancy, dosages may be based on the body weight at day 0 or 6 of the pregnancy, if desired.

1.6.3.2. Limit test

In the case of substances of low toxicity, if a dose level of at least 1000 mg/kilogram produces no evidence of interference with reproductive performance, studies at other dose levels may not be considered necessary. If a preliminary study at the high-dose level, with definite evidence of maternal toxicity, shows no adverse effects on fertility, studies at other dose levels may not be considered necessary.

1.6.3.3. Performance of the test

Experimental schedules

Daily dosing of the parental (P) males should begin when they are about five to nine weeks of age, after they have been weaned and acclimatised for at least five days. In rats, dosing is continued for 10 weeks prior to the mating period (for mice, eight weeks). Males should be killed and examined either at the end of the mating period or, alternatively, males may be retained on the test diet for the possible production of a second litter and should be killed and examined at some time before the end of the study. For parental (P) females dosing should begin after at least five days of acclimatisation and continue for at least two weeks prior to mating. Daily dosing of the p females should continue throughout the three-week mating period, pregnancy and up to the weaning of the Fl offspring. Consideration should be given to modification of the dosing schedule based on other available information on the test substance, such as induction of metabolism or bioaccumulation.

Mating procedure

Either 1:1 (one male to one female) or 1:2 (one male to two females) mating may be used in reproduction toxicity studies.

Based on 1:1 mating, one female should be placed with the same male until pregnancy occurs or three weeks have elapsed. Each morning the females should be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug or sperm is found.

Those pairs that fail to mate should be evaluated to determine the cause of the apparent infertility.

This may involve such procedures as providing additional opportunities to mate with other proven sires or dams, microscopic examination of the reproductive organs, and examination of the oestrous cycle or spermatogenesis.

Litter sizes

Animals dosed during the fertility study are allowed to litter normally and rear their progency to the stage of weaning without standardisation of litters.

Where standardisation is done, the following procedure is suggested. Between day 1 and day 4 after birth, the size of each litter may be adjusted by eliminating extra pups by selection to yield, as nearly as possible, four males and four females per litter.

Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is acceptable. Adjustments are not applicable for litters of less than eight pups.

1.6.4. Observations

Throughout the test period, each animal should be observed at least once daily. Pertinent behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity, including mortality, should be recorded. During pre-mating and mating periods, food consumption may be measured daily. After parturition and during lactation, food consumption measurements (and water consumption measurements when the test substance is administered in the drinking water) should be made on the same day as the weighing of the litter. P males and females should be weighed on the first day of dosing and weekly thereafter. These observations should be reported individually for each adult animal.

The duration of gestation should be calculated from day 0 of pregnancy. Each litter should be examined as soon as possible after delivery to establish the number and sex of pups, still births, live births and the presence of gross anomalies.

Dead pups and pups sacrificed at day 4 should be preserved and studied for possible defects. Live pups should be counted and litters weighed on the morning after birth and on days 4 and 7 and weekly thereafter until the termination of the study, when animals should be weighed individually.

Physical or behavioural abnormalities observed in the dams or offspring should be recorded.

1.6.5. Pathology

1.6.5.1. Necropsy

At the time of sacrifice or death during the study the animals of the P generation should be examined macroscopically for any structural abnormalities or pathological changes, with special attention being paid to the organs of the reproductive system. Dead or moribund pups should be examined for defects.

1.6.5.2. Histopathology

The ovaries, uterus, cervix, vagina, testes, epididymes, seminal vesicles, prostate, coagulating gland, pituitary gland and target organ(s) of all P animals should be preserved for microscopic examination. In the event that these organs have not been examined in other multiple-dose studies, they should be microscopically examined in all high-dose and control animals and animals which die during the study where practicable.

Organs showing abnormalities in these animals should then be examined in all other P animals. In these instances, microscopic examination should be made of all tissues showing gross pathological changes. As suggested under mating procedures, reproductive organs of animals suspected of infertility may be subjected to microscopic examination.

2. DATA

Data may be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of fertile males, the number of pregnant females, the types of changes and the percentage of animals displaying each type of change.

When possible, numerical results should be evaluated by an appropriate statistical method. Any generally accepted statistical method may be used.

3. REPORTING

TEST REPORT

The test report shall, if possible, contain the following information:

- species/strain used,

- toxic response data by sex and dose, including fertility, gestation and viability,

- time of death during the study or whether animals survived to time of scheduled sacrifice or to termination of the study,

- table presenting the weights of each litter, the mean: pup weights and the individual weights of the pups at termination,

- toxic or other effects on reproduction, offspring and postnatal growth,

- the day of observation of each abnormal sign and its subsequent course,

- bodyweight data for P animals,

- necropsy findings,

- a detailed description of all microscopic findings,

- statistical treatment of results, where appropriate,

- discussion of the results,

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.35. TWO-GENERATION REPRODUCTION TOXICITY STUDY

1. METHOD

This method is a replicate of the OECD TG 416 (2001).

1.1. INTRODUCTION

This method for two-generation reproduction testing is designed to provide general information concerning the effects of a test substance on the integrity and performance of the male and female reproductive systems, including gonadal function, the oestrus cycle, mating behaviour, conception, gestation, parturition, lactation, and weaning, and the growth and development of the offspring. The study may also provide information about the effects of the test substance on neonatal morbidity, mortality, and preliminary data on prenatal and postnatal developmental toxicity and serve as a guide for subsequent tests. In addition to studying growth and development of the F1 generation, this test method is also intended to assess the integrity and performance of the male and female reproductive systems as well as growth and development of the F2 generation. For further information on developmental toxicity and functional deficiencies, either additional study segments can be incorporated into this protocol, consulting the methods for developmental toxicity and/or developmental neurotoxicity as appropriate, or these endpoints could be studied in separate studies, using the appropriate test methods.

1.2. PRINCIPLE OF THE TEST METHOD

The test substance is administered in graduated doses to several groups of males and females. Males of the P generation should be dosed during growth and for at least one complete spermatogenetic cycle (approximately 56 days in the mouse and 70 days in the rat) in order to elicit any adverse effects on spermatogenesis. Effects on sperm are determined by a number of sperm parameters (e.g. sperm morphology and motility) and in tissue preparation and detailed histopathology. If data on spermatogenesis are available from a previous repeated dose study of sufficient duration, e.g. a 90-day study, males of the P generation need not be included in the evaluation. It is recommended, however, that samples or digital recordings of sperm of the P generation are saved, to enable later evaluation. Females of the P generation should be dosed during growth and for several complete oestrus cycles in order to detect any adverse effects on oestrus cycle normality by the test substance. The test substance is administered to parental (P) animals during their mating, during the resulting pregnancies, and through the weaning of their F1 offspring. At weaning the administration of the substance is continued to F1 offspring during their growth into adulthood, mating and production of an F2 generation, until the F2 generation is weaned.

Clinical observations and pathological examinations are performed on all animals for signs of toxicity with special emphasis on effects on the integrity and performance of the male and female reproductive systems and on the growth and development of the offspring.

1.3. DESCRIPTION OF THE TEST METHOD

1.3.1. Selection of animal species

The rat is the preferred species for testing. If other species are used, justification should be given and appropriate modifications will be necessary. Strains with low fecundity or well-known high incidence of developmental defects should not be used. At the commencement of the study, the weight variation of animals used should be minimal and not exceed 20 % of the mean weight of each sex.

1.3.2. Housing and feeding conditions

The temperature in the experimental animal room should be 22 oC (± 3o). Although the relative humidity should be at least 30 % and preferably not exceed 70 % other than during room cleaning, the aim should be 50-60 %. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method.

Animals may be housed individually or be caged in small groups of the same sex. Mating procedures should be carried out in cages suitable for the purpose. After evidence of copulation, mated females shall be single-caged in delivery or maternity cages. Mated rats may also be kept in small groups and separated one or two days prior to parturition. Mated animals shall be provided with appropriate and defined nesting materials when parturition is near.

1.3.3. Preparation of animals

Healthy young animals, which have been acclimated to laboratory conditions for at least five days and have not been subjected to previous experimental procedures, should be used. The test animals should be characterised as to species, strain, source, sex, weight and/or age. Any sibling relationships among the animals should be known so that mating of siblings is avoided. The animals should be randomly assigned to the control and treated groups (stratification by body weight is recommended). Cages should be arranged in such a way that possible effects due to cage placement are minimised. Each animal should be assigned a unique identification number. For the P generation, this should be done before dosing starts. For the F1 generation, this should be done at weaning for animals selected for mating. Records indicating the litter of origin should be maintained for all selected F1 animals. In addition, individual identification of pups as soon after birth as possible is recommended when individual weighing of pups or any functional tests are considered.

Parental (P) animals shall be about five to nine weeks old at the start of dosing. The animals of all test groups shall, as nearly as practicable, be of uniform weight and age.

1.4. PROCEDURE

1.4.1. Number and sex of animals

Each test and control group should contain a sufficient number of animals to yield preferably not less than 20 pregnant females at or near parturition. For substances that cause undesirable treatment related effects (e.g. sterility, excessive toxicity at the high dose) this may not be possible. The objective is to produce enough pregnancies to assure a meaningful evaluation of the potential of the substance to affect fertility, pregnancy and maternal behaviour and suckling, growth and development of the F1 offspring from conception to maturity, and the development of their offspring (F2) to weaning. Therefore, failure to achieve the desired number of pregnant animals (i.e. 20) does not necessarily invalidate the study and should be evaluated on a case-by-case basis.

1.4.2. Preparation of doses

It is recommended that the test substance be administered orally (by diet, drinking water or gavage) unless another route of administration (e.g. dermal or inhalation) is considered more appropriate.

Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water, the toxic characteristics of the vehicle must be known. The stability of the test substance in the vehicle should be determined.

1.4.3. Dosage

At least three dose levels and a concurrent control shall be used. Unless limited by the physical-chemical nature or biological effects of the test substance, the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. In case of unexpected mortality, studies with a mortality rate of less than approximately 10 % in the parental (P) animals would normally still be acceptable. A descending sequence of dose levels should be selected with a view to demonstrating any dosage related effect and no-observed-adverse-effects levels (NOAEL). Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of 10) between dosages. For the dietary studies the dose interval should be not more than three fold. Dose levels should be selected taking into account any existing toxicity data, especially results from repeated dose studies. Any available information on metabolism and kinetics of the test compound or related materials should also be considered. In addition, this information will also assist in demonstrating the adequacy of the dosing regimen.

The control group shall be an untreated group or a vehicle-control group if a vehicle is used in administering the test substance. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used, the control group shall receive the vehicle in the highest volume used. If a test substance is administered in the diet, and causes reduced dietary intake or utilisation, then the use of a pair-fed control group may be considered necessary. Alternatively data from controlled studies designed to evaluate the effects of decreased food consumption on reproductive parameters may be used in lieu of a concurrent pair-fed control group.

Consideration should be given to the following characteristics of vehicle and other additives: effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.

1.4.4. Limit test

If an oral study at one dose level of at least 1000 mg/kg body weight/day or, for dietary or drinking water administration, an equivalent percentage in the diet or drinking water using the procedures described for this study, produces no observable toxic effects in either parental animals or their offspring and if toxicity would not be expected based upon data from structurally and/or metabolically related compounds, then a full study using several dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher oral dose level to be used. For other types of administration, such as inhalation or dermal application, the physical-chemical properties of the test substance, such as solubility, often may indicate and limit the maximum attainable level of exposure.

1.4.5. Administration of doses

The animals should be dosed with the test substance on a 7-days per week basis. The oral route of administration (diet, drinking water, or gavage) is preferred. If another route of administration is used, justification shall be provided, and appropriate modifications may be necessary. All animals shall be dosed by the same method during the appropriate experimental period. When the test substance is administered by gavage, this should be done using a stomach tube. The volume of liquid administered at one time should not exceed 1 ml/100 g body weight (0,4 ml/100 g body weight is the maximum for corn oil), except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Except for irritant or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels. In gavage studies, the pups will normally only receive test substance indirectly through the milk, until direct dosing commences for them at weaning. In diet or drinking water studies, the pups will additionally receive test substance directly when they commence eating for themselves during the last week of the lactation period.

For substances administered via the diet or drinking water, it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the body weight of the animal may be used; the alternative used must be specified. For a substance administered by gavage, the dose should be given at similar times each day, and adjusted at least weekly to maintain a constant dose level in terms of animal body weight. Information regarding placental distribution should be considered when adjusting the gavage dose based on weight.

1.4.6. Experimental schedules

Daily dosing of the parental (P) males and females shall begin when they are five to nine weeks old. Daily dosing of the F1 males and females shall begin at weaning; it should be kept in mind that in cases of test substance administration via diet or drinking water, direct exposure of the F1 pups to the test substance may already occur during the lactation period. For both sexes (P and F1), dosing shall be continued for at least 10 weeks before the mating period. Dosing is continued in both sexes during the two week mating period. Males should be humanely killed and examined when they are no longer needed for assessment of reproductive effects. For parental (P) females, dosing should continue throughout pregnancy and up to the weaning of the F1 offspring. Consideration should be given to modifications in the dosing schedule based on available information on the test substance, including existing toxicity data, induction of metabolism or bioaccumulation. The dose to each animal should normally be based on the most recent individual body weight determination. However, caution should be exercised when adjusting the dose during the last trimester of pregnancy.

Treatment of the P and F1 males and females shall continue until termination. All P and F1 adult males and females should be humanely killed when they are no longer needed for assessment of reproductive effects. F1 offspring not selected for mating and all F2 offspring should be humanely killed after weaning.

1.4.7. Mating procedure

1.4.7.1. Parental (P) mating

For each mating, each female shall be placed with a single male from the same dose level (1:1 mating) until copulation occurs or twq weeks have elapsed. Each day, the females shall be examined for presence of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug or sperm are found. In case pairing is unsuccessful, re-mating of females with proven males of the same group could be considered. Mating pairs should be clearly identified in the data. Mating of siblings should be avoided.

1.4.7.2. F1 mating

For mating the F1 offspring, at least one male and one female should be selected at weaning from each litter for mating with other pups of the same dose level but different litter, to produce the F2 generation. Selection of pups from each litter should be random when no significant differences are observed in body weight or appearance between the litter mates. In case these differences are observed, the best representatives of each litter should be selected. Pragmatically, this is best done on a body weight basis but it may be more appropriate on the basis of appearance. The F1 offspring should not be mated until they have attained full sexual maturity.

Pairs without progeny should be evaluated to determine the apparent cause of the infertility. This may involve such procedures as additional opportunities to mate with other proven sires or dams, microscopic examination of the reproductive organs, and examination of the oestrous cycles or spermatogenesis.

1.4.7.3. Second mating

In certain instances, such as treatment-related alterations in litter size or the observation of an equivocal effect in the first mating, it is recommended that the P or F1 adults be remated to produce a second litter. It is recommended to remate females or males, which have not produced a litter with proven breeders of the opposite sex. If production of a second litter is deemed necessary in either generation, animals should be remated approximately one week after weaning of the last litter.

1.4.7.4. Litter size

Animals shall be allowed to litter normally and rear their offspring to weaning. Standardisation of litter sizes is optional. When standardisation is done, the method used should be described in detail.

1.5. OBSERVATIONS

1.5.1. Clinical observations

A general clinical observation should be made each day and, and in the case of gavage dosing its timing should take into account the anticipated peak period of effects after dosing. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity should be recorded. An additional, more detailed examination of each animal should be conducted on at least a weekly basis and could conveniently be performed on an occasion when the animal is weighed. Twice daily, during the weekend once daily when appropriate, all animals should be observed for morbidity and mortality.

1.5.2. Body weight and food/water consumption of parent animals

Parental animals (P and Fl) shall be weighed on the first day of dosing and at least weekly thereafter. Parental females (P and F1) shall be weighed at a minimum on gestation days 0, 7, 14, and 20 or 21, and during lactation on the same days as the weighing of litters and on the day the animals are killed. These observations should be reported individually for each adult animal. During the premating and gestation periods food consumption shall be measured weekly at a minimum. Water consumption shall be measured weekly at a minimum if the test substance is administered in the water.

1.5.3. Oestrus cycle

Estrous cycle length and normality are evaluated in P and F1 females by vaginal smears prior to mating, and optionally during mating, until evidence of mating is found. When obtaining vaginal/cervical cells, care should be taken to avoid disturbance of mucosa and subsequently, the induction of pseudopregnancy (1).

1.5.4. Sperm parameters

For all P and F1 males at termination, testis and epididymis weight shall be recorded and one of each organ reserved for histopathological examination (see Section 1.5.7, 1.5.8.1). Of a subset of at least 10 males of each group of P and F1 males, the remaining testes and epididymides should be used for enumeration of homogenisation-resistant spermatids and cauda epididymal sperm reserves, respectively. For this same subset of males, sperm from the cauda epididymides or vas deferens should be collected for evaluation of sperm motility and sperm morphology. If treatment-related effects are observed or when there is evidence from other studies of possible effects on spermatogenesis, sperm evaluation should be conducted in all males in each dose group; otherwise enumeration may be restricted to control and high-dose P and F1 males.

The total number of homogenisation-resistant testicular spermatids and cauda epididymal sperm should be enumerated (2)(3). Cauda sperm reserves can be derived from the concentration and volume of sperm in the suspension used to complete the qualitative evaluations, and the number of sperm recovered by subsequent mincing and/or homogenising of the remaining cauda tissue. Enumeration should be performed on the selected subset of males of all dose groups immediately after killing the animals unless video or digital recordings are made, or unless the specimens are freezed and analysed later. In these instances, the controls and high dose group may be analysed first. If no treatment-related effects (e.g. effects on sperm count, motility, or morphology) are seen the other dose groups need not be analysed. When treatment-related effects are noted in the high-dose group, then the lower dose groups should also be evaluated.

Epididymal (or ductus deferens) sperm motility should be evaluated or video taped immediately after sacrifice. Sperm should be recovered while minimising damage, and diluted for motility analysis using acceptable methods (4). The percentage of progressively motile sperm should be determined either subjectively of objectively. When computer-assisted motion analysis is performed (5)(6)(7)(8)(9)(10) the derivation of progressive motility relies on user-defined thresholds for average path velocity and straightness or linear index. If samples are videotaped (11) or the images are otherwise recorded at the time of necropsy, subsequent analysis of only control and high-dose P and F1 males may be performed unless treatment-related effects are observed; in that case, the lower dose groups should also be evaluated. In the absence of a video or digital image, all samples in all treatment groups should be analysed at necropsy.

A morphological evaluation of an epididymal (or vas deferens) sperm sample should be performed. Sperm (at least 200 per sample) should be examined as fixed, wet preparations (12) and classified as either normal or abnormal. Examples of morphologic sperm abnormalities would include fusion, isolated heads, and misshapen heads and/or tails. Evaluation should be performed on the selected subset of males of all dose groups either immediately after killing the animals, or, based on the video or digital recordings, at a later time. Smears, once fixed, can also be read at a later time. In these instances, the controls and high dose group may be analysed first. If no treatment-related effects (e.g. effects on sperm morphology) are seen the other dose groups need not be analysed. When treatment-related effects are noted in the high-dose group, then the lower dose groups should also be evaluated.

If any of the above sperm evaluation parameters have already been examined as part of a systemic toxicity study of at least 90 days, they need not necessarily be repeated in the two-generation study. It is recommended, however, that samples or digital recordings of sperm of the P generation are saved, to enable later evaluation, if necessary.

1.5.5. Offspring

Each litter should be examined as soon as possible after delivery (lactation day 0) to establish the number and sex of pups, stillbirths, live births, and the presence of gross anomalies. Pups found dead on day 0, if not macerated, should preferably be examined for possible defects and cause of death and preserved. Live pups should be counted and weighed individually at birth (lactation day 0) or on day 1, and on regular weigh days thereafter, e.g. on days 4, 7, 14, and 21 of lactation. Physical or behavioural abnormalities observed in the dams or offspring should be recorded.

Physical development of the offspring should be recorded mainly by body weight gain. Other physical parameters (e.g. ear and eye opening, tooth eruption, hair growth) may give supplementary information, but these data should preferably be evaluated in the context of data on sexual maturation (e.g. age and body weight at vaginal opening or balano-preputial separation) (13). Functional investigations (e.g. motor activity, sensory function, reflex ontogeny) of the F1 offspring before and/or after weaning, particularly those related to sexual maturation, are recommended if such investigations are not included in separate studies. The age of vaginal opening and preputial separation should be determined for F1 weanlings selected for mating. Anogenital distance should be measured at postnatal day 0 in F2 pups if triggered by alterations in F1 sex ratio or timing of sexual maturation.

Functional observations may be omitted in groups that otherwise reveal clear signs of adverse effects (e.g. significant decrease in weight gain, etc.). If functional investigations are made, they should not be done on pups selected for mating.

1.5.6. Gross necropsy

At the time of termination or death during the study, all parental animals (P and F1), all pups with external abnormalities or clinical signs, as well as one randomly selected pup/sex/litter from both the F1 and F2 generation, shall be examined macroscopically for any structural abnormalities or pathological changes. Special attention should be paid to the organs of the reproductive system. Pups that are humanely killed in a moribund condition and dead pups, when not macerated, should be examined for possible defects and/or cause of death and preserved.

The uteri of all primiparous females should be examined, in a manner which does not compromise histopathological evaluation, for the presence and number of implantation sites.

1.5.7. Organ weights

At the time of termination, body weight and the weight of the following organs of all P and F1 parental animals shall be determined (paired organs should be weighed individually):

- uterus, ovaries,

- testes, epididymides (total and cauda),

- prostate,

- seminal vesicles with coagulating glands and their fluids and prostate (as one unit),

- brain, liver, kidneys, spleen, pituitary, thyroid and adrenal glands and known target organs.

Terminal body weights should be determined for F1 and F2 pups that are selected for necropsy. The following organs from the one randomly selected pup/sex/litter (see Section 1.5.6) shall be weighed: Brain, spleen and thymus.

Gross necropsy and organ weight results should be assessed in context with observations made in other repeated dose studies, when feasible.

1.5.8. Histopathology

1.5.8.1. Parental animals

The following organs and tissues of parental (P and F1) animals, or representative samples thereof, shall be fixed and stored in a suitable medium for histopathological examination.

- Vagina, uterus with cervix, and ovaries (preserved in appropriate fixative),

- one testis (preserved in Bouin's or comparable fixative), one epididymis, seminal vesicles, prostate, and coagulating gland,

- previously identified target organ(s) from all P and F1 animals selected for mating.

Full histopathology of the preserved organs and tissues listed above should be performed for all high dose and control P and F1 animals selected for mating. Examination of the ovaries of the P animals is optional. Organs demonstrating treatment-related changes should also be examined in the low- and mid-dose groups to aid in the elucidation of the NOAEL. Additionally, reproductive organs of the low-and mid-dose animals suspected of reduced fertility, e.g. those that failed to mate, conceive, sire, or deliver healthy offspring, or for which oestrus cyclicity or sperm number, motility, or morphology were affected, should be subjected to histopathological evaluation. All gross lesions such as atrophy or tumours shall be examined.

Detailed testicular histopathological examination (e.g. using Bouin's fixative, paraffin embedding and transverse sections of 4-5 μm thickness) should be conducted in order to identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen (14). Examination of the intact epididymis should include the caput, corpus, and cauda, which can be accomplished by evaluation of a longitudinal section. The epididymis should be evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm. PAS and haematoxylin staining may be used for examination of the male reproductive organs.

The postlactational ovary should contain primordial and growing follicles as well as the large corpora lutea of lactation. Histopathological examination should detect qualitative depletion of the primordial follicle population. A quantitative evaluation of primordial follicles should be conducted for F1 females; the number of animals, ovarian section selection, and section sample size should be statistically appropriate for the evaluation procedure used. Examination should include enumeration of the number of primordial follicles, which can be combined with small growing follicles, for comparison of treated and control ovaries (15)(16)(17)(18)(19).

1.5.8.2. Weanlings

Grossly abnormal tissue and target organs from all pups with external abnormalities or clinical signs, as well as from the one randomly selected pup/sex/litter from both the F1 and F2 generation which have not been selected for mating, shall be fixed and stored in a suitable medium for histopathological examination. Full histopathological characterisation of preserved tissue should be performed with special emphasis on the organs of the reproductive system.

2. DATA

2.1. TREATMENT OF RESULTS

Data shall be reported individually and summarised in tabular form, showing for each test group and each generation the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons, the time of any death or humane kill, the number of fertile animals, the number of pregnant females, the number of animals showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the types of parental and offspring observations, the types of histopathological changes, and all relevant litter data.

Numerical results should be evaluated by an appropriate, generally accepted statistical method; the statistical methods should be selected as part of the design of the study and should be justified. Dose-response statistical models may be useful for analysing data. The report should include sufficient information on the method of analysis and the computer program employed, so that an independent reviewer/statistician can re-evaluate and reconstruct the analysis.

2.2. EVALUATION OF RESULTS

The findings of this two-generation reproduction toxicity study should be evaluated in terms of the observed effects including necropsy and microscopic findings. The evaluation will include the relationship, or lack thereof, between the dose of the test substance and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, affected fertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects. The physico-chemical properties of the test substance, and when available, toxicokinetics data should be taken into consideration when evaluating test results.

A properly conducted reproduction toxicity test should provide a satisfactory estimation of a no-effect level and an understanding of adverse effects on reproduction, parturition, lactation, postnatal development including growth and sexual development.

2.3. INTERPRETATION OF RESULTS

A two-generation reproduction toxicity study will provide information on the effects of repeated exposure to a substance during all phases of the reproductive cycle. In particular, the study provides information on the reproductive parameters, and on development, growth, maturation and survival of offspring. The results of the study should be interpreted in conjunction with the findings from subchronic, prenatal developmental and toxicokinetic and other available studies. The results of this study can be used in assessing the need for further testing of a chemical. Extrapolation of the results of the study to man is valid to a limited degree. They are best used to provide information on no-effect-levels and permissible human exposure (20)(21)(22)(23).

3. REPORTING

3.1. TEST REPORT

The test report must include the following information:

Test substance:

- physical nature and, where relevant, physicochemical properties,

- identification data,

- purity.

Vehicle (if appropriate):

- ustification for choice of vehicle if other than water.

Test animals:

- species/strain used,

- number, age and sex of animals,

- source, housing conditions, diet, nesting materials, etc.,

- individual weights of animals at the start of the test.

Test conditions:

- rationale for dose level selection,

- details of test substance formulation/diet preparation, achieved concentrations,

- stability and homogeneity of the preparation,

- details of the administration of the test substance,

- conversion from diet/drinking water test substance concentration (ppm) to the achieved dose (mg/kg body weight/day), if applicable,

- details of food and water quality.

Results:

- food consumption, and water consumption if available, food efficiency (body weight gain per gram of food consumed), and test material consumption for P and F1 animals, except for the period of cohabitation and for at least the last third of lactation,

- absorption data (if available),

- body weight data for P and F1 animals selected for mating,

- litter and pup weight data,

- body weight at sacrifice and absolute and relative organ weight data for the parental animals,

- nature, severity and duration of clinical observations (whether reversible or not),

- time of death during the study or whether animals survived to termination,

- toxic response data by sex and dose, including indices of mating, fertility, gestation, birth, viability, and lactation; the report should indicate the numbers used in calculating these indices,

- toxic or other effects on reproduction, offspring, post-natal growth, etc.,

- necropsy findings,

- detailed description of all histopathological findings,

- number of P and F1 females cycling normally and cycle length,

- total cauda epididymal sperm number, percent progressively motile sperm, percent morphologically normal sperm, and percent of sperm with each identified abnormality,

- time-to-mating, including the number of days until mating,

- gestation length,

- number of implantations, corpora lutea, litter size,

- number of live births and post-implantation loss,

- number of pups with grossly visible abnormalities, if determined the number of runts should be reported,

- data on physical landmarks in pups and other post natal developmental data, physical landmarks evaluated should be justified,

- data on functional observations in pups and adults, as applicable,

- statistical treatment of results, where appropriate.

Discussion of results.

Conclusions, including NOAEL values for maternal and offspring effects.

4. REFERENCES

(1) Sadleir, R.M.F.S., (1979) Cycles and Seasons, In: Reproduction in Mammals: I. Germ Cells and Fertilisation, C.R. Auston and R.V. Short (eds.), Cambridge, New York.

(2) Gray, L.E. et al., (1989) A Dose-Response Analysis of Methoxychlor-Induced Alterations of Reproductive Development and Function in the Rat. Fundamental and Applied Toxicology, 12, p. 92-108.

(3) Robb, G.W. et al., (1978). Daily Sperm Production and Epididymal Sperm Reserves of Pubertal and Adult Rats. Journal of Reproduction and Fertility 54:103-107.

(4) Klinefelter, G.R. et al., (1991) The Method of Sperm Collection Significantly Influences Sperm Motion Parameters Following Ethane Dimethanesulfonate Administration in the Rat. Reproductive Toxicology, 5, p. 39-44.

(5) Seed, J. et al., (1996). Methods for Assessing Sperm Motility, Morphology, and Counts in the Rat, Rabbit, and Dog: a Consensus Report. Reproductive Toxicology, 10(3), p. 237-244.

(6) Chapin, R.E. et al., (1992) Methods for Assessing Rat Sperm Motility. Reproductive Toxicology, 6, p. 267-273

(7) Klinefelter, G.R. et al., (1992) Direct Effects of Ethane Dimethanesulphonate on Epididymal Function in Adult Rats: an In Vitro Demonstration. Journal of Andrology, 13, p. 409-421.

(8) Slott, V.L. et al., (1991) Rat Sperm Motility Analysis: Methodologic Considerations. Reproductive Toxicology, 5, p. 449-458.

(9) Slott, V.L. and Perreault, S.D., (1993) Computer-Assisted Sperm Analysis of Rodent Epididymal Sperm Motility Using the Hamilton-Thorn Motility Analyzer. In: Methods in Toxicology, Part A., Academic, Orlando, Florida, p. 319-333.

(10) Toth, G.P. et al., (1989) The Automated Analysis of Rat Sperm Motility Following Subchronic Epichlorhydrin Administration: Methodologic and Statistical Considerations. Journal of Andrology, 10, p. 401-415.

(11) Working, P.K. and M. Hurtt, (1987) Computerised Videomicrographic Analysis of Rat Sperm Motility. Journal of Andrology, 8, p. 330-337.

(12) Linder, R.E. et al., (1992) Endpoints of Spermatoxicity in the Rat After Short Duration Exposures to Fourteen Reproductive Toxicants. Reproductive Toxicology, 6, p. 491-505.

(13) Korenbrot, C.C. et al., (1977) Preputial Separation as an External Sign of Pubertal Development in the Male Rat. Biological Reproduction, 17, p. 298-303.

(14) Russell, L.D. et al., (1990) Histological and Histopathological Evaluation of the Testis, Cache River Press, Clearwater, Florida.

(15) Heindel, J.J. and R.E. Chapin, (eds.) (1993) Part B. Female Reproductive Systems, Methods in Toxicology, Academic, Orlando, Florida.

(16) Heindel, J.J. et al., (1989) Histological Assessment of Ovarian Follicle Number in Mice As a Screen of Ovarian Toxicity. In: Growth Factors and the Ovary, A.N. Hirshfield (ed.), Plenum, New York, p. 421-426.

(17) Manson, J.M. and Y.J. Kang, (1989) Test Methods for Assessing Female Reproductive and Developmental Toxicology. In: Principles and Methods of Toxicology, A.W. Hayes (ed.), Raven, New York.

(18) Smith, B.J. et al., (1991) Comparison of Random and Serial Sections in Assessment of Ovarian Toxicity. Reproductive Toxicology, 5, p. 379-383.

(19) Heindel, J.J., (1999) Oocyte Quantitation and Ovarian Histology. In: An Evaluation and Interpretation of Reproductive Endpoints for Human Health Risk Assessment, G. Daston,. and C.A. Kimmel, (eds.), ILSI Press, Washington, DC.

(20) Thomas, J. A., (1991) Toxic Responses of the Reproductive System. In: Casarett and Doull's Toxicology, M.O. Amdur, J. Doull, and C.D. Klaassen (eds.), Pergamon, New York.

(21) Zenick, H. and E.D. Clegg, (1989) Assessment of Male Reproductive Toxicity: A Risk Assessment Approach. In: Principles and Methods of Toxicology, A.W. Hayes (ed.), Raven Press, New York.

(22) Palmer, A.K., (1981) In: Developmental Toxicology, Kimmel, C.A. and J. Buelke-Sam (eds.), Raven Press, New York.

(23) Palmer, A.K., (1978) In Handbook of Teratology, Vol. 4, J.G. Wilson and F.C. Fraser (eds.), Plenum Press, New York.

B.36. TOXICOKINETICS

1. METHOD

1.1. INTRODUCTION

See General introduction Part B.

1.2. DEFINITIONS

See General introduction Part B.

1.3. REFERENCE SUBSTANCES

None.

1.4. PRINCIPLE OF THE TEST METHOD

The test substance is administered by an appropriate route. Depending on the purpose of the study, the substance may be administered in single or repeated doses over defined periods to one or several groups of experimental animals. Subsequently, depending on the type of study, the substance and/or metabolites are determined in body fluids, tissues and/or excreta.

Studies may be done with "unlabelled" or "labelled" forms of the test substance. Where a label is used it should be positioned in the substance in such a way to provide the most information about the fate of the compound.

1.5. QUALITY CRITERIA

None.

1.6. DESCRIPTION OF THE TEST METHOD

1.6.1. Preparations

Healthy young adult animals are acclimatised to the laboratory conditions for at least five days prior to the test. Before the test, animals are randomised and assigned to the treatment groups. In special situations, very young, pregnant or pre-treated animals may be used.

1.6.2. Test conditions

1.6.2.1. Experimental animals

Toxicokinetic studies may be carried out in one or more appropriate animal species and should take account of the species used or intended to be used in other toxicological studies on the same test substance. Where rodents are used in a test the weight variation should not exceed ± 20 % of the mean weight.

1.6.2.2. Number and sex

For absorption and excretion studies, there should be four animals in each dose group initially. Sex preference is not mandatory, but under some circumstances both sexes may need to be studied. If there are sex differences in response, then four animals of each sex should be tested. In the case of studies with non-rodents fewer animals may be used. When tissue distribution is being studied, the initial group size should take into account both the number of animals to be sacrificed at each time point and the number of time points to be examined.

When metabolism is being studied, the group size is related to the needs of the study. For multiple-dose and multiple-time-point studies, the group size should take into account the number of time points and planned sacrifice(s), but may not be smaller than two animals. The group size should be sufficient to provide an acceptable characterisation of uptake, plateau and depletion (as appropriate) of the test substance and/or metabolites.

1.6.3. Dose levels

In the case of single-dose administration, at least two dose levels should be used. There should be a low dose at which no toxic effects are observed and a high dose at which there might be changes in toxicokinetic parameters or at which toxic effects occur.

In the case of repeated-dose administration the low dose is usually sufficient, but under certain circumstances a high dose may also be necessary.

1.6.4. Route of administration

Toxicokinetic studies should be performed using the same route and, where appropriate, the same vehicle as that used or intended to be used in the other toxicity studies. The test substance is usually administered orally by gavage or in the diet, applied to the skin, or administered by inhalation for defined periods to groups of experimental animals. Intravenous administration of the test substance may be useful in determining relative absorption by other routes. In addition, useful information may be provided on the pattern of distribution soon after the intravenous administration of a substance.

The possibility of interference of the vehicle with the rest substance should be taken into consideration. Attention should be given to differences in absorption between the administration of the test substance by gavage and in the diet and the need for an accurate determination of dose particularly when the test substance is given in the diet.

1.6.5. Observation period

All the animals should be observed daily and signs of toxicity and other relevant clinical features recorded, including time of onset, degree and duration.

1.6.6. Procedure

After weighing test animals, the test substance is administered by an appropriate route. If considered relevant, animals may be fasted before the test substance is administered.

Absorption

The rate and extent of absorption of the administered substance can be evaluated using various methods, with and without reference groups [8], for example by:

- determination of the amount of test substance and/or metabolites in excreta, such as urine, bile, faeces, exhaled air and that remaining in the carcase,

- comparison of the biological response (e.g. acute toxicity studies) between test and control and/or reference groups,

- comparison of the amount of renally excreted substance and/or metabolite in test and reference groups,

- determination of the area under the plasma-level/time curve of the lest substance and/or metabolites and comparison with data from a reference group.

Distribution

Two approaches are available at present, one or both of which may be used for analysis of distribution patterns:

- useful qualitative information is obtained using whole body autoradiographic techniques,

- quantitative information is obtained by sacrificing animals at different times after exposure and determining the concentration and amount of the test substance and/or metabolites in tissues and organs.

Excretion

In excretion studies, urine, faeces and expired air and, in certain circumstances, bile are collected. The amount of test substance and/or metabolites in these excreta should be measured several times after exposure, either until about 95 % of the administered dose has been excreted or for seven days, whichever comes first.

In special cases, the excretion of the test substance in the milk of lactating test animals may need to be considered.

Metabolism

To determine the extent and pattern of metabolism, biological samples should be analysed by suitable techniques. Structures of metabolites should be elucidated and appropriate metabolic pathways proposed where there is a need to answer questions arising from previous toxicological studies. It may be helpful to perform studies in vitro to obtain information on metabolic pathways.

Further information on the relationship of metabolism to toxicity may be obtained from biochemical studies, such as the determination of effects on metabolising enzyme systems, depletion of endogenous non-protein sulphydryl compounds and binding of the substance with macromolecules.

2. DATA

According to the type of study performed, data should be summarised in tabular form supported by graphical presentation whenever appropriate. For each test group, mean and statistical variations of measurements in relation to time, dosage, tissues and organs should be shown when appropriate. The extent of absorption and the amount and rates of excretion should be determined by appropriate methods. When metabolism studies are performed, the structure of identified metabolites should be given and possible metabolic pathways presented.

3. REPORTING

3.1. TEST REPORT

According to the type of study performed, the test report shall, if possible, contain the following information:

- species, strain, source, environmental conditions, diet,

- characterisation of labelled materials, when used,

- dosage levels and intervals used,

- route(s) of administration and any vehicles used,

- toxic and other effects observed,

- methods for determination of test substance and/or metabolites in biological samples, including expired air,

- tabulation of measurements by sex, dose, regimen, time, tissues and organs,

- presentation of the extent of absorption and excretion with time,

- methods for the characterisation and identification of metabolites in biological samples,

- methods for biochemical measurements related to metabolism,

- proposed pathways for metabolism,

- discussion of the results,

- interpretation of the results.

3.2. EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.37. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES FOLLOWING ACUTE EXPOSURE

1. METHOD

1.1. INTRODUCTION

In the assessment and evaluation of the toxic effects of substances, it is important to consider the potential of certain classes of substances to cause specific types of neurotoxicity that might not be detected in other toxicity studies. Certain organophosphorus substances have been observed to cause delayed neurotoxicity and should be considered as candidates for evaluation.

In vitro screening tests could be employed to identify those substances which may cause delayed polyneuropathy; however, negative findings from in vitro studies do not provide evidence that the test substance is not a neurotoxicant.

See General introduction Part B.

1.2. DEFINITIONS

Organophopsphorus substances include uncharged organophosphorus esters, thioesters or anhydrides of organophosphoric, organophosphonic or organophosphoramidic acids or of related phosphorothioic, phosphonothioic or phosphorothioamidic acids, or other substances that may cause the delayed neurotoxicity sometimes seen in this class of substances.

Delayed neurotoxicity is a syndrome associated with prolonged delayed onset of ataxia, distal axonopathies in spinal cord and peripheral nerve, and inhibition and aging of neuropathy target esterase (NTE) in neural tissue.

1.3. REFERENCE SUBSTANCES

A reference substance may be tested with a positive control group as a means of demonstrating that under the laboratory test conditions, the response of the tested species has not changed significantly.

An example of a widely used neurotoxicant is tri-o-tolyl phosphate (CAS 78-30-8, Einecs 201-103-5, CAS nomenclature: phosphoric acid, tris(2-methylphenyl)ester), also known as tris-o-cresylphosphate.

1.4. PRINCIPLE OF THE TEST METHOD

The test substance is administered orally in a single dose to domestic hens which have been protected from acute cholinergic effects, when appropriate. The animals are observed for 21 days for behavioural abnormalities, ataxia, and paralysis. Biochemical measurements, in particular neuropathy target esterase inhibition (NTE), are undertaken on hens randomly selected from each group, normally 24 and 48 hours after dosing. Twenty-one days after exposure, the remainder of the hens are killed and histopathological examination of selected neural tissues is undertaken.

1.5. DESCRIPTION OF THE TEST METHOD

1.5.1. Preparations

Healthy young adult hens free from interfering viral diseases and medication and without abnormalities of gait should be randomised and assigned to treatment and control groups and acclimatised to the laboratory conditions for at least five days prior to the start of the study.

Cages or enclosures which are large enough to permit free mobility of the hens, and easy observation of gait should be used.

Dosing with the test substance should normally be by the oral route using gavage, gelatine capsules, or a comparable method. Liquids may be given undiluted or dissolved in an appropriate vehicle such as corn oil; solids should be dissolved if possible since large doses of solids in gelatine capsules may not be absorbed efficiently. For non-aqueous vehicles the toxic characteristics of the vehicle should be known, and if not known should be determined before the test.

1.5.2. Test conditions

1.5.2.1. Test animals

The young adult domestic laying hen (Gallus gallus domestícus), aged eight to 12 months, is recommended. Standard size breeds and strains should be employed and the hens normally should have been reared under conditions which permitted free mobility.

1.5.2.2. Number and sex

In addition to the treatment group, both a vehicle control group and a positive control group should be used. The vehicle control group should be treated in a manner identical to the treatment group, except that administration of the test substance is omitted.

Sufficient number of hens should be utilised in each group of birds so that at least six birds can be killed for biochemical determination (three at each of two time points) and six can survive the 21-day observation period for pathology.

The positive control group may be run concurrently or be a recent historical control group. It should contain at least six hens, treated with a known delayed neurotoxicant, three hens for biochemistry and three hens for pathology. Periodic updating of historical data is recommended. New positive control data should be developed when some essential element (e.g. strain, feed, housing conditions) of the conduct of the test has been changed by the performing laboratory.

1.5.2.3. Dose levels

A preliminary study using an appropriate number of hens and dose levels groups should be performed to establish the level to be used in the main study. Some lethality is typically necessary in this preliminary study to define an adequate main study dose. However, to prevent death due to acute cholinergic effects, atropine or another protective agent, known to not interfere with delayed neurotoxic responses, may be used. A variety of test methods may be used to estimate the maximum non-lethal dose of test substances (See method B.1bis). Historical data in the hen or other toxicological information may also be helpful in dose selection.

The dose level of the test substance in the main study should be as high as possible taking into account the results of the preliminary dose selection study and the upper limit dose of 2000 mg/kg body weight. Any mortality which might occur should not interfere with the survival of sufficient animals for biochemistry (six) and histology (six) at 21 days. Atropine or another protective agent, known to not interfere with delayed neurotoxic responses, should be used to prevent death due to acute cholinergic effects.

1.5.2.4. Limit test

If a test at a dose level of at least 2000 mg/kg body weight/day, using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from structurally related substances, then a study using a higher dose may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.

1.5.3. Observation period

Observation period should be 21 days.

1.5.4. Procedure

After administration of a protective agent to prevent death due to acute cholinergic effect, the test substance is administered in a single dose.

General observation

Observations should start immediately after exposure. All hens should be carefully observed several times during the first two days and thereafter at least once daily for a period of 21 days or until scheduled kill. All signs of toxicity should be recorded, including the time of onset, type, severity and duration of behavioural abnormalities. Ataxia should be measured on an ordinal grading scale consisting of at least four levels, and paralysis should be noted. At least twice a week the hens selected for pathology should be taken outside the cages and subjected to a period of forced motor activity, such as ladder climbing, in order to facilitate the observation of minimal toxic effects. Moribund animals and animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.

Body weight

All hens should be weighed just prior to administration of the test substance and at least once a week thereafter.

Biochemistry

Six hens randomly selected from each of the treatment and vehicle control groups, and three hens from the positive control group (when this group is run concurrently), should be killed within a few days after dosing, and the brain and lumbar spinal cord prepared and assayed for neuropathy target esterase inhibition activity. In addition, it may also be useful to prepare and assay sciatic nerve tissue for neuropathy target esterase inhibition activity. Normally, three birds of the control and each treatment group are killed after 24 hours and three at 48 hours, whereas the three hens of the positive controls should be killed at 24 hours. If observation of clinical signs of intoxication (this can often be assessed by observation of the time of onset of cholinergic signs) indicates that the toxic agent may be disposed of very slowly then it may be preferable to sample tissue from three birds at each of two times between 24 and as late as 72 hours after dosing.

Analyses of acetylcholinesterase (AChE) may also be performed on these samples, if deemed appropriate. However, spontaneous reactivation of AChE may occur in vivo, and so lead to underestimation of the potency of the substance as an AChE inhibitor.

Gross necropsy

Gross necropsy of all animals (scheduled killed and killed when moribund) should include observation of the appearance of the brain and spinal cord.

Histopathological examination

2. DATA

Negative results on the endpoints selected in this method (biochemistry, histopathology and behavioural observation) would not normally require further testing for delayed neurotoxicity. Equivocal or inconclusive results for these endpoints may require further evaluation.

Individual data should be provided. Additionally, all data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, behavioural or biochemical effects, the types and severity of these lesions or effects, and the percentage of animals displaying each type and severity of lesion or effect.

The findings of this study should be evaluated in terms of the incidence, severity, and correlation of behavioural, biochemical and histopathological effects and any other observed effects in the treated and control groups.

Numerical results should be evaluated by appropriate and generally acceptable statistical methods. The statistical methods used should be selected during the design of the study.

3. REPORTING

TEST REPORT

The test report shall, if possible, include the following information:

3.1. Test animals:

- strain used,

- number and age of animals,

- source, housing conditions, etc.,

- individual weights of animals at the start of the test.

3.2. Test conditions:

- details of test substance preparation, stability and homogeneity, where appropriate,

- justification for choice of vehicle,

- details of the administration of the test substance,

- details of food and water quality,

- rationale for dose selection,

- specification of doses administered, including details of the vehicle, volume and physical form of the material administered,

- identity and details of the administration of any protective agent.

3.3. Results:

- body weight data,

- toxic response data by group, including mortality,

- nature, severity and duration of clinic observations (whether reversible or not),

- a detailed description of biochemical methods and findings,

- necropsy findings,

- a detailed description of all histopathological findings,

- statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

4. REFERENCES

This method is analogius to OECD TG 418.

B.38. DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS SUBSTANCES 28-DAY REPEATED DOSE STUDY

1. METHOD

1.1. INTRODUCTION

This 28-day delayed neurotoxicity test provides information on possible health hazards likely to arise from repeated exposures over a limited period of time. It will provide information on dose response and can provide an estimate of a no-observed-adverse effect level, which can be of use for establishing safety criteria for exposure.

See also General introduction Part B.

1.2. DEFINITIONS

Organophosphorus substances include uncharged organophosphorus esters, thioesters or anhydrides of organophosphoric, organophosphonic or organophosphoramidic acids or of related phosphorothioic, phosphonothioic or phosphorothioamidic acids or other substances that may cause the delayed neurotoxicity sometimes seen in this class of substances.

Delayed neurotoxicity is a syndrome associated with prolonged delayed onset of ataxia, distal axonopathies in spinal cord and peripheral nerve, and inhibition and ageing of neuropathy target esterase (NTE) in neural tissue.

1.3. PRINCIPLE OF THE TEST METHOD

Daily doses of the test substance are administered orally to domestic hens for 28 days. The animals are observed at least daily for behavioural abnormalities, ataxia and paralysis until 14 days after the last dose. Biochemical measurements, in particular neuropathy target esterase inhibition (NTE), are undertaken, on hens randomly selected from each group, normally 24 and 48 hours after the last dose. Two weeks after the last dose, the remainder of the hens are killed and histopathological examination of selected neural tissues is undertaken.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparations

Healthy young adult hens free from interfering viral diseases and medication, and without abnormalities of gait should be randomised and assigned to treatment and control groups and acclimatised to the laboratory conditions for at least five days prior to the start of the study.

Cages or enclosures which are large enough to permit free mobility of the hens and easy observation of gait should be used.

Oral dosing each day, seven days per week, should be carried out, preferably by gavage or administration of gelatine capsules. Liquids may be given undiluted or dissolved in an appropriate vehicle such as corn oil; solids should be dissolved if possible since large doses of solids in gelatine capsules may not be absorbed efficiently. For non-aqueous vehicles the toxic characteristics of the vehicle should be known, and if not known should be determined before the test.

1.4.2. Test conditions

1.4.2.1. Test animals

The young adult domestic laying hen (Gallus gallus domesticus), aged eight to 12 months, is recommended. Standard size, breeds and strains should be employed and the hens normally should have been reared under conditions which permitted free mobility.

1.4.2.2. Number and sex

Generally at least three treatment groups and a vehicle control group should be used. The vehicle control group should be treated in a manner identical to the treatment group, except that administration of the test substance is omitted.

Sufficient number of hens should be utilised in each group of birds so that at least six birds can be killed for biochemical determinations (three at each of two timepoints) and six birds can survive the 14-day post-treatment observation period for pathology.

1.4.2.3. Dose levels

Dose levels should be selected taking into account the results from an acute test on delayed neurotoxicity and any other existing toxicity or kinetic data available for the test compound. The highest dose level should be chosen with the aim of inducing toxic effects, preferably delayed neurotoxicity, but not death nor obvious suffering. Thereafter, a descending sequence of dose levels should be selected with a view to demonstrate any dose-related response and no-observed-adverse effects at the lowest dose level.

1.4.2.4. Limit test

If a test at a dose level of at least 1000 mg/kg body weight/day, using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data from structurally related substances, then a study using a higher dose may not be considered necessary. The limit test applies except when expected human exposure indicates the need for a higher dose level to be used.

1.4.2.5. Observation period

All the animals should be observed at least daily during the exposure period and 14 days after, unless scheduled necropsy.

1.4.3. Procedure

Animals are dosed with the test substance on seven days per week for a period of 28 days.

General observations

Observations should start immediately after treatment begins. All hens should be carefully observed at least once daily on each of the 28 days of treatment, and for 14 days after dosing or until scheduled kill. All signs of toxicity should be recorded including their time of onset, type, severity and duration. Observations should include, but not be limited to, behavioural abnormalities. Ataxia should be measured on an ordinal grading scale consisting of at least four levels, and paralysis should be noted. At least twice a week the hens should be taken outside the cages and subjected to a period of forced motor activity, such as ladder climbing, in order to facilitate the observation of minimal toxic effects. Moribund animals in severe distress or pain should be removed when noticed, humanely killed and necropsied.

Body weight

All hens should be weighed just prior to the first administration of the test substance and at least once a week thereafter.

Biochemistry

Six hens randomly selected from each of the treatment and vehicle control groups should be killed within a few days after the last dose, and the brain and lumbar spinal cord prepared and assayed for neuropathy target esterase (NTE) inhibition activity. In addition, it may also be useful to prepare and assay sciatic nerve tissue for neuropathy target esterase (NTE) inhibition activity. Normally, three birds of the control and each treatment group are killed after 24 hours and three at 48 hours after the last dose. If data from the acute study or other studies (e.g. toxicokinetics) indicate that other times of killing after final dosing are preferable then these times should be used and the rationale documented.

Gross necropsy

Gross necropsy of all animals (scheduled killed and killed when moribund) should include observation of the appearance of the brain and spinal cord.

Histopathological examination

Neural tissue from animals surviving the observation period and not used for biochemical studies should be subjected to microscopic examination. Tissues should be fixed in situ, using perfusion techniques. Sections should include cerebellum (mid longitudinal level), medulla oblongata, spinal cord and peripheral nerves. The spinal cord sections should be taken from the upper cervical segment, the mid-thoracic and the lumbo-sacral regions. Sections of the distal region of the tibial nerve and its branches to the gastrocnemial muscle and of the sciatic nerve should be taken. Sections should be stained with appropriate myelin and axon-specific stains. Initially, microscopic examination should be carried out on the preserved tissues of all animals in the control and high dose group. When there is evidence of effects in the high dose group, microscopic examination should also be carried out in hens from the intermediate and low dose groups.

2. DATA

Numerical results should be evaluated by appropriate and generally acceptable statistical methods. The statistical methods should be selected during the design of the study.

3. REPORTING

TEST REPORT

The test report shall, if possible, include the following information:

3.1. Test animals:

- strain used,

- number and age of animals,

- source, housing conditions, etc.,

- individual weights of animals at the start of the test.

3.2. Test conditions:

- details of test substance preparation, stability and homogeneity, where appropriate,

- justification for choice of vehicle,

- details of the administration of the test substance,

- details of food and water quality,

- rationale for dose selection,

- specification of doses administered, including details of the vehicle, volume and physical form of the material administered,

- rationale for choosing other times for biochemical determination, if other than 24 and 48 h.

3.3. Results:

- body weight data,

- toxic response data by dose level, including mortality,

- no-observed adverse effect level,

- nature, severity and duration of clinic observations (whether reversible or not),

- a detailed description of biochemical methods and findings,

- necropsy findings,

- a detailed description of all histopathological findings,

- statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

4. REFERENCES

This method is analogous to OECD TG 419.

B.39. UNSCHEDULED DNA SYNTHESIS (UDS) TEST WITH MAMMALIAN LIVER CELLS IN VIVO

1. METHOD

This method is a replicate of the OECD TG 486, Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo (1997).

1.1. INTRODUCTION

The purpose of the unscheduled DNA Synthesis (UDS) test with mammalian liver cells in vivo is to identify test substances that induce DNA repair in liver cells of treated animals (see 1,2,3,4).

This in vivo test provides a method for investigating genotoxic effects of chemicals in the liver. The end-point measured is indicative of DNA damage and subsequent repair in liver cells. The liver is usually the major site of metabolism of absorbed compounds. It is thus an appropriate site to measure DNA damage in vivo.

If there is evidence that the test substance will not reach the target tissue, it is not appropriate to use this test.

The end-point of unscheduled DNA synthesis (UDS) is measured by determining the uptake of labelled nucleosides in cells that are not undergoing scheduled (S-phase) DNA synthesis. The most widely used technique is the determination of the uptake of tritium-labelled thymidine (3H-TdR) by autoradiography. Rat livers are preferably used for in vivo UDS tests. Tissues other than the livers may be used, but are not the subject of this method.

The detection of a UDS response is dependent on the number of DNA bases excised and replaced at the site of the damage. Therefore, the UDS test is particularly valuable to detect substance-induced "longpatch repair" (20-30 bases). In contrast, "shortpatch repair" (1-3 bases) is detected with much lower sensitivity. Furthermore, mutagenic events may result because of non-repair, misrepair or misreplication of DNA lesions. The extent of the UDS response gives no indication of the fidelity of the repair process. In addition, it is possible that a mutagen reacts with DNA but the DNA damage is not repaired via an excision repair process. The lack of specific information on mutagenic activity provided by the UDS test is compensated for by the potential sensitivity of this endpoint because it is measured in the whole genome.

See also General introduction Part B.

1.2. DEFINITIONS

Cells in repair: a net nuclear grain (NNG) higher than a preset value, to be justified at the laboratory conducting the test.

Net nuclear grains (NNG): quantitative measure for UDS activity of cells in autoradiographic UDS tests, calculated by subtracting the average number of cytoplasmic grains in nucleus-equivalent cytoplasmic areas (CG) from the number of nuclear grains (NG): NNG = NG - CG. NNG counts are calculated for individual cells and then pooled for cells in a culture, in parallel cultures, etc.

Unscheduled DNA Synthesis (UDS): DNA repair synthesis after excision and removal of a stretch of DNA containing a region of damage induced by chemical substances or physical agents.

1.3. PRINCIPLE OF THE TEST METHOD

The UDS test with mammalian liver cells in vivo indicates DNA repair synthesis after excision and removal of a stretch of DNA containing a region of damage induced by chemical substances or physical agents. The test is usually based on the incorporation of 3H-TdR into the DNA of liver cells which have a low frequency of cells in the S-phase of the cell cycle. The uptake of 3H-TdR is usually determined by autoradiography, since this technique is not as susceptible to interference from S-phrase cells as, for example, liquid scintillation counting.

1.4. DESCRIPTION OF THE METHOD

1.4.1. Preparations

1.4.1.1. Selection of animal species

Rats are commonly used, although any appropriate mammalian species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. At the commencement of the study the weight variation of animals should be minimal and not exceed ± 20 % of the mean weight for each sex.

1.4.1.2. Housing and feeding conditions

General conditions referred in the General introduction to Part B are applied although the aim for humidity should be 50-60 %.

1.4.1.3. Preparation of the animals

1.4.1.4. Test substance/Preparation

1.4.2. Test conditions

1.4.2.1. Solvent/Vehicle

1.4.2.2. Controls

Concurrent positive and negative controls (solvent/vehicle) should be included in each independently performed part of the experiment. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the animals in the treated groups.

Positive controls should be substances known to produce UDS when administered at exposure levels expected to give a detectable increase over background. Positive controls needing metabolic activation should be used at doses eliciting a moderate response (4). The doses may be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. Examples of positive control substances include:

Sampling Times | Substance | CAS No | EINECS No |

Early sampling times (2-4 hours) | N-Nitrosodimethylamine | 62-75-9 | 200-249-8 |

Late sampling times (12-16 hours) | N-2-Fluorenylacetamide (2-AAF) | 53-96-3 | 200-188-6 |

Other appropriate positive control substances may be used. It is acceptable that the positive control should be administered by a route different from the test substance.

1.5. PROCEDURE

1.5.1. Number and sex of animals

An adequate number of animals should be used, to take account of natural biological variation in test response. The number of animals should be at least three analysable animals per group. Where a significant historical database has been accumulated, only one or two animals are required for the concurrent negative and positive control groups.

If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences in toxicity between sexes, then testing in a single sex, preferably males, will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.

1.5.2. Treatment schedule

Test substances are generally administered as a single treatment.

1.5.3. Dose levels

Normally, at least two dose levels are used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. In general, the lower dose should be 50 % to 25 % of the high dose.

Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. If a range finding study is performed because there are no suitable data available, it should be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study.

The highest dose may also be defined as a dose that produces some indication of toxicity in the liver (e.g. pyknotic nuclei).

1.5.4. Limit test

If a test at one dose level of at least 2000 mg/kg body weight, applied in a single treatment, or in two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected, based upon data from structurally related substances, then a full study may not be necessary. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.

1.5.5. Administration of doses

The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula. Other routes of exposure may be acceptable where they can be justified. However, the intraperitoneal route is not recommended as it could expose the liver directly to the test substance rather than via the circulatory system. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100 g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

1.5.6. Preparation of liver cells

Liver cell are prepared from treated animals normally 12-16 hours after dosing. An additional earlier sampling time (normally two to four hours post-treatment) is generally necessary unless there is a clear positive response at 12-16 hours. However, alternative sampling times may be used when justified on the basis of toxicokinetic data.

Short-term cultures of mammalian liver cells are usually established by perfusing the liver in situ with collagenase and allowing freshly dissociated liver cells to attach themselves to a suitable surface. Liver cells from negative control animals should have a viability (5) of at least 50 %.

1.5.7. Determination of UDS

Freshly isolated mammalian liver cells are incubated usually with medium containing 3H-TdR for an appropriate length of time, e.g. 3-8 hours. At the end of the incubation period, medium should be removed from the cells, which may then be incubated with medium containing excess unlabelled thymidine to diminish unincorporated radioactivity ("cold chase"). The cells are then rinsed, fixed and dried. For more prolonged incubation times, cold chase may not be necessary. Slides are dipped in autoradiographic emulsion, exposed in the dark (e.g. refrigerated for 7-14 days), developed, stained, and exposed silver grains are counted. Two to three slides are prepared from each animal.

1.5.8. Analysis

The slide preparations should contain sufficient cells of normal morphology to permit a meaningful assessment of UDS. Preparations are examined microscopically for signs of overt cytotoxicity (e.g. pyknosis, reduced levels of radiolabelling).

Slides should be coded before grain counting. Normally 100 cells are scored from each animal from at least two slides; the scoring of less than 100 cells/animal should be justified. Grain counts are not scored for S-phase nuclei, but the proportion of S-phase cells may be recorded.

The amount of 3H-TdR incorporation in the nuclei and the cytoplasm of morphologically normal cells, as evidenced by the deposition of silver grains, should be determined by suitable methods.

Grain counts are determined over the nuclei (nuclear grains, NG) and nucleus equivalent areas over the cytoplasm (cytoplasmic grains, CG). CG counts are measured by either taking the most heavily labelled area of cytoplasm, or by taking an average of two to three random cytoplasmic grain counts adjacent to the nucleus. Other counting methods (e.g. whole cell counting) may be used if they can be justified (6).

2. DATA

2.1. TREATMENT OF RESULTS

Individual slide and animal data should be provided. Additionally, all data should be summarised in tabular form. Net nuclear grain (NNG) counts should be calculated for each cell, for each animal and for each dose and time by subtracting CG counts from NG counts. If "cells in repair" are counted, the criteria for defining "cells in repair" should be justified and based on historical or concurrent negative control data. Numerical results may be evaluated by statistical methods. If used, statistical tests should be selected and justified prior to conducting the study.

2.2. EVALUATION AND INTERPRETATION OF RESULTS

Examples of criteria for positive/negative responses include:

positive | (i) | NNG values above a pre-set threshold which is justified on the basis of laboratory historical data; or |

| (ii) | NNG values significantly greater than concurrent control; |

negative | (i) | NNG values within/below historical control threshold; or |

| (ii) | NNG values not significantly greater than concurrent control. |

The biological relevance of data should be considered: i.e. parameters such as inter-animal variation, dose-response relationship and cytotoxicity should be taken into account. Statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

A positive result from the UDS test with mammalian liver cells in vivo indicate that a test substance induces DNA damage in mammalian liver cells in vivo that can be repaired by unscheduled DNA synthesis in vitro. A negative result indicates that, under the test conditions, the test substance does not induce DNA damage that is detectable by this test.

The likelihood that the test substance reaches the general circulation or specifically the target tissue (e.g. systemic toxicity) should be discussed.

3. REPORTING

TEST REPORT

The test report must include the following information:

Solvent/Vehicle:

- justification for choice of vehicle,

- solubility and stability of the test substance in solvent/vehicle, if known.

Test animals:

- species/strain used,

- number, age and sex of animals,

- source, housing conditions, diet, etc.,

- individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group,

Test conditions:

- positive and negative vehicle/solvent controls,

- data from range-finding study, if conducted,

- rationale for dose level selection,

- details of test substance preparation,

- details of the administration of the test substance,

- rationale for route of administration,

- methods for verifying that test agent reached the general circulation or target tissue, if applicable,

- conversion from diet/drinking water test substance concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable,

- details of food and water quality,

- detailed description of treatment and sampling schedules,

- methods for measurement of toxicity,

- method of liver cell preparation and culture,

- autoradiographic technique used,

- number of slides prepared and numbers of cells scored,

- evaluation criteria,

- criteria for considering studies as positive, negative or equivocal,

Results:

- individual slide, animal and group mean values for nuclear grains, cytoplasmic grains, and net nuclear grains,

- dose-response relationship, if available,

- statistical evaluation if any,

- signs of toxicity,

- concurrent negative (solvent/vehicle) and positive control data,

- historical negative (solvent/vehicle) and positive control data with range, means and standard deviations,

- number of "cells in repair" if determined,

- number of S-phase cells if determined,

- viability of the cells.

Discussion of results.

Conclusions.

4. REFERENCES

(1) Ashby, J., Lefevre, P.A., Burlinson, B. and Penman, M.G., (1985) An Assessment of the In Vivo Rat Hepatocyte DNA Repair Assay. Mutation Res., 156, p. 1-18.

(2) Butterworth, B.E., Ashby, J., Bermudez, E., Casciano, D., Mirsalis, J., Probst, G. and Williams, G., (1987) A Protocol and Guide for the In Vivo Rat Hepatocyte DNA-Repair Assay. Mutation Res., 189, p. 123-133.

(3) Kennelly, J.C., Waters, R., Ashby, J., Lefevre, P.A., Burlinson, B., Benford, D.J., Dean, S.W. and Mitchell, I. de G., (1993) In Vivo Rat Liver UDS Assay. In: Kirkland D.J. and Fox M., (Eds) Supplementary Mutagenicity Tests: UKEM Recommended Procedures. UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report. Part II revised. Cambridge University Press, Cambridge, New York, Port Chester, Melbourne, Sydney, p. 52-77.

(4) Madle, S., Dean, S.W., Andrae, U., Brambilla, G., Burlinson, B., Doolittle, D.J., Furihata, C., Hertner, T., McQueen, C.A. and Mori, H., (1993) Recommendations for the Performance of UDS Tests In Vitro and In Vivo. Mutation Res., 312, p. 263-285.

(5) Fautz, R., Hussain, B., Efstathiou, E. and Hechenberger-Freudl, C., (1993) Assessment of the Relation Between the Initial Viability and the Attachment of Freshly Isolated Rat Hepatocytes Used for the In Vivo/In Vitro DNA Repair Assay (UDS). Mutation Res., 291, p. 21-27.

(6) Mirsalis, J.C., Tyson, C.K. and Butterworth, B.E., (1982) Detection of Genotoxic Carcinogens in the In Vivo/In Vitro Hepatocyte DNA Repair Assay. Environ. Mutagen, 4, p. 553-562.

B.40. IN VITRO SKIN CORROSION: TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST (TER)

1. METHOD

This testing method is equivalent to the OECD TG 430 (2004).

1.1. INTRODUCTION

Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test material (as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)) (1). This method provides a procedure by which the assessment of corrosivity is not carried out in live animals.

The assessment of skin corrosivity has typically involved the use of laboratory animals (2). Concern for the pain and suffering of animals involved with this procedure has been addressed in the revision of testing method B.4 that allows for the determination of skin corrosion by using alternative, in vitro, methods, avoiding pain and suffering.

A first step towards defining alternative tests that could be used for skin corrosivity testing for regulatory purposes was the conduct of prevalidation studies (3). Following this, a formal validation study of in vitro methods for assessing skin corrosion (4)(5) was conducted (6)(7)(8). The outcome of these studies and other published literature led to the recommendation that the following tests could be used for the assessment of in vivo skin corrosivity (9)(10)(11): the human skin model test (see tesing method B.40bis) and the transcutaneous electrical resistance test (this method).

A validation study and other published studies have reported that the rat skin transcutaneous electrical resistance (TER) assay (12)(13) is able to reliably discriminate between known skin corrosives and non-corrosives (5)(9).

The test described in this method allows the identification of corrosive chemical substances and mixtures. It further enables the identification of non-corrosive substances and mixtures when supported by a weight of evidence determination using other existing information (e.g. pH, structure-activity-relationships, human and/or animal data) (1)(2)(11)(14). It does not provide information on skin irritation, nor does it allow the sub-categorisation of corrosive substances as permitted in the Globally Harmonised Classification System (GHS) (1).

For a full evaluation of local skin effects after a single dermal exposure, it is recommended to follow the sequential testing strategy as appended to testing method B.4 (2) and provided in the Globally Harmonised System (1). This testing strategy includes the conduct of in vitro tests for skin corrosion (as described in this method) and skin irritation before considering testing in live animals.

1.2. DEFINITIONS

Skin corrosionin vivo: is the production of irreversible damage of the skin: namely, visible necrosis through the epidermis and into the dermis, following the application of a test subsance for up to four hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of the observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia, and scars. Histopathology should be considered to evaluate questionable lesions.

Transcutaneous Electrical Resistance (TER): is a measure of the electrical impendance of the skin, as a resistance value in kilo Ohms. A simple and robust method of assessing barrier function by recording the passage of ions through the skin using a Wheatstone bridge apparatus.

1.3. REFERENCE SUBSTANCES

Table 1

Reference chemicals

Name | EINECS No | CAS No | |

1,2-Diaminopropane | 201-155-9 | 78-90-0 | Severely corrosive |

Acrylic Acid | 201-177-9 | 79-10-7 | Severely Corrosive |

2-tert. Butylphenol | 201-807-2 | 88-18-6 | Corrosive |

Potassium hydroxide (10 %) | 215-181-3 | 1310-58-3 | Corrosive |

Sulfuric acid (10 %) | 231-639-5 | 7664-93-9 | Corrosive |

Octanoic acid (caprylic acid) | 204-677-5 | 124-07-02 | Corrosive |

4-Amino-1,2,4-triazole | 209-533-5 | 584-13-4 | Not corrosive |

Eugenol | 202-589-1 | 97-53-0 | Not corrosive |

Phenethyl bromide | 203-130-8 | 103-63-9 | Not corrosive |

Tetrachloroethylene | 204-825-9 | 27-18-4 | Not corrosive |

Isostearic acid | 250-178-0 | 30399-84-9 | Not corrosive |

4-(Methylthio)-benzaldehyde | 222-365-7 | 3446-89-7 | Not corrosive |

Most of the chemicals listed are taken from the list of chemicals selected for the ECVAM international validation study (4). Their selection is based on the following criteria:

(i) equal number of corrosive and non-corrosive substances;

(ii) commercially available substances covering most of the relevant chemical classes;

(iii) inclusion of severely corrosive as well as less corrosive substances in order to enable discrimination based on corrosive potency;

(iv) choice of chemicals that can be handled in a laboratory without posing other serious hazards than corrosivity.

1.4. PRINCIPLE OF THE TEST METHOD

The test material is applied for up to 24 hours to the epidermal surfaces of skin discs in a two-compartment test system in which the skin discs function as the separation between the compartments. The skin discs are taken from humanely killed rats aged 28-30 days. Corrosive materials are identified by their ability to produce a loss of normal stratum corneum integrity and barrier function, which is measured as a reduction in the TER below a threshold level (12). For rat TER, a cut-off value of 5 kΏ has been selected based on extensive data for a wide range of chemicals where the vast majority of values were either clearly well above (often > 10 kΩ), or well below (often < 3 kΩ) this value (12). Generally, materials which are non-corrosive in animals but are irritating or non-irritating do not reduce the TER below this cut-off value. Furthermore, use of other skin preparations or other equipment may alter the cut-off value, necessitating further validation.

A dye-binding step is incorporated into the test procedure for confirmation testing of positive results in the TER including values around 5 kΩ. The dye-binding step determines if the increase in ionic permeability is due to physical destruction of the stratum corneum. The TER method utilising rat skin has shown to be predictive of in vivo corrosivity in the rabbit assessed under Testing Method B.4 (2). It should be noted that the in vivo rabbit test is highly conservative with respect to skin corrosivity and skin irritation when compared with the human skin patch test (15).

1.5. DESCRIPTION OF THE TEST METHOD

1.5.1. Animals

Rats are the species of choice because the sensitivity of their skin to chemicals in this test has been previously demonstrated (10). The age (when the skin is collected) and strain of the rat is particularly important to ensure that the hair follicles are in the dormant phase before adult hair growth begins.

The dorsal and flank hair from young, approximately 22 day-old, male or female rats (Wistar-derived or a comparable strain), is carefully removed with small clippers. Then, the animals are washed by careful wiping, whilst submerging the clipped area in antibiotic solution (containing, for example, streptomycin, penicillin, chloramphenicol, and amphotericin, at concentrations effective in inhibiting bacterial growth). Animals are washed with antibiotics again on the third or fourth day after the first wash and are used within three days of the second wash, when the stratum corneum has recovered from the hair removal.

1.5.2. Preparation of the skin discs

Animals are humanely killed when 28-30 days old; this age is critical. The dorso-lateral skin of each animal is then removed and stripped of excess subcutaneous fat by carefully peeling it away from the skin. Skin discs, with a diameter of approximately 20 mm each, are removed. The skin may be stored before disks are used where it is shown that positive and negative control data are equivalent to that obtained with fresh skin.

Each skin disc is placed over one of the ends of a PTFE (polytetrafluoroethylene) tube, ensuring that the epidermal surface is in contact with the tube. A rubber "O" ring is press-fitted over the end of the tube to hold the skin in place and excess tissue is trimmed away. Tube and "O" ring dimensions are shown in Figure 2. The rubber "O" ring is then carefully sealed to the end of the PTFE tube with petroleum jelly. The tube is supported by a spring clip inside a receptor chamber containing MgSO4 solution (154 mM) (Figure 1). The skin disc should be fully submerged in the MgSO4 solution. As many as 10-15 skin discs can be obtained from a single rat skin.

Before testing begins, the electrical resistance of two skin discs is measured as a quality control procedure for each animal skin. Both discs should give resistance values greater than 10 kΩ for the remainder of the discs to be used for the test. If the resistance value is less than 10 kΩ, the remaining discs from that skin should be discarded.

1.5.3. Application of the test and control substances

Concurrent positive and negative controls should be used for each study to ensure adequate performance of the experimental model. Skin discs from a single animal should be used. The suggested positive and negative control substances are 10 M hydrochloric acid and distilled water, respectively.

Liquid test substances (150 μL) are applied uniformly to the epidermal surface inside the tube. When testing solid materials, a sufficient amount of the solid is applied evenly to the disc to ensure that the whole surface of the epidermis is covered. Deionised water (150 μL) is added on top of the solid and the tube is gently agitated. In order to achieve maximum contact with the skin, solids may need to be warmed to 30 oC to melt or soften the test substance, or ground to produce a granular material or powder.

Three skin discs are used for each test and control substance. Test substances are applied for 24 hours at 20-23 oC. The test substance is removed by washing with a jet of tap water at up to 30 oC until no further material can be removed.

1.5.4. TER measurements

The skin impedance is measured as TER is measured by using a low-voltage, alternating current Wheatstone databridge (13). General specifications of the bridge are 1-3 Volt operating voltage, a sinus or rectangular shaped alternating current of 50 – 1000 Hz, and a measuring range of at least 0,1 – 30 kΩ. The databridge used in the validation study measureds inductance, capacitance and resistance up to values of 2000 H, 2000 μF, and 2 MΩ, respectively at frequencies of 100 Hz or 1 kHz, using series or parallel values. For the purposes of the TER corrosivity assay measurements are recorded in resistance, at a frequency of 100 Hz and using series values. Prior to measuring the electrical resistance, the surface tension of the skin is reduced by adding a sufficient volume of 70 % ethanol to cover the epidermis. After a few seconds, the ethanol is removed from the tube and the tissue is then hydrated by the addition of 3 mL MgSO4 solution (154 mM). The databridge electrodes are placed on either side of the skin disc to measure the resistance in kΩ/skin disc (Figure 1). Electrode dimensions and the length of the electrode exposed below the crocodile clips are shown in Figure 2. The clip attached to the inner electrode is rested on the top of the PTFE tube during resistance measurement to ensure that a consistent length of electrode is submerged in the MgSO4 solution. The outer electrode is positioned inside the receptor chamber so that it rests on the bottom of the chamber. The distance between the spring clip and the bottom of the PTFE tube is maintained as a constant (Figure 2), because this distance affects the resistance value obtained. Consequently, the distance between the inner electrode and the skin disc should be constant and minimal (1-2 mm).

If the measured resistance value is greater than 20 kΩ, this may be due to the remains of the test substance coating the epidermal surface of the skin disc. Further removal of this coating can be attempted, for example, by sealing the PTFE tube with a gloved thumb and shaking it for approximately 10 seconds; the MgSO4 solution is discarded and the resistance measurement is repeated with fresh MgSO4.

The properties and dimensions of the test apparatus and the experimental procedure used may influence the TER values obtained. The 5 kΩ corrosive threshold was developed from data obtained with the specific apparatus and procedure described in this method. Different threshold and control values may apply if the test conditions are altered or a different apparatus is used. Therefore, it is necessary to calibrate the methodology and resistance threshold values by testing a series of reference standards chosen from the chemicals used in the validation study (4)(5), or from similar chemical classes to the chemicals being investigated. A set of suitable reference chemicals is shown in Table 1.

1.5.5. Dye binding methods

Exposure of certain non-corrosive materials can result in a reduction of resistance below the cut-off of 5 kΩ allowing the passage of ions through the stratum corneum, thereby reducing the electrical resistance (5). For example, neutral organics and chemicals that have surface-active properties (including detergents, emulsifiers and other surfactants) can remove skin lipids making the barrier more permeable to ions. Thus, if the TER values of test substances are less than or around 5 kΩ in the absence of visual damage, an assessment of dye penetration should be carried out on the control and treated tissues to determine if the TER values obtained were the result of increased skin permeability, or skin corrosion (3)(5). In case of the latter where the stratum corneum is disrupted, the dye sulforhodamine B, when applied to the skin surface rapidly penetrates and stains the underlying tissue. This particular dye is stable to a wide range of chemicals and is not affected by the extraction procedure described below.

1.5.5.1. Sulforhodamine B dye application and removal

Following TER assessment, the magnesium sulfate is discarded from the tube and the skin is carefully examined for obvious damage. If there is no obvious major damage, Sulforhodamine B dye (Acid Red 52; C.I. 45100; EINECS Number 222-529-8; CAS number 3520-42-1), 150 μL of a 10 % (w/v) dilution in distilled water, is applied to the epidermal surface of each skin disc for two hours. These skin discs are then washed with tap water at up to room temperature for approximately 10 seconds to remove any excess/unbound dye. Each skin disc is carefully removed from the PTFE tube and placed in a vial (e.g. a 20 mL glass scintillation vial) containing deionised water (8 mL). The vials are agitated gently for five minutes to remove any additional unbound dye. This rinsing procedure is then repeated, after which the skin discs are removed and placed into vials containing 5 ml of 30 % (w/v) sodium dodecyl sulphate (SDS) in distilled water and are incubated overnight at 60 oC.

After incubation, each skin disc is removed and discarded and the remaining solution is centrifuged for eight minutes at 21 oC (relative centrifugal force ~175 × g). A 1 ml sample of the supernatant is diluted 1 in 5 (v/v) [i.e. 1 mL + 4 mL] with 30 % (w/v) SDS in distilled water. The optical density (OD) of the solution is measured at 565 nm.

1.5.5.2. Calculation of dye content

The sulforhodamine B dye content per disc is calculated from the OD values (5) (sulforhodamine B dye molar extinction coefficient at 565 nm = 8,7 × 104; molecular weight = 580). The dye content is determined for each skin disc by the use of an appropriate calibration curve and a mean dye content is then calculated for the replicates.

2. DATA

Resistance values (kΩ) and mean dye content values (μg/disc), where appropriate, for the test material, as well as for positive and negative controls should be reported in tabular form (individual trial data and means ± S.D.), including data for replicates/repeat experiments, mean and individual values.

2.1. INTERPRETATION OF RESULTS

The mean TER results are accepted if the concurrent positive and negative control values fall within the acceptable ranges for the method in the testing laboratory. The acceptable resistance ranges for the methodology and apparatus described above are given in the following table:

Control | Substance | Resistance range (kΩ) |

Positive | 10M Hydrochloric acid | 0,5-1,0 |

Negative | Distilled water | 10-25 |

The mean dye binding results are accepted on condition that concurrent control values fall within the acceptable ranges for the method. Suggested acceptable dye content ranges for the control substances for the methodology and apparatus described above are given below:

Control | Substance | Dye content range (μg/disc) |

Positive | 10M Hydrochloric acid | 40-100 |

Negative | Distilled water | 15-35 |

The test substance is considered to be non-corrosive to skin:

(i) if the mean TER value obtained for the test substance is greater than 5 kΩ; or

(ii) the mean TER value is less than or equal to 5 kΩ; and

- the skin disc is showing no obvious damage, and

- the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.

The test substance is considered to be corrosive to skin:

(i) if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged; or

(ii) the mean TER value is less than or equal to 5 kΩ; and

- the skin disc is showing no obvious damage, but

- the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.

3. REPORTING

3.1. TEST REPORT

The test report must include the following information:

Test and control substances:

- chemical name(s) such as IUPAC or CAS name and CAS number, if known,

- purity and composition of the substance or preparation (in percentage(s) by weight) and physical natur,

- physico-chemical properties such as physical state, pH, stability, water solubility, relevant to the conduct of the study,

- treatment of the test/control substances prior to testing, if applicable (e.g. warming, grinding),

- stability, if known.

Test animals:

- strain and sex used,

- age of the animals when used as donor animals,

- source, housing condition, diet, etc,

- details of the skin preparation.

Test conditions:

- calibration curves for test apparatus,

- calibration curves for dye binding test performance,

- details of the test procedure used for TER measurements,

- details of the test procedure used for the dye binding assessment; if appropriate,

- description of any modification of the test procedures,

- description of evaluation criteria used.

Results:

- tabulation of data from the TER and dye binding assay (if appropriate) for individual animals and individual skin samples;

- description of any effects observed.

Discussion of the results.

Conclusions.

4. REFERENCES

(1) OECD, (2001) Harmonised Integrated Classification System for Human Health and Environmental Hazards of Chemical Substances and Mixtures. OECD Series on Testing and Assessment Number 33. ENV/JM/MONO(2001)6, Paris. http://www.olis.oecd.org/olis/2001doc.nsf/LinkTo/env-jm-mono(2001)6.

(2) Testing Method B.4. Acute Toxicity: Dermal Irritation/Corrosion.

(3) Botham, P.A., Chamberlain, M., Barratt, M.D., Curren, R.D., Esdaile, D.J., Gardner, J.R., Gordon, V.C., Hildebrand, B., Lewis, R.W., Liebsch, M., Logemann, P., Osborne, R., Ponec, M., Regnier, J.F., Steiling, W., Walker, A.P., and Balls, M., (1995) A prevalidation study on in vitro skin corrosivity testing. The report and recommendations of ECVAM Workshop 6. ATLA 23, p. 219-255.

(4) Barratt, M.D., Brantom, P.G., Fentem, J.H., Gerner, I., Walker, A.P., and Worth, A.P. (1998). The ECVAM international validation study on in vitro tests for skin corrosivity. 1. Selection and distribution of the test chemicals. Toxicology. In Vitro 12, p. 471-482.

(5) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Holzhütter, H.-G., and Liebsch, M., (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology. In Vitro 12, p. 483- 524.

(6) OECD, (1996) Final Report of the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods, p. 62.

(7) Balls, M., Blaauboer, B.J., Fentem. J.H., Bruner. L., Combes, R.D., Ekwall, B., Fielder. R.J., Guillouzo, A., Lewis, R.W., Lovell, D.P., Reinhardt, C.A., Repetto, G., Sladowski. D., Spielmann, H., and Zucco, F., (1995) Practical aspects of the validation of toxicity test procedures. The report and recommendations of ECVAM workshops. ATLA 23, p. 129-147.

(8) ICCVAM, (Interagency Coordinating Committee on the Validation of Alternative Methods)., (1997) Validation and Regulatory Acceptance of Toxicological Test Methods. NIH Publication No 97-3981. National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA. http://iccvam.niehs.nih.gov/docs/guidelines/validate.pdf.

(9) ECVAM, (1998) ECVAM News & Views. ATLA 26, 275-280.

(10) ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods)., (2002) ICCVAM evaluation of EpiDermTM, EPISKINTM (EPI-200), and the Rat Skin Transcutaneous Electrical Resistance (TER) assay: In Vitro test methods for assessing dermal corrosivity potential of chemicals. NIH Publication No 02-4502. National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA. http://iccvam.niehs.nih.gov/methods/epiddocs/epis_brd.pdf.

(11) OECD (2002) Extended Expert Consultation Meeting on The In Vitro Skin Corrosion Test Guideline Proposal, Berlin, 1st–2nd November 2001, Secretariat's Final Summary Report, 27th March 2002, OECD ENV/EHS, available upon request from the Secretariat.

(12) Oliver, G.J.A., Pemberton, M.A., and Rhodes, C., (1986) An in vitro skin corrosivity test -modificationsand validation. Fd. Chem. Toxicol. 24, 507-512.

(13) Botham, P.A., Hall, T.J., Dennett, R., McCall, J.C., Basketter, D.A., Whittle, E., Cheeseman, M., Esdaile, D.J., and Gardner, J., (1992) The skin corrosivity test in vitro: results of an interlaboratory trial. Toxic. In Vitro 6, p. 191-194.

(14) Worth A.P., Fentem J.H., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile D.J., Liebsch, M., (1998) An Evaluation of the Proposed OECD Testing Strategy for Skin Corrosion. ATLA 26, p. 709-720

(15) Basketter, D.A., Chamberlain, M., Griffiths, H.A., Rowson, M., Whittle, E., York, M., (1997) The classification of skin irritants by human patch test. Fd. Chem. Toxicol. 35, p. 845-852.

(16) Oliver G.J.A, Pemberton M.A and Rhodes C., (1988) An In Vitro model for identifying skin-corrosive chemicals. I. Initial Validation. Toxicology In Vitro. 2, p. 7-17.

Figure 1

Apparatus for the rat skin TER assay

inner (thick) electrode

crocodile clip

outer (thin) electrode

PTFE tube

crocodile clip

spring clip

receptor chamber (disposable tube)

magnesium sulphate (154 mM)

epidermis of skin disc

dermis of skin disc

rubber 'O' ring

magnesium sulphate (154 mM)

+++++ TIFF +++++

Figure 2

Dimensions of the polytetrafluoroethylene (PFTE) and receptor tubes and electrodes used

Crocodile clip

+++++ TIFF +++++

Critical factors of the apparatus shown above:

- the inner diameter of the PTFE tube,

- the length of the electrodes relative to the PTFE tube and receptor tube, such that the skin disc is not touched by the electrodes and that a standard length of electrode is in contact with the MgSO4 solution,

- the amount of MgSO4 solution in the receptor tube should give a depth of liquid, relative to the level in the PFTE tube, as shown in Figure 1,

- the skin disk should be fixed well enough to the PFTE tube, such that the electrical resistance is a true measure of the skin properties.

B.40 BIS. IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST

1. METHOD

This testing method is equivalent to the OECD TG 431 (2004).

1.1. INTRODUCTION

Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test material [as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)] (1). This Testing Method does not require the use of live animals or animal tissue for the assessment of skin Corrosivity.

The assessment of skin corrosivity has typically involved the use of laboratory animals (2). Concern for the pain and suffering involved with this procedure has been addressed in the revision of testing method B.4 that allows for the determination of skin corrosion by using alternative, in vitro, methods, avoiding pain and suffering of animals.

A first step towards defining alternative tests that could be used for skin corrosivity testing for regulatory purposes was the conduct of prevalidation studies (3). Following this, a formal, validation study of in vitro methods for assessing skin corrosion (4)(5) was conducted (6)(7)(8). The outcome of these studies and other published literature (9) led to the recommendation that the following tests could be used for the assessment of the in vivo skin corrosivity (10)(11)12)(13): the human skin model test (this method) and the transcutaneous electrical resistance test (see testing method B.40).

Validation studies have reported that tests employing human skin models (3)(4)(5)(9) are able to reliably discriminate between known skin corrosives and non-corrosives. The test protocol may also provide an indication of the distinction between severe and less severe skin corrosives.

The test described in this method allows the identification of corrosive chemical substances and mixtures. It further allows the identification of non-corrosive substances and mixtures when supported by a weight of evidence determination using other existing information (e.g. pH, structure-activity relationships, human and/or animal data) (1)(2)(13)(14). It does not normally provide adequate information on skin irritation, nor does it allow the subcategorisation of corrosive substances as permitted in the Globally Harmonised Classification System (GHS) (1).

For a full evaluation of local skin effects after single dermal exposure, it is recommended to follow the sequential testing strategy as appended to testing method B.4 (2) and provided in the Globally Harmonised System (GHS) (1). This testing strategy includes the conduct of in vitro tests for skin corrosion (as described in this method) and skin irritation before considering testing in live animals.

1.2. DEFINITIONS

Cell viability: parameter measuring total activity of a cell population (e.g. ability of cellular mitochondrial dehidrogenases to reduce the vital dye MTT), which, depending on the end point measured and the test design used, correlates with the total number and/or vitality of the cells.

1.3. REFERENCE SUBSTANCES

Table 1

Reference chemicals

Name | EINECS No | CAS No | |

1,2-Diaminopropane | 201-155-9 | 78-90-0 | Severely corrosive |

Acrylic Acid | 201-177-9 | 79-10-7 | Severely corrosive |

2-tert. Butylphenol | 201-807-2 | 88-18-6 | Corrosive |

Potassium hydroxide (10 %) | 215-181-3 | 1310-58-3 | Corrosive |

Sulfuric acid (10 %) | 231-639-5 | 7664-93-9 | Corrosive |

Octanoic acid (caprylic acid) | 204-677-5 | 124-07-02 | Corrosive |

4-Amino-1,2,4-triazole | 209-533-5 | 584-13-4 | Not corrosive |

Eugenol | 202-589-1 | 97-53-0 | Not corrosive |

Phenethyl bromide | 203-130-8 | 103-63-9 | Not corrosive |

Tetrachloroethylene | 204-825-9 | 27-18-4 | Not Corrosive |

Isostearic acid | 250-178-0 | 30399-84-9 | Not corrosive |

4-(Methylthio)-benzaldehyde | 222-365-7 | 3446-89-7 | Not corrosive |

Most of the chemicals listed are taken from the list of chemicals selected for the ECVAM international validation study (4). Their selection is based on the following criteria:

(i) equal number of corrosive and non-corrosive substances;

(ii) commercially available substances covering most of the relevant chemical classes;

(iii) inclusion of severely corrosive as well as less corrosive substances in order to enable discrimination based on corrosive potency;

(iv) choice of chemicals that can be handled in a laboratory without posing other serious hazards than corrosivity.

1.4. PRINCIPLE OF THE TEST METHOD

The test material is applied topically to a three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum. Corrosive materials are identified by their ability to produce a decrease in cell viability (as determined, for example, by using the MTT reduction assay (15)) below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.

1.4.1. Procedure

1.4.1.1. Human skin models

Human skin models can be constructed or obtained commercially (e.g. the EpiDermTM and EPISKINTM models) (16)(17)(18)(19) or be developed or constructed in the testing laboratory (20)(21). It is recognised that the use of human skin is subject to national and international ethical considerations and conditions. Any new model should be validated (at least to the extent described under 1.4.1.1.2). Human skin models used for this test must comply with the following:

1.4.1.1.1. General model conditions:

Human keratinocytes should be used to construct the epithelium. Multiple layers of viable epithelial cells should be present under a functional stratum corneum. The skin model may also have a stromal component layer. Stratum corneum should be multi-layered with the necessary lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic markers. The containment properties of the model should prevent passage of material around the stratum corneum to the viable tissue. Passage of test chemicals around the stratum corneum will lead to poor modeling of the exposure to skin. The skin model should be free of contamination with bacteria (including mycoplasma) or fungi.

1.4.1.1.2. Functional model conditions:

The magnitude of viability is usually quantified by using MTT or the other metabolically converted vital dyes. In these cases the optical density (OD) of the extracted (solubilised) dye from the negative control tissue should be at least 20 fold greater than the OD of the extraction solvent alone (for an overview, see (22)). The negative control tissue should be stable in culture (provide similar viability measurements) for the duration of the test exposure period. The stratum corneum should be sufficiently robust to resist the rapid penetration of certain cytotoxic marker chemicals (e.g. 1 % Triton X-100). This property can be estimated by the exposure time required to reduce cell viability by 50 % (ET50) (e.g. for the EpiDermTM and EPISKINTM models this is > 2 hours). The tissue should demonstrate reproductivity over time and preferably between laboratories. Moreover it should be capable of predicting the corrosive potential of the reference chemicals (see Table 1) when used in the testing protocol selected.

1.4.1.2. Application of the test and control substances

Two tissue replicates are used for each treatment (exposure time), including controls. For liquid materials, sufficient test substance must be applied to uniformly cover the skin surface: a minimum of 25 μL/cm2 should be used. For solid materials, sufficient test substance must be applied evenly to cover the skin, and it should be moistened with deionised or distilled water to ensure good contact with the skin. Where appropriate, solids should be ground to a powder before application. The application method should be appropriate for the test substance (see e.g. reference 5). At the end of the exposure period, the test material must be carefully washed from the skin surface with an appropriate buffer, or0,9 % NaCl.

Concurrent positive and negative controls should be used for each study to ensure adequate performance of the experimental model. The suggested positive control substances are glacial acetic acid or 8N KOH. The suggested negative controls are 0,9 % NaCl or water.

1.4.1.3. Cell viability measurements

Only quantitative, validated, methods can be used to measure cell viability. Furthermore, the measure of viability must be compatible with use in a three-dimensional tissue construct. Non-specific dye binding must not interfere with the viability measurement. Protein binding dyes and those which do not undergo metabolic conversion (e.g. neutral red) are therefore not appropriate. The most frequently used assay is MTT (3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue: EINECS number 206-069-5, CAS number 298-93-1)) reduction, which has been shown to give accurate and reproducible results (5) but others may be used. The skin sample is placed in an MTT solution of appropriate concentration (e.g. 0,3-1 mg/mL) at appropriate incubation temperature for three hours. The precipitated blue formazan product is then extracted using a solvent (isopropanol), and the concentration of the formazan is measured by determining the OD at wavelength between 540 and 595 nm.

Chemical action by the test material on the vital dye may mimic that of cellular metabolism leading to a false estimate of viability. This has been shown to happen when such a test material is not completely removed from the skin by rinsing (9). If the test material directly acts on the vital dye, additional controls should be used to detect and correct for the test substances interference with the viability measurement (9)(23).

2. DATA

For each tissue, OD values and calculated percentage cell viability data for the test material, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.

2.1. INTERPRETATION OF RESULTS

The OD values obtained for each test sample can be used to calculate a percentage viability relative to the negative control, which is arbitrarily set at 100 %. The cut-off percentage cell viability value distinguishing corrosive from non-corrosive test materials (or discriminating between different, corrosive classes), or the statistical procedure(s) used to evaluate the results and identify corrosive materials, must be clearly defined and documented, and be shown to be appropriate. In general, these cut-off values are established during test optimisation, tested during a prevalidation phase, and confirmed in a validation study. As an example, the prediction of Corrosivity associated with the EpiDermTM model is (9):

The test substance is considered to be corrosive to skin:

(i) if the viability after three minutes exposure is less than 50 %; or

(ii) if the viability after three minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.

The test substance is considered to be non-corrosive to skin:

(i) if the viability after three minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.

3. REPORTING

3.1. TEST REPORT

The test report must include the following information:

Test and control substance:

- chemical names(s) such as IUPAC or CAS name and CAS number, if known,

- purity and composition of the substance or preparation (in percentage(s) by weight),

- physico-chemical properties such as physical state, pH, stability, water solubility relevant to the conduct of the study,

- treatment of the test/control substances prior to testing, if applicable (e.g. warming, grinding),

- stability, if known.

Justification of the skin model and protocol used.

Test conditions:

- cell system used,

- calibration information for measuring device used for measuring cell viability (e.g. Spectrophotometer),

- complete supporting information for the specific skin model used including its validity,

- details of the test procedure used,

- test doses used,

- description of any modifications of the test procedure,

- reference to historical data of the model,

- description of evaluation criteria used.

Results:

- tabulation of data from individual test samples,

- description of other effects observed.

Discussion of the results.

Conclusion.

4. REFERENCES

(1) OECD (2001) Harmonised Integrated Classification System for Human Health and Environmental Hazards of Chemical Substances and Mixtures. OECD Series on Testing and Assessment Number 33. ENV/JM/MONO(2001)6, Paris. http://www.olis.oecd.org/olis/2001doc.nsf/LinkTo/env-jm-mono(2001)6.

(2) Testing Method B.4. Acute Toxicity: Dermal Irritation/Corrosion.

(3) Botham, P.A., Chamberlain, M., Barratt, M.D., Curren, R.D., Esdaile, D.J., Gardner, J.R., Gordon, V.C., Hildebrand, B., Lewis, R.W., Liebsch, M., Logemann, P., Osborne, R., Ponec, M., Regnier, J.F., Steiling, W., Walker, A.P., and Balls, M., (1995) A prevalidation study on in vitro skin corrosivity testing. The report recommendations of ECVAM Workshop 6 ATLA 23, p. 219-255.

(5) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Holzhutter, H.G. and Liebsch, M., (1998) The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology In Vitro 12, p. 483-524.

(6) OECD, (1996) Final Report of the OECD Workshop on Harmonisation of Validation and Acceptance Criteria for Alternative Toxicological Test Methods, p. 62.

(7) Balls, M., Blaauboer, B.J., Fentem, J.H., Bruner, L., Combes, R.D., Ekwall, B., Fielder, R.J., Guillouzo, A., Lewis, R.W., Lovell, D.P., Reinhardt, C.A., Repetto, G., Sladowski, D., Spielmann, H., and Zucco, F., (1995) Practical aspects of the validation of toxicity test procedures. Test report and recommendations of ECVAM workshops. ATLA 23, p. 129-147.

(8) ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods)., (1997) Validation and Regulatory Acceptance of Toxicological Test Methods. NIH Publication No 97-3981. National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA. http://iccvam.niehs.nih.gov/docs/guidelines/validate.pdf.

(9) Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Uphill, P., Wilkins, S., McPherson, J.P., Wiemann, C., Kaufmann, T., Remmele, M. and Holzhutter, H.G., (2000) The ECVAM prevalidation study on the use of EpiDerm for skin Corrosivity testing. ATLA 28, p. 371-401.

(10) ECVAM, (1998) ECVAM News & Views. ATLA 26, p. 275-280.

(11) ECVAM, (2000) ECVAM News & Views. ATLA 28, p. 365-67.

(12) ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods)., (2002) ICCVAM evaluation of EpiDermTM, EPISKINTM (EPI-200), and the Rat Skin Transcutaneous Electrical Resistance (TER) assay: In Vitro test methods for assessing dermal corrosivity potential of chemicals. NIH Publication No 02-4502. National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA. http://iccvam.niehs.nih.gov/methods/epiddocs/epis_brd.pdf.

(13) OECD, (2002) Extended Expert Consultation Meeting on The In Vitro Skin Corrosion Test Guideline Proposal, Berlin, 1st–2nd November 2001, Secretariat's Final Summary Report, 27th March 2002, OECD ENV/EHS, available upon request from the Secretariat

(14) Worth, A.P., Fentem, J.H., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Liebsch, M., (1998) An Evaluation of the Proposed OECD Testing Strategy for Skin Corrosion. ATLA 26, p. 709-720.

(15) Mosmann, T., (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity asssays. J.Immunol. Meth. 65, p. 55-63.

(16) Cannon, C.L., Neal, P.J., Southee, J.A., Kubilus, J., and Klausner, M., 1994. New epidermal model for dermal irritancy testing. Toxicology In Vitro 8, p. 889-891.

(17) Ponec, M., Boelsma, E., Weerheim, A., Mulder, A., Boutwstra, J., and Mommaas, M., 2000. Lipid and ultrastructural characterisation of reconstructed skin models. International Journal of Pharmaceutics. 203, p. 211-225.

(18) Tinois, E., Gaetani, Q., Gayraud, B., Dupont, D., Rougier, A., Pouradier, D.X., (1994) The Episkin model: Successful reconstruction of human epidermis in vitro. In In vitro Skin Toxicology. Edited by A Rougier, AM Goldberg and HI Maibach, p. 133-140

(19) Tinois E, Tiollier J, Gaucherand M, Dumas H, Tardy M, Thivolet J (1991). In vitro and post–transplantation differentiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute. Experimental Cell Research 193, p. 310-319

(20) Parentau, N.L., Bilbo, P., Molte, C.J., Mason, V.S., and Rosenberg, H., (1992) The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function. Cytotechnology 9, p. 163-171.

(21) Wilkins, L.M., Watson, S.R., Prosky, S.J., Meunier, S.F., Parentau, N.L., (1994) Development of a bilayered living skin construct for clinical applications. Biotechnology and Bioengineering 43/8, p. 747-756.

(22) Marshall, N.J., Goodwin, C.J., Holt, S.J., (1995) A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. Growth Regulation 5, p. 69-84.

(23) Fentem, J.H., Briggs, D., Chesne’, C., Elliot, G.R., Harbell, J.W., Heylings, J.R., Portes, P., Rouget, R., and van de Sandt, J.J.M., and Botham, P.A., (2001) A prevalidation study on in vitro tests for acute skin irritation: results and evaluation by the Management Team. Toxicology In Vitro 15, p. 57-93.

B.41. IN VITRO 3T3 NRU PHOTOTOXICITY TEST

1. METHOD

This method is equivalent to OECD TG 432 (2004).

1.1. INTRODUCTION

Phototoxicity is defined as a toxic response from a substance applied to the body which is either elicited or increased (apparent at lower dose levels) after subsequent exposure to light, or that is induced by skin irradiation after systemic administration of a substance.

The in vitro 3T3 NRU phototoxicity test is used to identify the phototoxic potential of a test substance induced by the excited chemical after exposure to light. The test evaluates photo-cytotoxicity by the relative reduction in viability of cells exposed to the chemical in the presence versus absence of light. Substances identified by this test are likely to be phototoxic in vivo following systemic application and distribution to the skin, or after topical application.

Many types of chemicals have been reported to induce phototoxic effects (1)(2)(3)(4). Their common feature is their ability to absorb light energy within the sunlight range. According to the first law of photochemistry (Grotthaus-Draper Law), photoreaction requires sufficient absorption of light quanta. Thus, before biological testing is considered, a UV/vis absorption spectrum of the test chemical must be determined according to OECD Test Guideline 101. It has been suggested that if the molar extinction/absorption coefficient is less than 10 litre × mol-1 × cm-1 the chemical is unlikely to be photoreactive. Such chemical may not need to be tested in the in vitro 3T3 NRU phototoxicity test or any other biological test for adverse photochemical effects (1)(5). See also Appendix 1.

The reliability and relevance of the in vitro 3T3 NRU phototoxicity test was recently evaluated (6)(7)(8) (9). The in vitro 3T3 NRU phototoxicity test was shown to be predictive of acute phototoxicity effects in animals and humans in vivo. The test is not designed to predict other adverse effects that may arise from combined action of a chemical and light, e.g. it does not address photogenotoxicity, photoallergy, or photocarcinogenicity, nor does it allow an assessment of phototoxic potency. In addition, the test has not been designed to address indirect mechanisms of phototoxicity, effects of metabolites of the test substance, or effects of mixtures.

Whereas the use of metabolising systems is a general requirement for all in vitro tests for the prediction of genotoxic and carcinogenic potential, up to now, in the case of phototoxicology, there are only rare examples where metabolic transformation is needed for the chemical to act as a phototoxin in vivo or in vitro. Thus, it is neither considered necessary nor scientifically justified for the present test to be performed with a metabolic activation system.

1.2. DEFINITIONS

Irradiance: the intensity of ultraviolet (UV) or visible light incident on a surface, measured in W/m2 or mW/cm2.

Dose of light: the quantity (= intensity × time) of ultraviolet (UV) or visible radiation incident on a surface, expressed in Joules (= W × s) per surface area, e.g. J/m2 or J/cm2.

UV light wavebands: the designations recommended by the CIE (Commission Internationale de L'Eclairage) are: UVA (315-400 nm), UVB (280-315 nm) and UVC (100-280 nm). Other designations are also used; the division between UVB and UVA is often placed at 320 nm, and the UVA may be divided into UV-A1 and UV-A2 with a division made at about 340 nm.

Cell viability: parameter measuring total activity of a cell population (e.g. uptake of the vital dye Neutral Red into cellular lysosomes), which, depending on the endpoint measured and the test design used, correlates with the total number and/or vitality of the cells.

Relative cell viability: cell viability expressed in relation of solvent (negative) controls which have been taken through the whole test procedure (either +Irr or -Irr) but not treated with test chemical.

PIF (Photo-Irritation-Factor): factor generated by comparing two equally effective cytotoxic concentrations (IC50) of the test chemical obtained in the absence (-Irr) and in the presence (+Irr) of a non-cytotoxic irradiation with UVA/vis light.

IC50: the concentration of the test chemical by which the cell viability is reduced by 50 %.

MPE (Mean-Photo-Effect): measurement derived from mathematical analysis of the concentration response curves obtained in the absence (-Irr) and in the presence (+Irr) of a non-cytotoxic irradiation with UVA/vis light.

Phototoxicity: acute toxic response that is elicited after the first exposure of skin to certain chemicals and subsequent exposure to light, or that is induced similarly by skin irradiation after systemic administration of a chemical.

1.3. PRINCIPLE OF THE TEST METHOD

The in vitro 3T3 NRU phototoxicity test is based on a comparison of the cytotoxicity of a chemical when tested in the presence and in the absence of exposure to a non-cytotoxic dose of simulated solar light. Cytotoxicity in this test is expressed as a concentration-dependent reduction of the uptake of the vital dye Neutral Red when measured 24 hours after treatment with the test chemical and irradiation (10). NR is a weak cationic dye that readily penetrates cell membranes by non-diffusion, accumulating intracellulary in lysosomes. Alterations of the surface of the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes brought about by the action of xenobiotics result in a decreased uptake and binding of NR. It is thus possible to distinguish between viable, damaged or dead cells, which is the basis of this test.

Balb/c 3T3 cells are maintained in culture for 24 h for formation of monolayers. Two 96-well plates per test chemical are pre-incubated with eight different concentrations of the test substance for 1 h. Thereafter one of the two plates is exposed to the highest non-cytotoxic irradiation dose whereas the other plate is kept in the dark. In both plates the treatment medium is then replaced by culture medium and after another 24 h of incubation cell viability is determined by Neutral Red uptake. Cell viability is expressed as percentage of untreated solvent controls and is calculated for each test concentration. To predict the phototoxic potential, the concentration responses obtained in the presence and in the absence of irradiation are compared, usually at the IC50 level, i.e., the concentration reducing cell viability to 50 % compared to the untreated controls.

1.4. DESCRIPTION OF THE TEST METHOD

1.4.1. Preparations

1.4.1.1. Cells

A permanent mouse fibroblast cell line, Balb/c 3T3, clone 31, either from the American Type Culture Collection (ATCC), Manassas, VA, USA, or from the European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, was used in the validation study, and therefore is recommended to obtain from a well qualified cell depository. Other cells or cell lines may be used with the same test procedure if culture conditions are adapted to the specific needs of the cells, but equivalency must be demonstrated.

Cells should be checked regularly for the absence of mycoplasma contamination and only used if none is found (11).

It is important that UV sensitivity of the cells is checked regularly according to the quality control procedure described in this method. Because the UVA sensitivity of cells may increase with the number of passages, Balb/c 3T3 cells of the lowest obtainable passage number, preferably less than 100, should be used. (See Section 1.4.2.2.2 and Appendix 2).

1.4.1.2. Media and culture conditions

Appropriate culture media and incubation conditions should be used for routine cell passage and during the test procedure, e.g. for Balb/c 3T3 cells these are DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10 % new-born calf serum, 4 mM glutamine, penicillin (100 IU), and streptomycin (100 μg/mL), and humidified incubation at 37 oC, 5-7,5 % CO2 depending on the buffer (See Section 1.4.1.4, second paragraph.). It is particularly important that cell culture conditions assure a cell cycle time within the normal historical range of the cells or cell line used.

1.4.1.3. Preparation of cultures

Cells from frozen stock cultures are seeded in culture medium at an appropriate density and subcultured at least once before they are used in the in vitro 3T3 NRU phototoxicity test.

Cells used for the phototoxicity test are seeded in culture medium at the appropriate density so that cultures will not reach confluence by the end of the test, i.e., when cell viability is determined 48 h after seeding of the cells. For Balb/c 3T3 cells grown in 96-well plates, the recommended cell seeding density is 1 × 104 cells per well.

For each test chemical cells are seeded identically in two separate 96-well plates, which are then taken concurrently through the entire test procedure under identical culture conditions except for the time period where one of the plates is irradiated (+Irr) and the other one is kept in the dark (-Irr).

1.4.1.4. Preparation of test substance

Test substances must be prepared fresh, immediately prior to use unless data demonstrate their stability in storage. It is recommended that all chemical handling and the initial treatment of cells be performed under light conditions that would avoid photoactivation or degradation of the test substance prior to irradiation.

Test chemicals shall be dissolved in buffered salt solutions, e.g. Earle's Balanced Salt Solution (EBSS), or other physiologically balanced buffer solutions, which must be free from protein components, light absorbing components (e.g. pH-indicator colours and vitamins) to avoid interference during irradiation. Since during irradiation cells are kept for about 50 minutes outside of the CO2 incubator, care has to be taken to avoid alkalisation. If weak buffers like EBSS are used this can be achieved by incubating the cells at 7,5 % CO2. If the cells are incubated at 5 % CO2 only, a stronger buffer should be selected.

Test chemicals of limited solubility in water should be dissolved in an appropriate solvent. If a solvent is used it must be present at a constant volume in all cultures, i.e. in the negative (solvent) controls as well as in all concentrations of the test chemical, and be noncytotoxic at that concentration. Test chemical concentrations should be selected so as to avoid precipitate or cloudy solutions.

Dimethylsulphoxide (DMSO) and ethanol (ETOH) are the recommended solvents. Other solvents of low cytotoxicity may be appropriate. Prior to use, all solvents should be assessed for specific properties, e.g. reaction with the test chemical, quenching of the phototoxic effect, radical scavenging properties and/or chemical stability in the solvent.

Vortex mixing and/or sonication and/or warming to appropriate temperatures may be used to aid solubilisation unless this would affect the stability of the test chemical.

1.4.1.5. Irradiation conditions

1.4.1.5.1. Light source

The choice of an appropriate light source and filters is a crucial factor in phototoxicity testing. Light of the UVA and visible regions is usually associated with phototoxic reactions in vivo (3)(12), whereas generally UVB is of less relevance but is highly cytotoxic; the cytotoxicity increases 1000-fold as the wavelength goes from 313 to 280 nm (13). Criteria for the choice of an appropriate light source must include the requirement that the light source emits wavelengths absorbed by the test chemical (absorption spectrum) and that the dose of light (achievable in a reasonable exposure time) should be sufficient for the detection of known photocytotoxic chemicals. Furthermore, the wavelengths and doses employed should not be unduly deleterious to the test system, e.g. the emission of heat (infrared region).

Simulation of sunlight with solar simulators is considered the optimal artificial light source. The irradiation power distribution of the filtered solar simulator should be close to that of outdoor daylight given in (14). Both, Xenon arcs and (doped) mercury-metal halide arcs are used as solar simulators (15). The latter has the advantage of emitting less heat and being cheaper, but the match to sunlight is less perfect compared to that of xenon arcs. Because all solar simulators emit significant quantities of UVB they should be suitably filtered to attenuate the highly cytotoxic UVB wavelengths. Because cell culture plastic materials contain UV stabilisers the spectrum should be measured through the same type of 96-well plate lid as will be used in the assay. Irrespective of measures taken to attenuate parts of the spectrum by filtering or by unavoidable filter effects of the equipment the spectrum recorded below these filters should not deviate from standardised outdoor daylight (14). An example of the spectral irradiance distribution of the filtered solar simulator used in the validation study of the in vitro 3T3 NRU phototoxicity test is given in (8)(16). See also Appendix 2 Figure 1.

1.4.1.5.2. Dosimetry

The intensity of light (irradiance) should be regularly checked before each phototoxicity test using a suitable broadband UV-meter. The intensity should be measured through the same type of 96-well plate lid as will be used in the assay. The UV-meter must have been calibrated to the source. The performance of the UV-meter should be checked, and for this purpose the use of a second, reference UV-meter of the same type and identical calibration is recommended. Ideally, at greater intervals, a spectroradiometer should be used to measure the spectral irradiance of the filtered light source and to check the calibration of the broadband UV-meter.

A dose of 5 J/cm2 (as measured in the UVA range) was determined to be non-cytotoxic to Balb/c 3T3 cells and sufficiently potent to excite chemicals to elicit phototoxic reactions, (6) (17) e.g. to achieve 5 J/cm2 within a time period of 50 min, irradiance was adjusted to 1,7 mW/cm2. See Appendix 2 Figure 2. If another cell line or a different light source are used, the irradiation dose may have to be calibrated so that a dose regimen can be selected that is not deleterious to the cells but sufficient to excite standard phototoxins. The time of light exposure is calculated in the following way:

t(min)=irradiation dose (J/cm2)×1000irradiance (mW/cm2)×60 | (1 J = 1 Wsec) |

1.4.2. Test conditions

1.4.2.1. Test substance concentrations

The ranges of concentrations of a chemical tested in the presence (+Irr) and in the absence (-Irr) of light should be adequately determined in dose range-finding experiments. It may be useful to assess solubility initially and at 60 min (or whatever treatment time is to be used), as solubility can change during time or during the course of exposure. To avoid toxicity induced by improper culture conditions or by highly acidic or alkaline chemicals, the pH of the cell cultures with added test chemical should be in the range 6,5 - 7,8.

The highest concentration of the test substance should be within physiological test conditions, e.g. osmotic and pH stress should be avoided. Depending on the test chemical, it may be necessary to consider other physico-chemical properties as factors limiting the highest test concentration. For relatively insoluble substances that are not toxic at concentrations up to the saturation point the highest achievable concentration should be tested. In general, precipitation of the test chemical at any of the test concentrations should be avoided. The maximum concentration of a test substance should not exceed 1000 μg/mL; osmolarity should not exceed 10 mmolar. A geometric dilution series of eight test substance concentrations with a constant dilution factor should be used (See Section 2.1, second paragraph).

If there is information (from a range finding experiment) that the test chemical is not cytotoxic up to the limit concentration in the dark experiment (-Irr), but is highly cytotoxic when irradiated (+Irr), the concentration ranges to be selected for the (+Irr) experiment may differ from those selected for the (-Irr) experiment to fulfill the requirement of adequate data quality.

1.4.2.2. Controls

1.4.2.2.1. Radiation sensitivity of the cells, establishing of historical data:

Cells should be checked regularly (about every fifth passage) for sensitivity to the light source by assessing their viability following exposure to increasing doses of irradiation. Several doses of irradiation, including levels substantially greater than those used for the 3T3 NRU Phototoxicity test should be used in this assessment. These doses are easiest quantitated by measurements of UV parts of the light source. Cells are seeded at the density used in the in vitro 3T3 NRU phototoxicity test and irradiated the next day. Cell viability is then determined one day later using Neutral Red uptake. It should be demonstrated that the resulting highest non-cytotoxic dose (e.g. in the validation study: 5 J/cm2 [UVA]) was sufficient to classify the reference chemicals (Table 1) correctly.

1.4.2.2.2. Radiation sensitivity, check of current test:

The test meets the quality criteria if the irradiated negative/solvent controls show a viability of more than 80 % when compared with non-irradiated negative/solvent.

1.4.2.2.3. Viability of solvent controls:

The absolute optical density (OD540 NRU) of the Neutral Red extracted from the solvent controls indicates whether the 1×104 cells seeded per well have grown with a normal doubling time during the two days of the assay. A test meets the acceptance criteria if the mean OD540 NRU of the untreated controls is ≥ 0,4 (i.e. approximately 20 times the background solvent absorbance).

1.4.2.2.4. Positive control:

A known phototoxic chemical shall be tested concurrently with each in vitro 3T3 NRU phototoxicity test. Chlorpromazine (CPZ) is recommended. For CPZ tested with the standard protocol in the in vitro 3T3 NRU phototoxicity test, the following test acceptance criteria were defined: CPZ irradiated (+Irr): IC50 = 0,1 to 2,0 μg/ml, CPZ non-irradiated (-Irr): IC50 = 7,0 to 90,0 μg/mL. The Photo Irritation Factor (PIF), should be > 6. The historical performance of the positive control should be monitored.

Other phototoxic chemicals, suitable for the chemical class or solubility characteristics of the chemical being evaluated, may be used as the concurrent positive controls in place of chlorpromazine.

1.4.3. Test procedure (6)(7)(8)(16)(17):

1.4.3.1. 1st day:

Dispense 100 μL culture medium into the peripheral wells of a 96-well tissue culture microtiter plate (= blanks). In the remaining wells, dispense 100 μL of a cell suspension of 1×105 cells/mL in culture medium (= 1×104 cells/well). Two plates should be prepared for each series of individual test substance concentrations, and for the solvent and positive controls.

Incubate cells for 24 h (See Section 1.4.1.2) until they form a half confluent monolayer. This incubation period allows for cell recovery, adherence, and exponential growth.

1.4.3.2. 2nd day:

After incubation, decant culture medium from the cells and wash carefully with 150 μL of the buffered solution used for incubation. Add 100 μL of the buffer containing the appropriate concentration of test chemical or solvent (solvent control). Apply eight different concentrations of the test chemical. Incubate cells with the test substance in the dark for 60 minutes (See Section 1.4.1.2 and 1.4.1.4 second paragraph).

From the two plates prepared for each series of test substance concentrations and the controls, one is selected, generally at random, for the determination of cytotoxicity (-Irr) (i.e., the control plate), and one (the treatment plate) for the determination of photocytotoxicity (+Irr).

To perform the +Irr exposure, irradiate the cells at room temperature for 50 minutes through the lid of the 96-well plate with the highest dose of radiation that is non-cytotoxic (see also Appendix 2). Keep non-irradiated plates (-Irr) at room temperature in a dark box for 50 min (= light exposure time).

Decant test solution and carefully wash twice with 150 μL of the buffered solution used for incubation, but not containing the test material. Replace the buffer with culture medium and incubate (See Section 1.4.1.2.) overnight (18-22 h).

1.4.3.3. 3rd day:

1.4.3.3.1. Microscopic evaluation

Cells should be examined for growth, morphology, and integrity of the monolayer using a phase contrast microscope. Changes in cell morphology and effects on cell growth should be recorded.

1.4.3.3.2. Neutral Red uptake test

Wash the cells with 150 μL of the pre-warmed buffer. Remove the washing solution by gentle tapping. Add 100 μL of a 50 μg/mL Neutral Red (NR) (3-amino-7-dimethylamino-2-methylphenazine hydrochloride, EINECS number 209-035-8; CAS number 553-24-2; C.I. 50040) in medium without serum (16) and incubate as described in paragraph 1.4.1.2., for 3 h. After incubation, remove the NR medium, and wash cells with 150 μL of the buffer. Decant and remove excess buffer by blotting or centrifugation.

Add exactly 150 μL NR desorb solution (freshly prepared 49 parts water + 50 parts ethanol + 1 part acetic acid).

Shake the microtiter plate gently on a microtiter plate shaker for 10 min until NR has been extracted from the cells and has formed a homogeneous solution.

Measure the optical density of the NR extract at 540 nm in a spectrophotometer, using blanks as a reference. Save data in an appropriate electronic file format for subsequent analysis.

2. DATA

2.1. QUALITY AND QUANTITY OF DATA

The test data should allow a meaningful analysis of the concentration-response obtained in the presence and in the absence of irradiation, and if possible the concentration of test chemical by which cell viability is reduced to 50 % (IC50). If cytotoxicity is found, both the concentration range and the intercept of individual concentrations shall be set in a way to allow the fit of a curve to the experimental data.

For both clearly positive and clearly negative results (See Section 2.3, first paragraph), the primary experiment, supported by one or more preliminary dose range-finding experiment(s), may be sufficient.

Equivocal, borderline, or unclear results should be clarified by further testing (see also section 2.4, second paragraph). In such cases, modification of experimental conditions should be considered. Experimental conditions that might be modified include the concentration range or spacing, the pre-incubation time, and the irradiation-exposure time. A shorter exposure time may be appropriate for water-unstable chemicals.

2.2. EVALUATION OF RESULTS

To enable evaluation of the data, a Photo-Irritation-Factor (PIF) or Mean Photo Effect (MPE) may be calculated.

For the calculation of the measures of photocytotoxicity (see below) the set of discrete concentration-response values has to be approximated by an appropriate continuous concentration-response curve (model). Fitting of the curve to the data is commonly performed by a non-linear regression method (18). To assess the influence of data variability on the fitted curve a bootstrap procedure is recommended.

A Photo-Irritation-Factor (PIF) is calculated using the following formula:

PIF=

(-Irr)

(+Irr)

If an IC50 in the presence or absence of light cannot be calculated, a PIF cannot be determined for the test material. The mean photo effect (MPE) is based on comparison of the complete concentration-response curves (19). It is defined as the weighted average across a representative set of photo effect values

MPE=

The photo effect PEc at any concentration C is defined as the product of the response effect REc and the dose effect DEc i.e. PEc = REc × DEc. The response effect REc is the difference between the responses observed in the absence and presence of light, i.e. REc = Rc (-Irr) - Rc (+Irr). The dose-effect is given by

C/C

-1

C/C

where C* represents the equivalence concentration, i.e. the concentration at which the +Irr response equals the –Irr response at concentration C. If C* cannot be determined because the response values of the +Irr curve are systematically higher or lower than RC(-Irr) the dose effect is set to 1. The weighting factors wi are given by the highest response value, i.e. wi = MAX {Ri (+Irr), Ri (-Irr) }. The concentration grid Ci is chosen such that the same number of points falls into each of the concentration intervals defined by the concentration values used in the experiment. The calculation of MPE is restricted to the maximum concentration value at which at least one of the two curves still exhibits a response value of at least 10 %. If this maximum concentration is higher than the highest concentration used in the +Irr experiment the residual part of the +Irr curve is set to the response value "0". Depending on whether the MPE value is larger than a properly chosen cut-off value (MPEc = 0,15) or not, the chemical is classified as phototoxic.

A software package for the calculation of the PIF and MPE is available from (20).

2.3. INTERPRETATION OF RESULTS

Based on the validation study (8), a test substance with a PIF < 2 or an MPE < 0,1 predicts: "no phototoxicity". A PIF > 2 and < 5 or an MPE > 0,1 and < 0,15 predicts: "probable phototoxicity"; and a PIF > 5 or an MPE > 0,15 predicts: "phototoxicity".

For any laboratory initially establishing this assay, the reference materials listed in Table 1 should be tested prior to the testing of test substances for phototoxic assessment. PIF or MPE values should be close to the values mentioned in Table 1.

Table 1

Amiodarone HCL | 243-293-2 | [19774-82-4] | > 3,25 | 0,2-0,54 | 242 nm 300 nm (shoulder) | ethanol |

Choloropromazine HCL | 200-701-3 | [69-09-0] | > 14,4 | 0,33-0,63 | 309 nm | ethanol |

Norfloxacin | 274-614-4 | [70458-96-7] | > 71,6 | 0,34-0,90 | 316 nm | acetonitrile |

Anthracene | 204-371-1 | [120-12-7] | > 18,5 | 0,19-0,81 | 356 nm | acetonitrile |

Protoporphyrin IX, Disodium | 256-815-9 | [50865-01-5] | > 45,3 | 0,54-0,74 | 402 nm | ethanol |

L-Histidine | | [7006-35-1] | no PIF | 0,05-0,10 | 211 nm | water |

Hexacholorophene | 200-733-8 | [70-30-4] | 1,1-1,7 | 0,00-0,05 | 299 nm 317 nm (shoulder) | ethanol |

Sodium lauryl sulphate | 205-788-1 | [151-21-3] | 1,0-1,9 | 0,00-0,05 | no absorption | water |

2.4. INTERPRETATION OF DATA

If phototoxic effects are observed only at the highest test concentration, (especially for water soluble test chemicals) additional considerations may be necessary for assessment of hazard. These may include data on skin absorption, and accumulation of the chemical in the skin and/or data from other tests, e.g. testing of the chemical in in vitro animal or human skin, or skin models.

If no toxicity is demonstrated (+Irr and -Irr), and if poor solubility limited the concentrations that could be tested, then the compatibility of the test substance with the assay may be questioned and confirmatory testing should be considered using, e.g. another model.

3. REPORTING

TEST REPORT

The test report must include at least the following information:

Test substance:

- identification data, common generic names and IUPAC and CAS number, if known,

- physical nature and purity,

- physicochemical properties relevant to conduct of the study,

- UV/vis absorption spectrum,

- stability and photostability, if known.

Solvent:

- justification for choice of solvent,

- solubility of the test chemical in solvent,

- percentage of solvent present in treatment medium.

Cells:

- type and source of cells,

- absence of mycoplasma,

- cell passage number, if known,

- Radiation sensitivity of cells, determined with the irradiation equipment used in the in vitro 3T3 NRU phototoxicity test.

Test conditions (1); incubation before and after treatment:

- type and composition of culture medium,

- incubation conditions (CO2 concentration; temperature; humidity),

- duration of incubation (pre-treatment; post-treatment).

Test conditions (2); treatment with the chemical:

- rationale for selection of concentrations of the test chemical used in the presence and in the absence of irradiation,

- in case of limited solubility of the test chemical and absence of cytotoxicity: rationale for the highest concentration tested,

- type and composition of treatment medium (buffered salt solution),

- duration of the chemical treatment.

Test conditions (3); irradiation:

- rationale for selection of the light source used,

- manufacturer and type of light source and radiometer,

- spectral irradiance characteristics of the light source,

- transmission and absorption characteristics of the filter(s) used,

- characteristics of the radiometer and details on its calibration,

- distance of the light source from the test system,

- UVA irradiance at this distance, expressed in mW/cm2,

- duration of the UV/vis light exposure,

- UVA dose (irradiance × time), expressed in J/cm2,

- temperature of cell cultures during irradiation and cell cultures concurrently kept in the dark.

Test conditions (4); Neutral Red viability test:

- composition of Neutral Red treatment medium,

- duration of Neutral Red incubation,

- incubation conditions (CO2 concentration; temperature; humidity),

- Neutral Red extraction conditions (extractant; duration),

- wavelength used for spectrophotometric reading of Neutral Red optical density,

- second wavelength (reference), if used,

- content of spectrophotometer blank, if used.

Results:

- cell viability obtained at each concentration of the test chemical, expressed in percent viability of mean, concurrent solvent controls,

- concentration response curves (test chemical concentration vs. relative cell viability) obtained in concurrent +Irr and -Irr experiments,

- analysis of the concentration-response curves: if possible, computation/calculation of IC50 (+Irr) and IC50 (-Irr),

- comparison of the two concentration response curves obtained in the presence and in the absence of irradiation, either by calculation of the Photo-Irritation-Factor (PIF), or by calculation of the Mean-Photo-Effect (MPE),

- test acceptance criteria; concurrent solvent control:

- absolute viability (optical density of Neutral Red extract) of irradiated and non-irradiated cells,

- historic negative and solvent control data; means and standard deviations,

- test acceptance criteria; concurrent positive control,

- IC50(+Irr) and IC50(-Irr) and PIF/MPE of positive control chemical,

- historic positive control chemical data: IC50(+Irr) and IC50(-Irr) and PIF/MPE; means and standard deviations.

Discussion of the results.

Conclusions.

4. REFERENCES

(1) Lovell W.W., (1993) A scheme for in vitro screening of substances for photoallergenic potential. Toxicology In Vitro 7, p. 95-102.

(2) Santamaria, L. and Prino, G., (1972) List of the photodynamic substances. In "Research Progress in Organic, Biological and Medicinal Chemistry" Vol. 3 part 1. North Holland Publishing Co. Amsterdam. p. XI-XXXV.

(3) Spielmann, H., Lovell, W.W., Hölzle, E., Johnson, B.E., Maurer, T., Miranda, M.A., Pape, W.J.W., Sapora, O., and Sladowski, D., (1994) In vitro phototoxicity testing: The report and recommendations of ECVAM Workshop 2. ATLA, 22, p. 314-348.

(4) Spikes, J.D., (1989) Photosensitisation. In "The science of Photobiology" Edited by K.C. Smith. Plenum Press, New York. 2nd edition, p. 79-110.

(5) OECD, (1997) Environmental Health and Safety Publications, Series on Testing and Assessment No 7 "Guidance Document On Direct Phototransformation Of Chemicals In Water" Environment Directorate, OECD, Paris.

(6) Spielmann, H., Balls, M., Döring, B., Holzhütter, H.G., Kalweit, S., Klecak, G., L'Eplattenier, H., Liebsch, M., Lovell, W.W., Maurer, T., Moldenhauer. F. Moore. L., Pape, W., Pfannbecker, U., Potthast, J., De Silva, O., Steiling, W., and Willshaw, A., (1994) EEC/COLIPA project on in vitro phototoxicity testing: First results obtained with a Balb/c 3T3 cell phototoxicity assay. Toxic. In Vitro 8, p. 793-796.

(7) Anon, (1998) Statement on the scientific validity of the 3T3 NRU PT test (an in vitro test for phototoxicity), European Commission, Joint Research Centre: ECVAM and DGXI/E/2, 3 November 1997, ATLA, 26, p. 7-8.

(8) Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., Pechovitch, G. De Silva, O., Holzhütter, H.G., Clothier, R., Desolle, P., Gerberick, F., Liebsch, M., Lovell, W.W., Maurer, T., Pfannenbecker, U., Potthast, J. M., Csato, M., Sladowski, D., Steiling, W., and Brantom, P., (1998) The international EU/COLIPA In vitro phototoxicity validation study: results of phase II (blind trial), part 1: the 3T3 NRU phototoxicity test. Toxicology In Vitro 12, p. 305-327.

(9) OECD, (2002) Extended Expert Consultation Meeting on The In Vitro 3T3 NRU Phototoxicity Test Guideline Proposal, Berlin, 30th-31th October 2001, Secretariat's Final Summary Report, 15th March 2002, OECD ENV/EHS, available upon request from the Secretariat.

(10) Borenfreund, E., and Puerner, J.A., (1985) Toxicity determination in vitro by morphological alterations and neutral red absorption. Toxicology Lett., 24, p. 119-124.

(11) Hay, R.J., (1988) The seed stock concept and quality control for cell lines. Analytical Biochemistry 171, p. 225-237.

(12) Lambert L.A, Warner W.G., and Kornhauser A., (1996) Animal models for phototoxicity testing. In "Dermatotoxicology", edited by F.N. Marzulli and H.I. Maibach. Taylor & Francis, Washington DC. 5th Edition, p. 515-530.

(13) Tyrrell R.M., Pidoux M., (1987) Action spectra for human skin cells: estimates of the relative cytotoxicity of the middle ultraviolet, near ultraviolet and violet regions of sunlight on epidermal keratinocytes. Cancer Res., 47, p. 1825-1829.

(14) ISO 10977., (1993) Photography — Processed photographic colour films and paper prints — Methods for measuring image stability.

(15) Sunscreen Testing (UV.B) TECHNICALREPORT, CIE, International Commission on Illumnation, Publication No 90, Vienna, 1993, ISBN 3 900 734 275

(16) ZEBET/ECVAM/COLIPA — Standard Operating Procedure: In Vitro 3T3 NRU Phototoxicity Test. Final Version, 7 September, 1998. p. 18.

(17) Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., De Silva, O., Holzhütter, H.G., Gerberick, F., Liebsch, M., Lovell, W.W., and Pfannenbecker, U., (1998) A study on UV filter chemicals from Annex VII of the European Union Directive 76/768/EEC, in the in vitro 3T3 NRU phototoxicity test. ATLA 26, p. 679-708.

(18) Holzhütter, H.G., and Quedenau, J., (1995) Mathematical modeling of cellular responses to external signals. J. Biol. Systems 3, p. 127-138.

(19) Holzhütter, H.G., (1997) A general measure of in vitro phototoxicity derived from pairs of dose-response curves and its use for predicting the in vivo phototoxicity of chemicals. ATLA, 25, p. 445-462.

(20) http://www.oecd.org/document/55/0,2340,en_2649_34377_2349687_1_1_1_1,00.html

Appendix 1

Role of the 3T3 NRU PT in a sequential approach to the phototoxicity testing of chemicals

Initial Evaluation of the Physical, Chemical, and Toxicological Properties of the Test Substance

Physico-chemical properties

Chemical structure, structural alerts

UV/vis - absorption

QSAR - photochemistry

General toxicity (including kinetics and metabolism)

UV/vis absorption spectra in appropriate solvent (e.g. OECD TG 101)

No Absorption

Phototoxicity Testing not considered necessary

Absorption

In Vitro 3T3 NRU Phototoxicity Test and/or other methods if necessary

+++++ TIFF +++++

Appendix 2

Figure 1

Spectral power distribution of a filtered solar simulator

Irradiance [mW/cm2]

Wavelength [nm]

SOL 500 + H2 Filter

SOL 500 + H1 Filter

SOL 500 + H1 Filter + lid of 96-well plate

+++++ TIFF +++++

(see Section 1.4.1.5, second paragraph)

Figure 1 gives an example of an acceptable spectral irradiance distribution of a filtered solar simulator. It is from the doped metal halide source used in the validation trial of the 3T3 NRU PT (6)(8)(17). The effect of two different filters and the additional filtering effect of the lid of a 96-well cell culture plate are shown. The H2 filter was only used with test systems that can tolerate a higher amount of UVB (skin model test and red blood cell photo-haemolysis test). In the 3T3 NRU-PT the H1 filter was used. The figure shows that additional filtering effect of the plate lid is mainly observed in the UVB range, still leaving enough UVB in the irradiation spectrum to excite chemicals typically absorbing in the UVB range, like Amiodarone (see Table 1).

Figure 2

Irradiation sensivity of Balb/c 3T3 cells (as measured in the UVA range)

Cell viability (% Neutral Red uptake of dark controls)

5 J/cm2

Figure 2

irradiation time (minutes)

10 min = 1 joule UVA/cm2)

+++++ TIFF +++++

(see Sections 1.4.1.5.2 second paragraph; 1.4.2.2.1, 1.4.2.2.2)

Sensitivity of Balb/c 3T3 cells to irradiation with the solar simulator used in the validation trial of the 3T3NRU-phototoxicity test, as measured in the UVA range. Figure shows the results obtained in seven different laboratories in the pre-validation study (1). While the two curves with open symbols were obtained with aged cells (high number of passages), that had to be replaced by new cell stocks the curves with bold symbols show cells with acceptable irradiation tolerance.

From these data the highest non-cytotoxic irradiation dose of 5 J/cm2 was derived (vertical dashed line). The horizontal dashed line shows in addition the maximum acceptable irradiation effect given in paragraph 1.4.2.2.

B.42. SKIN SENSITISATION: LOCAL LYMPH NODE ASSAY

1. METHOD

This test method is equivalent to the OECD TG 429 (2002)

1.1. INTRODUCTION

The Local Lymph Node Assay (LLNA) has been sufficiently validated and accepted to justify its adoption as a new Method (1)(2)(3). This is the second method for assessing skin sensitisation potential of chemicals in animals. The other method (B.6) utilises guinea pig tests, notably the guinea pig maximisation test and the Buehler test (4).

The LLNA provides an alternative method for identifying skin sensitising chemicals and for confirming that chemicals lack a significant potential to cause skin sensitisation. This does not necessarily imply that in all instances the LLNA should be used in place of guinea pig test, but rather that the assay is of equal merit and may be employed as an alternative in which positive and negative results generally no longer require further confirmation.

The LLNA provides certain advantages with regard to both scientific progress and animal welfare. It studies the induction phase of skin sensitisation and provides quantitative data suitable for dose response assessment. The details of the validation of the LLNA and a review of the associated work have been published (5)(6)(7)(8). In addition, it should be noted that the mild/moderate sensitisers, which are recommended as suitable positive control substances for guinea pig test m, are also appropriate for use with the LLNA (6)(8)(9).

The LLNA is an in vivo method and, as a consequence, will not eliminate the use of animals in the assessment of contact sensitising activity. It has, however, the potential to reduce the number of animals required for this purpose. Moreover, the LLNA offers a substantial refinement of the way in which animals are used for contact sensitisation testing. The LLNA is based upon consideration of immunological events stimulated by chemicals during the induction phase of sensitisation. Unlike guinea pig tests the LLNA does not require that challenged-induced dermal hypersensitivity reactions be elicited. Furthermore, the LLNA does not require the use of an adjuvant, as is the case for the guinea pig maximisation test. Thus, the LLNA reduces animal distress. Despite the advantages of the LLNA over traditional guinea pig tests, it should be recognised that there are certain limitations that may necessitate the use of traditional guinea pigs tests (e.g. false negative findings in the LLNA with certain metals, false positive findings with certain skin irritants)(10).

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