EN

ANNEX

The Annex to Regulation (EC) No 440/2008 is amended as follows:

(1)    In part B, Chapter B.4 is replaced by the following:

"B.4 ACUTE DERMAL IRRITATION/CORROSION

INTRODUCTION

1. This test method is equivalent to OECD test guideline (TG) 404 (2015). OECD guidelines for testing of Chemicals are periodically reviewed to ensure that they reflect the best available science. In the review of OECD TG 404, special attention was given to possible improvements in relation to animal welfare concerns and to the evaluation of all existing information on the test chemical in order to avoid unnecessary testing in laboratory animals. The updated version of OECD TG 404 (originally adopted in 1981, revised in 1992, 2002 and 2015) includes reference to the Guidance Document on Integrated Approaches to Testing and Assessment (IATA) for Skin Irritation/Corrosion (1), proposing a modular approach for skin irritation and skin corrosion testing. The IATA describes several modules which group information sources and analysis tools, and provides guidance on (i) how to integrate and use existing testing and non-testing data for the assessment of the skin irritation and skin corrosion potentials of chemicals and (ii) proposes an approach when further testing is needed (1). In addition, where needed, the successive, instead of simultaneous, application of the three test patches to the animal in the initial in vivo test is recommended in that Guideline.

2. Definitions of dermal irritation and corrosion are set out in the Appendix to this test method.

INITIAL CONSIDERATIONS

3.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 test chemical have been evaluated in a weight-of-the-evidence (WoE) analysis as presented in the Guidance Document on Integrated Approaches to Testing and Assessment for Skin Corrosion and Irritation, i.e. over the three Parts of this guidance and their corresponding modules (1). Briefly, under Part 1 existing data is addressed over seven modules covering human data, in vivo data, in vitro data, physico-chemical properties data (e.g. pH, in particular strong acidity or alkalinity) and non-testing methods. Under Part 2, WoE analysis is performed. If this WoE is still inconclusive, Part 3 should be conducted with additional testing, starting with in vitro methods, and in vivo testing is used as last resort. This analysis should therefore decrease the need for in vivo testing for dermal corrosivity/irritation of test chemicals for which sufficient evidence already exists from other studies as to those two endpoints.

PRINCIPLE OF THE IN VIVO TEST

4.The test chemical 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.

5.Animals showing continuing signs of severe distress and/or pain at any stage of the test should be humanely killed, and the test chemical assessed accordingly. Criteria for making the decision to humanely kill moribund and severely suffering animals are the subject of a separate Guidance Document (2).

PREPARATIONS FOR THE IN VIVO TEST

Selection of animal species

6.The albino rabbit is the preferable laboratory animal, and healthy young adult rabbits are used. A rationale for using other species should be provided.

Preparation of the animals

7.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.

8.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.

Housing and feeding conditions

9.Animals should be individually housed. The temperature of the experimental animal room should be 20°C (± 3°C) 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

TEST PROCEDURE

Application of the test chemical

10.The test chemical 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 chemical 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 chemical 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 test chemical on the skin. Access by the animal to the patch and ingestion or inhalation of the test chemical should be prevented.

11.Liquid test chemicals are generally used undiluted. When testing solids (which may be pulverised, if considered necessary), the test chemical 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 chemical should be minimal, if any.

12.At the end of the exposure period, which is normally 4 hours, residual test chemical should be removed, where practicable, using water or an appropriate solvent without altering the existing response or the integrity of the epidermis.

Dose level

13.A dose of 0.5 ml of liquid or 0.5 g of solid or paste is applied to the test site.

Initial test (In vivo dermal irritation/corrosion test using one animal)

14.When a test chemical has been judged to be corrosive, irritant or non-classified on the basis of a weight of evidence analyses or of previous in vitro testing, further in vivo testing is normally not necessary. However, in the cases where additional data are felt warranted, the in vivo test is performed initially using one animal and applying the following approach. Up to three test 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 at a different site 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.

15.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.

16.In those cases in which the test chemical is not expected to produce corrosion but may be irritating, a single patch should be applied to one animal for four hours.

Confirmatory test (In vivo dermal irritation test with additional animals)

17.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.

Observation period

18.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.

Clinical observations and grading of skin reactions

19.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.

20.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 (3).

DATA AND REPORTING

21.Study results should be summarised in tabular form in the final test report and should cover all items listed in paragraph 24.

Evaluation of results

22.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.

23.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 chemical should be considered an irritant.

Test report

24.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 chemical:

-Mono-constituent substance: chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc;

-Multi-constituent substance, mixture and substances of unknown or variable composition, complex reaction products or biological materials (UVCB): characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant physico-chemical properties of the constituents;

-Physical appearance, water solubility, and additional relevant physico-chemical properties;

-Source, lot number if available;

-Treatment of the test chemical/control substance prior to testing, if applicable (e.g. warming, grinding);

-Stability of the test chemical, limit date for use, or date for re-analysis if known;

-Storage conditions.

Vehicle:

-    Identification, concentration (where appropriate), volume used;

-    Justification for choice of vehicle.

Test animal(s):

-    Species/strain used, rationale for using animal(s) other than albino rabbit;

-    Number of animal(s) of each sex;

-    Individual animal weight(s) at start and conclusion of test;

-    Age at start of study;

-    Source of animal(s), housing conditions, diet, etc.

Test conditions:

-    Technique of patch site preparation;

-    Details of patch materials used and patching technique;

-    Details of test chemical 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

Conclusions

Literature

(1)OECD (2014). Guidance document on Integrated Approaches to Testing and Assessment for Skin Irritation/Corrosion. Environmental Health and Safety Publications, Series on Testing and Assessment, (No 203), Organisation for Economic Cooperation and Development, Paris.

(2)OECD (1998) Harmonized 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 .

(3)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 Publications, Series on Testing and Assessment (No 19), Organistion for Economic Cooperation and Development, Paris.

TABLE: 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

Definitions

Chemical is a substance or a mixture.

Dermal irritation is the production of reversible damage of the skin following the application of a test chemical for up to 4 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 chemical for up to four hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs, and, by the end of 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.

Test chemical is any substance or mixture tested using this test method"

(2) In Part B, Chapter B.17 is replaced by the following:

"B.17 IN VITRO MAMMALIAN CELL GENE MUTATION TESTS USING THE HPRT AND XPRT GENES

INTRODUCTION

1.This test method (TM) is equivalent to the OECD test guideline 476 (2016). Test methods are periodically reviewed in the light of scientific progress, changing regulatory needs and animal welfare. This current revised version of TM B.17 reflects nearly thirty years of experience with this test and also results from the development of a separate new method dedicated to in vitro mammalian cell gene mutation tests using the thymidine kinase gene. TM B.17 is part of a series of test methods on genetic toxicology. A document that provides succinct information on genetic toxicology testing and an overview of the recent changes that were made to genetic toxicity OECD test guidelines has been developed by OECD (1).

2.The purpose of the in vitro mammalian cell gene mutation test is to detect gene mutations induced by chemicals. The cell lines used in these tests measure forward mutations in reporter genes, specifically the endogeneous hypoxanthine-guanine phosphoribosyl transferase gene (Hprt in rodent cells, HPRT in human cells; collectively referred to as the Hprt gene and HPRT test in this test method), and the xanthine-guanine phosphoribosyl transferase transgene (gpt) (referred to as the XPRT test). The HPRT and XPRT mutation tests detect different spectra of genetic events. In addition to the mutational events detected by the HPRT test (e.g. base pair substitutions, frameshifts, small deletions and insertions) the autosomal location of the gpt transgene may allow the detection of mutations resulting from large deletions and possibly mitotic recombination not detected by the HPRT test because the Hprt gene is located on the X-chromosome (2) (3) (4) (5) (6) (7). The XPRT is currently less widely used than the HPRT test for regulatory purposes.

3.Definitions used are provided in Appendix 1.

INITIAL CONSIDERATIONS AND LIMITATIONS

4.Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. The exogenous metabolic activation system does not entirely mimic in vivo conditions.

5.Care should be taken to avoid conditions that would lead to artefactual positive results, (i.e. possible interaction with the test system), not caused by direct interaction between the test chemicals and the genetic material of the cell; such conditions include changes in pH or osmolality (8) (9) (10), interaction with the medium components (11) (12), or excessive levels of cytotoxicity (13). Cytotoxicity exceeding the recommended top cytotoxicity levels as defined in paragraph 19 is considered excessive for the HPRT test.

6.Before use of the test method on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose. Such considerations are not needed when there is a regulatory requirement for testing of the mixture.

PRINCIPLE OF THE TEST

7.Mutant cells deficient in Hprt enzyme activity in the HPRT test or xprt enzyme activity in the XPRT test are resistant to the cytostatic effects of the purine analogue 6-thioguanine (TG). The Hprt (in the HPRT test) or gpt (in XPRT test) proficient cells are sensitive to TG, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TG, whereas normal cells, which contain the Hprt (in the HPRT test) or gpt (in XPRT test) enzyme, are not.

8.Cells in suspension or monolayer cultures are exposed to the test chemical, both with and without an exogenous source of metabolic activation (see paragraph 14), for a suitable period of time (3-6 hours), and then sub-cultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection (14) (15) (16) (17). Cytotoxicity is determined by relative survival (RS), i.e. cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control (paragraph 18 and Appendix 2). The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each cell type, to allow near-optimal phenotypic expression of induced mutations (typically a minimum of 7-9 days). Following phenotypic expression, mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant colonies, and in medium without selective agent to determine the cloning efficiency (viability). After a suitable incubation time, colonies are counted. Mutant frequency is calculated based on the number of mutant colonies corrected by the cloning efficiency at the time of mutant selection.

DESCRIPTION OF THE METHOD

Preparations

Cells

9.The cell types used for the HPRT and XPRT tests should have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency, a stable karyotype, and a stable spontaneous mutant frequency. The most commonly used cells for the HPRT test include the CHO, CHL and V79 lines of Chinese hamster cells, L5178Y mouse lymphoma cells, and TK6 human lymphoblastoid cells (18) (19). CHO-derived AS52 cells containing the gpt transgene (and having the Hprt gene deleted) are used for the XPRT test (20) (21); the HPRT test cannot be performed in AS52 cells because the hprt gene has been deleted. The use of other cell lines should be justified and validated.

10.Cell lines should be checked routinely for the stability of the modal chromosome number and the absence of Mycoplasma contamination (22) (23), and cells should not be used if contaminated or if the modal chromosome number has changed. The normal cell cycle time used in the testing laboratory should be established and should be consistent with the published cell characteristics. The spontaneous mutant frequency in the master cell stock should also be checked, and the stock should not be used if the mutant frequency is not acceptable.

11.Prior to use in this test, the cultures may need to be cleansed of pre-existing mutant cells, e.g.by culturing in HAT medium for HPRT test and MPA for XPRT test (5) (24) (See Appendix 1). The cleansed cells can be cryopreserved and then thawed to use as working stocks. The newly thawed working stock can be used for the test after normal doubling times are attained. When conducting the XPRT test, routine culture of AS52 cells should use conditions that assure the maintenance of the gpt transgene (20).

Media and culture conditions

12.Appropriate culture medium and incubation conditions (culture vessels, humidified atmosphere of 5% CO2, and incubation temperature of 37°C) should be used for maintaining cultures. Cell cultures should always be maintained under conditions that ensure that they are growing in log phase. It is particularly important that media and culture conditions be chosen to ensure optimal growth of cells during the expression period and optimal cloning efficiency for both mutant and non-mutant cells.

Preparation of cultures

13.Cell lines are propagated from stock cultures, seeded in culture medium at a density such that the cells in suspensions or in monolayers will continue to grow exponentially through the treatment and expression periods (e.g. confluence should be avoided for cells growing in monolayers).

Metabolic activation

14.Exogenous metabolising systems should be used when employing cells which have inadequate endogenous metabolic capacity. The most commonly used system, that is recommended by default, unless otherwise justified, is a co-factor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents (generally rats) treated with enzyme-inducing agents such as Aroclor 1254 (25) (26) (27) (28) or a combination of phenobarbital and β-naphthoflavone (29) (30) (31) (32). The latter combination does not conflict with the Stockholm Convention on Persistent Organic Pollutants (33) and has been shown to be as effective as Aroclor 1254 for inducing mixed-function oxidases (29) (31). The S9 fraction typically is used at concentrations ranging from 1 to 2% (v/v) but may be increased to 10% (v/v) in the final test medium. The choice of the type and concentration of exogenous metabolic activation system or metabolic inducer employed may be influenced by the class of substances being tested (34) (35) (36).

Test chemical preparation

15.Solid test chemicals should be prepared in appropriate solvents and diluted, if appropriate, prior to treatment of the cells (see paragraph 16). Liquid test chemicals may be added directly to the test system and/or diluted prior to treatment of the test system. Gaseous or volatile test chemicals should be tested by appropriate modifications to the standard protocols, such as treatment in sealed culture vessels (37) (38). Preparations of the test chemical should be made just prior to treatment unless stability data demonstrate the acceptability of storage.

Test conditions

Solvents

16.The solvent should be chosen to optimise the solubility of the test chemicals without adversely impacting the conduct of the test e.g. changing cell growth, affecting the integrity of the test chemical, reacting with culture vessels, impairing the metabolic activation system. It is recommended that, wherever possible, the use of an aqueous solvent (or culture medium) should be considered first. Well established solvents are for example, water and dimethyl sulfoxide. Generally, organic solvents should not exceed 1% (v/v) and aqueous solvents (saline or water) should not exceed 10% (v/v) in the final treatment medium. If the solvents used are not well-established (e.g. ethanol or acetone), their use should be supported by data indicating their compatibility with the test chemicals and the test system, and their lack of genetic toxicity at the concentration used. In the absence of that supporting data, it is important to add untreated controls (see Appendix 1) to demonstrate that no deleterious or mutagenic effects are induced by the chosen solvent.

Measuring cytotoxicity and choosing exposure concentrations

17.When determining the highest test chemical concentration, concentrations that have the capability of producing artefactual positive responses, such as those producing excessive cytotoxicity (see paragraph 20), precipitation in the culture medium (see paragraph 21), or marked changes in pH or osmolality (see paragraph 5) should be avoided. If the test chemical causes a marked change in the pH of the medium at the time of addition, the pH might be adjusted by buffering the final treatment medium so as to avoid artefactual positive results and to maintain appropriate culture conditions.

18.Concentration selection is based on cytotoxicity and other considerations (see paragraphs 20-22). While the evaluation of cytotoxicity in an initial test may be useful to better define the concentrations to be used in the main experiment, an initial test is not required. Even if an initial cytotoxicity evaluation is performed, the measurement of cytotoxicity for each culture is still required in the main experiment. Cytotoxicity should be evaluated using RS, i.e. cloning efficiency (CE) of cells plated immediately after treatment, adjusted by any loss of cells during treatment, based on cell count, as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%) (see Appendix 2 for the formula).

19.At least four test concentrations (not including the solvent and positive controls) that meet the acceptability criteria (appropriate cytotoxicity, number of cells, etc.) should be evaluated. While the use of duplicate cultures is advisable, either replicate or single treated cultures may be used at each concentration tested. The results obtained in the independent replicate cultures at a given concentration should be reported separately but can be pooled for the data analysis (17). For test chemicals demonstrating little or no cytotoxicity, concentration intervals of approximately 2 to 3 fold will usually be appropriate. Where cytotoxicity occurs, the test concentrations selected should cover a range from that producing cytotoxicity to concentrations at which there is moderate and little or no cytotoxicity. Many test chemicals exhibit steep concentration response curves and in order to cover the whole range of cytotoxicity or to study the concentration response relationship in detail, it may be necessary to use more closely spaced concentrations and more than four concentrations, in particular in situations where a repeat experiment is required (see paragraph 43). The use of more than 4 concentrations may be particularly important when using single cultures.

20.If the maximum concentration is based on cytotoxicity, the highest concentration should aim to achieve between 20 and 10% RS. Care should be taken when interpreting positive results only found at 10% RS or below (paragraph 43).

21.For poorly soluble test chemicals that are not cytotoxic at concentrations below the lowest insoluble concentration, the highest concentration analysed should produce turbidity or a precipitate visible by eye or with the aid of an inverted microscope at the end of the treatment with the test chemical. Even if cytotoxicity occurs above the lowest insoluble concentration, it is advisable to test at only one concentration producing turbidity or with a visible precipitate because artefactual effects may result from the precipitate. At the concentration producing a precipitate, care should be taken to assure that the precipitate does not interfere with the conduct of the test. The determination of solubility in the culture medium prior to the experiment may be useful.

22.If no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2 mg/ml or 2 µl/ml, whichever is the lowest (39) (40). When the test chemical is not of defined composition, e.g. substance of unknown or variable composition, complex reaction products or biological materials (i.e. Chemical Substances of Unknown or Variable Composition (UVCBs)) (41), environmental extracts, etc., the top concentration may need to be higher (e.g. 5 mg/mL), in the absence of sufficient cytotoxicity, to increase the concentration of each of the components. It should be noted however that these requirements may differ for human pharmaceuticals (42).

Controls

23.Concurrent negative controls (see paragraph 16), consisting of solvent alone in the treatment medium and handled in the same way as the treatment cultures, should be included for every experimental condition.

24.Concurrent positive controls are needed to demonstrate the ability of the laboratory to identify mutagens under the conditions of the test protocol used and the effectiveness of the exogenous metabolic activation system, when applicable. Examples of positive controls are given in Table 1 below. Alternative positive control substances can be used, if justified. Because in vitro mammalian cell tests for genetic toxicity are sufficiently standardised, tests using treatments with and without exogenous metabolic activation may be conducted using only a positive control requiring metabolic activation. In this case, this single positive control response will demonstrate both the activity of the metabolic activation system and the responsiveness of the test system. Each positive control should be used at one or more concentrations expected to give reproducible and detectable increases over background in order to demonstrate the sensitivity of the test system, and the response should not be compromised by cytotoxicity exceeding the limits specified in this test method (see paragraph 20).

Table 1: Reference substances recommended for assessing laboratory proficiency and for selection of positive controls.

PROCEDURE

Treatment with test chemical

25.Proliferating cells are treated with the test chemical in the presence and absence of a metabolic activation system. Exposure should be for a suitable period of time (usually 3 to 6 hours is adequate).

26.The minimum number of cells used for each test (control and treated) culture at each stage in the test should be based on the spontaneous mutant frequency. A general guide is to treat and passage sufficient cells as to maintain 10 spontaneous mutants in every culture in all phases of the test (17). The spontaneous mutant frequency is generally between 5 and 20 x 10-6. For a spontaneous mutant frequency of 5 x 10-6 and to maintain a sufficient number of spontaneous mutants (10 or more) even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment (10% RS), it would be necessary to treat at least 20 x 106 cells. In addition a sufficient number of cells (but never less than 2 million) must be cultured during the expression period and plated for mutant selection (17).

Phenotypic expression time and measuring mutant frequency

27.After the treatment period, cells are cultured to allow expression of the mutant phenotype. A minimum of 7 to 9 days generally is sufficient to allow near optimal phenotypic expression of newly induced Hprt and xprt mutants (43) (44). During this period, cells are regularly sub-cultured to maintain them in exponential growth. After phenotypic expression, cells are re-plated in medium with and without selective agent (6-thioguanine) for the determination of the number of mutants and cloning efficiency at the time of selection, respectively. This plating can be accomplished using dishes for monolayer cultures or microwell plates for cells in suspension. For mutant selection, cells should be plated at a density to assure optimum mutant recovery (i.e. avoid metabolic cooperation) (17). Plates are incubated for an appropriate length of time for optimum colony growth (e.g. 7-12 days) and colonies counted. Mutant frequency is calculated based on the number of mutant colonies corrected by the cloning efficiency at the time of mutant selection (see Appendix 2 for formulas).

Proficiency of the laboratory

28.In order to establish sufficient experience with the test prior to using it for routine testing, the laboratory should have performed a series of experiments with reference positive substances acting via different mechanisms (at least one active with and one active without metabolic activation selected from the substances listed in Table 1) and various negative controls (using various solvents/vehicles). These positive and negative control responses should be consistent with the literature. This is not applicable to laboratories that have experience, i.e. that have an historical data base available as defined in paragraphs 30 to 33.

29.A selection of positive control substances (see Table 1 in paragraph 25) should be investigated in the absence and in the presence of metabolic activation, in order to demonstrate proficiency to detect mutagenic chemicals, to determine the effectiveness of the metabolic activation system and to demonstrate the appropriateness of the cell growth conditions during treatment, phenotypic expression and mutant selection and of the scoring procedures. A range of concentrations of the selected substances should be chosen so as to give reproducible and concentration-related increases above the background in order to demonstrate the sensitivity and dynamic range of the test system.

Historical control data

30.The laboratory should establish:

-A historical positive control range and distribution,

-A historical negative (untreated, solvent) control range and distribution.

31.When first acquiring data for an historical negative control distribution, concurrent negative controls should be consistent with published control data (22). As more experimental data are added to the control distribution, concurrent negative controls should ideally be within the 95% control limits of that distribution (17) (45) (46).

32.The laboratory’s historical negative control database should initially be built with a minimum of 10 experiments but would preferably consist of at least 20 experiments conducted under comparable experimental conditions. Laboratories should use quality control methods, such as control charts (e.g. C-charts or X-bar charts (47)), to identify how variable their positive and negative control data are, and to show that the methodology is 'under control' in their laboratory (46). Further recommendations on how to build and use the historical data (i.e. criteria for inclusion and exclusion of data in historical data and the acceptability criteria for a given experiment) can be found in the literature (45).

33.Negative control data should consist of mutant frequencies from single or preferably replicate cultures as described in paragraph 23. Concurrent negative controls should ideally be within the 95% control limits of the distribution of the laboratory’s historical negative control database (17) (45) (46). Where concurrent negative control data fall outside the 95% control limit they may be acceptable for inclusion in the historical control distribution as long as these data are not extreme outliers and there is evidence that the test system is ‘under control’ (see above) and there is evidence of no technical or human failure.

34.Any changes to the experimental protocol should be considered in terms of their consistency with the laboratory’s existing historical control databases. Any major inconsistencies should result in the establishment of a new historical control database.

DATA AND REPORTING

Presentation of the results

35.The presentation of results should include all of the data needed to calculate cytotoxicity (expressed as RS). The data, for both treated and control cultures, should include the number of cells at the end of treatment, the number of cells plated immediately following treatment, and the colony counts (or number of wells without colonies for the microwell method). RS for each culture should be expressed as a percentage relative to the concurrent solvent control (refer to Appendix 1 for definitions).

36.The presentation of results should also include all of the data needed to calculate the mutant frequency. Data for both treated and control cultures, should include: (1) the number of cells plated with and without selective agent (at the time the cells are plated for mutant selection), and (2) the number of colonies counted (or the number of wells without colonies for the microwell method) from the plates with and without selective agent. Mutant frequency is calculated based on the number of mutant colonies (in the plates with selective agent) corrected by the cloning efficiency (from the plates without selective agent). The mutant frequency should be expressed as the number of mutant cells per million viable cells (refer to Appendix 1 for definitions).

37.Individual culture data should be provided. Additionally, all data should be summarised in tabular form.

Acceptability Criteria

38.Acceptance of a test is based on the following criteria:

-The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database as described in paragraph 33.

-Concurrent positive controls (see paragraph 24) should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.

-Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results (see paragraph 25).

-Adequate number of cells and concentrations are analysable (paragraphs 25, 26 and 19).

-The criteria for the selection of top concentration are consistent with those described in paragraphs 20, 21 and 22.

Evaluation and interpretation of results

39.Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

-at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,

-the increase is concentration-related when evaluated with an appropriate trend test,

-any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limit; see paragraph 33).

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system. Recommendations for the most appropriate statistical methods can be found in the literature (46) (48).

40.Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:

-none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,

-there is no concentration-related increase when evaluated with an appropriate trend test,

-all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limit; see paragraph 33).

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

41.There is no requirement for verification of a clearly positive or negative response.

42.In cases when the response is neither clearly negative nor clearly positive as described above, or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions [i.e. S9 concentration or S9 origin]) could be useful.

43.In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results. Therefore the test chemical response should be concluded to be equivocal (interpreted as equally likely to be positive or negative).

Test report

44.The test report should include the following information:

Test chemical:

-source, lot number, limit date for use, if available;

-stability of the test chemical itself, if known;

-solubility and stability of the test chemical in solvent, if known;

-measurement of pH, osmolality and precipitate in the culture medium to which the test chemical was added, as appropriate.

Mono-constituent substance:

- physical appearance, water solubility, and additional relevant physicochemical properties;

-chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc.

Multi-constituent substance, UVCBs and mixtures:

-characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant physicochemical properties of the constituents.

Solvent:

-justification for choice of solvent;

-percentage of solvent in the final culture medium.

Cells:

For Laboratory master cultures:

-type, source of cell lines;

-number of passages, if available, and history in the laboratory;

-karyotype features and/or modal number of chromosomes;

-methods for maintenance of cell cultures;

-absence of mycoplasma;

-cell doubling times.

Test conditions:

-rationale for selection of concentrations and number of cultures including, e.g. cytotoxicity data and solubility limitations;

-composition of media, CO2 concentration, humidity level;

-concentration of test chemical expressed as final concentration in the culture medium (e.g. µg or mg/ml or mM of culture medium);

-concentration (and/or volume) of solvent and test chemical added in the culture medium;

-incubation temperature;

-incubation time;

-duration of treatment;

-cell density during treatment;

-type and composition of metabolic activation system (source of S9, method of preparation of the S9 mix, the concentration or volume of S9 mix and S9 in the final culture medium, quality controls of S9);

-positive and negative control substances, final concentrations for each condition of treatment;

-length of expression period (including number of cells seeded, and subcultures and feeding schedules, if appropriate);

-identity of the selective agent and its concentration;

-criteria for acceptability of tests;

-methods used to enumerate numbers of viable and mutant cells;

-methods used for the measurements of cytotoxicity;

-any supplementary information relevant to cytotoxicity and method used;

-duration of incubation times after plating;

-criteria for considering studies as positive, negative or equivocal;

-methods used to determine pH, osmolality and precipitation.

Results:

-number of cells treated and number of cells sub-cultured for each culture;

-cytotoxicity measurements and other observations if any;

-signs of precipitation and time of the determination;

-number of cells plated in selective and non-selective medium;

-number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequencies;

-concentration-response relationship, where possible;

-concurrent negative (solvent) and positive control data (concentrations and solvents);    

-historical negative (solvent) and positive control data, with ranges, means and standard deviations and confidence interval (e.g. 95%) as well as the number of data;

-statistical analyses (for individual cultures and pooled replicates if appropriate), and p-values if any.

Discussion of the results.

Conclusion

LITERATURE

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(28)Maron D.M. and Ames B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, 173, 215.

(29)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. Mutagen. 7, 175-177.

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(35)O’Neill J.P., Machanoff R., San Sebastian J.R., Hsie A.W. (1982). Cytotoxicity and Mutagenicity of Dimethylnitrosamine in Cammalian Cells (CHO/HGPRT system): Enhancement by Calcium Phosphate. Environ. Mol. Mutation., 4, 7-18.

(36)Li, A.P. (1984). Use of Aroclor 1254-Induced Rat Liver Homogenate in the Assaying of Promutagens in Chinese Hamster Ovary Cells. Environ. Mol. Mutation, 4, 7-18.

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(38)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. Environ. Mutagen.,5, 795-801.

(39)OECD (2014). Document Supporting the WNT Decision to Implement Revised Criteria for the Selection of the Top Concentration in the In Vitro Mammalian Cell Assays on Genotoxicity (Test Guidelines 473, 476 and 487). Available upon request from the Organisation for Economic Cooperation and Development.

(40)Brookmire L., Chen J.J. and Levy D.D. (2013). Evaluation of the Highest Concentrations Used in the In Vitro Chromosome Aberrations Assay, Environ. Mol. Mutation, 54, 36-43.

(41)EPA, Office of Chemical Safety and Pollution Prevention. (2011). Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials: UVCB Substances,

(42)USFDA (2012). International Conference on Harmonisation (ICH) Guidance S2 (R1) on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. Available at: [https://federalregister.gov/a/2012-13774].

(43)O’Neill J.P. and Hsie A.W. (1979). Phenotypic Expression Time of Mutagen-Induced 6-thioguranine resistance in Chinese hamster ovary cells (CHO/HGPRT system), Mutation, Res., 59, 109-118.

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Appendix 1

DEFINITIONS

Base pair substitution mutagens: chemicals that cause substitution of base pairs in the DNA. 

Chemical: A substance or a mixture.

Cloning efficiency: The percentage of cells plated at a low density that are able to grow into a colony that can be counted.

Concentrations: refer to final concentrations of the test chemical in culture medium

Cytotoxicity: For the assays covered in this test method, cytotoxicity is identified as a reduction in relative survival of the treated cells as compared to the negative control (see specific paragraph).

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 or the function of the encoded protein.

Frameshift mutagens: chemicals which cause the addition or deletion of single or multiple base pairs in the DNA molecule.

Genotoxic: a general term encompassing all types of DNA or chromosomal damage, including DNA breaks, adducts, rearrangements, mutations, chromosome aberrations, and aneuploidy. Not all types of genotoxic effects result in mutations or stable chromosomal damage

HAT medium: medium containing Hypoxanthine, Aminopterin and Thymidine, used for cleansing of Hprt mutants.

Mitotic recombination: during mitosis, recombination between homologous chromatids possibly resulting in the induction of DNA double strand breaks or in a loss of heterozygosity.

MPA medium: medium containing Xanthine, Adenine, Thymidine, Aminopterin and Mycophenolic acid, used for cleansing of Xprt mutants.

Mutagenic: produces a heritable change of DNA base-pair sequences(s) in genes or of the structure of chromosomes (chromosome aberrations).

Mutant frequency (MF): the number of mutant colonies observed divided by the number of cells plated in selective medium, corrected for cloning efficiency (or viability) at the time of selection.

Phenotypic expression time: The time after treatment during which the genetic alteration is fixed within the genome and any preexisting gene products are depleted to the point that the phenotypic trait is altered.

Relative survival (RS): RS is used as the measure of treatment-related cytotoxicity. RS is cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in negative controls (assigned a survival of 100%).

S9 liver fractions: supernatant of liver homogenate after 9000g centrifugation, i.e. raw liver extract

S9 mix: mix of the liver S9 fraction and cofactors necessary for metabolic enzyme activity.

Solvent control: General term to define the control cultures receiving the solvent alone used to dissolve the test chemical.

Test chemical: Any substance or mixture tested using this test method.

Untreated control: cultures that receive no treatment (i.e. neither test chemical nor solvent) but are processed concurrently and in the same way as the cultures receiving the test chemical

UVCB: Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials

Appendix 2

FORMULAS FOR ASSESSMENT OF CYTOTOXICITY AND MUTANT FREQUENCY

Cytotoxicity is evaluated by relative survival, i. e., cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%) (see RS formula below).

Adjusted CE for a culture treated by a test chemical is calculated as:

RS for a culture treated by a test chemical is calculated as:

Mutant frequency is the cloning efficiency of mutant colonies in selective medium divided by the cloning efficiency in non-selective medium measured for the same culture at the time of selection.

When plates are used for cloning efficiency:

CE = Number of colonies / Number of cells plated.

When micro-well plates are used for cloning efficiency:

The number of colonies per well on micro-wells plates follows a Poisson distribution.

Cloning Efficiency = -LnP(0) / Number of cells plated per well

Where -LnP(0) is the probable number of empty wells out of the seeded wells and is described by the following formula

LnP(0)= -Ln (number of empty wells / number of plated wells)"

(3) In Part B, Chapter B.22 is replaced by the following:

"B.22 RODENT DOMINANT LETHAL TEST

INTRODUCTION

1.This test method (TM) is equivalent to the OECD test guideline (TG) 478 (2016). Test methods are periodically reviewed in the light of scientific progress, changing regulatory needs, and animal welfare considerations. This modified version of the test method reflects more than thirty years of experience with this test and the potential for integrating or combining this test with other toxicity tests such as developmental, reproductive toxicity, or genotoxicity studies; however due to its limitations and the use of a large number of animals this assay is not intended for use as a primary method, but rather as a supplemental test method which can only be used when there is no alternative for regulatory requirements. Combining toxicity testing has the potential to spare large numbers of animals from use in toxicity tests. A document that provides succinct information on genetic toxicology testing and an overview of the recent changes that were made to genetic toxicity OECD test guidelines has been developed by OECD (1).

2.The purpose of the Dominant lethal (DL) test is to investigate whether chemicals produce mutations resulting from chromosomal aberrations in germ cells. In addition, the dominant lethal test is relevant to assessing genotoxicity because, although they may vary among species, factors of in vivo metabolism, pharmacokinetics and DNA-repair processes are active and contribute to the response. Induction of a DL mutation after exposure to a test chemical indicates that the chemical has affected germinal tissue of the test animal.

3.DL mutations cause embryonic or foetal death. Induction of DL mutation after exposure to a test chemical indicates that the chemical has affected the germ cells of the test animal.

4.A DL assay is useful for confirmation of positive results of tests using somatic in vivo endpoints, and is a relevant endpoint for the prediction of human hazard and risk of genetic diseases transmitted through the germline. However, this assay requires a large number of animals and is labour-intensive; as a result, it is very expensive and time-consuming to conduct. Because the spontaneous frequency of dominant lethal mutations is quite high, the sensitivity of the assay for detection of small increases in the frequency of mutations is generally limited.

5.Definitions of key terms are set out in Appendix 1.

INITIAL CONSIDERATIONS

6.The test is most often conducted in mice (2) (3) (4) but other species, such as rats (5) (6) (7) (8), may in some cases be appropriate if scientifically justified. DLs generally are the result of gross chromosomal aberrations (structural and numerical abnormalities) (9) (10) (11), but gene mutations cannot be excluded. A DL mutation is a mutation occurring in a germ cell per se, or is fixed post fertilisation in the early embryo, that does not cause dysfunction of the gamete, but is lethal to the fertilised egg or developing embryo.

7.Individual males are mated sequentially to virgin females at appropriate intervals. The number of matings following treatment is dependent on the ultimate purpose of the DL study (Paragraph 23) and should ensure that all phases of male germ cell maturation are evaluated for DLs (12).

8.If there is evidence that the test chemical, or its metabolite(s), will not reach the testis, it is not appropriate to use this test.

PRINCIPLE OF THE TEST

9.Generally, male animals are exposed to a test chemical by an appropriate route of exposure and mated to untreated virgin females. Different germ cell types can be tested by the use of sequential mating intervals. Following mating, the females are euthanised after an appropriate period of time, and their uteri are examined to determine the numbers of implants and live and dead embryos. The dominant lethality of a test chemical is determined by comparing the live implants per female in the treated group with the live implants per female in the vehicle/solvent control group. The increase of dead implants per female in the treated group over the dead implants per female in the control group reflects the test-chemical-induced post-implantation loss. The post-implantation loss is calculated by determining the ratio of dead to total implants in the treated group compared to the ratio of dead to total implants in the control group. Pre-implantation loss can be estimated by comparing corpora lutea counts minus total implants or the total implants per female in treated and control groups.

VERIFICATION OF LABORATORY PROFICIENCY

10.Competence in this assay should be established by demonstrating the ability to reproduce dominant lethal frequencies from published data (e.g. (13) (14) (15) (16) (17) (18)) with positive control substances (including weak responses) such as those listed in Table 1, and vehicle controls and obtaining negative control frequencies that are consistent acceptable range of data (see references above) or with the laboratory’s historical control distribution, if available.

DESCRIPTION OF THE METHOD

Preparations

Selection of animal species

11.Commonly used laboratory strains of healthy sexually mature animals should be employed. Mice are commonly used but rats may also be appropriate. Any other appropriate mammalian species may be used, if scientific justification is provided in the report.

Animal housing and feeding conditions

12.For rodents, the temperature in the animal room should be 22oC (±3oC). Although the relative humidity ideally should be 50-60%, it should be at least 40% and preferably not exceed 70%, other than during room cleaning. Lighting should be artificial, the sequence being 12 hours light, followed by 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 chemical when administered by this route. Prior to treatment or mating, rodents should be housed in small groups (no more than five) of the same sex if no aggressive behaviour is expected or observed, preferably in solid cages with appropriate environmental enrichment. Animals may be housed individually if scientifically justified.

Preparation of the animals

13.Healthy and sexually mature male and female adult animals are randomly assigned to the control and treatment groups. The individual animals are identified uniquely using a humane, minimally invasive method (e.g. by ringing, tagging, micro-chipping, or biometric identification, but not toe and ear clipping) and 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. Cross contamination by the positive control and the test chemical should be avoided. 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.

Preparation of doses

14.Solid test chemicals should be dissolved or suspended in appropriate solvents or vehicles or admixed in diet or drinking water prior to dosing of the animals. Liquid test chemicals may be dosed directly or diluted prior to dosing. For inhalation exposures, test chemicals can be administered as gas, vapour, or a solid/liquid aerosol, depending on their physicochemical properties. Fresh preparations of the test chemical should be employed unless stability data demonstrate the acceptability of storage and define the appropriate storage conditions.

Test Conditions

Solvent/vehicle

15.The solvent/vehicle should not produce toxic effects at the dose volumes used, and should not be suspected of chemical reaction with the test chemical. 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. Examples of commonly used compatible solvents/vehicles include water, physiological saline, methylcellulose solution, carboxymethyl cellulose sodium salt solution, olive oil and corn oil.

Positive controls

16.Concurrent positive control animals should always be used unless the laboratory has demonstrated proficiency in the conduct of the test and has used the test routinely in the recent past (e.g. within the last 5 years). However, it is not necessary to treat positive control animals by the same route as animals receiving the test chemical, or sample all the mating intervals. The positive control substances should be known to produce DLs under the conditions used for the test. Except for the treatment, animals in the control groups should be handled in an identical manner to animals in the treated groups.

17.The doses of the positive control substances should be selected so as to produce weak or moderate effects that critically assess the performance and sensitivity of the assay, but which consistently produce positive dominant lethal effects. Examples of positive control substances, and appropriate doses, are included in Table 1.

Table 1: Examples of Positive Control Substances.

Negative controls

18.Negative control animals, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups, should be included for every sampling time (20). In the absence of historical or published control data showing that no DLs or other deleterious effects are induced by the chosen solvent/vehicle, untreated control animals should also be included for every sampling time in order to establish acceptability of the vehicle control.

PROCEDURE

Number of Animals

19.Individual males are mated sequentially at appropriate predetermined intervals (e.g. weekly intervals, Paragraphs 21 & 23) preferably to one virgin female. The number of males per group should be predetermined to be sufficient (in combination with the number of mated females at each mating interval) to provide the statistical power necessary to detect at least a doubling in DL frequency (Paragraph 44).

20.The number of females per mating interval should also be predetermined by statistical power calculations to permit the detection of at least a doubling in the DL frequency (i.e. sufficient pregnant females to provide at least 400 total implants) (20) (21) (22) (23) and that at least one dead implant per analysis unit (i.e. mating group per dose) is expected (24).

Administration Period and Mating Intervals

21.The number of mating intervals following treatment is governed by the treatment schedule and should ensure that all phases of male germ cell maturation are evaluated for DL induction (12) (25). For a single treatment up to five daily dose administrations, there should be 8 (mouse) or 10 (rat) matings conducted at weekly intervals following the last treatment. For multiple dose administrations, the number of mating intervals may be reduced in proportion to the increased time of the administration period, but maintaining the goal of evaluating all phases of spermatogenesis (e.g. after a 28-day exposure, only 4 weekly matings are sufficient to evaluate all phased of spermatogenesis in the mouse). All treatment and mating schedules should be scientifically justified.

22.Females should remain with the males for at least the duration of one oestrus cycle (e.g. one week covers one oestrus cycle in both mice and rats). Females that did not mate during a one-week interval can be used for a subsequent mating interval. Alternatively, until mating has occurred, as determined by the presence of sperm in the vagina or by the presence of a vaginal plug.

23.The exposure and mating regimen used is dependent on the ultimate purpose of the DL study. If the goal is to determine whether a given chemical induces DL mutations per se, then the accepted method would be to expose an entire round of spermatogenesis (e.g. 7 weeks in the mouse, 5-7 treatments per week) and mate once at the end. However, if the goal is to identify the sensitive germ cell type for DL induction, then a single or 5 day exposure followed by weekly mating is preferred.

Dose Levels

24.If a preliminary range-finding study is performed because there are no suitable data already available to aid in dose selection, it should be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study (26). The study should aim to identify the maximum tolerated dose (MTD), defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, abnormal behaviour or reactions, minor body weight depression or hematopoietic system cytotoxicity), but not death or evidence of pain, suffering or distress necessitating humane euthanasia (27).

25.The MTD must also not adversely affect mating success (21).

26.Test chemicals with specific biological activities at low non-toxic doses (such as hormones and mitogens), and chemicals which exhibit saturation of toxicokinetic properties may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis.

27.In order to obtain dose response information, a complete study should include a negative control group and a minimum of three dose levels generally separated by a factor of 2, but not greater than 4. If the test chemical does not produce toxicity in a range-finding study, or based on existing data, the highest dose for a single administration should be 2000 mg/kg body weight. However, if the test chemical does cause toxicity, the MTD should be the highest dose administered and the dose levels used should preferable cover a range from the maximum to a dose producing little or no toxicity. For not-toxic chemicals, the limit dose for an administration period of 14 days or more is 1000 mg/kg body weight/day, and for administration periods of less than 14 days the limit dose is 2000 mg/kg body weight/day.

Administration of Doses

28.The anticipated route of human exposure should be considered when designing an assay. Therefore, routes of exposures such as dietary, drinking water, subcutaneous, intravenous, topical, inhalation, oral (by gavage), or implantation may be chosen as justified. In any case, the route should be chosen to ensure adequate exposure of the target tissue(s). Intraperitoneal injection is not normally recommended since it is not an intended route of human exposure, and should only be used with specific scientific justification. If the test chemical is admixed in diet or drinking water, especially in case of single dosing, care should be taken that the delay between food and water consumption and mating should be sufficient to allow detection of the effects (paragraph 31). 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 normally exceed 1 ml/100g body weight except in the case of aqueous solutions where a maximum of 2 ml/100g may be used. The use of volumes greater than this (if permitted by animal welfare legislation) should be justified. Variability in test volume should be minimised by adjusting the concentration to ensure a constant volume in relation to body weight at all dose levels.

Observations

29.General clinical observations of the test animals should be made and clinical signs recorded at least once a day, preferably at the same time(s) each day and considering the peak period of anticipated effects after dosing. At least twice daily during the dosing period, all animals should be observed for morbidity and mortality. All animals should be weighed at the beginning of the study and at least once a week during repeated dose studies, and at the time of euthanasia. Measurements of food consumption should be made at least weekly. If the test chemical is administered via the drinking water, water consumption should be measured at each change of water and at least weekly. Animals exhibiting non-lethal indicators of excess toxicity should be euthanised prior to completion of the test period (27).

Tissue Collection and Processing

30.Females are euthanised in the second half of pregnancy at gestation day (GD) 13 for mice and GD 14-15 for rats. Uteri are examined for dominant lethal effects to determine the number of implants, live and dead embryos, and corpora lutea.

31.The uterine horns and ovaries are exposed for counting of corpora lutea, and fetuses are removed, counted, and weighted. Care should be taken to examine the uteri for resorptions obscured by live fetuses and to ensure that all resorptions are enumerated. Fetal mortality is recorded. The number of successfully impregnated females and the number of total implantations, pre-implantation losses, and post-implantation mortality (included early and late resorptions) also are recorded. In addition, the visible fetuses may be preserved in Bouin’s fixative for at least 2 weeks followed by examination for major external malformations (28) to provide additional information on the reproductive and developmental effects of the test agent.

DATA AND REPORTING

Treatment of Results

32.Data should be tabulated to show the number of males mated, 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. The mating interval, dose level for treated males, and the numbers of live implants and dead implants should be enumerated for each female.

33.The post-implantation loss is calculated by determining the ratio of dead to total implants from the treated group compared to the ratio of dead to total implants from the vehicle/solvent control group.

34.Pre-implantation loss is calculated as the difference between the number of corpora lutea and the number of implants, or as a reduction in the average number of implants per female in comparison with control matings. Where pre-implantation loss is estimated, it should be reported.

35.The Dominant Lethal factor is estimated as: (post-implantation deaths/total implantations per female) x 100.

36.Data on toxicity and clinical signs (as per Paragraph 29) should be reported.

Acceptability Criteria

37.The following criteria determine the acceptability of a test.

-Concurrent negative control is consistent with published norms for historical negative control data, and the laboratory's historical control data if available (see Paragraphs 10 and 18).

-Concurrent positive controls induce responses that are consistent with published norms for historic positive control data, or the laboratory’s historical positive control database, if available, and produce a statistically significant increase compared with the negative control (see Paragraphs 17 and 18).

-Adequate number total implants and doses have been analysed (Paragraph 20).

-The criteria for the selection of top dose are consistent with those described in Paragraphs 24 and 27.

Evaluation and Interpretation of Results

38.At least three treated dose groups should be analysed in order to provide sufficient data for dose-response analysis.

39.Providing that all acceptability criteria are fulfilled, a test chemical is considered a clear positive if:

-at least one of the test doses exhibits a statistically significant increase compared with the concurrent negative control;

-the increase is dose-related in at least one experimental condition (e.g. a weekly mating interval) when evaluated with an appropriate test; and,

-any of the results are outside of the acceptable range of negative control data, or the distribution of the laboratory’s historical negative control data (e.g. Poisson-based 95% control limit) if available.

The test chemical is then considered able to induce dominant lethal mutations in germ cells of the test animals. Recommendations for the most appropriate statistical methods are described in Paragraph 44; other recommend statistical approaches can also be found in the literature (20) (21) (22) (24) (29). Statistical tests used should consider the animal as the experimental unit.

40.Providing that all acceptability criteria are fulfilled, a test chemical is considered a clear negative if:

-none of the test doses exhibits a statistically significant increase compared with the concurrent negative control;

-there is no dose-related increase in any experimental condition; and

-all results are within acceptable range of negative control data, or the laboratory’s historical negative control data (e.g. Poisson-based 95% control limit), if available.

The test chemical is then considered unable to induce dominant lethal mutations in germ cells of the test animals.

41.There is no requirement for verification of a clear positive or a clear negative response.

42.If the response is not clearly negative or positive, and in order to assist in establishing the biological relevance of a result (e.g. a weak or borderline increase), the data should be evaluated by expert judgment and/or further investigations using the existing experimental data, such as consideration whether the positive result is outside the acceptable range of negative control data, or the laboratory's historical, negative control data (30).

43.In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and will therefore be concluded as equivocal.

44.Statistical tests used should consider the male animal as the experimental unit. While it is possible that count data (e.g. number of implants per female) may be Poisson distributed and/or proportions (e.g. proportion of dead implants) may be binomially distributed, it is often the case that such data are overdispersed (31). Accordingly, statistical analysis should first employ a test for over- underdispersion using variance tests such as Cochran’s binomial variance test (32) or Tarone’s C(α) test for binomial overdispersion (31) (33). If no departure from binomial dispersion is detected, trends in proportions across dose levels may be tested using the Cochran-Armitage trend test (34) and pairwise comparisons with the control group may be tested using Fisher’s exact test (35). Likewise, if no departure from Poisson dispersion is detected, trends in counts may be tested using Poisson regression (36) and pairwise comparisons with the control group may be tested within the context of the Poisson model, using pairwise contrasts (36). If significant overdispersion or underdispersion is detected, nonparametric methods are recommended (23) (31). These include rank-based tests, such as the Jonckheere-Terpstra test for trend (37) and Mann-Whitney tests (38) for pairwise comparisons with the vehicle/solvent control group, as well as permutation, resampling, or bootstrap tests for trend and pairwise comparisons with the control group (31) (39).

45.A positive DL assay provides evidence for the genotoxicity of the test chemical in the germ cells of the treated male of the test species.

46.Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response (40).

Test Report

47.The test report should include the following information.

Summary.

Test chemical:

-source, lot number, limit date for use, if available;

-stability of the test chemical itself, if known;

-solubility and stability of the test chemical in solvent, if known;

-measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical was added, as appropriate.

Mono-constituent substance:

-physical appearance, water solubility, and additional relevant physicochemical properties;

-chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc.

Multi-constituent substance, UVCBs and mixtures:

-characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant physicochemical properties of the constituents.

Test chemical preparation:

-justification for choice of vehicle;

-solubility and stability of the test chemical in the solvent/vehicle, if known;

-preparation of dietary, drinking water or inhalation formulations;

-analytical determinations on formulations (e.g. stability, homogeneity, nominal concentrations) when conducted.

Test animals:

-species/strain used and justification for the choice;

-number, age and sex of animals;

-source, housing conditions, diet, etc.;

-method of uniquely identifying the animals;

-for short-term studies: individual body weight of the male animals at the start and end of the test; for studies longer than one week: individual body weights during the study and food consumption. Body weight range, mean and standard deviation for each group should be included.

Test conditions:

-positive and negative (vehicle/solvent) control data;

-data from the range-finding study;

-rationale for dose level selection;

-details of test chemical preparation;

-details of the administration of the test chemical;

-rationale for route of administration;

-methods for measurement of animal toxicity, including, where available, histopathological or hematological analyses and the frequency with which animal observations and body weights were taken;

-methods for verifying that the test chemical reached the target tissue, or general circulation, if negative results are obtained;

-actual dose (mg/kg body weight/day) calculated from diet/drinking water test chemical concentration (ppm) and consumption, if applicable;

-details of food and water quality;

-details on cage environment enrichment;

-detailed description of treatment and sampling schedules and justifications for the choices;

-method of analgesia

-method of euthanasia;

-procedures for isolating and preserving tissues;

-source and lot numbers of all kits and reagents (where applicable);

-methods for enumeration of DLs;

-mating schedule;

-methods used to determine that mating has occurred;

-time of euthanasia;

-criteria for scoring DL effects, including, corpora lutea, implantations, resorptions and pre-implantation losses, live implants, dead implants.

Results:

-animal condition prior to and throughout the test period, including signs of toxicity;

-male body weight during the treatment and mating periods;

-number of mated females;

-dose-response relationship, where possible;

-concurrent and historical negative control data with ranges, means and standard deviations;

-concurrent positive control data;

-tabulated data or each dam including: number of corpora lutea per dam; number of implantations per dam; number of resorptions and pre-implantation losses per dam; number of live implants per dam; number of dead implants per dam; fetus weights;

-the above data summarised for each mating period and dose, with Dominant Lethal frequencies;

-statistical analyses and methods applied.

Discussion of the results.

Conclusion.

LITERATURE

(1)OECD (2016). Overview of the set of OECD Genetic Toxicology Test Guidelines and updates performed in 2014-2015. ENV Publications. Series on Testing and Assessment, No 234, OECD, Paris.

(2)Bateman, A.J. (1977). The Dominant Lethal Assay in the Male Mouse, in Handbook of Mutagenicity Test Procedures B.J. Kilbey et. al.(Eds.) pp. 235-334, Elsevier, Amsterdam

(3)Ehling U.H., Ehling, U.H., Machemer, L., Buselmaier, E., Dycka, D., Frohberg, H., Kratochvilova, J., Lang, R., Lorke, D., Muller, D., Pheh, J. , Rohrborn, G., Roll, R., Schulze-Schencking, M., and Wiemann, H. (1978). Standard Protocol for the Dominant Lethal Test on Male Mice. Set up by the Work Group “Dominant lethal mutations of the ad hoc Committee Chemogenetics, Arch. Toxicol., 39, 173-185

(4)Shelby M.D. (1996). Selecting Chemicals and Assays for Assessing Mammalian Germ Cell Mutagenicity. Mutation Res,. 352:159-167.

(5)Knudsen I., Knudsen, I., Hansen, E.V., Meyer, O.A. and Poulsen, E. (1977). A proposed Method for the Simultaneous Detection of Germ-Cell Mutations Leading to Fetal Death (Dominant Lethality) and of Malformations (Male Teratogenicity) in Mammals. Mutation Res., 48:267-270.

(6)Anderson D., Hughes, J.A., Edwards, A.J. and Brinkworth, M.H. (1998). A Comparison of Male-Mediated Effects in Rats and Mice Exposed to 1,3-Butadiene. Mutation Res., 397:77-74.

(7)Shively C.A., C.A., White, D.M., Blauch, J.L. and Tarka, S.M. Jr. (1984). Dominant Lethal Testing of Theobromine in Rats. Toxicol. Lett. 20:325-329.

(8)Rao K.S., Cobel-Geard, S.R., Young, J.T., Hanley, T.R. Jr., Hayes, W.C., John, J.A. and Miller, R.R. (1983). Ethyl Glycol Monomethyl Ether II. Reproductive and dominant Lethal Studies in Rats. Fundam. Appl. Toxicol., 3:80-85.

(9)Brewen J.G., Payne, H.S., Jones, K.P., and Preston, R.J. (1975). Studies on Chemically Induced Dominant Lethality. I. The Cytogenetic Basis of MMS-Induced Dominant Lethality in Post-Meiotic Male Germ Cells, Mutation Res., 33, 239-249.

(10)Marchetti F., Bishop, J.B., Cosentino, L., Moore II, D. and Wyrobek, A.J. (2004). Paternally Transmitted Chromosomal Aberrations in Mouse Zygotes Determine their Embryonic Fate. Biol. Reprod., 70:616-624.

(11)Marchetti F. and Wyrobek, A.J. (2005). Mechanisms and Consequences of Paternally Transmitted Chromosomal Aberrations. Birth Defects Res., C 75:112-129.

(12)Adler I.D. (1996). Comparison of the Duration of Spermatogenesis Between Rodents and Humans. Mutation Res., 352:169-172.

(13)Favor J., and Crenshaw J.W. (1978). EMS-Induced Dominant Lethal Dose Response Curve in DBA/1J Male Mice, Mutation Res., 53: 21–27.

(14)Generoso W.M., Witt, K.L., Cain, K.T., Hughes, L. Cacheiro, N.L.A, Lockhart, A.M.C. and Shelby, M.D. (1995). Dominant Lethal and Heritable Translocation Test with Chlorambucil and Melphalan. Mutation Res., 345:167-180.

(15)Hastings S.E., Huffman K.W. and Gallo M.A. (1976). The dominant Lethal Effect of Dietary Triethylenemelamine, Mutation Res., 40:371-378. 

(16)James D.A. and Smith D.M. (1982). Analysis of Results from a Collaborative Study of the Dominant Lethal Assay, Mutation Res., 99:303-314.

(17)Shelby M.D., Cain, K.T., Hughes, L.A., Braden, P.W. and Generoso, W.M. (1986). Dominant Lethal Effects of Acrylamide in Male Mice. Mutation Res., 173:35-40.

(18)Sudman P.D., Rutledge, J.C., Bishop, J.B. and Generoso W.M. (1992). Bleomycin: Female-Specific Dominant Lethal Effects in Mice, Mutation Res., 296: 143-156.

(19)Holstrom L.M., Palmer A.K. and Favor, J. (1993). The Rodent Dominant Lethal Assay. In Supplementary Mutagenicity Tests. Kirkland D.J. and Fox M. (Eds.), Cambridge University Press, pp. 129-156.

(20)Adler I-D., Bootman, J., Favor, J., Hook, G., Schriever-Schwemmer, G., Welzl, G., Whorton, E., Yoshimura, I. and Hayashi, M. (1998). Recommendations for Statistical Designs of In Vivo Mutagenicity Tests with Regard to Subsequent Statistical Analysis, Mutation Res., 417:19–30.

(21)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., 312:313-318.

(22)Generoso W.M. and Piegorsch W.W. (1993). Dominant Lethal Tests in Male and Female Mice. Methods, Toxicol., 3A:124-141.

(23)Haseman J.K. and Soares E.R. (1976).The Distribution of Fetal Death in Control Mice and its Implications on Statistical Tests for Dominant Lethal Effects. Mutation. Res., 41: 277-288.

(24)Whorton E.B. Jr. (1981). Parametric Statistical Methods and Sample Size Considerations for Dominant Lethal Experiments. The Use of Clustering to Achieve Approximate Normality, Teratogen. Carcinogen. Mutagen., 1:353 – 360.

(25)Anderson D., Anderson, D., Hodge, M.C.E., Palmer, S., and Purchase, I.F.H. (1981). Comparison of Dominant Lethal and Heritable Translocation Methodologies. Mutation. Res., 85:417‑429.

(26)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. Mutagen., 7:313-319.

(27)OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environment, Health and Safety Publications, Series on Testing and Assessment (No.19.), Organisation for Economic Cooperation and Development, Paris.

(28)Barrow M.V., Taylor W.J and Morphol J. (1969). A Rapid Method for Detecting Malformations in Rat Fetuses, 127, 291–306.

(29)Kirkland D.J., (Ed.)(1989) . Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press. 

(30)Hayashi, M., Dearfield, K., Kasper P., Lovell D., Martus H.-J. and Thybaud V. (2011). “Compilation and Use of Genetic Toxicity Historical Control Data”, Mutation. Res., 723:87-90. 

(31)Lockhart A.C., Piegorsch W.W. and Bishop J.B. (1992). Assessing Over Dispersion and Dose-Response in the Male Dominant Lethal Assay. Mutation. Res., 272:35-58.

(32)Cochran W.G. (1954). Some Methods for Strengthening the Common χ2 Tests. Biometrics, 10: 417-451.

(33)Tarone R.E. (1979). Testing the Goodness of Fit of the Binomial Distribution. Biometrika, 66: 585-590.

(34)Margolin B.H. (1988). Test for Trend in Proportions. In Encyclopedia of Statistical Sciences, Volume 9, Kotz S. and Johnson N. L. (Eds.), pp. 334-336. John Wiley and Sons, New York.

(35)Cox D.R., Analysis of Binary Data. Chapman and Hall, London (1970).

(36)Neter J.M., Kutner, H.C., Nachtsheim, J. and Wasserman, W. (1996). Applied Linear Statistical Models, Fourth Edition, Chapters 14 and 17. McGraw-Hill, Boston

(37)Jonckheere R. (1954). A Distribution-Free K-Sample Test Against Ordered Alternatives. Biometrika, 41:133-145.

(38)Conover W.J. (1971). Practical Nonparametric Statistics. John Wiley and Sons, New York

(39)Efron, B. (1982). The Jackknife, the Bootstrap and Other Resampling Plans. Society for Industrial and Applied Mathematics, Philadelphia, PA.

(40)Fleiss J. (1973). Statistical Methods for Rates and Proportions. John Wiley and Sons, New York.

Appendix 1

DEFINITIONS

Chemical: A substance or a mixture

Corpora luteum (lutea): the hormonal secreting structure formed on the overy at the site of a follicle that has released the egg. The number of corpora lutea in the ovaries corresponds to the number of eggs that were ovulated.

Dominant Lethal Mutation: a mutation occurring in a germ cell, or is fixed after fertilisation, that causes embryonic or foetal death.

Fertility rate: the number of mated pregnant female over the number of mated females.

Mating interval: the time between the end of exposure and mating of treated males. By controlling this interval, chemical effects on different germ cell types can be assessed. In the mouse mating during the 1, 2, 3, 4, 5, 6, 7 and 8 week after the end of exposure measures effects in sperm, condensed spermatids, round spermatids, pachytene spermatocytes, early spermatocytes, differentiated spermatogonia, differentiating spermatogonia and stem cell spermatogonia.

Preimplantation loss: the difference between the number of implants and the number of corpora lutea. It can also be estimated by comparing the total implants per female in treated and control groups.

Postimplantation loss: the ratio of dead implant in the treated group compared to the ratio of dead to total implants in the control group.

Test chemical: Any substance or mixture tested using this test method.

UVCB: Chemical Substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials

Appendix 2

TIMING OF SPERMATOGENESIS IN MAMMALS

Fig.1: Comparison of the duration (days) of male germ cell development in mice, rats and humans. DNA repair does not occur during the periods indicated by shading.

A schematic of spermatogenesis in the mouse, rat and human is shown above (taken from Adler, 1996). Undifferentiated spermatogonia include: A-single; A-paired; and A-aligned spermatogonia (Hess and de Franca, 2008). A-single is considered the true stem cells; therefore, to assess effects on stem cells at least 49 days (in the mouse) must pass between the last injection of the test chemical and mating.

References

Adler, ID (1996). Comparison of the duration of spermatogenesis between rodents and humans. Mutat Res, 352:169-172.

Hess, RA, De Franca LR (2008). Spermatogenesis and cycle of the seminiferous epithelium. In: Molecular Mechanisms in Spermatogenesis, C. Yan Cheng (Ed), Landes Biosciences and Springer Science&Business Media:1-15."

(4) In Part B, Chapter B.23 is replaced by the following:

"B.23 MAMMALIAN SPERMATOGONIAL CHROMOSOMAL ABERRATION TEST

INTRODUCTION

1.This test method (TM) is equivalent to the OECD test guideline 483 (2016). Test methods are periodically reviewed in the light of scientific progress, changing regulatory needs, and animal welfare considerations. This modified version of the test method reflects many years of experience with this assay and the potential for integrating or combining this test with other toxicity or genotoxicity studies. Combining toxicity studies has the potential to reduce the numbers of animals used in toxicity testing. This test method is part of a series of test methods on genetic toxicology. A document that provides succinct information on genetic toxicology testing and an overview of the recent changes that were made to genetic toxicity OECD test guidelines has been developed by OECD (1).

2.The purpose of the in vivo mammalian spermatogonial chromosomal aberration test is to identify those chemicals that cause structural chromosomal aberrations in mammalian spermatogonial cells (2) (3) (4). In addition, this test is relevant to assessing genetoxicity because, although they may vary among species, factors of in vivo metabolism, pharmacokinetics and DNA-repair processes are active and contribute to the response. This test method is not designed to measure numerical abnormalities; the assay is not routinely used for this purpose.

3.This test measures structural chromosomal aberrations (both chromosome- and chromatid-type) in dividing spermatogonial germ cells and is, therefore, expected to be predictive of induction of heritable mutations in these germ cells.

4.Definitions of key terms are set out in the Appendix.

INITIAL CONSIDERATIONS

5.Rodents are routinely used in this test but other species may in some cases be appropriate if scientifically justified. Standard cytogenetic preparations of rodent testes generate mitotic (spermatogonia) and meiotic (spermatocyte) metaphases. Mitotic and meiotic metaphases are identified based on the morphology of the chromosomes (4). This in vivo cytogenetic test detects structural chromosomal aberrations in spermatogonial mitoses. Other target cells are not the subject of this test method.

6.To detect chromatid-type aberrations in spermatogonial cells, the first mitotic cell division following treatment should be examined before these aberrations are converted into chromosome-type-aberrations in subsequent cell divisions. Additional information from treated spermatocytes can be obtained by meiotic chromosome analysis for chromosomal structural aberrations at diakinesis-metaphase I and metaphase II.

7.A number of generations of spermatogonia are present in the testis (5), and these different germ cell types may have a spectrum of sensitivity to chemical treatment. Thus, the aberrations detected represent an aggregate response of treated spermatogonial cell populations. The majority of mitotic cells in testis preparations are B spermatogonia, which have a cell cycle of approximately 26 hr (3).

8.If there is evidence that the test chemical, or its metabolite(s), will not reach the testis it is not appropriate to use this test.

PRINCIPLE OF THE TEST METHOD

9.Generally, animals are exposed to the test chemical by an appropriate route of exposure and are euthanised at appropriate times after treatment. Prior to euthanasia, animals are treated with a metaphase-arresting agent (e.g. colchicine or Colcemid®). Chromosome preparations are then made from germ cells and stained, and metaphase cells are analysed for chromosome aberrations.

VERIFICATION OF LABORATORY PROFICIENCY

10.Competency in this assay should be established by demonstrating the ability to reproduce expected results for structural chromosomal aberration frequencies in spermatogonia with positive control substances (including weak responses) such as those listed in Table 1 and obtaining negative control frequencies that are consistent with acceptable range of control data in the published literature (e.g. (2)(3)(6)(7)(8)(9)(10)) or with the laboratory’s historical control distribution, if available.

DESCRIPTION OF THE METHOD

Preparations

Selection of animal species

11.Commonly used laboratory strains of healthy young adult animals should be employed. Male mice are commonly used; however, males of other appropriate mammalian species may be used when scientifically justified and to allow this test to be run in conjunction with another test method. The scientific justification for using species other than rodents should be provided in the report.

Animal Housing and feeding conditions

12.For rodents, the temperature in the animal room should be 22°C (±3°C). Although the relative humidity ideally should be 50-60%, it should be at least 40% and preferably not exceed 70% other than during room cleaning. 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 chemical when administered by this route. Rodents should be housed in small groups (no more than five per cage) if no aggressive behaviour is expected, preferably in solid floor cages with appropriate environmental enrichment. Animals may be housed individually if scientifically justified.

Preparation of the animals

13.Healthy young adult male animals (8-12 weeks old at start of treatment) are normally used, and are randomly assigned to the control and treatment groups. The individual animals are identified uniquely using a humane, minimally invasive method (e.g. by ringing, tagging, micro-chipping or biometric identification, but not ear or toe clipping) and 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. Cross contamination by the positive control and test chemical should be avoided. At the commencement of the study, the variation between individual animal weights should be minimal and not exceed ± 20%.

Preparation of doses

14.Solid test chemicals should be dissolved or suspended in appropriate solvents or vehicles or admixed in diet or drinking water prior to dosing of the animals. Liquid test chemicals may be dosed directly or diluted prior to dosing. For inhalation exposures, test chemicals can be administered as gas, vapour, or a solid/liquid aerosol, depending on their physicochemical properties. Fresh preparations of the test chemical should be employed unless stability data demonstrate the acceptability of storage and define the appropriate storage conditions.

Test conditions - Solvent/vehicle

15.The solvent/vehicle should not produce toxic effects at the dose levels used, and should not be capable of chemical reaction with the test chemicals. 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. Examples of commonly used compatible solvents/vehicles include water, physiological saline, methylcellulose solution, carboxymethyl cellulose sodium salt solution, olive oil and corn oil. In the absence of historical or published control data showing that no structural chromosomal aberrations and other deleterious effects are induced by a chosen atypical solvent/vehicle, an initial study should be conducted in order to establish the acceptability of the solvent/vehicle control.

Positive controls

16.Concurrent positive control animals should always be used unless the laboratory has demonstrated proficiency in the conduct of the test and has used the test routinely in the recent past (e.g. within the last 5 years). When a concurrent positive control group is not included, scoring controls (fixed and unstained slides) should be included in each experiment. These can be obtained by including within the scoring of the study appropriate reference samples that have been obtained and stored from a separate positive control experiment conducted periodically (e.g. every 6-18 months) in the laboratory where the test is performed; for example, during proficiency testing and on a regular basis thereafter, where necessary.

17.Positive control substances should reliably produce a detectable increase in the frequencies of cells with structural chromosomal aberrations over the spontaneous levels. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded samples to the scorer. Examples of positive control substances are included in Table 1.

Table 1: Examples of positive control substances.

Negative controls

18.Negative control animals, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups, should be included for every sampling time. In the absence of historical or published control data showing that no chromosomal aberrations or other deleterious effects are induced by the chosen solvent/vehicle, untreated control animals also should be included for every sampling time in order to establish acceptability of the vehicle control.

PROCEDURE

Number of animals

19.Group sizes at study initiation should be established with the aim of providing a minimum of 5 male animals per group. This number of animals per group is considered to be sufficient to provide adequate statistical power (i.e. generally able to detect at least a doubling in chromosomal aberration frequency when the negative control level is 1.0% or greater with 80% probability at a significance level of 0.05) (3) (11). As a guide to typical maximum animal requirements, a study at two sampling times with three dose groups and a concurrent negative control group, plus a positive control group (each composed of five animals per group), would require 45 animals.

Treatment schedule

20.Test chemicals are usually administered once (i.e. as a single treatment); other dose regimens may be used, provided they are scientifically justified.

21.In the highest dose group two sampling times after treatment are used. Since the time required for uptake and metabolism of the test chemical(s), as well as its effect on cell cycle kinetics, can affect the optimum time for chromosomal aberration detection, one early and one late sampling time approximately 24 and 48 hours after treatment are used. For doses other than the highest dose, an early sampling time of 24 hours (less than or equal to the cell cycle time of B spermatogonia and thus optimising the probability of scoring first post-treatment metaphases) after treatment should be taken, unless another sampling time is known to be more appropriate and justified.

22.Other sampling times may be used. For example in the case of chemicals that exert S-independent effects, earlier sampling times (i.e. less than 24 hr) may be appropriate.

23.A repeat dose treatment regimen can be used, such as in conjunction with a test on another endpoint that uses a 28 day administration period (e.g., TM B.58); however, additional animal groups would be required to accommodate different sampling times. Accordingly, the appropriateness of such a schedule needs to be justified scientifically on a case-by-case basis.

24.Prior to euthanasia, animals are injected intraperitoneally with an appropriate dose of a metaphase arresting chemical (e.g. Colcemid® or colchicine). Animals are sampled at an appropriate interval thereafter. For mice and rats, this interval is approximately 3 - 5 hours.

Dose levels

25.If a preliminary range-finding study is performed because there are no suitable data already available to aid in dose selection, it should be performed in the same laboratory, using the same species, strain, and treatment regimen to be used in the main study, according to recommendations for conducting dose range-finding studies (12). This study should aim to identify the maximum tolerated dose (MTD), defined as the dose inducing slight toxic effects relative to the duration of the study period (for example, abnormal behaviour or reactions, minor body weight depression or hematopoietic system cytotoxicity) but not death or evidence of pain, suffering or distress necessitating euthanasia of the animals (13).

26.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%.

27.Test chemicals with specific biological activities at low non-toxic doses (such as hormones and mitogens), and chemicals which exhibit saturation of toxicokinetic properties may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis.

28.In order to obtain dose response information, a complete study should include a negative control group (paragraph 18) and a minimum of three dose levels generally separated by a factor of 2, but by no greater than 4. If the test chemical does not produce toxicity in a range-finding study or based on existing data, the highest dose for a single administration should be 2000 mg/kg body weight. However, if the test chemical does cause toxicity, the MTD should be the highest dose administered, and the dose levels used should preferably cover a range from the maximum to a dose producing little or no toxicity. When target tissue (i.e. testis) toxicity is observed at all dose levels tested, further study at non-toxic doses is advisable. Studies intending to more fully characterise the quantitative dose-response information may require additional dose groups. For certain types of test chemicals (e.g. human pharmaceuticals) covered by specific requirements, these limits may vary. If the test chemical does produce toxicity, the limit dose plus two lower doses (as described above) should be selected. The limit dose for an administration period of 14 days or more is 1000 mg/kg body weight/day, and for administration periods of less than 14 days, the limit dose is 2000 mg/kg/body weight/day.

Administration of doses

29.The anticipated route of human exposure should be considered when designing an assay. Therefore, routes of exposure such as dietary, drinking water, topical subcutaneous, intravenous, oral (by gavage), inhalation, or implantation may be chosen as justified. In any case, the route should be chosen to ensure adequate exposure of the target tissue. Intraperitoneal injection is not normally recommended unless scientifically justified since it is not usually a physiologically relevant route of human exposure. If the test chemical is admixed in diet or drinking water, especially in case of single dosing, care should be taken that the delay between food and water consumption and sampling should be sufficient to allow detection of the effects (see paragraph 33). 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 normally exceed 1 ml/100g body weight except in the case of aqueous solutions where a maximum of 2 ml/100g body weight may be used. The use of volumes greater than this (if permitted by animal welfare legislation) should be justified. Variability in test volume should be minimised by adjusting the concentration to ensure a constant volume in relation to body weight at all dose levels.

Observations

30.General clinical observations of the test animals should be made and clinical signs recorded at least once a day, preferably at the same time(s) each day and considering the peak period of anticipated effects after dosing. At least twice daily, all animals should be observed for morbidity and mortality. All animals should be weighed at study initiation, at least once a week during repeated-dose studies, and at euthanasia. In studies of at least one-week duration, measurements of food consumption should be made at least weekly. If the test chemical is administered via the drinking water, water consumption should be measured at each change of water and at least weekly. Animals exhibiting non-lethal indicators of excess toxicity should be euthanised prior to completion of the test period (13).

Chromosome preparation    

31.Immediately after euthanasia, germ cell suspensions are obtained from one, or both, testes, exposed to hypotonic solution and fixed following established protocols (e.g. (2) (14) (15). The cells are then spread on slides and stained (16) (17). All slides should be coded so that their identity is not available to the scorer.

Analysis

32.At least 200 well spread metaphases should be scored for each animal (3) (11). If the historical negative control frequency is < 1%, more than 200 cells/animal should be scored to increase the statistical power (3). Staining methods that permit the identification of the centromere should be used.

33.Chromosome and chromatid-type aberrations should be recorded separately and classified by sub-types (breaks, exchanges). Gaps should be recorded, but not considered, when determining whether a chemical induces significant increases in the incidence of cells with chromosomal aberrations. Procedures in use in the laboratory should ensure that analysis of chromosomal aberrations is performed by well-trained scorers. Recognising that slide preparation procedures often result in the breakage of a proportion of metaphases with a resulting loss of chromosomes, the cells scored should, therefore, contain a number of centromeres not less than 2n±2, where n is the haploid number of chromosomes for that species.

34.Although the purpose of the test is to detect structural chromosomal aberrations, it is important to record the frequencies of polyploid cells and cells with endoreduplicated chromosomes when these events are seen (see Paragraph 44).

DATA AND REPORTING

Treatment of results

35.Individual animal data should be presented in tabular form. For each animal the number of cells with structural chromosomal aberration(s) and the number of chromosome aberrations per cell should be evaluated. Chromatid- and chromosome-type aberrations classified by sub-types (breaks, exchanges) should be listed separately with their numbers and frequencies for experimental and control groups. Gaps are recorded separately. The frequency of gaps is reported but generally not included in the analysis of the total structural chromosomal aberration frequency. Percentage of polyploidy and cells with endoreduplicated chromosomes are reported when seen.

36.Data on toxicity and clinical signs (as per Paragraph 30) should be reported.

Acceptability Criteria

37.The following criteria determine the acceptability of a test.

-Concurrent negative control is consistent with published norms for historical negative control data, which are generally expected to be > 0% and  ≤ 1.5% cells with chromosomal aberrations, and the laboratory's historical control data if available (see Paragraphs 10 and 18).

-Concurrent positive controls induce responses that are consistent with published norms for historical positive control data, or the laboratory’s historical positive control database, if available, and produce a statistically significant increase compared with the negative control (see Paragraphs 17, 18).

-Adequate numbers of cells and doses have been analysed (see Paragraphs 28 and 32).

-The criteria for the selection of top dose are consistent with those described in Paragraphs 25, and 26.

38.If both mitosis and meiosis are 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. If only mitosis is observed, the mitotic index should be determined in at least 1000 cells for each animal.

Evaluation and interpretation of results

39.At least three treated dose groups should be analysed in order to provide sufficient data for dose-response analysis.

40.Providing that all acceptability criteria are fulfilled, a test chemical is considered a clear positive if:

-at least one of the test doses exhibits a statistically significant increase compared with the concurrent negative control;

-the increase is dose-related at least at one sampling time; and,

-any of the results are outside acceptable range of negative control data, or the distribution of the laboratory’s historical negative control data (e.g. Poisson-based 95% control limit) if available.

The test chemical is then considered able to induce chromosomal aberrations in spermatogonial cells of the test animals. Recommendations for the most appropriate statistical methods can also be found in the literature (11) (18). Statistical tests used should consider the animal as the experimental unit.

41.Providing that all acceptability criteria are fulfilled, a test chemical is considered a clear negative if:

-none of the test doses exhibits a statistically significant increase compared with the concurrent negative control;

-there is no dose-related increase in any experimental condition; and,

-all results are within acceptable range of negative control data, or the laboratory’s historical negative control data (e.g. Poisson-based 95% control limit), if available.

The test chemical is then considered unable to induce chromosomal aberrations in the spermatogonial cells of the test animals. Recommendations for the most appropriate statistical methods can also be found in the literature (11) (18). A negative result does not exclude the possibility that the chemical may induce chromosomal aberrations at later developmental phases not studied, or gene mutations.

42.There is no requirement for verification of a clear positive or clear negative response.

43.If the response is not clearly negative or positive, and in order to assist in establishing the biological relevance of a result (e.g. a weak or borderline increase), the data should be evaluated by expert judgment and/or further investigations using the existing experimental data, such as consideration whether the positive result is outside the acceptable range of negative control data, or the laboratory's historical negative control data (19).

44.In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and will therefore be concluded as equivocal.

45.An increase in the number of polyploid cells may indicate that the test chemical has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations (20). An increase in the number of cells with endoreduplicated chromosomes may indicate that the test chemical has the potential to inhibit cell cycle progress (21) (22), which is a different mechanism of inducing numerical chromosome changes than inhibition of mitotic processes (see Paragraph 2). Therefore incidence of polyploid cells and cells with endoreduplicated chromosomes should be recorded separately.

Test report

46.The test report should include the following information:

Summary.

Test chemical:

-source, lot number, limit date for use, if available;

-stability of the test chemical itself, if known;

-solubility and stability of the test chemical in solvent, if known;

-measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical was added, as appropriate.

Mono-constituents substance:

-physical appearance, water solubility, and additional relevant physicochemical properties;

-chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc.

Multi-constituent substance, UVCBs and mixtures:

-characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant physicochemical properties of the constituents.

Test chemical preparation:

-justification for choice of vehicle;

-solubility and stability of the test chemical in solvent/vehicle.

-preparation of dietary, drinking water or inhalation formulations;

-analytical determinations on formulations (e.g. stability, homogeneity, nominal concentrations)

-when conducted.

Test animals:

-species/strain used and justification for use;

-number and age of animals;

-source, housing conditions, diet, etc.;

-method for uniquely identifying the animals

-for short-term studies: individual weight of the animals at the start and end of the test; for studies longer than one week: individual body weights during the study and food consumption. Body weight range, mean and standard deviation for each group should be included.

Test conditions:

-positive and negative (vehicle/solvent) control data;

-data from range finding study, if conducted;

-rationale for dose level selection;

-rationale for route of administration;

-details of test chemical preparation;

-details of the administration of the test chemical;

-rationale for sacrifice times;

-methods for measurement of animal toxicity, including, where available, histopathological or hematological analyses and the frequency with which animal observations and body weights were taken;

-methods for verifying that the test chemical reached the target tissue, or general circulation, if negative results are obtained;

-actual dose (mg/kg body weight/day) calculated from diet/drinking water test chemical concentration (ppm) and consumption, if applicable;

-details of food and water quality;

-detailed description of treatment and sampling schedules and justifications for the choices;

-method of euthanasia;

-method of analgesia (where used)

-procedures for isolating tissues;

-identity of metaphase arresting chemical, 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:

-animal condition prior to and throughout the test period, including signs of toxicity;

-body and organ weights at sacrifice (if multiple treatments are employed, body weights taken during the treatment regimen);

-signs of toxicity;

-mitotic index;

-ratio of spermatogonial mitoses cells to first and second meiotic metaphases, or other evidence of exposure to the target tissue;

-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;

-dose-response relationship, where possible;

-statistical analyses and methods applied;

-concurrent negative control data;

-historical negative control data with ranges, means, standard deviations, and 95% confidence interval (where available), or published historical negative control data used for acceptability of the test results;

-concurrent positive control data;

-changes in ploidy, if seen, including frequencies of polyploidy and/or endoreduplicated cells.

Discussion of the results

Conclusion

LITERATURE

(1)OECD (2016). Overview of the set of OECD Genetic Toxicology Test Guidelines and updates performed in 2014-2015. ENV Publications. Series on Testing and Assessment, No 234, OECD, Paris

(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, pp. 275-306.

(3)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, 313-318.

(4)Russo, A. (2000). In Vivo Cytogenetics: Mammalian Germ Cells. Mutation Res., 455, 167-189.

(5)Hess, R.A. and de Franca L.R. (2008). Spermatogenesis and Cycle of the Seminiferous Epithelium. In: Molecular Mechanisms in Spermatogenesis, Cheng C.Y. (Ed.) Landes Bioscience and Springer Science+Business Media, pp. 1-15.

(6)Adler, I.-D. (1974). Comparative Cytogenetic Study after Treatment of Mouse Spermatogonia with Mitomycin C, Mutation. Res., 23(3): 368-379.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, pp. 477-484.

(7)Cattanach, B.M., and Pollard C.E. (1971). Mutagenicity Tests with Cyclohexylamine in the Mouse, Mutation Res., 12, 472-474.

(8)Cattanach, B.M., and Williams, C.E. (1971). A search for Chromosome Aberrations Induced in Mouse Spermatogonia by Chemical Mutagens, Mutation Res., 13, 371-375.

(9)Rathenburg, R. (1975). Cytogenetic Effects of Cyclophosphamide on Mouse Spermatogonia, Humangenetik 29, 135-140.

(10)Shiraishi, Y. (1978). Chromosome Aberrations Induced by Monomeric Acrylamide in Bone Marrow and Germ Cells of Mice, Mutation Res., 57(3): 313–324.

(11)Adler I-D., Bootman, J., Favor, J., Hook, G., Schriever-Schwemmer, G., Welzl, G.,, Whorton, E., Yoshimura, I. and Hayashi, M. (1998). Recommendations for Statistical Designs of In Vivo Mutagenicity Tests with Regard to Subsequent Statistical Analysis, Mutation Res., 417, 19–30.

(12)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, 313-319.

(13)OECD (2000). Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation, Series on Testing and Assessment, (No 19.), Organisation for Economic Cooperation and Development, Paris.

(14)Yamamoto, K. and Kikuchi, Y. (1978). A New Method for Preparation of Mammalian Spermatogonial Chromosomes. Mutation Res., 52, 207-209.

(15)Hsu, T.C., Elder, F. and Pathak, S. (1979). Method for Improving the Yield of Spermatogonial and Meiotic Metaphases in Mammalian Testicular Preparations. Environ. Mutagen., 1, 291-294.

(16)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, 289-294.

(17)Richold, M., Ashby, J., Bootman, J., Chandley, A., Gatehouse, D.G. and Henderson, L. (1990). In Vivo Cytogenetics 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, pp. 115-141.

(18)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, pp. 184-232.

(19)Hayashi, M., Dearfield, K., Kasper, P., Lovell, D., Martus, H.-J. and Thybaud, V. (2011). Compilation and Use of Genetic Toxicity Historical Control Data. Mutation Res., 723, 87-90.

(20)Warr T.J., Parry E.M. and Parry J.M. (1993). A Comparison of Two In Vitro Mammalian Cell Cytogenetic Assays for the Detection of Mitotic Aneuploidy Using 10 Known or Suspected Aneugens, Mutation Res., 287, 29-46.

(21)Huang, Y., Change, C. and Trosko, J.E. (1983). Aphidicolin-Induced Endoreduplication in Chinese Hamster Cells. Cancer Res., 43, 1362-1364. 

(22)Locke-Huhle, C. (1983). Endoreduplication in Chinese Hamster Cells during Alpha-Radiation Induced G2 Arrest. Mutation Res., 119, 403-413.

Appendix

Definitions

Aneuploidy: any deviation from the normal diploid (or haploid) number of chromosomes by a single chromosome or more than one, but not by entire set(s) of chromosomes (polyploidy).

Centromere: 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.

Chemical: A substance or a mixture

Chromosome diversity: diversity of chromosome shapes (e.g. metacentrique, acrocentriques, etc) and sizes.

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.

Clastogen: any chemical which causes structural chromosomal aberrations in populations of cells or organisms.

Gap: an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatids.

Genotoxic: a general term encompassing all types of DNA or chromosome damage, including breaks, deletions, adducts, nucleotides modifications and linkages, rearrangements, mutations, chromosome aberrations, and aneuploidy. Not all types of genotoxic effects result in mutations or stable chromosome damage.”

Mitotic index (MI): 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.

Mitosis: division of the cell nucleus usually divided into prophase, prometaphase, metaphase, anaphase, and telophase.

Mutagenic: produces a heritable change of DNA base-pair sequence(s) in genes or of the structure of chromosomes (chromosome aberrations).

Numerical abnormality: 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 and fragments, exchanges.

Test chemical: Any substance or mixture tested using this test method.

UVCB: Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials"

(5) In Part B, Chapter B.40 is replaced by the following:

"B.40 IN VITRO SKIN CORROSION: TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST METHOD (TER)

INTRODUCTION

1.This test method (TM) is equivalent to OECD test guideline (TG) 430 (2015). Skin corrosion refers to the production of irreversible damage to the skin manifested as visible necrosis through the epidermis and into the dermis, following the application of a test chemical [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (1) and the European Union (EU) Regulation 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures (CLP) 1 ]. This updated test method B.40 provides an in vitro procedure allowing the identification of non-corrosive and corrosive substances and mixtures in accordance with UN GHS (1) and CLP

2.The assessment of skin corrosivity has typically involved the use of laboratory animals (TM B.4, equivalent to OECD TG 404 originally adopted in 1981, and revised in 1992, 2002 and 2015) (2). In addition to the present TM B.40, other in vitro test methods for testing of skin corrosion potential of chemicals have been validated and adopted as TM B.40bis (equivalent to OECD TG 431) (3) and TM B.65 (equivalent to OECD TG 435) (4), that are also able to identify sub-categories of corrosive chemicals when required. Several validated in vitro test methods have been adopted as TM B.46 (equivalent to OECD TG 439 (5), to be used for the testing of skin irritation. An OECD guidance document on Integrated Approaches to Testing and Assessment (IATA) for Skin Corrosion and Irritation describes several modules which group various information sources and analysis tools and provides guidance on (i) how to integrate and use existing testing and non-testing data for the assessment of skin irritation and skin corrosion potentials of chemicals and (ii) proposes an approach when further testing is needed (6).

3.This test method addresses the human health endpoint skin corrosion. It is based on the rat skin transcutaneous electrical resistance (TER) test method, which utilises skin discs to identify corrosives by their ability to produce a loss of normal stratum corneum integrity and barrier function. The corresponding OECD test guideline was originally adopted in 2004 and updated in 2015 to refer to the IATA guidance document.

4.In order to evaluate in vitro skin corrosion testing for regulatory purposes, pre-validation studies (7) followed by a formal validation study of the rat skin TER test method for assessing skin corrosion were conducted (8) (9) (10) (11). The outcome of these studies led to the recommendation that the TER test method (designated the Validated Reference Method – VRM) could be used for regulatory purposes for the assessment of in vivo skin corrosivity (12) (13) (14).

5.Before a proposed similar or modified in vitro TER test method for skin corrosion other than the VRM can be used for regulatory purposes, its reliability, relevance (accuracy), and limitations for its proposed use should be determined to ensure its similarity to the VRM, in accordance with the requirements of the Performance Standards (PS) (15). OECD Mutual Acceptance of Data will only be guaranteed after any proposed new or updated test method following the PS have been reviewed and included in the corresponding OECD test guideline.

DEFINITIONS

6.Definitions used are provided in the Appendix.

INITIAL CONSIDERATIONS

7.A validation study (10) and other published studies (16) (17) have reported that the rat skin TER test method is able to discriminate between known skin corrosives and non-corrosives with an overall sensitivity of 94% (51/54) and specificity of 71% (48/68) for a database of 122 substances.

8.This test method addresses in vitro skin corrosion. It allows the identification of non-corrosive and corrosive test chemicals in accordance with the UN GHS/CLP. A limitation of this test method, as demonstrated by the validation studies (8) (9) (10) (11), is that it does not allow the sub-categorisation of corrosive substances and mixtures in accordance with the UN GHS/ CLP. The applicable regulatory framework will determine how this test method will be used. While this test method does not provide adequate information on skin irritation, it should be noted that TM B.46 specifically addresses the health effect skin irritation in vitro (5). For a full evaluation of local skin effects after a single dermal exposure, the OECD Guidance Document on IATA should be consulted (6).

9.A wide range of chemicals representing mainly substances has been tested in the validation underlying this test method and the empirical database of the validation study amounted to 60 substances covering a wide range of chemical classes (8) (9). On the basis of the overall data available, the test method is applicable to a wide range of chemical classes and physical states including liquids, semi-solids, solids and waxes. However, since for specific physical states test items with suitable reference data are not readily available, it should be noted that a comparably small number of waxes and corrosive solids were assessed during validation. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. In cases where evidence can be demonstrated on the non-applicability of the test method to a specific category of substances, the test method should not be used for that specific category of substances. In addition, this test method is assumed to be applicable to mixtures as an extension of its applicability to substances. However, due to the fact that mixtures cover a wide spectrum of categories and composition, and that only limited information is currently available on the testing of mixtures, in cases where evidence can be demonstrated on the non-applicability of the test method to a specific category of mixtures (e.g. following a strategy as proposed by Eskes et al., 2012) (18), the test method should not be used for that specific category of mixtures. Before use of the test method on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose. Such considerations are not needed, when there is a regulatory requirement for testing of the mixture. Gases and aerosols have not been assessed yet in validation studies (8) (9). While it is conceivable that these can be tested using the TER test method, the current test method does not allow testing of gases and aerosols.

PRINCIPLE OF THE TEST

10.The test chemical 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 chemicals 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 (16) (see paragraph 32). For rat skin TER, a cut-off value of 5kΩ has been selected based on extensive data for a wide range of substances where the vast majority of values were either clearly well above (often > 10 kΩ), or well below (often < 3 kΩ) this value (16). Generally, test chemicals that are non-corrosive in animals but are irritant or non-irritant 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.

11.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 TM B.4 (2).

DEMONSTRATION OF PROFICIENCY

12.Prior to routine use of the rat skin TER test method that adheres to this test method, laboratories should demonstrate technical proficiency by correctly classifying the twelve Proficiency Substances recommended in Table 1. In situations where a listed substance is unavailable or where justifiable, another substance for which adequate in vivo and in vitro reference data are available may be used (e.g. from the list of reference chemicals (16)) provided that the same selection criteria as described in Table 1 is applied.

Table 1: List of Proficiency Substances1

Abbreviations: aq = aqueous; CASRN = Chemical Abstracts Service Registry Number;; VRM = Validated Reference Method; ND = Not Determined.

1The proficiency substances, sorted first by corrosives versus non-corrosives, then by corrosive subcategory and then by chemical class, were selected from the substances used in the ECVAM validation study of the rat skin TER test method (8) (9). Unless otherwise indicated, the substances were tested at the purity level obtained when purchased from a commercial source (8). The selection included, to the extent possible, substances that: (i) are representative of the range of corrosivity responses (e.g. non-corrosives; weak to strong corrosives) that the VRM is capable of measuring or predicting; (ii) are representative of the chemical classes used in the validation study; (iii) reflect the performance characteristics of the VRM; (iv) have chemical structures that are well-defined; (v) induce definitive results in the in vivo reference test method; (vi) are commercially available; and (vii) are not associated with prohibitive disposal costs.

2Chemical class assigned by Barratt et al. (8).

3The corresponding UN Packing groups are I, II and III, respectively, for the UN GHS/CLP categories 1A, 1B and 1C.

4The pH values were obtained from Fentem et al. (9) and Barratt et al. (8).

PROCEDURE

13.Standard Operating Procedures (SOP) for the rat skin TER skin corrosion test method are available (19). The rat skin TER test methods covered by this test method should comply with the following conditions:

Animals

14.Rats should be used because the sensitivity of their skin to substances in this test method has been previously demonstrated (12) and is the only skin source that has been formally validated (8) (9). 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.

15.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 3 days of the second wash, when the stratum corneum has recovered from the hair removal.

Preparation of the skin discs

16.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 discs are used where it is shown that positive and negative control data are equivalent to that obtained with fresh skin.

17.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. 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. Tube and ‘O’ ring dimensions are shown in Figure 2.

18.Before testing begins, the TER of two skin discs are measured as a quality control procedure for each animal skin. Both discs should give electrical resistance values greater than 10 kΩ for the remainder of the discs to be used for the test method. If the resistance value is less than 10 kΩ, the remaining discs from that skin should be discarded.

Application of the test chemical and control substances

19.Concurrent positive and negative controls should be used for each run (experiment) to ensure adequate performance of the experimental model. Skin discs from a single animal should be used in each run (experiment). The suggested positive and negative control test chemicals are 10M hydrochloric acid and distilled water, respectively.

20.Liquid test chemicals (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 300 C to melt or soften the test chemical, or ground to produce a granular material or powder.

21.Three skin discs are used for each test and control chemical in each testing run (experiment). Test chemicals are applied for 24 hours at 20-230 C. The test chemical is removed by washing with a jet of tap water at up to room temperature until no further material can be removed.

TER measurements

22.The skin impedance is measured as TER by using a low-voltage, alternating current Wheatstone bridge (18). 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 measured inductance, capacitance and resistance up to values of 2000H, 2000 μF, and 2 MΩ, respectively at frequencies of 100Hz or 1kHz, 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 (154mM). 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).

23.If the measured resistance value is greater than 20 kΩ, this may be due to the remains of the test chemical 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.

24.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 test 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 Proficiency Substances chosen from the substances used in the validation study (8) (9), or from similar chemical classes to the substances being investigated. A set of suitable Proficiency Substances is identified in Table 1.

Dye Binding Methods

25.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 (9). For example, neutral organics and substances that have surface-active properties (including detergents, emulsifiers and other surfactants) can remove skin lipids making the barrier more permeable to ions. Thus, if TER values produced by such chemicals are less than or around 5 kΩ in the absence of visually perceptible damage of the skin discs, 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 (7) (9). 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 substances and is not affected by the extraction procedure described below.

Sulforhodamine B dye application and removal

26.Following TER assessment, the magnesium sulphate is discarded from the tube and the skin is carefully examined for obvious damage. If there is no obvious major damage (e.g. perforation), 150 μl of a 10% (w/v) dilution in distilled water of the dye sulforhodamine B (Acid Red 52; C.I. 45100; CAS number 3520-42-1), is applied to the epidermal surface of each skin disc for 2 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 5 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 5ml of 30% (w/v) sodium dodecyl sulphate (SDS) in distilled water and are incubated overnight at 600 C.

27.After incubation, each skin disc is removed and discarded and the remaining solution is centrifuged for 8 minutes at 210 C (relative centrifugal force ~175 x g). A 1ml sample of the supernatant is diluted 1 in 5 (v/v) [i.e. 1ml + 4ml] with 30% (w/v) SDS in distilled water. The optical density (OD) of the solution is measured at 565 nm.

Calculation of dye content

28.The sulforhodamine B dye content per disc is calculated from the OD values (9) (sulforhodamine B dye molar extinction coefficient at 565nm = 8.7 x l04; molecular weight = 580). The dye content is determined for each skin disc by the use of an appropriate calibration curve and mean dye content is then calculated for the replicates.

Acceptability Criteria

29.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:

30.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 in the following table:

Interpretation of results

31.The cut-off TER value distinguishing corrosive from non-corrosive test chemicals was established during test method optimisation, tested during a pre-validation phase, and confirmed in a formal validation study.

32.The prediction model for rat skin TER skin corrosion test method (9) (19), associated with the UN GHS/CLP classification system, is given below:

   The test chemical is considered to be non-corrosive to skin:

I)if the mean TER value obtained for the test chemical is greater than (>) 5 kΩ, or

II)the mean TER value obtained for the test chemical is less than or equal to (≤) 5 kΩ, and

-the skin discs show no obvious damage(e.g. perforation), and

-the mean disc dye content is less than (<) the mean disc dye content of the 10M HCl positive control obtained concurrently (see paragraph 30 for positive control values).

   The test chemical is considered to be corrosive to skin:

I)if the mean TER value obtained for the test chemical is less than or equal to (≤) 5 kΩ and the skin discs are obviously damaged(e.g. perforated), or

II)the mean TER value obtained for the test chemical is less than or equal to (≤) 5 kΩ, and

-the skin discs show no obvious damage(e.g. perforation), 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 (see paragraph 30 for positive control values).

33.A testing run (experiment) composed of at least three replicate skin discs should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean TER equal to 5 ± 0.5 kΩ, a second independent testing run (experiment) should be considered, as well as a third one in case of discordant results between the first two testing runs (experiments).

DATA AND REPORTING

Data

34.Resistance values (kΩ) and dye content values (µg/disc), where appropriate, for the test chemical, as well as for positive and negative controls should be reported in tabular form, including data for each individual replicate disc in each testing run (experiment) and mean values ± SD. All repeat experiments should be reported. Observed damage in the skin discs should be reported for each test chemical.

Test report

35.The test report should include the following information:

Test Chemical and Control Substances:

-Mono-constituent substance: chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc;

-Multi-constituent substance, UVCB and mixture: characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant physico-chemical properties of the constituents;

-Physical appearance, water solubility, and additional relevant physico-chemical properties;

-Source, lot number if available;

-Treatment of the test chemical/control substance prior to testing, if applicable (e.g. warming, grinding);

-Stability of the test chemical, limit date for use, or date for re-analysis if known;

-Storage conditions.

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, band pass used for measuring OD values, and OD linearity range of measuring device (e.g. spectrophotometer), if appropriate;

-Details of the test procedure used for TER measurements;

-Details of the test procedure used for the dye binding assessment, if appropriate;

-Test doses used, duration of exposure period(s) and temperature(s) of exposure;

-Details on washing procedure used after the exposure period;

-Number of replicate skin discs used per test chemical and controls (positive and negative control);

-Description of any modification of the test procedure;

-Reference to historical data of the model. This should include, but is not limited to:

I) Acceptability of the positive and negative control TER values (in kΩ) with reference to positive and negative control resistance ranges

II)     Acceptability of the positive and negative control dye content values (in µg/disc) with reference to positive and negative control dye content ranges

III)Acceptability of the test results with reference to historical variability between skin disc replicates

-Description of decision criteria/prediction model applied.

Results:

-Tabulation of data from the TER and dye binding assays (if appropriate) for individual test chemicals and controls, for each testing run (experiment) and each skin disc replicate (individual animals and individual skin samples), means, SDs and CVs;

-Description of any effects observed;

-The derived classification with reference to the prediction model/decision criteria used.

Discussion of the results

Conclusions

LITERATURE

(1)United Nations (UN) (2013). Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Second Revised Edition, UN New York and Geneva, 2013. Available at: [http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html].

(2)Chapter B.4 of this Annex, Acute Dermal Irritation, Corrosion.

(3)Chapter B.40bis of this Annex, In Vitro Skin Model.

(4)Chapter B.65 of this Annex, In Vitro Membrane Barrier Test Method.

(5)Chapter B.46 of this Annex, In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.

(6)OECD (2014). Guidance document on Integrated Approaches to Testing and Assessment for Skin Irritation/Corrosion. Environment, Health and Safety Publications, Series on Testing and Assessment, (No 203), Organisation for Economic Cooperation and Development, Paris.

(7)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, 219-255.

(8)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. Toxic.In Vitro 12, 471-482.

(9)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. Toxic.In Vitro12, 483- 524.

(10)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.ATLA23, 129-147.

(11)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.

(12)EC-ECVAM (1998). Statement on the Scientific Validity of the Rat Skin Transcutaneos Electrical Resistance (TER) Test (an In Vitro Test for Skin Corrosivity), Issued by the ECVAM Scientific Advisory Committee (ESAC10), 3 April 1998.

(13)ECVAM (1998). ECVAM News & Views. ATLA 26, 275-280.

(14)ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) (2002). ICCVAM Evaluation of EpiDermTM (EPI-200), EPISKINTM (SM), 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.    

(15)OECD (2015). Performance Standards for the Assessment of Proposed Similar or Modified In Vitro Transcutaneous Electrical Resistance (TER) Test Method for Skin Corrosion in Relation to TG 430. Environmental Health and Safety Publications, Series on Testing and Assessment No 218. Organisation for Economic Cooperation and Development, Paris.

(16)Oliver G.J.A., Pemberton M.A., and Rhodes C. (1986). An In Vitro Skin Corrosivity Test -Modifications and Validation. Fd. Chem. Toxicol.24, 507-512.

(17)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. Toxicol. In Vitro 6,191-194.

(18)Eskes C., Detappe V., Koëter H., Kreysa J., Liebsch M., Zuang V., Amcoff P., Barroso J., Cotovio J., Guest R., Hermann M., Hoffmann S., Masson P., Alépée N., Arce L.A., Brüschweiler B., Catone T., Cihak R., Clouzeau J., D'Abrosca F., Delveaux C., Derouette J.P., Engelking O., Facchini D., Fröhlicher M., Hofmann M., Hopf N., Molinari J., Oberli A., Ott M., Peter R., Sá-Rocha V.M., Schenk D., Tomicic C., Vanparys P., Verdon B., Wallenhorst T., Winkler G.C. and Depallens O. (2012). Regulatory Assessment of In Vitro Skin Corrosion and Irritation Data Within the European Framework: Workshop Recommendations. Regul.Toxicol.Pharmacol. 62, 393-403.

(19)TER SOP (December 2008). INVITTOX Protocol (No 115) Rat Skin Transcutaneous Electrical Resistance (TER) Test.

(20) OECD (2005). Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment. Environment, Health and Safety Publications, Series on Testing and Assessment (No 34), Organisation for Economic Cooperation and Devlopment, Paris.

Figure 1: Apparatus for the rat skin TER assay

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 should not be 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 PTFE tube, as shown in Figure 1,

-The skin disc should be fixed well enough to the PTFE tube, such that the electrical resistance is a true measure of the skin properties.

Appendix

DEFINITIONS

Accuracy: The closeness of agreement between test method results and accepted reference values. It is a measure of test method performance and one aspect of relevance. The term is often used interchangeably with “concordance” to mean the proportion of correct outcomes of a test method (20).

C: Corrosive.

Chemical: A substance or a mixture.

Concordance: A measure of test method performance for test methods that give a categorical result, and is one aspect of relevance. The term is sometimes used interchangeably with accuracy, and is defined as the proportion of all chemicals tested that are correctly classified as positive or negative. Concordance is highly dependent on the prevalence of positives in the types of test chemical being examined (20).

GHS (Globally Harmonized System of Classification and Labelling of Chemicals (UN)): A system proposing the classification of chemicals (substances and mixtures) according to standardised types and levels of physical, health and environmental hazards, and addressing corresponding communication elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data sheets, so that to convey information on their adverse effects with a view to protect people (including employers, workers, transporters, consumers and emergency responders) and the environment (1).

IATA: Integrated Approach on Testing and Assessment.

Mixture: A mixture or solution composed of two or more substances.

Mono-constituent substance: A substance, defined by its quantitative composition, in which one main constituent is present to at least 80% (w/w).

Multi-constituent substance: A substance, defined by its quantitative composition, in which more than one main constituent is present in a concentration ≥ 10% (w/w) and < 80% (w/w). A multi-constituent substance is the result of a manufacturing process. The difference between mixture and multi-constituent substance is that a mixture is obtained by blending of two or more substances without chemical reaction. A multi-constituent substance is the result of a chemical reaction.

NC: Non corrosive.

OD: Optical Density.

PC: Positive Control, a replicate containing all components of a test system and treated with a substance known to induce a positive response. To ensure that variability in the positive control response across time can be assessed, the magnitude of the positive response should not be excessive.

Performance standards (PS): Standards, based on a validated test method, that provide a basis for evaluating the comparability of a proposed test method that is mechanistically and functionally similar. Included are; (i) essential test method components; (ii) a minimum list of Reference Chemicals selected from among the chemicals used to demonstrate the acceptable performance of the validated test method; and (iii) the similar levels of reliability and accuracy, based on what was obtained for the validated test method, that the proposed test method should demonstrate when evaluated using the minimum list of Reference Chemicals.

Relevance: Description of relationship of the test method to the effect of interest and whether it is meaningful and useful for a particular purpose. It is the extent to which the test method correctly measures or predicts the biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test method (20).

Reliability: Measures of the extent that a test method can be performed reproducibly within and between laboratories over time, when performed using the same protocol. It is assessed by calculating intra- and inter-laboratory reproducibility (20).

Sensitivity: The proportion of all positive/active chemicals that are correctly classified by the test method. It is a measure of accuracy for a test method that produces categorical results, and is an important consideration in assessing the relevance of a test method (20).

Skin corrosion in vivo: The production of irreversible damage of the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test chemical 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.

Specificity: The proportion of all negative/inactive chemicals that are correctly classified by the test method. It is a measure of accuracy for a test method that produces categorical results and is an important consideration in assessing the relevance of a test method (20).

Substance: A chemical element and its compounds in the natural state or obtained by any production process, inducing any additive necessary to preserve its stability and any impurities deriving from the process used, but excluding any solvent which may be separated without affecting the stability of the substance or changing it composition.

(Testing) run: A single test chemical concurrently tested in a minimum of three replicate skin discs.

Test chemical: Any substance or mixture tested using this test method.

Transcutaneous Electrical Resistance (TER): is a measure of the electrical impedance 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.

UVCB: Substances of unknown or variable composition, complex reaction products or biological materials. "

(6) In Part B, Chapter B.40bis is replaced by the following:

"B.40bis IN VITRO SKIN CORROSION: RECONSTRUCTED HUMAN EPIDERMIS (RhE) TEST METHOD

INTRODUCTION

1.This test method (TM) is equivalent to OECD test guideline (TG) 431 (2016). Skin corrosion refers to the production of irreversible damage to the skin manifested as visible necrosis through the epidermis and into the dermis, following the application of a test chemical [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (1) and the European Union (EU) Regulation 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures (CLP) 2 ]. This updated test method B.40bis provides an in vitro procedure allowing the identification of non-corrosive and corrosive substances and mixtures in accordance with UN GHS and CLP. It also allows a partial sub-categorisation of corrosives.

2.The assessment of skin corrosion potential of chemicals has typically involved the use of laboratory animals (TM B.4, equivalent to OECD TG 404; originally adopted in 1981 and revised in 1992, 2002 and 2015) (2). In addition to the present test method B.40bis, two other in vitro test methods for testing corrosion potential of chemicals have been validated and adopted as TM B.40 (equivalent to OECD TG 430) (3) and TM B.65 (equivalent to OECD TG 435) (4). Furthermore the in vitro TM B.46 (equivalent to OECD TG 439) (5) has been adopted for testing skin irritation potential. A OECD guidance document on Integrated Approaches to Testing and Assessment (IATA) for Skin Corrosion and Irritation describes several modules which group information sources and analysis tools, and provides guidance on (i) how to integrate and use existing testing and non-testing data for the assessment of skin irritation and skin corrosion potentials of chemicals and (ii) proposes an approach when further testing is needed (6).

3.This test method addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The corresponding OECD test guideline was originally adopted in 2004 and updated in 2013 to include additional test methods using the RhE modelsand the possibility to use the methods to support the sub-categorisation of corrosive chemicals, and updated in 2015 to refer to the IATA guidance document and introduce the use of an alternative procedure to measure viability.

4.Four validated commercially available RhE models are included in this test method. Prevalidation studies (7), followed by a formal validation study for assessing skin corrosion (8)(9)(10) have been conducted (11) (12) for two of these commercially available test models, EpiSkin™ Standard Model (SM) and EpiDerm™ Skin Corrosivity Test (SCT) (EPI-200) (referred to in the following text as the Validated Reference Methods - VRMs). The outcome of these studies led to the recommendation that the two VRMs mentioned above could be used for regulatory purposes for distinguishing corrosive (C) from non-corrosive (NC) substances, and that the EpiSkin™ could moreover be used to support sub-categorisation of corrosive substances (13)(14)(15). Two other commercially available in vitro skin corrosion RhE test models have shown similar results to the EpiDerm™ VRM according to PS-based validation (16)(17)(18). These are the SkinEthic™ RHE 3 and epiCS® (previously named EST-1000) that can also be used for regulatory purposes for distinguishing corrosive from noncorrosive substances (19)(20). Post validation studies performed by the RhE model producers in the years 2012 to 2014 with a refined protocol correcting interferences of unspecific MTT reduction by the test chemicals improved the performance of both discrimination of C/NC as well as supporting subcategorisation of corrosives (21)(22). Further statistical analyses of the post-validation data generated with EpiDerm™ SCT, SkinEthic™ RHE and EpiCS® have been performed to identify alternative predictions models that improved the predictive capacity for sub-categorisation (23).

5.Before a proposed similar or modified in vitro RhE test method for skin corrosion other than the VRMs can be used for regulatory purposes, its reliability, relevance (accuracy), and limitations for its proposed use should be determined to ensure its similarity to the VRMs, in accordance with the requirements of the Performance Standards (PS) (24) set out in accordance with the principles of OECD guidance document No 34 (25). The Mutual Acceptance of Data will only be guaranteed after any proposed new or updated test method following the PS have been reviewed and included in the corresponding test guideline. The test models included in that test guideline can be used to address countries’ requirements for test results on in vitro test method for skin corrosion, while benefiting from the Mutual Acceptance of Data.

DEFINITIONS

6.Definitions used are provided in Appendix 1.

INITIAL CONSIDERATIONS

7.This test method allows the identification of non-corrosive and corrosive substances and mixtures in accordance with the UN GHS and CLP. This test method further supports the sub-categorisation of corrosive substances and mixtures into optional sub-category 1A, in accordance with the UN GHS (1), as well as a combination of sub-categories 1B and 1C (21)(22)(23). A limitation of this test method is that it does not allow discriminating between skin corrosive sub-category 1B and sub-category 1C in accordance with the UN GHS and CLP due to the limited set of well-known in vivo corrosive sub-category 1C chemicals. EpiSkin™, EpiDerm™ SCT, SkinEthic™ RHE and epiCS® test models are able to sub-categorise (i.e. 1A versus 1B-and-1C versus NC)

8.A wide range of chemicals representing mainly individual substances has been tested in the validation supporting the test models included in this test method when they are used for identification of non-corrosives and corrosives; the empirical database of the validation study amounted to 60 chemicals covering a wide range of chemical classes (8)(9)(10). Testing to demonstrate sensitivity, specificity, accuracy and within-laboratory-reproducibility of the assay for sub-categorisation was performed by the test method developers and results were reviewed by the OECD (21) (22) (23). On the basis of the overall data available, the test method is applicable to a wide range of chemical classes and physical states including liquids, semi-solids, solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. Whenever possible, solids should be ground to a fine powder before application; no other prior treatment of the sample is required. In cases where evidence can be demonstrated on the non-applicability of test models included in this test method to a specific category of test chemicals, they should not be used for that specific category of test chemicals. In addition, this test method is assumed to be applicable to mixtures as an extension of its applicability to substances. However, due to the fact that mixtures cover a wide spectrum of categories and composition, and that only limited information is currently available on the testing of mixtures, in cases where evidence can be demonstrated on the non-applicability of the test method to a specific category of mixtures (e.g. following a strategy as proposed in (26)), the test method should not be used for that specific category of mixtures. Before use of the test method on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose. Such considerations are not needed, when there is a regulatory requirement for testing of the mixture. Gases and aerosols have not been assessed yet in validation studies (8)(9)(10). While it is conceivable that these can be tested using RhE technology, the current test method does not allow testing of gases and aerosols.

9.Test chemicals absorbing light in the same range as MTT formazan and test chemicals able to directly reduce the vital dye MTT (to MTT formazan) may interfere with the tissue viability measurements and need the use of adapted controls for corrections. The type of adapted controls that may be required will vary depending on the type of interference produced by the test chemical and the procedure used to measure MTT formazan (see paragraphs 25-31).

10.While this test method does not provide adequate information on skin irritation, it should be noted that TM B.46 specifically addresses the health effect skin irritation in vitro and is based on the same RhE test system, though using another protocol (5). For a full evaluation of local skin effects after a single dermal exposure, the OECD Guidance Document on Integrated Approaches for Testing and Assessment should be consulted (6). This IATA approach includes the conduct of in vitro tests for skin corrosion (such as described in this test method) and skin irritation before considering testing in living animals. It is recognised that the use of human skin is subject to national and international ethical considerations and conditions.

PRINCIPLE OF THE TEST

11.The test chemical is applied topically to a three-dimensional RhE model, comprised of non- transformed, human-derived epidermal keratinocytes, which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.

12.The RhE test method is based on the premise that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the cells in the underlying layers. Cell viability is measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS number 298-93-1], into a blue formazan salt that is quantitatively measured after extraction from tissues (27). Corrosive chemicals are identified by their ability to decrease cell viability below defined threshold levels (see paragraphs 35 and 36). The RhE-based skin corrosion test method has shown to be predictive of in vivo skin corrosion effects assessed in rabbits according to the TM B.4 (2).

DEMONSTRATION OF PROFICIENCY

13.Prior to routine use of any of the four validated RhE test models that adhere to this test method, laboratories should demonstrate technical proficiency by correctly classifying the twelve Proficiency Substances listed in Table 1. In case of the use of a method for sub-classification, also the correct sub-categorisation should be demonstrated. In situations where a listed substance is unavailable or where justifiable, another substance for which adequate in vivo and in vitro reference data are available may be used (e.g. from the list of reference chemicals (24)) provided that the same selection criteria as described in Table 1 is applied.

Table 1: List of Proficiency Substances1

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; VRM = Validated Reference Method; NC = Not Corrosive

1 The proficiency substances, sorted first by corrosives versus non-corrosives, then by corrosive sub-category and then by chemical class, were selected from the substances used in the ECVAM validation studies of EpiSkin™ and EpiDerm™ (8) (9) (10) and from post-validation studies based on data provided by EpiSkin™ (22), EpiDerm™, SkinEthic™ and epiCS® developers (23). Unless otherwise indicated, the substances were tested at the purity level obtained when purchased from a commercial source (8) (10). The selection includes, to the extent possible, substances that: (i) are representative of the range of corrosivity responses (e.g. noncorrosives; weak to strong corrosives) that the VRMs are capable of measuring or predicting; (ii) are representative of the chemical classes used in the validation studies; (iii) have chemical structures that are well-defined; (iv) induce reproducible results in the VRM; (v) induce definitive results in the in vivo reference test method; (vi) are commercially available; and (vii) are not associated with prohibitive disposal costs.

2 Chemical class assigned by Barratt et al. (8).

3 The corresponding UN Packing groups are I, II and III, respectively, for the UN GHS/CLP 1A, 1B and 1C.

4 The VRM in vitro predictions reported in this table were obtained with the EpiSkin™ and the EpiDerm™ test models (VRMs) during post-validation testing performed by the test method developers.

5 The viability values obtained in the ECVAM Skin Corrosion Validation Studies were not corrected for direct MTT reduction (killed controls were not performed in the validation studies). However, the post-validation data generated by the test method developers that are presented in this table were acquired with adapted controls (23).

14.As part of the proficiency exercise, it is recommended that the user verifies the barrier properties of the tissues after receipt as specified by the RhE model manufacturer. This is particularly important if tissues are shipped over long distance/time periods. Once a test method has been successfully established and proficiency in its use has been demonstrated, such verification will not be necessary on a routine basis. However, when using a test method routinely, it is recommended to continue to assess the barrier properties in regular intervals.

PROCEDURE

15.The following is a generic description of the components and procedures of the RhE test models for skin corrosion assessment covered by this test method. The RhE models endorsed as scientifically valid for use within this test method, i.e. the EpiSkin™ (SM), EpiDerm™ (EPI-200), SkinEthic™ RHE and epiCS® models (16)(17)(19)(28)(29)(30)(31)(32)(33), can be obtained from commercial sources. Standard Operating Procedures (SOPs) for these four RhE models are available (34)(35)(36)(37), and their main test method components are summarised in Appendix 2. It is recommended that the relevant SOP be consulted when implementing and using one of these models in the laboratory. Testing with the four RhE test models covered by this test method should comply with the following:

RHE TEST METHOD COMPONENTS

General Conditions

16.Non-transformed human keratinocytes should be used to reconstruct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) should be present under a functional stratum corneum. The stratum corneum should be multi-layered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic benchmark chemicals, e.g. sodium dodecyl sulphate (SDS) or Triton X-100. The barrier function should be demonstrated and may be assessed either by determination of the concentration at which a benchmark chemical reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the benchmark chemical at a specified, fixed concentration (see paragraph 18). The containment properties of the RhE model should prevent the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The RhE model should be free of contamination by bacteria, viruses, mycoplasma, or fungi.

Functional Conditions

Viability

17.The assay used for quantifying tissue viability is the MTT-assay (27). The viable cells of the RhE tissue construct reduce the vital dye MTT into a blue MTT formazan precipitate, which is then extracted from the tissue using isopropanol (or a similar solvent). The OD of the extraction solvent alone should be sufficiently small, i.e., OD < 0.1. The extracted MTT formazan may be quantified using either a standard absorbance (OD) measurement or an HPLC/UPLC-spectrophotometry procedure (38). The RhE model users should ensure that each batch of the RhE model used meets defined criteria for the negative control. An acceptability range (upper and lower limit) for the negative control OD values should be established by the RhE model developer/supplier. Acceptability ranges for the negative control OD values for the four validated RhE test models included in this test method are given in Table 2. An HPLC/UPLC- Spectrophotometry user should use the negative control OD ranges provided in Table 2 as the acceptance criterion for the negative control. It should be documented that the tissues treated with negative control are stable in culture (provide similar OD measurements) for the duration of the exposure period.

Table 2: Acceptability ranges for negative control OD values to control batch quality

Barrier function

18.The stratum corneum and its lipid composition should be sufficient to resist the rapid penetration of certain cytotoxic benchmark chemicals (e.g. SDS or Triton X-100), as estimated by IC50 or ET50 (Table 3). The barrier function of each batch of the RhE model used should be demonstrated by the RhE model developer/vendor upon supply of the tissues to the end user (see paragraph 21).

Morphology

19.Histological examination of the RhE model should be performed demonstrating multi-layered human epidermis-like structure containing stratum basale, stratum spinosum, stratum granulosum and stratum corneum and exhibits lipid profile similar to lipid profile of human epidermis. Histological examination of each batch of the RhE model used demonstrating appropriate morphology of the tissues should be provided by the RhE model developer/vendor upon supply of the tissues to the end user (see paragraph 21).

Reproducibility

20.Test method users should demonstrate reproducibility of the test methods over time with the positive and negative controls. Furthermore, the test method should only be used if the RhE model developer/supplier provides data demonstrating reproducibility over time with corrosive and non-corrosive chemicals from e.g. the list of Proficiency Substances (Table 1). In case of the use of a test method for subcategorisation, the reproducibility with respect to sub-categorisation should also be demonstrated.

Quality control (QC)

21.The RhE model should only be used if the developer/supplier demonstrates that each batch of the RhE model used meets defined production release criteria, among which those for viability (paragraph 17), barrier function (paragraph 18) and morphology (paragraph 19) are the most relevant. These data are provided to the test method users, so that they are able to include this information in the test report. Only results produced with QC accepted tissue batches can be accepted for reliable prediction of corrosive classification. An acceptability range (upper and lower limit) for the IC50 or the ET50 is established by the RhE model developer/supplier. The acceptability ranges for the four validated test models are given in Table 3.

Table 3: QC batch release criteria

Application of the Test Chemical and Control Chemicals

22.At least two tissue replicates should be used for each test chemical and controls for each exposure time. For liquid as well as solid chemicals, sufficient amount of test chemical should be applied to uniformly cover the epidermis surface while avoiding an infinite dose, i.e. a minimum of 70 μl/cm2 or 30 mg/cm2 should be used. Depending on the models, the epidermis surface should be moistened with deionised or distilled water before application of solid chemicals, to improve contact between the test chemical and the epidermis surface (34)(35)(36)(37). Whenever possible, solids should be tested as a fine powder. The application method should be appropriate for the test chemical (see e.g. references (34-37). At the end of the exposure period, the test chemical should be carefully washed from the epidermis with an aqueous buffer, or 0.9% NaCl. Depending on which of the four validated RhE test model is used, two or three exposure periods are used per test chemical (for all four valid RhE models: 3 min and 1 hour; for EpiSkinTM an additional exposure time of 4 hours). Depending on the RhE test model used and the exposure period assessed, the incubation temperature during exposure may vary between room temperature and 37°C.

23.Concurrent negative and positive controls (PC) should be used in each run to demonstrate that viability (with negative controls), barrier function and resulting tissue sensitivity (with the PC) of the tissues are within a defined historical acceptance range. The suggested PC chemicals are glacial acetic acid or 8N KOH depending upon the RhE model used. It should be noted that 8N KOH is a direct MTT reducer that might require adapted controls as described in paragraphs 25 and 26. The suggested negative controls are 0.9% (w/v) NaCl or water.

Cell Viability Measurements

24.The MTT assay, which is a quantitative assay, should be used to measure cell viability under this test method (27). The tissue sample is placed in MTT solution of appropriate concentration (0.3 or 1 mg/ml) for 3 hours. The precipitated blue formazan product is then extracted from the tissue using a solvent (e.g. isopropanol, acidic isopropanol), and the concentration of formazan is measured by determining the OD at 570 nm using a filter band pass of maximum ± 30 nm, or by an HPLC/UPLC- spectrophotometry procedure (see paragraphs 30 and 31)(38).

25.Test chemicals may interfere with the MTT assay, either by direct reduction of the MTT into blue formazan, and/or by colour interference if the test chemical absorbs, naturally or due to treatment procedures, in the same OD range of formazan (570 ± 30 nm, mainly blue and purple chemicals). Additional controls should be used to detect and correct for a potential interference from these test chemicals such as the non-specific MTT reduction (NSMTT) control and the non-specific colour (NSC) control (see paragraphs 26 to 30). This is especially important when a specific test chemical is not completely removed from the tissue by rinsing or when it penetrates the epidermis, and is therefore present in the tissues when the MTT viability test is performed. Detailed description of how to correct direct MTT reduction and interferences by colouring agents is available in the SOPs for the test models (34)(35)(36)(37).

26.To identify direct MTT reducers, each test chemical should be added to freshly prepared MTT medium (34) (35) (36) (37). If the MTT mixture containing the test chemical turns blue/purple, the test chemical is presumed to directly reduce the MTT, and further functional check on non-viable epidermis should be performed, independently of using the standard absorbance (OD) measurement or an HPLC/UPLC-spectrophotometry procedure. This additional functional check employs killed tissues that possess only residual metabolic activity but absorb the test chemical in similar amount as viable tissues. Each MTT reducing chemical is applied on at least two killed tissue replicates per exposure time, which undergo the whole skin corrosion test. The true tissue viability is then calculated as the percent tissue viability obtained with living tissues exposed to the MTT reducer minus the percent non-specific MTT reduction obtained with the killed tissues exposed to the same MTT reducer, calculated relative to the negative control run concurrently to the test being corrected (%NSMTT).

27.To identify potential interference by coloured test chemicals or test chemicals that become coloured when in contact with water or isopropanol and decide on the need for additional controls, spectral analysis of the test chemical in water (environment during exposure) and/or isopropanol (extracting solution) should be performed. If the test chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, further colorant controls should be performed or, alternatively, an HPLC/UPLC- spectrophotometry procedure should be used in which case these controls are not required (see paragraphs 30 and 31). When performing the standard absorbance (OD) measurement, each interfering coloured test chemical is applied on at least two viable tissue replicates per exposure time, which undergo the entire skin corrosion test but are incubated with medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSCliving) control. The NSCliving control needs to be performed concurrently per exposure time per coloured test chemical (in each run) due to the inherent biological variability of living tissues. The true tissue viability is then calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent nonspecific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently to the test being corrected (%NSCliving).

28.Test chemicals that are identified as producing both direct MTT reduction (see paragraph 26) and colour interference (see paragraph 27) will also require a third set of controls, apart from the NSMTT and NSCliving controls described in the previous paragraphs, when performing the standard absorbance (OD) measurement. This is usually the case with darkly coloured test chemicals interfering with the MTT assay (e.g., blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT as described in paragraph 26. These test chemicals may bind to both living and killed tissues and therefore the NSMTT control may not only correct for potential direct MTT reduction by the test chemical, but also for colour interference arising from the binding of the test chemical to killed tissues. This could lead to a double correction for colour interference since the NSCliving control already corrects for colour interference arising from the binding of the test chemical to living tissues. To avoid a possible double correction for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) needs to be performed. In this additional control, the test chemical is applied on at least two killed tissue replicates per exposure time, which undergo the entire testing procedure but are incubated with medium instead of MTT solution during the MTT incubation step. A single NSCkilled control is sufficient per test chemical regardless of the number of independent tests/runs performed, but should be performed concurrently to the NSMTT control and, where possible, with the same tissue batch. The true tissue viability is then calculated as the percent tissue viability obtained with living tissues exposed to the test chemical minus %NSMTT minus %NSCliving plus the percent non-specific colour obtained with killed tissues exposed to the interfering test chemical and incubated with medium without MTT, calculated relative to the negative control run concurrently to the test being corrected (%NSCkilled).

29.It is important to note that non-specific MTT reduction and non-specific colour interferences may increase the readouts of the tissue extract above the linearity range of the spectrophotometer. On this basis, each laboratory should determine the linearity range of their spectrophotometer with MTT formazan (CAS # 57360-69-7) from a commercial source before initiating the testing of test chemicals for regulatory purposes. In particular, the standard absorbance (OD) measurement using a spectrophotometer is appropriate to assess direct MTT-reducers and colour interfering test chemicals when the ODs of the tissue extracts obtained with the test chemical without any correction for direct MTT reduction and/or colour interference are within the linear range of the spectrophotometer or when the uncorrected percent viability obtained with the test chemical already defined it as a corrosive (see paragraphs 35 and 36). Nevertheless, results for test chemicals producing %NSMTT and/or %NSCliVing > 50% of the negative control should be taken with caution.

30.For coloured test chemicals which are not compatible with the standard absorbance (OD) measurement due to too strong interference with the MTT assay, the alternative HPLC/UPLC- spectrophotometry procedure to measure MTT formazan may be employed (see paragraph 31) (37). The HPLC/UPLC-spectrophotometry system allows for the separation of the MTT formazan from the test chemical before its quantification (38). For this reason, NSCliVing or NSCkilled controls are never required when using HPLC/UPLC-spectrophotometry, independently of the chemical being tested. NSMTT controls should nevertheless be used if the test chemical is suspected to directly reduce MTT or has a colour that impedes the assessment of the capacity to directly reduce MTT (as described in paragraph 26). When using HPLC/UPLC-spectrophotometry to measure MTT formazan, the percent tissue viability is calculated as percent MTT formazan peak area obtained with living tissues exposed to the test chemical relative to the MTT formazan peak obtained with the concurrent negative control. For test chemicals able to directly reduce MTT, true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the test chemical minus %NSMTT. Finally, it should be noted that direct MTT-reducers that may also be colour interfering, which are retained in the tissues after treatment and reduce MTT so strongly that they lead to ODs (using standard OD measurement) or peak areas (using UPLC/HPLC- spectrophotometry) of the tested tissue extracts that fall outside of the linearity range of the spectrophotometer cannot be assessed, although these are expected to occur in only very rare situations.

31.HPLC/UPLC-spectrophotometry may be used also with all types of test chemicals (coloured, non-coloured, MTT-reducers and non-MTT reducers) for measurement of MTT formazan (38). Due to the diversity of HPLC/UPLC-spectrophotometry systems, qualification of the HPLC/UPLC- spectrophotometry system should be demonstrated before its use to quantify MTT formazan from tissue extracts by meeting the acceptance criteria for a set of standard qualification parameters based on those described in the U.S. Food and Drug Administration guidance for industry on bio-analytical method validation (38)(39). These key parameters and their acceptance criteria are shown in Appendix 4. Once the acceptance criteria defined in Appendix 4 have been met, the HPLC/UPLC-spectrophotometry system is considered qualified and ready to measure MTT formazan under the experimental conditions described in this test method.

Acceptability Criteria

32.For each test method using valid RhE models, tissues treated with the negative control should exhibit OD reflecting the quality of the tissues as described in table 2 and should not be below historically established boundaries. Tissues treated with the PC, i.e. glacial acetic acid or 8N KOH, should reflect the ability of the tissues to respond to a corrosive chemical under the conditions of the test model (see Appendix 2). The variability between tissue replicates of test chemical and/or control chemicals should fall within the accepted limits for each valid RhE model requirements (see Appendix 2) (e.g. the difference of viability between the two tissue replicates should not exceed 30%). If either the negative control or PC included in a run fall out of the accepted ranges, the run is considered as not qualified and should be repeated. If the variability of test chemicals falls outside of the defined range, its testing should be repeated.

Interpretation of Results and Prediction Model

33.The OD values obtained for each test chemical should be used to calculate percentage of viability relative to the negative control, which is set at 100%. In case HPLC/UPLC-spectrophotometry is used, the percent tissue viability is calculated as percent MTT formazan peak area obtained with living tissues exposed to the test chemical relative to the MTT formazan peak obtained with the concurrent negative control. The cut-off percentage cell viability values distinguishing corrosive from non-corrosive test chemical (or discriminating between different corrosive sub-categories) are defined below in paragraphs 35 and 36 for each of the test models covered by this test method and should be used for interpreting the results.

34.A single testing run composed of at least two tissue replicates should be sufficient for a test chemical when the resulting classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements, a second run may be considered, as well as a third one in case of discordant results between the first two runs.

35.The prediction model for the EpiSkin™ skin corrosion test model (9)(34)(22), associated with the UN GHS/CLP classification system, is shown in Table 4:

Table 4: EpiSkinTM prediction model

*) According to the data generated in view of assessing the usefulness of the RhE test models for supporting subcategorisation, it was shown that around 22 % of the sub-category 1A results of the EpiSkin™ test model may actually constitute sub-category 1B or sub-category 1C substances/mixtures (i.e. over classifications) (see Appendix 3).

36.The prediction models for the EpiDerm™ SCT (10)(23)(35), the SkinEthic™ RHE (17)(18) (23) (36), and the epiCS® (16)(23)(37) skin corrosion test models, associated with the UN GHS/CLP classification system, are shown in Table 5:

Table 5: EpiDermTM SCT, SkinEthic™ RHE and epiCS®

DATA AND REPORTING

Data

37.For each test, data from individual tissue replicates (e.g. OD values and calculated percentage cell viability for each test chemical, including classification) should be reported in tabular form, including data from repeat experiments as appropriate. In addition, means and ranges of viability and CVs between tissue replicates for each test should be reported. Observed interactions with MTT reagent by direct MTT reducers or coloured test chemicals should be reported for each tested chemical.

Test report

38.The test report should include the following information:

Test Chemical and Control Chemicals:

-Mono-constituent substance: chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc;

-Multi-constituent substance, UVCB and mixture: characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant physicochemical properties of the constituents;

-Physical appearance, water solubility, and any additional relevant physicochemical properties;

-Source, lot number if available;

-Treatment of the test chemical/control substance prior to testing, if applicable (e.g. warming, grinding);

-Stability of the test chemical, limit date for use, or date for re-analysis if known;

-Storage conditions.

RhE model and protocol used and rationale for it (if applicable)

Test Conditions:

-RhE model used (including batch number);

-Calibration information for measuring device (e.g. spectrophotometer), wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device;

-Description of the method used to quantify MTT formazan;

-Description of the qualification of the HPLC/UPLC-spectrophotometry system, if applicable;

-Complete supporting information for the specific RhE model used including its performance. This should include, but is not limited to:

I)Viability;

II)Barrier function;

III)Morphology;

IV)Reproducibility and predictive capacity;

V)Quality controls (QC) of the model;

-Reference to historical data of the model. This should include, but is not limited to

-acceptability of the QC data with reference to historical batch data;

-Demonstration of proficiency in performing the test method before routine use by testing of the proficiency substances.

Test Procedure:

-Details of the test procedure used (including washing procedures used after exposure period);

-Doses of test chemical and control chemicals used;

-Duration of exposure period(s) and temperature(s) of exposure;

-Indication of controls used for direct MTT-reducers and/or colouring test chemicals, if applicable;

-Number of tissue replicates used per test chemical and controls (PC, negative control, and NSMTT, NSCliving and NSCkilled, if applicable), per exposure time;

-Description of decision criteria/prediction model applied based on the RhE model used;

-Description of any modifications of the test procedure (including washing procedures).

Run and Test Acceptance Criteria:

-Positive and negative control mean values and acceptance ranges based on historical data;

-Acceptable variability between tissue replicates for positive and negative controls;

-Acceptable variability between tissue replicates for test chemical.

Results:

-Tabulation of data for individual test chemicals and controls, for each exposure period, each run and each replicate measurement including OD or MTT formazan peak area, percent tissue viability, mean percent tissue viability, differences between replicates, SDs and/or CVs if applicable;

-If applicable, results of controls used for direct MTT-reducers and/or colouring test chemicals including OD or MTT formazan peak area, %NSMTT, %NSCliving, %NSCkilled, differences between tissue replicates, SDs and/or CVs (if applicable), and final correct percent tissue viability;

-Results obtained with the test chemical(s) and control chemicals in relation to the defined run and test acceptance criteria;

-Description of other effects observed;

-The derived classification with reference to the prediction model/decision criteria used.

Discussion of the results

Conclusions

LITERATURE

(1)UN (2013). United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS). Fifth Revised Edition, UN    New York    and    Geneva. Available at: http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html

(2)Chapter B.4 of this Annex, Acute Dermal Irritation, Corrosion.

(3)Chapter B.40 of this Annex, In Vitro Skin Corrosion.

(4)Chapter B.65 of this Annex, In Vitro Membrane Barrier Test Method.

(5)Chapter B.46 of this Annex, In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. 

(6)OECD (2014). Guidance Document on Integrated Approaches to Testing and Assessment of Skin Irritation/Corrosion. Environment, Health and Safety Publications, Series on Testing and Assessment, (No 203) Organisation for Economic Cooperation and Development, Paris.

(7)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 23:219-255.

(8)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. Toxicol.In Vitro 12:471-482.

(9)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 SkinCorrosivity. 2. Results and Evaluation by the Management Team. Toxicol.in Vitro 12:483-524.

(10)Liebsch M., Traue D., Barrabas C., Spielmann H., Uphill, P., Wilkins S., Wiemann C., Kaufmann T., Remmele M. and Holzhütter H. G. (2000). The ECVAM Prevalidation Study on the Use of EpiDerm for Skin Corrosivity Testing, ATLA 28: 371-401.

(11)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. et Zucco F. (1995). Practical Aspects of the Validation of Toxicity Test Procedures. The Report and Recommendations of ECVAM Workshops, ATLA 23:129-147.

(12)ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) (1997). Validation and Regulatory Acceptance of Toxicological TestMethods. NIH Publication No 97-3981. National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.

(13)ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) (2002). ICCVAM evaluation of EpiDerm™ (EPI-200), EPISKIN™ (SM), 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.

(14)EC-ECVAM (1998). Statement on the Scientific Validity of the EpiSkin™ Test (an In Vitro Test for Skin Corrosivity), Issued by the ECVAM Scientific Advisory Committee (ESAC10), 3 April 1998.

(15)EC-ECVAM (2000). Statement on the Application of the EpiDerm™ Human Skin Model for Skin Corrosivity Testing, Issued by the ECVAM Scientific Advisory Committee (ESAC14), 21 March 2000.

(16)Hoffmann J., Heisler E., Karpinski S., Losse J., Thomas D., Siefken W., Ahr H.J., Vohr H.W. and Fuchs H.W. (2005). Epidermal-Skin-Test 1000 (EST-1000)-A New Reconstructed Epidermis for In Vitro Skin Corrosivity Testing. Toxicol.In Vitro 19: 925-929.

(17)Kandárová H., Liebsch M., Spielmann,H., Genschow E., Schmidt E., Traue D., Guest R., Whittingham A., Warren N, Gamer A.O., Remmele M., Kaufmann T., Wittmer E., De Wever B., and Rosdy M. (2006). Assessment of the Human Epidermis Model SkinEthic RHE for In Vitro Skin Corrosion Testing of Chemicals According to New OECD TG 431. Toxicol.In Vitro 20: 547-559.

(18)Tornier C., Roquet M. and Fraissinette A.B. (2010). Adaptation of the Validated SkinEthic™ Reconstructed Human Epidermis (RHE) Skin Corrosion Test Method to 0.5 cm2 Tissue Sample. Toxicol. In Vitro 24: 1379-1385.

(19)EC-ECVAM (2006). Statement on the Application of the SkinEthic™ Human Skin Model for Skin Corrosivity Testing, Issued by the ECVAM Scientific Advisory Committee (ESAC25), 17 November 2006.

(20)EC-ECVAM (2009). ESAC Statement on the Scientific Validity of an In-Vitro Test Method for Skin Corrosivity Testing: the EST-1000, Issued by the ECVAM Scientific Advisory Committee (ESAC30), 12 June 2009.

(21)OECD (2013). Summary Document on the Statistical Performance of Methods in OECD Test Guideline 431 for Sub-categorisation. Environment, Health, and Safety Publications, Series on Testing and Assessment (No 190). Organisation for Economic Cooperation and Development, Paris.

(22)Alépée N., Grandidier M.H., and Cotovio J. (2014). Sub-Categorisation of Skin Corrosive Chemicals by the EpiSkin™ Reconstructed Human Epidermis Skin Corrosion Test Method According to UN GHS: Revision of OECD Test Guideline 431. Toxicol. In Vitro 28:131-145.

(23)Desprez B., Barroso J., Griesinger C., Kandárová H., Alépée N., and Fuchs, H. (2015). Two Novel Prediction Models Improve Predictions of Skin Corrosive Sub-categories by Test Methods of OECD Test Guideline No 431. Toxicol. In Vitro 29:2055-2080.

(24)OECD (2015). Performance Standards for the Assessment of Proposed Similar or Modified In Vitro Reconstructed Human Epidermis (RHE) Test Methods For Skin Corrosion in Relation to OECD TG 431. Environmental Health and Safety Publications, Series on Testing and Assessment (No 219). Organisation for Economic Cooperation and Development, Paris

(25)OECD (2005). Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment. . Environment, Health and Safety Publications, Series on Testing and Assessment (No 34), Organisation for Economic Cooperation and Development, Paris.

(26)Eskes C. et al. (2012). Regulatory Assessment of In Vitro Skin Corrosion and Irritation Data Within the European Framework: Workshop Recommendations. Regul.Toxicol.Pharmacol. 62:393-403.

(27)Mosmann T. (1983). Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 65:55-63.

(28)Tinois E., et al. (1994). The Episkin Model: Successful Reconstruction of Human Epidermis In Vitro. In: In Vitro Skin Toxicology. Rougier A.,. Goldberg A.M and Maibach H.I. (Eds): 133-140.

(29)Cannon C. L., Neal P.J., Southee J.A., Kubilus J. and Klausner M. (1994), New Epidermal Model for Dermal Irritancy Testing. Toxicol.in Vitro 8:889 - 891.

(30)Ponec M., Boelsma E, Weerheim A, Mulder A, Bouwstra J and Mommaas M. (2000). Lipid and Ultrastructural Characterization of Reconstructed Skin Models. Inter. J. Pharmaceu. 203:211 - 225.

(31)Tinois E., Tillier, J., Gaucherand, M., Dumas, H., Tardy, M. and 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. Exp. Cell Res. 193:310-319.

(32)Parenteau N.L., Bilbo P, Nolte CJ, Mason VS and Rosenberg M. (1992). The Organotypic Culture of Human Skin Keratinocytes and Fibroblasts to Achieve Form and Function. Cytotech. 9:163-171.

(33)Wilkins L.M., Watson SR, Prosky SJ, Meunier SF and Parenteau N.L. (1994). Development of a Bilayered Living Skin Construct for Clinical Applications. Biotech. Bioeng. 43/8:747-756.

(34)EpiSkin™ SOP (December 2011). INVITTOX Protocol (No 118). EpiSkin™ Skin Corrosivity Test.

(35)EpiDerm™ SOP (February 2012). Version MK-24-007-0024 Protocol for: In Vitro EpiDerm™ Skin Corrosion Test (EPI-200-SCT), for Use with MatTek Corporation's Reconstructed Human Epidermal Model EpiDerm.

(36)SkinEthic™ RHE SOP (January 2012).  INVITTOX Protocol SkinEthic™ Skin Corrosivity Test.

(37)EpiCS® SOP (January 2012). Version 4.1 In Vitro Skin Corrosion: Human Skin Model Test Epidermal Skin Test 1000 (epiCS® ) CellSystems.

(38)Alépée N., Barroso J., De Smedt A., De Wever B., Hibatallah J., Klaric M., Mewes K.R., Millet M., Pfannenbecker U., Tailhardat M., Templier M., and McNamee P. Use of HPLC/UPLC- spectrophotometry for Detection of MTT Formazan in In Vitro Reconstructed Human Tissue (RhT)- based Test Methods Employing the MTT Assay to Expand their Applicability to Strongly Coloured Test Chemicals. Toxicol. In Vitro 29: 741-761.

(39)US FDA (2001). Guidance for Industry: Bioanalytical Method Validation. U.S. Department of Health and Human Services, Food and Drug Administration. (May 2001). Available at: [http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf].

Appendix 1

DEFINITIONS

Accuracy: The closeness of agreement between test method results and accepted reference values. It is a measure of test method performance and one aspect of relevance. The term is often used interchangeably with “concordance” to mean the proportion of correct outcomes of a test method (25).

Cell viability: Parameter measuring total activity of a cell population e.g. as ability of cellular mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Thiazolyl blue), which depending on the endpoint measured and the test design used, correlates with the total number and/or vitality of living cells.

Chemical: A substance or a mixture.

Concordance: This is a measure of test method performance for test methods that give a categorical result, and is one aspect of relevance. The term is sometimes used interchangeably with accuracy, and is defined as the proportion of all chemicals tested that are correctly classified as positive or negative. Concordance is highly dependent on the prevalence of positives in the types of test chemical being examined (25).

ET50: Can be estimated by determination of the exposure time required to reduce cell viability by 50% upon application of the benchmark chemical at a specified, fixed concentration, see also IC50.

GHS (Globally Harmonized System of Classification and Labelling of Chemicals): A system proposing the classification of chemicals (substances and mixtures) according to standardized types and levels of physical, health and environmental hazards, and addressing corresponding communication elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data sheets, so that to convey information on their adverse effects with a view to protect people (including employers, workers, transporters, consumers and emergency responders) and the environment (1).

HPLC: High Performance Liquid Chromatography.

IATA: Integrated Approach on Testing and Assessment.

IC50: Can be estimated by determination of the concentration at which a benchmark chemical reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, see also ET50.

Infinite dose: Amount of test chemical applied to the epidermis exceeding the amount required to completely and uniformly cover the epidermis surface.Mixture: A mixture or solution composed of two or more substances in which they do not react.

Mono-constituent substance: A substance, defined by its quantitative composition, in which one main constituent is present to at least 80% (w/w).

MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide.

Multi-constituent substance: A substance, defined by its quantitative composition, in which more than one main constituent is present in a concentration > 10% (w/w) and < 80% (w/w). A multi-constituent substance is the result of a manufacturing process. The difference between mixture and multi-constituent substance is that a mixture is obtained by blending of two or more substances without chemical reaction. A multi-constituent substance is the result of a chemical reaction.

NC: Non corrosive.

NSCkilled control: Non-Specific Colour control in killed tissues.

NSClivingcontrol : Non-Specific Colour control in living tissues.

NSMTT: Non-Specific MTT reduction.

OD: Optical Density

PC: Positive Control, a replicate containing all components of a test system and treated with a chemical known to induce a positive response. To ensure that variability in the positive control response across time can be assessed, the magnitude of the positive response should not be excessive.

Performance standards (PS): Standards, based on a validated test method, that provide a basis for evaluating the comparability of a proposed test method that is mechanistically and functionally similar. Included are; (i) essential test method components; (ii) a minimum list of Reference Chemicals selected from among the chemicals used to demonstrate the acceptable performance of the validated test method; and (iii) the similar levels of reliability and accuracy, based on what was obtained for the validated test method, that the proposed test method should demonstrate when evaluated using the minimum list of Reference Chemicals (25).

Relevance: Description of relationship of the test method to the effect of interest and whether it is meaningful and useful for a particular purpose. It is the extent to which the test method correctly measures or predicts the biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test method (25).

Reliability: Measures of the extent that a test method can be performed reproducibly within and between laboratories over time, when performed using the same protocol. It is assessed by calculating intra- and inter-laboratory reproducibility (25).

Run: A run consists of one or more test chemicals tested concurrently with a negative control and with a PC.

Sensitivity: The proportion of all positive/active chemicals that are correctly classified by the test method. It is a measure of accuracy for a test method that produces categorical results, and is an important consideration in assessing the relevance of a test method (25).

Skin corrosion in vivo: The production of irreversible damage of the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test chemical 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.

Specificity: The proportion of all negative/inactive chemicals that are correctly classified by the test method. It is a measure of accuracy for a test method that produces categorical results and is an important consideration in assessing the relevance of a test method (25).

Substance: A chemical element and its compounds in the natural state or obtained by any production process, inducing any additive necessary to preserve its stability and any impurities deriving from the process used, but excluding any solvent which may be separated without affecting the stability of the substance or changing it composition. Test chemical: Any substance or mixture tested using this test method.

UPLC: Ultra-High Performance Liquid Chromatography.

UVCB: substances of unknown or variable composition, complex reaction products or biological materials.

Appendix 2

MAIN COMPONENTS OF THE RhE TEST MODELS VALIDATED FOR SKIN CORROSION TESTING

Appendix 3

PERFORMANCE OF TEST MODELS FOR SUB-CATEGORISATION

The table below provides the performances of the four test models calculated based on a set of 80 chemicals tested by the four test developers. Calculations were performed by the OECD Secretariat, reviewed and agreed by an expert subgroup (21) (23).

EpiSkin™, EpiDerm™ , SkinEthic™ and epiCS® test models are able to sub-categorise (i.e. 1A versus 1B-and-1C versus NC)

Performances, overclassification rates, underclassification rates, and accuracy (Predictive capacity) of the four test models based on a set of 80 chemicals all tested over 2 or 3 runs in each test model:

NC: Non-corrosive

Appendix 4

Key parameters and acceptance criteria for qualification of an HPLC/UPLC-spectrophotometry system for measurement of MTT formazan extracted from RhE tissue

1LLOQ: Lower Limit of Quantification, defined to cover 1-2% tissue viability, i.e., 0.8 µg/ml.

2ULOQ: Upper Limit of Quantification, defined to be at least two times higher than the highest expected MTT formazan concentration in isopropanol extracts from negative controls i.e., 200 µg/ml."

(7) In Part B, Chapter B.46 is replaced by the following:

"B.46 IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS TEST METHOD

INTRODUCTION

1.This test method (TM) is equivalent to OECD test guideline (TG) 439 (2015). Skin irritation refers to the production of reversible damage to the skin following the application of a test chemical for up to 4 hours [as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS)](1) and the European Union (EU) Regulation 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures (CLP) 4 . This test method provides an in vitro procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) in accordance with UN GHS/CLP Category 2 (2). In regions that do not adopt the optional UN GHS Category 3 (mild irritants), this test method can also be used to identify non-classified chemicals. Therefore, depending on the regulatory framework and the classification system in use, this test method may be used to determine the skin irritancy of chemicals either as a stand-alone replacement test for in vivo skin irritation testing or as a partial replacement test within a testing strategy (3).

2.The assessment of skin irritation has typically involved the use of laboratory animals [TM B.4, equivalent to OECD TG 404 originally adopted in 1981 and revised in 1992, 2002 and 2015] (4). For the testing of corrosivity, three validated in vitro test methods have been adopted as OECD TM B.40 (equivalent to OECD TG 430), TM B.40bis (equivalent to OECD TG 431) and TM B.65 (equivalent to OECD TG 435) (5) (6) (7). An OECD guidance document on Integrated Approaches to Testing and Assessment (IATA) for Skin Corrosion and Irritation describes several modules which group information sources and analysis tools, and provides guidance on (i) how to integrate and use existing test and non-test data for the assessment of skin irritation and skin corrosion potentials of chemicals and (ii) proposes an approach when further testing is needed (3).

3.This test method addresses the human health endpoint skin irritation. It is based on the in vitro test system of reconstructed human epidermis (RhE), which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture. Performance Standards (PS) are available to facilitate the validation and assessment of similar and modified RhE-based test methods, in accordance with the principles of the OECD guidance document No 34 (8) (9). The corresponding test guideline was originally adopted in 2010, updated in 2013 to include additional RhE models, and updated in 2015 to refer to the IATA guidance document and introduce the use of an alternative procedure to measure viability.

4.Pre-validation, optimisation and validation studies have been completed for four commercially available in vitro test models (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) based on the RhE test system (sensitivity 80%, specificity 70%, and accuracy 75%). These four test models are included in this TG and are listed in Appendix 2, which also provides information on the type of validation study used to validate the respective test methods. As noted in Appendix 2, the Validated Reference Method (VRM) have been used to develop the present test method and the Performance Standards (8).

5.OECD Mutual Acceptance of Data will only be guaranteed for test models validated according to the Performance Standards (8), if these test models have been reviewed and adopted by OECD. The test models included in this test method and the corresponding OECD TG can be used indiscriminately to address countries’ requirements for test results from in vitro test methods for skin irritation, while benefiting from the Mutual Acceptance of Data.

6.Definitions of terms used in this document are provided in Appendix 1.

INITIAL CONSIDERATIONS AND LIMITATIONS

7.A limitation of the test method, as demonstrated by the full prospective validation study assessing and characterising RhE test methods (16), is that it does not allow the classification of chemicals to the optional UN GHS Category 3 (mild irritants) (1). Thus, the regulatory framework in member countries will decide how this test method will be used. For the EU, Category 3 has not been taken up in CLP. For a full evaluation of local skin effects after a single dermal exposure, the OECD Guidance Document on Integrated Approaches for Testing Assessment should be consulted (3). It is recognised that the use of human skin is subject to national and international ethical considerations and conditions.

8.This test method addresses the human health endpoint skin irritation. While this test method does not provide adequate information on skin corrosion, it should be noted that TM B.40bis (equivalent to OECD TG 431) on skin corrosion is based on the same RhE test system, though using another protocol (6). This test method is based on RhE-models using human keratinocytes, which therefore represent in vitro the target organ of the species of interest. It moreover directly covers the initial step of the inflammatory cascade/mechanism of action (cell and tissue damage resulting in localised trauma) that occurs during irritation in vivo. A wide range of chemicals has been tested in the validation underlying this test method and the database of the validation study amounted to 58 chemicals in total (16) (18) (23). The test method is applicable to solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. Whenever possible, solids should be ground to a fine powder before application; no other pre-treatment of the sample is required. Gases and aerosols have not been assessed yet in a validation study (29). While it is conceivable that these can be tested using RhE technology, the current test method does not allow testing of gases and aerosols.

9.Before use of the test method on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose. Such considerations are not needed, when there is a regulatory requirement for testing of the mixture. However, due to the fact that mixtures cover a wide spectrum of categories and composition, and that only limited information is currently available on the testing of mixtures, in cases where evidence can be demonstrated on the non-applicability of the test method to a specific category of mixtures (e.g. following a strategy as proposed in Eskes et al. 2012 (30)), the test method should not be used for that specific category of mixtures. Similar care should be taken in case specific chemical classes or physico-chemical properties are found not to be applicable to the current test method.

10.Test chemicals absorbing light in the same range as MTT formazan and test chemicals able to directly reduce the vital dye MTT (to MTT formazan) may interfere with the cell viability measurements and need the use of adapted controls for corrections (see paragraphs 28-34).

11.A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5%, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

PRINCIPLE OF THE TEST

12.The test chemical is applied topically to a three-dimensional RhE model, comprised of non-transformed human-derived epidermal keratinocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.

13.Chemical-induced skin irritation, manifested mainly by erythema and oedema, is the result of a cascade of events beginning with penetration of the chemicals through the stratum corneum where they may damage the underlying layers of keratinocytes and other skin cells. The damaged cells may either release inflammatory mediators or induce an inflammatory cascade which also acts on the cells in the dermis, particularly the stromal and endothelial cells of the blood vessels. It is the dilation and increased permeability of the endothelial cells that produce the observed erythema and oedema (29). Notably, the RhE-based test methods, in the absence of any vascularisation in the in vitro test system, measure the initiating events in the cascade, e.g. cell / tissue damage (16) (17), using cell viability as readout.

14.Cell viability in RhE models is measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1], into a blue formazan salt that is quantitatively measured after extraction from tissues (31). Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels (i.e. ≤ 50%, for UN GHS/CLP Category 2). Depending on the regulatory framework and applicability of the test method, test chemicals that produce cell viabilities above the defined threshold level, may be considered non-irritants (i.e. > 50%, No Category).

DEMONSTRATION OF PROFICIENCY

15.Prior to routine use of any of the four validated test models that adhere to this test method (Appendix 2), laboratories should demonstrate technical proficiency, using the ten Proficiency Substances listed in Table 1. In situations where, for instance, a listed substance is unavailable, another substance for which adequate in vivo and in vitro reference data are available may be used (e.g. from the list of reference chemicals (8)) provided that the same selection criteria as described in Table 1 are applied. Using an alternative proficiency substance should be justified.

16.As part of the proficiency testing, it is recommended that users verify the barrier properties of the tissues after receipt as specified by the RhE model producer. This is particularly important if tissues are shipped over long distance/time periods. Once a test method has been successfully established and proficiency in its use has been acquired and demonstrated, such verification will not be necessary on a routine basis. However, when using a test method routinely, it is recommended to continue to assess the barrier properties at regular intervals.

Table 1: Proficiency Substances1

1 The Proficiency Substances are a subset of the substances used in the validation study and the selection is based on the following criteria; (i), the chemicals substances are commercially available; (ii), they are representative of the full range of Draize irritancy scores (from non-irritant to strong irritant); (iii), they have a well-defined chemical structure; (iv), they are representative of the chemical functionality used in the validation process; (v) they provided reproducible in vitro results across multiple testing and multiple laboratories; (vi) they were correctly predicted in vitro, and (vii) they are not associated with an extremely toxic profile (e.g. carcinogenic or toxic to the reproductive system) and they are not associated with prohibitive disposal costs.

2 In vivo score in accordance with TM B.4 (4).

3 Under this test method, the UN GHS optional Category 3 (mild irritants) (1) is considered as No Category.

PROCEDURE

17.The following is a description of the components and procedures of a RhE test method for skin irritation assessment (See also Appendix 3 for parameters related to each test model). Standard Operating Procedures (SOPs) for the four models complying with this test method are available (32) (33) (34) (35).

RhE TEST METHOD COMPONENTS

General conditions

18.Non -transformed human keratinocytes should be used to reconstruct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) should be present under a functional stratum corneum. Stratum corneum should be multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic benchmark chemicals, e.g. sodium dodecyl sulphate (SDS) or Triton X-100. The barrier function should be demonstrated and may be assessed either by determination of the concentration at which a benchmark chemical reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the benchmark chemical at a specified, fixed concentration. The containment properties of the RhE model should prevent the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure. The RhE model should be free of contamination by bacteria, viruses, mycoplasma, or fungi.

Functional conditions

Viability

19.The assay used for quantifying viability is the MTT-assay (31). The viable cells of the RhE tissue construct can reduce the vital dye MTT into a blue MTT formazan precipitate which is then extracted from the tissue using isopropanol (or a similar solvent). The optical density (OD) of the extraction solvent alone should be sufficiently small, i.e. OD< 0.1. The extracted MTT formazan may be quantified using either a standard absorbance (OD) measurement or an HPLC/UPLC-spectrophotometry procedure (36). The RhE model users should ensure that each batch of the RhE model used meets defined criteria for the negative control. An acceptability range (upper and lower limit) for the negative control OD values (in the Skin Irritation test method conditions) are established by the RhE model developer/supplier. Acceptability ranges for the four validated RhE models included in this test method are given in Table 2. An HPLC/UPLC-Spectrophotometry user should use the negative control OD ranges provided in Table 2 as the acceptance criterion for the negative control. It should be documented that the tissues treated with the negative control are stable in culture (provide similar viability measurements) for the duration of the test exposure period.

Table 2: Acceptability ranges for negative control OD values of the test models included in this TM

Barrier function

20.The stratum corneum and its lipid composition should be sufficient to resist the rapid penetration of cytotoxic benchmark chemicals, e.g. SDS or Triton X-100, as estimated by IC50 or ET50 (Table 3).

Morphology

21.Histological examination of the RhE model should be provided demonstrating human epidermis-like structure (including multilayered stratum corneum).

Reproducibility

22.The results of the positive and negative controls of the test method should demonstrate reproducibility over time.

Quality control (QC)

23.The RhE model should only be used if the developer/supplier demonstrates that each batch of the RhE model used meets defined production release criteria, among which those for viability (paragraph 19), barrier function (paragraph 20) and morphology (paragraph 21) are the most relevant. These data should be provided to the test method users, so that they are able to include this information in the test report. An acceptability range (upper and lower limit) for the IC50 or the ET50 should be established by the RhE model developer/supplier. Only results produced with qualified tissues can be accepted for reliable prediction of irritation classification. The acceptability ranges for the four test models included in this TG are given in Table 3.

Table 3: QC batch release criteria of the test models included in this TM

Application of the Test Chemical and Control Chemicals

24.At least three replicates should be used for each test chemical and for the controls in each run. For liquid as well as solid chemicals, sufficient amount of test chemical should be applied to uniformly cover the epidermis surface while avoiding an infinite dose, i.e. ranging from 26 to 83 μl/cm2 or mg/cm2 (see Appendix 3). For solid chemicals, the epidermis surface should be moistened with deionised or distilled water before application, to improve contact between the test chemical and the epidermis surface. Whenever possible, solids should be tested as a fine powder. A nylon mesh may be used as a spreading aid in some cases (see Appendix 3). At the end of the exposure period, the test chemical should be carefully washed from the epidermis surface with aqueous buffer, or 0.9% NaCl. Depending on the RhE test models used, the exposure period ranges between 15 and 60 minutes, and the incubation temperature between 20 and 37°C. These exposure periods and temperatures are optimised for each individual RhE test method and represent the different intrinsic properties of the test models (e.g. barrier function) (see
Appendix 3).

25.Concurrent negative control (NC) and positive control (PC) should be used in each run to demonstrate that viability (using the NC), barrier function and resulting tissue sensitivity (using the PC) of the tissues are within a defined historical acceptance range. The suggested PC is 5% aqueous SDS. The suggested NCs is either water or phosphate buffered saline (PBS).

Cell Viability Measurements

26.According to the test procedure, it is essential that the viability measurement is not performed immediately after exposure to the test chemical, but after a sufficiently long post-treatment incubation period of the rinsed tissue in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects. A 42 hours post-treatment incubation period was found optimal during test optimisation of two of the RhE-based test models underlying this test method (11) (12) (13) (14) (15).

27.The MTT assay is a standardised quantitative method which should be used to measure cell viability under this test method. It is compatible with use in a three-dimensional tissue construct. The tissue sample is placed in MTT solution of appropriate concentration (e.g. 0.3 - 1 mg/ml) for 3 hours. The MTT is converted into blue formazan by the viable cells. The precipitated blue formazan product is then extracted from the tissue using a solvent (e.g. isopropanol, acidic isopropanol), and the concentration of formazan is measured by determining the OD at 570 nm using a filter band pass of maximum ± 30 nm or, by using an HPLC/UPLC-spectrophotometry procedure (see paragraph 34) (36).

28.Optical properties of the test chemical or its chemical action on MTT (e.g. chemicals may prevent or reverse the colour generation as well as cause it) may interfere with the assay leading to a false estimate of viability. This may occur when a specific test chemical is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If a test chemical acts directly on the MTT (e.g. MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test chemical interference with the viability measurement technique (see paragraphs 29 and 33). Detailed description of how to correct direct MTT reduction and interferences by colouring agents is available in the SOPs for the four validated models included in this test method (32) (33) (34) (35).

29.To identify direct MTT reducers, each test chemical should be added to freshly prepared MTT solution. If the MTT mixture containing the test chemical turns blue/purple, the test chemical is presumed to directly reduce MTT and a further functional check on non-viable RhE tissues should be performed, independently of using the standard absorbance (OD) measurement or an HPLC/UPLC-spectrophotometry procedure. This additional functional check employs killed tissues that possess only residual metabolic activity but absorb the test chemical in a similar way as viable tissues. Each MTT reducing test chemical is applied on at least two killed tissue replicates which undergo the entire testing procedure to generate a non-specific MTT reduction (NSMTT) (32) (33) (34) (35). A single NSMTT control is sufficient per test chemical regardless of the number of independent tests/runs performed. The true tissue viability is then calculated as the percent tissue viability obtained with living tissues exposed to the MTT reducer minus the percent non-specific MTT reduction obtained with the killed tissues exposed to the same MTT reducer, calculated relative to the negative control run concurrently to the test being corrected (%NSMTT).

30.To identify potential interference by coloured test chemicals or test chemicals that become coloured when in contact with water or isopropanol and decide on the need for additional controls, spectral analysis of the test chemical in water (environment during exposure) and/or isopropanol (extracting solution) should be performed. If the test chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, further colorant controls should be performed or, alternatively, an HPLC/UPLC-spectrophotometry procedure should be used in which case these controls are not required (see paragraphs 33 and 34). When performing the standard absorbance (OD) measurement, each interfering coloured test chemical is applied on at least two viable tissue replicates, which undergo the entire testing procedure but are incubated with medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSCliving) control. The NSCliving control needs to be performed concurrently to the testing of the coloured test chemical and in case of multiple testing, an independent NSCliving control needs to be conducted with each test performed (in each run) due to the inherent biological variability of living tissues. The true tissue viability is then calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently to the test being corrected (%NSCliving).

31.Test chemicals that are identified as producing both direct MTT reduction (see paragraph 29) and colour interference (see paragraph 30) will also require a third set of controls, apart from the NSMTT and NSCliving controls described in the previous paragraphs, when performing the standard absorbance (OD) measurement.. This is usually the case with darkly coloured test chemicals interfering with the MTT assay (e.g. blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT as described in paragraph 29. These test chemicals may bind to both living and killed tissues and therefore the NSMTT control may not only correct for potential direct MTT reduction by the test chemical, but also for colour interference arising from the binding of the test chemical to killed tissues. This could lead to a double correction for colour interference since the NSCliving control already corrects for colour interference arising from the binding of the test chemical to living tissues. To avoid a possible double correction for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) needs to be performed. In this additional control, the test chemical is applied on at least two killed tissue replicates, which undergo the entire testing procedure but are incubated with medium instead of MTT solution during the MTT incubation step. A single NSCkilled control is sufficient per test chemical regardless of the number of independent tests/runs performed, but should be performed concurrently to the NSMTT control and, where possible, with the same tissue batch. The true tissue viability is then calculated as the percent tissue viability obtained with living tissues exposed to the test chemical minus %NSMTT minus %NSCliving plus the percent non-specific colour obtained with killed tissues exposed to the interfering test chemical and incubated with medium without MTT, calculated relative to the negative control run concurrently to the test being corrected (%NSCkilled).

32.It is important to note that non-specific MTT reduction and non-specific colour interferences may increase the readouts of the tissue extract above the linearity range of the spectrophotometer. On this basis, each laboratory should determine the linearity range of their spectrophotometer with MTT formazan (CAS # 57360-69-7) from a commercial source before initiating the testing of test chemicals for regulatory purposes. The standard absorbance (OD) measurement using a spectrophotometer is appropriate to assess direct MTT-reducers and colour interfering test chemicals when the ODs of the tissue extracts obtained with the test chemical without any correction for direct MTT reduction and/or colour interference are within the linear range of the spectrophotometer or when the uncorrected percent viability obtained with the test chemical is already ≤ 50%. Nevertheless, results for test chemicals producing %NSMTT and/or %NSCliving ≥ 50% of the negative control should be taken with caution as this is the cut-off used to distinguish classified from not classified chemicals (see paragraph 36).

33.For coloured test chemicals which are not compatible with the standard absorbance (OD) measurement due to too strong interference with the MTT assay, the alternative HPLC/UPLC-spectrophotometry procedure to measure MTT formazan may be employed (see paragraph 34) (36). The HPLC/UPLC-spectrophotometry system allows for the separation of the MTT formazan from the test chemical before its quantification (36). For this reason, NSCliving or NSCkilled controls are never required when using HPLC/UPLC-spectrophotometry, independently of the chemical being tested. NSMTT controls should nevertheless be used if the test chemical is suspected to directly reduce MTT or has a colour that impedes the assessment of the capacity to directly reduce MTT (as described in paragraph 29). When using HPLC/UPLC-spectrophotometry to measure MTT formazan, the percent tissue viability is calculated as percent MTT formazan peak area obtained with living tissues exposed to the test chemical relative to the MTT formazan peak obtained with the concurrent negative control. For test chemicals able to directly reduce MTT, true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the test chemical minus %NSMTT. Finally, it should be noted that direct MTT-reducers that may also be colour interfering, which are retained in the tissues after treatment and reduce MTT so strongly that they lead to ODs (using standard OD measurement) or peak areas (using UPLC/HPLC-spectrophotometry) of the tested tissue extracts that fall outside of the linearity range of the spectrophotometer cannot be assessed, although these are expected to occur in only very rare situations.

34.HPLC/UPLC-spectrophotometry may be used also with all types of test chemicals (coloured, non-coloured, MTT-reducers and non-MTT reducers) for measurement of MTT formazan (36). Due to the diversity of HPLC/UPLC-spectrophotometry systems, qualification of the HPLC/UPLC-spectrophotometry system should be demonstrated before its use to quantify MTT formazan from tissue extracts by meeting the acceptance criteria for a set of standard qualification parameters based on those described in the U.S. Food and Drug Administration guidance for industry on bio-analytical method validation (36) (37). These key parameters and their acceptance criteria are shown in Appendix 4. Once the acceptance criteria defined in Appendix 4 have been met, the HPLC/UPLC-spectrophotometry system is considered qualified and ready to measure MTT formazan under the experimental conditions described in this test method.

Acceptability Criteria

35.For each test method using valid RhE model batches (see paragraph 23), tissues treated with the negative control should exhibit OD reflecting the quality of the tissues that followed shipment, receipt steps and all protocol processes. Control OD values should not be below historically established boundaries. Similarly, tissues treated with the PC, i.e. 5% aqueous SDS, should reflect their ability to respond to an irritant chemical under the conditions of the test method (see Appendix 3 and for further information SOPs of the four test models included in this TG (32) (33) (34) (35)). Associated and appropriate measures of variability between tissue replicates, i.e. standard deviations (SD) should fall within the acceptance limits established for the test model used (see Appendix 3).

Interpretation of Results and Prediction Model

36.The OD values obtained with each test chemical can be used to calculate the percentage of viability normalised to the negative control, which is set to 100%. In case HPLC/UPLC-spectrophotometry is used, the percent tissue viability is calculated as percent MTT formazan peak area obtained with living tissues exposed to the test chemical relative to the MTT formazan peak obtained with the concurrent negative control. The cut-off value of percentage cell viability distinguishing irritant from non-classified test chemicals and the statistical procedure(s) used to evaluate the results and identify irritant chemicals should be clearly defined, documented, and proven to be appropriate (see SOPs of the test models for information). The cut-off values for the prediction of irritation are given below:

-The test chemical is identified as requiring classification and labelling according to UN GHS/CLP (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%. Since the RhE test models covered by this test method cannot resolve between UN GHS/CLP Categories 1 and 2, further information on skin corrosion will be required to decide on its final classification [see also the OECD Guidance Document on IATA (3)]. In case the test chemical is found to be non-corrosive (e.g. based on TM.40, B.40bis or B.65), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS/CLP Category 2.

-Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant to skin in accordance with UN GHS/CLP No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

DATA AND REPORTING

Data

37.For each run, data from individual replicate tissues (e.g. OD values and calculated percentage cell viability data for each test chemical, including classification) should be reported in tabular form, including data from repeat experiments as appropriate. In addition means ± SD for each run should be reported. Observed interactions with MTT reagent and coloured test chemicals should be reported for each tested chemical.

Test Report

38.The test report should include the following information:

Test Chemical and Control Chemicals:

-Mono-constituent substance: chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc;

-Multi-constituent substance, UVCB and mixture: characterised as far as possible by chemical identity (see above), quantitative occurrence and relevant physicochemical properties of the constituents;

-Physical appearance, water solubility, and any additional relevant physicochemical properties;

-Source, lot number if available;

-Treatment of the test chemical/control chemicals prior to testing, if applicable (e.g. warming, grinding);

-Stability of the test chemical, limit date for use, or date for re-analysis if known;

-Storage conditions.

RhE model and protocol used (and rationale for the choice, if applicable)

Test Conditions:

-RhE model used (including batch number);

- Calibration information for measuring device (e.g. spectrophotometer), wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device; Description of the method used to quantify MTT formazan;

-Description of the qualification of the HPLC/UPLC-spectrophotometry system, if applicable; Complete supporting information for the specific RhE model used including its performance. This should include, but is not limited to;

i) Viability;

ii) Barrier function;

iii) Morphology;

iv) Reproducibility and predictivity;

v) Quality controls (QC) of the model;

-Reference to historical data of the model. This should include, but is not limited to acceptability of the QC data with reference to historical batch data.

-Demonstration of proficiency in performing the test method before routine use by testing of the proficiency substances.

Test Procedure:

- Details of the test procedure used (including washing procedures used after exposure period); Dose of test chemical and controls used;

- Duration and temperature of exposure and post-exposure incubation period;

-Indication of controls used for direct MTT-reducers and/or colouring test chemicals, if applicable;

-Number of tissue replicates used per test chemical and controls (PC, negative control, and NSMTT, NSCliving and NSCkilled, if applicable);

-Description of decision criteria/prediction model applied based on the RhE model used;

-Description of any modifications to the test procedure (including washing procedures).

Run and Test Acceptance Criteria:

- Positive and negative control mean values and acceptance ranges based on historical data; Acceptable variability between tissue replicates for positive and negative controls;

- Acceptable variability between tissue replicates for test chemical.

Results:

- Tabulation of data for individual test chemical for each run and each replicate measurement including OD or MTT formazan peak area, percent tissue viability, mean percent tissue viability and SD; 

- If applicable, results of controls used for direct MTT-reducers and/or colouring test chemicals including OD or MTT formazan peak area, %NSMTT, %NSCliving, %NSCkilled, SD, final correct percent tissue viability;

-Results obtained with the test chemical(s) and controls in relation to the defined run and test acceptance criteria;

- Description of other effects observed;

-The derived classification with reference to the prediction model/decision criteria used.

Discussion of the results

Conclusions

LITERATURE

(1)United Nations (UN) (2013). Globally Harmonized System of Classification and Labelling of Chemicals (GHS), Second Revised Edition, UN New York and Geneva, 2013. Available at: http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html.

(2)EURL-ECVAM (2009). Statement on the “Performance Under UN GHS of Three In Vitro Assays for Skin Irritation Testing and the Adaptation of the Reference Chemicals and Defined Accuracy Values of the ECVAM Skin Irritation Performance Standards”, Issued by the ECVAM Scientific Advisory Committee (ESAC31), 9 April 2009. Available at: https://eurl-ecvam.jrc.ec.europa.eu/about-ecvam/archive-publications/publication//ESAC31_skin-irritation-statement_20090922.pdf 

(3)OECD (2014). Guidance Document on Integrated Approaches to Testing and Assessment for Skin Irritation/Corrosion. Environment, Health and Safety Publications, Series on Testing and Assessment (No 203), Organisation for Economic Cooperation and Development, Paris.

(4)Chapter B.4 of this Annex, Acute Dermal Irritation, Corrosion.

(5)Chapter B.40 of this Annex, In Vitro Skin Corrosion: Transcutaneous Electrical Resistance (TER).

(6)Chapter B.40bis of this Annex, In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) test method.

(7)Chapter B.65 of this Annex, In Vitro Membrane Barrier Test Method.

(8)OECD (2015). Performance Standards for the Assessment of Proposed Similar or Modified In Vitro Reconstructed Human Epidermis (RhE) Test Methods for Skin Irritation in Relation to TG 439. Environment, health and Safety Publications, Series on Testing and Assessment (No 220). Organisation for Economic Cooperation and Development, Paris.

(9)OECD (2005). Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment. Environment, Health and Safety Publications, Series on Testing and Assessment (No 34) Organisation for Economic Cooperation and Development, Paris.

(10)Fentem, J.H., Briggs, D., Chesné, C., Elliot, G.R., Harbell, J.W., Heylings, J.R., Portes, P., Roguet, R., van de Sandt, J.J. M. and Botham, P. (2001). A Prevalidation Study on In Vitro Tests for Acute Skin Irritation, Results and Evaluation by the Management Team, Toxicol. in Vitro 15, 57-93.

(11)Portes, P., Grandidier, M.-H., Cohen, C. and Roguet, R. (2002). Refinement of the EPISKIN Protocol for the Assessment of Acute Skin Irritation of Chemicals: Follow-Up to the ECVAM Prevalidation Study, Toxicol. in Vitro 16, 765–770.

(12)Kandárová, H., Liebsch, M., Genschow, E., Gerner, I., Traue, D., Slawik, B. and Spielmann, H. (2004). Optimisation of the EpiDerm Test Protocol for the Upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests, ALTEX 21, 107–114.

(13)Kandárová, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D. and Spielmann, H. (2005), The EpiDerm Test Protocol for the Upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests – An Assessment of the Performance of the Optimised Test, ATLA 33, 351-367.

(14)Cotovio, J., Grandidier, M.-H., Portes, P., Roguet, R. and Rubinsteen, G. (2005). The In Vitro Acute Skin Irritation of Chemicals: Optimisation of the EPISKIN Prediction Model Within the Framework of the ECVAM Validation Process, ATLA 33, 329-349.

(15)Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., Elliot, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van De Sandt, J.J.M., Wiemann, C. and Worth, A. (2002). Follow-Up to the ECVAM Prevalidation Study on In Vitro Tests for Acute Skin Irritation, The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2, ATLA 30, 109-129.

(16)Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R., Cotovio, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kandárová, H., Gamer, A., Remmele, M., Curren, R., Raabe, H., Cockshott, A., Gerner, I. and Zuang, V. (2007). The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test, ATLA 35, 559-601.

(17)Hoffmann S. (2006). ECVAM Skin Irritation Validation Study Phase II: Analysis of the Primary Endpoint MTT and the Secondary Endpoint IL1-α.

(18)Eskes C., Cole, T., Hoffmann, S., Worth, A., Cockshott, A., Gerner, I. and Zuang, V. (2007). The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Selection of Test Chemicals, ATLA 35, 603-619.

(19)Cotovio, J., Grandidier, M.-H., Lelièvre, D., Roguet, R., Tinois-Tessonneaud, E. and Leclaire, J. (2007). In Vitro Acute Skin Irritancy of Chemicals Using the Validated EPISKIN Model in a Tiered Strategy - Results and Performances with 184 Cosmetic Ingredients, ALTEX, 14, 351-358.

(20)EURL-ECVAM (2007). Statement on the Validity of In Vitro Tests for Skin Irritation, Issued by the ECVAM Scientific Advisory Committee (ESAC26), 27 April 2007. Available at: https://eurl-ecvam.jrc.ec.europa.eu/about-ecvam/archive-publications/publication//ESAC26_statement_SkinIrritation_20070525_C.pdf

(21)EURL-ECVAM. (2007). Performance Standards for Applying Human Skin Models to In Vitro Skin Irritation Testing. N.B. These are the original PS used for the validation of two test methods. These PS should not be used any longer as an updated version (8) is now available.

(22)EURL-ECVAM. (2008). Statement on the Scientific Validity of In Vitro Tests for Skin Irritation Testing, Issued by the ECVAM Scientific Advisory Committee (ESAC29), 5 November 2008. https://eurl-ecvam.jrc.ec.europa.eu/about-ecvam/archive-publications/publication/ESAC_Statement_SkinEthic-EpiDerm-FINAL-0812-01.pdf 

(23)OECD (2010). Explanatory Background Document to the OECD Draft Test Guideline on In Vitro Skin Irritation Testing. Environment, Health and Safety Publications. Series on Testing and Assessment, (No 137), Organisation for Economic Cooperation and Development, Paris.

(24)Katoh, M., Hamajima, F., Ogasawara, T. and Hata K. (2009). Assessment of Human Epidermal Model LabCyte EPI-MODEL for In Vitro Skin Irritation Testing According to European Centre for the Validation of Alternative Methods (ECVAM)-Validated Protocol, J Toxicol Sci, 34, 327-334

(25)Katoh, M. and Hata K. (2011). Refinement of LabCyte EPI-MODEL24 Skin Irritation Test Method for Adaptation to the Requirements of OECD Test Guideline 439, AATEX, 16, 111-122

(26)OECD (2011). Validation Report for the Skin Irritation Test Method Using LabCyte EPI-MODEL24. Environment, Health and Safety Publications, Series on Testing and Assessment (No 159), Organisation for Economic Cooperation and Development, Paris.

(27)OECD (2011). Peer Review Report of Validation of the Skin Irritation Test Using LabCyte EPI-MODEL24. Environment, Health and Safety Publications, Series on Testing and Assessment (No 155), Organisation for Economic Cooperation and Development, Paris.

(28)Kojima, H., Ando, Y., Idehara, K., Katoh, M., Kosaka, T., Miyaoka, E., Shinoda, S., Suzuki, T., Yamaguchi, Y., Yoshimura, I., Yuasa, A., Watanabe, Y. and Omori, T. (2012). Validation Study of the In Vitro Skin Irritation Test with the LabCyte EPI-MODEL24, Altern Lab Anim, 40, 33-50.

(29)Welss, T., Basketter, D.A. and Schröder, K.R. (2004). In Vitro Skin Irritation: Fact and Future. State of the Art Review of Mechanisms and Models, Toxicol. In Vitro 18, 231-243.

(30)Eskes, C. et al. (2012). Regulatory Assessment of In Vitro Skin Corrosion and Irritation Data within the European Framework: Workshop Recommendations. Regul.Toxicol.Pharmacol. 62, 393-403).

(31)Mosmann, T. (1983). Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays, J. Immunol. Methods 65, 55-63.

(32)EpiSkin™ (February 2009). SOP, Version 1.8ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ Test Method 15 min - 42 hours for the Prediction of acute Skin Irritation of Chemicals

(33)EpiDerm™ (Revised March 2009). SOP, Version 7.0, Protocol for: In Vitro EpiDerm™ Skin Irritation Test (EPI-200-SIT), for Use with MatTek Corporation's Reconstructed Human Epidermal Model EpiDerm (EPI-200).

(34)SkinEthic™ RHE (February 2009) SOP, Version 2.0, SkinEthic Skin Irritation Test-42bis Test Method for the Prediction of Acute Skin Irritation of Chemicals: 42 Minutes Application + 42 Hours Post-Incubation.

(35)LabCyte (June 2011). EPI-MODEL24 SIT SOP, Version 8.3, Skin Irritation Test Using the Reconstructed Human Model “LabCyte EPI-MODEL24”

(36)Alépée, N., Barroso, J., De Smedt, A., De Wever, B., Hibatallah, J., Klaric, M., Mewes, K.R., Millet, M., Pfannenbecker, U., Tailhardat, M., Templier, M., and McNamee, P. Use of HPLC/UPLC-Spectrophotometry for Detection of MTT Formazan in In Vitro Reconstructed Human Tissue (RhT)-Based Test Methods Employing the MTT Assay to Expand their Applicability to Strongly Coloured Test Chemicals. Manuscript in preparation.

(37)US FDA (2001). Guidance for Industry: Bioanalytical Method Validation. U.S. Department of Health and Human Services, Food and Drug Administration. May 2001. Available at: [http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf].

(38)Harvell, J.D., Lamminstausta, K., and Maibach, H.I. (1995). Irritant Contact Dermatitis, in: Practical Contact Dermatitis, pp 7-18, (Ed. Guin J. D.). Mc Graw-Hill, New York.

(39)EURL-ECVAM (2009). Performance Standards for In Vitro Skin Irritation Test Methods Based on Reconstructed Human Epidermis (RhE). N.B. This is the current version of the ECVAM PS, updated in 2009 in view of the implementation of UN GHS. These PS should not be used any longer as an updated version (8) is now available related to the present TG.

(40)EURL-ECVAM. (2009). ESAC Statement on the Performance Standards (PS) for In Vitro Skin Irritation Testing Using Reconstructed Human Epidermis, Issued by the ECVAM Scientific Advisory Committee (ESAC31), 8 July 2009.

(41)EC (2001). Commission Directive 2001/59/EC of 6 August 2001 Adapting to Technical Progress for the 28th Time Council Directive 67/548/EEC on the Approximation of Laws, Regulations and Administrative Provisions Relating to the Classification, Packaging and Labelling of Dangerous Substances, Official Journal of the European Union L225, 1-333.

Appendix 1

DEFINITIONS

Accuracy: The closeness of agreement between test method results and accepted reference values. It is a measure of test method performance and one aspect of relevance. The term is often used interchangeably with “concordance” to mean the proportion of correct outcomes of a test method (9).

Cell viability: Parameter measuring total activity of a cell population e.g. as ability of cellular mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue), which depending on the endpoint measured and the test design used, correlates with the total number and/or vitality of living cells.

Chemical: means a substance or a mixture.

Concordance: This is a measure of performance for test models that give a categorical result, and is one aspect of relevance. The term is sometimes used interchangeably with accuracy, and is defined as the proportion of all chemicals tested that are correctly classified as positive or negative. Concordance is highly dependent on the prevalence of positives in the types of test chemical being examined (9).

ET50: Can be estimated by determination of the exposure time required to reduce cell viability by 50% upon application of the benchmark chemical at a specified, fixed concentration, see also IC50.

GHS (Globally Harmonized System of Classification and Labelling of Chemicals by the United Nations (UN)): A system proposing the classification of chemicals (substances and mixtures) according to standardised types and levels of physical, health and environmental hazards, and addressing corresponding communication elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data sheets, so that to convey information on their adverse effects with a view to protect people (including employers, workers, transporters, consumers and emergency responders) and the environment (1).

HPLC: High Performance Liquid Chromatography.

IATA: Integrated Approach on Testing and Assessment

IC50: Can be estimated by determination of the concentration at which a benchmark chemical reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, see also ET50.

Infinite dose:  Amount of test chemical applied to the epidermis exceeding the amount required to completely and uniformly cover the epidermis surface.

Mixture: A mixture or a solution composed of two or more substances.

Mono-constituent substance: A substance, defined by its quantitative composition, in which one main constituent is present to at least 80% (w/w).

MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide.

Multi-constituent substance: A substance, defined by its quantitative composition, in which more than one main constituent is present in a concentration ≥ 10% (w/w) and < 80% (w/w). A multi-constituent substance is the result of a manufacturing process. The difference between mixture and multi-constituent substance is that a mixture is obtained by blending of two or more substances without chemical reaction. A multi-constituent substance is the result of a chemical reaction.

NSCkilled: Non-Specific Colour in killed tissues.

NSC: Non-Specific Colour in living tissues.

NSMTT: Non-Specific MTT reduction.

Performance standards (PS): Standards, based on a validated test method, that provide a basis for evaluating the comparability of a proposed test method that is mechanistically and functionally similar. Included are; (i) essential test method components; (ii) a minimum list of Reference Chemicals selected from among the chemicals used to demonstrate the acceptable performance of the validated test method; and (iii) the comparable levels of accuracy and reliability, based on what was obtained for the validated test method, that the proposed test method should demonstrate when evaluated using the minimum list of Reference Chemicals (9).

PC: Positive Control, a replicate containing all components of a test system and treated with a chemical known to induce a positive response. To ensure that variability in the positive control response across time can be assessed, the magnitude of the positive response should not be excessive.

Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful and useful for a particular purpose. It is the extent to which the test correctly measures or predicts the biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test method (9).

Reliability: Measures of the extent that a test method can be performed reproducibly within and between laboratories over time, when performed using the same protocol. It is assessed by calculating intra- and inter-laboratory reproducibility (9).

Replacement test: A test which is designed to substitute for a test that is in routine use and accepted for hazard identification and/or risk assessment, and which has been determined to provide equivalent or improved protection of human or animal health or the environment, as applicable, compared to the accepted test, for all possible testing situations and chemicals (9).

Run: A run consists of one or more test chemicals tested concurrently with a negative control and with a PC.

Sensitivity: The proportion of all positive/active test chemicals that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results, and is an important consideration in assessing the relevance of a test method (9).

Skin irritation in vivo: The production of reversible damage to the skin following the application of a test chemical for up to 4 hours. Skin irritation is a locally arising reaction of the affected skin tissue and appears shortly after stimulation (38). It is caused by a local inflammatory reaction involving the innate (non-specific) immune system of the skin tissue. Its main characteristic is its reversible process involving inflammatory reactions and most of the clinical characteristic signs of irritation (erythema, oedema, itching and pain) related to an inflammatory process.

Specificity: The proportion of all negative/inactive test chemicals that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results and is an important consideration in assessing the relevance of a test method (9).

Substance: A chemical element and its compounds in the natural state or obtained by any production process, inducing any additive necessary to preserve its stability and any impurities deriving from the process used, but excluding any solvent which may be separated without affecting the stability of the substance or changing it composition.

Test chemical: Any substance or mixture tested using this test method..

UPLC: Ultra-High Performance Liquid Chromatography.

UVCB: substances of unknown or variable composition, complex reaction products or biological materials.

Appendix 2

TEST MODELS INCLUDED IN THIS TEST METHOD

*) The original ECVAM Performance Standards (PS) (21) were developed in 2007 upon completion of the prospective validation study (16) which had assessed the performance of test models Nr 1 and 2 in reference to the classification system as described in the 28th amendment to the EU Dangerous Substances Directive (41). In 2008 the UN GHS (1) and EU CLP were introduced, effectively shifting the cut-off value for distinguishing non-classified from classified substances from an in vivo score of 2.0 to 2.3. To adapt to this changed regulatory requirement, the accuracy values and reference chemical list of the ECVAM PS were updated in 2009 (2) (39) (40). As the original PS, also the updated PS were largely based data from models Nr. 1 and 2 (16), but additionally used data on reference chemicals from model Nr. 3. In 2010, the updated ECVAM PS were used for stipulating the PS related to this TG (8). For the purpose of this test method, EpiSkin™ is considered the VRM, due to the fact that it was used to develop all the criteria of the PS.. Detailed information on the validation studies, a compilation of the data generated as well as background to the necessary adaptations of the PS as a consequence of the UN GHS/CLP implementation can be found in the ECVAM/BfR explanatory background document to the corresponding OECD TG 439 (23).

SIT: Skin Irritation Test

RHE: Reconstructed Human Epidermis

Appendix 3

PROTOCOL PARAMETERS SPECIFIC TO EACH OF THE TEST MODELS
INCLUDED IN THIS TEST Method

The RhE models do show very similar protocols and notably all use a post-incubation period of 42 hours (32) (33) (34) (35). Variations concern mainly three parameters relating to the different barrier functions of the test models and listed here: A) pre-incubation time and volume, B) Application of test chemicals and C) Post-incubation volume.

RT: Room temperature

DW: distilled water

DPBS: Dulbecco’s Phosphate Buffer Saline

Appendix 4

Key parameters and acceptance criteria for qualification of an HPLC/UPLC-spectrophotometry system for measurement of MTT formazan extracted from RhE tissues

1LLOQ: Lower Limit of Quantification, defined to cover 1-2% tissue viability, i.e. 0.8 µg/ml.

2ULOQ: Upper Limit of Quantification, defined to be at least two times higher than the highest expected MTT formazan concentration in isopropanol extracts from negative controls i.e. 200 µg/ml."

(8) In Part B, the following Chapters are added:

"B.63 REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST

INTRODUCTION

1.This test method is equivalent to OECD test guideline (TG) 421 (2016). OECD guidelines for the testing of chemicals are periodically reviewed in the light of scientific progress. The original screening test guideline 421 was adopted in 1995, based on a protocol for a "Preliminary Reproduction Toxicity Screening Test" discussed in two expert meetings, in London in 1990 (1) and in Tokyo in 1992 (2).

2.This test method has been updated with endocrine disruptor relevant endpoints, as a follow up to the high-priority activity initiated at OECD in 1998 to revise existing test guidelines and to develop new test guidelines for the screening and testing of potential endocrine disruptors (3). OECD TG 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents, Chapter B.7 of this Annex) for example, was enhanced in 2008 by parameters suitable to detect endocrine activity of test chemicals. The objective in updating TG 421 was to include some endocrine disruptor relevant endpoints in screening TGs where the exposure periods cover some of the sensitive periods during development (pre- or early postnatal periods).

3.The selected additional endocrine disrupter relevant endpoints, also part of TG 443 (Extended One Generation Reproductive Toxicity Study, Chapter B.56 of this Annex), were included in TG 421 based on a feasibility study addressing scientific and technical questions related to their inclusion, as well as possible adaptations of the test design needed for their inclusion (4).

4.This test method is designed to generate limited information concerning the effects of a test chemical on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. It is not an alternative to, nor does it replace the existing test methods B.31, B.34, B.35 or B.56.

INITIAL CONSIDERATIONS

5.This screening test method can be used to provide initial information on possible effects on reproduction and/or development, either at an early stage of assessing the toxicological properties of chemicals, or on chemicals of concern. It can also be used as part of a set of initial screening tests for existing chemicals for which little or no toxicological information is available, as a dose range finding study for more extensive reproduction/developmental studies, or when otherwise considered relevant. In conducting the study, the guiding principles and considerations outlined in the OECD guidance document no 19 on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluations (5) should be followed.

6.This test method does not provide complete information on all aspects of reproduction and development. In particular, it offers only limited means of detecting post-natal manifestations of pre-natal exposure, or effects that may be induced during post-natal exposure. Due (amongst other reasons) to the relatively small numbers of animals in the dose groups, the selectivity of the end points, and the short duration of the study, this method will not provide evidence for definite claims of no effects. Moreover, in the absence of data from other reproduction/developmental toxicity tests, positive results are useful for initial hazard assessment and contribute to decisions with respect to the necessity and timing of additional testing.

7.The results obtained by the endocrine related parameters should be seen in the context of the “OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupting Chemicals” (6). In this Conceptual Framework, the enhanced OECD TG 421 is contained in level 4 as an in vivo assay providing data on adverse effects on endocrine relevant endpoints. An endocrine signal might not however be considered sufficient evidence on its own that the test chemical is an endocrine disruptor.

8.This test method assumes oral administration of the test chemical. Modifications may be required if other routes of exposure are used.

9.Before use of the test method on a mixture for generating data for an intended regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose. Such considerations are not needed, when there is a regulatory requirement for testing of the mixture.

10.Definitions used are given in Appendix 1.

PRINCIPLE OF THE TEST

11.The test chemical is administered in graduated doses to several groups of males and females. Males should be dosed for a minimum of four weeks and up to and including the day before scheduled kill (this includes a minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post-mating). In view of the limited pre-mating dosing period in males, fertility may not be a particular sensitive indicator of testicular toxicity. Therefore, a detailed histological examination of the testes is essential. The combination of a pre-mating dosing period of two weeks and subsequent mating/fertility observations with an overall dosing period of at least four weeks, followed by detailed histopathology of the male gonads, is considered sufficient to enable detection of the majority of effects on male fertility and spermatogenesis.

12.Females should be dosed throughout the study. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and at least thirteen days after delivery, up to and including the day before scheduled kill.

13.Duration of study, following acclimatisation and pre-dosing oestrous cycle evaluation, is dependent on the female performance and is approximately 63 days, [at least 14 days premating, (up to) 14 days mating, 22 days gestation, 13 days lactation].

14.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 period are necropsied and, at the conclusion of the test, surviving animals are killed and necropsied.

DESCRIPTION OF THE METHOD

Selection of animal species

15.This test method is designed for use with the rat. If the parameters specified within this test method are investigated in another rodent species a detailed justification should be given. In the international validation program for the detection of endocrine disrupters in OECD TG 407 (corresponding to Chapter B.7 of this Annex), the rat was the only species used. Strains with low fecundity or well-known high incidence of developmental defects should not be used. Healthy virgin animals, not subjected to previous experimental procedures, should be used. The test animals should be characterised as to species, strain, sex, weight and age. 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 study to a long-term or a full-generation study, it is preferable that animals from the same strain and source are used in both studies.

Housing and feeding

16.All procedures should conform to local standards of laboratory animal care. The temperature in the experimental animal room should be 22 °C (± 3°). 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 photoperiod 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 chemical when administered by this method.

17.Animals should be group housed in small groups of the same sex; animals may be housed individually if scientifically justified. For group caging, no more than five animals should be housed per cage. Mating procedures should be carried out in cages suitable for the purpose. Pregnant females should be caged individually and provided with nesting materials. Lactating females will be caged individually with their offspring.

18.The feed should be regularly analysed for contaminants. A sample of the diet should be retained until finalisation of the report.

Preparation of the animals

19.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 uniquely identified 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.

Preparation of doses

20.It is recommended that the test chemical be administered orally unless other routes of administration are considered more appropriate. When the oral route is selected, the test chemical is usually administered by gavage; however, alternatively, test chemicals may be administered via the diet or drinking water.

21.Where necessary, the test chemical 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 should be known. The stability and homogeneity of the test chemical in the vehicle should be determined.

PROCEDURE

Number and sex of animals

22.It is recommended that each group be started with at least 10 males and 12-13 females. Females will be evaluated pre-exposure for oestrous cyclicity and animals that fail to exhibit typical 4-5 day cycles will not be included in the study; therefore, extra females are recommended in order to yield 10 females per group. Except in the case of marked toxic effects, it is expected that this will provide at least 8 pregnant females per group which normally is the minimum acceptable number of pregnant females per group. The objective is to produce enough pregnancies and offspring to assure a meaningful evaluation of the potential of the test chemical to affect fertility, pregnancy, maternal and suckling behaviour, and growth and development of the F1 offspring from conception to day 13 post-partum.

Dosage

23.Generally, at least three test groups and a control group should be used. Dose levels may be based on information from acute toxicity tests or on results from repeated dose studies. Except for treatment with the test chemical, 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 chemical, the control group should receive the vehicle in the highest volume used.

24.Dose levels should be selected taking into account any existing toxicity and (toxico-) kinetic data available. It should also be taken into account that there may be differences in sensitivity between pregnant and non-pregnant animals. 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 (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 10) between dosages.

25.In the presence of observed general toxicity (e.g. reduced body weight, liver , heart, lung or kidney effects, etc.) or other changes that may not be toxic responses (e.g. reduced food intake, liver enlargement), observed effects on endocrine sensitive endpoints should be interpreted with caution.

Limit test

26.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 and if toxicity would not be expected based upon data from structurally related substances, 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 chemicals often may dictate the maximum attainable concentration.

Administration of doses

27.The animals are dosed with the test chemical daily for 7 days a week. When the test chemical 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 test chemicals 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.

28.For test chemical administered via the diet or drinking water, it is important to ensure that the quantities of the test chemical involved do not interfere with normal nutrition or water balance. When the test chemical 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 should be specified. For a test chemical 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.

Experimental schedule

29.Dosing of both sexes should begin at least 2 weeks prior to mating, after they have been acclimatised for at least five days and females have been screened for normal oestrous cycles (in a 2 weeks pre-treatment period). The study should be scheduled in such a way that oestrous cycle evaluation begins soon after the animals have attained full sexual maturity. This may vary slightly for different strains of rats in different laboratories, e.g. Sprague Dawley rats 10 weeks of age, Wistar rats about 12 weeks of age. Dams with offspring should be killed on day 13 post-partum, or shortly thereafter. The day of birth (viz. when parturition is complete) is defined as day 0 post-partum. Females showing no-evidence of copulation are killed 24-26 days after the last day of the mating period. Dosing is continued in both sexes during the mating period. Males should further be dosed after the mating period at least until the minimum total dosing period of 28 days has been completed. They are then killed, or, alternatively, are retained and continued to be dosed for the possible conduction of a second mating if considered appropriate.

30.Daily dosing of the parental females should continue throughout pregnancy and at least up to, and including, day 13 post-partum or the day before sacrifice. For studies where the test chemical is administered by inhalation or by the dermal route, dosing should be continued at least up to, and including, day 19 of gestation, and dosing should be re-initiated as soon as possible and not later than PND 4.

31.A diagram of the experimental schedule is given in Appendix 2.

Mating procedure

32.Normally, 1:1 (one male to one female) matings should be used in this study. Exceptions can arise in the case of occasional deaths of males. The female should be placed with the same male until evidence of copulation is observed or two weeks have elapsed. Each morning the females should be examined for the presence of sperm or a vaginal plug. Day 0 of pregnancy is defined as the day on which mating evidence is confirmed (a vaginal plug or sperm is found). In case pairing is unsuccessful, re-mating of females with proven males of the same group could be considered.

Litter size

33.On day 4 after birth, the size of each litter may be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four or five pups per sex per litter depending on the normal litter size in the strain of rats used. Blood samples should be collected from two of the surplus pups, pooled, and used for determination of serum T4 levels. Selective elimination of pups, e.g. based upon body weight, or anogenital distance (AGD) is not appropriate. Whenever the number of male or female pups prevents having four or five of each sex per litter, partial adjustment (for example, six males and four females) is acceptable. No pups will be eliminated when litter size will drop below the culling target (8 or 10 pups/litter). If there is only one pup available above the culling target, only one pup will be eliminated and used for blood collection for possible serum T4 assessments. 

34.If litter size is not adjusted, two pups per litter are sacrificed on day 4 after birth and blood samples are taken for measurement of serum thyroid hormone concentrations. If possible the two pups per litter should be female pups to reserve male pups for nipple retention evaluations except in the event that removing these pups leaves no remaining females for assessment at termination. No pups will be eliminated when litter size will drop below 8 or 10 pups/litter (depending on the normal litter size in the strain of rats used). If there is only one pup available above the normal litter size, only one pup will be eliminated and used for blood collection for possible serum T4 assessments.

In life observations

Clinical observations

35.Throughout the test period, general clinical observations should be made at least once a day, and more frequently when signs of toxicity are observed. They should be made preferably at the same time(s) each day, considering the peak period of anticipated effects after dosing. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, should be recorded. These records should include time of onset, degree and duration of toxicity signs.

Body weight and food/water consumption

36.Males and females should be weighed on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females should be weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and at least day 4 and 13 post-partum. These observations should be reported individually for each adult animal.

37.During pre-mating, pregnancy and lactation, food consumption should be measured at least weekly. The measurement of food consumption during mating is optional. Water consumption during these periods should also be measured when the test chemical is administered via drinking water.

Oestrous cycles

38.Oestrous cycles should be monitored before treatment starts to select for the study females with regular cyclicity (see paragraph 22). Vaginal smears should also be monitored daily from the beginning of the treatment period until evidence of mating. If there is concern about acute stress effects that could alter oestrous cycles with the initiation of dosing, laboratories can expose test animals for 2 weeks, then collect vaginal smears daily to monitor oestrous cycle for a minimum of two weeks during the pre-mating period with continued monitoring into the mating period until there is evidence of mating. When obtaining vaginal/cervical cells, care should be taken to avoid disturbance of mucosa, which could induce pseudopregnancy (7) (8).

Offspring parameters

39.The duration of gestation should be recorded and is 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, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups) and the presence of gross abnormalities.

40.Live pups should be counted and sexed and litters weighed within 24 hours of parturition (day 0 or 1 post-partum) and at least on day 4 and 13 post-partum. In addition to the observations described in paragraph 35, any abnormal behaviour of the offspring should be recorded.

41.The AGD of each pup should be measured on the same postnatal day between PND 0 through PND 4. Pup body weight should be collected on the day the AGD is measured and the AGD should be normalised to a measure of pup size, preferably the cube root of body weight (9). The number of nipples/areolae in male pups should be counted on PND 12 or 13 as recommended in OECD GD 151 (10).

Clinical biochemistry

42.Blood samples from a defined site are taken based on the following schedule:

-    from at least two pups per litter on day 4 after birth, if the number of pups allows (see paragraphs 33-34)

-    from all dams and at least two pups per litter at termination on day 13, and

-    from all adult males, at termination,

All blood samples are stored under appropriate conditions. Blood samples from the day 13 pups and the adult males are assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups is done if relevant. As an option other hormones may be measured if relevant. Pup blood can be pooled by litter for thyroid hormone analyses. Thyroid hormones (T4 and TSH) should preferably be measured as ‘total’.

43.The following factors may influence the variability and the absolute concentrations of the hormone determinations:

-time of sacrifice because of diurnal variation of hormone concentrations

-method of sacrifice to avoid undue stress to the animals that may affect hormone concentrations

-test kits for hormone determinations that may differ by their standard curves.

44.Plasma samples specifically intended for hormone determination should be obtained at a comparable time of the day. The numerical values obtained when analysing hormone concentrations differ with various commercial assay kits.

Pathology

Gross necropsy

45.At the time of sacrifice or death during the study, the adult animals should be examined macroscopically for any abnormalities or pathological changes. Special attention should be paid to the organs of the reproductive system. The number of implantation sites should be recorded. Vaginal smears should be examined in the morning on the day of necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of ovaries.