TABLE 1: TEST METHODS FOR PHYSICOCHEMICAL PROPERTIES OF THE SUBSTANCE

Endpoint

Test method

Corresponding chapter in Part A of this Annex (numbers in brackets indicate that a test method has been deleted from Part A; empty cell: no corresponding test method in Part A of this Annex)

Melting point/freezing point

OECD Test Guideline 102: Melting Point/ Melting Range (1995)

A.1.

Boiling point

OECD Test Guideline 103: Boiling point (1995)

A.2.

Density

OECD Test Guideline 109: Density of Liquids and solids (2012)

(A.3.)

Vapour pressure

OECD Test Guideline 104: Vapour Pressure (2006)

(A.4)

Surface tension

OECD Test Guideline 115: Surface tension of aqueous solutions (1995)

A.5.

Water solubility

OECD Test Guideline 105: Water Solubility (1995)

A.6.

Partition coefficient
n-octanol/water

OECD Test Guideline 107: Partition coefficient (n-octanol/water): shake-flask method (1995)

(A.8.)

OECD Test Guideline 123: Partition Coefficient (1-Octanol/Water): Slow-Stirring Method (2006)

A.23.

OECD Test Guideline 117: Partition Coefficient (n-octanol/water): HPLC Method (2004)

A.24.

Dissociation constant

OECD Test Guideline 112: Dissociation Constants in Water. (1981)

A.25.

Viscosity

OECD TG 114: Viscosity of Liquids (2012)

Flashpoint

Test methods according to table 2.6.3 of Annex I, Part 2 of Regulation (EC) No 1272/2008

(A.9)

Lower and upper explosion limit

EN 1839:2017 – Determination of the explosion limits and the limiting oxygen concentration (LOC) for flammable gases and vapours

Flammability

Test methods according to section 2.2.4.1. of Annex I, Part 2 of Regulation (EC) No 1272/2008

(A.11)

Test L.2: sustained combustibility test, Part III, section 32 of the UN RTDG, Manual of Tests and Criteria

Test N.1: test method for readily combustible solids, Part III, sub-section 33.2.1.4 of the UN RTDG Manual of Tests and Criteria

(A.10)

Test N.5: test method for substances which in contact with water emit flammable gases, Part III, sub-section 33.4.1.4 of the UN RTDG Manual of Tests and Criteria

(A.12)

Self-ignition temperature (solids)

Test N.4: test method for self-heating substances, Part III, sub-section 33.3.1.6 of the UN RTDG Manual of Tests and Criteria

EN 15188:2020 – Determination of the spontanous ignition behaviour of dust accumulations

Auto-ignition temperature (liquids, gases)

EN 14522:2005 – Determination of the auto ignition temperature of gases and vapours

(A.15)

Decomposition temperature

Test Series H, part II, section 28, of the UN RTDG Manual of Tests and Criteria

Explosive properties

Test methods according to Test series 1-3, Part I, sections 11-13 of the UN RTDG Manual of Tests and Criteria

(A.14)

Oxidising properties

Test method according to section 2.4.4. of Annex I, Part 2 of Regulation (EC) No 1272/2008

Test O.2: test for oxidizing liquids, Part III, sub-section 34.4.2 of the UN RTDG Manual of Tests and Criteria

(A.21)

Test O.1: Test for oxidizing solids, Part III, sub-section 34.4.1 of the UN RTDG Manual of Tests and Criteria

(A.17)

Test O.3 Gravimetric test for oxidizing solids, Part III, sub-section 34.4 3 of the UN RTDG Manual of Tests and Criteria

(A.17)

Pyrophoricity

Test N.3: test method for pyrophoric liquids, Part III, sub-section 33.3.1.5 of the UN RTDG Manual of Tests and Criteria

Test N.2: test method for pyrophoric solids, Part III, sub-section 33.3.1.4 of the UN RTDG Manual of Tests and Criteria

Granulometry/ particle characteristics

EU test method A.22. Length weighted geometric mean diameter of fibres

A.22.

ISO 13318 - Determination of particle size distribution by centrifugal liquid sedimentation methods

ISO 21501 - Determination of particle size distribution - Single particle light interaction methods

pH

OECD TG 122: Determination of pH, Acidity and Alkalinity (2013)

Properties of polymers

OECD TG 118: Determination of the Number-Average Molecular Weight and the Molecular Weight Distribution of Polymers using Gel Permeation Chromatography (1996)

A.18.

OECD TG 119: Determination of the Low Molecular Weight Content of a Polymer Using Gel Permeation Chromatography (1996)

A.19.

OECD TG 120: Solution/Extraction Behaviour of Polymers in Water (2000)

(A.20.)

TABLE 2: TEST METHODS FOR TOXICOLOGICAL PROPERTIES

Endpoint

Test method

Corresponding chapter in Part B of this Annex (numbers in brackets indicate that a test method has been deleted from Part B; empty cell: no corresponding EU test method in Part B of this Annex)

Skin corrosion/irritation

In vitro:

OECD Test Guideline 430: In vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (2015)

B.40.

OECD Test Guideline 431: In vitro Skin Corrosion: Human Skin Model Test (2019)

(B.40bis.)

OECD Test Guideline 435: In vitro Membrane Barrier Test Method for Skin Corrosion (2015)

B.65.

OECD Test Guideline 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2021)

(B.46.)

In vivo:

OECD Test Guideline 404: Acute Dermal Irritation/Corrosion (2015)

B.4.

Serious eye damage/eye irritation

In vitro:

OECD Test Guideline 437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (2020)

(B.47.)

OECD Test Guideline 438: Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants (2018)

(B.48.)

OECD Test Guideline 460: Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants (2012, corrected in 2017)

(B.61.)

OECD test guideline 491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2020)

(B.68.)

OECD test guideline 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (2019)

(B.69.)

OECD test guideline 494: Vitrigel Eye Irritancy Test (2021)

OECD test guideline 496: In vitro Macromolecular Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2019)

In vivo:

OECD Test Guideline 405: Acute eye irritation/corrosion (2021)

(B.5.)

Skin sensitisation

In vitro:

OECD Test Guideline 442C: In chemico skin sensitisation: Direct peptide reactivity assay (DPRA) (2021)

(B.59.)

OECD Test Guideline 442D: In vitro skin sensitisation: ARE-NRF2 Luciferase test method (2018)

(B.60.)

OECD Test Guideline 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT) (2017, corrected 2018)

B.71.

OECD Test Guideline 497: Defined Approaches on Skin Sensitisation (2021)

In vivo:

OECD Test Guideline 429: Skin Sensitisation - Local Lymph Node Assay (2010)

B.42.

OECD Test Guideline 442A: Skin Sensitisation - Local Lymph Node Assay: DA (2010)

B.50.

OECD Test Guideline 442B: Skin Sensitisation - Local Lymph Node Assay: BrdU-ELISA (2018)

(B.51.)

OECD Test Guideline 406: Skin sensitisation (2021)

(B.6.)

Mutagenicity

In vitro:

OECD Test Guideline 471: Bacterial Reverse Mutation Test (1997, corrected 2020)

(B.13./14.)

OECD Test Guideline 476: In vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes (2015, corrected 2016)

B.17.

OECD Test Guideline 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene (2016)

B.67.

OECD Test Guideline 473: In vitro Mammalian Chromosome Aberration Test (2014, corrected 2016)

B.10.

OECD Test Guideline 487. In vitro Mammalian Cell Micronucleus Test (2016)

B.49.

In vivo:

OECD    Test    Guideline    475: Mammalian Bone Marrow Chromosome Aberration Test (2014, corrected 2016)

B.11.

OECD    Test    Guideline    474: Mammalian Erythrocyte Micronucleus Test (2014, corrected 2016)

B.12.

OECD Test Guideline 483: Mammalian Spermatogonial Chromosome Aberration Test (2015, corrected 2016)

B.23.

OECD Test Guideline 488: Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays (2020)

(B.58.)

OECD Test Guideline 489: In Vivo Mammalian Alkaline Comet Assay (2016)

B.62.

Acute toxicity

Oral:

OECD Test Guideline 420: Acute oral toxicity: fixed dose procedure (2002)

B.1 bis.

OECD Test Guideline 423: Acute oral toxicity: acute toxic class method (2002)

B.1 tris.

OECD Test Guideline 425: Acute oral toxicity: up-and-down procedure (2006, corrected 2008)

Dermal:

OECD Test Guideline 402: Acute Dermal Toxicity (2017)

(B.3.)

Inhalation:

OECD Test Guideline 403: Acute Inhalation Toxicity (2009)

B.2.

OECD Test Guideline 436: Acute Inhalation Toxicity - Acute Toxic Class Method (2009)

B.52.

OECD test guideline 433: Acute Inhalation Toxicity: Fixed Concentration Procedure (2017, corrected 2018)

Repeated dose toxicity

OECD Test Guideline 407: Repeated dose 28-day oral toxicity study in rodents (2008)

B.7.

OECD Test Guideline 412: Subacute inhalation toxicity: 28-day study (2017, corrected 2018)

(B.8.)

OECD Test Guideline 410: Repeated dose dermal toxicity: 21/28-day study (1981)

B.9.

OECD Test Guideline 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2016)

B.64.

OECD Test Guideline 408: Repeated dose 90-day oral toxicity study in rodents (2018)

(B.26.)

OECD Test Guideline 409: Repeated dose 90-day oral toxicity study in non-rodents (1998)

B.27.

OECD Test Guideline 413: Subchronic inhalation toxicity: 90-day study (2017, corrected 2018)

(B.29.)

OECD Test Guideline 411: Subchronic dermal toxicity: 90-day study (1981)

B.28.

OECD Test Guideline 452: Chronic Toxicity Studies (2009, corrected 2018)

(B.30.)

OECD Test Guideline 453: Combined Chronic Toxicity/Carcinogenicity Studies (2009, corrected 2018)

(B.33.)

Reproductive/developmental toxicity

OECD Test Guideline 443: Extended One-generation Reproduction Toxicity Study (2012, corrected in 2018)

(B.56.)

OECD Test Guideline 421: Reproduction/ Developmental Toxicity Screening Test (2016)

B.63.

OECD Test Guideline 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2016)

B.64.

OECD Test Guideline 414: Prenatal developmental toxicity study (2018)

(B.31.)

Toxicokinetics

OECD Test Guideline 417: Toxicokinetics (2010)

B.36.

OECD Test Guideline 428: Skin Absorption: In Vitro Method (2004)

B.45.

OECD Test Guideline 427: Skin Absorption: In Vivo Method (2004)

B.44.

Carcinogenicity

OECD Test Guideline 451: Carcinogenicity Studies (2009, corrected 2018)

(B.32.)

OECD Test Guideline 453: Combined Chronic Toxicity/Carcinogenicity (2009, corrected 2018)

(B.33.)

In vitro mammalian cell transformation test 

B.21.

(Developmental) Neurotoxicity

OECD Test Guideline 424: Neurotoxicity study in rodents (1997)

B.43.

OECD Test Guideline 426: Developmental neurotoxicity study (2007)

B.53.

OECD Test Guideline 418: Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure (1995)

B.37.

OECD Test Guideline 419: Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study (1995)

B.38.

Endocrine disrupting properties

In vitro

OECD Test Guideline 455: Stably Transfected Human Estrogen Receptor-alpha Transcriptional Activation Assay for Detection of Estrogenic Agonist-Activity of Chemicals (2016)

B.66.

OECD Test Guideline 456: H295R Steroidogenesis Assay (2011)

B.57.

OECD Test Guideline 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals (2020)

OECD Test Guideline 493: Performance-Based Test Guideline for Human Recombinant Estrogen Receptor (hrER) In Vitro Assays to Detect Chemicals with ER Binding Affinity (2015)

B.70.

In vivo

OECD Test Guideline 440: Uterotrophic Bioassay in Rodents A short-term screening test for oestrogenic properties (2007)

B.54.

OECD Test Guideline    441: Hershberger Bioassay in Rats, A Short-term Screening Assay for (Anti)Androgenic Properties (2009)

B.55.

Phototoxicity

OECD Test Guideline 432: In vitro 3T3 NRU Phototoxicity Test (2019)

(B.41.)

OECD Test Guideline 495: Ros (Reactive Oxygen Species) Assay for Photoreactivity (2019)

OECD Test Guideline 498: In vitro phototoxicity test method using the Reconstructed human Epidermis (RhE) (2021)

TABLE 3: TEST METHODS FOR ECOTOXICOLOGICAL PROPERTIES

Endpoint

Test method

Corresponding chapter in Part C of this Annex (numbers in brackets indicate that a test method has been deleted from Part C; empty cell: no corresponding EU test method in Part C of this Annex)

Aquatic toxicity

OECD Test Guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test (2006, corrected 2011)

C.3.

OECD test guideline 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) (2010)    

C.11.

OECD test guideline 224: Determination of the Inhibition of the Activity of Anaerobic Bacteria (2007)    

C.34.

OECD test guideline 244: Protozoan Activated Sludge Inhibition Test (2017)    

OECD test guideline 221: Lemna sp. Growth Inhibition Test (2006)

C.26.

OECD Test Guideline 202: Daphnia sp. Acute Immobilisation Test (2004)

C.2.

OECD Test Guideline 211: Daphnia magna Reproduction Test (2012)

C.20.

OECD Test Guideline 203: Fish, Acute Toxicity Test (2019)

(C.1.)

OECD test guideline 210: Fish, Early-life Stage Toxicity Test (2013)

C.47.

OECD Test Guideline 215: Fish, Juvenile Growth Test (2000)

C.14.

OECD test guideline 236: Fish Embryo Acute Toxicity (FET) Test (2013)

C.49.

OECD test guideline 249: Fish cell line acute toxicity - the RTgill-W1 cell line assay (2021)

OECD test guideline 242: Potamopyrgus antipodarum Reproduction Test (2016)

OECD test guideline 243: Lymnaea stagnalis Reproduction Test (2016)

OECD test guideline 319A: Determination of in vitro intrinsic clearance using cryopreserved rainbow trout hepatocytes (RT-HEP) (2018)

OECD test guideline 319B: Determination of in vitro intrinsic clearance using rainbow trout liver S9 sub-cellular fraction (RT-S9) (2018)

Degradation

OECD test guideline 111: Hydrolysis as a Function of pH (2004)

C.7.

OECD test guideline 301: Ready Biodegradability (1992, Part 301F corrected 2013)

(C.4.)

OECD test guideline 302A: Inherent Biodegradability: Modified SCAS Test (1981)

C.12.

OECD test guideline 302B: Inherent Biodegradability: Zahn-Wellens/ EMPA Test (1992)

(C.9).

OECD test guideline 302C: Inherent Biodegradability: Modified MITI Test (II) (1981, corrected 2009)

OECD test guideline 303: Simulation Test - Aerobic Sewage Treatment -- A: Activated Sludge Units; B: Biofilms (2001)

C.10.

OECD test guideline 304A: Inherent Biodegradability in Soil (1981)

OECD test guideline 306: Biodegradability in Seawater (1992)

C.42.

OECD test guideline 307: Aerobic and Anaerobic Transformation in Soil (2002)

C.23.

OECD test guideline 308: Aerobic and Anaerobic Transformation in Aquatic Sediment Systems (2002)

C.24.

OECD test guideline 309: Aerobic Mineralisation in Surface Water – Simulation Biodegradation Test (2004)

C.25.

OECD test guideline 310: Ready Biodegradability - CO2 in sealed vessels (Headspace Test) (2006, corrected 2014)

C.29.

OECD test guideline 311: Anaerobic Biodegradability of Organic Compounds in Digested Sludge: by Measurement of Gas Production (2006)

C.43.

OECD test guideline 314: Simulation Tests to Assess the Biodegradability of Chemicals Discharged in Wastewater (2008)

OECD test guideline 316: Phototransformation of Chemicals in Water – Direct Photolysis (2008) 

EU test method C.5. Degradation – biochemical oxygen demand

C.5.

EU test method C.6. Degradation – chemical oxygen demand

C.6.

Fate and behavior in the environment

OECD Test Guideline 305: Bioaccumulation in Fish: Aqueous and Dietary Exposure (2012)

C.13.

OECD test guideline 315: Bioaccumulation in Sediment-dwelling Benthic Oligochaetes (2008)

C.46.

OECD test guideline 317: Bioaccumulation in Terrestrial Oligochaetes (2010)

C.30.

OECD test guideline 318: Dispersion Stability of Nanomaterials in Simulated Environmental Media (2017)

OECD test guideline 121: Estimation of the Adsorption Coefficient (Koc ) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC) (2001)

C.19.

OECD test guideline 106: Adsorption - Desorption Using a Batch Equilibrium Method (2000)

C.18.

OECD test guideline 312: Leaching in Soil Columns (2004)

C.44.

OECD test guideline 313: Estimation of Emissions from Preservative - Treated Wood to the Environment (2007)

C.45.

Effects on terrestrial organisms

OECD test guideline 216: Soil Microorganisms: Nitrogen Transformation Test (2000)

C.21.

OECD test guideline 217: Soil Microorganisms: Carbon Transformation Test (2000)

C.22.

OECD test guideline 207: Earthworm, Acute Toxicity Tests (1984)

C.8.

OECD test guideline 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) (2016)

(C.33.)

OECD test guideline 220: Enchytraeid Reproduction Test (2016)

(C.32.)

OECD test guideline 226: Predatory mite (Hypoaspis (Geolaelaps) aculeifer) reproduction test in soil (2016)

(C.36.)

OECD test guideline 232: Collembolan Reproduction Test in Soil (2016)

(C.39.)

OECD test guideline 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test (2006)

C.31.

OECD test guideline 227: Terrestrial Plant Test: Vegetative Vigour Test (2006)

Effects on sediment organisms

OECD test guideline 218: Sediment-Water Chironomid Toxicity Using Spiked Sediment (2004)

C.27.

OECD test guideline 219: Sediment-Water Chironomid Toxicity Using Spiked Water (2004)

C.28.

OECD test guideline 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment (2010)

C.40.

OECD test guideline 235: Chironomus sp., Acute Immobilisation Test (2011)

OECD test guideline 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment (2007)

C.35.

OECD test guideline 238: Sediment-Free Myriophyllum Spicatum Toxicity Test (2014)    

C.50.

OECD test guideline 239: Water-Sediment Myriophyllum Spicatum Toxicity Test (2014)

C.51.

Effects on birds

OECD test guideline 205: Avian Dietary Toxicity Test (1984)

OECD test guideline 206: Avian Reproduction Test (1984)

OECD test guideline 223: Avian Acute Oral Toxicity Test (2016)

Effects on insects

OECD test guideline 213: Honeybees, Acute Oral Toxicity Test (1998)

C.16.

OECD test guideline 214: Honeybees, Acute Contact Toxicity Test (1998)

C.17.

OECD test guideline 237: Honey Bee (Apis Mellifera) Larval Toxicity Test, Single Exposure (2013)

OECD test guideline 245: Honey Bee (Apis Mellifera L.), Chronic Oral Toxicity Test (10-Day Feeding) (2017)

OECD test guideline 246: Bumblebee, Acute Contact Toxicity Test (2017)

OECD test guideline 247: Bumblebee, Acute Oral Toxicity Test (2017)

OECD test guideline 228: Determination of Developmental Toxicity to Dipteran Dung Flies(Scathophaga stercoraria L. (Scathophagidae), Musca autumnalis De Geer (Muscidae)) (2016)

Endocrine disrupting properties

OECD test guideline 230: 21-day Fish Assay (2009)

C.37.

OECD test guideline 229: Fish Short Term Reproduction Assay (2012)

C.48.

OECD test guideline 231: Amphibian Metamorphosis Assay (2009)

C.38.

OECD test guideline 234: Fish Sexual Development Test (2011)

C.41.

OECD test guideline 240: Medaka Extended One Generation Reproduction Test (MEOGRT) (2015)

C.52.

OECD test guideline 241: The Larval Amphibian Growth and Development Assay (LAGDA) (2015)

C.53.

OECD test guideline 248: Xenopus Eleutheroembryonic Thyroid Assay (XETA) (2019)

OECD test guideline 250: the EASZY assay (2021)’