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

• 55.Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained from the selected five males and five females of each group. Overnight fasting of the animals prior to blood sampling is recommended ([^E0048]). Investigations of plasma or serum should include sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, at least two enzymes indicative of hepatocellular effects (such as alanin aminotransferase, aspartate aminotransferase and sorbitol dehydrogenase) and bile acids. Measurements of additional enzymes (of hepatic or other origin) and bilirubin may provide useful information under certain circumstances.

• 56.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 40-41)

• — 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’.

• 57.Optionally, the following urinalysis determinations could be performed in five randomly selected males of each group during the last week of the study using timed urine volume collection; appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells.

• 58.In addition, studies to investigate serum markers of general tissue damage should be considered. Other determinations that should be carried out if the known properties of the test chemical may, or are suspected to, affect related metabolic profiles include calcium, phosphate, fasting triglycerides and fasting glucose, specific hormones, methaemoglobin and cholinesterase. These need to be identified on a case-by-case basis.

• 59.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.

• 60.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.

• 61.If historical baseline data are inadequate, consideration should be given to determination of haematological and clinical biochemistry variables before dosing commences or preferably in a set of animals not included in the experimental groups. For females, the data have to be from lactating animals.

PATHOLOGY

Gross necropsy

• 62.All adult animals in the study should be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. 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 on the day of necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.

• 63.The testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of all male adult animals should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying. In addition, optional organ weights could include levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis in males and paired ovaries (wet weight) and uterus (including cervix) in females; if included, these weights should be collected as soon as possible after dissection. The ovaries, testes, epididymides, accessory sex organs, and all organs showing macroscopic lesions of all adult animals, should be preserved.

• 64.From all adult males and females and one male and female day 13 pup from each litter thyroid glands should be preserved in the most appropriate fixation medium for the intended subsequent histopathological examination. The thyroid weight could be determined after fixation. Trimming should also be done very carefully and only after fixation to avoid tissue damage. Tissue damage could compromise histopathology analysis. Blood samples should be taken from a named site just prior to or as part of the procedure for euthanasia of the animals, and stored under appropriate conditions (see paragraph 56).

• 65.In addition, for a least five adult males and females, randomly selected from each group (apart from those found moribund and/or euthanised prior to the termination of the study), the liver, kidneys, adrenals, thymus, spleen, brain and heart should be trimmed of any adherent tissue, as appropriate and their wet weight taken as soon as possible after dissection to avoid drying. The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, eye, stomach, small and large intestines (including Peyer's patches), liver, kidneys, adrenals, spleen, heart, thymus, trachea and lungs (preserved by inflation with fixative and then immersion), gonads (testis and ovaries), accessory sex organs (uterus andcervix, epididymides, prostate, seminal vesicles plus coagulating glands), vagina, urinary bladder, lymph nodes (besides the most proximal draining node, another lymph node should be taken according to the laboratory’s experience (16)), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, skeletal muscle and bone, with bone marrow (section or, alternatively, a fresh mounted bone marrow aspirate). It is recommended that testes be fixed by immersion in Bouin’s or modified Davidson’s fixative (16) (17) (18); formalin fixation is not recommended for these tissues. The tunica albuginea may be gently and shallowly punctured at the both poles of the organ with a needle to permit rapid penetration of the fixative. The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test chemical should be preserved.

• 66.The following tissues may give valuable indication for endocrine-related effects: Gonads (ovaries and testes), accessory sex organs (uterus including cervix, epididymides, seminal vesicles with coagulation glands, dorsolateral and ventral prostate), vagina, pituitary, male mammary gland and adrenal gland. Changes in male mammary glands have not been sufficiently documented but this parameter may be very sensitive to substances with estrogenic action. Observation of organs/tissues that are not listed in paragraph 65 is optional.

• 67.Dead pups and pups killed at day 13 post-partum, or shortly thereafter, should, at least, be carefully examined externally for gross abnormalities. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development.

Histopathology

• 68.Full histopathology should be carried out on the preserved organs and tissues of the selected animals in the control and high dose groups (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure). The thyroid gland from pups and from the remaining adult animals may be examined when necessary. These examinations should be extended to animals of other dosage groups, if treatment-related changes are observed in the high dose group. The Guidance on histopathology (10) details extra information on dissection, fixation, sectioning and histopathology of endocrine tissues.

• 69.All gross lesions should be examined. To aid in the elucidation of NOAELs, target organs in other dose groups should be examined, particularly in groups claimed to show a NOAEL.

• 70.When a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the treated groups.

DATA AND REPORTING

Data

• 71.Individual animal data should be provided. Additionally, all data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or euthanised for humane reasons, the time of any death or euthanasia, the number of fertile animals, the number of pregnant females, the number of animals showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the types of histopathological changes, and all relevant litter data. A tabular summary report format, which has proven to be very useful for the evaluation of reproductive/developmental effects, is given in Appendix 3.

• 72.When possible, numerical results should be evaluated by an appropriate and general acceptable statistical method. Comparisons of the effect along a dose range should avoid the use of multiple t-tests. The statistical methods should be selected during the design of the study. Statistical analysis of AGD and nipple retention should be based on individual pup data, taking litter effects into account. Where appropriate, the litter is the unit of analysis. Statistical analysis of pup body weight should be based on individual pup data, taking litter size into account. Due to the limited dimensions of the study, statistical analyses in the form of tests for ‘significance’ are of limited value for many endpoints, especially reproductive endpoints. Some of the most widely used methods, especially parametric tests for measures of central tendency, are inappropriate. If statistical analyses are used then the method chosen should be appropriate for the distribution of the variable examined and be selected prior to the start of the study.

Evaluation of results

• 73.The findings of this toxicity study should be evaluated in terms of the observed effects, necropsy and microscopic findings. The evaluation will include the relationship between the dose of the test chemical and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, infertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects.

• 74.Because of the short period of treatment of the male, the histopathology of the testes and epididymides should be considered along with the fertility data, when assessing male reproduction effects. The use of historic control data on reproduction/development (e.g. for litter size, AGD, nipple retention, serum T4 levels), where available, may also be useful as an aid to the interpretation of the study.

• 75.For quality control it is proposed that historical control data are collected and that for numerical data coefficients of variation are calculated, especially for the parameters linked with endocrine disrupter detection. These data can be used for comparison purposes when actual studies are evaluated.

Test report

• 76.The test report should include the following information:

Test chemical:

• — source, lot number, limit date for use, if available

• — stability of the test chemical, if known.

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

.

Vehicle (if appropriate):

• — justification for choice of vehicle, if other than water.

Test animals:

• — species/strain used;

• — number, age and sex of animals;

• — source, housing conditions, diet, etc.;

• — individual weights of animals at the start of the test.

• — justification for species if not rat

Test conditions:

• — rationale for dose level selection;

• — details of test chemical formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation;

• — details of the administration of the test chemical;

• — conversion from diet/drinking water test chemical concentration (ppm) to the actual dose (mg/kg body weight/day), if applicable;

• — details of food and water quality;

• — detailed description of the randomisation procedure to select pups for culling, if culled.

Results:

• — body weight/body weight changes;

• — food consumption and water consumption, if applicable;

• — toxic response data by sex and dose, including fertility, gestation, and any other signs of toxicity;

• — gestation length;

• — toxic or other effects on reproduction, offspring, postnatal growth, etc.;

• — nature, severity and duration of clinical observations (whether reversible or not);

• — sensory activity, grip strength and motor activity assessments;

• — haematological tests with relevant baseline values;

• — clinical biochemistry tests with relevant base-line values;

• — number of adult females with normal or abnormal oestrous cycle and cycle duration;

• — number of live births and post implantation loss;

• — number of pups with grossly visible abnormalities; gross evaluation of external genitalia, number of runts;

• — time of death during the study or whether animals survived to termination;

• — number of implantations, litter size and litter weights at the time of recording;

• — pup body weight data

• — AGD of all pups (and body weight on day of AGD measurement)

• — nipple retention in male pups,

• — thyroid hormone levels, day 13 pups and adult males (and dams and day 4 pups if measured)

• — body weight at sacrifice and organ weight data for the parental animals;

• — necropsy findings;

• — a detailed description of histopathological findings;

• — absorption data (if available);

• — statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

Interpretation of Results

• 77.The study will provide evaluations of reproduction/developmental toxicity associated with administration of repeated doses. In particular, since emphasis is placed on both general toxicity and reproduction/developmental toxicity endpoints, the results of the study will allow for the discrimination between reproduction/developmental effects occurring in the absence of general toxicity and those which are only expressed at levels that are also toxic to parent animals (see paragraphs 7-11). It could provide an indication of the need to conduct further investigations and could provide guidance in the design of subsequent studies. OECD Guidance Document 43 should be consulted for aid in the interpretation of reproduction and developmental results (19). OECD Guidance Document 106 on Histologic Evaluation of Endocrine and Reproductive Tests in Rodents (16) provides information on the preparation and evaluation of (endocrine) organs and vaginal smears that may be helpful for this test method.

LITERATURE

• (1)OECD (1990). Room Document No 1 for the 14th Joint Meeting of the Chemicals Group and Management Committee. Available upon request at Organisation for Economic Cooperation and Development, Paris

• (2)OECD (1992). Chairman's Report of the ad hoc Expert Meeting on Reproductive Toxicity Screening Methods, Tokyo, 27th-29th October, 1992. Available upon request at Organisation for Economic Cooperation and Development, Paris

• (3)Mitsumori K., Kodama Y., Uchida O., Takada K., Saito M. Naito K., Tanaka S., Kurokawa Y., Usami, M., Kawashima K., Yasuhara K., Toyoda K., Onodera H., Furukawa F., Takahashi M. and Hayashi Y. (1994). Confirmation Study, Using Nitro-Benzene, of the Combined Repeat Dose and Reproductive/ Developmental Toxicity Test Protocol Proposed by the Organization for Economic Cooperation and Development (OECD). J. Toxicol, Sci., 19, 141-149.

• (4)Tanaka S., Kawashima K., Naito K., Usami M., Nakadate M., Imaida K., Takahashi M., Hayashi Y., Kurokawa Y. and Tobe M. (1992). Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test (OECD): Familiarization Using Cyclophosphamide. Fundam. Appl. Toxicol., 18, 89-95.

• (5)OECD (1998). Report of the First Meeting of the OECD Endocrine Disrupter Testing and Assessment (EDTA) Task Force, 10th-11th March 1998, Available upon request at Organisation for Economic Cooperation and Development, Paris

• (6)OECD (2015). Feasibility Study for Minor Enhancements of TG 421/422 with ED Relevant Endpoints. Environment, Health and Safety Publications, Series on Testing and Assessment (No 217), Organisation for Economic Cooperation and Development, Paris.

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

• (8)Goldman J.M., Murr A.S., Buckalew A.R., Ferrell J.M.and Cooper R.L. (2007). The Rodent Estrous Cycle: Characterization of Vaginal Cytology and its Utility in Toxicological Studies, Birth Defects Research, Part B, 80 (2), 84-97.

• (9)Sadleir R.M.F.S. (1979). Cycles and Seasons, in Auston C.R. and Short R.V. (Eds.), Reproduction in Mammals: I. Germ Cells and Fertilization, Cambridge, New York.

• (10)IPCS (1986). Principles and Methods for the Assessment of Neurotoxicity Associated with Exposure to Chemicals. Environmental Health Criteria Document (No 60).

• (11)Moser V.C., McDaniel K.M. and Phillips P.M. (1991). Rat Strain and Stock Comparisons Using a Functional Observational Battery: Baseline Values and Effects of Amitraz. Toxicol. Appl. Pharmacol., 108, 267-283.

• (12)Meyer O.A., Tilson H.A., Byrd W.C. and Riley M.T. (1979). A Method for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats and Mice. Neurobehav. Toxicol., 1, 233-236.

• (13)Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W., Tilson H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor Activity Experiments: Implication for Neurotoxicological Assessments. Neurotoxicol. Teratol. 13, 599-609.

• (14)Gallavan R.H. Jr, J.F. Holson, D.G. Stump, J.F. Knapp and V.L. Reynolds. (1999). ‘Interpreting the Toxicologic Significance of Alterations in Anogenital Distance: Potential for Confounding Effects of Progeny Body Weights’, Reproductive Toxicology, 13: 383-390.

• (15)OECD (2013). Guidance Document in Support of the Test Guideline on the Extended One Generation Reproductive Toxicity Study. Environment, Health and Safety Publications, Series on Testing and Assessment (No 151). Organisation for Economic Cooperation and Development, Paris.

• (15)OECD (2009).Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents. Environment, Health and Safety Publications, Series on Testing and Assessment (No. 106) Organisation for Economic Cooperation and Development, Paris.

• (17)Hess RA and Moore BJ. (1993). Histological Methods for the Evaluation of the Testis. In: Methods in Reproductive Toxicology, Chapin RE and Heindel JJ (Eds.). Academic Press: San Diego, CA, pp. 52-85.

• (18)Latendresse JR, Warbrittion AR, Jonassen H, Creasy DM. (2002). Fixation of Testes and Eyes Using a Modified Davidson's Fluid: Comparison with Bouin's Fluid and Conventional Davidson's fluid. Toxicol. Pathol. 30, 524-533.

• (19)OECD (2008). Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment. Environment, Health and Safety Publications, Series on Testing and Assessment (No 43), Organisation for Economic Cooperation and Development, Paris.

• (20)OECD (2011), Guidance Document on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption (No 150), Organisation for Economic Cooperation and Development, Paris.

Appendix 1

DEFINITIONS (SEE ALSO (20) OECD GD 150)

Androgenicity is the capability of a chemical to act like a natural androgenic hormone (e.g. testosterone) in a mammalian organism.

Antiandrogenicity is the capability of a chemical to suppress the action of a natural androgenic hormone (e.g. testosterone) in a mammalian organism.

Antioestrogenicity is the capability of a chemical to suppress the action of a natural oestrogenic hormone (e.g. oestradiol 17ß) in a mammalian organism.

Antithyroid activity is the capability of a chemical to suppress the action of a natural thyroid hormone (e.g. T3) in a mammalian organism.

Chemical is a substance or a mixture.

Developmental toxicity: the manifestation of reproductive toxicity, representing pre-, peri- post-natal, structural, or functional disorders in the progeny.

Dose is the amount of test chemical administered. The dose is expressed as weight of test chemical per unit body weight of test animal per day (e.g. mg/kg body weight/day), or as a constant dietary concentration.

Dosage is a general term comprising dose, its frequency and the duration of dosing.

Evident toxicity is a general term describing clear signs of toxicity following administration of test chemical. These should be sufficient for hazard assessment and should be such that an increase in the dose administered can be expected to result in the development of severe toxic signs and probable mortality.

Impairment of fertility represents disorders of male or female reproductive functions or capacity.

Maternal toxicity: adverse effects on gravid females, occurring either specifically (direct effect) or not specifically (indirect effect) and being related to the gravid state.

NOAEL is the abbreviation for no-observed-adverse-effect level. This is the highest dose level where no adverse treatment-related findings are observed due to treatment.

Oestrogenicity is the capability of a chemical to act like a natural oestrogenic hormone (e.g. oestradiol 17ß) in a mammalian organism.

Reproduction toxicity represents harmful effects on the progeny and/or an impairment of male and female reproductive functions or capacity.

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

Thyroid activity is the capability of a chemical to act like a natural thyroid hormone (e.g. T3) in a mammalian organism.

Validation is a scientific process designed to characterise the operational requirements and limitations of a test method and to demonstrate its reliability and relevance for a particular purpose.

Appendix 2

DIAGRAM OF THE EXPERIMENTAL SCHEDULE, INDICATING THE MAXIMUM STUDY DURATION, BASED ON A FULL 14-DAY MATING PERIOD

Image omitted; see official source Image omitted; see official source

Appendix 3

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

OBSERVATIONS	VALUES	VALUES	VALUES	VALUES	VALUES
Dosage (units).......	0 (control)	...	...	...	...
Pairs started (N)
Oestrus cycle (at least mean length and frequency of irregular cycles)
Females showing evidence of copulation (N)
Females achieving pregnancy (N)
Conceiving days 1 - 5 (N)
Conceiving days 6 -... [^E0049] (N)
Pregnancy ≤ 21 days (N)
Pregnancy = 22 days (N)
Pregnancy ≥ 23 days (N)
Dams with live young born (N)
Dams with live young at day 4 pp (N)
Implants/dam (mean)
Live pups/dam at birth (mean)
Live pups/dam at day 4 (mean)
Sex ratio (m/f) at birth (mean)
Sex ratio (m/f) at day 4 (mean)
Litter weight at birth (mean)
Litter weight at day 4 (mean)
Pup weight at birth (mean)
Pup weight at the time of AGD measurement(mean males, mean females)
Pup AGD on the same postnatal day, birth- day 4 (mean males, mean females, note PND)
Pup weight at day 4 (mean)
Pup weight at day 13 (mean)
Male pup nipple retention at day 13 (mean)
ABNORMAL PUPS	ABNORMAL PUPS	ABNORMAL PUPS	ABNORMAL PUPS	ABNORMAL PUPS	ABNORMAL PUPS
Dams with 0
Dams with 1
Dams with ≥ 2
LOSS OF OFFSPRING	LOSS OF OFFSPRING	LOSS OF OFFSPRING	LOSS OF OFFSPRING	LOSS OF OFFSPRING	LOSS OF OFFSPRING
Pre-natal (implantations minus live births)	Pre-natal (implantations minus live births)	Pre-natal (implantations minus live births)	Pre-natal (implantations minus live births)	Pre-natal (implantations minus live births)	Pre-natal (implantations minus live births)
Females with 0
Females with 1
Females with 2
Females with ≥ 3
Post-natal (live births minus alive at post natal day 13)	Post-natal (live births minus alive at post natal day 13)	Post-natal (live births minus alive at post natal day 13)	Post-natal (live births minus alive at post natal day 13)	Post-natal (live births minus alive at post natal day 13)	Post-natal (live births minus alive at post natal day 13)
Females with 0
Females with 1
Females with 2
Females with ≥ 3

last day of the mating period

B.65.   IN VITRO MEMBRANE BARRIER TEST METHOD FOR SKIN CORROSION

INTRODUCTION

• 1.This test method is equivalent to OECD test guideline (TG) 435 (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) ([^E0050]) This test method, equivalent to the updated OECD test guideline 435 provides an in vitro membrane barrier test method that can be used to identify corrosive chemicals. The test method utilises an artificial membrane designed to respond to corrosive chemicals in a manner similar to animal skin in situ.

• 2.Skin corrosivity has traditionally been assessed by applying the test chemical to the skin of living animals and assessing the extent of tissue damage after a fixed period of time (2). Besides the present test method, a number of other in vitro test methods have been adopted as alternatives (3)(4) to the standard in vivo rabbit skin procedure (Chapter B.4 of this Annex, equivalent to OECD TG 404) used to identify corrosive chemicals (2). The UN GHS tiered testing and evaluation strategy for the assessment and classification of skin corrosivity and the OECD Guidance Document on Integrated Approaches to Testing and Assessment (IATA) for Skin Irritation/Corrosion recommend the use of validated and accepted in vitro test methods under modules 3 and 4 (1)(5). The IATA describes several modules which group information sources and analysis tools and (i) provides guidance on how to integrate and use existing test and non-test data for the assessment of the skin irritation and skin corrosion potentials of chemicals and (ii) proposes an approach when further testing is needed, including when negative results are found (5). In this modular approach, positive results from in vitro test methods can be used to classify a chemical as corrosive without the need for animal testing, thus reducing and refining the use of animals in and avoiding the pain and distress that might occur if animals were used for this purpose.

• 3.Validation studies have been completed for the in vitro membrane barrier model commercially available as Corrositex® (6)(7)(8), showing an overall accuracy to predict skin corrosivity of 79 % (128/163), a sensitivity of 85 % (76/89), and a specificity of 70 % (52/74) for a database of 163 substances and mixtures (7). Based on its acknowledged validity, this validated reference method (VRM) has been recommended for use as part of a tiered testing strategy for assessing the dermal corrosion hazard potential of chemicals (5)(7). Before an in vitro membrane barrier model for skin corrosion can be used for regulatory purposes, its reliability, relevance (accuracy), and limitations for its proposed use should be determined to ensure that it is similar to that of the VRM (9), in accordance with the pre-defined performance standards (PS) (10). The OECD Mutual Acceptance of Data will only be guaranteed after any proposed new or updated method following the PS have been reviewed and included in the equivalent OECD test guideline. Currently, only one in vitro method is covered by OECD test guideline 435 and this test method, the commercially available Corrositex^® model.

• 4.Other test methods for skin corrosivity testing are based on the use of reconstituted human skin (OECD TG 431) (3) and isolated rat skin (OECD TG 430) (4). This Test Guideline also provides for subcategorisation of corrosive chemicals into the three UN GHS Sub-categories of corrosivity and the three UN Transport Packing Groups for corrosivity hazard. This Test Guideline was originally adopted in 2006 and updated in 2015 to refer to the IATA guidance document and update the list of proficiency substances.

DEFINITIONS

• 5.Definitions used are provided in the Appendix.

INITIAL CONSIDERATIONS AND LIMITATIONS

• 6.The test described in this test method allows the identification of corrosive test chemicals and allows the sub-categorisation of corrosive test chemicals according to UN GHS/CLP (Table 1). In addition, such a test method may be used to make decisions on the corrosivity and non-corrosivity of specific classes of chemicals, e.g. organic and inorganic acids, acid derivatives ([^E0051]), and bases for certain transport testing purposes (7)(11)(12). This test method describes a generic procedure similar to the validated reference test method (7). While this test method does not provide adequate information on skin irritation, it should be noted that TM B.46 (equivalent to OECD TG439) specifically addresses the health effect skin irritation in vitro (13). 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 (5).

Table 1

The UN GHS Skin Corrosive Category and Subcategories [^E0052]

Corrosive Category (category 1) (for authorities not using subcategories)	Potential Corrosive Subcategories [^E0052] (for authorities using subcategories, including the CLP Regulation)	Corrosive in ≥ 1 of 3 animals	Corrosive in ≥ 1 of 3 animals
Corrosive Category (category 1) (for authorities not using subcategories)	Potential Corrosive Subcategories [^E0052] (for authorities using subcategories, including the CLP Regulation)	Exposure	Observation
Corrosive	Corrosive subcategory 1A	≤ 3 minutes	≤ 1 hour
Corrosive	Corrosive subcategory 1B	> 3 minutes /≤ 1 hour	≤14 days
Corrosive	Corrosive subcategory 1C	> 1 hour /≤ 4 hours	≤ 14 days

For the EU, the CLP Regulation applies the three skin corrosion subcategories 1A, 1B and 1C.

• 7.A limitation of the validated reference method (7) is that many non-corrosive chemicals and some corrosive chemicals may not qualify for testing, based on the results of the initial compatibility test (see paragraph 13). Aqueous chemicals with a pH in the range of 4.5 to 8.5 often do not qualify for testing; however, 85 % of chemicals tested in this pH range were non-corrosive in animal tests (7). The in vitro membrane barrier method may be used to test solids (soluble or insoluble in water), liquids (aqueous or non-aqueous), and emulsions. However, test chemicals not causing a detectable change in the compatibility test (i.e. colour change in the Chemical Detection System (CDS) of the validated reference test method) cannot be tested with the membrane barrier method and should be tested using other test methods.

PRINCIPLE OF THE TEST

• 8.The test system comprises two components: a synthetic macromolecular bio-barrier and a chemical detection system (CDS); this test method detects via the CDS membrane barrier damage caused by corrosive test chemicals after the application of the test chemical to the surface of the synthetic macromolecular membrane barrier (7), presumably by the same mechanism(s) of corrosion that operate on living skin.

• 9.Penetration of the membrane barrier (or breakthrough) might be measured by a number of procedures or CDS, including a change in the colour of a pH indicator dye or in some other property of the indicator solution below the barrier.

• 10.The membrane barrier should be determined to be valid, i.e. relevant and reliable, for its intended use. This includes ensuring that different preparations are consistent in regard to barrier properties, e.g. capable of maintaining a barrier to non-corrosive chemicals, able to categorise the corrosive properties of chemicals across the various UN GHS Sub-categories of corrosivity (1). The classification assigned is based on the time it takes a chemical to penetrate through the membrane barrier to the indicator solution.

DEMONSTRATION OF PROFICIENCY

• 11.Prior to routine use of the in vitro membrane barrier method, adhering to this test method, laboratories should demonstrate technical proficiency by correctly classifying the twelve Proficiency Substances recommended in Table 2. 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 (10)) provided that the same selection criteria as described in Table 1 is applied.

Table 2

Proficiency Substances [^E0053]

Substance [^E0054]	CASRN	Chemical Class	In Vivo UN GHS Sub-category [^E0055]	In Vitro UN GHS Sub-category [^E0055]
Boron trifluoride dihydrate	13 319 -75-0	Inorganic acids	1A	1A
Nitric acid	7 697 -37-2	Inorganic acids	1A	1A
Phosphorus pentachloride	10 026 -13-8	Precursors of inorganic acids	1A	1A
Valeryl chloride	638-29-9	Acid chlorides	1B	1B
Sodium Hydroxide	1 310 -73-2	Inorganic bases	1B	1B
1-(2-Aminoethyl) piperazine	140-31-8	Aliphatic amines	1B	1B
Benzenesulfonyl chloride	98-09-9	Acid chlorides	1C	1C
N,N-Dimethyl benzylamine	103-83-3	Anilines	1C	1C
Tetraethylenepentamine	112-57-2	Aliphatic amines	1C	1C
Eugenol	97-53-0	Phenols	NC	NC
Nonyl acrylate	2 664 -55-3	Acrylates/methacrylates	NC	NC
Sodium bicarbonate	144-55-8	Inorganic salts	NC	NC

The twelve substances listed above contain three substances from each of the three UN GHS subcategories for corrosive substances and three non-corrosive substances, are readily available from commercial suppliers, and the UN GHS subcategory is based on the results of high-quality in vivo testing. These substances are taken from the list of 40 reference substances that are included in the minimum list of chemicals identified for demonstrating the accuracy and reliability of test methods that are structurally and functionally similar to the validated reference test method, and were selected from the 163 reference chemicals that were originally used to validate the reference test method (Corrositex^®) (7) (10) (14). The goal of this selection process was to include, to the extent possible, chemicals that: were representative of the range of corrosivity responses (e.g. non-corrosives; UN Packing Groups I, II, and III corrosives) that the validated reference test method is capable of measuring or predicting; were representative of the chemical classes used during the validation process; have chemical structures that were well-defined; induced reproducible results in the validated reference test method; induced definitive results in the in vivo reference test; were commercially available; and were not associated with prohibitive disposal costs (14).

Substances tested neat or with purity 90 %

The corresponding UN Packing groups are I, II and III, respectively, for the UN GHS Sub-categories 1A, 1B and 1C. NC; Non-corrosive.

PROCEDURE

• 12.The following paragraphs describe the components and procedures of an artificial membrane barrier test method for corrosivity assessment (7)(15), based on the current VRM, i.e. the commercially available Corrositex^®. The membrane barrier and the compatibility/indicator and categorisation solutions can be constructed, prepared or obtained commercially such as in the case of the VRM Corrositex^®. A sample test method protocol for the validated reference test method is available (7). Testing should be performed at ambient temperature (17-25^oC) and the components should comply with the following conditions.

Test Chemical Compatibility Test

• 13.Prior to performing the membrane barrier test, a compatibility test is performed to determine if the test chemical is detectable by the CDS. If the CDS does not detect the test chemical, the membrane barrier test method is not suitable for evaluating the potential corrosivity of that particular test chemical and a different test method should be used. The CDS and the exposure conditions used for the compatibility test should reflect the exposure in the subsequent membrane barrier test.

Test Chemical Timescale Category Test

• 14.If appropriate for the test method, a test chemical that has been qualified by the compatibility test should be subjected to a timescale category test, i.e. a screening test to distinguish between weak and strong acids or bases. For example, in the validated reference test method a timescale categorisation test is used to indicate which of two timescales should be used based on whether significant acid or alkaline reserve is detected. Two different breakthrough timescales should be used for determining corrosivity and UN GHS skin corrosivity Sub-category, based on the acid or alkali reserve of the test chemical.

MEMBRANE BARRIER TEST METHOD COMPONENTS

Membrane Barrier

• 15.The membrane barrier consists of two components: a proteinaceous macromolecular aqueous gel and a permeable supporting membrane. The proteinaceous gel should be impervious to liquids and solids but can be corroded and made permeable. The fully constructed membrane barrier should be stored under pre-determined conditions shown to preclude deterioration of the gel, e.g. drying, microbial growth, shifting, cracking, which would degrade its performance. The acceptable storage period should be determined and membrane barrier preparations not used after that period.

• 16.The permeable supporting membrane provides mechanical support to the proteinaceous gel during the gelling process and exposure to the test chemical. The supporting membrane should prevent sagging or shifting of the gel and be readily permeable to all test chemicals.

• 17.The proteinaceous gel, composed of protein, e.g. keratin, collagen, or mixtures of proteins, forming a gel matrix, serves as the target for the test chemical. The proteinaceous material is placed on the surface of the supporting membrane and allowed to gel prior to placing the membrane barrier over the indicator solution. The proteinaceous gel should be of equal thickness and density throughout, and with no air bubbles or defects that could affect its functional integrity.

Chemical Detection System (CDS)

• 18.The indicator solution, which is the same solution used for the compatibility test, should respond to the presence of a test chemical. A pH indicator dye or combination of dyes, e.g. cresol red and methyl orange that will show a colour change, in response to the presence of the test chemical, should be used. The measurement system can be visual or electronic.

• 19.Detection systems that are developed for detecting the passage of the test chemical through the barrier membrane should be assessed for their relevance and reliability in order to demonstrate the range of chemicals that can be detected and the quantitative limits of detection.

TEST PERFORMANCE

Assembly of the Test Method Components

• 20.The membrane barrier is positioned in a vial (or tube) containing the indicator solution so that the supporting membrane is in full contact with the indicator solution and with no air bubbles present. Care should be taken to ensure that barrier integrity is maintained.

Application of the Test Chemical

• 21.A suitable amount of the test chemical, e.g. 500 μl of a liquid or 500 mg of a finely powdered solid (7), is carefully layered onto the upper surface of the membrane barrier and evenly distributed. An appropriate number of replicates, e.g. four (7), is prepared for each test chemical and its corresponding controls (see paragraphs 23 to 25). The time of applying the test chemical to the membrane barrier is recorded. To ensure that short corrosion times are accurately recorded, the application times of the test chemical to the replicate vials are staggered.

Measurement of Membrane Barrier Penetrations

• 22.Each vial is appropriately monitored and the time of the first change in the indicator solution, i.e. barrier penetration, is recorded, and the elapsed time between application and penetration of the membrane barrier determined.

Controls

• 23.In tests that involve the use of a vehicle or solvent with the test chemical, the vehicle or solvent should be compatible with the membrane barrier system, i.e. not alter the integrity of the membrane barrier system, and should not alter the corrosivity of the test chemical. When applicable, solvent (or vehicle) control should be tested concurrently with the test chemical to demonstrate the compatibility of the solvent with the membrane barrier system.

• 24.A positive (corrosive) control with intermediate corrosivity activity, e.g. 110 ± 15 mg sodium hydroxide (UN GHS Corrosive Sub-category 1B) (7), should be tested concurrently with the test chemical to assess if the test system is performing in an acceptable manner. A second positive control that is of the same chemical class as the test chemical may be useful for evaluating the relative corrosivity potential of a corrosive test chemical. Positive control(s) should be selected that are intermediate in their corrosivity (e.g. UN GHS Sub-category 1B) in order to detect changes in the penetration time that may be unacceptably longer or shorter than the established reference value, thereby indicating that the test system is not functioning properly. For this purpose, extremely corrosive (UN GHS Sub-category 1A) or non-corrosive chemicals are of limited utility. A corrosive UN GHS Sub-category 1B chemical would allow detection of a too rapid or too slow breakthrough time. A weakly corrosive (UN GHS Sub-category 1C) might be employed as a positive control to measure the ability of the test method to consistently distinguish between weakly corrosive and non-corrosive chemicals. Regardless of the approach used, an acceptable positive control response range should be developed based on the historical range of breakthrough times for the positive control(s) employed, such as the mean ± 2-3 standard deviations. In each study, the exact breakthrough time should be determined for the positive control so that deviations outside the acceptable range can be detected.

• 25.A negative (non-corrosive) control, e.g. 10 % citric acid, 6 % propionic acid (7), should also be tested concurrently with the test chemical as another quality control measure to demonstrate the functional integrity of the membrane barrier.

Study Acceptability Criteria

• 26.According to the established time parameters for each of the UN GHS corrosivity Sub-categories, the time (in minutes) elapsed between application of a test chemical to the membrane barrier and barrier penetration is used to predict the corrosivity of the test chemical. For a study to be considered acceptable, the concurrent positive control should give the expected penetration response time (e.g. 8-16 min breakthrough time for sodium hydroxide if used as a positive control), the concurrent negative control should not be corrosive, and, when included, the concurrent solvent control should neither be corrosive nor should it alter the corrosivity potential of the test chemical. Prior to routine use of a method that adheres to this test method, laboratories should demonstrate technical proficiency, using the twelve substances recommended in Table 2. For new ‘me-too’ methods developed under this test method that are structurally and functionally similar to the validated reference method (14) the pre-defined performance standards should be used to demonstrate the reliability and accuracy of the new method prior to its use for regulatory testing (10).

Interpretation of Results and Corrosivity Classification of Test Chemicals

• 27.The time (in minutes) elapsed between application of the test chemical to the membrane barrier and barrier penetration is used to classify the test chemical in terms of UN GHS corrosive Sub-categories (1) and, if applicable, UN Packing Group (16). Cut-off time values for each of the three corrosive subcategories are established for each proposed test method. Final decisions on cut-off times should consider the need to minimise under-classification of corrosive hazard (i.e. false negatives). In the present test guideline, the cut-off times of Corrositex® as described in table 3 should be used as it represents the only test method currently falling within the test guideline (7).

Table 3

Corrositex^® prediction model

Mean breakthrough time (min.)	Mean breakthrough time (min.)	UN GHS prediction [^E0058]
Category 1 test chemicals ^E0056	Category 2 test chemicals [^E0057] (determined by the method’s categorisation test)	UN GHS prediction [^E0058]
0-3 min.	0-3 min.	Corrosiveoptional Sub-category 1A
> 3 to 60 min.	> 3 to 30 min.	Corrosiveoptional Sub-category 1B
> 60 to 240 min.	> 30 to 60 min.	Corrosiveoptional Sub-category 1C
> 240 min.	> 60 min.	Non-corrosive

Test chemicals with high acid/alkaline reserve (6)

Test chemicals with low acid/alkaline reserve (6)

UN GHS Subcategories 1A, 1B and 1C correspond to UN packing groups I, II and III respectively

DATA AND REPORTING

Data

• 28.The time (in minutes) elapsed between application and barrier penetration for the test chemical and the positive control(s) should be reported in tabular form as individual replicate data, as well as means ± the standard deviation for each trial.

Test Report

• 29.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 physicochemical properties of the constituents;

• — Physical appearance, water solubility, and 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.

Vehicle:

• — Identification, concentration (where appropriate), volume used;

• — Justification for choice of vehicle.

In vitro membrane barrier model and protocol used, including demonstrated accuracy and reliability

Test Conditions:

• — Description of the apparatus and preparation procedures used;

• — Source and composition of the in vitro membrane barrier used;

• — Composition and properties of the indicator solution;

• — Method of detection;

• — Test chemical and control substance amounts;

• — Number of replicates;

• — Description and justification for the timescale categorisation test;

• — Method of application;

• — Observation times.

• — Description of the evaluation and classification criteria applied;

• — Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals.

Results:

• — Tabulation of individual raw data from individual test and control samples for each replicate;

• — Descriptions 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), Fifth 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 corrosion: reconstructed human epidermis (RHE) test method.

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

• (5) OECD (2015). 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.

• (6) Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Esdaile, D.J., Holzhutter, H.-G. and Liebsch, M. (1998). The ECVAM International Validation Study on In Vitro Tests for Skin Corrosivity. 2. Results and Evaluation by the Management Team. Toxicology In Vitro 12, 483-524.

• (7) ICCVAM (1999). Corrositex®. An In Vitro Test Method for Assessing Dermal Corrosivity Potential of Chemicals. The Results of an Independent Peer Review Evaluation Coordinated by ICCVAM, NTP and NICEATM. NIEHS, NIH Publication (No 99-4495.)

• (8) Gordon V.C., Harvell J.D. and Maibach H.I. (1994). Dermal Corrosion, the Corrositex® System: A DOT Accepted Method to Predict Corrosivity Potential of Test Materials. In vitro Skin Toxicology-Irritation, Phototoxicity, Sensitization. Alternative Methods in Toxicology 10, 37-45.

• (9) OECD (2005). Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment. Environmental, Health and Safety Publications. Series on testing and Assessment (No 34).

• (10) OECD (2014). Performance Standards for the Assessment of Proposed Similar or Modified In Vitro Membrane Barrier Test Method for Skin Corrosion in Relation to TG 435. Organisation for Economic Cooperation and Development, Paris. Available at: http://www.oecd.org/chemicalsafety/testing/PerfStand-TG430-June14.pdf.

• (11) ECVAM (2001). Statement on the Application of the CORROSITEX Assay for Skin Corrosivity Testing. 15th Meeting of ECVAM Scientific Advisory Committee (ESAC), Ispra, Italy. ATLA 29, 96-97.

• (12) U.S. DOT (2002). Exemption DOT-E-10904 (Fifth Revision). (September 20, 2002). Washington, D.C., U.S. DOT.

• (13) Chapter B.46 of this Annex, In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. ICCVAM (2004). ICCVAM Recommended Performance Standards for In Vitro Test Methods for Skin Corrosion. NIEHS, NIH Publication No 04-4510. Available at: http://www.ntp.niehs.nih.gov/iccvam/docs/dermal_docs/ps/ps044510.pdf.

• (14) U.S. EPA (1996). Method 1120, Dermal Corrosion. Available at: http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/1120.pdf.

• (15) United Nations (UN) (2013). UN Recommendations on the Transport of Dangerous Goods, Model Regulations, 18th Revised Edition (Part, Chapter 2.8), UN, 2013. Available at: http://www.unece.org/fileadmin/DAM/trans/danger/publi/unrec/rev18/English/Rev18_Volume1_Part2.pdf.

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 (9).

Chemical : A substance or a mixture.

Chemical Detection System (CDS) : A visual or electronic measurement system with an indicator solution that responds to the presence of a test chemical, e.g. by a change in a pH indicator dye, or combination of dyes, that will show a colour change in response to the presence of the test chemical or by other types of chemical or electrochemical reactions.

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 (9).

GHS (Globally Harmonized System of Classification and Labelling of Chemicals) : 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.

Performance standards : 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 (9).

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 (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).

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 (9).

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 (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.

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

B.66.   STABLY TRANSFECTED TRANSACTIVATION IN VITRO ASSAYS TO DETECT ESTROGEN RECEPTOR AGONISTS AND ANTAGONISTS

The full description of this test method has been deleted. The equivalent international test method appears in Table 2 of Part 0.

B.67.   IN VITRO MAMMALIAN CELL GENE MUTATION TESTS USING THE THYMIDINE KINASE GENE

INTRODUCTION

• 1.This test method (TM) is equivalent to the OECD test guideline 490 (2016). Test methods are periodically reviewed and revised in the light of scientific progress, regulatory needs and animal welfare. The mouse lymphoma assay (MLA) and TK6 test using the thymidine kinase (TK) locus were originally contained in test method B.17. Subsequently, the MLA Expert Workgroup of the International Workshop for Genotoxicity Testing (IWGT) has developed internationally harmonised recommendations for assay acceptance criteria and data interpretation for the MLA (1)(2)(3)(4)(5), and these recommendations are incorporated into this new test method B.67. This test method is written for the MLA and, because it also utilises the TK locus, the TK6 test. While the MLA has been widely used for regulatory purposes, the TK6 has been used much less frequently. It should be noted that in spite of the similarity between the endpoints the two cell lines are not interchangeable and regulatory programs may validly express a preference for one over the other for a particular regulatory use. For instance, the validation of the MLA demonstrated its appropriateness for detecting not only gene mutation, but also, the ability of a test chemical to induce structural chromosomal damage. 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 (6).

• 2.The purpose of the in vitro mammalian cell gene mutation tests is to detect gene mutations induced by chemicals. The cell lines used in these tests measure forward mutations in reporter genes, specifically the endogeneous thymidine kinase gene (TK for human cells and Tk for rodent cells, collectively referred to as TK in this test method). This test method is intended for use with two cell lines: the L5178Y TK^+/- -3.7.2C mouse lymphoma cell line (generally called L5178Y) and the TK6 human lymphoblastoid cell line (generally called TK6). Although the two cell lines vary because of their origin, cell growth, p53-status, etc., the TK gene mutation tests can be conducted in a similar way in both cell types as described in this test method.

• 3.The autosomal and heterozygous nature of the thymidine kinase gene enables the detection of viable colonies whose cells are deficient in the enzyme thymidine kinase following mutation from TK ^+/- to TK ^-/-. This deficiency can result from genetic events affecting the TK gene including both gene mutations (point mutations, frame-shift mutations, small deletions, etc.) and chromosomal events (large deletions, chromosome rearrangements and mitotic recombination). The latter events are expressed as loss of heterozygosity, which is a common genetic change of tumor suppressor genes in human tumorigenesis. Theoretically, loss of the entire chromosome carrying the TK gene resulting from spindle impairment and/or mitotic non-disjunction can be detected in the MLA. Indeed, a combination of cytogenetic and molecular analysis clearly shows that some MLA TK mutants are the result of nondisjunction. However, the weight of evidence shows that the TK gene mutation tests cannot reliably detect aneugens when applying standard cytotoxicity criteria (as described in this test method) and therefore, it is not appropriate to use these tests to detect aneugens (7)(8)(9).

• 4.In the TK gene mutation tests, two distinct phenotypic classes of TK mutants are generated; the normal growing mutants that grow at the same rate as the TK heterozygous cells, and slow growing mutants which grow with prolonged doubling times. The normal growing and slow growing mutants are recognised as large colony and small colony mutants in the MLA and as early appearing colony and late appearing colony mutants in the TK6. The molecular and cytogenetic nature of both large and small colony MLA mutants has been explored in detail (8)(10)(11)(12)(13). The molecular and cytogenetic nature of the early appearing and late appearing TK6 mutants has also been extensively investigated (14)(15)(16)(17). Slow growing mutants for both cell types have suffered genetic damage that involves putative growth regulating gene(s) near the TK locus which results in prolonged doubling times and the formation of late appearing or small colonies (18). The induction of slow growing mutants has been associated with chemicals that induce gross structural changes at the chromosomal level. Cells whose damage does not involve the putative growth regulating gene(s) near the TK locus grow at rates similar to the parental cells and become normal growing mutants. The induction of primarily normal growing mutants is associated with chemicals primarily acting as point mutagens. Consequently it is essential to count both slow growing and normal growing mutants in order to recover all of the mutants and to provide some insight into the type(s) of damage (mutagens vs. clastogens) induced by the test chemical (10)(12)(18)(19).

• 5.The test method is organised so as to provide general information that applies to both MLA and TK6 and specialised guidance for the individual tests.

• 6.Definitions used are provided in Appendix 1.

INITIAL CONSIDERATIONS AND LIMITATIONS

• 7.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.

• 8.Care should be taken to avoid conditions that could lead to artefactual positive results (i.e. possible interaction with the test system) not caused by interaction between the test chemical and the genetic material of the cell; such conditions include changes in pH or osmolality, interaction with the medium components (20)(21), or excessive levels of cytotoxicity (22)(23)(24). Cytotoxicity exceeding the recommended top cytotoxicity levels as defined in paragraph 28 is considered excessive for the MLA and TK6. In addition, it should be noted that test chemicals that are thymidine analogues, or behave like thymidine analogues can increase the mutant frequency by selective growth of the spontaneous background mutants during cell treatment and require additional test methods for adequate evaluation (25).

• 9.For manufactured nanomaterials, specific adaptations of this test method may be needed but are not described in this test method.

• 10.Before using the test method for testing a mixture to generate 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 the mixture.

• 11.Mutant cells deficient in thymidine kinase enzyme activity because of a mutation TK ^+/- to TK ^-/- are resistant to the cytostatic effects of the pyrimidine analogue trifluorothymidine (TFT). The TK proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TFT and form visible colonies, whereas cells containing the TK enzyme are not.

PRINCIPLE OF THE TEST

• 12.Cells in suspension are exposed to the test chemical, both with and without an exogenous source of metabolic activation (see paragraph 19), for a suitable period of time (see paragraph 33), and then sub-cultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection. Cytotoxicity is determined by relative total growth (RTG—see paragraph 25) for the MLA and by relative survival (RS—see paragraph 26) for TK6. The treated cultures are maintained in growth medium for a sufficient period of time, characteristic of each cell type (see paragraph 37), to allow near-optimal phenotypic expression of induced mutations. 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

• 13.For MLA: Because the MLA was developed and characterised using the TK ^+/- -3.7.2C subline of L5178Y cells, this specific subline has to be be used for the MLA. The L5178Y cell line was derived from a methylcholanthrene-induced thymic lymphoma from a DBA-2 mouse (26). Clive and co-workers treated L5178Y cells (designated by Clive as TK^+/+ -3) with ethylmethane sulfonate and isolated a TK^-/- (designated as TK^-/- -3.7) clone using bromodeoxyuridine as the selective agent. From the TK^-/- clone a spontaneous TK^+/- clone (designated as TK^+/- -3.7.2.) and asubclone (designated as TK^+/--3.7.2C) were isolated and characterised for use in the MLA (27). The karyotype for the cell line has been published (28)(29)(30)(31). The modal chromosome number is 40. There is one metacentric chromosome (t12;13) that should be counted as one chromosome. The mouse TK locus is located on the distal end of chromosome 11. The L5178Y TK ^+/- -3.7.2C cell line has mutations in both p53 alleles and produces mutant-p53 protein (32) (33). The p53 status of the TK^+/--3.7.2C cell line is likely responsible for the ability of the test to detect large-scale damage (17).