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)

• 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.

Substances [CAS No] (reference no)
Cyclophosphamide (monohydrate) [CAS no. 50-18-0 (CAS no. 6055-19-2)] (9)
Cyclohexylamine [CAS no. 108-91-8] (7)
Mitomycin C [CAS no. 50-07-7] (6)
Monomeric acrylamide [CAS 79-06-1] (10)
Triethylenemelamine [CAS 51-18-3] (8)

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 2 000 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 1 000 mg/kg body weight/day, and for administration periods of less than 14 days, the limit dose is 2 000 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 1 000 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

B.25.   MOUSE HERITABLE TRANSLOCATION

This test method has been deleted, as it is no longer recognised as being appropriate for generating information on the toxicological properties of chemicals for the purpose of Regulation (EC) No 1907/2006. The applicable test methods for the endpoint in question appear in Table 2 of Part 0.

B.26.   SUB-CHRONIC ORAL TOXICITY TESTREPEATED DOSE 90 – DAY ORAL TOXICITY STUDY IN RODENTS

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

B.27.   SUB-CHRONIC ORAL TOXICITY TEST REPEATED DOSE 90-DAY ORAL TOXICITY STUDY IN NON-RODENTS

1. METHOD

This sub-chronic oral toxicity test method is a replicate of the OECD TG 409 (1998).

1.1.   INTRODUCTION

In the assessment and evaluation of the toxic characteristics of a chemical, the determination of sub-chronic oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained from acute or repeated dose 28-day toxicity tests. The 90-day study provides information on the possible health hazards likely to arise from repeated exposure over a period of rapid growth and into young adulthood. The study will provide information on the major toxic effects, indicate target organs and the possibility of accumulation, and can provide an estimate of a no-observed-adverse-effect level of exposure which can be used in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.

The test method allows for the identification in non-rodent species of adverse effects of chemical exposure and should only be used:

• — where effects observed in other studies indicate a need for clarification/characterisation in a second, non-rodent species, or

• — where toxicokinetic studies indicate that the use of a specific non-rodent species is the most relevant choice of laboratory animal, or

• — where other specific reasons justify the use of a non-rodent species.

See also General introduction Part B.

1.2.   DEFINITIONS

Dose: is the amount of test substance administered. Dose is expressed as weight (g, mg) or as weight of test substance per unit weight of test animal (e.g. mg/kg), or as constant dietary concentrations (ppm).

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

NOAEL: is the abbreviation for no-observed-adverse-effect level and is the highest dose level where no adverse treatment-related findings are observed.

1.3.   PRINCIPLE OF THE TEST METHOD

The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 90 days. During the period of administration the animals are observed closely for signs of toxicity. Animals, which die or are killed during the test are necropsied and at the conclusion of the test surviving animals are also killed and necropsied.

1.4.   DESCRIPTION OF THE TEST METHOD

1.4.1.   Selection of animal species

The commonly used non-rodent species is the dog, which should be of a defined breed; the beagle is frequently used. Other species, e.g. swine, mini-pigs, may also be used. Primates are not recommended and their use should be justified. Young, healthy animals should be employed, and in the case of the dog, dosing should begin preferably at four to six months and not later than nine months of age. Where the study is conducted as a preliminary to a long-term chronic toxicity study, the same species/breed should be used in both studies.

1.4.2.   Preparation of animals

Healthy young animals, which have been acclimated to laboratory conditions and have not been subjected to previous experimental procedures, should be used. The duration of acclimatisation will depend upon the selected test species and their source. At least five days for dogs or purpose bred swine from a resident colony and at least two weeks for these animals if from external sources are recommended. The test animals should be characterised as to species, strain, source, sex, weight and/or age. Animals should be randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. Each animal should be assigned a unique identification number.

1.4.3.   Preparations of doses

The test substance may be administered in the diet or in the drinking water, by gavage or in capsules. The method of oral administration is dependent on the purpose of the study, and the physical-chemical properties of the test material.

Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g. corn oil) and then by possible solution in other vehicles. For vehicles other than water the toxic characteristics of the vehicle must be known. The stability of the test substance under the conditions of administration should be determined.

1.5.   PROCEDURE

1.5.1.   Number and sex of animals

At least eight animals (four female and four male) should be used at each dose level. If interim kills are planned, the number should be increased by the number of animals scheduled to be killed before the completion of the study. The number of animals at the termination of the study must be adequate for a meaningful evaluation of toxic effects. Based on previous knowledge of the substance or a close analogue, consideration should be given to including an additional satellite group of eight animals (four per sex) in control and in top dose group for observation after the treatment period of reversibility or persistence of any toxic effects. The duration of this post-treatment period should be fixed appropriately with regard to the effects observed.

1.5.2.   Dosage

At least three dose levels and a concurrent control shall be used, except where a limit test is conducted (see 1.5.3). Dose levels may be based on the results of repeated dose or range finding studies and should take into account any existing toxicological and toxicokinetic data available for the test compound or related materials. Unless limited by the physical-chemical nature or biological effects of the test substance, the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. A descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a no-observed-adverse-effect level (NOAEL) at the lowest dose level. Two to fourfold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals (e.g. more than a factor of about 6–10) between dosages.

The control group shall be an untreated group or a vehicle-control group if a vehicle is used in administering the test substance. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to those in the test groups. If a vehicle is used, the control group shall receive the vehicle in the highest volume used. If a test substance is administered in the diet, and causes reduced dietary intake, then a pair-fed control group may be useful in distinguishing between reductions due to palatability or toxicological alterations in the test model.

Consideration should be given to the following characteristics of the vehicle and other additives, as appropriate: effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status of the animals.

1.5.3.   Limit test

If a test at one dose level, equivalent to at least 1 000 mg/kg body weight/day, using the procedures described for this study, produces no-observed-adverse-effects and if toxicity would not be expected based upon data from structurally related substances, then a full study using three dose levels may not be considered necessary. The limit test applies except when human exposure indicates the need for a higher dose level to be used.

1.5.4.   Administration of doses

The animals are dosed with the test substance daily seven days each week for a period of 90 days. Any other dosing regime, e.g. five days per week, needs to be justified. When the test substance is administered by gavage, this should be done in a single dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. Normally the volume should be kept as low as possible. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels.

For substances administered via the diet or drinking water it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animal's body weight may be used; any alternative used must be specified. For a substance administered by gavage or by capsule, the dose should be given at similar times each day, and adjusted as necessary to maintain a constant dose level in terms of animal body weight. Where the 90 day study is used as a preliminary to a long term chronic toxicity study, a similar diet should be used in both studies.

1.5.5.   Observations

The observation period should be at least 90 days. Animals in a satellite group scheduled for follow-up observations should be kept for an appropriate period without treatment to detect persistence of, or recovery from toxic effects.

General clinical observations should be made at least once a day, preferably at the same time(s) each day, taking into consideration the peak period of anticipated effects after dosing. The clinical condition of the animals should be recorded. At least twice daily, usually at the beginning and end of each day, all animals should be inspected for signs of morbidity and mortality.

At least once prior to the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations should be made in all animals. These observations should be made, where practical outside the home cage in a standard arena and preferably at similar times on each occasion. Effort should be made to ensure that variations in the observation conditions are minimal. Signs of toxicity should be carefully recorded, including time of onset, degree and duration. Observations should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or any bizarre behaviour should also be recorded.

Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to the administration of the test substance and at the termination of the study, preferably in all animals but at least in the high dose and control groups. If treatment related changes in the eyes are detected all animals should be examined.

1.5.5.1.   Body weight and food/water consumption

All animals should be weighed at least once a week. Measurements of food consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly. Water consumption may also be considered for dietary or gavage studies during which drinking activity may be altered.

1.5.5.2.   Haematology and clinical biochemistry

Blood samples should be taken from a named site and stored, if applicable, under appropriate conditions. At the end of the test period, samples are collected just prior to or as part of the procedure for killing the animals.

Haematology, including haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count and a measure of clotting potential such as clotting time, prothrombin time, or thromboplastin time should be investigated at the start of the study, then either at monthly intervals or midway through the test period and finally at the end of the test period.

Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver, should be performed on blood samples obtained from all animals at the start, then either at monthly intervals or midway through the test and finally at the end of the test period. Test areas, which should be considered are electrolyte balance, carbohydrate metabolism, and liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the test substance. Animals should be fasted for a period appropriate to the species prior to blood sampling. Suggested determinations include calcium, phosphorus, chloride, sodium, potassium, fasting glucose, alanine aminotransferase, aspartate aminotransferase, ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements.

Urinalysis determinations should be performed at least at the start, then midway and finally at the end of the study using timed urine volume collection. Urinalysis determinations include appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells. Additional parameters may be employed where necessary to extend the investigation of observed effect(s).

In addition, studies to investigate markers of general tissue damage should be considered. Other determinations, which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methaemoglobin, and cholinesterase inhibition. Additional clinical biochemistry may be employed where necessary to extend the investigation of observed effects. These need to be identified for chemicals in certain classes or on a case-by-case basis.

Overall, there is a need for a flexible approach, depending on the species and the observed and/or expected effect from a given substance.

1.5.5.3.   Gross necropsy

All animals in the study shall be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver with gall bladder, kidneys, adrenals, testes, epididymides, ovaries, uterus, thyroid (with parathyroids), thymus, spleen, brain and heart of all animals (apart from those found moribund and/or inter-currently killed) should be trimmed of any adherent tissue, as appropriate, and their wet weight taken as soon as possible after dissection to avoid drying.

The following tissues should be preserved in the most appropriate fixation medium for both the type of tissue and the intended subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and medulla/pons), spinal cord (at three levels: cervical, mid-thoracic and lumbar), pituitary, eyes, thyroid, parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines (including Peyer's patches), liver, gall bladder, pancreas, kidneys, adrenals, spleen, heart, trachea and lungs, aorta, gonads, uterus, accessory sex organs, female mammary gland, prostate, urinary bladder, lymph nodes (preferably one lymph node covering the route of administration and another one distant from the route of administration to cover systemic effects), peripheral nerve (sciatic or tibial) preferably in close proximity to the muscle, a section of bone marrow (and/or a fresh bone marrow aspirate) and skin. The clinical and other findings may suggest the need to examine additional tissues. Also any organs considered likely to be target organs based on the known properties of the test substance should be preserved.

1.5.5.4.   Histopathology

Full histopathology should be carried out on the preserved organs and tissues in at least all animals in control and high dose group. The examination should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group.

All gross lesions should be examined.

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

2. DATA AND REPORTING

2.1.   DATA

Individual data should be provided. Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or killed for humane reasons and the time of any death or humane kill, the number showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion.

When applicable, numerical results should be evaluated by an appropriate and generally acceptable statistical method. The statistical methods and the data to be analysed should be selected during the design of the study.

2.2.   TEST REPORT

The test report must include the following information:

2.2.1.   Test substance:

• — physical nature, purity and physico-chemical properties,

• — identification data,

• — vehicle (if appropriate): justification for choice of vehicle, if other than water.

2.2.2.   Test species:

• — species and strain used,

• — number, age and sex of animals,

• — source, housing conditions, diet etc.,

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

2.2.3.   Test conditions:

• — rationale for dose level selection,

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

• — details of the administration of the test substance,

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

• — details of food and water quality.

2.2.4.   Results:

• — body weight/body weight changes,

• — food consumption, and water consumption, if applicable,

• — toxic response data by sex and dose level, including signs of toxicity,

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

• — ophthalmological examination,

• — haematological tests with relevant base-line values,

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

• — terminal body weight, organ weights and organ/body weight ratios,

• — necropsy findings,

• — a detailed description of all histopathological findings,

• — absorption data if available,

• — statistical treatment of results, where appropriate.

Discussion of results.

Conclusions.

B.28.   SUB-CHRONIC DERMAL TOXICITY STUDY 90-DAY REPEATED DERMAL DOSE STUDY USING RODENT SPECIES

1. METHOD

1.1.   INTRODUCTION

See General introduction Part B.

1.2.   DEFINITIONS

See General introduction Part B.

1.3.   REFERENCE SUBSTANCES

None.

1.4.   PRINCIPLE OF THE TEST METHOD

The test substance is applied daily to the skin in graduated doses to several groups of experimental animals, one dose per group for a period of 90 days. During the period of application the animals are observed daily to detect signs of toxicity. Animals, which die during the test are necropsied, and at the conclusion of the test surviving animals are necropsied.

1.5.   QUALITY CRITERIA

None.

1.6.   DESCRIPTION OF THE TEST METHOD

1.6.1.   Preparations

The animals are kept under the experimental housing and feeding conditions for at least five days prior to the test. Before the test healthy young animals are randomised and assigned to the treated and control groups. Shortly before testing fur is clipped from the dorsal area of the trunk of the test animals. Shaving may be employed but it should be carried out approximately 24 hours before the test. Repeat clipping or shaving is usually needed at approximately weekly intervals. When clipping or shaving the fur, care must be taken to avoid abrading the skin. Not less than 10 % of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of the covering. When testing solids, which may be pulverised if appropriate, the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle to ensure good contact with the skin. Liquid test substances are generally used undiluted. Daily application on a five to seven-day per week basis is used.

1.6.2.   Test conditions

1.6.2.1.   Experimental animals

The adult rat, rabbit or guinea pig may be used. Other species may be used but their use would require justification. At the commencement of the test the range of the weight variation should be ± 20 % of the mean weight. Where a sub-chronic dermal study is conducted as a preliminary to a long-term study, the same species and strain should be used in both studies.

1.6.2.2.   Number and sex

At least 20 animals (10 female and 10 male) with healthy skin should be used at each dose level. The females should be nulliparous and non-pregnant. If interim sacrifices are planned the number should be increased by the number of animals scheduled to be sacrificed before the completion of the study. In addition, a satellite group of 20 animals (10 animals per sex) may be treated with the high-dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for 28 days post-treatment.

1.6.2.3.   Dose levels

At least three dose levels are required with a controle or a vehicle control if a vehicle is used. The exposure period should be at least six hours per day. The application of the test substance should be made at similar times each day, and the amount of substance applied adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body weight. Except for treatment with the test substance, animals in the control group should be handled in an identical manner to the test group subjects. Where a vehicle is used to facilitate dosing, the vehicle control group should be dosed in the same way as the treated groups, and receive the same amount as that received by the highest dose level group. The highest dose level should result in toxic effects but produce no, or few, fatalities. The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest level should exceed this. Ideally, the intermediate dose level should produce minimal observable toxic effects. If more than one intermediate dose is used, the dose levels should be spaced to produce a gradation of toxic effects. In the low and intermediate groups, and in the controls, the incidence of fatalities should be low, in order to permit a meaningful evaluation of the results.

If application of the test substance produces severe skin irritation the concentrations should be reduced and this may result in a reduction in, or absence of, other toxic effects al: the high-dose level. If the skin has been badly damaged it may be necessary to terminate the study and undertake a new study at lower concentrations.

1.6.3.   Limit test

If a preliminary study at a dose level of 1 000 mg/kilograms, or a higher dose level related to possible human exposure where this is known, produces no toxic effects, further testing may not be considered necessary.

1.6.4.   Observation period

The experimental animals should be observed daily for signs of toxicity. The time of death and the time at which signs of toxicity appear and disappear should be recorded.

1.6.5.   Procedure

Animals should be caged individually. The animals are treated with the test substance, ideally on seven days per week, for a period of 90 days.

Animals in any satellite groups scheduled for follow-up observations should be kept for a further 28 days without treatment to detect recovery from, or persistence of, toxic effects. Exposure time should be six hours per day.

The test substance should be applied uniformly over an area, which is approximately 10 % of the total body surface area. With highly toxic substances, the surface area covered may be less but as much of the area should be covered with as thin and uniform a film as possible.

During exposure the test substance is held in contact with the skin with a porous gauze dressing and non-irritating tape. The test site should be further covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance. Restrainers may be used to prevent the ingestion of the test substance but complete immobilisation is not a recommended method.

At the end of the exposure period, residual test substance should be removed where practicable using water or some other appropriate method of cleansing the skin.

All the animals should be observed daily and signs of toxicity recorded, including the time of onset, their degree and duration. Cageside observations should include changes in skin and fur, eyes and mucous membranes, as well as respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Measurements should be made of food consumption weekly and the animals weighed weekly. Regular observations of the animals are necessary to ensure that animals are not lost from the study due to causes such as cannibalism, autolysis of tissues or misplacement. At the end of the study period all survivors in the non-satellite treatment groups are necropsied. Moribund animals should be removed and necropsied when noticed.

The following examinations are customarily made on all animals including the controls:

• (a) ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, should be made prior to exposure to the test substance and at the termination of the study, preferably in all animals but at least in the high-dose and control groups. If changes in the eyes are detected all animals should be examined.

• (b) haematology, including haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count, and a measure of clotting potential, such as clotting time, prothrombin time, thromboplastin time, or platelet count, should be investigated at the end of the test period.

• (c) clinical biochemistry determination on blood should be carried out at the end of the test period. Test areas, which are considered appropriate to all studies are electrolyte balance, carbohydrate metabolism, liver and kidney function. The selection of specific tests will be influenced by observations on the mode of action of the substance. Suggested determinations are calcium, phosphorus, chloride, sodium, potassium, fasting glucose (with period of fasting appropriate to the species), sercum glutamic pyruvic transaminase ([^E0015]), serum glutamic oxaloacetic transaminase ([^E0016]), ornithine decarboxylase, gamma glutamyl transpeptidase, urea nitrogen, albumin, blood creatinine, total bilirubin and total serum protein measurements. Other determinations which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methaemoglobin and choliensterase activity. Additional clinical biochemistry may be employed, where necessary, to extend the investigation of observed effects.

• (d) urinalysis is not required on a routine basis but only when there is an indication based on expected or observed toxicity.

If historical baseline data are inadequate, consideration should be given to determination of haem a to logical and clinical biochemistry parameters before dosing commences.

Gross necropsy

All animals should be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals and testes must be weighed wet as soon as possible after dissection to avoid drying. The following organs and tissues should be preserved in a suitable medium for possible future histopathological examination: all gross lesions, brain — including sections of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/parathyroid, any thymic tissue, (trachea), lungs, heart, aorta, salivary glands, liver, spleen, kidneys, adrenals, pancreas, gonads, uterus, accessory genital organs, gall bladder (if present), oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, representative lymph node, (female mammary gland), (thigh musculature), peripheral nerve, (eyes), (sternum with bone marrow), (femur — including articular surface), (spinal cord at three levels — cervical, mid-thoracic and lumbar), and (exorbital lachrymal glands). The tissues mentioned between brackets need only be examined if indicated by signs of toxicity or target organ involvement.

Histopathological examination

• (a) Full histopathology should be carried out on normal and treated skin and on organs and tissues of animals in the control and high-dose groups.

• (b) all gross lesions should be examined.

• (c) target organs in other dose groups should be examined.

• (d) where rats are used, lungs of animals in the low- and intermediate-dose groups should be subjected to histopathological examination for evidence of infection, since this provides a convenient assessment of the state of health of the animals. Further histopathological examination may not be required routinely on the animals in these groups, but must always be carried out in organs, which show evidence of lesions in the high-dose group.

• (e) when a satellite group is used, histopathology should be performed on tissues and organs identified as showing effects in the other treated groups.

2. DATA

Data should be summarised in tabular form, showing for each test group the number of animals at the start of the test, the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion. Results should be evaluated by an appropriate statistical method. Any recognised statistical method may be used.

3. REPORTING

3.1.   TEST REPORT

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

• — species, strain, source, environmental conditions, diet,

• — test conditions,

• — dose levels (including vehicle, if used) and concentrations,

• — toxic response data by sex and dose,

• — no-effect level, where possible,

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

• — description of toxic or other effects,

• — the time of observation of each abnormal sign and its subsequent course,

• — food and bodyweight data,

• — ophthalmological findings,

• — haematological tests employed and all results,

• — clinical biochemistry tests employed and all results (including results of any urinalysis),

• — necropsy findings,

• — a detailed description of all histopathological findings,

• — statistical treatment of results where possible,

• — discussion of the results,

• — interpretation of the results.

3.2.   EVALUATION AND INTERPRETATION

See General introduction Part B.

4. REFERENCES

See General introduction Part B.

B.29.   SUBCHRONIC INHALATION TOXICITY: 90-DAY STUDY

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

B.30.   CHRONIC TOXICITY STUDIES

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

B.31.   PRENATAL DEVELOPMENTAL TOXICITY STUDY

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

B.32.   CARCINOGENICITY STUDIES

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

B.33.   COMBINED CHRONIC TOXICITY/CARCINOGENICITY STUDIES

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

B.34.   ONE-GENERATION REPRODUCTION TOXICITY TEST

This test method has been deleted, as it is no longer recognised as being appropriate for generating information on the toxicological properties of chemicals for the purpose of Regulation (EC) No 1907/2006. The applicable test methods for the endpoint in question appear in Table 2 of Part 0.

B.35.   TWO-GENERATION REPRODUCTION TOXICITY STUDY

This test method has been deleted, as it is no longer recognised as being appropriate for generating information on the toxicological properties of chemicals for the purpose of Regulation (EC) No 1907/2006. The applicable test methods for the endpoint in question appear in Table 2 of Part 0.

B.36.   TOXICOKINETICS

INTRODUCTION

• 1.This Test Method is equivalent to OECD TG 417 (2010). Studies examining the toxicokinetics (TK) of a test chemical are conducted to obtain adequate information on its absorption, distribution, biotransformation (i.e. metabolism) and excretion, to aid in relating concentration or dose to the observed toxicity, and to aid in understanding its mechanism of toxicity. TK may help to understand the toxicology studies by demonstrating that the test animals are systemically exposed to the test chemical and by revealing which are the circulating moieties (parent chemical/metabolites). Basic TK parameters determined from these studies will also provide information on the potential for accumulation of the test chemical in tissues and/or organs and the potential for induction of biotransformation as a result of exposure to the test chemical.

• 2.TK data can contribute to the assessment of the adequacy and relevance of animal toxicity data for extrapolation to human hazard and/or risk assessment. Additionally, toxicokinetic studies may provide useful information for determining dose levels for toxicity studies (linear vs. non-linear kinetics), route of administration effects, bioavailability, and issues related to study design. Certain types of TK data can be used in physiologically based toxicokinetic (PBTK) model development.

• 3.There are important uses for metabolite/TK data such as suggesting possible toxicities and modes of action and their relation to dose level and route of exposure. In addition, metabolism data can provide information useful for assessing the toxicological significance of exposures to exogenously produced metabolites of the test chemical.

• 4.Adequate toxicokinetic data will be helpful to support the further acceptability and applicability of quantitative structure-activity relationships, read-across or grouping approaches in the safety evaluation of chemicals. Kinetics data may also be used to evaluate the toxicological relevance of other studies (e.g. in vivo/in vitro).

• 5.Unless another route of administration is mentioned (see in particular paragraphs 74-78), this Test Method is applicable to oral administration of the test chemical.

INITIAL CONSIDERATIONS

• 6.Regulatory systems have different requirements and needs regarding the measurement of endpoints and parameters related to toxicokinetics for different classes of chemicals (e.g. pesticides, biocides, industrial chemicals). Unlike most Test Methods this Test Method describes toxicokinetics testing, which involves multiple measurements and endpoints. In the future, several new Test Methods, and/or guidance document(s), may be developed to describe each endpoint separately and in more detail. In the case of this Test Method, which tests or assessments are conducted is specified by the requirements and/or needs of each regulatory system.

• 7.There are numerous studies that might be performed to evaluate the TK behaviour of a test chemical for regulatory purposes. However, depending on particular regulatory needs or situations, not all of these possible studies may be necessary for the evaluation of a test chemical. Flexibility, taking into consideration the characteristics of the test chemical being investigated, is needed in the design of toxicokinetic studies. In some cases, only a certain set of questions may need to be explored in order to address test chemical-associated hazard and risk concerns. In some situations, TK data can be collected as part of the evaluation in other toxicology studies. For other situations, additional and/or more extensive TK studies may be necessary, depending on regulatory needs and/or if new questions arise as part of test chemical evaluation.

• 8.All available information on the test chemical and relevant metabolites and analogues should be considered by the testing laboratory prior to conducting the study in order to enhance study quality and avoid unnecessary animal use. This could include data from other relevant Test Methods (in vivo studies, in vitro studies, and/or in silico evaluations). Physicochemical properties, such as octanol-water partition coefficient (expressed as log POW), pKa, water solubility, vapour pressure, and molecular weight of a chemical may be useful for study planning and interpretation of results. They can be determined using appropriate methods as described in the relevant Test Methods.

LIMITATIONS

• 9.This Test Method is not designed to address special circumstances, such as the pregnant or lactating animal and offspring, or to evaluate potential residues in exposed food-producing animals. However, the data obtained from a B.36 study can provide background information to guide the design of specific studies for these investigations. This Test Method is not intended for the testing of nanomaterials. A report on preliminary review of OECD Test Guidelines for their applicability to nanomaterials indicates that TG 417 (equivalent to this Test Method B.36) may not apply to nanomaterials (1).

DEFINITIONS

• 10.Definitions used for the purpose of this Test Method are provided in Appendix.

ANIMAL WELFARE CONSIDERATIONS

• 11.Guidance on humane treatment of animals is available in OECD Guidance Document (GD) 19 (2). It is recommended that OECD GD 19 be consulted for all in vivo and in vitro studies described in this Test Method.

DESCRIPTION OF THE METHODS

Pilot Studies

• 12.The use of pilot studies is recommended and encouraged for the selection of experimental parameters for the toxicokinetics studies (e.g. metabolism, mass balance, analytical procedures, dose-finding, exhalation of CO2, etc.). Characterisation of some of these parameters may not necessitate the use of radiolabelled chemicals.

Animal Selection

Species

• 13.The animal species (and strain) used for TK testing should preferably be the same as that used in other toxicological studies performed with the test chemical of interest. Normally, the rat should be used as it has been used extensively for toxicological studies. The use of other or additional species may be warranted if critical toxicology studies demonstrate evidence of significant toxicity in these species or if their toxicity/toxicokinetics is shown to be more relevant to humans. Justification should be provided for the selection of the animal species and its strain.

• 14.Unless mentioned otherwise, this Test Method refers to the rat as the test species. Certain aspects of the method might have to be modified for the use of other test species.

Age and Strain

• 15.Young healthy adult animals (normally 6-12 weeks at the time of dosing) should be used (see also paragraphs 13 and 14). Justification should be provided for the use of animals that are not young adults. All animals should be of similar age at the outset of the study. The weight variation of individual animals should not exceed ± 20 % of the mean weight of the test group. Ideally, the strain used should be the same as that used in deriving the toxicological database for the test chemical.

Number and Sex of Animals

• 16.A minimum of four animals of one sex should be used for each dose tested. Justification should be provided for the sex of the animals used. The use of both sexes (four males and four females) should be considered if there is evidence to support significant sex-related differences in toxicity.