799.9538—TSCA mammalian bone marrow chromosomal aberration test.
(a) Scope.
This section is intended to meet the testing requirements under section 4 of TSCA. The mammalian bone marrow chromosomal aberration test is used for the detection of structural chromosome aberrations induced by test compounds in bone marrow cells of animals, usually rodents. Structural chromosome aberrations may be of two types, chromosome or chromatid. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. With the majority of chemical mutagens, induced aberrations are of the chromatid-type, but chromosome-type aberrations also occur. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumor suppressor genes are involved in cancer in humans and experimental systems.
(b) Source.
The source material used in developing this TSCA test guideline is the OECD guideline 475 (February 1997). This source is available at the address in paragraph (g) of this section.
Chromatid-type aberration is structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids.
Chromosome-type aberration is structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
Endoreduplication is a process in which after an S period of DNA replication, the nucleus does not go into mitosis but starts another S period. The result is chromosomes with 2,4,8,...chromatids.
Gap is an achromatic lesion smaller than the width of one chromatid, and with minimum misalignment of the chromatids.
Numerical aberration is a change in the number of chromosomes from the normal number characteristic of the animals utilized.
Polyploidy is a multiple of the haploid chromosome number (n) other than the diploid number (i.e., 3n, 4n and so on).
Structural aberration is a change in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, observed as deletions and fragments, intrachanges or interchanges.
(d) Initial considerations.
(1)
Rodents are routinely used in this test. Bone marrow is the target tissue in this test, since it is a highly vascularised tissue, and it contains a population of rapidly cycling cells that can be readily isolated and processed. Other species and target tissues are not the subject of this section.
(2)
This chromosome aberration test is especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in vivo metabolism, pharmacokinetics and DNA-repair processes although these may vary among species and among tissues. An in vivo test is also useful for further investigation of a mutagenic effect detected by an in vitro test.
(3)
If there is evidence that the test substance, or a reactive metabolite, will not reach the target tissue, it is not appropriate to use this test.
(e) Test method—
(1) Principle.
Animals are exposed to the test substance by an appropriate route of exposure and are sacrificed at appropriate times after treatment. Prior to sacrifice, animals are treated with a metaphase-arresting agent (e.g., colchicine or Colcemid ® ). Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells are analyzed for chromosome aberrations.
(2) Description—
(i) Preparations—
Rats, mice and Chinese hamsters are commonly used, although any appropriate mammalian species may be used. Commonly used laboratory strains of young healthy adult animals should be employed. At the commencement of the study, the weight variation of animals should be minimal and not exceed ±20% of the mean weight of each sex.
(B) Housing and feeding conditions.
The temperature in the experimental animal room should be 22 °C ±3 °C). Although the relative humidity should be at least 30% and preferably not exceed 70% other than during room cleaning, the aim should be 50-60%. Lighting should be artificial, the sequence being 12 hrs light, 12 hrs dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. The choice of diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method. Animals may be housed individually, or be caged in small groups of the same sex.
(C) Preparation of the animals.
Healthy young adult animals shall 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 minimized. The animals are identified uniquely. The animals are acclimated to the laboratory conditions for at least 5 days.
(D) Preparation of doses.
Solid test substances shall be dissolved or suspended in appropriate solvents or vehicles and diluted, as appropriate, prior to dosing of the animals. Liquid test substances may be dosed directly or diluted prior to dosing. Fresh preparations of the test substance should be employed unless stability data demonstrate the acceptability of storage.
(ii) Test conditions—
(A) Solvent/vehicle.
The solvent/vehicle shall not produce toxic effects at the dose levels used, and shall not be suspected of chemical reaction with the test substance. If other than well-known solvents/vehicles are used, their inclusion should be supported with data indicating their compatibility. It is recommended that wherever possible, the use of an aqueous solvent/vehicle should be considered first.
(B) Controls.
(1) Concurrent positive and negative (solvent/vehicle) controls shall be included for each sex in each test. Except for treatment with the test substance, animals in the control groups should be handled in an identical manner to the animals in the treated groups.
(2) Positive controls shall produce structural chromosome aberrations in vivo at exposure levels expected to give a detectable increase over background. Positive control doses should be chosen so that the effects are clear but do not immediately reveal the identity of the coded slides to the reader. It is acceptable that the positive control be administered by a route different from the test substance and sampled at only a single time. The use of chemical class related positive control chemicals may be considered, when available. Examples of positive control substances include:
Chemical | CAS No. |
---|---|
Triethylenemelamine | [CAS no. 51-18-3] |
Ethyl methanesulphonate | [CAS no. 62-50-0] |
Ethyl nitrosourea | [CAS no. 759-73-9] |
Mitomycin C | [CAS no. 50-07-7] |
Cyclophosphamide (monohydrate) | [CAS no. 50-18-0][CAS no. 6055-19-2] |
(3) Negative controls, treated with solvent or vehicle alone, and otherwise treated in the same way as the treatment groups, shall be included for every sampling time, unless acceptable inter-animal variability and frequencies of cells with chromosome aberrations are available from historical control data. If single sampling is applied for negative controls, the most appropriate time is the first sampling time. In the absence of historical or published control data demonstrating that no deleterious or mutagenic effects are induced by the chosen solvent/vehicle, untreated animals should be used.
(3) Procedure—
(i) Number and sex of animals.
Each treated and control group shall include at least 5 analyzable animals per sex. If at the time of the study there are data available from studies in the same species and using the same route of exposure that demonstrate that there are no substantial differences in toxicity between sexes, then testing in a single sex will be sufficient. Where human exposure to chemicals may be sex-specific, as for example with some pharmaceutical agents, the test should be performed with animals of the appropriate sex.
(ii) Treatment schedule.
(A)
Test substances are preferably administered as a single treatment. Test substances may also be administered as a split dose, i.e. two treatments on the same day separated by no more than a few hrs, to facilitate administering a large volume of material. Other dose regimens should be scientifically justified.
(B)
Samples shall be taken at two separate times following treatment on one day. For rodents, the first sampling interval is 1.5 normal cell cycle length (the latter being normally 12-18 hr) following treatment. Since the time required for uptake and metabolism of the test substance as well as its effect on cell cycle kinetics can affect the optimum time for chromosome aberration detection, a later sample collection 24 hr after the first sample time is recommended. If dose regimens of more than one day are used, one sampling time at 1.5 normal cell cycle lengths after the final treatment should be used.
(C)
Prior to sacrifice, animals shall be injected intraperitoneally with an appropriate dose of a metaphase arresting agent (e.g. Colcemid ® or colchicine). Animals are sampled at an appropriate interval thereafter. For mice this interval is approximately 3-5 hrs; for Chinese hamsters this interval is approximately 4-5 hrs. Cells shall be harvested from the bone marrow and analyzed from chromosome aberrations.
(iii) Dose levels.
If a range finding study is performed because there are no suitable data available, it shall be performed in the same laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study (an approach to dose selection is presented in the reference under paragraph (g)(5) of this section). If there is toxicity, three dose levels shall be used for the first sampling time. These dose levels shall cover a range from the maximum to little or no toxicity. At the later sampling time only the highest dose needs to be used. The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. Substances with specific biological activities at low non-toxic doses (such as hormones and mitogens) may be exceptions to the dose-setting criteria and should be evaluated on a case-by-case basis. The highest dose may also be defined as a dose that produces some indication of toxicity in the bone marrow (e.g. greater than 50% reduction in mitotic index).
(iv) Limit test.
If a test at one dose level of at least 2,000 mg/kg body weight using a single treatment, or as two treatments on the same day, produces no observable toxic effects, and if genotoxicity would not be expected based on data from structurally related compounds, then a full study using three dose levels may not be considered necessary. For studies of a longer duration, the limit dose is 2,000 mg/kg/body weight/day for treatment up to 14 days, and 1,000 mg/kg/body weight/day for treatment longer than 14 days. Expected human exposure may indicate the need for a higher dose level to be used in the limit test.
(v) Administration of doses.
The test substance is usually administered by gavage using a stomach tube or a suitable intubation cannula, or by intraperitoneal injection. Other routes of exposure may be acceptable where they can be justified. The maximum volume of liquid that can be administered by gavage or injection at one time depends on the size of the test animal. The volume should not exceed 2 ml/100g body weight. The use of volumes higher than these must be justified. Except for irritating or corrosive substances which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimized by adjusting the concentration to ensure a constant volume at all dose levels.
(vi) Chromosome preparation.
Immediately after sacrifice, bone marrow shall be obtained, exposed to hypotonic solution and fixed. The cells shall be then spread on slides and stained.
(vii) Analysis.
(A)
The mitotic index should be determined as a measure of cytotoxicity in at least 1,000 cells per animal for all treated animals (including positive controls) and untreated negative control animals.
(B)
At least 100 cells should be analyzed for each animal. This number could be reduced when high numbers of aberrations are observed. All slides, including those of positive and negative controls, shall be independently coded before microscopic analysis. Since slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, the cells scored should therefore contain a number of centromeres equal to the number 2n ±2.
(f) Data and reporting—
(1) Treatment of results.
Individual animal data shall be presented in tabular form. The experimental unit is the animal. For each animal the number of cells scored, the number of aberrations per cell and the percentage of cells with structural chromosome aberration(s) shall be evaluated. Different types of structural chromosome aberrations shall be listed with their numbers and frequencies for treated and control groups. Gaps shall be recorded separately and reported but generally not included in the total aberration frequency. If there is no evidence for a difference in response between the sexes, the data may be combined for statistical analysis.
(2) Evaluation and interpretation of results.
(i)
There are several criteria for determining a positive result, such as a dose-related increase in the relative number of cells with chromosome aberrations or a clear increase in the number of cells with aberrations in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results (some statistical methods are described in the reference under paragraph (g)(6) of this section). Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.
(ii)
An increase in polyploidy may indicate that the test substance has the potential to induce numerical chromosome aberrations. An increase in endoreduplication may indicate that the test substance has the potential to inhibit cell cycle progression. This phenomenon is described in the references under paragraphs (g)(7) and (g)(8) of this section.
(iii)
A test substance for which the results do not meet the criteria described in paragraph (f)(2)(i) of this section is considered non-mutagenic in this test.
(iv)
Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgment about the activity of the test substance. Results may remain equivocal or questionable regardless of the number of experiments performed.
(v)
Positive results from the in vivo chromosome aberration test indicate that a substance induces chromosome aberrations in the bone marrow of the species tested. Negative results indicate that, under the test conditions, the test substance does not induce chromosome aberrations in the bone marrow of the species tested.
(vi)
The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g., systemic toxicity) should be discussed.
(D)
Individual weight of the animals at the start of the test, including body weight range, mean and standard deviation for each group.
(G)
Methods for verifying that the test substance reached the general circulation or target tissue, if applicable.
(H)
Conversion from diet/drinking water test substance concentration parts per million (ppm) to the actual dose (mg/kg body weight/day), if applicable.
(g) References.
For additional background information on this test guideline, the following references should be consulted. These references are available for inspection at the TSCA Nonconfidential Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday, except legal holidays.
(1)
Adler, I.D. Eds. S. Venitt and J.M. Parry. Cytogenetic Tests in Mammals. Mutagenicity Testing: A Practical Approach. (IRL Press, Oxford, Washington DC, 1984) pp. 275-306.
(2)
Preston, R.J., Dean, B.J., Galloway, S., Holden, H., McFee, A.F., and Shelby, M. Mammalian In Vivo Cytogenetic Assays: Analysis of Chromosome Aberrations in Bone Marrow Cells. Mutation Research. 189, 157-165 (1987).
(3)
Richold, M., Chandley, A., Ashby, J., Gatehouse, D.G., Bootman, J., and Henderson, L. Ed. D.J. Kirkland. In Vivo Cytogenetic Assays. Basic Mutagenicity Tests, UKEMS Recommended Procedures. UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report. Part I revised. (Cambridge University Press, Cambridge, NY, Port Chester, Melbourne, Sydney, 1990) pp. 115-141.
(4)
Tice, R.R., Hayashi, M., MacGregor, J.T., Anderson, D., Blakey, D.H., Holden, H.E., Kirsch-Volders, M., Oleson Jr., F.B., Pacchierotti, F., Preston, R.J., Romagna, F., Shimada, H., Sutou, S., and Vannier, B. Report from the Working Group on the In Vivo Mammalian Bone Marrow Chromosomal Aberration Test. Mutation Research. 312, 305-312 (1994).
(5)
Fielder, R.J., Allen, J.A., Boobis, A.R., Botham, P.A., Doe, J., Esdaile, D.J., Gatehouse, D.G., Hodson-Walker, G., Morton, D.B., Kirkland, D. J., and Richold, M. Report of British Toxicology Society/UK Environmental Mutagen Society Working Group: Dose Setting in In Vivo Mutagenicity Assays. Mutagenesis. 7, 313-319 (1992).
(6)
Lovell, D.P., Anderson, D., Albanese, R., Amphlett, G.E., Clare, G., Ferguson, R., Richold, M., Papworth, D.G., and Savage, J.R.K. Ed. Kirkland,D. J. Statistical Analysis of In Vivo Cytogenetic Assays. UKEMS Sub-Committee on Guidelines for Mutagenicity Testing. Report Part III. Statistical Evaluation of Mutagenicity Test Data (Cambridge University Press, Cambridge, 1989) pp. 184-232.
(7)
Locke-Huhle, C. Endoreduplication in Chinese Hamster Cells During Alpha-Radiation Induced G2 Arrest. Mutation Research. 119, 403-413 (1983).
(8)
Huang, Y., Change, C., and Trosko, J. E. Aphidicolin-Induced Endoreduplication in Chinese Hamster Cells. Cancer Research. 43, 1362-1364 (1983).