§ 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.

(c) Definitions. The following definitions apply to 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—(A) Selection of animal species. 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.