§ 798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.

(a) Purpose. The in vivo bone marrow cytogenetic test is a mutagenicity test for the detection of structural chromosomal aberrations. Chromosomal aberrations are generally evaluated in first post-treatment mitoses. With the majority of chemical mutagens, induced aberrations are of the chromatid type but chromosome type aberrations also occur.

(b) Definitions. (1) Chromosome-type aberrations are changes which result from damage expressed in both sister chromatids at the same time.

(2) Chromatid-type aberrations are damage expressed as breakage of single chromatids or breakage and/or reunion between chromatids.

(c) Reference substances. Not applicable.

(d) Test method—(1) Principle. Animals are exposed to test chemicals by appropriate routes and are sacrificed at sequential intervals. Chromosome preparations are made from bone marrow cells. The stained preparations are examined and metaphase cells are scored for chromosomal aberrations.

(2) Description. The method employs bone marrow of laboratory rodents which have been exposed to test chemicals. Prior to sacrifice, animals are further treated with a spindle inhibitor, (e.g., colchicine or Colcemid ®) to arrest the cells in c-metaphase. Chromosome preparations from the cells are stained and scored for chromosomal aberrations.

(3) Animal selection—(i) Species and strain. Any appropriate mammalian species may be used. Examples of commonly used rodent species are rats, mice, and hamsters.

(ii) Age. Healthy young adult animals shall be used.

(iii) Number and sex. At least five female and five male animals per experimental and control group shall be used. Thus, 10 animals would be sacrificed per time per group treated with the test compound if several test times after treatment are included in the experimental schedule. The use of a single sex or smaller number of animals should be justified.