§ 798.5395 In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay.

(a) Purpose. The micronucleus test is a mammalian in vivo test which detects damage of the chromosomes or mitotic apparatus by chemicals. Polychromatic erythrocytes in the bone marrow of rodents are used in this assay. When the erythroblast develops into an erythrocyte the main nucleus is extruded and may leave a micronucleus in the cytoplasm. The visualization of micronuclei is facilitated in these cells because they lack a nucleus. Micronuclei form under normal conditions. The assay is based on an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow of treated animals.

(b) Definition. Micronuclei are small particles consisting of acentric fragments of chromosomes or entire chromosomes, which lag behind at anaphase of cell division. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm.

(c) Reference substances. Not applicable.

(d) Test method—(1) Principle. (i) Animals are exposed to test substance by an appropriate route. They are sacrificed, the bone marrow extracted and smear preparations made and stained. Polychromatic erythrocytes are scored for micronuclei under the microscope.

(ii) Micronuclei may also be detected in other test systems:

(A) Tissue culture.

(B) Plants.

(C) Blood smears.

(D) Fetal tissues.

(E) Meiotic cells.

(F) Hepatic cells.

(iii) The present guideline is based on the mammalian bone marrow assay.

(2) Description. The method employs bone marrow of laboratory mammals which are exposed to test substances.

(3) Animal selection—(i) Species and strain. Mice are recommended. However, any appropriate mammalian species may be used.

(ii) Age. 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 if several test times after treatment were included in the experimental schedule. The use of a single sex or a smaller number of animals should be justified.