Table 2: Major in vivo and in vitro testing models for chromosome damage.

Test model

Test guidance (Adopted)

Design (taken from the OECD test guidance)

Mammalian chromosome aberration test

OECD TG 475 [39] - in vivo

Animals are exposed to the test substance (liquid or solid) 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. Chromosome preparations are then made from the bone marrow cells and stained, and metaphase cells are analysed for chromosome aberrations. Each treated and control group must include at least 5 analysable animals per sex. The limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days.

OECD TG 473 [44] - in vitro

The in vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. Cell cultures are exposed to the test substance (liquid or solid) both with and without metabolic activation during about 1.5 normal cell cycle lengths. At least three analysable concentrations of the test substance should be used. At each concentration duplicate cultures should normally be used. At predetermined intervals after exposure of cell cultures to the test substance, the cells are treated with a metaphase-arresting substance, harvested, stained. Metaphase cells are analysed microscopically for the presence of chromosome aberrations.

Mammalian micronucleus test

OECD TG 474 [46] - in vivo

Animals are exposed to the test substance by an appropriate route. Bone marrow and/or blood cells are collected, prepared and stained. Preparations are analyzed for the presence of micronuclei. Each treated and control group must include at least 5 analysable animals per sex. Administration of the treatments consists of a single dose of test substance or two daily doses (or more). The limit dose is 2000 mg/kg/body weight/day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days.

OECD TG 487 [48] - in vitro

Cell cultures of human or other mammalian origin are exposed to the test chemical both with and without an exogenous source of metabolic activation. During or after exposure to the test chemical, the cells are grown for a period sufficient to allow chromosome damage or other effects on cell cycle/cell division to lead to the formation of micronuclei in interphase cells. For induction of aneuploidy, the test chemical should ordinarily be present during mitosis. Harvested and stained interphase cells are analysed for the presence of micronuclei. Ideally, micronuclei should only be scored in those cells that have completed mitosis during exposure to the test chemical or during the post-treatment period, if one is used. For all protocols, it is important to demonstrate that cell proliferation has occurred in both the control and treated cultures, and the extent of test chemical-induced cytotoxicity or cytostasis should be assessed in all of the cultures that are scored for micronuclei.