Introduction to
GENETIC TOXICOLOGY

Micronucleus Test

The micronucleus test is traditionally performed in mice, where the bone marrow or peripheral blood is analyzed for the presence of micronuclei. Other rodents species, such as rats, may be used for the micronucleus test when, for example, toxicokinetic data is available).

Micronulei are formed as a result of chromosomal breakage or spindle damage. Fragments of whole chromosomes may not be included in the nuclei of the daughter cells following cell division and form single, or multiple micronuclei in the cytoplasm of these cells. These micronuclei are also known as Howell-Jolly bodies. Erythrocytes are chosen for examination, since micronuclei are not obscured by the main nucleus are are easily detected. Virtually all the chromosome damafe detected in immature erythrocytes (less than 24 hours old) will have occurred during recent exposure to the test substance.

Immature erythrocytes can be differentiated using a variety of staining techniques that rely on their relatively high content of residual DNA. Giemsa is used for mouse bone marrow/peripheral blood and stained immature erythrocytes blue, while the mature erythrocytes that have a low nucleic acid content, appear pinkish orange. Due to the presence of mast cell granules that closely ressemble in rat bone marrow, an acridine orance fluorescence staining method is used for the rat, as it specifically stains DNA-containing bodies a deep purple. The rat peripheral blood method is generally less sensitive than the equivalent mouse system, because micronucleated blood cells tend to be rapidly filtered out of circulation. Therefore, a special, supravital staining method is used for rat blood to identify the youngest immature erythrocyte population sensitivity of the technique.

Groups of male and female animals are treated with the test substance at three dose levels, the highest dose level being the estimated maximum tolerated dose (a dose porducing clinicla signs of toxicity, such that a slightly higher dose would be expected to cause lethality or unacceptably severe clinical signs) or the standard limit dose for the micronucleus test, whichever is least. Concurrent negative and positive control groups are also treated. Bone marrow is usually sampled at 24 and 48 hours after a single treatment (peripheral blood is usually sampled 24 hours later). Two sampling times are employed to allow for variations in the rate of absorption and metabolism of test substances and to allow for any delay in erythrocyte production as a result of cytostatic or cytotoxic effects.

The smears thus prepared are examined by light microscopy for the incidence of micronucleated erythrocytes (MIE). The proportion of immature erythrocytes (IE) and the number of micronucleated mature erythrocutes are also recorded. Since no substantial increase in the incidence of micronucleated mature erythrocytes (micronucleated ME) would be expected 24 hours after administration of a chromosome-damaging agent, nay micronucleus-like artifact are readily distinguishable.

Genotoxic activity is indicated by a statistically significant, dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group. Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significan dose-related decrease in the proportion of immature erythrocytes; a very large decrease in the proportion would be indicative of a cytostatic or cytotoxic effect.

Considerations of target organ exposure in the micronucleus test are very important. The usual route of administration is normally the expected maximum route of human exposure, but where is it known or suspected that the compound is poorly absorbed by this route, another route may be chosen to increase absorption. Supporting evidence for absorption and systemic distribution should be included in the study.