Taken from the NTP web site.

The Micronucleus Test

Testing chemicals for the ability to induce numerical or structural chromosomal damage is easily accomplished by using the micronucleus assay. A "micronucleus" is literally a small nucleus. The nucleus is the organelle in the cell that contains the genetic material (DNA) that directs normal cellular function and cellular reproduction. In cells of eukaryotic organisms, the nucleus contains DNA packaged into chromosomes. Chromosome shape, size, and number are constant for a species. During cell division, the genetic material replicates and then divides equally between the two daughter cells that are produced. If the process is disrupted, or the chromosomes are broken or damaged by chemicals or radiation, then the distribution of genetic material between the two daughter nuclei during cell division may be affected and pieces or entire chromosomes may fail to be included in either of the two daughter nuclei. When this occurs, the genetic material that is not incorporated into a new nucleus may form its own "micronucleus" which is clearly visible with a microscope. Thus, in the micronucleus test, animals are treated with a chemical and then the frequency of micronucleated cells is determined at some specified time after treatment. If a treated group of animals shows significantly higher frequencies of micronucleated cells than do the untreated control animals, then the chemical is considered to be capable of inducing structural and/or numerical chromosomal damage.

The NTP is interested in determining a chemical's ability to induce chromosomal damage because it has been shown that most, if not all, cancers are characterized by chromosomal changes that are frequently specific to a particular tumor-type. Also, induction of aneuploidy (an abnormal chromosome count) or chromosomal rearrangements in germ cells (eggs and sperm) is a cause of birth defects, fetal deaths, and infertility in animals; therefore, a chemical that can produce chromosomal damage in somatic cells, perhaps presenting a risk of carcinogenicity, may also carry a risk of producing damage in germ cells, resulting in adverse reproductive outcomes.

The micronucleus test is performed in a variety of ways, depending upon the questions the investigator is attempting to answer, the test organism, the cell type that is assayed, and the mode of action of the chemical. The NTP conducts micronucleus tests in rats or mice, and the tissues most often assessed for frequency of micronuclei are bone marrow and peripheral blood. Erythrocytes (red blood cells) are the cells that are scored in the bone marrow or the blood for presence of micronuclei. Micronucleus tests must be performed on cells that are dividing. Erythrocytes arise from "stem cells" in the bone marrow and are produced by a series of divisions in a precursor cell population. The constant, rapid turnover of precursor cells makes erythrocytes an ideal cell type for a micronucleus test. Another unique feature of the erythrocyte is that immediately after formation of the fully differentiated erythrocyte, the nucleus is pushed out of the cell: erythrocytes are the only mammalian cell type that does not contain a nucleus, and therefore, the differentiated erythrocyte cannot further divide. Thus the bone marrow stem cells are continuously producing new erythrocytes to replace the ones that eventually die. If a stem cell is damaged by a chemical and a micronucleus is formed as a consequence of this damage, the micronucleus remains in the cell after the main nucleus has been pushed out and is very easy to observe microscopically.

Rat and mouse bone marrow micronucleus tests typically employ 1 to 3 treatments of the chemical under study; treatments are administered at 24 hr. intervals, and there are normally 5 male animals per treatment group. Doses extend up to the maximum tolerated dose. The route of administration in these short-term tests is usually either intraperitoneal injection or oral gavage. Based on the cell cycle and maturation times of the erythrocytes, harvesting of the bone marrow usually occurs 24 hours after the final dosing. At that time, about 50% of the erythrocytes in the bone marrow are immature, newly formed erythrocytes, and these are the cell types that are checked for presence of micronuclei. The animals are killed by CO2 inhalation and the femurs are removed. The bone marrow is flushed from the femurs and spread onto slides. The slides are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present. 2000 polychromatic erythrocytes (PCEs, or immature erythrocytes) are scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as a measure of toxicity. If the production of erythrocytes in the bone marrow is reduced due to chemical toxicity, then the %PCE in the bone marrow will drop from the typical 50-60%. If erythrocyte production is stimulated, then a higher percentage of immature erythrocytes may be observed. These figures tell the investigator if the chemical is toxic to the bone marrow.

The NTP also routinely conducts peripheral blood micronucleus tests on mice that are treated in the 13-week toxicity studies as part of the bioassay program. At the end of the 13-week exposure period (routes of exposure: inhalation, dosed-feed, drinking water, oral gavage, skin painting, intraperitoneal injection), a blood sample is obtained from male and female mice in each dose group (usually 10 animals per treatment group per sex) and slides are prepared, fixed and stained as for the bone marrow studies. At least 1,000, and sometimes up to 10,000 mature erythrocytes (normochromatic erythrocytes or NCEs) are scored per animal for presence of micronuclei. These mature erythrocytes represent about 95% or more of the circulating erythrocytes. The percent PCE is determined in the blood as a measure of chemical-induced toxicity to the bone marrow. All data are analyzed separately for male and female mice.

The acridine orange staining procedure that is used for micronucleus slides allows the scorer to differentiate between the recently formed, immature erythrocytes (polychromatic or PCE) that are less than 48 hr old, and mature erythrocytes 2-35 days old (normochromatic or NCE) based on their staining characteristics. PCE contain residual RNA and thus they stain differently than the NCE that no longer have residual RNA. MN in PCE's arise from damage that occurred recently (within the past 48 hr) and the NCE population shows the result of damage accumulated over the past month, with the NCE population being in steady state equilibrium in the peripheral blood (newly damaged or undamaged erythryocytes are moving from bone marrow to blood at the same rate as old erythrocytes -- the NCEs-- are being removed from the blood). Thus, for longer-term peripheral blood MN studies, it is usually more appropriate to score MN in the NCE population. For acute studies, particularly those in which bone marrow tissue is analyzed, PCEs are scored.

A formal statistical analysis of the data is performed, including a trend test, to determine if there is an overall increase across all doses in the frequency of cells containing micronuclei, and a pairwise comparison of each dose group to the corresponding control, to see if any one dose group is statistically different from the control group in frequency of micronucleated cells. Data are typically presented as the mean number of micronucleated cells per 1,000 cells for each treatment group. A positive trend test is one in which the P value is equal to or less than 0.025. For the micronucleus frequency in any dose group to be considered significantly elevated over the control group, the P value must be equal to or less than 0.025 divided by the number of chemical-treatment groups. Thus, if the number of treated groups is 3, then the required pairwise P value is 0.008. In the short-term studies, tests that give positive results are repeated to confirm the response. The 13-week micronucleus tests are not repeated, since they are included as part of the NTP subchronic toxicity tests which are not repeated.

Factors which must be considered in analyzing micronucleus test data include number of animals per dose group (a minimum of 3 is required), dose levels and number of doses administered, route of administration, tissue and cell type analyzed, sample time (interval between last dosing and harvesting of cells for analysis), frequencies of micronucleated cells in the negative and positive controls, and the results of the statistical analyses.

The micronucleus test provides information that is complementary to the gene mutation information that is obtained from a Salmonella assay. However, there does not seem to be an increased ability to define the carcinogenicity of a chemical by conducting a micronucleus test in addition to a Salmonella assay. Despite the lack of increased predictive value for carcinogenicity, the micronucleus test provides valuable information about the chemical's ability to disrupt mammalian chromosome structure and function. Most human carcinogens are positive in mammalian micronucleus tests.