Mouse Lymphoma Assay
Chromosome Aberration Test
Rat Liver UDS Test
ICH and FDA recommendations
Chromosome Aberration TestTreatment of cells with DNA-damaging agents can result in unrepairable lesions in both strands of DNA. This leads to chromosome breakage, a phenomena that can be visually detected with the help of a microscope in metaphase cells. As with the mammalian cell mutation tests, various cultures cell lines (such as CHL and CHO) have been used to perform this assay. However, cultured cells lines tend to lose and gain chromosomes spoontaneously, showing a high and unpredictable rate of chromosome aberrations. Primary cultures of human lymphocytes are therefore recommended, since their chromosomes are far more stable.
Primary lymphocyte cultures are prepared by dilution of human blood with culture medium. Lymphocytes are stilmulated to divide by the addition of a plant lectin (PHA), sometimes referred to as a mitogen. Two sets of cultures are grown for 48 hours before adition of the test substance. S9 mix is added to one set of cultures. Cells are washed 4 hours later to minimize toxic effects of the S9. All cultures are then incubated for a further 17 hours (or 1.5 cell cycles). Two hours prior to harvesting, the cultures are treated with colcemid to arrest cells in metaphase when chromosome structure is best seen.
The cells are separated by centrifugation, then resuspended in dilute (hypotonic) potassium chloride solution. This causes the cells to swell and enhances eventual separation of the chromosomes to facilitate visual analysis. The cells are fixed and washed in a mixture of methanol and acetic acid, then dropped onto glass microscope slides. Humidity is an important factor for spreading the chromosomes properly, and the methanol and acetic acid are best evaporated in a humid environment. A second test is performed using similar methodology, except that the cells are treated in the absence of S9 only for a continuous period of 21 hours. Slides are stained with Giemsa, then mounted iwth coverslips and examined by light microscopy by trained personel.
Slides from duplicate cultures of at least three dose leels of the test substance are analyzed with 100 cells being analyzed per culture. The highest does analyzed being that producing at least a 50% reduction in the mitotic index, a level that produces precipitation in the culture medium or 5 mg/ml, whichever is least. The mitotic index is the percentage of the cell population in metaphase, although other measures of toxicity may be applied. Different types of chromosome aberrations are tabulated, but all results from chromosome breakage, and the important result is considered to be the percentage of cells showing aberrations. Gaps are listed separately from other types of damage, since it is not clear whether these represent real chromosome breakage.