Genetic Toxicology: In Vitro Micronucleus Test in Mammalian Cells
Micronucleus Test in Culture CHO Cells and Human Peripheral Blood Lymphocytes (OECD TG 487)
The in vitro micronucleus (MNvit) assay is a method that uses cultured human or rodent cells. It provides a comprehensive basis for investigating chromosome damaging potential in vitro because both aneugens and clastogens can be detected. The test will be performed with the actin polymerisation inhibitor cytochalasin B (Cyto B). The study design follows the current OECD Guideline for Testing of Chemicals – TG 487, In Vitro Mammalian Cell Micronucleus Test (MNvit) (2014). The assay can be performed in accordance with Good Laboratory Practices.
A preliminary toxicity test may be performed to help design the concentrations. In the main experiment (short treatment), three analyzable concentrations in duplicate flasks are designed. Cells are exposed to the test article with and without exogenous drug-metabolic enzymes from liver (S9 fraction). Positive control cultures will be treated with the clastogen Cyclophosphamide (presence of S9) and the aneugen Colchicine (absence of S9). Negative control cultures will be treated with vehicle. Cultures are treated for three hours, rinsed with buffered saline and incubated for a total of 18 hours (approximately 1.5 times a normal cell cycle). At an appropriate time, cells receive Cyto B to allow for the identification and selective analysis of micronucleus frequency in cells that have completed one mitosis (binucleated cells). Slides are prepared for analysis and coded. Cytokinesis-Block Proliferation Index is determined to assess cell proliferation using at least 500 cells per culture. At least 4000 bi-nucleated cells from each concentration (2000 cells from each of the duplicates) are examined.
A positive response shows a concentration-related increase or a reproducible increase in the number of cells containing micronuclei over that of the solvent controls. Positive results indicate that the test article induces micronuclei in the cultured cells. A negative response presents no statistically significant increase in the percentage of cells with micronuclei. Negative results indicate that, under the test conditions, the test article does not induce micronuclei in the cultured cells. In the case of a negative result, a confirmatory experiment is required. In the confirmatory assay, cells are exposed for 18 hours to the test article without exogenous drug-metabolic enzymes. A positive result will not usually need confirmation.
Experimentation will start after receipt of signed protocol and the test substance. A draft report will be ready for review within 8-12 weeks after the experimental start date.
For further information, to receive a quotation, or to schedule a test, please contact NUCRO-TECHNICS.