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Genetic Toxicology: Sister Chromatid Exchange Assay in CHO Cells

Sister Chromatid Exchange Assay in CHO Cells (OECD – 479)

The objective of this assay is to evaluate the test article and its metabolites for their potential to cause sister chromatid exchanges (SCE) in cultured mammalian cells. Bromo-deoxy-uridine (BrdU ) is added to cultures to generate a differential staining of both sister chromatids. Chromatids in which only one strand of DNA incorporated BrdU show a normal dark Giemsa staining, whereas those with two substituted strands, stain less darkly. The exchange process presumably involves DNA breakage and reunion. If an exchange occurred, this can be seen as the dark part changes to the other arm: “harlequin chromosomes” and therefore the number of scored exchanges correlates to the number of DNA breakage and reunion events. The study design follows the current OECD Guideline for Testing of Chemicals – 479, Genetic Toxicology: In vitro Sister Chromatid Exchange Assay in Mammalian Cells (1986). The assay will be performed in accordance with Good Laboratory Practices.

A preliminary toxicity test may be performed to help design the concentrations. In the main experiment, 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 Cyclophosphamide (presence of S9) and Mitomycin C (absence of S9). Negative control cultures will be treated with vehicle.

CHO cells are exposed in vitro to the test chemical with and without rat S9 mix for metabolic activation and cultured for two rounds of replication in BrdU containing medium. After treatment with a spindle inhibitor, cells are harvested and chromosome preparations are made. Cells are analyzed in their second mitotic division after initiation of treatment. Usually, 25 well-spread metaphases per culture are analysed for SCEs, but the number may be reduced for obvious positive results. Only metaphases containing 21 +/- 2 centromeres will be analyzed. Slides are coded before analysis.

A positive response shows a concentration-related response and a statistically significant increase in the incidence of SCE per chromosome at one or more concentrations over that of the solvent controls.  Positive results indicate that the test article induces SCE in the cultured cells. A negative response presents no statistically significant increase in the percentage of cells with aberrations. Negative results indicate that, under the test conditions, the test article does not induce SCEs in the cultured cells.

The experiment will start after receipt of signed protocol and the test substance. A draft report will be ready for review within 8-10 weeks after the experimental start date. Documents and slides will be archived for six years.

For further information about these services, to receive a quotation, or to schedule a test, please contact NUCRO-TECHNICS.