Influence of cytotoxicity and compound precipitation on test results in the alkaline comet assay

We use the comet assay as part of our genotoxicity screening battery for ne wly synthesized drug candidates. A dataset of more than 250 tests carried o ut with 75 drug candidates of various chemical classes was analyzed to eluc idate the influence of cytotoxicity and compound precipitation on DNA migra tion in the comet assay. Using a V79 Chinese hamster cell line, 38 of the c ompounds were negative and 37 were positive in the comet assay. The reprodu cibility of test results between repeat experiments was 85%. Data on 72 tes ts with a negative call in which the compounds were tested up to highly cyt otoxic concentrations demonstrated that cytotoxicity, as determined by Tryp an blue dye exclusion and occurrence of cells with completely fragmented ch romatin, did not lead to false positive test results. The majority (64.2%) of compounds with a positive call induced elevated DNA migration in the abs ence of excessive cytotoxicity. Compound precipitation was observed in 84 t ests. In 88.1% of these cases, the test result at the precipitating concent ration did not differ from that found at the highest soluble concentration. Half of the remaining 11.9% of contrary results (most of them weak effects ) were not reproducible in the respective repeat experiment, indicating no or only a negligible influence of precipitation on test results. The data i ndicate that using V79 cells, the comet assay specifically detects genotoxi c effects and is not confounded by cytotoxicity or compound precipitation u nder the conditions used. (C) 2001 Elsevier Science B.V. All rights reserve d.