Mutation Research 497 (2001) 199–212 Influence of cytotoxicity and compound precipitation on test results in the alkaline comet assay Andreas Hartmann ∗ , Evangelos Kiskinis, Ann Fjällman, Willi Suter Novartis Pharma AG, Genetic and Experimental Toxicology, WSH2881.5.14, CH-4002 Basel, Switzerland Received 10 April 2001; received in revised form 19 June 2001; accepted 26 June 2001 Abstract We use the comet assay as part of our genotoxicity screening battery for newly synthesized drug candidates. A dataset of more than 250 tests carried out with 75 drug candidates of various chemical classes was analyzed to elucidate the influence of cytotoxicity and compound precipitation on DNA migration in the comet assay. Using a V79 Chinese hamster cell line, 38 of the compounds were negative and 37 were positive in the comet assay. The reproducibility of test results between repeat experiments was 85%. Data on 72 tests with a negative call in which the compounds were tested up to highly cytotoxic concentrations demonstrated that cytotoxicity, as determined by Trypan blue dye exclusion and occurrence of cells with completely fragmented chromatin, did not lead to false positive test results. The majority (64.2%) of compounds with a positive call induced elevated DNA migration in the absence of excessive cytotoxicity. Compound precipitation was observed in 84 tests. In 88.1% of these cases, the test result at the precipitating concentration 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 indicate that using V79 cells, the comet assay specifically detects genotoxic effects and is not confounded by cytotoxicity or compound precipitation under the conditions used. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Alkaline comet assay; Cytotoxicity; Compound precipitation 1. Introduction The alkaline comet assay was first introduced by Singh et al. [1] is increasingly used in genotoxicity testing and has various applications [2]. Sensitivity and specificity of the test are considered to be very high and guidelines for the conduct of the comet assay have recently been published [3]. However, it has yet not well been established whether cytotoxicity influences DNA migration in ∗ Corresponding author. Tel.: +41-61-32-41951; fax: +41-61-32-41274. E-mail address: andreas.hartmann@pharma.novartis.com (A. Hartmann). the comet assay. It is known that strand break as- says, such as alkaline elution, alkaline unwinding or sucrose-gradients have the potential problem to distinguish between strand breaks induced by geno- toxicity or excessive cytotoxicity [4]. Similarly, an important issue in genotoxicity testing with the comet assay is the possible influence of cytotoxicity on test results. Dead or dying cells can undergo rapid DNA fragmentation which should be expected to increase DNA migration in the comet assay. It is, therefore, mandatory to perform concurrent viability tests to control for excess cytotoxicity [3]. For the alkaline unwinding technique (which measures a similar end point) it was recommended not to test concentra- tions of compounds decreasing viability to less that 1383-5718/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S1383-5718(01)00256-X