A pre-validation trial - testing genotoxicity of several chemicals using standard, medium- and high-throughput comet formats.

As part of the EU-project COMICS, high throughput versions of the comet assay was developed. In order to test the performance of the new formats; the novel 12-gel slide and the 96-minigel film were compared with standard 3-gel slides for their ability to detect effects of genotoxic chemicals on cellular DNA with limited cytotoxicity. Among the chemicals were negative controls, a non-carcinogen that causes DNA damage through cytotoxicity, and carcinogens that are known to be hard to detect by simple DNA strand break analysis. Chemicals requiring metabolic activation were preincubated with rat liver S9 fraction. TK-6 human lymphoblastoid cells were treated with a range of concentrations of each chemical, above and below the expected cytotoxic concentration. Trials were carried out in 3 centres applying all three formats. Results obtained with the three systems (standard, medium- and high-throughput) were essentially the same. The 96-minigel format was analysed with the fully automated scoring system IMSTAR and comparable results were achieved with the semi-automated scoring system from Perceptives. The known genotoxic chemicals MNU, B(a)P, 4-NQO and cyclophosphamide showed little consistent sign of genotoxicity at concentrations causing limited cytotoxicity. D-mannitol and Triton X-100 were, as expected, non-genotoxic (though Triton X-100, at high concentrations, caused DNA breaks as an apparent secondary effect of cytotoxicity). Etoposide and bleomycin gave significant increase in DNA strand break at borderline cytotoxic concentrations. The limitation of the assay to detect damaged bases by known genotoxins may be overcome by incorporating a DNA repair enzyme, such as formamidopyrimidine-DNA-glycosylase (FPG), to convert damaged bases into breaks as shown by Azqueta A et al., Mutagenesis vol. 28 no. 3 pp. 271–277, 2013 .