The comet assay as an indicator test for germ cell genotoxicity.

The in vivo comet assay is a well-established genotoxicity test. It is currently mainly performed with somatic cells from different organs to detect a genotoxic activity of potential carcinogens. It is regarded as a useful test for follow-up testing of positive or equivocal in vitro test results and for the evaluation of local genotoxicity. However, the comet assay also has the potential to detect germ cell genotoxicity and may be used for demonstrating the ability of a substance or its metabolite(s) to directly interact with the genetic material of gonadal and/or germ cells. Such results are important for the classification of germ cell mutagens, e.g. in the context of the "Globally Harmonized System of Classification and Labelling of Chemicals" (GHS). This review summarizes and discusses available information on the use of the comet assay with germ cells and cells from the gonads in genetic toxicology. The literature contains results from in vitro studies, ex vivo studies and in vivo studies. With regard to the assessment of germ cell genotoxicity, only in vivo studies are relevant but the other kind of studies provided important information on various aspects of the methodology. Many comet assay studies with human sperm have been performed in the context of male infertility and assisted fertilization. The results of these studies are not reviewed in detail here but various aspects of the assay modifications used are discussed. Measuring DNA effects by the comet assay in sperm requires additional steps for chromatin decondensation. Many different modifications of the alkaline and the neutral comet assay are in use but a standard protocol has not been established yet. High and variable background levels of DNA effects were reported and there is still need for standardization and validation of the comet assay with sperm. Some human biomonitoring studies with human sperm were published, but it seems to be premature to use these data for hazard identification and classification of chemicals. In contrast, the standard alkaline in vivo comet assay can easily be adapted to investigations with cells from reproductive organs. Tests with cells from the gonads (testis and ovary) seem to be most appropriate and a promising tool for demonstrating that a test compound reaches the gonads and is able to interact with the genetic material of germ cells. However, studies to standardize and validate these methods are necessary before the comet assay can be usefully applied in risk assessment of germ cell mutagens.