Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing

At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidel ines for the use of the single-cell gel (SCG)/Comet assay in genetic toxico logy. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH, 13) version of the assay developed by Singh et al. [1988]. The pH > 1 3 version is capable of detecting DNA single-strand breaks (SSB), alkali-la bile sites (ALS), DNA-DNA/DNAprotein cross-linking, and SSB associated with incomplete excision repair sites. Relative to at her genotoxicity tests, t he advantages of the SCG assay include its demonstrated sensitivity for det ecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, ifs ease of application, and t he short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guide lines developed for preparing slides with agarose gels, lysing cells to lib erate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkal ine conditions, alkali neutralization, DNA staining, comet visualization, a nd data collection. Based on the current state of knowledge, the expert pan el developed guidelines for conducting in vitro or in vivo Comet assays. Th e goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submiss ion, The expert panel used the current Organization for Economic Go-operati on and Development (OECD) guidelines Far in vitro and in vivo genetic toxic ological studies as guides during the development of the corresponding in v itro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consi deration was given by the expert panel to the potential adverse effect of D NA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies th at might be useful in the interpretation of positive Comet data, The relate d methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS (2 ) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/nec rotic cells; and (4) the use of the acellular SCG assay to evaluate the abi lity of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work i n progress. Additional information is needed before the assay can be critic ally evaluated for its utility in genetic toxicology. The information neede d includes comprehensive data on the different sources of variability (e.g. , cell to cell, gel to gel, run to run, culture to culture, animal to anima l, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing us ing these guidelines, and the results of appropriately designed multilabora tory international validation studies. (C) 2000 Wiley-Liss, Inc.