Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25–26, 1999, an expert panel met to develop guidelines for the use of the single‐cell gel (SCG)/Comet assay in genetic toxicology. 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 > 13 version is capable of detecting DNA single‐strand breaks (SSB), alkali‐labile sites (ALS), DNA‐DNA/DNA‐protein cross‐linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the 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 guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single‐stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co‐operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro 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 consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related 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/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the as...

The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.

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