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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The in vivo comet assay (single cell gel electrophoresis assay) in its alkaline version (pH >13) is being increasingly used in genotoxicity testing of substances such as industrial chemicals, biocides, agrochemicals, food additives and pharmaceuticals. Recommendations for an appropriate performance of the test using OECD guidelines for other in vivo genotoxicity tests have been published. In this review, we critically discuss the biological significance of comet assay effects in general and the status of the test in current strategies for genotoxicity testing. Examples for practical applications of the in vivo comet assay and potential consequences of positive and negative test results are given. The significance of comet assay results for hazard identification and risk assessment is discussed. In accordance with international guidelines for genotoxicity testing the in vivo comet assay is recommended for follow-up testing of positive in vitro findings. It is particularly useful as a tool for the evaluation of local genotoxicity, especially for organs/cell types which cannot easily be evaluated with other standard tests. A positive result in an appropriately performed in vivo comet assay indicates genotoxicity of the test compound in the tissue tested and gains particular significance when a mutagenic potential of the test compound has already been demonstrated in vitro. Such findings will have practical consequences in the risk assessment processes and further development of substances.

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