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Genotoxicity testing in vitro - development of a higher throughput analysis method based on the comet assay

: Ritter, D.; Knebel, J.


Toxicology in vitro 23 (2009), Nr.8, S.1570-1575
ISSN: 0887-2333
International Congress on In Vitro Toxicology <15, 2008, Stockholm>
Zeitschriftenaufsatz, Konferenzbeitrag
Fraunhofer ITEM ()
genetic toxicology testing; high-throughput; comet assay; automation; regulatory toxicology; GLP compliance; comet slide; comet protocol; comet analysis; in vitro; in vivo; standardization; DNA damage

Higher throughput methods, high content analysis and automated screening methods are of highest demand in drug development today. In toxicology, these strategies are becoming increasingly important, as well. Therefore, an integrated higher throughput method for the comet assay is addressed by the development presented here.
The sensitivity, specificity and relevance of the comet assay as a method for determination of DNA damage in vivo and in vitro have been highlighted in many studies. Actually, efforts are made to include it in a panel of genotoxicity tests for regulatory purposes. However, the standard comet assay is a time consuming procedure due to the specific methods needed. The improvements presented here lead to a faster and easier slide-production, a smaller amount of cells needed, a higher amount of comets quantified, a fully automated analysis of comets including reanalysis, storing, visualisation and documentation possibilities using standard comet quantification models such as tail length or tail moment, and – by introduction of clearly definable selection criteria based on image analysis algorithms – clearly improve objectivity and standardization of the analysis procedure.
Results prove the high reproducibility, flexibility, efficiency and suitability of the procedure as a fully automated analysis method in higher throughput genotoxicity testing in vitro.