title: Validation of a high throughput flow cytometric in vitro micronucleus assay including assessment of metabolic activation in TK6 cells.
abstract: Genotoxicity is an unacceptable property for new drug candidates and we employ three screening assays during the drug discovery process to identify genotoxicity early and optimize chemical series. One of these methods is the flow cytometric in vitro micronucleus assay for which protocol optimizations have been described recently. Here, we report further validation of the assay in TK6 cells including assessment of metabolic activation. We first optimized assay conditions to allow for testing with and without metabolic activation in parallel in a 96-well plate format. Then, we tested a set of 48 compounds carefully selected to contain known in vivo genotoxins, nongenotoxins and drugs. Avoidance of irrelevant positives, a known issue with mammalian cell-based genotoxicity assays, is important to prevent early deselection of potentially promising compounds. Therefore, we enriched the validation set with compounds that were previously reported to produce irrelevant positive results in mammalian cell-based genotoxicity assays. The resulting dataset was used to set the relevant cut-off values for scoring a compound positive or negative, such that we obtained an optimal balance of high sensitivity (88%) and high specificity (87%). Finally, we tested an additional set of 16 drugs to further probe assay performance and 14 of them were classified correctly. To our knowledge, the present study is the most comprehensive validation of the in vitro flow cytometric micronucleus assay and the first to report parallel assessment with metabolic activation in reasonable throughput. The assay allows for rapidly screening novel compounds for genotoxicity and is therefore well-suited for use in early drug discovery projects. Environ.
authors.affiliation: Department of Exploratory Toxicology, H. Lundbeck A/S, Ottiliavej 9, DK-2500, Valby, Denmark.
authors.forename: Annemette V
authors.initials: AV
authors.lastname: Thougaard
authors.forename: Joan
authors.initials: J
authors.lastname: Christiansen
authors.forename: Tomas
authors.initials: T
authors.lastname: Mow
authors.forename: Jorrit J
authors.initials: JJ
authors.lastname: Hornberg
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D015162:Micronucleus Tests / Q000379:methods*; D051381:Rats
keywords: drug discovery; genotoxicity; micronucleus assay