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Validation of a high throughput flow cytometric in vitro micronucleus assay including assessment of metabolic activation in TK6 cells
Author(s) -
Thougaard Annemette V.,
Christiansen Joan,
Mow Tomas,
Hornberg Jorrit J.
Publication year - 2014
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.21891
Subject(s) - genotoxicity , micronucleus test , micronucleus , false positive paradox , in vitro toxicology , computational biology , in vitro , flow cytometry , biology , drug discovery , high throughput screening , chemistry , pharmacology , biochemistry , microbiology and biotechnology , toxicity , computer science , organic chemistry , machine learning
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. Mol. Mutagen. 55:704–718, 2014. © 2014 Wiley Periodicals, Inc.