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A key step in the risk assessment process of a substance is the assessment of its genotoxic potential. Irrespective of the industry involved, current approaches rely on combinations of two or three in vitro tests and while highly sensitive, their specificity is thought to be limited. A refined in vitro genotoxicity testing strategy with improved predictive capacity would be beneficial and “3R” friendly as it helps to avoid unnecessary in vivo follow‐up testing. Here, we describe a proof of concept study evaluating a balanced set of compounds that have in vivo negative or positive outcomes, but variable in vitro data, to determine if we could differentiate between direct and indirect acting genotoxicants. Compounds were examined in TK6 cells using an approach in which the same sample was used to evaluate both early genomic markers (Affymetrix analysis 4 hr post treatment), and the genotoxic outcome (micronuclei [MN] after 24 hr). The resulting genomic data was then analyzed using the TGx‐DDI biomarker, Connectivity mapping and whole genome clustering. Chemicals were also tested in the ToxTracker assay, which uses six different biomarker genes. None of the methods correctly differentiated all direct from indirect acting genotoxicants when used alone, however, the ToxTracker assay, TGx‐DDI biomarker and whole genome approaches provided high predictive capacity when used in combination with the MN assay (1/18, 2/18, 1/18 missed calls). Ultimately, a “fit for purpose” combination will depend on the specific tools available to the end user, as well as considerations of the unique benefits of the individual assays.

environmental and molecular mutagenesis

A comparison of classical and 21st century genotoxicity tools: A proof of concept study of 18 chemicals comparing in vitro micronucleus,  ToxTracker  and genomics‐based methods ( TGx‐DDI , whole genome clustering and connectivity mapping)