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)

Abstract

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.

Interlaboratory validation of the ToxTracker assay: An in vitro reporter assay for mechanistic genotoxicity assessment

“…The secondary objective of this interlaboratory validation was to investigate whether the additional biological information provided by ToxTracker provides insight into the MoA of compounds that have either conflicting in vitro genotoxicity test results or discordant test results when moving from in vitro to in vivo genotoxicity tests. Insight into MoA is critical to not only understand DNA reactivity, but also to explain the differences between test results empirically and improve overall genotoxicity predictions (Allemang et al, 2021; Brandsma et al, 2020).…”

“…The MoA is critical to not only understand DNA reactivity, but also to explain the differences between test results empirically and improve overall genotoxicity predictions (Allemang et al, 2021;Brandsma et al, 2020).…”

Quantitative in vitro to in vivo extrapolation of genotoxicity data provides protective estimates of in vivo dose

“…For these 31 chemicals, an AED (in vitro POD based on BMC 100 and IVIVE) could be compared against an in vivo POD (i.e., POD based on in vivo BMD 100 or LOGEL). There were published MicroFlow data for 12 chemicals (Allemang et al, 2021), published MultiFlow data for 13 chemicals with raw data provided by Litron Laboratories (Dertinger et al, 2019; Bryce et al, 2017), published or PrediTox‐supplied PrediScreen data for 22 chemicals (Khoury et al, 2013; 2016a; 2016b; Kopp et al, 2018), published TGR data for 18 chemicals identified by White, Luijten, et al (2019), and published or Toxys‐supplied ToxTracker data for 21 chemicals (Allemang et al, 2021; Boisvert, 2020).…”

Use of transcriptomics in hazard identification and next generation risk assessment: A case study with clothianidin

“…Thus, our results provide further evidence that toxicogenomics may be used combination with other methods (cp. ( Allemang et al, 2021 )) to classify potentially genotoxic compounds.…”