In vitro genotoxicity has historically served a hazard identification role, with simple binary outcomes provided for each of several single endpoint assays. This will need to change, given: i) efforts to curtail animal testing, ii) the increased use of multiplexed in vitro assays and the ongoing development of NAMS, and iii) the desire to holistically consider quantitative results from multiple biomarkers/endpoints that take potency into consideration. To help facilitate more quantitative analyses of multiple biomarkers and/or assay streams, we explored the combined use of PROAST and Toxicological Prioritization Index (ToxPi) software. As a proof-of-concept, this investigation employed the MultiFlow® DNA damage assay, focusing on γH2AX and p53 biomarkers at two time points, whereby 10 genotoxicants were evaluated in the presence and absence of rat liver S9 metabolic activation. Whereas PROAST was used to calculate BMD point estimates and confidence intervals (CI), ToxPi synthesized the BMD results into visual, quantitative summaries conveying genotoxic potency and metabolic properties. Our analyses suggest that ToxPi’s data synthesis and visualization modules provide useful insights into compound potency, chemical grouping, and genotoxic mechanisms. By integrating multiple data sources, we find that ToxPi offers a powerful complementary approach to traditional BMD CI potency graphs, particularly for the simultaneous analysis of multiple biomarkers enhancing chemical potency analysis of complex datasets.

Quantitative risk assessments of chemicals are routinely performed in rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.

Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points-of-departure (PODs) for setting exposure limits. The more recent higher-throughput in vitro genotoxicity assays, many of which also provide mechanistic information, offer a powerful approach for determining high-precision PODs for potency ranking and risk assessment. In order to obtain relevant human dose context from the in vitro assays, in vitro to in vivo extrapolation (IVIVE) models are required to determine what dose would elicit a concentration in the body demonstrated to be genotoxic using in vitro assays. Previous work has demonstrated that application of IVIVE models to in vitro bioactivity data can provide PODs that are protective of human health, but there has been no evaluation of how these models perform with in vitro genotoxicity data. Thus, the Genetic Toxicology Technical Committee, under the Health and Environmental Sciences Institute, conducted a case study on 31 reference chemicals to evaluate the performance of IVIVE application to genotoxicity data. The results demonstrate that for most chemicals (20/31), the PODs derived from in vitro data and IVIVE are highly health protective relative to in vivo PODs from animal studies. PODs were also protective by individual assay type: mutations (8/13 chemicals), micronuclei (9/12) and aneugenicity markers (4/4). It is envisioned that this novel testing strategy could enhance prioritization, rapid screening, and risk assessment of genotoxic chemicals.