Stefan Pfuhler

There is growing interest in in vitro‐based new approach methodologies (NAMs) for assessing genotoxicity. These approaches are desirable for industries like cosmetics which are prohibited from animal testing due to regulatory restrictions, as well as “3R” considerations to avoid in vivo testing. One NAM is the adaptation of the in vitro micronucleus assay for metabolically competent human hepatic HepaRG cells. However, per OECD Test Guideline 487, the use of non‐validated cell types such as these needs to be justified based on their proficiency in the assay. To this end, we have tested 28 chemicals in HepaRG and TK6 human lymphoblast cells, a standard cell line considered validated for use in the in vitro micronucleus assay. HepaRG cells showed a high overall accuracy of 86%, compared to 75% for TK6 cells across a balanced set of expected in vivo positives and negatives across various genotoxic modes of action. The improved overall performance of HepaRG cells was predominantly based on their lower susceptibility towards compounds known to cause “false positive” responses (positive results in vitro, but are known to be negative in vivo), particularly those related to oxidative stress as demonstrated by the ToxTracker assay. The study indicates a high predictive capacity of HepaRG cells, suggesting that with further examination of chemicals requiring metabolic activation, they may be suitable for regulatory testing. These findings advocate for the incorporation of HepaRG cells into genotoxicity assessment frameworks, providing a more human‐relevant approach that has the potential to improve risk assessment outcomes.

This interlaboratory evaluation of HepaRG CometChip was conducted to assess transferability and reproducibility of this new approach methodology (NAM) across four laboratories. Concentrations inducing up to ~70% relative cytotoxicity were determined by the organizing laboratory, and frozen chemical formulation blocks were sent to each participant. When noncytotoxic, 10 mM was the maximum dose. Cultures were exposed once daily for three consecutive days, and both cytotoxicity assessment, via ATP quantification, and comet analysis, commenced 3–4 h after initiation of the final exposure. Positive response was statistical pairwise significance ( p < 0.05) with concentration‐related increases in %Tail DNA across ≥ 2 consecutive exposures. For 8 of 11 compounds, all four labs generated unanimous test results, with four negative compounds (2‐acetylaminofluorene [2‐AAF], 2,4‐dichlorophenol, eugenol and hydroquinone) and four positive compounds (azidothymidine, benzo(a)pyrene [BP], cyclophosphamide [CP], ethyl methanesulfonate).For the remaining chemicals, three of four labs generated negative calls for amitrole, cadmium chloride, and DMBA. In cases where bulky lesions were anticipated, the magnitude of %Tail DNA was low, due to the inherent insensitivity of the alkaline comet assay (not the CometChip per se) to detect bulky adducts repaired by nucleotide excision repair. This is supported by the small magnitude in %Tail DNA induced by BP and CP. Taken together, for all compounds there was majority agreement in CometChip results across participating laboratories supporting that the endpoint is readily transferable to new labs. Overall, this platform is a promising human‐relevant NAM, with a physiologically relevant detoxification process that could be incorporated into rodent replacement strategies.

In human health risk assessment of chemicals and pharmaceuticals, identification of genotoxicity hazard usually starts with a standard battery of in vitro genotoxicity tests, which is needed to cover all genotoxicity endpoints. The individual tests included in the battery are not designed to pick up all endpoints. This explains why resulting data can appear contradictory, thereby complicating accurate interpretation of the findings. Such interpretation could be improved through application of mathematical modeling. One of the advantages of mathematical modeling is that the strengths and weaknesses of each test are taken into account. Furthermore, the generated predictions are objective and convey the associated uncertainties. This approach was explored by the working group “Predictivity of In Vitro Genotoxicity Testing,” convened in the context of the 8th International Workshop on Genotoxicity Testing (IWGT). Specifically, we applied mathematical modeling to a database with publicly available in vitro and in vivo data for genotoxicity. The results indicate that a mammalian in vitro clastogenicity test and a mammalian cell gene mutation test together provide strong predictive weight‐of‐evidence for evaluating genotoxic hazard of a substance, although they are better in predicting absence of genotoxic potential than in predicting presence of genotoxic potential. Remarkably, the bacterial reverse mutation (Ames) test did not significantly change these predictions when used in combination with in vitro mutagenicity and clastogenicity tests using cells of mammalian origin. However, in case only data from a bacterial reverse mutation test are available for the assessment of genotoxic potential, these do bear weight of evidence and thus can be used. Genotoxicity assays are generally executed in tiers, in which the bacterial reverse mutation test often is the starting point. Thus, it is reasonable to suspect that early in development test results from the bacterial reverse mutation test have influenced the composition of the database studied here. We performed several tests on the robustness of the database used for the analyses presented here, and the forthcoming results do not indicate a strong bias. Further research comparing in vitro genotoxicity data with in vivo data for additional compounds will provide more insights whether it is indeed time to reconsider the composition of the standard in vitro genotoxicity battery.

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose–response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An “effect severity” AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health‐based guidance value (HBGV) when a “severe” toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose–response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub‐group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease‐causing mutations by error‐corrected next generation sequencing clarifies selection of ESAF values.

Quantitative risk assessments of chemicals are routinely performed using in vivo data from 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.

ToxTracker is a mammalian cell reporter assay that predicts the genotoxic properties of compounds with high accuracy. By evaluating induction of various reporter genes that play a key role in relevant cellular pathways, it provides insight into chemical mode‐of‐action (MoA), thereby supporting discrimination of direct‐acting genotoxicants and cytotoxic chemicals. A comprehensive interlaboratory validation trial was conducted, in which the principles outlined in OECD Guidance Document 34 were followed, with the primary objectives of establishing transferability and reproducibility of the assay and confirming the ability of ToxTracker to correctly classify genotoxic and non‐genotoxic compounds. Reproducibility of the assay to predict genotoxic MoA was confirmed across participating laboratories and data were evaluated in terms of concordance with in vivo genotoxicity outcomes. Seven laboratories tested a total of 64 genotoxic and non‐genotoxic chemicals that together cover a broad chemical space. The within‐laboratory reproducibility (WLR) was up to 98% (73%–98% across participants) and the overall between‐laboratory reproducibility (BLR) was 83%. This trial confirmed the accuracy of ToxTracker to predict in vivo genotoxicants with a sensitivity of 84.4% and a specificity of 91.2%. We concluded that ToxTracker is a robust in vitro assay for the accurate prediction of in vivo genotoxicity. Considering ToxTracker's robust standalone accuracy and that it can provide important information on the MoA of chemicals, it is seen as a valuable addition to the regulatory in vitro genotoxicity battery that may even have the potential to replace certain currently used in vitro battery assays.

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.

To determine the utility of the ToxTracker assay in animal alternative testing strategies, the genotoxic potential of four fragrance materials (2‐octen‐4‐one, lauric aldehyde, veratraldehyde, and p‐methoxy cinnamaldehyde) were tested in the ToxTracker assay. These materials have been previously evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per OECD guidelines. In addition to these studies, reconstructed human skin micronucleus studies were conducted on all four materials. All four materials were positive in an in vitro micronucleus assay but were negative in both in vivo and 3D skin micronucleus assays. The ToxTracker assay, in combination with in silico methods to predict metabolism was used to identify mechanisms for the misleading positive outcomes observed in the in vitro micronucleus assays. The results show that the ToxTracker assay, in conjunction with in silico predictions, can provide the information needed to aid in the identification of an appropriate animal alternative follow‐up assay, for substances with positive results in the standard in vitro test battery. Thus, the ToxTracker assay is a valuable tool to identify the genotoxic potential of fragrance materials and can aid with replacing animal‐based follow‐up testing with appropriate animal alternative assay(s).

Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, and aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points‐of‐departures (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 defined 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 considered here (20/31), the PODs derived from in vitro data and IVIVE are health protective relative to in vivo PODs from animal studies. PODs were also protective by assay target: 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.