Duplex Sequencing (DS) is an ultra-accurate, error-corrected next generation sequencing (ecNGS) technology for mutation analysis. A working group (WG) within Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee is investigating the suitability of ecNGS for regulatory mutagenicity testing, using DS as a model. Initial steps to promote acceptance require demonstrating technical reproducibility across DS-experienced and inexperienced laboratories and establishing the method's sensitivity relative to conventional tests. Thus, the WG conducted a 'reconstruction experiment' to evaluate the transferability, reproducibility, and sensitivity of DS. TwinStrand Biosciences first applied DS to establish mutation frequency (MF) in DNA samples extracted from the livers of an untreated Sprague Dawley rat, or rats treated with either 100 mg/kg/day benzo[a]pyrene (B[a]P) for ten days or 40 mg/kg/day N-ethyl-N-nitrosourea (ENU) for three days. Using the measured MF in these original samples, mixtures were then constructed using the B[a]P- and ENU-treated samples to create "MF standards" with target MFs 1.2-, 1.5-, and 2-fold greater than the untreated control. Aliquots of these standards were distributed to seven laboratories in North America and Europe. DS libraries were prepared by each laboratory and TwinStrand. All eight laboratories met library preparation and assay performance metrics to yield high quality sequencing data with MF in the expected 'MF standard' range. The measured MF and mutation spectra were nearly identical across the laboratories and a 2-fold increase in MF could readily be identified in all labs relative to the untreated controls. The results confirm the high reproducibility and sensitivity of DS for mutagenicity assessment.

Agrochemical active ingredients are among the most toxicologically evaluated chemical substances, and genetically modified (GM) crops must be evaluated for safety and nutritional adequacy. Traditionally, these evaluations are conducted in vivo. There are concerted efforts in the agrochemical sector to reduce animal testing, but there is also an emphasis on updating test guidelines and fulfilling new data package requirements, which can both result in increased animal testing. The purpose of this project was to generate benchmarks for the numbers of vertebrate animals used in: a) evaluating agrochemical pesticidal active ingredients for human health hazards; and b) assessing GM crops for safety and nutritional adequacy, based on guideline studies for data package requirements. To achieve this, guideline studies employing vertebrates, as required by regulatory bodies for developing global data packages for new active ingredients and for GM crops, were listed. These listed guideline studies were reviewed, in terms of the study details and the required animal-use, which was determined based on best testing practices. For historical animal-use benchmarking, Corteva's six most recent agrochemical pesticidal active ingredients and four most recent GM crop events were evaluated. Across the six most recently developed active ingredients, an average of approximately 10,000 mammals were used for the testing of each (range: 5500-19,000); across the four most recently developed GM crops, the average number of vertebrates similarly used for each was approximately 1200 (range: 1000-1500). Though regulatory testing requirements are likely to change with time, as new technologies become available, this project has established a theoretical minimum requirement to help drive aspirational animal reduction goals, identified regulatory challenges associated with the reduction of animal-use, and helped to refine Corteva's vertebrate animal-use tracking approaches.

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.

The two-test in vitro battery for genotoxicity testing (Ames and micronucleus) has in the majority of cases replaced the three-test battery (as two-test plus mammalian cell gene mutation assay) for the routine testing of chemicals, pharmaceuticals, cosmetics, and agrochemical metabolites originating from food and feed as well as from water treatment. The guidance for testing agrochemical groundwater metabolites, however, still relies on the three-test battery. Data collated in this study from 18 plant protection and related materials highlights the disparity between the often negative Ames and in vitro chromosome aberration data and frequently positive in vitro mammalian cell gene mutation assays. Sixteen of the 18 collated materials with complete datasets were Ames negative, and overall had negative outcomes in in vitro chromosome damage tests (weight of evidence from multiple tests). Mammalian cell gene mutation assays (HPRT and/or mouse lymphoma assay (MLA)) were positive in at least one test for every material with this data. Where both MLA and HPRT tests were performed on the same material, the HPRT seemed to give fewer positive responses. In vivo follow-up tests included combinations of comet assays, unscheduled DNA synthesis, and transgenic rodent gene mutation assays, all gave negative outcomes. The inclusion of mammalian cell gene mutation assays in a three-test battery for groundwater metabolites is therefore not justified and leads to unnecessary in vivo follow-up testing.

Several New Approach Methodologies (NAMs) for hazard assessment of skin sensitisers have been formally validated. However, data regarding their applicability on certain product classes are limited. The purpose of this project was to provide initial evidence on the applicability domain of GARD™skin and GARD™potency for the product class of agrochemical formulations.For this proof of concept, 30 liquid and 12 solid agrochemical formulations were tested in GARDskin for hazard predictions. Formulations predicted as sensitisers were further evaluated in the GARDpotency assay to determine GHS skin sensitisation category. The selected formulations were of product types, efficacy groups and sensitisation hazard classes representative of the industry's products.The performance of GARDskin was estimated by comparing results to existing in vivo animal data. The overall accuracy, sensitivity, and specificity were 76.2% (32/42), 85.0% (17/20), and 68.2% (15/22), respectively, with the predictivity for liquid formulations being slightly higher compared to the solid formulations. GARDpotency correctly subcategorized 14 out of the 17 correctly predicted sensitisers. Lack of concordance was justifiable by compositional or borderline response analysis.In conclusion, GARDskin and GARDpotency showed satisfactory performance in this initial proof-of-concept study, which supports consideration of agrochemical formulations being within the applicability domain of the test methods.

The in vitro H295R steroidogenesis assay (OECD TG 456) is used to determine a chemical's potential to interfere with steroid hormone synthesis/metabolism. As positive outcomes in this assay can trigger significant higher tiered testing, we compiled a stakeholder database of reference and test item H295R data to characterize assay outcomes. Information concerning whether a Level 5 reproductive toxicity study was triggered due to a positive outcome in the H295R assay was also included. Quality control acceptance criteria were not always achieved, suggesting this assay is challenging to conduct within the guideline specifications. Analysis of test item data demonstrated that pairwise significance testing to controls allowed for overly sensitive statistically significant positive outcomes, which likely contribute to the assay's high positive hit rate. Complementary interpretation criteria (e.g., 1.5-fold change threshold) markedly reduced the rate of equivocal and positive outcomes thus improving identification of robust positive effects in the assay. Finally, a case study (positive H295R outcome and no endocrine adversity in vivo) is presented, which suggests that stricter data interpretation criteria could refine necessary in vivo follow-up testing. Overall, the described additional criteria could improve H295R data interpretation and help inform on how to best leverage this assay for regulatory purposes.

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.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools. Additionally, early data suggest that ecNGS can measure clonal expansion of mutations as a mechanism-agnostic early marker of carcinogenic potential and can evaluate mutational load directly in human biomonitoring studies. In this review, we discuss promising applications, challenges, limitations, and key data initiatives needed to enable regulatory testing and adoption of ecNGS - including for advancing safety assessment, augmenting weight-of-evidence for mutagenicity and carcinogenicity mechanisms, identifying early biomarkers of cancer risk, and managing human health risk from chemical exposures.

Error-corrected next-generation sequencing (ecNGS) is an emerging technology with the potential to revolutionize the field of genetic toxicology. Here, we present recommendations from an expert working group convened to discuss potential applications, advantages and challenges associated with implementing ecNGS in nonclinical safety studies.

Skin sensitization testing is a regulatory requirement for safety evaluations of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is recommended as a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are accepted; however, the methods that comprise them have been evaluated using monoconstituent substances rather than mixtures or formulations. To address this data gap, we tested 27 agrochemical formulations in the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay, and the human cell line activation test (h-CLAT). These data were used as inputs to evaluate three DAs for hazard classification of skin sensitization potential and two DAs for potency categorization. When compared to historical animal results, balanced accuracy for the DAs for predicting in vivo skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) ranged from 56 to 78%. The best performing DA was the “2 out of 3 (2o3)” DA, in which the hazard classification was based on two concordant results from the DPRA, KeratinoSens, or h-CLAT. The KE 3/1 sequential testing strategy (STS), which uses h-CLAT and DPRA results, and the integrated testing strategy (ITSv2), which uses h-CLAT, DPRA, and an in silico hazard prediction from OECD QSAR Toolbox, had balanced accuracies of 56–57% for hazard classification. Of the individual test methods, KeratinoSens had the best performance for predicting in vivo hazard outcomes. Its balanced accuracy of 81% was similar to that of the 2o3 DA (78%). For predicting potency categories defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the correct classification rate of the STS was 52% and that of the ITSv2 was 43%. These results demonstrate that non-animal test methods have utility for evaluating the skin sensitization potential of agrochemical formulations as compared to animal reference data. While additional data generation is needed, testing strategies such as DAs anchored to human biology and mechanistic information provide a promising approach for agrochemical formulation testing.

Efforts are underway to develop and implement nonanimal approaches which can characterize acute systemic lethality. A workshop was held in October 2019 to discuss developments in the prediction of acute oral lethality for chemicals and mixtures, as well as progress and needs in the understanding and modeling of mechanisms of acute lethality. During the workshop, each speaker led the group through a series of charge questions to determine clear next steps to progress the aims of the workshop. Participants concluded that a variety of approaches will be needed and should be applied in a tiered fashion. Non-testing approaches, including waiving tests, computational models for single chemicals, and calculating the acute lethality of mixtures based on the LD50 values of mixture components, could be used for some assessments now, especially in the very toxic or non-toxic classification ranges. Agencies can develop policies indicating contexts under which mathematical approaches for mixtures assessment are acceptable; to expand applicability, poorly predicted mixtures should be examined to understand discrepancies and adapt the approach. Transparency and an understanding of the variability of in vivo approaches are crucial to facilitate regulatory application of new approaches. In a replacement strategy, mechanistically based in vitro or in silico models will be needed to support non-testing approaches especially for highly acutely toxic chemicals. The workshop discussed approaches that can be used in the immediate or near term for some applications and identified remaining actions needed to implement approaches to fully replace the use of animals for acute systemic toxicity testing.

We present a hypothetical case study to examine the use of a next-generation framework developed by the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute for assessing the potential risk of genetic damage from a pharmaceutical perspective. We used etoposide, a genotoxic carcinogen, as a representative pharmaceutical for the purposes of this case study. Using the framework as guidance, we formulated a hypothetical scenario for the use of etoposide to illustrate the application of the framework to pharmaceuticals. We collected available data on etoposide considered relevant for assessment of genetic toxicity risk. From the data collected, we conducted a quantitative analysis to estimate margins of exposure (MOEs) to characterize the risk of genetic damage that could be used for decision-making regarding the predefined hypothetical use. We found the framework useful for guiding the selection of appropriate tests and selecting relevant endpoints that reflected the potential for genetic damage in patients. The risk characterization, presented as MOEs, allows decision makers to discern how much benefit is critical to balance any adverse effect(s) that may be induced by the pharmaceutical. Interestingly, pharmaceutical development already incorporates several aspects of the framework per regulations and health authority expectations. Moreover, we observed that quality dose response data can be obtained with carefully planned but routinely conducted genetic toxicity testing. This case study demonstrates the utility of the next-generation framework to quantitatively model human risk based on genetic damage, as applicable to pharmaceuticals.

We present a hypothetical case study to examine the use of a next-generation framework developed by the Genetic Toxicology Technical Committee of the Health and Environmental Sciences Institute for assessing the potential risk of genetic damage from a pharmaceutical perspective. We used etoposide, a genotoxic carcinogen, as a representative pharmaceutical for the purposes of this case study. Using the framework as guidance, we formulated a hypothetical scenario for the use of etoposide to illustrate the application of the framework to pharmaceuticals. We collected available data on etoposide considered relevant for assessment of genetic toxicity risk. From the data collected, we conducted a quantitative analysis to estimate margins of exposure (MOEs) to characterize the risk of genetic damage that could be used for decision-making regarding the predefined hypothetical use. We found the framework useful for guiding the selection of appropriate tests and selecting relevant endpoints that reflected the potential for genetic damage in patients. The risk characterization, presented as MOEs, allows decision makers to discern how much benefit is critical to balance any adverse effect(s) that may be induced by the pharmaceutical. Interestingly, pharmaceutical development already incorporates several aspects of the framework per regulations and health authority expectations. Moreover, we observed that quality dose response data can be obtained with carefully planned but routinely conducted genetic toxicity testing. This case study demonstrates the utility of the next-generation framework to quantitatively model human risk based on genetic damage, as applicable to pharmaceuticals.

The regulatory community is transitioning to the use of nonanimal methods for dermal sensitization assessments; however, some in vitro assays have limitations in their domain of applicability depending on the properties of chemicals being tested. This study explored the utility of epidermal sensitization assay (EpiSensA) to evaluate the sensitization potential of complex and/or "difficult to test" chemicals. Assay performance was evaluated by testing a set of 20 test chemicals including 10 methacrylate esters, 5 silicone-based compounds, 3 crop protection formulations, and 2 surfactant mixtures; each had prior in vivo data plus some in silico and in vitro data. Using the weight of evidence (WoE) assessments by REACH Lead Registrants, 14 of these chemicals were sensitizers and, six were nonsensitizers based on in vivo studies (local lymph node assay [LLNA] and/or guinea pig studies). The EpiSensA correctly predicted 16/20 materials with three test materials as false positive and one silane as false negative. This silane, classified as weak sensitizer via LLNA, also gave a "false negative" result in the KeratinoSens™ assay. Overall, consistent with prior evaluations, the EpiSensA demonstrated an accuracy level of 80% relative to available in vivo WoE assessments. In addition, potency classification based on the concentration showing positive marker gene expression of EpiSensA was performed. The EpiSensA correctly predicted the potency for all seven sensitizing methacrylates classified as weak potency via LLNA (EC3 ≥ 10%). In summary, EpiSensA could identify dermal sensitization potential of these test substances and mixtures, and continues to show promise as an in vitro alternative method for dermal sensitization.

The need to develop new tools and increase capacity to test pharmaceuticals and other chemicals for potential adverse impacts on human health and the environment is an active area of development. Much of this activity was sparked by two reports from the US National Research Council (NRC) of the National Academies of Sciences, Toxicity Testing in the Twenty-first Century: A Vision and a Strategy (2007) and Science and Decisions: Advancing Risk Assessment (2009), both of which advocated for “science-informed decision-making” in the field of human health risk assessment. The response to these challenges for a “paradigm shift” toward using new approach methodologies (NAMS) for safety assessment has resulted in an explosion of initiatives by numerous organizations, but, for the most part, these have been carried out independently and are not coordinated in any meaningful way. To help remedy this situation, a framework that presents a consistent set of criteria, universal across initiatives, to evaluate a NAM's fit-for-purpose was developed by a multi-stakeholder group of industry, academic, and regulatory experts. The goal of this framework is to support greater consistency across existing and future initiatives by providing a structure to collect relevant information to build confidence that will accelerate, facilitate and encourage development of new NAMs that can ultimately be used within the appropriate regulatory contexts. In addition, this framework provides a systematic approach to evaluate the currently-available NAMs and determine their suitability for potential regulatory application. This 3-step evaluation framework along with the demonstrated application with case studies, will help build confidence in the scientific understanding of these methods and their value for chemical assessment and regulatory decision-making.

Cell line-based in vitro testing has been widely used as an important component of the genotoxicity testing battery; however, the use of cell lines is constrained by several limitations, including the genetic drift and variability. A study recently reported in the literature comprehensively examined genomic changes in a large number of cell lines and reported extensive genetic variations within the same cell lines across passage numbers and laboratories, even for single-cell derived subclones. The primary objective of this communication is to raise awareness and stimulate discussion within the genotoxicity testing community of the extent of genetic variability of cell lines in general and how these variables could potentially influence the results and reproducibility of genotoxicity testing. Meanwhile, some recommendations for good cell culture practices are highlighted to mitigate, at least to some extent, the concern about genetic variation. Environ. Mol. Mutagen. 60:559-562, 2019. © 2019 Wiley Periodicals, Inc.

We previously demonstrated that the Connectivity Map (CMap) (Lamb et al., 2006) concept can be successfully applied to a predictive toxicology paradigm to generate meaningful MoA-based connections between chemicals (De Abrew et al., 2016). Here we expand both the chemical and biological (cell lines) domain for the method and demonstrate two applications, both in the area of read across. In the first application we demonstrate CMap's utility as a tool for testing biological relevance of source chemicals (analogs) during a chemistry led read across exercise. In the second application we demonstrate how CMap can be used to identify functionally relevant source chemicals (analogs) for a structure of interest (SOI)/target chemical with minimal knowledge of chemical structure. Finally, we highlight four factors: promiscuity of chemical, dose, cell line and timepoint as having significant impact on the output. We discuss the biological relevance of these four factors and incorporate them into a work flow.