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Bisphenol A (BPA), a widely used chemical in the production of plastics and epoxy resins, has garnered significant attention due to its association with adverse health effects, particularly its endocrine-disrupting properties. Regulatory measures aimed at reducing human exposure to BPA have led to a proliferation of alternative chemicals used in various consumer and industrial products. While these alternatives serve to reduce BPA exposure, concerns have arisen regarding their safety and potential toxicity as regrettable substitutes. Previous efforts have demonstrated that in vitro high-throughput transcriptomics (HTTr) studies can be used to assess the endocrine-disrupting potential of BPA alternatives, and this strategy produces transcriptomic points-of-departure (tPODs) that are protective of human health when compared to the PODs from traditional rodent studies. In this study, we used in vitro HTTr to assess the potential for toxicity of eleven data-poor legacy chemicals sharing structural similarities to BPA. Human breast cancer MCF-7 cells were exposed to BPA and 11 alternatives at concentrations ranging from 0.1 to 25 μM to assess toxicity. Analysis of global transcriptomic changes and a previously characterized estrogen receptor alpha (ERα) transcriptomic biomarker signature revealed that 9 of 11 chemicals altered gene expression relative to controls. One of the chemicals (2,4'-Bisphenol A) activated the ERα biomarker at the same concentration as BPA (i.e., 4,4'-BPA) but was deemed to be more potent as it induced global transcriptomic changes at lower concentrations. These results address data gaps in support of ongoing screening assessments to identify BPA alternatives with hazard potential and help to identify potential candidates that may serve as safer alternatives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

Computational toxicology models have been successfully implemented to prioritize and screen chemicals. There are numerous in silico (quantitative) structure-activity relationship ([Q]SAR) models for the prediction of a range of human-relevant toxicological endpoints, but for a given endpoint and chemical, not all predictions are identical due to differences in their training sets, algorithms, and methodology. This poses an issue for high-throughput screening of a large chemical inventory as it necessitates several models to cover diverse chemistries but will then generate data conflicts. To address this challenge, we developed a consensus modeling strategy to combine predictions obtained from different existing in silico (Q)SAR models into a single predictive value while also expanding chemical space coverage. This study developed consensus models for nine toxicological endpoints relating to estrogen receptor (ER) and androgen receptor (AR) interactions (i.e., binding, agonism, and antagonism) and genotoxicity (i.e., bacterial mutation, in vitro chromosomal aberration, and in vivo micronucleus). Consensus models were created by combining different (Q)SAR models using various weighting schemes. As a multi-objective optimization problem, there is no single best consensus model, and therefore, Pareto fronts were determined for each endpoint to identify the consensus models that optimize the multiple-criterion decisions simultaneously. Accordingly, this work presents sets of solutions for each endpoint that contain the optimal combination, regardless of the trade-off, with the results demonstrating that the consensus models improved both the predictive power and chemical space coverage. These solutions were further analyzed to find trends between the best consensus models and their components. Here, we demonstrate the development of a flexible and adaptable approach for in silico consensus modeling and its application across nine toxicological endpoints related to ER activity, AR activity, and genotoxicity. These consensus models are developed to be integrated into a larger multi-tier NAM-based framework to prioritize chemicals for further investigation and support the transition to a non-animal approach to risk assessment in Canada., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Collins, Mailloux, Kulkarni, Gagné, Long and Barton-Maclaren.)

The growing number of chemicals in the current consumer and industrial markets presents a major challenge for regulatory programs faced with the need to assess the potential risks they pose to human and ecological health. The increasing demand for hazard and risk assessment of chemicals currently exceeds the capacity to produce the toxicity data necessary for regulatory decision making, and the applied data is commonly generated using traditional approaches with animal models that have limited context in terms of human relevance. This scenario provides the opportunity to implement novel, more efficient strategies for risk assessment purposes. This study aims to increase confidence in the implementation of new approach methods in a risk assessment context by using a parallel analysis to identify data gaps in current experimental designs, reveal the limitations of common approaches deriving transcriptomic points of departure, and demonstrate the strengths in using high-throughput transcriptomics (HTTr) to derive practical endpoints. A uniform workflow was applied across six curated gene expression datasets from concentration-response studies containing 117 diverse chemicals, three cell types, and a range of exposure durations, to determine tPODs based on gene expression profiles. After benchmark concentration modeling, a range of approaches was used to determine consistent and reliable tPODs. High-throughput toxicokinetics were employed to translate in vitro tPODs (µM) to human-relevant administered equivalent doses (AEDs, mg/kg-bw/day). The tPODs from most chemicals had AEDs that were lower (i.e., more conservative) than apical PODs in the US EPA CompTox chemical dashboard, suggesting in vitro tPODs would be protective of potential effects on human health. An assessment of multiple data points for single chemicals revealed that longer exposure duration and varied cell culture systems (e.g., 3D vs. 2D) lead to a decreased tPOD value that indicated increased chemical potency. Seven chemicals were flagged as outliers when comparing the ratio of tPOD to traditional POD, thus indicating they require further assessment to better understand their hazard potential. Our findings build confidence in the use of tPODs but also reveal data gaps that must be addressed prior to their adoption to support risk assessment applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Reardon, Farmahin, Williams, Meier, Addicks, Yauk, Matteo, Atlas, Harrill, Everett, Shah, Judson, Ramaiahgari, Ferguson and Barton-Maclaren.)

Use of molecular data in human and ecological health risk assessments of industrial chemicals and agrochemicals has been anticipated by the scientific community for many years; however, these data are rarely used for risk assessment. Here, a logic framework is proposed to explore the feasibility and future development of transcriptomic methods to refine and replace the current apical endpoint-based regulatory toxicity testing paradigm. Four foundational principles are outlined and discussed that would need to be accepted by stakeholders prior to this transformative vision being realized. Well-supported by current knowledge, the first principle is that transcriptomics is a reliable tool for detecting alterations in gene expression that result from endogenous or exogenous influences on the test organism. The second principle states that alterations in gene expression are indicators of adverse or adaptive biological responses to stressors in an organism. Principle 3 is that transcriptomics can be employed to establish a benchmark dose-based point of departure (POD) from short-term, in vivo studies at a dose level below which a concerted molecular change (CMC) is not expected. Finally, Principle 4 states that the use of a transcriptomic POD (set at the CMC dose level) will support a human health-protective risk assessment. If all four principles are substantiated, this vision is expected to transform aspects of the industrial chemical and agrochemical risk assessment process that are focused on establishing safe exposure levels for mammals across numerous toxicological contexts resulting in a significant reduction in animal use while providing equal or greater protection of human health. Importantly, these principles and approaches are also generally applicable for ecological safety assessment., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology.)

An area of ongoing concern in toxicology and chemical risk assessment is endocrine disrupting chemicals (EDCs). However, thousands of legacy chemicals lack the toxicity testing required to assess their respective EDC potential, and this is where computational toxicology can play a crucial role. The US (United States) Environmental Protection Agency (EPA) has run two programs, the Collaborative Estrogen Receptor Activity Project (CERAPP) and the Collaborative Modeling Project for Receptor Activity (CoMPARA) which aim to predict estrogen and androgen activity, respectively. The US EPA solicited research groups from around the world to provide endocrine receptor activity Qualitative (or Quantitative) Structure Activity Relationship ([Q]SAR) models and then combined them to create consensus models for different toxicity endpoints. Random Forest (RF) models were developed to cover a broader range of substances with high predictive capabilities using large datasets from CERAPP and CoMPARA for estrogen and androgen activity, respectively. By utilizing simple descriptors from open-source software and large training datasets, RF models were created to expand the domain of applicability for predicting endocrine disrupting activity and help in the screening and prioritization of extensive chemical inventories. In addition, RFs were trained to conservatively predict the activity, meaning models are more likely to make false-positive predictions to minimize the number of False Negatives. This work presents twelve binary and multi-class RF models to predict binding, agonism, and antagonism for estrogen and androgen receptors. The RF models were found to have high predictive capabilities compared to other in silico modes, with some models reaching balanced accuracies of 93% while having coverage of 89%. These models are intended to be incorporated into evolving priority-setting workflows and integrated strategies to support the screening and selection of chemicals for further testing and assessment by identifying potential endocrine-disrupting substances., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Collins and Barton-Maclaren.)

New approach methodologies (NAMs) are increasingly being used for regulatory decision making by agencies worldwide because of their potential to reliably and efficiently produce information that is fit for purpose while reducing animal use. This article summarizes the ability to use NAMs for the assessment of human health effects of industrial chemicals and pesticides within the United States, Canada, and European Union regulatory frameworks. While all regulations include some flexibility to allow for the use of NAMs, the implementation of this flexibility varies across product type and regulatory scheme. This article provides an overview of various agencies' guidelines and strategic plans on the use of NAMs, and specific examples of the successful application of NAMs to meet regulatory requirements. It also summarizes intra- and inter-agency collaborations that strengthen scientific, regulatory, and public confidence in NAMs, thereby fostering their global use as reliable and relevant tools for toxicological evaluations. Ultimately, understanding the current regulatory landscape helps inform the scientific community on the steps needed to further advance timely uptake of approaches that best protect human health and the environment., Competing Interests: Author JH was employed by the company Corteva Agriscience. Authors CH and JM-H were employed by the company JT International SA. Authors EN and TS were employed by the law firm Bergeson & Campbell PC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stucki, Barton-Maclaren, Bhuller, Henriquez, Henry, Hirn, Miller-Holt, Nagy, Perron, Ratzlaff, Stedeford and Clippinger.)

Risk assessments are increasingly reliant on information from in vitro assays. The in vitro micronucleus test (MNvit) is a genotoxicity test that detects chromosomal abnormalities, including chromosome breakage (clastogenicity) and/or whole chromosome loss (aneugenicity). In this study, MNvit datasets for 292 chemicals, generated by the US EPA's ToxCast program, were evaluated using a decision tree-based pipeline for hazard identification. Chemicals were tested with 19 concentrations (n = 1) up to 200 µM, in the presence and absence of Aroclor 1254-induced rat liver S9. To identify clastogenic chemicals, %MN values at each concentration were compared to a distribution of batch-specific solvent controls; this was followed by cytotoxicity assessment and benchmark concentration (BMC) analyses. The approach classified 157 substances as positives, 25 as negatives, and 110 as inconclusive. Using the approach described in Bryce et al. (Environ Mol Mutagen 52:280-286, 2011), we identified 15 (5%) aneugens. IVIVE (in vitro to in vivo extrapolation) was employed to convert BMCs into administered equivalent doses (AEDs). Where possible, AEDs were compared to points of departure (PODs) for traditional genotoxicity endpoints; AEDs were generally lower than PODs based on in vivo endpoints. To facilitate interpretation of in vitro MN assay concentration-response data for risk assessment, exposure estimates were utilized to calculate bioactivity exposure ratio (BER) values. BERs for 50 clastogens and two aneugens had AEDs that approached exposure estimates (i.e., BER < 100); these chemicals might be considered priorities for additional testing. This work provides a framework for the use of high-throughput in vitro genotoxicity testing for priority setting and chemical risk assessment., (© 2022. Crown.)

The replacement of regulated brominated flame retardants and plasticizers with organophosphate esters (OPEs) has led to their pervasive presence in the environment and in biological matrices. Further, there is evidence that exposure to some of these chemicals is associated with reproductive toxicity. Using a high-content imaging approach, we assessed the effects of exposure to 9 OPEs on cells related to reproductive function: KGN human granulosa cells, MA-10 mouse Leydig cells, and C18-4 mouse spermatogonial cells. The effects of OPEs were compared with those of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a legacy brominated flame retardant. Alterations in several important cell features, including cell survival, mitochondrial dynamics, oxidative stress, lysosomes, and lipid droplets, were analyzed. Most of the OPEs tested displayed higher cytotoxicity than BDE-47 in all 3 cell lines. Effects on phenotypic parameters were specific for each cell type. Several OPEs increased total mitochondria, decreased lysosomes, increased the total area of lipid droplets, and induced oxidative stress in KGN cells; these endpoints were differentially affected in MA-10 and C18-4 cells. Alterations in cell phenotypes were highly correlated in the 2 steroidogenic cell lines for a few triaryl OPEs. Potency ranking using 2 complementary approaches, Toxicological Prioritization Index analyses and the lowest benchmark concentration/administered equivalent dose method, revealed that while most of the OPEs tested were more potent than BDE-47, others showed little to no effect. We propose that these approaches serve as lines of evidence in a screening strategy to identify the potential for reproductive and endocrine effects of emerging chemicals and assist in regulatory decision-making., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The developmental and reproductive toxicity associated with exposure to phthalates has motivated a search for alternatives. However, there is limited knowledge regarding the adverse effects of some of these chemicals. We used high-content imaging to compare the effects of mono (2-ethylhexyl) phthalate (MEHP) with six alternative plasticizers: di-2-ethylhexyl terephthalate (DEHTP); diisononyl-phthalate (DINP); di-isononylcyclohexane-1,2-dicarboxylate (DINCH); 2-ethylhexyl adipate (DEHA); 2,2,4-trimethyl 1,3-pentanediol diisobutyrate (TXIB) and di-iso-decyl-adipate (DIDA). A male germ spermatogonial cell line (C18-4), a Sertoli cell line (TM4) and two steroidogenic cell lines (MA-10 Leydig and KGN granulosa) were exposed for 48 h to each chemical (0.001-100 μM). Cell images were analyzed to assess cytotoxicity and effects on phenotypic endpoints. Only MEHP (100 μM) was cytotoxic and only in C18-4 cells. However, several plasticizers had distinct phenotypic effects in all four cell lines. DINP increased Calcein intensity in C18-4 cells, whereas DIDA induced oxidative stress. In TM4 cells, MEHP, and DINCH affected lipid droplet numbers, while DEHTP and DINCH increased oxidative stress. In MA-10 cells, MEHP increased lipid droplet areas and oxidative stress; DINP decreased the number of lysosomes, while DINP, DEHA, and DIDA altered mitochondrial activity. In KGN cells, MEHP, DINP and DINCH increased the number of lipid droplets, whereas DINP decreased the number of lysosomes, increased oxidative stress and affected mitochondria. The Toxicological Priority Index (ToxPi) provided a visual illustration of the cell line specificity of the effects on phenotypic parameters. The lowest administered equivalent doses were observed for MEHP. We propose that this approach may assist in screening alternative plasticizers., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Globally, regulatory authorities grapple with the challenge of assessing the hazards and risks to human and ecosystem health that may result from exposure to chemicals that disrupt the normal functioning of endocrine systems. Rapidly increasing number of chemicals in commerce, coupled with the reliance on traditional, costly animal experiments for hazard characterization - often with limited sensitivity to many important mechanisms of endocrine disruption -, presents ongoing challenges for chemical regulation. The consequence is a limited number of chemicals for which there is sufficient data to assess if there is endocrine toxicity and hence few chemicals with thorough hazard characterization. To address this challenge, regulatory assessment of endocrine disrupting chemicals (EDCs) is benefiting from a revolution in toxicology that focuses on New Approach Methodologies (NAMs) to more rapidly identify, prioritize, and assess the potential risks from exposure to chemicals using novel, more efficient, and more mechanistically driven methodologies and tools. Incorporated into Integrated Approaches to Testing and Assessment (IATA) and guided by conceptual frameworks such as Adverse Outcome Pathways (AOPs), emerging approaches focus initially on molecular interactions between the test chemical and potentially vulnerable biological systems instead of the need for animal toxicity data. These new toxicity testing methods can be complemented with in silico and computational toxicology approaches, including those that predict chemical kinetics. Coupled with exposure data, these will inform risk-based decision-making approaches. Canada is part of a global network collaborating on building confidence in the use of NAMs for regulatory assessment of EDCs. Herein, we review the current approaches to EDC regulation globally (mainly from the perspective of human health), and provide a perspective on how the advances for regulatory testing and assessment can be applied and discuss the promises and challenges faced in adopting these novel approaches to minimize risks due to EDC exposure in Canada, and our world., (Copyright © 2021. Published by Elsevier Inc.)

Modeling developmental toxicity has been a challenge for (Q)SAR model developers due to the complexity of the endpoint. Recently, some new in silico methods have been developed introducing the possibility to evaluate the integration of existing methods by taking advantage of various modeling perspectives. It is important that the model user is aware of the underlying basis of the different models in general, as well as the considerations and assumptions relative to the specific predictions that are obtained from these different models for the same chemical. The evaluation on the predictions needs to be done on a case-by-case basis, checking the analogues (possibly using structural, physicochemical, and toxicological information); for this purpose, the assessment of the applicability domain of the models provides further confidence in the model prediction. In this chapter, we present some examples illustrating an approach to combine human-based rules and statistical methods to support the prediction of developmental toxicity; we also discuss assumptions and uncertainties of the methodology., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Internationally, there are thousands of existing and newly introduced chemicals in commerce, highlighting the ongoing importance of innovative approaches to identify emerging chemicals of concern. For many chemicals, there is a paucity of hazard and exposure data. Thus, there is a crucial need for efficient and robust approaches to address data gaps and support risk-based prioritization. Several studies have demonstrated the utility of in vitro bioactivity data from the ToxCast program in deriving points of departure (PODs). ToxCast contains data for nearly 1,400 endpoints per chemical, and the bioactivity concentrations, indicative of potential adverse outcomes, can be converted to human-equivalent PODs using high-throughput toxicokinetics (HTTK) modeling. However, data gaps need to be addressed for broader application: the limited chemical space of HTTK and quantitative high-throughput screening data. Here we explore the applicability of in silico models to address these data needs. Specifically, we used ADMET predictor for HTTK predictions and a generalized read-across approach to predict ToxCast bioactivity potency. We applied these models to profile 5,801 chemicals on Canada’s Domestic Substances List (DSL). To evaluate the approach’s performance, bioactivity PODs were compared with in vivo results from the EPA Toxicity Values database for 1,042 DSL chemicals. Comparisons demonstrated that the bioac­tivity PODs, based on ToxCast data or read-across, were conservative for 95% of the chemicals. Comparing bioactivity PODs to human exposure estimates supports the identification of chemicals of potential interest for further work. The bioac­tivity workflow shows promise as a powerful screening tool to support effective triaging of chemical inventories.

Assessment of potential human health risks associated with environmental and other agents requires careful evaluation of all available and relevant evidence for the agent of interest, including both data-rich and data-poor agents. With the advent of new approach methodologies in toxicological risk assessment, guidance on integrating evidence from mul-tiple evidence streams is needed to ensure that all available data is given due consideration in both qualitative and quantitative risk assessment. The present report summarizes the discussions among academic, government, and private sector participants from North America and Europe in an international workshop convened to explore the development of an evidence-based risk assessment framework, taking into account all available evidence in an appropriate manner in order to arrive at the best possible characterization of potential human health risks and associated uncertainty. Although consensus among workshop participants was not a specific goal, there was general agreement on the key consider-ations involved in evidence-based risk assessment incorporating 21st century science into human health risk assessment. These considerations have been embodied into an overarching prototype framework for evidence integration that will be explored in more depth in a follow-up meeting.

In 2012, the Council of Canadian Academies published the expert panel on integrated testing of pesticide's report titled: Integrating emerging technologies into chemical safety assessment. This report was prepared for the Government of Canada in response to a request from the Minister of Health and on behalf of the Pest Management Regulatory Agency. It examined the scientific status of the use of integrated testing strategies for the regulatory health risk assessment of pesticides while noting the data-rich/poor dichotomy that exists when comparing pesticide formulations to most industrial chemicals. It also noted that the adoption of integrated approaches to testing and assessment (IATA) strategies may refine and streamline testing of chemicals, as well as improve results in the future. Moreover, the experts expected to see an increase in the use of integrated testing strategies over the next decade, resulting in improved evidence-based decision-making. Subsequent to this report, there has been great advancements in IATA strategies, which includes the incorporation of adverse outcome pathways (AOPs) and new approach methodologies (NAMs). This perspective provides the first Canadian regulatory update on how Health Canada is also advancing the incorporation of alternative, non-animal strategies, using a weight of evidence approach, for the evaluation of pest control products and industrial chemicals. It will include specific initiatives and describe how this work is leading to the creation of next generation risk assessments. It also reflects Health Canada's commitment towards implementing the 3Rs of animal testing: reduce, refine and replace the need for animal studies, whenever possible., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bhuller, Ramsingh, Beal, Kulkarni, Gagne and Barton-Maclaren.)

Next generation risk assessment (NGRA) is an exposure-led, hypothesis-driven approach that has the potential to support animal-free safety decision-making. However, significant effort is needed to develop and test the in vitro and in silico (computational) approaches that underpin NGRA to enable confident application in a regulatory context. A workshop was held in Montreal in 2019 to discuss where effort needs to be focussed and to agree on the steps needed to ensure safety decisions made on cosmetic ingredients are robust and protective. Workshop participants explored whether NGRA for cosmetic ingredients can be protective of human health, and reviewed examples of NGRA for cosmetic ingredients. From the limited examples available, it is clear that NGRA is still in its infancy, and further case studies are needed to determine whether safety decisions are sufficiently protective and not overly conservative. Seven areas were identified to help progress application of NGRA, including further investments in case studies that elaborate on scenarios frequently encountered by industry and regulators, including those where a 'high risk' conclusion would be expected. These will provide confidence that the tools and approaches can reliably discern differing levels of risk. Furthermore, frameworks to guide performance and reporting should be developed., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Concerns about the potential adverse effects of bisphenol A (BPA) have led to an increase in the use of replacements, yet the toxicity data for several of these chemicals are limited. Using high-content imaging, we compared the effects of BPA, BPAF, BPF, BPS, BPM, and BPTMC in germ (C18-4 spermatogonial) and steroidogenic (MA-10 Leydig and KGN granulosa) cell lines. Effects on cell viability and phenotypic markers were analyzed to determine benchmark concentrations (BMCs) and estimate administered equivalent doses (AEDs). In all 3 cell lines, BPA was one of the least cytotoxic bisphenol compounds tested, whereas BPM and BPTMC were the most cytotoxic. Interestingly, BPF and BPS were cytotoxic only in MA-10 cells. Effects on phenotypic parameters, including mitochondria, lysosomes, lipid droplets, and oxidative stress, were both bisphenol- and cell-line specific. BPA exposure affected mitochondria (BMC: 1.2 μM; AED: 0.09 mg/kg/day) in C18-4 cells. Lysosome numbers were increased in MA-10 cells exposed to BPA or BPAF but decreased in KGN cells exposed to BPAF or BPM. Lipid droplets were decreased in C18-4 cells exposed to BPF and in MA-10 cells exposed to BPTMC but increased in BPF, BPM, and BPTMC-exposed KGN cells. BPA and BPM exposure induced oxidative stress in MA-10 and KGN cells, respectively. In summary, structurally similar bisphenols displayed clear cell-line-specific differences in BMC and AED values for effects on cell viability and phenotypic endpoints. This approach, together with additional data on human exposure, may aid in the selection and prioritization of responsible replacements for BPA. ., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Determination of the no observed adverse effect level (NOAEL) of a substance is an important step in safety and regulatory assessments. Application of conventional in silico strategies, for example, quantitative structure-activity relationship (QSAR) models, to predict NOAEL values is inherently problematic. Whereas QSAR models for well-defined toxicity endpoints such as Ames mutagenicity or skin sensitization can be developed from mechanistic knowledge of molecular initiating events and adverse outcome pathways, QSAR is not appropriate for predicting a NOAEL value, a concentration at which "no effect" is observed. This paper presents a chemoinformatics approach and explores how it can be further refined through the incorporation of toxicity endpoint-specific information to estimate confidence bounds for the NOAEL of a target substance, given experimentally determined NOAEL values for one or more suitable analogues. With a sufficiently large NOAEL database, we analyze how a difference in NOAEL values for pairs of structures depends on their pairwise similarity, where similarity takes both structural features and physicochemical properties into account. The width of the estimate NOAEL confidence interval is proportional to the uncertainty. Using the new threshold of toxicological concern (TTC) database enriched with antimicrobials, examples are presented to illustrate how uncertainty decreases with increasing analogue quality and also how NOAEL bounds estimation can be significantly improved by filtering the full database to include only substances that are in structure categories relevant to the target and analogue.

Background: Decades of research to understand the impacts of various types of environmental occupational and medical stressors on human health have produced a vast amount of data across many scientific disciplines. Organizing these data in a meaningful way to support risk assessment has been a significant challenge. To address this and other challenges in modernizing chemical health risk assessment, the Organisation for Economic Cooperation and Development (OECD) formalized the adverse outcome pathway (AOP) framework, an approach to consolidate knowledge into measurable key events (KEs) at various levels of biological organisation causally linked to disease based on the weight of scientific evidence (http://oe.cd/aops). Currently, AOPs have been considered predominantly in chemical safety but are relevant to radiation. In this context, the Nuclear Energy Agency's (NEA's) High-Level Group on Low Dose Research (HLG-LDR) is working to improve research co-ordination, including radiological research with chemical research, identify synergies between the fields and to avoid duplication of efforts and resource investments. To this end, a virtual workshop was held on 7 and 8 October 2020 with experts from the OECD AOP Programme together with the radiation and chemical research/regulation communities. The workshop was a coordinated effort of Health Canada, the Electric Power Research Institute (EPRI), and the Nuclear Energy Agency (NEA). The AOP approach was discussed including key issues to fully embrace its value and catalyze implementation in areas of radiation risk assessment., Conclusions: A joint chemical and radiological expert group was proposed as a means to encourage cooperation between risk assessors and an initial vision was discussed on a path forward. A global survey was suggested as a way to identify priority health outcomes of regulatory interest for AOP development. Multidisciplinary teams are needed to address the challenge of producing the appropriate data for risk assessments. Data management and machine learning tools were highlighted as a way to progress from weight of evidence to computational causal inference.

In silico toxicology (IST) approaches to rapidly assess chemical hazard, and usage of such methods is increasing in all applications but especially for regulatory submissions, such as for assessing chemicals under REACH as well as the ICH M7 guideline for drug impurities. There are a number of obstacles to performing an IST assessment, including uncertainty in how such an assessment and associated expert review should be performed or what is fit for purpose, as well as a lack of confidence that the results will be accepted by colleagues, collaborators and regulatory authorities. To address this, a project to develop a series of IST protocols for different hazard endpoints has been initiated and this paper describes the genetic toxicity in silico (GIST) protocol. The protocol outlines a hazard assessment framework including key effects/mechanisms and their relationships to endpoints such as gene mutation and clastogenicity. IST models and data are reviewed that support the assessment of these effects/mechanisms along with defined approaches for combining the information and evaluating the confidence in the assessment. This protocol has been developed through a consortium of toxicologists, computational scientists, and regulatory scientists across several industries to support the implementation and acceptance of in silico approaches., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Current global efforts are aiming to increase use of mechanistic information in regulatory testing. In tiered testing paradigms, in vitro, in silico, and in vivo studies are employed progressively to identify and classify health hazards, which are then compared against human equivalent doses. We used data from three companion papers on the brominated flame retardant hexabromocyclododecane (HBCD) to conduct a case study on tiered testing. We included ToxCast™ and in vitro-in vivo extrapolation (Tier 1), rat liver transcriptomic (Tier 2), and conventional rat (Tier 3) data. Bioactivity-exposure ratios (BERs) were derived by comparing human administered dose equivalents of the measured effects to Canadian exposure levels. Biological perturbations were highly aligned between Tiers 1/2, and consistent with apical effects in Tier 3. Tier 1 had the smallest BERs, and Tiers 2/3 were similar. The study demonstrates the promise of using physiologically-based pharmacokinetic modeling and mechanistic analyses in a tiered framework to identify pathways through which chemicals exert toxicological effects; however, they also point to some shortcomings associated with in vitro and in silico approaches. Additional case studies of chemicals from multiple classes are required to define optimal tiered screening procedures to reduce future in vivo requirements in health hazard assessments., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Traditional approaches for chemical risk assessment cannot keep pace with the number of substances requiring assessment. Thus, in a global effort to expedite and modernize chemical risk assessment, New Approach Methodologies (NAMs) are being explored and developed. Included in this effort is the OECD Integrated Approaches for Testing and Assessment (IATA) program, which provides a forum for OECD member countries to develop and present case studies illustrating the application of NAM in various risk assessment contexts. Here, we present an IATA case study for the prediction of estrogenic potential of three target phenols: 4-tert-butylphenol, 2,4-di-tert-butylphenol and octabenzone. Key features of this IATA include the use of two computational approaches for analogue selection for read-across, data collected from traditional and NAM sources, and a workflow to generate predictions regarding the targets' ability to bind the estrogen receptor (ER). Endocrine disruption can occur when a chemical substance mimics the activity of natural estrogen by binding to the ER and, if potency and exposure are sufficient, alters the function of the endocrine system to cause adverse effects. The data indicated that of the three target substances that were considered herein, 4-tert-butylphenol is a potential endocrine disruptor. Further, this IATA illustrates that the NAM approach explored is health protective when compared to in vivo endpoints traditionally used for human health risk assessment., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Changes in chemical regulations worldwide have increased the demand for new data on chemical safety. New approach methodologies (NAMs) are defined broadly here as including in silico approaches and in chemico and in vitro assays, as well as the inclusion of information from the exposure of chemicals in the context of hazard [European Chemicals Agency, " New Approach Methodologies in Regulatory Science ", 2016]. NAMs for toxicity testing, including alternatives to animal testing approaches, have shown promise to provide a large amount of data to fill information gaps in both hazard and exposure. In order to increase experience with the new data and to advance the applications of NAM data to evaluate the safety of data-poor chemicals, demonstration case studies have to be developed to build confidence in their usability. Case studies can be used to explore the domains of applicability of the NAM data and identify areas that would benefit from further research, development, and application. To ensure that this science evolves with direct input from and engagement by risk managers and regulatory decision makers, a workshop was convened among senior leaders from international regulatory agencies to identify common barriers for using NAMs and to propose next steps to address them. Central to the workshop were a series of collaborative case studies designed to explore areas where the benefits of NAM data could be demonstrated. These included use of in vitro bioassays data in combination with exposure estimates to derive a quantitative assessment of risk, use of NAMs for updating chemical categorizations, and use of NAMs to increase understanding of exposure and human health toxicity of various chemicals. The case study approach proved effective in building collaborations and engagement with regulatory decision makers and to promote the importance of data and knowledge sharing among international regulatory agencies. The case studies will be continued to explore new ways of describing hazard (i.e., pathway perturbations as a measure of adversity) and new ways of describing risk (i.e., using NAMs to identify protective levels without necessarily being predictive of a specific hazard). Importantly, the case studies also highlighted the need for increased training and communication across the various communities including the risk assessors, regulators, stakeholders (e.g., industry, non-governmental organizations), and the general public. The development and application of NAMs will play an increasing role in filling important data gaps on the safety of chemicals, but confidence in NAMs will only come with learning by doing and sharing in the experience.

There is increasing interest in the use of quantitative transcriptomic data to determine benchmark dose (BMD) and estimate a point of departure (POD) for human health risk assessment. Although studies have shown that transcriptional PODs correlate with those derived from apical endpoint changes, there is no consensus on the process used to derive a transcriptional POD. Specifically, the subsets of informative genes that produce BMDs that best approximate the doses at which adverse apical effects occur have not been defined. To determine the best way to select predictive groups of genes, we used published microarray data from dose-response studies on six chemicals in rats exposed orally for 5, 14, 28, and 90 days. We evaluated eight approaches for selecting genes for POD derivation and three previously proposed approaches (the lowest pathway BMD, and the mean and median BMD of all genes). The relationship between transcriptional BMDs derived using these 11 approaches and PODs derived from apical data that might be used in chemical risk assessment was examined. Transcriptional BMD values for all 11 approaches were remarkably aligned with corresponding apical PODs, with the vast majority of toxicogenomics PODs being within tenfold of those derived from apical endpoints. We identified at least four approaches that produce BMDs that are effective estimates of apical PODs across multiple sampling time points. Our results support that a variety of approaches can be used to derive reproducible transcriptional PODs that are consistent with PODs produced from traditional methods for chemical risk assessment.

One of the key challenges of Canada's Chemicals Management Plan (CMP) is assessing chemicals with limited/no empirical hazard data for their risk to human health. In some instances, these chemicals have not been tested broadly for their toxicological potency; as such, limited information exists on their potential to induce human health effects following exposure. Although (quantitative) structure activity relationship ((Q)SAR) models are able to generate predictions to address data gaps for certain toxicological endpoints, the confidence in predictions also needs to be addressed. One way to address this issue is to apply a chemical space approach. This approach uses international toxicological databases, for example, those available in the Organisation for Economic Co-operation and Development (OECD) QSAR Toolbox. The approach,assesses a model's ability to predict the potential hazards of chemicals that have limited hazard data that require assessment under the CMP when compared to a larger, data-rich chemical space that is structurally similar to chemicals of interest. This evaluation of a model's predictive ability makes (Q)SAR analysis more transparent and increases confidence in the application of these predictions in a risk-assessment context. Using this approach, predictions for such chemicals obtained from four (Q)SAR models were successfully classified into high, medium and low confidence levels to better inform their use in decision-making.

Existing Quantitative Structure-Activity Relationship (QSAR) models have limited predictive capabilities for aromatic azo compounds. In this study, 2 new models were built to predict Ames mutagenicity of this class of compounds. The first one made use of descriptors based on simplified molecular input-line entry system (SMILES), calculated with the CORAL software. The second model was based on the k-nearest neighbors algorithm. The statistical quality of the predictions from single models was satisfactory. The performance further improved when the predictions from these models were combined. The prediction results from other QSAR models for mutagenicity were also evaluated. Most of the existing models were found to be good at finding toxic compounds but resulted in many false positive predictions. The 2 new models specific for this class of compounds avoid this problem thanks to a larger set of related compounds as training set and improved algorithms., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Application of in silico models to predict developmental toxicity has demonstrated limited success particularly when employed as a single source of information. It is acknowledged that modelling the complex outcomes related to this endpoint is a challenge; however, such models have been developed and reported in the literature. The current study explored the possibility of integrating the selected public domain models (CAESAR, SARpy and P&G model) with the selected commercial modelling suites (Multicase, Leadscope and Derek Nexus) to assess if there is an increase in overall predictive performance. The results varied according to the data sets used to assess performance which improved upon model integration relative to individual models. Moreover, because different models are based on different specific developmental toxicity effects, integration of these models increased the applicable chemical and biological spaces. It is suggested that this approach reduces uncertainty associated with in silico predictions by achieving a consensus among a battery of models. The use of tools to assess the applicability domain also improves the interpretation of the predictions. This has been verified in the case of the software VEGA, which makes freely available QSAR models with a measurement of the applicability domain., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Modeling developmental toxicity has been a challenge for (Q)SAR model developers due to the complexity of the endpoint. Recently, some new in silico methods have been developed introducing the possibility to evaluate the integration of existing methods by taking advantage of various modeling perspectives. It is important that the model user is aware of the underlying basis of the different models in general, as well as the considerations and assumptions relative to the specific predictions that are obtained from these different models for the same chemical. The evaluation on the predictions needs to be done on a case-by-case basis, checking the analogs (possibly using structural, physicochemical, and toxicological information); for this purpose, the assessment of the applicability domain of the models provides further confidence in the model prediction. In this chapter, we present some examples illustrating an approach to combine human-based rules and statistical methods to support the prediction of developmental toxicity; we also discuss assumptions and uncertainties of the methodology.

Systematic consideration of scientific support is a critical element in developing and, ultimately, using adverse outcome pathways (AOPs) for various regulatory applications. Though weight of evidence (WoE) analysis has been proposed as a basis for assessment of the maturity and level of confidence in an AOP, methodologies and tools are still being formalized. The Organization for Economic Co-operation and Development (OECD) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD 2014a; hereafter referred to as the OECD AOP Handbook) provides tailored Bradford-Hill (BH) considerations for systematic assessment of confidence in a given AOP. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulatory agencies worldwide are committed to the objectives of the Strategic Approach to International Chemicals Management to ensure that by 2020 chemicals are used and produced in ways that lead to the minimization of significant adverse effects on human health and the environment. Under the Government of Canada's Chemicals Management Plan, the commitment to address a large number of substances, many with limited data, has highlighted the importance of pursuing alternative hazard assessment methodologies that are able to accommodate chemicals with varying toxicological information. One such method is (Quantitative) Structure Activity Relationships ((Q)SAR) models. The current investigation into the predictivity of 20 (Q)SAR tools designed to model bacterial reverse mutation in Salmonella typhimurium is one of the first of this magnitude to be carried out using an external validation set comprised mainly of industrial chemicals which represent a diverse group of aromatic and benzidine-based azo dyes and pigments. Overall, this study highlights the value in challenging the predictivity of existing models using a small but representative subset of data-rich chemicals. Furthermore, external validation revealed that only a handful of models satisfactorily predicted for the test chemical space. The exercise also provides insight into using the Organisation for Economic Co-operation and Development (Q)SAR Toolbox as a read across tool.