From vision toward best practices: Evaluating in vitro transcriptomic points of departure for application in risk assessment using a uniform workflow.

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.

Development of an Evidence-Based Risk Assessment Framework.

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.

Use of new approach methodologies (NAMs) to meet regulatory requirements for the assessment of industrial chemicals and pesticides for effects on human health.

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.

Comprehensive interpretation of in vitro micronucleus test results for 292 chemicals: from hazard identification to risk assessment application.

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.

Hexabromocyclododecane (HBCD): A case study applying tiered testing for human health risk assessment.

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.

Predicting estrogen receptor activation by a group of substituted phenols: An integrated approach to testing and assessment case study.

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.

Accelerating the Pace of Chemical Risk Assessment.

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.

Recommended approaches in the application of toxicogenomics to derive points of departure for chemical risk assessment.

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.