Commentary: Value of information case study strongly supports use of the Threshold of Toxicological Concern (TTC).

A Value of Information (VOI) analysis can play a key role in decision-making for adopting new approach methodologies (NAMs). We applied EPA's recently developed VOI framework to the Threshold of Toxicological Concern (TTC). Obtaining/deriving a TTC value for use as a toxicity reference value (TRV) for substances with limited toxicity data was shown to provide equivalent or greater health protection, immense return on investment (ROI), greater net benefit, and substantially lower costs of delay (CoD) compared with TRVs derived from either traditional human health assessment (THHA) chronic toxicity testing in lab animals or the 5-day in vivo EPA Transcriptomic Assessment Product (ETAP). For all nine exposure scenarios examined, the TTC was more economical terms of CoD and ROI than the ETAP or the THHA; expected net benefit was similar for the TTC and ETAP with both of these more economical than the THHA The TTC ROI was immensely greater (5,000,000-fold on average) than the ROI for THHA and the ETAP ROI (100,000-fold on average). These results support the use of the TTC for substances within its domain of applicability to waive requiring certain in vivo tests, or at a minimum, as an initial screening step before conducting either the ETAP or THHA in vivo studies.

Quantitative Structure Use Relationships: Highlights from a technical summit meeting.

The Quantitative Structure Use Relationship (QSUR) Summit, held on November 2-4, 2022, focused on advancing the development, refinement, and use of QSURs to support chemical substance prioritization and risk assessment and mitigation. QSURs utilize chemical structures to predict the function of a chemical within a formulated product or an industrial process. This presumed function can then be used to develop chemical use categories or other information necessary to refine exposure assessments. The invited expert meeting was attended by 38 scientists from Canada, Finland, France, the UK, and the USA, representing government, business, and academia, with expertise in exposure science, chemical engineering, risk assessment, formulation chemistry, and machine learning. Workshop discussions emphasized the importance of collection and sharing of data and quantification of relative chemical quantities to progress QSUR development. Participants proposed collaborative approaches to address key challenges, including mechanisms for aggregating information while still protecting proprietary product composition and other confidential business information. Discussions also led to proposals for applications beyond exposure and risk modeling, including sustainable formulation discovery. In addition, discussions continue to construct, conduct, and circulate case studies tied to various specific problem formulations in which QSURs supply or derive information on chemical functions, concentrations, and exposures.

Best practices for exposure model peer review - A SciPinion advisory panel report.

The extent and rigor of peer review that a model undergoes during and after development influences the confidence of users and managers in model predictions. A process for determining the breadth and depth of peer review of exposure models was developed with input from a panel of exposure-modeling experts. This included consideration of the tiers and types of models (e.g., screening, deterministic, probabilistic, etc.). The experts recommended specific criteria be considered when evaluating the degree to which a model has been peer reviewed, including quality of documentation and the model peer review process (e.g., internal review with a regulatory agency by subject matter experts, expert review reports, formal Scientific Advisory Panels, and journal peer review). In addition, because the determination of the confidence level for an exposure model's predictions is related to the degree of evaluation the model has undergone, irrespective of peer review, the experts recommended the approach include judging the degree of model rigor using a set of specific criteria: (1) nature and quality of input data, (2) model verification, (3) model corroboration, and (4) model evaluation. Other key areas considered by the experts included recommendations for addressing model uncertainty and sensitivity, defining the model domain of applicability, and flags for when a model is used outside its domain of applicability. The findings of this expert engagement will help developers as well as users of exposure models have greater confidence in their application and yield greater transparency in the evaluation and peer review of exposure models.

Developing an internal threshold of toxicological concern (iTTC).

BACKGROUND: Threshold of Toxicological Concern (TTC) approaches are used for chemical safety assessment and risk-based priority setting for data poor chemicals. TTCs are derived from in vivo No Observed Effect Level (NOEL) datasets involving an external administered dose from a single exposure route, e.g., oral intake rate. Thus, a route-specific TTC can only be compared to a route-specific exposure estimate and such TTCs cannot be used for other exposure scenarios such as aggregate exposures. OBJECTIVE: Develop and apply a method for deriving internal TTCs (iTTCs) that can be used in chemical assessments for multiple route-specific exposures (e.g., oral, inhalation or dermal) or aggregate exposures. METHODS: Chemical-specific toxicokinetics (TK) data and models are applied to calculate internal concentrations (whole-body and blood) from the reported administered oral dose NOELs used to derive the Munro TTCs. The new iTTCs are calculated from the 5th percentile of cumulative distributions of internal NOELs and the commonly applied uncertainty factor of 100 to extrapolate animal testing data for applications in human health assessment. RESULTS: The new iTTCs for whole-body and blood are 0.5 nmol/kg and 0.1 nmol/L, respectively. Because the iTTCs are expressed on a molar basis they are readily converted to chemical mass iTTCs using the molar mass of the chemical of interest. For example, the median molar mass in the dataset is 220 g/mol corresponding to an iTTC of 22 ng/L-blood (22 pg/mL-blood). The iTTCs are considered broadly applicable for many organic chemicals except those that are genotoxic or acetylcholinesterase inhibitors. The new iTTCs can be compared with measured or estimated whole-body or blood exposure concentrations for chemical safety screening and priority-setting. SIGNIFICANCE: Existing Threshold of Toxicological Concern (TTC) approaches are limited in their applications for route-specific exposure scenarios only and are not suitable for chemical risk and safety assessments under conditions of aggregate exposure. New internal Threshold of Toxicological Concern (iTTC) values are developed to address data gaps in chemical safety estimation for multi-route and aggregate exposures.

Identifying the landscape of developmental toxicity new approach methodologies.

BACKGROUND: The dynamics and complexities of in utero fetal development create significant challenges in transitioning from lab animal-centric developmental toxicity testing methods to assessment strategies based on new approach methodologies (NAMs). Nevertheless, considerable progress is being made, stimulated by increased research investments and scientific advances, such as induced pluripotent stem cell-derived models. To help identify developmental toxicity NAMs for toxicity screening and potential funding through the American Chemistry Council's Long-Range Research Initiative, a systematic literature review was conducted to better understand the current landscape of developmental toxicity NAMs. METHODS: Scoping review tools were used to systematically survey the literature (2010-2021; ~18,000 references identified), results and metadata were then extracted, and a user-friendly interactive dashboard was created. RESULTS: The data visualization dashboard, developed using Tableau® software, is provided as a free, open-access web tool. This dashboard enables straightforward interactive queries and visualizations to identify trends and to distinguish and understand areas or NAMs where research has been most, or least focused. CONCLUSIONS: Herein, we describe the approach and methods used, summarize the benefits and challenges of applying the systematic-review techniques, and highlight the types of questions and answers for which the dashboard can be used to explore the many different facets of developmental toxicity NAMs.

Microphysiological Systems Evaluation: Experience of TEX-VAL Tissue Chip Testing Consortium.

Much has been written and said about the promise and excitement of microphysiological systems, miniature devices that aim to recreate aspects of human physiology on a chip. The rapid explosion of the offerings and persistent publicity placed high expectations on both product manufacturers and regulatory agencies to adopt the data. Inevitably, discussions of where this technology fits in chemical testing paradigms are ongoing. Some end-users became early adopters, whereas others have taken a more cautious approach because of the high cost and uncertainties of their utility. Here, we detail the experience of a public-private collaboration established for testing of diverse microphysiological systems. Collectively, we present a number of considerations on practical aspects of using microphysiological systems in the context of their applications in decision-making. Specifically, future end-users need to be prepared for extensive on-site optimization and have access to a wide range of imaging and other equipment. We reason that cells, related reagents, and the technical skills of the research staff, not the devices themselves, are the most critical determinants of success. Extrapolation from concentration-response effects in microphysiological systems to human blood or oral exposures, difficulties with replicating the whole organ, and long-term functionality remain as critical challenges. Overall, we conclude that it is unlikely that a rodent- or human-equivalent model is achievable through a finite number of microphysiological systems in the near future; therefore, building consensus and promoting the gradual incorporation of these models into tiered approaches for safety assessment and decision-making is the sensible path to wide adoption.

Recommendations for further revisions to improve the International Agency for Research on Cancer (IARC) Monograph program.

In 2019, the International Agency for Research on Cancer (IARC) "Preamble to the IARC Monographs" expanded guidance regarding the scientific approaches that should be employed in its monographs. These amendments to the monograph development process are an improvement but still fall short in several areas. While the revised Preamble lays out broad methods and approaches to evaluate scientific evidence, there is a lack of specificity with regard to how IARC Working Groups will conduct consistent evaluations in a standardized, objective, and transparent manner; document systematic review and evidence integration actions, and substantiate how these actions and decisions inform the ultimate classifications. Furthermore, no guidance is provided to ensure Working Groups consistently incorporate mechanistic evidence in a robust manner using a defined approach in the context of 21st century knowledge of modes of action. Nor are the conclusions of the working groups subjected to outside, independent scientific peer review. Continued improvements and modernization of the procedures for evaluating, presenting, and communicating study quality, and in the methods used to conduct and peer-review evidence-based decision making will benefit the Working Group members, the IARC Monographs Programme overall, and the international regulatory community and public who rely upon the monographs.

21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances: Workshop highlights.

The June 2019 workshop 21st Century Approaches for Evaluating Exposures, Biological Activity, and Risks of Complex Substances, co-organised by the International Council of Chemical Association's Long-Range Research Initiative and the European Commission's Joint Research Centre, is summarised. Focus was the need for improved approaches to evaluate the safety of complex substances. Approximately 10% and 20% of substances registered under the EU chemicals legislation are 'multi-constituent substances' and 'substances of unknown or variable compositions, complex reaction products and biological substances' (UVCBs), respectively, and UVCBs comprise approximately 25% of the U.S. Toxic Substances Control Act Inventory. Workshop participants were asked to consider how the full promise of new approach methodologies (NAMs) could be brought to bear to evaluate complex substances. Sessions focused on using NAMs for screening, biological profiling, and in complex risk evaluations; improving read-across approaches employing new data streams; and methods to evaluate exposure and dosimetry. The workshop concluded with facilitated discussions to explore actionable steps forward. Given the diversity of complex substances, no single 'correct' approach was seen as workable. The path forward should focus on 'learning by doing' by developing and openly sharing NAM-based fit-for-purpose case examples for evaluating biological activity, exposures and risks of complex substances.

Internal Threshold of Toxicological Concern (iTTC): Where We Are Today and What Is Possible in the Near Future.

The Threshold of Toxicological Concern (TTC) is a risk assessment tool for evaluating low-level exposure to chemicals with limited toxicological data. A next step in the ongoing development of TTC is to extend this concept further so that it can be applied to internal exposures. This refinement of TTC based on plasma concentrations, referred to as internal TTC (iTTC), attempts to convert the chemical-specific external NOAELs (in mg/kg/day) in the TTC database to an estimated internal exposure. A multi-stakeholder collaboration formed, with the aim of establishing an iTTC suitable for human safety risk assessment. Here, we discuss the advances and future directions for the iTTC project, including: (1) results from the systematic literature search for metabolism and pharmacokinetic data for the 1,251 chemicals in the iTTC database; (2) selection of ~350 chemicals that will be included in the final iTTC; (3) an overview of the in vitro caco-2 and in vitro hepatic metabolism studies currently being generated for the iTTC chemicals; (4) demonstrate how PBPK modeling is being utilized to convert a chemical-specific external NOAEL to an internal exposure; (5) perspective on the next steps in the iTTC project.

The role of fit-for-purpose assays within tiered testing approaches: A case study evaluating prioritized estrogen-active compounds in an in vitro human uterotrophic assay.

Chemical risk assessment relies on toxicity tests that require significant numbers of animals, time and costs. For the >30,000 chemicals in commerce, the current scale of animal testing is insufficient to address chemical safety concerns as regulatory and product stewardship considerations evolve to require more comprehensive understanding of potential biological effects, conditions of use, and associated exposures. We demonstrate the use of a multi-level new approach methodology (NAMs) strategy for hazard- and risk-based prioritization to reduce animal testing. A Level 1/2 chemical prioritization based on estrogen receptor (ER) activity and metabolic activation using ToxCast data was used to select 112 chemicals for testing in a Level 3 human uterine cell estrogen response assay (IKA assay). The Level 3 data were coupled with quantitative in vitro to in vivo extrapolation (Q-IVIVE) to support bioactivity determination (as a surrogate for hazard) in a tissue-specific context. Assay AC50s and Q-IVIVE were used to estimate human equivalent doses (HEDs), and HEDs were compared to rodent uterotrophic assay in vivo-derived points of departure (PODs). For substances active both in vitro and in vivo, IKA assay-derived HEDs were lower or equivalent to in vivo PODs for 19/23 compounds (83%). Activity exposure relationships were calculated, and the IKA assay was as or more protective of human health than the rodent uterotrophic assay for all IKA-positive compounds. This study demonstrates the utility of biologically relevant fit-for-purpose assays and supports the use of a multi-level strategy for chemical risk assessment.

Developing context appropriate toxicity testing approaches using new alternative methods (NAMs).

In the past 10 years, the public, private, and non-profit sectors have found agreement that hazard identification and risk assessment should capitalize on the explosion of knowledge in the biological sciences, moving away from in life animal testing toward more human-relevant in vitro and in silico methods, collectively referred to as new approach methodologies (NAMs). The goals for implementation of NAMs are to efficiently identify possible chemical hazards and to gather dose-response data to inform more human-relevant safety assessment. While work proceeds to develop NAMs, there has been less emphasis on creating decision criteria or showing how risk context should guide selection and use of NAMs. Here, we outline application scenarios for NAMs in different risk contexts and place different NAMs and conventional testing approaches into four broad levels. Level 1 relies solely on computational screening; Level 2 consists of high throughput in vitro screening with human cells intended to provide broad coverage of possible responses; Level 3 focuses on fit-for-purpose assays selected based on presumptive modes of action (MOA) and designed to provide more quantitative estimates of relevant dose responses; Level 4 has a variety of more complex multi-dimensional or multi-cellular assays and might include targeted in vivo studies to further define MOA. Each level also includes decision-appropriate exposure assessment tools. Our aims here are to (1) foster discussion about context-dependent applications of NAMs in relation to risk assessment needs and (2) describe a functional roadmap to identify where NAMs are expected to be adequate for chemical safety decision-making.

Toward the Development and Application of an Environmental Risk Assessment Framework for Microplastic.

Emissions of plastic waste to the environment and the subsequent degradation into microplastic particles that have the potential to interact with biological organisms represent a concern for global society. Current understanding of the potential impacts on aquatic and terrestrial population stability and ecosystem structure and function associated with emissions of microplastic particles is limited and insufficient to fully assess environmental risks. Multistakeholder discussions can provide an important element in helping to identify and prioritize key knowledge gaps in assessing potential risks. In the present review, we summarize multistakeholder discussions from a 1-d International Council of Chemical Associations-sponsored symposium, which involved 39 scientists from 8 countries with representatives from academia, industry, and government. Participants were asked to consider the following: discuss the scientific merits and limitations of applying a proposed conceptual environmental risk assessment (ERA) framework for microplastic particles and identify and prioritize major research needs in applying ERA tools for microplastic particles. Multistakeholder consensus was obtained with respect to the interpretation of the current state of the science related to effects and exposure to microplastic particles, which implies that it is unlikely that the presence of microplastic in the environment currently represents a risk. However, the quality and quantity of existing data require substantial improvement before conclusions regarding the potential risks and impacts of microplastic particles can be fully assessed. Research that directly addresses the development and application of methods that strengthen the quality of data should thus be given the highest priority. Activities aimed at supporting the development of and access to standardized reference material were identified as a key research need. Environ Toxicol Chem 2019;38:2087-2100. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Development of an adverse outcome pathway for chemically induced hepatocellular carcinoma: case study of AFB1, a human carcinogen with a mutagenic mode of action.

Adverse outcome pathways (AOPs) are frameworks starting with a molecular initiating event (MIE), followed by key events (KEs) linked by KE relationships (KERs), ultimately resulting in a specific adverse outcome. Relevant data for the pathway and each KE/KER are evaluated to assess biological plausibility, weight-of-evidence, and confidence. We aimed to describe an AOP relevant to chemicals directly inducing mutation in cancer critical gene(s), via the formation of chemical-specific pro-mutagenic DNA adduct(s), as an early critical step in tumor etiology. Such chemicals have mutagenic modes-of-action (MOA) for tumor induction. To assist with developing this AOP, Aflatoxin B1 (AFB1) was selected as a case study because it has a rich database and is considered to have a mutagenic MOA. AFB1 information was used to define specific KEs, KERs, and to inform development of a generic AOP for mutagen-induced hepatocellular carcinoma (HCC). In assessing the AFB1 information, it became clear that existing data are, in fact, not optimal and for some KEs/KERs, the definitive data are not available. In particular, while there is substantial information that AFB1 can induce mutations (based on a number of mutation assays), the definitive evidence - the ability to induce mutation in the cancer critical gene(s) in the tumor target tissue - is not available. Thus, it is necessary to consider the patterns of results in the weight-of-evidence for KEs and KERs. It was important to determine whether there was sufficient evidence that AFB1 can induce the necessary critical mutations early in the carcinogenic process, which was the case.

Utilizing Threshold of Toxicological Concern (TTC) with High Throughput Exposure Predictions (HTE) as a Risk-Based Prioritization Approach for thousands of chemicals.

Regulatory agencies across the world are facing the challenge of performing risk-based prioritization of thousands of chemicals in commerce. Here, we present an approach using the Threshold of Toxicological Concern (TTC) combined with heuristic high-throughput exposure (HTE) modelling to rank order chemicals for further evaluation. Accordingly, for risk-based prioritization, chemicals with exposures > TTC would be ranked as higher priority for further evaluation whereas substances with exposures < TTC would be ranked as lower priority. An initial proof of concept, using a dataset of 7986 substances with previously modeled median and upper 95% credible interval (UCI) total daily median exposure rates showed fewer than 5% of substances had UCI exposures > the Cramer Class III TTC (1.5 µg/kg-day). We extended the analysis by profiling the same dataset through the TTC workflow published by Kroes et al (2004) which accounts for known exclusions to the TTC as well as structural alerts. UCI exposures were then compared to the appropriate class-specific TTC. None of the substances categorized as Cramer Class I or Cramer Class II exceeded their respective TTC values and no more than 2% of substances categorized as Cramer Class III or acetylcholinesterase inhibitors exceeded their respective TTC values. The modeled UCI exposures for the majority of the 1853 chemicals with genotoxicity structural alerts did exceed the TTC of 0.0025 µg/kg-day, but only 79 substances exceeded this TTC if median exposure values were used. For substances for which UCI exposures exceeded relevant TTC values, we highlight possible approaches for consideration to refine the HTE : TTC approach. Overall, coupling TTC with HTE offers promise as a pragmatic first step in ranking substances as part of a risk-based prioritization approach.

How well can carcinogenicity be predicted by high throughput "characteristics of carcinogens" mechanistic data?

IARC has begun using ToxCast/Tox21 data in efforts to represent key characteristics of carcinogens to organize and weigh mechanistic evidence in cancer hazard determinations and this implicit inference approach also is being considered by USEPA. To determine how well ToxCast/Tox21 data can explicitly predict cancer hazard, this approach was evaluated with statistical analyses and machine learning prediction algorithms. Substances USEPA previously classified as having cancer hazard potential were designated as positives and substances not posing a carcinogenic hazard were designated as negatives. Then ToxCast/Tox21 data were analyzed both with and without adjusting for the cytotoxicity burst effect commonly observed in such assays. Using the same assignments as IARC of ToxCast/Tox21 assays to the seven key characteristics of carcinogens, the ability to predict cancer hazard for each key characteristic, alone or in combination, was found to be no better than chance. Hence, we have little scientific confidence in IARC's inference models derived from current ToxCast/Tox21 assays for key characteristics to predict cancer. This finding supports the need for a more rigorous mode-of-action pathway-based framework to organize, evaluate, and integrate mechanistic evidence with animal toxicity, epidemiological investigations, and knowledge of exposure and dosimetry to evaluate potential carcinogenic hazards and risks to humans.

Quantitative weight of evidence to assess confidence in potential modes of action.

The evolved World Health Organization/International Programme on Chemical Safety mode of action (MOA) framework provides a structure for evaluating evidence in pathways of causally linked key events (KE) leading to adverse health effects. Although employed globally, variability in use of the MOA framework has led to different interpretations of the sufficiency of evidence in support of hypothesized MOAs. A proof of concept extension of the MOA framework is proposed for scoring confidence in the supporting data to improve scientific justification for MOA use in characterizing hazards and selecting dose-response extrapolation methods for specific chemicals. This involves selecting hypothesized MOAs, and then, for each MOA, scoring the weight of evidence (WOE) in support of causality for each KE using evolved Bradford Hill causal considerations (biological plausibility, essentiality, dose-response concordance, consistency, and analogy). This early proof of concept method is demonstrated by comparing two potential MOAs (mutagenicity and peroxisome proliferator activated receptor-alpha) for clofibrate, a rodent liver carcinogen. Quantitative confidence scoring of hypothesized MOAs is shown to be useful in characterizing the likely operative MOA. To guide method refinement and future confidence scoring for a spectrum of MOAs, areas warranting further focus and lessons learned, including the need to incorporate a narrative discussion of the weights used in the evaluation and an overall evaluation of the plausibility of the outcome, are presented.

FutureTox III: Bridges for Translation.

Future Tox III, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in November 2015. Building upon Future Tox I and II, Future Tox III was focused on developing the high throughput risk assessment paradigm and taking the science of in vitro data and in silico models forward to explore the question-what progress is being made to address challenges in implementing the emerging big-data toolbox for risk assessment and regulatory decision-making. This article reports on the outcome of the workshop including 2 examples of where advancements in predictive toxicology approaches are being applied within Federal agencies, where opportunities remain within the exposome and AOP domains, and how collectively the toxicology community across multiple sectors can continue to bridge the translation from historical approaches to Tox21 implementation relative to risk assessment and regulatory decision-making.

Does GLP enhance the quality of toxicological evidence for regulatory decisions?

There is debate over whether the requirements of GLP are appropriate standards for evaluating the quality of toxicological data used to formulate regulations. A group promoting the importance of non-monotonic dose responses for endocrine disruptors contend that scoring systems giving primacy to GLP are biased against non-GLP studies from the literature and are merely record-keeping exercises to prevent fraudulent reporting of data from non-published guideline toxicology studies. They argue that guideline studies often employ insensitive species and outdated methods, and ignore the perspectives of subject-matter experts in endocrine disruption, who should be the sole arbiters of data quality. We believe regulatory agencies should use both non-GLP and GLP studies, that GLP requirements assure fundamental tenets of study integrity not typically addressed by journal peer-review, and that use of standardized test guidelines and GLP promotes consistency, reliability, comparability, and harmonization of various types of studies used by regulatory agencies worldwide. This debate suffers two impediments to progress: a conflation of different phases of study interpretation and levels of data validity, and a misleading characterization of many essential components of GLP and regulatory toxicology. Herein we provide clarifications critical for removing those impediments.