ICCVAM Biennial Report 2022-2023

A contaminant of emerging concern is a material for which there is no regulatory standard, that may have toxicity at lower levels of exposure than previously characterized, is challenging to address across jurisdictions, and for which it is difficult to share information with stakeholders in a timely manner. Many contaminants of emerging concern are of interest to multiple federal agencies, which has given rise to efforts to coordinate addressing them. Initiatives by the White House Office of Science and Technology Policy and directives of the 2020 omnibus National Defense Authorization Act led to establishment of the National Emerging Contaminants Research Initiative to improve the identification, analysis, monitoring, and treatment methods for contaminants of emerging concern. The Contaminants of Emerging Concern Interagency Working Group coordinates federal programs and activities to address contaminants of emerging concern; 18 agencies are involved including eight ICCVAM member agencies. The working group’s 2022 report summarizes current government activities and future needs. Specifically, the report articulated a vision to provide access to clean and plentiful drinking water for everyone in the U.S. and specified five goals to achieve this. Three coordination teams will focus on (1) non-targeted analysis and effects-based monitoring to discover and screen contaminants of emerging concern; (2) characterizing risk by assessing the potential hazards and exposure; and (3) formulating joint solicitations across agencies. This last activity will involve making a plan for how to work together to craft solicitations and manage them. A draft implementation plan, which was under agency review in fall 2023, outlines a series of short- and long-term activities that are anchored to success metrics. NAMs are seen as a critical tool for potential hazard characterization.

DoD and the Veterans’ Administration have a responsibility to care for its service members who bravely put themselves in harm’s way. The Promise to Address Comprehensive Toxics (PACT) Act of 2022 provided resources and intent to improve and expand healthcare to veterans who were exposed to toxic substances during their military service. Understanding past exposure events and their potential influence on health is a requirement for providing informed care and to reduce the probability of future adverse events. This need drove the establishment of an interagency group to outline a strategy to fill research and data gaps associated with toxic exposures during military service. The Toxic Exposures Research Working Group, which includes representatives of seven ICCVAM agencies, was initiated to help scope the problem, outline goals, objectives, and tasks that could be reasonably accomplished in five years to help address some of these gaps. The working group’s goals include:

1. Improving understanding of the military exposome.
2. Prioritizing and linking military exposures to toxicity and adverse health outcomes, including understanding modes-of-action and AOPs.
3. Investigating associations and interplay between priority military toxic exposures, toxicological endpoints, and adverse health outcomes.
4. Preventing and mitigating military adverse health outcomes from military exposures.
5. Communicate toxic exposure risk and adverse health outcomes to relevant stakeholders to enhance exposure-informed care.

Objectives and tasks were developed with the intention of developing interagency cooperation to establish a federal infrastructure framework for coordinating implementation of this strategic plan. At the end of 2023, the working group is focusing on its final report, which includes contributions from all federal agency partners. The report will be issued in 2024 and address requirements of the PACT Act to outline goals in this area over the next five years.

In 2021, NICEATM, the National Institute of Allergy and Infectious Diseases, the U.S. Army Combat Capabilities Development Command Chemical Biological Center, and NCATS established the MPS for COVID Research (MPSCoRe) Working Group in collaboration with NC3Rs. The MPSCoRe Working Group facilitates open communication among stakeholders to maximize the impact of MPS technologies in understanding disease mechanisms and treatments and reducing animal use while improving human health. In this way, the group aims to promote adoption of MPS for studying COVID-19 and future emerging infectious diseases (Kleinstreuer and Holmes 2021). These efforts will accelerate the development and adoption of MPS in infectious disease research and may reduce the reliance on animal models in future studies.

The MPSCoRe Working Group held a virtual workshop in May-June 2023 to facilitate discussion and collaboration about current regulatory approaches and to raise awareness of opportunities for accelerating the integration of MPS models for infectious diseases in the regulatory framework. The working group is planning to organize future webinars on topics of interest.

Model species have been used for decades in guideline tests to generate survival, growth, and reproductive data for prospective ecological risk assessments. While such endpoints are central to hazard assessment, nonstandard measures of toxicity at multiple levels of biological organization (e.g., molecular, cellular, tissue, organ, organism, population, community, ecosystem) may enhance the relevance of prospective and retrospective wildlife risk assessments. The incorporation of NAMs into ecological risk assessment frameworks to reduce or replace animal tests require robust validations against in vivo responses. To address these issues and develop a plan for applying NAMs to ecological risk assessments, the Society of Environmental Toxicology and Chemistry convened a 2021 virtual workshop, “Wildlife Risk Assessment in the 21st Century: Integrating Advancements in Ecology, Toxicology, and Conservation,” for which the DOI’s USGS was among the sponsors. Two reports from that workshop describe many of the activities that are needed to support application of NAMs to ecological risk assessment. Bean et al. (2023) explores how existing test guidelines for in vivo studies could be improved to provide better data to support NAMs validation. Rattner et al. (2023) discusses how laboratory- and field-derived data along with modeling approaches will likely need to be combined to apply NAMs to ecological risk assessment.

There are currently several peer-reviewed and publicly available methods and tools, such as EPA’s SeqAPASS, to facilitate cross-species extrapolation. However, because no individual method can advance the application of these types of data in regulatory decision-making, collaboration is needed to effectively advance these approaches. EPA participates in the International Consortium to Advance Cross-Species Extrapolation (LaLone et al. 2021), which was founded to advance cross-species extrapolation and uphold regulatory goals for assessing human and ecological health without animal testing. This is a global, cross-sector consortium that includes researchers, regulators, and other advocates working to integrate bioinformatics approaches. The consortium aims to review and bring the available bioinformatic techniques together to advance the science of cross-species extrapolation using to inform regulatory needs.

EPA computational toxicology researchers are developing and using new approaches to evaluate the potential health effects of chemicals. As part of this effort, EPA’s Center for Computational and Exposure and Unilever’s Safety and Environmental Assurance Centre have established a cooperative research and development agreement aimed at development and evaluation of NAMs for use in next-generation risk assessment. Currently 40 case study chemicals representing common natural ingredients found in consumer products are being used to explore the utility of a battery of NAMs for evaluating the safety and potential hazards. Research topics in this project include (1) exploring the use of combinations of approaches for determining molecular points-of-departure, (2) comparing molecular PODs to hazardous and non-hazardous human exposure scenarios, (3) evaluating the impact of in vitro metabolism on bioactivity assay readouts, (4) exploring methods for metabolite determination, and (5) piloting the use of fish-derived cell lines to evaluate ecotoxicity hazard. The cooperative research and development agreement was initiated in 2021 and has recently been extended to May of 2027. The project’s current status will be discussed in a webinar planned for April 2024.

ICCVAM members from NIEHS and NIST served on a committee convened by the National Academies of Sciences, Engineering, and Medicine for the consensus study, “Variability and Relevance of Current Laboratory Mammalian Toxicity Tests and Expectations for New Approach Methods (NAMs) for use in Human Health Risk Assessment.” This study was undertaken by the National Academies in response to a request from EPA. It reviewed the variability and relevance of existing mammalian toxicity tests, specifically in the context of addressing challenges to applying NAMs to human health risk assessment. The committee held two public meetings in 2021. Proceedings from the second of these, a workshop to address the potential utility and expectations for the future use of NAMs in risk assessment and to reflect on the challenges to their implementation, were published in 2022.

While the promise and need for NAMs is clear, many barriers to their use remain. The final report of the study, issued in 2023, aims to bridge the gap between the potential of NAMs and their practical application in human health risk assessment. Lessons learned from laboratory mammalian toxicity tests are used as a basis to help inform approaches for building scientific confidence in NAMs and for incorporating such data into risk assessment and decision-making. Overall, the report recommendations aim to ensure a seamless handoff from the evaluation of NAM-based testing strategies in the laboratory to the incorporation of NAM data into modern, systematic-review-based risk assessments.

At the request of EPA, an ad hoc planning committee of the National Academies of Sciences, Engineering, and Medicine organized two workshops on topics pertinent to their assessment of human health effects. The workshops were designed to assist EPA with increasing the quality, transparency, and confidence in its chemical assessments by addressing scientific issues related to systematic review, hazard identification, and dose-response analysis. Scientists from NCI and NIEHS served on the committee. Topics included:

• Evaluating elements of the systematic review process (e.g., development and application of specialized tools, methods for evaluating mechanistic data).
• Hazard identification approaches (e.g., techniques for integrating evidence).
• Incorporating NAMs for testing chemicals into chemical assessments.
• Research on quantitative analyses for evidence integration and dose-response analyses.

The first workshop, “Triangulation of Evidence in Environmental Epidemiology,” considered approaches to integrating results from a variety of data streams to inform and strengthen causal inferences. The second workshop, “Artificial Intelligence and Open Data Practices in Chemical Hazard Assessment,” focused on approaches to automating and streamlining data extraction and evidence synthesis for systematic reviews. Videos of the workshops and meeting materials are available on each of the meeting webpages.

In February 2022, EPA launched a new program under the TSCA to modernize the process and bring innovative science to the review of new chemicals before they can enter the marketplace. Under the New Chemicals Collaborative Research Program, EPA’s Office of Research and Development is working with the Office of Chemical Safety and Pollution Prevention to advance five key research areas:

• Updates and refinements to chemical analogue and category approaches.
• Development and expansion of databases containing TSCA chemical information.
• Development and refinement of predictive models for physicochemical properties, environmental fate/transport, hazard, exposure, and toxicokinetics.
• Integration and application of in vitro NAMs.
• Development of a TSCA new chemicals decision support tool to modernize the process.

Each of these five research areas represents translation and extension of computational toxicology research that has been in development under the vision of the CompTox Blueprint (Thomas et al. 2019) and the 2021 EPA NAMs Work Plan, which together form a strategic roadmap for developing and integrating NAMs to fill information gaps, establishing scientific confidence of NAM application, and engaging with stakeholders. EPA announced the program in February 2022. A public meeting in April 2022 provided an overview of the program and gave individual stakeholders an opportunity to provide input. Subsequently a 2023-2026 research plan was reviewed by the Board of Scientific Counselors in October 2022 with a final report posted in early 2023. In 2023, the program advanced multiple research objectives, including publications of research evaluation of chemical and analogue approaches, cheminformatics-driven selection of chemicals to include in an in vitro NAM proof-of-concept, and the beginning of screening work on that proof-of-concept.

An AOP is a conceptual framework for organizing information and evidence on toxicity. It defines a causal relationship between perturbation of a biological pathway at the molecular, biochemical, or cellular level and one or more resulting adverse outcomes to human or ecosystem health. AOPs are useful in supporting both the development and application of NAMs and DAs in chemical safety assessment because they can:

• Organize information about biological interactions and toxicity mechanisms into sequential steps that describe how exposure to a substance might cause illness or injury.
• Suggest cell- or biochemical-based tests for pathway elements that could be used to develop testing strategies for targeted toxicity.
• Identify steps in a toxicity mechanism that need improved characterization.

EPA is an active partner in the OECD adverse outcome pathways development workplan, managed under OECD’s Working Party for Hazard Assessment and Working Party of the National Test Guidelines program. EPA is also an active participant in the Society for the Advancement of Adverse Outcome Pathways, which promotes and advances scientific research that fosters the development and use of AOPs. The Society hosts the AOP-Wiki, which is one component of a larger OECD-coordinated AOP Knowledgebase, also supported by EPA. EPA scientists were among the speakers at a 2023 workshop on AOP-Wiki 3.0, which was presented in two sessions in July and August 2023.

EPA and NIEHS also participate with international partners in the Methods2AOP Collaboration, which seeks to develop an infrastructure to accommodate the integration of defined method details with key events in AOPs, including molecular initiating events. This connection between specific test methods/assays and key events in AOPs can facilitate improved interpretation of likely chemical hazards of substances shown to elicit a given assay or endpoint response. A poster describing the Methods2AOP Collaboration (Wittwehr et al.) was presented at the 2023 annual meeting of the Society of Toxicology, and collaboration leader Clemens Wittwehr was profiled in an article in the September 2022 NIEHS Environmental Factor newsletter. Ongoing activities of the collaboration include development of a prototype for the next version of the AOP-Wiki to demonstrate functionalities and data model improvements that better reflect the importance of test method information in the AOP Framework. This includes an increased focus on method validation and readiness status, better communication of various stakeholder roles, and the introduction and implementation of ontologies to tag methods in a way that makes them better identifiable in neighboring initiatives (e.g., the OECD test guidelines program). An outreach event for regulators is planned April 2024 and a second version of the M2AOP prototype is under development. The collaboration will provide recommendations to OECD beyond information and communications technology aspects, as well as a publication summarizing activities and outcomes to be submitted to a peer-reviewed journal.

ICCVAM agency scientists participate in programs coordinated by the Health and Environmental Sciences Institute (HESI). HESI collaboratively identifies and helps to resolve global health and environmental challenges through the engagement of scientists from academia, government, industry, nongovernmental organizations, and other strategic partners. During 2022 and 2023, ICCVAM agency scientists participated in these HESI projects.

• The Emerging Systems Toxicology for the Assessment of Risk (eSTAR) Committee was established to develop and deliver innovative systems toxicology approaches for risk assessment. In 2023, this committee launched the Omics for Assessing Signatures for Integrated Safety Consortium to increase confidence in the use of techniques such as imaging-based phenomics, transcriptomics, and proteomics for chemical safety assessment. The consortium has assembled a list of over 1000 candidate molecules for testing in Cell Painting, high-throughput transcriptomics and high-throughput proteomics assays. These molecules have liver toxicity data from traditional animal studies and/or human clinical studies. Data generation from in vitro screening studies is scheduled to begin in summer of 2024.
• The eSTAR Committee is also coordinating an effort to develop and qualify biomarker gene expression panels that measure widely accepted molecular pathways linked to tumorigenesis and their activation levels to predict tumorigenic doses of chemicals from short-term exposures (Corton et al. 2022). Success from these efforts will facilitate the transition from current heavy reliance on conventional two-year rodent carcinogenicity studies to more rapid animal- and resource-sparing approaches for mechanism-based carcinogenicity evaluation supporting internal and regulatory decision-making.
• The HESI eSTAR Molecular Point-of-Departure workgroup published a paper that establishes the scientific principles underpinning the genomic dose-response approach being investigated by the group (Johnson et al. 2022). Ongoing efforts are focused on a manuscript that describes the current standards for data analysis and identifies points of uncertainty in relation to analysis pipeline parameter selections and definition of terms (e.g., concerted molecular change). The data analysis standards paper will provide background information and a starting point for a workshop, tentatively planned for late 2025, with the goal of codifying best practices in genomic dose-response analysis and molecular POD determination.
• The HESI eSTAR microRNA biomarkers workgroup continued to develop biomarkers based on extracellular microRNA released by region-specific nephron cells that are indicative of toxicity due to chemical or drug exposure. The current project builds on foundational work in a rat model that established kidney microRNA release into urine corresponding to subregion-specific expression that indicated nephrotoxicity due to drug exposure (Chorley et al. 2021). A manuscript currently in review describes a project started in 2022 studying temporal modulation of primate urinary microRNA biomarkers after mild nephrotoxicity. NAMs development work has focused on proximal tubule microRNA release in a human primary cell culture media after nephrotoxicant exposure.
• The Genetic Toxicology Technical Committee is working to improve the scientific basis of the interpretation of results from genetic toxicology tests for more accurate hazard identification and assessment of human risk and develop follow-up strategies for determining the relevance of test results to human health. The committee’s In Vitro Working Group is critically evaluating NAMs for in vitro genotoxicity testing and envisioning how NAMs could expand current in vitro genetic toxicology testing strategies. A recent publication reports on a case study on 31 reference chemicals to evaluate the performance of IVIVE application to genotoxicity data (Beal et al. 2023). The group will make recommendations for creating an “in vitro only” approach for genetic toxicology testing that would meet the needs of various regulatory decision-makers.
• The Transforming the Evaluation of Agrochemicals Committee seeks to develop fit-for-purpose safety evaluation for agrochemicals that is applicable to changing global, as well as local, needs for evaluation and regulatory decisions that can incorporate relevant evolving science inputs. The group has been assessing the use of various test guidelines across different regulatory agencies in the context of agrochemical safety assessment and compiling information from industry members on the types of NAMs typically used.
• The HESI PBPK Models Committee aims to address key needs related to PBPK modeling practices and applications that could facilitate use of PBPK models more consistently within the risk assessment context. The group has been working to develop PBPK templates and frameworks, as well as evaluate and address commonly raised technical and scientific issues related to the use of toxicokinetic data for dose selection and/or interpretation of results in toxicity testing. The PBPK Models Committee is also collaborating with the Transforming the Evaluation of Agrochemicals Committee to evaluate the utility of the subchronic dog study for agrochemical safety evaluation.

For over four years, FDA has maintained an Alternative Methods Working Group with representatives from all of FDA. The goals of this working group are to:

• Strengthen FDA’s long commitment to promoting the development and use of new technologies and to reduce animal testing.
• Discuss new alternative in vitro/in silico/in vivo methods across FDA.
• Interact with federal government partners and other global stakeholders to facilitate discussion and development of draft performance criteria for such assays.

During 2022 and 2023, the working group continued to coordinate an internal webinar series on alternative methods that provided test method developers the opportunity to present their new methods to FDA scientists. The group also updated FDA webpages on alternative methods to provide a more comprehensive and user-friendly resource for this information. Two 2022 reports are available from these pages:

• The Focus Areas of Regulatory Science (FARS) report, updated in 2022, outlines areas FDA has identified as needing continued targeted investment in regulatory science research to foster the development of innovative products, provide data and methods to inform regulatory decision-making, and improve guidance to sponsors. One focus area is novel technologies to improve predictivity of nonclinical studies and replace, reduce, and refine animal testing.
• The November 2022 report, Successes and Opportunities in Modeling & Simulation for FDA, elucidates in part how and where modeling and simulation are used across FDA.

Animal safety studies are usually performed with three groups of animals where increasing amounts of the test chemical are given to the animals and one control group where the animals do not receive the test chemical. The design of such studies, the characteristics of the animals, and the measured parameters are often very similar from study to study. Therefore, it has been suggested that measurement data from the control groups could be reused from study to study to lower the total number of animals per study. This could reduce animal use by up to 25% for such standardized studies. FDA and NIEHS scientists joined a broad range of stakeholder groups at a March 2023 workshop to discuss the pros and cons of such a concept and what would have to be done to implement it without threatening the reliability of the study outcome or the resulting human risk assessment (Golden et al. 2023). Workshop participants concluded that implementation of virtual control groups could support a better understanding of nonclinical data, increased study power, and reduced animal use, making such an approach an attractive alternative to running concurrent controls. However, before use of virtual control groups can be fully implemented, there is work to be done to increase confidence in their utility. In particular, any implementation must be fully validated in a manner that does not compromise study interpretation. It became evident during the workshop that overcoming the identified hurdles and challenges will require a joint effort of all stakeholders, ideally in a consortium approach.

Complex in vitro models such as MPS offer the potential to improve pharmaceutical clinical drug attrition due to safety and/or efficacy concerns. For this technology to have an impact, robust characterization and qualification plans constructed around specific contexts of use need to be establish. To address this need, FDA and the pharmaceutical industry Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate convened a 2020 workshop. The workshop considered through various case studies both how these technologies are currently being applied by pharmaceutical companies during drug development and how they are being tested at the FDA. The goal was to identify hurdles (real or perceived) to the adoption of MPS technologies and to address evaluation/qualification pathways for these technologies. Output from the workshop (Baran et al. 2022) included a working definition of MPS, a detailed description of 11 case studies, and in-depth analysis. The report also includes key take aways from breakout sessions on ADME, pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of data from complex in vitro models. IQ MPS Affiliate and FDA scientists reached a general consensus on the need for animal models for preclinical species to better determine species concordance. Furthermore, there was acceptance that technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific context of use.

In 2019, FDA, NIEHS, and HESI signed a memorandum of understanding establishing the Botanical Safety Consortium. The Consortium includes participants from industry, academia, and government. It considers how to use cutting-edge toxicology tools, including alternatives to animal testing, to promote scientific advances in evaluating the safety of botanical ingredients and mixtures in dietary supplements.

The Botanical Safety Consortium held virtual annual meetings in 2022 and 2023. The 2022 meeting considered topics including supplement-induced liver injury, approaches to screening complex mixtures, and the role of analytical methods. In addition to another look at screening approaches, speakers at the 2023 meeting discussed global research and regulatory activities. The Consortium also presented webinars in January and November 2022, and participants gave presentations at meetings of SOT, Environmental Genomics and Mutagen Society, and other toxicology organizations.

MPS comprise several bioengineering breakthroughs that reproduce organ architecture and function in vitro. Fueled by stem cell technologies, a broad variety of human models and test systems have emerged. Convening of global conferences on MPS was identified by opinion leaders in the field as a key step forward in the maturation and harmonization of the area. In response, international companies and organizations teamed up in 2021 to initiate annual MPS World Summits to present the latest scientific achievements, discuss advances and challenges, and enable communication between young and newly interested scientists and pioneers of the MPS field. Scientists from FDA, NIEHS, and NIH spoke at the 2022 and 2023 MPS World Summits.

The 2021 and 2022 MPS World Summits also laid the groundwork for establishment in 2023 of the International MPS Society. The Society’s mission is to connect, exchange, and educate to promote international standardization and harmonization of MPS, establish a global training environment, and maximize the potential of MPS to advance the life sciences. ICCVAM co-chair Suzanne Fitzpatrick (FDA) serves on the Society’s governing board, as does Danilo Tagle of NCATS (NIH). ICCVAM member Donna Mendrick (FDA) and FDA scientist Carolina Romero serve on the Society’s scientific advisory committee.

NIEHS and EPA are fostering a community-driven initiative, the Environmental Health Language Collaborative, to advance integrative environmental health research by developing and promoting adoption of a harmonized language. This initiative will facilitate answering large-scale complex research questions that require integration of multiple disparate data sources by developing language standards for describing data and biomedical knowledge. The collaborative’s website has resources including a glossary, bibliography, links to ontologies, and overview videos.

The collaborative presented six webinars during 2022 and 2023 and held a three-day workshop in early 2023 on “Sharing Your Environmental Health Sciences Data: Metadata, Standards, and Tools.” Videos and materials from the webinars and workshops are available on the collaborative’s website. The collaborative also established use-case working groups to explore language solutions that will enhance the findability, reuse, and/or operability of environmental health and safety data. Current working groups are focused on the use cases of “Biomarkers and Biological Processes of Exposure,” “Data Discovery,” and “Data Harmonization.” All working groups meet periodically and welcome new members.