Trust your gut: Establishing confidence in gastrointestinal models - An overview of the state of the science and contexts of use.

The webinar series and workshop titled "Trust Your Gut: Establishing Confidence in Gastrointestinal Models ­ An Overview of the State of the Science and Contexts of Use" was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)- related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and pro­cesses occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from dif­ferent countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.

Defined approaches to classify agrochemical formulations into EPA hazard categories developed using EpiOcularTM reconstructed human corneal epithelium and bovine corneal opacity and permeability assays.

Many sectors have seen complete replacement of the in vivo rabbit eye test with reproducible and relevant in vitro and ex vivo methods to assess the eye corrosion/irritation potential of chemicals. However, the in vivo rabbit eye test remains the standard test used for agrochemical formulations in some countries. Therefore, two defined approaches (DAs) for assessing conventional agrochemical formulations were developed, using the EpiOcularTM Eye Irritation Test (EIT) [Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 492] and the Bovine Corneal Opacity and Permeability (OECD TG 437; BCOP) test with histopathology. Presented here are the results from testing 29 agrochemical formulations, which were evaluated against the United States Environmental Protection Agency's (EPA) pesticide classification system, and assessed using orthogonal validation, rather than direct concordance analysis with the historical in vivo rabbit eye data. Scientific confidence was established by evaluating the methods and testing results using an established framework that considers fitness for purpose, human biological relevance, technical characterisation, data integrity and transparency, and independent review. The in vitro and ex vivo methods used in the DAs were demonstrated to be as or more fit for purpose, reliable and relevant than the in vivo rabbit eye test. Overall, there is high scientific confidence in the use of these DAs for assessing the eye corrosion/irritation potential of agrochemical formulations.

Weight of evidence for cross-species conservation of androgen receptor-based biological activity.

The U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP) is tasked with assessing chemicals for their potential to perturb endocrine pathways, including those controlled by androgen receptor (AR). To address challenges associated with traditional testing strategies, EDSP is considering in vitro high-throughput screening assays to screen and prioritize chemicals more efficiently. The ability of these assays to accurately reflect chemical interactions in nonmammalian species remains uncertain. Therefore, a goal of the EDSP is to evaluate how broadly results can be extrapolated across taxa. To assess the cross-species conservation of AR-modulated pathways, computational analyses and systematic literature review approaches were used to conduct a comprehensive analysis of existing in silico, in vitro, and in vivo data. First, molecular target conservation was assessed across 585 diverse species based on the structural similarity of ARs. These results indicate that ARs are conserved across vertebrates and are predicted to share similarly susceptibility to chemicals that interact with the human AR. Systematic analysis of over 5000 published manuscripts was used to compile in vitro and in vivo cross-species toxicity data. Assessment of in vitro data indicates conservation of responses occurs across vertebrate ARs, with potential differences in sensitivity. Similarly, in vivo data indicate strong conservation of the AR signaling pathways across vertebrate species, although sensitivity may vary. Overall, this study demonstrates a framework for utilizing bioinformatics and existing data to build weight of evidence for cross-species extrapolation and provides a technical basis for extrapolating hAR-based data to prioritize hazard in nonmammalian vertebrate species.

Data curation to support toxicity assessments using the Integrated Chemical Environment.

Humans are exposed to large numbers of chemicals during their daily activities. To assess and understand potential health impacts of chemical exposure, investigators and regulators need access to reliable toxicity data. In particular, reliable toxicity data for a wide range of chemistries are needed to support development of new approach methodologies (NAMs) such as computational models, which offer increased throughput relative to traditional approaches and reduce or replace animal use. NAMs development and evaluation require chemically diverse data sets that are typically constructed by incorporating results from multiple studies into a single, integrated view; however, integrating data is not always a straightforward task. Primary study sources often vary in the way data are organized and reported. Metadata and information needed to support interoperability and provide context are often lacking, which necessitates literature research on the assay prior to attempting data integration. The Integrated Chemical Environment (ICE) was developed to support the development, evaluation, and application of NAMs. ICE provides curated toxicity data and computational tools to integrate and explore available information, thus facilitating knowledge discovery and interoperability. This paper describes the data curation workflow for integrating data into ICE. Data destined for ICE undergo rigorous harmonization, standardization, and formatting processes using both automated and manual expert-driven approaches. These processes improve the utility of the data for diverse analyses and facilitate application within ICE or a user's external workflow while preserving data integrity and context. ICE data curation provides the structure, reliability, and accessibility needed for data to support chemical assessments.

Analysis of variability in the rabbit skin irritation assay.

The in vivo rabbit test is the benchmark against which new approach methodologies for skin irritation are usually compared. No alternative method offers a complete replacement of animal use for this endpoint for all regulatory applications. Variability in the animal reference data may be a limiting factor in identifying a replacement. We established a curated data set of 2624 test records, representing 990 substances, each tested at least twice, to characterize the reproducibility of the in vivo assay. Methodological deviations from guidelines were noted, and multiple data sets with differing tolerances for deviations were created. Conditional probabilities were used to evaluate the reproducibility of the in vivo method in identification of U.S. Environmental Protection Agency or Globally Harmonized System hazard categories. Chemicals classified as moderate irritants at least once were classified as mild or non-irritants at least 40% of the time when tested repeatedly. Variability was greatest between mild and moderate irritants, which both had less than a 50% likelihood of being replicated. Increased reproducibility was observed when a binary categorization between corrosives/moderate irritants and mild/non-irritants was used. This analysis indicates that variability present in the rabbit skin irritation test should be considered when evaluating nonanimal alternative methods as potential replacements.

Exploring current read-across applications and needs among selected U.S. Federal Agencies.

Read-across is a well-established data gap-filling technique applied for regulatory purposes. In US Environmental Protection Agency's New Chemicals Program under TSCA, read-across has been used extensively for decades, however the extent of application and acceptance of read-across among U.S. federal agencies is less clear. In an effort to build read-across capacity, raise awareness of the state of the science, and work towards a harmonization of read-across approaches across U.S. agencies, a new read-across workgroup was established under the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). This is one of several ad hoc groups ICCVAM has convened to implement the ICCVAM Strategic Roadmap. In this article, we outline the charge and scope of the workgroup and summarize the current applications, tools used, and needs of the agencies represented on the workgroup for read-across. Of the agencies surveyed, the Environmental Protection Agency had the greatest experience in using read-across whereas other agencies indicated that they would benefit from gaining a perspective of the landscape of the tools and available guidance. Two practical case studies are also described to illustrate how the read-across approaches applied by two agencies vary on account of decision context.

Skin sensitization testing needs and data uses by US regulatory and research agencies.

United States regulatory and research agencies may rely upon skin sensitization test data to assess the sensitization hazards associated with dermal exposure to chemicals and products. These data are evaluated to ensure that such substances will not cause unreasonable adverse effects to human health when used appropriately. The US Consumer Product Safety Commission, the US Environmental Protection Agency, the US Food and Drug Administration, the Occupational Safety and Health Administration, the National Institute for Occupational Safety and Health, and the US Department of Defense are member agencies of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). ICCVAM seeks to identify opportunities for the use of non-animal replacements to satisfy these testing needs and requirements. This review identifies the standards, test guidelines, or guidance documents that are applicable to satisfy each of these agency's needs; the current use of animal testing and flexibility for using alternative methodologies; information needed from alternative tests to fulfill the needs for skin sensitization data; and whether data from non-animal alternative approaches are accepted by these US federal agencies.

International regulatory requirements for skin sensitization testing.

Skin sensitization test data are required or considered by chemical regulation authorities around the world. These data are used to develop product hazard labeling for the protection of consumers or workers and to assess risks from exposure to skin-sensitizing chemicals. To identify opportunities for regulatory uses of non-animal replacements for skin sensitization tests, the needs and uses for skin sensitization test data must first be clarified. Thus, we reviewed skin sensitization testing requirements for seven countries or regions that are represented in the International Cooperation on Alternative Test Methods (ICATM). We noted the type of skin sensitization data required for each chemical sector and whether these data were used in a hazard classification, potency classification, or risk assessment context; the preferred tests; and whether alternative non-animal tests were acceptable. An understanding of national and regional regulatory requirements for skin sensitization testing will inform the development of ICATM's international strategy for the acceptance and implementation of non-animal alternatives to assess the health hazards and risks associated with potential skin sensitizers.