ICCVAM Biennial Report 2024-2025

PFAS are organofluorine chemicals that have been manufactured and used for decades in products and applications including firefighting foams, food packaging, and stain- and water-resistant fabrics and carpeting. PFAS are highly persistent and bioaccumulate in the environment, biota, and humans, but understanding PFAS hazards remains challenging. There is no consensus definition of PFAS, and their large variety of chemical structures and toxicological and physiochemical properties makes hazard prediction extremely challenging.

ICCVAM established a workgroup in 2023 to provide expertise in identifying and evaluating NAMs to predict PFAS toxicity. The workgroup includes members from nine ICCVAM member agencies. The group’s charges include:

• Evaluate current PFAS definitions and groupings
• Assess the utility of new approach methodologies (NAMs) currently being used by national and international regulatory agencies for testing and risk assessment of specific PFAS based on case examples of successful application of certain NAMs
• Identify research challenges and data gaps in the use of specific NAMs for testing and risk assessment of different PFAS
• Identify NAMs that have not been used for PFAS and explore opportunities for expanding their application for PFAS testing and risk assessment

The workgroup drafted a white paper that identifies specific challenges and data gaps that need to be addressed for NAMs implementation for PFAS testing. It notes that NAMs encompassing a range of in vitro and computational approaches will be important sources of data for initial characterization and prioritization of PFAS, and makes specific recommendations on how initial efforts should be directed. The white paper is being reviewed by participating agencies and will be published in 2026.

The ICCVAM PFAS Workgroup will host a workshop to address issues identified in the white paper, with findings from the workshop presented in a published manuscript.

While MPS technologies are gaining traction as a viable alternative model for toxicity studies, further characterization is necessary to explore their full translational potential as well as the utility of these platforms to serve as a diagnostic tool. For human-relevant omics data to be truly reliable, samples must be collected and analyzed from patients or individuals infected by or exposed to the drugs or agents in question. U.S. Army CBC scientists investigated how well bioinformatic data collected from organ chips match bioinformatic data collected from human samples, with the aim of using organ chips as an alternative approach for biomarker discovery in toxicity assessment and exposure identification. To do this, TissUse Chip3 multiorgan chips seeded with kidney organoids, liver organoids, and full-thickness lung tissue were exposed to acetaminophen and analyzed to characterize effects on protein content and metabolism. Data from the organ chips largely matched the data in the published literature, including the identification of several known acetaminophen metabolites and biotransformation products. These data suggest that organ chips may be a suitable surrogate for human biomarker identification and drug exposure diagnosis. A paper describing this work is currently under review with the Journal of Proteome Research. Follow-up work that aims to establish animal organ chips and examine their translational potential was recently funded. Currently, CBC is developing rat and nonhuman primate multiorgan chip systems and will compare the effects of acetaminophen on the incorporated tissues to results reported in historic and published literature.

The individuals listed on this page served as representatives from ICCVAM member agencies in 2024 and 2025. ICCVAM includes three types of representatives:

• The Principal Agency Representative is the primary source of communication from his or her agency to the committee and NICEATM. He or she regularly attends all ICCVAM meetings and teleconferences, coordinates participation of other representatives from the agency, and casts the agency’s vote on occasions when formal voting is required.
• Alternate Agency Representatives participate in ICCVAM committee activities in place of the principal when necessary.
• An agency may designate one or more Other Agency Representatives to participate in ICCVAM to provide broader representation or expertise.

Rapid advancements in engineering have driven the need to understand human exposure to ultrafine particles and volatiles. To this end, U.S. Air Force (USAF) scientists developed an in vitro inhalation model to quickly assess toxicity. An alveolus-on-a-chip platform was developed using primary alveolar epithelial and microvascular endothelial cells cultivated at the air–liquid interface. The platform incorporates cyclical stretch and breathing mechanics to better emulate human physiology in a laboratory model. This model has been utilized to identify unique biomarkers of soot-based exposures, an emerging respiratory global concern to human health and performance. Initial results after treating the alveolus-on-a-chip platform with diesel particulate matter resulted in increased cytotoxicity and a statistically significant decrease in lung–blood barrier integrity. Extracellular vesicles isolated from particulate matter-exposed alveolus-on-a-chip platforms exhibited increased RNA content compared to the controls, suggesting that exposure alters the signaling cargo released by the lung. These exosomes were successfully labeled and traced for uptake by recipient cells. Multiple downstream models, including liver, skin, and immune cell systems, showed significant exosome uptake, supporting a mechanism for systemic communication beyond the lung following alveolar exposure. RNA from the recipient models after exosome exposure was isolated for global transcriptomic profiling. Investigators now aim to characterize pathways by which soot-based exposures drive whole-body effects through exosome-mediated transfer of molecular information to aid in biomarker identification for countermeasure development.

DOT regulates hazardous materials to ensure safe packaging and handling of these materials in transport. Recent updates to DOT regulations align them with international regulations that enable the reduction of animal use for the purposes of classifying potential Class 8 (corrosive) materials. These updates resulted from ongoing collaborations with the United Nations to expand options for NAMs for classifying hazardous materials for transportation.

The Final Rule for HM-215Q (89 FR 25434) was published in April 2024 and incorporates in vitro test methods described in Test Guideline (TG) 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” issued by the Organisation for Economic Co-operation and Development (OECD) to allow a material to be classified or not as Class 8. DOT also revised 49 CFR 173.137 in the U.S. Code of Federal Regulations to allow corrosive materials to be assigned to the most conservative applicable packing group without further testing when classified using test methods that cannot distinguish between each packing group. This followed the 2022 publication of the Final Rule for HM-215P (87 FR 44944), which allowed the use of in vitro test methods described in OECD TG 431 to classify materials as corrosives.

The 1985 amendments to the Animal Welfare Act established the Animal Welfare Information Center (AWIC), an information service at USDA's National Agricultural Library. AWIC’s mission is to provide information pertinent to employee training that could prevent unintended duplication of animal experimentation and on improved research methods that could reduce or replace animal use or minimize pain and distress to animals (7 USC § 2143(e)).

In 2024 and 2025, AWIC continued to strengthen its support for the toxicology community by offering information on alternative methods in ecotoxicity testing and by maintaining resources that highlight mandatory and regulatory testing guidelines across federal agencies. The 3Rs in Ecotoxicity Testing webpage features curated searches for NAMs in the PubAg and PubMed databases, as well as a bibliography that was updated in September 2025 and provides access to nearly 800 abstracts on alternative testing approaches.

AWIC’s website received substantial use during this two-year period, with 308,733 visitors generating 542,858 page views. These users accessed a variety of materials designed to facilitate the identification and application of animal use alternatives, especially within toxicology and related research disciplines.

In addition to online resources, AWIC engaged directly with the scientific and regulatory communities through presentations, posters, workshops, webinars, and conference exhibits. Across 40 outreach events in 2024 and 2025, AWIC interacted with approximately 3,191 animal researchers, veterinarians, Institutional Animal Care and Use Committee members, and others. These engagements emphasized best practices for conducting comprehensive literature searches, locating current research on alternative methods, and applying 3Rs principles within testing and experimentation, including toxicology contexts.

AWIC also continued to offer its workshops on meeting the requirements of the Animal Welfare Act. These sessions provided attendees with guidance on where to find literature and resources on the 3Rs, instruction on database literature search techniques (e.g., search syntax), and demonstrations of how to conduct literature searches to address requirements for consideration of NAMs. Four workshops were presented during 2024 and 2025, reaching a total of 927 participants. For those unable to attend live sessions, AWIC maintained access to online, on-demand training through its events webpage.

To reduce animal use in the investigation of human-relevant inhalation toxicity, the NIEHS Division of Translational Toxicology is evaluating the use of in vitro lung models, including air–liquid interface airway tissues. Potential applications of these models include screening-level assessments to help predict the adverse airway effects of inhaled substances and prioritize them for further testing.

Initially, proof-of-concept studies were conducted to characterize and optimize the VITROCELL 48 2.0 Plus exposure system with human and rat tracheobronchial-derived air–liquid interface airway tissues. The chemical used was 2,3-pentanedione, a highly volatile component of artificial butter flavorings that has been associated with airway injury and fibrosis via occupational inhalation exposure. The studies were designed to explore the validity of extrapolations among rat in vivo, rat in vitro, and human in vitro airway effects in these models. They showed that exposure of the air–liquid interface tissues to 2,3-pentanedione vapor induces concentration-dependent changes in relevant airway toxicity endpoints. Changes in transepithelial electrical resistance, cytotoxicity, histopathology, and secreted biomarkers were comparable between species for most of the endpoints. Measurements of 2,3-pentanedione concentration were also performed on apical wash, basolateral culture media, and tissue lysate samples as indicators of dosimetry. One paper describing these studies has been submitted for publication and a second is in preparation. Further method development and optimization work are also in progress, including the use of alveolar-derived air–liquid interface models and incorporation of transcriptomics.

To help better assess the utility and application of in vitro lung (including air–liquid interface) models, NIEHS issued a Request for Information in April 2024 to gather information on the use of these models in inhalation toxicology research including knowledge gaps and technical challenges and limitations. Most responders indicated that they currently use in vitro lung models for inhalation/respiratory toxicity screening, although many also use them for chemical-specific mechanistic studies or IVIVE modeling. There is also interest in developing and validating the models for regulatory decision-making. The primary challenge or limitation identified in the use of these models was reproducibility (encompassing both data variability and method standardization), but other concerns included dosimetry (including test article generation and exposure), physiological/mechanistic relevance (model complexity, or lack thereof), model validation, and translational relevance/prediction (correlating in vitro model findings to data in vivo and in exposed humans).

To address the reproducibility concern, the NIEHS Division of Translational Toxicology has assembled a steering committee to oversee an interlaboratory reproducibility study that will involve multiple independent testing facilities. The steering committee is determining test article generation and exposure systems and the source of air–liquid interface airway tissues used, appropriate chemistry and toxicological endpoints, standardizing the protocol according to the approach used by the Division of Translational Toxicology testing facility, formulating a plan for statistical analyses, and selecting three to five partnering facilities (federal or nonfederal) to conduct testing using 2,3-pentanedione.

EPA currently relies on animal testing, specifically rat studies, to determine the LD50 (the dose of a substance that causes death in 50% of test animals) for pesticide safety assessments. This information is used for pesticide toxicity labeling and environmental risk assessment. To reduce animal use, a consortium of international researchers led by NICEATM developed a computer-based tool called the Collaborative Acute Toxicity Modeling Suite (CATMoS; Mansouri et al. 2021). CATMoS uses QSAR modeling to predict acute oral toxicity (LD50) values for chemicals.

EPA and NIEHS researchers compared CATMoS predictions to real-world animal data for 177 pesticides (Bishop et al. 2024). The tool was evaluated for its ability to classify chemicals into the EPA’s acute toxicity categories and accurately predict LD50 values. Results showed that CATMoS was highly reliable at categorizing substances into EPA toxicity Categories III (moderate toxicity, 500–5000 mg/kg) and IV (low toxicity, over 5000 mg/kg), with an impressive 88% agreement between CATMoS predictions and animal test data for chemicals with LD50 values over 500 mg/kg. Additionally, for chemicals with LD50 values of 2000 mg/kg or higher, CATMoS predictions closely matched outcomes from high-dose animal tests.

These findings demonstrate that CATMoS is a reliable tool for predicting pesticide toxicity, particularly for less-toxic substances. By using CATMoS, regulators and industry can reduce the need for animal testing, making pesticide safety evaluations more ethical while still ensuring accurate risk assessments. This work represents a significant step forward in modernizing toxicity testing methods.

Organophosphorus flame retardants (OPFRs) are widely used chemicals that persist in the environment, yet limited information regarding their potential toxicity exists. Their structural similarity to organophosphate pesticides raises concerns about developmental neurotoxicity (DNT), particularly for human health. However, traditional in vivo testing methods are not suitable for evaluating the wide range of OPFRs that lack safety data.

To address this gap, NIEHS researchers applied an in vitro testing battery designed to assess key processes in neurodevelopment and function (Kreutz et al. 2024). Behavioral data from small model organisms were also included. This study used an IATA combining data from experimental assays, the NIH Integrated Chemical Environment (NICE) database, and existing literature. Human exposure data and physiologically based toxicokinetic models were used to compare in vitro toxicity thresholds with predicted human plasma concentrations.

Eight OPFRs, including aromatic OPFRs and halogenated flame retardants, were evaluated alongside two brominated flame retardants known to cause DNT. Results showed that several aromatic OPFRs (e.g., triphenyl phosphate and isodecyl diphenyl phosphate) demonstrated activity at concentrations comparable to the toxic brominated flame retardants, suggesting a need for further investigation. While the in vitro assays highlighted potential hazards, they provided limited insights into mechanisms of toxicity. By integrating additional information, endocrine disruption was identified as a potential mechanism of action for some OPFRs.

This study underscores the utility of an IATA for prioritizing chemicals of concern. It also found that human exposure to some OPFRs may result in plasma concentrations similar to those that trigger activity in vitro, raising concerns about potential human health risks. These findings provide a foundation for future investigations into OPFR safety and the mechanisms driving their neurotoxicity.

PFAS are organofluorine chemicals manufactured and used for decades in products and applications such as firefighting foams. PFAS are highly persistent and bioaccumulative in the environment, biota, and humans, and concern about their health effects has driven efforts to identify and evaluate possible substitutes. Scientists at the Defense Centers for Public Health used in silico and in vitro approaches to predict toxicity of six fluorine-free firefighting formulations were assessed for toxicity. Ingredient information provided by the manufacturers missed up to 70% of the constituents per formulation. QSARs were completed for some of the known ingredients, and in vitro assays (the Ames test for genotoxicity, the Microtox test for cytotoxicity, and the EpiDerm assay for skin irritation) were conducted to fill data gaps to help rank the toxicity of these formulations before progression to in vivo tests. Direct comparisons of these products through these QSARs and in vitro screens provided early data to prevent potential regrettable substitutions when selecting alternative PFAS-free firefighting foams.

Neurotoxicity is of particular concern for the military due to the potential for cognitive, behavioral, and physiological effects. With an ever-evolving operational landscape, de novo generation of in vivo data for all potential neurotoxicants is not feasible. USAF Predictive Risk Team (PRT) evaluated the utility of high-throughput new approach methodologies (NAMs) for rapid risk assessment of 220 potentially neurotoxic chemicals. Chemical-specific point-of-departure (PODs) were derived from novel in vitro neuronal assay data and ToxCast bioactivity concentrations. The AC50 (concentration with 50% maximum activity) was derived for each chemical/assay combination and PODs were calculated from the 5th percentile of the AC50 values. In vitro to in vivo extrapolation (IVIVE) was performed using the EPA httk model to estimate the equivalent administered dose (EAD) for each POD. Monte Carlo calculations from httk were modified to reflect USAF active-duty demographics. Neurotoxicity-associated endpoints produced higher PODs compared to all CompTox in vitro endpoints combined. In vitro-derived EADs were lower than in vivo-derived PODs for most of the chemicals in both the U.S. and USAF populations. In vitro-derived EADs were also lower than 45% of provisional reference doses calculated using one-year Military Exposure Guideline procedures. An uncertainty factor of 1000 results in conservative estimated exposure limits (vs. Military Exposure Guidelines) for almost all the chemicals. This case study supports the use of NAMs to derive conservative PODs for risk assessment. A manuscript is currently being prepared for publication in 2024.

Cardiovascular disease is the leading cause of death for people of most ethnicities in the United States. In a pioneering effort to minimize animal testing to evaluate chemicals for potential cardiotoxicity, NIEHS scientists applied new approach methodologies (NAMs) that blend in vitro, in chemico, and in silico methods to investigate potential cardiotoxicity of over 800 substances. These substances, characterized by widespread human exposure, included personal care product ingredients, flame retardants, herbicides, pesticides, pharmaceuticals, and industrial byproducts. A systems-based modeling workflow including PBPK models was used to transform data from molecular and cellular assays relevant to cardiovascular endpoints into human daily equivalent administered doses (EADs). The study compared these in vitro-derived EADs against both human exposure predictions and in vivo toxicological data to gauge human-relevant risks. It also integrated geospatial analyses to evaluate the compounded risks across diverse U.S. populations, spotlighting communities at disproportionate risk. Through this comprehensive approach, which merged high-throughput screening (HTS) assays, PBPK modeling, and exposure data, the project aimed to refine human health risk assessments for chemicals posing cardiovascular hazards. This endeavor marks a significant stride in transitioning from molecular insights to actionable public health interventions, striving to replace animal testing with human biology-based strategies for chemical safety evaluation. A paper describing this work will be submitted for publication in 2024.

Formaldehyde is an irritating, highly reactive aldehyde that is widely believed to cause asthma. Additionally, it is classified as a Group 1 carcinogen by NTP and the International Agency for Research on Cancer, and airborne formaldehyde exposure is associated with nasal cancer in animals and humans. FDA-regulated products may release formaldehyde fumes and present toxicity risks to patients and healthcare workers. Airway epithelium is a key boundary between the environment and mammalian systemic circulation. To study tissue responses to formaldehyde fumes in a non-animal system, FDA scientists used an in vitro human ALI airway tissue model (Ren et al. 2022). In the model, exposure to formaldehyde induced functional changes in cells as well as possible squamous differentiation. Although DNA damage was not detected in a comet assay, formaldehyde exposures lowered the rate of DNA repair enzymes suggesting that it interferes with DNA repair ability. A general agreement was observed between in vitro responses to formaldehyde fumes and the reported in vivo toxicity of formaldehyde, supporting the application of the ALI airway system as a potential in vitro alternative for screening and evaluating the respiratory toxicity of inhaled substances.

FDA’s Center for Food Safety and Applied Nutrition (CFSAN) partnered with organ chip developer Emulate to evaluate the utility of their Beta Human Liver Emulation System for its regulatory science program (Eckstrum et al. 2022). The platform's performance was evaluated using both known hepatotoxic compounds and compounds that have no reported human cases of liver toxicity. Toxicity was assessed by albumin secretion, urea and lactate dehydrogenase release, nuclei number, mitochondrial membrane potential, and apoptosis. System/platform performance was evaluated in terms of sensitivity and specificity, power, and variability and repeatability. Chemical interactions with the chip material were also assessed. Preliminary findings suggested that for the model test compounds selected, the system accurately predicted toxicity, demonstrated high sensitivity and specificity, high power, and low variability. This evaluation of the Beta Human Liver Emulation System demonstrated that it was easily transferred to the CFSAN laboratory. However, some compounds interacted with the chip material resulting in variable exposure levels that should be accounted for when planning experimentation.

The EPA’s Endocrine Disruptor Screening Program employs a two-tiered approach. Tier 1 uses 11 screening assays to identify the potential of a chemical substance to interact with the estrogen, androgen, or thyroid pathways. Tier 2 testing is designed to identify any adverse endocrine-related effects caused by the substance and establish a dose-response relationship for any adverse estrogen, androgen, or thyroid effect. Given the large number of chemical substances covered by the EDSP, research efforts have focused on the development and evaluation of new approach methodologies (NAMs) for use as alternatives to the current suite of assays in the EDSP Tier 1 battery to accelerate the pace of screening, add efficiencies, decrease costs, and reduce animal testing.

In January 2023, EPA released a draft whitepaper for public comment on the availability of NAMs in the EDSP that describes validated NAMs, including those incorporated into the estrogen receptor and androgen receptor pathway models, that EPA may now accept as alternatives for certain EDSP tests. Additional NAMs were identified for potential use in prioritization or for consideration as Other Scientifically Related Information in weight-of-evidence evaluations. After considering public comments, EPA will issue a finalized document.

Additionally, in October 2023, EPA announced the availability of near-term strategies to help the agency meet its obligations and commitments relating to the EDSP. As part of these strategies, results from the estrogen receptor and androgen receptor pathway models were utilized as part of a prioritization framework.

The Botanical Safety Consortium is a public-private partnership advancing the scientific study of botanical ingredients to improve safety assessments. The Consortium combines expertise from federal agencies, academia, and industry to address gaps in the reliable evaluation of botanical substances, which are gaining popularity as dietary supplements and consumer products. Recent updates from the Consortium highlight efforts to establish robust methods and frameworks for assessing botanical safety. These achievements reflect the Consortium’s commitment to improving the scientific foundation of botanical safety evaluation while supporting principles of replacement, reduction, and refinement of animal use.

• Identification of best practices for in vitro botanical safety testing: A group of Consortium researchers, including NIEHS scientists, reviewed methodologies for assessing botanical ingredients in vitro (Waidyanatha et al. 2024). The study underlined the importance of standardized protocols to reduce variability in test results and emphasized the critical role of understanding the complex interactions within botanicals to develop reliable testing strategies.
• Chemical identification to support botanical identity: A key activity of the Consortium is the compilation of data to support the identification of botanicals. These data are available via the National Toxicology Program’s Chemical Effects in Biological Systems database. Updates to the dataset in July 2024 added data for Ephedra sinica, and October 2024 updates added data on Nerium oleander and Aconitum napellus.
• Collaborative efforts and new resources: The Botanical Safety Consortium continues to expand its initiatives through Technical Working Group projects that integrate scientific advancements and promote data sharing. Currently active working groups focus on Chemical Analysis, Developmental and Reproductive Toxicity, Hepatotoxicity, Genotoxicity, Dermal Toxicity, Data Analysis, Cardiotoxicity, Neurotoxicity, and Pharmacognosy.

Skin sensitization is an allergic reaction triggered by chemical exposure, which can cause significant health issues. To ensure safety, regulatory frameworks rely on methods to assess both hazard, the likelihood of a chemical to be a sensitizer, and potency, a prediction of the severity of sensitization that could be caused by the chemical. Traditionally, sensitization hazard and potency have been estimated using animal models, such as the LLNA. However, advances in nonanimal methods, such as in chemico, in vitro, and in silico tests, have enabled more ethical and human-relevant approaches through integrated strategies such as DAs, which combine data from multiple nonanimal tests to predict sensitization potential.

A May 2025 reportsummarized a NICEATM evaluation of test substances nominated by ICCVAM member agencies. In the study, 181 “difficult” test substances of regulatory interest were tested in individual in chemico and in vitro methods, and the results applied to three DAs for predicting skin sensitization hazard or potency. The in vitro methods evaluated were the direct peptide reactivity assay, the KeratinoSens™ assay, and the human cell line activation test. The three DAs used were the Key Event 3/1 Sequential Testing Strategy, the Two-out-of-Three strategy, and the Integrated Testing Strategy. Predictions were compared against reference data from LLNAs and human predictive patch tests (HPPTs) to assess concordance.

Results showed varying concordance with human and LLNA reference data. Individual methods had concordance rates ranging from 30%–93% and 52%–78% with LLNA data and human data, respectively. The DAs demonstrated better agreement among themselves (70%–92%) and showed higher concordance with human reference data (42%–47%) compared to the LLNA (24%–51%). Importantly, the DAs proved to be more accurate predictors of human skin sensitization hazard and potency than LLNA methods.

These findings confirm that DAs provide a more reliable, efficient, and ethical alternative to animal models for assessing skin sensitization. By integrating multiple nonanimal methods, DAs improve human health predictions, supporting regulatory decision-making and reducing reliance on animal-based testing.

USAF Predictive Risk Team (PRT) is developing a new approach methodologies-based approach to support rapid risk assessment of novel and poorly characterized chemicals. A tiered approach is being evaluated, where lower tiers use in silico and high-throughput in vitro models to prioritize hazards, while higher tiers use organotypic in vitro models to perform quantitative risk assessments. To test the utility of this NAM-based approach on operationally relevant scenarios, researchers conducted a case study using several chemicals with known liver and central nervous system toxicity. The chemicals were assessed using the Edwards et al. 2022 in silico model (Tier 0) to evaluate mechanism of action and acute systemic toxicity. In vitro tests were then performed using assays appropriate for the mechanism of action identified by the in silico model (Tier 1A). Additional in vitro tests evaluated cell health (cell viability, cytokines, oxidative stress) in glial and liver monoculture models (Tier 1B). Finally, Tier 2 human liver microtissue (3DInsight) and central nervous system (CNS-3D, 28bio) organoid models were used to evaluate tissue functional effects. Points-of-departure (PODs) were derived using benchmark dose modeling, converted to human-equivalent doses using toxicokinetic modeling, and compared to traditional testing data. Human-equivalent doses were generally more conservative than in vivo acute oral toxicity values, indicating that use of this metric to set exposure limits would be at least as health-protective as using metrics derived from traditional approaches. Neurotoxicity human-equivalent doses were generally similar to, or more conservative than, literature-derived traditional PODs. Liver microtissue testing results were also qualitatively representative of in vivo effects, and quantitative analysis of human-equivalent doses is ongoing. Findings from the project were presented at the 2025 and 2026 Society of Toxicology meetings and the 2025 Military Health Systems Research Symposium, and two publications are planned for 2026.

The public is often aware of and interested in fish and wildlife diseases, particularly those that are highly visible, change the appearance of animals, and affect species of high recreational or commercial value like black bass (Micropterus spp.). “Blotchy bass syndrome” is a term used to describe external hyperpigmentation (melanosis) in black basses. This condition has received increased attention from anglers and resource managers in recent years and is a popular topic of discussion and reporting on angling websites and blogging platforms. Crowdsourced data collection can be used to increase community engagement and buy-in, as well as expand geographical and temporal sampling beyond those provided by state agencies. Recognizing a need to understand the geographical extent, seasonality, and biological threat of blotchy bass syndrome, USGS established crowdsourcing efforts to monitor this condition. Approaches used included traditional solicitation, smartphone applications, and virtual fishing events. Multiple discrete but overlapping efforts were undertaken starting in 2021. In March 2022, state agency partners solicited reports of blotchy bass syndrome via social media, requesting that anglers submit photos and information on catch location. Between June and November 2022, a virtual BioBlitz was conducted using the Angler’s Atlas MyCatch smartphone application, incentivized with prizes. A “Blotchy Bass Bonanza” participatory science effort was launched in July 2022 and encompassed all freshwater waterbodies within the United States and Canada. The Bonanza was reinitiated in March 2023 and will continue through February 2024. Overall, efforts yielded data from 31 states, six Canadian provinces, and some submissions from Mexico, Spain, and South Africa. Anglers submitted a total of 1,077 digital photographs of individual fish with presumptive blotchy bass syndrome for scientific review. USGS valued the 52,399 donated personnel hours by participating anglers at $1,153,000.

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 Toxicology and Exposure and Unilever’s Safety and Environmental Assurance Centre established a cooperative research and development agreement aimed at the development and evaluation of NAMs for use in next-generation risk assessment. A set of 40 case study chemicals representing common natural ingredients found in consumer products were used to explore the utility of a battery of NAMs for evaluating their safety and potential hazards. Research topics in this project include:

• Exploring how combinations of screening approaches using biologically diverse human cell lines can be applied to determining molecular points-of-departure (PODs). Screening approaches included safety pharmacology screening, cell stress assay panels, and high-throughput transcriptomic and imaging-based phenotypic profiling assays.
• Comparing molecular PODs to human exposure levels in hazardous and nonhazardous exposure scenarios.
• Incorporating metabolic competence into high-throughput profiling assays. This study (Jurgelewicz et al. 2025) demonstrated that high-throughput profiling assays can reproducibly detect changes in chemical bioactivity between parent compounds and metabolites generated using the previously developed Alginate Immobilization of Metabolic Enzymes platform.

The project was discussed in an April 2024 webinar presented by Physicians Committee for Responsible Medicine.

An AOP is a conceptual framework for organizing information and evidence of 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 defined approaches 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 AOP Development Programme, which is managed by the Organisation for Economic Co-operation and Development (OECD) Advisory Group on Emerging Science in Chemicals Assessment. 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, previously supported by EPA. In 2025, it was announced that all members of the Society should transition to becoming either members of the American Society for Cellular and Computational Toxicology or the European Society for Toxicology In Vitro and continue work to support development of AOPs via interest groups within each of the broader NAMs-focused societies.

EPA and NIEHS also work with international partners in the Methods2AOP Collaboration, which seeks to develop infrastructure to accommodate the integration of defined method details with key events in AOPs. 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 paper describing the Methods2AOP Collaboration’s activities (Karmaus et al. 2026) describes outreach events held in 2024 to facilitate stakeholder engagement. The Collaboration proposes the establishment of standardized fields for AOP documentation that leverage controlled vocabularies/ontologies alongside existing free-text options. They noted the continuing need for guidance for method developers embarking on validation studies and encouraged consideration of how changes to the AOP Knowledgebase and AOP-Wiki might impact third-party tools.

In a parallel effort, the FAIR Cluster Workgroup, an international collaboration in which EPA also participated, considered how available AOP resources might better support FAIR principles (findability, accessibility, interoperability, and reusability of data). Their recommendations (Mortensen et al. 2025) included coordination of mechanistic process mapping to molecular identifiers, and they identified a need for a workflow that stacks the curation process with domain expertise and tools. They also identified ongoing data standardization efforts that should support the application of FAIR principles to AOPs. The topic was explored further at the NIEHS-EPA coordinated Environmental Health Language Collaborative AOP Standards Workshop (Mortensen et al. 2026), where AOP and biomedical data experts deliberated on how to incorporate AOP associated biomedical data into existing or prototype AOP-Wiki data frameworks.

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. In 2024 and 2025, ICCVAM agency scientists participated in the following HESI projects.

• tPODs for Chemical Risk Assessments Working Group: This working group is developing a comprehensive, multilayered methodological framework for deriving transcriptomic points-of-departure (tPODs) from short-term in vivo toxicity studies for ecological and human health chemical assessments. The goal is to develop tPODs that are stable, biologically meaningful, and suitable for use in chemical risk assessment. A 2025 workshop advanced this effort by addressing study design considerations, establishing upstream quality control practices to improve data harmonization, defining standardized approaches for transcriptomic benchmark dose modeling, and evaluating both gene level and gene set based summarization strategies for consistent tPOD derivation. Outcomes from the workshop are being prepared as several manuscripts. The working group has also emphasized opportunities to reduce and refine animal use by demonstrating how mechanistic studies that incorporate transcriptomics and other molecular data could help replace traditional animal-intensive developmental and reproductive toxicity (DART) studies. These concepts will be featured in a roundtable at the 2026 Society of Toxicology annual meeting, “Towards a New Paradigm: Can Mechanistic In Vivo Studies Replace Traditional DART Guideline Studies?” that will be summarized into a short communication for publication.
• Cell Painting - OASIS Consortium: The HESI-sponsored Omics for Assessing Signatures for Integrated Safety (OASIS) Consortium is advancing next-generation risk assessment by integrating multiomics technologies, including transcriptomics, proteomics, and high-content cell painting, across a range of human-relevant biological systems. With an initial focus on liver safety, the consortium is combining molecular and phenotypic data from rat and human cell line exposures to well-characterized reference chemicals to improve translation from in vitro findings to in vivo and clinical outcomes. Chemical testing and data acquisition have recently been completed, and analysis is now underway to compare transcriptomic responses with phenotypic profiles generated through cell painting. Through this work, the consortium aims to build more predictive, efficient, and human-relevant approaches to chemical safety assessment, while supporting reduced reliance on animal models and strengthening regulatory science across sectors.
• eSTAR MicroRNA Biomarkers Working Group: Previous work within the Emerging Systems Toxicology for the Assessment of Risk (eSTAR) MicroRNA Biomarkers Working Group identified candidate microRNA biomarkers for kidney toxicity (Chorley et al. 2021; Dasgupta et al. 2024). More recent work focused on NAMs development by measuring extracellular release of these biomarkers in human primary proximal tubule cell culture media after exposure to kidney toxicants. Results from the study, now in preparation for publication, are part of the working group’s long-term efforts to develop biomarkers based on extracellular microRNA released by region-specific nephron cells that are indicative of toxicity due to chemical or drug exposure. These biomarkers can be utilized as nondestructive, time-course measures in cell culture media.
• eSTAR Carcinogenomics Working Group: This working group is developing transcriptomic signatures to detect early molecular events that are associated with liver tumorigenic outcomes in short-term rodent studies. Using publicly available rat liver gene expression datasets, machine learning, expert biological review, and HESI-sponsored studies from wild-type and receptor-null rat models, the group is building concise gene sets that are sensitive and specific for the activation of key tumor-associated pathways, including AhR, CAR, PPARα, ER, PXR, p53, and cytotoxicity. In particular, the receptor-null rat models were leveraged to confirm signature gene responses that require the tumor-associated receptor. The goal is to support mechanistically informed, weight-of-evidence approaches for assessing carcinogenic potential in short-term studies and to help reduce the need for traditional two-year rodent carcinogenicity assays in alignment with guidance S1B(R1) issued by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use.

Animal tests have traditionally been used to identify acute inhalation toxicants and define safety standards that protect human health. NAMs that include in vitro and computational approaches have been proposed as complementary resources. These approaches have the potential to identify toxicants and elucidate toxicity mechanisms in a manner that is more relevant to human biology than animal models. However, developing and evaluating these NAMs requires robust, well-curated, and chemically diverse reference data.

Using data from open-access sources, NICEATM compiled a database of in vivo rat acute inhalation data for approximately 1,200 unique substances. In addition to LC50 values (exposure concentration of a toxic substance estimated to be lethal to half of the test animals), metadata collected for each entry included exposure type, exposure route, species, sex, and number of animals tested, when available. The chemical set was found to represent a diversity of physicochemical properties and functional use categories represented in the EPA Chemical and Products Database. EPA hazard categories (e.g., nontoxic, toxic, highly toxic) were assigned based on LC50 values. While all hazard categories are represented in the database, about two-thirds of the chemicals were classified as Category IV (least toxic/nontoxic). The final curated database contains 2,565 entries for 1,209 unique substances. Of these, 1,025 unique chemicals (2,109 entries) have chemical structures standardized for quantitative structure–activity relationship (QSAR) modeling.

A variability analysis was conducted on the 231 chemicals with two or more LC50 values by calculating mean absolute deviation, a measure of variability based on the absolute distance between each data value and the mean of the dataset. Principal component analysis was then used to evaluate any associations with inhalation LC50 variability, but no associations were found between LC50 values and either physicochemical properties or functional use categories. Conditional probabilities were also calculated for these chemicals to determine the probability that a chemical would be assigned a specific EPA hazard category given that it was previously assigned the same or another category on the basis of an LC50 from another study. This analysis showed Category IV categorization to be most consistent and Category III (mildly toxic) to be least consistent. The database can be downloaded from NICE, and a paper describing the database is in preparation.

Flavor chemicals contribute to the appeal and toxicity of e-cigarettes and other tobacco products, and the assortment of flavor chemicals available for use in tobacco products is extensive. FDA scientists used a chemistry-driven computational approach to evaluate flavor chemicals based on intrinsic hazardous structures and reactivity of chemicals (Goel et al. 2022). A library of 3,012 unique flavor chemicals was evaluated based on physicochemical properties, GHS health hazard classification, structural alerts linked to the chemical’s reactivity, instability, or toxicity, and substructures shared with known respiratory toxicants. Computational analysis of the constructed flavor library flagged 638 chemicals with GHS classified respiratory health hazards, 1,079 chemicals with at least one structural alert, and 2,297 chemicals with substructures of concern. From further analysis of a subset of 173 chemicals, four general structures with an increased potential for respiratory toxicity were identified. This study indicated that computational methods are efficient tools for hazard identification and understanding structure-toxicity relationship. With appropriate context of use and interpretation, in silico methods may provide scientific evidence to support toxicological evaluations of chemicals in or emitted from tobacco products.

EPA’s Endocrine Disruptor Screening Program (EDSP) is responsible for determining the potential for certain chemicals to cause adverse effects in humans and wildlife via endocrine pathways. One goal of the EDSP is to evaluate how broadly results can be concluded across taxa. Two EPA projects are using computational approaches to assess how potential endocrine disruptors might affect a diversity of mammalian and nonmammalian species.

EPA scientists and collaborators assessed the cross-species conservation of androgen receptor-modulated pathways by using computational analyses and systematic literature review approaches to conduct a comprehensive analysis of existing in silico, in vitro, and in vivo data (Vliet et al. 2023). Results indicate that androgen receptors are conserved across vertebrate species, which could thus be predicted to share similarly susceptibility to chemicals that interact with the human androgen receptor. This study demonstrated a framework for utilizing bioinformatics and existing data to build weight-of-evidence for cross-species extrapolation and provides a technical basis for extrapolating human androgen receptor-based data to prioritize hazard in nonmammalian vertebrate species.

Thyroid hormone system-disrupting compounds are considered potential threats for human and environmental health. Multiple AOPs for thyroid hormone system disruption are being developed in different taxa. Combining these AOPs results in a cross-species AOP network for thyroid hormone system disruption which may provide an evidence-based foundation for extrapolating these data across vertebrate species and bridging the gap between human and environmental health. A review by EPA scientists and collaborators (Haigis et al. 2023) aimed to advance the description of the taxonomic domain of applicability in the network to improve its utility for cross-species extrapolation. The study focused on molecular initiating events and adverse outcomes, evaluating both the taxa they are likely applicable to and where evidence for applicability to various taxa exists in the context of thyroid system disruption. The evaluation showed that all molecular initiating events in the AOP network are applicable to mammals. There was some evidence of structural conservation across vertebrate taxa, especially for fish and amphibians and to a lesser extent for birds. The results of this evaluation are summarized in a conceptual AOP network that helps prioritize specific parts of AOPs for a more detailed evaluation.

Cardiovascular disease is the leading cause of death for people of most ethnicities in the United States. The hERG potassium channel plays a pivotal role in cardiac rhythm regulation, and drug molecules and environmental chemicals can potentially induce cardiotoxicity via hERG inhibition. An evaluation of the effect of environmental chemicals on hERG channel function can help inform the potential public health risks of these compounds. NICEATM and NCATS scientists employed several machine-learning approaches to develop QSAR prediction models for the assessment of hERG inhibition for drug-like and environmental chemicals screened in the Tox21 federal research program (Krishna et al. 2022). The data and scripts used to generate the hERG prediction models are provided in an open-access format as key in vitro and in silico tools that can be applied in a translational toxicology pipeline for drug development and environmental chemical screening.

Flavor chemicals contribute to the appeal and toxicity of tobacco products, and the assortment of flavor chemicals available for use in tobacco products is extensive. However, potential harms from inhaling these substances and their byproducts have not been extensively studied. To help address this data gap, FDA scientists (Goel et al. 2022) used a chemistry-driven computational approach to evaluate flavor chemicals based on intrinsic hazardous structures and reactivity of chemicals. A library of 3,012 unique flavor chemicals was compiled from publicly available information, and a structure-based analysis was done to characterize their (1) physicochemical properties, (2) GHS health hazard classification, (3) structural alerts linked to the chemical’s reactivity, instability, or toxicity, and (4) substructures shared with chemicals characterized by FDA as respiratory toxicants. Computational analysis of the constructed flavor library flagged 638 chemicals with GHS classified respiratory health hazards, 1,079 chemicals with at least one structural alert, and 2297 chemicals with substructural similarity to chemicals on the respiratory toxicant list. A subsequent analysis was performed on a subset of 173 chemicals in the flavor library, from which four general structures with an increased potential for respiratory toxicity were identified. This study indicated that computational methods are efficient tools for hazard identification and understanding structure-toxicity relationship. With appropriate context of use and interpretation, in silico methods may provide scientific evidence to support toxicological evaluations of chemicals in or emitted from tobacco products.

Seizures have been reported among e-cigarette users, in particular youth or young adults. FDA scientists used chemoinformatic computational models to compare chemicals documented to be present in e-cigarettes with known neuroactive compounds, with the goals of predicting blood-brain barrier penetration potential, central nervous system activity, and structural similarities (Stratford et al. 2024). The e-cigarette chemicals identified showed structural similarity to neuroactive compounds based on chemical fingerprint similarity analyses. Most chemicals studied were predicted to cross the blood-brain barrier and were also predicted to have central nervous system activity. This study showed that computer-based models can be useful to screen e-cigarette chemicals, allowing for prioritization for further possible in vitro and in vivo testing and potential early identification of central nervous system toxicity.

To fully characterize the potential human health risk of a substance, data regarding the substance’s toxicokinetics are often needed, including how a substance is absorbed, distributed, metabolized, and eliminated in the body. Traditional approaches for obtaining toxicokinetic data use animal models, but alternative approaches are being developed to computationally estimate relevant parameters. EPA researchers have developed toxicokinetic models within an R software package called high-throughput toxicokinetics (httk) to estimate chemical concentrations in humans and support IVIVE. The package currently uses human in vitro data to predict the fate of chemicals in humans, rats, mice, dogs, and rabbits. The latest version of httk, v. 2.7.4, was released in December 2025. This version provides quantitative structure–property relationship (QSPR) predictions of pharmacokinetic parameters for several thousand volatile organic chemicals. Other updates during 2024 and 2025 added models that describe absorption through the skin (Meade et al. 2026), partial oral absorption (Honda et al. 2025), and human pregnancy (Truong et al. 2025), as well as in vitro data and QSPR predictions of pharmacokinetic parameters for PFAS. More information is available on the EPA httk website.

EPA’s Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) is a fast online screening tool that allows researchers to extrapolate toxicity information across species.

There were four updates to SeqAPASS in 2024 and 2025, the most recent being the release of SeqAPASS v. 9.0 in September 2025. Improvements to SeqAPASS in these releases included:

• Protein structure generation and alignment capabilities to support detailed analyses of protein structural biology
• Incorporation of data from the World Register of Marine Species database to allow filtering of marine species
• Addition of a Focus/Filter toggle to customize results displays to either highlight optional selections from a full list of species or filter results to only display species that match the optional selection
• Data and executable updates

Training on new SeqAPASS capabilities was provided at an EPA virtual training session in May 2025. SeqAPASS v. 9.1 is scheduled for release in early 2026 and will include additional data and executable updates; v. 10.0 will be released later in 2026.

ICCVAM member agencies participate in the development and review of chemical testing guidelines issued by the Test Guidelines Programme of the Organisation for Economic Co-operation and Development (OECD) and collaborate on projects supporting the Integrated Approaches to Testing and Assessment (IATA) case studies program under the Working Party for Hazard Assessment. OECD test guidelines (TGs) and related documents are used by government, industry, and independent laboratories of the 38 OECD member countries to assess chemical safety. The U.S. National Coordinator for the OECD Test Guidelines Programme, who is a member of ICCVAM, facilitates the development, review, and clearance of a comprehensive U.S. interagency position for all draft test guidelines, guidance documents, project proposals, workshop reports, and other documents of the supported programs. The National Coordinator also coordinates expert group membership and project leadership for U.S. sponsored projects. The National Coordinator heads the delegation that represents the United States at the annual meeting of the Working Group of National Coordinators and in other test guideline development activities. One or more ICCVAM subject matter experts may join the National Coordinator at this meeting.

In 2024 and 2025, ICCVAM agencies actively participated and commented on draft OECD documents through the National Coordinator. ICCVAM members and/or NICEATM staff also supported a wide range of OECD activities during 2024 and 2025, such as:

• Supporting OECD activities to revise Test Guidelines Programme resources and processes to better support the uptake of emerging technologies, particularly for in silico methods. Efforts are ongoing to develop criteria for evaluation of in silico methods in defined approaches.
• Co-leading a project and participating in an OECD expert group coordinating revision of Guidance Document 34 on validation and acceptance of new or updated test methods for hazard assessment. ICCVAM experts participated in one in-person meeting for the drafting of the document in December 2025, in addition to ongoing virtual meetings. ICCVAM members are leading drafting of revised chapters on the essential elements of validation, the design and conduct of validation studies, and developing readiness criteria for methods undergoing validation. It is anticipated that the revised guidance document will be submitted for approval in the 2027 review cycle.
• Participating in a workshop on operation and financial aspects of validation under the OECD Test Guidelines Programme. The workshop was part of broader discussions aimed at strengthening regulatory confidence and modernizing validation processes for new and emerging chemical safety testing methods. Key points included the need for increased public funding for validation activities, particularly independent peer review, and improved visibility of existing validation resources and expertise to better support and guide method developers in facilitating validation efforts.
• Contributing to a proposal to update OECD TG 496 for in vitro test methods to identify eye irritants. TG 496 was updated in 2024 to add the OptiSafe test method.
• Leading a project to update OECD TG 497 for defined approaches to identify skin sensitizers. TG 497 was updated in 2025 to allow in vitro and in chemico methods in TGs 442C, 442D, and 442E to be used as alternate sources of information. The update also expands TG 497 to include Skin Allergy Risk Assessment–Integrated Chemical Environment (SARA-ICE), a defined approach for the determination of points-of-departure for skin sensitization potential.
• Leading a project to develop a Detailed Review Paper to “Facilitate the Development of Non-animal Test Methods to Predict the Respiratory Sensitisation Potential of Low Molecular Weight Chemicals.” The paper will support the development of an IATA for respiratory sensitization. The Detailed Review Paper was submitted to the Working Group of National Coordinators for the 2026 review cycle. An IATA project was submitted to the Working Party on Hazard Assessment for inclusion on the 2026 work plan. 
• Co-leading a project with the OECD and the European Food Safety Authority to develop an IATA framework template specifically for developmental neurotoxicity. The project will build on the existing developmental neurotoxicity IATA case studies, support the development of new ones, and provide guidance on in vitro to in vivo extrapolation, as well as standardizing uncertainty analysis and data integration within weight-of-evidence assessment. The project was approved by the Working Party on Hazard Assessment in June 2024.
• Participating in a group developing a case study on the use of an IATA for identifying systemic toxicity, which has been submitted to the OECD Working Party on Hazard Assessment.
• Serving as members and cochairs of the Developmental Neurotoxicity Expert Group. In December 2024, a “Workshop on Critical Innovations in Pesticides Safety Testing and Chemical Risk Assessment for Developmental Neurotoxicity” was convened, cochaired by U.S. experts alongside representatives from OECD and the European Food Safety Authority. The workshop examined key challenges identified through regulatory case studies, with the goal of defining priority questions and action items to be addressed in the short to medium term for regulatory implementation. Its overall aim was to support a more efficient regulatory framework by advancing the use of NAMs for assessing developmental neurotoxicity risks, particularly for chemicals and pesticides. 
• Participating in expert groups that are evaluating methods to assess thyroid disruption, skin sensitization, respiratory sensitization, and defined approaches for skin sensitization.
• Participating in an advisory group on emerging science in chemicals assessment.

In June 2023, CPSC updated its Policy on Animal Testing webpage. It has been redesigned to be more user-friendly and have direct access to necessary information and documents.

One of the resources available on the CPSC webpage is CPSC’s “Guidance for Industry and Test Method Developers: CPSC Staff Evaluation of Alternative Test Methods and Integrated Testing Approaches and Data Generated from Such Methods to Support FHSA Labeling Requirements.” CPSC developed this guidance, building on its Animal Testing Policy, to assist stakeholders in determining what test methods are deemed reliable for determining compliance with the labeling requirements under the Federal Hazardous Substances Act. This includes clarification of CPSC informational requirements and process for evaluating new approach methodologies and integrated approaches to testing and assessment.

Regulatory agencies require that chemical products be evaluated for their potential to cause skin sensitization. Traditionally, such evaluations involved animal models, but more recently, nonanimal methods have been developed. Defined approaches for skin sensitization (DASS) use standardized interpretation procedures to combine data from multiple nonanimal tests, such as human cell-based experiments, molecular studies, and computational models, to determine if a chemical has potential to cause allergic dermatitis.

While regulatory agencies accept several DASS methods, applying these approaches manually can be complex, slow, and prone to errors. To address this, NICEATM researchers created the DASS App (To et al. 2024), a free, open-source web application that simplifies the use of three regulatory testing strategies: Two-out-of-Three, Integrated Testing Strategy, and Key Event 3/1 Sequential Testing Strategy. This web tool, first launched in 2023, allows users to upload their own data, identify inconsistencies, and generate predictions without needing advanced computational skills or special software installation. DASS App provides a data template and the option to select the assays from which the data are taken. Reference data can be imported from NICE. Outputs are provided in a user-friendly, downloadable format as tables and interactive graphics. By making the application freely accessible online, the developers ensure the tool is available for wide adoption and future improvements. This innovation supports faster, more reliable hazard assessments while advancing the goal of replacing the use of animals with humane and efficient alternatives.

Updates in 2024 and 2025 made the app more user-friendly, including simplified handling of borderline calls, as well as adding new test method inputs to cover similar updates to OECD Test Guideline 497, which contains the Two-out-of-Three and Integrated Testing Strategy defined approaches. DASS App now allows the user to evaluate performance using their own reference data or data from NICE chemical reference lists.

FDA is required to assess the environmental impact of its tobacco regulatory actions per the National Environmental Policy Act. Increased use and sales of e-cigarettes raises concerns about the potential environmental impacts throughout their life cycle. However, few available research studies focus on the environmental impacts and ecotoxicity of e-cigarettes. Using a combination of available laboratory data and structure‒activity relationship models, FDA scientists compiled a list of e-liquid chemicals to be considered for future environmental impact and risk assessments (Venugopal et al. 2023). Characteristics considered included environmental persistence, bioaccumulation, and aquatic toxicity. Of the 421 unique e-liquid chemicals considered, 35 are considered hazardous constituents by EPA, 42 are considered by FDA to be harmful or potentially harmful constituents in tobacco products and smoke, and 20 were included on both lists. The study ultimately identified 81 chemicals that should be considered for future environmental impact and risk assessments, including tobacco-specific compounds, polycyclic aromatic hydrocarbons, flavors, metals, phthalates, and flame retardants. This study underscores the importance of awareness and education when handling or disposing of e-liquids/e-cigarettes and aim to inform strategies to prevent and reduce hazards from e-cigarettes.

EPA, in collaboration with NICEATM and nongovernmental organizations, is developing a new framework for identifying eye irritation and corrosion hazards for new chemicals reviewed under TSCA. With this new framework, EPA will prioritize data from non-animal test methods that are more reproducible and provide results more relevant to humans than the corresponding in vivo animal model test. Under the framework, information is collected and evaluated in the following order: (1) data from human cell/tissue-based test methods that have been demonstrated to be reproducible and relevant to eye irritation; (2) data from in chemico or non-human in vitro and/or ex vivo test methods that have been demonstrated to be reproducible and provide information on the mechanisms of toxicity relevant to eye irritation in humans; and (3) data from in vivo animal studies. If no acceptable information on eye irritation is available, the framework allows for consideration of skin irritation data that predict irritating or corrosive properties to make inferences about eye irritation hazard of the new chemical substance. Evaluation of skin irritation and corrosion data follows the same prioritization order as presented above for eye irritation information. This framework will streamline the decision-making process and increase efficiency through a standard process for EPA to use each time it evaluates eye irritation or corrosion hazards test data. The new framework supports EPA’s mandate under TSCA to promote the development and implementation of alternative test methods and strategies that can provide information on chemical hazards without vertebrate animal testing. This framework also supports EPA’s ongoing efforts to reduce the use of animal testing and make the Agency’s review of new chemicals more efficient, helping to bring new chemicals to market more quickly while protecting human health.

Dysregulation of VEGF and its receptor VEGFR contributes to the development of atherosclerosis and cardiovascular disease. This makes the VEGF pathway a potential target for cardiovascular risk assessment of pharmaceuticals and environmental chemicals. AOPs represent a logical sequence of biological responses that contribute to toxicity phenomena and are useful in informing chemical risk assessments. The advent of high-throughput screening (HTS) has made available large-scale in vitro bioassay data that provides mechanistic information that can help assess chemical toxicity and identify AOP molecular initiating events. This in turn can enable the development of human-relevant new approach methodologies (NAMs) for assessing toxicity without the need for extensive animal experimentation. NIEHS scientists applied AOP frameworks to gain a better understanding of the relationship between VEGFR signaling and the development of atherosclerosis. A data-driven approach was developed to find environmental chemicals linked to the bioactivity of the VEGF signaling pathway, and to investigate their links to other regulatory proteins like estrogen receptor alpha and endpoints like atherosclerosis. ToxCast, Tox21, and PubChem data were evaluated to obtain bioprofiles of 4,165 compounds with bioactivity in assays targeting different VEGFR. An AOP hypothesis was developed by coupling the mechanistic relationships highlighted by HTS data with literature review findings. These linked estrogen, serotonin, and vasopressin receptor targets with VEGFR activity mediated by several endocrine-disrupting chemicals, such as bisphenols, triclosan, dichlorodiphenyltrichloroethane, and polychlorinated biphenyls. Structure-based clustering was performed on relevant bioactive chemicals to evaluate potential molecular initiating events and analyze associations with use-case classes. Computational toxicology profiling of in vitro HTS bioassay facilitates the development of mechanism-driven AOPs and associated chemical perturbants to better understand the link between environmental chemical exposures and potential adverse cardiovascular outcomes. A paper describing this project is in preparation for submission in 2024.

The U.S. Army Chemical Biological Center (CBC) has established cooperative research and development agreements with the tissue chip manufacturers Emulate, Inc., and TissUse GmbH to apply their products to investigations of aerosol toxicity.

Aerosolization of chemical agents is a health concern that presents unique technical challenges. When aerosols are generated in specific environments, the agent can be deposited on surfaces and then reaerosolized into the air due to environmental factors, foot traffic, or passing vehicles. This is particularly likely with substances that are easily disseminated, persist in the environment, and are hydrophobic and thus remain in the surface soil environment. To investigate this concept, CBC scientists developed a novel system to simulate reaerosolization of chemical agents in soil with a high sand content. A vibration system with air inlet and outlet ports was developed to allow sampling of the reaerosolized soil-agent to determine a worst-case scenario concentration. Reaerosolized agent concentrations were identified and tested for toxicity in Emulate organ-chip models.

Toxicity of substances of concern will also be assessed in collaboration with TissUse using an open-top multiorgan chip system that can be exposed to an aerosolized agent or compound of interest. The system functions with an established TissUse microfluidic delivery system and a novel aerosol chip exposure apparatus called the Independent Holistic Air–Liquid Exposure System (InHALES). This system accurately replicates the entire human respiratory tract in vitro. The InHALES system will be implemented at CBC in early 2026, and feasibility studies will begin in summer 2026 to better understand aerosol maturation and dissemination.

Additionally, CBC leveraged success with bespoke aerosol organ-chip delivery systems to establish a cooperative agreement with Emulate. The focus of the collaboration is to provide user input on Emulate’s A1 open-top lung chip and to adapt the chip to CBC’s existing aerosol generation infrastructure.

CBC recently began work that aims to characterize exposure routes in vitro. Human lung chips will be exposed to toxic compounds in liquid directly on the tissue or through aerosols generated by the VITROCELL® Cloud in vitro exposure system (VITROCELL GmbH).

In 2025, NICEATM, PETA Science Consortium International e.V., EPA, and CropLife America member companies completed a collaborative effort to develop DAs for predicting United Nations Globally Harmonized System of Classification and Labelling (GHS) and EPA eye irritancy hazard classifications.

Using data from 29 agrochemical formulations tested in a common set of in vitro or ex vivo methods, four DAs were developed:

• The bovine corneal opacity and permeability assay (BCOP; OECD TG 437) with or without histopathology.
• The EpiOcular eye irritation test (OECD TG 492), followed by the BCOP with or without histopathology.
• The SkinEthic time-to-toxicity test for liquids (OECD TG 492B), followed by the BCOP with or without histopathology.
• The EyeIRR-IS assay (Cottrez et al. 2021), followed by the BCOP with or without histopathology.

Rather than evaluating direct concordance with historical in vivo rabbit eye test data, GHS and EPA predictions derived using the DAs were assessed using orthogonal validation and weight-of-evidence. Results showed that, for both classification systems, the predictions from each of the four DAs were as protective or more protective of human health than the in vivo test (Daniel et al. 2025). This study refines and builds upon two DAs described in a previous publication (van der Zalm et al. 2024). This work underscores the limitations of current animal-based reference data and highlights the need for a more human-relevant approach to eye irritation hazard classification. By improving integrated testing strategies, this research lays the groundwork for safer, more ethical, and scientifically meaningful alternatives to animal testing in regulatory toxicology.

During 2022 and 2023, several ICCVAM agencies published new webpages summarizing activities to advance alternative methods.

• In June 2023, CPSC updated its Policy on Animal Testing web page. It has been redesigned to be more user-friendly and have direct access to necessary information and documents. One of the resources available on this page is CPSC’s “Guidance for Industry and Test Method Developers: CPSC Staff Evaluation of Alternative Test Methods and Integrated Testing Approaches and Data Generated from Such Methods to Support FHSA Labeling Requirements.”
• In 2020, FDA launched “Advancing Alternative Methods at FDA,” which highlights activities of its Alternative Methods Working Group. A 2023 update expanded this site from a single page to a site with pages providing information on:
  • FDA partnerships to advance alternatives.
  • FDA implementation of alternative methods.
  • The FDA alternatives webinar series.
  • Relevant publications and events.
• EPA maintains a number of webpages describing efforts to replace and reduce animal use for chemical safety testing. The Office of Research and Development’s “New Approach Methods Research” site links to the EPA’s 2021 New Approach Methods Work Plan, which describes objectives and strategies to implementing new approach methodologies (NAMs). Other links from this page lead to EPA’s Catalog of NAMs Training Materials and materials from EPA NAMs Conferences. The EPA Office of Pesticide Programs provides a webpage describing its “Strategic Vision for New Approach Methodologies – Metrics.” This page includes data compiled by the Hazard and Science Policy Council, the Chemistry and Acute Toxicology Science Advisory Council, and other groups about how EPA activities and guidance documents have reduced the number of animals used in testing and saved EPA and stakeholder resources. The Office of Pollution Prevention and Toxics “Predictive Models and Tools for Assessing Chemicals” site links to models and tools that can be used to assess hazards, exposure, and environmental fate of new chemicals.
• DOI’s USGS is preparing a public webpage titled “Reducing Animal Use in Ecotoxicity Testing and Biomonitoring.” It briefly describes the types of ecotoxicological work conducted by the DOI, the 6R Principles, and ongoing efforts to reduce animal use and increase use of non-animal alternatives. It also provides some animal alternative reports and training opportunities.
• A major update of the USDA National Agricultural Library’s Animal Welfare Information Center (AWIC) website in 2022 improved user experience and streamlined information discovery; a new introductory video gives an overview of AWIC resources.

Certain chemicals used in commercial products and present in the environment have the potential to interfere with biological systems. Identifying human health effects using in vitro new approach methodologies (NAMs) is challenged by the absence of metabolism in most test systems, which may lead to the under- or overestimation of potential health hazards. To retrofit existing high-throughput assays with metabolic competence, the EPA developed the alginate immobilization of metabolic enzymes platform (AIME; Deisenroth et al. 2020) and used it to screen the ToxCast library to evaluate chemicals’ estrogenic potential (Hopperstad et al. 2022). The AIME platform improves upon many conventional high-throughput screening assays by incorporating encapsulated hepatic S9-alginate microspheres to allow for consideration of the effects of liver metabolism. The ToxCast library data demonstrated a range of metabolism-dependent effects across a diverse chemical library. These results support the utility of the AIME platform for identifying false positive and false negative target assay effects and reprioritizing hazards based on metabolism-dependent bioactivity. They also highlight the need to evaluate the role of intrinsic xenobiotic metabolism for endocrine and other toxicity-related health effects.

Throughput and accessibility of the lid-based AIME method were improved by incorporating automated bioprinting to deposit S9‐encapsulated microspheres directly into standard microplates with requisite cofactors for phase I and II hepatic metabolism (Hopperstad and Deisenroth 2023). The AIME bioprinting metabolism method will be a useful tool for the tiered testing paradigm outlined in the CompTox Blueprint (Thomas et al. 2019), and is also more amenable to method transfer because it uses commercial hardware rather than custom proprietary lids. Planned activities for 2024 and beyond include an active cooperative research and development agreement with Proctor & Gamble to transfer the AIME method to a commercial contract research organization, application of the AIME method to additional endocrine assays, and application of the method to broad high-throughput phenotypic assays and transcriptomic profiling.

Machine-learning methods enable data-driven development of predictive models for health-effects screening of novel chemicals. Single-task machine-learning models train on one endpoint and lack transferability to similar endpoints. These models also require large, homogeneous data sets. Health-effects screening needs machine-learning tools that can handle multiple small, noisy data sets. USAF Predictive Risk Team (PRT) is collaborating with a team from Johns Hopkins University Applied Physics Laboratory to investigate a novel machine-learning pipeline for molecular representation learning based on a multitask machine-learning paradigm. ToxCast assays with the same target were consolidated to minimize missing entries, and a machine-learning model was trained simultaneously on multiple tasks from moderate-sized data sets. To predict novel tasks from small data sets, the pretrained multitask model was combined with a dimension-reducing map and a task-specific predictor. The multitask model performed better than single-task models on six of 11 training tasks and six of seven distinct non-training tasks. This novel machine-learning pipeline generates molecular representations, leverages reduced dimensionality for greater efficiency, and combines information on multiple effects, including highly specific ligand binding and nonspecific systemic responses, to provide a more generalizable chemical risk model. The pipeline is being completed and prepared for publication in 2024.

BioBricks (Insilica) is an application that makes large bioinformatics databases programmatically accessible. Because of the potential usefulness of this tool for toxicological data acquisition and analysis, NICEATM began working with Insilica in 2022 to provide support and testing of the BioBricks platform. The NICEATM team contributed to building numerous bricks (units that encapsulate datasets), including those derived from NICE datasets. NICEATM also supported the testing and expansion of the BioBricks interface through activities such as the development of a Python version. A complete description of BioBricks has been published in Frontiers in Artificial Intelligence (Gao et al. 2025), with NICEATM team members serving as coauthors. All databases integrated into BioBricks are fully and freely available; the code is open-source and available via a GitHub repository.

Evaluation of chemical risk requires understanding of both the hazard presented by a chemical, via toxicity, irritation, or other harmful effects, and how much or how often the organism of interest is exposed to the chemical. ChemExpo is a publicly accessible data search and visualization tool for exploring chemical data relevant to exposure assessment that have been curated from public documents. It provides data collected by EPA about how chemicals are used in commerce and how they occur in consumer and industrial products. The ChemExpo team actively works to curate these data to harmonize consumer and occupational product categories, chemical functional categories, and exposure-relevant keywords, as well as to substance identifiers (DTXSIDs) used by EPA and the CompTox Chemicals Dashboard. These curated data are collectively known as EPA's Chemicals and Products Database (CPDat).

The beta version of ChemExpo was released in September of 2023. The beta release allows users to explore, search, and download CPDat data, aligning with bulk releases to enhance accessibility and integration with the CompTox Chemicals Dashboard.

Inhalation is the most relevant route of VOC exposure. However, due to unique challenges posed by properties of VOCs and their poor solubility in aqueous solutions, in vitro chemical safety testing is predominantly performed using direct application dosing or submerged exposures. To address the difficulties in screening toxic effects of VOCs, scientists in the U.S. EPA Office of Research and Development developed a cell culture system that permits cells to be exposed to multiple concentrations at air-liquid interface (ALI) in a 24-well format. A pilot study with eight volatile compounds was conducted to determine whether high-throughput transcriptomics (HTTr) using the TempO-Seq assay would predict toxicity at sub-cytotoxic exposure concentrations delivered to human airway cells at the ALI. The publication describing these results (Speen et al. 2022) was recognized as a “Top 10 Best Paper Demonstrating an Application of Risk Assessment” in March 2023 by the Society of Toxicology Risk Assessment Specialty Section. To expand the applications of the ALI exposure system, computer-aided design and computational fluid dynamics modeling were conducted to optimize operational parameters. Results indicate that a redesign of the system would improve VOC delivery and expand its applications to include aerosols. Planned 2024 activities include the evaluation of a broad array of VOCs and nonvolatile compounds with a focus on direct quantification of deposition and cellular uptake to improve in vitro to in vivo extrapolation (IVIVE). Furthermore, comparisons between ALI and direct liquid application studies are underway to determine best practices for in vitro new approach methodologies (NAMs).

QSAR models may be used to assess the potential acute toxicity of chemicals in fish for pesticides or pesticide degradation products for which there are little or no toxicity data available. However, data used to develop QSARs often do not represent the broad range of pesticidal structures and modes-of-action that contribute to pesticide toxicity. EPA Office of Pesticide Programs and Office of Research and Development scientists compared the performance of three existing QSAR models with a random forest model newly developed for this purpose. The new random forest model predicted toxicity better than the existing models, likely because it was trained using only pesticide data and a targeted predictor set, and because its algorithm accounts for predictor interactions and non-linear relationships. The new random forest model is being incorporated into a graphical user interface tool with the goal of making it publicly available to web users.

In recent years, USDA’s Animal Welfare Information Center (AWIC) has been developing PubMed search strategies, known as “hedges,” for topics specific to alternatives. Hedges are complex search queries that can be used on their own or in combination with other terms to return the most current literature relevant to that query available in PubMed. The focus of AWIC’s first hedges was refinement of animal use, and these hedges tended to be focused on animal species. Since 2023, NICEATM and AWIC have been collaborating to develop hedges on topics focused on replacement of animal use. In 2023 and 2024, hedges were developed for the topics MPS, NAMs for DNT, NAMs for acute inhalation toxicity, and IVIVE. These hedges were published in spring 2024 and are available on the National Toxicology Program website. All AWIC hedges are also available on their website. Additional search hedges on the topics of NAMs for developmental and reproductive toxicology, NAMs for skin sensitization, QSAR models for chemical hazard, and reduction alternatives for the two-year rodent cancer bioassay were developed in 2025 and will be published on the National Toxicology Program and AWIC websites in 2026.

Increases in the incidence of neurodevelopmental disorders, such as attention deficit/hyperactivity disorder, dyslexia, and autism spectrum disorder, have prompted scientific interest in the potential contribution of environmental toxicants to the development of these disorders. To support these investigations, the NIEHS Division of Translational Toxicology launched the Developmental NeuroToxicity Data Integration and Visualization Enabling Resource (DNT-DIVER). DNT-DIVER allows users to analyze, compare, and visualize multiple DNT assays in an interactive web-based application. The application provides public access to and visualization of all data from compounds screened by the division’s Developmental Neurotoxicity Health Effects Innovation Program. The current version (v. 2.2) includes different tabs for experimental design summary, quality control, chemical-specific concentration–response curves, ranking of chemical toxicity per laboratory/assay, and comparison of results across assays. DNT-DIVER will also accept data from novel screening assays as they become available and are accepted by the international community of researchers investigating DNT.

Ecological Structure Activity Relationships (ECOSAR) is a computerized predictive system that estimates aquatic toxicity. The program estimates a chemical's acute and chronic toxicity to aquatic organisms, such as fish, aquatic invertebrates, and aquatic plants, by using computerized structure–activity relationships (SARs). ECOSAR software is available for free without licensing requirements. Key characteristics of the program include:

• Grouping of structurally similar organic chemicals with available experimental effect levels that are correlated with physicochemical properties to predict toxicity of new or untested industrial chemicals.
• Programming of a classification scheme to identify the most representative class for new or untested chemicals.
• Continuous update of aquatic QSARs based on collected or submitted experimental studies from both public and confidential sources.

ECOSAR version 2.2 was released in March 2022. Updates included two new chemical classes, a module to predict toxicity of cationic polymers, user interface updates, and user input of melting point, octanol–water partition coefficient, and water solubility.

In 2020, EPA announced a partnership with DoD, international governments, and industry to sponsor the EcoTox TARGET innovation challenge. The challenge awarded $300,000 to the applicant who successfully developed a low-cost high-throughput technology for measuring global gene expression in samples from four common aquatic toxicity test organisms. The winner, BioSpyder Technologies, Inc., was announced in spring 2022. An abstract describing the challenge and outcome (Villeneuve et al.) has been accepted for a poster presentation at the Advances in Genome Biology and Technology general meeting in February 2024. There are no plans to repeat the challenge in the foreseeable future but research to apply the technology is ongoing.

DOI's USGS develops and tests environmental DNA (eDNA) tools for the detection of both invasive and native species in the nation’s waterways. Using eDNA for surveying or monitoring aquatic species can reduce costs and be less intrusive than methods that require direct capture of an organism. Throughout 2022 and 2023, efforts focused on the development and implementation of eDNA methods for native freshwater mussel species, including those that are federally listed. These activities are providing insight into best practices for eDNA survey design, including optimal sampling time for mussel eDNA and identification of factors affecting eDNA detection. USGS also assisted with a study of eDNA detection of the Missouri state-listed longnose darter that indicated possible expansion of the species’ range of distribution.

Animal models are currently used to predict whether a chemical might cause human neurotoxicity, which can lead to adverse effects such as seizures. However, research suggests that animal models have limited reliability, particularly in predicting drug safety for the central nervous system. The prediction of whether a drug might cause seizures, specifically, is unreliable due to a significant failure rate of novel drugs in human clinical trials caused by unforeseen toxicity, revealing the inadequacy of these models. To address this issue, a collaboration between NIEHS and industry was initiated to identify potential biological targets associated with seizures. By combining targets from established AOPs and drug discovery databases, a seizure-specific AOP network was generated. Resources including NICEATM’s Integrated Chemical Environment and the European Molecular Biology Laboratory’s ChEMBL database were employed to identify new approach methodologies (NAMs) that could measure identified targets. The search identified both compounds likely to induce seizure and compounds that have tested negative for seizure effects. A proof-of-concept evaluation to assess in vitro mechanistic assay data availability across the seizure-relevant targets was conducted. Through this comprehensive approach, the current landscape of seizure risk-informative testing assay availability was assessed, laying the groundwork for future predictive modeling efforts with the ultimate goal of enhancing the ability to anticipate and mitigate seizure-related adverse effects. This project was presented at the 2023 annual meeting of the American College of Toxicology (Behl et al.), and a manuscript is in preparation to be submitted in 2024.

NIEHS and EPA are fostering a community-driven initiative, the Environmental Health Language Collaborative. The Collaborative is a community for those who generate, analyze, or manage environmental health science (EHS) data and want to ensure the data are formatted and annotated using common EHS language standards to promote data discovery, interoperability, and reuse. The Collaborative is addressing this mission through three high-level strategic goals:

• Foster community-based extension and development of vocabularies, terminologies, and ontologies, and develop methods and tools to apply those resources
• Apply language standards and best practices for accurate environmental health data and knowledge representation
• Cultivate a vocabulary-aware environmental health community through training and education

The Collaborative’s website has resources including a glossary, bibliography, links to ontologies, and overview videos.

The Collaborative presented six webinars during 2024 and 2025 and held a two-day international workshop in June 2025 on the standardization of adverse outcome pathways. Videos and materials from the webinars and workshops are available on the Collaborative’s website. The Collaborative also maintains 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. The Data Harmonization Use Case working group submitted a manuscript in 2025 (Stingone et al. 2026) and is working on two additional papers. Other use cases have manuscripts in preparation.

The EPA Ecotoxicology Knowledgebase (ECOTOX) provides public access to comprehensive information on adverse effects of single chemical stressors to ecologically relevant aquatic and terrestrial species. ECOTOX includes curated data from over 55,000 references in the scientific literature and over 1.2 million test records, covering more than 14,000 aquatic and terrestrial species and 13,000 chemicals.

There were eight updates to ECOTOX during 2024–2025. Recent additions include information on 6PPD-quinone, 6PPD-alternatives, arsenic, cyanotoxins, metolachlor, quizalofop, and per- and polyfluoroalkyl substances (PFAS). In addition to the data updates, the Explore Publications module launched in 2024 enables queries by citation information (author, title, ECOTOX ECOREF number, DOI) and displays toxicity data by reference. In 2024 and 2025, ECOTOX had an average of over 16,000 monthly page views and over 2,000 active monthly users. An ECOTOX training session was presented in April 2024 as part of a broader in-person EPA NAMs tools training workshop. Additional training resources for ECOTOX are available on the EPA NAMs Training webpage.

EPA created its New Approach Methods (NAMs) Work Plan to prioritize agency efforts and resources toward activities that aim to reduce the use of vertebrate animals while continuing to protect human health and the environment. The Work Plan was updated in December 2021.

To implement the Work Plan’s objective of “Engage and Communicate with Stakeholders,” EPA developed and implemented a NAMs training program. This program provides users courses and workshops with the appropriate level of oversight to enable them to use EPA NAMs tools and approaches in their own work.

In April 2024, EPA presented a comprehensive two-day NAMs Tools Training Workshop in person at their Research Triangle Park, NC, campus. It gave participants the opportunity to advance their knowledge about 10 different EPA NAMs tools through overview sessions, in-depth learning, interactions with tool and data owners, and tool-specific case studies. Materials from the workshop are available on the NAMs training program webpage.

EPA also presented several virtual training sessions during 2024 and 2025:

• June 2024: Chemical Transformation Simulator, a web-based tool for predicting transformation pathways and physicochemical properties of organic chemicals
• September 2024: Collaborative AOP Wiki
• May 2025: SeqAPASS online screening tool to extrapolate toxicity information across species
• June 2025: CompTox Chemicals Dashboard, the primary web-based application that provides access to data and algorithms from the EPA Center for Computational Toxicology and Exposure
• July 2025: httk R software package providing toxicokinetic models to estimate internal chemical concentrations from exposure and support in vitro to in vivo extrapolation (IVIVE)

Videos and other materials from these events are on the NAMs training program webpage. Other resources available on this page include links to online user guides and tool webpages.

New approach methodologies (NAMs) can provide human-relevant information that may be challenging to obtain from whole-animal tests. EPA’s Office of Pesticide Programs is interested in using NAMs to reduce the reliance on default assumptions for risk assessment, including the application of 10X default uncertainty factors each for interspecies and intraspecies extrapolations. To this end, the Office of Pesticide Programs collaborated with EPA’s Office of Research and Development, academia, and industry to use NAM data to inform extrapolation/uncertainty and safety factors. As a part of this effort, EPA held a public meeting of the Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel in September 2020 to consider and review the use of NAMs to derive extrapolation factors and evaluate developmental neurotoxicity (DNT) for human health risk assessment. The panel was supportive of using the DNT NAM battery in weight-of-evidence evaluations of DNT potential, while recognizing that the battery should be a “living and evolving process.” The panel also supported the use of chemical-specific in vitro data to inform interspecies extrapolation factors for organophosphate pesticides, but provided recommendations to reanalyze the data in a manner more consistent with the EPA’s “Guidance for Applying Quantitative Data to Develop Data-Derived Extrapolation Factors.” EPA responded to the panel’s recommendations in a March 2023 memorandum, available with related documents on the Regulations.gov website.

Scientists in the USAF Force Health Protection program’s Predictive Risk Team (PRT) performed a study using NAMs to characterize the risk of allergic contact dermatitis from occupational chemical exposures (Keebaugh et al. 2025). The goals of the study were to evaluate the sensitization potential of chemicals used by fabricators and maintainers and to develop preliminary candidate surface guidelines, i.e., guidelines for maximum surface concentrations to prevent the induction of sensitization. The kinetic direct peptide reactivity, KeratinoSensTM, and human cell line activation test assays were used to predict LLNA effective concentration values using the regression models published in Natsch et al. (2022a) and Natsch et al. (2022b). In vitro assay results, in silico model predictions, and available human and animal data were used to predict sensitizer status using a weight-of-evidence approach. Preliminary candidate surface guidelines, derived from predicted effective concentration values for predicted sensitizers, can be used to evaluate the presence of these chemicals on work surfaces to better understand the risk of USAF personnel developing allergic contact dermatitis from occupational exposures.

Skin permeation is a major consideration in the safety assessment of cosmetics, topical drugs, and veterinary medicine products. FDA, NIH, and NIEHS scientists conducted a study to explore the usefulness of alternative skin barrier models to replace excised human skin or animal models to assess skin penetration (Salminen et al. 2023). A standardized dermal absorption testing protocol was developed to predict skin absorption in humans. Caffeine, salicylic acid, and testosterone were used in side-by-side assessments of a reconstructed human epidermis model, a synthetic barrier membrane model, and an excised human skin model. Transepidermal water loss and histology of the biological models were compared. Based on the results of this study, authors made specific recommendations about how to evaluate and use both alternative skin barrier models and excised human skin to assess skin penetration. Evaluating novel models in the manner outlined in this study has the potential to reduce the time from basic science discovery to regulatory impact.

NICEATM scientists have been working to replace the use of animal models in skin sensitization studies with reliable and ethical alternatives. A June 2025 NICEATM report summarized an evaluation of the performance of a nonanimal test method called the Genomic Allergen Rapid Detection (GARD™skin) assay, which tests whether chemicals can cause allergic skin reactions. The study specifically assessed the GARDskin assay’s ability to predict both hazard, the likelihood of a chemical to be a sensitizer, and potency, a prediction of the severity of sensitization that could be caused by the chemical, utilizing defined approaches (DAs) for skin sensitization. Results were compared against traditional animal test results, including LLNA and HPPT data.

The study examined 31 substances using the GARDskin assay, in conjunction with results from other methods, such as the direct peptide reactivity assay, the human cell line activation test, and the KeratinoSens assay. Predictions from these tests were compared against human and LLNA data to evaluate accuracy (an overall assessment of performance), sensitivity (ability to identify true positives), and specificity (ability to identify true negatives). The GARDskin assay performed well, ranking highest among the methods in terms of accuracy (63%), balanced accuracy (62%), and sensitivity (81%) when compared to LLNA data. It also had the lowest rate of false negatives (19%). Inclusion of GARDskin data improved the performance of DAs, which combine data from multiple nonanimal tests to predict sensitization potential. For potency, the DA containing GARDskin had higher accuracy than other DAs (53%) and was less likely to overpredict or erroneously assign a more restrictive hazard classification compared to other approaches.

While only 31 substances were tested, limiting broader conclusions, this evaluation shows promising results for the GARDskin assay in providing human-relevant, reliable predictions. Combining multiple tests into integrated approaches adds confidence in safety assessments, helping advance nonanimal methods for regulatory use. This study supports the shift toward modern, ethical testing alternatives for chemical safety evaluations.

The modern battlefield is consistently evolving, and the USAF Force Health Protection program must account for new technologies and chemical threats. While much progress has been made in the development of computational methods for predicting public health risk, the warfighter is expected to experience exposures under conditions that are substantially different from those experienced by the general U.S. population. Thus, USAF Predictive Risk Team (PRT) is evaluating the utility of existing models for operational exposure scenarios and modifying existing models when necessary.

The Air Force population differs from the general U.S. population with respect to age and gender distribution. PRT constructed an approach to use published demographic data (Mullenger and Zehner, 2020) to customize the httk pipeline to adequately model Air Force population. A Monte Carlo simulation was performed to generate 48 anthropometric characteristics that closely resemble the USAF population. Upon completion, an analysis was compiled to compare previous oral-equivalent doses for the general population to those derived for the military. This pipeline is being used as a foundation to perform a high throughput risk analysis on military-relevant chemicals and will continue to be expanded as necessary as the population changes in the future. Additional areas for development in 2024 include consideration of occupational and acute dosing scenarios and physiological changes due to extreme environments.

To date, Tox21 high-throughput screening (HTS) assays have focused primarily on selected nuclear receptor and stress response pathways. This relatively limited focus suggests that activity in other toxicity pathways has not been adequately assessed; it is likely that some unexplored pathways relate to unanticipated adverse drug effects. Therefore, expanding the coverage of biological responses by adding assays that probe underrepresented pathways in the current Tox21 assay portfolio may improve the predictivity of Tox21 data. Scientists at FDA, NCATS, and NIEHS are systematically identifying these underrepresented pathways in a data-driven approach and nominating assays for development and Tox21 chemical screening. The data generated will be used to build models for human toxicity prediction, focusing on common adverse drug effects such as drug-induced liver injury and cardiotoxicity. The initial survey identified targets and pathways of interest, including CYP450 metabolic enzymes and G protein-coupled receptors. In 2022 and 2023, HTS assays for the proposed new targets and pathways were screened against the Tox21 10K chemical library. Predictive models for hepatotoxicity and cardiotoxicity were built using chemical structure and Tox21 in vitro assay data (Ye et al. 2022). The impact of the newly generated assay data on improving model performance was evaluated, and predictions from the models were validated in relevant assays. The expanded Tox21 assay panel was found to significantly enhance the capacity of assay data for in vivo toxicity prediction, underscoring the importance of a diverse and comprehensive in vitro assay portfolio in advancing safety assessment (Xu et al. 2025).

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.

As part of FDA’s commitment to promote novel technologies for potential regulatory use, FDA gives developers and others the opportunity to present their new methods and methodologies directly to FDA scientists. Educational opportunities to hear more about new predictive in vitro, in vivo, and in silico methods is vital to ensuring that FDA regulators and researchers have a broad skill set and remain current with cutting-edge science and technology. Developers presented 29 webinars during 2022 and 2023; presentations described in silico models and in vitro platforms such as MPS, organoids, and engineered tissues relevant to contexts including drug-induced liver injury, neurotoxicity, inhalation toxicity, and veterinary medicine. Information for prospective presenters is available on the FDA website.

Tissue chips have emerged as an in vitro alternative to animal use with the potential to be more predictive of human response in the safety and efficacy assessment of leading therapeutics. In 2022 and 2023, NCATS and FDA offered small business innovation research and business technology transfer grants for the development of neuromuscular junction tissue chips to replace the mouse lethality bioassay as a potency assay for botulinum toxin. A main objective for this funding opportunity was to position these tissue chips as an alternative test method as a standalone replacement for mouse lethality bioassays.

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 a November 2022 Federal Register notice (87 FR 66195), FDA announced availability of a final guidance for industry, “S1B(R1) Addendum to S1B Testing for Carcinogenicity of Pharmaceuticals.” The guidance was prepared under the auspices of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, and offers an integrative approach that provides specific weight-of-evidence criteria that inform whether a 2-year rat study is likely to add value in completing a human carcinogenicity risk assessment. This final guidance considered comments received from the public in response to release of the draft guidance in October 2021.

In November 2023 FDA published guidance on “Assessing the Credibility of Computational Modeling and Simulation in Medical Device Submissions.” Computational modeling and simulation can be used in a variety of ways in medical device applications, including to perform in silico device testing or as part of software embedded in a device. This guidance provides a risk-informed framework for credibility assessment of computational modeling and simulation used in medical device regulatory submissions. The guidance is intended to promote consistency and facilitate efficient review of medical device submissions, to increase confidence in the use of computational modeling and simulation in regulatory submissions, and to facilitate improved interpretation of computational modeling and simulation credibility evidence submitted in regulatory submissions. The guidance will be discussed in a presentation at the January 2024 ICCVAM Communities of Practice webinar.

In June 2023 FDA finalized the guidance document, “Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals.” The purpose of this guidance is to assist sponsors in the nonclinical evaluation of the immunotoxic potential of drug products and biopharmaceuticals.

The guidance addresses evaluation of topical pharmaceuticals for skin sensitization potential. Specifically, the guidance states that, for individual chemicals, “FDA will consider a battery of studies (e.g., in silico, in chemico, in vitro) that have been shown to adequately predict human skin sensitization with an accuracy similar to existing in vivo methods.”

In 2020, FDA launched the Innovative Science and Technology Approaches for New Drugs (ISTAND) program to support the development of innovative tools that can improve drug development and regulatory decisions. ISTAND focuses on qualifying cutting-edge technologies including super intelligence (SI)-based algorithms; novel advanced testing methods, including NAMs such as microphysiological systems (MPS) that reduce reliance on animals; and digital health tools, such as wearables. As of 2025, ISTAND has transitioned from a pilot program to a permanent part of the FDA’s Drug Development Tools Qualification Program.

The ISTAND qualification process involves three steps: submitting a Letter of Intent, a Qualification Plan, and a Full Qualification Package. Accepted tools and their status are made public, ensuring transparency as required by the 21st Century Cures Act. Once a tool is qualified, it can be used in regulatory submissions without requiring further re-evaluation for its qualified context of use.

Since its launch, ISTAND has accepted several innovative tools, including SI-based tools, methods for assessing safety without using animals, and technologies involving human tissues, into the program. These tools provide modern, science-driven approaches to support drug safety and effectiveness evaluations.

ISTAND complements other FDA initiatives aimed at accelerating drug development while reducing the need for animal models. By promoting and qualifying innovative approaches, ISTAND plays a crucial role in advancing drug development and fostering regulatory acceptance of modern scientific methods.

FDA provides a Catalog of Regulatory Science Tools to Help Assess New Medical Devices. This peer-reviewed resource assists medical device companies in evaluating new medical devices in contexts where standards and qualified medical device development tools do not yet exist. The catalog collates a variety of regulatory science tools developed by the FDA's Center for Devices and Radiological Health's Office of Science and Engineering Labs. The tools use the most innovative science to support medical device development and patient access to safe and effective medical devices. Tools categories include laboratory methods, computer models, and data sets, and they are applicable to program areas including cardiovascular, neurology, and biocompatibility and toxicology. The catalog was updated in 2024.

In 2021, FDA published “Advancing New Alternative Methodologies at FDA,” which provided updates on activities to advance development of alternatives to animal use and their application to regulatory decision-making. The report was developed by the FDA Alternative Methods Working Group to highlight the progress made by FDA scientists in laying the groundwork to integrate alternatives to animal testing into FDA regulatory programs. Two reports issued in 2022 expanded on topics discussed in the report:

• The 2022 “Update to the Focus Areas of Regulatory Science (FARS) Report” outlines topics FDA has identified as needing continued targeted investment. FDA reviewed each of the focus areas from the 2021 report, and this document provides important updates to the examples highlighted in the report.
• A November 2022 report on “Successes and Opportunities in Modeling & Simulation for FDA” was prepared by the FDA Modeling and Simulation Working Group. The report:
  • Elucidates how and where modeling and simulation is used across FDA, and the type and purpose of modeling and simulation used.
  • Presents a selection of modeling and simulation case studies from across nearly all FDA centers, which demonstrate how modeling and simulation is playing a tangible role in FDA fulfilling its mission.
  • Identifies opportunities for FDA to better harness modeling and simulation in upcoming years by embracing computational advances and new (and big) data streams to develop improved public health solutions.

FDA scientists also gave presentations on activities to advance alternatives at several public meetings during 2022 and 2023. Details of these presentations and presentation slides are available on the FDA website.

Engineered nanomaterials (ENMs) come in a wide array of shapes, sizes, surface coatings, and compositions, and often possess novel or enhanced properties compared to larger sized particles of the same elemental composition. To ensure the safe commercialization of products containing ENMs, it is important to thoroughly understand their potential risks. Given that ENMs can be created in an almost infinite number of variations, it is not feasible to conduct in vivo testing on each type of ENM. Instead, new approach methodologies (NAMs) such as in vitro or in chemico test methods may be needed, given their capacity for high-throughput testing, lower cost, and ability to provide information on toxicological mechanisms. However, the different behaviors of ENMs compared to dissolved chemicals may challenge safety testing of these substances using NAMs. The ICCVAM Nanomaterials Workgroup queried ICCVAM member agencies about what types of ENMs are of agency interest and whether there is agency-specific guidance for ENMs toxicity testing (Petersen et al. 2022). To support the ability of NAMs to provide robust results in ENM testing, two key issues in the usage of NAMs, namely dosimetry and interference/bias controls, are thoroughly discussed.

OECD has developed harmonized templates for standardized reporting of chemical information including potential human health and environmental effects. OECD Harmonised Template (OHT) 201: Intermediate Effects aims to capture mechanistic information from chemical test results generated by NAMs, such as in vitro testing or in silico methods.

NICEATM developed a workflow for curating HTS assay data from EPA’s invitrodb (v. 4.2) that included an annotation scheme to provide biological context to cHTS data and enable toxicological interpretation. The scheme incorporates annotations from the OBO Foundry, a harmonized and interoperable database consisting of multiple knowledge areas to encompass a broader range of biologic and toxicologic processes. These annotations are available through the NIH Integrated Chemical Environment. Assay description documents based on OECD Guidance Document 211, which outlines ways to harmonize nonguideline in vitro methods, have been made available for ToxCast assay endpoints through invitrodb. These documents describe the details of each assay technology, performance metrics, and data analysis procedures, which align with information requested for OHT 201 fields.

Ongoing annotation efforts by NICEATM, EPA, and the European Joint Research Centre include:

• Identification of HTS-relevant OHT 201 fields
• Alignment of OBO Foundry annotations and mapping information from NICE and invitrodb to OHT 201 fields
• Development of automation workflows to upload information to the International Uniform Chemical Information Database

Toxic responses are specific to cell types, and the gene expression patterns of cell types are highly conserved in evolution. The diversity in gene expression among cell types in a single individual is far greater than gene sequence diversity in populations. Thus, any cell type-restricted study using either cell lines or physiological systems such as organoids will fail to capture the full diversity of human exposure risk. Invertebrate organisms such as insects are exempt from most animal regulations and have thousands of cell types, many of which are shared between these organisms and humans. The European Union PrecisionTox project is an international consortium that explores the replacement of traditional mammalian chemical safety testing by comparative toxicology in fruit flies, nematodes, water fleas, clawed frog and zebrafish embryos, and human cell lines.

NIH is participating in a PrecisionTox study that is supplementing comparative toxicology data using more traditional terminal endpoints with data from gene expression and metabolite profiling induced by low subphenotypic doses of chemicals to map conserved AOPs. The study is leveraging fruit fly genetics to inform testing of key human genes and allelic variants, as well as counteracting chemicals that might change susceptibility in people. These data are expected to provide mechanistic insight and feed predictive models useful for regulating groups of chemicals. During 2022 and 2023, institutions participating in the study assembled information on a 250-chemical library, including chemical class, diversity in terms of structure, physicochemical properties, toxicity modes-of-action if known, and database/literature-derived associations with disease pathology, genes, and putative metabolic biomarkers. Harmonized toxicity testing experiments were conducted on about 90 substances, with 54 substances having sufficient comparative toxicology results for a first “phylogenetic toxicity analysis” to allow cross-species extrapolation. RNA expression and metabolite profiling is in progress for the 90-substance set, and the consortium is also conducting genome-wide screenings for genetic variation in toxicity. Study data will be made available in a manner supporting FAIR (findable, accessible, interoperable, and reusable) data standards.

PrecisionTox participating institutions are also engaging with government stakeholders to advance the use of new approach methodologies (NAMs) in chemical regulation. Outcomes of the studies will be applied to support the development of cost-effective NAMs to assess chemical hazard and exposure that can be applied by regulatory bodies and industry.

High-throughput transcriptomics (HTTr) has the potential to support efforts to reduce or replace some animal tests. EPA and NIEHS scientists developed a computational approach utilizing MCF-7 breast cancer cells and a biomarker assessing expression of 46 genes to predict estrogen receptor activity after chemical exposure (Ryan et al. 2016). To further explore the utility of this model, they investigated whether it could identify estrogen receptor activities of chemicals examined by Endocrine Disruptor Screening Program (EDSP) Tier 1 screening assays (Corton et al. 2022). For the 62 chemicals examined, the estrogen receptor biomarker model accuracy was 97% for in vitro reference chemicals and at least 76% for in vivo assays. These accuracies were similar or slightly better for the same chemicals than those of a previously described ToxCast estrogen receptor model based on 18 in vitro assays. These results indicate that the HTTr biomarker model can correctly identify active and inactive estrogen receptor reference chemicals, and is potentially useful to rapidly identify chemicals with potential estrogen receptor bioactivities for additional screening and testing.

Read-across is a computational technique that uses toxicity data from one or more known (source) chemicals to predict toxicity for another (target) chemical, usually but not always on the basis of structural similarity. EPA’s Generalized Read-Across (GenRA) application is a standalone tool linked to the CompTox Chemicals Dashboard that performs read-across algorithmically, helping researchers to make informed decisions about chemicals with little toxicity data available.

GenRA had two major updates during 2022-2023.

• In September 2022, a new update allowed users to evaluate the relevance of similar substances using predicted physical property information. It provided predictions of toxicity and bioactivity that users could download for further analysis.
• Version 3.2 was released in March 2023. This update included data updates and over 30 minor improvements and bug fixes. Improved indexing in this release enabled increased processing speed.

In 2023, EPA presented virtual training on GenRA. Approximately 725 people attended the main session and 215 attended the breakouts, representing a record for attendance at an EPA NAMs training session. Additional training resources for GenRA are available on the EPA NAMs Training webpage.

Traditional risk assessments based on in vivo animal studies typically use a chemical-by-chemical approach and apical disease endpoints. However, in the real world, individuals are exposed to chemicals from sources that vary over space and time. EPA and NIEHS scientists collaborated to develop a workflow (Eccles et al. 2022) to integrate human exposure data for 41 chemicals in the EPA National Air Toxics Assessment with curated high-throughput screening (cHTS) assays to identify counties where exposure to the local chemical mixture may perturb a common biological target. The workflow used the estimated blood plasma concentration and the concentration–response curve from the cHTS data to determine the chemical-specific effects of the mixture components. Three mixture modeling methods were used to estimate the joint effect from exposure to the chemical mixture on the activity levels, which were geospatially mapped. This workflow demonstrates how new approach methodologies (NAMs) can be used to predict early-stage biological perturbations that can lead to adverse health outcomes that result from exposure to chemical mixtures. As a result, this work will advance mixture risk assessment and other early events in the effects of chemicals.

OSHA is leading efforts in the U.S. to update and expand the use of nonanimal test data for the United Nations Globally Harmonized System for Classification and Labelling of Chemicals (GHS). OSHA participated in meetings convened by the United Nations Subcommittee of Experts on the GHS in 2024 and 2025 to harmonize international standards on safe handling of chemicals. OSHA held multiple public meetings to receive input from U.S. stakeholders in advance of the international meetings. The most recent hazard classes to be updated for use of nonanimal methods include skin sensitization and germ cell mutagenicity. Updates were also made to the introductory chapter that provides overarching principles to be considered when developing chemical hazard classifications. The update of the GHS chapter on skin sensitization was finalized in 2024, which included new in vitro tests and nontesting methods for classifying hazards. Guidance on the use of in silico methods and defined approaches was also included in the updated chapter. The update to the germ cell mutagenicity chapter is expected to be finalized by the end of 2026 and will include enhanced use of in vitro methods and other NAMs. Guidance of the use of these methods is being included in this update.

To support participation in its Cancer Tissue Engineering Collaborative Research Program, NCI offers grants to support the development and characterization of state-of-the-art biomimetic tissue-engineered technologies for cancer research. The goals of the program are to (1) catalyze the advancement of innovative, well-characterized in vitro and ex vivo systems available for cancer research, (2) expand the breadth of these systems to several cancer types, and (3) promote the exploration of cancer phenomena with biomimetic tissue-engineered systems. The program awards grants of up to $400,000 to fund projects that can continue up to five years. Eligibility for these grants is open to for-profit and nonprofit institutions within and outside the United States. NCI began accepting applications for funding in May 2017; applications will be accepted and considered through February 2025.

Gene expression profiling with alternative toxicological cell systems is a valuable tool for exploring mechanisms of chemical interactions in biological systems. However, interpretation of these data for decision-making is challenging. EPA and NIEHS scientists are addressing this gap by building interpretable bridges among environmental chemicals and specific molecular targets associated with biological response such as nuclear receptors, enzymes, and transporters. Over 300 reference chemicals have been identified and procured to build robust transcriptomic datasets in various cell systems, including the U-2-OS human osteosarcoma cell line and differentiated cultures of HepaRG and 3D microtissues of primary human hepatocytes, using the TempO-Seq™ technology for next-generation sequencing of cell lysates. Experiments covered a wide range of exposures to capture the initial stages of biological responses to environmental chemicals, using transcriptomic patterns to define similarity and help infer mechanisms for data-poor chemicals. This efficient strategy has the potential to greatly increase the mechanistic understanding of adaptive responses to environmental chemicals at the gene expression level and inform next steps in toxicology research investigations. The research teams have completed data generation for all three cell systems, and manuscripts for each cell system are in preparation. Furthermore, these reference chemicals were shared with colleagues at the University of Leiden to add renal proximal tubule epithelial cells to the list of cell types. A manuscript by the researchers involved in this project has been prepared for submission in 2026.

Researchers at EPA are using two high-throughput profiling assays, high-throughput transcriptomics (HTTr) with the human whole transcriptome TempO-Seq assay and high-throughput phenotypic profiling (HTPP) with the Cell Painting assay to characterize the biological activity of chemicals across a variety of human-derived cell types. Computational workflows have been established for determining biological pathway-altering concentrations and phenotype-altering concentrations from these two assay types (Harrill et al. 2021; Nyffeler et al. 2023). The high-dimensionality data have been organized for display and distribution to the public through the CompTox Chemicals Dashboard, under the “Bioactivity, HTTr: Summary” and “Bioactivity, HTPP:Summary” views. HTTr and HTPP results for hundreds of chemicals in a variety of cell types (MCF-7 breast adenocarcinoma, U-2 OS osteosarcoma and the 2-D differentiated HepaRG liver cell model) became available on the Dashboard in 2022 and will be updated in future releases.

During 2024 and 2025, NICEATM, an office within the NIEHS Division of Translational Toxicology (DTT), provided technical and scientific support for ICCVAM and ICCVAM workgroup activities, peer review panels, expert panels, workshops, and validation efforts.

In addition to providing support for ICCVAM, NICEATM:

• Supported activities of the NIEHS Division of Translational Toxicology, especially those contributing to the U.S. government’s interagency Tox21 initiative
• Conducted analyses and evaluations and coordinated independent validation studies on novel and high-priority alternative testing approaches
• Provided information, data, and tools to test method developers, regulators, and regulated industry through its website and workshops on topics of interest

NICEATM staff (as of November 2025)

NIEHS

Helena Hogberg, Ph.D., Acting Director
Kamel Mansouri, Ph.D., Computational Chemist

NICEATM Contract Staff (Inotiv, Inc.)

Emily Reinke, Ph.D., DABT, Principal Investigator
Steven Morefield, M.D., Project Manager
Michaela Blaylock, B.A.
Alexandre Borrel, Ph.D. (subcontractor, Sciome LLC)
Bryant Chambers, Ph.D.
Xiaoqing Chang, Ph.D., DABT
Shail Choksi, B.S. (subcontractor, Sciome LLC)
Amber Daniel, M.S. Tox
Sherif Farag, Ph.D.
Adrian Green, Ph.D. (subcontractor, Sciome LLC)
Jon Hamm, Ph.D.
Bridgett Hill, M.S.
Victoria Hull, M.S.
Bridget Knapp, M.S.
Anna Kreutz, Ph.D.
Tripp LaPratt, B.S.
Eric McAfee (subcontractor, Sciome LLC)
Shri Mokkapatti, B.S. (subcontractor, Sciome LLC)
Jason Phillips, B.S. (subcontractor, Sciome LLC)
Brad Reisfeld, Ph.D.
Ruchir Shah, Ph.D. (subcontractor, Sciome LLC)
Catherine Sprankle, M.S.
Arpit Tandon, M.S. (subcontractor, Sciome LLC)
Getachew Tedla, Ph.D.
Aswani Unnikrishnan, M.S.

Under a proposal announced in late 2025, in 2026 NICEATM will become a division of the Office of Research Innovation, Validation, and Application within the Division of Program Coordination, Planning, and Strategic Initiatives, National Institutes of Health Office of the Director.

Appropriate evaluation of new approach methodologies (NAMs) requires reference data for assessing the method’s ability to predict an outcome of interest. Human data provide the most relevant basis for such comparisons, but they are rarely available due to obvious ethical issues associated with toxicology testing in humans. One exception is data from skin sensitization tests, which that are routinely conducted using a wide range of materials. For this project, CPSC, FDA, and NICEATM scientists and collaborators in the German Federal Institute of Risk Assessment collected data from human predictive patch tests conducted under two protocols: the human repeat insult patch test and the human maximization test. Data were collected from more than 1,500 publications. The data collection process also captured protocol elements and positive or negative outcomes, calculated traditional and non-traditional dose metrics, and developed a scoring system to evaluate each test for reliability. The resulting database (Strickland et al. 2023), which represents the largest set of human data ever assembled for the purpose of evaluating non-animal approaches for chemical safety testing, was characterized for physicochemical properties, chemical structure categories, and protein binding mechanisms. The data are publicly available via the Integrated Chemical Environment to serve as a resource for the development and evaluation of NAMs for skin sensitization testing.

The ICCVAM Authorization Act established the Scientific Advisory Committee on Alternative Toxicological Methods (SACATM). SACATM advises the Director of NIEHS, NICEATM, and ICCVAM about NICEATM and ICCVAM activities.

SACATM, which is directed by its charter to meet at least once each fiscal year, met in September 2024 and September 2025.

Roster of SACATM Members 2024–2025

The Scientific Advisory Committee on Alternative Toxicological Methods (SACATM) is a federally chartered advisory group that advises NICEATM, ICCVAM, and the Director of NIEHS about ICCVAM activities. SACATM is directed by its charter to meet at least once each fiscal year.

The September 17–18, 2024, SACATM meeting was held at NIH in Bethesda, MD. About 40 people attended in person and over 100 people viewed each day of the webcast. Advisory committee members were briefed on federal government and international activities to support validation of new methods, including new ICCVAM guidance on validation, establishment of the Method Developers Forums, updates to OECD guidance on validation, and launch of the NIH Complement-ARIE program. Advisory committee members welcomed the publication of the ICCVAM guidance and encouraged continued progress on the other activities. The other focus topic of the meeting was DNT testing, and scientists from NIEHS, FDA, EPA, and the European Food Safety Authority presented on advances in the use of NAMs for this endpoint.

The September 11–12, 2025, SACATM meeting was hosted at NIEHS in Research Triangle Park, NC, and presented virtually. The meeting attracted over 300 viewers on the first day and nearly 200 on the second day. Many of the presentations at this meeting focused on recently announced initiatives by NIH and FDA to support implementation of NAMs in research and regulatory contexts. Advisory committee members welcomed these efforts and agreed that they would support efficiency in method validation. Data standards and harmonization were both noted as key to this effort. Updates were also provided on development of online tools such as the Collection of Alternative Methods for Regulatory Application, NIH Integrated Chemical Environment, and NICEATM-developed apps for predicting skin sensitization potential. A session on “ICCVAM Public Outreach and Education” presented retrospectives of the scope and impact of the ICCVAM Public Forum meetings and ICCVAM Communities of Practice webinars, as well as a summary of NICEATM training and outreach activities during 2024 and 2025.

ICCVAM agencies convened workshops and webinars from the beginning of 2024 and continuing into 2026, summarized in the table below, to foster collaboration and provide information about alternative testing methods. 

In 2015, ICCVAM initiated a series of Communities of Practice webinars to provide detailed presentations on a current topic relevant to alternative test method development. ICCVAM chair Natalia Vinas, Ph.D., presented a retrospective of ten years of Communities of Practice webinars at the 2025 meeting of the Scientific Advisory Committee on Alternative Toxicological Methods (SACATM). She reviewed the topics covered in all of the webinars and noted those that had set the stage for subsequent activities including collaborations and workshops.

• The January 29, 2024, Communities of Practice webinar was titled Implementing Computational Approaches for Regulatory Safety Assessments. It focused on how to establish confidence in computational approaches for regulatory applications. Michael Lowit, Ph.D., presented an overview of EPA Office of Pesticide Programs goals for applying new approach methodologies (NAMs), including computational models, to ecological risk assessments and described ongoing projects in support of those goals. Pras Pathmanathan, Ph.D., summarized recent FDA Center for Devices and Radiological Health guidance on reporting of computational modeling studies in medical device submissions. Jon Arnot, Ph.D., of Arnot Research and Consulting and the University of Toronto, described tools and approaches for bioactivity and exposure modeling and provided examples of how they can be used to support regulatory decision-making.
• The 2025 webinar was presented on July 21 and focused on Evolution of Complex In Vitro Models. Three scientists from government and academic laboratories and the private sector described how they are approaching validation of complex in vitro models such as microphysiological systems (MPS). Danilo Tagle, Ph.D., of NCATS presented an overview of their Tissue Chips for Drug Screening program. Ivan Rusyn, M.D., Ph.D., described activities of the Texas A&M University’s Tissue Chip Testing Center, which is funded through a public–private consortium. Remi Villenave, Ph.D., from Roche, summarized pharmaceutical industry perspectives on NAMs use, including perceived barriers to NAMs usage.

The ICCVAM Authorization Act directs ICCVAM to carry out the following duties:

• Coordinate the technical review and evaluation of new, revised, or alternative test methods
• Foster interagency and international harmonization of test protocols that encourage replacing, reducing, and refining animal test methods
• Assist with and provide guidance on validation criteria and processes
• Promote the acceptance of scientifically valid test methods
• Promote awareness of accepted test methods
• Submit ICCVAM test method recommendations to appropriate U.S. federal agencies
• Consider requests from the public to review and evaluate new, revised, or alternative test methods that have evidence of scientific validity
• Ensure ICCVAM’s final test recommendations are available to the public
• Prepare reports on ICCVAM progress and accomplishments under the Act and ensure they are available to the public

U.S. regulatory agencies are charged with protecting human and animal health and the environment. To do this, agencies must determine hazards presented by substances such as pesticides, consumer products, and workplace chemicals. Evaluating these substances provides information about possible hazards and enables informed decisions about responsible use, storage, and disposal.

Many currently accepted test methods use laboratory animals. Alternative test methods are methods that replace animal use with nonanimal test systems or lower species, reduce the number of animals required for a specific test procedure, or refine animal use to enhance animal well-being and lessen or avoid pain and distress. Collectively, the principles of replacement, reduction, or refinement of animal use for scientific research or product safety testing are referred to as the 3Rs. More recently, the term “new approach methodologies” has been adopted as a broadly descriptive reference to any technology, methodology, approach, or combination of these that support the 3Rs and can be used to obtain information about chemical hazards and risk assessments.

The ICCVAM Authorization Act of 2000 (42 U.S.C. 285l-3) established the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) to

“Establish, wherever feasible, guidelines, recommendations, and regulations that promote the regulatory acceptance of new or revised scientifically valid safety testing methods that protect human and animal health and the environment while reducing, refining, and replacing animal tests and ensuring human safety and product effectiveness.”

The ICCVAM Authorization Act states that the purposes of ICCVAM are to:

• Increase the efficiency and effectiveness of federal agency test method review
• Eliminate unnecessary duplicative efforts and share experiences between federal regulatory agencies
• Optimize utilization of scientific expertise outside the federal government
• Ensure that new and revised test methods are validated to meet the needs of federal agencies
• Reduce, refine, and replace the use of animals in testing, where feasible

The ICCVAM Authorization Act established ICCVAM as a permanent interagency committee of NIEHS under NICEATM.

• Agency for Toxic Substances and Disease Registry (ATSDR)
• National Center for Advancing Translational Sciences (NCATS)
• National Cancer Institute (NCI)
• National Institute for Occupational Safety and Health (NIOSH)
• National Institute of Environmental Health Sciences (NIEHS)
• National Institute of Standards and Technology (NIST)
• National Institutes of Health (NIH)
• National Library of Medicine (NLM)
• Occupational Safety and Health Administration (OSHA)
• U.S. Consumer Product Safety Commission (CPSC)
• U.S. Department of Agriculture (USDA)
• U.S. Department of Energy (DOE)
• U.S. Department of the Interior (DOI)
• U.S. Department of Transportation (DOT)
• U.S. Department of War (DoW)
• U.S. Department of Veterans Affairs Office of Research and Development (VA ORD)
• U.S. Environmental Protection Agency (EPA)
• U.S. Food and Drug Administration (FDA)

ICCVAM’s goals include promotion of national and international partnerships between governmental and nongovernmental groups, including academia, industry, advocacy groups, and other key stakeholders. To foster these partnerships, ICCVAM holds public forums annually to share information and facilitate direct communication of ideas and suggestions from stakeholders.

The first ICCVAM Public Forum was held in 2014. At the 2025 SACATM meeting, ICCVAM member Anna Lowit, Ph.D. (EPA principal agency representative) described how both agency participation and audience engagement with the event has grown over time. She noted how public forum meetings provide a venue for agencies to announce and discuss projects and initiatives. They also facilitate engagement with specific stakeholder groups such as nongovernmental organizations and method developers.

The 2024 ICCVAM Public Forum was held in person on May 20 and 21 at NIH in Bethesda, MD, with an option for remote viewing. About 40 people attended in person, and nearly 300 viewed the webcast. The program featured 18 presentations from seven of the 17 ICCVAM member agencies describing activities both to advance new approaches to safety testing of chemicals and medical products and to reduce the amount of testing required. Updates were also provided on ICCVAM local and international activities. Public comments ranged from general suggestions about areas of focus for ICCVAM to specific discussions on the status of replacing animals for endpoints such as inhalation toxicity or developmental neurotoxicity (DNT). Specific comments recognized ICCVAM for publication of the document “Validation, Qualification, and Regulatory Acceptance of New Approach Methodologies” and asked ICCVAM agencies to play an active role in the application of super intelligence in toxicology.

Due to uncertainty about travel and public communications, the 2025 ICCVAM Public Forum was held virtually on July 20 and 21 rather than in May as in the past. Between 150 and 180 people viewed the webinar over two days. Eight presentations provided updates on NICEATM and ICCVAM activities and activities of six ICCVAM member agencies. Five organizations submitted written comments, and six organizations presented public comments. Commenters recognized that federal agencies and stakeholders are undergoing a major transition toward NAMs that is driven by broader regulatory momentum and specifically noted new NIH policies and the 2025 FDA roadmap. However, they also noted uneven progress due to unclear guidance, inconsistent terminology, limited data sharing, and lack of standardized tracking of animal use. Organizations including advocacy groups and scientific consortia agreed on the need for better infrastructure, including training, transparent reporting, harmonized definitions, and validated methods, alongside increased international coordination. While some groups pushed for rapid replacement of animal testing where alternatives exist, others advocated a balanced approach that continues improving animal models in parallel to ensure scientific rigor and public health protection during the transition.

Although new approach methodologies (NAMs) are being increasingly developed and used, their adoption for regulatory decisions has been slow, and awareness outside certain scientific communities remains limited. To address this, the ICCVAM Consideration of Alternative Methods Workgroup gathered input from stakeholders in industries such as agrochemicals, consumer products, and pharmaceuticals as well as from academic researchers and animal care committees.

The review (Oyetade et al. 2025) found that while all stakeholders use or consider NAMs, adoption levels vary. Five key barriers were identified: perception, regulatory acceptance, scientific and technical limitations, education, and financial challenges. Stakeholders highlighted the need for clear regulatory criteria, better awareness of NAM capabilities, and resources for education and funding as critical points for improvement.

To overcome these barriers, several solutions were proposed. These include tailored education programs to inform diverse audiences, proactive collaboration across sectors, harmonized national and international standards for NAM acceptance, and the creation of centralized repositories, such as the Collection of Alternative Methods for Regulatory Application (CAMERA), for NAM-related information. Other recommendations involve increasing awareness of NAM strengths and limitations and ensuring NAMs are used in ways that suit specific purposes.

This effort provides a roadmap for advancing NAM adoption, emphasizing the importance of communication, education, and collaboration. By addressing these challenges, the use of NAMs can expand, supporting ethical and scientifically robust methods for chemical testing and regulatory decisions.

ICCVAM establishes ad hoc workgroups to perform specific tasks important for the development or validation of alternatives to animal models. The workgroups are composed of representatives from agencies that use or require data from the topic of interest. Representatives of partner organizations in the International Cooperation on Alternative Test Methods may also be invited to participate in a workgroup.

ICCVAM workgroups play a key role in carrying out ICCVAM activities, including implementing the goals of the Strategic Roadmap for Establishing New Approaches to Evaluate the Safety of Chemicals and Medical Products in the United States. When a workgroup completes its charge tasks, it transitions to an expert group to facilitate continued communication and engagement in that area of interest.

During 2024 and 2025, ICCVAM had active workgroups focused on the following areas:

• Acute toxicity: co-chairs Kristen Fulbright, M.S. (DoW) and Grace Patlewicz, Ph.D. (EPA)
• Consideration of alternative methods: transitioned to expert group status in 2025; co-chairs Jessie Carder, M.S. (USDA) and Shannon Marko, D.V.M. (DoW)
• Ecotoxicology: co-chairs William Eckel, Ph.D. (EPA) and Natalia Garcia-Reyero Vinas, Ph.D. (EPA)
• PFAS: co-chairs Natalia Garcia-Reyero Vinas, Ph.D. (EPA), Kelly Carstens, Ph.D. (EPA), and Shruti Kabadi, Ph.D. (FDA)
• Validation: transitioned to expert group status in 2024; co-chairs Suzanne Fitzpatrick, Ph.D. (FDA), John Gordon, Ph.D. (CPSC), and Elijah Petersen, Ph.D. (NIST)

Aircrew piloting high-performance aircraft face environmental stressors during missions that could reduce efficiency and compromise success. One of those stressors is hypoxia. The military has addressed the issue of hypoxia by supplying high levels of oxygen to the pilots. Cognitive symptoms have been observed that may be related to pilot oxygenation, leading to concern over negative impacts directly from hyperoxia or from hypoxia that may be caused by a mismatch between the pilots and the equipment. To study the mechanisms that may be affected, U.S. Air Force (USAF) scientists used a neurovascular-unit-on-a-chip model built in Emulate microfluidic chips and exposed it to either hypoxia or hyperoxia. Oscillatory hypoxia and hyperoxia exposures were included to address the potential mismatch between pilot breathing and oxygen supply from the equipment. Both hypoxic and hyperoxic conditions were found to disrupt the blood-brain barrier, although viability was not affected very much by hypoxia or hyperoxia in the vascular compartment. In the brain compartment, both hypoxia and hyperoxia appeared to improve viability of the cells.

Developmental neurotoxicity (DNT) testing is traditionally done using animals, which is resource-intensive and fails to provide information on cellular processes affected by chemicals. EPA scientists have been evaluating use of new approach methodologies (NAMs) to assess DNT potential (Carstens et al. 2022), focusing on the microelectrode array neuronal network formation assay and high-content imaging to evaluate proliferation, apoptosis, neurite outgrowth, and synapse formation. Data for 92 chemicals with a range of potential DNT activities screened in 57 assay endpoints were sourced from publicly available data. From these data, a proposed DNT NAM battery was constructed that provides a sensitive marker of DNT bioactivity, particularly for evaluating cytotoxicity and network connectivity. This multi-dimensional assay suite may also provide insight into specific functional processes affected by chemical exposure.

The potential for neurotoxicity in children following exposure to environmental chemicals is of concern due to recent increases in the prevalence of neurological disorders such as attention deficit hyperactivity disorder and autism. Neurotoxicity risk for an individual can be influenced both by genetic background and by exposures to neurotoxic chemicals in the environment. To investigate the role of genetic diversity in susceptibility to neurotoxicity, scientists at EPA and FDA are using a genetically diverse set of cells to evaluate a curated set of chemicals with neurotoxic potential. Neural progenitor cells were derived from a set of mice bred to maximize genetic diversity, and a set of 135 cell lines was established, considered to be the minimum number of cell lines needed to quantitatively assess diversity in population responses. The panel of cell lines was exposed to eight concentrations of a 12-chemical test set. The intracellular structure of each treated cell was visualized using a high-content imaging assay called Cell Painting, which uses six fluorescent dyes to quantitatively describe cell features such as cell membranes, mitochondria, DNA and RNA, cytoskeleton, and Golgi bodies (Bray et al. 2016). Data analysis has enabled derivation of points-of-departure for chemical-induced intracellular morphometric endpoints and characterization of the variability in these points-of-departure across cell lines for each chemical. Analysis is ongoing to utilize a probabilistic framework to inform data-driven uncertainty factors that describe interindividual variability, allowing for adequate protection of genetically sensitive subpopulations. A paper describing the project is planned for submission in early 2025.

The Frank R. Lautenberg Chemical Safety in the 21st Century Act, enacted in 2016, amended the Toxic Substances Control Act of 1976. Section 4 of the updated law authorizes EPA to issue test orders to chemical manufacturers (including importers) and processors to develop information on chemical toxicity, subject to specific requirements to reduce vertebrate animal use. One such requirement, described in Section 4(a)(4), is to employ a tiered-testing strategy. Other requirements, described in Section 4(h), directs EPA to review all reasonably available toxicity information to avoid unnecessary or duplicative testing, and to consider non-vertebrate methods where appropriate.

As of the end of 2023, EPA has issued three test orders on PFAS and plans to issue more in the near future. The PFAS test orders include in vitro tests in the tiered-testing strategy, including a respiratory tract epithelial cell culture and genotoxicity tests. One of the test orders, on a chemical known as HFPO-DAF (a precursor to GenX), contains no vertebrate testing at all. Vertebrate testing is included in Tier 2 tests for the other two PFAS test orders, but the tiered testing provides for waiving of these tests based on Tier 1 test results. Taken together, the orders demonstrate EPA’s commitment to employ non-vertebrate testing to the extent practicable and scientifically justified.

Animal test data from evaluations of the toxicity of inhaled chemicals have limited applicability to humans due to anatomical differences between human and rodent respiratory tracts and complexities in the transport kinetics of inhaled chemicals through these organs. New approach methodologies (NAMs) have the potential to provide more human-relevant, mechanistically driven approaches to identify potential inhalation toxicity hazards. While in vitro assay data can provide valuable information about the effects of chemicals on biological systems, it is necessary to translate in vitro results to an in vivo context to determine safe exposure levels in real-life situations. In vitro to in vivo extrapolation (IVIVE) estimates the equivalent administered dose that would result in plasma concentrations equivalent to in vitro activity concentrations.

ATSDR and NICEATM scientists applied IVIVE to in vitro assay data from 20 volatile organic compounds with available pharmacokinetic and in vivo toxicity data covering multiple target organs via inhalation exposure. For each chemical, equivalent administered doses that would result in plasma or lung concentrations equivalent to the in vitro activity concentrations were estimated and compared to the in vivo points-of-departure (PODs) or ATSDR Minimal Risk Levels. While equivalent administered doses for most tested chemicals either underpredicted or were within 5-fold of in vivo PODs, substantial deviations from in vivo PODs warrant further investigation into the sources of uncertainty. However, most equivalent administered doses based on in vitro data were at least an order of magnitude higher than the corresponding Minimal Risk Levels, suggesting that an empirical or statistical modifying factor may be required to align equivalent administered dose-derived thresholds with public health-based guidance values. Physicochemical properties, particularly volatility, were found to significantly influence the reliability of in vitro assay results, and recognizing this and applying proper controls could improve results. There is also the possibility that incorporating experimental toxicokinetic data where available could improve the precision of IVIVE predictions and strengthen alignment with in vivo PODs. A paper describing this study is in preparation for submission in 2026.

Development of military-specific substances can include substances used in new weapons systems and platforms, medications for countermeasures, insect repellants, fire extinguishing agents, warfighter gear, propellants, smokes, simulators, and specialized coatings. All of these must be shown to be safe for warfighter use and be sustainable for manufacture, use, and demilitarization. DoD has developed an effective approach to providing environmental, safety, and occupational health information early in the research, development, testing, and evaluation stages for new and emerging materials (Johnson and Adams 2023). Research begins with modeling promising chemical structures in silico. These quantum mechanical models can also provide information useful for understanding environmental fate and transport and assist in understanding relevant exposure pathways. Once chemical structure information is available, toxicologists provide interpretation on toxicity using in silico models (e.g., QSARs) and read-across techniques. These data are provided to chemists selecting the optimal chemical solutions. Prospective substances are then tested using a variety of in vitro assays useful for suspected toxicity targets based on structural read-across or in silico predictions. Data from these studies are used as the basis for recommendations on progression to in vivo studies focusing on identifying toxic thresholds to protect human health and the environment, and subsequent studies for specific endpoints as needed.

Guideline in vivo developmental neurotoxicity (DNT) studies are conditionally required for pesticide registration with need determined on a case-by-case basis taking into consideration toxicological effects, biological plausibility, and an understanding of the mode-of-action. However, the guideline DNT study is time-consuming and costly both financially and in terms of animal use. While such studies have been conducted on the herbicide DL-GLF, no such data exist for L-GLF acid and L-GLF ammonium, compounds that have the same molecular composition as DL-GLF but are structurally different. This situation presented an opportunity for EPA scientists to explore whether toxicokinetic assessments based on in vitro assay data could be used to support a decision for the need for guideline DNT studies (Dobreniecki et al. 2022). DL-GLF, L-GLF acid, and L-GLF ammonium were screened using in vitro assays for network formation and neurite outgrowth, and toxicokinetic assessments were conducted to derive administered equivalent doses for the in vitro testing concentrations. The results indicated that the available guideline study would be protective of potential DNT due to L-GLF exposure, and were thus used in a weight-of-evidence evaluation to support the decision not to require L-GLF isomer guideline DNT studies, providing a case study for a useful application of DNT screening assays. Similarly, results from in vitro DNT studies were used in a weight-of-evidence evaluation to support the need for additional DNT data for the pesticide dicloran. EPA has also continued its work to analyze data from the DNT new approach methodology battery for use in chemical-specific weight-of-evidence analyses to evaluate the DNT potential of organophosphate pesticides with the weight-of-evidence evaluation for the organophosphate insecticide acephate released in 2023 as part of registration review.

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.

With the increased availability of chemical data in public databases, innovative techniques and algorithms have emerged for the analysis, exploration, visualization, and extraction of information from these data. One such technique is chemical grouping, where chemicals with common characteristics are categorized into distinct groups based on physicochemical properties, use, biological activity, or a combination. However, existing tools for chemical grouping often require specialized programming skills or the use of commercial software packages. To address these challenges, NIEHS scientists developed a user-friendly chemical grouping workflow implemented in KNIME, a free open-source low/no-code and data analytics platform. The workflow serves as an all-encompassing tool, expertly incorporating a range of processes such as molecular descriptor calculation, feature selection, dimensionality reduction, hyperparameter search, and incorporates supervised and unsupervised machine learning methods, enabling effective chemical grouping and visualization of results. The workflow also has tools for interpretation, identifying key molecular descriptors for the chemical groups, and using natural language summaries to clarify the rationale behind these groupings. The workflow was designed to run seamlessly in both the KNIME local desktop version and KNIME Server WebPortal as a web application. It incorporates interactive interfaces and guides to assist users in a step-by-step manner. The workflow is being implemented as part of the Modeling and Visualization (MoVIZ) Pipeline, which is described in an abstract (Moreira-Filho et al.) accepted for a poster presentation at the 2024 Society of Toxicology meeting.

Over the past decade, there has been a worldwide trend toward earlier breast development in girls. The rapid pace of this trend suggests the involvement of environmental factors. Scientists at NCATS and NIEHS examined the potential effects of endocrine-disrupting chemicals on important components of the biochemical pathway responsible for pubertal timing (Yang et al. 2024). In these studies, chemicals in the Tox21 10K collection were tested in human cell-based assays that measure activation or inhibition of the gonadotropin-releasing hormone receptor and kisspeptin receptor. These receptors are expressed in the hypothalamus and are important players in the control of pubertal timing. The 58 chemicals that exhibited activity included chemicals known to be active in these pathways and chemicals for which this activity was novel; these were characterized further in other cell-based assays and molecular docking studies. Further studies are necessary to investigate whether exposure to these chemicals might play a role in the increasing prevalence of early puberty in girls.

Key accomplishments of ICCVAM, ICCVAM member agencies, and NICEATM in support of the ICCVAM mission during 2024 and 2025 include:

• Updated validation guidance by ICCVAM and ICCVAM Method Developers Forum. “Validation, Qualification, and Regulatory Acceptance of New Approach Methodologies: A Report of the ICCVAM Validation Workgroup,” developed by ICCVAM with input from 17 federal agencies, offers recommendations on how to establish confidence in new approach methodologies (NAMs). Publication of the report was followed by the August 2024 ICCVAM Method Developers Forum, which provided an opportunity for NAMs developers to present new methods to identify potential carcinogens to relevant stakeholders.
• ICCVAM review proposing approaches to addressing barriers to adoption of NAMs. A review by the ICCVAM Consideration of Alternative Methods Workgroup (Oyetade et al. 2025) identifies five key barriers to the adoption of NAMs encountered by a variety of stakeholder groups and proposes approaches to address these barriers.
• Launch of the NIH Complement Animal Research in Experimentation (Complement-ARIE) program. Complement-ARIE will accelerate the development, standardization, validation, and use of human-based NAMs and significantly advance the understanding of human health and disease by providing a range of ready and standardized biomedical research models.
• Shift toward adoption of alternatives by FDA and NIH. Policy shifts by the U.S. Food and Drug Administration and the National Institutes of Health (Scheufen Tieghi et al. 2025) will prioritize the development of predictive systems that use computer modeling and in vitro organ and cell testing.
• International acceptance of a defined approach to estimate skin sensitization potency. The Skin Allergy Risk Assessment – Integrated Chemical Environment (SARA-ICE) defined approach for skin sensitization (Reinke et al. 2025) was accepted by OECD in an update to its Guideline 497 for defined approaches to identify skin sensitizers. SARA-ICE is the first defined approach that provides a numerical estimate of potency to be accepted as an OECD guideline test.
• Validation of a thyroid microtissue model. NICEATM coordinated an interlaboratory validation study led by the U.S. Environmental Protection Agency of the utility of a thyroid 3D microtissue model for evaluating chemical effects on thyroid hormone synthesis and tissue viability (Deisenroth et al. 2026). The study established standard operating procedures that were successfully applied in multiple laboratories, and consistent results were generated among the testing laboratories.
• Launch of the CAMERA resource. CAMERA is an interactive database and user interface that provides online access to validated alternative methods for U.S. regulatory and other applications. It is being developed by NICEATM, with an ICCVAM steering committee.

FDA is required to assess the environmental impact of its tobacco regulatory actions per the National Environmental Policy Act. Increases in use of waterpipe tobacco products raises concerns about environmental impacts from waterpipe waste disposal. To characterize the scope of this issue, FDA scientists compiled a comprehensive list of waterpipe wastewater chemical concentrations from literature (Termeh-Zonoozi et al. 2023). Chemicals were then selected for risk assessment by estimating persistence, bioaccumulation, and aquatic toxicity characteristics and hazardous concentration values. Of 38 chemicals with concentration data, 20 were found to be listed as harmful or potentially harmful constituents in tobacco smoke and tobacco products by FDA, and 15 are EPA hazardous waste substances. Six metals on both lists were selected for future risk assessments, as were three non-metals because of their persistence and/or toxicity. The presence of multiple hazardous compounds in waterpipe wastewater highlights the importance of awareness on the proper disposal of waterpipe wastewater in residential and retail settings. Future studies can build on the hazard characterization provided in this study through fate and transport modeling, exposure characterization and risk assessments of waterpipe wastewater chemicals.

High-throughput screening (HTS) programs such as Tox21 gather data on chemical bioactivity from a variety of targets and activities to prioritize assays for further testing or better understand mechanisms of toxicity. However, interpreting such data is complicated by nonspecific chemical reactivity. To help address this problem, specific HTS assays have been developed to measure autofluorescence, and data from these assays have been used to train machine learning models to predict reactivity.

The fluorescence-based MSTI ((E)-2-(4-mercaptostyryl)-1,3,3-trimethyl-3H-indol-1-ium) HTS assay identifies thiol-reactive compounds. EPA and NCATS scientists used this assay that identifies thiol-reactive compounds to screen 7,872 unique chemicals in the Tox21 10K chemical library (Patlewicz et al. 2023). Active chemicals were compared to profiling outcomes using structural alerts encoding electrophilic information. A random forest classification model based on chemical fingerprints was then developed to predict assay outcomes. The classification model shows promise as a tool to screen untested chemicals for their potential electrophilic reactivity based solely on chemical structure. MSTI assay data were subsequently evaluated for their utility in predicting compound promiscuity and cytotoxicity by comparing the activity of the Tox21 10K compounds in the MSTI assay to their activities across the entire Tox21 assay panel (Kruger et al. 2024). The predictive performance showed a sensitivity of 55% and specificity of 97%; chemical features such as halides and carboxamides resulted in false negatives and false positives, respectively. While this high-throughput assay allows researchers to identify whether a compound will be cytotoxic and/or promiscuous in an efficient manner, this study also reveals potential challenges in its use.

Key characteristics of carcinogens (KCCs) provide a foundation for organizing data on cancer mechanisms, with the potential to better characterize cancer hazard. To apply this framework to HTS assay data, a workgroup mapped assay data from EPA ToxCast invitrodb v. 4.2 to KCCs (Cuomo et al. 2026). The workgroup was made up of experts with diverse subject matter knowledge from NIEHS; EPA; California EPA; University of California, Berkeley; Texas A&M University; and the International Agency for Research on Cancer. Previous KCC assay mappings using the International Agency for Research on Cancer and NIH Integrated Chemical Environment databases served as the starting point for this work. Reviewers developed a harmonized mapping approach to relate different assay technologies and endpoints to carcinogenicity. The workgroup’s approach was to (1) clarify the contexts of the defined KCCs, considering directionality and biological coverage of the in vitro HTS assay space; (2) develop a tiered approach to mapping assays to integrate key considerations about the direct relevance of assay platforms to carcinogenesis; and (3) map relevant assays to one or more KCC, each with a detailed documented rationale for the attribution. Of the 1,570 assay endpoints available in the database, 838 were identified as mappable to at least one KCC. For each mapped KCC, assays were assigned as either “Tier A”, assays measuring a direct effect on the biology defined by a KCC or “Tier B”, assays measuring an indirect or downstream effect on the biological process underlying a KCC. This comprehensive mapping, validated through thorough manual review and documentation by a panel of experts, enhances the utility of HTS data for integration into carcinogenicity evaluation and ultimately contributes to a deeper understanding of the complex mechanisms underlying chemical-induced cancer. This effort will be incorporated into NICE and the International Agency for Research on Cancer Monographs for use in weight-of-evidence analyses and other integrative approaches for the identification of cancer hazards.

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.

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.

A contaminant of emerging concern is a material with no regulatory standard, that may induce toxicity at lower levels of exposure than previously characterized, is challenging to address across jurisdictions, and presents challenges for timely information‑sharing with stakeholders. Many contaminants of emerging concern are of interest to multiple federal agencies, which has given rise to efforts to coordinate addressing them. The Contaminants of Emerging Concern Interagency Working Group coordinated federal programs and activities to address contaminants of emerging concern; 18 agencies were involved, including eight ICCVAM member agencies. A 2022 report issued by the working group summarized 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 were established to focus on (1) nontargeted analysis and effects-based monitoring to discover and screen contaminants of emerging concern, (2) characterizing risk by assessing potential hazards and exposure, and (3) formulating joint solicitations across agencies. A 2024 implementation plan to address this last activity outlined a series of short- and long-term activities that are anchored to success metrics and identified NAMs as a critical tool for potential hazard characterization.

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.

The NIH Integrated Chemical Environment (NICE) provides data and tools to help develop, assess, and interpret chemical safety tests. The March 2024 NICE v. 4.0.2 release improved support for chemical name searching in the NICE REST API. The September 2024 NICE v. 4.1 release added versioning information to datasets and added applicability domain information for chemical property predictions from the Open (Quantitative) Structure–activity/property Relationship App (OPERA). The July 2025 NICE v. 4.2 release updated curated high-throughput screening (cHTS) data to align with data in the EPA ToxCast invitrodb v. 4.2 release. Other updates and improvements to NICE datasets during 2024–2025 include:

• Links to the NTP Chemical Effects in Biological Systems database and the EPA CompTox Chemicals database in data downloads
• Data annotation aligned with Open Biological and Biomedical Ontology Foundry ontologies
• Ability of users to download ClassyFire (Djoumbou Feunang et al. 2016) chemical taxonomies for over one million chemicals
• A new “PFAS” Chemical Quick List; a “Demo List of Chemicals” Chemical Quick List was also added to help new users explore NICE functionality
• An updated “RoC Classifications” Chemical Quick List that includes new data from the NTP’s 15th Report on Carcinogens
• An update of the “Tox21” Chemical Quick List
• Continued harmonization of skin sensitization and eye irritation data

The NIH Integrated Chemical Environment (NICE), developed by NICEATM, provides data and tools to help develop, assess, and interpret chemical safety tests. All tool inputs now provide a “filter-as-you-type” function to help users find Chemical Quick Lists of interest. NICE tool help videos were updated in the September 2024 NICE v. 4.1 release and the February 2025 NICE v. 4.1.1 release. The updates also implemented improvements to many specific NICE tools.

• Major updates to Search result data visualizations in the March 2024 v. 4.0.2 release added immersive visualizations and functional use pagination; this release also updated filter chain options for Search results.
• The March 2024 update of the In Vitro to In Vivo Extrapolation (IVIVE) tool added new options for data overlays to contextualize model results. The July 2025 NICE v. 4.2 release also enabled users to provide their own physicochemical and absorption, distribution, metabolism, and excretion (ADME) data to Physiologically Based Pharmacokinetics (PBPK) and IVIVE tool queries.
• The July 2025 release revised the principal component analysis plots in the Chemical Characterization tool.
• Multiple improvements were made to the Curve Surfer tool, which allows users to view and interact with concentration–response curves from curated high-throughput screening (HTS) data. The tool now allows users to overlay AC50 or ACC values and includes technological interference flags and points-of-departure in results. Curve Surfer data are also now available via the NICE REST API.

Cardiotoxicity, or toxicity to the heart or cardiovascular system, is a major cause of failure of new drugs in mid- to late-stage development. Chemicals found in these drugs or in the environment may also contribute more broadly to human cardiovascular disease. The NIEHS Division of Translational Toxicology (DTT) conducts testing and research to determine potential human health effects of chemicals, drugs, food additives, dietary supplements, or environmental agents. Activities during 2022 and 2023 in the DTT’s Cardiovascular Health Effects Innovation Program focused on how environmental factors can affect human susceptibility to or development of cardiovascular disease.

• In vitro to in vivo extrapolation (IVIVE) was used to derive human-equivalent administered doses (EADs) from test chemical concentrations that induce effects in in vitro cardiotoxicity assays. These EADs were then compared with U.S. human exposure biomonitoring, prediction models, and data from geospatial mapping to prioritize chemicals for further study. Reports on this project (Krishna et al.) were presented at the 2023 annual meeting of the SOT and the 12th World Congress on Alternatives and Animal Use in the Life Sciences. A publication describing this work is in preparation for submission in 2024.
• In vitro assays representing a broad range of cardiovascular-relevant activities were used to characterize potential cardiotoxicity hazard of a group of chemicals including botanicals, flame-retardants, insecticides, polycyclic aromatic hydrocarbons, quaternary ammonium salts, and PFAS. A report on this project (Ramaiahgari et al.) was presented at the SOT 2023 annual meeting.
• Program scientists developed an interactive systematic evidence map to integrate data from human, animal, and in vitro studies of effects of environmental exposures on cardiotoxicity to support prioritization of future cardiotoxicity studies. A publication describing this systematic evidence map is in preparation for submission in 2024. A similar map was constructed to integrate data from human and animal studies on hypertensive disorders of pregnancy.
• In collaboration with the NCATS, the program is testing chemicals with potential vascular toxicity in human umbilical vein endothelial cells and in coculture and flow models.

Future activities include collaborative efforts with external cardiovascular researchers using various models such as tissue-engineered blood vessels and cardiomyocytes to evaluate potentially cardiotoxic chemical activity.

To reduce and replace the use of animals in immunotoxicity assessments, the NIEHS Division of Translational Toxicology collaborated with NICEATM to develop a comprehensive human whole-blood NAM battery for in vitro assessment of immune function. The system uses human whole blood diluted in culture media, to which an immune stimulant such as anti-CD3/CD28 antibodies or viral peptides may be added. The model immunotoxicants dexamethasone and benzo[a]pyrene were used to evaluate the utility of the screening assay. Following in vitro culture, immune cells were identified and counted by flow cytometric immunophenotyping and assessed for natural killer cell activity and T cell activation. Proinflammatory cytokine levels were measured in culture supernatants. In vitro treatment with dexamethasone resulted in concentration-dependent suppression of cytokine production, natural killer cell activity, and T cell activation. Treatment with benzo[a]pyrene caused suppression of cytokine production, but immunosuppression only occurred following metabolic activation by addition of S9 fraction enzymes. Advantages of this NAM battery include the ability to assess adaptive immunity and relate immunotoxicity to resistance to infectious diseases. These studies demonstrate that in vitro immunotoxicity assays can measure effects that reflect known outcomes from in vivo studies. This multiendpoint human whole-blood NAM battery may be useful for screening compounds for immunotoxicity hazard identification in a manner that reduces reliance on animal systems and increases translatability to humans.

Carcinogenesis is a multistep process in which healthy cells acquire properties that allow them to form tumors or malignant cancers. The concept of key characteristics of carcinogens has been developed to describe 10 properties that are shared by viruses and chemicals that induce human cancers. QSAR models that rely on structural or physicochemical properties to predict carcinogenesis potential endpoints usually perform poorly, likely because they lack sufficient information on the complex mechanisms involved in carcinogenicity. NICEATM scientists and collaborators combined a novel imputation profile QSAR modeling approach with modern machine learning to analyze data on 10,000 Tox21/ToxCast chemicals and 2,000 in vitro assay endpoints associated with key characteristics of carcinogens. Because limited experimental data were available, data gaps were filled by imputing assay results for the Tox21/ToxCast inventory using structural and physicochemical properties and novel artificial intelligence modeling. Various machine-learning approaches including a multitask deep learning model were applied to predict each chemical’s likelihood of inducing cancer based on the imputed in vitro data. Results included output metrics on the quality of imputation, defined by grouping of assays, and performance computed per chemical. Work is ongoing to validate the prediction model results against literature data, develop confidence scores for the imputation modeling, and map assay data to the key characteristics of carcinogens.

The Open (Quantitative) Structure–activity/property Relationship App (OPERA) is a free and open-source/open-data suite of QSAR models developed to support a range of research and regulatory purposes. In addition to physicochemical and environmental fate properties, OPERA offers a number of models to predict ADME endpoints that are important to PBPK modeling and IVIVE studies. All OPERA models were built using curated datasets split into training and test sets and molecular descriptors calculated based on standardized QSAR-ready chemical structures. Modeling adhered to the five principles for QSAR model development adopted by OECD. These principles support the development of scientifically valid, highly accurate models with minimal complexity that support mechanistic interpretation, when possible. For consistency and transparency, OPERA provides a tool for standardizing chemical structures, an estimate of prediction accuracy, an assessment of applicability domain, and incorporation of experimental values when available. Technical and performance details are described in OECD-compliant QSAR Model Reporting Format reports.

Existing OPERA models are updated regularly when new experimental data become available. In 2024 and 2025, OPERA models were expanded with newly curated datasets covering a number of physicochemical properties and ADME parameters, as well as new input/output features and bug fixes. OPERA predictions are available through the EPA CompTox Chemicals Dashboard and NICE. The OPERA application can also be downloaded from the NIEHS GitHub repository as a command-line or graphical user interface for Windows and Linux operating systems. It is also available as an extension to OECD’s QSAR Toolbox, a resource provided by OECD and the European Chemicals Agency to support animal-free chemical hazard assessment and on the FDA’s Precision platform.

There is a need for reliable and easy-to-use navigation tools capable of browsing, mining, and utilizing large and diverse repositories of chemical and biological data. To address this need, NIEHS in collaboration with Sciome, LLC, developed OrbiTox. OrbiTox uniquely offers an interactive and immersive 3D environment for the visualization of millions of chemical data entries, including both experimental and predicted biological activities toward various gene targets tested in vitro and/or in vivo. By organizing those data entries in concentric “orbits” (e.g., chemicals, genes, pathways, species), OrbiTox facilitates translational discovery by inferring knowledge from connections across multiple data domains and sources (Ross et al. 2025). Through an intuitive graphical user interface, OrbiTox offers rapid and interactive navigation in the chemical space along with extensive gap-filling capabilities. Currently, OrbiTox employs over 150 predictive and cross-validated QSAR models, including individual models for Tox21 assays, bacterial mutagenicity, cardiotoxicity failure modes, and carcinogenicity classification and potency. These models use Saagar molecular fingerprints (Sedykh et al. 2021), which provide interpretable, chemistry-backed reasoning for each prediction with state-of-the-art atom contributions. As a result, users can hypothesize on the substructural origins and/or mechanistic steps highlighting why a given chemical exhibits a particular activity. Such identification of favorable versus unfavorable chemotypes for a given biological property can be of critical importance for prioritizing experimental testing. OrbiTox also provides rich, structurally relevant analogs for read-across assessments, thanks to Saagar-based structural similarity and its vast database of bioactivity and metabolic profiles. Finally, OrbiTox provides users with opportunities to directly compare animal versus nonanimal data, which is important for building confidence in and establishing the relevance of new approach methodologies, ultimately supporting the transition to human-based safety testing. The fall 2026 release will include an extended set of new QSAR models and advanced chemical filtering features to further refine chemical search results and predictions.

The International Cooperation on Alternative Test Methods (ICATM) was created to foster dialog among national validation organizations. This dialog facilitates international cooperation in the critical areas of validation studies, independent peer review, and development of harmonized recommendations. ICATM includes member organizations from the European Union, United States, Japan, Canada, and South Korea. In recent years Brazil, China, Singapore, and Taiwan have participated in ICATM as observers.

ICATM Participant Organizations

• ICCVAM is an interagency committee of the U.S. government that coordinates technical reviews of alternative test methods and cross-agency activities relating to validation, acceptance, and harmonization of test methods. NICEATM administers ICCVAM and provides scientific support for its activities.
• EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) is a unit within the Institute of Health and Consumer Protection in the European Union’s Joint Research Centre. EURL ECVAM coordinates the validation of alternative test methods in the European Union.
• JaCVAM (Japanese Center for the Validation of Alternative Methods) coordinates the evaluation of alternative test methods for the Japanese National Institute of Health Sciences, its parent organization.
• Health Canada’s Environmental Health Science and Research Bureau coordinates the evaluation of alternative test methods in Canada. The Canadian Centre for Alternatives to Animal Methods and its subsidiary, the Canadian Centre for the Validation of Alternative Methods, participate as partners with Health Canada in ICATM activities.
• KoCVAM (Korean Center for the Validation of Alternative Methods) is part of the National Institute of Food and Drug Safety Evaluation of the South Korean Food and Drug Administration.
• BraCVAM (Brazilian Center for the Validation of Alternative Methods) functions as the focal point within Brazil to identify or receive requests for test method validation. It works to implement appropriate validation studies with the National Network of Alternative Methods to the Use of Animals within the Brazilian Ministry of Science, Technology, Innovations, and Communications.

In most years, ICATM member organizations hold at least one coordination meeting. The most recent coordination meeting was convened in August 2023 in Niagara Falls, Canada, in conjunction with the 12th World Congress on Alternatives and Animal Use in the Life Sciences. Representatives from the United States, European Union, Canada, Japan, South Korea, Taiwan, Brazil, and the OECD Test Guidelines Programme met to discuss ongoing efforts to update OECD’s “Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment,” informally known as “Guidance Document 34.”

Participants in the August 2023 ICATM coordination meeting felt that the traditional validation approach of conducting multi-laboratory ring trials to assess transferability and performance may not be practical for many new technologies upon which NAMs are based. Elements identified as key to successful validation included well-defined protocols that incorporate quality management principles; a diverse set of reference chemicals; clear acceptance criteria that consider standards other than prediction of in vivo animal data (e.g., human biological relevance); and coordinated peer review that minimizes duplication of effort.

ICATM representatives also attend or participate in meetings of the Scientific Advisory Committee on Alternative Toxicological Methods.

NICEATM participated in a peer review of a JaCVAM-coordinated validation study of the EpiSensA skin sensitization test method. The method utilizes a reconstructed human epidermis model to evaluate changes in gene expression of four markers of keratinocyte response to skin sensitizers, the second key event of the adverse outcome pathway for skin sensitization. A poster presented at the 2023 annual meeting of the Society of Toxicology (Reinke et al., Alternatives to Mammalian Models II session) summarized the validation study. The method will be under evaluation at OECD for inclusion in TG 442D in the 2024 review cycle. NICEATM and JaCVAM scientists and members of the EURL ECVAM advisory committee also participated in a peer review of the SENS-IS test method for skin sensitization.

The following ICCVAM workgroups had ICATM member liaison representatives during 2022 and 2023.

Microphysiological systems (MPS), also frequently referred to as “tissue chips” or “organs-on-chips” are in vitro platforms composed of cells, explants derived from tissues or organs, or organoid cell formations. The cells and tissues, which may be of human or animal origin, are maintained in a microenvironment that provides and supports the biochemical, electrical, and mechanical responses to model the specific properties that define the function of the organ or tissue of interest. Over the past decade, a broad variety of MPS models and systems have emerged. To facilitate the collaboration and cooperation needed to appropriately support further development and acceptance of MPS, international companies and organizations teamed up in 2021 to initiate annual MPS World Summits. These meetings present the latest scientific achievements, discuss advances and challenges, and enable communication between young and newly interested scientists and pioneers in the MPS field.

The World Summits are organized by the International MPS Society, which was established in 2023 to connect, exchange, and educate to promote the international standardization and harmonization of MPS, establish a global training environment, and maximize the potential of MPS to advance the life sciences. At the 2024 World Summit, NIST organized a “Workshop for Advancing Standards and Regulatory Science for MPS and Microfluidic-based Medical Products” that included members of the Microfluidics Association and members of the International MPS Society Standardization Interest Group. The 1½-day workshop gathered participants and speakers from over 15 countries and was attended by over 120 people. It covered a wide range of topics and featured 25 speakers discussing their work in areas of microfluidics-based medical devices as well as on research and standardization efforts in MPS.

The 2026 MPS World Summit will be held in Washington, DC, and scientists in the FDA Human Foods Program (HFP) are on the organizing committee.

The FDA Center for Drug Evaluation and Research pursues international harmonization of nonclinical recommendations for pharmaceutical development through its engagement with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). ICH brings together regulatory authorities and the pharmaceutical industry to discuss scientific and technical aspects of drug registration. ICH's mission is to achieve greater harmonization worldwide to ensure that safe, effective, and high-quality medicines are developed and registered in the most resource-efficient manner. These activities prevent repetition of studies and reduce and refine animal use in overall drug development. New guidance recently approved will continue to contribute to the 3Rs as will future guidance currently being developed or revised.

FDA published guidance aligned with ICH documents on carcinogenicity testing of pharmaceuticals as discussed elsewhere in this report.

PBPK modeling can be applied to high-throughput applications to facilitate decision-making in drug discovery and risk assessment. However, high-throughput pharmacokinetic modeling is limited to organs and organ systems represented by compartments included in the model. Many PBPK models lack brain and adipose compartments due to the complexity of chemical transport, partitioning, and release in these tissues. However, estimating brain and adipose tissue concentrations is important to predict neurotoxicity and effects of long-term exposure to persistent chemicals. To address these challenges, NICEATM scientists and collaborators developed a PBPK model using the EPA open-source httk R package (v. 2.2.2) that incorporates brain and adipose tissue compartments. The brain compartment accounts for chemical transfer across the blood–brain barrier. The concentration–time profiles generated by the revised model showed general concordance for both brain and adipose compartments in comparison to experimental data. For example, the maximum plasma concentration (Cmax) values predicted by the model for brain tissue were within 2-fold of experimental measurements for both water-soluble chemicals such as acetaminophen (predicted Cmax = 54.01 µM, observed Cmax = 74.03 µM) and hydrophobic chemicals such as benzo[a]pyrene (predicted Cmax = 11.21 µM, observed Cmax = 9.59 µM). This alignment between the httk-based brain–adipose model predictions and predictions from experimental data demonstrates the robustness of the model and its potential applicability for both drug development and chemical risk assessment. A paper describing this work is in preparation, and the model will be made publicly available.

To use data generated by high-throughput screening (HTS) initiatives such as Tox21 and ToxCast in regulatory applications, the assays and models built from the assays must be validated based on their performance against the biological targets they query. This requires developing sets of reference chemicals that consistently yield reproducible results when assayed for these biological targets. Furthermore, the development of reference chemical sets needs to be streamlined and rapid enough to manage the tens to hundreds of assays that can help inform regulatory toxicity endpoints. To address this need, scientists at EPA and NIEHS developed a process to identify reference chemicals that consistently produce positive or negative results when tested in defined assays (Judson et al. 2019). Current work under this project in partnership with NCATS seeks to automate the generation of reference chemical lists without the need for new experimental tests or upfront literature review. The targets being examined are androgen receptor, estrogen receptor, glucocorticoid receptor, peroxisome proliferator-activated receptor gamma, progesterone receptor, retinoic acid receptors, thyroid hormone receptor, tumor protein p53, mitochondria toxicity, and cell stress pathways. The list of assays and reference chemicals generated through this process will be used to validate new assays or AOPs, which will improve chemical screening abilities when predicting toxicity. The project will produce a reference chemical selection workflow and an associated paper to be submitted for publication in 2024.

EPA and NICEATM researchers (Karmaus et al. 2026) reviewed the consistency of repeated traditional animal toxicity studies used in safety assessments. They examined published analyses covering various safety tests including eye and skin irritation, acute systemic toxicity, cancer studies, and developmental toxicity. The results showed that replicability, getting the same result when repeating a study, varied widely depending on the type of test and how the results are interpreted. For instance, a study could report a dose that caused toxicity, or it could be used in a categorical classification format with varying levels of concern. One recurring finding was that extreme results (finding that the chemical was either highly toxic or nontoxic) were more consistent than moderate effects. Evaluating replicability was difficult because studies are not frequently repeated for the same chemical, and the approach used in a subsequent study is not always the same as the approach used in the first. This variability comes from multiple sources, including differences in animal lines, experimental procedures, natural biological variation, and analysis methods. These findings are important because regulatory agencies are increasingly accepting new test methods that do not use animals, but these alternatives are often required to perform "as well as or better than" traditional animal test methods. Understanding that animal studies themselves have significant variability helps set realistic performance standards for new methods and suggests that some alternative approaches may be more reliable than the animal tests they are meant to replace.

Results from in vitro assays used in the Tox21 and EPA ToxCast HTS programs may be confounded by overt cell stress and cytotoxicity, such that a decrease in viable cells could erroneously be attributed to a chemical’s mechanistic effects. Integration of cytotoxicity assessment with assay endpoints can bolster confidence in the interpretation of assay outcomes. To address this, concurrent cytotoxicity readouts from Tox21/ToxCast assays were mapped for hundreds of assay endpoints, allowing direct comparison of bioactivity potency against cytotoxicity. For chemicals tested in HTS assays that lack directly relevant cytotoxicity data, NICEATM scientists are developing machine learning algorithms to predict chemical- and cell type-specific cytotoxicity concentrations across time trajectories. The models’ predictions will be integrated with bioactivity data to strengthen confidence in assay outcome interpretation by identifying mechanistic vs. nonspecific/cytotoxicity-confounded bioactivities. The comparisons and predictions will be integrated into future versions of concentration–response visualizations for the curated HTS data in the NICE Curve Surfer tool. The direct comparisons will also be incorporated into the NICEATM project to harmonize curated HTS data to the OECD Harmonised Template 201, providing additional context for bioactivity calls to help facilitate the international adoption of standardized NAMs.

The developing child is vulnerable to genetic, pharmacological, or chemical disruption of the retinoid biochemical pathway, especially during early growth and differentiation of embryonic tissues. Susceptibility of this pathway to chemical disruption is an important regulatory concern for developmental and reproductive hazard identification. In this study, which involves all four Tox21 participating offices, Tox21 data are being mined and modeled to identify potential retinoid pathway disruptors. Results from a dozen Tox21 molecular targets mapping to retinoid pathway targets identified over 100 structurally diverse chemicals with relevant bioactivity. Computational tools and approaches are now being built to integrate these data with embryological knowledge and construct data-driven models for developmental hazard prediction. Earlier publications from the project:

• Identified candidate reference chemicals for use in in vitro assays relevant to the pathway (Baker et al. 2022).
• Described a computational model for fetal skeletal defects potentially linked to disruption of retinoic acid signaling (Pierro et al. 2022).
• Applied in vitro to in vivo extrapolation to characterize potency of chemicals interacting with the pathway (Chang et al. 2022).
• Developed and validated a high-throughput assay measuring activity of the metabolic enzyme CYP26A1 (Sakamuru et al. 2024); this assay was then used to first screen a library of pharmacologically active chemicals and then the Tox21 10K compound collection. Chemicals with inhibitory activity were selected for follow-up assays planned for 2026.

More recently, a high-throughput cytokine detection platform using human induced pluripotent stem cell-derived microglia was developed and validated to identify potential neurotoxicants (Yang et al. 2025) by testing nine environmental chemicals with evidence of cytokine-inducing potential in other in vitro assays.

Regulatory agencies use data from in vivo fish acute toxicity tests to assess the potential risk of substances to aquatic vertebrates. The ICCVAM Ecotoxicology Workgroup is preparing a review to help guide the prioritization of the development and application of alternatives to the fish acute toxicity test, focusing on regulatory use of fish acute toxicity data and alternatives. The review will be submitted for publication in 2026.

The Ecotoxicology Workgroup asked each agency:

• What does your agency do with acute fish toxicity data?
• What is your flexibility to use NAMs?
• What should the data submitter know about your agency’s process?
• Are there legal or regulatory impediments to the adoption of NAMs? For example, are live-animal data specifically called for in your agency’s regulations?

Responses were included in the draft manuscript, which will also address performance of the fish embryo toxicity test, an alternative test method described in OECD test guidelines.

Profiling chemical effects on transcription factor activity can help characterize the mechanisms by which chemicals may disrupt biological systems. The Attagene cis-FACTORIAL™ assay uses a reporter system to detect transactivation of 46 transcription factors, providing a quantitative assessment of chemical effects. A later version of this assay, CYP-FACTORIAL™, added nine key CYP450 enzymes to enable the evaluation of chemical effects on transcription factor activity with and without CYP-mediated Phase 1 metabolism. These two complementary assay formats support a better understanding of whether CYP-mediated oxidation results in altered bioactivity profiles between parent and metabolite compounds.

To explore the application of this system to toxicity prediction, NIEHS and Attagene collaborators tested 24 chemicals of regulatory concern using both systems. Profiling across all 46 transcription factors produced chemical toxicity signatures, and applying a biological read-across approach identified chemicals with similar effects. Additionally, comparing the profiles of “toxic” vs. “nontoxic” chemicals yielded insights into the potential biological mechanisms underlying adverse outcomes. Dose–response modeling using the tcplfit2 R package (Sheffield et al. 2022) further revealed how the addition of CYP enzymes altered bioactivity and response sensitivity across chemicals and transcription factors. This study exemplifies how integrating metabolism into a multiplexed in vitro assay system can provide additional mechanistic insights needed to understand chemical-elicited bioactivity, thereby facilitating the development of human-relevant test systems. A manuscript is in preparation and is planned for submission in 2026.

Reviewing in vivo toxicity studies to assess the risk chemical exposures pose to human health is an important process. A foundational part of this process involves extracting and organizing information into a structured format. However, the scientific language used across studies may vary widely, requiring substantial manual work to standardize descriptions before the data can be analyzed effectively. To address this, EPA and NIEHS scientists developed and applied a semiautomated workflow to map pre-existing controlled vocabularies (standardized systems of terminology) to ensure consistency in how toxicological effects are described.

In this study, the team reviewed over 40,000 toxicological data points extracted from prenatal developmental toxicity studies conducted by NTP and the European Chemicals Agency. They used a harmonized controlled vocabulary system that combined terms from three sources: Unified Medical Language System codes as implemented in the EPA Toxicity Reference Database; German Federal Institute for Risk Assessment DevTox harmonized terms; and endpoint vocabularies used by OECD. Leveraging a programmatic approach, standardized terms were successfully matched to 75% of the NTP data and 57% of the European Chemicals Agency data. Approximately half of the standardized matches required manual review to ensure accuracy. Data points that could not be matched automatically were often too general or required human judgment to interpret.

This semiautomated workflow saved over 350 hours of manual work and created useful resources, including reproducible code for implementing the mapping workflow and a comprehensive, accessible dataset of harmonized vocabulary for developmental and reproductive toxicity. This crosswalk effort can be readily adapted to other study types. The results demonstrate how automation can advance the organization of toxicological data by facilitating semantic interoperability, an essential prerequisite for integrating legacy datasets to support analyses and decision-making.

Warfighters are subject to a variety of chemical exposures, and PBK models can be used to predict chemical metabolism and potential toxicity given a specific exposure concentration. However, most in silico techniques for estimation of metabolism are only suitable for low exposure concentrations. Saturable metabolism is a key component for extending current high-throughput PBK models beyond environmental exposures to occupationally relevant concentrations. To adequately support human health decision-making, USAF Predictive Risk Team (PRT) scientists identified a need for in silico approaches to estimate chemical metabolism parameters suitable for describing the rate of compound biotransformation over a wide range of exposure concentrations. Sweeney and Sterner (2022) describe the reconstruction of published, insufficiently validated QSAR and the application of these QSARs to jet fuel components. A subsequent publication (Sweeney 2022) used these QSAR estimates, along with other techniques, to demonstrate the value of smaller families of highly similar compounds for generating reliable QSAR-derived metabolism estimates and their application to internal dosimetry-based risk assessment for both low dose (chronic) and higher dose (acute) risk assessment activities. PRT scientists are coordinating a newly established DoD QSAR interest group and are exploring future collaborations that will expand the application of these tools. Additionally, the team is assessing the feasibility of adding saturable metabolism into the EPA httk model.

Identifying and applying mitigation strategies for oxidative stress can reduce adverse health effects from operational stressors and improve USAF aircrew and guardian readiness. NRF2 is a key element of the cellular antioxidant defense system, because it regulates transcription of antioxidant proteins and detoxifying enzymes. Scientists in the USAF Force Health Protection program’s Predictive Risk Team (PRT) developed a computational approach to predict potential activators of NRF2 using structural alerts and machine-learning QSAR modeling (Chushak and Clewell 2024). Once developed, the approach was used to screen a list of approved drugs collected in DrugBank to identify potential novel NRF2 activators. Currently, PRT scientists are working on development of QSAR models to identify inhibitors of PPARg. Chemicals that inhibit PPARg increase the risk of long-term lung damage from inhaled toxins. The developed models will be used together with in vitro data and other mechanistic models to predict whether exposure to chemicals in the operational environment may cause acute or chronic lung injury.

NIEHS scientists developed of a set of computational models to predict eye irritation and corrosion (Sedykh et al. 2022). The models were developed using a curated database of in vivo eye irritation studies from the scientific literature and stakeholder-provided data. The database contained over 500 unique substances, including many mixtures, tested at different concentrations. Substances were categorized according to GHS and EPA hazard classifications. Two modeling approaches were used to predict classification of mixtures. A conventional approach generated predictions based on the chemical structure of the most prominent component of the mixture. A mixture-based approach generated predictions by using weighted feature averaging to consider all known components in the mixture. Results suggest that these models are useful for screening compounds for eye irritation potential. Future efforts to increase the models’ utility will focus on expanding their applicability domains and using them in conjunction with other input variables (e.g., in vitro data) to establish defined approachs (DAs) for eye irritation testing.

Traditional research on the effects of chemicals on embryos and fetuses often relies on animal models, but use of human-relevant NAMs has the potential to improve safety assessments. An example is the Stemina devTOX quickPredict (devTOXqP) assay, which uses pluripotent stem cells to measure a chemical’s potential to cause developmental toxicity. In a preliminary study using the known developmental toxicant valproic acid and its analogs (Chang et al. 2022), NIEHS, EPA, FDA, and Stemina demonstrated that the devTOXqP assay, when used with advanced pharmacokinetic models, could estimate safe exposure limits and rank chemicals based on toxicity risks.

To better understand the applicability domain and performance of the devTOXqP assay, NICEATM and Stemina evaluated the accuracy of the assay in predicting in vivo developmental toxicity in humans and rodents for a broad range of 198 chemicals: 117 developmental toxicants, 67 developmental nontoxicants, and 14 chemicals with unknown developmental toxicity in vivo. For chemicals with the highest level of in vivo evidence, the devTOXqP assay performed well in predicting in vivo outcomes. The study results suggest that the devTOXqP assay may be useful for the identification of substances likely to cause perturbations in early development, with a negative result in the test considered along with other evidence in decisions to prioritize chemicals for in vivo testing. As the devTOXqP assay informs on a specific biochemical aspect of fetal development, it may also be useful as part of research strategies to clarify adverse outcome pathways for developmental neurotoxicity (DNT). A paper describing the study is in preparation for submission in 2026.

Progress in developing new tools, assays, and approaches to assess human hazard and health risk provides an opportunity to re-evaluate the need of dog studies for the safety evaluation of agrochemicals. EPA and NIEHS scientists and a diverse group of international collaborators met at a 2021 workshop to discuss the strengths and limitations of past use of dogs for pesticide evaluations and registrations (Bishop et al. 2023). Opportunities were identified to support alternatives to the 90-day dog study for answering human safety questions. Participants proposed developing a decision tree for determining when the dog study might not be necessary to inform pesticide safety and risk assessment. Such a process will require global regulatory authority participation to lead to its acceptance. The identification of unique effects in dogs that are not identified in rodents will need further evaluation and determination of their relevance to humans. The establishment of in vitro and in silico approaches that can provide critical data on relative species sensitivity and human relevance will be important in the decision. Novel tools including in vitro comparative metabolism studies, in silico models, and high-throughput assays able to identify metabolites and mechanisms of action could potentially support development of adverse outcome pathways; such tools show promise but will need further development. To replace or eliminate the 90-day dog study, a collaborative, multidisciplinary, international effort that goes beyond organizations and regulatory agencies will be needed in order to develop guidance on when the study would not be necessary for human safety and risk assessment.

The EPA Toxicity Forecasting (ToxCast) program makes in vitro medium- and high-throughput screening (HTS) assay data publicly available for prioritization and hazard characterization of thousands of chemicals. The assays employ a variety of technologies to evaluate the effects of chemical exposure on diverse biological targets, from distinct proteins to more complex cellular processes like mitochondrial toxicity, nuclear receptor signaling, immune responses, and developmental toxicity. The ToxCast data pipeline (tcpl) is an open-source R package that stores, manages, curve-fits, and visualizes ToxCast data and populates the linked MySQL database, invitroDB.

In 2022-2023, major updates were made to tcpl and invitroDB to accommodate a new curve-fitting approach (Feshuk et al. 2023). The original tcpl curve-fitting models (constant, Hill, and gain-loss models) have been expanded to include Polynomial 1 (linear), Polynomial 2 (quadratic), Power, Exponential 2, Exponential 3, Exponential 4, and Exponential 5, which are based on BMDExpress and encoded by the R package dependency, tcplfit2. Inclusion of these models impacted invitroDB (beta version v4.0) and tcpl v3 in several ways: (1) long-format storage of generic modeling parameters to permit additional curve-fitting models; (2) updated logic for winning model selection; (3) continuous hit calling logic; and (4) removal of redundant endpoints as a result of bidirectional fitting. The tcpl and invitroDB resources provide a standard for consistent and reproducible curve-fitting and data management for diverse, targeted in vitro assay data with readily available documentation, thus enabling sharing and use of these data in myriad toxicology applications.

The CompTox Chemicals Dashboard v2.3.0 (release planned for 2024) will display data from invitroDB version 4.1, which was released in September 2023. In 2024, EPA will continue iteratively improving the tcpl software, releasing new data in invitroDB, and providing additional ways to access ToxCast data

Rodent cancer bioassays have long been required for regulatory assessment of human cancer hazard and risk. However, decades of information from chronic and carcinogenicity studies have revealed some limitations of these studies. For example, certain aspects of the assay may lack human relevance. In addition, long-term rodent bioassays are costly, time-consuming, require large numbers of animals, and can be difficult to reproduce. Human-relevant new approach methodologies (NAMs) based on an improved understanding of human cancer biology may provide a less resource-intensive and more rapid method to assess chronic toxicity and carcinogenicity when making health-protective decisions. The use of NAMs in chemical safety assessment can be achieved by way of regulatory flexibilities, including a weight-of-evidence-based approach in which studies can be waived when alternative methods or existing data are sufficient. To streamline and support approval of new technologies, scientists from government, academia, nongovernmental organizations, and industry stakeholders convened the Rethinking Carcinogenicity Assessment for Agrochemicals Project. The project developed a reporting framework (Hilton et al. 2022) to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode of action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

High-throughput screening (HTS) assays that have been used in the Tox21 testing program to date generally lack characteristics of metabolic activity found in living systems, which can potentially increase or decrease the toxicity evaluation of chemicals. As a result, HTS results may not accurately reflect in vivo activity. Scientists at EPA, NCATS, and NIEHS used several approaches to address this problem: adding human or rat liver microsomes into the existing assays (Ooka et al. 2022), transfecting cells with mRNAs encoding human metabolic enzymes, or using metabolically capable human HepaRG cells. The retrofitted assays were then used to screen the Tox21 10K chemical library to identify chemicals that are either bioactivated or detoxified by metabolic activity. Chemicals that exhibit metabolism-mediated shifts in bioactivity were further screened to identify the responsible metabolic enzymes, including the primary cytochrome P450 (CYP) drug-metabolizing enzymes CYP1A2, 3A4, 2C9, 2C19, and 2D6. The screening data were analyzed by grouping chemicals based on structural similarity, which enabled the identification of specific and nonspecific CYP inhibition patterns (Sakamuru et al. 2025). This information ultimately supports the development of predictive models for CYP inhibitory activities.

Retrofitted assays are also being used to explore how metabolism might affect interaction of chemicals with the androgen receptor pathway. Rat liver microsomes—small, membrane-bound vesicles formed from fragments of the endoplasmic reticulum—were incorporated into androgen pathway assays for screening the Millipore–Sigma Library of Pharmacologically Active Compounds (Lynch et al. 2025). Known androgenically active compounds, as well as at least one novel chemical, were identified using this newly optimized and physiologically relevant model. The 10K compound collection was screened during 2024 and 2025 to identify novel compounds requiring metabolism for androgen receptor antagonist activity; follow-up studies are ongoing in 2026.

EPA uses data from the in vivo fish acute toxicity test to assess potential risk of substances to aquatic vertebrates. The test is typically conducted on a cold and a warm freshwater species and a saltwater species for a conventional pesticide registration, potentially requiring upwards of 200 or more fish. EPA and NIEHS scientists conducted a retrospective data evaluation to explore the potential for using fewer fish species to support conventional pesticide risk assessments (Ceger et al. 2023). LC50 values and experimental details were extracted and curated from 718 studies on fish acute toxicity submitted to EPA. The LC50 data were analyzed to determine, when possible, the relative sensitivity of the tested species to each pesticide. In 85% of those cases, one of the tested freshwater species was identified as the most sensitive, with the cold freshwater species being the most sensitive overall among cases with established relative sensitivity. The results support potentially using fewer than three fish species to conduct ecological risk assessments for the registration of conventional pesticides.

EPA considers chronic risk to birds when evaluating ecological risk to wildlife from the application of pesticides. These risk assessments are supported by ecotoxicity data required for pesticide registration, including data on avian reproductive toxicity (OCSPP 850.2300) in two bird species: an upland game species (typically bobwhite quail, Colinus virginianus) and a waterfowl species (typically mallard duck, Anas platyrhynchos). EPA is collaborating with nongovernmental organizations and industry stakeholders on a retrospective analysis to assess species differences in sensitivity to conventional pesticides, which can help determine how testing only two standard avian species influences ecological risk assessments. The analysis examines patterns in species sensitivity across chemical groups. When species differences are identified, an additional analysis will be conducted to determine if differences in species sensitivities could be reliably accounted for without testing of a second species. The goal of this project is to determine if there are factors that can be considered when determining testing requirements for conventional pesticides and if chronic risk to birds can be confidently assessed using a reduced number of test species. Results will be described in a paper projected for submission to a peer-reviewed journal in 2026.

EPA ecological risk assessments for pesticide registration include a summary of potential risks to bees. Toxicity data are submitted to support these assessments, and EPA maintains a database containing acute and chronic toxicity data on adult and larval honeybees (Apis mellifera), which EPA considers a surrogate for Apis and non-Apis bees. EPA compared these toxicity data to explore possible trends and assess whether efficiencies can be gained in targeting the specific studies needed for risk assessment (Farruggia et al. 2022). The analysis provided evidence that compounds with a low acute (single exposure) toxicity for adults through contact and oral exposure pathways may still be toxic for adults and larvae following chronic (repeat) oral exposure. For example, the acute toxicity of herbicides and fungicides was lower for adult versus larval honeybees, and chronic toxicity studies resulted in more sensitive endpoints in larvae than adults, indicating increased sensitivity of larvae.

The use of animals in toxicity testing and in scientific research more broadly has been a subject of increasing discussion in recent years. Objections to the use of dogs for this purpose focus both on ethical and humanitarian grounds and practical concerns, which note both the expense and time needed for these studies and the value and relevance of dog studies in human risk assessment. To explore alternatives to animal testing for food and color additives, scientists within the FDA Center for Food Safety and Applied Nutrition analyzed a sample of food additive and color additive petitions submitted to FDA (Flannery et al. 2023). The analysis indicated that most safety evaluations of food and color additives did not rely upon dog studies to set an acceptable daily intake, although dog study data were used in making safety decisions in roughly one-third of petitions. Future research should include the development and use of qualified alternative studies to replace the use of animal testing for food and color additive safety assessment while ensuring human safety.

Biologics such as vaccines, cytokines, enzymes, and hormones are tested routinely after approval to ensure the safety and potency of products. Many of these tests use animals, and thus a large number of animals are used for biologics testing each year.

FDA and NIEHS are supporting a project led by the World Health Organization and NC3Rs to carry out an independent and comprehensive review of animal use requirements within World Health Organization guidelines for biologics and to make recommendations for where non-animal approaches can be adopted. The project began with a survey of biologics manufacturers to better understand the opportunities and barriers to adoption of replacement, reduction, and refinement (3Rs) alternatives and use of non-animal approaches in quality control, batch, and lot release testing of biologics. A similar survey of national regulatory authorities and national control laboratories was conducted in early 2022. A September 2022 virtual workshop focused on requirements in North and South America. Biologicals regulatory agencies and manufacturers from the region discussed their perspectives on current testing strategies and how changes to WHO guidelines could be implemented to support wider adoption of 3Rs and non-animal approaches. The findings from this project were presented to the World Health Organization Expert Committee on Biological Standardization in October 2023.

In vitro toxicity assays are being applied to transform toxicology from an observational to a predictive science, improve throughput, and reduce costs. The qualitative linkage between in vitro and in vivo toxicity endpoints can be strengthened via application of in vitro to in vivo extrapolation (IVIVE) of dosimetry, which relates an in vitro concentration associated with bioactivity to an equivalent external exposure level. In some contexts, applications of IVIVE have advanced past the exploratory research stage and are beginning to gain acceptance for risk assessment of chemicals. The ICCVAM IVIVE Workgroup requested information from ICCVAM member agencies regarding the extent and context of their use of IVIVE. Surveyed agencies were also asked about programmatic needs, data gaps, and agency-specific guidance documents or publications related to IVIVE, as well as for information about modeling tools or software they had used or may use for facilitating IVIVE analysis and decision-making. This information was compiled into a review summarizing the workgroup’s findings, current challenges, and future needs (Chang et al. 2022). The review also proposes operational definitions for IVIVE, presents literature examples for several common toxicity endpoints, and highlights implications of IVIVE use in decision-making processes. This well-received paper was recognized by the journal Toxics as an "Annual Recommended Review for 2022."

U.S. regulatory and research agencies use ecotoxicity test data to assess the hazards and risks associated with substances that may be released into the environment, including but not limited to industrial chemicals, pharmaceuticals, pesticides, food additives, and cosmetics. These data are used to conduct hazard assessments and evaluate potential risks to non-target animals and plants. To identify opportunities for regulatory uses of non-animal replacements for ecotoxicity tests, the needs and uses for these types of test data must first be clarified. The ICCVAM Ecotoxicology Workgroup prepared a review (Ceger et al. 2022) that identifies the ecotoxicity test data relied upon by U.S. federal agencies. The review describes the standards, test guidelines, guidance documents, and endpoints that are used to address each of the responding agencies’ regulatory and research needs regarding ecotoxicity testing in the context of their application to decision-making. This information will be useful for coordinating efforts to develop and implement alternative test methods to reduce, refine, or replace animal use in chemical safety evaluations.

Chemical regulatory authorities around the world require systemic toxicity data from acute exposures via the oral, dermal, and inhalation routes for human health risk assessment. To identify opportunities for regulatory uses of non-animal replacements for these tests, NICEATM scientists and collaborators reviewed acute systemic toxicity testing requirements for jurisdictions that participate in the International Cooperation on Alternative Test Methods (ICATM): Brazil, Canada, China, the European Union, Japan, South Korea, Taiwan, and the United States (Strickland et al. 2023). The chemical sectors included in review of each jurisdiction were cosmetics, consumer products, industrial chemicals, pharmaceuticals, medical devices, and pesticides. The study demonstrated acute systemic toxicity data were most often required for hazard assessment, classification, and labeling, and to a lesser extent quantitative risk assessment. Where animal methods were required, animal reduction methods were typically recommended. For many jurisdictions and chemical sectors, non-animal alternatives are not accepted, but several jurisdictions provide guidance to support the use of test waivers to reduce animal use for specific applications. An understanding of international regulatory requirements for acute systemic toxicity testing will inform ICATM’s strategy for the development, acceptance, and implementation of non-animal alternatives to assess the health hazards and risks associated with acute toxicity.

Preclinical safety assessment for CAR T cell-based therapies is necessary because non-targeted binding can have serious adverse consequences in healthy tissues. Three potential scenarios are of concern:

• A cell that binds to its intended target on the tumor (“on-target/on-tumor”) could potentially trigger cytokine release syndrome or tumor lysis syndrome.
• A cell that binds to its intended target off the tumor (“on-target/off-tumor”) could destroy healthy cells that express the target of interest.
• A cell that binds to an unintended target off the tumor (“off-target/off-tumor”) could damage healthy cells that do not express the target of interest.

For CAR T cell-based therapies, safety testing in animal models is generally limited or impossible; therefore, robust in vitro assays that address on- or off-target binding and subsequent cytotoxic consequences are necessary as part of the preclinical safety information for these therapies. To evaluate the safety of CAR T cell drug candidates, NCI scientists are performing co-culture experiments using human pluripotent stem cell-derived cell types as models of healthy human cells that broadly represent various cell types that could be targeted by CAR T cell therapy. Potential readouts from such co-culture experiments include cell imaging for morphologic signs of cell stress/cytotoxicity, cytotoxicity assays, cytokine release assays, and impedance monitoring. Special consideration is given to the setup and execution of each readout to ensure that each assay is adequately controlled. The orthogonal data generated by these co-culture experiments will collectively support a good weight-of-evidence approach for assessing on- and off-target binding and potential cytotoxicity of CAR T cells in healthy human tissues. A paper describing this project is in preparation for submission in 2024.

Assessing the potential of chemicals to cause skin sensitization (allergic reaction) is essential for protecting public health. While nonanimal methods have been developed, single tests alone are not sufficient to fully determine a chemical’s sensitization risk or potency. Defined approaches (DAs) such as those described in OECD TG 497 (OECD 2025) combine multiple data sources for more reliable evaluations. However, existing DAs lack the ability to calculate a specific dose threshold, known as a point-of-departure (POD), for use in risk assessments.

The Skin Allergy Risk Assessment–Integrated Chemical Environment (SARA-ICE) DA for skin sensitization (Reinke et al. 2025) was developed by NICEATM and the consumer products company Unilever specifically to address this challenge. SARA-ICE employs statistical modeling to estimate a human-relevant metric called the ED01. The ED01 is the probability distribution for the dermal dosage at which there is a 1% chance of inducing sensitization, making it directly applicable to risk assessment. SARA-ICE integrates data from various in vitro tests as well as data from the local lymph node assay and HPPT, drawing upon historical data from 434 chemicals, and accounts for variability and uncertainty in input data. In addition to the ED01, SARA-ICE provides probabilities for GHS skin sensitization classifications. Specifically, the model calculates the probability that a chemical would be placed into a GHS subcategory: severe sensitizers (1A), moderate sensitizers (1B), or Not Classified (NC). In a case study focused on isothiazolinones, SARA-ICE demonstrated its ability to integrate diverse data inputs and calculate ED01 values for six isothiazolinone compounds that compared favorably with results based on historical datasets (Reinke et al. 2025).

In 2025, OECD revised TG 497 to include a more limited guideline-specific version of the SARA-ICE model, which provides a POD as the geometric mean of the ED01 and does not provide hazard or GHS classification predictions. It also assumes that any chemical evaluated is presumed to be a skin sensitizer due to model restrictions.

To broaden the accessibility of the SARA-ICE model, in 2025 NICEATM developed the SARA-ICE app. The app provides access to the two SARA-ICE models: the "OECD TG 497 DA”, which aligns with TG 497 criteria, and the “Extended Model”, which allows the user to make a hazard determination and predict GHS subcategorization and alternative “EDx” outputs (e.g. ED05, the dose at which 5% of a population would be sensitized). The Extended Model does not assume a chemical is a skin sensitizer and allows the user to adjust the GHS decision threshold depending on desired stringency. The OECD TG 497 DA is static because it is based on the guideline; however, the Extended Model could be modified in the future to add in more test methods or outputs. Current outputs include a graphic representation of the uncertainty distribution and a tabular report of the probability distributions for various classifications. Future development may add methods to the Extended Model, in silico reactivity structural alerts, extrapolation of population-level effects, a user input feature, and a downloadable local version.

PFAS are a distinct set of commercial chemicals widely found in humans and the environment. However, only a small number of PFAS have epidemiological or experimental data to characterize any potential hazard they might pose. Using in vitro new approach methodologies (NAMs), EPA scientists tested 160 PFAS to characterize their potential to induce developmental neurotoxicity (DNT) (Carstens et al. 2023). The DNT NAMs battery used evaluated proliferation, apoptosis, neurite outgrowth, and neural network formation. While most of the PFAS tested were inactive or equivocal in the DNT NAMs, specific chemical structures were identified that appeared to correlate with PFAS bioactivity in the NAMs. These data demonstrate that a subset of PFAS perturb neurodevelopmental processes in vitro and suggest focusing future studies of DNT on PFAS with specific structural features. Analytical quality control indicated that significant numbers of both inactive PFAS and active PFAS samples were degraded. This indicates a need for careful interpretation of test results of these substances, as some negatives may have been due to loss of the parent PFAS and some active results may have been caused by PFAS degradants.

In 2023, the OECD published the guidance document “Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity (DNT) In-Vitro Testing Battery” (OECD 2023). The DNT in vitro battery consists of multiple new approach methodologies (NAMs) that evaluate key processes of neurodevelopment such as proliferation, migration, differentiation, neuronal network formation, and function and locomotor activity in zebrafish (Danio rerio) embryos.

To explore a broader chemical space and increase the applicability domain, there is a need to screen additional compounds. The DNT Health Effects Innovation program within the NIEHS Division of Translational Toxicology is generating screening-level information on chemicals using the DNT in vitro battery and behavioral assays in small model organisms. Currently about 220 chemicals have been selected and distributed for testing with half of them being finalized and analyzed using a unified data analysis pipeline to combine data from the individual assays. To further build confidence in using the DNT in vitro battery for regulatory applications, the screening level information is being used to develop specific case studies of integrated approaches to testing and assessment.

Scientists at NIEHS are using zebrafish (Danio rerio) embryos as a test model to study how chemical exposure might impact development. Zebrafish are useful for developmental studies because their embryos are transparent, develop quickly, and respond similarly to humans. However, different laboratories use different testing methods, and these discrepancies make it difficult to compare results, limiting the wider use of zebrafish embryos for regulatory chemical safety testing. To address the discrepancies in testing methods, the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was created. This project aimed to improve understanding of what aspects of experimental protocols, data analysis, and reporting causes variability among zebrafish testing across laboratories and help identify ways to make methods more consistent.

In a study involving three laboratories (Hamm et al. 2024), 42 chemicals were tested under different conditions; for example, using embryos with the chorion intact versus removed or using static versus static renewal chemical exposure. The study compared how these methods influenced observed developmental toxicity. The results showed good agreement across laboratories, with 33 of the 42 chemicals (~79%) producing the same activity outcomes. However, differences in chemical potency linked to varying laboratory protocols underscored the need for method standardization. The findings of this study provide valuable insights into the application of standardized ontologies for cataloging, comparing, and sharing developmental toxicity data. This work could help improve the reliability and regulatory acceptance of zebrafish embryos as a tool for evaluating developmental toxicity.

An interactive web resource, SEAZIT Data Integration and Visualization Enabling Resource (SEAZIT-DIVER), has been developed to allow the public to explore and visualize SEAZIT data. This platform also provides methodological details, information on test chemicals, tools to evaluate data quality, and options to download data for additional exploration.

Accessing and organizing information from an increasing number of scientific studies poses a major challenge for researchers, especially in fields such as toxicology that rely on structured data from a variety of studies. Manual data extraction from scientific publications is time-consuming and labor-intensive. To address this issue, DOE and NIEHS researchers developed an automated workflow that uses large language models within the KNIME platform to extract structured data from scientific papers and general PDF files (Moreira-Filho et al. 2025). This workflow combines advanced document processing tools with large language models to extract variables from publications. The workflow has two modes: text mode, which extracts structured text and tables from sources such as peer-reviewed journal articles, and image mode, which processes more complex layouts such as nonstandardized reports or graphical content. The workflow achieved 81.14% accuracy in extracting data from scientific papers using text mode and up to 98.54% accuracy in handling general PDF files using image mode. The free and open-source KNIME data analytics platform, designed with user-friendly features, makes these cutting-edge tools accessible to researchers without specialized expertise. By automating data extraction, this approach saves time and effort while ensuring critical toxicological data are organized and ready for use in chemical assessments, study evaluations, and validation of NAMs. The success of this workflow demonstrates how advanced technologies such as large language models can revolutionize biomedical research by making data retrieval faster and more efficient. By democratizing access to these tools, the platform empowers researchers to synthesize knowledge more effectively, driving progress in toxicology and other fields.

Throughout 2024 and 2025, NIEHS provided funding for small businesses developing technologies of interest to Institute goals. The funding was offered as part of the 2024 and 2025 Omnibus Solicitations of NIH, Centers for Disease Control and Prevention, and FDA for small business grant applications to support the development and commercialization of innovative technologies. Areas of high priority included the development of:

• Informatics tools and platforms to organize, store, retrieve, extract, and integrate information on exposures and health effects data
• Machine learning methods and natural language processing for extracting and integrating diverse data types and for generating causal networks from experimental data and public knowledgebases
• New methods and tools for automating environmental health-related literature and systematic reviews, including article selection and prioritization, data extraction, study quality evaluation, and summarization of environmental health impacts
• Mid- to high-throughput and high-content assays using in vitro or tissue chip technologies to screen and rank toxicity of emerging engineered nanomaterials for cytotoxicity, genotoxicity, and metabolic toxicity
• Human and rodent organotypic culture models and microphysiological systems (MPS)
• Approaches to characterize and integrate key molecular and cellular changes related to effects of toxicant exposures in carcinogenicity, developmental neurotoxicity (DNT), or cardiotoxicity
• Screening systems that incorporate genetic diversity into toxicology testing
• Short-term tests, assays, or systems designed specifically to reduce or replace existing regulatory animal studies for acute toxicity (oral or inhalation), reproductive or developmental toxicity, carcinogenicity, or ocular toxicity

In 2024 NIEHS provided funding to small businesses as part of the RFA-ES-23-008: Novel Approaches for Developmental Neurotoxicity Testing funding announcement. The funding aimed to support the development of novel and innovative approaches and new approach methodologies (NAMs) that can be applied in testing strategies to better understand the role of environmental chemicals in the etiology of neurodevelopmental disorders. The overall goal was to develop effective ways to address current challenges and gaps in the assessment of DNT. These screening systems or technologies, when fully developed and validated with appropriate throughput and controls, were expected to provide cost-effective, quicker and/or comprehensive results to understand the underlying role of environmental chemicals in this endpoint.

NIEHS participated in the Applicant Assistance Program conducted by the Small Business Programs of the National Institutes of Health. This free 10-week program assisted small businesses in preparing a Small Business Innovation Research or Small Business Technology Transfer grant application. Participants received services such as application needs assessment, mentoring, application preparation support, and application review to help applicants maximize their chances of success.

It is not unusual for Air Force pilots to experience cognitive fatigue, which has the potential to compromise mission success. U.S. Air Force (USAF) scientists are developing transcranial alternating current or direct current therapies that use electrical stimulation to promote neural plasticity and mitigate fatigue. A 3D-printed platform using human induced pluripotent stem cell-derived neurons and astrocytes is being used to investigate the impact of applying direct and alternating currents on stress levels and gene expression. Transcriptional alterations, viability, and morphological changes were assessed to determine the impact of low current stimulation, emulating transcranial therapies. Extrapolation of this technique to include high-density microelectrode arrays will enable an understanding of how low current stimulation impacts neuronal firing, bursting, networking, and synaptic plasticity. Taken together, this in vitro platform allows for both functional and molecular analyses of electrical stimuli representing transcranial therapies with the goal of optimizing future therapies to combat military cognitive fatigue.

Reducing, refining, and replacing the use of animals (applying 3Rs principles) is a long-standing goal in chemical toxicology. To support this effort, NICEATM and collaborators developed STopTox (Systemic and Topical Chemical Toxicity), an in silico platform that uses advanced computer models to predict chemical toxicity without the need for animal studies (Borba et al. 2022). STopTox uses QSAR models, which analyze chemical structures to estimate toxicity. Designed as a user-friendly, accessible tool, STopTox provides a comprehensive alternative for identifying hazards.

During its initial development, STopTox was rigorously tested using large publicly available datasets, ensuring it met international standards, including principles for QSAR model development adopted by the Organisation for Economic Co-operation and Development (OECD). The models demonstrated high internal accuracy and strong external predictive capability. To further build confidence in STopTox as a substitute for animal testing, researchers conducted external validation tests using independent datasets. They assessed the ability of STopTox to predict toxicity across six major categories: acute oral, dermal, and inhalation toxicity, as well as skin sensitization, skin irritation/corrosion, and eye irritation/corrosion. The validation results were highly encouraging, as STopTox performed reliably across all endpoints (Scheufen Tieghi et al. 2025). This supports the platform's potential as a dependable tool for regulatory toxicology decision-making. By offering accurate predictions and reducing the reliance on animal studies, STopTox represents an important step forward in advancing humane and efficient approaches to hazard identification in chemical testing. It highlights the promise of computational methods in revolutionizing toxicological assessments and contributing to animal replacement efforts.

As part of ongoing assessments of wildlife health, DOI is investigating potential chemical effects on the cardiovascular system and general health, with measured endpoints including pericardial area, circulation, heart rate, body length, median lethal concentration, and mode-of-action. Chemicals of interest include pesticides and pharmaceuticals detected in surface waters and fish tissues, as well as polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs from a subsurface oil spill by assaying groundwater samples. DOI’s USGS conducts high-content screening of compounds to formulate hypotheses and prioritize contaminants for further toxicity testing. This approach reduces animal use, quantity of test compound, and waste by utilizing pre-feeding zebrafish embryos in a microtiter plate format. The high-content screening is also being coupled with behavioral assays and in silico approaches to characterize toxicity of algal toxins associated with harmful algal blooms, which continue to pose health concerns to the public and natural resources. These assays can provide evidence to justify larger-scale studies to determine actual versus perceived risk of contaminants.

In May 2021, NASA announced a multiagency initiative, “Extended Longevity of 3D Tissues and Microphysiological Systems for Modeling of Acute and Chronic Exposures to Stressors.” The initiative is focused on adapting existing 3D tissues and MPS to extend their longevity to at least 6 months. Among the sponsors of the initiative are several ICCVAM agencies: NIH; NCATS and National Institute of Allergy and Infectious Diseases (NIAID), NCI, and FDA. Proposals for projects to be funded under the initiative were accepted through September 2021, with awardees announced in March 2022.

The Promise to Address Comprehensive Toxics 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 reducing 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 Exposure Research Working Group, which included 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 included:

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 adverse outcome pathways.
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 fostering interagency cooperation to establish a federal infrastructure framework for coordinating implementation of this strategic plan. The working group’s final report, issued in 2024, addressed requirements of the Promise to Address Comprehensive Toxics Act to outline goals in this area over the next five years. ICCVAM co-chair Natalia Garcia-Reyero Vinas, Ph.D., served on the working group’s Strategy Team.

Chemicals that enter the body are metabolized via several pathways. Rates of metabolism can vary across human populations due to genetic variability of metabolic enzymes, such that some populations are more sensitive to effects of parent chemicals or metabolites. Risk assessors apply PBK models to predict the dynamics of tissue concentrations for parent chemicals and their metabolites, but it is difficult to use these models to characterize the effects of enzymatic pathway-related variability. NIEHS scientists developed a generalized workflow to incorporate pathway-related variability for specific metabolic enzymes across human populations into PBK models. The workflow includes metabolite structures generated using SimulationsPlus ADMET Predictor®, PBK models from EPA’s httk package, estimates of interindividual enzyme variability from European Food Safety Authority reports, and parameter predictions from OPERA (v2.8). Parent chemical dynamics were simulated following initial exposure, and the amount of parent metabolized was scaled by percent yield to provide an intravenous time series for metabolite models. Ranges of parent and metabolite concentrations were estimated by Monte Carlo sampling of enzymatic variability in intrinsic clearance. A case study to demonstrate the utility of the workflow used 10 parent chemicals and their metabolites, and efforts are ongoing to incorporate additional chemicals. In quantifying the range of tissue concentrations resulting from metabolic pathway variability, this work facilitates a more health-protective risk assessment for susceptible population groups. The case study was described in an oral presentation (Hull et al.) at the 12th World Congress on Alternatives and Animal Use in the Life Sciences.

“Qualification of Medical Device Development Tools: Guidance for Industry, Tool Developers, and FDA Staff,” was issued in July 2023. This document, an update of guidance issued is 2017, provides guidance on a voluntary program for qualification of medical device development tools for use in evaluating devices subject to regulation by FDA’s Center for Devices and Radiological Health. Medical device development tools are methods, materials, or measurements used to assess the safety, effectiveness, or performance of medical devices. They can include new approach methodologies, biomarker tests, and clinical outcome assessments.

Access to visualization tools to navigate chemical space has become increasingly important due to the increasing size and diversity of publicly accessible compendiums of HTS and other descriptor and effects data. Construction of such tools relies on complex projection techniques using molecular descriptors. However, application of these techniques requires advanced programming skills that are beyond the capabilities of many stakeholders.

Inspired by the Google Maps application, NICEATM developed ChemMaps.com to easily navigate chemical space. The first version of ChemMaps.com was limited to exploration of drugs and drug candidates. ChemMaps.com v. 2.0, released in 2022, added data on approximately one million environmental chemicals from the EPA Distributed Structure-Searchable Toxicity (DSSTox) inventory (Borrel et al. 2023). ChemMaps.com v. 2.0 incorporated mapping to HTS assay data from the U.S. federal Tox21 research collaboration, which includes results from approximately 2,000 assays tested on up to 10,000 chemicals. Users can visualize chemical activity by both assay and target directly on the map and compare chemical spaces occupied by active and inactive chemicals. The latest update of ChemMaps.com (v. 2.1), released in October 2024, refined the chemical preparation pipeline to address previously unsupported chemistry. Chemical predictions were also updated to utilize the latest version of OPERA (v. 2.9).

The CompTox Chemicals Dashboard is the primary web-based application for access to data and algorithms from the EPA Center for Computational Toxicology and Exposure. The Dashboard is a widely used resource for chemistry, toxicity, and exposure information for over a million chemicals. There were eight updates to the Dashboard released in 2024 and 2025, increasing the number of chemicals to 1,246,399 with all related data. These related data include mass spectrometry-ready/quantitative structure–activity relationship (QSAR)-ready structures, linked substances, and property predictions generated by the Open (Quantitative) Structure–activity/property Relationship App (OPERA) and EPA Toxicity Estimation Software Suite tools. The EPA Toxicity Forecaster (ToxCast) invitrodb v. 4.2 dataset was released in September 2024, and an updated version of the Chemical and Products Database was released via the Dashboard in June 2025 (Handa et al. 2025). The next Dashboard update is planned for August 2026. 

A June 2025 virtual training on the Dashboard gave an overview of Dashboard functions along with its risk evaluation components, navigation, and a case study. Additional training resources for the Dashboard are available on the EPA NAMs Training webpage.

The CompTox Chemicals Dashboard is the primary web-based application that provides access to data and algorithms from EPA Center for Computational Toxicology and Exposure (CCTE). The Dashboard is a widely used resource for chemistry, toxicity, and exposure information for over a million chemicals. There were seven updates to the Dashboard released during 2022 and 2023. Data updates included in these releases added data on 300,000 new chemicals and enabled users to explore exposure predictions, production volumes, and analytical quality control data for PFAS. The next Dashboard update is planned for April 2024. A October 2022 virtual training on the Dashboard gave an overview of Dashboard functions and highlighted new features from the 2022 updates. Additional training resources for the Dashboard are available on the EPA NAMs Training webpage.

APIs allow programmatic access to EPA’s computational toxicology and exposure data resources. APIs provided by EPA enable users to extract specific data from various databases and integrate them into their applications. APIs can effectively automate the process of accessing and downloading the data that populates the CompTox Chemicals Dashboard. As part of EPA’s commitment to provide “open data,” all Computational Toxicology and Exposure APIs and computational toxicology data resources are publicly available for anyone to access and use. These APIs are hosted on cloud.gov, a secure cloud environment managed by the General Services Administration specifically for U.S. federal government applications. Data are free of all copyright restrictions and are fully and freely available for both non-commercial and commercial use. These APIs are documented on the EPA's Center for Computational Toxicology and Exposure (CCTE) API webpage and all data are also available for download on EPA’s Downloadable Computational Toxicology Data webpage.

Naloxone was the first opioid antagonist specifically indicated for prophylaxis and treatment of overdose from highly potent opioids such as fentanyl and its analogues. Due to ethical considerations, clinical trials evaluating overdose reversal in patients are unfeasible. To support a regulatory review of a naloxone autoinjector 10 mg product under development for use by military personnel and chemical incident responders, FDA and Veterans’ Administration scientists and collaborators evaluated whether an in vitro/in silico quantitative systems pharmacology model could be used to support approval (Mann et al. 2022). The model was used to evaluate the effects of this naloxone product and contributed confirmatory evidence in support of its approval. The modeling approach was also used to support approval of another opioid antagonist product, nalmefene intranasal 3 mg. This approach is described in a December 2022 post on the FDA Regulatory Science in Action website. Since then, the model has been used to evaluate different dosing strategies for intramuscular and intranasal naloxone products and to support pediatric dosing. A subset of results was presented at a March 2023 FDA workshop on “Understanding Fatal Overdoses to Inform Product Development and Public Health Interventions to Manage Overdose.” The model is being used to optimize intranasal naloxone repeat dosing strategies, and a paper adapting this model to pediatric patients has been accepted and will be published in 2024 (Strauss et al. 2024). In addition, the modeling is being expanded to assess the safety of opioid antagonists by simulating precipitated withdrawal, an adverse effect of concern in patients with heavier opioid dependency.

Next-generation textile-based wearable sensing systems will require flexibility and strength to maintain capabilities over a wide range of deformations. However, materials currently used for textile-based skin contacting electrodes lack these key properties, hindering applications such as electrophysiological sensing. U.S Air Force (USAF) scientists and collaborators developed a facile spray coating approach to integrate liquid metal nanoparticle systems into textile form factors for conformal, flexible, and robust electrodes (Li et al. 2022). The system employs functionalized liquid metal nanoparticles that provide a simple "peel-off to activate" means of imparting conductivity. The spray coating approach combined with the functionalized liquid metal system creates long-term reusable textile-integrated liquid metal electrodes. To perform risk assessment on these materials, USAF investigators utilized an in vitro reconstructed tissue model (EpiDerm FT). They demonstrated biocompatibility of textile-integrated liquid metal electrodes in an in vivo skin environment and improved sensing performance compared to previously reported textile-based dry electrodes. The "spray on dry—behave like wet" characteristics of the textile-integrated liquid metal electrodes opens opportunities for textile-based wearable health monitoring and augmented/virtual reality applications that require the use of flexible and conformable dry electrodes.

Synbiotics are a new class of live therapeutics employing engineered genetic circuits. The rapid adoption of genetic editing tools has catalyzed the expansion of possible synbiotics, exceeding traditional testing paradigms in terms of both throughput and model complexity. U.S Air Force (USAF) conducted two projects using gut-on-a-chip models to evaluate candidate synbiotics.

• SYN is a cortisol sensing tryptamine-producing synbiotic for cognitive performance sustainment. It can sense cortisol at physiological concentrations and activates a genetic circuit that produces tryptophan decarboxylase and converts bioavailable tryptophan to tryptamine. The gut-chip provided a stable environment to characterize the sensitivity of the cortisol sensor and dynamic range by altering cortisol and tryptophan dosimetry. Collectively, the human gut-chip provided human-relevant apparent permeability to assess tryptophan and tryptamine metabolism, production, and transport, and enabled host analyses of cellular viability and proinflammatory cytokine secretion, providing a successful efficacy test of a novel synbiotic (Nelson et al. 2023).
• The ECN synbiotic is a medical countermeasure taken to prevent traveler’s diarrhea, an illness caused by Shigella flexneri infection that affects nearly 80% of all deployed warfighters, impacting readiness and performance. In an ongoing study, USAF used a gut-on-a-chip to model traveler’s diarrhea infection. Host assessments of barrier integrity, cellular viability, cytokine production, and transcriptomic responses were conducted, showing that S. flexneri challenges reduced barrier integrity by 2-fold, and increased host-cell cytotoxicity by 1.5-fold. In addition, high-resolution confocal microscopy of host tissue morphology and phenotypic alterations in tight-junctions and mucin production were evaluated in response to S. flexneri challenge, showing that the pathogen was able to effectively invade and replicate within the intestine-chip. When the ECN synbiotic was administered as a prophylactic, S. flexneri was unable to infect, and was undetectable in samples after 4 hours.

Since the 1940s, human predictive patch tests (HPPTs) have been used to study chemicals that can cause skin sensitization, which occurs when the immune system overreacts to certain substances after skin exposure. These tests involve applying a chemical to healthy volunteers to observe reactions. Using a previously compiled database of 2,277 HPPT results for 1,366 unique chemicals (Strickland et al. 2024), scientists from CPSC, FDA, NICEATM, and the German Federal Institute of Risk Assessment conducted an analysis (Herzler et al. 2024) focused on how HPPT data can help classify chemicals as skin sensitizers under the United Nations Globally Harmonized System for Classification and Labelling of Chemicals. They suggested that the standard dose per skin area metric could be refined to include improved descriptors, such as the dose per skin area that causes sensitization in one person or 5% of tested individuals. These new measures could provide more accurate assessments of the risks posed by chemicals.

While HPPT data alone are not always enough for clear classifications, their use in combination with data from other sources, such as the local lymph node assay, improves assessment accuracy. The study also introduced standardized workflows for analyzing individual HPPT results, combining multiple results, and incorporating them into a broader weight-of-evidence approach with local lymph node assay data. Overall, this research demonstrates that while HPPT data have limitations, their use remains a valuable tool for chemical classification when combined with other methods. Integration of these data into modern approaches enhances reliability and reproducibility, paving the way for safer chemical regulations and improved skin sensitization assessments.

DOI’s USGS applies and modifies microbial and eukaryotic reporter bioassays to detect the presence of bioactive chemicals in water samples. These analyses compliment traditional analytical chemistry and biological datasets, which detect specific analytes, by documenting synergistic or antagonistic activities detected by in vitro assays. Additionally, bioreactivity can be measured by bioassays, where it might be missed by traditional methods targeting suspected, but unconfirmed, compounds. USGS is using the bioluminescent yeast estrogen screen to estimate total estrogenicity of a test substance. Relative net agonistic activity per liter is calculated based on sample concentration. Similar assays utilizing other yeast strains can be conducted to determine the presence of androgens and measure cytotoxicity of chemical compounds collected in water or other matrices.

As part of its human health risk assessments, EPA evaluates potential health effects from different routes of exposures based on the pattern or conditions of use. Subchronic studies performed with laboratory animals (typically rats) are used to evaluate route-specific inhalation effects. However, human and animal respiratory tracts differ to an extent that may affect the ability of animal test results to accurately predict effect in humans. Furthermore, there are specific challenges associated with testing irritating and corrosive compounds. The EPA Office of Pesticide Programs previously used a refined inhalation approach that employs an in vitro inhalation model to derive a point-of-departure (POD) for inhalation toxicity for the fungicide chlorothalonil, which is a direct contact irritant. The EPA Office of Pesticide Programs has since been working with staff in the Office of Research and Development and external researchers to investigate the use of in vitro data to apply similarly for other contact irritants or to provide additional information for other chemicals.

The use of pesticides to control invasive species is a key component of integrated pest management plans. Traditionally, the development of new chemical controls required testing on numerous animals. In vitro cytotoxicity testing, which reduces animal use, is becoming more common to prioritize candidate toxicants and permits high-throughput testing. However, it remains unknown whether in vitro cytotoxicity values (i.e., effective concentrations or EC50 values) are representative of in vivo toxicity values (i.e., lethal concentration for 50% of organisms or LC50). DOI’s USGS has begun proof-of-concept studies on the use of fish cytotoxicity screening assays to prioritize new candidate pesticides to control nuisance fishes. Recent studies have quantified the cytotoxicity EC50 values of the fish toxicant Antimycin-A using a commercially available gill cell line (RTgill-W1) from rainbow trout (Oncorhynchus mykiss). This study utilized the procedure described in OECD Test Guideline 249 and the CellTiter-Glo 2.0 (Promega) cell viability assay for assessing RTgill-W1 cytotoxicity. In vitro to in vivo extrapolation (IVIVE) was applied to assay results and indicated that, for most valuable metrics, the ratio of in vitro to in vivo toxicity was approximately 1. These results demonstrate that toxicity as measured in rainbow trout gill cell lines is predictive of whole-animal toxicity and shows promise for the development of additional fish gill cell lines for screening of pesticide candidates.

Virus transmission from handling of fish has not been adequately studied, but damage to a fish’s external slime coat may facilitate entry of pathogens. Blotchy bass syndrome is a condition characterized by visible, variable, and discrete areas of hyperpigmentation on the external surface of black basses. This condition has received increased attention from anglers and resource managers in recent years and is a popular topic of discussion and reporting on angling websites and blogging platforms. Advances in sequencing technology and diagnostics led to the discovery that blotchy bass syndrome is associated with viruses of the family Adomaviridae. Sampling of the epidermis of affected fish is necessary to identify presence of adomaviruses. Typical sample collection for suitable molecular biology methods requires removal of fish scales or clips of fin tissue to collect DNA. Recently, USGS has begun using non-invasive skin swabs in lieu of traditional sampling methods. Skin swabbing does not require the use of anesthetics, is fast (<10 seconds), and reduces changes in behavior and physiology associated with tissue clips. It is a more refined technique for DNA and RNA collection with the potential to improve animal health and welfare. By using swabs and shelf-stable collection buffers, USGS was able to undertake a nationwide effort using both state agencies as well as live-display animals at public aquaria to collect minimally invasive samples. Such collections have been used to generate complete genomes and has resulted in the identification of at least five novel viruses.

An AOP is a model that identifies the sequence of molecular and cellular events required to produce a toxic effect when an organism is exposed to a substance. As most AOPs are defined using a single or small number of species, they have a narrow taxonomic domain of applicability (tDOA). Defining the tDOA of an AOP is critical for use in regulatory decision-making for ecotoxicity, particularly when considering protection of untested species. Structural and functional conservation are two elements that can be considered when defining the tDOA. Publicly accessible bioinformatics approaches, such as the SeqAPASS tool, take advantage of existing and growing databases of protein sequence and structural information to provide lines of evidence toward structural conservation of key events and key event relationships of an AOP. It is anticipated that SeqAPASS results could readily be combined with data derived from empirical toxicity studies to provide evidence of both structural and functional conservation to define the tDOA for AOPs and elements of AOPs. Such data could be incorporated into resources such as the AOP-Wiki as lines of evidence toward biological plausibility for the tDOA. EPA scientists developed a case study describing the process of using bioinformatics to define the tDOA of an AOP using an AOP linking the activation of the nicotinic acetylcholine receptor to colony death/failure in the honeybee (Apis mellifera). Although the AOP was developed to gain a particular biological understanding relative to honeybee health, applicability to other Apis bees, as well as non-Apis bees, has yet to be defined. The EPA study demonstrates how bioinformatics can be utilized to rapidly take advantage of existing protein sequence and structural knowledge to enhance and inform the tDOA and elements of AOPs, focusing on providing evidence of structural conservation across species.

Chemical risk evaluations under TSCA are often conducted with limited test data, as TSCA does not have minimum data requirements. EPA is evaluating how this limitation can be addressed using Web-based Interspecies Correlation Estimation (Web-ICE), a model that can predict toxicity values for environmental species absent from a dataset and provide a more robust dataset to estimate toxicity thresholds. Web-ICE is an online tool that estimates acute toxicity values for aquatic and terrestrial species using surrogate species data and least-squared regressions. Each Web-ICE model represents the relationship of inherent sensitivity between a surrogate species and a predicted taxon (species, genus, or family). Web-ICE models use surrogate species sensitivity as an input to estimate the sensitivity of all available taxa. For the industrial chemicals examined so far under TSCA, Web-ICE predictions increased the number of aquatic species represented in the dataset and provided more species representation. Additionally, the use of Web-ICE predictions with empirical data to create species sensitivity distributions provided a data-driven way of accounting for uncertainties in aquatic hazard characterization. A poster describing the project (Koehrn et al.) was presented at the 2022 meeting of SETAC North America.

Skin sensitization, or an allergic skin reaction, is an important health concern in chemical testing. Assessment of skin sensitization hazard traditionally relies on animal models, such as the local lymph node assay (LLNA). Efforts to reduce animal use have led to the development of alternative methods based on the skin sensitization adverse outcome pathway (AOP).

The electrophilic allergen screening assay (EASA) is a promising in chemico method to identify substances that covalently bind to skin proteins, Key Event 1 in the AOP for skin sensitization. The assay was converted from a cuvette-based platform into a 96-well platform and characterized previously (Petersen et al. 2022). A multilaboratory validation study assessed intra- and interlaboratory reproducibility of the 96-well EASA. In addition to the CPSC/NIST lead laboratory, laboratories sponsored by FDA, DoW, and NIEHS participated in the validation study. Each participating laboratory confirmed transferability and assessed 20 reference chemicals. Results showed that the method is reproducible and reliable, supporting its potential inclusion in regulatory frameworks. A report of the validation study was published in August 2025, and a paper summarizing the study has been submitted for publication (Reinke et al. 2026).

Key Event 1 assays are incorporated in defined approaches (DAs) for skin sensitization hazard and potency assessments described in Test Guideline (TG) 497 issued by the Organisation for Economic Co-operation and Development (OECD; OECD 2025a). These DAs offer structured, fixed procedures for data interpretation, reducing reliance on expert judgment. By meeting performance standards for in chemico Key Event 1 assays, described by OECD in TG 442C (OECD 2025b), the EASA is a promising alternative to existing Key Event 1 assays. The EASA is currently under consideration by OECD for inclusion in TG 442C. Compared to the current TG 442C assays, the EASA offers higher throughput and requires less specialized equipment, making it more accessible and efficient. To support its inclusion in TG 442C, the OECD Skin Sensitization Expert Group has requested additional testing for predictive capacity and confirmation of molecular interaction between the probe and reference chemicals; this testing is in progress by NIST and the NIEHS-sponsored laboratory.

Current regulatory decision-making for potential thyroid-disrupting chemicals is based on in vivo apical endpoints, such as alterations in thyroid hormone levels. New approach methodologies (NAMs) to complement or replace traditional in vivo tests are being developed to enable higher-throughput mechanistic understanding of potential endocrine-disrupting chemicals.

A key element in the acceptance of NAMs in regulatory contexts is to establish confidence through validation studies that evaluate a NAM’s reliability and relevance for a specific application. NICEATM coordinated an interlaboratory validation study of the utility of a thyroid 3D microtissue model for evaluating chemical effects on thyroid hormone synthesis and tissue viability. The lead laboratory was within the EPA Office of Research and Development, with three agrochemical industry and commercial laboratories participating in the transferability phase (Deisenroth et al. 2026). Goals of this effort included (1) development of the study design, (2) test method harmonization and standardization and demonstration of intralaboratory reproducibility, and (3) method transferability, reference chemical testing, and demonstration of interlaboratory reproducibility. Reference chemical selection was based on previous work demonstrating disruption of thyroid hormone synthesis. These included one “true negative” and three “true positive” compounds. The study established standard operating procedures that were successfully transferred to multiple laboratories with no experience conducting the assay. Consistent results were generated among the testing laboratories using the same four blinded reference chemicals and among the three donor pairs. Independent human thyrocyte donors were matched between a testing laboratory and the lead laboratory to account for biological variability. In addition to the publication describing the study, a detailed report of the study is being prepared. A follow-up study to test the model with a larger chemical set and potentially improve replicability is currently being designed.

Comparing in vitro results to in vivo data is necessary to establish suitability of the in vitro models as a replacement for in vivo acute lung toxicity for volatile chemical hazards. USAF PRT, in collaboration with the Air Force Research Laboratory’s MPS team and the Naval Medical Research Unit Dayton, are evaluating the mechanistic relevance and the quantitative predictivity of in vitro lung models by comparing dose responses for key events to in vivo data following acute exposure to sulfur dioxide. Key events in the acute lung injury AOP, including oxidative stress, cytotoxicity, inflammation, and barrier integrity, are being measured in monoculture cell models (A549, HuArlo) and human reconstructed tissue models (MatTek EpiAirway™ and EpiAlveolar™; Epithelix MucilAir™) using gas exposure at the air–liquid interface. In vitro data collection is nearly complete. Concurrent studies in a rat model with corresponding sulfur dioxide exposures and correlated endpoints were recently completed at the Naval Medical Research Unit Dayton. Dose–response modeling is being used to identify points-of-departure from each endpoint, and in vitro to in vivo extrapolation (IVIVE) is being used to describe gas disposition in the lung (Tsujino et al. 2005; Nowak et al. 2025). Assays and measured endpoints that are most predictive of in vivo response will be incorporated into a recommended rapid chemical testing protocol. These NAMs are expected to facilitate scientifically sound chemical risk assessments on a timescale that would realistically support operational and acquisition decisions.

NAMs incorporate advanced technologies to aid in the investigation of biological mechanisms of toxicity, evaluation of product safety, and determination of hazard levels and dose–response relationships needed for health and environmental risk assessments.

“Validation, Qualification, and Regulatory Acceptance of New Approach Methodologies: A Report of the ICCVAM Validation Workgroup,” developed by ICCVAM with input from 17 federal agencies, offers recommendations on how to establish confidence in NAMs. Confidence is key for methodology acceptance and use in regulatory and nonregulatory settings. The report emphasizes using "fit-for-purpose” validation approaches that align with each NAM's specific application rather than relying on outdated “one-size-fits-all” strategies. Concepts such as context of use, biological relevance, and technical characterization are highlighted as essential components for the effective validation of NAMs.

The report also reviews regulatory acceptance processes by U.S. federal agencies and explores the potential for international harmonization of NAMs. Best practices for ensuring quality and technical reliability are outlined, and the importance of collaboration and communication between developers, users, and regulatory agencies to streamline adoption is stressed.

ICCVAM plays a critical role by coordinating validation efforts, fostering research collaboration, and advising stakeholders on NAM implementation. This report serves as a key resource for advancing NAMs and supporting their integration into health and environmental decision-making processes, helping reduce reliance on animal testing while improving safety assessments.

Value-of-information analysis is a systematic approach to determine the “value of information” in economic terms. It allows comparison of “what we already know” and “what we will know” to determine which data generation methodologies are most valuable for decision-making. While the potential application of value-of-information analysis in toxicology has been discussed for several years, practical applications of such an analysis to real-world toxicological problems are lacking.

EPA and collaborators developed a value-of-information framework for assessing the trade-offs associated with uncertainty, duration, and cost of chemical toxicity testing (Hagiwara et al. 2023). A novel feature of this framework is the inclusion of a time dimension that permits incorporation of the cost of delay in additional information. A case study applied this framework to compare a short-term in vivo transcriptomic assay approach for the development of protective reference doses with the traditional chronic rodent bioassay and human health assessment process. The analysis found that the transcriptomic assay was the preferred testing method in the majority of scenarios considered. The analysis evaluated various combinations of exposure conditions, health endpoints, exposure control costs, population characteristics, and decision types using a range of values informed by real-world data. The value-of-information analysis results indicated that reducing the time to decision, realized by using the shorter-duration transcriptomic assay, has significant public health and economic benefits when compared to longer traditional toxicity testing and the human health assessment process. The final case study was published in 2024.

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.

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.