ICCVAM Biennial Report 2022-2023

The electrophilic allergen screening assay (EASA) is a promising in chemico method to identify substances that covalently bind to skin proteins, the first key event in the AOP for skin sensitization. This assay assesses the depletion of either of two probe molecules in the presence of a test compound. The initial version of the EASA, developed by the NIOSH, used a cuvette format, which presented multiple measurement challenges such as low throughput and the inability to include adequate control measurements. Scientists with the NIST, CPSC, and NIEHS redesigned the EASA into a 96-well plate format that incorporates in-process control measurements to quantify key sources of variability each time the assay is run (Petersen et al. 2022). The paper also describes a measurement science approach that provides steps that can be taken during assay development to increase confidence of in chemico assays and other new approach methodologies (NAMs) by characterizing sources of variability and potential biases and incorporating in-process control measurements.

A subsequent validation study assessed intra- and interlaboratory of the 96-well EASA assay. In addition to the CPSC/NIST lead lab, laboratories sponsored by FDA, DoD, and NIEHS participated in the validation study. Each participating laboratory tested 10 positive and negative control substances and 20 reference chemicals. Within- and between-laboratory reproducibility met performance criteria established by the validation management team. A report of the validation study has been prepared and a peer review of the study will be conducted in 2024.

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.

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.

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.

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. This model has been utilized to identify unique biomarkers of soot-based exposures, an emerging respiratory global concern to human health and performance. An alveolus-on-a-chip platform was developed using primary alveolar epithelial and microvascular endothelial cells cultivated at an ALI. The platform incorporates cyclical stretch and breathing mechanics to better emulate human physiology in a laboratory model. A primary goal of this effort is to establish and optimize extracellular vesicle isolation, enumeration, and molecular cargo analysis workflows using a relatively low number of cells and conditioned medium. Extracellular vesicles carry cellular material such as microRNAs to facilitate cell-to-cell communications in response to a variety of stress conditions. These are found in nearly every biological fluid, providing a rich opportunity for investigating tissue specific biosignatures of exposure. Preliminary results 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. These preliminary findings drove the development of a “mini-jet engine” soot generator, an in vitro aerosol deposition platform, and the integration of the alveolus-on-a-chip to form a platform termed AERO-TOX (Aerosol Exposure of Respiratory Organ-on-a-Chip for Toxicology). The soot generator has been optimized to produce soot of relevant size distribution and mixtures with common combustion reaction gases such as nitric dioxide and carbon monoxide, and has been statistically shown to emulate F-22 jet exhaust soot profiles. Investigators now aim to utilize the AERO-TOX platform to discover novel extracellular vesicle-derived biomarkers of exposure to aid in next-generation diagnostic and countermeasure development.

U.S. Army Chemical Biological Center (CBC) scientists are validating a highly complex and human-relevant multiorgan chip in its ability to serve as a model system for antimicrobial drug efficacy and safety and to recapitulate physiologically based pharmacokinetic parameters.

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. CBC scientists investigated how well bioinformatic data collected from organ chips matches the bioinformatic data collected from human samples, with the hope that organ chips will provide an alternative approach for improved biomarker discovery for toxicity assessment and exposure identification. 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 what was found 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.

CBC is also incorporating immune components into commercially available chip systems. The pulmonary system is an ideal exposure route for chemical and biological agents making mast cells a prime choice to assimilate into a lung-on-a-chip. Mast cells rapidly release a variety of mediators such as histamine, heparin, proinflammatory cytokines, and chemokines in response to foreign substances or endogenous damage associated molecular patterns. These factors facilitate immune cell recruitment and play a key role in pulmonary toxicity due to mast cells location within airways and mucosal surfaces of the lungs. CBC is collaborating with the University of Colorado Anschutz Medical Campus to integrate a mast cell immune component into the Emulate system. This work will allow substantial future work to incorporate other immune cells into the system to build toward a complete model of immune system infection prevention and pathogen defense.

A comparison of 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. U.S. Air Force (USAF) Predictive Risk Team (PRT) is evaluating the mechanistic predictivity of respiratory in vitro models by comparing the in vitro dose-response curves for key events in acute respiratory to in vivo rodent and human data following exposure to prototype respiratory toxicants. Key events in the acute lung injury AOP are being measured in a monoculture cell model (A549) and human reconstructed tissue models (EpiAirway, EpiAlveolar). In vitro to in vivo extrapolation (IVIVE) will be performed using in silico models describing gas disposition in the lung. Assays and measured endpoints that are determined to be most predictive of in vivo response will be incorporated into a rapid chemical testing protocol. These new approach methodologies (NAMs) are expected to facilitate scientifically sound chemical risk assessments on a timescale that would realistically support operational and acquisitions decisions. The project was selected for funding by the Military Operational Medical Research Program in 2023, with experimental work planned to begin in 2024.

USAF Predictive Risk Team (PRT) is developing a new approach methodologies-based rapid approach to better support risk assessment of operational exposure to novel and poorly characterized chemicals. This is a tiered process where lower tiers use in silico and high-throughput in vitro models to identify hazards and prioritize testing, and higher tiers use organotypic in vitro models to perform quantitative risk assessments. PRT is also building models and validating assays to support this approach, focusing initially on acute toxicity. To test the utility of this NAM-based approach and developed models, the PRT is performing case studies with several occupationally relevant chemicals. These chemicals are being tested with available in silico models (Tier 0) to evaluate mode-of-action and acute systemic toxicity. Based on in silico results, the chemicals are being tested in high-throughput (Tier 1) and organotypic (Tier 2) in vitro assays, which will be used with in vitro to in vivo extrapolation to derive an equivalent administered dose (EAD). EADs will be compared to in vivo point-of-departure (PODs) and published exposure limits to assess the utility of this process for estimating safe limits for acute operational exposures.

USAF Predictive Risk Team (PRT) performed a study to characterize the risk of allergic contact dermatitis from occupational chemical exposures using new approach methodologies. 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 induction of sensitization. The kinetic direct peptide reactivity and KeratinoSens(TM) assays were used to predict mouse local lymph node assay effective concentration values. In vitro assay results, in silico model predictions, and available human and animal data were used in an integrated approach to testing and assessment to predict sensitizer status using a weight-of-evidence approach. Preliminary candidate surface guidelines were derived from predicted effective concentration values for predicted sensitizers. Findings from this study were published by DoD as a Defense Technical Information Center report. Additional data from the in vitro human cell line activation test and updated analyses will be presented at the 2024 SOT annual meeting. A manuscript describing this work is planned for peer-reviewed publication in 2024.

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.

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.

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.

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.

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.

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.

Organophosphorus flame retardants are abundant and persistent in the environment due to their extensive use in industrial processes and products. The structural similarity of these flame retardants to organophosphate pesticides has prompted concern that they could potentially cause both acute neurotoxicity and developmental neurotoxicity (DNT).

DNT testing is traditionally done using animals, which is resource-intensive and fails to provide information on cellular processes affected by chemicals. OECD has published a series of case studies to support application of integrated approaches to testing and assessment (IATAs) to identification of potential developmental neurotoxicants. One of these case studies, developed by EPA and NIEHS scientists, used an in vitro testing battery to prioritize a class of organophosphorus flame retardants. The case study was published in September 2022.

Since the development of the IATAs, new data have become available that could refine the testing approach and provide a stronger case study. NIEHS is currently extracting these data and incorporating additional parameters such as exposure data, toxicokinetics, and endpoints and mechanisms relevant to DNT but not included in the original data. The revised dataset is described in an abstract (Oyetade et al.) accepted for a poster presentation at the 2024 annual meeting of the Society of Toxicology. The original case study submitted to OECD will be revised into a manuscript that will include and integrate the new data and updated resources into the information generated in the first project.

NICEATM, PETA Science Consortium International e.V., EPA, and CropLife America member companies are collaborating to develop an in vitro defined approach for hazard classifications of eye irritation potential of agrochemical formulations. A three-phased prospective evaluation was designed to (1) assess the applicability of seven in vitro eye irritation/corrosion protocols to agrochemical formulations and (2) develop defined approaches (DAs) for agrochemical formulations testing for prediction of U.S. and international irritancy classifications. Agrochemical formulations were selected for prospective testing based on availability of historical rabbit test data, to represent common agrochemical formulation types, and to span the full range of ocular irritation hazard. Test methods were included based on their relevance to mechanisms of human eye irritation, and the results were assessed to determine which methods should advance to potential incorporation in a DA. Twenty-nine formulations were tested in up to five methods: bovine corneal opacity and permeability, EpiOcular, SkinEthic time-to-toxicity for liquids, in vitro depth of injury, and EyeIRR-IS.

In a project led by PETA Science Consortium International, two DAs were developed to predict eye irritation potential in the context of the EPA pesticide classification system (van der Zalm et al. 2023). Predictions derived using the DAs were assessed using orthogonal validation and weight-of-evidence, rather than direct concordance analysis with the historical in vivo rabbit eye data. Both DAs were demonstrated to be as or more fit-for-purpose, reliable, and relevant than the in vivo rabbit eye test.

A separate project led by NICEATM focused on predicting eye irritation potential based on the GHS classification system. A preliminary analysis of alignment across the five in vitro methods and historical rabbit test data was conducted to determine consensus predictions for each formulation. Four methods were then used in the development of four DAs to predict GHS classifications. All four proposed DAs may have high utility for predicting eye irritation classification of agrochemical formulations, as the hazard labeling associated with the predictions are as or more protective of human health compared with the in vivo rabbit test. Furthermore, using the consensus prediction as the reference standard, some standalone in vitro methods can predict the human eye irritation hazard of agrochemical formulations as well as or better than the rabbit test. Results of the study will be described in a poster at the 2024 annual meeting of the Society of Toxicology (Daniel et al., Ocular Toxicology session) and in a paper to be submitted for publication in 2024.

Current regulatory decision-making for potential thyroid-disrupting chemicals is based on in vivo apical endpoints including 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 to 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 is coordinating 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 is within the EPA Office of Research and Development, with three agrochemical industry and commercial labs participating in the transferability phase. Goals of this effort include (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. Refinement of standard operating procedures, tissue procurement, method standardization in the main laboratory, and an initial assessment of method transferability were completed in 2023. Method transfer and model validation are currently being completed in the additional laboratories, to be followed by method performance evaluation by all laboratories. Results of the study will be described in a study validation report.

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.

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.

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.

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.

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.

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.

Skin sensitization testing is a regulatory requirement for the safety of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are internationally accepted, specifically via OECD Guideline 497. However, these DAs were evaluated with mono-constituent substances, which may limit their applicability to multi-constituent substances such as pesticides. An analysis by NIEHS scientists and collaborators evaluated rule-based DAs for hazard and/or potency categorization of skin sensitization for agrochemical formulations (Strickland et al. 2022). The data set for the analysis included 27 formulations, each tested using the direct peptide reactivity assay, the KeratinoSens™ assay, and the human cell line activation test. The KeratinoSens assay had the highest performance for predicting in vivo hazard outcomes and performed better than any of the DAs. The analysis demonstrates that non-animal test methods are useful for evaluating the skin sensitization potential of agrochemical formulations. Further investigation is necessary to determine whether DAs can outperform individual assays for predicting in vivo sensitization hazard of pesticide formulations in general.

In a separate study, NIEHS and EPA scientists and collaborators evaluated the use of DAs to evaluate sensitization potential of isothiazolinones. Isothiazolinones are used as preservatives in a range of consumer products but are known to cause skin sensitization and irritation. The skin sensitization potential of six isothiazolinones was evaluated using three internationally harmonized non-animal test methods: the direct peptide reactivity assay, KeratinoSens™, and the human cell line activation test. Results from these test methods were then applied to two versions of the Shiseido Artificial Neural Network defined approach. Hazard or potency predictions showed high concordance with those produced by reference animal test data with less variability. The application of in silico models to in chemico and in vitro skin sensitization data is a promising data integration procedure for DAs to support hazard and potency classification and quantitative risk assessment.

To explore broadening the number of test methods that can be used in defined approaches to identify skin sensitizers, NIEHS scientists used the GARD®skin assay (SenzaGen AB) to test 31 substances nominated by several U.S. federal agencies. GARDskin results were applied as a substitute for results from the human cell line activation test within two accepted defined approaches (DAs). Both assays measure the same endpoint, mobilization of dendritic cells and induction of inflammatory cytokines and surface molecules, which represents Key Event 3 in the AOP for skin sensitization. Results were evaluated both for prediction of skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) and potency classification according to GHS categories. Concordance and performance of the GARDskin results were also compared to existing DAs from OECD Guideline 497 and individual test methods, including murine local lymph node assay reference data. While GARDskin tended to overpredict sensitization hazard when compared to reference data skin sensitization hazard classification, concordance against the local lymph node assay was higher for DAs that incorporated GARDskin than those that did not. In vitro testing and DAs provide a useful alternative to animal testing for skin sensitization hazard and potency classification of substances relevant to a wide range of federal agency programs.

Another SenzaGen new approach methodologies (NAM) model, the GARDair assay, shows promise to assess respiratory sensitization, an endpoint for which there is no good animal model. NIEHS and collaborators are testing about 100 substances nominated by U.S federal agencies using GARDair. Results for chemicals tested thus far indicate that hazard classification using the GARDair assay shows promising concordance with hazard classifications based on existing occupational human reference data and results from skin sensitization assays. Both studies are described in an abstract accepted for a poster presentation (Johnson et al.) at the 2024 SOT annual meeting.

Results of in vitro assays used in the Tox21 and ToxCast high-throughput screening (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. However, many chemicals tested in HTS assays lack directly relevant cytotoxicity data needed to ensure overt toxicity does not confound mechanistic outputs. Chemicals may also vary in their potency for eliciting cytotoxicity across cell types and time trajectories. NIEHS scientists are investigating applying multiple machine-learning algorithms to predict chemical- and cell type-specific cytotoxicity concentrations to provide context for flagging nonspecific in vitro chemical-elicited bioactivity using cytotoxicity assays included in Tox21 and ToxCast. Cell type- and time point-specific machine-learning models were developed to predict chemical concentrations likely to induce cytotoxicity to provide context for assays without concurrent cytotoxicity data. After being further refined, the predictive models will be integrated with bioactivity data to provide context and bolster confidence in assay outcome interpretation for identifying specific vs. nonspecific/cytotoxicity-confounded bioactivities. Concurrent cytotoxicity readouts from Tox21/ToxCast assays were mapped for 492 assay endpoints, allowing direct comparison of bioactivity potency against cytotoxicity. These comparisons will be integrated into future versions of concentration–response visualizations for the curated HTS data in the Integrated Chemical Environment Curve Surfer tool. This project was described in an abstract accepted for a poster presentation (Tedla et al.) at the 2024 SOT annual meeting.

To reduce animal use for investigating human-relevant inhalation toxicity, the Occupational and Inhalation Exposures Program in the NIEHS Division of Translational Toxicology (DTT) is evaluating the use of in vitro lung models, including air-liquid interface (ALI) airway tissues. Potential applications include screening-level assessments to help predict the adverse airway effects of inhaled substances and prioritize them for further testing. A proof-of-concept study was conducted to characterize and optimize a VITROCELL 48 2.0 Plus exposure system with ALI airway tissues. The chemical used was 2,3-pentandione, a highly volatile component of artificial butter flavorings that has been associated with airway injury and fibrosis via occupational inhalation exposure. NIEHS scientists first performed method development and validation of the VITROCELL system using 2,3-pentandione vapor. Then, both normal human and rat tracheaobronchial epithelial cell-derived ALI tissues were treated to evaluate 2,3-pentandione-induced airway toxicity in vitro. These studies were designed to explore the validity of extrapolations among rat in vivo, rat in vitro, and human in vitro effects in these models. Preliminary results show that exposure of the ALI tissues to 2,3-pentandione induces concentration-dependent changes in relevant airway toxicity endpoints that are comparable between species. Specific observations included decreased transepithelial electrical resistance, cytotoxicity, and histopathologic effects characteristic of the airway injury caused by in vivo exposure to artificial butter flavorings. Results from this work were presented at the SOT annual meeting in 2022 (Gupta et al.) and further results are described in an abstract (Gwinn et al.) accepted for a poster presentation at the 2024 SOT annual meeting. Further work planned for 2024-2025 will focus on expanding the capabilities applicable to this exposure system and tissue models, including supporting transcriptomic analysis, informing on quantitative AOPs, supporting testing of different types of chemicals, and evaluating reproducibility in other laboratories.