ICCVAM Biennial Report 2022-2023

The U.S. Army Chemical Biological Center (CBC) has established CRADAs with the tissue chip manufacturers Emulate and TissUse to apply their products to investigation of toxicity of aerosols.

Aerosolization of chemical agents is a health concern that presents specific technical challenges in addressing. Generation of aerosols in specific environments raises the possibility that the agent could be deposited on surfaces and then be reaerosolized into the environment due to the elements, 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. In a first-of-its-kind study, CBC scientists investigated the potential of reaerosolization of pharmaceutical-based agents (PBAs) in a soil with high sand content. Investigation into this concept required novel experimental system design and unique approaches for data collection. A vibration system with air inlet and outlet ports was developed to allow sampling of the reaerosolized soil-PBA, and work is ongoing to determine a worst-case scenario concentration. Once identified, the PBA concentration will be assessed using receptor-based in vitro screening tools. Toxicity of substances of concern will be assessed using an open-top TissUse organ-on-a-chip system that is amenable to exposures from an aerosolized agent. The system functions with an established Emulate 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. Experimental work with the Emulate and TissUse systems is planned for 2024.

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.

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).

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.

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.

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.

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.

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.

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. One DTT area of study is how environmental factors that alter immune responses may contribute to human disease. Changes in immune function can affect susceptibility to infectious disease or cancer, contribute to the development of respiratory or dermal allergic responses resulting from xenobiotic exposures, and induce or exacerbate autoimmune disease. The DTT immunotoxicity testing and research program investigates the ability of xenobiotics to alter the normal structure and/or function of the immune system. Current efforts within the research program focus on using in vitro approaches to assessing potential immunotoxicity. A major effort during 2022 and 2023 used an in vitro human whole blood culture system to investigate how interindividual susceptibility factors and environmental risk factors impact the response to viral infection. Over 200 individual human samples have been screened, and data are being analyzed to examine how intrinsic factors such as age, gender, and ethnicity influence the response of peripheral blood leukocytes to influenza and SARS-CoV-2 antigens. Preliminary results suggest that males have a higher natural killer cell activity in peripheral blood than females, and data are being further analyzed to determine if this effect is due to males having higher abundance of natural killer cells or more active natural killer cells than females. A second phase of this study is investigating responses to influenza and SARS-CoV-2 antigens following in vitro exposure to known immunotoxicants and how exposure to these environmental agents may affect susceptibility to viral infection. As proof of concept, whole blood cultures were unstimulated or stimulated with anti-T-cell receptor antibodies or viral peptide pools in the presence of dexamethasone, a known immunosuppressive drug. Dexamethasone treatment resulted in inhibition of natural killer activity, cytokine production, and T-cell activation following stimulation with the positive control. This work demonstrated that the in vitro immunotoxicity platform could detect immune suppression and alterations in responses to SARS-CoV-2 peptides. A second proof-of-concept study to examine the effect of benzo(a)pyrene exposure in the presence of metabolizing enzymes resulted in potent suppression in immune endpoints. Work planned for 2024 will develop additional endpoints for this culture system to facilitate interrogation of antibody-mediated responses and cytotoxic T-cell driven immunity. This in vitro toolbox will be critically important for providing direct human relevance of methods used to identify chemicals that have the potential to modulate immune function and reduce the use of animals in toxicology testing.

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.

Profiling chemical effects on transcription factor activity can help characterize the mechanisms by which chemicals may perturb biological systems. The Attagene cis-FACTORIAL™ assay uses a reporter system to detect transactivation of 46 transcription factors to provide a quantitative assessment of chemical effects. A new version of this assay, CYP-FACTORIAL, adds 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 an altered bioactivity profile between parent and metabolite compounds. To explore the application of this system to predicting toxicity, NIEHS and Attagene collaborators tested 24 chemicals of regulatory concern in both systems. Profiling across all 46 transcription factors produced toxicity signatures for chemicals, and applying a biological read-across approach identified chemicals with similar effects. Additionally, profiles for “toxic” vs. “non-toxic” chemicals yielded insight into the biological mechanisms underlying adversity. This study exemplifies how integrating metabolism into a multiplexed in vitro assay system can provide additional mechanistic insight needed to understand chemical-elicited bioactivity, thereby facilitating the development of human-relevant test systems. A poster describing this work (Karmaus et al.) was presented at the 12th World Congress on Alternatives and Animal Use in the Life Sciences, and a manuscript is under development.