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ICCVAM Biennial Report 2020-2021

Biennial Progress Report 2020-2021 Interagency Coordinating Committee on the Validation of Alternative Methods

Assay Development

Many ICCVAM member agencies are developing new in vitro technologies and resources intended to replace animal use for chemical safety testing. Many of these include technologies to address important endpoints such as carcinogenicity, inhalation toxicity, and neurotoxicity.

Two-dimensional Cellular and Three-dimensional Bioprinted Skin Models to Screen Topical-use Compounds for Irritation Potential

Assessing skin irritation potential is critical for the safety evaluation of topical drugs and other consumer products such as cosmetics. Use of animals for these evaluations is prohibited in some sectors, with advanced in vitro cellular models considered as possible replacements. To compare the utility of different cellular skin models for this purpose, scientists at NCATS, the National Eye Institute, and NIEHS assessed irritation potential in different cellular skin models compatible with high-throughput screening platforms (Wei et al. 2020). The study tested 46 compounds related to topical products on monolayer keratinocytes, reconstructed human epidermis, and full-thickness skin models, and evaluated performance of the models using generally accepted cellular and molecular indicators of irritant activity. The study indicated that using human cells to generate bioprinted tissue is a quick and reliable method to model human skin in a high-throughput manner, and that this approach could be used as evidence for hazard labeling in a variety of hazard classification systems.

Human Neuronal Cell Line as a Model for Organophosphorus Pesticide-induced Neurotoxicity

Non-animal screening methods that can rapidly and accurately characterize organophosphorus compound-induced neurotoxicity are needed. Scientists at the U.S. Army Engineer Research and Development Center examined molecular and cellular responses characteristic of this neurotoxicity pathway via an in vitro model using the SYSY5Y human neuroblastoma cell line and the model organophosphorus compound ethyl parathion. In these studies, ethyl parathion was found to increase reactive oxygen species and cell membrane lipid peroxidation and reduce mitochondrial membrane potential, contributing ultimately to cell death. Overall, the mechanistic responses observed in the SYSY5Y cells corresponded closely with in vivo mammalian results, demonstrating potential for this non-animal model to provide accurate neurotoxicology screening for organophosphorus compounds. These investigations also provide data to support development of the AOP for this endpoint. In 2022, the research team will use these methods to investigate time-dynamic neurotoxicity responses to ethyl parathion. They will also begin developing in vitro methods that provide mechanistic toxicology indicators characteristic of mammalian acute ammonia inhalation exposures.

Development of a Non-animal-based Vaccine Test for RHDV2

Rabbit hemorrhagic disease is a devastating disease affecting wild, farmed, and companion rabbits. The virus causing the disease is closely related to human norovirus. However, unlike norovirus, which rarely causes severe disease, rabbit hemorrhagic disease causes a syndrome culminating in the death of affected rabbits. The disease was historically not detected in the United States, but in 2020, RHDV2 was isolated from rabbits in Canada and has since caused significant outbreaks and mortality in the United States. While vaccines are available to prevent the disease, USDA licensing of an RHDV2 vaccine requires testing of each lot in live animals to confirm vaccine potency. To provide a non-animal alternative method for testing of a novel RNA-based vaccine, in 2021 NIEHS provided funding to optimize and qualify an in vitro potency assay that uses a monoclonal antibody specific for the RHDV2 target protein. Evaluation of the potency assay will be completed in 2022.

Genetic Toxicity Screening Approach for Flavoring Chemicals

A variety of compounds are used as flavorings for electronic nicotine delivery systems, or e-cigarettes. Scientists in the FDA Center for Tobacco Products conducted a study (Hung et al. 2020) using alternative methods as prioritization tools to study the genotoxic mode of action of these compounds. The approach specifically examined clastogen-sensitive and aneugen-sensitive biomarkers of DNA damage in human TK6 cells. These biomarkers were aggregated through a supervised three-pronged ensemble machine learning prediction model to prioritize chemicals based on genotoxicity. In addition, in silico QSAR models were used to predict genotoxicity and carcinogenic potential. The parallel use of these predictive technologies to clarify modes of action for potential genetic damage might be helpful for screening chemicals in tobacco products.

Integration of Transcriptome Analysis with Pathophysiological Endpoints to Evaluate Cigarette Smoke Toxicity in an In Vitro Human Airway Tissue Model

To assess the effects of cigarette smoke on the function and phenotype of airway epithelial cells, scientists at the FDA Center for Tobacco Products and the FDA National Center for Toxicological Research developed a novel repeated-treatment protocol to evaluate the progression of molecular, functional, and structural abnormalities induced by cigarette smoke in a human in vitro air–liquid-interface airway tissue model (Xiong et al. 2021a). Transcriptomics analysis was combined with in vitro measurements to demonstrate cigarette smoke-mediated effects on a variety of cell functions. These measurements revealed effects that were highly consistent with abnormalities observed in airways of smokers. Enrichment analysis on the transcriptomic profiles of the air–liquid-interface cultures revealed key molecular pathways, such as xenobiotic metabolism, oxidative stress, and inflammatory responses, that were perturbed in response to cigarette smoke exposure. A subsequent study (Xiong et al. 2021b) described the transcriptome analyses of these cultures and noted that cultures exposed to the high concentration of cigarette smoke exhibited 5,090 differentially expressed genes and 551 differentially expressed microRNAs. Expression of genes involved in ciliary development and function were significantly perturbed by repeated cigarette smoke exposures, leading to changes in protein content and cilia beating frequency. In particular, the expression of miR-449a, a conserved miRNA highly enriched in ciliated airway epithelia and implicated in motile ciliogenesis, showed a time-dependent decrease following cigarette smoke, an effect consistent with observations reported in smokers with chronic obstructive pulmonary disease. Investigating the effects of cigarette smoke on the transcriptome profile of human air–liquid-interface cultures may provide both mechanistic insights and potential early biomarkers for harm from cigarette smoke exposure.

In Vitro Dosimetry Analyses for Acrolein Exposure in Human Cells

Establishing accurate dosimetry is important for assessing the toxicity of xenobiotics as well as for comparing responses between different test systems. Scientists at the FDA Center for Tobacco Products and the FDA National Center for Toxicological Research used acrolein as a model toxicant and defined the concentration–response relationships of the key adverse responses in both normal human bronchial epithelial cells and human pulmonary carcinoma cells (Xiong et al. 2021). The researchers developed a new method for indirectly estimating the intracellular uptake of acrolein based on the chemical’s key alkylation reactions. Responses, including protein carbonylation, glutathione depletion, and glutathione–acrolein adduct formation, were all linearly correlated with acrolein uptake in both cell types. The study demonstrated that normal human bronchial epithelial cells were more sensitive to acrolein exposure than the pulmonary carcinoma cells and provided mechanistic information about acrolein-induced cytotoxicity. The dosimetric analysis presented in this study may provide useful information for computational modeling and risk assessment of acrolein using different test systems.

Development of Cellular and Molecular Approaches to Evaluate Effects of Environmental Contaminants and Stressors in Fish

Aquatic exposure to endocrine disruptors, pesticides, and cyanobacteria affects the health of fish in the environment. Most of the affected species are not typically used in the laboratory and their specific responses to such exposure cannot always be inferred from data from laboratory species. During 2020-2021, USGS scientists (DOI) developed and applied methods to evaluate species-specific cellular and molecular responses to environmental contaminants and thermal stress using primary tissue culture and non-lethal biopsy methods. Specifically, this research focused on brook trout, largemouth bass, and smallmouth bass collected from the upper Chesapeake Bay watershed. Methods used included evaluation of gill biopsy tissue and collection of primary hepatocytes and leukocytes from species that inhabit aquatic ecosystems vulnerable to environmental contamination and thermal stress. Analytical approaches included in vitro exposures, image analysis-based flow cytometry, and transcriptional profiling of gill, hepatic, and immune-responsive genes using RNA-seq and nCounter technologies. These approaches are used to investigate specific mechanistic questions in environmentally relevant fishes to minimize the use of vertebrates.