https://ntp.niehs.nih.gov/go/articles2023

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Fruit fly models for assessment of population variability in toxicity

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.

AOP, Small Model Organisms NIH
ICCVAM Communities of Practice webinars

In 2015, ICCVAM initiated a series of Communities of Practice webinars to provide opportunities for detailed presentations on a current topic relevant to alternative test method development.

  • The 2022 webinar, presented on Jan. 25, was titled New Approach Methodologies to Assess (Developmental) Neurotoxicity. In this webinar, two scientists discussed new approach methodologies (NAMs) that are being considered or developed for assessing potential effects of chemicals on the nervous system. NICEATM scientist Helena Hogberg, Ph.D., discussed use of in vitro assays for developmental neurotoxicity assessment. Jyotshnabala Kanungo, Ph.D., of the FDA National Center for Toxicological Research summarized their studies using zebrafish to assess the effects of dietary supplements on drug safety and drug–drug interactions.
  • The Jan. 30, 2023, Communities of Practice webinar focused on Emerging Approaches for Anchoring Biological Relevance of New Approach Methodologies. In this webinar, three scientists from the academic and private sectors discussed approaches to evaluating NAMs that focus on the biological relevance of the NAM to the species of regulatory interest. Lorna Ewart, Ph.D., of Emulate Inc. discussed their work on using MPS to predict liver toxicity. James McKim, Ph.D., IONTOX/LifeNet Health LifeSciences summarized studies using integrated organ models of absorption, distribution, metabolism, and excretion. Finally, Tamara Tal, Ph.D., Helmholtz-Centre for Environmental Research UFZ explained their approach to building confidence in larval zebrafish behavior assays.
Developmental Neurotoxicity, Hepatotoxicity, Metabolism, MPS, Neurotoxicity, Small Model Organisms FDA, ICCVAM, NIEHS
Screening-level information for developmental neurotoxicity using new approach methodologies

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.

Defined Approaches/IATA, Developmental Neurotoxicity, Small Model Organisms EPA, NIEHS
SEAZIT ontologies, database, and data analysis pipeline

The zebrafish embryo is a useful alternative research model for assessing the effects of substances on growth and development. However, cross-laboratory developmental toxicity outcomes can vary due to lack of standardization both in laboratory procedures and terminology used to describe outcomes. Thus, reported developmental defects in zebrafish may not be directly comparable between laboratories. To enable broader adoption of zebrafish for toxicological screening NIEHS established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) program.

Discussions among scientists participating in SEAZIT considered how variability in results could be addressed by implementing standardized nomenclature systems known as ontologies. A collaborative exercise was conducted to evaluate how the application of ontologies improved data consistency (Thessen et al. 2022). Analysis of the results suggested that the use of ontology terms increased consistency and decreased ambiguity, and that utilizing a common data standard should reduce the heterogeneity of reported terms and potentially increase agreement and repeatability between different laboratories.

A key element of SEAZIT is an interlaboratory study to investigate how experimental protocol differences can influence chemical-mediated effects on developmental toxicity. Three laboratories were provided a common and blinded set of 42 substances to evaluate chemical effects on developmental toxicity in the embryonic zebrafish model. Laboratory work was completed in 2022, and a paper has been published (Hsieh et al. 2023) describing the relational database developed to store the data, which features harmonization of the above-described ontologies for altered phenotype endpoints, and the data analysis pipeline. Data are available in the NIEHS CEBS data resource, and a web application is being developed to allow users to interactively explore the data. A second paper describing the study design is being prepared for publication in 2024.

Developmental Toxicity, Small Model Organisms NIEHS
Strategy using in silico methods and zebrafish assays to characterize algal toxins and contaminants

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.

Cardiotoxicity, Ecotoxicity, Mixtures Toxicity, Small Model Organisms DOI
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