SEAZIT: Systematic Evaluation of the Application of Zebrafish in Toxicology

The small size and rapid development of the zebrafish make it a useful vertebrate model for assessing the potential effects of substances on growth and development using high-throughput screening methods (as reviewed in Planchart et al. 2016). The embryonic zebrafish model has been used for this purpose in pharmaceutical development and in high-throughput screening programs at NTP and EPA. However, broader adoption of the zebrafish model for toxicological screening is hindered by deficits in:

• Consistent experimental protocol elements.
• Clear understanding of mechanisms of chemical absorption, distribution, metabolism, and excretion in zebrafish.
• Consistent informatics approaches used for classification of outcomes.

To enable the broader adoption of zebrafish for toxicological screening, NTP established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) program, jointly led by NTP and NICEATM scientists. This webpage summarizes SEAZIT program activities in:

• Information gathering with a focus on protocols and laboratory practice.
• Webinar series exploring:
  • Use of informatics to improve data analysis for zebrafish screening studies (2017).
  • Case studies that illustrate the utility of zebrafish models for toxicology (2018).
• An interlaboratory zebrafish study (testing completed in 2022).
• A zebrafish best practices workshop.

Information Gathering

In 2016, SEAZIT team members conducted a series of interviews with experts in the use of zebrafish in toxicology studies. These interviews identified areas key to developing a harmonized testing protocol for zebrafish studies, as well as sources of variability among laboratories.

A meeting with zebrafish researchers and data scientists was convened in 2017 to discuss the use of ontologies (standardized nomenclature systems) to improve zebrafish screening data analyses. Participants agreed that a comprehensive group analysis of a compiled data set would help demonstrate the advantages of applying a standardized phenotype terminology.

• A flash report of the meeting summarized the presentations, discussions, and recommendations.
• Information about current research practices gathered from this effort are summarized in Hamm et al. 2018.

Webinar Series

Key issues being addressed by SEAZIT include the variability among laboratories in the endpoints measured and the nomenclature used for each endpoint. On behalf of SEAZIT, NICEATM presented a webinar series in 2017 that considered how these issues might be addressed by implementing ontologies (i.e., standardized nomenclature systems).

The goals of the webinar series were to:

• Summarize some of the barriers to routine use of zebrafish in toxicological evaluations.
• Review the concept of ontologies.
• Consider how ontologies could be applied to harmonize procedures used for zebrafish screening studies.

A second webinar series in fall 2018 examined three case studies that illustrated the utility of zebrafish models for toxicology.

Ontology Needs for Zebrafish Screening

Building on the findings of the 2017 webinar series and discussions among scientists, SEAZIT is coordinating collaborative projects to address ontology needs for zebrafish screening. Three laboratories tested the same 90-chemical set using similar study designs and morphology assessments. Data from these studies are being curated and assessed to:

• Establish a web portal to collect and display zebrafish data.
• Identify genes associated with similar phenotypes resulting from chemical exposure.
• Identify limitations in zebrafish phenotype screening data.

To further define ontologies for zebrafish screening, NICEATM coordinated an online evaluation of heterogeneity in terms used to describe zebrafish phenotypes following chemical exposure (Thessen et al. 2022). Zebrafish images were posted online and researchers asked to evaluate the phenotypes using their in-lab terminology. Results were compiled and terms mapped to the Zebrafish Phenotype Ontology. A second online evaluation employed controlled vocabulary.

Interlaboratory Study

The interlaboratory study conceived during the Information Gathering phase was completed in 2022. A paper has been published describing the data analysis pipeline (Hsieh et al. 2023); a second paper describing the study design will be published in 2024.

The study was designed to determine the effect of chorion removal and exposure media renewal on study outcomes. Participating laboratories used in-house protocols to test a defined chemical set while varying the two protocol elements under investigation. The chemical set, which was designed to provide overlap with other NTP studies, included chemicals with a range of physicochemical properties and developmental effects. Many of the chemicals have in vivo reference data available from rodent and other zebrafish studies. The interlaboratory study included a pilot effort on chemical kinetics in support of future studies of absorption, distribution, metabolism, and excretion in zebrafish.

A primary goal of SEAZIT is to develop best practices for data analysis. To this end, the data generated in this study will be made publicly available, so that all study data may be used by investigators to estimate consensus toxicity values for each chemical.

Best Practices Workshop

Once the data have been generated and analyzed, a best practices workshop is planned as a public forum where experts from various fields can discuss continued development and standardization of assays, as well as practices for collecting, analyzing, and reporting data. A report from the workshop will be published in the peer-reviewed literature and will provide recommendations on how to conduct zebrafish screening assays and report the data.