Regulatory Compliance and The Zebrafish Model (Part II)

Zebrafish for regulatory purposes in ecotoxicology and safety pharmacology

By Miriam Martínez, Marketing Manager at ZeClinics.

Miriam is a Human Biologist expert in neuropharmacology. After a master’s degree in Pharmaceutical and Biotech Industry, she obtained her PhD in Biomedicine from the Pompeu Fabra University (Barcelona). During her doctorate, she focused her research on the behavioral analysis of animal models for neurophenotypical characterization. Since then, she has been working in the healthcare marketing and publicity sector, where she has contributed to developing marketing campaigns for several pharmaceutical brands. This year she joined ZeClinics with a branding and marketing strategy focus.

ZeClinics in global initiatives for zebrafish model validation

ZeClinics is participating in several initiatives led by international Institutions and Agencies investigating zebrafish as a new alternative methodology (NAM) to assess environmental and human risk assessment.

It is a work carried out for years, in an effort to generate fundamental knowledge and sufficient scientific evidence for broader adoption of the zebrafish model in toxicological screening, fully supported by regulatory agencies. 

These initiatives are directed to ultimately position zebrafish as a valid model for the preclinical regulatory roadmap, and lead the segment of alternative models with high predictivity in preclinical phases. Below we describe the different projects in which ZeClinics is involved.

A. Inter-laboratory Study to investigate Protocol Variables on Toxicity Measures in Zebrafish, led by SEAZIT (Systematic Evaluation of the Application of Zebrafish in Toxicology) Program - National Toxicology Program (NTP) NIH [2019-to current day].

STAKEHOLDERS: SEAZIT (study sponsor); Battelle (contractor). ZeClinics and other undisclosed parties.

SEAZIT is a multipronged and multiyear initiative to support the generation of fundamental knowledge for the use of zebrafish in toxicology research and provide the scientific basis for NTP’s future use of zebrafish in chemical toxicity screening. The initial phase of SEAZIT focused on cataloging standard practices currently used in zebrafish assay protocols and involved interviews with zebrafish researchers in academic, federal, and industry laboratories [1,2]. Interviews revealed a high degree of variability across design parameters, data collected, and analysis procedures. This variability was recognized as a barrier to research by preventing the efficient integration of large quantities of data.

The aim of the SEAZIT program was to standardize zebrafish-based:

  1. Protocols (2019-2022): To accomplish this mission, ZeClinics, along with other stakeholders and in an interlaboratory validation effort, assesses the toxicological and teratogenic impact of 38 chemicals that have in vivo data available from rodent and other studies on zebrafish embryos under different conditions (Chorion/No Chorion; Static /Repeated Dose…). Concluded in June 2022.
  2. Ontology (2021-2022): Cover the lack of consistent data annotation resulting from terminological harmonization. Results from this work have been recently published [3].
  3. Data analysis & statistics (to be performed)
zebrafish-ontology-standarization-1

Reducing data heterogeneity with ontologies. Different laboratories test the same chemical and observe the same endpoint but report their observations differently according to each laboratory’s internal standard. Mapping these terms to an ontology reduces this heterogeneity and aids in data integration across laboratories. Source: Thessen AE, et al., Front Toxicol, 2022.

The following two initiatives are addressed to improve the current chemical Developmental NeuroToxicity (DNT) testing

DNT is defined as potential functional and morphological hazards to the nervous system which may arise in the offspring from exposure of the mother during pregnancy and lactation. The developing Central Nervous System (CNS) has a different susceptibility to chemical-induced injury than the adult CNS, thus, a specific methodology for DNT assessment is required.

Zebrafish-vs-human_nervous-system
High conservation in development and function between the human and zebrafish brain.

B. Developmental NeuroToxicity Data Integration and Visualization Enabling Resource (DNT-DIVER), led by NTP NIH [2017-2019]

STAKEHOLDERS: NIH (leading team); University of California, San Diego; University of California, Davis;  Oregon State University; Biobide; ZeClinics; US EPA; University of Konstanz; Molecular Devices; Boston University.

The National Toxicology Program (NTP) sponsored this collaborative initiative to investigate new and rapid screening approaches using alternative models such as Zebrafish to identify environmental compounds that may be linked with neurodevelopmental disorders.

The ultimate goals are to protect public health by early identification of environmental chemicals with DNT potential in order to prioritize further in vivo testing and provide timely information to stakeholders for regulatory decision-making. DNT consortia started with an interlaboratory effort to integrate data for the community.

DNT-DIVER: Interlaboratory and multi-assay study by the U.S. Department of Health and Human Services to assess the contribution of environmental toxicants to neurodevelopmental disorders onset [4].

As a result, a publicly-available interactive web-application tool known as DNT-DIVER has been developed to compare data across divergent assays and laboratories.

Example of the information one can obtain using this powerful platform.

C. New Guidance Document using Integrated Approaches to Testing and Assessment (IATA) as part of OECD DNT Experts group (WNT program) [2019- current day]

STAKEHOLDERS: Organization for Economic Co-operation and Development (OECD) (leading team); European Food Safety Authority (EFSA) (leading team); U.S. Environmental Protection Agency (U.S. EPA)(leading team); Joint Research Centre (JRC) European Commission (leading team); Danish EPA (leading team);  University of Konstanz; Leibniz Research Institute for Environmental Medicine, Düsseldorf.

Zebrafish Expert Group Stakeholders: U.S. EPA; Oregon State University; Division of the National Toxicology Program, NIH; Health Canada / Government of Canada; National Research Council of Canada; ZeClinics; Biobide; Helmholtz Centre for Environmental Research GmbH - UFZ; VU University Amsterdam; National Institute for Public Health and the Environment (RIVM) (leading team).

The Test Guidelines program of the Organization for the Economic Cooperation and Development (OECD) took the initiative to coordinate an international collaboration between diverse stakeholders to develop DNT Integrated Approaches to Testing and Assessment (IATA) [5]. To do so, Adverse Outcome Pathways (AOP) are being developed to increase the mechanistic understanding of DNT effects. This aspect is expected to increase scientific confidence in the use of in vitro methods, thus facilitating regulatory uptake.

The goal is to develop a new guidance document for DNT assessment using an in vitro battery (DNT-IVB) including zebrafish that could be used immediately for the screening of all chemicals. CNS development involves many events within strictly controlled time frames, creating different windows of vulnerability to chemical exposure. For this reason, the integration of several methods to reach a highly predictive hit scoring system is essential.

Developmental Neurotoxicity (DNT)

3Rs-aligned method for high throughput in vivo DNT screening.

The proposed DNT-IVB includes 13 cell-based assays and 2 whole organism assays (in zebrafish) to evaluate key events in the neurodevelopmental process including oligodendrocytes differentiation, myelin formation, radial and astroglia development, differentiation, microglia interaction/activation, and functional responses to chemical exposure.

Neurobehavioral tests currently represent one of the most powerful tools to estimate the potential functional and morphological effects on the developing nervous system. In this context, zebrafish are used as a whole organism assay to complete and boost the predictability of DNT-IVB. The aim of the zebrafish group of experts is to demonstrate the added value of the gold standard zebrafish light/dark transition behavioral assay inside the IVB.

After the recognition and reduction of its uncertainties, OECD regulatory and scientific efforts to develop guidance on the development and implementation of a DNT-IVB will surely gain support from the regulatory community.

REFERENCES

[1] Planchart A, Mattingly CJ, Allen D, Ceger P, Casey W, Hinton D, Kanungo J, Kullman SW, Tal T, Bondesson M, Burgess SM, Sullivan C, Kim C, Behl M, Padilla S, Reif DM, Tanguay RL, Hamm J. Advancing toxicology research using in vivo high throughput toxicology with small fish models. ALTEX. 2016;33(4):435-452. doi: 10.14573/altex.1601281.

[2] Hamm JT, Ceger P, Allen D, Stout M, Maull EA, Baker G, Zmarowski A, Padilla S, Perkins E, Planchart A, Stedman D, Tal T, Tanguay RL, Volz DC, Wilbanks MS, Walker NJ. Characterizing sources of variability in zebrafish embryo screening protocols. ALTEX. 2019;36(1):103-120. doi: 10.14573/altex.1804162.

[3] Thessen AE, Marvel S, Achenbach JC, Fischer S, Haendel MA, Hayward K, Klüver N, Könemann S, Legradi J, Lein P, Leong C, Mylroie JE, Padilla S, Perone D, Planchart A, Prieto RM, Muriana A, Quevedo C, Reif D, Ryan K, Stinckens E, Truong L, Vergauwen L, Vom Berg C, Wilbanks M, Yaghoobi B, Hamm J. Implementation of Zebrafish Ontologies for Toxicology Screening. Front Toxicol. 2022 Mar 11;4:817999. doi: 10.3389/ftox.2022.817999.

[4] National Toxicology Program. (2018). Data Release: Developmental NeuroToxicity Data Integration and Visualization Enabling Resource (DNT-DIVER). Last Updated: December 3, 2018. https://doi.org/10.22427/NTP-DATA-002-00062-0001-0000-1

[5] Sachana, M.; Shafer, T.J.; Terron, A. Toward a Better Testing Paradigm for Developmental Neurotoxicity: OECD Efforts and Regulatory Considerations. Biology 2021, 10, 86. https://doi.org/10.3390/biology10020086.

3Rsacute toxicityalternative modelsChemical toxicitydevelopmental neurotoxicityecotoxicologyenvironmental toxicologyNAMspredictive toxicologyREACHregulatory complianceteratogenicityZebrafish