Miriam Martínez - 30 June 2022
Regulatory Compliance and The Zebrafish Model (Part I)
New trends and updated availability for zebrafish studies in EU and US
The growing market of toxicology: Vertebrate use prediction under REACH
In the 27 countries comprising the EU, global chemicals research & innovation (R&I) spending has been steadily growing, especially for the last 10 years, getting to the highest level in 2020. If this evolution continues, more research efforts and resources should be expected in the future, which would bring a bigger need for toxicity testing for newly generated substances.
In the European Union, a piece of legislation called REACH entered into force in 2007. REACH stands for the Registration, Evaluation, and Authorisation and restriction of Chemicals. Its purpose is to establish whether all existing and new chemicals are safe for humans and the environment and to control the use of those judged to present a risk.
All companies that manufacture and market chemicals in the EU need to submit registrations to European Chemical Agency (ECHA). ECHA elaborates a registrations statistics report every 5 years which analyzes all the dossiers submitted.
A higher number of dossier submissions is directly associated with the higher use of vertebrate animals to comply with the required toxicity endpoints.
In this context, safety testing and chemical risk assessment need to innovate in order to reduce dependency on animal testing and to improve the quality, efficiency, and speed of assessments.
Consolidated role of zebrafish in environmental toxicology
Many years ago, the National Institutes of Health (NIH) already ranked zebrafish as the second most important animal model behind the mouse  (Goldsmith & Solari, 2003). During the last decades, the zebrafish model has heavily contributed to a paradigm shift in toxicity testing.
On the current day, several fish toxicity tests are a standard information requirement under REACH (Registration, Evaluation, Authorisation, and Restriction of Chemicals) regulation: among them, the AFT (acute fish toxicity) is one of the most spreadly used. The testing methods for regulatory purposes need to respond to strict internationally recognized standards that are developed under the OECD Test Guideline programme.
These Guidelines are elaborated with the assistance of experts from regulatory agencies, academia, industry, environmental, and animal welfare organizations and are published as “Series on Testing and Assessment”, to measure the potential effects of chemicals on human health and the environment.
We summarize in Table 1 the ones where the chemical safety is assessed using zebrafish.
Table 1. Current OECD guidelines using Zebrafish Model. Publicly consultable at https://www.oecd-ilibrary.org/.
In this framework, the OECD TG 236, based on the use of fish embryos, has been an important innovation since it provides a predictivity similar to the testing in adults fish (AFT) responding, at the same time, to the REACH and EU (Directive 2010/63/EU) requirement for reducing the animal testing. This test method is already used as weight-of-evidence under REACH Regulation.
Fish Embryo Acute Test (FET)
3Rs-aligned method for fast and high throughput acute toxicity assessment in vivo.
In addition to the listed TG, several other guidance documents rely on the use of zebrafish.
The OECD is also considering including thyroid endpoints for fish test guidance to improve them and be even more informative (Project 2.64 OECD TG WORK PLAN).
In 2017, the OECD published a conceptual framework for Testing and Assessment of Endocrine Disrupting Chemicals (OECD Revised Guidance Document 150 on Standardized Test Guidelines for Evaluating Chemicals for Endocrine Disruption) where fish test guidance represents the majority of non-mammalian testing methods (levels 3,4,5).
We would like to underline that the data generated in accordance with OECD Testing and GLP standards are accepted from all member countries (38 on the current date) for the purpose of safety assessment (Mutual Acceptance of Data).
As presented in the previous report, ZeClinics set its quality standards on OECD principles to be authorized as a GLP test facility. A Quality Management System has been put in place to control organizational processes and conditions under which non-clinical health and environmental studies will be conducted.
Advances of zebrafish in the regulatory context
In addition to its consolidated role in environmental toxicology, zebrafish is currently one of the remarkable new alternative methodologies (NAMs) for new golden standard methods in the regulatory context.
The model constitutes the experimental system of a validation study called ZEOGRT (project 2.59 ”New Test Guideline on Zebrafish Extended One Generation Reproduction Test” OECD WORK PLAN) expected to provide a new test guideline by 2023. Additionally, the OECD Expert Group on Developmental Neurotoxicity, of which ZeClinics is part, is drafting a new guidance document (Project 4.124: New Guidance Document on Developmental neurotoxicity (DNT) in vitro assays), as explained in the following section.
Zebrafish is also crucial for an innovative testing strategy called Integrated Approaches to Testing and Assessment (IATA). The IATA, under the OECD umbrella, is a new pragmatic, science-based approach for chemical hazard characterization that relies on an integrated analysis of existing information coupled with the generation of new information using alternative testing methods. IATA can include multiple and different methodological approaches [(Q)SAR, read-across, in chemico, in vitro, ex vivo, in vivo] or omic technologies.
This kind of integrated approach improves interspecies translation and toxicity prediction: zebrafish-based assays can be used to reduce the uncertainties of in vitro assays and increase the sensitivity of the proposed test battery. As a whole organism assay, zebrafish offer mechanistic understanding in addition to behavioral and pathology endpoints.
Additionally, the Adverse Outcome Pathway (AOP) concept, formalized by OECD in 2012, can be applied as a framework to develop IATAs. AOPs are a conceptual scheme for organizing, synthesizing, and presenting scientific knowledge regarding the linkage between the perturbation of a specific biological target, pathway, or process by a stressor and a consequent adverse outcome(s) considered relevant to risk assessment, regulatory decision-making, and/or environmental management. The AOP Wiki is a publicly available tool that serves as the primary repository of qualitative information and it serves as one component of a larger OECD-sponsored AOP Knowledgebase (AOP-KB) effort. Also there, zebrafish confirms its relevant contribution: several AOPs (also WNT Endorsed) include zebrafish data for hazard and risk assessment of stressors.
Last but not least, in February 2020 a new version of the ICH S5 (R3) guideline  was published which described the qualification and use of alternative methods for embryo-fetal developmental (EFD) toxicity assessment of chemicals intended for human use.
This step definitively constitutes the entrance of NAMs into the pharmaceutical sector to screen and assess the teratogenicity of candidate drugs for regulatory acceptance.
In 2021, a validation study for the application of the Zebrafish Developmental Toxicity Assay for pharmaceuticals under ICH S5 (R3) was published .
Other laboratories have recently tested the compound list proposed in the guidance and they already confirmed the very high sensitivity and specificity of the zebrafish assay to predict mammalian embryotoxicity exposure.
More studies in the world are currently being conducted to definitively confirm the zebrafish as a preclinical model for safety studies during drug development.
 13 April 2003 , By Dr Paul Goldsmith and Roberto Solari , Spring 2003.
 Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (Text with EEA relevance)Text with EEA relevance.
 OECD (2018), Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption, OECD Series on Testing and Assessment, OECD Publishing, Paris. https://doi.org/10.1787/9789264304741-en.
 ICH S5 (R3) guideline on reproductive toxicology: Detection of Toxicity to Reproduction for Human Pharmaceuticals.
 Song YS, Dai MZ, Zhu CX, Huang YF, Liu J, Zhang CD, Xie F, Peng Y, Zhang Y, Li CQ, Zhang LJ. Validation, Optimization, and Application of the Zebrafish Developmental Toxicity Assay for Pharmaceuticals Under the ICH S5(R3) Guideline. Front Cell Dev Biol. 2021 Sep 14;9:721130. doi: 10.3389/fcell.2021.721130.
By Miriam Martínez
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 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. In 2021, she joined ZeClinics with a branding and marketing strategy focus.