Reproductive Toxicity Assessment Using Zebrafish: Enhancing Early-Stage Drug Development with Predictive In Vivo Models

The ICH S5(R3) guideline sets international standards for assessing reproductive and developmental toxicity in pharmaceuticals. While it emphasizes in vivo mammalian studies, it also recognizes the value of alternative models. Qualified alternative assays, including non-mammalian in vivo systems like zebrafish, can support hazard identification and, in certain contexts, defer definitive mammalian studies when used as part of an integrated strategy.

As an alternative to traditional mammalian models, zebrafish have emerged as a compelling in vivo system for the pharmaceutical industry's developmental and reproductive toxicity testing.  Their biological proximity to mammals, transparency during early development, and rapid lifecycle make them particularly suitable for high-throughput toxicity testing.

ICH S5(R3): Evaluating Pharmaceuticals for Developmental and Reproductive Toxicity

What is the ICH S5(R3)?

ICH S5(R3) is a regulatory guideline that provides a framework for assessing the potential risks of pharmaceuticals on reproductive and developmental health. It establishes criteria for evaluating developmental and reproductive toxicity (DART) and is essential for regulatory submissions in drug development.

When Does ICH S5(R3) Apply in the Drug Development Process?

Developmental and reproductive toxicity studies are conducted during the preclinical phase for pharmaceuticals that could impact reproduction and development. These include:

• Drugs absorbed systemically
• Compounds affecting hormonal pathways
• Gene and biological therapies
• Vaccines in specific cases

ICH S5(R3) studies form a critical part of the safety data package required by regulatory agencies such as the FDA and EMA for:

• Investigational New Drug (IND) applications are necessary before initiating human clinical trials (Phase I).
• Marketing Authorization Applications (MAA) are required when seeking drug approval for commercialization.

Purpose of ICHS5(R3) Studies

ICH S5(R3) studies aim to assess potential adverse effects on:

• Fertility
• Embryonic and fetal development
• Pre-/Postnatal development

Developmental toxicity involves the study of embryonic and fetal development, focusing primarily on malformations or embryo-fetal lethality (MEFL). This can be evaluated by directly exposing fertilized eggs and embryos to test compounds. According to ICH S5(R3), new alternative methods can be qualified for MEFL risk assessment and prediction. This opens the door to incorporating alternative models such as zebrafish embryos, which offer significant advantages over mammalian models in terms of throughput, cost-efficiency, and ethical considerations.

When assessing the impact of compounds on adult fertility and the pre- and postnatal development of their offspring, we refer to reproductive toxicity. Male and female fertility are evaluated using parameters such as egg production, egg viability (reflecting gamete function and fertilization), embryo viability, and reproductive organ health. Molecular markers, including vitellogenin (Vtg) expression and the gonadosomatic index, are useful for detecting endocrine-disrupting activity. These outputs are assessed through phenotypic scoring, gonadal histology (Figures 1 and 2), and gene expression analysis. Mating performance is evaluated independently, based on the frequency and success of spawning events during exposure.

Pre- and postnatal development studies may also involve monitoring the development and reproductive capacity of offspring from exposed adults, assessing whether these offspring are viable and if they can successfully reproduce once they reach maturity.

Figure 1. Histological observations of male gonads of zebrafish exposed to DMSO or 17β-estradiol for 21 days. Quantification of spermatid cells normalized against the area. Estradiol induces a reduction in spermatid cell count.

Figure 2. Histological observations of female gonads of zebrafish exposed to DMSO or 17β-estradiol for 21 days. Quantification of different follicle stages normalized against the area. Pre-vitellogenic oocyte (PO), Vitellogenic oocyte (VO), and the late or post-vitellogenic oocyte (LO). Estradiol induces an increase of LO follicles.

Zebrafish in Reproductive Toxicity Testing: Advantages and Limitations

Zebrafish are a model organism that offers significant benefits in male and female reproductive toxicity studies. They share many organs and tissues with humans, including reproductive systems with ovaries and testes, regulated by endocrine and paracrine signals. Their external fertilization and transparency enable direct observation of female gametogenesis, egg fertilization, and embryonic development. In addition, embryogenesis is completed in the first 72h, allowing for rapid and cost-effective screening of numerous compounds in developmental toxicity. This timing is crucial, as it precedes the threshold after which zebrafish are considered protected animals under European legislation (Directive 2010/63/EU). Besides their rapid development, their small size and the small amount of the tested compound required are compatible with high-throughput screening approaches, offering a compelling advantage to bridge the gap between in vitro models and in vivo conventional mammalian assays, which are laborious, costly, and time-consuming.

Nonetheless, some limitations are present. Unlike mammals, zebrafish lack a placenta, which means certain maternal-fetal interactions cannot be studied. Their aquatic environment also introduces variability in how the drugs are exposed (direct versus indirect), which can affect drug absorption and bioavailability. Despite these differences, zebrafish assays remain highly predictive, especially when used alongside mammalian data. 

Integrating Zebrafish Reproductive Toxicity Data into Early Drug Development

When robustly validated, zebrafish assays are particularly useful in the early stages of the drug development process to flag potential adverse effects on reproduction and embryonic development. Their use can reduce animal consumption and improve the mechanistic understanding of compound toxicity. However, these models are not yet considered a full replacement for regulatory purposes and must be complemented by mammalian data to support clinical trial authorization. They must be seen as a bridge between in vitro models and mammalian ones, aligned with ICH guidelines. 

The ICH guideline permits flexibility, allowing pharmaceutical developers to use alternative models to support decision-making before Phase 3, especially when combined with preliminary embryo-fetal development (pEFD) data from mammalian species. This progressive stance opens the door for zebrafish assays to gain further traction as regulatory authorities seek to balance scientific rigor with the 3Rs principles in drug discovery. 

Incorporating zebrafish reproductive toxicity data into early-stage drug development research offers multiple strategic advantages. These models enable rapid and cost-effective triage of drug candidates before advancing to more resource-intensive studies. By identifying compounds that disrupt key reproductive pathways, such as hormone signaling or gametogenesis, developers can de-risk their pipelines and optimize lead selection.

As regulatory frameworks continue to evolve and place greater emphasis on translational relevance and humane science, zebrafish are poised to play a more central role in reproductive toxicity assessment, which complements developmental toxicity screenings. Their predictive power, scalability, and compatibility with early-phase testing make them indispensable screening solutions for drug discovery. 

At ZeClinics, we have developed a regulatory-aligned Reproduction Toxicity assay for identifying reproductive toxicity risks early in zebrafish. 

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References

He, J. H., Gao, J. M., Huang, C. J., & Li, C. Q. (2014). Zebrafish models for assessing developmental and reproductive toxicity. Neurotoxicology and Teratology, 42, 35–42. https://doi.org/10.1016/j.ntt.2014.01.006

International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. (2020). ICH S5(R3) guideline on detection of reproductive and developmental toxicity for human pharmaceuticals (EMA/CHMP/ICH/544278/1998). European Medicines Agency. https://www.ema.europa.eu/en/ich-s5-r3-guideline-detection-reproductive-developmental-toxicity-human-pharmaceuticals-scientific-guideline

Organisation for Economic Co-operation and Development. (2012). Test No. 229: Fish Short Term Reproduction Assay. OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing. https://doi.org/10.1787/9789264185265-en Van den Bulck, K., Hill, A., Mesens, N., Diekman, H., De Schaepdrijver, L., & Lammens, L. (2011). Zebrafish developmental toxicity assay: A fishy solution to reproductive toxicity screening, or just a red herring? Reproductive Toxicology, 32(2), 213–219. https://doi.org/10.1016/j.reprotox.2011.06.119

By Miriam Martínez

Miriam is a Human Biologist with a strong background in neuropharmacology and a passion for bridging science and innovation. After earning a master’s degree in the Pharmaceutical and Biotech Industry, she completed her PhD in Biomedicine at Pompeu Fabra University (Barcelona), where her research focused on the behavioral analysis of animal models for neurophenotypical characterization. Following her doctoral studies, Miriam transitioned into the healthcare marketing and communication sector, where she played a key role in developing impactful marketing strategies and educational campaigns for leading pharmaceutical brands. She now leverages her scientific expertise, strategic thinking, and creative communication skills in her current role at ZeClinics.