Zebrafish in Developmental Toxicity Screening for Pharmaceuticals

The detection of reproductive and developmental toxicity is a critical step in preclinical drug development, ensuring that pharmaceuticals are safe for human use, particularly during pregnancy. The ICH S5 (R3) Guideline provides a comprehensive framework for assessing these risks, traditionally relying on mammalian models such as rats, mice, and rabbits. However, advances in research models and a growing emphasis on the 3Rs principle (Replacement, Reduction, and Refinement) open new avenues for alternative approaches, including the use of the zebrafish model. 

This versatile organism offers unique advantages that align with the guidelines' objectives while minimizing the use of higher-order animals. In this article, we explore the ICH S5 (R3) framework, the current reliance on mammalian models, and how zebrafish can complement this process to improve efficiency, cost-effectiveness, and ethical compliance.

Understanding Developmental Toxicity in Drug Development

Developmental toxicity refers to any adverse effect on a developing organism that results from exposure to a substance before conception (via either parent), during prenatal development, or postnatally until sexual maturity. These effects can manifest as structural malformations, growth retardation, functional impairments, or embryo-fetal lethality.

In the context of the drug development process, it is estimated that developmental and reproductive toxicity issues are responsible for more than 10% of preclinical toxicology-related attrition. Here, alternative models such as zebrafish can play an essential role. Zebrafish data can provide predictive insights into teratogenic potential, allowing for more informed and efficient preclinical pipelines.

Overview of the ICH S5 (R3) Guideline and Its Role in Toxicity Screening

The ICH S5 (R3) Guideline is a regulatory guideline that gives recommendations on the strategy of developmental and reproductive toxicity testing of pharmaceutical compounds.  

 It provides a standardized framework to evaluate:

• Fertility and Early Embryonic Development: Assessing the impact on gamete production, fertilization, and preimplantation stages.
• Embryo-Fetal Development (EFD): Identifying potential teratogenic effects during organogenesis.
• Pre- and Postnatal Development (PPND): Evaluating adverse outcomes from pregnancy to offspring maturity.

The guideline outlines testing approaches using rodents (rats, mice) and non-rodents (rabbits) to achieve these endpoints. While effective, these predictive in vivo models involve high costs, lengthy timelines, and ethical considerations related to animal use.

→ Check out the purpose of ICHS5(R3) studies for evaluating pharmaceuticals for reproductive and developmental toxicity.

Why Zebrafish Are Valuable for Reproductive and Developmental Toxicity Screening

Traditional mammalian models, while valuable, are costly, slow, ethically controversial, and poorly suited for high-throughput screening, which limits their efficiency in early drug development.

These challenges underscore the need for innovative approaches, such as the zebrafish model, to complement traditional methods.

Although not yet included in the ICH S5 (R3) Guideline, zebrafish present a compelling case as an alternative in vivo model for developmental toxicity studies. Here’s why:

1. Rapid Development and Cost-Effective Maintenance

• Zebrafish embryos develop externally and complete major organogenesis within 72 hours post-fertilization, drastically reducing study timelines.
• Maintenance costs for zebrafish are significantly lower than those for mammals, enabling cost-effective scalability.

2. High-Throughput and Transparent Embryo Screening

• Zebrafish are ideal for screening large libraries of compounds, with hundreds of embryos generated per breeding pair each week.
• Automated imaging systems can analyze developmental defects in zebrafish embryos in a high-throughput format (Figure 1).

Figure 1. Automated phenotype extraction by deep learning to analyze developmental defects in zebrafish embryos. 

3. Ethical Compliance through the 3Rs Principle

• Incorporating zebrafish aligns with the 3Rs principle, reducing the number of mammals used in preclinical research and refining the drug discovery process.
• The transparency of zebrafish embryos allows for non-invasive observation of organ development and teratogenic effects in real-time.

4. Predictive Alignment with Mammalian Models

• Zebrafish share approximately 84% of human disease-related genes, providing relevant insights into the mechanisms of developmental toxicity.
• Studies demonstrate that zebrafish can predict teratogenic effects observed in mammals, supporting their potential as a complementary model. 

In a study carried out by Weiner et al., 32 compounds classified as teratogens or non-teratogens in mammals were analyzed in zebrafish. The 3 negative compounds were properly categorized, while 26 compounds out of the 29 reference ones were classified as teratogenic in zebrafish (Figure 1).

Figure 2. Results obtained by Weiner et al. when analyzing the power of the zebrafish model to predict Malformation or Embryo-Fetal Lethality (MEFL) of 32 compounds classified as teratogens or non-teratogens in mammals. 

The developmental toxicity assay in zebrafish gave a:

• Sensitivity of 89.7% 
• Specificity of 100% 
• Accuracy of 90.6% 
• Repeatability of 100% (three independent blinded biological replicates were performed)

…compared to mammals. Similar results were obtained in a second study performed by Mori et al.:

• Sensitivity of 82% 
• Specificity of 66% 
• Accuracy of 80% 

Therefore, this is a well-integrated strategy using New Alternative Methods to minimize the use of animals in developmental toxicity studies.

Applications of Zebrafish in Reproductive and Developmental Toxicology

Zebrafish are particularly effective for early-stage screening of:

• Teratogenicity: Identifying morphological defects caused by chemical exposure during embryogenesis.
• Endocrine Disruption: Evaluating the impact of compounds on hormonal pathways affecting fertility and reproduction.
• Dose-Response Relationships: Assessing the concentration at which developmental toxicity is triggered.

By using zebrafish as a preliminary screening tool, researchers can identify the most promising compounds for further testing in mammalian models, narrowing down candidates and reducing overall resource consumption.

At ZeClinics, we have developed a zebrafish developmental toxicity assay that is structured to maximize data extraction within the first five days post-fertilization (dpf), before ethical limitations apply under EU law. 

The assay involves exposing zebrafish embryos to the test compound starting at the blastula stage (typically around 6 hours post-fertilization) and monitoring their development until 5 dpf. A solubility test and a Dose Range Finding assay are previously performed to define the concentration range for developmental toxicity testing.

Once the concentration range is defined, 17 teratogenic phenotypes are analyzed using automated deep-learning-based phenotype extraction for morphological abnormalities, delays in growth, edema, cardiac function, and viability. Lethal Concentration 50 (LC50), Effective Concentration 50 (EC50 - for the most sensitive phenotype), and the Teratogenic Index (TI) are calculated to predict the teratogenic potential of test compounds.

Figure 3. Phenotypical panel: examples of developmental defects in embryos exposed to different toxic compounds

→ This assay can be complemented with the zebrafish Reproductive Toxicity service for full ICH S5(R3) coverage.

Conclusion

The ICH S5 (R3) Guideline lays the foundation for rigorous developmental and reproductive toxicity testing. While zebrafish are not yet recognized within this framework, their unique features make them a valuable complementary model for early-stage screening.

By integrating zebrafish in preclinical drug discovery, pharmaceutical companies can:

• Reduce costs by up to 60%.
• Save time by up to 40%.
• Align with ethical principles by minimizing mammalian use.

Zebrafish do not replace mammalian models but provide a critical intermediary step to streamline drug discovery processes. As the demand for ethical, efficient, and scalable models grows, zebrafish are poised to play an increasingly prominent role in preclinical research.

References

Brannen KC, Chapin RE, Jacobs AC, Green ML. Alternative Models of Developmental and Reproductive Toxicity in Pharmaceutical Risk Assessment and the 3Rs. ILAR J. 2016 Dec;57(2):144-156. doi: 10.1093/ilar/ilw026.

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

Weiner AMJ, Irijalba I, Gallego MP, Ibarburu I, Sainz L, Goñi-de-Cerio F, Quevedo C, Muriana A. Validation of a zebrafish developmental defects assay as a qualified alternative test for its regulatory use following the ICH S5(R3) guideline. Reprod Toxicol. 2024 Jan;123:108513. doi: 10.1016/j.reprotox.2023.108513. Epub 2023 Nov 26.Mori K, Aoki Y, Mikashima F, Maki K, Tanaka T, Hayashi M, Sugimoto W, Ono M, Umekita S, Niino T, Fujiwara M, Ebata T, Hirata H, Kojima H. Validation of a new protocol for a zebrafish MEFL (malformation or embryo-fetal lethality) test method that conforms to the ICH S5 (R3) guideline. J Toxicol Sci. 2024;49(8):337-348. doi: 10.2131/jts.49.337.

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.