Animal Testing Pros and Cons in Preclinical R&D: How Teams Reduce Mammalian Use Without Losing Translational Confidence

Every year, millions of animals are used in preclinical research, and yet more than 90% of drugs that pass those tests still fail in humans. Something is not adding up. As regulators in the US, UK, and EU begin to question the dominance of mammalian models, a quiet shift is underway: one that is forcing the industry to rethink not just which models it uses, but why it has relied on them for so long —and what comes next.

Animal Testing Remains a Regulatory and Scientific Requirement in Drug Development?

In short, yes. Animal testing remains the gold standard in the preclinical stages of the drug discovery and development pipeline from a regulatory standpoint. Most major agencies, including the European Medicines Agency (EMA), the US Food and Drug Administration (FDA), and other international authorities aligned with ICH guidelines, still require in vivo data before a new drug can enter human clinical trials. These requirements primarily assess systemic toxicity, pharmacokinetics, reproductive effects, carcinogenicity risks, and safety margins that cannot yet be fully predicted by in vitro or computational models alone. 

Nevertheless, the landscape is evolving. The US Senate has recently cleared the FDA Modernization Act 3.0. The Act directs the FDA to revise regulatory language that still defaults to animal testing, replacing it with the broader term “nonclinical methods.” This aligns regulations with how preclinical research is already being conducted in many fields and removes a key regulatory barrier to the adoption of New Approach Methodologies (NAMs). It follows the FDA roadmap announced in April 2025 to make animal studies the “exception rather than the norm” in drug safety and toxicity. During the first quarter of 2026, the EMA is expected to publish a similar roadmap to reduce animal testing. It will follow the initiative from the UK government published in November 2025 to phase out animal testing, including skin irritation tests in 2026.  

Although the use of animals in research remains a common practice, Directive 2010/63/EU has been in force across the European Union since 2010. This regulation governs the use of animals for scientific purposes and requires the application of the 3Rs principles: Replacement, Reduction, and Refinement. One of its core principles is that no procedure may be carried out if a scientifically validated alternative method exists that can achieve the same result without the use of animals.

Scientific and Operational Limitations of Traditional Mammalian Models

From a scientific perspective, the main challenge lies in species translation. Although mammals share substantial biological similarities with humans, relatively small differences in metabolism, gene expression, immune responses, or physiology can significantly alter how a drug behaves. Many drug candidates that appear safe or effective in animals ultimately fail in human clinical trials, illustrating that traditional animal models do not always reliably predict clinical outcomes. This translational gap reduces their predictive value and contributes to the high attrition rates observed in late-stage drug development.

It can also happen the other way around. Potential therapies may never reach patients because they fail in preclinical studies. Paracetamol is a great example to illustrate this idea. It is toxic to dogs, which means that if tests had been performed in this species, it would have never been developed further. 

From an operational point of view, studies involving mammals are time-consuming and costly. Maintaining animal colonies, complying with regulatory requirements, conducting long-term toxicology studies, and analyzing outcomes can take months or years, slowing decision-making during critical stages of drug discovery. This pace contrasts with NAMs, such as zebrafish, which can generate comparable datasets in substantially shorter timeframes, reducing the number of candidates that advance to subsequent phases.

Scalability represents another important limitation. Mammalian studies usually involve relatively small sample sizes due to ethical and financial constraints, which reduces statistical power and limits the ability to test multiple variables simultaneously. By comparison, automated in vitro and in vivo platforms allow the evaluation of thousands of compounds or experimental conditions in parallel, which is impossible with traditional animal studies.

Beyond the Debate: In VitroSystems, Ex VivoModels, and Emerging In VivoAlternatives

Advanced in vitro, ex vivo, and alternative in vivo models are rapidly expanding to address the ethical, cost, and scientific limits of mammalian animal testing. These alternatives to animal testing aim to better recapitulate human biology, improve prediction of drug response, and support 3Rs-driven reduction of animal use.

In vitro systems 

Modern in vitro platforms now extend far beyond 2D cell lines to include 3D cultures, organoids, and microphysiological systems (MPS), often derived from human induced pluripotent stem cells (iPSCs) or primary cells. These systems improve metabolic competence, tissue architecture, and long‑term function, enabling more predictive studies of ADME, toxicity, and disease mechanisms in liver, intestine, kidney, brain, and other organs. 

Ex vivo models 

Ex vivo approaches use intact human or animal tissues, such as intestinal explants, cryopreserved mucosa, or tissue slices kept vital in a controlled laboratory environment. They preserve native multicellularity, extracellular matrix, and immune components, offering highly realistic barriers (e.g., intestinal, vaginal, reproductive) and tumor microenvironments for drug absorption, toxicity, and efficacy testing. 

In vivo alternatives

Non‑mammalian organisms (Danio rerio, Caenorhabditis elegans, Drosophila, Galleria mellonella) and other alternative animals are increasingly used for in vivo screening and predictive toxicology due to low cost, high throughput, and substantial genetic homology to humans. These systems are particularly valuable for target validation, early toxicity, and disease modeling before moving to mammals. 

Zebrafish: A 3Rs-Based Strategy to Reduce Mammalian Use Without Compromising Translational Confidence

Among the non-mammalian in vivo alternatives currently available, zebrafish (Danio rerio) stand out as one of the most scientifically validated and operationally mature options for preclinical drug development. 

From a genetic standpoint, approximately 70% of human genes have at least one zebrafish ortholog, and around 84% of human disease-related genes are conserved in this species. This level of homology, combined with their vertebrate biology, means that zebrafish studies capture organ-level and systemic interactions that cell-based models simply cannot replicate. At the same time, their small size, external development, and high reproductive capacity, with up to 300 eggs per spawning and a new clutch every 10 days, make them compatible with high-throughput workflows. Hundreds of embryos can be screened in parallel in multi-well plate formats, combining the scalability of in vitro assays with the physiological complexity of a living organism.

Summary of the advantages of Danio rerio as an animal model in research. Adapted from: Fontana, C.M., Van Doan, H. Zebrafish xenograft as a tool for the study of colorectal cancer: a review. (2024). Cell Death Dis. 15:23.

From an ethical and regulatory perspective, EU Directive 2010/63 does not classify zebrafish embryos as protected animals until 5 days post-fertilization, providing an additional advantage for early-stage, high-throughput experimentation. This makes zebrafish particularly well-aligned with the 3Rs framework. 

Zebrafish are not positioned as a replacement for mammalian models but as a strategic complement that strengthens the preclinical package. By integrating zebrafish between in vitro screening and rodent studies, development teams can generate whole-organism toxicity and efficacy data, covering cardiac, hepatic, neurological, or developmental endpoints, within days and at a fraction of the cost. This early, physiologically relevant evidence supports better go/no-go decisions, reduces late-stage attrition, and helps ensure that only the most promising candidates advance to resource-intensive mammalian studies.

Zebrafish possess a translation track record that justifies their inclusion in the drug discovery and development pipeline. Indeed, the inclusion of zebrafish could bring multiple benefits, ultimately reducing the preclinical-clinical gap:

1. Eliminating species blind spots: No single animal perfectly mimics human biology. Combining diverse species (like rodents and zebrafish) triangulates data, increasing confidence that the drug's mechanism will translate to humans.

2. Capturing biological complexity: Different models answer different questions. Layering cellular assays (in vitro) with whole-body systems (in vivo) builds a complete picture of how the drug behaves systemically.

3. Improving PK/PD predictions: Because drugs are metabolized differently across species, using multiple models provides richer data. This allows for highly accurate predictions of human dosing and clearance rates.

4. "Failing fast" on toxicity: Early screening in high-throughput models catches critical flaws like cardiotoxicity right away. This weeds out unsafe compounds before you spend millions on mammalian trials.

5. Strengthening regulatory packages: Agencies like the FDA strongly encourage modern, multi-model approaches. A diverse data package builds a much more convincing and resilient Investigational New Drug (IND) submission.

Compounds validated in zebrafish have advanced into clinical trials across multiple therapeutic areas, including oncology, neurology, hematology, rare diseases, and immunology. These examples reflect a growing body of evidence that zebrafish data can reliably inform and accelerate clinical development programs. As regulatory frameworks continue to evolve toward embracing NAMs, zebrafish are well-positioned to become a standard component of the modern preclinical toolkit.

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Fontana CM, Van Doan H. Zebrafish xenograft as a tool for the study of colorectal cancer: a review. Cell Death Dis. 2024 Jan 9;15(1):23. doi: 10.1038/s41419-023-06291-0.

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By Javier Terriente

Javier is the co-founder of ZeClinics and ZeCardio Therapeutics, two biotech firms specializing in zebrafish-based preclinical drug discovery for cardiovascular, neural, and toxicology applications. He combines scientific leadership with business acumen, having successfully driven fundraising efforts and strategic partnerships.

Currently leading scientific efforts at ZeCardioTx (and formerly CSO at ZeClinics), Javier also serves on the Board of Directors of AseBio, where he advocates for industry collaboration. His academic background includes a PhD in Molecular Biology and a Marie Curie Fellowship. Recognized as an expert in zebrafish models, he has published extensively and has supervised five industrial PhD theses.