Immunotoxicity Testing with Zebrafish: Detecting Drug-Induced Immune System Effects in Preclinical Safety Screening

What Is Immunotoxicity and Why It Matters for Preclinical Drug Safety

Immunotoxicity refers to any adverse effect a compound has on the structure or function of the immune system, whether or not that compound was designed to act on immunity at all. The immune system is, in fact, one of the common targets of both drug toxicity and drug efficacy: the same pathways that make immunotherapies work are the ones that, when disrupted unintentionally, can suppress or over-activate immune function in ways regulators need to see characterized before approval.

The regulatory framework for evaluating these effects is anchored in ICH S8 (Immunotoxicity Studies for Human Pharmaceuticals), first adopted in 2005 and still the reference guideline used by the FDA, EMA, and PMDA. ICH S8 requires that every new human pharmaceutical be evaluated for immunotoxic potential, but it does not mandate dedicated functional testing (such as TDAR or immunophenotyping) for every compound. It asks sponsors to build a weight-of-evidence case from standard toxicity studies, supplemented with dedicated immunotoxicity studies only when findings, pharmacology, patient population, or structural similarity to known immunomodulators raise a flag. ICH S8 is complemented by ICH S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals.  

In June 2023, the FDA finalized a complementary guidance, Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals, intended to supplement both ICH S8 and ICH S6(R1). It replaces a withdrawn 2002 guidance, broadening that earlier scope to explicitly include pharmaceuticals intended to affect the immune system, biopharmaceuticals, and oligonucleotides. It fills in the endpoints of hypersensitivity and adverse immunostimulation. It adds guidance on assessing carcinogenicity risk from immunomodulators, non-animal methods for dermal sensitization, and effects on pregnancy and developmental immunotoxicity.

Core Immunotoxicity Endpoints: Immunophenotyping, Cytokine Release, and Immunogenicity Assays

Immunotoxicity uses a tiered framework that starts with routine pathology and hematology screening and moves to functional immune assays only when earlier findings raise concern. According to Nandre and Terse (2025): 

• Tier 1 covers hematology, serum globulins, lymphoid organ weight, and histopathology.
• Tier 2 covers immunophenotyping, TDAR, and cell-mediated immunity.
• Tier 3 covers host resistance to infection or tumor challenge.

A parallel set of endpoints addresses the opposite risk, immunostimulation: cytokine release assays, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and cell activation/proliferation assays. 

Because dedicated rodent studies for Tier 2 and Tier 3 are resource-intensive, the field has also been developing New Approach Methodologies (NAMs) to generate immunotoxicity-relevant data earlier and with fewer animals: in silico and AI/ML models, immune organ-on-chip systems (lymph node, bone marrow, skin), and 3D organoid or spheroid cultures. These tools are valuable for mechanism- and target-specific questions, but on their own they lack the systemic, whole-organism context needed to capture effects that depend on organ crosstalk, circulation, and the coordinated response of multiple immune compartments, which is where an in vivo model can still add distinct value.

Zebrafish Models for Immunotoxicity: Faster Detection of Drug-Induced Immune Effects

Zebrafish are a strong preclinical model for immunotoxicity in drug discovery, especially for early-stage screening of whole-organism toxicity, innate immune effects, and mechanistic liabilities before rodent studies. Their main value is combining vertebrate physiology with throughput, live imaging, transgenics, and simultaneous readouts of efficacy, ADME-tox, and off-target tissue effects.

Zebrafish occupy the space between in vitro systems and mammalian models, giving faster and cheaper in vivo screening while preserving organism-level biology that cell culture misses. This positioning supports early elimination of unsafe compounds, prioritization of leads, and iterative medicinal chemistry guided by toxicity mechanisms.

Immunotoxicity is a substantial and established zebrafish toxicology domain, accounting for roughly 11% of the 20,291 zebrafish toxicology publications indexed between 2014 and 2024, the third most-studied category after acute and neurotoxicity. The model is especially useful for early-life immune screening because embryos and larvae are transparent, highly fecund, experimentally tractable, and compatible with high-throughput assays. 

Distribution of publications indexed between 2014 and 2024 among the main toxicity areas evaluated in zebrafish. Source: Lima C., et al. 2026. 

For immunotoxicity specifically, zebrafish are strongest for innate immune biology. For roughly their first three weeks of life, they rely almost exclusively on a fully functional innate immune system; macrophages become phagocytically active around 24–26 hours post-fertilization, neutrophils appear at 48 hpf, while the adaptive immune system only becomes fully functional weeks later. Macrophage functions, leukocyte lineages, key signaling pathways driving immune activation, and cytokine repertoire are well conserved. 

Fluorescent immune-cell lines allow direct in vivo visualization, supporting real-time measurement of inflammatory responses, neutrophil and macrophage behavior, and host-pathogen interactions during compound exposure. 

→ It may interest you: Zebrafish Models for Immunology and Inflammation Research

Integrating Immunotoxicity Screening Earlier in Drug Discovery to Reduce Late-Stage Attrition

The ICH S8 weight-of-evidence approach was designed precisely to catch warning signs early from standard toxicity studies, but those studies are performed using rodents and happen relatively late in a program, after significant investment in chemistry, formulation, and early efficacy work.

Adding an in vivo zebrafish screen earlier in discovery before committing to full rodent immunotoxicity studies creates an additional checkpoint where innate immune liabilities can be flagged while a chemical series is still flexible. A compound that unexpectedly triggers disproportionate inflammatory activation can be deprioritized or structurally modified before it reaches costlier stages of development.

This doesn't replace the mammalian studies that ICH S8 and FDA guidance require for regulatory submission. But as an early, high-throughput, whole-organism filter that is pharmacologically validated against known immunomodulators, zebrafish immunotoxicity screening helps arrive at rodent studies with a better-characterized, lower-risk candidate, reducing both the number of animals used downstream and the risk of late-stage, immune-related attrition.

If you are looking to add immunotoxicity endpoints to your preclinical safety package, or to screen candidates for drug-induced immune effects earlier in your pipeline, ZeClinics can help you. 

References

Belo MAA, et al. Zebrafish as a model to study inflammation: A tool for drug discovery. Biomed Pharmacother. 2021 Dec;144:112310. doi: 10.1016/j.biopha.2021.112310.

FDA. Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals. Guidance for Industry, June 2023. fda.gov.

ICH Harmonised Tripartite Guideline. S8: Immunotoxicity Studies for Human Pharmaceuticals. Step 4 version, 15 September 2005. ICH database.

Lima C, et al. The zebrafish in toxicology: a bibliometric analysis reveals current trends and future avenues for predictive safety assessment. Front Toxicol. 2026 Jan 12;7:1700031. doi: 10.3389/ftox.2025.1700031. 

Nandre RM, Terse PS. An overview of immunotoxicity in drug discovery and development. Toxicol Lett. 2025 Jan;403:66-75. doi: 10.1016/j.toxlet.2024.11.007.

Smith C. The potential of zebrafish as drug discovery research tool in immune-mediated inflammatory disease. Inflammopharmacology. 2024 Jun 26;32:2219-2233. doi: 10.1007/s10787-024-01511-1.

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.