Reproductive Toxicity Assessment in Zebrafish

Reproductive Toxicity Assessment of Pharmaceuticals

Identifying reproductive toxicity risks early is crucial for pharmaceutical development. Regulatory guidelines (ICH S5(R3) for human drugs and VICH GL43 for veterinary drugs) mandate reproductive toxicity assessments before clinical trials or market approval. However, traditional mammalian models are costly and time-consuming.

Our zebrafish-based assay provides a fast, high-throughput, and regulatory-aligned alternative for evaluating fertility, mating performance, and endocrine disruption effects. By bridging the gap between in vitro assays and full regulatory studies, we help you de-risk candidates sooner and optimize preclinical development.

Two Services, One Complete Solution

Our Reproductive Toxicity assay complements the Developmental Toxicity service for an Extended One-Generation DART evaluation.

Reproductive Toxicity Assessment of Agrochemicals and Industrial Chemicals

Our Reproductive Toxicity assay also supports endocrine disruption assessment for pesticides, biocides, and—soon—industrial chemicals.

In 2018, EFSA and ECHA published guidance on identifying endocrine-disrupting properties in pesticides and biocides (Regulation EU No 528/2012). This framework follows a tiered testing approach:

✔ Level 1: Review of existing data and in silico approaches.
✔ Level 2: Battery of in vitro assays.
✔ Level 3: In vivo assays assessing endocrine mechanisms.
✔ Level 4: In vivo assays evaluating adverse effects across life stages.
✔ Level 5: In vivo assays assessing long-term effects over full life cycles or generations.

The Fish Short-Term Reproduction Assay (OECD TG 229) is a Level 3 in vivo assay required for endocrine disruption testing.

Although regulatory frameworks for industrial chemicals are still under development, Level 3 assays will soon be required for industrial chemical registration.

Our zebrafish-based assay aligns with OECD TG 229, providing a cost-effective, high-throughput solution for endocrine disruption testing in agrochemical and industrial chemical development.

Additionally, the development and reproductive capacity of offspring can be assessed in an extended version of the assay (Two-Generation DART available upon request).

Why Choose Our Zebrafish-Based Reproductive Toxicity Assay?

Regulatory-aligned with ICH S5(R3), VICH GL43, and OECD TG 229.

Faster and more cost-effective than traditional mammalian models.

Bridges the gap between in vitro assays and full regulatory studies.

High-throughput screening for early de-risking of candidate compounds.

Method description

1. Solubility test

• Ensures the test compound does not precipitate.
• Confirms treatment at the intended concentration.

2. Dose-Range-Finding

• Zebrafish adults are exposed to the compound for a week.
• Mortality is assessed at the end of the exposure.
• The Benchmark Dose (BMD) is calculated to define the concentration range for reproductive toxicity testing.

3. Reproductive Toxicity

• Sexually mature zebrafish (males and spawning females) are exposed to three concentrations of the test compound for 21 days.
• Male and female fertility is evaluated, considering effects on egg production, fertility rate (embryo viability), and reproductive organs.
• Mating performance is also assessed based on spawning events during the exposure.
• (OPTIONAL) Development and reproductive capacity of offspring (Two-Generation DART available upon request).

Readouts

• Daily fish survival and well-being – Monitors overall health throughout the assay.
• Growth tracking – Weekly body weight and length at exposure's beginning and end.
• Egg Production – Measures laid eggs as a measure of reproductive capacity.
• Fertility Rate – As another measure of reproductive capacity, it measures the embryo viability (the number of live eggs out of the total) at 24 hpf.
• Number of spawning events – As a measure of mating performance.
• Histological analysis of gonads – Identifies structural alterations in reproductive tissues.
• Vitellogenin Analysis (ELISA) – A well-established bioindicator of estrogen-mediated endocrine activity.
• Gonadosomatic Index (GSI) – Assesses gonad maturation and serves as an indicator of endocrine disruption effects.

Figure 1. Gonadosomatic index (GSI (%) determined in adult male and female zebrafish exposed to two different concentrations of 17β-estradiol and control (DMSO). GSI = (Gonad weight/body weight)x100. A significant increase were measured after 17β-estradiol exposure suggesting an endocrine disruption affecting gonadal function. In females reflect an induction of vitellogenesis and an increase of mature oocytes. In males increase of gonadal mass as potential edemas and/or disruption of normal tissue development.

Figure 2. 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 of spermatid cell count.

Figure 3. 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 (LO). Estradiol induces an increase of LO follicle number.

References

1. OECD. 2012. Test No. 229: Fish Short Term Reproduction Assay.