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Chemicals such as pesticides, biocides, pharmaceuticals, or industrial chemicals enter water bodies, where they can impair the growth, development, and reproduction of fish. Even in early stages of development, subtle disturbances in hormone balance or metabolism can have long-term consequences for entire populations. Classic long-term tests with fish provide robust data, but they are time-consuming, costly, and involve the use of large numbers of vertebrates. Against the backdrop of regulatory requirements and the 3R principles of replacement, refinement, and reduction of animal testing, there is a growing need for alternative strategies that provide early indications of potential hazards without further increasing the number of laboratory animals used.

Dr. Sebastian Eilebrecht, Head of the department Exotoxicogenomics at Fraunhofer IME, and his colleagues are using toxicogenomics to close this gap. Toxicogenomics describes the comprehensive analysis of gene expression changes caused by chemical exposure. In fish embryo models, this allows characteristic “molecular fingerprints” to be detected at an early stage, indicating harmful mechanisms of action even before classic apical toxicological effects become apparent. Working with zebrafish embryos is considered 3R-compliant because these developmental stages are not yet classified as protected, independently feeding animals under EU animal welfare legislation. This approach combines modern molecular biology methods with ecotoxicological expertise and enables precise, 3R-compliant screening of environmental chemicals.

The focus is on mechanistic screening of modes of action (MoA), which reveals a wide variety of substance-specific interventions in the organism's central regulatory circuits. Many chemicals can impair the reproduction, development, or behavior of fish in the long term without immediately leading to increased mortality but still affecting populations in the long term.

In sublethal zebrafish embryo tests, we exposed reference substances with defined action profiles and used transcriptomics to record their characteristic gene expression patterns. We assign these molecular fingerprints to steps within adverse outcome pathways (AOPs) – from initial receptor binding or enzyme inhibition to cellular and organ-related changes. Endocrine disruptors serve as a prominent example: they attack estrogen/androgen receptors or enzymes involved in thyroid hormone synthesis, for example, thereby distinguishing estrogen- and androgen-receptor-mediated effects and disturbances in thyroid hormone homeostasis from one another (publications 1-3). These can then be linked to AOP building blocks such as altered gonadal development, hatching rates, or swim bladder function. Phenotypic parameters recorded in parallel help to link the causal chains visible in gene expression with organismic endpoints.

Molecular fingerprints as a screening tool

The transcriptome data generated in the course of this work was compiled in a central database of toxicogenomic fingerprints. This database contains characteristic gene expression patterns for numerous reference substances that are linked to specific mechanisms of action and AOPs. New substances can be compared with this database based on their expression profiles and assigned to known types of effects.

A practical example: If a new chemical shows an expression pattern in a zebrafish embryo test that closely resembles a known estrogen receptor-mediated fingerprint, it is reasonable to suspect that it is a potential endocrine disruptor. Conversely, a profile that shows no clear matches with established problematic mechanisms of action can help to lower the risk assessment of a substance and focus resources on more critical candidates.

This approach enables broad-based, mechanistically sound screening early in the chemical development process. Companies can test substance libraries before costly animal studies or large-scale environmental studies become necessary. Substances with distinct fingerprints can be sorted out early on or investigated further, while indistinct candidates can be given higher priority in downstream development steps.

tPOD: Early warning thresholds for chronic toxicity

In addition to the qualitative classification of mechanisms of action, the derivation of quantitative thresholds plays a central role. In the ECHA project E-FET, we are further developing the transcriptomic point of departure (tPOD) for this purpose – a concept that identifies the concentration at which systematic, concentration-dependent changes in gene expression occur in zebrafish embryos, thus providing an early molecular warning point for chronic toxicity. In an extended Fish Embryo Test (E-FET), embryos are exposed to industrial chemicals across several concentration levels, after which the RNA is sequenced and modeled for numerous genes to determine how strongly their expression increases or decreases depending on the concentration. Benchmark doses are derived from these concentration-response relationships, and the tPOD is determined as a conservative low range of the BMD distribution (publication 4).

Systematic comparison with classic Fish Early Life Stage (FELS) studies shows that substances that cause significant effects on growth or survival in long-term tests usually also produce clear, concentration-dependent gene expression changes, and thus tPOD signals in the E-FET. While substances without chronic FELS effects generally do not show relevant tPOD responses. tPOD values are often in the same order of magnitude as NOEC values (No Observed Effect Concentration) from OECD TG 210 studies and often are conservative. Borderline cases—such as poorly soluble or unstable chemicals with exposure that is difficult to control—highlight the importance of careful exposure control and, where necessary, optimized test systems. Overall, the experience gained from the E-FET project to date suggests that, with well-controlled exposure, tPOD values are a robust measure of threshold concentration for chronic toxicity and are suitable for planning subsequent tests and prioritizing substances in risk assessment.

Added value for regulation, industry, and the environment

The combination of mechanistic effect screening and tPOD derivation offers a wide range of applications. For regulation, it opens up the prospect of basing decisions with greater emphasis on mechanistic evidence while reducing the number of laboratory animals used. In future testing strategies, toxicogenomic endpoints could help shorten test batteries, avoid redundant studies, and make the planning of long-term studies more targeted.

For industry, the approach provides a powerful tool for early risk assessment. Even in the substance design phase, candidates with problematic fingerprints can be identified and sorted out before high development investments are made. At the same time, tPOD values can help to define realistic but precautionary concentration ranges for further testing. This saves resources and increases planning reliability.

The toxicogenomic approach also offers clear advantages from an environmental and animal welfare perspective. The use of embryo models, combined with highly informative molecular measures, allows for a significantly more efficient use of biological resources. Fewer animals provide more information, and many questions can be addressed at very early stages of development without using legally restricted fish.

In the coming years, toxicogenomics will continue to evolve from a primarily research-driven approach to an integral part of regulatory testing strategies. Ongoing work at Fraunhofer IME shows how molecular fingerprints and tPOD concepts can be specifically integrated into assessment processes. At the same time, we are expanding collaborations with authorities, industry, and academic partners in order to harmonize methods, expand reference data, and demonstrate use cases.

In the long term, a situation could arise in which toxicogenomic data from fish embryo models is sufficient for certain groups of substances, making it possible to dispense with classic long-term studies or significantly reduce their scope. This requires a solid evidence base showing that tPOD values and mechanistic profiles reliably protect against chronic effects. The results so far are promising and underscore the potential for effectively combining ecological risk assessment and 3R principles.

With our toxicogenomic program, we are making an important contribution to the development of innovative, sustainable, and scientifically sound strategies for the protection of aquatic ecosystems, and to the sustainable design of chemical regulation and drug development.