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Challenge 41


Launched Phase 1 awarded Phase 2 awarded Completed

The aim of this Challenge is to develop a suite of innovative, scalable bioassays for key adverse outcome pathways to replace in vivo fish studies in chemical safety screening and regulatory environmental risk assessment.

Challenge Partners collaborate with the NC3Rs to provide additional resources to successful applicants to help deliver the Challenge. The Challenge Partners for this Challenge are DEFRA, the Health and Safety Executive and the Environment Agency. 

Challenges briefing webinar

View the Challenges briefing webinar to find out more about this Challenge.

Challenge launched

Sponsored by AstraZeneca, Bayer AG and Unilever, and in Partnership with DEFRA, HSE and the Environment Agency, this Challenge aims to develop a suite of innovative, scalable bioassays for key adverse outcome pathways to replace in vivo fish studies in chemical safety screening and regulatory environmental risk assessment.


Environmental Risk Assessment (ERA) evaluates the likelihood that the environment may be impacted as a result of exposure to one or more chemical stressors by addressing the relationship between the exposure of a specific environmental compartment to a chemical (e.g. emissions resulting from consumer use or from industrial processes) and the inherent hazard of that chemical (e.g. potential for it to cause harm to relevant species). Traditionally, the ERA framework relies on in vivo testing across three trophic levels: unicellular algae, invertebrates (most commonly Daphnia) and vertebrates (fish). Manufacturers use the concepts that underpin ERA when designing new chemicals, aiming to reduce potential environmental impact, but rigorous safety assessment is still required for regulatory approval.

In vivo fish testing is frequently required for regulatory purposes, but global research efforts into the development and application of New Approach Methodologies (NAMs), broadly defined as any non-animal technology, methodology, approach, or combination thereof, aim to provide improved information on chemical hazard and risk assessment and reduce the reliance on in vivo studies (1,2). For example, the RTgill-W1 cell line assay (3) has been accepted by regulators under OECD (OECD TG 249)  ISO (ISO 21115:2019) to predict fish acute toxicity. NAMs using chemical read-across (4) and biological cross-species extrapolation (5) are also increasingly accepted by regulators to replace in vivo studies. These approaches, however, are limited by their reliance on the identification of an analogue chemical or species for which similar characteristics can be demonstrated and the presence of associated hazard data. These methods can therefore only be used for pathways that are known to be highly conserved across species and lead to a common adverse outcome.

There is a need for the development of innovative NAMs-based testing strategies that can holistically assess the risk and impacts of chemicals on fish and replace their use in safety assessment. These approaches will require:

The AOPs framework collates existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organisation relevant to risk assessment. The use of AOPs can improve toxicity testing by identifying species and endpoint selection, enhancing cross-chemical extrapolation, and supporting the prediction of mixture effects. AOPs can also facilitate the use of molecular or biochemical endpoints (biomarkers) to predict chemical impacts on individuals and populations (6). Using the AOP approach in combination with the development of novel bioassays to interrogate biomarkers could fill data gaps in cases where finding a suitable, data-rich chemical or biological analogue may be not feasible and expand the use of NAMs in ERA.

The Challenge

This Challenge aims to develop a suite of scalable bioassays to permit reliable chemical screening and risk assessment for relevant fish adverse outcomes that can be standardised and, in the future, accepted within global regulatory frameworks. Information generated by these assays coupled with the capability to understand fish internal kinetics (TK) dynamics (TD) of compounds (7), reverse dosimetry calculations, and the use of quantitative in vitro to in vivo extrapolation (qIVIVE) models will permit optimised environmental protection without the need for further animal testing.

Examples of relevant pathways that are in scope are shown in Tables 1 and 2 (pathways in scope are not limited to this list). Development of this technology will represent a major step forward in the use of NAM-based protection of the environment, while maximising the potential to design safer chemicals early in the development pipeline.

Table 1: Examples of fish-specific adverse outcomes. Population level adverse outcome: survival.

Individual level adverse outcomes

Mode of action

Swim bladder inflation

Thyroid signalling


Oestrogen signalling

Lateral line impairment

Oestrogen signalling

Olfactory rosette

Oestrogen signalling

Fin regeneration

Oestrogen signalling

Oocyte maturation


Table 2: Examples of fish-specific adverse outcomes. Population level adverse outcome: reproduction.

Individual level adverse outcomes

Mode of action


Oestrogen signalling

Oestrogen signalling

11 keto-testosterone enzymes

Poor quality sperm

Inhibited synthesis of androgens

Decreased E2 levels/decreased synthesis of VTG


Oocyte maturation


3Rs Benefits

Between 2015 to 2017, approximately 1.3 million procedures were performed on fish in the EU (190,000 in the United Kingdom), of which a significant number were to support regulatory safety requirements (8). As an example, the most frequently used bioassay for chronic fish toxicity — the fish early life stage test (OECD 210) — requires a minimum of 480 fish individuals from a recommended species, for example zebrafish, excluding breeding stock and animals used for dose range‐finding. The early life stage test assesses lethal and sub-lethal effects, including mortality, abnormal behaviour and morphology, and the majority of these tests are conducted at moderate severity (e.g. likely to cause short-term moderate pain, suffering or distress for the animals used). Dose range finding studies are conducted at the highest severity and there is a significant risk of mortality, with animals likely to experience severe pain, suffering and distress.

There are very few validated in vitro assays that have been accepted within regulatory frameworks, particularly those addressing potential long-term sub-lethal effects. Identification, development and validation of new bioassays able to detect potential impairment due to chemical exposure in fish will represent a leap forward in the ability to perform refined ERA without the need to rely on vertebrate testing.

Completion of this Challenge will deliver a reliable, widely available NAM-based platform to address chemical safety in fish. In the short-term, this will result in a reduction in the number of in vivo fish studies carried out for safety purposes at candidate early screening stages and ERA. In the longer term, with increasing acceptance of the validity and reliability of NAMs, there will be an opportunity to fully replace in vivo animal studies for regulatory purposes in ERA.


  1. ICCVAM (2018). A Strategic Roadmap for Establishing New Approaches to Evaluate the Safety of Chemicals and Medical Products in the United States. National Toxicology Program, National Institute of Environmental Health. [Online - accessed 06 April 2021]. 

  1. Richard AM et al. (2016). ToxCast chemical landscape: paving the road to 21st century toxicology. Chemical research in toxicology. 29: 1225-1251. 

  1. Tanneberger K et al. (2013). Predicting Fish Acute Toxicity Using a Fish Gill Cell Line-Based Toxicity Assay. Environmental Science & Technology 47: 1110-1119. 

  1. ECHA (2017). Read-Across Assessment Framework (RAAF) [Online - accessed 06 April 2021]. 

  1. Rivetti C et al. (2020). Vision of a near future: Bridging the human health–environment divide. Toward an integrated strategy to understand mechanisms across species for chemical safety assessment. Toxicology in Vitro 62: 104692. 

  1. Ankely GT et al. (2010) Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment. Environmental Toxicology and Chemistry. 29, 730-741. 

  1. Margiotta-Casaluci L et al. (2016). Internal exposure dynamics drive the Adverse Outcome Pathways of synthetic glucocorticoids in fish. Scientific Reports 6: 21978. 

  1. REPORT FROM THE COMMISSION TO THE EUROPEAN PARLIAMENT AND THE COUNCIL 2019 report on the statistics on the use of animals for scientific purposes in the Member States of the European Union in 2015-2017. COM/2020/16 final. [Online - accessed 23 April 2021]. 

Full Challenge Information

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Assessment information

Review Panel membership

Name Institution
Dr Martino Picardo (Chair) Independent
Dr Bruno Campos (Sponsor) Unilever
Dr Claudia Rivetti  (Sponsor) Unilever
Dr Stewart Owen (Sponsor) AstraZeneca
Dr Martin Blank (Sponsor) Bayer
Dr Nina Hallmark (Sponsor) Bayer
Dr Tobias Pamminger (Sponsor) Bayer
Dr Michelle Bloor University of Glasgow 
Professor Michael Capaldi Newcastle University
Dr Elke Eilebrecht Fraunhofer Institute for Molecular Biology and Applied Ecology IME
Professor Raymond Pieters University of Applied Sciences Utrecht