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Many regulations are beginning to explicitly require investigation of a chemical's endocrine‐disrupting properties as a part of the safety assessment process for substances already on or about to be placed on the market. Different jurisdictions are applying distinct approaches. However, all share a common theme requiring testing for endocrine activity and adverse effects, typically involving in vitro and in vivo assays on selected endocrine pathways. For ecotoxicological evaluation, in vivo assays can be performed across various animal species, including mammals, amphibians, and fish. Results indicating activity (i.e., that a test substance may interact with the endocrine system) from in vivo screens usually trigger further higher‐tier in vivo assays. Higher‐tier assays provide data on adverse effects on relevant endpoints over more extensive parts of the organism's life cycle. Both in vivo screening and higher‐tier assays are animal‐ and resource‐intensive and can be technically challenging to conduct. Testing large numbers of chemicals will inevitably result in the use of large numbers of animals, contradicting stipulations set out within many regulatory frameworks that animal studies be conducted as a last resort. Improved strategies are urgently required. In February 2020, the UK's National Centre for the 3Rs and the Health and Environmental Sciences Institute hosted a workshop ("Investigating Endocrine Disrupting Properties in Fish and Amphibians: Opportunities to Apply the 3Rs"). Over 50 delegates attended from North America and Europe, across academia, laboratories, and consultancies, regulatory agencies, and industry. Challenges and opportunities in applying refinement and reduction approaches within the current animal test guidelines were discussed, and utilization of replacement and/or new approach methodologies, including in silico, in vitro, and embryo models, was explored. Efforts and activities needed to enable application of 3Rs approaches in practice were also identified. This article provides an overview of the workshop discussions and sets priority areas for follow‐up. Integr Environ Assess Manag 2022;18:442–458. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). KEY POINTS: In vivo screening and higher‐tier assays to evaluate a chemical's potential endocrine‐disrupting properties and potential impacts on ecosystems are animal‐ and resource‐intensive and can be technically challenging to conduct.There has been much recent work to develop new approach methodologies (NAMs)—in silico and in vitro approaches—that can be applied in this space.An understanding of the opportunities and challenges with the current in vivo approaches, coupled with the ongoing efforts to expand the development, use, and application of NAMs, will help focus short‐ and long‐term goals.International, multisector science‐based activities provide the framework to improve ecological safety as it relates to compounds with endocrine‐disrupting properties. [ABSTRACT FROM AUTHOR]

ABSTRACT The ecotoxicity testing of chemicals for prospective environmental safety assessment is an area in which a high number of vertebrates are used across a variety of industry sectors. Refining, reducing, and replacing the use of animals such as fish, birds, and amphibians for this purpose addresses the ethical concerns and the increasing legislative requirements to consider alternative test methods. Members of the UK-based National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) Ecotoxicology Working Group, consisting of representatives from academia, government organizations, and industry, have worked together over the past 6 y to provide evidence bases to support and advance the application of the 3Rs in regulatory ecotoxicity testing. The group recently held a workshop to identify the areas of testing, demands, and drivers that will have an impact on the future of animal use in regulatory ecotoxicology. As a result of these discussions, we have developed a pragmatic approach to prioritize and realistically address key opportunity areas, to enable progress toward the vision of a reduced reliance on the use of animals in this area of testing. This paper summarizes the findings of this exercise and proposes a pragmatic strategy toward our key long-term goals-the incorporation of reliable alternatives to whole-organism testing into regulations and guidance, and a culture shift toward reduced reliance on vertebrate toxicity testing in routine environmental safety assessment. Integr Environ Assess Manag 2016;12:417-421. © 2015 SETAC [ABSTRACT FROM AUTHOR]

Highlights: [•] Feasibility of the threshold approach for formulated PPPs fish acute toxicity tests. [•] Database analysis demonstrates that a reduction of approximately 38% of the fish might be achieved. [•] Proposal for the threshold approach in the development of a regulatory dataset. [Copyright &y& Elsevier]

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines. • Vitellogenin (VTG) is an endocrine activity biomarker in fish regulatory studies. • A survey of laboratories revealed technical challenges for VTG assessment. • These include methodological, data handling and data acceptability issues. • The results provide a basis for improving and harmonizing the measurement of VTG. • Implementation of the recommendations will support decision-making processes. [ABSTRACT FROM AUTHOR]

The European Plant Protection Products Regulation 1107/2009 requires that registrants establish whether pesticide metabolites pose a risk to the environment. Fish acute toxicity assessments may be carried out to this end. Considering the total number of pesticide (re-) registrations, the number of metabolites can be considerable, and therefore this testing could use many vertebrates. EFSA's recent “Guidance on tiered risk assessment for plant protection products for aquatic organisms in edge-of-field surface waters” outlines opportunities to apply non-testing methods, such as Quantitative Structure Activity Relationship (QSAR) models. However, a scientific evidence base is necessary to support the use of QSARs in predicting acute fish toxicity of pesticide metabolites. Widespread application and subsequent regulatory acceptance of such an approach would reduce the numbers of animals used. The work presented here intends to provide this evidence base, by means of retrospective data analysis. Experimental fish LC 50 values for 150 metabolites were extracted from the Pesticide Properties Database ( http://sitem.herts.ac.uk/aeru/ppdb/en/atoz.htm ). QSAR calculations were performed to predict fish acute toxicity values for these metabolites using the US EPA's ECOSAR software. The most conservative predicted LC 50 values generated by ECOSAR were compared with experimental LC 50 values. There was a significant correlation between predicted and experimental fish LC 50 values (Spearman r s = 0.6304, p < 0.0001). For 62% of metabolites assessed, the QSAR predicted values are equal to or lower than their respective experimental values. Refined analysis, taking into account data quality and experimental variation considerations increases the proportion of sufficiently predictive estimates to 91%. For eight of the nine outliers, there are plausible explanation(s) for the disparity between measured and predicted LC 50 values. Following detailed consideration of the robustness of this non-testing approach, it can be concluded there is a strong data driven rationale for the applicability of QSAR models in the metabolite assessment scheme recommended by EFSA. As such there is value in further refining this approach, to improve the method and enable its future incorporation into regulatory guidance and practice. [ABSTRACT FROM AUTHOR]

Fish bioconcentration test guidelines generally require that bioconcentration factors (BCFs) are determined at two exposure concentrations. However, recent revisions to the OECD test guideline for bioconcentration testing (TG 305) provide the option to use only one exposure concentration, when justification is provided, although two concentrations may still be required for some regulatory purposes. Recently, this justification has been demonstrated for plant protection product active ingredients. To determine whether this justification has a broader validity for general chemicals, an analysis of 236 BCF studies on general chemicals was conducted. The results presented here again demonstrate that BCF values do not significantly differ between concentrations when more than one concentration is used. This relationship is particularly strong for BCFs ⩾1000 L/kg, which is beneficial, since only chemicals with BCFs >2000 L/kg may require regulatory action. This analysis therefore provides a data-driven rationale for using the one test concentration approach for general chemical substances and thus could contribute to a substantial reduction in the use of fish in bioconcentration tests. [ABSTRACT FROM AUTHOR]

Abstract: Fish bioconcentration tests are time consuming, expensive, and use many animals. Alternative methods that replace, reduce or refine the use of fish for BCF testing would therefore be of value. Test guidelines generally require that bioconcentration factors (BCFs) are determined at two exposure concentrations. However, recent revisions to the OECD Test Guideline for BCF testing (TG 305) provide the option to use only one exposure concentration, when justification is provided, although two concentrations may still be required for some regulatory purposes. Analysis of 55 studies on plant protection products demonstrates that BCF values do not significantly differ between the two exposure concentrations. This analysis therefore provides evidence to support the revision of OECD TG 305, and in particular provides justification for using the one test concentration approach for plant protection product active substances. [Copyright &y& Elsevier]