Big Question to Developing Solutions: A Decade of Progress in the Development of Aquatic New Approach Methodologies from 2012 to 2022.

In 2012, 20 key questions related to hazard and exposure assessment and environmental and health risks of pharmaceuticals and personal care products in the natural environment were identified. A decade later, this article examines the current level of knowledge around one of the lowest-ranking questions at that time, number 19: "Can nonanimal testing methods be developed that will provide equivalent or better hazard data compared with current in vivo methods?" The inclusion of alternative methods that replace, reduce, or refine animal testing within the regulatory context of risk and hazard assessment of chemicals generally faces many hurdles, although this varies both by organism (human-centric vs. other), sector, and geographical region or country. Focusing on the past 10 years, only works that might reasonably be considered to contribute to advancements in the field of aquatic environmental risk assessment are highlighted. Particular attention is paid to methods of contemporary interest and importance, representing progress in (1) the development of methods which provide equivalent or better data compared with current in vivo methods such as bioaccumulation, (2) weight of evidence, or (3) -omic-based applications. Evolution and convergence of these risk assessment areas offer the basis for fundamental frameshifts in how data are collated and used for the protection of taxa across the breadth of the aquatic environment. Looking to the future, we are at a tipping point, with a need for a global and inclusive approach to establish consensus. Bringing together these methods (both new and old) for regulatory assessment and decision-making will require a concerted effort and orchestration. Environ Toxicol Chem 2024;43:559-574. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Are changes in vitellogenin concentrations in fish reliable indicators of chemical-induced endocrine activity?

Vitellogenin (VTG), a biomarker for endocrine activity, is a mechanistic component of the regulatory assessment of potential endocrine-disrupting properties of chemicals. This review of VTG data is based on changes reported for 106 substances in standard fish species. High intra-study and inter-laboratory variability in VTG concentrations was confirmed, as well as discrepancies in interpretation of results based on large differences between fish in the dilution water versus solvent control, or due to the presence of outlier measurements. VTG responses in fish were ranked against predictions for estrogen receptor agonist activity and aromatase inhibition from bioactivity model output and ToxCast in vitro assay results, respectively. These endocrine mechanisms explained most of the VTG responses in the absence of systemic toxicity, the magnitude of the VTG response being proportional to the in vitro potency. Interpretation of the VTG data was sometimes confounded by an alternative endocrine mechanism of action. There was evidence for both false positive and negative responses for VTG synthesis, but overall, it was rare for substances without endocrine activity in vitro to cause a concentration-dependent VTG response in fish in the absence of systemic toxicity. To increase confidence in the VTG results, we recommend improvements in the VTG measurement methodologies and greater transparency in reporting of VTG data (including quality control criteria for assay performance). This review supports the application of New Approach Methodologies (NAMs) by demonstrating that endocrine activity in vitro from mammalian cell lines is predictive for in vivo VTG response in fish, suggesting that in vitro mechanistic data could be used more broadly in decision-making to help reduce animal testing.

An international cross-laboratory survey on fish vitellogenin analysis: Methodological challenges and opportunities for best practice.

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.

New Approach Methodologies for the Endocrine Activity Toolbox: Environmental Assessment for Fish and Amphibians.

Multiple in vivo test guidelines focusing on the estrogen, androgen, thyroid, and steroidogenesis pathways have been developed and validated for mammals, amphibians, or fish. However, these tests are resource-intensive and often use a large number of laboratory animals. Developing alternatives for in vivo tests is consistent with the replacement, reduction, and refinement principles for animal welfare considerations, which are supported by increasing mandates to move toward an "animal-free" testing paradigm worldwide. New approach methodologies (NAMs) hold great promise to identify molecular, cellular, and tissue changes that can be used to predict effects reliably and more efficiently at the individual level (and potentially on populations) while reducing the number of animals used in (eco)toxicological testing for endocrine disruption. In a collaborative effort, experts from government, academia, and industry met in 2020 to discuss the current challenges of testing for endocrine activity assessment for fish and amphibians. Continuing this cross-sector initiative, our review focuses on the current state of the science regarding the use of NAMs to identify chemical-induced endocrine effects. The present study highlights the challenges of using NAMs for safety assessment and what work is needed to reduce their uncertainties and increase their acceptance in regulatory processes. We have reviewed the current NAMs available for endocrine activity assessment including in silico, in vitro, and eleutheroembryo models. New approach methodologies can be integrated as part of a weight-of-evidence approach for hazard or risk assessment using the adverse outcome pathway framework. The development and utilization of NAMs not only allows for replacement, reduction, and refinement of animal testing but can also provide robust and fit-for-purpose methods to identify chemicals acting via endocrine mechanisms. Environ Toxicol Chem 2023;42:757-777. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Investigating endocrine-disrupting properties of chemicals in fish and amphibians: Opportunities to apply the 3Rs.

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).

Dying for change: A roadmap to refine the fish acute toxicity test after 40 years of applying a lethal endpoint.

The fish acute toxicity test (TG203; OECD, 2019) is frequently used and highly embedded in hazard and risk assessment globally. The test estimates the concentration of a chemical that kills 50% of the fish (LC50) over a 96 h exposure and is considered one of the most severe scientific procedures undertaken. Over the years, discussions at the Organisation for Economic Co-operation and Development (OECD) have resulted in changes to the test which reduce the number of fish used, as well as the development of a (potential) replacement test (TG236, OECD, 2013). However, refinement of the mortality endpoint with an earlier (moribundity) endpoint was not considered feasible during the Test Guideline's (TG) last update in 2019. Several stakeholders met at a UK-based workshop to discuss how TG203 can be refined, and identified two key opportunities to reduce fish suffering: (1) application of clinical signs that predict mortality and (2) shortening the test duration. However, several aspects need to be addressed before these refinements can be adopted. TG203 has required recording of major categories of sublethal clinical signs since its conception, with the option to record more detailed signs introduced in the 2019 update. However, in the absence of guidance, differences in identification, recording and reporting of clinical signs between technicians and laboratories is likely to have generated piecemeal data of varying quality. Harmonisation of reporting templates, and training in clinical sign recognition and recording are needed to standardise clinical sign data. This is critical to enable robust data-driven detection of clinical signs that predict mortality. Discussions suggested that the 96 h duration of TG203 cannot stand up to scientific scrutiny. Feedback and data from UK contract research organisations (CROs) conducting the test were that a substantial proportion of mortalities occur in the first 24 h. Refinement of TG203 by shortening the test duration would reduce suffering (and test failure rate) but requires a mechanism to correct new results to previous 96 h LC50 data. The actions needed to implement both refinement opportunities are summarised here within a roadmap. A shift in regulatory assessment, where the 96 h LC50 is a familiar base for decisions, will also be critical.

The Extended Amphibian Metamorphosis Assay: A Thyroid-Specific and Less Animal-Intensive Alternative to the Larval Amphibian Growth and Development Assay.

The amphibian metamorphosis assay (AMA; US Environmental Protection Agency [USEPA] test guideline 890.1100 and Organisation for Economic Co-Operation and Development test guideline 231) has been used for more than a decade to assess the potential thyroid-mediated endocrine activity of chemicals. In 2013, in the context of the Endocrine Disruptor Screening Program of the USEPA, a Scientific Advisory Panel reviewed the results from 18 studies and recommended changes to the AMA test guideline, including a modification to a fixed-stage design rather than a fixed-time (i.e., 21-d) design. We describe an extended test design for the AMA (or EAMA) that includes thyroid histopathology and time to metamorphosis (Nieuwkoop-Faber [NF] stage 62), to address both the issues with the fixed-time design and the specific question of thyroid-mediated adversity in a shorter assay than the larval amphibian growth and development assay (LAGDA; Organisation for Economic Co-Operation and Development test guideline 241), using fewer animals and resources. A demonstration study was conducted with the EAMA (up to NF stage 58) using sodium perchlorate. Data analyses and interpretation of the fixed-stage design of the EAMA are more straightforward than the fixed-time design because the fixed-stage design avoids confounded morphometric measurements and thyroid histopathology caused by varying developmental stages at test termination. It also results in greater statistical power to detect metamorphic delays than the fixed-time design. By preferentially extending the AMA to NF stage 62, suitable data can be produced to evaluate thyroid-mediated adversity and preclude the need to perform a LAGDA for thyroid mode of action analysis. The LAGDA remains of further interest should investigations of longer term effects related to sexual development modulated though the hypothalamus-pituitary-gonadal axis be necessary. However, reproduction assessment or life cycle testing is currently not addressed in the LAGDA study design. This is better addressed by higher tier studies in fish, which should then include specific thyroid-related endpoints. Environ Toxicol Chem 2021;40:2135-2144. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Opportunities and Challenges for Integrating New In Vitro Methodologies in Hazard Testing and Risk Assessment.

Nanomaterials are defined as materials with at least one dimension of 100 nm or less. Their small size confers unique properties that may alter the toxicity profile when compared to larger forms of the same material, requiring additional considerations for safety assessment. There has been a rise in the development of nanomaterials for many applications, and although traditional approaches for toxicity testing may address some of the new toxicity concerns, many may not be directly applicable to nanomaterials and new tools or approaches may need to be developed. Since nanomaterials can exist in many different forms, each of which may cause different adverse biological effects, reliance on traditional in vivo models for safety assessment will simply not be feasible or sustainable, given the volume of materials that may need to be tested. It is essential to consider and develop new in vitro methods that can be applied for hazard identification and risk assessment. Many challenges are associated with using alternative approaches to ensure they are as robust and reliable as traditional in vivo approaches, but by overcoming these issues and adopting new testing strategies there are opportunities to improve safety assessments and reduce the reliance on animal-based toxicity testing strategies.

Key Opportunities to Replace, Reduce, and Refine Regulatory Fish Acute Toxicity Tests.

Fish acute toxicity tests are conducted as part of regulatory hazard identification and risk-assessment packages for industrial chemicals and plant protection products. The aim of these tests is to determine the concentration which would be lethal to 50% of the animals treated. These tests are therefore associated with suffering in the test animals, and Organisation for Economic Co-operation and Development test guideline 203 (fish, acute toxicity) studies are the most widely conducted regulatory vertebrate ecotoxicology tests for prospective chemical safety assessment. There is great scope to apply the 3Rs principles-the reduction, refinement, and replacement of animals-in this area of testing. An expert ecotoxicology working group, led by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research, including members from government, academia, and industry, reviewed global fish acute test data requirements for the major chemical sectors. The present study highlights ongoing initiatives and provides an overview of the key challenges and opportunities associated with replacing, reducing, and/or refining fish acute toxicity studies-without compromising environmental protection. Environ Toxicol Chem 2020;39:2076-2089. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

In silico toxicology protocols.

The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information.

The future trajectory of adverse outcome pathways: a commentary.

The advent of adverse outcome pathways (AOPs) has provided a new lexicon for description of mechanistic toxicology, and a renewed enthusiasm for exploring modes of action resulting in adverse health and environmental effects. In addition, AOPs have been used successfully as a framework for the design and development of non-animal approaches to toxicity testing. Although the value of AOPs is widely recognised, there remain challenges and opportunities associated with their use in practise. The purpose of this article is to consider specifically how the future trajectory of AOPs may provide a basis for addressing some of those challenges and opportunities.

Aligning nanotoxicology with the 3Rs: What is needed to realise the short, medium and long-term opportunities?

Nanomaterials convey numerous advantages, and the past decade has seen a considerable rise in their development and production for an expanse of applications. While the potential advantages of nanomaterials are clear, concerns over the impact of human and environmental exposure exist. Concerted, science-led efforts are required to understand the effects of nanomaterial exposure and ensure that protection goals are met. There is much on-going discussion regarding how best to assess nanomaterial risk, particularly considering the large number of tests that may be required. A plethora of forms may need to be tested for each nanomaterial, and risk assessed throughout the life cycle, meaning numerous acute and chronic toxicity studies could be required, which is neither practical nor utilises the current evidence-base. Hence, there are scientific, business, ethical and legislative drivers to re-consider the use of animal toxicity tests. An expert Working Group of regulators, academics and industry scientists were gathered by the UK's NC3Rs to discuss: i) opportunities being offered in the short, medium and long-terms to advance nanosafety, ii) how to align these advances with the application of the 3Rs in nanomaterial safety testing, and iii) shifting the focus of risk assessment from current hazard-based approaches towards exposure-driven approaches.

Maximizing the success of bile duct cannulation studies in rats: recommendations for best practice.

Bile duct cannulation (BDC) studies are usually carried out in the rat to support the absorption, distribution, metabolism and excretion profiling of novel agrochemicals and pharmaceuticals. The different aspects of these studies (e.g. surgical preparation, dosing and collection of bile) can be intricate and/or technically complex. The animals are often kept singly housed for the duration of the studies following surgical implantation of the cannulas. The generation of insufficient data to meet the study objectives, for example due to failure in cannula patency, can result in the need to repeat these studies. A working group of contract research organizations that routinely carry out BDC studies was brought together by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) to share their experiences, and to establish the key factors necessary to ensure routinely high success rates. Through these discussions the group has identified opportunities for best practice across various aspects of the studies. The aim of these recommendations is to support all staff involved in conducting BDC studies to maximize the amount of useful data generated using the fewest animals possible, while ensuring the highest possible standards of animal welfare.

The current status of exposure-driven approaches for chemical safety assessment: A cross-sector perspective.

For the purposes of chemical safety assessment, the value of using non-animal (in silico and in vitro) approaches and generating mechanistic information on toxic effects is being increasingly recognised. For sectors where in vivo toxicity tests continue to be a regulatory requirement, there has been a parallel focus on how to refine studies (i.e. reduce suffering and improve animal welfare) and increase the value that in vivo data adds to the safety assessment process, as well as where to reduce animal numbers where possible. A key element necessary to ensure the transition towards successfully utilising both non-animal and refined safety testing is the better understanding of chemical exposure. This includes approaches such as measuring chemical concentrations within cell-based assays and during in vivo studies, understanding how predicted human exposures relate to levels tested, and using existing information on human exposures to aid in toxicity study design. Such approaches promise to increase the human relevance of safety assessment, and shift the focus from hazard-driven to risk-driven strategies similar to those used in the pharmaceutical sectors. Human exposure-based safety assessment offers scientific and 3Rs benefits across all sectors marketing chemical or medicinal products. The UK's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) convened an expert working group of scientists across the agrochemical, industrial chemical and pharmaceutical industries plus a contract research organisation (CRO) to discuss the current status of the utilisation of exposure-driven approaches, and the challenges and potential next steps for wider uptake and acceptance. This paper summarises these discussions, highlights the challenges - particularly those identified by industry - and proposes initial steps for moving the field forward.

Steps towards the international regulatory acceptance of non-animal methodology in safety assessment.

The current animal-based paradigm for safety assessment must change. In September 2016, the UK National Centre for Replacement, Refinement and Reduction of Animals in Research (NC3Rs) brought together scientists from regulatory authorities, academia and industry to review progress in bringing new methodology into regulatory use, and to identify ways to expedite progress. Progress has been slow. Science is advancing to make this possible but changes are necessary. The new paradigm should allow new methodology to be adopted once it is developed rather than being based on a fixed set of studies. Regulatory authorities can help by developing Performance-Based Standards. The most pressing need is in repeat dose toxicology, although setting standards will be more complex than in areas such as sensitization. Performance standards should be aimed directly at human safety, not at reproducing the results of animal studies. Regulatory authorities can also aid progress towards the acceptance of non-animal based methodology by promoting "safe-haven" trials where traditional and new methodology data can be submitted in parallel to build up experience in the new methods. Industry can play its part in the acceptance of new methodology, by contributing to the setting of performance standards and by actively contributing to "safe-haven" trials.

Alternative approaches to vertebrate ecotoxicity tests in the 21st century: A review of developments over the last 2 decades and current status.

The need for alternative approaches to the use of vertebrate animals for hazard assessment of chemicals and pollutants has become of increasing importance. It is now the first consideration when initiating a vertebrate ecotoxicity test, to ensure that unnecessary use of vertebrate organisms is minimized wherever possible. For some regulatory purposes, the use of vertebrate organisms for environmental risk assessments has been banned; in other situations, the number of organisms tested has been dramatically reduced or the severity of the procedure refined. However, there is still a long way to go to achieve a complete replacement of vertebrate organisms to generate environmental hazard data. The development of animal alternatives is based not just on ethical considerations but also on reducing the cost of performing vertebrate ecotoxicity tests and in some cases on providing better information aimed at improving environmental risk assessments. The present Focus article provides an overview of the considerable advances that have been made toward alternative approaches for ecotoxicity assessments over the last few decades. Environ Toxicol Chem 2016;35:2637-2646. © 2016 SETAC.