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.

Assessable learning outcomes for the EU Education and Training Framework core and Function A specific modules: Report of an ETPLAS WORKING Group.

Article 23(2) of the European Union Directive 2010/63/EU, which regulates welfare provisions for animals used for scientific purposes, requires that staff involved in the care and use of animals for scientific purposes be adequately educated and trained before they undertake any such work. However, the nature and extent of such training is not stipulated in the Directive. To facilitate Member States in fulfilling their education and training obligations, the European Commission developed a common Education and Training Framework, which was endorsed by the Member States Competent Authorities. An Education & Training Platform for Laboratory Animal Science (ETPLAS) Working Group was recently established to develop further guidance to the Learning Outcomes in the Framework, with the objective to clarify the levels of knowledge and understanding required by trainees, and to provide the criteria by which these Learning Outcomes should be assessed. Using the Framework document as a starting point, assessment criteria for the Learning Outcomes of the modules required for Function A persons (carrying out procedures on animals) for rats, mice and zebrafish were created with sufficient detail to enable trainees, providers and assessors to appreciate the level of knowledge, understanding and skills required to pass each module. Adoption and utilization of this document by training providers and accrediting or approving bodies will harmonize introductory education and training for those involved in the care and use of animals for scientific purposes within the European Union, promote mutual recognition of training within and between Member States and therefore free movement of personnel.

Refining rodent models of spinal cord injury.

This report was produced by an Expert Working Group (EWG) consisting of UK-based researchers, veterinarians and regulators of animal experiments with specialist knowledge of the use of animal models of spinal cord injury (SCI). It aims to facilitate the implementation of the Three Rs (Replacement, Reduction and Refinement), with an emphasis on refinement. Specific animal welfare issues were identified and discussed, and practical measures proposed, with the aim of reducing animal use and suffering, reducing experimental variability, and increasing translatability within this critically important research field.

An International Perspective on the Tools and Concepts for Effluent Toxicity Assessments in the Context of Animal Alternatives: Reduction in Vertebrate Use.

Since the 1940s, effluent toxicity testing has been used to assess potential ecological impacts of effluents and help determine necessary treatment options for environmental protection prior to release. Strategic combinations of toxicity tests, analytical tools, and biological monitoring have been developed. Because the number of vertebrates utilized in effluent testing is thought to be much greater than that used for individual chemical testing, there is a new need to develop strategies to reduce the numbers of vertebrates (i.e., fish) used. This need will become more critical as developing nations begin to use vertebrates in toxicity tests to assess effluent quality. A workshop was held to 1) assess the state of science in effluent toxicity testing globally; 2) determine current practices of regulators, industry, private laboratories, and academia; and 3) explore alternatives to vertebrate (fish) testing options and the inclusion of modified/new methods and approaches in the regulatory environment. No single approach was identified, because of a range of factors including regulatory concerns, validity criteria, and wider acceptability of alternatives. However, a suite of strategies in a weight-of-evidence approach would provide the flexibility to meet the needs of the environment, regulators, and the regulated community; and this "toolbox" approach would also support reduced reliance on in vivo fish tests. The present Focus article provides a brief overview of wastewater regulation and effluent testing approaches. Alternative methodologies under development and some of the limitations and barriers to regulatory approaches that can be selected to suit individual country and regional requirements are described and discussed. Environ Toxicol Chem 2018;37:2745-2757. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Refinement of the use of food and fluid control as motivational tools for macaques used in behavioural neuroscience research: report of a Working Group of the NC3Rs.

This report provides practical guidance on refinement of the use of food and fluid control as motivational tools for macaques used in behavioural neuroscience research. The guidance is based on consideration of the scientific literature and, where data are lacking, expert opinion and professional experience, including that of the members of a Working Group convened by the United Kingdom National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). The report should be useful to researchers, veterinarians and animal care staff responsible for the welfare of macaques used in food and fluid control protocols, as well as those involved with designing, performing and analysing studies that use these protocols. It should also assist regulatory authorities and members of local ethical review processes or institutional animal care and use committees concerned with evaluating such protocols. The report provides a framework for refinement that can be tailored to meet local requirements. It also identifies data gaps and areas for future research and sets out the Working Group's recommendations on contemporary best practice.

Regulatory aspects on the use of fish embryos in environmental toxicology.

Animal alternative tests are gaining serious consideration in an array of environmental sciences, particularly as they relate to sound management of chemicals and wastewater discharges. The ILSI Health and Environmental Sciences Institute and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test in March, 2008. This relatively young discipline is following advances in animal alternatives for human safety sciences, and it is advisable to develop a broad comparison of how animal alternative tests involving fish are viewed in a regulatory context over a wide array of authorities or advising bodies. These include OECD, Western Europe, North America, and Japan. This paper summarizes representative practices from these regions. Presently, the global regulatory environment has varying stances regarding the protection of fish for use as an experimental animal. Such differences have a long-term potential to lead to a lack of harmony in approaches to fish toxicity testing, especially for chemicals in commerce across multiple geographic regions. Implementation of alternative methods and approaches will be most successful if accepted globally, including methods of fish toxicity testing. An important area for harmonization would be in the interpretation of protected and nonprotected life stages of fish. Use of fish embryos represent a promising alternative and allow bridging to more technically challenging alternatives with longer prospective timelines, including cell-based assays, ecotoxicogenomics, and QSARs.

Knee extension strength cutpoints for maintaining mobility.

OBJECTIVES: To identify levels of knee extensor strength that are associated with high and low risk of incident severe mobility limitation (SML) in initially well-functioning older adults. DESIGN: Prospective cohort study. SETTING: University clinic center. PARTICIPANTS: One thousand three hundred fifty-five men and 1,429 women (aged 73.6+/-2.85) who reported no mobility limitation. MEASUREMENTS: Unilateral knee extensor isokinetic strength of participants was obtained. Participants were followed over a median of 5.90 years for the onset of SML, defined as two consecutive reports of a lot of difficulty or inability to walk one-quarter of a mile or climb 10 steps. Deciles of knee extension strength relative to body weight were evaluated to identify cutpoints most predictive of incident SML. Cutpoints were then compared with prevalence of having slow gait speed (<1.22 m/s) and mortality. RESULTS: Two sex-specific knee extension strength cutpoints were found. High and low risk of SML corresponded to less than 1.13 newton-meters (Nm)/kg (1st decile) and more than 1.71 Nm/kg (6th decile) in men and less than 1.01 Nm/kg (3rd decile) and more than 1.34 Nm/kg (7th decile) in women, respectively. Moderate risk was defined as being between the low- and high-risk cutpoints. Individuals with knee extension strength in the high- and moderate-risk categories were more likely to have a gait speed less than 1.22 m/s (hazard ratio (HR)=7.00, 95% confidence interval (CI)=5.47-8.96 and HR=2.14 7.00, 95% CI=1.73-2.64, respectively) and had a higher risk of death (HR=1.77, 95% CI=1.41-2.23 and HR=1.51, 95% CI=1.24-1.84, respectively) than individuals in the low-risk category. Adjustment for demographic factors, health behaviors, and medical conditions did not alter these associations. CONCLUSION: Knee extensor strength cutpoints provide objective markers to identify initially well-functioning older adults at high and low risk of future mobility limitation.