A global initiative to refine acute inhalation studies through the use of 'evident toxicity' as an endpoint: Towards adoption of the fixed concentration procedure.

Acute inhalation studies are conducted in animals as part of chemical hazard identification and characterisation, including for classification and labelling purposes. Current accepted methods use death as an endpoint (OECD TG403 and TG436), whereas the fixed concentration procedure (FCP) (draft OECD TG433) uses fewer animals and replaces lethality as an endpoint with 'evident toxicity.' Evident toxicity is defined as clear signs of toxicity that predict exposure to the next highest concentration will cause severe toxicity or death in most animals. A global initiative including 20 organisations, led by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) has shared data on the clinical signs recorded during acute inhalation studies for 172 substances (primarily dusts or mists) with the aim of making evident toxicity more objective and transferable between laboratories. Pairs of studies (5 male or 5 female rats) with at least a two-fold change in concentration were analysed to determine if there are any signs at the lower dose that could have predicted severe toxicity or death at the higher concentration. The results show that signs such as body weight loss (>10% pre-dosing weight), irregular respiration, tremors and hypoactivity, seen at least once in at least one animal after the day of dosing are highly predictive (positive predictive value > 90%) of severe toxicity or death at the next highest concentration. The working group has used these data to propose changes to TG433 that incorporate a clear indication of the clinical signs that define evident toxicity.

Animal Research beyond the Laboratory: Report from a Workshop on Places Other than Licensed Establishments (POLEs) in the UK.

Research involving animals that occurs outside the laboratory raises an array of unique challenges. With regard to UK legislation, however, it receives only limited attention in terms of official guidelines, support, and statistics, which are unsurprisingly orientated towards the laboratory environment in which the majority of animal research takes place. In September 2019, four social scientists from the Animal Research Nexus program gathered together a group of 13 experts to discuss nonlaboratory research under the Animals (Scientific Procedures) Act (A(SP)A) of 1986 (mirroring European Union (EU) Directive 2010/63/EU), which is the primary mechanism for regulating animal research in the UK. Such nonlaboratory research under the A(SP)A often occurs at Places Other than Licensed Establishments (POLEs). The primary objective of the workshop was to assemble a diverse group with experience across a variety of POLEs (e.g., wildlife field sites, farms, fisheries, veterinary clinics, zoos) to explore the practical, ethical, and regulatory challenges of conducting research at POLEs. While consensus was not sought, nor reached on every point of discussion, we collectively identified five key areas that we propose require further discussion and attention. These relate to: (1) support and training; (2) ethical review; (3) cultures of care, particularly in nonregulated research outside of the laboratory; (4) the setting of boundaries; and (5) statistics and transparency. The workshop generated robust discussion and thereby highlighted the value of focusing on the unique challenges posed by POLEs, and the need for further opportunities for exchanging experiences and sharing best practice relating to research projects outside of the laboratory in the UK and elsewhere.

Prospective severity classification of scientific procedures in cephalopods: Report of a COST FA1301 Working Group survey.

Cephalopods are the first invertebrate class regulated by the European Union (EU) under Directive 2010/63/EU on the protection of animals used for scientific purposes, which requires prospective assessment of severity of procedures. To assist the scientific community in establishing severity classification for cephalopods, we undertook a web-based survey of the EU cephalopod research community as represented by the participants in the European COoperation on Science and Technology (COST) Action FA1301, CephsInAction'. The survey consisted of 50 scenarios covering a range of procedures involving several cephalopod species at different life stages. Respondents (59 people from 15 countries) either allocated a severity classification to each scenario or indicated that they were unable to decide (UTD). Analyses evaluated score distributions and clustering. Overall, the UTD scores were low (7.0 ± 0.6%) and did not affect the severity classification. Procedures involving paralarvae and killing methods (not specified in Annexe IV) had the highest UTD scores. Consensus on non-recovery procedures was reached consistently, although occasionally non-recovery appeared to be confused with killing methods. Scenarios describing procedures above the lower threshold for regulation, including those describing behavioural studies, were also identified and allocated throughout the full range of severity classifications. Severity classification for scenarios based on different species (e.g. cuttlefish vs. octopus) was consistent, comparable and dependent on potentially more harmful interventions. We found no marked or statistically significant differences in the overall scoring of scenarios between the demographic subgroups (age, sex, PhD and cephalopod experience). The COST Action FA1301 survey data provide a basis for a prospective severity classification for cephalopods to serve as guide for researchers, project assessors and regulators.

A Good Death? Report of the Second Newcastle Meeting on Laboratory Animal Euthanasia.

Millions of laboratory animals are killed each year worldwide. There is an ethical, and in many countries also a legal, imperative to ensure those deaths cause minimal suffering. However, there is a lack of consensus regarding what methods of killing are humane for many species and stages of development. In 2013, an international group of researchers and stakeholders met at Newcastle University, United Kingdom to discuss the latest research and which methods could currently be considered most humane for the most commonly used laboratory species (mice, rats and zebrafish). They also discussed factors to consider when making decisions about appropriate techniques for particular species and projects, and priorities for further research. This report summarises the research findings and discussions, with recommendations to help inform good practice for humane killing.

Cephalopods in neuroscience: regulations, research and the 3Rs.

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.

A roadmap for hazard monitoring and risk assessment of marine biotoxins on the basis of chemical and biological test systems.

Aquatic food accounts for over 40% of global animal food products, and the potential contamination with toxins of algal origin--marine biotoxins--poses a health threat for consumers. The gold standards to assess toxins in aquatic food have traditionally been in vivo methods, i.e., the mouse as well as the rat bioassay. Besides ethical concerns, there is also a need for more reliable test methods because of low inter-species comparability, high intra-species variability, the high number of false positive and negative results as well as questionable extrapolation of quantitative risk to humans. For this reason, a transatlantic group of experts in the field of marine biotoxins was convened from academia and regulatory safety authorities to discuss future approaches to marine biotoxin testing. In this report they provide a background on the toxin classes, on their chemical characterization, the epidemiology, on risk assessment and management, as well as on their assumed mode of action. Most importantly, physiological functional assays such as in vitro bioassays and also analytical techniques, e.g., liquid chromatography coupled mass spectrometry (LC-MS), as substitutes for the rodent bioassay are reviewed. This forms the basis for recommendations on methodologies for hazard monitoring and risk assessment, establishment of causality of intoxications in human cases, a roadmap for research and development of human-relevant functional assays, as well as new approaches for a consumer directed safety concept.

Oxidation of essential amino acids by the ovine gastrointestinal tract.

It is not known if the ruminant animal gastrointestinal tract (GIT) can oxidise essential amino acids (AA) other than leucine. Therefore, the oxidation of four essential AA (leucine, lysine, methionine and phenylalanine), supplied systemically as labelled 1-13C forms, was monitored across the mesenteric-drained viscera (MDV; small intestine) and portal-drained viscera (PDV; total GIT), as part of a Latin square design, in four wether sheep (35-45 kg) fed at 1.4 x maintenance. Oxidation was assessed primarily by appearance of 13CO2, corrected for sequestration of [13C]bicarbonate. The GIT contributed 25 % (P<0.001) and 10 % (P<0.05) towards whole-body AA oxidation for leucine and methionine respectively. This reduced net appearance across the PDV by 23 and 11 % respectively. The contribution of MDV metabolism to total PDV oxidation was 40 % for leucine and 60 % for methionine. There was no catabolism of systemic lysine or phenylalanine across the GIT. Production and exchange of secondary metabolites (e.g. 4-methyl-2-oxo-pentanoate, homocysteine, 2-aminoadipate) across the GIT was also limited. Less AA appeared across the PDV than MDV (P<0.001), indicative of use by tissues such as the forestomach, large intestine, spleen and pancreas. The PDV: MDV net appearance ratios varied (P<0.001) between AA, e.g. phenylalanine (0.81), lysine (0.71), methionine (0.67), leucine (0.56), histidine (0.71), threonine (0.63) and tryptophan (0.48). These differences probably reflect incomplete re-absorption of endogenous secretions and, together with the varied oxidative losses measured, will alter the pattern of AA net supply to the rest of the animal.