Results interpretation and statistical analysis of animal study data is challenging, since the sample sizes involved are usually very small. The application of frequently used approaches to statistical hypothesis testing, e.g. -tests or ANOVA methods, rely on specific distributional assumptions being satisfied. It can be hard to reliably assess these assumptions in animal studies with group sizes of usually less than ten animals. Non-parametric analysis methods might be considered as an alternative, but it is well-known that these approaches have lower statistical power in some situations. Following the Three Rs principles, it would be desirable to apply a class of statistical tests that is able to deal with a small number of observations, without the need for specific distributional assumptions. Thus, in this paper, we assess the application of permutation tests which seem to be able to meet both the above requirements. The performance of these permutation tests was compared with standard statistical tests by means of four real-world data examples from animal studies. The results demonstrated that permutation tests have good computational properties, leading to the conclusion that they could be a useful alternative approach when analysing small sample size animal study data for which distributional assumptions may not hold.

Agrochemical active ingredients are among the most toxicologically evaluated chemical substances, and genetically modified (GM) crops must be evaluated for safety and nutritional adequacy. Traditionally, these evaluations are conducted There are concerted efforts in the agrochemical sector to reduce animal testing, but there is also an emphasis on updating test guidelines and fulfilling new data package requirements, which can both result in increased animal testing. The purpose of this project was to generate benchmarks for the numbers of vertebrate animals used in: a) evaluating agrochemical pesticidal active ingredients for human health hazards; and b) assessing GM crops for safety and nutritional adequacy, based on guideline studies for data package requirements. To achieve this, guideline studies employing vertebrates, as required by regulatory bodies for developing global data packages for new active ingredients and for GM crops, were listed. These listed guideline studies were reviewed, in terms of the study details and the required animal-use, which was determined based on best testing practices. For historical animal-use benchmarking, Corteva's six most recent agrochemical pesticidal active ingredients and four most recent GM crop events were evaluated. Across the six most recently developed active ingredients, an average of approximately 10,000 mammals were used for the testing of each (range: 5500-19,000); across the four most recently developed GM crops, the average number of vertebrates similarly used for each was approximately 1200 (range: 1000-1500). Though regulatory testing requirements are likely to change with time, as new technologies become available, this project has established a theoretical minimum requirement to help drive aspirational animal reduction goals, identified regulatory challenges associated with the reduction of animal-use, and helped to refine Corteva's vertebrate animal-use tracking approaches.

Research involving the use of animals follows the Three Rs principles of , and . Based on the principle of , UK and EU legislation requires that technologies or alternative approaches directly replacing or avoiding the use of animals in experiments must be used wherever scientifically possible. However, is often not thoroughly considered, and failures in the existing system of checks and balances are widespread. Existing guidance and advice on searching for and identifying alternative approaches and replacement techniques is confusing and misleading, and this contributes to the lack of knowledge and confidence in addressing and the structural and procedural barriers around it. In this paper, we propose simple improvements to existing processes and a basic practical checklist, to help researchers identify and assess scientifically satisfactory replacement approaches. This tool will also support members of funding review panels, Animal Welfare and Ethical Review Bodies (AWERBs), Animal Welfare Bodies (AWBs), Animal Ethics Committees, and those editing and reviewing scientific journals in their scrutiny of applications, applicants and publications - particularly with regard to the use of alternative approaches and how these potential approaches were explored.

In 2020, the EU Commission's Joint Research Centre published the . In advance of the publication of this EURL ECVAM Recommendation, Landsforeningen Forsøgsdyrenes Værn arranged for an expert presentation on non-animal-derived antibodies to the Animal Experimentation Council, which is the body responsible for the review and approval of projects involving animals in Denmark. The main concerns of Council Members following the presentation were the widespread unfamiliarity with phage display methodology in Denmark, and how it would be possible to adapt licensing decisions. Before deciding how to approach the implementation of the Recommendation, the animal experimentation authority Dyreforsøgstilsynet, of which the Animal Experimentation Council and its secretariat are part, probed the wider scientific community and requested an opinion from the National Committee on Laboratory Animals and Alternatives. Wider scientific opinion in Denmark spanned those who were, to a certain extent, positive toward the non-animal methodology and those who were sceptical. Consequently, the approach chosen by the Danish authority is to encourage and monitor the uptake of non-animal methods, while allowing time for adjustment. Change has been slow, but the seeds of change are sown.

This report is a result of an interdisciplinary workshop held at the Collegium Helveticum in Zurich, Switzerland in February 2024, in which ideas for accelerating NAMs (New Approach Methodologies) in Swiss universities were shared and discussed. Due to regional differences in university organisation and funding structures, not all recommendations will be transferable to all regions worldwide. All participants were qualified to contribute to the discussion, due to their knowledge and experience of the Three Rs, in particular with regard to their implementation. The workshop participants believed that universities, which play a pioneering role in so many other areas, should also exploit their innovative potential in the field of animal-free research. The workshop uncovered four areas that would need to be addressed in order to achieve a significant change in university science culture and do more justice to the Three Rs, namely: language - innovative framing (pro-replacement framing in official university statements); knowledge transfer - communicating innovative findings in teaching (redirecting curriculum); change of values within science faculties; and structured implementation and well-coordinated planning of the transformation (establishment of a 'transition unit'). Specific strategies for implementing these four areas are outlined. In addition, we discuss why the replacement of animal testing should be an essential goal for universities, why this goal has not yet been achieved, and why concerted efforts toward change are required.

The first ONTOX Hackathon of the EU Horizon 2020-funded ONTOX project was held on 21-23 April 2024 in Utrecht, The Netherlands (https://ontox-project.eu/hackathon/). This participatory event aimed to collectively advance innovation for human safety through the use of Artificial Intelligence (AI), and hence significantly reduce reliance on animal-based testing. Expert scientists, industry leaders, young investigators, members of animal welfare organisations and academics alike, joined the hackathon. Eight teams were stimulated to find innovative solutions for challenging themes, that were selected based on previous discussions between stakeholders, namely: How to drive the use of AI in chemical risk assessment?; To predict or protect?; How can we secure human health and environmental protection at the same time?; and How can we facilitate the transition from animal tests to full implementation of human-relevant methods? The hackathon ended with a pitching contest, where the teams presented their solutions to a jury. The most promising solutions will be presented to regulatory authorities, industry, academia and non-governmental organisations at the next ONTOX Stakeholder Network meeting and taken up by the ONTOX project in order to tackle the above-mentioned challenges further. This report comprises two parts: The first part highlights some of the lessons learnt during the planning and execution of the hackathon; the second part presents the outcome of the ONTOX Hackathon, which resulted in several innovative and promising solutions based on New Approach Methodologies (NAMs), and outlines ONTOX's intended way forward.

Increasing the use of microphysiological systems (MPS) in Three Rs and regulatory applications is a nuanced but important goal, which would also help increase their scientific impact. There are three distinct and important stakeholder groups that each play a unique role in expediting the use of MPS for regulatory purpose - namely, commercial MPS developers, end-users and regulators. Additionally, non-profit organisations, such as the 3Rs Collaborative (3RsC), can help coordinate these efforts. This paper introduces the MPS Initiative, as organised by the 3RsC, and clarifies the potential for MPS to benefit all Three Rs. Key differences in the use of MPS-derived data for regulatory evidence of efficacy versus safety, and for various other contexts of use, are discussed. Finally, the results are presented from a survey of primarily commercial MPS developers, that collected their views on the realistic responsibilities of each stakeholder group. The results also highlight their key perspectives on the use of MPS, in the context of Three Rs and regulatory applications.

Currently for engineered nanomaterials (ENMs), there are no alternatives to bioaccumulation testing in fish as prescribed by OECD TG 305. New approaches are being sought, in order to reduce the numbers of animals used and address the ethical concerns associated with the use of vertebrates in such testing. This study aimed to demonstrate the value of an alternative method - the digestibility assay - as a tool to assess the bioaccumulation potential of ENMs. Titanium dioxide ENMs, or their equivalent bulk form, were incorporated onto a commercial fish diet and the fish digestion process was simulated at pH 2 (simulated stomach phase) and pH 7.8 (simulated intestinal phase). Only the 1000 mg/kg dry weight feed treatment showed measurable Ti release in the stomach phase, with the results being similar for the TiO ENM and the bulk form. More Ti from TiO was released from the feed during the intestinal phase of digestion compared to the stomach phase, but there were no statistically significant differences between either the ENM or bulk form. There was higher incidental Ti release from untreated control feed in the intestinal phase compared to the stomach phase. On a percentage basis, the stomach generally showed a statistically significant digestibility of around 3% of the total Ti in the feed, compared to around 7% in the intestine. The digestibility assay shows potential for use in the determination of the bioaccessible fraction of ENMs, and could be a useful screening tool to identify ENMs of concern for bioaccumulation.

The use of adverse effect data from animals as the gold standard in regulatory toxicology has a long tradition dating back to the 1960s. It has also been increasingly criticised, based on both scientific and animal welfare concerns, and yet, animal studies remain the gold standard in most areas of toxicology to this very day. In the 1980s, when the first generation of non-animal methods were evaluated as alternatives to animal testing, it was logical to compare the 'new' data obtained with historical animal data. This worked reasonably well for simple endpoints, such as skin and eye irritation, but became problematic for the more complex systemic endpoints, since in these cases, the effects are not directly comparable to those observed in systems. While the need to redefine the gold standard is not new, there is still no consensus on how to do so. We propose a consistent principle that avoids the need for animal reference data, while also ensuring an equivalent or better level of protection. We argue that the gold standard can be redefined, or rather bypassed, by focusing on risk management outcomes rather than the outputs of animal methods. This allows us to more efficiently protect human health and the environment, ensuring the safe use of chemicals while also identifying less hazardous chemicals for use as substitutes. We describe how this might work out for two main contexts of use: classification and labelling, and risk assessment. This has implications for the implementation of the EU Commission Roadmap toward the phasing out of animal testing in chemical safety assessments.

The acquisition of skills to perform an ovariohysterectomy (OVH) is crucial for veterinary medicine students. It has been demonstrated that the most effective way to develop these skills is through repetitive training on simulators. Unfortunately, commercial simulators are expensive, limiting their use and highlighting the need for the development of more cost-effective alternatives. This study aimed to assess the effectiveness of a low-cost, easily constructed textile-based simulator for the canine ovaries and uterus in training veterinary students on the OVH technique. The impact of tutor guidance on student learning was also assessed. Participants were divided into two groups: simulator and tutor guidance (SG) and simulator only (SO). Each student performed 20 repetitions of the three-clamp OVH technique, and the number of errors and execution time were quantified. The SG group reached the learning curve plateau in terms of minimum errors on the 7th repetition and attained the fastest time on the 6th repetition. The SO group reached the learning curve plateau in terms of minimum errors and attained the fastest time both on the 15th repetition. From individuals in the SG group, there were no requests for tutor guidance from the 11th attempt. This low-cost simulator is ideal for training veterinary students in the early stages of surgical learning, as it effectively facilitates learning the OVH procedure without the use of live animals.