Toxicological testing seeks to enable robust regulatory decisions while reducing uncertainty. Advances in modern biomedical technologies now allow the generation of data at an unprecedented scale and granularity. However, the proliferation of assays and datasets does not in itself translate into decision-relevant knowledge. This Comment article examines when testing turns into a case of frustra laborare - i.e. expending effort without proportional epistemic gain. We argue that exposure realism and toxicokinetic plausibility function as gatekeepers of relevance: mechanistic effects observed only at implausible internal concentrations rarely alter risk characterisation. Likewise, the act of expanding batteries of new approach methodologies (NAMs) without establishing predefined integration rules, inflates false-positive accumulation and amplifies uncertainty. Reproducibility limits and borderline outcomes are structural features of both animal and non-animal methods, and these factors must be quantified rather than obscured. A value-of-information (VoI) view brings much-needed discipline: the value of a test lies in its ability to shift decisions; those that cannot do so, do not meaningfully contribute. Across all domains discussed, reproducibility stands out as the essential constraint - exposure anchoring, probabilistic integration and uncertainty quantification all depend on assays that perform consistently across laboratories. In order for toxicologists to avoid frustra laborare, testing should be aligned with internal exposure, reproducible, and embedded within decision frameworks that yield actionable outcomes.

The non-publication of negative or null results contributes to increased animal use in biomedical research and experimentation, by encouraging the unnecessary repetition of studies. This practice also delays the development, validation and uptake of alternative methods, including New Approach Methodologies (NAMs). This Comment article discusses that recognising and disseminating negative results is important, in terms of accelerating the implementation of alternative methods and supporting replacement, reduction and refinement (the Three Rs).

The preclinical evaluation of novel imaging agents, such as contrast agents for computed tomography (CT), still largely relies on rodent models or simplified in vitro systems, each carrying limitations with regard to ethical burden, physiological relevance or translational value. To address this gap, we propose Galleria mellonella larvae as a scalable, ethically favourable and physiologically relevant in vivo model for use in the early-stage screening of novel CT contrast agents. We established a standardised imaging protocol utilising isoflurane-based sedation and longitudinal micro-CT scanning, and used it to evaluate three commercially available contrast agents: one clinically approved molecular agent and two nanoparticulate formulations that are used for preclinical studies. All agents were systemically distributed, well-tolerated and enabled consistent visualisation of internal anatomy over eight days. While differences in clearance kinetics and route related to particle size or formulation were less pronounced compared to vertebrate models, this larval system reliably reflected biodistribution trends and allowed non-invasive tracking of agent functionality and safety. The use of G. mellonella in this context enables early elimination of less promising candidates and reduces reliance on vertebrates in downstream (pre)clinical development, aligning with the Three Rs principles. Our findings support the use of G. mellonella as a practical intermediate model, bridging in vitro cell-based assays and in vivo rodent studies in the development of next-generation CT and imaging contrast agents.

Risk decision-making has evolved from a vernacular focused on risk assessment and management to fully integrated approaches designed to inform risk acumen. Consequently, there has been a renewed interest, particularly in areas with significant paradigm shifts, to understand the complex nature of the underlying intersections between data, ethics and risk. For example, building more awareness of the risk and ethical implications for applying artificial intelligence for generative (content creation) and agentic (decision-making) purposes or relying on next generation risk assessments grounded in models reflective of the Three Rs principles (i.e. the replacement, reduction and refinement of animal studies). Global thinkers in risk science and analysis have also developed frameworks and models, such as the Projector Model, to show the complex nature of these intersections relevant to public health and regulatory risk decision-making. This Comment article builds on this work by sharing real-life examples from an expert panel discussion, which occurred during the Chief Data & Analytics Officer (CDAO) Canada Public Sector 2025 meeting. These panel members relied on the Projector Model to navigate the discussion in a session titled 'Data-driven science - Transforming risk assessment and regulation in the public sector'. The examples showed how institutional values and norms serve as foundational elements for risk decision-making, and highlighted that data-driven science, especially that based on novel approaches and technologies, needs careful consideration from an ethical and risk perspective.

The UK's 2025 Replacing Animals in Science strategy introduces a commitment to end polyclonal antibody reagent production by 2030. While this represents a significant milestone, focusing exclusively on polyclonal reagents raises scientific, ethical and implementation challenges, given that monoclonal and other recombinant antibody formats frequently remain dependent on animal immunisation. This study examines whether current UK licensing practice and reporting frameworks are positioned to deliver genuine replacement. A structured, Three Rs-aligned replacement framework is articulated, reflecting the principles set out in the UK Animals in Science Committee (ASC) 2022 review. This framework is applied to UK Non-Technical Summaries (NTS), to assess how antibody reagent production is justified at the project licence stage and the extent to which applications align with ASC expectations. In parallel, UK Home Office Annual Statistics of Scientific Procedures on Living Animals are analysed, to evaluate how antibody-related procedures are categorised, and to determine whether this national reporting enables progress toward the 2030 commitment to be monitored. The analysis identifies limited implementation of ASC replacement guidance in post-2022 licences, with few applications providing evidence-based interrogation of non-animal alternatives, documented use of validated in vitro discovery platforms, or robust justification for immunisation. The national statistics lack sufficient resolution to clearly capture immunisation-dependent antibody reagent production across commercial, basic and translational research contexts. In addition, inconsistent terminology complicates the interpretation of genuine replacement. Importantly, the findings indicate that, without complementary regulatory, reporting and infrastructural measures, the 2030 commitment risks being met through increased reliance on other immunisation-dependent antibody formats, geographical outsourcing, or reduced visibility of antibody production that is embedded within broader research categories, rather than through genuine replacement. Achieving the intended reduction in animal use will therefore require clearer terminology, improved reporting resolution, wider access to non-animal discovery infrastructure and the integration of a Three Rs-aligned decision framework into licensing expectations.

One Health initiatives are modern paradigms for research and health care practices in various fields. Concrete definitions of the One Health framework, however, remain heterogeneous, leading to conceptual problems and uncertainties in the application of the framework. This article discusses several approaches to the One Health concept, and their associated consequences, with special focus on animal experimentation. The first issue addressed is how One Health should be defined, as well as what (and who) should be considered within a One Health approach. In order to shed further light on this, we explore the history of animals in biomedical science, highlighting historical milestones in the use of animal models, as well as the development and current state of ethical considerations in the field of animal experimentation. The second issue comes with the inclusion of animal experimentation per se as part of the One Health concept. Therefore, particular attention is paid to bioethical principles and the resulting problems that can arise when applying them to the One Health concept. Arguments such as the idea of inequality between humans and non-human animals, and the premise that all actions are done for the benefit of humans, are raised and then used to explore the question of whether the One Health concept is compatible with existing bioethical principles. Based on the bioethical principles of protecting the environment, the biodiversity and biosphere, this paper seeks an inclusive perspective of the One Health concept. Successful solutions will be based on this concept, which embraces all living beings. The authors conclude that a multispecies ethics approach could help create a more ethical ecosystem that is aligned with the wellbeing of all life on a shared planet.

This paper critically examines the legal coherence of the EU's chemical safety regulations, REACH and CLP, in light of the EU's commitment to the phasing out of animal testing. While both instruments express normative support for non-animal approaches, their operational provisions remain structurally biased toward animal-based evidence. This bias persists despite scientific concern about the predictive value of animal data, and the demonstrable inefficiency of animal testing for chemical safety assessment, as well as the growing availability of scientifically sound non-animal methodologies. Alongside cultural and institutional barriers, such as regulatory conservatism and a rigid validation system, legal obstacles embedded in the legislative design of REACH and CLP also impede a paradigm shift away from animal testing. Applying the principle of proportionality, the article argues that the animal-centric operational provisions of these regulations are: (i) neither suitable nor necessary to ensure a high level of human health protection; and (ii) that they disproportionately encroach upon animal welfare, in relation to their benefits. The article therefore calls for a legislative recalibration, to realign REACH and CLP with primary Union law. Without such changes, the EU Roadmap toward the phasing out of animal testing in chemical safety assessment, risks having limited practical effect.