The Institute for In Vitro Sciences (IIVS) has sponsored a series of workshops with the aim of developing recommendations for scientific and technical approaches to conducting in vitro assays to assess potential toxicity within and across tobacco and nicotine products. As well as providing a background overview of oral tobacco and nicotine products, this report focuses on the methods used to generate extracts from these products for in vitro testing, the recommended best testing practices, and suggested areas for future research. This publication was developed by a working group of workshop members, in conjunction with the ninth IIVS in vitro workshop entitled 'In Vitro Models for Testing Oral Tobacco and Nicotine Products and Continued Discussion of In Vitro Models of Toxicity and Disease', which was held in January 2024.
The development of antidepressants has traditionally relied on vertebrate models. Drosophila melanogaster offers a promising alternative approach, yet evidence of antidepressant-like effects is fragmented. The aim of this systematic review was to analyse the literature on the effects of antidepressants on D. melanogaster behaviours. A comprehensive search of a number of bibliographic databases (i.e. PubMed, Web of Science, Scopus and EMBASE) was followed by a further screening of the retrieved articles (i.e. inclusion of studies on any D. melanogaster strain, sex, age, dose or outcome, tested in any behavioural assay; exclusion of reviews, studies on co-treatments and/or on non-prototypical antidepressants, and studies lacking control groups). This resulted in the final selection of 12 articles, comprising 146 studies. Most studies involved adult flies (k = 131), males (k = 56), fluoxetine treatment (k = 109) and courtship outcomes (k = 85). Some studies involved larvae (k = 15), females (k = 39), treatment with other antidepressants (citalopram, k = 12; desipramine, k = 6; amitriptyline, k = 2; imipramine, k = 2) and other outcomes (locomotion, k = 11; sucrose preference, k = 5; light preference, k = 2; aggression, k = 2; climbing, k = 1). Studies on larvae had, on average, larger sample sizes than studies on adult flies, resulting in more conclusive effect sizes. The effect sizes indicated that most studies presented inconclusive effects. However, sucrose preference, courtship and light preference were outcomes with conclusive findings, depending on the conditions (sex, treatment, stress), and thus these may represent more reliable indicators of antidepressant effects. However, the lack of consistency among studies precluded drawing any definitive conclusions on reliable indicators. In addition, incomplete reporting precluded an accurate risk-of-bias assessment. These findings highlight the need for standardisation and transparent reporting, in order to establish flies as a reproducible, ethically acceptable alternative to vertebrates in antidepressant research.
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.
Gabapentin is a structural analogue of gamma-aminobutyric acid. Initially introduced as an anti-epileptic drug, it has also been shown to be effective in the treatment of pain in humans and non-human animals. The main purpose of this study was to test the suitability of the cockroach Gromphadorhina portentosa (Blattodea: Blaberidae) as a non-vertebrate in vivo model for evaluation of the antinociceptive properties of gabapentin. After the intersegmental abdominal membrane administration of different doses of gabapentin (50, 150 and 250 mg/kg), two acute thermal pain tests were performed: the hot plate test and the hot box escape test. In the hot plate test, 50 mg/kg gabapentin was the most potent dose in terms of reducing thermal stimulus-associated movement of the cockroaches; in the hot box escape test, 150 mg/kg gabapentin was the most efficacious dose. Thus, when comparing the two different tests, gabapentin exerted effective antinociception effects in cockroaches at different doses. This is similar to the effects observed in mice and rats exposed to thermal stimuli. Our results therefore demonstrate that G. portentosa could be a suitable non-vertebrate in vivo model for the study of pain, and suggest that it could be effectively used for the evaluation and screening of other analgesic compounds.
The acquisition of surgical skills is an essential component of veterinary training. The use of live animals or cadavers for early surgical skills development is limited by ethical, logistical and financial constraints, highlighting the need for accessible and animal welfare-oriented training alternatives. The objective of this study was to evaluate the effectiveness of a low-cost, balloon-based simulator for teaching basic surgical knot techniques to undergraduate veterinary students with no prior surgical experience. A cohort of 20 students practised slip knots, Miller's knots and transfixation knots, using a low-fidelity balloon simulator under a structured training protocol. Performance was assessed by using standardised rubrics, i.e. execution time and a binary success/failure classification based on air leakage. From the 13th repetition onward, all students successfully executed slip knots and Miller's knots, while competency in the transfixation knot was achieved by the 14th repetition. Execution time decreased significantly from the 11th repetition for all three techniques, indicating progressive improvement in technical efficiency. It was evident that the balloon-based simulator facilitated the acquisition of basic surgical knot-tying skills, and represents an accessible, ethical and effective tool for early-stage surgical training in veterinary education.
The ex vivo Human Organotypic Skin Explant Culture (hOSEC) model utilises skin fragments, mainly obtained from plastic surgery procedures, as the primary source of material. The model is used for testing that requires human tissue, and as the basis for various research models. The objectives of the current study were to review, analyse and summarise the published literature on the use of ex vivo human skin explants in experimental studies in Brazil. The literature search was conducted within three databases, using terms related to explants and study location combined with defined eligibility criteria. Sixteen studies were considered eligible for further detailed analysis in the review. The geographical location of these studies was concentrated in the South-Eastern region of Brazil, and demonstrated a prevalence of explants obtained during plastic surgery procedures. Five main focus areas were identified in the studies, namely: disease pathology, cellular ageing, pharmacological testing of cosmetic ingredients, wound healing, and cell culture optimisation - with pathology-focused models being the most common. Despite the search strategy being capable of identifying the diverse characteristics of the studies, the research protocols used in the studies were heterogeneous. This is an intrinsic limitation of the published literature in general, which prevents direct comparisons and hinders reproducibility. Human skin explants were shown to represent versatile tools, with potential for expansion into other areas. The creation and dissemination of standardised methodologies and guidelines for human skin explant-based research are essential. This information, as well as the promotion of the use of such models, will contribute to a reduction in the use of animals in experiments.
Drosophila melanogaster has been used as a model organism in biomedical research for over a century, facilitating a number of fundamental breakthroughs and Nobel Prize-winning discoveries in genetics, developmental biology and disease mechanisms. This bibliometric analysis assessed 140,962 Drosophila-related publications indexed in the Web of Science Core Collection from 1984 to 2024. Publication output increased rapidly until the late 1990s, with an average annual growth rate of 13%, before stabilising. Co-authorship analysis revealed extensive international collaborative networks spanning multiple continents, while keyword co-occurrence analysis identified seven major research clusters: developmental biology, cell biology, circadian biology, ageing, evolutionary biology, molecular biology and immunology. Temporal trend analyses demonstrated sustained growth in fields such as neuroscience, neurodegenerative diseases, ageing, circadian biology, immunology, pharmacology, toxicology and environmental sciences. These findings provide a comprehensive overview of contemporary Drosophila-based research, highlighting its ongoing global relevance, evolving applications and emerging research frontiers. The unique advantages of Drosophila for investigating complex biological processes establish it as a more ethical and efficient alternative to vertebrate models in modern biomedical science.
We established a 3D model of human NT2/D1-derived early neural progenitor cells immobilised in alginate microfibres as a system for testing the neurotoxicity of energy drinks and their components, either alone, together, or in combination with alcohol. The in vitro system supports the retinoic acid-induced neurogenesis of NT2/D1 cells, and the proliferative capacity of the NT2/D1-derived early neural progenitor cells was maintained in the 3D environment. Cell cycle distribution and the expression of pluripotency markers (SOX2, OCT4 and NANOG), early neural markers (SOX3, PAX6 and miR-219), and Cyclin D1 (a marker of neural commitment), showed profiles characteristic of early neural progenitors. Treatments with an energy drink and its major components (caffeine and taurine) - either alone, together, or in combination with alcohol - had different effects on the proliferative capacity of the NT2/D1-derived early neural progenitor cells in the 2D and 3D models. In the 2D-cultured cells, all treatments except for caffeine led to a significant decrease, while cells within the 3D model exhibited a significant increase after treatment with caffeine, or after combined treatment with energy drink and alcohol. Preliminary findings suggesting that there were no treatment effects on OCT4 and PAX6 protein expression in either model, should be further confirmed. This human cell-based 3D model could potentially represent a rapid and cost-effective system for assessing the acute and long-term neurotoxicity of various compounds.
Traditionally, animal testing has been considered the 'gold standard' in determining potential effects of chemicals on humans. However, this dogma is increasingly being questioned, not only due to ethical and financial implications, but also because of the poor translatability of the results of animal tests to humans. Therefore, there is a need to modernise the concept of the gold standard and ensure that any new approach is flexible, adaptable and as future-proof as possible. Herein, we reflect on recent suggestions for updating, or redefining, the traditional gold standard of animal testing and propose a new definition. This proposal focuses more on the process of answering a specific question, using all available tools, rather than seeking to recapitulate an animal test. New Approach Methodologies (NAMs) provide an ever-expanding array of tools that can assist product development and play a key role in chemical assessment strategies. Ten recommendations for developing fit-for-purpose NAMs, to increase their acceptance and accelerate their adoption, are presented.
Recent global initiatives are accelerating the shift toward human-centric approaches, reducing reliance on animal models in preclinical research and other domains. In this changing landscape, objectively evaluating the scientific relevance and merit of research involving animal models, and assessing their translational relevance is increasingly critical. Over the past decade, several tools have been developed to assess translational relevance, accuracy/appropriateness and efficacy of preclinical animal models, evaluate risk-of-bias in preclinical research, support harm-benefit analyses, and facilitate the adoption of non-animal replacement strategies. However, the uptake of such tools remains limited. To address this, a Biomedical Research for the 21st Century (BioMed21) Collaboration workshop on 'Evaluating translational value of animal models in preclinical research - Tools, challenges, and strategies', was convened by Humane World for Animals (30 June-1 July 2025). The event brought together tool developers and diverse global interest-holders to review current assessment tools, discuss their strengths, complementarity, limitations and feasibility, and explore opportunities for cross-sector collaboration. This paper summarises key outcomes of these presentations and discussions, highlighting knowledge gaps and barriers to the adoption of these tools and frameworks by researchers, funders and regulators. Strategies to raise awareness and promote the use of the tools and frameworks, to better inform funding decisions, regulatory approval and the appraisal of preclinical research, are also proposed.
Toxicological profiling is a critical component of the regulatory process required to inform the hazard characterisation and labelling of chemicals, thus guiding their safe handling and use. Skin irritation is a key endpoint for regulatory registrations, traditionally assessed by using the Draize rabbit test, which was introduced in 1944. Advances in New Approach Methodologies (NAMs), and particularly those based on reconstructed skin models, have led to more human-relevant alternatives, as compared to the in vivo test method. Here, we report on some groundbreaking high-throughput adaptations to the validated in vitro Skin Irritation Test (SIT; OECD TG 439) with the commercially available EpiDerm™ reconstructed human epidermis (RhE) model from MatTek Corporation. These minor adaptations enabled the safety evaluation of over 3000 chemicals in just ten months. This was necessary due to the large number of chemicals that needed to be pre-screened for dermal safety, after the insect repellent or insecticidal activity was predicted and assessed. The data obtained in the SIT were then used to train a new Artificial Intelligence (AI) tool, designed to predict the skin irritation potential of large libraries of molecules within (but not limited to) the chemical space which the AI tool is based upon. Our work exemplifies the integration of AI with existing non-animal-based approaches, in order to further develop chemical safety assessment by taking advantage of this new technology.
One Welfare is an emerging concept that seeks to integrate the welfare of animals, humans and the environment. This systematic review examines the role of laboratory animal veterinarians in promoting One Welfare within the context of animal use for scientific research. By analysing 65 publications, we explored the diverse roles of laboratory animal veterinarians in advancing animal welfare, supporting human health and promoting environmental sustainability. The reviewed literature described veterinarians' involvement in areas such as animal welfare oversight, implementation of the Three Rs principles, regulatory compliance and ethical review processes. Several themes emerged that are related to the One Welfare framework, namely: veterinary oversight, welfare assessment and implementation, emotional wellbeing, professional support, and contribution to education and training. The literature highlights the potential need for specialised training in laboratory animal medicine, and outlines the challenges veterinarians encounter regarding ethical considerations and cognitive dissonance in research environments. Based on the identified gaps, we propose measures to reinforce the principles of One Welfare, including interdisciplinary collaboration and the adoption of strategies to safeguard the environment (e.g. the use of composting for minimally-infectious waste and digesters for high-impact waste). Additionally, we recommend establishing institutional sustainability committees to oversee governance and resource management across research institutions.
Assessment of the Culture of Care (CoC) within an institution is essential in understanding the gaps in current CoC practices and highlighting what needs to be further improved. As the first responders in laboratory animal facilities, laboratory animal care staff (i.e. laboratory animal technicians and laboratory animal attendants) play a key role in establishing and sustaining a CoC. We employed the previously introduced Capability Maturity Model (CMM) to assess the CoC among Sri Lankan laboratory animal facility staff. A questionnaire-based survey was distributed, in order to assess the current state of a particular facility's CoC, based on the CMM. The survey also included questions to help identify the challenges faced by staff and evaluate the impact of Laboratory Animal Science education and training on improving the CoC. To identify further issues, a roundtable discussion involving a number of laboratory animal care staff was also conducted. This study revealed that most Sri Lankan laboratory animal facilities are currently at Level 1 of the CMM, with some showing early signs of transitioning to Level 2. Lack of management commitment and insufficiency of funds were identified as the key challenges. Lack of knowledge and training, and inadequate understanding and co-operation between co-workers and also with researchers, were stated as additional issues during the roundtable discussion. Thus, we urge training providers, such as the Sri Lanka Association for Laboratory Animal Science (SLALAS), to conduct more targeted training programmes, focusing on good CoC practices, laboratory animal welfare standards and ethical decision-making.
Minimally invasive catheter-based techniques for cardiac diseases, such as patent ductus arteriosus occlusion, pulmonic stenosis balloon valvuloplasty, atrial septal defect occlusion and transseptal approaches, have gained popularity in veterinary cardiology owing to their high success and low complication rates. However, the technical complexity of these procedures and the lack of accessible training opportunities have limited their widespread adoption in clinical practice. In this study, we evaluated the effectiveness of a 3D-printed canine cardiac model as a simulator-based training tool for improving proficiency in these interventional procedures. The 3D model was developed by using computed tomography (CT) data of a dog diagnosed with type IIa patent ductus arteriosus. A virtual prototype of the model was created by segmenting the CT data. A hard-type photopolymer resin was used for 3D printing the model to ensure durability. To enhance the structural stability and applicability of the model, iterative optimisation was carried out by incorporating feedback from testers who used it for the different procedural simulations. The model accurately replicated cardiovascular structures and artificial defects, such as atrial and ventricular septal defects. Patent ductus arteriosus occlusion, pulmonic stenosis balloon valvuloplasty, atrial septal defect occlusion and transseptal approaches were performed, in order to evaluate the compatibility of the model with fluoroscopy. To assess the effectiveness of the model as a training tool, time-trial assessments and pre- and post-training evaluations involving six participants were conducted. The 3D-printed canine cardiac model improved procedural efficiency and understanding of cardiac anatomy. The 3D-printed model of canine cardiac diseases introduced here is scalable and valuable as a teaching tool in veterinary interventional cardiology.
