It's Time to Review the Three Rs, to Make them More Fit for Purpose in the 21st Century.

The Three Rs have become widely accepted and pursued, and are now the go-to framework that encourages the humane use of animals in science, where no other option is believed to exist. However, many people, including scientists, harbour varying degrees of concern about the value and impact of the Three Rs. This ranges from a continued adherence to the Three Rs principles in the belief that they have performed well, through a belief that there should be more emphasis (or indeed a sole focus) on replacement, to a view that the principles have hindered, rather than helped, a critical approach to animal research that should have resulted in replacement to a much greater extent. This critical review asks questions of the Three Rs and their implementation, and provides an overview of the current situation surrounding animal use in biomedical science (chiefly in research). It makes a case that it is time to move away from the Three Rs and that, while this happens, the principles need to be made more robust and enforced more efficiently. To expedite a shift from animal use in science, toward a much greater and quicker adoption of human-specific New Approach Methodologies (NAMs), some argue for a straightforward focus on the best available science.

Bridging toxicological properties of environmental chemicals between animals and humans using healthy organoid systems.

The application of organoids derived from animal tissues and human-induced pluripotent stem cells to safety assessments of environmental chemicals has been introduced over the last decade. One of the objectives of this approach is to develop an alternative method for animal toxicological studies, while another is to focus on the local reactions of chemicals in each organ/tissue. One of the most important goals is bridging the toxicological properties of chemicals between animals and humans, which may be compared on a level playing field using healthy organoids derived from both animals and humans in vitro, excluding species difference in the absorption, distribution, metabolism, and excretion properties of chemicals in vivo. An overview of the application of organoid systems to safety assessments of environmental chemicals, including general toxicology, developmental toxicology, carcinogenicity, and mutagenicity, was provided herein, and bridging strategies using both animal and human organoids are proposed as a future perspective.

Trends in the use of animals and non-animal methods over the last 20 years.

Despite the importance of the animal testing issue, there has been little presentation in the scientific literature of the trends in animal use. This is crucial to resolve, particularly if we are to measure the impact of initiatives to reduce and replace animal experiments that were recently announced in Europe and the USA. For the first time, the number of animals used between 2002 and 2022 are presented for the EU, key animal-using countries in Europe (the UK, France and Germany), and North America (the USA and Canada). Animal testing is on a slow decrease in the EU, 11% in the last 20 years, but animal use in the UK, France and Germany is at similar levels as it was in 2002. Notably there has been a decrease in the production of genetically altered animals in the UK and a decrease in regulatory testing in the EU. Animal use in Canada has been steadily growing, and figures for the USA are still incomplete as laboratory-bred rodents and some other species are not counted. However, globally, the use of non-animal methods in biomedical research is increasing exponentially; this accelerated in the mid-2010s. The UK appears to be the leader in this field. The technological, regulatory, political and economic factors that might explain these trends are discussed.

Animal testing is an important scientific and ethical issue. Many countries count the numbers of animals they use each year, but it has not been reported recently how the numbers are developing. We need this information if we are to measure the success of initiatives to reduce and replace animal tests that have been recently announced in Europe and the USA. Here, I present the number of animals used in Europe and North America in the last 20 years between 2002 and 2022. There has been little change in the use of animals over this time period. I argue that there have been few regulatory or political drivers over this period that would have influenced change. However, based on the scientific literature, the uptake of non-animal methods is rapidly increasing, which is positive news.

The validation of regulatory test methods - Conceptual, ethical, and philosophical foundations.

Validation establishes the reproducibility and relevance of regulatory test methods, particularly for new approach methods (NAMs) as alternatives to animal testing. While validation concepts provide a framework to assess method suitability, they rarely undergo method-critical assessment. This paper explores the philosophical and ethical foundations of the validation process, drawing from various philosophical traditions and contemporary ethical frameworks. How validation intersects with utilitarian principles, ethics of responsibility, and post-modern critiques is examined, offering a multifaceted perspective on its role in scientific progress and societal values. The paper argues for a paradigm shift in validation, moving beyond traditional animal-based comparisons towards more flexible, fit-for-purpose approaches that embrace emerging technologies and ethical con-siderations. Key ethical principles guiding NAM validation are discussed, including beneficence, non-maleficence, justice, and respect for animal welfare. Integrating these principles with scientific rigor can create a more holistic validation framework that balances human safety, animal welfare, and technological innovation. By critically examining the philosophical underpinnings of validation, this paper aims to stimulate dialogue on reforming the process to better align with contemporary scientific knowledge, ethical standards, and societal expectations. It calls for a more adaptive, transparent, and ethically grounded approach to validation that can accelerate the adoption of innovative and human-relevant toxicological methods while maintaining scientific integrity and public trust.

How do we know if new methods for testing chemical safety are reliable and relevant? This process, called validation, is crucial for protecting public health and reducing animal testing. This paper explores the ethical and philosophical ideas behind validation, asking important questions about fairness, animal welfare, and scientific progress. It is argued that current validation methods need updating to keep pace with new technologies and changing social values. By examining different philosophical viewpoints, ways to make validation more flexible, transparent, and ethically sound are suggested. This matters because better validation can lead to safer products, less animal suf­fering, and more effective environmental protection. The goal is to spark a conversation about how we can improve the way we evaluate new safety testing methods, balancing scientific rigor with ethical considerations and public trust.

Leveraging biomarkers and translational medicine for preclinical safety - Lessons for advancing the validation of alternatives to animal testing.

This article explores the potential of principles established in translational medicine for the use of bio-markers to advance the validation of alternatives to animal testing in preclinical safety assessment. It examines especially how such principles can enhance the predictive power, mechanistic under-standing, and human relevance of new approach methodologies (NAMs). Key concepts from translational medicine, such as fit-for-purpose validation, evidence-based approaches, and inte-grated testing strategies, are already being applied to the development and validation of NAMs. The article discusses challenges in implementing biomarker-based approaches, including standardi-zation, demonstration of relevance, regulatory acceptance, and addressing biological complexity. It also highlights opportunities for advancement through collaborative efforts, technological inno-vations, and regulatory evolution. Case studies demonstrate successful applications of biomarkers in preclinical safety, while future perspectives explore emerging trends like multi-omics integration, microphysiological systems, and artificial intelligence. The article emphasizes the potential of bio-markers and translational science approaches in creating more predictive, efficient, and ethical preclinical safety assessment paradigms in the use of NAMs. Use of biomarkers can enable the mechanistic validation of human-relevant models and provide a means to relate changes in NAMs to animal or clinical study results. By leveraging these tools, the field can work towards reducing reliance on animal testing while improving the accuracy and human relevance of safety predictions.

This article examines how biomarkers and translational science principles can improve safety testing without using animals. Biomarkers are quantifiable indicators of biological processes. Some of these can predict disease progression or drug effects. Translational science aims to apply laboratory findings towards clinical benefits. The article explores how combining these approaches can create better, more human-relevant and validated alternatives to animal testing. It discusses challenges that the field faces, including standardization of methods and getting regulatory acceptance. It also highlights opportunities, like integration with emerging technologies and increased global collabo­ration. The ultimate goal is to improve human health by streamlining NAM validation processes, i.e., show that new safety tests are more accurate, efficient, and ethical than current animal-based methods.

E-validation - Unleashing AI for validation.

The validation of new approach methods (NAMs) in toxicology faces significant challenges, including the integration of diverse data, selection of appropriate reference chemicals, and lengthy, resource-intensive consensus processes. This article proposes an artificial intelligence (AI)-based approach, termed e-validation, to optimize and accelerate the NAM validation process. E-vali-dation employs advanced machine learning and simulation techniques to systematically design validation studies, select informative reference chemicals, integrate existing data, and provide tailored training. The approach aims to shorten current decade-long validation timelines, using fewer resources while enhancing rigor. Key components include the smart selection of reference chemicals using clustering algorithms, simulation of validation studies, mechanistic validation powered by AI, and AI-enhanced training for NAM education and implementation. A centralized dashboard interface could integrate these components, streamlining workflows and providing real-time decision support. The potential impacts of e-validation are extensive, promising to accel-erate biomedical research, enhance chemical safety assessment, reduce animal testing, and drive regulatory and commercial innovation. While the integration of AI and machine learning offers sig-nificant advantages, challenges related to data quality, complexity of implementation, scalability, and ethical considerations must be addressed. Real-world validation and pilot studies are crucial to demonstrate the practical benefits and feasibility of e-validation. This transformative approach has the potential to revolutionize toxicological science and regulatory practices, ushering in a new era of predictive, personalized, and preventive health sciences.

Validating new methods to replace traditional animal testing for chemicals can be slow and costly, often taking up to ten years. This article introduces e-validation, an artificial intelligence (AI)- powered approach designed to speed up and improve this process. By using advanced computer techniques, e-validation selects the best chemicals for testing, designs efficient studies, and inte­grates existing data. This approach would cut validation time and use fewer resources. E-validation includes a smart system for choosing test chemicals, virtual simulations to predict study outcomes, and AI tools to understand the biological effects of chemicals. It also provides training in these new methods. E-validation could accelerate medical research, improve chemical safety, reduce the need for animal testing, and help create safer products faster. While promising, this new approach will need real-world testing to prove its benefits and address potential challenges.

Building knowledge of NAMs through risk science.

The 12th World Congress on Alternatives and Animal Use in the Life Sciences provided a platform for mobilizing and exchanging knowledge on the advancements in science and technology. It also provided an opportunity for experts to discuss how to accelerate the adoption of new strategies and tools. One of these recommendations advocated the need to bridge the gap between the next generation of scientists who have yet to learn about 'New Approach Methodologies' (NAMs) and the current generation of thought leaders who have pioneered the development and validation of these non-animal approaches to toxicological risk assessment. Consequently, a mini-course, held at Canada's University of Ottawa, was developed for students, aged 13-16 years, interested in learning about risk science and how NAMs can be used to inform human health risk assessment. This course also served as a platform for creating a virtual training roadmap, provided in this paper, thereby bringing this knowledge to a broader audience of learners who are establishing their careers in the field of risk science.

The new paradigm in animal testing - "3Rs alternatives".

Regulatory studies have revolutionised over time. Today, the focus has shifted from animal toxicity testing to non-animal for regulatory safety testing. This move is in line with the international 3Rs (Replacement, Reduction, and Refinement) principle and has also changed the regulator's perspective. The 3R principle has stimulated changes in policy, regulations, and new approaches to safety assessment in drug development in many countries. The 3Rs approach has led to the discovery and application of new technologies and more human-relevant in vitro approaches that minimise the use of animals including non-human primates, in research and improve animal welfare. In 2016, the European Medicines Agency published the Guidelines on the principles of regulatory acceptance of 3Rs testing approaches, followed by a conceptual paper in 2023 to align with current 3R standards. Additionally, the United States Food and Drug Administration passed new legislation in 2023 that no longer requires all new human drugs to be tested on animals, which will change the current testing paradigm. This review paper provides the adoption of the 3Rs and the current regulatory perspective regarding their implementation.

Lessons for the Future of NAMs from History, Philosophy and Social Studies of Science.

This paper explores what we can learn from the humanities and social sciences about how standards operate in and around science, in order to understand more about how 'the gold standard' can be shifted away from the use of animals in research and testing, and toward New Approach Methodologies (NAMs). These fields allow us to consider potential futures of NAMs as alternatives, replacements, or complements to animal use in testing and research. As we demonstrate, the questions that we pose and how they are framed are as important as the answers that result. Rather than asking how to 'redefine the gold standard', norms and expectations for NAMs must be actively debated and transparently defined. These considerations would be based, in part, on what has been learned in the past from non-human animal models and systems, but also use the norms within the fields from which the NAMs derive in light of the rich broader contexts within which they are being developed. As we argue, notions such as 'a gold standard' are limited and must be replaced by contextualised standards that depend on the scientific, sociocultural and other factors that contribute to our understanding of a particular method (new or otherwise) as 'good' for a particular purpose.

With What Should We Replace Nonhuman Animals in Biomedical Research Protocols?

Historically, most discussions about nonhuman animal experimentation consider what has become known as the 3 R's: refinement, reduction, and replacement. Refinement and reduction receive the most attention, but recent modeling advances suggest that suitable replacement of nonhuman animal testing would bolster human research and increase translatability to human health outcomes. This article discusses these modeling advances and advocates their use, especially as replacements to nonpredictive nonhuman animal protocols, and discusses growing momentum in biomedical research communities and federal agencies that favors replacement of animal testing.

Development of a monoclonal antibody sandwich ELISA for the quality control of human and animal tetanus vaccines.

Antigen identity, quantity and integrity are key factors to be evaluated as part of consistency testing of tetanus vaccines. Here we have developed a monoclonal antibody sandwich ELISA to measure the relative amount and quality of tetanus toxoid (TTxd) in human and animal tetanus vaccines. The ELISA is highly specific, has good dilutional linearity, and is suitable for detecting TTxd in a range of different products. We have demonstrated the ability of the assay to discriminate between batches of different content, using vaccine batches that had been prepared to contain differing amounts of TTxd, and of different quality, using samples of non-adjuvanted TTxd that had been exposed to sonication and final lot vaccines that had been exposed to heat or oxidative stress. We have also demonstrated successful transfer of the method to other laboratories and have shown that different tetanus antigen materials may be able to serve as a reference antigen for standardization of the method. The results show this test has the potential to play a key role in a control strategy no longer including an in vivo potency test.

Tetanus vaccines help to protect against tetanus infection. Currently, animal tests are used to ensure the potency of such vaccines. Since these tests were first introduced, there have been improvements in non-animal technologies that can be used to ensure consistent production of potent vaccine batches. To demonstrate that a new batch of tetanus vaccine is consistent with a previous batch of known potency, the quality and amount of the component that stimulates the immune response upon vaccination must be assessed in comparison. We have developed an assay that can measure the quality of a range of different tetanus vaccine product types. The assay is very specific and reliable, and different laboratories obtained comparable results, showing that the assay is suited for routine use. Once validated by manufacturers and accepted by regulators, this assay will greatly reduce the number of animals needed for batch release of tetanus vaccines.

An alternative method for comparing hemorrhagic activity of snake venoms following one of the 3R's principles.

Venom-induced hemorrhage analysis usually is performed by Minimum Hemorrhagic Dose (MHD), however a similar method can be used to compare venoms with fewer laboratory animals. Our work compared the MHD of five different venoms, with the size of hemorrhagic spot, finding good correlations in the results. Considering the 3Rs principle, we propose the use of the hemorrhagic spot method to compare hemorrhagic activity of snake venoms, rather than using the MHD method, since the first one needs 5 times less animals than the other.

An update on nonhuman primate usage for drug and vaccine evaluation against filoviruses.

INTRODUCTION: Due to their faithful recapitulation of human disease, nonhuman primates (NHPs) are considered the gold standard for evaluating drugs against Ebolavirus and other filoviruses. The long-term goal is to reduce the reliance on NHPs with more ethical alternatives. In silico simulations and organoid models have the potential to revolutionize drug testing by providing accurate, human-based systems that mimic disease processes and drug responses without the ethical concerns associated with animal testing. However, as these emerging technologies are still in their developmental infancy, NHP models are presently needed for late-stage evaluation of filovirus vaccines and drugs, as they provide critical insights into the efficacy and safety of new medical countermeasures. AREAS COVERED: In this review, the authors introduce available NHP models and examine the existing literature on drug discovery for all medically significant filoviruses in corresponding models. EXPERT OPINION: A deliberate shift toward animal-free models is desired to align with the 3Rs of animal research. In the short term, the use of NHP models can be refined and reduced by enhancing replicability and publishing negative data. Replacement involves a gradual transition, beginning with the selection and optimization of better small animal models; advancing organoid systems, and using in silico models to accurately predict immunological outcomes.

The dimer effect: A refinement approach towards skin sensitization assessment in-chemico using Amino acid Derivative Reactivity Assay.

Skin sensitization is a key endpoint for safety assessment, especially for cosmetics and personal care products. The adverse outcome pathway for skin sensitization and the chemical and biological events driving the induction of human skin sensitization are now well understood. Several non-animal test methods have been developed to predict sensitizer potential by measuring the impact of chemical sensitizers on these key events. In this work, we have focused on Key Event 1 (the molecular initiating step), which is based on formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. There exists three in-chemico assays approved by the Organization for Economic Co-operation and Development-(1) Direct Peptide Reactivity Assay (DPRA), (2) Amino Acid Derivative Reactivity Assay (ADRA), and (3) Kinetic Direct Peptide Reactivity Assay (kDPRA) to quantify peptide/amino acid derivative depletion after incubation with test chemicals. However, overestimated depletion of the cysteine-based peptide/amino acid derivatives is known in such assays because of the dimerization of the thiol group. In this present work, we report the synthesis and structural confirmation of the dimer of N-(2-[1-naphthyl]acetyl)-L-cysteine (NAC) from the ADRA assay to allow simultaneous determination of (a) peptide depletion by quantifying NAC monomer and (b) peptide dimerization by quantifying NAC dimer thereby eliminating the overestimation. We present a case study with three chemicals to demonstrate the importance of this approach. Thus, this simultaneous assay gives a more informed view of the peptide reactivity of chemicals to better identify skin sensitizers.

An Evaluation of the Replacement of Animal-derived Biomaterials in Human Primary Cell Culture.

The likelihood that potential new drugs will successfully navigate the current translational pipeline is poor, with fewer than 10% of drug candidates making this transition successfully, even after their entry into clinical trials. Prior to this stage, candidate drugs are typically evaluated by using models of increasing complexity, beginning with basic in vitro cell culture studies and progressing through to animal studies, where many of these candidates are lost due to lack of efficacy or toxicology concerns. There are many reasons for this poor translation, but interspecies differences in functional and physiological parameters undoubtedly contribute to the problem. Improving the human-relevance of early preclinical in vitro models may help translatability, especially when targeting more nuanced species-specific cell processes. The aim of the current study was to define a set of guidelines for the effective transition of human primary cells of multiple lineages to more physiologically relevant, translatable, animal-free in vitro culture conditions. Animal-derived biomaterials (ADBs) were systematically replaced with non-animal-derived alternatives in the in vitro cell culture systems, and the impact of the substitutions subsequently assessed by comparing the kinetics and phenotypes of the cultured cells. ADBs were successfully eliminated from primary human dermal fibroblast, uterine fibroblast, pulmonary fibroblast, retinal endothelial cell and peripheral blood mononuclear cell culture systems, and the individual requirements of each cell subtype were defined to ensure the successful transition toward growth under animal-free culture conditions. We demonstrate that it is possible to transition ('humanise') a diverse set of human primary cell types by following a set of simple overarching principles that inform the selection, and guide the evaluation of new, improved, human-relevant in vitro culture conditions.

Development of Sensitive In Vitro Protocols for the Biocompatibility Testing of Medical Devices and Pharmaceuticals Intended for Contact with the Eyes: Acute Irritation and Phototoxicity Assessment.

This study introduces a novel in vitro methodology that employs the 3-D reconstructed tissue model, EpiOcular, to assess the irritation and phototoxicity potential of medical devices and drugs in contact with the eye. Our study evaluated diverse test materials, including medical devices, ophthalmological solutions and an experimental drug (cemtirestat), for their potential to cause eye irritation and phototoxicity. The protocols used in this study with the EpiOcular tissue model were akin to those used in the ultra-mildness testing of cosmetic formulations, which is challenging to predict with standard in vivo rabbit tests. To design these protocols, we leveraged experience gained from the validation project on the EpiDerm skin irritation test for medical devices (ISO 10993-23:2021) and the OECD TG 498 method for photo-irritation testing. The predictions were based on the tissue viability and inflammatory response, as determined by IL-1α release. By developing and evaluating these protocols for medical devices, we aimed to expand the applicability domain of the tests referred to in ISO 10993-23. This will contribute to the standardisation and cost-effective safety evaluation of ophthalmic products, while reducing reliance on animal testing in this field. The findings obtained from the EpiOcular model in the photo-irritation test could support its implementation in the testing strategies outlined in OECD TG 498.

Collaborative study for the characterisation of the BINACLE Assay for in vitro detection of tetanus toxicity in toxoids - Part 1.

For several decades the European Pharmacopoeia monographs Tetanus vaccine (adsorbed) (0452) and Tetanus vaccine for veterinary use (0697) required that Specific toxicity and Absence of toxin and irreversibility of the toxoidof each bulk of tetanus toxoids had to be tested by an in vivo toxicity test in guinea pigs before it could be included in vaccines for human or veterinary use. In line with the 3Rs concept of replacing, reducing and refining animal experiments, an in vitro method for the detection of active tetanus neurotoxin (TeNT) has been developed at the Paul-Ehrlich-Institut (PEI, Germany). This method, the so-called BINACLE (binding and cleavage) assay, uses the receptor-binding and proteolytic properties of TeNT for the specific detection of active toxin molecules. Successful in-house validation studies as well as a small-scale transferability study had demonstrated that this method may represent a suitable alternative to the compendial in vivo toxicity test. As a follow up, an international collaborative study aimed at verifying the suitability of the BINACLE assay as a potential alternative to the guinea pig toxicity test for tetanus toxoids was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) under the aegis of its Biological Standardisation Programme (BSP). Within the framework of this study, coded BSP136, a feasibility phase - also referred to as Phase 1 - was run to select and qualify critical study reagents and samples and to assess the performance of the BINACLE Standard Operating Procedure developed by the project leaders. Then the international collaborative study aimed at evaluating the BINACLE, referred to as BSP136 Phase 2, was started. A total of 19 international laboratories (comprising vaccine manufacturers as well as national control laboratories) were supplied with a detailed assay protocol, critical reagents required for the assay, three samples consisting of three different bulk tetanus toxoids donated by major European vaccine manufacturers and one international standard toxoid. Each of the participants was asked to perform three independent BINACLE assays following the provided protocol. The statistical analysis of the results showed that most of the participating laboratories were able to perform the BINACLE assay according to the provided protocol. However, the results obtained by the participants varied widely, and not all the laboratories were able to achieve a sensitive detection of active TeNT. Multiple factors may have contributed to the elevated variability of the BSP136 study results. From an analysis of these factors, strategies were developed to help increase the standardisation of the BINACLE assay and obtain more consistent results in a follow-up validation study, BSP 136 Phase 3 (Part 2), for which the experimental phase took place in 2023. The present manuscript summarises the outcome of Phases 1 and 2, which constitute Part 1 of the BSP136 project.

Endeavours made by trade associations, pharmaceutical companies and regulators in the replacement, reduction and refinement of animal experimentation in safety testing of pharmaceuticals.

Following the European Commission decision to develop a roadmap to phase out animal testing and the signing of the US Modernisation Act, there is additional pressure on regulators and the pharmaceutical industry to abandon animal experimentation in safety testing. Often, endeavours already made by governments, regulators, trade associations, and industry to replace, reduce and refine animal experimentation (3Rs) are unnoticed. Herein, we review such endeavours to promote wider application and acceptance of 3Rs. ICH guidelines have stated 3Rs objectives and have enjoyed many successes driven by global consensus. Initiatives driven by US and European regulators such as the removal of the Abnormal Toxicity Test are neutralised by reticent regional regulators. Stream-lined testing requirements have been proposed for new modalities, oncology, impurity management and animal pharmacokinetics/metabolism. Use of virtual controls, value of the second toxicity species, information sharing and expectations for life-threatening diseases, human specific or well-characterised targets are currently being scrutinised. Despite much effort, progress falls short of the ambitious intent of decisionmakers. From a clinical safety and litigation perspective pharmaceutical companies and regulators are reluctant to step away from current paradigms unless replacement approaches are validated and globally accepted. Such consensus has historically been best achieved through ICH initiatives.

Rethinking the Three Rs: Growing Momentum Toward a New Gold Standard in Australian Research.

Recent years have seen increasing recognition of the scientific, economic and ethical benefits of the use of non-animal models in advancing preclinical research, giving reason to rethink the application and framework of the Three Rs. However, to benefit from the economic advantages of shifting to such alternative methods, and to realise Australia's drug development potential, legislative reform is essential. Such reform should be responsive to international regulations that encourage the use of animal-free methods, and be coupled with a corresponding re-evaluation of current Three Rs frameworks and principles. If these supportive changes, and the recommendations from the 2023 Australian Commonwealth Scientific and Industrial Research Organisation (CSIRO) Futures Non-animal models report, are implemented concurrently - with government support paramount- then a new gold standard for scientific research in Australia could be created in which the use of non-animal models and animal-free methods is the default.