I. Introduction

Animal Experimentation has a long history in science, with examples of the earliest experimental studies using animals dating back to ancient Greece and Rome. However, its systematic use in modern scientific research began largely in the 19th century, with the development of vaccines such as rabies and anthrax by the eminent French chemist Louis Pasteur. Since then, the use of animals in laboratory research has expanded significantly and has become a common part of the research and drug development processes.^[1]

Concern and attention to the welfare of laboratory animals are also not new. The debate over the use of animals in scientific research is a long-standing issue, as reflected in the activities of various animal-welfare and anti-vivisectionist groups dating back to the 19th century.^[2] It is a deeply controversial topic, with some calling for the complete abolition of all animal experimentation and others expressing their strong support for it. Opponents usually base their view on the suffering of the animals involved, while proponents emphasize the scientific benefits of the experiments and the lack of viable alternatives.^[3]

Therefore, although animal experimentation has contributed to many discoveries and understandings of various aspects of life sciences, increasing awareness of animal sentience and their experience of pain and suffering has led to strong opposition to animal experimentation among many scientists and the general public.^[4] Furthermore, “the usefulness of extrapolating animal data to humans has been questioned”^[5] in the literature “because of differences between the species.”^[6] It has been argued that “[s]everalstudies have shown that even the most promising findings from animal research often fail in human trials and are rarely adopted into clinical practice.”^[7] For example, “in stroke medicine, despite decades of immense human, animal, and financial investment, animal models have failed to yield a single neuroprotective treatment for humans.”^[8] However, a significant portion of animal model-based research suffers from methodological quality deficiencies. Also, the discrepancy between the results of animal experiments and clinical trials can be due to factors such as bias, random error, or the inability of these models to accurately and adequately represent human diseases.^[9]

Efforts to replace, reduce, and refine the use of animals in research have been increasing for decades, and in some areas, animal use is declining,^[10] as a growing number are calling for a complete halt to animal research. The use of animals in research and experimentation is a major ethical and scientific issue worldwide, and the and there is a growing movement to reduce or ultimately end animal testing through the replacement of animal models with alternative methods.^[11] For example, “both the European Medicines Agency (EMA)^[12] and the Center for Drug Evaluation and Research (CDER), which is part of the Food and Drug Administration of the United States (FDA), fully support the 3Rs” (replacement, reduction, and refinement).^[13] However, these agencies simultaneously emphasize the necessity of animal studies and continue to consider them a prerequisite for the initiation of human clinical trials.^[14] 

This article examines the European Union (EU) legal framework on animal testing and analyses the extent to which it has, in practice, been able to limit the use of animals in scientific research and pave the way for the use of New Approach Methodologies (NAMs). NAMs “are defined as any technology, methodology, approach, or combination thereof that can be used to replace, reduce, or refine animal toxicity testing and may allow for more rapid or effective prioritization and/or assessment of chemicals.”^[15]

In Europe, Directive 2010/63/EU^[16] establishes a set of measures aimed at the protection of animals used for scientific purposes, including animals used in basic and applied research, in particular for the production of medical products for humans and animals, as well as for the safety and efficacy testing of products and substances.^[17] Therefore, the study focuses on Directive 2010/63/EU^[18] and its amendments, Delegated Directive (EU) 2024/1262,^[19] while also addressing some of the reported implementation challenges in practice.

The European Union has been chosen as an advanced regulatory case study to provide “lessons learned” for countries and legal systems that still lack comprehensive regulations on animal testing or are in the process of designing modern laws by examining the strengths, challenges, and limitations of this framework.

This article argues that the EU legal framework governing the use of animals in scientific research is not merely a set of technical rules for a specialized field but an example of how ethical values can be integrated into the EU’s public law system. The main question is whether the EU has managed to make the 3Rs binding and effectively enforceable through its coordination and monitoring instruments, or implementation gaps and institutional diversity among Member States have made this goal challenging.

The study seeks to identify areas that can be considered as grey spots, areas that, within the existing legal framework and guidance documents, may give rise to animal welfare concerns, but could potentially be addressed through targeted reform. The analysis is presented in a constructive manner and is not intended to be a purely critical position, but rather to present the EU regulatory framework as a model that can be exploited by other legal systems.

II. Methodology

This article adopts a doctrinal legal research approach and examines the European Union (EU) legal framework governing the use of animals in scientific research. The analysis relies on binding legal instruments, official interpretative materials, and selected scientific literature regarding animal welfare regulation.

The study examines legal obligations and mechanisms for implementation, oversight, and accountability, and analyses the relationship between the normative design of regulations and the implementation challenges at the EU level. It does not use quantitative methods, statistical comparisons, or country-by-country analysis; instead, it focuses on a structural doctrinal analysis of the gap between normative design and the practical implementation of regulations, and the analysis of implementation differences is based primarily on secondary literature and reported examples, rather than a comprehensive dataset.

III. Historical Background of Animal Use and Experimentation Regulation in the European Union

As this article does not intend to provide a comprehensive historical analysis of the evolution of animal experimentation regulations in the European Union, the aim of this section is simply to provide a brief historical overview of some of the important developments and legal instruments that have contributed to the formation of the EU regulatory framework for the use of animals for scientific purposes. Therefore, only some of the most important legislative milestones and related developments are briefly mentioned to provide the necessary context for examining the current legal framework.

The first national law in the world to regulate the use of animals in scientific research was the Cruelty to Animals Act (1876),^[20] enacted in the United Kingdom. This law is considered the starting point for the formation of legal frameworks in the field of Animal Welfare in Europe.^[21]

Concerns about animal welfare led to the formation of ethical and legal frameworks for animal welfare and paved the way for the 3Rs principles proposed by William Russell and Rex Burch in 1959.^[22] These principles, developed with the support of the Universities Federation for Animal Welfare (UFAW)^[23] and the scientific guidance of Sir Peter Medawar, established an ethical framework that gradually became a normative reference for scientists and policymakers.^[24]

Figure 1. The 3Rs Principles^[25]

The 3Rs approach includes three fundamental principles in animal-based research, as illustrated in Figure 1: Replacement, meaning the use of alternative research methods instead of animal experiments whenever possible; Reduction, to minimize the number of animals used in research; and Refinement, which refers to the use of methods and strategies to reduce pain, suffering, and stress of animals during experiments.^[26] In recent literature, concepts such as Responsibility and Refusal (rejection of projects that lack sufficient scientific justification) have also been added to these principles, reflecting the increasing demand for more rigorous ethical accountability.^[27]

The European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (ETS No. 123), adopted in 1986 within the framework of the Council of Europe, is an intergovernmental instrument that sets common minimum standards for animal welfare for the Member States.^[28] In the same year, Directive 86/609/EEC^[29] was adopted as the first EU binding directive aimed at harmonization across Member States of national regulations on the use of animals in scientific research. It included minimum requirements for the welfare and husbandry of animals, including the minimization of pain, and required authorization for persons and institutions involved in animal research. The Directive emphasized the use of alternative non-animal methods wherever possible and practical, and encouraged the development of new methods for the replacement of animal testing. In cases where the use of animals was still necessary, the Directive called for the use of a minimum number of animals and for the minimisation of pain in the research process.

However, the text of the Directive did not explicitly refer to the principles of the 3Rs.^[30] Although overlapping in time, these two instruments operate within different institutional frameworks and have played complementary roles in shaping the European regulatory system in the field of animal experimentation.^[31]

The European Union subsequently took important steps towards restricting and progressively phasing out animal testing in certain areas, in particular in cosmetics and chemicals. In this context, Directive 2003/15/EC,^[32] by imposing specific restrictions on the marketing of cosmetic products tested on animals, marked the gradual adoption of an alternative approach in EU regulation. This trend was further strengthened by the adoption of Regulation (EC) No 1223/2009,^[33] which, in line with the 3Rs principle, prohibited animal testing for both the finished product and its ingredients and, at the same time, prohibited the import and placing on the EU market of products whose final formulation had been tested on animals. In the field of chemical regulations, Commission Regulation (EC) No 440/2008^[34] established the test methods used to determine the physicochemical properties, toxicity, and ecotoxicity of chemicals under Regulation (EC) No 1907/2006^[35] (REACH^[36]).^[37]

Ultimately, the main basis for the regulation of animal experimentation in the European Union was established by Directive 2010/63/EU on the protection of animals used for scientific purposes.^[38]

IV. Directive 2010/63/EU and the 2024 Commission Delegated Directive Amendment

The Directive 2010/63/EU has a systematic structure, with each chapter addressing one of the main aspects of the use of animals in scientific research. “Chapter I – general provisions” establishes the circumstances in which the Directive applies and the purposes for which animals can be used, defines key terms, and establishes the principle of the 3Rs as a guiding framework for animal experiments. “Chapter II – provisions on the use of certain animals for procedures” sets out restrictions on the use of endangered species, non-human primates, animals taken from the wild, and stray and feral animals of domesticated species, and introduces the use of purpose-bred animals as the standard approach for common laboratory species. “Chapter III – procedures” contains more detailed provisions for the practical implementation of the 3Rs, including through experimental methods, anaesthesia, severity classification, reuse of animals, endpoints, sharing organs and tissues, and rehoming. “Chapter IV – authorization” covers the requirements for animal breeders, suppliers, and users in terms of conditions of care, responsibilities, training of personnel and advisory bodies, and also addresses inspections and requirements for project evaluation, authorization, and retrospective assessment. “Chapter V – avoidance of duplication and alternative approaches” focuses on avoiding duplication of testing and the recognition, development, and validation of NAMs, and “Chapter VI – final provisions” governs the rules for implementation, adaptation to technical progress, delegated powers, reporting obligations, safeguard clauses, review mechanisms, competent authorities, and transitional and final provisions. Finally, seven annexes provide additional and detailed information that supports and complements some of the provisions of this Directive.^[39]

In EU law, the scope of legal protection for animals is limited in scope and applicable only under specific conditions. According to Article 1(3) of Directive 2010/63/EU, this framework applies to animals used in “procedures”, intended for such use, or bred for such use. In terms of species, the system is mainly limited to (1) live non-human vertebrate animals, including independently feeding larval forms, and foetal forms of mammals as from the last third of their normal development; and (2) live cephalopods. Therefore, not all animal species and all forms of animal use fall under this protection system.^[40]

The use of invertebrates as alternatives to animal research has also raised specific ethical questions. Although invertebrates are widely used in experiments due to their relatively simple maintenance and culture, shorter life cycle, and usefulness as biological models, they are often not covered by animal welfare legislation (except cephalopods), and their welfare is largely ignored. This situation, especially given the widespread use of some species on a scale of millions of specimens in a laboratory, has raised concerns about ethical and regulatory gaps in the research use of invertebrates.^[41] “Cephalopods have been included within the scope of the (…) Directive on the grounds that, as for vertebrates, there is scientific evidence of their ability to experience pain, suffering, distress and lasting harm.” (Recital 8 of the Directive 2010/63/EU).^[42] However, “[i]nvertebrates, with the exception of cephalopods, are not included in this description arguably due to the belief that they do not experience pain, suffering, or distress.”^[43]

According to Article 5 of Directive 2010/63/EU, the use of animals in scientific research is only permitted if the purpose of the research falls within one of the specified areas. These purposes include basic research, applied and translational research aimed at the prevention, diagnosis, or treatment of diseases, assessment or regulation of physiological conditions, animal welfare, environmental protection, species preservation, education and training, and forensic inquiries. Article 5 is directly related to the 3Rs and the development and acceptance of NAMs and plays an important role in limiting the scope of animal testing and strengthening the replacement approach.^[44] The importance of Article 5 lies in the fact that it defines the scope of permitted purposes for the use of animals, thereby establishing a link between scientific necessity and ethical legitimacy. It restricts the use of animals in research, which aligns with a utilitarian approach to determining whether an act is ethical. This approach focuses on the consequences of an act, arguing that the act is acceptable if its overall consequences are beneficial. Therefore, in a utilitarian approach, the aim may justify the means.^[45]

Commission Delegated Directive (EU) 2024/1262^[46] of 13 March 2024 amended Directive 2010/63/EU of the European Parliament and of the Council as regards the requirements for establishments and for the care and accommodation of animals, and as regards the methods of killing animals.

The main legal requirements illustrated include:

A. Provisions under Directive 2010/63/EU

• Replacement,^[47] Reduction and Refinement (3Rs) (Article 4)^[48]
• Requirements for Projects (Project Authorisation, Application for Project Authorisation, Project Evaluation, and Granting of Project Authorization) (Articles 36, 37, 38, 40)^[49]
• Retrospective Assessment (Article 39)^[50]
• Classification of Severity of Procedures (Article 15)^[51]
• Requirements for Personnel Competence and Training (Competence of Personnel, Specific Requirements for Personnel, Designated Veterinarian) (Articles 23–25)^[52]
• Transparency through Non-Technical Project Summaries (NTPS) (Article 43)^[53]

B. Provisions of Directive 2010/63/EU Amended by Commission Delegated Directive (EU) 2024/1262

• Requirements for Establishments and for the Care and Accommodation of Animals (Annex III to Directive 2010/63/EU, amended by Commission Delegated Directive (EU) 2024/1262)^[54]
• Methods of Killing Animals (Annex IV to Directive 2010/63/EU, amended by Commission Delegated Directive (EU) 2024/1262)^[55]

Within the framework of Directive 2010/63/EU, animals used in scientific experiments must receive appropriate care and treatment, and several main requirements are defined in this regard. Animals must be kept in cages that are big enough, and their environment must be species-specific and in accordance with the standards set out in Annex III.^[56] Social animals must also be kept together, and environmental enrichment techniques must be used to increase their range of activities, in particular to encourage physical activity, exploration, and cognitive activities in line with their natural behaviour.^[57] In addition, an animal welfare body must be established to advise on animal welfare issues as well as on new methods of replacing, reducing, and refining the use of animals (the 3Rs principle). This body should include a person responsible for the welfare of animals and should be advised by a designated veterinarian, and its work should be supported by national committees in each EU country. The methods of killing should also be such as to minimise pain, suffering, and distress to the animals and should only be carried out by persons with the required skills, at the breeder, supplier, or user establishment, in accordance with the methods set out in Annex IV.^[58] Finally, in general, all persons working with animals should have appropriate training and practice, and their competence should be assessed before working unsupervised.^[59]

Directive 2010/63/EU reflects the ethical principles that underpin animal welfare legislation, in that animal welfare is considered a public good, and legal protection is based on the recognition that sentient animals^[60] are capable of experiencing both suffering and positive experiences.^[61] However, the successful implementation of the standards depends on the proactive and coordinated engagement of multiple stakeholders, from the breeding, housing, and handling of experimental animals to the design and reporting of experiments.^[62]

V. Challenges and Limitations of the EU Legal Framework

Examining the challenges and limitations of the EU legal framework on animal testing is an integral part of assessing the practical functioning of this regulatory system. Even in frameworks that are legally structured and ethically advanced, there is a gap between normative objectives and how regulations are implemented in practice. The identification of these gaps is not intended to question the existing legal foundations, but to better understand the obstacles that can affect the realization of the 3Rs principles and animal welfare. Despite recent structural reforms, including Directive 2010/63/EU and Delegated Directive (EU) 2024/1262, there are still gaps and limitations that prevent the full realization of the legal objectives, in particular compliance with the 3Rs principles and ensuring animal welfare.

These gaps are briefly categorized in the following sections.

1. Implementation Gap – Variation between Member States

Non-compliance with EU rules may be broadly divided into two categories. The first category includes cases where Member States do not implement directives correctly and promptly into their domestic law (legal implementation/transposition). Since directives are the main instrument for harmonizing EU policies, in order to become effective, they must first be implemented in the domestic legal systems of Member States by adopting new laws, amending existing laws or removing incompatible provisions.^[63] The second category concerns non-compliance in the application of EU rules; that is, cases where directives have been legally implemented into the domestic system, but are not properly applied in practice. In this case, the issue is not the lack of legal implementation, but the non-compliance of Member States’ behaviour with the rules and requirements of EU law in the implementation phase.^[64]

Directive 2010/63/EU lays down common EU measures for the protection of animals used for scientific purposes, but the way in which this Directive is implemented varies between EU Member States, particularly in areas where it lacks specific and detailed provisions.^[65] For example, Article 23(2) of the Directive 2010/63/EU,^[66] which specifies the competence of personnel for the care and use of animals for scientific purposes, requires that personnel involved in the care and use of animals have received adequate education and training prior to undertaking any procedure. However, the nature and extent of such training are not specified in detail. To facilitate the fulfilment of the education and training obligations of Member States, the European Commission has developed a common EU Education and Training^[67] Framework.^[68] According to Eurogroup for Animals, “[m]ost Member States mentioned that they follow the EU Education and Training Framework document, but did not explicitly explain how competence should be demonstrated beyond having obtained the required education.”^[69]

Furthermore, as the Directive does not prescribe how competence must be demonstrated, only that a person is named as being responsible for ensuring competence, Member States can decide independently how such competence can be demonstrated.^[70]Moreover, the structure of the competent authority differs across countries. For example, Directive 2010/63/EU stipulates that the processes relating to authorization of establishments, inspections, project evaluation, project authorization, and retrospective assessment must be carried out by a competent authority or competent authorities.^[71] However, in practice there are significant differences between Member States in the allocation of these tasks; some countries have only one competent authority for all five tasks (such as Denmark, Ireland, Cyprus, Malta, Portugal and Slovenia), while in others the number of authorities is very high (e.g., 112 authorities in Spain, 245 in Italy, 328 in Poland and 573 in Germany).^[72] This institutional fragmentation may create challenges for the implementation of a coherent and uniform approach at the Union level, as the multiplicity of authorities can increase the risk of inconsistencies and differences in the interpretation and application of the rules.^[73]

If the legal implementation or practical application of Directive 2010/63/EU and Delegated Directive (EU) 2024/1262 differ across Member States, the Directive’s harmonizing function may be undermined, particularly concerning the objective^[74] of achieving uniform rules on the use of animals for scientific purposes. It should be ensured that differences arising from various implementation methods are not too wide to prevent harmonization.^[75]

2. Insufficient Attention to the Psychological Well-Being of Animals

Although Directive 2010/63/EU addresses the behavioural, social and psychological welfare needs of animals through requirements such as social housing, environmental enrichment and consideration of the natural behaviours of the species,^[76] current minimum legal requirements for animal housing are still insufficient in meeting all the needs of all animals. Animals’ lives in captivity are monotonous, which leads to boredom, learned helplessness, depression, and abnormal behaviours.^[77]

The concept of stress is central to the concept of animal welfare.^[78] According to Bailey, “[r]ecurrent acute and/or chronic stress can affect all vertebrate species, and can have serious consequences. It is increasingly and widely appreciated that laboratory animals experience significant and repeated stress, which is unavoidable and is caused by many aspects of laboratory life, such as captivity, transport, noise, handling, restraint, and other procedures, as well as the experimental procedures applied to them.”^[79]For example, if a young animal is separated from its mother, it may experience undernutrition and cold exposure, which trigger a series of physiological and behavioural responses, including decreased metabolism due to undernutrition and shivering due to cold exposure. In addition, the absence of the mother can trigger an emotional response such as fear and psychological distress. In the long term, feelings of maternal absence may lead to a negative affective state such as anxiety. This simple example illustrates the complex relationship between psychological and physiological components in response to stressors.^[80] Furthermore, although animals may experience stressors in their natural environments, the stressors encountered in the laboratory are different, unavoidable, and potentially more chronic than in nature. Exposure to these stressors can not only affect the well-being of animals but also reduce the validity and generalizability of the results of animal experiments.^[81]

3. Insufficient Transparency and Data Quality in Non-Technical Summaries (NTSs)

A Non-Technical Summary (NTS) is a short, easy-to-understand description of a project. It is written in simple language and is intended to help the public understand the reasons and methods for the project, including how the 3Rs principles will be implemented.^[82] As Taylor and others state, “[a]ll countries of the European Union (EU) are required to publish ‘non-technical summaries’ (NTS) of research projects that use animals. (…) While there has been some improvement in reporting, many NTS still fail to adequately describe the harm that animals will experience.”^[83] This weakness may make it difficult to independently assess and monitor the success of the 3Rs.

4. Lengthy Validation Processes and Delayed Regulatory Acceptance of NAMs

The validation process of alternative methods is inherently time-consuming due to the need for accurate, impartial, and scientific evaluation, as both the reliability and relevance of the method to the intended effects must be confirmed; this includes examining stability and reproducibility in one laboratory and then transferability and repeatability in at least two other independent laboratories (inter-laboratory reproducibility/ring trials). In addition, pre-validation stages, selection and distribution of test materials, training of laboratory staff, and finally independent peer review by EURL ECVAM Scientific Advisory Committee (ESAC)^[84] are also performed, and the totality of these technical and administrative requirements prolongs the validation process.^[85]

Fragmented validation approaches and regulatory acceptance criteria are often inconsistent or unclear. This situation slows progress towards animal-free testing and prevents the full potential of NAMs in transforming toxicological safety assessment.^[86] If regulatory agencies do not consider the NAM to be appropriate for the intended application, the validation process may be halted or delayed. Also, lack of awareness of the existence or precise functioning of a model, the complexity of the validation process, and the need to develop specialized guides for the use of these methods are among the challenges posed to the regulatory acceptance of these methods.^[87]

5. Cross-Border Cooperation with Non-EU Countries

Directive 2010/63/EU does not have extraterritorial application and therefore cannot be directly implemented outside the EU. While Article 4 (the 3Rs Principles) emphasizes the principles of Replacement, Reduction, and Refinement, its implementation outside the EU is difficult to monitor.^[88] When the projects are being transferred to third countries with lower standards, companies may need to perform testing to comply with these markets, regardless of legal requirements within the EU.^[89] The international cooperation of regulatory frameworks and acceptance criteria for NAMs is necessary because even if a single country or region requires traditional animal testing for product approval, the overall goal of reducing animal use will not be achieved.^[90]

6. Financial Pressures and Resource Constraints

Financial pressures and resource constraints can have a major impact on research institutions’ decisions to use animal testing and adopt NAMs. While larger institutions are more able to invest in and deploy NAMs due to access to greater financial resources and government and international support, smaller institutions may face challenges in meeting the costs of animal care and investing in validation and development of alternative methods due to budget constraints.^[91] The level of funding allocated to NAMs should also be considered. Lack of funding can reduce incentives for researchers to switch to non-animal models. Also, in low- and middle-income countries, the government may not have allocated specific funding for NAMs, and the necessary support may not be available for researchers working with non-animal methods.^[92] In addition to a lack of funding, economic barriers to adopting NAMs can include uncertainty about regulatory acceptance and concerns about Return On Investment (ROI). In many cases, research institutions, sponsors, and Contract Research Organizations (CROs) may be reluctant to invest heavily in NAM-based approaches because of the possibility that data generated from these approaches may not be accepted by regulatory authorities in some sectors or jurisdictions.^[93] Also, the transition from animal-based systems to NAMs may require high costs for infrastructure transition, development of specialized expertise, personnel training, and production of dual data packages during the transition period.^[94] In addition, NAMs face entrenched resistance rooted in infrastructure inertia and economic vested interests. Institutions that have previously invested heavily in animal-testing infrastructure may resist the shift to NAM-based systems due to concerns about short-term financial sustainability and reduced economic returns on existing assets.^[95]

VI. Recommendations

Given the gaps and limitations in the EU regulatory framework for animal experimentation in scientific research, the following strategies can help improve animal welfare and strengthen ethical compliance. These recommendations aim to highlight possible paths to improve the implementation of existing regulatory frameworks and are not intended to address each of the identified challenges in a linear manner, but rather focus on broader governance conditions and mechanisms that affect the overall effectiveness of the regulatory system. These recommendations can serve as reference points for policymakers, regulators, and research institutions.

• Enhancing Regulatory Coherence Across Member States:

To reduce inconsistencies in the implementation of EU directives related to animal welfare in scientific research, the following measures can help to strengthen alignment across Member States:

• Develop Harmonized Guidelines: In general, the more detailed and less ambiguous a subject is in a Directive, the more likely it is that the transposition process will lead to similar standards in the Member States. In contrast, harmonisation is less successful when the provisions of the Directive are general and lack detail, and when implementation depends on variable infrastructures and areas that lack sufficient common regulation.^[96] The European Commission should develop specific, detailed harmonised guidelines in areas such as training, project evaluation, transparency, the application of the 3Rs, and inspections to provide interpretative clarity and reduce differences in transposition and implementation across Member States.^[97]
• Strengthening and Harmonisation of Training and Competence Requirements: Each breeder, supplier, and user must, before employment, determine the qualifications required to perform the tasks of Articles 23, 24, and 25 of the Directive 2010/63/EU^[98] and establish a training system with clear standards for theoretical knowledge and practical skills. Initial training and continuing professional development must be provided to ensure consistency in the performance of tasks and that staff are kept up to date. Additional training or supervision is required in the event of intermittent or rare procedures or the introduction of new methods. During inspections, staff qualifications and training records are checked, and each facility should have monitoring mechanisms to maintain competence. It is also beneficial to appoint a competent officer, provide certified training with examination, and keep records for inspection purposes. Finally, training of members of competent authorities should be ensured in areas such as project evaluation, ethics, animal welfare, and the 3Rs under the EU Education and Training Framework document^[99] and the Education and Training Platform form Laboratory Animal Science (ETPLAS) modules^[100] that can provide a good basis for developing these essential skills.^[101]
• Development of Peer Review Mechanisms: Mutual monitoring and review mechanisms between Member States could enhance transparency, reduce differences in implementation, and enable continuous improvement in national systems.^[102]Such peer review-like mechanisms could be linked to the institutional framework established under Article 49 of the Directive 2010/63/EU, which requires Member States to establish national committees for the protection of animals used for scientific purposes. According to Article 49, these national committees exchange information on the functioning of animal-welfare bodies and project evaluation at the Union level and share best practices. Such mechanisms can contribute to greater coordination across Member States and strengthen the implementation of the 3Rs principle.^[103] Moreover, such peer review mechanisms could allow experts from competent authorities in different Member States to assess specific aspects of national enforcement systems. This could allow for the sharing of experiences and lessons learned and the identification of solutions to common enforcement issues and challenges. Peer reviews could lead to recommendations for improving enforcement systems and facilitate mutual learning and the exchange of good practices. This could improve the effectiveness of national regulatory and enforcement systems across the EU and support compliance with Directive 2010/63/EU.^[104]

• Addressing the Psychological & Social Well-Being Dimensions of Animal Welfare:

To improve animal welfare, psychological and social aspects (stress, anxiety, social behaviour) should be systematically assessed and improved:

• Welfare Assessment Tools: “Employ advanced analytical tools”^[105] and the development of standard instruments to assess stress, anxiety, and social behaviour of animals in laboratory settings.
• Environmental Enrichment: Legal requirements to create conditions similar to the natural environment, such as controlled social interaction, toys, and environmental modifications.^[106]
• Integration of interdisciplinary scientific developments: Future revisions of EU directives should adopt a more comprehensive approach to animal welfare, taking into account not only physical health but also the psychological well-being and social behaviour of animals.

• Transparency & Data Quality:

To ensure data quality and transparency in project implementation, the following should be considered:

• Reporting Consistency and Data Quality: Member States should develop and communicate clear and detailed reporting requirements to all breeders, suppliers, and users to ensure consistent and accurate reporting. This can improve data quality, reduce discrepancies in reporting practices, and allow for better comparison of information between Member States.^[107]
• Expansion of Knowledge through Wider Public Access to Private Data: Access to private data by public and independent institutions should be provided to expand the knowledge base for implementing NAMs and, as a result, improve the development and reliability of computational toxicology tools and predictive models.^[108]
• AI-Supported Quality Control: Limited and complementary use of Artificial Intelligence (AI) as a data quality control tool in the laboratory data recording and analysis process: These tools can help identify inconsistencies, data recording errors, and unusual patterns in welfare reports, without replacing existing standards or human evaluation.^[109]

• Accelerating the Acceptance of NAMs (Validation & Uptake of Alternatives):

To accelerate the adoption and expansion of alternative methods (NAMs), the following can be considered:

• Addressing Personal, Cultural and Societal Barriers: To facilitate the adoption of NAMs, personal, cultural, and societal barriers must be addressed, including reliance on established methods and a tendency to use familiar approaches. Also, the mindset shifts necessary to accept and trust new methods must be supported.^[110]
• Pilot and Implementation Projects: Support for pilot-type and implementation projects to accelerate the validation, practical implementation, and uptake of NAMs at an operational scale.^[111]
• Public–Private Partnerships: Partnerships between the pharmaceutical and biotechnology industries and universities to accelerate the development and validation of alternatives.^[112]

• Cooperation with Non-EU Countries

To promote global animal welfare standards, cooperation with non-member countries and compliance with Directive 2010/63/EU are essential, so the following should be considered:^[113]

• Global Agreements: Achieving a global agreement with Non-EU Countries on uniform standards for animal welfare.^[114]
• Increased International Comparability: Share data and accessibility for NAMs that have shown good results so that international comparisons can be made and the regulators across the globe have access to this information in one place and can make informed decisions.^[115]
• International Training and workshops: Training to raise awareness and to inform third countries about animal welfare.^[116]

• Financial Pressures & Resource Constraints:

To overcome resource constraints and financial pressures, targeted support and economic facilitation are needed:

• Dedicated EU Funding: Allocate targeted funding to small and medium-sized institutions that cannot independently invest in NAMs.^[117]
• Expansion and Networking of Shared Facilities: Strengthening capacity and access to shared research facilities and research infrastructures to avoid unnecessary duplication of resources and costs.^[118]
• Financial Incentives: Strengthening competitive grants and creating targeted financial incentives for projects based on NAMs to accelerate the transition to these methods in the European Union.^[119]

VII. Conclusion

The analysis of the EU legal framework on the use of animals in scientific research shows that Directive 2010/63/EU and its recent amendments, including Delegated Directive (EU) 2024/1262, set high standards for the protection of animal welfare and the observance of the 3Rs principles. This framework is considered an advanced model of animal testing regulation compared to many existing legal systems. However, differences in the implementation of the regulations between Member States, the variable quality of NTSs, and the delays in the adoption and application of NAMs still prevent the full achievement of the envisaged legal and ethical objectives.

This situation is an example of the structural tension in EU public law between the objectives of harmonization and the realities of institutional diversity of Member States. Therefore, studying this framework can lead to a deeper understanding of the capacities and limitations of the EU in institutionalizing ethical values through legal instruments.

The experience of the European Union shows that combining a strong legal framework with a commitment to ethical principles, data transparency, and the promotion of education and awareness can play an important role in improving animal welfare and increasing the quality of scientific research. Examining this experience, beyond the European context, can also be instructive for legal systems that still lack comprehensive frameworks on animal experimentation and help develop more effective and ethical regulatory and legal frameworks at the international level. The findings of this study can be useful for policymakers and regulatory bodies, because by identifying implementation gaps and institutional challenges in the European Union legal framework, it provides a set of analytical considerations for designing or amending regulatory frameworks in the field of animal experimentation and animal welfare governance.

VIII. Bibliography

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