Different narcotic gases and concentrations for immobilization of ostrich embryos for in-ovo imaging.

In-ovo imaging using avian eggs has been described as a potential alternative to animal testing using rodents. However, imaging studies are hampered by embryonal motion producing artifacts. This study aims at systematically comparing isoflurane, desflurane and sevoflurane in three different concentrations in ostrich embryos. Biomagnetic signals of ostrich embryos were recorded analyzing cardiac action and motion. Ten groups comprising eight ostrich embryos each were investigated: Control, isoflurane (2%, 4%, and 6%), desflurane (6%, 12%, and 18%) and sevoflurane (3%, 5%, and 8%). Each ostrich egg was exposed to the same narcotic gas and concentration on development day (DD) 31 and 34. Narcotic gas exposure was upheld for 90 min and embryos were monitored for additional 75 min. Toxicity was evaluated by verifying embryo viability 24 h after the experiments. Initial heart rate of mean 148 beats/min (DD 31) and 136 beats/min (DD 34) decreased over time by 44-48 beats/minute. No significant differences were observed between groups. All narcotic gases led to distinct movement reduction after mean 8 min. Embryos exposed to desflurane 6% showed residual movements. Isoflurane 6% and sevoflurane 8% produced motion-free time intervals of mean 70 min after discontinuation of narcotic gas exposure. Only one embryo death occurred after narcotic gas exposure with desflurane 6%. This study shows that isoflurane, desflurane and sevoflurane are suitable for ostrich embryo immobilization, which is a prerequisite for motion-artifact free imaging. Application of isoflurane 6% and sevoflurane 8% is a) safe as no embryonal deaths occurred after exposure and b) effective as immobilization was observed for approx. 70 min after the end of narcotic gas exposure. These results should be interpreted with caution regarding transferability to other avian species as differences in embryo size and incubation duration exist.

Replacing Animal Testing with Stem Cell-Organoids : Advantages and Limitations.

Various groups including animal protection organizations, medical organizations, research centers, and even federal agencies such as the U.S. Food and Drug Administration, are working to minimize animal use in scientific experiments. This movement primarily stems from animal welfare and ethical concerns. However, recent advances in technology and new studies in medicine have contributed to an increase in animal experiments throughout the years. With the rapid increase in animal testing, concerns arise including ethical issues, high cost, complex procedures, and potential inaccuracies.Alternative solutions have recently been investigated to address the problems of animal testing. Some of these technologies are related to stem cell technologies, such as organ-on-a-chip, organoids, and induced pluripotent stem cell models. The aim of the review is to focus on stem cell related methodologies, such as organoids, that can serve as an alternative to animal testing and discuss its advantages and limitations, alongside regulatory considerations.Although stem cell related methodologies has shortcomings, it has potential to replace animal testing. Achieving this requires further research on stem cells, with potential societal and technological benefits.

[Current regulations in the Animal Welfare Act and the significance for animal research]. / Aktuelle Regelungen im Tierschutzgesetz und Bedeutung für die tierexperimentelle Forschung.

Due to the legal implantation of the 3R principle, the number of laboratory animals decreased significantly over the past 10 years. In this article, the historical development of animal experiments over the last decade will be presented in the context of the current regulations of the Animal Welfare Act. It points out bureaucratic obstacles to the approval of animal experiments, which jeopardize Germany as a research location for both academia and industry. The article presents constructive proposals for solutions. This should be done in accordance with the DFG recommendation to ensure efficient biomedical research while maintaining the highest animal welfare standards.

Phase-out planning for animal experimentation.

Since the late 2010s, the idea of phase-out planning for animal experimentation (PPAE) has come to the foreground of political debates, but central notions and arguments are understood differently by different participants and stand in need of clarification. This article draws on public communications on ten political projects related to PPAE to propose a philosophical explication of PPAE and to articulate proponents' central moral argument. According to the argument, the phase-out of animal experimentation is morally desirable and planned interventions are both necessary and sufficient to achieve it. The normative and descriptive premises of the argument are stated and discussed, flagging questions that need answering for a more thorough assessment of the argument. This results in a series of seven action points for researchers and stakeholders of phase-out planning for animal experimentation. The overall goal is to enable an open and productive discussion about PPAE in public, political, and academic settings.

In recent years, a new demand has entered the political arena: that the phase-out of animal experimentation should be planned. But it is important to understand exactly what this means. This article draws on ten documents from governments, parliaments, and NGOs to tease out what they mean by "planning the phase-out of animal experimentation." It also discusses the main argument that is advanced in favor of phase-out planning and highlights seven gaps in our knowledge that we should try to fill in order to move the discussion forward. In sum, the article is the first to explicitly define phase-out planning for animal experimentation and to directly discuss its pros and cons from a philosophical point of view. This is helpful in avoiding misunderstandings and talking past each other, enabling an open and productive debate.

Proof of concept testing of a positive reference material for in vivo and in vitro sensitization testing of medical devices.

In vivo skin sensitization tests are required to evaluate the biological safety of medical devices in contact with living organisms to provide safe medical care to patients. Negative and positive reference materials have been developed for biological tests of cytotoxicity, implantation, hemolysis, and in vitro skin irritation. However, skin sensitization tests are lacking. In this study, polyurethane sheets containing 1 wt/wt % 2,4-dinitrochlorobenzene (DNCB-PU) were developed and evaluated as a positive reference material for skin sensitization tests. DNCB-PU sheet extracts prepared with sesame oil elicited positive sensitization responses for in vivo sensitization potential in the guinea pig maximization test and the local lymph node assay. Furthermore, DNCB-PU sheet extracts prepared with water and acetonitrile, 10% fetal bovine serum-containing medium, or sesame oil elicited positive sensitization responses as alternatives to animal testing based on the amino acid derivative reactivity assay, human cell line activation test, and epidermal sensitization assay, respectively. These data suggest that the DNCB-PU sheet is an effective extractable positive reference material for in vivo and in vitro skin sensitization testing in medical devices. The formulation of this reference material will lead to the development of safer medical devices that contribute to patient safety.

No evidence of real-world equivalence in chickens (Gallus gallus domesticus) categorizing visually diverse images of natural stimuli presented on LCD monitors.

Category learning is often tested with similar images that have no significance outside of the experiment for the subjects. By contrast, in nature animals often need to generalize a behavioral response like "eat" across visually distinct stimuli, such as spiders and seeds. Forming functional categories like "food" and "predator" may require conceptual rather than purely perceptual generalization. We trained free-range chickens to classify images assigned to one of four categories based on putative functional significance: inanimate objects, predators, food, and non-competing vertebrates. Images were visually diverse within each category, discouraging classification by perceptual similarity alone. In Experiment 1, chickens classified 80 images into four categories. Chickens then generalized to 80 new exemplars in each of three successive generalization tests. In Experiment 2, chickens saw new types of images to test whether their generalization was perceptual or functional. For example, chickens saw images of skunks for the predator category after training with images of hawks and snakes. Chickens used the "predator" response with these new images for both predators and non-threatening vertebrates, but not for objects or food, and did not successfully generalize any category other than predator. In Experiment 3, chickens categorized fractals as "food," and three of four chickens categorized a range of vertebrates they had not previously encountered as "predators," suggesting that chickens did not see the images as representing real world objects and animals. These results highlight constraints on the use of computer-generated images to assess categorization of natural stimuli in chickens.

Engineering Sensory Ganglion Multicellular System to Model Tissue Nerve Ingrowth.

Discogenic pain is associated with deep nerve ingrowth in annulus fibrosus tissue (AF) of intervertebral disc (IVD). To model AF nerve ingrowth, primary bovine dorsal root ganglion (DRG) micro-scale tissue units are spatially organised around an AF explant by mild hydrodynamic forces within a collagen matrix. This results in a densely packed multicellular system mimicking the native DRG tissue morphology and a controlled AF-neuron distance. Such a multicellular organisation is essential to evolve populational-level cellular functions and in vivo-like morphologies. Pro-inflammatory cytokine-primed AF demonstrates its neurotrophic and neurotropic effects on nociceptor axons. Both effects are dependent on the AF-neuron distance underpinning the role of recapitulating inter-tissue/organ anatomical proximity when investigating their crosstalk. This is the first in vitro model studying AF nerve ingrowth by engineering mature and large animal tissues in a morphologically and physiologically relevant environment. The new approach can be used to biofabricate multi-tissue/organ models for untangling pathophysiological conditions and develop novel therapies.

Analysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity.

Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.

The last resort requirement under REACH: From principle to practice.

REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) is a European Union regulation that aims to protect human health and the environment from the risks posed by chemicals. Article 25 clearly states that: "[i]n order to avoid animal testing, testing on vertebrate animals for the purposes of this Regulation shall be undertaken only as a last resort." In practice, however, the standard information requirements under REACH are still primarily filled using animal studies. This paper presents examples illustrating that animal testing is not always undertaken only as a last resort. Six over-arching issues have been identified which contribute to this: (1) non-acceptance of existing animal or non-animal data, (2) non-acceptance of read-across, (3) inflexible administrative processes, (4) redundancy of testing, (5) testing despite animal welfare concerns and (6) testing for cosmetic-only ingredients. We, members of the Animal-Free Safety Assessment (AFSA) Collaboration, who work together to accelerate the global adoption of non-animal approaches for chemical safety assessment, herein propose several recommendations intended to aid the European Commission, the European Chemicals Agency and registrants to protect human health and the environment while avoiding unnecessary animal tests - truly upholding the last resort requirement in REACH.

Prediction of pharmacokinetics of an anaplastic lymphoma kinase inhibitor in rat and monkey: application of physiologically based pharmacokinetic model as an alternative tool to minimise animal studies.

The pharmacokinetic (PK) and toxicokinetic profile of a drug from its preclinical evaluation helps the researcher determine whether the drug should be tested in humans based on its safety and toxicity.Preclinical studies require time and resources and are prone to error. Moreover, according to the United States Food and Drug Administration Modernisation Act 2, animal testing is no longer mandatory for new drug development, and an animal-free alternative, such as cell-based assay and computer models, can be used.Different physiologically based PK models were developed for an anaplastic lymphoma kinase inhibitor in rats and monkeys after intravenous and oral administration using its physicochemical properties and in vitro characterisation data.The developed model was validated against the in vivo data available in the literature, and the validation results were found within the acceptable limit. A parameter sensitivity analysis was performed to identify the properties of the compound influencing the PK profile.This work demonstrates the application of the physiologically based PK model to predict the PKs of a drug, which will eventually assist in reducing the number of animal studies and save time and cost of drug discovery and development.

Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals: Responses from a survey of National Control Laboratories and National Regulatory Authorities.

The UK National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) is reviewing World Health Organization (WHO) manuals, guidelines and recommendations for vaccines and biotherapeutics to identify the extent to which animal-based testing methods are described. The aim is to recommend where updates to these documents can lead to an increased and more harmonised adoption of 3Rs principles (i.e. Replacement, Reduction and Refinement of animal tests) in the quality control and batch release testing requirements for vaccines and biotherapeutics. Improved adoption of 3Rs principles and non-animal testing strategies will help to reduce the delays and costs associated with product release testing. Developing recommendations that are widely applicable by both the manufacturers and national regulatory authorities for vaccines and biological therapeutics globally requires a detailed understanding of how different organisations view the opportunities and barriers to better integration of the 3Rs. To facilitate this, we developed and distributed a survey aimed at individuals who work for national regulatory authorities (NRAs) and/or national control laboratories (NCLs). In this paper, we present the key findings from this survey and how these will help inform the recommendations for wider integration of 3Rs approaches by WHO in their guidance documents applicable to the quality control and batch release testing of vaccines and biotherapeutics.

[Alternative methods to animal testing: the need for funding and dissemination]. / Méthodes alternatives à l'expérimentation animale: un besoin de financement et de diffusion.

The use of animals for scientific purposes brings a moral conflict between the necessity for human health and animal rights. Indeed, science has shown that most animals used in research are sentient beings, capable of suffering. Based on the principle of the 3Rs, the European legislation encourages the development of alternative methods to animal testing. French and European public opinions support the development of alternatives and broadly reject the use of animals for scientific purposes when alternatives exist. However, alternative methods to animal testing are still lacking. In order to drastically reduce the use of animals to this end, significant fundings are necessary. As far as this matter is concerned, France comes at the bottom of the class, but the creation of its 3Rs centre gathering all major scientific institutes and launching calls for fundings is a step into the right direction. The European Union funds projects of alternative methods, so do other private stakeholders, such as companies and NGOs. Another matter is the dissemination of alternative methods to scientists so they are aware of these methods. Some French research teams develop innovative methods and try to disseminate them. French and European platforms bring together creators, users and regulators to that end. Funding and disseminating alternative methods to animal testing should be a priority.

Title: Méthodes alternatives à l'expérimentation animale: un besoin de financement et de diffusion. Abstract: L'utilisation des animaux pour la recherche, l'enseignement et les tests de toxicité des produits engendre un conflit moral entre sa nécessité pour la santé humaine et le respect dû aux animaux. Sur la base du concept des 3R, la législation européenne promeut le développement des méthodes alternatives à l'expérimentation. Le développement de ces méthodes est soutenu par l'opinion publique française et européenne, qui s'oppose globalement à l'expérimentation animale lorsque d'autres méthodes existent. Cependant, les méthodes alternatives sont encore insuffisantes. Pour réduire drastiquement le nombre d'animaux utilisés à des fins scientifiques, des financements importants sont nécessaires. Dans ce domaine, la France fait figure de mauvais élève. Toutefois, la création de son centre 3R, qui réalise des appels à projets, est un signal dans le bon sens. Quant à l'Union européenne, elle finance des projets de méthodes alternatives. D'autres acteurs privés, ONG et entreprises y participent. Un autre enjeu est la diffusion des méthodes alternatives pour que la communauté scientifique s'en empare. En France, des équipes de recherche développent des techniques innovantes. Des plateformes françaises et européennes permettent de rapprocher les concepteurs, les utilisateurs et les régulateurs pour diffuser les méthodes alternatives. Le financement et la diffusion des méthodes doivent se poursuivre et s'accentuer.

[Legal aspects of alternative methods to animal testing]. / Aspects juridiques des méthodes alternatives à l'expérimentation animale.

While the total replacement of animal experimentation was the goal set by the European Directive of 22 September 2010 on the protection of animals used for scientific purposes, it has to be said that it is still far from being achieved. The number of animals is not decreasing and alternative methods are struggling to be used. Under pressure from the citizens, the European Commission has just made new commitments to define the stages and specific actions to be put in place to reduce animal testing, a prerequisite for the transition to an animal-free regulatory system. Given the shortcomings and lack of coherence in European policy, mobilising the public is an essential lever for speeding up the implementation of alternative methods.

Title: Aspects juridiques des méthodes alternatives à l'expérimentation animale. Abstract: Si le remplacement total de l'expérimentation animale était l'objectif visé par la directive européenne du 22 septembre 2010 relative à la protection des animaux utilisés à des fins scientifiques, force est de constater qu'il est encore loin d'être atteint. Le nombre d'animaux ne diminue pas et les méthodes alternatives peinent à être utilisées. La Commission européenne, sous la pression citoyenne, vient de prendre de nouveaux engagements pour définir les étapes et les actions spécifiques à mettre en place pour réduire l'expérimentation animale, condition préalable à la transition vers un système réglementaire sans animaux. Entre dysfonctionnements et manque de cohérence dans la politique européenne, la mobilisation des citoyens représente un levier indispensable à l'accélération de la mise en œuvre des méthodes alternatives.

Intra-aortic band impairs transapical device implantation in a pig: a case report.

BACKGROUND: Anatomic anomalies in the ascending aorta may impair the implantation and testing of cardiovascular devices in humans and animal models. CASE PRESENTATION: We present the rare case of an intra-aortic band in a German Landrace pig. During terminal animal testing, the band hindered the implantation of a left ventricular assist device (LVAD) with transventricular outflow graft across the aortic valve. After lower partial sternotomy, epicardial echocardiography displayed an intraluminal echogenic structure at the sinotubular junction causing unspecific flow turbulences. Under cardiopulmonary bypass, coring of the left ventricular apex was performed. Due to strong resistance in the proximal aorta, accurate positioning of the transventricular LVAD outflow graft was impossible. After euthanasia, necropsy revealed a fibrous band located at the sinotubular junction, dividing the lumen of the ascending aorta. CONCLUSIONS: The occurrence of an intra-aortic band represents an extremely rare case of a most likely congenital anomaly. Awareness of such anomalies is important for planning and performing animal testing. Perioperative echocardiography may help to either remove such anomalies or allow discontinuing the procedure prior to device implantation.

Correlative CD4 and CD8 T-cell immunodominance in humans and mice: Implications for preclinical testing.

Antigen-specific T-cell recognition is restricted by Major Histocompatibility Complex (MHC) molecules, and differences between CD4 and CD8 immunogenicity in humans and animal species used in preclinical vaccine testing are yet to be fully understood. In this study, we addressed this matter by analyzing experimentally identified epitopes based on published data curated in the Immune Epitopes DataBase (IEDB) database. We first analyzed SARS-CoV-2 spike (S) and nucleoprotein (N), which are two common targets of the immune response and well studied in both human and mouse systems. We observed a weak but statistically significant correlation between human and H-2b mouse T-cell responses (CD8 S specific (r = 0.206, p = 1.37 × 10-13); CD4 S specific (r = 0.118, p = 2.63 × 10-5) and N specific (r = 0.179, p = 2.55 × 10-4)). Due to intrinsic differences in MHC molecules across species, we also investigated the association between the immunodominance of common Human Leukocyte Antigen (HLA) alleles for which HLA transgenic mice are available, namely, A*02:01, B*07:02, DRB1*01:01, and DRB1*04:01, and found higher significant correlations for both CD8 and CD4 (maximum r = 0.702, p = 1.36 × 10-31 and r = 0.594, p = 3.04-122, respectively). Our results further indicated that some regions are commonly immunogenic between humans and mice (either H-2b or HLA transgenic) but that others are human specific. Finally, we noted a significant correlation between CD8 and CD4 S- (r = 0.258, p = 7.33 × 1021) and N-specific (r = 0.369, p = 2.43 × 1014) responses, suggesting that discrete protein subregions can be simultaneously recognized by T cells. These findings were confirmed in other viral systems, providing general guidance for the use of murine models to test T-cell immunogenicity of viral antigens destined for human use.

The future of pyrogenicity testing: Phasing out the rabbit pyrogen test. A meeting report.

The rabbit pyrogen test (RPT) was the benchmark for pyrogenicity testing, but scientific advancements have provided innovative and humane methods, such as the in vitro monocyte-activation test (MAT). However, transitioning from the RPT to the MAT has been challenging. The European Directorate for the Quality of Medicines & HealthCare, the Council of Europe, and the European Partnership for Alternative Approaches to Animal Testing jointly hosted an international conference entitled "The future of pyrogenicity testing: phasing out the rabbit pyrogen test". The conference aimed to show how the European Pharmacopoeia intends to remove the RPT from its texts by 2026, facilitate the use of MAT, and identify gaps in the suppression of RPT. The events contributed to a better understanding of the barriers to RPT replacement and acceptance of in vitro alternatives. Participants comprised stakeholders from Asia, Europe, and North America, including vaccine developers, contract laboratories, and regulators. Participants shared their replacement strategies and experiences with MAT implementation. They emphasised the need for continued cooperation between stakeholders and stressed the importance of international harmonisation of regulatory requirements to help accelerate MAT acceptance outside Europe. Despite the challenges, the willingness to eliminate the unnecessary use of RPT was common across all participants.

Author Guide for Addressing Animal Methods Bias in Publishing.

There is growing recognition that animal methods bias, a preference for animal-based methods where they are not necessary or where nonanimal-based methods may already be suitable, can impact the likelihood or timeliness of a manuscript being accepted for publication. Following April 2022 workshop about animal methods bias in scientific publishing, a coalition of scientists and advocates formed a Coalition to Illuminate and Address Animal Methods Bias (COLAAB). The COLAAB has developed this guide to be used by authors who use nonanimal methods to avoid and respond to animal methods bias from manuscript reviewers. It contains information that researchers may use during 1) study design, including how to find and select appropriate nonanimal methods and preregister a research plan, 2) manuscript preparation and submission, including tips for discussing methods and choosing journals and reviewers that may be more receptive to nonanimal methods, and 3) the peer review process, providing suggested language and literature to aid authors in responding to biased reviews. The author's guide for addressing animal methods bias in publishing is a living resource also available online at animalmethodsbias.org, which aims to help ensure fair dissemination of research that uses nonanimal methods and prevent unnecessary experiments on animals.

FDA Modernization Act 2.0: An insight from nondeveloping country.

Animal testing is required in drug development research and is crucial for assessing the efficacy and safety of medications before they are commercialized. However, the newly furnished Food and Drug Administration Modernization Act 2.0 has given new insight into drug development. It opens a new door by offering an alternative testing method for developing a new drug without using animals. This newly proposed system may potentially significantly impact nondeveloped countries worldwide. In this study, we explore the alternative testing options such as in silico modeling, human tissue-on-chip engineering, animal-free recombinant antibodies, tissue engineering, and artificial intelligence presented by this act and discuss its implications for nondeveloped countries.

The role of extracellular vesicles in non-small-cell lung cancer, the unknowns, and how new approach methodologies can support new knowledge generation in the field.

Extracellular vesicles (EVs) are nanosized particles released from most human cell types that contain a variety of cargos responsible for mediating cell-to-cell and organ-to-organ communications. Current knowledge demonstrates that EVs also play critical roles in many aspects of the progression of Non-Small-Cell Lung Cancer (NSCLC). Their roles range from increasing proliferative signalling to inhibiting apoptosis, promoting cancer metastasis, and modulating the tumour microenvironment to support cancer development. However, due to the limited availability of patient samples, intrinsic inter-species differences between human and animal EV biology, and the complex nature of EV interactions in vivo, where multiple cell types are present and several events occur simultaneously, the use of conventional preclinical and clinical models has significantly hindered reaching conclusive results. This review discusses the biological roles that EVs are currently known to play in NSCLC and identifies specific challenges in advancing today's knowledge. It also describes the NSCLC models that have been used to define currently-known EV functions, the limitations associated with their use in this field, and how New Approach Methodologies (NAMs), such as microfluidic platforms, organoids, and spheroids, can be used to overcome these limitations, effectively supporting future exciting discoveries in the NSCLC field and the potential clinical exploitation of EVs.

3Rs implementation in veterinary vaccine batch-release testing: Current state-of-the-art and future opportunities. A webinar and workshop report.

Regulatory authorities require veterinary batch-release testing to confirm vaccine potency and safety, but these tests have traditionally relied on large numbers of laboratory animals. Advances in vaccine research and development offer increasing opportunities to replace in vivo testing, and some stakeholders have made significant progress in incorporating 3Rs elements in quality control strategies. A three-part event series entitled "3Rs Implementation in Veterinary Vaccine Batch-Release Testing: Current state-of-the-art and future opportunities" was jointly organized by the Animal-Free Safety Assessment Collaboration, HealthforAnimals, and the International Alliance of Biological Standardization. Two webinars and a workshop aimed to outline the state-of-the-art non-animal approaches for veterinary batch-release testing. The events included information on the state of the deletion of obsolete safety testing and the current initiatives implemented by European, North American, and Asian-Pacific stakeholders on 3Rs implementation and regulatory acceptance. The events contributed to a better understanding of the barriers to 3Rs implementation. Participants highlighted the need for open communication, continued collaboration between stakeholders, and international harmonization of regulatory requirements to help accelerate acceptance. Despite the challenges, the countries represented at this three-part event have shared their commitments to advancing the acceptance of alternative methods.