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The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA’s 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU ‘on the ground’ in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Current in vitro thrombosis models utilise simplistic 2D surfaces coated with purified components of the subendothelial matrix. The lack of a realistic humanised model has led to greater study of thrombus formation in in vivo tests in animals. Here we aimed to develop 3D hydrogel-based replicas of the medial and adventitial layers of the human artery to produce a surface that can optimally support thrombus formation under physiological flow conditions. These tissue-engineered medial- (TEML) and adventitial-layer (TEAL) hydrogels were developed by culturing human coronary artery smooth muscle cells and human aortic adventitial fibroblasts within collagen hydrogels, both individually and in co-culture. Platelet aggregation upon these hydrogels was studied using a custom-made parallel flow chamber. When cultured in the presence of ascorbic acid, the medial-layer hydrogels were able to produce sufficient neo-collagen to support effective platelet aggregation under arterial flow conditions. Both TEML and TEAL hydrogels possessed measurable tissue factor activity and could trigger coagulation of platelet-poor plasma in a factor VII-dependent manner. Biomimetic hydrogel replicas of the subendothelial layers of the human artery are effective substrates for a humanised in vitro thrombosis model that could reduce animal experimentation by replacing current in vivo models.

In 2010, the European Parliament and the European Union adopted a directive on the protection of animals used for scientific purposes. The directive aims to protect animals in scientific research, with the final goal of complete replacement of procedures on live animals for scientific and educational purposes as soon as it is scientifically viable. Furthermore, the directive announces the implementation of the 3Rs principle: "When choosing methods, the principles of replacement, reduction and refinement should be implemented through a strict hierarchy of the requirement to use alternative methods." The visibility, accessibility, and the rapid growth of the chorioallantoic membrane (CAM) offers a clear advantage for various manipulations and for the simulation of different Bio-Barriers according to the 3R principle. The extensive vascularisation on the CAM provides an excellent substrate for the cultivation of tumour cells or tumour xenografts which could be used for the therapeutic evaluation of nanoscale drug delivery systems. The tumour can be targeted either by topical application, intratumoural injection or i.v. injection. Different application sites and biological barriers can be examined within a single model.

Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two‐dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species‐specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell‐ and organ‐based assays for more accurate representation of human susceptibility to drug response. Among others, the three‐dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs‐on‐chips, and from single‐cell type static 3D models to cell co‐culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver‐, intestine‐, kidney‐, and neuron‐based 3D cellular models.

The aim of this study was to develop a useful antibody PK evaluation tool using a combination of cassette-dosing and microsampling in mice and monkeys in order to reduce the number of animals used. Cetuximab, denosumab, infliximab, and a mixture of the three antibodies, i.e., cassette-dosing, were administered intravenously to cynomolgus monkeys, C57BL/6J mice, and homozygous human neonatal Fc-receptor transgenic (Tg32) mice. Mouse blood was collected from one animal continuously via the jugular vein at nine points. In cynomolgus monkeys, infliximab showed faster elimination in the cassette-dosing group than in the single-dose group. Anti-drug antibody production was observed, but the PK parameters of the clearance and distribution volume were similar in both groups. In C57BL/6J and Tg32 mice, each of the plasma concentrations–time profiles after cassette-dosing were similar to those after single dosing. PK evaluation using a combination of cassette-dosing and microsampling in mice may reduce the number of mice used by approximately 90% compared with the conventional method. The combination of antibody cassette-dosing and microsampling is a promising PK evaluation method as a high-throughput and reliable with reduced numbers of mice and cynomolgus monkeys.

Antibody induction test (AIT) is a promising candidate as a refinement of the troublesome National institutes of Health (NIH) test in the sense of animal welfare 3R approach for determination of potency of inactivated rabies vaccines for veterinary and human use. In this study, we initially try to develop AIT as a suitable alternative to NIH test, to achieve a reduction of test duration and diminish animal suffering by omitting intracerebral CVS infection and measuring humoral immunity upon vaccination. Designs of both multi-dose and single-dose AIT were examined. Biological reference preparation, batch 5 with assigned titer of 10 IU/vial, was taken as both standard and test vaccine. Six consecutive AITs were performed and eight pools of sera in each AIT were tested in triplicate by rapid fluorescent focus inhibition test. We estimated the upper detection limit and calculated test variability for individual dilutions. For multi-dose AIT, we estimated the dose-response function and performed calculations of final test results and statistical validity parameters for both linear and sigmoidal model using CombiStats program. Sigmoidal 4-parameter dose-response model was found optimal. Presented design of multi-dose AIT showed a satisfactory detection limit for testing of inactivated rabies vaccines for both veterinary and human use. However, due to nonconformity of obtained results with statistical validity criteria, we concluded that the presented model of multi-dose AIT was unsuitable for introduction in routine practice. However, we concluded that there was a realistic option for introduction of two versions of single-dose AIT. The first version would be with two standard vaccine controls and could be introduced immediately, while the second version would include testing of the sample only and rely on comparison of the induced rabies antibody level with absolute cut-off limits set in advance.

Replicability of experimental results and optimal use of experimental animals are everybody's concern. Current efforts towards increased replicability include guidelines and checklists as tools for experimenters, referees, editors and publishers. Guidelines are also provided for appropriate use of animals. To ensure the quality of experimental results, the number of animals must be adequate, that is, sufficiently large, for the purpose of the given experiment. To comply with current ethical recommendations, the use of animals should be reduced as much as possible. Therefore, determination of the number of animals for a given scientific objective includes contrasting considerations. Current guidelines for animal experimentation, notably from the National Institute of Health, mandate (with very few exceptions) inclusion of animals of both sexes in experimental designs statistically powered to address the difference between the two groups. Notably, absence of evidence for sex differences between the organ or system functions under study does not qualify as an exception. Mandatory, equal representation of both sexes raises several questions including ethical ones. Other guidelines, by public regulators and major publishers, do not seem to have a similar selective focus on sex differences. In summary, current concerns about replicability of scientific results are justified. Concomitantly, the knowledge of sex differences also between non-reproductive, non-endocrine organ functions is increasing. In principle, sex matters in any experimental context. However, an indiscriminate demand for inclusion of both sexes in all experimental protocols seems a waste of animals, money and time, violating traditional principles of animal experimentation, particularly that of reduction.

Complementing the human genome with an exposome reflects the increasingly obvious impact of environmental exposure, which far exceeds the role of genetics, on human health. Considering the complexity of exposures and, in addition, the reactions of the body to exposures - i.e., the exposome - reverses classical exposure science where the precise measurement of single or few exposures is associated with specific health or environmental effects. The complete description of an individual's exposome is impossible; even less so is that of a population. We can, however, cast a wider net by foregoing some rigor in assessment and compensating with the statistical power of rich datasets. The advent of omics technologies enables a relatively cheap, high-content description of the biological effects of substances, especially in tissues and biofluids. They can be combined with many other rich data-streams, creating big data of exposure and effect. Computational methods increasingly allow data integration, discerning the signal from the noise and formulating hypotheses of exposure-effect relationships. These can be followed up in a targeted way. With a better exposure element in the risk equation, exposomics - new kid on the block of risk assessment - promises to identify novel exposure (interactions) and health/environment effect associations. This may also create opportunities to prioritize the more relevant chemicals for risk assessment, thereby lowering the burden on hazard assessment in an expo-sure-driven approach. Technological developments and synergies between approaches, quality assurance (ultimately as Good Exposome Practices), and the integration of mechanistic thinking will advance this approach. published

Over the years, much criticism against animal use for physiology teaching has been made. Hence, replacement by suitable alternatives has increased in several pedagogical approaches. This study examined students’ perceptions of animal versus virtual (video/computer) laboratory classes in physiological sciences associated with the effectiveness of the problem-based learning (PBL) hybrid curriculum. Three cohorts of medical students from the University of Ribeirão Preto, who participated in animal or virtual physiology classes or both, were asked to fill out a 5-point Likert questionnaire about knowledge acquisition/motivation, importance to PBL learning goals, skills acquired, need for animal use, academic formation, learning impairment, and alternative methods. We also assessed their grades in the final exam. A total of 350 students were included, in which 108 participated only in virtual classes, 120 only in practical animal laboratory classes, and 122 in both approaches. The majority agreed that the two methods improved their knowledge acquisition/motivation and helped to reinforce tutorial goals and to acquire skills. However, the cohort who experienced both approaches favored animal laboratory. Students believe animal use is needed and did not impair their learning. Conversely, their opinion about academic formation without animal laboratory classes was divided, as was whether this approach inspired them to seek alternative methods. Despite the different perceptions, there was no difference among the groups’ final grades (7.3 ± 1 vs. 7.2 ± 1 vs. 7.2 ± 2 for virtual or practical animal laboratory classes or both, respectively). Therefore, virtual activities are not as effective as animal use in the opinions of the students, but they are successful strategies in physiology learning that can be used in practical classes in a hybrid PBL curriculum.

Recent recommendations from the European Union Reference Laboratory regarding the generation of antibodies using animals have stimulated significant debate. Here, four of the scientists who served on the Scientific Advisory Committee provide clarification of their views regarding the use of animals and in vitro platforms in antibody generation. Abbreviations: EURL ECVAM, European Union Reference Laboratory for alternatives to animal testing. ESAC, EURL ECVAM Scientific Advisory Committee

An integrated rearing system for

This study was financed by Animalfree Research–Switzerland, and by Portuguese national funds through FCT–Fundação para a Ciência e a Tecnologia, within the CFCUL Unit funding UID/FIL/00678/2013. TAM received partial support from CEAUL (funded by FCT–Fundação para a Ciência e a Tecnologia, Portugal, through the project UID/MAT/00006/2019). Open access publication costs were covered by Animalfree Research–Switzerland and by funding provided by the Ketty and Leif Hjordt Foundation. Major depressive disorder (MDD) is the most severe form of depression and the leading cause of disability worldwide. When considering research approaches aimed at understanding MDD, it is important that their effectiveness is evaluated. Here, we assessed the effectiveness of original studies on MDD by rating their contributions to subsequent medical papers on the subject, and we compared the respective contribution of findings from non-human primate (NHP) studies and from human-based in vitro or in silico research approaches. For each publication, we conducted a quantitative citation analysis and a systematic qualitative analysis of the citations. In the majority of cases, human-based research approaches (both in silico and in vitro) received more citations in subsequent human research papers than did NHP studies. In addition, the human-based approaches were considered to be more relevant to the hypotheses and/or to the methods featured in the citing papers. The results of this study suggest that studies based on in silico and in vitro approaches are taken into account by medical researchers more often than are NHP-based approaches. In addition, these human-based approaches are usually cheaper and less ethically contentious than NHP studies. Therefore, we suggest that the traditional animal-based approach for testing medical hypotheses should be revised, and more opportunities created for further developing human-relevant innovative techniques. Publisher PDF

There are currently three in vitro methods adopted by the Organization for the Economic Co-operation and Development for testing chemicals based on the third key event of the skin sensitization adverse outcome pathway, the activation of dendritic cells. All of them use culture medium supplemented with fetal bovine serum (FBS), which brings technical disadvantages and animal welfare concerns. The objective of this study was to analyze the possibility of eliminating the use of FBS in the human Cell Line Activation Test (h-CLAT). After successful implementation of the h-CLAT using THP-1 cells cultured in FBS-containing medium, several attempts to adapt THP-1 cells to four different serum-free media were made. The best results were obtained with gradual adaptation to RPMI-1640 medium with HL-1™ Supplement and to X-VIVO™ 10. Adapted cells were cryopreserved and submitted to the reactivity check. After being approved, they were used in dose finding and proficiency assays. Despite minor adjustments in the original protocol, it was possible to correctly predict the sensitizing potential of the ten proficiency substances using THP-1 cells adapted to X-VIVO™ 10, which indicates that it is possible to eliminate the use of FBS in the h-CLAT, using a chemically defined medium.

Fish embryos are increasingly being utilized in aquatic toxicity testing, as evidenced by the Organisation for Economic Co-operation and Development's approval of the fish embryo acute toxicity (FET) test. However, the FET test only allows for the estimation of acute toxicity, whereas other test methods such as the larval growth and survival (LGS) test allow for the estimation of both acute and chronic toxicity. Additionally, it has been demonstrated that the FET test is less sensitive than other test methods for some neurotoxic compounds. To address these limitations, efforts to identify sublethal endpoints that increase FET test sensitivity and allow for the prediction of sublethal adverse effects have begun. As such, the objectives of the current study were 1) to compare estimated LC50 values from the FET and LGS test for three known neurotoxicants: fluoride (F), nickel (Ni), and cadmium (Cd) and 2) to evaluate the responsiveness of potential sublethal endpoints for the FET test related to growth (i.e., wet weight and snout-vent length), neurological development (i.e., spontaneous contraction frequency and eye size), and cardiovascular function (i.e., heart rate and pericardial area). The calculated LC50 values from the F and Cd FET test were significantly higher than those from the LGS test, demonstrating that the FET test is less sensitive than the LGS test for neurotoxic compounds. Only Cd exposure resulted in alterations in any of the sublethal endpoints investigated. Embryos/eleutheroembryos exposed to Cd displayed alterations in length, eye size, and pericardial area at concentrations five-fold less than the estimated LC50 value, suggesting that for Cd the inclusion of these sublethal endpoints would improve the sensitivity of the FET test. Overall, these results provide evidence that for some neurotoxicants, the inclusion of sublehtal endpoints may improve the utility of the FET test; however, further research utilizing a broader range of neurotoxicants with differing mechanisms of action, is needed to fully establish such endpoints in the context of routine FET test.

Research endeavors aiming to understand the maternal immune adaptation to pregnancy significantly rely on the use of animal models, such as mice and rats. These models have provided important insights into the pathophysiology of a number of pregnancy disorders in humans. However, the use of animal models in scientific research is a vividly debated and emotive topic. The 3R principles - replacement, reduction and refinement of research animals - have been propagated a few decades ago. The present review advocates a forward-thinking consciousness to address the 3R principles in research projects in the field of reproductive biology and immunology. Specific measures and alternative methods are being proposed to replace research animals by using e.g. tissue engineering approaches, biobank-derived tissue, 'placenta-on-a-chip' devices or in silico methods. The latter may involve data queries from repositories now available to provide single cell sequencing information on reproductive tissues. Reduction of research animals by gestational imaging and a wealth of suggestions for refinement are proposed. Taken together, the measures and guidelines introduced in this review are expected to spark a reconsideration of experimental designs in the area of reproductive biology and immunology in order to implement 3R principle where applicable.

For many years in vivo assays have been a corner stone in safety testing of vaccines for human use. However, there is now an increasing regulatory focus on replacement, reduction and refinement of methods involving animal use. Accordingly, European Pharmacopoeia (Ph.Eur.) monographs and chapters are currently being revised to reduce or discontinue the use of animals in safety and other testing, when such in vivo tests are not absolutely necessary to facilitate risk mitigation. In the current study, a risk assessment of extraneous agents in viral vaccine production has been carried out and it is concluded that only the handling procedures carried out by the technical personnel pose a risk for extraneous viral contamination. A list of named, potentially virulent contaminating viruses, which may have been introduced by these procedures, has been generated. Each of the viruses on this list has been evaluated for possible persistence during the production processes, and it has for all of these been concluded that, if at all present, they only present a negligible risk of introducing extraneous agents in the final product. The overall conclusion of the risk assessment of our vaccine production process is that it justifies the discontinuation of the current in vivo testing, and furthermore demonstrates that there is no need to substitute these in vivo assays with novel in vitro methods.

Depression is the world's predominant mental health problem and a leading cause of disability. Neuropharmacological research has not yet advanced treatments to sufficiently meet clinical need, largely due to the failure of animal models to predict clinical efficacy. The forced swim test (FST) has been extensively used in the field of antidepressant research but has been under scrutiny due to its perceived severity to animals. Any use of animals in experiments and testing must have a scientific or regulatory purpose and researchers need to ensure that there is no scientifically valid alternative. However, regulatory requirements have been incorrectly cited as a reason to support the use of the FST. More research is required on tests that do not involve stressing animals as replacements for the FST. Non-behavioural neurochemical measures might provide a means to advance neuropharmacological developments while reducing animal suffering. For example, brain-derived neurotrophic factor (BDNF) may be promising.

Insulin activity is generally determined by an in vivo rabbit blood glucose drop assay in research and industriel laboratories. The humane experimental techniques imply the use of alternative invertebrate organisms in place of animals, known as replacement rule of the 3Rs. In this study, we report an alternative in vivo extracellular glucose drop assay using unicellular invertebrate Tetrahymena thermophila to replace the use of rabbit and mouse. This assay has four major steps; growing cells, starving cells, treatment of cells and measurement of glucose drop. In this assay, 0.2 mg/ml of human, porcine and bovine insulins dropped extracellular glucose level to 16%, 14% and 12%, respectively in ten minutes. In addition, mammalian insulins respectively increased the cell area about 19%, 15%, and 16% at 6th hour with statistically significant effect on the cell growth, but not in the cell viability. The results showed that the in vivo Tetrahymena thermophila extracellular glucose drop assay could be used as an alternative assay to replace the mouse or the rabbit insulin blood glucose drop assay.

The database of practical examples where toxicokinetic (TK) data has benefitted all stages of the human health risk assessment process are increasingly being published and accepted. This review aimed to highlight and summarise notable examples and to describe the "state of the art" in this field. The overall recommendation is that for any in vivo animal study conducted, measurements of TK should be very carefully considered for inclusion as the numerous benefits this brings continues to grow, particularly during the current march towards animal free toxicology testing and ambitions to eventually conduct human health risk assessments entirely based upon non-animal methods.

OBJECTIVE(S) To create and validate a synthetic simulator for teaching tracheostomy and laryngotracheal reconstruction (LTR) using anterior costal cartilage and thyroid ala cartilage grafts. METHODS A late adolescent/adult neck and airway simulator was constructed based on CT scans from a cadaver and a live patient. Images were segmented to create three-dimensional printed molds from which anatomical parts were casted. To evaluate the simulator, expert otolaryngologists - head and neck surgeons performed tracheostomy and LTR using anterior costal cartilage and thyroid ala cartilage grafts on a live anesthetized porcine model (gold standard) followed by the synthetic simulator. They evaluated each model for face validity (realism and anatomical accuracy) and content validity (perceived effectiveness as a training tool) using a five-point Likert scale. For each expert, differences for each item on each simulator were compared using Wilcoxon Signed-Rank tests with Sidak correction. RESULTS Nine expert faculty surgeons completed the study. Experts rated face and content validity of the synthetic simulator an overall median of 4 and 5, respectively. There was no difference in scores between the synthetic model and the live porcine model for any of the steps of any of the surgical procedures. CONCLUSION The synthetic simulator created for this study has high face and content validity for tracheostomy and LTR with anterior costal cartilage and thyroid ala cartilage grafts and was not found to be different than the live porcine model for these procedures. LEVEL OF EVIDENCE 5 Laryngoscope, 131:E2378-E2386, 2021.

In May 2020, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) published a recommendation report entitled "Recommendation on nonanimal-derived antibodies". In this report, the EURL ECVAM specifically states: "Therefore, taking into consideration the ESAC Opinion on the scientific validity of replacements for animal-derived antibodies, EURL ECVAM recommends that animals should no longer be used for the development and production of antibodies for research, regulatory, diagnostic and therapeutic applications. The provisions of Directive 2010/63/EU should be respected, and EU countries should no longer authorise the development and production of antibodies through animal immunisation, where robust, legitimate scientific justification is lacking." (1). Here, we are providing the American Association of Pharmaceutical Scientists (AAPS) opinion on the EURL ECVAM recommendation report. In brief, there has been a clear and strong progress in reduction of animal use in the drug discovery and development process, including significant reduction of animal use in production of antibody reagents. Yet, it is proposed that more data need to be generated, shared and discussed within the scientific community before a decision to implement the change to non-animal derived antibodies is made.

Magnetic resonance imaging is playing a significant role in applying the 3Rs to neuroscience studies using non-human primates. MRI scans are contributing to refinement by enhancing the selection and assignment of animals, guiding the manufacture of custom-fitted recording and head fixation devices, and assisting with the diagnosis of health issues and their treatment. MRI is also being used to better understand the impact of neuroscience procedures on the welfare of NHPs. MRI has helped to optimise NHP use and make greater scientific progress than would otherwise be made using larger numbers of animals. Whilst human fMRI studies have replaced some NHP studies, their potential to directly replace NHP electrophysiology is limited at present. Given the considerable advantages of MRI for electrophysiology experiments, including improved welfare of NHPs, consideration should be given to focusing NHP electrophysiology laboratories around MRI facilities. Greater sharing of MRI data sets, and improvements in MRI contrast and resolution, are expected to further advance the 3Rs in the future.

The use of laboratory animals in biomedical research is a matter of intense public debate. Recent statistics indicates that about half of the western population, sensitive to this discussion, would be in favor of animal testing while the other half would oppose it. Here, outlining scientific, historical, ethical, and philosophical aspects, we provide an integrated view explaining the reasons why biomedical research can hardly abandon laboratory animal testing. In this paper, we retrace the historical moments that mark the relationship between humans and other animal species. Then starting from Darwin's position on animal experimentation, we outline the steps that over time allowed the introduction of laws and rules that regulate animals' use in biomedical research. In our analysis, we present the perspectives of various authors, with the aim of delineating a theoretical framework within which to insert the ethical debate on laboratory animals research. Through the analysis of fundamental philosophical concepts and some practical examples, we propose a view according to which laboratory animals experimentation become ethically acceptable as far as it is guided by the goal of improving humans and other animal species (i.e., pets) life. Among the elements analyzed, there is the concept of responsibility that only active moral subjects (humans) have towards themselves and towards passive moral subjects (other animal species). We delineate the principle of cruelty that is useful to understand why research in laboratory animals should not be assimilated to a cruel act. Moreover, we touch upon the concepts of necessity and "good cause" to underline that, if biomedical research would have the possibility to avoid using animals, it would surely do that. To provide an example of the negative consequences occurring from not allowing laboratory animal research, we analyze the recent experience of Covid-19 epidemic. Finally, recalling the principle of "heuristics and biases" by Kahneman, we discuss why scientists should reconsider the way they are conveying information about their research to the general public.