For centuries, animals have been used in research due to their genetic and physiological similarities to humans. However, significant differences exist between humans and animals, which have the potential to confound res...For centuries, animals have been used in research due to their genetic and physiological similarities to humans. However, significant differences exist between humans and animals, which have the potential to confound results obtained from such experiments. These differences result in reduced translatability of animal data to humans, which is a major contributing factor to the 92% failure rate for novel therapies in clinical trials. Advances in scientific research have enabled the development of human-focused New Approach Methodologies (NAMs), which include and 3-D models. By harnessing these novel approaches, greater predictive power for human biology, human diseases and assessment of novel therapies could be achieved. However, several obstacles remain to their wider adoption, including potential financial constraints, publication bias, and some concerns about the reliability of NAMs due to the novelty of this field, compared to animal studies. Here, we outline the differences between humans and animals used in research, discuss in detail the obstacles to the greater adoption of NAMs in research, and provide recommendations on how to accelerate a shift toward human-focused research.
The overall aim of the 'Improving the Quality of Biomedical Science with 3Rs Concepts' (IMPROVE) COST Action (CA21139) is to establish an interdisciplinary network dedicated to refining, harmonising and promoting the ado...The overall aim of the 'Improving the Quality of Biomedical Science with 3Rs Concepts' (IMPROVE) COST Action (CA21139) is to establish an interdisciplinary network dedicated to refining, harmonising and promoting the adoption of the Three Rs concepts, to enhance the quality of biomedical research. The 'IMPROVE' COST Action has assembled an Ethics Crossover Group composed of volunteer members from each of IMPROVE's four established Working Groups: 'Quality and translatability of science', 'Implementation', 'Dissemination' and 'Education'. This report relates to a two-day workshop, held in Acibadem University in Istanbul on 2-3 September 2024. The workshop was organised by the Ethics Crossover Group in line with the aims and the scope of the 'IMPROVE' COST Action, to address the ethical dimension of research and experiments on laboratory animals.
Azhakesan A, Seiz E, Kern J
… +10 more, Hendricks L, Fleckenstein J, Jungbauer F, Ludwig S, Brochhausen C, Bugia L, Scherl C, Lammert A, Rotter N, Affolter A
Patient-derived head and neck squamous cell carcinoma (HNSCC) explant models have been shown to retain the original tumour microenvironment and morphological characteristics. To enhance the human relevance of models and...Patient-derived head and neck squamous cell carcinoma (HNSCC) explant models have been shown to retain the original tumour microenvironment and morphological characteristics. To enhance the human relevance of models and improve clinical outcomes, there is an emerging move toward preclinical models that are cultured in xeno-free media (i.e. media containing no non-human animal-derived components). Fetal bovine serum (FBS) has been the standard cell culture medium supplement in most systems. However, growing emphasis on ethical concerns, animal welfare considerations and reproducibility, as well as the need to implement the Three Rs principles, have driven substantial efforts to identify viable xeno-free alternatives to FBS. In this study, an culture model for HNSCC was developed, based on the use of xeno-free media. Human platelet lysate (hPL)-supplemented medium and a commercially available xeno-free human mesenchymal stromal cell (MSC) expansion medium were evaluated, comparing HNSCC explant model growth to that in the 'standard' FBS-supplemented medium, over a 10-day culture period. To best reflect clinical conditions, the tissues were treated with radiochemotherapy (RCT) comprising cisplatin and fractionated irradiation. After 10 days, the tissues were formalin-fixed, paraffin-embedded, and assessed for the expression of key biomarkers, including PD-L1, Ki-67 and vimentin. The upregulation of PD-L1, as well as the downregulation of Ki-67 and vimentin, were consistent across all media, thus validating hPL-supplemented medium and MSC medium as viable alternatives to FBS-supplemented medium, for use in the culture of humanised HNSCC preclinical models.
Uropathogenic (UPEC) is responsible for the majority of urinary tract infections, including those in catheterised patients. , an insect species, has been proposed as an alternative model to study the pathogenicity of ....Uropathogenic (UPEC) is responsible for the majority of urinary tract infections, including those in catheterised patients. , an insect species, has been proposed as an alternative model to study the pathogenicity of . The aim of this study was to determine the pathogenicity of 48 UPEC strains using the larval disease score obtained from larvae model. The bacteria were also classified according to their phylogenetic group and the ability to form biofilms. The larvae were inoculated with the bacteria and observed for 72 hours. The LD, mortality rate, and survival curve for each UPEC strain were assessed. A larval disease score (0-10) was determined, which considered mortality, larval activity, cocoon formation and melanisation. Phylogenetic groups were determined by using PCR, and biofilm formation was assessed by using the crystal violet assay. The larval disease score permitted the classification of the bacteria into three pathogenicity groups (low, intermediate and high). Bacteria were classified into six of the seven phylogenetic groups tested (i.e. A, B1, B2, C, D and F), but none were classified into group E. Biofilm-producing strains were detected at both incubation times used. There was no significant association between the larval disease score and the phylogenetic group. The larvae were susceptible to infection by all UPEC strains and the larval disease score deserves attention as a potential means of assessing the pathogenicity of UPEC isolates.
As new approach methodologies (NAMs) are increasingly explored to identify dependable and accurate non-animal alternatives to predict human toxicities, several 3-D test systems have emerged as excellent models of the hum...As new approach methodologies (NAMs) are increasingly explored to identify dependable and accurate non-animal alternatives to predict human toxicities, several 3-D test systems have emerged as excellent models of the human respiratory tract. Among these, human precision-cut lung slices (hPCLS) are considered highly relevant, as they contain many cell types (including key immune cells), feature small airway structures and boast native respiratory parenchymal architecture. However, a lack of long-term preservation methods has hampered the use of the hPCLS model for repeat and mainstream testing. In the current study, a range of potential methods were progressively evaluated for the optimum recovery of hPCLS after thawing and multi-week culturing. These methods featured: five different cryopreservation buffer (CB) recipes; freezing either before or after culture initiation; two culture media (based on E-199 and DMEM/F12); and two culture maintenance methods (submerged and air-liquid interface (ALI)). Endpoints used for the assessment of hPCLS culture health included the WST-8 viability assay, protein content and H&E histology of slice sections. Two of the CBs and immediate cryopreservation after slicing produced hPCLS with higher post-thaw viability. While both media recipes and culture methods maintained high slice viability for approximately 2 weeks, the use of DMEM-F12-based medium in ALI culture was superior for the 3-week cultures. Applying due diligence to hPCLS cryopreservation and post-thaw method development provides researchers with an underutilised human respiratory model. Studies making use of cryopreserved banks of normal or diseased tissues (from a diverse demographic pool of donors) can now be initiated as desired, repeated, or expanded upon to interrogate numerous aspects of physiology, toxicology and drug efficacy. These can be applied as routine screening applications or complex evaluations, including those benefitting a regulatory setting.
In animal-based toxicology studies, oral gavage is the predominant technique for administering compounds directly into the stomach. Recognised as a stressful procedure for animals, gavage is associated with accidents and...In animal-based toxicology studies, oral gavage is the predominant technique for administering compounds directly into the stomach. Recognised as a stressful procedure for animals, gavage is associated with accidents and 'gavage-related reflux' (GRR), which may lead to respiratory distress and/or death. This paper estimates the prevalence of incidents (deaths and/or non-fatal clinical signs) attributed to the gavage procedure in a representative sample of repeated dose toxicity test reports from the last 20 years, sourced from the ECHA CHEM database of REACH-registered chemicals. In 21% of the 300 reviewed studies, at least one death or clinical sign in rats was attributed to the gavage procedure in the study report. An additional 16% of these studies reported clinical signs associated with GRR in the animals, with no other adverse effects, but without attribution to the gavage procedure. Among those studies with incidents attributed to the gavage procedure, incidents were three times more common in 90-day studies than they were in 28-day studies. Incidents were also more likely to have occurred with the test substance rather than with the control, with highly viscous substances, and in more recently conducted studies. There was no association between the number of studies with a gavage-attributed incident and the strain, size or age of the rats, the dose volume or the testing of irritant substances. The current study suggests that incidents attributed to gavage are more prevalent than previously thought, and that a review of the gavage procedure is urgently needed.
As innovators who made non-animal-derived antibodies available to researchers starting 20 years ago, we have generated more than 60,000 antibodies during this time span. Here, we review and highlight the key challenges t...As innovators who made non-animal-derived antibodies available to researchers starting 20 years ago, we have generated more than 60,000 antibodies during this time span. Here, we review and highlight the key challenges that hampered the adoption of research antibodies generated by phage display. We describe the advantages of this method over traditional immunisation-based methods, and provide examples of specific projects. As active participants in attesting the scientific validity of non-animal-derived antibodies, we have contributed and supported key advancements in phage display for the generation of research antibodies. Such advancements have paved the way for the scientific community to switch to animal-free antibodies at a faster pace, as a goal for the near future.
Results interpretation and statistical analysis of animal study data is challenging, since the sample sizes involved are usually very small. The application of frequently used approaches to statistical hypothesis testing...Results interpretation and statistical analysis of animal study data is challenging, since the sample sizes involved are usually very small. The application of frequently used approaches to statistical hypothesis testing, e.g. -tests or ANOVA methods, rely on specific distributional assumptions being satisfied. It can be hard to reliably assess these assumptions in animal studies with group sizes of usually less than ten animals. Non-parametric analysis methods might be considered as an alternative, but it is well-known that these approaches have lower statistical power in some situations. Following the Three Rs principles, it would be desirable to apply a class of statistical tests that is able to deal with a small number of observations, without the need for specific distributional assumptions. Thus, in this paper, we assess the application of permutation tests which seem to be able to meet both the above requirements. The performance of these permutation tests was compared with standard statistical tests by means of four real-world data examples from animal studies. The results demonstrated that permutation tests have good computational properties, leading to the conclusion that they could be a useful alternative approach when analysing small sample size animal study data for which distributional assumptions may not hold.
Deckha M, Michel M, Azilagbetor D
… +20 more, Blattner C, Cajiga Morales RM, Davies G, Elger B, Faizee S, Fox M, Gerritsen V, Heuss A, Kämpfen L, Louis-Maerten E, Lüthi N, Milford A, Müller ND, Persson K, Ritskes-Hoitinga M, Rothen-Rutishauser B, Rüttimann A, Stoykova K, Stucki S, Zemanova MA
This report is a result of an interdisciplinary workshop held at the Collegium Helveticum in Zurich, Switzerland in February 2024, in which ideas for accelerating NAMs (New Approach Methodologies) in Swiss universities w...This report is a result of an interdisciplinary workshop held at the Collegium Helveticum in Zurich, Switzerland in February 2024, in which ideas for accelerating NAMs (New Approach Methodologies) in Swiss universities were shared and discussed. Due to regional differences in university organisation and funding structures, not all recommendations will be transferable to all regions worldwide. All participants were qualified to contribute to the discussion, due to their knowledge and experience of the Three Rs, in particular with regard to their implementation. The workshop participants believed that universities, which play a pioneering role in so many other areas, should also exploit their innovative potential in the field of animal-free research. The workshop uncovered four areas that would need to be addressed in order to achieve a significant change in university science culture and do more justice to the Three Rs, namely: language - innovative framing (pro-replacement framing in official university statements); knowledge transfer - communicating innovative findings in teaching (redirecting curriculum); change of values within science faculties; and structured implementation and well-coordinated planning of the transformation (establishment of a 'transition unit'). Specific strategies for implementing these four areas are outlined. In addition, we discuss why the replacement of animal testing should be an essential goal for universities, why this goal has not yet been achieved, and why concerted efforts toward change are required.
In 2020, the EU Commission's Joint Research Centre published the . In advance of the publication of this EURL ECVAM Recommendation, Landsforeningen Forsøgsdyrenes Værn arranged for an expert presentation on non-animal-de...In 2020, the EU Commission's Joint Research Centre published the . In advance of the publication of this EURL ECVAM Recommendation, Landsforeningen Forsøgsdyrenes Værn arranged for an expert presentation on non-animal-derived antibodies to the Animal Experimentation Council, which is the body responsible for the review and approval of projects involving animals in Denmark. The main concerns of Council Members following the presentation were the widespread unfamiliarity with phage display methodology in Denmark, and how it would be possible to adapt licensing decisions. Before deciding how to approach the implementation of the Recommendation, the animal experimentation authority Dyreforsøgstilsynet, of which the Animal Experimentation Council and its secretariat are part, probed the wider scientific community and requested an opinion from the National Committee on Laboratory Animals and Alternatives. Wider scientific opinion in Denmark spanned those who were, to a certain extent, positive toward the non-animal methodology and those who were sceptical. Consequently, the approach chosen by the Danish authority is to encourage and monitor the uptake of non-animal methods, while allowing time for adjustment. Change has been slow, but the seeds of change are sown.
Research involving the use of animals follows the Three Rs principles of , and . Based on the principle of , UK and EU legislation requires that technologies or alternative approaches directly replacing or avoiding the...Research involving the use of animals follows the Three Rs principles of , and . Based on the principle of , UK and EU legislation requires that technologies or alternative approaches directly replacing or avoiding the use of animals in experiments must be used wherever scientifically possible. However, is often not thoroughly considered, and failures in the existing system of checks and balances are widespread. Existing guidance and advice on searching for and identifying alternative approaches and replacement techniques is confusing and misleading, and this contributes to the lack of knowledge and confidence in addressing and the structural and procedural barriers around it. In this paper, we propose simple improvements to existing processes and a basic practical checklist, to help researchers identify and assess scientifically satisfactory replacement approaches. This tool will also support members of funding review panels, Animal Welfare and Ethical Review Bodies (AWERBs), Animal Welfare Bodies (AWBs), Animal Ethics Committees, and those editing and reviewing scientific journals in their scrutiny of applications, applicants and publications - particularly with regard to the use of alternative approaches and how these potential approaches were explored.