A Brief Review of the European Directive on 3Rs and Facilitating Animal Experimentation



  • Omeralfaroug Ali Magyar Agrár- és Élettudományi Egyetem / Agrobiotechnology and Precision Breeding for Food Security National Laboratory, Institute of Physiology and Animal Nutrition, Department of Animal Physiology and Health, Hungarian University of Agriculture and Life Sciences, 40 Guba Sándor Street, 7400 Kaposvár, Hungary




animal welfare, reduction, refinement, replacement, research


The value of animals to humankind extends beyond their provision of food and physical resources; they also serve a wide range of purposes, including their role in experimentation. Given that animals play a substantial part in experimentation, it becomes imperative to improve their living conditions, at the very least as a form of compensation. As a result, the driving force behind the enactment of various legislations, including the European Union Directive (2010/63/EU), is human morality. This directive primarily pertains to specific European member states and is fundamentally aimed at safeguarding the welfare of animals used in experimentation by advocating for the implementation of the 3Rs: replacement, reduction, and refinement. Nevertheless, concerns have arisen, particularly regarding how the provisions of the directive might constrain or influence the advancement of experimentation, notably within the biomedical field. This review explores the nexus between Directive 2010/63/EU and the facilitation of animal experimentation, with a specific focus on the 3Rs. Findings reveal that the judicious application of the 3Rs markedly enhances the progress of experimentation without compromising the quality of outcomes. Furthermore, recent studies propose that the 3Rs alone may not suffice, suggesting the need to introduce additional relevant Rs. Hence, the current decrees are deemed adequate for a certain period, with potential modifications in the future. In order to encourage the advancement of experimentation without jeopardizing animal welfare, it is imperative to conduct periodic, routine reviews.





. 6Roundtable (2022). Available at: https://www.berliner-kompaktkurse.de/6r-roundtable.html. Accessed on 15 January, 2024.

Abelson, K. S., Chambers, C., De La Cueva, T., et al. (2023). Harmonisation of education, training and continuing professional development for laboratory animal caretakers, technicians and tech-nologists: Report of the FELASA-EFAT Working Group. Lab Anim., 57, 599-610. https://doi.org/10.1177/00236772231175550.

Akhila, J., Shyamjith, D., Alwar, M. (2007). Acute toxicity studies and determination of median lethal dose. Curr. Sci., 93, 917-920. Available at: http://www.jstor.org/stable/24099255.%20Accessed%2018%20Apr.%202023. Accessed on 15 January, 2024.

Akkermans, A., Chapsal, J. M., Coccia, E. M., et al (2020). Animal testing for vaccines. Implementing replacement, reduction and refinement: challenges and priorities. Biologicals, 68, 92–107. https://doi.org/10.1016/j.biologicals.2020.07.010.

Australian National Health and Medical Research Council (NHMRC). (2013). Australian code for the care and use of animals for scientific purposes, 8th ed. Canberra: National Health and Medical Re-search Council, Australia. Available at: https://www.nhmrc.gov.au/about-us/publications/australian-code-care-and-use-animals-scientific-purposes#block-views-block-file-attachments-content-block-1. Accessed on 15 January, 2024.

Barbee, R.W., Turner, P.V. (2019). Incorporating laboratory animal science into responsible biomed-ical research. ILAR J., 60, 9-16. https://doi.org/10.1093/ilar/ilz017.

Basketter, D.A., Gerberick, G.F. (2022). Skin Sensitization Testing: The Ascendancy of Non-Animal Methods. Cosmetics, 9, 38. https://doi.org/10.3390/cosmetics9020038.

Bayne, K. A., Bayvel, A. C. D., Williams, V. (2013). Laboratory animal welfare: international issues. In Bayne KA, Turner PV, eds. Laboratory Animal Welfare, Ed 1. New York: Academic Press. p 55-76.

Bonafos, L., Simonin, D., Gavinelli, A. (2010). Animal welfare: European legislation and future per-spectives. J. Vet. Med. Educ., 37, 26–29. https://doi.org/10.3138/jvme.37.1.26.

Bracken, M. B. (2009). Why animal studies are often poor predictors of human reactions to expo-sure. J. R. Soc. Med., 102, 120–122. https://doi.org/10.1258%2Fjrsm.2008.08k033.

Brannen, K. C., Chapin, R. E., Jacobs, A. C, Green, M. L. (2016). Alternative models of developmental and reproductive toxicity in pharmaceutical risk assessment and the 3Rs. ILAR J., 57, 144-156. https://doi.org/10.1093/ilar/ilw026.

Bundesrat (2022). Popular initiative of March 18, 2019 “Yes to the ban on animal and human experi-ments – Yes to research paths with impulses for safety and progress”. Available at: https://www.bk.admin.ch/ch/d/pore/va/20220213/can651.htmlhttps://www.bk.admin.ch/ch/d/pore/va/20220213/can651.html. Accessed on 15 January, 2024

Campbell, B. R., Trotter G. D., Hines, C.D., et al. (2016). In vivo imaging in pharmaceutical development and its impact on the 3Rs. ILAR J., 57, 212–220. https://doi.org/10.1093/ilar/ilw019.

Canadian Council on Animal Care (CCAC). (1989). CCAC policy statement on ethics of animal investi-gation. Ottawa: CCAC. 1989. Available at: https://www.ccac.ca/Documents/Standards/Policies/Ethics_of_animal_investigation.pdf. Ac-cessed on 15 January, 2024.

Clark, J. M., Clifford, P., Jarrett, W., Pekow, C. (2019). Communicating about animal research with the public. ILAR J., 60, 34-42. https://doi.org/10.1093/ilar/ilz007.

Curzer, H. J., Wallace, M. C., Perry, G., Muhlberger, P. J., Perry, D. (2013). The ethics of wildlife re-search: a nine R theory, ILAR Journal, 54,52–57. https://doi.org/10.1093/ilar/ilt012.

Czubala, M. A., Eilles, E., Staubi, A., et al. (2022). 3R Blackboard: A platform for animal and organ shar-ing. Laboratory Animals, 56, 292-296. https://doi.org/10.1177/00236772211067456.

Das, R. G., Fry, D., Preziosi, R., Hudson, M. (2009). Planning for reduction. Altern Lab Anim., 37, 27–32. https://doi.org/10.1177/026119290903700106.

DeGrazia, D., Beauchamp, T. L. (2019). Beyond the 3 Rs to a more comprehensive framework of principles for animal research ethics. ILAR Journal, ilz011, 1-10. https://doi.org/10.1093/ilar/ilz011.

Department for Environment, Food and Rural Affairs (DEFRA). (2010). Review of the animal welfare research programme 2005-2010. DEFRA, London, UK, p.13. available at: https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/69385/pb13556-welfare-research-review-110128.pdf. Accessed on 15 January, 2024.

Dolan, K. (1999). Ethics, Animals, and Science. Blackwell: Oxford, UK.

Ecuer, E., Boxall, J., Louwerse, A. L., et al. (2023). FELASA recommendations for the rehoming of ani-mals used for scientific and educational purposes. Lab Anim., 57, 572-582. https://doi.org/10.1177/00236772231158863.

Eggel, M., Grimm, H. (2018a). Necessary, but not sufficient. The benefit concept in the project evalua-tion of animal research in the context of Directive 2010/63/EU. Animals-Basel, 8, 34. https://doi.org/10.3390/ani8030034.

Eggel, M., Grimm, H. (2018b). The logic, methodological and practical flaws of the harm-benefit-analysis in Directive 2010/63/EU. Conference Proceedings of Professionals in food chains. Aus-tria, 13 June 2018 - 16 June 2018, Wageningen Academic Publishers, pp.407-411. https://doi.org/10.3920/978-90-8686-869-8_64.

ESAC, 2008. Statement on the scientific validity of in-vitro tests for skin irritation testing. http://ecvam.jrc.it/index.htm. Accessed on 15 January, 2024.

European Animal Research Association (EARA) (2023). 2020 EU numbers on animals used in re-search show the impact of Covid-19. Available at: https://www.eara.eu/post/2020-eu-figures-on-animals-used-in-research-show-the-impact-of-covid-19. Accessed on 15 January, 2024.

European Commission (EC) (2015). Commission replies to "Stop Vivisection" European Citizens' Initiative. Available at: https://ec.europa.eu/commission/presscorner/detail/en/IP_15_5094. Accessed on 15 January, 2024.

European Union (EU) (1986). European Convention for the protection of vertebrate animals used for experimental and other scientific purposes/Convention européenne sur la protection: Protec-tion of Vertebrate Animals. Council of Europe, 123. Available at: https://www.coe.int/en/web/conventions/full-list/-/conventions/rms/090000168007a67b. Accessed on 15 January, 2024.

European Union (EU) (2009). 22nd Report of Session 2008–09: The revision of the EU Directive on the protection of animals used for scientific purposes. Volume II; Evidence. House of Lords, London, UK. Available at: https://publications.parliament.uk/pa/ld200809/ldselect/ldeucom/164/164ii.pdf. Accessed on 15 January, 2024.

European Union (EU) (2010). Directive 2010/63/EU of the European Parliament and of the council: on the protection of animals used for scientific purposes. Official Journal of the European Union. 276: 33-79. Available at: https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:en:PDF. Accessed on 15 January, 2024.

Felsmann, M. Z., Szarek, J., Felsmann, M., Strzyzewska, E. (2014). Protection of animals used in exper-iments in Polish law – history, present day and perspective: a review. Vet Med (Praha), 59, 117–123. Available at: https://www.agriculturejournals.cz/pdfs/vet/2014/03/01.pdf. Accessed on 15 January, 2024.

Ferdowsian, H. R., Beck, N. (2011). Ethical and scientific considerations regarding animal testing and research. PLoS One, 6, e24059. http://doi.org/10.1371/journal.pone.0024059.

Ferrara, F., Hiebl, B., Kunzmann, P., et al. (2022). Culture of care in animal research – Expanding the 3Rs to include people. Lab. Ani., 56, 511-518. http://doi.org/10.1177/00236772221102238.

Ford, K. A. (2016). Refinement, reduction, and replacement of animal toxicity tests by computational methods. ILAR J., 57, 226-233. https://doi.org/10.1093/ilar/ilw031.

Franco, N. H., Olsson, I. A. S. (2013). Scientists and the 3Rs: attitudes to animal use in biomedical research and the effect of mandatory training in laboratory animal science. Lab. Anim., 48, 50–60. https://doi.org/10.1177/0023677213498717.

Genzel, L., Adan, R., Bernes, A., et al. (2020). How the COVID-19 pandemic highlights the necessity of animal research. Curr. Biol., 30, R1014–R1018. https://doi.org/10.1016%2Fj.cub.2020.08.030.

Giles, J. M., Whitaker, J. W., Moy, S. S., Fletcher, C. A. (2018). Effect of environmental enrichment on aggression in BALB/cJ and BALB/cByJ mice monitored by using an automated system. J Am As-soc Lab Anim Sci., 57, 236–43. https://doi.org/10.30802/aalas-jaalas-17-000122.

Goh, J. Y., Weaver, R., Dixon, L., Platt, N., Roberts, R. (2015). Development and use of in vitro alterna-tives to animal testing by the pharmaceutical industry 1980-2013. Toxicol. Res., 4, 1297–1307. https://doi.org/10.1039/C5TX00123D.

Grimm, H., Biller-Andorno, N., Buch, T., et al. (2023). Advancing the 3Rs: innovation, implementation, ethics and society. Front Vet Sci., 10:1185706. https://doi.org/10.3389%2Ffvets.2023.1185706.

Grimm, H., Eggel, M., Deplazes-Zemp, A., Biller-Andorno, N. (2017). The road to hell is paved with good intentions: why harm–benefit analysis and its emphasis on practical benefit jeopardizes the credibility of research. Animals-Basel, 7, 70. https://doi.org/10.3390/ani7090070.

Gruen, L. (2011). Ethics and Animals. Cambridge University Press: Cambridge, MA, USA, 2011, p.118.

Hackam, D. G., Redelmeier, D. A. (2006). Translation of research evidence from animals to humans. JAMA, 296, 1727-1732. https://doi.org/10.1001/jama.296.14.1731.

Han J. J. (2023). FDA Modernization Act 2.0 allows for alternatives to animal testing. Artif Organs, 47, 449-450. https://doi.org/10.1111/aor.14503.

Harding, J. (2017). Genomic tools for the use of non-human primates in translational research. ILAR J., 58, 59–68. https://doi.org/10.1093%2Filar%2Filw042.

Hartung, T. (2010). Comparative Analysis of the Revised Directive 2010/63/EU for the Protection of Laboratory Animals with its Predecessor 86/609/EEC – a t4 Report. Altex. 27, 285-303. https://doi.org/10.14573/altex.2010.4.285.

Hassan, Z. A., Schattner, P., Mazza, D. (2006). Doing a pilot study: why is it essential?. Malays. Fam. Physician. 1, 70–73. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453116/pdf/MFP-01-70.pdf. Accessed on 15 January, 2024.

Hawkins, P., Bertelsen, T. (2019). 3Rs-related and objective indicators to help assess the culture of care. Animals, 9, 5–11. https://doi.org/10.3390/ani9110969.

Hehemann, L. (2019). The approval of animal experiments in the context of animal protection and freedom of research. A legal comparison between Germany, Austria and Switzerland: Work from the legal seminar at the University of Freiburg Switzerland. Schulthess Publishing, Switzer-land. pp. 305–325. (The book is in German language).

Hooijmans, C. R., IntHout, J., Ritskes-Hoitinga, M., Rovers, M. M. (2014). Meta-analyses of animal studies: an introduction of a valuable instrument to further improve healthcare. ILAR Jl., 55, 418–426. https://doi.org/10.1093/ilar/ilu042.

Hopper, L. D. (2016). Automated microsampling technologies and enhancements in the 3Rs. ILAR J., 57, 166-177. https://doi.org/10.1093/ilar/ilw020.

Hudson, M. (2006). The EU Physical Agents (EMF) directive and its impact on MRI imaging in animal experiments: a submission by FRAME to the HSE. Altern. Lab. Anim. 34, 343–347. https://doi.org/10.1177/026119290603400302.

Hungary (1998). XXVIII of 1998 Act on the Protection and Welfare of Animals (Article in Hungarian language). Available at: https://net.jogtar.hu/jogszabaly?docid=99800028.tv. Accessed on 15 January, 2024.

Hungary (2013). 40/2013. (II. 14.) Government decree on animal experiments (Article in Hungarian language). Available at: https://net.jogtar.hu/jogszabaly?docid=a1300040.kor. Accessed on 15 January, 2024.

Hungary (2014). The Government 96/2014. (III. 25.) Government Decree on the taking over by the central budget body of the tasks carried out through the participating organizations (Article in Hungarian language). Available at: http://www.kozlonyok.hu/nkonline/MKPDF/hiteles/MK14043.pdf. Accessed on 15 January, 2024.

Johnson, V. E. (2013). Revised standards for statistical evidence. Proc. Nat. Acad. Sci., 110, 19313–19317. https://doi.org/10.1073/pnas.1313476110.

Kattan, M. W., Gerds, T. A. (2018). The index of prediction accuracy: an intuitive measure useful for evaluating risk prediction models. Diagn. Progn. Res., 2: 7. https://doi.org/10.1186/s41512-018-0029-2.

Kendall, L. V., Owiny, J. R., Dohm, E. D., et al. (2019). Replacement, refinement, and reduction in animal studies with biohazardous agents. ILAR J., 59, 177-194. https://doi.org/10.1093/ilar/ily021.

Kiani, A. K., Pheby, D., Henehan, G. (2022). Ethical considerations regarding animal experimentation. J. Prev. Med. Hyg., 63, E255–E266. https://doi.org/10.15167%2F2421-4248%2Fjpmh2022.63.2S3.2768.

Kirby, P. (2004). A guide to actively involving young people in research: for researchers, research commissioners, and managers. INVOLVE. Available at: https://www.invo.org.uk/wp-content/uploads/2012/01/InvolvingYoungPeople2004.pdf. Accessed on 15 January, 2024.

Krebs, C. E., Lam, A., McCarthy, J., Constantino, H., Sullivan, K. (2022). A survey to assess animal methods bias in scientific publishing. bioRxiv. https://doi.org/10.1101/2022.03.24.485684.

Kroeger, M. (2006). How omics technologies can contribute to the “3R” principles by introducing new strategies in animal testing. Trends in Biotechnology, 24, 343-346. http://doi.or/10.1016/j.tibtech.2006.06.003.

Laroche, C., Aggarwal, M., Bender, H., et al (2018). Finding synergies for 3Rs – toxicokinetics and read-across: report from an EPAA partners’ Forum. Regul Toxicol Pharmacol., 99, 5–21. https://doi.org/10.1016/j.yrtph.2018.08.006.

Louhimies, S. (2012). EU Directive 2010/63/EU: “Implementing the three Rs through policy”. ALTEX Proc., 1, 27–33. Available at: https://proceedings.altex.org/data/2012-01/027033_Louhimies31.pdf. Accessed on 15 January, 2024.

Macleod, M., Mohan, S. (2019). Reproducibility and rigor in animal-based research. ILAR J., 60, 17-23. https://doi.org/10.1093/ilar/ilz015.

Mahony, C., Ashton, R. S., Birk, B., et al (2020). New ideas for non-animal approaches to predict re-peated-dose systemic toxicity: Report from an EPAA Blue Sky Workshop. Regul. Toxicol. Phar-macol., 114, 104668. https://doi.org/10.1016/j.yrtph.2020.104668.

Mandal, J., Parija, S. C. (2013). Ethics of involving animals in research. Trop Parasitol., 3, 4-6. http://doi.org/10.4103/2229-5070.113884.

McCormick-Ell, J., Connell, N. (2019). Laboratory safety, biosecurity, and responsible animal use. ILAR J., 60, 24-33. https://doi.org/10.1093/ilar/ilz012.

Mikhaylova, A.V., Thornton, T. A. (2019). Accuracy of gene expression prediction from genotype data with PrediXcan varies scross and eithin continental populations. Front. Genet., 10, 261. https://doi.org/10.3389/fgene.2019.00261.

Olsson, I. A. S., da Silva, S. P., Townend, D., Sandøe, P. (2016). Protecting animals and enabling research in the European Union: an overview of development and implementation of Directive 2010/63/EU. ILAR J., 2016; 57(3): 347-357.

Olsson, I. A. S., Nicol, C. J., Niemi, S. M., Sandøe, P. (2020). From unpleasant to unbearable—why and how to implement an upper limit to pain and other forms of suffering in research with animals. ILAR J., ilz018: 1-11. https://doi.org/10.1093/ilar/ilz018.

Ormandy, E. H., Schuppli, C. A. (2014). Public attitudes toward animal research: a review. Animals-Basel. 4, 391-408. https://doi.org/10.3390/ani4030391.

Polli, J. E. (2008). In vitro studies are sometimes better than conventional human pharmacokinetic in vivo studies in assessing bioequivalence of immediate-release solid oral dosage forms. AAPS J., 10, 289–299. https://doi.org/10.1208/s12248-008-9027-6.

Richter, S. H., Garner, J. P., Würbel, H. (2009). Environmental standardization: cure or cause of poor reproducibility in animal experiments?. Nat. Methods, 6, 257–261. https://doi.org/10.1038/nmeth.1312.

Ritskes-Hoitinga, M. (2022). Medical regulators: look beyond animal tests. Nature, 604, 599. https://doi.org/10.1038/d41586-022-01110-6.

Russell, W. M. S., Burch, R. L. (1959). The principles of humane experimental technique. Methuen & CO LTD, London, UK.

Saeidnia, S., Manayi, A., Abdollahi, M. (2015). From in vitro experiments to in vivo and clinical stud-ies; pros and cons. Curr Drug Discov Technol., 12, 218-24. https://doi.org/10.2174/1570163813666160114093140.

Slokenberga, S. (2017). Country report: an overview of animal protection in Latvia. In: Il comitato scientifico del Progetto (ed.), Scritti su etica e legislazione medica e veterinaria. Bari: Cacucci Edi-tore, pp.149-158. Available at: http://uu.diva-portal.org/smash/get/diva2:1158663/FULLTEXT01.pdf. Accessed on 15 January, 2024.

Spencer, C. C. A., Su, Z., Donnelly, P., Marchini, J. (2019). Designing genome-wide association studies: sample size, power, imputation, and the choice of genotyping chip. PLoS Genet., 5, e1000477. https://doi.org/10.1371/journal.pgen.1000477.

Statista (2020). Annual number of animals used in research and testing in selected countries world-wide as of 2020. Available at: https://www.statista.com/statistics/639954/animals-used-in-research-experiments-worldwide/. Accessed on 15 January, 2024.

Taylor K., Alvarez, L. R. (2019). An estimate of the number of animals used for scientific purposes worldwide in 2015. Alternatives to Laboratory Animals, 47, 196-213. https://doi.org/10.1177/0261192919899853.

Van der Meulen-Frank, M., Prins, J. B., Waarts, B. L., Hofstra, W. (2017). Vertebrate animals used for experimental and other scientific purposes: principles and practice for legislation and protec-tion. In: Glaudemans A., Medema J., van Zanten A., Dierckx R., Ahaus C. (eds). Quality in nuclear medicine. Springer, Cham, pp 91-105.

Van der Worp, H. B., Howells, D. W, Sena, E. S., et al. (2010). Can animal models of disease reliably inform human studies? PLoS Med., 7, e1000245. https://doi.org/10.1371/journal.pmed.1000245.

Van Norman, G. A. (2019). Limitations of animal studies for predicting toxicity in clinical trials: is it time to rethink our current approach?. JACC: Basic to Translational Science, 4, 845-854. https://doi.org/10.1016/j.jacbts.2019.10.008.

Wells, D. J. (2011). Animal welfare and the 3Rs in European biomedical research. Ann. N. Y. Acad. Sci., 1245, 14–16. https://doi.org/10.1111/j.1749-6632.2011.06335.x.

Wong, J. C., Zidar, J., Ho, J., et al. (2017). Assessment of several machine learning methods towards reliable prediction of hormone receptor binding affinity. Chemical Data Collections, 9-10, 114-124. https://doi.org/10.1016/j.cdc.2017.05.002




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A Brief Review of the European Directive on 3Rs and Facilitating Animal Experimentation: Review. (2023). ACTA AGRARIA KAPOSVARIENSIS, 27(1-2), 7-28. https://doi.org/10.31914/aak.4191