Ethics of animal experimentation

All staff working with animals should be suitably qualified through initial training and completion of a specific training course on animal experimentation within one year after starting to work, and thereafter. The use of animals for scientific purposes also implies maintaining skills and keeping abreast of technical and scientific developments. This notion of “skills” for personnel involved in animal experimentation is defined in the European Directive 2010/63/EU (https://eur-lex.europa.eu/legal-content/FR/TXT/?uri=CELEX:32010L0063).

Initial training is required only for staff who design experimental procedures (designers). It corresponds to a Masters level (in a scientific discipline relevant to the work to be performed, or an Associate Degree (level diploma with a minimum of 5 years’ professional experience) validated by one or more designers. In addition to this initial training, all personnel handling animals in the context of experimentation (designers, experimenters and caretakers) are required to undergo specific training in animal experimentation. This specific training should be accompanied by training in surgery for anyone designing or applying experimental surgical procedures on animals. The training program is to be approved in France by the Ministry of Agriculture, following approval from the National Commission for the Protection of Animals Used for Scientific Purposes (CNEA) (https://www.enseignementsup-recherche.gouv.fr/fr/commission-nationale-pour-la-protection-des-animaux-utilises-des-fins-scientifiques-dite-cnea-84416).

In addition to these mandatory training courses, and in order to maintain their accreditation, laboratory animal handlers are required, throughout their career, to take part in a continuing education program (https://www.enseignementsup-recherche.gouv.fr/sites/default/files/2022-05/recommandation-concernant-la-formation-continue-dans-le-cadre-de-la-r-glementation-relative-l-utilisation-des-animaux-des-fins-scientifiques-17-mai-20-18446.pdf) in areas related to their professional practice, amounting to at least the equivalent of three days’ training over a six-year period. Continuing education enables staff to update their knowledge and skills in the following areas:

• protection of animals used for scientific purposes
• ensuring the welfare of the animals
• the ethics of the use of animals for scientific purposes
• application of the 3Rs (Reduce, Refine and Replace) principle (https://nc3rs.org.uk/)

Staff working with animals of non-domestic species must hold a specific certificate of competence for the care and breeding of the species in question.

For this purpose, they are required to complete a training course in animal care: https://formationsoigneuranimalier.fr/blogs/infos/construire-un-bon-parcours-jusquau-certificat-de-capacite

The concept of the 3Rs (Reduce, Refine and Replace) is being progressively adopted by many international institutions in order to set guidelines for animal experimentation: the Canadian Council on Animal Care, https://ccac.ca/fr/trois-r/; the U.S. Department of Agriculture, https://www.nal.usda.gov/animal-health-and-welfare/animal-welfare-act; and the UK Government: https://www.gov.uk/guidance/research-and-testing-using-animals. For the Council of Europe, directives can be found in the Convention ETS No. 123, https://rm.coe.int/168007a682; for the European Union in directive n° 2010/63/UE, https://eur-lex.europa.eu/legal-content/FR/TXT/PDF/?uri=CELEX:32010L0063&from=DE; and for France in Legislative Decree No. 2013-118, https://www.legifrance.gouv.fr/loda/id/JORFTEXT000027037840 and the Decree of February 1st, 2013, https://www.legifrance.gouv.fr/loda/id/JORFTEXT000027037983/.

More specific information on the 3Rs principle, https://www.inserm.fr/modeles-animaux/qu-est-regle-3-r/ and substitutive methods can be found on the websites of the french 3Rs center (FC 3R) https://www.fc3r.com/ and of the European 3Rs center https://joint-research-centre.ec.europa.eu/eu-reference-laboratory-alternatives-animal-testing-eurl-ecvam/knowledge-sharing-three-rs/knowledge-networks/european-3rs-centres_en.

The National Centre for Replacement, Reduction and Refinement in animal research (NC3Rs) offers a range of resources classified by theme: https://www.nc3rs.org.uk/3rs-resources.

The french national research infrastructure “Creation, breeding, phenotyping, distribution and archiving of model organisms” (CELPHEDIA), the “Infrastructures en Biologie Santé et Agronomie” (IBiSA), the “European research infrastructure for modeling human diseases” (INFRAFRONTIER) and private companies (Charles River, Janvier-labs, The Jackson Laboratory, …) provide a wide range of expertise in the creation, functional exploration, conservation and dissemination of model organisms (rodents, non-human primates, aquatic vertebrates, etc) for preclinical research.

https://celphedia.eu

https://www.ibisa.net/plateformes/index.php?sor=0%7CD&srch=&dom%5B%5D=Animalerie%2C+exploration+fonctionnelle#resultats

https://www.criver.com

https://janvier-labs.com

https://www.jax.org

https://www.infrafrontier.eu

Since the publication of the French Decree n°2013-118, Animal welfare entities (SBEA) have been set up in establishments using and/or breeding laboratory animals: https://www.sbea-c2ea.fr/. These structures play a role in checking and auditing all procedures included in projects involving the use of animals in accredited establishments.

In France, any research project using animals for scientific purposes needs a favorable assessment from the “animal experimentation ethics committee” (CEEA) of the establishment in which it will be carried out, followed by an authorization from the French Ministry of Higher Education and Research.

There are a number of guidelines available to help you prepare for the authorization process:

The Interprofessional think tank on ethics committees applied to animal experimentation, “Groupe de réflexion interprofessionnel sur les comités d’éthique appliqués à l’expérimentation animale” (GRICE), is a working group belonging to Gircor, has drawn up a guide for the ethical evaluation of animal studies: https://www.gircor.fr/guide-devaluation-ethique-2/

The checklist “Animal Research: Reporting of In Vivo Experiments“ (ARRIVE) : https://arriveguidelines.org/ of NC3Rs proposes 10 steps to follow to organize and improve a project using animals.

Norecopa “Planning Research and Experimental Procedures on Animals: Recommendations for Excellence“ (PREPARE) checklist is also available for download in French, and proposes 20 items to help build a project: https://norecopa.no/media/7893/prepare_checklist_english.pdf.

A network created from the “Enhancing Quality in Preclinical Data” (EQIPD) project offers a comprehensive framework for the design, conduct, analysis and documentation of animal experiments: https://www.nature.com/articles/s41592-022-01615-y.

What is the best in vitro, in silico or in vivo model to address a scientific question?

Various resources are available to guide scientists in their choice: in vitro GDR Organoïdes (https://gdr-organoides.cnrs.fr/), GDR Microfluidique (https://www.gdrmicrofluidique.com), Re-Place (https://www.re-place.be), ECVAM, EPAA and model organisms (vertebrates and et invertebrates) EFOR (https://efor.fr/), Celphedia (https://celphedia.eu/en/).

Numerous efforts have been made in recent years to reduce the number of animals used in experimental procedures.

To ensure rational animal breeding, it is important to consider first and foremost the nature of the strain under consideration, in order to adapt the breeding strategy while ensuring optimization of strain-specific parameters.

For non-inbred rodent strains, maintaining genetic heterogeneity is essential, but it can quickly become a challenge, leading to the production of larger numbers of animals. To avoid this, there are simple tools available for setting up mating schemes that keep the inbreeding rate below 1% when using small batches of animals. These include the Poiley, Robertson, Falconer and HAN mating schemes.

In the case of inbred strains, the opposite reasoning should be adopted, as the aim is to achieve maximum genetic stability. To achieve this, breeding stock with the highest genetic homology should be selected in order to limit genetic drift. DNA sequencing or microsatellite research may be necessary to check the conformity of the strain and that of the model, the absence of genetic drift, and the absence of genetic contamination. Beware of “false backcrossing”, often presented as a way of “refreshing” the genetic background of a strain. This is a common practice, but from a genetic point of view it makes no sense and is of little interest.

When maintaining transgenic strains, it is essential to customize the breeding strategy according to the strain characteristics and your experimental needs. To achieve this, it is imperative to define project objectives and priorities, collect data to monitor breeding performance and adjust breeding accordingly, and manage breeding as a project.

Generally speaking, you should always choose the optimum mating scheme, the type of breeding (monogamous, continuous, etc.), consider the genetic background and its impact, and take into account the objectives set (number, genotype, age, sex, frequency, controls, etc.).

Never neglect environmental factors and their control (health status, rearing conditions, enrichment, in some cases seasonality, etc.) which can have a major impact on animal production and lead to unsuitable production.

Never forget:

• Planning is essential,
• Breeding colonies of genetically altered animals can be complex,
• That reproductive performance is specific to each species and each farm,
• Record as much data as possible and analyse it regularly.

Think about stabilizing your lines and making them safe by opting for sperm or embryo cryopreservation. What is described here for rodents can also be applied to other species such as fish.

National platforms (PPA-UMS28, CREFRE, CIPHE) and/or professionals (Charles River,Janvier-labs, The Jackson Laboratory) can support you in the production and conservation of your animal lines.

https://infrastructures.inserm.fr/Pages/ficheinfrastructure.aspx?infrastructureId=2294

https://anexplo.genotoul.fr/zootechnie/

https://ciphe.marseille.inserm.fr/en/

https://www.criver.com/products-services/research-models-services/genetically-engineered-model-services/mouse-rat-breeding?region=3696

https://janvier-labs.com/elevage/gestion-des-colonies/

https://www.jax.org/jax-mice-and-services/colony-management/cryopreservation/sperm-cryo-kit

The reproducibility of research findings is a key factor in ensuring their validity and credibility. However, a number of studies have highlighted a lack of reproducibility of findings in most disciplines. In studies involving animals, it is essential to ascertain reproducibility, from both a scientific and an ethical point of view. A number of issues affecting the reproducibility in vivo results can and must be improved, particularly in the experimental design.

With this in mind, to help researchers design their in vivo experiments, NC3Rs has developed an open-access web platform: the Experimental Design Assistant  (EDA). This tool is currently used by over 7,000 researchers worldwide, and is recommended by a growing number of funding bodies (MRC, https://www.ukri.org/councils/mrc/guidance-for-applicants/proposals-involving-animal-use/; BBSRC, https://www.ukri.org/councils/bbsrc/guidance-for-applicants/animal-use-in-research; Wellcome Trust, https://wellcome.org/grant-funding/guidance/responsible-conduct-research; Cancer Research UK, https://www.cancerresearchuk.org/sites/default/files/cruk_animal_research_policy.pdf; Academy of Medical Sciences, https://acmedsci.ac.uk/grants-and-schemes/grant-schemes/information-for-applicants/start-your-application; NIH, https://grants.nih.gov/policy/reproducibility/resources.htm ; the National Science Foundation, https://www.nsf.gov/pubs/2019/nsf19028/nsf19028.jsp; the Australian National Health and Medical Research Council, https://research.unsw.edu.au/document/ea20_best_practice_methodology_in_the_use_of_animals_for_scientific_purposes_2017.pdf.

NC3Rs itself, and now Inserm, are offering support in using the tool.

Other project design support tools include the SHiny Application for Designing Experiments (SHADE) freely available from the Institut Pasteur: https://research.pasteur.fr/fr/software/shade-shiny-application-for-designing-experiments/.

The pre-registration of protocols is the process of depositing the protocol of your research project on a registry before it begins. This ensures that the final publication of results is in line with what was planned. It also ensures compliance with the highest standards of research integrity, with the aim of promoting the reproducibility of the study. It also enables any changes to be traced transparently. There are several pre-registration websites for studies using animals for scientific purposes, such as the animal study registry (https://www.animalstudyregistry.org/asr_web/index.action) and preclinical trials (https://preclinicaltrials.eu/). More generically, the Open Science Framework also enables these registrations (https://osf.io/).

In addition to registries, it is possible to submit your project for peer review to a journal, even before conducting any scientific experimentation. In this case, the project is evaluated by peers on the basis of the relevance of the research question and the quality of the method. A project judged to be of high quality will then be accepted in advance for publication, provided that the method implemented is indeed the one envisaged. This new format of ‘registered reports’ offers two additional advantages: peer review by experts guarantees 1/ the best possible methodological and analytical approaches, and 2/ an editorial decision based solely on the quality of the research and the importance of the scientific question, and not on the outcome of the results. This approach is thus intended to limit publication bias. Currently, over 300 scientific journals accept registered reports. A list of these journals, as well as many other resources related to the pre-registration of research projects, can be found on the «Center for open science» website.

Although they are an essential component of research work, most negative results are not published or disseminated. However, as an editorial in the journal Nature points out, the fact that a result does not validate the hypothesis formulated at the outset does not mean that this result is unimportant and should disappear from the scientific community. Sharing knowledge and information within the scientific community avoids unnecessary and unethical repetitions. Subject to reliability (like any other result), many initiatives have been launched to reinstate negative results. https://www.datacc.org/bonnes-pratiques/diffuser-des-resultats-negatifs/les-donnees-negatives-la-partie-immergee-de-liceberg-des-publications-scientifiques/#part-6.

The GIS FC3R was assigned the task of setting up a platform of this type in France: https://www.fc3r.com/donnees-non-publiees.php.

Sharing genetically modified tissues, cells and organisms reduces the number of animals used, increases the amount of knowledge acquired per animal, and optimizes the use of financial resources. The sharing of biological resources is encouraged and financially supported by the GIS FC3R.

Animal experiments are a highly sensitive issue, and one that rightly concerns and mobilizes public opinion. Petitions, citizens initiatives and requests for referendums on the topic are increasing in number, even though opinion polls show that the public as a whole knows little about animal experimentation. This ignorance or misunderstanding from the general public means that we need to provide more reliable, accurate and factual information on current animal research, so that everyone can form its own opinion, refocus the debate on the issues that make sense, and support research as it moves forward (https://experimentation-animale.info/opinion-publique/).

This need for transparency and better communication is also evident within the scientific community, for instance to avoid unnecessary repetition of experiments or to facilitate the transition from existing methods to alternatives. The most common reason for using laboratory animals to address a specific research question is that alternative methods do not yet exist or cannot be used to study the complex interactions in an entire organism. But in some cases, the replacement of a methodology by non-animal research approaches may exist without having been identified. This can involve using a combination of methods to obtain the same data as the animal experiment, rather than a single “replacement”.

It therefore seems necessary to provide more information on:

• Why and how animals are used for scientific research in Europe,
• Which areas still require the use of animals,
• Which fields use the most animals, which are the procedures that involve moderate or severe suffering, and which regulatory requirements entail the use of animals,
• R&D efforts and funding to accelerate the development of alternative approaches.

Numerous initiatives have been implemented to make animal research more transparent, such as the publication of annual statistics and other information at https://www.enseignementsup-recherche.gouv.fr/fr/resumes-non-techniques-des-dossiers-notifies-46563. In line with the 2010 Directive, the EU has also developed ALURES (Animal Use Reporting – EU System), the first free and open public database on the use of animals for scientific purposes. It provides reliable statistics on the number of animals used in research throughout Europe, and lists complete and detailed data on each use of animals and the procedures implemented.

A series of initiatives has also been set up to support and accompany research institutes in this opening-up process, which can be difficult – and even distressing – given the stigma attached to animal research. For example, several EU countries have an association specializing in animal research communication, such as the “Groupe interprofessionnel de réflexion et de communication sur la recherche” (Gircor) in France. The EARA association also works internationally to communicate on the interests of biomedical and health research in Europe. In particular, it has supported numerous institutions in signing the now widespread ‘transparency agreements’, which commit them to being more open on the reasons and circumstances surrounding the use of animals for scientific purposes, to communicating more with the public and the media, and producing an annual report on progress made.

Find out more about the French Transparency Charter: Transparency Charter

Animals could be adopted, released or placed in specialized facilities (zoos, shelters, educational farms, etc.) of laboratory animals whose state of health allows it. More and more laboratories are turning to this practice, which fits in perfectly with the 3Rs approach, providing healthy laboratory animals with a practical and ethical alternative to euthanasia.

The placement of laboratory animals after experiments is authorized by Article 19 of Directive 2010/63/UE of the European Parliament and of the Council of September 22, 2010 on the protection of animals used for scientific purposes, and by Article R.214-112 of the French Rural Code. The placement of a laboratory animal after experiments is possible under 3 cumulative conditions:

• that the animal is in good health and is not suffering from any physical or psychological after-effects,
• that the animal presents no risk to the environment or public health,
• that rehoming is in the animal’s best interests.

Animals that can be rehomed are selected on the basis of these criteria within research institutes by authorized personnel (researchers, veterinarians, animal handlers, etc), and then contracted out to associations or structures capable of caring for them.

Since 2005, the GRAAL association has been facilitating the placement of laboratory animals by acting as a mediator between laboratories and numerous shelters (SPA, zoos, etc.).

Since 2014, the White Rabbit association has made it possible to extend rehoming to the species most commonly used in laboratories – rodents, fish, rabbits – notably by offering solutions for fostering and caring for these small animals, as well as facilitating administrative procedures for laboratories.