G R O U P E M E N T D I N T E R E T S C I E N T I F I Q U E

G R O U P E M E N T D I N T E R E T S C I E N T I F I Q U E Plateforme française pour le développement des méthodes alternatives en expérimentation animale RAPPORT A MADAME LA MINISTRE DE L ENSEIGNEMENT SUPERIEUR ET DE LA RECHERCHE ETAT DES LIEUX DES METHODES ALTERNATIVES DANS LE DOMAINE DE L EXPERIMENTATION ANIMALE EN FRANCE Le 30 mars 2010 1

ABSTRACT This report is the result of a collaborative work of the French platform for the development of alternative methods to animal experiments" conducted from August 2008, following a letter Minister of High Education and Research, requesting an up-to-date assessment report on alternative methods to animal experiments. The French platform (FRANCOPA) used several resources to obtain the information necessary for this overview, the first one performed in France: Sending a questionnaire to institutions involved in testing on animals and also sent to institutions dedicated to research and expertise. A collective work of two technical committees of the platform. A collection of information from various actors in this field as the National Committee for ethical reflection on animal experiments and scientific societies that are mentioned in this report. The objectives of the report: alternative methods The methods considered are alternative methods to animal testing as defined by Russell & Burch in 1959, where there has been established the so-called 3Rs. These are methods that can replace the use of animals (Replacement), but also those that reduce the number of animals used (reduction) or to reduce pain or distress imposed to animals without affecting the high level of information of the study (Refinement). This definition of 3R is that adopted by all EU regulations. Alternative methods available The investigation the following methods used: - The replacement methods are, in silico (expression became common for computer calculations) based on data modelling, in vitro methods using biological materials, tissues, cells, organelles, molecules, analytical chemistry methods. - The reduction methods are, ex vivo (cells, tissues, organs from living organisms) or to reduce the number of animals used in experimental studies. The reduction of animal use can be obtained by expanding the use of technology "Omics" (more frequently hight-throughput measurement of genetic material, proteins, metabolites) and databases, the downsizing of experimental lots using relevant statistical tests or by implementing reduction strategies such as integrative approaches. - The refinement methods are for example the use of less sensitive species and the use of noninvasive scanning techniques (small animal imaging, in vivo NMR, clinical examination, behavioural assessment, telemetry) knowing that these methods are also methods of reduction. Unfortunately, to date, few alternative methods can be used as stand alone method. Alternative methods are most often used in integrated approaches combining all the alternative tools available (in vitro, ex vivo, in silico) to use the animal as a last resort when other methods have failed to provide sufficient information. The use of methods and constraints There are two major uses of alternative methods which correspond to areas of use of animal testing, but which are not subject to the same constraints: - Basic research develops and uses alternative tools for understanding the mechanistic aspects of cell functions, in cellular and molecular biology as well as in completion of animal testing for studying integrated biological mechanisms (study of disease, behaviour, etc...). At the beginning, the alternative methods used in this field were tools for research. They don t have to be subject to validation for regulatory checks, but a scientific validation or standardization or certification may be necessary to ensure the credibility and reproducibility of experimental protocols. 1

- Studies conducted in a regulatory context are intended to give targeted answers to specific questions, for example, toxicity of a substance or quality control of a batch of vaccine. In this field, validation is very restrictive in that a method acceptable by regulatory authorities must be validated to prove its relevance, its ability to detect the effects observed in animals and humans and reproducibility. Regulatory validations are mainly conducted at the European level by ECVAM Joint Research Centre (JRC) in Ispra for chemicals, the EDQM for pharmaceutical product and at the international level by ICCVAM (USA). The validation process as defined by ECVAM must be improved to reduce the duration and cost. Indeed the length of regulatory validation studies (> 10 years) and cost (200 000 / study) are an obstacle to the practical use of alternative methods. Note that some regulations (see REACh) have eased the process, in certain circumstances, by accepting "recognized" methods (scientifically by not regulatory adoption). The actors In 1986, Europe progressed in the field of animal protection, issuing a directive (86/609/EEC) to oversee the protection of animals used in experimental studies. The application of this Directive was accompanied, at the European level, by the creation of many structures dedicated to the development and validation of alternative methods: ECVAM in 1991 to conduct validation studies, ECOPA, coordinating the activities of the National Consensus Platforms in 1999 (ECOPA-NCP), EPAA established in 2005, a partnership between the EC and industry to foster communication and technology transfer between industries. In France, the scientific community, ahead of the regulation, set up ethics committees responsible for evaluating any study performed on animals. This was structured at a national level through the National Committee for ethical reflection on animal experimentation (CNREEA). Furthermore, the lessons for the ethical practice are provided through regulatory training for the implementation of animal experiments, approval of these trainings is provided by the National Commission on animal experimentation (CNEA). Currently, collaborations have been set up at international level to achieve a harmonization of practices and regulations. Due to these collaboration projects, in 2007, the creation of the French platform for the development of alternative methods to animal testing" enabled France to join ECOPA. The up-to-date assessment in France, issues (the stakes involved in up-to-date assessment in France) It emerges from the x survey responses received from national actors solicited, that several findings identify areas for improvement and enhancement of national expertise in the field: Public research developed numerous tools and concepts that have a high application potential for industry, however, technology transfer from basic research to applied research is not sufficiently effective. There is a confused perception of regulations by the researchers: for example they tend to confuse the regulatory requirement to use alternative methods in research (Directive 86/609 applies in all areas of research and R&D) and this same obligation with regard to demonstrations in the regulations applied to products (eg studies supporting MMA or safety of products). Some areas seem to be "orphans" in the sense that the incentive to develop alternative methods is low. They must be identified and solutions proposed. These remarks are clearly identified before this study: The national expertise in the field of toxicology is inadequate and poorly organized in terms of new issues and regulations at the European level. The transfer of expertise and data is not smooth. Accompanying actions of the transfer by the government are expected. The passage of information between basic research and industry is presently lacking. Fundamental applied research as carried out by academic and government institutes could be bridged to help industry improve their present methods. 2

Results The summary of results combines the contributions of the investigation, analysis of the literature and inputs from experts of the scientific Committees. The wide variety of research areas involved (in vitro, ex vivo, in silico, analytical chemistry, statistics, etc.), but also non-invasive imaging have already been described. France is involved in all these fields of research (one European project out of two, but only 8% of teams). If in vitro methods are already regularly accepted for toxicity, in silico models are also well advanced. Artificial organs and small animal imaging have prospects of short/medium term validation. The generalization of "Omics" for testing remains more uncertain, while an integrative approach is a realistic research goal. While corrosion and irritation tests have already been validated in regulations, reproductive toxicity and repeated toxicity are more upstream research objectives, while effects like carcinogenesis may be intermediaries. These assessments can be qualified by the Committees (see ECHA, EFSA) or methods. Alternative methods for toxicology and control are already validated in regulations, but they are only few: 15 for toxicity, 6 types of control tests set by the European Pharmacopoeia. During the investigation it appeared that the list of used methods is three times as long as the list of regulatory methods because the alternative methods are not used only for regulatory purposes. It was noted the large (relative to other methods!) use of in vitro methods and to a lesser extend in silico for the substitution. Contributions to the reduction and refinement are not well known. In any case, studies on the toxicity and control represent only a minority of experiments. The actors implementing these methods are varied, from research institutes to start-ups. Among the financial supports, Europe has well identified alternative methods in the context of FPRD (estimated at 200 millions which must be added in 2010, 50 million committed in sharing with COLIPA). In France, this financial project targetting is rarely possible. The National Research Agency, ANR, particularly through programs on imaging on small animal, gives financial supports on projects concerning alternative methods. AFSSET (en anglais?), conducted an investigation on evaluation of projects relative to this theme. The theme, however, is explicit only for the sole funding of the "Grenelle de l Environnement" on the platform in toxicology and ecotoxicology, the first part of 5 million (about 15 millions planned in 3 years) was released in 2009 via INERIS and ANTIOPES partnership. In the same way, it is difficult to categorize among these topics. AFSSAPS (Anglais) and INERIS (Anglais), through their actions involving 6 to 15 scientists, have targeted research to regulatory issues. Within CEA, INRA, AFSSA, INRS, INSERM and CNRS,(anglais) the investigation elicited responses and showed many activities in the 3Rs. However, those conducting these research activities have some difficulties recognizing themselves as actors in the field of the 3Rs, not only because no funding is dedicated to this theme (see investigation at the CNRS), but also because objectives of research focus on the development of new tools for investigating in vivo, substitution or reduction are just additional advantages. Researchers consider foremost the interest of these methods as tools of investigation and not as alternatives to animal testing. Thus, the CNRS, through the internal investigation of teams with approximately 370 employees and 1.9 million, reported practice of the 'alternative', without saying that this is their main objective. It's the same in the INSERM, for some 700 employees that were involved in the internal investigation. These researchers are using alternative methods, but institutes insist on the fact that it would be wrong to think that the creation of alternative methods per se is an objective of fundamental research. Among the difficulties met, we note the absence of a nomenclature of the methods allowing actors to identify themselves as actors in the field, particularly for upstream actors. It also notes the already known difficulty to go from upstream research to operational tools, and the issue of public-private partnership. 3

Recommendations The GIS has stated recommendations in the following areas: To abandon the unnecessary tests, - To promote the rules of waiving envisaged by the regulations, - To accelerate the optimization of control process, - To use more simulations in education - To optimize the terms of exchange of acquired results with an exemplary State (country). To incorporate alternative methods in the teaching of life sciences, - A systematic educational contribution, - Specific scheme set up. To focus research on innovative tools, - To put integrative approaches in toxicology at the top of the agenda - To identify needs in research and development of alternative methods, - To develop incentive programs in the area of 3R. To build an infrastructure of finalized research, - To create an IT-platform Infocentre to make known the activities and share results across sectors: - To establish a system of transfer research supported by a network of resources, - To create a center to guarantee the quality of methods (standardization, voluntary certification, etc..) - To create a center to organize the regulatory validation, - To support the development of a network of operators within industrial clusters, - To ensure integration of national strategies for research in toxicology, ecotoxicology and life sciences. To define policies among fields and evaluate their progress, - To identify and build sector policies based on reduction potential, - To refine and enhance knowledge of the type of use of animal testing and identify targets for its reduction. - Build a nomenclature allowing identification of alternative methods. Conclusions and prospects This work constitutes a first analysis on the subject. It can be improved to the extent that methodology and investigation tools are not available and that their development has to be completed (see recommendations). The depth of the planning for the development is the aim. However, this work has already benefited from active participation in the scientific community. Mobilization has been good for ethical reasons but also because these methods are linked to major advances in the understanding of in vivo mechanisms, whether in biology, therapy and toxicology. In this latter field, synergy with the renewal of toxicology is noteworthy (see action on the structuring of the field). The GIS intends to work to deepen this interaction and to facilitate the transfer from developments to upstream applications. However, GIS, in the spirit of the platforms under of umbrella of ECOPA must remember that the alternatives to animal testing are not just scientific breakthroughs, but that the spread of good practices and dissemination of information have to be taken into account. The GIS will work with means that will be developed to monitor implementation of the methods, and follow the recommendations proposed. 4