Existing approaches incorporating replacement, reduction and refinement of animal testing: applicability in food and feed risk assessment

The founding Regulation of EFSA3 requires the Authority to contribute to a high level of protection of human life and health, and in this respect to take account of animal health and welfare. EFSA is committed to a proactive animal welfare approach, based on sound scientific principles and the need to ensure that adequate data are available for a reliable risk assessment. In this context, EFSA and its Scientific Committee recognise the importance to stimulate the use of food and feed assessment approaches that would not only minimise the number of experimental animals and any suffering, but also work towards their replacement. The present Opinion gives an overview of the legislation and guiding principles on the use of animals for experimental purposes and explains the principles of scientific validation of toxicity tests for regulatory purposes. It summarises possibilities for replacement, reduction and refinement (Three Rs) of toxicological animal testing within the different areas of EFSA's activities. Finally, integrated testing and risk assessment strategies are considered and recommendations are given to implement better animal welfare within EFSA's activities. Most of the assessments conducted by EFSA's Scientific Committee and Scientific Panels require experimental data. The complete replacement of animal experiments while maintaining the same level of food and feed safety is not possible at present. However in the meantime, considerable improvements can be made to promote better animal welfare. In this Opinion, the existing approaches for incorporating replacement, reduction and refinement methods for each toxicological endpoint are reviewed, and recent and future developments are discussed. The status of the science can be summarised as follows: Toxicokinetic studies: several in vitro methods have been developed, however they cannot at present replace in vivo methods. Acute toxicity testing: the classical oral test has been replaced by in vivo alternatives that require fewer animals and reduce pain and suffering. In vitro methods are available for estimating starting doses. Skin irritation and corrosion testing: for skin irritation, at present only an in vivo method is accepted by regulatory authorities4; however it is anticipated that an in vitro method will be accepted soon for regulatory purposes at OECD level. For corrosion, in vitro methods are available and must be used in Europe. Skin sensitisation testing: only in vivo methods are accepted by regulatory authorities among which the Local Lymph Node Assay, LLNA, is the preferred refined method. An alternative in vivo method for the LLNA, and in silico and in vitro methods, are being developed. Eye irritation testing: the in vivo Draize rabbit eye test is still the standard for eye irritation testing in Europe. However, four in vitro methods can be used to classify severe eye irritants without further testing on animals. Testing for acute systemic and local toxicity is still required for plant protection products and animal feed additives to assess risk to workers. EFSA does not require such tests for risk assessment of food additives, food contact material or newly expressed proteins in GMO. Genotoxicity testing: initial testing is often done using in vitro methods, but positive results may need to be confirmed by in vivo testing. In other areas of toxicology, particularly those involving complex endpoints, such as those that are investigated in repeated dose toxicity, reproduction and developmental toxicity studies, the development of alternative methods is more difficult. The same applies for ecotoxicity endpoints such as acute and chronic toxicity in fish and birds and bioconcentration in fish. A number of methods, based on the Three Rs, are under development and some are undergoing validation. At present, they cannot yet provide the information that can be derived from currently used in vivo methods. In these areas, modifications of the individual tests and the implementation of integrated testing and risk assessment strategies can contribute to a reduction in the number of animal studies needed and, by the choice of test selected, may result in use of fewer animals. Various EFSA approaches implementing the Three Rs are described. They relate for example to tiered testing, which has been developed for food contact materials, and to application of the threshold of toxicological concern for flavouring substances and metabolites of plant protection products in groundwater. Another approach is the implementation of the qualified presumption of safety which will lead to a significant reduction in the need for animal testing in the assessment of microorganisms. The EFSA Scientific Committee notes that the legislation in force since 1986 requires that when validated, practical and accepted alternative methods, as described by the Three Rs are available they must be used. This should be communicated to the applicants that submit dossiers to EFSA as well as being fully reflected in any guidelines developed by EFSA. The Scientific Committee also recommends that in EFSA evaluations, all existing data should be reviewed before any additional in vivo studies are requested. Recognising that the Commission and Member States have responsibility for agreement of new testing methods, it is important that the existing communication between EFSA and the corresponding Commission Services that lead in this area continue to be improved, so that EFSA is informed of latest developments on the validation and acceptance of new testing methods. Communication on implementing the Three Rs with other EU Agencies dealing with chemical risk assessment is also important. The Scientific Committee recommends that EFSA follows up this Opinion with a review of progress in the field of alternatives in 3 years time.