EU research activities in alternative testing strategies: current status and future perspectives

T. Vanhaecke 0 1 2 3 S. Snykers 0 1 2 3 V. Rogiers 0 1 2 3 B. Garthoff 0 1 2 3 J. V. Castell 0 1 2 3 J. G. Hengstler 0 1 2 3 S. Snykers e-mail: 0 1 2 3 V. Rogiers e-mail: 0 1 2 3 0 J. V. Castell Departmento de Bioquimica Facultad de Medicina, Centro de Investigacion, Hospital Universitario La Fe , Avenida de Campanar 21, 46009 Valencia, Spain 1 B. Garthoff Bayer AG DE, Kaiser-Wilhelm-Allee, W11, 51368 Leverkusen, Germany 2 T. Vanhaecke (&) S. Snykers V. Rogiers Department of Toxicology, Vrije Universiteit Brussel , Laarbeeklaan 103, 1090 Brussels, Belgium 3 J. G. Hengstler Leibniz-Institut fur Arbeitsforschung an der TU Dortmund, Leibniz Research Centre for Working Environment and Human Factors (IfADo) , Ardeystrasse 67, 44139 Dortmund, Germany - Current EU research initiatives Alternative testing methods have become increasingly important within EU funding policy. The driving force behind this push for alternative testing methods is the urgent need for effective and robust alternative toxicity tests in order to make the tools available that are necessary to comply with the 3R-strategy (Replacement, Reduction, Refinement) that has been built into the European Cosmetic (Directive 2003/15/EC) and Chemical (REACH) (Regulation EC No 1907/2006) legislations. Indeed, a general European policy has to take into consideration the growing ethical concern of the public with respect to the use of experimental animals in the risk assessment process of consumer products. It is reasonable to believe that further development and strategic incorporation of recent progress in different areas of scientific research could lead to a better and more mechanistically based approach of studying toxicological problems. In line with this, the pharmaceutical industry is confronted with unexpected failures during the drug development process, even late during the clinical phase. These could not be properly anticipated. Reasons are in particular safety (hepato- and cardiotoxicity) as well clinical efficacy of the new chemical entities. Therefore, incorporation of novel alternative methods, human based and metabolically functional, is high on the priority list since the competitiveness of the European pharmaceutical industry is at stake. An overview of the different EU initiatives in the context of the 3Rs funded research is given in EUR22846 (European Commission 2008) and in the update EUR23886 (European Commission 2009). The EU offers four different funding schemes: (1) Collaborative/Integrated projects, which are medium/large-sized ambitious projects, usually involving several areas of expertise, (2) Specific Targeted Research Projects (STREP), representing smaller networks focussed on specific research issues, (3) SME-STREP, aimed at supporting research of small- and medium-sized enterprises (SMEs), and (4) Specific Support Actions (SSA), for scientific training of personal as well as organization of conferences, workshops and expert meetings (EUR22846, EUR23886). The research networks fall into the categories of high-throughput techniques, specific toxicity tests and organization of forums and workshops. Limitations of one-to-one strategies A substantial part of current research focuses on one-toone strategies aimed at replacing a particular in vivo test by a specific alternative technique. This is currently the case both in regulatory toxicology as well as in efficacy testing of substances. In regulatory toxicology, the risk assessment process of chemical substances and hazard identification are usually carried out using experimental animals. A typical example of a replacement methodology is Episkin aimed at replacing in vivo skin irritation assays (EU 2009a B.46; OECD 2009a). The up-and-down method can reduce the number of animals used in acute toxicity testing (OECD 2001; OECD 2009b), and a refinement test is the LLNA test (Local Lymph Node Assay) (EU 2009b B.42; OECD 2002) for sensitisation testing. Examples can also be provided for efficacy testing of new chemical entities that rely solely on animal experiments. Alternatives currently used and presently investigated include the robotised enzyme testing aimed at identifying candidates acting at different stages on a specific enzymatic pathway, or ligand-based assays (such as e.g. radioimmunoassays in the past) developed for assessing effects on a particular target. Unfortunately, despite the efforts of numerous individuals, including many highly respected researchers, and the investment of millions of euros by the European Commission, various industries, and private institutions, no real breakthrough has been accomplished yet. Indeed, although the one-toone replacement strategy has contributed towards the development of the alternative strategies used today and/ or taken up in the EU regulatory framework, full implementation of 3R-alternatives has not been achieved. The in vivo scenario is thus significantly more complex than envisaged, and substitution by the one-to-one in vitro strategy seems unrealistic. Therefore, it is of utmost importance to bring innovative ideas to the field of the 3Rs and not limit experimental strategies to simple test systems and endpoints that are unable to provide a scientifically satisfactory answer to the complex in vivo reality. Hence, more consideration should be given to the complexity of the organism, which allows for the better integration of the information generated into a more solid decision-making strategy. Integration of new crosscutting technologies Important areas for deeper exploitation in alternative testing research correspond to the topics that were identified by top scientists during the meeting on New Perspectives on Safety organized by epaa (European Partnership for Alternative Approaches to Animal Testing) in April 2008 Progress in this field has been achieved by the identification of algorithms that allow classification of toxic mechanisms from gene expression data. Examples of such mechanisms include oxidative stress response, DNA damage response, and responses mediated by activation of specific receptors. One of the limitations of this approach is that toxic mechanisms can be identified only qualitatively. Currently, no clear concept is available on how quantitative information on in vivo doseresponse relationships and identification of NOELs (no observable effect levels) are to be obtained. Perhaps, a combination of toxicogenomics with kinetic modelling may be a perspective and will help guide in vitro testing at in vivo relevant concentrations (Thum and Borlak 2008; Glahn et al. 2008; Degen and Hengstler 2008; Dewa et al. 2007; Sul et al. 2007; Schug et al. 2008; Ryan et al. 2008; Sistare and Degeorge 2008; Ellinger-Ziegelbauer et al. 2009). The major advantage of using technologies that screen across many different cellular metabolic parameters is their ability to produce metabolic fingerprints of toxicants. Because changes in these metabolites are thought to precede toxic (...truncated)