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In Deutschland und allen EU Mitgliedsstaaten wird die Verträglichkeitsprüfung von Kosmetika in Tierversuchen in der Öffentlichkeit stark kritisiert. Dieser Kritik, die von Tierschutzverbänden ausging, haben sich inzwischen die Verbraucher und die Behörden und Regierungen der EU Mitgliedsstaaten angeschlossen. Nach mehrjähriger Diskussion wurde die vom EU Parlament vorgeschlagene 7. Änderung der EU Kosmetikrichtlinie 76/768/EEC (EU Kommission 1976) am 27. Februar 2003 vom Ministerrat der EU Mitgliedsstaaten akzeptiert (EU Richtlinie 2003/15/EG), die eine stufenweise Einschränkung von Tierversuchen für Kosmetika und ein Verbot in 10 Jahren vorsieht. Grund für das abgestufte Vorgehen ist die Tatsache, dass noch nicht alle zur Prüfung von Kosmetika vorgeschriebenen Tierversuche durch tierversuchsfreie Methoden ersetzt werden können. Gleichzeitig wurden bei der Entwicklung, Validierung und internationalen behördlichen Anerkennung neuer, tierversuchsfreier Prüfmethoden in den letzten 10 Jahren erhebliche Fortschritte erzielt, so daß der Ersatz der Tierversuche bei der Verträglichkeitsprüfung von Kosmetika in naher Zukunft möglich sein wird. Es werden deshalb die Fortschritte vorgestellt, die aufgrund der gemeinsamen Anstrengungen von Wissenschaftlern in Laboratorien der Kosmetikindustrie und anderen Forschungsinstituten beim der Entwicklung von Alternativmethoden zu Tierversuchen erzielt wurden.The tolerability test of cosmetics in animal experiments is subject to severe criticism in the public in Germany and all EU member states. This criticism, which emanated from animal protection associations, to begin with has been taken up in the meantime by consumers, authorities and governments of the EU member states. After several years' discussion, the 7th amendment of the EU Cosmetics Directive 76/768/EEC (EU Commission 1976), proposed by the EU Parliament, was accepted (EU directive 2003/15/EG) on 27 February 2003 by the Council of Ministers of the EU member states. This amendment designates a gradual restriction of animal experiments for cosmetics and a ban in 10 years. The reason for this gradual way of proceedings is the fact that at the time being not all animal experiments provided for testing of cosmetics can be replaced by animal-experiment-free methods. At the same time considerable progress has been achieved in the development, validation and international regulatory approval of novel, animal-experiment-free test methods in the last 10 years so that the replacement of animal experiments in the tolerability testing of cosmetics will be feasible in the near future. Therefore, the progress attained is introduced which is based on mutual efforts of scientists in laboratories of the cosmetics industry and other research institutes in the development of alternative methods for animal experiments.

An overview is provided of the legal framework in Europe for the use of experimental animals set by European Union (EU) Directive 86/609/EEC and of the activities of EU member states to implement this directive for regulatory testing in animals. The use of animal data in the safety assessment of chemicals by services of the EU Commission and by EU member states is described. Specific examples are given for the current use of animal tests in Europe for the safety evaluation of industrial chemicals, pesticides and biocides, cosmetics, endocrine disrupters, and existing chemicals. Recent progress in implementing the 3Rs concept of Russell and Burch into regulatory guidelines of the EU are described, with particular reference to acute systemic and local toxicity testing. Progress in implementing the 3Rs concept in the EU is compared with the situation outside Europe, particularly with the incentive initiated by the Organisation for Economic Co-operation and Development, to reduce regulatory testing in animals. The harmonization of toxicity test guidelines initiated by the Organisation for Economic Co-operation and Development in 1982 has been the most successful measure to reduce pain and distress of laboratory animals in regulatory testing. From the animal welfare perspective, the international harmonization of test guidelines and the mutual acceptance of data are, therefore, the way forward for all areas of chemicals testing

An overview is given on the legal framework in Europe for the use of experimental animals set by EU (European Union) Directive 86/609/EEC and on the activities of EU member states to implement this directive in the field of regulatory testing in animals. The significant decrease in the number of experimental animals in Germany during the past decade is described with particular reference to the recent increase that is due to transgenic animal models. From the regulatory and the animal welfare perspective the international harmonisation of test guidelines and the mutual acceptance of data are the way forward for chemical safety testing. The recent White Paper of the EU Commission for the future chemicals policy calls for an immediate increase in the number of validated in vitro toxicity tests to be accepted for regulatory purposes in the EU. In addition, deficits of properly educating young scientists in Germany in conducting animal experiments and implementing the 3-Rs concept of Russel and Burch are described. It is therefore, quite urgent to establish new chairs on animals and alternatives at universities in Europe. They should focus on both education young students of the biomedical sciences in the humane use of laboratory animals according to the 3-Rs concept and on developing new toxicity tests to be validated for regulatory purposes under the new EU chemicals policy by the established validation centres in Europe

The ECVAM validation concept, which was defined at two validation workshops held in Amden (Switzerland) in 1990 and 1994, and which takes into account the essential elements of prevalidation and biostatistically defined prediction models, has been officially accepted by European Union (EU) Member States and by the Federal regulatory agencies of the USA and the OECD. The ECVAM validation concept was introduced into the ongoing ECVAM/COLIPA validation study of in vitro phototoxicity tests, which ended successfully in 1998. The 3T3 neutral red uptake in vitro phototoxicity test was the first experimentally validated in vitro toxicity test recommended for regulatory purposes by the ECVAM Scientific Advisory Committee (ESAC). It was accepted by the EU into the legislation for chemicals in the year 2000. From 1996 to 1998, two in vitro skin corrosivity tests were successfully validated by ECVAM, and they were also officially accepted into the EU regulations for chemicals in the year 2000. Meanwhile, in 2002, the OECD Test Guidelines Programme is considering the worldwide acceptance of the validated in vitro phototoxicity and corrosivity tests. Finally, from 1997 to 2000, an ECVAM validation study on three in vitro embryotoxicity tests was successfully completed. Therefore, the three in vitro embryotoxicity tests, the whole embryo culture (WEC) test on rat embryos, the micromass (MM) test on limb bud cells of mouse embryos, and the embryonic stem cell test (EST) including a permanent embryonic mouse stem cell line, are considered for routine use in laboratories of the European pharmaceutical and chemicals industries

The European Union's Registration, Evaluation and Authorisation of Chemicals (REACH) legislation mandates testing and evaluation of approximately 30,000 existing substances within a short period of time, beginning with the most widely used "high production volume" (HPV) chemicals. REACH testing requirements for the roughly 3000 HPV chemicals specify three separate tests for reproductive toxicity: two developmental toxicity studies on different animal species (OECD Test Guideline 414) and a two-generation reproduction toxicity study (OECD TG 416). These studies are highly costly in both economic and animal welfare terms. OECD TG 416 is a fertility study intended to evaluate reproductive performance of animals in the P and F1-generations following repeated exposure to a test substance. It can also be used to detect adverse effects on structural and functional development. Thus, it has conventionally been preferred to the one-generation study (OECD TG 415). Recently, the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) proposed that routine two-generation studies could in most cases be replaced with an "enhanced" one-generation study (Reuter et al. [1]). The flexible design proposed by HESI-ACSA allows for the addition of one or more specialised modules, if triggered (e.g. production of a second generation or the investigation of classical developmental toxicity or developmental neuro- or immunotoxicity). Significantly, however, the HESI-ACSA proposal was designed for use in the safety assessment of pesticidal, as opposed to industrial, chemicals. Thus for the purposes of REACH, a streamlined one-generation study that also examines structural development would be the most efficient means of addressing current information requirements for HPV chemicals. This study represents a flexible testing system that can be modified to meet regulatory needs in a variety of sectors

In the beginning of the year 2005, the European Commission published the fourth statistical report on the number of laboratory animals used in the EU Member States in the year 2002. A total of 10,731,020 experimental animals was reported, the majority of which were mice, rats and fish. In comparison to 1999, the year of the previous data collection, this is an increase by over 900,000 animals, which is mainly attributed to an increased use in fish. As in the previous years, France, the United Kingdom and Germany were the Member States that reported the highest numbers of laboratory animals. 4.5 million animals were used for the development, production and quality control of products for human medicine, dentistry and veterinary medicine and 1 million animals in toxicological and safety evaluations. 3.7 million animals were used in fundamental studies, and therefore are not covered by the scope of Council Directive 86/609/EEC on the protection of laboratory animals. This regulatory gap leaving more than a third of the laboratory animals used in the European Union without protection by EU legislation is unacceptable and should be closed during a revision of the Directive. All national authorities submitted some kind of explanatory notes alongside their national tables. In the article, an extensive summary and discussion of the contents of these notes is presented. Germany, Austria, Finland report on providing financial support for the development of alternative methods. The numbers put forward in the fourth EU statistical show that such support is desperately needed

A brief overview is given of the data published in the third report on the number of animals used for scientific purposes in the European Union (EU), in 1999. In comparison to the second EU statistical report, the meaningfulness of the data has been greatly improved, since 14 EU Member States have now used the "EU tables". Even though the total number of animals officially reported in the EU for 1999 (9.81 million animals) is lower than the number reported for 1996 in the second report (11.65 million animals), an increase in animal numbers was observed in several Member States. Repeatedly, the use of transgenic animals was considered to be the cause for this. Most national authorities provided some comments on the data they submitted. However, the quality of these accompanying notes varies considerably, with the comments provided by The Netherlands, Finland, Sweden, Austria, Denmark and the United Kingdom containing the most-detailed remarks on their data

Congenital abnormalities represent a severe problem, given that approximately 3% of all human newborns are affected. About 20% of these defects are associated with gene mutations and another 5% with chromosomal aberrations. Approximately 5-10% of the remaining abnormalities are known to be caused by teratogenic agents. It is thus mandatory in toxicological safety assessment of chemicals and drugs to evaluate adverse effects on reproduction and embryonic development according to certain OECD test guidelines or so-called segment studies encompassing three crucial periods of pre- and postnatal development and fertility (ICH, 1993). All of these guidelines generally specify time-consuming and expensive in vivo experiments mostly performed with mammalian species such as rats or rabbits. The implementation of the new chemical legislation REACH adopted in the European Union is expected to create a dramatic increase in the number of toxicological studies that rely on animal experimentation. It has been estimated that reproductive and developmental toxicity testing alone would account for approximately 60% of all animals used for safety assessments under REACH. Thus, for both economical and ethical reasons there is a global demand for alternatives to living mammals in testing of chemical-induced adverse effects on reproduction and development. Over the past 30 years, a wide spectrum of in vitro models has been developed to detect teratogenic effects of chemicals. These test systems either utilize dissociated cells from limb buds and brains of rat embryos (micromass test) or whole embryos of frogs (Frog Embryo Teratogenesis Assay-Xenopus (FETAX)) and rats (whole embryo culture test). In recent years, embryonic stem (ES) cells became more important and shifted into focus. The ability to differentiate into a wide range of different cell types has made ES cells a popular system to study gene function and developmental processes during differentiation in vitro. For instance, the embryonic stem cell test (EST) established by Spielmann et al. (1997), makes use of this capacity to detect developmental toxicants during stem cell differentiation into cardiomyocytes. Currently, many research groups are committed to propose and to work-out additional murine and human stem cell based approaches to discover predictive biomarkers of developmental toxicity. An overview of the current activities in this field will be presented.

The oral acute toxic class method (ATC method) was developed as an alternative to replace the oral LD50 test. The ATC method is a sequential testing procedure using only three animals of one sex per step at any of the defined dose levels. Depending on the mortality rate three but never more than six animals are used per dose level. This approach results in the reduction of numbers of animals used in comparison to the LD50 test by 40-70%. The principle of the oral ATC method is based on the Probit model and it was first evaluated on a biometric basis before a national and subsequently an international ring study were conducted. The results demonstrated an excellent agreement between the toxicity and the animal numbers predicted biometrically and observed in the validation studies. The oral ATC method was adopted as an official test guideline by OECD in 1996 and was slightly amended in 2001. The ATC method has been successfully used in Germany and in 2003 >85% of all tests on acute oral toxicity testing was conducted as oral ATC tests. In member states of the European Union the ATC method is used in the range of 50% of all tests conducted. Meanwhile the oral LD50 test has been deleted by OECD, by the European Union and by the USA, making the use of alternatives to the oral LD50 test mandatory

Testing for embryotoxicity in vitro is an attractive alternative to animal experimentation. The embryonic stem cell test (EST) is such a method, and it has been formally validated by the European Centre for the Validation of Alternative Methods. A number of recent studies have underscored the potential of this method. However, the EST performed well below the 78% accuracy expected from the validation study using a new set of chemicals and pharmaceutical compounds, and also of toxicity criteria, tested to enlarge the database of the validated EST as part of the Work Package III of the ReProTect Project funded within the 6th Framework Programme of the European Union. To assess the performance and applicability domain of the EST we present a detailed review of the substances and their effects in the EST being nitrofen, ochratoxin A, D-penicillamine, methylazoxymethanol, lovastatin, papaverine, warfarin, +¦-aminopropionitrile, dinoseb, furosemide, doxylamine, pravastatin, and metoclopramide. By delineation of the molecular mechanisms of the substances we identify six categories of reasons for misclassifications. Some of these limitations might also affect other in vitro methods assessing embryotoxicity. Substances that fall into these categories need to be included in future validation sets and in validation guidelines for embryotoxicity testing. Most importantly, we suggest conceivable improvements and additions to the EST which will resolve most of the limitations.

One of the main requirements of the current animal welfare legislation in Europe is to prove the necessity of performing a given experiment with animals. Thus, a study using animals should not proceed, if another scientifically reliable method is available to obtain the desired results that either avoids animal experiments altogether, minimises pain and suffering of animals or reduces the number of animals needed. Scientists are legally required to search the literature and other relevant sources for alternatives prior to any experimental study with animals. Access to information has become much easier since the introduction of the Internet as a standard tool. Today, a variety of online sources is available, e.g. web-based bibliographic databases and specialised web sites providing details about alternatives to animal studies. However, scientists still need to determine the most appropriate searching strategies, depending on the objectives of the relevant web sites and their own line of research. A critical discussion of this issue takes into account the objectives of both the information providers and the information retrieval systems

An ECVAM-funded prevalidation study (PV) was conducted during 1999 and 2000 to identify in vitro tests capable of reliably distinguishing between skin irritants (I) and non-irritants (NI) according to European Union risk phrases ("R38" or no classification). The tests evaluated were EpiDerm, EPISKIN, PREDISKIN, the non-perfused pig ear method, and the mouse skin integrity function test (SIFT). Whereas reproducibility of the two human skin model tests and SIFT was acceptable, none of the methods was deemed ready to enter a formal validation study due to their low predictivity. The ECVAM Skin Irritation Task Force therefore suggested improvements of protocols and prediction models for these tests. Furthermore, it was agreed that experience gained with the two human-skin models be shared, and a common protocol should be developed for EpiDerm and EPISKIN (Zuang et al., 2002). When we applied an improved EPISKIN protocol (Portes et al., 2002) to the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was far too low. In 2003, the EPISKIN protocol was further refined by extension of the post-incubation period following chemical exposure. In the current study, we evaluated this EPISKIN refinement by applying it to EpiDerm. In addition, we developed technical improvements for the application of the test chemicals and rinsing procedure, which reduced the variability of results and increased the percentage of correct predictions. A set of twenty non-coded reference substances from the ECVAM prevalidation study phase III (Fentem et al., 2001) was tested with the final protocol in three independent runs. Both high sensitivity (80%) and high specificity (78%) were achieved, and the statistical probability of correct classifications was high, so that the test is now regarded ready for formal validation

During the past 20 years the EU legislation for the notification of chemicals has focussed on new chemicals and at the same time failed to cover the evaluation of existing chemicals in Europe. Therefore, in a new EU chemicals policy (REACH, Registration, Evaluation and Authorization of Chemicals) the European Commission proposes to evaluate 30,000 chemicals within a period of 15 years. We are providing estimates of the testing requirements based on our personal experiences during the past 20 years. A realistic scenario based on an in-depth discussion of potential toxicological developments and an optimised "tailor-made" testing strategy shows that to meet the goals of the REACH policy, animal numbers may be significantly reduced below 10 million if industry would use in-house data from toxicity testing, which are confidential, if non-animal tests would be used, and if information from quantitative structure activity relationships (QSARs) would be applied in substance-tailored testing schemes. The procedures for evaluating the reproductive toxicity of chemicals have the strongest impact on the total number of animals bred for testing under REACH. We are assuming both an active collaboration with our colleagues in industry and substantial funding of the development and validation of advanced non-animal methods by the EU Commission, specifically in reproductive and developmental toxicity

A review of publications on pesticides assessing the need for 1-year toxicity studies in dogs was performed. Four key peer-reviewed papers with different approaches investigated the value of a 1-year dog study in addition to a 3-month study. Despite different databases and approaches, each concluded with the recommendation to limit the testing of pesticides in dogs to a duration of 3 months. The combined weight of evidence presented in this review reinforces these independent conclusions. Therefore, the routine inclusion of a 1-year dog study as a mandated regulatory requirement for the safety assessment of pesticides is no longer justifiable and a globally harmonized approach should be taken to match the latest legislation of the European Union and the US EPA

From 1996 to 2000, ZEBET (Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments at the BgVV, Berlin, Germany) coordinated the European Centre for the Validation of Alternative Methods (ECVAM) prevalidation and validation study on three embryotoxicity tests: a) a test employing embryonic stem cell lines (EST); b) the micromass (MM) test; and c) the postimplantation rat whole-embryo culture assay (WEC test). The main objectives of the study were to assess the performance of these three in vitro tests in discriminating between non- embryotoxic, weakly embryotoxic and strongly embryotoxic compounds. Phase I of the study (1997) was designed as a prevalidation phase, for test protocol optimisation, and for the establishment of a comprehensive database of in vivo and in vitro data on embryotoxic compounds. Phase II (1998-2000) involved a formal validation trial, conducted under blind conditions on 20 test compounds selected from the database, which were coded and distributed to the participating laboratories. In the preliminary phase of the validation study, six chemicals out of the 20, which showed embryotoxic potential, were tested. These results were used to define new biostatistically based prediction models (PMs) for the MM and WEC tests, and to evaluate those developed previously for the EST. As a next step, the PMs were evaluated by using the results for the remaining 14 chemicals of the definitive phase of the validation study. The three in vitro embryotoxicity tests proved to be applicable to testing a diverse group of chemicals with different embryotoxic potentials (non-embryotoxic, weakly embryotoxic, and strongly embryotoxic). The reproducibility of the three in vitro embryotoxicity tests were acceptable according to the acceptance criteria defined by the Management Team. The concordances between the embryotoxic potentials derived from the in vitro data and from the in vivo data were good for the EST and the WEC (PM2) test, and sufficient for the MM test and the WEC (PM1) tests according to the performance criteria defined by the Management Team before the formal validation study. When applying the PM of the EST to the in vitro data obtained in the definitive phase of the formal validation study, chemicals were classified correctly in 78% of the experiments. For the MM and the WEC tests, the PMs provided 70% and 80% (PM2) correct classifications, respectively. And, very importantly, an excellent predictivity (100%, except for PM1 of the WEC test, with 79%, considered as good) was obtained with strong embryotoxic chemicals in each of the three in vitro tests