Current Pharmaceutical Design, 2004, 10, 2733-2747 2733 1381-6128/04 $45.00+.00 © 2004 Bentham Science Publishers Ltd. The Use of Embryonic Stem Cells for Regulatory Developmental Toxicity Testing In Vitro – The Current Status of Test Development S. Bremer * and T. Hartung T.P.580, ECVAM, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, 21020 Ispra (VA), Italy Abstract: The future chemical policy of the European Union as well as the 7 th amendment of the cosmetic directive is calling for the development of alternative tests to animal experimentation for toxicological safety testing. In the field of embryotoxicity one of the most promising in vitro models are based on embryonic stem cells. The embryonic stem cell test has already been validated in comparison to in vivo results in an international blind collaboration study. The presented review is discussing the use, limitations and further needs for the test in order to be fully suitable for regulatory acceptance. In this context, it is summarized which requirements for an in vitro embryotoxicity test have to be fulfilled for regulatory toxicity testing. In addition, an overview about the current status of test development of other embryonic stem cell tests is presented. Several workshops have reviewed the progress of in vitro tests for developmental toxicity testing. A general consensus of these workshops has been that one single test will not be sufficient to cover all manifestations of developmental toxicity. The establishment of a test battery for developmental toxicity is required. This will be even more challenging since the principles of a composing test strategy and its validation have not been defined yet. Finally, the unique possibility of combining the human embryonic stem cell technology and the microarray techniques might lead to a deeper understanding of the toxicological mechanisms of human developmental toxicants. Key Words: Embryonic stem cells, developmental toxicity, alternative method, chemical policy, test strategy. POLITICAL BACKGROUND FOR THE DEVELOP- MENT OF ALTERNATIVE METHODS IN THE AREA OF DEVELOPMENTAL TOXICITY TESTING On the 21 st of January 2003, the European Commission reported to the Council and the European Parliament on the number of animals used for experimental and other scientific purposes in the member states of the European Union [1]. Due to the introduction of harmonized tables a more detailed presentation of the animal use in the member states was possible for the first time. The report provided an overview about the current use of animals in the area of developmental toxicity testing. 8 % (781866) of the total number of animals have been used for toxicological and other safety evaluations including the safety evaluation of products and devices for human medicine, dentistry and for veterinary medicine. 4.8 % of these animals are used for developmental toxicity testing. An additional 10.6 % have been used for the detection of reproductive toxicants, which are also providing information on a possible chemical hazard to the offspring. For developmental toxicity testing the following animals have been used in the European Member States: rodents (44.4.%), rabbits (14%), pigs (0.1%), birds (19.1%) and fishes (22.5%). Developmental toxicity testing has been performed in order to evaluate the safety for human medicines (45%), products for agriculture (22.6.%), products in industry (8.7%) and in cosmetics (1.7%). *Address correspondence to this author at the T.P.580, ECVAM, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, 21020 Ispra (VA), Italy; Tel: +39/0332/785914; Fax: +39/0332/785336; E-mail: Susanne.Bremer@jrc.it However, due to the current ongoing implementation of the new chemical policy of the European Union, a dramatic increase of animal consumption is expected in the area of toxicity safety testing of chemicals as soon as the REACH programme (Registration, Evaluation and Authorisation of new and existing Chemicals) is in place [2]. This programme aims for the assessment of existing chemicals by requesting the essential test data in a relatively short period of time. Consequently, this will result in a substantial increase of toxicological risk assessment for about 30.000 chemicals produced or marketed at more than 1 ton per year. For regulatory developmental toxicity testing only guidelines based on in vivo experiments are available (OECD TG 414, Directive 67/548/EEC B31). These experiments do not only require many animals, they are also costly, laborious, time- consuming and thus represent a bottleneck for the completion of the chemical dossiers. Beside ethical aspects and public acceptance, these economic and social problems call for the timely development and validation of in vitro alternatives. This is in line with the White Paper on a Strategy for Future Chemicals Policy published by the European Commission in 2001, which requests to employ in vitro alternatives as much as possible. Beside the Chemical Policy, the 7 th Amendment of the Cosmetic Directive 2003/15/EC has imposed fixed deadlines for phasing out any animal experiments for safety toxicity testing [3]. From 2013 onwards a marketing ban on cosmetics tested on animals for repeated-dose toxicity, reproductive toxicity and toxic kinetics has been decided, but already now a testing ban of final products and from 2009 ingredients is forseen.