Developmental toxicology is the study of undesirable effects on the development of the organism, which may result from exposure before conception, from the period of prenatal development, or postnatally during the time of sexual maturation. The principal manifestations of developmental toxicity include: embryolethality, malformations, growth retardation, and functional impairment. In 2001, the European Commission published the future chemicals policy entitled "White Paper: Strategy for a Future Chemicals Policy". The new regulation requires a toxicological evaluation with strong emphasis on reproductive toxicity, by using in vitro methods, especially for those chemicals marketed at more than I ton per year. For this reason, the establishment of in vitro models capable of detecting major undesirable manifestations in the fetus, are urgently required. The aim of the present review is to explore the capacity of existing in vitro systems, based on embryonic stem (ES) cells, to identify embryotoxicity with a focus on specific effects such as teratogenicity and growth retardation. In addition, we discuss the possibility to adapt the mouse ES cells based tests to human ES cells, avoiding inter-species variations in developmental toxicity studies and address related ethical issues. Considering the different manifestations of developmental toxicity, only a battery of in vitro tests will provide the necessary information for regulatory developmental toxicity assessment.

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.

After its standardisation at the national level in Germany (DIN 38415-6, 2001; DIN, 2001), the 48 h sewage testing assay with zebrafish (Danio rerio) embryos has been submitted for standardisation to ISO. As an alternative to the conventional acute (96 h) fish test, a modified fish embryo test will be submitted to the OECD for chemical testing in late 2005. For this, a protocol originally designed for zebrafish was adapted to fit also the requirements of other OECD species, namely medaka (Oryzias latipes) and fathead minnow (Pimephales promelas). Results document that the transfer of the protocol is possible with only minor modifications. Data obtained from embryo tests with the three species are comparable. Statistical analysis of existing zebrafish embryo toxicity data resulted in the conclusions (1) that there is a reliable correlation between the fish embryo test and the acute fish test, (2) that the confidence belt of the regression line was relatively small, but that the prediction range was relatively wide. The regression thus seems appropriate to describe the relationship between acute fish and embryo LC50 with good confidence, but is less appropriate as a prediction model. Investigations into oxygen requirements of zebrafish embryos reveal that they adapt to a broad range of oxygen levels and survive at concentrations of 2 mg/l without malformations. Zebrafish embryos can thus be exposed in very small toxicant volumes (100 μl), which is of particular interest for the testing of metabolites. Dechorionation studies with 48 h old zebrafish embryos indicate that the barrier function of the chorion increases with the lipophilicity of the test compound. Finally, examples are given as to how additional endpoints can be incorporated into the fish embryo test protocol to extend its scope, e.g. to sediment toxicity assessment or genotoxicity and mutagenicity testing.

Cell lines have wide applications as model systems in the medical and pharmaceutical industry. Much drug and chemical testing is now first carried out exhaustively on in vitro systems, reducing the need for complicated and invasive animal experiments. The basis for any research, development or production program involving cell lines is the choice of an authentic cell line. Microsatellites in the human genome that harbour short tandem repeat (STR) DNA markers allow individualisation of established cell lines at the DNA level. Fluorescence polymerase chain reaction amplification of eight highly polymorphic microsatellite STR loci plus gender determination was found to be the best tool to screen the uniqueness of DNA profiles in a fingerprint database. Our results demonstrate that cross-contamination and misidentification remain chronic problems in the use of human continuous cell lines. The combination of rapidly generated DNA types based on single locus STR and their authentication or individualisation by screening the fingerprint database constitutes a highly reliable and robust method for the identification and verification of cell lines.

Escherichia coli (E. coli) related diarrhoea poses a major problem in piglet production. Vaccination of dams increases the antibody titre in their milk against relevant adhesion antigens. Transfer of maternal antibodies via the milk can serve to protect the suckling piglets from the disease. However, a marketing authorisation for veterinary use of these vaccines requires potency determination as assayed in infection trials in piglets. Enzyme immunoassays testing the antibodies in blood serum and colostrum samples from vaccinated dams against the relevant adhesion antigens, F4ab, F4ac, F6 and F41, were developed to replace the very distressing infection experiments by in vitro methods. Eight vaccines authorised in Germany were included in the trials. Vaccinated sows showed significantly higher antibody titres in blood and colostrum than unvaccinated control animals. Thus the enzyme immunoassays appear to be suitable to detect antibodies against four important adhesion antigens, and can be used to replace the distressing infection trials in piglets.

According to the 7th Amendment of the Cosmetics Directive the European Commission had to establish timetables for the phasing out of the various animal tests for the safety evaluation of ingredients used in cosmetics. However; the published timetables do not reflect the objectives of the 7th Amendment but contain longer deadlines for the ban on animal experiments of several endpoints. The European Commission also had to draw up a Directive for establishing an Annex IX that should list validated alternative methods which are not already listed in Annex V of the Dangerous Substances Directive. Although various alternative methods could have been listed in this Annex IX, the Commission published an empty table. From the point of view of the German Animal Welfare Federation amendments of the timetables and the Directive establishing Annex IX are urgently required. Additionally, the Commission has to provide optimal conditions for the replacement of alternative methods.