vitro, including the removal of the maternal environment, the ablation or transplantation of tissues and cells, labeling and tracking of cells or molecules, biochemical and gene manipulations by the use of inhibitors and anti-sense RNA, and real-time physiological monitoring of the embryo. The types of information generated include the identification of proximate developmental toxicants, exact tissue sites of accumulation, initial biochemical insults, gene expression changes, intrinsic SARs (of the parent compound), and identification of disrupted developmental pathways.

The search for alternatives for testing purposes is driven by the need to assess a larger number of chemicals than that allowed by using available resources for in vivo methods and also by the desire to reduce or replace the use of experimental mammals. Two levels of testing should be distinguished: secondary and primary. Secondary testing is the assessment of chemicals that have some known potential developmental toxicity. Most commonly, secondary testing involves analogs of prototype chemicals that have known in vivo developmental toxicity. The objective is to replicate the observed developmental toxicity in a simple system. The approach has been successful, especially for pharmaceuticals and particularly with the use of isolated mammalian embryos and embryonic cells in culture. For example, the approach has been used for testing retinoids (Kistler and Howard 1990) and triazoles (Flint and Boyle 1985). For that type of use, a universal validation of the method is not required. It is sufficient to show that the method replicates a specific in vivo effect for the particular chemicals under study.

Primary testing, in contrast, is the testing of chemicals that have no known potential toxicity, the aim being to predict in vivo actions. There must be confidence that the test outcome will accurately classify most chemicals by their potential to cause human developmental toxicity. Furthermore, the required sensitivity and selectivity will vary, depending on the purpose of the test. Sensitivity is the proportion of in vivo toxicants that are positive in the test, and selectivity is the proportion of inactive chemicals that are negative in the test. In some contexts, for example, in drug discovery by combinatorial chemistry, the aim is the early elimination of potential toxicants. False-positive results are not problematic, because there are many other chemicals from which to choose. Conversely, if the context is hazard identification and the aim is to set priorities for further in vivo testing, then a high rate of false-positive results would be inappropriate. Thus, there is a drive to validate tests for screening purposes by measuring their sensitivity and selectivity (Lave and Omenn 1986). Regardless of all the testing-related problems about to be discussed, it is worth bearing in mind that some countries have already banned the use of mammals for testing in certain situations, so there is an obligation to continue to refine in vitro approaches.

Alternative testing for developmental toxicity has a long history, encompassing regular international conferences (Ebert and Marois 1976; Kimmel et al. 1982; Schwetz 1993), comprehensive reviews (Brown and Freeman 1984; Faustman 1988; Welsch 1992; Brown et al. 1995), and much debate in print (Mirkes 1996;



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