Daston 1996). Alternative tests for development toxicity are not currently used by any regulatory agency.

Intrinsic Limitations

Alternatives to in vivo testing will never detect all the developmental toxicants that have actions in pregnant mammals. This is true for several reasons. First, some toxicants initiate their effects outside the embryo and in the maternal or placental compartments. Second, some effects are mediated by physiological changes only represented within the intact embryo (e.g., peripheral vascular perfusion). Third, known mechanisms of developmental toxicity are diverse, so it is unlikely that all targets will be present in a simple system. Fourth, some adverse outcomes are only observable as functional impairment postnatally. Finally, most alternative systems are static and have neither the dynamic changes in concentration associated with physiological disposition in vivo nor the metabolic transformation of the test agent.

Validation

Validation is complex and includes protocol standardization, interlaboratory consistency, and statistical prediction models, but the fundamental question remains: how well does the system mimic the susceptibility of human development? This has yet to be answered for any system, and there are a number of problems that are discussed below.

In Vitro Test for What Outcome? The type of adverse outcome induced by a chemical in vivo often varies between individuals, across species, and sometimes with routes or schedules of administration. Thus, although the initial aim of alternative tests was to predict the overall induction of congenital malformations, it is more appropriate to consider that in vitro tests can help to predict specified developmental toxicity and to identify potential mechanisms of disruption to particular cell-signaling and genetic regulatory pathways.

General Versus Specific Toxicity. Presumably, all chemicals would disturb development, if a high enough concentration were delivered to the embryo, even though such concentrations might be unattainable in mammals because of maternal toxicity. However, chemicals vary widely in their intrinsic hazard to development. For example, high-affinity ligands for some nuclear-hormone receptors cause irreversible developmental defects (see Chapter 4). It would be helpful to be able to discriminate such chemicals from those that affect development (D) only at exposures that are simultaneously toxic to the adult (A). The A/D ratio (the ratio of adult toxic dose to developmentally toxic dose) attempts to measure that specificity. However, use of that value has been tempered by the demonstra-



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