The efficacy of three non‐mammalian test systems in the identification of chemical teratogens

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Abstract
This report concerns an evaluation of three in vitro teratogenesis tests: the Dugesia regeneration assay, the Hydra reaggregation assay and the Xenopus embryo assay. Our approach involves the establishment and/or refinement of test protocols, definition of endpoints, and evaluation of test performance by comparison with available results of in vivo mammalian studies. Chemicals used for preliminary evaluation studies were the known mammalian teratogens, vinblastine sulfate (VIN) and hydroxyurea (HU), a coeffective teratogen, cadmium chloride (Cd), and an National Toxicology Program priority chemical, 9-aminoacridine hydrochloride (9AA). The Dugesia assay takes advantage of the ability of beheaded flatworms to regenerate and can be completed in 7–14 days. Concentrations of VIN of 3.2 mg 1−1 inhibited auricle formation and further regeneration observed in 3–6 days. Similarly, eyespot and auricle formation was blocked by HU (180 mg 1−1). The duration of regeneration, measured as the time elapsed between decapitation and eyespot formation (control =5 days), was extended by 1–4 days during exposure to 9AA. Sublethal Cd had little effect on regeneration. The Hydra assay is an evaluation of the ability of dissociated cells to regenerate complete organisms when randomly reassociated. A teratogenic test substance is detected by observing the ratio of the minimal effective concentrations of a substance between intact organisms and regenerates. Compounds with a ratio > 2.0 are potential teratogens. The ratios determined for 9AA, VIN, HU and Cd, respectively, were 10.0, 4.0, 2.7 and 1.2. These results indicate that the first three compounds tested positive for teratogenesis. The Xenopus assay has several endpoints, all of which can be compared with embryolethality in order to index teratogenic hazard. A 4-day test encompasses development from the blastula stage, through hatching, to formation of swimming tadpoles. Reduced growth was noted during exposure to HU concentrations above 50 mg 1−1 (embryolethality of HU was above 200 mg1−1). Significant skeletal abnormalities, recorded as moderate to severe spinal curvature and resultant loss of swimming ability, were observed during exposure to all four test chemicals. However, this phenomenon was apparent at non-embryolethal concentrations only for HU and VIN. Further abnormalities, such as altered skin pigmentation and osmoregulatory failure were also recorded.