Developmental toxicity of dinocap in the mouse is not due to two isomers of the major active ingredients.

Technical-grade dinocap, a complex-mixture fungicide, is teratogenic in the CD-1 mouse, causing cleft palate and otolith defects. In this study we compared the developmental toxicities of 2,4-dinitro-6-(1-methylheptyl)phenyl crotonate and 2,6-dinitro-4-(1-methylheptyl)phenyl crotonate, model isomers of the major active ingredients of technical dinocap, to the known teratogenicity of the technical compound. Individual isomers, both isomers combined, or technical dinocap were administered to pregnant mice on days 7-16 of gestation. Some dams were killed at term and litters were removed, dead fetuses and resorptions were counted, and live fetuses were weighed and preserved in Bodian's fixative for examination for cleft palate. Other treated dams were allowed to give birth: postnatal viability and growth, development of swimming behavior, and otolith formation were evaluated. As in previous studies, technical-grade dinocap caused cleft palate and weight deficits in fetuses at term and increased neonatal mortality and abnormal swimming behavior, torticollis, and deficient otolith formation in surviving pups. Neither of the purified isomers exhibited any developmental toxicity when administered under identical conditions. Thus, it is concluded that these isomers are not the active teratogenic component(s) in technical-grade dinocap.

Co-culture of rat embryos and hepatocytes: in vitro detection of a proteratogen.

The technique of whole embryo culture developed by New [Environ Health Perspect 18:105-110, 1976] provides a sensitive assay to evaluate the effects of a test chemical on embryo development independent of maternal influences. To detect proteratogens, this assay must be coupled with an exogenous metabolic activation system. We have developed methods for the co-cultivation of rat embryos with primary hepatocytes, which offers several advantages over subcellular fractions when providing metabolic activation for in vitro assays. In the present study, rat embryos removed from the dam on day 10 of pregnancy were co-cultivated in vitro with primary cultures of rat, rabbit, or hamster hepatocytes. Embryos co-cultivated with hepatocytes developed normally, as did embryos exposed to a test chemical, cyclophosphamide (CP) in the absence of hepatocytes. When embryos were co-cultivated with hepatocytes and exposed to CP, a dose-related embryotoxicity was observed, indicating metabolic activation of the proteratogen. Using hepatocytes isolated from rats pretreated in vivo with phenobarbital, we observed an increase in CP-induced malformations and embryotoxicity compared to those of embryos exposed to CP in the presence of uninduced hepatocytes. The teratogenic bioactivation of CP was inhibited in vitro by the addition of metyrapone. When similar numbers of hepatocytes were used for metabolic activation of CP the induced embryotoxicity was greater in the presence of rabbit and hamster hepatocytes than with rat hepatocytes. Development of procedures for the culture of rat embryos with hepatocytes from other species suggests the utility of this in vitro system for the investigation of species differences in sensitivity to chemical teratogens.

Perinatal toxicity of maneb, ethylene thiourea, and ethylenebisisothiocyanate sulfide in rodents.

The potential of the fungicide maneb and two of its metabolites, ethylenebisisothiocyanate sulfide (EBIS) and ethylene thiourea (ETU), to induce perinatal toxicity in four species of rodents was investigated. The compounds were admininistered to rats and mice during the period of organogenesis, and ETU was also administered to rats and mice during the period of organogenesis, and ETU was also administered by oral gavage for a similar period to hamsters and guinea pigs. Treatment also continued through the lactational period in groups of rats that were allowed to give birth. Fetuses were examined for signs of toxicity, including terata, and neonates for reflex developement and open-field behavior. Maneb produced hydrocephalus in fetuses in litters of rats receiving 480 mg/kg . d. No fetotoxic effects were noted in litters of rats receiving EBIS at doses at high as 30 mg/kg . d. ETU proved to be a potent teratogen in the rat. Among the effects seen at doses of 40 mg/kg . d or greater were hydrocephalus, encephalocele, kyphosis, and various defects of the digits. Neither maneb (up to 1500 mg/kg . d), ETU (up to 200 mg/kg . d), nor EBIS (up to 200 mg/kg . d) elicited signs of fetal toxicity in the mouse. ETU also failed to result in fetal toxicity when administered to the hamster (100 mg/kg . d) or the guinea pig (100 mg/kg . d). Neither maneb nor EBIS produced significant dose-related alterations in the behavioral development of perinatally exposed rat neonates. At doses that also produced neonatal hydrocephalus, ETU produced significant increases in the open-field activity of the neonates. In addition to the perinatal effects noted above, both maneb and EBIS caused maternal limb paralysis in the rat, an effect not noted in the mouse at much higher doses.