No treatment-related effects with aryloxyalkanoate dioxygenase-12 in three 28-day mouse toxicity studies.

The aryloxyalkanoate dioxygenase-12 (AAD-12) protein is expressed in genetically modified soybean events DAS-68416-4 and DAS-444Ø6-6. Expression of the AAD-12 protein in soybeans confers tolerance to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) providing an additional herbicide choice to farmers. This enzyme acts by catalyzing the degradation of 2,4-D into herbicidally inactive metabolites. To meet evolving interpretation of regulations in the European Union, three separate 28-day repeat-dose oral mouse studies were conducted at increasing doses of up to 1100 mg AAD-12 protein/kg bw/day. No treatment-related effects were seen in any of these three studies.

Comparative response of rat and rabbit conceptuses in vitro to inhibitors of histiotrophic nutrition.

Histiotrophic nutrition via the visceral yolk sac is an essential nutritional pathway of the rodent conceptus, and inhibition of this pathway may cause growth retardation, malformations, and death in rodent embryos. Morphologic differences among species during early development indicate that the visceral yolk sac histiotrophic nutrition pathway may be of lesser importance in nonrodent species, including humans. Here, comparative studies were conducted with inhibitors of different steps in the visceral yolk sac histiotrophic nutrition pathway to determine whether the rabbit is similarly responsive to the rat. Early somite stage New Zealand White rabbit and Crl:CD(SD) rat conceptuses (gestation day 9, rabbits; gestation day 10, rats) were exposed for 48 hr to three different histiotrophic nutrition pathway inhibitors using whole embryo culture techniques, after which they were evaluated for growth and malformations. Cubilin antibody, an inhibitor of endocytosis, reduced growth and development and increased malformations in both rat and rabbit embryos, although the rabbit appeared more sensitive. Leupeptin, a lysosomal cysteine protease inhibitor, also impaired growth and development and increased malformations in rat embryos, while in the rabbit it induced malformations and a slight decrease in morphology score but had no effect upon growth. Trypan blue, an inhibitor of endocytosis and endosome maturation, affected all measures in both species to a similar degree at the highest concentration (2500 µg/ml), but rat embryos responded to a greater extent at lower concentrations. Although the specific adverse outcomes appear to be different, these results demonstrate that rabbits, like rats, are sensitive to inhibitors of the histiotrophic nutrition pathway.

Toxicokinetic profile of N-(2-aminoethyl)ethanolamine in the female Wistar rat and distribution into the late gestation fetus and milk.

N-(2-aminoethyl)ethanolamine (AEEA) caused aneurysms of the great vessels in rats exposed in utero and during the first days post partum, exacerbated by postnatal treatment of the lactating dams (Moore et al., 2012). The purpose of this work was to examine the systemic availability of AEEA during gestation and early lactation. The absorption of AEEA was determined following oral administration to nonpregnant and pregnant female Wistar rats. A single dose administered by gavage (0.5 or 50 mg/kg) on gestation day 18 was rapidly and extensively (>90%) absorbed from the gastrointestinal tract (absorption t(1/2) = 0.1-0.2 hr). Elimination from the plasma followed a biphasic pattern, with a rapid elimination phase (t(1/2 α) = 1.6-1.8 hr) followed by a slower phase (t(1/2 ß) = 16.7-17.3 hr). Following repeated gavage administration during gestation day 17 to 19, (14) C-AEEA-derived radioactivity readily partitioned into the fetus and was evenly distributed therein, but cleared approximately twofold slower from the fetal blood and tissues than the maternal blood and chorioallantoic placenta. When administered to lactating dams during lactation days 1 to 12, (14) C-AEEA-derived radioactivity preferentially partitioned into the milk reaching levels that were between 1.6- and 2.5-fold higher than the maternal blood. Although the concentration of AEEA equivalents in the maternal blood remained quite consistent, the concentration in the milk fell by almost 40% between lactation days 4 and 12, probably reflecting an increase in milk production over this same period. We confirm exposure of the offspring to AEEA both in utero and during lactation, but that AEEA does not appear to specifically concentrate in the great vessels.

Dietary Route of Exposure for Rabbit Developmental Toxicity Studies.

Dietary administration is a relevant route of oral exposure for regulatory toxicity studies of agrochemicals as it mimics potential human intake of the chemical via treated crops and commodities. Moreover, dietary administration of test compounds during a developmental toxicity study can deliver a prolonged and stable systemic exposure to the embryo or fetus at all stages of development. In this study, strategies were employed to optimize rabbit test material consumption via diet. Comparative toxicokinetic profiles of gavage versus dietary administration were evaluated in pregnant or non-pregnant New Zealand White rabbits for 2 novel agrochemicals with different plasma half-lives of elimination (sulfoxaflor, t½ = 13.5 h and halauxifen, t½ = 1 h). Dietary administration of sulfoxaflor resulted in stable 24-h plasma concentrations, whereas gavage administration resulted in a 3-fold fluctuation in plasma levels between Cmax and Cmin Dietary administration of sulfoxaflor resulted in a 2-fold higher nominal and diurnal systemic dose when compared with gavage dosing due to Cmax-related maternal toxicity following gavage. Results with the shorter half-life molecule, halauxifen, were more striking with a 6-fold diurnal fluctuation by the dietary route compared with a 368-fold fluctuation between Cmax and Cmin by gavage. Furthermore, plasma halauxifen was detectable only up to 12 h following gavage but up to 24 h following dietary administration. Finally, the presence of these compounds in fetal blood samples was demonstrated, confirming that dietary exposure is appropriate for achieving fetal exposure. Collectively, the results of these studies support the use of dietary exposure in rabbit developmental toxicity studies.

Disposition of glycolic acid into rat and rabbit embryos in vitro.

High dose gavage administration of ethylene glycol (EG) induces teratogenicity in rodents, but not in rabbits, resulting from saturation of intermediate EG metabolism and glycolic acid (GA) accumulation. In vivo, rat embryos sequester GA 2-4-fold higher than maternal blood, a phenomenon absent in rabbits and proposed not to occur in humans. This research explored the mechanisms of GA disposition into rat and rabbit conceptuses using whole embryo culture (WEC). Rat and rabbit embryos concentrated GA from the culture medium. In vitro to in vivo discordance in the rabbit plausibly stemmed from anatomical differences between these models. GA sequestration was attenuated at 4°C in both species. Rat embryos further demonstrated pH-dependence of GA sequestration and inhibition by D-lactic acid. These data suggest GA disposition into rat and rabbit embryos is energy- and pH-dependent, and carrier-mediated. Anatomical and maternal-to-conceptal pH gradient differences likely underlie the lack of enhanced GA disposition in non-rodent species.

Rabbit whole embryo culture.

Although the rabbit is used extensively in developmental toxicity testing, relatively little is known about the fundamental developmental biology of this species let alone mechanisms underlying developmental toxicity. This paucity of information about the rabbit is partly due to the historic lack of whole embryo culture (WEC) methods for the rabbit, which have only been made available fairly recently. In rabbit WEC, early somite stage embryos (gestation day 9) enclosed within an intact amnion and attached to the visceral yolk sac are dissected from maternal tissues and placed in culture for up to 48 h at approximately 37°C and are continuously exposed to an humidified gas atmosphere mixture in a rotating culture system. During this 48 h culture period, major phases of organogenesis can be studied including cardiac looping and segmentation, neural tube closure, and development of anlagen of the otic system, eyes and craniofacial structures, somites and early phases of limb development (up to bud stage), as well as expansion and closure of the visceral yolk sac around the embryo. Following completion of the culture period, embryos are evaluated based on several growth and development parameters and also are assessed for morphological abnormalities. The ability to sustain embryo development independent of the maternal system allows for exposure at precise development stages providing the opportunity study the direct action of a teratogen or one of its metabolites on the developing embryo. Rabbit WEC is perhaps most useful when used in conjunction with rodent WEC methods to investigate species-specific mechanisms of developmental toxicity.

Refinement of a morphological scoring system for postimplantation rabbit conceptuses.

BACKGROUND: The rabbit is used extensively in developmental toxicity testing, yet basic information on rabbit embryo development is lacking. The goals of this study were to refine a rabbit embryo morphology scoring system, and use it to evaluate rabbit whole embryo cultures (WEC). METHODS: A total of 265 conceptuses were harvested between GD 8.0 and 12.0 (coitus = GD 0) at 6-hr intervals and examined in detail. Discreet developmental landmarks were then established for 18 morphological features and assigned scores ranging from 0 up to 6. The scoring system was then validated on a subset of randomly selected in vivo conceptuses, and was used to evaluate conceptuses grown for 12, 24, 36, or 48 hr in WEC beginning from GD 9.0 or 10.0. A few embryos also were examined using microscopic computed tomography (microCT)-based virtual histologytrade mark to assess the utility of this technology. RESULTS: Morphology scores of in vivo developed conceptuses increased linearly (r2 = 0.98) with advancing gestational age, from means of 0.0 on GD 8.0 to 67.9 on GD 12.0. Application of the scoring system, supplemented with evidence from Virtual histologytrade mark, indicated that the WEC system supported normal morphological development of rabbit conceptuses. However, when explanted at GD 9, the rate of development was about 20% slower than in vivo, whereas the rate of development in WEC from GD 10 was indistinguishable from in vivo. CONCLUSIONS: This work enhances the evaluation tools available to study mechanisms of normal and abnormal development in this widely used animal testing species.