A Novel Mode-of-Action Mediated by the Fetal Muscle Nicotinic Acetylcholine Receptor Resulting in Developmental Toxicity in Rats

Abstract

Sulfoxaflor (X11422208), a novel agricultural molecule, induced fetal effects (forelimb flexure, hindlimb rotation, and bent clavicle) and neonatal death in rats at high doses (≥ 400 ppm in diet); however, no such effects occurred in rabbit dietary studies despite achieving similar maternal and fetal plasma exposure levels. Mode-of-action (MoA) studies were conducted to test the hypothesis that the effects in rats had a single MoA induced by sulfoxaflor agonism on the fetal rat muscle nicotinic acetylcholine receptor (nAChR). The studies included cross-fostering and critical windows of exposure studies in rats, fetal ((α1)(2)β1γδ) and adult ((α1)(2)β1δε) rat and human muscle nAChR in vitro agonism experiments, and neonatal rat phrenic nerve-hemidiaphragm contracture studies. The weight of evidence from these studies supported a novel MoA where sulfoxaflor is an agonist to the fetal, but not adult, rat muscle nAChR and that prolonged agonism on this receptor in fetal/neonatal rats causes sustained striated muscle contracture resulting in concomitant reduction in muscle responsiveness to physiological nerve stimulation. Fetal effects were inducible with as little as 1 day of exposure at the end of gestation, but were rapidly reversible after birth, consistent with a pharmacological MoA. With respect to human relevance, sulfoxaflor was shown to have no agonism on human fetal or adult muscle nAChRs. Taken together, the data support the hypothesis that the developmental effects of sulfoxaflor in rats are mediated via sustained agonism on the fetal muscle nAChR during late fetal development and are considered not relevant to humans.

Human relevance framework evaluation of a novel rat developmental toxicity mode of action induced by sulfoxaflor

“…In order for a single MoA to be plausible, the fi ndings should have a similar incidence across dose levels from all studies. The similarity in the R 2 values between the non-linear regression analyses of the individual dose -response curves for death ( R 2 ϭ 0.93) and limb abnormalities ( R 2 ϭ 0.91), respectively, support a single MoA responsible for diff erent developmental eff ects rather than separate MoAs for each of the two major eff ects in rats (Rasoulpour et al 2012). …”

“…The regulatory guideline and MoA toxicity studies with sulfoxafl or described above revealed fi ndings for which an MoA had not previously been described (Rasoulpour et al 2012). In the past decade, frameworks to evaluate the MoA of adverse toxicological eff ects have been developed through the International Programme on Chemical Safety (IPCS) (Boobis et al 2006) and by the International Life Sciences Institute Risk Science Institute (Meek et al 2003).…”

“…Based upon this hypothesis, a series of studies were designed and conducted (Rasoulpour et al 2012). This MoA is proposed to progress through the following key events: (1) sulfoxafl or binding and (2) agonism on the rat fetal-type muscle nAChR, resulting in (3) sustained agonism/sustained muscle contracture in rat neonatal off spring (fetus and pup).…”

“…In order to examine this question, rat fetal and adult muscle nAChRs were expressed as recombinant receptors through microinjection of cRNA in Xenopus oocytes. Recombinant rat nAChR subunits (fi ve subunits total: fetal subtype α (2), β , δ , γ or adult subtype α (2), β , δ , ε ) were expressed in Xenopus oocytes to determine the functional nAChR activity (two-electrode voltage-clamp recording of membrane currents as described in Rasoulpour et al 2012). The nAChRs expressed in Xenopus oocytes form fully functional pentameric nAChRs expressed on the cell surface.…”

“…During the regulatory guideline mammalian toxicology studies, developmental eff ects were observed in rats following dietary sulfoxafl or exposure, including off spring death and near-term fetal abnormalities (forelimb fl exure, hindlimb rotation, and bent [misshapen] clavicle bones; Table 1; Rasoulpour et al 2012). More specifi cally, the rat limb eff ects were without changes in the associated skeletal bone structure while the off spring death occurred prior to postnatal day (PND) 4.…”