Prenatal developmental toxicity of nitrous oxide: results of a GLP-compliant OECD TG 414 study in rats and implications for hazard classification

Abstract

Nitrous oxide (N₂O) is classified as a reproductive toxicant (Repr. 1B, H360D) under the EU CLP Regulation based on developmental toxicity findings in non-guideline rat studies conducted in the 1970s and 1980s. Those studies reported malformations and embryolethality after continuous 24-h exposures to high and anesthetic concentrations of N2O, but provided limited data on maternal toxicity and may have been confounded by hypoxia. A new prenatal developmental toxicity study was conducted in Wistar rats according to OECD Test Guideline 414 and following Good Laboratory Practice (GLP). Mated females (21–24 per group) were exposed nose-only to target concentrations of 0 (air control), 150,000; 380,000; or 750,000 ppm N₂O for 6 h/day from gestation day (GD) 6 to GD 19. All test atmospheres contained ≥ 25% oxygen and blood oxygen saturation was monitored to preclude hypoxia. No maternal mortality or clinical signs were observed. Body weight gain in dams was reduced at 750,000 ppm (− 15%); however, terminal body weights and gravid uterus-corrected body weight gains remained comparable to controls. Reproductive parameters were unaffected. No treatment-related malformations were detected at external, visceral or skeletal examinations in any group. Fetal body weight was reduced (− 8%) and the incidence of unossified sternebrae, a skeletal variation, increased only at the high dose, consistent with maternal toxicity and growth retardation. These results show that N₂O does not induce selective developmental toxicity in rats when tested under current OECD guideline conditions.