Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder. Although there is no established treatment for the core symptoms of ASD, recent research has indicated a potentially therapeutic effect of intranasally administered oxytocin. TTA-121 is a novel oxytocin nasal spray with high bioavailability and is expected to increase oxytocin delivery to the brain by adjusting osmolality and viscosity of the formulation. As nonclinical safety studies to support the conduct of the Phase 1 and Phase 2 studies of TTA-121, a 6-week repeated intranasal dose toxicity study of TTA-121 in rats was conducted. In the present study, TTA-121 was administered intranasally to male and female rats at 0 (placebo), 1.2, 6, and 30 U/body/day once daily for 6 weeks followed by a 4-week recovery period to evaluate potential toxicity and systemic exposure to oxytocin. The toxicokinetic analysis indicated that systemic exposure of oxytocin increased with a dose ranging from 1.2 to 30 U/body/day at the first and last dosing. No deaths or moribundity were observed. There were no toxicologically significant changes in clinical signs, functional observational battery, body weight, food consumption, water consumption, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, necropsy or histopathology at any dose during the dosing or recovery period. Based on these results, the no-observed-adverse-effect level of TTA-121 was 30 U/body/day for male and female rats, suggesting that there is a sufficient safety margin. We believe that TTA-121 is expected to be sufficiently safe to treat males and females with ASD.