Bixa orellana L. is a plant popularly known as "ucurum", "annatto", and "achiote". It is native to South America, and its seeds are an abundant source of geranylgeraniol and tocotrienols. Nanoencapsulation is a valuable technique that can decrease the drug needed to achieve an effect, decreasing potential toxicity and side effects. This study aimed to evaluate the acute toxicity of an intramuscular application of a nanodispersion containing a standardized extract (Chronic®) from the seeds of Bixa orellana (NBO) in Wistar rats. The chemical evaluation showed δ-tocotrienol at 0.725 ± 0.062 mg/ml (72.6 ± 0.9%). The stability study showed the nanoparticles had an average size from 53.15 ± 0.64 nm to 59.9 ± 3.63 nm, with a polydispersity index ranging from 0.574 ± 0.032 to 0.574 ± 0.32, Zeta potential from 18.26 ± 0.59 mV to 19.66 ± 1.45 mV. After testing the intramuscular application of NBO with doses from 1 to 5 mg/kg in animals, it was observed that the acute treatment did not elicit any toxic effects within this range. The dose of 10 mg/kg, although not affecting hematological and biochemical parameters (CPK, LDH, myoglobin, AST, ALT), could induce some muscle tissue changes, including leukocyte in ltration, morphological chances, and potentially necrosis. In conclusion, the results showed that the treatments devoided toxicity between 1 mg/kg and 5 mg/kg.