Following the previous study, which investigated the pharmacological properties of the Technekitty injection (Tc-99m), the toxicity of a single intravenous administration of the Technekitty injection (Tc-99m) and the side effects that may occur at the diagnostic dose were confirmed. The Technekitty injection (Tc-99m) was administered intravenously once at a dose of 0, 0.67, 2.0, and 6.0 mCi/kg to 5 male and female rats per group. Mortality, general symptom observation, and weight measurement were performed for 2 weeks, followed by observation of autopsy findings. There were no deaths, and no statistically significant weight change was observed. No abnormal systemic signs related to the Technekitty injection (Tc-99m) were observed. These results confirmed that Technekitty injection (Tc-99m) can be safely administered intravenously at doses up to 6.0 mCi/kg. Additionally, technetium-99m at an average dose of 2 mCi (74 MBq) has been verified as a diagnostic dose without adverse effects, allowing the Technekitty injection (Tc-99m) to be used safely without side effects at this dosage. This study demonstrates that the Technekitty injection (Tc-99m) has a wide safety margin, supporting its potential for clinical application. Moreover, these findings align with the nonclinical safety standards for radiopharmaceuticals, reinforcing its utility in veterinary medicine. The Technekitty injection (Tc-99m) is expected to be applicable for clinical diagnosis as a veterinary drug in Korea.