Hydrogen peroxide (H
2
O
2
) is a reactive chemical used in a wide range of applications. Most importantly, it is used for sterilization process in health care environment. In the present study, safety assessment of 0.5% of H
2
O
2
and its mist intended to be used in the disinfection gateway for COVID-19 was evaluated. Skin irritation and repeated-dose inhalation toxicity studies were carried out in rabbits and rats, respectively. In Skin irritation study, New Zealand white rabbits were exposed topically with 0.5% H
2
O
2
solution and observed for 24 h, 48 h, and 72 h. For repeated-dose inhalation toxicity study, Wistar rats (both male and female) were exposed (whole body exposure) to 0.5% of H
2
O
2
mist, at a concentration of 11.022 (low dose—2-min exposure), 22.044 (medium dose—4-min exposure), and 55.11mg/kg (high dose/high dose recovery—10-min exposure) body weight, daily for 7 days. Rats in the high-dose recovery group (55.11mg/kg—10-min exposure) were kept for another 7 days without any exposure. A toxicological evaluation was done based on general health parameters, hematology, serum biochemistry, gross necropsy, and histopathological data. The results of the study indicated that there was no skin irritation potential induced on exposure of 0.5% of H
2
O
2
to rabbits. Similarly, the inhalation toxicity of 0.5% of H
2
O
2
mist imparts no evidence of hematological, biochemical, gross pathology, or histopathological abnormalities in rats. Further, at the laboratory condition stimulated, the NOEL was found to be 55.11mg/kg body weight. Hence, the present study concluded that 0.5% H
2
O
2
or its mist used in the disinfection gateway for COVID-19 failed to induce any skin irritation in rabbits or inhalation toxicity in rats.
Graphical abstract
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