Objective To investigate the acute toxicity, skin hypersensitivity and external auditory canal skin irritation reactions of Cangai Volatile Oil administered via external auditory canal, in order to evaluate the preclinical safety of Cangai Volatile Oil aqueous solution. Methods ① Acute toxicity test in external auditory canal: guinea pigs with intact skin and broken skin models were used respectively, and Cangai Volatile Oil was administered at a concentration of 15% and observed continuously for 7 d after 4 times intra-ear canal administrations in 1 d; ② Dermal allergenicity test: guinea pigs with intact skin and broken skin models were used respectively, and different concentrations(1%, 5%, 15%) of Cangai Volatile Oil were applied for 7 d, and polysorbate 80 solution as the blank control; ③ External auditory canal skin irritation test: guinea pigs with intact skin and broken skin models were used respectively, and cotton balls soaked with different concentrations(1%, 5%, 15%) of Cangai Volatile Oil were placed in the ear canal for 7 d, and polysorbate 80 as the blank control, and removed after 6 h administration, and treatment lasted for 7 d. Results ① In the acute toxicity test, there were no significant changes in external auditory canal, tympanic membrane and body weight in all guinea pigs, and no systemic toxic reactions observed; ② No erythema and edema were observed in the Cangai Volatile Oil and polysorbate 80 in all guinea pigs, which were evaluated as non-sensitizing; ③ No erythema, edema and tympanic membrane changes were observed in the intact and damaged external auditory canal skin groups which were evaluated as non-irritating. Conclusion Cangai Volatile Oil at lower concentration (less than 15%) is safe, non-irritating and non-sensitizing to the external auditory canal and tympanic membrane of guinea pigs, a topical Chinese medicine with high safety, which could be used for topical application of external auditory canal treatment in clinical practice.