The tissue optical clearing technique based on immersion of tissues into optical clearing agents (OCAs) can enhance the penetration depth of light in tissues, thus improve the capabilities of noninvasive optical diagnosis and therapeutic treatment. However, the optical clearing efficiency of skin caused by topical application of OCAs usually suffers from the barrier of stratum corneum and epithelium. The addition of chemical penetration enhancers to OCAs could significantly improve the optical clearing of skin, but the investigations on their safety are seldom concerned. In this study, based on the direct observation and histological examinations, the short-term and long-term effects of the mixture of PEG-400 and Thiazone on morphology and microstructure of in vivo rat skin were investigated. The results demonstrated that, in the first week, the hair growth was stunted, some degree of abnormal thickens and vacuoles occurred at the epidermis layer, and the collagen arranged denser. After 2 week, the morphology and microstructure of rat skin was completely recovered. It means that there is no long-term effect on rat skin by the mixture of PEG-400 and Thiazone. In addition, this work provides a useful method for assessing the safety of OCAs to skin.© (2010) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.