Petrolatum is widely used in cosmetics, topical pharmaceuticals and also as a vehicle in dermal toxicity studies. New Zealand white rabbits treated with white petrolatum (vehicle control) in a 2-week dermal irritation study exhibited moderate to severe erythema starting on Day 7 that subsided towards the end of the study. Histological examination of abraded and non-abraded petrolatum-treated skin obtained at termination (Day 15) revealed mild acanthosis, hyperkeratosis, dermal edema with mixed inflammatory cells in the dermis. Macroscopic and microscopic features noted in rabbits were consistent with dermal irritation to petrolatum. Wistar-Han rats, CD1 mice, C57/Bl/6J mice and Göttingen minipigs treated topically with white petrolatum did not exhibit clinical or histologic evidence of dermal irritation. Therapeutic agents developed for topical application are generally tested in rabbits during some point in development. Interpretation of skin irritation data from a single species can impact risk assessment for humans and on product labeling. Copyright © 2013 John Wiley & Sons, Ltd.