Are contact allergens stable in patch test preparations? Investigation of the degradation of d-limonene hydroperoxides in petrolatum

Several of the products formed after oxidation of d-limonene exhibit strong contact allergenic properties. Some, e.g., the hydroperoxides, are unstable compounds. In this study, we have examined whether the limonene hydroperoxides are chemically stable in white petrolatum used for patch testing. We found that the stability of the hydroperoxides was strongly dependent on whether or not the petrolatum was stabilized with alpha-tocopheryl acetate. In the presence of this antioxidant, the hydroperoxides were degraded to a greater extent. The hydroperoxides were shown to be directly reduced to the corresponding alcohols by this agent. On the other hand, the compounds where shown to be stable in non-stabilized petrolatum throughout clinical patch testing for a period of 6 weeks, provided that the preparations were stored in a refrigerator when not used. Thus, it is recommended that vehicles without alpha-tocopheryl acetate are used when peroxy or hydroperoxy compounds are patch tested or used in sensitization experiments. However, it is important to limit the storage time so that optimal conditions are at hand. A fast method was developed to enable isolation and quantification of the hydroperoxides in white petrolatum. This analytical method may also be applicable to other compositions of patch test preparations.