Patch testing with oxidized R-(+)-limonene and its hydroperoxide fraction

R-(+)-Limonene is an ubiquitous allergen in our environment. It is one of the most widely used fragrance materials not only in fine fragrances but also most often incorporated in domestic and occupational products. Although the non-oxidized R-(+)-limonene itself is not allergenic, it easily forms allergenic products due to autoxidation during handling and storage. 2273 patients at 4 dermatological clinics in Europe were patch tested between 1997 and 1999 in 2 steps. First, the oxidation mixture of R-(+)-limonene and 1 selected allergen fraction of the mixture, the limonene hydroperoxides, were tested in 2 different vehicles in consecutive patients. A diverging frequency of positive patch test reactions was observed in the 4 clinics. 3.8% of the consecutive patients tested reacted to oxidized R-(+)-limonene in 2 clinics, 6.5% in the 3rd, whereas 0.3% in the 4th clinic. In 2 of the centres, different but significant concomitant positive response rates to other allergens were observed; e.g. to fragrance materials and to colophonium. However, in the total test population, 57% of the limonene-allergic subjects did not react to any of the fragrance allergy markers used in the standard series. In the 2nd step, patients showing positive reactions were retested, also including additional separate allergens of the limonene oxidation mixture (carvone and limonene oxide). 60% of the limonene-allergic patients showed positive results at retesting. The limonene hydroperoxide fraction was proved to be the most important allergen of the oxidation mixture, showing positive reactions in around 60% of the limonene-allergic patients at both test sessions. Testing limonene oxide and carvone separately resulted in very few positive reactions. 3% oxidized R-(+)-limonene in non-stabilized petrolatum is most suitable when using only 1 test preparation for diagnosis of contact allergy to oxidized limonene. Our data give clinical support to the European classification of R-(+)-limonene, containing oxidation products, as a skin sensitizer.