Ann-Therese Karlberg

Selected oxidized fragrance terpenes are common contact allergens

Terpenes are widely used fragrance compounds in fine fragrances, but also in domestic and occupational products. Terpenes oxidize easily due to autoxidation on air exposure. Previous studies have shown that limonene, linalool and caryophyllene are not allergenic themselves but readily form allergenic products on air‐exposure. This study aimed to determine the frequency and characteristics of allergic reactions to selected oxidized fragrance terpenes other than limonene. In total 1511 consecutive dermatitis patients in 6 European dermatology centres were patch tested with oxidized fragrance terpenes and some oxidation fractions and compounds. Oxidized linalool and its hydroperoxide fraction were found to be common contact allergens. Of the patients tested, 1.3% showed a positive reaction to oxidized linalool and 1.1% to the hydroperoxide fraction. About 0.5% of the patients reacted to oxidized caryophyllene whereas 1 patient reacted to oxidized myrcene. Of the patients reacting to the oxidized terpenes, 58% had fragrance‐related contact allergy and/or a positive history for adverse reaction to fragrances. Autoxidation of fragrance terpenes contributes greatly to fragrance allergy, which emphasizes the need of testing with compounds that patients are actually exposed to and not only with the ingredients originally applied in commercial formulations.

Contact allergy to oxidized d-limonene among dermatitis patients.

d‐Limonene. obtained as a by‐product from the citrus juice industry was introduced on the market as a more environmentally friendly defatting and cleaning agent than the traditionally used organic solvents. Autoxidation of d‐limonene readily occurs to give a variety of oxygenated monocyclic terpenes that are strong contact allergens. The aim of the present study was to investigate the prevalence of contact allergy to air exposed d‐limonene among dermatitis patients. A fraction consisting of d‐limonene hydroperoxides was also tested. Screening with oxidized d‐limonene will detect cases of allergic contact dermatitis. Additional cases were detected when testing with the fraction of limonene hydroperoxides. The proportion of positive patch test reactions to oxidized d‐limonene was comparable to that seen for several of the allergens within the standard series. An increased UAT of d‐limonene containing allergenic oxidation products in industry where high concentrations are used, as well as in domestic exposure, might result in contact sensitization and dermatitis. Patients reacting to d‐limonene often reacted to fragrance mix, balsam of Peru and colophony in the standard series.

Studies on the autoxidation and sensitizing capacity of the fragrance chemical linalool, identifying a linalool hydroperoxide

Fragrances are among the most common causes of allergic contact dermatitis. The two monoterpenes linalool and d‐limonene are the most frequently incorporated fragrance chemicals in scented products. Previous studies on d‐limonene show that this monoterpene oxidizes on air exposure (autoxidation) and that allergenic oxidation products are formed. Due to structural similarities, linalool might also form allergenic oxidation products on air exposure. The aim of the present study was to study the autoxidation of linalool and to investigate the sensitizing potential of linalool before and after air exposure. Linalool was oxidized for 10 weeks and gas chromatographic analyses showed that the content of linalool decreased to about 80%. The chromatograms revealed the formation of other compounds during oxidation. One of the major oxidation products was isolated and identified as 7‐hydroperoxy‐3,7‐dimethyl‐octa‐1,5‐diene‐3‐ol. This substance is, to the best of our knowledge, described for the first time. In sensitization studies in guinea pigs, linalool of high purity gave no reactions, while linalool that had been oxidized for 10 weeks sensitized the animals. It is concluded that autoxidation of linalool is essential for its sensitizing potential.

Autoxidation of linalyl acetate, the main component of lavender oil, creates potent contact allergens.

Background:  Fragrances are among the most common causes of allergic contact dermatitis. We have in previous studies shown that linalool, present in lavender oil, autoxidizes on air exposure, forming allergenic oxidation products. Oxidized linalool was found to be a frequent cause of contact allergy in a patch test study on consecutive dermatitis patients. Linalyl acetate, the main component of lavender oil is commonly used as a fragrance chemical in scented products. Because of structural similarities, linalyl acetate should also be susceptible to oxidation on air exposure, forming similar oxidation products as linalool.

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.

Is abietic acid the allergenic component of colophony

In order to investigate whether abietic acid itself is the allergenic component of Colophony, 2 commercial samples were extensively purified and tested in guinea pies and in colophony‐sensitive patients. In the modified FCAT method, sensitization was obtained with Portuguese colophony and when challenged with puritied abietic acid, the animals showed no reaction. In the GPMT method, the animals were exposed to purified abietic acid. Challenging with 2 different samples of it gave no significant reaction. When patch tested, patients sensitive to colophony showed no reactions to abietic acid which had been purified immediately before the test. It is concluded that abietic acid itself is not a contact allergen

Formation of formaldehyde and peroxides by air oxidation of high purity polyoxyethylene surfactants

Ethoxylated alcohols are non‐ionic surfactants. The majority are used in household cleaners, laundry products, toiletries and in industrial and institutional cleaners. In previous studies, an ethoxylated non‐ionic surfactant of technical quality showed allergenic activity in guinea pig experiments. Chemical analysis revealed a content of formaldehyde, a well‐known contact allergen, and peroxides in the surfactant. Most cases of occupational contact dermatitis are considered to be of irritant origin, caused by contact with water and surfactants, but if allergenic autoxidation products can be formed, allergic contact dermatitis cannot be excluded. The sensitizing potential of a chemically defined high purity ethoxylated alcohol was investigated and oxidation under various storage and handling conditions was studied for this and a homologous product. The pure surfactant showed no significant allergenic activity on predictive testing in guinea pigs. When ethoxylated alcohols were stored in the refrigerator, their deterioration was limited. At room temperature, their content of peroxides and formaldehyde increased with time. Levels of formaldehyde above those capable of causing positive patch test reactions were found. Since such surfactants have wide applications, resulting exposure to formaldehyde could be more frequent than is generally realized, contributing to persistence of dermatitis in individuals allergic to formaldehyde.

Not only oxidized R-(+)- but also S-(-)-limonene is a common cause of contact allergy in dermatitis patients in Europe

Limonene, one of the most often used fragrance terpenes in any kind of scented products, is prone to air‐oxidation. The oxidation products formed have a considerable sensitizing potential. In previous patch test studies on consecutively tested dermatitis patients, oxidized R‐limonene has been proven to be a good and frequent indicator of fragrance‐related contact allergy. The current study extends these investigations to 6 European clinics of dermatology, where the oxidation mixture of both enantiomers of limonene (R and S) have been tested in 2411 dermatitis patients. Altogether, 63 out of 2411 patients tested (2.6%) reacted to 1 or both the oxidized limonene preparations. Only 2.3% reacted to the oxidized R‐limonene and 2.0% to the oxidized S‐limonene. In 57% of the cases, simultaneous reactions were observed to both oxidation mixtures. Concomitant reactions to the fragrance mix, colophonium, Myroxylon pereirae, and fragrance‐related contact allergy were common in patients reacting to 1 or both the oxidized limonene enantiomers. Our study provides clinical evidence for the importance of oxidation products of limonene in contact allergy. It seems advisable to screen consecutive dermatitis patients with oxidized limonene 3% petrolatum, although this patch test material is not yet commercially available.

Cytochrome P450-mediated activation of the fragrance compound geraniol forms potent contact allergens

Contact sensitization is caused by low molecular weight compounds which penetrate the skin and bind to protein. In many cases, these compounds are activated to reactive species, either by autoxidation on exposure to air or by metabolic activation in the skin. Geraniol, a widely used fragrance chemical, is considered to be a weak allergen, although its chemical structure does not indicate it to be a contact sensitizer. We have shown that geraniol autoxidizes and forms allergenic oxidation products. In the literature, it is suggested but not shown that geraniol could be metabolically activated to geranial. Previously, a skin-like CYP cocktail consisting of cutaneous CYP isoenzymes, was developed as a model system to study cutaneous metabolism. In the present study, we used this system to investigate CYP-mediated activation of geraniol. In incubations with the skin-like CYP cocktail, geranial, neral, 2,3-epoxygeraniol, 6,7-epoxygeraniol and 6,7-epoxygeranial were identified. Geranial was the main metabolite formed followed by 6,7-epoxygeraniol. The allergenic activities of the identified metabolites were determined in the murine local lymph node assay (LLNA). Geranial, neral and 6,7-epoxygeraniol were shown to be moderate sensitizers, and 6,7-epoxygeranial a strong sensitizer. Of the isoenzymes studied, CYP2B6, CYP1A1 and CYP3A5 showed high activities. It is likely that CYP1A1 and CYP3A5 are mainly responsible for the metabolic activation of geraniol in the skin, as they are expressed constitutively at significantly higher levels than CYP2B6. Thus, geraniol is activated through both autoxidation and metabolism. The allergens geranial and neral are formed via both oxidation mechanisms, thereby playing a large role in the sensitization to geraniol.

Linalool - a significant contact sensitizer after air exposure

Background: Linalool is a widely used fragrance terpene. Pure linalool is not allergenic or a very weak allergen, but autoxidizes on air exposure and the oxidation products can cause contact allergy. Oxidized (ox.) linalool has previously been patch tested at a concentration of 2.0% in petrolatum (pet.) in 1511 patients, and 1.3% positive patch test reactions were observed.