Anna Börje

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.

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.

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.

Limonene hydroperoxide analogues differ in allergenic activity

Background:  The fragrance terpene R‐limonene is a very weak sensitizer but forms allergenic oxidation products upon contact with air. Oxidized (ox.) limonene is a frequent cause of contact allergy in clinical testing.

Lavender oil lacks natural protection against autoxidation, forming strong contact allergens on air exposure.

Background:  Lavender oil is an essential oil frequently used as a fragrance ingredient and in traditional herbal medicine. We have previously studied the effect of air oxidation on the skin sensitizing potency of the monoterpenes linalyl acetate, linalool and β‐caryophyllene, the main constituents of lavender oil.

Categorization of fragrance contact allergens for prioritization of preventive measures: clinical and experimental data and consideration of structure–activity relationships

Contact allergy to fragrances is still relatively common, affecting ∼ 16% of patients patch tested for suspected allergic contact dermatitis, considering all current screening allergens. The objective of the review is to systematically retrieve, evaluate and classify evidence on contact allergy to fragrances, in order to arrive at recommendations for targeting of primary and secondary prevention. Besides published evidence on contact allergy in humans, animal data (local lymph node assay), annual use volumes and structure–activity relationships (SARs) were considered for an algorithmic categorization of substances as contact allergens. A total of 54 individual chemicals and 28 natural extracts (essential oils) can be categorized as established contact allergens in humans, including all 26 substances previously identified as contact allergens (SCCNFP/0017/98). Twelve of the 54 individual chemicals are considered to be of special concern, owing to the high absolute number of reported cases of contact allergy (> 100). Additionally, 18 single substances and one natural mixture are categorized as established contact allergens in animals. SARs, combined with limited human evidence, contributed to the categorization of a further 26 substances as likely contact allergens. In conclusion, the presence of 127 single fragrance substances and natural mixtures should, owing to their skin sensitizing properties, be disclosed, for example on the label. As an additional preventive measure, the maximum use concentration of 11 substances of special concern should be limited to 100 ppm. The substance hydroxyisohexyl 3‐cyclohexene carboxaldehyde and the two ingredients chloroatranol and atranol in the natural extracts Evernia prunastri and Evernia furfuracea should not be present in cosmetic products.

Activation of non-sensitizing or low-sensitizing fragrance substances into potent sensitizers - prehaptens and prohaptens.

Experimental and clinical studies have shown that fragrance substances can act as prehaptens or prohaptens. They form allergens that are more potent than the parent substance by activation outside or in the skin via abiotic (chemical and physical factors) and/or biotic activation, thus, increasing the risk of sensitization.

Hydroperoxides form specific antigens in contact allergy

Concomitant positive reactions to colophonium, oxidized limonene, and/or oxidized linalool are recorded in patch test studies. The main allergens in these patch test mixtures are hydroperoxides, which form antigens by a radical pathway. Theoretically, concomitant reactions can be explained not only by concomitant sensitization or by true cross‐reactions but also by the hydroperoxides acting as oxidizing agents on skin proteins to form non‐specific antigens without hapten–protein binding. The aim of this study was to explore concomitant reactions and cross‐reactivity patterns among hydroperoxide haptens. We investigated whether individuals allergic to the main allergen in colophonium, 15‐hydroperoxyabietic acid, would also react to limonene hydroperoxide or linalool hydroperoxide. Only 1 of 29 individuals reacted to more than 1 hydroperoxide. The cross‐reactivity pattern among cumene hydroperoxide, limonene hydroperoxide, 1‐(1‐hydroperoxy‐1‐methylethyl) cyclohexene (cyclohexene hydroperoxide), and 15‐hydroperoxydehydroabietic acid was investigated in guinea‐pigs. No general cross‐reactivity was observed. Cross‐reactions between cumene hydroperoxide and cyclohexene hydroperoxide show that similarity in the overall structure and the way of antigen formation are needed. Quantum calculations were used to determine the formation energies of the intermediary radicals. We concluded that hydroperoxides form specific antigens and that formation of non‐specific antigens is unlikely. The concomitant patch test reactions described in the literature are best explained as a result of multiple sensitizations.

α-Terpinene, an antioxidant in tea tree oil, autoxidizes rapidly to skin allergens on air exposure.

The monoterpene α-terpinene is used as a fragrance compound and is present in different essential oils. It is one of the components responsible for the antioxidant activity of tea tree oil. α-Terpinene is structurally similar to other monoterpenes, e.g., limonene, known to autoxidize on air exposure and form allergenic compounds. The aim of the present study was to investigate the possible autoxidation of α-terpinene at room temperature. To investigate the sensitization potency of air-exposed α-terpinene and the oxidation products formed, the murine local lymph node assay was used. Chemical analysis showed that α-terpinene degrades rapidly, forming allylic epoxides and p-cymene as the major oxidation products and also hydrogen peroxide. Thus, the oxidation pathway differs compared to that of, e.g., limonene, which forms highly allergenic hydroperoxides as the primary oxidation products on autoxidation. The sensitization potency of α-terpinene was increased after air-exposure. The allylic epoxides and a fraction, in which only an α,β-unsaturated aldehyde could be identified, were shown to be strong sensitizers in the local lymph node assay. Thus, we consider them to be the major contributors to the increased sensitization potency of the autoxidized mixture. We also investigated the presence of α-terpinene and its oxidation products in four different tea tree oil samples of various ages. α-Terpinene and its oxidation products were identified in all of the tea tree oil samples. Thus, from a technical perspective, α-terpinene is a true antioxidant since it autoxidizes rapidly compared with many other compounds, preventing these from degradation. However, as it easily autoxidizes to form allergens, its suitability can be questioned when used in products for topical applications, e.g., in tea tree oil but also in cosmetics and skin care products.