Limonene Hydroperoxides Research Articles

Can contact allergy to p-phenylenediamine explain the high rates of terpene hydroperoxide allergy? - An epidemiological study based on consecutive patch test results.

Contact allergy to linalool hydroperoxides (Lin-OOHs) and limonene hydroperoxides (Lim-OOHs) is common. Similarly to what occurs with the terpene hydroperoxides, reactive intermediates formed from p-phenylenediamine (PPD) can cause oxidative modifications of tryptophan residues on proteins in mechanistic studies. To test the hypothesis that patients sensitized to PPD are at increased risk of concomitant reactivity to either of the terpene hydroperoxides, owing to a 'common pathway' of skin protein oxidation. A database study of consecutively patch tested eczema patients (n = 3843) from 2012 to 2015, tested concomitantly with PPD, Lim-OOHs and Lin-OOHs, was performed. Associations were examined by level of concordance and odds ratios (ORs) adjusted for age, sex, and contact allergy to fragrance mix I and fragrance mix II. Concomitant reactions to PPD were seen in 2.2% of Lim-OOH-positive patients and in 4.9% of Lin-OOH-positive patients. Neither proportion was higher than expected by chance. No association existed between PPD and Lim-OOH patch test reactivity. In a multiple logistic regression analysis, PPD allergy was associated with an insignificantly increased risk (OR 2.11, 95%CI:0.92-4.80) of a positive patch test reaction to Lin-OOHs. PPD sensitization cannot explain the high rates of sensitization to Lin-OOHs and/or Lim-OOHs. Contact allergy to oxidized linalool is more strongly associated with fragrance allergy than with PPD allergy.

Oxidized limonene and oxidized linalool - concomitant contact allergy to common fragrance terpenes.

Limonene and linalool are common fragrance terpenes. Both oxidized R-limonene and oxidized linalool have recently been patch tested in an international setting, showing contact allergy in 5.2% and 6.9% of dermatitis patients, respectively. To investigate concomitant reactions between oxidized R-limonene and oxidized linalool in consecutive dermatitis patients. Oxidized R-limonene 3.0% (containing limonene hydroperoxides 0.33%) and oxidized linalool 6% (linalool hydroperoxides 1%) in petrolatum were tested in 2900 consecutive dermatitis patients in Australia, Denmark, Singapore, Spain, Sweden, and the United Kingdom. A total of 281 patients reacted to either oxidized R-limonene or oxidized linalool. Of these, 25% had concomitant reactions to both compounds, whereas 29% reacted only to oxidized R-limonene and 46% only to oxidized linalool. Of the 152 patients reacting to oxidized R-limonene, 46% reacted to oxidized linalool, whereas 35% of the 200 patients reacting to oxidized linalool also reacted to oxidized R-limonene. The majority of the patients (75%) reacted to only one of the oxidation mixtures, thus supporting the specificity of the reactions. The concomitant reactions to the two fragrance allergens suggest multiple sensitizations, which most likely reflect the exposure to the different fragrance materials in various types of consumer products. This is in accordance with what is generally seen for patch test reactions to fragrance materials.

Contact allergy to essential oils cannot always be predicted from allergy to fragrance markers in the baseline series.

Essential oils are fragrance substances that are labelled on cosmetic products by their INCI names, potentially confusing consumers. To establish whether contact allergy to essential oils might be missed if not specifically tested for. We tested 471 patients with 14 essential oils and 2104 patients with Melaleuca alternifolia oil between January 2008 and June 2014. All patients were tested with fragrance mix I, fragrance mix II, hydroxyisohexyl 3-cyclohexene carboxaldehyde, and Myroxylon pereirae. Three hundred and twenty-six patients were tested with hydroperoxides of limonene and linalool. Thirty-four patients had a +/++/+++ reaction to at least one essential oil. Eleven had no reaction to any of the six marker fragrance substances. Thus, 4 of 11 positive reactions to M. alternifolia oil, 2 of 7 reactions to Cymbopogon flexuosus oil, 1 of 5 reactions to Cananga odorata oil, 3 of 4 reactions to Santalum album oil and 2 of 3 reactions to Mentha piperita oil would have been missed without individual testing. A small number of patients who are allergic to essential oils could be missed if these are not specifically tested. Labelling by INCI names means that exposure may not be obvious. Careful inspection of so-called 'natural' products and targeted testing is recommended.

Acute Limonene Toxicity in Escherichia coli Is Caused by Limonene Hydroperoxide and Alleviated by a Point Mutation in Alkyl Hydroperoxidase AhpC.

Limonene, a major component of citrus peel oil, has a number of applications related to microbiology. The antimicrobial properties of limonene make it a popular disinfectant and food preservative, while its potential as a biofuel component has made it the target of renewable production efforts through microbial metabolic engineering. For both applications, an understanding of microbial sensitivity or tolerance to limonene is crucial, but the mechanism of limonene toxicity remains enigmatic. In this study, we characterized a limonene-tolerant strain of Escherichia coli and found a mutation in ahpC, encoding alkyl hydroperoxidase, which alleviated limonene toxicity. We show that the acute toxicity previously attributed to limonene is largely due to the common oxidation product limonene hydroperoxide, which forms spontaneously in aerobic environments. The mutant AhpC protein with an L-to-Q change at position 177 (AhpC(L177Q)) was able to alleviate this toxicity by reducing the hydroperoxide to a more benign compound. We show that the degree of limonene toxicity is a function of its oxidation level and that nonoxidized limonene has relatively little toxicity to wild-type E. coli cells. Our results have implications for both the renewable production of limonene and the applications of limonene as an antimicrobial.

An HPLC method for hydroperoxides derived from limonene and linalool in citrus oils, using post‐column luminol‐mediated chemiluminescence detection

AbstractLimonene and linalool are unsaturated terpenes commonly found as major components in many essential oils, and both are easily oxidized by atmospheric oxygen to form hydroperoxides. The hydroperoxides of both limonene and linalool are known to be sensitizers capable of causing allergic contact dermatitis, but with different potency. In addition, positional isomers of limonene hydroperoxide have been demonstrated to have different allergenic potencies. This creates a need for an analytical method that is capable of differentiating hydroperoxides derived from different terpenes, including the various positional isomers. The standard iodometric titration methods [peroxide value (POV) methods] typically used to measure hydroperoxide levels in essential oils provide only a total level of all oxidizing species, including hydroperoxides. These POV methods are not capable of species differentiation and therefore may not reliably correlate well with the skin sensitizing potency of a particular sample. A high‐performance liquid chromatographic (HPLC) method using a post‐column reaction to produce chemiluminescence via luminol oxidation was developed to address the need for a species‐differentiating method. Copyright © 2015 John Wiley & Sons, Ltd.

Occupational contact dermatitis caused by D-limonene.

Limonene is widely used as a fragrance substance and solvent in cleansing products. Oxidized limonene is a frequent contact allergen among consumers of cosmetics, personal care products, and scented household cleaning products. Less is known about the sources of occupational exposure and occupational contact dermatitis caused by limonene. To report 14 patients with occupational contact allergy to limonene. The patients were examined in 2008-2013. An in-house preparation of oxidized limonene was patch tested as 3% and 5% in petrolatum from 2008 to August 2010, and after this as 3%, 1% and 0.3% pet. From 2012 onwards, a commercial test substance of limonene hydroperoxides was also used. We assessed the patients' occupational and domestic exposure to limonene. Occupational limonene allergy was observed in workers who used limonene-containing machine-cleaning detergents and hand cleansers, and in workers who used limonene-containing surface cleaners and dishwashing liquids similar to those used by consumers. In 3 cases, the occupational limonene allergy resulted from work-related use of limonene-containing, leave-on cosmetic products. Limonene is a frequent occupational sensitizer in hand cleansers and cleaning products. Occupational limonene contact allergy may also be caused by exposure to cosmetic products scented with limonene.

Positive patch test reactions to oxidized limonene: exposure and relevance.

R-Limonene is a common fragrance terpene found in domestic and industrial products. R-Limonene autoxidizes on air exposure, and the oxidation products can cause contact allergy. In a recent multicentre study, 5.2% (range 2.3-12.1%) of 2900 patients showed a positive patch test reaction to oxidized R-limonene. To study the exposure to limonene among consecutive dermatitis patients reacting to oxidized R-limonene in an international setting, and to assess the relevance of the exposure for the patients' dermatitis. Oxidized R-limonene 3.0% (containing limonene hydroperoxides at 0.33%) in petrolatum was tested in 2900 consecutive dermatitis patients in Australia, Denmark, the United Kingdom, Singapore, Spain, and Sweden. A questionnaire assessing exposure to limonene-containing products was completed. Overall, exposure to products containing limonene was found and assessed as being probably relevant for the patients' dermatitis in 36% of the limonene-allergic patients. In Barcelona and Copenhagen, > 70% of the patients were judged to have had an exposure to limonene assessed as relevant. Oxidized R-limonene is a common fragrance allergen, and limonene was frequently found in the labelling on the patients' products, and assessed as relevant for the patients' dermatitis. A large number of domestic and occupational sources for contact with R-limonene were identified.

Acetylcholine as a catalyst of hydroperoxide decomposition to free radicals

In organic solvents, acetylcholine chloride accelerates lipid oxidation and hydroperoxide decomposition to free radicals similarly to cationic surfactants. The rates of radical formation in the reaction of acetylcholine with cumyl, tert-butyl, and limonene hydroperoxides and hydrogen peroxide at 37 °C were estimated by the inhibitor method using different radical acceptors. In an aqueous medium, hygroscopic acetylcholine does not react with hydroperoxides.

Allergy to oxidized limonene and linalool is frequent in the U.K.

The oxidized forms of the fragrance terpenes limonene and linalool are known to cause allergic contact dermatitis. Significant rates of contact allergy to these fragrances have been reported in European studies and in a recent worldwide study. Patch testing to oxidized terpenes is not routinely carried out either in the U.K. or in other centres internationally. To investigate the prevalence of contact allergy to oxidized limonene and linalool in the U.K. Between 1 August 2011 and 31 December 2012, 4731 consecutive patients in 13 U.K. dermatology departments were tested for hydroperoxides of limonene 0·3% pet., hydroperoxides of linalool 1·0% pet., stabilized limonene 10·0% pet. and stabilized linalool 10·0% pet. Doubtful (?+) and equivocal (±) reactions were grouped together as irritant reactions. Two hundred and thirty-seven patients (5·0%) had a positive patch test reaction to hydroperoxides of limonene 0·3% pet. and 281 (5·9%) to hydroperoxides of linalool 1·0% pet. Irritant reactions to one or both oxidized terpenes were found in 242 patients (7·3%). Eleven patients (0·2%) had a positive patch test reaction to the stabilized terpenes alone. This large, multicentre U.K. audit shows a significant rate of allergy to the hydroperoxides of limonene and linalool plus a high rate of irritant reactions. Testing to the oxidized forms alone captures the majority (97·0%; 411 of 422) of positive reactions; testing to nonoxidized terpenes appears to be less useful. We recommend that the hydroperoxides of limonene and linalool be added to an extended baseline patch test series.

Stability of limonene and monitoring of a hydroperoxide in fragranced products

ABSTRACTUpon prolonged exposure to air, limonene is prone to oxidation. Hydroperoxides formed upon oxidation are strong skin sensitizers in animal tests and give positive patch tests in human dermatitis patients. However, there is no analytical evidence indicating potential sources from which the public can be exposed to sufficient quantities of limonene hydroperoxide for induction of skin sensitization to occur. It has been proposed that fragranced products might lead to such exposure. Here, we developed analytical methods using mass spectrometry (MS) in combination with gas chromatography (GC)‐MS and liquid chromatography (LC)‐MS to detect limonene‐2‐OOH. GC‐MS has a low sensitivity, whereas LC‐MS was found to be a sensitive method to detect this hydroperoxide. However, selectivity of LC‐MS is low in complex fine fragrances owing to limited separation and unspecific ions. We developed an additional method based on selective reduction of limonene‐2‐OOH to carveol by triphenylphosphine (PPh3). Quantitative carveol formation from the hydroperoxide in the presence of PPh3 could be verified in hydroperoxide‐spiked fragrances. These methods were then applied to stability studies. We found a high stability of limonene in eaux de toilette and the PPh3‐reduction assay gave no indication of hydroperoxide formation in repeatedly opened, half‐empty bottles stored for 9 months. Aged fine fragrances retrieved from consumers contained an average of 1990 µg/g limonene, but only traces of carveol were formed in a minority of the samples in the presence of PPh3. Control samples were spiked with trans‐limonene‐2‐OOH. Quantitative trans‐carveol formation in the presence of PPh3 indicated that the method would detect the hydroperoxide in these consumer products if it was present at higher concentrations. In conclusion, limonene‐2‐OOH may be detected in complex fragrance samples by selective reduction followed by GC‐MS. However, we could not find evidence for significant limonene‐2‐OOH formation in aged fine fragrance samples. Copyright © 2014 John Wiley & Sons, Ltd.

Limonene hydroperoxide analogues show specific patch test reactions

The fragrance terpene R-limonene is a very weak sensitizer, but forms allergenic oxidation products upon contact with air. The primary oxidation products of oxidized limonene, the hydroperoxides, have an important impact on the sensitizing potency of the oxidation mixture. One analogue, limonene-1-hydroperoxide, was experimentally shown to be a significantly more potent sensitizer than limonene-2-hydroperoxide in the local lymph node assay with non-pooled lymph nodes. To investigate the pattern of reactivity among consecutive dermatitis patients to two structurally closely related limonene hydroperoxides, limonene-1-hydroperoxide and limonene-2-hydroperoxide. Limonene-1-hydroperoxide, limonene-2-hydroperoxide, at 0.5% in petrolatum, and oxidized limonene 3.0% pet. were tested in 763 consecutive dermatitis patients. Of the tested materials, limonene-1-hydroperoxide gave most reactions, with 2.4% of the patients showing positive patch test reactions. Limonene-2-hydroperoxide and oxidized R-limonene gave 1.7% and 1.2% positive patch test reactions, respectively. Concomitant positive patch test reactions to other fragrance markers in the baseline series were frequently noted. The results are in accordance with the experimental studies, as limonene-1-hydroperoxide gave more positive patch test reactions in the tested patients than limonene-2-hydroperoxide. Furthermore, the results support the specificity of the allergenic activity of the limonene hydroperoxide analogues and the importance of oxidized limonene as a cause of contact allergy.

An international multicentre study on the allergenic activity of air‐oxidized R‐limonene

Limonene is a common fragrance terpene that, in its pure form, is not allergenic or is a very weak allergen. However, limonene autoxidizes on air exposure, and the oxidation products can cause contact allergy. Oxidized R-limonene has previously been patch tested in multicentre studies, giving 2-3% positive patch test reactions in consecutive patients. To investigate whether oxidized R-limonene 3.0% in petrolatum, with a stable concentration of the main haptens, limonene hydroperoxides (Lim-OOHs), could be a useful tool for the detection of contact allergy in an international setting. Oxidized R-limonene 3.0% (Lim-OOHs 0.33%) pet. was tested in 2900 consecutive dermatitis patients in Denmark, the United Kingdom, Singapore, Spain, Sweden, and Australia. Overall, 5.2% (range 2.3-12.1%) of the patients showed a positive patch test reaction to oxidized R-limonene. Doubtful reactions were found in 7.0% of the patients (range 0-24%). Few irritant reactions were seen. Oxidized R-limonene at 3.0% pet. with a specified content of Lim-OOHs 0.33% is a standardized and useful tool for the detection of contact allergy in dermatitis patients. Many patients showing positive patch test reactions to oxidized R-limonene would not be informed of their fragrance allergy if this specific test had not been performed.

Structural Influence on Radical Formation and Sensitizing Capacity of Alkylic Limonene Hydroperoxide Analogues in Allergic Contact Dermatitis

Hydroperoxides are known to be strong contact allergens and a common cause of contact allergy. They are easily formed by the autoxidation of, for example, fragrance terpenes, compounds that are common in perfumes, cosmetics, and household products. A requirement of the immunological mechanisms of contact allergy is the formation of an immunogenic hapten-protein complex. For hydroperoxides, a radical mechanism is postulated for this formation. In our previous investigations of allylic limonene hydroperoxides, we found that the formation of carbon- and oxygen-centered radicals, as well as the sensitizing capacity, is influenced by the structure of the hydroperoxides. The aim of the present work was to further investigate the connection between structure, radical formation, and sensitizing capacity by studying alkylic analogues of the previously investigated allylic limonene hydroperoxides. The radical formation was studied in radical-trapping experiments employing 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride as an initiator and 1,1,3,3-tetramethylisoindolin-2-yloxyl as a radical trapper. We found that the investigated hydroperoxides initially form carbon- and oxygen-centered radicals that subsequently form alcohols and ketones. Trapped carbon-centered radicals and nonradical products were isolated and identified. Small changes in structure, like the omission of the endocyclic double bond or the addition of a methyl group, resulted in large differences in radical formation. The results indicate that alkoxyl radicals seem to be more important than carbon-centered radicals in the immunogenic complex formation. The sensitizing capacities were studied in the murine local lymph node assay (LLNA), and all hydroperoxides tested were found to be potent sensitizers. For two of the hydroperoxides investigated, the recently suggested thiol-ene reaction is a possible mechanism for the formation of immunogenic complexes. For the third investigated, fully saturated, hydroperoxide, the thiol-ene mechanism is not possible for immunogenic complex formation. This strongly indicates that several radical reaction pathways for immunogenic complex formation of limonene hydroperoxides are active in parallel.

Limonene hydroperoxide analogues differ in allergenic activity

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. This study investigates the sensitizing potencies of ox. and non-ox. limonene and of structurally closely related limonene hydroperoxides. The clinical importance of the difference in sensitizing potency of two hydroperoxides in autoxidized limonene was studied. Ox. and non-ox. limonene were investigated in the murine local lymph node assay (LLNA). Limonene hydroperoxides were investigated using a modified LLNA involving non-pooled lymph nodes and statistical calculations; patch testing of patients with known contact allergy to ox. limonene was performed. A marked increase in the sensitizing potency of ox. limonene compared with that of pure limonene was observed in the LLNA. One analogue, limonene-1-hydroperoxide, was a significantly more potent sensitizer than the other hydroperoxides and gave more positive test reactions in the allergic patients. The results support that hydroperoxides have a specific reactivity indicating that oxygen-centred radicals are important in hapten-protein complex formation of hydroperoxides. The primary oxidation products of ox. limonene, the hydroperoxides, have an important impact on the sensitizing capacity of the oxidation mixture.

Evaluation of ionization techniques for mass spectrometric detection of contact allergenic hydroperoxides formed by autoxidation of fragrance terpenes

Hydroperoxides formed by autoxidation of common fragrance terpenes are strong allergens and known to cause allergic contact dermatitis (ACD), a common skin disease caused by low molecular weight chemicals. Until now, no suitable methods for chemical analyses of monoterpene hydroperoxides have been available. Their thermolability prohibits the use of gas chromatography and their low UV-absorption properties do not promote sensitive analytical methods by liquid chromatography based on UV detection. In our study, we have investigated different liquid chromatography/mass spectrometry (LC/MS) ionization techniques, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI), for detection of hydroperoxides from linalool and limonene.Flow injection analysis was used to evaluate the three different techniques to ionize the monoterpene hydroperoxides, linalool hydroperoxide and limonene hydroperoxide, by estimating the signal efficacy under experimental conditions for positive and negative ionization modes. The intensities for the species [M+H]+ and [M+H-H2O]+ in positive ionization mode and [M-H]- and [M-H-H2O]- in negative ionization mode were monitored. It was demonstrated that the mobile phase composition and instrumental parameters have major influences on the ionization efficiency of these compounds. ESI and APCI were both found to be appropriate as ionization techniques for detection of the two hydroperoxides. However, APPI was less suitable as ionization technique for the investigated hydroperoxides.

Involvement of Limonene Hydroperoxides Formed after Oil Gland Injury in the Induction of Defense Response against Penicillium digitatum in Lemon Fruit

The effects of wounding oil glands of lemon [Citrus limon (L.) Burm.] fruit were investigated. Young mature-green lemons demonstrated significantly lower decay incidence than older yellow fruit when their oil glands were punctured in the presence of postharvest wound pathogen Penicillium digitatum Sacc. Contact with the released gland content on the green lemon surface reduced the viability of P. digitatum spores approximately twice. Wounding caused rapid production of limonene hydroperoxides that persisted for only a few minutes. The magnitude depended on the physiological maturity of the fruit; mature-green fruit produced much higher levels than did yellow lemons. Furthermore, wounding of the oil glands or injection of limonene hydroperoxides into the lemon peel elicited the production of the citrus fruit phytoalexins, scoparone and scopoletin, to levels known to be effective in reducing decay caused by P. digitatum. The mature-green fruit produced about twice as much of these phytoalexins as the older yellow fruit. This induced defensive elicitation of phytoalexin production, as well as the direct effects of these antifungal compounds, markedly inhibited the pathogen in mature-green fruits but was ineffective in older yellow ones.

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.

DEVELOPING A NOVEL ENVIRONMENTALLY FRIENDLY MICROBIOCIDAL FORMULATION FROM PEEL OF CITRUS FRUIT

ISHS IV International Conference on Managing Quality in Chains - The Integrated View on Fruits and Vegetables Quality DEVELOPING A NOVEL ENVIRONMENTALLY FRIENDLY MICROBIOCIDAL FORMULATION FROM PEEL OF CITRUS FRUIT

Effect of reaction conditions on limonene epoxidation with H 2O 2 catalyzed by supported Keggin heteropolycompounds

Limonene epoxidation by hydrogen peroxide in the presence of Keggin-type heteropolycompound supported catalysts was studied. Catalysts containing molybdenum or tungsten heteropolycompounds were prepared by pore filling impregnation of carbon or alumina, and were characterized by diffuse reflectance spectroscopy (DRS), Fourier transform infrared (FT-IR) and potentiometric titration. During the reaction, limonene oxide, carvone, carveol, diols and limonene hydroperoxide were produced. Different reaction conditions have been studied, such as nitrogen atmosphere, absence of light, and radical scavenger addition. A catalytic process of oxygen transference could be assumed to occur when a lacunar tungstophosphate supported on carbon is used as catalyst, forming a peroxometalate intermediate. A free radical competitive mechanism was found in the presence of alumina-supported heteropolyacids.

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.