Cross-reactivity between citral and geraniol can it be attributed to oxidized geraniol?

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1 Contact Dermatitis riginal Article CD Contact Dermatitis Cross-reactivity between citral and geraniol can it be attributed to oxidized geraniol? Lina Hagvall 1 and Johanna Bråred Christensson 1,2 1 Department of Dermatology, Sahlgrenska Academy, University of Gothenburg, Gröna Stråket 16, Gothenburg, Sweden and 2 Dermatochemistry and Skin Allergy, Department of Chemistry and Molecular Biology, University of Gothenburg, Kemigården 4, Gothenburg, Sweden doi: /cod Summary Background. The fragrance compound geraniol is susceptible to autoxidation when in contact with air, and to cutaneous metabolism. In both processes, the isomeric aldehydes geranial and neral are formed. Citral consists of geranial and neral. Among patients with positive reactions to citral, we have previously detected concomitant reactions to geraniol in 85% of cases and to oxidized geraniol in 73% of cases. bjective. To study the pattern of concomitant reactions to geraniol and citral and its isomers geranial and neral, and to determine whether these isomers are important sensitizers in contact allergy to geraniol and oxidized geraniol. Patients and methods. The irritancy of geranial and citral was studied. Six hundred and fifty-five patients were patch tested with geranial, neral and citral at 3.5% pet., pure geraniol at 6.0% and 11.0% pet., and oxidized geraniol at 6.0% pet. Results. Twenty-six per cent of citral-positive patients reacted to oxidized geraniol, and 10.5% reacted to pure geraniol. Citral and/or its isomers gave positive reactions in 25% of the patients who reacted to pure geraniol. Conclusions. There is little cross-reactivity between pure geraniol and citral; however, concomitant reactions to citral and oxidized geraniol were common, owing to geranial. Geranial was also the main sensitizer in the mixture citral. Key words: allergic contact dermatitis; autoxidation; CAS no ; CAS no ; CAS no ; CAS no ; citral; geraniol; metabolism; patch test. Geraniol is a widely used fragrance terpene with a flowery odour, and is used commercially in many different types of products. Studies of consumer products have shown that geraniol was declared or detected in 40 60% of Correspondence: Lina Hagvall, Department of Dermatology, Sahlgrenska Academy, University of Gothenburg, Gröna Stråket 16, Gothenburg, Sweden. Tel: ; Fax: lina.hagvall@gu.se Funding: The Edvard Welander Foundation, the Swedish Asthma and Allergy Association s Research Foundation, and the Agreement Concerning Research and Education of Doctors (ALF), Västra Götalandsregionen, Sweden. Conflict of interest: The authors declare no conflict of interest. Accepted for publication 15 July 2014 the investigated products (1 3). In a recent assessment of consumer exposure to terpene alcohols used as fragrance ingredients, geraniol was found to be one of the most common fragrances in cosmetic products, thus providing one of the highest maximum daily exposures to consumers among the studied terpenes (4). Geraniol is part of Fragrance Mix I (FM I), and is included in the European baseline series. In a large screening study, the frequency of positive reactions to FM I was found to be 6.6% (5). Approximately 5% of these positive reactions were attributable to geraniol (5), indicating that 0.3% (5% of 6.6%) of the tested patients would have had contact allergy to geraniol if tested separately. A large study has been conducted on contact allergy to the 26 fragrances to be declared according to the EU directive, and in this 280 Contact Dermatitis, 71,

2 study, 0.5% of the tested patients showed positive patch test reactions to geraniol, tested at 1% in petrolatum (pet.) (6). Geraniol, like other fragrance terpenes such as limonene and linalool, has been shown to autoxidize when in contact with air, thus forming sensitizing oxidation products (7 10). In the autoxidation of most studied terpenes, the major oxidation products and the main sensitizers of the oxidation mixtures were shown to be the hydroperoxides (8 10). Limonene hydroperoxides have been identified in natural sweet orange oil at the point of delivery from the producer, and further oxidation was observed after storage and handling, with increasing amounts of limonene hydroperoxides (11). Likewise, linalyl acetate hydroperoxides and linalool hydroperoxides were identified in petitgrain oil (11). As these oils are intended for use in consumer products, oxidation products can most likely be found in formulated products, with the oxidation occurring either before the fragrance components are added to the product, or during storage and handling. An added antioxidant will prevent the degradation of components, but, after the antioxidant is consumed, the oxidation will proceed, as has been shown for limonene and butylated hydroxytoluene (BHT) (12). Recent multicentre studies, including assessments of exposure and relevance for contact allergy, have been performed for oxidized linalool and oxidized limonene. In these studies, frequent exposure to linalool and/or limonene was recorded among the allergic patients, and the exposure was judged as likely to be relevant for the patients dermatitis in 42% and 36% of the patients, respectively. At some test centres, > 70% of the allergic patients were judged to have a relevant exposure to the respective fragrance chemical (13 15). In the case of geraniol, the major oxidation products in the autoxidation mixture were found to be the cis/trans-isomeric aldehydes geranial and neral (Fig. 1), which are formed from an unstable hydroperoxide, geraniol-1-hydroperoxide (Fig. 1) (7, 16). A more stable geraniol hydroperoxide (geraniol-7-hydroperoxide; Fig. 1) analogous to the most prominent linalool hydroperoxide was detected in the autoxidation mixture, but at a lower concentration than in a corresponding autoxidation mixture of linalool (7, 9). Quantum calculations showed that the autoxidation pathway resulting in the formation of geranial and neral was more favourable than that resulting in the formation of geraniol-7-hydroperoxide (Fig. 1) (16). In metabolism studies of geraniol with a skin-like CYP450 cocktail, we identified geranial and neral as metabolites (17). Epoxides of geraniol (Fig. 1), most of which were not found in the autoxidation mixture, were also identified. H Geraniol-7- hydroperoxide H H Geraniol epoxides H Geraniol-1- hydroperoxide H H Geraniol Autoxidation H Skin metabolism Geranial Geranial Citral Neral Citral Neral Fig. 1. Scheme of formation for sensitizing oxidation products resulting from autoxidation and cutaneous metabolism of geraniol. Geraniol-1-hydroperoxide is too unstable to be detected, but is considered to be an intermediate in the autoxidation pathway leading to the formation of geranial and neral (7, 11). Citral is a common fragrance and flavour substance, consisting of a 2:1 mixture of the two isomers geranial and neral, the same aldehydes that are formed by autoxidation and metabolic activation of geraniol. Citral is the major component of lemongrass (Cymbopogon citratus) and Litsea cubeba oil (18, 19), and one of the major components in lemon and lime leaf oils (20). It has been identified in 25% of domestic and occupational products (1), which indicates that skin exposure to citral and its isomers is frequent. Citral is a well-known allergen, and has been used for patch testing in several studies. At 2% pet., % of tested dermatitis patients showed positive test reactions (5, 21, 22). Citral is included in Fragrance Mix II (FM II) at 1% pet. in the European baseline series (23, 24). FM II has, in recent studies, given 4.9% positive patch test reactions among dermatitis patients (25). When tested separately, citral was found to be the second most important fragrance allergen in FM II, as 16.1% of patients with positive reactions to FM II showed positive reactions to citral 1.0% pet., indicating a frequency of positive patients of 0.8% (16% of the 4.9% positive patients). This is in good Contact Dermatitis, 71,

3 agreement with the above-mentioned study on the 26 fragrances to be labelled according to the EU regulations, in which citral was tested at 2% pet., and gave positive patch test reactions in 0.6% of the patients (6). An earlier study reported a high degree of irritation (14%) when citral was tested at 2% pet. (21). In later studies, a lower degree of irritancy was reported, i.e. 0.5% (6). In this large European multicentre patch test study, a connection between allergy to geraniol and allergy to citral was shown (6). We confirmed this connection in a patch test study in which patients with positive reactions to citral also, to a large extent, reacted to both pure and oxidized geraniol (26). The aim of the present study was to further investigate the connection between contact allergy to geraniol and contact allergy to citral. We wanted to determine whether either of the two isomers of citral (geranial and neral) could be better tools for the diagnosis of contact allergy than the mixture citral. Concomitant reactions to geranial, neral, citral, geraniol and oxidized geraniol were also studied, to obtain a clearer picture of the complex pattern of positive reactions to geraniol, which can act as both a prehapten and a prohapten. Prior to the patch test study, the skin irritancy of citral, geranial and neral was investigated, in order to determine whether the previously used patch test concentrations could be increased. Patients and Methods Chemicals Geraniol (98%) was purchased from Sigma Aldrich (St Louis, M, USA). Citral (66% geranial and 34% neral), geranial (98%) and neral (> 99%) were obtained from Bedoukian Research Inc. (Danbury, CT, USA). All chemicals were distilled prior to use. Instrumentation and mode of analysis High-performance liquid chromatography (HPLC) analyses were conducted on an Agilent 1260 HPLC system with a 1260 DAD VL+ detector. An Eclipse Plus C8 column (3.5 μm, mm 2 ; Agilent Technologies, Santa Clara, CA, USA) was used. Analysis of the test chemicals was conducted with external standards as previously described (26). Air exposure of geraniol Pure geraniol was air exposed for 10 weeks as previously described (7), and found to contain 48% geraniol, 3.9% geranial, 1.0% neral, and 3.2% geraniol-7- hydroperoxide. Table 1. Visual assessment of erythema grading scale according to Basketter et al. (24) 0 No reaction 1 Marginal reaction 2 Slight perceptible erythema 3 A greater than slight reaction that is not sufficient to be classified as distinct 4 Distinct erythema 5 A greater than distinct reaction that is insufficient to be classed as well developed 6 Well-developed, possibly spreading erythema 7 A greater reaction that is not sufficient to be classed as strong 8 Strong, deep erythema that may extend beyond the treatment site 9 A more intense reaction than above Patch test materials All chemicals were analysed prior to preparation of the patch test materials. n the basis of previous experience (27), non-stabilized pet. (Prolabo, Chester, PA, USA) was used for all patch test preparations. Patch test preparations were stored at 20 C until use. Each preparation was used for a test period of 6 months at most, according to previous experience (13). During this time, the preparations were stored in the refrigerator. In the study of irritancy, citral and geranial were tested at 5.0% and 2.5% pet. As the cis/trans-isomers were thought to show the same irritant properties, neral was not tested in the irritation study. In the subsequent study of contact allergy, citral, geranial and neral were tested at 3.5% pet. The patients were tested concomitantly with pure and oxidized geraniol at 4.0%, 6.0% and 11.0% pet., as described previously (28). Patients: irritation study Dermatitis patients [n = 22, 14 women and 8 men, mean age 46.5 years, standard deviation (SD) ±18.2] were asked to participate in the irritation study in addition to their regular patch testing. The irritation study was performed during the period February to April Patients 1 5 were tested in February 2010, and patients 6 22 were tested in April Exclusion criteria were previously diagnosed contact allergy to fragrances or a history of previous subjective reactions to fragranced products. The test substances were applied together with the ordinary patch test, and irritant reactions were evaluated on D3 D4. The reactions were evaluated visually, with a scale described by Basketter et al. (29) (Table 1). Informed consent was obtained. The study was approved by the local ethics committee. 282 Contact Dermatitis, 71,

4 Patients: study of contact allergy Consecutive patients patch tested with the Swedish baseline series at the Department of Dermatology, Sahlgrenska University Hospital, Gothenburg, Sweden, during the period September 2010 up to and including December 2011, were included in the study. Six hundred and fifty-five patients participated in the study (200 men, 455 women, mean age 45.2 years, SD ±17.8). Patch test preparations of 20 mg (30) were applied in small Finn Chambers (diameter 8 mm, inner area of 0.5 cm 2 ; Epitest Ltd y, Tuusula, Finland) on Scanpor tape (Norgesplaster A/S, Vennesla, Norway) to the back of the patient, left under occlusion for 2 days, and then removed by the patient. Readings were performed according to the ICDRG recommendations (31) on D3 D4 and D7. A positive patch test reaction was assessed as likely to be relevant if the patient used products in which geraniol and/or citral were declared among the contents at the site of eczema, or if the patient applied products containing essential oils or products declaring essential oils known to contain geraniol and/or citral at the site of eczema. In many cases, the patient brought in products to the clinic for inspection. Alternatively, the patient searched for the INCI names geraniol or citral on the labelling of their products after being given information about the contact allergy, and subsequently reported findings to the clinic. The assessments of exposure and probable relevance were performed at the second reading on D7, so information on improvement of eczema after avoidance of the allergen was not available. Statistical analysis Analysis was carried out with R version 3.0.3: A language and environment for statistical computing (R Foundation for Statistical Computing, Vienna, Austria). The visual readings with increasing concentrations of geranial and citral were compared as paired values by the use of Wilcoxons s signed rank test at the level of p < McNemar s test was used to evaluate differences in frequencies of positive patch test reactions to the study materials. Results Study of irritancy The mean values from the visual readings of irritant reactions to geranial and citral are shown in Table 2. The irritancy was low at the lowest test concentration for both geranial and citral; however, it increased at the higher test concentration. The irritant reactions overall ranged from 0 to 4 on the visual evaluation grading scale (29) (Fig. 2). Table 2. Data from irritation study, showing numerical readings of irritant reactions to geranial and citral on a visual scale from 0 to 9 (Table 1) at D3 D4 Test material Concentration (% pet.) Mean score SD Added score (n = 22) Geranial Geranial Citral Citral SD, standard deviation. Readings are presented with mean score and SD, as well as the cumulative score for 22 patients. The differences between the two test concentrations were found to be statistically significant, with p < for both geranial and citral. There was no statistically significant difference in irritation between equal concentrations of geranial and citral. Fig. 2. Sunflower plot of visual readings of irritancy (scale 0 9) of geranial and citral according to the erythema grading scale (24). A dot represents 1 patient, and each petal of the flower represents 1 additional patient showing the same result. The results obtained with the mixture citral are not significantly different from those obtained with the pure compound geranial. There was no difference in irritation caused by geranial and citral (isomeric mixture of geranial 66% and neral 34%) at equal concentrations. As the results for citral and geranial were very similar, neral was assumed to have the same irritant properties as geranial. A concentration of 3.5% pet. was chosen for further separate testing of geranial, neral, and citral, on the basis of the results from the study. Study of contact allergy The results of the patch testing in terms of total number of tested patients and frequencies of positive and doubtful reactions to citral, geranial and neral are shown in Table 3. No irritant reactions were recorded. Geranial 3.5% pet. yielded the highest frequency of positive reactions of these three test materials, giving 13 positive patch test reactions (2.0%) in 655 patients. In total, 19 of 655 patients reacted to one or more of the preparations of citral, geranial, and neral. These patients Contact Dermatitis, 71,

5 Table 3. The numbers and frequencies of positive and doubtful reactions, and the distribution of strength of the reactions (+ or ++/+++), for patients tested in the screening study with geraniol and oxidized geraniol at each patch test concentration; in all, 655 patients were tested 14 Geranial, neral or citral positive Positive Doubtful + ++/+++ Test material n % n % n % n % Citral 3.5% pet Geranial 3.5% pet Neral 3.5% pet xidized geraniol positive Geraniol positive Citral positive Fig. 4. Venn diagram representing the pattern of concomitant reactions to oxidized geraniol, geraniol and citral or its components among the 33 patients showing positive reactions to any of the test materials of the study Geranial positive Neral positive Fig. 3. Venn diagram representing the pattern of concomitant reactions to citral, geranial and neral among the 19 patients with positive reactions to any of these test materials. are described in detail in Table 4. Thirteen of 19 reacted to geranial, 6 of 19 reacted to neral, and 6 of 19 reacted to citral. Two patients reacted to all three test materials (geranial, neral, and citral). A Venn diagram representing the pattern of concomitant reactions to citral and the isomers geranial and neral is shown in Fig. 3. Concomitant reactions to geraniol and oxidized geraniol (6.0% pet.) among the patients reacting to citral, geranial or neral are shown in Table 4 and Fig. 4. A total of 33 patients reacted to one or more of the test materials of the study. xidized geraniol 6.0% gave the most reactions (15 patients). Details of these patients have been published previously (28). f the 33 patients reacting to the separate patch test materials of the study (geranial, neral, citral, geraniol, and oxidized geraniol), 26 would not have been detected with only FM I and FM II in the baseline series. FM I (containing geraniol) was positive in 7 of 33 patients (Table 4). f these 7 patients, 2 also reacted to geraniol, 4 reacted to geranial, and 4 reacted to oxidized geraniol. In 3 of the 7 cases, concomitant reactions were seen to oxidized geraniol, geranial, and FM I. FM II (containing citral) was positive in only 2 cases. In both cases, a reaction to citral (+) and strong reactions to geranial (++) and to oxidized geraniol (++) were seen. In 12 (36%) of the 33 cases, the reactions were assessed as most likely relevant at D7, as these patients were using products containing geraniol or citral on the dermatitis areas (Table 4). The Venn diagram (Fig. 4) shows the pattern of concomitant reactions to oxidized geraniol 6.0% pet., geraniol 11% pet., and neral, citral and geranial 3.5% pet. Concomitant reactions were most commonly seen between citral, the isomers geranial and neral, and oxidized geraniol. No concomitant reactions solely between citral and pure geraniol were observed. In the statistical analysis, the difference between the combined frequency of positive reactions to citral, neral or geranial and the frequency of positive reactions to oxidized geraniol was not statistically significant. However, on comparison of the combined frequency of positive reactions to citral, neral or geranial with the frequency of positive reactions to pure geraniol, a statistically significant difference could be seen (p = 0.035). During the study period, the overall frequencies of positive reactions at the clinic to the fragrance markers of the Swedish baseline series were: FM I, 6.5%; Myroxylon pereirae resin, 4.5%; FM II, 1.4%; and hydroxyisohexyl 3-cyclohexene carboxaldehyde, 0.7%. Discussion This study has identified geranial as an important allergen in its own right. f the patients reacting to any of the patch test preparations citral, geranial, or neral, 13 of 19 reacted to geranial. nly 3 of the 13 geranial-allergic patients were identified by the isomeric mixture citral, which may be because, if citral is tested at 3.5% pet., the concentrations of geranial and neral in the citral test 284 Contact Dermatitis, 71,

6 Table 4. Concomitant reactions to citral, geranial, and neral, as well as to fragrance markers of the baseline series, in patients with positive reactions to any of the test materials of the study; exposure to citral-containing or geraniol-containing products at the site of the patients dermatitis has been used as an approximation of exposure to the fragrance components, including oxidized geraniol Patient Sex Age (years) Citral 3.5% Geranial 3.5% Neral 3.5% Geraniol a 6.0% Geraniol a 11.0% xidized geraniol a 6.0% Relevance FM II FM I MP HICC Colophonium 1 F 18 +? 2 F F 57 + R ++ 4 M F R? M R NT NT + NT 7 F R F 24 + NT 9 F NT + ++ R F R 11 F 23 +? 12 F F F F F M M 69 +? R F F F R F 34 + R 23 F 54 + R 24 F F F 27 + R 27 F M 66? F ? 30 F F F 22 + R F 69 + F, female; FM, fragrance mix; M, male; MP, Myroxylon pereirae resin (balsam of Peru); HICC, hydroxyisohexyl 3-cyclohexene carboxaldehyde; NT, not tested; R, probable relevance of the reaction. a Data previously published (28). preparation are 2.3% and 1.2%. Thus, the concentrations of geranial and neral in the test preparation of citral could be too low to elicit a reaction in these patients. The irritancy of the two isomers combined in citral, however, prevents testing at a higher concentration of citral. Moreover, these patients were not identified by the low concentration of citral in FM II. Thus, patch testing with pure geranial at a higher concentration could be more useful than patch testing with the isomeric mixture citral, and may open the possibility of increasing detection rates for contact allergy to these fragrance compounds. Exposure to geranial and neral in the general population is probably mainly attributable to exposure to citral; however, it could also result from contact with oxidized geraniol. xidized geraniol has recently been shown to be an important test material, giving % positive patch test reactions in consecutive patients, and thus many more positive patch test reactions than pure geraniol (28). As citral consists of a 2:1 mixture of geranial and neral, the exposure concentration of geranial is always higher than that of neral (32). Similar proportions of geranial and neral are detected in oxidized geraniol (7). Exposure to geranial is thus expected to be higher, which would explain the higher frequency of positive reactions to geranial than of positive reactions to neral, as the sensitization potencies of geranial and neral are similar (7). Contact Dermatitis, 71,

7 Geranial is a main product of both autoxidation and cutaneous metabolism of geraniol, which could explain the high frequency of concomitant reactions to geranial and pure and oxidized geraniol in a previous study (26). To investigate this, we tested pure and oxidized geraniol in parallel with geranial, neral, and citral, and showed that 33% of patients (5 of 15) who reacted to oxidized geraniol also reacted to citral, geranial, or neral (Fig. 4). Thus, patch testing with citral and the isomers geranial and neral detects some, but not all, cases of contact allergy to oxidized geraniol. Geraniol-7-hydroperoxide might be the culprit sensitizer in cases of positive reactions to oxidized geraniol and negative reactions to citral and the isomers. Although geraniol-7-hydroperoxide is detected at a slightly lower concentration than geranial in the autoxidation mixture, it is a strong sensitizer (7). Reactions to citral or the isomers but not to oxidized geraniol could be explained by the lower concentrations of geranial and neral in the preparations of oxidized geraniol than when they are tested alone. Thus, patch testing with citral and its isomers geranial and neral, as separate test materials, is important. The fact that most patients who reacted to geranial did not react to neral indicates that there is no general crossreactivity between geranial and neral. Although the only difference between the two molecules is the cis/trans-conformation of the α,β-double bond, this is most likely enough to induce separate immunological responses. This is in accordance with results from our earlier studies exploring the cross-reactivity of enantiomers of carvone, in which the R-enantiomer and S-enantiomer were found not to cross-react in guinea-pigs (33). In an experimental setting, it has been shown that geranial can be converted to neral and vice versa in a reaction catalysed by amino acids (34). The reaction is favoured by a high ph, and is thus expected to be slow, and of little importance in the acidic environment of the skin, or on the acidic skin surface. Geranial and neral have been shown to be the main metabolites of the prehapten and prohapten geraniol (17). However, no patients in this study showed concomitant reactions to pure geraniol and citral or its isomers geranial and neral only (Fig. 4). Also, pure geraniol was the only test material giving a statistically significant lower frequency of reactions. No clear evidence for this metabolic activation in patients could therefore be established in the present study. However, in earlier clinical studies on fragrance contact allergy, high frequencies of concomitant reactions to geraniol and citral have been observed (6, 24). However, in those studies, the purity of the used test preparations of geraniol was not declared. As the composition of patch test materials may vary because of autoxidation or degradation, both in the raw material used for production and during storage and handling, the presence of oxidation products such as geranial in the test preparation of geraniol is possible. In order to study patterns of concomitant reactions, it is important to use test materials of known composition and high purity. A higher frequency of concomitant reactions to geraniol and oxidized geraniol than of concomitant reactions to geraniol and citral and its isomers was observed. ne explanation for this could be the formation of sensitizing epoxides, which have been identified as metabolites of geraniol (17). These epoxides could contribute to the sensitization to geraniol, and, as geraniol is still present in the preparations of oxidized geraniol, metabolism to epoxides in the skin is possible. Thus, these epoxides might be important in contact allergy to geraniol. Assessment of clinical relevance is difficult. In this study, exposure to citral-containing or geraniolcontaining products at the site of dermatitis has been used as an approximation of exposure to the fragrance components, including oxidized geraniol. As citral (geranial and neral) is added to fragranced products, this approximation is straightforward. However, in the case of oxidized geraniol, there is currently insufficient knowledge about the presence, qualitatively and quantitatively, of the oxidation products geraniol-7-hydroperoxide, geranial and neral in consumer products. The identification and quantification of hydroperoxides in formulated products are difficult, for technical reasons. Among the 19 patients reacting to citral or its isomers, only 2 (11%) reacted to FM II, containing 1% citral (Table 4). This indicates that many patients with contact allergy to citral or its isomers may be missed in routine testing with the baseline patch test material FM II. Conclusion This study explored the complex relationship between contact allergies to chemically and metabolically related compounds, which in turn are common fragrance ingredients. The results suggest that geranial is the main sensitizer in the mixture citral, and that there is little cross-reactivity between pure geraniol and citral. However, concomitant reactions to citral and oxidized geraniol were common, owing to the presence of geranial. This means that the important oxidized form of geraniol and its oxidation product and metabolite geranial can be useful in combination for the diagnosis of contact allergy to geraniol and citral. 286 Contact Dermatitis, 71,

8 Acknowledgements Professor Ann-Therese Karlberg is gratefully acknowledged for fruitful and constructive discussions. Martin Gillstedt is gratefully acknowledged for statistical analysis. Marie Norbäck is gratefully acknowledged for collocation of patient data. This study was performed within the Centre for Skin Research (SkinResQU) in Gothenburg. References 1 Rastogi S C, Heydorn S, Johansen J D, Basketter D A. Fragrance chemicals in domestic and occupational products. Contact Dermatitis 2001: 45: Rastogi S C, Johansen J D, Frosch P et al. Deodorants on the European market: quantitative chemical analysis of 21 fragrances.contact Dermatitis 1998: 38: Rastogi S C, Johansen J D, Menné T. Natural ingredients based cosmetics. Content of selected fragrance sensitizers. Contact Dermatitis 1996: 34: Belsito D, Bickers D, Bruze M et al. A toxicologic and dermatologic assessment of cyclic and non-cyclic terpene alcohols when used as fragrance ingredients. 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