Contact sensitization to hydroperoxides of limonene and linalool: Results of consecutive patch testing and clinical relevance

Background Hydroperoxides of limonene and linalool are potent sensitizers. Objectives To investigate the prevalence of contact allergy to both hydroperoxides of limonene and hydroperoxides of linalool, to report clinical relevance, and to investigate patient demographics. Methods A total of 821 patients (35.6% male, mean age 42.4 years ± 17.8 years) were consecutively patch tested with our departmental baseline series and our fragrance series, including hydroperoxides of limonene 0.3% pet. and hydroperoxides of linalool 1.0% pet. The clinical relevance was assessed for all positive reactions. Results Positive patch test reactions to hydroperoxides of limonene and to hydroperoxides of linalool were observed in 77 patients (9.4%, 95% confidence interval [CI]: 7.4%‐11.4%) and in 96 patients (11.7%, 95%CI: 9.5%‐13.9%), respectively; 38 of these patients (4.6%, 95%CI: 3.2%‐6.0%) reacted to both. Most reactions were considered to be possibly or probably clinically relevant (66.3% and 68.8%, respectively), and a small proportion were deemed to be of certain clinical relevance (18.2% and 19.8%, respectively). Conclusion As compared with previous studies, high numbers of positive reactions to both hydroperoxides of limonene and hydroperoxides of linalool were observed, the majority of which were clinically relevant, supporting their inclusion in the European baseline series.


| INTRODUCTION
Limonene (D-limonene) is the main ingredient of pressed oil from the peel of citrus fruits, and linalool is present in many herbs, flowers, woods, etc. 1,2 Both are common ingredients in household products and cosmetics, such as hygiene products, perfumes, and detergents, as well as industrial products. 3,4 Limonene and linalool are ubiquitous fragrance terpenes with low sensitizing potential. 1,2,5 However, upon air exposure, oxidation occurs, during which different oxidation products are formed. Of these oxidation products, the hydroperoxides are potent sensitizers. High prevalences of contact allergy to these hydroperoxides of limonene and linalool have been reported. [6][7][8][9] The aim of the current study was to investigate the prevalences of contact allergy to hydroperoxides of limonene and hydroperoxides of linalool, and to characterize patients allergic to either or both hydroperoxides of limonene and hydroperoxides of linalool, with respect to patient characteristics and concomitant fragrance contact allergies.

| METHODS
A database study was performed on all patients who were at least patch tested with both hydroperoxides of limonene 0.3% pet. and hydroperoxides of linalool 1.0% pet. All patients referred to our tertiary referral centre with suspected allergic contact dermatitis (ACD) are consecutively patch tested with our departmental extended European baseline series (EBS), TRUE Test panels 1 and 2 (SmartPractice Europe, Reinbek, Germany) supplemented with additional investigator-loaded allergens, and a fragrance series. When specific contact allergies are suspected, additional series are patch tested. All investigator-loaded allergens were tested in Van  Consecutively patch tested dermatitis patients from December 1, 2015 to December 15, 2017 were included in the current analysis.
All patch tests were read and interpreted by the same dermatologist, with potential back-up from a dermatologist also trained in reading and interpreting patch test results, according to ICDRG/ESCD criteria, with the possible outcomes being: negative, irritant, doubtful (?+), weak positive (+), strong positive (++), and extreme positive (+++) reactions. 10 Reactions were considered to be irritant if margins were sharply demarcated and the surface of the test area showed a silk paper structure or a shiny skin. Reactions were considered to be doubtful if erythema and infiltration did not cover the whole test area.
Readings were performed on day (D) 3

| Clinical relevance and additional contact allergies
For all positive patch test reactions, the current and/or past clinical relevance was determined based on patient history and exposure, with possible outcomes being unlikely/not, possible, probable, and certain. "Unlikely/not" suggested that there was no suspected ACD, "possible" suggested that there was some suspicion of a relationship between the allergen and the dermatitis (between 1% and 49% convinced), "probable" suggested that this suspicion was stronger (between 50% and 99% convinced), and "certain" meant that the relationship was proven (100% convinced) by the presence of allergen in a product to which there was exposure at the body site where there was dermatitis, with a clear temporal relationship. For hydroperoxides of limonene and hydroperoxides of linalool, patients were instructed to review the labelling of their products for either limonene (or D-limonene, also known as R-limonene, and its enantiomer S-limonene), or linalool, respectively, as ingredients in their products. Their findings were subsequently discussed at our outpatient clinic; if patients were unsure or unable to review their products, they were instructed to bring all of their suspected products for review by the dermatologist.
Clinical relevance is presented for the same groups as described above for strength of patch test reaction. The types of product for which exposure caused ACD in patients with a contact allergy to hydroperoxides of limonene and/or hydroperoxides of linalool of certain clinical relevance are presented.
To evaluate concomitant reactions in patients with contact allergy to hydroperoxides of limonene, hydroperoxides of linalool, or both, the proportion of patients with at least ≥1 additional contact allergies apart from allergy to either hydroperoxides of limonene or hydroperoxides of linalool, and the proportion of patients with at least ≥1 additional non-fragrance allergies (excluding colophonium), are presented.
Additional contact allergies were not limited to EBS allergens; that is, any contact allergy was considered.  Table S1 provides an overview of all fragrance allergens tested, including the tested concentration and vehicle, in the current cohort of patients. Patient demographics for these groups were described according to the MOAHLFA index. 11

| Statistics
Statistical analyses were performed according to pertinent guidelines. 12

| Strength of reactions
As can be deduced from the calculated PRs shown above, the majority of positive reactions to both hydroperoxides of limonene and hydroperoxides of linalool were weak (+) positive. Only 1 extreme (+++) positive reaction to hydroperoxides of linalool was observed. Table 2 shows

| Clinical relevance
The clinical relevance of the positive reactions to both hydroperoxides of limonene and hydroperoxides of linalool were evaluated according to the groups described above (Table 3). Positive reactions to hydroperoxides of limonene/hydroperoxides of linalool in patients who were allergic to both, with or without additional fragrance allergies, were more frequently of certain clinical relevance (ranging from 27.3% to 37.5%, not statistically significant). The majority of reactions were evaluated as being of possible or probable clinical relevance. For patients with "certainly relevant" positive patch test reactions, the product type responsible for the allergic contact dermatitis are shown in Table S2. In the majority of patients (15/21, 71.4%), the responsible product types were rinse-off products such as soap and shampoo,

| Patient characteristics
The patient characteristics according to the MOAHLFA index are shown in Table 4 for each of the defined groups ( Figure 1). When A regression analysis was performed for each of these groups (except for group F, all members of which were not allergic to a fragrance); the results are shown in

| DISCUSSION
In our cohort of consecutively patch tested dermatitis patients, 9.4% and 11.7% showed positive patch test reactions to hydroperoxides of limonene and hydroperoxides of linalool, respectively, supporting the TABLE 6 The proportion of patients with at least 1 additional contact allergy other than to hydroperoxides of limonene and hydroperoxides of linalool, the proportion of patients with at least 1 additional non-fragrance allergy, the median additional (non-fragrance) reactions in group A (patients allergic to hydroperoxides of limonene and/or hydroperoxides of linalool (Figure 1), and the subsets of patients allergic to only hydroperoxides of limonene, patients allergic to only to hydroperoxides of linalool, and patients allergic to both hydroperoxides of limonene and hydroperoxides of linalool  Univariable and multivariable regression analysis performed for each group of patients as shown in Figure 1, with the exception of group F, with the following variables: sex, age (<40 years vs ≥40 years), (a history of ) atopic dermatitis, occupational dermatitis, and primary site of dermatitis (generalized, trunk, hand, leg, face, and other) recent proposal to include them in the EBS. 15 This is a higher number than found in recent studies, in which prevalences of contact allergy to hydroperoxides of limonene ranged from 2.5% to 5.4%, and prevalences of contact allergy to hydroperoxides of linalool ranged from 3.9% to 7.7%. 6,8,9 However, in multicentre studies, a large variation in positive patch test reactions between centres was observed; for example, in one study, prevalences of contact allergy to hydroperoxides of limonene ranged from 0% to 24.8%. 8 A possible explanation for the large number of positive reactions to the hydroperoxides could be that our centre is a tertiary referral centre, so more patients with TABLE 7 The number of patients with positive reactions to each of the fragrance (markers) allergens and/or colophonium, presented for patients allergic to hydroperoxides of limonene and/or hydroperoxides of linalool, for the subsets of patients allergic to only hydroperoxides of limonene, patients allergic to only to hydroperoxides of linalool, and patients allergic to both hydroperoxides of limonene and hydroperoxides of linalool, and for all other patients not allergic to either hydroperoxides of limonene or hydroperoxides of linalool  On the basis of our results, a low RI (<0) and a high PR (>80%) for both hydroperoxides of limonene and hydroperoxides of linalool were calculated, indicative of a problematic patch test concentration. 13 tration. An additional concern regarding testing at higher concentrations is the higher risk of active sensitization, as studies have shown that an irritant effect can increase this risk. 19  Clinical relevance is generally difficult to ascertain, as it depends on how well and how diligent a patient reads product labels and identifies the presence of contact allergens in the product. For hydroperoxides of limonene and hydroperoxides of linalool, an additional limitation is that these are not mentioned as such on labels.
Therefore, patients have to look for limonene and/or linalool, as these are among the 26 fragrances for which labelling is required on cosmetic and detergent products in the EU. 22 Studies have shown that fine fragrances and essential oils, which often contain limonene and/or linalool, also contain hydroperoxides as a result of autoxidation. 23,24 In the current study, the designation "certain" clinical relevance was reserved for patients who showed a clear temporal relationship between body site-specific exposure to a product containing limonene and/or linalool and dermatitis at that body site, even

CONFLICT OF INTEREST
The authors declare no potential conflict of interests.