Severely compromised supply of patch test allergens in Europe hampers adequate diagnosis of occupational and non-occupational contact allergy. A European Society of Contact Dermatitis (ESCD), European Academy of Allergy and Clinical Immunology (EAACI), European Academy of Dermatology and Venereology (EADV) task forces 'Contact Dermatitis' and 'Occupational Skin Disease' position paper.

Patch testing is the only clinically applicable diagnostic method for Type IV allergy. The availability of Type IV patch test (PT) allergens in Europe, however, is currently scarce. This severely compromises adequate diagnostics of contact allergy, leading to serious consequences for the affected patients. Against this background, the European Society of Contact Dermatitis (ESCD) has created a task force (TF) (i) to explore the current availability of PT substances in different member states, (ii) to highlight some of the unique characteristics of Type IV vs. other allergens and (iii) to suggest ways forward to promote and ensure availability of high-quality patch testing substances for the diagnosis of Type IV allergies throughout Europe. The suggestions of the TF on how to improve the availability of PT allergens are supported by the ESCD, the European Academy of Allergy and Clinical Immunology, and the European Academy of Dermatology and Venereology and intend to provide potential means to resolve the present medical crisis.

Sensitization properties of acetophenone azine, a new skin sensitizer identified in textile.

BACKGROUND: Acetophenone azine (CAS no. 729-43-1) present in sports equipment (shoes, socks and shin pads) has been suspected to induce skin allergies. Twelve case reports of allergy in children and adults from Europe and North America were published between 2016 and 2021. OBJECTIVES: The objective of this study was to confirm that acetophenone azine is indeed a skin sensitizer based on in vitro/ in vivo testings derived from the Adverse Outcome Pathway (AOP) built for skin sensitization by OECD in 2012. METHODS: Acetophenone azine was tested in vitro according to the human cell line activation test (h-CLAT) and the ARE-Nrf2 Luciferase Test (KeratinoSens) and in vivo using the Local Lymph Nodes Assay (LLNA). RESULTS: Both the h-CLAT and the KeratinoSens were positive whereas the LLNA performed at 5, 2.5 and 1% (wt/vol) of acetophenone azine, was negative. CONCLUSION: Based on these results, acetophenone azine was considered as a skin sensitizer. This was recently confirmed by its classification under the CLP regulation.

Synthesis and In Situ Behavior of 1,4- and 2,5-(13C) Isotopomers of p-Phenylenediamine in Reconstructed Human Epidermis Using High Resolution Magic Angle Spinning NMR.

p-Phenylenediamine (PPD) has been classified as a strong skin allergen, but when it comes to toxicological concerns, benzoquinone diamine (BQDI), the primary oxidation derivative of PPD, is frequently considered and was shown to covalently bind nucleophilic residues on model peptides. However, tests in solution are far from providing a reliable model, as the cutaneous metabolism of PPD is not covered. We now report the synthesis of two 13C substituted isotopomers of PPD, 1,4-(13C)p-phenylenediamine 1 and 2,5-(13C)p-phenylenediamine 2, and the investigation of their reactivity in reconstructed human epidermis (RHE) using the high resolution magic angle spinning (HRMAS) NMR technique. RHE samples were first treated with 1 or 2 and incubated for 1 to 48 h. Compared to the control, spectra clearly showed only the signals of 1 or 2 gradually decreasing with time to disappear after 48 h of incubation. However, the culture media of RHE incubated with 1 for 1 and 24 h, respectively, showed the presence of both monoacetylated- and diacetylated-PPD as major products. Therefore, the acetylation reaction catalyzed by N-acetyltransferase (NAT) enzymes appeared to be the main process taking place in RHE. With the aim of increasing the reactivity by oxidation, 1 and 2 were treated with 0.5 equiv of H2O2 prior to their application to RHE and incubated for different times. Under these conditions, new peaks having close chemical shifts to those of PPD-cysteine adducts previously observed in solution were detected. Under such oxidative conditions, we were thus able to detect and quantify cysteine adducts in RHE (maximum of 0.2 nmol/mg of RHE at 8 h of incubation) while no reaction with other nucleophilic amino acid residues could be observed.

One hundred years of allergic contact dermatitis due to oxidized terpenes: What we can learn from old research on turpentine allergy.

Although in recent years the focus on sensitizing terpene oxidation products has been on oxidized limonene and linalool, the autoxidation of terpenes in relation to allergic contact dermatitis is not new and dates back to the early part of the 20th century with the use of turpentine causing occupational contact dermatitis in painters. This review is written in a way as to allow us to get closer to the work of the scientists in earlier days, to participate in the successes, and also to observe the weak points. The researchers concluded that the main culprit in Scandinavian turpentine was Δ3 -carene hydroperoxides. This explains its high sensitizing effect compared with French turpentine which is of the Iberian type with no or only traces of Δ3 -carene. Historical exposure to turpentine showed that ending the industrial exposure stopped the occupational skin sensitization. Patch test studies demonstrated that monoterpene hydroperoxides, far from being an obsolete source of contact allergy solely related to turpentine, is a common cause of contact allergy in the population. A hundred years of extensive chemical and clinical studies worldwide should be sufficient to meet the evidence requirement regarding allergic contact dermatitis caused by terpenes.

Chemical Modifications Induced by Phthalic Anhydride, a Respiratory Sensitizer, in Reconstructed Human Epidermis: A Combined HRMAS NMR and LC-MS/MS Proteomic Approach.

Chemical skin and respiratory allergies are becoming a major health problem. To date our knowledge on the process of protein haptenation is still limited and mainly derived from studies performed in solution using model nucleophiles. In order to better understand chemical interactions between chemical allergens and the skin, we have investigated the reactivity of phthalic anhydride 1 (PA), a chemical respiratory sensitizer, toward reconstructed human epidermis (RHE). This study was performed using a new approach combining HRMAS NMR to investigate the in situ chemical reactivity and LC-MS/MS to identify modified epidermal proteins. In RHE, the reaction of PA appeared to be quite fast and the major product formed was phthalic acid. Two amide type adducts on lysine residues were observed and after 8h of incubation, we also observed the formation of an imide type cyclized adducts with lysine. In parallel, RHE samples topically exposed to phthalic anhydride (13C)-1 were analyzed using the shotgun proteomics method. Thus, 948 different proteins were extracted and identified, 135 of which being modified by PA, i.e., 14.2% of the extracted proteome. A total of 211 amino acids were modified by PA and validated by fragmentation spectra. We thus identified 154 modified lysines, 22 modified histidines, 30 modified tyrosines, and 5 modified arginines. The rate of modified residues, as a proportion of the total number of modifiable nucleophilic residues in RHE, was rather low (1%). At the protein level, modified proteins were mainly type I and type II keratins and other proteins which are abundant in the epidermis such as protein S100A, Caspase 14, annexin A2, serpin B3, fatty-acid binding protein 5, histone H2, H3, H4, etc. However, the most modified protein, mainly on histidine residues, was filaggrin, a protein that is of low abundance (0.0266 mol %) and rich in histidine.

In Situ Alkylation of Reconstructed Human Epidermis by Methyl Methanesulfonate: A Quantitative HRMAS NMR Chemical Reactivity Mapping.

Allergic contact dermatitis (ACD) is a reaction of the immune system resulting from skin sensitization to an exogenous hazardous chemical and leading to the activation of antigen-specific T-lymphocytes. The adverse outcome pathway (AOP) for skin sensitization identified four key events (KEs) associated with the mechanisms of this pathology, the first one being the ability of skin chemical sensitizers to modify epidermal proteins to form antigenic structures that will further trigger the immune system. So far, these interactions have been studied in solution using model nucleophiles such as amino acids or peptides. As a part of our efforts to better understand chemistry taking place during the sensitization process, we have developed a method based on the use of high-resolution magic angle spinning (HRMAS) NMR to monitor in situ the reactions of 13C substituted chemical sensitizers with nucleophilic amino acids of epidermal proteins in reconstructed human epidermis. A quantitative approach, developed so far for liquid NMR applications, has not been developed to our knowledge in a context of a semisolid nonanisotropic environment like the epidermis. We now report a quantitative chemical reactivity mapping of methyl methanesulfonate (MMS), a sensitizing methylating agent, in reconstructed human epidermis by quantitative HRMAS (qHRMAS) NMR. First, the haptenation process appeared to be much faster in RHE than in solution with a maximum concentration of adducts reached between 4 and 8 h. Second, it was observed that the concentration of cysteine adducts did not significantly increase with the dose (2.07 nmol/mg at 0.4 M and 2.14 nmol/mg at 1 M) nor with the incubation time (maximum of 2.27 nmol/mg at 4 h) compared to other nucleophiles, indicating a fast reaction and a potential saturation of targets. Third, when increasing the exposure dose, we observed an increase of adducts up to 12.5 nmol/mg of RHE, excluding cysteine adducts, for 3112 µg/cm2 (1 M solution) of (13C)MMS. This methodology applied to other skin sensitizers could allow for better understanding of the potential links between the amount of chemical modifications formed in the epidermis in relation to exposure and the sensitization potency.

Mechanistic Insights on Skin Sensitization to Linalool Hydroperoxides: EPR Evidence on Radical Intermediates Formation in Reconstructed Human Epidermis and 13C NMR Reactivity Studies with Thiol Residues.

Linalool is one of the most commonly used fragrance terpenes in consumer products. While pure linalool is considered as non-allergenic because it has a very low skin sensitization potential, its autoxidation on air leads to allylic hydroperoxides that have been shown to be major skin sensitizers. These hydroperoxides have the potential to form antigens via radical mechanisms. In order to obtain in-depth insights of such reactivity, we first investigated the formation of free radicals derived from linalool hydroperoxides in situ in a model of human reconstructed epidermis by electron paramagnetic resonance combined with spin trapping. The formation of carbon- and oxygen-centered radical species derived from the hydroperoxides was especially evidenced in an epidermis model, mimicking human skin and thus closer to what may happen in vivo. To further investigate these results, we synthesized linalool hydroperoxides containing a 13C-substitution at positions precursor of carbon radicals to elucidate if one of these positions could react with cysteine, its thiol chemical function being one of the most labile groups prone to react through radical mechanisms. Reactions were followed by mono- and bidimensional 13C NMR. We validated that carbon radicals derived from allylic hydrogen abstraction by the initially formed alkoxyl radical and/or from its ß-scission can alter directly the lateral chain of cysteine forming adducts via radical processes. Such results provide an original vision on the mechanisms likely involved in the reaction with thiol groups that might be present in the skin environment. Consequently, the present findings are a step ahead toward the understanding of protein binding processes to allergenic allylic hydroperoxides of linalool through the involvement of free radical species and thus of their sensitizing potential.

Contact Allergy-Emerging Allergens and Public Health Impact.

Contact allergy (sensitisation) and allergic contact dermatitis (ACD) resulting from it have a considerable public health impact. For the present review, all pertinent articles were systematically searched via Medline and Web of Science™; additionally, all available issues of the journals "Contact Dermatitis" and "Dermatitis" were manually searched, covering the years 2018-2019, thereby extending and re-focusing a previous similar review. New allergens, or previously described allergens found in a new exposure context or of other current importance, are described in sections according to substance classes, e.g., metals, preservatives, fragrances. As a common finding in many investigations, a lack of information on product composition has been noted, for instance, regarding a newly described allergen in canvas shoes (dimethylthiocarbamylbenzothiazole sulfide) and, most notably, absence of co-operation from manufacturers of glucose-monitoring devices and insulin pumps, respectively. These latter devices have been shown to cause severe ACD in a considerable number of diabetic patients caused by the liberation of isobornyl acrylate and N,N'-dimethylacrylamide, respectively, as demonstrated by an international collaboration between dermatologists and chemists. Improved and complete ingredient labelling for all types of products, and not just cosmetics, must be put on the legislative agenda.

Modifications induced by chemical skin allergens on the metabolome of reconstructed human epidermis: A pilot high-resolution magic angle spinning nuclear magnetic resonance study.

BACKGROUND: High-resolution magic angle spinning (HRMAS) is a nuclear magnetic resonance (NMR) technique that enables the characterization of metabolic phenotypes/metabolite profiles of cells, tissues, and organs, under both normal and pathological conditions, without resorting to time-consuming extraction techniques. OBJECTIVES: To assess the impact of chemical skin sensitizers vs non-sensitizers on the metabolome of three-dimensional reconstructed human epidermis (RHE) by HRMAS NMR. METHODS: Based on the SENS-IS assay, 12 skin sensitizers and five non-sensitizing chemicals were investigated and applied on EpiSkin RHE at the published maximal non-irritating concentrations under the conditions of the test. The metabolome of RHE samples was then analyzed by HRMAS NMR. RESULTS: A total of 32 different metabolites were identified; 20 of these were quantified for all samples. Statistical univariate analysis showed that the tissue content of most measured metabolites (with the exception of acetate and glucose) was different in the untreated, treated with non-sensitizers, and treated with sensitizers samples. In RHE samples in contact with sensitizing chemicals, concentrations of 18 metabolites were significantly decreased. Alanine and tyrosine could not discriminate between sensitizer- and non-sensitizer-treated groups. A multivariate partial least-squares-discriminant analysis was performed on the two treated groups, discriminating sensitizing and non-sensitizing chemicals with a very good R2Y value of 0.87 and a good Q2Y value of 0.70. CONCLUSIONS: Data suggest that HRMAS NMR could be used to monitor the impact of chemicals, skin allergens vs non-sensitizers, on the metabolome of three-dimensional RHE.

Formation of methyl radicals derived from cumene hydroperoxide in reconstructed human epidermis: an EPR spin trapping confirmation by using 13C-substitution.

Dermal exposure to cumene hydroperoxide (CumOOH) during manufacturing processes is a toxicological issue for the industry. Its genotoxicity, mutagenic action, ability to promote skin tumour, capacity to induce epidermal hyperplasia, and aptitude to induce allergic and irritant skin contact dermatitis are well known. These toxic effects appear to be mediated through the activation to free radical species such as hydroxyl, alkoxyl, and alkyl radicals characterised basically by electron paramagnetic resonance (EPR) and spin-trapping (ST) techniques. To be a skin sensitiser CumOOH needs to covalently bind to skin proteins in the epidermis to form the antigenic entity triggering the immunotoxic reaction. Cleavage of the O-O bond allows formation of unstable CumO•/CumOO• radicals rearranging to longer half-life specific carbon-centred radicals R• proposed to be at the origin of the antigen formation. Nevertheless, it is not still clear which R• is precisely formed in the epidermis and thus involved in the sensitisation process. The aim of this work was to elucidate in conditions closer to real-life sensitisation which specific R• are formed in a 3D reconstructed human epidermis (RHE) model by using 13C-substituted CumOOH at carbon positions precursors of potentially reactive radicals and EPR-ST. We demonstrated that most probably methyl radicals derived from ß-scission of CumO• radicals occur in RHE through a one-electron reductive pathway suggesting that these could be involved in the antigen formation inducing skin sensitisation. We also describe a coupling between nitroxide radicals and ß position 13C atoms that could be of an added value to the very few examples existing for the coupling of radicals with 13C atoms.

Sensitization potential and potency of terpene hydroperoxides in the cocultured activation test method.

BACKGROUND: Positive patch test reactions to mixtures of oxidized terpenes containing allergenic hydroperoxides are frequently reported. However, human sensitization data for these hydroperoxides are not available. OBJECTIVES: To analyse and evaluate the human sensitization potential and potency of hydroperoxides in vitro by using human cells. MATERIALS/METHODS: Limonene-1-hydroperoxide, limonene-2-hydroperoxide, citronellol-7-hydroperoxide, cumene hydroperoxide, 1-(1-hydroperoxy-1-methylethyl)cyclohexene and mixtures of citronellol hydroperoxides (isomers at positions 6 and 7) and linalool hydroperoxides (isomers at positions 6 and 7) were studied. All compounds were synthesized except for cumene hydroperoxide, which was commercially available. Their potential and potency to activate dendritic cells (DCs) was evaluated by measuring the upregulation of CD86 and CD54 on THP-1 cells upon exposure in the cocultured activation test (COCAT) consisting of HaCaT cells (human keratinocyte cell line) and THP-1 monocytes (as a surrogate for DCs). RESULTS: Hydroperoxides upregulated CD86 and/or CD54 on cocultured THP-1 cells in a concentration-dependent manner. The results are comparable with their sensitization potency ranking in predictive animal models. CONCLUSIONS: For the first time, the human sensitization potential and potency of several hydroperoxides were determined by the use of human cells and the COCAT method.

Understanding the skin sensitization capacity of ascaridole: a combined study of chemical reactivity and activation of the innate immune system (dendritic cells) in the epidermal environment.

To improve the prediction of the possible allergenicity of chemicals in contact with the skin, investigations of upstream events are required to better understand the molecular mechanisms involved in the initiation of allergic reactions. Ascaridole, one of the compounds responsible for skin sensitization to aged tea tree oil, degrades into intermediates that evolve via different mechanisms involving radical species. We aimed at broadening the knowledge about the contribution of radical intermediates derived from ascaridole to the skin sensitization process by assessing the reactivity profile towards amino acids, identifying whether free radicals are formed in a reconstructed human epidermis (RHE) model and their biological properties to activate the immune system, namely dendritic cells in their natural context of human HaCaT keratinocytes and RHE. Electron paramagnetic resonance combined to spin-trapping in EpiSkin™ RHE confirmed the formation of C-radicals in the epidermal tissue from 10 mM ascaridole concentration, while reactivity studies toward amino acids showed electrophilic intermediates issued from radical rearrangements of ascaridole as the main reactive species. Activation of THP-1 cells, as surrogate for dendritic cells, that were cocultured with HaCaT was significantly upregulated after treatment with low micromolar concentrations based on cell surface expression of the co-stimulatory molecule CD86 and the adhesion molecule CD54. Placing THP-1 cells underneath the RHE allowed us to monitor which of the concentrations that produce radical(s) and/or protein antigens in the epidermal skin environment promote the activation of dendritic cells. We detected no significant upregulation of CD86/CD54 after topical RHE application of concentrations up to 30 mM ascaridole (t = 24 h) but clear upregulation after 60 mM.

Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept.

The first step in the development of skin sensitisation to a chemical, and in the elicitation of further allergic contact dermatitis (ACD), is the binding of the allergen to skin proteins after penetrating into the epidermis. The so-formed antigenic adduct is then recognised by the immune system as foreign to the body. Sensitising organic hydroperoxides derived from autoxidation of natural terpenes are believed to form antigens through radical-mediated mechanisms, although this has not yet been established. So far, in vitro investigations on reactive radical intermediates derived from these skin sensitisers have been conducted in solution, yet with experimental conditions being far away from real-life sensitisation. Herein, we report for the first time, the potential use of EPR spin-trapping to study the in situ generation of free radicals derived from cumene hydroperoxide CumOOH in a 3D reconstructed human epidermis (RHE) model, thus much closer to what may happen in vivo. Among the undesirable effects associated with dermal exposure to CumOOH, it is described to cause allergic and irritant dermatitis, being reported as a significant sensitiser. We considered exploiting the usage of spin-trap DEPMPO as an extensive view of all sort of radicals derived from CumOOH were observed all at once in solution. We showed that in the EpiskinTM RHE model, both by incubating in the assay medium and by topical application, carbon radicals are mainly formed by redox reactions suggesting the key role of CumOOH-derived carbon radicals in the antigen formation process.

Isothiazolinones are still widely used in paints purchased in five European countries: a follow-up study.

BACKGROUND: An increasing incidence of contact allergy to methylisothiazolinone (MI) has been seen, caused, in particular, by cosmetic products and paints. A study from 2015 showed that 93.0% of paints bought in five European countries contained MI. New regulations have been discussed for paints in the EU, which may have influenced this market. OBJECTIVES: To re-evaluate the use and concentrations of MI and four other isothiazolinones in water-based wall paints. METHODS: Water-based white wall paints (n = 60) were purchased in retail stores in five European countries: Denmark, France, Germany, Sweden, and the United Kingdom. The paints were analysed for isothiazolione content by the use of high-performance liquid chromatography coupled to ultraviolet detection, and the results were confirmed with high-performance liquid chromatography-tandem mass spectrometry. RESULTS: MI was identified in 55 (91.7%) of the paints, with concentrations ranging from 1.1 to 142.7 ppm. The other isothiazolinones were identified in 20.0% [methylchloroisothiazolinone (MCI)] to 88.3% [benzisothiazolinone (BIT)] of the paints. BIT concentrations varied significantly between countries, whereas MI and MCI concentrations did not. There were no statistically significant differences in MI, MCI and BIT concentrations between the current study and the 2015 study. CONCLUSIONS: MI and other isothiazolinones are widely used in paints available in Europe. Their use does not seem to be decreasing.

Editor's Highlight: Fragrance Allergens Linalool and Limonene Allylic Hydroperoxides in Skin Allergy: Mechanisms of Action Focusing on Transcription Factor Nrf2.

Allergic contact dermatitis is regarded as the most frequent expression of immunotoxicity in humans. Many odorant terpenes commonly used in fragrance compositions are considered as weak skin sensitizers, whereas some of their autoxidation products, allylic hydroperoxides, are classified as strong sensitizers according to the local lymph node assay. However, the mechanism of their effects on the immune system remains unclear. Since dendritic cells play a key role in allergic contact dermatitis, we studied their activation by the frequently used linalool (LINA) and limonene (LIMO), and their respective sensitizing allylic hydroperoxides (LINA-OOH, LIMO-OOH). The THP-1 cell-line was used as a surrogate for dendritic cells, the model currently employed in the validated h-CLAT in vitro test. Our data showed that allylic hydroperoxides behave differently. Both LINA-OOH and LIMO-OOH oxidized cell surface thiols 30 min after stimulation. However, the oxidative stress induced by LINA-OOH was stronger, with a higher decreased GSH/GSSG ratio and a stronger reactive species production. Moreover, LINA-OOH induced a stronger Nrf2 accumulation in correlation with nqo1 and ho-1 gene expression, 2 Nrf2 target genes. Regarding signaling pathways involved in these effects, P38 mitogen-activated protein kinase and P-ERK were activated in response to LINA-OOH but not with LIMO-OOH. CD54 and CD86 were induced 24-h postexposure. In contrast, LINA and LIMO did not modify THP-1 phenotype. This work underlies that autoxidation forming allylic hydroperoxide (ROOH) does not lead to equal chemical reactivity since LINA-OOH appears to be a stronger activator than LIMO-OOH, in regard to oxidative stress and Nrf2 pathway activation.

Immunological, chemical and clinical aspects of exposure to mixtures of contact allergens.

Allergic contact dermatitis is one of the most frequent forms of skin inflammation. Very often, we are exposed to mixtures of allergens with varying potencies, doses/areas, and exposure times. Therefore, improved knowledge about immune responses to combinations of contact allergens is highly relevant. In this article, we provide a general introduction to immune responses to contact allergens, and discuss the literature concerning immune responses to mixtures of allergens. According to the existing evidence, increased responses are induced following sensitization with combinations of allergens as compared with single allergens. The response to a mixture of allergens can be both additive and synergistic, depending on the dose and combination of allergens. Importantly, sensitization with combinations of either fragrance allergens or metal salts can result in increased challenge responses to specific allergens within the mixture. Taken together, the immune responses to mixtures of allergens are complex, and further studies are required to obtain the necessary knowledge to improve consumer safety.

Contact allergy caused by isothiazolinone derivatives: an overview of non-cosmetic and unusual cosmetic sources.

The isothiazolinone derivatives, methylchloroisothiazolinone (MCI), methylisothiazolinone (MI), benzisothiazolinone (BIT), and octylisothiazolinone (OIT), owing to their strong bactericide, fungicide and algicide properties, are widely used in non-cosmetic products, such as chemical (industrial) products, household detergents, and water-based paints, and the former two derivatives are also used in cosmetic products. However, given their inherent sensitization potential (with MCI > MI > BIT > OIT), allergic contact dermatitis is frequently observed, both in consumers as well as workers in various industries. In this review, we provide an update on the use of MCI/MI and MI in cosmetics, highlighting certain aspects of MI; the use of excessive concentrations, the presence in some less familiar cosmetic products, and the association with unusual clinical manifestations. Furthermore, the use of isothiazolinones in dish-washing and washing-machine liquids, cleaning agents for dental care, and their general presence in multi-purpose household detergents, which may elicit (airborne) allergic contact dermatitis, is discussed. Finally, we provide a brief overview of the use of isothiazolinone derivatives in the paint and textile industry, and of OIT in the leather industry in particular.

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

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.