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Background: Contact allergy rates of linalool and limonene hydroperoxides (HPs) have increased., Objectives: To demonstrate the patterns of simultaneous positive patch test (PT) reactions and prevalences of multiple contact allergies (MCAs) in patients with contact allergy to linalool and/or limonene HPs., Methods: A retrospective analysis of consecutive dermatitis patients in 2015-2020 was performed., Results: Of all 4192 patients, 1851 had at least one positive PT reaction. Of these, 410 (22.2%) had MCAs, significantly related to a higher age (p-value = 0.003). Patients with an exclusively positive reaction to linalool HPs but not limonene HPs were shown to have MCAs (p-value <0.001, odds ratio (95% confidence interval) = 4.15 (3.01-5.73)). Patients with simultaneous contact allergies to both linalool and limonene HPs had contact allergies to many other screening and fragrance allergens., Conclusions: Simultaneous positive PT reactions to allergens in baseline series and fragrances are common in patients with the HPs contact allergy, especially linalool HPs. The pattern of simultaneous PT reactions principally suggested the co-sensitization of the cosmetic allergens., (© 2023 The Authors. Contact Dermatitis published by John Wiley & Sons Ltd.)

Women with premature ovarian insufficiency (POI) may be more vulnerable to a variety of health risks. To seek a new method to treat the disease, the effects of low-intensity pulsed ultrasound (LIPUS) on promoting repair of ovarian injury in female SD rats induced by 4-vinylcyclohexene diepoxide (VCD) were explored in this research. A total of 24 female SD rats were subjected to intraperitoneal injection of VCD to induce POI. Successful modeling was achieved in 22 rats, which were then randomized into VCD + LIPUS group (n = 13) and VCD group (n = 9). The control group (n = 5) was injected with equal normal saline. Hematoxylin and eosin staining, enzyme-linked immunosorbent assay, Western blot analysis, scanning electron microscope, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated nick end labeling assay were applied to detect the results. The results indicated that rats in the VCD group showed disorder in the estrous cycle, the number of atresia follicles and apoptosis granulosa cells increased (p < .05). After the LIPUS treatment, the estrous cycle recovered, the number of follicles increased (p < .05), the level of E2 and anti-Müllerian hormone enhanced (p < .05), and the follicle-stimulating hormone decreased (p < .05). The expression of NF-κB p65, TNFα, Bax, ATF4, and caspase-3 in ovarian tissue was significantly decreased (p < .05). These findings showed that LIPUS could promote the repair of the VCD-induced ovarian damage in SD rats, which has the potential to be further applied in the clinic., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Hyperthyroidism is associated with impairment in the neurotransmission and severe tissue damage in the brain. The present study explored the potential deleterious effects of experimentally-induced hyperthyroidism on the neurotransmitters, oxidative homeostasis, apoptosis and DNA fragmentation in cerebral cortex, thalamus & hypothalamus, and hippocampus in rats. Methods and Results: The ameliorative effects of N-acetylcysteine (NAC; 50 mg/kg, oral) and safranal (50 mg/kg, intraperitoneal) against hyperthyroidism (L-T4 500 µg/kg, subcutaneous) were investigated. All treatments continued daily over three weeks. Hyperthyroidism was manifested by significant elevations in serum fT3 and fT4 levels and a decline in serum TSH level and body weight. It was also characterized by significant elevations in the levels of dopamine, serotonin, and 5-hydroxyindole acetic acid, and monoamine oxidase activity to varying degrees in the brain regions examined and a significant reduction in norepinephrine in hippocampus only. Hyperthyroidism resulted in a significant oxidative stress in brain typified by elevations in malondialdehyde and nitric oxide content and reductions in glutathione level and SOD and catalase activities. This led to elevations in Caspases 9 and 3 and a reduction in Bcl2 resulting in DNA damage and confirmed by the histopathology of brain tissue. The administration of NAC or safranal with L-T4 prevented these deleterious effects by reducing the oxidative load and improving the brain antioxidant status. Conclusions: Hyperthyroidism disrupted the neurotransmitters in the brain which aggravated the oxidative stress and resulted in apoptosis. N-Acetylcysteine and safranal prevented these deleterious effects by enhancing the poor antioxidant milieu of the brain.

Background: Hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC) has been the most reported fragrance chemical for two decades and will be prohibited in cosmetic products from August 2021., Objectives: To describe the time trend of HICC contact allergy in European patients with dermatitis in 2009 to 2019, and the added value of testing HICC separately in the baseline series., Methods: Data were reviewed for 124 472 patients with dermatitis who were patch tested with HICC 5% pet. in the baseline series in the European Surveillance System on Contact Allergy (ESSCA) network (2009 to 2018) and at the Herlev-Gentofte Hospital Department of Dermatology and Allergy (2009 to 2019)., Results: Contact allergy to HICC was found in 1.98% of 9865 patients in Gentofte and 1.62% of 114 607 patients in the ESSCA network. Overall, the prevalence decreased annually, with 0.156 percentage points (P = .001) in Gentofte and 0.051 percentage points (P = .0002) in ESSCA. The frequency of missed contact allergy to HICC when testing only with fragrance mix II (FMII) was 0.17% (17/9865) and 0.35% (405/114607) of the whole test population in the Gentofte and ESSCA populations, respectively., Conclusions: This is the first study to demonstrate a significant decline in HICC allergy in European patients with dermatitis, most likely attributed to the upcoming European ban., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

TAC-302 stimulates neurite outgrowth activity and is expected to restore urinary function in patients with lower urinary tract dysfunction. We conducted 2 phase 1, randomized, placebo-controlled studies to confirm the safety and pharmacokinetics (PK) of TAC-302 in healthy adult Japanese male volunteers. In the first-in-human single-dose study (n = 60), TAC-302 was administered at doses from 100 to 1200 mg after an overnight fast. The effects of a meal on the PK of TAC-302 400 mg were also examined. A multiple-dose study (n = 36) evaluated the effects of meal fat content on the PK of single doses of TAC-302 (100, 200, or 400 mg) and multiple doses of TAC-302 administered for 5 days (100, 200, and 400 mg twice daily). TAC-302 showed linear PK up to doses of 1200 mg in the fasting state, and across the dose range of 100-400 mg in the fed state. No accumulation of TAC-302 was observed. Food, particularly with high fat content, increased TAC-302 plasma concentrations. No differences were observed in the adverse event incidence between the TAC-302 and placebo groups in either study. TAC-302 showed a wide safety margin., (© 2020 The Authors. Clinical Pharmacology in Drug Development published by Wiley Periodicals, Inc. on behalf of American College of Clinical Pharmacology.)

Background: Fragrance mix II (FM II) is included in the baseline patch test series recommended by the International Contact Dermatitis Research Group (ICDRG). Hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC) is the most important sensitizer of the 6 fragrance materials included in FM II. Besides being a part of FM II, HICC is also tested separately in the ICDRG baseline series., Objectives: The aim of the study was to investigate the prevalence of contact allergy to FM II and HICC in 2012-2016 with a focus on simultaneous reactions and the percentage of missed contact allergy to HICC provided that only FM II had been tested., Patients and Methods: A total of 25,019 consecutive dermatitis patients in 13 dermatology clinics representing 12 countries in 5 continents were patch tested with FM II and HICC in the baseline series., Results: Contact allergy to FM II and HICC was found in 3.9% and 1.6%, respectively. For FM II, the frequency varied from 1.5% to 7.6% in different centers. The corresponding range for HICC was 0.2% to 3.6%. Simultaneous contact allergy to FM II and HICC was noted in 1.4% with the range 0.2% to 2.6%. Seventy-seven patients (0.31%) with contact allergy to HICC did not test positively to FM II. The range for missed HICC allergy by testing only FM II in the different centers would be 0.04% to 0.74%. The ratio between the contact allergy rates for FM II and HICC was similar for all centers, except for Montreal having significantly more contact allergy to FM II than to HICC., Conclusions: The frequency of missed contact allergy to HICC when testing only with FM II was less than 0.5%, therefore questioning the need to test HICC separately in the ICDRG baseline series.

This article gives an overview of fragrance allergy. The following subjects are discussed: composition of perfumes, contact with fragrances, diagnosing fragrance allergy, frequency of allergy, clinical picture of allergic contact dermatitis, culprit products, occupational contact dermatitis, and other adverse effects of fragrances. For diagnosing fragrance sensitization, personal products and a fragrance series may need to be tested in addition to the baseline series. In the general adult population, up to 4.5% may be allergic to fragrance materials, and in consecutive patients patch tested for suspected contact dermatitis, the frequency may reach 20% to 25%. More than 150 fragrances have caused contact allergy. The most frequent sensitizers are linalool and limonene hydroperoxides, hydroxyisohexyl 3-cyclohexene carboxaldehyde, treemoss and oakmoss absolute, isoeugenol, cinnamyl alcohol, and cinnamal. Culprit products for induction of sensitization are often deodorants, fine fragrances, and aftershaves. Occupational contact dermatitis from fragrances is seen occasionally. Other adverse effects are all discussed but occur infrequently.

Sex and ovarian function contribute to hypertension susceptibility, however, the mechanisms are not well understood. Prior studies show that estrogens and neurogenic factors, including hypothalamic glutamatergic NMDA receptor plasticity, play significant roles in rodent hypertension. Here, we investigated the role of sex and ovarian failure on AMPA receptor plasticity in estrogen-sensitive paraventricular nucleus (PVN) neurons in naïve and angiotensin II (AngII) infused male and female mice and female mice at early and late stages of accelerated ovarian failure (AOF). High-resolution electron microscopy was used to assess the subcellular distribution of AMPA GluA1 in age-matched male and female estrogen receptor beta (ERβ) enhanced green fluorescent protein (EGFP) reporter mice as well as female ERβ-EGFP mice treated with 4-vinylcyclohexene diepoxide. In the absence of AngII, female mice at a late stage of AOF displayed higher levels of GluA1 on the plasma membrane, indicative of functional protein, in ERβ-expressing PVN dendrites when compared to male, naïve female and early stage AOF mice. Following slow-pressor AngII infusion, males, as well as early and late stage AOF females had elevated blood pressure. Significantly, only late stage-AOF female mice infused with AngII had an increase in GluA1 near the plasma membrane in dendrites of ERβ-expressing PVN neurons. In contrast, prior studies reported that plasmalemmal NMDA GluN1 increased in ERβ-expressing PVN dendrites in males and early, but not late stage AOF females. Together, these findings reveal that early and late stage AOF female mice display unique molecular signatures of long-lasting synaptic strength prior to, and following hypertension., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Fragrance chemicals constitute the second most frequent cause of contact allergy in Spain. There are no data available concerning the individual fragrances that are most frequently involved., Objectives: To describe the diagnostic contribution provided by specific fragrance series to the results obtained with baseline series fragrance markers by correlating the results of both series., Materials and Methods: We performed a 5-year retrospective study of fragrance marker-positive patients tested with specific fragrance series in 23 Spanish centres. We collected the demographic and clinical characteristics, and compared the results of patch tests obtained from different suppliers., Results: Of 19 588 patients patch tested with the Spanish baseline series, 1590 (8.1%) reacted positively to a fragrance marker. Of these, 1013 (63.7%) were patch tested with a fragrance series, and 664 patients reacted positively to at least one individual fragrance other than hydroxyisohexyl 3-cyclohexene carboxaldehyde. Geraniol was the most frequent allergen. Positive reactions to substances not included in fragrance mix (FM) I or FM II were found in 230 patients. Of the 436 FM I-positive patients and the 419 FM II-positive patients, 184 (42%) and 64 (39.1%), respectively, had no positive reactions to fragrance series. In the case of FM I, negative results were more common when individual fragrances were patch tested at low concentrations., Conclusions: We recommend patch testing all patients positive for any fragrance marker with a specific fragrance series. The correlation between the results of baseline series and fragrance series could be improved by increasing the concentrations of individual fragrances., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Di (2-ethylhexyl) phthalate (DEHP), a typical plasticizer used for polyvinyl chloride (PVC), is eluted from PVC-made blood containers and protects against red blood cell (RBC) hemolysis. However, concerns have arisen regarding the reproductive and developmental risks of DEHP in humans, and the use of alternative plasticizers for medical devices has been recommended worldwide. In this study, we propose that the use of a novel plasticizer, 4-cyclohexene-1,2-dicarboxylic acid dinonyl ester (DL9TH), could help produce more useful and safe blood containers. PVC sheet containing DL9TH and di (2-ethylhexyl) 4-cyclohexene-1,2-dicarboxylate (DOTH) provides comparable or superior protective effects to RBCs relative to PVC sheet containing DEHP or di-isononyl-cyclohexane-1,2-dicarboxylate (DINCH ® , an alternative plasticizer that has been used in PVC sheets for blood containers). The total amount of plasticizer eluted from DOTH/DL9TH-PVC sheets is nearly the same as that eluted from DEHP-PVC sheets. In addition, DOTH/DL9TH-PVC has better cold resistance than DEHP- and DINCH ® -PVC sheets. In vitro and in vivo tests for biological safety based on International Organization for Standardization guidelines (10993 series) suggest that the DOTH/DL9TH-PVC sheet can be used safely. Subchronic toxicity testing of DL9TH in male rats in accordance with the principles of Organisation for Economic Co-operation and Development Test Guideline 408 showed that DL9TH did not induce adverse effects up to the highest dose level tested (717 mg/kg body weight/day). There were no effects on testicular histopathology and sperm counts, and no indications of endocrine effects: testosterone, thyroid-stimulating hormone, follicle-stimulating hormone, and 17β-estradiol were unchanged by the treatment, compared with the control group. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1052-1063, 2018., (© 2017 Wiley Periodicals, Inc.)

Background: For cosmetics, it is mandatory to label 26 fragrance substances, including all constituents of fragrance mix I (FM I) and fragrance mix II (FM II). Earlier reports have not included oxidized R-limonene [hydroperoxides of R-limonene (Lim-OOH)] and oxidized linalool [hydroperoxides of linalool (Lin-OOH)], and breakdown testing of FM I and FM II has mainly been performed in selected, mix-positive patients., Objectives: To report the prevalence of sensitization to the 26 fragrances, and to assess concomitant reactivity to FM I and/or FM II., Methods: A cross-sectional study on consecutive dermatitis patients patch tested with the 26 fragrances and the European baseline series from 2010 to 2015 at a single university clinic was performed., Results: Of 6004 patients, 940 (15.7%, 95%CI: 14.7-16.6%) were fragrance-sensitized. Regarding the single fragrances, most patients were sensitized to Lin-OOH (3.9%), Evernia furfuracea (3.0%), Lim-OOH (2.5%), and hydroxyisohexyl 3-cyclohexene carboxaldehyde (2.1%). Significantly fewer patients were 'FM I-positive and constituent-positive' than 'FM II-positive and constituent-positive' (32.7% versus 57.0%, p < 0.0001). Additionally, significantly more patients were 'FM II-negative but constituent-positive' than 'FM I-negative but constituent-positive' (12.4% versus 3.2%, p = 0.0008)., Conclusions: Non-mix fragrances are the most important single fragrance allergens among consecutive patients. The test concentration of the single FM I constituents should be increased when possible., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Linalool and D-limonene are common fragrance ingredients that readily oxidize on exposure to air. The resulting hydroperoxides of linalool and D-limonene have been shown to have high frequencies of positive patch test reactions in several European and international studies., Objective: The aim of the study was to investigate the prevalence of contact allergy to the hydroperoxides of linalool and D-limonene in a US population., Methods: In this retrospective study, 103 patients with suspected fragrance allergy were patch tested to linalool 10% petrolatum (pet), hydroperoxides of linalool 1% pet, D-limonene 10% pet, and/or the hydroperoxides of D-limonene 0.3% pet between July 9, 2014, and October 25, 2016., Conclusions: In this study, the frequency of positive patch test reactions to the hydroperoxides of linalool is 20% (19/96), and the frequency of positive reactions to the hydroperoxides of D-limonene is 8% (7/90). These high frequencies suggest that patch testing to the hydroperoxides of linalool and limonene should be performed in all patients with suspected fragrance allergy.

Metastasis causes approximately 90% of breast cancer-related deaths in women. Previously, we have demonstrated that 2-dodecyl-6-methoxycyclohexa-2,5-diene- 1,4-dione (DMDD) remarkably inhibited the growth of human breast cancer cells with little toxicity. In this study, we investigated the toxicity and efficacy of DMDD to treat metastatic breast tumors using an in vivo mouse model of the 4T1 mammary carcinoma. DMDD caused no observable toxicity and significantly extended the survival of 4T1 tumor-bearing mice. DMDD effectively inhibited the growth of 4T1 cells in vitro, and suppressed the growth and metastasis of mammary tumor in vivo. The levels of inflammatory cytokines in the serum (TNF-α, IL-6, IL-12, TGF-β, and VEGF) were down regulated by DMDD. Immunohistochemical analysis demonstrated that the inhibition of tumor growth and metastasis was associated with activation of Bax, cleaved caspases-3 and -9, and down-regulation of Bcl-2, MMP-2 and -9, NF-κB and IκBα. We speculate that DMDD inhibits cytokine production in the tumor cells in mice, which leads to deactivation of NF-κB pathway, and consequently inhibits the expression of many anti-apoptosis and metastasis-promoting genes, such as Bcl-2 and MMPs. Collectively, our results demonstrate the potential of DMDD as a safe and effective antitumor agent in the treatment of late-stage breast cancer.

Background: Limonene and linalool are common fragrance terpenes widely used in cosmetic, household and hygiene products. Their primary oxidation products formed after air exposure, the hydroperoxides, have been recognized as important contact haptens., Objectives: To investigate the prevalence of contact allergy to hydroperoxides of limonene (Lim-OOHs) and hydroperoxides of linalool (Lin-OOHs) in Spain, and to define the optimal concentration for screening in consecutive patients., Methods: Three different concentrations of Lim-OOHs (0.1%, 0.2% and 0.3% pet.) and Lin-OOHs (0.25%, 0.5% and 1.0% pet.) were simultaneously tested in 3639 consecutive patients at 22 departments of dermatology in Spain., Results: Lim-OOHs at 0.1%, 0.2% and 0.3% yielded positive patch test reactions in 1.4%, 3.4% and 5.1% of the tested patients, respectively; and Lin-OOHs at 0.25%, 0.5% and 1.0% yielded positive reactions in 1.3%, 2.9% and 4.9% of the tested patients, respectively. Few irritant (1.5-1.9%) and doubtful reactions (0.4-0.5%) to both terpene hydroperoxides were registered at the highest concentrations tested., Conclusions: Lim-OOHs and Lin-OOHs can be considered as common causes of contact allergy, and their inclusion in an extended baseline patch test series therefore seems to be appropriate. The patch test preparations of Lim-OOHs 0.3% pet. and Lin-OOHs 1.0% pet. are useful tools for screening of contact sensitization., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

4-vinilcyclohexene diepoxide (4-VCD) causes premature ovarian failure and may result in estrogen deficiency, characterizing the transition to estropause in rodents (equivalent to menopause in women). Estropause/menopause is associated with metabolic derangements such as glucose intolerance and insulin resistance. Glucocorticoids (GCs) are known to exert diabetogenic effects. Thus, we aimed to investigate whether rats with premature ovarian failure are more prone to the diabetogenic effects of GC. For this, immature female rats received daily injections of 4-VCD [160mg/kg body weight (b.w.), intraperitoneally (i.p.)] for 15 consecutive days, whereas control rats received vehicle. After 168days of the completion of 4-VCD administration, rats were divided into 4 groups: CTL-received daily injections of saline (1mL/kg, b.w., i.p.) for 5days; DEX-received daily injections of dexamethasone (1mg/kg, b.w., i.p.) for 5days; VCD-treated as CTL group; VCD+DEX-treated as DEX group. Experiments and euthanasia occurred one day after the last dexamethasone injection. 4-VCD-treated rats exhibited ovary hypotrophy and reduced number of preantral follicles (p<0.05). Premature ovarian failure had no impact on the body weight gain or food intake, but both were reduced by the effects of dexamethasone. The increase in blood glucose, plasma insulin and triacylglycerol levels as well as the reduction in insulin sensitivity caused by dexamethasone treatment was not exacerbated in the VCD+DEX group of rats. Premature ovarian failure did change neither the hepatic content of glycogen and triacylglycerol nor the glycerol release from perigonadal adipose tissue. Glucose intolerance was observed in the VCD group after an ipGTT (p<0.05), but not after an oral glucose challenge. Glucose intolerance and compensatory pancreatic β-cell mass caused by GC were not modified by ovarian failure in the VCD+DEX group. We conclude that reduced ovarian function has no major implications on the diabetogenic effects promoted by GC treatment, indicating that other factors related to aging may make rats more vulnerable to GC side effects on glucose metabolism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: In 2014, the fragrance hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC) was excluded from the Swedish baseline series., Objectives: To study (i) whether fragrance mix (FM) II with 5% HICC detects more positive reactions than usual FM II with 2.5% HICC, and (ii) the reproducibility of patch testing with HICC., Methods: Two thousand one hundred and eighteen dermatitis patients at five Swedish dermatology departments were consecutively tested with FM II 14% pet., FM II 16.5% pet., and duplicate preparations of HICC 5% pet., Results: Of the patients, 3.2% reacted to FM II 14%, and 1.5% reacted to HICC. Separate testing with HICC detected 0.3% reactions without concomitant reactivity to FM II. FM II with 5% HICC did not give rise to more irritant reactions or signs of active sensitization than FM II with 2.5% HICC. Patch testing with duplicate applications of HICC increased the overall prevalence of HICC contact allergy to 1.9%., Conclusion: FM II with 5% HICC does not detect more positive reactions than FM II with 2.5% HICC. Separate testing with HICC does not detect a sufficient proportion of patients who react only to HICC, without concomitant reactions to FM II, to warrant its inclusion in a baseline series., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In this article, contact allergy to, and the chemical composition of, tea tree oil (TTO) are reviewed. This essential oil is a popular remedy for many skin diseases, and may be used as neat oil or be present in cosmetics, topical pharmaceuticals and household products. Of all essential oils, TTO has caused most (published) allergic reactions since the first cases were reported in 1991. In routine testing, prevalences of positive patch test reactions have ranged from 0.1% to 3.5%. Nearly 100 allergic patients have been described in case reports and case series. The major constituents of commercial TTO are terpinen-4-ol, γ-terpinene, 1,8-cineole, α-terpinene, α-terpineol, p-cymene, and α-pinene. Fresh TTO is a weak to moderate sensitizer, but oxidation increases its allergenic potency. The major sensitizers appear to be ascaridole, terpinolene, α-terpinene, 1,2,4-trihydroxymenthane, α-phellandrene, and limonene. The clinical picture of allergic contact dermatitis caused by TTO depends on the products used. Most reactions are caused by the application of pure oil; cosmetics are the culprits in a minority of cases. Patch testing may be performed with 5% oxidized TTO. Co-reactivity to turpentine oil is frequent, and there is an overrepresentation of reactions to fragrance mix I, Myroxylon pereirae, colophonium, and other essential oils., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Data on the chemistry of essential oils which have caused contact allergy are provided. The largest group of chemicals found in essential oils consists of terpenes. The number of identified components usually ranges from 100 to 250, but in some oils (lavender, geranium, rosemary) 450 to 500 chemicals have been found. Many chemicals are present in a large number of oils, up to 98% for β-caryophyllene and 97% for limonene. Chemicals that are important constituents of >20 oils are limonene, linalool, and α-pinene. In many essential oils, there are 2 to 5 components which together constitute over 50% to 60% of the oil. In some oils, however, there is one dominant ingredient, making up more than 50% of the oil, including (E)-anethole in aniseed and star anise oil, carvone in spearmint oil, 1,8-cineole (eucalyptol) in Eucalyptus globulus oil, and (E)-cinnamaldehyde in cassia oil. The most important chemicals in 93 individual oils are specified.

Background: 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., Objective: To investigate concomitant reactions between oxidized R-limonene and oxidized linalool in consecutive dermatitis patients., Methods: 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., Results: 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., Conclusions: 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., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Essential oils are fragrance substances that are labelled on cosmetic products by their INCI names, potentially confusing consumers., Objectives: To establish whether contact allergy to essential oils might be missed if not specifically tested for., Methods: 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., Results: 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., Conclusion: 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., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Preclinical Research Premature ovarian failure (POF) is defined by the WHO as the loss of physiological ovarian function before the age of 40. The effect of American ginseng and its underlying mechanisms in preventing and treating premature ovarian failure (POF) was studied in female Sprague-Dawley rats where POF was induced by ip administration of 4-vinylcyclohexene diepoxide (VCD). Rat behavior, serum hormone levels, ovarian and uterine size, pathological features, and ovarian tissue expression of genes associated with POF were assessed in controls, untreated POF model rats, and POF model rats treated with low- (1.125 g/kg), medium- (2.25 g/kg), and high-dose (4.5 g/kg) American ginseng. Compared with untreated POF model rats, those treated with medium- and high-dose American ginseng had more stable behavior and better coat appearance as well as serum hormone levels closer to those in control rats. Moreover, treatment with medium- or high-dose American ginseng increased ovarian and uterine size. Hematoxylin and eosin-staining revealed mature follicles and endometrium with an alternating concave/convex surface structure with visible capillaries and glands in ginseng- treated POF rats. PLA2G4A expression was positively correlated with POF, while the expression levels of PAPPA, STC2, CCL2, and NELL1 were negatively correlated with POF. Our study showed that American ginseng may effectively prevent POF and alleviate POF symptoms by regulating serum hormone levels and altering the expression levels of genes related to POF in ovarian tissue., (© 2014 Wiley Periodicals, Inc.)

Background: 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., Objective: To report 14 patients with occupational contact allergy to limonene., Methods: 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., Results: 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., Conclusions: 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., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: 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., Objective: 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., Methods: 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., Results: 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., Conclusions: 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., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The NMDAR glutamate receptor subtype mediates various vital physiological neuronal functions. However, its excessive activation contributes to neuronal damage in a large variety of acute and chronic neurological disorders. NMDAR antagonists thus represent promising therapeutic tools that can counteract NMDARs' overactivation. Channel blockers are of special interest since they are use-dependent, thus being more potent at continuously activated NMDARs, as may be the case in pathological conditions. Nevertheless, it has been established that NMDAR antagonists, such as MK801, also have unacceptable neurotoxic effects. Presently only Memantine is considered a safe NMDAR antagonist and is used clinically. It has recently been speculated that antagonists that preferentially target extrasynaptic NMDARs would be less toxic. We previously demonstrated that the phencyclidine derivative GK11 preferentially inhibits extrasynaptic NMDARs. We thus anticipated that this compound would be safer than other known NMDAR antagonists. In this study we used whole-genome profiling of the rat cingulate cortex, a brain area that is particularly sensitive to NMDAR antagonists, to compare the potential adverse effects of GK11 and MK801. Our results showed that in contrast to GK11, the transcriptional profile of MK801 is characterized by a significant upregulation of inflammatory and stress-response genes, consistent with its high neurotoxicity. In addition, behavioural and immunohistochemical analyses confirmed marked inflammatory reactions (including astrogliosis and microglial activation) in MK801-treated, but not GK11-treated rats. Interestingly, we also showed that GK11 elicited less inflammation and neuronal damage, even when compared to Memantine, which like GK11, preferentially inhibits extrasynaptic NMDAR. As a whole, our study suggests that GK11 may be a more attractive therapeutic alternative in the treatment of CNS disorders characterized by the overactivation of glutamate receptors.

Background: The common practice of preparing patch tests in advance has recently been called into question by researchers. It has been established that fragrance compounds are volatile and their testing efficacy may be affected by storage conditions and preparation. Allergens in fragrance mix I rapidly decrease in concentration after preapplication to test chambers., Objectives: This study aimed to investigate the volatility of hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC) in petrolatum when stored in test chambers and to explore the correlation between vapor pressure and allergen loss in petrolatum during preparation and storage., Methods: Standardized HICC in petrolatum was prepared and stored in IQ Chambers and Finn Chambers with covers at 5°C, 25°C, and 35°C, and concentration was analyzed at intervals for up to 9 days using gel permeation chromatography., Results: Changes in HICC concentrations were not statistically significant at 8 hours at 5°C, 25°C, and 35°C. After 9 days, HICC concentrations were found to fall approximately 30% when stored at 35°C, 10% at 25°C, and less than 5% at 5°C. There was no significant difference between IQ and Finn chambers., Conclusions: Hydroxyisohexyl 3-cyclohexene carboxaldehyde concentrations are more stable in petrolatum than many other studied fragrance allergens, but HICC is still at risk for decreasing concentration when exposed to ambient air or heat for prolonged periods.

Background: Hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC) is a synthetic fragrance chemical and an important contact allergen, at least in Europe. Despite this importance, little is known about risk factors associated with this allergen., Objective: To examine factors from the history and clinical presentation of patch tested patients associated with HICC sensitization., Methods: Contact allergy surveillance data of 95 637 patients collected by the Information Network of Departments of Dermatology (IVDK, www.ivkd.org) in 2002-2011 were analysed. Point and interval estimates of the relative risk were derived from multifactorial logistic regression modelling., Results: The overall prevalence of HICC sensitization was 2.24%. The strongest risk factors were polysensitization and dermatitis of the axillae, followed by dermatitis at other sites. No consistent and significant time trend was observed in this analysis. As compared with the youngest patients, the odds of HICC sensitization increased approximately three-fold in the 52-67-year age group, and strongly declined with further increasing age., Conclusions: The risk pattern with regard to age and affected anatomical site differed from that observed with other fragrance screening allergens. Cosmetic exposure, as broadly defined here, was a stronger and more prevalent individual risk factor than occupational exposure., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The frequency of fragrance contact allergy has shown a fluctuating trend over the years., Objectives: To describe the frequency of positive reactions to the baseline screening agents and fragrance mix (FM) 1 and 2 components, to determine trends of the latter over the years, and to evaluate simultaneous reactions., Patients and Methods: This was a cross-sectional study on patch test results of 13 332 patients from January 1990 to December 2011., Results: Of the total population, 9.6% reacted positively to FM 1, and 6% of 3416 tested with FM 2 reacted positively. Of those tested with both, 30.4% of 349 FM 1-positive patients reacted to FM 2, and 51.7% of 205 FM 2-positive patients reacted to FM 1. Hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC) and FM 2 were tested simultaneously in 3401 patients: 6 reacted to HICC alone. Nine hundred and forty patients were tested with FM 1 ingredients and 205 with FM 2 ingredients; Evernia prunastri was the most frequent FM 1 allergen, and HICC was the most frequent FM 2 allergen. Simultaneous reactions were frequently observed., Conclusions: Fragrance-allergic subjects often show multiple positive reactions, some of which are highly significantly associated. Recently, there has been a decreasing trend in positivity for both Evernia prunastri and HICC, whereas a slight increase for cinnamyl alcohol has been observed., (© 2013 John Wiley & Sons A/S.)

Background: 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., Objectives: 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., Methods: 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., Results: 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., Conclusions: 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., (© 2013 John Wiley & Sons A/S.)

The fate of dermally applied [(14)C]d-limonene was evaluated in humans and Long-Evans rats. In rats, 5 mg/kg body weight of [(14)C]d-limonene applied dermally to the shaved back under occlusion, resulted in the absorption of approximately 12% of the dose. The absorbed d-limonene was completely metabolized and excreted rapidly, primarily from the urine (80%) with a small fraction (20%) excreted in the feces. There was no long-term retention of the test material in body tissues. In humans, following dermal application of 12 mg of [(14)C]d-limonene in ethanol (1 mL) to the back under nonocclusive conditions (for 1 h after application to allow the material to dry, thereafter under occlusion), only 0.16% of the dose was absorbed and the radioactivity was recovered from the urine. Radioactivity in human feces was below the limit of detection. These results indicate that under conditions of simulated use of fragrances and cosmetics, d-limonene has a low potential for dermal absorption and tissue accumulation, and the d-limonene that is absorbed is rapidly excreted in the urine. Based upon these findings and the knowledge that d-limonene possesses a low-systemic toxicity profile, it is reasonable to conclude that dermal exposure to d-limonene from fragrance and cosmetic applications is highly unlikely to result in any clinically significant human toxicity.

Background: Fragrance ingredients are a frequent cause of allergic contact dermatitis. The EU Cosmetics Directive states that 26 specific fragrance ingredients, known to cause allergic contact dermatitis, must be declared on the ingredient lists of cosmetic products., Objectives: To investigate frequencies of sensitization to the 26 individual fragrances and evaluate their importance as screening markers of fragrance allergy., Method: This was a retrospective study based on data from the Department of Dermato-Allergology, Copenhagen University Hospital Gentofte. Eczema patients (n = 1508) were patch tested (January 2008 to July 2010) with the 26 fragrance ingredients., Results: Sensitization to the 26 fragrances was identified in 115 (7.6%) subjects. The most frequent allergens were Evernia furfuracea (n = 50), Evernia prunastri (n = 31), and hydroxyisohexyl 3-cyclohexene carboxaldehyde (n = 24). Including fragrance mix I, fragrance mix II and Myroxylon pereirae, 196 (13.0%) had a fragrance allergy. Testing with the 26 fragrances additionally identified 23 subjects who would otherwise have gone undetected. The majority (75.7%) of positive reactions to the 26 fragrances were of clinical relevance., Conclusions: Sensitization to the 26 individual fragrance ingredients was identified in 7.6% of the subjects patch tested. Most reactions were of clinical relevance. Fragrance-allergic subjects would be missed if testing with the individual fragrance ingredients was not performed., (© 2011 John Wiley & Sons A/S.)

Introduction: Hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC)-Lyral® is a widely used synthetic fragrance found in personal care and household products. It is an aldehyde, lipophilic enough to penetrate the skin and is a frequent cause of contact sensitization., Objective: Describe the frequency of contact allergy to HICC in a Contact Dermatitis Unit, after its inclusion in baseline patch test series., Methods: A retrospective study including all patients submitted to patch test, from January 2007 to December 2009., Results: Over a 3-year period, 629 consecutive patients were patch tested. The frequency of positive reactions to HICC was 2.7% (17/629). Of the positive patients, 35% (6/17) gave a history of atopy, 58.8% (10/17) had eczema on the face and neck, 23.5% (4/17) on the hands, 23.5% (4/17) in the axillae, 17.6% (3/17) on the trunk and 6% (3/17) had generalized eczema. All patients were patch positive for more than one allergen: all (17/17) positive to fragrance Mix 2 (FM2); 47% of the patients (8/17) positive to fragrance Mix 1 (FM1); and 23.5% of the patients (4/17) positive to Balsam of Peru. In 94% (16/17) of cases, the reaction was judged to be of current relevance., Discussion: The frequency of positive reactions to HICC of 2.7% found in our population is according to what is described in several European reports, where HICC is still widely used as a fragrance ingredient. In contrast, in North America, the prevalence is lower. All the patients were positive also to FM2. The association found between reactions to FM1 and HICC is also commonly reported and could represent a concomitant sensitization following increased exposure to fragrance allergens. These data confirm the importance of HICC introduction in the baseline patch test series.

Background: Fragrance mix II (FM II) is a relatively new screening marker for fragrance contact allergy. It was introduced in the patch test baseline series in Denmark in 2005 and contains six different fragrance chemicals commonly present in cosmetic products and which are known allergens., Aim: To investigate the diagnostic contribution of including FM II in the baseline series by comparing it with other screening markers of fragrance allergy: fragrance mix I (FM I), Myroxylon pereirae and hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC)., Method: Retrospective study of 12 302 patients consecutively patch tested with FM II by members of the Danish Contact Dermatitis Group 2005-2008., Results: FM II gave a positive patch test in 553 patients (4.5%), and in 72.2% of these patients the reaction was judged to be clinically relevant. FM II ranked second in detecting fragrance allergy, after FM I. If FM II had not been included as a screening marker in the baseline series, 15.6% (n = 202) of individuals with fragrance allergy would not have been identified by the other fragrance screening markers (FM I, M. pereirae or HICC)., Conclusion: FM II contributes substantially to detecting fragrance allergy. It ranked second among the fragrance screening markers tested in the baseline series and detects individuals with an allergy who otherwise would not have been identified., (© 2010 John Wiley & Sons A/S.)

Background: Fragrance chemicals are the second most frequent cause of contact allergy. The mandatory labelling of 26 fragrance chemicals when present in cosmetics has facilitated management of patients allergic to fragrances., Objectives: The study was aimed to define the characteristics of the population allergic to perfumes detected in our hospital district, to determine the usefulness of markers of fragrance allergy in the baseline GEIDAC series, and to describe the contribution made by the fragrance series to the data obtained with the baseline series., Material and Methods: We performed a 4-year retrospective study of patients tested with the Spanish baseline series and/or fragrance series. There are four fragrance markers in the baseline series: fragrance mix I (FM I), Myroxylon pereirae, fragrance mix II (FM II), and hydroxyisohexyl 3-cyclohexene carboxaldehyde., Results: A total of 1253 patients were patch tested, 117 (9.3%) of whom were positive to a fragrance marker. FM I and M. pereirae detected 92.5% of the cases of fragrance contact allergy. FM II and hydroxyisohexyl 3-cyclohexene carboxaldehyde detected 6 additional cases and provided further information in 8, enabling improved management. A fragrance series was tested in a selected group of 86 patients and positive results were obtained in 45.3%. Geraniol was the allergen most frequently found in the group of patients tested with the fragrance series., Conclusions: Classic markers detect the majority of cases of fragrance contact allergy. We recommend incorporating FM II in the Spanish baseline series, as in the European baseline series, and using a specific fragrance series to study patients allergic to a fragrance marker.

Allergic contact dermatitis due to citrus fruits is rare, but has been reported in cooks and bartenders. We report an interesting case of a bartender with hand dermatitis who had an allergic contact sensitivity to lime peel, fragrance mix I, and fragrance mix II. Most reported cases of citrus peel allergy are due to d-limonene, which makes up the majority of the peel oil. However, our patient had an allergic reaction to geraniol, which is a minor component of the peel oil and is present in fragrance mix I. It is important to consider a contact sensitivity to citrus in patients who have positive reactions to fragrance mix I and II and who are occupationally exposed to citrus fruits. An initial positive reaction to fragrance mixes should prompt further testing to citrus in these individuals.

Background: Previously, a quantitative risk assessment suggested there was no risk of induction of fragrance allergy from minor residues of fragrance chemicals on washed fabrics., Objective: To investigate whether there was any risk of the elicitation of contact allergy from fragrance chemical residues on fabric in individuals who were already sensitized., Methods: Thirty-six subjects with a positive patch test to isoeugenol (n = 19) or hydroxyisohexyl 3-cyclohexene carboxaldehyde (n = 17) were recruited. Dose-response and fabric patch tests were performed, respectively, with filter paper and a cotton sample loaded with fragrance in ethanol-diethylphthalate (DEP) and applied in a Finn Chamber or a Hill Top Chamber., Results: Only two subjects reacted to an isoeugenol patch test concentration of 0.01% (>20x the estimated likely skin exposure level), none reacted to lower concentrations. Of 36 subjects, 18 reacted to the fabric patch treated with ethanol-DEP vehicle alone and 20 to the fragrance-chemical-treated fabric patch. These were only minor non-specific skin reactions. They were also quite evenly distributed between the two fragrance chemical allergic groups., Conclusions: On the basis of the examples studied, fragrance chemical residues present on fabric do not appear to present a risk of the elicitation of immediate or delayed allergic skin reactions on individuals already sensitized.

Background: Hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC) is a synthetic fragrance ingredient. Case reports of allergy to HICC appeared in the 1980s, and HICC has recently been included in the European baseline series. Human elicitation dose-response studies performed with different allergens have shown a significant relationship between the patch-test threshold and the repeated open application test (ROAT) threshold, which mimics some real-life exposure situations. Fragrance ingredients are special as significant amounts of allergen may evaporate from the skin., Objectives: The study aimed to investigate the relationship between elicitation threshold doses at the patch test and the ROAT, using HICC as the allergen. The expected evaporation rate was calculated., Materials and Methods: Seventeen HICC-allergic persons were tested with a dilution series of HICC in a patch test and a ROAT (duration up to 21 days). Seventeen persons with no HICC allergy were included as control group for the ROAT. Results The response frequency to the ROAT (in microg HICC cm(-2) per application) was significantly higher than the response frequency to the patch test at one of the tested doses. Furthermore the response rate to the accumulated ROAT dose was significantly lower at half of the doses compared with the patch test. The evaporation rate of HICC was calculated to be 72% over a 24-h period., Conclusions: The ROAT threshold in dose per area per application is lower than the patch test threshold; furthermore the accumulated ROAT threshold is higher than the patch test threshold, which can probably be explained by the evaporation of HICC from the skin in the open test.