In Situ Metabolism of Cinnamyl Alcohol in Reconstructed Human Epidermis: New Insights into the Activation of This Fragrance Skin Sensitizer.

Chemical modification of epidermal proteins by skin sensitizers is the molecular event which initiates the induction of contact allergy. However, not all chemical skin allergens react directly as haptens with epidermal proteins but need either a chemical (prehaptens) or metabolic (prohaptens) activation step to become reactive. Cinnamyl alcohol has been considered a model prohapten, as this skin sensitizer has no intrinsic reactivity. Therefore, the prevailing theory is that cinnamyl alcohol is enzymatically oxidized into the protein-reactive cinnamaldehyde, which is the sensitizing agent. Knowing that reconstructed human epidermis (RHE) models have been demonstrated to be quite similar to the normal human epidermis in terms of metabolic enzymes, use of RHE may be useful to investigate the in situ metabolism/activation of cinnamyl alcohol, particularly when coupled with high-resolution magic angle spinning nuclear magnetic resonance. Incubation of carbon-13 substituted cinnamyl derivatives with RHE did not result in the formation of cinnamaldehyde. The metabolites formed suggest the formation of an epoxy-alcohol and an allylic sulfate as potential electrophiles. These data suggest that cinnamyl alcohol is inducing skin sensitization through a route independent of the one involving cinnamaldehyde and should therefore be considered as a skin sensitizer on its own.

Matrix Effect of Human Reconstructed Epidermis on the Chemoselectivity of a Skin Sensitizing α-Methylene-γ-Butyrolactone: Consequences for the Development of in Chemico Alternative Methods.

Adoption of new legislations and social pressure are pushing toward the development of alternative methods to the use of animals for the assessment of most toxicological end-points including skin sensitization. To that respect, much efforts have been put in the first step of the adverse outcome pathway focusing on chemical interactions taking place between sensitizing chemicals or haptens and epidermal proteins. However, these in chemico approaches have been so far only based on the use of model nucleophiles, amino acids, peptides, or proteins in water/buffer solution and focused mainly on thiol reactivity. These studies even if bringing a valuable set of information are very far from reflecting chemical interactions that may happen between a xenobiotic and nucleophiles present in a complex heterogeneous tissue such as the epidermis. Recently, we have shown that using a high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) technique it was possible to characterize chemical interactions taking place between a skin sensitizer and nucleophilic amino acids present in a 3-D reconstructed human epidermis (RHE). We have now compared the chemical reactivity and chemoselectivity of a sensitizing α-methylene-γ-butyrolactone toward human serum albumin used as a model protein and RHE. Using this technique, we showed that amino acid modifications by this hapten was different according to the model used and that in RHE histidine residues seem to have an important role in the formation of adducts. Obviously, the role of histidine in the induction of skin sensitization has been so far neglected and should probably be taken into account for the refinement of in chemico approaches for the detection and potency classification of skin sensitizers.

Risk assessment of dimethylfumarate residues in dwellings following contamination by treated furniture.

Recently, numerous cases of dermatitis induced by dimethylfumarate (DMFu) have been reported in Europe. DMFu has been used to prevent mold development in various items, although it is not registered as a biocide. In France, from October 2008 to December 2009, more than 100 cases were reported. Despite a ban on articles containing DMFu and the removal of potentially contaminated products, some people were still suffering from dermatitis or other health problems. The French Agency for Food, Environmental and Occupational Health & Safety was mandated to assess whether the existence in the past of DMFu-contaminated items in dwellings could continue to pose a threat to the health of inhabitants. A risk assessment was performed based on the classical risk analysis approach for environmental contaminants. Hazard assessment of DMFu with regard to its sensitizing properties was performed, based on human case reports collected in France between January 2009 and February 2010. For around half of the 132 individual cases reported, the causal link to DMFu was considered at least probable. An Organisation for Economic Co-Operation and Development (OECD) local lymph node assay performed in a study on mice showed strong sensitizing potential for DMFu. Exposure was assessed by measuring DMFu in items sampled in preselected dwellings. These investigations demonstrated that DMFu exposure can persist after removal of the primary contaminated items. We therefore concluded that there was clearly a risk of skin reactions in patients previously sensitized to DMFu. Furthermore, the available data do not support the existence of significant health effects through the respiratory route.

Allergic contact dermatitis to plants: understanding the chemistry will help our diagnostic approach.

Allergic contact dermatitis due to plants is common. Potentially allergenic plants and plant products are found in many everyday environments, such as the home, the garden, the workplace, and recreational settings. By improving our knowledge of allergenic plant-derived chemical compounds, we will be better positioned to identify novel allergens. We review the most relevant chemical allergens that contribute to plant allergic contact dermatitis and propose a clinical classification system based on 5 major families of chemical sensitizers: α-methylene-γ-butyrolactones, quinones, phenol derivatives, terpenes, and miscellaneous structures (disulfides, isothiocyanates, and polyacetylenic derivates). We also describe the different clinical pictures of plant allergic contact dermatitis and review currently available patch test materials. A better understanding of the specific allergens involved in plant allergic contact dermatitis will help to predict cross-reactivity between different plant species or families.

Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. Current knowledge assembled at an international workshop at BfR, Germany.

Contact allergies are complex diseases, and one of the important challenges for public health and immunology. The German 'Federal Institute for Risk Assessment' hosted an 'International Workshop on Contact Dermatitis'. The scope of the workshop was to discuss new discoveries and developments in the field of contact dermatitis. This included the epidemiology and molecular biology of contact allergy, as well as the development of new in vitro methods. Furthermore, it considered regulatory aspects aiming to reduce exposure to contact sensitisers. An estimated 15-20% of the general population suffers from contact allergy. Workplace exposure, age, sex, use of consumer products and genetic predispositions were identified as the most important risk factors. Research highlights included: advances in understanding of immune responses to contact sensitisers, the importance of autoxidation or enzyme-mediated oxidation for the activation of chemicals, the mechanisms through which hapten-protein conjugates are formed and the development of novel in vitro strategies for the identification of skin-sensitising chemicals. Dendritic cell cultures and structure-activity relationships are being developed to identify potential contact allergens. However, the local lymph node assay (LLNA) presently remains the validated method of choice for hazard identification and characterisation. At the workshop the use of the LLNA for regulatory purposes and for quantitative risk assessment was also discussed.

Deodorants: an experimental provocation study with hydroxycitronellal.

Axillary dermatitis is a common problem, particularly in individuals with contact allergy to fragrances. Many individuals suspect their deodorant to be the causal product of their fragrance allergy. It has been shown that deodorants containing cinnamic aldehyde (cinnamal) can elicit axillary dermatitis in patients sensitized to this substance. The aim of the present investigation was to evaluate the importance of hydroxycitronellal used in deodorants for the development of axillary dermatitis, when applied by individuals with and without contact allergy to this fragrance chemical. Patch tests with deodorants and ethanolic solutions containing hydroxycitronellal, as well as repeated open application tests (ROAT) with roll-on deodorants with and without hydroxycitronellal at different concentrations, were performed in 14 dermatitis patients, 7 with and 7 without contact allergy to hydroxycitronellal. A positive ROAT was noted only in the patients hypersensitive to hydroxycitronellal (P < 0.001) and only in the axilla to which the deodorants containing hydroxycitronellal had been applied (P < 0.001). Deodorants containing hydroxycitronellal in the concentration range of 0.032-0.32% used twice daily on healthy skin in individuals hypersensitive to hydroxycitronellal can elicit axillary dermatitis in a few weeks.

Routine patch testing with frullanolide mix: an European Environmental and Contact Dermatitis Research Group multicentre study.

Contact sensitivity to plants containing 1 or more sesquiterpene lactones (SLs) is difficult to diagnose. The mixture of SLs (SL mix) has been shown to detect only about 60% of sensitized individuals. In order to improve the diagnosis of sensitization to plants containing SLs, we have tested a mixture of frullanolides contained in Frullania dilatata and Frullania tamarisci at 3 different concentrations (0.01%, 0.033% and 0.1% in petrolatum). 8605 consecutive eczema patients in 1 North American and 15 European dermatology departments were tested with this mix, and 0.35% of positive cases to the different concentrations were found. Routine use of this mix permitted detection of only a small percentage of extra cases and did not improve the SL mix score. The frullanolide mix should therefore be restricted to investigations in particular geographical zones and/or in particular occupations.

Deodorants: an experimental provocation study with cinnamic aldehyde.

BACKGROUND: Axillary dermatitis is common and overrepresented in individuals with contact allergy to fragrances. Many individuals suspect their deodorants to be the incriminating products. OBJECTIVE: Our aim was to investigate the significance of cinnamic aldehyde in deodorants for the development of axillary dermatitis when used by individuals with and without contact allergy to cinnamic aldehyde. METHODS: Patch tests with deodorants and ethanol solutions with cinnamic aldehyde, and repeated open application tests with roll-on deodorants without and with cinnamic aldehyde at different concentrations, were performed in 37 patients with dermatitis, 20 without and 17 with contact allergy to cinnamic aldehyde. RESULTS: A repeated open application test with positive findings was noted only in patients hypersensitive to cinnamic aldehyde (P <.001) and only in the axilla to which the deodorants containing cinnamic aldehyde had been applied (P <.001). CONCLUSION: Deodorants containing cinnamic aldehyde in the concentration range 0.01% to 0.32%, used twice daily on healthy skin, can elicit axillary dermatitis within a few weeks.

Structure-activity relationships for selected fragrance allergens.

Fragrance substances represent a very diverse group of chemicals, a proportion of them providing not only desirable aroma characteristics, but also being associated with adverse effects, notably the ability to cause allergic reactions in the skin. However, efforts to find substitute materials are hampered by the need to undertake animal testing to evaluate both the presence and the degree of skin sensitization hazard. One potential route to avoid such testing is to understand the relationships between chemical structure and skin sensitization. In the present work we have evaluated two groups of fragrance chemicals, saturated aldehydes (aryl substituted and aliphatic aldehydes) and alpha,beta-unsaturated aldehydes. Data on their skin sensitization potency defined using the local lymph node assay has been evaluated in relation to their physicochemical properties. The initial outcome has been consistent with the concept that alpha,beta-unsaturated aldehydes react largely via Michael addition, whilst the group of saturated aldehydes form Schiff bases with proteins. Simple models of chemical reactivity based on these mechanisms suggest that it may be possible to predict allergenic potency. Accordingly, the evaluation of an additional group of similar aldehydes is now underway to assess the robustness of these models, with some emphasis being based on ensuring a wider spread of chemical reactivity.

Further important sensitizers in patients sensitive to fragrances.

The aim of this study was to determine the frequency of responses to selected fragrance materials in consecutive patients patch tested in 6 dermatological centres in Europe. 1855 patients were evaluated with the 8% fragrance mix (FM) and 14 other frequently used well-defined fragrance chemicals (series I). Each patient was classified regarding a history of adverse reactions to fragrances: certain, probable, questionable, none. Reactions to FM occurred in 11.3% of the subjects. The 6 substances with the highest reactivity following FM were Lyral (2.7%), citral (1.1%), farnesol P (0.5%), citronellol (0.4%), hexyl cinnamic aldehyde (0.3%), and coumarin (0.3%). 41 (2.2%) of the patients reacted only to materials of series I and not to FM. 6.6% of 1855 patients gave a history of adverse reactions to fragrances which was classified as certain. This group reacted to FM only in 41.1%, to series I and FM in 12.0% and to series I only in 7.2%. 74.3% of the 39 patients reacting to both FM and 1 of the materials of series I had any type of positive fragrance history, which was significantly higher in comparison to those with isolated reactions to series I (53.6% of 41), p = 0.04. The study identified further sensitizers relevant for patch testing of patients with contact dermatitis, of which Lyral is the most important single chemical.

Principles and methodology for identification of fragrance allergens in consumer products.

Fragrances contain several hundreds of different chemicals, a few major and many minor, which are responsible for the complexity of the odour. Fragrances are a major cause of allergic contact dermatitis. As a diagnostic tool, the current fragrance mix is very useful though not ideal. A 50-year-old woman presented with a pruriginous, erythematous eruption, characterized by papules, vesicles, exudation and crusting over the neck and chest. With the suspicion of fragrance allergy, patch testing was performed. Initially, the only positive reaction observed was with her own eau de toilette named Woman. The TRUE Test fragrance mix patch test was negative. Chemical fractionation of Woman perfume concentrate was combined with a sequenced patch testing procedure and with structure-activity relationship studies. Ingredients supplied by the manufacturer were also included in the study. Benzophenone-2, Lyral, alpha-hexyl cinnamic aldehyde and alpha-damascone were found to be responsible for the patient's contact allergy to the commercial product. These substances contain chemical structural alerts giving them antigenic ability. The common use of new chemicals to manufacture fragrances, and the increased number of patients sensitive to them but with negative fragrance mix reactions, makes it necessary to identify new potential fragrance sensitizers in commercial products.

Further important sensitizers in patients sensitive to fragrances.

In order to find sensitizers additional to the current fragrance mix (FM) a series of fragrance materials (series II) was evaluated in 6 dermatological centres in Europe. 11 of the test materials were essential oils, the remaining 7 being either mixtures of isomers or simple chemicals of frequent usage in the perfume industry. 1606 patients were consecutively tested with series II and 8% FM. Each patient was classified regarding a history of adverse reactions to scented products: certain, probable, questionable, none. Reactions to FM occurred most frequently in 11.4% of the subjects. The 6 materials with the highest reactivity after the FM were ylang-ylang oil (YY) I (2.6%), YY II (2.5%), lemongrass oil (1.6%), narcissus absolute (1.3%), jasmine absolute (1.2%) and sandalwood oil (0.9%). 48 (3.0%) of the patients reacted only to materials of series II and not to FM. 6.0% of 1606 patients gave a history of adverse reactions to fragrances which was classified as certain. This group reacted to FM only in 22.9%, to series II and FM in 15.6% and to series II only in 5.2%. 63.5% of the patients reacting to both FM and 1 of the materials of series II had some type of positive fragrance history, which was higher in comparison to those with isolated reactions to FM (46.2% of 121) or to series II, respectively, (45.8% of 48). However, this difference was not statistically significant. In conclusion, the materials of series II identified a further subset of patients with a fragrance problem, which would have been missed by the current FM as the single screening tool for patch testing.

Patch testing with serial dilutions of budesonide, its R and S diastereomers, and potentially cross-reacting substances.

BACKGROUND: Budesonide, a marker for corticosteroid allergy, is a 1:1 mixture of 2 diastereomers, the R and S, present in all commercial formulations. Budesonide is said to cross-react with group B substances through the R and S diastereomer and some group D substances only through the S diastereomer. OBJECTIVE: To investigate the cross-reactivity pattern between the R and S diastereomers and 4 potentially cross-reacting substances, 2 from group B and 2 from group D. METHODS: By patch testing 10 patients hypersensitive to budesonide with a serial dilution of budesonide, the R and S diastereomer, triamcinolone acetonide, amcinonide, prednicarbate, and hydrocortisone-17-butyrate. RESULTS: Nine of 10 patients reacted to budesonide and the S diastereomer. Seven of 9 to the R diastereomer. Each of the 9 patients with S diastereomer allergy reacted to the group B and/or group D substances. Five patients reacted to triamcinolone acetonide, not to 1.0% but only to 0.0010% and 0.00010%. CONCLUSION: The R and S diastereomers can induce positive patch test reactions in budesonide-hypersensitive individuals. The potential of budesonide to cross-react with substances from group B and D might be explained by the presence of the 2 diastereomers. When patch testing with triamcinolone acetonide, much lower concentrations than recommended should be used.

The time-dose-response relationship for elicitation of contact dermatitis in isoeugenol allergic individuals.

The elicitation response in allergic contact dermatitis is dose dependent, but the time-concentration relationship for elicitation has not previously been described. In this study 27 isoeugenol-sensitive patients participated in serial dilution patch tests with isoeugenol and a double-blinded Repeated Open Application Test (ROAT) using two concentrations of isoeugenol, 0.2 and 0.05%. Seven controls without isoeugenol allergy were also included. The participants applied 3.72 +/- 1.57 (mean +/- SD) mg/cm(2) of coded isoeugenol solutions twice a day to a 3 x 3 cm(2) area on the volar aspect of the right and left arm, respectively. For each test site the applications continued until a reaction appeared or for a maximum of 28 days. The minimal criteria for a positive reaction regarded as allergic contact dermatitis was persistent erythema at the ROAT test site. All controls were negative and 16/24 (66.7%) of the included isoeugenol-sensitive subjects showed a positive ROAT to the 0.2% solution within the study period (Fisher's test, p = 0.0024). Ten of the positive patients also reacted to the 0.05% solution. The median number of days until a positive reaction to the 0.2% solution was 7 days and was 15 days for the 0.05% solution. There was a highly significant correlation between the patients' patch test threshold and the number of days until a positive ROAT. In conclusion, the time until an isoeugenol allergic individual reacts in a ROAT depends on the individual sensitivity as well as the exposure concentrations; for low concentrations of the allergen or low degree of sensitivity, the allergic contact dermatitis may develop after several weeks of exposure. Therefore, a negative ROAT after 7 days may be a false negative.

Tixocortol pivalate contact allergy in the GPMT: frequency and cross-reactivity.

In spite of their intrinsic anti-inflammatory properties, corticosteroids can induce contact allergy. When studying the allergenic properties of corticosteroids it has to be considered that both the allergenic and anti-inflammatory effect may influence the induction phase as well as the elicitation phase and that such effects may be dose-dependent. A multiple dose guinea pig maximization test (GPMT) was therefore used to study the dose-response relationship of tixocortol pivalate. The GPMT was conducted according to OECD guideline #406, using a multiple-dose design and test results were analysed with logistic regression analysis. There was a significant tixocortol pivalate sensitization of the test animals compared to the control group (p<0.05), after both challenge and re-challenge. The challenge with 1% tixocortol pivalate gave more positive reactions than the challenge with 3%. The highest frequency of positive animals was observed when the animals were treated with low to intermediate induction concentrations and intermediate to high challenge concentrations with tixocortol pivalate in the TRUE Test. Cross-reactivity was found between tixocortol pivalate and hydrocortisone, which was expected from their close molecular resemblance, whereas no cross-reactivity was seen between tixocortol pivalate and the 3 other corticosteroids: amcinonide, budesonide, and hydrocortisone-17-butyrate.