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Fragrance chemical allergy: a major environmental and consumer health problem in europe

Deliverables

A method has been validated for the identification of sensitizers in complex natural mixtures based on the combination of bioassay-guided chemical fractionation, gas chromatography-mass spectrometry analysis and structure-activity relationship studies. This method has been developed using the model of oak moss absolute, a natural extract (from the lichen Evernia prunastri (L.) Arch.) used in the perfume industry, which is considered a major contact sensitizer and is included in the fragrance mix used for diagnosing perfume allergy. The first results show that atranol and chloroatranol, formed by transesterification and decarboxylation of the lichen depsides atranorin and chloroatranorin during the preparation of oak moss absolute, are strong elicitants in most patients sensitised to oak moss. Methyl-b-orcinol carboxylate, another despite degradation product and the most important monoaryl derivative of oak moss from an olfactive standpoint, was also found to elicit reactions in some patients although to a lower extent.
The study aimed to determine whether simple reduction in the use of isoeugenol, known as an important fragrance allergen, by industry has had an effect on the prevalence of allergy to this substance and whether allergen substitution with isoeugenol derivatives is possible or not. Reactivity to isoeugenol derivatives in isoeugenol allergic subjects: implications for allergen substitution: Isoeugenol is an important fragrance allergen for consumers. Industry has recommended a reduction in its levels in cosmetic products without guidance on how it should be substituted. We looked at reactivity to isoeugenol derivatives in isoeugenol allergic individuals. Such derivatives may be proposed as replacements for isoeugenol: allergen substitution. 2261 subjects attending contact dermatitis clinics were patch tested to isoeugenol and its derivatives. Concomitant contact allergy between isoeugenol and its derivatives may occur through chemical cross reactivity or local skin metabolism of the derivatives. The data suggest that isoeugenol ether derivatives may be a safer substitute than ester derivatives in isoeugenol allergic subjects. Isoeugenol acetate has been proposed as an alternative to isoeugenol but there is a high degree of concomitant reactivity with isoeugenol so such a strategy may be hazardous. The rate of isoeugenol allergy has not yet shown a consistent decline since the reduction in levels in cosmetic products. Knowledge gained could contribute to EU policy on fragrance allergen substitution and would be useful to regulators in industry.
Fragrance allergens giving rise to significant problems in the consumer have been identified in the FRAGRANCE ALLERGY project. Experimental studies in sensitised patients have shown that these allergens currently are used at unacceptable levels in consumer products causing significant disease in the consumer. This programme has provided data on no-effect levels that if considered in risk assessment will lead to safer environment and decreased disease. The results will be submitted to the EU-Commission for taking legislative action.
QSARs (quantitative structure-activity relationships) were developed for two different classes of aldehydes, Schiff base and a,b-unsaturated aldehydes. The QSARs were tested and explored for their domain applicability and scope. The QSAR developed for Schiff base aldehydes was explored to investigate its general applicability. Reasonable predictions were made for 1,2-diketones as well as other carbonyl groups such as esters. Knowledge derived from these QSARs could be incorporated into expert toxicity prediction systems e. g. DEREK (LHASA Ltd, UK) to improve existing rules for chemicals containing carbonyl groups. Knowledge derived could also be useful to risk assessors in Industry and Regulatory Authorities, particularly in their initial assessment of the potential of novel chemicals to behave as allergens.
The common fragrance chemicals linalool, b-caryophyllene and b-myrcene are all terpenes. Other terpenes have been shown to autoxidise at air exposure. We wanted to investigate if the actual compounds autoxidise in the same manner and if this affects their contact allergenic activity. Their oxidative degradation at air exposure was monitored as a decrease in the concentration of original terpenes over time. The concentrations of all terpenes started to decrease immediately. 50% of the original compounds remained after 30 weeks for linalool, and after 8 weeks for caryophyllene and myrcene. Different oxidation products were identified in the oxidised samples e. g. hydroperoxides, alcohols and epoxides. The effect of antioxidant on air oxidation was investigated by adding 0.2% BHT (butylated hydroxy toluene) before air exposure. The oxidation was prevented for linalool for one year and for myrcene for 15 weeks. Caryophyllene did not decompose for at least one year. The results indicate that the normal population is exposed to oxidation products of the actual fragrance chemicals in scented products such as cosmetics and domestic and occupational products. Experimental studies have shown that linalool and caryophyllene themselves are not allergenic while oxidised linalool and caryophyllene oxide cause sensitisation. These results indicate a risk of contact allergy in the population caused by the oxidised fragrance chemicals studied.
Development of a new fragrance mix for diagnostic patch testing has been developed. As the currently used fragrance mix (FM-I) does not identify all patients with a contact allergy to fragrances a new fragrance mix (FM-II) was evaluated as an improved diagnostic tool in patch testing. FM-II contains 6 fragrances of wide usage: 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene carboxaldehyde, citral, farnesol, citronellol, a-hexyl cinnamic aldehyde and coumarin. FM-II was patch tested in 3 concentrations (28%, 14%, 2.8% in petrolatum) on 1272 consecutive patients of 6 dermatological centres in Europe. Positive reactions to FM-II were found in decreasing frequencies in regard to test concentration: 4% to FM-II 28%, 2.8% to FM-II 14%, 1.3% to FM-II 2.8%. 34.8% of the patients reacting to FM-II were negative to FM-I which produced positive reactions in 6.1% of the total population. Testing of constituents revealed 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene carboxaldehyde, citral and farnesol to be the main sensitizers (4% to 1.6%). Patients positive to FM-II had a certain history of adverse reactions to scented products in 42.3%. The new FM-II identifies additional patients sensitive to fragrances, which would have been missed by using only the FM-I. The FM-II is a candidate for inclusion into the European standard series for patch testing and thus improves the diagnostic value of this procedure. The consumer/patient benefits from detailed knowledge by avoiding cosmetics containing the sensitizer(s).
The reactivity patterns of representative sensitising fragrance aldehydes were studied. Hydroxycitronellal and citral were chosen as the representative compounds for the families of aliphatic aldehydes and a,b-unsaturated aldehydes, respectively. Carbon 13 labelled haptens were used to study, at the molecular level and by nuclear magnetic resonance (NMR), the mechanisms of interaction between these haptens and macromolecules such as skin proteins. Hydroxycitronellal and citral were synthesised 13C labelled at their reactive sites and their interactions with model amino acids were studied by 13C-NMR. Only adducts of the Schiff base kind were identified.
A new method for the identification of sensitizers in complex mixtures has been developed. Chemical fractionation of oak moss and patch testing of the fractions in sensitised oak moss patients allowed the identification of the most eliciting fractions. Their chemical composition was analysed by gas chromatography-mass spectrometry and screened for the presence of molecules containing a hazard by using SARs analysis. The principle of SARs is that properties of a chemical with respect to how it will interact with a defined system are inherent in its molecular structure. It is possible, with chemical knowledge, to describe relationships between chemical structures and the ability to form conjugates with proteins, which is the first step for the induction and elicitation of contact allergy. This knowledge relating chemical structure to skin sensitisation, can be decomposed in a series of "structural alerts", essentially molecular substructures, which have previously been found to correlate with skin sensitisation capacity. Molecules identified in oak moss containing at least one structural alert were patch tested in the patients and major sensitizers were found. Results obtained by testing fractions were fully in agreement with those obtained by testing constituents of the fractions containing structural alerts. The method was therefore validated and could be extended to the analysis of essential oils.
Prior experimental and clinic studies have shown that the oxidation products of the fragrance chemical R-limonene, obtained at air exposure, are relevant and frequently observed contact allergens, while limonene itself is non allergenic. Samples of oxidised R- and S-limonene were diluted in petrolatum and tested in consecutive dermatitis patients in 6 European dermatological clinics to investigate the frequency of contact allergy in dermatitis patients in a large European population. We also wanted to address the question of relevancy of oxidised limonene allergy in fragrance contact allergy. In total 2411 patients were tested. 2.3% reacted to oxidised R-limonene, 2% reacted to oxidised S-limonene and 1.6% to both. The results obtained showed variations between the test centres. This is in accordance with the results observed for R-limonene as well as for 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene carboxaldehyde in earlier investigations. A significant correlation with contact allergy to other fragrance materials was observed. The results demonstrate that oxidised limonene is a common cause of contact allergy. It confirms the EU classification of limonene as a sensitizer by skin contact (R43) due to formation of allergens when air exposed, which is based on our previous experimentally obtained data.
Allergenicity of fragrance terpenes with respect to their degradation/oxidation over time was studied. The prevalence of contact sensitisation to the oxidative degradation products of monoterpenes frequently used as fragrance chemicals (limonene, linalool, caryophyllene, myrcene) in patients with suspected fragrance allergy and in patients with hand eczema due to contact with scented household products was investigated. The aim was also to identify the potential allergenic oxidation/degradation products. The fragrance chemicals linalool, caryophyllene and myrcene easily autoxidise when air exposed. Experimental studies have shown that these terpenes themselves are not allergenic. To test if autoxidation can cause a change in the allergenic activity, the oxidised terpenes were diluted in petrolatum and patch tested in consecutive dermatitis patients in 6 European dermatological clinics. The preparations tested were: oxidised linalool, a hydroperoxide fraction of linalool, oxidised caryophyllene, caryophyllene oxide and oxidised myrcene. Positive reactions were observed in 1.8% of in total 1343 patients tested. Most reactions were seen to oxidised linalool (1.2%) and its hydroperoxide fraction (1%). This could, at least partly, be explained by a greater usage of linalool (lavender oil) compared to the usage of the other fragrance chemicals studied. The reaction pattern and the frequency of positive results differed between the different clinics. A significant correlation with contact allergy to other fragrance materials was observed. Results showed that the observations from the limonene study also apply to other fragrance terpenes. It further implies that the EU regulation regarding limonene (classified as a sensitizer by skin contact R43- due to formation of allergens when air exposed) can be applicable on the chemicals studied, especially on linalool.
This study investigates whether the exposure from fragrance chemicals included in many household products is a risk factor for the elicitation and maintenance of chronic hand eczema in patients with fragrance allergy. The 10 most important fragrance chemicals in household products relevant to hand eczema have been identified. In the context of secondary prevention, the specific scientific aim was to establish measures aiming to avoid the elicitation of contact allergy in already sensitised patients, by the standardisation of diagnostic methods, the determination of threshold concentrations and the evaluation of cross-reactivity and of allergenic fragrance chemical substitution. Hand exposure to fragrance allergens was assessed by chemical analysis of domestic and occupational products. The product contents of fragrance allergens were different from those of cosmetic products. The current diagnostic test is based on fragrance allergens, which frequently occur in cosmetic products. Thus this method may not identify cases with allergic hand eczema from fragrance allergens present in domestic and occupational products. Based on this exposure assessment a diagnostic screening series was established and tested on a large number of patients with hand eczema. It was shown that 10% of the patients gave a positive test to one or more of the allergens in the new series, less that half of these were picked up by the current diagnostic test for fragrance allergy. It is suggested to test patients with hand eczema with the supplementary fragrance allergen series. The results from this study add important steps to primary prevention by identifying exposures relevant to hand eczema and which should be considered for risk assessment. Improved diagnostic tools are important for secondary prevention. The results are disseminated by scientific publications and lectures and will in this way be implemented in clinical practice.

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