Methylisothiazolinone contact allergy and dose-response relationships

Background. Methylisothiazolinone (MI) used alone is a new preservative causing a high prevalence of contact allergy. The eliciting threshold of MI is unknown. The combination of MI and phenoxyethanol enhances the antimicrobial efficacy of MI. Objectives. The eliciting doses of MI contact allergy in a patch test and a repeated open application test (ROAT) were investigated. In the patch test, it was determined whether phenoxyethanol influenced the reactivity to MI. Methods. Eleven MI‐allergic individuals were patch tested with two dilution series of 12 doses of MI and the same 12 doses with phenoxyethanol. The ROAT mimicked the use of a cream preserved with 100, 50 and 5 ppm MI (corresponding to 0.21, 0.105 and 0.0105 µg MI/cm²). Results. Phenoxyethanol had no influence on the reactions to MI. The lowest eliciting dose in the patch test was 1.47 µg MI/cm². In the ROAT, 7 patients (64%) reacted to 0.21 and 0.105 µg MI/cm² and 2 patients (18%) reacted to 0.0105 µg MI/cm², corresponding to a cream preserved with 5 ppm MI. Conclusions. A maximum of 100 ppm MI is permitted in cosmetic products. Eighteen per cent of MI‐allergic patients reacted to a concentration 20 times lower in a ROAT. The amounts used in cosmetics should be reduced, and the development of MI contact allergy should be monitored closely.     

Investigation of the threshold for allergic reactivity to chromium

Allergy to chromium is relatively common, often in association with exposure to cement or in leather manufacture. However, in certain locations, there appears to be a relatively large cohort of chromium-sensitive individuals whose allergy cannot be explained by these common sources. In particular, this group include Israeli housewives with persistent hand eczema and concomitant patch test positivity to chromium. The causation of their allergy has been linked with relatively high levels of chromium contamination in household products. To provide further information in respect of the definition of safe levels for such products, we examined 17 chromium-allergic individuals to determine their threshold for reaction under closed patch test and repeated open application test (ROAT) conditions. The data derived indicated that, on normal skin, the patch test threshold was 10 ppm chromium; in the presence of an irritant (sodium lauryl sulfate) the threshold was closer to 1 ppm, 2/17 subjects giving 1+ reactions at this concentration. In the more realistic exposure conditions of the ROAT, 8/14 individuals failed to react to 50 ppm, whilst 3/15 reacted to 5 ppm. Interestingly, there was very poor correlation between patch test sensitivity and ROAT sensitivity. To ensure the large majority of chromium-allergic individuals do not suffer elicitation of their allergy, as well as to limit the development of new chromium-sensitive subjects, it is recommended that household products adhere to a previously published standard of a maximum limit of 5 ppm, with an ultimate target of 1 ppm contamination by chromium.     

FS04.1Formaldehyde allergy – clinically relevant threshold reactions

The objective of the study was to establish eliciting threshold concentrations of diazolidinyl urea (Germall II)– derived formaldehyde in formaldehyde and/or in diazolidinyl urea – sensitive patients, using a leave‐on face cream formulation in a repeated open application test (ROAT) applied to different anatomical regions.
150 patients with known formaldehyde allergy were reviewed for inclusion in the study. 108 patients were contacted and in 65 patients the formaldehyde sensitisation was reconfirmed by a patch test. Four groups of 10 formaldehyde allergic subjects were exposed to 0.05%, 0.15%, 0.3% and 0.6% diazolidinyl urea, corresponding to approximately 50, 100, 200 and 400 ppm free formaldehyde, respectively.
Additional 10 individuals allergic to the formaldehyde donor – diazolidinyl urea itself – were exposed to 0.15% diazolidinylurea, corresponding to approximately 100 ppm free formaldehyde and 10 healthy non‐allergic individuals were exposed to 0.6% of diazolidinylurea (approximately 400 ppm free formaldehyde).
A ROAT was performed in a scheduled sequence: upper arm, neck and face.
Contact allergy reactions were elicited in 39 out of 58 formaldehyde‐sensitive and in 5 out of 7 diazolidinyl urea‐sensitive individuals.
Elicitation responses were dose‐ and anatomical region – dependent.
No reactions were observed at the lowest dose, suggesting that an elicitation threshold was attained in the study.     

The repeated open application test (ROAT)

Repeated open application tests (ROATs) were performed with common ingredients of vehicles in 86 patients with contact dermatitis. The substances were applied twice daily for 7 days to the flexor aspect of the forearm near the cubital fossa, unless dermatitis appeared earlier. Of the patients with a questionable (?+) patch test result, 44% were positive in ROATs. The corresponding figure was 80% in the patients with B positive (+ or ++) response in the patch lest, when the results of ROAT with propylene glycol were excluded. Only 5 of 14 patients reacting lo 30% or to 10% propylene glycol but not in 15 in water in patch testing, showed a positive result to a cream containing 5% propylene glycol in ROAT. All 5 patients with a positive patch test reaction to 1% propylene glycol reacted to 5% propylene glycol in ROAT. The results suggest that ROATs should be performed more often, especially in patients in whom little known or new allergens are suspected as being the cause of allergic contact dermatitis.     

Allergic contact dermatitis from formaldehyde

Formaldehyde is a common contact allergen. The prognosis of formaldehyde-sensitive patients is generally considered to be bad because of widespread exposure to formaldehyde. 11 patients with eczema and a positive patch test to formaldehyde were interviewed by a dermatologist and a toxicologist/chemist and instructed to fill in a questionnaire on exposure to chemical products. The content of formaldehyde and formaldehyde releasers in such products was examined using the database of the Danish Product Register (PROBAS) and by supplemental inquiries of manufacturers or importers. All the patients used one or more products containing formaldehyde or formaldehyde releasers. Sources of exposure were cosmetics and personal care products, dishwashing liquids, water-based paints, photographic products, etc. Patients were advised to use alternatives to those products containing formaldehyde or formaldehyde releasers. The status of 10 out of the 11 patients' eczema at follow-up was about 1/3 healed, 1/3 improved and in 1/3 no change. When the relevance of positive patch test reactions to formaldehyde was based on information obtained on exposure, a very high rate of current relevance was found. Computerized data on product composition allows the screening of products for contact allergens and also generates lists of contact allergens indicated for patch testing, based on the patients' own products.     

Rapid increase in contact allergy to Kathon® CG in Finland

In unselected eczema patients subjected to routine patch testing, the number with positive reactions to Kathon CG 100 ppm increased from none in 1983 to 0.7% in January-August 1985, and to 4.6% in September 1985 to March 1986. Repeated open application tests (ROAT) with creams containing 7-15 ppm of the isothiazolinones were positive in 12 of 24 patients tested. 2 of the ROAT-positive cases had negative patch tests to 100 ppm Kathon CG, but 1 was positive with 200 ppm. Atopic dermatitis, chronic hand dermatitis and lower leg dermatitis were the most common disorders in the positive patients. The cause of the rapid increase of Kathon CG allergy in Finland during the winter of 1985-1986 was the use of a popular moisturizing cream containing first 19 ppm, then 7 ppm of a mixture of 2 isothiazolinones (Euxyl K 100).     

Provocative use tests in CAPB-allergic subjects with CAPB-containing product

Cocamidopropyl betaine (CAPB) has been identified as a cause of contact allergy in personal care products. Furthermore, it has been suggested that chemicals responsible are impurities, especially dimethylaminopropylamine (DMAPA). However, skin contact concentrations with these impurities, especially DMAPA, are very low. The aim of the study was to analyse whether subjects with previous positive patch tests to CAPB would react in provocative use tests of a product containing CAPB. 10 individuals with a clinical history of contact allergy to CAPB (by positive patch test and history) took part in a ROAT which used a CAPB-based shower gel at 25% (DMAPA concentration < 1 ppm). None of the subjects showed positive allergic reactions. 1 of the test subjects did experience a flare of atopic dermatitis at the treatment site. Later, all 10 subjects were patch tested to 3 different concentrations of CAPB and DMAPA (0.1%, 0.3%, 1%) to verify the threshold that was capable of inducing a positive test reaction. 5/10 showed clear + reactions to 1% CAPB (typically at D3), whilst a further 3 gave marginal and/or irritant reactions. Only 1 of the subjects showed an allergic reaction to DMAPA. Finally, in uncontrolled use testing with the shower gel, none of the test subjects reported any adverse skin reactions. Thus, the study confirmed that CAPB-sensitive individuals can use a CAPB-based rinse-off product without the risk of experiencing an allergic reaction to CAPB.     

Minimum eliciting patch test concentrations of cobalt

In sensitized subjects, minimum eliciting levels of cobalt were estimated using patch tests with aqueous cobalt chloride on both normal skin and on skin pretreated in various ways to enhance penetration and reactivity. 6 reacted to 10,000 ppm and 1 gave an equivocal reaction to 1000 ppm aq. cobalt. Pretreatment of the patch test site for 24 h with surfactant enhanced reactivity, reducing the minimum eliciting concentration to 1000 ppm cobalt chloride in 1 subject, to 100 ppm in 2 subjects, and in 3 subjects to 10 ppm aqueous cobalt chloride. No reactions were obtained at 1 ppm. EDTA was effective in reducing the response to aqueous cobalt.     

Contact sensitization to 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (MCI/MI)

The frequency of positive reactions to 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (MCI/MI) were studied at 22 European contact dermatitis clinics over a period of 1 year. A total of 4713 patients participated. All the patients were patch tested with nickel sulphate, formaldehyde, paraben-mix, and MCI/MI. 19.4% of the patients had positive patch tests to nickel, making this the most common allergen. 3% of the patients reacted to 100 ppm MCI/MI, while 2.6% reacted to formaldehyde and 1.1% to parabens. There was great variation in the frequency of MCI/MI sensitivity among the 22 centres. MCI/MI contact allergy was most common among women and in patients with facial dermatitis, while it was rarely seen in patients with dermatitis on the lower legs. There were no fluctuations in the number of positive patch tests to MCI/MI on a monthly basis when the results from all centres were combined. 117 of the 141 MCI/MI sensitized patients included in the study were retested. 88% had positive patch tests when retested. 101 of the MCI/MI-sensitive patients participated in a double-blind, placebo-controlled product use test. This test showed that 31% of the MCI/MI-sensitive patients had a positive reaction to a MCI/MI-preserved product. Only a few patients reacted to a control product. It is concluded that the preservative MCI/MI is an important new contact allergen.     

Nickel allergy: relationship between patch test and repeated open application test thresholds

BACKGROUND: The frequency of nickel allergy varies between different population groups. Exposure regulation has proven effective in decreasing the frequency. Experimental studies with other allergens have shown a significant relation between patch test reactivity and repeated open application test (ROAT) reactivity. OBJECTIVES: This study was aimed at determining the elicitation threshold in nickel-allergic individuals in a patch test and a ROAT, and comparing the threshold from these two test methods. METHODS: Twenty nickel-allergic persons were tested with a dilution series of 19 concentrations in a patch test and a dilution series of three concentrations in a ROAT, with duration of up to 21 days. Eighteen persons with no nickel allergy were included as control group for the ROAT. RESULTS: The predicted dose which will elicit a reaction in 10% of allergic individuals was calculated to be 0.78 microg nickel cm(-2) in the patch test. The threshold for the ROAT (in microg nickel cm(-2) per application) was significantly lower than the threshold for the patch test, while the dose-response for the accumulated ROAT dose at 1 week, 2 weeks and 3 weeks was very similar to the patch test dose-response; indeed, there was no statistically significant difference. CONCLUSIONS: For elicitation of nickel allergy the elicitation threshold for the patch test is higher than the elicitation threshold (per application) for the ROAT, but is approximately the same as the accumulated elicitation threshold for the ROAT. This may be important for risk assessment based on dose-response results from allergic patients.     

Repeated open application test with methyldibromo glutaronitrile, a multicentre study within the EECDRG

Contact allergy to and allergic contact dermatitis from methyldibromo glutaronitrile (MDBGN) have frequently been reported. This study was initiated to help determine the optimal patch test preparation for MDBGN. In 51 patients with a doubtful or a positive patch test reaction to at least 1 of 4 test preparations with MDBGN in petrolatum at 1.0% w/w, 0.5%, 0.3% and 0.1%, a repeated open application test (ROAT) with moisturizers with and without MDBGN at 0.03% w/w was performed on the upper arms for 2 weeks. 18 of the 51 (35.3%) patients developed a positive ROAT. In all patients, there was a positive ROAT only to the moisturizer with MDBGN (P < 0.001). A statistically significant association was also found between the patch test reactivity (PTRL) and the outcome of the ROAT (P < 0.001). If only considering those with a PTRL above 0.3%, thus with negative or doubtful test reactions to 0.1% and 0.3%, there were still statistically significantly more patients with a positive ROAT to the moisturizer with MDBGN than to the moisturizer without MDBGN. The study demonstrates that patch testing with MDBGN at 0.3% and 0.1% will miss clinically relevant patch test reactions to MDBGN.     

Methyldibromoglutaronitrile allergy: relationship between patch test and repeated open application test thresholds

Background Methyldibromoglutaronitrile (MDBGN) is a preservative, which was approved for use in cosmetics in the mid‐1980s. The incidence of allergy to MDBGN rose during the 1990s, but is now decreasing due to regulatory intervention. Experimental studies with other allergens have shown a significant relationship between the patch test and the repeated open application test (ROAT) reactivity. Objectives To study the relationship between elicitation threshold doses at single occluded exposure and repeated open application, using MDBGN as the allergen. Methods Eighteen subjects allergic to MDBGN were tested with a dilution series of MDBGN in a patch test and a ROAT (duration up to 21 days). Seventeen people with no MDBGN allergy were included as a control group for the ROAT. Results The response frequency for the ROAT (in μg MDBGN cm^?2 per application) was significantly higher than the response frequency for the patch test, while the response frequency for the accumulated ROAT dose, at 1, 2 and 3 weeks was very similar to the patch test response frequency; indeed there was no statistical significant difference. Conclusions For elicitation of MDBGN allergy the response frequency for the patch test is lower than the response frequency (per application) for the ROAT, but approximately the same as the response frequency for the accumulated ROAT doses. This is important for risk assessment in general.     

Hydroxyisohexyl 3-cyclohexene carboxaldehyde allergy: relationship between patch test and repeated open application test thresholds

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 μg 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.     

Contact sensitization to methylisothiazolinone in Finland--a multicentre study

Background. Antimicrobials constitute the second most common cause of contact allergy to cosmetics. Methylisothiazolinone (MI), previously always used together with methylchloroisothiazolinone (MCI), has recently been approved in the EU for use on its own in cosmetics and also various industrial products. MCI has been classified as an extreme–strong and MI as a strong–moderate sensitizer. Objectives. To study the frequency of positive patch test reactions to MI, and its relevance and relation to MCI/MI sensitivity, in Finland. Methods. Over a period of 3 years (2006–2008), MI 0.1% (1000 ppm) and 0.03% (300 ppm) were patch tested in 10 821 patients at eight Finnish dermatological clinics. During 2008, patients with positive reactions to MI were asked to take part in a repeated open application test (ROAT). Results. Of the patients tested, 1.4% and 0.6% showed positive patch test reactions to 0.1% and 0.03% MI, respectively. Sixty‐six per cent of those who were MI‐positive were also positive to 100 ppm MCI/MI. Thirty‐three agreed to undergo the use test, and 10 of these gave positive results (30%). Conclusions. Our data show that MI used alone also potentially induces contact allergy. Careful monitoring is needed to determine whether or not this antimicrobial is safe to use in cosmetics.     

Quantitative patch and repeated open application testing in methyldibromo glutaronitrile-sensitive patients

Contact allergy to methyldibromo glutaronitrile (MDBGN), often combined with phenoxyethanol (PE) (e.g., Euxyl K 400), increased throughout the 1990s in Europe. Consequently, in 2003, the European Commission banned its use in leave-on products, where its use concentration was considered too high and the non-sensitizing use concentration as yet unknown. The 2 objectives of the study are (a) to find a maximum non-eliciting concentration in a leave-on product in MDBGN/PE-sensitized patients, which could possibly also be considered safe regarding induction and (b) to find the best patch test concentration for MDBGN. We, therefore, performed a use-related test (ROAT) in patients sensitized to MDBGN/PE (n = 39) with 3 concentrations of MDBGN/PE (50, 100 and 250 p.p.m. MDBGN, respectively). A subset of these patients (n = 24) was later patch-tested with various concentrations (0.1, 0.2, 0.3 and 0.5% MDBGN, respectively). 15 patients (38%, 95% confidence interval (CI) = 23-55%) had a negative and 24 (62%; 95% CI = 45-77%) a positive overall repeated open application test (ROAT) result. 13 reacted to the lowest (50 p.p.m.), 8 to the middle (100 p.p.m.) and 3 to the highest concentration (250 p.p.m.) only. In those 13 reacting to the lowest ROAT concentration, dermatitis developed within a few days (1-7). The strength of the initial and the confirmatory patch test result, respectively, and the outcome of the ROAT were positively associated. Of the 24 patients with a use and confirmatory patch test, 15 reacted to 0.1% MDBGN, 16 to 0.2%, 17 to 0.3% and 22 to 0.5%. With the patch test concentration of 0.5%, the number of ROAT-negative patients but patch-test-positive patients increases considerably, particularly due to + reactions. A maximum sensitivity of 94% (95% CI = 70-100%) is reached with a patch test concentration of 0.2%, and is not further improved by increasing the concentration. However, the specificity decreases dramatically from 88 (95% CI = 47-100%) with 0.2% to a mere 12.5% (95% CI = 0-53%) with 0.5%. It can be concluded (a) that for MDBGN 0.2% is very likely the best patch test concentration and (b) that 50 p.p.m. in a leave-on product can elicit contact dermatitis in sensitized persons. We were, therefore, unable to find a safe, still microbicidal, concentration for leave-on products. By contrast, with other contact allergens, dose-response use tests may be able to identify a non-eliciting concentration, which could give valuable clues to a non-inducing (i.e., safe) concentration in products.     

Patch test reactivity to DMDM hydantoins

The relationship between contact allergy to formaldehyde and positive patch test reactions to DMDM hydantoin was investigated. 35 formaldehyde-allergic patients were patch tested with serial dilutions of formaldehyde (0.1%-0.3%-1.0% aq.) and DM hydantoin (the non-formaldehyde-containing parent compound of DMDM hydantoin). 21 were also patch tested with MDM hydantoin (1 molecule formaldehyde) in serial dilutions: 7 (33%) reacted to 1 or more concentrations. The other 14 were also tested with DMDM hydantoin (2 molecules formaldehyde) in serial dilutions: 8 (57%) reacted to 1 or more concentrations. Patients patch-test-positive to formaldehyde 0.1% and/or 0.3% tended to show more patch test reactivity to (D)MDM hydantoin than those who reacted only to 1%. Aqueous solutions of (D)MDM hydantoin in concentrations as used in cosmetic products therefore contain enough free formaldehyde to cause dermatitis in a patch test system in some formaldehyde-allergic patients: 12 such patients applied a cream containing 1% DMDM hydantoin to the flexor aspect of the lower arm twice daily for 1 week; 4 (33%) developed dermatitis. The use of a cream containing 0.25% DMDM hydantoin in these 4 patients still caused dermatitis in 1 and provoked itching in another. An increase in the use of DMDM hydantoin in cosmetic products will also inevitable increase the risk of cosmetic dermatitis in consumers allergic to formaldehyde.     

Testing with fragrance mix

In a multicentre study, the value of adding sorbitan sesquioleate (SSO) to the constituents of the 8% fragrance mix (FM) was investigated. In 7 centres, 709 consecutive patients were tested with 2 types of FM from different sources, its 8 constituents with 1% SSO, its 8 constituents without SSO, and 20% SSO. 5 patients (0.71%) reacted to the emulsifier SSO itself, read as definitely allergic on day 3/4. 53 patients reacted to either one of the mixes with an allergic type of reaction. When tested with the constituents without SSO, 41.5% showed an allergic reaction versus 54.7% with SSO. If both types of reactions were considered (allergic and irritant) 38.3% of 73 patients showed a positive “breakdown” result without SSO, versus 54.8% with SSO. The differences were statistically significant. Reactivity to FM constituents was changed in a specific pattern by addition of SSO-irritant reactions increased, particularly for cinnamic alcohol, eugenol, geraniol, oak moss and hydroxycitronellal, whereas others showed only a slight change. Allergic reactions were also increased by SSO, but the rank order of the top 3 sensitizers (isoeugenol, oak moss and eugenol) did not change. Cinnamic alcohol was the only constituent with decreased reactivity after addition of SSO. A positive history of fragrance sensitivity (HFS) was clearly associated with a positive allergic reaction to either the mix or 1 of its constituents (51% versus 28.6% with a negative HFS). Irritant reactions were linked to a negative HFS in a high proportion (64.3%). In 17 patients, a repeated open application test (ROAT) was performed with a total of 43 patch-test-positive materials. The ROAT was positive in 20/31 (64.5%) tests in 11 patients with a positive HFS, but negative in all 6 patients with a negative HFS (0/12 tests). In conclusion, addition of SSO to the constituents of FM increases both irritant and allergic reactions, though the difference from the results obtained without SSO is not as high as previously reported. The ROAT is a valuable tool in validating such patch test results.     

Comparison between visual score and erythema index (DermaSpectrometer) in evaluation of allergic patch tests

Background/aims: During the last decade several new bioengineering methods have been proposed for evaluation of patch test reactions in a more objective manner. The aim of the present study was to investigate the usefulness of erythema index (DermaSpectrometer) in a clinical setting, i.e., reading of allergic patch tests. Methods: Twenty patients with known allergy to formaldehyde participated in the study. Each patient had patch tests for 2 days with formaldehyde solutions from 0 to 10,000 p.p.m. applied. Clinical reading of the test sites and measurement of the erythema index by the DermaSpectrometer were done 24 h after removal. A control group of 20 volunteers with no allergy to formaldehyde were tested in a similar way. Results: Erythema indices were significantly higher for visually rated positive patch tests than for negative tests (P<0.05). The single categories of visually positive reactions (+?, +, ++, +++) could not be unambiguously separated by the DermaSpectrometer. The correlation between clinical readings and the formaldehyde concentration (Spearman's rank correlation coefficient, r=0.60) was higher than between DermaSpectrometer readings and the formaldehyde concentration (r=0.35). Conclusion: In a dilution series of formaldehyde patch testing, readings from a DermaSpectrometer were not found to give better information than visual readings.     

Threshold responses in formaldehyde-sensitive subjects.

Patch tests with three low concentrations of formaldehyde were applied continuously in formaldehyde-sensitive subjects for 1 wk. The closed patch test method produced a response to 30 parts per million (ppm) in some subjects by 120 continuous hours of testing. Thirteen subjects then sprayed a 30-ppm formaldehyde solution in an axilla on a double-blind, controlled, 2-wk-use test. Their responses to 30 ppm aqueous formaldehyde indicate that levels below this concentration should be tolerated by sensitive subjects if repeatedly applied to normal skin. Popular formaldehyde-releasing preservatives can be above or below this threshold-eliciting response.     

Ethosome formulation of contact allergens may enhance patch test reactions in patients*

Background: Ethosomes and liposomes are ultra‐small vesicles capable of encapsulating drugs and cosmetic ingredients for topical use, thereby potentially increasing bioavailability and clinical efficacy. So far, few reports have suggested that formulation of cosmetic ingredients in vesicular carrier systems may increase the allergenicity potential. Objectives: To investigate the effect of ethosome formulation of isoeugenol and methyldibromo glutaronitrile on the elicitation response under patch test conditions and by repeated open applications. Patients/Materials/Methods: A total of 27 volunteer patients with a previous positive patch test reaction to either isoeugenol or methyldibromo glutaronitrile were included in the study. In all patients, a serial dilution patch test was performed with the allergen in question formulated in ethosomes and in an ethanol/water solution. In addition, a repeated open application test (ROAT) was performed in a subset of 16 patients, and lag time until a positive response was recorded. Results: Both contact allergens encapsulated in ethosomes showed significantly enhanced patch test reactions as compared with the allergen preparation in ethanol/water without ethosomes. No significant difference in the median lag time was recorded between preparations in the ROAT. Conclusions: Encapsulating potential contact allergens in ethosomes may increase the challenge response as compared with the same concentrations in an ethanol/water base without ethosomes.