Repeated Open Application Test Research Articles

A Dermatological Safety Test of a Face Serum Formulation Derived from Honey and Propolis of Stingless Bee from East Kalimantan

Skincare, which is a type of cosmetics, is an essential component in the process of maintaining and caring for the skin. It is known that the skin quickly absorbs the tiny molecular structure of serum. Stingless bees, widespread in East Kalimantan and Indonesia, benefit the skin's health by producing honey and propolis. This research aims to demonstrate that a face serum derived from honey and propolis of stingless bees from East Kalimantan is safe for use on the skin using dermatological standards. Here we will see whether there are allergic reactions and irritation from using facial serum. Using the Repeated Open Application Test (ROPT) technique, 20 volunteers were subjected to applying a face serum containing honey and stingless bee propolis. The serum was given to the upper arm twice daily for seven days. As part of the test, allergic reactions were evaluated using the system developed by the International Contact Dermatitis Research Group (ICDRG), and irritating reactions were evaluated using the Primary Irritation Index (PII). The results showed that on the first day, four of the volunteers reported moderate irritation reactions. Consequently, they scored 0.7, indicating they suffered minor irritation. On the other hand, the reactions of the remaining 16 volunteers were typical. Twenty participants had normal skin from the second treatment until the seventh day. The overall findings show that the facial serum based on honey and kelulut propolis from stingless bees from East Kalimantan showed a mild irritation reaction at the beginning of use and was safe to use in subsequent uses.

Allergic Contact Dermatitis to Linalool Hydroperoxides: Pitfalls in the Diagnostic Process-Findings from a Repeated Open Application Test Study.

Background: Increasing trends of oxidized linalool contact allergy have been reported. However, the impact of reactivity and dose in eliciting allergic contact dermatitis caused by linalool hydroperoxides is insufficiently investigated. Objectives: To perform repeated open application tests (ROATs) using the real-world concentrations of linalool hydroperoxides in patients and control participants. Materials and Methods: Patients who previously had a positive (patients) and a negative (controls) patch test reaction to linalool hydroperoxides 1.0% in petrolatum were patch tested with a dilution series of linalool hydroperoxides preparations and asked to perform ROAT twice daily with 3 concentrations of linalool hydroperoxides creams and a negative control cream for 28 days. The creams contain 44, 140, and 440 PPM of linalool hydroperoxides, representing real-world doses reported in consumer products. Results: Of all 47 participants, 31 were linalool hydroperoxides contact allergy patients, and 16 were controls. One patient had a positive ROAT reaction in the area where cream at the highest concentration of linalool hydroperoxides was applied for 28 days. Conclusions: Repeated exposure to creams containing linalool hydroperoxides at real-life concentrations could rarely elicit an allergic reaction on intact skin after 4 weeks.

In Situ Patch Test and Repeated Open Application Test for Fixed Drug Eruption: A Multicenter Study

Few studies have evaluated allergy workup in fixed drug eruption (FDE) in a large population. To evaluate the sensitivity of a standardized allergy workup for diagnosing the cause of FDE, with a focus on in situ repeated open application tests (ROATs). In a retrospective multicenter study, we analyzed the practice of conducting a complete allergy workup for the etiological diagnosis of FDE. It consisted of 3 steps: in situ patch tests (PTs) for all cases except pure mucosal involvement, followed by in situ ROAT if in situ PT results were negative, and finally a drug challenge (DC). The in situ ROAT involved daily application of the suspected drug on a previously affected FDE site for 7 days. Of 98 suspected FDE cases, 61 patients (median age 61 y; male-to-female ratio 1.8) with a complete allergy workup were included. In 4 cases, even the DC yielded negative results. Among the remaining 57 patients with a positive workup, implicated drugs included paracetamol (12 cases), β-lactams (11 cases), imidazoles (9 cases, including 5 with metronidazole), nonsteroidal anti-inflammatory drugs (8 cases), iodinated contrast media (4 cases), cotrimoxazole (3 cases), and various other drugs in 10 patients. The diagnosis was confirmed by in situ PT in 17 of 54 cases (31.5%), in situ ROAT in 14 of 40 cases (35%) (with 4 cases showing remote reactivation of FDE sites), and DC in 26 cases. The sequential allergy workup involving successively in situ PT, in situ ROAT, and DC is a reliable and safe method for diagnosing the cause of FDE. In situ tests exhibited a sensitivity of over 50%.

Allergic Contact Dermatitis From 100% Mineral Oil Supported by Repeated Open Application Test.

Allergic Contact Dermatitis From 100% Mineral Oil Supported by Repeated Open Application Test.

It is not always chlorhexidine: Identification of benzoxonium chloride and lauramine oxide as culprit allergens in a popular antiseptic in Switzerland.

A popular antiseptic spray in Switzerland (Merfen spray), containing chlorhexidine digluconate, benzoxonium chloride and lauramine oxide, is frequently used to treat skin wounds. However, it is also increasingly reported as a major cause of adverse skin reactions, including allergic contact dermatitis (ACD). To investigate the contact allergens responsible for ACD from this antiseptic. Patch tests were performed on seven patients with a clinical history compatible with contact dermatitis from this antiseptic mixture. All patients presented with acute eczematous reactions following contact with either Merfen spray alone, or with multiple products including this spray. Patients showed positive reactions to this product in both patch tests and repeated open application tests (ROATs). Four patients showed dose-dependent reactions to both benzoxonium chloride and lauramine oxide. One patient showed a dose-dependent reaction to the former and a non-dose-dependent reaction to the latter. Finally, two subjects showed responses only to lauramine oxide. One patient reacted to chlorhexidine digluconate 0.5% aq. in addition to both other allergens. Two commercially unavailable allergens, that is, benzoxonium chloride and/or lauramine oxide were identified as major causes of ACD from Merfen antiseptic spray, whereas chlorhexidine digluconate was a contributing culprit in only one patient.

Allergic reactivity for different dilutions of eugenol in repeated open application test and patch testing.

Eugenol is a known contact sensitiser included in fragrance mix I. To assess the allergic reactivity to eugenol in different concentrations using patch test as well as repeated open application test (ROAT). Overall 67 subjects from 6 European dermatology clinics participated in the study. The ROAT was performed for 21 days twice a day, applying 3 dilutions of eugenol (2.7%-0.5%) and a control. Before and after the ROAT, patch testing with 17 dilutions of eugenol (2.0%-0.00006%) and controls was performed. Out of the 34 subjects with contact allergy to eugenol, 21 (61.8%) showed a positive patch test before ROAT was performed, the lowest positive concentration was 0.031%. The ROAT was positive in 19 (55.9%) of the 34 subjects, the time until a positive reaction occurred was negatively associated with the concentration of the ROAT solution, as well as with the allergic reactivity of the subjects as defined by patch testing. In the patch test after ROAT, 20 of the 34 test subjects (58.8%) showed a positive reaction. In 13 (38.2%) of the 34 test subjects, the patch test result was not reproduceable, still 4 (31.0%) of these 13 subjects developed a positive ROAT. Eugenol can provoke a positive patch test reaction in a very low dose; besides, this hypersensitivity may persist even if a former positive patch test is not reproduceable.

Teaching Patients to Teach Themselves (Not by Rote but) via Repeated Open Application Test.

Dermatitis®Vol. 34, No. 2 Pearls & ZebrasTeaching Patients to Teach Themselves (Not by Rote but) via Repeated Open Application TestMalgorzata K. Nowakowska, Tiffany Sierro, and Ponciano D. Cruz Jr.Malgorzata K. NowakowskaAddress reprint requests to Malgorzata K. Nowakowska, BS, School of Medicine, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030-3411, USA, E-mail Address: mknowako@bcm.eduhttps://orcid.org/0000-0001-5756-1878From the *School of Medicine, Baylor College of Medicine, Houston, Texas, USA.Search for more papers by this author, Tiffany SierroFrom the *School of Medicine, Baylor College of Medicine, Houston, Texas, USA.Search for more papers by this author, and Ponciano D. Cruz Jr.From the *School of Medicine, Baylor College of Medicine, Houston, Texas, USA.Search for more papers by this authorPublished Online:16 Mar 2023https://doi.org/10.1089/derm.2022.0001AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Teaching Patients to Teach Themselves (Not by Rote but) via Repeated Open Application Test." Dermatitis®, 34(2), pp. 154–155FiguresReferencesRelatedDetails Volume 34Issue 2Apr 2023 Information© 2023 American Contact Dermatitis Society. All Rights Reserved.To cite this article:Malgorzata K. Nowakowska, Tiffany Sierro, and Ponciano D. Cruz Jr..Teaching Patients to Teach Themselves (Not by Rote but) via Repeated Open Application Test.Dermatitis®.Apr 2023.154-155.http://doi.org/10.1089/derm.2022.0001Published in Volume: 34 Issue 2: March 16, 2023Online Ahead of Print:January 25, 2023PDF download

What is the optimal duration for a ROAT? The experience of the French Dermatology and Allergology group (DAG).

The repeated open application test (ROAT) is an adjuvant investigation measure to patch testing in the diagnosis of allergic contact dermatitis. ESCD recommends a 15 days duration but its overall duration varies according to publications and patients hardly adhere to prolonged ROAT duration beyond 1 week. The Dermatology and Allergy Group of the French Society of Dermatology performed a prospective study with the aim of determining the best duration for the ROAT. A total of 328 ROAT results were collected for topical products, including cosmetics (60%) and topical medications (31.1%). Fifty-nine (18%) ROATs were positive, and 16 (5%) were doubtful. All the positive ROATs occurred within 10 days, with a median time to positivity of 3 days. According to our results, a minimum duration of 10 days is necessary to achieve a positive ROAT to a topical product.

Allergic contact dermatitis to phenoxyethanol: A rare, but possible cause of hand dermatitis.

Phenoxyethanol (PE; Syn. 2-Phenyoxethanol; CAS no. 122-99-6) is one of the most commonly used preservatives in cosmetics and is also found in industrial products, including metalworking fluids. It is considered to have a low sensitization potential.1-3 A 59-year-old male toolmaker presented with therapy-resistant, 1-year lasting hand dermatitis suspected to be of occupational origin. Despite being on work leave and treatment with alitretinoin for the past 5 months, severe dermatitis of hands and lower arms was observed (Figure 1). We performed patch tests and readings according to the guidelines of the German Contact Dermatitis Research Group (DKG)4 using the DKG baseline series; the DKG series for topical preparations, preservatives, rubber, disinfectants, acrylates, industrial biocides, and metalworking fluids; and the patient's own metalworking fluid. Patch tests were removed at day (D) 2 with the exception of the metalworking fluid, which was removed already at D1 and reactions were evaluated at D2, D3, D4, and D7. Solely, a 2+ reaction to PE 1% pet. (SmartPractice, Greven, Germany) from D2 to D7 was noted (Figure 2A). PE was not found in any of the available safety datasheets of the patient's workplace products, including the metalworking fluid. However, according to the product labels, it was an ingredient of the patient's skin care products “Eucerin UreaRepair PLUS 5% Hand Creme” and “Nivea 3IN1 Repair Hand Creme” (both from Beiersdorf AG, Hamburg, Germany), which he had regularly applied on his hands and lower arms after the onset of dermatitis. Repeated open application test (ROAT) with “Eucerin UreaRepair PLUS 5% Hand Creme,” two times daily for 6 days on the inner sides of the patient's upper arm resulted in a positive reaction (Figure 2B). Dermatitis of hands and lower arms improved significantly after avoidance of these skin care products, indicating clinical relevance of the sensitization to PE as the contributing factor to the initially occupational irritant contact dermatitis. Despite its widespread use as preservative in cosmetic and pharmaceutical products as well as for other applications, only very few cases of allergic contact dermatitis and contact urticaria to PE have been reported.2, 5-7 Accordingly, a retrospective study of the Information Network of Departments of Dermatology (IVDK) revealed a very low sensitization rate of 0.24% among 6932 individuals patch tested in Germany, Austria, and Switzerland between 1996 and 2009.1 Similarly, in 681 cutting metalworkers patch tested with the DKG metalworking fluids series in IVDK centres from 2010 to 2018, sensitization to PE was rarely observed (0.3%).8 Although rare, allergic contact dermatitis to PE should be considered and if undiscovered may lead to longstanding disease and inefficient treatments. The present case illustrates the importance of performing early patch tests to avoid delays in diagnosis and prevention measures. Diagnostic workup of patients with suspected occupational hand dermatitis should include patch testing and in doubtful cases also ROAT with patient's own skin care products. The authors declare no conflicts of interest. Michaela Kolodziej: Conceptualization (equal); investigation (equal); writing – original draft (equal); writing – review and editing (equal). Alexander Kiewert: Investigation (supporting); writing – review and editing (equal). Christoph Skudlik: Investigation (supporting); writing – review and editing (equal). Richard Brans: Conceptualization (equal); investigation (equal); supervision (lead); writing – original draft (equal); writing – review and editing (equal).

Does aluminium in sunscreens cause dermatitis in children with aluminium contact allergy: A repeated open application test study

Parents report that children with aluminium contact allergy and vaccination granulomas may react to aluminium-containing sunscreen following application. To evaluate whether contact dermatitis develops following repeated application of aluminium-containing sunscreens in children with aluminium sensitization and vaccination granulomas. Sixteen children aged 2-9 years (mean age 5 years) with vaccination granulomas and a positive patch test reaction to aluminium chloride hexahydrate 2%/10% petrolatum completed a blinded repeated open application test (ROAT) with two daily applications of two sunscreens for 14 days. One cream contained aluminium and the other did not. The children served as their own controls. Sixteen children completed the study. Only one child (6%) had a positive skin reaction during ROAT on day 2 to the sunscreen with aluminium. None reacted to the sunscreen without aluminium. Use of aluminium-containing sunscreens may on a case basis lead to allergic contact dermatitis in aluminium allergic children.

Eczéma palpébral à la norfloxacine en collyre chez un enfant : intérêt du ROAT

Eyelid contact dermatitis to antibiotic eye drops is increasingly reported, but the fluoroquinolone is exceptionally involved. Patch tests are used to confirm the nature of the allergen, however, false-negative results are increasingly found. Alternative tests such as ROAT (Repeated Open Application Test) may be useful. We report the case of a 6 year old child with norfloxacin-induced palpebral eczema with negative patch tests, where the ROAT was of great interest.

ETIOLOGY OF CONTACT DERMATITIS ON FACE IN INDIAN PATIENTS

Introduction: Facial contact dermatitis has exponentially increased in the past decade because of the freely available over the counter topical medications and the innumerable cosmetics. Patch testing is still the gold standard for diagnosis. Aims and Objectives: To elicit the patterns and specic allergens causing facial dermatitis on face in Indian patients. 50 patients of predominantly facial dermatitis with Materials and methods: suspected contact dermatitis (CD) were taken up for patch testing. The patients were tested with the Indian standard series (ISS) and Indian cosmetic series and the results were read at 48 and 96 hours. Repeated open application test (ROAT) was performed with the suspected allergens in 33 patients, and the results were read on Days 2, 4, and 7 using the Johansen's Modied scale. Results: The mean age of the patients was 48.3 years with M: F ratio of 3:7. The mean duration of the complaints was 20 months, while the mean duration of exposure to the implicated allergen was 10.6 years. Patch test with ISS and cosmetic series were positive in 34% of people with 4-Phenylenediamine base PPD (1%) being the most common allergen in 14 patients. ROAT was positive in 18 patients, out of which 78% were patch test negative. Patch test has stood the test of Discussion: time as an invaluable diagnostic aid in patients with dermatitis. Sindoor dermatitis is a commonly encountered ACD in our clinics but the implicated allergens are not a part of ISS or cosmetic series. In our study, ROAT was positive in 8 patients of sindoor dermatitis who were patch test negative. Thus, ROAT should also be ideally be performed in these patients.

Improving povidone-iodine and iodine preparations for patch testing.

Allergy evaluation by patch testing with povidone-iodine (PVP-I) or iodine remains challenging, because current patch test preparations frequently lead to false-positive or irritant skin reactions. To investigate different preparations for iodine patch tests and to assess their clinical relevance with repeated open application tests (ROATs). We monocentrically analyzed 95 patients with suspected allergy to disinfectants in retrospect who underwent parallel iodine patch testing with four preparations: PVP-I 2% aq., 5% aq., 10% aq., and iodine 0.5% pet. In 27 of 95 patients (28.4%), we found positive reactions to one of the four test preparations. After ROATs in 22 of these 27 positively tested individuals, only one patient was diagnosed with iodine allergy. In contrast, 31 of 95 patients (32.6%) showed irritant or questionable patch test reactions on day 2 (D2) and/or D3 and/or D7 to one or more test preparations. Testing with PVP-I 2% aq. resulted in the lowest number of doubtful skin reactions while detecting the single allergic patient. PVP-I 2% aq. was found to be the optimal patch test preparation. In general, iodine allergy appears to be substantially overestimated, and positive patch test responses to iodine should prompt an urgent ROAT for confirmation before diagnosing iodine allergy.

Practical suggestions to improve standardization of repeated open application testing (ROAT) for daily use products.

IntroductionRepeated open application testing (ROAT) is an important adjuvant in the evaluation of allergic contact dermatitis. There are variations in the way ROAT is used by practitioners and standardizing the method, in terms of the area and amount of the antigen applied, could help improve the validity of the process.AimOur study attempts to address ways in which part of the ROAT technique can be standardized – with respect to the area and amount of application and validation of the same through consensus opinion.Material and methodsTwo proposed modifications – one for the area of application (using waterproof surgical markers and a stencil to mark the area of application) and one for daily amount applied (using easily available 1 cm3 syringes), for the ROAT technique were drafted. The same was discussed with a total of 10 dermatologists. The participants were given four statements and were asked to choose one option for each statement (strongly agree – agree – neutral – disagree – strongly disagree). All the respondents were also asked to give their opinion on possible limitations and problems of the proposed modifications and possible solutions for the same.ResultsThere was generally a consensus among the 10 dermatologists, with the majority agreeing that the proposed modifications were both effective and practical.ConclusionsSimple practical modifications of the ROAT technique like marking the area to be applied using a waterproof skin marker and stencil, and the use of 1 cm3 syringes to dispense the test product, can improve the standardization of the process.

Clinical relevance of positive patch test reactions to lanolin: A ROAT study

BackgroundLanolin is often included when patch testing for common contact allergens. The clinical relevance of a positive patch test reaction to lanolin markers is, however, still a subject for debate.ObjectivesTo evaluate Amerchol L101 as a marker of lanolin allergy and investigate the clinical impact of lanolin‐containing moisturizers on healthy and damaged skin using the repeated open application test (ROAT).MethodsTwelve test subjects and 14 controls were patch tested with Amerchol L 101 and additional lanolin markers. Subsequently, a blinded ROAT was performed on the arms of the study participants for 4 weeks. Each participant applied a lanolin‐free cream base and two different lanolin‐containing test creams twice daily on one arm with intact skin and on the other arm with irritant dermatitis, induced by sodium lauryl sulfate (SLS).ResultsEleven test subjects (92%) had positive patch test reactions to Amerchol L 101 when retested and one test subject (8%) had a doubtful reaction. None of the study participants had any skin reactions to the ROAT on intact skin and all participants healed during the ROAT on damaged skin.ConclusionsLanolin‐containing emollients do not cause or worsen existing dermatitis when performing ROAT in volunteers patch test positive to Amerchol L101.

Skin irritation potential of cosmetic preservatives: An exposure-relevant study.

Preservatives represent one of the main causes of skin irritation and contact allergies. To comprehensively evaluate the skin irritation potential of phenoxyethanol, methylparaben, propylparaben, imidazolidinyl urea, and DMDM hydantoin under regulatory acceptable concentrations. A patch test and repeated open application test (ROAT) were applied to evaluate skin irritation in vivo. In vitro alternative methods consisting of the keratinocyte cytotoxicity assay, red blood cell (RBC) test, and hen's egg test-chorioallantoic membrane (HET-CAM) were performed to elucidate the mechanism of preservative-induced irritation responses. The patch test showed that all test substances showed a weak erythema response. Propylparaben had the highest occlusive irritancy potential in the patch test, owing to damage to the cell membrane. The two formaldehyde releasers showed noticeable skin irritation potential in the ROAT through their cytotoxicity to keratinocytes, while a visible response was observed after applying phenoxyethanol and the two parabens. No filtration was noticed in the in vivo tests, which might be attributed to the failure of subcutaneous vessel alteration by the preservatives. Commonly used cosmetic preservatives have minor skin irritation potential with mild erythema reaction under practical use, especially formaldehyde releasers and propylparaben.

Stepwise approach of development of dermo-cosmetic products in healthy and atopic dermatitis paediatric population: safety evaluation, clinical development and postmarket surveillance.

Background/objectivesPaediatric skin, considered sensitive, and infant skin, more susceptible to percutaneous toxicity, require specially formulated cosmetic products. As recently shown, early use of emollients in infants “at risk” of developing atopic dermatitis has shown controversial results in reducing the incidence of atopic dermatitis. Development of dermo‐cosmetic products for this specific population should especially ensure tolerance and safety. In absence of good clinical practice guideline, we propose here a stepwise approach for the development of paediatric cosmetic skincare products.MethodsOur stepwise methodology for cosmetics aimed at paediatrics, starts with in vitro assessment of product's ingredients safety, followed by preclinical and clinical evaluations of the final product, including sequentially: (1) Repeated Open Application Test (ROAT), (2) Human Repeated Insult Patch Test (HRIPT), (3) In‐use dermatological and ophthalmological tolerance studies (sequentially in 3a: healthy adults, 3b: healthy paediatric subjects and finally 3c: paediatric patients). We also describe the integrated cosmetovigilance–toxicological surveillance during the clinical development phase and postmarketing.ResultsAs illustrated with one dermo‐cosmetic product intended to be used as a preventative/maintenance treatment for atopic dermatitis in paediatric population, we show that using this stepwise methodology to test a product reduces potential risks of irritation and contact dermatitis in this sensitive population.ConclusionStandardized ethical stepwise development approach is needed to ensure the commercialization of safe and well‐tolerated dermo‐cosmetics for paediatrics. The approach described here could potentially serve as guidance for evaluation of new paediatric cosmetic products.

Validation of a questionnaire algorithm based on repeated open application testing with the constituents of fragrance mix I.

In a European study on contact allergy in the general population, it was hypothesized that the combination of contact allergy to a fragrance together with a history indicating dermatitis at exposure, and thereafter subsequent avoidance of scented products, implied a diagnosis of allergic contact dermatitis. The primary aim of this study was to validate this hypothesis and algorithm. The secondary aim was to investigate whether there was any association between the outcome of the repeated open application test (ROAT) and the patch test reactivity. In total, 109 patients with and without contact allergy to fragrance mix (FM) I were recruited. Volunteers from six European dermatology clinics participated in the study including a patch test and a ROAT. Positive ROAT reactions were noted in 26 of the 44 volunteers with contact allergy to FM I. None of the volunteers reacted to the vehicle (P < 0·001). More individuals with a positive algorithm had positive ROATs than those with a negative algorithm. However, the difference was not statistically significant. The lower the patch test concentration eliciting a positive test reaction, the more likely a positive ROAT and the more likely that the positive ROAT appeared early during the investigative period. The algorithm used in this study was not substantiated in this ROAT set-up. The stronger the patch test reactivity the more likely was a positive ROAT and the more likely it was that the positive ROAT appeared early during the application period. What's already known about this topic? To the best of our knowledge, a scientifically designed and conducted repeated open application test (ROAT) has never been performed before to validate a diagnosis of allergic contact dermatitis partly based on a questionnaire. What does this study add? This is the largest controlled, randomized and blinded ROAT performed to date. Higher patch test reactivity to fragrance mix I indicated a greater likelihood of a positive ROAT. What are the clinical implications of this work? Further refinement of the questions is required in order to diagnose allergic contact dermatitis from fragrances based on a questionnaire.

Positive Occluded Patch Test in the Face of Negative Repeat Open Application Test.

Positive Occluded Patch Test in the Face of Negative Repeat Open Application Test.

Allergic contact dermatitis caused by hydroperoxides of limonene and dose-response relationship-A repeated open application test (ROAT) study.

Contact allergy to oxidized limonene, with hydroperoxides of limonene (Lim-OOHs) as the main allergens, is common. However, high proportions of weak positive and doubtful patch test reactions have been reported. To determine the clinical relevance, elicitation threshold and dose-response relationship of Lim-OOHs in individuals with a positive or doubtful patch test reaction to standard Lim-OOHs 0.3% pet. A multicentre 3-week double-blind vehicle-controlled repeated open application test (ROAT) study with a simulated fine fragrance containing Lim-OOHs at 1260, 420 and 140 ppm, equal to a dose/area per application of Lim-OOHs of 3.0, 0.99 and 0.33 μg/cm2 , was performed. Among 11 subjects allergic to Lim-OOHs, 11 (100%), 7 (64%), and 3 (27%), respectively, reacted to the applied doses. No reactions were seen in 17 healthy controls exposed to the highest dose. This difference in reactivity was statistically significant (P < 0.0001). Among 13 subjects with doubtful patch test reactions to Lim-OOHs, two (15%) had positive ROAT reactions to the highest Lim-OOH dose applied (P = 0.36 as compared with controls). Contact allergy to Lim-OOHs is of clinical relevance in patients with positive patch test reactions. A doubtful patch test reaction to Lim-OOHs 0.3% pet. can be of clinical relevance.