University of Groningen

Skin problems related to Indonesian leather & shoe production and the use of footwear inIndonesiaFebriana, Sri Awalia

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Citation for published version (APA):Febriana, S. A. (2015). Skin problems related to Indonesian leather & shoe production and the use offootwear in Indonesia. [Groningen]: Rijksuniversiteit Groningen.

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R 8 Thin layer chromatography and gas chromatography-mass spectrometry (GCMS) examination of footwear materials from patients with shoe dermatitis

Sri Awalia Febriana1,2, Erik Zimerson3 , Cecilia Svedman3, Winarto Haryadi4, Pieter-Jan Coenraads2, Marie-Louise Anna Schuttelaar2

1 Department of Dermatology & Venereology, Gadjah Mada University, Yogyakarta, Indonesia; 2 Department of Dermatology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; 3

Department of Occupational and Environmental Dermatology, SUS Malmö, University of Lund, Malmo, Sweden; 4 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Gadjah Mada University, Yogyakarta, Indonesia Abridge version accepted for publication in Contact Dermatitis

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Keywords: shoe dermatitis; thin layer chromatography (TLC); gas chromatography-mass spectrometry (GCMS); patch testing; shoe materials; 2(3H)-benzothiazolone; methyl dehydroabietate; 7-oxodehydroabietic acid methyl ester; colophony

Introduction

Shoe materials are known to be a common source of contact dermatitis, but the actual causative allergens are often unknown. Patch testing with allergens in a standard screening tray containing chemicals used in shoe manufacturing has been found to be insufficient.1, 2 Negative reactions to those allergens do not necessarily exclude shoe dermatitis; 25 - 50% of reactions are due to allergens not present in the screening tray, often because modern shoes are manufactured from unknown not defined components provided by outside suppliers.3 Therefore, when the clinical picture strongly suggests shoe dermatitis but the patient does not reacted to materials from the screening tray, one must extract and identify individual substances from the suspected shoes and patch test to possibly prove a contact allergy.

Case 1.

A 50-year-old male with shoe dermatitis had symmetrical eczematous skin lesions on the dorsum of both feet (Figure 1). After wearing imitation leather sandals for about 6 months his feet began to itch and skin lesions appeared. His lesions healed when he was not wearing the sandals. Upon re-exposure the itch and lesions recurred (Figure 1). The patient had no history of atopic manifestations but his grandfather had suffered from recurrent eczema

Figure 1. Eczematous lesion on the dorsum of the feet of patient 1 (left). Upper part of

the patient 1’s sandal which in direct contact with skin lesion (right).

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Case 2

A 53-year-old female was diagnosed with shoe dermatitis on her dorsum and plantar surfaces of the feet (Figure 2). The dermatitis appeared every time she wore sandals made from imitation leather. Her lesions healed when she stopped wearing the sandals and reccured afrer re-exposure to the same sandals. When she had first started using the sandals, she felt itchiness and a mild burning sensation. The lesions worsened when her feet got wet. Upon performing a skin examination, we also found atypical clinical features of shoe dermatitis possibly due to herbal medication and antiseptic soap used to treat the current symptoms of shoe dermatitis.

Figure 2. Skin lesion on the dorsum of the feet of patient 2

Patch testing

Both patients were patch tested with allergens from the European Baseline Series, Shoe Series and additional series, based on our own earlier studies in Indonesian leather and shoe factories.4 Test preparations were supplied by Chemotechniques Diagnostics®, Vellinge, Sweden and by the laboratory

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of the Dermatology Department, Faculty of Medicine, Gadjah Mada University, Indonesia. Patch test procedures were performed on patients’ upper backs using Finn chambers® (Epitest Ltd., Helsinki, Finland) mounted on acrylate-based adhesive tape (Scanpor Alpharma AS, Norgesplaster Facility, Norway). The patches were removed after 48 hours and read on D2, D3 and D4 as recommended by the International Contact Dermatitis Research Group. Result were negative in both patients.

Both patients were then patch tested with suspected shoe materials (imitation leather) taken from the upper parts of the sandals which were directly in contact with the corresponding areas of the skin lesions. Each patients was also patcch tested with the material of the other patient. These materials were cut into pieces 1 cm2 and 2 mm thick and were moistened with saline before being applied. Both patients reacted to the inner part of the sandalas uppers of patient 1 (Table 1).

Shoe materials from both patiens were examined. Extract of suspected materials of both patients were prepared at the Organic Chemistry Laboratory, Faculty of Mathematics and Natural Sciences, Gadjah Mada University, Indonesia by adding 80 ml ethanol to 8 gr shoe material, then extracted with an ultrasonic bath for 2 hours, and centrifuged for 5 minutes at 500 r.p.m. The supernatant was then concentrated using a 'Buchi' vacuum evaporator.5 Both patients reacted to the shoe extract of patient 1 but not to the extract of patient 2. The extract of patient 1 was thus suspected of containing interesting allergens. We patch tested the extracts on 10 healthy volunteers as controls and showed negative reactions to both shoe extracts. The extract of patient 1 was thus suspected to contain possible interesting shoe allergens.

Thin layer chromatography (TLC) was performed in order to separate the components of the extracts of the shoe materials of patient 1 and to obtain a TLC strip for patch testing (TLC plastic roll, Silica Gel 60F 254, Merck KGaA, Darmstadt, Germany). The TLC was prepared at the Dept. of Occupational and Environmental Dermatology, Malmö, Sweden. The TLC strips were applied to the back using Scanpor tape. The position of the TLC strip and its spot were carefully marked. The TLC strip was removed after 48 hours and read on D2, D4 and D7 according to ICDRG criteria.6 Both patients reacted to spots A and B on the TLC strip and patient 2 also reacted to spot C and spot D (Figure 3). Areas corresponding to the positive

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reactions were scraped off the silica gel for each spot, respectively, from an exact copy of the tested TLC strip and extracting the gel with a minimal amount of ethyl acetate. The resulting solutions were analyzed by gas chromatography-mass spectrometry (GCMS).7 It was found that positive TLC spots contained methyl dehydroabietate (Spot A), 7-oxodehydroabietic acid methyl ester (Spot B), and 2(3H)-benzothiazolone (Spot C) (Figure 3 and 4) The main substance in spot D was unidentifiable. Both patients were patch tested with 2(3H)-benzothiazolone 0.1% and 1.0% in petrolatum. Patient 1 showed a doubtful reaction and patient 2 was found positive to both concentrations (Figure 3).

Figure 3. Patient 2 reacted to Spots A, B, C and D in TLC strip and to 2(3H)-benzothiazolone 0.1% and 1.0% pet

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S

HN

O

2(3H)-Benzothiazolone CAS: 934-34-9 MW 151

O

O O

7-Oxodehydroabietic acid methyl ester CAS 110936-78-2 MW 328

O O

Methyl dehydroabietate 1235-74-1 MW 314

Figure 3. Chemical structures, names, CAS numbers and molecular weights of confirmed and suspected allergens.

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Discussion

Commercially available patch test kits are helpful for testing patients, but the constantly growing number of substances in everyday modern products make such tests inadequate. To improve accuracy patch testing with the patient´s own products is often reccomended. However, in a product an allergen may be present in a concentration so low that it gives a negative patch test result. One solution would be to get content information from the producer on the ingredients and then patch test these ingredients. We could also patch test directly with an extract of the product.5 In an extract the chemical components of a product will be tested at a higher concentration, and therefore the patch test will be more sensitive and also compensate for repeated exposure to a product that is often needed to give allergic eczema. A positive reaction to an extract can be verified by patch testing controls. The patient can be advised to avoid the product and sometimes, as often the case with cosmetics, this is sufficient. With regard to some everyday objects, for example further investigation is often needed in order to establish the identity of the allergen and make it possible for the dermatologist give advice on how to avoid the allergen in the future. Patch testing with TLC strips of the positive extract is often helpful for identification of allergens.6 The chemical substances in the extract are separated onto the TLC strip and this greatly facilitates chemical analysis. Identification the substances present in a positive TLC strip patch can often be done with GCMS (or a similar method).

The 2 cases described here illustrate that neither purchased shoe series or extracts (extract patient 1) may be enough. The clinical symptoms of our patients led to a suspicion of contact dermatitis, but routine investigation indicated no contact allergens. Since both patients arrived simultaneously at the clinic the shoe materials of each were patch tested on both. Shoe material no.1 (from shoe patient 1) was positive in both patients whereas shoe material no. 2 (from shoe patient 2) was negative in both. Extracts and further TLC testing (with TLC from shoe extract no 1) gave similar results (Table 1).

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Table 1: Test results from patch testing of patch test series, materials, extracts and thin layer chromatography strips (TLC strips)

Test material Concentration Vehicle Patient 1 Patient 2

Shoe material pat 1 (upper) as is + + Shoe material pat 2 as is - - Shoe extract pat 1 ethanol + + Shoe extract pat 2 ethanol - - TLC spot A TLC strip (+) + TLC spot B TLC strip (+) + TLC spot C TLC strip - + TLC spot D TLC strip - + 2(3H)-Benzothiazolone 1.0% pet (+) + 2(3H)-Benzothiazolone 0.10% pet (+) + Triclosan 2.0% pet - - European standard (15 allergens) - - Shoes series (22 allergens) including colophony - -

Additional allergens (12 allergens) - - Reactions:

(+) = doubtful reaction

+ = non vesicular reaction, palpable erythema and slight infiltration

Because the extracts were to be sent to an external laboratory and the clinical investigation was to occur in a geographically remote area, both extracts were actually analysed before patch testing (Table 2). Analysis indicated the presence of several possible allergens in the extracts. Based on these findings we patch tested 2(3H)-benzothiazolone and the preservative triclosan on both patients. Triclosan gave negative results for both but patient 2 reacted positively to 2(3H)-benzothiazolone whuc (according to GCMS) was present in the shoe extract of patient 1.

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Table 2 Substances found in the analysis of shoe extracts.

Patient Substances Patient 1 Cinnamic acid (CAS 140-10-3)

2(3H)-Benzothiazolone (CAS 934-34-9) Triclosan (CAS 3380-34-5) Colophony Diethylhexyl phthalate (CAS 117-81-7) Diisobutyl phthalate (CAS 84-69-5) 2,4-Di-tert-butylphenol (CAS 96-76-4) 1-Dodecanol

Patient 2 Cinnamic acid (CAS 140-10-3) Diisobutyl phthalate (CAS 84-69-5) Diethylhexyl phthalate (CAS 117-81-7) Nonylphenol ethoxylates 1-Dodecanol 1-Chlordodecan

Patch testing with the TLC strip showed that patient 2 reacted to four spots on the TLC (spots A, B, C and D). 2(3H)-Benzothiazolone was identified on spot C. In spots A and B, respectively, methyl dehydroabietate and 7-oxodehydroabietic acid methyl ester were found, both derivatives of colophony. The main component in spot D was unidentifiable and thus not patch tested. Also spot C contained some substances that could not be identified.

Patient 1, whose shoe extract provided important information on patient 2, reacted only to the shoe extract and the shoe material. The TLC spot C with 2(3H)-benzothiazolone was negative, the patch testing with this allergen doubtful, as was the spot containing colophony derivatives (Spot A and B). What do we conclude from this? With regard to patient 1 we can only be certain that he had an allergic contact reaction to the extract and his shoe material (controls were patch tested).

Patient 2, had no reaction to either his own shoe material or its extract, but he did react to shoe material extract patient 1 and the allergens found there. We have thus not been able to prove a contact allergy with regard to the shoe. This does, however, indirectly emphasize the need for an updated shoe series; allergens giving positive reactions may not be common but could very well be present in shoes. 2(3H)-Benzothiazolone is a rare allergen which we have not previously encountered. However, an Indian paper reported it as a common sensitizer used as a corrosion inhibitor.8

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This substance could have been added to the product, or it could be a degradation product from the rubber chemical mercaptobenzothiazole or an oxidation product from benzothiazole. We previously discovered the latter substance as an antimicrobial agent used in bras (unpublished findings).

Methyl dehydroabietate and 7-oxodehydroabietic acid methyl ester indicate the presence of colophony or modified colophony in shoe materials, probably as a component of glue. As colophony can be produced either from pines or other plants it may contain different allergens. The best approach is to patch test the colophony to which the patient was exposed, which in this case was not possible; the two detected colophony substances were not available at the time. At least with the inforamtion from the TLC patch testing patient 2 can be advised not wearing shoes possibly containing colophony derived glues.

References

1. Lynde CW, Warshawski L, Mitchell JC. Patch test results with a shoewear screening tray in 119 patients, 1977-80. Contact Dermatitis 1982; 8: 423-5.

2. Angelini G, Vena GA, Meneghini CL. Shoe contact dermatitis. Contact Dermatitis 1980: 6(4): 279-83.

3. Romaguera C. Shoe contact dermatitis. Int J Dermatol 1987; 26: 532-5.

4. Febriana SA, Jungbauer F, Soebono H, Coenraads PJ. Inventory of the chemicals and the exposure of the workers'skin to these at two leather factories in Indonesia. Int. Arch Occup Environ Health 2012; 85: 517-526

5. Bruze M. The use of ultrasonic bath extracts in the diagnosis of contact allergy and allergic contact dermatitis. In: Patch testing tips. Lachapelle JM, Bruze M, Elsner P, eds. Springer-Verlag, Berlin Heidelberg, 2014: 129-42.

6. Bruze M, Frick M, Persson L. Patch testing with thin-layer chromatograms. Contact Dermatitis 2003; 48: 278-9.

7. Svedman C, Zimerson E, Bruze M. Allergic contact dermatitis caused by benzanthrone in a pair of trousers. Contact Dermatitis 2014; 71: 54-7.

8. Mathur AK, Khanna SK. Dermal toxicity due to industrial chemicals. Skin Pharmacol Appl Skin Physiol 2002; 15: 147-53.