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Speciation of chromium in tanned leather gloves and relapse of chromium allergy from tanned leather samples
Olle Nygren*a and Jan E. Wahlbergb a National Institute for Working Life, P.O. Box 7654, S-907 13 Ume, Sweden b National Institute for Working Life, S-171 84 Solna and Karolinska Hospital, S-171 76 Stockholm, Sweden

There are concerns that wearing chromium tanned leather gloves can provoke a relapse of hand dermatitis in chromium sensitive patients. Various forms of chromium have different biological effects and the determination of total chromium (both soluble and insoluble), which often is used in clinical studies, may therefore not be an adequate measure of the adverse exposure. However, investigations, where dissolution of hexavalent chromium from leather gloves has been experimentally studied, could not been found in the literature. The aim of this pilot study was therefore to determine the total amount of chromium and the amount of sweat leachable chromium in different leather glove materials and also to apply samples of these materials in a patch test to chromium sensitive patients to see if they could provoke a reaction. The results showed that chromium tanned leather contained about 3% (m/m) chromium. Chromium can also be found in dyed vegetable tanned leather, probably emanating from the leather dye. Small amounts (average 0.08% m/m) of leachable hexavalent chromium were found in both chromium and vegetable tanned leathers. Thus, the risk of relapse of chromium dermatitis cannot be disregarded and further studies on the causes of relapse of chromium dermatitis are necessary. Keywords: Allergy; chromium speciation; tanned leather

Experimental Chemicals All chemicals were of analytical reagent grade and water was purified in a Milli-R/Q Water Purifier (Millipore, Bedford, MA, USA). Aqua regia was prepared by mixing concentrated hydrochloric acid and nitric acid (1 : 1; Suprapure acids, Merck, Darmstadt, Germany). The diphenylcarbazide (DPC) complexing reagent was prepared by dissolving 0.25 g of DPC in 50 ml of acetone in a 100 ml volumetric flask and adding water to volume.4 Standards for determination of total chromium were prepared from a commercial atomic absorption spectrometry (AAS) standard solution (Cr 1.000 g ml21, BDH, Poole, Dorset, UK). Standards of hexavalent chromium were diluted from a stock solution (1.000 g ml21) made by dissolving potassium dichromate in water. Synthetic sweat was made by dissolving sodium chloride (5 g l21), lactic acid (1 g l21) and urea (1 g l21) in water and adjusting the pH to 6.5 using ammonia solution.5 Leather samples Eleven different brands of leather protective gloves were purchased from local retailers. Five brands were made from chromium tanned leathers and six from various vegetable tanned leathers. The leathers were made of skin from different animals and were subject to different treatments besides tanning (see Table 1). From each pair of gloves 15 discs (10 mm in diameter, mean weight 0.152 g, range 0.1000.190 g) were punched out. Ten discs from each pair of gloves were coded and packed in plastic bags and were used for patch testing on chromium sensitive patients. The remaining five discs from each pair of gloves were used for determination of the total chromium content and leachable chromium. Determination of chromium in tanned leather Leachable hexavalent chromium was determined by leaching three discs (three of the five remaining discs) in synthetic sweat. The mean value of the three discs was used. Each disc was weighed and placed in a 20 ml test tube and 5 ml synthetic sweat solution was added. Leaching was performed by ultra-sonication for 60 min. The sample solutions were filtered using microfilters (Millex-SR, Millipore). Hexavalent chromium was determined using spectrophotometry after DPC complexation.4 A 2 ml aliquot was withdrawn from each sample and transferred to a 15 ml calibrated flask. Complexing reagent (1 ml), 0.5 m sulfuric acid (5 ml) and water to volume were added and the sample was left to stand for 10 min to allow formation of the complex. The absorbance at 540 nm was measured using 40 mm cuvettes and hexavalent chromium was quantified using standards prepared in the synthetic sweat solution. The total chromium content was determined in duplicate (the remaining two discs) and the mean value was used. In short, the

There are some anecdotal reports implying that wearing chromium tanned leather gloves can provoke a relapse of hand dermatitis in patients sensitive to chromates and other soluble hexavalent chromium compounds. Primary sensitisation is rare, but has been observed in workers using wet (water soaked/ impregnated) gloves in jobs where other sources of chromium exposure have been excluded (own clinical observations). Various forms of chromium have different biological effects and determination of total chromium, which often is used in clinical studies,1 may therefore not be an adequate measure of the adverse exposure. Hexavalent chromium is more toxic than the trivalent form.23 Consequently, speciation of chromium is a more suitable procedure to assess possible hazardous exposure. On the other hand, investigations, where dissolution of hexavalent chromium from leather gloves has been experimentally studied, could not been found in the literature. The aim of this pilot study was therefore to determine the total amount of chromium and the amount of sweat leachable hexavalent chromium in different leather glove materials and also to apply samples of these materials to chromate sensitive patients to see if they could provoke a reaction at patch testing.

Presented at the Third International Symposium on Speciation of elements in Toxicology and in Environmental and Biological Sciences, Port Douglas, Australia, September 1519, 1997.

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discs were weighed and placed in 100 ml beakers and acid digested in aqua regia on a hot plate until completely decomposed. The acid extract was filtered through a microfilter (Millex-SR, Millipore) and the total chromium content in the clear solution was determined by AAS (SP1900 Atomic Absorption Spectrophotometer, Pye Unicam, Cambridge, UK) employing nitrogen oxideacetylene flame and matrix matched standards. Patch testing Occlusive tests were carried out in patients with previously established chromium sensitivity. Their thresholds of sensitivity to bichromate had previously been determined by serial dilution tests6 and the observed threshold concentration was applied as positive control using Finn Chambers (K2Cr2O7 in petrolatum). One disc of each sample, moistened in water as well as the positive control were applied on the back of the subjects for two days using Scanpore tape (Norgesplaster AG, Norway) and the results were recorded at day 3 (24 h after removal of the patches). Eight chromate sensitive males (age 5472 years) and two healthy volunteers participated in the study, which was approved by the local ethical committee. Results and discussion Determination of chromium in tanned leather The total chromium content was detemined by flame AAS, after an acid decomposition of the leather samples, according to general AAS procedures.7 Hexavalent chromium, leachable from the leather samples into synthetic sweat, was determined by spectrophotometry, after complexation with DPC. This is a method frequently used for selective determination of hexavalent chromium.4,8 These two analytical methods have different detection limits, i.e., 0.01 and 0.0001 mg g21 for total chromium and soluble hexavalent chromium, respectively. One consequence of the difference in detection limits between the two analytical methods is, however, that low levels of soluble hexavalent chromium might be detected, although the level is below the detection limit for total chromium. This is evident for sample nos. 7 and 911.

Table 2 shows the results of the chromium determinations. The average total chromium content in chromium tanned leather was 29.7 mg g21 with a relative standard deviation (RSD) of 13%. The level of total chromium was below 0.01 mg g21 in non-dyed vegetable tanned leather, while some total chromium could be found in the dyed vegetable tanned leather (0.97 mg g21), as well as in the natural tanned leather (0.17 mg g21). The latter was made in China and of unknown origin. These results show that dyed vegetable tanned leather may contain chromium, probably from the dye. Small amounts of soluble hexavalent chromium were leached from all samples. The average amount of leachable hexavalent chromium from chromium tanned leather was 0.0008 mg g21 with a RSD of 90% and the amount leached from vegetable tanned leather was similar (mean = 0.0009 mg g21; RSD = 83%). During the tanning process hexavalent chromium is absorbed in the leather and is reduced to trivalent chromium. In contact with organic materials, hexavalent chromium is also known to be reduced to trivalent chromium.8 This indicates that the amount of hexavalent chromium reflects a background chromium level in leather. These results also show that the variation of the amounts of leachable hexavalent chromium was higher than for total chromium in chromium tanned leather. For each tanning process, no differences in total or leachable chromium could be found between skin from different animals. The ratios of hexavalent chromium versus total chromium in the leachates were also determined by analysing the leachates using the AAS method. However, only six samples (nos. 2, 3, 5, 8, 10, and 11), gave results above the detection limit. These results showed that most of the soluble chromium was present in the hexavalent form (mean 92%; range 75100%; n = 6).

Patch testing All eight patients showed positive reactions to the positive control (K2Cr2O7 in petrolatum), which confirmed their bichromate sensitivity, while none of the reference subjects reacted. None of the patients or reference subjects, however, showed any positive reactions to the leather samples. The analytical results showed that soluble chromium is released from all kinds of tanned leather and, although the patch testing gave negative results, further studies on possible relapse of dermatitis among chromium sensitive subjects are necessary. Such studies can be performed by glove finger provocation on chromium sensitive subjects and the results correlated to the

Table 1 Specification of the various leather protective gloves used in this study* Sample no. 1 2 3 4 5 6 7 8 9 10 11
*

Leather Goat skin Swine grain Goat skin Swine grain Cow skin Unknown Cow skin Reindeer skin Cow skin Cow skin Cow skin

Tanning process Cr tanning Cr tanning Cr tanning Cr tanning Cr tanning Chrome free tanning Bark tanning Vegetable tanning Vegetable tanning Vegetable tanning Vegetable tanning, hydrophobic

Other treatment Impregnation Natural colour Natural colour Natural colour Black surface dye Natural colour Natural colour Light brown dye Natural colour Natural colour Natural colour

Table 2 Analytical results* Glove no. 1 2 3 4 5 6 7 8 9 10 11 Tanning Cr Cr Cr Cr Cr Chrome free Bark Vegetable Vegetable Vegetable Vegetable Soluble Cr6+ 0.0002 0.0011 0.0024 0.0004 0.0010 0.0002 0.0001 0.0015 0.0003 0.0010 0.0009 Total Cr 27.8 34.3 28.7 24.7 32.9 0.17 < 0.01 0.97 < 0.01 < 0.01 < 0.01

Cr tanning is industrial tanned leather using chromic acid. Bark tanning is a vegetable tanning made in an extract of alder bark, which gives a reddish brown leather. Impregnation or hydrophobic treatment of leather (sample 1 and 11) was made using silicone to improve water resistance. Leather with natural colour means that no dye was used. Leather with black surface dye had the dye printed on to the surface of the leather. The light brown dyed leather was coloured using a penetrating dye. One pair of gloves (sample no. 6) was made in China and no other information on the origin of the leather other than natural tanned and chromium free could be obtained.

* Hexavalent chromium, leachable to synthetic sweat (Soluble Cr6+) and the total chromium content determined after acid decomposition (Total Cr) in leather samples from protective gloves are expressed in mg g21. The results are the mean of three (leaching) and two (acid digestion) samples from each glove.

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release of total and hexavalent chromium from the glove fingers. Conclusion Chromium tanned leather contain about 3% (m/m) total chromium while vegetable tanned leathers contain less than 0.001% (m/m) of chromium. However, small amounts of chromium can be found in dyed vegetable tanned leather, probably emanating from the leather dye. Small amounts of hexavalent chromium were leached from both chromium tanned and vegetable tanned leather and no significant difference between the tanning processes was found. Since chromium is leached from both chromium tanned and dyed vegetable tanned leather, the risk for relapse among chromium sensitised persons wearing chromium tanned protective gloves cannot be disregarded. Further studies on the causes of relapse of chromium dermatitis are necessary. The participation of the volunteers is acknowledged as well as the chromium determinations made by E. Kragh.

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Paper 7/07458A Received October 16, 1997 Accepted December 11, 1997