The ‘non-allergenicity’ of NR dry rubber products, with reference to type 1 protein allergy*
Introduction
NATURAL RUBBER PRODUCTS, from both latex and dry rubber, have been widely used all over the world for many years. Recently, the use of some latex-dipped articles, such as latex gloves, catheters and condoms, has been reported to have given rise to Type 1 hypersensitivity in some individuals.1-4 Symptoms for this allergic reaction include urticaria, rhinitis, conjunctivitis, asthma and less frequently, anaphylaxis. The onset of this type of 1gE-medicated allergy is believed to be due to a number of factors, one of which is the sudden demand in the late 1980s for latex products such as gloves and condoms, which are very good protective barriers against viral diseases, particularly AIDS. It is thought that the increased exposure to latex has resulted in sensitization of, especially, atopic individuals.1
This allergic reaction has been shown to be due to be due to a very small fraction of residual soluble proteins (EP) containing the allergens found in latex products.4-8 Research findings9-13 have shown that the amount of this protein fraction in different latex products prepared from the same latex source varies, depending on the processing procedure employed during their manufacturing. For example, it increases10 when latex is compounded, vulcanized or dried at an elevated temperature of 100° C. It decreases,9,11-13 on the other hand, when the products are washed/leached in water or chlorinated. The ability of the product to cause the allergic reaction, or its allergenicity, is very much influenced by the quantity of this protein fraction present, as shown by Yip et al14 who demonstrated that both the total residual extractable proteins and the allergenicity are well correlated, that is high EP contents are always associated with positive allergic reaction when skin-tested on latex hypersensitive persons, and vice versa.
Although some inhibition activity of IgE binding was detected in extract of fragments from a worn tyre contaminated with road pollutants,15 there is however, no such allergy incidence reported involving the use of dry rubber products which are prepared somewhat differently from the latex-dipped goods. Nevertheless, it is learned that there is a certain ‘fear campaign’ launched against natural rubber threads, capitalizing on the latex protein issue. Work was therefore carried out to study the residual extractable proteins in NR dry rubbers and their products. Their allergen activity, as measured by a serological method, and their allergic responses, if any, elicited in latex protein hypersensitive subjects were investigated.
Methods
Quantitation of extractable proteins - RRIM modified Lowry method
Extraction of soluble proteins: The NR dry rubber sample was cut into small pieces (of about 1mm3), which were extracted in 0.01M phosphate buffered saline at pH7 (5ml/g of rubber) at 23°C for 3 hours using a polypropylene container. The extract was centrifuged at 3000 x g for 15 minutes to remove any particular matter that might be present. The clear extract was then immediately subjected to protein precipitation.
Protein precipitation: 6ml volume of the extract in a polypropylene tube was treated with 1ml trichloroacetic acid (35%, w/v) and 1ml phosphotungstic acid (1.6% w/v). The content was mixed and allowed to stand for 20 minutes. The resulting precipitated proteins were sedimented by centrifugation at 10 000 x g for 30 minutes, and were redissolved in 1ml of 0.2M sodium hydroxide.
Colorimetric measurement:Protein concentration was then determined using the RRIM modified Lowry microassay.16 Procedures involved essentially the addition of 300µl of fresh mixture containing sodium carbonate (6%) and a solution of 1.5% copper sulphate in 3% sodium citrate (mixed in the ratio of 10.0:0.2) to 800µl of redissolved protein test sample. After standing for 10 minutes, a volume of 100µl Folin reagent (72%, Sigma Chemical) was introduced. Colour was allowed to develop at room temperature for 30 minutes. Absorbance readings at 750nm were recorded and read against a calibrated curve using bovine serum albumin (BSA) standard.
RAST-inhibition immunoassay:
The total in-vitro allergenic protein activity was measured using the procedure according to Yman et al.17
Solid-phase allergens: Activated paper discs (Immobilon Affinity MembraneR, Millipore, Bedford, MA) were coupled with an optimal amount (1:100, v/v) of latex serum prepared by centrifuging non-ammoniated Hevea latex after freezing and thawing. The same latex serum was also used as a reference with a given arbitrary activity of 100 000 relative latex units (RLU/ml).
Latex-specific IgE antibodies: The source of these antibodies was a pool of sera from more than 30 patients with confirmed allergy to latex and with a high latex specific IgE test results using RASTR (Pharmacia, Uppsala, Sweden). The patients concerned comprised children and adults, healthcare workers and lay people.
Inhibition immunoassay: Each rubber sample was cut into pieces and extracted (1:5 weight per volume) in physiological saline in a shaker overnight. Several serial dilutions (1:2 or 1:10) were used from the reference and sample extracts. 30µl of each dilution was incubated with 20µl of the calibrated IgE serum pool in a tube for 3 hours in a shaker, after which one latex disc was added to each tube. Contents of the tubes were then allowed to incubate overnight. The tubes were washed three times and 50µl of a radio-labelled anti-IgE (Pharmacia, Uppsala, Sweden) was introduced to each tube. After an overnight incubation, the tubes were washed again, and the activity measured in a gamma-counter.
Percentage of inhibition was calculated from the control discs, one with no added inhibitor, and the other for background binding. The allergy activity of the sample was calculated relative to the reference using the parallel line assay method.18 The sensitivity of the method is 0.1µg/ml protein as measured by the Lowry method: the inter-assay coefficient of variation is 20%.
Skin prick test:
The test solution was prepared by extracting 1g of the rubber test sample, cut in small pieces of about 1mm cubes, in 5ml of physiologival saline (pH7) for 15 minutes at room temperature.
A drop of the test extract was first placed on the skin of the patient’s forearm and pierced through the drop with the tiny one-mm peak of a sterile lancet, creating a small break in the epidermis. The size of the wheal developed was measured 15 minutes after application. A positive control using histamine dihydrochloride (10mg/ml) and a negative control with the physiologival saline were also included in the test battery.
test reactions or responses were evaluated in relation to the histamine wheal. Reaction size of twice that or more of the histamine control is a strong positive reaction and is denoted as 4+; same size as that of histamine control is 3+ (a clear positive); at leas one-half of that of histamine is 2+ (a weak positive). Very small wheals were not considered to be positive.
Results
Residual extractable proteins (EP)
The preparations of dry rubbers and dry rubber products are different from those of latex-dipped products such as gloves. In the dipping process, the formers are usually first dipped in a coagulant such as calcium nitrate, and then in the compounded latex concentrate (derived from Hevea latex. The wet-gel gloves so formed are then leached in water for a few minutes, dipped in a cornstarch slurry, and finally vulucanized/dried at 100° -120°C. It may be mentioned that depending on the extent of leaching, or if the gloves had been chlorinated or polymer coated, the if the gloves had been chlorinated or polymer coated, the EP content can vary from as high as more than 1000µg/g to as low as below 20µg/g.
The processing of dry rubber and products, on the other hand takes a different route. Usually Hevea latex is converted directly into raw rubber by acid coagulation. After removal of the unwanted latex serum, the coagulated rubber is crumbled/creped and then dried. Except for drying, continuous washing with water is employed generously throughout the entire procedure. To fabricate into its products, the dry rubber is compounded and vulcanized, at temperatures sometimes as high as 160°C.
In view of the extensive washing employed during processing, it would bot be surprising if most of the EP in the raw rubber has been removed. This is indeed found to be so when a total of twenty seven raw dry rubber samples from nine differently processed dry rubber grades were analysed. All the rubber grades were commercially produced, with the exception of the steam-coagulated rubber. Results, shown, in Table 1, revealed that all samples have consistently very low EP contents of about 20µ/g of rubber and less, which are, in fact, at levels reaching the limit of measurements by the method used.
Subsequent vulcanization and fabrication processes of the dry rubber into its products, which often involve high temperatures, do not appear to have any adverse effect on the EP contents, which remain low. This is evident in Table 3, which shows EP levels of both raw and compounded rubbers as well as vulcanizates from five different grades and some final rubber products. In all cases, no values exceeded 35µ/g, which were extremely low. Such low EP levels have been indicated by Yip et al14 in the case of gloves, to elicit very little or no allergic response in latex hypersensitive persons when clinically tested. Therefore, dry rubbers and dry rubber products may be expected to display minimal or no allergic activity.
Allergenicity
To ascertain the very low or non-allergenicity of dry rubbers and their products, as suggested by their extremely low EP contents, their allergen activity and allergic response, if any, elicited in latex protein hypersensitive patients were investigated. While the allergen activity was measured using the in-vitro method of radioallergosorbent inhibition test (RAST-inhibition),17 the allergic response was assessed by the in-vitro skin-prick test.19 which is most commonly used for evaluating the Type 1 allergy of immediate hypersensitivity.20
Radioimmunoassay of RAST-inhibition. In this method, latex allergens were quantitated by allowing the soluble latex allergens in the sample extract to compete with a reference allergen mixture on a solid phase for the binding sites of human IgE antibodies. The amount of latex specific antibodies bound to the solid phase was determined, and was inversely proportional to the quantity of latex allergens in the test sample. Using this technique eleven dry rubber samples were examined. These included three commercial grades of SMR rubber (both raw and compounded), and five different rubber products. For controls, two samples of latex gloves known to show positive allergenicity and sample of vinyl non-NR gloves were also analysed.
Results in Table 2 showed that except for one sample which indicated a slightly higher value of 6 RUL/ml, all others gave values less than 5 RLU/ml, showing very low or no allergen activity at all. Their EP contents were as anticipated, extremely low. These are in stark contrast with those of glove samples containing considerable quantities of EP (allergen activity 438 and 431 RLU/ml).
Skin-prick test. This is a simple and rapid test of high sensitivity for IgE-mediated allergy. The allergic response to the allergens in the sensitized persons can be easily measured. Besides being used for identifying sensitized patients, the test is also used for detecting the presence of protein allergens in latex products.6,14
Extracts from 14 dry rubber samples of various grades and five different rubber products with pre-determined EP contents were skin-tested on latex protein hypersensitive subjects. The samples included both the raw and compounded (ACS1 mix) rubbers, vlucanizates (with ACS1 mix and cured at 140°C for 40 minutes) and rubber products such as cut threads, hot water bottles and diver’s flippers. A total of 31 patients shown to be sensitive to latex proteins were clinically tested in three groups. Results are shown in Table 3.
There was very little or no allergic response shown by the latex protein hypersensitive patients tested in all cases. These negative observations were substantiated by the strong positive reactions elicited in the same patients by extracts from a certain brand of latex gloves known for their allergenicity.
Discussion
Although it is not possible to test all the dry rubber products available in the market, the present study has examined most of the major rubber grades used in the dry rubber product manufacturing industry, wither in their raw, compounded or vulcanized forms, as well as several finished products. Findings have shown that in all cases, dry rubbers and their products have insignificant amount of residual extractable protein fraction containing the allergens. Their removal apparently occurred mainly during processing of the raw rubbers, whereby these allergenic proteins were either rendered insoluble by the acid treatment or leached out by the extensive and continuous washing employed throughout the procedure. Subsequently processes converting them into products such as compounding, vulucanization and product fabrication all of which were usually conducted in dry rubber state, did not induce any marked changes to either their low EP content or their ‘non-allergenicity’. This is unlike the latex gloves, where their EP increases9 when the ‘wet gel’ gloves are vulcanized/dried at elevated temperature, due to migration of more soluble allergenic proteins along with considerable amounts of water to the surface of the latex film as it is being dried.21
Assessments of both the in-vitro and in-vivo allergen activities of the test samples by the RASR-inhibition immunoassay and the skin-prick test respectively, have been shown to be consistent with the ‘non-allergenicity’ of these products as suggested by their remarkably low EP contents. It may be of interest to know that these two methods of assessment are very well correlated.22 It is also noteworthy that these findings confirm the association of low EP contents with low allergen activity and the near absence of allergen activity or non-allergenicity related to EP levels less than 100µg/g, as reported earlier.14 However, it may be pointed out that there may be an extremely small number of individuals who are highly atopic, and who may develop sensitivity to a great number of things atopic, and who may contact with. Such people should be identified, treated specially and allergen avoidance should be recommended.
It may be of interest to mention that the inhibition of IgE binding to latex proteins reported for extract of fragments from a worn and contaminated tyre,15 may not necessarily be due to latex antigens. The possibility of some other antigens in the extract effecting such an interaction due to cross-reaction23,24 cannot be excluded.
It is hence reasonable to conclude that, as tested by the best methods available, low residual extractable protein contents, but also very low or negligible allergenicity. This is not withstanding the fact that there are relatively fewer dry rubber products used in the healthcare sector where prevalence of Type 1 hypersensitivity has been reported. Furthermore, products such as the cut threads which are often used as medical bandages, are not likely to pose any problem since they are generally covered by fabric thereby minimising any contact with the human skin. Therefore NR dry rubber products are essentially not affected by the protein allergy.
Acknowledgements
The authors wish to thank the Director of the Rubber Research Institute of Malaysia for permission to publish this paper. The capable technical assistance of Mr Alias Jaedin and Ms Päivi Hanhiney is also gratefully acknowledged. Thanks are also extended to Mr Ng Yu Tong of the SMR Quality Control Laboratory for the dry rubber samples and to the Assistant Director of Chemistry and Technology Department of the RRIM for his helpful comments on the manuscript.
References