Laboratory for Pathology and Immunobiology, National Institute for Public Health and the Environment, RIVM, P.O.Box 1, 3720 BA Bilthoven, The Netherlands
Received October 1, 2001; accepted December 4, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: allergy; latex; chemicals; LLNA.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Few studies have dealt with quantitative determinations of rubber chemicals in rubber products (Emmet et al., 1994; Hansson et al., 1997; Kaniwa et al., 1994a
; Knudsen et al., 1993
). An extraction procedure and quantitative analysis for the most common rubber chemicals in rubber gloves has been described recently (Knudsen et al., 2000a
). There was no clear correlation between chemical content in latex gloves and positive patch-test reactivity in patients (Knudsen et al., 2000b
). However, a tendency was seen for gloves with a low combined amount of chemicals in the extracts to give few positive reactions in the patients, while gloves with a high amount of chemicals gave many positive reactions (Knudsen et al., 2000b
). In addition, cross reactivity can occur between various chemicals, i.e., between thiurams and carbamates (Knudsen et al., 2000b
; Knudsen and Menné, 1996
), and mixtures of chemicals can be present in the gloves.
The chemicals most often reported as contact sensitizers belong to the thiuram, carbamate, and mercaptobenzothiazole groups, but also guanidines and several antioxidants have occasionally been reported as sensitizers (Estlander et al., 1994). The risk for sensitization and the resulting contact hypersensitivity reactions can theoretically be eliminated by a reduction of exposure and/or a complete substitution of sensitizers with nonsensitizers (Cardin et al., 1986
; Rycroft et al., 1992
). This is, however, not compatible with the use of natural rubber products. The use of new technologies such as irradiation instead of the traditional vulcanization process may, however, decrease the demand for accelerators (Wan Manshol, 1998
). Also the use of products manufactured from alternative materials will reduce induction of allergy to rubber chemicals. However, this does not completely prevent sensitization by rubber chemicals, since the same accelerators and antioxidants may be used as in latex production. Substitution of strong allergens with weaker allergens also is a possibility. Comparison and rating of sensitizers has been performed using experimental models in guinea pigs (Andersen et al., 1995
; Wang and Suskind, 1988
) and mice (Basketter et al., 1999b
; Ikarashi et al., 1994
; Van Och et al., 2000
). However, experiments systematically investigating a wide range of chemicals used in natural rubber latex products have not been reported.
The local lymph node assay (LLNA) is now commonly used for the identification of sensitizing activity of chemicals, and the assay has been validated to the guinea pig maximization test (Basketter and Scholes, 1992; Basketter et al., 1993
; Kimber et al., 1995
). Test chemicals are applied to the dorsum of the ear and lymphocyte activation is determined by measuring cell proliferation in the draining auricular lymph nodes, which can be done either by in vivo or ex vivo tritium-thymidine labeling of the cells (Kimber et al. 1995
; Kimber and Weisenberger 1989
, Van Och et al. 2000
). Analysis of 134 chemicals tested in the local lymph node assay (LLNA), guinea pig maximization test (GPMT), and/or with clear clinical evidence for human skin sensitization potential, revealed that an EC3 (effective concentration causing a stimulation index of 3 compared to vehicle control) in the LLNA is an acceptable threshold value for hazard identification (Basketter et al., 1999b
). In the local lymph node assay, quantitative data are obtained on the induction phase of the immune response. So, based on the EC3 value of sensitizing chemicals, an accurate assessment of sensitizing potency is possible (Basketter et al., 1999b
; Van Och et al., 2000
).
In the LLNA, responses to chemicals can be compared and a ranking order of weak, moderate, or strong sensitizers can be established (Basketter et al., 1999a,b
; Van Och et al., 2000
). This offers the opportunity to use the LLNA in selecting chemicals with low or minimal allergenic potency. In this study, a modified LLNA (sodium dodecyl sulfate pretreatment and ex vivo cell labeling) was used for determination of the sensitizing potential of 15 chemicals used during natural rubber latex production, and known to induce contact hypersensitivity reactions in man.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All other husbandry conditions were maintained according to all applicable provisions of the following national laws: Experiments on Animals Decree, and Experiments on Animals Act.
Chemicals.
The chemicals investigated (Table 1) belong to different groups of compounds used during latex rubber glove production, such as thiurams, carbamates, benzothiazoles, and amines. TMTD, TETD, ZDEC, MBT, MBI, MBTS, and DEA were obtained from Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands. ZDMC was obtained from Fluka, Zwijndrecht, The Netherlands. TMTM was obtained from Acros Organics, Geel, Belgium. TBTD, PTD, ZDBC, and ZPC (Robinson Brothers, Ltd., West Bromwich, UK), PTT (ICN Pharmaceuticals, Costa Mesa, CA) and ZMBT (Semperit, Vienna, Austria) were kindly provided by Dr. C. Hametmer, Östereichisches Institut für biomedizinische Werkstofftechnik, Vienna, Austria.
|
Experimental design.
The sensitizing potency of the chemicals was investigated in a modified local lymph node assay using ex vivo labeling of the proliferating lymph node cells (Kimber and Weisberger 1989, Vandebriel et al., 2000; Van Och et al.. 2000
). One hour before the chemicals were applied, 25 µl of 1% sodium dodecyl sulfate (SDS, Merck BV, Amsterdam, The Netherlands) was applied epicutaneously to the dorsum of both ears to enhance responses to weak sensitizers (De Jong et al., manuscript in preparation). Additionally, 25 µl of test solution or vehicle control was applied to the dorsum of both ears (50 µl per animal) of female BALB/c mice daily for 3 consecutive days (days 0, 1, and 2). At day 5 following start of treatment, animals were sacrificed and draining (auricular) lymph nodes (LN) were excised. Isolated left and right LNs from each mouse were weighed, and single-cell suspensions prepared using a cell strainer (Falcon, Franklin Lakes, NJ, USA). Cells were washed twice and suspended in RPMI 1640 (Gibco, Grand Island, NY, USA) culture medium supplemented with 10% heat inactivated fetal calf serum (PAA, Linz, Austria), 100 IU/ml penicillin, and 100 µg/ml streptomycin, referred to as supplemented medium. Cells were counted in a Coulter Counter (Coulter Electronics, Mijdrecht, the Netherlands) and adjusted to a concentration of 1 x 107 cells/ml. When necessary, cell suspensions of several animals were pooled in order to obtain cell concentrations of 1 x 107 cells/ml, notably so for vehicle (AOO)-treated controls.
Lymphocyte stimulation test.
LN cell suspensions, 2 x 106 cells in 200 µl, were cultured in RPMI 1640-supplemented medium in triplicate in round-bottomed, 96-well microtiter plates (Greiner, Alphen aan de Rijn, The Netherlands). An aliquot of 10 µl of 3H-methylthymidine (3H-TdR, Amersham International, Buckinghamshire, UK), 37 kBq/well, 3.7 MBq/ml, 100 µCi/ml), specific activity 185 GBq/mmol (5 Ci/mmol in 217.8 µg/ml cold thymidine in PBS) was added to the wells of the cell culture directly after initiation of culture. Cultures were maintained for 24 h at 37°C in a humidified atmosphere of 5% CO2 in air. The cellular DNA was harvested on glass fiber filters using an automatic cell harvester (Harvester 96® Tomtec, Orange, CT), scintillation liquid was added, and incorporation of 3H-TdR into the DNA was measured by liquid scintillation in a ß plate counter (1205 BetaplateTM, Wallac, Turku, Finland). Proliferation per animal was determined by calculating the 3H-TdR incorporation for the total cell number harvested (left and right lymph nodes combined).
Statistical analysis.
The EC3 (effective concentration inducing a 3-fold increase in 3H-thymidine incorporation in the harvested lymph node cells of treated animals compared to vehicle-treated animals) was estimated by the benchmark approach, by fitting a nonlinear regression model to the data of all individual animals. The choice of the model for deriving the EC3 follows from a procedure of applying likelihood ratio tests on the members of the following nested family of models.
For each data set (compound), one of these models was selected by choosing a more complicated model when the increase in number of parameters resulted in a significantly better fit to the dose-response data. The selected model was used to estimate the EC3 (point estimate). Additionally, an estimate of the uncertainty (90% confidence interval) associated with the estimated EC3 was determined using a (parametric) bootstrap method (Slob and Pieters, 1998), as follows. Once a model is selected for describing the dose-response data, this fitted model is used as a basis for generating 200 artificial data sets (according to the experimental design) by Monte Carlo sampling. For each generated data set, the EC3 is re-estimated. Taking all these EC3s together results in a distribution representing the uncertainty associated with the EC3 estimate. The 5th and 95th percentiles of this empirical distribution were determined, serving as a 90% confidence interval for the EC3. Full statistical details are given in Slob (2001, in press). Other applications can be found in Slob (1999), Piersma et al. (2000), Van Och et al. (2000), and Woutersen et al. (2001).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There was no group of chemicals, as a whole, that showed either a low or high allergenicity for all compounds as indicated by the EC3 value. Our results suggest that for the thiuram compounds, an increase in EC3 value, and thus a decrease in sensitizing potency, occurs with an increase in the length of the side chains. For TMTD (methyl side chains) the EC3 was 0.7%, for TETD (ethyl side chains) 1.4%, and for TBTD (butyl side chains) 34.8%. For the corresponding carbamates, a similarity could be observed for ZDBC (butyl side chains) being nonsensitizing (EC3 value could not be determined). In contrast ZDMC (methyl side chains, EC3 = 2.7%) was less potent than ZDEC (ethyl side chains, EC3 = 0.4%). The benzothiazoles showed EC3 values from low (MBTS EC3 = 2.9%) to high (ZMBT EC3 = 30.3%). For TMTD, ZDMC, MBT, and DEA, a decreasing order in sensitizing potential has already been reported (Van Och et al., 2000). Ikarashi et al. (1993) reported a decreasing order in sensitizing activity for IPPD (N-isopropyl-N'-phenyl-p-phenylenediamine), TMTD, MBT, and ZDEC. In contrast to their results we found ZDEC to be more potent than MBT. Furthermore, for none of the doses investigated was an SI = 3 reached for TMTD and ZDEC by Ikarashi et al. (1993). The comparison of sensitizing potency was based on the highest proliferation index obtained. In our view this is less accurate than comparison on the basis of the EC3 value. Also the difference in vehicle used for ZDEC may have contributed to the low reactivity in the study of Ikarashi et al. (1993). We used acetone-olive oil, which is commonly used in the LLNA, whereas Ikarashi et al. used chloroform. Although for strong sensitizers the influence of the vehicle is probably limited, it is known that the vehicle has an effect on the performance of the LLNA (Edwards et al., 1994
; Montelius et al., 1996
; Warbrick et al., 1999
).
Besides the qualification of a compound as sensitizer, the use of nonlinear regression analysis for calculation of the EC3 offers the advantage of a quantitative estimation of the confidence limits of the estimated EC3. Nonlinear regression analysis as applied in this study, uses all available individual data, and not just the mean of the treated animals. Thus, a 90% confidence interval could be calculated. For the stronger sensitizers in general, the 90%-percentile confidence interval is smaller than for the weak sensitizers (also on the log-scale, i.e. in a relative sense). The 5th percentile level may be interesting from the point of risk management, as it indicates that below this level there is only a 5% probability that positive reactions do occur. So the 5th percentile of the EC3 is very similar to the BMDL (lower confidence limit of the benchmark dose) and may be used as the lowest acceptable dose (or concentration for exposure) for risk management purposes. Comparing our results with clinical data (Knudsen et al., 2000b) for the weakest identified chemicals a low frequency of positive reactions was found, while for the strongest identified chemicals a high frequency of positive reactions was observed.
In conclusion, a ranking is presented for rubber chemicals known to induce contact hypersensitivity reactions in man. When using these chemicals for the production of natural rubber latex products, the preference would be to use chemicals with low sensitizing activity or no such activity. Our results indicate that the chemicals of choice are for the thiuram compounds, TBTD; for the carbamates, ZDBC; and for the benzothiazoles, ZMBT.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Basketter, D. A., Lea, L. J., Cooper, K., Stocks, J., Dickens, A., Pate, I., Dearman, R. J., and Kimber, I. (1999a). Threshold for classification as a skin sensitizer in the local lymph node assay: A statistical evaluation. Food Chem. Toxicol. 37, 11671174.[ISI][Medline]
Basketter, D. A., Lea, L. J., Dickens, A., Briggs, D., Pate, I., Dearman, R. J., and Kimber, I. (1999b). A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses. J. Appl. Toxicol. 19, 261266.[ISI][Medline]
Basketter, D. A., and Scholes, E. W. (1992). Comparison of the local lymph node assay with the guinea-pig maximization test for the detection of a range of contact allergens. Food Chem. Toxicol. 30, 6569.[ISI][Medline]
Basketter, D. A., Selbie, E., Scholes, E. W., Lees, D., Kimber, I., and Botham, P. A. (1993). Results with OECD recommended positive control sensitizers in the maximization: Buehler and local lymph node assays. Food Chem. Toxicol. 31, 6367.[ISI][Medline]
Cardin, C. W., Weaver, J. E., and Bailey, P. T. (1986). Dose-response assessments of Kathon biocide: II. Threshold prophetic patch testing. Contact Dermatitis 15, 1016.[ISI][Medline]
Conde-Salazar, L., del Rio, E., Guimaraens, D., and Gonzalez-Domingo, A. (1993). Type IV allergy to rubber additives: A 10-year study of 686 cases. J. Am. Acad. Dermatol. 29, 176180.[ISI][Medline]
Cronin, E. (1980) Rubber. In Contact Dermatitis (E. Cronin, Ed.), pp 714740. Churchill Livingstone, New York.
Duarte, I., Nakano, J. T., and Lazzarini, R. (1998). Hand eczema: Evaluation of 250 patients. Am. J. Contact. Dermatol. 9, 216223.
Edwards, D. A., Soranno, T. M., Amoruso, M. A., House, R. V., Tummey, A. C., Trimmer, G. W., Thomas, P. T., and Ribeiro, P. L. (1994). Screening petrochemicals for contact hypersensitivity potential: A comparison of the murine local lymph node assay with guinea pig and human test data. Fundam. Appl. Toxicol. 28, 179187.
Emmett, E. A., Risby, T. H., Taylor, J., Chen, C. L., Jiang, L., and Feinman, S. E. (1994). Skin elicitation thresholds of ethylbutyl thiourea and mercaptobenzothiazole with relative leaching from sensitizing products. Contact Dermatitis 30, 8590.[ISI][Medline]
Estlander, T., Jolanki, R., and Kanerva, L. (1986). Dermatitis and urticaria from rubber and plastic gloves. Contact Dermatitis 14, 2025.[ISI][Medline]
Estlander, T., Jolanki, R., and Kanerva, L. (1994). Allergic contact dermatitis to rubber and plastic gloves. In Protective Gloves for Occupational Use (G. A. Mellström, J. Wahlberg, and H. I. Maibach, Eds.), pp. 221239. CRC, Boca Raton, FL.
Fregert, S., Hjorth, N., Magnusson, B., Bandmann, H. J., Calnan, C. D., Cronin, E., Malten, K., Meneghini, C. L., Pirila, V., and Wilkinson, D. S. (1969). Epidemiology of contact dermatitis. Trans. St. Johns Hosp. Dermatol. Soc. 55, 1735.[Medline]
Hansson, C., Bergendorff, O., Ezzelarab, M., and Sterner, O. (1997). Extraction of mercaptobenzothiazole compounds from rubber products. Contact Dermatitis 36, 195200.[ISI][Medline]
Ikarashi, Y., Ohno, K., Momma, J., Tsuchiya, T., and Nakamura, A. (1994). Assessment of contact sensitivity of four thiourea rubber accelerators: Comparison of two mouse lymph node assays with the guinea pig maximization test. Food Chem. Toxicol. 32, 10671072.[ISI][Medline]
Ikarashi, Y., Tsuchiya, T., and Nakamura, A. (1993). Evaluation of contact sensitivity of rubber chemicals using the murine local lymph node assay. Contact Dermatitis 28, 7780.[ISI][Medline]
Kaniwa, M.-A., Isama, K., Nakamura, A., Kantoh, H., Itoh, M., Ichikawa, M., and Hayakawa, R. (1994a). Identification of causative chemicals of allergic contact dermatitis using a combination of patch testing in patients and chemical analysis. Application to cases from industrial rubber products. Contact Dermatitis 30, 2025.[ISI][Medline]
Kaniwa, M.-A., Isama, K., Nakamura, A., Kantoh, H., Itoh, M., Miyoshi, K., Saito, S., and Shono, M. (1994b). Identification of causative chemicals of allergic contact dermatitis using a combination of patch testing in patients and chemical analysis. Application to cases from rubber footwear. Contact Dermatitis 30, 2634.[ISI][Medline]
Kimber, I., Hilton, J., Dearman, R. J., Gerberick, G. F., Ryan, C. A., Basketter, D. A., Scholes, E. W., Ladics, G. S., Loveless, S. E., and House, R. V. (1995). An international evaluation of the murine local lymph node assay and comparison of modified procedures. Toxicology 103, 6373.[ISI][Medline]
Kimber, I., and Weisenberger, C. (1989). A murine local lymph node assay for the identification of contact allergens: Assay development and results of an initial validation study. Arch. Toxicol. 63, 274282.[ISI][Medline]
Kligmann, A. (1966). The identification of contact allergens by human assay. J. Invest. Dermatol. 47, 393408.[ISI][Medline]
Knudsen, B. B., Hametner, C., Seycek, O., Heese, A., Koch, H.-U., and Peters, K.-P. (2000a). Allergologically relevant rubber accelerators in single-use medical gloves. Contact Dermatitis 43, 915.[ISI][Medline]
Knudsen, B. B., Hametmer, C., Seycek, O., Heese, A., Koch, H.-U., and Peters, K.-P. (2000b). Bioavailability of rubber accelerators in rubber gloves and patch test reactivity. Dermatosen 48, 127133.
Knudsen, B. B., Larsen, E., Egsgåard, H., and Menné, T. (1993). Release of thiurams and carbamates from rubber gloves. Contact Dermatitis 28, 6369.[ISI][Medline]
Knudsen, B. B., and Menné, T. (1996). Contact allergy and exposure patterns to thiurams and carbamates in consecutive patients. Contact Dermatitis 35, 9799.[ISI][Medline]
Magnusson, B., and Kligman, A. M. (1969). The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Dermatol. 52, 268276.[ISI][Medline]
Maurer, T. H., Thomann, P., Weirich, E. G., and Hess, R. (1979). Predictive evaluation in animals of the contact allergenic potential of medically important substances: II. Comparison of different methods of cutaneous sensitization with "weak" allergens. Contact Dermatitis 5, 110.[ISI][Medline]
Montelius, J., Boman, A., Wahlkvist, H., and Wahlberg, J. E. (1996). The murine local lymph node assay: Search for an alternative, more adequate, vehicle than acetone/olive oil (4:1). Contact Dermatitis 34, 428430.
Piersma, A. H., Verhoef, A., te Biesebeek, J. D., Pieters, M. N., and Slob, W. (2000). Developmental toxicity of butyl benzyl phthalate in the rat using a multiple dose study design. Reprod. Toxicol. 14, 417425.[ISI][Medline]
Rycroft, R. J. G., Menné, T., Frosch, P. J, and Benezra, C. (1992). Textbook of Contact Dermatitis. Springer-Verlag, Berlin.
Slob, W. (1999) Deriving safe exposure levels for chemicals from animal studies using statistical methods: Recent developments. In Statistics for the Environment 4: Statistical Aspects of Health and the Environment (V. Barnett, A. Stein, and K. F. Turkman, Eds.), pp. 153174. John Wiley, New York.
Slob, W. (2001). Dose-response modeling of continuous endpoints. Toxicol. Sci. (in press).
Slob, W., and Pieters, M. N. (1998). A probabilistic approach for deriving acceptable human intake limits and human health risks from toxicological studies: General framework. Risk Anal. 18, 787798.[ISI][Medline]
Taylor, J. S. (1995). Allergy to rubber. In Fisher's Contact Dermatitis, 4th ed, pp. 697752. Williams and Wilkins, Baltimore.
Vandebriel, R. J., De Jong, W. H., Spiekstra, S. W., Van Dijk, M., Fluitman, A., Garssen, J., and Van Loveren, H. (2000). Assessment of preferential T-helper 1 or T-helper 2 induction by low molecular weight compounds using the local lymph node assay in conjunction with RT-PCR and ELISA for interferon-gamma and interleukin-4. Toxicol. Appl. Pharmacol. 162, 7785.[ISI][Medline]
Van Och, F. M., Slob, W., De Jong, W. H., Vandebriel, R. J., and Van Loveren, H. (2000). A quantitative method for assessing the sensitizing potency of low molecular weight chemicals using a local lymph node assay: Employment of a regression method that includes determination of the uncertainty margins. Toxicology 146, 4959.[ISI][Medline]
Wan Manshol bin, W. Z. (1998). Radiation vulcanized natural rubber latex (RVNRL). Malaysian Rubber Seminar, pp 2831. Lembaga Getah Malaysia (Malaysian Rubber Board), Kuala Lumpur.
Wang, X. S., and Suskind, R. R. (1988). Comparative studies of the sensitization potential of morpholine, 2 mercaptobenzothiazole, and 2 of their derivatives in guinea pigs. Contact Dermatitis 19, 1115.[ISI][Medline]
Warbrick, E. V., Dearman, R. J., Basketter, D. A., and Kimber, I. (1999). Influence of application vehicle on skin sensitization to methylchloroisothiazolinone/methylisothiazolinone: An analysis using the local lymph node assay. Contact Dermatitis 41, 325329.[ISI][Medline]
Woolhiser, M. R., Munson, A. E., and Meade, B. J. (2000). Comparison of mouse strains using the local lymph node assay. Toxicology 146, 221227.[ISI][Medline]
Woutersen, R. A., Jonker, D., Stevenson, H., te Biesebeek, J. D., and Slob, W. (2001). The benchmark approach applied to a 28-day toxicity study with Rhodorsil Silane in rats: The impact of increasing the number of dose groups. Food Chem. Toxicol. 39, 796707.
Wrangsjö, K., and Meding, B. (1994). Occupational contact allergy to rubber chemicals. Dermatosen 42, 184189.[ISI]