Ranking of Allergenic Potency of Rubber Chemicals in a Modified Local Lymph Node Assay

Wim H. De Jong,1, François M. M. Van Och, Constance F. Den Hartog Jager, Sander W. Spiekstra, W. Slob, Rob J. Vandebriel and Henk Van Loveren

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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A modified local lymph node assay (LLNA) with ex vivo tritium thymidine (3H-TdR) labeling of the proliferating lymph node cells was used for determination of the allergenic potency of chemicals used in the production of rubber for latex medical gloves. Fifteen chemicals known to induce contact hypersensitivity reactions in man, including various thiuram, carbamate, and benzothiazole compounds, and one amine were tested. The EC3 (effective concentration inducing a 3-fold increase in proliferation of lymph node cells [Stimulation Index, SI = 3]) was calculated with nonlinear regression analysis, including a bootstrap method for determination of the 5–95% confidence interval of the EC3 value. This procedure identified 14 out of the 15 chemicals tested as sensitizers, while for one chemical, ZDBC, no EC3 could be calculated due to low responses and a lack of a dose-response relationship in the data obtained. The ranking order of the chemicals with increasing EC3 values (and thus decreasing allergenic potency) was found to be in the following order: ZDEC < TMTD < TETD < ZPC < ZDMC < MBTS < PTD < TMTM < MBT < MBI < PTT < ZMBT < TBTD < DEA < ZDBC. Our results indicate that the chemicals of choice for use in the production of natural rubber latex products would be for the thiuram compounds, TBTD; for the carbamates, ZDBC; and for the benzothiazoles, ZMBT. However, one has to be aware that besides potency, the total amount of residual chemical present in the final product is also important for allergy induction.

Key Words: allergy; latex; chemicals; LLNA.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The use of natural rubber latex products poses risks for the development of allergy. Latex products can induce both immediate-type (IgE-mediated, type I) hypersensitivity due to the latex proteins, and/or delayed type (cellular-mediated, type IV) hypersensitivity due to the chemicals present as residues. Chemicals are added to rubber to improve it: to prolong its life and to give it special properties. Some of the chemicals are added directly at harvesting of the latex sap as preservatives, others are added as vulcanizers, accelerators, retardants, and/or antioxidants (Cronin, 1980Go). The added chemicals have been well documented as contact sensitizers in experimental studies in animals and humans, as well as in clinical studies (Cronin, 1980Go; Duarte et al., 1998Go; Estlander et al., 1986Go; Kaniwa et al., 1994aGo; Kligmann, 1966Go; Magnusson and Kligmann, 1969; Maurer et al., 1979Go; Taylor, 1995Go; Wrangsjö and Meding, 1994Go). Rubber polymer itself is not allergenic (Cronin, 1980Go). In most cases sensitization by rubber chemicals is related to the use of natural rubber latex gloves (Conde-Salazar et al., 1993Go), but sensitization from rubber shoes also has been reported (Kaniwa et al., 1994bGo). Causality has been demonstrated by identifying the particular chemical in the rubber material and the presence of a positive patch test in humans to the rubber material, concomitantly with a positive patch test to the pure chemical (Fregert et al., 1969Go; Rycroft et al., 1992Go).

Few studies have dealt with quantitative determinations of rubber chemicals in rubber products (Emmet et al., 1994; Hansson et al., 1997Go; Kaniwa et al., 1994aGo; Knudsen et al., 1993Go). An extraction procedure and quantitative analysis for the most common rubber chemicals in rubber gloves has been described recently (Knudsen et al., 2000aGo). There was no clear correlation between chemical content in latex gloves and positive patch-test reactivity in patients (Knudsen et al., 2000bGo). 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., 2000bGo). In addition, cross reactivity can occur between various chemicals, i.e., between thiurams and carbamates (Knudsen et al., 2000bGo; Knudsen and Menné, 1996Go), 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., 1994Go). 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., 1986Go; Rycroft et al., 1992Go). 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, 1998Go). 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., 1995Go; Wang and Suskind, 1988Go) and mice (Basketter et al., 1999bGo; Ikarashi et al., 1994Go; Van Och et al., 2000Go). 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, 1992Go; Basketter et al., 1993Go; Kimber et al., 1995Go). 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. 1995Go; Kimber and Weisenberger 1989Go, Van Och et al. 2000Go). 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., 1999bGo). 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., 1999bGo; Van Och et al., 2000Go).

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., 1999aGo,bGo; Van Och et al., 2000Go). 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Young adult (6–8 weeks of age) female BALB/c mice were obtained from the Central Animal Laboratory of the Institute. BALB/c mice show similar responses in the LLNA compared to CBA/Ca mice (Woolhiser et al, 2000Go). The animals were bred under specified pathogen-free (SPF) conditions. During the experiments the animals were housed barrier-maintained under conventional conditions in light-, humidity-, and temperature-controlled rooms. All animals were housed in macrolon cages. The mice were fed chow pellets (Hope Farms, Woerden, The Netherlands) and water ad libitum.

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 1Go) 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.


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TABLE 1 Chemicals Investigated for Their Sensitizing Potency in a Modified Local Ly mph Node Assay (LLNA)
 
All chemicals were dissolved in 4:1 acetone/olive oil (AOO), and dose response studies were performed with the LLNA, as previously reported (Van Och et al., 2000Go).

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., 2000Go; Van Och et al.. 2000Go). 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.

Model 1: y = a
Model 2: y = a exp(bx)
Model 3: y = a exp(bxd)
Model 4: y = a(c – (c 1)exp(bx))
Model 5: y = a(c – (c – 1)exp(bxd)),
where y is the response, and x denotes the applied concentration. The parameter a represents the level of response at concentration zero, and b can be considered as the parameter reflecting the efficacy of the chemical. At high doses, models 4 and 5 level off to the value ac, so the parameter c can be interpreted as the maximum relative change compared to the background. Models 3 and 5 have the flexibility to mimic threshold-like responses. All these models are nested to each other, except models 3 and 4, which both have 3 parameters. Therefore, these 2 models cannot be (formally) compared to each other by a likelihood-ratio test.

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, 1998Go), 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dose-response evaluations of 2 compounds are presented in Table 2Go. ZDEC induced a strong response for all parameters determined, while ZMBT induced a weak response. For ZDEC the lymph node weight, number of cells isolated per animal, and the 3H-thymidine incorporation per cell culture (2 x 106 cells) and per animal (left and right lymph node combined) increased with higher dosages. For ZMBT, only moderate changes were observed. Especially for the lower dosages of chemicals and the AOO control, lymph node cell populations had to be pooled in order to obtain a sufficient number of cells for the in vitro 3H-thymidine labeling.


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TABLE 2 Dose-Response Studies with the Strong Sensitizer ZDEC and the Weak Sensitizer ZMBT
 
All individual animal data (expressed as proliferation per animal) were used to estimate the best fitting, nonlinear regression model as determined with the maximum likelihood method. For ZDEC and ZMBT, the best fitting curve is presented in Figures 1A and 1BGo, respectively. The EC3 concentration (concentration of the chemical inducing an SI = 3) was estimated using a benchmark approach (see Materials and Methods), by fitting a dose-response model to the observed 3H-thymidine incorporation as a function of the concentration of the chemical (Figs. 1A and 1BGo). The EC3 value for ZDEC (EC3 = 0.4%) is shown in Figure 1AGo, while for ZMBT the EC3 value (EC3 = 30.3%) was calculated by extrapolation of the experimental results shown in Figure 1BGo.



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FIG. 1. (A) Observations (cpm) as a function of concentration for ZDEC together with fitted regression function. SI = 3 with corresponding EC3 value (0.4%) of ZDEC is indicated. (B). Observations (cpm) as a function of concentration for ZMBT together with fitted regression function. SI = 3 not reached with concentrations investigated (EC3 in Table 3Go calculated with regression function).

 
The EC3 value is an estimated value from a best fitting curve. To account for the noise in the data, a parametric bootstrap method was used for further evaluation of the EC3 value. The results of 200 bootstrap runs, indicating possible EC3 values based on the experimental data obtained for ZDEC and ZMBT, are presented in Figures 2A and 2BGo. For ZDEC, the EC3 value ranges from 0.1 to 0.8%, while for ZMBT the EC3 values ranges from 20 to 50%. With the 200 parametric bootstrap runs, the 5 and 95% confidence limits of the EC3 can be estimated. Table 3Go shows the calculated EC3 values and their 90% confidence intervals for 15 chemicals evaluated in the modified LLNA. For 14 out of the 15 chemicals tested an EC3 was determined, identifying these chemicals as sensitizers. For ZDBC, the curve fitting and bootstrap method did not result in an EC3 value, due to low responses and the lack of a dose-response relationship of the data obtained in the LLNA. Based on the EC3 values, the chemicals are ranked from high to low reactivity in the LLNA (Table 3Go).



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FIG. 2. (A) Distribution of EC3 value of ZDEC (n = 200). (B) Distribution of EC3 value of ZMBT (n = 200).

 

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TABLE 3 Ranking of Chemicals Used for Latex Production
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We tested 15 different chemicals used in latex medical-glove production. The chemicals were chosen because of their known sensitizing capacity as they were found to induce contact hypersensitivity reactions in humans (Knudsen et al., 2000bGo). Fourteen of the 15 chemicals were positively identified, while the assay failed to detect ZDBC, a carbamate compound, as a sensitizer. Using the LLNA, we quantified the sensitizing potency and found ZDEC the strongest sensitizer and DEA the weakest. The associated 90% confidence interval gives an indication of the reliability of the estimated EC3 concentration. Use of the EC3 values makes it possible to rank various chemicals used within a single production process, with regard to their sensitizing potency (Basketter et al., 1999bGo; Van Och et al., 2000Go). This offers the possibility for manufacturers to choose chemicals with low sensitizing properties. In the production process of natural rubber latex several different types of chemicals are used, each with their own function in the production process (Cronin, 1980Go). Our results may provide information to make a choice between these chemicals. Especially, chemicals failing to induce an SI = 3 or needing rather high concentrations to induce a stimulation index of 3 seem warranted for use in latex production facilities. Our results indicate that in particular the chemicals of choice are for the thiuram compounds TBTD, for the carbamates ZDBC, and for the benzothiazoles ZMBT. In addition to sensitizing activity, the total amount present and the bioavailability also determine the sensitizing potency of a medical glove manufactured using natural rubber latex. The total amount of chemicals in glove extracts varies but is in the same order of magnitude (approximately 2–15 µmol/g glove), although 10-fold differences between different manufacturers may occur (Knudsen et al., 2000aGo).

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., 2000Go). 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., 1994Go; Montelius et al., 1996Go; Warbrick et al., 1999Go).

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., 2000bGo) 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
 
We gratefully acknowledge the excellent technical assistance of Diane Kegler and Piet Van Schaaik. We thank Prof. J. G. Vos for his critical reading of the manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Fax: ++ 31-30-2744437. E-mail: w.de.jong{at}rivm.nl. Back


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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