Industrial Health, 1977, 15, 87. EXPERIMENTAL STUDY ON CROSS-CONTACT ALLERGY DUE TO DITHIOCARBAMATE FUNGICIDES Toshio MATSUSHITA õ, Mitsuki YOSHIOKA õ, Yoshiki ARIMATSU õ õ and Shigeru NOMURA õ õ õd epartment of Hygiene, Faculty of Medicine, Kagoshima University, Usuki-cho, Kagashima 890 Japan õ õd epartment of Public Health, Kumamoto University Medical School, Honjo, Kumamoto 860 Japan (Received March 14, 1977) Cross sensitizations among dithiocarbamate fungicides were investigated using the guinea pig maximization test. Extreme potency of cross-reaction was noted among ethylene bis-dithiocarbamates, maneb, mancozeb and zineb. Such degree of potentialities, however, was not revealed in cases of dimethyldithiocarbamates, ferbam, thiram, ziram and dimethyldithiocarbamic acid sodium salt. Cross-contact allergy among other combinations of dithiocarbamates and their related compounds were tested and discussed. One of the most interesting aspects of allergic contact dermatitis is the phenomenon of cross-sensitization, in which one chemical is associated with allergic sensitization to one or more related chemicals. Some chemicals with such potency are listed up in an Adams' book on occupational contact dermatitis.1) Though some cross sensitizations have been indicated for carbamate fungicide thiram and its derivatives2) and the authors have demonstrated preliminary about cross-reactions among some dithiocarbamate fungicides,3,4) but scanty information have been reported on cross-sensitivity of the dithiocarbamate fungicides. The present investigation was undertaken to serve this problem. MATERIALS AND METHODS The chemicals tested in the present study were follows : ethylene bis-dithiocarbamic acid group meneb (Maneb dithane M ôr), mancozeb (Ziman dithane ôr) and zineb (Dithane ôr) ; dimethyldithiocarbamic acid derivatives ferbam, ziram and dimethyldithiocarbamic acid sodium salt ; other related compound-lime sulfur mixture. The chemicals were diluted with distilled water, except for dimethyldithiocarbamic 87
T. MATSUSHITA, M. YOSHIOKA, Y. ARIMATSU & S. NOMURA acid derivatives with ethanol 70%, and their solutions or emulsions were prepared for each experiment. Threshold limits of primary irritancy by the test chemicals on skin of guinea pigs In order to decide challenging topical concentrations of the test chemicals used in the latter experiment, threshold limits of primary irritancy by test chemicals on the skin of guinea pigs were determined. The animals used for this experiment were matured female Hartley strain weighing about 500 g. Six animals each were used in a group. The application area on the flank was clipped free of hair and shaved clean with an electric razor about 4 hr before treatment. The test preparations were closely patched using Testpflaster ôr for 24 hr around the flank. When erythema was revealed on the skin, the lowest concentrations of the test chemicals were determined to be threshold limits for irritating reaction. Allergenicity of test chemicals predicted by guinea pig maximization test To evaluate allergenicity of the test chemicals, the guinea pig maximization test5) was applied. Hartly strain female guinea pigs weighing 300-500 g were used throughout the study. Ten animals were served in each group. The induction and challenge were conducted as the original method. The topical concentrations of test chemicals applied for challenge were below the threshold levels obtained from the former experiment. The concentrations of the chemicals used for induction and challenge are to be found together with results in tables. Using a procedure developed by Kligman6), the results of the maximization test read at 24 hr after removal of the patch, were devided into five classes, ranging from weak (grade I) to extreme (grade V), according to the percentage of animals sensitized. Cross sensitizations among dithiocarbamates After guinea pigs were inducted by test chemicals alike the former experiment, the animals were challenged by a few another preparations applied for the induction and the cross sensitizations were evaluated by reading of challenged reactions. RESULTS Threshold limits of primary irritating effect by test chemicals on skin of guinea pigs The values were 5% or more in maneb, mancozeb, zineb and thiram, 10% and/or more in ferbam, ziram and dimethyldithiocarbamic acid sodium salt, and 20% or more in lime sulfur mixture. From the results of this experiment, the challenging topical concentrations of these compounds were decided below the concentration of those values, namely, such combinations as 5% and 1%, or 2% and 0.5%, respectively. Allergenicity of dithiocarbamates predicted by guinea pig maximization test 88 As indicated in Table 1, the allergenicity ratings for the ethylene bis-dithicarbamates
CROSS SENSITIZATION AMONG DITHIOCARBAMATES tested were grade V. Those of ferbam, thiram, ziram and lime sulfur mixture were almost grade III. Nevertheless, no sensitized animals were observed in case of dimethyldithiocarbamic acid sodium salt. Table 1. Allergenicity of dithiocarbamate fungicides predicted by guinea pig maximization test. ranging weak (grade I) to extreme (grade V), using a procedure developed by Kligman (1967). The numerals inside parentheses indicate the percentage for sensitized animals. Table 2. Cross sensitizations among the same group of ethylene bis-dithiocarbamates. 25%, respectively. 89
T. MATSUSHITA, M. YOSHIOKA, Y. ARIMATSU & S. NOMURA Cross sensitizations among dithiocarbamates As seen in Table 2, the allergenicity ratings for the cross sensitization among the same group of ethylene bis-dithiocarbamates were estimated as extreme in potency of causing contact allergy. But, as shown in Table 3, such degree of potencialities was not observed in cases of dimethyldithiocarbamates. Comparatively high degree of cross-reactions were noted between ferbam with thiram of ziram, thiram with ferbam, ziram or dimethyldithiocarbamic acid sodium salt. Nextly, cross-reactions between ethylene bis-dithiocarbamates and dimethyldithiocarbamates were observed in some combinations tested (see Table 4). The most high potency of causing cross sensitization was noted between the ethylene bis-dithiocarbamates and ziram. Though moderate degree of potency of causing cross-contact allergy was evaluated generally between mancozeb and zineb with ferbam, thiram, ziram and lime sulfur mixture, such degree of potentiality was not observed in case of the former chemicals with dimethyldithiocarbamic acid sodium salt. When dimethyldithiocarbamates were treated with the induction, cross-reactions with Table 3. Cross sensitivity among the same group of dimethyldithiocarbamates. 90 respectively.
CROSS SENSITIZATION AMONG DITHIOCARBAMATES Table 4. Cross-reactions between ethylene bis-dithiocarbamates and dimethyldithiocarbamates. 25%, respectively. ethylene bis-dithiocarbamates were comparatively lower than that of the results obtained in the former experiment (see Table 5). Cross-reactions between lime sulfur mixture and dithiocarbamates investigated, however, were mild to moderate in potency of causing contact sensitization. DISCUSSION Most chemicals involved in allergy are complex antigens and some cross-antigenic. Such chemicals as sulfonamides, organic color compounds (p-phenylenediamine, azo-compounds), thiram sulfides, and so forth have been known to cause cross sensitization7-10). 91
T. MATSUSHITA, M. YOSHIOKA, Y. ARIMATSU & S. NOMURA Table 5. Cross-reactions between dimethyldithiocarbamates and ethylene bis-dithiocarbamates. 25%, respectively. In the present study, considerable theoretical interest was found in the problem allergenicity of dithiocarbamate fungicides in connection with their chemical structure and physicochemical properties. Among two types of the dithiocarbamates (see Fig. 1), ethylene bis-dithiocarbamates seemed to be stronger sensitizers than dimethyldithiocarbamates, which were examined using the guinea pig model for contact hypersensitivity. As well known, the greater the degree of hypersensitivity to the primary allergen, the marked is the tendency to develop cross-sensitization to additional substance in general. Ethylene bis-dithiocarbamates seemed to have such potentiality of causing cross sensitization. In 92
CROSS SENSITIZATION AMONG DITHIOCARBAMATES ( a ) ( b ) Fig. 1. The chemical formula for two types of dithiocarbamates. this case, the degradation products of these chemicals might be similar and played the role of antigenic determinant. With two dithiocarbamate groups, weaker cross-reactions were obtained in the dimethyldithiocarbamate group, e.g., among ferbam, thiram, ziram and dimethyldithiocarbamic acid sodium salt. Very low rates of cross-reaction were noted especially between dimethyldithiocarbamic acid sodium salt and other dithiocarbamates tested. On the other hand, some cross sensitization was noted between sulfuric compound and the dithiocarbamates. From the results, cross-contact allergy for the dithiocarbamates may be caused partially by sulfuric atoms in association, but its role must not be major problem. As the skin is highly selective in its ability to detect antigenic groups and this specificity is so great that even optical isomers can be differentiated"), further study is needed to clarify the exact mode of allergenicity and cross-reactivity of dithiocarbamate fungicides. ACKNOWLEDGEMENTS The authors wish to express their gratitude for skilled technique to Mrs. T. Takahashi and Misses Y. Nishina and M. Kawanabe. This study was supported, in part, by grant from the Japanese Ministry of Education and the Ministry of Public Health Welfare. REFERENCES 1) Adams, R.M. (1969). Occupational Contact Dermatitis. p. 21. Lippincott, Philadelphia. 2) Fisher, A.A. (1973). Contact Dermatitis. 2nd Ed. p. 300. Lea & Febiger, Philadelphia. 3) Matsushita, T., Arimatsu, Y. and Nomura, S. (1976). Mt. Arch. Occup. Environ. Health, 37, 169. 4) Matsushita, T., Arimatsu, Y. and Nomura, S. (1977). Mt. Arch. Occup. Environ. Health, in press. 5) Magnusson, B. and Kligman, A.M. (1969). J. Invest. Dermatol., 52, 268. 93
T. MATSUSHITA, M. YOSHIOKA, Y. ARIMATSU & S. NOMURA 6) Kligman, A.M. (1967). J. Invest. Dermatol., 47, 369. 7) Baer, R.L., Leider, M. and Mayer, R.L. (1948). Proc. Soc. Exp. Biol. Med., 67, 489. 8) Fregert, S. (1974). Manual of Contact Dermatitis. p. 16. Munksgaad, Copenhagen. 9) Kleniewska, D. (1975). Berufsdermatosen, 23, 31. 10) Rudzki, E. and Krajewska, D. (1976). Contact Dermatitis, 2, 311. 11) Rostenberg, A., Jr. (1964). J. Asthma Res., 2, 25. 94