Dermal Oncogenicity Studies on Two Methoxysilanes and Two Ethoxysilanes in Male C3H Mice

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FUNDAMENTAL AND APPLIED TOXICOLOGY,79-83(989) Dermal Oncogenicity Studies on Two Methoxysilanes and Two Ethoxysilanes in Male C3H Mice LINVAL R. DEPASS,' * BRYAN BALLANTYNE,! EDWARD H.

1 FUNDAMENTAL AND APPLIED TOXICOLOGY,79-83(989) Dermal Oncogenicity Studies on Two Methoxysilanes and Two Ethoxysilanes in Male C3H Mice LINVAL R. DEPASS,' * BRYAN BALLANTYNE,! EDWARD H. FOWLER,* AND CARROL S. WEIL* * Bushy Run Research Center, Mellon Institute-Union Carbide Corporation, Export, Pennsylvania 3, and \Union Carbide Corporation, Applied Toxicology Department, Danbury, Connecticut 87 Received May , accepted November Dermal Oncogenicity Studies on Two Methoxysilanes and Two Ethoxysilanes in Male C3H Mice. DEPASS, L. R., BALLANTYNE, B., FOWLER, E. H., AND WEIL, C. S. (989). Fundam. Appl Toxtcol., The dermal oncogenic potential of -{3,4-epoxycyclohexyl)ethyltrimethoxysilane (EEMS), y-glycidoxypropyltrimethoxysilane (GPMS), -(3,4-epoxycyclohexyl)ethyltriethoxysilane (EEES), and y-glycidoxypropyltriethoxysilane (GPES) was assessed by applying -jtl aliquots of acetone solutions to the skin of 4 male C3H/HeJ mice. The concentrations applied were,,, and % by volume for EEMS, GPMS, EEES, and GPES, respectively. Applications were made thrice weekly until the death of the animals. A negative control group received acetone (solvent) only. No treatment-related skin tumors were observed, nor was there evidence of increased incidence of any internal tumor in the groups that received GPMS, EEES, or GPES. In the group treated with EEMS, four mice were observed with squamous cell carcinomas of the treated skin and two mice had subcutaneous sarcomas outside of the treated area. No skin tumors were observed in the group treated with acetone, but two mice had subcutaneous sarcomas outside of the treated area. The mean survival times were 9, 48, 4,49, and days for the EEMS, GPMS, EEES, GPES, and acetone control groups, respectively. In no case was the mortality rate significantly different from that of the controls. The results indicate that only EEMS was oncogenic under the conditions of these studies, o 989 Society of To Organosilanes are industrially important ity (rabbit LD,.3 ml/kg), did not produce chemicals with a wide range of applications, signs of toxicity on exposure of rats for 8 hr to They are used primarily as coupling agents or a vapor-saturated atmosphere, and is a minor as starting materials for the attachment of skin and eye irritant (Mellon Institute Special organic groups in the synthesis of silicone Report, 9a). EEMS has been shown to be polymers. Four typical organosilanes are (3- mutagenic in an Ames bacterial assay (Litton (3,4-epoxycyclohexyl)ethyltrimethoxysilane Bionetics, 977), increased sister chromatid (EEMS: CAS No ), 7-glycidoxy- exchanges in L78Y mouse lymphoma cells propyltrimethoxysilane (GPMS: CAS No. (Litton Bionetics, 979), increased unsched ), /9-(3,4-epoxycyclohexyl)ethyltri- uled DNA synthesis in human WI-38 cells ethoxysilane (EEES: CAS No ), (Litton Bionetics, 978a), and increased muand 7-glycidoxypropyltriethoxysilane (GPES). tation frequency in a mouse lymphoma for- EEMS is oflowperoral toxicity (rat LD, ward mutation assay (Litton Bionetics,.3 g/kg) and low acute percutaneous toxic- 978b). Given by gavage over the period of major organogenesis, EEMS was not embry- Present address: Syntex Research, 34 HiUview Av- otoxic or teratogenic in rats or rabbits (Tyl el enue, Palo Alto, CA al, 988). Downloaded from at Pennsylvania State University on September 8, /89 $3. Copyright l989bythesoci«yoftoiico4ofy Allrighoofreproductionin «ny form reiervtd.

2 8 DE PASS ET AL. GPMS has low acute peroral toxicity (rat LD, 8.4 g/kg) and slight acute percutaneous toxicity (rabbit LD, 3.97 ml/kg), did not produce signs of toxicity on exposure of rats for 8 hr to a vapor-saturated atmosphere, and is a slight skin and moderate eye irritant (Mellon Institute Special Report, 9b; Palazzolo, 93). A 8-day oral gavage study revealed no evidence of toxicity among rats exposed to doses of up to mg/kg/day of GPMS (Siddiqui and Hobbs, 983). With rats exposed for 9 days ( hr/day) to a respirable aerosol of GPMS at 77,, and 743 mg/m 3, there were deaths at the highest concentration, and at the mid- and low concentrations there were dose-related signs of irritancy and body weight depression (Allied Report, 98a). GPMS has been shown to be weakly mutagenic in an Ames bacterial assay and L78Y mouse lymphoma assay (Litton Bionetics, 97a,b), but not active in a mammalian cell transformation assay (Isquith, 98). It has been demonstrated that the in vitro mutagenic activity of GPMS can be reduced in a dose-related manner by treatment with tissue homogenates (Allied Report, 98), and in vitro studies showed GPMS not to increase the incidence of lymphocyte sister chromatid exchanges (Allied Report, 98b). GPMS was not embryotoxic or teratogenic in rats at dose levels up to mg/kg/day administered during the period of major organogenesis (Siddiqui and Hobbs, 984). EEES was not mutagenic in an Ames bacterial assay (Bushy Run Research Center Report, 983a), did not increase the incidence of sister chromatid exchanges or cytogenetic effects in Chinese hamster ovary cells in vitro, and did not increase the level of unscheduled DNA synthesis in primary cultures of rat hepatocytes (Bushy Run Research Center Report, 983b). No other information is available on EEES, and none is available on GPES. In view of the results of genotoxicity studies and because of the absence of direct information on the potential oncogenicity of these organosilanes, studies were conducted to assess tumorigenicity by chronic cutaneous bioassay procedures. Skin was chosen as the route of exposure because this is a major occupational site of contamination. METHODS Test substances. All test samples were supplied by Union Carbide Corporation, Sistersville, West Virginia. They were analyzed by gas chromatography prior to shipping and at four times during the studies. Analyses were conducted at -9, -3, 8-, and 4-8 months of treatment For EEMS, which was applied undiluted, the purity was 97.7, 98.8, 98., and 97.8% at the four sampling periods. For GPMS, which was applied as a % solution by volume, the purity was 9., 9., 9.4, and 9.% at the four sampling periods. For EEES, the purity was 93.,, 93.4,9., and 9.8% at the four sampling periods. For GPES, the purity was 94., 94.3, 93., and 9.4% at the four sampling periods. EEES and GPES were applied as % solutions by volume. Impurities were not identified. The stability of all acetone dilutions for the period of use was demonstrated during these studies, and the concentrations were verified analytically. High-purity acetone from Fisher Scientific Company (Pittsburgh, PA) or J. T. Baker (Phillipsburg, NJ) was used as the solvent and negative control substance. Experimental design. Forty male C3H/HeJ mice from Jackson Laboratories, Bar Harbor, Maine, were randomly assigned to each test group. The animals were housed five per cage in stainless-steel cages with wiremeshfloors.all mice were housed in the same room with controlled lighting. Ziegler Bros. NIH 7 pellets (Gardners, PA) and water from an automatic watering system were provided ad libitum. The mice were treated three times weekly for their complete life span with /il per application of each substance. The substances were applied with an Eppendorf pipet to the back of each mouse from which the fur was clipped once weekly. All mice were examined daily, and the presence and progress of skin tumor growth were recorded monthly. Complete necropsies were performed on all mice. The dorsal skin from all animals plus all tissues with suspect tumors were examined histologically after sectioning and staining with hematoxylin and eosin. The concentrations and approximate doses of each test substance are shown in Table. The concentration of each substance was selected in preliminary -week studies in which various concentrations ( to %) were applied daily. The skin was closely observed for signs of irritation, and the mice were weighed several times to assess any effects on weight gain. The concentrations chosen for the lifetime studies were the highest ones that resulted in neither unacceptable grossly visible local irritation, such as ulceration orflakingand peeling of the skin, nor reduced weight gain. Downloaded from at Pennsylvania State University on September 8,

ALKOXYSILANE ONCOGENICITY STUDIES 8 TABLE RESULTS OF DERMAL ONCOGENICITY STUDIES ON VARIOUS ALKOXYSILANES EEMS GPMS EEES GPES Acetone Concentration (%) Dose(mg)' Mean survival time (days)*

3 ALKOXYSILANE ONCOGENICITY STUDIES 8 TABLE RESULTS OF DERMAL ONCOGENICITY STUDIES ON VARIOUS ALKOXYSILANES EEMS GPMS EEES GPES Acetone Concentration (%) Dose(mg)' Mean survival time (days)* Skin/subcutis Lesions'' Carcinoma Fibrosarcoma Lymphosareoma Hyperplasia Dermatitis Hyperkeratosis Necrosis Melanosis Fibrosis OO.. " Approximate dose per mouse per application; mice were treated three times per week with test substances diluted in acetone (except for EEMS); see text for explanation of abbreviations. * No significant differences by Mantel-Cox or Breslow statistics. ' Number of animals with lesion out of 4 treated animals. Statistical analysis. Mortality rates and skin tumor incidence were compared by the product-limit method (Kaplan and Meier, 98). The Mantel-Cox and Breslow statistics were used to test the equality of the survival and time-to-tumor curves (Mantel, 9; Breslow, 97). RESULTS The results of these studies are summarized in Table. The only epidermal tumors seen in these studies were in the EEMS-treated group. Four mice were diagnosed with squamous cell carcinomas in the treatment area. Since epidermal tumors are very rare in male C3H mice based on many years of study at this laboratory (/74 acetone controls), these tumors are considered to be treatment related. Statistical analysis of these data indicated a statistically significant difference from the control incidence ( tumors, p <.) by the Mantel-Cox test but not by the Breslow test (p =.8), which gives greater weight to early tumors. The first skin tumor was observed after 7 days on study. Other skin lesions occurred in EEMStreated mice which were considered attributable to treatment. These included three cases of epidermal hyperplasia, five animals with hyperkeratosis, and two with dermal fibrosis. Two mice in the EEMS-treated group had subcutaneous tumors which were not considered to be related to treatment. One had a fibrosarcoma of the left foreleg and the second had a myxofibrosarcoma of the ventral abdomen. These tumors were not considered treatment related since they were not in the treatment area and since similar tumors occurred in the acetone control group. In the group treated with GPMS, eight mice had epidermal hyperkeratosis, suggestive of irritation, but no tumors were observed. One mouse also had dermal fibrosis. Among the mice treated with EEES, one animal had epidermal hyperplasia; there were three cases of ulcerative dermatitis, five cases of hyperkeratosis, five with dermal fibrosis, and one animal with epidermal necrosis. No skin tumors were observed. Among the mice treated with GPES, a poorly differentiated fibrosarcoma was found on the right front leg of one mouse. This tumor was not considered to be biologically sig- Downloaded from at Pennsylvania State University on September 8,

4 8 DE PASS ET AL. nificant because sarcomas of various types have been diagnosed in acetone control mice (/74) at this laboratory. Indeed, one fibrosarcoma was found on an acetone control mouse in this study. Another GPES-treated mouse had a subcutaneous nodule in the inguinal area which was diagnosed as an area of caseous necrosis, without evidence of neoplasm. Many additional lesions were found on the skin of GPES-treated mice. These included hyperkeratosis, melanosis, epidermal hyperplasia, epidermal necrosis, and dermatitis (Table ). These skin lesions were attributed to treatment with GPES. In the acetone control group, two animals were diagnosed with subcutaneous mesenchymal neoplasms (a fibrosarcoma and a lymphosarcoma). Two animals in this group had ulcerative dermatitis, one had epidermal hyperplasia, and one had hyperkeratosis. A similar group of mice from the same source and housed in the same room was treated with methylcholanthrene as a positive control group. As expected, a very high incidence of skin tumors was observed (DePass et ai, 984). There were no other treatment-related pathologic findings in these studies. In addition, statistical comparison of the mean survival times (Table ) and the survival curves indicated that none of these substances significantly affected the life span of the mice in these studies. DISCUSSION These studies were performed to assess the potential oncogenicity of four industrially important alkoxysilanes. The results indicate no evidence of oncogenicity for three of the silanes when these substances were applied to the skin of mice for their lifetime. A local carcinogenic effect was observed for EEMS as indicated by four squamous cell carcinomas on the treated skin. Since biological effects in the form of epidermal changes were seen with the other three silanes, the doses and concentrations used are considered to be close to the "maximum tolerated concentrations" for these substances. The available data do not provide an adequate explanation for the carcinogenic effect of EEMS in contrast to the lack of oncogenicity of the other alkoxysilanes, especially its ethoxylated analog (EEES). Thus, EEMS has been shown to be mutagenic in both the presence and the absence of a metabolic activating system, whereas GPMS was a direct-acting mutagen in vitro but not in the presence of metabolic activation or in vivo, and EEES was devoid of mutagenic potential in vitro. The results may have been related to the difference in concentrations applied, since the concentration of EEMS was substantially higher than those of the other silanes. The concentrations were determined by the relative irritancy of the substances. For example, the concentration of EEES applied in this study was determined in preliminary rangefinding studies in which this compound had greater skin irritation potential than did EEMS. Whereas undiluted EEMS was well tolerated, a % dilution of EEES produced an unacceptable degree of skin irritation. Signs of irritation, such as desquamation, were observed even with the % solution which was selected for the oncogenicity bioassay. The results of the bioassays have shown that the group treated with % EEES had a similar incidence of hyperkeratosis and a higher incidence of dermalfibrosisthan the group that received EEMS. In any oncogenicity bioassay, the probability of demonstrating oncogenic activity is dependent to some degree on the toxicity or irritancy of the test substance, since these properties often determine the maximum dose administered. Since the doses applied produced histopathologic changes in the skin, the negative results obtained for the three alkoxysilanes, EEES, GPMS, and GPES, are considered to be valid demonstrations of the lack of cutaneous oncogenic activity of these Downloaded from at Pennsylvania State University on September 8,

5 ALKOXYSILANE ONCOGENICITY STUDIES 83 compounds at maximum tolerated concentrations. In conclusion, there was no evidence of an oncogenic effect of three alkoxysilanes under the conditions of these studies. However, p- (3,4- epoxycyclohexyl)ethyltrimethoxysi]ane was carcinogenic when applied to the skin of mice. ACKNOWLEDGMENTS The authors are grateful to L. G. Peterson for supervising the conduct of these studies, to D. R. Meckley for providing excellent technical assistance, and to C. R. Thrash for analyzing the samples. We also thank R. H. Garraan for his helpful suggestions in the preparation of the manuscript. These studies were supported by Union Carbide Corporation. REFERENCES Allied Report (98). Evaluation of Silane, Trimethoxy[3-(,3-epoxypropyl)propyl], for Its Sensitivity to Detoxicating Enzymes from Mammalian Tissues in the Salmonetla typhimurium Assay, Report No. MA Allied Corp., Morristown, NJ. Allied Report (98a). A Two- Week Inhalation Toxicity Study ofprylog in the Rat, Report No. MA Allied Corp., Morristown, NJ. Allied Report (98b). Analysis of Sister Chromatid Exchange (SCE) Frequencies in Peripheral Lymphocytes of Rats and Rabbits Exposed via Inhalation to Prylog, Report No. MA Allied Corp., Morristown, NJ. BRESLOW, N. (97). A generalized Kruskal-Wallis test for comparing samples subject to unequal patterns of censorship. Biometrics 7, Bushy Run Research Center Report (983a). Silane Y- 43. Salmonella/Microsome (Ames) Bacterial Mutagenicity Assay. Project Report 4-33 (May, 983). Bushy Run Research Center, Export, PA. Bushy Run Research Center Report (983b). Silane Y- 43: In vitro Mutagenesis Studies: 3-Test Battery, Project Report 4- (January 7, 983). Bushy Run Research Center, Export, PA. DEPASS, L. R., FOWLER, E. H., MECKLEY, D. R., AND WEIL, C. S. (984). Dermal oncogenicity bioassays of acrylic acid, ethyl acrylate, and butyl acrylate. J. Toxicol. Environ. Health 4, -. ISQUFTH, A. J. (98). In vitro Mammalian Cell Transformation Assay of gamma-glycidoxypropyltrimeihoxysilane, unpublished Dow Corning U.S.A. report. KAPLAN, E. L., AND MEIER, Q. (98). Nonparametric estimation from incomplete observations. /. Amer. Stat.Assoc. 3, Litton Bionetics Report (97a). Mutagenicity Evaluation of Union Carbide A-87: gamma -Glycidoxypropyltrimethoxysilane, Project No. 47 (July 4, 97). Litton Bionetics. Inc., Kensington, MD. Litton Bionetics Report (97b). Mutagenicity Evaluation ofa-87 and N-343 in the Mouse Lymphoma Assay and a Microbial Suspension Assay, Project No. 84 (Dec. 3, 97). Litton Bionetics, Inc., Kensington, MD. Litton Bionetics Report (977). Mutagenicity Evaluation ofchf-4-3, Project No. 838 (Nov. 977). Litton Bionetics, Inc., Kensington, MD. Litton Bionetics Report (978a). Mutagenicity Evaluation ofchf-4-4 in the Unscheduled DNA Synthesis in Human Wl-38 Cell Assay, Project No. 84 (July 978). Litton Bionetics, Inc., Kensington, MD. Litton Bionetics Report (978b). Mutagenicity Evaluation ofchf-4-4 in the Mouse Lymphoma Forward Mutation Assay, Project No. 839 (June 978). Litton Bionetics, Inc., Kensington, MD. Litton Bionetics Report (979). Mutagenicity Evaluation ofchf-4-4 in the Sister Chromatid Exchange Assay in L78Y Mouse Lymphoma Cells, Project No. 99 (March 979). Litton Bionetics, Inc., Kensington, MD. MANTEL, N. (9). Evaluation of survival data and two rank order statistics arising in its consideration. Cancer Chemother. Rep., 3-7. Mellon Institute Special Report (9a). Range Finding Tests on 3,4-Epoxycyclohexyltrimethoxysilane. Report - (June 4, 9). Mellon Institute of Industrial Research, Pittsburgh, PA. Mellon Institute Special Report (9b). Range finding tests on glycidoxypropyllrimelhoxysilane, Report - (June, 9). Mellon Institute of Industrial Research, Pittsburgh, PA. PALAZZOLO, R. J. (93). Acute Toxicity Studies with gamma - Glycidoxypropyltrimethoxysilane, unpublished Dow Corning U.S.A. Report. SIDDIQUI, W. H., AND HOBBS, E. J. (983). Subchronic toxicity study of gamma-glycidoxypropyltrimethoxysilane in rats. Drug Chem. Toxicol.,487-. SIDDIQUI, W. H., AND HOBBS, E. J. (984). Teratological evaluation of gamma-glycidoxypropyltrimethoxysilanein rats. Toxicology 3, -8. TYL, R. W., BALLANTYNE, B., FISHER, L. C, AND FRANCE, K. A. (988). Evaluation of the developmental toxicity of beta-{3,4-epoxycyclohexyl) ethyltrimethoxysilane in Fisher 344 rats and New Zealand White rabbits. Fundam. Appl. Toxicol., Downloaded from at Pennsylvania State University on September 8,

DIBROMOACETONITRILE. 1. Exposure Data

DIBROMOACETONITRILE. 1. Exposure Data DIBROMOACETONITRILE Data were last evaluated in IARC (1991). 1. Exposure Data 1.1 Chemical and physical data 1.1.1 Nomenclature Chem. Abstr. Serv. Reg. No.: 3252-43-5 Chem. Abstr. Name: Dibromoacetonitrile