Carcinogenicity and Chronic Toxicity in Rats and Mice Exposed by Inhalation to 1,2-Dichloroethane for Two Years

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1 J Occup Health 2006; 48: Journal of Occupational Health Carcinogenicity and Chronic Toxicity in Rats and Mice Exposed by Inhalation to 1,2-Dichloroethane for Two Years Kasuke NAGANO, Yumi UMEDA, Hideki SENOH, Kaoru GOTOH, Heihachiro ARITO, Seigo YAMAMOTO and Taijiro MATSUSHIMA Japan Bioassay Research Center, Japan Industrial Safety and Health Association, Japan Abstract: Carcinogenicity and Chronic Toxicity in Rats and Mice Exposed by Inhalation to 1,2- Dichloroethane for Two Years: Kasuke NAGANO, et al. Japan Bioassay Research Center, Japan Industrial Safety and Health Association Carcinogenicity and chronic toxicity of 1,2- dichloroethane (DCE) were examined by inhalation exposure of groups of 50 F344 rats and 50 BDF1 mice of both sexes to DCE vapor or clean air as control for 6 h/d, 5 d/wk and 104 wk. The rats were exposed to 10, 40 or 160 ppm (v/v) DCE, while the mice were exposed to 10, 30 or 90 ppm. The 2-yr exposure to DCE produced a dose-dependent increase in incidences of benign and malignant tumors, including subcutaneous fibroma, mammary gland fibroadenoma and peritoneal mesothelioma in male rats; subcutaneous fibroma and mammary gland adenoma, fibroadenoma and adenocarcinoma in female rats; and bronchiolo-alveolar adenoma and carcinoma, endometrial stromal polyp, mammary gland adenocarcinoma and hepatocellular adenoma in female mice. No exposure-related change in the incidence of non-neoplastic lesions or in any hematological, blood biochemical or urinary parameter occurred in any DCE-exposed rat or mouse group. The types of tumors and their target organs found in this study were consistent with those observed in rats and mice administered DCE by gavage in a NCI study. Selection of the exposure concentrations was considered appropriate with reference to the maximum tolerated dose for the highest doses and an occupational exposure limit of DCE for the lowest dose. The present findings suggest that those carcinogenic responses be primarily considered for standard setting of occupational and environmental exposure to DCE. (J Occup Health 2006; 48: ) Received Feb 9, 2006; Accepted Jul 4, 2006 Correspondence to: K. Nagano, Japan Bioassay Research Center, Japan Industrial Safety and Health Association, 2445 Hirasawa, Hadano, Kanagawa , Japan ( k-nagano@jisha.or.jp) Key words: 1,2-Dichloroethane, Inhalation, Carcinogenicity, Rat, Mouse, Chronic toxicity 1,2-Dichloroethane (DCE) has been used primarily as an intermediate in the production of vinyl chloride 1). It has also been used as an intermediate in the production of tri- and tetrachloroethylene, ethyleneamines and trichloroethane, and as a solvent, metal degreaser and finish remover 1). Annual production of DCE in Japan was reported to be 3,352,000 tons in ). According to the Pollutant Release and Transfer Register Report of the Japan Ministry of the Environment, in ), 603 and 5 tons of DCE were released annually into the atmosphere and public water, respectively, mainly by chemical industries. The Japan Ministry of the Environment 4) reported that DCE was detected in outdoor air, ranging from to 0.62 µg/m 3 ( ppb) and indoor air, ranging from to 0.30 µg/m 3 ( ppb) in Mean air concentrations of DCE at three production plants in the UK were reported to be 0.7, 0.8 and 1.7 ppm, while the respective maximum exposures were 4.5, 20 and 40 ppm 5). Medical case reports have revealed that while ingestion of DCE affects the stomach, liver, kidney, lung, heart and central nervous system 6), inhalation of DCE causes irritation and inflammation of the respiratory tract, damage to the lung, liver and kidneys, and death due to cardiac toxicity 7). Experimental toxicology studies showed that DCE primarily affects the liver, kidneys and respiratory system 1, 5, 8 14). Five cohort studies examining the risk of cancer among workers with potential exposure to DCE were not able to link the excesses of cancer incidence to occupational exposure to DCE 15 19). The National Cancer Institute (NCI) reported that 78-wk administration of DCE by gavage to rats and mice of both sexes significantly increased incidences of tumors in several organs 13). Maltoni et al. 20) reported that inhalation exposure of rats and mice of either sex to up to 250 ppm DCE for 78 wk did not induce any definite

2 Kasuke NAGANO, et al.: Inhalation Carcinogenicity of 1,2-Dichloroethane in Rodents 425 carcinogenic effects. However, the International Agency for Research on Cancer (IARC) 14) noted that their study was considered to be inadequate for evaluating the carcinogenicity of DCE because of the low and variable survival rates. Cheever et al. 21) reported that no tumor was induced after inhalation exposure of rats of both sexes to 50 ppm DCE for 2 yr. DCE is thought to be genotoxic in in vitro and in vivo mutation assays and binds to DNA in vivo 22 25). Consequently, the IARC classified the carcinogenicity of DCE as being possibly carcinogenic to humans (Group 2B) in consideration of inadequate evidence in humans and sufficient evidence in experimental animals for the DCE carcinogenicity 14). The Japan Society for Occupational Health (JSOH) also assessed DCE carcinogenicity as Group 2B (possibly carcinogenic to humans) 26), however, the American Conference of Governmental Industrial Hygienists (ACGIH) assigned DCE carcinogenicity as A4 (not classifiable as a human carcinogen) 27), because of the limited evidence derived from studies employing gavages as the route of DCE administration. The primary route of exposure of workers to DCE is inhalation of its vapor, and an occupational exposure limit (OEL) of DCE has been recommended as 10 ppm by ACGIH 27) and JSOH 28). In the UK, the OEL was proposed as 5 ppm 5). This study was designed to provide dose response data of rodent carcinogenicity and chronic toxicity of inhaled DCE to make health risk assessments of occupational and environmental exposure of humans to DCE, since sufficient experimental animal data by the inhalation route are not available for evaluating the carcinogenicity of DCE. F344 rats and BDF1 mice were exposed by inhalation to DCE vapor at concentrations of 10, 40 or 160 ppm, and 10, 30 or 90 ppm (v/v), respectively, for 2 yr. The range of the concentrations of inhalation exposure to DCE was selected in consideration of the OEL 5, 27, 28) for the lowest dose, and of the maximum tolerated dose (MTD) stipulated by both the NCI and IARC guidelines 29, 30) for the highest dose in this 2-yr rodent carcinogenicity study. Materials and Methods This study was conducted with reference to the Organisation for Economic and Co-operation and Development (OECD) Guideline for Testing of Chemicals 453, Combined Chronic Toxicity/Carcinogenicity Studies 31), and in accordance with the OECD Principles of Good Laboratory Practice (GLP) 32). The animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals 33). This study was approved by the ethics committee of the Japan Bioassay Research Center (JBRC). Chemical DCE of reagent grade (greater than 99% pure, less than 0.03% water, less than % chlorine, and less than 0.002% non-volatile chemicals) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Each lot of DCE used in this study was analyzed for its purity and stability by gas chromatography before and after its use. Neither decomposition products nor other impurities were detected. Animals F344/DuCrj (SPF) rats and Crj:BDF1 (SPF) mice of both sexes were obtained at 4 wk of age from Charles River Japan, Inc. (Kanagawa, Japan). The animals were quarantined and acclimated for 2 wk, and then divided by stratified randomization into 4 body weight matched groups, each comprising 50 rats and 50 mice of both sexes. The animals were housed individually in stainlesssteel wire hanging cages (150 mm [W] 220 mm [D] 176 mm [H] for rats and 100 mm [W] 120 mm [D] 120 mm [H] for mice) in stainless steel inhalation exposure chambers maintained at a temperature of 23 ± 2 C and at a relative humidity of 55 ± 10% with 12 ± 1 air changes/h. Fluorescent lighting was controlled automatically to give a 12-h light/dark cycle. All rats and mice had free access to sterilized water and a γ- irradiation-sterilized commercial pellet diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan). The average body weight measured immediately before the first exposure to DCE or clean air was 120 ± 5 (mean ± SD) g for male rats, 100 ± 3 g for female rats, 23.4 ± 0.8 g for male mice and 19.8 ± 0.7 g for female mice. Experimental design Groups of 50 male and 50 female rats and mice were exposed to airflow containing DCE vapor at a target concentration of 10, 40, or 160 ppm for rats and 10, 30 or 90 ppm for mice for 6 h/d, 5 d/wk and for 104 wk (2 yr). Fifty rats and 50 mice of both sexes, serving as respective controls, were handled in the same manner as the DCE-exposed groups, but were exposed to clean air in the inhalation exposure chambers. The lowest exposure concentration of 10 ppm was selected in consideration of the OEL of 10 ppm for DCE 27, 28). Selection of the highest concentrations of 160 ppm for rats and 90 ppm for mice was based on both subchronic toxicity and body weight decrement from a preliminary 13-wk inhalation exposure study conducted at the JBRC. All rats died during the first week of 13-wk exposure to 320 ppm DCE, but 13-wk exposure of rats to 160 ppm DCE did not cause any deaths, overt toxic signs or body weight decrements. Six out of 10 female mice died during a period of 13-wk exposure to 160 ppm DCE, but 13-wk exposure of mice to 80 ppm DCE did not cause either death or overt toxic manifestation, although body weight decrements of 9% for males and 7% for females were observed with respect to the controls. Therefore, the highest exposure concentrations of 160 ppm for rats and 90 ppm for mice

3 426 J Occup Health, Vol. 48, 2006 were predicted not to exceed the MTD 29, 30) from the results of the 13-wk inhalation exposure study. Exposure to DCE Airflow containing DCE vapor at a target concentration for rats of 10, 40 or 160 ppm, and for mice of 10, 30 or 90 ppm, was prepared by a vaporization technique. The saturated vapor-air mixture was generated by bubbling clean air through liquid DCE in a temperature-regulated glass flask (25 C), and by cooling it through a thermostatted condenser at 18 C. The airflow containing the saturated vapor was diluted with clean air, and then warmed to 25 C in a thermostatted circulator which served to stabilize the vapor concentration by complete gasification of DCE. The flow rate of vapor-air mixture was regulated with a flow meter, further diluted with humidity- and temperature-controlled clean air in a spiraling line mixer, and then supplied to the inhalation exposure chambers. Four inhalation exposure chambers of 7,600 l in volume for rats and four inhalation exposure chambers of 3,700 l for mice were used in this study. Each exposure chamber accommodated 100 individual cages for 50 males and 50 females of either rats or mice. Chamber concentrations of DCE were monitored by gas chromatography every 15 min, and maintained constant at 10.0 ± 0.1 (mean ± SD), 39.8 ± 0.6 and ± 2.1 ppm for the exposure of rats, and at 10.0 ± 0.2, 30.0 ± 0.4 and 89.8 ± 1.2 ppm for the exposure of mice throughout the 2-yr exposure period. Clinical observations and analysis, and pathological examinations The animals were observed daily for clinical signs and mortality. Body weights and food consumption were measured once a week for the first 14 wk, and every 4 wk thereafter. All the rats and mice which died or were killed in a moribund state during the 2-yr exposure period, or survived to the end of the 2-yr period received complete necropsy. Urinary parameters were measured from urine sampled with Ames Reagent Strips (Multistix for rats, Uro-Labstix for mice: Bayer Corporation, NY, USA) in the last week of the 2-yr exposure period. For hematology and blood biochemistry, the surviving animals were bled under ether anesthesia at the terminal necropsy after they were fasted overnight. The blood samples were analyzed on an automatic blood cell analyzer Coulter Counter SP (Coulter Electronics, Luton, UK) for hematology, and an automatic analyzer Hitachi 705 and a flame analyzer Hitachi 750 (Hitachi, Tokyo, Japan) for blood biochemistry. Organs were removed, weighed, and examined for macroscopic lesions at the necropsy. Tissues for microscopic examinations were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections 5 µm thick were prepared and stained with hematoxylin and eosin. Statistics and data analysis Incidences of neoplastic lesions were analyzed for a dose response relationship indicated by a significant positive trend by Peto s test 34), and for a significant difference from the concurrent control group by Fisher s exact test. Incidences of non-neoplastic lesions and urinary parameters were analyzed by Chi-square test. Survival curves were plotted according to the Kaplan- Meier method 35), and the log-rank test 36) was used to test statistical significance of the difference between any DCE-exposed rat or mouse group of either sex and the respective control. Body weight, organ weight, hematological and blood biochemical parameters were analyzed by Dunnett s test. The method for application of the statistical tests to the data was described in detail in a previous paper 37). When tumor incidences were increased in a doserelated manner as indicated by Peto s test and when the tumor incidence in each of the DCE-exposed groups was increased but not statistically significant as compared with the concurrent, matched-control group by Fisher s exact test, the borderline increase in the tumor incidence was tested as to whether or not it was biologically meaningful, using a range of minimum and maximum tumor incidences in the JBRC historical control data. Use of the historical control data was similar to that described by Haseman et al. 38, 39). The historical control data were compiled from the 2-yr inhalation studies of rodent carcinogenicity conducted at the JBRC during the 18-yr period from 1987 to 2004, consisting of data for 749 male and 747 female F344/DuCrj (SPF) rats from 15 studies, and 748 male and 749 female Crj:BDF1 (SPF) mice from 15 studies. All the animals used were obtained from the same supplier (Charles River Japan, Inc). The experimental conditions were similar throughout all the studies with respect to the air supply system for inhalation exposure to clean air, diet and drinking water, accommodation, including caging regimens and inhalation exposure chambers, and various environmental parameters in the same barrier system area of the JBRC test facility. The nomenclature conventions, diagnostic criteria for neoplastic lesions, diagnostic processes by pathologists and finalization of the diagnoses were also essentially identical. The studies and raw data were assured by an independent quality assurance unit of JBRC in compliance with the OECD GLP 32) and the Standard Operating Procedures of JBRC. The number of tumorbearing animals/total number of animals examined and the range of minimum and maximum incidences of each tumor in the single study are shown in Tables 1 and 2. Results Rat study Survival, body weight, food consumption and clinical observations and analyses: There was no significant

4 Kasuke NAGANO, et al.: Inhalation Carcinogenicity of 1,2-Dichloroethane in Rodents 427 Fig. 1. Survival curves of male and female rats exposed to DCE vapor at 10, 40 or 160 ppm and clean air as control for 2 yr. difference in the survival rate at any time point of the 2- yr exposure period between any DCE-exposed group of either sex and the respective control (Fig. 1). At the end of 2-yr exposure period, the survival rates of the 0 (clean air as control), 10, 40 and 160 ppm DCE exposure groups were 74, 70, 64 and 74% for males, and 70, 82, 74 and 76% for females, respectively. Neither growth rate nor food consumption was suppressed in any DCE-exposed group of either sex as compared with the respective control. The body weights of the 0, 10, 40 and 160 ppm DCE exposure groups at the end of 2-yr exposure period were 434 ± 46, 459 ± 59, 448 ± 41 and 467 ± 85 g for males, and 317 ± 46, 329 ± 41, 33 ± 45 and 336 ± 56 g for females, respectively. Incidences of subcutaneous masses, which were found in the breast, back, and abdominal and perigenital areas by clinical observation, tended to increase in the DCEexposed groups of both sexes. No exposure-related change in any hematological, blood biochemical or urinary parameter was found in any DCE-exposed group of either sex. Pathology: Macroscopic examination at necropsy revealed that incidences of subcutaneous masses were increased in an exposure concentration-related manner (for males: 8 /50 cases in control, 13/50 at 10 ppm, 20/50 at 40 ppm and 18/50 at 160 ppm, and for females: 12/50 in control, 12/50 at 10 ppm, 14/50 at 40 ppm and 30/50 at 160 ppm). There was no significant difference in organ weight between any DCE-exposed group of either sex and the respective control. Table 1 presents tumor incidences in rats of both sexes exposed by inhalation to DCE or clean air for 2 yr, with reference to the tumor incidences of the JBRC historical control data. In male rats, incidences of fibromas in the subcutis, fibroadenomas in the mammary gland and mesotheliomas in the peritoneum showed a significant positive trend. The incidences of subcutaneous fibromas in both the 40 and 160 ppm DCE exposed male groups exceeded the maximum tumor incidence of the historical control data, although those tumor incidences were not statistically increased as compared with the concurrent control. The fibromas were non-invasive and expansile benign tumors composed of fibroblasts and abundant collagenous stroma. The incidence of mammary gland fibroadenomas in the 160 ppm DCE exposed male group was statistically increased as compared with the concurrent control (p 0.05), and also exceeded the maximum tumor incidence of the historical control data. The mammary fibroadenomas were benign tumors composed of both glandular epithelium and fibrous connective tissue. Mammary gland adenomas were also found as benign tumors mainly composed of glandular epithelium, but their tumor incidence was not increased in any DCE-exposed male group. However, the combined incidences of mammary adenomas and fibroadenomas exhibited a significant positive trend, and the combined mammary tumor incidence in the 160 ppm DCE exposure group was statistically increased (p 0.05), and exceeded the maximum tumor incidence of the historical control data. These mammary gland tumors were diagnosed according to the criteria of Boorman et al. 40). The incidence of peritoneal mesotheliomas in the 160 ppm DCE exposed male group exceeded the maximum tumor incidence of the historical control data, although the tumor incidence was not statistically increased as compared with the concurrent control. The mesotheliomas were malignant tumors involving the surface of the peritoneal cavity, especially the scrotal sac, and were composed of one to several layers of neoplastic mesothelial cells covering pedunculated fibrovascular stalks. In female rats, incidences of fibromas in the subcutis and adenomas, fibroadenomas and adenocarcinomas in the mammary gland showed a significant positive trend. The incidence of subcutaneous fibromas in the 160 ppm DCE exposed female group was statistically increased (p 0.05), and also exceeded the maximum tumor incidence of the historical control data. The incidences of mammary gland adenomas and fibroadenomas in the 160 ppm DCE exposed female group and the combined incidence of those two benign mammary tumors were

5 428 J Occup Health, Vol. 48, 2006 Table 1. Number of tumor-bearing rats of both sexes exposed by inhalation to DCE or clean air for 2 yr (A) Male Group Name Control 10 ppm 40 ppm 160 ppm Peto s JBRC historical control data Number of animals test Incidence a) Min. Max. b) (%) (%) (%) (%) (%) (%) Subcutis Fibroma /749 1/50 10/50 (12.0) (18.0) (24.0) (30.0) (7.3) ( ) Mammary gland Adenoma /749 0/50 2/50 (2.0) (4.0) (0.0) (4.0) (0.9) ( ) Fibroadenoma * 13/749 0/50 3/50 (0.0) (0.0) (2.0) (10.0) (1.7) ( ) Combined adenoma and fibroadenoma * 19/749 0/50 4/50 (2.0) (4.0) (2.0) (14.0) (2.5) ( ) Peritoneum Mesothelioma /749 0/50 4/50 (2.0) (2.0) (2.0) (10.0) (2.1) ( ) (B) Female Group Name Control 10 ppm 40 ppm 160 ppm Peto s JBRC historical control data Number of animals test Incidence a) Min. Max. b) (%) (%) (%) (%) (%) (%) Subcutis Fibroma * 8/747 0/50 4/50 (0.0) (0.0) (2.0) (10.0) (1.1) ( ) Mammary gland Adenoma * 28/747 0/50 9/50 (6.0) (10.0) (10.0) (22.0) (3.7) ( ) Fibroadenoma * 76/747 0/50 8/50 (8.0) (2.0) (12.0) (26.0) (10.2) ( ) Combined adenoma and fibroadenoma * 103/747 2/50 10/50 (14.0) (12.0) (22.0) (44.0) (13.8) ( ) Adenocarcinoma /747 0/50 2/50 (2.0) (4.0) (0.0) (10.0) (0.7) ( ) Combined adenoma, fibroadenoma and adenocarcinoma ** 104/747 2/50 10/50 (16.0) (16.0) (22.0) (50.0) (13.9) ( ) * and **: Significantly different from the control group at p 0.05 and p 0.01 by Fisher s exact test, respectively. and : Significantly different at p 0.05 and p 0.01 by Peto s test, respectively. a : Number of animals bearing tumor / number of animals examined in the 15 historical inhalation studies. b : Number of animals bearing tumor / number of animals examined in a single historical study. The underlined values indicate the tumor incidences exceeding the maximum tumor incidence in the JBRC historical control data. statistically increased (p 0.05), and also exceeded the maximum tumor incidences of the historical control data. Besides, the combined incidence of mammary gland adenomas and fibroadenomas in the 40 ppm DCE exposed female group exceeded the maximum tumor incidence of the historical control data, but did not attain statistical significance as compared with the concurrent control. Furthermore, the incidence of mammary gland adenocarcinomas in the 160 ppm DCE exposed female group exceeded the maximum tumor incidence of the historical control data without significant increase as compared with the concurrent control. The neoplastic

6 Kasuke NAGANO, et al.: Inhalation Carcinogenicity of 1,2-Dichloroethane in Rodents 429 Fig. 2. Survival curves of male and female mice exposed to DCE vapor at 10, 30 or 90 ppm and clean air as control for 2 yr. epithelia of the adenocarcinomas were arranged in a ductular structure, forming multiple layers indicative of malignancy, but did not metastasize to any other organ. The combined incidence of all the mammary gland tumors showed a significant positive trend. The combined incidence of those three tumors in the 160 ppm DCE exposed female group was statistically increased (p 0.01), and also exceeded the maximum tumor incidence of the historical control data. No exposure-related, non-neoplastic lesions were histopathologically observed in any DCE-exposed rat group of either sex. Mouse study Survival, body weight, food consumption and clinical observations and analyses: Survival rates of DCEexposed male groups were not significantly different from those of the male control at any time point of the 2-yr exposure period (Fig. 2). At the end of the 2-yr exposure period, the survival rates of males of the 0, 10, 30 and 90 ppm DCE exposure groups were 78, 65, 70 and 74%, respectively. On the other hand, the survival rates of DCE-exposed female mice tended to be decreased (69% in female control, 56% at 10 ppm, 38% at 30 ppm and 52% at 90 ppm), and the 30 ppm DCE exposure group females exhibited a significant decrease in the survival rate as compared with the female control (p 0.01). The numbers of animals which died of malignant lymphoma before the end of the 2-yr exposure period were 19 cases (38%) for the 30 ppm DCE exposure females, as compared with 6 cases (12.2%) for the female control, 12 (24%) at 10 ppm and 9 (18%) at 90 ppm. Therefore, the significantly decreased survival rate of females of the 30 ppm DCE exposure group was attributed to the significantly increased malignant lymphoma deaths (19 cases). However, neither the lowered survival rates nor the increased incidences of malignant lymphoma deaths were DCE exposure-related. Neither growth rate nor food consumption was suppressed in any DCE exposure group of either sex as compared with the respective control. The body weights of the 0, 10, 30 and 90 ppm DCE exposure groups at the end of the 2-yr exposure period were 50.8 ± 6.5, 51.7 ± 6.1, 48.1 ± 8.2 and 50.7 ± 6.6 g for males, and 36.6 ± 5.2, 35.8 ± 4.1, 37.4 ± 4.9 and 34.1 ± 4.0 g for females, respectively. The incidence of subcutaneous masses, which were found in the back, breast, and the abdominal area by clinical observation, was increased in females of the 90 ppm DCE exposure group (2 cases in female control, 0 at 10 ppm, 3 at 30 ppm and 9 at 90 ppm). No exposurerelated change in any hematological, blood biochemical or urinary parameter was found in any DCE-exposed group of either sex. Pathology: Macroscopic examination at necropsy revealed that numbers of tumor-associated nodules in the lung (7 cases in female control, 2 at 10 ppm, 4 at 30 ppm and 13 at 90 ppm), uterus (12 cases in control, 13 at 10 ppm, 7 at 30 ppm and 20 at 90 ppm) and subcutis (3 cases in control, 3 at 10 ppm and 3 at 30 ppm, and 9 at 90 ppm) were increased in females of the 90 ppm DCE exposure group mice. The relative lung weight of females of the 90 ppm DCE exposure group (0.783 ± 0.525% as mean ± SD) was significantly increased as compared with that of the female control (0.625 ± 0.123%) (p 0.05). Table 2 presents tumor incidences in mice of both sexes exposed by inhalation to DCE or clean air for 2 yr, with reference to the tumor incidences of the JBRC historical control data. In male mice, incidences of hemangiosarcomas in the liver of both 30 and 90 ppm DCE exposure groups were statistically increased as compared with the concurrent control (p 0.05), and the tumor incidence of 30 ppm DCE exposed males exceeded the maximum incidence of the historical control data. However, those tumor incidences did not show any significant positive trend. In female mice, a significant positive trend was shown for incidences of bronchioloalveolar adenomas and carcinomas in the lung, endometrial stromal polyps in the uterus, adenocarcinomas in the mammary gland, and hepatocellular adenomas as well as combined incidences

7 430 J Occup Health, Vol. 48, 2006 Table 2. Number of tumor-bearing mice of both sexes exposed by inhalation to DCE or clean air for 2 yr (A) Male Group Name Control 10 ppm 30 ppm 90 ppm Peto s JBRC historical control data Number of animals a) test Incidence b) Min. Max. c) (%) (%) (%) (%) (%) (%) Liver Hemangiosarcoma 0 4 6* 5* 27/748 0/50 5/50 (0.0) (8.2) (12.0) (10.0) (3.6) ( ) (B) Female Group Name Control 10 ppm 30 ppm 90 ppm Peto s JBRC historical control data Number of animals 49 a) test Incidence b) Min. Max. c) (%) (%) (%) (%) (%) (%) Lung Bronchiolo-alveolar adenoma /749 0/50 5/50 (8.2) (2.0) (6.0) (16.0) (3.9) ( ) Bronchiolo-alveolar carcinoma /749 0/50 3/50 (2.0) (0.0) (2.0) (6.0) (2.8) ( ) Combined bronchiolo-alveolar adenoma /749 0/50 6/50 and bronchiolo-alveolar carcinoma (10.2) (2.0) (8.0) (22.0) (6.5) ( ) Uterus Endometrial stromal polyp /748 0/50 4/50 (4.1) (0.0) (2.0) (12.0) (3.5) ( ) Mammary gland Adenocarcinoma /749 0/50 4/50 (2.0) (4.0) (2.0) (12.0) (2.7) ( ) Liver Hepatocellular adenoma /749 1/50 4/50 (2.0) (2.0) (2.0) (12.0) (4.4) ( ) Hepatocellular carcinoma /749 0/50 4/50 (2.0) (0.0) (2.0) (0.0) (3.1) ( ) Combined hepatocellular adenoma and /749 1/50 6/50 hepatocellular carcinoma (4.1) (2.0) (4.0) (12.0) (7.2) ( ) Lymph node Malignant lymphoma 6 17* 22** /749 7/50 23/50 (12.2) (34.0) (44.0) (24.0) (28.6) ( ) * and **: Significantly different from the control group at p 0.05 and p 0.01 by Fisher s exact test, respectively. and : Significantly different at p 0.05 and p 0.01 by Peto s test, respectively. a : One mouse died accidentally during the 2-yr exposure period. b : Number of animals bearing tumor / number of animals examined in the 15 historical inhalation studies. c : Number of animals bearing tumor / number of animals examined in a single historical study. The underlined values indicate the tumor incidences exceeding the maximum tumor incidence in the JBRC historical control data. of bronchiolo-alveolar adenomas and carcinomas, and of hepatocellular adenomas and carcinomas. Both the incidence of bronchiolo-alveolar adenomas and the combined incidences of bronchiolo-alveolar adenomas and carcinomas in the 90 ppm DCE exposed female group exceeded the respective maximum tumor incidences of the historical control data, although those two tumor incidences were not statistically increased as compared with the concurrent control. The bronchiolo-alveolar carcinoma was characterized by destruction of the pulmonary parenchyma indicative of malignancy, but the malignant tumor incidences were within the range of minimum and maximum tumor incidences in the historical control data. No metastasis of the bronchiolo-

8 Kasuke NAGANO, et al.: Inhalation Carcinogenicity of 1,2-Dichloroethane in Rodents 431 alveolar carcinoma to any other organ was observed. The incidence of endometrial stromal polyps in the uterus of 90 ppm DCE exposure group exceeded the maximum tumor incidence of the historical control data without statistical significance as compared with the concurrent control. These endometrial stromal polyps were polypoid benign tumors protruding into the uterine lumen, and characterized by nodular proliferation of endometrial cells overlying the cuboidal to columnar epithelium. The incidence of mammary gland adenocarcinomas in the 90 ppm DCE exposed female group exceeded the maximum tumor incidence of the historical control data without statistical significance as compared with the concurrent control. The adenocarcinomas showed tubular or acinar growth patterns and they were diagnosed as malignant tumors according to the criteria of Squartini 41), but no metastasis to any other organ was observed. The incidence of hepatocellular adenomas in the 90 ppm DCE exposed female group exceeded the maximum tumor incidence of the historical control data without statistical significance as compared with the concurrent control. The hepatocellular adenomas were characterized by a wellcircumscribed lesion compressing adjacent parenchyma and absence of normal lobular architecture, and were composed of well-differentiated hepatocytes. Although the incidences of malignant lymphomas in both the 10 and 30 ppm DCE exposed female groups were statistically increased (p 0.05 and p 0.01, respectively), a significant positive trend was not found for the tumor incidences. The statistically increased incidences of malignant lymphomas were within the range of minimum and maximum tumor incidences in the historical control data. No exposure-related, non-neoplastic lesions occurred in any DCE-exposed mouse group of either sex. Discussion It was found in this study that 2-yr inhalation exposure to DCE vapor significantly increased incidences of benign and malignant tumors in several organs of both rats and mice. In rats, subcutaneous fibromas and mammary gland fibroadenomas in both males and females, peritoneal mesotheliomas in males, and mammary gland adenomas and adenocarcinomas in females were induced by the 2- yr exposure to DCE on the following basis of evidence. First, those tumor incidences were increased in an exposure concentration-dependent manner, as indicated by a significant positive trend. Second, statistical significance as compared with the concurrent control group was seen in the increases in those tumor incidences at 160 ppm DCE exposure, except for the subcutaneous fibroma and peritoneal mesothelioma in males and the mammary adenocarcinoma in females. Additionally, the incidences of those tumors in the 160 ppm DCE exposure groups of both sexes, as well as the incidence of subcutaneous fibromas in the 40 ppm DCE exposed male group and the combined incidence of mammary gland adenomas and fibroadenomas in the 40 ppm DCE exposed female groups, exceeded the respective maximum tumor incidences of the JBRC historical control data. In female mice, an exposure concentration-dependent increase in the incidences of bronchiolo-alveolar adenomas and carcinomas, uterine endometrial stromal polyps, mammary gland adenocarcinomas and hepatocellular adenomas was evidenced by a significant positive trend. The incidences of those tumors in the 90 ppm DCE exposed female group exceeded the maximum tumor incidence of the historical control data except for bronchiolo-alveolar carcinomas, although the increases in those tumor incidences did not attain statistical significance as compared with the concurrent control. Therefore, those mouse tumors were also induced in an exposure concentration-dependent manner by the 2-yr exposure of female mice to DCE vapor. On the other hand, neither hemangiosarcomas in the liver of male mice nor malignant lymphomas in the lymph node of female mice were likely to be causally related to the DCE exposure, since no significant dose response relationship was found in the observed incidences of hemangiosarcomas or malignant lymphomas in the DCEexposed groups. The present results are in sharp contrast with those of the long-term rodent studies by Maltoni et al. 20) and Cheever et al. 21). Maltoni et al. 20) reported no definite carcinogenic effects were induced by inhalation exposure to 5, 50 or ppm DCE vapor for 78 wk of Sprague-Dawley rats and Swiss mice, while the survival rates were low and variable 14). Their exposure duration was shorter than that used in the present study. Cheever et al. 21) reported no significant increase in incidences of any tumor in Sprague-Dawley rats exposed to 50 ppm DCE vapor for 2 yr. Their exposure concentration seems to have been too low to clearly demonstrate tumor induction by DCE exposure. In the present study, however, it was recognized that the incidences of subcutaneous fibromas and mammary gland tumors were respectively increased in male and female F344 rats exposed to 40 ppm DCE. The present results are in essential agreement with the types of tumors and their target organs in the Osborne- Mendel rats and B6C3F1 mice of both sexes orally administered DCE by gavage for 78 wk in a NCI study 13), although there were differences in the exposure route, the exposure period and the animal strains used between the two studies (Table 3). There were similarities in tumor occurrences between the NCI study and the present study, as increased incidences of subcutaneous fibromas in male rats, mammary gland adenocarcinomas in female rats, bronchiolo-alveolar adenomas, endometrial stromal tumors and mammary gland adenocarcinomas in female mice were noted in both studies. In addition, the effective

9 432 J Occup Health, Vol. 48, 2006 Table 3. Comparison of the tumors induced by administration of DCE between the NCI and JBRC studies NCI JBRC <Rats> Route Gavage Inhalation Administration period 78 wk 104 wk Strain of animal Osborne-Mendel F344/DuCrj Male Female Male Female Forestomach Squamous cell carcinoma + Circulatory system Hemangiosarcoma + Subcutis Fibroma Mammary gland Adenoma + Fibroadenoma + + Adenocarcinoma + + Peritoneum Mesothelioma + <Mice> Route Gavage Inhalation Administration period 78 wk 104 wk Strain of animal B6C3F1 Crj:BDF1 Male Female Male Female Lung Bronchiolo-alveolar adenoma Bronchiolo-alveolar carcinoma + Uterus Endometrial stromal polyp Endometrial stromal sarcoma + a) + Mammary gland Adenocarcinoma + + Liver Hepatocellular adenoma + +: An increase in tumor incidence is causally related to administration of DCE. a : An increase in combined incidence of endometrial stromal polyp and sarcoma. dose to produce a carcinogenic response to DCE was thought to be similar on the following calculation of body uptake of DCE through oral administration and inhalation. In the NCI study, the time-weighted average oral doses to significantly increase the tumor incidences were 47 and 95 mg/kg body weight/day for male and female rats, respectively, and 149 mg/kg/d for female mice. In this study, the concentration of inhalation exposure to DCE that caused the increased tumor incidences was found to be 40 ppm for male and female rats and 90 ppm for female mice. It can be estimated that the 6-h inhalation exposure of both male and female rats to 40 ppm corresponded to a daily DCE uptake of 33 mg/kg body weight, assuming a minute volume of 561 ml/min 42) and a lung absorption ratio of DCE of 100%. The daily uptake of DCE by the 6-h inhalation exposure of female mice to 90 ppm can

10 Kasuke NAGANO, et al.: Inhalation Carcinogenicity of 1,2-Dichloroethane in Rodents 433 also be estimated as 165 mg/kg body weight, assuming a minute volume of 1,239 ml/min 43) and a lung absorption ratio of DCE of 100%. It can also be estimated from the comparison of area-under-curves (AUC) for blood DCE levels following 6-h inhalation and gavage dose in a pharmacokinetic study by Reitz et al. 44) that the daily uptake of DCE through the 6-h inhalation exposure of male and female rats to 40 ppm was equivalent to that through oral administration of 31 mg/kg body weight by gavage. These two different estimates which were calculated on the basis of the estimated daily intake and the pharmacokinetic data can be taken to indicate that the tumors were induced at approximately similar amounts of daily DCE uptakes through both inhalation and oral gavage, although there was a difference in the exposure route between the two studies. A notable difference between the NCI study and the present study was that the administration of DCE by gavage induced squamous cell carcinomas of the forestomach and hemangiosarcomas of the circulatory system in male rats. The forestomach squamous cell carcinoma observed in the NCI gavage study 13) might have been caused by the route-specific gavage dosage in which the forestomach mucosa was exposed to far higher levels of DCE than the levels reaching the mucosa through the bloodstream following DCE inhalation, since long-term repetitive gavage administration of some chemicals to rodents is known to induce the forestomach proliferative lesions by direct action on the forestomach mucosa 45). It is striking that the 2-yr exposure of rats and mice of both sexes to DCE vapor at up to 160 ppm and 90 ppm, respectively, did not produce any overt toxic responses as evidenced by no exposure-related change in the incidence of histopathologically-observed non-neoplastic lesions or in any hematological, blood biochemical or urinary parameter. It has been reported that repeated exposure of rats and mice to DCE not far above the observed no-effect level produced severe effects and deaths, suggesting a steep dose response curve 1, 5). The present carcinogenic responses of the two rodent species to DCE and the reported genotoxicity of DCE 22 25), together with the rodent carcinogenicity data of orally administered DCE in the NCI study 13), suggest that those carcinogenic and genotoxic responses should be primarily considered as animal experimental data for the health risk assessment of DCE. Species and gender differences in the DCE-induced carcinogenicity were noted in the present study as well as in the NCI study 13). In the present study, the species difference was specific to the occurrence of the subcutis and peritoneum tumors in rats, and lung, uterus and liver tumors in mice. The gender difference seen in the present study was tumor induction at various sites in female mice, whereas there was no tumor induction in male mice. However, sufficient data for elucidating these species and gender differences are not yet available. A clue to understanding these species and gender differences might exist in the reported evidence of DCE metabolism, DNA adduct and mutagenesis 1, 25, 46, 47). DCE was metabolized via two principal pathways involving microsomal oxidation mediated by hepatic cytochrome P-450 and direct conjugation of DCE with glutathione. The reaction products from the direct glutathione conjugation pathway gave rise to DNA adducts in vitro and positive bacterial mutagenicity. Further study will be needed for plausible explanations of the species and gender differences in the carcinogenicity in the term of hepatic DCE-metabolizing capacities of P450 isozymes and glutathione S- transferases in rats and mice of both sexes. Selection of the highest concentrations of inhalation exposure to DCE vapor used in this study was considered appropriate for evaluation of the carcinogenic potency of DCE for the following reasons. First, terminal body weights of 160 ppm DCE exposed rats and 90 ppm DCE exposed mice of both sexes were decreased by less than 10% as compared with those of respective controls. Second, there was no significant difference in the survival rate between any of the DCE-exposed rat and mouse groups of both sexes and the respective controls except for the terminal survival rate of 30 ppm DCE exposed female mice. The significantly decreased terminal survival rate was attributed to an increase in the number of malignant lymphoma deaths. According to the MTD criteria set up by both the NCI 29) and IARC guidelines 30) for the 2-yr rodent carcinogenicity study, the highest dose should not induce toxic manifestations that are predicted to reduce the lifespan of the animals except as the result of neoplastic development or a 10% or greater retardation of body weight gain, compared with the control. Therefore, the present data of both the 2-yr survival rates and the terminal body weight decrements without any overt manifestation of chronic non-neoplastic lesions in the DCE-exposed animals fulfills the MTD criteria, indicating that the highest concentrations of 160 ppm DCE for rats and 90 ppm for mice did not exceed the MTD in accordance with the prediction from the prechronic 13-wk inhalation exposure study. Moreover, the highest and middle dose levels used in this study were not far high above the air concentrations that would be encountered in workplaces, since the maximum concentrations of DCE at 3 production plants in the UK were reported to be 4.5, 20 and 40 ppm 5). The lowest dose of 10 ppm for rats and mice was close or equivalent to the existing OEL for DCE 5, 27, 28). It can be inferred, therefore, that the concentrations of inhalation exposure to DCE used in this study are relevantly low enough to allow extrapolation of the present data to the DCE levels to which humans would be exposed in workplaces. It was found in the present study that 2-yr inhalation exposure of rats and mice to DCE vapor at

11 434 J Occup Health, Vol. 48, 2006 levels relevant to human exposure produced a dosedependent increase in the incidences of benign and malignant tumors in various organs of two rodent species. Notably, the most sensitive response to the long-term inhalation exposure to DCE was the increased incidences of subcutaneous fibromas and mammary gland tumors in the 40 ppm DCE exposed male and female rats, respectively, without any accompanying exposure-related change in the incidence of non-neoplastic lesions or in any hematological, blood biochemical or urinary parameter in any DCE exposure group rat or mouse in this study. Therefore, the present carcinogenic responses of two rodent species to DCE at inhalation exposure levels relevant to human occupational exposure would provide novel information about DCE as a human carcinogen for standard setting of occupational and environmental exposures to DCE in the absence of sufficient data either for epidemiologically linking cancer incidence to occupational exposure to DCE or for a carcinogenic mode of action for DCE. In addition, the present OEL value of 10 ppm 28) for DCE recommended in 1984 by the JSOH might need to be reconsidered, since the OEL recommendation was primarily based on both the negative carcinogenicity by inhalation exposure of rats and mice to DCE and the positive carcinogenicity by oral administration of DEC to rats and mice, as well as the hepatic and renal toxicities in the DCE-exposed workers. 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