Summary of Inhalation Carcinogenicity Study. of Tetrachloroethylene. in BDF 1 Mice

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1 Summary of Inhalation Carcinogenicity Study of Tetrachloroethylene in BDF 1 Mice March 1993 Japan Bioassay Laboratory Japan Industrial Safety and Health Association

2 PREFACE The tests were contracted and supported by the Ministry of Labour of Japan. The tests were conducted by Japan Bioassay Laboratory (JBL) and the report was prepared by JBL and peer reviewed by outside expert pathologist. Complete report was submitted to Ministry of Labour of Japan on March 31, This English Summary was translated by JBL from Japanese complete report.

3 (Study No.0105) Summary of Inhalation Carcinogenicity Study of Tetrachloroethylene in BDF1 Mice Purpose, materials and methods Tetrachloroethylene (CAS No ) is a colorless liquid with a boiling point of C. It is insoluble in water and soluble ethanol, ether, chloroform and benzene. The carcinogenicity and chronic toxicity of tetrachloroethylene (purity : 99.0%) were examined by inhalation exposure using Crj:BDF1 mice. Groups of test animals were exposed to tetrachloroethylene vapors at target concentrations of 0 (clean air), 10, 50 or 250 ppm (v/v) for 6 hours/day, 5 days/week for 2 years (104 weeks). Each group of test animals consisted of either 50 male or 50 female mice. Both sexes were exposed to each concentration of tetrachloroethylene vapor. The highest dose level was chosen so as not to exceed the maximum tolerated dose (MTD), based on both growth rate and toxicity in a previous 13-week toxicity study. The identity of the tetrachloroethylene used in these experiments was confirmed by both infrared spectrometry and mass spectrometry. The chemical was analyzed by both infrared spectrometry and gas chromatography before and after use to affirm its stability. Stainless-steel inhalation exposure chambers (volume: 3.7 m 3 ) were used throughout the 2-year exposure period. Tetrachloroethylene vapor-air mixtures were generated by bubbling clean air through tetrachloroethylene liquid and the mixtures delivered to the inhalation exposure chambers. Air concentrations of the tetrachloroethylene in the inhalation exposure chambers were monitored at 15 min intervals by gas chromatography. The animals were observed daily for clinical signs and mortality. Body weight and food consumption were measured once a week for the first 14 weeks and every 2 weeks thereafter. All animals, including those found dead or in a moribund state as well as those surviving to the end of the 2-year exposure period, underwent complete necropsy. Urinalysis was performed near the end of the exposure period. Hematology and blood biochemistry analysis were performed at the terminal necropsy: surviving animals were fasted overnight and bled under anesthesia. Organs and tissues were removed, weighed and examined for macroscopic lesions at necropsy. The organs and tissues were then fixed and embedded in paraffin. Five μm thick tissue sections were prepared and stained with hematoxylin and eosin and examined microscopically. Incidences of neoplastic lesions were statistically analyzed by Fisher s exact test. Any positive dose-response trends of tetrachloroethylene induction of neoplastic lesions were analyzed by Peto s test. Incidences of non-neoplastic lesions and urinalysis were analyzed by the Chi-square test. Changes in body weight, food consumption, hematological and blood biochemical parameters, and organ weights were analyzed by Dunnett s test. The present studies were conducted with reference to the Organisation for 1

4 (Study No.0105) Economic Co-operation and Development (OECD) Good Laboratory Practice and the OECD Guideline for Testing of Chemicals 451 Carcinogenicity Studies. Results Survival rates of the tetrachloroethylen-exposed groups of both sexes tended to be decreased as compared with the respective controls in a dose-related manner. The decreased survival rates in both sexes were causally related to the increased incidences of hepatocellular carcinoma. Body weights was significantly suppressed 6th week and afterward of 250 ppm-exposed males. Body weights was significantly suppressed 80th week and afterward of 250 ppm-exposed females. Food consumption of the 250 ppm-exposed of both sexes tended to be decreased as compared with the respective controls. No overt clinical sign was observed in any tetrachloroethylen-exposed mice of both sexes. AST and ALT were significantly increased in the 50 and 250 ppm-exposed males and 250 ppm-exposed females. Total bilirubin, LDH and ALP were significantly increased in the 250 ppm-exposed mice of both sexes. Total protein and total cholesterol were significantly increased in the 50 ppm-exposed males. Glucose and triglyceride were significantly decreased in the 50 ppm-exposed males. The incidences of selected neoplastic lesions in male and female rats are presented in the tables below. The incidences of hepatocellular adenomas and hepatocellular carcinomas were significantly increased in the 250 ppm-exposed mice of both sexes, and were increased dosedependently as indicated by a significant positive trend by Peto s test in both sexes. The incidence of hemangioendothelioma in the liver was increased dose-dependently as indicated by a significant positive trend by Peto s test in males. In the Harderian gland, the incidence of adenoma was increased dose-dependently as indicated by a significant positive trend by Peto s test in males. In the spleen, the incidences of hemangioendothelioma was increased dosedependently as indicated by a significant positive trend by Peto s test in males. The incidence of hemangioendothelioma in all organs including spleen and liver was increased dose-dependently as indicated by a significant positive trend by Peto s test in males and females. As non-neoplastic lesions in the liver, the incidence of angiectasis was increased in males exposed to 50 ppm and above and females exposed to 250 ppm. The incidence of centrolobular degeneration were increased in the 250 ppm-exposed males and females. The incidence of focal necrosis were increased in the 250 ppm-exposed males. In the kidney, nuclear enlargement in the proximal tubule was increased in males exposed to 50 ppm and above and females exposed to 250 ppm. Atypical tubular dilation in the proximal tubule was increased in the 250 ppmexposed females. 2

5 (Study No.0105) Conclusions In mice, there was clear evidence of carcinogenic activity of tetrachloroethylene in males and females based on the increased incidences of hepatocellular carcinomas, hepatocellular adenomas in males and females and increased incidence of Harderian gland adenomas in males. 3

6 (Study No. 0105) Incidences of selected neoplastic lesions of male mice in the 2-year inhalation carcinogenicity study of tetrachloroethylene Dose (ppm) Peto test Cochran- Armitage test Number of examined animals benign tumor lung bronchiolar-alveolar adenoma liver hepatocellular adenoma ** Harderian gland adenoma malignant tumor lung bronchiolar-alveolar carcinoma lymph node malignant lymphoma spleen hemangioendothelioma liver hepatocellular carcinoma ** hemangioendothelioma histiocytic sarcoma seminal vesicle histiocytic sarcoma All Site hemangioendothelioma liver hepatocellular adenoma+ hepatocellular carcinoma ** Tumors occurred more than 5% of examined animals at least in one group were presented. Significant difference *: p 0.05 **: p 0.01 (Fisher test) : p 0.05 increase : p 0.01 increase (Peto, Cochran-Armitage test) : p 0.05 decrease : p 0.01 decrease (Cochran-Armitage test) 4

7 (Study No. 0105) Incidences of selected neoplastic lesions of female mice in the 2-year inhalation carcinogenicity study of tetrachloroethylene Dose (ppm) Number of examined animals benign tumor lung bronchiolar-alveolar adenoma Peto test Cochran- Armitage test liver hepatocellular adenoma ** 1) pituitary adenoma ) 9 Harderian gland adenoma malignant tumor lymph node malignant lymphoma spleen malignant lymphoma ) 3 liver hepatocellular carcinoma ** uterus histiocytic sarcoma All Site hemangioendothelioma liver hepatocellular adenoma+ hepatocellular carcinoma ** Tumors occurred more than 5% of examined animals at least in one group were presented. 1):Number of examined animals is 49. 2):Number of examined animals is 48. Significant difference *: p 0.05 **: p 0.01 (Fisher test) : p 0.05 increase : p 0.01 increase (Peto, Cochran-Armitage test) : p 0.05 decrease : p 0.01 decrease (Cochran-Armitage test) 5

8 (Study No.0105) SELECTED TABLES TABLE 29 TABLE 30 TABLE 31 TABLE 32 TABLE 33 TABLE 34 TABLE 35 TABLE 36 TABLE 37 TABLE 38 TABLE 39 TABLE 40 TABLE 41 TABLE 42 SURVIVAL ANIMAL NUMBERS AND BODY WEIGHT CHANGES IN MALE MOUSE (TWO-YEAR STUDIES) SURVIVAL ANIMAL NUMBERS AND BODY WEIGHT CHANGES IN FEMALE MOUSE (TWO-YEAR STUDIES) INCIDENCE AND TIME OF MASS OCCURRENCE (CLINICAL OBSERVATION): MOUSE: MALE INCIDENCE AND TIME OF MASS OCCURRENCE (CLINICAL OBSERVATION): MOUSE: FEMALE FOOD CONSUMPTION IN MALE MOUSE (TWO-YEAR STUDIES) FOOD CONSUMPTION IN FEMALE MOUSE (TWO-YEAR STUDIES) NEOPLASTIC LESIONS (SPLEEN) INCIDENCE AND STATISTICAL ANALYSIS: MOUSE :MALE NEOPLASTIC LESIONS (LIVER) INCIDENCE AND STATISTICAL ANALYSIS: MOUSE :MALE NEOPLASTIC LESIONS (LIVER) INCIDENCE AND STATISTICAL ANALYSIS: MOUSE :FEMALE NUMBER OF MOUSE WITH SELECTED LIVER LESIONS NUMBER OF MOUSE WITH SELECTED KIDNEY LESIONS NEOPLASTIC LESIONS (HARDERIAN GLAND) INCIDENCE AND STATISTICAL ANALYSIS: MOUSE :MALE NEOPLASTIC LESIONS (PITUITARY GLAND) INCIDENCE AND STATISTICAL ANALYSIS: MOUSE :FEMALE CAUSE OF DEATH : MOUSE 6

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20 (Study No.0105) SELECTED FIGURES FIGURE 9 SURVIVAL ANIMAL RATE : MOUSE MALE (TWO-YEAR STUDIES) FIGURE 10 SURVIVAL ANIMAL RATE : MOUSE FEMALE (TWO-YEAR STUDIES) FIGURE 11 BODY WEIGHT CHANGES : MOUSE MALE (TWO-YEAR STUDIES) FIGURE 12 BODY WEIGHT CHANGES : MOUSE FEMALE (TWO-YEAR STUDIES) FIGURE 13 FOOD CONSUMPTION : MOUSE MALE (TWO-YEAR STUDIES) FIGURE 14 FOOD CONSUMPTION : MOUSE FEMALE (TWO-YEAR STUDIES) 18

21 (Study Nor.P104,0105) leo 7' " l' co:..ncl SO PP:! Pr): 250 PPl o 1 FIGURE 9 SURVIVAL ANIMAL RATE MOUSE:MALE(TWO-YEAR STUDIES) (1) " J- " CDXi2Cl _... _...._, Pf'.1 50 P?~ Pi'.1 FIGURE 10 SURVIVAL ANIMAL RATE MOUSE:FEMALE(TWO-YEAR STUDIES) 19

22 (Study No.0104,Ol05) (e) - J I - 15 I - I I B-1 as 9 96 I 104 COIt"TROl. 10 PPI\ 50 P?:I 250 PPX - 10 FIGURE 11 BODY WEIGHT CHANGES MOUSE:MALE(TWO-YEAR STUDIES) (0) (0) :J5 '" < ~r- <I- y- IIlIDS Q).'f[ROI. 10 r ':1 50 PI'~ 250 I'I'~ FIGURE 12 BODY WEIGHT CHANGES MOUSE:FEMALE(TWO-YEAR STUDIES) 20

23 (Study No.Ol04,Ol05) (Q i-,- t- lo,ll~ ~ ~/ t- v.-'w~ I- l- I~~ i7'" [/-I-" '}.-1 /:' ~ ~~ ~ ~. r-.>-~t-b /j! f..., '"<;W I'VV-U I') r-;- ~ '-', f:i... ~ f7 ~, - ~~ I- I I-.- I i- t- I- I 4... '" COIITR<lI. 10 rpl'l m 0 (e) r- FIGURE 13 FOOD CONSUMPTION MOUSE:MALE(TWO-YEAR STUDIES) I i I I I I i- I I I. I i I I ill I I - -V: I.-1.~~ I 1- ~~/.h7$~!.a-1~0:t/~~' I I' ~Ii ~k--'. f.!«k,: ~~i,~~ f- oo'~ t,~~ I I '"- t.'''' \{... I. I i I Jll I I I f- 0'1: I I I I-, i I I I I I, I f- I I I I I I I- I I I I I I 4.2 OEl'J(S i I II I - I I II \ill 1 I,1'I I \ I I I I. I I I M &! 12 Jb fiij M 12 to \ CON'TROL ~- )0 PP:I ===:::' = Jg ~~~ FIGURE 14 FOOD CONSUMPTION MOUSE:FEMALE(TWO-YEAR STUDIES) (e) 21

24 (Study No 0105) PHOTOGRAPHS PHOTOGRAPH 7 LIVER, HEPATOCELLULAR ADENOMA :A TWO-YEAR STUDY, MOUSE, MALE, 250 ppm, ANIMAL NO (H. E., X152) PHOTOGRAPH 8 LIVER, HEPATOCELLULAR CARCINOMA TWO-YEAR STUDY, MOUSE, MALE, 250 ppm, ANIMAL NO (H. E., X152) PHOTOGRAPH 9 LIVER, HEMANGIOENDOTHELIOMA TWO-YEAR STUDY, MOUSE, MALE, 250 ppm, ANIMAL NO (H. E., X152) PHOTOGRAPH 10 LIVER, ANGIECTASIS :A TWO-YEAR STUDY, MOUSE, MALE, 250 ppm, ANIMAL NO (H. E., X60) PHOTOGRAPH 11 SPLEEN, HEMANGIOENDOTHERIOMA :A TWO-YEAR STUDY, MOUSE, MALE, 250 ppm, ANIMAL NO (H. E., X152) PHOTOGRAPH 12 KIDNEY, NUCLEAR ENLARGMENT:PROXIMAL TUBULE :A TWO-YEAR STUDY, MOUSE, MALE, 250 ppm, ANIMAL NO (H. E., X304) 22

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Summary of Inhalation Carcinogenicity Study. of Isopropyl Acetate. in F344 Rats

Summary of Inhalation Carcinogenicity Study of Isopropyl Acetate in F344 Rats March 2009 Japan Bioassay Research Center Japan Industrial Safety and Health Association PREFACE The tests were contracted