Carcinogenicity Test of 2-Ethoxy-2-methylpropane in Rats (Inhalation Study)

Transcription

1 (Study No. 0686) FINAL REPORT Carcinogenicity Test of 2-Ethoxy-2-methylpropane in Rats (Inhalation Study) Study No.: 0686 March 25, 2010 <Translation from Japanese original into English> Japan Industrial Safety and Health Association Japan Bioassay Research Center

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3 (Study No. 0686) Study title Carcinogenicity test of 2-ethoxy-2-methylpropane in rats (inhalation study) Objective This test was conducted to investigate the potential of carcinogenesis of 2-ethoxy-2-methylpropane by whole-body (inhalation) exposure in rats for 104 weeks. Guideline The present test was performed in accordance with OECD Guideline for testing chemicals 451 (carcinogenicity study dated May 12, 1981). GLP The present test was performed in compliance with the good laboratory practice (GLP) Standards of the "The basis which related to the examination institution which carried out an examination to modify new chemical substance", Notification Yakushoku No dated November 21, 2003, Notification Seikyoku No.3 dated November 17, 2003, Notification Kanhoki No , and Ministry of Health, Labour and Welfare medicine food chief of the bureau, Ministry of Economy, Trade and Industry production industry chief of the bureau, Ministry of the Environment synthesis habitat policy chief of the bureau joint signature notice applied about, and also performed in compliance with "OECD Principles of Good Laboratory Practice" (November 26, 1997). Sponsor Japan Petroleum Energy Center Sumitomo Shin- Toranomon Building, 4-3-9, Toranomon, Minato-ku, Tokyo, Japan Test facility and facility manager Japan Bioassay Research Center, Japan Industrial Safety and Health Association Vice-director : Kasuke Nagano, D.V.M., Ph, D Hirasawa, Hadano, Kanagawa, Japan i

4 (Study No. 0686) Schedule of the Test Commencement of test : May 1, 2007 Receipt of animals : May 7, 2007 Allocation of animals : May 21, 2007 Initiation of test material treatment : May 22, 2007 Termination of test material treatment : May 18, 2009 Scheduled necropsy : May 19, 20, 21, 22 and 25, 2009 Completion of test : March 25, 2010 Study personnel Study director: Tomoshi Nishizawa (Inhalation Test Section, Department of Experimental Toxicology) Analysis, administration and management of test substance : Tomoshi Nishizawa (Inhalation Test Section, Department of Experimental Toxicology) Kaoru Goto (Department of Experimental Toxicology) Tatsuya Kasai (Inhalation Test Section, Department of Experimental Toxicology) Arata Saito (Inhalation Test Section, Department of Experimental Toxicology)) Toshiaki Sasaki (Inhalation Test Section, Department of Experimental Toxicology) Makoto Ohnishi (Analytical Chemistry Section, Department of Experimental Toxicology) Makoto Take (Analytical Chemistry Section, Department of Experimental Toxicology) Animal care: Tadashi Noguchi (Microbe Examination Section, Department of Pathology) Taku Katagiri (Animal Care Section, Department of Experimental Toxicology) Tomoyuki Kamigaito (Microbe Examination Section, Department of Pathology) Pathology examination: Shigetoshi Aiso (Pathology Examination Section, Department of Pathology) Hideki Senoh (Pathology Examination Section, Department of Pathology) Yumi Umeda (Pathology Examination Section, Department of Pathology) Misae Saito (Pathology Examination Section, Department of Pathology) Takayoshi Noguchi (Hematological and Biochemical Examination Section, Department of Pathology) Hitomi Kondo (Hematological and Biochemical Examination Section, Department of Pathology) Data processing and statistics: Naoki Ikawa (Data Processing Section, Department of Administration and Finance) Hiroaki Ishikawa (Data Processing Section, Department of Administration and Finance) Takashi Mine (Data Processing Section, Department of Administration and Finance) Positions in parenthesis are at March 25, ii

5 (Study No. 0686) Archives of documents and samples Protocol, specimens, raw data, test material, record documents, final report, certificate of quality assurance, the other documents and samples related to this test are to be stored in archives. Furthermore, 50 g of test compound are to be stored in the storage. The retention period is for ten years (as principle) after submission of the final report. However, storage of specimens and test material will be restricted to the period to be maintain for their quality. Signature, seal and date of study director (person prepared the final report) <Signatured and Sealed in original> < Dated in original> Tomoshi Nishizawa March 25, 2010 Inhalation Test Section, Department of Experimental Toxicology iii

6 (Study No. 0686) Statement Study title: Carcinogenicity test of 2-ethoxy-2-methylpropane in rats (inhalation study) The present test was performed in compliance with the good laboratory practice (GLP) Standards of the "The basis which related to the examination institution which carried out an examination to modify new chemical substance", Notification Yakushoku No dated November 21, 2003, Notification Seikyoku No.3 dated November 17, 2003, Notification Kanhoki No , and Ministry of Health, Labour and Welfare medicine food chief of the bureau, Ministry of Economy, Trade and Industry production industry chief of the bureau, Ministry of the Environment synthesis habitat policy chief of the bureau joint signature notice applied about, and also performed in compliance with "OECD Principles of Good Laboratory Practice" (November 26, 1997). We consider the data generated by Japan Bioassay Research Center during the course of this test to be valid and that the final report fully and accurately reflect this raw data. Japan Bioassay Research Center, Japan Industrial Safety and Health Association Study director Date <Signatured and Sealed in original> <Dated in original> Facility manager Date <Signatured and Sealed in original> <Dated in original> iv

7 (Study No. 0686) Certificate of Quality Assurance Study title: Carcinogenicity test of 2-ethoxy-2-methylpropane in rats (inhalation study) Study No.: 0686 Test Substance: 2-ethoxy-2-methylpropane (ETBE) The study was conducted essentially in compliance with "The Standards Concerning Test Facilities Conducting the Tests Relating to New Chemical Substances" of the Joint Notification of No of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, Heisei No.3 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry and No of the Environmental Policy Bureau, Ministry of the Environment, Japan, November 21, 2003 and "OECD Principles of Good Laboratory Practice" (November 26, 1997). We herewith certify that the study was properly performed and the methods and procedures were correctly recorded in the final report and this presents accurately all data generated. Items Inspection or audit Dates Protocol May 1, 2007 May 1, 2007 Protocol (Amendment) June 26, 2007 June 26, 2007 Report to management and study director April 22, September 5, 2008 April 22, September 10, 2008 Animal receipt/quarantine May 7, 2007 May 7, 2007 Acclimation/Allocation May 21, 2007 May 21, 2007 May 21, August 6, November 19, 2007 May 21, August 6, November 19, 2007 Animal husbandry February 4, May 26, August 25, 2008 February 4, May 26, August 25, 2008 November 17, 2008 November 17, 2008 February 9, May 18, 2009 February 9, May 18, 2009 May 22, August 6, November 19, 2007 May 22, August 6, November 19, 2007 Inhalation study system/management February 4, May 26, August 25, 2008 February 4, May 26, August 25, 2008 of the test material November 17, 2008 November 17, 2008 February 9, May 18, 2009 February 9, May 18, 2009 Urinalysis May 14, 2009 May 14, 2009 Necropsy May 19, 2009 May 19, 2009 Hematology/Blood biochemistry May 19&20, 2009 May 20, 2009 Management of the test material January 20, 2010 January 20, 2010 Management of raw data January 4-20, 2010 January 20, 2010 Draft report March 18-24, 2010 March 24, 2010 Final report March 25, 2010 March 25, 2010 March 25, 2010 Quality Assurance Director Attached to: Japan Bioassay Research Center Japan Industrial Safety and Health Association Title :Quality Assurance Manager <Signatured and Sealed in original> v

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12 (Study No. 0686) Abstract The carcinogenic potential of 2-ethoxy-2-methylpropane (ethyl tert-butyl ether, ETBE) was investigated in a two year (104 weeks) inhalation carcinogenicity test using F344/DuCrlCrlj rats. A total of 400 rats (6-week-old at the start of treatment) including one control and 3 treated groups, each comprising 50 males and 50 females, were used in the present test. The administration of test material, ETBE, was performed by inhalation for six hours/day, five days/week, for 104 weeks. The administration levels were 0 (control group), 500, 1500 and 5000 ppm (volume ratio v/v) for both sexes. Observations and examinations carried out in the present test were as follows: general condition, body weight, food consumption, hematology, blood chemistry, urinalysis, gross pathology, organ weights and histopathology. As a result of exposure of ETBE, statistically significant decreases of survival rates were noted in males of the 5000 ppm group and females receiving 1500 ppm or 5000ppm. The final survival rate in males of the 5000 ppm group was 60% as compared to 88 % in control males, and those in females of 1500 and 5000 ppm were both 60% as compared to 76 % in control females. The decrease in survival rate of 5000 ppm males appeared related to chronic nephropathy. Although treatment-related change in general condition was not found for animals exposed to ETBE, significant inhibition of body weight gain was noted in males of the 5000 ppm group and females of the 1500 and 5000 ppm groups. Final body weights in 5000 ppm males were 75% of control male value, while in in females of the 1500 ppm and 5000 ppm groups they were 91% and 78%, respectively. Food consumption by both sexes of treated groups were comparable to the control values, except in the early phase of the experiment (weeks 1 to 7), when males in the treated groups exhibited lower values. Regarding neoplastic lesions, the incidence of hepatocellular adenomas was increased in males of the 5000 ppm group, along with the incidences of acidophilic and basophilic cell foci. Treatment-related increases in incidences of neoplastic lesions were not found in females. Treatment-related non-neoplastic lesions were observed in the kidney, stomach and arteries. In the kidney, the degree of chronic nephropathy was increased in both sexes of the 5000 ppm group, as was that of urothelial hyperplasia in the renal pelvis of males exposed to 1500 ppm or more, and that of mineral deposition in the papilla at 5000 ppm. In addition, incidences of mineral deposition in the stomach and arteries were significantly increased in males of the 5000 ppm group. In the present 2 year (104 weeks) carcinogenicity test (inhalation study) of ETBE in the F344/DuCrlCrlj rats, the following was concluded. An increased incidence of hepatocellular adenomas was found in males, indicating some evidence of ETBE carcinogenicity in male rats. The two-year inhalation exposure to ETBE did not exert carcinogenicity in female rats. 1

13 (Study No. 0686) Incidences of major tumors in male rats in the present carcinogenicity test of 2-ethoxy-2-methylpropane Administration concentration (ppm) Peto test Cochran- Armitage test Numbers of animals (Benign tumors) Subcutis Fibroma * Liver Hepatocellular adenoma ** Pancreas Islet cell adenoma Pituitary Adenoma Thyroid C-cell adenoma Adrenal Pheochromocytoma Testis Interstitial cell tumor Preputial gl. Adenoma (Malignant tumors) Lung Bronchiolar alveolar carcinoma Spleen Mononuclear cell leukemia Liver Hepatocellular carcinoma Thyroid C- cell carcinoma Peritoneum Mesothelioma Liver Hepatocellular adenoma / Hepatocellular carcinoma ** Incidences of major tumors in female rats in the present carcinogenicity test of 2-ethoxy-2-methylpropane Administration concentration (ppm) Peto test Cochran- Armitage test Numbers of animals (Benign tumors) Pituitary Adenoma a Thyroid C- cell adenoma * 3 Uterus Endometrial stromal polyp Mammary gl. Fibroadenoma * (Malignant tumors) Spleen Mononuclear cell leukemia Uterus Adenocarcinoma Uterus Endometrial stromal sarcoma a: significant by only Peto test (death analysis) * : P=0.05 ** : P=0.01 (Fisher exact test) : significant increase, P=0.05 :significant increase, P=0.01 (Peto, Cochran-Armitage test) : significant decrease, P=0.05 : significant decrease, P=0.01 (Cochran-Armitage test) 2

14 (Study No. 0686) I Test Materials I-1 Properties of the test material I-1-1 Names Chemical name : 2-Ethoxy-2- methylpropane Synonym : Ethyl tert butyl ether (ETBE) CAS No. : I-1-2 Structural formula and molecular weight Structural formula: CH 3 H 3 C C O CH 2 CH 3 CH 3 Molecular weight; I-1-3 Physical and chemical properties Appearance : Colorless transparent liquid Melting point : 70 ºC Vapor pressure : 17kPa (25 ºC) Solubility : Slightly soluble in water (1.2g /100g, 20 ºC) Storage condition : In a dark place at room temperature I-2 Lot of the test material Manufacturer : Nippon Refine Co., Ltd., Gifu, Japan Purity : More than 99wt% (measured by Toray Research Center Co., Ltd., Tokyo, Japan) Lo t number : L

15 (Study No. 0686) I-3 Characteristics, identity and stability of the test material I-3-1 Characteristics and identity The identity of test material was analyzed by electron impact mass spectrometry (Agilent Technologies 5973N) and confirmed by comparison with reported values. The mass spectrum of the test material showed the expected fragment peak (Reference 1), and confirmed identity as ETBE. The results are shown in Appendix A 1. I-3-2 Stability Stability of test material was measured using gas chromatograph (Agilent Technologies 5890A) before the beginning and at the end of the test material administration, and confirmed by comparison of the data from the two time points. There was no difference between the results at the beginning and the end of test material use, and the stability of the test material during the treatment period was confirmed. The results are shown in Appendix A 2. I-4 Animals Both sexes of F344/DuCrlCrlj rat (SPF animal) from Charles River Laboratories Japan Inc. (Atsugi breeding center, 795 Shimofurusawa, Atsugi, Kanagawa) were used in the present test. At four weeks of age, totals of 227 animals of each sex were obtained and allowed one week of quarantine and a one week acclimation period, and then 200 males and 200 females (body weight range; males: g, females: g) were selected for the test. These animals showed no abnormality in general condition and body weight gain, and near to the median body weight values. The F344/DuCrlCrlj rat (SPF animal) was selected because of its genetic stability and routine use and known sensitivity for carcinogenicity studies of chemicals. 4

16 (Study No. 0686) II Test methods II-1 Administration II-1-1 Route of administration Inhalation was selected as the route of administration. II-1-2 Exposure method for the test compound Air including the test compound adjusted to the set concentration was introduced into the inhalation chambers accommodating the animals for examination, and administration was performed systematically. II-1-3 Duration of administration The duration of administration by exposure was for six hours/day, five days/week, 104 weeks, for a total of 520 treatments. II-1-4 Administered concentrations The administration concentrations were set at three levels of 500, 1500 and 5000 ppm (volume ratio v/v). In addition, a control group was maintained with clean air ventilation. II-1-5 Rationale for the route of administration, the duration of administration and the choice of administration concentrations Systemic exposure by inhalation was selected as the administration route of one main human exposure route. The duration of administration was 2 years (104 weeks) according to OECD chemistry product test guideline 451 (a carcinogenicity test) (Reference 2). The administration time was determined to be six hours/day according to OECD chemistry product test guideline 451. The administration concentrations were set in discussion with the sponsor taking account of the results of inhalation toxicity examination for 13 weeks [preliminary study for the caricinogenicity test] (Study No. 0666) carried out in a Japan Bioassay Research Center (Reference 3). In the preliminary study, both sexes of F344/DuCrlCrlj rats were given 0 ppm (control), 50, 150, 500, 1500 and 5000 ppm ETBE by systemic exposure (inhalation) for 13 weeks, and general condition, body weight, food consumption, hematology, blood biochemistry, urinalysis, gross pathology, organ weight and histopathology were investigated. No mortality was found in any group. Inhibition of body weight gain was found in 5000 ppm group males. Average body weights at the end of the treatment in males of the 50, 150, 500, 1500 and 5000 ppm groups were 94 %, 95 %, 95 %, 97 % and 89 %, respectively, and those in female 50, 150, 500, 1500 and 5000 ppm groups were 95 %, 97 %, 96 %, 94 % and 95 %, respectively, of the control values. Food consumption by 5000 ppm group males was low in the early phase of the exposure period. In hematology, slight, but significant, decreases in red blood cell count and hemoglobin 5

17 (Study No. 0686) concentration, and slight, but significant, increase in platelet counts were observed in 5000 ppm group males. In blood biochemistry, protein, parameters related to lipids, and urea nitrogen were significantly altered in 5000 ppm group males. In organ weights, significant increases in kidney weights were observed in both sexes of rats given 1500 ppm ETBE or more, and significant increases in liver weights were noted in both sexes at 5000 ppm. On histopathological examination, an increased incidence of regeneration of proximal renal tubules in the kidney was observed in 5000 ppm males. For the selection of dose levels in carcinogenicity test, many guidelines recommend that the highest dose should induce minimal toxic effects and cause less than 10% inhibition of body weight gain, and the lowest dose should be not lower than 1/10 of the highest dose level. In the preliminary 13-week study, males given 5000 ppm showed inhibition of body weight gain (to 89% of control value). In addition, significant increases of kidney and liver weights were also found in both sexes receiving 5000 ppm, and histopathological lesions were observed in 5000 ppm males. Therefore, the highest dose for the carcinogenicity test was decided as 5000 ppm. In the preliminary 13-week study, no treatment related changes were found in either sex at 500 ppm. Therefore, the lowest dose for the carcinogenicity test, was set at 500 ppm, with 1500 ppm as the mid level with a proportional factor of 3. II-1-6 Vaporization methods and adjustment of test material levels ETBE was placed in the container of an organic solvent vapor generator (model; specially made to order, Shibata Scientific Technology Ltd., Tokyo, Japan), and vaporized by bubbling of clean air with heating by a thermostatted circulator. The air flow containing the saturated vapor was diluted with clean air, cooled through a thermostatted condensor, then warmed in a thermostatted circulator, which served to stabilize the vapor concentration. The flow rate of the vapor-air mixture was regulated with a flow meter, for supply to the line mixer in the inhalation exposure chambers. Chamber concentrations of the test material were monitored by gas chromatography, and supply volume of test material to the inhalation chamber was regulated to adjust to the intended concentrations based on the data obtained. II-1-7 Measurement of test material concentrations Chamber concentrations of ETBE were analyzed by gas chromatography with an automatic sampler (model GC-14B, Shimadzu Corporation, Kyoto, Japan) every 15 minutes during the exposure period. The analytical results are shown in Table A. For each treated group, differences between the found and intended concentrations ((found concentration intended concentration) / intended concentration x100) were less than 0.1 %, and variation indexes (standard deviation / mean value x100) were less than 1.0 %. Thus, it was shown that ETBE concentrations in the chamber were maintained with high precision. 6

18 (Study No. 0686) II-2 Animal husbandry II-2-1 Numbers of animals in each group Three treatment groups and one control group, for a total of 4 groups (50 rats/sex/group) were employed in the present test.. Group name The number of the animals (animal numbers) Male Female Control group 50 ( ) 50 ( ) 500 ppm 50 ( ) 50 ( ) 1500 ppm 50 ( ) 50 ( ) 5000 ppm 50 ( ) 50 ( ) II-2-2 Allocation and individual identification methods Allocation was carried out by a method to minimize the deviation of body weights among groups (proper stratification method) (Reference 4). At first, animals, which were normal for general condition and body weight gain, were allotted in order of heaviest body weight. Secondly, comparing with the total weight of each group, heaviest weight animals were, in turn, allotted to the lightest weight groups,, this procedure then being repeated. During the quarantine/acclimation period, individual animals were identified by tail marking with a felt-pen. During the treatment period, individual animals were identified by ear punch. In addition, a label carrying individual animal numbers was affixed to each cage. The animals were housed in an independent room (Room No.: 712) in a barrier area, and the study number, animal species and animal numbers were displayed on the door of the room to allow distinction from other studies and different species of animals. II-2-3 Animal husbandry conditions (1) Housing condition Animals were housed in quarantine rooms (Room No.: 716 and 717) during quarantine and in inhalation chambers of the inhalation test room (Room No.: 712) during the acclimation and administration periods. Environmental conditions of the quarantine room, inhalation test room, inhalation chambers and cages were as shown below. The actual values (mean standard deviation) for temperature and humidity of quarantine room and inhalation test room are shown in < >, and the measured results for the inhalation chamber internal environment are given in Appendix B. No major changes affecting health conditions of animals were found in the environments of the quarantine inspection room, inhalation test room and inhalation chambers. Temperature: Quarantine room; 23±2ºC <Room No.716; 22.7±0.0ºC, room No.717;23.4±0.0> Inhalation test room; 22±3ºC<Room No. 712; 21.6±1.0ºC> Inhalation chambers; 22±2ºC 7

19 (Study No. 0686) Humidity: Quarantine room; 55±15% < Room No.716; 53±1%, Room No. 717; 55±1% > Inhalation chambers; 50±20% Lighting: artificially illuminated for 12 h on (8:00 to 20:00) and 12 h off (20:00 to 8:00) Ventilation frequency: Quarantine room; times/hour Inhalation test room; 7-9 times/hour Inhalation chambers; 13±1 times /hour Pressure power: In inhalation chamber; 0~ -15x10 Pa Accommodation methods: group housing (5 animals/cage) during quarantine period one animal to a cage during the acclimation and treatment periods Materials/shape/dimensions of cages: Quarantine period; stainless steel mesh cage [340(W)x294(D)x176(H) mm/5 animals] Acclimation period; six-linked stainless steel mesh cage [125(W)x216(D)x176(H) mm/animal] Treatment period; five-linked stainless steel mesh cage [150(W)x270(D)x176(H) mm/animal] (2) Food Rats had ad libitum access to CRF-1 pellet diet (sterilized by 30kGy- γ rays irradiation) obtained from Oriental Yeast Co., Ltd. (Chiba factory: 8-2 Shinminato, Mihama-ku, Chiba, Japan) placed in feeders for pellets throughout the experimental period. However, animals were fasted from the evening of the previous day for scheduled necropsy. Analytical data for nutrients for each lot used in the present test were obtained from Oriental Yeast Co., Ltd., and archived. Analytical data for concomitants in diet for each lot used in the present test were also received from Japan Food Research Laboratories, Foundation (52-1 Motoyoyogi-cho, Shibuya-ku, Tokyo, Japan) and Eurofin Scientific Japan, K.K. ( Shimouma, Setagaya-ku, Tokyo, Japan), and contaminant levels in diet were confirmed to be within acceptable ranges (see protocol), and then archived. (3) Water Animals had ad libitum access via automatic water suppliers, to filtered and ultraviolet irradiated tap water (Hadano, Kanagawa water service station supply) as drinking water throughout the experimental period. The drinking water was routinely sampled and shipped to Hatano Research Institute, Food and Drug Safety Center, Japan (729-5 Ochiai, Hadano, Kanagawa, Japan) for analyses performed in accordance with the law for tap water. Analytical results were received, and contaminant levels in drinking water were confirmed to be within acceptable ranges, and then archived. II-3 Observation and laboratory investigation, and methods II-3-1 Observations of fate and general condition Animals were observed for their status (alive, dead and in extremis) daily, and also observed for detailed general condition once a week. 8

20 (Study No. 0686) II-3-2 Measurement of body weights Animals were weighed weekly for the first 14 weeks, and thereafter once every four weeks (additionally at week 104). In addition, animals were weighed when found dead, killed in extremis and at scheduled necropsy (when carried out from the animal room). II-3-3 Measurement of food consumption Supplied and remaining amounts of food were measured weekly for the first 14 weeks and once four weeks thereafter (additionally at week 104), and daily food consumption values per rat were calculated. II-3-4 Hematology Hematological examinations were performed for surviving animals, with collected blood samples, at the termination of the experiment. Blood was collected into sample tubes including EDTA-2 potassium via the abdominal aorta under ether anesthesia just before necropsy. Methods for hematological estimations are shown in Appendix C for the following. Parameters: red blood cell count, hemoglobin concentration, hematocrit, mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), platelet count, reticulocyte count, white blood cell count, differential white blood cell percentages II-3-5 Blood biochemistry At scheduled necropsy, blood was collected into heparin lithium treated sample tubes via abdominal aorta under anesthesia just before autopsy and examined for the following items. Examination methods are given in Appendix C. Analysis items: total protein, albumin, A/G ratio, total bilirubin, glucose, total cholesterol, triglyceride, phospholipid, AST, ALT, LDH, ALP, -GTP, CK, urea nitrogen, creatinine, sodium, potassium, chlorine, calcium, inorganic phosphorus II-3-6 Urinalysis From animal that lived until week 104, fresh urine was obtained and analyzed for the following items using urine test paper (Multisticks, Siemens Healthcare Dyagnostics Inc.). Analysis item: ph, protein, glucose, ketone, bilirubin, occult blood, urobilinogen 9

21 (Study No. 0686) II-3-7 Pathology (1) Gross pathology All animals were observed macroscopically. (2) Organ weights For all animals that lived until scheduled necropsy, organ wet weights (absolute organ weights) were measured. In addition, percentages of each organ weight to final body weights (organ weight/body weight ratio) were calculated. Organs weighed: adrenal gland, testis, ovary, heart, lung, kidney, spleen, liver, and brain (3) Histopathological examination The following organs and tissues of all animals were excised, and fixed with 10% neutral buffered formalin solution. After fixation, they were routinely processed for embedding in paraffin, sectioned, stained with hematoxylin and eosin, and examined histopathologically. The nasal cavity was sliced into three sections (Reference 5) at the incisal end (level 1), the incisal papilla (level 2) and the front edge (level 3) of the first molar tooth for examination. Organs and tissues examined: skin, nasal cavity, nasopharynx, larynx, trachea, lung, bone marrow (femur), lymph node (axilla, abdominal wall, etc.), thymus, spleen, heart, tongue, salivary gland, esophagus, stomach, small intestine (including the duodenum), large intestine, liver, pancreas, kidney, urinary bladder, pituitary gland, thyroid gland, parathyroid, adrenal gland, testis, epididymis, seminal vesicle, prostate, ovary, uterus, vagina, mammary gland, brain, spinal cord, peripheral nerve (sciatic nerve), eyeball, Hadrian gland, muscle, bone (femur), and any other tissues with an abnormal appearance macroscopically II-4 Numerical value processing and statistical methods II-4-1 The handling of numerical values All numerical data are presented depending on the precision of the measuring apparatus. Concentrations (ppm as the unit) of test material in inhalation exposure chambers were measured to three decimal places, rounded off to two decimal places and shown as one decimal place. Body weights (grams as the unit) were measured to 1 digit of an integer value and shown as such. Food consumption (grams as the unit) was measured with reference to initial and remaining weights of food to one decimal place. Values were divided by days of measurement, and rounded off to two decimal places, and shown as one decimal place. Absolute organ weights (grams as the unit) were measured to three decimal places, and shown as such. Organ to body weight ratios (% as the unit) were calculated by dividing absolute organ weights by final body weights, rounded off to four decimal places, and shown as three decimal places. 10

22 (Study No. 0686) Measured values for hematology and blood biochemistry were shown dependent on units and the precision as noted in Appendix C. Furthermore, the means and standard deviations of numerical data were rounded off in order to consistent with the aforementioned digits, and shown in the summary tables. II-4-2 Statistical processing The effective numbers of animals in each group was the initial total number of animals with subtraction of the number of animals excluded for reasons of accidents. For the histopathological examination, the numbers of organs and animals were those for which it was possible to carry out the examination. Statistical comparisons between control and treated groups for numerical data obtained for body weights, food consumption, hematology, blood biochemistry and organ weights were conducted using the the Bartlett s test. If homogenous, the data were analyzed by one-way layout variance analysis, and if significant differences were found with the Dunnett s multiple comparison test. If not homogenous, the data were ranked through each group, and analyzed by the Kruskal-Wallis s test, and further by a multiple comparison test of Dunnett s type when significant differences were found between groups. For non-neoplastic lesions in the histopathological examination, the animals with no change were graded 0, and animals with lesions were classified into grades 1-4 dependent on the degree and range of alterations. The data were statistically analyzed with the Chi-square test. Data for urinalyses were also analyzed with the Chi-square test between control and treated groups. For neoplastic lesions, numbers of animals with tumors in each organ for each neoplastic lesion were analyzed by the Peto test (Reference 6), Cochran-Armitage test and Fisher exact test. Furthermore, Peto tests, using contex (see Notes), were performed by the death analysis (test for neoplasms given contex 3 and 4), a incidental tumor test (test for neoplasms given contex 0, 1 and 2), and a death analysis plus a incidental tumor test (analyzed by a total of contex 0-4). The significance of differences for each parameter was analyzed in each test, and evaluated at P<0.05. The Peto and Fisher exact test were carried out one sided, and other tests were performed two-sided, with analytical results were shown at P<0.05 and Notes: Contex used for Peto test 0: tumor found in a scheduled necropsy animal 1: tumor found in an animal which died or was killed in an moribund condition, and not related to the death 2: tumor classified as probably 1, but not confirmed 3: tumor classified as probably 4, but not confirmed 4: tumor found in an animal which died or was killed in an moribund condition, and related to the death 11

23 (Study No. 0686) III Test results III-1 Survival animal numbers Survival animal numbers are shown in Tables B 1 and 2 and Figures 1 and 2. - Males - Significant decrease of the survival rate was found in the 5000 ppm group. At week 104, numbers of survivors (survival rate) in the male 0, 500, 1500 and 5000 ppm groups were 44 (88 %), 38 (76 %), 39 (80 %) and 30 (60 %), respectively. Furthermore, effective numbers of animals in 1500 ppm group became 49, since one animal (number 1213) was found dead on the 5th day of week Females - Significant decrease of survival rate was found in the 1500 and 5000 ppm groups. At weeks 104, numbers of survivals (survival rate) in female 0, 500, 1500 and 5000 ppm groups were 38 (76 %), 39 (78 %), 30 (60 %) and 30 (60 %), respectively. III-2 General condition Observation results for general condition are shown in Tables C 1 and 2 and Appendices D 1 and 2. - Males and females - No treatment related changes were found in any group. III-3 Body weights Changes of body weights are shown in Tables D 1 to 4, Figures 3 and 4 and Appendices E 1 and 2. - Males - Inhibition of body weight gain was observed in the 5000 ppm group throughout the course of the test. Body weights in the 500 and 1500 ppm groups were comparable to the control values, although they showed tendencies for lowering during the late phase of the test. Body weights on the last measurement day (104 weeks) were 94%, 94% and 75 % of the control value for the 500, 1500 and 5000 ppm groups. - Females - Inhibition of body weight gain was observed in the 5000 ppm group from week 10 to the termination, and in the 1500 ppm group from week 30 to the termination. Body weights on the last measurement day (104 weeks) were 95%, 91% and 78 % of the control value for the 500, 1500 and 5000 ppm groups. 12

24 (Study No. 0686) III-4 Food consumption Food consumption data are shown in Tables E 1 to 4, Figures 5 and 6 and Appendices F 1 and 2. - Males - Food consumption in the treated groups was significantly lower than the control values during the early phase of (1-7 weeks) of treatment period. Thereafter, those in treated groups were comparable to control values, with the exception of lowered values noted sporadically. - Females - Food consumption in the treated groups was comparable to control values, with the exception of lowered values noted sporadically. III-5 Hematological examination Results of hematology are shown in Tables F 1 and 2 and Appendices G 1 and 2. - Males - No consistent treatment related changes were found in any group. A significantly elevated lymphocyte ratio in differentiation of white blood cells was noted in the 1500 ppm group, but without dose-relation. - Females - Significant lowered MCV values were observed in the 5000 ppm group. Although slight, a significant lowered value for MCH was observed in the 5000 ppm group. These alterations did not clearly correlate with the administration level. III-6 Blood biochemistry Results of blood biochemistry are shown in Tables G 1 and 2 and Appendices H 1 and 2. - Males - Significant elevation of urea nitrogen, creatinine, and inorganic phosphorus was observed in all treated groups. And, significant elevation of total cholesterol, triglyceride, phospholipid, -GTP and calcium and significant lower value of A/G ratio were observed at 1500 and 5000 ppm. Furthermore, significantly increased values for potassium and significant lowered values for total protein, albumin, AST and chlorine were noted at 5000 ppm. Although slight, significant lowering of values for sodium were observed in the 5000 ppm group, but the correlation with the administration level was not clear. Furthermore, significant lowering of total protein and albumin was noted at 500 ppm, and of ALP at 1500 ppm, but these changes were not dose-related. - Females - Significantly elevated values for total cholesterol, phospholipid, urea nitrogen and potassium and significant lowering of values for albumin, the A/G ratio, AST, ALT and sodium were observed in the 5000 ppm group. Average AST and ALT values in the 5000 ppm group were higher than in the control group, but the changes were due to extremely high values for one rat. After exclusion of the abnormal data for the one rat, average AST and ALT values in the 5000 ppm group were significantly lower than in the controls. 13

25 (Study No. 0686) III-7 Urinalysis The results of urinalyses are shown in Tables H 1 and 2 and Appendices I 1and 2. - Males - Significant lowered values for urine ph were observed in all treated groups. - Females - Significant lowered values for urine ph and significant increase in amounts of urine protein were observed in the 5000 ppm group. III-8 Pathology III-8-1 Gross pathology Macroscopic findings are shown in Tables I 1 to 6 and Appendices J 1 and 2. - Males - Numbers of animals with granulated kidney surfaces were significantly increased in a dose-related manner (9, 16, 17 and 36 animals in the control, 500, 1500 and 5000 ppm groups, respectively). - Females - Granulated kidney surfaces were observed in 6 animals of the 5000 pm group, and this was relatively large as compared with the other groups (2, 1 and 0 animals in the control, 500 and 1500 ppm groups, respectively). III-8-2 Organ weights Absolute organ weights and organ to body weight ratios measured at scheduled necropsy are shown in Tables J 1 and 2, K 1 and 2 and Appendices K 1 and 2, L 1 and 2. - Males - Significantly elevated values for absolute and relative kidney weights were observed in all treated groups. Significantly elevated absolute liver weights were observed in the 1500 ppm group, but were not dose-related. Although significant lowering of absolute brain weights was noted in the 5000 ppm group, and significant elevated values for relative adrenal, testis, heart, lung, spleen, liver and brain weights were found in all treated groups, these were due to significantly lowered final body weights of treated groups. Average relative adrenal weights in the 5000 ppm group were lower than in the control group, but this was due to extremely high values for 2 rats in the controls. After exclusion of the abnormal data for the 2 rats, average relative adrenal weights in the 5000 ppm group were significantly higher than the control value. 14

26 (Study No. 0686) - Females - Significant elevation of absolute kidney weights was found in groups receiving 1500 or 5000 ppm, and significantly elevated relative kidney weights were noted in all treated groups. Significant lowering of absolute adrenal weights was observed in the 1500 ppm group, but this was not dose-related. Although significant reduction of absolute spleen weights was noted in the 5000 ppm group, and significant elevation of relative adrenal, ovary, heart, lung, liver and brain weights was found in all treated groups, these were due to significant lowered final body weights of the treated groups. III-8-3 Histopathological examination Data for non-neoplastic lesions are shown in Tables L 1 to 6. Numbers of animals with tumors and numbers of tumors are shown in Tables M 1 and 2, respectively. Incidences of neoplastic lesions are shown in Tables N 1 and 2, and the results of statistical analysis (Peto test, Cochran-Armitage test and Fisher test) are shown in Tables O 1 and 2. Data for metastatic lesions are shown in Tables P 1 and 2. Individual histopathological data are shown in Appendices M 1 and 2. Furthermore, for neoplastic lesions for which statistically significant alteration was found in the present test, historical control data [incidences (minimum % - maximum %) in each study, and average incidences (%), number of animals with tumors/total number of animals] of the Japan Bioassay Research Center are shown in Table Q. - Males - 1) Neoplastic lesions <Liver> Regarding development of hepatocellular adenomas [0/50 (0%) in the control group; 2/50 (4%) at 500 ppm; 1/49 (2%) at 1500 ppm;, 9/50(18%) at 5000 ppm], tendencies for increase were found by the Peto test (incidental tumor test) and the Cochran-Armitage test, and significant increase was evident for the 5000 ppm group by the Fisher exact test. The incidence of 18% (9/50) for hepatocellular adenomas in the 5000 ppm group exceeded the range in historical control data (incidences of hepatocellular adenomas in control groups from 48 carcinogenicity studies: minimum 0% ~ maximum 8%, average incidence 1.8%) in our laboratory. In addition, one hepatocellular carcinoma was observed at 5000 ppm, and combined incidences of hepatocellular adenoma or carcinoma [0/50 (0%) in the control group; 2/50 (4%) in the 500 ppm group; 1/49 (2%) in the 1500 ppm group; 10/50(20%) in the 5000 ppm group], demonstrated tendencies for increase by the Peto test (incidental tumor test) and the Cochran-Armitage test, and significant increase was evident in the 5000 ppm group by the Fisher exact test. Diagnoses of hepatocellular adenoma and hepatocellular carcinoma were based on the criteria of Guides for Toxicologic Pathology (Reference 7). In addition, for diagnosis of hepatocellular adenoma, account was taken of a tendency for disordering and multiple layers of hepatocyte cords as an indicator of tumor proliferation. In addition, the incidence of fibromas of the subcutis in the 5000 ppm group showed a tendency for decrease with the Cochran-Armitage test, and significant decrease with the Fisher exact test. 15

27 (Study No. 0686) 2) Non-neoplastic lesions <Liver> Significant increases in incidences of acidophilic and basophilic cell foci were observed in the 5000 ppm group. For development of acidophilic cell foci [31/50 (slight) in the control group; 28/50 (slight) in the 500 ppm group; 36/49 (slight) in the 1500 ppm group; 39/50(30 rats slight, 9 rats moderate) in the 5000 ppm group], a significantly increased incidence and enhancement of the degree were found in the 5000 ppm group. For development of basophilic cell foci [18/50 (slight) in the control group; 10/50 (slight) in the 500 ppm group; 13/49 (slight) in the 1500 ppm group; 33/50(31 rats slight, 2 rats moderate) in 5000 ppm group], a significantly increased incidence was found in the 5000 ppm group. Acidophilic and basophilic cell foci are considered to be preneoplastic lesions, and foci were sub-classified as acidophilic, basophili and clear cell, based on the criteria in Guides for Toxicological Pathology (Reference 7). In addition, with development of bile duct hyperplasias [48/50 (4 rats slight, 44 rats moderate) in the control group; 44/50 (9 rats slight, 35 rats moderate) in the 500 ppm group; 46/49 (7 rats slight, 39 rats moderate) in the 1500 ppm group; 41/50(17 rats slight, 24 rats moderate) in the 5000 ppm group], significant inhibition of the degree was found in the 5000 ppm group. A significantly increased incidence of hernias was noted in the 1500 ppm group, but this was not dose-related. <Kidney> Significant enhancement of the degree of chronic nephropathy was found in the 5000 ppm group. Significant increased incidences of urothelial hyperplasia of the pelvis were found at 1500 and 5000 ppm, and of mineral deposition in the renal papilla in the 5000 ppm group. Regarding chronic nephropathy [49/50 (1 rat slight, 24 rats moderate, 23 rats marked, 1 rat severe) in the control group; 50/50 (2 rats slight, 20 rats moderate, 24 rats marked, 4 rats severe) in the 500 ppm group; 49/49 (17 rats moderate, 31 rats marked, 1 rat severe) in the 1500 ppm group, 50/50 (12 rats moderate, 19 rats marked, 19 rats severe)] in the 5000 ppm group], lesions were found in almost all animals, and significant enhancement of their degree of development was found in the 5000 ppm group. Chronic nephropathy consisted of sclerosis of glomeruli, thickening of the renal tubular basement membranes, hyaline casts in renal tubules, regeneration of renal tubules, interstitial fibrosis and inflammatory cell infiltration. Assessment of the degree of chronic nephropathy was based on a previous publication (Reference 8). Classification was as: slight, less than 5 %; middle, 5~20 %; marked, 20~50%; and severe, more than 50 %, based on percentages of affected nephrons, judged from hyaline casts in the pars recta of proximal tubules and abnormal tubules. Regarding development of urothelial hyperplasia [2/50 (slight) in the control group; 5/50 (slight) in the 500 ppm group; 16/49 (slight) in the 1500 ppm group, 41/50(slight) in the 5000 ppm group], significant increased incidences were found in the groups receiving 1500 or 5000 ppm. For mineral deposition in the pelvis [1/49 (slight) in the 1500 ppm group, 6/50(slight) in the 5000 ppm group], a significantly increased incidence was found in the 5000 ppm group. Urothelial hyperplasia of the renal pelvis characteristically featured protrusion into the pelvis or multilayer thickening of papillary epithelium. In this test, the degree of this lesion was slight in almost all animals, found generally in small parts of the renal papilla. Mineral deposition was linear for basophilic material in the Henle s loop of papilla. 16

28 (Study No. 0686) <Stomach> Mineral deposition was found in 1/50 rats (slight) of the control group, 3 /50 rats (1 rat slight, 2 rats moderate) in the 500 ppm group, and 9 /50 rats (6 rats slight, 3 rats moderate) in the 5000 ppm group, with significant increase in lesion incidence and degree noted in the 5000 ppm group. Mineral deposition featured basophilic amorphous material deposition, but the site differed between the glandular stomach and forestomach. Although mineral deposition in the glandular stomach was primarily observed in the mucosal epithelium, deposition was also found in submucosal tissue and muscle layer in progressed (severe degree) case. Mineral deposition in the forestomach was mostly found in the muscle layer, rather than in the mucosal epithelium (stratified squamous epithelium). <Arteries / aorta> Mineral deposition in arteries was found in 2 /50 rats in the 500 ppm group (1 rat slight, 1 rat severe) and 6 /50 rats in the 5000 ppm group (slight), with a significantly increased incidence in the 5000 ppm group. Mineral deposition featured basophilic amorphous material in the walls of arteries efferent from the left ventricle of the heart, and lesion was mainly observed in the elastic lamina of the media. In addition, significant increase in the incidence of inflammation of respiratory epithelium in the nasal cavity was found in the 500 ppm group as compared to the control value, but this was not a dose-related change. - Females - 1 Neoplastic lesions <Pituitary > The incidence of pituitary adenomas in rats found dead and killed in extremis showed a tendency for increase with the Peto test (death analysis). However, the incidence of pituitary adenomas for all animals was 18/50 rats in the control group, 14/50 rats in the 500 ppm group, 11/50 rats in the 1500 ppm group and 13/50 rats in the 5000 ppm group, and there was no statistical significance between the control and any of the treated groups. Therefore, it was judged that the incidence of pituitary adenomas was not affected by administration of the test material. In addition, fibroadenomas of the mammary glands showed a tendency to decrease by the Cochran-Armitage test and significant decrease in the 5000 ppm group with the Fisher exact test. The incidence of C-cell adenomas of the thyroid in the 1500 ppm group demonstrated a significant decrease with the Fisher exact test. 2) Non-neoplastic lesions <Kidney> Significant enhancement of the degree of chronic nephropathy was observed in the 5000 ppm group. Chronic nephropathy was found in 32/50 rats in the control group ( 19 rats; slight, 11 rats; moderate, 2 rats; marked), 38/50 rats in the 500 ppm group ( 13 rats; slight, 24 rats; moderate, 1 rat; marked), 41/50 rats in the 1500 ppm group (19 rats; slight, 19 rats; moderate, 3 rats; marked) and 40/50 rats in the 5000 ppm group (10 rats; slight, 20 rats; moderate, 9 rats; marked, 1 rat; severe), with no significant variation in 17

29 (Study No. 0686) incidences noted among the groups, but again with a significant enhancement of degree at 5000 ppm. The diagnosis of chronic nephropathy was performed as in males. In addition, incidences of C-cell hyperplasia of the thyroid were significantly decreased in the 500 and 5000 ppm groups. III-8-4 Cause of deaths Causes of death and moribund condition, judged pathologically, are given in Tables R 1 and 2. - Males - Deaths related to chronic nephropathy were not found in control group, but accounted for 2, 1 and 11 rats of the 500, 1500 and 5000 ppm groups, respectively. - Females - Deaths related to pituitary tumors occurred in 6 rats in the 5000 ppm group, with significantly increase as compared to the control value (1 rat). 18

30 (Study No. 0686) IV Discussion and summary As a result of the present two-year inhalation carcinogenicity test of 2-ethoxy-2-methylpropane in rats performed at exposure levels of 0, 500, 1500 and 5000 ppm, an increased incidence of hepatocellular adenomas was found in male treated groups. IV-1 Survival, general condition, body weights, and food consumption Significant decrease of survival rates was noted in males of the 5000 ppm group and females given 1500 ppm or more. The survival rate was 60 % in 5000 ppm males as compared to 88 % in the control, and was 60 % in females of the 1500 and 5000 ppm groups as compared to 76 % in the control group at week 104. The decreased survival in males at 5000 ppm group appeared related to chronic nephropathy causing mortality. Mortality due to pituitary tumors in females given 5000 ppm was higher than in controls values, but the incidence of pituitary tumors did not appear to be directly related to ETBE exposure. Regarding general condition, no ETBE treatment related changes were found, although inhibition of body weight gain was noted in males of the 5000 ppm group and females receiving 1500 ppm or more. Namely, final body weights in 5000 ppm males were 75 % of the control value, and those in 1500 and 5000 ppm females were 91 and 78 %, respectively. Significant decrease of food consumption was noted in the male treated groups during the early phase (weeks 1 to 7) of the experiment, but recovery was evident thereafter. Food consumption in female treated groups was comparable to control values throughout the test. IV-2 Tumors and tumor-related lesions In males, the incidence of hepatocellular adenomas demonstrated a tendency for increase with the Peto test (incidental tumor test) and the Cochran-Armitage test, and significant increase in the 5000 ppm group with the Fisher exact test. It was thought that increased incidence was caused by exposure to ETBE, since the incidence of 18% (9/50 rats) of hepatocellular adenomas in the 5000 ppm group clearly exceeded the range (minimum value of 0 % ~ maximum value of 8 %, average incidence of 1.8 %) of historical control data in our laboratory. Thus, the increased incidence of hepatocellular adenomas, benign tumors, in the liver was considered to be evidence that ETBE exerts tumorigenicity in male rats. In addition, an increased incidence of altered cell foci (acidophilic cell and basophilic cell foci), which are considered as pre-neoplastic lesions for hepatocellular tumors (Reference 7), was also observed in the 5000 ppm group. On the other hand, increased incidences of hepatic tumors and cell foci were not found in any of the female treated groups. In both sexes, no increase in the incidence of neoplastic lesions due to exposure was found in any other organ. IV-3 Other influences In hematology, significant decrease of MCV was noted in females of the 5000 ppm group. In blood biochemistry, significant increases of urea nitrogen, creatinine and inorganic phosphorus were 19