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Final Report 1-1729.005 SIX WEEK REPEATED SMOKE INHALATION IN MICE TO COMPARE HISTOPATHOLOGY AND SHORT-TERM ENDPOINTS USING TEST (D6 and D10) AND REFERENCE CIGARETTES Final Report For Lorillard Research Center 420 English Street P.O. Box 21688 Greensboro, North Carolina 27420 September 25, 1985 By Microbiological Associates Inc. 5221 River Road Bethesda, Maryland 20816

Final Report I-1729.005 Table of Contents Page I. Data Page 3 II. Introduction 4 III. Purpose 6 IV. Reference and Test Cigarette Characterization 7 V. Test Description 9 VI. Materials and Methods 10 VII. Results and Discussion 16 A. General Toxicology 16 B. Clinical Chemistry and Hematology 19 VIII. Conclusions 21 IX. Figure and Tables 22 X. References 23 XI. Quality Assurance Statement 25 XII. Appendix A - Pathology Report 2

Final Report I-1729.005 I. DATA PAGE Cigarette Identity: Reference, Test Cigarettes D6 and D10 Dates Samples Received: June 9, 1982 (Reference) February 4,1983 (D6 and D10) Date Sample Returned: Retained in storage Initiation Date: March 24,1983 Completion Date: See Review Completed Date: Page 24 MA Experiment Number: 1-1729.005 MA Notebook Number: 1729.005 Archives Location: 5221 River Road, Bethesda, Maryland 20816 Sponsor: Lorillard Research Center 420 English Street P.O. Box 21688 Greensboro, North Carolina 27420 Authorized Representative: Harry Minnemeyer, Ph.D. Connie Stone, Ph.D. Testing Facility: Microbiological Associates Inc. 5221 River Road Bethesda, Maryland 20816 Lead Technician: Dj6vhd R. Dansie ~ Study Director: Rayny§nd M~-Bwid, Ph.D. 'r Z i S'j--- Da e 3

Final Report I-1729.005 II. INTRODUCTION Alterations in the morphology of the respiratory tract have been noted in animals exposed to whole cigarette smoke. The magnitude of the changes depend upon the species, the cigarette type, the smoke exposure regimen or dose, and the duration of the exposure. Recent reports have detailed procedures for standardized microscopic examination of the upper respiratory tract (Young, 1981) which complement the procedures for evaluation of the lower respiratory tract. These procedures permit visualization of the nasal passage, trachea, major bronchi, terminal airways and lung parenchyma (Dungworth et al, 1976). Thus a detailed evaluation will be performed on the following tissues from animals exposed to smoke for six weeks: 0 nasal cavity and nasal turbinates 0 oral cavity, larynx, and pharynx 0 trachea 0 lungs and bronchi 0 any other abnormal tissues In addition to the microscopic evaluation, the following 4 assays complement the histopathology endpoints and were performed on the same group of animals at the same time. These particular assays have been grouped together to most efficiently utilize the Walton Smoking Machine with 12 mice/machine. Clinical Chemistry: Changes in clinical chemistries can reflect generalized toxic effects to the lung, liver, kidney, heart or other organs by the alteration of specific blood enzyme levels (i.e., lactic dehydrogenase), blood glucose, blood urea nitrogen, ion or metal levels, and total protein levels. Hematology Assays: Hematologic changes can reflect the effects of a large number of chemicals or agents on the blood and blood-forming organs, either directly or indirectly. Decreased or increased numbers of erythrocytes or leukocytes in the peripheral blood can reflect diseased or toxic states. 4

Final Report I-1729.005 Metabolism - Microsomal Monooxygenase (MMO) Levels: Aryl hydrocarbon hydroxylase (AHH) is one of the microsomal monooxygenases and represents the enzymatic complex responsible for the metabolism of polycyclic hydrocarbons, such as benzo(a)pyrene (BaP), benz(a)anthracene, or 3-methylcholanthrene. It is present in most mammalian tissues and can be enhanced in animals by the administration of a variety of chemicals such as the polycyclic hydrocarbons themselves, 5,6-benzoflavone, or 2,3,7,8- tetrachlorodibenzo-p-dioxin (Kouri and Nebert, 1977). The enzyme is detected in a fluorometric assay which quantifies the conversion of BaP to 3-hydroxy-BaP. AHH levels will serve as internal markers in tissues for particulate deposition or redistribution after smoke exposure. Comparative levels of AHH in lung, liver, and kidney between cigarette types would allow differences in deposition, absorption, and redistribution to be observed. This may be particularly important if additives to the particulate phase involve AHH inducers. Cellular Proliferation: Cell turnover or replication can be estimated by either mitotic indices or incorporation of 3H-thymidine into DNA. These techniques are sensitive criteria indicating possible induced DNA or cellular damage, where no evidence of damage can be seen by histologic appearance (Lamb and Reid, 1968). Cigarette smoke has been shown to induce cellular proliferation in mice (Kanagalingam et al, 1982). Mitotic counts can be evaluated in the same slides (and animals) used for "general" histopathologic evaluations (Bertalanffy, 1964) and can be easily incorporated into these studies. 5

Final Report I-1729.005 III. PURPOSE The purpose of this study is to compare the response of animals exposed to smoke from the Reference cigarette with smoke from two test cigarettes using histopathology of the respiratory tract and selected short-term endpoints. 6

Final Report 1-1729.005 IV. CIGARETTE CHARACTERIZATION Approximately 2000 Reference cigarettes were received on June 9, 1982, and 1200 D6 test cigarettes, and 1200 D10 test cigarettes were received by Microbiological Associates Inc. on February 4, 1983. The Reference cigarette was assigned a registry number of 5, D6 a registry number of 14, and D10 a registry number of 15. Characterization of cigarette smoke was performed each week and included determination of resistance-to-draw (RTD), butt length, and wet total particulate matter (TPM) per cigarette. RTD was measured using a Filtrona Pressure Drop Tester (Cigarette Components UK Ltd., Wembley, England). Butt length was measured after burning cigarettes with 7 puffs/cigarette. TPM was collected onto Cambridge filter pads from each cigarette after burning on the Walton Horizontal Smoking Machine. The standardized conditions for burning cigarettes were the same as those used for exposing the animals (7 puffs). The following data were collected: Cigarette RTD Butt TPM Type (mm H20) Leygt~ (mg) mm Week 1 Reference 79.7 + 0.6 44.7 + 2.1 15.3 + 0.2 D6 101.7 + 6.0 47.3 + 2.3 13.8 + 0.4 D10 88.0 + 2.6 47.0 + 5.6 14.4 + 0.6 Week 2 Reference 80.0 + 1.4 44.0 + 4.2 14.9 + 0.1 D6 102.3 + 4.6 51.0 + 6.0 10.7 + 0.5 D10 95.5 + 10.6 49.0 + 2.8 12.3 + 0.1 Week 3 Reference 80.7 + 2.1 40.3 + 3.5 18.1 + 0.3 D6 95.3 + 11.0 47.7 + 2.5 13.0 + 0.3 D10 91.7 + 1.5 49.3 + 4.7 15.4 + 0.4 Continued... 7

Final Report I-1729.005 Cigarette RTD Butt Type (mm A20) Length T PM (mg) --------------------------------------------------------- Week 4 Reference 89.3 + 4.0 44.0 + 4.0 15.5 + 0.4 D6 101.5 + 2.1 45.5 + 0.7 15.3 + 0.1 D10 100.5 + 7.8 52.5 + 3.5 13.8 + 0.8 Week 5 Reference 83.5 + 0.1 40.0 + 5.7 14.5 + 0.2 D6 104.0 + 1.4 46.0 + 1.4 13.6 + 0.5 D10 95.5 + 7.8 47.5 + 0.7 16.7 + 2.8 Week 6 Reference 84.0 + 1.4 46.5 + 2.1 13.5 + 2.8 D6 103.5 + 0.7 45.5 + 0.7 15.8 + 0.4 D10 87.0 + 4.2 49.0 + 1.4 15.3 + 0.1 The Sponsor specified storage at room temperature and no expiration date was provided. For the purposes of this study, the test articles were stored in the original cellophane sealed boxes in a secured area at room temperature. 8

Final Report I-1729.005 V. TEST DESCRIPTION B6C3F1/Cr1BL mice were exposed to smoke from two test cigarettes and the Reference cigarette. The exposure regimen used (7 puffs, RSD1) was decreased from that used (8 puffs, RSD1) in the acute toxicity study (I-1729.003) to reduce toxicity and ensure adequate survival. At weekly intervals, mice were weighed and monitored for levels of carboxyhemoglobin (COHb). Three mice/group were sacrificed after 3 weeks exposure to determine microscopic appearance of the respiratory tract, clinical chemistry, and hematology. After 6 weeks exposure, 3 mice/group were sacrificed to determine microscopic appearance of the respiratory tract, clinical chemistry, and hematology; another 3 mice/group were sacrificed for respiratory tract histopathology and mitotic counts in the same respiratory tissues; and the remaining 3 mice/group were sacrificed to determine AHH levels in lung, liver, and kidney. 9

Final Report I-1729.005 VI. MATERIALS AND METHODS Materials Animals: B6C3F1/Cr1BL mice, females 8 weeks old Sendai vaccinated Charles River Breeding Farms Kingston, New York Smoke Generation Equipment: Walton Horizontal Smoking Machine (Walton) Stock-like neck holders Process and Instruments Corp. Brooklyn, New York Materials: Heparinized blood collecting tubes (250 ul) IL-CO-Oximeter Metofane anesthetic Methods Animals: Animals were obtained from a source monitored and known to be free of adventitious agents, vaccinated against Sendai virus and quarantined for at least 21 days. Stringent disease control procedures were followed during quarantine to assure the use of healthy animals. Mice were observed for signs of illness, unusual food and water consumption and other signs of poor health. The animals were 12 weeks old at initiation of test and were judged to be healthy prior to utilization. Animals were housed in an AAALAC-accredited facility with a controlled environment of 74 + 5 OF, 50 + 20% relative humidity, and a 12 hour light/dark cycle. Mice were housed 6 per cage in polycarbonate autoclavable cages with filter top cage lids. Corn-cob bedding was used and animals had free access to certified laboratory rodent chow which had been analyzed for environmental contaminants. Water and food were provided ad libitum, except during exposures on the smoking machines. 10