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1 PRODUCED IN BOTLER V. PHILIP NORRIS, ET AL. DISSEMINATION IS PRONIBITED BY PROTECTIVE ORDER ENTERED BY THE COURT.

, FOREWORD The material that follows is the complete and unedited final report of contract research performed from 1975 to 1983 by MicrobioloYieal Msociates, Bethea- da, MD, support approved by the Scientific Advisory Board to the Council for Tobaoco Rescarch-US.A., Inc. The ezperiments represented a determined effort to develop a suitable animal model involving chronic cigarette smoke exposure of inbred mouse strains known to develop various histopathologic types of lung carcinoma after intratracheal administration of pure chemical carcinogens. It is believed the report will be of interest to those engaged in inhalation research and in the field of investigatini human smoking and health relationships. Tbe results observed included these: none of the smoke-e:posed animals developed pulmonary squamous cell carcinoma; mice pretreated with a pure carcinogen did not have significantly increased lung cancers after prolonged smoke exposure; and the overall numbers of pulmonary neoplasms identified .rere not statistically significantly different in the smoke-exposed mice, compared to the sham smoke•ezposed or shelf control mice. Sheldon C. Sommers, MD Scientific Director, Council for Tobacco Research-US.A., Inc. ) ) •cm v sc~ ~ , CTR MN 043699.

I I I. INTRODUCTION/OBJECTIVES Chronic smoke inhalation studies were designed to determine if potential biological effects occur in Inbred strains of mice during long term exposure to whole cigarette smoke. In huqians, data to date suggest that cigarette smoke possesses weak biological activity. Beause of this weak biological potential and because of the conditions under which humans are exposed to cigarette smoke, it is a necessary requirement that any smoke exposure etudy use: 1) conditions whereby the smoke deposition and distribution parallel that of human smokers, 2) duration of exposure which parallels that of a human smoker, 3) an animal model which is capable of developing those types of biological lesions associated with cigarette smoking in humana, and 4) a number of animals sufficient to arrive at a significant conclusion. Several approaches were taken at Microbiological Associates (MA) to develop a murine model for lung earcinorenesis along with techniques and procedures for quantitative smoke exposure studies which take into account the above requirements. Essentially, three major smoke inhalation studies were performed. The first of these (CTR-100) utilized the Kentucky Reference 2A1 cigarettes characterized by high tar/low niootine content; the second (CTR-101A) used the Kentucky Reference 2R1 cigarettes characterized by high tar/high nicotine content; and in.the third experiment (CTR-101B), 2R1 cigarette smoke was used to expose one group of animals, and a second group was exposed to 3A1 cigarette smoke which, like 2A1, is characterized by high tar/low nicotine. The scope of the first two studies was the assessment of the carcinogenic potential of cigarette smoke either alone, or synergistically when a known chemical carcinogen was administered prior to long-term smoke exposure. The third study provided animals for the evaluation of short-term assays that had the potential to be early indicators of possible toxicologic consequences of exposure to 2R1 or 3A1 cigarette smoke. Corollary studies describe the deposition and distribution of smoke constituents and selected aerosolized chemicals after "nose-only" exposure. Over 10,000 mice were exposed to smoke from over 800,000 cigarettes at the Inhalation facility. The studies required the SEM 13 B and C smoking machines, a sham exposure machine, 3 animal containment units with 24 ezpoaure modules, 8 optical sensors with 8 strip chart recorders, and 400 animal restraint trays. Smoke exposure related manipulations Included indi- vidual animal identification (by ear tai), individual vaccination against Sendai vitus, individual loading and unloading of animals for daily smoke e:poeure, monthly weighing of each animal, and approximately 5-10 data entry points for each animal within any given experiment. Additional daily effort In technical support services included autoclaving food and bedding, washing cages and water bottles, and dismanteling and cleaning smoke exposure equipment each day (24 animal containment modules, 400 animal restraints, the smoke exposed surfaces of the SEM Il B or C, the Bow thermistors, and the optical sensors). Finally, another level of effort in professional support services C TR HN 043"`00

.• .. y."• . ,1 A tm 4 : .Kv. !'-~:~ya•..••. Introduction/Objectives ix was required for necropsy, histology, pathology, data evaluation, re-eva)ua- tion, and final analysea of each experiment. The )evel of effort required to perform these ttudiea amounted to approximately 11,000 animal related manipulationr each day. Tbe following report deacribee the experimental findings In five areas: facilitiea and equipment neceasary for "nose-on)y" exposure of mice to eisarette smoke (Section II), cocarciboaeneaiu and chronic inhalation of 2A1 cigarette smoke (Section III), chronic ezpaure of BC3F1/Cum mice to 2R1 eiprette amoke (Section IV), exposure of BC3F1/Cum mice to 2R1 and 3A1 eigarette smoke (Section V), the pharmacokinetia of inhaled materials (Sec- tion VI), and the references (Section VII). A list of publications from Microbiological Associates supported by The Council for Tobacco Reaearcb may be found in Section VIII. C! R 1 / I / V• 413l 0MA1 . J j