sample="quota" bates="89355946" isource="bw" decade="1980" class="ui" date="19860429" Draft Report I-5038.005 APPENDIX A PATHOLOGY REPORT SIX-WEEK REPEATED SMOKE INHALATION IN MICE TO COMPARE HISTOPATHOLOGY AND SHORT-TERM ENDPOINTS USING TEST AND REFERENCE CIGARETTES (D33, D34 AND D36) STUDY NO. I-5038.005 PATHOLOGY NARRATIVE PAI Pathology Associates, Inc. 10075 Tyler Place Hyatt Park II Ijamsville, Maryland 21754 (301) 663-1644 PATHOLOGY REPORT SIX WEEK REPEATED SMOKE INHALATION IN MICE TO COMPARE HISTOPATHOLOGY AND SHORT-TERM ENDPOINTS USING TEST AND REFERENCE CIGARETTES (D33, D34, AND D36) STUDY NO. I 5038.005 INTRODUCTION AND METHODS Female B6C3F1 mice were divided into sham controls, a reference cigarette group, and 3 test cigarette groups and exposed to repeated doses of smoke by nose-only inhalation for 4, 15, or 30 exposure days over a period of 4, 23, or 44 days, respectively. Two of the test cigarette groups (D33 and D34) were terminated during the study and the tissues discarded. Four days after initiation, three mice from the remaining groups (Reference, D36 cigarette and Sham) were given colchicine (1mg/kg). Four hours later they were anesthetized with Metofane and killed by exsanguination in order to determine mitotic indices of the tracheal and intrapulmonary airway epithelium. Three additional mice from each group were killed in the same manner following 15 exposure days; the remainder (6 per group) were killed after 30 exposure days and necropsied. Lungs of all mice were weighed to the nearest milligram and fixed by perfusion with formalin to approximate the normal expanded state. The nasal passages were flushed with formalin and immersed in fixative. The remaining organs and tissues were removed from the animal and fixed in 10% neutral buffered formalin. Nasal passages were decalcified and trimmed according to the procedures described by Young (1981). Lung lobes were sectioned and embedded longitudinally through the stem bronchus of each lobe (Dungworth, et al. 1976); trachea and mainstem bronchi were removed and embedded longitudinally. Three traverse sections were made of the larynx. All tissues were embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin, phloxine, and eosin. The mitotic mice index was determined for the trachea and intrapulmonary airways of rats exposed to smoke for 4 days by counting the number of mitotic figures per 10 high power fields of airway epithelium. Respiratory tissues and gross lesions from rats mice of the 15 and 30 day sacrifices were examined microscopically. RESULTS GROSS NECROPSY FINDINGS Treatment-related gross lesions characterized as brown discoloration of the lungs occurred in all smoke-exposed mice. The sham controls were free of discoloration. No difference was discerned between the reference and test cigarette groups. (Table of Gross vs. Micro Findings) MICROSCOPIC FINDINGS Mitotic indices were increased in the tracheal epithelium of the reference cigarette group compared with the test and sham groups. The indices were smaller among all groups for the intrapulmonary airways (Table I). Mice treated for 30 days with smoke had increased numbers of alveolar foam cells in pulmonary alveoli. Fewer neutrophils were associated with them. A majority of these cells had a brownish cytosol and were most often observed in alveoli located near small airways. Most smoke-exposed mice had this response. This cellular response was graded as minimal. Mice of group 5 (sham controls) were free of this response. It was attributed to smoke inhalation. A difference was not observed among reference and test cigarettes. Foam cells were not observed in the lungs of mice treated for 15 exposure days. Other responses observed were minimal perivascular and peribroncholar lyumphocytic infiltrates, and occasionally peribronchiolar infiltrates of other inflammatory cells consisting of a mixture of neutrophils and mononuclear leukocytes (leukocytic infiltrates). The infiltrates were of similar incidence among all groups including the sham controls. Thus, their occurrence is incidental and not related to smoke. Their cause could not be determined. Antibody was not detectable to any of the viral or mycoplasmal agents common to mice. In the nose, mucosal atrophy of olfactory epithelium (Sections III and IV) was observed in 1/6 reference cigarette-exposed mice, and respiratory epithelial metaplasia was observed focally in 1/6 D36 cigarette-exposed mice. The mucosal atrophy consisted of a focal reduction of cell numbers in the olfactory epithelium. Respiratory epithelial metaplasia was characterized by focal replacement of olfactory epithelium with respiratory mucociliary epithelium. The occurrence of the lesion in Sections III and/or IV mucociliary epithelium. The occurrence of the lesion in Sections III and/or IV of only 1/6 mice of each smoke-exposed group (reference and test) with no lesions in the more rostral Sections I And II suggest that they are incidental and unrelated to treatment. The cause of the lesions could not be determined. Lesions caused by exposure to cigarette smoke are most commonly observed in Sections I and II. SUMMARY The mitotic index was increased in the tracheal epithelium of mice exposed for 4 days to smoke from the reference cigarette (Group 1) compared with the test (D36) cigarette and sham controls (Groups 4 and 5, respectively). No difference in mitotic index was observed in bronchial epithelium. In the lung, pigmented alveolar foam cells were found in mice exposed for 30 exposure days to smoke from the reference and D36 cigarette types, but not sham controls. Similar lesions were not observed in mice exposed for 15 days. Lesions were accompanied by slight infiltration of neutrophils. Other inflammatory changes consisting of perivascular and peribronchiolar lymphoid infiltrates, and/or peribronchiolar leukocytic infiltrates (neutrophils and lymphoid cells) were found in mice from all groups of both sacrifices and considered incidental. Nasal lesions were considered incidental. Changes attributable to test article cigarette smoke were not discerned. William C. Hall, V.M.D., Ph.D. Diplomate, A.C.V.P. April 29, 1986 PROJECT SUMMARY TABLE TABULATED ANIMAL DATA GROSS VS. MICROSCOPIC FINDINGS QUALITY ASSURANCE REPORT QUALITY ASSURANCE STATEMENT This histopathology project has been conducted in compliance with Good Laboratory Practice regulations promulgated by the U.S. Food and Drug Administration and the U.S. Environmental Protection Agency. Pathology Associates, Incorporated has a functioning and responsive quality assurance unit which has conducted appropriate inspections and reported all findings to management. The following is a record of those inspections and reports: Six Week Repeated Smoke Inhalation in Mice to Compare Histopathology and Short-term Endpoints Using Test and Reference Cigarettes (D33, D34 and D36) Study No. I 5038.005 APPENDIX 1 Reports Code Table N Tissues within normal histological limits A Autolysis precluding adequate evaluation U Tissues unavailable/unsuitable for evaluation S Tissues not applicable to animal * Tissues not examined/not required by protocol. 1 minimal 2 mild 3 moderate 4 severe ) focal } diffuse > multifocal P Present B Neoplasm, Benign M Neoplasm, Malignant without Metastasis C Neoplasm, Malignant with Metastasis X Metastatic Site (+) APPENDIX 2 ABBREVIATIONS LIST ENDO T ENDOTURBINATE DNM DORSAL NASAL MEATUS RESP RESPIRATORY EPITH EPITHELIUM PERIBR PERIBRONCHIOLAR PERIVASC PERIVASCULAR Draft Report I-5038.005 APPENDIX B Draft Report I-5038.005