90-Day Inhalation Study with Cigarettes containing G7-25 NECROPSY AND HISTOPATHOLOGY Esophagus, Eyes/optic nerve, Heart, Ileum, Standard necropsy techniques will be Jejunum, Kidneys, Larynx, Liver, Lungs, used (Feldman & Seely, 1988) for each animal Lymph Nodes (thymic, mesenteric and in the study. The first 22 surviving animals peribronchial), Mammary glands, per group will be killed on the day following Nasopharynx, Nose/turbinates, Ovaries, the final inhalation exposure; the remaining Pancreas, Parathyroid, Pituitary, Prostate, animals in each group will be kept without any Rectum, Salivary gland (and associated lymph further treatment for an additional 14 weeks. nodes), Seminal vesicle, Skeletal muscle Necropsy (thigh), Skin (abdominal), Spinal Cord Feed will be removed from the animals (lumbar), Spleen, Stomach, Tail, Testes, during the day prior to necropsy. At each Thymus, Thyroid, Trachea, Urinary bladder, necropsy, animals will be weighed and then Uterus, Zymbal's gland. killed by first anesthetizing with 70% CO 2 in Additionally, organs showing gross air and then exsanguination prior to cessation lesions will be collected. The carcass and of heartbeat. remaining tissues will be incinerated. Any Animals will be subjected to a complete remaining sentinel animals will be killed and examination in the presence of a board- discarded. certified veterinary pathologist, with special Histopathology attention paid to the lungs and upper Respiratory tract tissues (nasal passages, respiratory tract. Animals from comparable larynx, trachea, conducting airways, deep groups will be killed on the same day. lung), heart and related lymph nodes (thymic Organ Weights and peribronchial) will be examined in each The lungs (complete with trachea and animal, along with any gross changes. The associated mediastinal tissue), liver, kidneys study pathologist will decide on the work to be (pair), brain, testes (pair, with epididymides performed in any "early death" animals. The attached), adrenals (pair), spleen and heart nasal tissues will be cut at four different (excluding major vessels) will be weighed at locations to obtain representative sections of necropsy, using Mettler PM 460 balances. the different epithelia (Young, 1981). The Organ weights and the terminal (fasted) body lungs will be sectioned so as to provide a weight at death will be used to calculate organ section along the main stem bronchus of each : body weight and organ : brain weight ratios. lung lobe. A precise anatomical site for The time from removal of the organ until cutting the larynx is required (Sagartz et al., weighing will be minimized and tissues will be 1991). Three sections of trachea will be kept in saline until weighing. No prepared. measurements will be made of organ weights In the high exposure groups and in in "early death" animals. controls, the following tissues will also be Tissue Collection examined in each animal: brain, liver, kidneys, Tissues will be removed from each spleen, adrenals and gonads. animal and fixed in 10% neutral buffered Sections will be stained with formalin (NBF), at an appropriate volume hematoxylin and eosin (H&E); duplicate slides dilution. The NBF will contain 20 ml of 1% of nasal tissues (level III only), larynx, lung eosin per 20 liters of 37% formalin, to identify and trachea will be stained with Periodic - the fluid as fixative. Acid - Schiff / Alcian Blue (PAS-AB) to After weights of the lungs have been facilitate evaluations of goblet cells. obtained, lungs will be infused with NBF Tissues will be read by a board-certified using gravity filling (25 cm water pressure). veterinary pathologist, initially with The trachea will be ligated after lung infusion. knowledge of the treatment groups. The larynx, with the tongue attached, will be separated from the trachea prior to weighing STATISTICAL ANALYSES and infusion of lungs. The following tissues In-life, organ weights. will be collected: Statistical evaluations will be made Adrenals, Aorta, Bone (marrow: femur), using Bartlett's test of homogeneity of Bone (sternum), Brain, Cecum, Colon, variance, followed by Fisher's least significant Cranium, Duodenum, Epididymides, difference (LSD) test. Labos DRAFT: DO NOT QUOTE. Page 6 Source: https://www.industrydocuments.ucsf.edu/docs/ffbb0103