“Clinical Assessments: The general condition of all animals (based on cage-side examinations) was checked and recorded daily. Cage-side observations included changes in: skin and fur, eyes, and mucous membranes, respiratory system, circulatory system, autonomic and central nervous system, somatomotor activity, and behavior pattern. Body weights and detailed clinical observations were measured and recorded within 1-2 days of arrival and weekly throughout the duration of the study.

Necropsy and Histopathology: On the day of each necropsy, rats were deeply anesthetized with sodium pentobarbital (intraperitoneal injection, approximately 60 mg/kg) and exsanguinated by transection of the abdominal aorta. The trachea was exposed and the lungs filled with 10% neutral buffered formalin (NBF) at approximately 30 cm H₂O pressure. The trachea was ligated to keep the lungs inflated during fixation. Following this procedure the nasal cavities were flushed (retrograde through the trachea) with NBF. Lungs with trachea and larynx attached were excised and placed in NBF. The heads were removed, skinned, trimmed of excess tissue, and placed in NBF. Respiratory tract tissues were fixed in NBF for approximately 48 hr with the exception of the recovery (65 + 60 day) group, which was fixed for 7 days. Following fixation, the heads were rinsed in running tap water for at least 30 minutes and decalcified in Immunocal™ Formic Acid Bone Decalcifier (Decal Chemical Corp., Tallman, NY) for 7 to 14 days. Noses were sectioned transversely to provide sections of the nasal cavity at six standard levels (Morgan, 1991Morgan K. T. Approaches to the identification and recording of nasal lesions in toxicology studies. Toxicol Pathol. 1991; 19: 337–351, (4 Pt 1), routinely processed and embedded in paraffin wax, sectioned (5 μ m), deparaffinized, and stained with hematoxylin and eosin (H&E). Post fixation in NBF the larynx, trachea, and lung were changed to 70% ethanol, gross trimmed, routinely processed to paraffin block, sectioned (5 μ m), and stained with H&E. H&E sections from all animals were examined histologically. Additional level II nasal sections from the 0.6 and 1.8 ppm (4, 14, and 30 day) acrolein treated rats were immuno-stained for proliferating cell nuclear antigen (PCNA) following high heat antigen retrieval with mouse monoclonal anti-proliferating cell nuclear antigen Clone PC10 diluted 1:1000 (Dako, Carpinteria, CA) and an Aminoethyl Carbazole (AEC, red) chromagen substrate kit (Zymed Laboratories, Inc., South San Francisco, CA) according to the manufacturer's instructions and standard procedures in place at CIIT. PCNA positive cells stained red.

Histological findings were recorded at each level. The majority of the findings were graded on a scale of 1–5 where 1 = minimal, 2 = slight or mild, 3 = moderate, 4 = moderately severe and 5 = severe/high. The entire area of the nasal epithelium for each of the six levels was examined. To aid in mapping the lesions the histological findings were recorded according to the site in which they occurred. Some sites for which histological findings were recorded included:

Nose tip. Dorsal meatus: area extending from the septum adjacent to the nasoturbinate (NT), up around the dorsal meatus and along the medial aspect of the NT to the tip of the NT. Lateral wall: area extending from the tip of the NT around the lateral wall to the tip of the maxilloturbinate.
Nose, level I. Dorsal meatus: extended from the septum adjacent to the NT, up around the dorsal meatus and down the medial aspect of the NT to the tip of the NT. Lateral wall: extended from the tip of the NT around the lateral wall to the tip of the maxilloturbinate. Maxilloturbinate: extended from the tip of the maxilloturbinate along its medial aspect.
Nose, level II. Dorsal meatus: extended from the septum adjacent to the NT, up around the dorsal meatus and down the medial aspect of the NT to the tip of the NT. Lateral wall: began from midventral midpoint of the NT and extended along the lateral aspect of the NT, continued along the lateral wall ventrally and extended up to the lateral aspect of the maxilloturbinate where it ended at the dorsal midpoint of the maxilloturbinate.
Nose levels III, IV and V. These levels were evaluated with respect to histological changes in the olfactory epithelium which was found at these levels.
Larynx, trachea and lung. These regions were evaluated with respect to histological changes in the airway epithelium.

Statistical Evaluation: The data for quantitative, continuous variables were inter-compared for the exposure and control groups by tests for homogeneity of variance, analysis of variance (ANOVA), and Dunnett's multiple comparison procedure for significant ANOVAs. When the ANOVA indicated statistical significance among experimental groups, the Dunnett's test was used to delineate which groups differ from the control group. When appropriate, Williams' test was also used to determine the lowest dose group which significantly differed from the control group. If the assumptions for a parametric ANOVA were not met, nonparametric procedures were used. Incidence data were compared using the appropriate statistical test, generally Pearson's chi-square test. Statistical analyses were performed using JMP Statistical Software (SAS, Cary, NC). Tests of homogeneity used a significance level of 0.01. A probability value of less than 0.05 was used as the critical level of significance for all other statistical tests. Unless otherwise noted data presented represent mean value (± SD).

Epithelial inflammation, trachea

Endpoint Details

Dataset

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