"MATERIALS AND METHODS

Chemical

Naphthalene (>99% pure) was obtained from Fisher Scientific (Fair Lawn, N.J.). The chemical was identified as naphthalene by appearance, melting point, and infrared, ultraviolet/visible spectra, and nuclear magnetic resonance spectroscopy. The purity of the chemical was determined by elemental analysis, Karl Fischer water analysis, thin-layer chromatography, gas chromatography, and high-performance liquid chromatography. The chemical was found to be stable for 2 wk at temperatures of up to 60 C.

Vapor Generation and Monitoring

Naphthalene vapor was generated in Hinners-type inhalation exposure chambers by direct sublimation from each of 3 exposure chambers and delivered through metering valves using nitrogen. Naphthalene chamber concentrations were monitored with a Miran model 80 infrared analyzer and were computer-adjusted to the desired concentration by a software controlled feedback arrangement. Individual monitors were used for each of the exposure chambers. Calibration was performed daily using 1.0-ml injections of a freshly prepared naphthalene standard into a closed-loop system. Air from each chamber was sampled every 40 s and averaged over 5-min intervals. Weekly average concentrations were within +/-20% of target concentrations (10 or 30 ppm), except for 1 wk when the mean in the 10 ppm chamber was 5.5 ppm.

Animals and Animal Care

Male and female B6C3F, mice (6-7 wk old) were obtained from Simonsen Laboratories (Gilroy, Calif.). Mice were housed by sex in groups of 5 throughout the study period in stainless steel wire-mesh cages (Lab Product, Inc., Maywood, N.J.) in stainless steel and glass Hinners-type inhalation chambers (Young & Berke Co., Cincinnati, Ohio) with a 28- to 35-day quarantine period prior to exposure initiation. Age of animals on first day of exposure was 12 wk. Cages were rotated weekly within the chambers. The animal room was provided with a 12-h light/dark cycle and 10 air changes per hour. NIH-31 diet (Zeigler Brothers, Gardners, Pa.) and deionized water were supplied ad libitum during nonexposure hours.

Study Design

Groups of 75 mice of each sex were exposed to naphthalene vapors at target concentrations of 0, 10, or 30 ppm (equivalent to 0, 50, or 150 mg/m) for 6 h/day, 5 days/wk for 103 wk. Two additional groups of 75 mice, one of males and one of females, were also exposed similarly to 30 ppm naphthalene. Twice as many animals received the high dose because of lack of information on the long-term toxicity of inhaled naphthalene and to ensure that a sufficient number of animals for statistical evaluation lived to study termination.

Clinical Observations and Pathology

Mice were observed for mortality and moribundity twice daily, 7 days/wk. Mice were weighed in groups of 5 by cage once weekly for the first 13 wk of the study, and monthly thereafter, Heparinized blood samples were collected from the vena cava of 5 mice of each sex from the control and 10 ppm dose group and 10 mice of each sex from the 30 ppm dose group on 14 days of study and used for hematological determinations. Hematology parameters measured were hematocrit, hemoglobin concentration, and total erythrocyte, reticulocyte, and leukocyte counts. Slit-lamp biomicroscopy and indirect ophthalmoscopic examinations were performed on five animals of each sex from each dose group at 6- mo intervals during the studies. The mice were euthanized with carbon dioxide, necropsied, and examined closely for visible lesions. A complete histopathologic examination inclusive of visible lesions was performed on all control and highdose mice and all animals dying or sacrificed moribund condition prior to 21 mo. Tissues for microscopic examination were fixed in 10% formalin, embedded, sectioned to a thickness of 4-6 mm, and stained with hematoxlyin and eosin. Tissues examined microscopically included adrenal glands, bone (femur including marrow), brain, epididymis, esophagus, gallbladder, heart, kidneys, large intestine (cecum, colon, and rectum), larynx, liver, lungs, lymph nodes (bronchial, mandibular, mediastinal, and mesenteric), mammary gland, nasal passages, ovaries, pancreas, parathyroid gland, pituitary gland, prostate, salivary glands, seminal vesicles, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach (glandular and forestomach), testes, thymus, thyroid gland, trachea, urinary bladder, and uterus. Only lungs and nasal tissues were examined from low-dose mice dying or sacrificed in moribund condition prior to 21 mo on study or surviving to study termination.

Statistical Analysis of Neoplastic and Nonneoplastic Lesions Incidence

The primary statistical method used was the logistic regression analysis of Dinse and Lagakos (1983), further described and illustrated by Dinse and Haseman (1986). Tests of significance included pairwise comparisons of each dose group with the controls and a test for an overall dose-response trend, Continuity-corrected tests were used in the analysis of tumor rates. Results were considered significant when the calculated p value was less than .05."

EPA calculated the exposure levels in mg/m3 from reported exposure levels in ppm using a molecular weight for naphthalene of 128.18 g/mol (NTP, 2000). Analytical concentrations; ranges (midpoint): 0, 29-59 (44), 128-163 (146) mg/m3

Inflammation, Chronic

Endpoint Details

Dataset

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