"MATERIALS AND METHODS

Chemical

Naphthalene (>99% pure) was obtained from Aldrich Chemical Co. (Milwaukee, WI). The identity of the chemical was confirmed by infrared and proton nuclear magnetic resonance spectroscopy and by gas chromatography/mass spectroscopy. The purity of naphthalene was determined by elemental analyses, gas chromatography/mass spectroscopy, and gas chromatography with flame ionization detection (FID). The stability of the bulk chemical was monitored during exposure by gas chromatography with FID. To ensure stability, the bulk chemical was stored under a nitrogen headspace at room temperature in metal drums lined with plastic. No degradation of the bulk chemical was detected.

Whole-Body Exposure System

Naphthalene vapor was generated using a 2-L glass reaction flask containing the solid chemical and surrounded by a heated mantle. Heated nitrogen metered into the flask carried the vapor out of the flask. The mantle and nitrogen temperatures were adjusted to maintain the temperature of the vapor above the bulk naphthalene between 66 and 71°C. The vapor was transported via a heated Teflon line to the exposure chambers and was diluted with heated HEPA- and charcoal-filtered air before entering the distribution manifold. The vapor was injected into the chamber as it was mixed and diluted with conditioned chamber air to obtain the target concentration.

Stainless steel Hazleton 2000 inhalation exposure chambers (Hartford Systems Division of Lab Products, Inc., Aberdeen, MD) with a total active mixing volume of 1.7 m 3 were used. The animals were housed in stainless-steel wire-bottom cages (space per animal: 14 × 11.7 × 15.2 cm) during exposure. The chamber airflow was 15 ± 2 air changes per hour. A small particle detector (Type CN, Gardner Associates, Schenectady, NY) was used with and without animals in the exposure chambers to ensure that naphthalene vapor, and not aerosol, was produced. A Type CN small particle detector was also used to determine whether naphthalene aerosols occurred at the concentrations studied. During exposure, no particle counts above the minimum resolvable level (approximately 200 particles/cm3 ) were detected.

Chamber concentrations of naphthalene were monitored by an online gas chromatograph; the average chamber concentrations were maintained within 1% of the target concentrations. Each chamber was sampled approximately every 24 min during the exposure period. The uniformity of naphthalene concentration with animals present in chamber was measured periodically throughout the study by gas chromatography. Chamber unformity was maintained; no vapor degradation was detected. The gas chromatograph was calibrated against gravimetrically prepared standards of naphthalene containing 1-phenylhexane as an internal standard in toluene monthly.

Experimental Design

Details of the experimental design are presented in the NTP technical report number 500 (National Toxicology Program, 2000). Four-week-old male and female Fischer 344/N rats were obtained from Taconic Laboratory Animals and Services (Germantown, NY). The rats were quarantined for 14 days before the beginning of the study. They were randomly distributed among groups of approximately equal weight.

Groups of 49 male and 49 female rats were exposed to naphthalene at concentrations of 0, 10, 30, or 60 ppm for 6 h plus T90 (12 min) per day, 5 days/wk for 105 wk. The animals were housed individually. NTP-2000 diet was available ad libitum, except during the exposure period; water was available ad libitum.

The two lowest exposure concentrations were the same as that used in the NTP 2-yr toxicity and carcinogenicity study (Abdo et al., 1992). Because mice are more sensitive than rats to naphthalene toxicity, an additional higher exposure group (60 ppm) was included. The 60 ppm exposure concentration is the maximum that can be generated without naphthalene condensation.

All animals were observed twice daily. Body weights were recorded on study day 1, every 4 wk beginning at wk 5, and every 2 wk beginning at wk 92. Clinical findings were recorded every 4 wk beginning at wk 5 and every 2 wk beginning at wk 92.

Pathology

Complete necropsies and microscopic examinations were performed on all core study animals. At necropsy, all organs and tissues were examined for grossly visible lesions. Tissues were fixed in 10% neutral buffered formalin, trimmed, processed, embedded in paraffin, sectioned at a thickness of 4 to 6 µm, and stained with hematoxylin and eosin for microscopic examination. Complete histopathology was performed on all core study rats. Tissues and organs examined microscopically included adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eyes, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung with mainstem bronchi, lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland (females), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistical Methods

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). The incidences of neoplasms and nonneoplastic lesions were evaluated by the Poly-3 test (Bailer & Portier, 1988; Portier & Bailer, 1989; Piegorsch & Bailer, 1997). This test is a survival-adjusted quantal-response procedure that modifies the Cochran– Armitage linear trend test to take survival differences into account. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure-related trend. Continuitycorrected Poly-3 tests were used in the analysis of lesion incidence, and reported p values are one-sided. In addition, body weight data, which have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Average severity values were analyzed for significance with the Mann–Whitney U-test (Hollander & Wolfe, 1973)."

Test subjects: rats, F344/N, 49/sex/group

Test article: Naphthalene (>99%)

Inhalation chamber: Dynamic whole-body chambers

Exposure regimen: Rats exposed to naphthalene vapor, 6 hours plus T₉₀ (12 min) per day, 5 d/wk for 2 yr

Analytical concs^a: 0, 52 ± 3.7, 158 ± 8.9, 316 ± 20.5 mg/m³

Female F344 Rats