“Animals and housing.

Six-week-old male and female CD- 1 ICR mice were obtained from Charles River Breeding Laboratories, Wilmington, Mass. Mice were quarantined for 4 days, randomized, and uniquely identified by ear punching and cage tag. The animals were maintained in plastic cages with hardwood bedding (PWI Hardwood Sawdust, Lowville, N.Y.) in temperature-controlled animal rooms at 21-24°C and a relative humidity of 40-60%. The light-dark cycle was maintained on 12-hr intervals. Male mice were caged in pairs and female mice in sets of four per cage with mice within any exposure group caged vertically to minimize both light and temperature differences between exposure groups. The mice were fed Purina Rodent Chow No. 5001 (Ralston Purina Co., St. Louis, MO.) and deionized water ad libitum.

Chemical.

Naphthalene (Lot No. EE09 1397) was obtained from Aldrich Chemical Company, Milwaukee, Wisconsin, and its purity confirmed at 99.3% employing gas chromatography. Throughout the reported studies, naphthalene was administered by oral gavage employing 19-gauge gavage needles. A corn oil (Mazola Pure Corn Oil, Best Food, Englewood Cliffs, N.J., Lot No. 6-0729) suspension of naphthalene was prepared daily and stirred continuously during dosing. Concentrations of the corn oil-naphthalene suspension were prepared such that all doses could be delivered in a volume of IO ml/kg body wt. Naive controls were not gavaged and vehicle controls received corn oil alone at a dose of IO ml/kg body wt.

Acute toxicity.

Five dose levels (200, 400, 600, 800, 1000 mg/kg) were employed to determine the acute oral LD50. Eight mice of each sex per dosage group were fasted for 18 hr prior to oral dosing, with food being returned 1 hr after dosing. The mice were continuously observed for 1 hr after dosing, hourly for the next 4 hr, and then twice each day for the next 14 days. All mice that died during the observation period and those that survived the 14-day period were necropsied. The LDIO, LDSO, and LD90 were computed for each sex by the Log Probit Analysis as described by Finney (1971).

14-Day repeated dosing study.

Male and female mice were divided into 5 groups consisting of 40- 112 mice per group. Each group, respectively, received no treatment (naive), corn oil (vehicle), and naphthalene at levels of 27 mg/kg, 53 mg/kg, and 267 mg/kg, representing 1/20, 1/10, and ½ the male LD50. Dosing took place between 10:30 AM and 12:30 PM each day for 14 consecutive days. The animals were observed twice daily for manifestations of toxicity. The terminal sacrifice was conducted 24 hr after the last administration and the various parameters chosen to assess toxicity measured. Positive controls for immunotoxicity were administered 25 mg/kg cyclophosphamide ip on Days 11, 12, 13, and 14.

90-Day subchronic toxicity.

For these studies, the mice were divided into five treatment groups, consisting of 40- 1 12 animals of each sex per group. The five treatment groups consisted of naive, corn oil vehicle, and naphthalene at doses of 5.3, 53, and 133 mg/kg, representing 1/100, 1/10, 1/4 the acute oral male LD50. Animals were dosed daily for 90 consecutive days. Positive controls for immunotoxicity were administered 50 mg/kg cyclophosphamide ip on days 87, 88, 89, and 90. Body weights were recorded on the first day of dosing and weekly thereafter. The mice were observed twice daily and any animals found moribund or dead were immediately necropsied. The experiment was terminated 24 hr after the last administration and the various parameters of toxicity determined.

Necropsy.

A randomly selected group of animals were anesthetized with chloroform and blood was collected by cardiac puncture into 3.2% sodium citrate. Gross pathological examination was performed, followed by removal and weighing of the brain, kidneys, liver, lungs, spleen, and thymus.

Hematology.

A Coulter counter (Model ZB) was used to determine leukocyte, erythrocyte, and platelet numbers. Microhematocrits were determined and hemoglobin was assayed as cyanomethemoglobin. Leukocyte differentials were evaluated by the standard Wright+Giemsa staining procedure. Bone marrow cells were flushed from a femur into a-minimal essential medium (Gibco, Grand Island, N.Y.) containing 5% fetal calf serum and enumerated with a Couher counter. Plasma extrinsic activity was determined by prothrombin time and intrinsic activity by activated partial thromboplastin time, employing reagents from General Diagnostics, Morris Plains, New Jersey. Plasma fibrinogen levels were determined using reagents from Dade Diagnostics, Inc., Miami, Florida. Serum enzyme chemistries. Serum enzyme levels were determined employing an Abbott Bichromatic Analyzer and Abbott diagnostic chemistry kits (Abbott Labs, N. Chicago, Ill.). Serum ion concentrations were determined with a flame photometer and a chloridometer.

Hexobarbital sleeping time.

Hexobarbital sleeping time was determined on a randomly selected group of mice in the 14&y study. Twenty-four hours after the last naphthalene administration the mice received an ip administration of 100 mg/kg hexobarbital and sleeping time was determined. Sleeping time was defined as the time between loss and return of the righting reflex.

Preparation and assay of hepatic microsomal activity.

The weighed livers were rinsed and homogenized at 0-4°C in 4 vol of 0.01 M potassium phosphate buffer (pH 7.4) containing 0.15 M KCl. Individual homogenates were prepared for each mouse. The homogenate was centrifuged at 9000g for 20 min and the supematant centrifuged at 100,000g for 1 hr. The microsomal pellet was resuspended in 0.05 M phosphate buffer (pH 7.4) at a concentration of 0.5 g liver/ml. Microsomal protein was determined by the method of Lowry et al. (195 1) and cytochrome P-450 by the method of Omura and Sato (1964). Glutathione levels, aminopyrine Ndemethylase activity, aniline hydroxylase, and henzo(a)pyrene hydroxylase activities were determined as previously described (Barnes et al., 1979). Immunotoxicity assays. The assays utilized to assess immunotoxicity were performed as previously described (Munson et al., 1982; Sanders et al., 1982). Antibody forming cells per spleen and antibody forming cells per lo6 spleen cells were determined to assess the humoral immune response. Lymphocyte responsiveness was determined by [‘*SI]iododeoxyuridine uptake by mitogen stimulated spleen cells. A stimulation index was calculated to assess the delayed-type hypersensitivity response and the popliteal lymph node response. Bone marrow function was assessed by measuring stem cell number and bone marrow DNA synthesis.

Statistical evaluation.

If a one-way analysis of variance of the means showed treatment effects when compared to vehicle, a Dunnett’s t test was performed. Those values which differed from the vehicle group at p < 0.05 were considered significant.”

Female CD-1 Mice