Rodney A. Miller

Histologic Methods and Interspecies Variations in the Laryngeal Histology of F344/N Rats and B6C3F1 Mice

The relatively high incidence and variety of lesions induced in the upper respiratory tract of rodents by inhalation of xenobiotics has resulted in considerable attention given to the microscopic anatomy of this area. Specific areas of the rodent laryngeal mucosa appear to be more sensitive to inhaled materials and more likely to contain cellular changes in response to injury. These include the epithelium covering the base of the epiglottis, ventral pouch, and the medial surfaces of the vocal processes of the arytenoid cartilages. There are few good landmarks for trimming rodent larynges to get consistent and accurate sections through these target areas. We have obtained consistently reproducible results by cutting transversely through the easily palpable cricothyroid notch and embedding the entire larynx anterior to this in paraffin with the cut surface against the face of the block. Multiple sections are cut from the caudal larynx toward the epiglottis, unstained sections examined microscopically for orientation, and sections from target areas selected for staining and histopathologic examination. Routine use of these methods for preparation and microscopic examination of sections of the larynx has revealed some variations in normal laryngeal anatomy between Fischer 344 (F344/N) rats and B6C3F1 mice.

Summary of Chemically Induced Pulmonary Lesions in the National Toxicology Program (NTP) Toxicology and Carcinogenesis Studies

The lung is the second most common target site of neoplasia of chemicals tested by the National Toxicology Program (NTP). Of all peer-reviewed NTP studies to date (N = 545), a total of sixty-four chemicals in sixty-six reports produced significant site-specific neoplasia in the lungs of rats and/or mice. Of the studies associated with lung tumor induction, approximately 35% were inhalation and 35% were gavage studies, with dosed-feed, dosed-water, topical, intraperitoneal, or in utero routes of chemical administration accounting for 18%, 6%, 3%, 1%, and 1% of the studies, respectively. The most commonly induced lung tumors were alveolar/bronchiolar (A/B) adenoma and/or carcinoma for both species. The most frequently observed nonneoplastic lesions included hyperplasia and inflammation in both species. The liver was the most common primary site of origin of metastatic lesions to the lungs of mice; however, skin was most often the primary site of origin of metastatic lesions to the lungs of rats. In summary, A/B adenoma and carcinoma were the most frequently diagnosed chemically induced tumors in the lungs of both rats and mice in the NTP toxicology and carcinogenesis bioassays, and hyperplasia and inflammation were the most common nonneoplastic changes observed.

Two-Year and Lifetime Toxicity and Carcinogenicity Studies of Ozone in B6C3F1 Mice

To evaluate the toxicity and carcinogenic potential of long-term exposure to ozone, B6C3F1 mice were exposed by whole-body inhalation to 0, 0.12, 0.5, or 1.0 ppm and 0, 0.5, or 1.0 ppm ozone for 24 or 30 mo (lifetime), respectively. The incidence of alveolar/ bronchiolar adenomas and carcinomas (combined) increased (p < 0.05) in female mice exposed to 1.0 ppm for 24 or 30 mo and marginally increased (p > 0.05) in male mice exposed to concentrations of 0.5 or 1.0 ppm. An increased incidence of nonneoplastic lesions were observed in the nasal cavities and in the centriacinar region of the lung of mice exposed to 0.5 or 1.0 ppm for 24 and 30 mo. Nasal cavity lesions were mild and included hyaline degeneration, hyperplasia, squamous metaplasia, fibrosis and suppurative inflammation of the transitional and respiratory epithelium of the lateral wall, and atrophy of the olfactory epithelium. Lung lesions included replacement of the epithelium of the alveolar ducts and adjacent alveolar septa with epithelium similar to that normally found in terminal bronchioles (metaplasia) and associated alveolar histiocytosis. Based on the results of these studies, we conclude that inhalation exposure of B6C3F1 mice to ozone for 24 or 30 mo (a) is carcinogenic in female B6C3F1 mice exposed to 1.0 ppm of ozone based on an increased incidence of alveolar/bronchiolar adenoma or carcinoma and (b) results in mild, site-specific, nonneoplastic lesions in the nasal cavity and centriacinar lung of male and female mice exposed to 0.5 or 1.0 ppm of ozone for 2 yrs, which persist with continued exposure to 30 mo. It is uncertain whether or not the marginal increase (p > 0.05) of alveolar/bronchiolar neoplasms in male B6C3F1 mice resulted from exposure to ozone.

Carcinogenesis Studies of Tetrahydrofuran Vapors in Rats and Mice

Tetrahydrofuran (THF) is a widely used industrial solvent and was selected for carcinogenesis studies by the National Toxicology Program (NTP) because of its potential for widespread occupational exposure in humans and a lack of information on animal toxicity and carcinogenicity. Groups of 50 male and 50 female F344/N rats and B6C3F1 mice were exposed to 0, 200, 600, or 1800 ppm THF by inhalation, 6 h per day, 5 days per week, for 105 weeks. Survival and mean body weights of male and female rats exposed to THF were comparable to that of the controls. No clinical findings or nonneoplastic lesions related to THF exposure were observed in male or female rats. The incidences of renal tubule epithelial adenoma or carcinoma (combined) in exposed male rats occurred with a positive trend, and in males exposed to 600 and 1800 ppm exceeded the historical range for controls in 2-year NTP inhalation studies. There were no other neoplastic lesions related to THF exposure observed in male or female rats. After week 36, the survival of male mice exposed to 1800 ppm was significantly lower than that of the controls. Mean body weights of male and female mice exposed to THF were similar to those of the controls throughout the study. Male mice exposed to 1800 ppm were observed in a state of narcosis during and up to 1 h after the exposure periods. Nonneoplastic lesions related to THF exposure were not observed in male or female mice. The neoplastic lesions related to THF exposure were seen in female mice only. In female mice exposed to 1800 ppm, the incidences of hepatocellular neoplasms were significantly greater than those in the controls. In conclusion, there was some evidence of carcinogenic activity of THF in male F344/N rats due to increased incidences of adenoma or carcinoma (combined) of the kidney at the 600 and 1800 ppm exposure levels. There was clear evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of hepatocellular neoplasms at the 1800 ppm exposure level. THF was not carcinogenic in female rats or male mice exposed at 200, 600, or 1800 ppm.

Toxicity of inhaled chloroprene (2-chloro-1,3-butadiene) in F344 rats and B6C3F1 mice

Chloroprene (2-chloro-1,3-butadiene) is a high production chemical used almost exclusively in the production of polychloroprene (neoprene) elastomer. Because of its structural similarity to isoprene (2-methyl-1,3-butadiene) and to 1,3-butadiene, a potent trans-species carcinogen, inhalation studies were performed on chloroprene to characterize its toxicological potential and to provide a basis for selecting exposure concentrations for chronic toxicity and carcinogenicity studies. Thirteen-week inhalation toxicology studies were conducted in male and female F344 rats and B6C3F(1) mice at exposure concentrations of 0, 5, 12, 32 or 80 ppm (6 h/day; 5 days/week). A 200 ppm exposure group was also included for rats only, because a previous study showed that this concentration of chloroprene is lethal to mice. In mice, exposure to 80 ppm chloroprene caused a marginal decrease in body weight gain in males and epithelial hyperplasia of the forestomach in males and females. This lesion has been observed in mice exposed to isoprene or 1,3-butadiene. In rats, exposure to 80 ppm chloroprene or higher concentrations caused degeneration and metaplasia of the olfactory epithelium and exposure to 200 ppm caused anemia, hepatocellular necrosis and reduced sperm motility. These lesions have not been observed in rats exposed to isoprene or 1,3-butadiene. The profile of toxic effects of chloroprene is considerably different from that of isoprene or 1,3-butadiene; this may be due to differences in exposure concentrations that were used in toxicology studies of these compounds and /or to the influence of the chlorine substitution on the toxicokinetics of these compounds, on their biotransformation, or on the reactivity of metabolic intermediates with tissue macromolecules.

Microscopic Anatomy of Toxicologically Important Regions of the Larynx of the Rat, Mouse, and Hamster

Laboratory rats, mice, and hamsters are the species most frequently used for subchronic and lifetime inhalation toxicity studies. The relatively high incidence of exposure-induced lesions in the upper respiratory tract in these species has generated considerable interest in the microscopic anatomy and histopathology of the nasal cavity (Brown et al. 1991; Harkema 1991; Morgan 1991) and the larynx (Brockmeyer et al. 1989; Gopinath et al. 1987; Lewis and Prentice 1980; Lewis 1981a,b; Lewis 1991; Nakano and Muto 1987; Renne et al. 1992a, 1994; Sagartz et al. 1992).

Long-term toxicity studies of ozone in F344/N rats and B6C3F1 mice.

The toxicity and carcinogenicity of ozone was evaluated in Fischer 344/N rats and B6C3F1 mice exposed to 0, 0.12 (2 years only), 0.5 or 1.0 ppm ozone by inhalation for 2-year and lifetime exposures. A 2-year cocarcinogenicity study (male rats only) included the subcutaneous administration of 0, 0.1 or 1.0 mg/kg/body wt. of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) for the first 20 weeks along with inhalation exposure to 0 or 0.5 ppm ozone followed by additional 84 weeks of ozone exposure alone. Ozone exposure in rats did not cause an increased incidence of lung neoplasms. In the cocarcinogenicity study, ozone exposure did not have an additive carcinogenic effect. Lifetime and 2-year ozone exposure was associated with a marginal increase in lung tumors in male B6C3F1 mice and a more pronounced increase in females. Unique mutations in the K-ras gene were found in the mouse lung neoplasms from the ozone-exposed mice.

Toxicity of Furfuryl Alcohol to F344 Rats and B6C3F1 Mice Exposed by Inhalation

Groups of F344 rats and B6C3F1 mice were exposed to furfuryl alcohol vapor for 6 hours per day, 5 days per week for 14 days (0, 16, 31, 63, 125, 250 ppm) or 13 weeks (0, 2, 4, 8, 16, 32 ppm). Reduced survival was observed in the 14‐day study at 250 ppm. Final mean body weights of rats and mice exposed to 125 ppm and of female mice exposed to 63 ppm were lower than controls at the end of the 14‐day study; there were no significant differences in mean body weight among chemical‐exposed and control groups in the 13‐week study. Exposure to furfuryl alcohol had no toxicologically significant effect on organ weights in either rats or mice, and did not cause any adverse changes in hematology or serum chemistry parameters evaluated in rats in the 13‐week study. Microscopic lesions associated with exposure to furfuryl alcohol were present in the nose of both rats and mice at all exposure concentrations in both the 14‐day and 13‐week studies. Lesions observed in the 14‐day study consisted of inflammation of the nasal turbinates accompanied by necrosis and squamous metaplasia of the respiratory epithelium and necrosis and degeneration of the olfactory epithelium. Similar lesions were observed in both rats and mice in the 13‐week study. In addition, squamous metaplasia and goblet cell hyperplasia of the respiratory epithelium, squamous metaplasia of the transitional epithelium and degeneration, hyperplasia and some respiratory metaplasia of the olfactory epithelium were also observed in rats in the 13‐week study, and hyaline droplets in the respiratory epithelium and chronic inflammation and respiratory metaplasia in the olfactory epithelium were observed in mice in the 13‐week study. In general the nasal passages of mice appeared less sensitive than those of rats at the concentrations used in the 13‐week study; a no‐observable‐effect level was not achieved in either the 14‐day or the 13‐week study. ©1997 by John Wiley & Sons, Ltd.

Inhalation toxicity and carcinogenicity of isoprene in rats and mice: comparisons with 1,3-butadiene

As with 1,3-butadiene (BD), inhalation exposure of B6C3F1 mice to isoprene (2-methyl-1,3-butadiene) caused a macrocytic anemia; induced increases in sister chromatid exchanges in bone marrow cells and in levels of micronucleated erythrocytes in peripheral blood; and produced degeneration of the olfactory epithelium, forestomach epithelial hyperplasia, and testicular atrophy. Most notable was the finding that like BD, isoprene induced neoplasms in the liver, lung, Harderian gland, and forestomach of mice. The carcinogenic effects of isoprene were observed after a 26-week exposure (6 h/day, 5 days/week) of male mice to 700 ppm or higher concentrations of isoprene followed by a 26-week recovery period. Unlike BD, isoprene did not induce lymphomas or hemangiosarcomas of the heart in mice under these conditions nor did it induce chromosomal aberrations in mouse bone marrow cells. No toxicological effects were evident in rats exposed for 13 weeks to either isoprene or BD at concentrations up to 7000 ppm or 8000 ppm, respectively. Interstitial cell hyperplasia of the testis was observed in male F344 rats exposed to 7000 ppm isoprene for 26 weeks, and following a 26-week recovery period, there was a marginal increase in benign testicular interstitial cell tumors.

Respiratory tract lesions in F344/N rats and B6C3F1 mice after inhalation exposure to 1,2-epoxybutane.

1,2-Epoxybutane, a short-chain epoxide used as a stabilizer in chlorinated hydrocarbon solvents, was administered by inhalation exposure as a vapor 6 h/day, 5 day/week, for 24 months at exposure concentrations of 0, 200 or 400 ppm to F344/N rats and 0, 50, or 100 ppm to B6C3F1 mice. Survival of all groups of rats was 50% or greater until week 98 but was reduced in exposed groups by the end of the study. Survival in male mice was comparable among groups. Survival in female mice was greater than 50% until week 86, but was then reduced in the high-exposure group of mice. Exposure-related inflammatory, degenerative, and proliferative lesions occurred in the nasal cavity of both rats and mice. Seven papillary adenomas occurred in the nasal passages of high-exposure male rats and 2 in the nasal passages of high-exposure female rats. Alveolar/bronchiolar adenoma or carcinoma (combined) occurred with increased incidence in exposed male rats relative to controls. No exposure-related neoplastic lesions were seen in mice. After inhalation exposure, 1,2-epoxybutane was carcinogenic in rodents as were other epoxides or related compounds including propylene oxide, 1,3-butadiene, and ethylene oxide. The site of carcinogenic activity was considered to be related to length of the carbon chain.