Comparative Incidences and Biological Outcomes for Thymoma in Various Rat Strains in National Toxicology Program Studies.

Thymomas from 277 Fischer 344/N (F344/N), 10 Sprague Dawley (HSD:Sprague Dawley SD) (SD), 129 Wistar Han [Crl:WI(Han)] (WH), and 4 Wistar Outbred (WO) rats were reviewed from long-term studies in the National Toxicology Program (NTP) database. The incidence of thymomas in F344/N rats was slightly higher in males than in females, while the incidences in SD and WH rats were higher in females than in males. Only male WO rats were used in NTP studies. Of the 277 thymomas in F344/N rats, 235 (84.8%) were benign and 42 (15.2%) malignant, 14 of which exhibited metastasis. Of the 10 thymomas in SD rats, 5 (50%) were benign and 5 (50%) were malignant, one of which exhibited metastasis. Of the 129 thymomas in WH rats, 126 (98%) were benign and 3 (2%) were malignant, 1 with metastasis. Of the 4 thymomas in WO rats, 3 (75%) were benign and 1 (25%) was malignant, with no metastases. Malignant thymomas in F344/N and WH rats showed a propensity to be the cause of death and to result in early mortality, whereas the benign thymomas were associated less often with decreased survival. No occurrences of this neoplasm were reported to be related to exposure to any test articles.

Utilizing Whole Slide Images for Pathology Peer Review and Working Groups.

This article describes the results of comparisons of digitally scanned whole slide images (WSIs) and glass microscope slides for diagnosis of tissues under peer review by the National Toxicology Program. Findings in this article were developed as a result of the data collected from 6 pathology working groups (PWGs), 1 pathology peer review, and survey comments from over 25 participating pathologists. For each PWG, 6-14 pathologists examined 10-143 tissues per study from 6- and 9-month perinatal studies and 2-year carcinogenicity studies. Overall it was found that evaluation of WSIs is generally equivalent to using glass slides. Concordance of PWG consensus diagnoses based upon review of WSIs versus glass slides ranged from 74% to 100% (median 86%). The intra- and interobserver diagnostic variation did not appear to influence the conclusions of any study. Based upon user opinions collected from surveys, WSIs may be less optimal than glass slides for evaluation of subtle lesions, large complex lesions, small lesions in a large section of tissue, and foci of altered hepatocytes. These results indicate that, although there may be some limitations, the use of WSIs can effectively accomplish the objectives of a conventional glass slide review and definitely serves as a useful adjunct to the conduct of PWGs.

Part 1. Assessment of carcinogenicity and biologic responses in rats after lifetime inhalation of new-technology diesel exhaust in the ACES bioassay.

The Health Effects Institute and its partners conceived and funded a program to characterize the emissions from heavy-duty diesel engines compliant with the 2007 and 2010 on-road emissions standards in the United States and to evaluate indicators of lung toxicity in rats and mice exposed repeatedly to 2007-compliant new-technology diesel exhaust (NTDE*). The a priori hypothesis of this Advanced Collaborative Emissions Study (ACES) was that 2007-compliant on-road diesel emissions "... will not cause an increase in tumor formation or substantial toxic effects in rats and mice at the highest concentration of exhaust that can be used ... although some biological effects may occur." This hypothesis was tested at the Lovelace Respiratory Research Institute (LRRI) by exposing rats by chronic inhalation as a carcinogenicity bioassay. Indicators of pulmonary toxicity in rats were measured after 1, 3, 12, 24, and 28-30 months of exposure. Similar indicators of pulmonary toxicity were measured in mice, as an interspecies comparison of the effects of subchronic exposure, after 1 and 3 months of exposure. A previous HEI report (Mauderly and McDonald 2012) described the operation of the engine and exposure systems and the characteristics of the exposure atmospheres during system commissioning. Another HEI report described the biologic responses in mice and rats after subchronic exposure to NTDE (McDonald et al. 2012). The primary motivation for the present chronic study was to evaluate the effects of NTDE in rats in the context of previous studies that had shown neoplastic lung lesions in rats exposed chronically to traditional technology diesel exhaust (TDE) (i.e., exhaust from diesel engines built before the 2007 U.S. requirements went into effect). The hypothesis was largely based on the marked reduction of diesel particulate matter (DPM) in NTDE compared with emissions from older diesel engine and fuel technologies, although other emissions were also reduced. The DPM component of TDE was considered the primary driver of lung tumorigenesis in rats exposed chronically to historical diesel emissions. Emissions from a 2007-compliant, 500-horsepower-class engine and after treatment system operated on a variable-duty cycle were used to generate the animal inhalation test atmospheres. Four groups were exposed to one of three concentrations (dilutions) of exhaust combined with crankcase emissions, or to clean air as a negative control. Dilutions of exhaust were set to yield average integrated concentrations of 4.2, 0.8, and 0.1 ppm nitrogen dioxide (NO2). Exposure atmospheres were analyzed by daily measurements of key effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors. components and periodic detailed physical-chemical characterizations. Exposures were conducted 16 hours/day (overnight, during the rats' most active period), 5 days/week. Responses to exposure were evaluated via hematology, serum chemistry, bronchoalveolar lavage (BAL), lung cell proliferation, histopathology, and pulmonary function. The exposures were accomplished as planned, with average integrated exposure concentrations within 20% of the target dilutions. The major components from exhaust were the gaseous inorganic compounds, nitrogen monoxide (NO), NO2, and carbon monoxide (CO). Minor components included low concentrations of DPM and volatile and semi-volatile organic compounds (VOCs and SVOCs). Among the more than 100 biologic response variables evaluated, the majority showed no significant difference from control as a result of exposure to NTDE. The major outcome of this study was the absence of pre-neoplastic lung lesions, primary lung neoplasia, or neoplasia of any type attributable to NTDE exposure. The lung lesions that did occur were minimal to mild, occurred only at the highest exposure level, and were characterized by an increased number and prominence of basophilic epithelial cells (considered reactive or regenerative) lining distal terminal bronchioles, alveolar ducts, and adjacent alveoli (termed in this report "Hyperplasia; Epithelial; Periacinar"), which often had a minimal increase in subjacent fibrous stroma (termed "Fibrosis; Interstitial; Periacinar"). Slight epithelial metaplastic change to a cuboidal morphology, often demonstrating cilia, was also noted in some animals (termed "Bronchiolization"). In addition to the epithelial proliferation, there was occasionally a subtle accumulation of pulmonary alveolar macrophages (termed "Accumulation; Macrophage") in affected areas. The findings in the lung progressed slightly from 3 to 12 months, without further progression between 12 months and the final sacrifice at 28 or 30 months. In addition to the histologic findings, there were biochemical changes in the lung tissue and lavage fluid that indicated mild inflammation and oxidative stress. Generally, these findings were observed only at the highest exposure level. There was also a mild progressive decrease in pulmonary function, which was more consistent in females than males. Limited nasal epithelial changes resulted from NTDE exposure, including increases in minor olfactory epithelial degeneration, hyperplasia, and/or metaplasia. Increases in these findings were present primarily at the highest exposure level, and their minor and variable nature renders their biologic significance uncertain. Overall, the findings of this study demonstrated markedly less severe biologic responses to NTDE than observed previously in rats exposed similarly to TDE. Further, the effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors.

Comparison of inhalation toxicity studies of gentamicin in rats and dogs.

Nebulized gentamicin solution was administered to rats (nose-only) and dogs (face mask) for 14 days with a 14-day recovery period. Control groups of each were exposed to saline aerosols. Mean estimated inhaled lung doses of gentamicin were 39, 123 and 245 mg/kg for rats (deposited doses 6, 17 and 34 mg/kg) over 30, 90 and 180 min, respectively. Since dogs do not tolerate exposures as long as rats, inhaled lung doses were limited to 7, 14 and 41 mg/kg (deposited doses of 1, 3 and 8 mg/kg) over 15, 30 and 60 min. Comparable doses were achieved at the low dose for rats and high dose for dogs. Serum AUCs (14 ± 2 µg/mL h (mean ± SD) at 6 mg/kg in rats and 11 ± 7 µg/mL h at 8 mg/kg in dogs) showed comparable exposure between the two, implying similar absorbed doses and confirming similar deposited lung doses. Rat exposures resulted in dose-related lung pathology (including low dose) manifested as upper respiratory tract irritant reactions with alveolar histiocytosis, inflammation, airway epithelial metaplasia and lymphomegaly in lung tissue. This was associated with high lung tissue gentamicin levels 24 h post-dose on Day 14 (375 ± 33 µg/g at deposited dose of 6 mg/kg). Dose-related kidney tubular necrosis (a well-known toxicity of parenteral gentamicin) was observed, but no test-article related effects on lung histopathology in dogs (even at highest deposited dose of 8 mg/kg) and low levels of lung tissue gentamicin (42 ± 11 µg/g) 24 h post-dose on Day 14.

Part 1. Biologic responses in rats and mice to subchronic inhalation of diesel exhaust from U.S. 2007-compliant engines: report on 1-, 3-, and 12-month exposures in the ACES bioassay.

The Health Effects Institute and its partners conceived and funded a program to characterize the emissions from heavy-duty diesel engines compliant with the 2007 and 2010 on-road emissions standards in the United States and to evaluate indicators of lung toxicity in rats and mice exposed repeatedly to diesel exhaust (DE*) from 2007-compliant engines. The preliminary hypothesis of this Advanced Collaborative Emissions Study (ACES) was that 2007-compliant on-road diesel emissions ". . . will not cause an increase in tumor formation or substantial toxic effects in rats and mice at the highest concentration of exhaust that can be used . . . although some biological effects may occur." This hypothesis is being tested at the Lovelace Respiratory Research Institute (LRRI) by exposing rats by chronic inhalation as a carcinogenicity bioassay, measuring indicators of pulmonary toxicity in rats after 1, 3, 12, and 24-30 months of exposure (final time point depends on the survival of animals), and measuring similar indicators of pulmonary toxicity in mice after 1 and 3 months of exposure. This report provides results of exposures through 3 months in rats and mice. Emissions from a 2007-compliant, 500-horsepower-class engine and aftertreatment system operated on a variable-duty cycle were used to generate the animal inhalation test atmospheres. Four treatment groups were exposed to one of three concentrations (dilutions) of exhaust combined with crankcase emissions, or to clean air as a negative control. Dilutions of exhaust were set to yield average integrated concentrations of 4.2, 0.8, and 0.1 ppm nitrogen dioxide (NO2). Exposure atmospheres were analyzed by daily measurements of key components and periodic detailed physical-chemical characterizations. Exposures were conducted 16 hr/dy (overnight), 5 dy/wk. Rats were evaluated for hematology, serum chemistry, bronchoalveolar lavage (BAL), lung cell proliferation, and histopathology after 1 month of exposure, and the same indicators plus pulmonary function after 3 months. Mice were evaluated for BAL, lung cell proliferation, and respiratory tract histopathology after 1 month of exposure, and the same indicators plus hematology and serum chemistry after 3 months. Samples from both species were collected for ancillary studies performed by investigators who were not at LRRI and were funded separately. Exposures were accomplished as planned, with average integrated exposure concentrations within 20% of the target dilutions. The major components were the gaseous inorganic compounds, nitrogen monoxide (NO), NO2, and carbon monoxide (CO). Minor components included low concentrations of diesel particulate matter (DPM) and volatile and semivolatile organic compounds (VOCs and SVOCs). There were no exposure-related differences in mortality or clinically evident morbidity. Among the more than 100 biologic response variables evaluated, the majority showed no significant difference from control as a result of exposure to DE. There was evidence of early lung changes in the rats, accompanied by a number of statistically significant increases in inflammatory and oxidative stress indicators, and some evidence of subtle changes in pulmonary function. In general, statistically significant effects were observed only at the highest exposure level. The mice did not have the same responses as the rats, but did have small but statistically significant increases in lavage neutrophils and the cytokine IL-6 at 1 month (but not at 3 months). These findings suggest that the rats were more sensitive than mice to the subchronic exposures.

Nasal epithelial lesions in F344 rats following a 90-day inhalation exposure to naphthalene.

Naphthalene (NA) was shown to be carcinogenic, causing respiratory epithelial adenoma in the nasal cavity of male F344 rats and olfactory epithelial neuroblastoma in female F344 rats at exposure concentrations of 10-60 ppm in a 2-year inhalation study conducted by the National Toxicology Program. To explore the exposure-response relationship and threshold for nasal epithelial effects in F344 rats, a 90-day (6 h/d, 5 d/wk) inhalation study was conducted at 0, 0.1, 1, 10 and 30 ppm NA vapor. Group size for nasal cavity histopathology was 10/sex with an additional 10/sex evaluated 4 wk post-exposure. NA exposure concentrations were measured by GC/MS, and aerosol testing verified that solid NA particles were not present. There were no NA exposure-related clinical observations and mild decreases in body weight (<10%) and food/water consumption were observed primarily in the 30 ppm rats. Rat heads were cross-sectioned at six levels for microscopic examination. There were no nasal cavity lesions related to NA exposure in rats of the 0.1 ppm group. Minimal hyperplasia was observed in the transitional/respiratory epithelium of rats exposed to 1 ppm. Mild hyperplasia and minimal squamous metaplasia were observed in the respiratory epithelium of rats exposed to 10 or 30 ppm. Lesions in the olfactory epithelium were observed only in rats of the 10 or 30 ppm groups and consisted of degeneration, necrosis, areas of re-epithelialization and basal cell hyperplasia. There was remarkable recovery of effects after 4 weeks, but residual olfactory epithelial degeneration and basal cell hyperplasia were still evident.

Proceedings of the 2010 National Toxicology Program Satellite Symposium.

The 2010 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri," was held in Chicago, Illinois, in advance of the scientific symposium sponsored jointly by the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP). The goal of the annual NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for voting or discussion. Some topics covered during the symposium included a comparison of rat and mouse hepatocholangiocarcinoma, a comparison of cholangiofibrosis and cholangiocarcinoma in rats, a mixed pancreatic neoplasm with acinar and islet cell components, an unusual preputial gland tumor, renal hyaline glomerulopathy in rats and mice, eosinophilic substance in the nasal septum of mice, INHAND nomenclature for proliferative and nonproliferative lesions of the CNS/PNS, retinal gliosis in a rat, fibroadnexal hamartoma in rats, intramural plaque in a mouse, a treatment-related chloracne-like lesion in mice, and an overview of mouse ovarian tumors.

Nasal olfactory epithelial lesions in F344 and SD rats following 1- and 5-day inhalation exposure to naphthalene vapor.

The exposure-response relationship and threshold for nasal epithelial effects of naphthalene (NP) vapor in F344 and SD rats were investigated in 1-day (6 hours) and 5-day (6 h/d) studies at concentration ranges of 0 to 30 ppm. Lesions related to 1-day exposure were predominantly necrosis of the olfactory epithelium (OE). The severity of OE lesions was concentration dependent and ranged from minimal (< or =1 ppm) to marked (10-30 ppm). In the 5-day study, degeneration of OE was observed in both strains, both sexes, with increasing incidence and severity that correlated with concentration. The epithelial degeneration lesion was minimal to moderate in severity. At 0.1 ppm, minimal OE lesions were observed in female SD rats only (20% incidence). Animals exposed to 10 ppm NP followed by 14 days without exposure also had OE lesions, but of lower severity, showing evidence of good recovery. In both studies, differences between sex or strain were not remarkable.

Proceedings of the 2009 National Toxicology Program Satellite Symposium.

The National Toxicology Program (NTP) Satellite Symposium is a one-day meeting that is held in conjunction with the annual Society of Toxicologic Pathology (STP) meeting. The topic of the 2009 Symposium was "Tumor Pathology and INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Nomenclature." The goal of this article is to provide summaries of each speaker's presentation, including the diagnostic or nomenclature issues that were presented, along with a few select images that were used for voting. The results of the voting process and interesting points of discussion that were raised during the presentation are also provided. A supplemental file with voting choices and voting results for each case presented at the symposium is available at http://tpx.sagepub.com/supplemental.

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.

Pulmonary lesions in female Harlan Sprague-Dawley rats following two-year oral treatment with dioxin-like compounds.

Dioxin and dioxin-related compounds have been associated with high incidences of pulmonary dysfunctions and/or cancers in humans. To evaluate the relative potencies of effects of these compounds, the National Toxicology Program completed a series of two-year bioassays which were conducted using female Harlan Sprague-Dawley rats. The rats were treated orally for up to 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and a ternary mixture of TCDD, PCB126 and PeCDF. In addition to treatment-related effects reported in other organs, a variety of pulmonary lesions were observed that were related to exposure. Pulmonary CYP1A1-associated 7-ethoxyresorufin-O-deethylase (EROD) activity was increased in all dosed groups. The most common non-neoplastic lesions, which occurred in all studies, were bronchiolar metaplasia and squamous metaplasia of the alveolar epithelium. Cystic keratinizing epithelioma was the most commonly observed neoplasm which occurred in all studies. A low incidence of squamous cell carcinoma was associated only with PCB126 treatment. Potential mechanisms leading to altered differentiation and/or proliferation of bronchiolar and alveolar epithelia may be through CYP1A1 induction or disruption of retinoid metabolism.

Inhalation toxicology and carcinogenesis studies of propylene glycol mono-t-butyl ether in rats and mice.

Propylene glycol mono-t-butyl ether (PGMBE) is used as a solvent in a variety of commercial applications. Male and female F344/N rats and B6C3F(1) mice were exposed to PGMBE by whole-body inhalation for 2 or 14 weeks (0, 75, 150, 300, 600, or 1200 ppm) or 2 years (0, 75, 300, or 1200 ppm); male NBR rats were exposed for 2 weeks. The kidney and the liver were targets of PGMBE toxicity in rats. Renal lesions suggestive of alpha(2u)-globulin nephropathy were observed in male F344/N, in the 2 and 14-week studies, no kidney lesions were seen in NBR rats. In the 2-year study, male rats displayed exposure-related nonneoplastic lesions in the kidney, and may have shown marginal increases in tubular neoplasms. In the liver, the incidences of hepatocellular adenomas occurred with a positive trend in male rats, and may have been related to PGMBE exposure. In mice of both sexes, the major target of PGMBE toxicity was the liver. In the 2-week study, liver weights and in the 14-week study, liver weights and the incidences of centrilobular hypertrophy were increased. In the 2-year study, the incidences of exposure-related hepatocellular adenoma, adenoma or carcinoma combined, and hepatoblastoma occurred with a positive trend, and were significantly increased in 1200 ppm groups. In summary, exposure to PGMBE resulted in nonneoplastic lesions of the kidney characteristic of alpha(2u)-globulin nephropathy, and may have increased renal tubular neoplasms in male rats. Exposure to PGMBE also produced increases in hepatic tumors in male and female mice.

Alpha 2u-globulin nephropathy and carcinogenicity following exposure to decalin (decahydronaphthalene) in F344/N rats.

Decalin (decahydronaphthalene) is a widely used industrial solvent known to cause male rat-specific alpha2u-globulin nephropathy. In this project, 13-week and two-year inhalation studies of decalin were conducted consecutively in both sexes of F344/N rats. The key objectives were to (1) characterize the 13-week toxicity of decalin in rats, with an emphasis on nephropathy in males; (2) compare the kidney concentrations of decalin, 2-decalone, and alpha2u-globulin in males over 2 to 13 weeks of decalin exposure; and (3) correlate male rat nephropathy observed in the 13-week study with renal carcinogenicity in the two-year study. F344 rats (M/F) were exposed via whole-body inhalation to 0, 25, 50, 100, 200, or 400 ppm decalin for 13 weeks. Urine was collected at weeks 2 and 6 for creatinine and decalol analyses and at week 12 for clinical urinalysis. Right kidneys were collected from male rats at weeks 2 and 6 and from both sexes at week 13, homogenates were prepared using the whole kidney, and these homogenates were analyzed for alpha2u-globulin, decalin, and 2-decalone. Left kidneys were evaluated for histopathology and cell proliferation utilizing a proliferating cell nuclear antigen technique and counting proximal renal tubular epithelial cells to determine cell labeling indices. Necropsies and histopathologic evaluations were performed at week 13. Decalin exposure caused increases in kidney weight, urinalysis parameters (protein, AST, LDH), kidney alpha2u-globulin concentration, and proximal convoluted renal tubular cell proliferation in males. These changes were accompanied by microscopic lesions (accumulation of hyaline droplets in cortical tubules, regeneration of proximal tubular epithelium, and granular casts in medullary tubules) clearly linked to alpha2u-globulin nephropathy. Both decalin and 2-decalone were related to increased alpha2u-globulin in male kidneys. Kidney concentrations of decalin, 2-decalone, and alpha2u-globulin in exposed females were negligible, while females excreted greater amounts of decalol metabolites in urine than males at weeks 2 and 6. There were no exposure-related microscopic lesions in females. For chronic exposure, F344 rats were exposed via whole-body inhalation to 0, 25, 50 (males only), 100, or 400 ppm decalin for two years. Chronic exposure induced a spectrum of nonneoplastic and neoplastic lesions in the renal cortex of males, ranging from regenerative lesions of chronic nephropathy to tubular carcinomas. Incidences of renal tubular adenoma, tubular carcinoma, combined tubular adenomas and carcinomas, cortical tubular hyperplasia, hyaline droplet accumulation, hyperplasia of pelvic epithelium, and mineralization in renal papilla were increased in exposed males compared to controls. There was a clear increase in the mean severity of chronic nephropathy in decalin-exposed males. It was concluded that the carcinogenic effect on the renal cortical epithelium of male rats exposed to decalin was related to increased turnover of this epithelium, resulting from the cytotoxic effects of alpha2u-globulin accumulation in the renal cortical tubular cell cytoplasm.

Mode of action and pharmacokinetic studies of 2-butoxyethanol in the mouse with an emphasis on forestomach dosimetry.

Chronic inhalation studies with 2-butoxyethanol (BE) conducted by the National Toxicology Program identified the forestomach and liver of B6C3F1 mice as target organs for tumorigenicity (NTP, 2000). Previous studies have shown that the liver tumors likely resulted from chronic hemolysis-induced oxidative stress. For the forestomach lesions seen in mice, chronic contact irritation (cytotoxicity) and regenerative hyperplasia are hypothesized to result in forestomach tumor development. To test this hypothesis, several experiments were conducted to address the sensitivity of the mouse forestomach to BE administered by various routes. Oral administration of undiluted BE was shown to cause irritation and a compensatory proliferative response in the mouse forestomach, confirming that direct contact between the forestomach and BE, which can occur via grooming of BE condensed on the fur during inhalation exposures, can cause irritation. However, only small amounts of BE (<10 mg/kg) were detected on the fur of mice at the end of 6-h, whole-body or nose-only inhalation exposures to the highest concentration used in the NTP chronic inhalation studies (250 ppm). Furthermore, no significant differences were detected in the end-exposure blood concentrations of BE and butoxyacetic acid (BAA) between these types of exposures. In addition, parenteral administration of BE (ip and sc injection) also resulted in forestomach lesions, indicating that there may be sources other than grooming for BE- or BAA-induced forestomach irritation. In the pharmacokinetic study, BE and, to a lesser extent, BAA was eliminated more slowly from the forestomach tissue of mice than from blood or other tissues, following either oral gavage or ip injection. The forestomach was the only tissue with detectable levels of BE at 24 h. BE and BAA were both excreted in the saliva and were present in stomach contents for a prolonged period of time following these routes of exposure, which may further contribute to forestomach tissue dosimetry. Thus, there appear to be multiple mechanisms behind the increased levels of BE and BAA in the forestomach tissue of mice, which together can contribute to a prolonged contact irritation, compensatory hyperplasia, and tumorigenicity in mice. The relevance of these effects in humans, who lack a forestomach, is questionable.