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Background In Japan, six workers handling cross-linked water-soluble acrylic acid polymer (CWAAP) at a chemical plant suffered from lung diseases, including fibrosis, interstitial pneumonia, emphysema, and pneumothorax. We recently demonstrated that inhalation of CWAAP-A, one type of CWAAP, causes pulmonary disorders in rats. It is important to investigate dose–response relationships and recoverability from exposure to CWAAPs for establishing occupational health guidelines, such as setting threshold limit value for CWAAPs in the workplace. Methods Male and female F344 rats were exposed to 0.3, 1, 3, or 10 mg/m3 CWAAP-A for 6 h/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. At 1 h, 4 weeks, and 13 weeks after the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses. In a second experiment, male rats were pre-treated with clodronate liposome or polymorphonuclear leukocyte-neutralizing antibody to deplete macrophages or neutrophils, respectively, and exposed to CWAAP-A for 6 h/day for 2 days. Results CWAAP-A exposure damaged only the alveoli. The lowest observed adverse effect concentration (LOAEC) was 1 mg/m3 and the no observed adverse effect concentration (NOAEC) was 0.3 mg/m3. Rats of both sexes were able to recover from the tissue damage caused by 13 weeks exposure to 1 mg/m3 CWAAP-A. In contrast, tissue damage caused by exposure to 3 and 10 mg/m3 was irreversible due to the development of interstitial lung lesions. There was a gender difference in the recovery from CWAAP-A induced pulmonary disorders, with females recovering less than males. Finally, acute lung effects caused by CWAAP-A were significantly reduced by depletion of alveolar macrophages. Conclusions Pulmonary damage caused by inhalation exposure to CWAAP-A was dose-dependent, specific to the lung and lymph nodes, and acute lung damage was ameliorated by depleting macrophages in the lungs. CWAAP-A had both a LOAEC and a NOAEC, and tissue damage caused by exposure to 1 mg/m3 CWAAP-A was reversible: recovery in female rats was less than for males. These findings indicate that concentration limits for CWAAPs in the workplace can be determined. Graphical Abstract

Objective In this study, in order to investigate the usefulness of intratracheal instillation in assessing the pulmonary toxicity of nanomaterials, intratracheal instillation of nickel oxide‐nanoparticles (NiO‐NP) was performed. Methods In this study, rats were administered test materials by intratracheal instillation at five different research institutions in order to assess the validity of using intratracheal instillation for hazard identification of nanomaterials. Eight‐week‐old male SD rats were administered NiO‐NP dispersed in deionized water by a single intratracheal instillation at doses of 0 (vehicle control), 0.2, 0.67, and 2 mg/kg BW. Three days after instillation, histopathological examination of the lungs was performed. Results NiO‐NP was distributed in the vicinity of hilus of the lung and in the alveoli around the bronchioles. Histopathological changes such as degeneration/necrosis of macrophages, inflammation, and proliferation of type II pneumocyte in the lung were observed, and their severity corresponded with increasing dose. The histopathological observations of pulmonary toxicity were almost similar at each institution. Conclusion The similarity of the histopathological changes observed by five independent groups indicates that intratracheal instillation can be a useful screening method to detect the pulmonary toxicity of nanomaterials.

Objectives In this study, we focused on the qualitative and quantitative differences of the lung lesions induced by single or multiple intratracheal administration of nickel oxide nanoparticles (NiO). Methods Male rats were randomized into groups receiving intratracheal administrations in a single dose or two to four divided doses of 2 mg/kg/bw. Bronchoalveolar lavage fluid (BALF) analyses were performed at 3 and 28 d post-dose. Histopathological analyses were performed at 28 and 91 d post-dose. Results BALF analyses revealed pulmonary injury, inflammation, and increases in the parameters indicating processing the foreign material in all the NiO-treated groups. Histopathological analyses showed the phagocytosis of NiO by alveolar macrophages, degeneration and necrosis of alveolar macrophages, and inflammatory responses. In the comparison between single and multiple administrations, the trend for stronger toxicity effects was observed after multiple application at 3 d post-dose, while the obvious toxicity effects were also seen in case of single administration. No particular differences of lung lesions depending on the frequency of administration at 28 and 91 d post-dose were evident. Conclusion Intratracheal NiO administration induced strong toxic response thoroughly even by single administration. Therefore, single administration was concluded to be applicable to assess the inhalation toxicity of nanomaterials and can be used in the screening test.

INTRODUCTION We investigated the 2-year survival rate and incidence of spontaneous tumors in F344/DuCrlCrlj rats used in carcinogenicity studies of chemical substances. Records for animals used in the control groups of carcinogenicity studies which were conducted during the last 10 years were obtained from the database of the Japan Bioassay Research Center (JBRC). Six hundred ninety-nine males and 550 females were used in 14 and 11 inhalation studies, respectively, and 500 animals of each sex were used in 10 male and 10 female oral studies. METHODS In each study, SPF (specific pathogen free) animals were housed for 2 years (104 weeks) as control groups in the carcinogenicity studies. All animals underwent necropsy and histopathological examination. Each study was conducted in accordance with the Good Laboratory Practice. RESULTS The incidence of interstitial cell tumors was highest in both inhalation studies and oral studies (inhalation studies 86.1%, oral studies 68.6%). Tumors which had an incidence of 6% or higher were adenoma of the pituitary, C-cell adenoma of the thyroid, and mononuclear cell leukemia (LGL leukemia) of the spleen in male and female rats; fibroma of the subcutaneous tissue, adrenal pheochromocytoma, and islet cell adenoma of the pancreas in male rats; and endometrial stromal polyps and fibroadenoma of the mammary gland in female rats. Tumors other than the above had rare incidence rates. A clear difference in the incidence of spontaneous tumors was not observed between the inhalation and oral studies. The incidences of spontaneous tumors in control groups of previous oral studies are similar to our findings. There are no other reports of the spontaneous tumor incidence in the control groups of inhalation studies using F344/DuCrlCrlj rats. The 2-year survival rate was about 77% in both the inhalation and oral studies, and a gender difference was not observed. The F344/DuCrlCrlj rats used at JBRC had a higher 2-year survival rate than F344/N rats. This difference is possibly due to the low incidence of LGL leukemia in the F344/DuCrlCrlj rat. CONCLUSIONS The incidences of spontaneous tumors in F344/DuCrlCrlj rats used in control groups of both inhalation and oral studies during the last 10 years at JBRC are similar to each other and similar to those reported in other studies. This is the first report on the incidence of spontaneous tumors in inhalation studies and contributes to the toxicological evaluation of studies using F344/DuCrlCrlj rats.

N,N-Dimethylformamide (DMF), a ubiquitous contaminant in living and working environments, enters the human body by inhalation, as well as by oral and dermal routes of exposure. In order to provide bioassay data for carcinogenic risk assessment of humans exposed to DMF by multiple routes of exposure, hepatocarcinogenic effect of combined inhalation and oral exposures of rats to DMF was examined. A group of 50 male F344 rats, 6-week-old, was exposed by inhalation to 0 (clean air), 200, or 400 ppm (v/v) of DMF vapor-containing air for 6 hr/day and 5 days/week during a 104-week period, and each inhalation group was given ad libitum DMF-formulated drinking water at 0, 800 or 1,600 ppm (w/w) for 104 weeks. Incidences of hepatocellular adenomas and carcinomas and their combined incidences were significantly increased in the combined-exposure groups compared with the untreated control group or each of the inhalation-alone and oral-alone groups with matching concentrations. Incidences of hepatocellular adenomas and carcinomas induced by the combined exposures were greater than the sum of the two incidences of the hepatocellular adenomas and carcinomas induced by the single-route exposures through inhalation and ingestion. The combined exposures enhanced tumor malignancy. It was concluded that the combined inhalation and oral exposures markedly enhance the incidences and malignancy of hepatocellular tumors, suggesting that the hepatocarcinogenic effect of the combined exposures is greater than the effect that would be expected under the assumption that the two effects of single-route exposures through inhalation and drinking are additive.

Subchronic oral toxicity of 1,4-dioxane was examined by administering 1,4-dioxane in drinking water at 6 different concentrations of 0 (control), 640, 1,600, 4,000, 10,000 or 25,000 ppm (wt/wt) to F344 rats and BDF(1)mice of both sexes for 13 weeks. Food and water consumption and terminal body weight were decreased dose-dependently in rats and mice. A dose-dependent increase in the relative weights of kidney and lung was noted in rats and mice, while the relative liver weight was increased only in rats. Increases in plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and a decrease in plasma glucose were noted primarily in the rats and mice dosed 25,000 ppm. Histopathological examination revealed that 1,4-dioxane affected the upper and lower respiratory tracts, liver, kidneys and brain in rats, while only the former two organs were affected in mice. Nuclear enlargement occurred in the respiratory, olfactory, tracheal and bronchial epithelia of the 1,4-dioxane-dosed rats and mice. The 1,4-dioxane-induced hepatic lesions were characterized by centrilobular swelling and necrosis in rats and mice and by glutathione S-transferase placental form (GST-P)-positive altered hepatocellular foci in rats, which are known as preneoplastic lesions. A no-observed-adverse-effect-level (NOAEL) was determined at 640 ppm for both rats and mice, since the nuclear enlargement in the nasal respiratory epithelium and the centrilobular swelling of hepatocytes in rats and the nuclear enlargement in the bronchial epithelium in mice were observed at 1,600 ppm. The NOAEL value corresponded to the estimated 1,4-dioxane intake of 52 mg/kg/day in rats and 170 mg/kg/day in mice.

Carcinogenicity and chronic toxicity of ortho-chloronitrobenzene (o-CNB) were examined by feeding groups of 50 F344 rats and 50 BDF(1) mice of both sexes o-CNB-containing diets for 2 years. The dietary concentration of o-CNB was 0, 80, 400 or 2000 ppm (w/w) for rats and 0, 100, 500 or 2500 ppm for mice. The 2-year administration of o-CNB produced a dose-dependent increase in incidences of hepatocellular adenomas and carcinomas in rats and mice of both sexes and hepatoblastomas in mice of both sexes. Incidences of altered cell foci in the liver were increased in the o-CNB-fed rats of both sexes. Metastasis from mouse malignant liver tumors occurred predominantly in the lung. The hepatocarcinogenic response to o-CNB was found to be more potent in mice than in rats. Marginally increased incidences of renal cell adenomas in the 2000 ppm-fed female rats and renal cell carcinomas in the 2000 ppm-fed male rats were noted, together with a significantly increased incidence of atypical tubule hyperplasias. Spontaneous, age-related chronic progressive nephropathy was exacerbated in a dose-related manner, and caused the death of 47 male rats fed 2000 ppm before the end of the 2-year administration period. The highest dose levels of o-CNB except for the administration of 2000 ppm to male rats were thought to meet the criteria of the maximum tolerated dose set by both NCI and IARC guidelines. Causative factors of o-CNB-induced carcinogenicity were discussed with reference to our previous rodent studies of subchronic toxicity of o-CNB and carcinogenicity and chronic toxicity of para-chloronitrobenzene.

Carcinogenicity and chronic toxicity of para-chloronitrobenzene (p-CNB) were examined by feeding diets containing p-CNB to rats and mice of both sexes for two years. The dietary concentration of p-CNB was 0 (control), 40, 200, or 1000 ppm (w/w) for rats and 0, 125, 500, or 2000 ppm for mice. Survival rates of the high-dosed male rats and male mice were significantly decreased compared with those of the respective controls, and this was attributed to the increased number of cancer deaths. Therefore, the high-dose levels were considered not to exceed the maximum tolerated dose. Significant decreases in red blood cell counts and hematocrit value and an increase in mean corpuscular volume were noted in the p-CNB-fed rats and mice. Nonneoplastic splenic lesions were characterized by capsule hyperplasia, fibrosis, fatty metamorphosis, and increased extramedullary hematopoiesis in rats, and congestion, increased extramedullary hematopoiesis, hemosiderin deposition, and ossification in mice. Incidences of fibromas, fibrosarcomas, osteosarcomas, sarcomas (NOS), and hemangiosarcomas in males and fibrosarcomas in females were significantly increased in the spleen of high-dosed rats. The most frequently observed splenic tumor was fibrosarcomas, followed by fibromas. The tumor incidences were increased in a dose-related manner and were more prevalent in males than in females. The malignant tumors metastasized mainly to the liver, peritoneum, and pancreas. Adrena/medullary hyperplasia and pheochromocytomas were significantly increased in the p-CNB-fed females. No tumor was induced in any of the p-CNB-fed mice of either sex except hepatic hemangiosarcomas in the 2000 ppm-fed females. Causative factors of p-CNB-induced carcinogenicity and chronic toxicity are discussed in light of the subchronic and chronic hematotoxicity reported in our present and previous studies and in the literature.

Carcinogenicity and chronic toxicity of N,N-Dimethylformamide (DMF) were examined by inhalation exposure of groups of 50 rats and 50 mice of both sexes to DMF vapor at a concentration of 0, 200, 400 or 800 ppm (v/v) for 6 h/d, 5 d/wk, for 104 wk. In rats, incidences of hepatocellular adenomas and carcinomas significantly increased in the 400 and 800 ppm-exposed groups and in the 800 ppm-exposed group, respectively. The hepatocellular adenoma did not increase significantly in the 400 ppm-exposed female rats, but its incidence exceeded a range of historical control data in the Japan Bioassay Research Center (JBRC). In mice, incidences of hepatocellular adenomas and carcinomas significantly increased in all the DMF-exposed groups. Incidence of hepatoblastomas significantly increased in the 200 and 400 ppm-exposed male mice, and 4 cases of hepatoblastomas in the 400 ppm-exposed female mice and the 800 ppm-exposed male mice exceeded the range of historical control data of the JBRC. Incidences of altered cell foci increased in the liver of exposed rats and mice in an exposure concentration-related manner, and those foci were causally related to the hepatocellular tumors. Liver weights increased in both rats and mice exposed to DMF at 200 ppm and above. Increased levels of gamma-GTP, ALT, AST and total bilirubin in exposed rats of both sexes and AST and ALT in exposed mice of both sexes were noted. It was concluded that 2-yr inhalation exposure to DMF increased incidences of hepatocellular adenomas and carcinomas in rats and incidences of hepatocellular adenomas, carcinomas and hepatoblastomas in mice, and that hepatocarcinogenicity of DMF was more potent in mice than in rats.

The unevenness of pulmonary nanoparticle (NP) distribution, which hinders the establishment of an absolute dose-response relationship, has been described as one of the limitations of intratracheal administration techniques for toxicological assessment of inhaled NPs. Quantification of the NP microdistribution would facilitate the establishment of a concentration-response relationship in localized regions of the lung; however, such quantitative methods have not been reported. Here, we established a quantitative method for evaluating pulmonary TiO2 NP microdistribution in rats using X-ray fluorescence microscopy. Ti intensity in lung sections from rats intratracheally administered 10 mg kg(-1) TiO2 NPs with a microsprayer was measured using X-ray fluorescence with a 100 µm beam size. Ti reference samples were prepared by dropping different concentrations of Ti solutions on glass slide or lung sections of untreated rat. Ti intensity increased linearly with Ti content in the reference samples on both substrates. The detection limit of TiO2 was estimated to be 6.3 ng mm(-2) . The reproducibility was confirmed for measurements done in the short- (2 weeks) and long-term (6 months). The quantitative results of TiO2 NP microdistribution suggested that more TiO2 NPs were distributed in the right caudal and accessory lobes, which are located downstream of the administration direction of the NP suspension, and the lower portion of each lobe. The detection rates of TiO2 NPs were 16.6-25.0%, 5.19-15.6%, 28.6-39.2%, 21.4-38.7% and 10.6-23.2% for lung sections from the right cranial, middle, caudal, accessory and left lobes, respectively.

Carcinogenicity and chronic toxicity of 2,4-dichloro-1-nitrobenzene (2,4-DCNB) were examined by dietary administration to F344/DuCrj rats and Crj:BDF(1) mice of both sexes for 2 years. Dietary administration commenced when the animals were 6 weeks old. The dietary concentration of 2,4-DCNB was 0 (control), 750, 1,500 and 3,000 ppm (w/w) for male and female rats; 0, 750, 1,500 and 3,000 ppm for male mice; and 0, 1,500, 3,000 and 6,000 ppm for female mice. In rats, there was a dose-dependent and significant induction of renal cell adenomas and carcinomas in both sexes and of preputial glands adenomas in males. In all the 2,4-DCNB-fed groups of both sexes, the incidence of atypical tubular hyperplasia, a pre-neoplastic lesion in the kidney, in the proximal tubule was significantly increased. In mice, there was a dose-dependent and significant induction of hepatocellular adenomas, hepatocellular carcinomas, hepatoblastomas and peritoneal hemangiosarcomas in both sexes. The incidence of acidophilic hepatocellular foci was also significantly increased in female mice. Thus, clear evidence of carcinogenic activity of 2,4-DCNB by 2-year feeding was demonstrated in both rats and mice.

Thirteen-week inhalation toxicity of 1,4-dioxane was examined by repeated inhalation exposure of male and female F344 rats to 0 (control), 100, 200, 400, 800, 1600, 3200, or 6400 ppm (v/v) 1,4-dioxane vapor for 6 h/day and 5 days/wk. All the 6400-ppm-exposed males and females died during the first week. Terminal body weight decreased, and relative weights of liver, kidney, and lung increased. AST increased in the 200 ppm-and 3200-ppm-exposed females, and ALT increased in the 3200-ppm-exposed males and females. Nuclear enlargement of nasal respiratory epithelial cells occurring in the 100-ppm-exposed males and females was the most sensitive, followed by the enlarged nuclei in the olfactory, tracheal, and bronchial epithelia. 1,4-Dioxane-induced liver lesions occurred at higher exposure concentrations than the nasal lesions did, and were characterized by single-cell necrosis and centrilobular swelling of hepatocytes in males and females. Glutathione S-transferase placental form (GST-P) positive liver foci were observed in the 1600-ppm-exposed females and 3200-ppm-exposed males and females, which are known as a preneoplastic lesion in rat hepatocarcinogenesis. Plasma levels of 1,4-dioxane increased linearly with an increase in the concentrations of exposure to 400 ppm and above. The enlarged nuclei in the nasal epithelia and the GST-P-positive liver foci were discussed in light of the possible development of nasal and hepatic tumors by long-term inhalation exposure to 1,4-dioxane. A lowest-observed-adverse-effect level (LOAEL) was determined at 100 ppm for the nasal endpoint in both male and female rats.

Occurrence of glutathione S-transferase placental form (GST-P)-positive hepatocytes was examined, using 15 Wistar rats of both sexes each orally administered 2,3,7,8-tetrabromo-dibenzo-p-dioxin (TBDD) by gavage at a single dose of 0, 10, 30, 100 or 300 microg/kg body weight. Liver tissues were stained with anti-GST-P antibody. Two different types of GST-P-positive hepatocytes were found in the TBDD-dosed rat. One type was of the hepatocytes stained homogeneously with anti-GST-P antibody and clearly distinguishable from the surrounding normal tissue. The foci were composed of 2 to 60 hepatocytes exhibiting morphologically focal and clonal proliferation. The GST-P-positive hepatocellular foci occurred at two higher dose levels and only on Day 36 after the single administration. Another type was of the area occupied by the positively but heterogeneously stained hepatocytes appearing predominantly in the centrilobular region, at lower dose levels and persistently on Day 2 through 36. The stained hepatocytes appeared to be neither focally nor clonally proliferating. Females were more susceptible to formation of the two differently stained hepatocytes than males. It is suggested that the GST-P-positive foci represent an early stage of hepatocarcinogenesis, while the GST-P-positive area is associated with the induction of detoxifying Phase II GSTs.

Carcinogenicity and chronic toxicity of 1,2-dichloroethane (DCE) were examined by inhalation exposure of groups of 50 F344 rats and 50 BDF1 mice of both sexes to DCE vapor or clean air as control for 6 h/d, 5 d/wk and 104 wk. The rats were exposed to 10, 40 or 160 ppm (v/v) DCE, while the mice were exposed to 10, 30 or 90 ppm. The 2-yr exposure to DCE produced a dose-dependent increase in incidences of benign and malignant tumors, including subcutaneous fibroma, mammary gland fibroadenoma and peritoneal mesothelioma in male rats; subcutaneous fibroma and mammary gland adenoma, fibroadenoma and adenocarcinoma in female rats; and bronchiolo-alveolar adenoma and carcinoma, endometrial stromal polyp, mammary gland adenocarcinoma and hepatocellular adenoma in female mice. No exposure-related change in the incidence of non-neoplastic lesions or in any hematological, blood biochemical or urinary parameter occurred in any DCE-exposed rat or mouse group. The types of tumors and their target organs found in this study were consistent with those observed in rats and mice administered DCE by gavage in a NCI study. Selection of the exposure concentrations was considered appropriate with reference to the maximum tolerated dose for the highest doses and an occupational exposure limit of DCE for the lowest dose. The present findings suggest that those carcinogenic responses be primarily considered for standard setting of occupational and environmental exposure to DCE.

In order to better characterize the toxicity of N,N-dimethylformamide (DMF) and to provide its basic toxicity data for risk assessment of workers exposed to DMF, F344 rats and BDF1 mice of both sexes were exposed by inhalation (6 h/d x 5 d/wk) to 100, 200, 400, 800 or 1,600 ppm DMF for 2 wk, and 50, 100, 200, 400 or 800 ppm DMF for 13 wk. Three male and 7 female rats died during the 2-wk exposure to 1,600 ppm DMF, but no death of the exposed rats or mice occurred under any other exposure conditions. Massive, focal and single cell necroses were observed in the liver of DMF-exposed rats and mice. The massive necrosis associated with the centrilobular fibrosis occurred at the highest exposure concentration. The single cell necrosis was associated with fragmentation of the nucleoli as well as an increased mitotic figure. The 13-wk exposures of rats and mice to DMF were characterized by increases in the relative liver weight and the incidence of the centrilobular hepatocellular hypertrophy as well as increased serum levels of AST, ALT, LDH, total cholesterol and phospholipid. Lower confidence limits of the benchmark dose yielding the response with a 10% extra risk (BMDL10) were determined for the relative liver weight and the incidence of hepatocellular hypertrophy of the 13-wk exposed animals. The BMDL10 resulted in 1 ppm for the increased relative liver weight of male rats and mice and 17 ppm for the hepatocellular hypertrophy of male mice.

Background Previously, we have developed and reported the method of measuring multi-walled carbon nanotube (MWCNT) in the lung from rats exposed to MWCNT intratracheally. The present research was performed to improve the analytical method of MWCNT to measure multiple samples in a short period of time. For the xanalysis of MWCNTs from tissues, the existence of carbon black may interfere. Therefore, it was examined whether or not carbon black interfere the determination of MWCNT in the standard solutions. Then, MWCNTs were administered to rats and the MWCNTs were determined in the rats by the new method and the recovery rates and time for determination were calculated. The standard solutions for MWCNTs and carbon black were prepared, and the concentrations in the solutions were determined by HPLC with checking their linearity between the concentrations and signal intensities. The reproducibility of the determination was also checked. Methods The concentrations of MWCMTs in the standard solutions were determined by HPLC with a fluorescent detector. Those of carbon black were also determined using the same method. The MWCNTs were administered to rats intratracheally. The MWCNTs in the lung were determined in a newly modified method including digestion of lung tissues by strong alkali solution and marking MWCNTs by benzo[ghi]perylene. The time for the determinations was recorded and the recovery rate of MWVNTs was calculated. Results MWCNT showed linearity in a range of 0.2 to 1.0 μg/mL. In contrast, carbon black demonstrated a very low slope, showing flat pattern. Regarding the reproducibility of the analysis, the coefficient of variation was lower than 10 %. The analysis of 20 samples were completed in 1.5 h. The recovery rates of MWCNT from the lung of rats receiving intratracheal MWCNT administration were 101 to 102 %. Conclusions The improved method for measuring MWCNT allows an efficient MWCNT quantitation in a short period of time. Also, a small amount of MWCNTs can be measured without influence of carbon black.