In vitro chromosomal aberrations induced by various shapes of multi-walled carbon nanotubes (MWCNTs).

OBJECTIVES: IARC has classified one type of multi-walled carbon nanotubes (MWCNTs), MWNT-7, as possibly carcinogenic to humans (Group 2B); however, other types of MWCNT were categorized as not classifiable as to their carcinogenicity to humans (Group 3). In vitro chromosomal aberration assays of MWNT-7 showed polyploid formation but not structural abnormalities. This study investigated the influence of the shape and size of MWCNT on in vitro induction of chromosomal aberrations. METHODS: Microscopic analysis and viable cell counting were used to assay for chromosomal aberrations and cytotoxicity induced in a Chinese hamster lung cell line (CHL/IU) exposed to different MWCNTs. RESULTS: Using scanning electron microscopy, seven MWCNTs were classified into three types: straight fibrous, curved fibrous, and tangled. The straight fibrous MWCNTs were the strongest inducers of polyploidy and the most cytotoxic among the three types of MWCNTs. The curved fibrous MWCNTs induced more polyploidy than the tangled MWCNTs, and the cytotoxicity of both types seemed to be a reflection of their induction of polyploidy. None of the seven MWCNTs induced structural chromosomal aberrations. CONCLUSION: The non-clastogenicity of the MWCNTs indicates that the MWCNTs may not interact directly with DNA. Since the straight fibrous MWCNTs, which exhibit a structure similar to asbestos, were the strongest inducers of polyploidy, MWCNT shape may be an important factor in induction of polyploidy. We hypothesize that CHL/IU cells endocytosed MWCNTs and formed endosomes with shapes corresponding to those of the endocytosed MWCNTs, and that the long axis diameter of the endosome is important in the capability of MWCNTs to induce polyploidy.

Carcinogenicity and chronic toxicity of hydrazine monohydrate in rats and mice by two-year drinking water treatment.

The carcinogenicity and chronic toxicity of hydrazine monohydrate was examined by administrating hydrazine monohydrate in drinking water to groups of 50 F344/DuCrj rats and 50 Crj:BDF1 mice of both sexes for two years. The drinking water concentration of hydrazine monohydrate was 0, 20, 40 or 80 ppm (wt/wt) for male and female rats and male mice; and 0, 40, 80 or 160 ppm for female mice. Survival rates of each group of males and females rats and mice were similar to the respective controls, except female rats administered 80 ppm. Two-year administration of hydrazine monohydrate produced an increase in the incidences of hepatocellular adenomas and carcinomas in rats of both sexes along with hepatic foci. In mice, the incidences of hepatocellular adenomas and carcinomas were increased in females, and significantly increased incidences of hepatocellular adenomas in females administered 160 ppm were observed. Thus, hydrazine monohydrate is carcinogenic in two species, rats and mice. Additionally, non-neoplastic renal lesions in rats and mice and non-neoplastic nasal lesions in mice were observed.

[Background data of spontaneous tumors in F344/DuCrlCrlj rats].

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.

Spontaneous harderian gland adenocarcinoma in a female f344 rat: a case report.

Harderian gland tumors are extremely rare in female F344 rats. An expansive enlarging lesion of the Harderian gland with compression, distortion and invasion of the surrounding muscle was found in a 110-week-old female F344/DuCrj rat, which was diagnosed as a Harderian gland adenocarcinoma. Epithelial growth patterns such as glandular, lobular, papillary and duct forming patterns were exhibited in most areas of the tumor. The tumor cells were pleomorphic and atypical. In one part of the tumor, poorly differentiated areas were found. This case was observed in the middle dose group of a carcinogenicity study of diphenylamine, which was not carcinogenic, we determine to be this case was a spontaneous tumor.

Development of a new multi-walled carbon nanotube (MWCNT) aerosol generation and exposure system and confirmation of suitability for conducting a single-exposure inhalation study of MWCNT in rats.

Because the primary route of human exposure to multi-walled carbon nanotube (MWCNT) is via inhalation, a new dry MWCNT aerosol generation and exposure system for whole-body inhalation exposure using a cyclone and sieve has been developed. The system was tested for operational performance at 0.2, 1 and 5 mg/m(3). Additionally, it was examined whether this system can be employed in animal whole-body inhalation studies by exposing rats to MWCNT aerosol for 6 h at 5 mg/m(3). The system could consistently provide aerosols with a similar particle size distribution and configuration at all the target exposure concentrations. Almost all MWCNTs were fibrous, and the presence of many well-dispersed, nano-sized particles was confirmed. Additionally, the animal study revealed that large amounts of MWCNTs were inhaled into the lung, resulting in an inflammatory response, with increased LDH and albumin levels, and granulomatous change. Therefore, the aerosol generation and exposure system appears useful for MWCNT inhalation studies using rats.

Lack of micronucleus induction activity of ethyl tertiary-butyl ether in the bone marrow of F344 rats by sub-chronic drinking-water treatment, inhalation exposure, or acute intraperitoneal injection.

Ethyl tertiary-butyl ether (ETBE) is an oxygenated gasoline additive synthesized from ethanol and isobutene that is used to reduce CO2 emissions. To support the Kyoto Protocol, the production of ETBE has undergone a marked increase. Previous reports have indicated that exposure to ETBE or methyl tertiary-butyl ether resulted in liver and kidney tumors in rats and/or mice. These reports raise concern about the effects of human exposure being brought about by the increased use of ETBE. The present study was conducted to evaluate the genotoxicity of ETBE using micronucleus induction of polychromatic erythrocytes in the bone marrow of male and female rats treated with ETBE in the drinking-water at concentrations of 0, 1,600, 4,000 or 10,000 ppm or exposed to ETBE vapor at 0, 500, 1,500 or 5,000 ppm for 13 weeks. There were no significant increases in micronucleus induction in either the drinking water-administered or inhalation-administered groups at any concentration of ETBE; although, in both groups red blood cells and hemoglobin concentration were slightly reduced in the peripheral blood in rats administered the highest concentration of ETBE. In addition, two consecutive daily intraperitoneal injections of ETBE at doses of 0, 250, 500 or 1,000 mg/kg did not increase the frequency of micronucleated bone marrow cells in either sex; all rats receiving intraperitoneal injections of ETBE at a dose of 2,000 mg/kg died after treatment day 1. These data suggest that ETBE is not genotoxic in vivo.

Inhalation carcinogenicity of 1,1,1-trichloroethane in rats and mice.

Carcinogenicity of 1,1,1-trichloroethane (TCE) was examined by an inhalation exposure of F344 rats and BDF1 mice of both sexes to TCE at 0, 200, 800 or 3200 ppm for 6 h/d, 5 d/week for 104 weeks. In male rats, the incidences of bronchiolo-alveolar adenomas and peritoneal mesotheliomas were significantly increased in the 800 and 3200 ppm-exposed groups, respectively. The incidence of bronchiolo-alveolar adenomas in the 3200 ppm-exposed groups exceeded the range of historical control data in the Japan Bioassay Research Center. In female rats, the tumor incidences were not increased in any organs of the TCE-exposed groups. In male mice, a significant positive trend with dose was shown for incidences of bronchiolo-alveolar carcinomas, combined incidences of bronchiolo-alveolar adenomas/carcinomas and hepatocellular adenomas. The incidence of Harderian gland adenomas was significantly increased in the 3200 ppm-exposed group, and malignant lymphomas of spleen at this highest dose exceeded the range of historical control data. In female mice, the combined incidence of bronchiolo-alveolar adenomas/carcinomas was significantly increased in the 3200 ppm-exposed group, and the incidences of hepatocellular adenomas and combined incidences of hepatocellular adenomas/carcinomas were significantly increased in the 200, 800 and 3200 ppm-exposed groups with dose dependence except the combined incidence of hepatocellular adenomas/carcinomas in the 200 ppm-exposed group. The incidences of bronchiolo-alveolar adenomas in the 3200 ppm-exposed group and combined incidences of hepatocellular adenomas/carcinomas in the 200 ppm-exposed groups exceeded the ranges of historical control data. Thus, this study provided clear evidence of inhalation carcinogenicity for TCE in both rats and mice.

Hepatotumorigenicity of ethyl tertiary-butyl ether with 2-year inhalation exposure in F344 rats.

Carcinogenicity of ethyl tertiary-butyl ether (ETBE) was examined with inhalation exposure using F344/DuCrlCrlj rats. Groups of 50 male and 50 female rats, 6 week old at commencement, were exposed to ETBE at 0, 500, 1,500 or 5,000 ppm (v/v) in whole-body inhalation chambers for 6 h/day, 5 days/week for 104 weeks. A significant increase in the incidence of hepatocellular adenomas was indicated in males exposed at 5,000 ppm, but not in females at any concentration. In addition, significantly increased incidences of eosinophilic and basophilic cell foci were observed in male rats at 5,000 ppm. Regarding non-neoplastic lesions, rat-specific changes were observed in kidney, with an increase in the severity of chronic progressive nephropathy in both sexes at 5,000 ppm. Increased incidences of urothelial hyperplasia of the pelvis were observed at 1,500 ppm and above, and mineral deposition was apparent in the renal papilla at 5,000 ppm in males. There were no treatment-related histopathological changes observed in any other organs or tissues in either sex. The present 2-year inhalation study demonstrated hepatotumorigenicity of ETBE in male, but not in female rats.

Two-year feed study of carcinogenicity and chronic toxicity of ortho-chloronitrobenzene in rats and 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.

Toxicity due to 2- and 13-wk inhalation exposures of rats and mice to N, N-dimethylformamide.

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.