Japan Bioassay Research Center Research Articles

Carcinogenic effect of potassium octatitanate (POT) fibers in the lung and pleura of male Fischer 344 rats after intrapulmonary administration

BackgroundPotassium octatitanate fibers (K2O•8TiO2, POT fibers) are used as an asbestos substitute. Their physical characteristics suggest that respirable POT fibers are likely to be carcinogenic in the lung and pleura. However, previous 2-year inhalation studies reported that respired POT fibers had little or no carcinogenic potential. In the present study ten-week old male F344 rats were left untreated or were administered vehicle, 0.25 or 0.5 mg rutile-type nano TiO2 (r-nTiO2), 0.25 or 0.5 mg POT fibers, or 0.5 mg MWCNT-7 by intra-tracheal intra-pulmonary spraying (TIPS), and then observed for 2 years.ResultsThere were no differences between the r-nTiO2 and control groups. The incidence of bronchiolo-alveolar cell hyperplasia was significantly increased in the groups treated with 0.50 mg POT and 0.50 mg MWCNT-7. The overall incidence of lung tumors, however, was not increased in either the POT or MWCNT-7 treated groups. Notably, the carcinomas that developed in the POT and MWCNT-7 treated rats were accompanied by proliferative fibrous connective tissue while the carcinomas that developed in the untreated rats and the r-nTiO2 treated rats were not (carcinomas did not develop in the vehicle control rats). In addition, the carcinoma that developed in the rat treated with 0.25 mg POT was a squamous cell carcinoma, a tumor that develops spontaneously in about 1 per 1700 rats. The incidence of mesothelial cell hyperplasia was 4/17, 7/16, and 10/14 and the incidence of malignant mesothelioma was 3/17, 1/16, and 2/14 in the 0.25 mg POT, 0.5 mg POT, and MWCNT-7 treated groups, respectively. Neither mesothelial cell hyperplasia nor mesothelioma developed in control rats or the rats treated with r-nTiO2. Since the incidence of spontaneously occurring malignant mesothelioma in rats is extremely low, approximately 1 per 1000 animals (Japan Bioassay Research Center [JBRC] historical control data), the development of multiple malignant mesotheliomas in the POT and MWCNT-7 treated groups was biologically significant.ConclusionThe incidence of pleural mesotheliomas in male F344 rats administered POT fibers and MWCNT-7 was significantly higher than the JBRC historical control data, indicating that the incidence of pleural mesothelioma in the groups administered POT fibers and MWCNT-7 fibers via the airway using TIPS was biologically significant. The incidence of type II epithelial cell hyperplasia and the histology of the carcinomas that developed in the POT treated rats also indicates that respirable POT fibers are highly likely to be carcinogenic in the lungs of male F344 rats.

Chronic inhalation/carcinogenicity studies and their applications to prevent the occupational cancers: A review of recent reports

There are many carcinogens in workplaces that could cause occupational cancers. Compared with acute toxicity, chronic toxicity is a property of a substance that has toxic effects on a living organism when that organism is exposed to the chemicals continuously or repeatedly. Due to the increasing awareness of the importance of chronic toxicity or carcinogenicity studies, this type of research is on the rise, as much information can be gained from investigating occupational cancers. This study involves a critical review of the recent literature relating to chronic-inhalation or carcinogenicity tests that have been reported regarding inhalation exposure in laboratory animals, including rats and mice. Many of the chronic-inhalation/carcinogenicity studies are performed by governmental and for-profit-organizations, such as the International Agency for Research on Cancer (IARC), the US National Toxicology Program (NTP), the Japan Bioassay Research Center (JBRC), and the Battelle Institute, etc. As research leads to the development of novel types of industrial chemicals, there is a growing need to understand and evaluate the health impacts of these new chemicals in terms of occupational cancers; thus it is essential that we undertake proper and informative research and decision-making. Future research is needed to better characterize the toxicity associated with the chronic inhalation of chemicals and to refine the development of toxicity values. The Occupational Safety and Health Research Institute (OSHRI) in Korea Occupational Safety and Health Agency (KOSHA) are attempting to develop a robust system for chronic-inhalation/carcinogenicity studies and our goal is to undertake multiple studies to prevent occupational cancers.

Background data of spontaneous tumors in F344/DuCrlCrlj rats

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. 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. 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. 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.

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.

International symposium on genotoxic and carcinogenic thresholds

New paradigms for the setting of tolerable exposure limits for carcinogenic chemicals are being discussed world wide, and there is a global discourse in this respect between toxicological science, regulatory bodies and stakeholders (Sweeney et al. 2006; Bader et al. 2007; Bolt and HuiciMontagud 2008). The traditionally accepted general dogma that health-based exposure limits cannot be derived for genotoxic carcinogens has been challenged by new research results (Hengstler et al. 2003). Against this background the Japanese National Institute of Health Science, in conjunction with the Japan Bioassay Research Center convened an international symposium in Tokyo, in order to foster the exchange of ideas between leading Japanese, American and European scientists in this Weld, and at the same time to communicate results of this discussion to Japanese authorities and stakeholders (Fig. 1). The main initiators of the symposium in particular, Shoji Fukushima and Takehiko Nohmi (Kanagawa and Tokyo), favoured a concept to diVerentiate “perfect” and “practical” thresholds for genotoxic compounds, as earlier deWned by Kirsch-Volders et al. (2000) and Hengstler et al. (2003). An analysis of Waddell et al. (2006) was cited comparing published data on GSTP-positive hepatic foci induced by N-nitroso-diethylamine. The foci were observed at doses below the threshold dose for the appearance of tumours. These results suggested that adducts and GSTP-positive foci appear at doses below those at which tumours appear. These results were interpreted to support the observations of the authors that thresholds for carcinogenicity of this genotoxin exist, and that adducts and altered foci appear at lower doses than the threshold for carcinogenicity. Potassium bromate (Kurokawa et al. 1990; Gultekin and Hicyilmaz 2007) and 1,4-dioxane (Stickney et al. 2003; Nannelli et al. 2005; Kuroiwa et al. 2007) were mentioned as examples of compounds with a “perfect threshold”. In total, the symposium covered a methodological spectrum reaching from bacterial mutagenicity assays (Nohmi 2000) up to modern transgenic mouse assays (Nohmi et al. 2000). The main focus of the symposium was on ingredients of food, e.g. heterocyclic amines, nitrosamines or mycotoxins (Huang et al. 2006; Schrickx et al. 2006; Nagashima et al. 2007). A controversial point of discussion was the evaluation of acrylamide detected recently in unexpectedly large amounts in starchy foods that had been cooked at high temperatures (Boettcher et al. 2006). This is biologically converted into the genotoxic epoxide glycidamide (Settels et al. 2008). The scientiWc evaluation of possible threshold mechanisms in this case is a matter of debate, so that mostly linear non-threshold risk extrapolation models are preferred. From the area of food toxicology parallels were drawn to the setting of occupational exposure limits and to concepts favoured by European scientiWc bodies, such as EUROTOX (Foth et al. 2005) and SCOEL (Bolt and Huici-Montagud 2008). The symposium revealed that the implementation of novel toxicological concepts within the regulatory context requires an intensive and well-founded dialogue between scientists, authorities and stakeholders, both on national and international levels. It will now be up to the scientists to further promote this dialogue. Key articles resulting from the symposium will be published in “Genes and Environment”. H. M. Bolt (&) Leibniz Research Centre for Working Environment and Human Factors, Institut fur Arbeitsphysiologie an der Universitat Dortmund, Ardeystr. 67, 44139 Dortmund, Germany e-mail: bolt@ifado.de

Effects of Inhalation Exposure to Propylene Oxide on Respiratory Tract, Reproduction and Development in Rats

Nasal, respiratory, reproductive and developmental toxicities of propylene oxide (PO) were examined by exposing male and female Sprague-Dawley rats to PO vapor by inhalation at a concentration of 0 (control), 125, 250, 500 or 1,000 ppm for 6 h/d, 7 d/wk, during a 5- to 6-wk period, including premating, mating and postmating or gestation. The inhalation exposure to 1,000 ppm PO seriously affected parental survival, the upper and lower respiratory tract, male and female reproductive systems, motor function, and fetal survival and development, whereas the exposure to 500 ppm or less primarily caused nasal lesions without any sign of reproductive or developmental toxicity. Because atrophy of the olfactory epithelium in the male rats exposed to 250 ppm was the most sensitive endpoint for PO toxicity, the NOAEL was determined to be 125 ppm for the nasal endpoint. An additional inhalation experiment was carried out to further examine developmental toxicity by exposing pregnant rats to 0, 125, 250, 500, 750 or 1,000 ppm PO during a 2-wk period of gestation, Day 6 through Day 19. The 2-wk inhalation experiment revealed that reduced fetal body weights and delayed ossification occurred in association with significantly reduced body weights of the dams exposed to 750 and 1,000 ppm, whereas neither fetal death nor teratogenicity occurred at those two exposure levels. It was concluded that the developmental toxicity of fetal death was manifested at parentally toxic exposure levels above 500 ppm, a level which seriously affected parental survival, the upper and lower respiratory tracts and reproductive system.

Carcinogenicity of Biphenyl in Mice by Two Years Feeding

Carcinogenicity and chronic toxicity of biphenyl was examined in the male and female BDF1 mice fed a diet containing biphenyl at 667, 2,000 or 6,000 ppm for 2 years. There was no difference in survival rate between any biphenyl-containing diet-fed group of either sex and the respective control. Body weights of the males and females fed 6,000 ppm diet were significantly lower than the respective control. Incidences of hepatocellular carcinomas and hepatocellular adenomas in the females fed diets containing biphenyl were significantly increased in a dose-related manner, and exceeded a range of the historical control data in the Japan Bioassay Research Center. Incidences of basophilic cell foci in the liver were increased in the females fed 2,000 and 6,000 ppm diets. There was no increase in tumor or tumor-related lesion in the males fed diets containing biphenyl. Chronic toxicity of biphenyl was characterized by increased incidences of urothelial desquamation in the renal pelvis in males and females and mineralization in the inner stripe of renal outer medulla in females, as well as changes in serum levels of BUN, ALP and some electrolytes in males and females. In conclusion, the 2-year oral administration of biphenyl-containing diets induced pre-neoplastic and neoplastic lesions in the liver of females and non-neoplastic lesions in the kidney of males and females. Causative factors for the biphenyl-induced hepatocarcinogenicity were discussed in light of our published finding of peroxisome proliferation.

Carcinogenicity and Chronic Toxicity after Inhalation Exposure of Rats and Mice to N,N‐Dimethylformamide

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.

Absence of β<i>-catenin</i> Mutations in Mouse Hepatoblastomas Induced by <i>N</i>, <i>N</i>-dimethylformamide

Abnormal β-catenin protein accumulation in cytoplasm or nucleus with somatic mutation of β-catenin has been reported to be very frequent in hepatoblastomas (HBs) both in humans and in experimental animals. Recently we noted HBs developing in an evaluation of the carcinogenicity of N, N-dimethylformamide (DMF) in a 104 week mouse inhalation study1 performed by the Japan Bioassay Research Center. In the present study we evaluated β-catenin mutations in mouse HBs induced by DMF, which is considered to be a non-genotoxic carcinogen. Immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analyses were performed using formalin-fixed and paraffin-embedded sections. No abnormal β-catenin accumulation or no apparent mutation was detected, and these findings point to functional normality of the Wnt signal pathway in these HBs induced by DMF. Our results raise questions as to the general nature of any relationship between HB and abnormal β-catenin accumulation.

Carcinogenicity and Chronic Toxicity in Rats and Mice Exposed to Chloroform by Inhalation

Carcinogenicity and Chronic Toxicity in Rats and Mice Exposed to Chloroform by Inhalation: Seigo Yamamoto, et al. Japan Bioassay Research Center—A bioassay study of carcinogenicity and chronic toxicity of chloroform was undertaken by inhalation exposures of groups of 50 F344 rats and 50 BDF1 mice of both sexes to chloroform for 6 h/d x 5 d/ wk X 104 wk. The exposure concentration was 0 (control), 10, 30 or 90 ppm for rats and 0, 5, 30 or 90 ppm for mice. Combined incidences of renal cell adenomas and carcinomas, and of hepatocellular adenomas and carcinomas increased in the exposed male and female mice, respectively. Incidences of atypical tubule hyperplasia, cytoplasmic basophilia and nuclear enlargement in the kidneys and fatty change in the liver increased in the exposed male mice. Increased incidence of altered cell foci in the exposed female mice was causally related to the hepatocellular adenomas and carcinomas. No significantly increased incidence of the kidney or liver tumors was observed in the exposed rats of either sex. Increased incidences of nuclear enlargement and dilatation of tubular lumen were found in the kidneys of exposed rats. No‐ observed‐adverse‐effect‐levels (NOAELs) for the biologically significant endpoint were determined from the dose‐response relationships of the present datasets. The NOAEL for the histopathological endpoint of the kidneys resulted in 5 ppm for mice and 10 ppm for rats. An occupational exposure limit for chloroform was discussed in light of the NOAELs.

Acute and Subchronic Inhalation Toxicity of Chloroform in Rats and Mice

Acute and Subchronic Inhalation Toxicity of Chloroform in Rats and Mice: Tatsuya Kasai, et al. Japan Bioassay Research Center—In order to better characterize acute and subchronic toxicities of chloroform and to provide its basic toxicity data for risk assessment of humans exposed to chloroform in work and living environments, mice and rats of both sexes were exposed by inhalation to chloroform at different concentrations from 500 to 8,000 ppm for 6 h/d x 5 d/ wk X 2 wk and from 12 to 400 ppm for 6 h/d x 5 d/wk x 13 wk. The kidneys, liver and nasal cavity were primarily damaged by the inhalation exposure. Acute death occurred at 12 ppm in male mice, 1,000 ppm in female mice and 2,000 ppm in male and female rats. The 13‐wk exposures induced renal lesions in male mice, hepatic and nasal lesions in female mice and renal, hepatic and nasal lesions in male and female rats. Susceptibility to the acute and subchronic toxicities was higher in male mice than female mice, and higher in mice than in rats. No‐observed‐adverse‐ effect‐levels (NOAELs) and lowest‐observed‐adverse‐ effect‐levels (LOAELs) were determined for the renal, hepatic and nasal endpoints of animals exposed to chloroform for 13 wk. For the hepatic endpoint, NOAEL was 100 ppm in male mice, 50 ppm in female mice and female rats and 100 ppm in male rats. For the renal endpoint, LOAEL was 12 ppm in male mice, and NOAEL was 25 ppm in male rats and 100 ppm in female rats. For the nasal endpoint, LOAEL was 12 ppm and 25 ppm in the mice and the rats of both sexes, respectively.

Two‐year Study of Carcinogenicity and Chronic Toxicity of Biphenyl in Rats

Two‐year Study of Carcinogenicity and Chronic Toxicity of Biphenyl in Rats: Yumi Umeda, et al. Japan Bioassay Research Center, Japan Industrial Safety and Health Association— Carcinogenicity and chronic toxicity of biphenyl were examined in 50 male and 50 female F344 rats exposed to 0, 500, 1,500 or 4,500 ppm biphenyl in the diet for 105 weeks. Bladder tumors were found in the 4,500 ppm males, as evidenced by significantly increased incidence of carcinoma (24/50) and papilloma (10/50) of the transitional cells as well as one rarely observed case both of carcinoma and papilloma of the squamous cells. The survival rate of the 4500 ppm males significantly decreased, due to the bladder tumors and the hematuria accompanied by bladder calculi. The bladder calculi were found in 43 males in the 4,500 ppm group, but in only 8 females. Urinary pH significantly increased in the males, and occult blood was observed both in males and females in the 4,500 ppm group. The pre‐neoplastic lesions were hyperplasia of transitional epithelium (simple, nodular and papillary hyperplasia) in the bladder of the 4,500 ppm males. Incidences of calculus formation and transitional cell hyperplasia in the renal pelvis also significantly increased in the 4,500 ppm males and females. On the other hand, the incidences of the transitional cell hyperplasia and the calculus formation in the bladder and the renal pelvis were far lower in females than in males, and no bladder tumors were observed in the females. Causative factors of the bladder tumors and their male predominance were discussed with reference to the findings reported in the literature and the previous study of biphenyl metabolism.

An Exposure System for Combined Administration of an Organic Solvent to Rodents by Inhalation and Water‐Drinking and its Operational Performance

An Exposure System for Combined Administration of an Organic Solvent to Rodents by Inhalation and Water‐Drinking and its Operational Performance: Hirokazu Kano, et al. Japan Bioassay Research Center, Japan Industrial Safety and Health Association—In order to obtain bioassay data for carcinogenicity and chronic toxicity of an organic solvent being absorbed into the body through multiple media by inhalation and ingestion, an exposure system for combined inhalation and oral exposures of rats to the organic solvent was constructed. Operational performance of the system was examined by monitoring air and water concentrations of chloroform or 1,4‐dioxane in both an inhalation exposure chamber and an automatic watering system, while groups of 50 male rats each were exposed to chloroform or 1,4‐dioxane by inhalation for 6 h/d, 5 d/wk and 104 wk and by water drinking for 24 h/d and 7 d/wk throughout the 104 wk. Day‐to‐day and time‐to‐time variations in concentrations in the exposure chamber were maintained at less than 0.8% C.V. and 4.8% C.V., respectively, in the case of chloroform, and less than 1.2% and 8.1 %, respectively, in the case of 1,4‐dioxane. The mean 6‐h averaged concentrations deviated by less than 0.4% from the respective target concentrations for all sets of the combined exposures. The watering system which was composed of a drinking valve, in‐between piping, a container and a solenoid drainage valve was used for giving ad libitum drinking water containing 1000 ppm chloroform or 1,4‐dioxane to rats. Mean water concentrations of chloroform and 1,4‐dioxane were attenuated by 3‐8% and by 1% in the container after 1 ‐week's supply, respectively. It was concluded that this exposure system can give accurate and reproducible air and water concentrations of chloroform and 1,4‐dioxane for long‐term combined inhalation and oral exposure.

A Pathological Study on Spontaneous Hepatic Neoplasms in BDF1 Mice

Neoplastic and proliferative hepatic lesions observed in 500 male and 499 female DBF1 mice used as untreated controls in the past ten 2-year carcinogenicity studies (each 50 males and 50 females for one study) for industrial chemicals at the Japan Bioassay Research Center. The incidences of altered cell foci (ACF) were 11.6% in males and 2.8% in females and those of each type of ACF were as follows; basophilic, eosinophilic, clear, vacuolated, and mixed cell foci were 4.0%, 1.2%, 4.6%, 0.2%, and 1.6% in male mice, respectively, and 1.2%, 0%, 0.8%, 0.4%, and 0.4% in females, hepatocellular adenoma (HCA) was 15% in males (minimum 4%, maximum 30%), and 4.4% in females (minimum 2%, maximum 8%), hepatocellular carcinoma (HCC) was 22.8% (minimum 2%, maximum 36%) in males and 2.0% (minimum 0%, maximum 4%) in females, vascular tumors were 6.6% (minimum 0%, maximum 6%) in male mice and 15.0% (minimum 6%, maximum 20%) in females. Compared with the data in rats, incidences of HCA and HCC in BDF1 mice were remarkably higher, however, the incidence of ACF in BDF1 mice was lower than those of rats. The different incidence of ACF noted in rats and in mice may suggest the different implications of ACF as the preneoplastic role in hepatocarcinogenesis.