Chemical-induced lung tumor in Tg-rasH2 mice: a novel mouse tumor model to assess immune checkpoint inhibitors combined with a chemotherapy drug.

In subcutaneous tumor models, changes in the tumor microenvironment can lead to differences in therapeutic treatment responses between the subcutaneous and parent tumors. Accordingly, we generated a lung carcinogenesis model that combines genetically modified mice (Tg-rasH2 mice) with two-stage chemical carcinogenesis as an alternative to the subcutaneous tumor model. In this model, Tg-rasH2 mice were treated with 1-ethyl-1-nitrosourea, followed by butylhydroxytoluene. Mice developed lung adenomas five weeks after treatment initiation. Subsequently, anti-mouse PD-1 antibody (α-mPD-1) or isotype control was administered intraperitoneally twice a week for 4 weeks. Tumor growth was examined by measuring the relative tumor area in serially sliced lung histopathological specimens. No statistically significant differences were observed in the relative lung tumor areas between treated and control groups. A second experiment then examined the antitumor efficacy of α-mPD-1 combined with gemcitabine in a mouse model. Mice were treated identically as in Experiment 1, except that the treated group received once-weekly intraperitoneal injections of 10 mg/kg gemcitabine. In contrast to Experiment 1, the combined treatment significantly reduced the relative tumor areas in the lungs. This result also resembles that of a phase III clinical trial (ORIENT-12), showing that patients with non-small-cell lung carcinoma benefited from combination treatment with gemcitabine and the anti-human PD-1 antibody sintilimab. Thus, this mouse model could be a feasible means to preclinically evaluate the antitumor efficacy of different immunotherapy and chemotherapy drug combinations.

Effects of administering different vehicles via single intratracheal instillation on responses in the lung and pleural cavity of Crl:CD(SD) rats.

Intratracheal instillation is the introduction of a substance directly into the trachea. Intratracheal instillation has been used to investigate the lung toxicity of several chemicals and requires the suspension or dissolution of test material in a vehicle for even dispersal throughout the lung. Importantly, the toxicities of vehicles used in intratracheal instillation studies are generally considered to be insignificant. Hence, evaluating the influence of different vehicles on the lung due to intratracheal instillation is crucial. We examined the toxic effects of pure water, saline, phosphate buffered saline (PBS), 0.5% Kolliphor® P188 (KP188), 0.1% Tween 20 in saline, and 1.0% BSA in PBS. These vehicles were administered to male Crl:CD(SD) rats by a single intratracheal instillation. On day 3, broncho-alveolar lavage fluid (BALF) from the right lung was collected and processed for cell counting and biochemical analysis, while the left lung was used for histopathological examination. Accumulation of alveolar macrophages was observed in all vehicle-treated groups but was minimal in the group administered saline, somewhat higher in the groups administered pure water, PBS, 0.1% Tween 20, and 1% BSA, and notably higher in the group administered 0.5% KP188. The results from BALF analysis indicated that intratracheal instillation of 0.5% KP188 also induced alveolar damage. Additionally, administering pure water did not appear to cause tissue damage. Eosinophil infiltration in the interstitial regions was histopathologically observed. Altogether, the results of this study are helpful for the selection of appropriate vehicles for use in intratracheal instillation studies.

Lung Tumor Induction by 26-week Dermal Application of 1,2-Dichloroethane in CB6F1-Tg rasH2 Mice.

Short-term alternatives to traditional 2-year carcinogenic studies in rodents are being actively pursued. Recently, a 26-week short-term carcinogenicity study using CB6F1-Tg rasH2@Jcl (rasH2) mice has become a worldwide standard for the evaluation of chemical carcinogenesis. However, an acceptable short-term carcinogenic study model for dermally applied products is still lacking. To investigate the suitability of using the rasH2 mouse to test carcinogenic potential, 1,2-dichloroethane (1,2-DCE) was dermally applied to rasH2 mice: 1,2-DCE is a known carcinogen that causes lung bronchiolo-alveolar adenomas and adenocarcinomas when administered topically, orally, or by inhalation exposure; 1,2-DCE at a dose level of 126 mg/mouse in 200 µl acetone or acetone alone (vehicle control) was applied to the dorsal skin of 10 mice of each sex 3 times a week for 26 weeks. As a positive control, 10 mice of each sex received a single intraperitoneal injection of 75 mg/kg of N-methyl- N-nitrosourea. Bronchiolo-alveolar adenomas and adenocarcinomas were significantly increased in 1,2-DCE-treated rasH2 mice of both sexes, and bronchiolo-alveolar hyperplasias were significantly increased in female mice. Overall, almost all mice of each sex developed adenomas and/or adenocarcinomas with 100% of female rasH2 mice developing bronchiolo-alveolar adenocarcinomas.

Promotion of liver and kidney carcinogenesis by ethyl tertiary-butyl ether (ETBE) in male Wistar rats.

Tumor-promoting effects of ethyl tertiary-butyl ether (ETBE) were investigated in a 2-stage carcinogenesis bioassay with regard to hepatic and renal carcinogenesis in rats. Male 6-week-old Wistar rats were given drinking water containing N-ethyl-N-(2-hydroxyethyl)nitrosamine (EHEN), as an initiator, at a dose of 500 ppm for 2 weeks. Starting one week thereafter, the animals were administered ETBE at dose levels of 0 (control), 100, 300, 500 or 1,000 mg/kg/day by gavage for 19 weeks from week 4 to 22. Necropsy of all rats was performed at week 23, and livers and kidneys were examined histopathologically. Incidences of hepatocellular adenomas, and those of combined hepatocellular adenomas and carcinomas were significantly elevated in rats given 1,000 mg/kg/day ETBE, but not 100‒500 mg/kg/day ETBE, and there was a significant increase in the average numbers of lesions. No significant differences in incidences and average numbers of renal tubule neoplasms were found in rats administered 100‒1,000 mg/kg/day ETBE. However, the average numbers of atypical tubule hyperplasias, considered to be preneoplastic lesions, were significantly increased in rats given ETBE at 1,000 mg/kg/day, but not in rats given 500 mg/kg/day or lower doses. Thus, these results imply that ETBE has hepatic and renal tumor-promoting activities that affect EHEN-induced carcinogenesis in male rats, and the no-observed-effect level is 500 mg/kg/day under the present experimental conditions.

Lack of Hepatocarcinogenicity of Combinations of Low Doses of 2-amino-3, 8-dimethylimidazo[4,5- f ]quinoxaline and Diethylnitrosamine in Rats: Indication for the Existence of a Threshold for Genotoxic Carcinogens.

The purposes of the present study were to evaluate the hepatocarcinogenicity of concurrent treatment of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and diethylnitrosamine (DEN) in rats and to determine whether no effect levels of combinations of these two different structural categories of genotoxic hepatocarcinogens exist. Two 16-week rat hepatocarcinogenesis assays were performed using a total of 790 male F344 rats. In experiment 1, we evaluated the effects of concurrent treatment of a subcarcinogenic dose of DEN on rat hepatocarcinogenesis induced by various doses of MeIQx. In experiment 2, we determined hepatocarcinogenicities of combinations of MeIQx and DEN at subcarcinogenic doses, low carcinogenic doses and high carcinogenic doses. Quantitative analyses of glutathione S-transferase placental form (GST-P)-positive foci, a preneoplastic lesion of the liver in rats, revealed that concurrent treatment with subcarcinogenic doses of DEN did not enhance MeIQx-induced rat hepatocarcinogenicity. We also found that concurrent treatment with combinations of subcarcinogenic doses of DEN and MeIQx was not hepatocarcinogenic, indicating that the combined effects of subcarcinogenic doses of DEN and MeIQx were neither additive nor synergistic. Moreover, concurrent treatment with low carcinogenic doses of these 2 carcinogens did not show additive or synergistic effects. Synergetic effects were observed only in rats coadministered high carcinogenic doses of the 2 carcinogens. These results demonstrate the existence of no effect levels of combinations of these 2 genotoxic hepatocarcinogens, and provide new evidence supporting our idea that there is a threshold, at least a practical threshold, that should be considered when evaluating the risk of genotoxic carcinogens. ( ; : -).

Arachidonate-enriched triglyceride oil does not promote tumor development in a rat medium-term multi-organ carcinogenesis model.

The modifying potential on tumor development of arachidonate-enriched triglyceride oil (ARA-oil) containing approximately 40% arachidonic acid was investigated in a medium-term multi-organ carcinogenesis bioassay using male and female F344 rats. The animals were sequentially given five carcinogens with different target sites in the first 4 weeks, and then administered ARA-oil for 24 weeks at dietary levels of 0% (control), 1.25%, 2.5% or 5.0%. No statistically significant differences in incidences and multiplicities of hyperplastic and neoplastic lesions were showed in the large intestine in either sex. In the liver, kidney, and lung in both sexes, and the mammary gland and uterus in females, tumor promoting potential was not evident with ARA-oil treatment. ARA-oil did not affect the quantitative data for glutathione S-transferase placental form positive foci of the liver. Increased induction of hyperplastic or neoplastic lesions in the urinary bladder and thyroid in ARA-oil-treated groups was without dose dependence. In addition, a second experiment with ARA-oil only administration for 8-week revealed no effects on cellular proliferation in the urinary bladder or thyroid in either sex. These results indicate that ARA-oil has no tumor promoting potential in any organs or tissues initiated with the five carcinogens applied in the present study.

Medium-term multi-organ carcinogenesis bioassay of ethyl tertiary-butyl ether in rats.

The modifying potential of ethyl tertiary-butyl ether (ETBE) on tumor development was investigated in a medium-term multi-organ carcinogenesis bioassay using male F344 rats. Animals were sequentially given 5 carcinogens with different target sites in the first 4 weeks for multi-organ initiation. After one week they received ETBE by gavage at dose levels of 0 (control), 300 or 1000mg/kg/day until experimental week 28. Further groups were also given ETBE at doses of 0 or 1000mg/kg/day without prior carcinogen application. Incidences and multiplicities of follicular cell hyperplasias and neoplasms in the thyroid were significantly increased at dose levels of more than 300mg/kg/day. Combined incidences of squamous cell hyperplasias and papillomas of the forestomach were also significantly increased at 300 and 1000mg/kg/day. Incidences and multiplicities of adenocarcinomas in the colon were increased at 1000mg/kg/day. The numbers and areas of glutathione S-transferase placental form (GST-P) positive foci per unit area of the liver sections, and the incidence of hepatocellular adenomas were also significantly increased at 1000mg/kg/day, along with multiplicities of atypical hyperplasias of renal tubules of the kidney and the incidence of papillomatosis of the urinary bladder. This latter lesion was also seen at low incidence at 1000mg/kg/day without initiation. Thus, the current results indicate that ETBE has tumor promoting potential for the thyroid and forestomach at dose levels of 300mg/kg/day and more, and for the colon, liver, kidney and urinary bladder at 1000mg/kg/day, under the present experimental conditions.

Concordance between Results of Medium-term Liver Carcinogenesis Bioassays and Long-term Findings for Carcinogenic 2-Nitropropane and Non-carcinogenic1-Nitropropane in F344 Rats.

This study was conducted to determine the concordance of results for a pair of structural isomers, 2-nitropropane (2-NP) and 1-nitropropane (1-NP), using the rat medium-term liver carcinogenesis bioassay (Ito test) and previously published long-term carcinogenicity tests. Male F344 rats were given a single intraperitoneal injection of DEN (200 mg/kg b.w.) to initiate hepatocarcinogenesis. After 2 weeks, they received per os 0, 0.8, 4 or 20 mg/kg/day of 2-NP or 1-NP six times a week and were subjected to two-thirds partial hepatectomy at week 3. Non-initiated groups receiving 0 or 20 mg/kg/day were also included. The animals were sacrificed for quantitative analysis of GST-P-positive foci at week 8. With the highest dose of 2-NP, significantly increased numbers and areas of GST-P-positive foci were demonstrated as compared with the respective control but were not noted with 1-NP. In the non-DEN-initiated groups, many small GST-P-positive foci of less than 0.2 mm in diameter were also induced in the rats treated with 2-NP at 20 mg/kg/day but were lacking with 1-NP. These results strongly support that 2-NP is a complete hepatocarcinogen with a potent initiation activity, whereas 1-NP is not.

Ninety-day oral toxicity study of rhamsan gum, a natural food thickener produced from Sphingomonas ATCC 31961, in Crl:CD(SD)IGS rats.

This study was designed to evaluate and characterize any subchronic toxicity of rhamsan gum, a polysaccharide produced from Sphingomonas strain ATCC 31961, when administered to both sexes of Crl:CD(SD)IGS rats at dietary levels of 0 (control), 0.5, 1.5, and 5.0% (10 rats/sex/group). During the study, the treatment had no adverse effects on clinical signs, survival, body weights and food and water consumption, or on findings of urinalysis, ophthalmology, hematology, or blood biochemistry. Examination of gross pathology and histopathology exhibited no differences of toxicological significance between control and treated rats. Increased relative cecum (filled) and cecum (empty) weights, evident in males of 1.5% group and both sexes of the 5.0% group, were considered to be a physiological adaptation. Thus, the results indicated the toxic level of rhamsan gum to be more than 5.0%, and the no-observed-adverse-effect level (NOAEL) was concluded to be 5.0% (3,362 mg/kg body weights/day for males, and 4,304 mg/kg body weights/day for males) from the present study.

Carcinogenesis risk assessment of chemicals using medium-term carcinogenesis bioassays.

There is a pressing need for medium term models as alternatives for two year testing of environmental compounds for carcinogenicity and toxicity. Optimally these should be of short duration in vivo, readily performed in the laboratory without the need for specialist equipment, be based on a priori reasoning and scientific principles and use effective surrogates for malignancies. The two models developed in DIMS Institute of Medical Science, the medium-term liver carcinogenesis bioassay and the medium-term multi-organ carcinogenesis bioassay, fulfil these criteria and have the massive advantage of already being used for testing of large numbers of agents.

A 28-day oral toxicity study of fermentation-derived cellulose, produced by Acetobacter aceti subspecies xylinum, in F344 rats.

This study was designed to evaluate any adverse effect of fermentation-derived cellulose, produced by Acetobacter aceti subspecies xylinum, when administered to both sexes of F344 rats at dietary levels of 0, 1.25, 2.5, and 5.0% for 28 days. The treatment had no adverse effects on clinical signs, mortality, body weights and food and water consumption, or on urinalysis, ophthalmology, hematology, blood biochemistry, and histopathology findings. At necropsy, slight increased absolute and relative cecum weights, evident in females ingesting 2.5% and 5.0% dietary levels, were considered to be a physiological adaptation to the poorly absorbed fermentation-derived cellulose. The non-observed-adverse-effect level (NOAEL) from the present study was concluded to be 5.0% in the diet (5,331 mg/kg body weights/day for males, and 5,230 mg/kg body weights/day for females).

Lack of adverse effects of whole-body exposure to a mobile telecommunication electromagnetic field on the rat fetus.

Abstract The recent steep increase in the number of users of cellular phones is resulting in marked increase of exposure of humans to radiofrequency electromagnetic fields (EMFs). Children are of particular concern. Our goal was to evaluate potential adverse effects of long-term whole-body exposure to EMFs simulating those from base stations for cellular phone communication. Pregnant rats were given low, high or no exposure. At the high level, the average specific absorption rate (SAR)for the dams was 0.066-0.093 W/kg. The SAR for the fetuses and the F(1) progeny was 0.068-0.146 W/kg. At the low level, the SARs were about 43% of these. The 2.14 GHz signals were applied for 20 h per day during the gestation and lactation periods. No abnormal findings were observed in either the dams or the F(1) generation exposed to the EMF or in the F(2) offspring. Parameters evaluated included growth, gestational condition and organ weights for dams and survival rates, development, growth, physical and functional development, hormonal status, memory function and reproductive ability of the F(1) offspring (at 10 weeks of age) along with embryotoxicity and teratogenicity in the F(2) rats. Thus, under our experimental conditions, whole-body exposure to 2.14 GHz for 20 h per day during gestation and lactation did not cause any adverse effects on pregnancy or the development of rats.

A twenty eight-day repeated dose toxicity study of black soybean extract in Sprague-Dawley rats.

This study was designed to evaluate any adverse effect of a hot water extract of black soybeans (Glycine max (L.) Merr.), when administered to both sexes of Crj:CD(SD)IGS rats at dietary levels of 0 (control), 0.5, 1.5 and 5.0% (6 rats/sex/group). During the study, the treatment had no adverse effects on clinical signs, survival, body weights, and food and water consumption, or on findings of ophthalmology, urinalysis, hematology, or blood biochemistry. Organ weights, gross pathology and histopathology exhibited no differences of toxicological significance between control and treated rats. Thus, the no-observed-adverse-effect level (NOAEL) of black soybean extract was concluded to be 5.0% (3,618 mg/kg body weight/day for males and 4,066 mg/kg body weight/day for females) from the present study.

Involvement of macrophage inflammatory protein 1alpha (MIP1alpha) in promotion of rat lung and mammary carcinogenic activity of nanoscale titanium dioxide particles administered by intra-pulmonary spraying.

Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.

A medium-term, rapid rat bioassay model for the detection of carcinogenic potential of chemicals.

The Ito Liver Model and the Ito Multi-organ Model are used in conjunction and constitute an efficient and rapid bioassay for the identification of both genotoxic and nongenotoxic carcinogenic chemicals. The Ito Liver Model is an 8-week bioassay system that uses the number and size of foci of hepatocytes positive for glutathione S-transferase placental form (GST-P) as the end-point marker. One hundred fifty-nine compounds were tested using the Ito Liver Model: 61 of 66 hepatocarcinogens tested positive, and 10 of 43 nonliver carcinogens were also positive. The false-positive detection of noncarcinogens was low; a single false-positive result was obtained from the 50 noncarcinogens tested. Since more than half of all known carcinogens are hepatocarcinogens in rodents, the initial 8-week bioassay is able to detect most carcinogens. The Ito Multi-organ Model is a 28-week bioassay system for the detection of carcinogens that were not identified by the Ito Liver Model. Results are evaluated by preneoplastic and neoplastic lesions in major organs. Forty-four compounds were tested using the Ito Multi-organ Model: 17 out of 17 liver carcinogens were positive, and 19 out of 22 (86%) nonliver carcinogens were positive. None of the five noncarcinogens tested positive.

Effects of gestational exposure to 1.95-GHz W-CDMA signals for IMT-2000 cellular phones: Lack of embryotoxicity and teratogenicity in rats.

The present study was designed to evaluate whether gestational exposure to an EMF targeting the head region, similar to that from cellular phones, might affect embryogenesis in rats. A 1.95-GHz wide-band code division multiple access (W-CDMA) signal, which is one applied for the International Mobile Telecommunication 2000 (IMT-2000) system and used for the freedom of mobile multimedia access (FOMA), was employed for exposure to the heads of four groups of pregnant CD(SD) IGS rats (20 per group) for gestational days 7-17. The exposure was performed for 90 min/day in the morning. The spatial average specific absorption rate (SAR) for individual brains was designed to be 0.67 and 2.0 W/kg with peak brain SARs of 3.1 and 7.0 W/kg for low (group 3) and high (group 4) exposures, respectively, and a whole-body average SAR less than 0.4 W/kg so as not to cause thermal effects due to temperature elevation. Control and sham exposure groups were also included. At gestational day 20, all dams were killed and fetuses were taken out by cesarean section. There were no differences in maternal body weight gain. No adverse effects of EMF exposure were observed on any reproductive and embryotoxic parameters such as number of live (243-271 fetuses), dead or resorbed embryos, placental weights, sex ratios, weights or external, visceral or skeletal abnormalities of live fetuses.

Lack of chronic oral toxicity of chemopreventive bovine lactoferrin in F344/DuCrj rats.

Studies were undertaken to determine whether bovine lactoferrin (bLF) and related compounds, shown to prevent carcinogenesis in the colon and other organs in rats, have any toxic effects in long-term feeding studies. In experiment I, male F344/DuCrj rats received a basal diet containing 0.2% bLF for 40 weeks. No adverse findings were noted, furthermore, serum triglyceride level was significantly decreased to 72% of the control level, suggesting preventive effects against the metabolic syndrome. In experiment II, male and female F344/DuCrj rats were fed a basal diet containing 0.02, 0.2, 2.0 and 5.0% bLF, 2.0% bLF hydrolysate (bLF-H) or 0.1% lactoferricin (LFcin), a peptide derived from bLF, for 60 weeks in males and 65 weeks in females. No toxicological effects, including carcinogenicity, were evident in either sex. The results of the studies provide subjective support for safety of clinical studies of bLF for supplement use.

Modifying effects of chitin, chitosan and their related compounds on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in a rat medium-term hepatocarcinogenesis model, and their post-initiation effects in a female rat 2-stage multi-organ carcinogenesis model.

The modifying effects of chitin, chitosan, chitin-oligo sugar, chitosan-oligo sugar and chlorophyllin-chitosan on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated in a rat medium-term hepatocarcinogenesis model. Male F344 rats were injected with diethylnitrosamine (DEN) and starting 2 weeks later, received 0.03% MeIQx alone, MeIQx plus each chemical (0.4%), or each chemical alone (0.1%) in diet for 6 weeks. Three weeks after the DEN injection, animals were subjected to 2/3 partial hepatectomy. The numbers and areas of glutathione S-transferase placental form (GST-P) positive foci given MeIQx plus test chemicals were similar to the MeIQx alone values. In a second experiment, post-initiation effects of chitin and chitosan on major organs were examined in female F344 rats after initiation with 1,2-dimethylhydrazine (DMH), 7,12-dimethylbenz[a]anthracene (DMBA) and 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN). In rats fed a diet containing 1.0% chitin for 36 weeks, the development of palpable mammary tumors tended to be delayed and the final incidence and multiplicity of adenocarcinomas, were significantly lowered. However, in the colon, the number of aberrant crypt foci (ACF) in the chitin and chitosan groups was significantly increased. These results indicate that chitin, chitosan and related compounds do not exert unequivocal chemopreventive effects on heterocyclic amine-induced hepatocarcinogenesis, and that effects in other organs may be tissue specific with possible inhibitory action in the mammary gland being offset by promotion of colon lesion development.

A 90-day oral toxicity study of purple corn color, a natural food colorant, in F344 rats.

A subchronic oral toxicity study of purple corn color (PCC), a natural food colorant, was performed with groups of 10 male and 10 female F344 rats fed the agent at dietary levels of 0%, 0.5%, 1.5% and 5.0% for 90 days. No mortalities occurred during the treatment period. No treatment-related changes in the body weight, food and water consumption, ophthalmology, hematology, organ weight data and histopathology were observed. Regarding general conditions and gross pathology, staining of fur and black feces were noted in rats of the 1.5% and 5.0% diet groups. Moreover, brown urine and black material in the stomach, small and large intestine were evident in rats receiving 5.0%. These changes were considered due to the anthocyanin content. On clinical chemistry analysis, total cholesterol, phospholipid and triglyceride were significantly lowered in both sexes of the 5.0% group, but these were not considered to be toxicologically significant. Thus, the No-observed-adverse-effect-level (NOAEL) was judged to be 5.0% in diet for both sexes (male: 3542 mg/kg/day, female: 3849 mg/kg/day) for PCC under the present experimental conditions.

No enhancing effects of diacylglycerol oil on tumor development in a medium-term multi-organ carcinogenesis bioassay using male F344 rats.

The modifying potential of diacylglycerol (DAG) oil on tumor development was investigated in a medium-term multi-organ carcinogenesis bioassay. DAG oil is a cooking oil that contains >80% diglycerides, <20% triglycerides and <5% monoglycerides. Male 6-week-old F344 rats (20 in each group) were sequentially treated with five carcinogens for initiation in different organ target sites for 4 weeks (DMBDD treatment), and then administered DAG oil at dietary levels of 0% (control), 1.375%, 2.75% or 5.5% [triacylglycerol (TGs), with the same fatty acid composition as DAG oil were also added at dietary levels of 5.5%, 4.125%, 2.75% and 0%, respectively, to maintain the same lipid level], or 5.5% high linoleic acid TG (HLTG), 5.5% high oleic acid TG (HOTG), or 5.5% medium-chain TG (MCTG) (as reference substances, mostly consisting of triacylglycerols) admixed into AIN-93G semi-synthetic diet, for an additional 24 weeks. Controls received standard diet without any supplementation as non-treated control. All animals were killed at the end of week 28, and the major organs were carefully examined for preneoplastic and neoplastic lesions. No DAG oil treatment-related changes were noted in survival, general conditions, body weights, food consumption and organ weights. Upon quantitative analysis of glutathione S-transferase placental form (GST-P) positive foci of the liver, DAG oil was not found to exert any effects. The incidence of colon adenomas was significantly increased in rats given 1.375% DAG oil, but not 2.75% and 5.5% DAG oil, when compared to the control (5.5% TG group) value. Furthermore, incidences and multiplicity of hyperplasias and adenomas and/or adenocarcinomas were comparable across all DAG oil-treated groups. In contrast, incidences of colon adenomas and/or adenocarcinomas were significantly increased in rats given 5.5% HOTG, and adenomas with MCTG, but not 5.5% HLTG, as compared to the 5.5% TG value. Preneoplastic and neoplastic lesions induced by DMBDD treatment in various organs other than the large intestine were comparable in all cases. Thus, the current results indicate that DAG oil may not exert modifying potential on tumor development, even in the colon because of the lack of dose-dependence. DAG oil was equivalent to HOTG (standard cocking oil composed of naturally occurring fatty acids), with regard to colon tumor development. Further dose-response study concerning HOTG may be needed to confirm whether the enhancing effect of large intestine carcinogenesis exert or not.