Toxicity and carcinogenicity studies of Ginkgo biloba extract in rat and mouse: liver, thyroid, and nose are targets.

Ginkgo biloba extract (GBE) is a popular herbal supplement that is used to improve circulation and brain function. In spite of widespread human exposure to relatively high doses over potentially long periods of time, there is a paucity of data from animal studies regarding the toxicity and carcinogenicity associated with GBE. In order to fill this knowledge gap, 3-month and 2-year toxicity and carcinogenicity studies with GBE administered by oral gavage to B6C3F1/N mice and F344/N rats were performed as part of the National Toxicology Program's Dietary Supplements and Herbal Medicines Initiative. The targets of GBE treatment were the liver, thyroid, and nose. These targets were consistent across exposure period, sex, and species, albeit with varying degrees of effect observed among studies. Key findings included a notably high incidence of hepatoblastomas in male and female mice and evidence of carcinogenic potential in the thyroid gland of both mice and rats. Various nonneoplastic lesions were observed beyond control levels in the liver, thyroid gland, and nose of rats and mice administered GBE. Although these results cannot be directly extrapolated to humans, the findings fill an important data gap in assessing risk associated with GBE use.

Complex histopathologic response in rat kidney to oral ß-myrcene: an unusual dose-related nephrosis and low-dose alpha2u-globulin nephropathy.

Oral gavage studies with ß-myrcene in male F344 rats showed a complex renal pathology comprising both alpha2u-globulin (α2u-g) nephropathy, an unusual nephrosis involving the outer stripe of outer medulla (OSOM), and an increased incidence of renal tubule tumors by 2 years. In the 90-day and 2-year studies, respectively, α2u-g nephropathy and linear papillary mineralization were observed in males at the two lower doses but were absent from the high dose. Nephrosis was characterized by dilation of the S3 tubules, nuclear enlargement (including karyomegaly), and luminal pyknotic cells, all in the outermost OSOM. Nephrosis was minimal at the higher doses in the 90-day study, but progressed to a severe grade in males dosed with 1,000 mg/kg for 2 years. Renal tubule tumors developed in treated groups with incidences up to 30% in the 250 and 500 mg/kg male dose groups. Tumors at the lower doses in males may have been associated with α2u-g nephropathy, while those at higher doses in both sexes may have been due to the nephrosis. Because ß-myrcene induced a complex spectrum of renal pathology, the α2u-g nephropathy mechanism cannot be the sole mechanism of carcinogenesis in these rats.

Liver toxicity and carcinogenicity in F344/N rats and B6C3F1 mice exposed to Kava Kava.

Kava Kava is an herbal supplement used as an alternative to antianxiety drugs. Although some reports suggest an association of Kava Kava with hepatotoxicity , it continues to be used in the United States due to lack of toxicity characterization. In these studies F344/N rats and B6C3F1 mice were administered Kava Kava extract orally by gavage in corn oil for two weeks, thirteen weeks or two years. Results from prechronic studies administered Kava Kava at 0.125 to 2g/kg body weight revealed dose-related increases in liver weights and incidences of hepatocellular hypertrophy. In the chronic studies, there were dose-related increases in the incidences of hepatocellular hypertrophy in rats and mice administered Kava Kava for up to 1g/kg body weight. This was accompanied by significant increases in incidences of centrilobular fatty change. There was no treatment- related increase in carcinogenic activity in the livers of male or female rats in the chronic studies. Male mice showed a significant dose-related increase in the incidence of hepatoblastomas. In female mice, there was a significant increase in the combined incidence of hepatocellular adenoma and carcinoma in the low and mid dose groups but not in the high dose group. These findings were accompanied by several nonneoplastic hepatic lesions.

Fourteen-week toxicity study of green tea extract in rats and mice.

The toxicity of green tea extract (GTE) was evaluated in 14-week gavage studies in male and female F344/NTac rats and B6C3F1 mice at doses up to 1,000 mg/kg. In the rats, no treatment-related mortality was noted. In the mice, treatment-related mortality occurred in male and female mice in the 1,000 mg/kg dose groups. The cause of early deaths was likely related to liver necrosis. Treatment-related histopathological changes were seen in both species in the liver, nose, mesenteric lymph nodes, and thymus. In addition, in mice, changes were seen in the Peyer's patches, spleen, and mandibular lymph nodes. The no adverse effect level (NOAEL) for the liver in both species was 500 mg/kg. In the nose of rats, the NOAEL in males was 62.5 mg/kg, and in females no NOAEL was found. No NOAEL was found in the nose of female or male mice. The changes in the liver and nose were considered primary toxic effects of GTE, while the changes in other organs were considered to be secondary effects. The nose and liver are organs with high metabolic enzyme activity. The increased susceptibility of the nose and liver suggests a role for GTE metabolites in toxicity induction.

Epididymal sperm granuloma induced by chronic administration of 2-methylimidazole in B6C3F1 mice.

Two-year mouse and rat bioassay studies of 2-methylimidazole (2-MI) conducted by the National Toxicology Program revealed that epididymal sperm granuloma(SG)s occurred only in male B6C3F1 mice in a dose-related manner. The present study characterized 2-MI-induced SGs in these epididymides. Groups of 50 male B6C3F1 mice were fed diets containing 0, 625, 1250, or 2500 ppm 2-MI for 105 weeks; the doses were equivalent to average daily doses of approximately 13, 40, or 130 mg/kg. Testes and epididymides were histopathologically reexamined. 2-Methylimidazole increased the incidence of epididymal SGs (0%, 0%, 6%, 12%, respectively). Histologically, most of the SGs exhibited rupture of epididymal ducts with focal aggregations of macrophages in interstitia. Lesions occurred in the proximal caput of the epididymis and/or efferent ducts, not in the corpus and cauda. In the testis, incidences of germinal epithelial atrophy (GEA) increased dose-relatedly (2%, 8%, 16%, 28%, respectively). All mice with epididymal SG developed testicular GEA. The grading scores of testicular GEA tended to be more severe in mice with SGs than those without. No epididymal SG or testicular GEA was observed in 6-month-interim-sacrificed mice. The results imply that 2-year treatment of B6C3F1 mice with 2-MI can induce epididymal SGs, primarily followed by more severe testicular GEA. The potential mechanism of SG induction by 2-MI is discussed.

NTP technical report on the toxicity studies of 1,1,2,2-tetrachloroethane (CAS No. 79-34-5) administered in microcapsules in feed to F344/N rats and B6C3F1 mice.

BACKGROUND: 1,1 ,2,2-Tetrachloroethane was widely used in the production of solvents and pesticides. Its production ended in the 1990s, but it is a major component of waste sites. We studied the effects of 1,1 ,2,2-tetrachloroethane on male and female rats and mice to identify potential toxic hazards to humans. METHODS: Because 1,1,2,2-tetrachloroethane can evaporate easily, we enclosed it in starch microcapsules and placed them in the feed of rats and mice for 14 weeks. Male and female rats received up to 4,600 parts per million (ppm) 1,1 ,2,2-tetrachloroethane (equivalent to 0.46%) and mice received up to 9,100 ppm (0.91%). Control animals received empty starch microcapsules in their feed. Tissues from more than 40 sites were examined in all control and high-dose animals; tissues with lesions were examined in the lower exposure groups until no lesions were observed. RESULTS: Rats receiving 1,180 ppm or more 1,1,2,2-tetrachloroethane and mice receiving 2,300 ppm or more weighed less than the control animals. Male and female rats given 1,1 ,2,2-tetrachloroethane had pale and diseased livers and also had atrophy of the bone marrow and of the genital systems. Male and female mice given 1,1,2,2-tetrachloroethane had lesions of the liver and the bile duct. CONCLUSION: We conclude that 1,1,2,2-tetrachloroethane at doses greater than 590 ppm in the feed was toxic to the liver of male and female rats. In mice, 1,1 ,2,2-tetrachloroethane was already known to cause cancer after long-term exposure. In these 14-week studies, 1,1 ,2,2-tetrachloroethane was toxic to the livers of male and female mice.

Toxicity and carcinogenicity of riddelliine in rats and mice.

These investigations of riddelliine analyzed potential carcinogenesis and the utility of the female-rat/male-mouse design in bioassays and dose-response. Groups of 50 Fischer rats and B6C3F1 mice were gavage-administered riddelliine 5 days per week for 105 weeks. The dose levels for male rats were 0 or 1.0 mg/kg body weight; female rats 0, 0.01, 0.033, 0.1, 0.33, or 1.0 mg/kg; male mice 0, 0.1, 0.3, or 1.0, 3.0 mg/kg; and female mice 0 or 3.0 mg/kg. The dose groups were purposely designed to evaluate the dose-response relationship only in female rats and male mice. In rats, liver hemangiosarcoma, hepatocellular adenoma, and mononuclear cell leukemia were significantly increased in the 1.0 mg/kg male and female dose groups. Non-neoplastic lesions occurred in the liver and kidney of male and female rats. In mice, hemangiosarcomas increased significantly in the liver of males in the 3.0 mg/kg dose group. Alveolar/bronchiolar neoplasms in the 3.0 mg/kg dose group of female mice were significantly increased. Hepatocellular neoplasms were significantly decreased in the 1.0 mg/kg dose group of male and 3.0 mg/kg dose groups of male and female mice. Non-neoplastic lesions occurred in the liver and kidney of male and female, and lung and arteries of female mice. These studies demonstrate toxicity and carcinogenicity of riddelliine in rats and mice, and a dose-response relationship in female rats and male mice under the experimental conditions employed.

Forestomach tumor induction by 2,4-hexadienal in F344N rats and B6C3F1 mice.

2,4-Hexadienal (2,4-Hx) was studied for its toxicity and carcinogenicity because of its alpha, beta-unsaturated aldehyde structure and potential link between exposure to lipid peroxidation products in the diet and human malignancies. Male and female F344N rats and B6C3F1 mice received 2,4-Hx in corn oil by gavage for 16 days, 14 weeks, or 2 years. In the 16-day studies 2,4-Hx induced forestomach necrosis and ulceration at 240 mg/kg and forestomach epithelial hyperplasia at 80 mg/kg in rats and mice. In the 14-week studies the chemical induced forestomach hyperplasia and nasal olfactory atrophy or necrosis at 120 mg/kg in rats and mice. In the 2-year studies 2,4-Hx induced squamous cell papilloma and carcinoma of the forestomach in male and female rats at 45 and 90 mg/kg and in male and female mice at 120 mg/kg. Two male mice in the 120 mg/kg group had uncommon squamous cell carcinoma of the oral cavity (tongue). Mechanistic studies indicated that the forestomach carcinogenesis in rats and mice may be due to depletion of glutathione as a result of oxidative stress induced by 2,4-Hx.

Single administration toxicokinetic studies of decalin (decahydronaphthalene) in rats and mice.

Decalin (decahydronaphthalene) is an industrial solvent known to cause alpha2u-globulin nephropathy in male rats. Studies were conducted using decalin (mixture of cis and trans isomers) to (1) characterize systemic elimination of decalin in rats and mice and (2) evaluate disposition of decalin, its metabolites, and kidney alpha2u-globulin in young and old rats of both sexes following a single 6-h whole-body inhalation exposure at up to 400 ppm decalin. Additionally, a separate group of young male F344/N rats were administered either cis- or trans-decalin iv at doses up to 20 mg/kg to assess disposition of each isomer, its metabolites, and kidney alpha2u-globulin. Decalin was eliminated from blood in a dose-dependent manner, regardless of sex, age, or species. C0 and AUC infinity increased supra-proportionally with exposure concentration. Mice were more efficient in eliminating decalin than rats at lower exposure concentrations, but nonlinear elimination kinetics were more noticeable at 400 ppm. Sex differences in blood decalin elimination were observed in rats; females had a consistently higher AUC infinity at all exposure concentrations. There was a dose-dependent increase in kidney decalin, decalone, and alpha2u-globulin in male rats exposed to decalin. Kidney alpha2u-globulin and decalone concentrations in old male rats were substantially lower than those in young males, but were similar to those observed in all (young and old) females. Compared to old males and all females, young male rats had significantly lower urinary decalol concentrations, but higher kidney decalin, decalone, and alpha2u-globulin concentrations. Administration of decalin to male rats as either the cis or trans isomer revealed that more cis -decalone is produced per unit dose as compared to trans-decalone, and that more trans-decalin accumulated in the kidney (as alpha2u-globulin-ligand complexes) compared to cis-decalin. These patterns of isomer-specific metabolism were also reflected in the cis/trans ratios of decalin in blood, as well as urinary decalol metabolites. The ratio of alpha2u-globulin to the total amount of decalin plus decalone measured in the male rat kidney was approximately 1.0. Therefore, alpha2u-globulin was a key factor in the accumulation of decalin and decalone in kidneys of young male rats, decalin and decalone were practically absent in all females and in old males.

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

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

The hepatic endothelial carcinogen riddelliine induces endothelial apoptosis, mitosis, S phase, and p53 and hepatocytic vascular endothelial growth factor expression after short-term exposure.

Riddelliineis a naturally occurring pyrrolizidine alkaloid found in certain poisonous rangeland plants of the western United States. In National Toxicology Program 2-year studies, riddelliine induced high incidences of hemangiosarcoma in the liver of F344/N rats (both sexes) and B6C3F1 mice (males). To understand this pathogenesis, we tested short-term effects of riddelliine. Three groups (control; 1.0 mg/kg/day, high dose used in the 2-year study; and 2.5 mg/kg/day) of seven male F344/N rats per group were terminated after 8 consecutive doses and 30 doses (6 weeks, excluding weekends). Serum vascular endothelial growth factor (VEGF), histological, immunohistochemical [factor VIII-related antigen/von Willebrand factor (fVIII-ra/vWf)], VEGF, VEGF receptor-2 (VEGFR2), glutathione S-transferase-pi, S-phase (BrdU), p53, apoptosis, and ultrastructural evaluations were performed on the liver. Following 8 doses of 1.0 and 2.5 mg/kg/day, increased numbers of apoptotic and S-phase nuclei appeared in hepatocytes and endothelial cells. Following 30 doses of 1.0 and 2.5 mg/kg/day, hepatocytes exhibited reduced mitosis, fewer S-phase nuclei, increased hypertrophy, and fatty degeneration, while endothelial cells showed karyomegaly, cytomegaly, decreased apoptosis, more S-phase nuclei, and p53 positivity. Hepatocytes of treated animals expressed higher VEGF immunopositivity. That altered endothelial cells were fVIII-ra/vWf and VEGFR2 positive confirmed their identity. These changes may have promoted hemangiosarcoma development upon long-term exposure through endothelial adduct formation, apoptosis, proliferation of endothelial cells having undamaged and/or damaged DNA, and mutation. Endothelial proliferation may also have been promoted through endothelial arrest at S phase, which was associated with endothelial karyo- and cytomegaly, resulting in hepatocytic hypoxia, triggering VEGF induction.

Toxicokinetics of riddelliine, a carcinogenic pyrrolizidine alkaloid, and metabolites in rats and mice.

Riddelliine is a representative pyrrolizidine alkaloid, a class of naturally occurring toxic phytochemicals present in plant species worldwide. Human exposure to pyrrolizidine alkaloids occurs through consumption of herbal dietary supplements, including comfrey, and through contaminated livestock products (e.g., milk). A recently completed 2-year bioassay of riddelliine carcinogenicity showed that male and female rats and male mice, but not female mice, developed liver tumors. The toxicokinetics of riddelliine and two metabolites, the N-oxide and retronecine, were determined in serum following an oral gavage dose in male and female rats and mice using a validated liquid chromatography-electrospray mass spectrometric method. The results are consistent with extensive metabolism of riddelliine and its more polar metabolites prior to excretion. It is concluded that factors other than toxicokinetics are responsible for the observed species/sex specificity of gross toxicity or liver tumor induction in rats and mice.