2-Week Studies in Rats and Mice
In the two-week studies, rats and mice were administered 0, 0.125, 0.25, 0.5, 1 and 2 g/kg Kava Kava extract by gavage. There were no treatment- related effects on survival or body weight in rats or mice. Clinical findings included abnormal breathing, ataxia, and lethargy in the high dose- groups of male and female rats and mice. No gross lesions related to Kava Kava extract administration were observed. The liver was found to be the major target organ of toxicity in both rats and mice. This was reflected by dose-related increases in absolute and relative liver weights in 1.0 and 2.0 g/kg male and 0.5 g/kg or greater female rats accompanied by significantly increased incidences of minimal hepatocellular hypertrophy in the 2.0 g/kg male and in 0.25 g/kg or greater female rats. In mice, liver weights were significantly increased in 2.0 g/kg males and females with accompanying increases in the incidence of hepatocellular hypertrophy in the 2.0 g/kg female mice (Data not shown). Since there were no treatment-related effects on survival or body weights, and histopathologic changes were minimal; the same doses were selected for the 3-month studies.
3-Month Studies in Rats
There were treatment- related decreases in survival and body weights in the highest dose group in male and female rats administered Kava Kava for 3- months (). Clinical findings included ataxia and lethargy in both sexes in the 1.0 g/kg groups during week 1 and persisted in the 2.0 g/kg groups throughout the study. No chemical-related gross lesions were seen in early death or terminal sacrifice animals. There were dose-related increases in the absolute and relative liver weights of 0.25 g/kg or greater males and 0.5 g/kg or greater females compared to the vehicle controls (). While there was no statistically significant increased incidence of hepatocellular hypertrophy in the males, the females showed significant dose-related increases in incidence and severity. Microscopically, the hypertrophy was minimal to mild in severity and consisted of a diffuse increase in cell size associated with glycogen depletion and amphophilic cytoplasm of the hepatocytes.
Survival, Body weight and Liver Pathology for Rats Administered Kava Kava for 3-months
Several alterations in clinical chemistry consistent with the histopathological lesions were observed (). The most prominent was a multiple-fold increase in serum gamma-glutamyltransferase (GGT) activity in the 2.0 g/kg males and females at all time-points. In addition, the 1.0 g/kg females were affected at week 14. In general, increases in serum GGT activity are used as a marker of cholestasis. Other markers of cholestasis, however, were either decreased (ALP activity) or unaffected (bile salts concentration) and did not support the increased GGT activities (data not shown). Dose-related increases in cholesterol concentrations occurred in the top three male and female dose groups (≥0.5, g/kg) at all time points. Lower-dose groups (0.125 and 0.25 g/kg) also demonstrated this alteration, but less consistently. Triglyceride concentration, another marker of lipid metabolism, was unaffected (data not shown). Total protein and albumin concentrations were minimally increased (<15%) in the top four female dose groups (0.25, 0.5, 1.0, and 2.0 g/kg) on day 23. By week 14, all female and the top three male dose groups (0.5, 1.0, and 2.0 g/kg) demonstrated these changes in serum proteins. The increases protein fractions were proportional. There were no changes in the hematology results considered attributable to Kava Kava extract administration (data not shown).
Selected Clinical Chemistry for Rats Administered Kava Kava for 3-months
The highest dose for the 2-year gavage study in rats was selected to be 1.0 g/kg per day. This was based on findings in the 3-month study including decreases in the survival and body weights of 2.0 g/kg males and females. The increases in liver weights and incidences of hepatocellular hypertrophy in 1.0 g/kg males and females were considered to be minimal and alterations in GGT activity at 1.0 g/kg were not considered dose-limiting.
3-Month Studies in Mice
There were treatment- related decreases in survival in the top- dose groups in mice administered Kava Kava for 3- months (). There was no effect on body weight between the control and treated groups. Clinical signs included transient ataxia and lethargy in males and females in the 1.0 and 2.0 g/kg groups. No chemical-related gross lesions were seen in early death or terminal sacrifice mice. There were no changes in the hematology data of mice that were considered attributable to Kava Kava extract administration. There were treatment –related increases in the absolute and relative liver weights in both sexes (). Non-neoplastic lesions included a dose-related increase in the incidence of centrilobular hypertrophy in both males and females with minimal to moderate severities. Microscopically, the lesion consisted of enlarged hepatocytes, primarily located in the centrilobular regions, and was characterized by increased hepatocellular size and ground-glass cytoplasmic eosinophilia and decreased cytoplasmic glycogen content. The no-observed-effect level for this lesion was 0.25 g/kg in males and females. The doses of 0.25, 0.5, and 1.0 g/kg were selected for the 2-year gavage study in mice based on decreased survival in 2.0 g/kg males and females.
Survival, Body weight and Liver Pathology in Male and Female Mice Administered Kava Kava for 3-months
2-Year Studies in Rats
Chronic administration of Kava Kava did not significantly affect survival or body weight in either males or females (). Clinical findings included ataxia and lethargy that occurred in the 1.0 g/kg groups and persisted randomly and intermittently throughout the study. Similar to the 3-month studies, there were increases in GGT activity which were statistically significant at 18- months in males and at 6, 12 and 18- months in females (). In addition, there was an increase in bile salt concentrations in both sexes.
Survival, Body Weight and Non-neoplastic Lesions of the Liver in Male and Female Rats Administered Kava Kava for 2-years
Selected Clinical Chemistry for Rats Administered Kava Kava for 2-years
There were increases in several non-neoplastic lesions in the livers of the rats following Kava Kava exposure (). Hepatocellular hypertrophy was noted in both males and females and was microscopically characterized by irregular increase in the size of hepatocytes, usually in a centrilobular distribution (, ). There was also a significant increase in the incidence of centrilobular fatty change in 0.1 and 1.0 g/kg males, and in 1.0 g/kg females. This lesion consisted of poorly demarcated areas of hepatocytes with large, clear cytoplasmic vacuoles, usually in the centrilobular and midzonal regions. Cystic degeneration was observed in all dosed groups of males, and the incidence in 1.0 g/kg males was significantly increased. Microscopically, cystic degeneration consisted of multilocular cystic areas containing a finely granular or flocculent eosinophilic material, apparently resulting from the distention and occasional rupture of adjacent hepatocytes (). In addition, a unique lesion was noted in the pancreas in the 1.0 g/kg males and females which included incidences of metaplasia of pancreatic acinar cells to a hepatocytic morphology. The increase was significant in the male rats (incidence: 0/49, 0/50, 0/50, 6/50). Microscopically, this lesion was characterized by the presence of small clusters of apparently normal hepatocytes adjacent to islets of Langerhans (). The etiology of this lesion in the current studies remains unknown.
Photomicrograph of a liver section from a control male rat from the 2 year Kava Kava study. Note (arrows), the normal aspect of the centrilobular hepatocytes. Compare with . X 32. H&E
Photomicrograph of a liver section from a female rat treated with 1 g/kg Kava Kava for 2 years. Note (arrows), mild (grade 2) centrilobular hypertrophy. Compare with . X 32. H&E
Photomicrograph of a liver section from a male rat treated with 1 g/kg Kava Kava for 2 years. Note (arrows), mild (grade 2) cystic degenerationX 16. H&E
Photomicrograph of minimal (grade 1) metaplasia of pancreatic acinar cells to a hepatocytic morphology (arrows) from a female rat treated with 1 g/kg Kava Kava for 2 years. X 16. H&E
2-Year Studies in Mice
Chronic administration to Kava Kava in male and female B6C3F1 mice did not affect survival (). There was a slight exposure-related reduction in body weight gain in the females in the highest exposure group. Clinical findings included ataxia and lethargy that occurred in both sexes in the 1.0 g/kg groups during the first week but persisted only in the females.
Survival, Body Weight, Neoplastic and Non-neoplastic Lesions of the Liver in Mice Administered Kava Kava for 2-years
There were several neoplastic lesions noted in the livers of mice in both sexes. The males revealed dose-related increases in hepatoblastomas. The incidences of hepatoblastomas in 0.5 and 1.0 g/kg males exceeded the concurrent and historical controls while the 0.25 g/kg males exceeded only historical controls. There were also statistically significant increases in hepatocellular adenomas (multiple) in the top-dose group males and in the 0.5 g/kg group in females. There was also an increase in hepatocellular carcinomas in all dosed group females which was statistically significant at the low dose (0.25 g/kg). Importantly, there was a statistically significant increase in the combined incidences of hepatocellular adenomas or carcinomas in the 0.25 and 0.5 g/kg groups. These values exceeded the historical range for corn oil gavage studies, but not for all routes combined. Histologically, the hepatocellular carcinomas were variably sized, nodular lesions composed of well-differentiated, neoplastic hepatocytes that typically compressed the adjacent hepatic parenchyma. Portal areas and central veins were typically absent, and mild cellular atypia was often present. Hepatocellular carcinomas were variably well demarcated from the surrounding hepatic parenchyma and were composed of neoplastic hepatocytes that displayed mild to marked cellular and nuclear pleomorphism and mitoses. The predominant pattern displayed by most neoplasms in this study was trabecular, although focal areas had glandular or solid patterns of growth (, ). Necrosis was occasionally quite extensive, and metastasis to the lung was frequently observed. Hepatoblastomas tended to arise within hepatocellular adenomas or carcinomas and were composed of small, basophilic fusiform cells with a high nucleus to cytoplasm ratio (, ). Mitoses, large, irregularly shaped cystic areas filled with blood, and areas of necrosis were common.
Figures 5 Photomicrographs of hepatocellular carcinoma from a male mouse treated with 0.25 mg/kg Kava Kava for 2 years. : Note (arrows), margins of the carcinoma compressing the adjacent normal tissue. X 2. : Higher magnification of (more ...)
Figures 6 Photomicrograph of hepatoblastoma from a male mouse treated with 1 mg/kg Kava Kava for 2 years (arrows). Note the hyperbasophilic appearance of the mass due to presence of basophilic fusiform cells with a high nucleus to cytoplasmic (N:C) ratio. (more ...)
The neoplastic lesions were accompanied by several non-neoplastic effects. There were dose-related increases in the incidences and severities of centrilobular hypertrophy in both sexes.
Microscopically, the hypertrophy was characterized by enlargement of centrilobular hepatocytes with increased amounts of eosinophilic cytoplasm and enlarged nuclei. This lesion was often variable in its presence and severity between lobes and within regions of the same lobe. There were significant increases in eosinophilic foci in the 0.5 g/kg males and in 1.0 g/kg males and females. The eosinophilic foci consisted of well-differentiated hepatocytes containing increased amounts of eosinophilic cytoplasm (). Portal areas and central veins were often present, and minimal compression of the adjacent parenchyma occurred occasionally. The incidences of angiectasis increased in a dose-related manner in males, and the increase in the 1.0 g/kg group was significant. This lesion was characterized by variably sized dilatations of the hepatic sinusoids, typically occurring in small clusters randomly arranged throughout the hepatic parenchyma and without a sub-anatomic orientation (). The sinusoids were lined by an attenuated to unapparent endothelium. The incidences of hepatocellular necrosis were significantly increased in 0.25 and 1.0 g/kg males. Microscopically, the hepatocellular necrosis was characterized by focal, widely scattered, randomly distributed areas of necrosis of hepatocytes often infiltrated by small numbers of mixed inflammatory cells. Necrosis was not diagnosed when it was deemed to be secondary to neoplasia.
Photomicrograph of eosinophilic focus (arrows) from a male mouse treated with 0.25 mg/kg Kava Kava for 2 years X 5. H&E
Photomicrograph of angiectasis (arrows) from a male mouse treated with 1 g/kg of Kava Kava for 2 years. X 10. H&E.