Twenty-eight-day Interim Study

All rats survived to the end of the study (Table 4). There were no significant treatment-related clinical observations in male or female rats. The final mean body weights of male and female rats in all dosed groups remained within 5% of the vehicle control body weights (Table 4).

Table 4

Survival and Body Weights of Rats in the Interim 28-day Gavage Study of Gum Guggul Extract^a.

Dose-related increases in absolute and relative liver weights were observed in male and female rats (Table 5). The mean absolute liver weights were significantly greater than those of vehicle controls in 1,000 mg/kg/day males (approximately 15%) and in 500 and 1,000 mg/kg/day females (approximately 16% and 30%, respectively). Relative liver weights were significantly greater than those of vehicle controls in 1,000 mg/kg/day males and females administered 125 mg/kg/day or greater. A dose-related increase in absolute kidney weights was observed in female rats. The mean absolute and relative kidney weights of 1,000 mg/kg/day females were significantly greater than those of vehicle controls. The mean absolute kidney weight of 1,000 mg/kg/day females was approximately 11% greater than that of vehicle controls. Mean absolute and relative thymus weights were significantly less than those of vehicle controls in 125 mg/kg/day males; however, this finding was not dose dependent and did not occur in both sexes, suggesting that it is a spurious finding and not treatment related (Appendix F ).

Table 5

Select Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the Interim 28-day Gavage Study of Gum Guggul Extract^a,b.

There were no dose-related microscopic findings (Appendix F ).

In female rats, on day 28, lymphocyte counts were significantly decreased in several dosed groups, relative to vehicle controls Appendix F). This particular change is most consistent with stress (i.e., corticosterone induced). All other significant hematological changes were mild and inconsistent and not considered related to treatment.

In males, alkaline phosphatase (ALP) activity was significantly decreased in the 500 and 1,000 mg/kg/day groups, relative to vehicle controls. In females, ALP activity was significantly decreased in all dosed groups, relative to vehicle controls (Table 6). The mechanism for these decreases is not known but indicates an alteration in ALP metabolism; there is no known toxicological relevance for decreases in ALP activity.

Table 6

Select Clinical Chemistry Data for Rats in the Interim 28-day Gavage Study of Gum Guggul Extract^a,b.

Cholesterol concentration was significantly decreased in 1,000 mg/kg/day males, and phospholipid concentrations were significantly decreased in 500 and 1,000 mg/kg/day males, relative to vehicle controls (Table 6). These changes were not observed in females (Appendix F ). Globulin concentrations were significantly increased in 1,000 mg/kg/day males and in 500 mg/kg/day and 1,000 mg/kg/day females, relative to vehicle controls, causing a significant increase in total protein and decrease in the albumin/globulin (A/G) ratio in 1,000 mg/kg/day females (Table 6). Bile acid concentrations were significantly decreased in 1,000 mg/kg/day males, relative to vehicle controls (Table 6). Other statistically significant biochemical changes were small or inconsistent relative to dose, and not considered to be due to the administration of the tested gum guggul extract (GGE) formulation.

A panel of thyroid hormones was measured in males and females. Thyroid stimulating hormone (TSH) was significantly increased in several female dosed groups, relative to vehicle controls; this was not accompanied by any changes in free thyroxine (T₄), total T₄, or triiodothyronine (T₃) (Appendix F ). No changes were observed in male thyroid hormones. The changes in TSH were not interpreted to be associated with the administration of the tested GGE formulation.

Both male and female rats exhibited dose-related increases in hepatic cytochrome P450 (CYP) 2B and CYP3A activity following administration of the GGE formulation, suggesting increased constitutive androstane receptor (CAR) and pregnane X receptor (PXR) activities (Table 7). A sex-related difference in the magnitude of induction of CYP2B was observed with up to a sixfold increase in males and a twofold increase in females, relative to vehicle controls. CYP3A activity was up to twofold higher in males and sixfold higher in females, relative to vehicle controls.

Table 7

Cytochrome P450 Measurements for Rats in the Interim 28-day Gavage Study of Gum Guggul Extract^a,b.

Three-month Study

All rats survived to the end of the study with the exception of three female rats, with a single death occurring in each of the 0, 125, and 500 mg/kg/day groups (Table 8). The three deaths occurred between days 50 and 57 and were not considered treatment related. There were no significant treatment-related clinical observations in male or female rats. The final mean body weights of male and female rats in all dosed groups were within 5% of the vehicle control body weights (Table 8; Figure 3).

Table 8

Survival and Body Weights of Rats in the Three-month Gavage Study of Gum Guggul Extract^a.

Figure 3

Growth Curves for Male and Female Rats Administered Gum Guggul Extract by Gavage for Three Months

Dose-related increases in absolute and relative liver weights were observed in male and female rats (Table 9). The mean absolute liver weights were significantly greater than those of vehicle controls in 1,000 mg/kg/day males (approximately 20%) and in 500 and 1,000 mg/kg/day females (approximately 25% and 43%, respectively). Relative liver weights were significantly greater than vehicle controls in males administered 250 mg/kg/day or greater and females administered 500 or 1,000 mg/kg/day. A dose-related increase in relative and absolute kidney weights was observed in female rats; a dose-related increase in relative kidney weights was observed in male rats. The relative kidney weight of 1,000 mg/kg/day males was significantly greater than that of vehicle controls. Dose-related increases in absolute and relative heart weights were observed in female rats, although no dose group exhibited significantly greater absolute or relative heart weights. The mean absolute and relative thymus weights of 1,000 mg/kg/day males were significantly less than those of vehicle controls. The mean absolute thymus weight of 1,000 mg/kg/day males was 23% lower than that of vehicle controls.

Table 9

Select Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the Three-month Gavage Study of Gum Guggul Extract^a,b.

There were no dose-related microscopic findings (Appendix F ).

Significant decreases in hematocrit and hemoglobin concentrations were observed in the 1,000 mg/kg/day male rats at day 92 (Appendix F ). These decreases were mild and not considered toxicologically relevant. All other significant hematological changes were mild and inconsistent and considered not due to treatment.

In males, ALP activity was significantly decreased in 250 mg/kg/day or greater groups. In females, ALP activity was significantly decreased in 125 mg/kg/day or greater groups (Table 10). The mechanism for these decreases is not known but indicates an alteration in ALP metabolism; there is no known toxicologic relevance for decreases in ALP activity.

Table 10

Select Clinical Chemistry Data for Rats in the Three-month Gavage Study of Gum Guggul Extract^a,b.

Globulin concentrations were significantly increased in 500 and 1,000 mg/kg/day females, relative to vehicle controls (Table 10), causing a significant decrease in the A/G ratio of the same dosed groups. Bile acid concentrations were significantly decreased in all dosed female groups (Table 10).

A panel of thyroid hormones was measured in males and females, and several statistically significant changes were observed in the male rats (Appendix F ). These changes were small or inconsistent relative to dose, or lacked a dose response, and therefore were not interpreted to be associated with administration of the tested GGE formulation.

Male rats administered 250 and 1,000 mg/kg/day displayed an increase (15–16%) in the mean total number of homogenization-resistant spermatids with a statistically significant difference between the 1,000 mg/kg/day group and the vehicle control group (Table 11). Male rats administered 1,000 mg/kg/day also displayed statistically higher (approximately 18%) mean total number of homogenization-resistant spermatids/mg. Both respective spermatid values in the 500 mg/kg/day group were similar to those of vehicle controls. Cauda epididymal sperm counts and motility of all dosed groups were similar to those of vehicle controls. There were no GGE-related effects on testicular weight, epididymal weights, or histopathology. Given the apparent increase in spermatid head counts was not dose-related, and the absence of concurrent increases in sperm counts or histopathological findings, these effects are considered not related to administration of the tested GGE formulation.

Table 11

Select Sperm Count Endpoints for Rats in the Three-month Gavage Study of Gum Guggul Extract^a.

Both male and female rats exhibited dose-related increases in hepatic CYP2B and CYP3A activity following administration of the tested GGE formulation, suggesting increased CAR and PXR activities (Table 12). CYP2B activity was up to approximately 11-fold higher in males and threefold higher in females, relative to vehicle controls. A sex-related difference in the magnitude of induction of CYP3A was observed with up to approximately twofold increase in males and ninefold increase in females, relative to vehicle controls. CYP2B and CYP3A activities were increased in males relative to females at all doses, including basal concentrations in vehicle controls.

Table 12

Cytochrome P450 Measurements for Rats in the Three-month Gavage Study of Gum Guggul Extract^a,b.

Twenty-eight-day Interim Study

All mice survived to the end of the study (Table 13). There were no significant treatment-related clinical observations in male or female mice. The final mean body weights of male and female mice in all dosed groups remained within 5% of the vehicle control body weights (Table 13).

Table 13

Survival and Body Weights of Mice in the Interim 28-day Gavage Study of Gum Guggul Extract^a.

Mean relative thymus weights were significantly decreased relative to the vehicle control groups in 125 mg/kg/day females; however, this finding was not dose dependent and did not occur in both sexes, suggesting that the finding is spurious and not treatment related. Therefore, no statistically significant organ weight changes were related to administration of the tested GGE formulation (Appendix F ).

There were no dose-related microscopic findings (Appendix F ).

No changes in the clinical chemistry values of male and female mice were attributable to administration of the tested GGE formulation (Appendix F ).

Both male and female mice exhibited a dose-related increase in hepatic CYP3A activity following GGE administration, suggesting increased PXR activity (Table 14). CYP3A activity was significantly increased in male mice administered 125 and 250 mg/kg/day, relative to the vehicle control group.

Table 14

Cytochrome P450 Measurements for Mice in the Interim 28-day Gavage Study of Gum Guggul Extract^a,b.

Three-month Study

All mice survived to the end of the study (Table 15). There were no significant treatment-related clinical observations in male or female mice. The final mean body weights of male and female mice in all dosed groups remained within 7% of the vehicle control body weights (Table 15; Figure 4).

Table 15

Survival and Body Weights of Mice in the Three-month Gavage Study of Gum Guggul Extract^a.

Figure 4

Growth Curves for Male and Female Mice Administered Gum Guggul Extract by Gavage for Three Months

A dose-related trend of decreased relative kidney weights with increasing dose was observed in female mice, although no dosed group exhibited significantly lower absolute or relative kidney weights, suggesting the finding is spurious and not treatment related. Therefore, there were no statistically significant organ weight changes related to administration of the tested GGE formulation (Appendix F ).

There were no dose-related microscopic findings (Appendix F ).

Cholesterol concentrations were significantly increased in 62.5 mg/kg/day or greater females and phospholipid concentrations were significantly increased in 250 mg/kg/day females, relative to vehicle controls (Table 16).

Table 16

Select Clinical Chemistry Data for Mice in the Three-month Gavage Study of Gum Guggul Extract^a,b.

Male mice administered 62.5, 125, and 250 mg/kg/day GGE formulation exhibited statistically significant lower mean total numbers of homogenization-resistant spermatids (23%, 18%, and 37% lower, respectively), and mean total numbers of homogenization-resistant spermatids/mg testis (21%, 17%, and 32% lower, respectively) relative to the vehicle control group (Table 17). These mice also displayed lower mean numbers of cauda epididymal sperm (15%, 13%, and 24% lower, respectively), and mean numbers of cauda sperm/mg (13%, 12%, and 26% lower, respectively). Testis weight in the 250 mg/kg/day group was lower (12%) relative to the vehicle control group, and the trend test was significant. The contralateral testicular weights were similar to vehicle control weights. There were no histopathological findings in the testes or epididymides. Given the concurrent responses (lower spermatid and sperm counts), GGE exhibited the potential to be a reproductive toxicant in male mice.

Table 17

Select Sperm Count Endpoints for Mice in the Three-month Gavage Study of Gum Guggul Extract^a.

Both male and female mice exhibited dose-related increases in hepatic CYP2B and CYP3A activity following administration of the tested GGE formulation, suggesting increased CAR and PXR activities (Table 18). CYP2B activity was significantly increased in 250 mg/kg/day female mice, relative to vehicle controls. CYP3A activity was significantly increased in 125 and 250 mg/kg/day male mice and in 250 mg/kg/day female mice, relative to the vehicle control groups.

Table 18

Cytochrome P450 Measurements for Mice in the Three-month Gavage Study of Gum Guggul Extract^a,b.