Berberine, and its salts (e.g. berberine chloride dihydrate, BCD) are yellow crystalline materials that are used in Chinese herbal medicine, and in the United States as a dietary supplement. In addition, berberine is a component of goldenseal, another popular herbal dietary supplement. This study was performed as a follow-up research study to a previously conducted developmental toxicity evaluation of berberine chloride dihydrate, administered in the feed (0, 3500, 5250, or 7000 ppm) to Swiss (CD-1®) mice on gestational days (gd) 6 through 17 (NTP, 2001a). In that study, an equivocal lowest-observed-adverse-effect level (LOAELs) for maternal and developmental toxicity, based on increased relative maternal water consumption and the incidence of cleft palate, respectively, was 5250 ppm (841 mg BCD/kg/day or 666 mg berberine chloride/kg/day). The no-observed-adverse-effect level (NOAELs) for maternal and developmental toxicity was 3500 ppm (569 mg BCD/kg/day or 450 mg berberine chloride/kg/day).
Thus, the present study sought to further define the maternal and developmental effects of 1000 mg/kg/day BCD when administered by gavage (a more accurate oral route of exposure) on gd 6 through 16. Administration by gavage was also chosen to eliminate changes in feed/water consumption observed in the previous study, that may have been due to altered palatability of the dosed feed.
Twenty-five timed-mated mice were assigned to the control and BCD-treated groups. Dams were monitored in-life for clinical signs of toxicity, feed and water intake, and body weight. At necropsy (gd 17), the following determinations were made: maternal clinical condition; body, liver, and gravid uterine weights; pregnancy status; and the number of corpora lutea. In the gravid uterus, the numbers of resorbed, dead, and live fetuses were recorded. Live fetuses were weighed, sexed, and examined for external, visceral, and skeletal morphological anomalies. Analysis of the dose formulation indicated that dams were exposed to 1068 -1071 mg BCD/kg/day.
Four dams in the 1000 mg/kg/day dose group were removed due to a dosing error, and seven additional dams were found dead or moribund due to causes other than technician error. There were no other remarkable clinical signs related to BCD exposure. In animals surviving to necropsy, BCD exposure had no effect on maternal body weight, maternal body weight change, gravid uterine weight, or maternal liver weight (absolute or relative to body weight).
Maternal relative feed consumption (g/kg/day) was unaffected by BCD treatment, with the exception of a small but significant (2.7%) increase for gd 9 to 12. Maternal relative water intake (g/kg/day) increased during the middle and latter portions of the treatment period (gd 12 to 15, 15 to 16; 19-21%) and was increased for the treatment period (gd 6 through 16; 13%), but not for the gestational period (gd 0 to 17).
At scheduled necropsy, pregnancy was confirmed in 24 (100%) control animals and the 14 (100%) surviving treated animals. Prenatal mortality was unaffected by treatment, as was average live litter size and the incidence of fetal morphological anomalies (external, visceral or skeletal malformations or variations).
In summary, CD-1® mice were dosed by gavage with BCD (0 or ~1000 mg/kg/day) in 0.5% aqueous methylcellulose from gd 6 through 16. Maternal mortality or moribundity occurred in 33% of the treated females. Maternal effects in surviving animals were limited to increased relative water consumption, as observed in the feed study. Live litter size in litters from surviving animals was not affected by BCD exposure. The average fetal body weight per litter of surviving fetuses was decreased 5-6% in the treated group. This difference was statistically significant for male fetuses only, but was considered biologically relevant for males, females, and the sexes combined, based on historical control data. The maternal deaths observed in the present study clearly indicate that 1000 mg/kg/day BCD is an unequivocal maternal effect level. The increase in relative maternal water intake, in the absence of a change in body weight, in the treated animals in the present study, supports the observation of increased relative maternal water consumption in the previous study. Thus, the maternal toxicity LOAEL remains at 841 mg/kg/day BCD or 666 mg/kg/day berberine chloride. The developmental toxicity LOAEL can be raised from 841 mg BCD/kg/day or 666 mg berberine chloride/kg/day as suggested by the feed study, to ~1070 mg/kg/day BCD or 938 mg/kg/day berberine chloride, based on the absence of an increased incidence of cleft palate, and the decrease in fetal body weight in the present study.