One-year chronic oral toxicity with combined reproduction toxicity study of a novel probiotic, Bacillus coagulans, as a food ingredient.

Some strains of Bacillus coagulans can survive extremes of heat, stomach acid and bile acids, to which commonly consumed probiotics are susceptible. A toxicological safety assessment was published in 2009 on a proprietary preparation of B. coagulans - GanedenBC(30)™ - a novel probiotic. It was concluded that GanedenBC(30)™ is safe for chronic human consumption based upon scientific procedures, supported by a safe history of use (Endres et al., 2009). A one-year chronic oral toxicity study combined with a one-generation reproduction study was conducted to further investigate safety of long-term consumption. The one-year study of GanedenBC(30)™ administered to male and female HsdBrlHan: Wistar rats in their diet showed no signs of toxicity at the highest dose tested. The conclusion from the reproduction toxicity study is that administration of GanedenBC(30)™ in the diet caused no signs of toxicity in the parental generation (male or female) nor the F1 offspring. Using the lowest NOEL of 1948 mg/kg concluded at the end of the 1-year feeding study, a 100-fold safety factor, a test article concentration of 6.88×10(10) CFU (colony forming units) per gram, and an average 70 kg human, it is determined that GanedenBC(30)™ is safe for chronic consumption at up to 9.38×10(10) CFUs per day.

Safety assessment of a novel ingredient for removable chewing gum.

Rev7 is an indigestible gum polymer used for the manufacturing of chewing gum. It allows for the formulation of chewing gum with low adhesion; thus can be readily removed from surfaces such as sidewalks, clothing, carpets and furniture. In a toxicological safety assessment, Rev7 was found to be non-mutagenic in the AMES assay. The highest concentration tested in a mouse lymphoma thymidine kinase locus gene mutation assay induced a slight but biologically relevant increase in mutations under non-metabolic activation conditions after 24h. Because of this finding, a mouse micronucleus assay was performed, and the test article was found to be negative for inducing chromosomal damage. A 28-day repeated oral toxicity study resulted in a NOAEL of 80,000 ppm; the highest concentration tested. Rev7 was found to be free from contaminants such as heavy metals, monomers, and solvents. Lastly, Rev7 did not demonstrate skin-sensitizing properties in the murine local lymph node assay.

Safety assessment of a proprietary preparation of a novel Probiotic, Bacillus coagulans, as a food ingredient.

It has been demonstrated that some strains of Bacillus coagulans can survive extremes of heat, acidity of the stomach, and bile acids, to which commonly consumed probiotics are susceptible. A toxicological safety assessment was performed on a proprietary preparation of B. coagulans - GanedenBC(30) - a novel probiotic. Seven toxicologic studies were conducted and included: in vitro bacterial reverse mutation assay; in vitro chromosomal aberration assay; micronucleus assay in mice; acute and 90 day subchronic repeated oral toxicity studies were conducted in Wistar Crl:(WI) BR rats; acute eye and skin irritation studies were conducted in rabbits. The results of this toxicological safety assessment indicate that GanedenBC(30)B. coagulans does not demonstrate mutagenic, clastogenic, or genotoxic effects. Furthermore, the results of the acute and 90-day subchronic oral toxicity studies in rats resulted in the conclusion of a NOAEL greater than 1000 mg/kg per day. Since the concentration of the cell mass used in the 90-day study was 1.36 x 10(11) CFUs/g, this corresponds to 95.2 x 10(11) CFUs for a 70 kg human and since the suggested human dose is in the range of 100 x 10(6) to 3 x 10(9) CFUs, this gives a safety factor ranging from 3173 to 95,200 times. Based upon scientific procedures and supported by history of use, GanedenBC(30) is considered safe for chronic human consumption.

Vitamin A levels in regenerating rat liver. Effects on microsomal drug metabolism.

After being maintained on a vitamin A-deficient or complete diet for a period of five weeks, male Sprague-Dawley rats were subjected to a two-thirds partial hepatectomy (PH) or sham operation. The vitamin A content of the liver of vitamin A-deficient, PH rats was below the limit of detection (less than 1 microgram/g liver). Rats fed the control diet and subjected to PH had hepatic levels of vitamin A that were 37% and 49% lower 48 and 72 hours after surgery, respectively, when compared with sham-operated controls. Hepatic microsomal cytochrome P-450 levels were significantly reduced in PH rats fed the complete diet 48 hours after PH and in PH rats fed either the deficient or complete diet 72 hours after. Vitamin A deficiency alone significantly reduced cytochrome P-450 levels. A combination of vitamin A deficiency and PH had the most dramatic effect on cytochrome P-450 and aminopyrine N-demethylase, which reduced the activity to approximately 50% of the activities found in the sham-operated control group. PH resulted in the greatest reduction in the rate of disappearance of benzo[a]pyrene in the presence of liver microsomes prepared from vitamin A-deficient rats.

Evaluation of purified 4-deoxynivalenol (vomitoxin) for unscheduled DNA synthesis in the primary rat hepatocyte-DNA repair assay.

Primary cultures of rat hepatocytes were used to determine unscheduled DNA synthesis (UDS) and cytotoxicity of purified 4-deoxynivalenol (vomitoxin), a trichothecene mycotoxin produced on cereal grains by fungi of the genus Fusarium. Nontoxic and toxic doses of deoxynivalenol, 0.1 to 1000 micrograms/ml, did not significantly increase UDS as measured by net grains per nucleus, net grains per nuclear area or percentage of cells incorporating greater than or equal to 5, 6, 10 or 20 grains per nucleus. Evidence of cytotoxicity, manifested as a reduction in cell number in autoradiographs, pyknotic nuclei or vacuolated cytoplasm, was observed in hepatocytes treated with deoxynivalenol concentrations of 5 micrograms/ml and above. These findings suggest that the cellular toxicity of deoxynivalenol may not be mediated by a DNA-damaging event in cultured hepatocytes. An increased percentage of large-sized nuclei was also found to be associated with toxic doses of deoxynivalenol as well as 2-acetylaminofluorene used as the positive control.

Vitamin A status and metabolism of benzo[a]pyrene in the rat.

Male Sprague-Dawley rats were maintained on a vitamin A-deficient diet for a period of five weeks. At the end of that time, hepatic cytochrome P450 levels in vitamin A-deficient rats were 65% that of rats fed a complete diet. However, the hepatic rate of benzo[a]pyrene metabolism was significantly greater (2 times) in vitamin A-deficient rats compared with those fed a complete diet. The pattern of metabolites separable by thin-layer chromatography was similar in both groups of rats. Benzo[a]pyrene induced its own metabolism by a slightly greater amount in the vitamin-sufficient rats, but it was not to the level of the deficient group, although the levels of cytochrome P450 were still below those of the deficient rats. In discussing lung microsomes, benzo[a]pyrene pre-treatment of deficient rats resulted in slightly elevated levels of cytochrome P450 and a slightly greater rate of metabolism of benzo[a]pyrene compared with rats fed the complete diet.

The differential effects of chemical carcinogens on vitamin A status and on microsomal drug metabolism in normal and vitamin A-deficient rats.

Male Sprague-Dawley rats were maintained on a vitamin A-deficient diet for 5 weeks. Although serum and hepatic levels of vitamin A were significantly lower at this time, no outward signs of vitamin A deficiency were present. Hepatic microsomal levels of cytochrome P-450 in the vitamin A-deficient animals were 70% that of the control animals. Of the three microsomal enzymes studied, ethylmorphine N-demethylase, aniline hydroxylase, and aminopyrine N-demethylase, only the last one was adversely affected by vitamin A deficiency. 3-Methylcholanthrene, phenobarbital, and 2-acetylaminofluorene had a greater inductive effect and cytochrome P-450 in vitamin A-deficient rats. 4-Dimethylaminoazobenzene treatment decreased in the level of cytochrome P-450 in control rats more than in deficieny rats. The hepatic concentration of vitamin A was significantly reduced in control rats that were given injections of 3-methylcholanthrene, 2-acetylaminofluorene, or phenobarbital. Benzo(a)pyrene and 4-dimethylaminoazobenzene had less effect.

Effects of different vitamin E-deficient basal diets on hepatic catalase and microsomal cytochromes P-450 and b5 in rats.

Male Sprague-Dawley rats maintained for a period of 6 or 12 weeks on a basal vitamin E-dificient diet consisting of 70% sucrose, 20% vitamin-free casein, 4% tocopherol stripped lard, 4% salt mixture, and 2% tocopherol-free vitamin fortification mixture were used to compare two sets of commonly used salt mixtures (salt mixtures USP XIV versus Briggs' salt mixture) and two sets of vitamin fortification mixtures (NBC vitamin fortification mixture versus that of Weglicki). Among the rats maintained on the deficient diets for 6 weeks, only those that received the combination of salt mixture USP XIV and vitamin fortification mixture of Weglicki showed a significantly lower level of hepatic catalase activity compared to the corresponding control animals. While there were no significant changes in microsomal cytochromes at this time period, after 12 weeks on the deficient diet, a significant depression in these cytochromes was noted in all experimental groups except the one on salt mixture USP XIV and NBC vitamin fortification mixture. A similar decrease in hepatic catalase was observed in deficient animals at 12 weeks. Since the most striking differences in these diets are in their content of iron and menaquinone, it appears that these two dietary constituents may interact in modulating the effect of vitamin E on hepatic hemeproteins.