Toxicological and safety evaluations of Weissella cibaria strain CMU in animal toxicity and genotoxicity

Weissella cibaria belongs to the Lactobacillaceae family and has been isolated from traditional fermented foods and saliva of children with good oral health. Previous investigations have shown that W. cibaria CMU (Chonnam Medical University) is expected to be safe based on results of in silico and in vitro analyses. However, there is a lack of studies assessing its safety in vivo. A toxicological safety evaluation of W. cibaria CMU was performed using an acute oral safety study in rats, a 14-day oral range finding study, a subsequent 13-week oral toxicity study in rats and a genetic toxicity battery (in vitro bacterial reverse mutation, in vitro chromosome aberration in Chinese Hamster Ovary cells and in vivo micronucleus study in mice). The results of the studies in rats showed that the acute lethal dose of W. cibaria CMU is > 5000 mg/kg body weight (bw)/day (1.8 × 109 CFU/kg bw/day) and the 14-day or 13-week no observed adverse effect level (NOAEL) is 5000 mg/kg bw/day (1.8 × 109 CFU/kg bw/day), the highest dose administered. W. cibaria CMU was non-mutagenic in the bacterial reverse mutation test and non-clastogenic or aneugenic in vitro and in vivo. In conclusion, the toxicological studies performed demonstrated W. cibaria CMU to be a safe strain to consume. This study is the first study examining the potential of a W. cibaria strain to cause genetic toxicity and subchronic toxicity in rats according to the Organization for Economic Cooperation and Development guidelines.

Assessment of genotoxicity of Ssanghwa-tang, an herbal formula, by using bacterial reverse mutation, chromosome aberration, and in vivo micronucleus tests

Objectives: Ssanghwa-tang (SHT) is a traditional herbal formula comprising nine medicinal herbs, and it is used for reducing fatigue in Korea. SHT exerts various effects such as anti-inflammatory, antioxidant, and anti-aging activities, and protection against acute hepatotoxicity. However, the genotoxicity of SHT has not yet been established.Methods: Ten components were identified in SHT water extract by using high-performance liquid chromatography analysis. We assessed the genotoxicity of SHT by using bacterial reverse mutation (Ames test), chromosome aberration, and in vivo micronucleus tests.Results: The contents of paeoniflorin, glycyrrhizin, and liquiritin apioside in SHT were 15.57, 6.94, and 3.48 mg/g extract, respectively. SHT did not increase the revertant colonies of Salmonella typhimurium and Escherichia coli strains in the presence or absence of metabolic activity. Although SHT did not induce structurally abnormal chromosomes in Chinese hamster lung (CHL) cells in the presence of metabolic activity, the number of structurally aberrated chromosomes increased dose-dependently in the absence of metabolic activity. In the in vivo micronucleus test, SHT did not affect the formation of micronuclei compared with the vehicle control.Conclusions: Genotoxicity of SHT was not observed in the Ames test and in vivo micronucleus test. However, based on the results of chromosome aberration test, it can be presumed that SHT has the potential to induce genotoxicity because it induced structurally abnormal chromosomes in the absence of metabolic activity.

Mutagenicity Assessment to Pesticide Adjuvants of Toluene, Chloroform and Trichloroethylene by Ames Test

Pesticide adjuvants (PAs) denote the general term for auxiliaries in pesticide preparations except for the active components. Toluene, chloroform, and trichloroethylene are the three most commonly used PAs as organic solvents. The residues of the three chemicals in the process of production and application of pesticides may endanger the ecosystem. In the present study, the mutagenicity of toluene, chloroform, and trichloroethylene as well the mixture of the three chemicals was tested by the Salmonella typhimurium reverse mutation test (Ames test) with TA97, TA98, TA100, and TA102 strains in the system with and without rat liver microsomal preparations (S9). The four tester strains have been used for more than 40 years to detect mutagenic compounds in chemicals, cosmetics, and environmental samples. The mutagenicity was detected on tester strains in the separated experiment from the three chemicals. The addition of S9 decreased the mutation ratios of toluene to four strains, except for the TA100 strain, but increased the mutation ratios of chloroform to four strains except for the TA98 strain. Trichloroethylene caused positive mutagenicity to become negative on the TA102 strain. In the mixed experiment, positive effects were detected only on the TA102 strain in the absence of S9. The addition of S9 increased the mutagenicity except for the TA102 strain. The mixture of toluene, chloroform, and trichloroethylene showed antagonism in mutagenicity to tester strains, except for the TA102 strain without S9. However, the mixture showed a synergistic effect to tester strains after adding S9 except for the TA98 strain.

Fermotein Does Not Exert Genotoxic Effects in Bacterial Reverse Mutation and in Vitro Mammalian Cell Micronucleus Tests

Aim: Fermotein is an innovative single-cell protein obtained from fermentation of the filamentous fungus Rhizomucor pusillus. Like other filamentous fungi, a lack of information on this species exists to assess its safety for human consumption. The capability to induce gene mutations or structural and numerical chromosomal aberrations of this fungus and derived products has never been studied before. The objective of the current study was to investigate the genotoxic effects of Fermotein using a bacterial reverse mutation test and an in vitro mammalian cell micronucleus test. Methodology: The bacterial reverse mutation test and in vitro mammalian cell micronucleus test were performed in accordance with GLP and concurrent OECD guidelines. Dose-range finding tests were used to select appropriate doses of Fermotein Dry. The highest doses in the genotoxicity experiments were determined by the solubility of the mycoprotein. Results: The bacterial reverse mutation test and in vitro mammalian cell micronucleus test were performed in accordance with GLP and concurrent OECD guidelines. Dose-range finding tests were used to select appropriate doses of Fermotein Dry. The highest doses in the genotoxicity experiments were determined by the solubility of the mycoprotein. Conclusion: No safety concerns regarding genotoxicity were identified for Fermotein and no further in vivo genotoxicity testing is required. Information from the current study contributes to the body of evidence for a novel food authorisation of Fermotein in the EU and a GRAS notification in the US.

Genotoxicity of Water Extract from Bark-Removed Rhus verniciflua Stokes

Rhus verniciflua Stokes (RVS) has been traditionally used as an herbal remedy to support the digestive functions in traditional Korean medicine. Additionally, the pharmacological effects of RVS, including antioxidative, antimicrobial and anticancer activities, have been well-reported. The genotoxicity of RVS, however, is elusive; thus, we evaluated the genotoxicity of RVS without bark (RVX) for safe application as a resource of functional food or a medical drug. To evaluate the genotoxicity of RVX, we used a bacterial reverse mutation test, chromosomal aberration test and comet assay, according to the “Organization for Economic Co-operation and Development” (OECD) guidelines. Briefly, for the reverse mutation test, samples (5000, 1667, 556, 185, 62 and 0 μg/plate of RVX or the positive control) were treated with a precultured strain (TA98, TA100, TA1535, TA1537 or WP2µvrA) with or without the S9 mix, in which RVX partially induced a reverse mutation in four bacterial strains. From the chromosomal aberration test and comet assay, the RVX samples (556, 185, 62, 20 and 0 μg/mL of RVX or the positive control) were treated in a Chinese hamster ovary cell line (CHO-K1 cells) in the conditions of the S9 mix absent or S9 mix present and in Chang liver cells and C2C12 myoblasts, respectively. No chromosomal aberrations in CHO-K1 or DNA damage in Chang liver cells and C2C12 myoblasts was observed. In conclusion, our results suggest the non-genotoxicity of RVX, which would be helpful as a reference for the safe application of bark-removed Rhus verniciflua Stokes as functional raw materials in the food, cosmetics or pharmaceutical fields.

Safety assessment of β-fructofuranosidase from Aspergillus brunneoviolaceus

β-Fructofuranosidase (β-D-fructofuranoside fructohydrolase; EC 3.2.1.26) is used in the production of fructo-oligosaccharides that are commonly used by the food industry as prebiotics for their purported health benefits. The β-fructofuranosidase discussed herein is obtained from a novel source organism that is a non-genetically modified strain of Aspergillus brunneoviolaceus, which phylogenetically belongs to the Aspergillus section Nigri. The safety of β-fructofuranosidase was evaluated in a series of toxicology studies as prescribed by Tier 1 toxicity testing by the European Food Safety Authority, including an evaluation of the mutagenicity and genotoxicity potential using the in vitro bacterial reverse mutation and mammalian chromosomal aberration assays, as well as systemic toxicity in a 90-day oral subchronic toxicity study in Sprague-Dawley rats. β-Fructofuranosidase was demonstrated to lack mutagenic or genotoxic potential based on the results of the in vitro assays due to absence of increased revertant colonies in the bacterial reverse mutation test and incidence of chromosome aberrations in the chromosomal aberration assay. Administration of β-fructofuranosidase by gavage at doses up to 1200 mg total organic solids (TOS)/kg body weight/day for 90 days did not elicit any systemic toxic effects in rats based on a lack of adverse effect in any study parameter, and therefore the no-observed-adverse-effect level of β-fructofuranosidase was concluded to be 1200 mg TOS/kg body weight/day, the highest dose tested. The results of the toxicology studies on β-fructofuranosidase from A. brunneoviolaceus demonstrate this species to be a safe and suitable source of enzymes for use by the food industry.