Genetic Toxicity Studies of the Ketogenic Ester Bis Hexanoyl (R)-1,3-Butanediol.

A series of studies was conducted to assess the genetic toxicity of a novel ketone ester, bis hexanoyl (R)-1,3-butanediol (herein referred to as BH-BD), according to Organization for Economic Co-operation and Development testing guidelines under the standards of Good Laboratory Practices. In bacterial reverse mutation tests, there was no evidence of mutagenic activity in any of the Salmonella typhimurium strains tested or in Escherichia coli strain WP2uvrA, at dose levels up to 5,000 µg/plate in the presence or absence of Aroclor 1254-induced rat liver (S9 mix) for metabolic activation. In the in vitro micronucleus test using human TK6 cells, BH-BD did not show a statistically significant increase in the number of cells containing micronuclei when compared with concurrent control cultures at all time points and at any of the concentrations analyzed (up to 100 µg/mL, final concentration in culture medium), with and without S9 mix activation. In the in vivo micronucleus test using Sprague Dawley rats, BH-BD did not show a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to the vehicle control group. Therefore, BH-BD was concluded to be negative in all 3 tests. These results support the safety assessment of BH-BD for potential use in food.

Genotoxicity assessment of the novel antitussive agent Benzonatate and its major metabolite.

Benzonatate (TESSALON®) is a peripherally acting oral antitussive. It undergoes rapid ester hydrolysis producing 4-(butylamino) benzoic acid (BBA) and methylated polyethylene glycol (MPG) metabolites, which are eliminated in urine and feces. The nonclinical and clinical efficacy of Benzonatate has been demonstrated over the last 60 years, but its safety was not fully assessed. In this study, we tested the genotoxicity of Benzonatate and its major metabolite BBA in an in vitro bacterial reverse mutation and in vivo micronucleus assays. A chromosomal aberration assay was also performed on Benzonatate and BBA. In the reverse mutation assay, Benzonatate and BBA doses 1.5-5000 µg/plate ±â€¯S9 metabolic activation were used and the numbers of revertants/plate were compared to various controls. Chromosomal aberration assays with human peripheral blood lymphocytes used Benzonatate and BBA concentrations 25-2000 and 62.5-1930 µg/mL, respectively. A CByB6F1 mouse bone marrow micronucleus assay was performed as part of a 28-day oral toxicology study at up to 250 mg/kg/day. The frequencies of micronuclei in polychromatic erythrocytes in treated groups were compared with the control group. Neither Benzonatate nor BBA induced significant mutagenicity in any of the bacterial strains, with or without metabolic activation. They also did not produce any biologically relevant structural or numerical aberrations in human chromosomes. Benzonatate and its BBA and MPG metabolites rapidly produced from esterase activity did not produce any significant increase in the incidence of micronucleated polychromatic erythrocytes. In conclusion, Benzonatate and its major metabolite BBA were not mutagenic and did not cause numerical or structural chromosome alterations. While the MPG metabolite was not tested, studies on structural analogues indicated it was also unlikely to be genotoxic. This was supported by oral rodent carcinogenicity assays showing no increase in malignancies.

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON).

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.

Bacterial mutagenicity assays: Vehicle and positive control results from the standard Ames assay, the 6- and 24-well miniaturized plate incorporation assays and the Ames II™ assay.

Bacterial mutation assays are conducted routinely as part of the safety assessment of new chemicals. The OECD Test Guideline (TG) 471 describes the conduct of the standard agar plate Ames assay, required for regulatory submissions. Higher throughput non-OECD 471 TG assays, such as the miniaturized plate incorporation and Ames II™ assays, can be used for prescreening purposes. We have compiled historical vehicle and positive control data generated using these methods. The historical database is comprised from experiments spanning 9 years and includes >1000 experiments from the standard Ames assay using the plate incorporation and pre-incubation methods (TA98, TA100, TA1535, TA1537, and WP2 uvrA), >50 experiments from the 6-well (TA98, TA100, TA1535, TA97a, and WP2 uvrA) and >100 experiments from the 24-well (TA98, TA100, TA102, TA1535, TA1537, and TA97a) plate incorporation assays, and >1000 experiments from the Ames II™ assay (TA98 and TAMix). Although miniaturization to a 24-well format made the measurement of control revertant colonies in TA1537 and TA1535 more difficult; this can be overcome by using an alternative strain with a higher spontaneous reversion rate (i.e., using TA97a instead of TA1537) or by increasing the number of replicate wells to 12 (for TA1535). All three miniaturized methods, including the Ames II™ assay, were responsive to known mutagens and the responses were reproducible over years of use. These data demonstrate the excellent reproducibility of the standard and miniaturized bacterial mutation assays using positive control chemicals. Environ. Mol. Mutagen. 57:483-496, 2016. © 2016 Wiley Periodicals, Inc.

Safety evaluation of the human-identical milk monosaccharide, l-fucose.

l-Fucose is a natural monosaccharide present in mammals where it is found predominantly as an O-glycosidically linked component of glycoproteins, glycolipids, and oligosaccharides. It is also present in its free form in human breast milk (human milk monosaccharide). l-Fucose plays important roles in the development of the immune and nervous systems and is involved in cognitive function and memory formation. The human-identical milk monosaccharide l-fucose is therefore proposed for use in infant formulas to better simulate the free saccharides present in human breast milk. As part of the safety evaluation of l-fucose, a subchronic dietary toxicity study preceded by an in utero phase was conducted in Sprague-Dawley rats. l-Fucose was without maternal toxicity or compound-related adverse effects on female reproduction and general growth and development of offspring at a maternal dietary level up to 1%, equivalent to a dose of 1655 mg/kg body weight (bw)/day. During the subchronic phase, no compound-related adverse effects were observed in first generation rats at dietary levels of up to 1% (highest level tested), corresponding to doses of 516 and 665 mg/kg bw/day in males and females, respectively. l-Fucose was non-genotoxic in a series of in vitro genotoxicity/mutagenicity tests. These results support the safe use of l-fucose in infant formula and as a food ingredient at levels equivalent to those present in human breast milk.

Toxicology studies with N-acetylglycine.

N-acetylglycine (NAGly) has been identified as a minor constituent of numerous foods. The current paper reports the outcome of in vitro and in vivo genotoxicity, acute oral and repeated dose dietary toxicology studies conducted with NAGly. No evidence of genotoxicity was observed with NAGly in vitro bacterial tester strains or in vivo bone marrow micronucleus studies conducted in mice. No mortalities or evidence of adverse effects were observed in Sprague-Dawley rats following acute oral gavage with NAGly at a dose of 2000 mg/kg of body weight or following repeated dose dietary exposure to NAGly at targeted doses of 100, 500, or 1000 mg/kg of body weight/day for 28 days. No biologically significant or test substance related differences were observed in body weights, feed consumption, or clinical pathology response variables in any of the treatment groups. Based on these results it was concluded that NAGly is not genotoxic or acutely toxic. Further, the no-observed adverse-effect-level (NOAEL) for systemic toxicity from repeated dose dietary exposure to NAGly was 898.9 mg/kg of body weight/day for male rats and 989.9 mg/kg of body weight/day for female rats.