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

Abstract 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 Benzonatatehas been demonstrated over the last 60 years, but its safety was not fully assessed. In this study, we tested the genotoxicity of Benzonatateand its major metabolite BBA in an in vitro bacterial reverse mutation and in vivo micronucleusassays. A chromosomal aberration assay was also performed on Benzonatateand BBA. In the reverse mutation assay, Benzonatateand BBA doses 1.5–5000 μg/plate ± 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 Benzonatateand BBA concentrations 25–2000 and 62.5–1930 μg/mL, respectively. A CByB6F1 mouse bone marrow micronucleusassay 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 Benzonatatenor 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. Benzonatateand 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, Benzonatateand 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.