An investigation into the formation of tebufenozide's toxic aromatic amine metabolites in human in vitro hepatic microsomes

Abstract

Tebufenozide is a nonsteroid ecdysone agonist that causes premature and incomplete molting in Lepidoptera. Studies conducted so far have shown the low toxicity of tebufenozide in mammals, birds and invertebrates. Tebufenozide potential metabolites such as aromatic amines are known to induce methemoglobinemia disorder in humans, most likely by the formation of N-hydroxy metabolites; therefore, the aim of this research is to investigate the formation of the potential toxic N-hydroxy derivatives in pooled human hepatic microsomal fractions. Analyses of metabolites by high performance liquid chromatography equipped by a time-of-flight detector (HPLC/TOF) indicated the formation of a hydroxylated metabolite (exact mass=369; retention time: 6.65min) and two de-dimethylethyl metabolites (exact masses=313; retention times: 5.76 and 6.22min). Hydroxylated tebufenozide metabolite resulted from hydroxylation at either the 3 or 5 position of the dimethylbenzoic acid moiety to form either 3-hydroxymethyl-5-methylbenzoic acid 1-(1,1-dimethylethyl)-2-(4-ethylbenzoyl) or 3-methyl-5-hydroxymethylbenzoic acid 1-(1,1-dimethylethyl)-2-(4-ethylbenzoyl), respectively. The two de-dimethylethyl-tebufenozide derivatives were 3,5-dimethylbenzoic acid-2-(4-hydroxyethylbenzoyl) and 3-hydroxymethyl-5-methylbenzoic acid-2-(4-ethylbenzoyl) or 3-methyl-5-hydroxymethylbenzoic acid-2-(4-ethylbenzoyl). Generally the metabolite formation rates increased with incubation time. The rate of hydroxylation of the dimethylbenzoic acid moiety was approximately 12 times higher than the hydroxylation of the ethylbenzoyl moiety. Tebufenozide does not appear to produce the toxic aromatic amine metabolites in human in vitro hepatic microsomes. This suggests that the fate of tebufenozide in humans is a process of detoxification rather than activation.