Structure Elucidation and in Vitro Reactivity of the Major Metabolite of 4,4′-Methylenebis(2-chloroaniline) (MBOCA) in Canine Urine

Abstract

Structure Elucidation and in Vitro Reactivity of the Major Metabolite of 4,4′-Methylenebis(2-chloroaniline) (MBOCA) in Canine Urine. Manis, M. O., and Braselton, W. E. Jr. (1984). Fundam. Appi. Toxicol. 4, 1000–1008. The structure of the major urinary metabolite of 4,4′-methylenebis(2-chloroaniline) (MBOCA) in dogs was identified and the reactivity of the metabolite was characterized in vitro. Arylsulfatase but not β-glucuronidase hydrolyzed the metabolite in a time- and enzyme concentration-dependent manner. Electron impact mass spectrometry following derivatization and transesterification indicated that the major metabolite was ring hydroxylated and fas atom bombardment mass spectrometry confirmed the molecular weight as a sulfate ester. Proton nuclear magnetic resonance studies indicated that the ring substitution was ortho to an arnine. These analytical and enzymatic data supported the proposed structure of the major urinary metabolite of MBOCA in dogs as 5-hydroxy-3,3′-dichloro-4,4′-diaminodiphenylmethane-5-sulfate. Protein and DNA binding in vitro and mutagenicity were investigated. During hydrolysis with arylsulfatase, time- and enzyme concentration-dependent protein binding and time-dependent DNA binding were observed. Mutagenicity during enzymatic hydrolysis in the presence of Salmonella typhimurium TA1538 with up to 20 μg metabolite/plate was negative and at 50 μ/plate the metabolite was cytotoxic. These results indicated that the metabolite was the sulfate conjugate of a reactive molecule. This study demonstrated that the major metabolite of MBOCA in canine urine is an ortho-hydroxysulfale and thus is similar to the major metabolites of bcnzkline, 2-naphthylamine, and 4-aminobiphenyl