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

Structure Elucidation and in Vitro Reactivity of the Major Metaboliteof 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 urinarymetabolite of 4,4'-methylenebis(2-chloroaniline) (MBOCA) indogs was identified and the reactivity of the metabolite wascharacterized in vitro. Arylsulfatase but not ß-glucuronidasehydrolyzed the metabolite in a time- and enzyme concentration-dependentmanner. Electron impact mass spectrometry following derivatizationand transesterification indicated that the major metabolitewas ring hydroxylated and fas atom bombardment mass spectrometryconfirmed the molecular weight as a sulfate ester. Proton nuclearmagnetic resonance studies indicated that the ring substitutionwas ortho to an arnine. These analytical and enzymatic datasupported the proposed structure of the major urinary metaboliteof 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-dependentprotein binding and time-dependent DNA binding were observed.Mutagenicity during enzymatic hydrolysis in the presence ofSalmonella typhimurium TA1538 with up to 20 µg metabolite/platewas negative and at 50 µ/plate the metabolite was cytotoxic.These results indicated that the metabolite was the sulfateconjugate of a reactive molecule. This study demonstrated thatthe major metabolite of MBOCA in canine urine is an ortho-hydroxysulfaleand thus is similar to the major metabolites of bcnzkline, 2-naphthylamine,and 4-aminobiphenyl