Characterization of metabolites of genotoxic 4,4′-aminoalkoxyazobenzene dyes

Abstract

Invariably these days the molecular design of non-genotoxic azo dyes takes into consideration the potential genotoxicity of aromatic amines arising from reductive cleavage of the azo bonds (–NN–), with those dyes producing carcinogenic amines no longer suitable for commerce. Much less is known about structural factors leading to genotoxic azo dyes having their –NN– bonds intact. This point came to the forefront when it was determined that hydrophobic 4,4′-aminoalkoxyazobenzene dyes such as 4-(3-(2-hydroxyethoxy-4-amino)phenylazo)-N,N-bis(2-hydroxyethyl)aniline are mutagenic but their reductive-cleavage products are not. Consequently, a study that was undertaken to unveil the basis for the mutagenic activity of such dyes. The initial study involved the synthesis and evaluation of the mutagenicity of a group of substituted 4,4′-diaminoalkoxyazobenzene dyes, to test our hypothesis which stated that the mutagenicity of 4-(3-(2-hydroxyethoxy-4-amino)phenylazo)-N,N-bis(2-hydroxyethyl)aniline arises from the metabolic cleavage of N-hydroxyethyl groups in the parent dye, leading ultimately to 4-(3-(2-hydroxyethoxy-4-amino)phenylazo)aniline. The key goal of the present work was to provide underpinning for results from mutagenicity testing, by isolating and identifying metabolites from S9 treatments. In this regard, title dyes such as 4-(3-(2-hydroxyethoxy-4-amino)phenylazo)-N,N-bis(2-hydroxyethyl)aniline were treated with rat or hamster liver S9 in the absence of Salmonella typhimurium and results from TLC and HPLC analysis of product mixtures from S9 treatments indicated that metabolism involved N-dehydroxyethylation to give 4-(3-(2-hydroxyethoxy-4-amino)phenylazo)-N-(2-hydroxyethyl)-aniline and 4-(3-(2-hydroxyethoxy-4-amino)phenylazo)aniline when rat liver S9 was used and reductive cleavage of the azo bonds to give the corresponding aromatic amines was also observed when hamster liver S9 was used. The analysis of product mixtures further indicated that rat liver S9 metabolism involved deacetylation of O-acetyl groups in the case of 4-(3-(2-hydroxyethoxy-4-amino)phenylazo)-N,N-bis(2-acetoxyethyl)aniline.