Investigating sensitization activity of azobenzene disperse dyes via the Direct Peptide Reactivity Assay (DPRA)

Azobenzene disperse dyes are the fastest-growing category of commercial dyestuffs and have been found in indoor house dust and in children's polyester apparel. Azobenzene disperse dyes are implicated as potentially allergenic; however, little experimental data is available on allergenicity of these dyes. Here, we examine the binding of azobenzene disperse dyes to nucleophilic peptide residues as a proxy for their potential reactivity as electrophilic allergenic sensitizers. The Direct Peptide Reactivity Assay (DPRA) was utilized via both a spectrophotometric method and a high-performance liquid chromatography (HPLC) method. We tested dyes purified from commercial dyestuffs as well as several known transformation products. All dyes were found to react with nucleophilic peptides in a dose-dependent manner with pseudo-first order kinetics (rate constants as high as 0.04 h^-1). Rates of binding reactivity were also found to correlate to electrophilic properties of dyes as measured by Hammett constants and electrophilicity indices. Reactivities of polyester shirt extracts were also tested for DPRA activity and the shirt extracts with high measured abundances of azobenzene disperse dyes were observed to induce greater peptide reactivity. Results suggest that azobenzene disperse dyes may function as immune sensitizers, and that clothing containing these dyes may pose risks for skin sensitization.