Characterizing Exposure and In Vitro Effects of Azobenzene Disperse Dyes in the Indoor Environment

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Azobenzene disperse dyes are the fastest-growing category of commercial dyestuffs, accounting for 70% of the 9.9 million tons of industrial dye colorants used annually. Azobenzene disperse dyes are intended to be applied to synthetic fabrics such as polyester, nylon, and acrylic; however, azo dyes may also be used in cosmetic products such as hair dyes, and in fashion accessories such as leather goods. Recently, our group and others have detected azobenzene disperse dyes in dust particles collected from the indoor environment, and raising concerns about the release of these chemicals from products and human exposure. Although extensive literature characterizes these chemicals as toxic contaminants in aquatic environments, to date there exists little data on levels, exposures, and hazards associated wit exposures to azobenzene disperse dyes in the indoor environment. The presence of these dyes in the indoor environment is concerning. House dust is a sink for many contaminants that leach out or off-gas from products in the home. Due to children’s unique behaviors (e.g. crawling and hand to mouth activity) they have higher exposure to chemicals associated with dust. Azobenzene disperse dyes are implicated in literature as potentially allergenic: they are known to be present in clothing that elicits allergic reactions such as skin sensitization. Therefore, it is of crucial importance to support research that seeks to characterize children’s exposure in the home environment, and evaluate the in vitro effects of azobenzene disperse dyes. The hypothesis of this research dissertation is that azobenzene disperse dyes are prevalent in dust collected from the indoor environment at concentrations of concern for human health. In the first aim of this thesis research, azobenzene disperse dyes were characterized in commercial mixtures and in children’s polyester clothing. Azo dyes were first purified from dyestuffs by Soxhlet extraction and flash chromatography and then analyzed using ultra-high-performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry (HRMS), as well as by 1H and 13C NMR for structural elucidations. Nineteen total azobenzene dyes were detected in dyestuffs via a non-targeted analysis approach, including Disperse Blue 79:1, Disperse Blue 183:1, Disperse Orange 44, Disperse Orange 73, Disperse Red 50, Disperse Red 73, and Disperse Red 354. Samples of children's polyester clothing (n=X) were then analyzed via UHPLC-HRMS. In clothing, 21 azobenzene disperse dyes were detected, 12 of which were confirmed and quantified via reference standards. Individual dyes in apparel were quantified at concentrations up to 9230 μg dye/g shirt, with geometric means ranging 7.91–300 μg dye/g shirt. Total dye load in apparel was quantified at up to 11,430 μg dye/g shirt. This research supported the development of reference standards and library mass spectra for azobenzene disperse dyes previously absent from standard and spectral libraries. This study was the first to confirm and quantify these azo compounds in children’s products, facilitating a more robust understanding of sources of azobenzene disperse dyes in the indoor environment. The second aim of this thesis research investigated the presences and quantities of azobenzene disperse dyes and related compounds in indoor house dust (n=188) collected from homes in Durham, NC. Using a targeted approach, we quantified 12 azo disperse dyes and quantified at least one dye in every house dust sample. Detection frequencies ranged from 11% to 89%; of the dyes that were detected in at least 50% of the samples, geometric mean levels ranged from 32.4 to 360 ng/g. HRMS suspect screening analysis identified an additional eight azobenzene compounds in dust that are present at high relative abundances. This study indicates that azo disperse dyes and related compounds are ubiquitous in the indoor environment. To support quality assurance and control during the analysis, a house dust Standard Reference Material (NIST SRM 2585) was extracted and analyzed with the samples. Based on the detection and abundance of azo dyes in SRM 2585, which was prepared from hundreds of dust samples collected in the mid 1990s, azo dye levels in the indoor environment may be increasing over time. To our knowledge, this is the most comprehensive quantitative study of azo disperse dyes in house dust to date. Future studies are needed to quantify additional dyes in dust, particularly those identified here via suspect screening, and to examine exposure pathways of dyes in the indoor environment where children are concerned. The third aim of this thesis research examined the binding reactivity of azobenzene disperse dyes to nucleophilic peptide residues in order to understand 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. Dyes isolated from the commercial dyestuffs, and several potential transformation products, were tested. All dyes were found to react with nucleophilic peptides in a dose-dependent manner with pseudo-first order (kobserved) activity, but overall to react more potently with cysteine than with lysine: EC10 values for cysteine binding were determined as low as 0.005mM and pseudo-first order rate constants as high as 0.04 hr-1 (as observed for Disperse Blue 79:1). Observed rate constants were correlated to metrics of structural features such as Hammett constants and electrophilicity indices, indicating that binding reactivity may be related to structural properties of azobenzene disperse dyes. In addition to examining dyes, the reactivity of extracts of polyester shirts were also examined; shirt extracts with high relative 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 azobenzene disperse dyes may pose risks for skin sensitization. Collectively, this thesis research suggests that azobenzene disperse dyes are common in clothing, and appear to be near ubiquitous in house dust. Given their reactivity in vitro, this may present health consequences, particularly for young children.






Overdahl, Kirsten Elise (2021). Characterizing Exposure and In Vitro Effects of Azobenzene Disperse Dyes in the Indoor Environment. Dissertation, Duke University. Retrieved from


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