Refining the Amino Reactivity-Based Identification of Respiratory Sensitizers

The sensitization of the respiratory tract may lead to various pulmonary diseases such as asthma. It can be triggered by the chemical reaction of organic electrophiles with nucleophiles of lung proteins with amino groups being of particular interest in this case. For assessing the dermal sensitization potential of chemicals, the direct peptide reactivity assay (DPRA) has become an OECD-accepted nonanimal test system. However, issues with the identification of known respiratory sensitizers such as isocyanates and anhydrides based on their amino reactivity in the DPRA have been reported. Hence, in this study the chemoassay employing glycine-para-nitroanilide (Gly-pNA) as model nucleophile is applied to eight iso(thio)cyanates, seven anhydrides, four dinitrobenzenes, one triazine, five acrylates, glutaraldehyde, and chloramine T to quantify their amino reactivity in terms of the second order rate constant k_Gly and the DPRA-like 24 h percent depletion D_Gly. A comparison of D_Gly with respective DPRA amino reactivity data (D_DPRA) showed that in particular iso(thio)cyanates and anhydrides are substantially more reactive toward Gly-pNA. This can be rationalized by the unintentional and so far not considered reaction of the test compounds with the ammonium acetate buffer used for DPRA testing. A detailed analysis of this reaction includes half-lives and analytically determined adduct patterns and indicates that it can hamper the envisaged depletion of the DPRA amino nucleophile. Finally, the obtained log k_Gly values range from -3.73 to ≥ 4.52 and allow for an improved identification of respiratory sensitizers. Hence, the Gly-pNA chemoassay may serve as a nonanimal screening method as one part of a mechanism-informed integrated testing and assessment strategy for respiratory sensitizers.