Rapid and Simple Kinetics Screening Assay for Electrophilic Dermal Sensitizers Using Nitrobenzenethiol

Abstract

The need for alternatives to animal based skin sensitization testing has spurred research on the use of in-vitro, in silico and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped flow techniques and conventional UV spectrophotometric measurements enabled determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for 7 extreme, 5 strong, 7 moderate and 4 weak/non-sensitizers. 17 out of the 23 tested chemicals were pseudo-first order and 3 were second order. In 3 out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, diones and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid and inexpensive absorbance based method has great potential for use as a preliminary screening tool for skin allergens.

Pyridoxylamine reactivity kinetics as an amine based nucleophile for screening electrophilic dermal sensitizers

“…Both NBT and PDA rate constants resulted in the regression equations for NBT (Chipinda et al, 2010) and PDA (equation (N10)) which were used to calculate the predicted pEC3 values for the chemicals listed in Table 4. Fig.…”

“…The use of 4-nitrobenzenethiol (NBT), which is a “soft” thiol based nucleophile, to quantify reactivity of more than 20 electrophilic skin sensitizers from different mechanistic domains was previously reported from our laboratory (Chipinda et al, 2010) where correlation of reactivity to LLNA potency was demonstrated across all domains. NBT reactivity to Schiff Base Formers and diones was predictably absent as these chemicals are harder electrophiles with preferential reactivity to amine based nucleophiles.…”

Criteria for the Research Institute for Fragrance Materials, Inc. (RIFM) safety evaluation process for fragrance ingredients

“…In Step 2, read-across is utilized for those substances for which there is sufficient sensitization test data on other close structural analogues. The validity of this read-across approach may be supported by in vitro studies such as the direct peptide reactivity assay (Gerberick et al, 2007, Gerberick et al, 2004, keratinocyte assays (Natsch et al, 2007, Natsch, 2010, Natsch, 2011 or other comparable in vitro assays (Aptula, 2006a, Aleksic et al, 2009, Natsch and Gfeller, 2008, Troutman et al, 2011, Goebel et al, 2012, Ade, 2006, Chipinda et al, 2010, Gerberick et al, 2007, Gerberick et al, 2009, Rousset, 2002. However, for the purpose of potency assessment, a quantitative relationship has to be demonstrated between the parameter measured and the known potency for the mechanistic domain being assessed.…”

In Vitro Approaches for Detection of Chemical Sensitization

“…Non-electrophilic binding occurs through disulfide exchange or co-ordination bonds [10]. The reaction rate and mechanism by which the hapten is reacting with the nucleophilic groups on the protein influence its allergenic potency [11,12]. In vitro studies suggest that the specificity of the covalent modification is time dependent and dose dependent and that the target proteins in vitro become more general and non-discriminative over time and with increasing concentrations of chemical [13].…”