The Incorporation of Lysine into the Peroxidase Peptide Reactivity Assay for Skin Sensitization Assessments

Abstract

To establish further a practical quantitative in chemico reactivity assay for screening contact allergens, lysine peptide was incorporated into a liquid chromatography and tandem mass spectrometry-based assay for reactivity assessments of hapten and pre-/pro-hapten chemical sensitizers. Loss of peptide was determined following 24 h coincubation with test chemical using a concentration-response study design. A total of 70 chemicals were tested in discrete reactions with cysteine or lysine peptide, in the presence and absence of horseradish peroxidase-hydrogen peroxide oxidation system. An empirically derived prediction model for discriminating sensitizers from nonsensitizers resulted in an accuracy of 83% for 26 haptens, 19 pre-/pro-haptens, and 25 nonsensitizers. Four sensitizers were shown to selectively react with lysine including two strong/extreme and two weak sensitizers. In addition, seven sensitizers were identified as having higher reactivity toward lysine compared with cysteine. The majority of sensitizing chemicals (27/45) were reactive toward both cysteine and lysine peptides. An estimate of the relative reactivity potency was determined based on the concentration of test chemical that depletes peptide at or above a threshold positive value. Here, we report the use of EC15 as one example to illustrate the use of the model for screening the skin sensitization potential of novel chemicals. Results from this initial assessment highlight the utility of lysine for assessing a chemical's potential to elicit sensitization reactions or induce hypersensitivity. This approach has the potential to qualitatively and quantitatively evaluate an important mechanism in contact allergy for hazard and quantitative risk assessments without animal testing.

A roadmap for the development of alternative (non-animal) methods for systemic toxicity testing

“…There are several assay systems from which to choose, including the following: peptide binding assays (Gerberick et al, 2004(Gerberick et al, , 2009Troutman et al, 2011) and based upon the Nrf2/Keap 1 electrophile-sensing pathway the KeratinoSens assay ; and the CeeTox Assay (McKim et al, between stimulatory and regulatory signals from DC, the balance achieved between effector T lymphocytes and Treg cells, and the impact of the kinetics and selectivity of the interaction of chemical allergens with skin proteins are all strong candidates with regard to influencing sensitizing potency.…”

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.…”

Advanced tests for skin and respiratory sensitization assessment

“…In addition to hazard identification (yes/no), the evaluation of the correlation between reactivity and sensitizing potency of chemicals seems promising (Gerberick et al, 2007b). Currently, there is ongoing research to further improve the DPRA by incorporating an oxidative activation step in order to allow for the identification of pro-haptens as well (Gerberick et al, 2009;Troutman et al, 2011) (Fig. 5), which did not show any activity in the DPRA so far.…”