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Due to the current ban on animal testing, the need for robust and reliable animal alternative test methods is critical. Reactivity of chemical allergens with proteins has long been established as a key step in induction of skin sensitization. Based on this, two in chemico approaches have been developed: the Direct Peptide Reactivity Assay (DPRA) and the Peroxidase Peptide Reactivity Assay (PPRA). Both assays utilize synthetic peptides which contain either a single cysteine or lysine amino acid as its nucleophilic center for assessing skin sensitization potential of chemicals. Chemical reactivity is determined by using analytical methods to measure the depletion of free peptide following a 24-h incubation of test chemical and peptide. By comparing reactivity data to local lymph node assay data, prediction models have been developed for both assays. These models allow for making hazard predictions and binning a chemical into reactivity categories. The DPRA has been thoroughly evaluated for its reproducibility, transferability, and accuracy under formal validation studies. The PPRA has not yet reached validated status but is undergoing interlaboratory evaluation. Although showing good correlation to established animal models, the data obtained from both of these assays should be considered in combination with other information in the context of integrated approaches such as weight of evidence or integrated testing strategies.

It is well known that some chemicals are capable of causing allergic diseases of the skin and respiratory tract. Commonly, though not exclusively, chemical allergens are associated with the selective development of skin or respiratory sensitization. The reason for this divergence is unclear, although it is hypothesized that the nature of interactions between the chemical hapten and proteins is influential. The direct peptide reactivity assay (DPRA) has been developed as a screen for the identification of skin-sensitizing chemicals, and here we describe the use of this method to explore whether differences exist between skin and respiratory allergens with respect to their peptide-binding properties. Known skin and respiratory sensitizers were reacted with synthetic peptides containing either lysine (Lys) or cysteine (Cys) for 24 h. The samples were analyzed by HPLC/UV, and the loss of peptide from the reaction mixture was expressed as the percent depletion compared with the control. The potential for preferential reactivity was evaluated by comparing the ratio of Lys to Cys depletion (Lys:Cys ratio). The results demonstrate that the majority of respiratory allergens are reactive in the DPRA, and that in contrast to most skin-sensitizing chemicals, preferentially react with the Lys peptide. These data suggest that skin and respiratory chemical allergens can result in different protein conjugates, which may in turn influence the quality of induced immune responses. Overall, these investigations reveal that the DPRA has considerable potential to be incorporated into tiered testing approaches for the identification and characterization of chemical respiratory allergens.

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.

Skin protein reactivity is a well established key step in the development of skin sensitization. Understanding the relationship between a chemical's ability to react with or modify skin protein and skin sensitization has led to the development of the Direct Peptide Reactivity Assay (DPRA) in our laboratory. A current limitation of the DPRA is that it cannot readily measure the reactivity of pro-hapten chemical sensitizers. Pro-haptens are chemical sensitizers that are not directly reactive and must be bioactivated in vivo to form an electrophilic intermediate(s). Results from this work demonstrate the utility of using horseradish peroxidase and hydrogen peroxide (HRP/P) for assessing the skin sensitization potential of pro-haptens. In comparison with "direct" reactivity assessments without HRP/P, statistically significant increases in peptide depletion for all pro-haptens examined were observed following coincubation with HRP/P. Conversely, the percent peptide depletion for all pre-haptens was equally high (> 40% depletion) with and without HRP/P demonstrating an auto-oxidation pathway. In contrast, peptide depletion for all nonsensitizing chemicals examined was low with and without HRP/P. The optimal HRP/P concentrations, incubation time and optimal peptide:chemical ratio were determined using a sensitive and selective high-performance liquid chromatography tandem mass spectrometry detection method. Dithiothreitol was incorporated to reverse the dimerization of the thiol-containing cysteine peptide nucleophile. This preliminary work shows the potential to incorporate an enzyme-mediated activation step for pro-haptens into an in chemico skin sensitization assay that results in the detection of all types of sensitizers.

Assessment of skin sensitization hazard of chemicals currently depends on in vivo methods. Considering the forthcoming European Union ban on in vivo testing of cosmetic/toiletry ingredients, the search for alternative non-animal approaches is an urgent challenge for investigators today. For the skin sensitization end-point the concept of protein/peptide haptenation, that could reflect the chemical modification of skin proteins, crucial to form immunogenic structures, has been used to develop in vitro assays to predict the sensitization potential of new chemicals. Using glutathione and nucleophile-containing synthetic peptides we confirmed previously the possibility to screen for skin sensitization potential by measuring peptide depletion following incubation with a set of allergens and non-allergens. In this paper, additionally to our model development work, we performed mechanistic based studies to confirm the peptide reactivity concept under the specific conditions used for haptens in the screening assay as they were somewhat different from the ones expected to happen in vivo. Following the reactivity toward the peptides of 13C labelled MI and MCI, models of true haptens, we showed that the initial step leading to the biological end-point was similar regardless the conditions used even if final adducts could be different. This confirmed the validity of the peptide reactivity concept as well as the choice made to look at peptide depletion rather than at adduct formation.

In the interest of reducing animal use, in vitro alternatives for skin sensitization testing are under development. One unifying characteristic of chemical allergens is the requirement that they react with proteins for the effective induction of skin sensitization. The majority of chemical allergens are electrophilic and react with nucleophilic amino acids. To determine whether and to what extent reactivity correlates with skin sensitization potential, 82 chemicals comprising allergens of different potencies and nonallergenic chemicals were evaluated for their ability to react with reduced glutathione (GSH) or with two synthetic peptides containing either a single cysteine or lysine. Following a 15-min reaction time with GSH, or a 24-h reaction time with the two synthetic peptides, the samples were analyzed by high-performance liquid chromatography. UV detection was used to monitor the depletion of GSH or the peptides. The peptide reactivity data were compared with existing local lymph node assay data using recursive partitioning methodology to build a classification tree that allowed a ranking of reactivity as minimal, low, moderate, and high. Generally, nonallergens and weak allergens demonstrated minimal to low peptide reactivity, whereas moderate to extremely potent allergens displayed moderate to high peptide reactivity. Classifying minimal reactivity as nonsensitizers and low, moderate, and high reactivity as sensitizers, it was determined that a model based on cysteine and lysine gave a prediction accuracy of 89%. The results of these investigations reveal that measurement of peptide reactivity has considerable potential utility as a screening approach for skin sensitization testing, and thereby for reducing reliance on animal-based test methods.