Peptide Reactivity Assay Research Articles

In chemico assessment of potential sensitizers: Stability and direct peptide reactivity of 24 fragrance ingredients.

Twenty-four pure fragrance ingredients of concern as potential skin sensitizers were previously subjected to degradation studies and evaluated using the high throughput with dansyl cysteamine (HTS-DCYA) method. The experimental results showed that two-thirds of the 24 fragrance ingredients underwent chemical degradation. In some cases, such degradation was accompanied by an increase in thio-reactivity. These results prompted us to investigate the reactivity of the same ingredients using the direct peptide reactivity assay (DPRA). In the present work, the 24 chemicals were subjected to forced degradation for 150days, and evaluated with both DPRA and HTS-DCYA methods. At the end of the study, four and eight compounds remained non-reactive in the DPRA and DCYA assay, respectively. Coumarin, benzyl salicylate, benzyl cinnamate and hexyl cinnamal were found unreactive in both assays, while cinnamal, cinnamyl alcohol, hydroxycitronellal and lilial were found negative in the DCYA but positive in the DPRA method. The incongruity in reactivity of these four compounds was attributed to a possible role of pro-oxidants formed upon degradation, resulting in depletion of peptide without formation of apparent covalent adducts with the test chemical. To validate this hypothesis, the effect of hydrogen peroxide as model pro-oxidant on both lysine- and cysteine-heptapeptide depletion in the DPRA method was thus investigated. The obtained results showed little effect of oxidative conditions on lysine depletion, while cysteine depletion was significantly affected by concentrations above 1.1mg/L of hydrogen peroxide. Overall, both in chemico methods confirmed chemical instability should be considered when assessing the skin sensitization potential of (un)known chemicals with alternative methods.

Cause of and countermeasures for oxidation of the cysteine-derived reagent used in the amino acid derivative reactivity assay.

The amino acid derivative reactivity assay (ADRA) is an in chemico alternative to animal testing for skin sensitization that solves certain problems found in the use of the direct peptide reactivity assay (DPRA). During a recent validation study conducted at multiple laboratories as part of the process to include ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in ADRA-N-(2-(1-naphthyl)acetyl)-l-cysteine (NAC)-was found to be susceptible to oxidation in much the same manner that the cysteine peptide used in DPRA was. Owing to this, we undertook a study to clarify the cause of the promotion of NAC oxidation. In general, cysteine and other chemicals that have thiol groups are known to oxidize in the presence of even minute quantities of metal ions. When metal ions were added to the ADRA reaction solution, Cu2+ promoted NAC oxidation significantly. When 0.25μm of EDTA was added in the presence of Cu2+ , NAC oxidation was suppressed. Based on this, we predicted that the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we tested 82 chemicals used in developing ADRA to determine whether EDTA affects ADRA's ability to predict sensitization. The results showed that the addition of EDTA has virtually no effect on the reactivity of NAC with a test chemical, yielding an accuracy of 87% for predictions of skin sensitization, which was roughly the same as ADRA.

Skin irritation and sensitization potential of oxidative hair dye substances evaluated with in vitro, in chemico and in silico test methods

Permanent oxidative hair dyes are widely used but their toxicity is not well-established. Here we aimed to evaluate the skin sensitization and irritation of nine hair dye substances (MAP, MRP-N, RS, PAOX, 2,4-DAPE, 2,6-PYR, PPD, Grey HED and PM) permitted for use in EU and Korea, using in vitro and in chemico and in silico test methods. Skin sensitization was evaluated by the KeratinoSens™ assay, Direct Peptide Reactivity Assay (DPRA) and DEREK. Six of nine dyes tested were determined as sensitizers in common. However, the decision for MAP, RS or PAOX was diverged across assays showing 2 positives and 1 negative. Skin irritation of hair dye substances was assessed with or without 6% H2O2 on a reconstructed human epidermis, Epiderm™, which demonstrated that H2O2 increased the skin irritation potential of some hair dyes. PPD and PM were determined to be irritants with H2O2. Epidermal damages by hair dye and H2O2 could be further confirmed through the histology of tissue remaining after MTT assay. Collectively, our study demonstrated that hair dyes possess potential skin sensitization and irritation issues which could be further aggravated by H2O2.

BASF, Givaudan seek animal-free testing

BASF and Givaudan say they have developed a test called the kinetic direct peptide reactivity assay to predict the potency of skin sensitizers without the use of animals. The two say other animal-free tests can give only a positive or negative indication but can’t determine potency, a requirement of some regulatory agencies. To validate the new test method, they have partnered with the Maryland-based nonprofit Institute for In Vitro Sciences, which should have results by the end of this year.

A highly sensitive and selective high pressure liquid chromatography with tandem mass spectrometry (HPLC/MS-MS) method for the direct peptide reactivity assay (DPRA)

While the HPLC/UV (high performance liquid chromatography coupled with ultra-violet spectrometry)-based DPRA (Direct Peptide Reactivity Assay) identifies dermal sensitizers with approximately 80% accuracy, the low selectivity and sensitivity of the HPLC/UV-based DPRA poses challenges to accurately identify the sensitization potential of certain chemicals. In this study, a high performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MS-MS)-based DPRA was developed and validated according to the test guideline (OECD TG 442C). The final results were compared with the results from the traditional HPLC/UV-based guideline DPRA. This HPLC/MS-MS-based DPRA demonstrated similar performance compared to HPLC/UV-based DPRA using known dermal sensitizers and non-sensitizers according to the test guideline (OECD TG 442C). Following the validation, a challenge set of chemicals with either overlapping retention time with peptides, or higher hydrophobicity or chemicals potentially forming non-covalent interactions with peptides were assessed for dermal sensitization potential using both methods and the results were compared to existing in vivo data. The HPLC/MS-MS-based DPRA correctly predicted these chemicals as sensitizers or non-sensitizers; however, the HPLC/UV-based DPRA resulted in false-positive predictions for hydrophobic substances, chemicals with UV peaks overlapping with those of the peptide(s), and compounds that non-covalently interact with the peptides. These findings demonstrate the broader applicability and better sensitivity and selectivity of the LC/MS-MS-based DPRA over the traditional HPLC/UV-based guideline DPRA.

Is a combination of assays really needed for non-animal prediction of skin sensitization potential? Performance of the GARD™ (Genomic Allergen Rapid Detection) assay in comparison with OECD guideline assays alone and in combination

To meet regulatory requirements, and avoid or minimize animal testing, there is a need for non-animal methods to assess the potential of chemicals to cause skin sensitization. It is widely assumed that no one test will be sufficient and that combined data from several assays spanning key events from the adverse outcome pathway will be required. This paper challenges that assumption. The predictive performance of a single assay, the Genomic Allergen Rapid Detection (GARD™) assay, was compared with the performance, singly and in combination, of three formally validated non-animal approaches that appear as OECD test guidelines: the direct peptide reactivity assay (DPRA), the ARE-Nrf2 luciferase test method, and the human cell line activation test (h-CLAT).It is shown here that GARD™ alone outperforms each of DPRA, ARE-Nrf2 luciferase or h-CLAT, alone or in any combination as a 2 out of 3 strategy, in terms of sensitivity, specificity and accuracy.Based on the datasets analysed here, the sensitivity and specificity of GARD™ alone are 90–92% and 79–84% (“2 out of 3”, 86% and 76%). Thus, in any situation where the 2 out of 3 strategy is considered adequate, GARD™ alone could be used with equal or better performance.

Development of a Profiler for Facile Chemical Reactivity Using the Open-Source Konstanz Information Miner

Abstract It is prudent to assess the chemical reactivity of compounds during early stages of product development, given that formation of covalent adducts between electrophilic compounds and biological nucleophiles serves as the molecular initiating event in a range of adverse outcome pathways (AOPs). In vitro assays such as the direct peptide reactivity assay (DPRA) have been developed to assess facile reactivity. However, these approaches still require test material and laboratory resources, are less conducive to rapid high-throughput screening than computational approaches, and have been developed with a primary focus on a single AOP (e.g., skin sensitization). Given the known set of specific mechanisms that drive facile chemical reactivity between electrophilic compounds and biological nucleophiles, we developed an in-house profiler to identify reactive moieties using an open-source Konstanz Information Miner (KNIME) workflow. This covered the chemical space for five known classes of chemical facile r...

A simple in chemico method for testing skin sensitizing potential of chemicals using small endogenous molecules

Among many of the validated methods for testing skin sensitization, direct peptide reactivity assay (DPRA) employs no cells or animals. Although no immune cells are involved in this assay, it reliably predicts the skin sensitization potential of a chemical in chemico. Herein, a new method was developed using endogenous small-molecular-weight compounds, cysteamine and glutathione, rather than synthetic peptides, to differentiate skin sensitizers from non-sensitizers with an accuracy as high as DPRA. The percent depletion of cysteamine and glutathione by test chemicals was measured by an HPLC equipped with a PDA detector. To detect small-size molecules, such as cysteamine and glutathione, a derivatization by 4-(4-dimethylaminophenylazo) benzenesulfonyl chloride (DABS-Cl) was employed prior to the HPLC analysis. Following test method optimization, a cut-off criterion of 7.14% depletion was applied to differentiate skin sensitizers from non-sensitizers in combination of the ratio of 1:25 for cysteamine:test chemical with 1:50 for glutathione:test chemical for the best predictivity among various single or combination conditions. Although overlapping HPLC peaks could not be fully resolved for some test chemicals, high levels of sensitivity (100.0%), specificity (81.8%), and accuracy (93.3%) were obtained for 30 chemicals tested, which were comparable or better than those achieved with DPRA.

Condition establishment study of the direct peptide reactivity assay in Korea

The purpose of this study is to set the method condition of the DPRA in Korea throughout reproducibility study. We conducted intra-lab (triplicate) and inter-lab (three labs of CRI, KTR and CU) validation using 20 chemicals (10 chemicals for the proficiency test listed in the OECD test guideline 442C and an additional 10 more chemicals from reference papers). The data from all three labs met the acceptance criteria. Upon intra-lab validation, two positive chemicals out of 20 total chemicals showed false negative, and one negative chemical showed false positive. In inter-lab validation of three labs, the sensitivity data were 83, 83 and 88% each, and the specificity data was 100, 100 and 88% each. So the accuracy of the three labs was equal to 90%. During these studies, we also checked and improved various limitations that arose. Taken together, the results indicated that the DPRA proposed by OECD test guideline 442C is expected to become a well-established method under Korean GLP applied system.

Development of an artificial neural network model for risk assessment of skin sensitization using human cell line activation test, direct peptide reactivity assay, KeratinoSens™ and in silico structure alert parameter.

It is important to predict the potential of cosmetic ingredients to cause skin sensitization, and in accordance with the European Union cosmetic directive for the replacement of animal tests, several in vitro tests based on the adverse outcome pathway have been developed for hazard identification, such as the direct peptide reactivity assay, KeratinoSens™ and the human cell line activation test. Here, we describe the development of an artificial neural network (ANN) prediction model for skin sensitization risk assessment based on the integrated testing strategy concept, using direct peptide reactivity assay, KeratinoSens™, human cell line activation test and an in silico or structure alert parameter. We first investigated the relationship between published murine local lymph node assay EC3 values, which represent skin sensitization potency, and in vitro test results using a panel of about 134 chemicals for which all the required data were available. Predictions based on ANN analysis using combinations of parameters from all three in vitro tests showed a good correlation with local lymph node assay EC3 values. However, when the ANN model was applied to a testing set of 28 chemicals that had not been included in the training set, predicted EC3s were overestimated for some chemicals. Incorporation of an additional in silico or structure alert descriptor (obtained with TIMES-M or Toxtree software) in the ANN model improved the results. Our findings suggest that the ANN model based on the integrated testing strategy concept could be useful for evaluating the skin sensitization potential.

Applicability of an Integrated Testing Strategy consisting of in silico, in chemico and in vitro assays for evaluating the skin sensitization potencies of isocyanates

The skin sensitization potential of chemicals has been traditionally assessed using regulatory accepted in vivo methods, such as guinea pig maximization test or mouse local lymph node assays (LLNAs). A huge effort to reduce and replace the use of animals for safety assessments of chemicals because of regulatory requirements and ethical issues is presently underway, and alternative non-animal methods have been greatly developed. So far, a few studies have investigated the sensitization potencies of isocyanates which is a group of highly reactive chemicals that are known to be occupational allergens. The present study evaluated nine commonly used isocyanates using an in vivo LLNA and assessed the applicability of an Integrated Testing Strategy (ITS) consisting of an in silico Derek Nexus prediction, an in chemico direct peptide reactivity assay (DPRA), and an in vitro human Cell Line Activation Test (h-CLAT) to isocyanates. All nine isocyanates were evaluated as positive using the LLNA, Derek Nexus and DPRA, whereas seven chemicals tested positive using the h-CLAT: hexamethylene diisocyanate tested negative, and 1,5-diisocyanatonaphthalene could not be examined because of a solubility issue. When assessed using the ITS, the positive/negative evaluations of skin sensitization hazard were consistent with those assessed using the LLNA for all nine chemicals. However, the potency prediction results of the ITS tended to be underestimated, compared with those of the LLNA. The data presented in this work provide insights into the performance of non-animal testing approaches for evaluating the skin sensitization potencies of isocyanates.

In chemico, in vitro and in vivo comparison of the skin sensitizing potential of eight unsaturated and one saturated lipid compounds

The applicability of the Direct Peptide Reactivity Assay (DPRA), the KeratinoSens™ assay and the human cell line activation test (OECD Test Guidelines 442C, 442D, 442E) in predicting the skin sensitising potential of nine lipid (bio)chemicals was investigated. The results from the three assays were integrated using a published prediction model (PM), by which skin sensitisation is predicted if at least two of the three assays yield positive results. Of the eight test substances that were classified as non-sensitisers using available Guinea Pig Maximisation Test (GPMT) data, only five were correctly predicted as ‘negative’ in the PM. (However, only two were correctly predicted as ‘negative’ in the murine Local Lymph Node Assay.) The one lipid (bio)chemical that tested positive in the GPMT was also positive applying the PM. Based upon the outcome of the present study, lipid (bio)chemicals with a log Kow up to 7–8 appear amenable to the three assays. However, solubility problems, that were not evident initially, affected the performance of the DPRA. Further investigations are merited to address the conclusiveness of negative test results with concurrent lack of cytotoxicity in the in vitro assays, to evaluate if poorly soluble substances come into contact with the cells.

Prediction of skin sensitization potency sub-categories using peptide reactivity data

While the skin sensitization hazard of substances can already be identified using non-animal methods, the classification of potency sub-categories GHS-1A and 1B is still challenging. Potency can be measured by the dose at which an effect is observed; since the protein-adduct formation is determining the dose of the allergen in the skin, peptide reactivity was used to assess the potency.The Direct Peptide Reactivity Assay (DPRA; one concentration and reaction-time) did not sufficiently discriminate between sub-categories 1A and 1B (56% accuracy compared to LLNA data, n=124). An extended protocol termed ‘quantitative DPRA’ (three concentrations and one reaction-time), discriminated sub-categories GHS 1A and 1B with an accuracy of 81% or 57% compared to LLNA (n=36) or human (n=14) data, respectively. The analysis of the Cys-adducts was already sufficient; additional analysis of Lys-adducts did not improve the predictivity. An additional modification, the ‘kinetic DPRA’ (several concentrations and reaction-times) was used to approximate the rate constant of Cys-peptide-adduct formation. 35 of 38 substances were correctly assigned to the potency sub-categories (LLNA data), and the predictivity for 14 human data was equally high.These results warrant the kinetic DPRA for further validation in order to fully replace in vivo testing for assessing skin sensitization including potency sub-classification.

Non‐animal assessment of skin sensitization hazard: Is an integrated testing strategy needed, and if so what should be integrated?

There is an expectation that to meet regulatory requirements, and avoid or minimize animal testing, integrated approaches to testing and assessment will be needed that rely on assays representing key events (KEs) in the skin sensitization adverse outcome pathway. Three non-animal assays have been formally validated and regulatory adopted: the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay and the human cell line activation test (h-CLAT). There have been many efforts to develop integrated approaches to testing and assessment with the "two out of three" approach attracting much attention. Here a set of 271 chemicals with mouse, human and non-animal sensitization test data was evaluated to compare the predictive performances of the three individual non-animal assays, their binary combinations and the "two out of three" approach in predicting skin sensitization potential. The most predictive approach was to use both the DPRA and h-CLAT as follows: (1) perform DPRA - if positive, classify as sensitizing, and (2) if negative, perform h-CLAT - a positive outcome denotes a sensitizer, a negative, a non-sensitizer. With this approach, 85% (local lymph node assay) and 93% (human) of non-sensitizer predictions were correct, whereas the "two out of three" approach had 69% (local lymph node assay) and 79% (human) of non-sensitizer predictions correct. The findings are consistent with the argument, supported by published quantitative mechanistic models that only the first KE needs to be modeled. All three assays model this KE to an extent. The value of using more than one assay depends on how the different assays compensate for each other's technical limitations. Copyright © 2017 John Wiley & Sons, Ltd.

In vitro assessment of skin sensitization, photosensitization and phototoxicity potential of commercial glyphosate-containing formulations

This study evaluated the applicability of a modified Direct Peptide Reactivity Assay (DPRA) (OECD N° 442C, 2015) through the 10-fold reduction of reaction volume (micro-DPRA, mDPRA) for skin sensitization evaluation of six commercial glyphosate-containing formulations. In addition, another modification of DPRA was proposed by adding a UVA (5J/cm2) irradiation step, namely photo-mDPRA, to better characterize (photo)sensitizer materials. The phototoxicity profile of pesticides was also evaluated using the 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) (OECD N° 432, 2004). The mDPRA could represent an environmentally acceptable test approach, since it reduces costs and organic waste. Peptide depletion was greater in photo-mDPRA and changed the reactivity class of each test material, in comparison to mDPRA. Thus, the association of mDPRA with photo-mDPRA was better for correctly characterizing human (photo)sensitizer substances and pesticides. In general, cysteine depletion was greater than that of lysine for all materials tested in both mDPRA and photo-mDPRA. Furthermore, while 3T3-NRU-PT is unable to predict (photo)sensitizers, it was capable of correctly identifying the phototoxic potential of the tested agrochemical formulations. In conclusion, mDPRA plus photo-mDPRA and 3T3-NRU-PT seem to be preliminary non-animal test batteries for skin (photo)sensitization/phototoxicity assessment of chemicals, agrochemical formulations and their ingredients.

Prediction of skin sensitization potency using machine learning approaches.

The replacement of animal use in testing for regulatory classification of skin sensitizers is a priority for US federal agencies that use data from such testing. Machine learning models that classify substances as sensitizers or non-sensitizers without using animal data have been developed and evaluated. Because some regulatory agencies require that sensitizers be further classified into potency categories, we developed statistical models to predict skin sensitization potency for murine local lymph node assay (LLNA) and human outcomes. Input variables for our models included six physicochemical properties and data from three non-animal test methods: direct peptide reactivity assay; human cell line activation test; and KeratinoSens™ assay. Models were built to predict three potency categories using four machine learning approaches and were validated using external test sets and leave-one-out cross-validation. A one-tiered strategy modeled all three categories of response together while a two-tiered strategy modeled sensitizer/non-sensitizer responses and then classified the sensitizers as strong or weak sensitizers. The two-tiered model using the support vector machine with all assay and physicochemical data inputs provided the best performance, yielding accuracy of 88% for prediction of LLNA outcomes (120 substances) and 81% for prediction of human test outcomes (87 substances). The best one-tiered model predicted LLNA outcomes with 78% accuracy and human outcomes with 75% accuracy. By comparison, the LLNA predicts human potency categories with 69% accuracy (60 of 87 substances correctly categorized). These results suggest that computational models using non-animal methods may provide valuable information for assessing skin sensitization potency. Copyright © 2017 John Wiley & Sons, Ltd.

Evaluation of in vitro assays for the assessment of the skin sensitization hazard of functional polysiloxanes and silanes

The skin sensitization potential of chemicals has traditionally been evaluated in vivo according to OECD testing guidelines in guinea pigs or the mouse local lymph node assay. There has lately been a great emphasis on establishing in vitro test methods reflecting the key biological events in the adverse outcome pathway (AOP) for skin sensitization as published by the OECD. Against this background, a group of 8 polysiloxanes and silanes, seven of them aminofunctionalised, for which in vivo data were already available, has been tested in vitro in the direct peptide reactivity assay (DPRA), the KeratinoSens™ and the human cell line activation test (h-CLAT) and in the modified myeloid U937 skin sensitization test (mMUSST) as far as technically feasible. The main objective of the programme was to determine the utility of these systems for this heterogeneous group of silicone-based substances, recognizing that some substances are outside the assays applicability domains. The presented data provided some interesting mechanistical insights into the performance of these assays for functionalised siloxanes and silanes. The data also allow for a preliminary evaluation of proposed integrated testing strategies (ITS) to determine the skin sensitization potential of chemicals which were not considered in the training sets of the respective ITS.

The use of peptide reactivity assays for skin sensitisation hazard identification and risk assessment.

Over the past 20 years or more, investigators have been developing non-animal test methods for use in assessing the skin sensitisation potential of chemicals. In parallel with this effort, the key biological events of skin sensitisation have been well-characterised in an Adverse Outcome Pathway (AOP) proposed by the Organisation for Economic Co-operation and Development (OECD). The key molecular initiating event of this AOP is haptenation or covalent modification of epidermal proteins. In this review, the strengths and limitations of the Direct Peptide Reactivity Assay (DPRA) are described, and the more recently developed Peroxidase Peptide Reactivity Assay (PPRA). The DPRA has been formally validated and incorporated into an OECD Test Guideline (TG442C). The DPRA shows promise for assisting in hazard identification as well as for assessing skin sensitisation potency when used in an integrated testing strategy.

Successful evaluation of sensitization by DPRA assay coupled with Mass Spectrometry detection

Sensitizing chemicals are commonly associated primarily with either skin or respiratory sensitization. In the Direct Peptide Reactivity Assay (DPRA), when compared with skin sensitizers, respiratory allergens have been demonstrated to selectively react with lysine rather than cysteine. The Peroxidase Peptide Reactivity Assay (PPRA) has been developed as a refinement to the DPRA. The PPRA incorporates dose–response analyses, mass spectroscopy for peptide detection and a horseradish peroxidase–hydrogen peroxide enzymatic system, increasing the potential to identify pro-haptens. In the investigations reported here, the PPRA was evaluated to determine whether it provides advantages for the identification of respiratory allergens. Twenty respiratory sensitizers, including five predicted to be pre-/pro-haptens were evaluated. The PPRA performed similarly to the DPRA with respect to identifying inherently reactive respiratory sensitizers. However, three respiratory sensitizers predicted to be pre-/pro-haptens (chlorhexidine, ethylenediamine and piperazine) were non-reactive and the general selectivity of the respiratory allergens for lysine was lost in the PPRA. Overall, the data indicate that the PPRA does not provide an advantage over the DPRA for discriminating allergens as either contact or respiratory sensitizers. Nevertheless, the PPRA provides a number of refinements to the DPRA that allow for an enhanced characterization of reactivity for both classes of chemical allergens.

Can currently available non-animal methods detect pre and pro-haptens relevant for skin sensitization?

Predictive testing to characterize substances for their skin sensitization potential has historically been based on animal tests such as the Local Lymph Node Assay (LLNA). In recent years, regulations in the cosmetics and chemicals sectors have provided strong impetus to develop non-animal alternatives. Three test methods have undergone OECD validation: the direct peptide reactivity assay (DPRA), the KeratinoSens™ and the human Cell Line Activation Test (h-CLAT). Whilst these methods perform relatively well in predicting LLNA results, a concern raised is their ability to predict chemicals that need activation to be sensitizing (pre- or pro-haptens). This current study reviewed an EURL ECVAM dataset of 127 substances for which information was available in the LLNA and three non-animal test methods. Twenty eight of the sensitizers needed to be activated, with the majority being pre-haptens. These were correctly identified by 1 or more of the test methods. Six substances were categorized exclusively as pro-haptens, but were correctly identified by at least one of the cell-based assays. The analysis here showed that skin metabolism was not likely to be a major consideration for assessing sensitization potential and that sensitizers requiring activation could be identified correctly using one or more of the current non-animal methods.