Skin Sensitization Potential Of Chemicals Research Articles

A rapid spectrophotometric test for assessing skin sensitization potential of chemicals by using N-acetyl-L-cysteine methyl ester in chemico

ABSTRACT During the key event 1 of skin sensitization defined as covalent binding or haptenization of sensitizer to either thiol or amino group of skin proteins, a sensitizer not only covalently binds with skin proteins but also interacts with nucleophilic small molecules such as glutathione (GSH). Although GSH would not be directly associated with skin sensitization, this interaction may be applied for developing an alternative test method simulating key event 1, haptenization. Thus, the aim of the present study was to examine whether N-acetyl-L-cysteine methyl ester (NACME), a thiol-containing compound, was selected as an electron donor to determine whether NACME reacted with sensitizers. Following a reaction of NACME with a sensitizer in a 96-well plate, the remaining NACME was measured spectrophotometrically using 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB). Following the optimization of test conditions with two different vehicles, such as acetonitrile (ACN) and dimethyl sulfoxide (DMSO), 64 test chemicals were tested to determine the predictive capacity of current NACME test method. The results obtained showed, the predictive capacity of 94.6% sensitivity, 88.9% specificity, and 92.2% accuracy utilizing DMSO as a vehicle with a cutoff NACME depletion of 5.85%. The three parameters were also over 85% in case of ACN. These values were comparable to or better than other OECD-approved test methods. Data demonstrated that a simple thiol-containing compound NACME might constitute as a reliable candidate for identifying reactive skin sensitizers, and that this method be considered as practical method as a screening tool for assessing a chemical’s tendency to initiate skin sensitization.

The inter-laboratory validation study of EpiSensA for predicting skin sensitization potential.

The Epidermal Sensitization Assay (EpiSensA) is a reconstructed human epidermis (RhE)-based gene expression assay for predicting the skin sensitization potential of chemicals. Since the RhE model is covered by a stratified stratum corneum, various kinds of test chemicals, including lipophilic ones and pre-/pro-haptens, can be tested with a route of exposure akin to an in vivo assay and human exposure. This article presents the results of a formally managed validation study of the EpiSensA that was carried out by three participating laboratories. The purpose of this validation study was to assess transferability of the EpiSensA to new laboratories along with its within- (WLR) and between-laboratory reproducibility (BLR). The validation study was organized into two independent stages. As demonstrated during the first stage, where three sensitizers and one non-sensitizer were correctly predicted by all participating laboratories, the EpiSensA was successfully transferred to all three participating laboratories. For Phase I of the second stage, each participating laboratory performed three experiments with an identical set of 15 coded test chemicals resulting in WLR of 93.3%, 93.3%, and 86.7%, respectively. Furthermore, when the results from the 15 test chemicals were combined with those of the additional 12 chemicals tested in Phase II of the second stage, the BLR for 27 test chemicals was 88.9%. Moreover, the predictive capacity among the three laboratories showed 92.6% sensitivity, 63.0% specificity, 82.7% accuracy, and 77.8% balanced accuracy based on murine local lymph node assay (LLNA) results. Overall, this validation study concluded that EpiSensA is easily transferable and sufficiently robust for assessing the skin sensitization potential of chemicals.

Development of an <i>in silico</i> consensus model for predicting the chemical reactivity to cysteine measured by the DPRA and its application to predict the skin sensitization potential of chemicals

Development of an <i>in silico</i> consensus model for predicting the chemical reactivity to cysteine measured by the DPRA and its application to predict the skin sensitization potential of chemicals

Theoretical Validation of In Chemico Skin Sensitization Assay "ADRA" Using the Products Formed by Nucleophilic Reagents and Chemicals.

Amino acid derivative reactivity assay (ADRA) is an in chemico assay for assessing the skin sensitization potential of chemicals by evaluating the reactivity of nucleophilic reagents that mimic skin proteins. N-(2-(1-Naphthyl)acetyl)-l-cysteine (NAC) and α-N-(2-(1-naphthyl)acetyl)-l-lysine (NAL), used as nucleophilic reagents, are small-molecule derivatives of two different amino acids, each with a naphthalene ring attached. The rate of decrease in the amount of NAC or NAL in the reaction solution is evaluated in this assay as an indicator of the test substance's skin sensitization ability. However, the products formed between the nucleophilic reagent and the test substance, which play an important role in vivo, are not directly identified. Therefore, six highly reactive chemicals, including the proficiency substances listed in the OECD Test Guidelines─squaric acid diethyl ester, 2-methyl-2H-isothiazol-3-one (MI), p-benzoquinone, palmitoyl chloride, diphenylcyclopropenone (DPCP), and imidazolidinyl urea (IU)─were used to determine each formed product. Samples were prepared according to the standard ADRA method, and the formed products were predicted on the basis of the reaction mechanism. Excluding DPCP, the estimated structures were validated using mass spectrometry and nuclear magnetic resonance spectrometry on the synthesized samples. In this manner, the products of each nucleophile were confirmed for all examined test substances. The estimated structure products were obtained through a series of reactions initiated by the nucleophilic attack of NAC's thiol group or NAL's amino group on the test substance's electron-deficient carbonyl carbon. However, contrary to expectations, disulfide-linked-type ring-opened products were detected in the case of MI, and products with free formaldehyde in solution were detected in the case of IU. In summary, all skin sensitizers tested herein reacted with NAC and/or NAL to give products. This supports the theoretical validity of ADRA, which provides an indirect evaluation of the formed products based on a decrease in nucleophilic reagents.

Evaluation of Skin Sensitization Potential of Chemicals and GHS Subcategorization by Local Lymph Node Assay: BrdU-ELISA

Evaluation of Skin Sensitization Potential of Chemicals and GHS Subcategorization by Local Lymph Node Assay: BrdU-ELISA

Pred-Skin: A Web Portal for Accurate Prediction of Human Skin Sensitizers.

Safety assessment is an essential component of the regulatory acceptance of industrial chemicals. Previously, we have developed a model to predict the skin sensitization potential of chemicals for two assays, the human patch test and murine local lymph node assay, and implemented this model in a web portal. Here, we report on the substantially revised and expanded freely available web tool, Pred-Skin version 3.0. This up-to-date version of Pred-Skin incorporates multiple quantitative structure-activity relationship (QSAR) models developed with in vitro, in chemico, and mice and human in vivo data, integrated into a consensus naïve Bayes model that predicts human effects. Individual QSAR models were generated using skin sensitization data derived from human repeat insult patch tests, human maximization tests, and mouse local lymph node assays. In addition, data for three validated alternative methods, the direct peptide reactivity assay, KeratinoSens, and the human cell line activation test, were employed as well. Models were developed using open-source tools and rigorously validated according to the best practices of QSAR modeling. Predictions obtained from these models were then used to build a naïve Bayes model for predicting human skin sensitization with the following external prediction accuracy: correct classification rate (89%), sensitivity (94%), positive predicted value (91%), specificity (84%), and negative predicted value (89%). As an additional assessment of model performance, we identified 11 cosmetic ingredients known to cause skin sensitization but were not included in our training set, and nine of them were accurately predicted as sensitizers by our models. Pred-Skin can be used as a reliable alternative to animal tests for predicting human skin sensitization.

Human keratinocytes and monocytes co-culture cell system: An important contribution for the study of moderate and weak sensitizers

In vitro approaches to address key events in chemical-induced skin sensitization have been developed, but there is still uncertain how they will be useful to predict the potency for an effective risk assessment. Keratinocytes (KCs) play a key role in all phases of skin sensitization. Dendritic cells (DCs) activation and maturation require the binding of cytokines produced by KC as a result of initial chemical exposure. We previously identified interleukin-18 (IL-18) as useful marker for determination of skin sensitization potential of chemicals. The aim of this paper was to mimic the interaction between KCs and DCs using a co-culture of NCTC 2544 and THP-1 cells. Three selected contact allergens of different potency (Bandrowski's base, diethyl maleate, and imidazolidinyl urea) were tested in time-course experiments (24, 48 and 72 h). Cell surface markers expression (CD80, CD86, and HLA-DR) was determined by flow cytometry analysis while IL-18 production was evaluated with specific sandwich ELISA. Results obtained from this simple in vitro co-culture system show the possibility to study the contribution of KCs in DCs activation through the analysis of HLA-DR expression. Results obtained demonstrate the ability of the KCs to favor the full maturation of the DCs in the presence of moderate and weak allergens, while the extreme allergen induced a complete maturation of DC alone without the need of KCs.

Theoretical studies to estimate the skin sensitization potential of chemicals of the Schiff base domain

AbstractSkin sensitization occurs when an exogenous chemical substance forms a covalent adduct with a dermal protein electrophile or nucleophile. This instigates an immune response which leads to inflammation. The local lymph node assay is an in vivo model used in the assessment of relative skin sensitizing potency of chemicals. The method is time consuming and expensive, as well as poses ethical questions given that a number of mice must be sacrificed for each compound assessed. In this work, we investigate the use of an inexpensive, rapid, and ethical method to predict the skin sensitization potential of Schiff base chemicals. We employ quantum chemical methods to rationalize the sensitization potential of 22 compounds with a diverse range of activities. To this end, we have evaluated the mechanistic profile associated with this type of reaction using gas‐phase models. We subsequently use the predicted rate determining barriers and key physico‐chemical parameters (such as logP) to establish stucture activity relationship (SAR) guidelines to predict the skin sensitization potential for new chemicals. We find that the predicted rate determining barriers for aldehydes, ketone, and 1,2 and 1,3 diones generally decrease in the given order, which concurs with the overall trends in sensitization. We find that lipophilicity also plays a role, with those chemicals displaying both low barriers to reaction, and lower lipophilicity (ie, diones), being more likely to display undesirable skin sensitization effects. These findings are in line with experiment‐based observations in the literature and point to the value 3D quantum chemical calculations could have if combined with other orthogonal approaches to estimate skin sensitization potential of chemicals.

High-throughput screening (HTS)-based spectrophotometric direct peptide reactivity assay (Spectro-DPRA) to predict human skin sensitization potential

Some cosmetic ingredients can act as a chemical hapten to induce an immune response; therefore, evaluating the sensitizing potential of cosmetic ingredients is essential. We previously developed a novel in chemico direct peptide reactivity assay involving a spectrophotometric evaluation (Spectro-DPRA) for animal skin sensitization tests (local lymph node assay; LLNA). Based on previous research, we expanded the test materials to confirm the effectiveness of the Spectro-DPRA method for predicting the animal skin sensitization potential, and further determined the feasibility of the method for estimating the human skin sensitization potential. Spectro-DPRA showed 83.1% or 89.1% accuracy compared to a conventional LLNA or prediction based on human data, respectively, with a combination model using both a cysteine peptide and lysine peptide cut-off. To identify the effect of the lipophilicity of a chemical on predicting the skin sensitization potential, we applied our prediction model to chemicals with a Log Pow range of ―1 to 4. Overall predictability was increased, and the accuracy compared to the LLNA and human data was 91.5% and 94.9%, respectively, in the combination cut-off prediction model. In conclusion, Spectro-DPRA serves as an easy, rapid, and high-throughput in chemico screening method with high accuracy to predict the human skin sensitization potential of chemicals.

Evaluation of skin sensitization potential of chemicals by local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry

The local lymph node assay using 5-bromo-2-deoxyuridine with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA used to identify skin sensitizers. This assay measures the proliferation of auricular lymph node cells (LNCs) during the induction phase of skin sensitization and the number of BrdU-positive LNCs using flow cytometry. We determined if LLNA: BrdU-FCM can evaluate the skin sensitization potential of 20 substances, including 16 sensitizers and 4 non-sensitizers, that were tested using LLNA: DA and LLNA: BrdU-ELISA but not listed in OECD TG 429. After selecting appropriate vehicles and conducting pre-screen tests in 2 phases, solvents and test concentrations for the main test were determined. In the main study, we measured changes in LN weight, the number of LNCs, and the proportion of BrdU incorporated into LNCs to calculate stimulation indexes (SI). SI was calculated based on the total number of LNCs and BrdU incorporation in LNCs. We found that all substances were correctly classified as sensitizers or non-sensitizers. Overall, we confirmed that the LLNA: BrdU-FCM can evaluate skin sensitization potential of the 20 substances. Additionally, our results of combining 22 reference substances listed in OECD TG 429 and 20 additional substances showed that concordance of LLNA: BrdU-FCM with the LLNA was higher than before.

A review of substances found positive in 1 of 3 in vitro tests for skin sensitization

There has been significant progress in recent years in the development and application of alternative methods for assessing the skin sensitization potential of chemicals. The pathways involved in skin sensitization have been described in an OECD adverse outcome pathway (AOP). To date, a single non-animal test method is not sufficient to address this AOP so numerous approaches involving the use of 2 or more assays are being evaluated for their performance. The 2 out of 3 approach is a simple approach that has demonstrated very good sensitivity, specificity and overall accuracy numbers for predicting the skin sensitization potential of chemicals. Chemicals with at least two positive results in tests addressing Key events 1–3 are predicted sensitizers, while chemicals with none or only one positive outcome are predicted non-sensitizers. In this report we have thoroughly reviewed the discordant results of 29 chemicals with 1 out of 3 positive results to understand better what led to the results observed and how this information might impact our hazard assessments of these chemicals. We initially categorized each chemical using a weight of evidence approach as positive, negative or indeterminate based on review of available human and animal data as well as what skin sensitization alerts were triggered using two versions of OECD Toolbox and DEREK Nexus. We determined that 4 of the 29 chemicals should be classified as indeterminate and not included in analysis of method performance based on insufficient, borderline and/or conflicting data to confidently categorized the chemicals as allergens or non-allergens. Of the 29 chemicals included in this analysis, 17 were classified as negative and would be correctly identified using a 2 out of 3 approach while 8 chemicals were classified as positive in vivo and would be false-negative with this approach. For some of these chemicals, the outcomes observed can be explained by in vitro borderline results (13 chemicals) or in some instances there is mechanistic understanding of why a chemical is positive or negative in a particular assay (9 chemicals). Thus, when comparing the performance of different defined approaches, one should attempt to only include chemicals which demonstrate clear evidence to be categorize as allergens or non-allergens. Finally, when interpreting the results obtained for an individual unknown chemical it is critical that the in vitro skin sensitization data is reviewed critically and there is a good understanding of the variance and applicability domain limitations for each assay being used.

Implementation of in vitro methods in the safety evaluation of the skin sensitization potential of chemicals under REACH regulation

Implementation of in vitro methods in the safety evaluation of the skin sensitization potential of chemicals under REACH regulation

Advancing the predictivity of skin sensitization by applying a novel HMOX1 reporter system.

Reporter cell lines are a particularly useful tool to screen for the skin sensitization potential of chemicals. Current cell models based on Keap1-Nrf2 mimic induction by conducting antioxidant response element-luciferase plasmids. However, plasmid-based reporters may ignore comprehensive aspects of induction, thus affecting the accuracy of hazard identification. Herein, we developed a novel HaCaT-based reporter system, EndoSens, whereby luciferase was specifically inserted into the cassette for heme oxygenase (decycling) 1 (HMOX1, the most consistent marker induced by skin sensitizers) by CRISPR/Cas9. Testing data from 20 coded substances showed an accuracy of 90%, sensitivity of 91.7%, and specificity of 87.5%, which exceeded the OECD requirement. Among the 35 chemicals examined, predictivity was better than reported for the validated KeratinoSens™. These results indicate that the EndoSens assay could advance the predictivity of skin sensitization, thus making it a promising tool for in vitro skin sensitization testing.

A Perspective and a New Integrated Computational Strategy for Skin Sensitization Assessment

Traditionally, the skin sensitization potential of chemicals has been assessed using animal models. Due to growing ethical, political, and financial concerns, sustainable alternatives to animal testing need to be developed. As publicly available skin sensitization data continues to grow, computational approaches, such as alert-based systems, read-across, and QSAR models, are expected to reduce or replace animal testing for the prediction of human skin sensitization potential. Herein, we discuss current computational approaches to predicting skin sensitization and provide future perspectives of the field. As a proof-of-concept study, we have compiled the largest skin sensitization data set in the public domain and benchmarked several methods for building skin sensitization models. We propose a new comprehensive approach, which integrates multiple QSAR models developed with in vitro, in chemico, animal, and human data, and a Naive Bayes model for predicting human skin sensitization. Both the data sets and t...

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.

Evaluation of in silico tools to predict the skin sensitization potential of chemicals

Public domain and commercial in silico tools were compared for their performance in predicting the skin sensitization potential of chemicals. The packages were either statistical based (Vega, CASE Ultra) or rule based (OECD Toolbox, Toxtree, Derek Nexus). In practice, several of these in silico tools are used in gap filling and read-across, but here their use was limited to make predictions based on presence/absence of structural features associated to sensitization. The top 400 ranking substances of the ATSDR 2011 Priority List of Hazardous Substances were selected as a starting point. Experimental information was identified for 160 chemically diverse substances (82 positive and 78 negative). The prediction for skin sensitization potential was compared with the experimental data. Rule-based tools perform slightly better, with accuracies ranging from 0.6 (OECD Toolbox) to 0.78 (Derek Nexus), compared with statistical tools that had accuracies ranging from 0.48 (Vega) to 0.73 (CASE Ultra – LLNA weak model). Combining models increased the performance, with positive and negative predictive values up to 80% and 84%, respectively. However, the number of substances that were predicted positive or negative for skin sensitization in both models was low. Adding more substances to the dataset will increase the confidence in the conclusions reached. The insights obtained in this evaluation are incorporated in a web database www.asopus.weebly.com that provides a potential end user context for the scope and performance of different in silico tools with respect to a common dataset of curated skin sensitization data.

An improvement of LLNA:DA to assess the skin sensitization potential of chemicals.

We developed a modified local lymph node assay based on ATP (LLNA:DA), termed the Two-Stage LLNA:DA, to further reduce the animal numbers in the identification of sensitizers. In the Two-Stage LLNA:DA procedure, 13 chemicals ranging from non-sensitizers to extreme sensitizers were selected. The first stage used reduced LLNA:DA (rLLNA:DA) to screen out sensitive chemicals. The second stage used LLNA:DA based on OECD 442 (A) to classify those potential sensitizers screened out in the first stage. In the first stage, the SIs of the methyl methacrylate, salicylic acid, methyl salicylate, ethyl salicylate, isopropanol and propanediol were below 1.8 and need not to be tested in the second step. Others continued to be tested by LLNA:DA. In the second stage, sodium lauryl sulphate and xylene were classified as weak sensitizers. a-hexyl cinnamic aldehyde and eugenol were moderate sensitizers. Benzalkonium chloride and glyoxal were strong sensitizers, and phthalic anhydride was an extreme sensitizer. The 9/9, 11/12, 10/11, and 8/13 (positive or negative only) categories of the Two-Stage LLNA:DA were consistent with those from the other methods (LLNA, LLNA:DA, GPMT/BT and HMT/HPTA), suggesting that Two-Stage LLNA:DA have a high coincidence rate with reported data. In conclusion, The Two-Stage LLNA:DA is in line with the "3R" rules, and can be a modification of LLNA:DA but needs more study.

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.

Comparative analysis of respiratory and skin sensitization potential of chemicals using Japanese GHS classification

Comparative analysis of respiratory and skin sensitization potential of chemicals using Japanese GHS classification