Deriving a point of departure for assessing the skin sensitization risk of wearable device constituents with in vitro methods.

Wearable devices are in contact with the skin for extended periods. As such, the device constituents should be evaluated for their skin sensitization potential, and a Point of Departure (PoD) should be derived to conduct a proper risk assessment. Without historical in vivo data, the PoD must be derived with New Approach Methods (NAMs). To accomplish this, regression models trained on LLNA data that use data inputs from OECD-validated in vitro tests were used to derive a predicted EC3 value, the LLNA value used to classify skin sensitization potency, for three adhesive monomers (Isobornyl acrylate (IBOA), N, N- Dimethylacrylamide (NNDMA), and Acryloylmorpholine (ACMO) and one dye (Solvent Orange 60 (SO60)). These chemicals can be used as constituents of wearable devices and have been associated with causing allergic contact dermatitis (ACD). Using kinetic DPRA and KeratinoSens™ data, the PoDs obtained with the regression model were 180, 215, 1535, and 8325 µg/cm2 for IBOA, SO60, ACMO, and NNDMA, respectively. The PoDs derived with the regression model using NAMs data will enable a proper skin sensitization risk assessment without using animals.

Identification and semi-quantification of protein allergens in complex mixtures using proteomic and AllerCatPro 2.0 bioinformatic analyses: a proof-of-concept investigation.

The demand for botanicals and natural substances in consumer products has increased in recent years. These substances usually contain proteins and these, in turn, can pose a risk for immunoglobulin E (IgE)-mediated sensitization and allergy. However, no method has yet been accepted or validated for assessment of potential allergenic hazards in such materials. In the studies here, a dual proteomic-bioinformatic approach is proposed to evaluate holistically allergenic hazards in complex mixtures of plants, insects, or animal proteins. Twelve commercial preparations of source materials (plant products, dust mite extract, and preparations of animal dander) known to contain allergenic proteins were analyzed by label-free proteomic analyses to identify and semi-quantify proteins. These were then evaluated by bioinformatics using AllerCatPro 2.0 (https://allercatpro.bii.a-star.edu.sg/) to predict no, weak, or strong evidence for allergenicity and similarity to source-specific allergens. In total, 4,586 protein sequences were identified in the 12 source materials combined. Of these, 1,665 sequences were predicted with weak or strong evidence for allergenic potential. This first-tier approach provided top-level information about the occurrence and abundance of proteins and potential allergens. With regards to source-specific allergens, 129 allergens were identified. The sum of the relative abundance of these allergens ranged from 0.8% (lamb's quarters) to 63% (olive pollen). It is proposed here that this dual proteomic-bioinformatic approach has the potential to provide detailed information on the presence and relative abundance of allergens, and can play an important role in identifying potential allergenic hazards in complex protein mixtures for the purposes of safety assessments.

Application of AllerCatPro 2.0 for protein safety assessments of consumer products.

Foreign proteins are potentially immunogenic, and a proportion of these are able to induce immune responses that result in allergic sensitization. Subsequent exposure of sensitized subjects to the inducing protein can provoke a variety of allergic reactions that may be severe, or even fatal. It has therefore been recognized for some time that it is important to determine a priori whether a given protein has the potential to induce allergic responses in exposed subjects. For example, the need to assess whether transgene products expressed in genetically engineered crop plants have allergenic properties. This is not necessarily a straightforward exercise (as discussed elsewhere in this edition), but the task becomes even more challenging when there is a need to conduct an overall allergenicity safety assessment of complex mixtures of proteins in botanicals or other natural sources that are to be used in consumer products. This paper describes a new paradigm for the allergenicity safety assessment of proteins that is based on the use of AllerCatPro 2.0, a new version of a previously described web application model developed for the characterization of the allergenic potential of proteins. Operational aspects of AllerCatPro 2.0 are described with emphasis on the application of new features that provide improvements in the predictions of allergenic properties such as the identification of proteins with high allergenic concern. Furthermore, the paper provides a description of strategies of how AllerCatPro 2.0 can best be deployed as a screening tool for identifying suitable proteins as ingredients in consumer products as well as a tool, in conjunction with label-free proteomic analysis, for identifying and semiquantifying protein allergens in complex materials. Lastly, the paper discusses the steps that are recommended for formal allergenicity safety assessment of novel consumer products which contain proteins, including consideration and integration of predicted consumer exposure metrics. The article therefore provides a holistic perspective of the processes through which effective protein safety assessments can be made of potential allergenic hazards and risks associated with exposure to proteins in consumer products, with a particular focus on the use of AllerCatPro 2.0 for this purpose.

Updating the Dermal Sensitisation Thresholds using an expanded dataset and an in silico expert system.

The Dermal Sensitisation Thresholds (DST) are Thresholds of Toxicological Concern, which can be used to justify exposure-based waiving when conducting a skin sensitisation risk assessment. This study aimed to update the published DST values by expanding the size of the Local Lymph Node Assay dataset upon which they are based, whilst assigning chemical reactivity using an in silico expert system (Derek Nexus). The potency values within the expanded dataset fitted a similar gamma distribution to that observed for the original dataset. Derek Nexus was used to classify the sensitisation activity of the 1152 chemicals in the expanded dataset and to predict which chemicals belonged to a High Potency Category (HPC). This two-step classification led to three updated thresholds: a non-reactive DST of 710 µg/cm2 (based on 79 sensitisers), a reactive (non-HPC) DST of 73 µg/cm2 (based on 331 sensitisers) and an HPC DST of 1.0 µg/cm2 (based on 146 sensitisers). Despite the dataset containing twice as many sensitisers, these values are similar to the previously published thresholds, highlighting their robustness and increasing confidence in their use. By classifying reactivity in silico the updated DSTs can be applied within a skin sensitisation risk assessment in a reproducible, scalable and accessible manner.

AllerCatPro 2.0: a web server for predicting protein allergenicity potential.

Proteins in food and personal care products can pose a risk for an immediate immunoglobulin E (IgE)-mediated allergic response. Bioinformatic tools can assist to predict and investigate the allergenic potential of proteins. Here we present AllerCatPro 2.0, a web server that can be used to predict protein allergenicity potential with better accuracy than other computational methods and new features that help assessors making informed decisions. AllerCatPro 2.0 predicts the similarity between input proteins using both their amino acid sequences and predicted 3D structures towards the most comprehensive datasets of reliable proteins associated with allergenicity. These datasets currently include 4979 protein allergens, 162 low allergenic proteins, and 165 autoimmune allergens with manual expert curation from the databases of WHO/International Union of Immunological Societies (IUIS), Comprehensive Protein Allergen Resource (COMPARE), Food Allergy Research and Resource Program (FARRP), UniProtKB and Allergome. Various examples of profilins, autoimmune allergens, low allergenic proteins, very large proteins, and nucleotide input sequences showcase the utility of AllerCatPro 2.0 for predicting protein allergenicity potential. The AllerCatPro 2.0 web server is freely accessible at https://allercatpro.bii.a-star.edu.sg.

Implementation of a dermal sensitization threshold (DST) concept for risk assessment: structure-based DST and in vitro data-based DST.

Skin sensitization resulting in allergic contact dermatitis represents an important toxicological endpoint as part of safety assessments. When available substance-specific sensitization data are inadequate, the dermal sensitization threshold (DST) concept has been proposed to set a skin exposure threshold to provide no appreciable risk of skin sensitization. Structure-based DSTs, which include non-reactive, reactive, and high potency category (HPC) DSTs, can be applied to substances with an identified chemical structures. An in vitro data-based "mixture DST" can be applied to mixtures based on data from in vitro test methods, such as KeratinoSens™ and the human Cell Line Activation Test. The purpose of this review article is to discuss the practical use of DSTs for conducting sound sensitization risk assessments to assure the safety of consumer products. To this end, several improvements are discussed in this review. For application of structure-based DSTs, an overall structural classification workflow was developed to exclude the possibility that "HPC but non-reactive" chemicals are misclassified as "non-reactive", because such chemicals should be classified as HPC chemicals considering that HPC rules have been based on the chemical structure of high potency sensitizers. Besides that, an extended application of the mixture DST principle to mixtures that either is cytotoxic or evaluated as positive was proposed. On a final note, we also developed workflows that integrate structure-based and in vitro-based mixture DST. The proposed workflows enable the application of the appropriate DST, which serves as a point of departure in the quantitative sensitization risk assessment.

Benchmarking performance of SENS-IS assay against weight of evidence skin sensitization potency categories.

Potency determination of potential skin sensitizers in humans is essential for quantitative risk assessment and proper risk management. SENS-IS is an in vitro test based on a reconstructed human skin model, that was developed to predict the hazard and potency of potential skin sensitizers. The performance of the SENS-IS assay in potency prediction for 174 materials was evaluated for this work. The potency used as a benchmark was determined based on the weight of evidence approach, by collectively considering all well-established test data, including human, animal, in chemico, in vitro, and in silico data. Based on this weight of evidence approach, the dataset was composed of 5, 19, 34, 54, and 38 extreme, strong, moderate, weak, and very weak sensitizers, respectively, as well as 24 non-sensitizers. SENS-IS provided good prediction of the skin sensitization potency for 85% of this dataset, with precise and approximate prediction on 46% and 39% of the 174 materials, respectively. Our evaluation showed that SENS-IS provides a good approximation of the skin sensitization potency.

Weight of Evidence Approach for Skin Sensitization Potency Categorization of Fragrance Ingredients.

BACKGROUND: Reliable human potency data are necessary for conducting quantitative risk assessments, as well as development and validation of new nonanimal methods for skin sensitization assessments. Previously, human skin sensitization potency of fragrance materials was derived primarily from human data or the local lymph node assay. OBJECTIVES: This study aimed to define skin sensitization potency of fragrance materials via weight of evidence approach, incorporating all available human, animal, in vitro, in chemico, and in silico data. METHODS: All available data on 106 fragrance materials were considered to assign each material into 1 of the 6 defined potency categories (extreme, strong, moderate, weak, very weak, and nonsensitizer). RESULTS: None of the 106 materials were considered an extreme sensitizer, whereas a total of 6, 23, 41, and 26 materials were categorized as strong, moderate, weak, and very weak sensitizers, respectively. Ten materials lacked evidence for the induction of skin sensitization. CONCLUSIONS: Skin sensitization potency categorization of the 106 fragrance materials based on the described weight of evidence approach can serve as a useful resource in evaluation of nonanimal methods, as well as in risk assessment.

The microbial gbu gene cluster links cardiovascular disease risk associated with red meat consumption to microbiota L-carnitine catabolism.

The heightened cardiovascular disease (CVD) risk observed among omnivores is thought to be linked, in part, to gut microbiota-dependent generation of trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the interrelationship of γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. In the present study, we show that plasma γBB levels in individuals from a clinical cohort (n = 2,918) are strongly associated with incident CVD event risks. Culture of human faecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine → γBB → TMA transformation, elevate TMAO levels and enhance thrombosis potential in recipients after arterial injury. RNA-sequencing analyses of E. timonensis identified a six-gene cluster, herein named the γBB utilization (gbu) gene cluster, which is upregulated in response to γBB. Combinatorial cloning and functional studies identified four genes (gbuA, gbuB, gbuC and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when coexpressed in Escherichia coli. Finally, reanalysis of samples (n = 113) from a clinical, randomized diet, intervention study showed that the abundance of faecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine → γBB → TMA → TMAO transformation in hosts and contributes to CVD risk.

Critical Evaluation of Low-Molecular Weight Respiratory Sensitizers and Their Protein Reactivity Potential Toward Lysine Residues.

Interest in the development of methods to evaluate the respiratory sensitization potential of low-molecular weight chemicals continues, but no method has yet been generally accepted or validated. A lack of chemical reference standards, together with uncertainty regarding relevant immunological mechanisms, has hampered method development. The first key event in the development of either skin or respiratory sensitization is the formation of stable adducts of the chemical with host proteins. This event is measured in the Direct Peptide Reactivity Assay using cysteine- and lysine-containing model peptides. It is hypothesized that protein reactivity and subsequent adduct formation may represent the earliest point of divergence in the pathways leading to either skin or respiratory sensitization. Direct Peptide Reactivity Assay data for 200 chemicals were compiled and grouped into respiratory, skin and nonsensitizers. Chemicals grouping was based on extensive literature research and expert judgment. To evaluate if chemical groups represent different peptide reactivity profiles, peptide reactivity data were clustered and compared with information on protein binding mechanisms and chemical categories available via the Organization for Economic Co-operation and Development. Toolbox. Respiratory sensitizers (n = 15) showed a significant (3-fold) higher lysine reactivity than skin sensitizers (n = 129). However, this difference was driven largely by the high representation of acid anhydrides among the respiratory sensitizers that showed clear lysine selectivity. Collectively, these data suggest that preferential reactivity for either cysteine or lysine is associated primarily with chemical structure, and that lysine preference is not a unifying characteristic of chemical respiratory allergens.

Identifying a reference list of respiratory sensitizers for the evaluation of novel approaches to study respiratory sensitization.

The induction of immunological responses that trigger bio-physiological symptoms in the respiratory tract following repeated exposure to a substance, is known as respiratory sensitization. The inducing compound is known as a respiratory sensitizer. While respiratory sensitization by high molecular weight (HMW) materials is recognized and extensively studied, much less information is available regarding low molecular weight (LMW) materials as respiratory sensitizers. Variability of symptoms presented in humans from such exposures, limited availability of (and access to) documented reports, and the absence of standardized and validated test models, hinders the identification of true respiratory sensitizers. This review aims to sort suspected LMW respiratory sensitizers based on available compelling, reasonable, inadequate, or questionable evidence in humans from occupational exposures and use this information to compose a reference list of reported chemical respiratory sensitizers for scientific research purposes. A list of 97 reported respiratory sensitizers was generated from six sources, and 52 LMW organic chemicals were identified, reviewed, and assigned to the four evidence categories. Less than 10 chemicals were confirmed with compelling evidence for induction of respiratory sensitization in humans from occupational exposures. Here, we propose the reference list for developing novel research on respiratory sensitization.

Refinement of the Peroxidase Peptide Reactivity Assay and Prediction Model for Assessing Skin Sensitization Potential.

A peptide reactivity assay with an activation component was developed for use in screening chemicals for skin sensitization potential. A horseradish peroxidase-hydrogen peroxide (HRP/P) oxidation system was incorporated into the assay for characterizing reactivity of hapten and pre-/prohapten sensitizers. The assay, named the Peroxidase Peptide Reactivity Assay (PPRA) had a predictive accuracy of 83% (relative to the local lymph node assay) with the original protocol and prediction model. However, apparent false positives attributed to cysteine depletion at relatively high chemical concentrations and, for some chemicals expected to react with the -NH2 group of lysine, little to no depletion of the lysine peptide were observed. To improve the PPRA, cysteine peptide reactions with and without HRP/P were modified by increasing the number of test concentrations and refining their range. In addition, removal of DL-dithiothreitol from the reaction without HRP/P increased cysteine depletion and improved detection of reactive aldehydes and thiazolines without compromising the assay's ability to detect prohaptens. Modification of the lysine reaction mixture by changing the buffer from 0.1 M ammonium acetate buffer (pH 10.2) to 0.1 M phosphate buffer (pH 7.4) and increasing the level of organic solvent from 1% to 25% resulted in increased lysine depletion for known lysine reactive chemicals. Refinement of the prediction model improved the sensitivity, specificity, and accuracy for hazard identification. These changes resulted in significant improvement of the PPRA making it is a reliable method for predicting the skin sensitization potential of all chemicals, including pre-/prohaptens and directly reactive haptens.

Application of the dermal sensitization threshold concept to chemicals classified as high potency category for skin sensitization assessment of ingredients for consumer products.

Skin sensitization evaluation is a key part of the safety assessment of ingredients in consumer products, which may have skin sensitizing potential. The dermal sensitization threshold (DST) concept, which is based on the concept of the thresholds of toxicological concern, has been proposed for the risk assessment of chemicals to which skin exposure is very low level. There is negligible risk of skin sensitization if a skin exposure level for the substance of interest was below the reactive DST which would protect against 95% of protein-reactive chemicals. For the remaining 5%, the substance with the defined knowledge of chemical structure (i.e., High Potency Category (HPC) rules) needs to be excluded from the application. However, the DST value for HPC chemicals has not yet been proposed. In this study, we calculated the 95th percentile probabilities estimate from distributions of skin sensitization potency data and derived a novel DST for HPC chemicals (HPC DST) of 1.5 µg/cm2. This value presents a useful default approach for unidentified substances in ingredients considering, as a worst-case scenario, that the unidentified compound may be a potent skin sensitizer. Finally, we developed a novel risk assessment workflow incorporating the HPC DST along with the previously published DSTs.

Determination of the relative allergenic potency of proteins: hurdles and opportunities.

The use of proteins and protein-containing materials in a variety of industrial and commercial products is increasing, with applications in pharmaceuticals, agrochemicals and consumer and personal care products. As a consequence there is a need to ensure potential and environmental risks are understood. One important requirement is an appreciation of the ability of proteins to induce allergic sensitization and allergic disease. However, there is currently no clear guidance for determination of whether or not to accept a new protein in a product based on potential allergenicity. A key requirement for effective risk assessment in this respect is an understanding of sensitizing potency. Here we describe issues and challenges associated with measurement of allergenic potency and explore emerging opportunities and possible ways forward. Effective assessment of the risk of allergy demands not only information about the likely conditions of exposure, but also an understanding of the sensitizing potency of protein allergens. For the purposes of this article sensitizing potency can be viewed as being the ease with which, and the concentration at which, proteins will induce sensitization in a previously non-sensitized subject. The immunological bases of protein allergy are summarized, and the properties that confer on proteins the ability to induce allergic sensitization are considered prior to a detailed exploration of the issues that have to be addressed for evaluation of sensitizing potency. Included among the important considerations are: the impact of route of exposure, identification of relevant dose metrics, and the requirement for reference standards. Finally, new and emerging opportunities to evaluate the sensitizing potency of allergenic proteins are reviewed, including the use of in silico modeling.

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.

Proteomic and Bioinformatic Analyses for the Identification of Proteins With Low Allergenic Potential for Hazard Assessment.

Use of botanicals and natural substances in consumer products has increased in recent years. Such extracts can contain protein that may theoretically represent a potential risk of IgE-mediated allergy. No method has yet been generally accepted or validated for assessment of the allergenic potential of proteins. For development of suitable methods datasets of allergenic and nonallergenic (or low allergenic) proteins are required that can serve, respectively, as positive and negative controls. However, data are unavailable on proteins that lack or have low allergenic potential. Here, low allergenic potential proteins are identified based on the assumption that proteins with established human exposure, but with a lack of an association with allergy, possess low allergenic potential. Proteins were extracted from sources considered to have less allergenic potential (corn, potato, spinach, rice, and tomato) as well as higher allergenic potential (wheat) regarding common allergenic foods. Proteins were identified and semi-quantified by label-free proteomic analysis conducted using mass spectrometry. Predicted allergenicity was determined using AllerCatPro (https://allercatpro.bii.a-star.edu.sg/). In summary, 9077 proteins were identified and semi-quantified from 6 protein sources. Within the top 10% of the most abundant proteins identified, 178 characterized proteins were found to have no evidence for allergenicity predicted by AllerCatPro and were considered to have low allergenic potential. This panel of low allergenic potential proteins provides a pragmatic approach to aid the development of alternative methods for robust testing strategies to distinguish between proteins of high and low allergenic potential to assess the risk of proteins from natural or botanical sources.

AllerCatPro-prediction of protein allergenicity potential from the protein sequence.

MOTIVATION: Due to the risk of inducing an immediate Type I (IgE-mediated) allergic response, proteins intended for use in consumer products must be investigated for their allergenic potential before introduction into the marketplace. The FAO/WHO guidelines for computational assessment of allergenic potential of proteins based on short peptide hits and linear sequence window identity thresholds misclassify many proteins as allergens. RESULTS: We developed AllerCatPro which predicts the allergenic potential of proteins based on similarity of their 3D protein structure as well as their amino acid sequence compared with a data set of known protein allergens comprising of 4180 unique allergenic protein sequences derived from the union of the major databases Food Allergy Research and Resource Program, Comprehensive Protein Allergen Resource, WHO/International Union of Immunological Societies, UniProtKB and Allergome. We extended the hexamer hit rule by removing peptides with high probability of random occurrence measured by sequence entropy as well as requiring 3 or more hexamer hits consistent with natural linear epitope patterns in known allergens. This is complemented with a Gluten-like repeat pattern detection. We also switched from a linear sequence window similarity to a B-cell epitope-like 3D surface similarity window which became possible through extensive 3D structure modeling covering the majority (74%) of allergens. In case no structure similarity is found, the decision workflow reverts to the old linear sequence window rule. The overall accuracy of AllerCatPro is 84% compared with other current methods which range from 51 to 73%. Both the FAO/WHO rules and AllerCatPro achieve highest sensitivity but AllerCatPro provides a 37-fold increase in specificity. AVAILABILITY AND IMPLEMENTATION: https://allercatpro.bii.a-star.edu.sg/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Quantitative risk assessment for skin sensitization: Success or failure?

Skin sensitization is unique in the world of toxicology. There is a combination of reliable, validated predictive test methods for identification of skin sensitizing chemicals, a clearly documented and transparent approach to risk assessment, and effective feedback from dermatology clinics around the world delivering evidence of the success or failure of the hazard identification/risk assessment/management process. Recent epidemics of contact allergy, particularly to preservatives, have raised questions of whether the safety/risk assessment process is working in an optimal manner (or indeed is working at all!). This review has as its focus skin sensitization quantitative risk assessment (QRA). The core toxicological principles of QRA are reviewed, and evidence of use and misuse examined. What becomes clear is that skin sensitization QRA will only function adequately if two essential criteria are met. The first is that QRA is applied rigourously, and the second is that potential exposure to the sensitizing substance is assessed adequately. This conclusion will come as no surprise to any toxicologist who appreciates the basic premise that "risk = hazard x exposure". Accordingly, use of skin sensitization QRA is encouraged, not least because the essential feedback from dermatology clinics can be used as a tool to refine QRA in situations where this risk assessment tool has not been properly used.

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.

Interleukin-1ß Processing Is Dependent on a Calcium-mediated Interaction with Calmodulin.

The secretion of IL-1ß is a central event in the initiation of inflammation. Unlike most other cytokines, the secretion of IL-1ß requires two signals: one signal to induce the intracellular up-regulation of pro-IL-1ß and a second signal to drive secretion of the bioactive molecule. The release of pro-IL-1ß is a complex process involving proteolytic cleavage by caspase-1. However, the exact mechanism of secretion is poorly understood. Here we sought to identify novel proteins involved in IL-1ß secretion and intracellular processing to gain further insights into the mechanism of IL-1 release. A human proteome microarray containing 19,951 unique proteins was used to identify proteins that bind human recombinant pro-IL-1ß. Probes with a signal-to-noise ratio of >3 were defined as biologically relevant. In these analyses, calmodulin was identified as a particularly strong hit, with a signal-to-noise ratio of ∼ 11. Using an ELISA-based protein-binding assay, the interaction of recombinant calmodulin with pro-IL-1ß, but not mature IL-1ß, was confirmed and shown to be calcium-dependent. Finally, using small molecule inhibitors, it was demonstrated that both calcium and calmodulin were required for nigericin-induced IL-1ß secretion in THP-1 cells and primary human monocytes. Together, these data suggest that, following calcium influx into the cell, pro-IL-1ß interacts with calmodulin and that this interaction is important for IL-1ß processing and release.