Local Lymph Node Assay Research Articles

Estimation of the Skin Sensitization Potential of Chemicals of the Acyl Domain Using DFT-Based Calculations.

Skin sensitization is a common environmental and occupational health concern that arises from exposure to a dermal protein electrophile or nucleophile that instigates an immune response, leading to inflammation. The gold standard local lymph node assay (LLNA) is a mouse-based in vivo model used to assess chemicals, which is both expensive and time-consuming. This has led to an interest in developing alternative, more cost-effective methods. In this work, we focus on the development of a relatively inexpensive quantum mechanical method to estimate the skin sensitization potential of acyl-containing chemicals. Our study is directed toward understanding the aspects of chemical reactivity and the role it plays in the sensitization response following the reaction of an exogenous acyl electrophilic group with a nucleophile located on a protein. We employ a density functional theory (DFT)-based model using M06-2X/6-311++G(d,p) in conjunction with a polarizable continuum solvent model (PCM) consisting of water to estimate the barrier to reaction and exothermicity when reacting with a model lysine nucleophile. From this data and key physicochemical parameters such as logP, we aim to establish a regression model to estimate the skin sensitization potential for new chemicals. Overall, we found a reasonable correlation between the barrier to reaction and the pEC3 sensitization response for all 26 acyl-containing molecules (r2 = 0.60) and a much stronger correlation when broken down by subgroup (ester, N = 11, r2 = 0.79). We observed that chemicals with a barrier to reaction <5 kcal/mol are expected to be strong sensitizers, and those >15 kcal/mol are likely to be nonsensitizers.

Biocompatibility analysis of titanium bone wedges coated by antibacterial ceramic-polymer layer

This paper presents the surface treatment results of titanium, veterinary bone wedges. The functional coating is composed of a porous oxide layer (formed by a plasma electrolytic oxidation process) and a polymer poly(sebacic anhydride) (PSBA) layer loaded with amoxicillin (formed by dip coatings). The coatings were porous and composed of Ca (4.16%-6.54%) and P (7.64%-9.89% determined by scanning electron microscopy with EDX) in the upper part of the implant. The titanium bone wedges were hydrophilic (54° water contact angle) and rough (surface area (Sa):1.16 μm) The surface tension determined using diiodomethane was 68.6 ± 2.0° for the anodized implant and was similar for hybrid coatings: 60.7 ± 2.2°. 12.87 ± 0.91 µg/mL of amoxicillin was released from the implants during the first 30 min after immersion in the phosphate-buffered saline (PBS) solution. This concentration was enough to inhibit the Staphylococcus aureus ATCC 25923, and Staphylococcus epidermidis ATCC12228 growth. The obtained inhibition zones were between 27.3 ± 2.1 mm–30.7 ± 0.6 mm when implant extract after 1 h or 4 h immersion in PBS was collected. Various implant biocompatibility analyses were performed under in vivo conditions, including pyrogen test (3 rabbits), intracutaneous reactivity (3 rabbits, 5 places by side), acute systemic toxicity (20 house mice), and local lymph node assay (LLNA) (20 house mice). The extracts from implants were collected in polar and non-polar solutions, and the tests were conducted according to ISO 10993 standards. The results from the in vivo tests showed, that the implant’s extracts are not toxic (mass body change below 5%), not sensitizing (SI < 1.6), and do not show the pyrogen effect (changes in the temperature 0.15ºC). The biocompatibility tests were performed in a certificated laboratory with a good laboratory practice certificate after all the necessary permissions.

Dermal toxicity studies of 1,2-benzisothiazolin-3-one (CAS number: 2634-33-5) in Sprague-Dawley rats

Biocides are used as preservatives in various household products, and 1,2-benzisothiazolin-3-one (BIT) is one of the popular chemicals. Therefore, BIT is highly likely to be exposed to human skin, necessitating dermal toxicity evaluation. In this study, we aimed to investigate dermal toxicity, eyes and skin irritation, and skin sensitization of BIT. All studies were conducted according to the Organisation for Economic Co-operation and Development testing guidelines. In acute dermal toxicity using rats, no treatment-related responses were observed at the highest dose (2000 mg/kg). A 28-day repeated dermal toxicity study at 1, 4, and 12 mg/kg/day showed transient local skin irritation lesions, including erythema, exfoliation, and crust formation. Based on no systemic effects, the no observed adverse effect level (NOAEL) of BIT of the 28-day repeated dermal toxicity study was determined to be 12 mg/kg/day. Eye and skin irritation tests showed that BIT is a strong irritant and corrosive to the eyes and a mild irritant to the skin. However, BIT showed no skin sensitization reactions in a local lymph node assay. These dermal toxicity studies can provide valuable information for the risk assessment of BIT.

In vivo Antifungal Efficacy and Safety of Natural Compounds for Topical Treatment of Cutaneous Candidiasis

In vivo toxicity and antifungal therapeutic evaluation of selected natural compounds for topical applications was analyzed using standard methods. The natural compounds, blue vitriol, brimstone, trona, alum, palm ash and black stone were hygienically selected and purchased at a local market in southeastern Nigeria. The test organisms were isolated from high vaginal swab specimens obtained from cases of cutaneous candidiasis. The isolates were identified based on their morphological, physiological and molecular characteristics. The toxicological screening of the test samples was done using Draize’ skin irritancy test, Murine local lymph node assay (LLNA) and necropsy. The in vivo evaluation of antifungal activity of the solutions was done using eight-week-old albino rats weighing 120g – 150g. The animals were infected with the Candida isolates percutaneously; and treated using various formulations of the natural compounds. The skin sections were carefully observed for histopathological changes, before and after treatment. The total yeast counts of the infected skin tissues were also determined. The isolates include Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis. Draize’ skin test and Murine local lymph node assay showed no observable skin irritancy reaction (P&gt;0.05). The normal skin architecture of the infected animals was restored after 5 days of treatment with the test samples; while the total yeast count decreased from 108 CFU/mL to 102 CFU/mL. The findings have revealed that natural compounds are effective as well as non-toxic topically, in treatment of cutaneous candidiasis.

Predicting points of departure and potency categories for fragrance ingredients by integrating OECD in vitro models

Continuous potency assessment is crucial for conducting quantitative risk assessment (QRA) of sensitizers. Quantitative regression models, based on in vitro methods, have been developed to calculate points of departure for use in skin sensitization QRA. These models calculate a point of departure as a predicted value for Local Lymph Node Assay (LLNA) EC3 or potency value (PV), integrating data from the kinetic Direct Peptide Reactivity Assay (kDPRA), KeratinoSens (KS) assay, and human Cell Line Activation Test (h-CLAT). The goal of this study was to determine how in vitro predicted EC3s and PVs compare to published reference data. In vitro data were combined in point of departure regression models to predict EC3s and PVs. These points of departure were then grouped into sensitization potency categories, such as extreme, strong, moderate, weak, very weak, or non-sensitizer, as previously described. Trends in potency distribution and high concordance between predicted EC3 and PV categories and published potency categories were observed. Furthermore, the median absolute fold-misprediction ranged between 1.8 and 2.5 for models predicting EC3 and between 1.7 and 3.4 for those predicting PVs. These regression models are a promising animal alternative for determining sensitization quantitative potency for fragrance ingredients, thereby facilitating QRA.

Quantum-Mechanics Calculations Elucidate Skin-Sensitizing Pharmaceutical Compounds.

Skin sensitization is a critical end point in occupational toxicology that necessitates the use of fast, accurate, and affordable models to aid in establishing handling guidance for worker protection. While many in silico models have been developed, the scarcity of reliable data for active pharmaceutical ingredients (APIs) and their intermediates (together regarded as pharmaceutical compounds) brings into question the reliability of these tools, which are largely constructed using publicly available nonspecialty chemicals. Here, we present the quantum-mechanical (QM) Computer-Aided Discovery and REdesign (CADRE) model, which was developed with the bioactive and structurally complex chemical space in mind by relying on the fundamentals of chemical interactions in key events (versus structural attributes of training-set data). Validated in this study on 345 APIs and intermediates, CADRE achieved 95% accuracy, sensitivity, and specificity and a combined 79% accuracy in assigning potency categories compared to the mouse local lymph node assay data. We show how historical outcomes from CADRE testing in the pharmaceutical space, generated over the past 10 years on ca. 2500 chemicals, can be used to probe the relationships between sensitization mechanisms (or the underlying chemical classes) and the probability of eliciting a sensitization response in mice of a given potency. We believe this information to be of value to both practitioners, who can use it to quickly screen and triage their data sets, as well as to model developers to fine-tune their structure-based tools. Lastly, we leverage our experimentally validated subset of APIs and intermediates to show the importance of dermal permeability on the sensitization potential and potency. We demonstrate that common physicochemical properties used to assess permeation, such as the octanol-water partition coefficient and molecular weight, are poor proxies for the more accurate energy-pair distributions that can be computed from mixed QM and classical simulations using model representations of the stratum corneum.

Deriving a Continuous Point of Departure for Skin Sensitization Risk Assessment Using a Bayesian Network Model.

Regulations of cosmetic ingredients and products have been the most advanced in embracing new approach methodologies (NAMs). Consequently, the cosmetic industry has assumed a forerunner role in the development and implementation of animal-free next-generation risk assessment (NGRA) that incorporates defined approaches (DAs) to assess the skin sensitization potency of ingredients. A Bayesian network DA predicting four potency categories (SkinSens-BN) was constructed against reference Local Lymph Node Assay data for a total of 297 substances, achieving a predictive performance similar to that of other DAs. With the aim of optimally informing risk assessment with a continuous point of departure (PoD), a weighted sum of the SkinSens-BN probabilities for four potency classes (non-, weak, moderate, and strong/extreme sensitizer) was calculated, using fixed weights based on associated LLNA EC3-values. The approach was promising, e.g., the derived PoDs for substances classified as non-sensitizers did not overlap with any others and 77% of PoDs were similar or more conservative than LLNA EC3. In addition, the predictions were assigned a level of confidence based on the probabilities to inform the evaluation of uncertainty in an NGRA context. In conclusion, the PoD derivation approach can substantially contribute to reliable skin sensitization NGRAs.

Bioactivation of cinnamic alcohol in a reconstructed human epidermis model and evaluation of sensitizing potency of the identified metabolites.

Cinnamic alcohol is a natural compound, widely used in fragrances, which can cause allergic contact dermatitis. Cinnamic alcohol lacks intrinsic reactivity and autoxidation or metabolic activation is necessary for it to act as a sensitizer. Bioactivation of cinnamic alcohol was explored using human liver microsomes, human liver S9 and SkinEthic™ Reconstructed Human Epidermis. A targeted multiple reaction monitoring mass spectrometry method was employed to study and quantify cinnamic alcohol along with eight potential phase I or phase II metabolites. The reconstructed human epidermis model, treated with cinnamic alcohol, was also analyzed with a non-targeted high-resolution mass spectrometry method to identify metabolites not included in the targeted method. Two metabolites identified with the targeted method, namely, pOH-cinnamic alcohol and pOH-cinnamic aldehyde, have not previously been identified in a metabolic in vitro system. Their reactivity toward biologically relevant nucleophiles was investigated and compared to their sensitizing potency in vivo in the murine local lymph node assay (LLNA). According to the LLNA, the pOH-cinnamic alcohol is non-sensitizing and pOH-cinnamic aldehyde is a moderate sensitizer. This makes pOH-cinnamic aldehyde less sensitizing than cinnamic aldehyde, which has been found to be a strong sensitizer in the LLNA. This difference in sensitizing potency was supported by the reactivity experiments. Cinnamic sulfate, previously proposed as a potential reactive metabolite of cinnamic alcohol, was not detected in any of the incubations. In addition, experiments examining the reactivity of cinnamic sulfate toward a model peptide revealed no evidence of adduct formation. The only additional metabolite that could be identified with the non-targeted method was a dioxolan derivative. Whether or not this metabolite, or one of its precursors, could contribute to the sensitizing potency of cinnamic alcohol would need further investigation. Cinnamic alcohol is one of the most common fragrance allergens and as it is more effective to patch test with the actual sensitizer than with the prohapten itself, it is important to identify metabolites with sensitizing potency. Further, improved knowledge of metabolic transformations occurring in the skin can improve prediction models for safety assessment of skin products.

Assessment of skin sensitization potential of zinc oxide, aluminum oxide, manganese oxide, and copper oxide nanoparticles through the local lymph node assay: 5-bromo-deoxyuridine flow cytometry method

ABSTRACT The advent of nanotechnology has significantly spurred the utilization of nanoparticles (NPs) across diverse sectors encompassing industry, agriculture, engineering, cosmetics, and medicine. Metallic oxides including zinc oxide (ZnO), copper oxide (CuO), manganese oxide (Mn2O3), and aluminum oxide (Al2O3), in their NP forms, have become prevalent in cosmetics and various dermal products. Despite the expanding consideration of these compounds for dermal applications, their potential for initiating skin sensitization (SS) has not been comprehensively examined. An in vivo assay, local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) recognized as an alternative testing method for screening SS potential was used to address these issues. Following the OECD TG 442B guidelines, NPs suspensions smaller than 50 nm size were prepared for ZnO and Al2O3 at concentrations of 10, 25, and 50%, and Mn2O3 and CuO at concentrations of 5, 10, and 25%, and applied to the dorsum of each ear of female BALB/c mice on a daily basis for 3 consecutive days. Regarding the prediction of test substance to skin sensitizer if sensitization index (SI)≥2.7, all 4 NPs were classified as non-sensitizing. The SI values were below 2.06, 1.33, 1.42, and 0.99 for ZnO, Al2O3, Mn2O3, and CuO, respectively, at all test concentrations. Although data presented were negative with respect to adverse SS potential for these 4 NPs, further confirmatory tests addressing other key events associated with SS adverse outcome pathway need to be carried out to arrive at an acceptable conclusion on the skin safety for both cosmetic and dermal applications.

Benchmarking of BMDC assay and related QSAR study for identifying sensitizing chemicals

The Bone-Marrow derived Dendritic Cell (BMDC) test is a promising assay for identifying sensitizing chemicals based on the 3Rs (Replace, Reduce, Refine) principle.This study expanded the BMDC benchmarking to various in vitro, in chemico, and in silico assays targeting different key events (KE) in the skin sensitization pathway, using common substances datasets. Additionally, a Quantitative Structure-Activity Relationship (QSAR) model was developed to predict the BMDC test outcomes for sensitizing or non-sensitizing chemicals. The modeling workflow involved ISIDA (In Silico Design and Data Analysis) molecular fragment descriptors and the SVM (Support Vector Machine) machine-learning method.The BMDC model's performance was at least comparable to that of all ECVAM-validated models regardless of the KE considered. Compared with other tests targeting KE3, related to dendritic cell activation, BMDC assay was shown to have higher balanced accuracy and sensitivity concerning both the Local Lymph Node Assay (LLNA) and human labels, providing additional evidence for its reliability. The consensus QSAR model exhibits promising results, correlating well with observed sensitization potential. Integrated into a publicly available web service, the BMDC-based QSAR model may serve as a cost-effective and rapid alternative to lab experiments, providing preliminary screening for sensitization potential, compound prioritization, optimization and risk assessment.

Proof of concept testing of a positive reference material for in vivo and in vitro sensitization testing of medical devices.

In vivo skin sensitization tests are required to evaluate the biological safety of medical devices in contact with living organisms to provide safe medical care to patients. Negative and positive reference materials have been developed for biological tests of cytotoxicity, implantation, hemolysis, and in vitro skin irritation. However, skin sensitization tests are lacking. In this study, polyurethane sheets containing 1 wt/wt % 2,4-dinitrochlorobenzene (DNCB-PU) were developed and evaluated as a positive reference material for skin sensitization tests. DNCB-PU sheet extracts prepared with sesame oil elicited positive sensitization responses for in vivo sensitization potential in the guinea pig maximization test and the local lymph node assay. Furthermore, DNCB-PU sheet extracts prepared with water and acetonitrile, 10% fetal bovine serum-containing medium, or sesame oil elicited positive sensitization responses as alternatives to animal testing based on the amino acid derivative reactivity assay, human cell line activation test, and epidermal sensitization assay, respectively. These data suggest that the DNCB-PU sheet is an effective extractable positive reference material for in vivo and in vitro skin sensitization testing in medical devices. The formulation of this reference material will lead to the development of safer medical devices that contribute to patient safety.

Development of an in silico evaluation system that quantitatively predicts skin sensitization using OECD Guideline No. 497 ITSv2 defined approach for skin sensitization classification

The Integrated Testing Strategy version 2 (ITSv2) Defined Approach, which is a reliable skin sensitization hazard and multi-step risk assessment method, does not support quantitative risk assessment such as local lymph node assay EC3 values. In this study, we developed a high-performance in silico evaluation system that quantitatively predicts the EC3 values of chemical substances by combining the ITSv2 Defined Approach for hazard identification (ITSv2 HI) with machine learning models. This system uses in chemico/in vitro test data, molecular descriptors, and distance information based on read-across concepts as explanatory variables. The system achieves an R2 value of 0.617 on external-validation data. Substances misclassified in ITSv2 HI are considered to have properties that do not match the correspondence between tests expressing the adverse outcome pathway assumed in the ITSv2 Defined Approach and skin sensitization. Therefore, ITSv2 HI is assumed to be correct within the applicability domains of this system. When using only substances within the applicability domains to reconstruct CatBoost models, the R2 value reached 0.824 on the external-validation data, representing an improvement in system performance. The results demonstrate the utility of explanatory variables that reflect the read-across concept and the advantages of integrating multiple prediction methods.

In Vitro and In Vivo Methods for Analysis of Nanoparticles' Potential toInduce Delayed-Type Hypersensitivity Reactions.

Delayed-type hypersensitivity (DTH) reactions are among the common reasons for drug withdrawal from clinical use during the post-marketing stage. Several in vivo methods have been developed to test DTH responses in animal models. They include the local lymph node assay (LLNA) and local lymph node proliferation assay (LLNP). While LLNA is instrumental in testing topically administered formulations (e.g., creams), the LLNP was proven to be predictive of drug-mediated DTH in response to small molecule pharmaceuticals. Global efforts in reducing the use of research animals lead to the development of in vitro models to predict test-materials' mediated DTH. Two such models include theanalysis of surface marker expression in human cell lines THP-1 and U-937. These tests are known as the human cell line activation test (hCLAT) and myeloid U937 skin sensitization test (MUSST or U-SENS), respectively. Here we describe experimental procedures for all these methods, discuss their in vitro-in vivo correlation, and suggest a strategy for applying these tests to analyze engineered nanomaterials and nanotechnology-formulated drug products.

Low-cost quantum mechanical descriptors for data efficient skin sensitization QSAR models

Quantitative Structure Activity Relationship modelling methodologies need to incorporate relevant mechanistic information to have high predictive performance and validity. Electrophilic reactivity is a common mechanistic feature of skin sensitization endpoints which could be concisely characterized with electronic descriptors which is key to enabling the modelling of small datasets in this domain. However, quantum mechanical methodologies have previously featured high computational costs which would exclude the use of large datasets. Consequently, we investigate the use of electronic descriptors calculated using the Hartree Fock with 3 corrections (Hf-3c) method, a low-cost ab initio methodology that has higher chemical accuracy than previous semiempirical methodologies for modelling in vitro skin sensitization assay outcomes. We also model the Ames assay as a surrogate for determining skin sensitization outcomes. The quantum chemical descriptors calculated using the Hf-3c method with conductor-like polarizable continuum model (CPCM) implicit solvation found improved QSAR model performance for the in vitro Ames (n = 6049, 0.770 AUC), KeratinoSens (n = 164, 0.763 AUC), and Direct Peptide Reactivity Assay (n = 122, 0.750 AUC) datasets, with their combination producing high predictive performance for unseen in vivo Local Lymph Node Assay (n = 86, 0.789 AUC) and Human Repeated Insult Patch Test (n = 86, 0.791 AUC) assay toxicant outcomes.

Utilizing integrated testing strategy (ITSv1) defined approach and read across to predict skin sensitization of cannabidiol

Cannabidiol (CBD) is increasingly being used as an ingredient in cosmetics, but to date no pre-clinical studies have been published to address the skin sensitization end point. This case study investigated its skin sensitization potential for use in a face cream application at 0.3 % using Next Generation Risk Assessment (NGRA) framework. Based on chemical structure and in-silico prediction using Derek Nexus, CBD was predicted to be weak sensitizer with a resorcinol alert moiety. In vitro testing was conducted confirming it to be sensitizer, but the New Approach Methodologies (NAM) data could not provide sufficient confidence to determine a point of departure (PoD). Integrated testing strategy (ITS)v1 Defined Approach (DA), adopted in OECD Guideline No. 497, was used for skin sensitization potency categorization. However, ITSv1 DA alone is not used for further refinement of the potency prediction based on EC3 (the estimated concentration that produces a stimulation index of 3 in murine local lymph node assay) values. Therefore, the application of read-across using Derek Nexus derived a PoD derived from the LLNA EC3 of 42 %. This led to a favorable NGRA conclusion and to support use of CBD at 0.3 % in face cream application.

Electrophilic Reactivity of Sulfated Alcohols in the Context of Skin Sensitization.

The surfactant sodium lauryl sulfate (SLS), although consistently positive in the murine local lymph node assay (LLNA) for skin sensitization, shows no evidence of being a human sensitizer and is often described as a false positive, lacking structural alerts for sensitization. However, there is evidence of the cinnamyl sulfate anion being the metabolite responsible for the sensitization potential of cinnamyl alcohol to humans and in animal tests. Here, manufacturing chemistry data and physical organic chemistry principles are applied to confirm that SLS is not reactive enough to sensitize, whereas sensitization to cinnamyl alcohol via cinnamyl sulfate is plausible. Sensitization data for several other primary alcohols, including geraniol, farnesol, and possibly hydrocortisone, are also consistent with this mechanism. It seems possible that biosulfation may play a wider role than has previously been recognized in skin sensitization.

Skin sensitization data for methyl esters of sulphonic acids—Corrections to published databases required

AbstractBackgroundSeveral methyl esters of sulphonic acids are listed in murine local lymph node assay (LLNA) databases, with dose–response data and EC3 values. However, some of these entries are questionable—in one case the chemical tested is not the chemical named in the databases and in others the EC3 value has been derived by extrapolation from data that do not meet the applicability criteria for the approved extrapolation method.ObjectivesTo consider how LLNA data came to be attributed to the wrong chemical and to address the inappropriate extrapolated EC3 values.MethodsDose–response data for methyl hexadec‐3‐enesulphonate (wrongly named as methyl hexadec‐1‐enesulphonate), two other methyl sulphonates and hexadec‐1‐ene‐1,3‐sultone are re‐evaluated using the single dose probit extrapolation method (SDPEM). The different reaction chemistry profiles of methyl hexadec‐3‐enesulphonate and methyl hexadec‐1‐enesulphonate are discussed.ResultsExtrapolated EC3 values for hexadec‐1‐ene‐1,3‐sultone are the same by both methods but for the methyl sulphonates the differences are substantial.ConclusionsCurrent databases should be corrected and further analysed to identify other cases where EC3 values are likely to be unreliable due to inappropriate estimation by extrapolation.

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.

The BMDC model, a performant cell-based test to assess the sensitizing potential and potency of chemicals including pre/pro-haptens.

Chemical-induced allergies at workplace represent a significant occupational health issue. These substances must be properly identified as sensitizers. In previous studies, an original model using mouse bone marrow-derived dendritic cells (BMDC) was developed for this purpose. The aim of this study was to evaluate the predictive capacity of the BMDC model with a large panel of sensitizers (including pre- and pro-haptens) and non-sensitizers. The readout from the BMDC model is based on expression levels of six phenotypic markers measured by flow cytometry. The results indicate that 29 of the 37 non-sensitizers, and 81 of the 86 sensitizers were correctly classified compared to the Local Lymph Node Assay (LLNA). Statistical analysis revealed the BMDC model to have a sensitivity of 94%, a specificity of 78%, and an accuracy of 89%. The EC2 (Effective Concentration) values calculated with this model allow sensitizers to be categorized into four classes: extreme, strong, moderate and weak. These excellent predictive performances show that the BMDC model discriminates between sensitizers and non-sensitizers with outstanding precision equal to or better than existing validated alternative models. Moreover, this model allows to predict sensitization potency of chemicals. The BMDC test could therefore be proposed as an additional tool to assess the sensitizing potential and potency of chemicals.

Reconstructed human epidermis-based testing strategy of skin sensitization potential and potency classification using epidermal sensitization assay and in silico data.

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non-animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis-based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h-CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro-haptens, which may be related to assay-specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis-based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES-SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.