The regression based defined approach for skin sensitization potency assessment based on NAM data from OECD accepted tests.

We review allergic contact dermatitis (ACD) with predominant Th2 type cytokine expression in the context of chronic cutaneous inflammation. While more has been written about Th1 skewed ACD due to potent allergens in the setting of healthy skin, this review highlights recognition of Th2 skewed ACD in both atopic dermatitis and systemic contact dermatitis and the role of allergen avoidance as an alternative to systemic therapies. Th2 skewed ACD rarely occurs to potent allergens. It more commonly occurs in response to allergens considered “non-sensitizers” in the local lymph node assay. These sensitizers include weaker allergens (e.g. propylene glycol), larger molecules (e.g. food proteins) and commensal micro-organisms. Importantly, group 2 innate lymphoid cells and natural killer T cells may contribute to these cutaneous memory responses without education of Th2 cells in the local lymph node and without downstream antigen-specific IgE. The resulting intrinsic atopic dermatitis may be food-triggered and better diagnosed with atopy patch testing than with tests for antigen specific-IgE used for immediate type hypersensitivity reactions. Chronic contact, atopic, and stasis dermatitis all occur in the setting of irritant dermatitis and microbial dysbiosis. Both Th1 and Th2 cytokines mediate ACD, although those cytokines may arise from innate immune pathways and not exclusively from T helper cells educated in the local lymph node. More refinement and use of atopy patch tests to identify less potent allergens and dietary avoidance of patient-specific allergens may reduce the number of patients who require systemic therapy for eczema.

Human health risk assessment of cosmetic ingredients must address skin sensitization due to their direct skin application. With the recent shift toward Next Generation Risk Assessment (NGRA), which utilizes animal-free alternative testing methods, the read-across (RAx) approach has gained prominence. RAx relies on information from structural analogues to fill data gaps and determine a point of departure (PoD). A major challenge in RAx is assessing "similarity" between the target chemical and its analogues, typically based on chemical structure, physicochemical properties, metabolic reactivity, in silico prediction profile, and biological activity. Structural similarity, often evaluated using the Tanimoto coefficient (Tc), is a common first step, and it relies on fingerprints that convert chemical structures into machine-readable formats. However, fingerprint choice significantly influences Tc calculations and subsequent analogue selection. Moreover, no guidelines exist for the use of specific fingerprints, and which fingerprint is most suitable for skin sensitization assessment by RAx has not yet been established. This study aimed to develop a novel fingerprint specifically optimized for skin sensitization assessment. A large dataset of chemicals with the murine local lymph node assay (LLNA)-positive data was used for performance evaluation of various fingerprints. Our findings revealed that a novel fingerprint incorporating Protein Binding Alert-based Fingerprint (PBAF) and Klekota-Roth fingerprint (KRFP) features into ToxPrint outperformed others in separating suitable from unsuitable chemical pairs for RAx. This study underscores the importance of endpoint-specific fingerprint development to improve analogue selection for skin sensitization risk assessments.

Development and evaluation of a novel consensus in silico model within the OECD GL497 defined approach for skin sensitization prediction.

OECD Guideline 497 includes defined approaches (DAs) that combine new approach methodologies (NAMs) to predict skin sensitization hazard and categorize potency according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). To increase flexibility, the OECD allows the substitution in the DAs of NAMs addressing the same key events (KEs) in the adverse outcome pathway (AOP). This study evaluated the implementation of the U-SENS™ assay for dendritic cell activation (KE3) within two integrated testing strategy (ITS) DAs. First, EC150 threshold values were determined via computational modeling to convert CD86 stimulation index data into ITS DA scores. The performance of the modified ITS DAs was then evaluated against available Local Lymph Node Assay (LLNA) and human reference data. For hazard identification, the ITS DAs achieved 79% balanced accuracy compared with LLNA data, and 73-74% compared with human data. For potency categorization, correct classification rates were 67-68% against LLNA and 65-67% against human data. In comparison, the LLNA showed lower performance, with 58% balanced accuracy for hazard identification and 60% for potency categorization against human data. Selected case studies illustrate the practical application of these DAs following the Guideline 497 decision flowchart. Overall, findings underscore that incorporating U-SENS™ into ITS DAs enhances flexibility without compromising predictive capacity and outperforms traditional animal testing in providing reliable skin sensitization classification outcomes.

This study investigated the phytochemical profile, in silico skin sensitization, cosmetic toxicity, and endocrine-disrupting potential of compounds from Amaranthus retroflexus identified through GC-MS analysis. Volatile constituents were identified using gas chromatography–mass spectrometry (GC–MS) analysis. The safety profiles of the major compounds were evaluated through in silico toxicity prediction models, including keratinosens, local lymph node assay (LLNA) and human cell line activation test (h-CLAT), along with molecular docking analysis to assess their endocrine-disrupting potential. A total of 39 distinct compounds were identified, with a predominant presence of unsaturated fatty acid methyl esters, including 9,12-octadecenoic acid, methyl ester (47.275 %) and 9-octadecenoic acid, methyl ester (22.790 %). Other notable compounds included methyl stearate (3.063 %), Squalene (9.687 %), and various alkanes. In silico skin sensitization analysis demonstrated that all five key compounds, including hexadecenoic acid methyl ester, methyl stearate, and squalene, fell within the applicability domain of the predictive models. High predictive confidence was observed in both the KeratinoSens and LLNA models, whereas the h-CLAT model exhibited lower predictive reliability . Cosmetic toxicity predictions revealed moderate to high risks for skin and eye irritation, particularly with Squalene, but the compounds were considered safe regarding systemic toxicity and photo-related effects. Furthermore, endocrine-disrupting potential was evaluated via in silico binding affinity analysis, which revealed minimal interaction with nuclear receptors. These findings highlight the potential of the plant extract for dermatological applications, while emphasizing the need for further evaluation of its safety in topical use.

Refining of spectrophotometric direct peptide reactivity assay (Spectro-DPRA) for in vitro skin sensitization: A novel predictive model based on decision tree.

An extended Reference Chemical Potency List (RCPL) for characterising the performance of New Approach Methodologies (NAMs) in measuring the skin sensitisation potency of fragrance chemicals.

New artificial neural network models for risk assessment of skin sensitization using amino acid derivative assay, KeratinoSens™, human cell line activation test and in silico structural alert parameter.

P32-13 Outside the validated range: Performing a Weight of Evidence (WoE) assessment of the skin sensitization potential for fragrance chemicals positive at high concentrations only in the Local Lymph Node Assay (LLNA)

Allergic contact dermatitis is one of the most common adverse events associated with cosmetic use. Accordingly, assessment of skin sensitisation hazard is required for safety evaluation of cosmetic ingredients. The transition to the use of alternative methods for testing has made skin sensitisation an intense field in the past decades. The first alternative methods have been in place for almost a decade, but none as stand-alone replacement for the reference murine Local Lymph Node Assay (LLNA). While strategies to combine data from several methods are being evaluated and refined, individual methods face technical limitations. These include issues related to their applicability to highly lipophilic substances and the lack of reliable potency estimation, which remain important obstacles to their widespread adoption as replacement for animal methods. The unique characteristics of in vitro skin models represented an attractive alternative, potentially overcoming these limitations and offering a more physiologically relevant environment for the assessment of the response in keratinocytes and dendritic cells. In this review, we recapitulate how reconstructed human skin models have been used as platforms for skin sensitisation testing, including the latest approaches using organ-on-a-chip and microfluidic technologies, aimed to develop next-generation organotypic skin models with increased complexity and monitoring capabilities.

Assays to predict the skin sensitisation potential of substances commonly address the molecular initiating event (MIE) of the adverse outcome pathway (AOP), which is the covalent binding of chemicals to proteins. This represents the first key event in the pathway. In chemico approaches have emerged as validated alternatives to conventional animal testing methods to determine this process. This review explores the integration of biosensor-based approaches to supplement other in chemico methods, for use as alternatives to the 'gold standard' Local Lymph Node Assay (LLNA) in skin sensitisation testing. While surface plasmon resonance-based biosensors have shown promise, they exhibit limitations such as poor sensitivity. To overcome such limitations, this review highlights the use of impedance-based biosensors in the study of hapten-skin protein interactions, which are associated with the MIE in skin sensitisation. Impedance-based biosensors have exhibited impressive performance, with a specificity of 97%, a sensitivity of 83% and an accuracy of 92% being obtained in one study (N = 49 substances). The advantages of impedance-based biosensors include label-free methodologies, high sensitivity, low cost and operational simplicity. This review also covers recent advancements in the use of biosensors in cosmetic studies and skin research, as well as comparisons of the limitations of the various in chemico methods and future perspectives for skin sensitisation assessment. The use of impedance-based biosensors as part of integrated testing approaches alongside other in chemico testing methods, can represent a reliable approach for skin sensitisation assessment, while supporting the reduction and replacement of animal use in toxicity testing.

Allergic contact dermatitis (ACD) is an immunologic reaction to a dermal chemical exposure that, once triggered in an individual, will result in an allergic response following subsequent encounters with the allergen. Air Force epidemiological consultations have indicated that aircraft structural maintenance workers may experience ACD at elevated rates compared to other occupations. We aimed to better understand the utility of non-animal testing methods in characterizing the sensitization potential of chemicals used during Air Force operations by evaluating the skin sensitization hazard of Air Force-relevant chemicals using new approach methodologies (NAMs) in a case study. We also evaluated the use of NAM data to develop preliminary candidate surface guidelines (PCSGs, maximum concentrations of chemicals on workplace surfaces to prevent induction of dermal sensitization) for chemicals identified as sensitizers. NAMs for assessing skin sensitization, including in silico models and experimental assays, were leveraged into an integrated approach to predict sensitization hazard for 19 chemicals. Local lymph node assay effective concentration values were predicted from NAM assay data via previously published quantitative models. The derived values were used to calculate PCSGs, which can be used to compare the presence of these chemicals on work surfaces to better understand the risk of Airmen developing ACD from occupational exposures.

Twenty-three single-ingredient substances, selected from materials labeled as allergens in cosmetic fragrances, were assessed for skin sensitization potential using a defined approach (DA) based on the OECD Guideline 497 Integrated Testing Strategy version 2 (ITSv2). According to the ITSv2, skin hypersensitivity data for these selected substances were integrated. In chemico data (from the Direct Peptide Reactivity Assay, DPRA) and in vitro data (from the human Cell Line Activation Test, h-CLAT) were obtained from existing databases, while in silico data were generated via the automated workflow of the OECD QSAR Toolbox. The DA for skin sensitization, which combines QSAR predictions with DPRA and h-CLAT test results, categorized 17 substances, including 2-benzylideneheptanal, as "Sensitizers"; 5 substances, including 4-methoxybenzyl alcohol, as "Inconclusive"; and citronellol alone was categorized as "Not Classified." The outcomes from the Local Lymph Node Assay (LLNA) for substances identified as skin sensitizers by the DA method demonstrated similar classification patterns to those from DA. These results underscore the necessity of integrating comprehensive in vitro, in chemico, and in silico data for accurate evaluation of potential skin sensitizers. This case study could aid in enhancing the safety assessments of potential skin sensitizers using DA; however, final categorization is subject to validation by regulatory authorities.

The global population is aging rapidly, posing new challenges for the safety evaluation of chemicals. Most toxicity studies use suitably aged animals that resemble healthy adults if they have similar responses. However, this assumption may not be valid because aging affects various physiological functions, such as immunity. The objective of this study was to compare the skin sensitization potential of chemicals in healthy adult mice, aged mice, and an aging mouse model using local lymph node assays (LLNA). Initially, eight-week-old female CBA/Ca, C3H/He (historical control for SAMP1 mice), senescence-accelerated mouse prone 1 (SAMP1), and C57BL6/N mice were compared to verify any differences among these strains. Next, 20-wk-old C3H/He and SAMP1 mice were compared with 80-wk-old C57BL6/N mice. Several concentrations (2.76, 8.3, and 25%) of α-hexyl cinnamaldehyde (HCA) were used as a positive substance for LLNA to determine the skin sensitization potential in each strain. The proliferation of T and B cells and related cytokine production were also measured. A dose-dependent correlation was observed, and a threshold of 1.8 for positive criteria for skin sensitization in LLNA, was surpassed in the CBA/Ca, C3H/He, and C57BL6/N strains, but not in the SAMP1 strain. In 20-wk-old mice, a positive response was observed only in C3H/He mice, whereas no positive response was observed in aged C57BL6 mice and SAMP1 mice. Our findings imply that senescence affects the skin sensitization potential of chemicals as measured using LLNA.

Using new approach methodologies for the identification of a sensitizing co-formulant in a plant protection product.

Weak Sensitizers May Be Associated with CD80 Polymorphisms: Implications for Systemic Contact Dermatitis

농약의 피부감작성 평가를 위한 Reduced Local Lymph Node Assay(rLLNA) 적용 연구

Positive patch test responses to benzoyl peroxide (BPO) have been reported from patients without and with known exposure. Up to 6.5% were found in the United States and 7.8% in a study including patients from Germany, Austria and Switzerland. We provide an overview of the skin sensitisation potential and potency of BPO based on animal experiments and non-animal methods. BPO tested positive in Guinea Pig Tests and the Local Lymph Node Assay. Application of the current OECD guideline to identify a skin sensitizer by combining non-animal method results gave differing outcomes. Moreover, patch test responses of patients to BPO were considered to determine the importance of BPO as a relevant occupational contact allergen. Another well-known BPO exposure is the topical application to treat acne. Despite widespread use, extensive and long-term skin exposure, we found for this group only studies reporting few positive patch test reactions. Further, occupational handling of BPO and contact allergy is reported by dental technicians. In-depth evaluation of the prevalence of contact allergy in different professions with suspected BPO exposure did not reveal an association with occupational handling of BPO. Consequently, a generally increased risk for those professions is not supported.

Potency and quantitative risk assessment are essential for determining safe concentrations for the for­mulation of potential skin sensitizers into consumer products. Several new approach methodologies (NAMs) for skin sensitization hazard assessment have been adopted in OECD test guidelines. Work is ongoing to develop NAMs for predicting skin sensitization potency on a quantitative scale for use as a point of departure in next generation risk assessment (NGRA). GARDskin Dose-Response (DR) is an adaptation of the GARDskin assay (OECD TG 442E); its readout is a quantitative potency pre­diction similar to the No Expected Sensitization Induction Level (NESIL) value (μg/cm2). Our goal was to evaluate the performance of the GARDskin DR for potency prediction of fragrance ingre­dients. One hundred fragrance ingredients from a reference database were tested in GARDskin DR. Materials tested had various protein-binding reactivity alerts, including Schiff base, Michael addition, SN2, and acylation. Potency categories were predicted with a total accuracy of 37% and an approximate accuracy (exact match or off by one category) of 81%. Combining predicted weak and very weak categories increased total accuracy to 53% and approximate accuracy to 98%. The mean prediction error for the NESIL and local lymph node assay (LLNA) EC3 was 3.15-fold and 3.36-fold, respectively. Based on the results of this study, GARDskin DR is a promising pre­dictor of skin sensitization potency with an applicability domain covering a wide range of fragrance ingredient reaction mechanisms, increasing the confidence in using the assay to conduct NGRA, ultimately reducing the need for animal testing. Plain language summary This study focused on testing a new in vitro method, GARDskin Dose-Response (DR), to predict the quantitative potency of fragrance ingredients in causing skin sensitization. This potency is important for setting safe levels of chemicals in consumer products. The GARDskin DR assay is based on an existing skin sensitization test (OECD TG 442E) and provides a quantitative measure of potency similar to the No Expected Sensitization Induction Level (NESIL). One hundred fragrance ingre­dients with different chemical structures and reactivity patterns were tested. The assay accurately distinguished between sensitizers and non-sensitizers for 81% of the materials, and also correctly predicted their approximate potency categories. The results show that GARDskin DR is a promising tool for predicting quantitative potency for skin sensitization risk, helping to reduce animal testing and support safer product development.