An in vitro human skin test for assessing sensitization potential.

Quantitative relationship between the local lymph node assay and human skin sensitization assays

An assessment of the skin sensitisation hazard of a group of polyfunctional silicones using a weight of evidence approach

The correlation of haptenation of gold nanoparticles and cysteine modified screen printed carbon electrode by impedance technique with local lymph node assay data

The assessment of risk is the deciding point in the toxicology of skin sensitization. Methods for the identification of skin sensitization hazards have been available for decades. Indeed, the majority of the clinically important chemical contact allergens are known. What is required to ensure that the incidence of allergic contact dermatitis is kept to a minimum is that these known hazards are properly assessed in the context of possible skin contact. A primary step in the risk assessment process is determination of the relative skin sensitizing potency of the substance in question in relation to that of other known, clinically relevant, contact allergens. Traditional test methodologies are not well suited to this task, not least because they lack any element of dose-response assessment. A newer approach, the local lymph node assay, offers an opportunity to obtain an objective and quantitative assessment of skin sensitizing potency. Armed with this information, the risk assessor must then evaluate the nature and extent of skin exposure to the substance, remembering the spectrum of habits and practices that may occur, as well as reasonably forseeable misuse. The approach then taken to skin sensitization risk assessment is usually that of comparative toxicology, in which the new chemical is compared and contrasted with known safe and unsafe materials. Reassurance of safety may be enhanced by carefully controlled human volunteer studies, including those which are negative, but where there has been a substantial exaggeration of exposure compared to what is actually expected.

In recent years, the local lymph node assay (LLNA) has emerged as a practical option for assessing the skin sensitization potential of chemicals. In addition to accurate identification of skin sensitizers, the LLNA can also provide a reliable measure of relative sensitization potency; information that is pivotal in successful management of human health risks. However, even with the significant animal welfare benefits provided by the LLNA, there is interest still in the development of non‐animal test methods for skin sensitization. Here, we have collected a large dataset of chemicals that have been tested in the LLNA, and the activity of which correspond with what is known of their potential to cause skin sensitization in humans. It is anticipated that this will be of value to other investigators in the evaluation and calibration of novel approaches to skin sensitization testing, in particular for the development of in silico methods. Prerequisite for the development of in silico models is always the availability of a large high quality data set, suitable for modeling. This dataset encompasses both the chemical and biological diversity of known chemical allergens, and provides also examples of negative controls. The data are a collection of published and non‐proprietary industry data. All materials were tested in standard vehicules following the standard LLNA protocol. It is hoped that this dataset will accelerate the development, evaluation and eventual validation of new approaches to skin sensitization testing.

There is compelling evidence that contact allergens differ substantially (by 4 or 5 orders of magnitude) with respect to their inherent skin-sensitizing potency. Relative potency can now be measured effectively using the mouse local lymph node assay (LLNA) and such data form the basis of risk assessment and risk management strategies. Such determinations also facilitate distinctions being drawn between the prevalence of skin sensitization to a particular contact allergen and inherent potency. The distinction is important because chemicals that are implicated as common causes of contact allergy are not necessarily potent sensitizers. One example is provided by nickel that is undoubtedly a common cause of allergic contact dermatitis, but is a comparatively weak sensitizer in predictive tests. In an attempt to explore other examples of contact allergens where there may exist a discrepancy between prevalence and potency, we describe here analyses conducted with methyl methacrylate (MMA). Results of LLNA studies have been interpreted in the context of historical clinical data on occupational allergic contact dermatitis associated with exposure to MMA.

Assessment of statistic analysis in non-radioisotopic local lymph node assay (non-RI-LLNA) with alpha-hexylcinnamic aldehyde as an example.

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.

Allergic contact dermatitis resulting from skin sensitization is a common occupational and environmental health problem. In recent years, the local lymph node assay (LLNA) has emerged as a practical option for assessing the skin sensitization potential of chemicals. In addition to accurate identification of skin sensitizers, the LLNA can also provide a reliable measure of relative sensitization potency; information that is pivotal in successful management of human health risks. However, even with the significant animal welfare benefits provided by the LLNA, there is still interest in the development of nonanimal test methods for skin sensitization testing. One characteristic of a chemical allergen is its ability to react with proteins prior to the induction of skin sensitization. The majority of chemical allergens is electrophilic and as such reacts with nucleophilic amino acids like cysteine or lysine. In order to determine if reactivity correlates with sensitization potential, 38 chemicals representing allergens of different potencies (weak to extreme) and nonsensitizers were evaluated for their ability to react with glutathione or three synthetic peptides containing either cysteine, lysine, or histidine. Following a 15-min reaction time for glutathione or a 24 h reaction period for the three synthetic peptides, the samples were analyzed by HPLC. UV detection was used to monitor the depletion of glutathione or the peptide following reaction. The results demonstrate that a significant correlation (Spearman correlation) exists between allergen potency and the depletion of glutathione (p = 0.001), lysine (p = 0.025), and cysteine (p = 0.020), but not histidine. The peptide with the highest sensitivity was cysteine (80.8%) whereas histidine was the least sensitive (11.5%). The data presented show that measuring peptide reactivity has utility for screening chemicals for their skin sensitization potency and thus potential for reducing our reliance on animal test methods.

Evaluation of skin sensitization potential of jet fuels by murine local lymph node assay

Twenty organic Salmonella mutagens, seven of which (including benzo[a]pyrene) are established skin carcinogens, and one of which (2-chloroethanol) is a well-defined noncarcinogen to skin, have been evaluated for skin-sensitizing activity using the local lymph node assay. The relative mutagenicity of the agents to Salmonella was also established. Fourteen of the chemicals were positive in the local lymph node assay, including the seven skin carcinogens. 2-Chloroethanol was inactive as a sensitizing agent. We suggest that a variety of factors contributes to the lack of sensitizing activity of the remaining six bacterial mutagens: extremes of intrinsic chemical reactivity, high water solubility reducing dermal translocation, and inappropriate dermal metabolism. Two reference skin-sensitizing agents (an oxazolinone and fluorescein isothiocyanate) were established as in vitro clastogens after their recognition as nonmutagens to Salmonella. These data imply that mutagenicity, rather than simply activity in the Salmonella assay, is a primary stimulus for electrophilic sensitization and carcinogenic initiation in the skin. We conclude that genotoxicity data for an agent can provide indications of the agent's potential to induce skin sensitization and that genotoxins which are skin-sensitizing agents have an enhanced potential to initiate skin carcinogenesis. We suggest that common, albeit individually distinct, structure-activity relationships underpin genotoxicity, skin sensitization, and the initiation of skin carcinogenesis. These relationships should simplify the hazard evaluation of chemicals and contribute to a reduction in animal usage. Several predictions of skin carcinogenicity are made based on the data presented.

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.

Strategies for identifying false positive responses in predictive skin sensitization tests

Allergic contact dermatitis (ACD) is to a considerable extent a preventable disease. Limitation can be achieved by correct identification of skin sensitizers, characterization of their potency, understanding human skin exposure and application of good risk assessment/management strategies. Various methods exist which are accurate for the predictive identification of chemicals that possess skin-sensitizing properties. These are enshrined in regulations that aim to provide a harmonized approach to hazard identification. One of the methods, the local lymph node assay, also delivers information on the relative potency of sensitizers. Efforts are continuing in the European Union and at the Organization for Economic Cooperation and Development to use elements of this information for regulatory categorization of skin sensitizers. However, greater use can be made of this potency information in the application of quantitative risk assessments. Such assessments depend also on the availability of accurate data on human skin exposure, one aspect where legislation has little role to play. Management of risks by restriction of skin exposure is, in contrast, a key point where legislation can play an important role, helping to establish a level playing field for industry and setting good standards based on the legislator's ability to access all data. Ultimately, the combination of accurate hazard identification, potency measurement, risk assessment and management, underpinned by enabling legislation, will lead to reduction of ACD. For individuals who do still develop contact allergy, avoidance of ACD should continue to be a goal, based on raising awareness of skin protection, allergen labelling and other skincare strategies.

The identification of potential skin sensitizing chemicals is a key step in the overall skin safety risk assessment process. Traditionally, predictive testing has been conducted in guinea pigs. More recently, the murine local lymph node assay (LLNA) has become the preferred test method for assessing skin sensitization potential. However, even with the significant animal welfare benefits provided by the LLNA, there is a need to develop non-animal test methods for skin sensitization. Mechanistic understanding of allergic contact dermatitis has increased substantially in recent years. For example, a number of changes are known to occur in epidermal Langerhans cells, the principal antigen-presenting dendritic cell in the skin, as a result of exposure to chemical allergens, including the internalization of surface major histocompatibility complex (MHC) class II molecules via endocytosis, the induction of tyrosine phosphorylation, the modulation of cell surface markers, and cytokine expression. The application of this knowledge to the design of predictive in vitro alternative tests provides both unique opportunities and challenges. In this review, we have focused specifically on the impact of chemical exposure on dendritic cells and the potential use of that information in the development of cell-based assays for assessing skin sensitization potential of chemicals in vitro.