Skin Sensitization Potential Of Chemicals Research Articles

Filling the concept with data: Integrating data from different in vitro and in silico assays on skin sensitizers to explore the battery approach for animal-free skin sensitization testing

Tests for skin sensitization are required prior to the market launch of new cosmetic ingredients. Significant efforts are made to replace the current animal tests. It is widely recognized that this cannot be accomplished with a single in vitro test, but that rather the integration of results from different in vitro and in silico assays will be needed for the prediction of the skin sensitization potential of chemicals. This has been proposed as a theoretical scheme so far, but no attempts have been made to use experimental data to prove the validity of this concept. Here we thus try for the first time to fill this widely cited concept with data. To this aim, we integrate and report both novel and literature data on 116 chemicals of known skin sensitization potential on the following parameters: (1) peptide reactivity as a surrogate for protein binding, (2) induction of antioxidant/electrophile responsive element dependent luciferase activity as a cell-based assay; (3) Tissue Metabolism Simulator skin sensitization model in silico prediction; and (4) calculated octanol-water partition coefficient. The results of the in vitro assays were scaled into five classes from 0 to 4 to give an in vitro score and compared to the local lymph node assay (LLNA) data, which were also scaled from 0 to 4 (nonsensitizer/weak/moderate/strong/extreme). Different ways of evaluating these data have been assessed to rate the hazard of chemicals (Cooper statistics) and to also scale their potency. With the optimized model an overall accuracy for predicting sensitizers of 87.9% was obtained. There is a linear correlation between the LLNA score and the in vitro score. However, the correlation needs further improvement as there is still a relatively high variation in the in vitro score between chemicals belonging to the same sensitization potency class.

Filling the Concept with Data: Integrating Data from Different In Vitro and In Silico Assays on Skin Sensitizers to Explore the Battery Approach for Animal-Free Skin Sensitization Testing

Tests for skin sensitization are required prior to the market launch of new cosmetic ingredients. Significant efforts are made to replace the current animal tests. It is widely recognized that this cannot be accomplished with a single in vitro test, but that rather the integration of results from different in vitro and in silico assays will be needed for the prediction of the skin sensitization potential of chemicals. This has been proposed as a theoretical scheme so far, but no attempts have been made to use experimental data to prove the validity of this concept. Here we thus try for the first time to fill this widely cited concept with data. To this aim, we integrate and report both novel and literature data on 116 chemicals of known skin sensitization potential on the following parameters: (1) peptide reactivity as a surrogate for protein binding, (2) induction of antioxidant/electrophile responsive element dependent luciferase activity as a cell-based assay; (3) Tissue Metabolism Simulator skin sensitization model in silico prediction; and (4) calculated octanol-water partition coefficient. The results of the in vitro assays were scaled into five classes from 0 to 4 to give an in vitro score and compared to the local lymph node assay (LLNA) data, which were also scaled from 0 to 4 (nonsensitizer/weak/moderate/strong/extreme). Different ways of evaluating these data have been assessed to rate the hazard of chemicals (Cooper statistics) and to also scale their potency. With the optimized model an overall accuracy for predicting sensitizers of 87.9% was obtained. There is a linear correlation between the LLNA score and the in vitro score. However, the correlation needs further improvement as there is still a relatively high variation in the in vitro score between chemicals belonging to the same sensitization potency class.

A Comparison of Reactivity Schemes for the Prediction Skin Sensitization Potential

Skin sensitization is an important toxic end point for both regulatory frameworks and safety assessment. There are many hurdles for a chemical to overcome in terms of inducing skin sensitization, although the binding of chemicals to skin protein is thought to be the rate-determining step. Current strategies to predict the skin sensitization potential of chemicals in silico is through the identification of electrophilic characteristics. A number of predictive schemes have been developed in recent years, some based on broad structural rules and some with a reaction chemistry mechanistic basis. This work compares two schemes that are based on reaction chemistry. The first scheme comprises a set of rules that characterize reaction mechanistic domains as proposed by Aptula and Roberts [(2006) Chem. Res. Toxicol. 19, 1097-1105]. The second is a set of structure-toxicity and structure-metabolism pathways that are encoded and embedded into the TIssue MEtabolism Simulator skin sensitization model (TIMES-SS) [(2005) Int. J. Toxicol. 24, 189-204]. Here, a comparison of these schemes has been made using a recently published data set of 210 chemicals that have been tested in the local lymph node assay. The similarities and differences of the schemes are highlighted, together with modifications that could be made to TIMES-SS to harmonize the two approaches.

Chemical reactivity indices and mechanism‐based read‐across for non‐animal based assessment of skin sensitisation potential

The skin sensitisation potential of chemicals is currently assessed using in vivo methods where the murine local lymph node assay (LLNA) is typically the method of first choice. Current regulatory initiatives are driving the impetus for the use of in vitro/in silico alternative approaches to provide the relevant information needed for the effective assessment of skin sensitisation, for both hazard characterisation and risk assessment purposes. A chemical must undergo a number of steps for it to induce skin sensitisation but the main determining step is formation of a stable covalent association with carrier protein. The ability of a chemical to react covalently with carrier protein nucleophiles relates to both its electrophilic reactivity and its hydrophobicity. This paper focuses on quantitative indices of electrophilic reactivity with nucleophiles, in a chemical mechanism-of-action context, and compares and contrasts the experimental approaches available to generate reactivity data that are suitable for mathematical modelling and making predictions of skin sensitisation potential, using new chemistry data correlated against existing in vivo bioassay data. As such, the paper goes on to describe an illustrative example of how quantitative kinetic measures of reactivity can be usefully and simply applied to perform mechanism-based read-across that enables hazard characterisation of skin sensitisation potential. An illustration of the types of quantitative mechanistic models that could be built using databases of kinetic measures of reactivity, hydrophobicity and existing in vivo bioassay data is also given.

Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals

The local lymph node assay (LLNA) is a murine model developed to evaluate the skin sensitization potential of chemicals. The LLNA is an alternative approach to traditional guinea pig methods and in comparison provides important animal welfare benefits. The assay relies on measurement of events induced during the induction phase of skin sensitization, specifically lymphocyte proliferation in the draining lymph nodes which is a hallmark of a skin sensitization response. Since its introduction the LLNA has been the subject of extensive evaluation on a national and international scale, and has been successfully validated and incorporated worldwide into regulatory guidelines. Experience gained in recent years has demonstrated that adherence to published procedures and guidelines for the LLNA (e.g., with respect to dose and vehicle selection) is critical for the successful conduct and eventual interpretation of the data. In addition to providing a robust method for skin sensitization hazard identification, the LLNA has proven very useful in assessing the skin sensitizing potency of test chemicals, and this has provided invaluable information to risk assessors. The primary method to make comparisons of the relative potency of chemical sensitizers is to use linear interpolation to estimate the concentration of chemical required to induce a stimulation index of three relative to concurrent vehicle-treated controls (EC3). In certain situations where there are available less than optimal dose response data a log-linear extrapolation method can be used to estimate an EC3 value which can reduce significantly the need for repeat testing of chemicals. The LLNA, when conducted according to published guidelines, provides a robust method for skin sensitization testing that not only provides reliable hazard identification information but also data necessary for effective risk assessment and risk management.

Evaluation of anex vivo murine local lymph node assay: multiple endpoint comparison

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity.

Compilation of Historical Local Lymph Node Data for Evaluation of Skin Sensitization Alternative Methods

Background: Within the toxicology community, considerable effort is directed toward the development of alternative methods for skin sensitization testing. The availability of high-quality, relevant, and reliable in vivo data regarding skin sensitization is essential for the effective evaluation of alternative methodologies. Ideally, data derived from humans would be the most appropriate source because the test methods are attempting to predict a toxicologic effect in humans. Unfortunately, insufficient human data of the necessary quality are available, so it is necessary to rely on the best available animal data. 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 accurately identifying skin sensitizers, the LLNA can also provide a reliable measure of relative sensitization potency, information that is pivotal to the successful management of human health risks. Objective: To provide a database of robust in vivo data to calibrate, evaluate, and eventually validate new approaches for skin sensitization testing. Methods: LLNA data derived from previously conducted studies were compiled from the published literature and unpublished sources. Results: We provide a database that comprises LLNA data on 211 individual chemicals. This extensive chemical data set encompasses both the chemical and biologic diversity of known chemical allergens. To cover the range of relative allergenic potencies, the data set includes data on 13 extreme, 21 strong, 69 moderate, and 66 weak contact allergens, classified according to each allergen's mathematically estimated concentration of chemical required to induce a threefold stimulation index. In addition, there are also 42 chemicals that are considered to be nonsensitizers. In terms of chemical diversity, the database contains data pertaining to the chemical classes represented by aldehydes, ketones, aromatic amines, quinones, and acrylates, as well as compounds that have different reactivity mechanisms. In addition to two-dimensional chemical structures, the physicochemical parameters included are log Kp, log KO/W, and molecular weight. Conclusions: The list of chemicals contained in the data set represents both the chemical and biologic diversity that is known to exist for chemical allergens and non-allergens. It is anticipated that this database will help accelerate the development, evaluation, and eventual validation of new approaches to skin sensitization assessment.

Interactions of Contact Allergens with Dendritic Cells: Opportunities and Challenges for the Development of Novel Approaches to Hazard Assessment

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.

Compilation of Historical Local Lymph Node Data for Evaluation of Skin Sensitization Alternative Methods

Within the toxicology community, considerable effort is directed toward the development of alternative methods for skin sensitization testing. The availability of high-quality, relevant, and reliable in vivo data regarding skin sensitization is essential for the effective evaluation of alternative methodologies. Ideally, data derived from humans would be the most appropriate source because the test methods are attempting to predict a toxicologic effect in humans. Unfortunately, insufficient human data of the necessary quality are available, so it is necessary to rely on the best available animal data. 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 accurately identifying skin sensitizers, the LLNA can also provide a reliable measure of relative sensitization potency, information that is pivotal to the successful management of human health risks. To provide a database of robust in vivo data to calibrate, evaluate, and eventually validate new approaches for skin sensitization testing. LLNA data derived from previously conducted studies were compiled from the published literature and unpublished sources. We provide a database that comprises LLNA data on 211 individual chemicals. This extensive chemical data set encompasses both the chemical and biologic diversity of known chemical allergens. To cover the range of relative allergenic potencies, the data set includes data on 13 extreme, 21 strong, 69 moderate, and 66 weak contact allergens, classified according to each allergen's mathematically estimated concentration of chemical required to induce a threefold stimulation index. In addition, there are also 42 chemicals that are considered to be nonsensitizers. In terms of chemical diversity, the database contains data pertaining to the chemical classes represented by aldehydes, ketones, aromatic amines, quinones, and acrylates, as well as compounds that have different reactivity mechanisms. In addition to two-dimensional chemical structures, the physicochemical parameters included are log Kp, log K(o/w), and molecular weight. The list of chemicals contained in the data set represents both the chemical and biologic diversity that is known to exist for chemical allergens and non-allergens. It is anticipated that this database will help accelerate the development, evaluation, and eventual validation of new approaches to skin sensitization assessment.

Development of a peptide reactivity assay for screening contact allergens.

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.

P79 A chemical dataset for evaluation of alternative approaches to skin sensitization testing

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.

Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line

It has been known that dendritic cells (DCs) including Langerhans cells (LCs) play a critical role in the skin sensitization process. Many attempts have been made to develop in vitro sensitization tests that employ DCs derived from peripheral blood mononuclear cells (PBMC-DC) or CD34+ hematopoietic progenitor cells (CD34+ HPC) purified from cord blood or bone marrow. However, the use of the DCs in in vitro methods has been difficult due to the nature of these cells such as low levels in the source and/or donor-to-donor variability. In our studies, we employed the human monocytic leukemia cell line, THP-1, in order to avoid some of these difficulties. At the start, we examined whether treatment of the cells with various cytokines could produce DCs from THP-1. Treatment of THP-1 cells with cytokines such as GM-CSF, IL-4, TNF-α, and/or PMA did induce some phenotypic changes in THP-1 cells that were characteristic of DCs. Subsequently, responses to a known sensitizer, dinitrochlorobenzene (DNCB), and a non-sensitizer, dimethyl sulfoxide (DMSO) or sodium lauryl sulfate (SLS), on the expression of co-stimulatory molecules, CD54 and CD86, were examined between the naive cells and the cytokine-treated cells. Interestingly, the naive THP-1 cells responded only to DNCB and the response to the sensitizer was more distinct than cytokine-treated THP-1 cells. Similar phenomena were also observed in the human myeloid leukemia cell line, KG-1. Furthermore, with treatment of DNCB, naive THP-1 cells showed augmented expression of HLA, CD80 and secretion of IL-1β. The response of THP-1 cells to a sensitizer was similar to that of LCs/DCs. Upon demonstrating the differentiation of monocyte cells in our system, we then evaluated a series of chemicals, including known sensitizers and non-sensitizers, for their potential to augment CD54 and CD86 expression on naive THP-1 cells. Indeed, known sensitizers such as PPD and 2-MBT significantly augmented CD54 and CD86 expression in a dose-dependent manner while non-sensitizers, such as SLS and methyl salicylate (MS), did not. To note, the metal allergens such as (NH 4) 2[PtCl 4], NiSO 4 and CoSO 4 augmented significantly only CD54 expression. Taking advantage of a cultured cell line, measurement of the co-stimulatory molecules, CD54 and CD86, on naive THP-1 cells following chemical exposure shows promise for the development of a simple, short-term in vitro sensitization test.

In vitro and in vivo testing techniques for allergic contact dermatitis

A variety of preclinical and clinical models have been developed for assessing the skin sensitization potential of chemicals. These models have been invaluable in identifying potential allergens and providing the information needed to conduct sound skin sensitization risk assessments. Of course, all models have inherent strengths and limitations. In the area of skin sensitization, significant advances have been made in understanding the underlying mechanisms of allergic contact dermatitis. This knowledge has been critical in the development of new in vitro and in vivo approaches for assessing the skin sensitization potential of chemicals. This article presents basic immunologic information that is being used to aid in the identification of cellular markers for differentiating irritant and allergic reactions in animals and humans. In addition, data are reviewed on the evaluation of skin equivalent cultures for the identification of skin allergens in vitro.

In Vitro and In Vivo Testing Techniques for Allergic Contact Dermatitis

A variety of preclinical and clinical models have been developed for assessing the skin sensitization potential of chemicals. These models have been invaluable in identifying potential allergens and providing the information needed to conduct sound skin sensitization risk assessments. Of course, all models have inherent strengths and limitations. In the area of skin sensitization, significant advances have been made in understanding the underlying mechanisms of allergic contact dermatitis. This knowledge has been critical in the development of newin vitroandin vivoapproaches for assessing the skin sensitization potential of chemicals. This article presents basic immunologic information that is being used to aid in the identification of cellular markers for differentiating irritant and allergic reactions in animals and humans. In addition, data are reviewed on the evaluation of skin equivalent cultures for the identification of skin allergensin vitro.

The performance of the local lymph node assay with chemicals identified as contact allergens in the human maximization test

For many years, tests in the guinea pig have been the favoured option for the identification of the skin sensitization potential of chemicals. However, the mouse has been used widely in immunology research and can represent a viable alternative. A variety of murine assays have been described, including several methods based on ear swelling as an endpoint. Another option is to assess induced lymph node cell proliferation and it is this which forms the basis of the murine local lymph node assay (LLNA). The LLNA has undergone several successful interlaboratory validations and compares well with standard guinea pig assays. In the present study, the performance of the LLNA was examined with chemicals tested previously in the human maximization test (HMT). 30 chemicals, 23 of which proved positive in the HMT and seven of which were negative, have been tested. All but four of the materials found positive in the HMT also tested positive in the LLNA. Of these four, sulfanilamide and paraben esters would not classify as skin sensitizers in the guinea pig maximization test and nickel has been found to yield variable results in a number of predictive animal tests. Of the seven substances which proved negative in the HMT, six were also negative in the LLNA, the exception being sodium dodecyl sulfate. These data demonstrate that the LLNA is able to identify accurately chemicals which have the potential to cause significant allergic contact dermatitis in humans.

Sulphanilic acid: divergent results in the guinea pig maximization test and the local lymph node assay.

The guinea pig maximization test (GPMT) has proven to be a valuable tool for the identification of the skin sensitization potential of chemicals. The method identifies a hazard which can lead in the EC to compulsory labelling of that chemical. In the present study, data on sulphanilic acid derived from the GPMT has been compared with results from a second guinea pig assay (the cumulative contact enhancement test) and the murine local lymph node assay, both of which require only topical application of chemical. Except for the GPMT, no test identified any sensitizing activity associated with exposure to sulphanilic acid. These latter results are consistent with the experience gained from substantial human exposure in an occupational setting and from which no cases of allergic contact dermatitis to sulphanilic acid have arisen over a 20-year period. In consequence, it is questioned which test protocol in practice has given the more accurate identification of sensitization hazard relevant to man.