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

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

A botanical reference set illustrating a weight of evidence approach for skin sensitization risk assessment.

Recent years have seen an increase in the use of botanicals and natural substances (BNS) in consumer products such as cosmetics and household care products. Most work conducted to date to assess botanicals for human safety has focused their use as dietary supplements and thus on systemic toxicity. However, the induction of skin sensitization is a possible adverse effect of natural products in particular those that come into skin contact, especially for cosmetics that remain on the skin and are not rinsed off following use. Assessments of BNS ingredients are often challenging for a number of reasons: the BNS are complex mixtures that can be of mostly unknown composition; the composition can be highly variable even within the same plant species and dependent on how processed; the physical form of the BNS raw material can vary from a highly concentrated powdered extract to a liquid extract containing only a small percentage of the BNS; testing of the BNS raw materials in New Approach Methods (NAM) has uncertainty as these methods are often not developed or validated for complex mixtures. In this study, a reference set of 14 selected BNS which span the range of skin sensitization potential was complied. These data were used in a Weight of Evidence (WoE) approach to evaluate their skin sensitization potential with each of the data rich BNS being classified as either having strong evidence of inducing skin sensitization based on human topical use history, animal data, clinical data, composition data and NAM data, or having some but more limited (weak) evidence of inducing skin sensitization, or having strong evidence of no skin sensitization potential. When available data have sufficient potency related information, sensitization potency assessment is also provided based on WoE, classifying these BNS as either strong, moderate, or weak sensitizers, or non-sensitizers. An outline for a BNS skin sensitization risk assessment framework is proposed starting with exposure-based waiving and WoE assessment for higher exposures. In addition to demonstrating the application of the WoE approach, the reference set presented here provides a set of 'data rich' botanicals which cover a range of sensitization potencies that could be used for evaluating existing test methods or aid in the development of new predictive models for skin sensitization.

Development of a peptide reactivity assay for screening botanicals and natural substances: Proof of concept studies.

Consumer products containing botanicals or natural substances (BNS) are often preferred because there is a perception that 'natural' is safe. As with any product ingredient, a thorough safety assessment must be conducted, including a determination of skin sensitization potential. A modification of the Peroxidase Peptide Reactivity Assay (PPRA) was explored for screening BNS (B-PPRA) for their reactivity to a model cysteine peptide. The PPRA incorporates a horseradish peroxidase­hydrogen peroxide (+HRP/P) oxidation system for the activation of potential pre- and pro-haptens. BNS test materials contained <2% botanical constituent in either glycerin/water or propylene glycol/water. Stock solutions prepared in acetonitrile were diluted to 8 working concentrations. Direct reactivity was determined in reaction mixtures containing peptide and deferoxamine in potassium phosphate buffer. Enzyme-mediated reactivity determinations were performed with addition of +HRP/P. Initial studies demonstrated that results were reproducible and impact of carrier low. To determine the sensitivity of the assay, experiments were conducted with chamomile extract spiked with three sensitizers. Peptide depletion was observed in the +HRP/P reaction mixtures with isoeugenol spikes as low as 0.05%. The B-PPRA shows promise as a screening method for skin sensitization potential and could become part of a framework for the skin sensitization safety assessment of BNS.

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

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

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

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

Updating exposure assessment for skin sensitization quantitative risk assessment for fragrance materials.

In 2008, a proposal for assessing the risk of induction of skin sensitization to fragrance materials Quantitative Risk Assessment 1 (QRA1) was published. This was implemented for setting maximum limits for fragrance materials in consumer products. However, there was no formal validation or empirical verification after implementation. Additionally, concerns remained that QRA1 did not incorporate aggregate exposure from multiple product use and included assumptions, e.g. safety assessment factors (SAFs), that had not been critically reviewed. Accordingly, a review was undertaken, including detailed re-evaluation of each SAF together with development of an approach for estimating aggregate exposure of the skin to a potential fragrance allergen. This revision of QRA1, termed QRA2, provides an improved method for establishing safe levels for sensitizing fragrance materials in multiple products to limit the risk of induction of contact allergy. The use of alternative non-animal methods is not within the scope of this paper. Ultimately, only longitudinal clinical studies can verify the utility of QRA2 as a tool for the prevention of contact allergy to fragrance materials.

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

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

The kinetic direct peptide reactivity assay (kDPRA): Intra- and inter-laboratory reproducibility in a seven-laboratory ring trial

While the skin sensitization hazard of substances can be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide are evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times. The reaction is stopped by addition of monobromobimane, which forms a fluorescent complex with the free cysteine of the model peptide. The relative remaining non-depleted amount of peptide is determined. Kinetic rate constants are derived from the depletion vs concentration and time matrix and used to distinguish between UN GHS sub-category 1A sensitizers and test substances in sub-category 1B/not classified test substances. In this study, we present a ring trial of the kDPRA with 24 blind-coded test substances in seven laboratories. The intra- and inter-laboratory reproducibility were 96% and 88%, respectively (both for differentiating GHS Cat 1A sensitizers from GHS Cat 1B/not classified). Following an independent peer review, the kDPRA was considered to be acceptable for the identification of GHS Cat 1A skin sensitizers. Besides GHS Cat 1A identification, the kDPRA can be used as part of a defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment. For this aim, next to reproducibility of classification, the quantitative reproducibility and variability of the rate constants were quantified in this study.

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

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

Allergic contact dermatitis: Adequacy of the default 10X assessment factor for human variability to protect infants and children.

When evaluating consumer products for safety, one must consider the heterogeneity of the population that might use those products, including the potential for different sensitivity based on factors such as age, gender, and genetics. For both systemic endpoints and allergic contact dermatitis (ACD), quantitative safety evaluations typically include a default 10-fold uncertainty/assessment factor to account for inter-individual variability. While this factor is intended to include age, the adequacy of the default 10-fold factor has been questioned for infants, for whom a precautionary assumption is often made that they are more sensitive. In-depth evaluations of the adequacy of the 10-fold factor have been published for systemic endpoints, but relatively little has been published to substantiate this for ACD. This paper reviews the state of the science regarding the etiology of ACD and factors that suggest an overall decreased sensitivity associated with early life exposures, thus confirming the sufficiency of the 10-fold inter-individual factor to provide protection for children and infants. While it remains prudent for all age groups to avoid contact with sensitizers, it is concluded that the quantitative methods used in safety evaluation to prevent the induction of skin sensitization are protective for infants, including neonates and premature infants.

Application of in vitro skin penetration measurements to confirm and refine the quantitative skin sensitization risk assessment of methylisothiazolinone.

Use of quantitative risk assessment (QRA) for assessing the skin sensitization potential of chemicals present in consumer products requires an understanding of hazard and product exposure. In the absence of data, consumer exposure is based on relevant habits and practices and assumes 100% skin uptake of the applied dose. To confirm and refine the exposure, a novel design for in vitro skin exposure measurements was conducted with the preservative, methylisothiazolinone (MI), in beauty care (BC) and household care (HHC) products using realistic consumer exposure conditions. A difference between measured exposure levels (MELs) for MI in leave-on versus rinse-off BC products, and lower MELs for MI in HHC rinse-off compared to BC products was demonstrated. For repeated product applications, the measured exposure was lower than estimations based on summation of applied amounts. Compared to rinse-off products, leave-on applications resulted in higher MELs, correlating with the higher incidences of allergic contact dermatitis associated with those product types. Lower MELs for MI in rinse-off products indicate a lower likelihood to induce skin sensitization, also after multiple daily applications. These in vitro skin exposure measurements indicate conservatism of default exposure estimates applied in skin sensitization QRA and might be helpful in future risk assessments.

Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.

Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.

Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy.

The presented Bayesian network Integrated Testing Strategy (ITS-3) for skin sensitization potency assessment is a decision support system for a risk assessor that provides quantitative weight of evidence, leading to a mechanistically interpretable potency hypothesis, and formulates adaptive testing strategy for a chemical. The system was constructed with an aim to improve precision and accuracy for predicting LLNA potency beyond ITS-2 (Jaworska et al., J Appl Toxicol 33(11):1353-1364, 2013) by improving representation of chemistry and biology. Among novel elements are corrections for bioavailability both in vivo and in vitro as well as consideration of the individual assays' applicability domains in the prediction process. In ITS-3 structure, three validated alternative assays, DPRA, KeratinoSens and h-CLAT, represent first three key events of the adverse outcome pathway for skin sensitization. The skin sensitization potency prediction is provided as a probability distribution over four potency classes. The probability distribution is converted to Bayes factors to: 1) remove prediction bias introduced by the training set potency distribution and 2) express uncertainty in a quantitative manner, allowing transparent and consistent criteria to accept a prediction. The novel ITS-3 database includes 207 chemicals with a full set of in vivo and in vitro data. The accuracy for predicting LLNA outcomes on the external test set (n = 60) was as follows: hazard (two classes)-100 %, GHS potency classification (three classes)-96 %, potency (four classes)-89 %. This work demonstrates that skin sensitization potency prediction based on data from three key events, and often less, is possible, reliable over broad chemical classes and ready for practical applications.

Evaluation of an optimized protocol using human peripheral blood monocyte derived dendritic cells for the in vitro detection of sensitizers: Results of a ring study in five laboratories.

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive have introduced a testing ban for cosmetic ingredients after March 2013. We have developed and optimized a stable and reproducible in vitro protocol based on human peripheral blood monocyte derived dendritic cells to assess the sensitization potential of chemicals. To evaluate the transferability and the predictivity of this PBMDCs based test protocol, a ring study was organized with five laboratories using seven chemicals with a known sensitization potential (one none-sensitizer and six sensitizers, including one pro-hapten). The results indicated that this optimized test protocol could be successfully transferred to all participating laboratories and allowed a correct assessment of the sensitization potential of the tested set of chemicals. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in the toolbox of in vitro methods for the evaluation of the skin sensitization potential of chemicals.

A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation.

Skin sensitization is a key endpoint for cosmetic ingredients, with a forthcoming ban for animal testing in Europe. Four alternative tests have so far been submitted to ECVAM prevalidation: (i) MUSST and (ii) h-Clat assess surface markers on dendritic cell lines, (iii) the direct peptide reactivity assay (DPRA) measures reactivity with model peptides and (iv) the KeratinoSens(TM) assay which is based on detection of Nrf2-induced luciferase. It is anticipated that only an integrated testing strategy (ITS) based on a battery of tests might give a full replacement providing also a sensitization potency assessment, but this concept should be tested with a data-driven analysis. Here we report a database on 145 chemicals reporting the quantitative endpoints measured in a U937- test, the DPRA and KeratinoSens(TM) . It can serve to develop data-driven ITS approaches as we show in a parallel paper and provides a view as to the current ability to predict with in vitro tests as we are entering 2013. It may also serve as reference database when benchmarking new molecules with in vitro based read-across and find use as a reference database when evaluating new tests. The tests and combinations thereof were evaluated for predictivity, and overall a similar predictivity was found as before on three-fold smaller datasets. Analysis of the dose-response parameters of the individual tests indicates a correlation to sensitization potency. Detailed analysis of chemicals false-negative and false-positive in two tests helped to define limitations in the tests but also in the database derived from animal studies.

The local lymph node assay (LLNA).

The murine local lymph node assay (LLNA) is a widely accepted method for assessing the skin sensitization potential of chemicals. Compared with other in vivo methods in guinea pig, the LLNA offers important advantages with respect to animal welfare, including a requirement for reduced animal numbers as well as reduced pain and trauma. In addition to hazard identification, the LLNA is used for determining the relative skin sensitizing potency of contact allergens as a pivotal contribution to the risk assessment process. The LLNA is the only in vivo method that has been subjected to a formal validation process. The original LLNA protocol is based on measurement of the proliferative activity of draining lymph node cells (LNC), as determined by incorporation of radiolabeled thymidine. Several variants to the original LLNA have been developed to eliminate the use of radioactive materials. One such alternative is considered here: the LLNA:BrdU-ELISA method, which uses 5-bromo-2-deoxyuridine (BrdU) in place of radiolabeled thymidine to measure LNC proliferation in draining nodes.

Historical perspective on the use of visual grading scales in evaluating skin irritation and sensitization.

Visual assessment of skin reactions has long been used to evaluate the safety of chemicals and preparations that contact the skin, and to meet regulatory requirements. This article reviews the history of visual grading scales, and the results of investigations into the reliability of the method. Some examples are provided to illustrate the diverse array of protocols that use visual scoring to evaluate skin irritation. Furthermore, as bioengineering methods are developed that can quantitate certain aspects of skin irritant and sensitization reactions, it is important to consider whether such measures should supplement or replace visual assessment. Examples of investigations comparing the outcomes of studies that use visual scoring and those that use bioengineering methods are discussed. These examples provide little evidence that bioengineering measures provide an improvement in overall quality in comparison with current testing methods that rely on visual assessment. In addition, such measuring techniques can add considerably to the complexity of testing protocols. When benefits and cost are weighed in the balance, the visual assessment scales popularized by Draize and others remain an effective, practical method of evaluation.

Chemical allergy: translating biology into hazard characterization.

The induction by chemicals of allergic sensitization and allergic disease is an important and challenging branch of toxicology. Skin sensitization resulting in allergic contact dermatitis represents the most common manifestation of immunotoxicity in humans, and many hundreds of chemicals have been implicated as skin sensitizers. There are far fewer chemicals that have been shown to cause sensitization of the respiratory tract and asthma, but the issue is no less important because hazard identification remains a significant challenge, and occupational asthma can be fatal. In all areas of chemical allergy, there have been, and remain still, intriguing challenges where progress has required a close and productive alignment between immunology, toxicology, and clinical medicine. What the authors have sought to do here is to exemplify, within the framework of chemical allergy, how an investment in fundamental research and an improved understanding of relevant biological and biochemical mechanisms can pay important dividends in driving new innovations in hazard identification, hazard characterization, and risk assessment. Here we will consider in turn three specific areas of research in chemical allergy: (1) the role of epidermal Langerhans cells in the development of skin sensitization, (2) T lymphocytes and skin sensitization, and (3) sensitization of the respiratory tract. In each area, the aim is to identify what has been achieved and how that progress has impacted on the development of new approaches to toxicological evaluation. Success has been patchy, and there is still much to be achieved, but the journey has been fascinating and there have been some very important developments. The conclusion drawn is that continued investment in research, if coupled with an appetite for translating the fruits of that research into imaginative new tools for toxicology, should continue to better equip us for tackling the important challenges that remain to be addressed.

Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials.

Regulatory policies in Europe prohibited the testing of cosmetic ingredients in animals for a number of toxicological endpoints. Currently no validated non-animal test methods exist for skin sensitization. Evaluation of changes in cell surface marker expression in dendritic cell (DC)-surrogate cell lines represents one non-animal approach. The human Cell Line Activation Test (h-CLAT) examines the level of CD86 and CD54 expression on the surface of THP-1 cells, a human monocytic leukemia cell line, following 24h of chemical exposure. To examine protocol transferability, between-lab reproducibility, and predictive capacity, the h-CLAT has been evaluated by five independent laboratories in several ring trials (RTs) coordinated by the European Cosmetics Association (COLIPA). The results of the first and second RTs demonstrated that the protocol was transferable and basically had good between-lab reproducibility and predictivity, but there were some false negative data. To improve performance, protocol and prediction model were modified. Using the modified prediction model in the first and second RT, accuracy was improved. However, about 15% of the outcomes were not correctly identified, which exposes some of the limitations of the assay. For the chemicals evaluated, the limitation may due to chemical being a weak allergen or having low solubility (ex. alpha-hexylcinnamaldehyde). The third RT evaluated the modified prediction model and satisfactory results were obtained. From the RT data, the feasibility of utilizing cell lines as surrogate DC in development of in vitro skin sensitization methods shows promise. The data also support initiating formal pre-validation of the h-CLAT in order to fully understand the capabilities and limitations of the assay.

Local lymph node data for the evaluation of skin sensitization alternatives: a second compilation.

BACKGROUND: Development, evaluation and validation of alternatives to skin sensitisation testing require the availability of reliable databases with which comparative analyses can be conducted to establish performance characteristics. To facilitate this we have published previously a database comprising results from local lymph node assays (LLNAs) conducted with 211 chemicals. That database embraced a substantial range of chemistry, and of relative skin sensitising potency, and has found application in the assessment of new or refined methods. OBJECTIVE: In this paper we describe a second compilation to extend the LLNA database. METHODS: This second data compilation was derived from previously conducted LLNA studies involving an additional 108 chemicals. In addition, the first database contained a small number of inaccuracies, affecting results recorded with a few chemicals. In this paper these have been corrected. RESULTS: The inclusion of 108 new substances has served to extend and consolidate the areas of chemistry covered by the database. In addition, the entire dataset was evaluated for pre and prohaptens which will facilitate the choice of chemicals for alternative assay developments. CONCLUSIONS: It is anticipated that the new revised and extended database totalling over 300 chemicals will now serve as the primary resource to support the development and evaluation of new approaches to hazard identification and potency assessment.