Assessing skin sensitization hazard in mice and men using non-animal test methods.

Sensitization, the prerequisite event in the development of allergic contact dermatitis, is a key parameter in both hazard and risk assessments. The pathways involved have recently been formally described in the OECD adverse outcome pathway (AOP) for skin sensitization. One single non-animal test method will not be sufficient to fully address this AOP and in many cases the use of a battery of tests will be necessary. A number of methods are now fully developed and validated. In order to facilitate acceptance of these methods by both the regulatory and scientific communities, results of the single test methods (DPRA, KeratinoSens, LuSens, h-CLAT, (m)MUSST) as well for a the simple '2 out of 3' ITS for 213 substances have been compiled and qualitatively compared to both animal and human data. The dataset was also used to define different mechanistic domains by probable protein-binding mechanisms. In general, the non-animal test methods exhibited good predictivities when compared to local lymph node assay (LLNA) data and even better predictivities when compared to human data. The '2 out of 3' prediction model achieved accuracies of 90% or 79% when compared to human or LLNA data, respectively and thereby even slightly exceeded that of the LLNA.

Bayesian integrated testing strategy to assess skin sensitization potency: from theory to practice.

Frameworks to predict in vivo effects by integration of in vitro, in silico and in chemico information using mechanistic insight are needed to meet the challenges of 21(st) century toxicology. Expert-based approaches that qualitatively integrate multifaceted data are practiced under the term 'weight of evidence', whereas quantitative approaches remain rare. To address this gap we previously developed a methodology to design an Integrated Testing Strategy (ITS) in the form of a Bayesian Network (BN). This study follows up on our proof of concept work and presents an updated ITS to assess skin sensitization potency expressed as local lymph node assay (LLNA) potency classes. Modifications to the ITS structure were introduced to include better mechanistic information. The parameters of the updated ITS were calculated from an extended data set of 124 chemicals. A detailed validation analysis and a case study were carried out to demonstrate the utility of the ITS for practical application. The improved BN ITS predicted correctly 95% and 86% of chemicals in a test set (n = 21) for hazard and LLNA potency classes, respectively. The practical value of using the BN ITS is far more than a prediction framework when all data are available. The BN ITS can develop a hypothesis using subsets of data as small as one data point and can be queried on the value of adding additional tests before testing is commenced. The ITS represents key steps of the skin sensitization process and a mechanistically interpretable testing strategy can be developed. These features are illustrated in the manuscript via practical examples.

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.

Nothing is perfect, not even the local lymph node assay: a commentary and the implications for REACH.

For many regulatory authorities, the local lymph node assay (LLNA) is the preferred assay for the predictive identification of skin-sensitizing chemicals. It is the initial requirement for sensitization testing within the new REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) regulations in the European Union. The primary reasons for the preferment of the LLNA are the animal welfare benefits it provides compared with traditional guinea-pig methods (refinement and reduction of animal usage) and the general performance characteristics of the assay with regard to overall reliability, accuracy, and interpretation. Moreover, a substantial published literature on the LLNA is available making it appropriate for use as a benchmark against which new approaches, including in vitro alternatives, can be evaluated and validated. There is, therefore, a view that the LLNA represents the 'gold standard' for skin sensitization testing. However, although this is probably correct, it is important to recognize and acknowledge that in common with all other predictive tests (whether they be validated or not), the LLNA has limitations, in addition to strengths, some of which were mentioned above. Arguably, it is the limitations (e.g., the occurrence of false positive and false negative results) of test methods that are most important to understand. With respect to the LLNA, these limitations are similar to those associated with guinea-pig skin sensitization methods. Among these are the occurrence of false positive and false negative results, susceptibility of results to changes in vehicle, and the possibility that interspecies differences may confound interpretation. In this commentary, these issues are reviewed and their impact on the utility of the LLNA for identification, classification, and potency assessment of skin sensitizers are considered. In addition, their relevance for the future development and validation of novel in vitro and in silico alternatives is explored.

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.

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.

Skin sensitisation: the Colipa strategy for developing and evaluating non-animal test methods for risk assessment.

Allergic contact dermatitis is a delayed-type hypersensitivity reaction induced by small reactive chemicals (haptens). Currently, the sensitising potential and potency of new chemicals is usually characterised using data generated via animal studies, such as the local lymph node assay (LLNA). There are, however, increasing public and political concerns regarding the use of animals for the testing of new chemicals. Consequently, the development of in vitro, in chemico or in silico models for predicting the sensitising potential and/or potency of new chemicals is receiving widespread interest. The Colipa Skin Tolerance task force currently collaborates with and/or funds several academic research groups to expand our understanding of the molecular and cellular events occurring during the acquisition of skin sensitisation. Knowledge gained from this research is being used to support the development and evaluation of novel alternative approaches for the identification and characterisation of skin sensitizing chemicals. At present three non-animal test methods (Direct Peptide Reactivity Assay (DPRA), Myeloid U937 Skin Sensitisation Test (MUSST) and human Cell Line Activation Test (hCLAT)) have been evaluated in Colipa interlaboratory ring trials for their potential to predict skin sensitisation potential and were recently submitted to ECVAM for formal pre-validation. Data from all three test methods will now be used to support the study and development of testing strategy approaches for skin sensitiser potency prediction. This publication represents the current viewpoint of the cosmetics industry on the feasibility of replacing the need for animal test data for informing skin sensitisation risk assessment decisions.

Determination of in vivo dose response and allergen-specific T cells in subjects contact-sensitized to squaric acid dibutyl ester.

BACKGROUND: Squaric acid dibutyl ester (SADBE) is a known contact sensitizer, but dose-response data are not defined. OBJECTIVE: To determine the relationship between sensitization dose and contact hypersensitivity (CHS) response to SADBE in human volunteers. The study also aimed to investigate whether SADBE-reactive blood T cells could be detected using ex vivo mature dendritic cells (DCs) as antigen-presenting cells. METHOD: Forty healthy volunteers were sensitized to either 12.5, 25, 50, or 250 microg of SADBE in a 48 microL volume. This was followed by elicitation 2 weeks later with five doses (0, 0.2, 2, 20, and 200 microg in 20 microL). An additional 10 subjects received the elicitation doses without prior sensitization. Blood samples obtained after sensitization were purified into T cells and mature DCs. RESULTS: A direct relationship between sensitization dose and in vivo CHS response was observed. The SADBE dose that effectively sensitized 50% of the population (ED50) was 22 microg/cm2. Significant SADBE-specific T-cell proliferation in vitro was not observed 2 weeks after sensitization but became evident after elicitation. CONCLUSION: This study establishes the in vivo dose-response characteristics of immune reactivity to SADBE and antigen-specific T-cell reactivity.

The local lymph node assay and the assessment of relative potency: status of validation.

For the prediction of skin sensitization potential, the local lymph node assay (LLNA) is a fully validated alternative to guinea-pig tests. More recently, information from LLNA dose-response analyses has been used to assess the relative potency of skin sensitizing chemicals. These data are then deployed for risk assessment and risk management. In this commentary, the utility and validity of these relative potency measurements are reviewed. It is concluded that the LLNA does provide a valuable assessment of relative sensitizing potency in the form of the estimated concentration of a chemical required to produce a threefold stimulation of draining lymph node cell proliferation compared with concurrent controls (EC3 value) and that all reasonable validation requirements have been addressed successfully. EC3 measurements are reproducible in both intra- and interlaboratory evaluations and are stable over time. It has been shown also, by several independent groups, that EC3 values correlate closely with data on relative human skin sensitization potency. Consequently, the recommendation made here is that LLNA EC3 measurements should now be regarded as a validated method for the determination of the relative potency of skin sensitizing chemicals, a conclusion that has already been reached by a number of independent expert groups.

Mechanistic applicability domain classification of a local lymph node assay dataset for skin sensitization.

The goal of eliminating animal testing in the predictive identification of chemicals with the intrinsic ability to cause skin sensitization is an important target, the attainment of which has recently been brought into even sharper relief by the EU Cosmetics Directive and the requirements of the REACH legislation. Development of alternative methods requires that the chemicals used to evaluate and validate novel approaches comprise not only confirmed skin sensitizers and non-sensitizers but also substances that span the full chemical mechanistic spectrum associated with skin sensitization. To this end, a recently published database of more than 200 chemicals tested in the mouse local lymph node assay (LLNA) has been examined in relation to various chemical reaction mechanistic domains known to be associated with sensitization. It is demonstrated here that the dataset does cover the main reaction mechanistic domains. In addition, it is shown that assignment to a reaction mechanistic domain is a critical first step in a strategic approach to understanding, ultimately on a quantitative basis, how chemical properties influence the potency of skin sensitizing chemicals. This understanding is necessary if reliable non-animal approaches, including (quantitative) structure-activity relationships (Q)SARs, read-across, and experimental chemistry based models, are to be developed.

Predictive identification of human skin sensitization thresholds.

For years, methods have been available for the predictive identification of chemicals that possess the intrinsic potential to cause skin sensitization. However, many have proven less suitable for the determination of relative sensitizing potency. In this respect, the local lymph node assay (LLNA) has been shown to have a number of important advantages. Through interpolation of LLNA dose-response data, the concentration of a chemical required to produce a threshold positive response (a 3-fold increase in activity compared with concurrent vehicle controls, the EC3 value) can be measured. The robustness of this parameter has been demonstrated rigorously in terms of inter- and intralaboratory reproducibility. Additionally, the relationship between potency estimates from the LLNA and an appreciation of human potency based on clinical experience has been reported previously. In the present investigations, we have sought to consolidate further our understanding of the association between EC3 values and human skin-sensitization potency by undertaking a thorough and extensive analysis of existing human predictive assays, particularly where dose-response information is available, from historical human repeated insult patch tests (HRIPTs). From these human data, information on the approximate threshold for the induction of skin sensitization in the HRIPT was determined for 26 skin-sensitizing chemicals. These data were then compared with LLNA-derived EC3 values. The results from each assay, expressed as dose per unit area (microg/cm(2)), revealed a clear linear relationship between the 2 values, thereby substantiating further the utility of LLNA EC3 values for prediction of the relative human sensitizing potency of newly identified skin sensitizers.