Extending the applicability domain of the human cell line activation test (h-CLAT).

Cosmetic ingredients must be toxicologically assessed to determine their skin sensitizing potential. The in vitro human cell line activation test (h-CLAT; OECD TG 442E) addresses the activation of dermal dendritic cells by analyzing specific protein expression after exposure of THP-1 cells to the test chemical. According to the protocol, FITC-labeled antibodies are used for protein detection. However, some chemicals show strong autofluorescence at FITC-specific wavelengths so that antibody-specific signals cannot be distinguished appropriately from autofluorescence background. This leads to inconclusive or false-negative predictions. Alternative fluorochromes can be used if their equivalence with the FITC-labeled antibodies is proven. In the current paper we describe the results of a proficiency exercise, based on the proficiency chemicals listed in the guideline, with FITC-labeled antibodies as the benchmark and APC-labeled anti­bodies as an alternative detection system. APC emits fluorescence at longer wavelengths, thus avoiding interference in the FITC spectrum. Irrespective of the employed fluorochrome, all chemicals were classified correctly, and the EC150 and 200 values were in the same order of magnitude. Hence, the equivalence in performance of FITC- and APC-labeled antibodies was demonstrated, and the respective demand of the guideline was fulfilled. In a case study, we then tested a proprietary oxidative hair dye using both fluorochromes. Using APC-labeled antibodies, the hair dye was unambiguously identified as a sensitizer, whereas no classification could be made with the FITC-labeled antibodies. With APC, fluorescence interference can be circumvented and the applicability domain of the h-CLAT extended to include autofluorescent chemicals.

Development of a next generation risk assessment framework for the evaluation of skin sensitisation of cosmetic ingredients.

All cosmetic products placed onto the market must undergo a risk assessment for human health to ensure they are safe for consumers, including an assessment of skin sensitisation risk. Historically, in vivo animal test methods were used to identify and characterise skin sensitisation hazard, however non-animal and other new approach methodologies (NAMs) are now the preferred and mandated choice for use in risk assessment for cosmetic ingredients. The experience gained over the last three decades on how to conduct risk assessments based upon NAMs has allowed us to develop a non-animal, next generation risk assessment (NGRA) framework for the assessment of skin sensitisers. The framework presented here is based upon the principles published by the International Cooperation on Cosmetic Regulation (ICCR) and is human relevant, exposure led, hypothesis driven and designed to prevent harm. It is structured in three tiers and integrates all relevant information using a weight of evidence (WoE) approach that can be iterated when new information becomes available. The initial tier (TIER 0) involves a thorough review of the existing information including; identification of the use scenario/consumer exposure; characterisation of the chemical purity and structure; in silico predictions; existing data pertaining to skin sensitisation hazard (historical or non-animal); the identification of suitable read-across candidates with supporting hazard identification/characterisation information and application of exposure-based waiving. Considering all information identified in TIER 0, the next step is the generation of a hypothesis (TIER 1). All data are considered in an exposure-led WoE approach, taking into account an initial view on whether a chemical is likely to be a skin sensitiser or not, choice of defined approach (DA) and availability of read-across candidates. If existing information is insufficient for concluding the risk assessment, the generation of additional information may be required to proceed (TIER 2). Such targeted testing could involve refinement of the exposure estimation or generation of data from in vitro or in chemico NAMs. Once sufficient information is available, the final stage of the NGRA framework is the determination of a point of departure (POD), characterising uncertainty and comparing to the consumer exposure in a WoE. Thorough evaluation of the sources of uncertainty is essential to ensure transparency and build trust in new risk assessment approaches. Although significant progress has been made, industry must continue to share its experience in skin sensitisation NGRA via case studies to demonstrate that this new risk assessment approach is protective for consumers. Dialogue and collaboration between key stakeholders, i.e. risk assessors, clinicians and regulators are important to gain mutual understanding and grow confidence in new approaches.

Non-animal methods to predict skin sensitization (II): an assessment of defined approaches *.

Skin sensitization is a toxicity endpoint of widespread concern, for which the mechanistic understanding and concurrent necessity for non-animal testing approaches have evolved to a critical juncture, with many available options for predicting sensitization without using animals. Cosmetics Europe and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods collaborated to analyze the performance of multiple non-animal data integration approaches for the skin sensitization safety assessment of cosmetics ingredients. The Cosmetics Europe Skin Tolerance Task Force (STTF) collected and generated data on 128 substances in multiple in vitro and in chemico skin sensitization assays selected based on a systematic assessment by the STTF. These assays, together with certain in silico predictions, are key components of various non-animal testing strategies that have been submitted to the Organization for Economic Cooperation and Development as case studies for skin sensitization. Curated murine local lymph node assay (LLNA) and human skin sensitization data were used to evaluate the performance of six defined approaches, comprising eight non-animal testing strategies, for both hazard and potency characterization. Defined approaches examined included consensus methods, artificial neural networks, support vector machine models, Bayesian networks, and decision trees, most of which were reproduced using open source software tools. Multiple non-animal testing strategies incorporating in vitro, in chemico, and in silico inputs demonstrated equivalent or superior performance to the LLNA when compared to both animal and human data for skin sensitization.

Non-animal methods to predict skin sensitization (I): the Cosmetics Europe database.

Cosmetics Europe, the European Trade Association for the cosmetics and personal care industry, is conducting a multi-phase program to develop regulatory accepted, animal-free testing strategies enabling the cosmetics industry to conduct safety assessments. Based on a systematic evaluation of test methods for skin sensitization, five non-animal test methods (DPRA (Direct Peptide Reactivity Assay), KeratinoSensTM, h-CLAT (human cell line activation test), U-SENSTM, SENS-IS) were selected for inclusion in a comprehensive database of 128 substances. Existing data were compiled and completed with newly generated data, the latter amounting to one-third of all data. The database was complemented with human and local lymph node assay (LLNA) reference data, physicochemical properties and use categories, and thoroughly curated. Focused on the availability of human data, the substance selection resulted nevertheless resulted in a high diversity of chemistries in terms of physico-chemical property ranges and use categories. Predictivities of skin sensitization potential and potency, where applicable, were calculated for the LLNA as compared to human data and for the individual test methods compared to both human and LLNA reference data. In addition, various aspects of applicability of the test methods were analyzed. Due to its high level of curation, comprehensiveness, and completeness, we propose our database as a point of reference for the evaluation and development of testing strategies, as done for example in the associated work of Kleinstreuer et al. We encourage the community to use it to meet the challenge of conducting skin sensitization safety assessment without generating new animal data.

State-of-the-art and new options to assess T cell activation by skin sensitizers: Cosmetics Europe Workshop.

Significant progress has been made in the development and validation of non-animal test methods for skin sensitization assessment. At present, three of the four key events of the Adverse Outcome Pathway (AOP) are assessable by OECD-accepted in vitro methods. The fourth key event describes the immunological response in the draining lymph node where activated dendritic cells present major histocompatibility complex-bound chemically modified peptides to naive T cells, thereby priming the proliferation of antigen-specific T cells. Despite substantial efforts, modelling and assessing this adaptive immune response to sensitizers with in vitro T cell assays still represents a challenge. The Cosmetics Europe Skin Tolerance Task Force organized a workshop, bringing together academic researchers, method developers, industry representatives and regulatory stakeholders to review the scientific status of T cell-based assays, foster a mutual scientific understanding and conceive new options to assess T cell activation. Participants agreed that current T cell assays have come a long way in predicting immunogenicity, but that further investment and collaboration is required to simplify assays, optimize their sensitivity, better define human donor-to-donor variability and evaluate their value to predict sensitizer potency. Furthermore, the potential role of T cell assays in AOP-based testing strategies and subsequent safety assessment concepts for cosmetic ingredients was discussed. It was agreed that it is currently difficult to anticipate uses of T cell assay data for safety assessment and concluded that experience from case studies on real-life risk assessment scenarios is needed to further consider the usefulness of assessing the fourth AOP key event.

In vitro eye irritation testing using the open source reconstructed hemicornea - a ring trial.

The aim of the present ring trial was to test whether two new methodological approaches for the in vitro classification of eye irritating chemicals can be reliably transferred from the developers' laboratories to other sites. Both test methods are based on the well-established open source reconstructed 3D hemicornea models. In the first approach, the initial depth of injury after chemical treatment in the hemicornea model is derived from the quantitative analysis of histological sections. In the second approach, tissue viability, as a measure for corneal damage after chemical treatment, is analyzed separately for epithelium and stroma of the hemicornea model. The three independent laboratories that participated in the ring trial produced their own hemicornea models according to the test producer's instructions, thus supporting the open source concept. A total of 9 chemicals with different physicochemical and eye-irritating properties were tested to assess the between-laboratory reproducibility (BLR), the predictive performance, as well as possible limitations of the test systems. The BLR was 62.5% for the first and 100% for the second method. Both methods enabled to discriminate Cat. 1 chemicals from all non-Cat. 1 substances, which qualifies them to be used in a top-down approach. However, the selectivity between No Cat. and Cat. 2 chemicals still needs optimization.

Alternatives for skin sensitisation: Hazard identification and potency categorisation: Report from an EPAA/CEFIC LRI/Cosmetics Europe cross sector workshop, ECHA Helsinki, April 23rd and 24th 2015.

In the two years since the last workshop report, the environment surrounding the prediction of skin sensitisation hazards has experienced major change. Validated non-animal tests are now OECD Test Guidelines. Accordingly, the recent cross sector workshop focused on how to use in vitro data for regulatory decision-making. After a review of general approaches and six case studies, there was broad consensus that a simple, transparent stepwise process involving non-animal methods was an opportunity waiting to be seized. There was also strong feeling the approach should not be so rigidly defined that assay variations/additional tests are locked out. Neither should it preclude more complex integrated approaches being used for other purposes, e.g. potency estimation. All agreed the ultimate goal is a high level of protection of human health. Thus, experience in the population will be the final arbiter of whether toxicological predictions are fit for purpose. Central to this is the reflection that none of the existing animal assays is perfect; the non-animal methods should not be expected to be so either, but by integrated use of methods and all other relevant information, including clinical feedback, we have the opportunity to continue to improve toxicology whilst avoiding animal use.

Systematic evaluation of non-animal test methods for skin sensitisation safety assessment.

The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitization potency prediction. The results of the first phase ­ systematic evaluation of 16 test methods ­ are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data,potential for throughput, transferability and accessibility in cooperation with the test method developers.A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy ­ combined with bioavailability and skin metabolism data and exposure consideration ­ is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients.

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.

Ageing processes influence keratin and KAP expression in human hair follicles.

In many cultures, a youthful look is strictly linked to strong and healthy hair. Source of the hair fibre is the hair follicle, a highly specialized skin appendage. Biological alterations because of intrinsic or extrinsic stimuli can destabilize this perfectly organized system, thus effecting hair growth or metabolism. Also, ageing could be characterized as a disturbance in this well-balanced machinery. Albeit the predominant symptom of hair ageing, greying, is addressed in a plurality of research activities, further age-related changes, e.g. related to hair structure, remain obscure. Therefore, we characterized hair follicles of two volunteer panels (below 25 years, above 50 years) on the molecular level, especially focussing on alterations influencing gene expression of keratins and keratin-associated proteins. We showed that concordantly to other biological systems the hair follicle undergoes several modifications during the ageing process associated among others with a significant decline in these structural proteins. Providing strategies to fight against these age-related changes is a challenge for hair science.