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.

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.

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), KeratinoSens, h-CLAT (human cell line activation test), U-SENS, 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.

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.

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.

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.

Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase II).

To replace the Draize skin irritation assay (OECD guideline 404) several test methods based on reconstructed human epidermis (RHE) have been developed and were adopted in the OECD test guideline 439. However, all validated test methods in the guideline are linked to RHE provided by only three companies. Thus, the availability of these test models is dependent on the commercial interest of the producer.

Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase I).

We have developed a new in vitro skin irritation test based on an open source reconstructed epidermis (OS-REp) with openly accessible protocols for tissue production and test performance. Due to structural, mechanistic and procedural similarity, a blinded catch-up validation study for skin irritation according to OECD Performance Standards (PS) was conducted in three laboratories to promote regulatory acceptance, with OS-REp models produced at a single production site only. While overall sensitivity and predictive capacity met the PS requirements, overall specificity was only 57%.

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.

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.

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.

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.

Unveiling the molecular basis of intrinsic skin aging(1).

The process of skin aging is a combination of an extrinsic and intrinsic aspect, and knowing the molecular changes underlying both is a prerequisite to being able to effectively counter it. However, despite its importance for a deeper understanding of skin aging as a whole, the process of intrinsic skin aging in particular has barely been investigated. In this study, the molecular changes of intrinsic skin aging were analyzed by applying 'Serial Analysis of Gene Expression' (SAGE(TM)) to skin biopsies of young and aged donors.

Safe water supply without disinfection in a large city case study: Berlin.

Berlin's water supplies originate exclusively from groundwater. For sustainable water management, river water is treated by flocculation and filtration and used either for artificial groundwater recharge (rivers Spree and Havel) or for bank filtration (Nordgraben and Lake Tegel). Drinking water chlorination was abandoned in Berlin (West) in 1978, and in Berlin (East) in 1992, following German unification.

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains.

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit.

Isolation and characterization of the rat huntingtin promoter.

Huntington's disease (HD) is a neurodegenerative disorder caused by a (CAG)>37 repeat expansion in a novel gene of unknown function. Although the huntingtin gene is expressed in neuronal and non-neuronal tissues, the disease affects nerve cells of selected regional areas of the central nervous system. To gain insight into the regulation of the HD gene we analysed 1348 bp of the rat huntingtin promoter region.

Cell-matrix interactions induce tyrosine phosphorylation of MAP kinases ERK1 and ERK2 and PLCgamma-1 in two-dimensional and three-dimensional cultures of human fibroblasts.

Using immunoprecipitation and phosphotyrosine detection by Western blotting, intracellular signaling intermediates were analyzed in human primary dermal fibroblasts, either seeded as monolayers on collagen I coats (2D) or seeded within three-dimensional collagen I lattices (3D). Previous results demonstrated that integrin activation in these systems resulted in a cascade of protein tyrosine phosphorylation, including focal adhesion kinase (D. Roeckel and T.

Role of zinc-finger proteins Sp1 and zif268/egr-1 in transcriptional regulation of the human synaptobrevin II gene.

Synaptobrevin II is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis. The 5'-flanking region of the human synaptobrevin II gene is very (G+C)-rich and contains a 13-bp motif that includes overlapping binding sites for the zinc finger transcription factors Sp1 and zif268/egr-1. To test whether Sp1 and zif268/egr-1 interact with this motif, gel retardation assays were performed.

pHIVTATA-CAT, a versatile vector to study transcriptional regulatory elements in mammalian cells.

We have constructed a vector, pHIVTATA-CAT, that contains the Escherichia coli cat gene, encoding chloramphenicol acetyltransferase, under the control of a minimal promoter consisting of the HIV TATA box and the adenovirus major late promoter initiator element. Putative transcriptional elements can be inserted either directly upstream from the TATA box or downstream from the reporter gene in an enhancer position. Transcription can be monitored enzymatically or by RNase protection mapping.

The human synapsin II gene promoter. Possible role for the transcription factor zif268/egr-1, polyoma enhancer activator 3, and AP2.

Synapsin II is a neuron-specific phosphoprotein that selectively binds to small synaptic vesicles in the presynaptic nerve terminal. Here we report the cloning and sequencing of the 5'-flanking region of the human synapsin II gene. This sequence is very GC-rich and lacks a TATA or CAAT box.

Regulation of synapsin I gene expression by the zinc finger transcription factor zif268/egr-1.

zif268/egr-1 is an immediate early response gene that is involved in regulation of growth and differentiation. Its mRNA encodes a sequence-specific transcriptional activator containing three zinc fingers that act as the DNA-binding domain. Although zif268/egr-1 is expressed in the nervous system during neuronal excitation, no target gene has yet been identified.