Non-animal Replacements for Acute Toxicity Testing

Carol Barker-Treasure, Kevin Coll, Nathalie Belot, Chris Longmore, Karl Bygrave, Suzanne Avey and Richard Clothier

Current approaches to predicting adverse effects in humans from acute toxic exposure to cosmetic ingredients still heavily necessitate the use of animals under EU legislation, particularly in the context of the REACH system, when cosmetic ingredients are also destined for use in other industries. These include the LD50 test, the Up-and-Down Procedure and the Fixed Dose Procedure, which are regarded as having notable scientific deficiencies and low transferability to humans. By expanding on previous in vitro tests, such as the animal cell-based 3T3 Neutral Red Uptake (NRU) assay, this project aims to develop a truly animal-free predictive test for the acute toxicity of cosmetic ingredients in humans, by using human-derived cells and a prediction model that does not rely on animal data. The project, funded by Innovate UK, will incorporate the NRU assay with human dermal fibroblasts in animal product-free culture, to generate an in vitro protocol that can be validated as an accepted replacement for the currently available in vivo tests. To date, the project has successfully completed an assessment of the robustness and reproducibility of the method, by using sodium lauryl sulphate (SLS) as a positive control, and displaying analogous results to those of the original studies with mouse 3T3 cells. Currently, the testing of five known ingredients from key groups (a surfactant, a preservative, a fragrance, a colour and an emulsifier) is under way. The testing consists of initial range-finding runs followed by three valid runs of a main experiment with the appropriate concentration ranges, to generate IC50 values. Expanded blind trials of 20 ingredients will follow. Early results indicate that this human cell-based test holds the potential to replace aspects of in vivo animal acute toxicity testing, particularly with reference to cosmetic ingredients.
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Cytotoxicity Studies of Anchorage-independent LS-L-929 Mouse Fibroblasts Using Membrane Integrity, ATP Content and ATP/ ADP Ratio as Determinants: Including the Xenobiotic Effects of Components in an Experimental Shampoo Formulation

Richard B. Kemp,

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Iron-induced Lipid Peroxidation in Human Skin-derived Cell Lines: Protection by a Red Orange Extract

Flora Morini, Fabiola Dusatti, Francesco P. Bonina, Antonella Saija and Margherita Ferro

Although photodamage and photoprotection have already been extensively studied in cultured cells, few data have been reported in the literature regarding the in vitro behaviour of skin cells toward a chemical stress, such as iron-induced lipid peroxidation. We investigated the susceptibilities of two human skin-derived cell lines (NCTC 2544 keratinocytes and HFFF2 fibroblasts) to lipid peroxidation induced by FeSO4/histidine, FeSO4/ascorbate and Fe2(SO4)3/ADP. NCTC 2544 cells were more susceptible than HFFF2 cells to lipid peroxidation (assessed by measuring the content of malondialdehyde [MDA]) with iron/ascorbate and iron/ADP as pro-oxidants whereas, with iron/histidine, the same level of MDA production was achieved (about 10nmol/mg protein) in the two cell populations. On the basis of these results, one experimental model (iron/histidine) was selected to assess the protective effect of a mixture of two classical antioxidants, Trolox C™ (50μM) and Vitamin C (1mM), added to the cell cultures according to various protocols. The maximal decrease of MDA production in both cell lines was obtained when the antioxidant mixture and the pro-oxidant were added simultaneously to the cultures. By using the same experimental design, NCTC 2544 and HFFF2 cells were exposed to a standardised extract of red oranges (ROE; 0.025–0.5mg/ml), the main active principles of which (anthocyanins 3.1%, hydroxycinnamic acid 2.07%, and flavanone glycosides 8.1%) possess antioxidant activity. Cells treated with ROE, that were still over 90% viable, as evaluated by means of neutral red uptake and tetrazolium salt reduction tests, showed a significant and dose-dependent inhibition of MDA production. This study provides new information about the behaviour of cultured skin cells exposed to chemically induced oxidative stress, and provides further support to the possibility of using skin-derived human cell lines in the evaluation of the effectiveness of antioxidant ingredients for new drugs and/or cosmetics.
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Development of a Cell-based Diabetic Wound Assay

Phil Stephens

Chronic wounds require prolonged healthcare and adversely affect the quality of life of patients. They are particularly prominent in patients with diabetes, and their relative numbers are set to increase with the rise of diabetes within our population. Research is still needed to understand the factors leading to such wounds, to understand why they persist for such long periods of time, and also to develop new and efficacious treatment strategies. One problem facing this research is a lack of adequate animal models, as the current models do not truly reflect the human condition and often lead to much animal suffering. Hence, over the past four years, our group has been trying to develop a human-based in vitro diabetic wound model, which could be used as a high-throughput screening system to pre-screen potential chronic diabetic wound healing agents and to reduce unnecessary animal pain and suffering. To this end, we have isolated healthy and diseased skin fibroblasts from patient tissue biopsies. Crucially, to create a cell reporter system that can be widely used in the future, the cells were immortalised in order to escape senescence. By using microarray analysis, gene expression pattern differences have been identified between healthy and diseased cells, and disease-specific ‘reporter’ genes have been selected for further studies. The promoters of these reporter genes have been coupled to fluorescent reporter constructs and inserted back into the diseased fibroblasts, so that we now have proof-of-concept for a real-time diabetic reporter system for future exploitation.
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