Hagino Shigenobu, Okazaki Yuuko, Itagaki Hiroshi
Shiseido Research Centre, Yokohama, Japan.
Altern Lab Anim. 2008 Dec;36(6):641-52. doi: 10.1177/026119290803600608.
A tier evaluation system was assessed as an alternative method for the identification of cosmetic ingredients which are not ocular irritants. The system employed monolayer cultures of SIRC cells, an established cell line originally derived from the rabbit cornea, and a three-dimensional living dermal model (LDM), MATREXtrade mark, which consists of human dermal fibroblasts in a contracted collagen lattice. Effects on the cell monolayer cultures were determined by using SIRC cell-Crystal Violet staining (SIRC-CVS), and effects on the LDM were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A non-irritating ingredient was defined as a compound having a maximal average total score (MAS) of 5 or less in the Draize eye test, as this is the criterion used in the Japanese draft guidance for evaluating cosmetic ingredients. Among 34 test substances with known characteristics, 30 were classified accurately. Based on these encouraging results, the possibility of simplifying the MTT assay on the LDM for more-practical use, by selecting only three concentration levels to discriminate non-irritants from irritants, was assessed. The simplified method, involving a three-dose set (the three-dose method), was confirmed as being suitable for the identification of non-irritating ingredients, with triethanolamine used as a negative reference standard. Finally, the LDM was used to evaluate compounds at similar concentrations to those tested in vivo, aiming to predict the concentration at which an ingredient can be formulated into products without causing eye irritation. On the basis of previous validation data and our additional results, it was found that test samples that resulted in a cell viability of 50% or more in this model, could be classified as non-irritating ingredients. In all, these results indicate that the tier evaluation system may be suitable for the evaluation of ingredients intended to be used in cosmetics and medicated cosmetics in Japan.
评估了一种分级评估系统，作为识别非眼部刺激性化妆品成分的替代方法。该系统采用SIRC细胞的单层培养物（一种最初源自兔角膜的成熟细胞系）和一种三维活体真皮模型（LDM，MATREX商标），其由收缩胶原晶格中的人真皮成纤维细胞组成。通过使用SIRC细胞-结晶紫染色（SIRC-CVS）确定对细胞单层培养物的影响，并通过使用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）测定法评估对LDM的影响。无刺激性成分被定义为在Draize眼试验中最大平均总分（MAS）为5或更低的化合物，因为这是日本化妆品成分评估草案中使用的标准。在34种具有已知特性的测试物质中，30种被准确分类。基于这些令人鼓舞的结果，评估了通过仅选择三个浓度水平来区分无刺激物和刺激物从而简化LDM上的MTT测定法以使其更具实用性的可能性。涉及三剂量组的简化方法（三剂量法）被确认为适用于识别无刺激性成分，使用三乙醇胺作为阴性参考标准。最后，使用LDM评估与体内测试浓度相似的化合物，旨在预测成分可配制成产品而不引起眼部刺激的浓度。根据先前的验证数据和我们的额外结果，发现在此模型中导致细胞活力达到50%或更高的测试样品可被分类为无刺激性成分。总之，这些结果表明分级评估系统可能适用于评估日本化妆品和药用化妆品中打算使用的成分。
