Uchiyama T, Akiyama J, Miyai E, Sakamoto K, Takino Y, Ohnuma M, Ohkosi K, Okamoto Y, Morito Y, Kojima H, Okumura H, Sawamura J, Ikeda N, Sumida Y, Chiba K, Makino I, Kawakami K, Yamamoto R, Torishima H, Yanase H, Miyajima A, Sunouchi M, Hayashi M, Ohno Y
Japan Cosmetic Industry Association, Hatsumei Bldg, 9-14 Toranomon 2-chome, Minato-ku, Tokyo, 105Japan; Reseach & Development Department, Kaminomoto Co., Ltd., 3-25, 3-chome, Kumochibashi-dori, Chuo-ku, Kobe, 651Japan.
Toxicol In Vitro. 1999 Feb;13(1):163-73. doi: 10.1016/s0887-2333(98)00070-8.
The cytotoxicity test of neutral red (NR) uptake in normal rabbit corneal epithelial cells (CornePack((R))) was validated as an alternative method to the Draize rabbit eye irritation test (Draize test). We tested 38 cosmetic ingredients as well as isotonic sodium chloride solution in three phases of the validation study. The test procedures were controlled among participating laboratories under a common standard operating procedure (SOP). The concentration of test substances that showed a 50% reduction in NR uptake relative compared with controls (median NR uptake concentration: NR(50), mug/ml) was determined and compared with in vivo Draize scores. Six laboratories participated in the first phase of the validation study, seven in the second, and five in the third. The average interlaboratory coefficient of variation (CV) was 32.9%. The correlation and rank correlation coefficients between the maximal average Draize total scores (MAS) and NR(50) were -0.583 and 0.587, respectively. When the anionic detergents were excluded from analysis, the correlation coefficient increased to -0.738. When the cut-off point for positive and negative irritation was set at MAS of 15 and the predictability of this method was assessed by liner regression line, six substances (two acids, two alkanolamines and two alcohols) were false negative. Through this project, it appeared that CornePack, supplied in kit form with frozen secondary cultured cell in serum-free medium, could provide an effective, highly sensitive and simple preliminary screen for determining the cytotoxicity of substances. These results suggested that CornePack might have the potential to predict the MAS if definite criteria can be established for the compounds to be applicable. However, it is important to understand the nature of CornePack responses since its NR(50) profile was quite different from other cytotoxicity assays.
中性红（NR）摄取法检测正常兔角膜上皮细胞（CornePack®）的细胞毒性试验，被确认为替代Draize兔眼刺激试验（Draize试验）的一种方法。在验证研究的三个阶段，我们对38种化妆品成分以及等渗氯化钠溶液进行了测试。测试程序在参与实验室中按照通用标准操作规程（SOP）进行控制。确定了与对照相比NR摄取降低50%的受试物浓度（中位NR摄取浓度：NR(50)，μg/ml），并与体内Draize评分进行比较。六个实验室参与了验证研究的第一阶段，七个参与了第二阶段，五个参与了第三阶段。实验室间平均变异系数（CV）为32.9%。最大平均Draize总分（MAS）与NR(50)之间的相关系数和等级相关系数分别为-0.583和0.587。当从分析中排除阴离子洗涤剂时，相关系数增至-0.738。当将阳性和阴性刺激的临界点设定为MAS为15，并通过线性回归线评估该方法的可预测性时，有六种物质（两种酸、两种烷醇胺和两种醇）为假阴性。通过该项目发现，以试剂盒形式提供、含有无血清培养基中冷冻的二次培养细胞的CornePack，可为确定物质的细胞毒性提供有效、高度灵敏且简单的初步筛选。这些结果表明，如果能为适用的化合物建立明确标准，CornePack可能有预测MAS的潜力。然而，了解CornePack反应的性质很重要，因为其NR(50)谱与其他细胞毒性试验有很大不同。
