| 摘要: | 在過去化妝品安全評估主要以動物測試為主,如皮膚刺激試驗就用兔子,天竺鼠,大白鼠等.這些試驗被動物保護團體批評為不人道. 在目前世界趨勢上，要以細胞層次來取代動物試驗。在本研究中我們計畫建立化妝品細胞毒性測試以取代數項傳統動物測試項目，以期用細胞毒性法搭配傳統方法以建立一套完善快速的化妝品安全性檢測模式。目前國內化妝品衛生管理條例暨有關法規，所定化妝品安全性，是以已知一成份和含量去訂定安全標準並沒有檢驗方法，但此法之缺失如化妝品含數種成份，每種劑量依目前法規皆在安全值內，但其混合是否安全，則不得而知。所以有須要建立一套完善的化妝品安全性檢測模式，只需將化妝品直接用生物檢測即知。如有新成份則更須有檢測方法方可知其是否安全。 在目前研究中已知用細胞毒性測試可有效，更快速的檢測化妝品毒性，方法中與皮膚刺激測試（draize test）有高度相關性的有用fibroblast纖維母細胞以Neutral Red uptake,Alamar blue，MTT，LDH release,L-6 release等方法去測細胞毒性。這些方法與動物試驗都有高度一致性，其中用Neutral Red uptake（IC 50）檢測結果與人類patch test有高度相關性。另外眼毒性測試也有數種方法可以取代如SIRC細胞用Neutral Red測，而另一種方法以epithelial-fibroblast 用MTT（Osbome等，1995）去測，或fibroblast 以Predicate kit( Neutral Red)去測（Garboard等，1994）。光毒性測試中可用Skin 2 Cell用MTT（Edwards等，1994）測。藉由這些以建立細胞毒性的測試法，我們計畫要去檢測目前已有的化妝品，包括已被認為是有毒的及無毒，以建立數據資料庫並定訂定出毒性標準值，我們利用細胞百分之五十的抑制濃度IC50做為評估毒性的指標，在本研究中我們計畫以現有化妝品毒性資料以我們要做的細胞毒性建立有刺激性與無刺激性的分界點閾值IC50。並且我們計畫以大白兔皮膚作原發性刺激實驗以了解在動物出現毒性時,此時細胞的毒性為何,求其相關性並尋找最敏感的細胞實驗,以取代動物實驗。在本研究中我們選用fibroblast（L929,3T3）及Keratinocyte，三種細胞，用MTT, Neutral Red, LDH三種方法去建立皮膚刺激性，眼毒性，及光毒性的細胞檢測模式。 在本計畫中我們預期以各種細胞毒性的檢測法建立一套皮膚刺激性，眼粘膜刺激性及光毒性的測試模式，並把目前各種化妝品有毒性，無毒性的建立數據庫，以利新化妝品安全性的檢測。
In the past, researcher routinely performed in vivo skin-irritancy studies on animal models-usually rabbits, guinea pigs or rats-and patch reasons. Animal-welfare groups criticize the use of animals in screening and testing, posing potential public-relations problems for companies using animal studies. Experiments using human volunteer, on the other hand, are time-consuming and expensive. Hence, researcher have been urgently working to develop appropriate alternatives to in vivo tests. In vitro models currently provide validates data applicable to the evaluation of human skin-irritation potentials to have found official approval for cosmetic safety testing. The close relationship is found between in vitro test and animal test (including skin irritation, eye irritation and phototoxicity) )(r>0.8). Therefore, the search for simple and reproducible in vitro screening procedures to estimate skin irritancy contimues in taiwan. In vitro cultures of submerged monolayers of keratinocytes or fibroblasts cell lines have been used in various in vitro irritation assays. In this study, we investigated whether the LDH leakage assay, neutral red uptake and MTT reduction test-methods for testing cytotoxicity in cultured fibroblasts-may br useful alternative models to replace the human patch and rabbit intradermal safe test. They are based in different endpoints: the neutral red uptake is based on incorporation of neutral red dye into lysosomes of vable cells; the MTT assay determines reduction of yellow MTT tetrazolium salt to a blue formazan by mitochondrial succinate dehydrogenase activity of uninjured cells; and the LDH assayuses the principle that lactate dehydrogenase leaks from cells after damage. The concentration that induced 50% inhibition relative to controls (IC50) is calculated for each test, ceel line and chemical. We developed protocols for cytotoxicity screening of cosmeticc representative of know skin irritants and set the data base in order to set up the model for in vitro screening procedures for cosmetic safety testing in taiwan. |
