Establishment Of Human Cell Line Activation Test And Its Application For Testing Skin Sensitization Of Chemicals

Background:Allergic Contact Dermatitis(ACD)is a delayed hypersensitivity triggered by skin contact with sensitizing substances.Skin sensitization testing is an important part of the safety assessment of skin contact products.In 2007,the US Federal Government-led 21st Century Toxicology Program in the’Toxicity Testing for the 21st Century:Prospects and Strategies’report stated that the goals of the toxicity testing strategy transformation need to be achieved include’reduce the cost and time required to toxicity testing.’And ’to minimize the number of animals used in the test.’In 2013,the 7th Amendment to the European Union’s Cosmetics Directive issued a ban on the complete ban on all cosmetic ingredients for animal testing.In 2017,the REACH(Registration,Evaluation,Authorisation and restriction of Chemicals)regulations also require the use of silico methods and in vitro methods as the primary method for skin sensitization testing.With the strengthening of international regulation and the implementation of animal ethics principles,it is essential to establish reliable skin sensitization in vitro methods in the laboratory.Objectives:To establish the human cell line activation test(h-CLAT)in laboratory by exploring the h-CLAT test conditions and carring out methodological studies.The skin sensitization of cosmetics was tested by the established h-CLAT test method and Local Lymph Nodes Assay:BrdU-ELISA at the same time.The results were compared to verify the applicability of the h-CLAT method for skin sensitization detection of hair dyes,sunscreens,and freckle cosmetics.Contents:1.Established the method according to OECD 442(E)and check the reliability by using 10 proficiency substances recommended in ANNEX Ⅱ2.Cytotoxicities of the 10 proficiency substances were measured by CCK-8 method and flow cytormetry method respectively,and comparisons between the two were further conducted to evaluate if CCK-8 method can be used in h-CLAT in the place of flow cytometry method3.Hair dyes(2),sunscreens products(3),freckle cosmetics(4)and raw materials from hair dyes(2)were subjected to h-CLAT method.7 out of 11 samples mentioned above were further tested according to OECD442(B):LLNA:BrdU-ELISA method Other results were obtained in laboratory(2 hair dyes products)before and by literature review(2 kinds of raw materials of hair dyes).The results of skin sensitization of cosmetic samples obtained via h-CLAT and LLNA:BrdU-ELISA were finally subjected to statistical consistency test to verify the applicability of the h-CLAT method for detecting cosmetics sensitization.Results:1.The h-CLAT test method has been established in the laboratory.The results of 10 proficiency substances were in accordance with OECD 442(E)requirements.2.Cytotoxicity results of CCK-8 and flow cy’tometry were both in accordance with the OECD reference range.The intra-group correlation coefficient(ICC)between the two methods was 0.993.3.8 samples were detected to be positive and 1 sample was detected to be negetive by h-CLAT and LLNA:BrdU-ELISA both methods.2 types of freckle cosmetics were judged to be negative by LLNA:BrdU-ELISA method and to be positive by h-CLAT method.The Kappa value of the two methods was 0.42.and the agreement was good.Fisher’s exact probability P value was 0.27(α=0.05).indicating that the difference between the two detection methods was not statistically significantConclusion:1.The results of the 10 verification materials are in line with the OECD reference range,which verifies the reliability of the h-CLAT method established.2.The h-CLAT method and the LLNA:BrdU-ELISA method have good consistency in the results of cosmetic sensitization test,indicating that the h-CLAT method is suitable for the cosmetic samples.It is conceivable to use the h-CLAT method as a screening method to detect the skin sensitization of cosmetics.