Abstract:Objective 　To establish a method for an in vitro test for skin sensitization based on ARE-Nrf2 (antioxidant responsive element - NF-E2-related factor 2) luciferase, and using this test to assess the sensitizing potency of chemicals and sample mixtures. Methods 　A stable transfection of the HaCaT cell line containing a selective plasmid encoding AKR1C2-ARE fused to the fluorescent luciferase was constructed (DSens), and the cells were incubated with samples for 48 hours at different concentrations. The expression of luciferase was measured by fluorescence detection. Skin sensitization was predicted for 16 known skin sensitizers and six mixtures. Results 　Sixteen reference substances were accurately distinguished by DSens. Among the six sample mixtures, the prediction result of five mixtures were consistent with the result of other detection method , and another unknown sample was predicted to be positive. Conclusions　The DSens in vitro testing method may be a useful replacement for some animal tests used to predict sensitivity to soluble skin products.

Abstract:Objective　To detect 25% cyhalofop-butyl emulsifiable concentrate(EC) skin sensitization by a local lymph node assay (BrdU-ELISA) in mice. Methods　Female BALB/ C mice were tested and the samples were set up to 100%, 50%, and 25%. The negative control group received acetone: olive oil (AOO)= 4 ∶1, and the positive control group received 25% basal cinnamaldehyde. Test samples were laid on the dorsum of bilateral mouse ears for three consecutive days, then the mice received an intraperitoneal injection of BrdU (5 mg/ mouse) on the sixth day. After 24 hours, the lymph nodes were removed, weighed, and mashed into a cell suspension. Lymphocyte proliferation was measured by BrdU-ELISA. Results　The body weights of female mice in all treated groups were similar to the control group. The increase of ear thickness and ear weight in each dose group was not more than 25%. The stimulus indexes of solvent and blank control groups were SI1and SI2< 1. 6, and the stimulation indexes of the positive control group (25% hexyl cinnamaldehyde) were SI1and SI2 > 1. 6. This indicated the test system was reliable. The stimulation indexes of 25%cyhalofop-butyl EC in the low, medium and high dose groups were SI1and SI2 < 1. 6, and there was no dose-response relationship. Conclusions 　The results of 25% cyhalofop-butyl EC in mouse local lymph node assays were negative,indicating that 25% cyhalofop-butyl does not cause skin sensitization.

Abstract:Allergy contact dermatitis is a type IV delayed type hypersensitivity that is induced by exogenous compounds and involves many cell types. Traditional animal testing uses guinea pigs or mice as a model. With progress of the adverse outcome pathway (AOP) on skin sensitization, a concept for development of alternative methods based on a molecular initiating event and key events is provided. Dendritic cell (DC) activation plays a key role in the AOP. Many alternative methods have been developed, with several methods validated and accepted as guidance for assays. This paper examines DC screening, characteristics of test parameters, and limitations and applicability of DC-derived methods. Progress on interactions between DCs and other cells, co?culture systems, and the human body-on-a-chip will also be introduced. Altogether, this paper will provide information for optimization of in vitro alternative methods for sensitization detection.

Abstract:【Abstract】: Objective Construction of h-CLAT testing method based on THP-1 cell line to build a assessment plans for skin sensitization powerful potency of chemicals, personal care products and plant extract. Method THP-1 cells was cultured in vitro and exposed to substances that were 11 reference skin sensitization chemicals and 9 samples,by monitoring the cell viability, cell surface marker CD54 /CD86 and relative fluorescence intensity of cells surface after the cells was exposures substances, to discover whether positive. Simultaneously, Buehler test was used to validate the results from h-CLAT. Results 11 reference chemicals were distinguished correctly by h-CLAT, subsequently, the 7 samples were recognized negative and 2 samples were perceived positive. The qualitative classification of sensitization was corresponded to the results of h-CLAT in 9 samples. Conclusion the h-CLAT-in vitro test methods can be used to replace some of the animal tests for the prediction of soluble skin allergy

Abstract:Objective To establish an in vitro skin sensitization test, human cell line activation test ( h-CLAT), based on THP-1 cell line (a human acute monocytic leukemia cell line), and to assess the sensitizing potency of plant raw materials of chemical and cosmetic products by this in vitro skin sensitization test. Method THP-1 cells were cultured in vitro and exposed to 11 reference skin sensitization chemicals and 9 samples, by monitoring the cell viability, cell surface marker CD54/CD86 and relative fluorescence intensity of cells surface after the cells was exposures to the substances, and to discover whether there is a positive reaction. At the same time, Buehler test was used to validate the results of samples tested by h-CLAT. Results 11 reference chemicals were distinguished correctly by h-CLAT. Among the 9 samples tested, 7 samples were recognized as negative sensitizer and 2 plant extracted substances were identified as suspicious skin sensitizer. The qualitative classification of the 9 samples by h-CLAT test was consistent with the results obtained by animal test. Conclusions The h-CLAT-in vitro test can be used to replace some animal tests for the prediction of soluble skin sensitizing substances.

Abstract:Skin sensitization (allergic contact dermatitis, ACD), is a serious condition caused by small reactive molecules and is characterized by a delayed-type hypersensitivity. Animal tests were usually used in the evaluation of sensitizing potential of chemical substances in the past. However, there is an increasing interest from the public for reducing and ultimately replacing animal tests. The European Union (EU) has posed a ban on animal testing of cosmetic ingredients that includes skin sensitization since 2013. Therefore, alternative in vitro tests are the main tendency in chemical substances and cosmetic sensitizing potential research in the future. In this study, different kinds of in vitro test methods that were adopted by OECD or on research(LLNA, DPRA, KeratinoSens TM, h-CLAT)were reviewed through recent years literature, comprehensive introduction and evaluation were made to obtain reliable hazard and potency information on potential skin sensitizers.