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
Objective To establish a detection method integrating DPRA ( direct peptide reactivity assay) with h?CLAT ( human cell line activation test) to screen the skin sensitization potency of chemicals and plant extracts. Methods 12 chemicals and 7 plant extracts were chosen as the test substances. Firstly, the test substances were incubated together with two different peptides ( cysteine and lysine) respectively for reaction for 24 h. The peptide consumptions were analyzed by HPLC. Simultaneously, THP?1 cells were cultured in vitro and then exposed to different concentrations of test sub?stances for 24 h to examine the cell viability, cell surface markers CD54 and CD86 were assessed by flow cytometry. The predicting results were compared further between DPRA and h?CLAT. Results 12 chemicals were distinguished correctly by DPRA classified as 2 non?sensitizers and 10 sensitizers. The results of DPRA were in accordance with h?CLAT. Predic?ting the sensitization potency of plant extracts by DPRA showed that 6 plant extracts were determined as suspected sensiti?zers except for green tea extract. But using the method of h?CLAT, 4 plant extracts were examined as suspected sensitizers except for green tea extract, herba portulacae extract and ginseng fruit extract. The coherence of DPRA and h?CLAT was 0?57. Conclusion This detection method integrating DPRA with h?CLAT can predict single compound accurately. As for complex compound, it can achieve preliminary prediction and need other integrating methods to make a further identifica?tion.