| ObjectiveTesting system was constructed for evaluating the skin sensitization of chemicals by using direct peptide reactivity assay(DPRA)and human cell line activation test(h-CLAT)and ROS redox pathway skin sensitizaition testing method was constructed.Sensitivity,specificity and accuracy of chemicals skin sensitization were predicted by single in vitro methods firstly than by integrated test strategy(ITS),which provided prediction and evaluation for unknown substance.MethodSubstance reacted with cysteine peptide and lysine peptide,respectively.Peak areas were detected by High Performance Liquid Chromatography and depletions were calculated.12 chemicals and 10 plant extracts were tested by DPRA.THP-1 cells were cultured.Cells were double-labeled by DCFH-DA and 7AAD and then were exposed to substance of different concentrations.ROS fluorescence intensity was detected.12 chemicals and 10 plant extracts were tested by ROS skin sensitizaition testing method.THP-1 cells were exposed to substance and then were double-labeled by FITC-CD86,PE-CD54 and 7AAD for the detection of fluorescence intensity and the expression of cell surface markers CD54 and CD86.12 chemicals and 10 plant extracts were tested by h-CLAT.Skin sensitization integrated methods were constructed by literature statisitcs.Sensitivity,specificity and accuracy of two integrated methods of battery of test and weight of evidence were analyzed.For the condition of more better for integrated methods for skin sensitizaiton prediction comparing to single in vitro methods,12 chemicals and 10 plant extracts for skin sensitization were classified and were evaluated by battery of test and weight of evidence.Result12 chemicals of 2 non-sensitizers and 10 sensitizers could be distinguished accurately by DPRA.It could be found that the concentration of substance had dose-response relationship with the depletion of peptide.Of 10 plant extracts,8 were sensitizers except brown rice extract which was non-sensitizer.Of 12 chemicals,4 chemicals were non-sensitizers and others were sensitizers by ROS skin sensitization testing method.Of 10 plant extracts,2 were sensitizers and others were non-sensitizers.h-CLAT could distinguish 12 chemicals accurately and 2 were non-sensitizers.For the prediction of plant extracts,4 were sensitizers andothers were non-sensitizers.Referring to the classification of literature and OECD guideline,skin sensitization of chemicals were predicted by two integrated methods by integrating three in vitro methods of DPRA,ROS and h-CLAT.The sensitivity,specificity and accuracy of battery of test were 85.19%,70.59% and 79.55% respectively by the analysis of 44 chemicals.For further classification,8 chemicals were extreme/strong sensitizers,7 were moderate sensitizers,12 were weak sensitizers.The accuracy was 61.36%.DPRA+ROS of weight of evidence had the best prediction potency.The sensitivity,specificity and accuracy were 92.59%,82.35% and 88.64%.The results of application for 12 chemicals and 10 plant extracts showed that 12 chemicals could be distinguished by battery of test for 2non-sensitizers and 10 sensitizers.The results for further classification presented that 3 chemicals were extreme/strong sensitizers,4 were moderate/weak sensitizers,2 were non-sensitizers.The accuracy was75%.All the “2 of 3”weight of evidence could distinguish 10 sensitizers and 2 non-sensitizers.The sensitivity,specificity and accuracy were the same of 100%.For the prediction of 10 plant extracts,the results of battery of test showed that 2 were non-sensitizers and 8 were sensitizers.Further classification,1 was strong sensitizer and 7 were weak sensitizers.ConclusionThe evaluation for skin sensitization of chemicals can be realized by single methods.But the sensitivity,specificity and accuracy for predicting skin sensitization of chemicals can be improved by integrated testing strategy,which shows that suitable integrated methods can improve the prediction potency of xenobiotics and make further risk assessment. |
PDF Full Text Request