| Micronucleus test is the most widely used method of in vivo genetic toxicity testing and can be used for the detection of genotoxicity of various target organs.The liver micronucleus test method is a target organ test method that is more in-depth than the hematopoietic system micronucleus test because the micronucleus test can detect metabolites as transient carcinogens compared to the hematopoietic system micronucleus test.Precursor carcinogens.At the same time,the liver is one of the most important metabolic organs in the human body.A variety of genetic toxicants can cause liver toxicity.Therefore,at the 6th International Conference on Genetic Toxicology in 2014,there were also more and more tests on the liver micronucleus test method as the technical requirement for the registration of human use drugs.The International Harmonization Association(ICH)S2(R1)The second combination test was available.Discussions of in vivo tests performed on tissues other than bone marrow and peripheral blood,as well as the recommended test procedures for hepatic micronucleus tests were also presented at the work conference.However,the liver micronucleus test needs a lot of validation before it enters into formal use.As early as 2004,the joint research group of the micronucleus test method in the environmental mutagenicity/mammalian mutagenicity research group in Japan has taken the lead in carrying out international joint validation of the micronucleus test method for juvenile rat liver.Completed in 2010.The joint verification of the repeated administration of hepatic micronucleus test method started in 2014 and most of the data have been reported,which laid the foundation for the future development of the guiding principles for this trial.Comparing with the verification of liver micronucleus test conducted by many laboratories in foreign countries,the domestic research on this test has just started.The purpose of this project is to establish a method for repeated administration of hepatic micronucleus in rats and to verify the feasibility of this method.At the same time,the ICH S2(R1)guidelines recommend that different genotoxic end points be combined in the same test in vivo and try to combine them with general toxicity tests to greatly increase test efficiency.The experimental design is close to the 3R principle.Therefore,this topic will be further tried.The hepatic micronucleus test method is combined with the liver comet assay,peripheral blood micronucleus test and Pig-a gene mutation test to conduct in vivo genetic toxicity testing,to improve the test efficiency and avoid false positive or false negative results.This topic is divided into two parts: The first part is based on the internationally validated method of repeated administration of hepatic micronucleus test.The rat is tested using the validated liver micronucleus positive compound N-nitrosodiethylamine(DEN).Repeated administration of hepatic micronucleus test method was established to initially verify the sensitivity and reproducibility of the method.At the same time,hepatic micronucleus test was combined with hepatic comet assay and peripheral blood micronucleus test in the same experiment to jointly characterize the genotoxicity information of DEN.Try to establish an in vivo genetic toxicity multi-end test method.The second part mainly uses four mechanisms with different mechanisms: auramine,o-aminoazotoluene,1,3-propane sultone,and methyl carbamate,and repeated administration of hepatic microinjection to rats with DEN as a positive control The nuclear test method is used for the verification;in the verification of the liver micronucleus method,combined with the liver comet assay,the peripheral blood micronucleus test orThe Pig-a gene mutation test was used to detect the genotoxicity of each test substance,to maximally characterize the genotoxicity information of the test substance,and to further verify the accuracy and reproducibility of the in vivo genetic multipleendpoint test.The main results obtained by this study are as follows:1.In the establishment phase of a repeated hepatic micronucleus test in rats,the establishment of the method was performed using a known genotoxic positive compound DEN.The enzymatically digested and fixed hepatocyte suspensions were stained and counted under a fluorescence microscope to count the number of micronuclei-containing cells.The results of the hepatic micronucleus test of DEN were consistent with the international joint verification: the DEN medium-dose and high-dose group liver The micronuclear rate(%MNHEP)in the cells showed a statistically significant increase compared to the negative control group and showed a dose-response relationship;the liver comet assay results of the DEN were also consistent with the results in the literature,compared to the negative control group.There was a statistically significant increase in mean DNA tail density in the DEN low,middle,and high dose groups,and in the peripheral blood micronucleus test,the micronucleus rate of reticulocytes in the DEN low,medium,and high dose groups(%MN-RET)was not statistically different from the negative control group and was consistent with the literature results.Therefore,this experiment successfully established a rat liver micronucleus test repeatedly,and proved the feasibility and reproducibility of the in vivo multi-terminal test method combined with liver micronucleus test,hepatic comet assay and peripheral blood micronucleus test.2.In the verification phase of repeated hepatic micronucleus test in rats,the amnion’s hepatic micronucleus test and hepatic comet assay were positive,and the results of peripheral blood micronucleus test and Pig-a gene mutation test were negative.The results of this substance suggest the necessity of hepatic micronucleus test compared with peripheral blood micronucleus;results of liver micronucleus test,liver comet assay and Pig-a gene mutation test of o-aminoazotoluene are negative,only The results of peripheral blood micronucleus were positive;the results of hepatic micronucleus test,hepatic comet assay,and peripheral blood micronucleus test of 1,3-propane sultone were all positive;the liver micronucleus test and hepatic comet test of methyl carbamate were The results of the peripheral blood micronucleus test were negative,so methyl carbamate was identified as a non-genotoxic compound.The preliminary conclusions are as follows:1.The result of liver micronucleus test of DEN is positive,which is consistent with the result of international joint verification.Therefore,this laboratory has initially established a method for repeated administration of hepatic micronucleus in rats.Meanwhile,hepatic micronucleus test,hepatic comet test and peripheral examination The micronucleus test combined with the evaluation of the genotoxicity of DEN,comet assay and peripheral blood micronucleus test results are consistent with the reports,initially established in vivo genetic toxicity multi-endpoint detection method.2.The method of repeated administration of hepatic micronucleus in rats using four different mechanisms was verified by the method.The result of hepatic micronucleus test was in line with the expectation,which proved that the method had higher sensitivity and repeatability.3.Simultaneous hepatic micronucleus test,hepatic comet assay,peripheral blood micronucleus test and Pig-a gene mutationThe test,except for the results of the comet assay of o-aminoazotoluene,is inconsistent with the reported results,probably due to low dosesThe sub-exposure test method had an effect on the results of the comet assay;the Pig-a gene mutation test was performed on the auramine and o-aminoazotoluene.The results were negative,and the metabolism of auramine and o-aminoazotoluene.liveThe fact that the sexual product is less exposed in peripheral blood is consistent with the fact that other known genotoxic end-point results are consistent with previous reports,demonstrating the accuracy and reproducibility of the multi-endpoint detection method for genotoxicity in vivo.In summary,(1)the study successfully established a rat liver micronucleus test method repeated;(2)selected four different mechanisms of the compound to further verify the rat repeated administration of hepatic micronucleus test method;(3)Simultaneous establishment of multiple endogenous genotoxicity assays in vivo,laying the foundation for the future in vivo genotoxicity testing of hepatic micronucleus tests with other endpoints or in combination with general toxicological studies. |
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