| Aim:The main test conditions of in vivo comet assay were discussed to establish an in vivo comet test method and standard operating procedures in GLP laboratory.The bacterial reverse mutation test,chromosome aberration test,in vivo micronucleus test and in vivo comet assay were used for comprehensive evaluation the genetic toxicity of typeⅠ innovative drug TJ1933 to provide a basis for preclinical safety evaluation and IND application data.Method:1.In vivo comet assay research: The livers of healthy male SD rats were used to prepare hepatocyte suspension,and select the most suitable test conditions by comparing three agarose concentrations,four unwinding times and three electrophoresis times.Healthy male SD rats were randomly divided into a vehicle control group and a positive drug group according to their body weight.The vehicle control group was given physiological saline,and the positive drug group was given ethyl methanesulfonate by gavage at 21 hours intervals.The drug was administered twice,and the hepatocyte suspension was prepared 3 hours after the last administration.The genotoxicity of EMS was tested according to the most suitable test conditions.2.Ames: We used plate incorporation method.Ames strains TA97,TA98,TA100,TA102 and TA1535 were used for non-metabolism activation(-S9)and metabolic activation(+S9)reversion tests of 5 test concentrations,1 vehicle control group and 1positive group.Each group had 3 parallel dishes and was repeated once.No reagents were added to the blank control group,dimethyl sulfoxide(DMSO)was added to the vehicle control group,and the positive drugs were Dexon 50 μg/dish,sodium azide(Na N3)1.5 μg/dish and 2-aminoanthracene(2-AA)5 μg/dish.We counted the number of colonies that were visible to the naked eye in each group.3.Chromosome aberration test: CHL cells were choosed to conduct 4-hour exposure non-metabolic(-S9)activation test(15 μg/m L,30 μg/m L and 60 μg/m L three doses),24-hour exposure non-metabolic activation(-S9)test(10 μg/m L,20 μg/m L and 40μg/m L three doses)and 4-hour metabolic activation(+S9)test(50 μg/m L,100 μg/m L and 200 μg/m L three doses).One vehicle control group and one positive control group(The 4-hour metabolic activation group was given 20 μg/m L cyclophosphamide and the other two groups were given 0.25 μg/mL mitomycin)were set up separately.After harvesting the cells,we prepared chromosome specimens.After staining,we observed300 well-dispersed metaphase cells in each dose group under the oil microscope,recorded the number of cells containing structural aberrant chromosomes and the type of aberration,and calculated the occurrence rate of aberrant cells.4.In vivo micronucleus test: Sixty healthy SD rats with half male and female were randomly divided into vehicle control group,low-dose group,medium-dose group,high-dose group and the positive drug group according to their body weight,twelve per group,half male and half female.In the vehicle control group,0.5% sodium carboxymethylcellulose(0.5% CMC-Na)solution and each dose group of TJ1933 were administered intragastrically for two days,once a day,with a volume of 10 ml/kg.The positive control group received a single intraperitoneal injection of 40 mg/kg of cyclophosphamide(CTX)with a dose volume of 5 ml/kg.The bone marrow smear specimens of positive drug group were made about 24-hour after the administration,and the other groups were made 18-24 hours after the last administration.We observed 4000 polychromatic erythrocytes(PCE)per animal,recorded the number of micronuclei and calculated the micronucleus rate of polychromatic erythrocytes.We observed 500 erythrocytes per animal and counted the number of polychromatic erythrocytes in the field of view(PCE)to determine the proportion of polychromatic red blood cells to red blood cells(PCE/500).5.In vivo comet assay: Twenty-three male rats were randomly divided into vehicle control group(0.5% CMC-Na solution),low-dose group,medium-dose group,high-dose group and the positive drug group(EMS)according to their body weight.The positive drug group had 3 rats,and the other groups had 5 rats.Each group was given intragastric administration twice with an interval of 21 hours.Hepatocyte suspension was prepared 3 hours after the last administration.After preparation,lysis,unwinding,electrophoresis,neutralization,and dehydration,it was stained and photographed under a fluorescence microscope.Use CASP analyze the percentage of tail DNA(Tail DNA%)to detect the DNA damage ability of TJ1933.Results: 1.Use 0.75% normal melting agarose and low melting agarose solutions to spread the sheet,unwind for 20 minutes and electrophoresis for 20 minutes are suitable test conditions.After testing,the result of EMS genotoxicity was positive.2.Under the conditions of metabolic activation and non-metabolic activation,the number of colonies in each dose group of TJ1933(concentration 20～2000 μg/dish)was within the normal range and there was no concentration-dependent increase and/or any a concentration can be increased repeatedly.3.4-hour of non-metabolic activation of low and medium doses,4-hour of metabolic activation of low,medium,and high doses and 24-hour of non-metabolic activation of low,medium and high doses did not cause significant chromosomal aberrations,while4-hour of non-metabolic activation high dose can cause chromosome aberrations.4.Compared with the vehicle control group,there is no significant difference between the ratio of bone marrow polychromatic erythrocytes and the micronucleus rate of bone marrow polychromatic erythrocytes in any dose of the innovative drug in female and male rats.5.Compared with the vehicle control group,the innovative drugs of any dose group have no significant difference in the damage of rat liver DNA.Conclusions: 1.This project successfully established an in vivo comet test and verified it successfully.2.After comprehensive evaluation of bacterial reverse mutation test,chromosome aberration test,in vivo micronucleus test and in vivo comet test,no potential genetic toxicity of TJ1933 has been found. |
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