| Reproductive development toxicity assessment is important for drug safety evaluation.Since manual operation takes a large proportion in reproduction toxicity evaluation,it may have the following issues-assay method is not standardized,test results contain many interference factors,evaluation is strongly affected by subjective factors and error of interpretation of results is big.With the development of GLP in our country and progress of advanced hardware facilities and software in domestic and overseas labs,more and more automated instruments are used in reproductive development toxicity evaluation.For example,automatic sperm analyzer to test fertility of males;autonomic activities monitor to evaluate animal behavior for F1 generation;shuttle box and water maze to evaluate learning and memory capabilities for F1 generation.By utilizing these instruments the obtained data is objective and compared with manual operation the operating is easier.In order to enhance the quality of drug safety evaluation and ensure the reliability and credibility of test data,results,conclusions and reports,it is necessary to ensure that those instruments are in control state,especially for the instruments which have complex function and customers can set parameters by themselves,and used for data generation,measurement,evaluation,handling and archival,complete qualification must be applied.In this study,in order to confirm whether expected use purpose can be met and reliable data can be obtained,qualification was done for 3 instruments under the condition of our labTOX IVOS sperm analyzer,shuttle box and autonomic activities test system.And qualified instruments were used to conduct reproductive development toxicity evaluation for a monomeric compound NCS-010 derived from Traditional Chinese Medicines.Pare ? Related instruments qualification in reproduction toxicity safety evaluation study under GLP requirementsStudy I,TOX IVOS sperm analyzer:in this study,in order to ensure the instrument is operating properly and reliable test data can be obtained,installation,operation and performance qualification were performed for TOX IVOS sperm analyzer.Installation Qualification:verify whether the instrument is installed properly and verify whether working environment can meet the instrument operating requirements through check the instrument transportation list and test the lab environment.Operation Qualification:ensure operating personnel can handle the instrument properly through provide training on hardware and software and check training attendance list.Performance Qualification:use standard sample and rat sperm separately,to verify whether TOX IVOS's performance is proper.In order to verify whether the instrument's basic counting is accurate or not,standard sample test was conducted.2 levels of concentration of accu-beads were obtains(35mol/mL and 18mol/mL),for each level 2 samples were collected,sample concentration was tested and mean value was calculated.Then compared the test results with the standard values;10 adult male rats,obtained sperm from epididymis,tested the concentration and dynamics of rat sperm.To verify the instrument's accuracy of sperm counting and rate calculation by comparing manual count with machine reading and comparing manual rate calculation with instrument calculation.In addition,after low temperature treatment of samples,compared them with the ones had not been low temperature treated,in order to verify whether the instrument has the capacity to test or not.Qualification results:after verified instrument components against the transportation checklist,the instrument components were found to be complete;the installation environment was-temperature 25?,humidity 39%,power 220V,ventilation status was good.The installation standards were met.The instrument engineer had provided an introduction of relative hardware and given a training session to the staff members who were going to use the instrument on software,including rat mortality,animal mortality,rat matrix and oval matrix.The scale length was 10?m,and the instrument measurement was accurate.It can document operation related records.In the range between0.035 mol/Lą0.005 and 0.018 mol/Lą0.0025,3 tests were done for standard samples,and tested results were in the normal concentration range.It indicated that the count function of the instrument was accurate.The error ranges for the tested 10 rats' sperm total concentration,movement sperm concentration,total sperm count and movement sperm count were 0.23?0.49%,0.19?0.74%,1.75?6.67%and 0.00?4.00%separately.The error ranges were acceptable.Based on motion trail,the results of manual calculated VSL,VCL,VAP,ALH and BCF were the same with the ones calculated by the instrument,and all errors were 0%.It indicated that the instrument's calculation on relative parameters of sperm movement rate was accurate.Compared with the control group,movement rate for the sperms which were low temperature treated was significantly decreased,approximately decreased by 29.9%.It indicated that the instrument had the capability to test those factors which affect the movement of sperm.Study ?,performed 3Q qualification(IQ,OQ and PQ)for shuttle box in order to ensure the system was able to conduct animal active/passive avoidance reaction tests.System installation was completed properly under Microsoft Windows XP operating system.After confirming that the system or facility was in normal operation state,relevant information and data were documented to demonstrate that the system and facility can realize expected functions.In order to ensure that the instrument's overall performance was in normal state,4 SD rats which had been trained for 7 days were used to conduct performance qualification.Shutt Box system to be qualified in this study contained 4 shuttle boxes,during this study the instrument was used to record the number of times that animal suffered electronic shock in the 4 boxes separately,in the meanwhile human observation was applied as well.The results between instrument observed and human observed remained the same in general.It indicated that the qualification is successful.Qualification result for learning and memory model indicated that through 6-day shuttle Box training,3 rats can avoid electronic shock on the 7th day and another rat which cannot avoid electronic shock during the 6-day training but could actively avoid electronic shock on the 7th day.Study ?,performed operation and performance qualification for autonomic activities monitor.5 animals were chose and were tested separately in every time point,one time each and one by one.Removed the feed and water bottle from animal boxes,and placed animals in the boxes,one per each.Placed the boxes in the test position for 5 minutes.Then documented the times of activities for each mice in 5 minutes.The results showed that 'Activity Monitoring System' software can complete all the required operations by customers.However the data collected by No.8 probe had some problems and it was recommended not to use No.8 probe in the test.Conclusions were made through the above 3 studies:under the current lab conditions,TOX IVOS sperm analyzer and Shuttle Box can be operated properly and can generate reliable test data.7 out of 8 probes of autonomic activities monitor system can be used for autonomic activities test with the cooperation of Activity Monitoring System' software and can generate reliable data.Part ? NCS-010 reproduction toxicity safety evaluation studyNCS-010 is a monomeric compound derived from Traditional Chinese Medicines.The study conducted in a GLP lab is a reproductive toxicology safety evaluation study for the test article.It consists of 3 sub-studies.Sub-study ?-impact on embryo/fetal development of SD rats by oral perfusion of NCS-010.80 successfully mated SD rats were divided into 4 groups randomly,20 each.From gestation day 6(GD6)till GD17,they were exposed to NCS-010 0mg/kg,60mg/kg,250mg/kg,1000mg/kg respectively by oral perfusion,once per day.Autopsy was done on GD20.Observational indicators included:observation of clinical symptoms for maternal animal,body weight change,food ration,number of corpus luteum for maternal animal,number of implantation,embryo survival and death state,weight of survival fetal rats,gender,appearance,skeleton and internal organs test indicators.Results:Changes of embryo/fetal development for SD rats in relation to NCS-010 were not observed.Sub-study ?-impact on fertility and early embryo development of SD rats by oral perfusion of NCS-010.160 rats were divided into 4 groups(0mg/kg,30mg/kg,150mg/kg,750mg/kg),20 males and 20 females each.Females were exposed to the test article(NCS-010)from 2 weeks before mating till GD7 continuously.Males were exposed to the test article from 4 weeks before mating continuously.Impact on fertility and early embryo development of SD rats was observed.Observational indicators included:clinical symptoms,body weight,food ration,fertility state(number of sexual cycle,average days of sexual cycle,number of days before mating,mating rate,pregnancy rate for females,fertility rate for males,movement rate for sperm,total number of sperm and malformation rate for sperm),weight of internal organs(testis,epididymis,seminal vesicle,ovary)and early embryo development(number of corpus luteum,number of implantation,number of survival embryo,number of empty implantation,number of embryo fetal deaths,rate of implantation,rate of live fetuses,rate of embryonic death,pre-implantation loss rate and post-implantation loss rate)and etc.Through this study it was confirmed that maximum nontoxic dose for parental animal and nontoxic reaction dose for embryo were both 750mg/kg.Sub-study ?-evaluate toxicity on SD rats in perinatal period by oral perfusion of NCS-010.87 pregnant rats were divided into 4 groups-vehicle control group(21 rats),low dose group(22 rats),middle dose group(21 rats)and high dose group(23 rats).They were exposed to vehicle and test article NCS-010 Omg/kg,60mg/kg,250mg/kg,1000mg/kg respectively by oral perfusion from GD15 to the end of lactation period.Observational indicators included:body weight changes for female animals,food ration,clinical symptoms,delivery state,females implantation number,newborns(F1 generation)survival state,body weight of live newborns,gender,appearance,physical development,sense organ function,moto ability,sexual maturity,fertility,behavior and memory and etc.Resultes:Impact on the development of SD rats before and after birth in relation to NCS-010 was not observed.Conclusions:In this study,NCS-101 was administered to SD rats through oral perfusion from GD15 to the end of lactation period.Maternal toxicity and embryo/fetal development toxicity were not observed.In addition,for maternal toxicity and embryo/fetal development toxicity,the nontoxic reaction dose was 1000mg/kg. |
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