The Study Of Reproductive And Reratogenic Toxicity For A Newanticoccidial Triazine Drug Ethanamizuril

In order evaluate the safety of the ethanamizuril in reproductive toxicity, the paper conducted the following four studies: 1. The detection of ethanamizuril in rat feedA High Performance Liquid Chromatography(HPLC) method was developed to detect and quantify ethanamizuril in rat feed. Feed samples were first extracted with acetonitrile and degreased with n-hexane, then the extracts were separated with a C18 column and detected by a UV detector at 251 nm. Their contents were determined by normalizing with the internal standard(IS) 2-methyl-4-nitroanisole. Results show that ethanamizuril and 2-methyl-4-nitroanisole were well separated under our analytical conditions. The linear dynamic range of ethanamizuril is from 1 to 50 mg/kg and the limit of detection(LOD) is 1 mg/kg. The method is simple, rapid, accurate and reproducible for quantification of ethanamizuril in feed. 2. Two generation reproduction toxicity studyTwo generation reproduction test was performed to investigate the potential reproductive toxicity of Ethanamizuril. 240 SPF Wistar rats(50% male and 50% female) were divided into four groups, with each group containing 30 males and 30 female rats. Four groups were administrated with different dose of Ethanamizuril 0, 2, 8 and 30 mg/kg, respectively. The weight, fodder intake and reproductive indexes of parent and offspring rats were analyzed statistically, reproductive organs in parent rats were histopathologically examined as well. Results show that the body weights of rats were positively correlated to feed efficiency, no matter in treated groups or the control group, and also no drug-related pathological changes of reproductive organs were observed in treated rats. The reproductive indexes of these four groups were almost the same, except for some individual cases, such as the relatively low survival rate in F1 group. In the treated group with Ethanamizuril 30 mg/kg, the weights of offspring rats were obviously lower, suggesting that drugs may affect the development of offspring rats through breast milk. 3. Teratogenicity testFeeding teratogenicity test was combined with the second generation reproduction test in order to carry out teratogenicity test. 240 SPF Wistar rats(50% male and 50% female) were divided into four groups, with each group containing 30 males and 30 female rats. Four groups were administrated with different dose of Ethanamizuril 0, 2, 8 and 30 mg/kg, respectively. The anatomy of F1 with pregnancy for 20 days is conducted to check F2 b generation for teratogenicity test. Experimental results show that the fetal rats’ average weight in 30 mg/kg group is significantly lower than that of the blank group(P < 0.01), while other indexes did not differ distinctly with the control group. All groups do not reveal any bones or internal organs deformity associated with drugs, indicating that Ethanamizuril has no obvious influence in fetal development. 4. Determination of drug concentration in plasma of ratsWe have established an ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS) method to quantify ethanamizuril in rat plasma. Samples were extracted with acetonitrile, separated through BEH C18 column with acetonitrile-0.1% formic acid as the mobile phase. Ethanamizuril was measured by selected ion monitoring(SIM) mode under negative ion mode, and quantified with IS 2-methyl-4-nitroanisole. This approach is confirmed to meet the specificity, accuracy, precision stability and other requirements for ethanamizuril quantification. In a 30-day sub-chronic toxicity test, samples were collected at 0、1、2、3、28、29、30 and the 7th days(the recovery day). In reproductive toxicity test, samples were collected in recovery phase and then quantified the drug concentration in plasma. Results show that the drug concentration in rats could remain stable for a long time after being fed drugs. In reproductive test, the weights of weanling rats in 30 mg/kg group were obviously lower than that of the control group, which is ascribed to toxicity of drugs and that can be further proved by the higher drug concentrations in offspring rats than in their mothers’, indicating that lactation may be the main route for drug metabolism.To sum up, our preliminary results confirm that the new medicine ethanamizuril has no effect on the reproductive process and the development of organs and skeleton of offspring rats. Only when being fed with high-dosage drugs(30 mg/kg), the reproduction of mother rats may be affected and presented on the abnormal weight of offspring rats.