Determination Of Arprinocid In Feed And Acute, Subchronic Toxicity Of Arprinocid

Arprinocid is a nucleoside analogue applied as a coccidiostat in chickens and turkeys to prevent and treat coccidiosis. Arprinocid treatment protects broilers against the effects of Eimeria tenellum and has no cross resistance with other coccidiostat. There were no reports on sale and use of arprinocid in our country, little information is available about the toxicological effects of arprinocid, in order to evaluate the safety of clinical use of arprinocid, preclinical toxicity (acute and subchronic) studies were conducted. A HPLC method for the determination of arprinocid in feed was developed and validated. Acceptable daily intake (ADI) and Maximum residue limits (MRLs) were calculated based on the results of subchronic toxicity study. The results are as following:1. Acute toxicity studyTo obtain the LD50 of arprinocid in rats and mice,5 adult SD rats/sex/group and 5 adult Kunming mice/sex/group were orally given once by gavage of arprinocid suspended in 0.5% carboxymethylcellulose sodium(CMC-Na) after an overnight fast. After dosing the animals were observed for 14 days. LD50 values and 95% confidence intervals were calculated according to modified Karber’s method. The results showed that the LD50 values of arprinocid were 82.8,378.7 and 442.9 mg/kg b.w./day with 95% confidence intervals ranging from approximately 73.2 to 93.5,344.7 to 416.1 and 416.5 to 470.9 mg/kg b.w./day in mice, male and female rats, respectively. It was indicated that arprinocid is a moderately toxic substance.2. Development of a HPLC method for the analysis of arprinocid in feedA rapid, sensitive and reliable HPLC method for the determination of arprinocid in feed is developed and validated. Grinding feed is extracted by methanol and was cleaned up with n-hexane. Chromatographic separation is performed on a C18 reversed-phase column with UV detector at 261nm with the mobile phase of water containing 40% acetonitrile. The LOD and LOQ are 0.72 mg/kg and 1.8 mg/kg, respectively. The method has a good linearity in a range between 18～600 mg/kg, with related coefficient of r2=0.9996. The recoveries range from 85.7% to 99.2%, with the relative standard deviations between 1.9% and 3.8%.3. Subchronic toxicity studyTo investigate subchronic oral toxicity of arprinocid, male and female rats were fed with diets supplemented with 0 (control),25,187.5 or 500 mg/kg arprinocid for 13 weeks followed by a recovery period of 2 weeks.10 rats/sex/group were sacrificed under pentobarbital sodium anaesthesia on days 92,93. The rest was sacrificed after fed with basal diet for two weeks. Significantly lower cumulative body weights were noted in the 500 mg/kg group females. Significant differences in haematological and biochemical parameters as well as organ weights were detected between the test groups. Histopathological observations revealed that 500 and 187.5 mg/kg arprinocid could induce hepatic steatosis and necrosis of liver cells, protein tube of tubule and injury of tubular epithelial cells. The results of the present study demonstrated that the liver and kidney are possibly the main target organs of arprinocid, we conclude that the dietary no-observed-adverse-effect level (NOAEL) of arprinocid for 13 weeks is 25 mg/kg in SD rats (approximately 2 mg/kg b.w./day).4. Determination of ADI and MRLsWith NOAEL of the aforementioned subchronic toxicity study, ADI and Maximum residue limits were calculated according to method of FDA. ADI was calculated to be 0.002 mg/kg b.w. MRLs of arprinocid for muscle is 0.4μg/g, and that for liver is 1.2μg/g and both that for kidney and fat are 2.4μg/g. Tissues residues of arprinocid detected in tissues of animals given arprinocid in diet were lower than the corresponding MRLs of all the above tissues. Therefore, arprinocid possess a good safety.The aforementioned toxicity studies were basically designed and conducted according to the Technological Requirement for General Toxicity Test of New Veterinary Drug published by MO A in 1991 and corresponding national standards published by National Standardization Management Committee, and were standardized and improved by referring to Toxicological Principles for the Safety Assessment of Food Ingredients published by FDA. The acute and subchronic oral toxicity of arprinocid were systematically evaluated, A HPLC method for the determination of arprinocid in feed is developed. The results showed that arprinocid is a moderately toxic substance and possess a good safety. Results of these studies can be used as scientific evidence for approval and application of arprinocid in food-producing animals.