| Albendazole(ABZ)and ivermectin(IVM)are antiparasitic agents commonly used in livestock husbandry industry.With the clinical application of many years,the drug resistance of parasites is increasing gradually.In order to cope with the drug resistance of parasites and improve the efficiency of the two drugs,ABZ and IVM was combined as a good clinical strategy,and good effects were obtained.On this basis,a series of compound contained ABZ and IVM were developed,such as compound tablet(each 0.36g compound containing 350 mg ABZ and 10 mg IVM),compound powder(each 100g compound containing 10g ABZ and 0.2g IVM),compound premix(each 100g compound containing 0.25 g ABZ and 6 g ivermectin).But the clinical adverse reactions like abortion and teratogenicity is frequently reported.So the compound premixed whose ratio of albendazole and ivermectin is fixed at 24:1 and widely utilized is selected to be investigated its dose-response of toxicity and compared with its components.The study is divided into the following two parts:1.The acute and accumulation toxicity study of compound ABZ and IVM(ABZ&VIM)and its components1.1 The acute toxicity study of ABZ&IVM and its componentsThe classical method of median lethal dose was selected to study the acute toxicity of ABZ&IVM and its components by oral administration.The test results showed that the mice in ABZ&IVM and IVM group displayed poisoning symptoms like specific performance is weak,confused haired,drooping eyelids and edema,tremor,coma and severe dehydration after administration of 2?4h and finally died of paralysis.The congestion in brain and lung,volume reducing in spleen was found in gross necropsy.LD50 calculated by the method of probit method(Bliss method)for ABZ&IVM and IVM was 1441.13 mg/kg+60.05 mg/kg and 50.77 mg/kg,and their 95%confidence limit was(1179.46 mg/kg+49.14 mg/kg)?(1722.43 mg/kg+71.77 mg/kg)and 54.03 mg/kg-79.23 mg/kg respectively.Albendazole was given to mice by 6000 mg/kg twice daily orally,6 hours in the period of two dosing,and there were no abnormal clinical manifestations and animal deaths in the period of 7 days.No abnormal was found in gross necropsy.Therefore,it was suggested that MTD>12000 mg/kg.In the acute toxicity comparesion of ABZ&IVM and the single drugs,No significant difference between ABZ&IVM and IVM was observed in statistically but ABZ.So,ABZ was lower toxicic than ABZ&IVM and IVM.1.2 The accumulated toxicity study of ABZ&IVM and its componentsThe fixed dose increasing method was selected to investigate the accumulated toxicity of ABZ&IVM and ivermectin.Cause LD50 of albendazole was unable to obtain,MTD replaced in the same method mentioned before.The accumulation coefficient of ABZ&IVM was calculated to be 4.30,showed a moderate accumulation,but accumulative coefficient of IVM was 2.94 indicated a significant accumulation.With the dose increasing,the mice in ABZ&IVM group and IVM group to appeared drinking decreasing,anorexia,lethargy,tremors,and after continued administration even to be coma and die.Meningeal hyperemia,edema,the smaller spleen and thymus were observed in gross necropsy,In the weight ratio of organ vesus brain,the ratio of liver/brain and testis/brain in the ABZ&IVM group showed increase and decrease respectively compared with control group,the same change of liver/brain was found in IVM single drug group,however,the ratio of spleen/brain and thymus/brain decreased compared with control group.Tissue morphology changes mainly occurred in the brain,spleen and thymus.No death occurred until to the end point in ABZ group except for anorexia and drinking decreasing.In necropsy,the volume of thymus and testis were observed to be smaller than that in control group.Through compared the weight ratio of organ versus brain with control group,the values of liver/brain in ABZ group was higher than the one in control group,and the values of spleen/brain and testis/brain had opposite trends,pathological changes mainly occurred in the thymus and testis,only total protein(TP)and globulin(GLB)of ABZ group in the all the blood biochemical parametres were displayed decreasing statistically.In the all blood biochemical parametres of IVM group,only Cr was observed to increase.The results showed that the accumulation of ivermectin mainly caused by CNS toxicity,spleen and thymus damage and the accumulation of albendazole had mild liver toxicity and serious toxicity in testis,thymus.According to 24:1(ABZ:IVM)prescription of ABZ&IVM can lead to serious accumulation in the central neurotoxicity,can produce toxic effects on the liver and testis mildly.2.The sub-acute toxicity study of ABZ&IVM and its components60 Kunming mice were divided into high dosage group of ABZ&IVM(240.19 mg/kg+10.01 mg/kg),middle dosage group of ABZ&IVM(80.06 mg/kg+3.34 mg/kg),low dosage group of ABZ&IVM(26.69 mg/kg+1.11 mg/kg),IVM group(10.01 mg/kg)and ABZ group(240.19 mg/kg),0.5%sodium carboxymethyl cellulose solution was set as control group.All mice were dosed orally for 30 consecutive days and measured food consumption,drinking water and bodyweight every 7 days.At the end of the experiment,the organ coefficient,clinical pathological parametres,cytokines level in spleen,pathological section were collected.The pexiels in red pulp and white pulp of spleen were as a paremetre W/R.The results showed that there was no abnormality in the mental state of mice in each group.Although there were significant differences in the average daily consumption of food intake and drinking water at different stages of administration,there was no significant difference in total daily food intake and drinking water consumption.In pathology parametres,the white blood cell in the albendazole group was significantly higher than those in the negative control group.The morphological observation showed that the lymphoid follicle in the high-dose group and the white pulp in IVM group developed poorly.The detection of cytokines in the spleen showed down-regulating of INF-? IL-4 and IL-1? and up-regulating of IL-10 in the high dose compound group,up-regulating of IL-1? in low dose compound group,the same trend of IL-1?,IL-4 and IL-10 was found in IVM group.INF-? was down-regulated,IL-4 and IL-1? up-regulated in ABZ group.The results of IgG showed that the level of IgG in the spleen only increased in ABZ group significantly.It was concluded that the mice administrated with ABZ&IVM for 30 days orally were not observed any abnormal reaction.The lymphoid follicle of spleen in high dose group developed poorly,and the mice were immunosuppression,it was because IVM exerted a more potent anti-inflammatory than the inflammatory and effect of ABZ in the ABZ&IVM.3.The genotoxicity study of ABZ&IVM and its componentsAmes test,the micronucleus test and mice sperm teratogenic test were conducted.The results indicated that the compound induced the micronucleus and sperm teratogenic rate to increase In the comparison of the combination and its components,the micronucleus and sperm teratogenic rate of components were higher than that of the combination,That indicated the toxicity was decreased in the combination of albendazole and ivermectin.Meanwhile,both combination and individual drug showed negative test result in Ames-test.These studies suggested that the combination had lower mutagenicity than its components,but it was still a positive mutagenic compound can cause increasing of the micronucleus and sperm teratogenic rate in mouse.Therefore,it is alert to the residue in food and the hazard to environment.4.The reproduction toxicity study of ABZ&IVM and its componentsA total of 168 Kunming mice,sex in half,bodyweight within 25?30g,were selected.The male and female mice were mated by 2:1 and at 16:00 each day,and the female rats were examined at 08:00 in the next morning,so as to confirm whether there was a vaginal plug or spearms were observed in a vaginal smear,the day was recorded as zeroth days(GO).The mating success mice randomized to ABZ&IVM high dose group(240.19 mg/kg+10.01 mg/kg,1/6 LD50),middle dose group(90.07 mg/kg+3.75 mg/kg,1/16 LD50),low dose group(22.52 mg/kg+0.94 mg/kg,1/64 LD50),IVM group(10.01 mg/kg)and ABZ group(240.19 mg/kg),negative control group(0.5%CMC),positive control group(aspirin 250 mg/kg).During the period of G6-G15,pregnant mice were administered by oral gavage,and were sacrificed on G18.All fetal were isolated to test related parametres,the maternal mice were also tested corresponding pareametres.The results showed that the mice during pregnancy were observed vaginal bleeding except IVM and control group.The bodyweight of mice in high dose and ABZ group were significantly lower than those in the negative control group at the eighteenth day of pregnancy,the placenta was significantly lower than that of the negative control group.In the reproductive prifiles of pregnant mice,only the high dose group had a significantly lower rate of live tire compared with control group.The appearance of fetal was mainly refers to tail anomalies,and the skeletal abnormality was sternum and spine deformity,occipital grade distribution and fontanelle width is significantly different from the negative control group,visceral examinations result showed high frequency of cleft palate in each administration except control group,IVM group even was observed high frequency brain hypoplasia.There are alos many cases of cerebral hemorrhage and no eyes IVM group.Histopathological examination revealed placenta labyrinth cells swelling and resulting labyrinth narrow even necrosis in ABZ&IVM high dose group,ABZ and aspirin group,but no obvious abnormality was seen in single drug group and low dose ABZ&IVM.The test results show that the ABZ&IVM at low dose levels was still able to induce abortion,so it should be forbidden for pregnant animal.High dose of ABZ&IVM can lead to live birth rate decline,fetal abnormal appearance,skeletal dysplasia or malformation,but its embryonic development toxicity is lower than that of dose of albendazole,reduced toxicity.It was shown only mild ivermectin variation in maternal toxicity,and no teratogenic effect. |
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