【作者】 张伟；

【导师】 袁宗辉；

【作者基本信息】 华中农业大学 ， 基础兽医， 2007， 硕士

【摘要】 喹噁啉类化合物是一类具有抗菌促生长作用的药物，自上世纪70年代以来被广泛的使用。喹乙醇和卡巴氧是本类的代表产品，主要作为饲料添加剂以50～100mg／kg的浓度用于食品动物，但因对动物具有不同程度的毒副作用而被禁用或被严格限制使用。喹烯酮为新一代喹噁啉类抗菌促生长剂。为评价喹烯酮的安全性，进行了系统的临床前毒性试验。测定喹烯酮的急性毒性，致突变性，亚慢性毒性，喂养致畸毒性和繁殖毒性，计算ADI（每日容许摄入量）和其在动物性食品中的最高残留限量。为喹烯酮的开发应用提供毒理学安全性评价依据。1急性毒性试验Wistar大鼠和昆明小鼠各60只，雌雄各半，喹烯酮用0.5％羧甲基纤维素钠（CMC）助溶制成混悬液，同时设同类药物喹乙醇、卡巴氧和痢菌净作为对照，采用一次性灌胃染毒，观察14d内的中毒反应，用寇氏法计算半数致死量和95％的可信限。结果表明，喹烯酮对Wistar大鼠和昆明小鼠的经口LD₅₀分别为8687.31和15848.93mg／kg b．w．；喹乙醇、卡巴氧和痢菌净对Wistar大鼠经口LD₅₀分别为1877.63、878.01和562.34 mg／kg b．w．，对昆明小鼠经口LD₅₀分别为3677.56、2815.51和694.44 mg／kg b．w．。按照急性毒性分级标准喹烯酮为实际无毒物质，喹乙醇、卡巴氧和痢菌净都为低毒物质。2遗传毒性研究鼠伤寒沙门氏菌回复突变（Ames）试验试验菌株为TA₉₇，TA₉₈，TA₁₀₀，TA₁₀₂，TA₁₅₃₅，TA₁₅₃₇。试验剂量为1.0，2.6，6.9，18.2，50.0μg／皿，用已知的阳性物作阳性对照，同时设溶剂对照（二甲亚砜（DMSO））和空白对照（蒸馏水），加和不加S₉混合液的对照。每个处理作3个平行平皿，采用平皿掺入法，计数各皿回变菌落数，结果评价为计算各处理平均每皿诱发回变菌落数与相应的空白对照组的平均每皿自发回变菌落数的比值（MR），如MR大于2为阳性，小于2为阴性。试验结果表明，喹烯酮对TA₁₀₂和TA₁₅₃₅加与不加S₉时都为阴性；TA₉₇加和不加S₉在剂量≥6.9μg／皿时均为阳性；TA₉₈加和不加S₉在剂量为50.0μg／皿时均为阳性；TA₁₀₀加和不加S₉在剂量≥18.2μg／皿时均为阳性；TA₁₅₃₇加S₉在剂量≥6.9μg／皿时为阳性，不加S₉在剂量≥18.2μg／皿时为阳性。作为对照药物，喹赛多的结果与喹烯酮基本相似，喹乙醇、卡巴氧和痢菌净对各菌株均为阳性结果，并呈一定的剂量-反应关系。小鼠骨髓细胞微核试验昆明种小鼠按体重随机分组，每组10只，雌雄各半，试验分组分别为溶剂对照组（0.5％CMC），喹烯酮组（1700，3600，7200mg／kg b．w．），阳性对照组（环磷酰胺（CP） 40mg／kg b．w．），并设药物对照喹赛多组（3600，6000，10000mg／kg b．w．）、喹乙醇组（400，700，1700mg／kg b．w．）、卡巴氧（350，700，1400mg／kgb．w．）和痢菌净（90，175，350mg／kg b．W．）。各组经口灌胃给药2次，间隔24h，于末次给药后6h采样制片。每只小鼠计数1000个嗜多染红细胞，计算含微核的嗜多染红细胞（MNPCE）数和含微核的嗜多染红细胞率，同时计数嗜多染红细胞（PCE）和正染红细胞（NCE）的比例。结果表明，溶剂对照组和喹烯酮、喹赛多各剂量组含微核嗜多染红细胞率与阴性对照组比较差异不显著（P＞0.05）。阳性对照组和喹乙醇组、卡巴氧组及痢菌净组微核率显著增加，与阴性对照组比较差异显著（P＜0.01）。各组小鼠的PCE／NCE均＞1.0，表明各药物对小鼠骨髓未呈明显的细胞毒性作用，红细胞的形成未受到抑制，不影响微核计数。阴性对照组含微核多染红细胞率＜3‰，符合一般自发率。喹烯酮、喹赛多微核试验结果为阴性，喹乙醇、卡巴氧和痢菌净微核试验结果为阳性。体外哺乳动物细胞染色体畸变试验以中国仓鼠肺成纤维细胞V79株为测试系统，设喹烯酮组（1.25、2.5、5.0、10.0μg／mL）、药物对照喹乙醇组、卡巴氧组、痢菌净组（剂量同喹烯酮组）和喹赛多组（0.625、1.25、2.5、5.0μg／mL），并设溶剂对照组（DMSO）和阳性对照组。实验分别在加或不加S₉混合物的条件下进行，不加S₉混合物在加入受试物24、48h后制片；加S₉混合物在加入受试物6h后换液，培养至24h制片。每一剂量计数200个中期分裂相细胞，计算畸变细胞率。试验结果表明，喹烯酮和喹赛多各剂量组在加与不加S₉时细胞畸变率均小于5％，为阴性。喹乙醇、卡巴氧和痢菌净在加与不加S₉时细胞畸变率均大于10％，为阳性。3 90d喂养试验SPF级Wistar大鼠400只，按体重随机分为5组，每组雌雄各40只。设喹烯酮试验组，剂量为50、300、1800mg／kg饲料：药物对照喹乙醇组，剂量为300mg／kg饲料；空白对照组，饲以不添加药物的饲料。雌雄分笼饲养。各组均连续饲喂14周。每周称量动物体重和饲料，计算饲料利用率。分别于用药后在第30d、60d、90d时每组扑杀大鼠雌雄各10只，余下大鼠给予基础饲料饲喂1周后扑杀。取全血作血常规检验，血清作生化指标检验，并进行病理剖检和病理组织学检查，主要脏器称重，计算脏器系数。结果表明，大鼠在90d内，喹烯酮50mg／kg和300mg／kg饲料组尿常规、血常规和生化指标与空白相比无显著差异（p＞0.05），组织病理学检查也未发现与药物有关的异常病变。1800mg／kg喹烯酮组大鼠的平均体重极显著低于对照组（p＜0.01）。喹烯酮1800mg／kg组和300mg／kg喹乙醇组大鼠的血清丙氨酸氨基转移酶活性显著低于空白组（p＜0.05），肾、肝、睾丸（带附睾）的脏器系数显著大于空白组（p＜0.05），尿常规、血常规和其他生化指标均没有异常变化。组织病理学观察表明，喹烯酮1800mg／kg和喹乙醇300mg／kg饲料组部分大鼠肝脏汇管区胆管增生。喹烯酮对Wistar大鼠亚慢性毒作用的靶器官为肝脏，与喹乙醇相同。喹烯酮亚慢性最大未观察的有害作用剂量（NOAEL）为300mg／kg饲料，相当于30mg／kg b．w．。4喂养致畸试验SPF级Wistar大鼠按体重随机分为5组，每组雌性24只，雄性12只。设喹烯酮试验组（50、300、1800mg／kg饲料），药物对照喹乙醇组（300mg╱kg饲料），空白对照组。各组均连续饲喂12周后进行交配。妊娠第20d剖腹进行畸胎检查。畸胎检查包括测窝重、活胎数、死胎数、胎鼠体重、体长、尾长等。试验结果表明，喹烯酮1800mg／kg组胎鼠体重、体长、尾长和窝重极显著低于空白对照组（p＜0.01），死胎数极显著高于空白对照组（p＜0.01）。各组均未出现明显的与药物相关的外观和内脏畸形。空白组、喹烯酮1800mg／kg饲料组与喹乙醇300mg╱kg饲料组各出现2例、1例和1例多肋变异，但与空白组比较差异不显著。除此之外未发现明显的骨骼畸形。喹烯酮致畸试验NOAEL为300mg／kg饲料，相当于30mg／kg b．w．。5二代繁殖试验SPF级Wistar大鼠按体重随机分为5组，第一代每组雌性30只，雄性15只：第二代每组雌性24只，雄性12只。设喹烯酮试验组（50、300、1800mg／kg饲料），药物对照喹乙醇组（300mg／kg饲料），空白对照组。各组均连续饲喂10周后进行交配。试验中观察亲代及子代动物的体重、饲料利用率、胎仔平均体重等指标，取其生殖器官进行病理组织学检查，并计算繁殖指数。结果表明，F₀代和F₁喹烯酮1800mg／kg组胎鼠体重和饲料利用率低于空白对照组，窝平均活崽数和第21d平均仔重与空白对照组相比差异极显著（p＜0.01）。组织病理学检查未见各组大鼠生殖器官受到损伤。喹烯酮对大鼠生殖发育毒性的NOAEL为300mg／kg饲料，相当于30mg／kg b．w．。6计算每日容许摄入量（ADI）及最高残留限量根据上述各试验结果得到喹烯酮的NOAEL为30mg／kg b．w．，按照FDA的ADI和最高残留限量公式，计算其ADI为0.03mg／kg b．w．；在肌肉、肝脏、肾脏和脂肪的最高残留限量分别为6.0、18.0、36.0和36.0mg／kg。实际给药时动物组织中原药及代谢物的残留量远远低于最高残留限量。所以，喹烯酮的安全性很高。上述试验均按照美国食品药品管理局（FDA）2000年颁布的食品安全性评价毒理学原则，参照我国农业部（MOA）颁布的《新兽药一般毒性试验技术要求》和《新兽药特殊毒性试验技术要求》进行试验设计，并按照卫生部（MOH）颁布食品安全性毒理学评价程序和方法，兽药注册国际协调局（VICH）的新兽药安全性评价要求进行规范和完善。在对喹烯酮的急性毒性、亚慢性毒性、致突变性、致畸性、繁殖毒性进行了系统评价，并与同类药物喹乙醇进行比较。本研究结果与相关研究基本一致，喹烯酮的毒性很低，明显弱于喹乙醇，其最高残留限量远远高于实际给药时动物组织中的残留量，具有较好的安全性。本研究可为喹烯酮的临床使用提供相关的科学依据。更多还原

【Abstract】 Quinoxalines are synthetic antimicrobial agents with growth-promoting activity, andhave been used extensively in food animals since 1970s. Olaquindox and carbadox are thewell-known members. They are supplied as 10％premix in feed for admixture in the finalfeed at 50～100 mg/kg for starter rations in pigs and 25～50 mg/kg in grower/fattener feeds.But they have been banned or strictly limited to use in food animals in some countries forthe genetic or potential toxicities. Quinocetone was a new quinoxaline antimicrobialpromoter in animal husbandry. Systematically preclinical toxicity tests were conducted toinvestigate the toxicity of quinocetone. Acceptable daily intake （ADI） and maximumresidue limits （MRLs） were caculated based on the results of toxicity tests.1 Acute toxicity test.To determine the LD₅₀ value of quinocetone in rats, 5 adult Wstar rats/sex/group and5 adult Kunming mice/sex/group were orally given by gavage of quinocetone suspendedin 0.5％carboxymethylcellulose sodium （CMC） at one time after an overnight fast.Meanwhile, olaquindox, carbadox and mequindox was given as drug control. After dosingthe rats were fed with blank diet for 14 days. The results showed that the LD₅₀ value ofquinocetone was 8687.31 mg/kg b.w. in Wistar rats and 15848.93mg/kg b.w. in Kunmingmice, which the LD₅₀ values of olaquindox, carbadox and mequindox were 1877.63,878.01 and 562.34 mg/kg b.w. in Wistar rats and 3677.56,2815.51 and 694.44 mg/kg b.w.in Kunming mice.It was indicated that quinocetone could be classified as a non-toxicsubstance and olaquindox, carbadox and mequindox could be low-toxic substance.2 Mutagenicity testsAmes test. The mutagenicity of quinocetone was evaluated in a reverse mutationassay using four histidine requiring strains of S. typhimurium (TA₉₇, TA₉₈, TA₁₀₀, TA₁₀₂,TA₁₅₃₅ and TA₁₅₃₇) with and without S₉ from PCB-induced rats as activation systemsusing triplicate plates. The tests were conducted with doses 1.0, 2.6, 6.9, 18.2, 50.0μg/plate quinocetone, while olaquindox, carbadox and mequindox were conductedwith the same doses as drugs control. The results were showed that no increase in thenumber of reventant colonies was found in TA₁₀₂ and TA₁₅₃₅ with and without S₉;6.9μg/plate and higher dose quinocetone induced mutagenic responses in TA97 with andwithout S₉, and 50.0μg/plate in TA₉₈, 18.2μg/plate and higher dose in TA₁₀₀ with andwithout S₉; 6.9μg/plate and higher dose quinocetone induced mutagenic responses inTA₁₅₃₇ with S₉, while 18.2μg/plate and higher dose quinocetone induced mutagenicresponses in TA₁₅₃₇ without S₉. The similar results were also found in cyadox groups.Olaquindox, carbadox and mequindox exhibited higher mutagenicity in each strains of S.typhimurium than quinocetone and cyadox.In viva mouse micronucleus test. The test was performed to test the induction ofmicronuclei in polychromatic erythrocytes from sternal bone marrow of Kunming miceresulting from exposure to quinocetone. Quinocetone was administered by gavage togroups of five male and five female mice at a single dose level of 1700, 3600, 7200mg/kgb.w. at one time. Meanwhile, cyadox, olaquindox, carbadox and mequindox was given asdrug control. The same volum 0.5％CMC was used as concurrent negative control.Cyclophosphamide （CP） was given at a dose level of 40mg/kg b.w. as concurrent positivecontrol. Mice were treatmented with the test substance twice at an interval of 24h, andthen were sacrificed and smear slides were made with sternal bone marrow after 6h afterthe second dosing. The slides were scored for micronuclei and for polychromatic （PCE）to normochromatic （NCE） cell ratio until 1000 cells （PCE and NCE） had been analyzed.Counting also continued until at least 1000 PCE had been observed. Quinocetone andcyadox is not mutagenic in all dose levels, olaquindox, carbadox and mequindox at alldoses and. CP at 40mg/kg b.w induced higher micronucleus rate relative to negativecontrol. So quinocetone and cyadox is not mutagenic in mice bone marrow micronucleusassay, and olaquindox, carbadox and mequindox showed higher mutagenicity thanquinocetone and cyadox.In vitro mammalian chromosome aberration test. To investigate the V79 cellsmutagenicity of quinocetone, chromosome aberration in vitro was studied. Quinocetonewas respectively administered to V79 cells with 1.25, 2.5, 5.0, 10.0μg/mL. Meanwhile,cyadox, olaquindox, carbadox and mequindox was given as drug control. The DMSOwas used as concurrent negative control. CP was given as positive control with S₉ andmitomycin C （MMC） without S₉. Cell cultures were treated with colchicine for 2h prior toharvesting.The results showed that quinocetone and cyadox did not cause any increase in aberrations relative to negative control. But olaquindox, carbadox and mequindox wouldgive clearly positive result.3 90-day feeding test.To investigate the potential subchronic toxicity of quinocetone, groups of 40 maleand 40 female Wistar rats were fed with the diets containing quinocetone （0, 50, 300 or1800mg/kg） or olaquindox （300mg/kg）, approximately equivalent to quinocetone 5, 30,180 or olaquindox 30mg/kg b.w./d, for 14w. 10 rats/sex/group were sacrificed underanesthesia with intraperitoneal sodium pentobarbital on days 30, 60, 90. The rest wassacrificed after fed with based diet for one week. The results showed that there were noquinocetone-related effects at 50 and 300 mg/kg dietary levels on clinical observations,hematology, blood biochemistry, urinalysis, relative organ weights and histopathologicalexaminations. During the experiment, body weights were significantly decreased at 1800mg/kg group. At the 90-day time point, significantly decreased serum alkalineamino-transferase values were observed in all treated groups; a significant decrease intotal protein and creatinine in females was recorded at 1800 mg/kg group; a significantincrease in alkaline phosphatase in males and relative weights of liver, kidney and testes（including epididymides） was also recorded at the high dose of quinocetone.Histopathological observations revealed that 1800 mg/kg quinocetone and 300 mg/kgolaquindox could induce proliferation of bile canaliculi in the portal area. In conclusion,the no-observed-adverse-effect level （NOAEL） of quinocetone for Wistar rats wasestimated to be 300 mg/kg dietary dose level.4 Feeding teratogenicity test.To investigate the potential teratogenic toxicity of quinocetone, groups of 12 maleand 24 female Wistar rats were fed with the diets containing quinocetone （0, 50, 300 or1800mg/kg） or olaquindox （300mg/kg） through a 12-week prebreed period. The pregnantrats were subjected to caesarean section on gestational day （GD） 20 for teratogenicexamination. Fetuses were examined for external, visceral, and skeletal abnormalities.The result showed that no test- material-related changes were seen in mortality, clinicalsigns, and macroscopic examinations throughout the study. Relative to concurrent controlgroup, body weights and feed efficiency of prebreed period for rats of 1800mg/kgquinocetone group were significantly lower. On 1800mg/kg quinocetone group, relativeto those of blank controls, body weight, body length and tail length of fetus and litterweight decreased and number of dead fetus increased significantly in the teratogenicity test. No obvious external and visceral abnormalities were found in all the groups in thistest. Except that extra little rib increased in the fetus of control group, 1800mg/kgquinocetone group and 300mg/kg olaquindon group, but no significant difference.1800mg/kg quinocetone depressed mildly the development of fetus and fertility of rats.No obvious teratogenicity toxicity was revealed. The NOAEL for teratogenic test ofquinocetone for rats was estimated to be 300mg/kg dietary dose level based on this study,which was equivalent to approximately 30mg/kg b.w./day.5 Two generation reproduction testTo investigate the potential reproductive toxicity of quinocetone, groups of 15 maleand 30 female Wistar rats （F₀） in the first generation were fed with the diets containingquinocetone （0, 50, 300 or 1800mg/kg） or olaquindox （300mg/kg） through a 10-weekprebreed period as well as during mating, gestation, parturition and lactation. At weaning,12 males and 24 females of F₁ generation weanlings per group were selected randomly asparents for the F₂ generation. Selected F₁ weanlings were exposed to the same diet andtreatment as their parents. Fetuses were examined for external, visceralandhistopathological examinations of reproductive organs. Body weights of the young ondays 0, 4, 7, 14 and 21 after birth were recorded. No test-material-related changes wereseen in mortality, clinical signs, and macroscopic examinations throughout the study.Relative to concurrent control group, body weights and feed efficiency of prebreed periodfor rats of 1800mg/kg quinocetone group were significantly lower in both sexes of F₀ andHistopathological observations revealed that no test-material-related changes were foundin reproductive organs in any group. On 1800mg/kg quinocetone group, relative to thoseof blank controls, body weight at the 21th day of fetus and number of survival fetussignificantly decreased in F₀ and F₁ generation. No obvious external and visceralabnormalities were found in all the groups in this study. So 1800mg/kg quinocetonedepressed mildly the development of fetus and fertility of rats. No obvious reproductivetoxicity was revealed, and results indicated that the NOAEL for reproduction test ofquinocetone for rats was estimated to be 300mg/kg dietary dose level based on this study,which was equivalent to approximately 30mg/kg b.w./day.6 Calculating ADI and MRLsWith NOAEL （30mg/kg b.w.） of the above subchronic toxicity test, feedingteratogenic test and two generation reproduction test, ADI was calculated to be 0.03mg/kgb.w. according to the method of FDA. MRLs of quinocetone for muscle, liver, kidney and fat are 6.0, 18.0, 36.0 and 36.0mg/kg. The MRLs of all the above tissues were muchlarger than residues of quinocetone detected in tissues of animals given quinocetone indiet. Therefore, quinocetone possess a good safety.Brief summary. All the above tests were basically designed and conductedaccording to Toxicological Principles for the Safety Assessment of Food Ingredientspublished by FDA, and were standarded and improved by the Technological Requirementfor General Toxicity Test of New Veterinary Drug and the Technological Requirement forSpecial Toxicity Test of New Veterinary Drug published by MOA in 1991, Procedures andmethods for toxicological assessment of Food published by MOH, Guidance for Industry:Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food publishedby VICH. The acute toxicity, mutagenicity, subchronic toxicity, teratogenicity andreproductive toxicity of quinocetone were systematically evaluated firstly. All the abovetests revealed that the toxicity of quinocetone was mild, which was much milder than thatof olaquindox. Results of these tests were consistent with other studies basically andprovided valuable toxicological information of quinocetone, which demonstrated thegood safety profile of quinocetone and can be used as scientific evidence for approvementand application of quinocetone in food-producing animals as a growth-promoting agent.更多还原