喹赛多临床前毒理学安全性评价
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摘要
喹喔啉类药物作为饲料添加用抗菌促生长剂，曾在世界范围内广范应用，给人类社会带来了巨大的物质财富和社会经济效益。但由于特殊毒性问题，本类中的主要品种卡巴氧和喹乙醇已在很多国家和地区被禁止使用或严格限制使用。喹赛多为新一代喹喔啉类抗菌促生长剂，对食品动物具有良好的促生长效果。如其具有好的安全性，开发上市代替卡巴氧和喹乙醇，必将对畜牧业生产和人民生活水平的提高起到巨大的推动作用。为评价喹赛多的安全性，进行了系统的临床前毒性试验。测定喹赛多的急性毒性，致突变性，致畸和繁殖毒性，亚慢性毒性，对其致癌性进行预测，计算ADI(每日容许摄入量)和其在动物性食品中的安全浓度。为喹赛多作为新药审批提供毒理学资料，为其过渡到临床试验和应用提供剂量选择和毒副反应监测的依据。
1急性毒性试验
Wistar大鼠20只，雌雄各半，喹赛多用0.5％羧甲基纤维素钠(CMC)助溶制成混悬液，以1mL／100g b．w．的容量在24h内灌胃3次。观察28d内的中毒反应。结果表明仅给药后大鼠粪便呈黄色，为黄色的试药随粪便排出所致。大鼠未出现明显的中毒反应和死亡。喹赛多对Wistar大鼠的经口MTD＞13g／kg b．w．，按照急性毒性分级标准喹赛多属实际无毒物质。
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TA_(97)，TA_(98)，TA_(100)，TA_(102)。进行了2次试验，第1次试验剂量为5，10，25，50，100，250μg／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S_9混合液的对照。每个处理作3个平行平皿，采用标准平皿掺入法，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。第2次试验剂量为0.2，1，5，25，100μg／皿，同时设同类药物对照(喹乙醇25μg／皿)，其余与第1次试验相同。
两次试验结果基本一致，TA_(97)和TA_(100)加和不加S_9在剂量≥5μg／皿时均为阳性；TA_(98)不加S_9在剂量≥10μg／皿时为阳性，加S_9在剂量≥5μg／皿时为阳性；TA_(102)加和不加S_9在剂量≥100μg／皿时均为阳性。阳性反应呈现一定的剂量反应关系。喹赛多100μg／皿对TA_(97)有轻微抑菌作用，250μg／皿对TA_(97)，TA_(98)，TA_(100)均有不同程度的抑菌作用。喹乙醇25μg／皿除TA_(102)不加S_9为阴性(MR=1.73)外，其余均为阳性。在本试验条件下喹赛多Ames试验结果为阳性。喹乙醇在TA_(102)上的诱变性高于喹赛多。
小鼠骨髓细胞微核试验第1次试验，取70只昆明种小鼠随机分为7组，雌雄各半，分别为空白对照组(蒸馏水)，溶剂对照组(0.5％CMC)，喹赛多组(0.016，0.16，1.6，16g／kg b.w．)，阳性对照组(环磷酰胺(CP)40mg／kg b.w.)。第2次试验，取80只昆明种小鼠随机分为8组，雌雄各半，分别为溶剂对照组(0.5％CMC)，喹赛多组(0.5，1，5，10，20g／kg b.w.)，喹乙醇组(0.5g／kg b.w.)，阳性对照组(CP 40mg／kgb.w.)。除阳性对照组外，其余各组均为每天灌胃给药1次，连续4d，第5d取胸骨骨髓制片。阳性对照组为腹腔注射给药2次，间隔24h，于末次给药后6h采样。每只小鼠计数1000个多染红细胞，计算含微核的嗜多染红细胞(MNPCE)数和含微核多染红细胞率，同时计数多染红细胞(PCE)和正染红细胞(NCE)的比例。
两次试验结果均表明，溶剂对照组和喹赛多各剂量组含微核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微核率与阴性对照组比较差异显著(P＜0.01)。各组小鼠的PCE／NCE均＞0.1，表明喹赛多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。喹赛多微核试验结果为阴性。喹乙醇的诱变性明显高于喹赛多。
小鼠睾丸生殖细胞染色体畸变试验第1次试验取45只雄性昆明种小鼠随机分为9组，分别为阴性对照组(蒸馏水)，溶剂对照组(0.5％CMC)，喹赛多组(0.016，0.16，1.6，16g／kg b.w.)，喹乙醇组(0.16，1.6g／kg b.w.)，阳性对照组(CP 40mg／kg b.w.)。除阳性对照组为腹腔注射给药外，其余各组均为灌胃给药，每天1次，连续5d，于第11，12d再连续给药2d，第13d杀鼠。第1次试验给药5d后，1.6g／kg b.w.喹乙醇组小鼠在6d内陆续死亡。表明喹乙醇的毒性较强。第2次试验取45只雄性昆明种小鼠随机分为8组，分别为溶剂对照组(0.5％CMC)，喹赛多组(0.5，1，5，10，20g／kg b.w.)，喹乙醇组(0.5g／kg b.w.)，阳性对照组(CP 40mg／kg b.w.)。给药方法和取样时间等与第1次试验相同。每只小鼠计数100个初级精母细胞中期相，进行染色体畸变分析。观察的染色体畸变的类型有断裂与断片、易位、性染色体早熟分离和常染色体早熟分离，并计算畸变细胞总数。
两次试验结果均表明，除阳性对照组的断裂与断片(P＜0.01)，畸变细胞总数(P＜0.01)，阳性对照组的易位(P＜0.05)和喹乙醇组的畸变细胞总数(P＜0.05)与阴性对照组比较差异显著外，溶剂对照组和喹赛多组的各项指标与阴性对照组比较差异均不显著(P＞0.05)。喹赛多对小鼠睾丸生殖细胞无致突变作用，喹乙醇的致突变性高于喹赛多。
3 90d喂养试验
动物和分组SPF级Wistar大鼠雌性175只，雄性125只。随机分为5组，每组雌性35只，雄性25只。设喹赛多试验组，剂量为50、150、2500mg／kg饲料；药物对照喹乙醇组，剂量为150mg／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90d。观察项目和指标每周称量动物体重和饲料，计算平均摄食量、增重和饲料利用率。分别于用药后30d，60d，90d各组随机抽取10只大鼠，雌雄各半，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。结果喹赛多最高剂量组大鼠粪便呈淡黄色，可能是药物(黄色)经粪便排出所致。给药2周后开始直至实验结束，喹赛多2500mg／kg饲料组大鼠体重显著低于空白对照组，喹乙醇组雌性大鼠在给药的第3周和第4周体重显著低于空白对照组。其他组与空白对照组比较差异不显著。各期杀鼠病理组织学观察表明喹赛多剂量为2500mg／kg饲料可引起大鼠肝细胞和肾脏近曲小管上皮细胞肿胀、变性。喹乙醇150mg／kg饲料组也出现相近的结果。结论喹赛多对Wistar大鼠亚慢性毒作用的靶器官为肝脏和肾脏，与喹乙醇相同。高剂量喹赛多引起的肝脏和肾脏病变为首次发现。喹赛多亚慢性无不良作用剂量(NOAEL)为150mg／kg饲料。相当于15mg／kg b.w.，这与本实验室进行的喹赛多大鼠长期毒性试验的结果基本一致。
4两代喂养繁殖与致畸试验
方法90d喂养试验除用于剖杀检查的大鼠之外，每组剩余的雌性20只，雄性10只，在90d喂养试验之后，进行交配。1／2孕鼠于妊娠20d剖腹进行畸胎检查，另1／2自然分娩，产生F_(1a)。F_(1a)断奶后处死，进行系统病理剖检。母鼠再次交配产生F_(1b)。F_(1b)断奶后处死F_0，进行病理剖检并取其生殖器官进行病理组织学检查。F_(1b)各组分别选留雌性28只，雄性15只，分别饲喂与亲代相同的饲料。饲喂30d时每组随机抽取雌雄各5只，扑杀，进行血常规、血清生化指标测定，计算脏体比，并进行系统病理剖检。余下F_(1b)大鼠饲喂90d后进行交配，1／2孕鼠用于畸胎检查，另1／2自然分娩，产生F_(2a)。F_(2a)断奶后处死全部大鼠，进行系统病理剖检。畸胎检查包括测窝重、胎盘重、子宫重、黄体数、活胎数、死胎数、胎鼠体重、体长、尾长、性别，检查外观畸形，制作骨骼标本和内脏标本进行骨骼和内脏畸形检查。对所有子鼠在出生时检查外观畸形、体重、体长、尾长、活胎数、死胎数和性别比率等。出生4d时每窝随机选留8只，尽量雌雄各半，称5d重和21d重。F_0和F_(1b)在13周喂养期内每周称体重和饲料，计算平均摄食量、增重和饲料利用率。
对亲代的影响喹赛多对孕鼠的健康状况，行为无明显不良影响，病理剖检未见明显的病变。组织病理学检查未见大鼠生殖器官受到损伤。体重：喹赛多2500mg／kg饲料组：雌雄F_0体重在13周的喂养期内均自第3周开始显著低于空白对照组；雌性F_(1b)在断奶后第2，5，11周显著低于空白对照组；雄F_(1b)在断奶后第1，2，4周显著低于空白对照组，在第8周显著高于空白对照组。其他各剂量组大鼠体重没有受到试药的明显影响。饲料利用率：与空白对照组比较，雄性F_0各剂量组均有所提高，雌性F_0各组无显著差异；F_(1b)各剂量组未出现明显的变化趋势。喹赛多2500mg／kg饲料组第2代致畸试验孕鼠体重显著低于空白对照组。病理组织学检查：F_(1b)喂养30d扑杀病理组织学观察表明喹赛多剂量为2500mg／kg饲料时引起大鼠肝细胞和肾近曲小管上皮细胞肿胀、变性。喹乙醇150mg／kg饲料组也出现相近的结果。其他组未发现与试药相关的变化。
对子代的影响喹赛多2500mg／kg饲料组：第1代致畸试验胎鼠体重、体长、尾长和窝重显著低于空白对照组，吸收胎数显著高于空白对照组；第2代致畸试验窝重和活胎数显著低于空白对照组，吸收胎数显著高于空白对照组：F_(1a)，F_(1b)和F_(2a)出生活胎数显著低于空白对照组；F_(2a)出生后21d体重显著低于空白对照组。喹赛多150mg／kg饲料组：F_(1b)出生后5d体重和21d体重显著高于空白对照组。喹赛多50mg／kg饲料组：F_(1b)出生后21d体重显著高于空白对照组。各组均未出现明显的与药物相关的外观和内脏畸形。喹赛多2500mg／kg饲料组F_(2a)多肋变异显著增加；喹乙醇150mg／kg饲料组F_(2a)多肋变异也明显增加，但差异不显著。除此之外未发现明显的骨骼畸形。
结论喹赛多在剂量为2500mg／kg饲料(临床剂量的50倍)时对大鼠胚胎发育有轻度抑制作用，对大鼠的生长发育和生殖机能有轻度抑制作用，无明显的繁殖毒性和致畸性。喹赛多的安全性较高。并初步判定同等剂量下喹乙醇对大鼠生殖发育的不良影响高于喹赛多。喹赛多对大鼠生殖发育毒性的NOAEL为150mg／kg饲料，相当于15mg／kg b.w.。
5预测致癌性和计算每日容许摄入量(ADI)及安全浓度
根据3项致突变试验的结果，运用致癌性预测和试验组合的选择(CPBS)法计算受试物的潜在致癌性概率。喹赛多可能是潜在致癌物的概率为16％，喹乙醇可能是潜在致癌物的概率为100％。根据目前通用的判别标准，θ~+＜30％判为非致癌物；30％＜θ~+＜70％，暂时无法下结论：θ~+＞70％，判为潜在致癌物。喹赛多可预测为非致癌物。喹乙醇可预测为致癌物。
根据上述各试验结果得到喹赛多的NOAEL=15mg／kg b.w.，按照FDA的ADI和安全浓度计算公式，计算其ADI=0.15mg／kg b.w.；在肌肉的安全浓度为30μg／g；在肝脏的安全浓度为90μg／g；在肾脏和脂肪的安全浓度为180μg／g。实际给药时动物组织中原药及代谢物的残留量远远低于安全浓度。所以，喹赛多的安全性很高。
上述试验基本按照我国农业部颁布的《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，并参照兽药注册国际协调局(VICH)的新兽药安全性评价要求，食品安全性毒理学评价程序，动物毒理学试验方法和卫生毒理学试验方法等相应指南和试验技术要求进行规范和完善。在国内外首次对喹赛多的急性毒性、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。试验结果与相关研究基本一致，喹赛多的毒性很低，且明显弱于喹乙醇，潜在致癌性预测结果为非致癌物。且其安全浓度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。可为喹赛多的临床使用提供相关的科学依据，也为喹赛多作为新兽药审评提供相关的资料。
(National Reference Laboratory of Veterinary Drug Residues, MO A Key Laboratory of Food Safety Evaluation, Huazhong Agricultural University, WuHan 430070, P. R. China) Quinoxalines were widely used as feed growth-promoting agent in the most areas of the world and produced large interests. However, the use of main quinoxalines drugs, carbadox and olaquindox, have been prohibited or limited attributing to their toxicity. Cyadox, a new member of the quinoxaline family, had the traits of obvious growth promotion in food-producing animals. If its safety is good it can be used to replace the old drugs and promote the development of animal husbandry, then improve people's lives largely. To investigate the toxicity of cyadox, systematically preclinical toxicity tests were conducted and potential carcinogenicity was predicted with CPBS method. Acceptable daily intake (ADI) and safe concentration were caculated based on the results of toxicity tests.1 Acute toxicity test. To determine the maximum tolerated dose (MTD) of cyadox in rats, 10 adult Wstar rats/sex were orally given 1mL/100g b.w. cyadox suspended in 0.5% carboxymethylcellulose sodium (CMC) three times within 24h. Concentration of cyadox suspension is 43.34% (larger will make intubation very difficult). After dosing the rats were fed with blank diet for 28 days. No death or obvious toxic effect related to cyadox was found other than yellow-colored feces in the first two days after dosing. The MTD of cyadox is above 13g/kg b.w. and cyadox belongs to practically nontoxic substance.2 Mutagenicity testsAmes assay. The mutagenicity of cyadox was evaluated in a reverse mutation assay using four histidine requiring strains of S. typhimurium (TA_(97), TA_(98), TA_(100), and TA_(102)) with and without S9 from PCB-induced rats as activation systems using triplicate plates. The tests were conducted with doses 5, 10, 25, 50, 100, 250μg/plate cyadox for the first time and 0.2, 1, 5, 25, 100μg/plate cyadox and 25μg/plate olaquindox for the second time. The results of two times were consistent basically. 5μg/plate and higher dose cyadox induced mutagenic responses in TA_(97) and TA_(100) with and without S_9, and TA_(98) with S_9; 10μg/plate and higher dose cyadox induced mutagenic responses in TA_(98) without S_9; 100μg/plate and higher dose cyadox induced mutagenic responses in TA_(102) with and without S_9. Mutagenic responses are dose related in all the four bacterial strains. 100μg/plate cyadox suppressed the growth of TA_(97) slightly; 250μg/plate cyadox suppressed the growth of TA_(97), TA_(98) and TA_(100) to a certain extent. 25μg/plate olaquindox induced mutagenic responses for all the four bacterial strains except TA_(102) without S_9. Olaquindox exhibited higher mutagenicity in TA_(102) than cyadox.
In vivo mouse micronucleus assay. The test was performed to test the induction of micronuclei in polychromatic erythrocytes from sternal bone marrow of Kunming mice resulting from exposure to cyadox. The highest technically feasible dose for cyadox in 0.5% CMC was 20g/kg b.w. at a dose volume of 0.5mL/10g b.w.. Therefore, in the first test cyadox was administered to groups of five male and five female mice at a single dose level of 0.016, 0.16, 1.6, 16g/kg b.w. once a day in constantly four days. The same volum distilled water or 0.5% CMC was used as concurrent negative control or solvent control. In the second test dose levels for cyadox are 0.5, 1, 5, 10, 20g/kg b.w., and 0.5g/kg b.w. olaquindox and 0.5% CMC was used as drug control or concurrent solvent control, respectively. Dose route, dose times and dose interval was the same as those of the first test. In both tests mice were sacrificed and smear slides were made with sternal bone marrow after 24hs from the last dosing. Cyclophosphamide (CP) was used via intraperitoneal injection at a dose level of 40mg/kg b.w. as concurrent positive control and sampled on 6h after the 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. Cyadox is not mutagenic in all dose levels. 40mg/kg b.w. CP and 0.5g/kg b.w. olaquindox induced higher micronucleus rate relative to negative control. So cyadox is not mutagenic in mice bone marrow micronucleus assay, and olaquindox showed higher mutagenicity than cyadox.
Mammalian spermatocyte of the first order chromosome aberration test. To investigate the potential germ cell mutagenicity of cyadox, groups of 5 male Kunming mice were given cyadox (0.016, 0.16, 1.6, 16g/kg b.w.) or olaquindox (0.16 and 1.6g/kg b.w.) by oral gavage once a day, both drugs suspended in 0.5% CMC, in concecutive five days. Mice were sacrificed in the thirteenth day from the first dosing and 100 metaphases of spermatocytes of the first order in each mouse were examined. In the testicle chromosome aberration test, cyadox did not cause any increase in aberrations. 1.6g/kg b.w. olaquindox group mice died within 6 days after 5 days dosing. A following study in male mice with cyadox (0.5, 1, 5, 10, 20g/kg b.w.) or olaquindox (0.5g/kg b.w.) with same sample times also did not reveal any increases of aberrations in cyadox group. 0.16 and 0.5g/kg olaquindox produced mild mutagenic effect.
3 90-day feeding test. To investigate the potential subchronic toxicity of cyadox, groups of 15 male and 15 female Wistar rats were fed with the diets containing cyadox (0, 50, 150 or 2500mg/kg) or olaquindox (150mg/kg), approximately equivalent to cyadox 5, 15, 250 or olaquindox 15mg/kg b.w./d, for 13 wk. 5 rats/sex/group were sacrificed on days 30, 60 and 90. No test-material-related changes were seen in mortality, clinical signs, hematology, clinical chemistry, organ weight data and macroscopic examinations. Except that body weights of both sexes of 2500mg/kg cyadox group were significantly lower than controls beginning from the second week of treatment. Body weights of females of 150mg/kg olaquindox group were significantly lower than those of the control group at weeks 3 and 4. Body weights of other groups were unaffected by treatments. Histopathological observations revealed that 2500mg/kg cyadox or 150mg/kg olaquindox induced swelling and fatty degeneration of the hepatocytes and proximal renal tubular epithelial cells. It is the first time that the changes of liver and kidneys were found in rats given high level of cyadox. The subchronic no-observed-adverse-effect level (NOAEL) of cyadox for rats was estimated to be 150mg/kg dietary dose level based on this study, which was equivalent to approximately 15mg/kg b.w./d and is significantly higher than that of olaquindox.
4 Two generation feeding reproduction test and teratogenicity test. Method: To investigate the potential teratogenic and reproductive toxicity of cyadox, groups of 10 male and 20 female Wistar rats (F_0) were fed with the diets containing cyadox (0, 50, 150 or 2500mg/kg) or olaquindox (150mg/kg) through a 13-week prebreed period as well as during mating, gestation, parturition and lactation. Half pregnant rats were subjected to caesarean section on gestational day (GD) 20 for teratogenic examination and another half produced the young (F_(1a)). F_(1a) were euthanized on day 21 after birth and autopsied. F_0 rats were mated again after 10 days following weaning of the F_(1a) generation to produce an F_(1b) generation. At weaning, 10 males and 25 females of F_(1b) weanlings per group were selected randomly as parents for the F_2 generation. Selected F_(1b) weanlings were exposed to the same diet and treatment as their parents. More than 12 pregnant rats were used to teratogenic examination and the rest produced the young (F_(2a)). F_(2a) were autopsied on day 21 after birth as well as their parents. Fetuses were examined for external, visceral, and skeletal abnormalities. The young were randomly selected on day 4 after birth and only 8 of each litter preferring equal number for female and male were kept. Body weights of the young on days 5 and 21 after birth were recorded. Body weight and feed intake of F_0 and F_(1b) throughtout 13wk feeding period were recorded and feed efficiency were caculated. Result: No test-material-related changes were seen in mortality, clinical signs, and macroscopic examinations throughout the study. Relative to concurrent control group, body weights of prebreed period for rats of 2500mg/kg cyadox group were significantly lower in both sexes of F_0 beginning from the third week of treatment, and in female F_(1b) at weeks 2, 5, 11 and male F_(1b) at weeks 1, 2, 4 after weaning. However, it is significantly higher in male F_(1b) at week 8. Body weights of other groups were not affected by treatment obviously. Feed efficiency of male F_0 increased to a certain extent in all the treatment groups and that of female F_0 showed no obvious drug-related effect. However, Feed efficiency in both sexes of F_(1b) showed no clear or obvious trend. Body weights of pregnant rats of 2500mg/kg cyadox group in F_(1b) were significantly lower relative to concurrent control group. Histopathological observations revealed that 2500mg/kg cyadox or 150mg/kg olaquindox induced swelling and degeneration of the hepatocytes and proximal renal tubular epithelial cells in F_(1b) rats on day 30. No test-material-related changes were found in other groups. On 2500mg/kg cyadox group, relative to those of blank controls, body weight, body length and tail length of fetus and litter weight decreased and number of resorption fetus increased significantly in the first generation teratogenicity test; litter weight, number of viable fetus decreased and number of resorption fetus increased significantly in the second generation teratogenicity test; number of viable fetus of F_(1a), F_(1b) and F_(2a) decreased in the reproduction test; Body weights of F_(2a) on day 21 after birth decreased significantly. On 150mg/kg cyadox group, body weights of F_(1b) on days 5 and 21 after birth increased significantly relative to those of blank controls. On 50mg/kg cyadox group, body weights of F_(1b) on day 21 after birth increased significantly relative to those of blank controls. No obvious external and visceral abnormalities were found in all the groups in both two generation teratogenic tests. Except that extra little rib in neck or waist significantly increased in the fetus of 2500mg/kg cyadox group in F_(2a), and obviously increased in those of 150mg/kg olaquindox group, but no significant difference. No obvious skeleton abnormalities were observed. Conclusion: 2500mg/kg cyadox depressed mildly the development of fetus and fertility of rats. No obvious teratogenicity or reproductive toxicity was revealed, and results indicated that olaquindox produced higher adverse effect on the reproduction and development of rats than cyadox at the same dose. The NOAEL for reproduction/development of cyadox for rats was estimated to be 150mg/kg dietary dose level based on this study, which was equivalent to approximately 15mg/kg b.w./day and significantly higher than that of olaquindox.
5 Preclinical safety evaluation for cyadox.
Predicting potential carcinogenicity. Based on the results of the above three mutagenicity tests probability of potential carcinogenicity for cyadox is 16% and that of olaquindox is 100% predicted with carcinogenicity prediction and battery selection (CPBS) method, and cyadox can be predicted to be non-carcinogen and olaquindox to be carcinogen.
Caeulating ADI (acceptable daily intake) and safe concentration. With NOAEL (15mg/kg b.w.) of the above subchronic toxicity test and two generation feeding reproduction test and teratogenic test, ADI was calculated to be 0.15mg/kg b.w. according to method of FDA. Safe concentration of cyadox for muscle is 30μg/g, and that for liver is 90μg/g and both that for kidney and fat are 180μg/g. The safe concentrations of all the above tissues were much larger than residues of cyadox or its metabolites detected in tissues of animals given cyadox in diet. Therefore, cyadox possess very good safety.
Brief summary. All the above tests were designed and conducted according to "the Technological Requirement for General Toxicity Test of New Veterinary Drug" and "the Technological Requirement for Special Toxicity Test of New Veterinary Drug" published by MOA in 1991 basically, and were standarded and improved by refering to "Guidance for Industry: Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food" published by International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH), "Toxicological Principles for the Safety Evaluation of New drug (western medicine)" published by Hygeian administration, "Toxicological Principles for the Safety Evaluation of Food", animal and hygeian toxicological test methods. The acute toxicity, subchronic toxicity, mutagenicity, teratogenicity and reproductive toxicity of cyadox were systematically evaluated firstly. All the above tests revealed that the toxicity of cyadox was mild, which was much milder than that of olaquindox. Results of these tests were consistent with other studies basically and provided valuable toxicological information of cyadox, which demonstrated the good safety profile of cyadox and can be used as scientific evidence for approvement and application of cyadox in food-producing animals as a growth-promoting agent.
1急性毒性试验
Wistar大鼠20只，雌雄各半，喹赛多用0.5％羧甲基纤维素钠(CMC)助溶制成混悬液，以1mL／100g b．w．的容量在24h内灌胃3次。观察28d内的中毒反应。结果表明仅给药后大鼠粪便呈黄色，为黄色的试药随粪便排出所致。大鼠未出现明显的中毒反应和死亡。喹赛多对Wistar大鼠的经口MTD＞13g／kg b．w．，按照急性毒性分级标准喹赛多属实际无毒物质。
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TA_(97)，TA_(98)，TA_(100)，TA_(102)。进行了2次试验，第1次试验剂量为5，10，25，50，100，250μg／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S_9混合液的对照。每个处理作3个平行平皿，采用标准平皿掺入法，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。第2次试验剂量为0.2，1，5，25，100μg／皿，同时设同类药物对照(喹乙醇25μg／皿)，其余与第1次试验相同。
两次试验结果基本一致，TA_(97)和TA_(100)加和不加S_9在剂量≥5μg／皿时均为阳性；TA_(98)不加S_9在剂量≥10μg／皿时为阳性，加S_9在剂量≥5μg／皿时为阳性；TA_(102)加和不加S_9在剂量≥100μg／皿时均为阳性。阳性反应呈现一定的剂量反应关系。喹赛多100μg／皿对TA_(97)有轻微抑菌作用，250μg／皿对TA_(97)，TA_(98)，TA_(100)均有不同程度的抑菌作用。喹乙醇25μg／皿除TA_(102)不加S_9为阴性(MR=1.73)外，其余均为阳性。在本试验条件下喹赛多Ames试验结果为阳性。喹乙醇在TA_(102)上的诱变性高于喹赛多。
小鼠骨髓细胞微核试验第1次试验，取70只昆明种小鼠随机分为7组，雌雄各半，分别为空白对照组(蒸馏水)，溶剂对照组(0.5％CMC)，喹赛多组(0.016，0.16，1.6，16g／kg b.w．)，阳性对照组(环磷酰胺(CP)40mg／kg b.w.)。第2次试验，取80只昆明种小鼠随机分为8组，雌雄各半，分别为溶剂对照组(0.5％CMC)，喹赛多组(0.5，1，5，10，20g／kg b.w.)，喹乙醇组(0.5g／kg b.w.)，阳性对照组(CP 40mg／kgb.w.)。除阳性对照组外，其余各组均为每天灌胃给药1次，连续4d，第5d取胸骨骨髓制片。阳性对照组为腹腔注射给药2次，间隔24h，于末次给药后6h采样。每只小鼠计数1000个多染红细胞，计算含微核的嗜多染红细胞(MNPCE)数和含微核多染红细胞率，同时计数多染红细胞(PCE)和正染红细胞(NCE)的比例。
两次试验结果均表明，溶剂对照组和喹赛多各剂量组含微核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微核率与阴性对照组比较差异显著(P＜0.01)。各组小鼠的PCE／NCE均＞0.1，表明喹赛多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。喹赛多微核试验结果为阴性。喹乙醇的诱变性明显高于喹赛多。
小鼠睾丸生殖细胞染色体畸变试验第1次试验取45只雄性昆明种小鼠随机分为9组，分别为阴性对照组(蒸馏水)，溶剂对照组(0.5％CMC)，喹赛多组(0.016，0.16，1.6，16g／kg b.w.)，喹乙醇组(0.16，1.6g／kg b.w.)，阳性对照组(CP 40mg／kg b.w.)。除阳性对照组为腹腔注射给药外，其余各组均为灌胃给药，每天1次，连续5d，于第11，12d再连续给药2d，第13d杀鼠。第1次试验给药5d后，1.6g／kg b.w.喹乙醇组小鼠在6d内陆续死亡。表明喹乙醇的毒性较强。第2次试验取45只雄性昆明种小鼠随机分为8组，分别为溶剂对照组(0.5％CMC)，喹赛多组(0.5，1，5，10，20g／kg b.w.)，喹乙醇组(0.5g／kg b.w.)，阳性对照组(CP 40mg／kg b.w.)。给药方法和取样时间等与第1次试验相同。每只小鼠计数100个初级精母细胞中期相，进行染色体畸变分析。观察的染色体畸变的类型有断裂与断片、易位、性染色体早熟分离和常染色体早熟分离，并计算畸变细胞总数。
两次试验结果均表明，除阳性对照组的断裂与断片(P＜0.01)，畸变细胞总数(P＜0.01)，阳性对照组的易位(P＜0.05)和喹乙醇组的畸变细胞总数(P＜0.05)与阴性对照组比较差异显著外，溶剂对照组和喹赛多组的各项指标与阴性对照组比较差异均不显著(P＞0.05)。喹赛多对小鼠睾丸生殖细胞无致突变作用，喹乙醇的致突变性高于喹赛多。
3 90d喂养试验
动物和分组SPF级Wistar大鼠雌性175只，雄性125只。随机分为5组，每组雌性35只，雄性25只。设喹赛多试验组，剂量为50、150、2500mg／kg饲料；药物对照喹乙醇组，剂量为150mg／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90d。观察项目和指标每周称量动物体重和饲料，计算平均摄食量、增重和饲料利用率。分别于用药后30d，60d，90d各组随机抽取10只大鼠，雌雄各半，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。结果喹赛多最高剂量组大鼠粪便呈淡黄色，可能是药物(黄色)经粪便排出所致。给药2周后开始直至实验结束，喹赛多2500mg／kg饲料组大鼠体重显著低于空白对照组，喹乙醇组雌性大鼠在给药的第3周和第4周体重显著低于空白对照组。其他组与空白对照组比较差异不显著。各期杀鼠病理组织学观察表明喹赛多剂量为2500mg／kg饲料可引起大鼠肝细胞和肾脏近曲小管上皮细胞肿胀、变性。喹乙醇150mg／kg饲料组也出现相近的结果。结论喹赛多对Wistar大鼠亚慢性毒作用的靶器官为肝脏和肾脏，与喹乙醇相同。高剂量喹赛多引起的肝脏和肾脏病变为首次发现。喹赛多亚慢性无不良作用剂量(NOAEL)为150mg／kg饲料。相当于15mg／kg b.w.，这与本实验室进行的喹赛多大鼠长期毒性试验的结果基本一致。
4两代喂养繁殖与致畸试验
方法90d喂养试验除用于剖杀检查的大鼠之外，每组剩余的雌性20只，雄性10只，在90d喂养试验之后，进行交配。1／2孕鼠于妊娠20d剖腹进行畸胎检查，另1／2自然分娩，产生F_(1a)。F_(1a)断奶后处死，进行系统病理剖检。母鼠再次交配产生F_(1b)。F_(1b)断奶后处死F_0，进行病理剖检并取其生殖器官进行病理组织学检查。F_(1b)各组分别选留雌性28只，雄性15只，分别饲喂与亲代相同的饲料。饲喂30d时每组随机抽取雌雄各5只，扑杀，进行血常规、血清生化指标测定，计算脏体比，并进行系统病理剖检。余下F_(1b)大鼠饲喂90d后进行交配，1／2孕鼠用于畸胎检查，另1／2自然分娩，产生F_(2a)。F_(2a)断奶后处死全部大鼠，进行系统病理剖检。畸胎检查包括测窝重、胎盘重、子宫重、黄体数、活胎数、死胎数、胎鼠体重、体长、尾长、性别，检查外观畸形，制作骨骼标本和内脏标本进行骨骼和内脏畸形检查。对所有子鼠在出生时检查外观畸形、体重、体长、尾长、活胎数、死胎数和性别比率等。出生4d时每窝随机选留8只，尽量雌雄各半，称5d重和21d重。F_0和F_(1b)在13周喂养期内每周称体重和饲料，计算平均摄食量、增重和饲料利用率。
对亲代的影响喹赛多对孕鼠的健康状况，行为无明显不良影响，病理剖检未见明显的病变。组织病理学检查未见大鼠生殖器官受到损伤。体重：喹赛多2500mg／kg饲料组：雌雄F_0体重在13周的喂养期内均自第3周开始显著低于空白对照组；雌性F_(1b)在断奶后第2，5，11周显著低于空白对照组；雄F_(1b)在断奶后第1，2，4周显著低于空白对照组，在第8周显著高于空白对照组。其他各剂量组大鼠体重没有受到试药的明显影响。饲料利用率：与空白对照组比较，雄性F_0各剂量组均有所提高，雌性F_0各组无显著差异；F_(1b)各剂量组未出现明显的变化趋势。喹赛多2500mg／kg饲料组第2代致畸试验孕鼠体重显著低于空白对照组。病理组织学检查：F_(1b)喂养30d扑杀病理组织学观察表明喹赛多剂量为2500mg／kg饲料时引起大鼠肝细胞和肾近曲小管上皮细胞肿胀、变性。喹乙醇150mg／kg饲料组也出现相近的结果。其他组未发现与试药相关的变化。
对子代的影响喹赛多2500mg／kg饲料组：第1代致畸试验胎鼠体重、体长、尾长和窝重显著低于空白对照组，吸收胎数显著高于空白对照组；第2代致畸试验窝重和活胎数显著低于空白对照组，吸收胎数显著高于空白对照组：F_(1a)，F_(1b)和F_(2a)出生活胎数显著低于空白对照组；F_(2a)出生后21d体重显著低于空白对照组。喹赛多150mg／kg饲料组：F_(1b)出生后5d体重和21d体重显著高于空白对照组。喹赛多50mg／kg饲料组：F_(1b)出生后21d体重显著高于空白对照组。各组均未出现明显的与药物相关的外观和内脏畸形。喹赛多2500mg／kg饲料组F_(2a)多肋变异显著增加；喹乙醇150mg／kg饲料组F_(2a)多肋变异也明显增加，但差异不显著。除此之外未发现明显的骨骼畸形。
结论喹赛多在剂量为2500mg／kg饲料(临床剂量的50倍)时对大鼠胚胎发育有轻度抑制作用，对大鼠的生长发育和生殖机能有轻度抑制作用，无明显的繁殖毒性和致畸性。喹赛多的安全性较高。并初步判定同等剂量下喹乙醇对大鼠生殖发育的不良影响高于喹赛多。喹赛多对大鼠生殖发育毒性的NOAEL为150mg／kg饲料，相当于15mg／kg b.w.。
5预测致癌性和计算每日容许摄入量(ADI)及安全浓度
根据3项致突变试验的结果，运用致癌性预测和试验组合的选择(CPBS)法计算受试物的潜在致癌性概率。喹赛多可能是潜在致癌物的概率为16％，喹乙醇可能是潜在致癌物的概率为100％。根据目前通用的判别标准，θ~+＜30％判为非致癌物；30％＜θ~+＜70％，暂时无法下结论：θ~+＞70％，判为潜在致癌物。喹赛多可预测为非致癌物。喹乙醇可预测为致癌物。
根据上述各试验结果得到喹赛多的NOAEL=15mg／kg b.w.，按照FDA的ADI和安全浓度计算公式，计算其ADI=0.15mg／kg b.w.；在肌肉的安全浓度为30μg／g；在肝脏的安全浓度为90μg／g；在肾脏和脂肪的安全浓度为180μg／g。实际给药时动物组织中原药及代谢物的残留量远远低于安全浓度。所以，喹赛多的安全性很高。
上述试验基本按照我国农业部颁布的《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，并参照兽药注册国际协调局(VICH)的新兽药安全性评价要求，食品安全性毒理学评价程序，动物毒理学试验方法和卫生毒理学试验方法等相应指南和试验技术要求进行规范和完善。在国内外首次对喹赛多的急性毒性、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。试验结果与相关研究基本一致，喹赛多的毒性很低，且明显弱于喹乙醇，潜在致癌性预测结果为非致癌物。且其安全浓度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。可为喹赛多的临床使用提供相关的科学依据，也为喹赛多作为新兽药审评提供相关的资料。
(National Reference Laboratory of Veterinary Drug Residues, MO A Key Laboratory of Food Safety Evaluation, Huazhong Agricultural University, WuHan 430070, P. R. China) Quinoxalines were widely used as feed growth-promoting agent in the most areas of the world and produced large interests. However, the use of main quinoxalines drugs, carbadox and olaquindox, have been prohibited or limited attributing to their toxicity. Cyadox, a new member of the quinoxaline family, had the traits of obvious growth promotion in food-producing animals. If its safety is good it can be used to replace the old drugs and promote the development of animal husbandry, then improve people's lives largely. To investigate the toxicity of cyadox, systematically preclinical toxicity tests were conducted and potential carcinogenicity was predicted with CPBS method. Acceptable daily intake (ADI) and safe concentration were caculated based on the results of toxicity tests.1 Acute toxicity test. To determine the maximum tolerated dose (MTD) of cyadox in rats, 10 adult Wstar rats/sex were orally given 1mL/100g b.w. cyadox suspended in 0.5% carboxymethylcellulose sodium (CMC) three times within 24h. Concentration of cyadox suspension is 43.34% (larger will make intubation very difficult). After dosing the rats were fed with blank diet for 28 days. No death or obvious toxic effect related to cyadox was found other than yellow-colored feces in the first two days after dosing. The MTD of cyadox is above 13g/kg b.w. and cyadox belongs to practically nontoxic substance.2 Mutagenicity testsAmes assay. The mutagenicity of cyadox was evaluated in a reverse mutation assay using four histidine requiring strains of S. typhimurium (TA_(97), TA_(98), TA_(100), and TA_(102)) with and without S9 from PCB-induced rats as activation systems using triplicate plates. The tests were conducted with doses 5, 10, 25, 50, 100, 250μg/plate cyadox for the first time and 0.2, 1, 5, 25, 100μg/plate cyadox and 25μg/plate olaquindox for the second time. The results of two times were consistent basically. 5μg/plate and higher dose cyadox induced mutagenic responses in TA_(97) and TA_(100) with and without S_9, and TA_(98) with S_9; 10μg/plate and higher dose cyadox induced mutagenic responses in TA_(98) without S_9; 100μg/plate and higher dose cyadox induced mutagenic responses in TA_(102) with and without S_9. Mutagenic responses are dose related in all the four bacterial strains. 100μg/plate cyadox suppressed the growth of TA_(97) slightly; 250μg/plate cyadox suppressed the growth of TA_(97), TA_(98) and TA_(100) to a certain extent. 25μg/plate olaquindox induced mutagenic responses for all the four bacterial strains except TA_(102) without S_9. Olaquindox exhibited higher mutagenicity in TA_(102) than cyadox.
In vivo mouse micronucleus assay. The test was performed to test the induction of micronuclei in polychromatic erythrocytes from sternal bone marrow of Kunming mice resulting from exposure to cyadox. The highest technically feasible dose for cyadox in 0.5% CMC was 20g/kg b.w. at a dose volume of 0.5mL/10g b.w.. Therefore, in the first test cyadox was administered to groups of five male and five female mice at a single dose level of 0.016, 0.16, 1.6, 16g/kg b.w. once a day in constantly four days. The same volum distilled water or 0.5% CMC was used as concurrent negative control or solvent control. In the second test dose levels for cyadox are 0.5, 1, 5, 10, 20g/kg b.w., and 0.5g/kg b.w. olaquindox and 0.5% CMC was used as drug control or concurrent solvent control, respectively. Dose route, dose times and dose interval was the same as those of the first test. In both tests mice were sacrificed and smear slides were made with sternal bone marrow after 24hs from the last dosing. Cyclophosphamide (CP) was used via intraperitoneal injection at a dose level of 40mg/kg b.w. as concurrent positive control and sampled on 6h after the 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. Cyadox is not mutagenic in all dose levels. 40mg/kg b.w. CP and 0.5g/kg b.w. olaquindox induced higher micronucleus rate relative to negative control. So cyadox is not mutagenic in mice bone marrow micronucleus assay, and olaquindox showed higher mutagenicity than cyadox.
Mammalian spermatocyte of the first order chromosome aberration test. To investigate the potential germ cell mutagenicity of cyadox, groups of 5 male Kunming mice were given cyadox (0.016, 0.16, 1.6, 16g/kg b.w.) or olaquindox (0.16 and 1.6g/kg b.w.) by oral gavage once a day, both drugs suspended in 0.5% CMC, in concecutive five days. Mice were sacrificed in the thirteenth day from the first dosing and 100 metaphases of spermatocytes of the first order in each mouse were examined. In the testicle chromosome aberration test, cyadox did not cause any increase in aberrations. 1.6g/kg b.w. olaquindox group mice died within 6 days after 5 days dosing. A following study in male mice with cyadox (0.5, 1, 5, 10, 20g/kg b.w.) or olaquindox (0.5g/kg b.w.) with same sample times also did not reveal any increases of aberrations in cyadox group. 0.16 and 0.5g/kg olaquindox produced mild mutagenic effect.
3 90-day feeding test. To investigate the potential subchronic toxicity of cyadox, groups of 15 male and 15 female Wistar rats were fed with the diets containing cyadox (0, 50, 150 or 2500mg/kg) or olaquindox (150mg/kg), approximately equivalent to cyadox 5, 15, 250 or olaquindox 15mg/kg b.w./d, for 13 wk. 5 rats/sex/group were sacrificed on days 30, 60 and 90. No test-material-related changes were seen in mortality, clinical signs, hematology, clinical chemistry, organ weight data and macroscopic examinations. Except that body weights of both sexes of 2500mg/kg cyadox group were significantly lower than controls beginning from the second week of treatment. Body weights of females of 150mg/kg olaquindox group were significantly lower than those of the control group at weeks 3 and 4. Body weights of other groups were unaffected by treatments. Histopathological observations revealed that 2500mg/kg cyadox or 150mg/kg olaquindox induced swelling and fatty degeneration of the hepatocytes and proximal renal tubular epithelial cells. It is the first time that the changes of liver and kidneys were found in rats given high level of cyadox. The subchronic no-observed-adverse-effect level (NOAEL) of cyadox for rats was estimated to be 150mg/kg dietary dose level based on this study, which was equivalent to approximately 15mg/kg b.w./d and is significantly higher than that of olaquindox.
4 Two generation feeding reproduction test and teratogenicity test. Method: To investigate the potential teratogenic and reproductive toxicity of cyadox, groups of 10 male and 20 female Wistar rats (F_0) were fed with the diets containing cyadox (0, 50, 150 or 2500mg/kg) or olaquindox (150mg/kg) through a 13-week prebreed period as well as during mating, gestation, parturition and lactation. Half pregnant rats were subjected to caesarean section on gestational day (GD) 20 for teratogenic examination and another half produced the young (F_(1a)). F_(1a) were euthanized on day 21 after birth and autopsied. F_0 rats were mated again after 10 days following weaning of the F_(1a) generation to produce an F_(1b) generation. At weaning, 10 males and 25 females of F_(1b) weanlings per group were selected randomly as parents for the F_2 generation. Selected F_(1b) weanlings were exposed to the same diet and treatment as their parents. More than 12 pregnant rats were used to teratogenic examination and the rest produced the young (F_(2a)). F_(2a) were autopsied on day 21 after birth as well as their parents. Fetuses were examined for external, visceral, and skeletal abnormalities. The young were randomly selected on day 4 after birth and only 8 of each litter preferring equal number for female and male were kept. Body weights of the young on days 5 and 21 after birth were recorded. Body weight and feed intake of F_0 and F_(1b) throughtout 13wk feeding period were recorded and feed efficiency were caculated. Result: No test-material-related changes were seen in mortality, clinical signs, and macroscopic examinations throughout the study. Relative to concurrent control group, body weights of prebreed period for rats of 2500mg/kg cyadox group were significantly lower in both sexes of F_0 beginning from the third week of treatment, and in female F_(1b) at weeks 2, 5, 11 and male F_(1b) at weeks 1, 2, 4 after weaning. However, it is significantly higher in male F_(1b) at week 8. Body weights of other groups were not affected by treatment obviously. Feed efficiency of male F_0 increased to a certain extent in all the treatment groups and that of female F_0 showed no obvious drug-related effect. However, Feed efficiency in both sexes of F_(1b) showed no clear or obvious trend. Body weights of pregnant rats of 2500mg/kg cyadox group in F_(1b) were significantly lower relative to concurrent control group. Histopathological observations revealed that 2500mg/kg cyadox or 150mg/kg olaquindox induced swelling and degeneration of the hepatocytes and proximal renal tubular epithelial cells in F_(1b) rats on day 30. No test-material-related changes were found in other groups. On 2500mg/kg cyadox group, relative to those of blank controls, body weight, body length and tail length of fetus and litter weight decreased and number of resorption fetus increased significantly in the first generation teratogenicity test; litter weight, number of viable fetus decreased and number of resorption fetus increased significantly in the second generation teratogenicity test; number of viable fetus of F_(1a), F_(1b) and F_(2a) decreased in the reproduction test; Body weights of F_(2a) on day 21 after birth decreased significantly. On 150mg/kg cyadox group, body weights of F_(1b) on days 5 and 21 after birth increased significantly relative to those of blank controls. On 50mg/kg cyadox group, body weights of F_(1b) on day 21 after birth increased significantly relative to those of blank controls. No obvious external and visceral abnormalities were found in all the groups in both two generation teratogenic tests. Except that extra little rib in neck or waist significantly increased in the fetus of 2500mg/kg cyadox group in F_(2a), and obviously increased in those of 150mg/kg olaquindox group, but no significant difference. No obvious skeleton abnormalities were observed. Conclusion: 2500mg/kg cyadox depressed mildly the development of fetus and fertility of rats. No obvious teratogenicity or reproductive toxicity was revealed, and results indicated that olaquindox produced higher adverse effect on the reproduction and development of rats than cyadox at the same dose. The NOAEL for reproduction/development of cyadox for rats was estimated to be 150mg/kg dietary dose level based on this study, which was equivalent to approximately 15mg/kg b.w./day and significantly higher than that of olaquindox.
5 Preclinical safety evaluation for cyadox.
Predicting potential carcinogenicity. Based on the results of the above three mutagenicity tests probability of potential carcinogenicity for cyadox is 16% and that of olaquindox is 100% predicted with carcinogenicity prediction and battery selection (CPBS) method, and cyadox can be predicted to be non-carcinogen and olaquindox to be carcinogen.
Caeulating ADI (acceptable daily intake) and safe concentration. With NOAEL (15mg/kg b.w.) of the above subchronic toxicity test and two generation feeding reproduction test and teratogenic test, ADI was calculated to be 0.15mg/kg b.w. according to method of FDA. Safe concentration of cyadox for muscle is 30μg/g, and that for liver is 90μg/g and both that for kidney and fat are 180μg/g. The safe concentrations of all the above tissues were much larger than residues of cyadox or its metabolites detected in tissues of animals given cyadox in diet. Therefore, cyadox possess very good safety.
Brief summary. All the above tests were designed and conducted according to "the Technological Requirement for General Toxicity Test of New Veterinary Drug" and "the Technological Requirement for Special Toxicity Test of New Veterinary Drug" published by MOA in 1991 basically, and were standarded and improved by refering to "Guidance for Industry: Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food" published by International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH), "Toxicological Principles for the Safety Evaluation of New drug (western medicine)" published by Hygeian administration, "Toxicological Principles for the Safety Evaluation of Food", animal and hygeian toxicological test methods. The acute toxicity, subchronic toxicity, mutagenicity, teratogenicity and reproductive toxicity of cyadox were systematically evaluated firstly. All the above tests revealed that the toxicity of cyadox was mild, which was much milder than that of olaquindox. Results of these tests were consistent with other studies basically and provided valuable toxicological information of cyadox, which demonstrated the good safety profile of cyadox and can be used as scientific evidence for approvement and application of cyadox in food-producing animals as a growth-promoting agent.
引文
蔡原．卫生毒理学基本概念．见：张桥主编，卫生毒理学基础(第三版)．北京：人民卫生出版社，2000，7，188
曹随忠，张力，梁剑平，刘宗平．喹噁啉-1，4-二氧化物类抗菌促生长剂特殊毒理学研究进展．动物医学进展，2001，22(2)：17-20
陈超然，范盛先，袁宗辉，陈昌福．喹赛多对鱼类常见致病菌的试管内抑菌作用．华中农业大学学报，2001，20(5)：466-468
陈红，陈杖榴．饲料中喹乙醇含量的高效液相色谱测定方法．养禽与禽病防治，1998，12：19-20
陈孝煊，袁宗辉，范盛先，袁军法．喹赛多对鲤、银鲫生长的影响．水利渔业，2004，24(3)：52-54
崔效亮．喹赛多放大试验与反应条件优化研究．[硕士学位论文]．武汉：华中农业大学图书馆，2004
丁明星．喹赛多对猪、羊的免疫促生长机理研究．[博士学位论文]．武汉：华中农业大学图书馆，2005
范盛先，袁宗辉，王大菊，孔科，陈品，马敬忠．喹赛多体外抑菌试验．华中农业大学学报，2000，20(1)：51-54
何庆华．喹赛多的皮肤光敏毒性和长期毒性研究．[硕士学位论文]．武汉：华中农业大学图书馆，2005
《化学药物急性毒性试验技术指导原则》课题研究组．化学药物急性毒性试验技术指导原则．2005
《化学药物长期毒性试验技术指导原则》课题研究组．化学药物长期毒性试验技术指导原则．2005
黄玲利，袁宗辉，范盛先，王玉莲，王国永．喹赛多对鸡大肠杆菌病的预防效果研究．华中农业大学学报，2002，21(1)：47-49
黄玲利，袁宗辉，范盛先，刘登才，陈品，王大菊．喹赛多对家禽常见病原茵的体外抗菌活性研究．华南农业大学学报(自然科学版)，2003，24(2)：81-83
黄玲利．喹赛多在肉鸡的有效性与安全性研究．[博士学位论文]．武汉：华中农业大学图书馆，2005
黄幸纾，朱心强．化学毒物的生殖毒性作用．见：张桥主编，卫生毒理学基础(第三版)．北京：人民卫生出版社，2000，167
李德培．我国法定饲用抗菌药物的生物学特性．兽药与饲料添加剂，2000，5(6)：9-10
李剑勇，李金善，徐忠赞，赵荣材，苗小楼，张继瑜，鲁润华．喹烯酮在猪、鸡体内的药代动力学研究．畜牧兽医学报，2002，34(1)：94-97
李剑勇，赵荣材，李金善，徐忠赞，杜小丽，苗小楼，薛飞群，柳军玺．猪血液中~(14)C标记喹烯酮的测定及药代动力学研究．动物医学进展，2003，24(3)：78-79
李剑勇，李金善，徐忠赞，赵荣材，苗小楼，张继瑜，鲁润华．喹烯酮在猪组织中的残留研究．动物医学进展，2004a，25(4)：117-120
李剑勇，李金善，徐忠赞，赵荣材，苗小楼，张继瑜，鲁润华．高效液相色谱仪检测鸡组织器官喹烯酮残留方法的建立．动物医学进展，2004b，25(5)：100-101
李寿祺．外源化学物致突变作用．见：张铣，刘毓谷主编，毒理学．北京：北京医科大学和中国协和医科大学联合出版社，1997，282-286
梁剑平，张力，曹随中，周丽霞，周学辉，魏春梅，苗小林，刘宗平，卫增泉．新
型饲料添加剂喹胺醇致突变性及其潜在致癌性预测．中国农业科学，2001，34(6)：679-685
刘毓谷主编．卫生毒理学基础(第二版)．北京：人民卫生出版社，1999，146
卢纹岱主编．SPSS for Windows统计分析(第2版)．北京：电子工业出版社，2002
秦长川．养鸡慎用痢菌净．中国家禽，2005，27(4)：26—27
邱银生，袁宗辉，范盛先，刘登才，黄玲利，殷居易．高效液相色谱法测定猪血浆及组织中喹赛多及其脱二氧代谢物．中国兽医学报，2003a，23(4)：363-365
邱银生，袁宗辉，殷居易．猪食用组织中喹喔啉-2-羧酸的高效液相色谱检测方法．中国兽医学报，2003b，23(3)：286—288
邱银生．喹赛多在猪体内的药动学和残留研究．[博士学位论文]．武汉：华中农业大学图书馆，2003c
石年．化学毒物致突变作用．见：张桥主编，卫生毒理学基础(第三版)．北京：人民卫生出版社，2000，130-131
孙永学，冯淇辉，董漓波，陈杖榴．喹乙醇在鸡体内的毒物动力学及其生化毒性和病理学研究．Ⅰ．喹乙醇在鸡体内的毒物动力学研究．畜牧兽医学报，1998a， 29(5)：438-442
孙永学，冯淇辉，董漓波，陈杖榴．喹乙醇在鸡体内的毒物动力学及其生化毒性和病理学研究．Ⅱ．喹乙醇对鸡的生化毒性和病理学研究．畜牧兽医学报，1998b，29(6)：525-530
孙展和，李文恩，王海刚，贾俊亮．群养鸡谨防痢菌净中毒．中国兽药杂志，2005，39(7)：55-56
孙振球主编．医学统计学(研究生用)北京：人民卫生出版社，2002
王继贵．临床生化检验(第2版)．长沙：湖南科学技术出版社，1996，286
王树槐，徐士新，郑定，孙玉梅．喹乙醇诱发小鼠骨髓嗜多染红细胞微核试验．中国兽药杂志，1991，25(1)：13-14
王树槐．喹乙醇诱发CHL细胞染色体畸变试验．中国兽药杂志，1993，27(4)：27-29
王树槐，徐士新，向德林，孙玉梅，黄齐颐，徐彦波，王明志．喹乙醇亚慢性毒性试验．中国兽药杂志，1991，25(4)：13-14。
王玉春，严相林，赵荣材，李金善，杜健．新型药物饲料添加剂喹烯酮的一般毒性研究，Ⅰ．急性毒性试验．中兽医医药杂志，1992，4：13-14
王玉春，严相林，赵荣材，李金善，薛飞群，梁建平，徐忠赞，杜小丽．新型饲料药物添加剂喹烯酮一般毒性研究，Ⅱ．蓄积毒性试验．中兽医医药杂志，1993，14
王玉春，赵荣材，严相林，李金善，杜小丽，张世珍，薛飞群，徐忠赞，梁建平，张继瑜．喹烯酮对小白鼠的致癌试验．中国兽医科技，1995，25(3)：24-25
王玉春，严相林，赵荣材，李金善，薛飞群，粱建平，杜小丽，徐忠赞．新型饲料药物添加剂喹烯酮一般毒性研究，Ⅳ．亚急性毒性试验．中兽医医药杂志，1994，5：10—11
王玉春，赵荣材，严相林，李金善，杜小丽，薛飞群，徐忠赞，粱建平，张继瑜，朱以萍．新型饲料药物添加剂喹烯酮的毒性研究——繁殖及致畸试验．中国兽医科技，1993，23(4)：25-26
王玉莲，袁宗辉，朱惠玲，邱银生，范盛先．喹赛多对断奶仔猪的生长性能和胴体品质的影响．华中农业大学学报，2005，24(1)：59-62
王泽民．当代结构药物全集．北京：北京科学技术出版社，1993，455-468
徐士新，郑定．喹乙醇蓄积性毒性试验．中国兽药杂志，1992，26(2)：22-24
徐叔云．药理实验方法学．第2版．北京：人民卫生出版社，1991，1084
严相林，李金善，王玉春，赵荣材，薛飞群．喹烯酮对小白鼠精子的畸变试验．中兽医医药杂志，1997，5：13-14。
严相林，李金善，王玉春，赵荣材，薛飞群．喹烯酮的Ames试验．中兽医医药杂志，1998，5：11-12
杨杏芬．化学毒物的一般毒性作用．见：张桥主编，卫生毒理学基础(第三版)．北京：人民卫生出版社，2000，77-78
殷居易．喹赛多在鲤体内的药动学和残留研究．[硕士学位论文]。武汉：华中农业大学图书馆，2003
张力，梁剑平，袁莉刚，周丽霞，魏春梅，周学辉，常寅龙．喹胺醇饲喂肉仔鸡急性毒性试验及病理形态学研究．中兽医医药杂志，2000，6：6-8
张力，梁剑平，周丽霞，周学辉，程富胜，苗小林，袁莉刚，张德寿，刘宗平．喹胺醇对肉用仔鸡亚急性毒性试验．中国兽医科技，2001a，1(1)：27-30
张力，粱剑平，程富胜，赵四喜，周学辉，牛晓荣，苗小林，袁莉刚，张德寿，刘宗平，石旭东．喹胺醇对断奶仔猪的亚急性毒性试验．中兽医医药杂志，2001b，2：11-12
张力，梁剑平，曹随中，刘宗平，周丽霞，魏春梅，周学辉，牛晓荣，苗小林．喹胺醇对小白鼠精子致畸性试验．中兽医医药杂志，2001c，5：11-12
张力，梁剑平，曹随中，刘宗平，周丽霞，魏春梅，周学辉，牛晓荣，苗小林．喹胺醇对小白鼠致畸作用的研究．中国兽医科技，2001d，31(7)：20-21
张桥主编．卫生毒理学基础(第三版)．北京：人民卫生出版社，2000，89-94，205-282
中华人民共和国国家标准：食品安全性毒理学评价程序和方法，GB15193-194
朱蓓蕾主编．动物性食品药物残留．上海：上海科学技术出版社，1994，141-143，143-144，146-151，151-155，166-172，172-175，183-184，184-189
Beutin L, Preller E, Kowalski B. Mutagenicity of quindoxin, its metabolites, and two substituted quinoxaline-di-N-oxides. Antimicrobial Agents and Chemotherapy, 1981, 20 (3): 336-343
Cihak R, Srb V. Cytogenetic effects of quinoxaline-1, 4-dioxide-type growth-promoting agents. I. Micronucleus test in rats. Mutat Res, 1983, 116: 129-135
Cihak R, Vontorkova M. Cytogenetic effects of quinoxaline-1, 4-dioxide-type growth- promoting agents. II. Metaphase analysis in mice. Mutation Research, 1983, 117: 311-316
Cihak R, Vontorkova M. Cytogenetic effects of quinoxaline-1,4-dioxide-type growth- promoting agents. III. Transplacental micronucleus test in mice. Mutation Research, 1985,144: 81-84
Devries H, Beyersbergen Van Henegouwen G M J, Kalloe F, Berkhuysen M H J. Phototoxicity of olaquindox in the rat. Research in Veterinary Science (London), 1990a, 48,240-244.
Devreis H, Bojarski J, Donker A A, Bakri A, Beyersbergen Van Henegouwen M J. Photochemical reactions of quindoxin, olaquindox, carbodox and cyandox with protein, indicating photoallergic properties. Toxicology, 1990b, 63: 85-95
EMEA (the European Agency for the Evaluation of Medicinal Products). Note for guidance on the establishment of maximum residue limits for minor animal species. 1997
EPA (United States Environmental Protection Agency). Health Effects Test Guidelines OPPTS 870.5380 Mammalian Spermatogonial Chromosome Aberration Test. 1998a
EPA (United States Environmental Protection Agency). Health Effects Test Guidelines OPPTS 870.7800 Immunotoxicity. 1998b
EPA (United States Environmental Protection Agency). Health Effects Test Guidelines OPPTS 870.1100 Acute Oral Toxicity. 2002
FDA (United States Food and Drug Administration). Good Laboratory Practice for Nonclinical Laboratory Study. 1993
FDA (United States Food and Drug Administration). Toxicological principles for the safety assessment of food ingredients. IV C 1 a, Bacterial Reverse Mutation Test. 2000a
FDA (United States Food and Drug Administration). Toxicological principles for the safety assessment of food ingredients. IV C 1 d, Mammalian Erythrocyte Micronucleus Test. 2000b
FDA (United States Food and Drug Administration). Toxixological Principles for the Safety Assessment of Food Ingredients. IV C 10, Neurotoxicity Studies. 2000c
FDA (United States Food and Drug Administration). Guidance for Industry #3. FDA General Principles for Evaluating the Safety of Compounds Used in Food-producing Animals. 2005
Geraut C, Tripodi D. New allergens in occupational dermatology (Fren) Les nouveaux allergens en dermatologee professionnelle. Rev Frallergol Immunol Clin, 1997, 37 (7): 832-837.
Haut K T, Stebbins K E, Johnson K A, Shabrang S N, Stott W T. Subchronic toxicity of ingested 1,3-Dichloropropene in rats and mice. Fundamental and Applied Toxicology, 1996, 32: 224-232.
Holsapple M P. The plaque-forming cell (PFC) response in Immunotoxicology: An approach to monitoring the primary effector function of B lymphocytes. In Methods in Immunotoxicology (Burleson G R, Dean J H, and Munson A E, Eds.), 1995, Vol. 1, pp. 71-108, Wiley-Liss, Inc., New York.
Huber L. Good Laboratory Practice USA. Hewlett-Packard, 1993
Hutchings M, Johnson I, Hayes E, Girling A E, Thain J, Thomas K, Benstead R, Whale G, Wordon J, Maddox R, and Chown P. Toxicity reduction evaluation, toxicityidentification evaluation and toxicity tracking in direct toxicity assessment. Ecotoxicology, 2004,13(5): 475-484
Jager L P, De Graaf G J, Widjaja-Greefkes H C, Accord-Burleson C C, Van Den Dungen H M, Baars A J. Effects of feed additives and veterinary drugs on aldosterone production and release by porcine adrenal cells in vitro. Journal of Veterinary Pharmacology & Therapeutics, 1994, 17 (3): 175-185
Keithly J, Brooker J A, DeForest D K, Wu B K, Brix K. Acute and chronic toxicity of nickel to a cladoceran (Ceriodaphnia dubia) and an amphipod (Hyalella azteca). Environmental Toxicology and Chemistry, 2004,23(3): 691-696
Lafranconi W M, Long P H, Atkinson J E, Knezevich A L, Wooding W L. Chronic oxicity and carcinogenicity of olestra in Swiss CD-I mice. Food and Chemical Toxicology, 1994, 32: 789-798
Lindamood C, Farnell D R, Giles H D, Prejean J D, Collins J J, Takahashi K, Maronpot R R. Subchronic toxicity studies of t-butyl alcohol in rats and mice. Fundamental and Applied Toxicology, 1992,19: 91-100
Lonceint J, Sassolas B, Guillet G. Photoallergic reactions to olaquindox in swine raisers: role of growth promotors used in feed. Annales de Dermatologie et de Venereologie, 2001, 128: 46-48
Lu F C. Acute, short-term and long-term toxicity studies. In: Basic Toxicology: Fundamentals, Target Organs, and Risk Assessment, 1985, pp. 99-104
Nabuurs M J A, Van der Molen B J, De Graaf G J, Jager L P. Clinical signs and performance of pigs treated with different doses of carbadox, cyadox and olaquindox. Journal of Veterinary Medicine, 1990, 37 (1): 68-76.
Nastuneak J, Klouch F, Vanova L, Dvorak M, Sevick B. Three month toxicity to laboratory Norway rats: Cyadox compared with olaquindox. Biology and Chemistry of Veterinery (Prague), 1986,22: 367-380
Nastuneak J, Dvorak M, Klouch F, Vanova L. Cyadox toxicity studies over twelve months on Laboratory rats. Journal of Biologizae a Chemizace, 1987, 23(6): 549-555
Negishi T, Tanaka K, Hayatsu H. Mutagenicity of carbadox and several quinoxaline 1, 4-dioxide derivatives. Chemical and Pharmaceutical Bulletin, 1980, 28: 1347-1349
NRC (National Research Council). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC. 1996
Schauder S, Schroder W, Geier J. Olaquindox-induced airborne photoallergic contact dermatitis followed by transient or persistent light reactions in 15 pig breeders. Contact Dermatitis, 1996, 35: 344-354
Scheutwinkel Reich M, Von der Hude W. Sister-chromatid exchange in Chinese hamster V79 cells exposed to quindoxin, carbadox and olaquindox. Mutation Research, 1984, 139: 199-202
Select Committee on European Scrutiny. Select Committee on European Scrutiny fifth report, antibiotics in animal feedingstuffs, Draft Council Regulation amending Council Directive 70/524/EC concerning additives in feedingstuffs as regards withdrawal of the authorisation of certain antibiotics, 1999, (19615) 14046/98 COM (98) 763
Sevcik B, Broz J, Strakova J. Toxicity and safety evaluation of new quinolaxine growth promotant cyadox. [Journal Article] Colloques de LInra. [Paris: Institut National de la Recherche Agronomique] 1982, 8: 243-244.
Sevcik B. Research of harmless and safety of application of the growth promoter cyadox. Workshop, 1986,1:93
Spierenburg Th J, Van der Molen E J, De Graaf G J, Jager L P, Baars A J. Adrenal toxicity of carbadox, olaquindox, and cyadox in weaned piglets. Workshop 1, 1986, 97
SCAN (Standing Committee for Animal Nutrition). Short report of the meeting of the standing committee for animal nutrition. 1998
Takeda K, Flood M. Chronic Toxicity and Carcinogenicity of Sucrose Fatty Acid Esters in Fisher 344/DuCrj Rats. Regulatory Toxicology and Pharmacology, 2002, 35: 157-164
Van Der Molen E J, De Greef G J, Baars A J. Persistence of Carbadox-induced Adrenal Lesions in Pigs Following Drug Withdrawal and Recovery of Aldosterone Plasma Concentration. Journal of Comparative Pathology, 1989,100: 295-304
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL23. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Genotoxicity Testing. 2001a
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL22. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Reproduction Toxicity Testing. 2001b
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL31. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Repeat-Dose (90-Day) Toxicity Testing. 2002a
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite
Guideline GL32. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Developmental Toxicity Testing. 2002b
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL28. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Carcinogenicity Testing. 2002c
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL33. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: General Approach to Testing. 2004a
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL37. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Repeat-Dose (Chronic) Toxicity Testing. 2004b
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL36. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: General Approach to Establish a Microbiological ADI. 2004c
Voogd C E, Van der Stel J J, Jacobs J J J A A. The mutagenic action of quindoxin, carbadox, olaquindox and some other N-oxides on bacteria and yeast. Mutation Research, 1980, 78 (3): 233-242
WHO (World Health Organization). Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 27, 1991, nos 694-705 on INCHEM.
WHO (World Health Organization). Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 33, 1994, nos 804-817 on INCHEM.
WHO (World Health Organization). Toxicological Evaluation of Certain Food Additives and Contaminants in Food. WHO Technical Report Series, No. 859, 1995, 129-138
WHO (World Health Organization). Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 51, 2003, carbadox (addendum).
Yoshimura H, Nakamura M, Koeda T, Yoshikawa K. Mutagenicities of carbadox and olaquindox—growth promoters for pigs. Mutation Research, 1981, 90 (1): 49-55.
Yoshimura H. Teratogenic assessment of carbadox in rats. Toxicology Letters, 2002, 129: 115-118.
Zhang Y L, Huang L L, Chen D M, Fan S X, Wang Y L, Tao Y F, Yuan Z H. Development of HPLC Methods for the Determination of Cyadox and Its Main Metabolites in Goat Tissues., Analytical Sciences, 2005a, 21 (12): 1495-1499
Zhang Y L, Wang Y L, Huang L L, Chen D M, Tao Y F, Yuan Z H. Effects of Cooking and Storage on Residues of Cyadox in Chicken Muscle. Journal of Agricultural and Food Chemistry, 2005b, 53(25): 9737-9341
曹随忠，张力，梁剑平，刘宗平．喹噁啉-1，4-二氧化物类抗菌促生长剂特殊毒理学研究进展．动物医学进展，2001，22(2)：17-20
陈超然，范盛先，袁宗辉，陈昌福．喹赛多对鱼类常见致病菌的试管内抑菌作用．华中农业大学学报，2001，20(5)：466-468
陈红，陈杖榴．饲料中喹乙醇含量的高效液相色谱测定方法．养禽与禽病防治，1998，12：19-20
陈孝煊，袁宗辉，范盛先，袁军法．喹赛多对鲤、银鲫生长的影响．水利渔业，2004，24(3)：52-54
崔效亮．喹赛多放大试验与反应条件优化研究．[硕士学位论文]．武汉：华中农业大学图书馆，2004
丁明星．喹赛多对猪、羊的免疫促生长机理研究．[博士学位论文]．武汉：华中农业大学图书馆，2005
范盛先，袁宗辉，王大菊，孔科，陈品，马敬忠．喹赛多体外抑菌试验．华中农业大学学报，2000，20(1)：51-54
何庆华．喹赛多的皮肤光敏毒性和长期毒性研究．[硕士学位论文]．武汉：华中农业大学图书馆，2005
《化学药物急性毒性试验技术指导原则》课题研究组．化学药物急性毒性试验技术指导原则．2005
《化学药物长期毒性试验技术指导原则》课题研究组．化学药物长期毒性试验技术指导原则．2005
黄玲利，袁宗辉，范盛先，王玉莲，王国永．喹赛多对鸡大肠杆菌病的预防效果研究．华中农业大学学报，2002，21(1)：47-49
黄玲利，袁宗辉，范盛先，刘登才，陈品，王大菊．喹赛多对家禽常见病原茵的体外抗菌活性研究．华南农业大学学报(自然科学版)，2003，24(2)：81-83
黄玲利．喹赛多在肉鸡的有效性与安全性研究．[博士学位论文]．武汉：华中农业大学图书馆，2005
黄幸纾，朱心强．化学毒物的生殖毒性作用．见：张桥主编，卫生毒理学基础(第三版)．北京：人民卫生出版社，2000，167
李德培．我国法定饲用抗菌药物的生物学特性．兽药与饲料添加剂，2000，5(6)：9-10
李剑勇，李金善，徐忠赞，赵荣材，苗小楼，张继瑜，鲁润华．喹烯酮在猪、鸡体内的药代动力学研究．畜牧兽医学报，2002，34(1)：94-97
李剑勇，赵荣材，李金善，徐忠赞，杜小丽，苗小楼，薛飞群，柳军玺．猪血液中~(14)C标记喹烯酮的测定及药代动力学研究．动物医学进展，2003，24(3)：78-79
李剑勇，李金善，徐忠赞，赵荣材，苗小楼，张继瑜，鲁润华．喹烯酮在猪组织中的残留研究．动物医学进展，2004a，25(4)：117-120
李剑勇，李金善，徐忠赞，赵荣材，苗小楼，张继瑜，鲁润华．高效液相色谱仪检测鸡组织器官喹烯酮残留方法的建立．动物医学进展，2004b，25(5)：100-101
李寿祺．外源化学物致突变作用．见：张铣，刘毓谷主编，毒理学．北京：北京医科大学和中国协和医科大学联合出版社，1997，282-286
梁剑平，张力，曹随中，周丽霞，周学辉，魏春梅，苗小林，刘宗平，卫增泉．新
型饲料添加剂喹胺醇致突变性及其潜在致癌性预测．中国农业科学，2001，34(6)：679-685
刘毓谷主编．卫生毒理学基础(第二版)．北京：人民卫生出版社，1999，146
卢纹岱主编．SPSS for Windows统计分析(第2版)．北京：电子工业出版社，2002
秦长川．养鸡慎用痢菌净．中国家禽，2005，27(4)：26—27
邱银生，袁宗辉，范盛先，刘登才，黄玲利，殷居易．高效液相色谱法测定猪血浆及组织中喹赛多及其脱二氧代谢物．中国兽医学报，2003a，23(4)：363-365
邱银生，袁宗辉，殷居易．猪食用组织中喹喔啉-2-羧酸的高效液相色谱检测方法．中国兽医学报，2003b，23(3)：286—288
邱银生．喹赛多在猪体内的药动学和残留研究．[博士学位论文]．武汉：华中农业大学图书馆，2003c
石年．化学毒物致突变作用．见：张桥主编，卫生毒理学基础(第三版)．北京：人民卫生出版社，2000，130-131
孙永学，冯淇辉，董漓波，陈杖榴．喹乙醇在鸡体内的毒物动力学及其生化毒性和病理学研究．Ⅰ．喹乙醇在鸡体内的毒物动力学研究．畜牧兽医学报，1998a， 29(5)：438-442
孙永学，冯淇辉，董漓波，陈杖榴．喹乙醇在鸡体内的毒物动力学及其生化毒性和病理学研究．Ⅱ．喹乙醇对鸡的生化毒性和病理学研究．畜牧兽医学报，1998b，29(6)：525-530
孙展和，李文恩，王海刚，贾俊亮．群养鸡谨防痢菌净中毒．中国兽药杂志，2005，39(7)：55-56
孙振球主编．医学统计学(研究生用)北京：人民卫生出版社，2002
王继贵．临床生化检验(第2版)．长沙：湖南科学技术出版社，1996，286
王树槐，徐士新，郑定，孙玉梅．喹乙醇诱发小鼠骨髓嗜多染红细胞微核试验．中国兽药杂志，1991，25(1)：13-14
王树槐．喹乙醇诱发CHL细胞染色体畸变试验．中国兽药杂志，1993，27(4)：27-29
王树槐，徐士新，向德林，孙玉梅，黄齐颐，徐彦波，王明志．喹乙醇亚慢性毒性试验．中国兽药杂志，1991，25(4)：13-14。
王玉春，严相林，赵荣材，李金善，杜健．新型药物饲料添加剂喹烯酮的一般毒性研究，Ⅰ．急性毒性试验．中兽医医药杂志，1992，4：13-14
王玉春，严相林，赵荣材，李金善，薛飞群，梁建平，徐忠赞，杜小丽．新型饲料药物添加剂喹烯酮一般毒性研究，Ⅱ．蓄积毒性试验．中兽医医药杂志，1993，14
王玉春，赵荣材，严相林，李金善，杜小丽，张世珍，薛飞群，徐忠赞，梁建平，张继瑜．喹烯酮对小白鼠的致癌试验．中国兽医科技，1995，25(3)：24-25
王玉春，严相林，赵荣材，李金善，薛飞群，粱建平，杜小丽，徐忠赞．新型饲料药物添加剂喹烯酮一般毒性研究，Ⅳ．亚急性毒性试验．中兽医医药杂志，1994，5：10—11
王玉春，赵荣材，严相林，李金善，杜小丽，薛飞群，徐忠赞，粱建平，张继瑜，朱以萍．新型饲料药物添加剂喹烯酮的毒性研究——繁殖及致畸试验．中国兽医科技，1993，23(4)：25-26
王玉莲，袁宗辉，朱惠玲，邱银生，范盛先．喹赛多对断奶仔猪的生长性能和胴体品质的影响．华中农业大学学报，2005，24(1)：59-62
王泽民．当代结构药物全集．北京：北京科学技术出版社，1993，455-468
徐士新，郑定．喹乙醇蓄积性毒性试验．中国兽药杂志，1992，26(2)：22-24
徐叔云．药理实验方法学．第2版．北京：人民卫生出版社，1991，1084
严相林，李金善，王玉春，赵荣材，薛飞群．喹烯酮对小白鼠精子的畸变试验．中兽医医药杂志，1997，5：13-14。
严相林，李金善，王玉春，赵荣材，薛飞群．喹烯酮的Ames试验．中兽医医药杂志，1998，5：11-12
杨杏芬．化学毒物的一般毒性作用．见：张桥主编，卫生毒理学基础(第三版)．北京：人民卫生出版社，2000，77-78
殷居易．喹赛多在鲤体内的药动学和残留研究．[硕士学位论文]。武汉：华中农业大学图书馆，2003
张力，梁剑平，袁莉刚，周丽霞，魏春梅，周学辉，常寅龙．喹胺醇饲喂肉仔鸡急性毒性试验及病理形态学研究．中兽医医药杂志，2000，6：6-8
张力，梁剑平，周丽霞，周学辉，程富胜，苗小林，袁莉刚，张德寿，刘宗平．喹胺醇对肉用仔鸡亚急性毒性试验．中国兽医科技，2001a，1(1)：27-30
张力，粱剑平，程富胜，赵四喜，周学辉，牛晓荣，苗小林，袁莉刚，张德寿，刘宗平，石旭东．喹胺醇对断奶仔猪的亚急性毒性试验．中兽医医药杂志，2001b，2：11-12
张力，梁剑平，曹随中，刘宗平，周丽霞，魏春梅，周学辉，牛晓荣，苗小林．喹胺醇对小白鼠精子致畸性试验．中兽医医药杂志，2001c，5：11-12
张力，梁剑平，曹随中，刘宗平，周丽霞，魏春梅，周学辉，牛晓荣，苗小林．喹胺醇对小白鼠致畸作用的研究．中国兽医科技，2001d，31(7)：20-21
张桥主编．卫生毒理学基础(第三版)．北京：人民卫生出版社，2000，89-94，205-282
中华人民共和国国家标准：食品安全性毒理学评价程序和方法，GB15193-194
朱蓓蕾主编．动物性食品药物残留．上海：上海科学技术出版社，1994，141-143，143-144，146-151，151-155，166-172，172-175，183-184，184-189
Beutin L, Preller E, Kowalski B. Mutagenicity of quindoxin, its metabolites, and two substituted quinoxaline-di-N-oxides. Antimicrobial Agents and Chemotherapy, 1981, 20 (3): 336-343
Cihak R, Srb V. Cytogenetic effects of quinoxaline-1, 4-dioxide-type growth-promoting agents. I. Micronucleus test in rats. Mutat Res, 1983, 116: 129-135
Cihak R, Vontorkova M. Cytogenetic effects of quinoxaline-1, 4-dioxide-type growth- promoting agents. II. Metaphase analysis in mice. Mutation Research, 1983, 117: 311-316
Cihak R, Vontorkova M. Cytogenetic effects of quinoxaline-1,4-dioxide-type growth- promoting agents. III. Transplacental micronucleus test in mice. Mutation Research, 1985,144: 81-84
Devries H, Beyersbergen Van Henegouwen G M J, Kalloe F, Berkhuysen M H J. Phototoxicity of olaquindox in the rat. Research in Veterinary Science (London), 1990a, 48,240-244.
Devreis H, Bojarski J, Donker A A, Bakri A, Beyersbergen Van Henegouwen M J. Photochemical reactions of quindoxin, olaquindox, carbodox and cyandox with protein, indicating photoallergic properties. Toxicology, 1990b, 63: 85-95
EMEA (the European Agency for the Evaluation of Medicinal Products). Note for guidance on the establishment of maximum residue limits for minor animal species. 1997
EPA (United States Environmental Protection Agency). Health Effects Test Guidelines OPPTS 870.5380 Mammalian Spermatogonial Chromosome Aberration Test. 1998a
EPA (United States Environmental Protection Agency). Health Effects Test Guidelines OPPTS 870.7800 Immunotoxicity. 1998b
EPA (United States Environmental Protection Agency). Health Effects Test Guidelines OPPTS 870.1100 Acute Oral Toxicity. 2002
FDA (United States Food and Drug Administration). Good Laboratory Practice for Nonclinical Laboratory Study. 1993
FDA (United States Food and Drug Administration). Toxicological principles for the safety assessment of food ingredients. IV C 1 a, Bacterial Reverse Mutation Test. 2000a
FDA (United States Food and Drug Administration). Toxicological principles for the safety assessment of food ingredients. IV C 1 d, Mammalian Erythrocyte Micronucleus Test. 2000b
FDA (United States Food and Drug Administration). Toxixological Principles for the Safety Assessment of Food Ingredients. IV C 10, Neurotoxicity Studies. 2000c
FDA (United States Food and Drug Administration). Guidance for Industry #3. FDA General Principles for Evaluating the Safety of Compounds Used in Food-producing Animals. 2005
Geraut C, Tripodi D. New allergens in occupational dermatology (Fren) Les nouveaux allergens en dermatologee professionnelle. Rev Frallergol Immunol Clin, 1997, 37 (7): 832-837.
Haut K T, Stebbins K E, Johnson K A, Shabrang S N, Stott W T. Subchronic toxicity of ingested 1,3-Dichloropropene in rats and mice. Fundamental and Applied Toxicology, 1996, 32: 224-232.
Holsapple M P. The plaque-forming cell (PFC) response in Immunotoxicology: An approach to monitoring the primary effector function of B lymphocytes. In Methods in Immunotoxicology (Burleson G R, Dean J H, and Munson A E, Eds.), 1995, Vol. 1, pp. 71-108, Wiley-Liss, Inc., New York.
Huber L. Good Laboratory Practice USA. Hewlett-Packard, 1993
Hutchings M, Johnson I, Hayes E, Girling A E, Thain J, Thomas K, Benstead R, Whale G, Wordon J, Maddox R, and Chown P. Toxicity reduction evaluation, toxicityidentification evaluation and toxicity tracking in direct toxicity assessment. Ecotoxicology, 2004,13(5): 475-484
Jager L P, De Graaf G J, Widjaja-Greefkes H C, Accord-Burleson C C, Van Den Dungen H M, Baars A J. Effects of feed additives and veterinary drugs on aldosterone production and release by porcine adrenal cells in vitro. Journal of Veterinary Pharmacology & Therapeutics, 1994, 17 (3): 175-185
Keithly J, Brooker J A, DeForest D K, Wu B K, Brix K. Acute and chronic toxicity of nickel to a cladoceran (Ceriodaphnia dubia) and an amphipod (Hyalella azteca). Environmental Toxicology and Chemistry, 2004,23(3): 691-696
Lafranconi W M, Long P H, Atkinson J E, Knezevich A L, Wooding W L. Chronic oxicity and carcinogenicity of olestra in Swiss CD-I mice. Food and Chemical Toxicology, 1994, 32: 789-798
Lindamood C, Farnell D R, Giles H D, Prejean J D, Collins J J, Takahashi K, Maronpot R R. Subchronic toxicity studies of t-butyl alcohol in rats and mice. Fundamental and Applied Toxicology, 1992,19: 91-100
Lonceint J, Sassolas B, Guillet G. Photoallergic reactions to olaquindox in swine raisers: role of growth promotors used in feed. Annales de Dermatologie et de Venereologie, 2001, 128: 46-48
Lu F C. Acute, short-term and long-term toxicity studies. In: Basic Toxicology: Fundamentals, Target Organs, and Risk Assessment, 1985, pp. 99-104
Nabuurs M J A, Van der Molen B J, De Graaf G J, Jager L P. Clinical signs and performance of pigs treated with different doses of carbadox, cyadox and olaquindox. Journal of Veterinary Medicine, 1990, 37 (1): 68-76.
Nastuneak J, Klouch F, Vanova L, Dvorak M, Sevick B. Three month toxicity to laboratory Norway rats: Cyadox compared with olaquindox. Biology and Chemistry of Veterinery (Prague), 1986,22: 367-380
Nastuneak J, Dvorak M, Klouch F, Vanova L. Cyadox toxicity studies over twelve months on Laboratory rats. Journal of Biologizae a Chemizace, 1987, 23(6): 549-555
Negishi T, Tanaka K, Hayatsu H. Mutagenicity of carbadox and several quinoxaline 1, 4-dioxide derivatives. Chemical and Pharmaceutical Bulletin, 1980, 28: 1347-1349
NRC (National Research Council). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC. 1996
Schauder S, Schroder W, Geier J. Olaquindox-induced airborne photoallergic contact dermatitis followed by transient or persistent light reactions in 15 pig breeders. Contact Dermatitis, 1996, 35: 344-354
Scheutwinkel Reich M, Von der Hude W. Sister-chromatid exchange in Chinese hamster V79 cells exposed to quindoxin, carbadox and olaquindox. Mutation Research, 1984, 139: 199-202
Select Committee on European Scrutiny. Select Committee on European Scrutiny fifth report, antibiotics in animal feedingstuffs, Draft Council Regulation amending Council Directive 70/524/EC concerning additives in feedingstuffs as regards withdrawal of the authorisation of certain antibiotics, 1999, (19615) 14046/98 COM (98) 763
Sevcik B, Broz J, Strakova J. Toxicity and safety evaluation of new quinolaxine growth promotant cyadox. [Journal Article] Colloques de LInra. [Paris: Institut National de la Recherche Agronomique] 1982, 8: 243-244.
Sevcik B. Research of harmless and safety of application of the growth promoter cyadox. Workshop, 1986,1:93
Spierenburg Th J, Van der Molen E J, De Graaf G J, Jager L P, Baars A J. Adrenal toxicity of carbadox, olaquindox, and cyadox in weaned piglets. Workshop 1, 1986, 97
SCAN (Standing Committee for Animal Nutrition). Short report of the meeting of the standing committee for animal nutrition. 1998
Takeda K, Flood M. Chronic Toxicity and Carcinogenicity of Sucrose Fatty Acid Esters in Fisher 344/DuCrj Rats. Regulatory Toxicology and Pharmacology, 2002, 35: 157-164
Van Der Molen E J, De Greef G J, Baars A J. Persistence of Carbadox-induced Adrenal Lesions in Pigs Following Drug Withdrawal and Recovery of Aldosterone Plasma Concentration. Journal of Comparative Pathology, 1989,100: 295-304
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL23. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Genotoxicity Testing. 2001a
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL22. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Reproduction Toxicity Testing. 2001b
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL31. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Repeat-Dose (90-Day) Toxicity Testing. 2002a
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite
Guideline GL32. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Developmental Toxicity Testing. 2002b
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL28. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Carcinogenicity Testing. 2002c
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL33. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: General Approach to Testing. 2004a
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL37. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: Repeat-Dose (Chronic) Toxicity Testing. 2004b
VICH (International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products). VICH Harmonized Tripartite Guideline GL36. Studies to Evaluate the Safety of Residues of Veterinary Drugs in Human Food: General Approach to Establish a Microbiological ADI. 2004c
Voogd C E, Van der Stel J J, Jacobs J J J A A. The mutagenic action of quindoxin, carbadox, olaquindox and some other N-oxides on bacteria and yeast. Mutation Research, 1980, 78 (3): 233-242
WHO (World Health Organization). Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 27, 1991, nos 694-705 on INCHEM.
WHO (World Health Organization). Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 33, 1994, nos 804-817 on INCHEM.
WHO (World Health Organization). Toxicological Evaluation of Certain Food Additives and Contaminants in Food. WHO Technical Report Series, No. 859, 1995, 129-138
WHO (World Health Organization). Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 51, 2003, carbadox (addendum).
Yoshimura H, Nakamura M, Koeda T, Yoshikawa K. Mutagenicities of carbadox and olaquindox—growth promoters for pigs. Mutation Research, 1981, 90 (1): 49-55.
Yoshimura H. Teratogenic assessment of carbadox in rats. Toxicology Letters, 2002, 129: 115-118.
Zhang Y L, Huang L L, Chen D M, Fan S X, Wang Y L, Tao Y F, Yuan Z H. Development of HPLC Methods for the Determination of Cyadox and Its Main Metabolites in Goat Tissues., Analytical Sciences, 2005a, 21 (12): 1495-1499
Zhang Y L, Wang Y L, Huang L L, Chen D M, Tao Y F, Yuan Z H. Effects of Cooking and Storage on Residues of Cyadox in Chicken Muscle. Journal of Agricultural and Food Chemistry, 2005b, 53(25): 9737-9341
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