Preclinical Toxicological Safety Evaluation Of Cyadox

(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 (TA₉₇, TA₉₈, TA₁₀₀, and TA₁₀₂) 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 TA₉₇ and TA₁₀₀ with and without S₉, and TA₉₈ with S₉; 10μg/plate and higher dose cyadox induced mutagenic responses in TA₉₈ without S₉; 100μg/plate and higher dose cyadox induced mutagenic responses in TA₁₀₂ with and without S₉. Mutagenic responses are dose related in all the four bacterial strains. 100μg/plate cyadox suppressed the growth of TA₉₇ slightly; 250μg/plate cyadox suppressed the growth of TA₉₇, TA₉₈ and TA₁₀₀ to a certain extent. 25μg/plate olaquindox induced mutagenic responses for all the four bacterial strains except TA₁₀₂ without S₉. Olaquindox exhibited higher mutagenicity in TA₁₀₂ 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 (F₀) 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. F₀ 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 F₂ 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 F₀ 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 F₀ 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 F₀ increased to a certain extent in all the treatment groups and that of female F₀ 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.