Preclinical Toxicology Of Quinocetone

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 10ï¼…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 0.5ï¼…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 0.5ï¼…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.