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Assessment of thirteen-week subchronic oral toxicity of cyadox in Beagle dogs
Regulatory Toxicology and Pharmacology 73 (2015) 652e659
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Assessment of thirteen-week subchronic oral toxicity of cyadox in Beagle dogs Xu Wang a, Wen Zhou b, Awais Ihsan d, Dongmei Chen b, Guyue Cheng b, Haihong Hao c, Zhenli Liu c, Yulian Wang c, **, Zonghui Yuan a, b, c, * a
National Reference Laboratory of Veterinary Drug Residues (HZAU) and MOA Key Laboratory for Detection of Veterinary Drug Residues, China MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei 430070, China c Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan, Hubei, China d Department of Biosciences, COMSATS Institute of Information Technology, Sahiwal, Pakistan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2015 Received in revised form 16 September 2015 Accepted 18 September 2015 Available online 25 September 2015
Cyadox (2-formylquinoxaline-N1,N4-dioxide cyanocetylhydrazone) is a new antimicrobial agent and growth-promoter to be used in food-producing animals. Although its toxicity has been clearly documented in rodents, no study is available in non-rodent animals. Therefore, we studied the subchronic effects of cyadox in Beagle dogs to provide additional information with which to establish safety criteria for human exposure. For this purpose, 36 Beagle dogs, 18 males and 18 females, were divided into four groups and fed diets containing 0, 100, 450 and 2500 mg/kg of cyadox, respectively, for 13 weeks. It was found that there were no significant changes among the examined parameters, except for an increase in the level of serum potassium (Kþ) in 2500 mg/kg cyadox group in males at week 13 of the study. However, the Kþ level returned to normal during the recovery period. In conclusion, cyadox showed slight effects in Beagle dogs in the subchronic oral toxicity study. The no-observed-adverse-effect level of cyadox was considered to be 450 mg/kg diet, which equates to approximately 15.3e15.4 mg/kg b.w./day. The study provided subchronic effects of cyadox in Beagle dogs, suggesting that cyadox might present mild toxicity in non-rodents. © 2015 Elsevier Inc. All rights reserved.
Keywords: Cyadox Quinoxaline Toxicity Subchronic toxicity Beagle dogs
1. Introduction Abbreviations: ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; b.w., body weight; BUN, blood urea nitrogen; Ca, calcium; CHO, total cholesterol; Cl, chloride; CREA, creatinine; FDA, Food and Drug Administration; GLU, glucose; HCT, hematocrit; HGB, hemoglobin; H&E, hematoxylin and eosin; i.m., intramuscular; Kþ, potassium; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MPV, mean platelet volume; Naþ, sodium; NOAEL, one-way analysis of variance; OECD, organization for economic co-operation and development; P, inorganic phosphorus; PCT, plateletcrit; PDW, platelet distribution width; PLT, platelet count; PMCol, pentamethylchromanol; QdNOs, quinoxaline 1,4-dioxide derivatives; RBC, red blood cell count; RDW, red cell volume distribution; TG, triglyceride; TP, total protein; URE, urea; VICH, international cooperation on harmonization of technical requirements for the registration of veterinary medicinal products; WBC, white blood cell count. * Corresponding author. National Reference Laboratory of Veterinary Drug Residues (HZAU) and MOA Key Laboratory for Detection of Veterinary Drug Residues, China. ** Corresponding author. E-mail addresses: [email protected] (Y. Wang), yuan5802@mail. hzau.edu.cn (Z. Yuan). http://dx.doi.org/10.1016/j.yrtph.2015.09.023 0273-2300/© 2015 Elsevier Inc. All rights reserved.
Quinoxaline 1,4-dioxide derivatives (QdNOs) are synthetic drugs with the wide range of biological activities like growth promoting, antibacterial, anti-candida, anti-tubercular, anticancer and antiprotozoal properties (Vicente et al., 2009; Zhang et al., 2015). Cyadox, 2-formylquinoxaline-N1,N4-dioxide cyanocetylhydrazone (CAS No. 65884-46-0, C12H9N5O3) (Fig. 1), is one of the QdNO member with outstanding antibacterial activity against pathogenic bacteria such as Pasteurella multocida and Escherichia coli (Ding et al., 2006a). It can also promote animal growth and improve feed efficiency with the potential to be used widely in husbandry in the future (Ding et al., 2006a, 2006b; Wang et al., 2005). QdNOs usually have serious hazards increased along with the increasing doses added as animal feed addictives. Compared with other QdNOs such as carbadox, olaquindox and mequindox, cyadox was considered to be less harmful and showed good safety in mutagenicity studies (Ihsan et al., 2013a, 2013b), 90-day feeding studies (Fang et al., 2006), teratogenicity and reproduction studies
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2002). The study will provide information on the major toxic effects, indicate target organs, provide an estimate of a no-observedadverse-effect (NOAEL) level of cyadox exposure, and can provide experimental evidence of the safety of cyadox for future veterinary clinical trials. 2. Materials and methods 2.1. Drug and chemicals Cyadox (molecular weight 271.23, purity 98%) was obtained from the Institute of Veterinary Pharmaceuticals, Huazhong Agricultural University (Wuhan, PR China). All other reagents were of analytical grade. 2.2. Animals Fig. 1. Structure of cyadox.
(Wang et al., 2011a), chronic studies (Wang et al., 2011b) and carcinogenicity studies (Sevcik, 1986). Carbadox and olaquindox were concluded to be mutagenic and carcinogenic with developmental and reproductive toxicities (Yoshimura, 2002; WHO, 1991a; WHO, 1991b). In rats, a dose-dependent decrease in weight gain and food consumption was noted when carbadox was administered at dose levels of 50 and 100 mg/kg b.w./day for 30 days (WHO, 1991b). In dogs, carbadox was found to be responsible of vomiting, weight loss and elevated serum alanine aminotransferase (ALT) at dose levels of 25 and 50 mg/kg b.w./day (WHO, 1991b). The adrenal cell atrophy was reported in the rats given olaquindox at doses of 60 and 180 mg/kg b.w./day for 5 days/week for 13 weeks. In dogs, fatty degeneration was observed in kidney tubules of dogs given 60 or 180 mg/kg b.w./day of olaquindox for 90 days (WHO, 1991a). Previously in a subchronic study, we have occasionally found that 275 mg/kg mequindox led changes in Naþ and Kþ levels in serum after 90 days of feeding in rats (Huang et al., 2009; Ihsan et al., 2010). Quinocetone showed mild toxicity and it at 1800 mg/kg dietary level in rats produced low toxicity for liver and depressed body weight gain in the subchronic oral study (Wang et al., 2010). Cyadox was thought to be a potential replacement for carbadox and olaquindox (Wang et al., 2011a). According to the toxicity guideline of the Food and Drug Administration (FDA), the Organization for Economic Co-operation and Development (OECD) and the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) for new drugs used in food animals, the subchronic 90-day feeding toxicity study should be performed in a rodent species and a non-rodent species (FDA, 2003; OECD, 1998; VICH, 2002; VICH, 2004). However, according to our limited knowledge, nearly all toxicity tests were performed in rodents and very few toxicological data are available in non-rodents, which is too limited to evaluate the safety of cyadox sufficiently and convincingly. As a potential replacement of olaquindox and carbadox, cyadox, with the potential to be used widely and over long-periods, must be studied systemically for toxicity. Therefore, it is necessary to perform the study in non-rodents. The 90-day study of cyadox in Beagle dogs was the main non-rodent test and could provide additional information with which to establish safety criteria for human exposure. To study the safety spectrum of cyadox, the present study was performed to evaluate the potential subchronic feeding toxicity test in Beagle dogs with a wide range of doses. The subchronic toxicity of cyadox was tested in 13-weeks feeding study in Beagle dogs according to OECD Guideline 409 and VICH Guideline 31 under modern Good Laboratory Practice Regulations (OECD, 1998; VICH,
Eighteen male and eighteen female Beagle dogs (5e6 months old), weighing 5.6e6.5 kg, were purchased from Guangdong National Beagles Resources Research Center (Guangzhou, PR China). The animal room was maintained at a temperature of 18e25 C, with a relative humidity of 42e69% and a 12-h light/dark cycle. The air ventilation was given once every 30 min. Prior to the initiation of dosing, the dogs were quarantined for 2 weeks and then acclimatized to the study environmental conditions before use. The dogs were fed 300 g certified commercial diet (Ke Ao Xie Li Co. Ltd., Beijing, PR China) at a fixed time per day and tap water ad libitum during the study. Each dog, with a unique ear tag number, was raised in a steel cage (1000 1000 900 mm). The animal numbers were also attached to each cage. All dogs enjoyed outdoor exercise at least once every week. The study was approved by the Ethical Committee of the Faculty of Veterinary Medicine at Huazhong Agricultural University and was in accordance with NIH Publication 85-23 ‘‘Guide for the Care and Use of Laboratory Animals’’ (NRC, 1996). 2.3. Experimental design Eighteen male and eighteen female dogs were randomly assigned to four groups based on their body weights using a randomized block model. The control, low-dose, middle and high-dose groups of cyadox included 5, 4, 4 and 5 dogs per sex, respectively. Each group of dogs was fed with the basal diets mixed with 0, 100, 450 and 2500 mg cyadox/kg diet. To ensure homogeneity, the drug was thoroughly mixed with the diet. The diets were mixed separately by group and placed in properly labeled containers. The diets were prepared every 4 weeks, and the stability and homogeneity were verified prior to the study. The dosage of 100 mg cyadox/kg diet was selected as the minimal dose as this was the maximal recommended concentration of cyadox incorporated as feed additive of pig for growth promoting (Wang et al., 2011b). Also, 2500 mg cyadox/kg diet inhibited the feed intake and body weight of rats in our previous study and was used here as the high dose (Fang et al., 2006). The dose level of 450 mg cyadox/kg diet was close to the geometric average of the high and low dose, and was used as the medium dose. At the end of 13 weeks, female dogs (2, 4, 4 and 3 dogs/group) and male dogs (3, 4, 4 and 3 dogs/group) in control, 100, 450 and 2500 mg cyadox/kg diet groups were sacrificed under anesthesia with intravenous sodium pentobarbital, respectively. The remaining dogs in the control and high-dose groups after 13 weeks were administered with control feed for 2 weeks and euthanized in the same manner to perform the examination. Clinical observations were performed on both a daily and weekly basis. Hematology, serum biochemistry and electrocardiogram of dogs were examined before the start of the study, at week 6 and at
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necropsy. Pathology findings were also determined at necropsy. 2.4. Clinical observations Each dog was observed at least twice a day throughout the study for mortality, signs of toxicity, any behavioral change in gait, posture, response to handling and changes in skin, fur, eyes, mucous membranes, occurrences of secretions and excretions. An ophthalmological examination was conducted by observing the appearance of the eyes with the naked eye before the administration of a drug and in the last week of the experiment. Individual body weight and food consumption were recorded in quarantine phase, and weekly during the treatment periods and fasted weights were assessed at necropsy. 2.5. Hematology
conducted by using a routine paraffin-embedding technique. Sections with a thickness of 5 mm stained with hematoxylin and eosin (H&E) were examined under a light microscope for morphological alterations. As a necessary step to determine the NOAEL in target organs, other tissues such as the liver and kidneys from the treatment groups were also examined histologically. 2.9. Calculations of feed efficiency and drug intake The equation of feed efficiency was (the increase weight of each dog for the week/weekly feed consumption) 100%. The dose levels (mg/kg b.w./day) were calculated by using the nominal concentration of drugs in the diet, the mean daily feed consumption, and the body weight for the week. The equation was (mean weekly feed consumption of each dog/7)/(the body weight for the week) nominal concentration of drugs in the diet.
Following an overnight fast, blood samples were collected from the radial vein. The anticoagulants were potassium EDTA for hematology tests. Hematological measurements and calculations were performed with Coulter HmX Hematology Analyzer (Beckman Coulter Inc., Fullerton, CA, USA). Hematological evaluations included red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), hemoglobin concentration (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), blood platelet count (PLT), red cell volume distribution (RDW), platelet distribution width (PDW), mean platelet volume (MPV) and white blood cell count (WBC).
2.10. Statistical analysis
2.6. Serum biochemistry
The intakes per kg bodyweight per day achieved in the study were calculated based on the food intakes and bodyweights. They are presented in Table 1.
For clinical biochemistry, blood samples were placed in serum tubes at room temperature for approximately 30 min to obtain the serum aliquots. After clotting, the blood tubes were centrifuged at 2500 rpm for 15 min with Himac CR 21 G centrifuge (Hitachi, Tokyo, Japan). Supernatants were decanted and stored at 20 C for further analysis. Serum biochemistry was assessed using the Synchron Clinical System CX4 (Beckman Coulter, Brea, CA USA) according to the manufacturer's instructions (Beijing Leadman Biochemistry Technology Co. Ltd, Beijing, China). Parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), glucose (GLU), urea (URE), total cholesterol (CHO), creatinine (CREA), triglyceride (TG), sodium (Na), potassium (K), chloride (Cl), calcium (Ca) and inorganic phosphorus (P). 2.7. Electrocardiogram PeR intervals, QeT intervals, QRS duration and heart rate of ECG Ⅱ lead in dogs were recorded by an ECG system (ECG-6511, Shanghai Kohden Medical Electronic Instrument Corp., Shanghai, China).
Statistical analyses were performed by comparing the treatment groups with the control group using the SPSS 13.0 program. Levene's test was used to examine variance homogeneity. One-way analysis of variance (ANOVA) was performed for data with significant dispersion, and this significance was confirmed with Dunnett's multiple comparison. The two-sided level of statistical significance was preset at p < 0.05 or 0.01. 3. Results
3.1. Survival and clinical signs All dogs survived, except one female in the control group who incidentally suffered severe intestinal volvulus on the fifth day of the study, and was euthanized under anesthesia with intravenous sodium pentobarbital. No other abnormal changes in appearance or behavior were observed in any of the groups. 3.2. Body weight, food efficiency and food consumption The body weights of both male and female dogs slightly increased in the group of 100 mg/kg cyadox in most of the weeks during the study, presenting a tendency to promote the effect of cyadox. However, no significant differences were noted in any of the groups when compared with the controls (Fig. 2 and Tables S12). Also, no significant differences in food efficiency and food consumption were found among the four groups (Tables S3-6). However, when compared with that of the 1st week and the 3rd week, significant decreases of feed efficiency in the 2nd week in all the groups were noted. Considering the calculation of food efficiency,
2.8. Pathology examinations All of the organs/tissues were carefully examined macroscopically, and gross lesions were recorded. The absolute and relative (organ to body weight ratios) weights of organs and tissues of each animal, including the heart, lungs, brain, kidneys, adrenal glands, liver, spleen, thymus, thyroid, pancreas, testes, ovaries and uterus were measured separately. The tissues from each animal were preserved in 10% neutral-buffered formalin and slides were prepared for histopathological examination. Testis and epididymes were fixed in Bouin's solution for histopathology as previously described (Shin et al., 2010). Histopathological examination was
Table 1 Dose levels calculated in the subchronic test. Dietary dose (mg/kg diet)
100 (n ¼ 8) 450 (n ¼ 8) 2500 (n ¼ 10)
Calculated dose level (mg/kg b.w./ day) Female
Male
3.4 ± 0.1 15.3 ± 0.5 87.0 ± 3.6
3.2 ± 0.1 15.4 ± 0.5 85.2 ± 3.2
Note: Values represent means ± SD, n ¼ number of animals. The 100, 450 and 2500 mg/kg diet groups of cyadox have 5, 4, 4 and 5 dogs per sex, respectively.
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Fig. 2. Mean body weights (A), food efficiency (B) and daily food intake of dogs (C) in the 90 days feeding study.
the much lower values of body weight gains in the 2nd week were suggested to be the main reason. However, the reason was still not clear. 3.3. Hematology and serum biochemistry There was a significant increase in PDW and WBC levels in the female dogs of the 100 and 450 mg/kg cyadox groups at weeks 6 and 13 when compared with the control group. However, the changes did not have a doseeresponse relationship. Significant decreases of MPV levels in the female dogs of the 2500 mg/kg cyadox group were observed at week 6. However, the change of MPV was within the physiological range, and did not appear at week 13. Therefore, these occasionally achieved statistical significances were not considered to be related to the treatment with cyadox (Tables 2 and 3). For serum biochemistry, serum Kþ level increased significantly in male dogs in the 2500 mg/kg cyadox group compared with the controls at week 13, but returned to normal during the recovery
phase. Other serum biochemical parameters were not affected by the treatment for either gender (Tables 4 and 5).
3.4. Temperature and electrocardiogram No significant changes were found in the temperature (Table S7), and electrocardiogram (Tables S8-9). 3.5. Organ weights and pathological findings No significant differences in absolute and relative organ weights were found among the four groups for both genders (Tables S1011). Necropsy and detailed histopathological examination did not reveal any lesions in any of the organs of the dogs. In particular, no microscopic changes were observed in the liver, kidney and adrenal tissues, which are known target organs of quinoxalines (Fig. 3) (Fang et al., 2006; Ihsan et al., 2010; Wang et al., 2011a, 2011b, 2010).
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Table 2 Hematology parameters of male dogs treated with cyadox (0, 100, 450 and 2500 mg/kg diet) for 13 weeks. Week (mg/kg diet)
WBC (109/l)
0 week 0 (n ¼ 5) 16.1 100 (n ¼ 4) 14.7 450 (n ¼ 4) 12.2 2500 (n ¼ 5) 11.8 6 weeks 0 (n ¼ 5) 14.9 100 (n ¼ 4) 15.0 450 (n ¼ 4) 12.7 2500 (n ¼ 5) 11.8 13 weeks 0 (n ¼ 5) 13.6 100 (n ¼ 4) 13.4 450 (n ¼ 4) 13.2 2500 (n ¼ 5) 13.9 Recovery periods 0 (n ¼ 2) 12.3 2500 (n ¼ 2) 12.4
RBC (1012/l)
HGB (g/l)
HCT (l/l)
MCV (fl)
MCH (pg)
MCHC (g/l)
RDW (%)
PLT (109/l)
MPV (fl)
PCT (%)
PDW (%)
± ± ± ±
4.0 4.8 2.4 2.0
5.95 6.02 6.06 5.78
± ± ± ±
0.41 0.30 0.57 0.95
136 137 135 127
± ± ± ±
9 7 14 21
0.372 0.382 0.374 0.341
± ± ± ±
0.028 0.020 0.039 0.046
62.6 63.5 61.6 61.3
± ± ± ±
1.8 1.2 1.2 1.5
22.8 22.8 22.1 21.7
± ± ± ±
0.6 0.3 0.1 0.7
365 359 362 355
± ± ± ±
5 2 5 4
15.1 14.4 14.1 15.2
± ± ± ±
1.0 1.0 0.8 1.1
347 311 345 293
± ± ± ±
92 52 76 93
8.4 8.4 8.3 8.5
± ± ± ±
0.6 0.5 0.3 0.3
0.278 0.260 0.297 0.237
± ± ± ±
0.092 0.039 0.054 0.118
15.8 16.0 16.0 16.2
± ± ± ±
0.7 0.4 1.0 0.8
± ± ± ±
2.4 3.7 2.4 1.7
6.37 5.88 5.94 5.51
± ± ± ±
0.51 0.39 0.18 0.62
143 133 137 125
± ± ± ±
10 10 5 11
0.398 0.358 0.372 0.348
± ± ± ±
0.027 0.023 0.010 0.038
62.5 60.9 62.7 64.2
± ± ± ±
2.0 0.2 2.0 1.6
22.7 22.6 23.0 23.4
± ± ± ±
0.9 0.6 0.8 0.5
332 371 368 364
± ± ± ±
65 9 5 6
13.0 14.0 13.1 12.5
± ± ± ±
0.6 1.3 0.4 0.3
271 298 314 301
± ± ± ±
73 64 98 69
8.6 8.3 8.8 8.5
± ± ± ±
0.8 0.4 0.8 0.4
0.252 0.229 0.246 0.251
± ± ± ±
0.081 0.082 0.097 0.131
15.6 16.1 16.5 15.9
± ± ± ±
1.0 1.0 1.4 1.1
± ± ± ±
1.4 2.2 4.0 4.0
6.27 5.69 6.06 5.46
± ± ± ±
0.74 0.47 0.55 0.52
143 131 141 128
± ± ± ±
16 9 12 12
0.389 0.351 0.380 0.333
± ± ± ±
0.045 0.022 0.027 0.033
62.1 61.8 62.8 64.7
± ± ± ±
2.4 1.5 2.0 1.1
22.8 23.1 23.2 23.7
± ± ± ±
1.0 0.5 0.4 0.5
367 372 370 367
± ± ± ±
3 6 6 4
13.4 13.8 13.1 12.9
± ± ± ±
0.6 0.3 0.5 0.3
292 347 355 295
± ± ± ±
58 64 63 109
8.8 8.5 8.1 8.8
± ± ± ±
1.1 0.4 0.3 1.2
0.254 0.292 0.287 0.253
± ± ± ±
0.036 0.047 0.042 0.079
15.4 15.0 15.1 15.1
± ± ± ±
0.7 0.3 0.3 0.4
± 0.2 ± 0.2
6.02 ± 0.23 5.49 ± 0.41
124 ± 6 130 ± 12
0.355 ± 0.023 0.313 ± 0.026
59.1 ± 1.3 62.6 ± 1.6
22.1 ± 0.6 23.0 ± 0.4
349 ± 6 367 ± 4
14.1 ± 1.1 13.2 ± 0.6
336 ± 8 363 ± 57
8.3 ± 0.1 8.1 ± 0.6
0.277 ± 0.008 0.271 ± 0.004
14.8 ± 0.1 15.5 ± 0.1
Note: Values represent means ± SD, n ¼ number of animals.
cyadox group (85.2e87.0 mg/kg b.w./day) in dogs showed almost no toxicological effects on liver function, behaviors and body weights. This conclusion was concordant with the acute toxicities in Wistar rats which showed that the acute toxicities of different quinoxalines were carbadox > olaquindox > cyadox (Wang et al., 2010). In previous studies, other known members of QdNOs such as olaquindox and carbadox were evaluated in beagle dogs. Inappetence and salivation were noted when the dogs were given oral doses of 60 mg/kg b.w./day olaquindox in gelatin capsules for 90 days. Histopathological observations revealed that 60 or 180 mg/kg b.w./day olaquindox induced fatty degeneration in liver and the kidney tubules (WHO, 1991a). The short-term toxicity of carbadox was studied by dosing animals 6 days/week for three weeks, using Beagle dogs (1 dog/sex/dose). The initial dose was 25 or 50 mg/kg b.w./day carbadox via oral capsule. It was found that the dogs lost weight and their serum ALT levels were elevated. It was concluded that carbadox was emetic and possibly hepatotoxic in dogs (WHO, 1991b). In the present study, the results indicated that cyadox was safer than olaquindox and carbadox in beagle dogs. In the present study, no effect in body weight was found in dogs
4. Discussion There were several studies in the literature which have evaluated the toxicity of cyadox in rodents, particularly in rats (Fang et al., 2006; Wang et al., 2011a, 2011b). However, no research on cyadox has been presented in non-rodents. Therefore, the subchronic toxicity study of cyadox in Beagle dogs was worth performing to further evaluate the potential effects of cyadox in nonrodents. The present study first demonstrated the toxicity of cyadox in dogs in a subchronic (90-day oral) trial and offered useful scientific knowledge about the administration of cyadox in a diets. In the present study, it was found that cyadox at 2500 mg/kg dietary level (85.2e87.0 mg/kg b.w./day) in dogs has no significant toxic effects on body weight gain, food intake, feed efficiency, body temperature, electrocardiographic, absolute and relative organ weights, hematologic and histological changes in the kidneys, adrenal glands and liver for both genders, except for an increase in the level of serum potassium (Kþ) in 2500 mg cyadox/kg diet group in males at week 13 of the study. Compared with olaquindox and carbadox, the 2500 mg/kg
Table 3 Hematology parameters of female dogs treated with cyadox (0, 100, 450 and 2500 mg/kg diet) for 13 weeks. Week (mg/kg diet)
WBC (109/l)
0 week 0 (n ¼ 5) 12.5 100 (n ¼ 4) 15.3 450 (n ¼ 4) 12.9 2500 (n ¼ 5) 14.7 6 weeks 0 (n ¼ 4) 12.2 100 (n ¼ 4) 15.3 450 (n ¼ 4) 13.1 2500 (n ¼ 5) 14.3 13 weeks 0 (n ¼ 4) 12.2 100 (n ¼ 4) 15.3 450 (n ¼ 4) 12.3 2500 (n ¼ 5) 11.2 Recovery periods 0 (n ¼ 2) 11.3 2500 (n ¼ 2) 11.6
RBC (1012/l)
HGB (g/l)
HCT (l/l)
MCV (fl)
MCH (pg)
MCHC (g/l)
RDW (%)
PLT (109/l)
MPV (fl)
PCT (%)
PDW (%)
± ± ± ±
0.9 2.8 4.0 3.5
6.06 5.76 5.87 5.65
± ± ± ±
0.49 0.12 0.51 0.60
138 130 132 128
± ± ± ±
12 4 7 16
0.385 0.362 0.368 0.353
± ± ± ±
0.039 0.011 0.019 0.041
63.5 62.8 62.8 62.4
± ± ± ±
2.5 1.2 2.6 1.4
22.7 22.6 22.6 22.7
± ± ± ±
0.2 0.5 1.0 0.6
358 359 359 363
± ± ± ±
11 2 3 3
14.5 15.0 14.0 14.3
± ± ± ±
1.3 0.2 1.1 1.5
299 332 407 380
± ± ± ±
93 64 37 66
8.9 8.6 8.3 8.2
± ± ± ±
0.8 1.1 0.3 0.5
0.284 0.278 0.338 0.293
± ± ± ±
0.046 0.022 0.020 0.103
16.2 15.7 15.5 15.4
± ± ± ±
0.6 0.5 0.3 0.7
± ± ± ±
0.3 2.4 3.6 3.0
5.60 6.29 5.99 5.92
± ± ± ±
1.09 0.13 0.31 0.35
139 142 137 136
± ± ± ±
16 8 7 8
0.363 0.394 0.374 0.378
± ± ± ±
0.080 0.015 0.024 0.022
64.5 62.6 62.4 63.7
± ± ± ±
2.4 1.8 1.8 1.5
23.3 23.1 22.8 22.9
± ± ± ±
1.1 0.4 0.7 0.7
356 333 366 360
± ± ± ±
20 61 6 2
13.2 13.5 13.5 12.7
± ± ± ±
0.6 1.0 0.7 0.6
324 322 283 302
± ± ± ±
57 87 55 82
9.2 8.9 8.2 8.0
± ± ± ±
0.2 0.7 0.7 0.5a
0.262 0.303 0.204 0.250
± ± ± ±
0.106 0.050 0.085 0.068
15.1 15.9 16.4 16.0
± ± ± ±
0.5 0.8 0.5a 0.7
± ± ± ±
0.9 1.3a 1.3 1.6
6.26 6.30 5.56 5.60
± ± ± ±
0.71 0.29 0.28 0.37
147 144 127 130
± ± ± ±
15 5 9 9
0.395 0.393 0.346 0.357
± ± ± ±
0.047 0.010 0.024 0.022
63.2 62.4 62.2 63.7
± ± ± ±
2.7 1.8 1.3 1.5
23.5 22.8 22.8 23.2
± ± ± ±
0.8 1.4 0.7 0.4
372 365 367 365
± ± ± ±
8 13 5 6
13.3 13.7 13.1 12.6
± ± ± ±
0.5 0.9 0.5 0.5
331 317 358 343
± ± ± ±
77 46 39 29
8.8 8.7 8.3 7.9
± ± ± ±
0.6 0.5 0.8 0.3
0.287 0.272 0.293 0.270
± ± ± ±
0.048 0.029 0.027 0.022
14.8 14.9 15.0 15.1
± ± ± ±
0.3 0.3 0.3 0.2
± 1.6 ± 1.2
5.32 ± 0.71 5.42 ± 0.13
121 ± 12 124 ± 2
0.340 ± 0.041 0.335 ± 0.003
Note: Values represent means ± SD, n ¼ number of animals. a Significantly different from control group at p < 0.05.
64.0 ± 1.0 62.8 ± 1.8
22.7 ± 0.8 22.8 ± 0.1
354 ± 7 369 ± 9
13.4 ± 0.4 12.6 ± 0.1
391 ± 19 355 ± 6
7.9 ± 04 8.2 ± 0.8
0.256 ± 0.069 0.242 ± 0.014
15.4 ± 0.5 15.4 ± 0.1
Table 4 Serum biochemical parameters of male dogs treated with cyadox (0, 100, 450 and 2500 mg/kg diet) for 13 weeks. Week (mg/kg diet)
GLU (mmol)
ALB (g/L)
ALP (U/L)
ALT (U/L)
AST (U/L)
URE (mmol/L)
CRE (mmol/L)
CHO (mmol/L)
TG (mmol/L)
Na (mmol/L)
K (mmol/L)
Ca (mmol/L)
CL (mmol/L)
PO4 (mmol/L)
± ± ± ±
0.30 0.13 0.27 0.46
66.3 67.5 66.1 68.2
± ± ± ±
2.9 4.6 6.0 6.2
31.3 30.8 30.5 32.5
± ± ± ±
1.5 1.4 2.4 2.0
82 86 90 84
± ± ± ±
3 8 2 5
29 30 34 35
± ± ± ±
6 4 8 9
30 35 38 41
± ± ± ±
4 5 9 18
4.50 3.88 3.98 3.85
± ± ± ±
0.73 0.74 0.51 0.86
37.1 37.9 44.1 34.4
± ± ± ±
5.5 5.9 6.1 7.6
3.19 2.68 2.45 3.58
± ± ± ±
0.22 0.45 0.53 0.89
0.46 0.40 0.44 0.46
± ± ± ±
0.06 0.09 0.04 0.07
147.5 146.5 148.4 147.7
± ± ± ±
2.0 2.6 2.0 2.9
5.45 5.48 5.48 5.42
± ± ± ±
0.26 0.32 0.31 0.35
2.48 2.52 2.50 2.45
± ± ± ±
0.03 0.07 0.06 0.11
98.1 98.8 100.8 100.8
± ± ± ±
3.7 2.7 5.7 3.8
2.24 2.19 2.20 2.30
± ± ± ±
0.10 0.25 0.22 0.17
± ± ± ±
0.42 0.48 0.25 0.23
62.7 60.1 65.6 68.2
± ± ± ±
7.8 2.4 5.2 5.5
28.4 27.6 29.2 30.2
± ± ± ±
3.5 1.2 1.4 1.2
90 91 94 92
± ± ± ±
6 2 1 1
34 25 26 26
± ± ± ±
4 6 8 1
40 41 46 36
± ± ± ±
14 6 11 2
5.72 5.65 6.49 5.70
± ± ± ±
1.49 0.55 1.31 1.76
49.1 45.0 48.9 51.5
± ± ± ±
6.7 7.4 7.1 10.1
2.29 2.12 1.99 2.37
± ± ± ±
0.52 0.22 0.28 0.36
0.45 0.48 0.47 0.50
± ± ± ±
0.15 0.05 0.04 0.07
146.8 147.8 149.6 150.7
± ± ± ±
2.6 2.8 3.8 5.4
5.44 5.65 5.52 5.52
± ± ± ±
0.25 0.03 0.28 0.23
2.35 2.44 2.45 2.41
± ± ± ±
0.20 0.01 0.03 0.02
99.5 96.5 96.9 101.4
± ± ± ±
5.1 1.3 0.6 1.8
2.07 2.27 2.37 2.25
± ± ± ±
0.35 0.15 0.12 0.27
± ± ± ±
0.85 0.82 0.62 1.00
69.7 62.7 67.3 68.6
± ± ± ±
5.3 1.0 7.0 3.6
31.0 29.4 31.0 31.4
± ± ± ±
1.0 1.7 4.3 0.9
144 149 112 139
± ± ± ±
35 13 18 69
37 41 31 25
± ± ± ±
9 8 7 4
49 52 46 38
± ± ± ±
4 12 7 6
4.77 4.42 5.17 4.31
± ± ± ±
0.51 0.91 1.60 0.79
50.6 51.2 55.8 52.0
± ± ± ±
5.8 8.5 3.9 10.3
2.52 2.15 2.18 2.42
± ± ± ±
0.39 0.36 0.47 0.34
0.57 0.59 0.47 0.46
± ± ± ±
0.09 0.07 0.09 0.07
147.2 145.8 146.0 146.2
± ± ± ±
0.7 1.3 1.0 0.6
5.31 5.71 5.77 6.08
± ± ± ±
0.36 0.46 0.14 0.41a
2.53 2.47 2.61 2.67
± ± ± ±
0.09 0.09 0.14 0.09
100.2 102.5 104.5 104.6
± ± ± ±
1.1 2.2 0.8 4.9
1.62 1.73 1.72 1.88
± ± ± ±
0.19 0.24 0.07 0.18
± 0.71 ± 0.63
62.9 ± 11.2 58.6 ± 2.1
28.2 ± 4.2 26.9 ± 1.0
92 ± 1 87 ± 13
41 ± 10 41 ± 8
38 ± 11 39 ± 1
4.62 ± 0.49 4.01 ± 0.81
59.9 ± 0.6 59.1 ± 0.4
2.19 ± 0.44 2.02 ± 0.13
0.32 ± 0.05 0.32 ± 0.01
146.5 ± 0.9 146.6 ± 0.1
5.43 ± 0.48 5.33 ± 0.43
2.50 ± 0.42 2.46 ± 0.16
108.6 ± 5.8 104.3 ± 4.7
1.93 ± 0.50 1.86 ± 0.38
CHO (mmol/L)
TG (mmol/L)
Na (mmol/L)
K (mmol/L)
Ca (mmol/L)
CL (mmol/L)
PO4 (mmol/L)
Note: Values represent means ± SD, n ¼ number of animals. a Significantly different from control group at p < 0.05.
Table 5 Serum biochemical parameters of female dogs treated with cyadox (0, 100, 450 and 2500 mg/kg diet) for 13 weeks. Week (mg/kg diet)
GLU (mmol/L)
0 week 0 (n ¼ 5) 5.95 100 (n ¼ 4) 6.05 450 (n ¼ 4) 5.94 2500 (n ¼ 5) 5.94 6 weeks 0 (n ¼ 4) 5.54 100 (n ¼ 4) 5.74 450 (n ¼ 4) 5.46 2500 (n ¼ 5) 5.62 13 weeks 0 (n ¼ 4) 3.65 100 (n ¼ 4) 2.85 450 (n ¼ 4) 3.34 2500 (n ¼ 5) 4.49 Recovery periods 0 (n ¼ 2) 5.65 2500 (n ¼ 2) 5.80
TP (g/L)
ALB (g/L)
ALP (U/L)
ALT (U/L)
AST (U/L)
URE (mmol/L)
CRE (mmol/L)
± ± ± ±
0.43 0.58 0.22 0.58
66.4 65.0 71.0 66.5
± ± ± ±
4.6 4.0 4.2 4.3
31.3 31.5 31.6 30.3
± ± ± ±
1.5 0.8 1.6 1.6
81 83 89 84
± ± ± ±
5 7 5 7
34 34 39 38
± ± ± ±
5 5 5 13
37 32 41 42
± ± ± ±
9 6 4 11
4.59 3.99 3.90 4.74
± ± ± ±
0.94 0.52 0.47 0.27
40.4 40.9 42.3 39.4
± ± ± ±
5.6 6.8 4.1 4.6
3.04 2.71 2.78 3.16
± ± ± ±
0.36 0.62 0.54 0.47
0.52 0.40 0.48 0.47
± ± ± ±
0.11 0.08 0.07 0.05
146.7 147.4 146.3 146.6
± ± ± ±
1.3 1.2 0.4 2.2
5.39 5.42 5.50 5.43
± ± ± ±
0.34 0.27 0.32 0.37
2.52 2.51 2.49 2.44
± ± ± ±
0.07 0.04 0.10 0.02
101.1 102.4 102.9 100.0
± ± ± ±
2.5 3.0 4.0 2.9
2.23 2.33 2.13 2.21
± ± ± ±
0.20 0.12 0.09 0.20
± ± ± ±
0.20 0.16 0.16 0.24
62.0 63.5 60.8 68.5
± ± ± ±
8.6 2.5 5.4 3.3
29.2 29.2 28.0 30.1
± ± ± ±
3.8 1.6 2.4 1.1
88 89 92 94
± ± ± ±
5 5 2 1
37 32 24 27
± ± ± ±
13 10 8 3
38 47 41 41
± ± ± ±
6 18 8 10
6.40 5.09 5.89 6.65
± ± ± ±
2.06 1.87 1.10 1.39
49.4 42.7 47.8 51.9
± ± ± ±
2.8 8.8 5.8 14.5
2.11 2.17 1.99 2.44
± ± ± ±
0.29 0.26 0.41 0.42
0.46 0.49 0.47 0.53
± ± ± ±
0.20 0.13 0.09 0.03
145.8 147.3 148.5 147.8
± ± ± ±
5.4 1.5 2.7 5.3
5.63 5.53 5.60 5.50
± ± ± ±
0.04 0.26 0.03 0.24
2.38 2.49 2.38 2.41
± ± ± ±
0.06 0.08 0.08 0.01
97.8 98.3 99.8 100.0
± ± ± ±
3.9 3.6 2.6 1.9
2.29 2.17 2.09 2.32
± ± ± ±
0.32 0.31 0.32 0.23
± ± ± ±
0.39 0.99 0.57 0.77
70.3 68.7 63.4 70.2
± ± ± ±
7.1 2.6 2.8 3.7
32.6 31.6 30.7 32.4
± ± ± ±
2.2 1.2 1.1 1.9
197 181 133 163
± ± ± ±
45 61 19 27
38 37 34 28
± ± ± ±
2 6 4 7
60 61 45 41
± ± ± ±
15 13 5 8
4.67 4.73 4.61 4.96
± ± ± ±
0.30 0.54 0.51 0.63
53.7 55.9 50.9 65.6
± ± ± ±
5.1 10.0 3.9 12.6
2.37 2.25 2.42 2.86
± ± ± ±
0.39 0.25 0.46 0.55
0.58 0.59 0.53 0.51
± ± ± ±
0.05 0.08 0.06 0.12
146.7 146.5 145.6 145.6
± ± ± ±
0.8 0.7 0.7 0.6
5.96 5.48 5.41 5.68
± ± ± ±
0.55 0.43 0.30 0.55
2.58 2.50 2.50 2.66
± ± ± ±
0.15 0.05 0.14 0.10
101.3 103.4 105.1 105.2
± ± ± ±
1.8 2.7 2.6 4.1
1.47 1.53 1.68 1.81
± ± ± ±
0.26 0.07 0.26 0.17
± 0.11 ± 0.21
66.5 ± 0.0 63.2 ± 0.9
28.0 ± 0.0 28.8 ± 0.7
139 ± 73 153 ± 8
51 ± 1 46 ± 6
41 ± 14 41 ± 11
5.45 ± 0.09 5.23 ± 0.88
58.0 ± 3.1 60.3 ± 0.1
1.93 ± 0.08 2.21 ± 0.20
0.33 ± 0.00 0.29 ± 0.08
146.2 ± 0.1 145.9 ± 0.7
4.98 ± 0.23 4.82 ± 0.84
2.46 ± 0.06 2.47 ± 0.11
108.1 ± 0.9 105.8 ± 4.0
X. Wang et al. / Regulatory Toxicology and Pharmacology 73 (2015) 652e659
0 week 0 (n ¼ 5) 5.85 100 (n ¼ 4) 5.74 450 (n ¼ 4) 6.06 2500 (n ¼ 5) 5.95 6 weeks 0 (n ¼ 5) 5.56 100 (n ¼ 4) 5.58 450 (n ¼ 4) 5.51 2500 (n ¼ 5) 5.75 13 weeks 0 (n ¼ 5) 3.48 100 (n ¼ 4) 3.46 450 (n ¼ 4) 3.63 2500 (n ¼ 5) 4.08 Recovery periods 0 (n ¼ 2) 6.13 2500 (n ¼ 2) 5.95
TP (g/L)
1.77 ± 0.06 1.76 ± 0.02
Note: Values represent means ± SD, n ¼ number of animals. 657
658
X. Wang et al. / Regulatory Toxicology and Pharmacology 73 (2015) 652e659
Fig. 3. Selected microphotographs for main related organs (40). A, B and C refer to liver, kidney and adrenal tissue in the control group, respectively. D, E and F refer to liver, kidney, adrenal tissue in the 2500 mg/kg dose group, respectively.
at the dose of 2500 mg/kg cyadox for 13 weeks subchronic toxicity. However, in the previous study, a significant decrease in body weight was observed at the dose of 2500 mg/kg cyadox for 13 weeks subchronic toxicity test in rats (Fang et al., 2006). One of the causes might be the fact that cyadox had an influence on food consumption in rats, while it had no effect in dogs. The reason for this might be that dogs are not sensitive to bitter materials, such as cyadox, when compared with rats (Michael, 2002). In the present study, the significant increase of serum Kþ in male dogs was noted, whereas the Kþ value was within the normal range (5.42e6.87 mmol/L), as reported by Wang et al. (2009). Additionally, there were no significant pathological findings in kidney and adrenal tissues. However, the significant increase of serum Kþ in male dogs might be treatment related in the current study, which is worthy of investigating in the future. In the previous studies, it was reported that the liver, kidneys and adrenal tissues were the main target organs for toxicity in quinoxaline group (WHO, 1991b). The toxicity presented as related serum biochemistry (such as AST, ALT, CREA, Naþ and Kþ) and organ weights were significantly changed following the pathological findings (Fang et al., 2006; Ihsan et al., 2010; Wang et al., 2011b, 2010; WHO, 1991a; WHO, 1991b).
Generally, the significant change of the serum Kþ level was considered to be associated with renal and adrenal toxicity and might be the common toxicity of QdNOs, as shown for most other members (Huang et al., 2009, 2010a, 2010b; Ihsan et al., 2010; WHO, 1991c). However, adrenal toxicity of cyadox was not found in the previous subchronic studies (Fang et al., 2006; Nastuneak et al., 1986) and chronic studies conducted in rats even in the 2500 mg/kg cyadox group (Nastuneak et al., 1987; Wang et al., 2011b). As the reason that the toxicity of cyadox is milder than other QdNOs, the lower bioavailability of cyadox than other QdNOs might play an important role. It was reported that the bioavailability of mequindox were 89.4% and 16.6% after intramuscular (i.m.) and oral administration of mequindox to chickens at a single dose of 10 (i.m.) or 20 mg/kg b.w. (oral) (Ding et al., 2012). In another study, the bioavailability of mequindox was 37% when rats were orally administered with mequindox at a single dose of 10 mg/kg b.w. (Li et al., 2012). The bioavailabilities were 21% and 53% when chickens were orally administered with quinocetone or olaquindox at a single oral dose of 30 mg/kg b.w., respectively (Zhang et al., 2011; Zhu et al., 1993). However, the bioavailability of cyadox was
X. Wang et al. / Regulatory Toxicology and Pharmacology 73 (2015) 652e659
reported to be 2% when pigs were administered with cyadox at a single oral dose of 1 mg/kg b.w. (Qiu, 2003). Compared with the bioavailabilities of mequindox, quinocetone, olaquindox and cyadox, it was suggested that the lowest bioavailability of cyadox was one of the reasons for its low toxicity. Base on the same quinoxaline structure and different toxicity of QdNOs with different side chains, the side chains were presumed to play crucial roles in their toxicity. As reported, their main metabolism in vivo was similar and it was N/O group reduction (Liu et al., 2008, 2009, 2010). A recent study found that the reduction of N/O group of cyadox is more easier than other ones, and the faster deoxidation of cyadox would result in its lower toxicity (Zhang et al., 2015). In summary, the toxicity of cyadox in non-rodents was carefully studied in a wide range of doses by subchronic evaluation. Although the increased Kþ value was within the normal range in males in the 2500 mg/kg cyadox group, the 450 mg/kg dietary level was selected as the NOAEL of cyadox for dogs, which approximates to 15.3e15.4 mg/kg b.w./day. The study confirmed the low toxicity of cyadox in non-rodents, but assessment of the safety of cyadox for veterinary clinical trials is still required prior to understanding the full risks of cyadox administration as a new antimicrobial and growth-promoting feed additive. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant no. 31272614 and 31502115), Grants from 2015 National Risk Assessment of Quality and Safety of Livestock and Poultry Products (GJFP2015008) and National 863 Program of China (2011AA10A214). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.yrtph.2015.09.023. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2015.09.023. References Ding, M.X., Wang, Y.L., Zhu, H.L., Yuan, Z.H., 2006a. Effects of cyadox and olaquindox on intestinal mucosal immunity and on fecal shedding of Escherichia coli in piglets. J. Animal Sci. 84, 2367e2373. Ding, M.X., Yuan, Z.H., Wang, Y.L., Zhu, H.L., Fan, S.X., 2006b. Olaquindox and cyadox stimulate growth and decrease intestinal mucosal immunity of piglets orally inoculated with Escherichia coli. J. Animal Physiol. Animal Nutr. Berl. 90, 238e243. Ding, H., Liu, Y., Zeng, Z., Si, H., Liu, K., Liu, Y., Yang, F., Li, Y., Zeng, D., 2012. Pharmacokinetics of mequindox and one of its major metabolites in chickens after intravenous, intramuscular and oral administration. Res. Vet. Sci. 93 (1), 374e377. Fang, G., He, Q., Zhou, S., Wang, D., Zhang, Y., Yuan, Z., 2006. Subchronic oral toxicity study with cyadox in Wistar rats. Food Chem. Toxicol. 44, 36e41. FDA, 2003. Subchronic Toxicity Studies with Non-rodents. Toxicological Principles for the Safety Assessment of Food Ingredients Redbook 2000. http://www. cfsan.fda.gov/~redbook/redIVC4b.html. Huang, X.J., Ihsan, A., Wang, X., Dai, M.H., Wang, Y.L., Su, S.J., Xue, X.J., Yuan, Z.H., 2009. Long-term dose-dependent response of Mequindox on aldosterone, corticosterone and five steroidogenic enzyme mRNAs in the adrenal of male rats. Toxicol. Lett. 191, 167e173. Huang, X.J., Wang, X., Ihsan, A., Liu, Q., Xue, X.J., Su, S.J., Yang, C.H., Zhou, W., Yuan, Z.H., 2010a. Interactions of NADPH oxidase, renin-angiotensinaldosterone system and reactive oxygen species in mequindox-mediated aldosterone secretion in Wistar rats. Toxicol. Lett. 198, 112e118. Huang, X.J., Zhang, H.H., Wang, X., Huang, L.L., Zhang, L.Y., Yan, C.X., Liu, Y.,
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Assessment of thirteen-week subchronic oral toxicity of cyadox in Beagle dogs Xu Wang a, Wen Zhou b, Awais Ihsan d, Dongmei Chen b, Guyue Cheng b, Haihong Hao c, Zhenli Liu c, Yulian Wang c, **, Zonghui Yuan a, b, c, * a
National Reference Laboratory of Veterinary Drug Residues (HZAU) and MOA Key Laboratory for Detection of Veterinary Drug Residues, China MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei 430070, China c Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan, Hubei, China d Department of Biosciences, COMSATS Institute of Information Technology, Sahiwal, Pakistan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2015 Received in revised form 16 September 2015 Accepted 18 September 2015 Available online 25 September 2015
Cyadox (2-formylquinoxaline-N1,N4-dioxide cyanocetylhydrazone) is a new antimicrobial agent and growth-promoter to be used in food-producing animals. Although its toxicity has been clearly documented in rodents, no study is available in non-rodent animals. Therefore, we studied the subchronic effects of cyadox in Beagle dogs to provide additional information with which to establish safety criteria for human exposure. For this purpose, 36 Beagle dogs, 18 males and 18 females, were divided into four groups and fed diets containing 0, 100, 450 and 2500 mg/kg of cyadox, respectively, for 13 weeks. It was found that there were no significant changes among the examined parameters, except for an increase in the level of serum potassium (Kþ) in 2500 mg/kg cyadox group in males at week 13 of the study. However, the Kþ level returned to normal during the recovery period. In conclusion, cyadox showed slight effects in Beagle dogs in the subchronic oral toxicity study. The no-observed-adverse-effect level of cyadox was considered to be 450 mg/kg diet, which equates to approximately 15.3e15.4 mg/kg b.w./day. The study provided subchronic effects of cyadox in Beagle dogs, suggesting that cyadox might present mild toxicity in non-rodents. © 2015 Elsevier Inc. All rights reserved.
Keywords: Cyadox Quinoxaline Toxicity Subchronic toxicity Beagle dogs
1. Introduction Abbreviations: ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; ANOVA, analysis of variance; AST, aspartate aminotransferase; b.w., body weight; BUN, blood urea nitrogen; Ca, calcium; CHO, total cholesterol; Cl, chloride; CREA, creatinine; FDA, Food and Drug Administration; GLU, glucose; HCT, hematocrit; HGB, hemoglobin; H&E, hematoxylin and eosin; i.m., intramuscular; Kþ, potassium; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MPV, mean platelet volume; Naþ, sodium; NOAEL, one-way analysis of variance; OECD, organization for economic co-operation and development; P, inorganic phosphorus; PCT, plateletcrit; PDW, platelet distribution width; PLT, platelet count; PMCol, pentamethylchromanol; QdNOs, quinoxaline 1,4-dioxide derivatives; RBC, red blood cell count; RDW, red cell volume distribution; TG, triglyceride; TP, total protein; URE, urea; VICH, international cooperation on harmonization of technical requirements for the registration of veterinary medicinal products; WBC, white blood cell count. * Corresponding author. National Reference Laboratory of Veterinary Drug Residues (HZAU) and MOA Key Laboratory for Detection of Veterinary Drug Residues, China. ** Corresponding author. E-mail addresses: [email protected] (Y. Wang), yuan5802@mail. hzau.edu.cn (Z. Yuan). http://dx.doi.org/10.1016/j.yrtph.2015.09.023 0273-2300/© 2015 Elsevier Inc. All rights reserved.
Quinoxaline 1,4-dioxide derivatives (QdNOs) are synthetic drugs with the wide range of biological activities like growth promoting, antibacterial, anti-candida, anti-tubercular, anticancer and antiprotozoal properties (Vicente et al., 2009; Zhang et al., 2015). Cyadox, 2-formylquinoxaline-N1,N4-dioxide cyanocetylhydrazone (CAS No. 65884-46-0, C12H9N5O3) (Fig. 1), is one of the QdNO member with outstanding antibacterial activity against pathogenic bacteria such as Pasteurella multocida and Escherichia coli (Ding et al., 2006a). It can also promote animal growth and improve feed efficiency with the potential to be used widely in husbandry in the future (Ding et al., 2006a, 2006b; Wang et al., 2005). QdNOs usually have serious hazards increased along with the increasing doses added as animal feed addictives. Compared with other QdNOs such as carbadox, olaquindox and mequindox, cyadox was considered to be less harmful and showed good safety in mutagenicity studies (Ihsan et al., 2013a, 2013b), 90-day feeding studies (Fang et al., 2006), teratogenicity and reproduction studies
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2002). The study will provide information on the major toxic effects, indicate target organs, provide an estimate of a no-observedadverse-effect (NOAEL) level of cyadox exposure, and can provide experimental evidence of the safety of cyadox for future veterinary clinical trials. 2. Materials and methods 2.1. Drug and chemicals Cyadox (molecular weight 271.23, purity 98%) was obtained from the Institute of Veterinary Pharmaceuticals, Huazhong Agricultural University (Wuhan, PR China). All other reagents were of analytical grade. 2.2. Animals Fig. 1. Structure of cyadox.
(Wang et al., 2011a), chronic studies (Wang et al., 2011b) and carcinogenicity studies (Sevcik, 1986). Carbadox and olaquindox were concluded to be mutagenic and carcinogenic with developmental and reproductive toxicities (Yoshimura, 2002; WHO, 1991a; WHO, 1991b). In rats, a dose-dependent decrease in weight gain and food consumption was noted when carbadox was administered at dose levels of 50 and 100 mg/kg b.w./day for 30 days (WHO, 1991b). In dogs, carbadox was found to be responsible of vomiting, weight loss and elevated serum alanine aminotransferase (ALT) at dose levels of 25 and 50 mg/kg b.w./day (WHO, 1991b). The adrenal cell atrophy was reported in the rats given olaquindox at doses of 60 and 180 mg/kg b.w./day for 5 days/week for 13 weeks. In dogs, fatty degeneration was observed in kidney tubules of dogs given 60 or 180 mg/kg b.w./day of olaquindox for 90 days (WHO, 1991a). Previously in a subchronic study, we have occasionally found that 275 mg/kg mequindox led changes in Naþ and Kþ levels in serum after 90 days of feeding in rats (Huang et al., 2009; Ihsan et al., 2010). Quinocetone showed mild toxicity and it at 1800 mg/kg dietary level in rats produced low toxicity for liver and depressed body weight gain in the subchronic oral study (Wang et al., 2010). Cyadox was thought to be a potential replacement for carbadox and olaquindox (Wang et al., 2011a). According to the toxicity guideline of the Food and Drug Administration (FDA), the Organization for Economic Co-operation and Development (OECD) and the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) for new drugs used in food animals, the subchronic 90-day feeding toxicity study should be performed in a rodent species and a non-rodent species (FDA, 2003; OECD, 1998; VICH, 2002; VICH, 2004). However, according to our limited knowledge, nearly all toxicity tests were performed in rodents and very few toxicological data are available in non-rodents, which is too limited to evaluate the safety of cyadox sufficiently and convincingly. As a potential replacement of olaquindox and carbadox, cyadox, with the potential to be used widely and over long-periods, must be studied systemically for toxicity. Therefore, it is necessary to perform the study in non-rodents. The 90-day study of cyadox in Beagle dogs was the main non-rodent test and could provide additional information with which to establish safety criteria for human exposure. To study the safety spectrum of cyadox, the present study was performed to evaluate the potential subchronic feeding toxicity test in Beagle dogs with a wide range of doses. The subchronic toxicity of cyadox was tested in 13-weeks feeding study in Beagle dogs according to OECD Guideline 409 and VICH Guideline 31 under modern Good Laboratory Practice Regulations (OECD, 1998; VICH,
Eighteen male and eighteen female Beagle dogs (5e6 months old), weighing 5.6e6.5 kg, were purchased from Guangdong National Beagles Resources Research Center (Guangzhou, PR China). The animal room was maintained at a temperature of 18e25 C, with a relative humidity of 42e69% and a 12-h light/dark cycle. The air ventilation was given once every 30 min. Prior to the initiation of dosing, the dogs were quarantined for 2 weeks and then acclimatized to the study environmental conditions before use. The dogs were fed 300 g certified commercial diet (Ke Ao Xie Li Co. Ltd., Beijing, PR China) at a fixed time per day and tap water ad libitum during the study. Each dog, with a unique ear tag number, was raised in a steel cage (1000 1000 900 mm). The animal numbers were also attached to each cage. All dogs enjoyed outdoor exercise at least once every week. The study was approved by the Ethical Committee of the Faculty of Veterinary Medicine at Huazhong Agricultural University and was in accordance with NIH Publication 85-23 ‘‘Guide for the Care and Use of Laboratory Animals’’ (NRC, 1996). 2.3. Experimental design Eighteen male and eighteen female dogs were randomly assigned to four groups based on their body weights using a randomized block model. The control, low-dose, middle and high-dose groups of cyadox included 5, 4, 4 and 5 dogs per sex, respectively. Each group of dogs was fed with the basal diets mixed with 0, 100, 450 and 2500 mg cyadox/kg diet. To ensure homogeneity, the drug was thoroughly mixed with the diet. The diets were mixed separately by group and placed in properly labeled containers. The diets were prepared every 4 weeks, and the stability and homogeneity were verified prior to the study. The dosage of 100 mg cyadox/kg diet was selected as the minimal dose as this was the maximal recommended concentration of cyadox incorporated as feed additive of pig for growth promoting (Wang et al., 2011b). Also, 2500 mg cyadox/kg diet inhibited the feed intake and body weight of rats in our previous study and was used here as the high dose (Fang et al., 2006). The dose level of 450 mg cyadox/kg diet was close to the geometric average of the high and low dose, and was used as the medium dose. At the end of 13 weeks, female dogs (2, 4, 4 and 3 dogs/group) and male dogs (3, 4, 4 and 3 dogs/group) in control, 100, 450 and 2500 mg cyadox/kg diet groups were sacrificed under anesthesia with intravenous sodium pentobarbital, respectively. The remaining dogs in the control and high-dose groups after 13 weeks were administered with control feed for 2 weeks and euthanized in the same manner to perform the examination. Clinical observations were performed on both a daily and weekly basis. Hematology, serum biochemistry and electrocardiogram of dogs were examined before the start of the study, at week 6 and at
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necropsy. Pathology findings were also determined at necropsy. 2.4. Clinical observations Each dog was observed at least twice a day throughout the study for mortality, signs of toxicity, any behavioral change in gait, posture, response to handling and changes in skin, fur, eyes, mucous membranes, occurrences of secretions and excretions. An ophthalmological examination was conducted by observing the appearance of the eyes with the naked eye before the administration of a drug and in the last week of the experiment. Individual body weight and food consumption were recorded in quarantine phase, and weekly during the treatment periods and fasted weights were assessed at necropsy. 2.5. Hematology
conducted by using a routine paraffin-embedding technique. Sections with a thickness of 5 mm stained with hematoxylin and eosin (H&E) were examined under a light microscope for morphological alterations. As a necessary step to determine the NOAEL in target organs, other tissues such as the liver and kidneys from the treatment groups were also examined histologically. 2.9. Calculations of feed efficiency and drug intake The equation of feed efficiency was (the increase weight of each dog for the week/weekly feed consumption) 100%. The dose levels (mg/kg b.w./day) were calculated by using the nominal concentration of drugs in the diet, the mean daily feed consumption, and the body weight for the week. The equation was (mean weekly feed consumption of each dog/7)/(the body weight for the week) nominal concentration of drugs in the diet.
Following an overnight fast, blood samples were collected from the radial vein. The anticoagulants were potassium EDTA for hematology tests. Hematological measurements and calculations were performed with Coulter HmX Hematology Analyzer (Beckman Coulter Inc., Fullerton, CA, USA). Hematological evaluations included red blood cell count (RBC), hematocrit (HCT), mean corpuscular volume (MCV), hemoglobin concentration (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), blood platelet count (PLT), red cell volume distribution (RDW), platelet distribution width (PDW), mean platelet volume (MPV) and white blood cell count (WBC).
2.10. Statistical analysis
2.6. Serum biochemistry
The intakes per kg bodyweight per day achieved in the study were calculated based on the food intakes and bodyweights. They are presented in Table 1.
For clinical biochemistry, blood samples were placed in serum tubes at room temperature for approximately 30 min to obtain the serum aliquots. After clotting, the blood tubes were centrifuged at 2500 rpm for 15 min with Himac CR 21 G centrifuge (Hitachi, Tokyo, Japan). Supernatants were decanted and stored at 20 C for further analysis. Serum biochemistry was assessed using the Synchron Clinical System CX4 (Beckman Coulter, Brea, CA USA) according to the manufacturer's instructions (Beijing Leadman Biochemistry Technology Co. Ltd, Beijing, China). Parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), glucose (GLU), urea (URE), total cholesterol (CHO), creatinine (CREA), triglyceride (TG), sodium (Na), potassium (K), chloride (Cl), calcium (Ca) and inorganic phosphorus (P). 2.7. Electrocardiogram PeR intervals, QeT intervals, QRS duration and heart rate of ECG Ⅱ lead in dogs were recorded by an ECG system (ECG-6511, Shanghai Kohden Medical Electronic Instrument Corp., Shanghai, China).
Statistical analyses were performed by comparing the treatment groups with the control group using the SPSS 13.0 program. Levene's test was used to examine variance homogeneity. One-way analysis of variance (ANOVA) was performed for data with significant dispersion, and this significance was confirmed with Dunnett's multiple comparison. The two-sided level of statistical significance was preset at p < 0.05 or 0.01. 3. Results
3.1. Survival and clinical signs All dogs survived, except one female in the control group who incidentally suffered severe intestinal volvulus on the fifth day of the study, and was euthanized under anesthesia with intravenous sodium pentobarbital. No other abnormal changes in appearance or behavior were observed in any of the groups. 3.2. Body weight, food efficiency and food consumption The body weights of both male and female dogs slightly increased in the group of 100 mg/kg cyadox in most of the weeks during the study, presenting a tendency to promote the effect of cyadox. However, no significant differences were noted in any of the groups when compared with the controls (Fig. 2 and Tables S12). Also, no significant differences in food efficiency and food consumption were found among the four groups (Tables S3-6). However, when compared with that of the 1st week and the 3rd week, significant decreases of feed efficiency in the 2nd week in all the groups were noted. Considering the calculation of food efficiency,
2.8. Pathology examinations All of the organs/tissues were carefully examined macroscopically, and gross lesions were recorded. The absolute and relative (organ to body weight ratios) weights of organs and tissues of each animal, including the heart, lungs, brain, kidneys, adrenal glands, liver, spleen, thymus, thyroid, pancreas, testes, ovaries and uterus were measured separately. The tissues from each animal were preserved in 10% neutral-buffered formalin and slides were prepared for histopathological examination. Testis and epididymes were fixed in Bouin's solution for histopathology as previously described (Shin et al., 2010). Histopathological examination was
Table 1 Dose levels calculated in the subchronic test. Dietary dose (mg/kg diet)
100 (n ¼ 8) 450 (n ¼ 8) 2500 (n ¼ 10)
Calculated dose level (mg/kg b.w./ day) Female
Male
3.4 ± 0.1 15.3 ± 0.5 87.0 ± 3.6
3.2 ± 0.1 15.4 ± 0.5 85.2 ± 3.2
Note: Values represent means ± SD, n ¼ number of animals. The 100, 450 and 2500 mg/kg diet groups of cyadox have 5, 4, 4 and 5 dogs per sex, respectively.
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Fig. 2. Mean body weights (A), food efficiency (B) and daily food intake of dogs (C) in the 90 days feeding study.
the much lower values of body weight gains in the 2nd week were suggested to be the main reason. However, the reason was still not clear. 3.3. Hematology and serum biochemistry There was a significant increase in PDW and WBC levels in the female dogs of the 100 and 450 mg/kg cyadox groups at weeks 6 and 13 when compared with the control group. However, the changes did not have a doseeresponse relationship. Significant decreases of MPV levels in the female dogs of the 2500 mg/kg cyadox group were observed at week 6. However, the change of MPV was within the physiological range, and did not appear at week 13. Therefore, these occasionally achieved statistical significances were not considered to be related to the treatment with cyadox (Tables 2 and 3). For serum biochemistry, serum Kþ level increased significantly in male dogs in the 2500 mg/kg cyadox group compared with the controls at week 13, but returned to normal during the recovery
phase. Other serum biochemical parameters were not affected by the treatment for either gender (Tables 4 and 5).
3.4. Temperature and electrocardiogram No significant changes were found in the temperature (Table S7), and electrocardiogram (Tables S8-9). 3.5. Organ weights and pathological findings No significant differences in absolute and relative organ weights were found among the four groups for both genders (Tables S1011). Necropsy and detailed histopathological examination did not reveal any lesions in any of the organs of the dogs. In particular, no microscopic changes were observed in the liver, kidney and adrenal tissues, which are known target organs of quinoxalines (Fig. 3) (Fang et al., 2006; Ihsan et al., 2010; Wang et al., 2011a, 2011b, 2010).
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Table 2 Hematology parameters of male dogs treated with cyadox (0, 100, 450 and 2500 mg/kg diet) for 13 weeks. Week (mg/kg diet)
WBC (109/l)
0 week 0 (n ¼ 5) 16.1 100 (n ¼ 4) 14.7 450 (n ¼ 4) 12.2 2500 (n ¼ 5) 11.8 6 weeks 0 (n ¼ 5) 14.9 100 (n ¼ 4) 15.0 450 (n ¼ 4) 12.7 2500 (n ¼ 5) 11.8 13 weeks 0 (n ¼ 5) 13.6 100 (n ¼ 4) 13.4 450 (n ¼ 4) 13.2 2500 (n ¼ 5) 13.9 Recovery periods 0 (n ¼ 2) 12.3 2500 (n ¼ 2) 12.4
RBC (1012/l)
HGB (g/l)
HCT (l/l)
MCV (fl)
MCH (pg)
MCHC (g/l)
RDW (%)
PLT (109/l)
MPV (fl)
PCT (%)
PDW (%)
± ± ± ±
4.0 4.8 2.4 2.0
5.95 6.02 6.06 5.78
± ± ± ±
0.41 0.30 0.57 0.95
136 137 135 127
± ± ± ±
9 7 14 21
0.372 0.382 0.374 0.341
± ± ± ±
0.028 0.020 0.039 0.046
62.6 63.5 61.6 61.3
± ± ± ±
1.8 1.2 1.2 1.5
22.8 22.8 22.1 21.7
± ± ± ±
0.6 0.3 0.1 0.7
365 359 362 355
± ± ± ±
5 2 5 4
15.1 14.4 14.1 15.2
± ± ± ±
1.0 1.0 0.8 1.1
347 311 345 293
± ± ± ±
92 52 76 93
8.4 8.4 8.3 8.5
± ± ± ±
0.6 0.5 0.3 0.3
0.278 0.260 0.297 0.237
± ± ± ±
0.092 0.039 0.054 0.118
15.8 16.0 16.0 16.2
± ± ± ±
0.7 0.4 1.0 0.8
± ± ± ±
2.4 3.7 2.4 1.7
6.37 5.88 5.94 5.51
± ± ± ±
0.51 0.39 0.18 0.62
143 133 137 125
± ± ± ±
10 10 5 11
0.398 0.358 0.372 0.348
± ± ± ±
0.027 0.023 0.010 0.038
62.5 60.9 62.7 64.2
± ± ± ±
2.0 0.2 2.0 1.6
22.7 22.6 23.0 23.4
± ± ± ±
0.9 0.6 0.8 0.5
332 371 368 364
± ± ± ±
65 9 5 6
13.0 14.0 13.1 12.5
± ± ± ±
0.6 1.3 0.4 0.3
271 298 314 301
± ± ± ±
73 64 98 69
8.6 8.3 8.8 8.5
± ± ± ±
0.8 0.4 0.8 0.4
0.252 0.229 0.246 0.251
± ± ± ±
0.081 0.082 0.097 0.131
15.6 16.1 16.5 15.9
± ± ± ±
1.0 1.0 1.4 1.1
± ± ± ±
1.4 2.2 4.0 4.0
6.27 5.69 6.06 5.46
± ± ± ±
0.74 0.47 0.55 0.52
143 131 141 128
± ± ± ±
16 9 12 12
0.389 0.351 0.380 0.333
± ± ± ±
0.045 0.022 0.027 0.033
62.1 61.8 62.8 64.7
± ± ± ±
2.4 1.5 2.0 1.1
22.8 23.1 23.2 23.7
± ± ± ±
1.0 0.5 0.4 0.5
367 372 370 367
± ± ± ±
3 6 6 4
13.4 13.8 13.1 12.9
± ± ± ±
0.6 0.3 0.5 0.3
292 347 355 295
± ± ± ±
58 64 63 109
8.8 8.5 8.1 8.8
± ± ± ±
1.1 0.4 0.3 1.2
0.254 0.292 0.287 0.253
± ± ± ±
0.036 0.047 0.042 0.079
15.4 15.0 15.1 15.1
± ± ± ±
0.7 0.3 0.3 0.4
± 0.2 ± 0.2
6.02 ± 0.23 5.49 ± 0.41
124 ± 6 130 ± 12
0.355 ± 0.023 0.313 ± 0.026
59.1 ± 1.3 62.6 ± 1.6
22.1 ± 0.6 23.0 ± 0.4
349 ± 6 367 ± 4
14.1 ± 1.1 13.2 ± 0.6
336 ± 8 363 ± 57
8.3 ± 0.1 8.1 ± 0.6
0.277 ± 0.008 0.271 ± 0.004
14.8 ± 0.1 15.5 ± 0.1
Note: Values represent means ± SD, n ¼ number of animals.
cyadox group (85.2e87.0 mg/kg b.w./day) in dogs showed almost no toxicological effects on liver function, behaviors and body weights. This conclusion was concordant with the acute toxicities in Wistar rats which showed that the acute toxicities of different quinoxalines were carbadox > olaquindox > cyadox (Wang et al., 2010). In previous studies, other known members of QdNOs such as olaquindox and carbadox were evaluated in beagle dogs. Inappetence and salivation were noted when the dogs were given oral doses of 60 mg/kg b.w./day olaquindox in gelatin capsules for 90 days. Histopathological observations revealed that 60 or 180 mg/kg b.w./day olaquindox induced fatty degeneration in liver and the kidney tubules (WHO, 1991a). The short-term toxicity of carbadox was studied by dosing animals 6 days/week for three weeks, using Beagle dogs (1 dog/sex/dose). The initial dose was 25 or 50 mg/kg b.w./day carbadox via oral capsule. It was found that the dogs lost weight and their serum ALT levels were elevated. It was concluded that carbadox was emetic and possibly hepatotoxic in dogs (WHO, 1991b). In the present study, the results indicated that cyadox was safer than olaquindox and carbadox in beagle dogs. In the present study, no effect in body weight was found in dogs
4. Discussion There were several studies in the literature which have evaluated the toxicity of cyadox in rodents, particularly in rats (Fang et al., 2006; Wang et al., 2011a, 2011b). However, no research on cyadox has been presented in non-rodents. Therefore, the subchronic toxicity study of cyadox in Beagle dogs was worth performing to further evaluate the potential effects of cyadox in nonrodents. The present study first demonstrated the toxicity of cyadox in dogs in a subchronic (90-day oral) trial and offered useful scientific knowledge about the administration of cyadox in a diets. In the present study, it was found that cyadox at 2500 mg/kg dietary level (85.2e87.0 mg/kg b.w./day) in dogs has no significant toxic effects on body weight gain, food intake, feed efficiency, body temperature, electrocardiographic, absolute and relative organ weights, hematologic and histological changes in the kidneys, adrenal glands and liver for both genders, except for an increase in the level of serum potassium (Kþ) in 2500 mg cyadox/kg diet group in males at week 13 of the study. Compared with olaquindox and carbadox, the 2500 mg/kg
Table 3 Hematology parameters of female dogs treated with cyadox (0, 100, 450 and 2500 mg/kg diet) for 13 weeks. Week (mg/kg diet)
WBC (109/l)
0 week 0 (n ¼ 5) 12.5 100 (n ¼ 4) 15.3 450 (n ¼ 4) 12.9 2500 (n ¼ 5) 14.7 6 weeks 0 (n ¼ 4) 12.2 100 (n ¼ 4) 15.3 450 (n ¼ 4) 13.1 2500 (n ¼ 5) 14.3 13 weeks 0 (n ¼ 4) 12.2 100 (n ¼ 4) 15.3 450 (n ¼ 4) 12.3 2500 (n ¼ 5) 11.2 Recovery periods 0 (n ¼ 2) 11.3 2500 (n ¼ 2) 11.6
RBC (1012/l)
HGB (g/l)
HCT (l/l)
MCV (fl)
MCH (pg)
MCHC (g/l)
RDW (%)
PLT (109/l)
MPV (fl)
PCT (%)
PDW (%)
± ± ± ±
0.9 2.8 4.0 3.5
6.06 5.76 5.87 5.65
± ± ± ±
0.49 0.12 0.51 0.60
138 130 132 128
± ± ± ±
12 4 7 16
0.385 0.362 0.368 0.353
± ± ± ±
0.039 0.011 0.019 0.041
63.5 62.8 62.8 62.4
± ± ± ±
2.5 1.2 2.6 1.4
22.7 22.6 22.6 22.7
± ± ± ±
0.2 0.5 1.0 0.6
358 359 359 363
± ± ± ±
11 2 3 3
14.5 15.0 14.0 14.3
± ± ± ±
1.3 0.2 1.1 1.5
299 332 407 380
± ± ± ±
93 64 37 66
8.9 8.6 8.3 8.2
± ± ± ±
0.8 1.1 0.3 0.5
0.284 0.278 0.338 0.293
± ± ± ±
0.046 0.022 0.020 0.103
16.2 15.7 15.5 15.4
± ± ± ±
0.6 0.5 0.3 0.7
± ± ± ±
0.3 2.4 3.6 3.0
5.60 6.29 5.99 5.92
± ± ± ±
1.09 0.13 0.31 0.35
139 142 137 136
± ± ± ±
16 8 7 8
0.363 0.394 0.374 0.378
± ± ± ±
0.080 0.015 0.024 0.022
64.5 62.6 62.4 63.7
± ± ± ±
2.4 1.8 1.8 1.5
23.3 23.1 22.8 22.9
± ± ± ±
1.1 0.4 0.7 0.7
356 333 366 360
± ± ± ±
20 61 6 2
13.2 13.5 13.5 12.7
± ± ± ±
0.6 1.0 0.7 0.6
324 322 283 302
± ± ± ±
57 87 55 82
9.2 8.9 8.2 8.0
± ± ± ±
0.2 0.7 0.7 0.5a
0.262 0.303 0.204 0.250
± ± ± ±
0.106 0.050 0.085 0.068
15.1 15.9 16.4 16.0
± ± ± ±
0.5 0.8 0.5a 0.7
± ± ± ±
0.9 1.3a 1.3 1.6
6.26 6.30 5.56 5.60
± ± ± ±
0.71 0.29 0.28 0.37
147 144 127 130
± ± ± ±
15 5 9 9
0.395 0.393 0.346 0.357
± ± ± ±
0.047 0.010 0.024 0.022
63.2 62.4 62.2 63.7
± ± ± ±
2.7 1.8 1.3 1.5
23.5 22.8 22.8 23.2
± ± ± ±
0.8 1.4 0.7 0.4
372 365 367 365
± ± ± ±
8 13 5 6
13.3 13.7 13.1 12.6
± ± ± ±
0.5 0.9 0.5 0.5
331 317 358 343
± ± ± ±
77 46 39 29
8.8 8.7 8.3 7.9
± ± ± ±
0.6 0.5 0.8 0.3
0.287 0.272 0.293 0.270
± ± ± ±
0.048 0.029 0.027 0.022
14.8 14.9 15.0 15.1
± ± ± ±
0.3 0.3 0.3 0.2
± 1.6 ± 1.2
5.32 ± 0.71 5.42 ± 0.13
121 ± 12 124 ± 2
0.340 ± 0.041 0.335 ± 0.003
Note: Values represent means ± SD, n ¼ number of animals. a Significantly different from control group at p < 0.05.
64.0 ± 1.0 62.8 ± 1.8
22.7 ± 0.8 22.8 ± 0.1
354 ± 7 369 ± 9
13.4 ± 0.4 12.6 ± 0.1
391 ± 19 355 ± 6
7.9 ± 04 8.2 ± 0.8
0.256 ± 0.069 0.242 ± 0.014
15.4 ± 0.5 15.4 ± 0.1
Table 4 Serum biochemical parameters of male dogs treated with cyadox (0, 100, 450 and 2500 mg/kg diet) for 13 weeks. Week (mg/kg diet)
GLU (mmol)
ALB (g/L)
ALP (U/L)
ALT (U/L)
AST (U/L)
URE (mmol/L)
CRE (mmol/L)
CHO (mmol/L)
TG (mmol/L)
Na (mmol/L)
K (mmol/L)
Ca (mmol/L)
CL (mmol/L)
PO4 (mmol/L)
± ± ± ±
0.30 0.13 0.27 0.46
66.3 67.5 66.1 68.2
± ± ± ±
2.9 4.6 6.0 6.2
31.3 30.8 30.5 32.5
± ± ± ±
1.5 1.4 2.4 2.0
82 86 90 84
± ± ± ±
3 8 2 5
29 30 34 35
± ± ± ±
6 4 8 9
30 35 38 41
± ± ± ±
4 5 9 18
4.50 3.88 3.98 3.85
± ± ± ±
0.73 0.74 0.51 0.86
37.1 37.9 44.1 34.4
± ± ± ±
5.5 5.9 6.1 7.6
3.19 2.68 2.45 3.58
± ± ± ±
0.22 0.45 0.53 0.89
0.46 0.40 0.44 0.46
± ± ± ±
0.06 0.09 0.04 0.07
147.5 146.5 148.4 147.7
± ± ± ±
2.0 2.6 2.0 2.9
5.45 5.48 5.48 5.42
± ± ± ±
0.26 0.32 0.31 0.35
2.48 2.52 2.50 2.45
± ± ± ±
0.03 0.07 0.06 0.11
98.1 98.8 100.8 100.8
± ± ± ±
3.7 2.7 5.7 3.8
2.24 2.19 2.20 2.30
± ± ± ±
0.10 0.25 0.22 0.17
± ± ± ±
0.42 0.48 0.25 0.23
62.7 60.1 65.6 68.2
± ± ± ±
7.8 2.4 5.2 5.5
28.4 27.6 29.2 30.2
± ± ± ±
3.5 1.2 1.4 1.2
90 91 94 92
± ± ± ±
6 2 1 1
34 25 26 26
± ± ± ±
4 6 8 1
40 41 46 36
± ± ± ±
14 6 11 2
5.72 5.65 6.49 5.70
± ± ± ±
1.49 0.55 1.31 1.76
49.1 45.0 48.9 51.5
± ± ± ±
6.7 7.4 7.1 10.1
2.29 2.12 1.99 2.37
± ± ± ±
0.52 0.22 0.28 0.36
0.45 0.48 0.47 0.50
± ± ± ±
0.15 0.05 0.04 0.07
146.8 147.8 149.6 150.7
± ± ± ±
2.6 2.8 3.8 5.4
5.44 5.65 5.52 5.52
± ± ± ±
0.25 0.03 0.28 0.23
2.35 2.44 2.45 2.41
± ± ± ±
0.20 0.01 0.03 0.02
99.5 96.5 96.9 101.4
± ± ± ±
5.1 1.3 0.6 1.8
2.07 2.27 2.37 2.25
± ± ± ±
0.35 0.15 0.12 0.27
± ± ± ±
0.85 0.82 0.62 1.00
69.7 62.7 67.3 68.6
± ± ± ±
5.3 1.0 7.0 3.6
31.0 29.4 31.0 31.4
± ± ± ±
1.0 1.7 4.3 0.9
144 149 112 139
± ± ± ±
35 13 18 69
37 41 31 25
± ± ± ±
9 8 7 4
49 52 46 38
± ± ± ±
4 12 7 6
4.77 4.42 5.17 4.31
± ± ± ±
0.51 0.91 1.60 0.79
50.6 51.2 55.8 52.0
± ± ± ±
5.8 8.5 3.9 10.3
2.52 2.15 2.18 2.42
± ± ± ±
0.39 0.36 0.47 0.34
0.57 0.59 0.47 0.46
± ± ± ±
0.09 0.07 0.09 0.07
147.2 145.8 146.0 146.2
± ± ± ±
0.7 1.3 1.0 0.6
5.31 5.71 5.77 6.08
± ± ± ±
0.36 0.46 0.14 0.41a
2.53 2.47 2.61 2.67
± ± ± ±
0.09 0.09 0.14 0.09
100.2 102.5 104.5 104.6
± ± ± ±
1.1 2.2 0.8 4.9
1.62 1.73 1.72 1.88
± ± ± ±
0.19 0.24 0.07 0.18
± 0.71 ± 0.63
62.9 ± 11.2 58.6 ± 2.1
28.2 ± 4.2 26.9 ± 1.0
92 ± 1 87 ± 13
41 ± 10 41 ± 8
38 ± 11 39 ± 1
4.62 ± 0.49 4.01 ± 0.81
59.9 ± 0.6 59.1 ± 0.4
2.19 ± 0.44 2.02 ± 0.13
0.32 ± 0.05 0.32 ± 0.01
146.5 ± 0.9 146.6 ± 0.1
5.43 ± 0.48 5.33 ± 0.43
2.50 ± 0.42 2.46 ± 0.16
108.6 ± 5.8 104.3 ± 4.7
1.93 ± 0.50 1.86 ± 0.38
CHO (mmol/L)
TG (mmol/L)
Na (mmol/L)
K (mmol/L)
Ca (mmol/L)
CL (mmol/L)
PO4 (mmol/L)
Note: Values represent means ± SD, n ¼ number of animals. a Significantly different from control group at p < 0.05.
Table 5 Serum biochemical parameters of female dogs treated with cyadox (0, 100, 450 and 2500 mg/kg diet) for 13 weeks. Week (mg/kg diet)
GLU (mmol/L)
0 week 0 (n ¼ 5) 5.95 100 (n ¼ 4) 6.05 450 (n ¼ 4) 5.94 2500 (n ¼ 5) 5.94 6 weeks 0 (n ¼ 4) 5.54 100 (n ¼ 4) 5.74 450 (n ¼ 4) 5.46 2500 (n ¼ 5) 5.62 13 weeks 0 (n ¼ 4) 3.65 100 (n ¼ 4) 2.85 450 (n ¼ 4) 3.34 2500 (n ¼ 5) 4.49 Recovery periods 0 (n ¼ 2) 5.65 2500 (n ¼ 2) 5.80
TP (g/L)
ALB (g/L)
ALP (U/L)
ALT (U/L)
AST (U/L)
URE (mmol/L)
CRE (mmol/L)
± ± ± ±
0.43 0.58 0.22 0.58
66.4 65.0 71.0 66.5
± ± ± ±
4.6 4.0 4.2 4.3
31.3 31.5 31.6 30.3
± ± ± ±
1.5 0.8 1.6 1.6
81 83 89 84
± ± ± ±
5 7 5 7
34 34 39 38
± ± ± ±
5 5 5 13
37 32 41 42
± ± ± ±
9 6 4 11
4.59 3.99 3.90 4.74
± ± ± ±
0.94 0.52 0.47 0.27
40.4 40.9 42.3 39.4
± ± ± ±
5.6 6.8 4.1 4.6
3.04 2.71 2.78 3.16
± ± ± ±
0.36 0.62 0.54 0.47
0.52 0.40 0.48 0.47
± ± ± ±
0.11 0.08 0.07 0.05
146.7 147.4 146.3 146.6
± ± ± ±
1.3 1.2 0.4 2.2
5.39 5.42 5.50 5.43
± ± ± ±
0.34 0.27 0.32 0.37
2.52 2.51 2.49 2.44
± ± ± ±
0.07 0.04 0.10 0.02
101.1 102.4 102.9 100.0
± ± ± ±
2.5 3.0 4.0 2.9
2.23 2.33 2.13 2.21
± ± ± ±
0.20 0.12 0.09 0.20
± ± ± ±
0.20 0.16 0.16 0.24
62.0 63.5 60.8 68.5
± ± ± ±
8.6 2.5 5.4 3.3
29.2 29.2 28.0 30.1
± ± ± ±
3.8 1.6 2.4 1.1
88 89 92 94
± ± ± ±
5 5 2 1
37 32 24 27
± ± ± ±
13 10 8 3
38 47 41 41
± ± ± ±
6 18 8 10
6.40 5.09 5.89 6.65
± ± ± ±
2.06 1.87 1.10 1.39
49.4 42.7 47.8 51.9
± ± ± ±
2.8 8.8 5.8 14.5
2.11 2.17 1.99 2.44
± ± ± ±
0.29 0.26 0.41 0.42
0.46 0.49 0.47 0.53
± ± ± ±
0.20 0.13 0.09 0.03
145.8 147.3 148.5 147.8
± ± ± ±
5.4 1.5 2.7 5.3
5.63 5.53 5.60 5.50
± ± ± ±
0.04 0.26 0.03 0.24
2.38 2.49 2.38 2.41
± ± ± ±
0.06 0.08 0.08 0.01
97.8 98.3 99.8 100.0
± ± ± ±
3.9 3.6 2.6 1.9
2.29 2.17 2.09 2.32
± ± ± ±
0.32 0.31 0.32 0.23
± ± ± ±
0.39 0.99 0.57 0.77
70.3 68.7 63.4 70.2
± ± ± ±
7.1 2.6 2.8 3.7
32.6 31.6 30.7 32.4
± ± ± ±
2.2 1.2 1.1 1.9
197 181 133 163
± ± ± ±
45 61 19 27
38 37 34 28
± ± ± ±
2 6 4 7
60 61 45 41
± ± ± ±
15 13 5 8
4.67 4.73 4.61 4.96
± ± ± ±
0.30 0.54 0.51 0.63
53.7 55.9 50.9 65.6
± ± ± ±
5.1 10.0 3.9 12.6
2.37 2.25 2.42 2.86
± ± ± ±
0.39 0.25 0.46 0.55
0.58 0.59 0.53 0.51
± ± ± ±
0.05 0.08 0.06 0.12
146.7 146.5 145.6 145.6
± ± ± ±
0.8 0.7 0.7 0.6
5.96 5.48 5.41 5.68
± ± ± ±
0.55 0.43 0.30 0.55
2.58 2.50 2.50 2.66
± ± ± ±
0.15 0.05 0.14 0.10
101.3 103.4 105.1 105.2
± ± ± ±
1.8 2.7 2.6 4.1
1.47 1.53 1.68 1.81
± ± ± ±
0.26 0.07 0.26 0.17
± 0.11 ± 0.21
66.5 ± 0.0 63.2 ± 0.9
28.0 ± 0.0 28.8 ± 0.7
139 ± 73 153 ± 8
51 ± 1 46 ± 6
41 ± 14 41 ± 11
5.45 ± 0.09 5.23 ± 0.88
58.0 ± 3.1 60.3 ± 0.1
1.93 ± 0.08 2.21 ± 0.20
0.33 ± 0.00 0.29 ± 0.08
146.2 ± 0.1 145.9 ± 0.7
4.98 ± 0.23 4.82 ± 0.84
2.46 ± 0.06 2.47 ± 0.11
108.1 ± 0.9 105.8 ± 4.0
X. Wang et al. / Regulatory Toxicology and Pharmacology 73 (2015) 652e659
0 week 0 (n ¼ 5) 5.85 100 (n ¼ 4) 5.74 450 (n ¼ 4) 6.06 2500 (n ¼ 5) 5.95 6 weeks 0 (n ¼ 5) 5.56 100 (n ¼ 4) 5.58 450 (n ¼ 4) 5.51 2500 (n ¼ 5) 5.75 13 weeks 0 (n ¼ 5) 3.48 100 (n ¼ 4) 3.46 450 (n ¼ 4) 3.63 2500 (n ¼ 5) 4.08 Recovery periods 0 (n ¼ 2) 6.13 2500 (n ¼ 2) 5.95
TP (g/L)
1.77 ± 0.06 1.76 ± 0.02
Note: Values represent means ± SD, n ¼ number of animals. 657
658
X. Wang et al. / Regulatory Toxicology and Pharmacology 73 (2015) 652e659
Fig. 3. Selected microphotographs for main related organs (40). A, B and C refer to liver, kidney and adrenal tissue in the control group, respectively. D, E and F refer to liver, kidney, adrenal tissue in the 2500 mg/kg dose group, respectively.
at the dose of 2500 mg/kg cyadox for 13 weeks subchronic toxicity. However, in the previous study, a significant decrease in body weight was observed at the dose of 2500 mg/kg cyadox for 13 weeks subchronic toxicity test in rats (Fang et al., 2006). One of the causes might be the fact that cyadox had an influence on food consumption in rats, while it had no effect in dogs. The reason for this might be that dogs are not sensitive to bitter materials, such as cyadox, when compared with rats (Michael, 2002). In the present study, the significant increase of serum Kþ in male dogs was noted, whereas the Kþ value was within the normal range (5.42e6.87 mmol/L), as reported by Wang et al. (2009). Additionally, there were no significant pathological findings in kidney and adrenal tissues. However, the significant increase of serum Kþ in male dogs might be treatment related in the current study, which is worthy of investigating in the future. In the previous studies, it was reported that the liver, kidneys and adrenal tissues were the main target organs for toxicity in quinoxaline group (WHO, 1991b). The toxicity presented as related serum biochemistry (such as AST, ALT, CREA, Naþ and Kþ) and organ weights were significantly changed following the pathological findings (Fang et al., 2006; Ihsan et al., 2010; Wang et al., 2011b, 2010; WHO, 1991a; WHO, 1991b).
Generally, the significant change of the serum Kþ level was considered to be associated with renal and adrenal toxicity and might be the common toxicity of QdNOs, as shown for most other members (Huang et al., 2009, 2010a, 2010b; Ihsan et al., 2010; WHO, 1991c). However, adrenal toxicity of cyadox was not found in the previous subchronic studies (Fang et al., 2006; Nastuneak et al., 1986) and chronic studies conducted in rats even in the 2500 mg/kg cyadox group (Nastuneak et al., 1987; Wang et al., 2011b). As the reason that the toxicity of cyadox is milder than other QdNOs, the lower bioavailability of cyadox than other QdNOs might play an important role. It was reported that the bioavailability of mequindox were 89.4% and 16.6% after intramuscular (i.m.) and oral administration of mequindox to chickens at a single dose of 10 (i.m.) or 20 mg/kg b.w. (oral) (Ding et al., 2012). In another study, the bioavailability of mequindox was 37% when rats were orally administered with mequindox at a single dose of 10 mg/kg b.w. (Li et al., 2012). The bioavailabilities were 21% and 53% when chickens were orally administered with quinocetone or olaquindox at a single oral dose of 30 mg/kg b.w., respectively (Zhang et al., 2011; Zhu et al., 1993). However, the bioavailability of cyadox was
X. Wang et al. / Regulatory Toxicology and Pharmacology 73 (2015) 652e659
reported to be 2% when pigs were administered with cyadox at a single oral dose of 1 mg/kg b.w. (Qiu, 2003). Compared with the bioavailabilities of mequindox, quinocetone, olaquindox and cyadox, it was suggested that the lowest bioavailability of cyadox was one of the reasons for its low toxicity. Base on the same quinoxaline structure and different toxicity of QdNOs with different side chains, the side chains were presumed to play crucial roles in their toxicity. As reported, their main metabolism in vivo was similar and it was N/O group reduction (Liu et al., 2008, 2009, 2010). A recent study found that the reduction of N/O group of cyadox is more easier than other ones, and the faster deoxidation of cyadox would result in its lower toxicity (Zhang et al., 2015). In summary, the toxicity of cyadox in non-rodents was carefully studied in a wide range of doses by subchronic evaluation. Although the increased Kþ value was within the normal range in males in the 2500 mg/kg cyadox group, the 450 mg/kg dietary level was selected as the NOAEL of cyadox for dogs, which approximates to 15.3e15.4 mg/kg b.w./day. The study confirmed the low toxicity of cyadox in non-rodents, but assessment of the safety of cyadox for veterinary clinical trials is still required prior to understanding the full risks of cyadox administration as a new antimicrobial and growth-promoting feed additive. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant no. 31272614 and 31502115), Grants from 2015 National Risk Assessment of Quality and Safety of Livestock and Poultry Products (GJFP2015008) and National 863 Program of China (2011AA10A214). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.yrtph.2015.09.023. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2015.09.023. References Ding, M.X., Wang, Y.L., Zhu, H.L., Yuan, Z.H., 2006a. Effects of cyadox and olaquindox on intestinal mucosal immunity and on fecal shedding of Escherichia coli in piglets. J. Animal Sci. 84, 2367e2373. Ding, M.X., Yuan, Z.H., Wang, Y.L., Zhu, H.L., Fan, S.X., 2006b. Olaquindox and cyadox stimulate growth and decrease intestinal mucosal immunity of piglets orally inoculated with Escherichia coli. J. Animal Physiol. Animal Nutr. Berl. 90, 238e243. Ding, H., Liu, Y., Zeng, Z., Si, H., Liu, K., Liu, Y., Yang, F., Li, Y., Zeng, D., 2012. Pharmacokinetics of mequindox and one of its major metabolites in chickens after intravenous, intramuscular and oral administration. Res. Vet. Sci. 93 (1), 374e377. Fang, G., He, Q., Zhou, S., Wang, D., Zhang, Y., Yuan, Z., 2006. Subchronic oral toxicity study with cyadox in Wistar rats. Food Chem. Toxicol. 44, 36e41. FDA, 2003. Subchronic Toxicity Studies with Non-rodents. Toxicological Principles for the Safety Assessment of Food Ingredients Redbook 2000. http://www. cfsan.fda.gov/~redbook/redIVC4b.html. Huang, X.J., Ihsan, A., Wang, X., Dai, M.H., Wang, Y.L., Su, S.J., Xue, X.J., Yuan, Z.H., 2009. Long-term dose-dependent response of Mequindox on aldosterone, corticosterone and five steroidogenic enzyme mRNAs in the adrenal of male rats. Toxicol. Lett. 191, 167e173. Huang, X.J., Wang, X., Ihsan, A., Liu, Q., Xue, X.J., Su, S.J., Yang, C.H., Zhou, W., Yuan, Z.H., 2010a. Interactions of NADPH oxidase, renin-angiotensinaldosterone system and reactive oxygen species in mequindox-mediated aldosterone secretion in Wistar rats. Toxicol. Lett. 198, 112e118. Huang, X.J., Zhang, H.H., Wang, X., Huang, L.L., Zhang, L.Y., Yan, C.X., Liu, Y.,
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