Beagle dog 90-day oral toxicity study of a novel coccidiostat – ethanamizuril

doi:10.21203/rs.3.rs-28217/v3

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Beagle dog 90-day oral toxicity study of a novel coccidiostat – ethanamizuril

https://doi.org/10.21203/rs.3.rs-28217/v3

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published 17 Nov, 2020

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Background: Triazine coccidiostats are widely used in chickens and turkeys for coccidiosis control. Ethanamizuril is a novel triazine compound that exhibits anticoccidial activity in poultry. This study evaluated the subchronic toxicity of ethanamuzuril in beagle dogs administered ethanamizuril by diet at doses of 12, 60 or 300 mg/kg/day for 90 days.

Results: Ethanamizuril was well tolerated at low and middle dosages and in these dose groups there were no ethanamizuril related effects on survival, clinical observations, clinical pathology parameters, organs weight, macroscopic or microscopic evaluations. The ethanamizuril related changes were limited to effects on food consumption and histologic changes of kidneys in the 300 mg/kg/day group in both sexes. However, renal lesions resolved in dogs given ethanamizuril at 300 mg/kg/day when evaluated four weeks after the end of exposure.

Conclusions: Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 60 mg/kg/day, the middle dosage level tested. These results add to the safety database for ethanamizuril with potential for use as a novel coccidiostat.

Small Animal Medicine

Ethanamizuril

Oral subchronic toxicity

Beagle dogs

Triazine coccidiostats

Nephrotoxicity

Coccidiosis is a detrimental disease caused by development and reproduction of multiple species of the Eimeria protozoa and affects numerous species. In the commercial broiler industry, Eimeria protozoan parasitizing on the intestinal epithelium of chicken resulted in significant increase in mortality and morbidity, and led to considerable impairment of growth and feed utilization, which is capable of inflicting devastating economic losses to poultry operations. It is estimated that the substantial economic burden caused by avian coccidiosis was more than US$3 billion annually to the industry worldwide [1, 2]. Due to the lack of efficacy and safety of coccidiosis vaccines, commercial synthetic anticoccidial drugs have been the main strategy of the control of coccidiosis since the late 1940s [3]. However, more and more clinical studies showed that the widespread resistances to commercial anticoccidial drugs have emerged in coccidian parasites for continuous use and misuse of anticoccidial drugs [2, 4]. Consequently, the resulting letdown of treatment promotes a constant demand for innovative products with safe and efficient. Triazines are benzene-aceto-nitrile compounds including diclazuril, toltrazuril, ponazuril and clazuril, which have been used globally in the interception and therapy of protozoal diseases, most commonly in coccidiosis of veterinary interest since 1980s [5]. However, the widely use of triazine coccidiostats for their clinical effects have generated resistance to the genus Eimeria in recent years [5, 6]. Fortunately, no cross-resistance was observed in triazine coccidiostats [5].

Ethanamizuril (Figure 1), a novel triazine coccidiostat, namely (N-(4-(4-(3,5-dioxo-4,5-dihydro-1,2,4-triazin-2-(3H)-yl)-2-methylphenoxy) phenyl)acetamide, has been independently established by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences in recent years and exert wide application prospective in future [5, 7]. In China and Japan Patents, the chemical synthesis of ethanamizuril and the chemical structure have been published [8]. Ethanamizuril has displayed excellent effectiveness against Eimeria protozoa such as Eimeria tenella, Eimeria. necatrix, Eimeria. acervulina, and Eimeria. maxima in broiler chickens at a dosage of 10 mg/kg in the feed or 10 mg/l in the drinking water [7, 8]. At the recommended dose, ethanamizuril could prominently improve feed conversion ratios and live weight, reduce oocyst excretion, mortality and lesions in broilers. ACIs (anticoccidia indexes) of ethanamizuril reached 185~190. Furthermore, the preclinical pharmacodynamic studies demonstrated ethanamizuril did not cause cross-resistance with diclazuril or toltrazuril resistant Eimeria. tenella in broilers.

Because of the diverse toxicities, the use of some coccidiostats, such as arprinocid, roxarsone and arsanilic acid has been forbidden in poultry industry [9, 10]. Up to now, a series of studies have been conducted to evaluate the safety of diclazuril and toltrazuril for use as coccidiostats. Increase of liver weight and swelling of the centrilobular hepatocytes were seen in diclazuril treated mice and rats [11, 12, 13]. The slight effects on haematological parameters, disturbances of the liver function and decreased on the weight gain and the daily feed intake were observed in toltrazuril treated rats [11, 12, 14]. In addition, in two teratogenicity studies, teratogenicity and embryotoxicity were observed at the toltrazuril highest dose in rats [14, 15]. With high anticoccidial effectiveness, ethanamizuril would be used widely in poultry industry in future. To the best of our knowledge, a series of toxicity evaluation of ethanamizuril has been carried out in rats and mice, and the NOAEL for the dietary administration of ethanamizuril in the 90-day oral toxicity study for rats was greater than 20 mg/kg. According to VICH's guidelines the dog is the default non-rodent species required for repeat-dose toxicity testing [16]. However, the toxicity evaluation in beagle dogs has not been reported. This is unfavourable for understanding the safety and clinical use of ethanamizuril. In order to improve our understanding of the toxicity of ethanamizuril, we conducted a 90-day toxicity study of ethanamizuril in beagle dogs in accordance with the guidelines of veterinary safety evaluation.

Survival and clinical observations

No unscheduled mortality occurred during the study. During the experimental period, there were no overt ethanamizuril related clinical signs of toxicity, abnormal behaviour or altered activity were noted. All observed phenomena in the study period were accepted findings for laboratory dogs of the breed and age. Ophthalmological examinations did not find noted ethanamizuril related changes.

Food consumption and body weights

Treated with ethanamizuril, the body weight changes of beagle dogs were shown in Figure 2. The body weights of males and females showed a steady growth trend and no significant statistical difference was noted at each time points. As shown in Table 1, the body weight of remaining dogs of control and high dose groups in convalescence showed no significant difference (the data were not analyze by statistics). No significant statistical difference was also observed for average body weight gains of all groups in each stage (Figure 3). The weight gains of day 1-45 were higher than that of day 46-90, which showed a normal growth pattern of animals. A statistically significant reduction in food consumption was observed in the 300 mg/kg/day groups of both sexes compared to the control group in the whole treatment time (Figure 4). Daily food consumed in the 300 mg/kg ethanamizuril group were 3%-11.0% less than untreated control group. No other noted ethanamizuril related effects on food consumption or body weights were observed.

Clinical pathology

The hematology, clinical biochemistry and urinalysis parameters of day 0, 45, 90 (scheduled necropsy), and 118 (end of convalescence) were analyzed in this study followed the test guideline 409 of OECD. The results showed that no consistent significant ethanamizuril related changes in hematology and clinical biochemistry were noted in either males or females (Table 2,3). In addition, there were no ethanamizuril related effects noted on the evaluation of urinalysis parameters. It's no statistically significant differences between the control and test ethanamizuril treated groups. Detection data of SG, pH and URO were presented in Table 4.

Macroscopic examination and organ weights

Dogs were euthanized on day 90 and 118 separately. The organs or tissues of the animals were necropsied with macroscopic inspection. No ethanamizuril related systemic changes were observed in macroscopic inspection when the animals treated with 12, 60 and 300 mg/kg/day ethanamizuril via diet. In addition, compared to the control group, there were no significant differences on the organ weights in either males or females when animals treated with 12, 60 and 300 mg/kg/day ethanamizuril. Summary data for organ relative weight were presented in Table 5.

Microscopic examination

Microscopic examination revealed increased incidence of slight congestion in renal tubulointerstitium for dogs of both sexes in the 300 mg/kg ethanamizuril group from day 90 after treatment (Figure 5). However, the histologic changes of kidneys were disappeared in dogs after drug withdrawal 4 weeks. There were no signs of ethanamizuril related histologic alterations appeared in other scheduled organs and tissues in the microscopic examination. The kidneys were identified as the targets of a potential toxicity of ethanamizuril based on the results of this study.

The present study is the first time to investigate a comprehensive toxicology of ethanamizuril in beagle dogs. It is advantageous to scientific assessments about the safety of ethanamizuril in the prevention and treatment of protozoal diseases in poultry industry.

In previous studies, we found that the NOAEL of ethanamizuril for rats was 20 mg/kg dietary dose level [10, 11]. Based on this result, we assumed that the dose of the dog would be greater than 60 mg/kg/day through the dose conversion with body surface area between different animals. In this study, dogs were administered ethanamizuril daily by diet for 90 consecutive days at dosage levels of 12, 60, and 300 mg/kg/day. During the experiment, no animal died and no obvious changes related to ethanamizuril were observed in behavior or external appearance of the animals. In addition, there were no ethanamizuril related differences observed in blood routine examination, serum chemistry, urine routine test, organ weights, and macroscopic evaluations. However, statistically significant reduced food consumption in the high-dose (300 mg/kg/day) group was observed. It's reasonable to speculate that a high concentration of ethanamizuril in the diet may cause the dogs feel discomfort and lead to slightly loss of appetite. Meanwhile, the histologic changes of kidneys noted in the high-dose (300 mg/kg/day) group in both sexes were considered to be related to treatment with ethanamizuril. Fortunately, the indicators related to renal function, such as BUN and creatinine, and urine analysis were not affected in these dose groups. Ethanamizuril primarily affected tubules and the renal lesions were repairable after drug withdrawal.

With distinguished anticoccidial effectiveness, both diclazuril and toltrazuril are widely used in the world to control coccidiosis of veterinary interest. Meanwhile, their toxicities have been concerned for a long time. In rat, there were slight effects on the haematological parameters and disturbances of the liver function in toxicity study of diclazuril and toltrazuril [11, 13, 14]. Beagle dogs administered 80 mg/kg bw/day diclazuril for 3 months displayed a fine granular, yellow to brown pigment in the cytoplasm of the hepatocytes [13]. In addition, the high dose of toltrazuril caused the mean weight of the testes and weight of the prostate decreased [13]. However, no obvious body weights, fetal body lengths, tail lengths, litter weights, number of viable fetuse, sexternal, skeletal or visceral malformations in fetuses were noted in any groups in two-generation reproduction and teratogenic test with ethanamizuril, and no adverse effects on the central nervous system, cardiovascular system, and respiratory system were showed in safety pharmacology test too [15, 17, 18]. Furthermore, the studies of 30 and 90-day subchronic toxicity with feeding ethanamizuril fed to SD rats revealed that the high dose of ethanamizuril, above 60 mg/kg dietary level, could cause minor damage to the liver, kidneys, and other organs, and induce alopecia [11, 12]. Moreover, the disorder on the hematologic and biochemical parameters was observed in rats when they were treated with high dose of ethanamizuril [11, 12]. Fortunately, the lesions induced by ethanamizuril in rats could be rehabilitated obviously after cessation of the drug [11, 12].

An enormous array of animal factors and environmental conditions affect the outcome of hematologic and clinical biochemistry analysis. In the present study, compared with the control group, there was no significant fluctuation in the indexes of hematological, serum chemistry, urinalysis, organ weights, macroscopic evaluations in beagle dogs. However, it should be to pay attention to the reduced food consumption and the histologic changes of kidneys that were observed in beagle dog when animal treated with the high-dose (300 mg/kg/day) ethanamizuril. In the rat subchronic toxicity study of ethanamizuril, there were renal tubular atrophy, tubule protein casts and interstitial cell hyperplasia in kidneys in 130 mg/kg dose groups [11,12]. The results of toxicity studies in different kinds of animals were mutually corroborated. It indicated that the kidneys is the main toxic target organs of ethanamizuril.

In conclusion, the results of the 90-day toxicity investigation described here provide a comprehensive toxicity profile of ethanamizuril. The limited toxicity changes of ethanamizuril related were reduced appetite and histologic lesions in kidneys at the 300 mg/kg/day. Nonetheless, oral administration of ethanamizuril for 90 consecutive days were well tolerated in mature beagle dogs, when the dosage of ethanamizuril was less than 300 mg/kg/day. For this reason, it was considered that the no-observed-adverse-effect level (NOAEL) is 60 mg/kg/day.

Test materials

Ethanamizuril (CAS:1560840-75-6, C₁₈H₁₆N₄O₄, molecular weight 352.3 g/mol, purity 98.6%), N-(4-(4-(3,5-dioxo-4,5-dihydro-1,2,4-triazin-2-(3H)-yl)-2-methylphenoxy)phenyl)acetamide, was synthesized by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science (Shanghai, P.R. China) and characterized by NMR, IR, LC-MS, and LC-UV methods (data were not shown).

Animal receipt, acclimation and husbandry

Forty beagle dogs (20 males and 20 females), approximately 4–6 months of age, were obtained in good health from Xinglong Laboratory Animal Breeding Plant, Haidian District, Beijing (Batch No. SCXK (Jing) 2016-0003). Each animal was immunized as planned prior to the study. It took 7 days to get acclimatized the testing facility conditions for dogs prior to dose administration. At the initiation of ethanamizuril administration, body weights of dog ranged from 6.0–7.0 kg. Each group of animals was housed in a separate room maintained at 18-25℃, with 30–70% relative humidity, natural ventilation, and a 12-h light-dark cycle. Each dog stayed and was fed in individual stainless steel cages measuring 100 cm in height, 100 cm in length, and 90 cm in width. Noise was controlled below 50 decibels. The animals were fed 2 times daily with a medicated diet at 9:00 and 15:00. Distilled water was available ad libitum throughout the study. Regular opportunity for exercise and social interaction were allowed for all animals. The study was approved (20160105) by the Institutional Animal Care and Use Committee at Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Diet preparation

According to the requirements of different doses in the beagle dog 90-day oral toxicity study, the diets were mixed separately by group and ethanamizuril was evenly incorporated in basal diet respectively. The processing of diet was in the charge of Beijing Keao Xieli Feed company (Beijing Keao Xieli Feed Co., Ltd., Beijing, China), and the components of the diet include: water content ≤10%, crude protein ≥20%, crude fat ≥8%, Crude fiber ≤4%, Crude ash ≤9%, calcium 0.7-1.0%, Total phosphorus 0.5-0.8%. To ensure the homogeneity and effectiveness, the drugs were weighed carefully and were thoroughly mixed and prepared every 4 weeks. In addition, the stability and homogeneity of the diets were verified prior to the study by HPLC method [18].

Assignment of animals to treatment groups

The animals were randomly divided into four groups by Excel software based on the body weight, and each group of dogs were fed basal diets mixed with 0, 12, 60 and 300 mg/kg ethanamizuril for a total period of 90 days, respectively. The low and middle dose groups each consisted of 4 males and 4 females, and the control and high dose groups each consisted of 6 males and 6 females. Animals were dosed for 90 days and four dogs/sex/group were sacrificed under anesthesia with sodium pentobarbital. The remaining dogs of control and high dose groups were administered control feed for a further 4 weeks (convalescence) after which they were killed in the same manner and subjected to examination.

Parameters evaluated

Clinical observations

All animals in the study were observed at least twice daily for any changes in appearance of coat, activity and respiration, food and water intake, micturition and stool excretion. The presence or absence of findings in each animal was recorded regularly.

Prior to the start of ethanamizuril administration and at the end of the treatment period, ophthalmological examinations with fluorescein sodium method were performed respectively in control and high dose groups. If there were the changes of ophthalmology in the high dose group, all animals in the other dosing groups should be examined.

Body weights, food consumption

The individual body weight and food consumption weights of animals were recorded every 5 days throughout the study period. Food consumption was calculated as g/animal/day. In addition, the body weight on the day of randomization was also recorded.

Clinical pathology/laboratory examinations

To detect hematology and serum chemistry parameters, blood samples of treated animals were collected during study day 0, 45, 90 (scheduled necropsy), and 118 (end of convalescence). Prior to blood collection, animals were fasted overnight. Hematological test parameters were basophil (BAS), eosinophil (EOS), erythrocyte count (RBC), hematocrit (HCT), hemoglobin (HGB), leukocyte count (WBC), lymphocyte (LYMPH), monocytes (MO), neutrophils (NEU), platelet count (PLT). Albumin (Alb), alanine aminotransferase (ALT), blood glucose (Glu), blood urea nitrogen (BUN), creatinine (Cr), glutathione aminotransferase (AST), total cholesterol (TCH), total protein (TP), triglyceride (TG) were included in clinical chemistry test parameters.

During study day 0, 45, 90 (scheduled necropsy), and 118 (end of convalescence), urine samples were also collected from all animals by using cage pans. Bilirubin (T-BIL), glucose (GLU), ketones (KET), occult blood (BLO), protein (PRO) and white blood cells (WBC) were detected using qualitative indicators of analyte concentration. Urine pH, specific gravity (SG) and urobilinogen (URO) were measured quantitatively.

Necropsy and pathology

After injected sodium pentobarbital with small saphenous vein, the dogs were euthanized by exsanguination via the abdominal aorta under anesthesia. Four dogs/sex/group were euthanized on day 90 and the remaining dogs of control and high dose groups were euthanized on day 118. The necropsy included, but was not limited to, examining the body external surface, all orifices, and all organs in coelom. All the organs from animals at the scheduled necropsy including the adrenals, brain, heart, kidney, liver, lung, spleen, stomach and intestine, testis and epididymides, ovaries and uterus were weighed. The organ relative weight (the percentage of organ weight to body weight) was calculated. Based on the SOPs of histopathology technical operation, the tissue sampling, paraffin embedding, sectioning, and hematoxylin-eosin staining were conducted for the above organs from each animal, and then evaluation via light microscopic for morphological alterations. Tissues from other groups were examined as necessary to determine NOAEL in target organs.

Statistical methods

The treated groups were compared to their respective control groups. To determine intergroup differences, the data in the study were applied to a parametric one-way analysis of variance (ANOVA) [20]. When statistically significant (p < 0.05) intergroup variance was revealed by ANOVA, Dunnett's test was applied to compare the groups.

-Ethics approval and consent to participate

The study was approved (20160105) by the Institutional Animal Care and Use Committee at Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

-Consent for publication

Not applicable

-Availability of data and material

All data generated or analysed during this study are included in this published article [and its supplementary information files].

-Competing interests

The authors declare that they have no competing interests.

-Funding

The design of the study and the collection, analysis, and interpretation of data were major supported by the National Key Research and Development Program of China (2018YFE0192600 & 2018YFD0500302) and the Special Fund for Agro-scientific Research in the Public Interest (201303038).

-Authors' contributions

KZ was responsible for designing experiments and writing papers. ST analyzed and interpreted the data and was a major contributor in writing the manuscript. XW analyzed and interpreted the data, SW analyzed and reviewed data, CW, MW and YL performed the examination, LZ and FX were responsible for monitoring feed and drug quality. All authors read and approved the final manuscript.

-Acknowledgements

Not Applicable

no-observed-adverse-effect level (NOAEL)，Association for Assessment and Accreditation of Laboratory Animal Care（AAALAC）, one-way analysis of variance (ANOVA)，protein (PRO), glucose (GLU), bilirubin (T-BIL), urobilinogen (URO), occult blood (BLO), white blood cells (WBC), ketones (KET)，hemoglobin (HGB), erythrocyte count (RBC), leukocyte count (WBC), platelet count (PLT), hematocrit (HCT), eosinophil (EOS), basophil (BAS), neutrophils (NEU), monocytes (MO), lymphocyte (LYMPH), albumin (Alb), alanine aminotransferase (ALT), glutathione aminotransferase (AST), blood urea nitrogen (BUN), total cholesterol (TCH), creatinine (Cr), blood glucose (Glu), total protein (TP), triglyceride (TG).

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Table 1 The rate during the recovery period versus raw values in body weight of control and high dose groups in convalescence (Mean ± SD, %).

group	male		female
group	Control (n=2)	High (n=2)	Control (n=2)	High (n=2)
Day 95	3.46±0.83	0.71±3.54	0.27±3.91	0.12±5.22
Day 100	4.59±1.42	1.91±4.68	1.01±3.55	1.40±7.08
Day 105	5.93±1.18	2.48±4.58	2.30±4.51	2.68±7.55
Day 110	6.85±0.31	3.13±4.42	3.03±4.59	2.91±8.01
Day 115	7.59±0.53	3.79±5.32	3.57±4.53	3.6±8.71
Day 120	7.88±0.34	4.93±5.93	4.93±5.86	4.21±8.99

Note: The rate= 100%*(body weight in the recovery period - body weight in Day 90)/ body weight in Day 90. Control and high refer to 0 and 300 mg/kg ethanamizuril dose treated. No significant statistical difference was observed for the body weight.

Table 2 Hematology of dogs on day 0, 45, 90 and 118.

Day 0

Day 45

day 90

day 118

High (n=6)

Middle (n=4)

Low(n=4)

Control(n=6)

High (n=6)

Middle(n=4)

Low(n=4)

Control(n=6)

High (n=6)

Middle(n=4)

Low(n=4)

Control (n=6)

High (n=2)

Control (n=2)

Male

HGB

(g/L)

159.70±6.69

163.01±6.39

163.72±9.32

158.80±6.46

160.35±6.68

165.09±5.45

167.66±13.93

156.32±9.10

158.97±8.14

166.93±0.75

167.66±13.9

155.09±10.17

166.52±1.67

156.55±5.20

RBC

(10¹²/L)

7.66±0.72

8.21±0.30

8.03±0.71

8.14±0.62

7.52±0.79

7.97±0.54

8.02±0.02

7.78±0.55

7.38±0.68

7.44±0.10

8.02±0.02

7.39±0.21

7.35±1.17

7.18±0.18

WBC

(10⁹/L)

13.21±1.44

12.28±1.44

12.29±1.24

12.71±1.51

12.70±1.82

12.06±1.39

12.14±1.19

13.09±1.52

11.99±2.19

12.82±1.52

12.14±1.19

10.73±0.45

13.90±1.56

12.55±2.40

PLT

(10⁹/L)

670.20±69.77

586.39±7.75

687.71±78.84

666.04±91.36

649.40±71.50

594.49±98.53

710.70±14.44

618.52±69.15

668.92±87.28

603.48±24.83

710.70±14.11

602.70±79.93

392.35±26.74

406.25±136.81

HCT

(%)

47.88±2.61

48.05±3.58

46.59±1.24

45.65±1.84

47.72±4.10

39.96±5.63

47.35±0.55

46.45±1.88

46.83±1.99

48.66±2.88

47.35±0.55

45.00±2.45

44.01±1.0

43.00±3.0

EOS

(10⁹/L)

1.11±0.08

1.13±0.03

1.11±0.02

1.09±0.04

1.13±0.07

1.14±0.08

1.26±0.09

1.19±0.15

1.16±0.11

1.13±0.05

1.26±0.09

1.19±0.25

1.69±0.06

1.92±0.09

BAS

(10⁹/L)

0.11±0.04

0.13±0.04

0.15±0.04

0.14±0.04

0.09±0.07

0.15±0.07

0.12±0.09

0.11±0.04

0.16±0.04

0.20±0.06

0.12±0.09

0.11±0.08

0.09±0.01

0.08±0.01

NEU

(109/L)

1.65±0.88

2.19±0.31

2.42±0.52

2.25±0.70

1.41±0.83

1.83±0.83

2.46±0.21

1.78±0.48

1.65±0.74

1.58±0.03

2.46±0.21

1.77±0.24

1.30±0.39

1.67±0.22

(109/L)

3.28±0.12

3.26±0.02

3.22±0.13

3.19±0.13

3.30±0.11

3.28±0.03

3.35±0.05

3.25±0.15

3.32±0.09

3.32±0.03

3.35±0.05

3.32±0.15

3.94±0.14

3.92±0.09

LYMPH

(10⁹/L)

15.02±0.44

14.53±0.63

14.87±0.44

14.69±0.82

15.23±0.46

14.70±0.58

14.73±0.83

14.77±0.87

15.29±0.47

14.60±0.18

14.73±0.83

14.43±1.23

15.05±0.81

14.82±0.77

Female

HGB

(g/L)

156.43±6.99

165.17±8.09

159.22±14.25

160.26±8.61

159.72±6.46

163.54±5.86

167.66±14.01

160.17±8.64

158.04±6.94

159.74±9.13

167.66±14.01

157.24±11.55

165.02±11.04

RBC

(10¹²/L)

7.61±0.74

7.90±0.78

8.02±0.72

7.90±0.60

7.72±0.76

8.01±0.50

7.54±0.68

7.91±0.59

7.60±0.42

7.67±0.87

7.54±0.68

7.84±0.33

7.51±0.73

7.20±0.16

WBC

(10⁹/L)

13.66±1.04

11.91±1.57

12.64±1.31

13.00±1.65

13.39±1.69

12.28±1.81

12.89±1.16

12.97±1.60

12.08±1.90

11.43±0.72

12.89±1.16

12.73±2.96

12.67±2.87

12.35±2.45

PLT

(10⁹/L)

701.46±72.18

669.34±90.59

667.61±62.86

667.79±83.58

682.98±87.40

618.30±59.51

659.86±71.32

639.10±71.15

653.87±89.75

688.37±91.20

659.86±71.32

604.53±61.83

427.35±148.73

395.86±133.91

HCT

(%)

47.34±2.69

46.51±2.54

48.63±2.74

46.61±2.45

46.93±2.28

39.66±5.44

45.11±4.37

46.45±1.95

46.13±3.83

45.93±4.04

45.11±4.37

45.96±1.57

42.01±5.0

41.00±2.0

EOS

(10⁹/L)

1.13±0.09

1.16±0.14

1.13±0.01

1.10±0.07

1.14±0.10

1.11±0.05

1.20±0.08

1.07±0.07

1.14±0.12

1.12±0.03

1.20±0.08

1.12±0.03

1.39±0.06

1.04±0.14

BAS

(10⁹/L)

0.11±0.06

0.16±0.14

0.14±0.03

0.13±0.03

0.09±0.06

0.15±0.05

0.11±0.02

0.12±0.14

0.10±0.06

0.22±0.11

0.11±0.02

0.19±0.11

0.09±0.10

0.05±0.03

NEU

(109/L)

1.71±0.97

1.69±0.34

2.37±1.15

2.13±0.63

1.66±0.7

1.72±0.12

2.43±0.18

2.07±0.66

1.74±0.79

1.81±0.46

2.43±0.18

2.18±0.64

2.00±1.22

1.59±0.21

(109/L)

3.33±0.9

3.29±0.07

3.25±0.08

3.26±0.15

3.28±0.08

3.24±0.08

3.23±0.13

3.26±0.10

3.26±0.06

3.25±0.15

3.23±0.13

3.24±0.14

4.05±0.15

4.04±0.14

LYMPH

(10⁹/L)

15.03±0.43

14.73±0.66

14.44±0.74

14.76±0.83

15.03±0.50

14.46±0.28

15.04±0.96

14.95±0.87

15.32±0.49

14.96±0.79

15.04±0.96

14.52±1.18

15.06±0.81

14.86±0.80

Note: Control, Low, Middle and High refer to 0, 12, 60 and 300 mg/kg ethanamizuril dose. There were no statistically significant differences when the control and test article-treated groups were compared.

Table 3 Clinical chemistry of dogs on day 0, 45, 90 and 118.

Day 0

Day 45

day 90

day 118

High (n=6)

Middle (n=4)

Low(n=4)

Control (n=6)

High (n=6)

Middle(n=4)

Low(n=4)

Control (n=6)

High (n=6)

Middle(n=4)

Low(n=4)

Control (n=6)

High (n=2)

Control (n=2)

Male

Alb

(mmol/L)

35.36±2.16

35.42±1.68

36.21±3.89

36.07±2.18

34.87±1.75

36.49±3.70

36.17±3.82

36.67±2.27

35.25±1.65

35.75±2.40

36.03±1.98

36.20±1.89

34.99±1.24

35.71±2.43

ALT

(U/L)

53.72±6.47

56.87±6.92

53.81±5.10

57.92±6.33

55.25±6.62

54.40±1.76

52.36±2.56

57.66±5.50

54.74±5.93

51.90±3.83

54.88±6.36

55.44±5.21

54.03±7.75

58.77±8.10

AST

(U/L)

219.07±26.45

206.39±36.90

253.82±31.42

248.25±37.59

226.79±27.84

218.28±12.53

255.55±28.06

233.24±32.73

220.40±30.74

235.21±49.83

235.09±30.69

225.90±37.02

275.27±34.93

226.48±58.58

BUN

(mmol/L)

5.06±1.21

5.25±1.39

4.85±0.51

4.22±0.88

5.25±1.19

5.16±1.09

5.33±0.67

4.56±0.74

5.63±0.61

4.52±0.94

5.07±0.53

5.08±0.71

4.22±1.85

4.87±1.13

TCH

(mmol/L)

1.41±0.25

1.58±0.33

1.54±0.28

1.59±0.23

1.37±0.29

1.35±0.28

1.44±0.26

1.61±0.25

1.31±0.35

1.64±0.17

1.49±0.23

1.64±0.11

1.44±0.60

1.65±0.20

(umoL/L)

42.93±3.31

40.01±9.07

47.35±2.92

45.48±2.92

45.00±2.29

39.76±4.47

47.33±2.92

45.60±2.50

44.70±2.61

42.92±8.47

45.02±6.74

46.32±4.36

42.67±0.70

45.72±4.54

Glu

(mmol/L)

4.50±0.91

4.27±1.07

5.61±1.01

5.22±0.86

4.29±1.20

4.46±1.26

5.56±1.03

5.12±0.59

4.17±1.00

4.78±0.61

4.89±0.50

4.91±0.53

5.55±0.21

5.05±0.46

(G/L)

75.05±2.72

76.01±1.66

75.02±4.60

72.64±3.10

75.07±1.28

75.70±3.35

74.99±4.52

73.35±2.66

74.70±0.74

74.99±2.84

76.01±3.14

75.24±3.29

75.09±1.49

72.49±0.60

(mmol/L)

1.42±0.06

1.40±0.04

1.43±0.03

1.39±0.06

1.43±0.06

1.42±0.10

1.42±0.05

1.38±0.05

1.46±0.10

1.43±0.08

1.43±0.09

1.39±0.06

1.46±0.02

1.40±0.04

Female

Alb

(mmol/L)

35.30±2.17

35.39±2.16

36.36±1.83

35.41±2.12

35.36±2.11

36.92±1.52

36.10±2.06

34.85±2.06

34.62±2.38

36.85±2.33

35.59±1.07

36.07±1.23

36.40±4.95

35.71±2.30

ALT

(U/L)

52.75±6.31

52.31±8.50

59.00±10.26

57.66±7.79

56.12±7.66

51.37±3.48

59.02±10.33

60.49±5.77

53.08±7.40

56.82±8.20

57.08±6.03

56.16±6.25

42.91±0.14

58.76±8.17

AST

(U/L)

224.24±30.90

225.92±38.31

286.14±39.24

239.81±38.82

212.03±14.14

247.89±35.59

239.62±5.35

240.87±32.28

214.61±19.90

226.69±36.42

230.00±33.50

242.82±30.21

247.70±46.71

226.45±48.51

BUN

(mmol/L)

5.35±1.14

5.06±1.16

4.71±0.16

4.65±0.66

5.11±1.18

4.85±0.81

3.68±1.51

4.18±0.87

5.27±0.90

4.81±0.49

4.35±0.60

4.90±0.58

5.67±0.58

4.85±1.09

TCH

(mmol/L)

1.56±0.50

1.49±0.36

1.52±0.46

1.64±0.28

1.42±0.43

1.43±0.24

1.48±0.54

1.60±0.28

1.35±0.16

1.44±0.27

1.58±0.15

1.66±0.23

1.40±0.24

1.65±0.12

(umoL/L)

44.75±2.73

46.15±3.65

44.45±3.59

44.17±4.06

43.90±2.06

46.51±4.38

44.38±3.59

45.14±3.15

42.57±3.34

43.85±5.03

46.65±1.93

45.38±2.46

40.09±2.18

45.76±4.44

Glu

(mmol/L)

4.40±1.16

4.29±1.20

5.97±0.42

5.37±0.84

4.56±0.63

5.00±1.58

5.94±0.51

5.16±0.86

4.31±1.12

4.93±0.89

5.28±0.70

4.74±0.46

4.70±2.13

5.03±0.48

(G/L)

76.22±3.76

75.17±1.60

73.14±3.68

74.29±3.56

75.77±2.03

76.31±2.58

73.07±3.61

73.35±2.81

75.46±1.26

74.11±3.32

72.87±2.04

71.97±2.37

74.89±0.98

72.46±0.51

(mmol/L)

1.44±0.09

1.43±0.06

1.48±0.01

1.40±0.07

1.40±0.14

1.36±0.01

1.46±0.05

1.42±0.07

1.40±0.03

1.36±0.13

1.39±0.05

1.34±0.04

1.40±0.04

1.41±0.08

Note: Control, Low, Middle and High refer to 0, 12, 60 and 300 mg/kg ethanamizuril dose. There were no statistically significant differences when the control and test article-treated groups were compared.

Table 4 Urinalysis of dogs on day 0, 45, 90 and 118.

	male			female
	SG	pH	URO	SG	pH	URO
Day 0
High (n=6)	1.08±0.03	7.60±0.32	3.11±0.13	1.04±0.04	7.44±0.51	3.15±0.08
Middle (n=4)	1.09±0.08	7.74±0.56	3.07±0.08	1.09±0.15	7.39±0.31	3.10±0.09
Low(n=4)	1.11±0.04	7.49±0.45	3.09±0.06	1.09±0.04	7.33±0. 42	3.17±0. 06
Control(n=6)	1.05±0.02	7.29±0.27	3.09±0.07	1.13±0.08	7.49±0.27	3.17±0.06
Day 45
High (n=6)	1.07±0.05	7.32±0.33	3.11±0.13	1.11±0.11	7.41±0.53	3.15±0.08
Middle (n=4)	1.09±0.05	7.30±0.14	3.10±0.07	1.03±0.10	7.42±0.34	3.12±0.03
Low(n=4)	1.00±0.03	7.47±0.38	3.13±0.09	1.06±0.10	7.34±0. 17	3.19±0. 03
Control(n=6)	1.05±0.06	7.31±0.25	3.08±0.10	1.15±0.08	7.37±0.28	3.17±0.10
Day 90
High (n=6)	1.09±0.05	7.46±0.42	3.13±0.12	1.06±0.03	7.26±0.53	3.13±0.07
Middle (n=4)	1.13±0.10	7.19±0.12	3.05±0.13	1.08±0.06	7.31±0.33	3.11±0.09
Low(n=4)	1.04±0.07	7.55±0.35	3.15±0.01	1.10±0.07	7.58±0.36	3.16±0. 04
Control(n=6)	1.08±0.07	7.39±0.12	3.12±0.10	1.18±0.04	7.35±0.25	3.13±0.02
Day 118
High (n=2)	1.10±0	7.48±0.12	3.08±0.06	1.03±0.06	7.29±0.80	3.17±0.04
Control(n=2)	1.08±0.07	7.35±0.30	3.08±0.06	1.09±0.01	7.74±0.01	3.15±0.02

Note: Control, Low, Middle and High refer to 0, 12, 60 and 300 mg/kg ethanamizuril dose. There were no statistically significant differences when the control and test article-treated groups were compared.

Table 5 Relative organ weights (g/100 g final bw) in dogs fed ethanamizuril on day 90 and 118 in subchronic toxicity study (Mean ± SD).

	Day 90				Day 118
	High (n=4)	Middle (n=4)	Low(n=4)	Control (n=4)	High (n=2)	Control (n=2)
male
liver	48.1±2.58	49.19±1.77	50.26±0.79	48.66±2.15	50.02±0.88	50.43±0.31
kidney	4.63±0.15	4.9±0.27	5.01±0.65	4.81±0.12	4.55±0.12	4.56±0.20
spleen	4.19±0.18	4.35±0.22	4.31±0.22	4.18±0.28	4.17±0.04	4.23±0.10
stomach and intestine	84.93±4.51	85.17±3.93	84.84±8.21	85.33±4.67	79.61±0.35	79.37±2.74
lung	8.92±0.22	9.12±0.16	9.27±0.45	9.08±0.35	9.04±0.13	9.22±0.43
heart	8.86±0.47	9.06±0.39	10.10±0.15	8.90±0.18	8.85±0.24	8.83±0.11
brain	7.81±0.23	8.16±0.72	8.04±0.25	7.97±0.37	7.67±0.12	7.60±0.26
adrenal gland	0.20±0.02	0.21±0.02	0.22±0.02	0.22±0.01	0.16±0.01	0.16±0.01
testes	0.94±0.09	0.93±0.04	0.95±0.04	0.94±0.06	1.08±0.06	1.01±0.15
epididymides	0.36±0.03	0.38±0.03	0.31±0.01	0.3±0.03	0.30±0.02	0.30±0
female
liver	47.81±1.49	48.65±2.26	48.8±1.43	48.15±2.12	46.13±0.95	47.26±2.66
kidney	4.88±0.26	4.89±0.19	5.15±0.31	4.91±0.20	4.88±0.06	4.93±0.39
spleen	3.83±0.10	3.9±0.16	3.89±0.19	3.86±0.12	3.66±0.05	3.78±0.46
stomach and intestine	89.78±2.10	91.43±3.18	88.14±4.81	88.79±4.65	86.06±0.55	84.43±2.90
lung	9.81±0.26	10.15±0.19	10.16±0.38	9.80±0.41	9.67±0.20	9.54±0.32
heart	9.47±0.42	9.61±0.28	9.14±0.55	9.57±0.55	9.34±0.34	9.59±0.57
brain	8.17±0.28	8.62±0.19	8.34±0.14	8.3±0.31	8.14±0.92	8.33±0.38
adrenal gland	0.19±0.02	0.19±0.01	0.17±0.02	0.19±0.01	0.18±0	0.18±0.01
uterus	0.15±0.02	0.15±0.01	0.15±0.01	0.15±0.01	0.13±0.02	0.13±0.01
ovaries	0.55±0.03	0.57±0.01	0.57±0.02	0.56±0.02	0.53±0.04	0.54±0

Note: Control, Low, Middle and High refer to 0, 12, 60 and 300 mg/kg ethanamizuril dose. No test article-related effects were noted on the organ weights of the male and female animals treated with ethanamizuril compared to the control group in either males or females.

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Beagle dog 90-day oral toxicity study of a novel coccidiostat – ethanamizuril

Status:

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Abstract

Figures

Background

Results

Survival and clinical observations

Food consumption and body weights

Clinical pathology

Macroscopic examination and organ weights

Microscopic examination

Discussion

Conclusions

Methods

Test materials

Animal receipt, acclimation and husbandry

Diet preparation

Assignment of animals to treatment groups

Parameters evaluated

Clinical observations

Body weights, food consumption

Clinical pathology/laboratory examinations

Necropsy and pathology

Statistical methods

Declarations

Abbreviations

References

Tables

Supplementary Files

Status:

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Version 3