Toxicity of diclofenac sodium salt in Yucatan minipigs (Sus scrofa) following 4 weeks of daily intramuscular administration

Highlights • Four-week repeated-dose toxicity of intramuscular DSS was studied in minipigs.• DSS administration at ≥10 mg/kg/day causes toxicity and injection-site reaction.• The NOAEL of DSS after 4-week administration was 2 mg/kg/day in minipigs.

Nonclinical studies in various species revealed that most adverse DSS-related side effects generally occurred in the renal, hepatic, and gastrointestinal systems. For example, hepatobiliary, nephro-, and GI toxicities were observed following 28 days of repeated oral administration in mice (9.5 mg/kg/day) [11]. In rats, the oral or intramuscular administration of DSS for 2-24 weeks (0.25− 40 mg/kg/day) resulted in GI toxicity, lymph node hypertrophy and hyperplasia, and anemia as well as changes in hepatic or renal function parameters (aspartate aminotransferase [AST], alanine aminotransferase [ALT], Alkaline phosphatase [AP], or blood urea nitrogen [BUN]) [1,[12][13][14]. Hematological effects of DSS in goats or chickens have also been reported [2,15]. In dogs, oral dosing for 30-90 days at 0.5− 2.5 mg/kg/day resulted in GI toxicity, anemia, extramedullary hematopoiesis, and lymphadenitis [14]. In rhesus monkeys, oral dosing for 30 days to 6 months at 1− 500 mg/kg/day resulted in mortality (>15 mg/kg/day), GI toxicity, and anemia [14]. These nonclinical data suggest that some toxicological responses observed in humans are also observed in animals. However, symptoms such as skin-related problems have not been observed in animals except in minipigs.
In minipigs (Yucatan), DSS administered dermally for 30 days to 6 months resulted in GI toxicity, renal and hepatic toxicity, and skin reactions, such as erythema at application sites [14]. Because no-observed-adverse-effect levels (NOAELs) and toxicokinetic values vary according to the route of administration [16,17], this study was performed to present the detailed toxicity and system exposure results of DSS after 28 days of intramuscular administration in minipigs.

Guidelines for experimental study
The study was designed to administer repeat-dose DSS intramuscularly in 32 minipigs (Yucatan) to investigate toxicity during a 4-week period. The study was conducted at the contract research organization

DSS
DSS (product No. D6899, CAS No. 15,307-79-6, Sigma-Aldrich, Saint Louis, Missouri, USA) and sterilized water (Lot no. 17S2 F21, Dai Han Pharm, Korea) were used as the test substance and vehicle control, respectively. DSS was formulated by suspending it in vehicle to prepare a stock solution (50 mg/mL). This formulation was prepared once a week and stored at room temperature in the dark. Analyses of formulation stability, homogeneity, and concentration were conducted.

Animals
All study procedures and animal care were performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines and approved by the Institutional Animal Care and Use Committee of the Korea Institute of Toxicology (KIT-1903− 0079). The animals used in this study were 3-5-month-old Yucatan Minipigs (Sus scrofa) weighing 10-15 kg. Specific pathogen-free minipigs and minipig diets, sterilized by gamma irradiation, were procured from Optipharm, Co., Ltd. (Korea). The animal room was maintained at the following conditions: temperature, 20-26 • C; relative humidity, 30-70 %; ventilation, 10-20 times/h; and light cycle of approximately 12 h at 300-700 lx.

Experimental design
All animals were randomized and assigned to study groups using the Pristima system (Version 7.4. Xybion Corporation, Lawrenceville, New Jersey, USA) based on body weight stratification after 7 days of acclimation. To obtain data before DSS administration, a 7-day pretreatment was set, followed by an administration period for 4 weeks and a recovery period for 2 weeks. A total of 24 male and female minipigs were assigned to a vehicle control group (three animals/sex/group) and three treatment groups (three animals/sex/group), namely low-, middle-, and high-dose groups that received 2, 10, and 20 mg/kg/day of DSS, respectively. The reversibility of any observed toxicities was assessed in a subset of eight additional minipigs (two animals/sex) in the vehicle control and 20 mg/kg/day dose groups, which were assigned to a 2week recovery period. Doses were administered at a volume of 0.4 mL/kg based on the most recently measured body weight. DSS was administered intramuscularly to the cervical muscle behind the ear once a day for 28 days.

Clinical signs
Clinical signs, including mortality, moribund state, general appearance, and behavioral changes, were observed and recorded once a day during the entire study period and twice a day during the administration period.

Body weight and food consumption
Body weight and food consumption of all animals were measured weekly. After separating the animals by installing partitions in the cages, feed was provided individually, and the partitions were removed after consumption. Considering the characteristics of the minipigs, the dropped feed on the floor of the cage was recorded as the feed amount remaining after consumption. However, feed falling on the floor outside the cage was not considered to be residual because it could not be accessed it for consumption.

Ophthalmological and electrocardiography examinations
All live animals were first sedated with ketamine (11− 12 mg/kg) and xylazine (2− 3 mg/kg) prior to ophthalmological and electrocardiographic examination during the pretreatment period, treatment period (day 24), and recovery period (day 10 or 11). Ophthalmological examinations were conducted by a veterinary ophthalmologist using a slit lamp (XL-1, Ohira Co., Ltd., Japan) and a binocular indirect ophthalmoscope (Vantage Plus Digital, Keeler Ltd., England) after the animals were administered one or two drop of a mydriatic agent (Mydriacyl oph soln 1%, Alcon, Geneva, Swiss) to both eyes.
Changes in the electrocardiograms of the anesthetized animals were recorded using an electrocardiograph (Cardio XP, Bionet Co., Ltd., Korea) by placing limb leads I, II, III and the augmented leads aVR, aVL, and aVF. Cardiac electrocardiogram intervals (QT, QTc, PR, and QRS) and heart rate were measured and analyzed by a veterinarian. QTc was derived at each time point using Bazett's formula [18,19].

Hematology and clinical chemistry
Blood samples for clinical pathology were obtained on the following days: before administration and on day 29 for all animals or day 43 for recovery animals. All animals were fasted for approximately 16 h prior to blood collection, but drinking water was provided ad libitum. Blood was collected from the jugular vein and placed into EDTA-2 K tubes for hematological assessment (0.5 mL), into tubes containing 3.2 % sodium citrate for plasma separation (1 mL), and into tubes without anticoagulant for serum preparation (1.5 mL). The parameters measured were as follows: hematological parameters, including total white blood cell (WBC) count, total red blood cell (RBC) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count absolute, reticulocyte count relative, neutrophil count absolute, neutrophil count relative, eosinophil count absolute, eosinophil count relative, basophil count absolute, basophil count relative, monocyte count absolute, monocyte count relative, lymphocytes absolute, lymphocytes relative, large unstained cells absolute, and large unstained cells relative; coagulation parameters, including prothrombin time and activated partial thromboplastin time; and clinical parameters, including glucose, BUN, creatinine (CREA), total protein (TP), albumin, albumin/globulin ratio, total cholesterol, triglyceride, phospholipid, AST, ALT, total bilirubin, AP, gamma-glutamyl transpeptidase (GGT), creatine phosphokinase, calcium, inorganic phosphorus, sodium, potassium and chloride.

Urinalysis and urine chemistry
Animals in the cage were separated and transferred to a metabolic cage or a cage equipped with a device for collection of urine on the day before administration as well as terminal sacrifice (day 29), and recovery sacrifice (day 43). Before urine collection, animals were fasted overnight; however, drinking water was made available. The urine volume was recorded using a measuring cylinder, and the following parameters were measured using a Cobas U411 urine analyzer (Roche, Switzerland) using a urine reagent strip: color, clarity, pH, specific gravity, bilirubin, proteins, urobilinogen level, nitrite level, glucose level, erythrocyte count, ketone, leukocyte count, urine potassium, urine chloride, urine sodium, urine cast and epithelial cell count.

Gross observation and organ weight
Pre-anesthesia was induced in all animals using ketamine (11− 12 mg/kg) and xylazine (2− 3 mg/kg) on the day of the terminal sacrifice (day 29) and recovery sacrifice (day 43) after fasting for at least 16 h. Following this, the animals were heavily sedated with thiopental sodium (75− 80 mg/kg) administered intravenously and then euthanized by exsanguination. Abnormalities in the external as well as in the abdominal, thoracic, and cranial cavities were observed by a veterinary pathologist, and full macroscopic examinations were recorded.

Histopathology
Histopathological examination was conducted for the following tissues: abnormal lesions, adrenal glands, animal ID, aorta (thoracic), brain, cecum, colon, duodenum, epididymis, esophagus, eyes with optic nerve, femur with marrow, heart, ileum, jejunum, kidneys, liver with gall bladder, lung with bronchi, mammary gland, uterus with cervix, vagina, injection sites, pancreas, prostate, pituitary gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscles, skin, thoracic spinal cord, spleen, sternum with marrow, stomach, testes, thymus, thyroids, tongue, trachea, urinary bladder, mesenteric lymph node, ovaries, and mandibular lymph nodes. The tissues from each animal were preserved in 10 % neutral buffered formalin, except the eyes with the attached optic nerve, which were fixed in Davidson's fixative, and the testes and epididymides, which were fixed in Bouin's fixative. Especially for the lung and urinary bladder, formalin was infused and fixed. After approximately 24-72 h of fixation, tissues preserved in Images were collected at ×200 or ×400 magnification, and microscopic evaluation was performed by a veterinary pathologist.

Bioanalysis
Approximately 1.0 mL of blood was collected from the jugular vein or vena cava on Day 1 and Week 4 from all available animals excluding the recovery animals. For vehicle control group, blood will be collected at pre-dose (0) and approximately 2 h after dosing (total 2 points). For treatment groups, blood will be collected at pre-dose (0), approximately 0.5, 1, 2, 4, 6, 10 and 24 h after dosing (total 8 points). The blood will be collected into blood collecting tubes containing potassium salt of EDTA. Blood samples will be mixed gently and placed on crushed wet-ice/ kryorack and then centrifuged (approximately 3000 rpm, 10 min, 4 • C). Following centrifugation, concentration of diclofenac sodium salt (DSS) in the plasma samples was analyzed according to the validated biological sample analysis method using LC-MS/MS (KIT Study No. G218052). The obtained samples were conducted protein removal using a methanol solution containing an internal standard (Amlodipine besylate) and centrifuged. A calibration curve was created with the concentration of DSS as the x-axis and the peak area ratio of DSS and the internal standard as the y-axis using 1/x2 weighted regression. The range of the diclofenac calibration curve was 10− 4000 ng/mL.

Toxicokinetic analysis
Non-compartmental method based on blood concentration curves was used for toxicokinetic analysis. Maximum plasma concentration (C max ) and Time to reach C max (T max ) were taken, and elimination rate constant (K el ) elimination half-life (T 1/2 ) were calculated from the plasma concentration versus time profile. In addition, area under plasma concentration-time curve (AUC) at the last quantifiable time point (AUC last ), actual volume of distribution at steady state (V ss ) and actual clearance (CL) were calculated using linear trapezoidal rule. All these toxicokinetic parameters were calculated and analyzed using the Phoenix® WinNonlin® (version 8.1, Centara Inc., USA)).

Statistical analysis
All data were statistically analyzed using the Pristima software (Version 7.4, Xybion Medical Systems Corporation, Lawrenceville, New Jersey, USA). Multiple comparison tests were performed to compare different dose groups. The data were analyzed for homogeneity of variance using Bartlett's test. Homogeneous data were analyzed using analysis of variance, and the significance of inter-group differences was analyzed using Dunnett's test. Heterogeneous data were analyzed using the Kruskal-Wallis test, and the significance of inter-group differences between the vehicle control and treated groups was assessed using Dunn's rank sum test. After performing the F-test for assessing homogeneity variance between the vehicle control and recovery groups, Student' s t-test was conducted to analyze significant differences between the homogeneous data of groups. The Wilcoxon rank-sum test was used for assessing differences between heterogeneous data of groups. A p value less than 0.05 was considered statistically significant

Formulation analysis
Solutions of DSS in the range of 1− 50 mg/mL were shown to be stable for 7 days under storage at room temperature in the dark. The low-, middle-, and high-dose DSS solutions, assessed at the start of dosing (week 1) and also at the last week (week 4), were found to have homogeneous distribution of DSS with coefficients of variation (CVs) within 10 % (0.3− 0.9%). The concentration analysis showed that acceptable stability was within 15 % CV (101.6-102.9 % on Day 1 and 97.0-102.7 % on week 4).

Body weight
As shown in Fig. 1, overall, body weight showed a tendency to increase during the administration and recovery periods. Body weight decreased slightly after 3 weeks of administration, but this decrease was not statistically significant.

Food consumption
In both sexes, there were no changes in food consumption following DSS administration

Ophthalmoscopy and electrocardiography
No DSS related changes in ophthalmoscopy and electrocardiography following DSS administration were observed.

Observations: survival and clinical signs
Following daily DSS administration, all minipigs were examined twice daily for clinical signs related to DSS administration using a standard operating procedure. Table 1 shows the number of minipigs  showing clinical signs related to DSS administration according to group. No animals died as a result of DSS treatment during the study. Low-dose group (2 mg/kg/day): Slight emesis was observed after 13-14 days of administration in one female minipig. Two male minipigs had consistent swelling (slight to moderate) of the lower forelimb, hindlimb, or pinna from day 5 of administration; in addition, these animals showed limping from day 16 to the day of necropsy. In one male animal, slight abscess was noted in the lower hindlimb from day 22 to the day of necropsy.
Middle-dose group (10 mg/kg/day): Emesis (slight to moderate) was sporadically observed after day 11. Limping was showed from at day 16 that persisted until the day of necropsy. In one male minipig, abscess of the lower hindlimb (slight to moderate) was observed from day 16 to the day of necropsy. A slight swelling of the neck was noted in two males and one female minipigs from day 18 to the day of necropsy. In two male animals, some amount of blood was noted intermittently in the urine after 2 weeks after administration. Ulcerations in the pinnae (slight to severe) appeared in two males at day 10 and were continuously present until the day of necropsy.
High-dose group (20 mg/kg/day): Chronic intermittent emesis (slight to moderate) was observed in all animals from day 7. One male developed a limp at day 16 that persisted until the day of necropsy. There was a slight swelling in the necks and lower forelimbs in both sexes (3/5, 3/5) from day 17 to the day of necropsy after the recovery period. Slight abscesses in the lower forelimbs and hindlimbs were observed in one male and one female for 2-3 days after 20 days of administration. Ulcerations (slight to moderate) of the lower forelimbs, upper/lower oral regions, and the pinnae were noted in two male minipigs and one female minipig from day 11 to the day of necropsy. In addition, swelling of the palate (slight to moderate) was observed in all male and in three female minipigs from day 18 to the day of necropsy day after the recovery period Blood was sporadically noted in the urine of three male and two female minipigs (slight to moderate) after 2 weeks of administration. Tooth loss occurred in three male animals and one female animal from day 18. Table 2 shows the hematology and coagulation parameters that were statistically significantly different in minipigs pre-and postadministration of DSS, and during the subsequent 2-week recovery period. Compared with those in the vehicle control group, males from the middle-dose group and females in the high-dose group receiving 10 and 20 mg/kg/day at day 29 had significant (p < 0.05− 0.01) decreases in mean RBC counts (0.53-fold), hemoglobin level (0.56-fold), and hematocrit level (0.61-fold), as well as significant (p < 0.05− 0.01) increases in WBC (2.55-fold), absolute and relative neutrophil (4.12-fold and 1.68-fold, respectively), and absolute monocyte (2.62-fold) counts. Moreover, significant (p < 0.05− 0.01) increases in mean corpuscular volume (1.15-fold), absolute and relative reticulocyte counts (5.57-fold and 8.66-fold), and platelet counts (2.21-fold) were noted in both sexes   The values are expressed as mean ± SD, *D: Dunnett LSD Test significant at the 0.05. **D: Dunnett LSD Test significant at the 0.01, *R: Dunn Rank Sum Test Significant at the 0.05, **R: Dunn Rank Sum Test Significant at the 0.01, *T: t-test Significant at the 0.05, **T: t-test Significant at the 0.01. CREA, creatinine; TP, total protein; ALB, albumin; A/G, albuminglobulin ratio; ALT, alanine aminotransferase; GGT, gamma glutamyl transpeptidase; ALP, alkaline phosphatase; TCHO, total cholesterol; TG, triglyceride; Ca, calcium; IP, inorganic phosphorus; K, potassium; PL, phospholipid. ERY, erythrocyte; URBC, red blood cell in urine; white blood cell in urine; Neg, negative.

Clinical pathology
Discoloration, bilateral, diaphragmatic lobe, dark, partial in the high-dose group receiving 20 mg/kg/day on day 29. In addition, the absolute eosinophil count significantly (p < 0.05) increased (3.76fold) in males in the high-dose group on day 29. These changes were partially or fully resolved after the recovery period.
The clinical chemistry parameters that were statistically significantly changed are shown in Table 3, along with the mean values and standard deviations. There were significant (p < 0.05− 0.01) decreases in the TP (0.62-fold), albumin (0.56-fold), GGT (0.63-fold), AP (0.54-fold), and calcium (0.8-fold) levels in both sexes in the high-dose group on day 29. Compared with those in the vehicle control group, males in the middledose group and females in the high-dose group had decreased ALT levels and males in the middle-dose group had decreased CREA levels on day 29. These were partially or fully resolved after the recovery period.
Selected urinalysis and urine sediment data are shown in Table 4. In the urinalysis, a score of 5 or greater was observed for males in the middle-dose group (2/3 and 2/5, respectively) and for females in the high-dose group (4/5). The urine sediment examination revealed the presence of RBCs and WBCs in the urine of males in the middle-dose group over (1/3 and 1/5, respectively) and of females in the high-dose group (3/5). Other statistically significant changes were not considered DSS-related symptoms as they did not show a dose-dependent effect. Table 5 summarizes the number of animals showing abnormal findings on macroscopic examination. Discoloration (brown, red, or yellow) at the injection site was observed in all animals except the vehicle control group at the end of the administration. A female in the vehicle control group and all animals in the high-dose group showed discoloration at the injection site after the recovery period. A swollen ear (1/3 males) or swollen lower hindlimbs (1/3 male and 1/3 female) was observed in the low-dose group receiving 2 mg/kg/day. Ulceration was noted around the mouth in the high-dose group receiving 20 mg/kg/day (3/3 males), and ulceration of the ear was noted in the middle-dose (2/3 males) and high-dose (1/3 female) groups receiving 10 and 20 mg/kg/ day, respectively. In addition, ulceration of the lower forelimb was observed in the middle-dose (1/3 male) and high-dose (1/3 male) groups receiving 10 and 20 mg/kg/day, respectively, as well as in the lower hindlimb of the high-dose (1/3 female) group receiving 20 mg/ kg/day. These findings were not observed in the recovery group and therefore appeared to be fully reversible. Enlarged and red discolored mesenteric lymph nodes and omental adhesions to the stomach were also observed in one male in the high-dose group. A cyst in the right kidney was observed in a male in the middle-dose group receiving 10 mg/kg/day. Pale discoloration of the kidneys (1/2 female) and reductions in thymus sizes (2/2 males) were observed in the high-dose recovery group receiving 20 mg/kg/day.

Organ weight
Organ weight (mean and standard deviation) are shown in Table 6. The absolute and relative (to terminal body weight) weight were significantly increased in the liver (1.59-fold and 1.72-fold, respectively) and kidney (1.93-fold and 1.79-fold) of males and females receiving 20 mg/kg/day compared with those in the vehicle control group. These changes showed could be fully resolved in the recovery phase. The values are expressed as mean ± SD, *D: Dunnett LSD Test significant at the 0.05, **D: Dunnett LSD Test significant at the 0.01, **R: Dunn Rank Sum Test Significant at the 0.01. TBW, terminal body weight. Table 7 provides the number of minipigs showing abnormal microscopic findings following administered DSS intramuscularly for 4 weeks and recovery period for 2 weeks.

Liver.
Mixed cells were minimally to moderately infiltrated into the hepatic lobular margin in the middle-dose (2/3 males and 2/3 females) and high-dose (all males and females) groups receiving 10 and 20 mg/kg/day, respectively. Slight eosinophil filtration was observed in the gallbladder of one female in the middle-dose group receiving 10 mg/ kg/day. Mixed cell infiltration was characterized by the invasion of numerous eosinophils and few mononuclear cells in the interstitial layer of the hepatic lobules and the hepatic portal vein (Fig. 2A). These changes were not observed in the recovery group.

Stomach.
Minimal-to-slight erosions/ulcers were noted in the middle-dose (1/3 female) and high-dose (1/3 male and 2/3 female) groups receiving 10 and 20 mg/kg/day, respectively (Fig. 2D). Marked submucosal edema in the stomach was observed in two animals of both sexes each at the high dose. Marked eosinophil and neutrophil infiltration was observed in the submucosa of males in the high-dose group (2/3 and 1/3, respectively) and of females in the middle dose (1/3 and 1/3, respectively) and high-dose group (2/3 and 1/3, respectively) receiving 10 and 20 mg/kg/day, respectively.

Intestine (cecum, colon, and ileum).
Erosions/ulcers (minimal to slight) were observed in the cecum, colon, or ileum (Fig. 2E) of two males in the high-dose group and in all females in the middle-dose and high-dose groups receiving 10 and 20 mg/kg/day, respectively. Eosinophil infiltration (minimal to slight) was observed in all animals in the middle-dose group receiving 10 mg/kg/day.

Skin.
A moderate number of erosions/ulcers were found in the ear, lower forelimbs/hindlimbs, or around the mouth (Fig. 2F) in the middle-dose (2/3 males) and high-dose (3/3 males and 2/3 females) groups receiving 10 and 20 mg/kg/day, respectively. Slight-to-marked granulomatous inflammation and minimal-to-moderate myonecrosis were observed at the site of intramuscular injection (Fig. 2G) in the middle-dose (1/3 and 3/3 males as well as 1/3 and 2/3 females, respectively) and high-dose (3/3 and 3/3 males as well as 2/3 and 3/3 females, respectively) groups receiving 10 and 20 mg/kg/day, respectively. In addition, moderate-to-marked chronic active inflammation was noted at the injection site in the middle-dose (2/3 males and 2/3 females) and high-dose (1/3 female) groups receiving 10 and 20 mg/kg/ day, respectively. These alterations were also retained during the recovery period.

Toxicokinetics
The mean value and standard deviations of plasma-levels of all minipigs receiving 2, 10, or 20 mg/kg/day on Day 1 and Day 28 are given in Fig. 3. Table 8 summarized the mean and standard deviation of the toxicokinetic parameters following the intramuscular administration of diclofenac sodium salt (DSS) at dose of 2, 10 and 20 mg/kg to minipigs for four weeks. Systemic exposure (AUC last ) was proportional to the dose by increasing about 1:5.2:10.1 in males (Fig. 3A) and 1:5.6:6.5 in females (Fig. 3B) as the doses increased to a ratio of 1:5:10 on Day1. There was a slightly lower rate of increase in the female high dose group receiving 20 mg/kg/day. The average value of T max on Day 1 was 0.5− 2 hours after administration. C max was measured in the ratio of 1:2.8:4.4 After repeated administration for 4 weeks, systemic exposure (AUC last ) to diclofenac sodium salt was 89-120 % at the low dose group receiving 2 mg/kg/day of both sexes compared to that on Day 1, and there was no difference according to diclofenac sodium salt repeated administration ( Fig. 3E-F). However, systemic exposure (AUC last ) at 10 mg/kg and 20 mg/kg dose groups showed a tendency to decrease to 68-70 % and 27-40 %, respectively ( Fig. 3E-F).
Systemic exposure (AUC last ) of diclofenac sodium salts in females was indicated in 56-94 % on Day 1 and 84-119 % on Day 28 compared to males. A gender difference was not observed (< 2 fold) in systemic exposure (AUC last ) of whole groups except for 20 mg/kg dose group on Day 1 (Fig. 3E-F).

Discussion
The toxicity of diclofenac sodium salt (DSS) has been assessed in various animal studies [2,11,12,14]. Gastrointestinal (GI)-related bleeding or anorexia were observed in acute toxicity study, and LD50 was between 95-1300 in mice, 53-1500 mg/kg in rats, 125-300 mg/kg in rabbits, 1110-1250 mg/kg in guinea pigs, 59-800 mg/kg in dogs and 3200 mg/kg in monkeys [14,20]. GI, hepatic, and renal dysfunctions were observed in chronic toxicity studies through oral, dermal or subcutaneous administration from about 1 week to 1 year, and No-observed-adverse-effect-level (NOAEL) was 2.5 mg/kg in rat administered DSS orally for 91 days, 7 mg/kg in rat administered DSS intravenously for 4 weeks and 3 mg/kg in monkey administered DSS intravenously for 4 weeks [20,21]. According to the results of dermal administration toxicity study for 9 weeks or 6 months in minipigs, not only the toxicity symptoms mentioned above, but also skin reactions not observed in other animal species were observed in pigs. However, sporadic erythema or dermatitis at the injection site was observed, and no other skin changes were observed [14].
Our study was conducted to evaluate the toxicity of DSS after 4 weeks of repeated intramuscular administration once daily in minipigs and to assess recovery for 2 weeks. A total of 32 minipigs were used in this study, and they were categorized into four dosing groups: 0, 2, 10, and 20 mg/kg/day. Each group comprised three males and females each, whereas the 0 mg/kg/day (vehicle control group) and 20 mg/kg/day groups, which were assigned as recovery groups, had additional two males and females. No deaths due to DSS administration were noted during the experimental period; however, significant changes in clinical signs; and hematological, clinical chemistry, and urinalysis parameters; organ weight; and macro/microscopic examination results were observed.
DSS-related renal parameters changes such as renal papillary necrosis, tubular dilation, and basophilia were observed at 10 mg/kg/day in males and 20 mg/kg/day in both sexes. These parameters included the colored urine observed in clinical analysis, reductions in total protein and albumin in clinical chemistry analysis, absolute or relative kidney weight increases in organ weight analysis, and paleness of the kidney in macroscopic examination. These changes were considered the adverse effects of DSS and showed a tendency to be resolved during the recovery period.
In the liver, mixed cell infiltration of the hepatic lobular margin was observed in both sexes in the 10 and 20 mg/kg/day groups, and eosinophil infiltration of the gallbladder was observed in females in the 10 mg/kg/day group. In this regard, increases in the number of leukocytes, monocytes, and eosinophils were observed in the hematological examination, as well as an increase in the absolute or relative weight of the liver. All these change were likely related to DSS administration, and there was a tendency for these to be resolved after the recovery period.
Histopathological examination of the GI tract (stomach, ileum, cecum, or colon) revealed erosions/ulcers in the 10 (female) and 20 mg/ kg/day (both sexes) groups. With regard to these changes in the GI tract, In A-D, the square represents the low-dose group (2 mg/kg/day), the circle represents the middledose group (10 mg/kg/day), and the triangle represents the high-dose group (20 mg/kg/day). The graph means the change in AUC last by dose according to the administration day in males (E) and females (F). In E-F, the AUC last on Day 1 after DSS administration was indicated by a square, and the AUC last on Day 28 was indicated by a circle.
vomiting, a reduction in the erythrocyte index (erythrocyte count, hemoglobin concentration, and hematocrit), and an increase in mean RBC volume and platelet count were observed. These findings were also considered to be adverse effects related to DSS and showed a tendency to be resolved after the recovery period.
In addition, although only one male in the high-dose group receiving 20 mg/kg/day showed an ulcer at the gastric retinal adhesion site, this finding was considered to be the effect of DSS because ulcer induction was consistently observed other animals administered DSS. Eosinophils or neutrophils infiltrated the GI tract and mesenteric lymph nodes in both sexes in the 10 and 20 mg/kg/day groups. These were considered to be associated with an increase in WBC count, neutrophil count and ratio, and eosinophil count observed in the hematological examination as well as with the red discoloration and enlargement of the mesenteric lymph nodes observed in the macroscopic analysis. These observations were induced by the administration of DSS and accompanied recovery.
In the bone marrow (sternum and femur), increased cellularity was observed in both sexes in the 10 and 20 mg/kg/day groups, as determined by the hematological examination. This was associated with an increase in the number and ratio of reticulocytes. These changes were assumed to be secondary hematopoietic changes in the RBC inflammation owing to the administration of DSS and were not adverse effects [22].
In the thymus, atrophy was observed in the thoracic cavity in males in the 10 mg/kg/day group and in males and females in the 20 mg/kg/ day group, and this persisted throughout the recovery period. These findings were correlated with a decreased thymus size, as determined by macroscopic analysis. This was considered a secondary change caused by stress rather than by DSS administration and was not considered an adverse effect of DSS [23].
At the injection site, granulomatous inflammation, muscle fiber necrosis, and chronic active inflammation, which is observed in histopathology, were observed in both male and females in the middle-and high-dose groups receiving 10 and 20 mg/kg/day, respectively. These were considered as toxicities of DSS associated with swelling of the neck observed via the clinical observation and discoloration observed via the macroscopic examination at the administration site in males and females in the 10 and 20 mg/kg/day groups. Similar changes were observed even after the recovery period.
In the skin (ear, limb, or mouth), erosions/ulcers were observed in males and females in the 10 and 20 mg/kg/day groups, and these were associated with the swelling of the skin, abscesses, ulcers, and swelling of the palate. Therefore, these symptoms were considered toxicities of DSS treatment [7,24]. Some animals showed evidence of limping; however, there were no changes in related blood clinical chemistry, and thus, this was considered to be a secondary change caused by skin damage, such as swelling, abscesses, and ulcers of the forelimb/hindlimb. In addition, loss of teeth was considered a secondary change caused by swelling of the palate and ulceration of the lips. Swelling of the palate was observed to a similar degree during the recovery period. Other symptoms such as scratches, scars, and scabs were considered unrelated to DSS administration. Rather, these were caused by fights between animals when two animals were housed in one cage. Subsequently, such symptoms were not observed or showed a tendency to recover when the animals were reared alone. In reports that DSS administered dermally for 30 days to 6 months in minipigs receiving 0, 3, 10 and 30/45 mg/kg, skin reactions such as sporadic erythema or dermatitis at only application sites were observed [14]. However, swelling, abscesses, and ulcers were observed not only at the site of administration but also at the sites such as the ear, limb or mouth. In toxicokinetics results, the systemic exposure (AUC last ) of the DSS administration intramuscularly on Day1 is about 380 times higher than dermal administration based on 10 mg/kg/day [14]. The AUC last following intramuscular administration at a dose 2.5 mg/kg/day similar to that of a low dose receiving 2 mg/kg in this study [25,26].
The systemic exposure (AUC last ) of the DSS on Day1 was similar to the increase rate of the dose in both sexes' animals or slightly lower in the 20 mg/kg dose group of female animals. Systemic exposure of the diclofenac sodium salt after four weeks of repeated administration showed a decreasing trend in the male and female animals with a dose of 10 mg/kg or more compared to Day 1. Systemic exposure of the diclofenac sodium salt was not significantly different between male and female animals in the other dose groups except for the 20 mg/kg dose group (56 %) on Day 1. It has been reported that the DSS was well tolerated after the IM injection of the DSS at a dose of 75 mg in humans (Leuratti et al., 2019). If the human effective dose (HED) used in the above literature converts to animal dose (minipig correction factor (Km), 35; human Km, 37), the animal dose is around 1.4 mg/kg. Also, the DSS was well tolerated in rats which are intravenously received the DSS at a dose of 7 mg/kg [21]. If the Km is applied to convert this dose, the effective mini pig dose is around 1.2 mg/kg. In this study, the DDS was well tolerated after IM injection of DDS at 2 mg/kg in mini pig, while the mini pig, which is received the DDS at dose of 10 and 20 mg/kg, exhibited toxicity. According to the EMEA report [20], DSS is intended for treatment in cattle and swine as an anti-inflammatory agent at doses of 2.5 mg/kg bw/day by intramuscular route for 1-3 days. As described above, no evidence of systemic toxicity was observed after IM injection of the DSS at 2 mg/kg for 28 days. However, the DDS treatment groups at 10 and 20 mg/kg showed systemic toxicity, thus, the increasing dose or extending treatment period of DDS should be cautioned in clinical condition.
In conclusion, intramuscular administration of DSS daily resulted in GI, renal, hepatic and skin toxicities as well as injection-site reactions in both males and females receiving 10 mg/kg/day or higher dose groups. These changes were observed systemically and considered to be adverse effects associated with DSS. The NOAEL of DSS in this 4-week repeatdose toxicity study was considered to be 2 mg/kg/day in both sexes of minipigs.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.no conflict of interest.