2.4. 28‐Day repeated‐dose oral toxicity study

The 28‐day repeated‐dose oral toxicity study was performed according to OECD TG407. The high dose was set at 5000 mg/kg/day as GHX02 is a mixture of many compounds of unknown concentration, and there were no toxic signs in the single‐dose study. Six‐week old Sprague‐Dawley rats (n = 10 per sex and group) were orally administered with GHX02 at 0, 625, 1250, 2500 and 5000 mg/kg/day for 28 days to male and female rats in this study. Five animals/sex were added to the vehicle control group and the high‐dose group (5000 mg/kg/day) to evaluate the recovery potential. The body weight ranges at the initiation of dosing were 192.42‐226.39 g for males and 150.23‐175.29 g for females, respectively.

Animals were checked once a day to observe any clinical signs and mortalities, and the type, date of occurrence and severity of signs were recorded individually. The body weights of all group rats were recorded before the initiation of dosing (day 1) and once a week during the experimental period. Before necropsy, all group rats were fasted overnight and the body weight of all group rats were recorded at necropsy. Food and water intake was checked on the same days as the body weight measurements were recorded. The ophthalmological observation examination was processed during the last week of the experimental period using a fundus camera (Keeler Instruments Inc.), and the eyes of all animals were macroscopically examined.

Urinalysis was performed during the last week of observation; all animals were individually housed in a stainless‐steel cage cleaned and disinfected with 70% alcohol. Urine samples were collected and 0.3 mL of fresh urine was taken for analysis. Urine samples were analyzed for glucose, bilirubin, ketone body, specific gravity, pH, protein, urobilinogen, nitrite, occult blood, urine color and clarity using an automatic analyzer (Clinitek Advantus; Siemens).

At necropsy, animals were anesthetized by inhalation of 3%‐5% isoflurane (Terel liquid; Kyongbo Pharma. Co., Ltd.). Blood samples were collected from the posterior vena cava for hematological and serum biochemical testing. Approximately 1 mL blood was placed in a CBC bottle (Vacutainer 3 mL; BD) with anticoagulant EDTA‐2K. Hematology parameters were measured using a Coulter counter (ADVIA 2120; Siemens), including red blood cell count (RBC), hemoglobin distribution width, hemoglobin concentration, hematocrit, platelet count (PLT), mean corpuscular volume, mean cell hemoglobin, mean cell hemoglobin concentration, red cell distribution width, mean platelet volume (MPV), reticulocytes, white blood cell count and white blood cell count differential count (neutrophils, basophils, monocytes, lymphocytes, eosinophils and large unstained cells).

Serum biochemical parameters were measured using a serum biochemistry analyzer (AU680; Beckman Coulter). About 2 mL of the blood sample was added into a 5 mL Vacutainer tube (SST™ II Advance; BD) that contained clot activator. The blood was coagulated by remaining at room temperature for 15‐20 minutes and then centrifuged for 10 minutes (3000 rpm, 1902 Relative Centrifugal Force (RCF), Combi‐514R; Hanil) to collect serum sample. The parameters examined were aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine phosphokinase, reticulocytes, total bilirubin, glucose, total cholesterol, triglyceride, total protein, albumin, albumin/globulin ratio, blood urea nitrogen (BUN), creatinine, inorganic phosphorus, calcium, sodium, potassium ion and chloride ion.

After the blood sampling, animals were killed by exsanguination from the abdominal aorta and posterior vena cava. Gross findings were recorded including the body surface, subcutis, head and all organs in the abdominal and thoracic cavities. Following this, organs were weighed using an electronic balance (Sartorius AG): kidney (both), spleen, liver, thymus, heart, lung, brain and adrenal gland. Organ weights (%) relative to the terminal body weights were also calculated. Tissue slides were prepared from fixed tissues with gross findings of all animals, and histopathological examination was performed. The histopathological findings were processed by the Pristima^® (xybion) program. The diagnostic terms in the lexicon of Pristima^® were used primarily.