A 13-Week Repeated Oral Dose Toxicity Study of ChondroT in Sprague-Dawley Rats

Background ChondroT, a new herbal medication, consists of Angelica grosseserrata Maxim., Lonicera japonica Thunb., Angelica gigas Nakai, Clematis terniflora var. manshurica (Rupr.) Ohwi, and Phellodendron amurense Rupr. (6:4:4:4:3). Our previous studies have shown that ChondroT exhibits significant anti-arthritic and anti-inflammatory effects. In this study, we aimed to assess the toxicological safety assessment of ChondroT. Methods This study was designed to assess the safety of ChondroT after repeated oral administration. Male and female Sprague-Dawley rats were treated with ChondroT at oral doses of 0, 500, 1000, and 2000 mg/kg for 13 weeks. Mortality, clinical signs, body weight changes, food consumption, ophthalmological findings, urinalysis, hematological and blood-chemical parameters, necropsy findings, organ weights, and histological markers were recorded throughout the study period. Rats were also monitored for an additional 4 weeks to determine the recovery time. Results No death occurred and no significant changes in food consumption, ophthalmologic findings, and urinalysis were found. Although there were alterations in clinical signs, body weights, hematological parameters, blood-chemical parameters, necropsy findings, organ weights, and histological markers, they were not considered to be toxicologically significant. Conclusions The results suggest that the no-observed adverse effects level (NOAEL) was 2000 mg/kg/day for the test substance. ChondroT, a new complex herbal medication composed of five plants, can therefore be used safely at the NOAEL.

ChondroT exerts chondroprotective effects and possesses multitarget mechanisms related to inflammation and arthritis [4][5][6]. In fact, the suppressive effect of ChondroT is greater than that of GHJT Y [7]. Moreover, ChondroT has shown therapeutic potential for the treatment of patients with hyperuricemia and gout [8]. Thus, it is currently undergoing clinical trials for approval from Ministry of Korean Food and Drug Safety [9].
Studies remain to be conducted on the toxicity of ChondroT. Although a 4-week repeated oral dose toxicity study had shown no adverse effects in Sprague-Dawley (SD) rats administered the maximum recommended dose of 2000 mg/kg/day via oral gavage [10], that study was performed primarily to determine the dosage range for a subsequent 13-week study. Thus, information regarding the oral toxicity of ChondroT during the subchronic period is insufficient.
In this 13-week repeated oral administration study on SD rats, we aimed to elucidate the toxic effects of ChondroT, identify its target organs, and determine its no-observed-adverse-effect-level (NOAEL) in order to provide toxicological data for assessing the safety of ChondroT.

Test facility
This study was conducted in compliance with the princi- The study was also conducted in accordance with the guideline for toxicity testing of pharmaceutical products by the Ministry of Food and Drug Safety (MFDS, Notification No. 2014-136, 2014-07-30).
After examining body weight changes and general conditions of animals, those deemed healthy were selected for this study (50 males, 50 females). Environmental conditions in the animal facility were maintained at 22 ± 3°C (20.5-23.27°C), 50 ± 20% (46.0-65.6%) relative humidity, 10-20 times/h ventilation frequency, 150-300 Lux luminous intensity, and 12-h light/12-h dark cycle. There were no influenceable variations with these conditions. Animals were given irradiation-sterilized pellet food (Rodent Diet 20 5053, Labdiet, USA) and ultrafiltration (reverse osmotic, R/O) water via a water bottle, ad libitum. The absence of contamination was confirmed by a periodical analysis of the food manufacturing facility and KTR Hwasun for the R/O water.
After group assignment, the remaining animals (five males, five females) were sacrificed using carbon dioxide (CO 2 ) in accordance with the American Veterinary Medical Association (AVMA) guidelines.

Group assignment and dosing
After the acclimatization period, 100 healthy rats (6 weeks old; male 181.7-203.9 g, female 53.0-177.5 g) were randomly divided according to average weight and standard deviation. For dose selection, we considered the result of previously reported repeated toxicity study (Study No.: TBW-0064-15, 4 weeks repeated toxicity; 0, 500, 1000, 2000 mg/kg) [10] where no significant toxicological changes were found at doses up to 2000 mg/kg. The dose 2000 mg/kg was selected as the high (maximum) dose and two-fold intervals were used for the lower dose levels in the present 13-week repeated dose toxicity study; 1000 and 500 mg/kg were set as the intermediate and low (minimum) doses, respectively.
Study animals were divided into the groups listed in Table 1. Eighty rats were randomly divided into four groups (20 animals per group) for each dose (treatment group). Twenty rats were allocated to the control and high dose groups for the identification of any toxic effects, whether persistent or delayed, at 4 weeks postexposure (recovery group).

Preparation of test substance and dosing
ChondroT (Lot No. 501) was supplied from Jung-woo Co. (Asan, Korea). The five herbs were combined in a 6: 4:4:4:3 ratio (Table 2). ChondroT was prepared by carrying out water extraction once, using 10-fold solvent at 100°C for 3 h, and then filtering it (180 mesh). The water extract solution was concentrated using a continuous vacuum evaporator (approximately 55-60°C, 670 mmHg), and this was followed by vacuum drying using a vacuum drier (720 mmHg) for 8 h. The yield was approximately 29.4%.
The ChondroT dried extract (light brown powder) was suspended in distilled water (D.W., Daihan Pharm Co., Ltd.) to achieve concentrations of 50, 100, and 200 mg/ mL for treatment. The administration volume was 10 mL/kg based on the most recently measured body weight. The test substance was administered once/day, 7 days/week, for 13 weeks by oral gavage using a stomach tube and was suspended using a magnetic stirrer during administration. The control group was administered vehicle (DW, Daihan Pharm Co., Ltd.).

High-Performance Liquid Chromatography (HPLC) analysis
Five marker components (chlorogenic acid, berberine chloride (Cl), oxypeucedanin hydrate, decursin, and decursinol angelate) were purchased from Acros Organics (Pittsburgh, PA, USA), Shanghai Sunny Biotech (Shanghai, China), ChemFaces (Wuhan, China), and NPC Bio Technology (Yeongi, Korea), respectively. Analytical method for quality control of ChondroT sample using HPLC was described in detail in previous studies [5,6]. In other words, the HPLC used for simultaneous analysis of the five marker components was the Shimadzu Prominence LC-20A Series (Shimadzu, Kyoto, Japan) coupled with photo-diode array (PDA) detector. A SunFire TM C 18 analytical column (4.6 × 250 mm, 5 μm; Waters, Milford, MA, USA) was used for the separation of the five marker components at a constant temperature of 40°C. The mobile phase was eluted with distilled water (A) and acetonitrile (B), both containing 0.1% (v/v) formic acid under the gradient elution mode. The parameters for the HPLC quantitative analysis were as follows: 0-30 min, 10-100% B; 30-40 min, 100% B; 40-50 min, 100-10% B. The flow rate and injection volume were 1.0 mL/min and 10 μL, respectively.

Clinical signs
All animals were observed daily before, during, and after oral treatment. Any treatment-related signs were individually recorded throughout the experimental period (13-week treatment and 4-week recovery period). In addition, mortality, development of clinical symptoms, and toxicity signs were recorded.

Body weight
Body weights of animals were measured on the day they were received, during group assignment, once per week, and the day before necropsy (13-week treatment and 4-week recovery period). Fasted weight was also measured on the day of necropsy to derive relative weight.

Food consumption
Food consumption was measured pre-dose and once per week for the 13-week treatment and 4-week recovery period. A weighed amount of food was placed in each cage and the amount remaining in each cage was measured the following day. Pre-dosing, food consumption was measured from group assignment to the first dosing day.

Ophthalmic examination and urinalysis
Ophthalmological examination and urinalysis were performed (five rats/sex/group) during the last week of treatment and the recovery period. Gross examination was performed followed by ophthalmological examination using mydriatic (Tropicamide 1%, Alcon, Belgium) and fundus camera (Genesis, Kowa, Japan). Urinalysis (i.e., appearance, volume, specific gravity, pH, protein, glucose, ketone body, bilirubin, urobilinogen, nitrite, blood, leukocyte, and urinary sediments) was performed with urine collected from a metabolic cage. Urinalysis items were analyzed using an automatic tester (CliniTek 500, Siemens, Germany), urine stick (Multistix 10 SG, Siemens, Germany) and a microscope (Leica, Germany).

Gross finding at necropsy
On the day scheduled for necropsy, all animals were anaesthetized by isoflurane inhalation (Forane, JW Pharmaceutical, Korea) followed by blood sampling. Animals were exsanguinated from the posterior vena cava and aorta prior to necropsy. Macroscopic examinations of the external surface, all orifices, all internal organs of the head, thoracic and abdominal cavities were performed on the dead animals.

Statistical Analysis
Data are presented as mean ± standard deviation (S.D.) for body weight, food consumption, organ weight, hematology, and biochemistry and were analyzed by SPSS software (ver. 19.0). The Leven's test was performed to derive the homogeneity of variance and one way ANOVA was performed to determine the significant differences between groups. If there were no significant differences, an additional analysis was not performed; however, if a significant difference was confirmed, posthoc test was used according to the result of variance homogeneity (homogeneity; Scheffe test, heterogeneity; Dunnett's T3 test). In the recovery group, data analysis was performed using independent t-test. A p value < 0.05 was considered significant.

Quality assessment of the five marker components in ChondroT Sample
Using the established HPLC method, simultaneous determination was conducted to evaluate the 13-week treatment of ChondroT sample. The five marker components (chlorogenic acid, berberine Cl, oxypeucedanin hydrate, decursin, and decursinol angelate) were eluted within 30 min and the retention times of these components were 8.94, 10.80, 15.95, 26.02, and 26.24 min, respectively (Fig. 1). The coefficient of determination for the calibration curve of the five components was ≥ 0.9996, showing a good linearity. The amounts of the five marker components ranged between 0.81 and 5.46 mg/g (Table 3).

Mortality and clinical signs
During the experimental period, treatment-related moribund, dead animals, and clinical signs were not observed for both the sexes in the control and treatment groups. However, loss of fur in posterior neck from day 12 to 91 and abdomen from day 25 to 91 (one female at middle dose) and a nodule (slightly hard with an egg-shape) from day 63 to 91 (one female administered the middle dose) were clinical signs observed. Salivation was transiently or intermittently observed in both the sexes when the high dose was administered (Table 4).

Body weight and food consumption
A gradual increase in body weight of males was observed over the treatment period, with significant differences (p < 0.05) at weeks 10 (ANOVA test: F value, 3.345; p value, 0.027; post-hoc test: Scheffe p value, 0.041) and 11 (ANOVA test: F value, 3.615; p value, 0.020; post-hoc test: Scheffe p value, 0.036) for animals administered the 2000 mg/kg dose. No significant differences were found for females among the groups (Fig. 2).
There was no significant difference in food consumption between the treatment groups and control group for both the sexes (Fig. 3).

Ophthalmological examination and urinalysis
There were no treatment-related changes during the treatment and recovery periods for the different dosage groups.

Hematology and serum biochemistry
There was no significant difference in the hematological items over the treatment period. However, some items displayed significant differences in the recovery period. There was a significant increase in mono (Levene's test: F value, 1.884; p value, 0.212; independent t-test: p value, 0.049) for males and neut (Levene's test: F value, 0.007; p value, 0.937, independent t-test: p value, 0.036) and baso (Levene's test: F value, 0.099, p value: 0.762; independent t-test: p value, 0.020) for females administered the 2000 mg/kg dose, and a significant decrease in Lymph (Levene's test: F value, 0.821; p value: 0.395, independent t-test: p value, 0.038) for females    Table 5). For serum biochemistry items, significant increases were found for sodium (ANOVA test: F value, 4.475; p value, 0.009; post-hoc test: Scheffe p value, 0.026) in males and triglycerides (ANOVA test: F value, 3.443, p value, 0.027, post-hoc test, Scheffe p value, 0.047) in females administered 2000 mg/kg. In the recovery group, no significant changes were found for both the sexes when administered 2000 mg/kg (Table 6).

Gross finding at necropsy
At the end of treatment, there were no treatmentrelated gross findings in the treatment groups over the 13-week treatment period and 4-week recovery period. However, the following gross findings were observed in the treatment group: reduced size of the left testis in one male in the control group, yellowish nodule on the prostate gland of one male in the low dose group and cysts and mass (3.0 × 4.5 × 3.0 cm) in the right submandibular region in one female administered the middle dose.

Organ weight
For absolute organ weight in the treatment group, there was a significant increase in the weight of the heart (low Fig. 2 Body weights of the rats used in the experiment dose, ANOVA test: F value, 6.205; p value, 0.002; posthoc test: Scheffe p value, 0.038), liver (high dose, ANOVA test: F value, 4.273; p value, 0.011; post-hoc test: Scheffe p value, 0.016) and lung (high dose, ANOVA test: F value, 4.559; p value, 0.008; post-hoc test, Scheffe p value, 0.021) of males; these changes did not resemble that of relative organ weight. For relative organ weight, a significant decrease was found for the brain (high dose, ANOVA test: F value, 4.248; p value, 0.11; post-hoc test : Scheffe p value, 0.018) of a male. There were no significant changes in the organ weight of females in the different treatment groups.
In the recovery group, there was a significant increase in the absolute organ weight of the thymus (high dose, Levene's test: F value, 0.469; p value, 0.513; independent t-test, p value, 0.013) of males and a significant decrease for that of the kidney (bilateral weight, high dose, Levene's test: F value, 4.007; p value, 0.080; independent t-test: p value, 0.029) and brain (high dose, Levene's test: F value, 0.063; p value, 0.808; independent t-test: p value, 0.021) of females. Similarly, a significant decrease in relative organ weight was found for the testis (bilateral weight, high dose, Levene's test: F value, 1.281; p value, 0.291; independent t-test: p value, 0.040).

Histopathological examination
The following histopathological lesions were observed in the high dose treatment group: inflammatory cell foci in Fig. 3 Food consumption of the rats used in the experiment the liver (one male), tubular basophilia (two males, one female), medullary mineralization (two females) in the kidney, cortical vacuolation of the adrenal gland (four males), inflammatory cell infiltration of the prostate gland (interstitial, one male), and cysts in the ovary (one female). Some of these lesions were also observed in the control group (i.e., inflammatory cell foci in the liver, tubular basophilia and medullary mineralization in the kidney, cortical vacuolation in the adrenal gland, and inflammatory cell infiltration/interstitial in the prostate gland).
The following histopathological lesions were observed in the high dose recovery group: medullary mineralization in the kidney (one female), cysts in the pituitary gland (pars distalis, one male), and inflammatory cell infiltration in the prostate gland (interstitial, one male). Similar to the treatment groups, some of these lesions were observed in the control group (i.e., medullary mineralization in the kidney and cysts in the pars distalis of the pituitary gland). Moreover, histopathological lesions were also observed in organs with gross lesions. These include tubular atrophy in the testis, oligospermia in the epididymis (reduced left testis in the control group, Fig. 4), abscess (yellowish nodule on the prostate gland of animals administered the middle dose, Fig. 5), and basal cell carcinoma (cysts and mass in the right submandibular region of animals administered the middle dose, Fig. 6). Lung metastasis was also observed following basal cell carcinoma (Fig. 7). The histopathological lesions are shown in Table 7.

Discussion
Osteoarthritis is emerging as an important issue resulting from the loss of cartilage associated with the shape of bones, such as osteophytes and subchondral bone sclerosis. It is caused by population aging, an increase in obesity, and a lack of definitive treatment [11][12][13]. Symptoms of osteoarthritis include progressive asymmetrical pain, joint stiffness, and restricted joint movement, which can hinder the performance of daily activities [14][15][16].
ChondroT is a new complex herbal medication developed for the treatment of osteoarthritis and its efficacy and safety have been demonstrated in several studies [4][5][6][7][8]. Although the safety of this herbal complex has been confirmed through animal studies, we sought to carry out the present study with additional doses and perform a rigorous toxicity study that could be translated to human clinical trials.
Herein, we aimed to evaluate the systemic toxicity response when ChondroT, the test substance, is administered via the oral route for 13 weeks, and determine its NOAEL. This 13-week repeated oral administration study included doses of 500, 1000, and 2000 mg/kg body weight.
During the test period, there were no treatmentrelated moribund or dead animals of any sex in the treatment groups. In addition, there were no significant changes in food consumption, urinalysis, and ophthalmological examination. Clinical signs included loss of fur in one female administered the 1000 mg/kg dose; however, this was not dose-dependent and was only observed in one animal. Although salivation was observed in some of the animals administered 2000 mg/kg of the test substance, continuous and transient or intermittent observations were not found for both sexes. Hence, these signs did not represent treatment-related toxicological effects and were considered to be incidental signs.
Body weight is a parameter affected by adverse effects exhibited by drugs and chemicals [17]. The increasing tendency of body weight in males administered the 2000 mg/kg dose was considered to be a treatment-related effect and not a toxicological effect. This was due to the changes observed in organ weight, hematological biochemistry parameters, and histopathological examination. A further study may however be needed to confirm whether or not toxic effects have an impact on body weight (e.g., chronic study).
There were no significant differences among the treatment groups based on the hematological analysis. Although significant differences were found when the 2000 mg/kg dose was administered, no histological lesions, organ weight changes, and gross findings were observed in the relevant organs. This indicates a normal biological variation and the lack of a treatment-related effect.
Based on the necropsy findings, there were no treatment-related effects for both sexes in the treatment groups. Nonetheless, histopathological examination revealed gross findings such as tubular atrophy with oligospermia of the epididymis and abscess in the control (reduced left testis) and treatment groups (nodule on the prostate gland at 500 mg/kg), respectively.
Previously, the incidence of spontaneous testicular atrophy in the testis of SD rats was reported to be 0.2%, 7.9%, and 10% at 4, 13, and 26 weeks, respectively. In addition, the epididymis with testicular atrophy has been reported to show low sperm density (oligospermia) [18]. Therefore, the observed lesion in this study may have been an aberration based on the lesion observed in the control group.
Male accessory sex glands are frequently observed as a site of inflammatory lesions and may appear as large abscesses or small suppurative lesions. Prostatitis is commonly observed in older rats but has been observed in young adult rats used in toxicity studies, especially in the dorsolateral prostate gland [19]. Therefore, the gross finding for the prostate gland in animals in the middle dose group might not be a treatment-related effect due   to the non-existent dose relationship and the frequency of necropsy. Organ weight could be a meaningful indicator of treatment-related changes with or without corresponding histopathological examination in repeated toxicity studies [20]. In this study, organ weight changes lacking a dose relationship were observed in some organs. In addition, a relationship between absolute weight and relative weight was not found. Additionally, treatmentrelated histological findings were not observed. Therefore, changes were considered to be normal biological variation.
In the histopathological examination, the lesions observed in animals administered the 2000 mg/kg were spontaneous incidence lesions, such as inflammatory cell foci (liver), tubular basophilia/medullary mineralization (kidney), cortical vacuolation (adrenal gland), inflammatory cell infiltration (prostate gland), and cysts (ovary, pars distalis of pituitary gland), which are common in repeated toxicity studies. These lesions were also observed in the control group but their frequency and grade were minor [19]. In addition, these lesions might have been background lesions and not a treatment-related effect.
Spontaneous basal cell carcinoma is very rare in rats, with an incidence rate of only 0.14% in aged animals. It is also rarely observed in young SD rats. Spontaneous basal cell carcinoma was reported recently in a subcutaneous mass found in the left inguinal region of a young (7-week) SD rat [21,22]. In the present study, this lesion was only observed in one animal in the middle dose group and did not result in a dose-dependent response. Therefore, it was considered to be a treatment-related effect and spontaneous lesion.
Sub-chronic exposure of rats to ChondroT did not result in treatment-related changes at doses up to 2000 mg/kg. This result suggests that ChondroT might be safe and is in agreement with the human equivalent dose (HED) of 324.32 mg/kg/day; animal dose (mg/kg) x (rat K m factor '6'/Human adult 60 kg K m factor '37')] using body surface area (BSA, mg/m 2 ) for initial clinical trials involving healthy adult volunteers [23,24].
In conclusion, the results of the present 13-week repeated oral dose toxicity study demonstrated that administering ChondroT did not adversely affect most of the toxicological factors in male and female SD rats. With the experimental conditions adopted in the present study, we found that the NOAEL of ChondroT was 2000 mg/kg/day and a target organ was not indicated.

Conclusions
In previous studies, ChondroT was demonstrated to be effective in arthritis via its chondroprotective and antiinflammatory mechanisms. Based on the results of this study, we found that its NOAEL was 2000 mg/kg, which reveals that ChondroT does not exhibit toxicity under the conditions employed in the study. As a new complex herbal medication composed of five plants, ChondroT can be used safely at its NOAEL