Acute and Subchronic Toxicological Evaluation of the Herbal Product HAD-B1 in Rats

This study evaluates acute and subchronic toxicity of a Korean herbal formula HAD-B1 in rat to investigate whether HAD-B1 has potential toxicity to humans. First, the study to assess the acute oral toxicity at dose levels of 0, 500, 1000, and 2000 mg/kg body weight (BW) was performed in male and female SD rats (Crl: CD, specific pathogen-free) (n = 5/group). Based on the result of the acute oral study, 4 weeks' dose range finding study and 13 weeks' subchronic study were performed (dose range finding study, DRF; n = 5/group) and 13 weeks (subchronic study; n = 10/group) in male and female SD rats. The control group was administered with distilled water (DW). Clinical signs, body weight, food consumption, ophthalmic examination, urinalysis, hematological/biochemical parameters, gross finding at necropsy, and histopathological examination were investigated and recorded. In the oral acute toxicity study of SD rats, no clinical signs, mortality, body weight changes, and gross findings were observed. Also, there were no treatment-related changes in the 4-week DRF study. Based on these results, a 13-week repeated-dose toxicity study (subchronic) in SD rats was performed. HAD-B1 showed temporal hypersalivation in clinical signs and an increased tendency in body weight at 2000 mg/kg BW. However, there were no treatment-related changes in mortality, food consumption, ophthalmology, urinalysis, hematology, biochemistry, gross finding at necropsy, organ weights, and histopathology in either sex of any group. Based on this toxicological evaluation of HAD-B1, we concluded that no target organ was determined, and the no observed adverse effect level (NOAEL) of HAD-B1 was determined to be > 2000 mg/kg B W. Therefore, we decided that consuming HAD-B1 is relatively nontoxic.


Introduction
Lung cancer is defined as the uncontrolled growth of cells originating in the lungs [1]. ere are different types of lung cancer, non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC accounts for 80-85% of all lung cancers, while SCLC is much infrequent, consisting of 10-15% of all lung cancers [2]. Lung cancer is the most common malignancies world. In Korea, lung cancer is the number one cause of cancer-related mortality (NCCN guidelines, 2012). erefore, efforts to decrease the lung cancer mortality rate are an important problem to solve. Advances in molecular biology have led to the discovery of various genetic mutations associated with the pathogenesis of non-small-cell lung cancer. Various targeted therapies have been developed based on those findings, leading to breakthroughs in lung cancer treatments [3]. However, these targeted therapies seem to only work for early-stage lung cancer and have limitations such as drug resistance and high cost [4,5]. erefore, we must find alternative therapies that can help lung cancer patients with various stages of cancer.
CAM (complementary and alternative medicine) refers to therapies such as acupuncture, herbal medicine, tai chi, and they are considered complementary and alternative methods to conventional medical treatment. Even though CAM medicine was used longer than conventional medicine, it is still considered to lack scientific validity. erefore, researchers worldwide are trying to build scientific data on the usage of CAM in helping cancer patients. e Daejeon University of Korean Medicine is one of them, and we have developed an herbal formula HAD-B1 to help lung cancer patients. rough previous research, HAD-B1 proved significant benefits in suppressing cancer cells' growth, reducing chemotherapy's side effects, and improving the quality of life of cancer patients by the formula itself or combined with the standard treatments [6,7]. is present study aims to further investigate the efficacy of HAD-B1 by evaluating acute and subchronic (4 weeks, 13 weeks) toxicological evaluation of HAD-B1 in the rat.

Overview.
e research protocols for this study were reviewed and assessed (IAC2015-0120, IAC2015-0418, IAC2015-1231) according to the regulation for the Institutional Animal Care and Use Committee at KTR based on the Laboratory Animal Act (Enforcement Date of July 30, 2013) (No. 11987 (July 30, 2013, partial revision)) and the Animal Protection Act (Enforcement Date of January 20, 2015) (No. 13023 (January 20, 2015, partial revision)). Also, all the tests performed in this paper are following Guidelines for toxicity testing of pharmaceuticals, Ministry of Food and Drug Safety, MFDS.

HAD-B1
. HAD-B1 used for both acute and subchronic trials was provided by Kyoung Bang pharmaceutical Co. (Namdong-daero, Namdong-gu, Incheon Metropolitan City, Korea). e ingredients of HAD-B1 are listed in Table 1. In order to prepare HAD-B1 powder, 4 herbs were soaked for 18 hr at 60°C in a soaking bath. After the fluid extracts had been dried twice by using a rotary vacuum evaporator and a flat evaporator at 60°C, the powder (recovery ratio: 27.3%) was gained for the experiments (Figure 1). is dark brown powder (test substance, HAD-B1) was dissolved in distilled water (DW, Daihan Pharm Co., Ltd.) and suspended for oral administration.

3D HPLC Analysis of HAD-B1
. HAD-B1 was prepared by extracting HAD-B1 powder with 9.72 g of powder in 10 ml of methanol at room temperature. en, it was centrifuged at 1000 × g for 30 min and filtered, applied to the C18 column, and eluted using acetonitrile mixed with DW. en, we closely measured 10 mg of the standard brand of cordycepin, 10 mg of notoginsenoside R1, 10 mg of each ginsenoside Rg1, and Rb1 then diluted them with ethanol until the mixture reached exactly 100 ml, using this as the base liquid. Next, we measured 20 μL of the test solution and base liquid each. We used these conditions to test using the liquid chromatography method, measuring each liquid's cordycepin, notoginsenoside R1, ginsenoside Rg1, and ginsenoside Rb1's peak area and finding the 3D HLPC. Closely measured 10 mg of alpha-boswellic acid and betaboswellic acid was diluted with ethanol until the mixture measures exactly 100 ml and was used as the base liquid. Measured 20 μL of the test solution and base liquid was used to test the liquid chromatography method, measuring each liquid's alpha-boswellic acid and beta-boswellic acid's peak area and finding the 3D HLPC. Six components were detected ( Figure 2).

Experimental Animals and Housing
Conditions. All of the rats used in tests presented in this experiment are SD rats (Crl: CD, specific pathogen-free) and Orient Bio Co., Ltd (Buk-myeon, Gapyeong-gun, Gyeonggi-do, Korea) supplied the animals. Room temperature was maintained at 22 ± 3°C, relative humidity of 50 ± 20%, 10 to 20 time air ventilation in an hour, and lux luminous intensity of 150 to 300 with a 12 hr light/dark cycle. Temperature and relative humidity were automatically monitored every 30 minutes by an automatic instrument, and environmental conditions were measured periodically during the examination period. e rats were fed with a pellet diet (Rodent diet 20 5053 (Labdiet, USA)), and given the R/O (reverse osmosis) water ad libitum.

Dosage and Administration.
Administration dosage was determined following the drug toxicity test standards of the Ministry of Food and Drug Safety. erefore, the highest dose given was 2000 mg/kg, and median and the low dose were determined using a standard ratio of 2 (1000 and 500 mg/kg), respectively, with a volume of 10 mL/kg BW. e control group was administered with distilled water ( Table 2). e rats had fasted for approximately 18 hr before the first dosing. e drug was delivered directly to the stomach using a sonde feeding needle catheter. Each animal was observed for signs of toxicity manifestations and behavioral changes at 0.5, 1, 2, 3, and 4 hr after the administration.

Observations and Measurement.
We inspected every rat once a day and 14 days after the administration. Weight measurement took place when at the initial import, before the grouping, before administrating, 7 and 14 days after the administration, and recorded every time. Fourteen days after the drug administration, a visual inspection was conducted and euthanized. Organs were checked with the naked eyes.  and Subchronic Toxicity  Study.

DRF
e study was conducted according to the guideline (Guidelines for toxicity testing of pharmaceuticals, Ministry of Food and Drug Safety, MFDS).

Grouping.
In the DRF study, Orient Bio Co., Ltd (Buk-myeon, Gapyeong-gun, Gyeonggi-do, Korea) supplied 44 5-week-old rats (22 females, 22 males). e rats had one week of acclimation. After that, 40 6-week-old healthy rats were chosen for each study. We divided them into 4 groups, including a control group (5 rats/sex/group). e rats were divided into four groups: three treatment groups with different doses and 1 control group. In the subchronic study, 110 5-week-old rats (55 females and 55 males) were supplied from Orient Bio Co., Ltd (Buk-myeon, Gapyeong-gun, Gyeonggi-do, Korea). After a week of the acclimation period, 100 rats (50 females and 50 males) were selected and separated into 4 groups: 3 treatment groups and 1 control group.

Dosage and Administration.
e animals consumed DW and the substance of 0, 500, 1000, and 2000 mg/kg BW was tested, orally once a day for 4-week and 13-week (DRF study; 5 rats/sex/group, subchronic study; 10 rats/sex/ group), respectively. Dosage administration for the subchronic test was determined to be the same based on the DRF study results, which showed toxic abnormalities. erefore, it was determined that the dosage could be the same. Additionally, 10 rats (5 females, 5 males) were added to the control group and the highest dose group in order to evaluate the reversibility of the drug. In the subchronic study, the doses were chosen based on the results of the DRF study, and additional recovery animals (5 rats/sex) in the control and the high-dose group were included for observation of reversibility or persistence of any toxic effects after the treatment period. During the study period, all animals were observed once a day for clinical signs and mortality.

Observation and Measurement
(1) Body Weight and Food Consumption. Weight measurement took place when at the initial import, before the grouping, before administrating, 7 and 14 days after the administration, and recorded every time. Furthermore, to Evidence-Based Complementary and Alternative Medicine 3 find out relative organ weight, fasting weight was measured. e amount of food consumed was measured before drug administration and once every week after the administration has started. Differences in food given and food consumed were recorded per cage.
(2) Ophthalmic Examination and Urinalysis. e ophthalmological examination (Genesis, Kowa, Japan) and urinalysis were not performed for the DRF study and were performed during the last week of the treatment and recovery period for the subchronic study. Fresh urine was collected through the metabolic cage and used for urinalysis (specific gravity, pH, protein, glucose, ketone body, bilirubin, urobilinogen, nitrite, blood, and leukocyte), urine sedimentation, and color test. Urinalysis was performed using an automatic tester (CliniTek 500, Siemens, Germany), urine stick (Multistix 10 SG, Siemens, Germany), and a microscope (Leica, Germany) for urine sediment test. An ophthalmic test was performed for 10 rats (5 females, 5 males) per group with naked eyes.

Statistical Analysis.
e data during the study period was analyzed by the SPSS program (ver. 19.0) and presented as mean ± SD (standard deviation) for body weight, food consumption, organ weight, hematology, and biochemistry. Leven's test was applied to the homogeneity of variance and one-way ANOVA was performed to evaluate the significant differences between groups. No further analysis was

Study of Acute Oral Toxicity.
ere were no deaths, clinical signs, and body weight changes in any dose groups (Table 2). Additionally, no gross lesions were found in any of the organs at necropsy. Based on these results of acute oral toxicity tests, approximate lethal dose (ALD) of HAD-B1 was considered to exceed 2000 mg/kg BW. for both sexes. erefore, the dosage levels of 500, 1000, and 2000 mg/ kg BW were selected for the DRF and subchronic toxicity studies.

Study of Dose Range Finding (DRF, 4-Week).
During the study period, treatment-related mortality, clinical signs, body weight change, and gross lesion were not found in any dose group in rats. In organ weight, significant differences were founded in the liver (2000 mg/kg, absolute weight) and prostate gland (500 mg/kg, absolute/relative weight) of males. In hematology and biochemistry, significant differences were observed in T-CHO (500 mg/kg) and MCV (2000 mg/kg) of females. However, the observed changes were minor levels within the historical data range and were not dose-dependent. Also, no test-substance-related histopathological examination was observed in the control and high-dose groups. Based on these results, the dosage levels of 500, 1000, and 2000 mg/kg BW were selected for the subchronic toxicity determined.

Clinical Signs and Mortality.
No clinical signs and mortality were observed in the treatment group (500, 1000, and 2000 mg/kg BW) for the 13-week treatment and 4-week recovery period. However, salivation was temporarily observed on days 89 to day 91 in one male and two female rats in the 2000 mg/kg BW group.

Body Weight and Food
Consumption. No significant differences in mean body weight and food consumption were observed in the treatment group (500, 1000, and 2000 mg/kg BW) for the 13-week treatment period and 4week recovery period. However, an increased tendency of body weight was observed during the treatment period and was continuously observed during the recovery period in 2000 mg/kg of males (Figures 2 and 3).

Ophthalmological and Urine Analysis.
No side effects related to the testing substance were observed in ophthalmological examination and urine test at any dose group.

Gross Finding at Necropsy.
At the termination of treatment, no treatment-related gross finding was observed in the control and treatment group (500, 1000, and 2000 mg/ kg BW) for 13-week study period including 4-week recovery period. However, reduced testis was observed in one animal at 2000 mg/kg of male and mass on the right mammary gland was observed in one animal in the control group of female (3.5 × 2.5 × 2.0) (Figure 4). e other gross findings were not observed.

Organ Weights.
In the treatment group, the absolute organ weight of the thymus was increased in 2000 mg/kg of males. In the recovery group, the relative organ weight of the spleen decreased in males, and the absolute/relative organ weight of the thymus was decreased in 2000 mg/kg of females. Other than that, no significant difference was observed (Tables 3 and 4 ).

Hematological and Biochemical Parameters.
ere were no statistically significant differences in the hematological examination for the 13-week treatment period and 4-week recovery period at any dose group in males and females. In terms of biochemistry, A/G level was increased at 500 mg/kg in male as compared with the control group, but this increase was not observed at 1000 mg/kg, 2000 mg/kg in male and any dose group in female treated with test substance for 13-week treatment period (Table 5). In the recovery group, significant decreases were observed in T-BIL, ALP, AST, and ALT in males, and increases were observed in ALB, A/G in females (Table 6). However, this parameter showed no significant difference in the 13-week.

Histopathological Examination.
e histopathological changes were observed in high-dose group: inflammatory cell infiltration of the liver, tubular basophilia of kidneys, cortical vacuolation of the adrenal gland, inflammatory cell infiltration of the prostate gland at treatment group, and tubular basophilia/medullary cyst of a kidney, cortical vacuolation in the adrenal gland (Tables 7 and 8 ). Some lesions were found in the control group and/or were low in frequency. In addition, histopathological lesions were observed in organs (testis, mammary gland) where gross lesions were observed at necropsy: tubular atrophy in the testis of male, adenocarcinoma in a mass of mammary gland of female ( Figures 5 and 6 ).

Discussion
Goals for any nonclinical safety study are identifying potential target organs for toxicity, evaluating potential reversibility toxicity, and determining the no observed adverse effect level (NOAEL) or no practical effect level [8]. NOAEL is the highest dose that produces no significant adverse effects in animals in a specific nonclinical toxicology study [9]. In this present research, acute toxicity, DRF, and subchronic toxicity studies for HAD-B1 were performed in SD rats (rodents) to investigate its potential toxicity in humans.
During the study, temporal hypersalivation was observed in 2000 mg/kg BW group in both male and female Evidence-Based Complementary and Alternative Medicine rats for acute toxicity and 4-week DRF studies. However, it was not a consistent manifestation and there was no sign of hypersalivation in any other animals. us, this observation resulted in a nonspecific response in those animals instead of a treatment-related effect.
Change in BW can be an adverse effect of drugs and chemicals [10]. As BW increased over time during both the treatment and recovering periods, this change was considered treatment-related. us, further study, e.g., a chronic toxicity study over 13 weeks, appears necessary to ascertain the toxic effects, if any, of HAD-B1 on BW.
Organ weight changes may occur when drugs or chemicals are used, and these changes could be meaningful indicators of test article-related changes, irrespective of corresponding microscopic findings, in repeated-dose rodent studies [11]. Although we observed a statistically significant increase in the absolute weight of the thymus in male rats in the 2000 mg/kg BW group during the subchronic toxicity study and statistically significant decreases during the 4-week recovery period in the relative organ weights of the thymus (female) and spleen (male) in rats in this group, other inter-group differences in organ weight either were minor or exhibited no dose-dependence. Furthermore, no treatment-related histological alterations were observed. erefore, these changes were attributed to normal biological variations.
Blood parameters analysis can be used to determine the extent of foreign compounds' adverse effects [12]. In this research, clinical biochemistry analyses were conducted to evaluate possible alterations in blood chemistry and detect possible treatment-related changes compared with the control findings. A previous study identified an increased risk of anemia during a subchronic toxicity test, in contrast to our findings of no significant blood parameter changes [13]. Similarly, the changes in parameters during the study period, despite reaching significance, were minor, within the historical ranges, or not dose-dependent, illustrating that HAD-B1 had no significant toxic effects in SD rats.
Gross necropsy findings revealed reduced testis size in one male rat in the 2000 mg/kg BW group and a mass in the  Figure 4: Food consumption of male (a) and female (b) rats treated with HAD-B1 for 13-week treatment period and 4-week recovery period (mean ± SD). All groups showed food and water consumption data, which indicated that there were no statistically significant differences among treated groups compared with the control group.  Figure 3: Body weight of male (a) and female (b) rats treated with HAD-B1 for 13-week treatment period and 4-week recovery period (mean ± SD). An increase tendency was showed in male (a). All groups showed increased body weight and there were no difference between each group (male). 6 Evidence-Based Complementary and Alternative Medicine       right mammary gland of a control female rat. Additionally, the histopathological findings revealed bilateral tubular atrophy of testis with oligospermia in the epididymis and adenocarcinoma (ductal subtype) in the mammary gland. Tubular atrophy is spontaneous [14], and oligospermia is considered a secondary effect of tubular atrophy. In general, classifying mammary adenocarcinomas by subtype (i.e., papillary, ductal, alveolar) in routine rodent carcinogenicity studies has not been demonstrated to be biologically meaningful. Besides, spontaneous mammary carcinomas typically occur in older rats, and they have been sporadically reported in control females as young as 10 weeks [9]. erefore, these lesions were not considered treatmentrelated.  Based on this study's results, the NOAEL for HAD-B1 was considered >2000 mg/kg BW. In terms of dose translation from animals to humans, the human equivalent dose (HED; animal dose (mg/kg) × (animal Km factor/human Km factor)) calculated using the body surface area (mg/m 2 ) for initial clinical trials in healthy adult volunteers [15,16] was 324.32 mg/kg/day for adults (60 kg) and 480 mg/kg/day for children (20 kg). In the future, this HED may be evaluated in a clinical study.

Conclusions
In this study, the oral administration of HAD-B1 on the acute toxicity study and subchronic toxicity study in SD rats caused neither mortality nor any adverse effects noticed in clinical signs, body weight (gain), organ weight, and biochemical parameters. Also, the food consumption, urine analysis, and hematological parameters of HAD-B1 treated rats showed no significant changes than control, and there are no findings related to treatment given in necropsy finding and histological examination. erefore, these results observed at the treatment doses (500, 1000, 2000 mg/ kg BW) suggest that HAD-B1 is not likely to produce any toxicological effects and is safe for herbal medicinal use. However, since the experiment resulted in some weight gain, further study is considered to ascertain this mechanism.
Data Availability e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.