Preclinical Safety of the Root Extract of Polygala tenuifolia Willdenow in Sprague-Dawley Rats and Beagle Dogs

The root of Polygala tenuifolia Willdenow has been used for the treatment of insomnia, depression, and amnesia. However, the toxicological properties of the herb have been overlooked, because it has been used for a long time for various purposes. In this study, we evaluated the preclinical safety of the root extract in rats and beagle dogs. First, the acute oral toxicity was tested in both rats and dogs. In the rats, only one female of 2 g/kg died, but no treatment-related death or clinical and gross findings were observed after the administration. No toxicological changes or mortalities related to the test substance were also observed after the administration in the dogs. Although vomiting, discoloration, or hemorrhage was found in some dogs, there were no serious abnormalities. Second, the subchronic toxicity was investigated in the rats. Two animals were found dead in the female group of 1,000 mg/kg/day, but there were no abnormal findings associated with the test substance. There also were no adverse effects on the clinical signs, body weight, and hematological and biochemical findings. Therefore, our results showed that the acute or subchronic toxicity of the root extract of Polygala tenuifolia might not be toxic to rats and dogs.


Introduction
Traditional oriental herbal prescriptions have regained popularity over the past decade. They are used widely for the treatment and prevention of various diseases [1,2], because their long history of clinical application and natural origin seem to guarantee that the prescriptions will be effective and nontoxic [3]. Each plant used in such herbal prescriptions can provide opportunities for the development of herbal food products, dietary supplements, and functional foods [4]. However, the toxicological aspects of medicinal herbs or naturally occurring functional foods have been neglected due to their long history of use. It has been demonstrated that medicinal plants may have undesirable properties [5], because some individuals taking herbal medicines have reported certain side effects. Therefore, the use of any plant for medicinal purposes by no means guarantees the safety of such a plant. This fact raises concerns about the potential toxic effects resulting from short-term and long-term use of such medicinal plants. Data from acute and subchronic toxicity studies on medicinal plants or naturally occurring functional foods should be obtained to increase confidence in the safety of their use, particularly in the development of pharmaceuticals [6]. Polygala tenuifolia Willdenow is a perennial herbaceous plant distributed widely in China and Korea. In Asia, it is a well-known traditional medicine for the treatment of phlegm and detumescence. Traditional medicines are prepared from the roots of P. tenuifolia because of its expectorant, tonic, tranquilizing, and antipsychotic properties [7][8][9]. In particular, the herb is used against insomnia, neurasthenia, amnesia, depression, anxiety-related palpitations, restlessness, disorientation, dementia, and memory failure [10,11]. Previously, we reported that the root extract of P. tenuifolia was shown to enhance memory and cognitive function in two animal models and two human models [12][13][14][15]. The extract has been shown to contain C-glycosides, triterpene saponins, sucrose esters, and oligosaccharide esters [16,17]. It also contains various substances, such as tenuigenin, tenuifolin, DISS (3,6disinapoyl sucrose), and TMCA (3,4,5-trimethoxycinnamic acid), which have been shown to have proliferative and protective effects on hippocampal neurons [18,19]. Several 2 Evidence-Based Complementary and Alternative Medicine researchers have demonstrated the effects of P. tenuifolia, but information on its safety is lacking. Therefore, systematic evaluation of the safety of the root extract of this herb is necessary for the development of new foods or drugs. In this study, an alcohol extract from the dried root of P. tenuifolia was prepared, and its safety was evaluated using an acute oral toxicity test and a subchronic oral toxicity test in Sprague-Dawley rats and beagle dogs in accordance with two standards: the Korea Food and Drug Administration Notification numbers 2005-60 and 2005-79.

Preparation of the Root Extract of Polygala tenuifolia.
The dried root extract of Polygala tenuifolia (500 g) was refluxed with 75% ethanol for 4 hr in a boiling water bath. This procedure was repeated twice and the ethanol solution was concentrated under a vacuum. The resulting concentrated ethanol fraction (125 g) of the plant root was used for this study [12].

Experimental Animals and Animal Husbandry. Six-
week-old specific pathogen-free Sprague-Dawley (HSD; SD) rats were used. Six animals of each sex for the acute oral toxicity study and 40 animals of each sex for the subchronic oral toxicity study were received from Koatech Co. (Pyeongtaeksi, Gyeonggi-do, Korea), Ltd. For the acute oral toxicity study, six beagle dogs of each sex at 6 months of age were obtained fromtheHanlim Laboratory Animals Company (Hwaseongsi, Gyeonggi-do, Korea). The animal room was maintained at a temperature of 23 ± 3 ∘ C, with a relative humidity of 55 ± 15%, air ventilation of 10-20 times/h, and ambient light (150-300 Lux) controlled to produce a 12 h light/dark cycle. Animals were given irradiation-sterilized pellet feed and ground water disinfected by an ultraviolet sterilizer and ultrafiltered ad libitum. The dogs were reared in stainless steel cages (8000 × 900 × 750 mm), and identification cards showing their study number and animal number were attached to the cages.

Study Design
Overview. This study was conducted in compliance with the Good Laboratory Practice (GLP) and Test Guidelines of the Organization for Economic Cooperation and Development [20,21] and the Korea Food and Drug Administration [22,23] at the GLP Institute approved by the KFDA (Korea Food and Drug Administration). The study protocol was approved by the Institutional Animal Care and Use Committee of the institute, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, 2010.

Acute Oral Toxicity Study in Rats.
After a 7-day quarantine and acclimatization period, ten healthy animals of each gender at 6 weeks of age were used. The study included two groups: a control group given no treatment and an experimental group given 2,000 mg/kg, based on a preliminary study (data not shown). Each group consisted of five rats of each sex, and body weight ranges at dosing were 161.61-168.38 g for males and 144.98-158.17 g for females. The test substance was administered orally to each group. Thereafter, clinical signs and body weight were observed throughout the 15-day experimental period, and gross findings were observed on the scheduled necropsy day. This study was carried out in accordance with the standard operating procedures of SNUH-GLP (Non-Clinical Research Center, Biomedical Research Institute, Seoul National University Hospital).

Acute
Oral Toxicity Study in Beagle Dogs. Six male and six female dogs were divided into three groups of four animals; then, two dogs of each gender were allocated. The study included three groups: a control group given no treatment and experimental groups exposed to 1,000 mg/kg and 2,000 mg/kg. The body weight ranges at dosing were 7.269-8.785 kg for males and 6.531-7.449 kg for females. The test substance was administered orally to each group. Thereafter, clinical signs, mortality, and body weight were observed throughout the 15-day experimental period, and gross findings were observed on the scheduled necropsy day. This study was carried out in accordance with the standard operating procedures of Preclinical Research Center, ChemOn (Yongin, Gyeonggi-do, Korea) Inc.

Subchronic Oral Toxicity Study in Rats.
After a 7-day quarantine and acclimatization period, forty healthy animals of each gender at 6 weeks of age were used. A dose of 1,000 mg/kg/day was set as the high dose considering the results of a preliminary 1-week subchronic oral toxicity study (data not shown) and the characteristics of the test substance. Doses of 600 and 360 mg/kg/day were set as the middle and low doses, respectively, using a common ratio of 1.67. The vehicle-treated group was administrated with sterile water for injection only. Each group consisted of 10 rats of each sex, and 10 rats of each sex and group were killed after a 90day treatment period. Body weight ranges at the beginning of dosing were 169. .56 g for males and 125.30-142.00 g for females. The test substance was administered repeatedly by the oral route to each group for 90 days. The following items were examined during the experimental period: clinical signs, body weight, food and water consumption, ophthalmic examination, urinalysis, hematology, blood clotting time, serum biochemistry, necropsy findings, organ weight, and histopathology. This study was carried out in accordance with the standard operating procedures of Preclinical Research Center, ChemOn Inc.

Necropsy.
In the dog studies, the animals were anesthetized with ketamine (Yuhan Yanghaeng) and rompun (Bayer Korea) and euthanized by exsanguinations from the axillary artery and vein. All organs in the thoracic and abdominal cavities were observed grossly and the gross findings were recorded.

Observation and Examination Items of the Subchronic
Oral Toxicity Study in Rats 2.6.1. Clinical Signs. All animals were observed daily for clinical signs and mortality. The type, date of occurrence, and the severity of the signs were recorded individually. The initial day of administration was set as day 1.

Body
Weight. Animals were weighed on the initial day of administration, once per week during the experimental period, and on the day of the necropsy. The body weight at the necropsy was measured after an overnight fast.

Food and Water Consumption.
Food and water consumption were measured on the initial day of administration and then once per week during the experimental period. The amounts of food and water were measured before they were supplied to each cage, and the food and water remaining the next day were measured to calculate the difference, which was regarded as daily food and water consumption (g/rat/day).

Ophthalmic Examination.
In the last week of observation, a mydriatic (Ocuhomapin, Lot number 013118, Samil Pharm Co., Korea) was dropped into both eyes of each animal to facilitate mydriasis after observing the external appearance of the eyes of five males and five females per group. The anterior parts of the eyes, the optic media, and the ocular fundus were then observed with an ophthalmoscope and a fundus camera (Genesis, Kowa Co., Tokyo, Japan). No abnormal signs were observed during the ophthalmic examination. Therefore, no eye photographs or further examinations were performed. : white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, hemoglobin distribution width, platelets, mean platelet volume, and WBC differential count (neutrophils, lymphocytes, monocytes, eosinophils, basophils, and large unstained cells). 1.8 mL of the blood taken during the necropsy was dispensed into a microtube containing 0.2 mL 3.2% sodium citrate, and plasma was obtained from centrifugation (Eppendorf, Hamburg, Germany) for 10 min to assess the blood clotting time. The prothrombin time and activated partial thromboplastin time were measured in seconds from plasma using the nephelometric analysis method (Woziwodzki, 1970) with a coagulation time analyzer (ACL 100, Instrumentation Laboratory, Bedford, MA, USA).

Serum Biochemistry.
More than 2 mL of the blood taken during the necropsy was added to a 5 mL Vacutainer tube (IMPROBE, Germany) containing a clot activator. The blood was coagulated by maintaining it at room temperature for 15-20 min and was then centrifuged (3,000 rpm, 1,500 RCF, MF300, Hanil, Korea) for 10 min. The following parameters were measured with a serum biochemistry analyzer (AU400, Olympus, Tokyo, Japan): aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine phosphokinase, total bilirubin, glucose, total cholesterol, triglycerides, total protein, albumin, albumin/globulin ratio, blood urea nitrogen, creatinine, inorganic phosphorus, and calcium ions. The electrolytes were measured with the electrolyte autoanalyzer (644 Na, K, Cl Analyzer, Ciba-Corning, USA).

Necropsy and Organ
Weight. Before the scheduled necropsy, all surviving animals were fasted overnight (for 16-20 h) and euthanized with isoflurane (Ifran liquid, Hana Pharm. Co., Korea) inhalation on the day of the necropsy. After anesthesia was confirmed, blood was taken from the posterior vena cava for hematology and serum biochemistry analyses. The abdominal aorta and posterior vena cava were cut to euthanize the animals. All organs of the body surface, subcutis, head, and all internal organs of the abdominal and thoracic cavities were observed grossly. Next, the pituitary gland, ovaries, uterus, adrenal glands, thymus, prostate gland, testes, epididymides, spleen, kidneys, heart, lung, brain, and liver were removed and weighed with an electronic balance (BP221S, Sartorius Co., Gottingen, Lower Saxony, Germany), and all paired organs were measured separately. The absolute organ weights were converted to relative organ weights based on the organ-to-fasted body weight ratios.
2.6.9. Histopathology. Microscopic examinations were performed on the preserved organs and tissues taken from all animals in the vehicle-treated and high dose-treated groups. All gross lesions as defined by the study pathologist were also included in the examination. The eyes and the optic nerves were preserved in Davidson's fixative, and the testes and epididymides were preserved in Bouin's fixative.

Statistical Analysis.
In the rat studies, data are presented as mean ± standard deviation. Body weight, food and water consumption, total volume of urine, hematology and serum biochemistry, and absolute and relative organ weights were assumed to be normally distributed and were analyzed by a one-way analysis of variance (ANOVA) [24].
The assumption of homogeneity was tested using Levene's test [25]. If the overall ANOVA was significant and the assumption of homogeneity of variance was met, Duncan's multiple-range test was used as a post hoc test to identify significantly different groups from the vehicle control group [26]. Scheffe's test was used if the sample size was unequal between the groups [27].
If the assumption of homogeneity of variance was not met, Dunnett's T3 test was used as the post hoc test [28]. Student's -test was used to analyze the differences in means between the two independent groups. The urinalysis data were ranktransformed and analyzed by the nonparametric Kruskal-Wallis -test [29]. If a statistically significant difference was observed between groups, the Mann-Whitney test was used to identify the groups that were significantly different from the vehicle control group [30]. SPSS 10.1K was used for all statistical analyses (Chicago, IL, USA). A < 0.05 was considered significant.
In the dog studies,data are presented as mean ± standard deviation. Body weight, food and water consumption, total volume of urine, hematology and serum biochemistry, and absolute and relative organ weights were assumed to be normally distributed and were analyzed by a one-way analysis of variance (ANOVA) [24]. SPSS 10.1K was used for all statistical analyses (Chicago, IL, USA). A < 0.05 was considered significant.

Acute Oral Toxicity Study in Rats.
The test substance was orally administered to a single group of five male and five female SD rats at 7 weeks of age in dosage levels of 2 g/mL/kg B.W. Only one female rat given 2 g/mL/kg displayed piloerection and activity decrease 1 day after the treatment and died within the same day. However, the gross necropsy results showed no treatment-related changes and revealed no evidence of specific toxicity related to the test substance. Also, there was a statistically significant decrease in the body weights at 1 day in all test group rats (Table 1). However, the body weights of the test groups recovered. Therefore, this was a temporary side effect from the test substance. But, all rats except the aforementioned female rat were not noted in the mortality, clinical, and gross findings.

Clinical Signs and Mortality.
No dogs died during the experimental period. After the administration of the test substance, vomiting was observed. In the male group given 1,000 mg/kg (Table 2), very slight vomiting of the test substance at 3 hr and vomiting of the food at 5 hr were observed in one male. In addition, in the male group given 2,000 mg/kg, severe vomiting was observed in two males at 30 min, and further vomiting of the food was observed in a male on day 5. As shown in Table 3, vomiting of the food was observed in one vehicle-treated female on day 1, moderate vomiting of the test substance was observed in one female given 1,000 mg/kg at 1 hr, and severe vomiting of the test substance was observed in two females given 2,000 mg/kg at 30 min. Vomiting of the food was observed in all female groups on day 7.

Body Weights.
The body weight was observed throughout the 15-day experimental period (Table 4). In the males, a loss of body weight was observed in a male given 1,000 mg/kg on days 1 and 7 and in a male given 2,000 mg/kg on day 3. In the females, a loss of the body weight was observed in a vehicle-treated female on day 3, in a female given 1,000 mg/kg on days 1 and 7, and in a female given 2,000 mg/kg on days 1 and 7. Table 5, hemorrhage of the mucosa of the duodenum and dark red discoloration in the right lobule of a lung were observed in one male. However, no abnormalities were observed in other males and females.

Necropsy. As shown in
Evidence-Based Complementary and Alternative Medicine 5

Clinical Signs and Mortality.
A rat was found dead on day 50 and another on day 67 after administration in the female group with 1,000 mg/kg/day. In both dead rats, abnormal fur, dirty noses, and hypothermia were observed on the day before death. There were no abnormal findings that were associated with the administration of the test substance other than minor observed signs such as scratched wounds, crust formation, scarring, and the loss of teeth, which occurred at a low frequency with no dose-relationship during the study period (data not shown).

Body Weight and Food and Water Consumption.
As a result of the observations of body weight changes and body weight gains for 13 weeks during the administration and observation periods, there were no statistically significant changes in all treatment groups of both sexes compared to the vehicle-treated group. The food consumption in the male group of 360 mg/kg/day was significantly increased at weeks 4 and 10 after the administration compared to the vehicletreated group (Tables 6 and 7) ( < 0.05 or < 0.01). There was no significant change in water consumption in all groups of both sexes compared to the vehicle-treated group.

Ophthalmic Examination.
In all groups, there were no particular signs observed from external eye examinations at grouping and ophthalmic examinations using a fundus camera in the final week of administration (data not shown).

Urinalysis.
There were no particular signs associated with the administration of the test substance as a result of urinalysis performed in the final administration week (data not shown).

Hematology.
The RDW (red cell distribution width) in the male group administered with 1,000 mg/kg/day significantly decreased compared to the vehicle-treated group (Tables 8 and 9) ( < 0.01), but no consistent changes were observed in the male or female groups in association with the administration of the test substance in other examination items. There were no significant changes between the groups as a result of measurement of PT (prothrombin time) and APTT (activated partial thromboplastin time) (data not shown).
3.3.6. Serum Biochemistry. As a result of serum biochemical examinations using the serum of collected blood during the necropsy (Tables 10 and 11), dose-related decreasing tendencies of ALT, BUN, GLU, AST, ALP, CHO, PRO, and CPK and dose-related increases of A/G, Na + , and Cl − were observed in the males. In addition, statistically significant dose-dependent decreases of ALT, BUN, and GLU were observed compared to the vehicle-treated group ( < 0.05 or < 0.01) and significant dose-dependent increases of A/G, Na + , and Cl − were observed compared to the vehicletreated group ( < 0.01). In the female groups, no statically significant changes were observed in all measurement items.

Necropsy.
As a result of the scheduled necropsy of live animals at 13 weeks after the administration, no notable doserelated changes were observed in either males or females. For males, enlargement of the spleen was observed in one case in the vehicle-treated group and one case in the 600 me/kg/daytreated group, a solid brown nodule of the prostate gland was observed in one case in the vehicle-treated group, and diffuse red spots or a flare-up of the thymus was observed in one case in the vehicle-treated group and one case in the test substance-treated groups. For the females, the retention 6 Evidence-Based Complementary and Alternative Medicine of a clear fluid in the uterus was observed in the vehicletreated group and the 360, 600, and 1,000 mg/kg/day-treated groups (2, 4, 3, and 2 cases, resp.) and diffuse red spots in the thymus and dark yellowish brown discoloration of the lung were observed in the 600 mg/kg/day-treated group in two cases and one case, respectively. Additionally, enlargement of the submandibular lymph node was observed in one case in the 1,000 mg/kg/day-treated group. As a result of the necropsy of two dead females of the 1,000 mg/kg/day-treated group, diffuse red spots or dark red coloration in the lung and diffuse red spots in the anterior stomach and in the thymus were observed along with gas retention due to postmortal changes (data not shown).

Organ Weight.
For males, the absolute and relative weights of the thymus showed significant increases in the 360 and 600 mg/kg/day-treated groups compared to the vehicletreated group (Tables 12 and 13) ( < 0.05 or < 0.01) and the relative weight of the left epididymis showed a significant decrease in the 600 mg/kg/day-treated group compared to the vehicle-treated group ( < 0.05), but these changes were not dose-related. For females, the relative weight of the liver showed a significant increase in the 1,000 mg/kg/day-treated group compared to the vehicle-treated group ( < 0.05).
3.3.9. Histopathology. As a result of histopathological examination of rats with a scheduled necropsy, in the males in the vehicle-treated group, severe granulomatous inflammation of the prostate gland, moderate pyelonephritis in the kidney, sight cysts in the pituitary gland, and moderate granuloma in the liver were observed in one case, along with moderate inflammatory cell infiltration in the Hadrian gland and slight inflammatory cell infiltration in the heart (one case each). For the females, two cases of slight tubular regeneration in the kidney were observed in one case each. In the males with a scheduled necropsy from the 1,000 mg/kg/day-treated group, slight tubular remnants in the pituitary gland, slight granuloma in the liver, and a slight dilation of the lumen in the uterus were observed in one case in each group. In the low-and medium-dose groups, pale yellowish brown discoloration of the left lobe of the lungs (identified as focal alveolitis) was observed in one female from the 600 mg/kg/daytreated group, and diffuse red spots in the thymus were observed in two cases (identified as focal hemorrhages). No abnormal findings were observed in the abnormal organs.  As a result of the histopathological examinations on the two dead female rats from the 1,000 mg/kg/day-treated group, it was found that the rats had suffered severe postmortal changes, and therefore an accurate evaluation of the lesions was difficult. However, in rats that died on day 67 after the administration, diffuse vacuolation in the adrenal gland, atrophy of the spleen, and inflammatory cell infiltration in the heart were observed to a minor extent, and no abnormal findings were observed in a dead animal on day 50 after the administration.

Discussion
Polygala tenuifolia root is a famous traditional medicine. Although many studies have reported the pharmacological efficacy of the root extract, there is no information on its safety, such as its acute and subchronic oral toxicity. First, we investigated the acute and subchronic oral toxicity of the root extract in rats. The root extract was administered orally at 0 or 2 g/kg body weight for the acute oral toxicity test and at 0, 360, 600, or 1,000 mg/kg body weight for the subchronic oral toxicity test. In the acute oral toxicity study, one female 8 Evidence-Based Complementary and Alternative Medicine  rat given 2 g/kg of the root extract exhibited piloerection and decrease in activity levels 1 day after treatment and died within the same day. The gross findings of the autopsy showed no evidence of specific toxicity related to the test substance. In addition, there was a statistically significant decrease in body weight at day 1 in all test-group rats, but the body weights later recovered. Hence, this finding may show a temporary effect of the test substance. However, all rats except the aforementioned female rat were not noted in the mortality, clinical, and gross findings. These results suggest that the approximate lethal dose for the root extract of P. tenuifolia may be ≥2 g/kg in male rats and ≤2 g/kg in female rats under the conditions used in this study. In the subchronic oral toxicity study, there were no abnormal signs associated with administration of the test substance. No specific findings for body weight, consumption of food and water, ophthalmic examination, urinalyses, and blood clotting time were observed in relation to administration of the test substance. In the female group of 1,000 mg/kg/day, rats that died on day 50 and day 67 showed normal clinical signs, body weight changes, and consumption of food and water during the study period but demonstrated abnormal fur, dirty noses or mouths, and hypothermia the day before death. Autopsies revealed dark red coloration or diffuse red spots in the lungs, as well  as diffuse red spots in the anterior stomach and thymus gland. A more accurate evaluation of the lesions was made difficult by severe postmortem changes in two rats during the histopathological studies. In the rat that died 67 days after administration of the root extract, diffuse vacuolation in the adrenal glands, atrophy of the spleen, and infiltration of the inflammatory cells in the heart were observed. In the rat that died on day 50, abnormal findings were not observed. As mentioned above, the clinical signs, body weight changes, food and water consumption, necropsy findings, and histopathological findings obtained for the dead rats did not indicate any toxicity of the test substance. Therefore, the deaths of the rats were not associated with the administration of the test substance and were judged to be accidental due to administration errors. This hypothesis is supported by the fact that no abnormal findings were observed in relation to the administration of the test substance in the remaining rats in the same dose group. We demonstrated that the test substance could be used as a food supplement, because some famous food ingredients were toxic at high doses, (e.g., lemongrass (Cymbopogon citratus) essential oil, Roystonea regia fruit, and -glycerylphosphorylcholine (AGPC)) [31][32][33]. The results of the serum biochemical examinations of the rat sera revealed reductions in the levels of AST and ALT in the male groups. AST and ALT are coenzymes of pyridoxal phosphate (PALP). PALP is a derivative of vitamin B 6 and AST and ALT are activated by union with PALP, but nonlinked aminotransferases are not activated. Hence, reductions in the levels of ALT and AST occur if there is low generation of aminotransferases or PALP or if the linkage between the   Body weight before necropsy and after fasting. * / * * Significant difference at P < 0.05/0.01 compared with the vehicle-treated group.
aminotransferases and PALP is disturbed. Cephalosporinlike antibiotics are known to induce reductions in ALT levels, but in toxicity studies, reductions have been mentioned in general and toxicological significance has not been attributed [34,35]. The blood urea nitrogen (BUN) levels in the blood represent the nitrogen content in relation to urea, which is a metabolite of nitrogen and is closely related to heart diseases. The BUN value might show a decrease, due to a reduction in urea synthesis during toxic hepatitis, severe hepatic impairment, and consuming a low protein diet. However, in the present study, no abnormal findings related to dose were observed. In general, the decrease in BUN level is not a critical factor in toxicity studies [34]. Moreover, these changes were not regarded as adverse effects because they remained within normal ranges [36,37]. Hematological examinations revealed reductions in red blood cell volume distribution width (RDW) in the male group given 1,000 mg/kg/day. Decreasing tendencies in the levels of GLU, ALP, CHO, PRO, ALB, and CPK and increments in the levels of A/G, Na + , and Cl − in male treatment groups were within the normal range [38,39] and also remained within the normal range when compared with the historical data [21] available at ChemOn Inc. In the vehicle-treated group, increments or reductions in some measurement items were also observed accidentally and were thought to be somewhat contributory to the above results [40]; therefore, it was considered that these changes were toxicological and were not associated with the administration of the test substance. RDW decreased