Preclinical study of safety of Dendropanax morbifera Leveille leaf extract: General and genetic toxicology.

ETHNOPHARMACOLOGY RELEVANCE: Dendropanax morbifera Leveille (DM) has been used in traditional medicines for infectious and skin diseases, and dysmenorrhea. It exhibits a diverse therapeutic potential including anti-cancer, anti-thrombotic, anti-diabetic, anti-oxidant, and anti-inflammatory activities. AIM OF THE STUDY: Despite promising health benefits of DM, knowledge of its potential adverse effects is very limited. The current study focused on the investigation of subchronic toxicity and genotoxicity of extract obtained from DM according to the test guidelines published by the Organization for Economic Cooperation and Development. MATERIALS AND METHODS: We conducted a toxicological evaluation of DM extracts using 14-day repeated-dose toxicity study and 13-week repeated-dose toxicity study in Sprague-Dawley rats administered orally at doses of 500, 1000, or 2000 mg/kg/day. The clastogenicity of DM extract was also evaluated by in vitro chromosome aberration assay and in vivo micronucleus assay. RESULTS: Assessment of subchronic toxicity of DM extract by oral administration in rats revealed unremarkable treatment-related findings with respect to food/water consumption, body weight, mortality, urinalysis, hematology, serum biochemistry, necropsy, organ weight and histopathology at doses of 500, 1000, and 2000 mg/kg. Accordingly, the level of no-observed-adverse-effect for DM extract in 13-week subchronic toxicity study was considered to be 2000 mg/kg/day in rats. The data observed from in vitro chromosome aberration assay and in vivo micronucleus assay exclude any clastogenicity of DM extract. CONCLUSION: The results suggest that the oral consumption of DM extract has no adverse effects in humans and represents a safe traditional medicine.

Assessment of acute, 14-day, and 13-week repeated oral dose toxicity of Tiglium seed extract in rats.

BACKGROUND: Seed of mature Croton tiglium Linne, also known as Tiglium seed (TS), has been widely used as a natural product due to its several health beneficial properties including anti-tumor and antifungal activities. Despite its ethnomedicinal beneficial properties, toxicological information regarding TS extract, especially its long-term toxicity, is currently limited. Therefore, the objective of the present study was to evaluate acute and subchronic toxicity of TS extract in rats after oral administration following test guidelines of the Organization for Economic Cooperation and Development (OECD). METHODS: Toxicological properties of TS extract were evaluated by toxicity assays to determine its single-dose acute toxicity (125, 250, 500, 1000, or 2000 mg/kg), 14-day repeated-dose toxicity (125, 250, 500, 1000, or 2000 mg/kg) and 13-week repeated-dose toxicity (31.25, 62.5, 125, 250, and 500 mg/kg) in Sprague-Dawley rats and F344 rats. Hematological, serum biochemical, and histopathological parameters were analyzed to determine its median lethal dose (LD50) and no-observed-adverse-effect-level (NOAEL). RESULTS: Oral single dose up to 2000 mg/kg of TS extract resulted in no mortalities or abnormal clinical signs. In 13-week toxicity study, TS extract exhibited no dose-related changes (mortality, body weight, food/water consumption, hematology, clinical biochemistry, organ weight, or histopathology) at dose up to 500 mg/kg, the highest dosage level suggested based on 14-day repeat-dose oral toxicity study. CONCLUSION: Acute oral LD50 of TS extract in rats was estimated to be greater than 2000 mg/kg. NOAEL of TS extract administered orally was determined to be 500 mg/kg/day in both male and female rats. Results from these acute and subchronic toxicity assessments of TS extract under Good Laboratory Practice regulations indicate that TS extract appears to be safe for human consumption.

In vitro and in vivo safety studies of cinnamon extract (Cinnamomum cassia) on general and genetic toxicology.

Cinnamomum cassia has been widely used as a natural product to treat diseases in Asia due to its diverse pharmacological functions including anti-inflammatory, anti-oxidant, anti-microbial, anti-diabetic, and anti-tumor effects. Despite its ethnomedicinal benefits, little information regarding its toxicity is currently available. The aim of this study was to evaluate its potential long-term toxicity and genotoxicity in compliance with test guidelines of the Organization for Economic Cooperation and Development. A 13-week repeat-dose oral toxicity study revealed that body weights of rats were normal after receiving cinnamon extract at up to 2000 mg/kg. High-dose intake of cinnamon extract (2000 mg/kg) showed potential nephrotoxicity and hepatotoxicity to both males and females as evidenced by obvious increases of kidney/liver weight along with a small but statistically elevation of total cholesterol level. Overall findings from genetic toxicity testing battery including Ames test, in vitro mammalian cell micronucleus assay, and in vivo bone marrow micronucleus assay indicated that cinnamon extract was not mutagenic or clastogenic. In conclusion, cinnamon extract may possess potential nephrotoxicity and hepatotoxicity at dose higher than its recommended daily safe dose. Further study is needed to clarify the mechanism involved in its induction of liver and kidney injury.

Enzymatic extract from Ecklonia cava: Acute and subchronic oral toxicity and genotoxicity studies.

Ecklonia cava (EC) is known to have antioxidant, anti-inflammatory, antidiabetic, and anticancer properties. Despite its wide use and beneficial properties, comprehensive toxicological information regarding EC extract is currently limited. Therefore, the purpose of this study was to investigate acute toxicity, subchronic toxicity, and genotoxicity of enzymatic EC extract according to test guidelines published by Organization for Economic Cooperation and Development. The acute oral LD50 values of this EC extract administered to rats and dogs were estimated to be more than 3000 mg/kg BW. In an oral 13-week toxicity study, changes in body weights of rats exposed to the EC extract up to 3000 mg/kg BW were found to be normal. In addition, repeated doses of EC extract failed to influence any systematic parameters of treatment-related toxic symptoms such as food/water consumption, mortality, urinalysis, hematology, serum biochemistry, organ weight, or histopathology. These results indicated that the no-observed-adverse-effect level for the EC extract was 3000 mg/kg/day for male and female rats. Data obtained from Ames test, chromosome aberration assay, and micronucleus assay indicated that EC extract was not mutagenic or clastogenic. Taken together, these results support the safety of enzymatic EC extract as a potential therapeutic for human consumption against various diseases.

Preclinical safety assessment of Angelica acutiloba using a 13-week repeated dose oral toxicity study in rats.

Angelica acutiloba (AA), a Japanese species of Danggui, has been used worldwide as a traditional herbal medicine with several bioactivities including anti-diabetic, anti-allergic, anti-inflammatory, anti-tumor, and anti-obesity. However, there is lack of toxicological data available to evaluate potential long-term toxicity and the no-observed-adverse-effect level (NOAEL) of AA extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In the 14-day repeat-dose toxicity study, no adverse effects on mortality, body weight change, clinical signs, and organ weights was found following repeat oral administration to rats for 14 days (125, 250, 500, 1000, and 2000 mg/kg body weight), leading that 2000 mg/kg is the highest recommended dose of AA extract for the 13-week repeat-dose oral toxicity study. In the 13-week repeat-dose oral toxicity study, the AA extract was orally administered to groups of rats for 13 weeks (125, 250, 500, 1000, and 2000 mg/kg body weight) to compare between control and AA extract groups. The administration of AA extract did not produce mortality or remarkable clinical signs during this 13-week study. And, the data revealed that there were no significant differences in food/water consumption, body weight, hematological parameters, clinical chemistry parameters, gross macroscopic findings, organ weight and histopathology in comparison to the control group. On the basis of these results, the subchronic NOAEL of the AA extract was more than 2000 mg/kg/day when tested in rats. And, the AA extract is considered safe to use orally as a traditional herbal medicine.

Evaluation of in vitro and in vivo genotoxicity of Angelica acutiloba in a standard battery of assays.

Among three representative species of Angelica found in Asian countries, including Korea, China, and Japan, Angelica acutiloba (AA) has been used as traditional herbal medicine with antitumor, anti-inflammatory, anti-obesity, and anti-diabetes activities. In this study, the potential genotoxicity and mutagenicity of the AA extract were examined in a battery of in vitro and in vivo tests (bacterial reverse mutation assay, in vitro chromosomal aberrations assay, and in vivo micronucleus assay) in accordance with the test guidelines for toxicity testing developed by the Organization for Economic Cooperation and Development. Upon testing in the bacterial mutation assay (Ames test) using five Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537, no significant increase the number of revertant colonies in the metabolic activation system and non-activation system was noted in the AA extract groups. Also, in the chromosome aberration test, the AA extract did not cause chromosomal aberration with or without metabolic activation by S9 mix. A bone marrow micronucleus test of mice demonstrated that the incidence of micronucleated polychromatic erythrocytes in the AA extract groups (500, 1000 and 2000 mg/kg BW) was equivalent to that of the negative control group. Based on these results from a standard battery of assays, the AA extract was concluded to have no genotoxic at the proper dose.

Absence of subchronic oral toxicity and genotoxicity of rice koji with Aspergillus terreus.

Koji products have been considered as an effective fermented food consumed in East Asia with many health benefits. Particularly, rice koji with Aspergillus terreus (RAT) has been reported to be able to prevent hyperlipidemia and hepatic steatosis through regulating cholesterol synthesis. Despite its biological activities, there is a lack of comprehensive information to give an assurance of its safety. Therefore, the objective of this study was to perform a series of toxicological studies (repeated dose oral toxicity and genotoxicity) according to test guidelines published by the Organization for Economic Cooperation and Development. Along with acute toxicity study using rats and beagle dogs, a 13-week toxicity study revealed no clear RAT-related toxic changes, including body weight, mortality, hematology, serum biochemistry, organ weight, and histopathology after oral administration at doses of 500, 1000, and 2000 mg/kg BW. The no-observed-adverse-effect level of RAT was considered to be more than 2000 mg/kg BW/day in rats of both genders. In addition, potential genotoxicity was evaluated using a standard battery of tests (Ames test, chromosome aberration assay, and micronucleus assay) which revealed that RAT showed no genotoxicity. Accordingly, these results suggest that RAT is a safe and non-toxic functional food for human consumption at proper dose.

A comprehensive study on in vitro and in vivo toxicological evaluation of Artemisia capillaris.

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.

Preclinical Biosafety Evaluation of Genetically Modified Human Adipose Tissue-Derived Mesenchymal Stem Cells for Clinical Applications to Brainstem Glioma.

Stem-cell based gene therapy is a promising novel therapeutic approach for inoperable invasive tumors, including brainstem glioma. Previously, we demonstrated the therapeutic potential of human adipose tissue-derived mesenchymal stem cells (hAT-MSC) genetically engineered to express a secreted form of tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) against brainstem glioma. However, safety concerns should be comprehensively investigated before clinical applications of hAT-MSC.sTRAIL. At first, we injected stereotactically low (1.2 × 10(5) cells/18 µL), medium (2.4 × 10(5)/18 µL), or high dose (3.6 × 10(5)/18 µL) of hAT-MSC.sTRAIL into the brainstems of immunodeficient mice reflecting the plan of the future clinical trial. Local toxicity, systemic toxicity, secondary tumor formation, and biodistribution of hAT-MSC.sTRAIL were investigated. Next, presence of hAT-MSC.sTRAIL was confirmed in the brain and major organs at 4, 9, and 14 weeks in brainstem glioma-bearing mice. In the 15-week subchronic toxicity test, no serious adverse events in terms of body weight, food consumption, clinical symptom, urinalysis, hematology, clinical chemistry, organ weight, and histopathology were observed. In the 26-week tumorigenicity test, hAT-MSC.sTRAIL made no detectable tumors, whereas positive control U-87 MG cells made huge tumors in the brainstem. No remaining hAT-MSC.sTRAIL was observed in any organs examined, including the brainstem at 15 or 26 weeks. In brainstem glioma-bearing mice, injected hAT-MSC.sTRAIL was observed, but gradually decreased over time in the brain. The mRNA of human specific GAPDH and TRAIL was not detected in all major organs. These results indicate that the hAT-MSC.sTRAIL could be applicable to the future clinical trials in terms of biosafety.

Pre-clinical in vitro and in vivo safety evaluation of Cimicifuga heracleifolia.

The rhizomes of Cimicifuga species, including Cimicifuga heracleifolia (CH), have been widely used as antipyretic, analgesic, and anti-inflammatory agents in oriental countries. However, information regarding its toxicity, especially long-term toxicity and genotoxicity, is limited. Therefore, we performed the subchronic toxicity and genotoxicity assays of the CH extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a 13-week repeat-dose oral toxicity study, the CH extract did not influence body weight, food/water consumption, mortality, clinical signs, and urinalysis throughout the study. Noteworthy, the CH extract groups exhibited increased liver weights along with serum alanine transaminase activity rise at doses of 667 and 2000 mg/kg in females. No-observed-adverse-effect-level of the CH extract administered orally was concluded to be 2000 mg/kg body weight/day for male rats and 222 mg/kg body weight/day for female rats. The CH extract did not exert a mutagenic or clastogenic effect in Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. Overall findings of the subchronic toxicity study indicate for the first time that the CH extract may possess hepatotoxic potential in female rats, suggesting that further mechanistic studies should be performed to have more conclusive results on hepatotoxic potential of the CH extract.

Safety evaluation of Angelica gigas: Genotoxicity and 13-weeks oral subchronic toxicity in rats.

As a well-known traditional medicine, Angelica gigas (AG) and its active constituents, including decursin and decursinol, have been shown to possess several health beneficial properties such as anti-bacterial, immunostimulating, anti-tumor, neuroprotective, anti-nociceptive and anti-amnestic activities. However, there is lack of toxicity studies to assess potential toxicological concerns, especially long-term toxicity and genotoxicity, regarding the AG extract. Therefore, the safety of AG extract was assessed in subchronic toxicity and genotoxicity assays in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a subchronic toxicity study for 13 weeks (125, 250, 500, 1000 and 2000 mg/kg body weight, delivered by gavage), data revealed no significant adverse effects of the AG extract in food consumption, body weight, mortality, hematology, biochemistry, necropsy, organ weight and histopathology throughout the study in male and female rats. These results suggest that no observed adverse effect level of the AG extract administered orally was determined to be greater than 2000 mg/kg/day, the highest dose tested. In addition, a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay suggested that the AG extract was not genotoxic. In conclusion, the AG extract appears to be safe as a traditional medicine for oral consumption.

Evaluation of subchronic (13week) toxicity and genotoxicity potential of vinegar-processed Genkwa Flos.

Genkwa Flos (GF) is a well-known traditional medicine that is used to treat tumors and to relieve inflammation-related symptoms. GF tends to be taken in repeated doses for a long period of time, and although many reports on the toxicity of raw GF have led to a processing method to remove the toxicity, little information is currently available with regards to the toxic effects of subchronic exposure to processed GF (PGF). The aim of this study was to assess the possible genotoxicity and subchronic toxicity of PGF extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. A 13-week repeat-dose oral toxicity study was carried out with rats, and the change in body weight observed in rats receiving PGF extract was normal. It is worth noting that the PGF extract groups exhibited an obvious increase in liver weight along with a significant increase in serum alkaline phosphatase activity at doses of 667 and 2000mg/kg, providing evidence of hepatotoxic potential. More importantly, the results of the Ames test indicated that the PGF extract presented a mutagenic potential. Altogether, these results are the first to determine the subchronic toxicity and genotoxicity of the PGF extract, indicating that when GF is used for medicinal purposes, the period of use should be considered despite the manner in which the extract is processed.

Comparative toxicity of silicon dioxide, silver and iron oxide nanoparticles after repeated oral administration to rats.

Although silicon dioxide (SiO2), silver (Ag) and iron oxide (Fe2O3) nanoparticles are widely used in diverse applications from food to biomedicine, in vivo toxicities of these nanoparticles exposed via the oral route remain highly controversial. To examine the systemic toxicity of these nanoparticles, well-dispersed nanoparticles were orally administered to Sprague-Dawley rats daily over a 13-week period. Based on the results of an acute toxicity and a 14-day repeated toxicity study, 975.9, 1030.5 and 1000 mg kg(-1) were selected as the highest dose of the SiO2 , Ag and Fe2O3 nanoparticles, respectively, for the 13-week repeated oral toxicity study. The SiO2 and Fe2O3 nanoparticles did not induce dose-related changes in a number of parameters associated with the systemic toxicity up to 975.9 and 1000 mg kg(-1) , respectively, whereas the Ag nanoparticles resulted in increases in serum alkaline phosphatase and calcium as well as lymphocyte infiltration in liver and kidney, raising the possibility of liver and kidney toxicity induced by the Ag nanoparticles. Compared with the SiO2 and Fe2O3 nanoparticles showing no systemic distribution in all tissues tested, the Ag concentration in sampled blood and organs in the Ag nanoparticle-treated group significantly increased with a positive and/or dose-related trend, meaning that the systemic toxicity of the Ag nanoparticles, including liver and kidney toxicity, might be explained by extensive systemic distribution of Ag originating from the Ag nanoparticles. Our current results suggest that further study is required to identify that Ag detected outside the gastrointestinal tract were indeed a nanoparticle form or ionized form.

Genotoxicity and subchronic toxicity of Sophorae radix in rats: hepatotoxic and genotoxic potential.

Although Sophorae radix (SR) has been traditionally used as a treatment for various clinical symptoms, a comprehensive investigation of its safety has not yet been carried out. Therefore, we present an evaluation of the toxicity of the SR extract that was performed according to the Organization for Economic Cooperation and Development test guidelines for subchronic toxicity and genotoxicity. In an oral subchronic study for 13 weeks, the repeated treatment of rats with 429 or 1500 mg/kg of the SR extract induced a dose-related change in body weight. In particular, the SR extract was observed to exert a significant increase in liver weight along with an increase in serum alkaline phosphatase and alanine transaminase. A small but statistically significant reductions in red blood cell, hemoglobin, and hematocrit levels in the SR extract-treated rats suggest the possibility that anemia, accompanied by liver injury, was at least partially induced. These findings indicate the no-observed-adverse-effect-level for the SR extract was considered to be 10mg/kg/d. And, the data obtained from the chromosome aberration assay showed that SR extract might be considered to be a weak clastogen although no significant micronucleus induction was observed in vivo. Despite the benefits that SR extract can exhibit, this study indicates that SR extract may possess hepatotoxic and genotoxic potential.

Rat pancreatitis produced by 13-week administration of zinc oxide nanoparticles: biopersistence of nanoparticles and possible solutions.

Zinc oxide (ZnO) nanoparticles (NPs) are used in diverse applications ranging from paints and cosmetics to biomedicine and food. Although micron-sized ZnO is a traditional food supplement, ZnO NPs are an unknown public health risk because of their unique physicochemical properties. Herein, we studied the 13-week subchronic toxicity of ZnO NPs administered via the oral route according to Organization for Economic Cooperation and Development (OECD) test guideline 408. Well-dispersed ZnO NPs were administered to Sprague-Dawley (SD) rats (11/sex/group) at doses of 67.1, 134.2, 268.4 or 536.8 mg kg(-1) per body weight over a 13-week period. The mean body weight gain in males given 536.8 mg kg(-1) ZnO NPs was significantly lower than that of control male rats, whereas no significant differences were observed between the other treatment groups and the controls. Male and female rats dosed at 536.8 mg kg(-1) ZnO NPs had significant changes in anemia-related hematologic parameters. Mild to moderate pancreatitis also developed in both sexes dosed at 536.8 mg kg(-1) , whereas no histological changes were observed in the other treatment groups. To evaluate the mechanism of toxicity, we performed a bio-persistence study and evaluated the effects of the ZnO NPs on cell proliferation. The treatment of a human gastric adenocarcinoma cell line with ZnO NPs resulted in a significant inhibition of cellular proliferation. The anti-proliferative effect of ZnO NPs or Zn(2+) was effectively blocked by treatment with chelators. These results indicate that the bio-persistence of ZnO NPs after ingestion is key to their toxicity; the no-observed-adverse effect level (NOAEL) of ZnO NPs was found to be 268.4 mg kg(-1) per day for both sexes.

Acute and subchronic toxicity of FCD, a soybean extract combined with L-carnitine, in Sprague-Dawley rats.

Soy products are primarily composed of proteins, phytochemicals such as isoflavones, soy lipids, and carbohydrates. Recently, soy isoflavones with L-carnitine were reported to exhibit anti-obesity effects in mice. FCD, a combination of soybean extract and L-carnitine, is a newly developed food substance. As a part of its safety assessment, acute and 13-week subchronic toxicity studies were performed in a total of 100 Sprague-Dawley (SD) rats. In the acute study, a single limit dose of 2000 mg/kg was orally administered to five male and five female rats. No adverse effects or mortality was observed during a 14-day period or upon gross pathological examination. In the subchronic study, FCD was orally administered in daily doses of 500, 1000, and 2000 mg/kg for 13 weeks, resulting in no mortality, and no changes in hematological and serum biochemistry parameters, gross pathology or histopathology. However, body weights of females were significantly decreased 10 weeks after treatment at an average of 2000 mg/kg. In addition, a slight decrease in mean food and water consumption was observed at the same dose level for 13 weeks. Therefore, the no-observed-adverse-effect-level (NOAEL) of FCD was considered to be 2000 mg/kg for male and 1000 mg/kg for female SD rats.

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation.

In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression increased as cation concentration of vehicle decreased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.