Chinese Hamster Lung Cells Research Articles

Chinese Hamster Lung Cell의 소핵시험을 이용한 식물추출물 유기농업자재의 유전독성평가

Abstract BACKGROUND: Azadirachta Indica extract(AIE) and Sophorae radix extract(SRE) are widely used as environment-friendly organic materials of plant origin in South Korea. METHODS AND RESULTS: In this study, the in vitro micronucleus(vitMN) tests of two samples of AIE and SRE were conducted to evaluate their genotoxicity using the Chinese hamster lung(CHL) cell. This study was composed of two parts; cytochalasin B(cyto B) test and non-cyto B test. Mitomycin C and colchicine were used as positive controls. As a result, the incidence of micronucleus(MN) in all AIE and SRE treated groups increased in dose- dependent manner, but were less than 2.2% in 1,000 binucleated cells. In addition, there were no significant increases of MN incidence in all AIE and SRE treated groups, compared with the negative control group. CONCLUSION: Therefore, we suggest that AIE samples and SRE samples used in this study may have no genotoxicity in the in vitro micronucleus test using the CHL cells. In our previous study, we reported that AIE and SRE did not cause genotoxicity in Ames test. According to the genotoxicity battery system, we concluded that AIE and SRE used in this study have no genotoxic effects to humans.

A comparison of three (67/68)Ga-labelled exendin-4 derivatives for β-cell imaging on the GLP-1 receptor: the influence of the conjugation site of NODAGA as chelator.

BackgroundVarious diseases derive from pathologically altered β-cells. Their function can be increased, leading to hyperinsulinism, or decreased, resulting in diabetes. Non-invasive imaging of the β-cell-specific glucagon-like peptide receptor-1 (GLP-1R) would allow the assessment of both β-cell mass and derived tumours, potentially improving the diagnosis of various conditions. We tested three new 67/68Ga-labelled derivatives of exendin-4, an agonist of GLP-1R, in vitro and in vivo. We determined the influence of the chelator NODAGA conjugated to resident lysines either at positions 12 and 27 or the C-terminally attached lysine at position 40 on the binding and kinetics of the peptide.MethodsBinding and internalisation of 67Ga-labelled Ex4NOD12, Ex4NOD27 and Ex4NOD40 were tested on Chinese hamster lung (CHL) cells stably transfected to express the GLP-1 receptor (GLP-1R). In vivo biodistribution of 68Ga-labelled peptides was investigated in CD1 nu/nu mice with subcutaneous CHL-GLP-1R positive tumours; the specificity of the binding to GLP-1R was determined by pre-injecting excess peptide.ResultsAll peptides showed good in vitro binding affinities to GLP-1R in the range of 29 to 54 nM. 67/68Ga-Ex4NOD40 and 67/68Ga-Ex4NOD12 show excellent internalisation (>30%) and high specific uptake in GLP-1R positive tissue, but high activity was also found in the kidneys.ConclusionsWe show that of the three peptides, Ga-Ex4NOD40 and Ga-Ex4NOD12 demonstrate the most favourable in vitro properties and in vivo binding to GLP-1R positive tissue. Therefore, we conclude that the lysines at positions 12 and 40 might preferentially be utilised for modifying exendin-4.

Evaluation of Genotoxicity of CSE1034 by Ames and <i>In vitro</i> Chromosomal Aberration Tests

Purpose: To evaluate the genotoxicity of CSE1034, a novel antibiotic adjuvant entity, using bacterial reverse mutation assay (Ames test) and in vitro chromosomal aberration test.Methods: Reverse mutation test was carried out using four strains of Salmonella typhimurium (TA 98, TA100, TA1535 and TA1537) and one strain of E. coli [WP2 (uvrA)], while chromosomal aberration test was done with cultured Chinese hamster lung (CHL) cells. Reverse mutation test was carried out in a dose range of 0.0015 to 0.16 ìg/plate in triplicate with and without S9 activation.Results: No significant increases in the number of revertants were observed at the dose levels where antibacterial effects were not noted. CSE1034 caused no increase in the number of chromosomal aberrants at dose levels of 0.34, 0.69, 1.37, 2.75 and 5.50 mg/ml in the absence and presence ofmetabolic activation.Conclusion: Based on the above observations, it can be concluded that CSE1034 has no mutagenic activity.Keywords: CSE1034, reverse mutation, antibiotic adjuvant entity, Chromosomal aberration, Mutagenic

Increased expression of sorcin is associated with multidrug resistance in leukemia cells via up-regulation of MDR1 expression through cAMP response element-binding protein

Sorcin, a 22kDa Ca2+ binding protein, was first identified in a vincristine-resistant Chinese hamster lung cell line, and was later demonstrated to be involved in the development of multidrug-resistance (MDR) phenotypes in a variety of human cancer cell lines. However, the exact role of sorcin in MDR cells is yet to be fully elucidated. Here we explored the role of sorcin in the development of MDR in leukemia cells, and revealed that the expression level of sorcin was directly correlated to the expression of MDR1/P-glycoprotein (P-gp). In addition, it was shown that sorcin induced the expression of MDR1/P-gp through a cAMP response element (CRE) between −716 and −709bp of the mdr1/p-gp gene. Furthermore, overexpression of sorcin increased the phosphorylation of CREB1 and the binding of CREB1 to the CRE sequence of mdr1/p-gp promoter, and induced the expression of MDR1/P-gp. These findings suggested that sorcin induces MDR1/P-gp expression markedly through activation of the CREB pathway and is associated with the MDR phenotype. The new findings may be helpful for understanding the mechanisms of MDR in human cancer cells, prompting its further investigation as a molecular target to overcome MDR.

Surfactant Uptake Dynamics in Mammalian Cells Elucidated with Quantitative Coherent Anti-Stokes Raman Scattering Microspectroscopy

The mechanism of surfactant-induced cell lysis has been studied with quantitative coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The dynamics of surfactant molecules as well as intracellular biomolecules in living Chinese Hamster Lung (CHL) cells has been examined for a low surfactant concentration (0.01 w%). By using an isotope labeled surfactant having CD bonds, surfactant uptake dynamics in living cells has been traced in detail. The simultaneous CARS imaging of the cell itself and the internalized surfactant has shown that the surfactant molecules is first accumulated inside a CHL cell followed by a sudden leak of cytosolic components such as proteins to the outside of the cell. This finding indicates that surfactant uptake occurs prior to the cell lysis, contrary to what has been believed: surface adsorption of surfactant molecules has been thought to occur first with subsequent disruption of cell membranes. Quantitative CARS microspectroscopy enables us to determine the molecular concentration of the surfactant molecules accumulated in a cell. We have also investigated the effect of a drug, nocodazole, on the surfactant uptake dynamics. As a result of the inhibition of tubulin polymerization by nocodazole, the surfactant uptake rate is significantly lowered. This fact suggests that intracellular membrane trafficking contributes to the surfactant uptake mechanism.

4-Hydroxynonenal induces an increase in expression of Receptor for Activating C Kinase 1 (RACK1) in Chinese hamster V79-4 lung cells

4-Hydroxy-trans-2-nonenal (HNE) is a cytotoxic α,β-unsaturated aldehyde implicated in the pathology of several diseases that have an oxidative stress mechanism, including atherosclerosis, diabetes, alcohol-induced liver disease, and neurodegenerative disorders. As the most toxic aldehydic product of lipid peroxidation, HNE is known to exert a range of biological effects in a concentration-dependent manner. In this study, the effect of HNE on the levels of proteins in V79-4 Chinese hamster lung cells was investigated using two-dimensional electrophoresis and mass spectrometry. The results revealed that the expression of 23 proteins was increased by at least 2-fold and the expression of 19 proteins was decreased by at least 2-fold after exposure to 10μM HNE for 24h. Decreased proteins included the metabolic enzyme phosphoglycerate kinase 1 (PGK1), levels of which were decreased by 47%. Levels of the apoptotic indicator Lamin C were decreased by 33%. In contrast, levels of the scaffolding protein Receptor for Activating C Kinase 1 (RACK1) were increased by 2-fold after treatment with 10μM HNE for 24h, and this was confirmed using quantitative PCR of reverse-transcribed mRNA and Western blots. The role of RACK1 in mediating the induction of apoptosis in response to 10μM HNE was confirmed using RACK1-specific siRNA. The results from this study provide new information on the mechanism of adaptive stress response to HNE and also identify potential new biomarkers of exposure to HNE.

Genotoxicity of styrene oligomers extracted from polystyrene intended for use in contact with food

Here, we conducted in vitro genotoxicity tests to evaluate the genotoxicity of styrene oligomers extracted from polystyrene intended for use in contact with food. Styrene oligomers were extracted with acetone and the extract was subjected to the Ames test (OECD test guideline No. 471) and the in vitro chromosomal aberration test (OECD test guideline No. 473) under good laboratory practice conditions. The concentrations of styrene dimers and trimers in the concentrated extract were 540 and 13,431 ppm, respectively. Extraction with acetone provided markedly higher concentrations of styrene oligomers compared with extraction with 50% ethanol aqueous solution, which is the food simulant currently recommended for use in safety assessments of polystyrene by both the United States Food and Drug Administration and the European Food Safety Authority. And these high concentrations of styrene dimers and trimers were utilized for the evaluation of genotoxicity in vitro. Ames tests using five bacterial tester strains were negative both in the presence or absence of metabolic activation. The in vitro chromosomal aberration test using Chinese hamster lung cells (CHL/IU) was also negative. Together, these results suggest that the risk of the genotoxicity of styrene oligomers that migrate from polystyrene food packaging into food is very low.

고삼추출물의 in vitro 항돌연변이원성과 유전독성 연구

Sophorae radix extract (SRE) has been registered as an environment-friendly organic material that is widely used in the cultivation of crops in Korea. Matrine, the active ingredient in SRE, was reported as a toxic substance in the nervous system in mice. However, no information is available on its toxic effects in other organisms. Therefore, antimutagenicity and two kinds of genotoxicity tests (bacterial reverse mutation and chromosome aberration test) of two samples of SRE were investigated in this study. Antimutagenicity test was experimented by using bacterial reverse mutation test. In the reverse mutation test, Salmonella Typhimurim TA98, TA1535 and TA1537 were used to evaluate the mutagenic potential of SRE. Bacterial reverse mutation test was also performed on positive and negative control groups in the presence of the metabolic activation system (with S-9 mix) and metabolic non-activation system (without S-9 mix). In the chromosome aberration test, Chinese hamster lung cells were exposed to SRE for 6 or 24 hours without S-9 mix, or for 6 hours with S-9 mix. Negative and positive control groups were experimented for chromosome aberration test. As a result, the number of mutated colonies induced by 4-NQO were reduced by SRE treatment in all strains, indicating that SRE may have antimutagenic effects. Reverse mutation was not shown at all concentrations of SRE, regardless of application of the metabolic activation system. In the chromosomal aberration test, one of the SRE sample gave a suspicious positive result at 250 µg/ml in the presence of S-9 mix. For the more adequate evaluation of the genotoxic potential of SRE samples, other in vivo genotoxicity study is needed.

In silico and in vitro genotoxicity evaluation of descarboxyl levofloxacin, an impurity in levofloxacin

It is important to establish the safety of impurities in drug substances or drug products. The assessment of genotoxicity of impurities and the determination of acceptable limits for genotoxic impurities was addressed in some recent guidances as a difficult issue. Descarboxyl levofloxacin is an impurity isolated from levofloxacin, which may impose a risk without associated benefit. However, there is insufficient toxic information about descarboxyl levofloxacin. This study investigated the genotoxicity of this impurity by in silico and in vitro methods. We used Derek, a commercial structure-activity relationship software package, as an in silico tool. The results showed that there was a structural alert (quinoline) in this impurity. Then, the in vitro genotoxicity of descarboxyl levofloxacin was investigated by a modified Ames test and by a chromosomal aberration test, using Chinese hamster lung (CHL) cells. Both assays were conducted in the presence or absence of S-9 mix. The results showed that the test impurity was not mutagenic in the Ames test (31.25–500 μg/plate). Whereas there was a statistically significant increase in the number of metaphase CHL cells with structural aberrations at the concentration of 1 mg/mL with S-9 mix, the aberrations rate was 7.5%. It did not significantly increase the number of structural aberration in CHL cells in the presence (at 250 and 500 μg/mL) or absence of S-9 mix. Based on these assays, descarboxyl levofloxacin could be controlled as a nongenotoxic impurity.

Evaluation of genotoxicity of Trois through Ames and In vitro chromosomal aberration tests

ObjectiveTo investigate the mutagenic potential of Trois using the bacterial reverse mutation assay (Ames test) and In vitro chromosomal aberration test. MethodsThe ability of Trois to induce reverse mutations was evaluated in Salmonella typhimurium (TA 98, TA100, TA1535 and TA1537) and Escherichia coli (WP2 uvrA) with and without metabolic activation system (S9 mix) at the dose range of 313 to 5000 μg/plate. Chromosomal aberrations were evaluated in Chinese hamster lung (CHL) cell line at the dose levels of 15, 7.5, 3.7, 1.9 and 0.9 mg/mL in the absence and presence of S9 mix. ResultsThere were no increases in the number of revertant colonies at any concentrations of Trois used in the study with and without S9 mix in all tester strains. Trois did not produce any structural aberration in CHL cells in the presence or absence of S9 mix. ConclusionsResults of this study suggest that Trois is non-mutagenic.

Genotoxicity of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™)

The genotoxic potential of pyrroloquinoline quinone (PQQ) disodium salt (BioPQQ™) was evaluated in a battery of genotoxicity tests. The results of the bacterial mutation assay (Ames test) were negative. Weak positive results were obtained in 2 separate in vitro chromosomal aberration test in Chinese hamster lung (CHL) fibroblasts. Upon testing in an in vitro chromosomal aberration test in human peripheral blood lymphocytes, no genotoxic activity of PQQ was noted. In the in vivo micronucleus assay in mice, PQQ at doses up to 2000mg/kg body weight demonstrated that no genotoxic effects are expressed in vivo in bone marrow erythrocytes. The weak responses in the in vitro test CHL cells were considered of little relevance under conditions of likely human exposure. PQQ disodium was concluded to have no genotoxic activity in vivo.

Cell Type-Dependent Induction of DNA Damage by 1800 MHz Radiofrequency Electromagnetic Fields Does Not Result in Significant Cellular Dysfunctions

BackgroundAlthough IARC clarifies radiofrequency electromagnetic fields (RF-EMF) as possible human carcinogen, the debate on its health impact continues due to the inconsistent results. Genotoxic effect has been considered as a golden standard to determine if an environmental factor is a carcinogen, but the currently available data for RF-EMF remain controversial. As an environmental stimulus, the effect of RF-EMF on cellular DNA may be subtle. Therefore, more sensitive method and systematic research strategy are warranted to evaluate its genotoxicity.ObjectivesTo determine whether RF-EMF does induce DNA damage and if the effect is cell-type dependent by adopting a more sensitive method γH2AX foci formation; and to investigate the biological consequences if RF-EMF does increase γH2AX foci formation.MethodsSix different types of cells were intermittently exposed to GSM 1800 MHz RF-EMF at a specific absorption rate of 3.0 W/kg for 1 h or 24 h, then subjected to immunostaining with anti-γH2AX antibody. The biological consequences in γH2AX-elevated cell type were further explored with comet and TUNEL assays, flow cytometry, and cell growth assay.ResultsExposure to RF-EMF for 24 h significantly induced γH2AX foci formation in Chinese hamster lung cells and Human skin fibroblasts (HSFs), but not the other cells. However, RF-EMF-elevated γH2AX foci formation in HSF cells did not result in detectable DNA fragmentation, sustainable cell cycle arrest, cell proliferation or viability change. RF-EMF exposure slightly but not significantly increased the cellular ROS level.ConclusionsRF-EMF induces DNA damage in a cell type-dependent manner, but the elevated γH2AX foci formation in HSF cells does not result in significant cellular dysfunctions.

Lack of Mutagenicity Potential of <i>Periploca sepium</i> Bge. in Bacterial Reverse Mutation (Ames) Test, Chromosomal Aberration and Micronucleus Test in Mice

ObjectivesThe root barks of Periploca sepium Bge. (P. sepium) has been used in traditional Chinese medicine for healing wounds and treating rheumatoid arthritis. However, toxicity in high-doses was often diagnosed by the presence of many glycosides. The potential mutagenicity of P. sepium was investigated both in vitro and in vivo.MethodsThis was examined by the bacterial reverse mutation (Ames) test using Escherichia coli WP2uvrA and Salmonella typhimurium strains, such as TA98, TA100, TA1535, and TA1537. Chromosomal aberrations were investigated using Chinese hamster lung cells, and the micronucleus test using mice.ResultsP. sepium did not induce mutagenicity in the bacterial test or chromosomal aberrations in Chinese hamster lung cells, although metabolic activation and micronucleated polychromatic erythrocytes were seen in the mice bone marrow cells.ConclusionsConsidering these results, it is suggested that P. sepium does not have mutagenic potential under the conditions examined in each study.

Genotoxicity of Zizyphi Spinosi Semen in Bacterial Reverse Mutation (Ames) Test, Chromosomal Aberration and Micronucleus Test in Mice

Zizyphi spinosi semen (Z. spinosi) has been used in traditional Chinese medicine for the treatment of rheumatoid arthritis and wounds. However, toxicity in high doses was often observed due to the presence of alka- loids. This study was conducted to investigate the potential genotoxicity of Z. spinosi in vitro and in vivo. This was examined by the Bacterial reverse mutation (Ames) test using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA, Chromosomal aberration was investigated using Chinese hamster lung cells and the micronucleus test using mice. Z. Spinosi did not induce mutagenicity in the Ames test, and it did not produce chromosomal aberration in Chinese hamster lung cells with and without metabolic activation, nor in the micronucle- ated polychromatic erythrocytes in the bone marrow cells in mice. Based on these results, it is concluded that Z. spi- nosi does not have mutagenic potential under the conditions examined in each study.

Increased risk of lung cancer associated with a functionally impaired polymorphic variant of the human DNA glycosylase NEIL2

Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6–7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients’ genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ∼5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant's lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.

Acute and subacute toxicity and genotoxicity of schizonepetin, a naturally occurring monoterpene with antiviral activity

In the present study, the acute, subacute and genetic toxicity of schizonepetin was assessed. The median lethal dose (LD50) of schizonepetin after oral administration was 478mg/kg body weight in mice. Studies on dose toxicity were repeatedly conducted at 0, 60, 120, and 240mg/kg bw/day in rats for 35days after oral administration. Based on the results of this study, a dose level of 120mg/kg bw/day is considered the no-observed-adverse-effect-level (NOAEL) in rats. Schizonepetin was negative in Salmonella typhimurium tester strains TA97, TA98, TA100, TA102 and TA1535, nonclastogenic in Chinese hamster lung (CHL) cells in the mammalian chromosome aberration test, and micronucleus formation were observed and no clinical signs or adverse effects were detected, and our results illustrated that schizonepetin is not genotoxic.

In vitro and in vivo evaluation of the genotoxicity of Gumiganghwal-tang, a traditional herbal prescription

Ethnopharmacological relevanceGumiganghwal-tang (GGT, known as Kumi-Kyokatsu-to in Japanese) is a traditional herbal prescription made from nine different herbs that is used for the treatment of the common cold, pain, and inflammatory diseases. Aim of the studyThis study evaluated the potential genotoxicity of an aqueous GGT extract using three standard battery of tests as part of a safety evaluation. Materials and methodsWe prepared GGT using a water extraction method and subsequently extracted six compounds from GGT by high performance liquid chromatography. GGT extract genotoxicity was assayed using three standard tests including the in vitro bacterial reverse mutation test, the in vitro chromosomal aberration test with Chinese hamster lung cells, and the in vivo micronucleus test using ICR mouse bone marrow recommended by the Korean Food and Drug Administration. ResultsThe bacterial reverse mutation assay showed that GGT extract doses ranging from 333.3 up to 5000mg/plate induced a greater than 2-fold increase in the number of revertant TA1537 strain colonies exhibiting metabolic activation (with S-9 mix), when compared with the vehicle control. The chromosomal aberration test showed that GGT extract induced an increase in the number of chromosomal aberrations after treatment for 6h with the S-9 mix and 22h without the S-9 mix, when compared with vehicle control. In contrast, the micronucleus test showed that GGT extract did not significantly increase the number of micronucleated polychromatic erythrocytes (MNPCEs) in ICR mouse bone marrow. ConclusionsBased on these results, it was concluded that GGT extract acted as a genotoxic material in our experimental conditions. We did not identify the compounds responsible for the induction of genotoxic effects, but it was significant that we provided a basic genotoxicity profile for GGT.

Evaluation on Cytotoxicity and Genotoxicity of the L-Glutamic Acid Coated Iron Oxide Nanoparticles

To evaluate the cytotoxicity and genotoxicity of L-glutamic acid (Glu) coated Fe2O3 nanoparticles (hereafter refer as Glu@MNPs) on Chinese Hamster Lung (CHL) cells using Trypan blue dye exclusion assay, Oxidative stress markers, Comet assay and micronucleus (MN) assay. Results showed a low cytotoxicity with an IC50 was 254.739 microg/ml 36 h post incubation period in CHL cells. Furthermore, Cell redox status is slightly disturbed: Glu@MNPs exposure cause reactive oxygen species production, glutathione depletion and inactivation of some antioxidant enzymes: glutathione reductase, superoxide dismutase, but not catalase. Moreover, no significant genotoxic response was observed in CHL cells over concentration ranges from 8 to 128 microg/mL for all exposure time periods. The results suggest that the Glu@MNPs show biocompatibility In Vitro.

Evaluation of genotoxicity of Bacillus mojavensis KJS-3 on culture supernatant for use as a probiotic

The genotoxicity of crude substances cultured in Bacillus mojavensis KJS-3 (B. mojavensis KJS-3) was investigated by using Ames tester strains of Salmonella typhimurium (S. typhimurium) with or without metabolic activation (S9 mix). No increase in the number of revertants was observed in response to any of the doses of the culture supernatant fermented by B. mojavensis KJS-3 (5, 10, 50, 100, and 500 μg/plate) in S. typhimurium TA 98, 100, 1535 and 1537 both in the absence and in the presence of S-9 metabolic activation system. The half-maximal inhibitory concentration (IC50) value of the culture supernatant was >83.33 μg/mL. Crude substances had no effect on the DNA repair system for mutagenesis. Furthermore, the cell number of S. typhimurium strains was observed to have decreased compared to the solvent control. These results showed dose-dependent activity on the mutagenicity of crude substances in Ames test. These results strongly indicate that antimutagenic compounds are produced during fermentation by B. mojavensis KJS-3. On using the Chinese hamster lung cell line in mammalian cell system, no clastogenicity of the culture supernatant fermented by B. mojavensis KJS-3 was observed in the absence or presence of the S9 mix at the concentrations of 1.66, 8.33, 16.67, and 83.33 μg/mL. No significant increase in chromosome aberrations was observed in response to treatment with any of these concentrations, regardless of activation of the metabolic system. Consequently, the mutagenic potential of the culture supernatant was not induced with respect to in vitro bacterial reverse mutation and clastogenicity in the range of concentrations evaluated in these experiments in our study.

Absence of Genotoxic and Mutagenic Effects of Zingiber zerumbet (L.) Smith (Zingiberaceae) Extract.

The present study evaluated the potential genotoxicity of the ethanol extracts from the rhizome of Zingiber zerumbet (L.) Smith (EEZZR) using a standard battery of tests. Chemical analysis with liquid chromatography-tandem mass spectrometry revealed that EEZZR contained Zerumbone (200.3 ± 0.37 μg/g) and 6-gingerol (102.5 ± 0.28 μg/g). There were no increases in the number of revertant colonies with EEZZR at concentrations of 150–5000 μg per plate, regardless of the metabolic activation system (S-9 mix) used in the histidine-dependent auxotrophic mutants of Salmonella typhimurium (strains TA97, TA98, TA100, TA102, and TA1535) compared to the vehicle control. Furthermore, EEZZR at doses of 150–5000 μg mL−1 did not increase the number of structural aberrations in Chinese hamster lung cells in the presence or absence of S-9 mix. An oral administration of EEZZR to ICR mice, with doses of up to 2000 mg/kg, caused no significant increases in the number of micronucleated polychromatic erythrocytes (MNPCEs) and mean ratio of polychromatic erythrocytes to total erythrocytes. Lastly, RZZEE did not increase the incidence of MNPCEs in bone marrow. Based on these findings, it may be concluded that the use of EEZZR in traditional medicine poses no risk of genotoxicity.