Plate Incorporation Method Research Articles

Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.

Mutagenicity Assessment of Homologous Series of Methyl Ester Sulphonates (MES) Using the Bacterial Reverse Mutation (Ames) Test.

Methyl ester sulphonate (MES) is an anionic surfactant that is suitable to be used as an active ingredient in household products. Four palm-based MES compounds with various carbon chains, namely C12, C14, C16 and C16/18 MES, were assayed by the in vitro bacterial reverse mutation (Ames) test in the Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and the Escherichia coli strain WP2 uvrA, with the aim of establishing the safety data of the compounds, specifically their mutagenicity. The test was also carried out on linear alkylbenzene sulphonate (LAS) for comparison. The plate incorporation method was conducted according to the Organization for Economic Cooperation and Development (OECD) Test Guideline 471. All compounds were tested at five analysable non-cytotoxic concentrations, varying from .001mg/plate to 5mg/plate, with and without S-9 metabolic activation. All tested concentrations showed no significant increase in the number of revertant colonies compared to revertant colonies of the negative control. The Ames test indicated that each concentration of C12, C14, C16, C16/18 MES, and LAS used in this study induced neither base-pair substitutions nor frame-shift mutations in the S. typhimurium strains TA98, TA100, TA1535, and TA1537 and the E. coli strain WP2 uvrA. The results showed that C12, C14, C16 and C16/18 MES have no potential mutagenic properties in the presence and absence of S-9 metabolic activation, similarly to LAS. Therefore, the MES is safe to be used as an alternative to petroleum-based surfactants for household cleaning products.

Mutagenicity assessment of two potential impurities in preparations of 5-amino-2,4,6 triiodoisophthalic acid, a key intermediate in the synthesis of the iodinated contrast agent iopamidol

5-Aminoisophthalic acid and 5-nitroisophthalic acid (5-NIPA) are potential impurities in preparations of 5-amino-2,4,6-triiodoisophthalic acid, which is a key intermediate in the synthesis of the iodinated contrast agent iopamidol. We have studied their mutagenicity in silico (quantitative structure-activity relationships, QSAR) and by the bacterial reverse mutation assay (Ames test). First, the compounds were screened with the tools Derek Nexus™ and Leadscope®. Both compounds were flagged as potentially mutagenic (class 3 under ICH M7). However, contrary to the in silico prediction, neither chemical was mutagenic in the Ames test (plate incorporation method) with or without S9 metabolic activation.

Phenolic, Flavonoids Content, Antioxidant Potential of Murraya koenigii L. (Curry Leave): in vitro and in vivo Protective Effects

In the current investigation, the proximate value, total phenolic content, toal flavonoid content, antioxidant potential and in vitro and in vivo protective effects of Murraya koenigii L. (curry leave) extracts were carried out. Flavonoids, phenols and alkaloids were recorded in ethanol, methanol, ethyl acetate, petroleum ether, benzene and acetone extracts. A higher level of total phenolic content was recorded in ethanolic fraction followed by benzene, ethyl acetate, methanol, petroleum ether and acetone fractions. The total flavonoids content was recorded in the range of 5.12 mg RE g−1 to 9.24 mg RE g−1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity was estimated in all the solvent fractions at the concentration ranging from 312.5 μg mL-1 to 5000 μg mL−1. Percentage inhibition was recorded in the range of 39.40 to 72.32. To study the antimutagenic property, the plate incorporation method was used to see the effects of the MKLE fraction on S. typhimurium trains TA 1537, TA 1535, TA 98, TA 100 and TA 102 both in the absence and presence of S9 mix (5% v/v). Higher antimutagenic property at a concentration of 5000 μg/plate with 2-nitrofluorene 7.5 μg/plate was observed against TA 98. In vivo study indicates the protective effects of the extract of the MKLE against chlorpyrifos-induced toxicity in brain and lungs of the experimental rats. Four animal groups 5 male and 5 females were selected, group I serve as a control, group II was given a single dose of MKLE as such for 28 consecutive days while group III was treated with chlorpyrifos at the dose level of 60 mg/kg B.wt and group IV was dosed with chlorpyrifos at the dose level of 60 mg/kg B.wt simultaneously treated with the MKLE as such twice in a day (B.D.). Chlorpyrifos administration (group III) to the rats resulted in reduced body weight. decrease Ache level increases, SGOT, SGPT, ALP were observed while no significant alterations were noticed in any parameter in the II group animals, Histopathology of brain and lung of the chlorpyrifos treated animals shows the cellular deficit in the brain tissue and mild congestion in the lung tissue. The present study concluded that curry leaves are a rich source of phenolic, alkaloids, flavonoids and exhibit antioxidant and antimutagenic activity and appeared to be beneficial to rats to a great extent in attenuating and resulting in the damage sustained by chlorpyrifos exposure.

New possibilities of the Ames test for evaluation of mutagenicity of technical products of active ingredients of pesticides

Introduction. The Ames test is the one of the most popular methods for mutagenicity evaluation of environmental factors. In some cases, this method is suggested to be the only and sufficient assay for the first stage of the equivalence assessment of pesticide technical grade active ingredients (TGAI) to the original products. A limitation of the Ames test is related to the impossibility of an objective equivalence assessment of some cytotoxic TGAIs, in particular, sulfonylureas, and triazolpyrimidines. Based on the mode of action of the pesticides belongs to these chemical classes, we suggested a modification of the plate incorporation method protocol of the Ames test to the increase of maximal non-cytotoxic concentration up to the 5 mg/plate recommended by regulatory documents. Materials and methods. The five strains of Salmonella typhimurium TA98, TA100, TA1535, TA97, TA102 were used. The modification of the protocol included a supplementation of the top agar with isoleucine (1-5 mM). Results. The maximum non-cytotoxic concentrations of thifensulfuron-methyl and florasulam using the standard top agar did not exceed 0.05-0.125 mg/plate. The enrichment of the top agar with isoleucine allowed evaluating the mutagenicity of the substances up to the maximal recommended concentration of 5.0 mg/plate. The number of spontaneous revertants was within the historical limits of the laboratory control obtained under standard conditions. Positive controls showed pronounced mutagenic effects in case of all strains with and without metabolic activation (p≤0.05). Limitations. Mutagenicity was evaluated only for TGAIs, which are acetohydroxyacid synthase inhibitors. Conclusion. The application of the modified Ames test protocol for mutagenicity assessment of TGAIs from the classes of sulfonylureas and triazolpyrimidines under supplementation of the top agar with isoleucine is a more objective way to evaluate their mutagenicity. The proposed protocol expands the possibilities of revealing dangerous mutagenic impurities that may occur in TGAIs in the small quantities, and after entering the environment can cause the gain in the mutation level in living organisms.

Evaluation of Mutagenic Potential of Ethanolic Extract of Leaves of <i>Couroupita guianensis</i> using <i>Salmonella typhimurium</i> Tester Strains

India has a golden resource and well-recorded traditionally practised incomparable awareness of herbal medicine. Not all herbals are considered safe as minimal data is available concerning the safety of Couroupita guianensis to cause mutations. Considering the enormous therapeutic potential and the absence of specific safety data, we planned this research to provide data on the mutagenic potential of Couroupita guianensis. Therefore, the present study was conducted with ethanolic extract of leaves of Couroupita guianensis using Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100, and TA102 at different concentrations (viz., 156.25, 312.5, 625, 1250, 2500, and 5000 μg/plate) along with the negative control and respective positive controls. The methodology followed was the standard plate incorporation method as per the internationally approved OECD TG 471 guidelines. The study was performed with and without S9 mix using the plate incorporation method. Results were evaluated after meeting the acceptance criteria for the study. The extracts did not show mutagenicity when compared to the negative control (spontaneous mutation) and respective positive controls. There was no concentration-response increase of revertant colonies in treatments from any strain. This is the first of its kind of research having been conducted following all the internationally set guidelines and evidenced the safety of the plant material. Therefore, it is concluded that ethanolic extract of leaves of Couroupita guianensis did not show mutagenic potential in bacterial reverse mutation test with and without S9 mix.

Evaluation of the in vitro and in vivo genotoxicity of a Dioscorea Rhizome water extract.

Dioscorea Rhizome is commonly used in traditional herbal medicines for the treatment of diabetes, hyperthyroidism, liver damage, neuropathy, and asthma. Here, we investigated the genotoxicity potential of D. Rhizome water extract (DRWE) using three standard battery systems in accordance with the test guidelines of the Organisation for Economic Cooperation and Development and Ministry of Food and Drug Safety as well as the principles of Good Laboratory Practice. A bacterial reverse mutation test (Ames test) was performed using the direct plate incorporation method in the presence or absence of a metabolic activation system (S9 mixture). The tester strains used included four histidine auxotrophic strains of Salmonella typhimurium, TA100, TA1535, TA98, and TA1537, along with a tryptophan auxotrophic strain of Escherichia coli, WP2 uvrA. An in vitro chromosome aberration test was performed using CHL/IU cells originally derived from the lung of a female Chinese hamster in the presence or absence of the S9 mixture. An in vivo mouse bone marrow micronucleus test was performed using male ICR mice. The micronucleus was confirmed after observation of the micro-nucleated polychromatic. The Ames test showed that DRWE did not induce gene mutations at any dose level in any of the tested strains. Additionally, DRWE did not result in any chromosomal aberrations specified in the in vitro chromosomal aberration and in vivo micronucleus tests. These results showed that DRWE exhibited neither mutagenic nor clastogenic potential in either the in vitro or in vivo test systems.

Antigenotoxicity, Cytogenetic Assay measuring Chromosomal Aberration and Antimicrobial Activity of Methanolic Extract of Microalgae: Nannochloropsis oculate

The aim of the present study was to investigate the anti-genotoxic effect of methanolic extract of microalgae-Nannochloropsis oculata and also to determine the antibacterial from marine microalga extract in methanol. Genotoxicity studies were conducted for screening of mutagenicity using Ames test, direct plate incorporation method using tester strain of Salmonella typhimurium viz., TA 98 and hence to prove this as an antigenotoxicant. The mutagenicity assay was performed with 3 dose levels (0.312, 1.25 and 5.0mg/ml) using all two tester strains without metabolic activation system (S9 fraction). Inhibition in the background lawn or reduction in revertant colonies was observed at tested dose concentration in two tester strains without metabolic activation system. Further, more works are to be carried out to find the exact mechanism of its Geno protective nature. The methanolic extract of Nannochloropsis oculata was also examined for antimicrobial activity by well-diffusion method. The extract showed high inhibition against the growth of Bacillus Subtilis when compared to the other tested strains. All microorganisms tested are susceptible to the treatment with methanolic extract of Nannochloropsis oculata in a concentration dependent manner.

Subacute Oral Toxicity and Bacterial Mutagenicity Study of a Mixture of Korean Red Ginseng (Panax ginseng C.A. Meyer) and Salvia plebeia R. Br. Extracts

As various populations are rapidly becoming an aging society worldwide and interest in health issues has increased, demand for functional foods including herbal products has increased markedly to maintain a healthy state which has led to safety issues about their intake as an inevitable result. The objective of this study was to identify the safety profile of a Korean red ginseng and Salvia plebeia R. Br. extract mixture (KGC-03-PS) which is a valuable ingredient that can be used as a functional food. In the present study, the subacute oral toxicity and bacterial reverse mutagenicity of KGC-03-PS were evaluated. Sprague Dawley rats were administered KGC-03-PS orally for 28 days by gavage. Daily KGC-03-PS dose concentrations were 0, 500, 1,000, or 2,000 mg/kg body weight (bw) per day. Bacterial reverse mutation test with KGC-03-PS dose levels ranging from 312.5 to 5,000 µg/plate was carried out by OECD test guideline No. 471. Five bacterial strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2) were tested in the presence or absence of metabolic activation by plate incorporation method. There were no toxicological effects related with test substance in the clinical evaluation of subacute oral toxicity test including clinical signs, body weight, and food consumption. Moreover, no toxicological changes related to KGC-03-PS were observed in the hematological and serum biochemical characteristics as well as in the pathological examinations, which included organ weight measurements and in the gross- or histopathological findings. KGC-03-PS did not induce an increase in the number of revertant colonies in all bacterial strains of the bacterial reverse mutation test. The no-observed-adverse-effect level of KGC-03-PS is greater than 2,000 mg/kg bw/day, and KGC-03-PS did not induce genotoxicity related to bacterial reverse mutations under the conditions used in this study.

Application of bacterial reverse mutation assay for detection of non-genotoxic carcinogens

Non-genotoxic carcinogens may play a significant role in development of cancer. Currently short-term assays for mutagenicity classify genotoxic carcinogens and lack the abilities to detect epigenetic carcinogens. The need to develop an endpoint always remains to recognize potentially carcinogenic agents employing rapid and practical bioassays. For this, the present study utilized TA98 and TA1537 tester strains of Salmonella typhimurium to evaluate four non-genotoxic carcinogenic agents (Coumarin, β-Myrcene, Bis(2-ethylhexyl) phthalate and trans-anethole). These chemicals were tested individually and in combination with promutagens 2-aminoanthracene (2AA) and benzo(a)pyrene (BP) in presence of metabolic activation system (S9) by plate incorporation method. Exposure to all four test chemicals revealed marked increase of revertant colonies in promutagen combined groups as compared to promutagens alone. However significantly greater fold responses were observed with 2AA combination groups (Coumarin +2AA, β-Myrcene +2AA, Bis(2-ethylhexyl) phthalate +2AA and trans-anethole +2AA) with TA98 strain as compared with TA1537, which seems to have enhanced the mutagenic response of 2AA in metabolically activated conditions. It is concluded that out of both tester strains TA98 strain of Salmonella typhimurium has the potential to detect non-genotoxic carcinogens when combined with potent promutgens either by inhibiting or modulating activities of liver microsomal enzymes biochemically which may indirectly contribute to neoplastic alterations. Further this simple, short-term alternative assay may provide rapid information during extrapolative toxicology for differentiating genotoxic and non-genotoxic carcinogens.

The micro-Ames test: A direct comparison of the performance and sensitivities of the standard and 24-well plate versions of the bacterial mutation test.

"Ames" bacterial mutation tests are widely performed for evaluation and registration of new materials including industrial chemicals, agrochemicals, medical devices, pharmaceuticals, pharmaceutical impurities and other materials. Tests are used to predict their potential long-term adverse health effects (including carcinogenicity). Given their importance, pre-screening 'miniaturized' versions have been developed which allow higher throughput and use less test material, including the widely-employed 24-well micro-Ames (µAmes) test which uses 20 times less material. However, little quantitative information has been published on the methodology or sensitivity of this system. We describe methods and results used in direct comparisons of the sensitivity of micro and standard systems using the same cultures, formulations, etc. Initial testing utilized the plate incorporation method and, later, the pre-incubation method. In a subsequent phase of testing, a four-way direct comparison was made between the pre-incubation and plate incorporation methods in both systems using some direct-acting mutagens. Tests used only those strain/S9/chemical combinations where a response was expected. Historical control results accumulated during testing are also presented. Spontaneous and induced revertant colony counts for the µAmes system were consistently proportionate and approximately 1/20th those for the standard Ames test. Sensitivities of the two systems were found to be nearly identical in almost all cases for a wide variety of weak and strong inorganic and organic mutagens. Standardized procedures and increased reliability of the estimate of the background revertant frequency in the µAmes system means that the two systems give equivalent results and are expected to be highly predictive of one another. Environ. Mol. Mutagen. 57:687-705, 2016. © 2016 Wiley Periodicals, Inc.

Safety of micronized palmitoylethanolamide (microPEA): lack of toxicity and genotoxic potential.

Palmitoylethanolamide (PEA) is a natural fatty acid amide found in a variety of foods, which was initially identified in egg yolk. MicroPEA of defined particle size (0.5–10 μm) was evaluated for mutagenicity in Salmonella typhimurium, for clastogenicity/aneuploidy in cultured human lymphocytes, and for acute and subchronic rodent toxicity in the rat, following standard OECD test protocols, in accordance with Good Laboratory Practice (GLP). PEA did not induce mutations in the bacterial assay using strains TA1535, TA97a, TA98, TA100, and TA102, with or without metabolic activation, in either the plate incorporation or liquid preincubation methods. Similarly, PEA did not induce genotoxic effects in human cells treated for 3 or 24 h without metabolic activation, or for 3 h with metabolic activation. PEA was found to have an LD50 greater than the limit dose of 2000 mg/kg body weight (bw), using the OECD Acute Oral Up and Down Procedure. Doses for the 90‐day rat oral toxicity study were based on results from the preliminary 14‐day study, that is, 250, 500, and 1000 mg/kg bw/day. The No Effect Level (NOEL) in both subchronic studies was the highest dose tested.

Bacterial mutagenicity assays: Vehicle and positive control results from the standard Ames assay, the 6- and 24-well miniaturized plate incorporation assays and the Ames II™ assay.

Bacterial mutation assays are conducted routinely as part of the safety assessment of new chemicals. The OECD Test Guideline (TG) 471 describes the conduct of the standard agar plate Ames assay, required for regulatory submissions. Higher throughput non-OECD 471 TG assays, such as the miniaturized plate incorporation and Ames II™ assays, can be used for prescreening purposes. We have compiled historical vehicle and positive control data generated using these methods. The historical database is comprised from experiments spanning 9 years and includes >1000 experiments from the standard Ames assay using the plate incorporation and pre-incubation methods (TA98, TA100, TA1535, TA1537, and WP2 uvrA), >50 experiments from the 6-well (TA98, TA100, TA1535, TA97a, and WP2 uvrA) and >100 experiments from the 24-well (TA98, TA100, TA102, TA1535, TA1537, and TA97a) plate incorporation assays, and >1000 experiments from the Ames II™ assay (TA98 and TAMix). Although miniaturization to a 24-well format made the measurement of control revertant colonies in TA1537 and TA1535 more difficult; this can be overcome by using an alternative strain with a higher spontaneous reversion rate (i.e., using TA97a instead of TA1537) or by increasing the number of replicate wells to 12 (for TA1535). All three miniaturized methods, including the Ames II™ assay, were responsive to known mutagens and the responses were reproducible over years of use. These data demonstrate the excellent reproducibility of the standard and miniaturized bacterial mutation assays using positive control chemicals. Environ. Mol. Mutagen. 57:483-496, 2016. © 2016 Wiley Periodicals, Inc.

Evaluation of Mutagenic ability of Volatile oils from Mentha longifolia in the Fungus Aspergillus amstelodami

This research involved testing the mutagenic ability of the volatile oil of Mentha longifolia on the conidia of fungus Aspergillus amstelodami .the inhibitory effect of different concentrations of volatile oil of Mentha were investigated so the percentage of inhibition were 20.89%-65.5% of concentrations(0.008-0.1) ml/ml. The mutagenicity of four sublethal concentrations (0.03 , 0.04 , 0.05 and 0.06 ml/ml) were tested by using three methods namly pretreatment, growth mediated and plate incorporation method, Non of the concentrations tested of volatile oil of M. longifolia were showed any mutagenic effect on conidia of fungus A. amstelodami ,so we can consider the volatile oil of M. longifolia is genetically safe .

International round‐robin study on the Ames fluctuation test

An international round-robin study on the Ames fluctuation test [ISO 11350, 2012], a microplate version of the classic plate-incorporation method for the detection of mutagenicity in water, wastewater and chemicals was performed by 18 laboratories from seven countries. Such a round-robin study is a precondition for both the finalization of the ISO standardization process and a possible regulatory implementation in water legislation. The laboratories tested four water samples (spiked/nonspiked) and two chemical mixtures with and without supplementation of a S9-mix. Validity criteria (acceptable spontaneous and positive control-induced mutation counts) were fulfilled by 92-100%, depending on the test conditions. A two-step method for statistical evaluation of the test results is proposed and assessed in terms of specificity and sensitivity. The data were first subjected to powerful analysis of variance (ANOVA) after an arcsine-square-root transformation to detect significant differences between the test samples and the negative control (NC). A threshold (TH) value based on a pooled NC was then calculated to exclude false positive test results. Statistically, positive effects observed by the William's test were considered negative, if the mean of all replicates of a sample did not exceed the calculated TH. By making use of this approach, the overall test sensitivity was 100%, and the test specificity ranged from 80 to 100%.

Mutagenicity, antimutagenicity and cytotoxicity evaluation of South African Podocarpus species

Ethnopharmacological relevanceFour species of Podocarpus are used in traditional medicine both in human and animal healthcare in South Africa. In vitro pharmacological screening of leaf and stem extracts of these species exhibited potent antimicrobial, anti-inflammatory, anti-tyrosinase, anthelmintic, acetylcholinesterase inhibitory and antioxidant activities. Aim of the studyTo investigate the mutagenicity, antimutagenicity and cytotoxicity effects of leaf and stem extract of South African Podocarpus species. Material and methodsThe mutagenicity and cytotoxic effects of extracts from four species of Podocarpus were tested using the Salmonella/microsome assay with and without metabolic activation, based on the plate-incorporation method and neutral red uptake (NRU) assay respectively. Five Salmonella typhimurium tester strains; TA98, TA100, TA102, TA1535 and TA1537 were used for mutagenicity testing. The relative cytotoxicity of the extracts was assessed by determining their NI50 values (50% inhibition of NRU). ResultsThe extracts did not show any mutagenic effects against all the tester strains with or without metabolic activation. All extracts demonstrated a strong antimutagenic effect on the mutations induced by 4NQO, decreasing its mutagenic effect in a dose-dependent manner. Strong cytotoxic effects were exhibited by petroleum ether extracts as compared to 80% ethanol extracts. When HepG2 cells were in contact with plant extracts in an increasing concentration, slopes of NRU decreased (highest–lowest %) following a concentration-dependent pattern. For 80% ethanol extracts, the most toxic extract in terms of percentage viability was leaves of Podocarpus falcatus whereby at 0.2mg/ml, the viability of the cells was 38.9%. Stem extract of Podocarpus latifolius was the most toxic among PE extracts, giving a percentage viability of 46.4 at 0.1mg/ml. ConclusionAbsence of mutagenicity does not indicate lack of toxicity, as was observed from these extracts. These findings will help in assessing the safety measures to be considered in the use of these species and also the need to determine the cytotoxic potential of these species against various forms of human cancer cells.

Scientific Opinion on Flavouring Group Evaluation 86, Revision 1 (FGE.86Rev1): Consideration of aliphatic and aromatic amines and amides evaluated by JECFA (65th meeting)

Scientific Opinion on Flavouring Group Evaluation 86, Revision 1 (FGE.86Rev1): Consideration of aliphatic and aromatic amines and amides evaluated by JECFA (65th meeting)

Mutagenicity and anti-mutagenicity of Acanthopanax divaricatus var. albeofructus

The beneficial effects of Acanthopanax divaricatus var. albeofructus (ADA) extracts have been assessed by mutagenic and anti-mutagenic activities by Ames test. Mutation of Salmonella typhimurium strains TA 98, TA 100, TA1535, TA1537, and Escherichia coli WP2 uvr A was assayed in duplicates by the procedure of Maron and Ames in the presence or absence of S9 mix. As a result, ADA extracts were not mutagenic for S. typhimurium strains TA 98, TA 100, TA1535, TA1537, and E. coli by the Ames assay. Anti-mutagenic activity was assayed by the Ames mutagenicity assay using histidine mutant of S. typhimurium strains TA 98 and TA 100, using the plate-incorporation method. 2-Aminoanthrancene (2-AA), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2), and sodium azide (NaN(3)) were used as the mutagens. ADA extracts showed a strong anti-mutagenic activity against 2-AA-induced mutagenesis which requires liver-metabolizing enzymes, and the same extract exhibited inhibitory effects on AF-2 and NaN(3)-induced mutagenesis in the absence of liver-metabolizing enzymes. The data indicate that ADA extracts contain anti-mutagenic activities against typical mutagens. The anti-mutagenic property of ADA provides additional health supplemental value to the other claimed therapeutic properties of the plant.

Evaluation of the genotoxicity of Orthosiphon stamineus aqueous extract

Orthosiphon stamineus, Benth, also known as Misai Kucing in Malaysia and Java tea in Indonesia, is traditionally used in Southeastern Asia to treat kidney dysfunctions, diabetes, gout and several other illnesses. Recent studies of Orthosiphon stamineus pharmacological profile have revealed antioxidant properties and other potentially useful biological activities thereby lending some scientific support to its use in folk medicine. So far the genotoxicity of Orthosiphon stamineus extracts has not been evaluated. In this study the genotoxic potential of Orthosiphon stamineus aqueous extract was investigated by the Salmonella/microsome mutation assay and the mouse bone marrow micronucleus test. Chemical composition of Orthosiphon stamineus aqueous extract was analyzed by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD). The Salmonella/microsome assay (TA97a, TA98, TA100 and TA1535; plate incorporation method) was performed in the presence or in the absence of extrinsic metabolic activation (S9 mixture). In the mouse micronucleus assay, Orthosiphon stamineus extract was administered by gavage (0, 500, 2000 and 4000 mg/kg body weight/day for 3 days) to male and female Swiss Webster mice (N=6 per dose per sex) and bone marrow cells were harvested 24 h after the last dose. Ethoxy-resorufin-O-dealkylase (EROD) and benzyloxy-resorufin-O-dealkylase (BROD) activities were determined in mouse liver microsomes. The chemical analysis revealed that the Orthosiphon stamineus extract contained flavonoids (sinensetin, eupatorin), caffeic acid, and rosmarinic acid (44.00±1.879 μg/mg), the latter seemed to be one of its major constituent. Tested at doses up to 5000 μg/plate, the Orthosiphon stamineus extract was not toxic to Salmonella tester strains and did not increase the number of revertant colonies over the background incidence. In the mouse bone marrow assay, the extract did not alter the polychromatic:normochromatic erythrocytes (PCE:NCE) ratio, nor did it increase the incidence of micronucleated polychromatic erythrocytes (MNPEs). No overt toxicity and no change of CYP1A (EROD) and 2B9/10 (BROD) activities were noted. Based on the aforementioned findings, it is concluded that the use of Orthosiphon stamineus in the traditional medicine poses no genotoxic risk.

Micronucleus Test in Bone Marrow Cells and Bacterial Reverse Mutation Assay of HMC05

Objectives：We investigated genetic toxicity of HMCO5 using the Micronucleus Test in bone marrow cells of mice and Bacterial Reverse Mutation Assay in plate incorporation method according to OECD Guidelines and KFDA Guidelines. Methods： 1. Micronucleus test: The male rats were divided into 5 groups, respectively; G(1), treated with distilled water: G(2), treated with 1250mg/kg HMC0 5: G(3), treated with 2500mg/kg HMC05, G(4), treated with 5,000mg/kg HMC05; G(5), treated with Cyclophosphamide·H2O. Sterilized distilled water and HMC05 were administered for two consecutive days. Cyclophosphamide·H2O was administered once on the day of 2nd administration. 2. Bacterial Reverse Mutation Aassay: Experimental groups were divided into two groups: with S-9mix(+S) or without S-9mix(-S). Each group treated with sterilized distilled water only, HMCO5(62, 185, 556, 1,667, 5,000㎍/plate) and, positive vehicles(Sodium azide, 2-Aminoanthracene, 4-Nitroquinoline N-oxide, ICR 191), respectively. Results：HMC05 did not show any changes in the number of micronucleated polychromatic erythrocytes(MNPCE) among 200 polychromatic erythrocytes compare to negative control. However, there were significant (p<0.01) increase with CPA in MNPCE. In Bacterial Reverse Mutation Aassay, no significant increases in the number of revertant colonies compared to (삭제) negative control were detected in all concentrations of HMC05. Conclusions：These results indicate that HMC05 did not show any genotoxicity against in Micronucleus test and Bacterial Reverse Mutation Aassay.