Absence of mutagenic activity of benorylate, paracetamol and aspirin in the Salmonella/mammalian microsome test

Benorylate and its two major hydrolysis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Absence of mutagenic and antimutagenic activities of fluoride in Ames salmonella assays

The mutagenicity of fluoride (as sodium fluoride, NaF) was investigated with Ames Salmonella/microsome assays in strains of TA97a, TA98, TA100, TA102 and TA1535. The concentrations of NaF tested ranged from 0.44 to 4421 micrograms/plate (0.1 to 1000 ppm F), both with and without microsome activation. In addition, the suggested antimutagenic effect of fluoride was evaluated with known mutagens at various concentrations of NaF (0.44-442.2 micrograms/plate, 0.1-100 ppm F). The data showed that NaF, in amounts from 0.44 to 442.2 micrograms/plate (0.1-100 ppm F), failed to significantly increase the number of the revertants over the number observed in the solvent (distilled deionized water) controls. Increases of NaF to, and beyond, 1100 micrograms/plate (250 ppm F) resulted in a toxic effect and a reduction of the revertants to various degrees among the strains. NaF in the presence of known mutagens did not significantly decrease the number of the revertants. The results of this study indicate that NaF does not have mutagenic or antimutagenic effects in the strains tested with Ames Salmonella assays.     

Lack of mutagenicity of some phytoestrogens in the salmonella/mammalian microsome assay

8 phytoestrogens were tested for mutagenicity using a variation of the Salmonella/mammalian microsome (or Ames) assay. Zearalenone is a mycotoxin produced by a grain contaminant, Fusarium graminearum (Gibberella zeae) and the isomers of zearalanol are reduced derivatives of this compound. The remaining compounds are all flavonoids which occur naturally at relatively high concentrations in many plants, particularly legumes. 4 of these flavonoids (daidzein, genistein, formononetin and biochanin-a) are isoflavones and the 5th, coumestrol, is a coumestan. Each compound was tested at several concentrations ranging from 1--500 micrograms per plate. The microsomal fracton was obtained from Aroclor 1254 (a PCB)-induced rat livers. None of the compounds tested was mutagenic to Salmonella strains TA1538, TA98 or TA100 at any concentration.     

Mutagenicity assessment of Salacia chinensis by bacterial reverse mutation assay using histidine dependent Salmonella typhimurium tester strains

Background and objectiveGenotoxicity analysis is one of the most important non-clinical environmental safety investigations required for pharmaceutical and agrochemical product registration. Any medicinal product must undergo a risk evaluation to determine its mutagenicity and carcinogenicity.Materials and methodsThe Ames test is a commonly used in vitro test for determining a test chemical's mutagenic activity. Histidine-dependent Salmonella typhimurium strains with a defective gene that causes the bacteria to synthesis the necessary amino acid histidine for life were tested for mutagenic potential. In order to reveal pro-mutagens and mutagens, the mutagenic potential of both plate integration and pre-incubation techniques was examined in the presence and absence of metabolizing system. Salacia chinensis has been widely used in ayurveda to treat various ailments. However, the information of mutagenicity of Salacia chinensis is scarce as per available literature.ResultsThe mutagenicity of a Salacia chinensis root extract was investigated utilizing the Ames assay with plate incorporation and pre-incubation protocols using the appropriate Salmonella typhimurium tester strains: TA98, TA100, TA1537, TA1535, and TA102 in the presence and absence of S9. The concentrations used were 0.3123, 0.625, 1.25, 2.5 and 5 mg/plate. The extract of Salacia chinensis root did not show any mutagenic effect in any of the Salmonella typhimurium strains at the concentrations tested in the absence or presence of metabolic activation.ConclusionThe root of Salacia chinensis was hence confirmed to be non-mutagenic and at least according to the results of this genotoxicity evaluation can be regarded as being safe for human use.     

Evaluation of diallate and triallate herbicides for genotoxic effects in a battery of in vitro and short-term in vivo tests

Commercial-grade preparations of two thiocarbamate herbicides, diallate and triallate, were evaluated for their mutagenic potential in a battery of short-term bioassays. All in vitro bioassays were performed with and without mammalian metabolic activation, and all such tests were repeated after an interval of at least 1 week. Diallate and triallate were tested in the Salmonella/microsome assay over dose ranges of 0.59 to 118.0 micrograms/plate and 6.37 to 1273 micrograms/plate, respectively. Both diallate and triallate gave positive results in S. typhimurium strains TA1535, TA98, and TA100 only in the presence of a rat-liver metabolic activation system. In Saccharomyces cerevisiae strain D7, diallate was tested at concentrations from 1.18 to 29.50 micrograms/ml, and triallate was tested at 0.955 to 9.548 micrograms/ml. Both diallate and triallate gave negative results for mitotic gene conversion, mitotic crossing-over, and reverse mutation. In the mouse lymphoma L5178Y TK+/- assay, diallate was tested at concentrations ranging from 1 to 72 micrograms/ml, and triallate was tested at 0.5 to 60 micrograms/ml. Both herbicides produced mutagenic responses in the mouse lymphoma assay in the presence of metabolic activation. In the Drosophila sex-linked recessive lethal test, flies were exposed to 0.0004% diallate and 0.001% triallate. In this assay, diallate was considered mutagenic, whereas triallate did not produce a detectable mutagenic response.     

Salmonella/mammalian microsome assay with tetranitromethane and 3-nitro-L-tyrosine

The nitrosating agent tetranitromethane (TNM) and the nitrosation product 3-nitro-L-tyrosine (NT) were tested for mutagenic activity in the Salmonella/mammalian microsome assay. TNM showed strong genotoxic activity: it was mutagenic in all tester strains used (TA97, TA98, TA100, and TA102). The maximum mutagenic activity was reached between 16 and 32 micrograms/plate using the standard plate test; higher amounts led to distinct bactericidal effects. The mutagenicity was independent of an in vitro activation system. In the preincubation assay an increased bactericidal effect was observed. In contrast to TNM, NT, the nitrosation product, was non-mutagenic and non-toxic in the standard plate test and with the preincubation method up to 5000 micrograms/plate with and without S9 mix and with all tester strains used. Although TNM is a strong direct-acting mutagen, its nitrosating effect on proteins does lead to nongenotoxic nitro products of tyrosine in proteins.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

Mutagenic effect of a tetrahydrodiazopyrene derivative on bacteria]

Mutagenic action of 3,7-diamino-4,9-dioxy-5,10-dioxo-4,5,9,10-tetrahydro-4,9-diazapiren (DDDTDP) was shown using indicator strains Salmonella typhimurium TA 1534, TA 1536, TA 1537, TA 1538. The drug-induced mutations in strains TA 1534 and TA 1538, and it can be used as a positive control in testing mutagens capable of inducing frameshift mutations. No significant differences was observed between DDDTDP effects on strains TA 1534 and TA 1538 which did or did not bear rfa mutation causing defects of cell wall lypopolysacharide complex. Within the range of concentrations tested DDDTDP had mutagenic effect without causing essential killing of bacteria. The mutagenic effect was decreased in the in vitro system of metabolic activation (Ames' plate test in Salmonella microsomes).     

Evaluation of the mutagenic potential of endosulfan using the Salmonella/mammalian microsome assay

The mutagenic potential of endosulfan, a polychlorinated insecticide, was assessed using the highly sensitive Salmonella tester strains TA97(a), TA98, TA100 and TA102. It exhibited a toxic effect at dose levels of 50 micrograms/plate and higher. Plate incorporation studies did not show mutagenic response with any of the tester strains used. A modification of the assay using a preincubation procedure showed mutagenic activity with and without metabolic activation with TA97(a) only. Increased toxicity was observed after addition of phenobarbital-induced S9 mix.     

Mutagenicity of the coccidiostat diaveridine in the Salmonella/mammalian microsome assay

The coccidiostat diaveridine was tested for mutagenicity in the Salmonella/microsome assay with tester strains TA100 and TA98. This compound was not mutagenic in either tester strain in the presence and absence of rat S9 mix, but was found to be mutagenic in strain TA100 after metabolic activation with hamster S9 mix.     

Mutagenic activity of airborne particulate matter as an indicative measure of atmospheric pollution

Mutagenic activity of organic extracts of airborne particulate matter at four different sites within the urban area of the city of Porto Alegre, Brazil, was investigated using the Salmonella/microsome assay, with the Kado microsuspension method. The extracts were obtained by sonication, sequentially extracted according to polarity, with cyclohexane (CX) and dichloromethane (DCM) solvents. The different fractions were tested for mutagenicity with the Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6, without S9 mix metabolic activation. A positive frameshift mutagenic response was observed for non-polar (CX) and/or moderately polar (DCM) compounds at the different sites. The responses varied at different seasons of the year, and the highest revertants per m3 (rev/m3) values were observed at the site subject to the strongest influence of automotive vehicles (site 3) in spring (17.13 rev/m3) in DCM fractions, and in summer (13.01 rev/m3) in CX fractions. The responses observed for the TA98NR and TA98/1,8-DNP6 strains suggest the contribution of nitrocompounds to the mutagenic activity observed. Although there appears to be an indicative association between the increased mass per unit volume of air (TSP) and the mutagenicity of organic extracts of airborne particulate matter in the present study, the Salmonella/microsome assay was a sensitive method to define areas contaminated by genotoxic compounds, even in samples that present TPS values acceptable by the environmental quality standards established by law.     

Mutagenic potential of toluidine blue evaluated in the Ames test.

Toluidine blue is a vital, metachromatic thiazine dye which is used as an adjunct in clinical examination for the early detection of asymptomatic recurrent or secondary primary carcinoma in individuals who are at high risk for developing oral cancer. Because available data on the mutagenicity of toluidine blue was limited and contradictory, this study was conducted to evaluate the mutagenic potential of toluidine blue in the in vitro Ames Salmonella test. Tester strains TA97a, TA98, TA100 and TA102 were used. Toluidine blue was tested at concentrations of 0.1, 1.0, 10, 50, 100, 250 and 500 micrograms/plate, with and without S9 microsomal activation, and positive and negative controls were included. Results from tests without S9 showed a significant increase (p less than 0.05) in number of revertants in TA102 and in TA97a with 50 and 100 micrograms toluidine blue/plate, respectively. In tests with S9 activation, doses of toluidine blue ranging from 10 to 250 micrograms/plate induced dose-related increases in the number of revertants in all 4 strains. The results of this study indicate that toluidine blue has a mutagenic effect in the Ames test.     

Evaluation of 1,3-pentadiene for mutagenicity by the Salmonella/mammalian microsome assay

1,3-Pentadiene, a food contaminant produced by some molds when they metabolize sorbic acid, was tested for mutagenicity, using variations of the Salmonella/mammalian microsome assay. The chemical was incorporated into the test system (with and without S9 mix) by 3 methods: (a) the standard plate incorporation assay, (b) a liquid preincubation procedure and (c) exposure of test bacteria in the soft agar overlay to gaseous 1,3-pentadiene. The chemical was extremely toxic to the test bacteria with amounts as low as 2.0 microgram/plate causing cell death. However, none of the nonlethal concentrations tested by any of the methods was mutagenic to Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 or TA1538.     

Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN)

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.     

Mutagenicity of anthraquinone and hydroxylated anthraquinones in the Ames/Salmonella microsome system.

The mutagenicity of anthracene, anthraquinone, and four structurally similar compounds of each was evaluated in the Ames/Salmonella microsome assay. Anthraquinone was shown to be mutagenic for strains TA1537, TA1538, and TA98 in the absence of rat liver homogenate. The four anthraquinone derivatives tested were mutagenic for TA1537 exclusively. None of the anthracenes exhibited mutagenic activity.     

Butylated hydroxyanisole, butylated hydroxytoluene and tert.-butylhydroquinone are not mutagenic in the Salmonella/microsome assay using new tester strains

The phenolic antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert.-butylhydroquinone (TBHQ) were reassessed for mutagenic activity using the recently developed Salmonella tester strains TA97, TA102 and TA104, and in addition TA100. None of the phenolic antioxidants showed mutagenic activity, either with or without metabolic activation. At doses of 100 micrograms/plate and higher all 3 phenolic antioxidants exhibited toxic effects. A modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Combinations of BHA and BHT, tested to detect possible synergistic effects, did not exert mutagenic activity.     

Genotoxicity of 6 oxime compounds in the Salmonella/mammalian-microsome assay and mouse lymphoma TK+/− assay

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 6 oxime compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK^+/− assay. All of the oximes were mutagenic in the mouse lymphoma assay in the absence of exogenous metabolic activation. Acetaldehyde oxime was also mutagenic in the presence of S9 activation. In contrast to these results, a positive response was noted only for 2-(hydroxyimino)-N-phenyl-acetamide oxime in strain TA1535 in the absence of activation in the Salmonella/microsome test.     

Aspects of chloroquine mutagenicity

Using the Ames plate reversion and fluctuation tests, the mutagenic activity of chloroquine was tested in the new tester strains of Salmonella typhimurium, TA97, TA102, and Escherichia coli strains WP2, WP2hcr, WP6 and WP67. The E. coli transconjugants obtained from the mating transfer of R-plasmid(s) in strains TA97 and TA102 respectively to E. coli WP2, i.e. EE97 and EE102, were also tested. Chloroquine reverted strain TA97 from histidine dependence to independence and also reverted E. coli strains EE97 and EE102 from tryptophan dependence to independence. The E. coli strains WP2, WP2hcr; WP6 and WP67 and S. typhimurium TA102 were not affected. S. typhimurium TA97 could be reverted with 250 ng/ml of chloroquine (therapeutic blood level of chloroquine is 300 ng/ml). Reversion generally occurred optimally at the relatively lower concentrations of chloroquine i.e. 25, 50 micrograms/ml than at higher concentrations. From the properties of the reverted tester strains, the results indicated that chloroquine per se mediated frameshift reversion.     

Validation of the Salmonella (SV50)/arabinose-resistant forward mutation assay system with 26 compounds

Mutagenic sensitivity of the Salmonella/arabinose-resistant (Ara^r) assay system using the tester strain SV50 was evaluated with 26 compounds both by the preincubation and the standard plate incorporation tests. The mutagenic activity of all 26 compounds was also tested with TA98 and/or TA100 of the Ames Salmonella/microsome assay system. The results indicate that 13 and 10 of 26 compounds were mutagenic and nonmutagenic, respectively, in both assay systems. PR toxin and hydrogen peroxide were mutagenic only in the Ara^r assay, while 2-nitrofluorene was mutagenic only in the Ames assay. The results also show that the mutagenic response of SV50 to 13 of 15 mutagenic compounds was much higher (2.1–154-fold) if the compounds were tested with the preincubation rather than the plate incorporation test. The mutagenic activity of 4 compounds (diethyl sulfate, niridazole, PR toxin and hydrogen peroxide) in the Ara^r assay was detected only with the preincubation test.Since the Ara^r assay using tester strain SV50 has similar mutagenic sensitivity as the Ames assay to chemicals with different modes of action and since it requires only one tester strain, we find this assay system to be useful for screening environmental mutagens. Based on the effectiveness of the preincubation test in this study, it is recommended that the preincubation test instead of the plate incorporation test be used for the Ara^r assay system with tester strain SV50.     

Mutagenicity testing of certified food colors and related azo, xanthene and triphenylmethane dyes with the Salmonella/microsome system.

Thirty-seven azo, xanthene and triphenylmethane dyes including FD and C colors currently approved for use in the U.S.A. and a number of delisted food colors, were tested in the Salmonella/microsome system. In addition to direct plate tests with five tester strains (TA1535, TA100, TA1537, TA1538, TA98), the azo dyes were also assayed after chemical reduction to their component amines. Also, a selected group of azo dyes was subjected to liquid tests (both aerobic with microsomes and anaerobic) and to plate tests involving initial 16 h anaerobic incubations to facilitate microbial reduction of the azo bond. None of the presently listed FD and C colors was mutagenic in any of the test modifications. Among formerly listed colors only Butter Yellow (p-dimethylaminoazobenzene), a recognized animal carcinogen, was mutagenic in the aerobic liquid test. Several other azo dyes were either directly mutagenic, viz. Acid Alizarin Yellow R and Alizarin Yellow GG; required microsomal activation, viz. Acid Alizarin Red B and Methyl Red; or required chemical reduction and microsomal activation, viz. Acid Alizarin Violet N and Sudan IV. Of the non-azo dyes tested only two xanthene dyes appeared to be mutagenic, viz. 9-(2-sulfophenyl)-6-hydroxy-3-isoxanthenone and its 2,4,5,7-tetrabromo derivative.