Mutagenicity monitoring of airborne particulate matter (PM10) in the Czech Republic.

The mutagenic activities associated with inhalable airborne particulate matter (PM10) collected over a year in four towns (Czech Republic) have been determined. The dichloromethane extracts were tested for mutagenicity using the Ames plate incorporation test and the Kado microsuspension test both with Salmonella typhimurium TA98 and its derivative YG1041 tester strains in the presence and absence of S9 mixture. The aim of this study was to assess the suitability of both bacterial mutagenicity tests and to choose the appropriate indicator strain for monitoring purposes. To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), the concentration of PAHs in the air samples were determined by GC/MS. In general, the significant mutagenicity was obtained in organic extracts of all samples, but differences according to the method and tester strain used were observed. In both mutagenicity tests, the extractable organic mass (EOM) exhibited higher mutagenicity in the YG1041 strain (up to 97 rev/microg in the plate incorporation and 568 rev/microg in the microsuspension tests) than those in TA98 (up to 2.2 rev/microg in the plate incorporation and 14.5 rev/microg in the microsuspension tests). In the plate incorporation test, the direct mutagenic activity in YG1041 was on average 60-fold higher and in microsuspension assay 45-fold higher with respect to strain TA98. In the presence of S9 mix, the mutagenic potency in YG1041 declined (P<0.001) in summer, but increased in TA98 (P<0.05) in samples collected during the winter season. The microsuspension assay provided higher mutagenic responses in both tester strains, but in both strains a significant decrease of mutagenic potency was observed in the presence of S9 mix (P<0.001 for YG1041, P<0.05 for TA98 in winter). The mutagenic potencies detected with both indicator strains correlated well (r=0.54 to 0.87) within each mutagenicity test used but not (for TA98) or moderately (r=0.44 to 0. 66 for YG1041) between both of the tests. The mutagenic activity (in rev/m(3)) likewise the concentration of benzo[a]pyrene and sum of carcinogenic PAHs showed seasonal variation with distinctly higher values during winter season. A correlation between the PAH concentrations and the mutagenicity results for the plate incorporation, but not for the microsuspension tests was found. In samples from higher industrial areas, the higher mutagenicity values were obtained in plate incorporation test with TA98 and in both tests with YG1041 in summer season (P<0.05). According to our results, plate incorporation test seems to be more informative than microsuspension assay. For routine ambient air mutagenicity monitoring, the use of YG1041 tester strain without metabolic activation and the plate incorporation test are to be recommended.     

Mutagenic activities of ten imidazole derivatives in <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

Ten imidazole derivatives were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 both in the absence and presence of metabolic activation by the microsomal fraction S9 mix. In a general manner, derivatives tested exhibited a greater mutagenic activity in the TA100 strain comparing to the responses in TA 98. In the standard plate incorporation assay, 8 of these substances (80%) were found to be mutagenic for at least one of the two strains in the presence or absence of metabolic activation. Two compounds showed positive results in TA98 and 6 compounds were also mutagenic in TA100 without S9. In the presence of S9 mix, all of the 10 substances were non-mutagenic in TA98, whereas 4 compounds were positive in TA100. The results suggested the mutagenic potentials of the imidazole derivatives particularly inducing the reversion of base-pair substitutions. According to the structure-activity relationships phenyl groups in position 2 with different substituents can confer the mutagenic activity of the tested compounds. Methyl groups in different positions of these phenyl substituents can cause different types of mutations. This mutagenic effect is observed more clearly when the phenyl group is inhibited with a nitro group.     

Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

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.     

Bacterial mutagenicity of aceanthrylene: a novel cyclopenta-fused polycyclic aromatic hydrocarbon of low molecular weight

Aceanthrylene, a non-alternant cyclopenta-fused hydrocarbon, was shown to be weakly mutagenic without S9 and strongly mutagenic with S9 in the Ames Salmonella plate incorporation assay. The compound was most active in strain TA100 (35 revertants/nmole in the presence of 0.3 mg of S9 protein), and less active in strains TA98, TA1537 and TA1538 (20, 10 and 3.1 rev/nmole respectively, + S9). Strain TA1535 was unresponsive, suggesting that this compound induces frameshift mutations rather than base-pair substitutions. The mutagenic potency of aceanthrylene is consistent with predictions of its activity based on the relatively large delocalization energy (delta E deloc/beta = 0.931) of the carbonium ion which would result from oxirane ring opening of the 1,2-epoxide, a potential active metabolite.     

Mutagenicity, comutagenicity, and antimutagenicity of erythrosine (FD and C red 3), a food dye, in the Ames/Salmonella assay

Erythrosine (diNa, tetraiodofluorescein) was nonmutagenic to the Ames/Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA104, to a concentration of 2 mg/plate. No mutative intermediates were detected on metabolism by rat caecal cell-free extracts or rat liver S9 mixture; or on incubation with the comutagens, harman and norharman (+/- S9). Instead, an unexpected dose-dependent suppression in spontaneous reversion frequencies was observed (maximum approximately equal to 35% decrease). Erythrosine was antimutagenic to benzo[a]pyrene, but it did not decrease the mutagenicity of the other adduct-forming mutagen, 4-nitroquinoline N-oxide. The food dye was strongly antimutagenic to the bifunctional alkylating agent, mitomycin C, though it did not exhibit a similar effect on the mutagenicity of the corresponding monofunctional agent, methyl methanesulphonate. It partially depressed the mutagenic potentials of sodium azide. The antimutagenic effect of erythrosine on an intercalating agent, ethidium bromide, was discernible only at the highest dose (2 mg/plate). These results have been interpreted in terms of a genointeractive role of erythrosine. Erythrosine produced differential toxic effects in repair-deficient (TA97a, TA98, TA100) and repair-proficient (TA102, TA104) Salmonella tester strains; survival of the repair-deficient strains was found to be decreased. Photoinduced potentiation of erythrosine toxicity was observed, although light irradiation in the presence of erythrosine did not modify the reversion frequencies of the tester strains. The evidence strongly suggests that erythrosine, which exhibits nonmutagenicity in the Ames/Salmonella test, can interact with DNA repair enzymes and/or with DNA.     

Genetic toxicology evaluation of commercial beers. II. Mutagenic activity of commercial beer products in Salmonella typhimurium strains TA98, TA100 and TA102

5 concentrated extracts of commercial beers were prepared using XAD-2 resin. The residues were subjected to evaluation for mutagenic activity in Salmonella typhimurium strains TA98, TA100 and TA102. The tests were conducted using preincubation protocols including provisions for S9 metabolic activation. Although the extracts did produce moderate toxicity to the Salmonella organisms used in the assays, none of the residues were found to induce mutation up to their maximum testable concentrations.     

Structural activity relationship between Salmonella-mutagenicity and nitro-orientation of nitroazaphenanthrenes

Nitroazaphenanthrenes (NAphs) and their N-oxides (NAphOs) were synthesized as derivatives with nitrogen atoms in the 1, 4, and 9 positions of phenanthrene rings, and as nitrated derivatives substituted at the 1, 2, 3, 4, 5, 6, 7, and 8 positions of phenanthrene rings. To determine the structure activity relationship of these derivatives, all 19 isomers were bioassayed with Salmonella tester strains. NAphs substituted at the 4, 6, 7 and 8 positions were mutagenic for TA98, and 1-, 2-, and 3-N-9-AphOs, 6-N-1-AphO and 6-N-4-AphO were mutagenic for TA98 and TA100 without the S9 mix, while 5-N-1-AphO and 5-N-9-AphO were non- or weakly mutagenic. Nitrated derivatives, 6-N-4-Aph, 6-N-9-Aph, 6-N-1-AphO, and 6-N-4-AphO, were powerful mutagens for TA98 and TA100. Mutagenicity was enhanced by mutant strains producing nitroreductase, such as YG1021 and 1026, and by those producing O-acetyltransferase, such as YG1024 and 1029. Nitro derivatives substituted at positions 4 and 5 in the phenanthrene rings were perpendicular, while those at positions 2, 3, 6 and 7 were coplanar to the phenanthrene rings. NAphs substituted at the 1 and 8 positions were noncoplanar due to steric hindrance of the aromatic proton at the peri position. On the other hand, 1,5- and 1,8-dinitro-4-azaphenanthrenes showed high mutagenicity for strains TA98 and TA100 in the absence of the S9 mix, and were strongly enhanced by nitroreductase and O-acetyltransferase, over-producing mutants. Therefore, it was found that the mutagenic potency of NAphs and NAphOs was closely associated with the chemical properties and orientation of nitro substitution of aromatic rings.     

Characterization of organic extracts from standard reference materials 1649, ‘urban dust/organics,’ and 1650, ‘diesel particulate matter’, using a microsuspension assay. A WHO/IPCS/CSCM study

Mutagenicity associated with replicate organic extracts from standard reference materials 1649 ‘urban dust/organics’ (air particles), and 1650, ‘diesel particulate matter’ (diesel particles), was determined using a Salmonella microsuspension assay. The results indicate that the mutagenicity of samples such as these can readily be determined using the microsuspension assay with only 5% of the mass required for the standard plate incorporation asssay.In general, 80% of the variation in mutagenic activity was due to the bioassay procedure and 20% to the extraction process. Extracts from both samples had primarily direct-acting mutagenicity as there were no significant differences in responses with and without metabolic activation (S9). The TA98 - S9 mean air particles mutagenic activities (C.V., %) based on mass of extractable organics or particles were 4.4 (4.7%) and 0.29 (3.6%) revertants/μg, respectively, and for the diesel particles were 66 (44%) and 12 (29%) revertants/μg, respectively. More of the observed direct-acting mutagenicity in the diesel particles extracts was due to nitro-substituted compounds because there were significant reductions in activity with TA98NR (45% of TA98 -S9) and TA98-1,8-DNP6 (21% of TA98 -S9). In the air particles extracts, the TA98NR activities were not significantly different from TA98 - S9 but the TA98-1,8-DNP6 levels were.     

Mutagenicity of beta-glucuronidase in the Ames test

The enzyme preparation beta-glucuronidase/arylsulphatase from Helix pomatia (Boehringer) caused base-pair substitutions in Salmonella typhimurium TA100 and TA1535 strains within the dose range of 0.50-50 microliter per plate. No effect was observed in the TA98 strain. The presence of S9 mix did not substantially affect the mutagenic potential of beta-G. The number of induced revertants decreased continually from experiment to experiment carried out in the course of 12 weeks.     

Sensitivity of different bacterial assays in detecting mutagens in urine of humans exposed to polycyclic aromatic hydrocarbons.

The urine mutagenicity and excretion of 1-hydroxypyrene (1-OH PYR) in non-smoking psoriatic patients treated topically with coal-tar-based ointments were analysed in order to find the most appropriate procedure for monitoring occupational PAH exposure. The bacterial mutagenicity assays used were the plate incorporation, macro-scale fluctuation and microsuspension tests, all on Salmonella typhimurium strain TA98 in the presence of S9 mix and beta-glucuronidase. The sensitivities of the three assays in detecting mutagenic urinary PAH metabolites were compared. The efficiencies of XAD-2 and C18 resins for concentrating PAH urinary mutagens were evaluated in the microsuspension assay. The plate and fluctuation tests on XAD-2 urine extracts were shown to be insufficiently sensitive to detect low urinary levels of mutagens, being positive on urine samples with very high PAH metabolite content, estimated as more than 30 micrograms/g of creatinine of 1-OH PYR. The microsuspension assay on XAD-2 or, even better, on C18 urine extracts was very sensitive in detecting up to 5 micrograms/g of creatinine of 1-OH PYR. It therefore seems to be applicable to the biological monitoring of most occupational low exposures to coal tar.     

Mutagenicity of processed bidi tobacco: possible relevance to bidi industry workers

The genotoxic potential of bidi tobacco was evaluated by mutagenicity testing of aqueous, aqueous: ethanolic, ethanolic and chloroform extracts of processed tobacco used in the manufacture of 'bidis', indigenous forms of cigarettes smoked in India. The Salmonella/mammalian microsome test (Ames assay) was used to detect mutagenicity in tester strains TA98, TA100 and TA102. The extracts were tested in the absence and presence of metabolic activation using liver S9 from rat and hamster, and following in vitro nitrosation with sodium nitrite at acidic pH. All the extracts were non-mutagenic in the absence of nitrosation. The nitrosated aqueous extract was mutagenic in strains TA98 and TA100. While weak mutagenicity was elicited by the nitrosated aqueous: ethanolic extract in TA100, the nitrosated ethanolic extract induced a 3-fold increase in the number of revertants in the same strain. Moreover both these extracts elicited a strong mutagenic response in TA102, while the chloroform extract was non-mutagenic even after nitrite treatment. The present study indicates that workers employed in the bidi industry are exposed to potentially mutagenic and genotoxic chemicals in the course of their occupation.     

Chemical investigation of different extracts and essential oil from the tubers of (Tunisian)Cyperus rotundus. Correlation with their antiradical and antimutagenic properties

The mutagenic potential of aqueous, Total Oligomers Flavonoids (TOF), ethyl acetate, and methanol extracts as well as essential oil (EO) obtained from tubers ofCyperus rotundus L. was assessed by “Ames assay”, usingSalmonella tester strains TA98 and TA100, and “SOS chromotest” usingEscherichia coli PQ37 strain with and without an exogenous metabolic activation system (S9). None of the different extracts showed a mutagenic effect. Likewise, the antimutagenicity of the same extracts was tested using the “Ames test” and the “SOS chromotest”. Our results showed thatC. rotundus extracts have antimutagenic effects withSalmonella typhimurium TA98 and TA100 strains towards the mutagen Aflatoxin B1 (AFB1), as well as withE. coli PQ37 strain against AFB1 and nifuroxazide mutagens. A free radical scavenging test was used in order to explore the antioxidant capacity of the extracts obtained from the tubers ofC. rotundus. TOF, ethyl acetate and methanol extracts showed an important free radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. These extracts showed IC50 values of respectively 5, 20 and 65 μg/ml. The beneficial effects of TOF, ethyl acetate, methanol and essential oil extracts ofC. rotundus have been assessed by antioxidant and antimutagenic activities.     

Evaluation of the mutagenicity of combustion particles from several common biomass fuels in the Ames/Salmonella microsome test

We have evaluated the mutagenicity of dichloromethane extracts of combustion particles from several biomass fuels that are commonly used in developing countries in Salmonella strains TA98 +/- S9 and TA100 +/- S9. Combustion-particle extracts from dried cow dung and crop residue exhibited mutagenic potencies similar to wood-smoke extracts (0.0-1.0 rev./microgram extract). However, extracts from coconut-shell-smoke particles showed relatively potent direct-acting mutagenicity (1.6 rev./micrograms, TA98-S9). Results from testing this sample in nitroreductase- and acetylase- deficient strains TA98NR and TA98 (1,8-DNP-6) revealed no contribution from nitroarenes.     

Activation of promutagenic chemicals by Streptomyces griseus containing cytochrome P-450soy

Streptomyces griseus cells containing cytochrome P-450soy oxidize a diverse array of xenobiotic compounds. This metabolic capability was exploited for activation of promutagenic chemicals such as polycyclic aromatic hydrocarbons, aromatic amines and small aliphatics in a modified Salmonella/Ames plate incorporation assay using tester strains TA98 and TA1538. In this assay promutagens such as 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine, benzidine, 2-acetylaminofluorene, 2-aminoanthracene, 2,4-diaminotoluene, 4-aminobiphenyl, benzo(a)pyrene, chloropicrin and N-nitrosodimethylamine were oxidized to mutagenic metabolites by S. griseus intact cells which mutated Salmonella tester strains (TA98 and TA1538). S. griseus failed to activate 7,12-dimethylbenzanthracene and 4-chloro-2-nitroaniline. In parallel tests performed with rat liver homogenate (S9), N-nitrosodimethylamine was not activated.     

Involvement of nitro-compounds in the mutagenicity of urban Pm2.5 and Pm10 in Turin

Fine particles can be active carriers of toxic compounds into the alveoli of the lungs. Among these compounds are numerous mutagens and carcinogens. The direct mutagenicity per unit mass of fine particulate matter (PM) is significantly higher than that of coarse particles, especially in urban areas. In this study, the mutagenic properties of urban PM2.5 and PM10 were evaluated, and the role of nitro-compounds was estimated. PM2.5 and PM10 samplings, and measurements of NOx and some PAHs were performed daily in 2007 in Turin, following a consolidated in vitro test - the Salmonella mutagenicity assay - conducted with organic extracts of PM2.5 and PM10. The mutagenic properties were assessed for each month of sampling with Salmonella typhimurium strain TA98 and TA98-derived strains: a nitroreductase-deficient mutant strain (TA98NR) and an additional nitroreductase-producing plasmid strain (YG1021). The annual measured mean levels of PM2.5 and PM10 were 34±20 and 48±18μg/m(3). The PM2.5/PM10 ratio ranged from 0.36 to 0.89. The Salmonella assay showed higher mutagenicity in autumn/winter (20±15 TA98NR; 54±39 TA98; 173±161 YG1021 net revertants/m(3)) compared with spring/summer (2±2 TA98NR; 7±8 TA98; 24±27 YG1021 net revertants/m(3)) (p<0.01). There are also statistically significant seasonal differences in the gravimetric analysis data. The number of TA98 net revertants per μg of PM2.5 is 6.5 times greater than per μg PM10. Moreover, the bioassay results showed an amplified response in the YG1021 strain and a reduced response in the TA98NR strain. The net revertant ratio TA98NR/YG1021 is 11±4 for organic extracts of PM2.5 and 13±6 for extracts of PM10 (p<0.01). There is a significant correlation between the NOx and PAH concentrations. These findings illustrate the relevant role of nitro compounds, and they underline the priority in improving preventive measures to reduce air pollution by nitrated molecules.     

Mutagenicity in salmonella of hazardous wastes and urine from rats fed these wastes

15 hazardous industrial waste samples were evaluated for mutagenicity in the Salmonella plate-incorporation assay using strains TA98 and TA100 in the presence and absence of Aroclor 1254-induced rat liver S9. Dichloromethane/methanol extracts of the crude wastes were also evaluated. 7 of the crude wastes were mutagenic, but only 2 of the extracts of these 7 wastes were mutagenic; extracts of 2 additional wastes also were mutagenic. In addition, 10 of the crude wastes were administered by gavage to F-344 rats, and 24-h urine samples were collected. Of the 10 raw urines evaluated, 3 were mutagenic in strain TA98 in the presence of S9 and beta-glucuronidase. The 3 crude wastes that produced these 3 mutagenic urines were, themselves, mutagenic. Adequate volumes of 6 of the 10 raw urines were available for extraction/concentration. These 6 urines were incubated with beta-glucuronidase and eluted through Sep-Pak C18 columns; the methanol eluates of 3 of the urines were mutagenic, and these were the same 3 whose raw urines also were mutagenic. In general, the C18/methanol extraction procedure reduced the cytotoxicity and increased the mutagenic potency of the urines. To our knowledge, this is the first report of the mutagenicity of urine from rodents exposed to hazardous wastes. Based on the present results, the use of only strain TA98 in the presence of S9 might be adequate for general screening of hazardous wastes or waste extracts for genotoxicity. The urinary mutagenesis assay does not appear to be a useful adjunct to the Salmonella assay for screening hazardous wastes. The problems associated with chemically fractionating diverse types of hazardous wastes for bioassay are also discussed.     

Bacterial mutagenicity of some tobacco aromatic nitrogen bases and their mixtures

The first aim was to compare the genotoxicities of two tobacco-specific nitrosamines (TSNA), 4-(methylnitrosamino)-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) in two types of tests, the Salmonella reverse mutation assay (250-2000 microg per plate) and the Mutatox test (up to 1000 microg/ml) using dark mutant M-169 of Vibrio fischeri. The second aim was to assess the effects of single other tobacco chemicals and metabolites (nicotine (NIC), cotinine (COT), trans-3-hydroxycotinine (3HC), cotinine-N-oxide (CNO) and nicotine-N-oxide (NNO)) on the mutagenic responses at relative concentrations observed physiologically. The Salmonella strains were TA100, TA7004, TA7005, and TA7006, all showing missense backmutations that are characteristic of the TSNA. NNN was a direct mutagen to strains TA100, TA7004, and in the Mutatox test, and was not mutagenic in the presence of rat or hamster S9. NNK was mutagenic only in strain TA7004 with rat and hamster S9, but not in TA100, but was directly mutagenic in the Mutatox test. While all the other tobacco chemicals were not mutagenic alone to strains TA100 and TA7004 in the presence and absence of rat or hamster S9, the Mutatox test produced direct mutagenicity for COT, 3HC, and NNO, but not CNO. The latter was mutagenic in the Mutatox test with rat or hamster S9, but only rat S9 was effective for COT, NNO and 3HC. Inhibitory potentiations of NNN by NIC and COT were observed on strain TA7004, and by NIC on strain TA100. There were no interactions on NNK in the presence of S9 for strain TA7004 or TA100. In contrast, a complex inhibition and enhancement behavior occurred in the Mutatox test for each interaction, but no effects were observed for CNO on NNK without S9, and few for NIC on NNK with hamster S9. Compounds which showed no activity alone modulated the genotoxicity of two potent TSNAs in both types of tests.     

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.     

Mutagenicity of five food additives in Ames/Salmonella/microsome test

The mutagenic activity of five food additives (K₂S₂O₅: potassium metabisulphite, KMB; K₂SO₄: potassium sulphate, KS; Na₂SO₃: sodium sulphite, SS; KNO₃: potassium nitrate, KN; NaNO₃: sodium nitrate, SN) were investigated using histidin auxotrophs TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix. The test substance were investigated for their mutagenic effects at non toxic concentrations of 0.83, 1.66, 3.33 and 5.00 mg/plate with and without S9 mix. All the test substances were not mutagenic on TA98 and TA100 strains ofSalmonella typhimurium in the presence or absence of S9 mix except KS and SN. KS and SN showed a weak mutagenic effect on TA100 strain in the absence of S9 mix.