Reduction in the mutagenicity of synthetic dyes by successive treatment with activated sludge and the ligninolytic fungus, Irpex lacteus

Synthetic dyes are released in wastewater from textile manufacturing plants, and many of these dyes are genotoxic. In the present study, the mutagenicity of azo, anthraquinone, and triphenyl methane dyes was investigated before and after successive biodegradation with activated sludge and the ligninolytic fungus, Irpex lacteus. Two biodegradation systems were used to reduce the genotoxicity of dyes that were not efficiently inactivated by activated sludge alone. Mutagenicity was monitored with the Salmonella reversion assay conducted with the base-pair substitution detector strains, TA100 and YG1042, and the frame-shift detector strains, TA98 and YG1041, with and without rat liver S9. All dyes except for Congo Red (CR) were mutagenic with S9 activation. Assays conducted with the dyes indicated that only the azo dye Reactive Orange 16 (RO16) was mutagenic in both TA98 and TA100. Methyl Red and Disperse Blue 3 (DB3) were mutagenic in TA98, YG1041 and YG1042, while Reactive Black 5 was mutagenic in YG1041 and YG1042. Remazol Brilliant Blue R (RBBR), Crystal violet (CV) and Bromophenol Blue (BPB) were mutagenic only in TA98, but the toxicity of the latter two dyes complicated the evaluation of their mutagenicity. CR was not mutagenic in any of the tester strains. Biodegradation studies conducted with RO16 and DB3 indicated that the two-step biodegradation process reduced the mutagenic potential of RO16 and DB3 to a greater extent than activated sludge alone; the mutagenicity of the two dyes was reduced by 95.2% and 77.8%, respectively, by the two-step process. These data indicate that the combined biodegradation process may be useful for reducing the mutagenicity associated with wastewater from textile factories that contain recalcitrant dyes.     

Mutagenicity of azo dyes following metabolism by different reductive/oxidative systems

The mutagenic activity of a group of diazo dyes based on benzidine and its congeners was compared following metabolic activation of the dyes through sequential reduction and oxidation. The dyes were reduced by incubating them with either a suspension of rat cecal flora or a hamster S9 mix supplemented with flavin mononucleotide. The products of dye reduction were then subjected to oxidative metabolism by either Aroclor-induced rat liver S9 or by hamster liver S9; the resultant mutagenic activity was assayed with Salmonella typhimurium TA1538. Fifteen of the 17 compounds tested were mutagenic, and the degree of mutagenicity was affected by the activity of both the reduction and oxidation systems used. Purified dyes required a reductive step to become mutagenic, but several of the crude dyes did not. All the positive compounds, however, were more mutagenic when the reduction step was included. The mutagenicity of the purified dyes was equal to or greater than that of an equimolar amount of benzidine or appropriate benzidine congener. For the crude dyes, there were no consistent quantitative relationships between the mutagenicity of the dye and that expected from the benzidine moiety.     

Structure-mutagenicity relationships of benzidine analogues

The mutagenic activities of benzidine, its dihydrochloride salt, and 12 of their analogues were compared in the Ames test using strains TA100 and TA98 with and without rat liver S9 activation. With the exceptions of 4,4'-methylenebis(3-nitroaniline) in both strains and 3,3-dichlorobenzidine in TA98, little or no mutagenicity was observed in the series when tested without S9 activation. All compounds, except tetramethylbenzidine, exhibited some activity in TA100 with S9 activation; dichlorobenzidine and 4-aminobiphenyl were significantly more mutagenic than the other compounds. This was in contrast to the TA98 results where the bridged diphenyl compounds, with the exception of the nitroaniline derivative, were only slightly mutagenic compared to the more planar biphenyl series. Only the nitroaniline compound was mutagenic in both strains in the presence or absence of S9 activation. For benzidine and the 3,3'-disubstituted benzidines (the dimethoxy-, diamino-, and dichloro- compounds), an increase in mutagenicity correlated to a decrease in basicity of the parent anilines in both TA100 and TA98.     

Mutagenic activity in synthetic pyrethroids in Salmonella typhimurium

Four pyrethroids, allethrin, resmethrin, permethrin and fen-valerate,were tested for mutagenicity in bacterial reversion assay systemswith seven strains (TA1535, TA100, TA1538, TA98, TA1537, TA97and TA104) of Salmonella typhimurium. Our results show thatthree pyrethroids, namely resmethrin, permethrin and fenvalerate,were not found to be mutagenic in S. typhimurium in the presenceor absence of a rat liver activation system. Allethrin was foundto be mutagenic with TA100, TA104 and TA97 strains and requiredmetabolic activation (S9 mix) in order to show its activity,mainly with TA100 and TA104 strains.     

In vitro genotoxicity of dyes present in colored smoke munitions

Genetic toxicology studies were conducted on organic dyes and mixtures used in colored smoke munitions. The dyes studied included Solvent Red 1; two different batches (Lot 1 and Lot 2) of Disperse Red 11; terephthalic acid; and a mixture of 25 parts Solvent Red 1, 5 parts Disperse Red 11, and 16 parts terephthalic acid. The dyes were evaluated for their ability to produce mutations in Salmonella bacterial strains and in Chinese hamster ovary (CHO) cells. The dyes were also tested in CHO cells to determine cytotoxicity and the induction of sister chromatid exchanges and chromosome aberration. None of the dyes were genotoxic in the standard Ames assay using bacterial strain TA1535 or TA100 with or without the addition of S-9 or in TA98 and TA1538 without S-9. With S-9, Disperse Red 11 (Lot 2) showed significant mutagenic activity in TA98 and TA1538 which increased as a function of S-9 concentration. However, the maximum level of mutagenic activity detected was low (3.8 revertants/micrograms). The azo dye Solvent Red 1 was also negative in a pre-incubation assay designed to reduce azo compounds to free amines. Solvent Red 1 was cytotoxic to mammalian cells, caused a significant increase in SCE, but was not mutagenic or clastogenic. Disperse Red 11 (Lot 1 and Lot 2) were not cytotoxic or clastogenic but produced an increase in cell cycle time and SCE frequency. Only Disperse Red 11 (Lot 2) increased mutations in the CHO/hypoxanthine-guanine phosphoribosyltransferase (HGPRT) assay. The mutagenic activity of the dye mixture was not significant, suggesting no synergistic interaction between the dyes. These studies demonstrated that none of the dyes was clastogenic and that a contaminant in Disperse Red 11 (Lot 2) may be responsible for the weak mutagenic activity in both mammalian and bacterial cell systems.     

Evaluation of laser dye mutagenicity using the ames/salmonella microsome test

Twenty-five laser dyes and four analogs were tested for mutagenicity in the Ames/Salmonella test. Seven dyes and two analogs gave positive mutagenic responses with bacterial strains TA1538 and TA98. Of two widely used families of laser dyes (coumarins and rhodamines), four coumarin samples, but none of the rhodamine samples, were mutagenic. All mutagenic compounds require enzyme activation for positive response except two terphenyl analogs, which are mutagenic with or without activation. Using high-performance liquid chromatography (HPLC), it was determined that five mutagenic dye samples had multiple components. The dyes themselves may not be the mutagenic agents in all cases (as with Nile Blue) but may contain impurities that are mutagenic. One dye, adicyanome-thylene (DCM) (≥95% pure), was mutagenic at doses below 0.5 μg/plate on strains TA1538 and TA98. DCM also induced reversions in strains TA96, TA97, TA100, TA102, and TA104, although less efficiently. This study indicates the need for further toxicological testing of these types of compounds. The mutagenic components of these dye mixtures, whether it is the dye or a contaminant, presents a possible hazard to those handling them. Therefore, practices and procedures for the safe handling of specific dyes should be reviewed in light of these findings.     

Structure--activity relationship studies on the mutagenicity of some azo dyes in the Salmonella/microsome assay

Analogs of Direct Black 19 and Direct Black 38 were synthesizedand tested in the Salmonella/microsome assay. Those dyes whichgave positive responses in strains TA98 and TA1538 would beexpected to be metabolized to p-phenyl-enediamine by the livermicrosomal enzymes (S9). Pure p-phenylenediamine is non-mutagenicin this assay but becomes mutagenic after it is oxidized. Thusthe positive response of our synthetic azo compounds are mostlikely due to the formation of oxidized p-phenylenediamine.Modification of the moieties that can be metabolized to p-phenylenediamineby sulfonation, carboxylation or copper complexation eliminatedthe mutagenic responses.     

Mutagenic activity of carbon black dyes used in the leather industry

Seven carbon black pastes used as commercial leather dyes weretested for their mutagenicity in the Salmonella/microsome test(TA98 and TA100 strains). All the samples assayed either directlyor after extraction with a 30-min sonication in benzene weredevoid of mutagenicity both in the presence and absence of ametabolic activation preparation. After a 48-h extraction withboiling toluene in a Soxhlet apparatus, four samples were mutagenicin TA98 strain in the presence of S9 mix. The activity rangedfrom 1.3 to 9.6 induced revertants/mg equivalent of extract.A weak direct mutagenic activity in strain TA98 was shown byone extract. Polycyclic aromatic hydrocarbons (PAH) were determinedin the toluene extracts by high resolution gas chromatography/massspectrometry. The presence of PAH could explain the mutagenicityof only one sample (8.79 µg of total PAH/100 mg equivalentsof extract), while low or undetectable levels of PAH were foundin the other mutagenic extracts. The mutagenic activity wasevident only after a vigorous extraction process, thus a lowbioavailability of the mutagens present in these compounds issuggested. ²To whom correspondence should be addressed     

Genotoxic activity in microorganisms of tetryl, 1,3-dinitrobenzene and 1,3,5-trinitrobenzene

N-Methyl-N,2,4,6-tetranitroaniline (tetryl), 1,3-dinitrobenzene, and 1,3,5-trinitrobenzene were subjected to DNA repair assays using the Escherichia coli W3110/polA⁺, p3478/polA^? system, reverse mutation assays with His^? Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, and mitotic recombinogenic tests with the yeast Saccharomyces cerevisiae D5. Tests were carried out in the absence of an exogenous activation system and in tissue homogenate-mediated assays using Aroclor 1254-induced, male rat-liver-derived S9 mix. Mutagenic activity of tetryl was demonstrated with S typhimurium strains TA1537, TA1538, TA98, and TA100. The responses were particularly strong in the absence of S9 mix. Tetryl also induced increases in recombinant numbers and frequencies in the S cerevisiae test without the S9 mix, but not in its presence. 1,3-Dinitrobenzene was demonstrated to be a mutagen with S typhimurium strains TA1538, TA98, and TA100. Slight activity was also seen with TA1537. The S9 mix reduced the magnitude of the responses. 1,3,5-Trinitrobenzene was also demonstrated to be mutagenic with S typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100. Again, the S9 mix reduced the magnitude of the responses. In this segment of a programme initiated by military authorities, the genotoxic potential of three nitroaromatic compounds, which have found significant use in explosive preparations, has been demonstrated. Twelve other compounds used in ordnance were not active in any of the test systems. These were octahydro-1-acetyl-3,5,7-trinitro-S-tetramine (SEX), hexahydro-1,3-dinitro-5-acetyl-S-triamine (TAX), ethyl centralite, 2-nitrodiphenylamine, N-nitrosodiphenylamine, diphenylamine, diethyleneglycoldinitrate, nitroguanidine, lead salicylate, lead resorcylate, red phosphorus, and zinc chloride.     

Propyldazine is mutagenic in Salmonella typhimurium and Escherichia coli: distinct specificity for strains TA1537 and TA97

The antihypertensive drug propyldazine (Atensil) was demonstrated to be mutagenic with auxotrophic mutants of Salmonella typhimurium and Escherichia coli. Addition of liver S9 mix (postmitochondrial supernatant fraction supplemented with an NADPH-generating system) had little, if any, effect on the mutagenicity. The mutagenicity showed an unusual pattern of strain specificity. Increased frequencies of reversion were observed with all strains whose auxotrophy was caused by frame-shift mutations: the number of revertant colonies per plate from S. typhimurium TA98, TA1538, TA97, and TA1537 was increased up to 5-, 9-, 43-, and 160-fold, respectively, above background. Among the strains that became auxotrophic by substitution mutations, S typhimurium TA102, E. coli WP2, and E coli WP2 uvrA yielded positive results (twofold above background). S. typhimurium TA1535 and TA100 were not reverted by propyldazine. It should be noted that propyldazine, due to its low toxicity and good solubility, could be tested up to very high doses. Hence, although quite impressive mutagenic effects occurred, the mutagenic potency was moderate even in the most responsive strains, TA1537 and TA97 (about 0.3 and 1.0 revertants per nmole, respectively). With the limitation that the strain specificities were different, the mutagenic potency of propyldazine was in the same order of magnitude as that of hydralazine and dihydralazine, two related antihypertensive drugs which were already known to be mutagenic. In our hands, both compounds were mutagenic in S typhimurium TA1535, TA100, TA1537, and TA98. These results differ from data in the literature in that we found clear but weak effects even with strains for which others have reported negative results.     

Comparisons of mutation induction by six monocyclic aromatic amines in Salmonella typhimurium tester strains TA97, TA1537, and TA1538

The mutagenicity of six monocyclic aromatic amines (2,4-diaminoanisole, 2,4-diaminoethoxybenzene, 2,4-diaminopropoxybenzene, m-phenylenediamine, 2,4-diaminotoluene, and nitro-p-phenylenediamine) was investigated in Salmonella typhimurium strains TA97, TA1537, and TA1538 in the presence of two different amounts of Aroclor 1254-induced S9 preparations. Strain TA1538 was found to be the most responsive of the three strains with this group of compounds. Regarding the other strains, TA1537 responded to three of the compounds better than strain TA97, if one calculates responsiveness as the fold-increase in numbers of revertants per plate. However, if one calculates the number of revertants per nanomole or compares the number of induced revertants per plate, TA97 was more responsive than TA1537 for all six compounds. Comparisons of mutagenesis from tests involving strain TA97 are complicated by the large variations in spontaneous mutation frequencies in this strain. The amount of S9 per plate is another important variable in tests of monocyclic aromatic amines; in general, more revertants are detected when the S9 mix contains 10% S9 than when it contains 4% S9. Nevertheless, in all our tests of 2,4-diaminoanisole, 2,4-diaminoethoxybenzene, and 2,4-diaminopropoxybenzene, the same relationship between chemical structure and mutagenic activity was observed. In all three strains, the mutagenic responses become less when the alkoxy substituent on the molecule becomes larger.     

Mutagenicity of resin acids identified in pulp and paper mill effluents using the salmonella/mammalian-microsome assay

Ten resin acids which have been identified as constituents of pulp and paper mill effluents have been examined for potential mutagenicity in the Salmonella/mammalian-microsome assay. Only neoabietic acid has been found to be mutagenic. Neoabietic acid showed dose-related increases in mutagenicity in strains TA1535, TA100, TA1538, and TA98, but not in strain TA1537. Metabolic activation with a preparation of Aroclor 1254-induced liver homogenate (S9) slightly reduced the mutagenic responses. Negative responses were found for abietic acid, dehydroabietic acid, levopimaric acid, 7-oxodehydroabietic acid, monochlorodehydroabietic acid, dichlorodehydroabietic acid, pimaric acid, isopimaric acid, and sandaracopimaric acid.     

Mutagenic evaluation of some triazino indoles using the Salmonella/mammalian microsome assay

The mutagenicity of ethyl l,2,3-triazino[5,4-b]indole-4-carb-oxylateN(3)-oxide (D3) and 2-chloroethyl 1,2,3-triazino-[5,4-b]indole-4-carboxylateN(3)-oxide (D4), heads of series of new products with considerableplatelet antiaggregating and hypotensive activity, and theirprecursors 2-ethoxy-carbonylmethyl-l-methyUndole-3-carboxylicacid (A₃) and 2-(2-chloroethoxycarbonyhnethyl)-l-methylindole-3-carb-oxylkacid (A₄) were tested in four strains of Salmonella typhimurium(TA98, TA100, TA97 and TA102) using the standard plate incorporationtechnique. A₃ and A₄ were not mutagenic whereas D3 was mutagenicto all the strains and D₄ was mutagenic to TA97, TA98 and TA100.The addition of 4 or 10% of S9 mix decreased the mutagenic activityof both compounds. This effect was independent of the concentrationof S9 in the S9 mix.     

Mutagenicity of resin acids identified in pulp and paper mill effluents using the Salmonella/mammalian-microsome assay.

Ten resin acids which have been identified as constituents of pulp and paper mill effluents have been examined for potential mutagenicity in the Salmonella/mammalian-microsome assay. Only neoabietic acid has been found to be mutagenic. Neoabietic acid showed dose-related increases in mutagenicity in strains TA1535, TA100, TA1538, and TA98, but not in strain TA1537. Metabolic activation with a preparation of Aroclor 1254-induced liver homogenate (S9) slightly reduced the mutagenic responses. Negative responses were found for abietic acid, dehydroabietic acid, levopimaric acid, 7-oxodehydroabietic acid, monochlorodehydroabietic acid, dichlorodehydroabietic acid, pimaric acid, isopimaric acid, and sandaracopimaric acid.     

A promising Ames battery for mutagenicity characterization of new dyes

When testing new products, potential new products, or their impurities for genotoxicity in the Ames test, the quantity available for testing can be a limiting factor. This is the case for a dye repository of around 98,000 substances the Max Weaver Dye Library (MWDL). Mutagenicity data on dyes in the literature, although vast, in several cases is not reliable, compromising the performance of the in silico models. In this report, we propose a strategy for the generation of high‐quality mutagenicity data for dyes using a minimum amount of sample. We evaluated 15 dyes from different chemical classes selected from 150 representative dyes of the MWDL. The purity and molecular confirmation of each dye were determined, and the microplate agar protocol (MPA) was used. Dyes were tested at the limit of solubility in single and concentration‐response experiments using seven strains without and with metabolic activation except for anthraquinone dyes which were tested with eight strains. Six dyes were mutagenic. The most sensitive was YG1041, followed by TA97a > TA98 > TA100 = TA1538 > TA102. YG7108 as well as TA1537 did not detect any mutagenic response. We concluded that the MPA was successful in identifying the mutagenicity of dyes using less than 12.5 mg of sample. We propose that dyes should be tested in a tiered approach using YG1041 followed by TA97a, TA98, and TA100 in concentration‐response experiments. This work provides additional information on the dye mutagenicity database available in the literature.     

Evaluation of the mutagenicity of an N-nitroso contaminant of the sunscreen Padimate O: N-nitroso-N-methyl-p-aminobenzoic acid, 2-ethylhexyl ester (NPABAO).

The nitrosamine contaminant, N-nitroso-N-methyl-p-aminobenzoic acid, 2-ethylhexyl ester (NPABAO), of the major sunscreen ingredient Padimate O (4-N,N'-dimethylamino-benzoic acid, 2-ethylhexyl ester) was synthesized and tested for mutagenicity in the Salmonella typhimurium and mouse lymphoma L5178Y TK +/- assays. In contrast to the previously reported positive responses in S. typhimurium tester strains TA100 and TA1535 [Loeppky et al., 1991], there were no increases in the number of revertants with strains TA98, TA100, TA1535, and TA1538 in either the Salmonella plate incorporation [Ames et al., 1975] or preincubation [Yahagi et al., 1977] assays. Additional testing with Salmonella, following the modified preincubation procedure [Rogan, 1990] that gave the initial positive response, was also negative. Data from the mouse lymphoma assays were also uniformly negative. During synthesis of NPABAO, small amounts of 4-N,N'-dimethylamino-3-nitrobenzoic acid, 2-ethylhexyl ester (DMANBAO) can be formed. To determine whether the reported positive mutagenicity response of NPABAO could be the result of trace amounts of DMANBAO in the NPABAO, that compound was also synthesized and tested for mutagenicity with Salmonella. Positive responses were obtained with tester strains TA98 and TA 1538 but not with TA100 and TA1535, indicating that DMANBAO was not responsible for the increase in revertants originally reported.     

Mutagenicity and antimutagenicity of hispidulin and hortensin, the flavonoids from Millingtonia hortensis L.

Studies of the mutagenicity and antimutagenicity of hispidulin and hortensin, the flavonoids from Millingtonia hortensis L. (Bignoniaceae), were performed using the liquid preincubation method of the Salmonella/microsome test. At the highest dose tested, 100 micrograms/plate, both compounds showed no mutagenicity and no cytotoxicity toward S. typhimurium strains TA98 and TA100 either in the presence or absence of S9 mix. However, these substances were antimutagens toward 2-aminoanthracene, aflatoxin B1 (in TA98), and dimethylnitrosamine (in TA100); but neither substance inhibited the direct mutagenic activity of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide nor that of sodium azide in strains TA98 and TA100, respectively.     

Mutagenicity tests of fabric-finishing agents in salmonella typhimurium: Fiber-reactive wool dyes and cotton flame retardants

Thirty-nine fabric-finishing agents were tested for mutagenic activity in Salmonella typhimurium. Twenty-four fiber-reactive wool dyes and three acid dyes (not fiber-reactive) were screened by spot tests in strains TA100, TA98, TA1535, and TA1537. Among these dyes, seven bromoacrylamide dyes and one vinyl sulfone dye were mutagenic. Additionally, one of the three acid dyes was mutagenic in spot tests. The mutagenicity of the acid dye was due to an impurity or breakdown product rather than to the dye itself; the origin of the activities of the other dyes is unknown. No mutagenicity was observed among five chlorotriazine or four sulfonyl-ethane sulfonic acid dyes. Eight phosphorus-containing flame retardants (phosphonium, phosphine, phosphine oxide, and phosphonic acid derivatives) and methyl-N-methylolcarbamate, which is employed to obtain a flame-retardant finish on cotton, were tested for mutagenicity in strains TA100, TA98, TA1535, and TA1537, using quantitative incorporation assays. All were nonmutagenic. Two of three bromoalkyl-substituted triazine flame retardants were mutagenic in strains TA100 and TA1535. It is unknown whether this activity is due to impurities or to the parent compound. The flame retardants tested were either in actual commercial use or in experimental development for potential commercial processes. These results indicate the need for early testing of potential fabric-finishing agents and processes.     

Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method

The volatile solvents carbon tetrachloride and chloroform are carcinogens that are often reported as nonmutagenic in bacterial mutagenicity assays. In this study, we evaluated the mutagenicity of these compounds in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, with and without S9 mix, using a gas exposure method. Tests were also conducted with a glutathione-supplemented S9 mix. Carbon tetrachloride was mutagenic in TA98 without S9 mix, and in WP2/pKM101 and WP2uvrA/pKM101 with and without S9 mix; carbon tetrachloride was not mutagenic in TA100, TA1535 or TA1537. Chloroform was mutagenic in WP2/pKM101, but only in the presence of glutathione-supplemented S9 mix. Chloroform was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 with or without S9 mix, and was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 in the presence of glutathione-supplemented S9 mix. The data indicate that carbon tetrachloride and chloroform are bacterial mutagens when adequate exposure conditions are employed and suggest that a genotoxic mode of action could contribute to the carcinogenicity of these compounds.     

Evidence that 4-aminobiphenyl, benzidine, and benzidine congeners produce genotoxicity through reactive oxygen species

4-Aminobyphenyl (4-Ab), benzidine (Bz), and Bz congeners were evaluated for their ability to induce genotoxicity through an oxidative mechanism. The mutagenicity of these compounds was tested in the presence and absence of Aroclor 1254-induced rat S9 mix using Salmonella typhimurium tester strain TA102, which is sensitive to agents producing reactive oxygen species (ROS). In the presence of S9, 4-Ab, Bz, N-acetyl-benzidine, and 3,3-dimethoxybenzidine were strongly mutagenic in TA102, whereas, 3,3,5,5-tetra-methylbenzidine, 3,3-dimethylbenzidine (O-tolidine), and N,N-diacetylbenzidine were not mutagenic. In addition, 3,3-dichlorobenzidine and 4,4-dinitro-2-biphenylamine were directly mutagenic in TA102. Incorporation of the free radical and metal scavengers, catalase, superoxide dismutase (SOD), butylated hydroxytolune (BHT), and ethylenediamine tetraacetic acid (EDTA) reduced the mutagenic responses of 4-Ab and Bz, whereas heat-inactivated catalase and SOD had no effect. 4-Ab and Bz also induced lipid peroxidation in the presence of S9 mix as shown using the thiobarbituric acid reactive substances assay. The results of this study indicate that 4-Ab and Bz induce mutations through the induction of ROS.