Mutagenicity testing of di(2-ethylhexyl)phthalate and related chemicals in Salmonella

Di(2-ethylhexyl)phthalate and 33 other phthalates, ethylhexanol derivatives, and related chemicals were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 without metabolic activation and in the presence of rat and hamster liver S-9 metabolic activation systems. No mutagenic activity was seen with any of the chemicals tested.     

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.     

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.     

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.     

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 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.     

Mutagenic screening of marker grenade dyes by the Salmonella reversion assay, L5178Y/TK+/- mouse lymphoma assay, and in vivo sister chromatid exchange analysis in mice

Two dyes (C.I. Solvent Yellow No. 33 and a mixture of C.I. Solvent Yellow No. 33 and C.I. Solvent Green No. 3) were tested for mutagenicity in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay, and also for sister chromatid exchange (SCE) induction in vivo in C57B1/6J mice. In addition, a greater than 99.9% pure sample of the yellow dye [2-(2'-quinolyl)-1,3-indandione] was tested with and without exogenous activation in the Salmonella reversion assay and the L5178Y/TK+/- mouse lymphoma assay. Neither C.I. Solvent Yellow No. 33 nor the C.I. Solvent Yellow No. 33 and Solvent Green No. 3 mixture was positive for inducing SCEs in vivo. All three dyes were tested in the standard plate incorporation test in seven Salmonella strains TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. The dyes were negative with and without exogenous activation in TA98, TA1535, and TA1538. One test with TA1537 was positive with the greater than 99.9% purified yellow dye. All three dyes gave weakly positive results (less than a twofold increase) with S-9 in TA100 and were clearly positive in TA102 and TA104 both with and without S-9. They also induced mutation at the thymidine kinase locus in mouse lymphoma cells, produced both large- and small-colony trifluorothymidine-resistant mutants, and were clastogenic. The purified yellow dye was further tested for SCE induction in mouse lymphoma cells and was determined to give a slightly positive response in the presence of S-9.     

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.     

Evaluation of biotoxicity of textile dyes using two bioassays

The toxicity of eight textile dyes was evaluated using two bioassays namely: Ames test and seed germination test. The Ames test is widely used for the evaluation of hazardous mutagenic effect of different chemicals, as a short-term screening test for environmental impact assessment. The eight-textile dyes and Eithidium bromide dye (as positive control) were tested with five "his" Salmonella typhimurium strains: TA 100; TA 98; TA 1535; TA 1537; TA 1538. Using six concentrations of each dye (2.5 microg/ml, 4.5 microg/ml, 9 microg/ml, 13.5 microg/ml, 18 microg/ml, and 22.5 microg/ml) revealed that, most of the dyes were mutagenic for the test strains used in this study. The high concentrations of dye eliminated microbial colonies due to the high frequency of mutation causing lethal effect on the cells.In this work the phytotoxicity of different soluble textile dyes was estimated by measuring the relative changes in seed germination of four plants: clover, wheat, tomato and lettuce. The changes in shooting percentages and root length as affected by dye were also measured. Seed germination percent and shoot growth as well as root length were recorded after 6 days of exposure to different concentrations of textile dyes in irrigation water. The results show that high concentrations of dyes were more toxic to seed germination as compared with the lower concentrations. However, the low concentrations of the tested dyes adversely affected the shooting percent significantly.     

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.     

Evaluation of the mutagenicity of azo dyes in Salmonella typhimurium: a study of structure-activity relationships

In order to explore structure-activity relationships, 4,4'-diaminoazobenzene and four structurally related azo dyes were tested for their ability to induce gene mutations in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98. Only 4,4'-diaminoazobenzene and 4,4'-N(beta-hydroxyethylamino)azobenzene were found to be active in the two frameshift strains TA1538 and TA98. Further tests were performed in strain TA98, both in the presence and in the absence of Aroclor 1254-induced rat or hamster liver S9 preparations. The amount of S9 used per plate was 50, 100, 150 or 300 microliters, which corresponds to 10, 20, 30 or 60% of S9 in S9 mix. 4,4'-Diaminoazobenzene was found to be mutagenic, and its mutagenicity depended on the percentage of S9 in S9 mix and the type of S9 fraction used. 4,4'-N-(beta-Hydroxyethylamino)azobenzene was less mutagenic than 4,4'-diaminoazobenzene, indicating a reduction in mutagenicity associated with the beta-hydroxyalkyl substituents. The other three azo dyes [4'-methyl-4-N,N-di(beta-hydroxyethylamino) azobenzene; 4'-amino-6-methyl-4-N,N-di(beta-hydroxyethylamino)azobenzene; and 4'-N(beta-hydroxyethyl-amino)4-N,N-di(beta-hydroxyethylamino)azobe nzene] were inactive, both in the presence and in the absence of the metabolic activation system. The use of the preincubation test did not alter the observed positive or negative response of these compounds. The importance of this finding is that the non-mutagenicity or decreased mutagenicity of these four compounds is predictable on the basis of their chemical structures. These azo dyes, like the non-mutagenic members of series of monocyclic aromatic amines, contain large substituents on one or both of the amino groups of the parent compound, in this case 4,4'-diaminoazobenzene. From our earlier data and the experiments discussed in this paper, we conclude that the study of structure--activity relationships can provide useful information for the prediction and interpretation of mutagenic responses.     

The SIMULTEST: a new approach to screening chemicals with the Salmonella reversion assay

A new Salmonella mutagenicity test method is under development to test a chemical with more than one strain simultaneously (the "SIMULTEST"), that is, different Salmonella typhimurium tester strains are used in combination on the same plate. Strains are combined in two sets: strains with plasmid pKM101 (TA97, TA98, TA100, and TA102) and strains without the plasmid (TA1535, TA1537, and TA1538). The SIMULTEST combinations successfully detect the mutagenic activity of five mutagens in different chemical classes. This approach may be useful in reducing the workload associated with mutagenicity testing with Salmonella.     

Mutagenic potential of bisphenol A diglycidyl ether (BADGE) and its hydrolysis-derived products in the Ames Salmonella assay.

The mutagenicity of bisphenol A diglycidyl ether (BADGE), its first and second hydrolysis products (the diol epoxide and bis-diol of BADGE, respectively) and the bis-chlorohydrin of BADGE were investigated using the Ames Salmonella assay with strains TA98, TA100, TA1535 and TA1537. The assays were performed in the absence and presence of various concentrations of rat liver S9 fraction. The results obtained confirm the mutagenic power of BADGE in strains TA100 and TA1535 and show a positive response to the diol epoxide of BADGE in these strains, although the latter compound was approximately 10 times less potent than the former. A lack of mutagenic activity of the bis-diol of BADGE and the chlorohydrin under study is also shown. These findings suggest that BADGE and, to a much lesser extent, the diol epoxide of BADGE may constitute a genotoxic hazard, but not the bis-diol or bis-chlorohydrin of BADGE.     

Mutagenicity of chili extract and capsaicin in short-term tests

Vanillin, capsaicin and chili extracts were tested for mutagenicity in Salmonella typhimurium histidine-deficient tester strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538. Vanillin was nonmutagenic, whereas chili extract and capsaicin were mutagenic with metabolic activation. Capsaicin, an active component of chili extract, was the more potent mutagen. The positive samples were also tested in two mammalian test systems: the micronucleus test and the 8-azaguanine-resistant mutagenesis assay that used V79 Chinese hamster cells. It was observed that both were negative for the latter test at the dose level tested, whereas in the micronucleus test, only capsaicin was positive near the LD50 dose. Capsaicin also inhibited DNA synthesis in the testes of Swiss mice injected at two dose levels.     

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.     

Microbial mutagenicity of isomeric two-, three-, and four-ring amino polycyclic aromatic hydrocarbons

The isomers of various two-, three-, and four-ring amino polycyclic aromatic hydrocarbons were tested for mutagenic activity using a microbial plate incorporation test with four Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537). All compounds were assayed with an S9 metabolic activating enzyme system. The two-ring compounds were tested only with TA98. All were weakly mutagenic (1-10 rev/micrograms) except 2-aminobiphenyl, which was not mutagenic under these test conditions. All except two of the 13 fused three-ring compounds (aminofluorenes, aminoanthracenes, and aminophenanthrenes) were active frame shift mutagens; only the aminophenanthrenes were active base-pair mutagens. The potency of this group of isomeric compounds ranged from moderately (approximately 20 rev/microgram) to strongly (greater than 5,000 rev/microgram) mutagenic. As a group, the pericondensed four-ring amino compounds were the most mutagenic of the three groups tested. All of the aminofluoranthene and aminopyrene isomers showed significant mutagenic activity with TA98, TA100, and TA1537. In general, the mutagenic potency of the amino polycyclic aromatic compounds tested was highly dependent on the structural position of the amino group.     

Formaldehyde is a bacterial mutagen in a range of Salmonella and Escherichia indicator strains

Formaldehyde was examined for bacterial mutagenicity using Escherichiacoli WP2(pKM101) and WP2uvrA(pKM101), and Salmonella typhimuriumTA1535, TA1537, TA1538, TA98, TA100 and TA102, in the absenceof any exogenous source of metabolic activation. Using pre-incubationexposure, clear mutagenicity was seen for TA98, TA100 and TA102,and both E.coli strains. In standard plate-incorporation assays,consistent mutagenicity was seen only for TA100 and WP2uvrA(pKM101).No evidence of mutagenicity was seen for TA1535, TA1537 or TA1538using either method of exposure. These data confirm the enhancedability of the pre-incubation method to detect the mutagenicityof formaldehyde both quantitatively, as expressed by numbersof revertant colonies, and qualitatively, in terms of the rangeof indicator strains reverted. The relatively greater sensitivityof the pre-incubation assay is probably due to better containmentof a volatile agent and/or lack of interaction with agar duringthe initial period of exposure. The findings are consistentwith the suggestion that formaldehyde induces lesions in bacteriawhich are, at least to some extent, excision-repairable, andindicate that the presence of the R-factor plasmid may be requiredfor the expression of its mutagenicity in excision repair-deficientSalmonella.     

Detection of mutagenic activity in the urine of rodents treated with p-rosaniline

p-Rosaniline was fed to male and female Fischer 344 rats and B6C3F1 mice at doses of 1,000 and 2,000 ppm for male rats and 500 and 1,000 ppm for female rats and mice of both sexes. Urine was collected overnight at 1-wk intervals over a 4-wk treatment period and frozen until its use in the mutagenicity assay. The neat urine was tested in triplicate without S-9 on Salmonella tester strains TA98, TA100, TA1535, and TA1537 at 0.75, 0.5, 0.2, and 0.05 ml per plate. When sufficient urine was available, samples were tested on TA100 in the presence of S-9. Either urine samples were pretreated for 18 hr at 37 degrees C with beta-glucuronidase, or the deconjugating enzyme was added to the top agar at the time of plating in the mutagenicity assay (non-pretreatment). Direct-acting mutagenic activity was detected on TA98 in the urine from male mice, but only when using the non-pretreatment deconjugation method. No direct-acting mutagenic activity was detected in the urine of male and female rats and female mice; however, in the presence of S-9, mutagenic activity was observed in the urine of male rats and in the urine of male and female mice regardless of the deconjugation method used. The non-pretreatment method was superior for detecting direct acting mutagenic activity, and the pretreatment method was superior for detecting mutagenic activity requiring metabolic activation by S-9.     

Studies on the induction of gene mutations in bacterial and mammalian cells by the ring-opened benzene metabolites trans,trans-muconaldehyde and trans,trans-muconic acid

t,t-Muconaldehyde and t,t-muconic acid have been investigated for the induction of gene mutations in Salmonella typhimurium (reversion of the his- strains TA97, TA98, TA100, TA102, TA104 and TA1535), Escherichia coli (reversion of the trp- strain WP2 uvrA) and Chinese hamster V79 cells (acquisition of resistance toward 6-thioguanine). t,t-Muconaldehyde proved weakly mutagenic in strain TA104 in the presence and absence of NADPH-fortified postmitochondrial fraction from rat liver homogenate (S9 mix). In strains TA97, TA100 and TA102, weak positive responses were observed only in the presence of S9 mix. In strains TA98, TA1535 and WP2 uvrA, the result was negative. In V79 cells, the mutation frequency was increased from approximately 7 X 10(-6) to 90 X 10(-6) in cultures exposed to t,t-muconaldehyde at optimal concentration (1.7-3 microM in separate experiments). The concentration-response curve showed pronounced hyperlinearity, with no mutagenic effect being observed at a third of the optimal concentration. t,t-Muconic acid was greater than 100 times less toxic than t,t-muconaldehyde in both bacteria and mammalian cells, and it did not show any mutagenic effect. These results complete a previous mutagenicity study, carried out on benzene and 13 metabolites. It is concluded that the newly investigated metabolites cannot account for the bacterial mutagenicity of bioactivated benzene and benzene-trans-1,2-dihydrodiol, since these compounds exhibited their strongest response in strain TA1535. t,t-Muconaldehyde showed similarities in its mutagenicity to p-benzoquinone and hydroquinone. All three compounds showed, at most, weak effects in bacteria, but were strongly mutagenic in V79 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancing bioremoval of textile dyes by eight fungal strains from media supplemented with gelatine wastes and sucrose

Eight Aspergillus strains were found to be successful in removing textile dyes from liquid media. These fungal strains were grown on medium containing: gelatine wastes and sucrose, as sources of nitrogen and carbon to test the possible speed up of the dyes removing while fungus biomass is building up in the media. The growth of fungal strains ranged from 10 to 110 mg biomass dry weight/100 ml medium. This growth induced high decolorization percentages, which ranged 33-95% within eight days. Two textile dyes Direct brown and Polar red were included in the study. The growth of the fungal strains as well as decolorization percentage of the dyes increased after 5, 6, and 8 days from incubation time with most tested strains. With Direct brown dye the strains number 2, 5, 31 and 37 recorded the highest percentage of decolorization (91, 92, 93 and 95 respectively) after incubation for 6 days. Fungal strains Aspergillus 5 and 31 gave the highest mycelium dry weight being 110 mg. Most of fungal strains induced 86 to 95 percentage of decolorization after 6 days incubation with Polar red dye. The possible toxicity of the remaining supernatant media after fungal biomass removal was tested by Ames test to assess the residual mutagenic agents remaining after dye removal, using three strains of Salmonella typhimurium (TA 1535, TA 1537, TA 1538). The results showed that the toxicity of the dyes, measured by Ames test could be removed by the dye absorption on the fungal biomass.