Comparative mutagenicity of aliphatic epoxides in Salmonella

37 aliphatic epoxides comprising 6 subclasses (unsubstituted aliphatic epoxides, halogenated aliphatic epoxides, glycidyl esters, glycidates, glycidyl ethers and diglycidyl ethers) were tested, under code, for mutagenicity in Salmonella strains TA98, TA100, TA1535 and TA1537 and/or TA97 with and without metabolic activation using a standardized protocol. The 4 halogenated aliphatic epoxides and the 4 diglycidyl ethers were all mutagenic. The 2 glycidates were negative in all strain/activation systems used while all 5 glycidyl esters were mutagenic. 3 of the 8 unsubstituted aliphatic epoxides and 11 of the 12 glycidyl ethers were mutagenic. Glycidol also was mutagenic whereas 9,10-epoxyoctadecanoic acid, 2-ethylhexyl ester was not mutagenic. Of the 28 mutagenic compounds, all but neodecanoic acid, 2,3-epoxypropyl ester and 2-ethylhexyl glycidyl ether were detected in TA100 without activation. The latter two were detected only with activation in TA100 and TA1535. The majority of the other 26 chemicals were also mutagenic in TA1535 without activation. Good intra- and interlaboratory reproducibility was seen in the results of each of the 4 chemicals tested in more than one set of experiments. The current results confirm and extend the observations of other investigators regarding structural effects on the mutagenicity of members of the aliphatic epoxide class of chemicals.     

Mutagenicity of N,N'-dimethylurea and methylamine hydrochloride in the Ames Salmonella/microsome test: absence of mutagenic response.

Methyl isocyanate (MIC) in aqueous solution forms methylamine (MA) and N,N'-dimethylurea (DMU). MA in buffered system further converts into its salt form, methylamine hydrochloride (MAH). Therefore, MAH and DMU were evaluated for their mutagenic activity in the in vitro Ames Salmonella/microsome mutagenicity test. The liquid preincubation protocol was followed, using tester strains TA98, TA100 and TA104 of Salmonella typhimurium, in the presence of 0, 5, 15 and 30% Aroclor 1254-induced rat liver S9 mixture. DMU and MAH did not induce a mutagenic response in any of the tester strains, both in the presence and in the absence of S9 mixture. The results therefore confirm that MIC in its native form or as its unknown metabolites is responsible for the mutagenic activity reported earlier by us in the his tester strains TA100 and TA104 of Salmonella typhimurium (Mutation Res., 204 (1988) 123-129) and not due to its hydrolysis products, MA or DMU.     

Statistical analysis of Salmonella test data and comparison to results of animal cancer tests

A quantitative framework for the analysis of results of the Salmonella (Ames) test is presented, and the relationship between mutagenesis and carcinogenesis is examined. Color graphics are used for the Salmonella data to describe variability, and trends across multiple chemicals and test conditions. Positivity in the Salmonella test, using statistical criteria to classify results, is compared to positivity in carcinogenesis bioassays for 48 chemicals tested in NCI/NTP-sponsored programs. Sensitivity of the Salmonella test across 5 tester strains was 91% (21/23), while specificity was only 36% (9/25). Results were most concordant for TA100 Aroclor-induced rat S9: sensitivity was 87%, specificity 64%. The correlation of mutagenic potency and carcinogenic potency was 0.41 (p less than 0.001) for 80 chemicals, using results from both the general published literature and the NCI/NTP-sponsored programs. After removal of 3 extreme values, the correlation was 0.24 (p = 0.04).     

A comparative study of mutagenic and SOS-inducing activity of biphenyls, phenanthrenequinones and fluorenones

A total of 23 chemicals--biphenyls, phenanthrenequinones and fluorenones--were tested for mutagenicity towards Salmonella typhimurium strains TA1538, TA1535 and TA98. SOS-inducing activity of the same chemicals was studied in terms of the SOS-inducing potency in Escherichia coli PQ37, using an automated instrument controlled by a dedicated computer program for the SOS Chromotest. Of the 23 chemicals studied 14 induced His+ revertants in S. typhimurium TA1538 hisD305 (-1 frameshift); none induced His+ reversions in TA1535 (base-pair substitution). The mutagenicity of the chemicals in S. typhimurium TA98 (pKM 101) was lower than in TA1538. There was a close correlation between mutagenicity and SOS-inducing activity of fluorenones and phenanthrenequinones. None of the biphenyls tested induced SOS response and this property does not depend upon the mutagenic activity of the chemicals. SOS Chromotest is particularly valid in detecting chemicals which give rise to base-pair substitutions through SOS induction. If positive results are obtained, the Salmonella assay may be omitted. However, this test cannot replace the Ames test especially for the primary screening of mutagenicity of chemicals with unknown structure.     

6-Dimethylaminopurine (6-DMAP), which is used to produce most cloned animals, is mutagenic in Salmonella typhimurium TA1535

6-Dimethylaminopurine (6-DMAP) and cycloheximide have recently been used for successful production of cloned mammals. We investigated whether 6-DMAP or cycloheximide are mutagenic agents in the Ames test. Whereas cycloheximide showed no differences compared to the negative control in any of the tester strains, 6-DMAP was clearly mutagenic in Salmonella typhimurium strain TA1535. Here, we strongly propose that innocuous chemicals be used in the production of cloned animals.     

Chemical purity and mutagenicity: case study of a drug in development.

During a routine Ames assay of a potential antipsychotic drug candidate, the compound appeared to be a frameshift mutagen in Salmonella typhimurium strains TA98 and TA1538. Additional testing indicated the mutagenic activity was due to one or more contaminants incurred during synthesis. While the compound was initially shown to be greater than 98% pure by high-performance liquid chromatography, the presence of small amounts (0.01-0.1%) of a highly mutagenic impurity produced positive mutagenicity results. The need to assess for chemical purity before discontinuing development of drug candidates found positive in the Ames assay is discussed.     

Response of the L-arabinose forward mutation assay of Salmonella typhimurium to frameshift-type mutagens

The present study was designed to evaluate the capacity of the L-arabinose resistance test of Salmonella typhimurium in the detection of frameshift-type mutagens. To this end the response of the Ara test was examined with respect to 15 chemicals which had been previously described as able to revert the Ames tester strain TA97. The mutagenicity of each compound was determined by the liquid test under experimental conditions which optimize the mutagenic response of the Ara test with the tester strain BA9. Strain TA97 was used simultaneously with BA9. The Ara forward-mutation assay efficiently detected the mutagenic activity of 14 out of the 15 chemicals assayed. PR toxin was the only compound which gave a weak dose response without doubling the spontaneous mutant level. In comparison with the Ara test, a total of 3 chemicals (HZ, PE and PR toxin) were not found to be mutagenic with strain TA97. In most cases (11/15) the mutagenic response of the Ara test was comparatively greater than that of strain TA97. Three chemicals (DEO, PRF and 9-AA) were detected with quite similar degrees of sensitivity by both mutation assays. ICR-191, which seems highly specific in reverting frameshift mutations with added cytosines in a run of cytosines, was the only chemical with a lower mutagenic activity in the Ara test than in strain TA97. The results enhance the interest of the L-arabinose forward-mutation assay as an alternative to the set of specific tester strains used by the histidine reverse-mutation assay in massive, general and primary screening for genotoxic agents.     

2-(p-Nitrophenyl)-2''-deoxyadenosine, a new type of mutagenic nucleoside.

A crude preparation of 2-phenyladenosine was found to be mutagenic in the Ames Salmonella assay. In the purification of this preparation, it was revealed that 2-phenyladenosine itself was nonmutagenic but that 2-(m- and p-nitrophenyl)-adenosines (5m,p) contaminating the sample were the mutagenic principles. A structure-activity relationship study was carried out, and it was found that 5p, 2-(p-nitrophenyl)-adenine (7p), and 2-(p-nitrophenyl)-2'-deoxyadenosine (15p) were strongly mutagenic toward S. typhimurium TA98 and TA100 without metabolic activation, the potency being in the order 15p greater than 7p greater than 5p. The potency of 15p in TA98 was one order of magnitude greater than that of 4-nitroquinoline N-oxide. 15p also showed mutagenicity in the mouse cell line FM3A in culture.     

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.     

Use of a Salmonella microsuspension bioassay to detect the mutagenicity of munitions compounds at low concentrations

Past production and handling of munitions has resulted in soil contamination at various military facilities. Depending on the concentrations present, these soils pose both a reactivity and toxicity hazard and the potential for groundwater contamination. Many munitions-related chemicals have been examined for mutagenicity in the Ames test, but because the metabolites may be present in low environmental concentrations, a more sensitive method is needed to elucidate the associated mutagenicity. RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), TNT (2,4,6-trinitrotoluene), tetryl (N-methyl-N-2,4,6-tetranitroaniline), TNB (1,3,5-trinitrobenzene) and metabolites were examined for mutagenicity in a microsuspension modification of the Salmonella histidine reversion assay with and without metabolic activation. TNB and tetryl were positive in TA98 (32.5, 5.2revertants/nmole) and TA100 (7.4, 9.5revertants/nmole) without metabolic activation and were more potent than TNT (TA98, 0.3revertants/nmole; TA100, 2.4revertants/nmole). With the exception of the tetranitroazoxytoluene derivatives, TNT metabolites were less mutagenic than TNT. RDX and two metabolites were negative in both strains, however, hexahydro-1,3,5-trinitroso-1,3,5-triazine was positive in TA100 with and without S9. Microsuspension bioassay results tend to correlate well with published Ames test data, however, there are discrepancies among the published data sets and the microsuspension assay results.     

Collaborative study using the preincubation Salmonella typhimurium mutation assay for airborne particulate matter in Japan. A trial to minimize interlaboratory variation.

A collaborative study has been performed over a period of 3 years to develop a suitable method for monitoring the mutagenicity of airborne particulate matter. The study was organized with 8 laboratories and performed in the following steps: (1) selection of a suitable technique for each process involved in the mutagenicity monitoring, (2) developing a tentative protocol by combining systematically the selected techniques, (3) evaluation of the protocol by intra- and inter-laboratory studies, (4) modification of the protocol according to the evaluation, and (5) evaluation of the modified protocol by conducting an interlaboratory study. We found a suitable method for mutagenicity monitoring of particles in the atmosphere. Airborne particles were sampled with a high-volume sampler, the samples were stored at -80 degrees C, extracted by sonication using dichloromethane, solvent-exchanged, and assayed by the preincubation method using Salmonella typhimurium TA98 and TA100. The observed mutagenic activity was normalized with that of an internal standard. Round robin tests revealed that the method resulted in excellent reproducibility. The coefficient of variation for mutagenic activities of airborne particulate samples collected in various districts of Japan were in the range of 14.7 +/- 6.6% to 19.6 +/- 4.0% for strains TA98 and TA100 with and without metabolic activation. We also found that the plate incorporation method was equivalent to the preincubation method for airborne particulate extracts.     

Mutagenicity of fume particles from metal arc welding on stainless steel in the Salmonella/microsome test.

Mutagenic activity of fume particles produced by metal arc welding on stainless steel (ss) is demonstrated by using the Salmonella/microsome mutagenicity test described by Ames et al., with strain TA100 (base-pair substitution) and TA98 (frame-shift reversion). Results of a representative but limited selection of processes and materials show that mutagenic activity is a function of process and process parameters. Welding on stainless steel produces particles that are mutagenic, whereas welding on mild steel (ms) produces particles that are not. Manual metal arc (MMA) welding on stainless steel produces particles of higher mutagenic activity than does metal inert gas (MIG) welding, and fume particles produced by MIG welding under short-arc transfer. Further studies of welding fumes (both particles and gases) must be performed to determine process parameters of significance for the mutagenic activity.     

6-Dimethylaminopurine (6-DMAP), which is used to produce most cloned animals, is mutagenic in Salmonella typhimurium TA1535

6-Dimethylaminopurine (6-DMAP) and cycloheximide have recently been used for successful production of cloned mammals. We investigated whether 6-DMAP or cycloheximide are mutagenic agents in the Ames test. Whereas cycloheximide showed no differences compared to the negative control in any of the tester strains, 6-DMAP was clearly mutagenic in Salmonella typhimurium strain TA1535. Here, we strongly propose that innocuous chemicals be used in the production of cloned animals.     

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.     

Chemical purity and mutagenicity: Case study of a drug in development

During a routine Ames assay of a potential antipsychotic drug candidate, the compound appeared to be a frameshift mutagen in Salmonella typhimurium strains TA98 and TA1538. Additional testing indicated the mutagenic activity was due to one or more contaminants incurred during synthesis. While the compound was initially shown to be > 98% pure by high-performance liquid chromatography, the presence of small amounts (0.01 – 0.1%) of a highly mutagenic impurity produced positive mutagenicity results. The need to assess for chemical purity before discontinuing development of drug candidates found positive in the Ames assay is discussed.     

Ames Salmonella/mammalian-microsome testing of peptides and peptide synthesis reagents

The Ames Salmonella/mammalian-microsome assay was used to evaluate the bacterial mutagenicity of 6 bioactive peptides and of 11 chemical reagents used in peptide synthesis. Samples of 2 reagents, bis(2-oxo-3-oxazolidinyl)phosphinic chloride and fluoren-9-ylmethyl chloroformate, showed mutagenic activity with strains TA100 and TA1535, and with TA1537, respectively. No mutagenic activity was found with the bioactive peptides or with the other 9 peptide synthesis reagents.     

Mutagenicity evaluation of HC Blue No. 1 and HC Blue No. 2. III. Effects in the Salmonella typhimurium/Escherichia coli reversion assay and the mouse lymphoma L5178Y TK+/- forward mutation assay

The hair-dye ingredients, HC Blue No. 1 (HCB1) and HC Blue No. 2 (HCB2), were tested for the induction of bacterial mutation using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100; and Escherichia coli strains WP2uvrA-. In addition, both dyes were evaluated in the mouse lymphoma L5178Y TK+/- assay (MLA) for the potential to induce forward mutation. A liver homogenate (S9) prepared from Aroclor 1254-induced male Fischer 344 rats was used to provide a means for metabolic activation. HCB1 was not mutagenic in the Ames assay, but was weakly mutagenic in the MLA only in the presence of metabolic activation. In contrast, HCB2 was a strong mutagen in the Ames assay in tester strain TA98 both in the presence and absence of metabolic activation. A positive response was also noted with HCB2 in the MLA, both in the presence and absence of metabolic activation. Negative findings from the Ames assay of this study agree with other published results where an identical lot of HCB1 was used. Using the same lot, a weak positive result was observed in the MLA, however, the activation requirements and magnitude of the response were different from that of a lot evaluated by the NTP. In contrast, HCB2 appears to be both a bacterial and mammalian cell mutagen independent of lot variability.     

Comparison of the mutagenic activity of 5-hydroxymethyldeoxyuridine with 5-substituted 2'-deoxyuridine analogs in the Ames Salmonella/microsome test

4 antiviral drugs 5-hydroxymethyldeoxyuridine (HMUdR), 5-trifluorothymidine (F3TdR), 5-methoxymethyldeoxyuridine (MMUdR) and 5-ethyldeoxyuridine (EtUdR) have been evaluated for mutagenic activity in the Ames Salmonella/microsome test. The antimetabolites F3TdR and HMUdR were mutagenic in a dose-dependent manner in strain TA100. F3TdR also was mutagenic in strain TA1535. Rat-liver post-mitochondrial supernatant (S9) was not required for mutagenicity.     

Metabolism of 2,4-dinitrotoluene by Salmonella typhimurium strains TA98, TA98NR and TA98/1,8-DNP6, and mutagenicity of the metabolites of 2,4-dinitrotoluene and related compounds to strains TA98 and TA100

The products detected in the incubation of 2,4-dinitrotoluene (2,4-DNT) with Salmonella typhimurium strains TA98 and TA98/1,8-DNP6 were nitrosonitrotoluenes, hydroxylaminonitrotoluenes, aminonitrotoluenes and dimethyl dinitroazoxybenzene. The capacity of TA98NR to reduce 2,4-DNT was much lower than that of TA98 and TA98/1,8-DNP6. The bacterial products showed no mutagenic activity in the Ames assay using TA98 and TA100. These results indicate that the lack of mutagenic activity of 2,4-DNT is not due to low reductive metabolism of 2,4-DNT by the bacteria, but to the lack of mutagenic activity of the bacterial reductive products of 2,4-DNT, including dimethyl dinitroazoxybenzene.     

Evaluation of secondary nitroalkanes, their nitronates, primary nitroalkanes, nitrocarbinols, and other aliphatic nitro compounds in the Ames Salmonella assay.

The secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane and nitrocyclopentane, as well as their anionic forms (nitronates); the primary nitroalkanes 1-nitropropane, 1-nitrobutane, and 1-nitropentane and their respective nitronates; the nitrocarbinols 2-nitro-1-propanol, 2-nitro-1-butanol, 3-nitro-2-butanol, and 3-nitro-2-pentanol and their respective nitronates; 2-methyl-2-nitropropane, and 2-nitroso-2-nitropropane were tested in the Ames Salmonella assay using strains TA98, TA100 and TA102. Nitronates of the secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane, and nitrocyclopentane were significantly mutagenic in Salmonella strains TA100 and TA102 at 10-80 mumoles/plate, but the parent compounds were mutagenic at only a single dose level or were not mutagenic at all in the same dose range. The primary nitroalkanes and the nitrocarbinols were not mutagenic, or only marginally so, at the concentrations tested. The nitronates of the primary nitroalkanes and the nitrocarbinols reprotonated too rapidly under the conditions of the assay for adequate evaluation of mutagenicity. 2-Methyl-2-nitropropane was not mutagenic in strains TA100 and TA102; 2-nitroso-2-nitropropane was also not mutagenic in strains TA100 and TA102, but induced an equivocal mutagenic response in TA98. The positive Salmonella mutation data for the nitronates of the secondary nitroalkanes studied correlate very well with the very slow rate of reprotonation of secondary nitroalkane nitronates at pH 7.7 (Conaway et al. (1991) Cancer Res., 51, 3143), and provide further evidence that nitronates of secondary nitroalkanes, rather than the neutral parent forms with which they may be in equilibrium, are the more proximate mutagenic species.