Mutagenicity of mono-nitrobenzene derivatives in the Ames test and rec assay

The mutagenicities of 37 mono-nitrobenzene derivatives, i.e. the ortho, meta, and para isomers of nitrotoluene, nitrophenol, nitroaniline, nitroanisole, nitrobenzaldehyde, nitrobenzyl chloride, nitrobenzonitrile, nitroacetophenone, nitrophenetol, nitrobenzoic acid, nitrophenylacetic acid, and nitrocinnamic acid and p-nitrothiophenol, were tested in the Ames test and rec assay. The rec assay gave more positive results than the Ames test. The para-substituted nitrobenzene derivatives were always positive in the rec assay as were all but 2 in the Ames test, while 5 out of 12 ortho and meta derivatives were mutagenic in the Ames test. The results of the present study, combined with those of the previous study, are discussed with special reference to the structure-mutagenicity relationships.     

Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone

3 ketone solvents (methyl ethyl ketone (MEK), methyl isobutyl ketone (MiBK), and isophorone) were tested for potential genotoxicity. The assays of MEK and MiBK included the Salmonella/microsome (Ames) assay, L5178Y/TK+/- mouse lymphoma (ML) assay, BALB/3T3 cell transformation (CT) assay, unscheduled DNA synthesis (UDS) assay, and micronucleus (MN) assay. Only the ML, UDS, and MN assays were conducted on samples of isophorone. No genotoxicity was found for MEK or isophorone. The presence of a marginal response only at the highest, cytotoxic concentration tested in the ML assay, the lack of reproducibility in the CT assay, and clearly negative results in the Ames assay, UDS and MN assays, suggest that MiBK is unlikely to be genotoxic in mammalian systems.     

Assessment of the performance of the Ames II assay: a collaborative study with 19 coded compounds

Nineteen coded chemicals were tested in an international collaborative study for their mutagenic activity. The assay system employed was the Ames II Mutagenicity Assay, using the tester strains TA98 and TAMix (TA7001-7006). The test compounds were selected from a published study with a large data set from the standard Ames plate-incorporation test. The following test compounds including matched pairs were investigated: cyclophoshamide, 2-naphthylamine, benzo(a)pyrene, pyrene, 2-acetylaminofluorene, 4,4'-methylene-bis(2-chloroaniline), 9,10-dimethylanthracene, anthracene, 4-nitroquinoline-N-oxide, diphenylnitrosamine, urethane, isopropyl-N(3-chlorophenyl)carbamate, benzidine, 3,3'-5,5'-tetramethylbenzidine, azoxybenzene, 3-aminotriazole, diethylstilbestrol, sucrose and methionine. The results of both assay systems were compared, and the inter-laboratory consistency of the Ames II test was assessed. Of the eight mutagens selected, six were correctly identified with the Ames II assay by all laboratories, one compound was judged positive by five of six investigators and one by four of six laboratories. All seven non-mutagenic samples were consistently negative in the Ames II assay. Of the four chemicals that gave inconsistent results in the traditional Ames test, three were uniformly classified as either positive or negative in the present study, whereas one compound gave equivocal results. A comparison of the test outcome of the different investigators resulted in an inter-laboratory consistency of 89.5%. Owing to the high concordance between the two test systems, and the low inter-laboratory variability in the Ames II assay results, the Ames II is an effective screening alternative to the standard Ames test, requiring less test material and labor.     

Absence of genotoxic activity of penbutolol in bacterial and mammalian cell screening systems

The genotoxic potential of the beta-adrenergic blocker penbutolol was assessed using the Ames and HGPRT tests, unscheduled DNA synthesis (UDS) and alkaline elution assays. In the Ames test, penbutolol was tested for cytotoxicity and genotoxic activity in concentration ranges of 0.8-500 micrograms/plate and 0.1-125 micrograms/ml in the HGPRT, UDS and alkaline elution assays. In the Ames test penbutolol showed significant toxicity above 500 micrograms/plate. In the mammalian cells (V79) used for the HGPRT test and A459 cells used for alkaline elution and UDS assays, penbutolol was cytotoxic at concentrations above 30 micrograms/ml. In another series of experiments, male Wistar rats were treated i.p. with penbutolol (1, 10 and 100 mg/kg) and after 2 h liver nuclei were isolated and formation of single DNA-strand breaks was measured. The results of the present study demonstrate the absence of genotoxic activity of penbutolol in the 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) and in the strain of Escherichia coli WP2 uvrA in the presence or absence of metabolic activation. In V79 cells, penbutolol showed no mutagenic effects at the HGPRT locus in the presence or absence of metabolic activation. Additionally, no significant incorporation of [3H]thymidine into the DNA in the UDS test or formation of DNA-strand breaks in the alkaline elution assay was detected in the non-toxic concentration range of penbutolol with or without metabolic activation. Furthermore, penbutolol did not cause DNA damage in liver nuclei isolated from penbutolol-treated rats.     

Screening of safrole, eugenol, their ninhydrin positive metabolites and selected secondary amines for potential mutagenicity.

The mutagenicity of safrole, eugenol, the secondary amines, with which they combine during metabolism, and the ninhydrin positive urinary metabolites of safrole and eugenol was tested. The panel of tests included the direct bacterial assay, a microsomal mutagenesis assay and a host-mediated assay. With the direct bacterial assay employing four mutant strains of Salmonella typhimurium (TA1530, TA1531, TA1532, TA1964), all the compounds gave negative results. In the microsomal mutagenesis assay, employing the same four mutant strains, safrole and safrole metabolite II were mutagenic with strains TA1530 and TA1532. Dimethylamine was also found to be a weak mutagen in the microsomal mutagenesis assay with strain TA1530. Safrole and safrole metabolite II were also mutagenic in the host-mediated assay with strains TA1950 and TA1952. Negative results were observed for safrole metabolites I and III, eugenol, eugenol metabolites I and II, piperidine, pipecolic acid, proline, and pyrrolidine in all three assay systems.     

Absence of mutagenic activity of WHR-1142A, lidamidine hydrochloride, in 4 short-term tests for mutagenic/carcinogenic potential

WHR-1142A, lidamidine hydrochloride, an antidiarrhoeal agent, was tested for possible mutagenic/carcinogenic activity in the Ames Salmonella typhimurium/metabolic activation test, the micronucleus test, by analysis of metaphase chromosomes obtained from human lymphocytes grown in culture and in a cell transformation assay. No evidence of mutagenic/carcinogenic activity due to WHR-1142A, lidamidine hydrochloride, was found in any of the 4 tests.     

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

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

Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts

The use of Syrian hamster embryo cells for assessing genotoxicity provides the unique opportunity to determine 5 different end-points (gene mutations, DNA-strand breaks, aneuploidy, DNA repair (unscheduled DNA synthesis, UDS) and neoplastic transformation) in the one cell system. This approach allows direct comparisons of results produced under identical conditions of dose at target, metabolism and bioavailability. We report here on the characterization of an additional end-point in the same cell system: the formation of micronuclei indicating chromosomal changes induced by chemicals. For a preliminary validation of this new test system we have investigated 14 carcinogens and 3 non-carcinogenic structural analogues in order to evaluate the significance of micronucleus induction for carcinogenic properties. All tested carcinogens induced micronuclei in a dose-dependent manner; all non-carcinogens yielded negative results. Correlations between the formation of micronuclei and the Ames test, induction of UDS, cell transformation and the in vivo bone marrow micronucleus test are demonstrated.     

Lack of mutagenicity of diphenylhydantoin in in vitro short-term tests

The mutagenicity of diphenylhydantoin (DPH) and its major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), has been re-evaluated by the Ames test using Salmonella typhimurium and, for DPH only, by an in vitro cytogenetic test with human lymphocytes and a turbidimetric assay of tubulin polymerization. As negative results were obtained in all test systems used here, one has to conclude that DPH is devoid of mutagenic properties.     

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.     

Interaction of Mesna (2-mercaptoethane sulfonate) with the mutagenicity of cyclophosphamide in vitro and in vivo

The effects of sodium 2-mercaptoethane sulfonate (Mesna) on the mutagenicity of cyclophosphamide (CP) were assessed in vitro by the Ames test and in vivo in rats by analyzing micronuclei in bone marrow and mutagenic activity in urine. Mesna alone was negative in all test systems, while CP gave a positive response in all of them. In a combined treatment there was no significant reduction of the CP-induced mutagenicity in Salmonella. In rats the frequency of bone marrow micronuclei was not diminished when Mesna was given together with CP. May-Grunwald-Giemsa staining and Hoechst-Pyronin fluorescent staining techniques for micronuclei yielded similar results. The urine of rats treated with CP was mutagenic to Salmonella and no significant difference was observed when the rats had received both Mesna and CP. The results give support to the theory that Mesna acts primarily by reducing the toxicity of metabolites of CP, particularly acrolein, in the urinary tract and not by suppressing the mutagenicity of the active metabolites of CP.     

Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo

There has been much discussion in recent years regarding the most appropriate follow-up testing in vivo when positive results are obtained in vitro but the in vivo micronucleus (MN) test (traditionally the most widely-used test) is negative. Not all rodent carcinogens give positive results in the micronucleus test, and so it has been common practice to include a second in vivo assay such as the unscheduled DNA synthesis (UDS) test. This has proved useful but is usually limited to analysis of rodent (usually rat) liver. With the increased evaluation and use of other in vivo assays, e.g. for transgenic mutations (TG) and DNA damage (Comet assay) it was important to investigate their usefulness. We therefore examined the published in vivo UDS, TG and Comet-assay results for 67 carcinogens that were negative or equivocal in the micronucleus test. Between 30 and 41 chemicals were evaluated in each of the three in vivo tests, with some overlap. In general, the UDS test was disappointing and gave positive results with <20% of these carcinogens, some of which induced tumours in rat liver and produced DNA adducts in vivo. The TG assay gave positive responses with >50% of the carcinogens, but the Comet assay detected almost 90% of the micronucleus-negative or equivocal carcinogens. This pattern of results was virtually unchanged when the in vitro profile (gene mutagen or clastogen) was taken into account. High sensitivity (ability to detect carcinogens as positive) is only really useful when the specificity (ability to give negative results with non-carcinogens) is also high. Based on small numbers of publications with non-carcinogens, the TG and Comet assays gave negative results with non-carcinogens on 69 and 78% of occasions, respectively. Although further evaluation of the Comet and TG assays, particularly with non-carcinogens, is needed, these data suggest that they both should play a more prominent role in regulatory testing strategies than the UDS test.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 microg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 microg/ml for 3 and 20 h in the absence of S9, and for 3h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 microg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 microg/kg, the last dose being administered 2h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Induction of DNA-repair synthesis in primary rat hepatocytes by epoxides

The genotoxicity of 10 epoxides was investigated in the UDS test with primary rat hepatocytes. The sensitivity of the assay was demonstrated using 2-acetylaminofluorence. The epoxides 1,2-epoxyoctane, 1,2-epoxydecane, epoxycyclooctane, epoxycyclododecane, (+)-limoneoxide, alpha-pinaneoxide, transstilbeneoxide, and cis-2,3-epoxysuccinic acid, which are known to be non-mutagenic in the Ames test, as well as the bacterial mutagen, 1,2-epoxyphenoxypropane did not induce UDS in primary hepatocytes of the rat. However, a positive UDS response obtained with glycidyltrimethylammonium chloride showed that metabolic inactivation of the oxirane ring in hepatocytes is influenced by further structural substituents.     

Drug residue formation from ronidazole, a 5-nitroimidazole. VI. Lack of mutagenic activity of reduced metabolites and derivatives of ronidazole

The potential toxicity of ronidazole residues present in the tissues of food-producing animals was assessed using the Ames mutagenicity test. Since ronidazole is activated by reduction, reduced derivatives of ronidazole and metabolites formed by enzymatic reduction of ronidazole were tested for mutagenicity. When tested at levels several orders of magnitude higher than that at which ronidazole was mutagenic, 5-amino-4-S-cysteinyl-1,2- dimethylimidazole , a product of the dithionite reduction of ronidazole in the presence of cysteine, the 5-N-acetylamino derivative of ronidazole and 5-amino-1,2- dimethylimidazole all lacked mutagenic activity in Ames strain TA100. The metabolites of ronidazole formed by the incubation of ronidazole with microsomes under anaerobic conditions were also not mutagenic. These data demonstrate that although ronidazole is a potent mutagen, residues from it which may be present in the tissues of food-producing animals lack any mutagenic activity.     

Assessing the cytotoxic and mutagenic effects of secondary metabolites produced by several fungal biological control agents with the Ames assay and the VITOTOX(®) test

The potential genotoxic effects of several pure secondary metabolites produced by fungi used as biological control agents (BCAs) were studied with the Ames Salmonella/microsome mutagenicity assay and the Vitotox test, with and without metabolic activation. A complete set of Salmonella tester strains was used to avoid false negative results. To detect possible mutagenic and/or cytotoxic effects of fungal secondary metabolites due to synergistic action, crude extracts and fungal cell extracts of the BCAs were also examined. Although the sensitivity of the methods varied depending on the metabolite used, clearly no genotoxicity was observed in all cases. The results of the two assays are discussed in the light of being used in a complementary fashion for a convincing risk-assessment evaluation of fungal BCAs and their secondary metabolites.     

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.     

Effect of food intake on urinary excretions of histamine, N tau-methylhistamine, imidazole acetic acid and its conjugate(s) in humans and mice

The urinary excretions by young healthy men of histamine and its metabolites, N tau-methylhistamine, imidazole acetic acid, and imidazole acetic acid conjugate(s), increased 1-3 h after food intake. The increase was seen even after the intake of konnyaku (mannan) as a protein-deficient food, suggesting that physical stimulation of the gastric mucosa by food is the main cause of histamine release. This suggestion was confirmed by the following findings in patients and mice. In patients with stomach diseases, gastrectomy resulted in decreases in the excretion of histamine and its metabolites in the urine, and patients subjected to intravenous hyperalimentation excreted less histamine and its metabolites in the urine than normal subjects. In mice, a correlation of histamine excretion with food intake was demonstrated experimentally. Namely, mice fed only during the night (21:00-0:00) showed increased excretions of histamine and its metabolites at 23:00-3:00, whereas those fed in the morning (9:00-12:00) showed increased excretions of those compounds at 11:00-15:00. All these results are consistent with the idea that urinary histamine and its metabolites mainly originate from the stomach.     

An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay. I. In vitro sensitivity of the bacteria to 61 compounds.

A differential DNA repair test was evaluated in vitro, using derivatives of E. coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+. The aim of this study was to characterize the sensitivity of the assay to different compounds in vitro and thereby provide information on the usefulness of this end-point as an indicator of genotoxicity in a host-mediated assay. Sixty-one compounds from diverse chemical groups were tested and of these 32 gave a positive result. The results obtained were compared with results from the Ames test and were in agreement for 49 out of the 61 compounds tested. Chemicals that were detected in this test but negative in the Ames test were 4-aminophenol, catechol, diethylstilbestrol, thioacetamide and thiourea. Seven of the compounds tested gave a negative result in E. coli but were positive in Salmonella. These were 4-aminobiphenyl, benzo[a]pyrene, cyclophosphamide, 1-naphthylamine, N-nitrosobutylpropylamine, quinoline and 2-toluidine. The performance of the in vitro test and reasons for the discrepant results with the Ames test are discussed. The overall concordance between the two tests was about 80%. On the basis of these results we consider these bacterial strains, and differential DNA repair as an end-point, to be sufficiently accurate as an indicator of genotoxicity in vitro and thereby also in vivo.