Evaluation of diallate and triallate herbicides for genotoxic effects in a battery of in vitro and short-term in vivo tests

Commercial-grade preparations of two thiocarbamate herbicides, diallate and triallate, were evaluated for their mutagenic potential in a battery of short-term bioassays. All in vitro bioassays were performed with and without mammalian metabolic activation, and all such tests were repeated after an interval of at least 1 week. Diallate and triallate were tested in the Salmonella/microsome assay over dose ranges of 0.59 to 118.0 micrograms/plate and 6.37 to 1273 micrograms/plate, respectively. Both diallate and triallate gave positive results in S. typhimurium strains TA1535, TA98, and TA100 only in the presence of a rat-liver metabolic activation system. In Saccharomyces cerevisiae strain D7, diallate was tested at concentrations from 1.18 to 29.50 micrograms/ml, and triallate was tested at 0.955 to 9.548 micrograms/ml. Both diallate and triallate gave negative results for mitotic gene conversion, mitotic crossing-over, and reverse mutation. In the mouse lymphoma L5178Y TK+/- assay, diallate was tested at concentrations ranging from 1 to 72 micrograms/ml, and triallate was tested at 0.5 to 60 micrograms/ml. Both herbicides produced mutagenic responses in the mouse lymphoma assay in the presence of metabolic activation. In the Drosophila sex-linked recessive lethal test, flies were exposed to 0.0004% diallate and 0.001% triallate. In this assay, diallate was considered mutagenic, whereas triallate did not produce a detectable mutagenic response.     

Mutagenicity of dipyridyls and their methylated derivatives in Salmonella typhimurium/rat liver microsome system

The Salmonella/microsome assay with strains TA97, TA98, TA100, TA1535, TA1537 and TA1538 was used to examine the potential mutagenicity of 5 dipyridyls, 1 tripyridyl, 3 dipyridinium diiodides and 2 pyridinium monoiodides. The widely used herbicide paraquat (1,1-dimethyl-4,4'-dipyridinium diiodide) and its precursor 4,4'-dipyridyl gave weak and marginal mutagenic activity to Salmonella typhimurium TA1535 and TA1538 in the presence of S9-mix. Significantly high mutagenicity was obtained with 2,2'-, 3,3'-, 2,3'-, and 2,4'-dipyridyls, 2,2',2"-tripyridyl, and 5 pyridinium salts under the same conditions. The positive mutagenic response of 2,2',2"-tripyridyl suggests that higher polymers of pyridine contaminating paraquat preparations might be mutagenic. The dose-response curves of 1,1-dimethyl-3,3'-dipyridinium diiodide and 1,1'-dimethyl-2,2'-dipyridinium diiodide revealed an exponential relationship between the number of induced revertants and the compound concentrations. The results suggested that the mechanism of mutation induced by these two compounds might be attributed to the chain reactions of their free-radicals with molecular oxygen.     

Mutagenicity of N-nitrosopyrrolidine derivatives in Salmonella (Ames) and Escherichia coli K-12 (343/113) assays

The mutagenicity of nitrosopyrrolidine (NPYR) and its derivatives was determined by use of the Ames Salmonella assay. A clear specificity to revert the missense stain of TA1535 and a requirement for the phenobarbital-induced rat-liver activation system (S9 mix) were noted. 3,4-Dichloronitrosopyrrolidine was more mutagenic than NPYR, whereas 3-hydroxynitrosopyrrolidine was weakly mutagenic. The carcinogenic nitroso-3-pyrrolidine was not mutagenic under the test conditions. The noncarcinogenic derivatives (2,5-dimethylnitrosopyrrolidine, nitrosoproline and 4-hydroxynitrosoproline) were not mutagenic. Liquid preincubation assays were not any more effective than the pour-plate assays. Selected derivatives of NPYR were tested in the Escherichia coli K-12 (343/113) assay A specificity to revert the missense mutation at the arg locus and a dependence on phenobarbital-induced rat-liver S9 mix were noted with NPYR and its derivatives. 3,4-Dibromonitrosopyrrolidine, which was not mutagenic in Salmonella, was effective in E. coli, and the weakly carcinogenic NPRL was a weak mutagen resulting in a 2-fold enhancement in the E. coli arginine reversion assay.     

The metabolic activation of dichloromethane and chlorofluoromethane in a bacterial mutation assay using Salmonella typhimurium

The metabolic activation and mutagenicity of dichloromethane and chlorofluoromethane were investigated using rat liver fractions and Salmonella typhimurium strain TA100. Both dihalomethanes gave a mutagenic response without the addition of rat-liver fractions. This response has been shown to be due to bacterial metabolism of the test compounds by pathways believed to be similar to those known in the rat. When rat-liver post-mitochondrial supernatant was added to the mutagenicity assay, there was no significant increase in the mutagenicity of dichloromethane, whereas a 2-fold increase was observed for chlorofluoromethane under the same conditions. This increase was derived both from glutathione conjugation and cytochrome P450 oxidative dehydrochlorination. A significant increase in dichloromethane mutagenicity could only be achieved by increasing the concentration of post-mitochondrial supernatant. Under these conditions the increase in mutagenicity was derived solely from glutathione conjugation of dichloromethane. The difference in mutagenic response after the addition of rat-liver fractions can be explained by differences in the half lives of the reactive intermediates rather than a difference in overall metabolic rate between the two compounds.     

Mutagenic evaluation of the monocyclic aromatic amine N-methylamino-2-nitro-4-N', N'-bis(2-hydroxyethyl)-aminobenzene in the Salmonella typhimurium/mammalian microsome test

The hair-dye component N-methylamino-2-nitro-4-N', N'-bis(2-hydroxyethyl) aminobenzene was investigated for mutagenic activity in Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98. The testing was performed in the absence and in the presence of a rat-liver microsomal activation system induced by Aroclor 1254. Our results indicate that N-methylamino-2-nitro-4-N', N'-bis(2-hydroxyethyl)aminobenzene does not induce mutations in Salmonella typhimurium strains, either in the absence or in the presence of the metabolic activation system. The purity of the compound was controlled by utilizing high-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC).     

Mutagenicity of methyl bromide in a series of short-term tests

Methyl bromide is commonly used as a soil fumigant in greenhouses. In the framework of a toxicological evaluation, it was tested for possible genotoxic properties in two bacterial test systems (the fluctuation test using Klebsiella pneumoniae and the plate test using Salmonella typhimurium TA100 and TA98), two systems using mammalian cells in vitro (forward mutations at the TK and HPRT loci in L5178Y mouse lymphoma cells and unscheduled DNA synthesis in primary rat-liver cells) and in the sex-linked recessive lethal test using Drosophila melanogaster. Methyl bromide was active in all tests except the DNA-repair assay. The results indicate a relatively low mutagenic efficiency of the compound, as expected from its alkylating properties.     

Mutagenicity of 7H-dibenzo[c,g]carbazole and metabolites in Salmonella typhimurium

7H-Dibenzo[c,g]carbazole (DBC) is a potent carcinogen of environmental import. Reverse-mutation plate-incorporation assays for mutagenicity were undertaken in Salmonella typhimurium strains TA98 and TA100. Results were negative when no exogenous activation system was used, as well as when assays incorporated liver homogenates (S9) from rats, mice and rabbits. By contrast DBC was mutagenic in a forward mutation assay in Salmonella strain TM677 using resistance to 8-azaguanine for selection. Metabolites of DBC were generated by incubation with rat-liver microsomes and separated by HPLC. Two of these metabolites were directly mutagenic for Salmonella strain TM 677 while two others were mutagenic upon addition of S9. Synthetic phenolic derivatives of DBC were also mutagenic in this assay when further metabolized. It is likely that metabolites of DBC phenols constitute the biologically active forms.     

The effects of S9 mix from rat oesophagus, salivary gland, and liver on the mutagenicity of the rat oesophageal carcinogen N-nitroso-N-methylaniline in the Salmonella typhimurium assay

The present study examines the feasibility of using the Salmonella typhimurium plate-incorporation assay of Ames for detecting target-organ specificity with N-nitroso-N-methylaniline (NMA), a compound for which the target site for tumour formation in the rat is the oesophagus. Thus it was anticipated that the oesophagus would bioactivate this compound. The compound has been investigated using S9 from Aroclor- and NMA-induced rat oesophagus, salivary gland and liver in the presence and absence of the co-mutagen, norharman. No response to NMA was seen with oesophageal S9 even though benzo[a]pyrene produced a dose-related increase in revertants in strain TA98 and TA100. No response to NMA was seen with salivary-gland S9. However, a response was produced with Aroclor-induced rat-liver S9 in the presence of norharman and with NMA-induced rat-liver S9 in the absence of norharman.     

Studies on the mutagenicity of p-phenylenediamine in Salmonella typhimurium. Presence of PCB's in rat-liver microsomal fraction induced by Aroclor.

The mutagenicity of fresh solutions of p-phenylenediamine (PPD) and Aroclor 1254 was investigated. The histidine-requiring strains of Salmonella typhimurium were used in the absence and presence of uninduced and/or Aroclor-induced rat-liver homogenate. The presence of polychlorinated biphenyls (PCBs) was also examined by chromatographic methods in Aroclor-induced rat-liver homogenate. In the absence of metabolic activation, as well as in the presence of uninduced rat-liver homogenate, PPD was not mutagenic in the strains used. In the presence of Aroclor-induced S9 a twofold increase (or less) was observed in the number of revertant colonies over those of the controls in TA1538 and TA98. There was no increase in the number of revertant colonies over those of the controls when PPD was dissolved in NH4OH solution and the solution mixed with H2O2 before the addition of S9 mix. Aroclor 1254 was not mutagenic in TA1538 or TA98. However, the presence of PCBs in Aroclor-induced rat-liver homogenate (induced S9) was identified by gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC) and gas--liquid chromatography/mass spectrometry (GC/MS).     

Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates

The mutagenic potential of three alkyl 2-cyanoacrylate adhesives, three commercial alkyl 2-cyanoacrylate adhesives and three methyl 2-cyano-3-phenylacrylates, was assessed using the Salmonella/microsome mutagenicity assay. Compounds were tested with and without Aroclor 1254-induced rat-liver homogenate (S9 mix). The methyl 2-cyanoacrylate adhesives were mutagenic in the standard plate test with S. typhimurium strain TA100 with and without S9 activation. Methyl 2-cyano-3-(2-bromophenyl)acrylate revealed a direct mutagenic action to S. typhimurium strain TA1535. The compounds most toxic towards the bacterium S. typhimurium, were the methyl 2-cyanoacrylate adhesives (greater than 500 micrograms/plate). All alkyl 2-cyanoacrylate adhesives were tested in a modified spot test for volatile compounds with tester strain TA100. Mutagenic and toxic effects were observed with the three methyl 2-cyanoacrylate adhesives. It can be concluded from the results that the bacterial toxicity and mutagenicity of methyl 2-cyanoacrylate adhesives may be due to the methyl 2-cyanoacrylate monomer.     

Absence of mutagenic activity of benorylate, paracetamol and aspirin in the Salmonella/mammalian microsome test

Benorylate and its two major hydrolysis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Absence of mutagenic activity of benorylate,paracetamol and aspirin in the Salmonella//mammalian microsome test

Benorylate and its two major hydroyssis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Mutagenicity of nitrovin--a nitrofuran feed additive.

Nitrovin, a nitrofuran feed additive, is shown to be directly mutagenic in Salmonella typhimurium TA 98 and TA 100 between 0.1 and 2.5 microgram per plate (0.09--2.3 micrometer). Addition of a rat-liver homogenate reduces the mutation rates. Nitrovin inhibits growth of the same bacteria in suspension cultures at concentrations above 0.09 micrometer.     

Inverse correlation between combined mutagenicity in Salmonella typhimurium and strength of coordinate bond in mixtures of cobalt(II) chloride and 4-substituted pyridines

Cobalt(II) chloride (CoCl₂), non-mutagenic by itself, has been tested for mutagenic activity in the presence of 4-substituted pyridines in the test strains of Salmonella typhimurium. CoCl₂ was found to be mutagenic in strains TA1537 and TA2637, when combined pyridine, with methyl isonicotinate, 4-methylpyridine, 4-ethylpyridine, 4-chloropyridine or 4-bromopyridine. Mixtures of CoCl₂ and isonicotinic acid, 4-cyanopyridine, 4-aminopyridine, or 4-dimethylaminopyridine exhibited no mutagenicity. Judging from the spectral observations, such combined mutagenicity may be due to the formation of moderate to weak complexes between these compounds and the Co(II) cation.     

Mutagenesis in Salmonella after metabolic activation of carcinogenic azo dyes and their isomers by liver S9 from rats, mice and hamsters

The mutagenicities of 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB) and 3'-CH2OH-DAB, potent hepatocarcinogens, activated by rat-liver S9 were compared with those of their isomers (2'- or 4'-substituted DAB) and with those obtained with liver S9 from mice, hamsters and man. All 6 aminoazo dyes showed positive mutagenicity on both strains TA98 and TA100 in the presence of liver S9 from rats pretreated with polychlorinated biphenyls (PCB) whereas 3'-Me-DAB and 3'-CH2OH-DAB were negative in the presence of S9 from other organs of rats and human liver. 3'-Me-DAB and 3'-CH2OH-DAB also showed negative or only a weak mutagenicity in the presence of liver S9 from non-treated animals. Treatment of the muta-carcinogens by liver S9 from PCB-treated mice or hamsters exerted mutagenicity on TA98, but less than that seen with rat-liver S9. The activity of 3'-Me-DAB in the presence of female rat-liver S9 was lower than that obtained with the male. Thus a specificity in the aminoazo dye carcinogenesis in regard to species, sex and organ was also observed in the mutagenic effects of 3'-Me-DAB on Salmonella.     

Mutagenicity of bithionol sulfoxide and its metabolites in the salmonella/mammalian microsome test

The mutagenic effects of bithionol sulfoxide and its two major metabolites, bithionol and bithionol sulfone, on 4 Salmonella typhimurium strains (TA97, TA98, TA100 and TA102) were investigated. Bithionol sulfoxide was found to be mutagenic to TA98 and TA100. However, mutagenicity was abolished in the presence of rat-liver S9 fractions.     

Inhibition of dinitropyrene mutagenicity in vitro and in vivo using Salmonella typhimurium and the intrasanguinous host-mediated assay

Dinitropyrenes (DNP), present in polluted air, are potent direct-acting mutagens in Salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of rat-liver S9 or microsomes. This has now been confirmed using mouse hepatic fractions. Since most in vitro test systems do not adequately simulate conditions encountered in the intact animal, we have investigated dinitropyrene mutagenicity to Salmonella in the host-mediated assay. 1,8-Dinitropyrene (1,8-DNP) given p.o. to BALB/c mice induced a weak mutagenic effect in S. typhimurium TA98 recovered from the liver 1 h after i.v. administration (optimum time). Over the entire dose range tested no toxicity to bacterial cells was detected. Mutation induction in vivo was dose-related with maximum response at 1 mg DNP/kg body weight. This optimum dose, however, was non-mutagenic to strains TA98/1,8-DNP6 (O-transacetylase-deficient) or TA98NR/1,8-DNP6 (nitroreductase- and O-transacetylase-deficient). 1,3-Dinitropyrene and 1,6-dinitropyrene were weakly mutagenic to TA98 at doses similar to 1,8-DNP. Studies with [14C]1,8-DNP showed that 1 h after oral dosing (1 mg/kg), over 100 ng of 1,8-DNP equivalents were present in the liver (= 0.73% dose). However, only about 5.5 ng were present in the bacterial pellet, suggesting that hepatic components in vivo, as in vitro, bind to DNP, thus interfering with its interaction with Salmonella.     

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.     

Results of a comparative study on the Salmonella pre-incubation and plate incorporation assays using test samples from the IPCS collaborative study.

Three complex mixtures (air particles, diesel particles and a coal tar fraction) and two pure compounds (benzo[a]pyrene and 1-nitropyrene) were tested in both the pre-incubation and the plate incorporation assay employing Salmonella typhimurium TA98 and TA100. Each experiment was conducted independently 2 or 4 times in duplicate in the presence and absence of metabolic activation. The mutagenic activities were calculated by least squares linear regression from the slope of the linear portion of each dose-response curve. Although slightly higher mutagenic activity was observed in the pre-incubation assay for the two pure compounds and with the plate incorporation assay for the diesel particulate sample, the overall data from both assays gave similar values and good correlations in TA100 and TA98. The results indicate that the pre-incubation assay could be used for these samples instead of the plate incorporation assay.     

Mutational studies with diquat and paraquat in vitro.

Diquat and paraquat were assayed in the following tests. (1) Ames test in Salmonella typhimurium (strains TA1535, TA1537, TA1538, TA98 and TA100) with and without rat-liver microsomal fractions. (2) Resistance to 8-azaguanine in Salmonella typhimurium (strain hisG46, TA92 and TA1535. (3) Repair test in Salmonella typhimurium (strains TA1538 and TA1978). (4) Gene mutations in Aspergillus nidulans: 8-AG resistance and methionine suppression (meth A1 locus). (5) Lethal recessive damage in Aspergillus nidulans. (6) Unscheduled DNA synthesis (UDS) in human epithelial-like cells (EUE). Diquat and paraquat were positive in S. typhimurium (in the repair test and the 8-AG resistance system), in A. nidulans (for gene mutations and lethal recessive damage induction) and in EUE cells (UDS induction).