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).     

Diethylstilbestrol and 11 derivatives: a mutagenicity study with Salmonella typhimurium.

Diethylstilbestrol was tested for mutagenicity with his- S. typhimurium strains under 10 different matabolic situations (no exogenous metabolizing system; S9 mix from liver homogenate of rats induced with Aroclor 1254, with or without inhibition of epoxide hydratase; liver and/or kidney S9 mix from control or hamsters treated with Aroclor 1254; horse-radish peroxidase + H2O2). Under none of these conditions did diethylstilbestrol give any indication of a mutagenic effect. Furthermore, 11 metabolites and other derivatives of diethylstilbestrol, 2 of them potent inducers of sister-chromatid exchange in cultured fibroblasts, were not mutagenic with any of the 4 tester strains (S. typhimurium TA100, TA98, TA1537, TA1535) in the presence or absence of S9 mix from liver homogenate of rats induced with Aroclor 1254. Thus, one of the few known human carcinogens is very resistant to detection by the mammalian enzyme-mediated Salmonella typhimurium mutagenicity test (Ames test). This is especially remarkable since the metabolizing systems used included: (1) some of very high metabolic activity (S9 mix from liver homogenate of rats and hamsters induced with Aroclor 1254); (2) metabolizing systems from organs susceptible to the carcinogenic activity of diethylstilbestrol (hamster kidney); as well as (3) a mixture of (1) and (2) in case both activities are required for the carcinogenic effect in the whole animal.     

Detection of mutagenic activity in particulate air pollutants.

Ames's strains of Salmonella typhimurium were used to evaluate the mutagenic activity of airbone particulate materials collected at six different points in the industrial area of Ohmuta and the residential area Fukuoka. Tests were done in presence of rat-liver S-9 fraction isolated from rats that had been treated with Aroclor 1254. When the number of revertant colonies per plate was plotted against the amount of methanol extract of particulate air pollutants, using strain TA98, approximately linear relationships were observed for active samples. Generally, mutagenic activity of the samples increased in proportion to the density of air pollutants. In our system, 38--349 microng of methanol extract, from 0.225--4.51 m3 of air from the factory districts in Ohmuta City gave 100 his+ revertants per plate. On the other hand, 54--2300 microng of air pollutants, from 1.29--14.1 m3 of air from the residential districts in Fukuoka City, gave a comparable activity. Every sample from each area had mutagenic activity. Chemicals in air pollutants were fractionated by alumina column chromatography and identified by gas chromatography and mass spectrometry. More than 28 compounds, including 12 unknown substances were identified as polycyclic hydrocarbons. Twelve of these compounds are already known to be carcinogens and to induce reversions to histidine independence in strain TA98 of Salmonella.     

Genotoxic activity of m-nitrobenzaldehyde

m-Nitrobenzaldehyde (MNB) was evaluated for mutagenic activity using the Ames microbial mutagenicity test and for its ability to induce DNA single-strand breaks in rat hepatocytes as measured by alkaline elution. MNB was tested in S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without pretreatment with liver microsomes (S9) isolated from rats pretreated with Aroclor 1254. MNB produced 2-fold or greater increases in revertants in TA1538, both with and without S9, and in TA100 with S9 only. A 2-fold increase in revertants was seen in TA98, but only at the highest dose tested which did not produce inhibition of background growth. MNB caused a greater than 3-fold increase in elution slope, with DNA alkaline elution assay, but only at highly cytotoxic doses and, therefore, is not considered genotoxic in this system. It is concluded that MNB possesses weak genotoxic activity.     

Evaluation of genotoxicity of N-nitrosodibenzylamine in Chinese hamster V79 cells and in Salmonella

Health concerns have arisen due to the formation of N-nitrosodibenzylamine (NDBzA; CAS No. 5336-53-8) in pork processed in a new type of rubber netting. In view of the potent carcinogenicity of related nitrosamines (e.g. N-nitroso-n-dibutylamine and N-nitrosodiethylamine), NDBzA was evaluated for genotoxicity in vitro in both Chinese hamster V79 cells and in Salmonella. In V79 cells, concentrations up to 25 micrograms/ml were tested with and without activation by rat or hamster hepatocytes. Significant elevation of SCE frequency was seen only at 25 micrograms/ml in the presence of uninduced hamster hepatocytes. Mutation to 6-thioguanine resistance was observed at 25 micrograms/ml, in the absence of hepatocytes and in the presence of induced (Aroclor 1254) or uninduced hamster hepatocytes, but not with rat hepatocytes. With uninduced rat hepatocytes, a small but significant (p less than 0.05) increase in the mutation frequency was seen with 10 micrograms/ml NDBzA. In the Salmonella assay, using a pre-incubation protocol and concentrations up to 1000 micrograms/ml, NDBzA was negative in strain TA98, and in TA100 with rat S9, but was positive at the highest dose in TA100 with hamster S9, and more strongly with Aroclor 1254-induced hamster S9. When activated by uninduced rat or hamster hepatocytes, as opposed to S9, NDBzA was negative with all tester strains. Hamster hepatocytes activated more than rat in the V79 studies, and hamster S9 was more strongly activating in the Salmonella assay. These results indicate that NDBzA is weakly mutagenic to both Salmonella and V79 cells.     

delta 1-Tetrahydrocannabinol and 1 alpha, 2 alpha-epoxyhexahydrocannabinol: mutagenicity investigation in the Ames test.

1,2-Epoxyhexahydrocannabinol is a metabolite of delta 1-tetrahydrocannabinol. Because many epoxides are mutagens, we investigated 1,2-epoxyhexahydrocannabinol as well as delta 1-tetrahydrocannabinol for mutagenicity with Salmonella typhimurium TA1535, TA1537, TA98 and TA100 in the presence and in the absence of S9 mix from liver homogenate of rats treated with Aroclor 1254. Additionally, an epoxide hydratase inhibitor was used in some experiments. Whereas several other epoxides and further positive controls, not requiring activation or activated under the same conditions, respectively, showed strong mutagenicity, no indications of a mutagenic hazard by 1,2-epoxyhexahydrocannabinol or by delta 1-tetrahydrocannabinol were found.     

Mutagenic activities of dictamnine and gamma-fagarine from dictamni radicis cortex (Rutaceae)

A methanol extract of Dictamni Radicis Cortex exhibited a mutagenic effect on Salmonella typhimurium TA100 and TA98 with S9 mix. Two mutagenic compounds in Dictamni Radicis Cortex were isolated on a Sephadex LH 20 column and silica gel column chromatography and by preparative TLC. These were identified as dictamnine and gamma-fagarine by UV, EI-Mass, 1H-NMR. Dictamnine and gamma-fagarine were mutagenic in strain TA100 and TA98 with S9 mix. The dose-response curves were linear in the range 10-40 micrograms. Dictamnine and gamma-fagarine had specific activities (His+/microgram) of about 50-70 revertant colonies in strain TA100, while in strain TA98 there were about 30-50 revertant colonies.     

Hepatocyte-mediated mutagenicity of mononitrobenzo[a]pyrenes in Salmonella typhimurium strains

The mononitro-substituted isomers of benzo[a]pyrene (B[a]P), 1-, 3- and 6-nitrobenzo[a]pyrene (NB[a]P), are environmental pollutants and are metabolized to mutagens in Salmonella by rat-liver homogenate postmitochondrial supernatant (S9) fractions. In this study, activation of these compounds to mutagens was investigated using the hepatocyte-mediated Salmonella mutagenicity assay. Hepatocytes from rats treated with Aroclor 1254 activated both 3-NB[a]P and 1-NB[a]P to mutagens, while 6-NB[a]P was not mutagenic. The positive mutagenicity responses were functions of both the chemical dose and the hepatocyte concentration. By using a nitroreductase-deficient strain (TA98NR) and a transesterificase-deficient strain (TA98/1,8-DNP6), it was verified that the direct-acting mutagenicities of 1- and 3-NB[a]P primarily were due to metabolic processes involving nitroreduction while the S9- and hepatocyte-mediated mutagenicity responses were also dependent on transesterification. When compared with the mutagenic responses produced with S9, the mutations induced by 1- and 3-NB[a]P in the presence of hepatocytes were relatively more dependent upon nitroreductase metabolism and less on transesterification. Thus, intact hepatocytes were capable of activating 1- and 3-NB[a]P to mutagenic metabolites and some of these metabolites appeared to be different from those produced by S9.     

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.     

Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test: activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources.

Over 70 naturally occurring and synthetic flavonoids were screened for mutagenicity with 5 tester strains in the Salmonella/mammalian microsome assay: TA1535, TA100, TA1537, TA1538 and TA98. Frameshift mutagenicity was confined to the flavonols (flavon-3-ols) in strain TA98, TA1537 and TA100. The two most mutagenic falvonols, namely, quercetin (3,3',4',5,7-pentahydroxyflavone) and kaempferol (3,4',5,7-tetrahydroxyflavone), exhibiting 12 and 7 revertants/nmol in TA98 respectively, are also the most common flavonols occurring in plants. Other flavonols exhibited less activity (revertants/nmol): galangin (2.0), rhamnetin (0.45), kaempferide (0.24), fisetin (0.14), myricetin (0.12), robinetin (0.06) and morin (0.05). All of these flavonols apparently exhibited significant activation by Aroclor 1254 induced rat-liver microsome preparations (S9). However, subsequent study revealed that only those flavonols either lacking or possessing one B ring hydroxyl group had an absolute requirement for microsomal activation. Alternatively, quercetin with two B-ring OH groups is not activated by microsomal enzymes, but by soluble (S100) enzymes from liver which are apparently constitutive and not subject to the usual chemical induction. 3 flavonol glycosides, namely, quercetrin (quercetin-3-O-rhamnoside), rutin (quercetin-3-O-rutinoside) and robinin (kaempferol-3-O-galactosido-rhamnoside-7-O-rhamnoside), were found to be nonmutagenic. They could, however, be activated by a variety of mixed glycosidases incorporated in the usual pour plate procedure. The most effective enzyme mixtures were obtained from rat cecal bacteria and from the snail Helix pomatia.     

Fluroxene mutagenicity.

The commercially available volatile anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) which contains the stabilizer N-phenyl-1-napthylamine, was tested for mutagenicity using four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100, and one strain of E. coli, WP2. In addition, purified fluroxene; N-phenyl-1-napthylamine; trifluoroethanol, a major metabolite of fluoroxene; and urine from rats anesthetized with fluroxene were tested. Several procedures were utilized including exposure of bacteria to vapor in desiccators and in liquid suspension. Results indicate that fluroxene, but not its stabilizer, was mutagenic to strains TA1535, TA100 and WP2 only in liquid suspension and only in the presence of a rat-liver enzyme system. Trifluoroethanol and urine from fluroxene-treated rat were not mutagenic to any strain of bacteria. These findings indicate that fluroxene is a promutagen which requires preincubation before it is recognized. Further experiments were performed with enzymes prepared from mouse, hamster and human liver. Fluroxene was mutagenic only in the presence of enzymes prepared from Aroclor 1254 pretreated rodents. Since fluroxene was not mutagenic in the presence of enzymes prepared from three human livers, the significance of these findings to man are unclear.     

The hair-dye reagent 2-(2',4'-diaminophenoxy)ethanol is mutagenic to Salmonella typhimurium

A new hair-dye ingredient, 2-(2',4'-diaminophenoxy)ethanol (2,4-DAPE), was described as being devoid of any genotoxic activity on the basis of a multi-laboratory study. Since 2,4-DAPE is a close analogue of 2,4-diaminoanisole (2,4-DAA), which is mutagenic and carcinogenic, we tested this claim by assaying 2,4-DAPE for bacterial mutagenicity. Two samples of 2,4-DAPE X 2HCl were synthesized by reduction of the corresponding dinitrophenoxyethanol and identity and purity were established by elemental analysis, NMR spectrometry, mass-spectrometry, UV-spectrophotometry, TLC and HPLC. Fresh aqueous solutions of 2,4-DAPE X 2HCl were assayed in several separate plate tests using S. typhimurium TA1538, TA97, TA98 and TA100, and E. coli WP2uvrA (pKM101), 3 plates per dose and 0%, 4%, 10% and 30% Aroclor 1254-induced rat-liver S9 in S9 mixes. We obtained negative results in TA100 and E. coli. Reproducible, statistically significant dose-related increases in revertants (up to 14 times the background) were obtained in frame-shift mutants of S. typhimurium in the dose range 10-80 micrograms per plate. Mutagenicity was S9-dependent, significant increases in revertants being obtained only with 50 microliter per plate or more of S9. 2,4-DAPE induced significant mutagenic effects at doses of less than 1 micrograms per ml in TA1538 and TA98 in fluctuation tests using 2% S9 in the S9 mix. In plate tests, 2,4-DAPE was less mutagenic (by a factor of about 8) than 2,4-DAA, which gave the highest mutant yields with 20 microliter S9 per plate (4% S9 in the S9 mix). 2,4-DAPE obtained commercially was about 8 times more mutagenic than our sample of 2,4-DAPE. After purification, the commercial product, now chromatographically identical with our own sample, gave plate-test results close to those obtained for our samples of 2,4-DAPE. A review of the published reports (in which 2,4-DAPE was claimed to be inactive in a variety of short-term tests) revealed: (a) the use of protocols for bacterial mutagenicity testing which, in the light of our own results, were probably too limited in scope, especially in the choice of conditions for metabolic activation; (b) insufficient information on the identification and purity of the samples of 2,4-DAPE tested in the published collaborative study.     

Biphenyl and fluorinated derivatives: liver enzyme-mediated mutagenicity detected in Salmonella typhimurium and Chinese hamster V79 cells.

Hepatocarcinogenic polychlorinated and polybrominated biphenyls usually show negative results in in vitro mutagenicity assays. Problems in their testing result from their low water solubility and their slow rate of metabolism. We therefore investigated better soluble model compounds, namely biphenyl and its 3 possible monofluorinated derivatives. In the direct test, these compounds proved to be nonmutagenic in Salmonella typhimurium TA98 and TA100 (reversion to histidine prototrophy) and in Chinese hamster V79 cells (acquisition of resistance to 6-thioguanine). However, when the exposure was carried out in the presence of NADPH-fortified postmitochondrial fraction of liver homogenate from Aroclor 1254-treated rats, all 4 compounds showed mutagenic activity in V79 cells. 3-Fluorobiphenyl produced strong mutagenic effects in S. typhimurium TA100 as well, whereas the other biphenyls were inactive. In strain TA98, 3- and 4-fluorobiphenyl showed mutagenic activity. This mutagenicity was enhanced in the presence of 1,1,1-trichloropropene 2,3-oxide, an inhibitor of microsomal epoxide hydrolase, thus suggesting that epoxides may be active metabolites.     

Mutagenic activation of 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole(Trp-P-1) and 3-amino-1-methyl-5H-pyrido(4,3-b)indole (Trp-P-2) by primary cultures of adult rat hepatocytes: effect of Aroclor induction in vitro

The mutagenic activation of tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, was studied in a Salmonella TA98/hepatocyte mutagenesis assay. Adult rat hepatocytes in primary culture were either untreated or induced by the addition of Aroclor 1254 (2 micrograms/ml) 18-20 h before the mutagenesis test which was performed at day 1 and at day 2 after the isolation of hepatocytes. The mutagenic activation of Trp-P-1 and Trp-P-2 was studied as a function of the time of incubation and of the concentration of chemical. Trp-P-1 and Trp-P-2 incubated for 20 min in the presence of untreated hepatocytes and bacteria gave rise to a weak number of revertants which doubled the level of spontaneous mutants. Aroclor-induced hepatocytes became highly competent in mutagenic activation of tryptophan pyrolysis products and the induction ratio reached 4.9 and 7.1 for Trp-P-1 and Trp-P-2, respectively, after 60 min of incubation, on day 2 of the experiment. It should be noted that the induction ratio was higher on day 2 than on day 1. When conditions were standardized, i.e. Aroclor-induced hepatocytes on day 2, final concentration of cellular protein about 1 mg/ml, 20 min of incubation, the Salmonella/hepatocyte assay produced a linear concentration-dependent mutagenic response for Trp-P-1 and Trp-P-2. By comparing the results obtained with Aroclor-induced hepatocytes and Aroclor-induced liver S9 fraction in the Salmonella test, it could be estimated that hepatocytes were 3 times less active than the S9 fraction with regard to mutagenic activation of both Trp-P-1 and Trp-P-2.     

The Salmonella/mammalian microsome mutagenicity test: comparison of human and rat livers as activating systems

The mutagenicity of several test compounds was verified by the Salmonella/microsome mutagenicity test (Ames test), using both human liver and rat liver (untreated or pretreated with Aroclor 1254) S9 under identical experimental conditions. Aflatoxin B1, 3-methylcholanthrene, and cigarette-smoke condensate were less mutagenic in the presence of human-liver S9 than in the presence of rat-liver S9 (particularly after treatment with Aroclor 1254). The opposite was observed with 2-aminonanthracene and to a lesser degree with 2-aminofluorene; correlation studies indicate that the two compounds were activated by the same or by very similar enzymes, probably cytochrome P-450s. These results clearly indicate that human-liver S9, as an activating system, behaves differently than rat-liver S9; therefore, it may constitute a useful, additional tool for the study of mutagenicity and probably, carcinogenicity in man.     

Mutagenicity of structurally related phenylazo-3-pyridines inSalmonella typhimurium

Studies were conducted to explore structure-activity relationships for 4'-N,Ndimethylamino-1'-phenylazo-3-pyridine and nine structurally related compounds in Salmonella typhimuriunz tester strains TA1535, TA100, TA1537, TA1538, TA98. Each compound was tested for mutagenicity at five or more concentrations that varied from 10-5000 pglplate. We used the standard plate test and the investigations were carried out both in the absence and presence of Aroclor-1254induced rat-liver homogenate and the components of the NADPHgenerating system. Negative response was observed for 4'-N,N-dimethylamino-1'-phenylazo-3-pyridine and five of its analogues (4'-N,N-diethylamino-1'phenylazo-3-pyridine; 4'-N,N-di-(-hydroxyethylamino)-1'phenylazo-3-pyridine; 4'-N-methylamino sulfonic acid-1'-phenylazo-3-pyrldine; 4'-N,N-dimethylamino-. 6'-acetamido-1'phenylazo-3-pyridine, and 4'-N,N-di-(-hydroxyethylamino)-6'-methyl-1'phenylazo-3-pyridine). When S9 induced by Aroclor-1254 was present, the compound 4'-N,N-dimethylamino-6-methoxy-1'phenylazo-3-pyridine exhibited mulagenic activity in the two strains TA1538 and TA98. The compound 4' ,6'-diamino-3-methyl-1'-phenylazo-3-pyridine was also mutagenic, both in the presence and in the absence of S9 mix. The two compounds 4'-NjVdimethylamino-6-butoxy-1'-phenylazo-3-pyridine and 4'N,N-di-(-hydroxyethylamino)-1'-phenylazo-3-[6-N,N-di-(-hydroxyethylamino)]-pyridine were either weakly mutagenic or nonmutagenic. On the basis of these data, it is concluded that the mutagenicity of phenylazo-3-pyrzdines, like monocyclic aromatic amines and azo dyes, is influenced by the nature of the substifuent chemical groups and their positions in the molecular structure of the compounds.     

Substrates and inhibitors of hepatic amine oxidase inhibit dimethylnitrosamine-induced mutagenesis in Salmonella typhimurium

The mutagenic effect of dimethylnitrosamine in Salmonella typhimurium TA100, in the presence of a rat-liver homogenate derived from animals treated with Aroclor 1254, was inhibited by substrates and inhibitors of monoamine oxidase. Substrates of diamine oxidase did not inhibit dimethylnitrosamine mutagenesis and, furthermore, monoamine oxidase inhibitors had no effects on mutagenesis by benzo[a]pyrene or aflatoxin B₁. The results suggest that monoamine oxidase participates in the activation of dimethylnitrosamine to a mustagen.     

Detection of 1-nitropyrene in yakitori (grilled chicken)

Pieces of raw chicken with or without a marinating sauce were grilled over a city gas flame, extracted with benzene-ethanol (4:1) by ultrasonication and fractionated into diethyl ether-soluble neutral, acidic and basic fractions. The mutagenicity of these fractions was measured with Salmonella typhimurium strains TA100, TA98, TA98NR and TA98/1,8-DNP6 in the presence and absence of a 9000 X g post-mitochondrial supernatant from Aroclor 1254-treated Sprague-Dawley rat liver (S9 mix). The basic fraction of yakitori without the sauce was more mutagenic than the other fractions for S. typhimurium strain TA98 in the presence of S9 mix. This is probably due to the presence of amino acid or protein pyrolysates. However, when the chicken was grilled with the sauce, the basic fraction showed lower mutagenicity for strain TA98 in the presence of S9 mix than did the same fraction without the sauce. The neutral fraction of yakitori with sauce showed high mutagenicity for strain TA98 in the absence of S9 mix, but low mutagenicity for strains TA98NR and TA98/1,8-DNP6, suggesting that this fraction might contain nitropyrenes (NPs). The neutral fraction of yakitori was analyzed by high-performance liquid chromatography (HPLC). The neutral fraction of the chicken grilled with the sauce for 3, 5 and 7 min contained 3.8, 19 and 43 ng, respectively, of 1-NP per gram of yakitori accounting for 3.0, 2.7 and 1.3%, respectively, of the total mutagenicity.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.