Mutagenicities of quinoline and its derivatives.

Quinoline, recently reported to be carcinogenic in rats [12], was mutagenic to Salmonella typhimurium tester strains TA100 and TA98 in the presence of the metabolic activation system S-9 mix. 2-Chloroquinoline, a non-carcinogen [12], was non-mutagenic with or without S-9 mix. 8-Hydroxyquinoline, which is t known to be carcinogenic, was mutagenic with S-9 mix to both bacterial strains. The mutagenicities of 17 other quinoline derivatives that are not known to be carcinogenic were tested, and 12 of these compounds were mutagenic.     

Mutagenic activity of 2-phenylbenzotriazole derivatives related to a mutagen, PBTA-1, in river water.

A mutagen, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]5-ami no-7-bromo-4-chloro-2H-benzotriiazole (PBTA-1), isolated from water of the Nishitakase River in Kyoto exhibits potent mutagenic activity in Salmonella typhimurium TA98 with S9 mix and has characteristic moieties, including bromo, chloro, acetylamino, bis(2-methoxyethyl)amino and primary amino groups on a 2-phenylbenzotriazole skeleton. The mutagenicities of PBTA-1, its congeners and five related 2-phenylbenzotriazoles were examined in S. typhimurium TA98 with S9 mix in order to elucidate the structure-activity relationships. The data obtained suggest that a primary amino group plays an essential role in the mutagenic activity as do aromatic amines including heterocyclic amines in cooked foods. The effect of planarity of the 2-phenylbenzotriazole ring was significant, and in addition, halogen groups of PBTA-1 influenced the enhancement of the mutagenic activity.     

Metabolic activation of 2,4-dinitrobiphenyl derivatives for their mutagenicity in Salmonella typhimurium TA98

In order to elucidate the mechanisms of mutagenic activation of nitroarenes, we tested the mutagenic potency of 18 kinds of nitroarenes including nitrated biphenyl, fluorene, phenanthrene and pyrene on Salmonella typhimurium TA98 in the absence and presence of S9 mix. The mutagenicities of 2,4-dinitrobiphenyl derivatives and 4-nitrobiphenyl were enhanced by the addition of S9. 2,4,6-Trinitrobiphenyl (3 net rev./10 micrograms without S9) was activated 60-fold by the mammalian metabolic system (181 net rev./10 micrograms with 10% S9). The mutagenic potency of 2,4,2',4'-tetranitrobiphenyl in TA98, TA98NR and TA98/1,8-DNP6 was also enhanced by the addition of 10% S9. But 1-nitropyrene and 1,3-dinitropyrene, which are well-known mutagens and carcinogens, were deactivated to 3% and 0.4%, respectively, by the addition of 10% S9. Separate addition of microsomal and cytosolic fractions slightly activated the mutagenicity of 2,4,6-trinitrobiphenyl, and 2,4,2',4'-tetranitrobiphenyl was activated not only by S9 but also by the cytosolic fraction.     

Study on mutagenicity and antimutagenicity of BHT and its derivatives in a bacterial assay

The mutagenicity of butylated hydroxytoluene (BHT) and its derivatives was investigated by the Ames method using Salmonella typhimurium TA98 and TA100 with or without S9 mix. The compounds were not mutagenic in either indicator strain at concentrations ranging from 50 to 330 micrograms/plate (SQ: 3,5,3',5'-tetra-tert-butylstilbenequinone; VI-III: unidentified), 500 micrograms/plate (BE: 3,5,3',5'-tetra-tert-4,4'-dihydroxy-1,2-diphenylethylene; VI: 2,6-di-tert-butyl-4-methyl-4-tert-butylperoxy-2,5-cyclohexadienone ; VI-I: unidentified; VI-II: 3-acetyl-2,5-di-tert-cyclopenta-2,4-dienone) and 1000 micrograms/plate (BHT). The antimutagenic effects of BHT and its derivatives on mutagenesis by chemical agents were investigated in Salmonella typhimurium TA98 and TA100 and Escherichia coli WP-2 hcr-. VI-II suppressed the mutagenesis induced in TA98 and TA100 by 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and that induced in WP-2 hcr- by 4-nitroquinoline-1-oxide (4NQO) without decreasing cell viability. In WP-2 hcr-, the mutagenesis induced by AF-2 and ethyl methanesulfonate was also suppressed significantly. Mutations induced by methyl methanesulfonate were slightly inhibited. However, VI-II had no effect on the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy-resin hardeners

3 epoxy-resin hardeners, 4,4'-diaminodiphenyl ether (DDE), 4,4'-diaminodiphenylmethane (DDM), and 4,4'-diaminodiphenylsulfone (DDS), and their N-acetyl and N,N'-diacetyl derivatives were examined for their mutagenicity using Salmonella typhimurium TA98 and TA100 as the tester stains and an S9 mix containing a rat-liver 9000 X g supernatant fraction as the metabolic activation system. DDE and DDM were mutagenic towards TA98 and TA100 in the presence of S9 mix while DDS exhibited no significant mutagenic activity towards these tester strains. These epoxy-resin hardeners were metabolized in vivo and their N-acetyl and N,N'-diacetyl metabolites were found in the urine. Among these acetyl metabolites, only N-acetyl-DDE was found to be mutagenic towards TA98 and TA100 in the presence of S9 mix. None of these acetyl metabolites exhibited significant mutagenic activity towards these tester strains in the absence of S9 mix.     

Mutagenic reactivities of 3,4-dinitrobiphenyl derivatives

3,4-Dinitrobiphenyl derivatives were mutagenic in Salmonella typhimurium TA98, TA98/1,8-DNP6 and in TA98NR. We describe here the specific reactivity of 3,4-dinitrobiphenyl derivatives with diluted sodium hydroxide solution and the determination of the amounts of released nitrous ion. 3,4-Dinitrobiphenyl derivatives begin to release nitrous ions when treated with NaOH solution at a concentration of 10(-3) N. The behavior of 4NQO and o-dinitrobenzene was the same as that of 3,4-dinitrobiphenyl derivatives. The residues of 3,4-dinitrobiphenyl derivatives, after releasing nitrous ions, were estimated to be hydroxy-nitrobiphenyls, as by GC/MS, we found the formation of o-nitrophenol in the reaction mixture of o-dinitrobenzene with aqueous NaOH solution. 3,4,4'-Trinitrobiphenyl, 3,4,3',4'-tetranitrobiphenyl and 4NQO had reduced mutagenic potency in Salmonella typhimurium TA98 following treatment with diluted NaOH. In order to elucidate the ultimate forms of 3,4-dinitrobiphenyl derivatives, we investigated the reaction of o-dinitrobenzene as a basic model substance of 3,4-dinitrobiphenyl, with nucleic bases in the presence of NaOH in nonaqueous solvent. o-Nitrophenyl guanine and adenine adducts were obtained.     

Structural activity relationship between Salmonella-mutagenicity and nitro-orientation of nitroazaphenanthrenes

Nitroazaphenanthrenes (NAphs) and their N-oxides (NAphOs) were synthesized as derivatives with nitrogen atoms in the 1, 4, and 9 positions of phenanthrene rings, and as nitrated derivatives substituted at the 1, 2, 3, 4, 5, 6, 7, and 8 positions of phenanthrene rings. To determine the structure activity relationship of these derivatives, all 19 isomers were bioassayed with Salmonella tester strains. NAphs substituted at the 4, 6, 7 and 8 positions were mutagenic for TA98, and 1-, 2-, and 3-N-9-AphOs, 6-N-1-AphO and 6-N-4-AphO were mutagenic for TA98 and TA100 without the S9 mix, while 5-N-1-AphO and 5-N-9-AphO were non- or weakly mutagenic. Nitrated derivatives, 6-N-4-Aph, 6-N-9-Aph, 6-N-1-AphO, and 6-N-4-AphO, were powerful mutagens for TA98 and TA100. Mutagenicity was enhanced by mutant strains producing nitroreductase, such as YG1021 and 1026, and by those producing O-acetyltransferase, such as YG1024 and 1029. Nitro derivatives substituted at positions 4 and 5 in the phenanthrene rings were perpendicular, while those at positions 2, 3, 6 and 7 were coplanar to the phenanthrene rings. NAphs substituted at the 1 and 8 positions were noncoplanar due to steric hindrance of the aromatic proton at the peri position. On the other hand, 1,5- and 1,8-dinitro-4-azaphenanthrenes showed high mutagenicity for strains TA98 and TA100 in the absence of the S9 mix, and were strongly enhanced by nitroreductase and O-acetyltransferase, over-producing mutants. Therefore, it was found that the mutagenic potency of NAphs and NAphOs was closely associated with the chemical properties and orientation of nitro substitution of aromatic rings.     

Mutagenicity of nitro- and amino-substituted phenazines in Salmonella typhimurium

The nitro- and amino-substituted phenazines were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98 and TA98NR. Of 7 tested nitrophenazines, 4 were mutagenic in the absence of a microsomal metabolic activation system (S9 mix) and were more mutagenic in TA98 than in TA98NR. The order of mutagenicity of nitrophenazines in TA98 is 1.7- less than 2- less than 2.8- less than 2.7-substituted phenazine. Of 7 tested amino derivatives, 4 exhibited mutagenic activity with S9 mix in TA98. 1-Nitro-, 1-amino, 1.6-dinitro-, 1.9-dinitro-, 1.6-diamino- and 1.9-diamino-phenazine were not mutagenic. As regards the relationship between mutagenic potency and chemical structure of the phenazines, the results suggested that structural requirements favoring mutagenic activity were the presence of substituents at the 2 and/or 7 position. Furthermore, 2.7-disubstituted phenazines were extremely mutagenic, 2.7-dinitrophenazine and 2.7-diaminophenazine induced 36,450 and 12,110 rev./nmole, respectively. In the preliminary study, 2.7-diaminophenazine was identified by gas chromatography/mass spectrometry from the reaction mixture of m-phenylenediamine and hydrogen peroxide.     

Mutagenic activity of the glutathione S-transferase substrate 1-chloro-2,4-dinitrobenzene (CDNB) in the Salmonella mutagenicity assay

The mutagenicity of the commonly used glutathione S-transferase substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) was investigated in the Salmonella mutagenicity assay. CDNB induced a concentration-dependent mutagenic response in Salmonella typhimurium strain TA98. Incorporation of an activation system derived from Aroclor 1254-induced rats did not influence mutagenic response. Under the same conditions DCNB failed to display mutagenic activity. The mutagenic activity of CDNB was attenuated in bacterial strains under-expressing nitroreductase or O-acetylase activity but, in contrast, it was exaggerated in an O-acetylase over-expressing strain. It is inferred that CDNB exhibits a mutagenic response following reduction of the nitro-group to the hydroxylamine, which is further acetylated to form the acetoxy derivative that presumably breaks down spontaneously to generate the nitrenium ion, the likely ultimate mutagen.     

Mutagenicities of indole and 30 derivatives after nitrite treatment

Indole and 7-derivatives, L- and D-tryptophan and 9 derivatives, and beta-carboline (norharman) and 11 derivatives were tested for mutagenicity to Salmonella typhimurium TA100 and TA98 after nitrite treatment. 1-Methylindole, which is present in cigarette smoke condensate (Grob and Voellmin, 1970; Hoffmann and Rathkamp, 1970), was the most mutagenic to TA100 without S9 mix after nitrite treatment, inducing 615,000 revertants/mg. 2-Methylindole, 1-methyl-DL-tryptophan, harmaline and (-)-(1S,3S)-1,2-dimethyl-1,2,3,4-tetrahydro-beta-carboline-3- carboxylic acid also showed strong mutagenicity after nitrite treatment, inducing 129,000, 184,000, 103,000 and 197,000 revertants/mg, respectively. These mutagenic potencies were comparable with those of benzo[alpha]pyrene, 3-methylcholanthrene and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) (Sugimura, 1982). Of 31 compounds tested, 22 were mutagenic after nitrite treatment. Since various indole compounds are ubiquitous in our environment, especially in plants, the presence of their mutagenicities after nitrite treatment warrants further studies, including those on their in vivo carcinogenicities.     

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.     

Mutagenicity of substituted phenanthrenes in Salmonella typhimurium

An extensive series of alkylated phenanthrenes was assayed for mutagenic activity in Salmonella typhimurium TA98 and TA100. Among the alkylated phenanthrenes assayed, 1-methylphenanthrene, 9-methylphenanthrene, 1,4-dimethylphenanthrene and 4,10-dimethylphenanthrene were active as mutagens. These studies suggest that the structural requirements favoring mutagenic activity among alkylated phenanthrenes are inhibition of 9,10-dihydrodiol formation and the presence of an unsubstituted angular ring adjacent to a free peri position. The mutagenic activities of 9-fluoro-, 9-chloro-, and 9-bromo-phenanthrene were also evaluated. The positive mutagenic response of these halogenated phenanthrenes further supports the observation that inhibition of 9,10-dihydrodiol formation among substituted phenanthrenes favors mutagenic activity.     

Mutagenicity of K-region derivatives of 1-nitropyrene; remarkable activity of 1- and 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one

The mutagenic activities toward S. typhimurium strains TA98 and TA100 of K-region derivatives of 1-nitropyrene and pyrene were determined. The compounds tested were trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene (Compound 3), trans-4,5-dihydro-4,5-dihydroxypyrene (Compound 4), 1-nitropyrene-4,5-quinone (Compound 5), 1-nitropyrene-9,10-quinone (Compound 6), pyrene-4,5-quinone (Compound 7), and the lactones, 1-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 8), 3-nitro-5H-phenanthro[4,5-bcd]pyran-5-one (Compound 9), and 5H-phenanthro[4,5-bcd]pyran-5-one (Compound 10). Neither pyrene nor any of its K-region derivatives was mutagenic, either in the absence or presence of S9 mix at the doses tested. Of the K-region derivatives of 1-nitropyrene, the lactones (Compounds 8 and 9) were generally the most active; 0.25 microgram/plate induced 900-2200 revertants in TA98 or TA100 without activation. The 4,5-dihydrodiol (Compound 3), an established mammalian metabolite of 1-nitropyrene, was less mutagenic than was 1-nitropyrene in TA98, but was more mutagenic than was 1-nitropyrene in TA100, regardless of the presence of S9 mix. The quinones (Compounds 5 and 6) were less mutagenic than was 1-nitropyrene in the absence of S9 mix in both strains, but their activities were increased in the presence of S9 mix. The mutagenic activities of the lactones (Compounds 8 and 9) were lower in strains TA98NR and TA98/1,8-DNP6 than in TA98, indicating that nitro-reduction and esterification are involved in their activation. The results of this study indicate that K-region derivatives of 1-nitropyrene may be important in its metabolic activation.     

The mutagenicity of halogenated alkanols and their phosphoric acid esters for Salmonella typhimurium.

9 halogenated alkanols, 9 corresponding tris (haloalkyl)phosphates, and 2 bis-(2,3-dibromopropyl)phosphate salts were evaluated for mutagenicity against Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538, with and without rat liver in vitro metabolic activation system (S9 mix). Most of the test samples showed mutagenic activity in the strains TA100 and TA1535, but not in the strains TA98, TA1537 and TA1538. In general, the mutagenic activities of the phosphates obtained with S9 mix were greater than the activities obtained without S9 mix. Among the phosphates, several structure--activity relationships were found; i.e., (i) the bromoalkyl derivatives were more mutagenic than the corresponding chloroalkyl derivatives, (ii) the beta-haloethyl derivatives were more mutagenic than the gamma-halopropyl derivatives, (iii) the phosphates having adjacent beta and gamma halogen atoms in the alkyl moiety, e.g., tris-(2,3-dibromopropyl)phosphate, were particularly potent mutagens, (iv) the branched carbon chain reduced the mutagenic activities in spite of the presence of beta-halogen atoms, e.g., tris(1-bromomethyl-2-bromoethyl)phosphate. However, such relations did not necessarily apply to the halogenated alkanols. It is concluded that the metabolic activation pathway via haloalkanols to mutagens must not be in common with all tris-BP-like phosphates.     

Mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solutions

The mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated naphthoresorcinol was mutagenic in TA97, TA98, TA100 and TA104 without S9 mix. By the addition of S9 mix, the mutagenic activity of ozonated naphthoresorcinol was markedly suppressed in TA98 and TA100, but became positive in TA102. High-performance liquid chromatography (HPLC) after derivatization to 2,4-dinitrophenylhydrazones demonstrated the formation of glyoxal as an ozonation product of naphthoresorcinol. Ion chromatographic technique also demonstrated the formation of o-phthalic acid, muconic acid, maleic acid, mesoxalic acid, glyoxylic acid and oxalic acid as ozonation products. The mutagenicity assays of these identified products with five Salmonella showed that glyoxal and glyoxylic acid were directly mutagenic; the former in TA100, TA102 and TA104, the latter in TA97, TA100 and TA104. In the presence of S9 mix, glyoxylic acid gave a positive response of mutagenicity for TA102. The experimental evidence supported that glyoxal and glyoxylic acid may contribute to the mutagenicity of ozonated naphthoresorcinol.     

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.     

Enhancement of the mutagenicity of IQ and MeIQ by nitrite in the Salmonella system.

The fried food mutagens IQ, MeIQ, Glu-P-1 and Trp-P-2 were treated with nitrite at pH 3.0 for 1 h at 37 degrees C. The resulting reaction mixtures were tested for mutagenicity towards Salmonella typhimurium TA97, TA98, TA100 and TA1535. Glu-P-1 and Trp-P-2 were readily converted to weak or non-mutagenic deaminated compounds, whereas IQ and MeIQ were converted to extremely strong mutagenic derivatives in both the presence and the absence of rat liver S9 mix. The mutagenicity of MeIQ in TA98 was enhanced by nitrite up to 3-fold, while that of nitrosated MeIQ was further enhanced by S9 mix up to 15-fold. The nitrosation products of MeIQ were resolved into 7 bands by TLC on silica gel plate. Bands I, III, V and VI were highly mutagenic to both TA98 and TA100. The experimental results suggest that the non-enzymatic formation of direct-acting mutagens from indirect-acting mutagens such as IQ or MeIQ might be physiologically important, especially with regard to the etiology of human gastrointestinal tract tumors.     

Evaluation of genotoxity and mutagenicity of DL-p-chlorophenylalanine, its methyl ester and some N-acyl derivatives

DL-p-chlorophenylalanine (PCPA) and its derivatives were evaluated for genotoxic effects using Escherichia coli and Bacillus subtilis strains lacking various DNA-repair mechanisms in spottest and in suspension test. The mutagenic activity of studied compounds was determined by the Ames test. Reverse mutation test was performed with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 without S9 mix. 0.02 M nitrosomethylurea (NMU) standard mutagen was used as a positive control. The results showed that the parent nonessential amino acid PCPA had no detectable genotoxic and mutagenic activities in bacteria. The methyl ester of this amino acid and its N-phenylacetyl derivative possessed weak genotoxicity. Meanwhile N-sec-butyloxycarbonyl, N-benzyloxycarbonyl, N-(p-nitrophenylacetyl) and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine exhibited appreciable genotoxicity. Among the seven tested compounds only N-benzyloxycarbonyl and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine have been found to be mutagenic. Only parent PCPA possessed antimutagenic properties in respect of nitrosomethylurea. The structural modification, which strongly affects genotoxicity and mutagenicity perhaps may be due to steric hydrance of the substituents, causing interference with enzyme and DNA interactions.     

Structural determination of a directly mutagenic amino-nitrobiphenyl as the S9 metabolite of 2,4,2',4'-tetranitrobiphenyl in Salmonella typhimurium TA98.

In order to elucidate the mechanisms of mutagenic activation of nitrobiphenyls by mammalian activation systems, 2,4,2',4'-tetranitrobiphenyl was incubated with S9 and its mutagenic metabolites were separated by SiO2 and Al2O3 column chromatography. The most mutagenic diamino-dinitrobiphenyl was isolated from the reaction mixture of 2,4,2',4'-tetranitrobiphenyl with S9 mix at 37 degrees C for 48 h, and its mutability was 4646 revertants/50 ng in Salmonella typhimurium TA98 without S9 mix. The deamination product of this most mutagenic metabolite was identical to 2,4'-dinitrobiphenyl by gas chromatography-mass spectrometry. Therefore, the structure of the metabolite was determined as 2,4'-diamino-2',4-dinitrobiphenyl by its chemical and physico-chemical properties.     

Phenazine derivatives as the mutagenic reaction product from o- or m-phenylenediamine derivatives with hydrogen peroxide

8 Kinds of o- and m-phenylenediamine (PD) derivatives, which are used as oxidative-type hair dyes, were treated with hydrogen peroxide (H2O2). Both before and after H2O2 treatment, their mutagenicity was tested by using Salmonella typhimurium TA98 in the presence or absence of a mammalian metabolic activation system (S9 mix). After H2O2 treatment, the mutagenic potencies of p-nitro-o-phenylenediamine, 3,4-diaminotoluene, p-nitro-m-phenylenediamine and 2,4-diaminophenol did not vary or slightly increased in comparison with those of the starting materials. The mutagenicity of o-PD, p-chloro-o-phenylenediamine (p-Cl-o-PD), m-PD and 2,4-diaminoanisole (p-OMe-m-PD) was enhanced remarkably by treatment with H2O2 and all the oxidation products required metabolic activation by S9 mix for their mutagenesis. In a gas chromatography/mass spectrometric study, 2,3-diaminophenazine and 2,7-diaminophenazine were identified with authentic samples in o-PD and m-PD oxidation mixture, respectively. The oxidation mixture obtained from p-Cl-o-PD and p-OMe-m-PD was separated into several fractions by repeated column chromatography. Brownish yellow crystals were isolated from oxidized p-Cl-o-PD and the structure of the compound was determined to be 2,3-diamino-7-chlorophenazine from physicochemical and chemical evidence. Two reddish yellow crystals, obtained from oxidized p-OMe-m-PD, were 2,7-diamino-3,8-dimethoxyphenazine and 2,7-diamino-3-methoxyphenazine. The number of revertants induced by 1 nmole of phenazines detected from oxidized PD derivatives was as follows; 2,3-diaminophenazine: 349 rev.; 2,3-diamino-7-chlorophenazine; 406 rev.: 2,7-diaminophenazine: 12 110 rev.; 2,7-diamino-3,8-dimethoxyphenazine: 4229 rev.; 2,7-diamino-3-methoxyphenazine: 24 640 rev. in S. typhimurium TA98 strain with 25 microliters S9 per plate.