Evaluation of the mutagenic and cytostatic potential of aristolochic acid (3,4-methylenedioxy-8-methoxy-10-nitrophenanthrene-1-carboxylic acid) and several of its derivatives

Aristolochic acid (1), a constituent of Aristolochia species, has been used for medicinal purposes since the Graeco-Roman period. Following the observation that the compound was mutagenic and carcinogenic, it was removed from pharmaceutical products. Consistent with previous reports, we have found that 1 serves as a direct-acting mutagen in Salmonella typhimurium strains TA100, TA102, TA1537 and TM677, but was not active in the nitroreductase-deficient strains TA98NR and TA100NR. However, aristolic acid (2), a compound that differs in structure only by the absence of the nitro group, was also found to be a direct-acting mutagen in Salmonella strains TA98, TA100, TA102, TA1537, and TM677, as well as strains TA98NR and TA100NR. Both compounds (1 and 2) were active mutagens when evaluated with cultured Chinese hamster ovary cells. Thus, in contrast to previous suggestions, the nitro group at position 10 is not required to induce a mutagenic response. Also, a series of structural relatives (the methyl esters of 1 and 2 (3 and 4, respectively), aristolochic acid-D (5), aristolactam (6), aristolactam A-II (7), and aristolactam-N-beta-D-glucoside (8)) were evaluated for mutagenic potential with Salmonella typhimurium strain TM677 and found to be inactive. Since compounds 3 and 4 were found to be active mutagens with Salmonella typhimurium strains TA98, TA100, TA102 and TA1537 (sufficient quantities of compounds 5-8 were not available for testing), differential sensitivity of the tester strains unrelated to mutagenic potential is suggested. Further, compounds 1, 2, and 6-8 were evaluated for potential to inhibit growth with cultured KB or P388 cells. P388 cells were substantially more sensitive, and compound 1 was the most active of the materials tested (ED5 = 0.58 microM). Compound 6 also demonstrated appreciable activity (ED50 = 4.2 microM), as did compound 8 (ED50 = 6.0 microM). It therefore appears that phenanthrene-ring substituents, in addition to the nitro group at position 10, serve important roles for biological potential. In considering the carcinogenic event induced by aristolochic acid, these functionalities should also be taken into account.     

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs

Quercetin, rhamnetin, isorhamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation.Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3′ and 4′ positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.     

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 dichloroethylamino derivatives of nitronaphthofuran and some nitrobenzofurans in the Salmonella/microsome assay.

The mutagenic activity of five dichloroethylamino 2-nitrobenzofuran derivatives and one dichloroethylamino 2-nitronaphthofuran derivative was analysed in the Salmonella/microsome assay. We investigated the influence of the position of the dichloroethylamino and/or the methoxy groups on the mutagenic activity of these nitro arenofurans in S. typhimurium strain TA100 and its variant TA100NR, deficient in nitroreductase. Without metabolic activation 7-[bis(2-chloroethyl)amino]-2-nitronaphtho[2,1-b]furan (1), 4-[bis(2-chloroethyl)amino]-7-methoxy-2-nitrobenzofuran (2), 7-[bis(2-chloroethyl)amino]-4-methoxy-2-nitrobenzofuran (5) and 6-[bis(2-chloroethyl)amino]-2-nitrobenzofuran (6) are mutagenic in TA100, while 4-[bis(2-chloroethyl)amino]-5-methoxy-2-nitrobenzofuran (4) is weakly mutagenic and 5-[bis(2-chloroethyl)-amino]-2-nitrobenzofuran (3) toxic. In the NR deficient strain compounds 1, 3 and 6 are strong mutagens and 4 is weakly positive. The two isomers 2 and 5 are negative in that strain. The naphthofuran derivative 1 is highly mutagenic in the absence of S9 mix in both strains considered, but less than R7000 (7). A decrease in the electronic polarity of compound 1 versus compound 7 according to the hypothesis developed by Royer et al. is a possible explanation. After exogenous metabolic activation by S9 mix all the compounds tested are highly mutagenic in both Salmonella strains. The position of the dichloroethylamino group and/or the presence of a methoxyl on the alpha-nitroarenofuran derivatives seem to modify the activity of bacterial as well as exogenous nitroreductases or other activating enzymes.     

The effects of 4'-alkyl substituents on the mutagenic activity of 4-amino- and 4-nitrostilbenes in Salmonella typhimurium

Six derivatives of trans-4-aminostilbene bearing different alkyl groups in the 4'-position and six of the corresponding nitro compounds were synthesized and tested for their mutagenic potency in Salmonella typhimurium strains TA98 and TA100. Regarding the test series in presence of S9-mix, maximum activity was observed for those trans-4-aminostilbenes and trans-4-nitrostilbenes bearing small alkyl substituents like methyl and ethyl. More bulky substituents reduced the mutagenic potential in the order iso-propylethyl>iso-propyl>sec-butyl>tert-butyl). These trends have been compared with quantitative structure activity relationship (QSAR) model predictions, leading to the conclusion that steric demand is an important factor for mutagenicity of substituted aminostilbenes and nitrostilbenes. The unexpected result for the tert-butyl nitrostilbene tested with metabolic activation may be attributed to a different metabolic pathway.     

Effect of various alkyl and unsaturated substituents on the mutagenicity of some nitrophenyl thioethers.

A variety of nitro-substituted phenyl alkyl/aryl thioethers and nitroso-substituted phenyl alkyl/aryl thioethers have been synthesized and tested for their mutagenicity towards Salmonella typhimurium strain TA100, TA98, TA98NR and TA98/1,8-DNP(6) in the absence of S9 mix. The relative order of mutagenicity in TA98 and TA100 among p-nitrophenyl thioethers having alkyl or aryl substituents is allyl>phenyl>benzyl>butyl>propyl>ethyl>methyl. Compounds having an alkyl chain C(6) to C(12) were found to be non-mutagenic. Among the various positional isomers (ortho, meta and para) of nitro-substituted diphenyl thioethers only the compounds having the -NO(2) function at the para position is mutagenic, whereas compounds having a -NO(2) function at ortho and meta are non-mutagenic. However, the reduced intermediate, ortho-nitroso derivative was found to be mutagenic in all the four strains but the meta-nitroso derivative was found to be non-mutagenic. All mutagens were found to be non-mutagenic when tested in nitroreductase deficient strain TA98NR, whereas their nitroso intermediates are found to be mutagenic. A substantial fall in the mutagenic activity is observed when some mutagens are tested in O-acetyltransferase deficient strain TA98/1,8-DNP(6).     

Mutagenic effect of furocoumarin monoadducts and cross-links on bacteriophage lambda

The mutagenicities of 17 closely related oxiranes were determined in 4 tester strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537). The test compounds comprised all possible oxides of benzene and its partially hydrogenated congeners. In TA100 and TA1535, 12 of the tested oxiranes were weak to moderate mutagens. 4 of these were also active in TA98. No mutagenicity was observed with the remaining 5 compounds in any of the 4 strains.The presence of a double bond in formal conjugation with the epoxide ring increased the mutagenicity relative to that of the saturated oxirane. Interestingly, additional epoxide rings within the same molecule did not markedly increase the mutagenic activity, and for the oxiranes that are not activated by a double bond, the relationship between mutagenic activity and the number of epoxide rings in the molecule was even inverse.The influence of bromo and hydroxyl substitution on oxirane mutagenicity is discussed. Most notably, a compound having a 4-hydroxyl group in syn position to a 1,2-epoxide ring fused to the cyclohexane ring, a structure which has been suggested to increase the electrophilic reactivity of dihydrodiol epoxides through hydrogen bonding, was almost inactive.     

Mutagenicity of natural naphthoquinones and benzoquinones in the Salmonella/microsome test

The mutagenicities of naturally occurring naphthoquinones and benzoquinones were tested by the pre-incubation method with Salmonella typhimurium strains TA98, TA100 and TA2637, which all contain plasmid pKM101. 6 of the 16 naphthoquinones tested, i.e., plumbagin, naphthazarin, 2-hydroxy-naphthoquinone, vitamin K3 (menadione), juglone and 7-methyljuglone, were mutagenic to strain TA2637 with metabolic activation. Except for juglone and 7-methyl-juglone, these compounds also had slight mutagenic effects on strain TA98 with S9 mix. All the mutagenic naphthoquinones contain one or two hydroxyl and/or methyl substituents. The naphthoquinone mompain, which has four hydroxyl groups, was not mutagenic. Unsubstituted beta-naphthoquinone, naphthoquinones with a prenyl side chain and all bi-naphthoquinone derivatives tested were non-mutagenic. None of the 13 benzoquinones examined was mutagenic to any of the strains used with or without metabolic activation. These results show that natural naphthoquinones are mutagenic when they have only one or two hydroxyl and/or methyl substituents.     

Mutagenic activity and structure-activity relationships of short-chain dialkyl N-nitrosamines in a hamster hepatocyte V79 cell-mediated system

A series of 19 short chain dialkyl N-nitrosamines was studied for mutagenic activity in an uninduced hamster hepatocyte V79 cell-mediated mutagenesis system. Ouabain was used as the selective agent to quantitatively analyze for chemically induced mutants. None of the nitrosamines was mutagenic in the absence of hamster hepatocyte activation. The relative mutagenic activities of the nitrosamines at an equimolar dose are presented. The results of the study indicated that: (a) increasing alkyl chain length decreased mutagenic activity; (b) oxidation of the carbon position to a carbonyl group increased the mutagenic activity of symmetrical and asymmetrical nitrosamines, whereas oxidation to a hydroxyl group only increased the mutagenic activity of the asymmetrical nitrosamines tested and (c) the carbon position at which oxidation occurred was important in determining mutagenic activity. The relationships between structure, metabolic activation, and mechanisms of mutagenic activity are discussed.     

Mutagenic activity of certain synthetic steroids: structural requirement for the mutagenic activity in Salmonella and E. coli

Eight steroids, structurally related to cholesterol, were tested for mutagenic activity in the Ames tester strains. All the test compounds were mutagenic without metabolic activation, although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA and lexA of Escherichia coli was observed as compared to their wild-type counterpart in the presence of the steroids. The role of recA and lexA genes gains further support from the lambda prophage induction in the lysogen as well as with Salmonella strains triggering the error-prone SOS response. Structural features which appear to be essential for mutagenic activity in these strains of the steroids are (1) reactive thio, sulfonyl or sulfinyl groups at the 6 position and (2) a halogen group at the 3 position of the steroidal nucleus. The mutagenicity appears to involve the formation of H2O2 as well as superoxide and hydroxyl radicals.     

Prophage induction and mutagenicity of a series of anti-tumour platinum(II) and platinum(IV) co-ordination complexes

11 platinum compounds with nitrogen donor ligands, previously tested for anti-tumour activity, were studied for induction of prophage lambda and for mutagenicity in the Ames assay, with various strains of Salmonella. The compounds included cis and trans isomers of Pt(II) and Pt(IV) complexes and were tested with and without metabolic activation. All the cis compounds elicited prophage induction, whereas the trans compounds were inactive. Mutagenicity was found only in strains containing the R factor, indicating that SOS-type repair processes are required for the conversion of initial DNA lesions into mutations. Mutation induction was also influenced by the excision-repair process. The 2 trans compounds were not, or only slightly, mutagenic; all other compounds were mutagenic in at least one strain, exhibiting a 2-20-fold increase over the spontaneous background level. Addition of liver homogenate had no significant effect on the number of mutants. One compound induced exclusively frameshift mutations. The other mutagenic compounds induced frameshift mutations as well as base-pair substitutions. 7 compounds were more mutagenic for the repair-proficient than for the repair-deficient strains; only one showed the opposite effect. This suggests that for mutagenicity testing of platinum compounds, repair-proficient strains are more sensitive indicators. The differences in response of the various strains are more sensitive indicators. The differences in response of the various strains toward the compounds suggest the formation of different DNA lesions and/or a selective action of repair processes on these lesions. In general, a good qualitative correlation was observed between prophage-inducing capacity, mutagenicity in bacterial and mammalian cells and anti-tumour activity.     

Mutagenic activities of ten imidazole derivatives in <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

Ten imidazole derivatives were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 both in the absence and presence of metabolic activation by the microsomal fraction S9 mix. In a general manner, derivatives tested exhibited a greater mutagenic activity in the TA100 strain comparing to the responses in TA 98. In the standard plate incorporation assay, 8 of these substances (80%) were found to be mutagenic for at least one of the two strains in the presence or absence of metabolic activation. Two compounds showed positive results in TA98 and 6 compounds were also mutagenic in TA100 without S9. In the presence of S9 mix, all of the 10 substances were non-mutagenic in TA98, whereas 4 compounds were positive in TA100. The results suggested the mutagenic potentials of the imidazole derivatives particularly inducing the reversion of base-pair substitutions. According to the structure-activity relationships phenyl groups in position 2 with different substituents can confer the mutagenic activity of the tested compounds. Methyl groups in different positions of these phenyl substituents can cause different types of mutations. This mutagenic effect is observed more clearly when the phenyl group is inhibited with a nitro group.     

Structural specificity of aromatic compounds with special reference to mutagenic activity in Salmonella typhimurium--a series of chloro- or fluoro-nitrobenzene derivatives

The mutagenicity of 21 chloro- or fluoronitrobenzene compounds and 9 chloro- or fluorobenzene compounds in Salmonella typhimurium (strains TA98, TA1538, TA1537, TA100 and TA1535) was examined. The tests were carried out under the conditions of absence and presence of liver microsomal activation. 15 nitro-group compounds had mutagenic activity; above all, compounds of fluoronitrobenzene were mutagenic for both types of strain. On the other hand, chloronitrobenzene compounds were mutagenic for base-pair substitution strains only. Mutagenic activity was exhibited by all compounds having a chloro or fluoro substituent at the para and ortho position in the nitrobenzene nucleus. All compounds without a nitro substituent showed no mutagenic activity.     

Lack of mutagenicity of some phytoestrogens in the salmonella/mammalian microsome assay

8 phytoestrogens were tested for mutagenicity using a variation of the Salmonella/mammalian microsome (or Ames) assay. Zearalenone is a mycotoxin produced by a grain contaminant, Fusarium graminearum (Gibberella zeae) and the isomers of zearalanol are reduced derivatives of this compound. The remaining compounds are all flavonoids which occur naturally at relatively high concentrations in many plants, particularly legumes. 4 of these flavonoids (daidzein, genistein, formononetin and biochanin-a) are isoflavones and the 5th, coumestrol, is a coumestan. Each compound was tested at several concentrations ranging from 1--500 micrograms per plate. The microsomal fracton was obtained from Aroclor 1254 (a PCB)-induced rat livers. None of the compounds tested was mutagenic to Salmonella strains TA1538, TA98 or TA100 at any concentration.     

Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium.

40 compounds structurally related to the plant flavonol quercetin were tested for mutagenic activity in Salmonella typhimurium strain TA98. 10 flavonols, quercetin, myricetin, rhamnetin, galangin, kaempferol, tamarixetin, morin, 3'-O-methylquercetin, 7,4'-di-O-methylquercetin and 5,7-di-O-methyl-quercetin, exhibited unequivocal mutagenic activity. 4 compounds, quercetin, myricetin, rhamnetin and 5,7-di-O-methylquercetin, were active without metabolic activation, although metabolic activation markedly enhanced their activity. All 4 have free hydroxyl groups at the 3' and 4' positions of the B ring. The other active compounds required an in vitro rat-liver metabolizing system for significant activity. Structural features which appear essential for mutagenic activity in this strain are a basic flavanoid ring structure with (1) a free hydroxyl group at the 3 position, (2) a double bond at the 2, 3 position, (3) a keto group at the 4 position, and (4) a structure which permits the proton of the 3-hydroxyl group to tautomerise to a 3-keto compound. The data are consistent with the requirement for a B ring structure that permits oxidation to quininoid intermediates. Free hydroxyl groups in the B ring are not essential for activity if a rat-liver metabolic activating system is employed. Data from 12 compounds which differ only at the essential sites described above indicate that the structural requirements for mutagenicity in strain TA100 are the same as those for activity in strain TA98. Based on the above structural requirements, a metabolic pathway for flavonol activation to DNA-reactive species is proposed.     

Mutagenicity of organophosphorus compounds in bacteria and drosophila

140 Organophosphorus compounds (OP's) have been tested for mutagenic activity in bacteria, principally by using two specially constructed sets of tester strains of the bacteria Salmonella typhimurium and Escherichia coli. It was found that 20% gave positive mutagenic responses and that this group of chemicals produce base substitutions rather than frame-shift mutations. In most cases the DNA repair genes exrA⁺ and recA⁺ were for mutagenic activity.Seven compounds were further tested in Drosophila melanogaster for the ability to induce recessive lethal mutations. In some of these cases the doses administered to the flies had to be very low due to the highly toxic nature of the compounds. To overcome this problem, the accumulation of recessive lethal mutations was measured in populations which were continually exposed to the compounds over a period of some 18 months. During this time the populations developed increased resistance to the compound and so the dose administered could gradually be increased. Six of the compounds were mutagenic.Of the compounds tested in both systems, those showing mutagenic activity in bacteria were also mutaganic in Drosophila, those mutagenic in bacteria were not mutagenic in Drosophila.     

Investigations on mutagenicity and genotoxicity of pentamidine and some related trypanocidal diamidines

Pentamidine, DAPI and some related compounds (DAI, 6-Br-AI, DPTN, DIPI, 3-Am-DAI, DiaPBF) were investigated in 2 different screening test systems for their potential mutagenic and cytotoxic effects, in the light of their binding to DNA. In the Ames test using Salmonella typhimurium strains TA98 and TA100 with and without metabolic activation no mutagenic effects could be observed. All diamidines tested, except DAI, were toxic at concentrations of 0.5 and 1.0 mumole/plate. In the sister-chromatid exchange (SCE) assay with human peripheral lymphocytes all compounds tested were growth-retarding particularly in the G0 phase. A significant induction of SCEs could only be seen after treatment with the monoamidino compound 6-Br-AI at a concentration of 100 mumole/l. It is concluded from the data obtained that pentamidine and related diamidines in the 2 assays tested show no mutagenic or genotoxic effects, in spite of their tight binding to DNA.     

Influence of the Uvr repair system on the mutagenicity of antiparasitic drugs

One amebicide (chloroquine diphosphate) and 2 anthelmintic compounds (niclosamide and pyrvinium pamoate) were found to be mutagenic for Salmonella typhimurium TA1537, TA1538, TA100 and TA98 Uvr- strains respectively. Drugs tested on homologous Uvr+ strains (TA1977, TA1978, UTH8414 and UTH8413) showed decreased mutagenic activity of the compounds. This indicates that premutational damage induced by the drugs was totally or partially repaired. Furthermore, results obtained in the present study suggest that niclosamide and pyrvinium pamoate induce premutational lesions by adduct formation, and that chloroquine diphosphate, known as an intercalating agent, behaves as an adduct-forming compound as regards its effects on Uvr- and Uvr+ S. typhimurium strains.     

Mutagenic activity of 6-aminoquinoxalines in Salmonella typhimurium

Mutagenicity of 6-aminoquinoxaline derivatives was tested with Salmonella typhimurium strains Ta98 and TA100 in the presence and absence of S9 mix from the viewpoint that the 6-aminoquinoxaline skeleton is a common unit of mutagenic imidazoquinoxalines. We tested nine compounds: 5-methyl-6-methylaminoquinoxaline (1), 3,5-dimethyl-6-methylaminoquinoxaline (2), 2,5-dimethyl-6-metnylaminoquinoxaline (3), 6-methylamino-2,3,5-trimethylquinoxaline (4), 2,3-diethyl-5-methyl-6-methylaminoquinoxaline (5), 5-methyl-6-methylamino 3-phenylquinoxaline (6), 6-amino-2,3,5-trimethylquinoxaline (7), 6-dimethylamino-2,3-5-trimethylaminoquinoxaline (8), 6-amino-2,3-dimethylquinoxaline (9). These compounds showed the mutagenic activity for both TA98 and TA100 in the presence of S9 mix, where they were more sensitive for TA100 strain. Methyl groups at the 2, 3 and/or 5 positions increased the potency of mutagenicity (1 < 2 < 3 ⪡ 4, 9 < 7). However, ethyl groups at the 2 and 3 positions lowered the mutagenicity of the methyl substitute but elevated it of the parental compound (1 < 5 < 4). A methyl group at the N⁶ position decreased the mutagenicity (7 > 4 > 8).     

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.