Microbial mutagenicity of 3- and 4-ring polycyclic aromatic sulfur heterocycles

The stable isomers of 3- and 4-ring polycyclic aromatic sulfur heterocycles were tested for mutagenicity in the Ames standard plate incorporation test and a liquid pre-incubation modification of the Ames test. Of the 4 three-ring compounds tested, only naphtho[1,2-b]thiophene was mutagenic. Of the four-ring compounds, 7 of 13 were mutagenic in the standard Ames or pre-incubation Ames test. The highest activity for the 4-ring compounds was observed for phenanthrol[3,4-b]thiophene, a compound of approximately the same mutagenic potency in the Ames test as benzo[a]pyrene. The other active 4-ring compounds were of considerable less mutagenic potency than phenanthrol[3,4-b]thiophene. Mutagenicity for two of the 4-ring aromatic thiophenes could only be detected in the liquid pre-incubation Ames test. Salmonella typhimurium TA100 was the most sensitive strain to mutagenesis by these compounds, followed by TA98. All mutagenesis was indirect, requiring metabolic activation.     

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.     

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

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

Evaluation of secondary nitroalkanes, their nitronates, primary nitroalkanes, nitrocarbinols, and other aliphatic nitro compounds in the Ames Salmonella assay.

The secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane and nitrocyclopentane, as well as their anionic forms (nitronates); the primary nitroalkanes 1-nitropropane, 1-nitrobutane, and 1-nitropentane and their respective nitronates; the nitrocarbinols 2-nitro-1-propanol, 2-nitro-1-butanol, 3-nitro-2-butanol, and 3-nitro-2-pentanol and their respective nitronates; 2-methyl-2-nitropropane, and 2-nitroso-2-nitropropane were tested in the Ames Salmonella assay using strains TA98, TA100 and TA102. Nitronates of the secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane, and nitrocyclopentane were significantly mutagenic in Salmonella strains TA100 and TA102 at 10-80 mumoles/plate, but the parent compounds were mutagenic at only a single dose level or were not mutagenic at all in the same dose range. The primary nitroalkanes and the nitrocarbinols were not mutagenic, or only marginally so, at the concentrations tested. The nitronates of the primary nitroalkanes and the nitrocarbinols reprotonated too rapidly under the conditions of the assay for adequate evaluation of mutagenicity. 2-Methyl-2-nitropropane was not mutagenic in strains TA100 and TA102; 2-nitroso-2-nitropropane was also not mutagenic in strains TA100 and TA102, but induced an equivocal mutagenic response in TA98. The positive Salmonella mutation data for the nitronates of the secondary nitroalkanes studied correlate very well with the very slow rate of reprotonation of secondary nitroalkane nitronates at pH 7.7 (Conaway et al. (1991) Cancer Res., 51, 3143), and provide further evidence that nitronates of secondary nitroalkanes, rather than the neutral parent forms with which they may be in equilibrium, are the more proximate mutagenic species.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

Mutagenicity testing with TA97 and TA102 of 30 DNA-damaging compounds, negative with other Salmonella strains

In a comparative study on 135 compounds of various chemical classes, 30 agents inducing direct nonreparable DNA damage in repair-deficient E. coli failed in reverting strains TA1535, TA1537, TA1538, TA98 and TA100 of S. typhimurium (De Flora et al., 1984b). These compounds were re-assayed in the Ames test using strains TA97 and TA102. A dose-dependent mutagenic response was detected with aminoantipyrine and p-rosaniline in TA97 and with streptomycin and formaldehyde in TA102. p-Rosaniline was the only mutagen requiring metabolic activation. 5 compounds, i.e. o-aminophenol in TA97 and methanol, ethanol, cadmium chloride and cadmium sulfate in TA102, induced a reproducible increase in revertants over controls, but this was less than 2-fold. The remaining 21 chemicals--including amino compounds, aliphatics, aromatics, heterocycles, hydrazine derivatives and inorganics--confirmed their inactivity in the Ames test. Overall data for 135 compounds, comparing the Ames test (7 strains) and the DNA-repair test (3 strains), are re-assessed on the basis of these findings.     

In vitro mutagenic,antimutagenic, and antioxidant activities evaluation and biotransformation of some bioactive 4‐substituted 1‐(2‐methoxyphenyl)piperazine derivatives

In vitro mutagenic, antimutagenic, and antioxidant potency evaluation and biotransformation of six novel 4‐substituted 1‐(2‐methoxyphenyl)piperazine derivatives demonstrating antidepressant‐like activity were investigated. Mutagenic and antimutagenic properties were assessed using the Ames test; free radical scavenging activity was evaluated with 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging assay and biotransformation was performed with liver microsomes. It was found that all tested compounds are not mutagenic in bacterial strains TA100 and TA1535 and exhibit antimutagenic effects in the Ames test. Noteworthy, compounds possessing propyl linker between phenoxyl and N‐(2‐methoxyphenyl)piperazine displayed more pronounced antimutagenic properties than derivatives with ethoxyethyl linker. Additionally, compounds 2 and 6 in vitro biotransformation showed that primarily their hydroxylated or O‐dealkylated metabolites are formed. Some of the compounds exhibited intrinsic clearance values lower than those reported previously for antidepressant imipramine. To sum up, the results of the present study might represent a valuable step in designing and planning future studies with piperazine derivatives.     

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.     

Mutagenicity studies of methyl-tert-butylether using the Ames tester strain TA102

Methyl-tert-butylether (MTBE) is an oxygenate widely used in the United States as a motor vehicle fuel additive to reduce emissions and as an octane booster [National Research Council, Toxicological and Performance Aspects of Oxygenated Motor Vehicle Fules, National Academy Press, Washington, DC, 1996]. But it is the potential for MTBE to enter drinking water supplies that has become an area of public concern. MTBE has been shown to induce liver and kidney tumors in rodents but the biochemical process leading to carcinogenesis is unknown. MTBE was previously shown to be non-mutagenic in the standard Ames plate incorporation test with tester strains that detect frame shift (TA98) and point mutations (TA100) and in a suspension assay using TA104, a strain that detects oxidative damage, suggesting a non-genotoxic mechanism accounts for its carcinogenic potential. These strains are deficient in excision repair due to deletion of the uvrB gene. We hypothesized that the carcinogenic activity of MTBE may be dependent upon a functional excision repair system that attempts to remove alkyl adducts and/or oxidative base damage caused by direct interaction of MTBE with DNA or by its metabolites, formaldehyde and tert-butyl alcohol (TBA), established carcinogens that are mutagenic in some Ames strains. To test our hypothesis, the genotoxicity of MTBE-induced DNA alterations was assayed using the standard Ames test with TA102, a strain similar to TA104 in the damage it detects but uvrB + and, therefore, excision repair proficient. The assay was performed (1) with and without Aroclor-induced rat S-9, (2) with and without the addition of formaldehyde dehydrogenase (FDH), and (3) with human S-9 homogenate. MTBE was weakly mutagenic when tested directly and moderately mutagenic with S-9 activation producing between 80 and 200 TA102 revertants/mg of compound. Mutagenicity was inhibited 25%-30% by FDH. TA102 revertants were also induced by TBA and by MTBE when human S-9 was substituted for rat S-9. We conclude that MTBE and its metabolites induce a mutagenic pathway involving oxidation of DNA bases and an intact repair system. These data are significant in view of the controversy surrounding public safety and the environmental release of MTBE and similar fuel additives.     

Mutagenic and genotoxic effects of theophylline and theobromine in Salmonella assay and in vivo sister chromatid exchanges in bone marrow cells of mice.

The mutagenic and genotoxic effects of two methylxanthines, theophylline (TH) and theobromine (TB), were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice. These are the two most commonly used nervous system stimulators throughout the world. TH is used in the long-term treatment of asthma. Bacterial mutagenicity assay showed very weak mutagenic effects of both drugs in Salmonella strains TA102 and TA104 only in certain concentrations when S9 was added to it. No mutagenic effects were observed in any other strains used in this assay either with or without metabolic activation. But results of in vivo SCE assay indicate that these two drugs can induce significant SCE in bone marrow cells of mice.     

Fate of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in Sprague-Dawley rats. I. Mutagens in the urine

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a potent bacterial mutagen formed during cooking of beef. IQ was administered intravenously to Sprague-Dawley rats at concentrations ranging from 7.5-50 mg/kg body weight. Urine was collected and analyzed for mutagenicity. Urinary mutagens were found which required activation by S9 mix, and reverted Ames test strains TA98 and TA100, but not TA1535 or TA1537. The amount of urinary mutagen(s) were related to IQ dose administered and were excreted within 48 h. Additional mutagenic activity was not released after incubation with beta-glucuronidase or aryl sulfatase. Analysis of urinary mutagens by HPLC indicates that the majority of mutagenic activity is due to unchanged IQ, but a small peak of mutagenic activity may correspond to N-acetyl or 3-N-demethylated metabolite. Since only 1% of the administered mutagenic activity is recovered in the urine, IQ may be readily detoxified in vivo.     

Comprehensive assessment of the photomutagenicity, photogenotoxicity and photo(cyto)toxicity of azulene

The polycyclic aromatic hydrocarbon azulene and its naturally occurring derivative guaiazulene (1,4-dimethyl-7-isopropylazulene) are known to absorb light in the UV-vis region of the spectrum. Both compounds were reported to be mutagenic in the Salmonella typhimurium bacterial mutagenicity assay (Ames test) in strain TA102, and to cause DNA damage in the comet assay in vitro upon exposure to UVA light. In contrast, another study reported a photoprotective effect in vitro of guaiazulene. We present here a comprehensive assessment of the photo(cyto)toxicity (3T3 fibroblast Neutral Red uptake test), the photomutagenicity (Ames test) and photogenotoxicity (comet assay and micronucleus test in L5178Y cells in vitro) of azulene. In the Ames test, the mutagenicity of azulene was assessed in the presence and absence of UV light by use of the Salmonella strains TA102, TA104, TA2638 and E. coli WP2. Azulene was irradiated before being plated with bacteria (pre-irradiation), or concomitantly with the bacteria either after plating or while in suspension. Guaiazulene was included in some of the experiments. Neither in the photo-Ames test nor in the other photogenotoxicity tests, azulene or guaiazulene showed any photomutagenic or photogenotoxic activity. Weak photo(cyto)toxicity (estimate of PIF≥1.67) was observed with azulene in the 3T3 NRU test, the Alamar Blue test and the relative cell count, which may be due to the generation of reactive oxygen species, as reported recently.     

Mutagenicity of the Alternaria metabolites altertoxins I, II, and III.

The Ames Salmonella typhimurium assay was used to demonstrate that an extract of the mold Alternaria alternata was mutagenic. The mutagenic extract was fractionated, and the Ames test was used to determine which fractions were mutagenic. Subsequently, altertoxins I and II and a new compound referred to as altertoxin III were isolated by liquid chromatography and shown to be hydroxyperylenequinone compounds by mass spectrometry and infrared, ultraviolet, and proton magnetic resonance spectroscopy. Altertoxins I, II, and III were mutagenic to S. typhimurium TA98, TA100, and TA1537 with and without metabolic activation.     

Mutagenicity of the Alternaria metabolites altertoxins I, II, and III

The Ames Salmonella typhimurium assay was used to demonstrate that an extract of the mold Alternaria alternata was mutagenic. The mutagenic extract was fractionated, and the Ames test was used to determine which fractions were mutagenic. Subsequently, altertoxins I and II and a new compound referred to as altertoxin III were isolated by liquid chromatography and shown to be hydroxyperylenequinone compounds by mass spectrometry and infrared, ultraviolet, and proton magnetic resonance spectroscopy. Altertoxins I, II, and III were mutagenic to S. typhimurium TA98, TA100, and TA1537 with and without metabolic activation.     

Relationships between structures and mutagenic potencies of 16 heterocyclic nitrogen mustards (ICR compounds) in Salmonella typhimurium

16 heterocyclic nitrogen mustards (ICR compounds), which were synthesized for use as possible antitumor agents by Creech and coworkers, were tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1536, TA1537, TA1538, TA98 and TA100. The compounds were incorporated into the top agar at 5 doses: 0.5, 1, 2.5, 5 and 10 micrograms/plate. All of the compounds were negative in TA1535 except ICR 449, which was positive in all 6 strains. The other 15 compounds were positive in the remaining strains with the following exceptions: ICR 371 and 355 were negative in TA100; ICR 445 was negative in TA98 and TA100; and ICR 360 was negative in TA1537, TA1538, TA98 and TA100. Good qualitative agreement was observed between the mutagenic and antitumor activities of the 16 compounds, and between the mutagenic and carcinogenic activities of the 5 compounds that have been tested for carcinogenicity by Peck and coworkers. However, no significant correlation was found between mutagenic potency in Salmonella and antitumor potency in mice for the 16 compounds. Also, for the 5 compounds that have been tested for carcinogenicity, no significant correlation was found between their mutagenic potency in Salmonella and their carcinogenic potency in mice. In Salmonella, the secondary (2 degrees) amines generally were more mutagenic than their tertiary (3 degrees) amine homologs, although the opposite result has been reported in certain eukaryotes. Relationships between structures and potencies for the different nuclei of the 16 ICR compounds are discussed, as are similarities and differences in strain sensitivities. We conclude that the Salmonella his reversion test is not a good predictor of the antitumor and carcinogenic potencies of these ICR compounds.     

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.     

The dependence of the mutagenicity of methanesulphonic acid esters in S. typhimurium TA100 on the alkylation mechanism

Four different model nucleophiles, 4-(p-nitrobenzyl)pyridine (NBP), N-methylmercaptoimidazole (MMI), trifluoroacetic acid (TFA) and H2O were tested with 22 methanesulphonates of widely varying structures for their suitability to predict mutagenic activities in S. typhimurium TA100. The soft nucleophiles NBP (N-alkylation) and MMI (S-alkylation) revealed as highly sensitive for SN2 reactivities whereas TFA (solvolysis at the O-atom) and H2O (hydrolysis) were very sensitive for SN1 reactivities. No correlation between the NBP or the MMI test and the Ames test was found. Quite good correlations could be demonstrated for the TFA test and the hydrolysis rates: with rising activities in the TFA solvolysis the mutagenic potencies were increasing up to a maximum at i-propyl methanesulphonate. After that due to the fast hydrolysis the mutagenicities were decreasing again despite increasing TFA solvolysis rates. In general the secondary methanesulphonates exerted high SN1 reactivities and distinct mutagenic activities, whereas the primary compounds showed no or very low SN1 reactivities and low mutagenic potentials. The "activated" compounds cyclopropylmethyl methanesulphonate, benzyl methanesulphonate and allyl methanesulphonate exerted high SN1 and SN2 reactivities. Methyl methanesulphonate displayed a high mutagenicity in spite of its lack in SN1 reactivity. This is probably due to the induction of the error prone repair (pkM 101 plasmid in TA100). The relation between the alkylating reactivities (SN1 and SN2) and the molecular mechanisms leading to back mutation is discussed.     

Screening complex hazardous wastes for mutagenic activity using a modified version of the TLC/Salmonella assay

10 complex hazardous wastes were tested for mutagenic activity using a modified version of the TLC/Salmonella assay developed by Bj?rseth et al. (1982). This fractionation/bioassay scheme couples thin-layer chromatography (TLC) with the Salmonella/mammalian-microsome (Ames) assay for the detection of mutagenic constituents in complex mixtures. Crude (unadulterated) hazardous wastes and selected hazardous waste extracts were fractionated on commercially available cellulose TLC plates. Mutagenicity testing was performed in situ by applying a single overlay of minimal growth agar, tester strain TA98 or TA100, and the optional metabolic activation system directly onto the developed chromatogram. A mutagenic effect was indicated either by the appearance of localized clusters of revertant colonies or by an increase in total revertant growth vis-à-vis control plates. 7 of 10 hazardous wastes (including tars, emulsions, sludges, and spent acids and caustics) demonstrated mutagenic activity when tested by this method. To assess the sensitivity of the modified TLC/Salmonella assay, 14 Salmonella mutagens from a wide range of chemical classes and polarities were tested. Selected compounds included heterocyclics, aromatic amines, alkylating agents, antitumor agents, a nitrosamine and a nitroaromatic. 11 of the 14 mutagens were positive in this test system. The 3 compounds refractory to analysis included a polycyclic aromatic hydrocarbon and two volatiles.     

An interlaboratory study of an EPA/Ames/Salmonella test protocol

7 laboratories participated in a collaborative study to evaluate an EPA standard protocol for the Ames test. The study utilized Salmonella typhimurium (strains TA98 and TA100) and 3 metabolic activation levels (0%, 2%, and 10% S9 in the S9 mix). 6 pure chemicals and 2 complex mixtures were tested as coded unknowns. Ability to obtain qualitative results in agreement with published data was less (% agreement) than that reported in an earlier study (% agreement) by de Serres and Ashby (1981) in which each laboratory used its own protocol. The conclusion from analysis of the quantitative data from this interlaboratory Ames study was that both intralaboratory and interlaboratory variations were substantial. Results for the same substance varied by an order of magnitude or more (CV of 115%) when the mutagenic response was measured as the slope of the dose response in revertants/microgram. Taking interlaboratory variation into account, one chemical must be more than an order of magnitude more mutagenic than another (ratio of slopes greater than 10) to have only an even chance of finding a statistically significant difference between the two chemicals at the 5% level. Such large variations must be taken into account when evaluating Ames/Salmonella data.     

Absence of mutagenic action of 5 beta-cholan-24-oic acid derivatives in the bacterial fluctuation and standard Ames tests

Published data on the mutagenicity of 3 bile acids in the bacterial fluctuation test are conflicting. Eleven 5 beta-cholanoic acids including 2 of the bile acids were assayed for mutagenicity in Salmonella typhimurium TA98 and TA100 in the fluctuation tests. In any of these bile acids at the doses tested, there were no dose-related statistically significant increases in mutagenicity compared with appropriate controls. Similarly, none of these compounds showed positive mutagenicity in both strains in the standard Ames test either with or without hepatic metabolic activation. Our results support the claim that 3 bile acids are not mutagenic, and indicate that the initiation activity of 5 beta-cholanoic acids is not demonstrable with a short-term assay using Salmonella strains.