Optimalization of rate adaptation using Holter functions in DDD/R pacemakers]

The genotoxicity of the organic peroxide 1,2,3,4-tetrahydronaphthaline-1-hydroperoxide (or tetraline-1-hydroperoxide, THP) was investigated in the Ames assay without a metabolic activating system using Salmonella typhimurium strains TA 98, TA 100, and TA 102. THP served as a model compound for higher organic peroxides, which can arise from autoxidation of hydrocarbons, e.g. in Diesel exhaust. While THP induced no mutagenic response in S. typhimurium TA 98, it was directly mutagenic in strains TA 100 and TA 102. These data, along with findings on mutagenic properties of other alkyl hydroperoxides, suggest that such compounds deserve further investigation regarding their genotoxic potential and occurrence in the environment.     

Clastogenic activity of sodium fluoride in great ape cells

Nitrobenzimidazole and nitroindole derivatives, related to oxiconazole and characterized by an oxyiminic function, have been synthesized as novel antimycotics and their mutagenic activity tested in Salmonella typhimurium strains TA100 and TA98 with and without an exogenous metabolizing system. TA98NR and TA98/1,8-DNP6 strains were employed to identify a specific metabolic reaction which governs the mutagenic potency. Active compounds are weak direct-acting mutagens. Only derivatives bearing a nitro group on the phenyl ring linked to the oxyiminic function and lacking halogenated substituents show mutagenic activity. Metabolism by bacterial enzyme systems is important to the expression of genotoxicity. The reductive activation of nitrobenzimidazoles and nitroindoles carried out by the 'classical' nitroreductase of Salmonella, which is defective in TA98NR, is required of mutagenicity. Similarly, the O-acetyltransferase defective in TA98/1,8-DNP6 is required for the efficient production of the ultimate electrophilic nitrogen species, which react with DNA. The role of bacterial metabolism in mutation induction needs careful consideration to assess the potential risk to humans from nitrobenzimidazole and nitroindole antimycotics.     

Recent update on the PPAR alpha-null mouse

The most important commercially available nitro- and aminobenzenes and the explosive trinitrobenzene were tested for mutagenicity in the Salmonella typhymurium TA 98 and TA 100 both in the absence and presence of S 9. Ten of the 14 compounds tested (71%) were mutagenic. All the substances showed positive results in TA 98 and 4 substances were also mutagenic in TA 100. The three diaminobenzenes and 4-nitroaniline were mutagenic only with metabolic activation. All other compounds did not require the addition of S 9. Only nitrobenzene, 1,2-dinitrobenzene, aniline and 2-nitroaniline were negative in both strains. In summary, all substances that are derived from nitrobenzene or aniline by addition of a nitro group in the meta- or para-position were mutagenic, whereas nitrobenzene and aniline themselves and their ortho-derivates were nonmutagenic. The possible relationships between the position of the substituents and the mutagenicity are discussed.     

Anthracene-9,10-diones as potential anticancer agents: bacterial mutation studies of amido-substituted derivatives reveal an unexpected lack of mutagenicity

Fifteen anthracene-9,10-dione ("anthraquinone") derivatives with (omega-aminoalkyl)carboxamido substituents at the 1-, 2-, 1,4-, or 2, 6-ring positions were tested for bacterial mutagenicity in reverse-mutation assays using Salmonella typhimurium frameshift strains TA1538, TA98, and TA97a, in the presence and absence of a metabolic activation system prepared from the livers of rats treated with Aroclor 1254. Six of the compounds were also tested in S. typhimurium TA100 and Escherichia coli WP2uvrApKM101 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution. Two structurally related compounds, mitoxantrone and bisantrene, were tested in parallel as positive controls. Mitoxantrone was mutagenic to S. typhimurium TA1538 and TA98, whereas bisantrene was weakly mutagenic to both these strains but strongly mutagenic toward the TA97a variant. By contrast, although they are also DNA-binding intercalators, none of the amide-functionalized anthracene-9,10-diones of the present study showed significant mutagenic activity in any of the bacterial strains examined. Further, neither substituent position nor systematic alterations in the nature of attached side chains appeared to induce mutagenicity with these agents, although other studies have shown that such structural factors markedly influence their cytotoxic potencies toward mammalian cells in vitro.     

Newer oral contraceptives and the risk of venous thromboembolism

Over the past 5 years, a large collaborative study of chemically-induced mutation has been performed using the four bacterial strains Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101 in order to compare the specific spectrum of response to chemicals and to evaluate the usefulness (sensitivity) of each strain. Following the two collaborative studies to test the chemicals in category 1, chemicals previously judged as positive only in E. coli WP2 strains and derivatives of these chemicals, and category 2, oxidative agents or crosslinking agents, 22 compounds of category 3 consisting of 10 nonmutagenic carcinogens and another 12 chemicals were selected in this study. Twenty participating laboratories tested each compound in the same method as previous reports. In the group of nonmutagenic carcinogens, no chemical induced revertant colonies of any strain tested. In the group of other chemicals, response to the chemicals was similar in TA102 and WP2 uvrA/pKM101. Overall, in the three collaborative studies, a total of 79 compounds were tested. No difference in qualitative response to the four strains was observed for 71% (56/79) of the test chemicals. The combination of strains providing the greatest number of positive responses was WP2 uvrA/pKM101 with TA102; 84% (66/79) of the test chemicals elicited the same qualitative response in these two strains. Therefore, it is suggested that WP2 uvrA/pKM101 and TA102 can be included as a part of the standard tester strains for detection of mutagenic activity of chemicals.     

Mutagenicity assay in Salmonella and in vivo sister chromatid exchange in bone marrow cells of mice for four pyrazolone derivatives

Phenylbutazone (PB), oxyphenbutazone (OPB), antipyrine (AP) and dipyrone (DP) are four important pyrazolone derivatives mainly used as anti-inflammatory, antipyretic and analgesic drugs. At present these are the most widely used pyrazolone derivatives throughout the world. The widespread use of these drugs are of great concern for human health problems. In the present study these four drugs were tested in mutagenicity assays in Salmonella strains TA97a, TA98, TA100 and TA102 using a plate incorporation assay both with and without S-9 mix and for in vivo sister chromatid exchanges (SCE) in bone marrow cells of mice. The first three drugs were negative in all the tester strains but dipyrone showed a weak mutagenic activity at higher concentrations in all four strains both with and without metabolic activation. In the in vivo SCE assay in male mice, all four drugs showed a statistically significant increase in SCE in bone marrow cells when compared with control.     

NMDA and AMPA receptors evoke transmitter release from noradrenergic axon terminals in the rat spinal cord

The organic extracts of soil collected at parks in residential areas in Osaka and neighboring cities in the Kansai area, Japan, showed mutagenicity in Salmonella typhimurium strain TA98 in the presence or absence of a mammalian metabolic activation system (S9 mix). The soil extracts from Ibaraki and two different sites in Osaka, i.e., Sumiyoshi-ku and Minato-ku, were mutagenic in strain TA100 as well as in strain TA98. Direct-acting mutagenicity of soil extracts from Sumiyoshi-ku and Minato-ku toward strain TA98 were 66 or more times higher than that of the other cities. Both extracts exerted stronger mutagenicity in strains YG1021 and YG1024 than TA98 and TA100, and the potency was especially high in strain YG1024: Sumiyoshi-ku, 153 000 revertants/g of soil; and Minato-ku, 246 000 revertants/g of soil. Two mutagenic compounds (I and II) were isolated from the Soxhlet extract of soil from the park in Sumiyoshi-ku by repetitive separation using normal-phase and reversed-phase column chromatography. By comparing the mass and UV spectra and retention times for HPLC on two individual ODS columns of compounds I and II with those of authentic chemicals, we identified these two compounds as 1,6- and 1,8-dinitropyrene (DNPy) isomers. Amounts of DNPy isomers in soil from Sumiyoshi-ku and Minato-ku were 1.7-2.2 ng/g. Forty-three percent and 40% of the mutagenicity of soil from Sumiyoshi-ku and Minato-ku could be attributed to these DNPy isomers, respectively.     

Application of the Salmonella mutagenicity assay and determination of polycyclic aromatic hydrocarbons in workplaces exposed to petroleum pitch and petroleum coke

Workplaces of an Italian carbon electrode factory, exposed to petroleum pitch and petroleum coke, were studied using a coupled chemical and biological approach to evaluate occupational mutagenic/carcinogenic hazards. Analytical procedures for the determination of polycyclic aromatic hydrocarbons (PAH) and Salmonella/microsome mutagenicity tests (with TA98 and TA100 strains) were performed on both industrial ingredients (pitch and coke) and airborne particulate matter of the working environment, after fractionating by sequential Soxhlet extractions with four organic solvents of increasing polarity (benzene, chloroform, methanol and acetone). The results showed: (a) the presence of extraordinarily high PAH (carcinogenic and non-carcinogenic) contents in the benzene extracts of petroleum pitch (3.6 wt% of total PAH) and of airborne particulate samples (up to 0.35 wt% of total PAH), in correlation with very high indirect (after metabolic activation) mutagenic responses of benzene extracts with strain TA98; (b) very high indirect mutagenic responses in the other extracts of the airborne particulate samples (especially with strain TA98); (c) the production during the processing at high temperatures of directly acting mutagens (without metabolic activation) which were absent in the starting materials and their release in the air of workplaces. The comparison of chemical analytical and mutagenicity data has proved to be an interesting approach for better defining the relative health hazards due to occupational exposure to potentially mutagenic/carcinogenic petroleum products.     

In vivo formation of mutagens by intraperitoneal administration of polycyclic aromatic hydrocarbons in animals during exposure to nitrogen dioxide

Consumption of fossil fuels has increased indoor and outdoor concentrations of polycyclic aromatic hydrocarbons (PAHs) and nitrogen dioxide (NO2). To study the combined effect of PAH administration and NO2 exposure on mutagenicity of urine from animals we injected 400 mg/kg body wt i.p. one of five kinds of PAH (pyrene, fluoranthene, fluorene, anthracene and chrysene) into ICR mice, Wistar rats, Syrian golden hamsters or Hartley guinea pigs after exposure to 20 p.p.m. NO2 gas for 24 h and then exposed the animals to NO2 gas for an additional 24 h. During the latter 24 h we collected the urine and assayed its mutagenicity with the Ames Salmonella strains after treatment with beta-glucuronidase and arylsulfatase and extraction with dichloromethane. The urine from mice treated with both PAH and NO2 showed high mutagenicity for Salmonella typhimurium strains TA98 and TA100, whereas the urine from mice treated with PAH and air showed almost no mutagenic activity. The mutagenicity was decreased in nitroreductase- and acetyltransferase-deficient strains TA98NR and TA98/1,8-DNP6 respectively. Treatment with a mixture of 20% of each of the five kinds of PAH and NO2 augmented the urinary mutagenicity of mice 1.5-fold. The urine from hamsters treated with pyrene or fluoranthene and NO2 was also highly mutagenic, but that from rats or guinea pigs was not very mutagenic. The mutagenicity was also decreased in strains TA98NR and TA98/1,8-DNP6. These results suggest that the urine contains nitro compounds and that the nitration of PAHs occurs in the body of animals under exposure to NO2 gas. Actually, the nitrated metabolites of pyrene, 1-nitro-6/8-hydroxypyrene and 1-nitro-3-hydroxypyrene, were detected in the urine from mice treated with pyrene under exposure to NO2 gas. To elucidate the mechanism of in vivo nitration, NO2 (20 p.p.m.) was bubbled through 50 mM Tris-HCl buffer (pH 7.4) or dichloromethane solution containing pyrene or 1-hydroxypyrene (10 microg/ml). Pyrene was not nitrated by NO2 in either aqueous or organic solutions. However, 1-hydroxypyrene was changed to nitrohydroxypyrenes by NO2 in the Tris-HCl buffer, but not in the organic solution. Ascorbic acid, alpha-tocopherol, glutathione oleic acid and hemoglobin were found to inhibit the nitration of 1-hydroxypyrene in aqueous solution. The urinary mutagenicity of mice treated with both pyrene and NO2 was also decreased by oral administration of ascorbic acid and alpha-tocopherol. These results suggest that 1-hydroxypyrene is nitrated by an ionic reaction in the animal body after hydroxylation of pyrene in the liver.     

Bile acids: co-mutagenic activity in the Salmonella-mammalian-microsome mutagenicity test: brief communication

Of 30 bile acids tested, none was mutagenic in the Salmonella-mammalian-microsome test with indicator strains G46, TA1530, TA1535, TA1536, TA1537, TA1538, TA98, or TA100. However, when lithocholic acid or one of its conjugates was tested with suboptimal amounts of 2-aminoanthracene and phenobarbital-stimulated rat liver homogenate, enhancement and co-mutagenesis were observed if TA1538 was the indicator strain.     

Genotoxicity of Farbasol and its component: 4-ethyltoluene

A combination of assays for gene mutations in Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains with and without rat liver activation, and for micronucleus and sister chromatid exchange (SCE) in bone marrow cells of Imp:Balb/c mice was used to provide data on the mutagenic and genotoxic properties of the mixture of aromatic solvents, known under the trade name of Farbasol. In addition, 4-ethyltoluene (the main ethylmethylbenzenic component of Farbasol) was also tested for muta- and genotoxicity. The results revealed that neither Farbasol nor 4-ethyltoluene induced an increased reverse mutation in bacterial cells or the formation of micronucleated polychromatic erythrocytes in bone marrow. However, those compounds were found to be active as sister chromatid exchange (SCE) agents.     

Thrombosis and malignancy: pathogenesis and prevention

Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine, were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchange (SCE) and chromosome aberration (CA) assays in bone marrow cells of mice. These are the most commonly used antimalarial drugs available at present throughout the world. The results of the bacterial mutagenicity assays showed a very weak mutagenic effect of all three drugs in Salmonella strains TA97a and TA100 both with and without S9 mix and in TA104 only with S9 mix. The results of the in vivo SCE and CA assays indicate that these three drugs are genotoxic in bone marrow cells of mice.     

Cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon ortho-quinones produced by dihydrodiol dehydrogenase

Eight polycyclic aromatic hydrocarbon (PAH) ortho-quinones that can be generated by dihydrodiol dehydrogenase (DD) were examined for their cytotoxicity in H-4-II-e (rat hepatoma) cells and for their mutagenicity in the Ames test. Seven of the PAH otrtho-quinones were potent cytotoxins yielding IC50 values for cell survival in the range 1-30 microns. PAH ortho-quinones were grouped into three classes based on their cytotoxicity profiles: group I contained ortho-quinones (e.g., naphthalene-1,2-dione and 7,12-dimethylbenz[alpha]anthracene-3,4-dione) which reduced cell viability and cell survival; group II contained ortho-quinones (e.g., benz[alpha]anthracene-3,4-dione and 5-methylchrysene-1,2-dione which reduced cell survival but had no effect on cell viability; and group III contained ortho-quinones (e.g., benzo[alpha]pyrene-7,8-dione) which had a pronounced effect on cell viability but minimal effects on cell survival. Using hepatoma cell suspensions and rat liver subcellular fractions, it was found that ortho-quinones underwent preferential enzymatic one-electron redox-cycling and produced superoxide anion radical (O2-.) and/or ortho-semiquinone anion or alternant radicals. ortho-Quinones that reduced cell viability produced O2-. and caused the most total free radical formation, while those that reduced cell survival produced ortho-semiquinone anion or alternant radicals only. PAH ortho-quinones were also tested as direct-acting mutagens in Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102 and TA104. They were found to be more mutagenic than the test mutagens used for each tester strain, and were predominantly frameshift mutagens. The presence of an activating system (Aroclor-induced rat liver S9 plus NADPH) did not increase the mutagenicity of ortho-quinones in tester strains that are sensitive to oxidative mutagens (TA102 and TA104). These data suggest that PAH ortho-quinones produced by DD are cytotoxic and mutagenic by different mechanisms. The mechanism of cytotoxicity involves the formation of reactive oxygen species and/or ortho-semiquinone anion or alternant radicals. The mechanism of mutagenicity is independent of free radical formation and is related to the ability of PAH orthooffinones to intercalate and covalently modify DNA.     

Replication of UV-irradiated DNA in human cell extracts: evidence for mutagenic bypass of pyrimidine dimers

We have examined the efficiency and fidelity of simian virus 40-origin-dependent replication of UV-irradiated double-stranded DNA in extracts of human cells. Using as a mutational target the alpha-complementation domain of the Escherichia coli lacZ gene in bacteriophage M13mp2 DNA, replication of undamaged DNA in HeLa cell extracts was highly accurate, whereas replication of DNA irradiated with UV light (280-320 nm) was both less efficient and less accurate. Replication was inhibited by irradiation in a dose-dependent manner. Nonetheless, covalently closed, monomer-length circular products were generated that were resistant to digestion by Dpn I, showing that they resulted from semiconservative replication. These products were incised by T4 endonuclease V, whereas the undamaged replication products were not, suggesting that pyrimidine dimers were bypassed during replication. When replicated, UV-irradiated DNA was used to transfect an E. coli alpha-complementation host strain to score mutant M13mp2 plaques, the mutant plaque frequency was substantially higher than that obtained with either unirradiated, replicated DNA, or unreplicated, UV-irradiated DNA. Both the increased mutagenicity and the inhibition of replication associated with UV irradiation were reversed by treatment of the irradiated DNA with photolyase before replication. Sequence analysis of mutants resulting from replication of UV-irradiated DNA demonstrated that most mutants contained C-->T transition errors at dipyrimidine sites. A few mutants contained 1-nt frameshift errors or tandem double CC-->TT substitutions. The data are consistent with the interpretation that pyrimidine dimers are bypassed during replication by the multiprotein replication apparatus in human cell extracts and that this bypass is mutagenic primarily via misincorporation of dAMP opposite a cytosine (or uracil) in the dimer.     

Structural determination of a mutagenic aminophenylnorharman produced by the co-mutagen norharman with aniline

Norharman (9H-pyrido[3,4-b]indole), widely distributed in our environment, including cigarette smoke and cooked foodstuffs, is not mutagenic to Salmonella strains, but becomes mutagenic to S.typhimurium TA98 and YG1024 with S9 mix in the presence of non-mutagenic aromatic amines such as aniline and o-toluidine. To elucidate the mechanisms of co-mutagenicity, we tried to isolate the mutagen(s) produced by a reaction between norharman and aniline with S9 mix. By HPLC purification, two mutagenic compounds (I and II), one (I) showing mutagenicity with and the other (II) without S9 mix, were isolated. The structure of compound I was deduced to be a coupled compound of norharman and aniline, 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman), by a variety of spectrometry techniques and this was confirmed by its chemical synthesis. The mutagenic activity of this novel heterocyclic amine was tested using the pre-incubation method and was found to induce 187,000 revertants in TA98 and 1,783,000 revertants in YG1024 per microg with S9 mix. Compound II was shown to be hydroxyaminophenylnorharman. Formation of the same DNA adducts was observed in YG1024 when aminophenylnorharman or a mixture of norharman plus aniline was incubated with S9 mix. The hydroxyamino derivative also yielded the same DNA adducts in YG1024. Thus, the appearance of mutagenicity by norharman with aniline in the presence of S9 mix suggests that the coupled mutagenic compound, aminophenylnorharman, is formed from norharman and aniline, then converted to the hydroxyamino derivative and forms DNA adducts to induce mutations in TA98 and YG1024.     

The role of the peripherin/RDS gene in retinal dystrophies

The effect of a potent endogenous antioxidant, the pineal gland indole melatonin (MLT) on the mutagenicity of twelve well-known mutagens and carcinogens has been investigated using two in vitro tests the Ames test and the single cell gel electrophoresis assay (SCGE assay or COMET assay). The 12 mutagens used were 7, 12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP), 2-aminofluorene (AF), 1,2-dimethylhydrazine (DMH), bleomycin, cyclophosphamide (CP), 4-nitroquinoline-N-oxide (NQO), 2,4, 7-trinitro-9-fluorenone (TNF), 9-aminoacridine (AA), N-nitrosomethylurea (NMU), mitomycin C and sodium azide tested in the absence or in the presence of S9 mix. MLT alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at the highest concentration tested (100 microM) in the SCGE assay. In four Salmonella typhimurium tester strains TA 97, TA 98, TA 100 and TA 102 MLT significantly reduced the mutagenicity of chemicals which require S9 activation. In the SCGE assay performed on CHO cells, preincubation with MLT led to a strong inhibition of clastogenic activities of DMBA and CP, and in a lesser extent with BP and NMU. With mitomycin C, MLT exacerbated responses in both tests. The possible mechanisms of MLT's inhibitory action are discussed.     

Mutagenic activity detected by the Ames test in river water under the influence of petrochemical industries

The present study was carried out on the waters of the Caí River (Rio Grande do Sul, Brazil) in an area under the influence of a petrochemical industrial complex, as the continuation of a study in which the mutagenic activity of water samples was evaluated in the internal area of this complex. In the previous study, the release of inducing substances was detected, revealing the need for a full analysis of the real ecological impact of the industrial complex on the river. Water samples from different sites along the Caí River were subjected to the Ames test during a study of 20 months duration for the detection of possible mutagens. Strains TA100 and TA98 were used for initial sample screening in the presence and absence of the S9 mix at a standard dose of 2000 microliters/plate. When positive activity (values equal to twice the spontaneous mutation rate) and/or cytotoxic activity (cell survival below 60%) was detected, the dose-response relationship was studied. Thirty-four percent of the samples tested were mutagenic, with different values according to collection site. Of the total number of positive responses, 6% were obtained for samples collected at the blank site upstream from the area studied, 82% at sites closest to the industrial complex, and 12% in downstream areas. Strain TA98 was the most sensitive in assays with no metabolic activation. A low frequency of induction (2%) was observed for strain TA102. Application of the Ames test permitted the delimitation of three areas of influence of the petrochemical industrial complex, and the test proved to be adequate for the detection of contaminants from the petrochemical industry.     

Mutagenicity of azo dyes used in foods, drugs and cosmetics before and after reduction by Clostridium species from the human intestinal tract

Various azo dyes currently approved by the US FDA for use in foods, drugs and cosmetics are reduced by anaerobic bacteria from the human intestinal tract. These bacteria with azoreductase activities include several Clostridium species. Seven of these azo dyes and their reduction products following incubation with a Clostridium sp. were evaluated for mutagenicity in Salmonella typhimurium strains TA98 and TA100. No mutagenicity was induced in either TA98 or TA100 by any of the seven azo dyes or the reduced metabolites when tested at concentrations as high as 200 microg/plate, with or without exogenous metabolic activation by rat liver fraction S-9.     

The genotoxicity and carcinogenic potential of gastrofenzin

Genotoxicity of the Bulgarian drug gastrophensin was studied by using a battery of two genotoxicity assays "in vitro" - Salmonella/mutation assay and "in vivo" - the rodent bone marrow micronucleus test. Mutagenicity of water solution of gastrophensin towards Salmonella "in vitro" - the rodent bone marrow micronucleus test. Mutagenicity of water solution of gastrophensin towards Salmonella "in vitro" was tested in five mutant, histidine auxotrophic strains - TA 1535, TA 1537, TA 1538, TA 98 and TA 100 without and in the presence of metabolic activation (+/- S9) at concentration of 0.4, 2 and 10 mg center dot ml-1. Gastrophensin did not induce mutagenic response in the Salmonella/mutation assay in a range of tested concentrations in both series of assays (+/- S9). Gastrophensin did not induce micronuclei in bone marrow cells of male C57Bl6 mice at 24, 48 and 72 hours after single oral treatment with 236 mg center dot kg-1 (80% DL50 oral, mice) and 118 mg center dot kg-1 (40% DL50 oral, mice). Based on the present data a conclusion of the lack of mutagenicity and of carcinogenic potency of gastrophensin was made.     

Clinical research: any future?

Monochloramine has been suggested as an alternative disinfectant to chlorine to reduce levels of trihalomethanes in treated drinking water, but little is known of the toxicological properties and potential health implications of by-products specific to the chloramination process. Model aqueous fulvic acid solutions (200-400 mg C/liter), serving as surrogates for humic surface waters, were chloraminated over a range of molar Cl:C ratios from 1:40 to 1:2. The resulting by-products were extracted into diethyl ether at pH 2 and investigated with the Ames plate incorporation assay. Extractable mutagenicity increased with increasing chlorine and carbon dose up to about 30,000 revertants/liter at Cl:C ratios of 1:2. Mutagenicity was higher in Salmonella typhimurium strain TA100 than in strain TA98, and was decreased in the presence of S9, indicating that the mutagens formed were direct-acting and induced predominantly base-pair substitutions. Bovine serum albumin decreased slightly, and glutathione reduced greatly, the mutagenic activity detected in extracts. HPLC fractionation of the by-products indicated that most of the mutagenic activity was found in the earliest-eluting (most polar) fraction. The mutagenic by-products appeared to be qualitatively similar to 3-chloro-4-dichloromethyl-5-hydroxy-2-(5H)-furanone (MX) in their chromatographic behavior and responses to glutathione and bovine serum albumin, but were less readily detoxified by S9 than was MX.