Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: II. With exogenous activation

In order to determine the usefulness of selected chemicals as potential reference materials for calibrating the Salmonella assay, two laboratories tested a series of Salmonella mutagens that require exogenous activation. When the variance for individual substances within a bioassay is sufficiently low and the rankings of those substances are of acceptable consistency, they can later be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The purpose of this project, therefore, was to evaluate the variability in the mutagenic response of potential reference chemicals that require exogenous metabolic activation in the standard plate-incorporation Salmonella mutagenicity assay, and to develop ranking criteria for mutagenic activity based on these data. Ten indirect-acting mutagens were tested in two laboratories using Salmonella typhimurium TA100 and an Aroclor-induced rat liver S9. Each laboratory conducted four definitive testing rounds. A different batch of S9 was utilized for every two rounds. Of the 10 chemicals tested only 2-anthramine had a mean slope value greater than 1000 revertants/micrograms. Three chemicals had slope values between 1000 and 100; and five chemicals had slope values between 100 and 10. The remaining compound, 9,10-dimethyl-1,2-benz[a]anthracene, could not be placed into a single category because it had slope values on either side of 100 revertants per mg. Coefficients of variance were low (i.e., below 25% in most cases). The low variability achieved in this study may be accounted for by two parameters of the study. First, based on Claxton et al. (1991a) and the S9 optimization for three compounds, the amount of S9 was calibrated to a set amount of protein per plate (1.1 mg/plate). Secondly, the 10 test doses were placed in the initial, linear, nontoxic portion of the dose-response curves. The use of ten closely spaced, nontoxic doses allowed for a more accurate estimate of the slope.     

Mutagenic potential of toluidine blue evaluated in the Ames test.

Toluidine blue is a vital, metachromatic thiazine dye which is used as an adjunct in clinical examination for the early detection of asymptomatic recurrent or secondary primary carcinoma in individuals who are at high risk for developing oral cancer. Because available data on the mutagenicity of toluidine blue was limited and contradictory, this study was conducted to evaluate the mutagenic potential of toluidine blue in the in vitro Ames Salmonella test. Tester strains TA97a, TA98, TA100 and TA102 were used. Toluidine blue was tested at concentrations of 0.1, 1.0, 10, 50, 100, 250 and 500 micrograms/plate, with and without S9 microsomal activation, and positive and negative controls were included. Results from tests without S9 showed a significant increase (p less than 0.05) in number of revertants in TA102 and in TA97a with 50 and 100 micrograms toluidine blue/plate, respectively. In tests with S9 activation, doses of toluidine blue ranging from 10 to 250 micrograms/plate induced dose-related increases in the number of revertants in all 4 strains. The results of this study indicate that toluidine blue has a mutagenic effect in the Ames test.     

Absence of mutagenic and antimutagenic activities of fluoride in Ames salmonella assays

The mutagenicity of fluoride (as sodium fluoride, NaF) was investigated with Ames Salmonella/microsome assays in strains of TA97a, TA98, TA100, TA102 and TA1535. The concentrations of NaF tested ranged from 0.44 to 4421 micrograms/plate (0.1 to 1000 ppm F), both with and without microsome activation. In addition, the suggested antimutagenic effect of fluoride was evaluated with known mutagens at various concentrations of NaF (0.44-442.2 micrograms/plate, 0.1-100 ppm F). The data showed that NaF, in amounts from 0.44 to 442.2 micrograms/plate (0.1-100 ppm F), failed to significantly increase the number of the revertants over the number observed in the solvent (distilled deionized water) controls. Increases of NaF to, and beyond, 1100 micrograms/plate (250 ppm F) resulted in a toxic effect and a reduction of the revertants to various degrees among the strains. NaF in the presence of known mutagens did not significantly decrease the number of the revertants. The results of this study indicate that NaF does not have mutagenic or antimutagenic effects in the strains tested with Ames Salmonella assays.     

Mutagenicity of polycyclic aromatic compounds (PAC) identified in source emissions and ambient air

Several polycyclic aromatic compounds (PAC) including nitrated and oxygenated derivatives of polycyclic aromatic hydrocarbons (PAH) were tested for mutagenic activity in the Salmonella/microsome assay. Among the compounds tested the isomer mix of nitro-1-hydroxypyrenes showed the highest direct mutagenic response in both the Salmonella strain TA98 and TA100 (1251 revertants/micrograms and 463 revertants/micrograms, respectively). The direct-acting mutagenicity of the nitro-1-hydroxypyrene isomer mix was dependent upon reduction of the nitro function as evidenced by the decrease in activity observed with the nitroreductase-deficient and arylhydroxylamine esterifying-deficient tester strains. The oxygenated derivatives of PAH containing aldehyde or keto groups showed weak or no mutagenic responses. In most cases addition of S9 was essential for any mutagenic activity and the strain TA100 was more sensitive than the strain TA98. Within this group, 7H-dibenzo[c,g]fluoren-7-one showed the highest mutagenic effect; 7 and 22 revertants/micrograms using the strains TA98 and TA100, respectively.     

Aliphatic halogenated hydrocarbons produce volatile Salmonella mutagens

Production of volatile mutagenic metabolites from 5 halogenated promutagens was examined by a simple modification of the conventional Salmonella/microsome mutagenicity assay. This method incorporates the taping together of 2 agar plates face to face during the initial portion of their incubation at 37 degrees C. By varying the contents of the soft agar in each of the two plates with respect to promutagen, S9 and tester strain cells, mutagenesis due to volatile promutagens and their metabolites could be quantitated separately. Using the taped plate assay, volatile mutagenic metabolites were detected from the promutagens 3-(2-chloroethoxy)-1,2-dichloropropene, the herbicides diallate, triallate and sulfallate, and the flame-retardant tris-(2,3-dibromopropyl) phosphate (Tris-BP). All compounds except Tris-BP were also found to be volatile promutagens. The mutagenic metabolites accounted for 50-80% of the activity of these compounds observed in the standard assay. Morever, our studies suggest that a small, but appreciable percentage of the mutagenic metabolites from all 5 compounds escaped detection in the conventional, untaped assay. Mutagenic activity of the volatile mutagenic metabolites from diallate was quenched by various Salmonella tester strains independent of their responsiveness to diallate mutagenesis. Detection of volatile mutagen formation from diallate was also prevented by cysteine and glutathione, but not by DNA or metyrapone. This taped plate method for the Salmonella assay should facilitate future investigations of the detection, isolation and identification of volatile mutagenic metabolites from other promutagenic compounds or mixtures.     

Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests

The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9), besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 μg/plate). On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/ μg). Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.     

Release of mutagens from finished leather

Extracts of a leather widely used in the furniture and dress-making industries were tested for their mutagenic activity in the Salmonella/microsome assay. Extracts obtained after vigorous treatment of leather samples in a Soxhlet apparatus with toluene or ethanol were mutagenic in strain TA98 of S. typhimurium in the absence of S9 mix. The analysis of extracts of leather at various intermediate stages of processing showed that the mutagenic activity appeared after the coloration process. The responsible compound was identified to be an azo dye (Color Index: Acid Brown 83) whose mutagenic potency was about 4 revertants/micrograms.     

Bacterial mutagenicity of aceanthrylene: a novel cyclopenta-fused polycyclic aromatic hydrocarbon of low molecular weight

Aceanthrylene, a non-alternant cyclopenta-fused hydrocarbon, was shown to be weakly mutagenic without S9 and strongly mutagenic with S9 in the Ames Salmonella plate incorporation assay. The compound was most active in strain TA100 (35 revertants/nmole in the presence of 0.3 mg of S9 protein), and less active in strains TA98, TA1537 and TA1538 (20, 10 and 3.1 rev/nmole respectively, + S9). Strain TA1535 was unresponsive, suggesting that this compound induces frameshift mutations rather than base-pair substitutions. The mutagenic potency of aceanthrylene is consistent with predictions of its activity based on the relatively large delocalization energy (delta E deloc/beta = 0.931) of the carbonium ion which would result from oxirane ring opening of the 1,2-epoxide, a potential active metabolite.     

Salmonella/mammalian microsome assay with tetranitromethane and 3-nitro-L-tyrosine

The nitrosating agent tetranitromethane (TNM) and the nitrosation product 3-nitro-L-tyrosine (NT) were tested for mutagenic activity in the Salmonella/mammalian microsome assay. TNM showed strong genotoxic activity: it was mutagenic in all tester strains used (TA97, TA98, TA100, and TA102). The maximum mutagenic activity was reached between 16 and 32 micrograms/plate using the standard plate test; higher amounts led to distinct bactericidal effects. The mutagenicity was independent of an in vitro activation system. In the preincubation assay an increased bactericidal effect was observed. In contrast to TNM, NT, the nitrosation product, was non-mutagenic and non-toxic in the standard plate test and with the preincubation method up to 5000 micrograms/plate with and without S9 mix and with all tester strains used. Although TNM is a strong direct-acting mutagen, its nitrosating effect on proteins does lead to nongenotoxic nitro products of tyrosine in proteins.     

Effect of phenolic antioxidants on the mutagenicity of aflatoxin B1.

The Ames assay employing Salmonella typhimurium TA100 and TA98 was used to investigate potential interactions between aflatoxin B1 (AFB1) and the phenolic antioxidants butylated hydroxytoluene, butylated hydroxyanisole, and propyl gallate. AFB1 doses were within the linear response range, and the antioxidants were used at levels of 0 to 50 micrograms per plate. All three antioxidants were nonmutagenic in either bacterial tester strain, with or without the hepatic S-9 enzyme preparation; toxic effects were observed at doses higher than 20 micrograms per plate. Butylated hydroxytoluene and butylated hydroxyanisole substantially increased AFB1-induced mutagenesis in the two tester strains with microsomal activation. The addition of 5 to 20 micrograms of butylated hydroxytoluene or hydroxyanisole to 5 to 20 ng of AFB1 per plate caused more than a twofold increase in the number of His+ revertants. Addition of propyl gallate resulted in only a moderate increase in the number of revertants. Whereas several anticarcinogenic and antimutagenic effects by phenolic antioxidants have been reported, particularly in studies with polycyclic aromatic hydrocarbons, the enhancement of mutagenic potency of AFB1 by these compounds suggests a specificity with respect to the chemical nature of AFB1.     

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.     

Evaluation of the mutagenic potential of endosulfan using the Salmonella/mammalian microsome assay

The mutagenic potential of endosulfan, a polychlorinated insecticide, was assessed using the highly sensitive Salmonella tester strains TA97(a), TA98, TA100 and TA102. It exhibited a toxic effect at dose levels of 50 micrograms/plate and higher. Plate incorporation studies did not show mutagenic response with any of the tester strains used. A modification of the assay using a preincubation procedure showed mutagenic activity with and without metabolic activation with TA97(a) only. Increased toxicity was observed after addition of phenobarbital-induced S9 mix.     

Cytogenetic and genotoxic effects of the insecticides, imidacloprid and methamidophos

We examined the cytogenetic and genotoxic effects of the neonicotinoid insecticide imidacloprid and the organophosphate insecticide methamidophos, when administered alone or in combination. These insecticides were tested with the bone marrow chromosome aberration assay and micronucleus test in rats and by the bacterial mutation assay (Salmonella/microsome mutagenicity assay). Wistar albino rats were orally fed daily with laboratory chow treated with various concentrations of insecticides, 50 and 100 mg/kg imidacloprid, 2.5 and 5 mg/kg methamidophos, and 2.5 and 5 mg/kg imidacloprid plus methamidophos, respectively, for 90 days. Numerical and structural chromosomal aberrations were evaluated. Significant differences were detected between all the insecticide-administered groups versus the control group and between the two concentrations of the pesticide-treated groups. Both concentrations of the insecticides induced a dose-related increase in the micronucleus frequency (P < 0.05). Dose-related increases in the number of revertants were observed with the two Salmonella strains (TA98 and TA100). All tested doses of the insecticides demonstrated mutagenic activity in the presence of S9 mix. These results lead us to the conclusion that the synergistic effect of methamidophos and imidacloprid causes an increase in potential damage to non-target organisms.     

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

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

Use of a Salmonella microsuspension bioassay to detect the mutagenicity of munitions compounds at low concentrations

Past production and handling of munitions has resulted in soil contamination at various military facilities. Depending on the concentrations present, these soils pose both a reactivity and toxicity hazard and the potential for groundwater contamination. Many munitions-related chemicals have been examined for mutagenicity in the Ames test, but because the metabolites may be present in low environmental concentrations, a more sensitive method is needed to elucidate the associated mutagenicity. RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), TNT (2,4,6-trinitrotoluene), tetryl (N-methyl-N-2,4,6-tetranitroaniline), TNB (1,3,5-trinitrobenzene) and metabolites were examined for mutagenicity in a microsuspension modification of the Salmonella histidine reversion assay with and without metabolic activation. TNB and tetryl were positive in TA98 (32.5, 5.2revertants/nmole) and TA100 (7.4, 9.5revertants/nmole) without metabolic activation and were more potent than TNT (TA98, 0.3revertants/nmole; TA100, 2.4revertants/nmole). With the exception of the tetranitroazoxytoluene derivatives, TNT metabolites were less mutagenic than TNT. RDX and two metabolites were negative in both strains, however, hexahydro-1,3,5-trinitroso-1,3,5-triazine was positive in TA100 with and without S9. Microsuspension bioassay results tend to correlate well with published Ames test data, however, there are discrepancies among the published data sets and the microsuspension assay results.     

Bacterial mutagenesis and hepatocyte unscheduled DNA synthesis induced by chrysoidine azo-dye components

5 azo dye components of Gurr chrysoidine 'Y' have been separated, synthesised and identified. Dyes with a methyl substitution (particularly between the two amino groups) were more mutagenic in Salmonella typhimurium strain TA100 with control rat liver S9 than the non-methylated counterpart (range 66-1992 revertants at 50 micrograms/plate). Mutagenicity was also catalysed by human-liver S9 and pre-treatment of rats with either phenobarbitone or beta-naphthoflavone enhanced the activation ability of S9 by greater than 4-fold. Using the most potent promutagenic component (2,4-diamino-3-methylazobenzene), the use of inhibitors of cytochrome P450 (metyrapone: 1.0 mM; alpha-naphthoflavone: 0.075 mM; DPEA: 0.125 mM) and of the flavin monooxygenase (methimazole: 0.75 mM) suggested a major role for cytochrome P448 in the activation of chrysoidine to mutagens. The ability of chrysoidine components to induce unscheduled DNA synthesis in rat hepatocytes in vitro was demonstrated and ranged between 11.92 and 23.5 net nuclear grains at a dose level of 2.5 micrograms/incubation. Since each dye was equi-potent, methyl substitution had little influence on genetic toxicity in hepatocytes.     

Genotoxicity tests with 6-acetyl-1,1,2,4,4,7-hexamethyltetraline and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyr an

6-Acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB), synthetic fragrance ingredients, were evaluated for potential genotoxicity in a battery of short-term tests. Salmonella typhimurium/Escherichia coli plate incorporation and liquid preincubation assays were conducted on AHTN using tester strains TA97, TA98, TA100, TA102, TA1535, TA1537 and WP2 uvrA +/- S9 activation at doses from 8 to 5000 micrograms/plate. The plate incorporation mutagenicity assay was conducted on HHCB using tester strains TA98, TA100, TA1535, TA1537, TA1538 and WP2 uvrA +/- S9 activation at doses from 10 to 5000 micrograms/plate. An in vitro cytogenetics assay in Chinese hamster ovary (CHO) cells was conducted with AHTN and HHCB at three concentrations each with +/- S9 activation. In the non-activated study, the exposure/harvest periods were 4/20-, 20/20- and 44/44-h. In the S9 activated study, the exposure/harvest periods were 4/20- and 4/44-h. In vitro unscheduled DNA synthesis (UDS) assays were conducted in primary rat hepatocytes at concentrations between 0.15 and 50 micrograms/ml for AHTN and HHCB. In vivo mouse micronucleus assays were conducted with high doses of 1600 mg AHTN/kg and of 1500 mg HHCB/kg in corn oil. No positive responses were observed in any of the tests with HHCB. With AHTN, no positive responses were observed except for cells with structural aberrations in the in vitro cytogenetics assay in CHO cells with S9 activation at the treatment/harvest time of 4/20 h. In initial studies with AHTN, the high dose of 7.8 micrograms/ml showed 0.5% aberrant cells, with the mitotic index at 41% relative to vehicle control and cell growth inhibition in the range of 25-50%. Thus the genotoxicity findings with AHTN were limited to this one positive response; all other genotoxicity tests with AHTN were considered as negative. In particular, the negative finding in the in vivo assay supports AHTN as not likely to be mutagenic in mammalian systems. These considerations, along with other negative published data, lead to the conclusion that both AHTN and HHCB do not have significant potential to act as genotoxic carcinogens.     

Butylated hydroxyanisole, butylated hydroxytoluene and tert.-butylhydroquinone are not mutagenic in the Salmonella/microsome assay using new tester strains

The phenolic antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert.-butylhydroquinone (TBHQ) were reassessed for mutagenic activity using the recently developed Salmonella tester strains TA97, TA102 and TA104, and in addition TA100. None of the phenolic antioxidants showed mutagenic activity, either with or without metabolic activation. At doses of 100 micrograms/plate and higher all 3 phenolic antioxidants exhibited toxic effects. A modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Combinations of BHA and BHT, tested to detect possible synergistic effects, did not exert mutagenic activity.     

Studies on the oxidation products from 2,4-diaminotoluene by hydrogen peroxide and their mutagenicities. II

2,4-Diaminotoluene (DAT) was reacted with hydrogen peroxide at room temperature for 2 days, and the resulting red precipitates were separated into 5 fractions on silica gel column chromatography. On the gas chromatographic (GC) study, the first fraction (Fr. 1), which is mutagenic (1425 and 1391 revertants/micrograms in the absence and presence of S9 respectively) in Salmonella typhimurium TA98, contained several peaks. Fr. 1 was further separated into 4 subfractions (Fr. 1-I-Fr. 1-IV) by silica gel column chromatography. The red crystals were separated from Fr. 1-III and the structure of the compound was determined to be 1,8-diamino-2,7-dimethylphenazine from physicochemical and chemical evidence. Further, o-nitro-p-toluidine, p-nitro-o-toluidine, 3,3'-diamino-4,4'-dimethylazobenzene and 3,3'-diamino-4,4'-dimethylazoxybenzene were identified with authentic and synthesized samples by gas chromatography/mass spectrometry. These compounds without nitrotoluidines were mutagenic, and phenazine, azo and azoxy compounds induced 49, 301 and 245 revertants/nmole in Salmonella typhimurium TA98 with 25 microliters S9 per plate, respectively.     

Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat tracheal epithelial cells.

The cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were studied by the assays of colony-forming efficiency (CFE), micronucleus formation (MN), and cell transformation in rat tracheal epithelial (RTE) cells both in vitro and in vivo. Liver S9, primary hepatocytes and RTE cells from normal and Aroclor-1254 induced rats were compared for bioactivation of NNK using Salmonella mutagenesis as the endpoint. Results from the in vitro experiments indicated that low concentrations of NNK (0.01-25 micrograms/ml) caused from 15% to greater than 100% increases in CFE of RTE cells. At high concentrations (100-200 micrograms/ml), NNK was significantly toxic to RTE cells. NNK treatment in vitro (50-200 micrograms/ml) increased MN frequency as much as 3-fold above background and significantly increased the transformation frequency (TF) in 4/5 (50 micrograms/ml) and 6/8 (100 micrograms/ml) experiments. The in vivo exposure of rats to NNK (150-450 mg/kg, given i.p.) resulted in a 60-85% reduction in CFE and a 3-5-fold increase in MN formation in RTE cells. In vivo treatment with cumulative doses of 150 and 300 mg/kg of NNK produced significant increases in TF of tracheal cells from 3/3 and 2/3 rats, respectively. Without activation, NNK was not mutagenic in Salmonella TA1535. The bioactivation of NNK to a mutagenic metabolite was achieved by incubation of NNK with liver S9 fraction from Aroclor-1254 induced rats or primary hepatocytes from both untreated and Aroclor-1254 pretreated rats. RTE cells did not produce sufficient quantities of mutagenic NNK metabolites to be detected by the Salmonella assay.