Mutagenicity of chloroolefins in the Salmonella/mammalian microsome test. III. Metabolic activation of the allylic chloropropenes allyl chloride, 1,3-dichloropropene, 2,3-dichloro-1-propene, 1,2,3-trichloropropene, 1,1,2,3-tetrachloro-2-propene and hexachloropropene by S9 mix via two different metabolic pathways

In the presence of S9 mix all allylic chloropropenes tested exert considerable indirect mutagenic activity which is most pronounced for 1,2,3-trichloropropene. Lower as well as higher chlorinated derivatives are clearly less mutagenic. Longer than standard incubation time (120 min instead of 20 min) at 37 degrees C always leads to an increase in mutagenic activity. An increase in concentration of rat-liver homogenate fraction (S9) in the metabolising system (S9 mix) enhances mutagenicity only for 1,3-dichloropropene, 2,3-dichloro-1-propene and for the cis isomer of 1,1,2,3-tetrachloro-2-propene. According to the effects of the enzyme inhibitors SKF525 1,1,1-trichloropropene-2,3-oxide and cyanamide the allylic chloropropenes fall into 3 groups distinguished by their mode of metabolic activation by S9 mix: (a) allyl chloride and 1,3-dichloropropene are hydrolysed to the corresponding allylic alcohols which can be oxidised to the respective acroleins (hydrolytic-oxidative pathway); (b) 2,3-dichloro-1-propene, 1,1,2,3-tetrachloro-2-propene and hexachloropropene are epoxidised in the C=C double bond, giving rise to reactive epoxides (epoxidative pathway); (c) only 1,2,3-trichloropropene is obviously activated by both these alternative metabolic pathways. Structural parameters like chloro-substitution of the central C atom of the C=C-C sequence and substituent-induced polarisation of the C=C double bond as well as cis/trans isomerism might be responsible for different substrate properties for the enzymes involved in allylic chloropropene metabolism, thus determining different degrees of activation by either one or both pathways.     

Allyl isothiocyanate is mutagenic in Salmonella typhimurium

Allyl isothiocyanate, a naturally occurring compound, component of oil of mustard and human food plants such as cabbage, cauliflower and horseradish, has up to now been regarded as nonmutagenic in bacterial mutagenicity testing systems. Recently, however, it was found to cause transitional-cell papillomas in the urinary bladder of male F344 rats. Contrary to earlier reports, in this study allyl isothiocyanate showed clear mutagenicity for Salmonella typhimurium TA100 in the preincubation assay after longer, non-standard preincubation times (greater than 20 min). The mutagenicity is expressed only in the presence of a rat-liver homogenate metabolising system, i.e. it is indirect. However, high concentrations of rat-liver homogenate suppress the mutagenicity of allyl isothiocyanate. SKF525, inhibitor of microsomal oxygenase, reduces the mutagenic potential which on the other hand is increased in the presence of 1,1,1-trichloropropene-2-oxide, inhibitor of epoxide hydrolase. This indicates the occurrence of an epoxide intermediate in allyl isothiocyanate metabolism. Another metabolic pathway, namely hydrolysis to allyl alcohol and oxidation to acrolein, a known mutagen, also seems possible as cyanamide, inhibitor of aldehyde dehydrogenase, can slightly increase the mutagenic potential. The reason(s) for allyl isothiocyanate's requirement for long preincubation times to express mutagenicity still requires elucidation, and the question arises: is allyl isothiocyanate a single, exceptional case or not?     

Mutagenicity of adsorbates to a copper-phthalocyanine derivative recovered from municipal river water

Blue cotton, bearing a covalently bound copper-phthalocyanine derivative capable of adsorbing polycyclic aromatic hydrocarbons (PAHs) over 3 rings, was applied to recover mutagens from the Katsura River which is a tributary of the Yodo River. The Ames Salmonella/microsome assay with TA98 and TA100 of the blue cotton concentrate recovered from the river water demonstrated indirect mutagenicity toward TA98. The subfractions separated by Sephadex G-25 gel chromatography also showed direct mutagenicity in strains YG1021 and YG1024, the nitroreductase- and O-acetyltransferase-overproducing derivatives of TA98; this activity was greatly increased by the addition of S9 mix, especially in YG1024. However, these subfractions were less mutagenic with TA98NR or TA98/1,8-DNP6, regardless of whether S9 mix was present or not. The behaviors of these mutagenic activities therefore suggested that frameshift mutagens of both directly mutagenic nitroarenes and indirectly mutagenic aminoarenes were present in the blue cotton concentrate from the river water.     

Mutagenicity of 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein in Salmonella typhimurium TA100. A comparative study.

The C2-alkylated acrolein derivatives 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein are mutagenic in Salmonella typhimurium TA100. They are direct mutagens, their mutagenic potency being inversely proportional to the size of the alkylating substituent in the C2 position. In the presence of S9 mix, the mutagenicity of all these substances is considerably reduced; the reduction in mutagenicity is inversely proportional to the direct mutagenic potential of the substance. As shown for 2-methylacrolein, the reduction in mutagenicity is dependent on the concentration of S9 in the S9 mix and is not significantly influenced by heat inactivation of the S9 mix or by addition of TCPO, an inhibitor of epoxide hydrolase, to the testing system. There are no indications of enzymatic activation by the metabolizing microsomal system.     

The dosing determines mutagenicity of hydrophobic compounds in the Ames II assay with metabolic transformation: Passive dosing versus solvent spiking

The Ames II bacterial mutagenicity assay is a new version of the standard Ames test for screening chemicals for genotoxic activity. However, the use of plastic micro-titer plates has drawbacks in the case of testing hydrophobic mutagens, since sorptive and other losses make it difficult to control and define the exposure concentrations, and they reduce availability for bacterial uptake or to the S9 enzymes. With passive dosing, a biocompatible polymer such as silicone is loaded with the test compound and acts as a partitioning source. It compensates for any losses and results in stable freely dissolved concentrations. Passive dosing using silicone O-rings was applied in the Ames II assay to measure PAH mutagenicity in strains TA98 and TAMix – a mixture of six different bacterial strains detecting six different base-pair substitutions – after metabolic activation by S9. Initially, 10 PAHs were tested with passive dosing from saturated O-rings, aiming at levels in the test medium close to aqueous solubility. Fluoranthene, pyrene and benzo(a)pyrene were mutagenic in both TA98 and TAMix, whereas benz(a)anthracene was mutagenic in TA98 only. The concentration-dependent mutagenic activity of benzo(a)pyrene was then compared for passive dosing and solvent spiking. With spiking, nominal concentrations greatly exceeded aqueous solubility before mutagenicity was observed, due to sorptive losses and limiting dissolution kinetics. In contrast, the passive dosing concentration-response curves were more reproducible, and shifted towards lower concentrations by several orders of magnitude. This study raises fundamental questions about how to introduce hydrophobic test substances in the Ames II assay with biotransformation, since the measured mutagenicity not only depends on the compound potency but also on its supply, sorption and consumption during the assay.     

Strategies and testing methods for identifying mutagenic risks

The evolution of testing strategies and methods for identification of mutagenic agents is discussed, beginning with the concern over potential health and population effects of chemical mutagens in the late 1940s that led to the development of regulatory guidelines for mutagenicity testing in the 1970s and 1980s. Efforts to achieve international harmonization of mutagenicity testing guidelines are summarized, and current issues and needs in the field are discussed, including the need for quantitative methods of mutagenic risk assessment, dose-response thresholds, indirect mechanisms of mutagenicity, and the predictivity of mutagenicity assays for carcinogenicity in vivo. Speculation is offered about the future of mutagenicity testing, including possible near-term changes in standard test batteries and the longer-term roles of expression profiling of damage-response genes, in vivo mutagenicity testing methods, and models that better account for differences in metabolism between humans and laboratory model systems.     

Isolation, identification and bacterial mutagenicity of 2-nitro-9-fluorenone from diesel-exhaust particle extracts

Organic extracts of diesel-exhaust particles show direct mutagenic activity in the Salmonella typhimurium bacterial mutagenicity assay. Nitro-aromatic compounds are believed to be responsible for part of the mutagenicity. A previously unidentified polyfunctional nitro-aromatic compound, 2-nitro-9-fluorenone (2N-Fone) was isolated from diesel-exhaust particles using a two-step fractionation scheme consisting of Sephadex LH20 chromatography and silica-gel thin-layer chromatography. Positive identification was by gas chromatography/mass spectroscopy and coelution with an authentic standard. Direct and indirect mutagenicities of 2N-Fone in several bacterial strains were also determined. The results indicated that 2N-Fone produces 60-70 rev/nmole of direct mutagenic activity, and is about 1/5 to 1/10 as mutagenic as 1-nitropyrene.     

Salmonella typhimurium--test system for detecting the mutagenic activity of environmental pollutants. II. Mutagenic effect of heavy metal salts in an in vitro system with metabolic activation]

Mutagenic effect of zinc chloride on Salmonella typhimurium strain was detected using in vitro metabolic activation system. Cadmium chloride showed no significant mutagenic activity in the same system. It is recommended to use both in vitro and in vivo metabolic activation systems in mutagenicity testing of chemicals.     

Mutagenicity studies on coffee. The influence of different factors on the mutagenic activity in the Salmonella/mammalian microsome assay

Recently, mutagenic activity on several strains of Salmonella typhimurium has been found in many heat-processed foodstuffs. The previously reported direct-acting mutagenic activity of coffee in Salmonella typhimurium TA100 (Ames assay) was confirmed in our study. In addition to TA100, a mutagenic effect of coffee was also found by using the newly developed strain TA102. The mutagenic activity was abolished by the addition of rat-liver homogenate. 10% S9 mix completely eliminated the mutagenic activity of 30 mg of coffee per plate. The addition of reduced glutathione to active S9 further decreased the mutagenic activity and also reduced the mutagenicity together with inactivated S9. The compound or compounds responsible for this inactivation are heat-labile and seem to be located in the cytosol fraction of the S9. Part of the mutagenicity of coffee was also lost spontaneously upon incubation at temperatures between 0 degrees and 50 degrees C. The loss of activity was dependent on temperature, being more pronounced at 50 degrees C compared to 0 degrees C (at 50 degrees C approximately 50% of the mutagenic activity was lost after 6 h). As anaerobic conditions prevented this loss of mutagenicity almost totally, oxidative processes are probably responsible for the inactivation. The stability of the mutagen was not influenced by incubation at low pH values (pH 1-3), with or without the addition of pepsinogen. The mutagenic properties of methylglyoxal, which to some extent could be responsible for the mutagenic activity of coffee, were compared with those of coffee. Methylglyoxal was strongly mutagenic towards Salmonella typhimurium TA100 and TA102. Its mutagenic activity was partially inactivated by the addition of 10% S9. Glyoxalase I and II together with reduced glutathione abolished the mutagenic activity of methylglyoxal but reduced the mutagenicity of coffee by only 80%. Since these enzymes occur in mammalian cells, the mutagenic compound(s) of coffee could also be degraded in vivo. This conclusion is supported by the fact that a long-term carcinogenicity study with rats was negative. These results clearly demonstrate that the effects observed in vitro do not necessarily also occur in vivo, but that in vitro experiments may contribute to the understanding of fundamental mechanisms of chemical carcinogenesis.     

Detection of the mutagenic activity of lead chromate using a battery of microbial tests.

The potential mutagenicity of the carcinogen lead chromate was tested by the following battery of microbial tests: the Escherichia coli PolA+/PolA- survival test; the Salmonella/microsome His+ reversion assay; the E. coli Trp+ reversion test as a plate assay; the E. coli Gal+ forward mutation test; and the Saccharomyces cerevisiae assay for mitotic recombination. Lead chromate is mutagenic in Salmonella and in Saccharomyces and is thus identified as a microbial mutagen by this battery. Metabolic activation by rat liver homogenate (S9) is not required for the mutagenic activity of lead chromate. The most statistically significant, positive result is found with a supplementary assay, the E. coli fluctuation test. To determine whether the lead ion and/or the chromate ion were responsible for the mutagenicity observed, lead chloride and chromium trioxide (chromic acid) were also tested. In E. coli fluctuation test, the ranges of maximal mutagenicity for chromium trioxide and lead chromate overlap at the concentration 10(-5)M, whereas lead chloride shows no mutagenicity and little lethality at concentrations up to 10(-3)M. Thus, it appears that the chromate ion is responsible for the mutagenicity of lead chromate.     

Genotoxic and mutagenic activity of environmental air samples from different rural, urban and industrial sites in Flanders, Belgium

The present study reports mutagenic and genotoxic activities associated with ambient air collected at 15 sites characteristic for urban, industrial or rural conditions in Flanders. Airborne particulates (PM10) and semi-volatile compounds were collected on quartz filters (QF) and polyurethane foam (PUF) cartridges using a high-volume sampling device. The mutagenic and genotoxic potency of the organic extracts--Soxhlet extraction with acetone--was determined by use of the Salmonella mutagenicity standard plate-incorporation assay and the Vitotox assay, respectively. Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC). Ambient air samples contained significant PAH levels and mutagenic activities at all 15 sites: direct mutagenicity of up to 47 revertants per cubic meter was found in the QF extracts and more limited activity of up to 11 rev m(-3) in the PUF extracts. Metabolic activation of PUF extracts resulted in an important increase in mutagenic activity, up to 30 rev m(-3), but no such increase was observed for QF extracts. The highest values were observed outside large cities at industrial sites and at a rural site contaminated by pollution from a chemical plant at a distance of 4 km. Also at the background location near the North Sea a significant mutagenic activity was measured in the QF extracts (+S9: 9 rev m(-3); -S9: 7 rev m(-3)). Apparently, there is in Flanders a significant background exposure level to airborne mutagenicity, even in areas with limited or no nearby pollution sources. Based on the concentrations of 10 mutagenic PAHs and supposing additivity of their specific mutagenicities, only a few percent (mean 3%) of the observed indirect mutagenic activity could be explained. This implies that most mutagenic activity originated from other substances that were not identified or measured in our chemical analysis. This underscores the importance of bio-monitoring measurements.     

Mutagenicity of electrophilic N-acyloxy-N-alkoxyamides.

N-acyloxy-N-alkoxybenzamides are mutagenic in TA100 without the need for metabolic activation with S9. Electronic effects of substituents on both the benzamide ring in N-acetoxy-N-butoxybenzamides or the benzyloxy ring in N-acetoxy-N-benzyloxybenzamides do not influence mutagenicity levels. For N-benzoyloxy-N-benzyloxybenzamides, mutagenicity levels are inversely related to the electron-withdrawing effect of substituents on the benzoyloxy leaving group. Since reactivities increase with increasing electron-withdrawing effects, mutagenicity correlates with stability rather than reactivity of these mutagens. Hydrophobicity is the dominant factor controlling mutagenicity levels and data for all mutagens correlate with computed logP values with a lower dependence (h=0.22) than that recorded for indirect mutagens (h=1.0), except where a sterically demanding p-tert-butyl substituent or a naphthyl group is present. N-acetoxy-N-butoxynaphthamide exhibits a much higher level of mutagenicity than predicted by its logP value and activity may be ascribed to an intercalative binding process with DNA rather than straightforward hydrophobic binding in the major or minor groove. Since these are direct-acting mutagens, structural factors influence binding and reactivity towards DNA.     

Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.     

Study on the mutagenicity of nifurtimox and eight derivatives with the L-arabinose resistance test of Salmonella typhimurium

The mutagenicity of nifurtimox (nfx) and 8 nfx analogues has been investigated with the L-arabinose forward-mutation assay of Salmonella typhimurium. The nfx analogues tested were obtained by replacing the 3-methyl-4-yl-tetrahydro-1,4-thiazine-1,1-dioxide group of the parent compound with the following other groups: indazol-1-yl (1); pyrazol-1-yl (2); benzimidazol-1-yl (3); 1,2,4-triazol-4-yl (4); 1-methyl-3-methylthio-1,2,4-triazol-4-yl-5-thione (5); 3,5-bis(methylthio)-1,2,4-triazol-4-yl (6); 1-adamantyl (7); 4,6-diphenylpyridin-1-yl-2-one (8). The mutagenic activity of each chemical was determined by the standard plate-incorporation test, in the presence or absence of the S9 activation mixture. The 9 compounds were mutagenic and exhibited linear dose-mutagenic response relationships. They were direct-acting mutagens and showed a nearly 1000-fold range in mutagenic potency from chemical 1 to nfx. In most cases, the addition of S9 mixture to the test plates decreased the mutagenicity of compounds. This effect was particularly noticeable in the case of chemicals 1-3, 5 and 7 where a more than 70% decrease in mutagenic activity was observed in the presence of the S9 mixture. The mutagenic potency of compounds in the Ara test showed a negative linear correlation with previously reported antitrypanosomal activity. Thus, chemicals 6 and 8 with in vitro activities against Trypanosoma cruzi clearly superior to that of nfx showed 2 of the lowest mutagenic potencies in the Ara test and these were only somewhat higher than the mutagenicity of the reference drug.     

Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity.

In previous studies on the mutagenicity of anisidine isomers, the ortho isomer was considered to be mutagenic towards standard Ames tester strains, while the para isomer gave equivocal results. In the present study we show that both para- and ortho-anisidine isomers are mutagenic in a Salmonella typhimurium tester strain containing elevated levels of N-acetyltransferase (YG1029). p-Anisidine gave a positive mutagenic response using either hamster S9 or ram seminal vesicle microsomes (RSVM) as an activating system, while o-anisidine gave a positive response only with the hamster S9 fraction. The mutagenic response from p-anisidine was greater than with o-anisidine in each case. In tests with p-anisidine and RSVM, the addition of arachidonic acid was not necessary to observe a mutagenic response. Catalase produced a dose-dependent decrease in the mutagenic response with p-anisidine and RSVM; this indicates that endogenous hydrogen peroxide from the bacteria acts as a substrate for the peroxidase activity of RSVM prostaglandin H synthase. These results demonstrate that both anisidine isomers are mutagenic and that N-acetyltransferase enzymes play an important role in their metabolism to mutagenic species.     

Mutagenicity in Salmonella typhimurium mutants of serum extracts from airborne particulates

Airborne particulates collected from urban and non-urban air were extracted with calf serum or benzene, and their mutagenic potencies were evaluated in the Salmonella reversion assay. The serum extracts were mutagenic to strains TA98 and TA100 and contained both direct- and indirect-acting mutagens. Mutagenic activities for TA98 recovered from the particulates by serum or benzene extraction were much less in the serum extracts than in the benzene extracts. There was no significant difference in mutagenic potencies of the extracts between the urban and non-urban particulates, irrespective of the presence of S9 mix. The calculated mutagenic activities per m3 of air, however, were greater for urban air than for non-urban air, because of higher concentration of particulates in urban air than in non-urban air. Serum effectively reduced both direct and indirect mutagenic activities of the benzene extracts except for an insufficient reduction in direct mutagenicity at a high dose of benzene extracts. These findings suggest that serum could contribute greatly to decrease the mutagenicity of airborne particulates by mechanisms such as less efficient solubilization of mutagenic components and inactivation by protein binding. Biological availability of mutagens, therefore, should be considered for evaluation of actual mutagenic hazard by airborne particulates.     

Assessment of the Mutagenicity of Sediments from Yangtze River Estuary Using Salmonella Typhimurium/Microsome Assay

Sediments in estuaries are of important environmental concern because they may act as pollution sinks and sources to the overlying water body. These sediments can be accumulated by benthic organisms. This study assessed the mutagenic potential of sediment extracts from the Yangtze River estuary by using the Ames fluctuation assay with the Salmonella typhimurium his (−) strain TA98 (frameshift mutagen indicator) and TA100 (baseshift mutagen indicator). Most of the sediment samples were mutagenic to the strain TA98, regardless of the presence or absence of exogenous metabolic activation (S9 induction by β-naphthoflavone/phenobarbital). However, none of the samples were mutagenic to the strain TA100. Thus, the mutagenicity pattern was mainly frameshift mutation, and the responsible toxicants were both direct (without S9 mix) and indirect (with S9 mix) mutagens. The mutagenicity of the sediment extracts increased when S9 was added. Chemical analysis showed a poor correlation between the content of priority polycyclic aromatic hydrocarbons and the detected mutagenicity in each sample. The concept of effect-directed analysis was used to analyze possible compounds responsible for the detected mutagenic effects. With regard to the mutagenicity of sediment fractions, non-polar compounds as well as weakly and moderately polar compounds played a main role. Further investigations should be conducted to identify the responsible components.     

Genotoxic and mutagenic activity of environmental air samples from different rural, urban and industrial sites in Flanders, Belgium

The present study reports mutagenic and genotoxic activities associated with ambient air collected at 15 sites characteristic for urban, industrial or rural conditions in Flanders. Airborne particulates (PM10) and semi-volatile compounds were collected on quartz filters (QF) and polyurethane foam (PUF) cartridges using a high-volume sampling device. The mutagenic and genotoxic potency of the organic extracts – Soxhlet extraction with acetone – was determined by use of the Salmonella mutagenicity standard plate-incorporation assay and the Vitotox^® assay, respectively. Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC).Ambient air samples contained significant PAH levels and mutagenic activities at all 15 sites: direct mutagenicity of up to 47 revertants per cubic meter was found in the QF extracts and more limited activity of up to 11 rev m⁻³ in the PUF extracts. Metabolic activation of PUF extracts resulted in an important increase in mutagenic activity, up to 30 rev m⁻³, but no such increase was observed for QF extracts. The highest values were observed outside large cities at industrial sites and at a rural site contaminated by pollution from a chemical plant at a distance of 4 km. Also at the background location near the North Sea a significant mutagenic activity was measured in the QF extracts (+S9: 9 rev m⁻³; −S9: 7 rev m⁻³). Apparently, there is in Flanders a significant background exposure level to airborne mutagenicity, even in areas with limited or no nearby pollution sources. Based on the concentrations of 10 mutagenic PAHs and supposing additivity of their specific mutagenicities, only a few percent (mean 3%) of the observed indirect mutagenic activity could be explained. This implies that most mutagenic activity originated from other substances that were not identified or measured in our chemical analysis. This underscores the importance of bio-monitoring measurements.     

Mutagenicity of benzotrichloride and related compounds.

Benzotrichloride (BTC), benzal chloride (BDC), benzyl chloride (BC) and benzoyl chloride (BOC) were surveyed for their mutagenicity in microbial systems such as rec-assay using Bacillus subtilis and reversion assays using E. coli WP2 and Ames Salmonella TA strains with or without metabolic activation in vitro. BTC and BDC required metabolic activation for their mutagenic activities in several strains of E. coli and Salmonella. The mutagenic metabolites of these compounds may not have been produced by hydrolysis. BC was weakly mutagenic without metabolic activation. Only BOC exhibited no mutagenic activity in the detection procedures used. The mutagenic metabolite of BTC might be very unstable under our experimental conditions. The strain E. coli WP2 try hcr was more sensitive than E. coli B/r WP2 try (hcr+) with regard to the mutagenicity of BTC.     

Mutagenicity of phenanthrene and phenanthrene K-region derivatives.

Phenanthrene and 9 K-region derivatives, most of them potential metabolites of phenanthrene, were tested for mutagenicity by the reversion of histidine-dependent Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100 and the rec assay with Bacillus subtilis H17 and M45. The strongest mutagenic effects in the reversion assay were observed with phenanthrene 9,10-oxide, 9-hydroxyphenanthrene and N-benzyl-phenanthrene-9,10-imine. Interestingly, the mutagenic potency of the arene imine was similar to that of the corresponding arene oxide. This is the first report on the mutagenicity of arene imine. The mutagenic effects of all these phenanthrene derivatives were much weaker than that of the positive control benzo[a]pyrene 4,5-oxide. Even weaker mutagenicty was found with cis-9,10-dihydroxy-9,10-dihydrophenanthrene and with trans-9,10-dihydroxy-9-10-dihydrophenanthrene. The other derivatives were inactive in this test. However, 9-10-dihydroxyphenanthrene and 9,10-phenanthrenequinone were more toxic to the rec- B. subtilis M45 strain than to the rec+ H17 strain. This was also true for phenanthrene 9,10-oxide and 9-hydroxyphenanthrene, but not with the other test compounds that reverted (9,10-dihydroxy-9,10-dihydrophenanthrenes; N-benzyl-phenanthrene 9,10-imine; benzo[a]pyrene 4,5-oxide) or did not revert (phenanthrene, 9,10-bis-(p-chlorophenyl)-phenanthrene 9,10-oxide, 9-10-diacetoxyphenanthrene) the Salmonella tester strains. Although the K region is a main site of metabolism and although all potential K-region metabolites were mutagenic, phenanthrene did not show a mutagenic effect in the presence of mouse-liver microsomes and an NADPH-generating system under standard conditions. However, uhen epoxide hydratase was inhibited, phenanthrene was activated to a mutagen that reverted his- S. typhimurium. This shows that demonstration of the mutagenic activity of metabolites together with the knowledge that a major metabolic route proceeds via these metabolites dose not automatically imply a mutagenic hazard of the mother compound, because the metabolites in question may not accumulate in sufficient quantities and therefore the presence and relative activities of enzymes that control the mutagenically active metabolites are crucial. N-Benzyl-phenanthrene 9.10-imine was mutagenic for the episome-containing S. typhimurium TA98 and TA100 but not for the precursor strains TA1538 and TA1535. This arene imine would therefore be useful as a positive control during routine testing to monitor in the former strains the presence of the episome which is rather easily lost.