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.     

Nitroreduction of 6-nitrobenzo[a]pyrene: a potential activation pathway in humans

6-Nitrobenzo[a]pyrene, an environmental pollutant, was metabolized by human intestinal microflora to 6-nitrosobenzo[a]pyrene and 6-aminobenzo[a]pyrene. The two-electron reduction product 6-nitrosobenzo[a]pyrene exhibited strong direct-acting mutagenicity in the Salmonella typhimurium assay. These results imply that 6-nitrobenzo[a]pyrene can be hazardous to human health via a nitroreduction activation pathway and opens the possibility that other nitro-polycyclic aromatic hydrocarbons that are not direct-acting mutagens may be activated in vivo by a similar mechanism.     

Relationship between mutagenic potency in Salmonella typhimurium and chemical structure of amino- and nitro-substituted biphenyls

All positional isomers of mononitro- and monoaminobiphenyls and those of dinitro-, diamino- and aminonitrobiphenyls, which have one substituent on each benzene ring, were assayed for mutagenicity in Salmonella typhimurium by the Ames method. The results suggest that the structural requirements favoring mutagenic activity are the presence of substituents at the 4-position and their absence at the 2'-position. The introduction of an amino group to the 3'- or 4'-position of 4-nitrobiphenyl or a nitro group to 3'- or 4'-position of 4-aminobiphenyl enhanced the mutagenicity. Among the mutagenic compounds, 4-nitro analogues were mutagenic in strains TA98 and TA100 in the absence of a microsomal metabolic activation system. Strain TA98NR was not reverted by the direct-acting mutagens, whereas strain TA98/1,8-DNP6 was as revertible as strain TA98; these results suggest that the direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes.     

Hepatocyte-mediated mutagenicity of mononitrobenzo[a]pyrenes in Salmonella typhimurium strains

The mononitro-substituted isomers of benzo[a]pyrene (B[a]P), 1-, 3- and 6-nitrobenzo[a]pyrene (NB[a]P), are environmental pollutants and are metabolized to mutagens in Salmonella by rat-liver homogenate postmitochondrial supernatant (S9) fractions. In this study, activation of these compounds to mutagens was investigated using the hepatocyte-mediated Salmonella mutagenicity assay. Hepatocytes from rats treated with Aroclor 1254 activated both 3-NB[a]P and 1-NB[a]P to mutagens, while 6-NB[a]P was not mutagenic. The positive mutagenicity responses were functions of both the chemical dose and the hepatocyte concentration. By using a nitroreductase-deficient strain (TA98NR) and a transesterificase-deficient strain (TA98/1,8-DNP6), it was verified that the direct-acting mutagenicities of 1- and 3-NB[a]P primarily were due to metabolic processes involving nitroreduction while the S9- and hepatocyte-mediated mutagenicity responses were also dependent on transesterification. When compared with the mutagenic responses produced with S9, the mutations induced by 1- and 3-NB[a]P in the presence of hepatocytes were relatively more dependent upon nitroreductase metabolism and less on transesterification. Thus, intact hepatocytes were capable of activating 1- and 3-NB[a]P to mutagenic metabolites and some of these metabolites appeared to be different from those produced by S9.     

The presence of the mutagenic polycyclic aromatic hydrocarbons benzo[a]pyrene and benz[a]anthracene in creosote P1

Several fractions of creosote P1 separated by TLC showed mutagenicity towards Salmonella typhimurium TA98. Thus mutagenicity is probably caused by the presence of mutagenic aromatic hydrocarbons. The mutagenic polycyclic aromatic hydrocarbons, benzo[a]pyrene and benz[a]anthracene, were detected in concentrations of 0.18 and 1.1% respectively. Because these compounds are probably not essential for the wood-preserving properties of creosote , a more selective composition of the product should be considered.     

Mutagenicity of benzylic acetates, sulfates and bromides of polycyclic aromatic hydrocarbons

Studies were performed to determine the direct mutagenicity of the acetates and some bromides and sulfates of hydroxymethyl polycyclic aromatic hydrocarbons in S. typhimurium strains TA98 and TA100. Benzylic acetates, bromides and sulfates were synthesized and characterized. The compounds tested were benzyl alcohol, 5-hydroxymethylchrysene, 1-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene, 6-(2-hydroxyethyl)benzo[a]pyrene, 6-hydroxymethylanthanthrene, 9-hydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene, 7-hydroxymethylbenz[a]anthracene, 7-(2-hydroxyethyl)benz[a]anthracene, 12-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, 12-hydroxymethyl-7-methylbenz[a]anthracene, 1-hydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene, 3-hydroxy-3, 4-dihydrocyclopental[cd]pyrene and 4-hydroxy-3, 4-dihydrocyclopental[cd]pyrene. The benzylic sulfate esters of 6-hydroxymethylbenzo[a]pyrene and 7-hydroxymethylbenz[a]anthracene were the most mutagenic compounds, whereas the aliphatic sulfate ester of 7-hydroxyethylbenz[a]anthracene did not cause an increase in mutations above background. All meso-anthracenic benzylic acetate esters were mutagenic in both strains with various degrees of activity, whereas the corresponding non-benzylic esters were inactive, as expected. Of the non-meso-benzylic acetate esters, only the 3-acetoxy-3, 4-dihydrocyclopenta[cd]pyrene was mutagenic. In the benzylic bromide series, only the eight mesoanthracenic were mutagenic, whereas benzyl bromide and 5-bromomethylchrysene were inactive. The aliphatic bromides, 6-(2-bromoethyl)benzo[a]pyrene and 7-(2-bromoethyl)benz[a]anthracene did not display significant activity. The potencies of the acetate esters more accurately reflect the mutagenicity because the rate of solvolysis did not compete with the reactivity of the esters with bacterial DNA. In the case of benzylic sulfates and bromides, the rate of solvolysis was very rapid and could have diminished the level of mutagenicity, depending on the assay conditions. These results demonstrate that meso-anthracenic benzylic acetates, sulfates and bromides are mutagenic, whereas benzylic acetate esters attached to other carbon atoms are inactive.     

Correlations between 15 polycyclic aromatic hydrocarbons (PAH) and the mutagenicity of the total PAH fraction in ambient air particles in La Spezia (Italy)

Airborne particulate matter has been monitored 4 times a month for 1 year (1988) in the city of La Spezia (Italy). The polycyclic aromatic hydrocarbon (PAH) fractions were extracted, purified and characterized for the content of 15 individual PAH. In general when concentrations of individual PAH were compared statistical correlation was obtained. Mutagenicity studies were performed by the use of the Ames plate test with the Salmonella strains TA98, TA100, TA98NR and TA98DNP6 with and without metabolic activation (S9 mix). The TA98 strain was by far the most responsive and the S9 mix was absolutely required as expected when PAH are assayed. Besides mutagenicity, toxicity was also considered and it proved to be correlated with mutagenicity in TA98, +S9. The TA98NR and TA98DNP6 strains showed no appreciable differences from the parental strain TA98 indicating the absence of significant amounts of direct-acting nitro derivatives in our PAH samples. Of the 15 PAH considered in this study the amounts of cyclopental[c,d]pyrene (CPP) correlated best with mutagenicity. The role of CPP in contributing to the indirect mutagenicity of urban air PAH samples is discussed.     

Mutagenicity of nitro-azabenzo[a]pyrene and its related compounds.

The mutagenicity of nitrated benzo[a]pyrene (BP) and the related compounds, 1- and 3-nitrobenzo[a]pyrene (NBP), 1- and 3-nitro-6-cyanobenzo[a]pyrene (N-6-CBP), 1- and 3-nitro-6-azabenzo[a]-pyrene (N-6-ABP), 1- and 3-nitro-6-azabenzo[a]-pyrene-N-oxide (N-6-ABPO) and 1,6- and 3,6-dinitrobenzo[a]-pyrene (DNBP), was investigated. The mutagenic activities of 3-N-6-CBP and 3-N-6-ABP were 117 and 76 times, respectively, that of 3-NBP. In addition, 3,6-DNBP was more mutagenic than 1,6-DNBP. It is suggested that the mutagenic activation differs with the position of NO2 substitution in the chemical structure. A nitro derivative with NO2 substitution at the 3 position of the aromatic ring of BP was more mutagenic than that with the substitution at the 1 or 6 position. The reducibility of DNBPs was then determined by detecting 1- or 3-amino-6-nitrobenzo[a]pyrene (A-6-NBP), a metabolite of DNBP; 3,6- and 1,6-DNBP were reduced to 3- and 1-A-6-NBP at frequencies of 958 +/- 26 and 79 +/- 8, respectively, pmole per mg of protein, when the compound was incubated anaerobically with rat liver S9 mix at 37 degrees C for 15 min. NO2 substituted at the 3 position of the aromatic ring of BP was readily reduced by a microsome enzyme to form an amino derivative. The result suggests that these compounds have a structure-activity relationship between mutagenicity and NO2 substitution of BP.     

From mutagenic to non-mutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of 4-nitrobiphenyl in Salmonella typhimurium. Part I. Substituents ortho to the nitro group and in 2'-position

Eleven alkyl substituted derivatives of 4-nitrobiphenyl (4NBp) and two corresponding nitroso compounds were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium. The mutagenicity of compounds substituted ortho to the nitro group (3-methyl-, 3-ethyl-, 3-isopropyl-, 3-tertbutyl-, 3, 5-diethyl-, 3,5-diisopropyl-, and 3,5-ditertbutyl-4NBp) decreased with growing steric demand of the alkyl substituents in both tester strains. The most sterically hindered compounds were non-mutagenic even at highest concentrations. This reduction of mutagenicity is correlated with deviations from the coplanar orientation of the nitro group relative to the aromatic plane. Since a comparable decrease of mutagenicity for the nitroso compounds (4NOBp and 3-tertbutyl-4NOBp) was not observed, the first reduction step of non-planar nitro groups must be inhibited.Alkyl groups in the 2'-position of 4NBp (2'-methyl-, 2'-ethyl-, 2'-isopropyl-, and 2',4', 6'-trimethyl-4NBp) also reduced mutagenic activity with increasing size and removed it completely for the most sterically hindered species (2'-isopropyl-, 2',4',6'-trimethyl-4NBp). In this case, the co-planarity of the nitro group is not affected but the twisting of the two aromatic rings, which is associated with a less effective charge delocalisation of the nitrenium ion.The experimental mutagenicities of all nitro compounds were compared to predicted values, that are based on recently developed empirical equations. While there was reasonable correspondence for the parent compound (4NBp), its ortho methylated derivative (3-methyl-4NBp) and two highly hydrophobic dialkylated species (3,5-diisopropyl- and 3, 5-ditertbutyl-4NBp), predictions for all other alkyl substituted compounds were too high. Thus, steric parameters should be included to improve the general validity of predictions by means of quantitative structure-activity relationships (QSAR).     

Relationship between mutagenic potency in Salmonella typhimurium strains and the chemical structure of nitro biphenyls

Most of the positional isomers of mono-, di-, tri- and tetranitrobiphenyls were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8DNP6 in the absence of S9 mix. In mono- and dinitrobiphenyls, the structure requirements favoring mutagenic activity are the presence of a nitro group at the 4-position and its absence at the 2-position. TA98 and TA98/1,8DNP6 were reverted by 2-position-free 4-nitro analogues, but TA98NR was not reverted. The results suggest that direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes. Some of the tri- and tetranitrobiphenyls e.g. 3,4,3'-, 3,4,4'-, 3,4,3',4'- and 3,4,2',4'-derivatives reverted not only TA98 and TA98/1,8DNP6 but also TA98NR. Those derivatives commonly have 2 nitro groups at an adjoining position (3,4-dinitro group), whereas 2,4,2',4'-tetranitrobiphenyl, which has strong potency not only in TA98 and TA98/1,8DNP6 but also in TA98NR, possesses 2 nitro groups at the 2-position of each benzene ring.     

Comparison of the mutagenic specificity induced by four nitro-group-containing aromatic amines in Salmonella typhimurium his genes

Four nitrated aromatic amines (2-nitro-p-phenylenediamine [2NPD], 3-nitro-o-phenylenediamine [3NPD], 4-nitro-o-phenylenediamine [4NPD] and 4,4'-dinitro-2-biphenylamine [DNBA]) are direct-acting mutagens in Salmonella typhimurium strain TA100. These compounds were tested further using the Xenometrix strains of S. typhimurium: TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006, with and without S9 mix in the plate incorporation assay. The direct-acting mutagenicity of 2NPD, 4NPD, and DNBA was detected with TA7002, TA7004 and TA7005. 2NPD and DNBA showed some activity in TA7006; DNBA also showed some activity in TA7003. Mutagenicity was generally decreased in these strains when S9 was added. 3NPD was mutagenic in TA7004 without S9 and in TA7005 with and without S9. These data suggest that 2NPD, 4NPD and DNBA induced TA-->AT and CG-->AT transversions as well as GC-->AT transitions in the his gene. 3NPD induced CG-->AT transversions and GC-->AT transitions. 2NPD and DNBA also induced a small portion of CG-->GC transversions.     

Mutagenicity of 2,6-dinitrotoluene and its metabolites, and their related compounds in Salmonella typhimurium

The mutagenic activities of 2,6-dinitrotoluene (2,6-DNT) and its 6 metabolites, and their 8 related compounds were examined using Salmonella typhimurium strains TA98 and TA100 in the absence or presence of S9 mix. 2,6-DNT itself showed no mutagenicity toward either strain, but 2,6-dinitrobenzaldehyde (2,6-DNBAl), one of the metabolites of 2,6-DNT, showed the highest mutagenic activity in strain TA100. 2,6-DNBAl was a direct-acting mutagen, not requiring metabolic activation. The other compounds containing nitro groups showed weak or no mutagenic activity. This result suggests that the direct-acting mutagenicity of 2,6-DNBAl is mainly due to the aldehyde group of the 2,6-DNBAl molecule.     

Mutagenicity of some commercially available nitro compounds for Salmonella typhimurium.

Benzoyl chloride and 53 commercially available aromatic heterocyclic and aliphatic nitro compounds were tested for mutagenicity in Salmonella typhimurium TA98 and TA100. 34 of 53 nitro compounds (64%) were mutagenic, 4 in TA100 only, 15 in TA98 only, and 15 in both strains. 13 of the heterocyclic derivatives of pyridine, indole, indazole, quinoline, and benzimidazole were mutagenic. 21 of 34 mutagenic nitro compounds were bactericidal. Nitromethane was the only aliphatic tested and was not mutagenic. Benzoyl chloride, a human carcinogen, was mutagenic for TA98.     

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.     

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.     

Photolysis primes biodegradation of benzo[a]pyrene

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Use of nitrous acid-dependent decrease in mutagenicity as an indication of the presence of mutagenic primary aromatic amines. Non-specific reactions with phenols and benzo[alpha]pyrene

Treatment of mutagenic primary aromatic amines with nitrous acid is known to decrease their mutagenicity. We examined some factors concerning the validity of using decreases in mutagenicity due to nitrous acid treatment as an indication of the presence of mutagenic primary aromatic amines in complex mixtures. We found that treatment of benzo[alpha]pyrene with nitrous acid for the extended periods of time previously employed leads to formation of three nitrobenzo[alpha]pyrene isomers. Some of the isomers are direct-acting mutagens for S. typhimurium with considerably greater mutagenicity than benzo[alpha]pyrene isomers. In attempts to minimize reaction of chemicals other than aromatic amines, we found that only very brief reaction periods are required for complete reaction of nitrous acid with representative aromatic amines, essentially eliminating their mutagenicity. During such brief reaction periods modification of benzo[alpha]pyrene is negligible, but phenols react readily. Chromatographic analysis indicated that reaction of nitrous acid with aromatic amines leads to the formation of families of products, thereby increasing the complexity of the mixtures in which the amines may occur. Thus, experiments examining the effects of nitrous acid on the mutagenic activity of complex mixtures must be carefully designed, and the results must be interpreted cautiously.     

Antimutagenic activity of some naturally occurring compounds towards cigarette-smoke condensate and benzo[a]pyrene in the Salmonella/microsome assay

Several compounds, occurring in food, were tested for antimutagenic activity towards cigarette-smoke condensate (CSC) and benzo[a]pyrene (BaP). Antimutagenicity was determined in the Salmonella/microsome test, with tester strain TA98, in the presence of rat-liver homogenate. Dose-response curves did show reduction of CSC- and BaP-induced mutagenicity by ellagic acid, riboflavin and chlorophyllin. Chlorophyll a and chlorophyll b, although less distinct, also inhibited CSC- and BaP-induced mutagenicity. Ascorbic acid, beta-carotene, tocopherol acetate, chlorogenic acid and butyl hydroxyanisole did not have any influence on the mutagenicity of CSC and BaP. The similarity in results for cigarette-smoke condensate and for BaP indicates that a general mechanism may be involved in the inhibition of CSC- and BaP-induced mutagenicity.     

Bacterial mutagenicity of new cyclopenta-fused cata-annelated polycyclic aromatic hydrocarbons, and identification of the major metabolites of benz[j]acephenanthrylene formed by Aroclor-treated rat liver microsomes

Three novel cyclopenta-fused polycyclic aromatic hydrocarbons were synthesized, benz[d]aceanthrylene, benz[k]aceanthrylene, and benz[j]acephenanthrylene, and evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. The two benzaceanthrylene derivatives were active at low S9 concentrations in strain TA98 (4 and 27 rev/nmole respectively), as had been predicted from the calculated delta Edeloc/beta values of the carbocations derived from opening of the cyclopenta-fused epoxide rings, but the majority of this mutagenicity appeared to be due to free-radical decomposition products of spontaneous endo-peroxide formation. These compounds were therefore not further investigated. Benz[j]acephenanthrylene was also an indirect-acting frameshift mutagen (8-12 rev/nmole in strain TA98), but unlike most of the previously assayed cyclopenta-fused polycyclic aromatic hydrocarbons exhibited no peak of activity at low S9 protein concentration. The principal metabolites formed from this compound by microsomes from Aroclor-treated rat liver were benz[j]acephenanthrylene-4,5-dihydro-4,5-diol (necessarily derived from hydration of benz[j]acephenanthrylene 4,5-oxide) and benz[j]acephenanthrylene-9,10-dihydro-9,10-diol (precursor to benz[j]acephenanthrylene-9,10-dihydrodiol 7,8-oxide, the bay-region diol-epoxide). Consideration of the reduced activity of this compound compared to the related structure chrysene, the S9 dependence curves, and the predicted delta Edeloc/beta values of the postulate active species, suggests that in contrast to most other cyclopenta-fused polycyclic aromatic hydrocarbons, bay-region diol-epoxide formation plays a greater role than epoxidation of the cyclopenta-fused ring in the metabolic activation of benz[j]acephenanthrylene.     

Mutagenicity of some methylated benzo[a]pyrene derivatives

The mutagenicity of benzo[a]pyrene (BP) and a number of methylated derivatives towards Salmonella typhimurium has been tested. The most mutagenic derivative tested was 6-methylbenzo[a]pyrene which produced about twice the number of revertants as did BP, 11-Methylbenzo[a]pyrene was slightly more mutagenic than BP. All the other compounds tested (7-, 8-, 9- and 10-methylbenzo[a]pyrene and 7,8- and 7,10-dimethylbenzo[a]pyrene) were significantly less active than benzo[a]pyrene. With the exception of 6-methylbenzo[a]pyrene, these results closely parallel the known carcinogenicity of the methylated benzo[a]pyrenes, and support the view that metabolic activation of BP may involve the 7-10 positions which are blocked in the methylated compounds.