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microsome prescreen to petroleum-derived complex mixtures and polynuclear aromatic hydrocarbons (PAH)
Mutation Research, 174 (1986) 247-253 Elsevier
247
MRLett. 0880
Application of modified Salmonella/microsome prescreen to petroleumderived complex mixtures and polynuclear aromatic hydrocarbons (PAH) J.H. Carver, M.L. Machado and J.A. MacGregor Chevron Environmental Health Center, Inc., P.O. Box 4054, Richmond, CA 94804-0054 (U.S.A.) (Accepted 25 March 1986)
Summary In some cases, the Salmonella mutagenicity assay may fail to predict the carcinogenic potential of PAH (and of complex mixtures containing PAH) because of nonoptimal in vitro metabolic activation parameters. In this study, 7 petroleum-derived complex mixtures, as well as a number of individual P A H which were representative constituents of such mixtures, were tested in a Salmonella prescreen using quadrant plates with rat or hamster $9 at concentrations approximately 2-8 times those used in the standard assay. Some PAH (perylene, quinoline, benzo[b]chrysene, phenanthrene, anthracene) were optimally activated to mutagens by $9 at 400 ~,l/plate. Rat $9 was similar to hamster $9 for most tested PAH, but anthracene and quinoline mutagenicity was enhanced by hamster $9. All 7 complex mixtures were more mutagenic with 200-400/~l/plate $9; rat was generally slightly more efficient than hamster. Modifying this assay to include a prescreen using a range of $9 concentrations (and perhaps from species other than rat) may improve prediction of the potential carcinogenicity of complex petroleum-derived mixtures.
Increasing evidence suggests that the failure of the Salmonella assay to predict the carcinogenic potential of some complex hydrocarbon mixtures or fractions containing polynuclear aromatic hydrocarbons (PAH) may be due, at least in part, to insufficient amounts of microsomes ($9). In some cases, $9 from hamster may be more effective (Blackburn et al., 1983, 1984; Carver and MacGregor, 1984; Carver et al., 1985). In this study, liver $9 from Aroclor-induced rats or hamsters was used in the Salmonella prescreen (Falcon 1009 quadrant plates, see Carver et al., 1985). A range of $9 concentrations approximately 2-8 times those suggested for the standard assay
(Maron and Ames, 1983) was applied. 29 individual PAH which were representative constituents of petroleum-derived complex mixtures (as determined by high-resolution mass spectroscopy and high-pressure liquid chromatography) were tested in Salmonella typhimurium TA100 with rat or hamster $9 at the equivalent of 50, 100, 200 and 400/d/plate. In addition, 7 complex mixtures known to be active in the mouse dermal carcinogenicity assay were tested: C-5d, D-5d, C-5a, D-5a, WPR1, WPR2, LD. The first four samples - - two petroleum distillates and their aromatic fractions separated by absorption chromatography - - have
0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Mutagenic activity in Salmonella typhimurium TAI00. Data from quadrant plates normalized and expressed as revertants per plate in the presence of 50, 100, 200 or 400/~1 $9 per plate plus I x NADP (2 x for 400/~g/plate $9). Spontaneous mutant frequencies did not differ significantly for rat and hamster $9; combined values are shown. Values for compounds tested without $9 at dose levels of 1000 #g/plate or 500/~g/plate (solubility limit) did not differ significantly from spontaneous mutant frequencies.
249 TABLE 1 COMPARISON OF PUBLISHED C A R C I N O G E N I C I T Y AND GENOTOX1CITY OF TESTED PAH W I T H M O D I F I E D S A L M O N E L L A / M I C R O S O M E PRESCREEN RESULTS NIOSH data a
Prescreen results
Number of rings
Car.
Neo.
Mut.
Chrysene, CHR
5 5 4 5 4
+ + + + +
+ + + + +
+ + + + +
+ + + + +
Anthracene, A Phenanthrene, P Quinoline, Q
3 3 2
+ + +
+ + +
+ + +
Benzo[b]chrysene, BC Benzo[b]anthracene, BbA 9-Methylanthracene, 9MA 2-Methylanthracene, 2MA Perylene, PER Benzo[ght]perylene, BPER Fluoranthene, FA Phenanthridine, P H D Benzo[e]pyrene, BeP Pyrene
5 4 3 3 5 5 3 3 5 4
+ + + + + + + + + +
+ + + + + + + + + -
Dibenzo[c,g]phenanthrene, DBP Dimethylbenzo[c]phenanthrene, DBMP
5 4 4 4 4 4
Benzo[a]pyrene, BaP Dibenz[a, h]anthracene, DBA Benzo[a]anthracene, BaA 3-Methylcholanthrene, MC
Benzo[c]phenanthrene, BP Benzo[ghOfluoranthene, BFA 2,3-Benzofluorene, 2,3BF 1,2-Benzofluorene, 1,2BF 3-Methylfluorene 3,6-Dimethylphenant hrene
+c
b
+ + + + + +
3 3
Hexabenzobenzene Dibenzopentacene
6 7
Dibenzo[g,p]chrysene
6
-
-
?d
a Registry of Toxic Effects. b Negative and non-toxic at 1000/zg/plate; weakly mutagenic at 1-3/zg/plate. ¢ Wood et al. (1980). d Mossandra et al. (1979).
been previously described (Carver and MacGregor, 1984). Three additional complex mixtures were also tested: waxy petroleum residuum, WPR, from two different crudes (WPR1, WPR2, boiling range 615-1000°F) and a lube distillate (LD; boiling range 550-900°F). As summarized by Carver and MacGregor (1984), samples C-5d, D-5d, C-5a and
D-5a produced a tumor incidence of 82%, 87%, 98% and 38°70 (with toxicity), respectively. The tumorigenic potential of the WPR and LD samples had been characterized in a similar in vivo dermal carcinogenicity assay in the mouse (unpublished data, Chevron Environmental Health Center, Inc.). One sample, WPRI, was weakly
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Fig. 2. Mutagenic activity as in Fig. 1. Complex mixtures shown are two high-boiling (700-1070°F) petroleum distillates (C-5d, D-5d) and their aromatic fractions (C-5a, D-5a) as previously described (Carver and MacGregor, 1984; Carver+ Machado and MacGregor, 1985); waxy petroleum residuum (WPRI, WPR2)
from two different crudes, boiling range 615-1000°F; lube distillate (LD), boilingrange 550-900"F.
tumorigenic (12°70 incidence). The other sample, WPR2, was more potent (5207o incidence). The LD sample was similar in potency, with an incidence of 4807o. These results are similar to those reported for other studies with crude shale oil, crude petroleum and synthetic fossil fuel materials (see refs. in Carver et al., 1985). However, initial mutagenicity experiments with these 7 complex mixtures were negative. The Salmonella prescreen was used to further test these 7 mixtures, to see if results obtained would more accurately reflect the samples PAH content and known oncogenic potential.
Materials and methods
Chemicals.
Most PAH were obtained in the highest purity available from a variety of commercial sources, i.e., Aldrich, Milwaukee, WI; Analabs, North Haven, CT, Biochem. Inst. for Environ. Carcinogenicity, Awrenceberg, Germany; Chem. Serv., West Chester, PA; Columbia Chem., Columbia, SC; ICN, Plalnview, NY; K&K, Red Bank, NJ. All were used as received. A
few materials were analytical standards obtained from the Chevron Research Company, Richmond, CA. Mutagenicity assays. Assay procedures, including solubilization of petroleum-derived mixtures, were previously described (Carver and MacGregor, 1984; Carver et al., 1985). Quantitation and analysis of results. The quadrant-plate prescreen is considered a semiquantitative assay. General trends as shown herein are reproducible from experiment to experiment, but mean values may show considerable variability, ca. 30-40070. Plates were counted on an Artek Automatic Colony Counter (Artek Systems, Corp., Farmingdale, NY); no correction factor was applied.
Results
In Table 1, qualitative results are shown for the 29 PAH tested. Preliminary experiments used rat $9; negative results were reproducible when repeated using hamster $9 (data not shown). Nonmutagenic PAH were tested to the limit of solubility, to cytotoxicity, or to 1000/zg/plate. In Fig. l, responses for 6 different PAH at 3 dose levels are shown. Rat or hamster $9 at the equivalent of 50, 100, 200 and 400/zl/plate (2 x NADP for the latter) was used. In general, the optimal concentration of $9 increased with increasing PAH dose levels. In some cases, higher concentrations of hamster $9 were needed to achieve the mutagenic response noted with rat. Perylene and quinoline were optimally activated at higher $9 concentrations (200-400 /~l/plate) with hamster somewhat more effective than rat. In Fig. 2, results for 10 PAH and 7 complex mixtures tested at a single dose level with rat $9 are compared to data obtained with hamster $9. Because PAH are frequently present in petroleum-derived complex mixtures in low concentrations, the comparison of rat vs. hamster $9 was made at PAH dose levels yielding detectable but not necessarily mutagenic activity. Preliminary experiments (data not shown) using rat $9 had indicated that the PAH dose levels
252
selected would result in weak mutagenic responses in the range of 2-3 times spontaneous background. Hamster $9 was optimal for activation of DBA (5 /zg/plate) over a larger range of $9 concentrations than was rat $9. PHD (100/~g/plate) was activated by rat $9, but would have been missed at any concentration of hamster $9. No significant differences between rat and hamster $9 were noted for CHR (5 #g/plate). For 2,3BF (15/~g/plate), rat $9 was effective over a larger concentration range. Rat $9 was clearly superior to hamster for activation of BeP (50 /zg/plate) to mutagenic metabolites. High concentrations (400/zl/plate) of either rat or hamster $9 activated 1,2BF (50 /zg/plate), but 2MA (50/zg/plate) showed a sharp concentration optimum for both rat and hamster. Both rat and hamster $9 activated BC (50/zg/plate) and P (50 ttg/plate), but hamster $9 was more effective for A (450 #g/plate). In general, promutagens in all 7 petroleum-derived complex mixtures (10 mg/plate) were optimally activated by high concentrations (400/~l/plate) of $9, with no highly significant differences between rat and hamster.
Discussion The Salmonella mutagenicity assay may not always reliably predict the potential carcinogenicity of energy-related materials. For example, results for coal-derived samples showed that the standard Ames assay had a high sensitivity to amino PAH but little sensitivity in general to neutral PAH in such complex mixtures, particularly when chemically-separated fractions were tested. The neutral PAH components contained the highest initiating activity, which was correlated with mutagenicity observed in cultured mammalian cells (Mahlum et al., 1984). Petroleum distillates tested in the Salmonella assay showed positive responses with elevated levels of $9, correlating at least qualitatively with the distillates' known tumorigenic activity (Carver and MacGregor, 1984). For complex mixtures derived from petroleum, at least, the evidence suggests that stan-
dard Salmonella protocols should be revised to include optimization of metabolic activation parameters (Blackburn et al., 1984; Carver et al., 1985). In the study reported here, rat or hamster $9 at concentrations approximately 2-8 times those routinely used enhanced the observed mutagenicity of selected, individual PAH as well as PAH components of complex mixtures. A prescreen using quadrant plates to predict, in a semiquantitative manner, the optimal $9 level for each complex mixture tested seems warranted. Testing a range of $9 concentrations from more than one species may improve the Salmonella assay's correlation with the carcinogenic potential of complex mixtures. However, with the exception of anthracene and quinoline, $9 from hamster had little or no advantage over that from rat for the PAHs and mixtures tested.
Acknowledgements We acknowledge the excellent technical assistance of M.T. Wininger and C.S. Fukuda in conducting the modified Salmonella prescreen assays; we thank A.E. Oranje for manuscript preparation. The authors are indebted to members of the Medical and Biological Science Department of the American Petroleum Institute (API) for contributing the two petroleum distillates and their aromatic fractions.
References Blackburn, G.R., R.A. Deitrich, T.A. Roy, C.A. Schreiner, J.R. Meeks and C.R. Mackerer (1983) Mutagenesis of used gasoline engine oil fractions, in: M. Cooke and A.J. Dennis (Eds.), Polynuclear Aromatic Hydrocarbons: Formation, Metabolism, and Meastirement, Battelle, Columbus, OH, pp. 113-122. Blackburn, G.R., R.A. Deitrich, C.A. Schreiner, M.A. Mehlman and C.R. Mackerer (1984) Estimation of the dermal carcinogenic activity of petroleum fractions using a modified Ames assay, Cell Biol. Toxieol., 1, 40-48. Carver, J.H., and J.A. MacGregor (1984) Mutagenicity and chemical characterization of two petroleum distillates, J. Appl. Toxicol., 4, 163-169.
253 Carver, J.H., M.L. Machado and J.A. MacGregor (1985) Petroleum distillates suppress in vitro metabolic activation: Higher [$9] required in the Salmonella/microsome mutagenicity assay, Environ. Mutagen., 7, 369-379. Mahlum, D.D., C.W. Wright, E.K. Chess and B.W. Wilson (1984) Fractionation of skin tumor-initiating activity in coal liquids, Cancer Res., 44, 5176-5181. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mossandra, K., F. Poncelet, A. Fouassin and M. Mercier
(1979) Detection of mutagenic polycyclic aromatic hydrocarbons in African smoked fish, Fd. Cosmet. Toxicol., 17, 141-143. Wood, A.W., R.L. Chang, W. Levin, D.E. Ryan, P.E. Thomas, M. Croisy-Delcey, Y. Ittah, H. Yagi, D.M. Jerina and A.H. Conney (1980) Mutagenicity of the dihydrodiols and bay-region diol-epoxides of benzo[c]phenanthrene in bacterial and mammalian cells, Cancer Res., 40, 2876-2883. Communicated by R.J. Preston
247
MRLett. 0880
Application of modified Salmonella/microsome prescreen to petroleumderived complex mixtures and polynuclear aromatic hydrocarbons (PAH) J.H. Carver, M.L. Machado and J.A. MacGregor Chevron Environmental Health Center, Inc., P.O. Box 4054, Richmond, CA 94804-0054 (U.S.A.) (Accepted 25 March 1986)
Summary In some cases, the Salmonella mutagenicity assay may fail to predict the carcinogenic potential of PAH (and of complex mixtures containing PAH) because of nonoptimal in vitro metabolic activation parameters. In this study, 7 petroleum-derived complex mixtures, as well as a number of individual P A H which were representative constituents of such mixtures, were tested in a Salmonella prescreen using quadrant plates with rat or hamster $9 at concentrations approximately 2-8 times those used in the standard assay. Some PAH (perylene, quinoline, benzo[b]chrysene, phenanthrene, anthracene) were optimally activated to mutagens by $9 at 400 ~,l/plate. Rat $9 was similar to hamster $9 for most tested PAH, but anthracene and quinoline mutagenicity was enhanced by hamster $9. All 7 complex mixtures were more mutagenic with 200-400/~l/plate $9; rat was generally slightly more efficient than hamster. Modifying this assay to include a prescreen using a range of $9 concentrations (and perhaps from species other than rat) may improve prediction of the potential carcinogenicity of complex petroleum-derived mixtures.
Increasing evidence suggests that the failure of the Salmonella assay to predict the carcinogenic potential of some complex hydrocarbon mixtures or fractions containing polynuclear aromatic hydrocarbons (PAH) may be due, at least in part, to insufficient amounts of microsomes ($9). In some cases, $9 from hamster may be more effective (Blackburn et al., 1983, 1984; Carver and MacGregor, 1984; Carver et al., 1985). In this study, liver $9 from Aroclor-induced rats or hamsters was used in the Salmonella prescreen (Falcon 1009 quadrant plates, see Carver et al., 1985). A range of $9 concentrations approximately 2-8 times those suggested for the standard assay
(Maron and Ames, 1983) was applied. 29 individual PAH which were representative constituents of petroleum-derived complex mixtures (as determined by high-resolution mass spectroscopy and high-pressure liquid chromatography) were tested in Salmonella typhimurium TA100 with rat or hamster $9 at the equivalent of 50, 100, 200 and 400/d/plate. In addition, 7 complex mixtures known to be active in the mouse dermal carcinogenicity assay were tested: C-5d, D-5d, C-5a, D-5a, WPR1, WPR2, LD. The first four samples - - two petroleum distillates and their aromatic fractions separated by absorption chromatography - - have
0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Mutagenic activity in Salmonella typhimurium TAI00. Data from quadrant plates normalized and expressed as revertants per plate in the presence of 50, 100, 200 or 400/~1 $9 per plate plus I x NADP (2 x for 400/~g/plate $9). Spontaneous mutant frequencies did not differ significantly for rat and hamster $9; combined values are shown. Values for compounds tested without $9 at dose levels of 1000 #g/plate or 500/~g/plate (solubility limit) did not differ significantly from spontaneous mutant frequencies.
249 TABLE 1 COMPARISON OF PUBLISHED C A R C I N O G E N I C I T Y AND GENOTOX1CITY OF TESTED PAH W I T H M O D I F I E D S A L M O N E L L A / M I C R O S O M E PRESCREEN RESULTS NIOSH data a
Prescreen results
Number of rings
Car.
Neo.
Mut.
Chrysene, CHR
5 5 4 5 4
+ + + + +
+ + + + +
+ + + + +
+ + + + +
Anthracene, A Phenanthrene, P Quinoline, Q
3 3 2
+ + +
+ + +
+ + +
Benzo[b]chrysene, BC Benzo[b]anthracene, BbA 9-Methylanthracene, 9MA 2-Methylanthracene, 2MA Perylene, PER Benzo[ght]perylene, BPER Fluoranthene, FA Phenanthridine, P H D Benzo[e]pyrene, BeP Pyrene
5 4 3 3 5 5 3 3 5 4
+ + + + + + + + + +
+ + + + + + + + + -
Dibenzo[c,g]phenanthrene, DBP Dimethylbenzo[c]phenanthrene, DBMP
5 4 4 4 4 4
Benzo[a]pyrene, BaP Dibenz[a, h]anthracene, DBA Benzo[a]anthracene, BaA 3-Methylcholanthrene, MC
Benzo[c]phenanthrene, BP Benzo[ghOfluoranthene, BFA 2,3-Benzofluorene, 2,3BF 1,2-Benzofluorene, 1,2BF 3-Methylfluorene 3,6-Dimethylphenant hrene
+c
b
+ + + + + +
3 3
Hexabenzobenzene Dibenzopentacene
6 7
Dibenzo[g,p]chrysene
6
-
-
?d
a Registry of Toxic Effects. b Negative and non-toxic at 1000/zg/plate; weakly mutagenic at 1-3/zg/plate. ¢ Wood et al. (1980). d Mossandra et al. (1979).
been previously described (Carver and MacGregor, 1984). Three additional complex mixtures were also tested: waxy petroleum residuum, WPR, from two different crudes (WPR1, WPR2, boiling range 615-1000°F) and a lube distillate (LD; boiling range 550-900°F). As summarized by Carver and MacGregor (1984), samples C-5d, D-5d, C-5a and
D-5a produced a tumor incidence of 82%, 87%, 98% and 38°70 (with toxicity), respectively. The tumorigenic potential of the WPR and LD samples had been characterized in a similar in vivo dermal carcinogenicity assay in the mouse (unpublished data, Chevron Environmental Health Center, Inc.). One sample, WPRI, was weakly
250
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(b) 2.3-BENZOFLUORENE.
CHRYSENE. PHENANTHRIDINE
BENZO(e)PYRENE
io0~JjLPiotl TA100, Rev/Plote hamE~9 PHD 600 lO
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800 700
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Fig. 2. Mutagenic activity as in Fig. 1. Complex mixtures shown are two high-boiling (700-1070°F) petroleum distillates (C-5d, D-5d) and their aromatic fractions (C-5a, D-5a) as previously described (Carver and MacGregor, 1984; Carver+ Machado and MacGregor, 1985); waxy petroleum residuum (WPRI, WPR2)
from two different crudes, boiling range 615-1000°F; lube distillate (LD), boilingrange 550-900"F.
tumorigenic (12°70 incidence). The other sample, WPR2, was more potent (5207o incidence). The LD sample was similar in potency, with an incidence of 4807o. These results are similar to those reported for other studies with crude shale oil, crude petroleum and synthetic fossil fuel materials (see refs. in Carver et al., 1985). However, initial mutagenicity experiments with these 7 complex mixtures were negative. The Salmonella prescreen was used to further test these 7 mixtures, to see if results obtained would more accurately reflect the samples PAH content and known oncogenic potential.
Materials and methods
Chemicals.
Most PAH were obtained in the highest purity available from a variety of commercial sources, i.e., Aldrich, Milwaukee, WI; Analabs, North Haven, CT, Biochem. Inst. for Environ. Carcinogenicity, Awrenceberg, Germany; Chem. Serv., West Chester, PA; Columbia Chem., Columbia, SC; ICN, Plalnview, NY; K&K, Red Bank, NJ. All were used as received. A
few materials were analytical standards obtained from the Chevron Research Company, Richmond, CA. Mutagenicity assays. Assay procedures, including solubilization of petroleum-derived mixtures, were previously described (Carver and MacGregor, 1984; Carver et al., 1985). Quantitation and analysis of results. The quadrant-plate prescreen is considered a semiquantitative assay. General trends as shown herein are reproducible from experiment to experiment, but mean values may show considerable variability, ca. 30-40070. Plates were counted on an Artek Automatic Colony Counter (Artek Systems, Corp., Farmingdale, NY); no correction factor was applied.
Results
In Table 1, qualitative results are shown for the 29 PAH tested. Preliminary experiments used rat $9; negative results were reproducible when repeated using hamster $9 (data not shown). Nonmutagenic PAH were tested to the limit of solubility, to cytotoxicity, or to 1000/zg/plate. In Fig. l, responses for 6 different PAH at 3 dose levels are shown. Rat or hamster $9 at the equivalent of 50, 100, 200 and 400/zl/plate (2 x NADP for the latter) was used. In general, the optimal concentration of $9 increased with increasing PAH dose levels. In some cases, higher concentrations of hamster $9 were needed to achieve the mutagenic response noted with rat. Perylene and quinoline were optimally activated at higher $9 concentrations (200-400 /~l/plate) with hamster somewhat more effective than rat. In Fig. 2, results for 10 PAH and 7 complex mixtures tested at a single dose level with rat $9 are compared to data obtained with hamster $9. Because PAH are frequently present in petroleum-derived complex mixtures in low concentrations, the comparison of rat vs. hamster $9 was made at PAH dose levels yielding detectable but not necessarily mutagenic activity. Preliminary experiments (data not shown) using rat $9 had indicated that the PAH dose levels
252
selected would result in weak mutagenic responses in the range of 2-3 times spontaneous background. Hamster $9 was optimal for activation of DBA (5 /zg/plate) over a larger range of $9 concentrations than was rat $9. PHD (100/~g/plate) was activated by rat $9, but would have been missed at any concentration of hamster $9. No significant differences between rat and hamster $9 were noted for CHR (5 #g/plate). For 2,3BF (15/~g/plate), rat $9 was effective over a larger concentration range. Rat $9 was clearly superior to hamster for activation of BeP (50 /zg/plate) to mutagenic metabolites. High concentrations (400/zl/plate) of either rat or hamster $9 activated 1,2BF (50 /zg/plate), but 2MA (50/zg/plate) showed a sharp concentration optimum for both rat and hamster. Both rat and hamster $9 activated BC (50/zg/plate) and P (50 ttg/plate), but hamster $9 was more effective for A (450 #g/plate). In general, promutagens in all 7 petroleum-derived complex mixtures (10 mg/plate) were optimally activated by high concentrations (400/~l/plate) of $9, with no highly significant differences between rat and hamster.
Discussion The Salmonella mutagenicity assay may not always reliably predict the potential carcinogenicity of energy-related materials. For example, results for coal-derived samples showed that the standard Ames assay had a high sensitivity to amino PAH but little sensitivity in general to neutral PAH in such complex mixtures, particularly when chemically-separated fractions were tested. The neutral PAH components contained the highest initiating activity, which was correlated with mutagenicity observed in cultured mammalian cells (Mahlum et al., 1984). Petroleum distillates tested in the Salmonella assay showed positive responses with elevated levels of $9, correlating at least qualitatively with the distillates' known tumorigenic activity (Carver and MacGregor, 1984). For complex mixtures derived from petroleum, at least, the evidence suggests that stan-
dard Salmonella protocols should be revised to include optimization of metabolic activation parameters (Blackburn et al., 1984; Carver et al., 1985). In the study reported here, rat or hamster $9 at concentrations approximately 2-8 times those routinely used enhanced the observed mutagenicity of selected, individual PAH as well as PAH components of complex mixtures. A prescreen using quadrant plates to predict, in a semiquantitative manner, the optimal $9 level for each complex mixture tested seems warranted. Testing a range of $9 concentrations from more than one species may improve the Salmonella assay's correlation with the carcinogenic potential of complex mixtures. However, with the exception of anthracene and quinoline, $9 from hamster had little or no advantage over that from rat for the PAHs and mixtures tested.
Acknowledgements We acknowledge the excellent technical assistance of M.T. Wininger and C.S. Fukuda in conducting the modified Salmonella prescreen assays; we thank A.E. Oranje for manuscript preparation. The authors are indebted to members of the Medical and Biological Science Department of the American Petroleum Institute (API) for contributing the two petroleum distillates and their aromatic fractions.
References Blackburn, G.R., R.A. Deitrich, T.A. Roy, C.A. Schreiner, J.R. Meeks and C.R. Mackerer (1983) Mutagenesis of used gasoline engine oil fractions, in: M. Cooke and A.J. Dennis (Eds.), Polynuclear Aromatic Hydrocarbons: Formation, Metabolism, and Meastirement, Battelle, Columbus, OH, pp. 113-122. Blackburn, G.R., R.A. Deitrich, C.A. Schreiner, M.A. Mehlman and C.R. Mackerer (1984) Estimation of the dermal carcinogenic activity of petroleum fractions using a modified Ames assay, Cell Biol. Toxieol., 1, 40-48. Carver, J.H., and J.A. MacGregor (1984) Mutagenicity and chemical characterization of two petroleum distillates, J. Appl. Toxicol., 4, 163-169.
253 Carver, J.H., M.L. Machado and J.A. MacGregor (1985) Petroleum distillates suppress in vitro metabolic activation: Higher [$9] required in the Salmonella/microsome mutagenicity assay, Environ. Mutagen., 7, 369-379. Mahlum, D.D., C.W. Wright, E.K. Chess and B.W. Wilson (1984) Fractionation of skin tumor-initiating activity in coal liquids, Cancer Res., 44, 5176-5181. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mossandra, K., F. Poncelet, A. Fouassin and M. Mercier
(1979) Detection of mutagenic polycyclic aromatic hydrocarbons in African smoked fish, Fd. Cosmet. Toxicol., 17, 141-143. Wood, A.W., R.L. Chang, W. Levin, D.E. Ryan, P.E. Thomas, M. Croisy-Delcey, Y. Ittah, H. Yagi, D.M. Jerina and A.H. Conney (1980) Mutagenicity of the dihydrodiols and bay-region diol-epoxides of benzo[c]phenanthrene in bacterial and mammalian cells, Cancer Res., 40, 2876-2883. Communicated by R.J. Preston