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Assessment of the utility of the micronucleus test for petroleum-derived materials
Mutation Research 438 Ž1999. 145–153
Assessment of the utility of the micronucleus test for petroleum-derived materials R.T. Przygoda ) , R.H. McKee, M.A. Amoruso, J.J. Freeman Exxon Biomedical Sciences, CN 2350, Mettlers Road, East Millstone, NJ 08875-2350, USA Received 25 June 1998; revised 22 October 1998; accepted 22 October 1998
Abstract The micronucleus test is a commonly used in vivo assay for chromosomal damage and is an integral part of many mutagenicity testing strategies. The present report describes an assessment of the micronucleus test for the detection of mutagenic potential of petroleum-derived materials. To this end, studies were conducted with catalytically cracked clarified oil ŽCCCO.. This material contains high levels of polycyclic aromatic constituents ŽPAC. and is a very potent inducer of mouse skin tumors. CCCO is also active in the Salmonella assay and other in vitro tests. As CCCO is the most potent of the various petroleum-derived materials in other assays, it was assumed to be the most easily detectable in the micronucleus test. CCCO was tested in standard mouse micronucleus tests utilizing oral and intraperitoneal injection for test material administration. All of these studies were negative, although DMBA, tested at roughly equivalent levels based on potency in the Salmonella assay, produced statistically significant increases in micronucleus frequency. In a second series of studies, aromatic fractions of CCCO were prepared and tested at up to acutely toxic levels. Results of these studies were also negative. Finally, another petroleum-derived material which is carcinogenic and contained PAC was tested in the micronucleus assay. It also produced negative results. Thus, it was concluded that petroleum-derived materials do not produce clastogenic effects in vivo in the mouse micronucleus test, despite the fact that some pure polycyclic aromatic hydrocarbons are quite active in this assay. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Micronucleus test; Petroleum-derived material; Catalytically cracked clarified oil
1. Introduction The micronucleus test is a commonly used in vivo assay for chromosomal damage and is an integral Abbreviations: CCCO, catalytically cracked clarified oil; CPP, cyclophosphamide; DMBA, 7,12-dimethylbenzŽ a.anthracene; NCE, normochromatic erythrocyte; PAC, polycyclic aromatic constituents; PCE, polychromatic erythrocyte; MNE, micronucleated polychromatic erythrocyte; IP, intraperitoneal; ULO, unrefined lubricating oil; MI, mutagenic index ) Corresponding author. Tel.: q1-732-873-6120; Fax: q1-732873-6009
part of many mutagenicity testing strategies w1–3x. Among its attractive features are the relative ease of performance and low cost, particularly by comparison to other in vivo mutagenicity assays and the perceived greater relevance of in vivo tests by comparison to in vitro assays. Possible negative aspects are the limited number of tissues examined, and an apparent lack of sensitivity. The extent to which this lack of sensitivity is due to pharmacodynamic processes is unknown w4,5x. In the United States the micronucleus test is part of the initial set of screening tests proposed for
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 6 6 - 1
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regulatory purposes w2x. When used in this context, selectivity may be more important than sensitivity, as lack of sensitivity may be compensated for, to some extent, by other tests in the battery w6,7x. The alternative use is as a second tier test, to determine whether in vitro mutagenic effects can also be produced under in vivo conditions w8x. This usage is more for hazard identification than for screening, and sensitivity is a more important consideration. The micronucleus test is also used for hazard identification purposes in Canada w1x and Europe w3x. We have used the micronucleus test for a number of years to test new and existing products. In our experience, no fuel, lubricant or blending stock has ever produced a positive response in this assay despite the fact that some were strongly mutagenic in other assays. Thus we were led to question whether the micronucleus test is a useful assay, at least for petroleum-derived materials. In the current study, we tested two mutagenic petroleum-derived materials in
standard micronucleus tests and also examined a number of alternative methodologies. The objective was to determine if the micronucleus test was a useful means of assessing the mutagenic potential of petroleum-derived materials.
2. Materials and methods 2.1. Materials The test materials were catalytically cracked clarified oil ŽCCCO, CAS no. 64741-62-4. and an unrefined lubricating oil feedstock derived from a naphthenic crude oil ŽCAS no. 64741-53-3.. CCCO contains high levels of polycyclic aromatic constituents ŽPAC. and is highly mutagenic or genotoxic in the Salmonella test, mouse lymphoma TK " test, Syrian hamster embryo cell transformation test and unscheduled DNA synthesis test w9,10x. CCCO is also a
Fig. 1. CCCO was administered in two consecutive daily doses, either by oral gavage or by IP injection. Corn oil was used as the vehicle. There were ten mice, five males and five females in each group. Results are for the 24-h harvest. No notable increases in micronuclei were observed using either method. Similar results were found for 48-h harvest Ždata not shown.. CCCO, catalytically cracked clarified oil; CPP, cyclophosphamide.
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potent inducer of skin tumors in the mouse epidermal carcinogenesis assay w11x. The unrefined lubricating oil feedstock also contains PACs, is active in a number of in vitro assays w9x and is also a dermal carcinogen in mouse skin w12x although substantially less so than CCCO. Also tested for comparative purposes was 7,12 dimethylbenzŽ a.anthracene ŽDMBA, Aldrich Chemical, Milwaukee, WI, CAS no. 57-97-6., a pure polycyclic aromatic hydrocarbon ŽPAH. which is also highly mutagenic and a potent inducer of skin tumors. Cyclophosphamide ŽAldrich Chemical, CAS no. 6055-19-2., a chemical previously shown to induce chromosomal aberrations and micronuclei w13,14x was used as a positive control in some of the micronucleus assays. Test materials were diluted into a highly refined mineral oil previously shown to be neither mutagenic nor carcinogenic w15x corn oil, dimethyl sulfoxide ŽDMSO. or water.
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2.2. Methods
2.2.1. Salmonella tests DMBA and CCCO were tested by a modification of the Salmonella test w16x using tester strain TA98. This modification was developed for testing certain types of petroleum-derived materials w17,18x. Both were diluted into DMSO. Each dilution of test material was plated in triplicate. The slope of the dose– response curve Ži.e., the mutagenic index, MI. was determined by the method of Myers et al. w19x. For petroleum-derived materials, the data are normally expressed as revertantsrml; however, since the treatment levels of DMBA were measured on a weight basis, its mutagenic potency was expressed as revertantsrmg. As the specific gravity of CCCO is approximately equal to 1.0 grml w20x, a microliter weighs approximately a milligram. Thus, the MI for CCCO was transformed to a revertantsrmg basis by dividing by 1000.
Fig. 2. DMBA was administered in two consecutive daily doses by oral gavage using either mineral oil or corn oil as the vehicle. Cells were harvested 24 h after the last administration of DMBA. There were four mice per group Žtwo males and two females.. A dose related increase was observed using either vehicle. The response when mineral oil was used as the vehicle was less than that observed for corn oil. DMBA, 7,12-dimethylbenzanthracene.
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2.2.2. Micronucleus tests Male and female CD-1 mice were obtained from Charles River Canada ŽQuebec, Canada.. Mice of both sexes were used in all studies. The mice were approximately 6–9 weeks old and weighed between 17–35 g at the time of dosing. They were held in quarantine for at least 17 days, and then randomly distributed by weight into dosing groups. The mice were singly housed in wire mesh cages and given ad libitum access to food ŽPurina Certified Rodent 5002 Chow. and water. Other matters of animal husbandry were consistent with the recommendations of the National Institutes of Health w21x. The micronucleus test was conducted essentially as described by Schmid w22x. The use of acridine orange for micronucleus evaluation was as described by Hayashi et al. w23x. Briefly, both femurs were removed from each treated mouse, and the proximal ends were cut to expose the bone marrow which was then aspirated with fetal bovine serum into a centrifuge tube. The cells were collected by centrifuga-
tion, and slides were prepared. After fixation in methanol, the slides were stained with acridine orange for approximately 1–2 min and evaluated at 400 = by fluorescence microscopy. A total of 1000 erythrocytes were counted from each animal; and the total number of polychromatic ŽPCE. and normochromatic ŽNCE. erythrocytes were tabulated. One thousand PCEs were evaluated for the presence of micronuclei. A range-finding study was performed to select doses for the initial oral gavage micronucleus study. Five separate micronucleus studies were conducted. All experiments were conducted with male and female mice. Initial studies used 10 animals per group ŽFig. 1.. Subsequent studies used four animals per group ŽFigs. 2–4 and Table 1.. Test material was administered in two consecutive daily doses, either by gavage or by intraperitoneal ŽIP. injection. In most cases the mice were sacrificed approximately 24 h after the second administration of test material, however, one study also utilized a 48 h collection
Fig. 3. CCCO was extracted using DMSO. The DMSO extracted material was administered by oral gavage in two consecutive daily doses. Cells were harvested 24 h after the last administration of CCCO. There were four mice per group, two males and two females. There were no notable increases in micronuclei. CCCO, catalytically cracked clarified oil; DMBA, 7,12-dimethylbenzanthracene.
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Fig. 4. Unrefined lubricating oil, either neat or DMSO extracted, was administered by oral gavage in two consecutive daily doses. There were four mice per group, two males and two females. Cells were harvested 24 h after the last administration of ULO. There were no notable increases in micronuclei using either the neat or extracted material. ULO, unrefined lubricating oil; DMBA, 7,12-dimethylbenzanthracene.
time to assure that delayed effects were not missed. Corn oil was normally used as the vehicle for CCCO, unrefined lubricating oil, and DMBA. In some stud-
Table 1 CCCO IP injection study Dose
N
% PCE ŽStd..r R
MNEr1000 PCE ŽStd..
3.0 grkg 1.5 grkg 0.75 grkg Corn oil
4 4 4 4
32.4 Ž10.1. 36.8 Ž7.7. 41.3 Ž10.1. 48.8 Ž2.7.
1.0 Ž0.8. 2.5 Ž1.7. 1.75 Ž1.3. 1.25 Ž1.0.
CCCO was administered in two consecutive daily doses by IP injection. There were two males and two females in each group. Bone marrow was removed 24 h after the last dose. The percentage of PCEs in all three treatment groups were below the normal historical range of the corn oil control Ž45 to 59%.. CCCO, catalytically cracked clarified oil; % PCE, the mean percentage of polychromatic erythrocytes in the total population of erythrocytes; MNEr1000 PCE, the mean number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes; Std., standard deviation; R, statistically significant regression coefficient Ž p- 0.01..
ies extracted materials were administered in DMSO and in one study DMBA was administered in mineral oil. Vehicle-treated controls were included in all studies. Cyclophosphamide Ž0.04 grkg. dissolved in water and administered by gavage was used as a positive control in some studies. Statistical analysis included calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period w24x. When the ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test w24x. A standard regression analysis was performed to test for a dose response w24x. Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion w25x. The residuals were normally distributed Žvalues were greater than 0.01 level of significance. in more than 75% of the analyses. Therefore non-parametric analysis was not performed. Sexes were analyzed separately in the first study ŽFig. 1.. In subsequent studies the sexes were not separated due to the smaller group sizes.
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3. Results
Table 3 CCCO oral gavage range-finding study
The Salmonella tests confirmed that CCCO was a relatively potent bacterial mutagen ŽTable 2.. The mutagenic index was approximately 600 revertantsr ml or 0.6 revertantsrmg. DMBA had a mutagenic index of approximately 2.3 revertantsrmg. Thus, on a per weight basis, the PAC constituents of CCCO were approximately 25% as active as DMBA in the Salmonella assay. In an initial range-finding study, CCCO was orally administered in doses of 1, 2, 3, or 4 grkg on two consecutive days to CD-1 mice in groups of four Žtwo males and two females, Table 3.. All mice in the highest dose group died as did one mouse in the 3 grkg group, and 2 mice in the 2 grkg group. All remaining mice in 2 grkg and 3 grkg groups were observed to be hypoactive or prostrate. All mice given 1 grkg survived. Based on these findings, 1.5 grkg was selected as the high dose for oral gavage studies. There were no notable increases in the mean number of micronucleated polychromatic erythrocytes in any of the dose groups in the range-finding study. All values were within the normal range of the corn oil control Ž0 to 4. for this laboratory. Orally administered CCCO was neither clastogenic nor toxic at levels up to 1.5 grkg ŽFig. 1.. The mean percentage of PCEs was between 49% and
Dose
N
% PCE ŽStd..
MNEr1000 PCE ŽStd..
3.0 grkg 2.0 grkg 1.0 grkg Corn Oil
3) 2)) 4 4
46.6 Ž1.2. 46.8 Ž4.7. 52.9 Ž2.9. 54.1 Ž5.5.
3.3 Ž1.5. 3.0 Ž1.4. 2.3 Ž1.9. 1.5 Ž1.0.
CCCO was administered in two consecutive daily doses by oral gavage. There were two males and two females in each group.Bone marrow was removed 24 h after the last dose. Similar results were observed in a repeat study. In this repeat study, the number of MNEr1000 PCEs was less 3.0 in all dose groups. CCCO, catalytically cracked clarified oil; % PCE, the mean percentage of polychromatic erythrocytes in the total population of erythrocytes; MNEr1000 PCE, the mean number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes; Std., standard deviation. )One animal died prior to sacrifice. ))Two animals died prior to sacrifice.
54%. These values were not considered different than the corn oil controls, 50.2% and 54.9%. In contrast, orally administered DMBA at a level of 0.15 grkg produced a greater than 10 fold increase in micronucleus frequency ŽFig. 2.. Thus, the study confirmed that DMBA was clastogenic, but CCCO was inactive under the same conditions even though the CCCO treatment level was 10 times higher than the DMBA dose.
Table 2 Results of Salmonella tests using TA98 for CCCO and DMBA CCCO ŽDMSO extract.
DMBA
Test material concentration Žml.
Mean revertantsrplate
1.5 1.0 0.6 0.5 0.4 0.3 0.2 0.15 0.10 0.05 DMSO
847 624 364 360 298 209 147 108 84 61 47
Test material concentration Žmg.
Mean revertantsrplate
24 20 16 12 8 6 4 2
110 87 81 80 83 60 55 54
DMSO
47
Mutagenic index: CCCO, 600 revertantsrml s 0.6 revertantsrmg; DMBA, 2.3 revertantsrmg control; mean of revertant counts from three plates; CCCO, catalytically cracked clarified oil; DMBA, 7,12-dimethylbenzanthracene. Historical range for the DMSO is between 30 and 60 revertantsrplate. DMBA and CCCO were tested using a modification of the Salmonella test w16x. The test materials were extracted and diluted using DMSO. Each dilution of test material was plated in triplicate. The slope of the dose response curve ŽMI, mutagenic index. was determined by the method of Myers et al. w19x.
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To test whether the lack of effect was related to the route of administration, CCCO was administered by intraperitoneal injection. No range-finding study was performed. Rather, literature data w13x which indicated that DMBA was substantially more potent following intraperitoneal injection than oral administration, was used as a basis for dose selection. Based on the literature information and the relative potency of DMBA and CCCO in the Salmonella test, we reasoned that CCCO doses between 0.15 and 0.75 mgrkg would produce substantial increases in micronucleus frequency. However, CCCO was neither clastogenic nor toxic when administered by IP injection at levels up to 0.75 grkg ŽFig. 1.. There were also no differences in response between the 24- and 48-h collections Ždata not shown.. Because 0.75 grkg was not overtly toxic, a further study was carried out to examine higher CCCO treatment levels but with fewer numbers of animals in each group ŽTable 1.. There was evidence of dose related decreases in the frequency of polychromatic erythrocytes but there was no evidence of increased frequencies of micronuclei at levels up to 3 grkg. This indicated that the bone marrow was affected by the test substance even though there was no clastogenic response. These studies demonstrated that the absence of effect was not related to either method of test material administration or collection time. DMBA was evaluated over a range of concentrations to collect dose–response information, and in two vehicles to determine whether vehicle influenced clastogenic response. Dose-related increases in clastogenic frequency for DMBA were observed at levels ranging from 0.075 to 0.3 grkg ŽFig. 2.. Dilution in mineral oil reduced the level of clastogenic activity by about 50% in comparison to dilution in corn oil. Thus, these data suggested the possibility of vehicle effects, although not to the degree necessary to explain the complete absence of response in the CCCO studies. Further tests were then conducted to determine whether the lack of effect might be due to the vehicle or to matrix effects related to other hydrocarbons in the CCCO. To test this, the PAC fraction of the CCCO was extracted with DMSO. The extract itself was tested at levels up to 5 grkg ŽFig. 3.. In this experiment, acutely lethal levels were achieved
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as one of four mice died after receiving 2.5 grkg and three of four died after receiving 5 grkg. Nevertheless, the PAC fraction of CCCO was inactive in this test, even at acutely lethal levels. An unrefined lubricating oil feedstock derived from a naphthenic crude oil was also tested. Among other things, unrefined lubricating oil is less viscous than CCCO. Thus, this study tested, to some extent, the effect of physical properties on clastogenic outcome. However, the unrefined base oil was inactive when tested either directly or following extraction ŽFig. 4.. Thus, these data suggested that the lack of sensitivity of the micronucleus test was not restricted to CCCO but could be generalized to other types of petroleum-derived materials.
4. Discussion The objective of this study was to evaluate the utility of the mouse micronucleus test to assess the clastogenic potential of petroleum-derived materials. To address that question, CCCO, a PAC containing material, was selected as the initial test material. As several pure PAC species including DMBA and benzoŽ a.pyrene are active in the micronucleus test w4x, it was expected that CCCO would also be active. Additionally, as CCCO is the most potent of the various petroleum-derived materials in other assays, it was assumed to be the most easily detectable. For this reason, it was planned to start with this material and to then test less active materials to assess assay sensitivity. For comparative purposes, DMBA, a pure PAC species, and a potent dermal carcinogen w26x was also tested. The mutagenic activity of these two materials was first compared in the Salmonella assay. Both produced positive responses; CCCO being approximately 25% as active as DMBA. Thus, as an initial prediction, it was assumed that CCCO would also be approximately 25% as active in the micronucleus test. As the high dose in the CCCO group received 10 times as much material as the DMBA comparison group, it was expected that positive results would be easily obtained. However, DMBA produced a greater than 10 fold increase in micronucleus frequency whereas CCCO was inactive, even at doses that were clearly toxic ŽFigs. 1 and 2, Tables 1 and 3..
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In subsequent tests, when either CCCO or unrefined base oil were tested in the micronucleus test, the results were consistently negative despite the fact that the test materials were dermal carcinogens in mice and had demonstrated high levels of mutagenic or genotoxic activity in the Salmonella test, the mouse lymphoma TK " test, Syrian hamster embryo cell transformation test and the unscheduled DNA synthesis test w9,10x. The various studies conducted and modifications evaluated suggested that the negative results were not due to reduced gastro-intestinal absorption, route of administration, collection time, or viscosity. The absence of effect may be related, at least in part, to the non-PAC constituents. However, studies in which the PAC constituents were extracted and then administered IP should have circumvented the majority of the matrix effects. The fact that these tests also produced negative results suggests that matrix effects alone are not sufficient to account for the complete absence of effects in all studies, although the comparison study of DMBA in corn oil and mineral oil suggested that matrix effects may play some role. A further possibility, not tested in the present study, is that there may be interactive effects between the PAC as these have been shown to reduce activity in the Salmonella assay w27x. It may be possible to generalize the results of this study to a broader range of petroleum-derived materials. The American Petroleum Institute conducted rat bone marrow chromosomal aberration tests on several petroleum derived materials as well as synthetic fuels: Retorted Shale Oils w28–30x, Light Catalytically Cracked Distillate w31,32x and CCCO w33x. None of these mutagenic and carcinogenic materials tested were positive. Thus, bone marrow assays in general may not be appropriate for petroleum-derived materials. In our experience, no fuel, lubricant or blending stock has ever produced a positive response in the micronucleus test even when some were strongly mutagenic in other tests. The current studies demonstrated that CCCO, a potent Salmonella mutagen was inactive in the micronucleus test even at levels which were acutely lethal andror provided clear evidence of bone marrow effects. In light of these findings, it is concluded that the in vivo micronucleus test is an insensitive method to evaluate the
mutagenic potential of petroleum-derived materials, despite the fact that some pure polycyclic aromatic hydrocarbons are quite active in these assays.
Acknowledgements The skilled technical assistance of Nancy Wojcik and Felicia Lynch is gratefully acknowledged. The authors also thank Dr. T.J. McDonagh ŽExxon. for his support of this work.
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Assessment of the utility of the micronucleus test for petroleum-derived materials R.T. Przygoda ) , R.H. McKee, M.A. Amoruso, J.J. Freeman Exxon Biomedical Sciences, CN 2350, Mettlers Road, East Millstone, NJ 08875-2350, USA Received 25 June 1998; revised 22 October 1998; accepted 22 October 1998
Abstract The micronucleus test is a commonly used in vivo assay for chromosomal damage and is an integral part of many mutagenicity testing strategies. The present report describes an assessment of the micronucleus test for the detection of mutagenic potential of petroleum-derived materials. To this end, studies were conducted with catalytically cracked clarified oil ŽCCCO.. This material contains high levels of polycyclic aromatic constituents ŽPAC. and is a very potent inducer of mouse skin tumors. CCCO is also active in the Salmonella assay and other in vitro tests. As CCCO is the most potent of the various petroleum-derived materials in other assays, it was assumed to be the most easily detectable in the micronucleus test. CCCO was tested in standard mouse micronucleus tests utilizing oral and intraperitoneal injection for test material administration. All of these studies were negative, although DMBA, tested at roughly equivalent levels based on potency in the Salmonella assay, produced statistically significant increases in micronucleus frequency. In a second series of studies, aromatic fractions of CCCO were prepared and tested at up to acutely toxic levels. Results of these studies were also negative. Finally, another petroleum-derived material which is carcinogenic and contained PAC was tested in the micronucleus assay. It also produced negative results. Thus, it was concluded that petroleum-derived materials do not produce clastogenic effects in vivo in the mouse micronucleus test, despite the fact that some pure polycyclic aromatic hydrocarbons are quite active in this assay. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Micronucleus test; Petroleum-derived material; Catalytically cracked clarified oil
1. Introduction The micronucleus test is a commonly used in vivo assay for chromosomal damage and is an integral Abbreviations: CCCO, catalytically cracked clarified oil; CPP, cyclophosphamide; DMBA, 7,12-dimethylbenzŽ a.anthracene; NCE, normochromatic erythrocyte; PAC, polycyclic aromatic constituents; PCE, polychromatic erythrocyte; MNE, micronucleated polychromatic erythrocyte; IP, intraperitoneal; ULO, unrefined lubricating oil; MI, mutagenic index ) Corresponding author. Tel.: q1-732-873-6120; Fax: q1-732873-6009
part of many mutagenicity testing strategies w1–3x. Among its attractive features are the relative ease of performance and low cost, particularly by comparison to other in vivo mutagenicity assays and the perceived greater relevance of in vivo tests by comparison to in vitro assays. Possible negative aspects are the limited number of tissues examined, and an apparent lack of sensitivity. The extent to which this lack of sensitivity is due to pharmacodynamic processes is unknown w4,5x. In the United States the micronucleus test is part of the initial set of screening tests proposed for
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 1 6 6 - 1
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regulatory purposes w2x. When used in this context, selectivity may be more important than sensitivity, as lack of sensitivity may be compensated for, to some extent, by other tests in the battery w6,7x. The alternative use is as a second tier test, to determine whether in vitro mutagenic effects can also be produced under in vivo conditions w8x. This usage is more for hazard identification than for screening, and sensitivity is a more important consideration. The micronucleus test is also used for hazard identification purposes in Canada w1x and Europe w3x. We have used the micronucleus test for a number of years to test new and existing products. In our experience, no fuel, lubricant or blending stock has ever produced a positive response in this assay despite the fact that some were strongly mutagenic in other assays. Thus we were led to question whether the micronucleus test is a useful assay, at least for petroleum-derived materials. In the current study, we tested two mutagenic petroleum-derived materials in
standard micronucleus tests and also examined a number of alternative methodologies. The objective was to determine if the micronucleus test was a useful means of assessing the mutagenic potential of petroleum-derived materials.
2. Materials and methods 2.1. Materials The test materials were catalytically cracked clarified oil ŽCCCO, CAS no. 64741-62-4. and an unrefined lubricating oil feedstock derived from a naphthenic crude oil ŽCAS no. 64741-53-3.. CCCO contains high levels of polycyclic aromatic constituents ŽPAC. and is highly mutagenic or genotoxic in the Salmonella test, mouse lymphoma TK " test, Syrian hamster embryo cell transformation test and unscheduled DNA synthesis test w9,10x. CCCO is also a
Fig. 1. CCCO was administered in two consecutive daily doses, either by oral gavage or by IP injection. Corn oil was used as the vehicle. There were ten mice, five males and five females in each group. Results are for the 24-h harvest. No notable increases in micronuclei were observed using either method. Similar results were found for 48-h harvest Ždata not shown.. CCCO, catalytically cracked clarified oil; CPP, cyclophosphamide.
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potent inducer of skin tumors in the mouse epidermal carcinogenesis assay w11x. The unrefined lubricating oil feedstock also contains PACs, is active in a number of in vitro assays w9x and is also a dermal carcinogen in mouse skin w12x although substantially less so than CCCO. Also tested for comparative purposes was 7,12 dimethylbenzŽ a.anthracene ŽDMBA, Aldrich Chemical, Milwaukee, WI, CAS no. 57-97-6., a pure polycyclic aromatic hydrocarbon ŽPAH. which is also highly mutagenic and a potent inducer of skin tumors. Cyclophosphamide ŽAldrich Chemical, CAS no. 6055-19-2., a chemical previously shown to induce chromosomal aberrations and micronuclei w13,14x was used as a positive control in some of the micronucleus assays. Test materials were diluted into a highly refined mineral oil previously shown to be neither mutagenic nor carcinogenic w15x corn oil, dimethyl sulfoxide ŽDMSO. or water.
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2.2. Methods
2.2.1. Salmonella tests DMBA and CCCO were tested by a modification of the Salmonella test w16x using tester strain TA98. This modification was developed for testing certain types of petroleum-derived materials w17,18x. Both were diluted into DMSO. Each dilution of test material was plated in triplicate. The slope of the dose– response curve Ži.e., the mutagenic index, MI. was determined by the method of Myers et al. w19x. For petroleum-derived materials, the data are normally expressed as revertantsrml; however, since the treatment levels of DMBA were measured on a weight basis, its mutagenic potency was expressed as revertantsrmg. As the specific gravity of CCCO is approximately equal to 1.0 grml w20x, a microliter weighs approximately a milligram. Thus, the MI for CCCO was transformed to a revertantsrmg basis by dividing by 1000.
Fig. 2. DMBA was administered in two consecutive daily doses by oral gavage using either mineral oil or corn oil as the vehicle. Cells were harvested 24 h after the last administration of DMBA. There were four mice per group Žtwo males and two females.. A dose related increase was observed using either vehicle. The response when mineral oil was used as the vehicle was less than that observed for corn oil. DMBA, 7,12-dimethylbenzanthracene.
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2.2.2. Micronucleus tests Male and female CD-1 mice were obtained from Charles River Canada ŽQuebec, Canada.. Mice of both sexes were used in all studies. The mice were approximately 6–9 weeks old and weighed between 17–35 g at the time of dosing. They were held in quarantine for at least 17 days, and then randomly distributed by weight into dosing groups. The mice were singly housed in wire mesh cages and given ad libitum access to food ŽPurina Certified Rodent 5002 Chow. and water. Other matters of animal husbandry were consistent with the recommendations of the National Institutes of Health w21x. The micronucleus test was conducted essentially as described by Schmid w22x. The use of acridine orange for micronucleus evaluation was as described by Hayashi et al. w23x. Briefly, both femurs were removed from each treated mouse, and the proximal ends were cut to expose the bone marrow which was then aspirated with fetal bovine serum into a centrifuge tube. The cells were collected by centrifuga-
tion, and slides were prepared. After fixation in methanol, the slides were stained with acridine orange for approximately 1–2 min and evaluated at 400 = by fluorescence microscopy. A total of 1000 erythrocytes were counted from each animal; and the total number of polychromatic ŽPCE. and normochromatic ŽNCE. erythrocytes were tabulated. One thousand PCEs were evaluated for the presence of micronuclei. A range-finding study was performed to select doses for the initial oral gavage micronucleus study. Five separate micronucleus studies were conducted. All experiments were conducted with male and female mice. Initial studies used 10 animals per group ŽFig. 1.. Subsequent studies used four animals per group ŽFigs. 2–4 and Table 1.. Test material was administered in two consecutive daily doses, either by gavage or by intraperitoneal ŽIP. injection. In most cases the mice were sacrificed approximately 24 h after the second administration of test material, however, one study also utilized a 48 h collection
Fig. 3. CCCO was extracted using DMSO. The DMSO extracted material was administered by oral gavage in two consecutive daily doses. Cells were harvested 24 h after the last administration of CCCO. There were four mice per group, two males and two females. There were no notable increases in micronuclei. CCCO, catalytically cracked clarified oil; DMBA, 7,12-dimethylbenzanthracene.
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Fig. 4. Unrefined lubricating oil, either neat or DMSO extracted, was administered by oral gavage in two consecutive daily doses. There were four mice per group, two males and two females. Cells were harvested 24 h after the last administration of ULO. There were no notable increases in micronuclei using either the neat or extracted material. ULO, unrefined lubricating oil; DMBA, 7,12-dimethylbenzanthracene.
time to assure that delayed effects were not missed. Corn oil was normally used as the vehicle for CCCO, unrefined lubricating oil, and DMBA. In some stud-
Table 1 CCCO IP injection study Dose
N
% PCE ŽStd..r R
MNEr1000 PCE ŽStd..
3.0 grkg 1.5 grkg 0.75 grkg Corn oil
4 4 4 4
32.4 Ž10.1. 36.8 Ž7.7. 41.3 Ž10.1. 48.8 Ž2.7.
1.0 Ž0.8. 2.5 Ž1.7. 1.75 Ž1.3. 1.25 Ž1.0.
CCCO was administered in two consecutive daily doses by IP injection. There were two males and two females in each group. Bone marrow was removed 24 h after the last dose. The percentage of PCEs in all three treatment groups were below the normal historical range of the corn oil control Ž45 to 59%.. CCCO, catalytically cracked clarified oil; % PCE, the mean percentage of polychromatic erythrocytes in the total population of erythrocytes; MNEr1000 PCE, the mean number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes; Std., standard deviation; R, statistically significant regression coefficient Ž p- 0.01..
ies extracted materials were administered in DMSO and in one study DMBA was administered in mineral oil. Vehicle-treated controls were included in all studies. Cyclophosphamide Ž0.04 grkg. dissolved in water and administered by gavage was used as a positive control in some studies. Statistical analysis included calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period w24x. When the ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test w24x. A standard regression analysis was performed to test for a dose response w24x. Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion w25x. The residuals were normally distributed Žvalues were greater than 0.01 level of significance. in more than 75% of the analyses. Therefore non-parametric analysis was not performed. Sexes were analyzed separately in the first study ŽFig. 1.. In subsequent studies the sexes were not separated due to the smaller group sizes.
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3. Results
Table 3 CCCO oral gavage range-finding study
The Salmonella tests confirmed that CCCO was a relatively potent bacterial mutagen ŽTable 2.. The mutagenic index was approximately 600 revertantsr ml or 0.6 revertantsrmg. DMBA had a mutagenic index of approximately 2.3 revertantsrmg. Thus, on a per weight basis, the PAC constituents of CCCO were approximately 25% as active as DMBA in the Salmonella assay. In an initial range-finding study, CCCO was orally administered in doses of 1, 2, 3, or 4 grkg on two consecutive days to CD-1 mice in groups of four Žtwo males and two females, Table 3.. All mice in the highest dose group died as did one mouse in the 3 grkg group, and 2 mice in the 2 grkg group. All remaining mice in 2 grkg and 3 grkg groups were observed to be hypoactive or prostrate. All mice given 1 grkg survived. Based on these findings, 1.5 grkg was selected as the high dose for oral gavage studies. There were no notable increases in the mean number of micronucleated polychromatic erythrocytes in any of the dose groups in the range-finding study. All values were within the normal range of the corn oil control Ž0 to 4. for this laboratory. Orally administered CCCO was neither clastogenic nor toxic at levels up to 1.5 grkg ŽFig. 1.. The mean percentage of PCEs was between 49% and
Dose
N
% PCE ŽStd..
MNEr1000 PCE ŽStd..
3.0 grkg 2.0 grkg 1.0 grkg Corn Oil
3) 2)) 4 4
46.6 Ž1.2. 46.8 Ž4.7. 52.9 Ž2.9. 54.1 Ž5.5.
3.3 Ž1.5. 3.0 Ž1.4. 2.3 Ž1.9. 1.5 Ž1.0.
CCCO was administered in two consecutive daily doses by oral gavage. There were two males and two females in each group.Bone marrow was removed 24 h after the last dose. Similar results were observed in a repeat study. In this repeat study, the number of MNEr1000 PCEs was less 3.0 in all dose groups. CCCO, catalytically cracked clarified oil; % PCE, the mean percentage of polychromatic erythrocytes in the total population of erythrocytes; MNEr1000 PCE, the mean number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes; Std., standard deviation. )One animal died prior to sacrifice. ))Two animals died prior to sacrifice.
54%. These values were not considered different than the corn oil controls, 50.2% and 54.9%. In contrast, orally administered DMBA at a level of 0.15 grkg produced a greater than 10 fold increase in micronucleus frequency ŽFig. 2.. Thus, the study confirmed that DMBA was clastogenic, but CCCO was inactive under the same conditions even though the CCCO treatment level was 10 times higher than the DMBA dose.
Table 2 Results of Salmonella tests using TA98 for CCCO and DMBA CCCO ŽDMSO extract.
DMBA
Test material concentration Žml.
Mean revertantsrplate
1.5 1.0 0.6 0.5 0.4 0.3 0.2 0.15 0.10 0.05 DMSO
847 624 364 360 298 209 147 108 84 61 47
Test material concentration Žmg.
Mean revertantsrplate
24 20 16 12 8 6 4 2
110 87 81 80 83 60 55 54
DMSO
47
Mutagenic index: CCCO, 600 revertantsrml s 0.6 revertantsrmg; DMBA, 2.3 revertantsrmg control; mean of revertant counts from three plates; CCCO, catalytically cracked clarified oil; DMBA, 7,12-dimethylbenzanthracene. Historical range for the DMSO is between 30 and 60 revertantsrplate. DMBA and CCCO were tested using a modification of the Salmonella test w16x. The test materials were extracted and diluted using DMSO. Each dilution of test material was plated in triplicate. The slope of the dose response curve ŽMI, mutagenic index. was determined by the method of Myers et al. w19x.
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To test whether the lack of effect was related to the route of administration, CCCO was administered by intraperitoneal injection. No range-finding study was performed. Rather, literature data w13x which indicated that DMBA was substantially more potent following intraperitoneal injection than oral administration, was used as a basis for dose selection. Based on the literature information and the relative potency of DMBA and CCCO in the Salmonella test, we reasoned that CCCO doses between 0.15 and 0.75 mgrkg would produce substantial increases in micronucleus frequency. However, CCCO was neither clastogenic nor toxic when administered by IP injection at levels up to 0.75 grkg ŽFig. 1.. There were also no differences in response between the 24- and 48-h collections Ždata not shown.. Because 0.75 grkg was not overtly toxic, a further study was carried out to examine higher CCCO treatment levels but with fewer numbers of animals in each group ŽTable 1.. There was evidence of dose related decreases in the frequency of polychromatic erythrocytes but there was no evidence of increased frequencies of micronuclei at levels up to 3 grkg. This indicated that the bone marrow was affected by the test substance even though there was no clastogenic response. These studies demonstrated that the absence of effect was not related to either method of test material administration or collection time. DMBA was evaluated over a range of concentrations to collect dose–response information, and in two vehicles to determine whether vehicle influenced clastogenic response. Dose-related increases in clastogenic frequency for DMBA were observed at levels ranging from 0.075 to 0.3 grkg ŽFig. 2.. Dilution in mineral oil reduced the level of clastogenic activity by about 50% in comparison to dilution in corn oil. Thus, these data suggested the possibility of vehicle effects, although not to the degree necessary to explain the complete absence of response in the CCCO studies. Further tests were then conducted to determine whether the lack of effect might be due to the vehicle or to matrix effects related to other hydrocarbons in the CCCO. To test this, the PAC fraction of the CCCO was extracted with DMSO. The extract itself was tested at levels up to 5 grkg ŽFig. 3.. In this experiment, acutely lethal levels were achieved
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as one of four mice died after receiving 2.5 grkg and three of four died after receiving 5 grkg. Nevertheless, the PAC fraction of CCCO was inactive in this test, even at acutely lethal levels. An unrefined lubricating oil feedstock derived from a naphthenic crude oil was also tested. Among other things, unrefined lubricating oil is less viscous than CCCO. Thus, this study tested, to some extent, the effect of physical properties on clastogenic outcome. However, the unrefined base oil was inactive when tested either directly or following extraction ŽFig. 4.. Thus, these data suggested that the lack of sensitivity of the micronucleus test was not restricted to CCCO but could be generalized to other types of petroleum-derived materials.
4. Discussion The objective of this study was to evaluate the utility of the mouse micronucleus test to assess the clastogenic potential of petroleum-derived materials. To address that question, CCCO, a PAC containing material, was selected as the initial test material. As several pure PAC species including DMBA and benzoŽ a.pyrene are active in the micronucleus test w4x, it was expected that CCCO would also be active. Additionally, as CCCO is the most potent of the various petroleum-derived materials in other assays, it was assumed to be the most easily detectable. For this reason, it was planned to start with this material and to then test less active materials to assess assay sensitivity. For comparative purposes, DMBA, a pure PAC species, and a potent dermal carcinogen w26x was also tested. The mutagenic activity of these two materials was first compared in the Salmonella assay. Both produced positive responses; CCCO being approximately 25% as active as DMBA. Thus, as an initial prediction, it was assumed that CCCO would also be approximately 25% as active in the micronucleus test. As the high dose in the CCCO group received 10 times as much material as the DMBA comparison group, it was expected that positive results would be easily obtained. However, DMBA produced a greater than 10 fold increase in micronucleus frequency whereas CCCO was inactive, even at doses that were clearly toxic ŽFigs. 1 and 2, Tables 1 and 3..
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In subsequent tests, when either CCCO or unrefined base oil were tested in the micronucleus test, the results were consistently negative despite the fact that the test materials were dermal carcinogens in mice and had demonstrated high levels of mutagenic or genotoxic activity in the Salmonella test, the mouse lymphoma TK " test, Syrian hamster embryo cell transformation test and the unscheduled DNA synthesis test w9,10x. The various studies conducted and modifications evaluated suggested that the negative results were not due to reduced gastro-intestinal absorption, route of administration, collection time, or viscosity. The absence of effect may be related, at least in part, to the non-PAC constituents. However, studies in which the PAC constituents were extracted and then administered IP should have circumvented the majority of the matrix effects. The fact that these tests also produced negative results suggests that matrix effects alone are not sufficient to account for the complete absence of effects in all studies, although the comparison study of DMBA in corn oil and mineral oil suggested that matrix effects may play some role. A further possibility, not tested in the present study, is that there may be interactive effects between the PAC as these have been shown to reduce activity in the Salmonella assay w27x. It may be possible to generalize the results of this study to a broader range of petroleum-derived materials. The American Petroleum Institute conducted rat bone marrow chromosomal aberration tests on several petroleum derived materials as well as synthetic fuels: Retorted Shale Oils w28–30x, Light Catalytically Cracked Distillate w31,32x and CCCO w33x. None of these mutagenic and carcinogenic materials tested were positive. Thus, bone marrow assays in general may not be appropriate for petroleum-derived materials. In our experience, no fuel, lubricant or blending stock has ever produced a positive response in the micronucleus test even when some were strongly mutagenic in other tests. The current studies demonstrated that CCCO, a potent Salmonella mutagen was inactive in the micronucleus test even at levels which were acutely lethal andror provided clear evidence of bone marrow effects. In light of these findings, it is concluded that the in vivo micronucleus test is an insensitive method to evaluate the
mutagenic potential of petroleum-derived materials, despite the fact that some pure polycyclic aromatic hydrocarbons are quite active in these assays.
Acknowledgements The skilled technical assistance of Nancy Wojcik and Felicia Lynch is gratefully acknowledged. The authors also thank Dr. T.J. McDonagh ŽExxon. for his support of this work.
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