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

Mutation Research, 253 (1991) 149-159 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-1161/91/$03.50 ADONIS 0165116191001012

149

MUTENV 08793

Assessing the use of known m u t a g e n s to calibrate the Salmonella typhimurium m u t a g e n i c i t y assay: II. W i t h exogenous activation Larry D. Claxton a, Virginia S. Houk a, Janet R. Warner b, Larry E. Myers b and Thomas J. Hughes b,, a MD-68A, Genetic Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 and b Research Triangle Institute, P.O. Box 12194, Research Triangle Park, NC 27709-2194 (U.S.A.) (Received 2 November 1990) (Accepted 1 March 1991)

Keywords: Salmonella mutagenicity assay, calibration; Calibration, mutagenicity assay; Exogenous activation; Reference compounds; Comparative bioassays; Reference standard; Audit materials; Reproducibility

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

* Present address: Environmental Health Research and Testing, P.O. Box 12199, Research Triangle Park, NC 27709 (U.S.A.).

Correspondence: Dr. Larry D. Claxton, MD-68A, Genetic Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (U.S.A.).

150 These data, along with the data generated previously (Claxton et al., 1991b) for 10 direct-acting chemicals, provide a basis for intralaboratory and interlaboratory comparison and ranking of Salmonella typhimurium TA100 mutagenicity data. Concurrent testing (same day, same laboratory, same technician, same bacteria, $9 and media) is a more acceptable method to control variance. However, this is not always possible. The ability to quantify Salmonella mutagenicity data by the use of standard reference materials will assist in potency comparisons of data generated in different laboratories or within the same laboratory on different days.

The Salmonella mutagenicity assay has been shown to detect known animal carcinogens as mutagens in a cost- and time-effective manner (McCann et al., 1975; Lave and Omenn, 1986). The assay has identified over 1500 pure chemicals as mutagens. The bioassay is sensitive and specific for detection of certain chemical classes of known mutagens and carcinogens (Claxton et al., 1988). Among these mutagens are components of numerous consumer products including: drinking water, processed food and drink, clothing, cosmetics, hair dyes, cigarette emissions, household items, fuels and photocopying ink. The Salmonella assay, therefore, provides a practical mutagenicity screen of environmental chemicals because it is relatively inexpensive and rapid compared to animal tests (Lave and Omenn, 1986). The Salmonella assay detects the frequency of mutational events, with the number of revertant colonies proportional to the mutagenic activity of the test compound (at nontoxic doses). Mutagenic activity between chemical compounds can vary a million-fold. Aflatoxin B 1 and 1,8-dinitropyrene represent the types of extremely potent mutagens; and saccharin is the weakest (Ames, 1979). As first shown by Mailing (1971), if the test chemical is a promutagen, it requires metabolic activation to become active. The most commonly used type of metabolizing system is a microsomecontaining subcellular fraction ($9), prepared from rat or hamster liver. In order to increase the level of activation enzymes in the liver (primarily P-448 and P-450 mixed-function oxidases), the animals often are injected with a mixture of polychlorinated biphenyls (Aroclor 1254) a week before sacrifice (Maron and Ames, 1983). The purpose of this project was to evaluate the variability in the mutagenic response in the Salmonella mutagenicity assay of potential refer-

ence standard chemicals that require $9 (indirect-acting mutagens) and to develop ranking criteria (boundaries) for mutagenic activity based on the data. By being able to rank newly tested compounds through comparison to compounds of known potency, it would be possible to calibrate the Salmonella assay. The concept of ranking criteria is closely related to concepts of relative mutagenicity advanced by de Serres (1983) and comparative potency described by Lewtas (1986). Through the use of an interlaboratory study, this is a testable hypothesis. The long-term goals of this research are to determine if chemicals tested with the assay could be ranked according to estimates of the slope (allowing comparison of resuits from laboratory to laboratory), to evaluate intralaboratory and interlaboratory variation of S9-requiring chemicals, and to develop an initial Salmonella mutagenicity data base to assist laboratories in evaluating their data. The purpose of this research, however, is not to produce a mandatory set of target values for other researchers, but to test the hypothesis that reference compounds can be used in the Salmonella assay in order to provide calibration standards for the assay. These goals will all be useful when evaluating bioassay data in a comparative manner. Materials and methods

Samples Information concerning the ten chemicals chosen, including catalogue and lot numbers, supplier, and molecular weight, is listed in Table 1. The study chemicals were of the highest purity commercially available. All the test chemicals were nonvolatile organic chemicals; therefore, these chemicals will remain stable unless they are

151 TABLE 1 IDENTIFICATION OF THE CHEMICALS CHOSEN FOR THE EXOGENOUS ACTIVATION DATA BASE STUDY Chemical I.D.

MW

Name

Supplier

Catalog No./Lot No.

Dose range: (/xg/plate)

MC

268.4

methylcholanthrene

Sigma

0.5- 5.0

AO

369.94

acridine orange

Aldrich

AAF

223.3

Sigma

2AN

193.2

2-acetamidofluorene 2-anthramine

BAA

228.3

benz[a]anthracene

Sigma

BAP

252.3

benzo[a]pyrene

Sigma

FG

225.3

Sigma

ABP

169.2

Fast Garnet or aminoazotoluene 4-aminobiphenyl

DBP

302.4

Sigma

DMBA

256.3

dibenzola,i]pyrene dimethylbenz[a]anthracene

M-6501 61F-0069 15,855-0 TT04530DT A-7015 97F-3527 A-1381 97F-3558 B-2750 129C-0520 B-1760 57F-3434 F-8504 27F-3656 A-2898 104F-3420 D-0253 96F-0615 D-3254 55F-0262

exposed to extreme heat or water. Samples were diluted in dimethylsulfoxide (DMSO), and a 100 /~1 solution of each dose was delivered to each bioassay plate. Because these chemicals were known indirect-acting mutagens, they were tested only in the presence of the exogenous activating system. The dose range for the definitive tests of each chemical is listed in Table 1. Ten doses of each chemical were analyzed with two plates per dose. The first round at the Research Triangle Institute (RTI) and the U.S. Environmental Protection Agency (EPA) laboratories was a dosefinding experiment, and this round was not included in the analysis of variance. The same lot of each chemical was tested at both R T I and EPA.

$9 preparations Four different lots of $9 (two at RTI and two at EPA) were utilized in this study. These $9 lots were prepared from Sprague-Dawley male rats, induced with Aroclor 1254 five days before sacrifice as described by Maron and Ames, 1983. The amount of $9 in 500 /zl of $9 m i x / p l a t e was

Sigma

Sigma

Sigma

0.5- 5.0 5.0-50.0 0.1- 1.0 1.0-10.0 0.2- 2.0 1.0-10.0 1.0-10.0 0.4- 4.0 1.0-10.0

adjusted so that the total amount of $9 was always the same, 1.1 mg of protein per plate. Protein levels were d e t e r m i n e d by the procedure of Lowry et al. (1951).

Bioassay procedures Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley, CA, supplied independently to the two participating laboratories the Salmonella typhimurium strain TA100 used in the study. The standard plate incorporation assay as described by Maron and Ames (1983) was used. At RTI, 10/xl of a stock culture of the bacterial strain TA100 were inoculated into 50 ml of nutrient broth ( O X O I D ®) and were grown for 10 h at 37°C with shaking at 100 rpm. At EPA, 20 ml of nutrient broth were inoculated with an isolated colony of TA100 and incubated for 16 h at 37°C with shaking at 120 rpm. The bacterial inoculum was utilized in the mutagenicity assay. Each assay plate contained 100/zl of sample, 100 /xl of bacteria and 500 /xl of $9 mix. Chemicals were tested at ten doses with duplicate p l a t e s / dose. In order to preclude toxicity to the bacteria,

152

solubility of the compound, or other factors from interfering with the determination of dose-response slope values, doses were chosen from the first one-half of the linear portion of the dose-response curve as determined in the preliminary range-finding tests for each compound. The solvent control plates each contained 100 ~1 of the solvent DMSO, 100/zl of bacteria and 500/zl of $9 mix. Four rounds were conducted at both RTI and EPA. Each laboratory used two different lots of $9 (a total of 4 $9 lots for the study) with the first lot being used in the initial two rounds and the second lot being used in the final two rounds. Assay plates were counted electronically with an Artek 880 (R) automatic cell counter. Electronic counts were periodically verified by hand to assure that no significant drifting of the machine calibration had occurred. Data were statistically analyzed with the statistical analysis program, Salmonel (Myers et al., 1991), which utilizes the model developed by Bernstein et al. (1982) to obtain linear slope values (revertants//~g). Spontaneous (solvent control) background counts of colony forming

units for the Salmonella strain were within the 95% confidence intervals of historical spontaneous background counts. These criteria are in agreement with the recommendations of de Serres and Shelby (1979) and Claxton et al. (1987). Results and discussion

The ten chemicals for this study (Table 1) were chosen because they were known indirect-acting mutagens that were readily accessible. From literature review and previous experience at RTI and EPA, these mutagens were known to have a wide range of mutagenic activity. Due to the volume of data, only the means of the revertant counts are presented (see the Appendix). The complete data set is available upon request from Larry D. Claxton (EPA). Results of the slope analysis and ranking for each round and a statistical summary by compound are contained in Table 2. It is readily apparent that the results for these 10 chemicals are highly reproducible both within and between laboratories. The slope values for BAA and FG

TABLE 2 SLOPE ANALYSIS (REVERTANTS//.~g) AND STATISTICAL SUMMARY OF THE SALMONELLA MUTAGENICITY DATA ( + $9) FROM RTI AND EPA Round a

AAF

2 3 4 5

RTI slope values (rank) 15 (1) 38 (3) 32 (2) 19 (1) 26 (2) 36 (3) 19 (1) 57 (3) 47 (2) 16 (1) 50 (2) 58 (3)

BAA

ABP

DMBA

DBP

MC

BAP

2AN

43 (4) 90 (4) 80 (4) 90 (4)

88 (6) 97 (5) 103 (6) 100 (5)

118 (7) 113 (6) 156 (7) 115 (6)

63 (5) 138 (7) 101 (5) 126 (7)

154 (8) 190 (8) 161 (8) 160 (8)

272 (9) 313 (9) 418 (9) 797 (10)

1066 (10) 1166 (10) 1 161 (10) 780 (9)

Mean S.D. C.V. Rank

17.3 2.0 0.12 1

43.3 11.7 0.27 3

75.8 22.3 0.29 4

97.0 6.5 0.07 5

125.5 20.4 0.16 7

107 33.1 0.31 6

166.3 16.1 0.10 8

450.0 239.4 0.53 9

1043.0 181.4 0.17 10

2 3 4 5

EPA slope values (rank) 25 (1) 57 (2) 63 (4) 29 (1) 41 (2) 45 (3) 21 (1) 52 (3) 45 (2) 26 (1) 48 (2) 58 (3)

73 (5) 57 (5) 68 (4) 105 (6)

97 (6) 110 (7) 75 (6) 84 (4)

60 (3) 82 (6) 71 (5) 99 (5)

144 (7) 49 (4) 194 (7) 160 (7)

247 (8) 245 (8) 226 (8) 249 (8)

319 248 228 282

1703 (10) 1420 (10) 1264 (10) 1 142 (10)

Mean S.D. C.V. Rank

25.3 3.3 0.13 1

75.8 20.6 0.27 4

91.5 15.3 0.17 6

78.0 16.6 0.21 5

136.8 62.1 0.45 7

241.8 10.6 0.04 8

269.3 39.9 0.15 9

42.8 13.7 0.32 2

49.5 6.8 0.14 2

FG

52.8 9.2 0.17 3

AO

(9) (9) (9) (9)

a Rounds with the same $9 lot: RTI (1 and 2) (3 and 4); EPA (1 and 2) (3 and 4); pilot round (1) not included.

1382.3 242.2 0.18 10

153

are almost identical. The slope values (revertants per/~g) for BAA and FG were 42.8 and 43.3 for RTI data and were 49.5 and 52.8 for EPA data. Therefore, the ranking of these two compounds was not perfectly consistent. Similarly, ABP, DMBA, and DBP gave similar slope values (means of 78 to 137 revertants per/zg). Table 3 contains a ranking of the ten chemicals based on their mean slope values from the four rounds. When a single compound (DMBA) is removed from the listing, nine of the ten chemicals had identical rankings in both laboratories. One should not over interpret this result, however. When examining the averaged slopes, BAA and FG did rank in the same order in both laboratories; however, this is obviously fortuitous because the slope values are so similar. BAA and FG, therefore, can be considered to have approximately the same potency. The other three chemicals with close slope values (78-137) had rankings that were different in the two laboratories (ABP, DBP and DMBA had rankings that varied from 5 to 7). These three compounds cannot be separated on the basis of potency as indicated by slope. If a + 40% difference in slope values between independent tests is considered an attainable goal in the Salmonella assay, the ratio between slope values in two laboratories should range between 0.6 and 1.4. Only DMBA did not meet this criterion of acceptability. Table 3 also

lists the coefficients of variance in percentage. It should be noted that 13 of the 20 listed CV values were below 25% and that the mean CV in both laboratories was below 25%. This is a statistically important observation. A doubling in slopes between two chemicals can be considered a true (i.e., real) mutagenic difference if the CV is below 25%. For example, it is reliable to say that chemical X with a slope of 25 revertants//~g is actually more mutagenic than chemical A with a slope of 12 revertants/~g if the CV is below 25% for the assay. This fact is extremely important when setting reliable ranking boundaries. One must remember, however, that the variation is dependent upon the bioassay, the precise protocol used, and the precision with which the protocol is followed. Compared to previous studies (Myers et al., 1987; Dunkel et al., 1984), these CV values are relatively small. Following the logic in the companion paper that deals with direct-acting mutagens (Claxton et al., 1991b), Table 4 divides potential slope value ranges on a log dose basis. Nine of the 10 chemicals fell into the same boundary ranges in both laboratories. DMBA was not included in Table 4 because of its highly variable results and because it ranked differently in the two laboratories. Using comparative bioassay efforts (Claxton et al., 1991b), slope values could be compared with the slope values concurrently generated for one or

TABLE 3 RANKING OF MUTAGENIC ACTIVITY BY AVERAGE SLOPE VALUES OF 10 INDIRECT-ACTING ( + $ 9 ) PURE CHEMICALS Chemical

AAF BAA FG AO DMBA ABP DBP MC BAP 2AN

Ranking (average slope value) EPA RTI Rounds Rounds 1 (25.3) 2 (49.5) 3 (52.8) 4 (75.8) 5 (78.0) 6 (91.5) 7 (136.8) 8 (241.8) 9 (269.3) 10 (1382.3)

1 (17.3) 2 (42.8) 3 (43.3) 4 (75.8) 7 (125.5) 5 (97.0) 6 (107.0) 8 (166.3) 9 (450.0) 10 (1043.0)

Ratio of slopes: RTI/ EPA

Coefficient of variation: Percent: EPA

RTI

0.7 0.9 0.8 1.0 1.6 1.1 0.8 0.7 1.7 0.8

13 14 17 27 21 17 45 4 15 18

12 32 27 29 16 7 31 10 53 17

a Slope in revertants per/zg from Salmonel statistical analysis program using the methods of Bernstein et al., 1982.

154 TABLE 4 POTENTIAL BOUNDARIES FOR COMPARATIVE ASSESSMENT ANALYSIS WITH SALMONELLA MUTAGENICITY DATA ( + $9) Boundaries in revertants/tzg (i.e., slopes)

Mutagenicity ranking

Chemicals that could be utilized to set boundaries a

< 0.1 0.11.0 1.0- 10.0 10.0- 100.0 100.0-1000.0 > 1000.0

equivocal extremely low low moderate high extremely high

ABP, FG, BAA, AAF, AO BAP, MC, DBP 2AN

a Data from this study. DMBA was ranked as moderate at EPA (78), and as high at RTI (126); therefore, it is not included in table (see text). more of these potential reference standards. If the potency consistently was found within one of these ranges, one would have a useful relative indicator of potency that could be c o m p a r e d to other results generated in a similar manner. The low variabilities in this study may have been achieved by two study parameters: selecting ten closely spaced doses that were nontoxic, and setting the $9 to an experimentally determined constant amount of protein. See Claxton et al. (1991a) for a discussion of factors affecting $9 variability and the choosing of appropriate $9 levels. The protein level for the $9 was determined by testing each lot of $9 (10 levels of $9) with single doses of benzo[a]pyrene, 2-anthramine, and 7,12-dimethylbenzanthracene (data not presented). The $9 levels providing the maximum response for each chemical using each lot of $9 was averaged. The resulting level of $9 (1.1 mg protein / plate) was then used in all experiments. The variance between the R T I and E P A laboratories was considerably lower than in other interlaboratory studies (see Myers et al., 1987 for a discussion of variation in the Salmonella mutagenicity assay). The low variance detected in this study was likely due to the fact that the chemicals were tested in the linear, nontoxic dose range, with more tightly spaced dose levels and more doses than are usually evaluated in routine testing. Interlaboratory variation in a large EPAsponsored study was 115% (Myers et al., 1987). In summary, chemicals requiring exogenous activation can be consistently ranked for mutagenic response in the Salmonella assay. This sup-

ports the potential use of mutagenicity bioassay data in a more quantitative manner when the data are calibrated like other bioassays used for quantitation. Pure compounds, therefore, can be used to place mutagens within ranges of activity as compared to a series of standard mutagens. This understanding in the future may allow for the comparison of results between laboratories in a more uniform fashion, allow for improved quality a s s u r a n c e / q u a l i t y control procedures for the assay, and help develop the quantitative comparison o f bioassay results. Concurrent testing (same day, same laboratory, same technician, same bacteria, $9 and media) is a more acceptable method to control variance and to compare the mutagenicity of two or more compounds; however, this is not always possible. The ability to quantify Salmonella mutagenicity data by the use of reference materials will assist in potency comparisons of data generated in different laboratories or within the same laboratory on different days.

Acknowledgements The authors want to thank Doctors Ferris Benson and Jerry Koenigs for their suggestion and administrative support. Thanks to Christine A. Rawn for her editorial assistance. This work was funded by E P A contract number 68-02-4186052. This manuscript has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the

155

genicity assay using strain TA100 (Maron and Ames, 1983). Values indicate the mean counts for the colony forming units at each dose. Chemicals were tested using two plates per dose during each round. Each dose was administered in 100/zl of dimethylsulfoxide. A full listing of the data with standard deviations is available from the authors. Data for the pilot rounds are not given but are available upon request.

contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Appendix The following tables contain the results for the ten chemicals tested in the Salmonella muta-

T A B L E A1 MEAN VALUES FROM SALMONELLA MUTAGENICITY DATA OF 2-ANTHRAMINE

Solvent

2-Anthramine doses (/zg)

0.00

0.10

0.20

0.30

0.40

RTI ROUNDS 2 187 3 192 4 145 5 152

230 264 216 187

360 369 414 273

487 526 577 360

602 619 803 506

EPA ROUNDS 2 130 3 120 4 118 5 93

284 221 198 191

469 367 353 317

669 554 503 441

878 757 602 559

Slope 0.60

0.70

0.80

0.90

1.00

Revertants//z g

737 852 958 616

916 986 1214 747

1116 1091 1541 941

1313 1440 1524 1016

1418 1500 1725 1099

1548 1616 1928 1 168

1066 1 166 1 161 780

1028 939 790 845

1087 1011 891 946

1497 1201 948 1 126

1558 1328 1 108 1229

1514 1574 1292 1413

1730 1643 1388 1378

1703 1420 1264 1 142

0.50

T A B L E A2 MEAN VALUES FROM SALMONELLA MUTAGENICITY DATA OF BENZO[a]PYRENE

Solvent 0.~

Benzo[ a]pyrene doses (p.g)

Slope

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

Revertant/~g

RTI ROUNDS 2 187 3 192 4 145 5 152

185 203 203 314

244 276 256 477

281 359 412 683

352 412 460 783

395 422 584 912

484 511 662 931

522 667 731 985

610 726 828 991

728 679 881 975

846 945 916 948

272 313 418 797

EPAROUNDS 2 130 3 120 4 118 5 93

201 182 174 173

279 208 242 217

347 260 295 268

451 298 313 305

455 372 364 299

517 429 418 385

538 442 456 453

622 533 467 430

677 567 489 420

727 643 474 474

319 248 228 282

156 TABLE A3 MEAN VALUES FROM SALMONELLA MUTAGENICITY D A T A OF METHYLCHOLANTHRENE SoNent 0.00

Methylcholanthrene doses (/zg)

Slope

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Revertants/p.g

RTI ROUNDS 2 187 3 192 4 145 5 152

194 228 234 249

275 410 318 321

322 464 338 405

509 798 446 498

646 791 568 577

773 775 679 619

908 1220 952 779

846 1206 822 726

907 1171 826 723

1045 1084 886 975

154 190 161 160

EPAROUNDS 2 130 3 120 4 118 5 93

238 257 269 217

410 383 394 363

514 624 507 525

613 652 541 579

731 783 640 711

688 882 669 949

660 945 618 845

733 954 653 676

792 951 644 711

894 968 710 768

247 245 226 249

TABLE A4 MEAN VALUES F R O M SALMONELLA MUTAGENICITY D A T A OF 9,10-DIMETHYL-1,2-BENZ[a]ANTHRACENE Solvent

9,10-Dimethyl-l,2-benz[a]anthracene doses (p.g)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Slope Revertants/~g

RTI ROUNDS 2 187 3 192 4 145 5 152

274 247 226 226

422 356 478 389

588 393 604 507

773 638 887 655

793 705 930 673

948 940 983 649

1 196 1084 752 943

1065 883 724 882

1155 860 997 996

1229 1125 1303 895

118 113 156 115

EPA ROUNDS 2 130 3 120 4 118 5 93

197 222 195 176

263 293 289 311

310 350 377 403

411 406 413 557

539 524 461 653

503 699 605 736

465 760 543 648

485 738 557 657

587 826 596 645

772 784 669 718

60 81 71 99

TABLE A5 MEAN VALUES F R O M SALMONELLA MUTAGENICITY D A T A OF DIBENZO[a,i]PYRENE Solvent

Dibenzo[a,i]pyrene doses (/zg)

0.00

Slope

0.40

0.80

1.20

1.60

2.00

2.40

2.80

3.20

3.60

4.00

Revertants/~zg

RTI ROUNDS 2 187 3 192 4 145 5 152

190 252 188 232

230 293 227 272

268 380 271 322

261 475 345 379

334 500 359 468

345 567 404 410

373 567 415 511

420 611 432 577

418 698 571 503

379 713 523 529

63 138 101 126

EPAROUNDS 2 130 3 120 4 118 5 93

179 162 190 182

229 166 245 218

318 162 356 243

463 190 386 359

614 224 526 423

603 239 652 499

575 252 638 579

566 273 648 598

691 289 655 660

793 362 707 614

144 49 194 160

157 TABLE A6 MEAN VALUES F R O M SALMONELLA MUTAGENICITY D A T A OF 4-AMINOBIPHENYL Solvent 0.00

4-Aminobiphenyl doses (/zg)

Slope

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Revertant/~g

RTI ROUNDS 2 187 3 192 4 145 5 152

301 309 303 257

367 463 382 359

439 486 495 488

591 632 646 567

632 687 690 677

699 790 671 702

800 914 766 738

779 910 886 774

922 974 957 909

958 1057 1050 909

88 97 103 100

EPAROUNDS 2 130 3 120 4 118 5 93

238 233 199 198

347 336 260 245

438 509 366 388

578 580 442 438

675 740 511 495

727 733 597 597

733 839 667 604

854 865 692 637

882 973 819 699

1118 1083 808 792

97 110 75 84

TABLE A7 MEAN VALUES FROM SALMONELLA MUTAGENICITY D A T A OF ACRIDINE O R A N G E Solvent

Acridine orange d o s e s ( ~ g )

0.00

Slope

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

RTI ROUNDS 2 187 3 192 4 145 5 152

180 207 140 190

211 232 193 222

222 273 217 292

237 325 271 309

274 370 343 378

275 466 369 442

315 509 417 484

369 537 438 498

362 544 477 568

391 652 528 571

43 90 80 90

EPAROUNDS 2 130 3 120 4 118 5 93

160 150 133 162

197 171 160 193

244 208 193 225

255 233 232 283

350 219 300 363

329 320 334 464

368 346 384 547

429 410 390 620

467 454 417 697

640 497 420 734

73 57 68 105

Revertants/lzg

TABLE A8 MEAN VALUES F R O M SALMONELLA MUTAGENICITY D A T A OF A M I N O A Z O T O L U E N E Solvent 0.00

Aminoazotoluene doses (/.rg)

Slope

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Revertants//z g

RTI ROUNDS 2 187 3 192 4 145 5 152

188 203 168 231

216 233 187 245

244 262 270 346

283 289 299 427

295 328 395 480

374 360 440 504

438 472 549 618

534 510 546 627

573 572 571 646

578 614 609 678

32 36 47 58

EPAROUNDS 2 130 3 120 4 118 5 93

157 156 157 163

216 183 176 195

273 250 223 229

335 324 274 321

425 467 341 428

501 509 395 427

598 585 483 518

606 601 474 586

742 684 556 585

748 693 545 666

63 45 45 58

158 TABLE A9 MEAN VALUES FROM SALMONELLA MUTAGENICITY DATA OF BENZ[a]ANTHRACENE Solvent 0.00

Benz[a]anthracene doses (/zg)

Slope

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

10.00

Revertants//z g

RTI ROUNDS 2 187 3 192 4 145 5 152

188 233 170 218

235 259 234 229

251 267 254 276

320 358 351 313

397 378 410 415

458 475 543 475

481 508 529 539

598 574 604 537

659 642 749 749

661 656 644 615

38 26 57 50

EPAROUNDS 2 130 3 120 4 118 5 93

166 165 154 144

232 202 188 174

279 229 240 216

336 274 292 293

461 352 364 352

547 494 449 433

552 478 554 505

655 556 619 615

705 575 757 710

897 684 808 827

57 41 52 48

TABLE A10 MEAN VALUES FROM SALMONELLA MUTAGENICITY DATA OF 2-ACETYLAMIDOFLUORENE So~ent

2-Acetylamidofluorene doses(~g)

0.00

Slope

5.00

10.00

15.00

20.00

25.00

30.00

35.00

RTI ROUNDS 2 187 3 192 4 145 5 152

275 310 251 247

326 376 343 343

435 496 443 395

480 581 534 486

596 610 635 539

639 732 750 680

EPAROUNDS 2 130 3 120 4 118 5 93

273 332 272 286

411 448 345 362

548 511 454 456

625 549 483 534

770 678 535 692

789 742 661 895

References Ames, B.N. (1979) Identifying environmental chemicals causing mutation and cancer, Science, 204, 587-593. Bernstein, L., J. Kaldor, J. McCann and M.C. Pike (1982) An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test, Mutation Res., 97, 267-281. Claxton, L.D., J. Allen, A. Auletta, K. Mortelmans, E. Nestmann and E. Zeiger (1987) Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutation Res., 189, 83-91. Claxton, L.D., A.G. Stead and D. Walsh (1988) An analysis by chemical class of Salmonella mutagenicity tests as predictors of animal carcinogenicity, Mutation Res., 205, 197225. Claxton, L.D., V.S. Houk, J.C. Allison and J. Creason (1991a) Evaluating the relationship of metabolic activation system

40.00

45.00

50.00

Revertants/~g

744 770 820 710

750 740 792 812

851 736 906 820

979 966 983 828

15 19 18 16

855 874 706 939

921 1027 698 945

1084 1103 869 950

1431 1224 962 1017

25 29 21 26

concentrations and chemical dose concentrations for the Salmonella spiral and plate assays, Mutation Res., 253, 127-136. Claxton, L.D., V.S. Houk, L.G. Monteith, L.E. Myers and T.J. Hughes (1991b) Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: I. Without exogenous activation, Mutation Res., 253, 137147. de Serres, F.J. (1983) The use of Neurospera in the evaluation of the mutagenic activity of environmental chemicals. Environ. Mutagen., 5, 341-351. de Serres, F., and M. Shelby (1979) Recommendations on data production and analysis using the Salmonella/ microsome mutagenicity assay, Mutation Res., 64, 159-165. Dunkel, V.C., E. Zeiger, D. Brusick, E. McCoy, D. McGregor, K. Mortelmans, H.S. Rosenkranz and V.F. Simmon (1984) Reproducibility of microbial mutagenicity assays: Test with Salmonella typhimurium and Escherichia coli

159 using a standardized protocol, Environ. Mutagen., 6, Suppl. 2, 1-254. Lave, L.B., and G.S. Omenn (1986) Cost-effectiveness of short-term tests for carcinogenicity, Nature (London), 324, 29-34. Lewtas, J. (1986) A quantitative cancer risk assessment methodology using short-term genetic bioassays: The comparative potency method, in: P. Oftedal and A. Brogger (Eds.), Risk and Reason: Risk Assessment in Relation to Environmental Mutagens and Carcinogens, Liss, New York, pp. 107-120. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with Folin phenol reagent, J. Biol. Chem., 193, 265-275. Mailing, H.V. (1971) Dimethylnitrosamine: Formation of mutagenic compounds by interaction with mouse liver microsomes, Mutation Res., 13, 425-429.

Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the Salmonella/ microsome test: Assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Myers, L.E., N.H. Adams, T.J. Hughes, L.R. Williams and L.D. Claxton (1987) An interlaboratory study of an EPA/ Ames/Salmonella test protocol, Mutation Res., 182, 121133. Myers, L.E., N. Adams, L. Kier, T.K. Rao, B. Shaw and L. Williams (1991) Microcomputer software for data management and statistical analysis of the Ames/Salmonella test, in: D. Krewski (Ed.), Statistical Methods in Toxicological Research, Gordon and Breech, New York, pp. 265-279.