Drug Metabolism Reviews
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Strategies for the Use of Genetic Toxicity Tests Errol Zeiger To cite this article: Errol Zeiger (1990) Strategies for the Use of Genetic Toxicity Tests, Drug Metabolism Reviews, 22:6-8, 765-775, DOI: 10.3109/03602539008991467 To link to this article: http://dx.doi.org/10.3109/03602539008991467
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DRUG METABOLISM REVIEWS, 22(6-8), 765-775 (1990)
STRATEGIES FOR THE USE OF GENETIC TOXICITY TESTS* ERROL ZEIGER Cellular and Genetic Toxicity Branch National Institute of Environmental Health Sciences P O . Box 12233 Research Triangle Park, North Carolina 27709
I.
INTRODUCTION ..........................................................
765
11.
METHODS .................................................................
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111.
RESULTS AND DISCUSSION ..........................................
768
References.. .................................................................
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I. INTRODUCTION Many short-term tests (STT) have been proposed for use as carcinogenicity prescreens. In 1979, Hollstein et al. [ I ] published a list of over 100, and the EPA in its Gene-Tox program in 1979 evaluated more than 75 test sys*This paper was refereed by Suzanne C. Fitzpatrick, Ph.D., Division of Chemistry, HFV- 140, Center for Veterinary Medicine, FDA, 5600 Fishers Lane, Rockville, MD 20857. 765 Copyright 0 1991 by Marcel Dekker. Inc
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tems that had been proposed for carcinogen identification. It was made very clear by those evaluations, although it was evident beforehand in many cases, that for many test systems there was little published test data. For example, in 1979, fewer than 20 assays had definitive results for more than 100 chemicals, and about 12 had definitive results for 50-100 chemicals. In many cases, the data published came only from the originating laboratory, and the test had only been run with a limited number of classical carcinogenic alkylating agents and a few model carcinogens that required metabolic activation. In 1977. before the formation of the National Toxicology Program (NTP), the National Institute of Environmental Health Sciences (NIEHS) initiated a genetic toxicology testing program to test chemicals of interest to the NIEHS, and later to the NTP and other agencies. An important part of this program was the testing of coded chemicals in standardized protocols. The tests selected for use at that time were the Salmonella/microsome mutagenicity assay (SAL), the induction of chromosome aberrations (ABS) and sister chromatid exchanges (SCE) in Chinese hamster ovary cells, the L5 l78Y mouse lymphoma tk+'- mutation assay (MLA), the Drosophila sex-linked recessive lethal assay, and other in virro and in vivo assays. The chemicals were tested in a number of laboratories under contract. Table I shows the progress of testing in some of the tests used. Other tests have been used to a lesser extent and are not presented here. Many of the test
TABLE I NTP Test Systems and Chemicals Tested" Test system and endpoint Salmonella mutagenicity I n virro chromosome aberrations (CHO cells) I n virro sister chromatid exchanges (CHO cells)
Mouse lymphoma cell mutagenicity Drosophila sex-linked recessive lethal test Drosophila reciprocal translocations Mouse bone marrow chromosome aberrations Mouse bone marrow sister chromatid exchanges
Chemicals tested
Samples tested'
1,566 567 567 35 1 255 62 I10 I10
2,064 62 I 62 I 445 324 78 I I5 115
aAs of November, 1988. 'Reflects repeat tests of chemicals under different codes in the same or different laboratories.
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results for the specific chemicals have been published or are in various stages of publication. They will all be published. Testing schemes using a number of STT have been proposed or enacted by various organizations. These schemes have included tier systems and the more widely used approach, the test battery. The results obtained at the various stages of these schemes dictate the future testing priorities for the chemical or, in some cases, whether the chemical will be considered for additional testing or development. These testing schemes were generally assembled according to early reports of the utility or advantages of the various assay systems, and what can be called “scientific intuition.” According to this intuition, different STT are needed to measure different genetic endpoints, and results from all endpoints are needed in order to make informed decisions regarding the potential carcinogenicity of the test chemical. Accordingly, test batteries assembled by regulatory agencies and other organizations included, as a rule, the Salmonella test, an in v i m chromosome aberration test, an in vivo chromosome aberration test, a gene mutation test in mammalian cells, and, because it was genetically well defined, a Drosophila sex-linked recessive lethal test. This was also the rationale behind the initial selection of test systems for the NIEHS testing program. Evaluations of the NTP STT database to determine the usefulness of these tests were begun in the early 1980s [2,3]. In 1984, a study to evaluate the effectiveness of four widely used STT (SAL, ABS, SCE. and MLA) for discriminating between carcinogens and noncarcinogens was initiated. The first segment of that study, comprising 73 chemicals, has been published 141. Additional chemicals are being evaluated. At the same time, the NTP started looking at the performances of the Drosophila sex-linked recessive lethal assay and in vivo cytogenetics assays with the same group of chemicals. These parts of the study are in progress. The results presented here summarize the results of the studies using SAL, ABS, SCE, and MLA on the initial 73 chemicals.
11. METHODS
The STT and protocols used in the evaluation study are listed in Table 2. All chemicals were tested under code in laboratories under contract to the NTI? The test chemicals were coded at the NTP Chemical Repository, which also sent them to the laboratories and decoded them after the results had been reviewed and evaluated. In a number of cases, the same chemical
ZEIGER
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TABLE 2 Short-Term Test Systems Used
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Salmonella typhimurium mutation (Ames test)
(SAL) [5,61 Chromosome aberrations in Chinese hamster ovary cells (ABS) [7] Sister chromatid exchanges in Chinese hamster ovary cells (SCE) 171 Mouse lymphoma L5178Y tk+’- mutation assay (MLN [81 was tested in more than one laboratory, or in the same laboratory at different times, to monitor the performance of the laboratories or to clarify a weak or equivocal response. When a chemical was tested more than once, different code numbers were used. The carcinogenicity tests were performed in male and female F344 rats and B6C3F1 mice, using 50 animals per group, treated for 2 years with solvent, a maximum tolerated dose, and one or two intermediate doses. The carcinogenesis evaluations were taken from the NTP Technical Reports for the particular chemicals. All equivocal responses in the carcinogenicity tests and STT were considered negative for the purposes of this evaluation. The following definitions were used for the evaluation of STT performance: sensitivity is the proportion of carcinogens that are positive in a test; specificity is the proportion of noncarcinogens that are not positive in a test; positive predictivity is the proportion of positives in a test that are carcinogens; negative predictivity is the proportion of negatives in a test that are noncarcinogens; concordance is the overall proportion of “correct” responses in the STT; and prevalence is the proportion of carcinogens in the population.
111. RESULTS AND DISCUSSION
It was anticipated, based on earlier studies (Table 3A) with the Salmonella test, that the test would correctly identify approximately 80-90% of the carcinogens and noncarcinogens. The SAL results derived from the NTP chemicals did not support this expectation (Tables 3B, 4). The concordance between SAL mutagenicity and rodent carcinogenicity in the NTP database was 62%. There are a number of explanations for this difference, the primary one being that the chemicals tested by the NTP belonged, for
GENETIC TOXICITY TESTS
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TABLE 3
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Historical Development of SAL Validation Studies No. chemicals
Prev.
Sens.
223 146 53 I20 112
.72 .67 .70 .48 .93
.88 .92 .68 .91 .72
87 60 224 73
.74 .55 .55
.47 .76 .54
.60
.45
Spec.
Conc .
(+I
(-1
Pred.
Pred .
A .75 .90 .77 .89 .88 .93 .94 .93 .63 .96 B [NTP studies] .70 .81 .59 .69 .70 .69 .86 .83
Ref.
.7 1 .82 .50 .92 .I5
.84 .87 .74 .93 .71
.32 .67
.53
14
.68
15
.55 .5 1
.6 I .62
3 4
9 10
II 12 13
Prev. = prevalence; Sens. = sensitivity; Spec = specificity; ( + ) Pred. = positive predictivity; (-1 Pred. = negative predictivity; Conc. = concordance (see text for definitions).
the most part, to different chemical classes than those tested earlier. It has been shown [3,16] that some classes of chemical carcinogens are efficiently detected by SAL (alkylating agents, nitroso compounds, nitroaromatics), whereas others (chlorinated hydrocarbons, hormones) are not. The carcinogenic responses in rodents also vary according to chemical class. This will be addressed in more detail below. The ability of SAL, ABS, SCE, and MLA to detect carcinogens (sensitivity) and discriminate between carcinogens and noncarcinogens is presented in Table 4. SAL and ABS had a low sensitivity and high specificity,
TABLE 4 Performance of Short-Term Tests with 73 Chemicals [4]
Sensitivity (%) Specificity (%) Positive predictivity (%) Negative predictivity (%) Concordance (%) Prevalence = 60%
SAL
ABS
SCE
MLA
45 86 83 51 62
55 69 73 50 60
73 45 67 52 62
70 45 66 50 60
ZEIGER
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and SCE and MLA had a high sensitivity and low specificity. SAL had the highest positive predictivity. However, the overall concordances for all four STT were equivalent (60-62%). SCE and MLA detected the most carcinogens, but their false positive rates (the proportion of noncarcinogens that were positive) were also the highest. Therefore, among these 4 STT, SAL would appear to perform the best (highest concordance) in a population of chemicals containing few carcinogens, and SCE and MLA would appear to perform best when there is a high proportion of carcinogens in the population. In order for a battery of tests to be more effective than a single test for predicting carcinogenicity and noncarcinogenicity. the tests in the battery must complement each other. That is, the other tests should correctly identify the carcinogens missed by the original test without misidentifying noncarcinogens. None of the STT complements SAL; they do not distinguish between a SAL-negative carcinogen and a SAL-negative noncarcinogen (Table 5 ) . This lack of complementarity extends to all 4 STT (data not shown). However, the four tests are confirmatory; a positive result in one test is likely to be confirmed in the other tests (data not shown). This is not surprising, because the tests measure mutagenesis and chromosome damage, not carcinogenesis. Therefore, a mutagen and chromosome-damaging agent would be expected to be active in all tests designed to measure these endpoints. The classes of chemicals studied affect the responses obtained in carcinogenicity and genetic toxicity tests. A recent report by Ashby and Tennant [ 171 divided test chemicals into two classes-those containing “structural alerts,” that is, substructures within the molecules that are capable of reacting with DNA, and those that do not contain DNA-reactive sites. Chemicals containing structural alerts are more likely to be carcinogens, tend to be carcinogenic in both rats and mice, produce tumors at multiple organ sites, and are mutagenic in Salmonella. Conversely, carcinogens without structural alerts tend to produce tumors in a single species, usually at a single site, and are not Salmonella mutagens. Tumors induced by structur-
TABLE 5 Responses in the ABS, SCE, and MLA Tests of Chemicals Negative in SAL [4] Carcinogenicity
+ -
+ 12 10
ABS -
+
23 34
20 23
SCE -
+
15 21
17 25
MLA -
18 19
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ally alerting chemicals can appear at all organ sites, whereas certain organ sites (e.g., lung; skin; Zymbal’s gland) appear to be unaffected by chemicals lacking structural alerts [ 171. Results with the 73 NTP chemicals are consistent with Ashby and Tennant’s findings (Table 6). The high correlation between the presence of structural alerts and mutagenicity is reflected in the increased sensitivities and concordances of the STT. Among these STT, SAL is the most responsive to the structural alerts; when this endpoint is used in lieu of carcinogenicity, all the measurements of association increase, and the overall concordance is 87%. This is similar to the concordances between carcinogenicity and mutagenicity reported in earlier articles (see Table 3A), where the majority of carcinogens tested contained these structural alerts. In a battery testing approach, a chemical is tested in a series of test systems designed to encompass a range of genetically relevant endpoints. In some test batteries it is necessary for the chemical to be positive in only one of the STT; in others, a positive result is required in more than one, or all, STT. Often, the results obtained at the various stages of these schemes dictate whether a chemical is assumed to be a potential carcinogen, and therefore a candidate for a lifetime carcinogenicity study in rodents, or a potential noncarcinogen and therefore given a lower priority for rodent carcinogenicity assays. One premise behind the use of test batteries is that the tests will complement each other and that combinations of tests will be more effective for detecting carcinogens than individual tests. The fact that this will not occur with the four STT studied here is reflected in the absence of complementarity among the tests (Table 5 ) . When the four tests are assembled into batteries, their positive predictivity for carcinogenicity does not improve over that for SAL, alone. SAL‘s positive predictivity is .83 and no combination of positive responses produces a greater value (Table 7). In addition, if only SAL is used, 20 carcinogens are correctly identified; the other test
TABLE 6 Short-Term Test Responses to Structural AlertsP SAL
ABS
SCE
MLA
75 95 91 85 87
75 14
89 49 53 88 65
93 60 60 93 73
~~
Sensitivity (%) Specificity (%) Positive predictivity (%) Negative predictivity (%) Concordance (%)
64 82 75
‘Based on 71 chemicals. Structural alerts as defined by Ref. 17.
ZEIGER
772
TABLE 7 Predictivity of SAL Results
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Predictivity For carcinogenicity: SAL positive SAL and at least one other STT positive SAL and at least two other STT positive SAL negative and at least one other STT positive SAL negative and at least two other STT positive All 4 STT positive For noncarcinogenicity: SAL negative SAL and at least one other STT negative SAL and at least two other STT negative All 4 STT negative
No. chems.a
.83
20
.83
19
.83
19
.55
18
.74 .82
12 14
.51
25
.53
21
.54 .63
14 10
“Correctly identified. combinations identify from 12 to 19. Conversely, a negative response in SAL predicts noncarcinogenicity 51% of the time, whereas a negative in all four STT has a predictivity of .63. However, only 10 noncarcinogens were negative in all four STT, whereas 25 noncarcinogens were negative in SAL. STT are recommended or rejected based on their ability to distinguish between carcinogens and noncarcinogens. The ideal STT to use as a routine screen would be one whose positive and negative predictivities approached 1 0 % and few, if any, false positives would be produced. None of the STT used here fit this requirement. The SAL positive predictivity of 83% suggests that few false positive responses will be produced as compared to the other STT. However, all four STT have similar negative predictivities (5052%). which means that a negative response in any one of the four STT, by itself, has no probative value for noncarcinogenicity. These results confirm the strong interrelationships among mutagenicity, DNA-reactive activity, and rodent carcinogenicity. Unfortunately for those who believe that STT will detect the great majority of carcinogens, and
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distinguish carcinogens from noncarcinogens, there is a high proportion of chemicals that are not mutagenic (in SAL) and whose chemical structures give no indication of potential DNA reactivity. We know that many of these chemicals are rodent carcinogens but we do not know their human carcinogenicity. However, for human health considerations, we must presume that they also have the potential to induce tumors in humans. A current major research goal is to develop STT that can identify these nonmutagenic, nonstructurally alerting carcinogens. On the basis of the results presented here, it is evident that a positive response in more than one test system does not strengthen the value of the original positive, and a positive response in SAL is not negated by negative results in the other tests. Combinations of these four tests do not improve upon the effectiveness of the individual tests, and none of these tests are complementary. These data, and the false-positive frequencies obtained, do not support the use of ABS, SCE, or MLA for the routine screening of chemicals for presumptive carcinogenicity. A positive result in SAL is sufficient for a prediction of carcinogenicity; positive or negative results in the other STT studied here do not change this prediction. We are currently examining the ability of mouse bone marrow cytogenetics analyses to identify carcinogens that are not positive in SAL, and also to discriminate between carcinogens and noncarcinogens within the population of chemicals that are mutagenic in Salmonella.
REFERENCES [ l ] Hollstein, M., McCann, J., Angelosanto, F. A., Nichols, W. W., Short-term tests for carcinogens and mutagens, Murat. Res.. 65, 133226 (1979). [2] Zeiger, E., Tennant, R. W.,Mutagenesis, clastogenesis, carcinogenesis: Expectations, correlations and relations, in Genetic Toxicology of Environmental Chemicals, Part B: Genetic Effects and Applied Mutagenesis (Ramel, C., Lambert, B., Magnussen, J., eds.), Alan R. Liss, Inc., New York, 1986, pp. 75-84. [3] Zeiger, E., Carcinogenicity of mutagens: Predictive capability of the Salmonella mutagenesis assay for rodent carcinogenicity, Cancer Res., 47, 1287-1296 (1987). [4] Tennant, R. W., Margolin, B. H.,Shelby, M. D., Zeiger, E., Haseman, J. K., Spalding, J., Caspary, W., Resnick, M., Stasiewicz, S., Anderson, B., Minor, R., Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays, Science, 236, 933-941 (1987).
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[5] Haworth, S . , Lawlor, T., Mortelmans, K., Speck, W., Zeiger, E., Salmonella mutagenicity test results for 250 chemicals. Environ. Muragen., S(Suppl I), 3-142 (1983). (61 Mortelmans, K., Haworth, S., Lawlor. T., Speck, W., Tainer, B., Zeiger, E., Salmonella mutagenicity tests. 11. Results from the testing of 270 chemicals, Environ. Muragen.. 8(Suppl 71, 1-119 (1986). 171 Galloway, S. M., Bloom, A. D., Resnick, M., Margolin, B. H., Nakamura. F., Archer, F?, Zeiger, E., Development of a standard protocol for in virro cytogenetic testing with CHO cells: Comparison of results for 22 compounds in two laboratories, Environ. Muragen., 7, 1-52 (1985). (81 Myhr, B., Bowers, L., Caspary, W., Assays for the induction of gene mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in culture, Prog. Mutar. Res., 5 , 555-568 (1985). (91 McCann, J., Choi, E., Yamasaki, E., Ames, B. N., Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Narl. Acad. Sci. USA, 72, 5135-5139 (1975). [lo] Sugimura, T., Sato, S . , Nagao, M., Yahagi, T., Matsushima, T., Seino, Y., Takeuchi, M.,Kawachi, T., Overlapping of carcinogens and mutagens, in Fundamentals of Cancer Prevention (Magee, F! N., Takayama, S . , Sugimura. T., Matsushima, T., eds.), University Park Press, Baltimore, 1976, pp. 191-215. [ I l l Heddle, J. A., Bruce, W. R., Comparison of tests for mutagenicity or carcinogenicity using assays for sperm abnormalities, formation of micronuclei, and mutations in Salmonella, in Origins of Human Cuncer, Book C, (Hiatt, H. H., Watson, J. D., Winsten, J. A., eds.), Cold Spring Harbor Laboratory, 1977, pp. 1549-1557. [I21 Purchase, I. F. H.. Longstaff, E., Ashby, J., Styles, J. A., Anderson, D., Lefevre, I? A., Westwood, F. R., An evaluation of 6 short-term tests for detecting organic chemical carcinogens, Br. J. Cancer, 37, 873-959 (1978). [ 131 Bartsch. H., Malaveille, C., Camus, A-M., Martel-Planche, G., Brun, G., Hautefeuille, A., Sabadie, N., Barbin, A., Kuroki, T., Drevon, C., Piccoli, C., Montesano, R., Validation and comparative studies on 180 chemicals with S. typhimurium strains and V79 Chinese hamster cells in the presence of various metabolizing systems, Mutat. Res., 76, 1-50 (1980). (141 Zeiger, E., Knowledge gained from the testing of large numbers of chemicals in a multi-laboratory, multi-system mutagenicity testing program, in Environmenral Muragens and Carcinogens (Sugimura, T., Kondo, S., Takebe, H., eds.), Univ. of Tokyo Press, Tokyo/ A. R. Liss, Inc., New York, 1982, pp. 337-344.
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[15] Dunkel, V. C., Zeiger, E., Brusick, D., McCoy, E., McGregor, D., Mortelmans, K., Rosenkranz, H. S., Simmon, V. F., Reproducibility of microbial mutagenicity assays: 11. Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli. Environ. Muragen., 7(Suppl 5 ) . 1-248 (1985). [16] Rinkus, S. J., Legator, M. S., Chemical characterization of 465 known or suspected carcinogens and their correlation with mutagenic activity in the Salmonella ryphimurium system, Cancer Res., 39, 3289-3318 (1979). [17] Ashby, J., Tennant, R. W., Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NCVNTP. Murar. Res., 204, 17- I15 (1988).
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Strategies for the Use of Genetic Toxicity Tests Errol Zeiger To cite this article: Errol Zeiger (1990) Strategies for the Use of Genetic Toxicity Tests, Drug Metabolism Reviews, 22:6-8, 765-775, DOI: 10.3109/03602539008991467 To link to this article: http://dx.doi.org/10.3109/03602539008991467
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 4
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [NUS National University of Singapore]
Date: 06 November 2015, At: 10:18
Downloaded by [NUS National University of Singapore] at 10:18 06 November 2015
DRUG METABOLISM REVIEWS, 22(6-8), 765-775 (1990)
STRATEGIES FOR THE USE OF GENETIC TOXICITY TESTS* ERROL ZEIGER Cellular and Genetic Toxicity Branch National Institute of Environmental Health Sciences P O . Box 12233 Research Triangle Park, North Carolina 27709
I.
INTRODUCTION ..........................................................
765
11.
METHODS .................................................................
.767
111.
RESULTS AND DISCUSSION ..........................................
768
References.. .................................................................
.773
I. INTRODUCTION Many short-term tests (STT) have been proposed for use as carcinogenicity prescreens. In 1979, Hollstein et al. [ I ] published a list of over 100, and the EPA in its Gene-Tox program in 1979 evaluated more than 75 test sys*This paper was refereed by Suzanne C. Fitzpatrick, Ph.D., Division of Chemistry, HFV- 140, Center for Veterinary Medicine, FDA, 5600 Fishers Lane, Rockville, MD 20857. 765 Copyright 0 1991 by Marcel Dekker. Inc
Downloaded by [NUS National University of Singapore] at 10:18 06 November 2015
766
ZElGER
tems that had been proposed for carcinogen identification. It was made very clear by those evaluations, although it was evident beforehand in many cases, that for many test systems there was little published test data. For example, in 1979, fewer than 20 assays had definitive results for more than 100 chemicals, and about 12 had definitive results for 50-100 chemicals. In many cases, the data published came only from the originating laboratory, and the test had only been run with a limited number of classical carcinogenic alkylating agents and a few model carcinogens that required metabolic activation. In 1977. before the formation of the National Toxicology Program (NTP), the National Institute of Environmental Health Sciences (NIEHS) initiated a genetic toxicology testing program to test chemicals of interest to the NIEHS, and later to the NTP and other agencies. An important part of this program was the testing of coded chemicals in standardized protocols. The tests selected for use at that time were the Salmonella/microsome mutagenicity assay (SAL), the induction of chromosome aberrations (ABS) and sister chromatid exchanges (SCE) in Chinese hamster ovary cells, the L5 l78Y mouse lymphoma tk+'- mutation assay (MLA), the Drosophila sex-linked recessive lethal assay, and other in virro and in vivo assays. The chemicals were tested in a number of laboratories under contract. Table I shows the progress of testing in some of the tests used. Other tests have been used to a lesser extent and are not presented here. Many of the test
TABLE I NTP Test Systems and Chemicals Tested" Test system and endpoint Salmonella mutagenicity I n virro chromosome aberrations (CHO cells) I n virro sister chromatid exchanges (CHO cells)
Mouse lymphoma cell mutagenicity Drosophila sex-linked recessive lethal test Drosophila reciprocal translocations Mouse bone marrow chromosome aberrations Mouse bone marrow sister chromatid exchanges
Chemicals tested
Samples tested'
1,566 567 567 35 1 255 62 I10 I10
2,064 62 I 62 I 445 324 78 I I5 115
aAs of November, 1988. 'Reflects repeat tests of chemicals under different codes in the same or different laboratories.
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GENETIC TOXICITY TESTS
767
results for the specific chemicals have been published or are in various stages of publication. They will all be published. Testing schemes using a number of STT have been proposed or enacted by various organizations. These schemes have included tier systems and the more widely used approach, the test battery. The results obtained at the various stages of these schemes dictate the future testing priorities for the chemical or, in some cases, whether the chemical will be considered for additional testing or development. These testing schemes were generally assembled according to early reports of the utility or advantages of the various assay systems, and what can be called “scientific intuition.” According to this intuition, different STT are needed to measure different genetic endpoints, and results from all endpoints are needed in order to make informed decisions regarding the potential carcinogenicity of the test chemical. Accordingly, test batteries assembled by regulatory agencies and other organizations included, as a rule, the Salmonella test, an in v i m chromosome aberration test, an in vivo chromosome aberration test, a gene mutation test in mammalian cells, and, because it was genetically well defined, a Drosophila sex-linked recessive lethal test. This was also the rationale behind the initial selection of test systems for the NIEHS testing program. Evaluations of the NTP STT database to determine the usefulness of these tests were begun in the early 1980s [2,3]. In 1984, a study to evaluate the effectiveness of four widely used STT (SAL, ABS, SCE. and MLA) for discriminating between carcinogens and noncarcinogens was initiated. The first segment of that study, comprising 73 chemicals, has been published 141. Additional chemicals are being evaluated. At the same time, the NTP started looking at the performances of the Drosophila sex-linked recessive lethal assay and in vivo cytogenetics assays with the same group of chemicals. These parts of the study are in progress. The results presented here summarize the results of the studies using SAL, ABS, SCE, and MLA on the initial 73 chemicals.
11. METHODS
The STT and protocols used in the evaluation study are listed in Table 2. All chemicals were tested under code in laboratories under contract to the NTI? The test chemicals were coded at the NTP Chemical Repository, which also sent them to the laboratories and decoded them after the results had been reviewed and evaluated. In a number of cases, the same chemical
ZEIGER
768
TABLE 2 Short-Term Test Systems Used
Downloaded by [NUS National University of Singapore] at 10:18 06 November 2015
Salmonella typhimurium mutation (Ames test)
(SAL) [5,61 Chromosome aberrations in Chinese hamster ovary cells (ABS) [7] Sister chromatid exchanges in Chinese hamster ovary cells (SCE) 171 Mouse lymphoma L5178Y tk+’- mutation assay (MLN [81 was tested in more than one laboratory, or in the same laboratory at different times, to monitor the performance of the laboratories or to clarify a weak or equivocal response. When a chemical was tested more than once, different code numbers were used. The carcinogenicity tests were performed in male and female F344 rats and B6C3F1 mice, using 50 animals per group, treated for 2 years with solvent, a maximum tolerated dose, and one or two intermediate doses. The carcinogenesis evaluations were taken from the NTP Technical Reports for the particular chemicals. All equivocal responses in the carcinogenicity tests and STT were considered negative for the purposes of this evaluation. The following definitions were used for the evaluation of STT performance: sensitivity is the proportion of carcinogens that are positive in a test; specificity is the proportion of noncarcinogens that are not positive in a test; positive predictivity is the proportion of positives in a test that are carcinogens; negative predictivity is the proportion of negatives in a test that are noncarcinogens; concordance is the overall proportion of “correct” responses in the STT; and prevalence is the proportion of carcinogens in the population.
111. RESULTS AND DISCUSSION
It was anticipated, based on earlier studies (Table 3A) with the Salmonella test, that the test would correctly identify approximately 80-90% of the carcinogens and noncarcinogens. The SAL results derived from the NTP chemicals did not support this expectation (Tables 3B, 4). The concordance between SAL mutagenicity and rodent carcinogenicity in the NTP database was 62%. There are a number of explanations for this difference, the primary one being that the chemicals tested by the NTP belonged, for
GENETIC TOXICITY TESTS
769
TABLE 3
Downloaded by [NUS National University of Singapore] at 10:18 06 November 2015
Historical Development of SAL Validation Studies No. chemicals
Prev.
Sens.
223 146 53 I20 112
.72 .67 .70 .48 .93
.88 .92 .68 .91 .72
87 60 224 73
.74 .55 .55
.47 .76 .54
.60
.45
Spec.
Conc .
(+I
(-1
Pred.
Pred .
A .75 .90 .77 .89 .88 .93 .94 .93 .63 .96 B [NTP studies] .70 .81 .59 .69 .70 .69 .86 .83
Ref.
.7 1 .82 .50 .92 .I5
.84 .87 .74 .93 .71
.32 .67
.53
14
.68
15
.55 .5 1
.6 I .62
3 4
9 10
II 12 13
Prev. = prevalence; Sens. = sensitivity; Spec = specificity; ( + ) Pred. = positive predictivity; (-1 Pred. = negative predictivity; Conc. = concordance (see text for definitions).
the most part, to different chemical classes than those tested earlier. It has been shown [3,16] that some classes of chemical carcinogens are efficiently detected by SAL (alkylating agents, nitroso compounds, nitroaromatics), whereas others (chlorinated hydrocarbons, hormones) are not. The carcinogenic responses in rodents also vary according to chemical class. This will be addressed in more detail below. The ability of SAL, ABS, SCE, and MLA to detect carcinogens (sensitivity) and discriminate between carcinogens and noncarcinogens is presented in Table 4. SAL and ABS had a low sensitivity and high specificity,
TABLE 4 Performance of Short-Term Tests with 73 Chemicals [4]
Sensitivity (%) Specificity (%) Positive predictivity (%) Negative predictivity (%) Concordance (%) Prevalence = 60%
SAL
ABS
SCE
MLA
45 86 83 51 62
55 69 73 50 60
73 45 67 52 62
70 45 66 50 60
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and SCE and MLA had a high sensitivity and low specificity. SAL had the highest positive predictivity. However, the overall concordances for all four STT were equivalent (60-62%). SCE and MLA detected the most carcinogens, but their false positive rates (the proportion of noncarcinogens that were positive) were also the highest. Therefore, among these 4 STT, SAL would appear to perform the best (highest concordance) in a population of chemicals containing few carcinogens, and SCE and MLA would appear to perform best when there is a high proportion of carcinogens in the population. In order for a battery of tests to be more effective than a single test for predicting carcinogenicity and noncarcinogenicity. the tests in the battery must complement each other. That is, the other tests should correctly identify the carcinogens missed by the original test without misidentifying noncarcinogens. None of the STT complements SAL; they do not distinguish between a SAL-negative carcinogen and a SAL-negative noncarcinogen (Table 5 ) . This lack of complementarity extends to all 4 STT (data not shown). However, the four tests are confirmatory; a positive result in one test is likely to be confirmed in the other tests (data not shown). This is not surprising, because the tests measure mutagenesis and chromosome damage, not carcinogenesis. Therefore, a mutagen and chromosome-damaging agent would be expected to be active in all tests designed to measure these endpoints. The classes of chemicals studied affect the responses obtained in carcinogenicity and genetic toxicity tests. A recent report by Ashby and Tennant [ 171 divided test chemicals into two classes-those containing “structural alerts,” that is, substructures within the molecules that are capable of reacting with DNA, and those that do not contain DNA-reactive sites. Chemicals containing structural alerts are more likely to be carcinogens, tend to be carcinogenic in both rats and mice, produce tumors at multiple organ sites, and are mutagenic in Salmonella. Conversely, carcinogens without structural alerts tend to produce tumors in a single species, usually at a single site, and are not Salmonella mutagens. Tumors induced by structur-
TABLE 5 Responses in the ABS, SCE, and MLA Tests of Chemicals Negative in SAL [4] Carcinogenicity
+ -
+ 12 10
ABS -
+
23 34
20 23
SCE -
+
15 21
17 25
MLA -
18 19
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ally alerting chemicals can appear at all organ sites, whereas certain organ sites (e.g., lung; skin; Zymbal’s gland) appear to be unaffected by chemicals lacking structural alerts [ 171. Results with the 73 NTP chemicals are consistent with Ashby and Tennant’s findings (Table 6). The high correlation between the presence of structural alerts and mutagenicity is reflected in the increased sensitivities and concordances of the STT. Among these STT, SAL is the most responsive to the structural alerts; when this endpoint is used in lieu of carcinogenicity, all the measurements of association increase, and the overall concordance is 87%. This is similar to the concordances between carcinogenicity and mutagenicity reported in earlier articles (see Table 3A), where the majority of carcinogens tested contained these structural alerts. In a battery testing approach, a chemical is tested in a series of test systems designed to encompass a range of genetically relevant endpoints. In some test batteries it is necessary for the chemical to be positive in only one of the STT; in others, a positive result is required in more than one, or all, STT. Often, the results obtained at the various stages of these schemes dictate whether a chemical is assumed to be a potential carcinogen, and therefore a candidate for a lifetime carcinogenicity study in rodents, or a potential noncarcinogen and therefore given a lower priority for rodent carcinogenicity assays. One premise behind the use of test batteries is that the tests will complement each other and that combinations of tests will be more effective for detecting carcinogens than individual tests. The fact that this will not occur with the four STT studied here is reflected in the absence of complementarity among the tests (Table 5 ) . When the four tests are assembled into batteries, their positive predictivity for carcinogenicity does not improve over that for SAL, alone. SAL‘s positive predictivity is .83 and no combination of positive responses produces a greater value (Table 7). In addition, if only SAL is used, 20 carcinogens are correctly identified; the other test
TABLE 6 Short-Term Test Responses to Structural AlertsP SAL
ABS
SCE
MLA
75 95 91 85 87
75 14
89 49 53 88 65
93 60 60 93 73
~~
Sensitivity (%) Specificity (%) Positive predictivity (%) Negative predictivity (%) Concordance (%)
64 82 75
‘Based on 71 chemicals. Structural alerts as defined by Ref. 17.
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TABLE 7 Predictivity of SAL Results
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Predictivity For carcinogenicity: SAL positive SAL and at least one other STT positive SAL and at least two other STT positive SAL negative and at least one other STT positive SAL negative and at least two other STT positive All 4 STT positive For noncarcinogenicity: SAL negative SAL and at least one other STT negative SAL and at least two other STT negative All 4 STT negative
No. chems.a
.83
20
.83
19
.83
19
.55
18
.74 .82
12 14
.51
25
.53
21
.54 .63
14 10
“Correctly identified. combinations identify from 12 to 19. Conversely, a negative response in SAL predicts noncarcinogenicity 51% of the time, whereas a negative in all four STT has a predictivity of .63. However, only 10 noncarcinogens were negative in all four STT, whereas 25 noncarcinogens were negative in SAL. STT are recommended or rejected based on their ability to distinguish between carcinogens and noncarcinogens. The ideal STT to use as a routine screen would be one whose positive and negative predictivities approached 1 0 % and few, if any, false positives would be produced. None of the STT used here fit this requirement. The SAL positive predictivity of 83% suggests that few false positive responses will be produced as compared to the other STT. However, all four STT have similar negative predictivities (5052%). which means that a negative response in any one of the four STT, by itself, has no probative value for noncarcinogenicity. These results confirm the strong interrelationships among mutagenicity, DNA-reactive activity, and rodent carcinogenicity. Unfortunately for those who believe that STT will detect the great majority of carcinogens, and
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distinguish carcinogens from noncarcinogens, there is a high proportion of chemicals that are not mutagenic (in SAL) and whose chemical structures give no indication of potential DNA reactivity. We know that many of these chemicals are rodent carcinogens but we do not know their human carcinogenicity. However, for human health considerations, we must presume that they also have the potential to induce tumors in humans. A current major research goal is to develop STT that can identify these nonmutagenic, nonstructurally alerting carcinogens. On the basis of the results presented here, it is evident that a positive response in more than one test system does not strengthen the value of the original positive, and a positive response in SAL is not negated by negative results in the other tests. Combinations of these four tests do not improve upon the effectiveness of the individual tests, and none of these tests are complementary. These data, and the false-positive frequencies obtained, do not support the use of ABS, SCE, or MLA for the routine screening of chemicals for presumptive carcinogenicity. A positive result in SAL is sufficient for a prediction of carcinogenicity; positive or negative results in the other STT studied here do not change this prediction. We are currently examining the ability of mouse bone marrow cytogenetics analyses to identify carcinogens that are not positive in SAL, and also to discriminate between carcinogens and noncarcinogens within the population of chemicals that are mutagenic in Salmonella.
REFERENCES [ l ] Hollstein, M., McCann, J., Angelosanto, F. A., Nichols, W. W., Short-term tests for carcinogens and mutagens, Murat. Res.. 65, 133226 (1979). [2] Zeiger, E., Tennant, R. W.,Mutagenesis, clastogenesis, carcinogenesis: Expectations, correlations and relations, in Genetic Toxicology of Environmental Chemicals, Part B: Genetic Effects and Applied Mutagenesis (Ramel, C., Lambert, B., Magnussen, J., eds.), Alan R. Liss, Inc., New York, 1986, pp. 75-84. [3] Zeiger, E., Carcinogenicity of mutagens: Predictive capability of the Salmonella mutagenesis assay for rodent carcinogenicity, Cancer Res., 47, 1287-1296 (1987). [4] Tennant, R. W., Margolin, B. H.,Shelby, M. D., Zeiger, E., Haseman, J. K., Spalding, J., Caspary, W., Resnick, M., Stasiewicz, S., Anderson, B., Minor, R., Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays, Science, 236, 933-941 (1987).
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[5] Haworth, S . , Lawlor, T., Mortelmans, K., Speck, W., Zeiger, E., Salmonella mutagenicity test results for 250 chemicals. Environ. Muragen., S(Suppl I), 3-142 (1983). (61 Mortelmans, K., Haworth, S., Lawlor. T., Speck, W., Tainer, B., Zeiger, E., Salmonella mutagenicity tests. 11. Results from the testing of 270 chemicals, Environ. Muragen.. 8(Suppl 71, 1-119 (1986). 171 Galloway, S. M., Bloom, A. D., Resnick, M., Margolin, B. H., Nakamura. F., Archer, F?, Zeiger, E., Development of a standard protocol for in virro cytogenetic testing with CHO cells: Comparison of results for 22 compounds in two laboratories, Environ. Muragen., 7, 1-52 (1985). (81 Myhr, B., Bowers, L., Caspary, W., Assays for the induction of gene mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in culture, Prog. Mutar. Res., 5 , 555-568 (1985). (91 McCann, J., Choi, E., Yamasaki, E., Ames, B. N., Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals, Proc. Narl. Acad. Sci. USA, 72, 5135-5139 (1975). [lo] Sugimura, T., Sato, S . , Nagao, M., Yahagi, T., Matsushima, T., Seino, Y., Takeuchi, M.,Kawachi, T., Overlapping of carcinogens and mutagens, in Fundamentals of Cancer Prevention (Magee, F! N., Takayama, S . , Sugimura. T., Matsushima, T., eds.), University Park Press, Baltimore, 1976, pp. 191-215. [ I l l Heddle, J. A., Bruce, W. R., Comparison of tests for mutagenicity or carcinogenicity using assays for sperm abnormalities, formation of micronuclei, and mutations in Salmonella, in Origins of Human Cuncer, Book C, (Hiatt, H. H., Watson, J. D., Winsten, J. A., eds.), Cold Spring Harbor Laboratory, 1977, pp. 1549-1557. [I21 Purchase, I. F. H.. Longstaff, E., Ashby, J., Styles, J. A., Anderson, D., Lefevre, I? A., Westwood, F. R., An evaluation of 6 short-term tests for detecting organic chemical carcinogens, Br. J. Cancer, 37, 873-959 (1978). [ 131 Bartsch. H., Malaveille, C., Camus, A-M., Martel-Planche, G., Brun, G., Hautefeuille, A., Sabadie, N., Barbin, A., Kuroki, T., Drevon, C., Piccoli, C., Montesano, R., Validation and comparative studies on 180 chemicals with S. typhimurium strains and V79 Chinese hamster cells in the presence of various metabolizing systems, Mutat. Res., 76, 1-50 (1980). (141 Zeiger, E., Knowledge gained from the testing of large numbers of chemicals in a multi-laboratory, multi-system mutagenicity testing program, in Environmenral Muragens and Carcinogens (Sugimura, T., Kondo, S., Takebe, H., eds.), Univ. of Tokyo Press, Tokyo/ A. R. Liss, Inc., New York, 1982, pp. 337-344.
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[15] Dunkel, V. C., Zeiger, E., Brusick, D., McCoy, E., McGregor, D., Mortelmans, K., Rosenkranz, H. S., Simmon, V. F., Reproducibility of microbial mutagenicity assays: 11. Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli. Environ. Muragen., 7(Suppl 5 ) . 1-248 (1985). [16] Rinkus, S. J., Legator, M. S., Chemical characterization of 465 known or suspected carcinogens and their correlation with mutagenic activity in the Salmonella ryphimurium system, Cancer Res., 39, 3289-3318 (1979). [17] Ashby, J., Tennant, R. W., Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NCVNTP. Murar. Res., 204, 17- I15 (1988).
