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2 THE MUTAGENICITY OF CARCINOGENIC COMPOUND S THE SALMONELLA/MI CROSOtIE: TE ST The work of Ames and colleagues}, 71, 73 has led to the development of microbial mutation assays to detect chemical carcinogens. Originally, analyses with the Salmonella/- microsome test were inductive; known animal carcinogens were tested for mutagenic activity for validation purposes. However, testing strategies have evolved, and short-term mutagenicity tests are now used to screen for chemicals with unknown carcinogenic properties. The Committee84 has reviewed the large number of mutagenicity tests that constitute a test strategy. The organisms in these tests span the phylo- genetic range from bacteria, such as salmonellae, to whole rodents. McCann and Ames72 have argued that most carcinogens are mutagens and that muta~enicitv screening tests would . . . ~ , . . .. . .. . ~ identity many chemical carcinogens without the need, an many cases, for expensive, lifetime rodent bioassays. Evidence presented in this section suggests a qualitative relationship between the mutagenic and carcinogenic properties of most classes of chemicals. What has been challenged is the degree of concordance between the processes and what causes this qualitative correlation to break down. McCann70 has discussed the use of short-term tests in relation to chemical carcinogenes is and cancer policy. She argued that short-term tests are valid experimental predictors of chemical carcin' genesis for four reasons: (~) although no test detects 811 care inogens, most carcinogens are detected in a relatively few mutagenicity tests; (2) although some carcinogens are not detected in any test, a combination of tests increases the proportion of carcinogens that are detected; (3) positive results from the Salmonella/microsome test are usually con- firmed by other tests, as are negative results and results witch weakly act ive chemicals o f ten conf irmed; and (4 ~ the percentage 8

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of true noncarcinogens that are falsely positive in short-term tests is likely to be lower than validation studies suggest. McCann and Ames72 asserted that approximately 90: of chemical carcinogens would be mutagenic in the Salmonelia/- micrOsOme assay and that most noncarcinogens would not be mutagenic. Despite the need for more data to substantiate the extent of the correlation, it is generally accepted, and the Committee84 concurs in the probable validity of these es timate s . In an exchange of views, Rinkus and Legatorl02, 103 and Ames and McCann6 discussed the success rate of the Salmonella/microsome test in detecting carcinogens. Rinkus and Legator contended that about 77: of carcinogens that had been tested in the assay were detected as mutagens, according to data available in 1979, and Ames and McCann estimated this value as slightly higher, 322. Most reviews e.g., Purchase et a1.,99 Brusick,3 Nagao et al.,82 and this Committees --have favored the c laim that approximately 80-90% of care ino- gens are mutagens, although such support is guarded by the perpetual call for more data. Ames and Ilaroun3 have also considered the criticisms of Lijinsky and colleagues8~l concerning the accuracy of the Salmonella/microsome test in detecting carcinogens. They reported that 80: of the carcinogenic nitrosamines and 58% of the carcinogenic hydrocarbons were detected in the assay. These f igures were rebut ted by Ames and Haroun largely on the basis of inadequacy of carcinogenicity data and were recal- culated to suggest that {383 of the carcinogenic polycyclic hydrocarbons were mutagens. Several potent carcinogens in both chemical groups were nonmutagenic in the test (i.e., false- negative s) . The disagreement involves several points. One point of contention is the quality of the cancer data. Often, the data used are from experiments that do not conform to guidelines, such as those suggested by the International Agency for Research on Cancer. 58 Ames and McCann6 discussed how the inclusion of questionable carcinogenicity data may influence the interpretation of mutagenicity testing. The accuracy and quantitation of carcinogenicity data are dealt with separately in Chapter 3. Analysis of chemicals by class influences the extent to which carcinogenicity and Salmonella/microsome test data are related e For example, Rinkus and Legatorl02 divided the 465 chemical s in their compendium into 38 classes . Some classes were constructed according to structural similarities, and others, such as the antifolates, were based on functional properties. Classes of carcinogens poorly detected in the Salmonella/microsome test included azonap~thols; carbonyis; phenyls; polychiorinated aromatics, cyclice, and aliphatics; and steroids. Ames and McCann6 have suggested that the 9

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classification scheme of Rinkus and Legator is poorly con- structed and its interpretation therefore misleading. However, hey encouraged attempts in general to categorize chemicals and suggested that an optimal scheme would require knowledge of the structure of ~ carcinogen, the enzyme system needed to transform it, and the structure and action of the proximate active forms. For strictly qualitative purposes, a short-te~m test battery of mutagenicity tests offers the best chance of detecting most carcinogens. Campbell35 suggested that the combination of the Salmonella/microsome test and a mammalian cell transformation test could theoretically detect 99.191 of carcinogens--a much higher detection rate than reported for any combination of tests by de Serres and Ashby.43 RECENT COMPARATIVE STUDIES - Several large, recent s tudies have examined the relation- ship between mutagenicity and carcinogenicity in a laboratory context. It is important to describe the new studies in detail and then to review earlier experimental data from which they were derived. Committee 2 of the International Commission for Protection against Environmental Mutagens and Carcinogens (ICPEMC) recently published its 3-year analysis of mutagenicity testing as an approach to carcinogenicity testing. 59 This group concluded that "the uncertainty about the mechanisms by which chemica 1 carcinogens induce cancer precludes the precise selection of predictive tests on the basis of similarities of mechanisms between the test system and the mammalian model of carcinogenesis." Committee 2 cautioned that the expansion of a test battery will produce an increase in false-positive results and thus necessitate the comparison of in vitro data with results of more protracted animal tests, which themselves are uncertain in predicting cancer in man. Because of the apparent complexity of carcinogenesis and the disparate modes of meta- bolic activation used in in vi-fro mutagenicity tests, the quantitative relationship between the two processes was con- idered very uncertain. As a member of the ICPEMC committee, Purchase97 defined three stages of validation for short-term tests in predicting carcinogenicity. Fly one "established" test was identified-- the Salmonella/microsome test. Nine "developed" tests were named, and 10 tests were described as "developing." Validation s tudies have suggested that the ability to predict carcinoma genicity will not exceed 90: for any test. Thus, a single mutagenicity test was thought to be inadequate for judging the potential carcinogenicity of a chemical. In another context, the ability of a single short-term mutagenicity test to predict 10

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clearly the activity of a chemical in other biologic systems was likewise questioned by the present Committee. 4 This lack of confidence in a single test result was extended in a quant itative sense . The Committee also concluded that the mutagenic potency of chemicals in one mutagenicity test cannot be directly compared with potency determined in other tests. Both as an ICPEMC member78 and independently, 79 Mohn discussed the use of bacterial tests for the ranking of chemical mutagens and the use of the tests as Screens for c arc inogenicity. Al though advocating the ranking of chemicals in a well-calibrated test, he discouraged attempts at quantification, because of the specificities of genetic end points to chemical classes and because ranking chemicals in bacterial strains with altered DNA repair or metabolism may be poor estimators of potency in wild-type organisms. Mohn described many carcinogens that are mutagenic in most test systems and those which react in only a few mutagenicity assays. He also listed a few animal and human carcinogens that appear to possess no mutagenic activity, such as dieldrin' saccharin, benzene, cadmium, carbon tetrachloride, and diethylstilbestrol. Venittll7 expressed similar doubts about the use of bacterial mutation as a quantitative indicator of carcino- genicity. Like others, he viewed imperfections in the short- term mutagenicity and long~term carcinogenicity data as barriers to establishing a strong conclusion Concerning the correlation. Several years earlier, Bartsch1 reviewed the ability to predict carcinogenicity from mutagenicity data. He, too, considered mutagenicity tests strictly as qualitative screens for potential care inogenicity. His demonstration of the variability of metabolic activation systems in several human liver extracts argued for a parallel between the vari- ability in the tests and the heterogeneity of the human population in susceptibility to particular chemical carcin' gens. Because of the variability in metabolic activation systems and the heterogeneity in cancer susceptibility, he did not consider it feasible at the time to correlate mutagenic and carcinogenic potencies across species. Two large experimental efforts have recently sought to validate short-term mutagenicity tests and explore their mutagenicity-carcinogenicity relationship. de Serres and Ashby43 directed an international ~ tudy designed to assess the effectiveness of short-term tests in detecting carcinogens and noncarc inogene as de fined by their activity in rodent systems. Forty-two chemicale--14 carcinogen~oncarcinogen pairs, Il carcinogens normally inactive in bacterialmutation as says, and thre e mis ce l laneous compound s-~were tee ted by 6 5 collaborators with 35 assays. The pairs of chemicals usually consisted of a well-established carcinogen and a noncarcinogen of similar chemical structure on which data were less sub- 11

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stantial. No assay gave positive results for all carcinogens and negative results for all noncarcinogens. However, a battery of tests was able to detect all carcinogens and all noncarcinogens. The tents were in five categories: bacterial mutation ssesys, yeast assays, in vitro mammalian cell assays, cytogenetic assays, and in Biro mammalian assays. The two most serious considerations in deriving tes t batteries were decreasing false-positive and false~negative results and creating maximal complementarily among tests. For example, mos ~ o f the care inogens f ound to be inac t ive in the Salmonel la/microsome assay were pos iodine in at leas t one in vitro eukaryotic system. The detail in this large study makes its total review difficult. However, from the data generated in the non- definitive report, a conclusion was reached concerning muta- genic and carcinogenic potencies; because of the inadequacy of the estimates of carcinogenic potency, any correlation was thought to have little general value. Some contradictions are evident in the preponderance of positive mutagenicit~test results versus negative carcinogenicity data. Of the 17 materials originally classified as noncarcinogens on the basis of negative animal carcinogenicity data, eight were reclassified as "true noncarcinogens" according to more extensive mutagenicity testing. False~positive or false- negative mutagenicity results are relative to their agreement with available animal carcinogenicity data, which appear to have a tendency toward fals~negatives. The second recent collaborative study comparing muta- genic ity and care inogenicity results was sponsored by the United Kingdom Environmenta 1 Mutagen Society. 90 It was conducted to supplement several aspects of the de Serres-Ashby international study, especially the development of quantitative data. Three chemicals were examined for mutagenicity and carcinogenicity: benzy~chloride (BC), 4-chioromethylbipherlyl (4CHB), and 4-hydroxymethylbiphenyl (4HMB). (A serious problem that developed in the course of the study was the unexpected instability of 4CMB.) Bacterial mutagenicity was determined in 17 laboratories; fungal mutagenicity in seven; mammalian cell mutagenesis, transformation, and DNA repair in 14; cytogenetic analysis in 11; in viva mutagenicity in 10; and mouse skin tumorigenesis in two. In most, all three chemicals were tested. The analysis of bacterial mutagenicity in Salmonella and E. c_ indicated rather wide differences among laboratories. All laboratorie s correct ly c las s if f ed 4HMB a s a nonmutagen, and a identified 4CHB as a mutagen. The results with BC were less consistent, because it appeared as a 'tweak" mutagen that acted at restricted sites. . The study grouping concluded that, although it is extremely unlikely that any useful relationship could be identified between mutagenic and carcinogenic potencies, the analysis of mutagenic potency among related , ~

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chemicals that were tested with the same bacterial ~ trains may provide useful quantitative information. The present Committee reached the same conclusion in its earlier report.84 The use of mammalian cells in culture (to study either mutation, transformation, or DNA repair) for 4CMB gave the greatest number of positive results: 17 of 26 tests were positive. Of 16 mammalian tests, only two (both measuring cellular transformation) were positive for 4HMB, and eight tests were positive for BC. The ma~alian cell study group could give no quantitative interpretation of the data. The sex-linked recessive lethal test in Drosophila was positive for 4CMB, BC, and probably 4HMB. All three chemicals gave positive results in the Drosophila somatic cell segregation assay. In contrast, the in viva mouse mutagenicity assays (micronucleus and dominant-lethal tests) gave no evidence of genotoxicity for any chemical. The mouse skin tumor tes t (conducted in two laboratorie s) failed to detect any neoplastic changes for any of the materials. An important genera 1 conclus ion that can be drawn from the UK ~ tudy is that, for the three chemicals tested, it was not possible to correlate the data quantitatively among the muta- genicity tests. Indeed, for the most consistent data (bacterial), the UK group recommended that potency be calcu- lated on the basis of the Salmonella/microsome test--and then only for specif ic bacterial strains . The carcinogenicity results were considered inadequate for deciding whether the consistent negative results accurately described ache activity of the compounds. Therefore, because of a general inconsis- tency in the mutagenicity results and a lack of confidence in the care inogenicity data, no correlation could be drawn between mutagenic and carcinogenic activities . As concluded by the Committee in the summary, the results of these large, recent analyses indicate a firm qualitative relationship between the two end points. Further s tudy is required to establish a quantitative correlation. EARLIER QUALITATIVE STUDIES Some perspective on efforts to determine the relationship between carcinogenicity and mutagenicity may be afforded by discussing a set of relevant reports in roughly chronologic order, not only to describe the evolution of an emerging consensus, but also to show how improved data may influence the result. Coombs et al. 41 in 1976 analyzed 54 polycyclic compounds, of which 29 were cyclopentata~phenanthrenes, 11 chrysenes, and 14 benz[~]anthracenes. Carcinogenic potency was described as the ratio of the incidence of skin tumors to latent period. Mutagenicity data were from the Salmonella/microsome test and were calculated from the elope of the linear portion of the 13

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dose~response curve. All 37 carcinogens were mutagenic, as were seven of 16 noncarc inogens. Despite the number of false-positive results, the authors were impressed with the qualitative correlation for these compounds. However, they concluded that their study did not show a quantitative relationship and questioned whether one could ever be demonstrated. This conclusion was also reached in a more recent analysis.52 In 1977, a multi-end-point studyl9 ~ imilar to that of Coombs et al . was pert armed with N-hydroxy- 2-aminof Luorene and other derivatives and the Salmonel la/- microsome test . lathe qual itative correspondence among electrophilicity, mutagenicity, DNA-repair induction, and care inogenicity (sarcomas following subcutaneous injection) was generally high. 2-N-Myristoyloxy derivatives lacked mutagenic activity, but had the other activities. Quantitatively, all the compounds differed markedly with respect to the four test end po int ~ . Bartsch and colleagues have conducted a series of studies on comparative mutagenicity. In 19Bo,18 they compared the carcinogenicity and mutagenicity (in Salmonella and CHO cells) of 180 chemicals. Over 90: of the chemical carcinogens were mutagenic in both tests. However, no quantitative correlation was found between mutagenic and carcinogenic activities of these chemicals. In a second study, Bartsch et al.20 com- pared hydrocarbon-derived barregion dihydrodiols for bacterial mutagenicity, Ibal 1 indexes (tumor incidence and latency) for carcinogenicity, and DNA-binding in mouse skin. The mutageni- cities of the dihydrodiols and the carcinogenicities of the hydrocarbons from which they were derived were much more closely associated than the mutagenicities of the hydrocarbons themselves and their carcinogenicities. The lack of a quantitative correlation between the mutagenicities of the polycyclic hydrocarbons and their carcinogenicities conf irms results with dibenzanthracenes73 and with cyclopenta- phenanthrenes.41 In 1979, Glatt et a1.49 examined 43 heterocyclic compounds for a mutagenicitrcarcinogenicity relationship. Mutagenicity was determined in the Salmonella/microsome test, and care inogenicity by subcutaneous injection of the compounds into mice . Of the 18 chemical 8 that produced tumors, one ~ a weak carcinogen) was not mutagenic in Salmonella. Of the 25 noncarcinogens, 13 were mutagenic to the same extent as the carcinogens. No quantitative correlation was reported. In 197B, Purchase and colleaguesl determined how well six short-term tests detected carcinogenicity of 120 organic chemica 18: 5 ~ ca rc inogens and 6 2 noncarc inogens from severe functional and structural classes, including polycyclice, alkylating agents, and aromatic amines. Compounds that were negative in studies that extended over most of the animals' lif e span were class if fed as noncarcinogens . The Salmonella/- 14

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microsome test correctly detected 91: of the carcinogens and 94% of the noncarc inogens. Approximately 942 of each type of compound were accurately detected in the cell-traneformation test. No attempt was made to determine potency. In 197 9, Poirier and de Serres93 described the results of an appraisal of mutagenicity screening tests for carcinoma genicity, which the National Cancer Institute (NCI )94 participated in and which involved ache comparison of data from several sources. Approximately 150 chemicals were surveyed, including aromatic amines, polycyclic hydrocarbons, nitros- amines and nitrosamides, alkylating agents, and other classe s. Each chemical had been classified as a procarcinogen, ultimate carcinogen, or noncarcinogen before to the beginning of the appraisal. Because between 25 and 40: of noncarcinogens in the NCI group were mutagenic, the collaborative group concluded that the Salmonella/microsome test, or any test, could not be used in isolation. The combination of the Salmonella/microsome test and one using an E. cold pal A-deficient strain detected 92: of the organic carcinogens in the NCI list. About 26: of the noncarc inogens were mutagenic in the two tes ts. The quality of the mutagenicity and carcinogenicity data was considered questionable, and no quantitative analysis was per- formed . Another review30 at the time surveyed a phylogenetically diverse set of mutagenicity tests for 54 carcinogens and noncarc inogens. Positive results were obtained with 100: ~17 /17 ~ of the ultimate carcinogens, 96: (27/28) of the procarcinogens, and 67: (6/9) of the noncarcinogens in at least one tes t . The Salmonel la/microsome assay detected 84% (37/44 of the carcinogens, but also found 25: (2/8) of the noncarcinogens to be pos itive . The Drosophila systems gave positive results in 90: (19/21) of the carcinogens and 50: (3/6) of noncarcinogens. No quantitative evaluation was pert armed. Although not strictly a genetic end point, DNA repair has also been used in correlations of carcinogenicity and mutagenicity. Rosenkranz and Poirierl04 compared the DNA~modifying ac tivities (E. cold DNA polymerase deficiency) and mutagenic activities (Salmonella/microsome assay) of 99 chemicals. The compounds were in eight chemical classes; 21 were noncarcinogens, 21 were ultimate carcinogens, 45 were procarcinogens, and the remainder could not be classified accurately. All ultimate carcinogens modified DNA, as assayed by growth inhibition of E. cold pot A-, and 79: were mutagenic; 52: of the procarcinogens were mutagenic and 67: modified DNA; and 39% of the noncarcinogens were mutagenic and 30: modif fed DNA. The noncarc inogens tested included aniline, anthracene, diphenylnitrosamine, and 5-bromo-2 '-deoxyuridine. The high proport ion of fals~positive results precluded quantitative analys i s . 15

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Brambilla and coworker studied the correlation of DNA damage with mutagenicity and carcinogenicity of N- .~itroso compounds. As calculated by regression analysis of logic values of the two activities, correlation was positive between carcinogenic and ANAL difvinP n~tiviti-~ hat hi ah 1 nor statistical significance (0.10 ~ ~ ~ 0.15) . , ~ _, ~ _ DNA damage was assayed by alkaline elusion of liver DNA from rats exposed in vivo. The mutagenic potency calculated from Salmonella/- microsome data correlated negatively with carcinogenic potency. Carcinogenic potency was defined by the formula, lOO(Kt - Kc)/(millimoles per kilogram per day), where K is a measure of transforming potency of the cellular environment, t is the exposure duration, and c is the control rate. For the 16 hydrazine derivatives, there was a positive correlation be tween DNA damage and ca rc inogenes i a, bu t no corre let ion between mutagenic and carcinogenic potencies or between mutagenic and DNA-dsmaging activities. In a later study,9b 218 procarcinogens, ultimate carcinogens, and noncarcinogens were compared for their correlation between bacterial mutagenicity and induction of unscheduled DNA synthesis (UDS) in rat hepatocyte~. For 67 compounds, the two systems gave positive results at similar dos e-respons e range s . ~~~ ~ nil ~rner`'l' - or! ' . towever, uos was tar less sensitive to a__ _~__.~, amides, ana other al~cylat~ng agents. Nega t ive results for 88 compounds were registered in both tests; most were noncarc inogens, but about 15-20: were care inogens. Of the 218 chemicals, 48 were positive only in the Salmonella/- microsome test and eight were positive only in the UDS system. 16