(CANCER RESEARCH 39, 3289-331 8, september 1979] 0008-5472/79/0039-0000$02.OO Chemical Characterization of 465 Known or Suspected Carcinogens and Their Correlation with Mutagenic Activity in the Salmonella typhimurium System1

Stephen J. Rinkus2 and Marvin S. Legator

Department of Preventive Medicine and Community Health, Division of Environmental Toxicology, University of Texas Medical Branch, Galveston, Texas 77550

ABSTRACT studieson groupsof carcinogenicand noncarcinogenicchem icals. Actually, the reported correlations between carcinogenic Since chemicals exhibiting mutagenic activity pose a poten ity and mutagenicity in Salmonella range from 63 to 92%. With tial hazard to their users, there is increasing acceptance of the exception of a brief comment by Odashima (172) on struc mutagenicity testing as an integral part of a premarketing ture-activity relationships apparent among false negatives and toxicological evaluation of chemicals. In vitro testing has gained false positives, there has been little indication that certain much notoriety as a quick and relatively inexpensive means to chemical categories of carcinogens are not well detected in assess the mutagenic potential of chemicals. However, the Salmonella. That the higher correlations do not necessarily innovative use of microsomes to simulate metabolism has not translateinto a correspondinglyhigh ability to detect carcino changed the fact that in vitro activation cannot duplicate faith gensfromamonganygroupof chemicalsisillustratedinTables fully the metabolism that occurs in vivo. This shortcoming will 1 and 2. Presented are the Salmonella testing results of 12 express itself by the production of false negatives and possibly pesticides judged positive for tumor induction by an expert false positives during mutagenicity screening. This assertion is panel of scientists reviewing the available studies (62). In this also borne out by a reanalysisof the ability of known animal case, from among a very relevant collection of chemicals, only carcinogens to cause mutations in the generally recognized 4 were mutagenic in Salmonella. Hence, it is important that the premier in vitro system, the Salmonel!a-S-9 system. Although reported correlation studies be seen in their proper perspec previous studies have suggested that a high percentage tive. The purposeof this paper is to presenta perspectiveon (>85%) of all carcinogens will be mutagenic in this system, the value and limitations of in vitro testing in general and with no indication that false negatives are associated with microbialtesting in particular. certain chemical types, these findings are of uncertain practical value due to the limited number of chemical types that were Background considered. An analysis of 465 compounds with known or suspected carcinogenic activity indicates that about 58% have The first 2 studies to examine the correlation between car been adequately tested in Salmonella, that the testing has cinogenicity and mutagenicity were conducted collaboratively concentrated on certain chemical types and has neglected in Japan and the United States. Unlike later studies, the Japa others, and that some categories of carcinogens exhibit mdi nese study did not test all of its chemicals with and without vidual correlations that are unsatisfactorily low by any stan S-9 activation. The American study did not use the Salmonella dard. Poorly detected categories of carcinogens include: azo strains carrying the R-factor, TA100 and TA98 (154); at least naphthols; carbamyls and thiocarbamyls; phenyls; benzodiox in the case of the American study, testing was performed with oles; polychlorinated aromatics, cyclics, and aliphatics; ste uncoded materials. In the Japanese study (172), 17 (63%) of roids; antimetabolites; and symmetrical hydrazmnes.Nonstand 27 carcinogens are detected in Salmonella, if one includes the ard procedures are necessary to optimize the testing of chem updates on N-nitrosodimethylamine and N,N-dimethyl-4-[(3- icals that are bactericidal, that are volatile, or that cross-link methylphenyl)azojbenzamide that were discussed in the text of DNA. False negatives appear to arise for two reasons: an the report. The mutagenicity ofthese 2 carcinogens is depend inability to devise an in vitro activation system that can be ent on metabolicactivation. Presumably,5-9 activation was reliably used in a standard way; and an inability to detect the also not attempted with 4-acetylaminobiphenyl which was reg entire spectrum of mutational events that can lead to the istered as a false negative in the Japanese study. Sugimura et inductionof cancer. al. (226) have shown this carcinogen to be a mutagen in the presence of 5-9. INTRODUCTION While a complete rendering of the American study has not In both scientific and science news publications, statements yet been published, 2 summary reportings (178, 180), which have appeared suggesting that a high percentage, e.g. , 85% do not list the chemicals per Se, indicate that 72% of about 70 or greater,of all chemicalcarcinogenswill be mutagenicinone carcinogens tested are mutagenic in Salmonella. The differ in vitro system, the Salmonella-S-9 system (7) (hereafter re ence in the correlations was thought to be due in part to the ferred to as Salmonella). These statements rely on several differences in the chemicals selected for testing in the 2 studies (172). The Japanese study claimed 12 classes of chemicals 1 This manuscript has been supported by EPA Grant R804621 020; Project and one miscellaneous class versus the 4 classes of chemicals Officer, Dr. J. F. Stara. and one miscellaneous class claimed in its American counter 2 To whom requests for reprints should be addressed. Received July 17, 1978; accepted April 19, 1979. part.

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1Mutagenicity Table Thus, 6 correlation studies have been conducted with Sal (Departmentoffindings previously reported for 6 Mrak Commission monella, but only 4 of these have been adequately reported in Health, Education, andtheSalmonella-S-9 Welfare (62)j carcinogenic pesticides in system1ig/ the literature to date. The lists of carcinogens in these 4 studies S-9 in- Ref. are not mutually exclusive. In fact, 78 carcinogens appear in 2 sourcePositiveBis(2-chloro-Chemical platea TA strains ducer or more of the lists of carcinogens in the 4 reported studies. This redundancy results from a duplication in both the testing NSC 100 (No S-9) 217 of some carcinogens and the reporting of test results for others. ethyl)ether―Negative Statistical Considerations of Correlating AIdrin 100 1535,d1538,100,98 1537, Aroclor 217p,p-DDT@ Let us assume that there is a set of chemicals which are able NS 1535,2171538,100,98 1537, Aroclor to cause cancer in the human species at some level of expo 104149Dieldrin sure. It should be clearly noted that whether the activity of 10,000 1535. 1537, 100, Aroclor 152 some or all of these human carcinogens has a threshold is not d149Heptachlor' 98 essential to the following argument, although it surely has other NS 1535,2171538,100.98 1537, Aroclor important implications. Chart 1 illustrates the overlap between 149Amitrole this set of carcinogens and the set of mutagens, some of which @@ 5,000 1535,15298 1537, 100, Aroclor are defined so, although not necessarily exclusively, from their mutagenicity in Salmonella. The more carcinogens and muta a The highest of several concentrations tested in the first cited source is pre@ented. gens overlap, the more mutagenicity testing as a screen for More mutagenic when assayed in a desiccator or in suspension than when carcinogens becomes appealing. Chart I also illustrates a incorporated into the agar. continuum of correspondences that could be expected. The C NS, not stated. T H. Connor, unpublished data. relationships between carcinogens and mutagens as well as e DDE (a metabolite of DDT) tested up to 5000 @zg/plate also is not active in mutagens and Salmonella could vary independently of each comparable testing (152). , Heptachlor epoxide (a metabolite of heptachlor) also is not mutagenic in a other (only 2 extremes are depicted in Chart 1 for the sake of phenobarbital-induced system, but this testing did not utilize TA100 and TA98 simplicity). In order to predict human carcinogenicity (and, (149). presumably, human mutagenicity) on the basis of results from testing that utilizes only Salmonella, the situation depicted on Four studies have utilized uniformly 5-9, TA100, and TA98 the extreme left of the continuum must be true. Similar Venn in their testing. McCann etal. (151, 152) tested 95 carcinogens diagrams offered by Sugimura et al. (226) depict a historical and combined their results with previously published findings merging of the carcinogens and mutagens but do not provide and unpublished works from other sources for 83 other carcin evidence of an extreme overlapping due to the few chemicals ogens. It was thus seen that 156 (88%) of the 178 carcinogens that were considered. (excluding cigarette smoke condensate) are mutagenic in Sal In Chart 2, the ‘‘true―correlationbetween carcinogenicity monella. Poirier and Simmon (180) have suggested that the and mutagenicity in Salmonella or population percentage, P, difference between the correlations of the American study and can be estimated reliably only in the form of p when the the reports of McCann et al. could be due to the use of the heterogeneity of this set of all carcinogens is representatively sensitive Salmonella strains TA100 and TA98. While it is not reproduced in the sample of carcinogens from which p is known how true this is, another aspect has undoubtedly influ calculated. When such is not observed, the correlation will be enced the outcome. Of the 95 carcinogens actually tested by either over- or underweighted. Equally important for a hetero McCann et al. in their study, 74 (78%) are positive in the Salmonella; this is in comparison to the 82 (99%) successful identifications of the 83 carcinogens for which results were obtained from other published and unpublished studies. Of the 240 chemicals that their group has investigated for mutagenicity in bacterial systems, Sugimura et al. (226) were able to detect 90 (92%) of the 98 carcinogens tested in Salmonella. Heddle and Bruce (104) have reported that 25 (69%) of 36 chemical carcinogens tested in Salmonella were mutagenic. @ This correlation does not include 5-iodo-2'-deoxyuridine which II(ts@ was mistakenlylisted as a carcinogen.3Interestingly,X-rays were shown not to be mutagenic in the system. This contrasts 90'@..OFCARCINOGENSARE SOMECARCINOGENSARE with the previously reported mutagenicity of X-rays in Salmo POSITIVE IN SALMONELLA; POSITIVE IN SALMONELLA; ne/la (3). THE SETSOF CARCINOGENS THE SETSOF CARCINOGENS AND MUTAGENSEXTENSIVELY AND MUTAGENSDO NOT Finally, Purchase et al. (187), in a preliminary report that OVERLAP EXTENSIVELY OVERLAP. also does not list all the chemicals, has obtained a 91% Chart 1. C. set of all human carcinogens; M, set of all mutagens; S, set of all correlation with 58 compounds representing 3 classes and one Salmonella mutagens. In I, many carcinogens are mutagens. In II, a spectrum of miscellaneous class of carcinogens. correspondences between C and M as well as M and S can be defined, only 2 extremes of which are shown. Only in II, left, does the bacterial system qualify as a qualitative test for carcinogens and mutagens In a systematic screening of 3 J. A. Heddle. personal communication. chemicals.

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Table Mutagenicity in Salmonella-S-9 system8of 6 Mrak Commission carcinogenic pesticides (Department(62))R,/R,Without of Health, Education, and Welfare

ToxicityChemical@og/pIateTA liver With liver ho mogenateQsg/plate)Chlorobenzilate1 strainshomogenate @ 150Mirex20, 0, 1005 strains‘-1 —1>3000Strobane1 200, 30005 strains‘-1 60, 1600, 48001 00 ±0.26 1.54 ±0.18 1.10±0.181600Di-allate 982.94 2.03±0.38 (Avadex)―25. 50, 1001535 ±0.29 19.98 ±3.48 ‘-1050N-(2@H@ydroxyethyl)hydrazineC 1000.82 1.06±0.10 839, 1119 ±0.27 5.41 ±0.79 500ControlsCyclophosphamide1PCNB560, 100, 5001535 5 strains2.74 ‘-1 ‘-1>1119

3.64NTeBenzo(a)pyrene51 001 5351 .42 ±0.33 8. 15 ± 2.81NT9-Aminoacridine1 5381 .08 ±0.29 3.96 ± NTNTMethyl 01 537>1 00 NTNTHycanthonemethanesulfonate64721 00>1 0 methanesulfonate1 0986.26 ±1.92 NTNT

a All pesticides were at least 97% pure preparations and were obtained from Chem seMce, West Chester, Pa., except for Strobane which was kindly donated by Tenneco Co., Piscataway, N. J., and N-(2-hydroxyethyl)hydrazine, which was obtained from Pfaltz and Bauer, Inc., Stamford, Conn. Cyclophosphamide was the injectable form obtained from Mead Johnson Laboratories, Evansville, Ind.; benzo(a)pyrene and @ 9-aminoacridine were obtained from Sigma Chemical Co., St. Louis, Mo. methyl methanesulfonate was obtained from Aldrich Chemical Co., Milwaukee, Wis.: hycanthone methanesulfonate was a gift from Dr. Ernest Bueding. All compounds were dissolved in 100% dimethyl sulfoxide except N-(2-hydroxyethyOhydrazine, which was dissolved in distilled water. Testing in the Salmonella-S-9 system utilized the 5 histidine auxotroph strains TA1535. TA1537, TA1538, TA100, and TA98 of S. typhimurium (6, 154). All 5 strains were involved in the testing of the pesticides except N-(2-hydroxyethyl)hydrazine which was tested only with TA1535. Liver homogenate was prepared according to the method of Ames et al. (4). Male Wistar rats were given Aroclor 1254 (Analabs Inc., New Haven, Conn.) in a single i.p. injection at a dosage of 500 mg/kg (200 mg/mI corn oil) 5 days prior to sacrifice. After removal and homogenization. the liver was centrifuged at 9000 x g for 10 mm, and the supernatant (the 5-9 fraction) was stored at —80°. Indicated amounts of each chemical were added to 2.0 ml molten agar at 50°containing approximately 2 x 1o@cellsof the tester strain; controls received 0. 1 ml of the solvent. Liver homogenate mix contained (per ml): 0.30 ml 5-9 fraction; 8 mmol MgCI2;33 mmol KCl; 5 mmol glucose 6-phosphate; 4 mmol NADP; 100 mmol sodium phosphate (pH 7.4). Liver homogenate (0.5 ml of the complete mix) was added to the molten agar as indicated. The mixture was then poured over plates of minimal agar supplemented with biotin (0.5 tog/mI) to which histidine (4 zg/ml) had been added to ensure several divisions of all bacteria. Each concentration of pesticide in an experiment was tested in duplicate. After incubation at 37°for 48 hr. the plates were scored for histidine revertants. Positive findings are presented as R1/R5±S.D., the ratio ± S.D. of the number of revertants on treated plates to the mean number of revertants on control plates of all the experiments for a given chemical at the last cited concentration in the specified strain(s); a dose respons@ was observed with each positive chemical. Negative results in all 5 strains are represented by: ‘-1.The spontaneous number of revertants on dimethyl sulfoxide control plates without liver homogenate for all experiments were: TA1535, 46.33 ±12.15; TA100. 239.74 ±40.65; TA1537, 12.50 ±3.60; TA1538, 37.94 ±8.64; TA98, 46.21 ± 7.96. Results with other control plates were comparable. Positive findings represent the results of at least 2 experiments. Toxicity was determined by direct observation or by visual comparison of bacterial lawns on treated and control plates under a dissecting microscope and was assigned if at least one strain showed reduced growth. If no toxicity was observed at the highest concentration used in the testing, it is denoted by:>. b Similar results have been reported by De Lorenzo et al. (60). C Similar results without the use of liver homogenate have been reported by Shirasu et a!. (213). d No mutagenicity was also observed by Simmon et al. (218).

0 NT, not tested.

geneous population like this set of carcinogens is the use of selves, or as derivatives resulting from their metabolism in the overstratification (i.e., overcategorizing) in the construction of host, of eliciting a carcinogenic response; they may or may not categories. This allows the determination of p,,, the correlation be related to presently existing chemical structures that are of an individual category. The practical value of p,'s is that, associated with carcinogenicity. It can be anticipated that the despite how high P is, when a given p@islow, it diminishes the creation of these future carcinogens will be influenced in part credibility that can be associated with negative results for by present-day trends in technological, cancer, and mutation chemicals tested in the given category. research. To estimate this ‘‘true'‘correlationrequires some under Only presently existing carcinogens lend themselves to any standing of what constitutes this set of carcinogens. It is analysis, but they are comprised of 3 parts: carcinogens tested beyond the scope of this paper to review the appropriateness and found positive (known carcinogens); carcinogens tested of interpreting the results of animal testing in terms of possible and not found positive (false negatives), and carcinogens not human experience. If it can be assumed, as Lijinsky (139) and tested. This implies then that the ultimate size of the set of Commoner (51) have essentially argued, that such can be done known carcinogens cannot be defined until all presently exist at least qualitatively, there still remain theoretical and practical ing chemicals have been adequately tested, which is an impos aspects to the calculation that confound the actual interpreta sibility. Since correlations can be calculated only with presently tion of the derived correlation. known carcinogens, it is not necessarily true that such corre First, this set of all carcinogens can be defined into 2 parts: lations are indicative of the more general set of presently carcinogens that presently exist, although not necessarily iden existing carcinogens or the general set of all carcinogens. How tified yet; and carcinogens that will be developed in the future. indicative a correlation is will depend on the representativeness The latter can be thought of as heretofore untried arrangements of the sample with which the estimation is made. of carbons, nitrogens, oxygens, etc. , that are capable in them Second, if correlations with the known carcinogens can be

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RANDOM SAMPLING AND TRUTHS ABOUT CARCINOGENS PROPORTIONAL ALLOCATION METHODS respectively, and that the percentages of some carcinogenic

PERCENTAGE POSITIVE CATEGORIES PERCENTAGE POSITIVE types, like haloaliphatics, are zero. However, this is not to IN SALMONELLA SIZE OF CARCINOGENS SAMPLE SIZE IN SALMONELLA suggest that there is anything intuitive about what constitutes @ (N P @ N1 C1 ] a random sample of carcinogens. The problem is unconven I \N11 I tional, and it does not lend itself comfortably to a statistical

@ P2 .@- N2 C2 /N2\ p2 approach. Given the circumstances surrounding the hitherto reported

p3 correlation studies (uncoded testing of chemicals, use of liter @ P3 -4.. N3 C3 n3―(i@;-)n ature searches, and other nonrandom selections of chemicals),

/N \ P the practical value of these overall correlations beyond the @ P N ICJ nii carcinogens tested is uncertain. However, this is not to insin N1—@N1 uate that, in some final analysis after more innovations in nI testing, the overall correlation would not be as high as these

@ PopuI@tIoniPercenlaqe P Eslimalorol Pip reports suggest. Rather, it is to underscore the point that such conclusions, at the least, are premature and not warranted by - i (@) the studies Conducted. Furthermore, as will be discussed be

@ —i(Ni\(positi@,psinN - ( ‘I(positives inn low, interest in this overall correlation becomes academic in @N1/@ N @N1/@ n view of the indication that certain categories of carcinogens -@ (@L) (vosdives inn n posilivesinN @E N exhibit individual correlations that are very low. , Ni —zpositivesin Construction of Table of 465 Compounds with Known or Suspected Carcinogenic Activity (Table 4) Chart 2. The set of all carcinogens has a total of N1chemicals that can be classified into several categories (C —°C@)thathave different sizes (N —.N,) Table 4 is an attempt to enumerate more of the carcinogens and percentages positive in Salmonella (P —@P,).The “true'‘correlationbetween known from animal testing. It is a compilation of 465 com carcinogenicity and mutagenicity in Salmonella or population percentage, P. is actually the summation of the products of the size of each of the categories pounds identified by several criteria. The table combines the relative to the total (N/N,) times the respective percentage positive (P,). In evaluations of chemicals for carcinogenicity published by property conducted random sampling and proportional allocation methods, the heterogeneity of N, is reconstructed in the sample, n,. Hence, the number of categories. their relative sizes (i.e., n,/n,) and their respective percentages Table 3 positive in Salmonella (pi —@p,)will estimate their counterparts for the set of all Construction of table of 465 compoundscarcinogenicactivity with known or suspected carcinogens. Likewise, p, which is also calculated as a summation, will be an (Table4)No. estimate of P. ofchemi

assumed to be satisfactory, there is the practical problem of calsidenti enumerating known carcinogens so that they can be tested. Saffiotti (203) has warned of the possible shortcomings of CommentsIARCSource tied correlations based on lists of carcinogens. Also, he estimates 14)a.vols. 1—13(1 184b.Animal carcinogens that, of the 6000 chemicals tested for carcinogenicity and activeonlyC.Bladder implantation (bi) 1 Strongly recorded in the Survey of Compounds Which Have Been Tested for Carcinogenicity (through 1972) (64), only one-half Epidemiological4(e) evidence onlyd. were probably tested with some degree of adequacy. Thus, the Possiblyin(ss) strain-specific 9 Those 7 chemicals reviewed some 1000 suggested carcinogens in this listing could be notrodentscarcinogenicity in vol. 12 were considered expected more reliably to be about 500. While this figure of evaluablee. Appearingofdiscussion in the 30 Documentation 500 serves as a rough estimate of the number of known fairlycarcinogenicityof the carcinogenicity is carcinogens (through 1972), the publication does not provide convincingmetabolitesof (M)Total any actual listing of these presumable carcinogens. Generally, 228EPA's the best lists of known carcinogens can be expected to be identifyingSuspectedOrdering of the NIOSH 202 Weakest criterion for those which represent the evaluations by expert Committees tableList Carcinogens chemicals for the positivein(78): reported reviewing the documentation for carcinogenicity for these mammaliansystemsIndividualat least 2 chemicals. Other sources are available, but they are only as credible as they are critical. chemicals 182 82IARCcommittees; were also reviewed by wereconsidered4 of these This practical problem of enumerating known carcinogens evaluable:ziram,not has had a biasing effect on the aforementioned correlation 8-quinolinol,chloromethyl ether,and methyl studies. That nonrandomly selected carcinogens have been hydrazideSecretarys maleic tested is best illustrated in the reporting of Sugimura et al. thePesticidesCommission on 36 IARC disagreed that (226) but is generally true of each of the studies. If their sample aldrin,Relationshipand Their carcinogenicity of IPC, wasEnvironmentalto and heptachlor of 98 carcinogens were randomly selected, the frequency of substantiatedpHealth (62): chemicals from a given category in the sample should reflect <0.054 their frequency in the population of carcinogens to which correlation234Salmonella-S-9 studies using system(104, @ inference is drawn (Chart 2, njn@ N@/N1).The sample of 226)Total 152, 172, Sugimura et al. indicates, probably erroneously, that 33 and variesinno. of individual chemicals 465 Proof of carcinogenicity 17% of all carcinogens are N-nitrosamines and 5-nitrofuryls, Table 3. from strong to marginal

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Chemical Structure Carcinogenicity and Salmonella Assay Table4 ChemicaltrendsandsomeSalmonellatestingresultsof 465 compoundswithknownor suspectedcarcinogenicactivity Carcinogenicity Documentation IARC(114). Thereference(volume:page)tothe appropriatemonographis presented.Thesymbolsthat can precedethe referenceare as follows:bi, bladderimplantationtestingis the only adequatelyconductedtestingthat hasshownthe chemicalto havea carcinogeniceffect;e, epidemiologicaldata ratherthanmammaliantestingindicatesthat the chemicalis associatedwithcarcinogenicity;M, the chemicalwasnot the subjectof a reviewper Se,but its documentationofcarcinogenicitywascited by an IARCstudygroupbecausethe chemicalis a metaboliteofor structurallysimilarto anotherchemical which that study group was specifically evaluating (the authors of this paper have reviewed these studies and have found them indicating a carcinogenic effect in the experimental design used); as, the observed carcinogenicity to date has been strain specific; —, chemical was concluded not to have been positivein all adequatetesting;?, the studygroupwasunableto evaluatethe documentationofcarcinogenicitydueto inadequatetestingor inadequate reportingof findingsin the availablestudies. EPA(78).The4-digitidentifiersummarizesthedocumentationofcarcinogenicityasitwouldhaveappearedinanupdatedversionofthe1975 Suspected Carcinogens(55).Thefirst digit (fromleft to right)representsthehighestphylogeneticspeciesin whichthe positiveresponsewasreported:7, humans;6, monkeys;5, cat, dog, pig, cattle,or domesticanimal;4, rat; 3, mouse;2, guineapig, gerbil, hamster,rabbit,squirrel,unspecifiedmammal;1, wild bird, bird, chicken, duck, pigeon,quail, or turkey; 0, frog. The seconddigit designatesthe numberof different speciesfor which a positiveresponsewas reported, up to a maximum of 9. The third digit describes the highest route of administration of all positive findings as such: 2, inhalation, ocular, or skin application;1, p.o.administration;0,all otherroutesof administration.Thefourthdigit is a countof thenumberof differentspecies-per-routecombinations reportedpositive,up to a maximumof9. Department of Health, Education, and Welfare (DHEW) (62). The judgment reached by the Technical Panel on Carcinogenesis is presented: B, chemicalincreasedtumor incidencein one or more mammalianspecies;the results are significantat the 0.01 level; Cl , chemicalsincreasedtumor incidencein one mammalianspeciessignificantat the 0.01 level but were consideredless active than the meanof a group of positivecontrols; C2, chemicalsincreasedtumorincidencein onemammalianspeciessignificantat the 0.02 levelandwereconsideredlessactivethanthe meanof a groupof positive controls; C3, chemicals increased tumor incidence in comparison to the negative controls, but the level of significance was less than 0.02 (the statisticalsignificancesof thesechemicalshas beencheckedand only those active at the 0.05 levelof significancehavebeen used in the table);C4, chemicalwastestedappropriatelyinonespeciesonly andjudgednot positivein that species. Salmon&Ia Tsting Documentation The testingresultsin the Salmonella-S-9systemas reportedby McCann et al. (152), Sugimuraet a!. (226), Heddle and Bruce (104), and Odashima (1 72) with the identifier assigned in the respective reporting are presented. For lack of space, the 5-character identifier from the report of Odashima (172), e.g. 73-20, is presentedin two parts.Thesymbolsthatcan precedean identifierare as follows:+ , positiveresults;w+ , weaklypositiveresults;?, toxic effect preventsproper testing or borderlineresults; —,negativeresults,minimallyin TA100 and TA98 with and without activation.Underlinedresults indicatethat the chemicalwasnot listedas a carcinogenin that reporting.For referencesof carcinogenicityina givenreporting,the readeris referredto the respective article. ‘‘Miscellaneous'‘containsSalmonella testing results from other sources. Testing results that are referenced as personal communicationsofT. Connorweredevelopedundera collaborativeprojectwith the Foodand DrugAdministration. Carc@nogenlcity SalmonellatestIng CaICInOgenICfty Salmonellatesting

Chsm@& 1 I@/@ @° @@@/I/:L@ - 0@ Cyanamide 19. 4-ft4.(5.((2-Hydroxyetttyl)su@. 1. Cyanamide 3111 1 fonyl)-2-methoxyphenyflezo}.5- Triazene hydroxy-3-methylpyrazol-1-yl) 2. 3-Monomethyl-1-phenyftrlazene 4223 3, 1-Phenyl-3,3-dlmethyttrlazene 41 13 +K1 0 td@@j (es. 4. 1-(4-Chlorophenyl).3,3-dlmethyl. 4101 +K9 lowG) m salt (RemazolYel trlazene (153) 3,3'-ft3,3'-DimethyKl ,1‘-bi Diazo phenyl)-4,4'-dlyIJls(azo))bie(5- 5. Dlazomethane 7:223 4223 arnino-4-hydroxy-2.7-naphtha 6. Azaeerine 10:73 +114 @ef@edlsuftoflicacid),tetrasodium 7. N-(DlazoacetyOglycineamide 3101 +115 @t(trypSflblue) 8. N-(DlazoacetyOglycinehydrazine 3101 I'Ll 6 (154) 6,6'-g3,3'-DImethyKl,1‘-bi ptwnyl)-4,4'-dlyIJle(azo))ble(4- @@@ M@;r:@ +L1 I 11. 1-(Phenytazo)-2-naphthalenol 8:2253112 —(4°) (Sudanl) : — Azoxy : @ 12 4-((4-Hydroxy-1-naphthalenyl)- 8 173 4101 (40) &zoxymettw.ne M4 14 4101 +2 azoJ@enzenesulfonlcacid, 21. Azoxyethane M4:15' 4101 monosodIumaalt(Orange I) 22. Methylazoxymethanol M1O:127 4202 +G22 13. 1.((2.Methylphenyl)szoj-2- 8:165 3101 23. Methylazoxymethanolacetate M1O:131 +G23 +14' + (126 naphthalenoI(0ll0rar@ge5S) 24. cyc@ 10:121 4313 —021 —5 14. 1.((2.4-Dlmethylphenyl)azo).2- b18:233 3101 +(90) naphthslsnol (Sudan m Hydrazk@e 15. 1-((2,5-Olmethoxyphenyl)ezo).2- 8:101 3101 ?(40) 25. Hydrazine 4:127 4213 w+K4 naphthalenol(Citrus Red No. 2) 26. HydrazlnecarboxamldeHO (earn- I 2:209 3111 16. 4-((2,4-Dlmethylphenyl)azo}.3- 8:189 4111 iCarbaZideHCI) hydroxy-2,7-naphthalenedlsul- 27. N-(2-Hydroxyethyl)hydrazlne 3111 B w+K6 tonic acid, dleodlumSaN(Pon- 28. leonicotinIcacid hydrazine (leone- 4:159 4214 _b ceau MX) azid) 1 7. 3-Hydroxy-4-((2.4,5-tr*methyl- 8:1 99 421 2 —(40) 29. 1 -Acetyl-2-isonicotlnoylhydrazlne M4:1 66 3111 phenyOezo).2,7-naphthalenedi- @o.2-Hydrazlno.4-(4-amlnopheny9. 4111 .113 [email protected] salt roacRodNo.1; @onceau3R) 31.2-Hydrazino-4-(4-nftrophenyl). 4212 ‘114 18. 5-Hydroxy-6-f[3-((2.hydroxy- 421@ ethyl)sultonyl)phenyl)azo)-1- 32. 1,1-Olmethy1hy&azlr@ 4:137 3111 + naphttialenesulfonlcacid, hydro- (1%) 2-Hydrazlno-4-(5-nltro.2-fury9- gen sulfate (eater),dlsodium saft thiazoie (RemazolRed B) 33. 1,2-Dimethylhydrazine 4:145 4313 —K5

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Table 4—Continued Carcsrtogen,c@ty Salmonellatesting / Carcinogenicily Salmonellatesting @/i;6/

@@/‘-@ @ ______Chemical /@/@‘/@/j@@$ @/ / ChemIcal 1V@y@/ ______4c)/@/.—@— / 34. 1,2-Oiethylhydrazine 4:153 4101 81. 1-NaphthalenolN-nltrosomethyl M12:45 35. 2-Phenylhydrazinecarboxamide12:177 3111 carbamate(N-nitrosocarbaryl) (@arbazide) 82. 1-Nitrosoazetidine 4212 @ 36. N-lsopropy$-a-(2-methylhydra 4214 —K7 - 83. N-Nitrosopyrrolidine 4111 w+G6 zino)-4-toluamideHt@l(procarba 84. 3-(1-Nitroso-2-pyrrniidinyl)- 4212 zine) pyridine (197) 2@2-Formylhydrazino)-4-(5-nltro 85. 1-Nitrosoplperidine 4315 w+G8 2-furyl)thiazole 86. Hexahydro-1-nltroso-1H-azeplne 4212 (198) 2-(2,2-Dimethylhydrazlno)-4-(5- 87. Octahydro-1-nitrosoazocine 4212 nitro-2-furyQthiazole 88. 1,4-Dkiftrosoplperazk@e 4214 89. N-Nitrosomorpholine 4314 w+G7 37. N-Nitrosodlmethylamine 1:95 4429 w+G1 +8@ - +7; b —2C Carbamyl,Ihiocarbamyl 38. N-Nitrososarcoslne 4212 90. Dimethylcarbamylchloride 12:77 3123 w+B19 + (187) 39. N-Methyl-N-n-butylnltrosamine 4122 + 141 91 . Acetamide 7.19 4111 -D28 40. N-Methyl-N.(4-hydroxybutyl)- +15@ 92. Thioacetamide 7:77 4212 —D27 — (126) nitrosamine 93. Cyclochlorotine 10:131 3111 41 . N-Methyl-N.(3-carboxypropyl)- +154 94. 2-EEhyl-4-pyridinecarbothioamide 13:83 3111 nitrosamine (Ethionamide) 42. N-Methvl-N-n-dodecvlnltrosamine 4111 + 15; (1 85) 2-(2-Furyl)-3-(5-nitro-2- 43. N-Methyl-N-benzytnltrosamine 4111 + 141 furyOacrylamide(AF-2) @ ?@‘ 44. N-Nitroeodlethylamine 1:107 8929 w+G2 +74 95. Ethyl carbamate(urethan) 7:111 4429 - D21 — (126) 45. Ethyl-2-hydroxyethylnlfrosamlne Mi :116 96. N-Hydroxyurethan M7: 121 4202 +12 46. N-Ethyl-N-nltrosovlnylamine 4213 97. n-Propyl carbamate 12:201 3112 47. N-Ethyl-N-nltrosobutylamine 4213 98. 4-(Dimethylamino)-3,5-dimethyl ssl2:23 3111 ci 48. N-Butyl-N-(2-hydroxyethyO +44 phenol methylcarbamate (ester) nitrosamine (Zectran) 49. N-Ethyl-N-(4-hydroxyethyl)- +9@ (291) Mitomycin C nitrosamine 99. Bis(dimethylcarbamodlthloato ?12:251 4213 C4 50. N-Ethyl-N.(3-carboxypropyl)- +91 S,S')zinc (Ziram) nitrosamine 1 00. Bis(dlmethylcarbamodilhioato ssl2:131 3101 51. N-Nltrosodlpropylamine 4212 w+G3 +8 S,S')lead (Ledate) 52. N-(2-Oxopropyl)-N-nitrosopro 4202 I 01 . Tetrakis(dielhylcarbamodithioato pylamine S,S')selenium (Ethyl Selenac) 53. N-Butyl-N-(2-oxopropyl)- +51 102. Tetrakis(diethylcarbamodilhioato nteosam@e S.S')tellurium(EthylTellurac) 54. N-Butyl-N-(2-carboxyethyl)- 1 03. Sodium diethyldithiocarbamate ssl2:2i7 nhtrosamine 1 04. Potassium bis(2-hydroxyethyl)- 12:183 3111 — (126) 55. ,v-re-@ropy$-N-n-butylnltrosamine +21 dithiocarbamate 56. N-Propyl-N-(4-hydroxybutyl)- +21 1 05. ([1 ,2-Ethanediylbis(carbamo ssi2:137 4112 C4 nitrosamine dithioato)X2—flmanganese 57. N-Propyl-N-(3-carboxypropy0- +21 (Maneb) nitrosamine 106. ([1 ,2-Ethanediylbis(carbamo ssl2:24 4112C3 58. N-Nltroso.dl-n-butylamlne 4:197 4418 +G4 + 7@ + +74 dithioato)X2—))zinc —10 (Zineb) 59. n-Butyl-(4-hydroxybutyl)- M4:204 4111 +4 + 73 107. 2-Benzothiazolethiol (Captax) 3101 C2 nitrosamine —22 108. Thiourea 7:95 4111 -019 — (126) 60. N-8utyl-N-(3-oxobutyO@ +5 109. N'.(4-Chlorophenyl)-N,N-dimeth 12:16 4212 Cl — (218) nitrosamine ylurea (Monuron) 61. n-Butyl'<3-carboxypropyU M4:205 4111 +3 1 1 0. 4,5-Dlhydroimidazole-2(3H)- 7:45 4212 + (231) nitrosamine thione (ETU) 62. N-n-Butyl-N-n-amylnltrosamlne +3 (1 86) 5-Nitro-2-furaldehyde semicarba 63. N-Amyl-N-(4-hydroxybutyl). zone (nitrofurazone) nitrosamine 64. Dl-n-pentylnftrosamO@e 4112 w+G5 Diaryl alkynyl carbamate 65. N-Methyl-N-nltrosoanillne 4212 111. 1-Phenyl-1 -(3,4-xylyl)-2-propynyl +D23 66. N-Methyl-N,4-dinitroeoanhline 1:141 4112 N-cyclohexylcarbamate 67. 1-Methyl-i -nitrosourea 1:125 5529 +018 +74 1 1 2. 1 , 1-Diphenyl-2-propynyl N-cyclo +D25 —14 hexylcarbamate as. i-@tt@yi-i-nitrosourea 1:135 4215 +019 113. 1,1-Diphenyl-2-butynyl N-cyclo +D24 69. 1-Butyl-1-nitrosourea 4213 +54 +73 hexylcarbamate —24 70. 1,3-Olmethyl-1-nitrosourea 4314 Aromatic amine 71 . N-o-Glucosyl-(2)-N'-nftroso 4:221 4101 +024 1 1 4. 2,4-Diaminotoluene 4112 +A42 methylurea(atreptozotocin) 1 1 5. 3-Hydroxyanthranilic acid 3102 —119 —74 72. N-Methy$-N'-nltro-N-nitrosoguani4:183 5529 +011 + 159 + —18 dW@e 1 1 6. lsopropyt N-phenylcarbamate —12:189 3111 C2 73. @EthyI-N'-nitro-N-nhtrosoguanl 4326 +012 +95 (IPC) dine I 1 7. N-(4-Ethoxyphenyl)acetamide e13:141 -205 —74 74. N-Propyl-N'-nltro-N-nitrosoguanl +013 +211 (Phenacetin) -06 dine 1 1 8. N-Hydroxyphenacetin M13:144 75. N-Butyl-N'-nitro-N-nltroeoguani +014 +52 1 1 9. 2,6-Dichloro-4-nitroaniline C2 dine 1 20. 2-Chloro-4,6..bis(ethylamino). 4202 —(218) 76. N-lsobutyl-N'-nltro-N-nitroso +015 +53 1 .3,5-triazine (Simazine) guanidine I 21 . 4-Aminobiphenyl 1:74 I 5416 +A26 ‘18 +73 77. N-Pentyl-N'-nltro-N-nltrosoguani +016 +204 -06 dine 1 22. 4-HydroxyaminobIphenyl M1:76 3101 78. N-Nltroso-N-methylurethan 4:211 4418 +020 I 23. 4-Acetylamlnobiphenyl 5213 +7 —73 79. N-Nitroso-N-ethylurethan 1213 —07 80. N-n-Sutyinltroeourethan 11 1 1 + 55 +74 I 24. N-Hydroxy-4-acetylaminobi Mi:76 4112 —12 phenyl

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1 25. 4-Amino-3-hydroxybiphenyl M1:76 3101 I 68. 3-Acetylaminophenanthrene E212 1 69. 2-Aminofluorene 4225 +A2 + I 26. 4,4'-Dlaminobiphenyl (benzidine) 1:80 7426 +A29 I+(187) 1 27. 4-Amino-4'-hydroxybiphenyl Mi :76 3101 1 70. N-Hydroxy-2-aminofluorene 4202 +A5 1 28. 4-Amino-4'-fluorobiphenyl 4213 I 71 . 2-Nitrosofluorene 4102 +A6 1 29. 4-Amioo-3,2'-dimethylblphenyl 4202 +A27 172. 2-Acetylaminofluorene 5529 +A3 +8 .73 +(187) 1 30. 3,3'-Dlmethylbenzidine (o-toli 1:87 4113 —11 dine) 173. N-Hydroxy-N-(2- 4519 +A4 +116@ 1 31 . 3,3'-Benzidinedicarboxylic acid 4213 fluorenyl)acetamide 1 32. 3,3'-Benzldlnediol 4226 1 74. 2-Diacetylaminofluorene 4223 1 33. 3,3'-Dimethoxybenzldine 4:41 4212 1 75. N-Acetoxy-2-acetylaminofluorene 3121 +A1 1 34. 3,3'-Dichlorobenzidine 4:49 @416 1 76. 2,7-Diaminofluorene 4111 +A13 1 35. 4,4'-Methylenebis(2-methylani 4:73 4111 1 77. 2,7-Bis(acetylamino)fluorene 4212 +A12 line) 1 78. 6-Aminochrysene +A14 1 36. 4,4'-Methylenebis(2-chloroani 4:65 4212 +A34 1 79. 4-(Hydroxyamino)Quinoline 1- 4225 +E7 118 line) (MOCA) oxide 1 37. 4,4'-(lmidocarbonyl)bis(N,N'-dl 1:69 4213 —A43 180. 3,6-Bis(dimethylamino)acridine 4222 +A17 methyl)anhline (auramine) (acridine orange) 1 38. Tris(4-aminophenyOmethane ?4:57 4101 —A35 (98) 4-(Dimethylamino)-3,5-dimethyl (pararosanlllne) phenol methylcarbamate (ester) 1 39. (4-(4-(Dlmethylamino)-a-(4- 4101 +11 (Zectran) ethy@3-suttony9amino)phenyl1- (1 09) N'@4-Chlorophenyl)-N,N-dimeth benzylldene-2.5-cyclohexadien ylurea (Monuron) 1-ylidene}ethyk3-sulfobenzyl)- (1 92) 2-Amino-.4-(5-nltro-2- ammoniumhydroxide, inner ash, furyl)thiazole sodium salt (Acid Violet 6B) (1 93) N-(4-(5-Nitro-2-fury9-2-thiazolyll +7 formamide (FANFT) 140. 4-(Phenylazo)benzenamine 8:53 0101 +L2 +15 (1 94) N-(4-(5-Nitro-2-fury9-2-thiazolyl)- 141. N-Methyl-4-(phenylazo)enlllne 4112 +L5 +145 acetamide 142. N,N-Dimethyl-4-(phenylazo)- 8:12!'4324 +16 +80 +(40) (1 95) 2,2,2-Trifiuoro-N44-(5-nitro-2-fu benzenamine (Butter Yellow) ryl)-2-thiazoly9acetamide 143. N-Benzoyloxy-4-methylaminoazo 4101 +111 +34 (1 99) 2-Amino-5-(5-nitro-2-furyl)-1 .3,4- benzene thiadlazole 144. 4-(Phenylazo)-1,3-benzenedi 8:91 3111 +(90) (200) N-(5-(5-Nitro-2-fury9-1,3,4-thiadi amine HCI(chrysoidine) azol-2-yl)acetamide 145. 2-Methyi-4-dlmethylamlnoazo +110 +151 + (126 (203) 4,@Diamino-2-(5-nitro-2-fury1)- benzene 1,3,5-triazlne +7 (204) 4-(2-Hydroxyethylamino)-2-(5-ni 1 46. N,N-Olmethyl-4-((3- 4212 +17 + 152 —1 tro-2-thienyl)guinazollne methylphenyl)azo@enzenamine (205) 4-Bis(2-hydroxyethyl)amino-2-(5- 147. 3-Methoxy-4-aminoazobenzene +14 + 13( nltro-2-thienyl)quinazoline I 48. 2-Methyl-4-((2-methylphenyO 8:61 4315 +13 +16 + (126 (208) 2-Formylamino-4-(4- azo@enzenamine nitrophenyt)thiazole 149. 2-(2-Tolylazo)-4-toluidlne 4212 (41 2) 4-Aminobenzenesulfamide (sulfa 150. N,N-Dlmethyl-4-phenylazoaniline 4212 nilamide) N-oxide (41 3) 2-(4-Aminobenzenesulfonamido)- 151 . 2-(4-Dlmethylamlno)-1 -naphtha 4222 thiazole leneazobenzene 1 52. 1 -(2-Methylphenyl)azo-2- 8:2W 4101 Nitroaromatic naphthalenamine(Yellow 08) 1 81 . 1 ,2-Dimethyt-5-nitrolmidazole 4111 +E1 1 1 53. 3,3'-{[3,3'-Dlmethyl(1 .1 ‘-bi 8:26@ 4102 ?(40) 1 82. 1 -(2-Hydroxyethyl)-2-methyi-5-ni 13:113 3111 +E10 @169 phenyl).4,4'-diyljbis(azo))bis(5- troimidazole (Metronidazole) amino.4-hydroxy-2.7-nsphtha 1 83. trans-2-{(Dlmethylamino)methyl 7:147 4111 +E21 +81 lenedisulfonic acid, tetrssodium imino)-5-(2-(5-nitro-2-furyl). salt (trypan blue) vlnyl)-1,3,4-oxadiazole I 54. 6,6'-((3,3'-Dimethyl(1 .1 ‘-bi 8:151 —(40) 184. Potassium 1-methyl-7-(2-(5-nitro +211 pheny9-4.4'-diyllbis(azo)}bis(4- 2-furyl)vlnyl)-4-oxo-1,4-dihydro amino-5-hydroxy-1 ,3-naphtha 1 ,8-naphthyridine-3-carboxylate lenedlsulfonic acid), tetrasodium 1 85. 2-(2-Furyl)-3-(5-nitro-2- +E16 +104 + salt (Evans blue) furyDacrylamide(AF-2) 1 55. trans-4-Aminostllbene 4212 +A32 186. 5-Nitro-2-furaidehyde aemicar ?7:171 4111 +E21 @181 + + 74 1 56. trens-4-Dlmethylaminostllbene 41 11 +A33 +82 + 74 bazone (nitrofurazone) —19 -04 1 87. 1 -((5-Nltrofurfurylidene)amlno)-2- 7:181 4111 I 57. N,N-Dlmethyl-4-(2-(1 - 4202 Imidazolidlnone naphthyl)vinyl@Bnillne 1 88. 4-Methyl-i -((5-nitrofurfuryli 4111 +E21 1155 1 58. 1 -Naphthylamine ?4:87 3101 +A22 +173 + + 74 — (187 dene)amlno).2-imidazolidinone —07 1 89. (-)-5-(Morphollnomethyl)-3-((5- 7:161 4111 159. N-(1 -Naphthyl)hydroxylamine M4:92 4101 +A24 nitrofurfurylidene)amino).2-oxa I 80. 1-Nitrosonaphthalene M4:92 4101 zolidinone 1 61 . 2-Naphthylamine 4:97 6517 +A21 +174 + + 74 + (187 1 90. 4-(5-Nitro-2-furyl)thiazole +E21 +191 —08 1 91 . 2-Methyl-4-(5-nitro-2- +E21 1156 1 62. N-(2-Naphthyl)hydroxylamine M4:1 0 4203 +A23 furyl)thiazole 1 63. 2-Nitrosonaphthalene M4:1 0 3101 +A25 1 92. 2-Amino-4-(5-nitro-2- 3111 +E21 +20 184. 3-Methyl-2-naphthylamine 4202 furyl)thiazole I 65. 1-Aminoanthracene 4111 +A20 1 93. N-(4-(5-Nftro-2-furyl)-2-thiazolyl). @414 +E17 + 193 + 168. 2-Aminoanthracene 4222 +A19 formamide(FANFT) 1 67. 1 -Amlno-9,1 0-dlhydro-4-(3-((2- 4212 1 94. N-(4-(5-Nftro-2-furyO-2-thlazolyl)- 7:185 5414 +E21 + 192 hydroxyethyl)sulfonyljanilino) acetamide 9.1 0.dloxo-2-anthracenesulfonic 1 95. 2,2.2-Trifluoro-N-(4-(5-nltro-2-fu 3111 +E21 + 237 acid.hydrogensulfate(ester),di ryl)-2-thiszolyl)acetamide sodium salt

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Table 4—Continued Carcinogenicity Salmonellatesting Carcinogenicity Salmonellatesting

@_/,$, ‘N /ii;/ 5.- ,@‘ c?,@:, a

@ Chemical @Jz/@':4'LL ______@@196. 2-Hydraz@o-4-(5-nitro-2-turyl)- 4212@//@234. a-(2-(24utoxyethoxy)ethoxy)- Chemical3112 1 j (126) thiazole 4,5-(methylenedioxy)-2-propylto. @ 197. 2-(2-Formylhydrazino).4-(5-nitro 7:151 431 +E21 +10 luene (Piperonyl Butoxide) 2-furyOthiazole 198. 2-(2,2-Dimethylhydrazino)-4-(5- 4212 +E21 +84 Substituted diphenylethane nitro-2-furyl)thlazole 235. Ethyl2-hydroxy-2,2-bis(4-chloro 5:75 3111 B 199. 2-Amlno-5.(5-nitro-2-furyl)- 7:143 4111 phenyl)acetate(Chlorobenzilate) 1.3.4-thiadiazole (355) 1.1,1-Trichloro-2,2-bls(4-chloro 200. N.(5.(5-Nftro-2-furyl)-1 ,3,4-thiadi 3111 +E21 +19 phenyl)ethane(p,p'-DDT) azol-2-yl)acetsmide (356) 1.1-Dichloro.2,2-bis(4-chloro. 201. N-([3.(5-Mtro-2-furyl).1 .2.4-ox +E21 +18 phenyflethane(p,p'-TDE; ODD) adlazol-5-yl)nethyl}acetamide (357) 1.1-Dichloro.2,2-bis(4-ethyi 202. 5-Acetamido-3-(5-nltro-2-furyl)- +E21 +3 phenyl)ethane(Perthane) SN-i ,2.4-oxadiazine (393) 1,1-Dlchloro.2,2-bis(4-chloro. 203. 4.6-Dismino-2-(5-nitro-2-turyfl 4111 +E21 +68 phenyOethylene(p,p'-DDE) 1,3,S-triszlne 204. 4-(2-Hydroxyethylsmino)-2-(5-ni +E14 Stilbenediol tro-2-thienyl)qulnazoline 236. trans-a,a'-Diethyl-4,4'-stilbene 6:55 4416 ?F19 — (126) 205. 4@s(2-hydroxyethyl)amlno-2-(5- +E1 5 diol (DES) nltro.2-thienyl)quinazoline 237. trans-a,a'-Diethyl-4,4'-stilbene 4202 206. 1,2-Dihydro-2-(5-nitro-2- +E13 did dipropionate thlenyl)quinszohn-4(3H).one 238. meso-3,4-Bia(4-hydroxyphenyl)- 3202 207. 1-(5-Nltro-2-thlazolyO-2-imidszo13:12@ +E12 +171 hexane(dihydrostilbestrol) lidinone (Niridazole) 208. 2-Fonaytamino-4-(4- + 102 Polyaromatic nitrophenyOthlazole 239. a-(((1-Methyl)amlno)isethyl)-2- 13:2273111 209. 4-Nitropyridine 1-oxide + 194 naphthalenemethanolHCI(Pro 210. 2-Methoxy-5-nitrotropone + 144 netelolHOt) 211. 4-Nitrobiphenyl 4:11@ 5111 +E2 (429) 3-Methoxy-16.17-secoestra 212. 2-Nltronaphthalene +E3 1.5.5,7.9-pentaen-1 7-oic acid 213. 5-Nitroacensphthene 4212 +E4 +177 240. Benz(a)anthracene 3:45 3124 +C16 214. 2-Nitrofluorene 41 2 +E5 241 . 4-Methylbenz(a)anthracene 4223 21 5. 4-Nitroquinoline + 195 242. 5-Methylbenz(a)anthracene 4223 216. 4-Nitroquinoline1-oxide(4..NQQ) 4521 +E6 +198 + 7 + (187) 243. 6-Methylbenz(a)anthracene 4224 —2 244. 7-Methylbenz(a)anthrscene 4225 +cl 8 217. 4-Mtro-6-quinolinecsrboxylic 4202 245. Benz(a)anthracen-7-yltrichloro 4202 acid1-oxide methyl ketone 218. 6.Chloro-4-nitroquinollne1-oxIde 4222 246. 3-Fluoro-7-methylbenz(a)- 4202 (31) 2-Hydrazlno-4-(4-nltrophenyl)- anthracene thiazole 247. 6-Fluoro-7-methylbenz(a)- 4223 (1 19) 2.6-Dlchloro-4-nitroanlllne anthracene (224) Pentachloronitrobenzene(P@NB) 248. 10-Fluoro-7-methylbenz(a)- 4223 (433) 6.((1-Methyl-4-nftroImidazol-5- anthracene yI)thio@urlne(Azathioprine) 249. 8-Methylbenz(a)anthracene 4223 250. 9-Methylbenz(a)anthracene 4222 Phenyl 251 . 1O-Methylbenz(a)anthracene 4223 219. Benzene e7:2033121 252. 12-Methylbenz(a)anthracene 4224 220. 5.5-Dlphenyl-2,4-imidazdidlne 13:201 4111 253. 7,1 2-Dlmethylbenz(a)anthracene 5729 +C19 +83 + +7; dione (Phenytoin) —01 221 . 5-EthYI-5-@*IenYI 13:157 3111 —Ji 254. 12-Methylbenz(a)anthracene-7- 4213 +020 2.4,8(1H.3H.5H)-pyrImldlnetrlone methanol (ph@ 255. 7-Formyl-12-methylbenz(a)- 4202 (446) TriphenyttInacetate anthracene (457) 7-Chloro.1 .3-dihydro-3-hydroxy 256. 7-Methoxy-12-methylbenz(a)- 4202 5-phenyt-2H-1.4-benzodiazepln anthracene 2-one (Oxazepam) 257. 7-Ethoxy-12-rnethylbenz(a)- 4202 222. 2-(2,4-Olchlorophenoxy)proplonic C3 anthracene acid 258. 7-Methylbenz(a)anthracene-12- 4212 223. 2.2'-ThIobia(4.6-dichlorophenol) 3111 Cl methanol (Vancide 81.) 259. 6,7-Oimethylbenz(a)anthracene 4202 224. Pentachloronltrobenzene(PCN8) ss5:21 1 3122B 260. 6,12-Dlmethylbenz(a)anthracene 4202 (324) Tannicacid:galllcacid —105 261. 7,8-Dlmethylbenz(a)anthracene 4202 225. Biphenyl 3101 02 — (126) 262. 7,11-Dimethylbenz(a)anthracene 4202 226. Polychlorinatedbiphenyls:Ks 7:261 4212 —210 —73 263. 8,12-Dlmethylbenz(a)anthracene 4202 nechl@500 and ArocI@ 1254 -09 264. 9,1O-Dlmethylbenz(a)anthracene 4222 265. 4,7,12-Trlmethylbenz(a)- 4223 Benzodloxole anthracene 227. 5-(2-Propenyl)-1.3-benzodloxole 10:231 4212 —F15 b 266. 7,8,12-Thmethylbenz(a)- 4223 (safrole) anthracene 228. 1‘-Hydroxysafrole Mi 0:240 —F16 267. 7,9,12-Trimethylbenz(a)- 4223 229. 1‘-Acetoxysafrole Mi 0:240 +F17 snthracene 230. 5-(1-Propenyl)-1.3-benzodioxole 10:2323111 268. Benz(a)phenanthrene(chrysene) 3:159 3122 +c14 (isosafrole) 269. 15,16-Dlhydro-11-methylcyclo +034 231 . 5-Propyl-1 .3-benzodloxole (dihy 10:233 3111 penta(a)phenanthrene-1 7-one drosafrole) 270. Daunomycin 10:145 4101 +H12 +65 232. 1,2,3,4-Tetrahydro-3-methyl-6,7- C3 271 . Adriamycin ?1O:43 4101 +H1 1 +13 methylenedioxynaphthalene-12- 272. Benzo(a)pyrene 3:91 6629 +C3 +32 + +74 dicarboxyllc acid, di-n.propyl es —02 tar (n.Propyl Isome) 273. 3-Hydroxybenzo(a)pyrene W+C6 233. 1@2-MethylenedIoxy-4-(2-(oct@ ci 274. 3-Methoxybenzo(a)pyrene 4202 sulfinyOpropyi@enzene(Pipero. 275. 6-Hydroxymethylbenzo(a)pyreneM3:1 14 4101 +C4 nyl Sulfoxide) 276. 7,8-Dihydrobenzo(a)pyrene +C7

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Table 4—Continued Carcinogenicity Se!monellatesting Carcinogenicity Salmonellatesting

:‘

@ Chemical ci@7@1n@:_,?i. 277. Benzo(e)pyrene 3:137 3121 +C8 324. Tannicacid:eIIaQiCacid 10:25 41011 @278. 3-Methyicholanthrene 582 +c13 + / Chemical 279. Benzo(b)fluoranthene 3:69 312 Anhydride 280. Benzo(j)ftuoranthene 3:82 3121 325. Succinicanhydnide 4101 ?D8 — (126) 281. Dibenz(a,c)anthracene +clO 326. (3aa,4$,7$,7ao)-Hexshydro 10:79 3121 282. Dibenz(a,h)anthracene 3:171 452 +011 3a,7a-dimethyl-4.7-epoxyisoben 283. Dibenzo(a,e)pyrene 3:201 312 +C1 zofuran-1 ,3-dione (canthanidin) 284. Dlbenzo(a,h)pyrene 3:207 3121 285. Oibenzo(a,s)pyrene 3:21f' 322 +C2 Pyrazoiinone 286. Dibenzo(a,I)pyrene 3:224 3122 327. 4-Amino-2,3-dlmethyl-1 -phenyl —J3 287. lndeno(1,2.3-cd)pyrene 3:229 3101 3-pyrazolin-5-one(4-aminoanti 288. Dibenzo(h,rst)pentaphene 3:197 3101 pyrine)

Aziridkw Pyrrolizidine 289. Aziridine 9:37 421 +K2 328. Lasiocarpine 10:2814101 290. 2-Methylaziridine 9:61 +K1 329. Monocrotaline 10:2914101 291 . Mitomycin C I 0:171 4202 1H1 4 + (154) 330. Retroraine 10:303 4112 292. 2-(l-Azirldlnyl)ethanol 9:47 3101 331 . Dehydroretronecine M1O:339 293. 1-Acetylaziridlne 4202 332. isatidine 10:269 4112 294. 1-Diethyiscetylazlridine 4202 295. 1-n-Butyrylsziridlne 4202 Hetenoaromatic 296. 1-Hexanoytazlridine 4202 333. Quinoline + 22: + (126) 297. 1-Nonanoytaziridlne 4203 334. 8-Quinolinol 113:101 4213 +124 298. 1-Myristoylazlridine 4203 335. Benz(c)acrldine 3:241 3121 299. Tris(1-azirldlnyl)phoaphinesulfide 9:85 4202 +K3 + 336. 7,9-Dimethylbenz(c)acridine 3101 +024 (thio-TEPA) 337. 7,10-Dlmethyibenz(c)acnidine 3101 +C25 300. Tris(2-methyl-1-aziridinyl). ?9:107 4111 + 338. Dibenz(a,h)acnidine 3:247 3124 phosphine oxide (METEPA) 339. Dibenz(aj)acridine 3:254 3122 +c9 301. Bis(1-aziridinyl)morphollno 9:55 3101 340. 1-(12-(Diethylsmino)ethyl)amino)- ?13:91 +J1 1 +112 + phosphine sulfide 4-(hydroxymethyOthioxsnthen-9- 302. 2,5-Bis(1 -aziridinyfl-3,6-bis(2- 9:51 3101 one, methanesuifonate(hycan methoxyethoxy)-4-benzoquinone thone methanesuifonate) 303. Tris(l -azlridlnyl).4-benzoquinone 9:67 4101 341. Actinomycin 0 10:29 4202 304. 2.4,6-Tris(1-azlridinyl)-1,3,5-tria 9:95 3122 342. Actinomycin L 10:29 zine(TEM) 343. Actinomycln 5 10:29 3101 344. Aflatoxln B 10:51 4214 +H1 + Oxirane, thllrane 345. Aflatoxin 82 10:51 4101 +H2 305. Propyleneoxide (methyloxirane) 11:191 346. Aflatoxin G, 10:51 4101 +H5 306. Oxirane carboxaidehyde (gly 11:17@ 422: +015 347. Aftatoxin M, Mi 0:62 +H4 cidsidehyde) 348. Aflatoxicol +H3 307. Chloromethyloxirane(epichloro 11:131 31 2 + (126) 10:2454112 +H8 +233 hydrin) 349. Stenigmatocystin 350. 7H-Dibenzo(c,g)carbazole 3:2605328 308. Benz(a)anthracene5,6-oxide +017 351 . Anthra(9,1 .2- 4212 309. Benzo(a)pyrene4,5-oxide +C5 +33 (326) (3aa.4$,7f1,7aa)-Hexahydro cde)benzo(h)cinnoiine 3a,7a-dlmethyl-4,7-epoxy-lso 352. Tricycioquinazoiine 4222 benzofuran-1 ,3-dione (canthari din) Halomethane,haloethane 353. Carbon tetnachlonide 1:53 4313 — (217) (401) 1,2,3,4,1O,1O-Hexachloro..6.7- —B2 epoxy-4a,5.6,7,8,8a-hexahydro 354. N-(Trichloromethyl)thio-4-cyclo 3112 2 +821 endo,exo-1,4:5.8-dimethano hexene-1,2-dicarboxyimide(Cap tan) naphthslene(Dieldnn) (245) Benz(a)anthracen-7-yl-trichloro 310. Dlepoxybutane (—,meso, and ±) 11:115 4223 +017 methylketone 31 1. 1,2,7,8-Diepoxyoctane 3121 +018 355. 1.1.1-Trlchloro-2.2-bis(4-chioro 5:83 3111 — (217) 312. 2,2'-(2,5,8,1 1-Tetradodecane 11:209 3101 phenyDethane(p.p'-DDI) 1.12-dIyl)bisoxirane 356. 1.1-Dichloro.2,2-bis(4-chloro 4212 313. 1-Epoxyethyl-3,4-epoxycycio 11:141 4222 M5:83 hexane phenyl)ethane(p,p'-TDE: ODD) 357. 1.1-Dichloro-2,2-bis(4-ethyl 3111 314. 3,4-Epoxy-6-methylcyclohexyl 11:147 3121 phenyl)ethane(Perthane) methyl-3,4-epoxy-6-methylcy 358. 10-Bromomethytanthracene clohexane carboxytate +C32 359. 10-Chloromethyl-9-methylanthra +030 31 5. Thllrane (ethylene sulfide) 11:257 4101 cane Dioxane 360. 10-Chloromethyl-9-chloroanthra +C31 316. 1,4-Dioxane 11:247 4111 cane 361 . 9.1 0-Dichloromethylanthracene +C29 Lactone 362. 7-Chloromethylbenz(a)- +C22 31 7. 2-Oxetanone ($-propiolactone) 4:259 4429 +09 +21 + (126) anthrscene 318. 4-Methyl-2-oxetanone (fl-butyro 11:225 4224 +010 363. 7-(Chloromethyl)-12- 4101 +021 lactone) methytbenz(a)anthracene 319. 3-Methoxy-5-methyi-4-oxo-2,5- 10:211 4202 -20@ 364. 7-(Bromomethyl)-12- 4222 +023 hexadlenoic acid (penicillic acid) methylbenz(a)anthracene 320. 4-Hydroxy-4H-furo(3,2-c@yran 10:205 4101 365. Benzylchloride 11:217 4101 w+B20 2(610-one(patulin) 366. lodomethane 4101 w+B1 + (217) 321. (s)-5.6-Dihydro-6-methyi-2H- 10:1994112 (397) Polychloninated terpenes: Stro pyran-2-one (parasorbic acid) bane 322. 2H-1-Benzopyran-2-one(cou 10:113 3121 367. 1,2-Dibromoethane w+B3 mann) 323. 2@,4aa,7-Tnlhydroxy-1-methyl-8- @2 —H9 N-, 5-. or 0-Mustard methylene-4b$-glbb-3-ene 368. 1.6-81s(2-chloroethylamino)- 9:157 4101 1a,1 Ofl-dIcsrboxytic acid 1,4a- 1,6.dideoxy-D-mannitol actone (gibbenellicacid; gibber cHin A3)

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Table 4—Continued Csrcinogenicity Salmonellatesting Carcinogenicily Salmonella testing

@ I /@ j /,@it1/ c

@ ______@/@/Chemical j/@4@ __ ‘@‘;/@Z@@L'/ ______i'@ __ @ 369. 2-0hloro.N-(2@chloroethyl)-N- 9:193 4101 +810 400. 1,2,3,4,10,iO-Hexachloro ?5:25 3 @@11— @@methylethanamineHCI(nitrogen /4a.5,6.7,8,8a-hexahydro-endo,- Chemical mustard HOt) exo-1,4:5,8-dimethanonaphtha 370. 4.{Bia(2-chloroethyl)amino)-t- 9:167 3121 +B15 lens (Aldnin) phenylsianine(Meiphalan) 401. 1,2,3,4,iO,iO-Hexachloro-6,7- 5:125 4212 3 —824 371 . 44Bis(2-chloroethy1)amino@en 9:125 3122 epoxy-4a.5.6,7,8.8a-hexahydro zenebutanoicacid (Chiorambucil) endo,exo-i ,4:5,8-dimethano 372. N,N-Bis(2-chloroethyl$etrahydro.9:135 4204 +B12 + naphthalene(Dieldrin) 2ff-i .3.2-oxaphoephonin-2- 402. 1,4,5,6,7,8,8-Heptachloro ?5:i73 B — (217) amine-2-oxidemonohydrate(cy 3a,4,7,7a-tetrahydro-4.7-meth clophoaphamide) anoindene(Heptachlor) 373. 3-(2-Ohloroethyl).2-((2-chlor +813 ethyl)[email protected],2-ox Sulfate, sulfonate, sultone azaphosphonine2-oxide(Iso 403. Dimethylsulfate 4:271 4122 ph@ide) 404. Diethyl sulfate 4:277 4112 +06 374. 5-(Bis(2-chloroethyl)amino)- 9:235 3101 +811 405. Methyl methaneaulfonate (MMS) 7:253 4213 +03 + 2,4(1H,3H).pynimidinedione(ura 406. Ethylmethanesulfonate(EMS) 7:245 4204 +04 + cii mustard) 407. 1,4-Butanedioldimethanesulfon 4:247 3122 + 375. N,N-Bis(2-chloroethyl)-2-naph 4:119 3101 +814 ate (Busulfan) thylamine 408. 2,4-Dichlorophenolbenzenesul 3101 :2 376. 2-Methoxy-6-chloro-9-(4-bis(2- +B16 fonate (Genite-R99) chloroethyl)amino-1-methylbu 409. 4-Chlorophenyl4-chlorobenzene 31 11 :2 tytaminojacnidine.2HCI sulfonate(Ovex) (ICR-i 0) 41 0. Ethyl 4-toluenesulfonate 4101 w+D5 377. Bis([4-(bis(2-chloroethyl). 9:217 41 1. 1 3-Propane sultone 4:253 4213 +012 +21: + (126) amino)phenyl)acetate)estra 1.3.5(1 0).tnlene-3,1 7/3-diol (as Sulfanilamide tradiot mustard) 41 2. 4-Aminobenzenesulfamide (sulfa 4202 378. 2-Chloro.N-(2-chloroethyl)-N- 9:2094202 nilamide) methylethanamineN-oxide HCI 41 3. 2-(4-Aminobenzenesulfonamido)- 4212 379. (2-Chloroethyl)trimethyl ammo 3111 Ci thiazole nium chloride (CCC) 380. N-(2-Chloroethy9-N-(1-methyi-2- 9:223 Phosphate phenoxyethylbenzenemethana 41 4. Tnimethylphosphate (TMP) + mine 381. 2-Methoxy-6-chloro.9-(3-(ethyt +B17 Steroid 2-chloroethyi)amlnopropylamino)- 415. Estrone 6:123 4304 acridine. 2HCI (IOR-i 70) 41 6. Estrone benzoate 4202 382. 1.1‘-Thiobis(2-chloroethane) 9:181 3102 41 7. 17fl-Estradiol 6:99 4304 (mustard gas) 418. 3-Benzoateestradiol 3203 383. Bia(2-chloroethy0ether 9:117 3111 B + (217) 41 9. Dipropionate estradiol 3203 384. 2-(4-tert-Butylphenoxy)isopropyl 5:39 3111 B 420. Ethinylestradiol 6:77 4111 —74 2-chloroethyl sulfite (Anamite) —23 421. Mestranol 6:87 3111 Haloalkyl ether 422. Progesterone 6:135 3101 385. Bie(chloromethyl)ether(BCME) 4:231 4224 +88 423. Testosterone 6:2093101 386. @hloromethyImethylether @4:2393223 424. Testosteronepropionate —23 —73 (OMME) —26 387. i-Chioro-2,2,2-tnifluoroethyl di Mli:289 425. Norethisterone 6:179 fluoromethyl ether (Isoflurane) 426. Ethynocjjoldiacetate 6:173 427. Norethynodrel 6:191 Chioroethylene 428. Cholesteryl-(+)-14-methylhexa MiO:10€ 388. Vinyl chloride 7:291 4222 w+84 -4 (217) decanoate(carcinolipin) 389. 1.1-Dlchioroethylene(vinylidene w+B5 + (217) 429. 3-Methoxy-i6,17-secoestra 2202 chloride) I ,5,5,7,9-pentaen-17-oic acid 390. Trichloroethylene 11:263 3111 +,(217) (15) Antimetabolite 391 . 2-Chloro-1 ,3-butadiene (chloro ?B7 prene) 430. 2.3-Dihydro-2-thioxo-4(1H)- 7:85 31 11 392. Bie(1-methylethyl)carbamothloic 12:69 3111 B pynimidinone (thiouracil) acid. S-(2,3-dichioro.2-propenyl) 431 . 6-Methylthiouracil 7:53 4111 eater, trans and cia isomers 432. 6-Propylthiouracil 7:67 3313 (Diallate) 433. 6-((i -Methyi-4-nitroimidazol-5- 3101 + 393. 1,llchloro.2,2-bis(4-chloro M5:83 3111 —823 — (217) yl)thio@urine(Azathioprine) phenyl)ethylene(p,p'-DDE) 434. Xanthine 4202 435. 3-Hydroxyxanthinehydrate 4213 Polychlorinatednonsromatic 436. 4-Aminofolicacid(aminopterin) 4222 394. a isomer of 1,2,3,4,5,6-hexa 5:47 31 1 1 —74 437. 5-(4-Chlorophenyl)-6-ethyl-2,4- 13:233 chiorocyclohexane —21 pyrimidinediamine(pynmetham 395. p isomerof 1,2,3,4,5,6-hexa 5:47 3111 —30 —74 ne) chlorocyclohexane —22 438. S-Ethylhomocysteine(ethionine) 4111 —F7—90 —74 396. ‘yisomerof 1,2,3,4,5,6-hexa 5:47 3111 —31 (L and Di.) —30 chlorocyclohexane(Lindane) 397. Polychlorinatedterpenes:Stro ss5:2i9 3111 B Polysaccharide bane 439. Carrageenan 10:181 398. 1,ls,2,2,3,3a,4,5,5a,5b,6-Dode 5:203 3111 440. Sodiumcarboxymethylcellulose 4101 —59 —73 cschlorooctahydro-1,3,4-meth —27 eno-1H-cyclobuta(c,dIpentalene 441. Dextran 10 4303 (Mirex) 442. Cellophane 4202 (93) Oyclochlorotine 399. 2.3,5.6-Tetrachloro-i ,4-benzo 3111 Polymer quinone (Chlorsnil) 443. Polyethylene 4202

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Table 4—Continued Carcinogenicity Salmonella testing !;/Zó5@/ f/i/i/i / @ I /4@ <@I Chemical Chemical #/@/i// /1 444. Polyoxyethylene8 monostearate 455.—J5456. 3-Amino-I,2,4-trlazole(Amitrole)7:31421 2B 445. Polyoxyethylene20sorbitsn (16)dioxidei,2-Benzisothiazolin-3-one1,1-4212—22— monoatearate(Tween 60) (saccharin)457. 7-Chloro-1,3-dihydro-3-hydroxy3:595-phenyl-2H-1 Mi Metalcomplex ,4-benzodiazepin 446. Trlphenyltinacetate (Oxazepam)Inorganic458.2-one 447. 1-Aurothio-o-glucopyranoae(au rothioglucose) 448. Iron-dextran complex (142)459. Arsenicacid,calciumsalt(2:3)e2:487101— 449. Iron-dextrin complex 77325anthophyllite,Asbestos(chrysotile.amosite,2: 1 450. Saccharatedironoxide crocidolite)460. (202)pounds461Berylliumandberylliumcorn 1:176222— Miscellaneous 451 . 3a,4a,5@-Trihydroxy-1 -cyclo (122)pounds462.. Cadmium and cadmium corn 11:394204—+ hexene-i -carboxylic acid (shlkimic acid) Chromiumandinorganicchro(142)mium 2:107222+ 452. Grlseofulvin compounds463. 453. Luteoskyrln 1:29464.Hematite(radon?)e 454. 1,2-Dihydropyrldazine-3,6-dione (202)465. Leadsalts1:404212—— (maleichydrazide) NiCkelandnickelcompounds1 1:754429 a5@T@2 b T. H. Connor, unpublished data. C Shakin (cited in Ref. 59).

IARC4expert committees(114) and by a similarly qualified the strain specificity that was observed. For 6 of these 7 committee of the Mrak Commission (62) with the carcinogens chemicals, the documentation of this strain-specific carcino identified by a discriminating search of the chemicals in Sus genicity is the same. These 6 chemicals were pesticides tested pected Carcinogens (78) prepared by the National Institute of in a large-scale study of about 130 chemicals. These chemicals Occupational Safety and Health and those carcinogens cited weretestedby a singles.c. injectionand by long-termfeeding in the 4 adequately reported correlation studies (104, 152, to 2 hybrid strains of mice of both sexes. While a partial 172, 226). The construction of Table 4 is outlined in Table 3 summary of the results was published by Innes et al. (1 13), the and discussed below. details of the testing can be seen only in a large document IARC. The IARC has convenedexpert committeesregularly available from NTIS (163). In this regard, it is also important to since 1971 to evaluate the evidence for carcinogenicity of appreciate that lnnes et al. highlighted only those 11 chemicals various chemicals. In its first 13 monographs (1 14), IARC that were carcinogenic at the 0.01 level of significance. In committees have reviewed 283 chemicals and, in doing so, contrast, 56 chemicals were considered carcinogenic by at briefly commented on the carcinogenicity testing of another 45 least one of the routes of administrationwith a 0.05 level of chemicalsthat are possiblemetabolitesof or are structurally significance in the NTIS document. Included among the 56 related to the chemicalsthat were being reviewed.Of these active chemicals were 5 of the 6 chemicals that were consid 283 chemicalsspecificallyreviewed,184 were consideredto ered not evaluable by an IARC committee because of their havebeencarcinogenicin at leastonesatisfactorilyconducted strain-specific results. animal study. One chemical was shown to be carcinogenic only Of the 45 chemicalsfor which carcinogenicitytesting was by the bladder implantationmethod,the other studies being briefly considered by the IARC committees, 30 have been used inadequately designed or reported. Four chemicals have epi in Table 4. The studies referenced (and often summarized) by demiologicalevidencefor their associationwith cancer but do the reviewingIARCcommitteeare consideredby the authors not havecorroboratinganimalstudiesto date. Ninechemicals of this paper to be adequate proof of the carcinogenicity of havehad a strain specificity in their carcinogenicityobserved these chemicals in the experimental designs used. These thus far. These latter 14 chemicals have the designations bi, e, chemicals have the designation M before their IARC reference and ss, respectively, before their IARC reference in Table 4 to in Table 4 so that they can be distinguished clearly from the highlight these aspects about their carcinogenicity testing. chemicalsthat were reviewedper se by the IARCcommittees. Sevenof the9 chemicalswithasbeforetheir IARCreference Otherchemicalsin Table4 with IARCreferenceswere iden were considered to be not evaluable by the IARC committee tified by the other criteria. (Vol. 12) that reviewed them. Apparently, this was because of EPA. The 1975 editionof Suspected Carcinogensprovides information on the carcinogenicity of 1545 chemicals. In the 1976 edition, this figure rose to 1905 chemicals (55). This 4 The abbreviations used are: IARC, International Agency for Research on Cancer; NTIS. National Technical Information Service; EPA. Environmental Pro publication only purports to present documentation of carci tection Agency; IPC, isopropyl phenylcarbamate; PCNB. pentachloronitroben nogenicity and does not judge the credibility of such findings. zene;DDT, 1•1,1-trfchloro-2,2-bls(4-chlorophenyl)ethane;DDE,1,1-dichloro 2,2-bis(4-chlorophenyl)ethylene; HMPA, hexamethylphosphoramlde; DBCP, dl However, EPA contracted the organization that prepares this bromochloropropane; 4-AAF, 4-acetylaminofluorene. publication to arrange by computer the chemicals in an updated

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version of the 1975 edition of Suspected Carcinogens to reflect stated only as being less than 0.02 (i.e., p > 0.02) (C3). the importance of their carcinogenicity documentation (78). A Apparently, the same pesticide, a-(2,4-dichlorophenoxy)- 4-digit identifier was computed for each suspected carcinogen propionic acid [also called 2-(2,4-DP)] was tested twice (113, to indicate the phylogenetically highest species of the test 163) and evaluated twice by the Technical Panel. Hence, the systems used, the number of different species tested, the actual number of carcinogens listed as C3 by the Technical routes of administration, and the total number of carcinogenic Panel is 8. The significance of 7 of these 8 chemicals has been responses reported. As fully described in the legend to Table recalculated by x2 analysis using the data presented in the 4, the speciesvary from frog to human;but some speciesare NTIS document (163). When both strains and sexes are corn actually collections of similar species; e.g., Species 1 is a bined or sex-strain subgroups are considered separately, 4 of collection of various birds. These identifiers have made possi the 8 chemicals have increased the incidence of tumor-bearing ble the enumeration of substances essentially contained in the mice relative to controls with significance at the 0.05 level: a- I 975 edition of Suspected Carcinogens which have been re (2,4-dichlorophenoxy)propionic acid; n-propyl isome; zineb; ported to be positive in 2 or more mammalian species. This and triphenyltin acetate. Only these 4 C3 carcinogenic pesti was accomplished by rejecting all chemicals with an identifier cides were used in Table 3. that indicated that one or no mammalian species had been Finally, 39 chemicals were considered not positive (C4) in tested; by immediately accepting all chemicals with an identifier the one animal system in which they were tested. The available that indicated that the number of species tested was at least 4, information on the remaining pesticides was so insufficient as and therefore included 2 mammalian species; and by looking not to allow comment in any respect. up the species in the toxic dose lines given in Suspected Those 36 chemicals which are active at the 0.05 level are Carcinogens (1 975) for the remaining chemicals to make cer presented in Table 4 under ‘‘DHEW'‘withtheir evaluations. tam that at least 2 mammalian species had been involved in Other chemicals in Table 4 with these evaluations were identi the testing. This approach yielded 202 entries: 190 were fled by the other criteria. chemicals; 7 entries were mixtures of chemicals; and 5 entries The IARC has also evaluated 23 pesticides reviewed by the were duplications of other chemicals. Only the 190 chemicals Technical Panel. The evaluations of the Technical Panel were were considered for use in Table 4. Consolidating 13 morgan consistent with those of the IARC committees, and most of the ics into their 5 respective compounds (asbestos and beryllium, discordance that does exist can be explained by the difference cadmium, chromium, and nickel compounds) reduces the total in the reports that were available to the 2 groups (e.g. , hep to I 82 chemicals that were reported positive in at least 2 tachlor). However, for 2 pesticides, both groups based their mammalian species. These 182 chemicals are presented in conflicting opinions on the same studies. The Technical Panel Table 4 under ‘‘EPA'‘alongwith other chemicals with these concluded that the testing in mice by p.o. administration of identifiers that are included in the table by other criteria. aldrin (58) was adequately Conducted and resulted in signifi The use of the chemicals thus identified in Suspected Car cant results (p < 0.01 ); in contrast, the IARC committee could cinogens is based on the assumption that it is not very likely not accept these positive findings due to the inadequate design that the testing in 2 or more mammalian species would have of the experiment. been inadequately conducted or would have given false-posi In the case of IPC, the Technical Panel rated the potency of tive results in each case. Some indication of the correctness of the carcinogenic response as C2 (p < 0.02); in contrast, the this assumption is seen by considering the 82 chemicals thus IARC committee concluded that no evidence of carcinogenicity identified that have also been evaluated by IARC expert corn was significant at the 0.05 level. The discrepancy in these 2 mittees. All but 4 of the 82 were evaluated as having been evaluations appears to be due to the difference in end points carcinogenic in at least one adequately conducted study. As that were analyzed. IPC was tested by a single s.c. injection would be expected, this criterion did not always exclude chern and by long-term feeding to 2 hybrid strains of mice of both icals with supposed carcinogenicity that is made less convinc sexes (113, 163). Only the latter mode of administration sug ing by considerations like a low survival rate associated with gested that IPC may be carcinogenic. When both strains and treatment (ziram), vehicle effects (cholesterol versus paraffin sexes are combined and the incidences of mice with tumors in wax pellets with 8-quinolinol), or the presence of carcinogenic the treated group (18 of 66) and in the 5 control groups (53 of contaminants in the test substance [chloromethyl methyl ether 338) are compared, the x2 statistic has a p value of 0.02. By a contaminated with bis(chloromethyl) ether; maleic hydrazide similar analysis, IPC was listed as ‘‘active'‘atthe 0.05 level in contaminated with hydrazine]. the NTIS document (163). Further analysis of the data suggests Department of Health, Education, and Welfare. In 1969, that the females of one of the strains contributed heavily to this the Technical Panel on Carcinogenesis, an advisory committee significance (5 of 18 in the treated group versus 8 of 87 in the to the Commission on Pesticides and Their Relationship to control group); the x2 statistic has a p value of 0.03. However, Environmental Health (62), published its evaluation of available in the IARC analysis of the data (see Ref. 114, Vol. 12, p. 189), reports on the carcinogenicity of over 100 pesticides. On the the incidences of tumor types were compared between the basis of testing for tumor induction conducted adequately in 2 treated and control groups. The differences in these incidences mammalian species, the panel listed 3 chemicals as not posi in any sex-strain subgroup, in the combined sexes of either tive. For similar testing that involved at least one mammalian strain, or in the combined strains are not significant at the 0.05 system, the panel listed 32 chemicals as having increased the level using a x2 statistic adjusted for continuity (Yates correc tumor incidence relative to controls with significance at the tion). 0.02 level (B, Cl , and C2). Correlation Studies. Among the 4 correlation studies re Nine chemicals were considered to have increased the tumor ported adequately to date (104, 152, 172, 226), 234 separate incidence relative to controls, but the level of significance was chemicals were considered as carcinogens and tested for

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Chemical Structure Carcinogenicity and Salmonella Assay mutagenicity. This figure excludes 5-iodo-2'-deoxyuridmne Table5 which was mistakenly presented in one of the correlation Synopsis of Table 4 studies as a carcinogen.3 This criterion provided 103 chemicals Chemi in in that had not been identified by any of the 3 previously de Activit?CategoryNot SalmonellaCyanamide10/10/0Substituted11/10/1diphenylethaneStllbenediol31calsTestedSalmonellaPositive scribed criteria. The documentation of carcinogenicity, given evaluable only in the reports of McCann et al. (152) and Heddle and Bruce (104), should be obtained from the respective reports. /30/1Dioxane10/10/0Anhydride21/20/1Pyrazolinone11 Thus, Table 4 is a listing of 465 compounds for which the documentation of carcinogenicity varies from strong (reviewed /10/1Pyrrolizidine50/50/0Haloalkyl by expert committees) to marginal (e.g. , those identified only by the EPA criterion that was used). As such, the reader should /31/1Sulfanilamide20/20/0Polysaccharide41ether31 appreciate the limitations of Table 4 which prevent it from being /40/1Polymer31/30/1Metal considered a listing of bona fide animal carcinogens. Further complex50/50/0Partial more, no attempt has been made to distinguish truly carcino genic chemicals from chemicals that may more properly be 13 cate9@oriesb 38 12/38 1/12 called promoters (e.g. , steroids) and solid-state carcinogens (8)HighsummariesMiscellaneous(33)C.7 (8)d5/7 (32)0/5 (e.g. , cellophane). However, it should be equally appreciated that these limitations do not prevent certain important conclu Diazo 4 3/4 3/3 sions being drawn from the results of the mutagenicity testing Azoxy 5 4/5 3/4 Nitroso 53 39/53 39/39 of these chemicals. In particular, it will be seen from the Diaryl alkynyl carbamate 3 3/3 3/3 following discussion of Table 4 that about 58% of the 465 Aromatic amine 67 43/67 36/43 Nitroaromatic 38 32/38 32/32 compounds have been adequately tested in Salmonella, that Polyaromatic 50 18/50 18/18 the testing has tended to concentrate on certain chemical types Aziridine 16 6/16 6/6 and neglect others, and that some categories of carcinogens Oxirane, thiirane 11 6/1 1 6/6 Heteroaromatic 20 13/20 12/13 exhibit individual correlations (Chart 2, ps's) that are unsatis Halomethane, 15 13/ 15 11/ 13 factorily low by any standard. haloethane N-, S-, or 0-mustard 17 10/ 17 10/10 Discussionof False Negatives from Table 45 Sulfate, sulfonate, 9 6/9 6/6 sultone Phosphate 1/1 1/1 A general synopsis of specifics is presented in Table 5. The Partial l5cateágories 312 199/312 188/199 465 compounds suggested 39 separate categories (including (94)MediumHydrazinesummariesTriazene(38)3 (67)d2/3 (64)2/2 a miscellaneous category) strictly on the basis of recurring chemical structures. The occurrence of many chemicals having Lactone 8 4/8 2/4 more than one functional group of importance made their 4/6Partial Chloroethylene12 68/12 6/65/8 ‘T@7'@ assignment more arbitrary than that of chemicals having only 4 cate9@ories 34 @7@4 one functional group. Chemicals for which a predominance of (57)LowAzosummariesInorganic(10)@.! (7)d5/8 (68)2/5 one functional group over the other(s) in the contribution to the carcinogenic activity of the chemical could not be readily Carbamyl, thiocarbamyl 21 9/21 3/9 assumed are repeated in the categories of presumedly second Phenyl 8 5/8 0/5 Benzodioxole 8 4/8 1/4 ary importance; these chemicals maintain that list number Polychlorinated cyclic 9 8/9 1/8 (enclosed by parentheses) of their primary assignment at which Steroid 15 2/15 0/2 their documentation of carcinogenicity and mutagenicity in 1/3Partial Antimetabolite11 96/11 3/92/6 Salmonella is presented. Consequently, 38 chemicals having categories (22)Final39summaries7 (18)81 (1 7)d37/81 (46)8/37 secondary groups repeat twice. Furthermore, tannic acid and tannins (Chemicals 324) appear twice: primarily as a lactone 0/271summaries(58)(77)categorlesb465271 /46521 on the basis of ellagic acid; and secondarily as a phenyl a Relative ability of the Salmonella-S-9 system (7) to detect carcinogens In compound on the basis of gallic acid. Both ellagic and gallic the given categories. ‘‘Notevaluable'‘indicateseither a small number of chemi acid are hydrolysis products of vegetable tannins, which are cals in the category or a lack of testIng; ‘‘high,―‘‘medium,'‘and‘‘low'‘indicate glucosides of phenolic acids. Since 1-nitrosonaphthalene that individual category correlations are in the upper, middle, and lower 33rd percentiles, respectively. (Chemical 160), 2-nitrosonaphthalene (Chemical 163), and 2- b Includes a miscellaneous category. nitrosofluorene (Chemical 171) represent higher oxidation C Numbers in parentheses, percentages. states of their corresponding amines, they have been classified d Percentages of respective total. with the aromatic amines. In fact, 2-nitrosonaphthalene is de tected as an urinary metabolite in dogs (but not various rodents) include known or presumable alkylating and acylating agents: treated with 2-naphthylamine (Chemical 161) (30). diazo compounds; nitrosamides; nitrosoureas; dimethylcarba The most salient commonality among the 21 0 carcinogens myl chloride; diaryl alkynyl carbamates (66, 102); aziridines; that are positive in Salmonella is the electrophilicity that is intrinsic to the molecule or introduced by enzymatic modifica oxiranes; thiirane; strained lactones; halogenated methanes tion. The former can be thought of as ultimate mutagens and and ethanes; ultimate mustards; sulfates; sulfonates; sultone; and trimethyl phosphate. Only a few intrinsically electrophilic

5 The chemicals appearing in Table 4 are followed by their chemical number carcinogens are not active in Salmonella. Penicillic acid (Chem for easy reference. ical 319), an aft-unsaturated ‘y-Iactone,couldact as an alkyl

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ating agent in a manner analogous to the reaction of such HH @ Ar—NN—Ar ‘2H Ar—N—N—Ar 2H 2 Ar—NH2 lactones with cysteine (120). Penicillin G, also not active in Salmonella (152), undergoes nucleophilic attack at the f3-Iac

tam carbonyl or rearranges in aqueous solution into an alkyl N-HYDROXYL.ATlca IjI ES1ER!FICATIce I;l Ar—NH2 a Ar—N—OH —a Ar—N—OR ating agent, benzylpenicillenic acid, which may account for the reported carcinogenicity of the antibiotic (1 43). Toxicity hinders testing of the acylating agent succinic anhydride (Chemical 325). Those carcinogens that are proximate mutagens in Salmo Araryl group; R@CH3 . S0 nella are chemicals which 5-9 can metabolize to reactive Chart 3. The reduction of aromatic azo compounds to their corresponding amines is mediated by Intestinal microflora. upon absorption, these amines would species like alkylating and arylating agents. Proximate muta be available for typical aromatic amine metabolism. Other metabolic reactions, gens generally activated by 5-9 include triazenes, azoxy corn e.g., aromatic hydroxylation. are possible but not illustrated. pounds, N-nitrosamines, aromatic amines, polyaromatics, het eroaromatics, and some proximate mustards. The nitroaromat anaerobic liquid testing in which the tester strains are prein ics, although active without activation per se, are metabolized cubated anaerobically for 16 hr at 37°in a salt solution con by bacterial nitroreductases to their reactive forms (201). taming dithiothreitol (a reducing agent) and test agent before In classifying carcinogens, overstratification emphasizes plating. In aerobic liquid testing, Citrus Red No. 2 is question their heterogeneity. The question of appropriateness of some ably mutagenic in TA100. Orange I yields ‘‘highlyvariable'‘but categories is not a concern, provided that the chemicals are apparently positive results only in TA98 when the agent is first properly categorized and their documentation of carcinogenic chemically reduced by treatment with sodium dithionite and ity is convincing. Twenty-nine of the carcinogens that have then activated with 5-9. Neither of these activities are due to been tested and not found mutagenic in Salmonella fall into the presence of contaminating impurities as evidenced by thin only 7 categories C‘low'‘categoriesin Table 4). A good majority layer chromatography. Garner and Nutman (90) tested 10 dyes (25 of 29) of these unresponsive carcinogens have been eval using only TA1538, including Sudan I, Orange I, Sudan II, uated by expert committees. Hence, the indication that 7 Ponceau MX, and FD and C Red No. 1. Only Sudan II is categories of carcinogens are poorly detected in Salmonella is mutagenic; thin-layer chromatography indicates that this activ not based on categories of questionable appropriateness. In ity is not due to contaminating impurities. many cases, the failure of these carcinogens to be active in The identification of aniline derivatives and benzidine (Chem Salmonella reflects the inadequacies of in vitro testing, in cal 126) as urinary metabolites of azobenzene administered to general, and of bacterial testing, in particular, for reasons to rats (76) and rabbits (32) indicates that azobenzene is reduced be discussed. in vivo to aniline by way of the corresponding hydrazine which Azo Compounds(Chemicals 9 to I 9). The azo carcinogens itself rearranges into benzidine upon acid extraction and (prob are dominated by the azonaphthols, some of which were once ably to a lesser extent) in the naturally acidic environments of used to color food. Citrus Red No. 2 is still an approved the whole animal. However, the predominance of hydroxylated coloring for orange skins in the United States (restricted to 2 derivatives in these same studies also indicates that oxidation, ppm). The metabolism of azo chemicals has been reviewed by presumably involving an arene-oxide intermediate, is an impor Walker (239); the toxicology of food colors in general has been tant metabolic pathway. It is not known which metabolism is reviewed by Radomski (190). Reduction in vivo of the azo occurring in the Salmonella testing that finds azobenzene ac moiety, although possible in the liver, has been largely attrib tive in TA100 with activation (152). Azoethane is an oxidative uted to the gutflora. This has been related to the more favorable metabolite of 1,2-diethylhydrazine; the metabolism of symmet anaerobic conditions in the intestine versus the liver (119) and rical hydrazines is discussed later. Phenylazoanilmnedyes may involve a nonenzymatic mechanism mediated by flavins (Chemicals 140 to 150) are presented as aromatic amines in (38, 39, 92). Accordingly, intestinal flora have been shown to Table 4 since the amino rather than the azo group is the metabolize in vitro Sudan I (48), Orange I (192), and Citrus Red understood site of metabolism (oxidation) of these chemicals No. 2 (189). Bisazo dye Evans blue (Chemical 154) is de (179). colored by intestinal bacteria, and the dye is not absorbed in Carbamyls and Thiocarbamyls (Chemicals 90 to 110). dogs if the intestinal tract is sterilized by pretreatment with The suggestion (151) that impurities may account for the antibiotics (224). When the dye is absorbed, it can be reduced carcinogenicity in rats fed acetamide can be equally said of all in the liver to benzidine and naphthylamine derivatives as chemicals of less than ultimate purity that are found to be evidenced from in vitro studies with liver microsomes and positive in such animal testing. If an impurity caused the hep trypan blue (Chemical 153), a congener of Evans blue (141). atomas in animals receiving 2.5% acetamide in their diet, the Reduction of azonaphthols yields aromatic amines that would impurity is powerfully carcinogenic but not mutagenic in Sal presumably be available for typical aromatic amine metabolism monella at its contamination level in acetamide. The observed (Chart 3) that is associated with carcinogenesis, e.g. , of the protection that equimolar amounts of L-argininyl-L-glutamate bladder (121). Brown et al. (40) tested over 37 dyes in Sal affords rats treated with acetamide may be indicative of some monella, including 4 azonaphthols that appear in Table 4. All metabolic specificity of action, the nature of which remains to 4 azonaphthols are not mutagenic in standard plate testing be investigated more fully (244). Ushioda (238) has determined with and without 5-9 activation. Sudan I, Citrus Red No. 2, and that tritiated acetamide is incorporated into DNA and, to a FO and C Red No. 1 are not mutagenic in similar testing that lesser extent, into RNA of the mutant bar larvae of Drosophila. involved a 16-hr anaerobic incubation of plates before the It was not stated if the label is an adduct product with bases standard (aerobic) incubation; Orange I is not mutagenic in (mainly thymine) or is part of the base nucleus. N-Hydroxyacet

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0 amide is known to be a teratogen in rats (47) and a clastogen 0 OOH 0CCH3 @ in mouse embryo cells in vitro (25). However, Poirier (177) H3cH2co—c—NH2— H;plf2co-c-NH •H,@CH2C0—@--NH found that N-hydroxyacetamide was not carcinogenic to rats Chart 5. Possible metabolic activation of urethan to acetyl derivative of N- at its highest tolerated dose and was not detectable in the urine hydroxyurethan [Nery (168)1. of rats fed acetamide. mode of action of urethan or its N-hydroxy metabolite may also Thioacetamide is almost entirely converted in vivo by rats to be as an antimetabolite in pyrimidine biosynthesis. Boyland acetamide, which is subsequently converted to acetate. Liver and Koller (29) found that the frequency of chromosome ab slices are 3 times more active than kidney slices in this metab normalities induced by urethan (but not nitrogen mustard) in olism. The ability of the liver to metabolize thioacetamide so Walker rat carcinoma is reduced and recovery is accelerated rapidly has been related to the hepatocarcinogenicity of the by simultaneous or previous treatment with thymine (but not chemical. However, the ultimate carcinogen is not thought to uracil or guanosine). Elion et al. (73) demonstrated that the be acetamide since thioacetamide causes hepatomas at a small inhibitory effect of urethan on the growth of mammary adeno fraction of the dose that is necessary for the carcinogenicity of carcinomas [implanted into axilla of male mice (74)] can be acetamide (196). In phenobarbital-pretreated rats, thioaceta reversed by treatment with various pyrimidines and pyrimidine mide, sodium diethyldithiocarbamate, thiourea, thiouracil anabolites. (Chemical 430), 6-methylthiouracil (Chemical 431 ), and 6-pro While the studies of Boyland and Koller and Elion et al. which pylthiouracil (Chemical 432) cause a loss in hepatic cyto were conducted in vivo do suggest that urethan has a specific chrome P-450 or inhibit the oxidative N-demethylation of benz ity for pyrimidine biosynthesis, Kaye (127) could not demon phetamine. This has been related to the mixed-function oxi strate in vitro any significant inhibition by urethan of several dase-catalyzed release and covalent binding of a reactive form enzymes involved in nucleic acid metabolism. Both urethan of sulfur to cellular components (112) (Chart 4). and its N-hydroxy metabolite bear a structural resemblance to Thiourea and the 3 thiouracils are considered to be antithy carbamyl phosphate and carbamyl-L-aspartate. The enzyme roid compounds because they induce thyroid cancer in rodents aspartate transcarbamylase begins pyrimidmnebiosynthesis by by inhibiting the organification of iodine and, therefore, the catalyzing the formation of carbamyl-L-aspartate from carbamyl synthesis of thyroxine. The resulting imbalance in the hypo phosphate and L-aspartate. Gin and Bhide (93) have reported thalamopituitary-thyroid hormonal system subjects the thyroid that in vivo administration of urethan decreased aspartate to continuous stimulation by thyroid-stimulating hormone, transcarbamylase activity of lung tissue of adult male and (to a which eventually causes a neoplastic condition in the gland lesser extent) female mice; no in vitro inhibition could be (see Ref. 114, Vol. 7, pp. 23—26).However,the production of demonstrated, a reactive species from oxidative desulfurization may be the The dithiocarbamates are subdivided into 2 structural cause of the concurrently observed carcinogenicity in the liver groups: the dialkyldithiocarbamates and the ethylene(bis) and other organs with these thiocarbamyl-containing antithy dithiocarbamates. Only one dithiocarbamate has been ade roid compounds. Thioacetamide and thiourea (as well as acet quately tested. The lack of mutagenicity of maneb (126) mdi amide) have been reported to be positive in transforming ham cates that its carcinogenic metabolite, 4,5-dihydroimidazole ster embryo cells (Pienta, cited in Ref. 65). 2(3H)-thione, which itself is weakly positive in TA1535 (but not That N-hydroxyurethan but not urethan is positive with S-9 TA100) without activation (231 ), is not adequately produced activation inicates that the 5-9 cannot adequately perform the by 5-9 activation. A common metabolite of both types of multiple-step activation of urethan (Chart 5). N-hydroxyurethan dithiocarbamates is carbon disulfide. Its carcinogenicity and and some acyl derivatives have been shown to acylate in vitro mutagenicity are essentially uninvestigated (63). However, pro the amino group of cytosine with no reactions occurring at longed inhalation of small concentrations of carbon disulfide other sites or with other bases. The resulting product is easily and hydrogen sulfide has been reported to induce aneuploidy deaminated by both acid and enzyme (phosphodiesterase) and polypoidy in bone marrow cells of male rats (14). The hydrolysis. Therefore, these conditions when used for the oxidative desulfurization of carbon disulfide by rat liver micro degradation of DNA treated in vivo will preclude the identifica somes is catalyzed by the mixed-function oxidase system and tion of the N-acylated product although a corresponding in results in the production of carbonyl sulfide and a reactive crease in 2'-deoxyuridylate should occur (168). However, sulfur atom (57, 61); the former is further metabolized to carbon Pound et al. (182) have shown the formation of alkyl esters dioxide and another reactive sulfur atom (56). with the phosphate groups of DNA in the liver of intact and Carbon disulfide, carbonyl sulfide, and the singlet form of partially hepatectomized mice treated with isotopically labeled atomic sulfur are electrophilic and should readily react with urethan. nucleophiles. The nucleophilic attack by protein amino groups Both of these activities contrast, although not necessarily on the electron-deficient carbon of carbon disulfide to yield a conflict, with the findings in adult (158) and newborn mice (23) dithiocarbamate that cyclizes into a thiazolidone has been that 70% of N-hydroxy['4C]urethan is converted in vivo to known for some time (e.g. , Ref. 50) (Chart 6). It would seem [‘4C]urethan.Thissuggests that urethan is metabolically closer unlikely that the short-lived, reactive species from oxidative to the ultimate species of the carcinogen than N-hydroxyure desulfurization in the endoplasmic reticulum could migrate to than in the carcinogenicity of both chemicals in mice. One the nucleus to react with nuclear targets (e.g. , DNA). The finding of mixed-function oxidase activity in nuclei (34, 35) has @ [.0.] ç:Ø ,@ the obvious importance of suggesting a nuclear source for @ c=s _c@ —@ _c—@ —@ _c=o S these (and other) short-lived species. It is not known if carbon Chart 4. Oxidative desulfurization of thiocarbamyl compounds leading to the disulfide is produced in the standard plate testing of maneb. release of electrophilic sulfur [modified from the report of Dalvi et al. (57)1. The metabolism of monuron in the rat consists of oxidative

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@ cs2 -H2O its reactivity with GMP (252), and its ability to induce unsched RCH-NH2 @, RçH—N—f S a @ HO-C HO-C :SH RçH—NH uled DNA repair synthesis (206). That neither safrole nor its 1 ‘- 0 0 0 S hydroxy metabolite are mutagenic in Salmonella with activation Chart 6. Reaction of carbon disulfide with nucleophilic center in an amino indicates that the S-9 cannot adequately metabolize these acid (modified from the report of Cohen et a!. (50)). chemicals to the 1‘-acetoxymetabolite(Chart 8). However, the importance of this metabolite in the carcinogenicity of 1‘-hy N-demethylations, ring hydroxylation, and, to a limited extent, droxysafrole, and therefore of safrole, is not clear. Further degradation of the urea residue to yield an aromatic amine more, dihydrosafrole and the pesticidal synergists, n-propyl (79). Using 4 microbial systems, Simmon et al. (218) tested 20 some, piperonyl sulfoxide, and piperonyl butoxide, lack the pesticides, including monuron, simazine (Chemical 120), and allyl group but are carcinogenic. Lawley (137) has postulated PCNB (Chemical 224); no positive results were reported for the production of a carbonium ion at the methylene group of these pesticides with or without S-9 in Salmonella or the other benzodioxoles in a fashion analogous to its metabolic reactions systems. with the mixed-function oxidases (105). Phenyls(Chemicals 219 to 226). Phenylsand polyhalogen Bactericides. McCann et al. (151) have noted that bacterial ated phenyls are metabolized to their hydroxy derivatives, each toxicity hinders the testing of 9 chemicals, including trans presumably through a benzene oxide intermediate (e.g., Refs. a,a'-diethyl-4,4'-stilbenediol (Chemical 236) and succinic an 89, 103, 125, 133, and 198) (Chart 7). Aromatic hydroxylation hydride (Chemical 325). Faced with toxicity, some researchers also occurs in the metabolism of oxazepam (Chemical 457) as have utilized cold liquid testing and pulse testing to demon evidenced by the production of hydroxyphenyl metabolites in strate the mutagenicity of the disinfectant povidone-iodine the urine of the rat and (to a lesser extent) pigs and humans (200) and 1,1,2,2-tetrabromoethane (33), respectively. These (221 ). Lutz and Schlatter (144) have demonstrated covalent studies indicate the sometimes close association between bac binding in vivo of a benzene metabolite with the DNA of livers tericidal and mutagenic activities and the need to investigate of rats exposed to isotopically labeled benzene in an inhalation this possibility by resorting to nonstandard procedures. chamber. In terms of alkylated nucleotides per mol of DNA Halogenated Compounds. As notedby McCannet al. (151), phosphate, the potency of benzene calculates to be several some halogenated compounds are apparently not adequately orders of magnitude lower than that of N-nitrosodimethylammne activated by S-9. However, many alkyl halides which are vola (Chemical 37), which itself is a proximate hepatocarcinogen. tile are demonstrably mutagenic only when tested in a desic A low rate of metabolism of benzene by microsomal and S-9 cator apparatus (217). Those compounds that are mutagenic preparations may be partially responsible for the lack of mu tend to be either monohalomethyl compounds (Chemicals 358 tagenicity in vitro (Shakin, cited in Ref. 59). However, since to 367) and ultimate mustards (Chemicals 369 to 371 , 374 to benzene oxide once formed is significantly stable (124), it may 376, 381 , 383), both of which are alkylating agents, or chlo be necessary to exclude competing nucleophiles present in the roethylenes (Chemicals 388 to 392) and proximate mustards S-9 in order to demonstrate mutagenicity in vitro. A sizable (Chemicals 372 and 373) which undergo metabolism to reac fraction of the phenol metabolites produced by a S-9 prepa tive epoxides (24, 97) and mustards (153), respectively. ration are conjugated with sulfate and glucuronic acid (229). Hexachlorocyclohexanes (Chemicals 394 to 396) undergo Harper et al. (101) observed the following order for the Vmaxof dehydrochlorinations to yield initially pentachlorocyclohexene the conversion of benzene to phenol by different microsomal and eventually a variety of polychlorobenzenes (123). The preparations: rabbit lung = hamster liver > rabbit liver > pentachlorocyclohexene derivative is almost entirely absorbed hamster lung > rat liver = rat lung. Tetrachlorobiphenyl is by the kidney but is not released into the urine (77). The metabolized by monkey liver microsomes to a species that can aromatized metabolites are also susceptible to hydroxylation react covalently with RNA (21 1). A relationship between in as evidenced by the presence of polychlorophenols in the urine creasing chlorination of biphenyl and decreasing metabolism of rats treated with either lindane, its pentachlorocyclohexene by microsomes isolated from the livers of phenobarbital-pre treated rats has been observed (91). The metabolism of PCNB (27) in the rat, dog, and bovine is rapid and does not involve storage of PCNB in fat, kidney, liver, or skeletal muscle. In contrast, the contaminants of technical grade PCNB, hexa- and pentachlorobenzene, do accumulate in these tissues. PCNB is reduced presumably in the liver to pentachloroaniline which probably is conjugated with glucuro nides and sulfate for excretion. Methyl pentachlorobenzene R1,R2 H,H (benzene) sulfide has also been identified as a metabolite. Al-Kassab et al. (2) have shown that rat liver supernatant contains a gluta I' thione S-aryltransferase system that catalyzes the replacement of the nitro group of PCNB (and other nitroaryls) with glutathi — ,H (blphenyl) one. Benzodioxoles (Chemicals 227 to 234). Benzodioxolesare ,\ more commonly known for the studies on safrole. Interest has ,CI (polychlorinated biphenyl) centered on the activation of the allyl group in safrole (252). Cl The mutagenicity of 1‘-acetoxysafrolewithoutactivation con Chart 7. Metabolic conversion of phenyl compounds to reactive benzene curs well with the enhanced electrophilicity of its allyl group, oxides.

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O—'CH2 radicals of chlorine and trichloromethyl and supports the hy 1@i pothesis that such species initiate an autocatalytic peroxidation of lipid membranes which results in the observed hepatotoxicity Ir@__ Ii @—I II (193, 235, 237). A similar scheme for radical formation and lipid destruction has been described by Reynolds and Molsen HT—cH=cH2 HJ—CHZCH2(197) for halothane. In contrast to the reductive dechlorination OH OgCH3 of carbon tetrachloride, the metabolism of chloroform to carbon Chart 8. Possible metabolic activation of allyl group in safrole [Wislocki et al. dioxide in vitro requires oxygen and produces carbonyl chlo (252)). ride (phosgene) as an intermediate (148, 176). That this also occurs in vivo is suggested by the similarity in production of derivative, or a trichlorobenzene (99) (Chart 9). The aromati isotopically labeled carbon dioxide upon acid hydrolysis of zation of a-hexachlorocyclohexane involves a cytosolic enzyme polylysine and albumin treated in vitro with carbonyl chloride that has a specific requirement for glutathione and is induced and liver protein from rats treated in vivo with carbon tetrachlo by pretreatment with substrate; the process proceeds by way ride (45) (Chart 11). of dehydrochlorinations to yield presumably chlorinated thio The metabolism of DDT (Chemical 355), and presumably its phenol(s) (181). This metabolic activity can also be demon analogs (Chemicals 356, 357, and 393), involves a series of strated in nuclear, mitochondrial, and microsomal subfractions reductive dechlorinations and dehydrochlorinations (174). Bu from liver when they are supplemented with glutathione; in selmaier et al. (43) have reported that 1,1-dichloro-2,2-bis(4- contrast to the cytosol, these fractions are not induced by chlorophenyl)ethane is mutagenic with Serratia marcescens pretreatment with substrate. Extrahepatic metabolism of a-hex but not with Salmonella strain G46 in the host-mediated assay; achlorocyclohexane is also widespread (135). DDT, DDE, and di(4-chlorophenyl)acetic acid were not positive Polychlorinated cyclodienes aldrin (Chemical 400) and hep in similar testing. tachlor (Chemical 402) are converted in vitro by rabbit liver Steroids (Chemicals 415 to 429). Steroidsare evolutionarily microsomes to their corresponding epoxides (Chart 10). The recent chemicals and reflect the necessity for multicellular epoxidase has the characteristics of the mixed-function oxi organisms to coordinate their cells for homeostasis. The bio dase system in that it requires both NADPH and oxygen and it logical action of steroids is mediated by their binding to a is inhibited by SKF 525-A (162). Dieldrin (Chemical 401 ) is cytoplasmic receptor in target cells and the translocation of detoxified in vivo in the rat through hydrolysis of the epoxy this complex to the nucleus for association with chromatin group to a trans-dihydrodiol which subsequently is oxidized to material; in some manner, this allows control of gene expres the dicarboxylic acid; other metabolites include 9-hydroxy sion. Some steroids can act as mutagens by causing chromo dieldrin and a pentachloroketone structure (13). some aberrations, aneuploidy, polyploidy, and dominant lethal Mirex (Chemical 398) is essentially metabolically inert (242, mutations (1 1, 17, 199, 212, 250, 251 ). Presumably, the 248), although reductive dechlorination can be expected as a cytoplasmic receptors of target cells which confer specificity pathway of degradation in vivo. In fact, Stein and Pittman (223) to the biological action of steroids should also confer specificity have identified a monohydro derivative of mirex in the feces of rhesus monkeys. The literature on mirex covering the years 1947 through 1976 has been abstracted by Waters and Black (241). Strobane (Chemical 397), a mixture of chlorinated cam :@ -@L@CuI@%COCl phene, pinene, and related polychlorinates, exhibited no mu tagenic activity with or without S-9 in TA1535, TA1537, or TA1538; but it tripled and doubled the mutation frequency in TA100 and TA98, respectively, when present at 4800 pg/plate without S-9. This activity was greatly reduced in the presence of 5-9 suggesting detoxification or protection by S-9 against a mutagenic species (possibly chloromethyl) in the heteroge H0ôCl [Cl@@@] neous mixture (Table 2). Saleh et al. (205) have proposed that the metabolic reductive dechlorinations and dehydrochlorina Chart 9. Metabolic aromatization and possible epoxidation of hexachlorocy tions of toxaphene, a mixture of polychlorinates of camphene, clohexane (lindane) [modified from the report of Grover and Sims (99)). leads to detoxification; a radical intermediate is thought to be generated in the reductive dechlorination by microsomal en zymes. Halomethyl compounds are subdivided into monohalome ::$cH2 _:iJ@Et@0 :@ —:*‘ thyls which are alkylating agents and polyhalomethyls which must be metabolized to an ultimate species. Reductive dechlor Chart 10. Metabolic epoxidation of aldrin (left) and heptachlor (right) (Nakat ination of carbon tetrachloride (Chemical 353) to chloroform sugawa et al. (162)).

by rabbit liver microsome parallels the concentration of cyto 0 @ chrome P.450 in the mcirosomes but requires anaerobic con Cd4 AftAERo@tc,[.Ccl3J -@ HCCI3 a [HO-cd3] CCI2 ditions and NADPH. The identification of hexachloroethane Chart 11. Reductive dechlorination of carbon tetrachloride and subsequent after incubation of NADPH-reduced microsomes with carbon oxidation of chloroform to reactive phosgene [Rechnagel and Glende (193); tetrachloride is indicative of homolytic formation of the free Mansuy et al. (148); PohI et al. (176)).

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. S. J. Rinkus and M. S. Legator to any mutagenic action of steroids. This model also predicts administration of L-ethionine suggests that ethylation is not that chemicals which are analogs of steroids, so-called anti mediated by methyltransferases that normally utilize S-adeno steroids, and chemicals which can induce oversecretion of syl-L-methionine and therefore some unknown mechanism of endogenous hormones may act as mutagens. While mutagen action is involved (228). L-Ethionine is naturally produced in icity may be limited to alterations in chromosome structure and some strains of bacteria including Escherichia coli B (81). If numbers, as Ohno (173) has proposed, such mutations could L-ethionine is also a natural metabolite for Salmonella, such still give rise to cancer if indeed the disease is the expression may explain its lack of mutagenicity. of a recessive trait. Whether the carcinogenicity of steroids is Shikimic acid (Chemical 451 ) may be one of probably several related to gene expression, some mechanism of promotion, or carcinogenic agents in bracken fern. It is a murine mutagen in mutation remains to be further investigated. However, the lack the dominant lethal test (reviewed in Ref. 222). However, of mutagenicity of ethinylestradiol and testosterone propionate Hirono et al. (109) contend that the carcinogenicity of bracken in Salmonella is not unexpected. fern is not due to its endogenous concentration of shikimic Antimetabolites (Chemicals 430 to 438). Azathioprine is a acid; rats did not develop tumors when fed shikimic acid at a conjugate of 6-mercaptopurine with a nitroimidazole carrier dosage twice that found in a bracken fern diet which induces designed to protect the sulfhydryl group from rapid methylation tumors in all feeding animals. Shikimic acid is also an anabolite which once was thought to inactivate 6-mercaptopurine. Aza in the synthesis of aromatic amino acids by those organisms thioprine is converted in vivo in the mouse, dog, and human to which are capable of such synthesis; this excludes rodents and free 6-mercaptopurine, which is further metabolized to the humans for whom phenylalanine and tryptophan are essential thioanalogs of IMP, GMP, and dGMP (75). 6-Thioinosine 5'- amino acids. In fact, shikimic acid serves as a nutrient for some monophosphate suppresses de novo biosynthesis of purines strains of E. coli, which may explain in part its lack of muta by ‘‘pseudofeedback',inhibition of several key enzymes (156). genicity in Salmonella testing. The observed mutagenicity of 6-mercaptopurine only in base The initial testing for carcinogenicity ofthe tryptophan catab pair mutants (107) concurs with the fact that 6-thiodeoxy olite 3-hydroxyanthranilic acid (Chemical 115) stemmed from guanosine 5'-monophosphate is incorporated into DNA (165). the once-held belief that o-hydroxylated derivatives were the However, Rosenkranz et al. (202) have shown that in TA100 ultimate carcinogens of aromatic amines. Therefore, it was the mutagenic activity of azathioprine is derived from the ni reasoned that the catabolism of tryptophan would serve as an troimidazole carrier; conditions which favor the reduction of endogenous source of carcinogens which presumably contrib the nitro group by bacterial enzymes are necessary to dem ute to the spontaneous appearance of cancer. The significance onstrate a dose response. Hence, these findings in vitro may of bladder cancer produced by surgically implanting choles not be entirely descriptive of the mutagenicity of azathioprine terol (but not paraffin wax) pellets containing this chemical into in vivo. Whether the carcmnogenicityof azathioprine is due to the bladder is embroiled in the controversy over the relevance the nitroimidazole carrier or liberated 6-mercaptopurine is dif of this mode of administration (see Ref. 49). Injection of this ficult to assess. While treatment with 6-mercaptopurine in chemical s.c. has been reported to cause myeloid leukemia in duced thymic lymphomas in neonatal mice (67) and hemato mice when compared to historical controls (71). poietic tumors in mice and rats (183), its immunosuppressive The aromatic ring of 3-hydroxyanthranilic acid is opened action may also play some part in the development of these enzymatically by a supernatant of rat liver homogenate. The tumors. resulting semialdehyde can internalize the amino nitrogen to Aminopterin, pyrimethamine, and the sulfanilamides (Chem form either quinolinic or picolinic acids; if no cyclization occurs, icals 412 and 413) are inhibitors of the enzyme dihydrofolate glutaric acid can be formed (170). Priest et al. (185) obtained reductase. As such, aminopterin and sulfanilamide can be used quantitative conversion to quinolinic acid with the supernatant to induce thymidine deficiency in bacterial testing. Freese (85) of rat liver homogenates. If 3-hydroxyanthranilic acid is carci predicted that thymine starvation may cause transversions. nogenic through typical aromatic amine metabolism, mutagen Thymine starvation of thymine auxotrophs of Salmonella also icity may not be observed because of the dominance in 5-9 of induced what appear to be deletions (110). Such activity may this alternative metabolism. Furthermore, any interpretation of be a cause of the clastogenicity observed in cell cultures carcinogenicity or mutagenicity testing results must take into treated with methotrexate (19). Like methotrexate, aminopterin account the instability of 3-hydroxyanthranilic acid under phys is probably not easily taken up by Salmonella tester strains iological conditions (175). Hence, the conclusion of Bowden et since they do not require folic acid to grow. Interestingly, in al. (28) that tryptophan metabolites including 3-hydroxyan Lactobacillus arabinosus, the uptake of pyrimethamine does thranilic acid ‘‘donot function as causative agents for chemical not appear to involve active transport, and that of aminopterin carcinogenesis in the large intestine' ‘asevidenced by their but not folic acid is mediated by an active system that normally lack of mutagenicity in Salmonella must certainly be qualified takes up thiamin (254). The pesticide simazine (Chemical 120) (as the authors noted) for reasons discussed (some of which also may be active antimetabolically as an analog of folate (12) were not noted by the authors). as well as pyrimidine (74). Two miscellaneous chemicals may also behave antimetabol The ethylation of hepatic DNA and RNA of rats given a large ically as their modes of action. The plant growth retardant dose of isotopically labeled L-ethiOfline (228) agrees with the maleic hydrazide (Chemical 454) may be regarded as either a reported heptocarcinogenicity of the amino acid in rats (80). pyrimidine or a purine analog on the basis of its structure in its S-Adenosyl-L-ethionine, an antimetabolite of S-adenosyl-L-me crystalline state. The geometry also suggests that, if the N- thionine, accumulates in the liver of rats treated with L-ethionifle nucleotide is formed and incorporated into nucleic acid, base (215). However, since 7-methylguanine is not generally found pairing with adenine and guanine are plausible (54). Maleic in DNA, the identification of only 7-ethylguanine in DNA after hydrazide inhibits the incorporation of tritiated thymidine and

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Chemical Structure Carcinogenicity and Salmonella Assay uridine into the nucleic acids of corn seedling roots without affecting their cellular uptake during the first 12 hr of treatment H2N@c7 :!NO H2N@@fl $20 (171). Isotopically labeled maleic hydrazide is incorporated into RNA, but not DNA, of young willow tree roots (53). These H Phosphoalbo.. Illbose findings have been likened to similar activities observed with Chart 12. Structural comparison (from left to right) of amitrole, 5'-phospho 5-fluorouracil and its deoxyribose nucleoside (171). The clas ribosyl-5-aminoimidazole-4-carboxylic acid, virazole. and 2-amino-i ,3,4-thiadi togenicity of maleic hydrazide in plant cells has been known azole. for some time (147), but apparently clastogenicity is not ob served in mammalian cells treated in vitro although the chemi Hydrazines (Chemicals 25 to 36) and N-Alkyl Compounds. cal is cytotoxic and inhibits mitosis (155). Toth (233) lists 9 symmetrical and 10 unsymmetrical hydra The herbicide amitrole (Chemical 455) has several docu zines as tumorigenic compounds. From their studies with ace mented effects on metabolism. In E. coli, histidine and adenine tylhydrazine and isopropylhydrazine, Nelson et al. (167) have together reverse the inhibition of growth by amitrole, whereas proposed that monoacyl and monoalkyl hydrazines are oxi adenine alone, but not histidine alone, partially relieves the dized by microsomal enzymes to diazenes which fragment into inhibition. In the yeast Torula cremoris, histidine alone, but not reactive radicals. The implication of radicals as the ultimate adenine alone, completely reverses the inhibition (246). A reactive species also had been made earlier in a study by metabolite identified as a conjugate of alanine and amitrole has Freese et al. (87) on the inactivation of transforming DNA by been shown to accumulate in the medium of E. coli cultured in various hydrazines in the presence of oxygen and transition the presence of amitrole; it apparently competes with histidine metals. The findings that hydrazine and N-(2-hydroxyethyl)- for incorporation into protein (249). Unlike amitrole, this me hydrazine are mutagenic without 5-9 to base-pair mutants tabolite does not inhibit the conversion of imidazole glycerol TA1530 (152) and TA1535 (see Table 2), respectively, suggest phosphate to imidazole acetol phosphate by imidazole glycerol that a nonenzymatic mechanism, possibly what Freese et al. phosphate dehydratase in Salmonella; the reaction is part of (87) observed or hydrazone formation with DNA, also occurs. the biosynthesis of histidine (108). In the presence of 5-9, the mutagenicity of N-(2-hydroxy The antithyroid effects of amitrole in the whole mammal result ethyl)hydrazmneis enhanced as predicted. The mutagenicity of from the inhibition of peroxidase, thereby preventing the or hydrazines in general has been reviewed by Kimball (132). ganification of iodine (1, 225). The resulting thyroxine defi According to the proposal of Nelson et al. (167), the carci ciency subjects the thyroid to continuous stimulation by thy nogenicity of isoniazid would be dependent on its acetylation roid-stimulating hormone that induces eventually a malignant which gives rise to acetylhydrazine (Chart 13). This then par change in the organ (234). However, as with other antithyroids, tially reverses the argument of Freese (86) and predicts that the hormonal imbalance does not necessarily explain the he ‘‘rapid inactivators,' ‘although less sensitive to the antidepres patocarcinogenicity that is also obseved (113, 163). sant effects of hydrazines by virtue of rapid acetylation, will be How amitrole affects purine metabolism was indirectly inves more apt to produce the reactive species and suffer mutagenic tigated by Rabinowitz and Pricer (1 88), who demonstrated that and carcinogenic events. An inability of 5-9 to produce and amitrole blocks the enzymatic degradation of 4-aminoimidazole oxidize acetylhydrazine from isoniazid may account for the lack in extracts of Clostridium cyclindrosporum. Since amitrole is of mutagenicity in Salmonella testing. However, Braun et al. an isostere of 4-aminoimidazole, it has been proposed that (31) have equated the mutagenicity of isoniazid in the host amitrole could interfere with purine catabolism (246). However, mediated assay with the release of the mutagenic hydrazine this activity cannot be related to mammalian carcinogenesis moiety. since that degradation scheme is not observed in mammals; Nelson et al. (1 67), elaborating on a postulation by Preuss purines are degraded to and excreted as uric acid in primates man et al. (184), have also proposed a reaction mechanism for and allantoin in mammals other than primates. However, there hydrazines symmetrically substituted with alkyl groups. Again, are several anabolic metabolites of purine metabolism to which a radical is produced for alkylation after the hydrazine has amitrole is practically isosteric, e.g., the base of 5-amino been oxidized to its azoxy derivative followed by oxidative imidazole-4-carboxylic acid ribonucleotide, a metabolite in the removal of one of the N-alkyl groups. One of these steps is biosynthesis of IMP. Tjalve (232) has noted that in mice amitrole apparently deficient in the activation by 5-9 of 1,2-dimethyl preferentially accumulates in tissues with rapid cell turnover hydrazine and procarbazmne.The importance of intestinal bac (DNA synthesis);only moderate accumulationoccurs in the teria in the metabolism of 1,2-dimethylhydrazmne has been thyroid. The 5-amino derivative of amitrole, guanazole, pos discussed (194). sesses antitumor activity; guanazole and (to a lesser extent) Oxidative N-dealkylation is implicated in the activation of amitrole are cytotoxic against a leukemic cell line in vitro (100). several N-dialkyl carcinogens. In the metabolism of N-nitroso The antiviral agent virazole is a ribonucleoside of 1,2,4-triazole dimethylamine (Chemical 37) as postulated by Magee (145), 3-carboxamide (216, 253) and a somewhat similar chemical, N-demethylation produces N-nitrosomethylammne which 2-amino-i ,3,4-thiadiazole, is anabolized in vitro into 2 NAD quickly rearranges first into a methyldiazohydroxide and sub analogs that are potent inhibitors of IMP dehydrogenase (166) sequently into a methyldiazonium cation that releases methyl (Chart i 2). carbonium ion for alkylation. MaIling (146) had to resort to a Interestingly, Rosenkranz et al. (202) have reported occa 20-mm incubation of N-nitrosodimethylamine with a bacterial sional false-positive results with amitrole and have attributed tester strain in a solution of postmitochondrial enzymes in order them to phenotypic curing. It is not known if this phenotypic to achieve this metabolism and demonstrate mutagenicity. curing is due to the action of amitrole as a histidine analog in However, without the incubation period, so-called liquid testing, protein synthesis or as a RNA analog in mRNA synthesis. mutagenicity in the presence of 5-9 is not observed, probably

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conformational change in the DNA, so-called base displace @ H2N@NH@@@4 AcEr@LAsF ‘-“@:c:çHO.L(@@@ ment, that is different from base substitution, deletion, and intercalation. The failure of classical intercalating agents to { @j.—[email protected]@2cH3cNH—NH1—cH3NH—NH2revert the frame-shift mutants in the absence of S-9 suggests a specificity for intercalation between base pairs. For instance, Chart 13. Formation of a reactive metabolite after acetylation of isoniazid (modified from the report of Nelson et al. (167)). agents with an affinity for thymine pairs might not be active in the Salmonella frame-shift mutants the mutated sites of which due to the short-lived nature of the reactive species and the consist of guanine-cytosine pairs (6). The lack of mutagenicity dilution that occurs with plating. Similarly, N-nitrosodiethyla with gibberellic acid (Chemical 323) might be related to such mine (Chemical 44), N-dimethyltriazenes (Chemicals 3 and 4), specificity. Gibberellin A7, a plant hormone that differs from and N,N-dimethyl-4-(phenylazo)benzenamine (Chemical 142) gibberellic acid (also called gibberellin A3)by lacking a hydroxyl and its congeners (Chemicals 141 and 145) require liquid group, changes the physical state of DNA in a way character testing to demonstrate weak mutagenicity (151). The lack of istic of intercalation. In contrast to the classical intercalating mutagenicity with auramine (Chemical 137) in plate testing also agents, the binding of gibberellin A7 requires the presence of may be related to this difficulty in activation by N-dealkylation. divalent magnesium cation and is quite specific for double Oxidative N-demethylation produces formaldehyde, a classic stranded DNA rich in adenine and thymidmne.Whether these mutagen (reviewed in Ref. 10). Butterworth6 has suggested findings are descriptive of the biological action of gibberellic that the lack of mutagenicity in Salmonella with HMPA, an acid may depend on whether gibberellic acid is dehydroxylated inhalation carcinogen in the rat (255), is related to the insen to gibberellin A7 since the presence of the hydroxy group sitivity of the bacteria to formaldehyde. HMPA, the hydroxy negates this activity. Interestingly, the observation that single methylpentamethyl analog of HMPA, and formaldehyde are all strand nicks in DNA increased its interaction with gibberellmn active in the mouse lymphoma system, the former only in the A7 led to a series of experiments in which it was found that presence of 5-9 (see also Ref. 9). Aldehyde formation may also DNA ligase catalyzes, in the presences of the gibberellin, the be of minor importance in the biological actions of the pesticide formation of duplex covalent circles (129—131). Gibberellic simazine (Chemical 120) and of the anticancer drug hexa acid also acts as a chemosterilant in a cotton leafworm (204). methylmelamine (22, 26), both of which are N-ethylated tria Chloroethylenes (Chemicals 388 to 393). In addition to zines. vinyl chloride and vinylidene chloride, di-allate is activated by Phenacetin (Chemical 117) is deethylated in vivo to aceta S-9 to a mutagen for TA1535 and TA100 but not for any of the minophen in humans (37) and in the rat (69). Oxidative 0- frame-shift mutants (see Table 2). These findings corroborate deethylation is inhibited in vivo in the rat and in S-9 from rat those reported by De Lorenzo et al. (60) who also found that liver by the mixed-function oxidase inhibitor, SKF 525-A (52). tri-allate (the 2,3,3-trichloro analog of di-allate) and sulfallate Phenacetin and acetaminophen can undergo N- or aromatic (2-chloroallyl N-diethyldithiocarbamate) are metabolized by hydroxylation, giving rise to species that are susceptible to S-9 to mutagens. Trichloroethylene is presumably metabolized attack by nucleophilic macromolecules (169) (Chart 14). Feed by rabbit liver microsomes to its epoxide (236), which rear ing of N-hydroxyphenacetin (Chemical 118) p.o. significantly ranges in vitro into dichloroacetyl chloride but in vivo into increased the incidence of hepatocellular carcinoma in male chloral (24). Trichloroethylene (analytical grade) caused a dou rats (44). Acetaminophen-induced hepatic necrosis in mice bling in the reversion to arginine prototrophy in E. coli Ki 2 only increased as covalent binding of isotopically labeled acetamin when microsomally activated (97). However, TA100 did not ophen increased; binding of the label was localized in the respond in plate testing with and without 5-9 activation (106) endoplasmic reticulum and to proteins in the cytoplasm (118). and maximally showed only about 100 net revertants in a However, binding of the metabolite(s) of acetaminophen to desiccator system when exposed to trichloroethylene with 5-9 hepatic macromolecules does not occur until the glutathione activation (21 7). The heretofore observed carcinogenicity of concentration in the liver has been depleted by conjugation trichloroethylene may be confounded by the presence of highly with the reactive species (159). Intercalating Agents. Rosenkranz et al. (202) have noted —S-er that 9-aminoacridine but not other intercalating agents like acridine orange (Chemical 180), proflavine, and ethidium bro mide can revert the frame-shift mutants of Salmonella without activation; the latter are only active in the presence of S-9. @:?@[@}—.-@ Similarly, aromatics like benzo(a)pyrene 4,5-oxide (Chemical ort 309) and N-acetoxy-2-acetylaminofluorene (Chemical 175), NARNAc_ @Ac which are known to form adduct products with DNA, prefer entially revert the frame-shift and not the base mutants (5). In the nomenclature of McCann et al. (154), these agents are

called ‘‘reactive'‘frame-shiftmutagens. Levine et al. (138) KNAc have postulated that the formation of the adduct results in a

OR 0 6 B. E. Butterworth. The value and limitations of the Salmonella microsomal Ac.CCH3 Et:cH2cH3 R.SO5. C6H906 and the L5178Y mouse lymphoma mutagenicity assays. Paper presented at the Eighth Annual Meeting of the Environmental Mutagen Society. Colorado Springs, Chart 14. Known and possible metabolic conversions of phenacetin [Brodie Cob., February 16, 1977. and Axelrod (37); Dubach and Raaflaub (69); Jollow et al. (118); Nery (169)].

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mutagenic contaminants, epichlorohydrin and 1,2-epoxybu in the testing of actinomycin D (Chemical 341 ). Whereas no tane, in the test substance used in the animal testing (106). response is seen in Salmonella with drug concentration as high Although Bartsch et al. (15) could activate chloroprene with as 10 @tg/plate(roughly, 0.5 @tg/mIagar),cytotoxicity is quite 5-9 to a highly active mutagen using a desiccator system, evident in hamster cells treated in vitro with concentrations of McCann et al. (152) listed its mutagenicity as questionable on 0.1 and 0.05 @ig/ml(18, 19). the basis of a personal communication of negative results by Pyrazolinone (Chemical 327). Kellermannet al. (128) have B. McKusick (du Pont). observed a good agreement between the magnitude of induc By virtue of its dichloroethylene group, DDE is listed in Table tion of an individual's mixed-function oxidase activity (meas 4 as a chloroethylene. Epoxidation across the double bond is ured as 3-methylcholanthrene induction of aryl hydrocarbon a plausible reaction for DDE if one postulates further that this hydroxylase in short-term lymphocyte cultures) and the plasma is followed by the formation of 1,1-dichloro-1 -hydroxy-2,2- half-lives of antipyrine and phenylbutazone in the same individ bis(4-chlorophenyl)ethane; this presumably unstable species ual. Antipyrine is metabolized to 3 reactive forms in the rabbit could dehydrochlorinate into an acetyl chloride which would which may contribute to its irreversible binding in vivo and in be quickly hydrolyzed to the corresponding carboxylic acid. vitro (microsomes) to liver proteins. They were the presumed Burchfield and Storrs (41) have suggested that epoxidation of 3,4-epoxide, the 3-formyl metabolite, and 1-phenyl-3-methyl 1-chloro-2,2-bis(4-chlorophenyl)ethylene,ametaboliteof DDT, 4-hydroxypyrazolin-5-one (and the corresponding tautomers). would produce a metabolite with alkylating power. By similar Amidopyrine (4-dimethylammnoantipyrine), and presumably 4- logic, 2,2-bis(4-chlorophenyl)ethylene, also a metabolite of aminoantipyrine, undergo similar conversions (230). DDT, may be made reactive by epoxidation. However, the fact Mitotic Poisons. The complexity with which the nucleic that the only commonality among the DDT analogs (Chemical material of mammalian cells is organized provides mechanisms 235) presented in Table 4 is the p-substituted diphenylethane for mutagenesis and presumably carcinogenesis that will not structure is reminiscent of the hypothesis of a DDT receptor to be assayable in microbial cells. Those agents which cause explain the pesticidal activity of this chemical type (reviewed in disfunctioning of the mitotic apparatus that segregates chro Ref. 88) (Chart 15). Interestingly, DDT and some of its analogs mosomes during division, so-called mitotic poisons, will not be behave estrogenically (70, 164, 220, 245), suggesting that mutagenic in bacteria. Accordingly, griseofulvin (Chemical such a receptor might be the cytosolic protein that normally 452), colchicine, and vinblastine are not active in Salmonella binds estrogen. (104). Membrane-specific Agents. Differences between bacterial Cross-linking Agents. MitomycmnC (Chemical 291), a DNA and mammalian cell membranes can be expected to be a factor cross-linking agent (1 15), is weakly mutgenic only in a Sal at times in bacterial testing. The suggestion (151) that the monella strain that has excision repair and the A-factor presence of ions and citrate (a chelator) in the minimal medium (hisG46/pKM1 01). As such, it is not detected in what can be prevents the entrance of metal carcinogens into the bacteria called standard testing. Presumably, excision repair of the does not agree with the since-observed sulfate—dependent cross-linking involves the production of nicks in the DNA mutagenicity of chromium(VI) (Chemical 462) in TA92 (142). strands which somehow leads to increased mutagenesis in the Similarly, cadmium chloride (Chemical 461 ) has been reported presence of the A-factor (154). Similar activity is observed in to be mutagenic in TA1950 in the host-mediated assay and, to E. coli with mitomycin C (134) and may be relevant for another a lesser extent, in TA1535 when preincubated for 30 mm class of cross-linking agents (247), the pyrrolizidines (Chemi (without S-9) (122); it is not active in standard plate testing cals 328 to 332). In those Salmonella strains that were used (1 04). What determines the entrance of a chemical into a cell (not stated), monocrotaline and heliotrine were not active with is the lipophilicity of the chemical or the presence of a transport or without 5-9; these alkaloids were activated by S-9 to a system in the membrane. Charged species like metal cations species that was toxic to 2 strains of repair-deficient E. coli do not readily diffuse through lipid membranes and must be (94). transported into the cell. Rosenkranz et al. (202) report that Summary. Table 4 is a listing of 465 compounds with known Salmonella is not reverted by either beryllium sulfate (Chemical or suspected carcinogenic activity that were identified by var 460) or lead acetate (Chemical 464); Heddle and Bruce (104) bus criterions. Fifty-eight % have been tested for mutagenicity corroborate the lack of effect for lead acetate. In testing with in Salmonella for an overall correlation of 77% (Table 5). At TA1535 and TA1538 with and without 5-9, asbestos fibers least 13 chemical categories (includes a miscellaneous cate were not mutagenic (46). Membrane specificity is also apparent gory) cannot be evaluated at this time due to the small number of chemicals or the lack of testing results within these cate gories. However, shikimic acid and griseofulvin appear to ex V w x y z V hibit a specificity for that metabolism and chromosomal orga Is? nization, respectively, found in the mammalian system. Trans @ chlorobenzllate CI OH c-o-cH2cH3 a,a'-Diethyl-4,4'-stilbenediol and succinic anhydride are too bactericidal to test (Table 6). pp-DOT w—c—c—y cI H Cl Cl Cl For 1 5 chemical categories, the collective correlation is quite high at 94%, based on testing of 64% of the 312 chemicals in p.p-T DE CI HH @l cI this grouping (high activity). These categories represent chem icals that are ultimate electrophiles or chemicals that can be V Perthens CH@CH3 H H CI Cl activated by 5-9 or bacterial nitroreductase to electrophilic

Chart 15. The differences in substituents (v —@Z)among 4 DOT-like carcin species. Presumably, this high correlation will continue to be ogens. true in further testing, although molecular size could pose a

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Table 6 Sixty-one false negatives from Table 4 Category Chemical Comments Not evaluable Substituted Chlorobenzilate (Chemical 235) Antihormone? (see text) diphenylethane Stilbenediol DES (Chemical 236) Antiestro9en Proximate alkylating agent? (21 , 157) Bactericidal action (151) Anhydride Succinic anhydride (Chemical 325) Bactericidal action (1 @1) Pyrazolinone 4-Aminoantipyrine (Chemical 327) Mixed-function oxidation of the 3,4-double bond? (230) Polysaccharide Sodium carboxymethyl cellulose Solid-state carcinogen? (116) (Chemical 440) Tumor promotor? (240) Polymer Tween 60 (Chemical 445) Carcinogenic contaminants? (111) Tumor promotor? (240) Miscellaneous Shikimic acid (Chemical 451) Anabolite in biosynthesis of phenylalanine, tyrosine, and tryptophan; xenobiotic to organisms for whom the aromatic amino acids are essential (222) Griseofulvin (Chemical 452) Mitotic poison (98) Maleic hydrazide (Chemical 454) Carcinogenicity possibly due to contaminating hydrazine (see Ref. 114, Vol. 4, p. 177) Antiuracil? (54, 171) Amitrole (Chemical 455) Antithyroid (1, 225) Antipurine? (see text) Conjugates with alanine to form an antihistidine in E. coli (249) Saccharin (Chemical 456) Metabolic activation? (urine of mice treated with highly purified material is mutagenic) Mutagenic impurities (16) High Azoxy Cycasin (Chemical 24) Azoxy aglycone liberated by gut bacterial f3-glucuronidase(136) Aromatic amine 3-Hydroxyanthranilic acid (Chemical 115) Catabolite of tryptophan that is decyclized enzymatically into a semialdehyde and recyclized into quinolinic or picolinic acids or is metabolized to glutaric acid (170) Phenacetin (Chemical 117) Mixed-function oxidation to acetaminophen which can be metabolized further to a protein-binding species (118, 169) Simazine (Chemical 120) Possible modes of chemosterilant action include aldehyde formation (26) and antimetabolic effects as analogs of pyrimidine (74) and folate (12) Auramine (Chemical 137) Mixed-function oxidation of N-dimethylamino groups? Pararosaniline (Chemical 138) N-Hydroxylation? Intercalating agent? (202) Trypan blue (Chemical 153) Reduced by liver microsomes to benzidine and napthylamine derivatives (1 41) Evans blue (Chemical 154) Decolorized by gut bacteria Not absorbed if gut is sterilized by pretreatment with antibiotics (224) Heteroaromatic Actinomycin 0 (Chemical 341) Membrane specificity (18) Halomethane, Carbon tetrachloride (Chemical 353) Cytochrome P-450-mediated reduction to trichloromethyl radical haloethane (235) p.p'-DDT (Chemical 355) Metabolic activation? [DOD, a metabolite of DOT, is mutagenic in a host-mediated assay (43)] Medium Hydrazine Isoniazid (Chemical 28) Formation of N-acetylhydrazine which is subsequently oxidized to reactive diazene (167) 1,2-Dimethylhydrazine (Chemical 33)) Metabolism by gut Conversion to monosubstituted hydrazine bacteria (194) which is subsequently oxidized to reactive Procarbazine (Chemical 36) diazene (1 67, 195) Lactone Penicillic acid (Chemical 319) Ultimate electrophile (120) Gibberellic acid (Chemical 323) Intercalating agent with adenine-thymine base pair specificity? (129—131) Chloroethylene Trichloroethylene (Chemical 390) Oxirane formation (236) Volatility (217) DDE(Chemical393) Oxirane formation? (see text) Inorganic Arsenate (Chemical 458) Beryllium sulfate (Chemical 460) Membrane specificity due to ionized state? (see text) Lead acetate (Chemical 464) Low Azo Sudan I (Chemical 11) Reduction by gut bacteria (e.g., Refs. 48, 189, 192) to aromatic Orange I (Chemical 12) amines which are then available for typical aromatic amine Citrus Red No. 2 (Chemical 15) metabolism FD&CRed No. 1 (Chemical 17) Carbamyl, Acetamide (Chemical 91) N-Hydroxylation? thiocarbamyl Appearance in DNA (238) Thioacetamide (Chemical 92) Reactive species of sulfur from mixed-function desulfurization (1 12) Urethan (Chemical 95) Esterification of N-hydroxyurethan (168) Antimetabolite in pyrimidine biosynthesis? (29) Maneb (Chemical 105) Conversion to several reactive metabolites including ETU and CS@ (210) Thiourea (Chemical 108) Reactive species of sulfur from mixed-function desulfurization (112) Monuron (Chemical 109) Conversion to several metabolites including aniline derivative Benzene-oxide formation? (79)

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6—Continued.CategoryChemicalCommentsPhenyl Table

(Chemical 219) 1 Phenobarbital (Chemical 221) j@ Benzene-oxide formation (103, 117) PCNB (Chemical 224) Reduction to aniline derivative (27); subsequent aromatic amine me tabolism? Biphenyl (Chemical 225) Aroclor 1254 (Chemical 226) B@nzene-Oxideformation(89) BenzodioxoleBenzene Safrole (Chemical 227) 1 Activation of allyl Formation of carbonium ion at methylene car (137)PolychlorinatedHexachlorocyclohexanes1 ‘-Hydroxysafrole (Chemical 228) j' group (252) ben? Piperonyl butoxide (Chemical 234). ancyclic394—396)otherwise (ChemicalsEpoxidation? (a major metabolite has a chloroethylene moiety in saturated ring) (99)Mirex Benzene-oxide formation (Chemical 398) Aidrin (Chemical 400) or epoxy In- Formation of radicals from dechlorination? (205)SteroidEthinylestradiolDleldrin (Chemical 401) termediate (162) Heptachlor (Chemical 402)Epoxy

(Chemical 420) 424)1HormonesAntimetaboliteAminopterinTestosterone propionate (Chemical (Chemical 436) not easily assimilated by non-folic acid-requiring bacteria (254) L-Ethionine (Chemical 438)Antifolate Naturally occurring amino acid in some strains of bacteria (81) Ethylating agent (228) problem for the high-molecular-weight polyaromatics and het testing of chemicals of these types are the most credible for eroaromatics that have not yet been tested. The 11 false extrapolating. negatives that have occurred within these 15 categories are restricted mostly to aromatic amines and polyhalogenated Validation of Salmonella Testing as a Predictor of Carcino methyls. As many as 7 of these false negatives may have genicity resulted from inadequate metabolic activation (i.e. , cycasin, phenacetin, auramine, trypan blue, Evans blue, carbon tetra Unlike correlation, validation is purely a subjective affair. chloride, and DOT). Ideally, one would want a mutagenicity test the sensitivity and Structure-activity relationships are also evident among the specificity of which are both unity and therefore the false 11 chemical categories that are not well detected (low and negative and false positive rates of which are both zero. This medium activity). An analysis of false negatives in these cate would afford maximum confidence in the test as an indicator of gories suggests specificities on the part of these chemicals for carcmnogenicity.As such things are rarely ideal, one can antic the metabolism and cellular membrane of the mammal. In ipate that the sensitivity or specificity of the test could vary particular, it appears that the Salmonella-S-9 system often independently of each other. Historically, the originally re cannot activate or detect chemicals the activations of which ported (151) high sensitivity (90%) and specificity (87%) of entail the formation of short-lived species (e.g. , from thiocar Salmonella have been well received. More recently, Purchase bamyls and polyhalogenated compounds) or benzene oxide et al. (i 87) have directed attention to the large number of false (i.e. , phenyls) or the activations of which consist of several positives possible in mass screening chemicals under certain enzymatic steps (e.g. , symmetrical hydrazines, azonaphthols, assumptions. However, the model for this demonstration must urethan, safrole). In the case of chemicals that are converted be seen with some reservations. First. there seems to be a tacit in vivo to several metabolites (e.g. , maneb, monuron), there assumption that the identification of bacterial mutagens which must be some accounting for how faithfully the 5-9 activation should be tested further for mutagenicity in the mammal is not corresponds to this metabolism. Other chemical categories an important finding in itself in the screening of chemicals. that do not exhibit high correlations include benzodioxoles, Secondly, the authors have not considered in their model that steroids, and antimetabolites. Chemicals that are too volatile to those carcinogens that are not mutagenic in Salmonella are not test in the standard plate assay (e.g. , alkyl halides) and DNA randomized over all the chemical categories of carcinogens. In cross-linking agents (e.g. , mitomycin C) may require optimizing fact, there is a discernible trend between the structure and rather than standard procedures to be detected. metabolism of a carcinogen and its ability or inability to cause Given the poor correlations for as many as 11 chemical reversions in Salmonella. For a given category, the parameter categories, it is concluded that the important parameter in of this trend is its individual correlation ( P@inChart 2). Thirdly, Salmonella testing is the individual category correlation, p@in the alarmingly large percentage of false positives occurs when Chart 2. The individual correlations of these 11 chemical cat the proportion of carcinogens in the chemicals that are egories are low enough to discourage extrapolation of negative screened is quite low. However, in some instances, a de facto results obtained in Salmonella testing of chemicals of these ‘‘enrichment' ‘for chemicals with a greater likelihood of being types. For at least another 13 categories, their individual cor mutagenic may occur. For example, in the testing of chemicals relations have not been evaluated. For the remaining 15 cate under the newly enacted Toxic Substances Control Act, se gories, which include the types with which Salmonella testing lecting chemicals on the basis of structure-activity relationships has been standardized, their individual correlations are the and existing toxicity data should enrich the population of chem most impressive; therefore, negative findings in Salmonella icals to be tested with mutagenic ones.

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Besides less than ideal sensitivity and specificity, another significant repair of alkylated DNA has occurred due to the aspect has emerged from an analysis of the various correlation presence of the A-factor. More recenlty, Teramoto et al. (231) studies in Salmonella. It has been argued in this paper that the have shown that, at concentrations up to 20 mg/plate, 4,5- sensitivity is overrated as a result of the bias makeup of the dihydroimidazole-2(3H)-thiol does not revert TA100 while it carcinogens that have been tested. This bias obscures the does revert TA1535. Hence, the possible effects of the A- poor response of some categories of carcinogens. Obviously, factor on mutagenesis with respect to non-plasmid-bearing this ‘‘restrictedsensitivity'‘doesnot invalidate Salmonella test strains include increasing the spontaneous mutation rate in ing provided the restrictions are appreciated. A more sophisti strains that are recA+ (capable of recombinational repair), cated way of interpreting Salmonella results and discussing masking the effects of at least one mutagen, and decreasing validation is indicated. It is necessary to see negative findings or enhancing enormously the effects of others. in Salmonella in light not of the overall sensitivity of the test but If the bacteria metabolize a chemical to a mutagen but of the sensitivities of the individual categories, some of which mammals do not, this would result in a false positive. One are very high and others of which are hardly investigated or so known difference between bacterial and mammalian metabo low as to be of questionable value in predicting noncarcino lism of chemicals involves those having a nitro group. Saz and genicity. Therefore, it is necessary to validate the test on a She (207) have studied this reduction in E. coli using various category by Category basis. A similar conclusion has been nitroaromatics and have shown that the nitroreductase is rela reached by Purchase et al. (187). tively nonspecific and distinct from the enzymes that reduce To extend this sophistication a step further, the occurrence nitrite and nitrate. The nitroreductases of Salmonella that acti of ‘‘restrictedspecificity'‘shouldalso be investigated. Ames vate nitroaromatic compounds to mutagens are not found in (3) originally discussed how mutagens to bacteria may not be mammalian cells, although nitro reduction can be performed similarly mutagenic to mammalian cells. Some of those argu by other means (201 ), e.g. , by xanthine oxidase (227). Hence, ments concerned a lack of quantitative agreement. Qualita there is a need to study the specificity of nitro compounds, tively, those factors that would produce false positives are e.g. , by testing noncarcinogenic nitroaliphatics and nitroaro similar to those that account for false negatives (Table 7). matics, presuming that such entities exist. The role of the A-factor in the enhanced sensitivity of TA100 That a difference in metabolism can produce a false positive and TA98 has not been sufficiently elucidated. It is believed is somewhat suggested by the weak mutagenicity of sodium that mutagens detected by these strains cause nicks in the nitrite (152). However, the carcinogenicity testing of sodium DNA, which leads to error-prone repair and mutagenesis (150, nitrite is not what can be called extensive. Outside of one early 154). The underlying assumption is that this same sequence of study involving 12 s.c. injections over a 90-day period to mice events occurs in mammalian cells in the absence of the A- (161), the testing of sodium nitrite has been done in the context factor. It remains to be clarified that this mutagenicity has a of a control for studies on the possible nitrosation of secondary counterpart in mammalian cells and is not merely associated amines to carcinogens. In such studies, the resulting N-nitroso with an episome which possibly supplies an aberrant nuclear compounds are expected to be so active as to require only a enzyme or otherwise is itself somehow a cause of the muta small latency period [e.g. , animals were sacrificed at 40 weeks genicity. A similar caution has been made by Bridges (36). This in the study of Greenblatt et al. (96)] and a small number of clarification will have to explain several peculiarities. As Mc rodents to show a positive effect (e.g. , Aefs. 95 and 140). Cann et al. (154) have discussed, certain chemicals (e.g. MMS) As previously mentioned, phenotypic curing was observed appear to cause nicks in the DNA. Mutagenicity is observed or with amitrole and resulted in what could be falsely interpreted enhanced in Salmonella if these nicks are repaired by an error as positive results. Similarly, bactericidal chemicals can pro prone process that is dependent on the presence of a nonmu duce false positives by cross-feeding of histidine released by tated, chromosomal recA gene and the A-factor. Mutagens killed bacteria to surviving bacteria; a large number of “pin whose activities are not increased by this combination (e.g. point' ‘Colonies@5suggestiveof this activity. This has prompted bis(2-chloroethyl)ammne)presumably then are mutagenic in a Aosenkranz et a!. (202) to recommend that routine testing direct way that does not involve recombinational repair. How include replica plating of some positive plates onto minimal ever, in the case of the alkylating agent, diethyl sulfate, the media supplemented with biotin in order to control for these mutagenic activity is attenuated dramatically in the presence phenotypic effects. of the R-factor. These results would indicate then that rather The possibility of contamination by mutagenic substances must be considered in the interpretation of testing results since Table 7 Salmonella can detect potent mutagens at minute concentra A priori expectationsmammaliansystemsI. for the action of mutagens in microbial and tions. McCann et al. (151, 152) have shown that the apparent mutagenicity of 2 hydroxy derivatives of 2-acetylaminofluorene systems.II.Mutagen is similarly active in both attributes.A.Mutagen is specific for mammalian system was attributable to trace amounts of the highly mutagenic poisonsB.Chromosomal organization, e.g., mitotic parent compound. Batzinger et al. (16) have demonstrated that MetabolismFalse less than highly purified preparations of saccharin contain nega- 1. Enzymatic, e.g., urethan, benzene fives steroids3.2. Hormonal, e.g. , mutagenic contaminants that are active in modified plate test acidC. Metabolic strategy, e.g., shikimic ing. The modifications include placing biotin and L-histidine in DIII.MutagenMembrane, e.g., antifolates, actinomycin the bottom agar (and not in the overlay) and adjusting the is specific for microbial system attributes. amounts of glucose and L-histidine for maximum response. plasmid-dependentFalseA. Chromosomal organization, e.g., posimutagens?tivesB. Apparently, the modifications are crucial to the demonstration Metabolism, e.g., nitro compounds? of mutagenicity in plate testing; Sugimura et al. (226) did not C. Membrane (theoretical) observe mutagenicity in TA1537, TA100, or TA98 with or

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Chemical Structure Carcinogenicity and Salmonella Assay without 5-9. The reported mutagenicity of DBCP (technical metabolized in the rat to a trans-dihydrodiol and mercapturic grade) without 5-9 in plate testing (186) is probably due to the acid (219), both of which are diagnostic for an arene oxide epichlorohydrin that is added to DBCP as a stabilizer; however, intermediate. in a desiccator system without S-9 and in plate testing with These examples substantiate the caution that Saffiotti (203) 5-9, purified DBCP is mutagenic (20).@ urged to be exercised in comparing the mutagenicities of However, not all mutagenic contaminants are categorically supposed carcinogen-noncarcinogen pairs. Preferably, assign detected in testing. Maleic hydrazide, which cannot be ob ment of noncarcinogenicity should be made on the basis of tamed free from hydrazine (214), is not active in Salmonella adequate animal testing that presently exists or is developed testing (152). Similarly, the unexpected carcinogenicity of poly concurrently. Some consideration of the electrophilicity of the oxyethylene polymers may be due to carcinogenic contami parent compound and its metabolites including presumed in nants (i 11) or decomposition products although it also has termediates should also be undertaken. been proposed that these substances are only tumor promoters Despite these various considerations, the specificity of Sal (240). The lack of mutagenicity in Salmonella of Tween 60 may monella appears quite high. While McCann et al. (151) found indicate insensitivity to or batch variation in the concentration that 14 (13%) of the 108 alleged noncarcinogens were muta of the carcinogenic species. Tween 80, a monooleate deriva genic, as argued, the classification of most of these false tive of Tween 60, has been shown to induce transformation of positives as noncarcinogens is based on testing that is made murine fetal skin cells; the transformed cells produce tumors quate by present standards. Hence, by the operational defini when transplanted back into syngeneic hosts (72). tion of the Technical Panel (62) for noncarcinogens (‘‘judged Finally, reproducibility can be another confounding factor in not positive for tumor induction on the basis of tests conducted Salmonella testing. In contrast to the 430 revertant colonies adequately in 2 or more species' ‘),mostof the chemicals are (140 revertant colonies with controls) in TA100 observed by excluded as noncarcinogens. It is on this basis, high specificity McCann et al. (152) with 1-naphthylamine tested at 100 zg/ with a restricted high sensitivity, that positive results in Sal plate, Purchase et al. (187) failed to find significant results in monella testing are validated as a qualitative predictor of car their testing. This fact was discussed as a hallmark of the cinogenesis. However, that mutagenic potency cannot be con Salmonella test since 1-naphthylammnewaspresented, possibly sidered a quantitative predictor of carcinogenicity is based on erroneously (191), as the noncarcinogenic analog of 2-naph the fact that mutagenicity can be both underestimated and thylamine, which is unquestionably mutagenic in Salmonella. overestimated in Salmonella. Underestimation ranges from Whether the conflict lies in differences in procedures (e.g., false negatives to positives that are the result of in vitro acti Aroclor versus phenobarbital induction of liver enzymes) is not vation that is incomplete or otherwise not representative of the known. total activatibn that occurs in vivo. Similarly, overestimation Interestingly, 2 more of the 8 supposed noncarcinogens ranges from false positives to positives with potency that has cited by Purchase et al. (187) have been reported occasionally not been attenuated by detoxificative metabolism in vivo, DNA as being carcinogenic. The noncarcinogenic nature of 4-AAF repair, or the protection afforded by the nuclear proteins that is hardly established. Neither the studies of Flaks and Lucas together with DNA make up the suprastructure of the mam (82—84)using6 or less rats per time interval of 4-AAF feeding malian chromosome (3). Furthermore, the fact that the host's nor those of Weisburger et al. (243) and Morris et al. (160) can immunological competency, about which mutagenicity testing be taken as adequate proof that 4-AAF is noncarcinogenic. In makes no evaluation, plays a role in the course of carcinogen fact, Morris et al. even suggested that the slight carcinogenic esis argues against equating mutagenic potency with carcino activity of 4-AAF might be substantiated by testing at a higher genic potency (see also Ref. 8). dose. Likewise, Schinz et al. (208) concluded (unjustifiably so) In closing, it should be noted that, in Burdette's (42) analysis from their study in which 1 of 10 rats fed 4-AAF had developed of the lack of correlation between mutagenicity and carcino colon cancer that there was weak carcinogenic activity. Again, genicity, a real appreciation for the importance of metabolic as in the case of 1-naphthylamine, there appears to be a activation of mutagens and carcinogens is conspicuously miss problem with reproducing the mutagenicity. McCann et al. ing. Since then, the consensus has grown and is continually (152) found a 10-fold increase in the number of TA1538 substantiated that the reactive metabolic products of proximate revertants after treatment with 200 @tg/pIatequantitiesof chro mutagens and carcinogens are electrophiles which are capable matographically pure material in the presence of phenobarbital of covalent reaction with genetic material. As discussed, many induced 5-9. In contrast, Purchase et al. (187) failed to see of the false negatives observed in the correlation studies are mutagenicity in their testing. understood to be metabolized in vivo to electrophilic species. The carcinogenicity testing of anthracene, which was paired Notwithstanding the possibility that somatic mutation is only with its carcinogenic 9, 10-dimethyl derivative, hardly conveys one of several initiating mechanisms in carcinogenesis, it would any confidence that the chemical has been adequately tested. thus appear that many false negatives are not demonstrably Although numerous, the studies that did not find carcinogenic mutagenic in vitro for lack of a proper test system to both activity suffer from the use of far too few test animals. Gener activate and detect them. Furthermore, this conclusion can ally, the route of administration has been limited to skin painting probably be generalized to all in vitro systems since in vitro and injection (i.p. , i.v., and s.c.). However, Druckrey and activation methods in general appear only crudely to simulate Schmähl(68) and Schmähl(209) have reported the induction what occurs in vivo for many chemicals. Hence, the primary of sarcomas at the site of s.c. injection in rats. Anthracene is deficiency of genetic toxicology as a discipline remains its inability to assay reliably for gene mutations in vivo. Not until

7 V. F. Simmon. A presentation at the Ninth Annual Meeting of the Environ such-procedures are developed can a final assessment of the mental Mutagen Society, San Francisco, Calif., March 10, 1978. relationship of mutation to carcinogenesis be rendered. In the

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. S. J. Rinkus and M. S. Legator meantime, by necessity, the qualitative identification of muta 22. Blum, A. H., Livingston, R. B., and Carter, S. K. Hexamethylmelamine—a new drug with activity in solid tumors. Eur. J. Cancer, 9: 195—202,1973. gens must rely on a battery of in vitro and in vivo procedures, 23. Boiato, L., Mirvish, S. S., and Berenblum, I. The carcinogenic action and the 2 complementing each other. metabolism of N-hydroxyurethane in newborn mice. Int. J. Cancer, 1: 265— 269, 1966. Acknowledgments 24. Bonse, G., Urban, T., Reichert, 0., and Henschler, 0. Chemical reactivity, metabolic oxirane formation and biological reactivity of chlorinated ethyl The authors are grateful to their many colleagues who provided criticisms, enes in the isolated perfused rat liver preparation. Blochem. Pharmacol., translations, and prepublication copies of their manuscripts during the writing of 24: 1829-1834, 1975. this manuscript. The authors would like to acknowledge Dr. E. B. Whorten for 25. Borenfreund, E., Krim, M.. and Bendich, A. Chromosomal aberrations suggesting Chart 2 and Dr. V. M. S. Ramanujam for his help in categorizing the induced by hyponitrite and hydroxylamine derivatives. J. NatI. Cancer Inst., chemicals in Table 4. Phyllis Sitra assisted in the testing of the pesticides in Table 32: 667-677, 1964. 2. Janie Unbehagen and Elizabeth Whiteman provided helpful assistance with 26. Borkovec, A. B., and DeMilo, A. B. Insect chemosterilants. V. Derivatives Table 4. The authors are especially appreciative of the untiring cooperation of of melamine. J. Med. Chem., 10: 457-461 , 1967. the Moody Library reference department. 27. Borzelleca, J. F., Larson, P. 5., Crawford, E. M., Hennigar, G. R., Jr., Kuchar, E. J., and Klein, H. H. Toxicologic and metabolic studies on References pentachloronitrobenzene. Toxicol. Appl. Pharmacol., 18: 522—534,1971. 28. Bowden, J. P., Chung. K.-T., and Andrews, A. W. Mutagenic activity of 1. Alexander, N. M. Iodide peroxidase in rat thyroid and salivary glands and tryptophan metabolites produced by rat intestinal microflora. J. NatI. Can its inhibition by antithyroid compounds. J. Biol. Chem., 234: 1530—1533, cer Inst., 57: 921—924,1976. 1959. 29. Boyland, E.. and KolIer, P. C. Effects of urethane on mitosis in the Walker 2. Al-Kassab, S.. Boyland, E., and Williams, K. An enzyme from rat liver rat carcinoma. Br. J. Cancer, 8: 677—684,1954. catalysing conjugations with glutathione. 2. Replacement of nitro groups. 30. Boyland, E., and Manson, 0. The biochemistry of aromatic amines. The Biochem. J., 87: 4-9, 1963. metabolism of 2-naphthylamine and 2-naphthylhydroxyamine derivatives. 3. Ames, B. N. A bacterial system for detecting mutagens and carcinogens. Biochem. J.. 101: 84-102, 1966. In: H. E. Sutton and M. I. Harris (eds.), Mutagenic Effects of Environmental 31. Braun, R., Schubert, J., and Schóneich,J.On the mutagenicity of isoniazid. Contaminants, pp. 57—66.NewYork: Academic Press, Inc., 1972. Biol. Zentralbl., 95: 423—436,1976. 4. Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. 0. Carcino9ens are 32. Bray, H. 0., Clowes, R. C., and Thorpe, W. V. The metabolism of azoben mutagens: a simple test system combining liver homogenates for activation zene and p-hydroxyazobenzene in the rabbit (Abstract). Biochem. J., 49: and bacteria for detection. Proc. Nati. Acad. Sci. U. S. A., 70: 2281-2285, lxv, 1951. 1973. 33. Brem, H., Stein, A. B., and Rosenkranz, H. S. The mutagenicity and DNA 5. Ames, B. N., Gurney, E. 0. . Miller, J. A., and Bartsch, H. Carcinogens as modifying effect of haloalkanes. Cancer Res., 34: 2576—2579,1974. frameshift mutagens: metabolites and derivatives of 2-acetylaminofluorene 34. Bresnick, E. Activation and inactivation of polycyclic hydrocarbons and and other aromatic amine carcinogens. Proc. NatI. Acad. Sci. U. S. A.. 69: their interaction with macromolecular components. In: F. J. deSerres, J. A. 3128-3132, 1972. Fouts, J. A. Bend, and A. M. Philpot (eds.), In Vitro Metabolic Activation in 6. Ames, B. N., Lee, F. 0., and Durston, W. E. An improved bacterial test Mutagenesis Testing, pp. 91—104.Amsterdam: Elsevier/North-Holland system for the detection and classification of mutagens and carcinogens. Biomedical Press, 1976. Proc. NatI. Acad. Sci. U. S. A., 70: 782-786, 1973. 35. Bresnick, E., Vaught, J. B., Chuang, A. H. L., Stoming, T. A., Bockman, 0.. 7. Ames, B. N., McCann, J., and Yamasaki, E. Methods for detecting carcin and Mukhtar, H. Nuclear aryl hydrocarbon hydroxylase and interaction of ogens and mutagens with the Salmonella/mammalian microsome muta polycyclic hydrocarbons with nuclear components. Arch. Biochem. Bio genicity test. Mutat. Res., 31: 347—363, 1975. phys., 181: 257—269,1977. 8. Ashby, J., and Styles, J. A. Does carcinogenic potency correlate with 36. Bridges, B. A. Short term screening tests for carcinogens. Nature (Lond.), mutagenic potency in the Ames assay? Nature (Lond.), 271: 452—456, 261: 195—200,1976. 1978. 37. Brodie, B. B., and Axelrod, J. The fate of acetophenetidin (Phenacetin) in 9. Ashby, J., Styles, J. A., and Anderson, 0. Selection of an in vitro carcino man and methods for the estimation of acetophenetidin and its metabolites genicity test for derivatives of the carcinogen hexamethylphosphoramide. in biological material. J. Pharmacol. Exp. Ther. , 97: 58—67, 1949. Br. J. Cancer, 36: 564-571, 1977. 38. Brown, J. P. Reduction of polymeric azo dyes by cell suspensions of 10. Auerbach, C., Moutschen-Dahmen, M., and Moutschen, J. Genetic and enteric bacteria (Abstract). In: Annual Meeting of the American Society for cytogenetical effects of formaldehyde and related compounds. Mutat. Res., Microbiology, p. 123. 1976. 39: 317-362, 1977. 39. Brown, J. P. Role of gut bacterial flora in nutrition and health: a review of 11. Badr, F. M., and Badr, R. S. Studies on the mutagenic effect of contracep recent advances in bacteriological techniques, metabolism, and factors tive drugs. I. Induction of dominant lethal mutations in female mice. Mutat. affecting flora composition. CRC Crit. Rev. Food Sd. Nutr. , 8: 229—336, Res., 26: 529-534, 1974. 1977. 12. Baker, B. R., and Ho, B-T. Analogs of tetrahydrofolic acid. XXX. Inhibition 40. Brown, J. P., Roehm, G. W., and Brown, R. J. Mutagenicity testing of of dihydrofolic reductase by some 6-substituted 2,4-diamino-s-triazines. J. certified food colors and related azo, xanthene, and triphenylmethane dyes Heterocycl. Chem., 2: 340-343, 1965. with the Salmonella/microsome system. Mutat. Res., 56: 249—271,1978. 13. Baldwin, M. K., Robinson, J., and Parke, 0. V. A comparison of the 41. Burchfield, H. P., and Storrs, E. E. Organohalogen carcinogens. In: H. F. metabolism of HEOD (Dieldrin) in the CF1 mouse with that in the CFE rat. Kraybill and M. A. Mehlman (eds.), Environmental Cancer, pp. 319—371. Food Cosmet. Toxicol., 10: 333—351,1972. New York: John Wiley and Sons, Inc., 1977. 14. Barilyak. I. R., and Vasil'eva, I. A. Antimitotic and cytogenetic activity of 42. Burdette, W. J. The significance of mutation in relation to the origin of carbon bisulfide and hydrogen sulfide in small concentrations. Tsitol. tumors: a review. Cancer Res., 15: 201—226,1955. Genet., 8: 126-129, 1974. 43. Buselmaier, W., Röhrborn,G., and Propping, P. Mutagenitäts-Untersu 15. Bartsch, H., Malaveille, C., Montesano, R., and Tomatis, L. Tissue-mediated chungen mit Pestiziden im Host-mediated assay und mit dem Dominanten mutagenicity of vinyl chloride and 2-chlorobutadiene in Salmonella typhi Letaltest an der Maus. Biol. Zentralbl., 91: 31 1-325, 1972. murium. Nature (Lond.), 255: 641—643,1975. 44. Calder, I. C.. Goss, 0. E.. Williams, P. J., Funder, C. C.. Green, C. R., Ham, 16. Batzinger, R. P., Ou, S-V. L., and Beuding, E. Saccharin and other K. N., and Tange, J. 0. Neoplasia in the rat induced by N-hydroxyphena sweeteners: mutagenic properties. Science, 198: 944—946,1977. cetin, a metabolite of phenacetin. Pathology, 8: 1—6,1976. 17. Beaconsfield, P.. and Ginsburg, J. Oral contraceptives and cell replication. 45. Cessi, C., Colombini, C., and Mameli, L. The reaction of liver proteins with Lancet, 1: 592, 1968. a metabolite of carbon tetrachloride. Biochem. J., 101: 46c—47c,1966. 18. Benedict, W. F., Baker, M. S., Haroun, L., Choi, E., and Ames, B. N. 46. Chamberlain, M., and Tarmy, E. M. Asbestos and glass fibres in bacterial Mutagenicity of cancer chemotherapeutic agents in the Salmonella/micro mutation tests. Mutat. Res., 43: 159—164,1977. some test. Cancer Res., 37: 2209—2213,1977. 47. Chaube, S., and Murphy, M. L. The effects of hydroxyurea and related 19. Benedict, W. F., Banerjee, A., Gardner, A., and Jones, P. A. Induction of compounds on the rat fetus. Cancer Res., 26 (Part 1): 1448—1457, 1966. morphological transformation in mouse C3H/ 1OT½clone 8 cells and 48. Childs, J. J., Nakajima, C.. and Clayson, 0. B. The metabolism of 1- chromosomal damage in hamster A(T1)CI-3 cells by cancer chemothera phenylazo-2-naphthol in the rat with reference to the action of the intestinal peutic agents. Cancer Res., 37: 2202—2208,1977. flora. Biochem. Pharmacol., 16: 1555- 1561 , 1967. 20. Biles, R. W., Connor, T. H., Trieff, N. M., and Legator, M. S. The influence 49. Clayson, 0. B., and Garner, R. C. Carcinogenic aromatic amines and of contaminants on the mutagenic activity of dibromochloropropane related compounds. In: C. E. Searle (ed), Chemical Carcinogens, ACS (DBCP). J. Environ. Pathol. Toxicol., 2: 301—312,1978. monograph 173, pp. 366—461.Washington, D. C.: American Chemical 21. Blackburn, G. M., Thompson, M. H., and King, H. W. S. Binding of Society, 1976. diethylstilboestrol to deoxyribonucleic acid by rat liver microsomal fractions 50. Cohen, A. E., Scheel, L. 0., Kopp, J. F., Stockell, F. R., Jr., Keenan, A. G., in vitro and in mouse foetal cells in culture. Biochem. J., 158: 643—646, Mountain, J. T., and Paulus, H. J. Biochemical mechanisms in chronic 1976. carbon disulfide poisoning. Am. Ind. Hyg. Assoc. J., 20: 303-323, 1959.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Chemical Structure Carcinogenicity and Salmonella Assay

51 . Commoner, B. Risks, benefits, and carcinogens. Chemistry, 50: 19—20, Carcinogens List Based Only on Data Contained in the List, PB-251851. 1977. Washington, 0. C.: National Technical Information Service, 1976. 52. Conney, A. H., Sansur, M., Soroko, F., Koster, A., and Burns, J. J. Enzyme 79. Ernst, W., and Bbhme, C. Uber den Stoffwechsel von Harnstoff-Herbiciden induction and inhibition in studies on the pharmacological actions of in der Ratte. 1. Mitteilung. Mohuron und Aresin. Food Cosmet. Toxicol. , 3: acetophenetidin. J. Pharmacol. Exp. Ther., 151: 133-138, 1966. 789-796, 1965. 53. Coupland, 0.. and Peel, A. J. Uptake and incorporation of “C-labeled 80. Farber, E. Ethionine carcinogenesis. Adv. Cancer Res., 7: 383—474.1963. maleic hydrazide into the roots of Salix viminalis. Physiol. Plant., 25: 141— 81 . Fisher, J. F. , and Mallette, M. F. The natural occurrence of ethionine in 144, 1971. bacteria. J. Gen. Physiol., 45: 1-1 3, 1961. 54. Cradwick, P. D. Is maleic hydrazide a pyrimidine or purine analogue? 82. Flaks, B. The effect of prolonged dietary administration of the non-carcin Nature (Lond.), 258: 774, 1975. ogen, 4-acetylaminofluorene. on the liver of the rat. An electron microscope 55. Christensen, H. E., and Fairchild, E. J. (eds.), Suspected carcinogens. study. Chem.-Biol. Interact., 7: 151—164,1973. Department of Health, Education, and Welfare (NIOSH) publications 75- 83. Flaks, B., and Lucas, J. Fine structural changes in rat pancreatic cells 188 and 77-149. Washington, 0. C.: Government Printing Office, 1975— during prolonged treatment with 4-acetylaminofluorene. Chem.-Biol. Inter 1976. act., 5: 217-225, 1972. 56. Dalvi, A. R., Hunter, A. L., and Neal, R. A. Toxicological implications of the 84. Flaks, B.. and Lucas, J. Persistent ultrastructural changes in pancreatic mixed-function oxidase catalyzed metabolism of carbon disulfide. Chem. acinar cells induced by 4-acetylaminofluorene. Chem.-Biol. Interact. . 6: BioI. Interact., 10: 347-361 , 197S. 91—98,1973. 57. Dalvi, R. A., Poore, R. E., and Neal, R. A. Studies of the metabolism of 85. Freese, E. The difference between spontaneous and base-analogue in carbon disulfide by rat liver microsomes. Life Sci., 14: 1785—1796,1974. duced mutations of phage T4. Proc. NatI. Acad. Sci. U. S. A.. 45: 622- 58. Davis, K. J., and Fitzhugh, 0. G. Tumorigenic potential of aldrin and dieldrin 633, 1959. for mice. Toxicol. Appi. Pharmacol., 4: 187- 189, 1962. 86. Freese, E. Genetic effects of isoniazid and other hydrazides. Environ. 59. Dean, B. J. Genetic toxicology of benzene, toluene, xylenes, and phenols. Mutagen Soc. Newslett., 4: 8-9, 1971. Mutat. Res., 47: 75—97,1978. 87. Freese, E., Sklarow, S., and Freese, E. B. DNA damage caused by 60. De Lorenzo, F., Staiano, N., Silengo, L., and Cortese, R. Mutagenicity of antidepressant hydrazines and related drugs. Mutat. Res.. 5: 343-348, diallate, sulfallate, and triallate and relationship between structure and 1968. mutagenic effects of carbamates used widely in agriculture. Cancer Res., 88. Fukuto, T. R. Physicochemical aspects of insecticidal action. In: C. F. 38: 13-15, 1978. Wilkinson (ed), Insecticide Biochemistry and Physiology, pp. 397—428. 61. De Matteis, F. Covalent binding of sulfur to microsomes and loss of New York: Plenum Publishing Corp., 1976. cytochrome P-450 during the oxidative desulfuration of several com 89. Gardner, A. M., Chen, J. T., Roach, J. A. G., and Ragelis, E. P. Polychlo pounds. Mol. Pharmacol., 10: 849-854, 1974. rinated biphenyls: hydroxylated urinary metabolites of 2,5,2',5'-tetrachlo 62. Department of Health, Education, and Welfare. The carcinogenicity of robiphenyl identified in rabbits. Biochem. Biophys. Res. Commun., 55: pesticides. In: Report of the Secretary's Commission on Pesticides and 1377—1384,1973. Their Relationship to Environmental Health, pp. 459-506. Washington, 0. 90. Garner, A. C., and Nutman, C. A. Testing of some azo dyes and their C.: Government Printing Office, 1969. reduction products for mutagenicity using Salmonella typhimurium 63. Department of Health, Education, and Welfare. Criteria for a recommended TA1538. Mutat. Res., 44: 9—19,1977. standard. Occupational exposure to carbon disulfide, Department of 91 . Ghiasuddin, S. M., Menzer, A. E., and Nelson, J. 0. Metabolism of 2,5,2'- Health, Education, and Welfare (NIOSH)Publication 77-156, p. 106. Wash trichloro-, 2,5,2',5'-tetrachloro-, and 2,4,5,2',S'-pentachlorobiphenyl in rat ington, 0. C.: Government Printing Office, 1977. hepatic microsomal systems. Toxical. AppI. Pharmacol., 36: 187-194, 64. Department of Health, Education, and Welfare. Survey of Compounds 1976. Which Have Been Tested for Carcinogenic Activity. USPHS Publication 92. Gingell, A., and Walker, R. Mechanisms of azo reduction by Streptococcus 149, 1957-1974. Washington, 0. C.: Government Printing Office, 1974. faecalis. II. The role of soluble flavins. Xenobiotica, 1: 231—239,1971. 65. de Serres, F. J. Perspective on the mutagenicity of chemical carcinogens. 93. Gin, C. P., and Bhide, S. V. Metabolic studies on the mechanism of urethan In: H. Bohme and J. SchOneich(eds.), Environmental Mutagens. proceed action; Part Ill—effect of urethan on aspartate and ornithine carbamoyl ings of the sixth annual meeting of the European environmental mutagen transferase activities in Swiss mice. Indian J. Exp. BioI., 6: 21—23,1968. society, pp. 25-33. Berlin: Akademie-Verlag, 1977. 94. Green, M. H. L., and Muriel, W. J. Use of repair-deficient strains of 66. Dillard, R. 0. , Poore, G., Cassady, 0. A., and Easton, N. A. Acetylenic Escherichia coli and liver microsomes to detect and characterize DNA carbamates. A new class of potential oncolytic agents. J. Med. Chem., 10: damage caused by the pyrrolizidine alkaloids heliotrine and monocrotaline. 40-44, 1967. Mutat. Res., 28: 331 -336, 1975. 67. Doell, A. G., St. Cyr, C. de V., and Grabar, P. Immune reactivity prior to 95. Greenblatt, M., and Mirvish, S. S. Dose-response studies with concurrent development of thymic Iymphoma in C57BL mice. lnt. J. Cancer, 2: 103— administration of piperazine and sodium nitrite to strain A mice. J. NatI. 108, 1967. Cancer Inst., 50: 119—124,1973. 68. Druckrey, H., and Schmähl,0. Cancerogene Wirkung von Anthracen. 96. Greenblatt, M., Mirvish, S., and So, B. T. Nitrosamine studies: induction of Naturwissenschaften, 42: 159- 160, 19@S. lung adenomas by concurrent administration of sodium nitrite and second 69. Dubach, U. C., and Raaftaub, J. Neue Aspekte zur Frage der Nephrotoxi ary amines in Swiss mice. J. Natl. Cancer Inst.. 46: 1029—1034,1971. zitätvon Phenacetin. Experientia, 25: 956—958, 1969. 97. Greim, H.. Bonse, G.. Radwan, Z., Reichert, 0., and Henschler, 0. Muta 70. Duby, A. T., Travis, H. F., and Terrill, C. E. Uterotropic activity of DOT in genicity in vitro and potential carcinogenicity of chlorinated ethylenes as a rats and mink and its influence on reproduction in the rat. Toxicol. AppI. function of metabolic oxirane formation. Biochem. Pharmacol., 24: 2013— Pharmacol., 18: 348—355,1971. 2017, 1975. 71 . Ehrhart, H. , Georgii, A., and Stanislawski, K. Untersuchungen überexpe 98. Grisham. L. M., Wilson, L., and Bensch, K. G. Antimitotic action of griseo rimentelle Leukämien.V.Mitteilung Uberdie leukämogeneWirkungvon 3- fulvin does not involve disruption of microtubules. Nature (Lond.), 244: Hydroxy-anthranilsäurebei RFH-Máusen.KIm. Wochenschr., 37: 1053- 294-296, 1973. 1059, 1959. 99. Grover, P. L., and Sims, P. The metabolism of y-2,3,4,5,6-pentachlorocy 72. Elias, P. M., Yuspa, S. H., Gullino, M., Morgan, 0. L., Bates, R. R., and clohex-1-ene and y-hexachlorocyclohexane in rats. Biochem. J.. 96: 521— Lutzner, M. A. In vitro neoplastic transformation of mouse skin cells: 525, 1965. morphology and ultrastructure of cells and tumors. J. Invest. Dermatol., 100. Hahn, M-A., and Adamson, A. H. Pharmacology of 3,5-diamino-1 .2,4- 62: 569-581, 1974. triazole (guanazole). 1 . Antitumor activity of guanazole. J. NatI. Cancer 73. Elion, G. B., Bieber, S., and Hitchings, G. H. Studies on the mechanism of Inst., 48: 783—790,1972. action of urethane on mammaryadenocarcinoma 755. Acta Unio mt. Contra 101 . Harper, C.. Drew, R. T., and Fouts, J. A. Species differences in benzene Cancrum, 16: 605-608, 1960. hydroxylation to phenol by pulmonary and hepatic microsomes. Drug 74, Elion, G. B., Bieber, S., Nathan, H., and Hitchings, G. H. Uracil antagonism Metab. Dispos., 3: 381-388, 1975. and inhibition of mammary adenocarcinoma 755. Cancer Res., 18: 802- 102. Harris, P. N., Gibson, W. A., and Dillard, A. 0. The oncogenicity of six 817, 1958. analogs of 1,1-diphenyl-2-propynyl N-cyclohexylcarbamate. Proc. Am. As 75. Elion, G. B., Callahan, S., Bieber, S., Hitchings, G. H., and Rundles, R. W. soc. Cancer Res., 12: 26, 1971. A summary of investigations with 6-((1-methyl-4-nitro-5-imidazolyl)thio]- 103. Harvey, 0. J., Glazener, L., Stratton, C.. Nowlin, J., Hill, R. M., and Horning. purine (B.W. 57-322). Cancer Chemother. Rep., 14: 93—98,1961. M. G. Detection of a 5-(3,4-dihydroxy-1 ,5-cyclohexadiene-1 -yl)-metabolite 76. Elson, L. A., and Warren, F. L. The metablism of azo compounds. 1. of phenobarbital and mephobarbital in rat, guinea pig, and human. Res. Azobenzene. Biochem. J., 38: 21 7-220, 1944. Commun. Chem. Pathol. Pharmacol., 3: 557-565, 1972. 77. Engst, A., Macholz, A. M., Kujawa, M., Lewerenz, H.-J., and Plass, R. The 104. Heddle, J. A., and Bruce, W. R. Comparison of tests for mutagenicity or metabolism of lindane and its metabolites gamma-2,3,4,5,6-pentachloro carcinogenicity using assays for sperm abnormalities, formation of micro cyclohexene, pentachlorobenzene, and pentachlorophenol in rats and the nuclei, and mutations in Salmonella. In: H. H. Hiatt, J. D. Watson, and J. A. pathways of lindane metabolism. J. Environ. Sci. Health Bull., 2: 95—117, Winsten (eds.). Origins of Human Cancer. Book C. pp. 1549-1 557. Cold 1976. Spring Harbor, N. Y.: Cold Spring Harbor Laboratory, 1977. 78. Environmental Protection Agency. An Ordering of the NIOSH Suspected 105. Hennessy, D. J. Hydride-transferring ability of methylenedioxybenzenes as

SEPTEMBER 1979 3315

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. S. J. Rinkus and M. S. Legator

a basis of synergistic activity. J. Agric. Food Chem., 13: 218—220,196S. cyclohexane. II. Glutathione-mediated conversion to hydrophilic substance 106. Henschler, 0., Eder, E., Neudecker, T.. and Metzler, M. Carcinogenicity of by particulate fractions of rat liver and by homogenates of various rat trichloroethylene: fact or artifact? Arch. Toxicol., 37: 233—236, 1977. organs. Naunyn-Schmiedebergs Arch. Pharmacol., 279: 199—202,1973. 107. Herbold, B.. and Buselmaier, W. Induction of point mutations by different 136. Laqueur, G. L., McDaniel, E. G., and Matsumoto, H. Tumor induction in chemical mechanisms in the liver microsomal assay. Mutat. Res., 40: 73— germfree rats with methylazoxymethanol (MAM) and synthetic MAM ace 84, 1976. tate. J. Natl. Cancer Inst., 39: 355—371,1967. 108. Hilton, J. L., Kearney, P. C., and Ames, B. N. Mode of action of the I 37. Lawley, P. D. Carcinogenesis by alkylating agents. In: C. E. Searle (ed), herbicide 3-amino-i ,2,4-triazole (amitrole): inhibition of an enzyme of his Chemical Carcinogens, ACS Monograph 173, pp. 83-244. Washington, tidine biosynthesis. Arch. Biochem. Biophys., 112: 544—547, 1965. 0. C.: American Chemical Society, 1976. 109. Hirono, I., Fushimi, K., and Matsubara, N. Carcinogenicity test of shikimic 138. Levine, A. F., Fink, L. M., Weinstein, I. B., and Grunberger, 0. Effect of N- acid in rats. Toxicol. Left. 1: 9—10,1977. 2-acetylaminofluorene modification on the conformation of nucleic acids. 110. Holmes. A. J.. and Eisenstark, A. The mutagenic effect of thymine-starva Cancer Res., 34: 319—327,1974. tion on Salmonella typhimurium. Mutat. Res., 5: 15—21,1968. 139. Lijinsky, W. In: Should the Delaney Clause be changed. Chem. Eng. News, 111. Hueper, W. C., and Payne, W. W. Polyoxyethylene (8)-stearate. Carcino June 27, 1977, pp. 25—33;45—46. genic studies. Arch. Environ. Health, 6: 484—494,1963. 140. Lijinsky, W., Greenblatt, M., and Kommineni, C. Feeding studies of nitrilo 112. Hunter, A. L., and Neal, R. A. Inhibition of hepatic mixed-function oxidase triacetic acid and derivatives in rats. J. NatI. Cancer Inst., 50: 1061 —1063, activity in vitro and in vivo by various thiono-sulfur-containing compounds. 1973. Biochem. Pharmacol., 24: 2199—2205, 1975. 141 . Lloyd, J. B., Beck, F., Griffiths, A., and Parry, L. M. The mechanism of 113. Innes, J. A. M., Ulland, B. M., Valerio, M. G., Petrucelli, L., Fishbien, L., action of acid bisazo dyes. In: P. N. Campbell (ed), The Interaction of Hart, E. R., Pallotta, A. J., Bates, A. A., Falk, H. L., Gart, J. J., Klein, M., Drugs and Subcellular Components in Animal Cells, pp. 171—203.Boston: Mitchell, I., and Peters, J. Bioassay of pesticides and industrial chemicals Little, Brown, and Company, 1968. for tumorigenicity in mice: a preliminary note. J. NatI. Cancer Inst. , 42: 142. Lôfroth,G. L., and Ames, B. N. Mutagenicity of inorganic compounds in 1101—1114. 1969. Salmonella typhimurium: arsenic, chromium, and selenium (abstract). Mu 114. International Agency for Research on Cancer. IARC Monographs on the tat. Res., 53: 65-66, 1978. evaluation of carcinogenic risk of chemicals to man, vols. 1 to 13. Lyon. 143. Longridge, J. L.. and Timms, 0. Nucleophilic attack on 4-aminomethyl France: International Agency for Research on Cancer, 1971—1977. eneoxazol-5(4H)-ones, a rationalization of penicillin carcinogenicity. J. 115. lyer, V. N., and Szybalski. W. A Molecular mechanism of mitomycin action: Chem. Soc. B, 848-851, 1971. linking of complementary DNA strands. Proc. NatI. Acad. Sci. U. S. A.. 50: 144. Lutz, W. K.. and Schlatter, C. Mechanism of the carcinogenic action of 355-362. 1963. benzene: irreversible binding to rat liver DNA. Chem.-Biol. Interact., 18: 116. Jasmin, G. Action cancérigènedeIa carboxyméthylcellulose.Rev.Can. 241—245,1977. Biol., 20: 701-707, 1961. 145. Magee, P. N. Nitrosamine activations. In: F. J. de Serres, J. A. Fouts, J. A. 117. Jerina, D. M. , and Daly, J. W. Arene oxides: a new aspect of drug Bend, and A. M. Philpot (eds.), In Vitro Metabolic Activation in Mutagenesis metabolism. Science, 185: 573—582,1974. Testing, pp. 21 3—216. Amsterdam: Elsevier/North-Holland Biomedical 118. Jollow, D. J., Mitchell, J. A., Potter, W. Z., Davis, 0. C., Gillette, J. A., and Press, 1976. Brodie, B. B. Acetaminophen-induced hepatic necrosis. II. Role of covalent 146. MaIling, H. V. Dimethylnitrosamine: formation of mutagenic compounds by binding in vivo. J. Pharmacol. Exp. Ther., 187: 195—202, 1973. interaction with mouse liver microsomes. Mutat. Res., 13: 425—429, 1971. 119. Jonen-Kern, R., Jonen, H. G., Schupp, R. A., Minck, K., Kahl, G. F., and 147. Mann, S. K. Enhancement of chromosomal aberrations and growth inhibi Netter, K. J. Reductive drug metabolism in isolated perfused rat liver under tion by maleic hydrazide when dissolved in dimethyl sulphoxide. Curr. Sci. restricted oxygen supply. Xenobiotica, 8: 271—280,1978. (Bangalore), 46: 83—84,1977. 120. Jones, J. B., and Young, J. M. Carcinogenicity of lactones. Ill. The reactions 148. Mansuy, 0., Beaune, P., Cresteil, T., Lange, M., and Leroux, J-P. Evidence of unsaturated y-lactones with L-cysteine. J. Med. Chem., 11: 1176—1182, for phosgene formation during liver microsomal oxidation of chloroform. 1968. Biochem. Biophys. Res. Commun., 79: 513-51 7, 1977. 121 . Kadlubar, F. F., Miller, J. A., and Miller, E. C. Hepatic microsomal N- 149. Marshall, T. C., Dorough, H. W., and Swim, H. E. Screening of pesticides glucuronidation and nucleic acid binding of N-hydroxy arylamines in rela for mutagenic potential using Salmonella typhimurium mutants. J. Agric. tion to urinary bladder carcinogenesis. Cancer Res., 37: 805—814, 1977. Food Chem., 24: 560-563, 1976. 122. Kalinina, L. M., Polukhina, G. N., and Lukasheva, L. I. Salmonella typhi 150. McCann, J., and Ames, B. N. A simple method for detecting environmental murium-test system for the detection of the mutagenic activity of environ carcinO9ens as mutagens. Ann. N. V. Acad. Sci., 2 71: 5—13, 1976. mental pollution. I. Mutagenic effect of heavy metal salts using in vivo and 151 . McCann, J. , and Ames, B. N. Detection of carcinogens as mutagens in the in vitro assays without metabolic activation. Genetika, 13: 1089—1092, Salmonella/microsome tests: assay of 300 chemicals: discussion. Proc. 1977. NatI. Acad. Sci. U. S. A., 73: 950-954, 1976. 123. Karapally, J. C. . Saha, J. G. , and Lee, Y. W. Metabolism of lindane-―C in 152. McCann, J., Choi, E., Yamasaki, E., and Ames, E. N. Detection of carcin the rabbit: ether-soluble urinary metabolites. J. Agric. Food Chem. , 21: ogens as mutagens in the Salmonella/microsome test: assay of 300 811-818, 1973. chemicals. Proc. NatI. Acad. Sci. U. S. A. 72: 51 35—5139, 1975. 124. Kasperek, G. J., and Bruice, T. C. The mechanism of the aromatization of 153. McCann, J. . Simmon, V. Streitwieser, 0. , and Ames, B. N. Mutagenicity of arene oxides. J. Am. Chem. Soc., 94: 198-202, 1972. chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane 125. Kaubisch, N., Daly, J. W., and Jerina, D. M. Arene oxide as intermediates (ethylene dichloride), chloroethanol (ethylene chlorohydrin), vinyl chloride, in the oxidative metabolism of aromatic compounds. Isomerization of and cyclophosphamide. Proc. NatI. Acad. Sci. U. S. A., 72: 3190-3193. methyl-substituted arene oxides. Biochemistry, 11: 3080—3088,1972. 1975. 126. Kawachi, T. [Development of new screening test for carcinogens mainly by 154. McCann, J., Spingarn, N. E., Kobori, J., and Ames, B. N. Detection of detecting mutagensj. Annual report of the cancer research, ministry of carcinogens as mutagens: bacterial tester strains with A factor plasmids. health and welfare, Japan, pp. 315—326,1975; pp. 744—755,1976. Proc. NatI. Acad. Sci. U. S. A., 72: 979-983, 1975. 127. Kaye, A. M. Urethan carcinogenesis and nucleic acid metabolism: in vitro 155. McCarthy, R. E., and Epstein, S. S. Cytochemical and cytogenetic effects interactions with enzymes. Cancer Res.. 28: 104 1—1046, 1968. of maleic hydrazide on cultured mammalian cells. Life Sci., 7: 1-6, 1968. 128. Kellermann, G., Luyten-Kellermann, M., Horning, M. G., and Stafford, M. 156. McCollister, A. J., Gilbert, W. A., Jr., Ashton, 0. M., and Wyngaarden, J. Correlation of aryl hydrocarbon hydroxylase activity of human lymphocyte B. Pseudofeedback inhibition of purine synthesis by 6-mercaptopurine cultures and plasma elimination rates for antipyrine and phenylbutazone. ribonucleotide and other purine analogues. J. Biol. Chem., 239: 1560— Drug Metab. Dispos., 3: 47-50. 1975. 1563, 1964. 129. Kessler, B. Interactions in vitro between hormones and DNA. III. Effects of 157. Metzler, M., Muller, W., and Hobson, W. C. Biotransformation of diethyl plant and animal hormones on the action of DNA ligase and its relationship stilbesterol in the rhesus monkey and the chimpanzee. J. Toxicol. Environ. to age. Biochim. Biophys. Acta, 230: 330-342, 1971. Health, 3: 439—450,1977. 130. Kessler, B. Interactions in vitro between gibberellins and DNA by optical 158. Mirvish, S. S. The metabolism of N-hydroxyurethane in relation to its rotatory profile of the thermal denaturation of ONA-gibberellin complexes. carcinogenic action: conversion into urethane and an N-hydroxyurethane Biochim. Biophys. Acta, 232: 61 1—613,1971. glucuronide. Biochim. Biophys. Acta, 117: 1—12,1966. 131 . Kessler. B., and Snir, I. Interactions in vitro between gibberellins and DNA. 159. Mitchell, J. R., Jollow, 0. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. Biochim. Biophys. Acta, 195: 207-21 8, 1969. Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. 132. Kimball, A. F. The mutagenicity of hydrazine and some of its derivatives. J.Pharmacol.Exp. Ther.,187:21 1—217,1973. Mutat.Res.,39: 111—126,1977. 160. Morris, H. P., velat, C. A., Wagner, B. P., Dahlgard, M., and Ray, F. E. 133. Kohli, J. , Jones, 0., and Safe, S. The metabolism of higher chlorinated Studies of carcinogenicity in the rate of derivatives of aromatic amines benzene isomers. Can. J. Biochem., 54: 203-208, 1976. related to N-2-fluorenylacetamide. J. NatI. Cancer Inst., 24: 149—180, 134. Kondo, S., Ichikawa, H., Iwo, K., and Kato, T. Base-change mutagenesis 1960. and prophage induction in strains of Escherichia coli with different DNA 161 . Nakahara, W., and Fukuoka, F. Study of carcinogenic mechanism based repair capacities. Genetics, 66: 187—217, 1970. on experiments with 4-nitroguinoline N-oxide. Gann, 50: 1—15,1959. 135. Kraus, P., Noack, 0. , and Portig, J. Biodegradation of alpha-hexachloro 162. Nakatsugawa, T., lshida, M., and Dahm, P. A. Microsomal epoxidation of

3316 CANCERRESEARCHVOL. 39

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. Chemical Structure Carcinogenicity and Salmonella Assay

cyclodiene insecticides. Biochem. Pharmacol., 14: 1853—1865,1965. detecting organic chemical carcinogens and recommendations for their 163. National Technical Information Service. Evaluation of Carcinogenic, Tera use. Nature (Lond.), 264: 624—627,1976. togenic, and Mutagenic Activities of Selected Pesticides and Industrial 188. Rabinowitz, J. C.. and Pricer, W. E., Jr. Purine fermentation by Clostridsum Chemicals, Vol. 1, publication PB-223-1 59, Washington, 0. C.: National cylindrosporum. V. Formininoglycine. J. Biol. Chem., 222: 537-554, 1956. Technical Information Service, 1968. 189. Radomski, J. L. The absorption, fate, and excretion of Citrus Red No. 2 164. Nelson, J. A. Effects of dichlorodiphenyltrichloroethane (DOT)analogs and (2,5-dimethoxyphenyl-azo-2-naphthol) and Ext. 0 and C Red No. i4 (1- polychlorinated biphenyl (PCB) mixtures on 17f1-(3Hjestradiolbinding to rat xylylazo-2-naphthol). J. Pharmacol. Exp. Ther.. 134: 100-1 09. 1961. uterine receptor. Biochem. Pharmacol., 23: 447—451, 1974. 190. Radomski, J. 1. Toxicology of food colors. Annu. Rev. Pharmacol., 14: 165. Nelson, J. A., Carpenter, J. W., Rose, 1. M., and Adamson, 0. J. Mecha 127—137,1974. nisms of action of 6-thioguanine. 6-mercaptopurine, and 3-azaguanine. 191 . Radomski, J. L., Brill, E., Deichmann, W. B., and Glass, E. M. Carcino Cancer Res., 35: 2872—2878,1975. genicity testing of N-hydroxy and other oxidation and decomposition prod 166. Nelson, J. A., Rose, L. M., and Bennett, L. L., Jr. Mechanism of action of ucts of 1- and 2-naphthylamine. Cancer Res., 31: 1461-1467, 1971. 2-amino-i ,3,4-thiadiazole (NSC 4728). Cancer Res., 37: 182—i87,1977. 192. Radomski, J. L., and Deichmann, W. B. Cathartic action and metabolism of 167. Nelson, S. 0., Snodgrass, W. A., and Mitchell, J. A. Chemical reaction certain coal tar food dyes. J. Pharmacol. Exp. Ther., 118: 322-327, 19S6. mechanisms responsible for the tissue injury caused by monosubstituted 193. Rechnagel. A. 0.. and Glende, E. A., Jr. Carbon tetrachloride hepatotox hydrazines and their hydrazide drug precursors. In: F. J. de Serres. J. R. icity: an example of lethal cleavage. CRC Crit. Rev. Toxicol., 2: 263-297, Fouts, J. A. Bend, and A. M. Philpot (eds.), In Vitro Metabolic Activation in 1974. Mutagenesis Testing, pp. 257-276. Amsterdam: Elsevier/North-Holland 194. Reddy, B. S., Narisawa, T., Wright, P., Vukusich, 0. , Weisburger, J. H.. Biomedical Press, 1976. and Wynder, E. L. Colon carcinogenesis with azoxymethane and dimeth 168. Nery, A. Acylation of cystosine by ethyl N-hydroxycarbamate and its acyl ylhydrazine in germ-free rats. Cancer Res., 35: 287-290. 1975. derivatives and the binding of these agents to nucleic acids and proteins. 195. Reed, 0. J. Procarbazine. In: A. C. Sartorelli and 0. G. Johns (eds.), J. Chem. Soc. C, 1860-1865, 1969. Handbook of Experimental Pharmacology, Vol. 38, Part 2, pp. 747-765. 169. Nery, A. The possible role of N-hydroxylation in the biological effects of New York: Springer-Verlag, 197g. phenacetin. Xenobiotica, 1: 339-343, 1971. 196. Rees, K. R.. Rowland, G. F.. and Varcoe, J. S. The metabolism of tritiated 170. Nishizuka, Y., Ichiyama, A., Gholson, A. K., and Hayaishi, 0. Studies on thioacetamide in the rat. Int. J. Cancer, 1: 197—206,1966. the metabolism of the benzene ring of tryptophan in mammalian tissues. I. 197. Reynolds, E. S.. and Moslen, M. T. Halothane hepatotoxicity: enhancement Enzymic formation of glutaric acid from 3-hydroxyanthranilic acid. J. Biol. by polychlorinated biphenyl pretreatment. Anesthesiology, 4 7: 19—27. Chem., 240: 733-739, 1965. 1977. 171. Nooden, L. 0. Inhibition of nucleic acid synthesis by maleic hydrazide. 198. Robinson, J. R., and Nebert, 0. W. Genetic expression of aryl hydrocarbon Plant Cell Physiol., 13: 609—621,1972. hydroxylase induction. Presence or absence of association with zoxaz 172. Odashima, S. The cooperative development in Japan of methods for olamine, diphenylhydantoin, and hexobarbital metabolism. Mol. Pharma screening chemicals for carcinogenicity. In: A. Montesano, M. Bartsch, col., 10: 484-493, 1974. and L. Tomatis (eds.), Screening Tests in Chemical Carcinogenesis. IARC 199. Röhrborn, G.. and Hansmann, I. Oral contraceptives and chromosome Scientific Publication No. 12, pp. 61—75.Lyon:International Agency for segregation in oocytes of mice. Mutat. Res., 26: 535-544, 1974. Research on Cancer, 1976. 200. Rosenkranz, H. S., Gutter, B., and Speck, W. T. Mutagenicity and DNA 173. Ohno, S. Aneuploidy as a possible means employed by malignant cells to modifying activity: a comparison of two microbial assays. Mutat. Res., 41: express recessive phenotypes. In: J. German (ed), Chromosomes and 61-70, 1976. Cancer, pp. 77-94. New York: John Wiley and Sons, Inc., 1974. 201 . Rosenkranz, H. S., and Speck, W. T. Mutagenicity of metronidazole: 174. Peterson, J. E., and Robison, W. H. Metabolic products of p,p'-OOT in the activation by mammalian liver microsomes. Biochem. Biophys. Res. Com rat. Toxicol. AppI. Pharmacol., 6: 321 -327, 1964. mun., 66: 520-525. 1975. 175. Pipkin, G. E., Nishimura. R., Banowsky, L., and Schlegel, J. U. Stabilization 202. Rosenkranz. H. S., Speck, W. T., and Gutter, B. Microbial assay proce of urinary 3-hydroxyanthranilic acid by oral administration of L-ascorbic dures: experience with two systems. In: F. J. de Serres, J. A. Fouts, J. R. acid. Proc. Soc. Exp. BioI. Med., 126: 702-704, 1967. Bend, and A. M. Philpot (eds.), In Vitro Metabolic Activation in Mutagenesis 176. PohI, L. A., Bhooshan, B., Whittaker, N. F., and Krishna, G. Phosgene: a Testing, pp. 337-363. Amsterdam: Elsevier/North-Holland Biomedical metabolite of chloroform. Biochem. Biophys. Res. Commun., 79: 684-691. Press, 1976. 1977. 203. Saffiotti, U. Validation of short-term bioassays as predictive screens for 177. Poirier, L. A. The carcinogenicity and metabolism of several aromatic chemical carcinogens. In: A. Montesano, H. Bartsch, and L. Tomatis (eds.), amines and their N-oxidized derivatives. Ph.D. Dissertation, University of Screening Tests in Chemical Carcinogenesis, IARC Scientific Publication Wisconsin, 1965. No. 12, pp. 3—13.Lyon: International Agency for Research on Cancer, 178. Poirier, L. A. Mutagenic screening tests for carcinogenicity. In: R. Monte 1976. sano, H. Bartsch, and L. Tomatis (eds.), Screening Tests in Chemical 204. Salama, H. S.. and El-Sharaby, A. M. Giberellic acid and fJ-sitosterol as Carcinogenesis, IARC Scientific Publication No. 12, pp. 15-24. Lyon: sterilants of the cotton leaf worm Spodoptera littoralis Boisduval. Experien International Agency for Research on Cancer, 1976. tia, 28: 413—414, 1972. 179. Poirier, L. A., Miller, J. A., Miller, E. C., and Sato, K. N-Benzoyloxy-N 205. Saleh, M. A., Turner, W. V., and Casida, J. E. Polychlorobornane compo methyl-4-aminazobenzene: its carcinogenic activity in the rat and its reac nents of toxapttene: structure-toxicity relations and metabolic reductive tions with proteins and nucleic acids and their constituents in vitro. Cancer dechlorination. Science, 198: 1256—i258, 1977. Res.,27: 1600—1613,1967. 206. San, R. H. C.. and Stick, H. F. DNA repair synthesis of cultured human 180. Poirier, L. A., and Simmon, V. F. Mutagenic-carcinogenic relationships and cells as a rapid bioassay for chemical carcinogens. Int. J. Cancer, 16: the role of mutagenic screening tests for carcinogenicity. Clin. Toxicol., 9: 284-291, 1975. 761—771,1976. 207. Saz. A. K., and She, A. B. The inhibition of organic nitro reductase by 181 . Portig, J., Kraus, P. . Sodomann, S. . and Noack, G. Biodegradation of aureomycin in cell-free extracts. II. Cofactor requirements for the nitro alpha-hexachlorocyclohexane. I. Glutathione-dependent conversion to a reductase enzyme complex. Arch. Biochem. Biophys., 51: 5-16, 1954. hydrophilic metabolite by rat liver cytosol. Naunyn-Schmiedebergs Arch. 208. Schinz, H. R., Fritz-Niggli, H., Campbell, T. W., and Schmid, H. Krebsbil Pharmacol., 279: 185-198, 1973. dung durch Aminofluorene und verwandte KOrper.Oncologia. 8: 233-245, 182. Pound, A. W., Franke, F., and Lawson, T. A. The binding of ethyl carbamate 1955. to DNA of mouse liver in vivo: the nature of the bound molecule and the 209. Schmähl,0. PrUfung von Naphthalin und Anthracen auf cancerogene site of binding. Chem.-BioI. Interact., 14: 149—163, 1976. Wirkung an Ratten. Z. Krebsforsch., 60: 697-710, 1955. 183. Prejean, J. D., Griswold, A. E., Casey, A. E., Peckham, J. C., Weisburger, 210. Seidler, H., Härtig.M., Schnaak, W., and Engst, R. Untersuchungen Uber J. H., Weisburger, E. K., and Wood, H. B., Jr. Carcinogenicity studies of den Metabolismus einiger lnsektizide und Fungizide in der Ratte. II. Ver clinically used anticancer agents. Proc. Am. Assoc. Cancer Res. , 13: 112, teilung und Abbau von “C-MarkiertemManeb. Nahrung, 14: 363-373, 1972. 1970. 184. Preussmann, A., Druckrey, H., Ivankovic, S., and von Hodenberg, A. 21 1. Seymour, J. L.. Schmidt, S. P., and Allen, J. R. In Vitro generation of a Chemical structure and carcinogenicity of aliphatic hydrazo, azo, and chemically reactive metabolite of 2,5,2',5'-tetrachlorobiphenyl by rhesus azoxy compounds and of triazenes, potential in vivo alkylating agents. Ann. monkey liver microsomes. Proc. Soc. Exp. Blol. Med., 152: 621-625, N. Y. Acad. Sci.,163:697-714, 1969. 1976. 185. Priest, A. E., Bokman, A. H., and Schweigert, B. S. 3-Hydroxyanthranilic 212. Shimazu, H., Shiraishi, N., Akematsu, T., Ueda, N., and Sugiyama, T. acid metabolism. V. Distribution of enzyme system in animal tissues. Proc. Carcinogenicity screening tests on induction of chromosomal aberrations Soc. Exp. BioI. Med., 78: 477-479, 1951. in rat bone marrow cell in vivo (Abstract). Mutat. Res. , 38: 347, 1976. 186. Prival, M. J., McCoy, E. C.. Gutter, B., and Rosenkranz, H. S. Tris(2,3- 213. Shirasu, V., Moriya, M., Kato, K., Furuhashi, A., and Kada, T. Mutagenicity dibromopropyl)phosphate: mutagenicity of widely used flame retardant. screening of pesticides in the microbial system. Mutat. Res., 40: 19—30, Science, 195: 76—78,1977. 1976. 187. Purchase, I. F. H., Longstaff, E., Ashby, J., Styles, J. A., Anderson, D.. 214. Shubik, P. Potential carcinogenicity of food additives and contaminants. Lefevre, P. A., and Westwood, F. A. Evaluation of six short term tests for CancerRes., 35:3475-3480, 1975.

SEPTEMBER1979 3317

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1979 American Association for Cancer Research. S. J. Rinkus and M. S. Legator

215. ShulI, K. H., McConomy, J., Vogt, M., Castillo, A., and Farber, E. On the hashi, M., and Ito, N. Tumorigenic effect of 3-amino-i H-i ,2,4-triazole on mechanism of induction of hepatic adenosine triphosphate deficiency by rat thyroid. J. Natl. Cancer Inst., 57: 861 —863,1976. ethionine. J. Biol. Chem., 241: 5060—5070, 1966. 235. Uehleke, H., Hellmer, K. H., and Tabarelli, S. Binding of “C-carbon 216. Sidwell, A. W., Huffman, J. H., Khare, G. P., Allen, L. B., Witkowksi, J. T., tetrachloride to microsomal proteins in vitro and formation of CHCI3 by and Robins, A. K. Broad-spectrum antiviral activity of virazole: 1-fI-o reduced liver microsomes. Xenobiotica, 3: 1—11, 1973. ribofuranosyl- 1 .2 ,4-triazole-3-carboxamide. Science, 177: 705—706, 236. Uehleke. H., Tabarelli-Poplawski, S., Bonse, G., and Henschler, 0. Spectral 1972. evidence for 2,2,3-trichloro-oxirane formation during microsomal trichlo 217. Simmon, V. F., Kauhanen, K., and Tardiff, A. G. Mutagenic activity of roethylene oxidation. Arch. Toxicol., 37: 95—105, 1977. chemicals identified in drinking water. In: 0. Scott, B. A. Bridges, and F. H. 237. Uehleke. H., Werner, T., Greim, H., and Kraemer, M. Metabolic activation Sobels (eds.), Progress in Genetic Toxicology, pp. 249-258. New York: of haloalkanes and tests in vitro for mutagenicity. Xenobiotica, 7: 393— Elsevier/North-Holland Biomedical Press, 1977. 400, 1977. 218. Simmon, V. F., Poole, D. C., and Newell, G. W. In vitro mutagenic studies 238. Ushioda, Y. Tritiated acetamide incorporation into DNA of the mutant bar of twenty pesticides (Abstract). Toxicol. AppI. Pharmacol., 37: 109, 1976. larvae in Drosophila melanogaster. Annot. Zool. Jpn., 49: 90—95,1976. 219. Sims, P. Metabolism of polycyclic compounds. 25. The metabolism of 239. Walker, A. The metabolism of azo compounds: a review of the literature. anthracene and some related compounds in rats. Biochem. J., 92: 621— Food Cosmet. Toxicol., 8: 659-676, i970. 631, 1964. 240. Walpole, A. L. Observations upon the induction of subcutaneous sarcomata 220. Singhal. A. L., Valadares, J. R. E., and Schwark, W. S. Metabolic control in rats. In: L. Seven (ed), The Morphological Precursors of Cancer, pp. mechanismsin mammaliansystems. IX. Estrogen-like stimulation of uterine 83—88.Perugia, Italy: Perugia University Department of Cancer Research, enzymes by o,p'-i ,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane. Biochem. 1962. Pharmacol., 19: 2145—2155,1970. 241 . Waters, E. M., and Black, S. A. Mirex II. An Abstracted Literature Collection, 221. Sisenwine, S. F., Tio, C. 0., Shrader, S. R., and Ruelius, H. W. The 1947-1976, ORNL/TIRC-76/4. Washington, 0. C.: National Technical biotransformation of oxazepam (7-chloro-i ,3-dihydro-3-hydroxy-5-phenyl Information Service, 1976. 2H-i ,4-.benzodiazepin-2-one)in man, miniature swine, and rat. Arzneim. 242. Waters. E. M., Huff, J. E., and Gerstner, H. B. Mirex. An overview. Environ. Forsch., 22: 682-687, 1972. Res., 14: 212—222.1977. 222. Stavric, B., and Stoltz, 0. A. Shikimic Acid. Food Cosmet. Toxicol., 14: 243. Weisburger, J. H. , Weisburger, E. K. , and Morris, H. P. Analogs of the 141—145,1976. carcinogen 2-acetylaminofluorene: the isomeric 4-acetylaminofluorene. J. 223. Stein, V. B., and Pittman, K. A. Identification of a mirex metabolite from Am. Chem. Soc., 74: 4540—4543, 1952. monkeys. Bull. Environ. Contam. Toxicol., 18: 425—427,1977. 244. Weisburger, J. H.. Yamamoto, A. S., Glass, R. M., and Frankel, H. H. 224. Stoelinga, G. B. A., and Van Munster, P. J. J. The behaviour of Evans blue Prevention by arginine glutamate of the carcinogenicity of acetamide in (azo-dye T-1 824) in the body after intravenous injection. Acta Physiol. rats. Toxicol. AppI. Pharmacol., 14: 163—i75, 1969. Pharmacol. Neerl., 14: 391-409, 1967. 245. Welch, R. M., Levin, W., and Conney, A. H. Estrogenic action of DOTand 225. Strum, J. M., and Karnovsky, M. J. Aminotriazole goiter. Fine structure and its analogs. Toxicol. AppI. Pharmacol., 14: 358—367, 1969. localization of thyroid peroxidase activity. Lab. Invest., 24: 1—12,1971. 246. Weyter, F. W., and Broquist, H. P. Interference with adenine and histidine 226. Sugimura, T., Sato, S., Nagao, M., Yahagi, T., Matsushima, T., Seino, Y., metabolism of microorganisms by aminotriazole. Biochim. Biophys. Acta, Takeuchi, M. , and Kawachi, T. Overlapping of carcinogens and mutagens. 40: 567-569, 1960. In: P. N. Magee, S. Takayama, T. Sugimura, and T. Matsushima (eds.), 247. White. I. N. H., and Mattocks, A. A. Reaction of dihydropyrrolizines with Fundamentals in Cancer Prevention, pp. 191—215.Baltimore: University deoxyribonucleic acids in vitro. Biochem. J., 128: 291 —297,1972. Park Press, 1976. 248. Wierner, M., Pittman, K. A., and Stein, V. Mirex kinetics in the rhesus 227. Sugimura, T., Yahagi, T., Nagao, M., Takeuchi, M., Kawachi, T., Hara, K., monkey. I. Disposition and excretion. Drug Metab. Dispos., 4: 281—287, Yamasaki, E., Matsushima, T. . Hashimoto, Y., and Okada, M. Validity of 1976. mutagenicity tests using microbes as a rapid screening method for envi 249. Williams, A. K., Cox, S. T., and Eagon, R. G. Conversion of 3-amino-i .2,4- ronmental carcinogens. In: A. Montesano, H. Bartsch, and L. Tomatis triazole into 3-amino-i ,2,4-triazolyl alanine and its incorporation into pro (eds.), Screening Tests in Chemical Carcinogenesis, IARC Scientific Pub tein by Escherichia coli. Biochem. Biophys. Res. Commun., 18: 250—254, lication No. 12, pp. 81 —101. Lyon. France: International Agency for Re 1965. search on Cancer, 1976. 250. Williams, D. L., Hagen, A. A., and Runyan, J. W., Jr. Chromosome altera 228. Swann, P. F., Pegg, A. E., Hawks, A., Farber, E., and Magee, P. H. tions produced in germ cells of dogs by progesterone. J. Lab. Clin. Med., Evidence for ethylation of rat liver deoxyribonucleic acid after administra 77: 417—429, 1971. tion ofethionine. Biochem. J., 123: 175—181, 1971. 251 . Williams, 0. L., Runyan, J. W., Jr., and Hagen, A. A. Progesterone-induced 229. Synder, R.. Uzuki. F.. Gonasun, L., Bromfeld, E., and Wells, A. The alterations of oogenesis in the Chinese hamster. J. Lab. Clin. Med., 79: metabolism of benzene in vitro. Toxicol. Appl. Pharmacol., 11: 346-360, 972—977,1972. 1967. 252. Wislocki, P. G., Borchert, P., Miller, J. A., and Miller, E. C. The metabolic 230. Tabarelli-Poplawski, S.. and Uehleke, H. Irreversible binding of 3-'C- activation of the carcinogen 1‘-hydroxysafrole in vivo and in vitro and the antipyrine to hepatic protein in vivo and in metabolizing liver microsomes. electrophilic reactivities of possible ultimate carcinogens. Cancer Res., 36; Naunyn-Schmiedebergs Arch. Pharmacol., 297: 105—110,1977. 1686-1695, 1976. 231 . Teramoto, S., Moriya, M., Kato, K., Tezuka, H., Nakamura, S., Shingu, A., 253. Witkowski, J. T., Robins, A. K., Sidwell, A. W., and Simon, L. N. Design, and Shirasu, Y. Mutagenicity testing on ethylenethiourea. Mutat. Res., 56: synthesis, and broad spectrum antiviral activity of 1-$-o-ribofuranosyl 121—129. 1977. 1,2,4-triazole-3-carboxamide and related nucleosides. J. Med. Chem., 15: 232. Tjalve, H. The distribution of labelled aminotriazole in mice. Toxicology, 3: 1150—1154,1972. 49-67, 1975. 254. Wood, R. C., Ferone, P., and Hitchings, G. H. The relationship of cellular 233. Toth. B. Synthetic and naturally occurring hydrazines as possible cancer permeability to the degree of inhibition by amethopterin and pyrimethamine causative agents. Cancer Res. , 35: 3693—3697, 1975. in several species of bacteria. Biochem. Pharmacol., 6: 1 13—i24, 1961. 234. Tsuda, H., Hananouchi, M., Tatematsu, M., Hirose, M., Hirao, K., Taka 255. Zapp, J. A., Jr., HMPA: a possible carcinogen. Science, 190: 422, 1975.

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