0008-5472/79/0039-0000$02.oo chemical characterization ......1mutagenicity table...

(CANCER RESEARCH 39, 3289-331 8, september 1979]0008-5472/79/0039-0000$02.OO

Chemical Characterization of 465 Known or Suspected Carcinogens andTheir Correlation with Mutagenic Activity in the Salmonellatyphimurium 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

studieson groupsof carcinogenicand noncarcinogenicchemicals. Actually, the reported correlations between carcinogenicity and mutagenicity in Salmonella range from 63 to 92%. Withthe exception of a brief comment by Odashima (172) on structure-activity relationships apparent among false negatives andfalse positives, there has been little indication that certainchemical categories of carcinogens are not well detected inSalmonella. That the higher correlations do not necessarilytranslateinto a correspondinglyhigh ability to detect carcinogensfromamonganygroupof chemicalsis illustratedinTables1 and 2. Presented are the Salmonella testing results of 12pesticides judged positive for tumor induction by an expertpanel of scientists reviewing the available studies (62). In thiscase, from among a very relevant collection of chemicals, only4 were mutagenic in Salmonella. Hence, it is important that thereported correlation studies be seen in their proper perspective. The purposeof this paper is to presenta perspectiveonthe value and limitations of in vitro testing in general andmicrobialtesting in particular.

Background

The first 2 studies to examine the correlation between carcinogenicity and mutagenicity were conducted collaborativelyin Japan and the United States. Unlike later studies, the Japanese study did not test all of its chemicals with and withoutS-9 activation. The American study did not use the Salmonellastrains carrying the R-factor, TA100 and TA98 (154); at leastin the case of the American study, testing was performed withuncoded materials. In the Japanese study (172), 17 (63%) of27 carcinogens are detected in Salmonella, if one includes theupdates on N-nitrosodimethylamine and N,N-dimethyl-4-[(3-methylphenyl)azojbenzamide that were discussed in the text ofthe report. The mutagenicity ofthese 2 carcinogens is dependent on metabolicactivation. Presumably,5-9 activation wasalso not attempted with 4-acetylaminobiphenyl which was registered as a false negative in the Japanese study. Sugimura etal. (226) have shown this carcinogen to be a mutagen in thepresence of 5-9.

While a complete rendering of the American study has notyet been published, 2 summary reportings (178, 180), whichdo not list the chemicals per Se, indicate that 72% of about 70carcinogens tested are mutagenic in Salmonella. The difference in the correlations was thought to be due in part to thedifferences in the chemicals selected for testing in the 2 studies(172). The Japanese study claimed 12 classes of chemicalsand one miscellaneous class versus the 4 classes of chemicalsand one miscellaneous class claimed in its American counterpart.

SEPTEMBER1979 3289

ABSTRACT

Since chemicals exhibiting mutagenic activity pose a potential hazard to their users, there is increasing acceptance ofmutagenicity testing as an integral part of a premarketingtoxicological evaluation of chemicals. In vitro testing has gainedmuch notoriety as a quick and relatively inexpensive means toassess the mutagenic potential of chemicals. However, theinnovative use of microsomes to simulate metabolism has notchanged the fact that in vitro activation cannot duplicate faithfully the metabolism that occurs in vivo. This shortcoming willexpress itself by the production of false negatives and possiblyfalse positives during mutagenicity screening. This assertion isalso borne out by a reanalysisof the ability of known animalcarcinogens to cause mutations in the generally recognizedpremier in vitro system, the Salmonel!a-S-9 system. Althoughprevious studies have suggested that a high percentage(>85%) of all carcinogens will be mutagenic in this system,with no indication that false negatives are associated withcertain chemical types, these findings are of uncertain practicalvalue due to the limited number of chemical types that wereconsidered. An analysis of 465 compounds with known orsuspected carcinogenic activity indicates that about 58% havebeen adequately tested in Salmonella, that the testing hasconcentrated on certain chemical types and has neglectedothers, and that some categories of carcinogens exhibit mdividual correlations that are unsatisfactorily low by any standard. Poorly detected categories of carcinogens include: azonaphthols; carbamyls and thiocarbamyls; phenyls; benzodioxoles; polychlorinated aromatics, cyclics, and aliphatics; steroids; antimetabolites; and symmetrical hydrazmnes.Nonstandard procedures are necessary to optimize the testing of chemicals that are bactericidal, that are volatile, or that cross-linkDNA. False negatives appear to arise for two reasons: aninability to devise an in vitro activation system that can bereliably used in a standard way; and an inability to detect theentire spectrum of mutational events that can lead to theinductionof cancer.

INTRODUCTION

In both scientific and science news publications, statementshave appeared suggesting that a high percentage, e.g. , 85%or greater,of all chemicalcarcinogenswill bemutagenicin onein vitro system, the Salmonella-S-9 system (7) (hereafter referred to as Salmonella). These statements rely on several

1 This manuscript has been supported by EPA Grant R804621 020; Project

Officer, Dr. J. F. Stara.2 To whom requests for reprints should be addressed.

Received July 17, 1978; accepted April 19, 1979.

Research. on November 11, 2020. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Table1Mutagenicityfindings previously reported for 6 Mrak Commission(Departmentof

Health, Education, and Welfare (62)j carcinogenic pesticides intheSalmonella-S-9system1ig/

S-9 in- Ref.Chemical platea TA strains ducersourcePositiveBis(2-chloro-

NSC 100 (No S-9) 217ethyl)etherâ€•Negative

AIdrin 100 1535, 1537, Aroclord1538,100,98217p,p-DDT@

NS 1535, 1537, Aroclor2171538,100,98104149Dieldrin

10,000 1535. 1537, 100, Aroclor 15298d149Heptachlor'

NS 1535, 1537, Aroclor2171538,100.98149Amitrole

@@ 5,000 1535, 1537, 100, Aroclor15298

S. J. Rinkus and M. S. Legator

Thus, 6 correlation studies have been conducted with Salmonella, but only 4 of these have been adequately reported inthe literature to date. The lists of carcinogens in these 4 studiesare not mutually exclusive. In fact, 78 carcinogens appear in 2or more of the lists of carcinogens in the 4 reported studies.This redundancy results from a duplication in both the testingof some carcinogens and the reporting of test results for others.

Statistical Considerations of Correlating

Let us assume that there is a set of chemicals which are ableto cause cancer in the human species at some level of exposure. It should be clearly noted that whether the activity ofsome or all of these human carcinogens has a threshold is notessential to the following argument, although it surely has otherimportant implications. Chart 1 illustrates the overlap betweenthis set of carcinogens and the set of mutagens, some of whichare defined so, although not necessarily exclusively, from theirmutagenicity in Salmonella. The more carcinogens and mutagens overlap, the more mutagenicity testing as a screen forcarcinogens becomes appealing. Chart I also illustrates acontinuum of correspondences that could be expected. Therelationships between carcinogens and mutagens as well asmutagens and Salmonella could vary independently of eachother (only 2 extremes are depicted in Chart 1 for the sake ofsimplicity). In order to predict human carcinogenicity (and,presumably, human mutagenicity) on the basis of results fromtesting that utilizes only Salmonella, the situation depicted onthe extreme left of the continuum must be true. Similar Venndiagrams offered by Sugimura et al. (226) depict a historicalmerging of the carcinogens and mutagens but do not provideevidence of an extreme overlapping due to the few chemicalsthat were considered.

In Chart 2, the â€˜â€˜trueâ€•correlation between carcinogenicityand mutagenicity in Salmonella or population percentage, P,can be estimated reliably only in the form of p when theheterogeneity of this set of all carcinogens is representativelyreproduced in the sample of carcinogens from which p iscalculated. When such is not observed, the correlation will beeither over- or underweighted. Equally important for a hetero

II(ts@@

Chart 1. C. set of all human carcinogens; M, set of all mutagens; S, set of allSalmonella mutagens. In I, many carcinogens are mutagens. In II, a spectrum ofcorrespondences between C and M as well as M and S can be defined, only 2extremes of which are shown. Only in II, left, does the bacterial system qualify asa qualitative test for carcinogens and mutagens In a systematic screening ofchemicals.

a The highest of several concentrations tested in the first cited source is

[email protected] mutagenic when assayed in a desiccator or in suspension than when

incorporated into the agar.C NS, not stated.

T H. Connor, unpublished data.e DDE (a metabolite of DDT) tested up to 5000 @zg/plate also is not active in

comparable testing (152)., Heptachlor epoxide (a metabolite of heptachlor) also is not mutagenic in a

phenobarbital-induced system, but this testing did not utilize TA100 and TA98(149).

Four studies have utilized uniformly 5-9, TA100, and TA98in their testing. McCann etal. (151, 152) tested 95 carcinogensand combined their results with previously published findingsand unpublished works from other sources for 83 other carcinogens. It was thus seen that 156 (88%) of the 178 carcinogens(excluding cigarette smoke condensate) are mutagenic in Salmonella. Poirier and Simmon (180) have suggested that thedifference between the correlations of the American study andthe reports of McCann et al. could be due to the use of thesensitive Salmonella strains TA100 and TA98. While it is notknown how true this is, another aspect has undoubtedly influenced the outcome. Of the 95 carcinogens actually tested byMcCann et al. in their study, 74 (78%) are positive in theSalmonella; this is in comparison to the 82 (99%) successfulidentifications of the 83 carcinogens for which results wereobtained from other published and unpublished studies.

Of the 240 chemicals that their group has investigated formutagenicity in bacterial systems, Sugimura et al. (226) wereable to detect 90 (92%) of the 98 carcinogens tested inSalmonella.

Heddle and Bruce (104) have reported that 25 (69%) of 36chemical carcinogens tested in Salmonella were mutagenic.This correlation does not include 5-iodo-2'-deoxyuridine whichwas mistakenlylisted as a carcinogen.3Interestingly,X-rayswere shown not to be mutagenic in the system. This contrastswith the previously reported mutagenicity of X-rays in Salmone/la (3).

Finally, Purchase et al. (187), in a preliminary report thatalso does not list all the chemicals, has obtained a 91%correlation with 58 compounds representing 3 classes and onemiscellaneous class of carcinogens.

3 J. A. Heddle. personal communication.

90'@..OF CARCINOGENSAREPOSITIVE IN SALMONELLA;THE SETSOF CARCINOGENSAND MUTAGENSEXTENSIVELYOVERLAP

SOMECARCINOGENSAREPOSITIVE IN SALMONELLA;THE SETSOF CARCINOGENSAND MUTAGENSDO NOTEXTENSIVELY OVERLAP.

3290 CANCERRESEARCHVOL. 39



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

liver With liver hoToxicityChemical@og/pIateTAstrainshomogenatemogenateQsg/plate)Chlorobenzilate1

0, 1005 strainsâ€˜-1@ 150Mirex20,

200, 30005 strainsâ€˜-1â€”1>3000Strobane160, 1600, 48001 00

982.94Â±0.26 1.54 Â±0. 18

2.03Â±0.381.10Â±0.181600Di-allate(Avadex)â€•25. 50, 1001535

1000.82Â±0.29 19.98 Â±3.48

1.06Â±0.10â€˜-1050N-(2@H@ydroxyethyl)hydrazineC

PCNB560,839, 1119

100, 5001535 5 strains2.74Â±0.27 5.41 Â±0.79

â€˜-1 â€˜-1>1119500ControlsCyclophosphamide1

001 5351 .42 Â±0.33 8.15 Â±3.64NTeBenzo(a)pyrene515381 .08 Â±0.29 3.96 Â±2.81NT9-Aminoacridine1

01 537>1 00NTNTMethylmethanesulfonate64721 00>1 0NTNTHycanthone

methanesulfonate1 0986.26 Â±1.92 NTNT

Chemical Structure Carcinogenicity and Salmonella Assay

Table

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 and9-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 sulfoxideexcept 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 withTA1535. 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 andhomogenization. the liver was centrifuged at 9000 x g for 10 mm, and the supernatant (the 5-9 fraction) was stored at â€”80Â°.

Indicated amounts of each chemical were added to 2.0 ml molten agar at 50Â°containing approximately 2 x 1o@cells of 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 mmolglucose 6-phosphate; 4 mmol NADP; 100 mmol sodium phosphate (pH 7.4). Liver homogenate (0.5 ml of the complete mix) was added to themolten agar as indicated. The mixture was then poured over plates of minimal agar supplemented with biotin (0.5 tog/mI) to which histidine (4zg/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 37Â°for 48 hr. the plates were scored for histidine revertants. Positive findings are presented as R1/R5Â±S.D., the ratio Â±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 givenchemical at the last cited concentration in the specified strain(s); a dose respons@ was observed with each positive chemical. Negative resultsin all 5 strains are represented by: â€˜-1. The spontaneous number of revertants on dimethyl sulfoxide control plates without liver homogenatefor all experiments were: TA1535, 46.33 Â±12.15; TA100. 239.74 Â±40.65; TA1537, 12.50 Â±3.60; TA1538, 37.94 Â±8.64; TA98, 46.21Â± 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 dissectingmicroscope and was assigned if at least one strain showed reduced growth. If no toxicity was observed at the highest concentration used inthe 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.

host, of eliciting a carcinogenic response; they may or may notbe related to presently existing chemical structures that areassociated with carcinogenicity. It can be anticipated that thecreation of these future carcinogens will be influenced in partby present-day trends in technological, cancer, and mutationresearch.

Only presently existing carcinogens lend themselves to anyanalysis, but they are comprised of 3 parts: carcinogens testedand found positive (known carcinogens); carcinogens testedand not found positive (false negatives), and carcinogens nottested. This implies then that the ultimate size of the set ofknown carcinogens cannot be defined until all presently existing chemicals have been adequately tested, which is an impossibility. Since correlations can be calculated only with presentlyknown carcinogens, it is not necessarily true that such correlations are indicative of the more general set of presentlyexisting carcinogens or the general set of all carcinogens. Howindicative a correlation is will depend on the representativenessof the sample with which the estimation is made.

Second, if correlations with the known carcinogens can be

3291

geneous population like this set of carcinogens is the use of selves, or as derivatives resulting from their metabolism in theoverstratification (i.e. , overcategorizing) in the construction ofcategories. This allows the determination of p,,, the correlationof an individual category. The practical value of p,'s is that,despite how high P is, when a given p@is low, it diminishes thecredibility that can be associated with negative results forchemicals tested in the given category.

To estimate this â€˜â€˜true'â€correlation requires some understanding of what constitutes this set of carcinogens. It isbeyond the scope of this paper to review the appropriatenessof interpreting the results of animal testing in terms of possiblehuman experience. If it can be assumed, as Lijinsky (139) andCommoner (51) have essentially argued, that such can be doneat least qualitatively, there still remain theoretical and practicalaspects to the calculation that confound the actual interpretation of the derived correlation.

First, this set of all carcinogens can be defined into 2 parts:carcinogens that presently exist, although not necessarily identified yet; and carcinogens that will be developed in the future.The latter can be thought of as heretofore untried arrangementsof carbons, nitrogens, oxygens, etc. , that are capable in them

SEPTEMBER1979



P@

Construction of table of 465 compounds with known or suspectedcarcinogenicactivity(Table4)No.

ofchemi

calsidenti

Source tiedCommentsIARC

vols. 1â€”13 (114)a.Animal carcinogens184b.Bladder implantation (bi) 1 StronglyactiveonlyC.

Epidemiological evidence4(e)

onlyd.Possibly strain-specific 9 Those 7 chemicals reviewedin(ss)

carcinogenicity in vol. 12 were considerednotrodentsevaluablee.

Appearing in the 30 Documentationofdiscussionof the carcinogenicity isfairlycarcinogenicity

ofconvincingmetabolites(M)Total

228EPA'sOrdering of the NIOSH 202 Weakest criterion foridentifyingSuspected

Carcinogens chemicals for thetableList(78): reportedpositivein

at least 2mammaliansystemsIndividual

chemicals 182 82 were also reviewed byIARCcommittees;4 of thesewereconsidered

notevaluable:ziram,8-quinolinol,chloromethyl

methylether,andmaleichydrazideSecretarys

Commission on 36 IARC disagreed thatthePesticidesand Their carcinogenicity of IPC,aldrin,Relationship

to and heptachlorwasEnvironmentalHealth (62):substantiatedp

<0.054correlation studies using234Salmonella-S-9

system(104,152, 172,226)Total

no. of individual chemicals 465 Proof of carcinogenicityvariesinTable 3. from strong to marginal


RANDOM SAMPLING ANDPROPORTIONAL ALLOCATION METHODS respectively, and that the percentages of some carcinogenic

types, like haloaliphatics, are zero. However, this is not tosuggest that there is anything intuitive about what constitutesa random sample of carcinogens. The problem is unconventional, and it does not lend itself comfortably to a statistical

p2 approach.

Given the circumstances surrounding the hitherto reportedp3 correlation studies (uncoded testing of chemicals, use of liter

ature searches, and other nonrandom selections of chemicals),P the practical value of these overall correlations beyond the

carcinogens tested is uncertain. However, this is not to insinuate that, in some final analysis after more innovations intesting, the overall correlation would not be as high as thesereports suggest. Rather, it is to underscore the point that suchconclusions, at the least, are premature and not warranted bythe studies Conducted. Furthermore, as will be discussed below, interest in this overall correlation becomes academic inview of the indication that certain categories of carcinogensexhibit individual correlations that are very low.

Construction of Table of 465 Compounds with Known orSuspected Carcinogenic Activity (Table 4)

Table 4 is an attempt to enumerate more of the carcinogensknown from animal testing. It is a compilation of 465 compounds identified by several criteria. The table combines theevaluations of chemicals for carcinogenicity published by

Table 3

TRUTHS ABOUT CARCINOGENS

PERCENTAGE POSITIVEIN SALMONELLA

P

CATEGORIESSIZE OF CARCINOGENS

N1@ C1 ]

P2 .@- N2@ C2

P3 -4.. N3@ C3@ n3â€•(i@;-)n

I C J

PERCENTAGE POSITIVEIN SALMONELLASAMPLE SIZE

(N@

I \N11 I

/N2\

/N \

nii

nI

assumed to be satisfactory, there is the practical problem ofenumerating known carcinogens so that they can be tested.Saffiotti (203) has warned of the possible shortcomings ofcorrelations based on lists of carcinogens. Also, he estimatesthat, of the 6000 chemicals tested for carcinogenicity andrecorded in the Survey of Compounds Which Have BeenTested for Carcinogenicity (through 1972) (64), only one-halfwere probably tested with some degree of adequacy. Thus, thesome 1000 suggested carcinogens in this listing could beexpected more reliably to be about 500. While this figure of500 serves as a rough estimate of the number of knowncarcinogens (through 1972), the publication does not provideany actual listing of these presumable carcinogens. Generally,the best lists of known carcinogens can be expected to bethose which represent the evaluations by expert Committeesreviewing the documentation for carcinogenicity for thesechemicals. Other sources are available, but they are only ascredible as they are critical.

This practical problem of enumerating known carcinogenshas had a biasing effect on the aforementioned correlationstudies. That nonrandomly selected carcinogens have beentested is best illustrated in the reporting of Sugimura et al.(226) but is generally true of each of the studies. If their sampleof 98 carcinogens were randomly selected, the frequency ofchemicals from a given category in the sample should reflecttheir frequency in the population of carcinogens to whichinference is drawn (Chart 2, njn@@ N@/N1).The sample ofSugimura et al. indicates, probably erroneously, that 33 and17% of all carcinogens are N-nitrosamines and 5-nitrofuryls,

N

N1â€”@N1

PopuI@tIoniPercenlaqe@ P

â€”i(Ni\(positi@,psinN@N1/@ N

n posilivesinN@E N

Eslimalorol Pip

- i (@)

-@ (@ â€˜I(positives inn

@N1/@ n

-@ (@L) (vosdives inn

, Ni

â€”zpositivesin

Chart 2. The set of all carcinogens has a total of N1chemicals that can beclassified into several categories (C â€”Â°C@)that have different sizes (N â€”.N,)and percentages positive in Salmonella (P â€”@P,). The â€œtrue'â€˜correlation betweencarcinogenicity and mutagenicity in Salmonella or population percentage, P. isactually the summation of the products of the size of each of the categoriesrelative to the total (N/N,) times the respective percentage positive (P,). Inproperty conducted random sampling and proportional allocation methods, theheterogeneity of N, is reconstructed in the sample, n,. Hence, the number ofcategories. their relative sizes (i.e., n,/n,) and their respective percentagespositive in Salmonella (pi â€”@p,) will estimate their counterparts for the set of allcarcinogens. Likewise, p, which is also calculated as a summation, will be anestimate of P.





Table4ChemicaltrendsandsomeSalmonellatestingresultsof 465 compoundswithknownor suspectedcarcinogenicactivity

Carcinogenicity DocumentationIARC(114). Thereference(volume:page)to the appropriatemonographis presented.Thesymbolsthat can precedethe referenceare as follows:bi,

bladderimplantationtestingis the only adequatelyconductedtestingthat hasshownthe chemicalto havea carcinogeniceffect;e, epidemiologicaldataratherthanmammaliantestingindicatesthat the chemicalis associatedwith carcinogenicity;M, the chemicalwasnot the subjectof a reviewper Se,butits documentationof carcinogenicitywascited by an IARCstudygroupbecausethe chemicalis a metaboliteof or structurallysimilarto anotherchemicalwhich that study group was specifically evaluating (the authors of this paper have reviewed these studies and have found them indicating a carcinogeniceffect in the experimental design used); as, the observed carcinogenicity to date has been strain specific; â€”, chemical was concluded not to have been

positivein all adequatetesting;?, the studygroupwasunableto evaluatethe documentationof carcinogenicitydue to inadequatetestingor inadequatereportingof findingsin the availablestudies.

EPA(78).The4-digitidentifiersummarizesthedocumentationofcarcinogenicityasitwouldhaveappearedinanupdatedversionofthe1975 SuspectedCarcinogens(55).Thefirst digit (fromleft to right)representsthe highestphylogeneticspeciesin 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 positiveresponsewasreported, 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 skinapplication;1, p.o.administration;0, all otherroutesof administration.Thefourthdigit is a countof thenumberof differentspecies-per-routecombinationsreportedpositive,up to a maximumof 9.

Department of Health, Education, and Welfare (DHEW) (62). The judgment reached by the Technical Panel on Carcinogenesis is presented: B,chemicalincreasedtumor incidencein one or moremammalianspecies;the results are significantat the 0.01 level; Cl , chemicalsincreasedtumorincidencein 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 groupofpositive controls; C3, chemicals increased tumor incidence in comparison to the negative controls, but the level of significance was less than 0.02 (thestatisticalsignificancesof thesechemicalshas beencheckedand only thoseactive at the 0.05 levelof significancehavebeenused in the table);C4,chemicalwastestedappropriatelyin onespeciesonlyandjudgednot 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.Thesymbolsthat can precedean identifierare as follows:+ , positiveresults;w+ , weaklypositiveresults;?, toxiceffect preventsproper testingor borderlineresults; â€”, negativeresults,minimallyin TA100 and TA98 with and without activation.Underlinedresultsindicatethat the chemicalwasnot listedas a carcinogenin that reporting.For referencesof carcinogenicityin a givenreporting,the readeris referredtothe respective article. â€˜â€˜Miscellaneous'â€contains Salmonella testing results from other sources. Testing results that are referenced as personalcommunicationsof T. Connorweredevelopedundera collaborativeprojectwith the Foodand DrugAdministration.

Carc@nogenlcity SalmonellatestIng CaICInOgenICfty

Chsm@& 1 I@/@ @Â° @@@/I/:L@ - 0@

Cyanamide 19. 4-ft4.(5.((2-Hydroxyetttyl)[email protected]. Cyanamide 3111 1 fonyl)-2-methoxyphenyflezo}.5-

Triazene hydroxy-3-methylpyrazol-1-yl)

2. 3-Monomethyl-1-phenyftrlazene 42233, 1-Phenyl-3,3-dlmethyttrlazene 4113 +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 ,1â€˜-biDiazo phenyl)-4,4'-dlyIJls(azo))bie(5-

5. Dlazomethane 7:223 4223 arnino-4-hydroxy-2.7-naphtha6. Azaeerine 10:73 +114 @ef@edlsuftoflicacid),tetrasodium7. N-(DlazoacetyOglycineamide 3101 +115 @t(trypSfl blue)8. N-(DlazoacetyOglycinehydrazine 3101 I'Ll 6 (154) 6,6'-g3,3'-DImethyKl,1â€˜-bi

ptwnyl)-4,4'-dlyIJle(azo))ble(4-

@@ M@;r:@ +L1@ I11. 1-(Phenytazo)-2-naphthalenol 8:2253112 â€”(4Â°)

(Sudanl) : â€” Azoxy :12 4-((4-Hydroxy-1-naphthalenyl)- 8 173 4101 (40)@ &zoxymettw.ne M4 14 4101 +2

azoJ@enzenesulfonlcacid, 21. Azoxyethane M4:15' 4101monosodIumaalt(Orange I) 22. Methylazoxymethanol M1O:127 4202 +G22

13. 1.((2.Methylphenyl)szoj-2- 8:165 3101 23. Methylazoxymethanolacetate M1O:131 +G23 +14' + (126naphthalenoI(0ll0rar@ge5S) 24. cyc@ 10:121 4313 â€”021 â€”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+K4naphthalenol(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+K6tonic acid, dleodlumSaN(Pon- 28. leonicotinIcacid hydrazine (leone- 4:159 4214 _bceau MX) azid)

1 7. 3-Hydroxy-4-((2.4,5-tr*methyl- 8:1 99 421 2 â€”(40) 29. 1 -Acetyl-2-isonicotlnoylhydrazlne M4:1 66 3111

phenyOezo).2,7-naphthalenedi- @o.2-Hydrazlno.4-(4-amlnopheny9. 4111 .113eu@onlcacid. disodlum saltroacRodNo.1; @onceau3R) 31. 2-Hydrazino-4-(4-nftrophenyl). 4212 â€˜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 â€”K5

Salmonella testing

SEPTEMBER1979 3293



Table 4â€”Continued

Carcsrtogen,c@ty Salmonellatesting / Carcinogenicily Salmonellatesting

Chemical@ j@@$ @/ /______________ /@/@â€˜/@/ ChemIcal@ 1V@y@/

@/i;6/@@/â€˜-@

_______________ ___ ______________ 4c)/@/ /.â€”@â€”

N-Methyl-N-benzytnltrosamineN-NitroeodlethylamineEthyl-2-hydroxyethylnlfrosamlneN-Ethyl-N-nltrosovlnylamineN-Ethyl-N-nltrosobutylamineN-Butyl-N-(2-hydroxyethyOnitrosamineN-Ethyl-N-(4-hydroxyethyl)-nitrosamineN-Ethyl-N.(3-carboxypropyl)-nitrosamineN-NltrosodlpropylamineN-(2-Oxopropyl)-N-nitrosopropylamineN-Butyl-N-(2-oxopropyl)-nteosam@eN-Butyl-N-(2-carboxyethyl)-nhtrosamine


34. 1,2-Oiethylhydrazine35. 2-Phenylhydrazinecarboxamide

(@arbazide)36. N-lsopropy$-a-(2-methylhydra

zino)-4-toluamideHt@l(procarbazine)

(197) 2@2-Formylhydrazino)-4-(5-nltro2-furyl)thiazole

(198) 2-(2,2-Dimethylhydrazlno)-4-(5-nitro-2-furyQthiazole

37. N-Nitrosodlmethylamine

38. N-Nitrososarcoslne39. N-Methyl-N-n-butylnltrosamine40. N-Methyl-N.(4-hydroxybutyl)-

nitrosamine41 . N-Methyl-N.(3-carboxypropyl)-

nitrosamine42. N-Methvl-N-n-dodecvlnltrosamine43.44.45.46.47.48.

49.

50.

51.

52.

53.

54.

55. ,v-re-@ropy$-N-n-butylnltrosamine56. N-Propyl-N-(4-hydroxybutyl)-

nitrosamine57. N-Propyl-N-(3-carboxypropy0-

nitrosamine58. N-Nltroso.dl-n-butylamlne

59. n-Butyl-(4-hydroxybutyl)-nitrosamine

60. N-8utyl-N-(3-oxobutyO@nitrosamine

61. n-Butyl'<3-carboxypropyUnitrosamine

62. N-n-Butyl-N-n-amylnltrosamlne63. N-Amyl-N-(4-hydroxybutyl).

nitrosamine64. Dl-n-pentylnftrosamO@e65. N-Methyl-N-nltrosoanillne66. N-Methyl-N,4-dinitroeoanhline67. 1-Methyl-i -nitrosourea

as. i -@tt@yi-i-nitrosourea69. 1-Butyl-1-nitrosourea

70. 1,3-Olmethyl-1-nitrosourea71 . N-o-Glucosyl-(2)-N'-nftroso

methylurea(atreptozotocin)72. N-Methy$-N'-nltro-N-nitrosoguani

dW@e73. @EthyI-N'-nitro-N-nhtrosoguanl

dine74. N-Propyl-N'-nltro-N-nitrosoguanl

dine75. N-Butyl-N'-nitro-N-nltroeoguani

dine76. N-lsobutyl-N'-nltro-N-nitroso

guanidine77. N-Pentyl-N'-nltro-N-nltrosoguani

dine78. N-Nltroso-N-methylurethan79. N-Nitroso-N-ethylurethan80. N-n-Sutyinltroeourethan

81. 1-NaphthalenolN-nltrosomethylcarbamate(N-nitrosocarbaryl)

82. 1-Nitrosoazetidine83. N-Nitrosopyrrolidine84. 3-(1-Nitroso-2-pyrrniidinyl)-

pyridine85. 1-Nitrosoplperidine86. Hexahydro-1-nltroso-1H-azeplne87. Octahydro-1-nitrosoazocine88. 1,4-Dkiftrosoplperazk@e89. N-Nitrosomorpholine

Carbamyl,Ihiocarbamyl90. Dimethylcarbamylchloride91 . Acetamide

92. Thioacetamide93. Cyclochlorotine94. 2-EEhyl-4-pyridinecarbothioamide

(Ethionamide)(1 85) 2-(2-Furyl)-3-(5-nitro-2-

furyOacrylamide(AF-2)95. Ethyl carbamate(urethan)96. N-Hydroxyurethan97. n-Propyl carbamate98. 4-(Dimethylamino)-3,5-dimethyl

phenol methylcarbamate (ester)(Zectran)

(291) Mitomycin C99. Bis(dimethylcarbamodlthloato

S,S')zinc (Ziram)100. Bis(dlmethylcarbamodilhioato

S,S')lead (Ledate)I 01 . Tetrakis(dielhylcarbamodithioato

S,S')selenium (Ethyl Selenac)102. Tetrakis(diethylcarbamodilhioato

S.S')tellurium(EthylTellurac)103. Sodium diethyldithiocarbamate

104. Potassium bis(2-hydroxyethyl)-

dithiocarbamate1 05. ([1 ,2-Ethanediylbis(carbamo

dithioato)X2â€”flmanganese(Maneb)

106. ([1 ,2-Ethanediylbis(carbamodithioato)X2â€”))zinc(Zineb)

107. 2-Benzothiazolethiol (Captax)108. Thiourea109. N'.(4-Chlorophenyl)-N,N-dimeth

ylurea (Monuron)1 10. 4,5-Dlhydroimidazole-2(3H)-

thione (ETU)(1 86) 5-Nitro-2-furaldehyde semicarba

zone (nitrofurazone)

Diaryl alkynyl carbamate111. 1-Phenyl-1 -(3,4-xylyl)-2-propynyl

N-cyclohexylcarbamate1 1 2. 1 , 1-Diphenyl-2-propynyl N-cyclo

hexylcarbamate113. 1,1-Diphenyl-2-butynyl N-cyclo

hexylcarbamate

Aromaticamine1 14. 2,4-Diaminotoluene

1 1 5. 3-Hydroxyanthranilic acid

1 1 6. lsopropyt N-phenylcarbamate

(IPC)I 1 7. N-(4-Ethoxyphenyl)acetamide

(Phenacetin)1 18. N-Hydroxyphenacetin

1 1 9. 2,6-Dichloro-4-nitroaniline

1 20. 2-Chloro-4,6..bis(ethylamino).

1 .3,5-triazine (Simazine)

I 21 . 4-Aminobiphenyl

1 22. 4-HydroxyaminobIphenyl

I 23. 4-Acetylamlnobiphenyl

I 24. N-Hydroxy-4-acetylaminobiphenyl

421241114212

43154212421242144314

3123411142123111

3111

4429420231123111

â€”K7 -

w+G1 +8@ -b

@ ?@â€˜

M12:45

12:777.19

7:7710:131

13:83

7:111M7: 12112:201

ssl2:23

?12:251 4213

ssl2:131 3101

ssl2:2i712:183

ssi2:137

3111

4112

ci

C4

C4

ssl2:24 4112C3

4:153 410112:177 3111

@ 4214

1:95 4429

42124122

41114111

1:107 8929Mi :116

42134213

42124202

4:197 4418

M4:204 4111

M4:205 4111

41124212

1:141 41121:125 5529

1:135 42154213

43144:221 4101

4:183 5529

4326

4:211 4418121311 1 1

w+G2

w+G3

+G4

w+G5

+018

+019

+024

+011

+012

+013

+014

+015

+016

+020

w+G6

w+G8

w+G7

w+B19-D28â€”D27

- D21

+12

-019

+D23

+D25

+D24

+A42

â€”119 â€”74â€”18

-205 â€”74-06

+A26 â€˜18 +73-06

+7 â€”73â€”07

+ 141

+15@

+154

+ 15;

+ 141+74

+44

+9@

+91

+8

+51

+21+21

+21

+ 7@

+4

+5

+3

+3

+54

+ 159

+95

+211

+52

+53

+204

+ 55

+

+

7:9512:16

7:45

â€”12:189

e13:141

M13:144

1:74 I

M1:76

Mi:76

C2

Cl

C2

C2

3101

4111

4212

4212

41123102

3111

4202

5416

31015213

4112

+ (187)

â€” (126)

â€” (126)

â€” (126)

â€” (126)

â€” (218)

+ (231)

â€”(218)

+7;â€”2C

+74â€”10

+ 73

â€”22

+74â€”14

+73â€”24

+74â€”12





Table 4â€”ContinuedCarcinogenicity Salmonella testing / Carcinogenicity Salmonella testing@//1@a@/ N/@

Chemical f@,@ Chemical @/i@

I

1 25. 4-Amino-3-hydroxybiphenyl

I 26. 4,4'-Dlaminobiphenyl (benzidine)

1 27. 4-Amino-4'-hydroxybiphenyl

1 28. 4-Amino-4'-fluorobiphenyl

1 29. 4-Amioo-3,2'-dimethylblphenyl

1 30. 3,3'-Dlmethylbenzidine (o-toli

dine)1 31 . 3,3'-Benzidinedicarboxylic acid

1 32. 3,3'-Benzldlnediol

1 33. 3,3'-Dimethoxybenzldine

1 34. 3,3'-Dichlorobenzidine

1 35. 4,4'-Methylenebis(2-methylani

line)1 36. 4,4'-Methylenebis(2-chloroani

line) (MOCA)1 37. 4,4'-(lmidocarbonyl)bis(N,N'-dl

methyl)anhline (auramine)1 38. Tris(4-aminophenyOmethane

(pararosanlllne)1 39. (4-(4-(Dlmethylamino)-a-(4-

ethy@3-suttony9amino)phenyl1-benzylldene-2.5-cyclohexadien1-ylidene}ethyk3-sulfobenzyl)-

ammoniumhydroxide, inner ash,sodium salt (Acid Violet 6B)

4-(Phenylazo)benzenamineN-Methyl-4-(phenylazo)enlllneN,N-Dimethyl-4-(phenylazo)-benzenamine (Butter Yellow)N-Benzoyloxy-4-methylaminoazobenzene4-(Phenylazo)-1,3-benzenediamine HCI(chrysoidine)2-Methyi-4-dlmethylamlnoazobenzene

1 46. N,N-Olmethyl-4-((3-

methylphenyl)azo@enzenamine147. 3-Methoxy-4-aminoazobenzeneI 48. 2-Methyl-4-((2-methylphenyO

azo@enzenamine149. 2-(2-Tolylazo)-4-toluidlne150. N,N-Dlmethyl-4-phenylazoaniline

N-oxide151 . 2-(4-Dlmethylamlno)-1 -naphtha

leneazobenzene1 52. 1 -(2-Methylphenyl)azo-2-

naphthalenamine(Yellow 08)1 53. 3,3'-{[3,3'-Dlmethyl(1 .1 â€˜-bi

phenyl).4,4'-diyljbis(azo))bis(5-amino.4-hydroxy-2.7-nsphthalenedisulfonic acid, tetrssodiumsalt (trypan blue)

I 54. 6,6'-((3,3'-Dimethyl(1 .1 â€˜-bi

pheny9-4.4'-diyllbis(azo)}bis(4-amino-5-hydroxy-1 ,3-naphthalenedlsulfonic acid), tetrasodiumsalt (Evans blue)

1 55. trans-4-Aminostllbene

1 56. trens-4-Dlmethylaminostllbene

I 57. N,N-Dlmethyl-4-(2-(1 -naphthyl)vinyl@Bnillne

1 58. 1 -Naphthylamine

159. N-(1 -Naphthyl)hydroxylamineI 80. 1-Nitrosonaphthalene1 61 . 2-Naphthylamine

1 62. N-(2-Naphthyl)hydroxylamine

1 63. 2-Nitrosonaphthalene

184. 3-Methyl-2-naphthylamineI 65. 1-Aminoanthracene168. 2-Aminoanthracene1 67. 1 -Amlno-9,1 0-dlhydro-4-(3-((2-

hydroxyethyl)sulfonyljanilino)9.1 0.dloxo-2-anthracenesulfonicacid.hydrogensulfate(ester),disodium salt

310174263101421342024113

421342264212@416

4111

4212

4213

4101

4101

+A32+A33 +82

I+(187)

+(40)

+(90)

+ (126

+ (126

?(40)

â€”(40)

â€” (187

+ (187

I 68. 3-Acetylaminophenanthrene1 69. 2-Aminofluorene

1 70. N-Hydroxy-2-aminofluorene

I 71 . 2-Nitrosofluorene

172. 2-Acetylaminofluorene

173. N-Hydroxy-N-(2-fluorenyl)acetamide

1 74. 2-Diacetylaminofluorene

1 75. N-Acetoxy-2-acetylaminofluorene

1 76. 2,7-Diaminofluorene

1 77. 2,7-Bis(acetylamino)fluorene

1 78. 6-Aminochrysene

1 79. 4-(Hydroxyamino)Quinoline 1-

oxide180. 3,6-Bis(dimethylamino)acridine

(acridine orange)(98) 4-(Dimethylamino)-3,5-dimethyl

phenol methylcarbamate (ester)(Zectran)

(1 09) N'@4-Chlorophenyl)-N,N-dimeth

ylurea (Monuron)(1 92) 2-Amino-.4-(5-nltro-2-

furyl)thiazole(1 93) N-(4-(5-Nitro-2-fury9-2-thiazolyll

formamide (FANFT)(1 94) N-(4-(5-Nitro-2-fury9-2-thiazolyl)-

acetamide(1 95) 2,2,2-Trifiuoro-N44-(5-nitro-2-fu

ryl)-2-thiazoly9acetamide(1 99) 2-Amino-5-(5-nitro-2-furyl)-1 .3,4-

thiadlazole(200) N-(5-(5-Nitro-2-fury9-1,3,4-thiadi

azol-2-yl)acetamide(203) 4,@Diamino-2-(5-nitro-2-fury1)-

1,3,5-triazlne(204) 4-(2-Hydroxyethylamino)-2-(5-ni

tro-2-thienyl)guinazollne(205) 4-Bis(2-hydroxyethyl)amino-2-(5-

nltro-2-thienyl)quinazoline(208) 2-Formylamino-4-(4-

nitrophenyt)thiazole(41 2) 4-Aminobenzenesulfamide (sulfa

nilamide)(41 3) 2-(4-Aminobenzenesulfonamido)-

Nitroaromatic1 81 . 1 ,2-Dimethyt-5-nitrolmidazole

182. 1 -(2-Hydroxyethyl)-2-methyi-5-ni

troimidazole (Metronidazole)183. trans-2-{(Dlmethylamino)methyl

imino)-5-(2-(5-nitro-2-furyl).vlnyl)-1,3,4-oxadiazole

184. Potassium1-methyl-7-(2-(5-nitro2-furyl)vlnyl)-4-oxo-1,4-dihydro1 ,8-naphthyridine-3-carboxylate

185. 2-(2-Furyl)-3-(5-nitro-2-

furyDacrylamide(AF-2)186. 5-Nitro-2-furaidehyde aemicar

bazone (nitrofurazone)1 87. 1 -((5-Nltrofurfurylidene)amlno)-2-

Imidazolidlnone1 88. 4-Methyl-i -((5-nitrofurfuryli

dene)amlno).2-imidazolidinone189. (-)-5-(Morphollnomethyl)-3-((5-

nitrofurfurylidene)amino).2-oxazolidinone

190. 4-(5-Nitro-2-furyl)thiazole

191 . 2-Methyl-4-(5-nitro-2-

furyl)thiazole1 92. 2-Amino-4-(5-nitro-2-

furyl)thiazole1 93. N-(4-(5-Nftro-2-furyl)-2-thiazolyl).

formamide(FANFT)1 94. N-(4-(5-Nftro-2-furyO-2-thlazolyl)-

acetamide195. 2,2.2-Trifluoro-N-(4-(5-nltro-2-fu

ryl)-2-thiszolyl)acetamide

E2124225420241025529

4519

422331214111

4212

4225

4222

M1:761:80

Mi :76

1:87

4:414:494:73

4:65

1:69

?4:57

+A2 ++A5+A6+A3 +8

+A4 +116@

+A1+A13+A12+A14+E7 118

+A17

+E1 1

+E10 @169

+E21 +81

+211

+E16 +104 +

+E21 @181+

+E21 1155

+A29

+A27

+A34

â€”A43

â€”A35

+L2+L5+16

+111

.73 +(187)â€”11

+ 74

â€”19

+11

+15+145+80

+34

8:53 01014112

8:12!'4324

4101

8:91 3111

4212

8:61 4315

42124212

4222

8:2W 4101

8:26@ 4102

8:151

140.141.142.

143.

144.

145. +110 +151

+17

+14+13

+ 152

+ 13(+16

thiazole

411113:113 3111

7:147 4111

?7:171 4111

7:181 4111

4111

7:161 4111

3111

@414

7:185 5414

3111

421241 11

4202

3101

410141016517

420331014202411142224212

+7

+7â€”1

+ 74

-04

+ 74

â€”07

+ 74

â€”08

?4:87

M4:92M4:924:97

M4:1 0M4:1 0

+A22

+A24

+A21

+A23+A25

+A20+A19

+173 +

+174 + +E21

+E21

+E21

+E17

+E21

+E21

+191

1156

+20

+ 193

+ 192

+ 237

+

3295SEPTEMBER1979





@//@ Chemical@@Carcinogenicity Salmonellatesting Carcinogenicity Salmonellatesting

/ii;/@_/,$, â€˜N

Chemical@ @Jz/@':4'LL ____________ ___

5.- ,@â€˜ c?,@:, a

3112 1234. a-(2-(24utoxyethoxy)ethoxy)-4,5-(methylenedioxy)-2-propylto.luene (Piperonyl Butoxide)

Substituted diphenylethane235. Ethyl2-hydroxy-2,2-bis(4-chloro

phenyl)acetate(Chlorobenzilate)(355) 1.1,1-Trichloro-2,2-bls(4-chloro

phenyl)ethane(p,p'-DDT)(356) 1.1-Dichloro.2,2-bis(4-chloro.

phenyflethane(p,p'-TDE; ODD)(357) 1.1-Dichloro.2,2-bis(4-ethyi

phenyl)ethane(Perthane)(393) 1,1-Dlchloro.2,2-bis(4-chloro.

phenyOethylene(p,p'-DDE)

Stilbenediol236. trans-a,a'-Diethyl-4,4'-stilbene

diol (DES)237. trans-a,a'-Diethyl-4,4'-stilbene

did dipropionate238. meso-3,4-Bia(4-hydroxyphenyl)-

hexane(dihydrostilbestrol)

Polyaromatic239. a-(((1-Methyl)amlno)isethyl)-2-

naphthalenemethanolHCI(PronetelolHOt)

(429) 3-Methoxy-16.17-secoestra1.5.5,7.9-pentaen-1 7-oic acid

240. Benz(a)anthracene241 . 4-Methylbenz(a)anthracene242. 5-Methylbenz(a)anthracene243. 6-Methylbenz(a)anthracene244. 7-Methylbenz(a)anthrscene245. Benz(a)anthracen-7-yltrichloro

methyl ketone246. 3-Fluoro-7-methylbenz(a)-

anthracene247. 6-Fluoro-7-methylbenz(a)-

anthracene248. 10-Fluoro-7-methylbenz(a)-

anthracene249. 8-Methylbenz(a)anthracene250. 9-Methylbenz(a)anthracene251 . 1O-Methylbenz(a)anthracene252. 12-Methylbenz(a)anthracene253. 7,1 2-Dlmethylbenz(a)anthracene

254. 12-Methylbenz(a)anthracene-7-methanol

255. 7-Formyl-12-methylbenz(a)-anthracene

256. 7-Methoxy-12-methylbenz(a)-anthracene

257. 7-Ethoxy-12-rnethylbenz(a)-anthracene

258. 7-Methylbenz(a)anthracene-12-methanol

259. 6,7-Oimethylbenz(a)anthracene260. 6,12-Dlmethylbenz(a)anthracene261. 7,8-Dlmethylbenz(a)anthracene262. 7,11-Dimethylbenz(a)anthracene263. 8,12-Dlmethylbenz(a)anthracene264. 9,1O-Dlmethylbenz(a)anthracene265. 4,7,12-Trlmethylbenz(a)-

anthracene266. 7,8,12-Thmethylbenz(a)-

anthracene267. 7,9,12-Trimethylbenz(a)-

snthracene268. Benz(a)phenanthrene(chrysene)269. 15,16-Dlhydro-11-methylcyclo

penta(a)phenanthrene-1 7-one270. Daunomycin271 . Adriamycin272. Benzo(a)pyrene

273. 3-Hydroxybenzo(a)pyrene274. 3-Methoxybenzo(a)pyrene275. 6-Hydroxymethylbenzo(a)pyrene276. 7,8-Dihydrobenzo(a)pyrene

196. 2-Hydraz@o-4-(5-nitro-2-turyl)-thiazole

197. 2-(2-Formylhydrazino).4-(5-nitro2-furyOthiazole

198. 2-(2,2-Dimethylhydrazino)-4-(5-nitro-2-furyl)thlazole

199. 2-Amlno-5.(5-nitro-2-furyl)-1.3.4-thiadiazole

200. N.(5.(5-Nftro-2-furyl)-1 ,3,4-thiadiazol-2-yl)acetsmide

201. N-([3.(5-Mtro-2-furyl).1 .2.4-oxadlazol-5-yl)nethyl}acetamide

202. 5-Acetamido-3-(5-nltro-2-furyl)-SN-i ,2.4-oxadiazine

203. 4.6-Dismino-2-(5-nitro-2-turyfl1,3,S-triszlne

204. 4-(2-Hydroxyethylsmino)-2-(5-nitro-2-thienyl)qulnazoline

205. 4@s(2-hydroxyethyl)amlno-2-(5-nltro.2-thienyl)quinazoline

206. 1,2-Dihydro-2-(5-nitro-2-thlenyl)quinszohn-4(3H).one

207. 1-(5-Nltro-2-thlazolyO-2-imidszolidinone (Niridazole)

208. 2-Fonaytamino-4-(4-nitrophenyOthlazole

209. 4-Nitropyridine 1-oxide210. 2-Methoxy-5-nitrotropone211. 4-Nitrobiphenyl212. 2-Nltronaphthalene213. 5-Nitroacensphthene214. 2-Nitrofluorene215. 4-Nitroquinoline216. 4-Nitroquinoline1-oxide(4..NQQ)

217. 4-Mtro-6-quinolinecsrboxylicacid1-oxide

218. 6.Chloro-4-nitroquinollne1-oxIde(31) 2-Hydrazlno-4-(4-nltrophenyl)-

thiazole(1 19) 2.6-Dlchloro-4-nitroanlllne(224) Pentachloronitrobenzene(P@NB)(433) 6.((1-Methyl-4-nftroImidazol-5-

yI)thio@urlne(Azathioprine)

Phenyl219. Benzene220. 5.5-Dlphenyl-2,4-imidazdidlne

dione (Phenytoin)221 . 5-EthYI-5-@*IenYI

2.4,8(1H.3H.5H)-pyrImldlnetrlone(ph@

(446) TriphenyttInacetate(457) 7-Chloro.1 .3-dihydro-3-hydroxy

5-phenyt-2H-1.4-benzodiazepln2-one (Oxazepam)

222. 2-(2,4-Olchlorophenoxy)proplonicacid

223. 2.2'-ThIobia(4.6-dichlorophenol)(Vancide 81.)

224. Pentachloronltrobenzene(PCN8)(324) Tannicacid:galllcacid225. Biphenyl226. Polychlorinatedbiphenyls:Ks

nechl@500 and ArocI@ 1254

Benzodloxole227. 5-(2-Propenyl)-1.3-benzodloxole

(safrole)228. 1â€˜-Hydroxysafrole229. 1â€˜-Acetoxysafrole230. 5-(1-Propenyl)-1.3-benzodioxole

(isosafrole)231 . 5-Propyl-1 .3-benzodloxole (dihy

drosafrole)232. 1,2,3,4-Tetrahydro-3-methyl-6,7-

methylenedioxynaphthalene-12-dicarboxyllc acid, di-n.propyl estar (n.Propyl Isome)

233. 1@2-MethylenedIoxy-4-(2-(oct@sulfinyOpropyi@enzene(Pipero.nyl Sulfoxide)

j (126)

â€” (126)

5:75 3111 B

6:55 4416

4202

3202

13:2273111

312442234223422442254202

4202

4223

4223

42234222422342245729

4213

4202

4202

4202

4212

4202420242024202420242224223

4223

4223

3122

410141016629

42024101

+83 +

+65+13+32 +

+7;â€”01

+74â€”02

+ (187)

â€” (126)

b

+ 7

â€”2

â€”73-09

7:151

7:143

13:12@

4:11@

e7:20313:201

13:157

ss5:21 1

7:261

10:231

Mi 0:240Mi 0:240

10:232

10:233

+E21 +10

+E21 +84

+E21 +19

+E21 +18

+E21 +3

+E21 +68

+E14

+E1 5

+E13

+E12 +171

+ 102

+ 194

+ 144

+E2+E3+E4 +177+E5

+ 195

+E6 +198

â€”Ji

â€”105

â€”210

â€”F15

â€”F16+F17

4212

431@

4212

4111

3111

4111

5111

4212412

4521

4202

4222

31214111

3111

3111

3122

3101

4212

4212

3111

3111

?F19

+C16

+cl 8

+C19

+020

+c14

+034

+H12

+H1 1+C3

W+C6

+C4+C7

3:45

3:159

10:145?1O:43

3:91

M3:1 14

C3

Cl

B

02

C3

ci






/ Chemical@Carcinogenicity Se!monellatesting Carcinogenicity Salmonellatesting

:â€˜

Chemical@ ci@7@1n@:_,?i.

3:137

3:693:82

3:1713:2013:2073:21f'3:2243:2293:197

421

420231014202420242024202420342034202

4111

3101

3101

41013122

31215823123121

4523123121322312231013101

324. Tannicacid:eIIaQiCacid

Anhydride325. Succinicanhydnide326. (3aa,4$,7$,7ao)-Hexshydro

3a,7a-dimethyl-4.7-epoxyisobenzofuran-1 ,3-dione (canthanidin)

Pyrazoiinone327. 4-Amino-2,3-dlmethyl-1 -phenyl

3-pyrazolin-5-one(4-aminoantipyrine)

Pyrrolizidine328. Lasiocarpine 10:281329. Monocrotaline 10:291330. Retroraine 10:303331 . Dehydroretronecine M1O:339332. isatidine 10:269

Hetenoaromatic333. Quinoline334. 8-Quinolinol 113:101335. Benz(c)acrldine 3:241336. 7,9-Dimethylbenz(c)acridine337. 7,10-Dlmethyibenz(c)acnidine338. Dibenz(a,h)acnidine 3:247339. Dibenz(aj)acridine 3:254340. 1-(12-(Diethylsmino)ethyl)amino)- ?13:91

4-(hydroxymethyOthioxsnthen-9-one, methanesuifonate(hycanthone methanesuifonate)

341. Actinomycin 0342. Actinomycin L343. Actinomycln 5344. Aflatoxln B345. Aflatoxin 82346. Aflatoxin G,347. Aftatoxin M,348. Aflatoxicol349. Stenigmatocystin350. 7H-Dibenzo(c,g)carbazole351 . Anthra(9,1 .2-

cde)benzo(h)cinnoiine352. Tricycioquinazoiine

Halomethane,haloethane353. Carbon tetnachlonide354. N-(Trichloromethyl)thio-4-cyclo

hexene-1,2-dicarboxyimide(Captan)

(245) Benz(a)anthracen-7-yl-trichloromethylketone

355. 1.1.1-Trlchloro-2.2-bis(4-chiorophenyDethane(p.p'-DDI)

356. 1.1-Dichloro.2,2-bis(4-chlorophenyl)ethane(p,p'-TDE: ODD)

357. 1.1-Dichloro-2,2-bis(4-ethylphenyl)ethane(Perthane)

358. 10-Bromomethytanthracene359. 10-Chloromethyl-9-methylanthra

cane360. 10-Chloromethyl-9-chloroanthra

cane361 . 9.1 0-Dichloromethylanthracene362. 7-Chloromethylbenz(a)-

anthrscene363. 7-(Chloromethyl)-12-

methytbenz(a)anthracene364. 7-(Bromomethyl)-12-

methylbenz(a)anthracene365. Benzylchloride366. lodomethane

(397) Polychloninated terpenes: Strobane

367. 1,2-Dibromoethane

N-, 5-. or 0-Mustard368. 1.6-81s(2-chloroethylamino)-

1,6.dideoxy-D-mannitol

277.278.279.280.281.282.283.284.285.286.287.288.

Aziridkw289. Aziridine290. 2-Methylaziridine291 . Mitomycin C292. 2-(l -Azirldlnyl)ethanol293. 1-Acetylaziridlne294. 1-Diethyiscetylazlridine295. 1-n-Butyrylsziridlne296. 1-Hexanoytazlridine297. 1-Nonanoytaziridlne298. 1-Myristoylazlridine299. Tris(1-azirldlnyl)phoaphinesulfide

(thio-TEPA)300. Tris(2-methyl-1-aziridinyl).

phosphineoxide (METEPA)301. Bis(1-aziridinyl)morphollno

phosphine sulfide302. 2,5-Bis(1 -aziridinyfl-3,6-bis(2-

methoxyethoxy)-4-benzoquinone303. Tris(l -azlridlnyl).4-benzoquinone304. 2.4,6-Tris(1-azlridinyl)-1,3,5-tria

zine(TEM)

Oxirane, thllrane305. Propyleneoxide (methyloxirane)306. Oxirane carboxaidehyde (gly

cidsidehyde)307. Chloromethyloxirane(epichloro

hydrin)308. Benz(a)anthracene5,6-oxide309. Benzo(a)pyrene4,5-oxide(326) (3aa.4$,7f1,7aa)-Hexahydro

3a,7a-dlmethyl-4,7-epoxy-lsobenzofuran-1 ,3-dione (cantharidin)

(401) 1,2,3,4,1O,1O-Hexachloro..6.7-epoxy-4a,5.6,7,8,8a-hexahydroendo,exo-1,4:5.8-dimethanonaphthslene(Dieldnn)

310. Dlepoxybutane (â€”,meso, and Â±)31 1. 1,2,7,8-Diepoxyoctane312. 2,2'-(2,5,8,1 1-Tetradodecane

1.12-dIyl)bisoxirane313. 1-Epoxyethyl-3,4-epoxycycio

hexane314. 3,4-Epoxy-6-methylcyclohexyl

methyl-3,4-epoxy-6-methylcyclohexane carboxytate

31 5. Thllrane (ethylene sulfide)

Lactone31 7. 2-Oxetanone ($-propiolactone)318. 4-Methyl-2-oxetanone (fl-butyro

lactone)319. 3-Methoxy-5-methyi-4-oxo-2,5-

hexadlenoic acid (penicillic acid)320. 4-Hydroxy-4H-furo(3,2-c@yran

2(610-one(patulin)321. (s)-5.6-Dihydro-6-methyi-2H-

pyran-2-one (parasorbic acid)322. 2H-1-Benzopyran-2-one(cou

mann)323. 2@,4aa,7-Tnlhydroxy-1-methyl-8-

methylene-4b$-glbb-3-ene1a,1 Ofl-dIcsrboxytic acid 1,4a-actone (gibbenellicacid; gibbercHin A3)

Benzo(e)pyrene3-MethyicholanthreneBenzo(b)fluorantheneBenzo(j)ftuorantheneDibenz(a,c)anthraceneDibenz(a,h)anthraceneDibenzo(a,e)pyreneDlbenzo(a,h)pyreneOibenzo(a,s)pyreneDibenzo(a,I)pyrenelndeno(1,2.3-cd)pyreneDibenzo(h,rst)pentaphene

41011

4101 ?D83121

â€”J3

+024+C25

+c9+J1 1

+H1+H2+H5+H4+H3+H8

â€”B22 +821

+C32+030

+C31

+C29+C22

+021

+023

w+B20w+B1

w+B3

+ (154)

+ (126)

+ (126)

41014101

4112

4112

421331213101310131243122

â€” (126)

+ (126)

â€” (217)

â€” (217)

+ (217)

9:379:61

I 0:1719:47

9:85

?9:107

9:55

9:51

9:679:95

11:19111:17@

11:131

11:115

11:209

11:141

11:147

+

+

+

+ 22:

+124

+112

+233

+

+

10:2910:2910:2910:5110:5110:51

Mi 0:62

10:2453:260

1:53

5:83

M5:83

11:217

9:157

4202

31014214

41014101

411253284212

4222

43133112

3111

4212

3111

4101

4222

41014101

4101

+33

+21

-20@

422:

312

422331213101

4222

3121

11:257 4101

Dioxane316. 1,4-Dioxane 11:247 4111

44294224

4202

4101

4112

3121

4:25911:225

10:211

10:205

10:199

10:113

+C8+c13

+clO+011+C1

+C2

+K2+K11H1 4

+K3

+015

+017+C5

+017+018

+09+010

@2 â€”H9

3297

10:25

10:79

SEPTEMBER1979




/ Chemical@@Csrcinogenicity Salmonellatesting Carcinogenicily Salmonella testing

______@/@/@ /

I@ /@ j /,@it1/ c

________ __ ________i'@Chemical j/@4@ â€˜@â€˜;/@Z@@L' __________ __?5:25@ 3 @@11â€”369. 2-0hloro.N-(2@chloroethyl)-N-

methylethanamineHCI(nitrogenmustard HOt)

370. 4.{Bia(2-chloroethyl)amino)-t-phenylsianine(Meiphalan)

371 . 44Bis(2-chloroethy1)amino@enzenebutanoicacid (Chiorambucil)

372. N,N-Bis(2-chloroethyl$etrahydro.2ff-i .3.2-oxaphoephonin-2-amine-2-oxidemonohydrate(cyclophoaphamide)

373. 3-(2-Ohloroethyl).2-((2-chlorethyl)[email protected],2-oxazaphosphonine2-oxide (Isoph@ide)

374. 5-(Bis(2-chloroethyl)amino)-2,4(1H,3H).pynimidinedione(uracii mustard)

375. N,N-Bis(2-chloroethyl)-2-naphthylamine

376. 2-Methoxy-6-chloro-9-(4-bis(2-chloroethyl)amino-1-methylbutytaminojacnidine.2HCI(ICR-i 0)

377. Bis([4-(bis(2-chloroethyl).amino)phenyl)acetate)estra1.3.5(1 0).tnlene-3,1 7/3-diol (astradiot mustard)

378. 2-Chloro.N-(2-chloroethyl)-N-methylethanamineN-oxide HCI

379. (2-Chloroethyl)trimethyl ammonium chloride (CCC)

380. N-(2-Chloroethy9-N-(1-methyi-2-phenoxyethylbenzenemethanamine

381. 2-Methoxy-6-chloro.9-(3-(ethyt2-chloroethyi)amlnopropylamino)-acridine. 2HCI (IOR-i 70)

382. 1.1â€˜-Thiobis(2-chloroethane)(mustardgas)

383. Bia(2-chloroethy0ether384. 2-(4-tert-Butylphenoxy)isopropyl

2-chloroethyl sulfite (Anamite)

Haloalkyl ether385. Bie(chloromethyl)ether(BCME)386. @hloromethyImethyl ether

(OMME)387. i-Chioro-2,2,2-tnifluoroethyl di

fluoromethyl ether (Isoflurane)

Chioroethylene388. Vinyl chloride389. 1.1-Dlchioroethylene(vinylidene

chloride)390. Trichloroethylene391 . 2-Chloro-1 ,3-butadiene (chloro

prene)392. Bie(1-methylethyl)carbamothloic

acid. S-(2,3-dichioro.2-propenyl)eater, trans and cia isomers(Diallate)

393. 1,llchloro.2,2-bis(4-chlorophenyl)ethylene(p,p'-DDE)

Polychlorinatednonsromatic394. a isomerof 1,2,3,4,5,6-hexa

chiorocyclohexane395. p isomerof 1,2,3,4,5,6-hexa

chlorocyclohexane396. â€˜yisomerof 1,2,3,4,5,6-hexa

chlorocyclohexane(Lindane)397. Polychlorinatedterpenes:Stro

bane398. 1,ls,2,2,3,3a,4,5,5a,5b,6-Dode

cschlorooctahydro-1,3,4-metheno-1H-cyclobuta(c,dIpentalene(Mirex)

(93) Oyclochlorotine399. 2.3,5.6-Tetrachloro-i ,4-benzo

quinone (Chlorsnil)

400. 1,2,3,4,10,iO-Hexachloro4a.5,6.7,8,8a-hexahydro-endo,-exo-1,4:5,8-dimethanonaphthalens (Aldnin)

401. 1,2,3,4,iO,iO-Hexachloro-6,7-epoxy-4a.5.6,7,8.8a-hexahydroendo,exo-i ,4:5,8-dimethanonaphthalene(Dieldrin)

402. 1,4,5,6,7,8,8-Heptachloro3a,4,7,7a-tetrahydro-4.7-methanoindene(Heptachlor)

Sulfate, sulfonate, sultone403. Dimethylsulfate404. Diethyl sulfate405. Methyl methaneaulfonate (MMS)406. Ethylmethanesulfonate(EMS)407. 1,4-Butanedioldimethanesulfon

ate (Busulfan)408. 2,4-Dichlorophenolbenzenesul

fonate (Genite-R99)409. 4-Chlorophenyl4-chlorobenzene

sulfonate(Ovex)41 0. Ethyl 4-toluenesulfonate41 1. 1 3-Propane sultone

Sulfanilamide41 2. 4-Aminobenzenesulfamide (sulfa

nilamide)41 3. 2-(4-Aminobenzenesulfonamido)-

Phosphate41 4. Tnimethylphosphate (TMP)

Steroid415. Estrone41 6. Estrone benzoate41 7. 17fl-Estradiol418. 3-Benzoateestradiol41 9. Dipropionate estradiol420. Ethinylestradiol

421. Mestranol422. Progesterone423. Testosterone424. Testosteronepropionate

425. Norethisterone426. Ethynocjjoldiacetate427. Norethynodrel428. Cholesteryl-(+)-14-methylhexa

decanoate(carcinolipin)429. 3-Methoxy-i6,17-secoestra

I ,5,5,7,9-pentaen-17-oic acid

Antimetabolite430. 2.3-Dihydro-2-thioxo-4(1H)-

pynimidinone (thiouracil)431 . 6-Methylthiouracil432. 6-Propylthiouracil433. 6-((i -Methyi-4-nitroimidazol-5-

yl)thio@urine(Azathioprine)434. Xanthine435. 3-Hydroxyxanthinehydrate436. 4-Aminofolicacid(aminopterin)437. 5-(4-Chlorophenyl)-6-ethyl-2,4-

pyrimidinediamine(pynmethamne)

438. S-Ethylhomocysteine(ethionine)(L and Di.)

Polysaccharide439. Carrageenan440. Sodiumcarboxymethylcellulose

441. Dextran 10442. Cellophane

Polymer443. Polyethylene

+

5:125 4212 3 â€”824

?5:i73 B

:2

:2

4:2714:2777:2537:2454:247

6:1796:1736:191

MiO:10â‚¬

41224112421342043122

3101

31 11

41014213

4202

4212

+06+03+04

+

+

+

+

+

w+D5+012 +21:

â€” (217)

+ (126)4:253

6:123

6:99

6:77

6:876:1356:209

thiazole

+810

+B15

+B12

+813

+811

+814

+B16

+B17

+88

430442024304320332034111

311131013101

31 11

411133133101

420242134222

+ (217)

-4 (217)

+ (217),(217)+ (15)

â€” (217)

â€”74â€”23

â€”73â€”26

â€”74â€”30

â€”73â€”27

9:193 4101

9:167 3121

9:125 3122

9:135 4204

9:235 3101

4:119 3101

9:217

9:2094202

3111 Ci

9:223

9:181 3102

9:117 3111 B5:39 3111 B

4:231 4224@4:2393223

Mli:289

7:291 4222

11:263 3111

12:69 3111 B

M5:83 3111

w+84w+B5

?B7

2202

â€”823

7:85

7:537:67

13:233

10:181

5:47

5:47

5:47

ss5:2i9

5:203

31 11

3111

3111

3111 B

3111

3111

â€”74â€”21â€”74â€”22

â€”30

â€”31

4111

4101

43034202

4202

â€”23

â€”F7â€”90

â€”59

CANCERRESEARCHVOL. 393298

S. J. Rinkusand M. S. Legator



455. 3-Amino-I,2,4-trlazole(Amitrole)7:31421 2Bâ€”J5456.i,2-Benzisothiazolin-3-one1,1-4212â€”22â€”(16)dioxide

(saccharin)457.7-Chloro-1,3-dihydro-3-hydroxyMi3:595-phenyl-2H-1

,4-benzodiazepin2-one(Oxazepam)Inorganic458.

Arsenicacid,calciumsalt(2:3)e2:487101â€”(142)459.

Asbestos(chrysotile.amosite,2: 177325anthophyllite,crocidolite)460.

Berylliumandberylliumcorn 1:176222â€”(202)pounds461

. Cadmium and cadmium corn 11:394204â€”+(122)pounds462.

Chromiumandinorganicchro 2:107222+(142)mium

compounds463.Hematite(radon?)e1:29464.Leadsalts1:404212â€”â€”(202)465.

NiCkelandnickelcompounds1 1:754429


Table 4â€”ContinuedCarcinogenicity Salmonella testing

!;/ZÃ³5@/f/i/i/i /

@ I@ /4@ <@I

#/@/i// /1ChemicalChemical

444. Polyoxyethylene8 monostearate445. Polyoxyethylene20 sorbitsn

monoatearate(Tween 60)

Metalcomplex446. Trlphenyltinacetate447. 1-Aurothio-o-glucopyranoae(au

rothioglucose)448. Iron-dextran complex449. Iron-dextrin complex450. Saccharatedironoxide

Miscellaneous451 . 3a,4a,5@-Trihydroxy-1 -cyclo

hexene-i -carboxylic acid(shlkimic acid)

452. Grlseofulvin453. Luteoskyrln454. 1,2-Dihydropyrldazine-3,6-dione

(maleichydrazide)

a5@T@2b T. H. Connor, unpublished data.

C Shakin (cited in Ref. 59).

IARC4expert committees(114) and by a similarly qualifiedcommittee of the Mrak Commission (62) with the carcinogensidentified by a discriminating search of the chemicals in Suspected Carcinogens (78) prepared by the National Institute ofOccupational Safety and Health and those carcinogens citedin the 4 adequately reported correlation studies (104, 152,172, 226). The construction of Table 4 is outlined in Table 3and discussed below.

IARC. The IARC has convenedexpert committeesregularlysince 1971 to evaluate the evidence for carcinogenicity ofvarious chemicals. In its first 13 monographs (114), IARCcommittees have reviewed 283 chemicals and, in doing so,briefly commented on the carcinogenicity testing of another 45chemicalsthat are possiblemetabolitesof or are structurallyrelated to the chemicalsthat were being reviewed.Of these283 chemicalsspecifically reviewed,184 were consideredtohavebeencarcinogenicin at leastonesatisfactorilyconductedanimal study. One chemical was shown to be carcinogenic onlyby the bladder implantationmethod, the other studies beinginadequately designed or reported. Four chemicals have epidemiologicalevidencefor their associationwith cancer but donot havecorroboratinganimalstudiesto date. Ninechemicalshavehad a strain specificity in their carcinogenicityobservedthus far. These latter 14 chemicals have the designations bi, e,and ss, respectively, before their IARC reference in Table 4 tohighlight these aspects about their carcinogenicity testing.

Sevenof the9 chemicalswithasbeforetheir IARCreferencewere considered to be not evaluable by the IARC committee(Vol. 12) that reviewed them. Apparently, this was because of

4 The abbreviations used are: IARC, International Agency for Research onCancer; NTIS. National Technical Information Service; EPA. Environmental Protection Agency; IPC, isopropyl phenylcarbamate; PCNB. pentachloronitrobenzene;DDT, 1â€¢1,1-trfchloro-2,2-bls(4-chlorophenyl)ethane;DDE, 1,1-dichloro2,2-bis(4-chlorophenyl)ethylene; HMPA, hexamethylphosphoramlde; DBCP, dlbromochloropropane; 4-AAF, 4-acetylaminofluorene.

the strain specificity that was observed. For 6 of these 7chemicals, the documentation of this strain-specific carcinogenicity is the same. These 6 chemicals were pesticides tested

in a large-scale study of about 130 chemicals. These chemicalsweretestedby a singles.c. injectionand by long-termfeedingto 2 hybrid strains of mice of both sexes. While a partialsummary of the results was published by Innes et al. (113), thedetails of the testing can be seen only in a large documentavailable from NTIS (163). In this regard, it is also important toappreciate that lnnes et al. highlighted only those 11 chemicalsthat were carcinogenic at the 0.01 level of significance. Incontrast, 56 chemicals were considered carcinogenic by atleast one of the routes of administrationwith a 0.05 level ofsignificance in the NTIS document. Included among the 56active chemicals were 5 of the 6 chemicals that were considered not evaluable by an IARC committee because of theirstrain-specific results.

Of the 45 chemicalsfor which carcinogenicitytesting wasbriefly considered by the IARC committees, 30 have been usedin Table 4. The studies referenced (and often summarized) bythe reviewingIARCcommitteeare consideredby the authorsof this paper to be adequate proof of the carcinogenicity ofthese chemicals in the experimental designs used. Thesechemicals have the designation M before their IARC referencein Table 4 so that they can be distinguished clearly from thechemicalsthat were reviewedper se by the IARCcommittees.

Otherchemicalsin Table4 with IARCreferenceswere identified by the other criteria.

EPA. The 1975 editionof Suspected Carcinogensprovidesinformation on the carcinogenicity of 1545 chemicals. In the1976 edition, this figure rose to 1905 chemicals (55). Thispublication only purports to present documentation of carcinogenicity and does not judge the credibility of such findings.

However, EPA contracted the organization that prepares thispublication to arrange by computer the chemicals in an updated

SEPTEMBER1979 3299




version of the 1975 edition of Suspected Carcinogens to reflectthe importance of their carcinogenicity documentation (78). A4-digit identifier was computed for each suspected carcinogento indicate the phylogenetically highest species of the testsystems used, the number of different species tested, theroutes of administration, and the total number of carcinogenicresponses reported. As fully described in the legend to Table4, the speciesvary from frog to human;but some speciesareactually collections of similar species; e.g. , Species 1 is acollection of various birds. These identifiers have made possible the enumeration of substances essentially contained in theI 975 edition of Suspected Carcinogens which have been reported to be positive in 2 or more mammalian species. Thiswas accomplished by rejecting all chemicals with an identifierthat indicated that one or no mammalian species had beentested; by immediately accepting all chemicals with an identifierthat indicated that the number of species tested was at least 4,and therefore included 2 mammalian species; and by lookingup the species in the toxic dose lines given in SuspectedCarcinogens (1 975) for the remaining chemicals to make certam that at least 2 mammalian species had been involved inthe testing. This approach yielded 202 entries: 190 werechemicals; 7 entries were mixtures of chemicals; and 5 entrieswere duplications of other chemicals. Only the 190 chemicalswere considered for use in Table 4. Consolidating 13 morganics into their 5 respective compounds (asbestos and beryllium,cadmium, chromium, and nickel compounds) reduces the totalto I 82 chemicals that were reported positive in at least 2mammalian species. These 182 chemicals are presented inTable 4 under â€˜â€˜EPA'â€along with other chemicals with theseidentifiers that are included in the table by other criteria.

The use of the chemicals thus identified in Suspected Carcinogens is based on the assumption that it is not very likelythat the testing in 2 or more mammalian species would havebeen inadequately conducted or would have given false-positive results in each case. Some indication of the correctness ofthis assumption is seen by considering the 82 chemicals thusidentified that have also been evaluated by IARC expert cornmittees. All but 4 of the 82 were evaluated as having beencarcinogenic in at least one adequately conducted study. Aswould be expected, this criterion did not always exclude chernicals with supposed carcinogenicity that is made less convincing by considerations like a low survival rate associated withtreatment (ziram), vehicle effects (cholesterol versus paraffinwax pellets with 8-quinolinol), or the presence of carcinogeniccontaminants in the test substance [chloromethyl methyl ethercontaminated with bis(chloromethyl) ether; maleic hydrazidecontaminated with hydrazine].

Department of Health, Education, and Welfare. In 1969,the Technical Panel on Carcinogenesis, an advisory committeeto the Commission on Pesticides and Their Relationship toEnvironmental Health (62), published its evaluation of availablereports on the carcinogenicity of over 100 pesticides. On thebasis of testing for tumor induction conducted adequately in 2mammalian species, the panel listed 3 chemicals as not positive. For similar testing that involved at least one mammaliansystem, the panel listed 32 chemicals as having increased thetumor incidence relative to controls with significance at the0.02 level (B, Cl , and C2).

Nine chemicals were considered to have increased the tumorincidence relative to controls, but the level of significance was

stated only as being less than 0.02 (i.e., p > 0.02) (C3).Apparently, the same pesticide, a-(2,4-dichlorophenoxy)-propionic acid [also called 2-(2,4-DP)] was tested twice (113,163) and evaluated twice by the Technical Panel. Hence, theactual number of carcinogens listed as C3 by the TechnicalPanel is 8. The significance of 7 of these 8 chemicals has beenrecalculated by x2 analysis using the data presented in theNTIS document (163). When both strains and sexes are cornbined or sex-strain subgroups are considered separately, 4 ofthe 8 chemicals have increased the incidence of tumor-bearingmice relative to controls with significance at the 0.05 level: a-(2,4-dichlorophenoxy)propionic acid; n-propyl isome; zineb;and triphenyltin acetate. Only these 4 C3 carcinogenic pesticides were used in Table 3.

Finally, 39 chemicals were considered not positive (C4) inthe one animal system in which they were tested. The availableinformation on the remaining pesticides was so insufficient asnot to allow comment in any respect.

Those 36 chemicals which are active at the 0.05 level arepresented in Table 4 under â€˜â€˜DHEW'â€with their evaluations.Other chemicals in Table 4 with these evaluations were identifled by the other criteria.

The IARC has also evaluated 23 pesticides reviewed by theTechnical Panel. The evaluations of the Technical Panel wereconsistent with those of the IARC committees, and most of thediscordance that does exist can be explained by the differencein the reports that were available to the 2 groups (e.g. , heptachlor). However, for 2 pesticides, both groups based theirconflicting opinions on the same studies. The Technical Panelconcluded that the testing in mice by p.o. administration ofaldrin (58) was adequately Conducted and resulted in significant results (p < 0.01 ); in contrast, the IARC committee couldnot accept these positive findings due to the inadequate designof the experiment.

In the case of IPC, the Technical Panel rated the potency ofthe carcinogenic response as C2 (p < 0.02); in contrast, theIARC committee concluded that no evidence of carcinogenicitywas significant at the 0.05 level. The discrepancy in these 2evaluations appears to be due to the difference in end pointsthat were analyzed. IPC was tested by a single s.c. injectionand by long-term feeding to 2 hybrid strains of mice of bothsexes (113, 163). Only the latter mode of administration suggested that IPC may be carcinogenic. When both strains andsexes are combined and the incidences of mice with tumors inthe treated group (18 of 66) and in the 5 control groups (53 of338) are compared, the x2 statistic has a p value of 0.02. By asimilar analysis, IPC was listed as â€˜â€˜active'â€˜at the 0.05 level inthe NTISdocument (163). Further analysis of the data suggeststhat the females of one of the strains contributed heavily to thissignificance (5 of 18 in the treated group versus 8 of 87 in thecontrol group); the x2 statistic has a p value of 0.03. However,in the IARC analysis of the data (see Ref. 114, Vol. 12, p. 189),the incidences of tumor types were compared between thetreated and control groups. The differences in these incidencesin any sex-strain subgroup, in the combined sexes of eitherstrain, or in the combined strains are not significant at the 0.05level using a x2 statistic adjusted for continuity (Yates correction).

Correlation Studies. Among the 4 correlation studies reported adequately to date (104, 152, 172, 226), 234 separatechemicals were considered as carcinogens and tested for




Activit?CategoryNot

evaluable


mutagenicity. This figure excludes 5-iodo-2'-deoxyuridmnewhich was mistakenly presented in one of the correlationstudies as a carcinogen.3 This criterion provided 103 chemicalsthat had not been identified by any of the 3 previously described criteria. The documentation of carcinogenicity, givenonly in the reports of McCann et al. (152) and Heddle andBruce (104), should be obtained from the respective reports.

Thus, Table 4 is a listing of 465 compounds for which thedocumentation of carcinogenicity varies from strong (reviewedby expert committees) to marginal (e.g. , those identified onlyby the EPA criterion that was used). As such, the reader shouldappreciate the limitations of Table 4 which prevent it from beingconsidered a listing of bona fide animal carcinogens. Furthermore, no attempt has been made to distinguish truly carcinogenic chemicals from chemicals that may more properly becalled promoters (e.g. , steroids) and solid-state carcinogens(e.g. , cellophane). However, it should be equally appreciated

that these limitations do not prevent certain important conclusions being drawn from the results of the mutagenicity testingof these chemicals. In particular, it will be seen from thefollowing discussion of Table 4 that about 58% of the 465compounds have been adequately tested in Salmonella, thatthe testing has tended to concentrate on certain chemical typesand neglect others, and that some categories of carcinogensexhibit individual correlations (Chart 2, ps's) that are unsatisfactorily low by any standard.

Discussionof False Negatives from Table 45

A general synopsis of specifics is presented in Table 5. The465 compounds suggested 39 separate categories (includinga miscellaneous category) strictly on the basis of recurringchemical structures. The occurrence of many chemicals havingmore than one functional group of importance made theirassignment more arbitrary than that of chemicals having onlyone functional group. Chemicals for which a predominance ofone functional group over the other(s) in the contribution to thecarcinogenic activity of the chemical could not be readilyassumed are repeated in the categories of presumedly secondary importance; these chemicals maintain that list number(enclosed by parentheses) of their primary assignment at whichtheir documentation of carcinogenicity and mutagenicity inSalmonella is presented. Consequently, 38 chemicals havingsecondary groups repeat twice. Furthermore, tannic acid andtannins (Chemicals 324) appear twice: primarily as a lactoneon the basis of ellagic acid; and secondarily as a phenylcompound on the basis of gallic acid. Both ellagic and gallicacid are hydrolysis products of vegetable tannins, which areglucosides of phenolic acids. Since 1-nitrosonaphthalene(Chemical 160), 2-nitrosonaphthalene (Chemical 163), and 2-nitrosofluorene (Chemical 171) represent higher oxidationstates of their corresponding amines, they have been classifiedwith the aromatic amines. In fact, 2-nitrosonaphthalene is detected as an urinary metabolite in dogs (but not various rodents)treated with 2-naphthylamine (Chemical 161) (30).

The most salient commonality among the 21 0 carcinogensthat are positive in Salmonella is the electrophilicity that isintrinsic to the molecule or introduced by enzymatic modification. The former can be thought of as ultimate mutagens and

5 The chemicals appearing in Table 4 are followed by their chemical numberfor easy reference.

Table5Synopsis of Table 4

ChemicalsTested

inSalmonellaPositive

inSalmonellaCyanamide10/10/0Substituted11/10/1diphenylethaneStllbenediol31

/30/1Dioxane10/10/0Anhydride21/20/1Pyrazolinone11

/10/1Pyrrolizidine50/50/0Haloalkyl

ether31/31/1Sulfanilamide20/20/0Polysaccharide41

/40/1Polymer31/30/1Metal

complex50/50/0Partial

summariesMiscellaneous13 cate9@oriesb

(33)C.7

38(8)d5/7

12/38(32)0/5

1/12(8)High

PartialsummariesTriazene

DiazoAzoxyNitrosoDiaryl alkynyl carbamateAromatic amineNitroaromaticPolyaromaticAziridineOxirane, thiiraneHeteroaromaticHalomethane,

haloethaneN-, S-, or 0-mustardSulfate, sulfonate,

sultonePhosphatel5cateÃ¡gories

(38)3

45

533

67385016112015

179

312(67)d2/3

3/44/5

39/533/3

43/6732/3818/506/166/1 1

13/2013/ 15

10/ 176/9

1/1199/312

(64)2/2

3/33/4

39/393/3

36/4332/3218/186/66/6

12/1311/ 13

10/106/6

1/1188/199

(94)MediumHydrazine

LactoneChloroethylene12

868/12

4/86/65/8

2/44/6Partial

summariesInorganic4 cate9@ories

(10)@.!34(7)d5/8

@7@4(68)2/5

â€˜T@7'@

(57)LowAzo

Carbamyl, thiocarbamylPhenylBenzodioxolePolychlorinated cyclicSteroidAntimetabolite11

21889

1596/11

9/215/84/88/92/153/92/6

3/90/51/41/80/2

1/3Partial

summaries7categories(18)81 (1 7)d37/81 (46)8/37(22)Final39

categorlesb465271 /465210/271summaries(58)(77)

a Relative ability of the Salmonella-S-9 system (7) to detect carcinogens Inthe given categories. â€˜â€˜Notevaluable' â€˜indicates either a small number of chemicals in the category or a lack of testIng; â€˜â€˜high,â€•â€˜â€˜medium,'â€˜and â€˜â€˜low'â€˜indicatethat individual category correlations are in the upper, middle, and lower 33rdpercentiles, respectively.

b Includes a miscellaneous category.

C Numbers in parentheses, percentages.d Percentages of respective total.

include known or presumable alkylating and acylating agents:diazo compounds; nitrosamides; nitrosoureas; dimethylcarbamyl chloride; diaryl alkynyl carbamates (66, 102); aziridines;oxiranes; thiirane; strained lactones; halogenated methanesand ethanes; ultimate mustards; sulfates; sulfonates; sultone;and trimethyl phosphate. Only a few intrinsically electrophiliccarcinogens are not active in Salmonella. Penicillic acid (Chemical 319), an aft-unsaturated â€˜y-Iactone,could act as an alkyl

SEPTEMBER1979 3301




ating agent in a manner analogous to the reaction of suchlactones with cysteine (120). Penicillin G, also not active inSalmonella (152), undergoes nucleophilic attack at the f3-Iactam carbonyl or rearranges in aqueous solution into an alkylating agent, benzylpenicillenic acid, which may account for thereported carcinogenicity of the antibiotic (1 43). Toxicityhinders testing of the acylating agent succinic anhydride(Chemical 325).

Those carcinogens that are proximate mutagens in Salmonella are chemicals which 5-9 can metabolize to reactivespecies like alkylating and arylating agents. Proximate mutagens generally activated by 5-9 include triazenes, azoxy cornpounds, N-nitrosamines, aromatic amines, polyaromatics, heteroaromatics, and some proximate mustards. The nitroaromatics, although active without activation per se, are metabolizedby bacterial nitroreductases to their reactive forms (201).

In classifying carcinogens, overstratification emphasizestheir heterogeneity. The question of appropriateness of somecategories is not a concern, provided that the chemicals areproperly categorized and their documentation of carcinogenicity is convincing. Twenty-nine of the carcinogens that havebeen tested and not found mutagenic in Salmonella fall intoonly 7 categories Câ€˜low'â€˜categories in Table 4). A good majority(25 of 29) of these unresponsive carcinogens have been evaluated by expert committees. Hence, the indication that 7categories of carcinogens are poorly detected in Salmonella isnot based on categories of questionable appropriateness. Inmany cases, the failure of these carcinogens to be active inSalmonella reflects the inadequacies of in vitro testing, ingeneral, and of bacterial testing, in particular, for reasons tobe discussed.

Azo Compounds(Chemicals 9 to I 9). The azo carcinogensare dominated by the azonaphthols, some of which were onceused to color food. Citrus Red No. 2 is still an approvedcoloring for orange skins in the United States (restricted to 2ppm). The metabolism of azo chemicals has been reviewed byWalker (239); the toxicology of food colors in general has beenreviewed by Radomski (190). Reduction in vivo of the azomoiety, although possible in the liver, has been largely attrib

uted to the gutflora. This has been related to the more favorableanaerobic conditions in the intestine versus the liver (119) andmay involve a nonenzymatic mechanism mediated by flavins(38, 39, 92). Accordingly, intestinal flora have been shown tometabolize in vitro Sudan I (48), Orange I (192), and Citrus RedNo. 2 (189). Bisazo dye Evans blue (Chemical 154) is decolored by intestinal bacteria, and the dye is not absorbed indogs if the intestinal tract is sterilized by pretreatment withantibiotics (224). When the dye is absorbed, it can be reducedin the liver to benzidine and naphthylamine derivatives as

evidenced from in vitro studies with liver microsomes andtrypan blue (Chemical 153), a congener of Evans blue (141).

Reduction of azonaphthols yields aromatic amines that wouldpresumably be available for typical aromatic amine metabolism(Chart 3) that is associated with carcinogenesis, e.g. , of thebladder (121). Brown et al. (40) tested over 37 dyes in Salmonella, including 4 azonaphthols that appear in Table 4. All4 azonaphthols are not mutagenic in standard plate testingwith and without 5-9 activation. Sudan I, Citrus Red No. 2, andFO and C Red No. 1 are not mutagenic in similar testing thatinvolved a 16-hr anaerobic incubation of plates before thestandard (aerobic) incubation; Orange I is not mutagenic in

HHArâ€”NNâ€”Ar â€2H@ Arâ€”Nâ€”Nâ€”Ar@ 2H@ 2 Arâ€”NH2

N-HYDROXYL.ATlca IjI ES1ER!FICATIce I;lArâ€”NH2 a Arâ€”Nâ€”OH â€”a Arâ€”Nâ€”OR

Araryl group; R@CH3 . S0

Chart 3. The reduction of aromatic azo compounds to their correspondingamines is mediated by Intestinal microflora. upon absorption, these amines wouldbe available for typical aromatic amine metabolism. Other metabolic reactions,e.g. , aromatic hydroxylation. are possible but not illustrated.

anaerobic liquid testing in which the tester strains are preincubated anaerobically for 16 hr at 37Â°in a salt solution contaming dithiothreitol (a reducing agent) and test agent beforeplating. In aerobic liquid testing, Citrus Red No. 2 is questionably mutagenic in TA100. Orange I yields â€˜â€˜highlyvariable' â€˜butapparently positive results only in TA98 when the agent is firstchemically reduced by treatment with sodium dithionite andthen activated with 5-9. Neither of these activities are due tothe presence of contaminating impurities as evidenced by thinlayer chromatography. Garner and Nutman (90) tested 10 dyesusing only TA1538, including Sudan I, Orange I, Sudan II,Ponceau MX, and FD and C Red No. 1. Only Sudan II ismutagenic; thin-layer chromatography indicates that this activity is not due to contaminating impurities.

The identification of aniline derivatives and benzidine (Chemcal 126) as urinary metabolites of azobenzene administered torats (76) and rabbits (32) indicates that azobenzene is reducedin vivo to aniline by way of the corresponding hydrazine whichitself rearranges into benzidine upon acid extraction and (probably to a lesser extent) in the naturally acidic environments ofthe whole animal. However, the predominance of hydroxylatedderivatives in these same studies also indicates that oxidation,presumably involving an arene-oxide intermediate, is an important metabolic pathway. It is not known which metabolism isoccurring in the Salmonella testing that finds azobenzene active in TA100 with activation (152). Azoethane is an oxidativemetabolite of 1,2-diethylhydrazine; the metabolism of symmetrical hydrazines is discussed later. Phenylazoanilmnedyes(Chemicals 140 to 150) are presented as aromatic amines inTable 4 since the amino rather than the azo group is theunderstood site of metabolism (oxidation) of these chemicals(179).

Carbamyls and Thiocarbamyls (Chemicals 90 to 110).The suggestion (151) that impurities may account for the

carcinogenicity in rats fed acetamide can be equally said of allchemicals of less than ultimate purity that are found to bepositive in such animal testing. If an impurity caused the hepatomas in animals receiving 2.5% acetamide in their diet, theimpurity is powerfully carcinogenic but not mutagenic in Salmonella at its contamination level in acetamide. The observedprotection that equimolar amounts of L-argininyl-L-glutamateaffords rats treated with acetamide may be indicative of somemetabolic specificity of action, the nature of which remains tobe investigated more fully (244). Ushioda (238) has determinedthat tritiated acetamide is incorporated into DNA and, to alesser extent, into RNA of the mutant bar larvae of Drosophila.It was not stated if the label is an adduct product with bases(mainly thymine) or is part of the base nucleus. N-Hydroxyacet





00 OOH 0CCH3

H3cH2coâ€”câ€”NH2â€” H;plf2co-c-NH@ â€¢H,@CH2C0â€”@--NH

Chart 5. Possible metabolic activation of urethan to acetyl derivative of N-hydroxyurethan [Nery (168)1.

mode of action of urethan or its N-hydroxy metabolite may alsobe as an antimetabolite in pyrimidine biosynthesis. Boylandand Koller (29) found that the frequency of chromosome abnormalities induced by urethan (but not nitrogen mustard) inWalker rat carcinoma is reduced and recovery is acceleratedby simultaneous or previous treatment with thymine (but noturacil or guanosine). Elion et al. (73) demonstrated that theinhibitory effect of urethan on the growth of mammary adenocarcinomas [implanted into axilla of male mice (74)] can bereversed by treatment with various pyrimidines and pyrimidineanabolites.

While the studies of Boyland and Koller and Elion et al. whichwere conducted in vivo do suggest that urethan has a specificity for pyrimidine biosynthesis, Kaye (127) could not demonstrate in vitro any significant inhibition by urethan of severalenzymes involved in nucleic acid metabolism. Both urethanand its N-hydroxy metabolite bear a structural resemblance tocarbamyl phosphate and carbamyl-L-aspartate. The enzymeaspartate transcarbamylase begins pyrimidmnebiosynthesis bycatalyzing the formation of carbamyl-L-aspartate from carbamylphosphate and L-aspartate. Gin and Bhide (93) have reportedthat in vivo administration of urethan decreased aspartatetranscarbamylase activity of lung tissue of adult male and (to alesser extent) female mice; no in vitro inhibition could bedemonstrated,

The dithiocarbamates are subdivided into 2 structuralgroups: the dialkyldithiocarbamates and the ethylene(bis)dithiocarbamates. Only one dithiocarbamate has been adequately tested. The lack of mutagenicity of maneb (126) mdicates that its carcinogenic metabolite, 4,5-dihydroimidazole2(3H)-thione, which itself is weakly positive in TA1535 (but notTA100) without activation (231 ), is not adequately producedby 5-9 activation. A common metabolite of both types ofdithiocarbamates is carbon disulfide. Its carcinogenicity andmutagenicity are essentially uninvestigated (63). However, prolonged inhalation of small concentrations of carbon disulfideand hydrogen sulfide has been reported to induce aneuploidyand polypoidy in bone marrow cells of male rats (14). Theoxidative desulfurization of carbon disulfide by rat liver microsomes is catalyzed by the mixed-function oxidase system andresults in the production of carbonyl sulfide and a reactivesulfur atom (57, 61); the former is further metabolized to carbondioxide and another reactive sulfur atom (56).

Carbon disulfide, carbonyl sulfide, and the singlet form ofatomic sulfur are electrophilic and should readily react withnucleophiles. The nucleophilic attack by protein amino groupson the electron-deficient carbon of carbon disulfide to yield adithiocarbamate that cyclizes into a thiazolidone has beenknown for some time (e.g. , Ref. 50) (Chart 6). It would seemunlikely that the short-lived, reactive species from oxidativedesulfurization in the endoplasmic reticulum could migrate tothe nucleus to react with nuclear targets (e.g. , DNA). Thefinding of mixed-function oxidase activity in nuclei (34, 35) hasthe obvious importance of suggesting a nuclear source forthese (and other) short-lived species. It is not known if carbondisulfide is produced in the standard plate testing of maneb.

The metabolism of monuron in the rat consists of oxidative

amide is known to be a teratogen in rats (47) and a clastogenin mouse embryo cells in vitro (25). However, Poirier (177)found that N-hydroxyacetamide was not carcinogenic to ratsat its highest tolerated dose and was not detectable in the urineof rats fed acetamide.

Thioacetamide is almost entirely converted in vivo by rats toacetamide, which is subsequently converted to acetate. Liverslices are 3 times more active than kidney slices in this metabolism. The ability of the liver to metabolize thioacetamide sorapidly has been related to the hepatocarcinogenicity of thechemical. However, the ultimate carcinogen is not thought tobe acetamide since thioacetamide causes hepatomas at a smallfraction of the dose that is necessary for the carcinogenicity ofacetamide (196). In phenobarbital-pretreated rats, thioacetamide, sodium diethyldithiocarbamate, thiourea, thiouracil(Chemical 430), 6-methylthiouracil (Chemical 431 ), and 6-propylthiouracil (Chemical 432) cause a loss in hepatic cytochrome P-450 or inhibit the oxidative N-demethylation of benzphetamine. This has been related to the mixed-function oxidase-catalyzed release and covalent binding of a reactive formof sulfur to cellular components (112) (Chart 4).

Thiourea and the 3 thiouracils are considered to be antithyroid compounds because they induce thyroid cancer in rodentsby inhibiting the organification of iodine and, therefore, thesynthesis of thyroxine. The resulting imbalance in the hypothalamopituitary-thyroid hormonal system subjects the thyroidto continuous stimulation by thyroid-stimulating hormone,which eventually causes a neoplastic condition in the gland(see Ref. 114, Vol. 7, pp. 23â€”26).However, the production ofa reactive species from oxidative desulfurization may be thecause of the concurrently observed carcinogenicity in the liverand other organs with these thiocarbamyl-containing antithyroid compounds. Thioacetamide and thiourea (as well as acetamide) have been reported to be positive in transforming hamster embryo cells (Pienta, cited in Ref. 65).

That N-hydroxyurethan but not urethan is positive with S-9activation inicates that the 5-9 cannot adequately perform themultiple-step activation of urethan (Chart 5). N-hydroxyurethanand some acyl derivatives have been shown to acylate in vitrothe amino group of cytosine with no reactions occurring atother sites or with other bases. The resulting product is easilydeaminated by both acid and enzyme (phosphodiesterase)hydrolysis. Therefore, these conditions when used for thedegradation of DNA treated in vivo will preclude the identification of the N-acylated product although a corresponding increase in 2'-deoxyuridylate should occur (168). However,Pound et al. (182) have shown the formation of alkyl esterswith the phosphate groups of DNA in the liver of intact andpartially hepatectomized mice treated with isotopically labeledurethan.

Both of these activities contrast, although not necessarilyconflict, with the findings in adult (158) and newborn mice (23)that 70% of N-hydroxy['4C]urethan is converted in vivo to[â€˜4C]urethan.This suggests that urethan is metabolically closerto the ultimate species of the carcinogen than N-hydroxyurethan in the carcinogenicity of both chemicals in mice. One

[.0.] Ã§:Ã˜ ,@@c=s@ _c@ â€”@ _câ€”@ â€”@ _c=o S

Chart 4. Oxidative desulfurization of thiocarbamyl compounds leading to therelease of electrophilic sulfur [modified from the report of Dalvi et al. (57)1.

SEPTEMBER1979 3303




its reactivity with GMP (252), and its ability to induce unschedRÃ§Hâ€”NH uled DNA repair synthesis (206). That neither safrole nor its 1 â€˜-

0 S hydroxy metabolite are mutagenic in Salmonella with activation

indicates that the S-9 cannot adequately metabolize thesechemicals to the 1â€˜-acetoxymetabolite (Chart 8). However, theimportance of this metabolite in the carcinogenicity of 1â€˜-hydroxysafrole, and therefore of safrole, is not clear. Furthermore, dihydrosafrole and the pesticidal synergists, n-propylsome, piperonyl sulfoxide, and piperonyl butoxide, lack theallyl group but are carcinogenic. Lawley (137) has postulatedthe production of a carbonium ion at the methylene group ofbenzodioxoles in a fashion analogous to its metabolic reactionswith the mixed-function oxidases (105).

Bactericides. McCann et al. (151) have noted that bacterialtoxicity hinders the testing of 9 chemicals, including transa,a'-diethyl-4,4'-stilbenediol (Chemical 236) and succinic anhydride (Chemical 325). Faced with toxicity, some researchershave utilized cold liquid testing and pulse testing to demonstrate the mutagenicity of the disinfectant povidone-iodine(200) and 1,1,2,2-tetrabromoethane (33), respectively. Thesestudies indicate the sometimes close association between bactericidal and mutagenic activities and the need to investigatethis possibility by resorting to nonstandard procedures.

Halogenated Compounds.As notedby McCannet al. (151),some halogenated compounds are apparently not adequatelyactivated by S-9. However, many alkyl halides which are volatile are demonstrably mutagenic only when tested in a desiccator apparatus (217). Those compounds that are mutagenictend to be either monohalomethyl compounds (Chemicals 358to 367) and ultimate mustards (Chemicals 369 to 371 , 374 to376, 381 , 383), both of which are alkylating agents, or chloroethylenes (Chemicals 388 to 392) and proximate mustards(Chemicals 372 and 373) which undergo metabolism to reactive epoxides (24, 97) and mustards (153), respectively.

Hexachlorocyclohexanes (Chemicals 394 to 396) undergodehydrochlorinations to yield initially pentachlorocyclohexeneand eventually a variety of polychlorobenzenes (123). Thepentachlorocyclohexene derivative is almost entirely absorbedby the kidney but is not released into the urine (77). Thearomatized metabolites are also susceptible to hydroxylationas evidenced by the presence of polychlorophenols in the urineof rats treated with either lindane, its pentachlorocyclohexene

cs2@ -H2ORCH-NH2 @, RÃ§Hâ€”Nâ€”f S a

HO-C HO-C@ :SH

0 0

N-demethylations, ring hydroxylation, and, to a limited extent,degradation of the urea residue to yield an aromatic amine(79). Using 4 microbial systems, Simmon et al. (218) tested 20pesticides, including monuron, simazine (Chemical 120), andPCNB (Chemical 224); no positive results were reported forthese pesticides with or without S-9 in Salmonella or the othersystems.

Phenyls(Chemicals 219 to 226). Phenylsand polyhalogenated phenyls are metabolized to their hydroxy derivatives, eachpresumably through a benzene oxide intermediate (e.g. , Refs.89, 103, 125, 133, and 198) (Chart 7). Aromatic hydroxylationalso occurs in the metabolism of oxazepam (Chemical 457) asevidenced by the production of hydroxyphenyl metabolites inthe urine of the rat and (to a lesser extent) pigs and humans(221 ). Lutz and Schlatter (144) have demonstrated covalentbinding in vivo of a benzene metabolite with the DNA of liversof rats exposed to isotopically labeled benzene in an inhalationchamber. In terms of alkylated nucleotides per mol of DNAphosphate, the potency of benzene calculates to be severalorders of magnitude lower than that of N-nitrosodimethylammne(Chemical 37), which itself is a proximate hepatocarcinogen.

A low rate of metabolism of benzene by microsomal and S-9preparations may be partially responsible for the lack of mutagenicity in vitro (Shakin, cited in Ref. 59). However, sincebenzene oxide once formed is significantly stable (124), it maybe necessary to exclude competing nucleophiles present in theS-9 in order to demonstrate mutagenicity in vitro. A sizablefraction of the phenol metabolites produced by a S-9 preparation are conjugated with sulfate and glucuronic acid (229).Harper et al. (101) observed the following order for the Vmaxofthe conversion of benzene to phenol by different microsomalpreparations: rabbit lung = hamster liver > rabbit liver >hamster lung > rat liver = rat lung. Tetrachlorobiphenyl ismetabolized by monkey liver microsomes to a species that canreact covalently with RNA (21 1). A relationship between increasing chlorination of biphenyl and decreasing metabolismby microsomes isolated from the livers of phenobarbital-pretreated rats has been observed (91).

The metabolism of PCNB (27) in the rat, dog, and bovine israpid and does not involve storage of PCNB in fat, kidney, liver,or skeletal muscle. In contrast, the contaminants of technicalgrade PCNB, hexa- and pentachlorobenzene, do accumulatein these tissues. PCNB is reduced presumably in the liver topentachloroaniline which probably is conjugated with glucuronides and sulfate for excretion. Methyl pentachlorobenzenesulfide has also been identified as a metabolite. Al-Kassab etal. (2) have shown that rat liver supernatant contains a glutathione S-aryltransferase system that catalyzes the replacementof the nitro group of PCNB (and other nitroaryls) with glutathione.

Chart 6. Reaction of carbon disulfide with nucleophilic center in an aminoacid (modified from the report of Cohen et a!. (50)).

R1,R2 H,H (benzene)

I'â€” ,H (blphenyl)

,\,CI (polychlorinated biphenyl)

Cl

Chart 7. Metabolic conversion of phenyl compounds to reactive benzeneoxides.

Benzodioxoles (Chemicals 227 to 234). Benzodioxolesaremore commonly known for the studies on safrole. Interest hascentered on the activation of the allyl group in safrole (252).The mutagenicity of 1â€˜-acetoxysafrolewithout activation concurs well with the enhanced electrophilicity of its allyl group,

3304 CANCER RESEARCH VOL. 39




radicals of chlorine and trichloromethyl and supports the hypothesis that such species initiate an autocatalytic peroxidationof lipid membranes which results in the observed hepatotoxicity(193, 235, 237). A similar scheme for radical formation andlipid destruction has been described by Reynolds and Molsen

(197) for halothane. In contrast to the reductive dechlorinationof carbon tetrachloride, the metabolism of chloroform to carbondioxide in vitro requires oxygen and produces carbonyl chloride (phosgene) as an intermediate (148, 176). That this alsooccurs in vivo is suggested by the similarity in production ofisotopically labeled carbon dioxide upon acid hydrolysis ofpolylysine and albumin treated in vitro with carbonyl chlorideand liver protein from rats treated in vivo with carbon tetrachloride (45) (Chart 11).

The metabolism of DDT (Chemical 355), and presumably itsanalogs (Chemicals 356, 357, and 393), involves a series ofreductive dechlorinations and dehydrochlorinations (174). Buselmaier et al. (43) have reported that 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane is mutagenic with Serratia marcescensbut not with Salmonella strain G46 in the host-mediated assay;DDT, DDE, and di(4-chlorophenyl)acetic acid were not positivein similar testing.

Steroids (Chemicals 415 to 429). Steroidsare evolutionarilyrecent chemicals and reflect the necessity for multicellularorganisms to coordinate their cells for homeostasis. The biological action of steroids is mediated by their binding to acytoplasmic receptor in target cells and the translocation of

this complex to the nucleus for association with chromatinmaterial; in some manner, this allows control of gene expression. Some steroids can act as mutagens by causing chromosome aberrations, aneuploidy, polyploidy, and dominant lethalmutations (11, 17, 199, 212, 250, 251 ). Presumably, thecytoplasmic receptors of target cells which confer specificityto the biological action of steroids should also confer specificity

:@ -@L@CuI@%COCl

H0Ã´Cl [Cl@@@]

Chart 9. Metabolic aromatization and possible epoxidation of hexachlorocyclohexane (lindane) [modified from the report of Grover and Sims (99)).

::$cH2 _:iJ@Et@0 :@ â€”:*â€˜

Chart 10. Metabolic epoxidation of aldrin (left) and heptachlor (right) (Nakatsugawa et al. (162)).

0

Cd4 AftAERo@tc, [.Ccl3J -@ HCCI3@ a [HO-cd3]@ CCI2

Chart 11. Reductive dechlorination of carbon tetrachloride and subsequentoxidation of chloroform to reactive phosgene [Rechnagel and Glende (193);Mansuy et al. (148); PohI et al. (176)).

SEPTEMBER1979 3305

Oâ€”'CH2

1@ir@__I Ii @â€”I II

HTâ€”cH=cH2 HJâ€”CHZCH2OH OgCH3

Chart 8. Possible metabolic activation of allyl group in safrole [Wislocki et al.(252)).

derivative, or a trichlorobenzene (99) (Chart 9). The aromatization of a-hexachlorocyclohexane involves a cytosolic enzymethat has a specific requirement for glutathione and is inducedby pretreatment with substrate; the process proceeds by wayof dehydrochlorinations to yield presumably chlorinated thiophenol(s) (181). This metabolic activity can also be demonstrated in nuclear, mitochondrial, and microsomal subfractionsfrom liver when they are supplemented with glutathione; incontrast to the cytosol, these fractions are not induced bypretreatment with substrate. Extrahepatic metabolism of a-hexachlorocyclohexane is also widespread (135).

Polychlorinated cyclodienes aldrin (Chemical 400) and heptachlor (Chemical 402) are converted in vitro by rabbit livermicrosomes to their corresponding epoxides (Chart 10). Theepoxidase has the characteristics of the mixed-function oxidase system in that it requires both NADPH and oxygen and itis inhibited by SKF 525-A (162). Dieldrin (Chemical 401 ) isdetoxified in vivo in the rat through hydrolysis of the epoxygroup to a trans-dihydrodiol which subsequently is oxidized tothe dicarboxylic acid; other metabolites include 9-hydroxydieldrin and a pentachloroketone structure (13).

Mirex (Chemical 398) is essentially metabolically inert (242,248), although reductive dechlorination can be expected as apathway of degradation in vivo. In fact, Stein and Pittman (223)have identified a monohydro derivative of mirex in the feces ofrhesus monkeys. The literature on mirex covering the years1947 through 1976 has been abstracted by Waters and Black(241).

Strobane (Chemical 397), a mixture of chlorinated camphene, pinene, and related polychlorinates, exhibited no mutagenic activity with or without S-9 in TA1535, TA1537, orTA1538; but it tripled and doubled the mutation frequency inTA100 and TA98, respectively, when present at 4800 pg/platewithout S-9. This activity was greatly reduced in the presenceof 5-9 suggesting detoxification or protection by S-9 against amutagenic species (possibly chloromethyl) in the heterogeneous mixture (Table 2). Saleh et al. (205) have proposed thatthe metabolic reductive dechlorinations and dehydrochlorinations of toxaphene, a mixture of polychlorinates of camphene,leads to detoxification; a radical intermediate is thought to begenerated in the reductive dechlorination by microsomal enzymes.

Halomethyl compounds are subdivided into monohalomethyls which are alkylating agents and polyhalomethyls whichmust be metabolized to an ultimate species. Reductive dechlorination of carbon tetrachloride (Chemical 353) to chloroformby rabbit liver microsome parallels the concentration of cytochrome P.450 in the mcirosomes but requires anaerobic conditions and NADPH. The identification of hexachloroethaneafter incubation of NADPH-reduced microsomes with carbontetrachloride is indicative of homolytic formation of the free




to any mutagenic action of steroids. This model also predictsthat chemicals which are analogs of steroids, so-called antisteroids, and chemicals which can induce oversecretion ofendogenous hormones may act as mutagens. While mutagenicity may be limited to alterations in chromosome structure andnumbers, as Ohno (173) has proposed, such mutations couldstill give rise to cancer if indeed the disease is the expressionof a recessive trait. Whether the carcinogenicity of steroids isrelated to gene expression, some mechanism of promotion, ormutation remains to be further investigated. However, the lackof mutagenicity of ethinylestradiol and testosterone propionatein Salmonella is not unexpected.

Antimetabolites (Chemicals 430 to 438). Azathioprine is aconjugate of 6-mercaptopurine with a nitroimidazole carrierdesigned to protect the sulfhydryl group from rapid methylationwhich once was thought to inactivate 6-mercaptopurine. Azathioprine is converted in vivo in the mouse, dog, and human tofree 6-mercaptopurine, which is further metabolized to thethioanalogs of IMP, GMP, and dGMP (75). 6-Thioinosine 5'-monophosphate suppresses de novo biosynthesis of purinesby â€˜â€˜pseudofeedback', inhibition of several key enzymes (156).The observed mutagenicity of 6-mercaptopurine only in basepair mutants (107) concurs with the fact that 6-thiodeoxyguanosine 5'-monophosphate is incorporated into DNA (165).However, Rosenkranz et al. (202) have shown that in TA100the mutagenic activity of azathioprine is derived from the nitroimidazole carrier; conditions which favor the reduction ofthe nitro group by bacterial enzymes are necessary to demonstrate a dose response. Hence, these findings in vitro maynot be entirely descriptive of the mutagenicity of azathioprinein vivo. Whether the carcmnogenicityof azathioprine is due tothe nitroimidazole carrier or liberated 6-mercaptopurine is difficult to assess. While treatment with 6-mercaptopurine induced thymic lymphomas in neonatal mice (67) and hematopoietic tumors in mice and rats (183), its immunosuppressiveaction may also play some part in the development of these

tumors.Aminopterin, pyrimethamine, and the sulfanilamides (Chem

icals 412 and 413) are inhibitors of the enzyme dihydrofolatereductase. As such, aminopterin and sulfanilamide can be usedto induce thymidine deficiency in bacterial testing. Freese (85)predicted that thymine starvation may cause transversions.Thymine starvation of thymine auxotrophs of Salmonella alsoinduced what appear to be deletions (110). Such activity maybe a cause of the clastogenicity observed in cell culturestreated with methotrexate (19). Like methotrexate, aminopterinis probably not easily taken up by Salmonella tester strainssince they do not require folic acid to grow. Interestingly, in

Lactobacillus arabinosus, the uptake of pyrimethamine doesnot appear to involve active transport, and that of aminopterin

but not folic acid is mediated by an active system that normallytakes up thiamin (254). The pesticide simazine (Chemical 120)also may be active antimetabolically as an analog of folate (12)as well as pyrimidine (74).

The ethylation of hepatic DNA and RNA of rats given a largedose of isotopically labeled L-ethiOfline (228) agrees with thereported heptocarcinogenicity of the amino acid in rats (80).

S-Adenosyl-L-ethionine, an antimetabolite of S-adenosyl-L-methionine, accumulates in the liver of rats treated with L-ethionifle(215). However, since 7-methylguanine is not generally foundin DNA, the identification of only 7-ethylguanine in DNA after

administration of L-ethionine suggests that ethylation is notmediated by methyltransferases that normally utilize S-adenosyl-L-methionine and therefore some unknown mechanism ofaction is involved (228). L-Ethionine is naturally produced insome strains of bacteria including Escherichia coli B (81). IfL-ethionine is also a natural metabolite for Salmonella, suchmay explain its lack of mutagenicity.

Shikimic acid (Chemical 451 ) may be one of probably severalcarcinogenic agents in bracken fern. It is a murine mutagen inthe dominant lethal test (reviewed in Ref. 222). However,Hirono et al. (109) contend that the carcinogenicity of brackenfern is not due to its endogenous concentration of shikimicacid; rats did not develop tumors when fed shikimic acid at adosage twice that found in a bracken fern diet which inducestumors in all feeding animals. Shikimic acid is also an anabolitein the synthesis of aromatic amino acids by those organismswhich are capable of such synthesis; this excludes rodents andhumans for whom phenylalanine and tryptophan are essentialamino acids. In fact, shikimic acid serves as a nutrient for somestrains of E. coli, which may explain in part its lack of mutagenicity in Salmonella testing.

The initial testing for carcinogenicity ofthe tryptophan catabolite 3-hydroxyanthranilic acid (Chemical 115) stemmed fromthe once-held belief that o-hydroxylated derivatives were theultimate carcinogens of aromatic amines. Therefore, it wasreasoned that the catabolism of tryptophan would serve as anendogenous source of carcinogens which presumably contribute to the spontaneous appearance of cancer. The significanceof bladder cancer produced by surgically implanting cholesterol (but not paraffin wax) pellets containing this chemical intothe bladder is embroiled in the controversy over the relevanceof this mode of administration (see Ref. 49). Injection of thischemical s.c. has been reported to cause myeloid leukemia inmice when compared to historical controls (71).

The aromatic ring of 3-hydroxyanthranilic acid is openedenzymatically by a supernatant of rat liver homogenate. Theresulting semialdehyde can internalize the amino nitrogen toform either quinolinic or picolinic acids; if no cyclization occurs,glutaric acid can be formed (170). Priest et al. (185) obtainedquantitative conversion to quinolinic acid with the supernatantof rat liver homogenates. If 3-hydroxyanthranilic acid is carcinogenic through typical aromatic amine metabolism, mutagenicity may not be observed because of the dominance in 5-9 ofthis alternative metabolism. Furthermore, any interpretation ofcarcinogenicity or mutagenicity testing results must take intoaccount the instability of 3-hydroxyanthranilic acid under physiological conditions (175). Hence, the conclusion of Bowden etal. (28) that tryptophan metabolites including 3-hydroxyanthranilic acid â€˜â€˜do not function as causative agents for chemicalcarcinogenesis in the large intestine' â€as evidenced by theirlack of mutagenicity in Salmonella must certainly be qualified(as the authors noted) for reasons discussed (some of whichwere not noted by the authors).

Two miscellaneous chemicals may also behave antimetabolically as their modes of action. The plant growth retardantmaleic hydrazide (Chemical 454) may be regarded as either apyrimidine or a purine analog on the basis of its structure in itscrystalline state. The geometry also suggests that, if the N-nucleotide is formed and incorporated into nucleic acid, basepairing with adenine and guanine are plausible (54). Maleichydrazide inhibits the incorporation of tritiated thymidine and





uridine into the nucleic acids of corn seedling roots withoutaffecting their cellular uptake during the first 12 hr of treatment(171). Isotopically labeled maleic hydrazide is incorporatedinto RNA, but not DNA, of young willow tree roots (53). Thesefindings have been likened to similar activities observed with5-fluorouracil and its deoxyribose nucleoside (171). The clastogenicity of maleic hydrazide in plant cells has been knownfor some time (147), but apparently clastogenicity is not observed in mammalian cells treated in vitro although the chemical is cytotoxic and inhibits mitosis (155).

The herbicide amitrole (Chemical 455) has several documented effects on metabolism. In E. coli, histidine and adeninetogether reverse the inhibition of growth by amitrole, whereasadenine alone, but not histidine alone, partially relieves theinhibition. In the yeast Torula cremoris, histidine alone, but notadenine alone, completely reverses the inhibition (246). Ametabolite identified as a conjugate of alanine and amitrole hasbeen shown to accumulate in the medium of E. coli cultured inthe presence of amitrole; it apparently competes with histidinefor incorporation into protein (249). Unlike amitrole, this metabolite does not inhibit the conversion of imidazole glycerolphosphate to imidazole acetol phosphate by imidazole glycerolphosphate dehydratase in Salmonella; the reaction is part ofthe biosynthesis of histidine (108).

The antithyroid effects of amitrole in the whole mammal resultfrom the inhibition of peroxidase, thereby preventing the organification of iodine (1, 225). The resulting thyroxine deficiency subjects the thyroid to continuous stimulation by thyroid-stimulating hormone that induces eventually a malignantchange in the organ (234). However, as with other antithyroids,the hormonal imbalance does not necessarily explain the hepatocarcinogenicity that is also obseved (113, 163).

How amitrole affects purine metabolism was indirectly investigated by Rabinowitz and Pricer (1 88), who demonstrated thatamitrole blocks the enzymatic degradation of 4-aminoimidazolein extracts of Clostridium cyclindrosporum. Since amitrole isan isostere of 4-aminoimidazole, it has been proposed thatamitrole could interfere with purine catabolism (246). However,this activity cannot be related to mammalian carcinogenesissince that degradation scheme is not observed in mammals;purines are degraded to and excreted as uric acid in primatesand allantoin in mammals other than primates. However, thereare several anabolic metabolites of purine metabolism to whichamitrole is practically isosteric, e.g. , the base of 5-aminoimidazole-4-carboxylic acid ribonucleotide, a metabolite in thebiosynthesis of IMP. Tjalve (232) has noted that in mice amitrolepreferentially accumulates in tissues with rapid cell turnover(DNA synthesis);only moderate accumulationoccurs in thethyroid. The 5-amino derivative of amitrole, guanazole, possesses antitumor activity; guanazole and (to a lesser extent)

amitrole are cytotoxic against a leukemic cell line in vitro (100).The antiviral agent virazole is a ribonucleoside of 1,2,4-triazole3-carboxamide (216, 253) and a somewhat similar chemical,2-amino-i ,3,4-thiadiazole, is anabolized in vitro into 2 NADanalogs that are potent inhibitors of IMP dehydrogenase (166)(Chart i 2).

Interestingly, Rosenkranz et al. (202) have reported occasional false-positive results with amitrole and have attributedthem to phenotypic curing. It is not known if this phenotypiccuring is due to the action of amitrole as a histidine analog inprotein synthesis or as a RNA analog in mRNA synthesis.

3307SEPTEMBER 1979

H2N@c7 :!NO H2N@@fl $20

H Phosphoalbo.. Illbose

Chart 12. Structural comparison (from left to right) of amitrole, 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid, virazole. and 2-amino-i ,3,4-thiadiazole.

Hydrazines (Chemicals 25 to 36) and N-Alkyl Compounds.Toth (233) lists 9 symmetrical and 10 unsymmetrical hydrazines as tumorigenic compounds. From their studies with acetylhydrazine and isopropylhydrazine, Nelson et al. (167) haveproposed that monoacyl and monoalkyl hydrazines are oxidized by microsomal enzymes to diazenes which fragment intoreactive radicals. The implication of radicals as the ultimatereactive species also had been made earlier in a study byFreese et al. (87) on the inactivation of transforming DNA byvarious hydrazines in the presence of oxygen and transitionmetals. The findings that hydrazine and N-(2-hydroxyethyl)-hydrazine are mutagenic without 5-9 to base-pair mutantsTA1530 (152) and TA1535 (see Table 2), respectively, suggestthat a nonenzymatic mechanism, possibly what Freese et al.(87) observed or hydrazone formation with DNA, also occurs.In the presence of 5-9, the mutagenicity of N-(2-hydroxyethyl)hydrazmneis enhanced as predicted. The mutagenicity ofhydrazines in general has been reviewed by Kimball (132).

According to the proposal of Nelson et al. (167), the carcinogenicity of isoniazid would be dependent on its acetylationwhich gives rise to acetylhydrazine (Chart 13). This then partially reverses the argument of Freese (86) and predicts thatâ€â€˜rapid inactivators,' â€ although less sensitive to the antidepres

sant effects of hydrazines by virtue of rapid acetylation, will bemore apt to produce the reactive species and suffer mutagenicand carcinogenic events. An inability of 5-9 to produce andoxidize acetylhydrazine from isoniazid may account for the lackof mutagenicity in Salmonella testing. However, Braun et al.(31) have equated the mutagenicity of isoniazid in the hostmediated assay with the release of the mutagenic hydrazinemoiety.

Nelson et al. (1 67), elaborating on a postulation by Preussman et al. (184), have also proposed a reaction mechanism forhydrazines symmetrically substituted with alkyl groups. Again,a radical is produced for alkylation after the hydrazine hasbeen oxidized to its azoxy derivative followed by oxidativeremoval of one of the N-alkyl groups. One of these steps isapparently deficient in the activation by 5-9 of 1,2-dimethylhydrazine and procarbazmne.The importance of intestinal bacteria in the metabolism of 1,2-dimethylhydrazmne has beendiscussed (194).

Oxidative N-dealkylation is implicated in the activation ofseveral N-dialkyl carcinogens. In the metabolism of N-nitrosodimethylamine (Chemical 37) as postulated by Magee (145),N-demethylation produces N-nitrosomethylammne whichquickly rearranges first into a methyldiazohydroxide and subsequently into a methyldiazonium cation that releases methylcarbonium ion for alkylation. MaIling (146) had to resort to a20-mm incubation of N-nitrosodimethylamine with a bacterialtester strain in a solution of postmitochondrial enzymes in orderto achieve this metabolism and demonstrate mutagenicity.However, without the incubation period, so-called liquid testing,mutagenicity in the presence of 5-9 is not observed, probably




H2N@NH@@@4 AcEr@LAsF@ â€˜-â€œ@:c:Ã§HO.L(@@@

{ @j.â€”[email protected] @2cH3cNHâ€”NH1â€” cH3NHâ€”NH2Chart 13. Formation of a reactive metabolite after acetylation of isoniazid

(modified from the report of Nelson et al. (167)).

due to the short-lived nature of the reactive species and thedilution that occurs with plating. Similarly, N-nitrosodiethylamine (Chemical 44), N-dimethyltriazenes (Chemicals 3 and 4),and N,N-dimethyl-4-(phenylazo)benzenamine (Chemical 142)and its congeners (Chemicals 141 and 145) require liquidtesting to demonstrate weak mutagenicity (151). The lack ofmutagenicity with auramine (Chemical 137) in plate testing alsomay be related to this difficulty in activation by N-dealkylation.

Oxidative N-demethylation produces formaldehyde, a classicmutagen (reviewed in Ref. 10). Butterworth6 has suggestedthat the lack of mutagenicity in Salmonella with HMPA, aninhalation carcinogen in the rat (255), is related to the insensitivity of the bacteria to formaldehyde. HMPA, the hydroxymethylpentamethyl analog of HMPA, and formaldehyde are allactive in the mouse lymphoma system, the former only in thepresence of 5-9 (see also Ref. 9). Aldehyde formation may alsobe of minor importance in the biological actions of the pesticidesimazine (Chemical 120) and of the anticancer drug hexamethylmelamine (22, 26), both of which are N-ethylated triazines.

Phenacetin (Chemical 117) is deethylated in vivo to acetaminophen in humans (37) and in the rat (69). Oxidative 0-deethylation is inhibited in vivo in the rat and in S-9 from ratliver by the mixed-function oxidase inhibitor, SKF 525-A (52).Phenacetin and acetaminophen can undergo N- or aromatichydroxylation, giving rise to species that are susceptible toattack by nucleophilic macromolecules (169) (Chart 14). Feeding of N-hydroxyphenacetin (Chemical 118) p.o. significantlyincreased the incidence of hepatocellular carcinoma in malerats (44). Acetaminophen-induced hepatic necrosis in miceincreased as covalent binding of isotopically labeled acetaminophen increased; binding of the label was localized in theendoplasmic reticulum and to proteins in the cytoplasm (118).However, binding of the metabolite(s) of acetaminophen tohepatic macromolecules does not occur until the glutathioneconcentration in the liver has been depleted by conjugationwith the reactive species (159).

Intercalating Agents. Rosenkranz et al. (202) have notedthat 9-aminoacridine but not other intercalating agents likeacridine orange (Chemical 180), proflavine, and ethidium bromide can revert the frame-shift mutants of Salmonella withoutactivation; the latter are only active in the presence of S-9.Similarly, aromatics like benzo(a)pyrene 4,5-oxide (Chemical309) and N-acetoxy-2-acetylaminofluorene (Chemical 175),which are known to form adduct products with DNA, preferentially revert the frame-shift and not the base mutants (5). Inthe nomenclature of McCann et al. (154), these agents arecalled â€˜â€˜reactive' â€˜frame-shift mutagens. Levine et al. (138)have postulated that the formation of the adduct results in a

6 B. E. Butterworth. The value and limitations of the Salmonella microsomaland the L5178Y mouse lymphoma mutagenicity assays. Paper presented at theEighth Annual Meeting of the Environmental Mutagen Society. Colorado Springs,Cob., February 16, 1977.

conformational change in the DNA, so-called base displacement, that is different from base substitution, deletion, andintercalation. The failure of classical intercalating agents torevert the frame-shift mutants in the absence of S-9 suggestsa specificity for intercalation between base pairs. For instance,agents with an affinity for thymine pairs might not be active inthe Salmonella frame-shift mutants the mutated sites of whichconsist of guanine-cytosine pairs (6). The lack of mutagenicitywith gibberellic acid (Chemical 323) might be related to suchspecificity. Gibberellin A7, a plant hormone that differs fromgibberellic acid (also called gibberellin A3)by lacking a hydroxylgroup, changes the physical state of DNA in a way characteristic of intercalation. In contrast to the classical intercalatingagents, the binding of gibberellin A7 requires the presence ofdivalent magnesium cation and is quite specific for doublestranded DNA rich in adenine and thymidmne.Whether thesefindings are descriptive of the biological action of gibberellicacid may depend on whether gibberellic acid is dehydroxylatedto gibberellin A7 since the presence of the hydroxy groupnegates this activity. Interestingly, the observation that singlestrand nicks in DNA increased its interaction with gibberellmnA7 led to a series of experiments in which it was found thatDNA ligase catalyzes, in the presences of the gibberellin, theformation of duplex covalent circles (129â€”131). Gibberellicacid also acts as a chemosterilant in a cotton leafworm (204).

Chloroethylenes (Chemicals 388 to 393). In addition tovinyl chloride and vinylidene chloride, di-allate is activated byS-9 to a mutagen for TA1535 and TA100 but not for any of theframe-shift mutants (see Table 2). These findings corroboratethose reported by De Lorenzo et al. (60) who also found thattri-allate (the 2,3,3-trichloro analog of di-allate) and sulfallate(2-chloroallyl N-diethyldithiocarbamate) are metabolized byS-9 to mutagens. Trichloroethylene is presumably metabolizedby rabbit liver microsomes to its epoxide (236), which rearranges in vitro into dichloroacetyl chloride but in vivo intochloral (24). Trichloroethylene (analytical grade) caused a doubling in the reversion to arginine prototrophy in E. coli Ki 2 onlywhen microsomally activated (97). However, TA100 did notrespond in plate testing with and without 5-9 activation (106)and maximally showed only about 100 net revertants in adesiccator system when exposed to trichloroethylene with 5-9activation (217). The heretofore observed carcinogenicity oftrichloroethylene may be confounded by the presence of highly

@ -@-@â€˜@@â€˜

@:?@[@}@Ac

KNAc

OR

â€”S-erâ€”.-@

ort

NARNAc_

0Ac.CCH3 Et:cH2cH3 R.SO5. C6H906

Chart 14. Known and possible metabolic conversions of phenacetin [Brodieand Axelrod (37); Dubach and Raaflaub (69); Jollow et al. (118); Nery (169)].





mutagenic contaminants, epichlorohydrin and 1,2-epoxybutane, in the test substance used in the animal testing (106).Although Bartsch et al. (15) could activate chloroprene with5-9 to a highly active mutagen using a desiccator system,McCann et al. (152) listed its mutagenicity as questionable onthe basis of a personal communication of negative results byB. McKusick (du Pont).

By virtue of its dichloroethylene group, DDE is listed in Table4 as a chloroethylene. Epoxidation across the double bond isa plausible reaction for DDE if one postulates further that thisis followed by the formation of 1,1-dichloro-1 -hydroxy-2,2-bis(4-chlorophenyl)ethane; this presumably unstable speciescould dehydrochlorinate into an acetyl chloride which wouldbe quickly hydrolyzed to the corresponding carboxylic acid.Burchfield and Storrs (41) have suggested that epoxidation of1-chloro-2,2-bis(4-chlorophenyl)ethylene,a metaboliteof DDT,would produce a metabolite with alkylating power. By similarlogic, 2,2-bis(4-chlorophenyl)ethylene, also a metabolite ofDDT, may be made reactive by epoxidation. However, the factthat the only commonality among the DDT analogs (Chemical235) presented in Table 4 is the p-substituted diphenylethanestructure is reminiscent of the hypothesis of a DDT receptor toexplain the pesticidal activity of this chemical type (reviewed inRef. 88) (Chart 15). Interestingly, DDT and some of its analogsbehave estrogenically (70, 164, 220, 245), suggesting thatsuch a receptor might be the cytosolic protein that normallybinds estrogen.

Membrane-specific Agents. Differences between bacterialand mammalian cell membranes can be expected to be a factorat times in bacterial testing. The suggestion (151) that thepresence of ions and citrate (a chelator) in the minimal mediumprevents the entrance of metal carcinogens into the bacteriadoes not agree with the since-observed sulfateâ€”dependentmutagenicity of chromium(VI) (Chemical 462) in TA92 (142).Similarly, cadmium chloride (Chemical 461 ) has been reportedto be mutagenic in TA1950 in the host-mediated assay and, toa lesser extent, in TA1535 when preincubated for 30 mm(without S-9) (122); it is not active in standard plate testing(1 04). What determines the entrance of a chemical into a cellis the lipophilicity of the chemical or the presence of a transport

system in the membrane. Charged species like metal cationsdo not readily diffuse through lipid membranes and must betransported into the cell. Rosenkranz et al. (202) report thatSalmonella is not reverted by either beryllium sulfate (Chemical460) or lead acetate (Chemical 464); Heddle and Bruce (104)corroborate the lack of effect for lead acetate. In testing withTA1535 and TA1538 with and without 5-9, asbestos fiberswere not mutagenic (46). Membrane specificity is also apparent

in the testing of actinomycin D (Chemical 341 ). Whereas noresponse is seen in Salmonella with drug concentration as highas 10 @tg/plate(roughly, 0.5 @tg/mIagar), cytotoxicity is quiteevident in hamster cells treated in vitro with concentrations of0.1 and 0.05 @ig/ml(18, 19).

Pyrazolinone (Chemical 327). Kellermannet al. (128) haveobserved a good agreement between the magnitude of induction of an individual's mixed-function oxidase activity (measured as 3-methylcholanthrene induction of aryl hydrocarbonhydroxylase in short-term lymphocyte cultures) and the plasmahalf-lives of antipyrine and phenylbutazone in the same individual. Antipyrine is metabolized to 3 reactive forms in the rabbitwhich may contribute to its irreversible binding in vivo and invitro (microsomes) to liver proteins. They were the presumed3,4-epoxide, the 3-formyl metabolite, and 1-phenyl-3-methyl4-hydroxypyrazolin-5-one (and the corresponding tautomers).Amidopyrine (4-dimethylammnoantipyrine), and presumably 4-aminoantipyrine, undergo similar conversions (230).

Mitotic Poisons. The complexity with which the nucleicmaterial of mammalian cells is organized provides mechanismsfor mutagenesis and presumably carcinogenesis that will notbe assayable in microbial cells. Those agents which causedisfunctioning of the mitotic apparatus that segregates chromosomes during division, so-called mitotic poisons, will not bemutagenic in bacteria. Accordingly, griseofulvin (Chemical452), colchicine, and vinblastine are not active in Salmonella(104).

Cross-linking Agents. MitomycmnC (Chemical 291), a DNAcross-linking agent (115), is weakly mutgenic only in a Salmonella strain that has excision repair and the A-factor(hisG46/pKM1 01). As such, it is not detected in what can becalled standard testing. Presumably, excision repair of thecross-linking involves the production of nicks in the DNAstrands which somehow leads to increased mutagenesis in thepresence of the A-factor (154). Similar activity is observed inE. coli with mitomycin C (134) and may be relevant for anotherclass of cross-linking agents (247), the pyrrolizidines (Chemicals 328 to 332). In those Salmonella strains that were used(not stated), monocrotaline and heliotrine were not active withor without 5-9; these alkaloids were activated by S-9 to aspecies that was toxic to 2 strains of repair-deficient E. coli(94).

Summary. Table 4 is a listing of 465 compounds with knownor suspected carcinogenic activity that were identified by varbus criterions. Fifty-eight % have been tested for mutagenicityin Salmonella for an overall correlation of 77% (Table 5). Atleast 13 chemical categories (includes a miscellaneous category) cannot be evaluated at this time due to the small numberof chemicals or the lack of testing results within these categories. However, shikimic acid and griseofulvin appear to exhibit a specificity for that metabolism and chromosomal organization, respectively, found in the mammalian system. Transa,a'-Diethyl-4,4'-stilbenediol and succinic anhydride are toobactericidal to test (Table 6).

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 inCI H H @l cI this grouping (high activity). These categories represent chem

icals that are ultimate electrophiles or chemicals that can beCH@CH3 H H CI Cl activated by 5-9 or bacterial nitroreductase to electrophilic

species. Presumably, this high correlation will continue to betrue in further testing, although molecular size could pose a

V w x y z

Is?chlorobenzllate CI OH@ c-o-cH2cH3

pp-DOT

p.p-T DE

Perthens

Chart 15. The differences in substituents (v â€”@Z) among 4 DOT-like carcinogens.

SEPTEMBER1979 3309

V

wâ€”câ€”câ€”y

V



Category Chemical Comments


Table 6Sixty-one false negatives from Table 4

Not evaluableSubstituted

diphenylethaneStilbenediol

Griseofulvin (Chemical 452)Maleic hydrazide (Chemical 454)

Amitrole (Chemical 455)

Saccharin (Chemical 456)

Cycasin (Chemical 24)3-Hydroxyanthranilic acid (Chemical 115)

Heteroaromatic Actinomycin 0 (Chemical 341)Halomethane, Carbon tetrachloride (Chemical 353)

haloethanep.p'-DDT (Chemical 355)

MediumHydrazine Isoniazid (Chemical 28)

1,2-Dimethylhydrazine (Chemical 33))

Procarbazine (Chemical 36)Lactone Penicillic acid (Chemical 319)

Gibberellic acid (Chemical 323)

Chloroethylene Trichloroethylene (Chemical 390)

DDE(Chemical393)Inorganic Arsenate (Chemical 458)

Beryllium sulfate (Chemical 460)Lead acetate (Chemical 464)

LowAzo Sudan I (Chemical 11)

Orange I (Chemical 12)Citrus Red No. 2 (Chemical 15)FD&CRed No. 1 (Chemical 17)

Carbamyl, Acetamide (Chemical 91)thiocarbamyl

Thioacetamide (Chemical 92)Urethan (Chemical 95)

Maneb (Chemical 105)

Thiourea (Chemical 108)Monuron (Chemical 109)

Antihormone? (see text)

Antiestro9enProximate alkylating agent? (21 , 157)Bactericidal action (151)Bactericidal action (1 @1)Mixed-function oxidation of the 3,4-double bond? (230)Solid-state carcinogen? (116)Tumor promotor? (240)Carcinogenic contaminants? (111)Tumor promotor? (240)Anabolite in biosynthesis of phenylalanine, tyrosine, and tryptophan;

xenobiotic to organisms for whom the aromatic amino acids areessential (222)

Mitotic poison (98)Carcinogenicity possibly due to contaminating hydrazine (see Ref.

114, Vol. 4, p. 177)

Antiuracil? (54, 171)Antithyroid (1, 225)Antipurine? (see text)

Conjugates with alanine to form an antihistidine in E. coli (249)Metabolic activation? (urine of mice treated with highly purified

material is mutagenic)Mutagenic impurities (16)

Azoxy aglycone liberated by gut bacterial f3-glucuronidase(136)Catabolite of tryptophan that is decyclized enzymatically into a

semialdehyde and recyclized into quinolinic or picolinic acids or ismetabolized to glutaric acid (170)

Mixed-function oxidation to acetaminophen which can bemetabolized further to a protein-binding species (118, 169)

Possible modes of chemosterilant action include aldehyde formation(26) and antimetabolic effects as analogs of pyrimidine (74) andfolate (12)

Mixed-function oxidation of N-dimethylamino groups?N-Hydroxylation?Intercalating agent? (202)Reduced by liver microsomes to benzidine and napthylamine

derivatives (1 41)Decolorized by gut bacteriaNot absorbed if gut is sterilized by pretreatment with antibiotics

(224)Membrane specificity (18)Cytochrome P-450-mediated reduction to trichloromethyl radical

(235)Metabolic activation? [DOD, a metabolite of DOT, is mutagenic in a

host-mediated assay (43)]

Formation of N-acetylhydrazine which is subsequently oxidized toreactive diazene (167)

Metabolism by gut Conversion to monosubstituted hydrazinebacteria (194) which is subsequently oxidized to reactive

diazene (1 67, 195)Ultimate electrophile (120)Intercalating agent with adenine-thymine base pair specificity?

(129â€”131)Oxirane formation (236)Volatility (217)Oxirane formation? (see text)

Membrane specificity due to ionized state? (see text)

Reduction by gut bacteria (e.g., Refs. 48, 189, 192) to aromaticamines which are then available for typical aromatic aminemetabolism

N-Hydroxylation?Appearance in DNA (238)Reactive species of sulfur from mixed-function desulfurization (1 12)Esterification of N-hydroxyurethan (168)Antimetabolite in pyrimidine biosynthesis? (29)Conversion to several reactive metabolites including ETU and CS@

(210)Reactive species of sulfur from mixed-function desulfurization (112)Conversion to several metabolites including aniline derivativeBenzene-oxide formation? (79)

Chlorobenzilate (Chemical 235)

DES (Chemical 236)

Succinic anhydride (Chemical 325)4-Aminoantipyrine (Chemical 327)Sodium carboxymethyl cellulose

(Chemical 440)Tween 60 (Chemical 445)

Shikimic acid (Chemical 451)

AnhydridePyrazolinonePolysaccharide

Polymer

Miscellaneous

HighAzoxyAromatic amine

Phenacetin (Chemical 117)

Simazine (Chemical 120)

Auramine (Chemical 137)Pararosaniline (Chemical 138)

Trypan blue (Chemical 153)

Evans blue (Chemical 154)




Table6â€”Continued.CategoryChemicalCommentsPhenyl

BenzodioxoleBenzene

(Chemical 219) 1Phenobarbital (Chemical 221) j@PCNB (Chemical 224)

Biphenyl (Chemical 225)Aroclor 1254 (Chemical 226)Safrole (Chemical 227) 11â€˜-Hydroxysafrole (Chemical 228) j'Piperonyl butoxide (Chemical 234).

Benzene-oxide formation (103, 117)

Reduction to aniline derivative (27); subsequent aromatic amine metabolism?

B@nzene-Oxideformation (89)

Activation of allyl Formation of carbonium ion at methylene cargroup (252) ben?(137)PolychlorinatedHexachlorocyclohexanes

(ChemicalsEpoxidation? (a major metabolite has a chloroethylene moiety inancyclic394â€”396)otherwisesaturated ring)

Benzene-oxide formation(99)Mirex(Chemical 398)

Aidrin (Chemical 400)Dleldrin (Chemical 401)Heptachlor (Chemical 402)Epoxy

or epoxy In- Formation of radicals from dechlorination?termediate (162)(205)SteroidEthinylestradiol

(Chemical 420)Testosterone propionate (Chemical424)1HormonesAntimetaboliteAminopterin

(Chemical 436)

L-Ethionine (Chemical 438)Antifolate

not easily assimilated by non-folic acid-requiring bacteria(254)

Naturally occurring amino acid in some strains of bacteria (81)Ethylating agent (228)


problem for the high-molecular-weight polyaromatics and heteroaromatics that have not yet been tested. The 11 falsenegatives that have occurred within these 15 categories arerestricted mostly to aromatic amines and polyhalogenatedmethyls. As many as 7 of these false negatives may haveresulted from inadequate metabolic activation (i.e. , cycasin,phenacetin, auramine, trypan blue, Evans blue, carbon tetrachloride, and DOT).

Structure-activity relationships are also evident among the

11 chemical categories that are not well detected (low andmedium activity). An analysis of false negatives in these categories suggests specificities on the part of these chemicals forthe metabolism and cellular membrane of the mammal. Inparticular, it appears that the Salmonella-S-9 system oftencannot activate or detect chemicals the activations of whichentail the formation of short-lived species (e.g. , from thiocarbamyls and polyhalogenated compounds) or benzene oxide(i.e. , phenyls) or the activations of which consist of severalenzymatic steps (e.g. , symmetrical hydrazines, azonaphthols,urethan, safrole). In the case of chemicals that are convertedin vivo to several metabolites (e.g. , maneb, monuron), theremust be some accounting for how faithfully the 5-9 activationcorresponds to this metabolism. Other chemical categoriesthat do not exhibit high correlations include benzodioxoles,steroids, and antimetabolites. Chemicals that are too volatile totest in the standard plate assay (e.g. , alkyl halides) and DNAcross-linking agents (e.g. , mitomycin C) may require optimizingrather than standard procedures to be detected.

Given the poor correlations for as many as 11 chemicalcategories, it is concluded that the important parameter inSalmonella testing is the individual category correlation, p@inChart 2. The individual correlations of these 11 chemical categories are low enough to discourage extrapolation of negativeresults obtained in Salmonella testing of chemicals of thesetypes. For at least another 13 categories, their individual correlations have not been evaluated. For the remaining 15 categories, which include the types with which Salmonella testinghas been standardized, their individual correlations are themost impressive; therefore, negative findings in Salmonella

testing of chemicals of these types are the most credible forextrapolating.

Validation of Salmonella Testing as a Predictor of Carcinogenicity

Unlike correlation, validation is purely a subjective affair.Ideally, one would want a mutagenicity test the sensitivity andspecificity of which are both unity and therefore the falsenegative and false positive rates of which are both zero. Thiswould afford maximum confidence in the test as an indicator ofcarcmnogenicity.As such things are rarely ideal, one can anticipate that the sensitivity or specificity of the test could varyindependently of each other. Historically, the originally reported (151) high sensitivity (90%) and specificity (87%) ofSalmonella have been well received. More recently, Purchaseet al. (i 87) have directed attention to the large number of falsepositives possible in mass screening chemicals under certainassumptions. However, the model for this demonstration mustbe seen with some reservations. First. there seems to be a tacitassumption that the identification of bacterial mutagens whichshould be tested further for mutagenicity in the mammal is notan important finding in itself in the screening of chemicals.Secondly, the authors have not considered in their model thatthose carcinogens that are not mutagenic in Salmonella are notrandomized over all the chemical categories of carcinogens. Infact, there is a discernible trend between the structure andmetabolism of a carcinogen and its ability or inability to causereversions in Salmonella. For a given category, the parameterof this trend is its individual correlation ( P@in Chart 2). Thirdly,the alarmingly large percentage of false positives occurs whenthe proportion of carcinogens in the chemicals that arescreened is quite low. However, in some instances, a de factoâ€â€˜enrichment' â€˜for chemicals with a greater likelihood of being

mutagenic may occur. For example, in the testing of chemicalsunder the newly enacted Toxic Substances Control Act, selecting chemicals on the basis of structure-activity relationshipsand existing toxicity data should enrich the population of chemicals to be tested with mutagenic ones.

3311SEPTEMBER 1979



A priori expectations for the action of mutagens in microbial andmammaliansystemsI.

Mutagen is similarly active in bothsystems.II.Mutagen is specific for mammaliansystemattributes.A.

Chromosomal organization, e.g. , mitoticpoisonsB.MetabolismFalse

nega- 1. Enzymatic, e.g. , urethan, benzenefives 2. Hormonal, e.g. ,steroids3.

Metabolic strategy, e.g. , shikimicacidC.Membrane, e.g., antifolates, actinomycinDIII.Mutagen

is specific for microbial system attributes.A. Chromosomal organization, e.g.,plasmid-dependentFalse

posimutagens?tivesB.Metabolism, e.g., nitro compounds?

C. Membrane (theoretical)


Besides less than ideal sensitivity and specificity, anotheraspect has emerged from an analysis of the various correlationstudies in Salmonella. It has been argued in this paper that thesensitivity is overrated as a result of the bias makeup of thecarcinogens that have been tested. This bias obscures thepoor response of some categories of carcinogens. Obviously,this â€˜â€˜restrictedsensitivity' â€˜does not invalidate Salmonella testing provided the restrictions are appreciated. A more sophisti

cated way of interpreting Salmonella results and discussingvalidation is indicated. It is necessary to see negative findingsin Salmonella in light not of the overall sensitivity of the test butof the sensitivities of the individual categories, some of whichare very high and others of which are hardly investigated or solow as to be of questionable value in predicting noncarcinogenicity. Therefore, it is necessary to validate the test on acategory by Category basis. A similar conclusion has beenreached by Purchase et al. (187).

To extend this sophistication a step further, the occurrenceof â€˜â€˜restrictedspecificity' â€˜should also be investigated. Ames(3) originally discussed how mutagens to bacteria may not besimilarly mutagenic to mammalian cells. Some of those arguments concerned a lack of quantitative agreement. Qualitatively, those factors that would produce false positives aresimilar to those that account for false negatives (Table 7).

The role of the A-factor in the enhanced sensitivity of TA100and TA98 has not been sufficiently elucidated. It is believedthat mutagens detected by these strains cause nicks in theDNA, which leads to error-prone repair and mutagenesis (150,154). The underlying assumption is that this same sequence ofevents occurs in mammalian cells in the absence of the A-factor. It remains to be clarified that this mutagenicity has acounterpart in mammalian cells and is not merely associatedwith an episome which possibly supplies an aberrant nuclearenzyme or otherwise is itself somehow a cause of the mutagenicity. A similar caution has been made by Bridges (36). This

clarification will have to explain several peculiarities. As McCann et al. (154) have discussed, certain chemicals (e.g. MMS)appear to cause nicks in the DNA. Mutagenicity is observed orenhanced in Salmonella if these nicks are repaired by an errorprone process that is dependent on the presence of a nonmutated, chromosomal recA gene and the A-factor. Mutagenswhose activities are not increased by this combination (e.g.bis(2-chloroethyl)ammne)presumably then are mutagenic in adirect way that does not involve recombinational repair. However, in the case of the alkylating agent, diethyl sulfate, themutagenic activity is attenuated dramatically in the presenceof the R-factor. These results would indicate then that rather

Table 7

significant repair of alkylated DNA has occurred due to thepresence of the A-factor. More recenlty, Teramoto et al. (231)have shown that, at concentrations up to 20 mg/plate, 4,5-dihydroimidazole-2(3H)-thiol does not revert TA100 while itdoes revert TA1535. Hence, the possible effects of the A-factor on mutagenesis with respect to non-plasmid-bearingstrains include increasing the spontaneous mutation rate instrains that are recA+ (capable of recombinational repair),masking the effects of at least one mutagen, and decreasingor enhancing enormously the effects of others.

If the bacteria metabolize a chemical to a mutagen butmammals do not, this would result in a false positive. Oneknown difference between bacterial and mammalian metabolism of chemicals involves those having a nitro group. Saz andShe (207) have studied this reduction in E. coli using variousnitroaromatics and have shown that the nitroreductase is relatively nonspecific and distinct from the enzymes that reducenitrite and nitrate. The nitroreductases of Salmonella that activate nitroaromatic compounds to mutagens are not found inmammalian cells, although nitro reduction can be performedby other means (201 ), e.g. , by xanthine oxidase (227). Hence,there is a need to study the specificity of nitro compounds,e.g. , by testing noncarcinogenic nitroaliphatics and nitroaromatics, presuming that such entities exist.

That a difference in metabolism can produce a false positiveis somewhat suggested by the weak mutagenicity of sodiumnitrite (152). However, the carcinogenicity testing of sodiumnitrite is not what can be called extensive. Outside of one earlystudy involving 12 s.c. injections over a 90-day period to mice(161), the testing of sodium nitrite has been done in the contextof a control for studies on the possible nitrosation of secondaryamines to carcinogens. In such studies, the resulting N-nitrosocompounds are expected to be so active as to require only asmall latency period [e.g. , animals were sacrificed at 40 weeksin the study of Greenblatt et al. (96)] and a small number ofrodents to show a positive effect (e.g. , Aefs. 95 and 140).

As previously mentioned, phenotypic curing was observedwith amitrole and resulted in what could be falsely interpretedas positive results. Similarly, bactericidal chemicals can produce false positives by cross-feeding of histidine released bykilled bacteria to surviving bacteria; a large number of â€œpinpoint' â€˜Colonies @5suggestive of this activity. This has promptedAosenkranz et a!. (202) to recommend that routine testinginclude replica plating of some positive plates onto minimalmedia supplemented with biotin in order to control for thesephenotypic effects.

The possibility of contamination by mutagenic substancesmust be considered in the interpretation of testing results sinceSalmonella can detect potent mutagens at minute concentrations. McCann et al. (151 , 152) have shown that the apparentmutagenicity of 2 hydroxy derivatives of 2-acetylaminofluorene

was attributable to trace amounts of the highly mutagenicparent compound. Batzinger et al. (16) have demonstrated thatless than highly purified preparations of saccharin containmutagenic contaminants that are active in modified plate testing. The modifications include placing biotin and L-histidine inthe bottom agar (and not in the overlay) and adjusting theamounts of glucose and L-histidine for maximum response.Apparently, the modifications are crucial to the demonstrationof mutagenicity in plate testing; Sugimura et al. (226) did notobserve mutagenicity in TA1537, TA100, or TA98 with or





without 5-9. The reported mutagenicity of DBCP (technicalgrade) without 5-9 in plate testing (186) is probably due to theepichlorohydrin that is added to DBCP as a stabilizer; however,in a desiccator system without S-9 and in plate testing with5-9, purified DBCP is mutagenic (20).@

However, not all mutagenic contaminants are categoricallydetected in testing. Maleic hydrazide, which cannot be obtamed free from hydrazine (214), is not active in Salmonellatesting (152). Similarly, the unexpected carcinogenicity of polyoxyethylene polymers may be due to carcinogenic contaminants (i 11) or decomposition products although it also hasbeen proposed that these substances are only tumor promoters(240). The lack of mutagenicity in Salmonella of Tween 60 mayindicate insensitivity to or batch variation in the concentrationof the carcinogenic species. Tween 80, a monooleate derivative of Tween 60, has been shown to induce transformation ofmurine fetal skin cells; the transformed cells produce tumorswhen transplanted back into syngeneic hosts (72).

Finally, reproducibility can be another confounding factor inSalmonella testing. In contrast to the 430 revertant colonies(140 revertant colonies with controls) in TA100 observed byMcCann et al. (152) with 1-naphthylamine tested at 100 zg/plate, Purchase et al. (187) failed to find significant results intheir testing. This fact was discussed as a hallmark of theSalmonella test since 1-naphthylammnewas presented, possiblyerroneously (191), as the noncarcinogenic analog of 2-naphthylamine, which is unquestionably mutagenic in Salmonella.Whether the conflict lies in differences in procedures (e.g.,Aroclor versus phenobarbital induction of liver enzymes) is notknown.

Interestingly, 2 more of the 8 supposed noncarcinogenscited by Purchase et al. (187) have been reported occasionallyas being carcinogenic. The noncarcinogenic nature of 4-AAFis hardly established. Neither the studies of Flaks and Lucas(82â€”84)using 6 or less rats per time interval of 4-AAF feedingnor those of Weisburger et al. (243) and Morris et al. (160) canbe taken as adequate proof that 4-AAF is noncarcinogenic. Infact, Morris et al. even suggested that the slight carcinogenicactivity of 4-AAF might be substantiated by testing at a higherdose. Likewise, Schinz et al. (208) concluded (unjustifiably so)from their study in which 1 of 10 rats fed 4-AAF had developedcolon cancer that there was weak carcinogenic activity. Again,as in the case of 1-naphthylamine, there appears to be aproblem with reproducing the mutagenicity. McCann et al.(152) found a 10-fold increase in the number of TA1538revertants after treatment with 200 @tg/pIatequantities of chromatographically pure material in the presence of phenobarbitalinduced 5-9. In contrast, Purchase et al. (187) failed to seemutagenicity in their testing.

The carcinogenicity testing of anthracene, which was pairedwith its carcinogenic 9,10-dimethyl derivative, hardly conveysany confidence that the chemical has been adequately tested.Although numerous, the studies that did not find carcinogenicactivity suffer from the use of far too few test animals. Generally, the route of administration has been limited to skin paintingand injection (i.p. , i.v. , and s.c.). However, Druckrey andSchmÃ¤hl(68) and SchmÃ¤hl(209) have reported the inductionof sarcomas at the site of s.c. injection in rats. Anthracene is

7 V. F. Simmon. A presentation at the Ninth Annual Meeting of the Environmental Mutagen Society, San Francisco, Calif., March 10, 1978.

metabolized in the rat to a trans-dihydrodiol and mercapturicacid (219), both of which are diagnostic for an arene oxideintermediate.

These examples substantiate the caution that Saffiotti (203)urged to be exercised in comparing the mutagenicities ofsupposed carcinogen-noncarcinogen pairs. Preferably, assignment of noncarcinogenicity should be made on the basis ofadequate animal testing that presently exists or is developedconcurrently. Some consideration of the electrophilicity of theparent compound and its metabolites including presumed intermediates should also be undertaken.

Despite these various considerations, the specificity of Salmonella appears quite high. While McCann et al. (151) foundthat 14 (13%) of the 108 alleged noncarcinogens were mutagenic, as argued, the classification of most of these falsepositives as noncarcinogens is based on testing that is madequate by present standards. Hence, by the operational definition of the Technical Panel (62) for noncarcinogens (â€˜â€˜judgednot positive for tumor induction on the basis of tests conductedadequately in 2 or more species' â€˜),most of the chemicals areexcluded as noncarcinogens. It is on this basis, high specificitywith a restricted high sensitivity, that positive results in Salmonella testing are validated as a qualitative predictor of carcinogenesis. However, that mutagenic potency cannot be considered a quantitative predictor of carcinogenicity is based onthe fact that mutagenicity can be both underestimated andoverestimated in Salmonella. Underestimation ranges fromfalse negatives to positives that are the result of in vitro activation that is incomplete or otherwise not representative of thetotal activatibn that occurs in vivo. Similarly, overestimationranges from false positives to positives with potency that hasnot been attenuated by detoxificative metabolism in vivo, DNArepair, or the protection afforded by the nuclear proteins thattogether with DNA make up the suprastructure of the mammalian chromosome (3). Furthermore, the fact that the host'simmunological competency, about which mutagenicity testingmakes no evaluation, plays a role in the course of carcinogenesis argues against equating mutagenic potency with carcinogenic potency (see also Ref. 8).

In closing, it should be noted that, in Burdette's (42) analysisof the lack of correlation between mutagenicity and carcinogenicity, a real appreciation for the importance of metabolicactivation of mutagens and carcinogens is conspicuously missing. Since then, the consensus has grown and is continually

substantiated that the reactive metabolic products of proximatemutagens and carcinogens are electrophiles which are capableof covalent reaction with genetic material. As discussed, manyof the false negatives observed in the correlation studies areunderstood to be metabolized in vivo to electrophilic species.Notwithstanding the possibility that somatic mutation is onlyone of several initiating mechanisms in carcinogenesis, it wouldthus appear that many false negatives are not demonstrablymutagenic in vitro for lack of a proper test system to bothactivate and detect them. Furthermore, this conclusion canprobably be generalized to all in vitro systems since in vitroactivation methods in general appear only crudely to simulatewhat occurs in vivo for many chemicals. Hence, the primarydeficiency of genetic toxicology as a discipline remains itsinability to assay reliably for gene mutations in vivo. Not untilsuch-procedures are developed can a final assessment of therelationship of mutation to carcinogenesis be rendered. In the

SEPTEMBER 1979 3313




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meantime, by necessity, the qualitative identification of mutagens must rely on a battery of in vitro and in vivo procedures,the 2 complementing each other.

Acknowledgments

The authors are grateful to their many colleagues who provided criticisms,translations, and prepublication copies of their manuscripts during the writing ofthis manuscript. The authors would like to acknowledge Dr. E. B. Whorten forsuggesting Chart 2 and Dr. V. M. S. Ramanujam for his help in categorizing thechemicals in Table 4. Phyllis Sitra assisted in the testing of the pesticides in Table2. Janie Unbehagen and Elizabeth Whiteman provided helpful assistance withTable 4. The authors are especially appreciative of the untiring cooperation ofthe Moody Library reference department.

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1979;39:3289-3318. Cancer Res Stephen J. Rinkus and Marvin S. Legator

SystemSalmonella typhimuriumthe Carcinogens and Their Correlation with Mutagenic Activity in Chemical Characterization of 465 Known or Suspected

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