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Mutag nic Activity of Ch mica! Carcinogens flnd Related Compounds in the Intraperitoneal Rost-Mediated Assay12 yinctflt P. Simmon, *3 41H*t*rb*o*r*t *S. Rosonkranz, s Errol Zolgnr, * and Liontl A. Poirier713 ABSTRACT--Tho mutaganle actlvKlaa T9 carcinogen*, non. (pUiwg*11*, and atructuraHy related compounds toward Saftno- typMmurtum strains TA1S30, TA1S3S, and TA153S and top^d Saecharomyeea oararNfaa 03 wara Iwuiatlgalid In tha Intrapaiiiowaai bool madiatad aaaay. Rawer than haM at tha earetnopnt wara mwtagante toward tha Saimonaffa strains. Tha Inaanall^lty at tha ayatam waa moot marttad with tha aromatic amino gd polycyclic hydrocarbon pracardnagsris. Under tha tact con* jBona. lower than 10% of tha catdnognna showed door mutalade acdvtty toward S. caraddaa 03. Howaaar. nona of tha nongaiUnoyana ama ilpnfflcantty mutagenic toward ofthor t typhfmMtum or S. earaafada D3. Overall, tha Intraparttonad boat* mediated aaaay dooa not aaam auitabla for routine prallndnary aaaardng of large numbers of potential carcinogens. Tha median Wtnal doaaa to miea of at eompounda ware determined. J Nad Canoor Inat U: 911-BIB, l7d. One of the methods proposed for routine prescreening of potential carcinogens has been the host-mediated bioassay (1). By this procedure sensitive microbial organisms or mammalian cells are implanted into animals and the animals are subsequently treated with the test chemical. The administered cells and their mutants are then recovered and counted. In this system the formation and transformation of the active form of the test chemical may be performed by the host. The host-mediated assay has been successfully used in the detection of a number of chemical carcinogens with widely varying structures. These include the nitro* aminw (2-4), the nitrosamides (5), hydrazine (6). aryldialkyltriazenes (7), alkylating agents (8-10), cyclo phosphamide (11), ethylene thiourea (12), and vinyl chloride (13). The detector organisms include yeast (14), Meurospora (15), Salmonella typhimurium (16), the murine leukemia cell line L5178y (17), Escherichia coli K12 and WP2 (8, 18), and bacterioptage T4 (19). Few studies have dealt with the mutagenic activities of the aromatic amines, the aminoazo dyes, and the polycyclic hydrocarbons in the host-mediated assay. Therefore, the mutagenic activities of a series of carcinogens and structurally related noncardnogens in the host-mediated assay were determined. MATERIALS AND METHODS The experiments were conducted by two groups at separate laboratories: group I (Herbert S. Rosenkranz at Columbia University) and group II (Vincent F. Simmon at SRI International). With the exception of minor modifications (noted below), the protocols were essentially identical to those previously published in (1). Group I generally used S. typhimurium strains TA1550 and TA1538, whereas group II used strains TA1535 and TA1538 as well as Satfcharomyces cerevtsiae D3. Animals, chemicals, and administration of com pounds.--Both groups used adult male Swiss-Webster mice (Division of Cancer Treatment, Mammalian Ge netics and Animal Production Section, National Cancer Institute) weighing 23-30 g. The mice were fed labora tory chow ad libitum until they were treated with the test chemicals. The sources of the chemicals are listed in (20). The compounds were administered at levels of 2-5,500 mg/kg (generally 125 mg/kg) (group I) or at 24-hour LD50 (group II). When the 24-hour LD50 were not available in the literature, group II determined the LD50 according to the method of Weil (21) (see table 1). Test organisms.--The strains of S. typhimurium and S. cerevisiae D3 were maintained and grown as described previously (22, 23). In each test group were 4-0 mice, and positive and negative control groups were used in each experiment. The test compound was given by im injection (group I) or as a single dose in 0.2 ml dimethyl sulfoxide by oral intubation (group II). Two milliliters of an overnight culture of the test mi croorganism was injected ip into the mouse after the skin on the abdomen was washed with alcohol. After 4 hours, the mice were killed by cervical dislocation, and 2 ml sterile saline was injected into the peritoneal AsmiyiaTtoms usu>; CFU>colony-forming units; LDJO-median lethal do*e<>). ' Received February 15. 1978: accepted August 8, 1978. 1 Supported by Public Health Service conmcts N0I-CP33394 and N01-CPS3S95 from the Division of Cause and Prevention, National Cancer Insutute. 1 Life Sciences Division. Department of Toxicology. SRI Iniernauonal, Menlo Park. Calif. 94025. 4 Department of Microbiology, Columbia University, New York, N.Y. 10032. 1 Present address; Department of Microbiology. New York Medical College. Valhalla. N.Y. 10595. * Environmental Mutagenesis Branch. National Institute ol Envi ronmental Health Sciences. Research Triangle Park, N.C 27709. 1 Nutrition and Metabolism Section. Laboratory of Carcinogen Metabolism. Division ol Cancer Cause and Prevention. National Cancer Insutute, National Institutes of Health, Public Health Service. US. Department of Health, Education, and Welfare, Bethesda. Md. 20014. 1 Address reprint requests to Dr. Posner. * We thank Dr. John Wattorn. Oak Ridge National Laboratory, Oak Ridge. Tennessee, for issuance with the preparauon of the references. VOL. 8L NO. 4, APRIL 1979 911 J NATL CANCER INST SL 066335 t12 cavity to aid in the recovery of the microorganisms. After' the skin on the abdomen was washed with alcohol, sterile focceps and surgical scissors were used to expose the peritoneum. The microorganisms were recovered with 1-ml tuberculin syringes (without needles). The peritoneal fluid was placed in sterile test tubes in an ice bath, and subsequent dilutions in saline were done' in the ice bath. For assays with Salmonella, undiluted 0.2-ml aliquots were spread on five minimal agar plates containing biotin to determine the number of histidine-indepen dent cells. An aliquot was diluted serially in saline to 10*T, and 0.2-ml aliquots were spread on bacterial complete agar to determine the survival (or recovery) of Salmonella. The minimal agar plates were incubated for 2 days at 37s C, and the number of histidineindependent revenants was determined. The bacterial complete agar plates were incubated overnight, and the number of CFU per milliliter was determined. In assays with 5. cerevisiae D3 as the test organism, the overnight culture was diluted to 5X107 CFU/ml before injection of 0.2 mi into each mouse. The recovered peritoneal fluid was diluted to 10*'. Aliquots (0.2 ml) of the lO'1 dilution were spread on 5 yeast agar plates (to determine the number of mitotic recombi nants), and 0.2-ml aliquots of the IQ"4 and 10'* dilutions were each spread on 3 yeast agar plates to determine the survival (or recovery) of the Saccharomyces. The plates were incubated for 2 days at 30s C. Colonies on plates spread with the 10~* or I O'* dilution were counted to determine the CFU per milliliter. Plates spread with the 10"* dilution were refrigerated to enhance the red pigment development indicative of mitotic recombination (adenine minus homozygosity). After refrigeration for 2-3 days, the number of red colonies or red segments per plate was determined by scanning the plates with a 10X dissecting microscope. The mutants per milliliter or mitotic recombinants per milliliter were then divided by the'CFU per milliliter to determine the mutants (or mitotic recombinants) per 10? survivors (for Salmonella) or per 10* survivors (for Saccharomyces). Thus a mutation frequency was deter mined tor cells recovered from each mouse. Treatment of data and calculation of results.--In each experiment the mutant organisms per milliliter and the total organisms per milliliter were measured for each mouse; the former was divided by the latter (o give an observed mutation frequency for each animal. The mean mutation frequency plus or minus standard error of the mean was thus calculated for each created group of mica and compared to their simultaneously run controls. Treated animals, from which the total number of organisms per milliliter was leu than 10% of the avenge total organisms per milliliter obtained from the negadve control animals, were not used in the determinations of mutation frequencies. With Salmo nella a compound was judged mutagenic if a twotailed /-test of statistical comparison showed P at 0.05 or less. With 5. cerevisiae, compounds were regarded as mutagenic only if the mutation frequency of the treated animals was at least ten times that of the Tabu l.--LDSO deUrntinatioee Compound Natulan S-Aminosaobonunt 3'- M#thyl-4*< dimothylsmino )izob*nxne 2'.3-Dimsthyl-4-minobiphenyl 1.58 zif. us l/k; AL(4d5-Nitro-2-furyl )-2-thiolyl Jformsmide 2-NitronsphthsIen* 4-Nitrobiphenyl 1-Anthramins >}-8 g/kg l'3 8/kg 847 n>g/kg If g/f, N- Hydroxy -S-(2 -fluorsny1) Acetamide Hydrazine sulfate Dimsthylearbamyl chloride 1,8,9-Trihydroxyanthrecent U2-Dimethyihydrazine Bromobenienc Acetamide Chryaene 1-Naphthylamin# 2-Naphthyiamine 2-Anthrmmine 1-5 %! kg 418 mg/k* 1 8/kg >1.6 g/kg 1 g/kg 1 8/kg 1 g/kg >320 tng/k* 167 mg/kg 867 mg/kg I S g/kj Methotrexate 2-Nitrofluoren# cCaprolaettne Glycidaldehyde Ethyl-p-toluenesulfonate 1.0 g/kg LS g/kg 1.3 g/ki 200 mg/kg 1 g/kg 5-Udo-2'-deoxyuridine 1 g/kg 1,4- Butaaeeultone 138 mg/kg 2- Methyl-4-<dimethylemino >azobenzene 345 mg/kg iV-Nitroeodiethylemine Thioaeetamide 132 mg/kg L13 yiw1 1-Phenyl-3,3-dimethyltriaaeM 232 mg/kg Thiourea 1 g/kg* S-Amino 1.2.4-triaxoie >5 g/kg Propytenimine B-Propioiactone 365 mg/kg 406 mg/kg Lead acetate 15 g/kg* Tttanoeene dichloride >680 mg/k| Beryllium sulfate A/-Methyl-iV'-iutro-iV-nitrooofUtniriine U g/kg 66 mg/kg 2-Fluorenamine 132 mg/kg' 4-NitroquinoliM N-aside 190 mg/kg L3-Properesultone 467 mg/kg Methylaaoxymethanol acetate 106-160 me) A/'Njtmeoethyiurw 600 mg/kg* N-Acetoxy-iV-2-fluorenylacetamide >660 mg/k| e-Naphthylisothioeytnate 162 mg/kg' Phenanthrene 700 mg/kg' a c1 I.*'' int let; ) f * Determined by Dr. Simmon, unless otherwise indicatsd. * Corrections from previous utimates. * From Dr. Bcunkranz. controls. The wide range of mutation frequencies (table 2) seen in the 5. cerevisiae obtained from control mice precluded our calling compounds "mutagenic" that produce a twofold to tenfold increase in mutation frequencies. RESULTS Aromatic Amlnaa The host-mediated assay was relatively insensitive in the detection of carcinogenic aromatic amines (table 31 Fewer than half of the carcinogenic amines tested demonstrated mutagenic activity toward S. typhtmunum TA1538 in this way, whereas none of 20 carono- J NATL CANcm INST vol a. so. t, Ann- SL 066336 Ijens tested were dearly recombinogenic in assays with jj. cerevinae D3. However, none of the noncardnogenic ,romatic amines tested showed dear mutagenic activity toward either S. cernmiae D3 or S. typhimurium fAl538 (table 3). Fourteen compounds were tested gainst 5. typhimurtum TA1538 in the two laboratories; j{ compounds (N-2-fluorenylacetamide, 1-naphthylIjtnine, 2-nitronaphthalene, and 2-nitrofluorene] were mutagenic toward 5. typhimurium TA1538 in one laboratory but not in the second (table 3). The different tesults may be due to the varying doses and modes of administration used by the two groups. A fifth com pound, 2-meihyl-4-(dimethylamino)azobenzene, was mutagenic toward S. typhimurium TA1538 in one test by group II but inactive in a second test by group II as veil as in a single test by group I (table 3). The remaining 9 compounds tested in common gave the same results in both laboratories. Polyeyellc Aromatic Hydrocarbons The host-mediated assay was also insensitive in detecting the carcinogenic polycyclic aromatic hydro carbons (table 4). Only one of eight carcinogenic hydro carbons tested showed mutagenic activity toward 5. typhtmurium TA1538, whereas none was active coward i cerevtsiae D3 (table 4). Neither of the two nonaircinogentc aromatic hydrocarbons tested showed clear mutagenic activity (table 4). However, anthracene did Mutagenesis in Vivo by Carcinogen* *13 give one positive response t ward S. typhimurium TA1538 in the host-mediated assay, but this result was not reproducible in the same or in the second laboratory. Nltroeemlnee, Nttroeamides, and Hydrazines The host-mediated assay was relatively successful in detecting this class of chemical carcinogens. Of seven such carcinogens investigated, five were mutagenic toward S. typhimurtum TA1550 or TA1535 in the hostmediated assay (table 5). However, group I detected four of five carcinogens, whereas group II detected two of six carcinogens tested against the base-pair substitution strains TA1530 and TA1535. Further, of four carcino gens tested in both laboratories, onlv ,V-nurosoethvlurea was found to be mutagenic to both S. typhimurium TA1530 and TA15S5. The only procarcinogens not detected by either of these two strains were .V-nurosodiethylamine and natulan (table b). Of the eight car cinogenic nitroso and hydrazine derivatives tested, only N-nitrosoethvlurea and l-phenvl-3.3-dimethyltrtazene were active toward S. cerevinae D3 in the host-mediated assay. Alkylating Agents Eight of ten alkylating carcinogens were mutagenic toward S. typhimurium TA1530 or TA1335 in the host- TaBLE 2.--Typical experiment* lifted to illuttrate variation in data collected Group Microbial strain Compound Dose, mg/kg P| Total population. CFU/ml. x 10'' Hie' rcvertanu/ml Hit' revertants/ 10* calls I 5. typhimurium Control TA1538 1 2 3 2-Anthraniine 125 1 2 3 II S. typhimurium Control TA1535 1 2 3 4 Hydrazine sulfate 420 1 2 3 4 II S. ctrtvinat D3 Control 2-Nitronapkthalene 1.300 1 2 3 4 5 6 I 2 3 4 5 9.3 8.4 6.2 7,4 7.9 9.1 4.32 2.62 2.88 2.88 4.20 1.72 1.36 4.45 Total population. CFU/ml. x 10"7 6.45 4.15 10.33 9.66 11.10 8.87 0.60 7.38 8.85 1.56 7.53 55 55 35 70 75 90 24 36 22 32 189 37 58 251 Ade' recombinants/ ml. x io*1 2 4 1 0 4 1 0.1 2 4 0.3 1 5.91 6.54 5.66 9.46 9.49 9.89 4.97 13.74 7.64 11.11 45.0 21.5 42.9 56.4 Ade' recombinants/ 101 ceils 3.10 9.64 0.97 0.00 3.60 L13 1.67 2.71 4.52 1.92 1.33 Average mutation frequency. +SEM, X 10* 6.03*0.26 9 61+0.14 q 92 41.5+7.1 Average mutation frequency, + SEM. X10' a Q7+ ] 42 2.43 0.57 VOL SZ. NO. 4. AMUL 1979 J NATL CANCER INST SL 066337 *14 mediated assay (able* 5). The alkylating carcinogens not muugenic toward these two Salmonella strains were benzyl chloride and ethyl-^-toluenesulfonate. 5. cerevisiae D3 was even less sensitive in host-mediated assays with alkylating cardnogens; only methylazoxymethanol acetate increased mitotic recombinations in this assay (table 5). No well-categorized noncarcinogens were studied in this assay. 1 MisceHaneoos Cardnogana These compounds have been arbitrarily subdivj^ into heterocyclic compounds; amides, ureas, and ating agents; anumetabolites; inorganic compout, and promoters. Few ol the cardnogens tested in any these categories showed cleai mutagenic activity towany of the strains tested (table 6). Table Z--Mutagenic activities of carcinogenic and noncarcinogenie aromatic aminct in the kcut-mediatid auay' Group I*_____________________ ____________ Group II' Compound Caretnoactivity' mg/kg Increase in mutation frequency.' S. typkimurinm TAisao TA1S38 Ham, mg/kg Af.Acetoxy-<V-2 -fluoreny1acetamide UC 25 0.9 28.6 ( 25.5*2.1 660 vi. 09*0.1/ 4-Aminoesobenzene PC 125 1.8 (1.39*9 0.9 500 vs. 0.73*0.05)' 2-AminobiphenyI 496 4-Aminobiphenyl PC 12 0.3 132 1-Anthramine 2-Anthramine Auramine Bi*-p-<dimethyleinino)diphenyl- 2'4-Dimethyl-4-eminobiphenyl 2-FIuorenamine Af-2-Fluorenylacetamide NC PC 125 1.0 PC PC 125 1.4 PC 125 1.1 PC 2 12 0.6 PC 125 1.0 1.6 (9 6*0.1 va. 6.010.3)' 2.7 (26.3*2.3 vs. 9.71.6)' 7.0 (5.1*0.3 va. 0.7*0.1/ 1,250 1,500 660 1,600 1,050 132 1.3 495 N-4-Fluorenylaeetamide N-Hydroxy-N-<2-fluornyl laeetamide S-Methoxy-4-aminoasobenxene 2-Methyl-4-( dimethylamino lasobensane 3'-Methyi-4/ dimethylamino )aaobenseno 1-Naphthylamine 2-Naphthylamine N-l-Naphthylhydroxyiamine Af-2-Naphthylhydroxylsmine a-Naphthylieothiocyanate 4-Nitrobiphenyi 2-NitmflwMwM 2-Nitnmaphthaieoe p-Boeaniline 4-( -Tolylaw 1-o-toluidine NC PC PC PC PC NC PC UC UC NC PC PC PC PC PC 125 2.7 (2.010.1 vs. 0.73*0.05/ 125 1.1 9.2 (34.0*8.8 vi. 3.7* 1.5)' 1.1 496 1,500 50 340 125 1.1 125 1.6 125 2.7 (15.1*4.4 vs. 5.5*0.6) 125 125 1.1 125 1.2 125 1.8 (9.8*0 4 va. 6.50.6/ 125 0.7 125 12 0.5 1.500 1.0 167 1.0 867 396 500 1.0 1.0 13.7 (49.2*3.2 vs. 3.6*0.2/ 6.5 (23.7*6.1 vs. 3.60.2)' 12 165 1,600 1,600 1,300 1,600 1,250 Increase in mutatu* frequaney' S. tyvkimurium S. a TA1538 5.9 (7.13ii.40 vs. 1.23*0.59/ 1.4 0. 0.7 1.8 (229*121 va 1.23*0.59) 0.6 11.5 (45.8*12.0 vi. 3.96*0.86/ Li 21 1 C 1/ 1 1.0 34.6 ( 50.1*17.1 va 1.46*0.77)' ( 1. 2.0 (4.80*0.23 va 2.40*1.00)' 0.4 9.5 (15.1*5.8 v*. 1.6*0.4) 6.3 (10.0*4.0 n. l.60-4)f i_2 0.3 m w Tr 3.1 (11.0*4.0 va 3.5*0.4)' 1.1 ,.2 '3 1.1 2.7 (2.70.5 va 1.0*0.2/ 0.8 3.1 (7.012.7 vs. 2.3* 1.0) 0.4 3 V,4 A' C 0.5 0.9 * Tba detector organisms war* injected ip, whams the chemicals wort sdministarod cithar im or orally. * Data tram Dr. Sosenkrantc 3 mica wara in saeh group, and the compounds were administered im. ' Data from Dr. Simmon; 44 mica wara in each (roup, and the compounds were administered orally. ` UC*ultimata earanogtn, PCprocarcinofen. NC*noncarcinogta. * Mutation frequency of the experimental group/mutation frequency of tba control group. The average mutation frequency (xiO*) for 5. tgpkimurutm TA1630. TA1536, and TA1S38 lor all positive or borderline compounds are in parenthue* < corresponding control values. Became of the excessively low mutation frequencies of the negative compounds, such values are r. for 5. ceremfio*. 1 PSO Ol in a two-tailed t-taat at statistical comparison. * 0.01SPS0.05 in s two-tailed {-test of statistical companion. J NATL CANCEX INST VOL 62. NO. 4. A] SL 066338 TABU 4---MtUaaenie actmtiee ef careinogneic and noneaminognic aromatic hydrocarbons in the hoot-mediated assay - and acyj ompoundj* d in any 0j ty toward ray' mutation eT_____ * S. Cfrj, wwpa 0.9 1.0 22 1.0 0.4 1.7 1.65* 0.9 1.1 Group I* Group II* Compound I Carcino genic activity' lacrosse in mutation frequency.* Dose. 5. typhrmunitm mg/kg TA1530 TA1538 mg/kg Increase in mutation frequency* 5. typAmunum TA1535 TA1538 S. cerevieiaeDZ anthracene NC gnwfojanthracena B*n*[tt]pyr,o Bn*t*]PSIT*J* T.BfOtnomfthyl1-12* narthylbenslajanthraeon* Chrytene 7,12-Dunethylbenirajanthractno 3-Hydroxyboni[ajpnane 74 Hydroxymethyl >12-methylbeni[ajanthncene Phcnaathrene PC PC PC UC PC PC PC PC NC 125 125 125 125 125 125 1.1 1.0 1,600 1.1 5.1 (50.1 1.5 vi. 9.9+1.9)7 1.1 0.9 1.600 1.0 1.1 496 500 500 0.9 0.5 0.9 12 330 1.600 23 500 0.5 0.9 700 0.9 0.9 2.9 2.7 (4.1 1.0 vs, 1.50.3)* 0.7 0.7 1.0 1.8 1.6 1.4 0.7 0.6 0.7 0.8 1.5 2.5 0.1 0.6 1.0 02 Tim detector organisms were injected ip. whereas the chemicala were administered either im or orally. > pita from Dr. Rossnkrantz; 3 mice were in each group, and the compounds were administered im. Data from Dr. Simmon; 4-6 mice were in each group, and the compounds were administered orally. NC soncarcinogen. PC procarcinogen, UC-ultimate carcinogen. < Mutation frequency of the experimental group/mutation frequency of the control group. The average mutation frequency sem (XlOa) for S. typhnanrinm TA1630, TA1S36, and TA1538 for all positive or borderline compounds are in parmtheess vs. the corrv- ponding control values. Because of the excaesively low mutation frequencies of the negative compounds, sueh values are not listed for I 1.0 1.0 2.1 1.1 0.8 0.9 1.2 1.3 4.0 0.4 0.3 1.2 0.7 0.8 2.1 0.3 :ncy jim m vs. the - not listed < PS0.01 in a two-tailed t-test of statistical comparison. 1 0.01SPS0.06 in a two-tailed t-test of statistical comparison. Among the heterocyclic compounds, only safrole, 4niuoquinoline N-oxide. A/-{4-(5-mtro-2-furyl)-2-thiaiolyi]formamide, and 4-{hydroxyamino)quinoline A/-oxide showed evident mutagenic activity toward Salmonella (able 6). Safrole, when administered at a dose of 5,500 mg/kg, was mutagenic to 5. typhimunum TA1538 I (able 5) but was inacdve at the lower dose of 869 mg/kg. Group I found that N-{4-(5-nitro*2*furyl)-2F chiazo1y1Jformamide (5-12 mg/kg) was mutagenic to S. k typhimunum TA1538; group II found that it was inactive even at a dose of 1,600 mg/kg. It is difficult to t reconcile this difference. 3-Amino 1,2,4-iriaxole appeared to be mutagenic to S. typhimunum TA1530 and TA1538 at low doses. It also appeared to possess i' borderline activity toward S. cerevisiae D3. Acridine or1 ange, aliatoxins Bi and Bi, and 7.9-dimethylbenz(c)acri- dine showed no mutagenic activity in any test studied. S Among the amides, ureas, and acyiacing agents, dimethyltarbamyl chloride was dearly mutagenic toward TA1535 at a dose of 1,000 mg/kg (able 6); however, at a dose of 125 mg/kg, it was inactive towatd S. typhimurium TA1530. Acetamide and urethan were com* 1 pletely inacdve toward 5. typhimunum TA1535 and S. I cerevisiae D3. Both thioacetamide and thiourea appeared * ' to be weakly mutagenic in the host-mediated assay. At doses of 125 mg/kg, both compounds produced signifi cant increases in the muurion frequences observed with S. typhimunum TA1530 and TA1538; however, doses of 1,000-1,430 mg/kg had no observable effect on the muta tion frequencies of 5. typhimunum TA1535 or the mitoric recombinauon frequency of S. cerevisiae D3 (able 6). None of the andmeabolites or metals showed any sig nificant mutagenic acuviry toward any of the test strains (able 6). The apparent recombinogenic acdvity of 5-iodo-2'-dcoxyuridine toward S. cerevisiae D3 was not sig nificant. Similarly, the promoter 1,8,9-trihydroxyanthracene showed no muugenic acuvity in the hostmediated assay toward either S. typhimunum TA1538 or S. cerevisiae D3. DISCUSSION The present results indicate that the host-mediatet assay is insuffidendy sensitive for the routine prelimi nary screening of large numbers of potential carcino gens- Even when the metals and promoters are excluded the host-mediated assay detected only about 50% of th carcinogens tested. Forty-three percent of the carrinc genic aromatic amines and 13% of the carrinogeni hydrocarbons were clearly mutagenic toward S. typh\ murium TA1538 in the host-mediated assay. Howevei 71% of the carcinogenic nitrosamine derivatives an 80% of the alkylating carcinogens were mutagenic to I typhimunum TA1530 or TA1535 in the same assa' vFXIL 1979 VOL. . NO. 4. AWL 1979 J NATL CANcut INS SL 066339 fit Tails a--J and noncarcinogtnic nttrorntmintt. nitrotamidtt, kydrtaintt. 3*4 (Ukyiatuuj -- tu Ms heat-mediaUd assay' Group 1* Compound Cardnogsnie activity' Does, rat/kg Increase in mutation frequency,' S. typAtmunum TA1S30 TA1538 Doe*, mg/kg Inerouo in mutation froquoney* S. TA1535 TA1538 Nitrusaminsa, nitro- saraides, and hydrasinm Ditnethylhydrsxine PC 125 2.5 (10.91.0 1.2 vs. 4.4*04/ 996 1.7 1.7 Dipbeuylnitro- mminc Hydroine sulfate NC PC 126 1.0 125 0.6 1.0 1.1 5,000 200 420 1.3 1.0 4.4 (41i7 04 A/-Msthyi-//'-nitroAf-nitroaoguanidint uc 5 3.6 (26.511.6 9.7 (3.210.6 v*. 7.3*04) va 0.310.1 / 30 44.3 (61.5*0.7 11.8 (3.910.1 vs. 1.4 *0.2/ vs, 0.310.1/ va 9l2)/ 66 04 66 0.1 Natulan PC 125 0.6 1.6 (0.5710.08 va 0.3510.05)' 600 0.7 14 Af-NitrosedisthylsmiiM N-Nitroeoethyl- uroa PC uc 377 0.8. 1.5 12 04 16 (0.810.1 va 0.310.1/ 120 27.4 (S7.80.S 132 0.6 602 324 ( 4101244 16.4 (180176 va 12.5114/ va 11.014.3/ 02 257 va 1.3104/ i! l-Fhehyi-3,3-di- PC 12 14.7 (127*17 L0 mothyltrisjsiM v 8.711.9/ 196 0.6 34.3 Alkylating agents Beaayi^ehJande 1,4-Butsueralnioe uc 4.400 1.0 U 600 1.0 1.1 uc 106.4 16J (13211 7.4 (63.4111.0 n. 8.010.3/ vs. 8.6104/ 460 MOO 188 1.1 164.0 (8881311 va 5.4114/ 0.3 04 t-Caprolaetons -- 432 1.1 0.9 UiM-Diopnay- uc 444 2.8 <20411.5 butiM va 7.5104/ Etfeyi-p-teluene- uc 468 1.0 0.9 1.300 56 1.000 1.4 0.6 U 4.7 14 Giyesdaidehyde Methylaaetymethanol acetate 1,3-Propanesuhone uc PC uc 8-PropiolactoM Prupytsaimine Uracil mustard UC UC uc 416 2.5 (18.5114 M va 7.5104/ 468 672 ( 666192 148 (260128 va 1.0104/ vs. 1410.7/ 12 646 ( 472140 64 (61118.9 va 04104/ va 9.9114 y 12 17 (41712.4 1.1 oo. 11410.4/ 200 24 (14414.7 va 6.311.5/ 132 467 269 (4861218 va 1.810.6/ 2-2 (3.910.7 va 1.810)* 406 3.9 (21.117.5 vs. 5.4114)' 365 3.1 (6.6104 va 1410.6/ 0.7 9.6 44 0.1 64 * The ornninM wars injected ip, wharms the chemicals wort sdministersd tithor im or orally. * Data from Dr. Ssoonkranta; S mica wart in Mch group, and tho compounds wer* administered im. ' Data from Dr. Simmon; 4-4 mica wort in mch group, and tho compounds wort administortd orally. ' POpruearcbana. NC-nonearcinogan. UC-ultimeta carcinogen. ' frannurr U tho experimental group/mutation froquoney at tho control group. Tho average mutation froquoney istn (X10*) ter S. tapttimaram TA1530, TA16S6. and TA1538 for ail poaitiaa or bordorlino compounds art in partntK*mt vs. tha corre- spmding emtrel vahssa. id tho rrrmsiTly low mutation frequencies of tho nagativa compounds, such values art not listed fur i Pso.Ol in a two-tailed t-tost of statistical comparison. ' 0.01SJ*S0.06 in a two-tailad t-taot of statistical comparison. S. cerevisiae D3 organisms were generally very insensi live to the activity of the carcinogens, especially the aromatic amines and the miscellaneous ardnogens. Also, the Salmonella strains used in the host-mediated assay detected a greater percentage of ultimate cardno j gens than procarcinogens (67 vs. 38%). The results from this study generally agree well with previous findings. The high percentage of the nitro- J NATL CANCL* INST VOL. St, NO, 4. AFRO. 191* *V, | SL 066340 MutagenMte In Vive by Carcinogens 917 T**tX 6.--Mntaponie activities of tamufiw and noneanrutognu luttmcpcHc compound*, trmuia. untinotaMito*. mttali. and pnrmottn tn Uu Sett-mediated auay' Group I* Group II' Compound Cardnog*nie activity' Doe*, mg/leg Inert*** in mutation frequency,' 5. (ypAimiirtwn TA1S38 IMR * mg/ kg Incrana* in mutation frequency' $. typAttnvrium TA1535 TA1538 S. cere* wia* D3 Heterocyclic* Acridine onne* Aflatoxin B> Aflatoxin Bi 3-Amino 1X4- tnsiol* PC PC PC PC 7,9-Dim*thylb*ni[e]*cndin* 4-1 Hydroxyunino)quinolin* V-oxid* .V-{4-( 5-Nitro-2furyl >-2-thia*olyl]formnmid* 4-Nitroquinolin* A'-oxid* Safroi* Amide* Acetamide Dimathylcarbamyi chloride Succinic anhydnd* Thioocttamida PC uc PC PC PC PC uc -- PC Thiouro* PC Ur*th*n Antimatabolite* Ethionin* S-Fluc*o-2'-d*oxyuridint $-Iodo-2'-daoxyuridina Methotrexate Inorganics Beryllium sulfat* Lead *c*tat* Titanoeene dichlond* Promoter 1.8.9-Trihydroxysnthr*c*n* PC PC NC NC NC 12 4.0 (3.5:14 LI v*. 2.1*0 217 125 1.8 (18:0.1 12 (1.1:0 0 V*. 1.0:02)' vs. 0 92: 0.09)' 250 1.0 1,0 125 1.0 1.0 12 3 7 (4 0:02 20.3 1 84 :23 v*. 1.1:0-21' v*. 4.1:0 3)' 5 12 12 5.500 21.1 (24 8:0.7 v*. 12:0.1)' 3.4 (72:5.5 vs 2.1*02) 02 231 (201:5 vs. 0.9:0 2)' 108 ( 368.4 :2.0 v*. 3.4:0.1)' 150 (132:10 vs, 0.9:0 5)' 22 (3.8:0.5 V*. 1.8:0 1)' 125 1.0 1.1 125 0.9 12 125 13 (1.9:0.3 2.8 (2.8:0 4 v*. 09:0.3)' vs. 0 9:0.0)' 13 (13:0.4 4.2 (7.5:0 7 *1. i.o:o.5)' vs i.8:o,0)' 1.6 (1.410 1 3.5 (2.3:0 3 v*. 0.9:0.0/ vs 0.7:0.Q)' 125 10 (10:0.5 2.0 (3.6:001 w i.o:o.5) vs. 1.8:0.02)' 1.3 1.4 (0.92:0.06 vs. 0.66:0.01)' 1.6 (1.64*0.13 1.7 (1.58*0.14 vs. 1.01*022) 4 vs. 0.92:0.04)' 495 33 2 1.585 0.9 5.000 500 1.600 1.600 1.8 (4.3:12 vs. 2.4:10) 3.2 (5.1:16 v*. 1.6*0 4) 0.7. 1.4 1.3 4.7 (10.8:5.7 vs. 2.3:1.0)' 1.6 190 11.4 (14.0:2.5 vs. 12:0.6)' 869 0.9 1.000 1.0 1,000 3.5, (22.1:6.9 w 6.4:05>' 869 06 . 1.130 0.5 1,000 0.7 495 0.6 125 0.8 125 12 125 1.1 125 1.1 25 1.3 25 1.1 25 1.1 11 1.0 0.9 1.1 1.1 07 0.5 1.600 1,5 1.?" 1.000 1200 0.8 3.500 4.5 (8.7:3 4 w 2.0=06) 660 0.6 1.6 1.0 125 0.8 02 1.585 1.000 05 07 IS 1.4 7,5 0.6 08 0.5 935 209 05 0.4 17 1.4 24 5.0 0.9 1.7 0.6 2.1 03 0.7 59 02 1.9 0,7 1.3 0.9 * Th* dateetor organisms w*r* injected ip, vhwai the ehemicali wer* administered either im or orally * Dou from Dr. Ronnkruitz: 3 mic* wer* in each group. snd th* compound! won admtnistvted im. ` Dmu from Dr. Simmon; 4-6 mic* wr* in mah croup, tnd th* compound* w*r* administered onlly ' PC"proeaiuiiMf*n. UCultim*t* carcinogen. NOnoncnrcinofon. ' Mutauoe frequency of th* experimental group/mutation fraqutncy of th* control croup. Th* *v*nf* muution frequency -3EM (xlO*) for S. tppkimuriitm TA1530, TA153S. *nd TA1538 for *11 pooltir* or bonforiin* compound* *r* t* par**thorn* vs. th* eorleeponding control valuta. Because of th* exceeeively low mutation froqucncm of th* negative compound*. *uch value* *r* not listed for S. cfr*m*Mi*. ' W0.01 in a two-tailed (-teat of stntiiticnl comparison. * Q.01SW0.Q5 in two-t*il*d t-tott of ttatiiticnl comparison VOL. 62. NO. 4. APRIL 1979 J NATL CANCER INST SL 06634 r lamine derivatives showing mutagenic activity by the hoi*tnediated assay in the present studies confirm* previous observations (2-5). Similarly, several alkylating dabgenu are mutagenic in the assay, both in the present dand previous studies (8-11). In this study thioacetamide and thiourea were weakly mutagenic a close structural analog, ethylene thiourea, is also mutagenic in the host-mediated assay (12). The two major classes of chemical carcinogens that show the least mutagenic activity in the host-mediated assay, the aromatic amines and the polycyclic hydrocarbons, had not been well invesdgated previously. REFERENCES (1) Legato* MS. Malung HV: The host-mediated uuy. a practi cal procedure lor evaluating potential mutagenic agenu m mammals. In Chemical Mutagens. Principles and Methods lor Their Detection (Hollaender A. ed), vol 2, New York; Plenum Press, 1971. pp 569-589 (2) ZuCE* E. LeCatok MS; Mutagenicity at IV-nitrosomorpholine in the host-mediated assay. Mutat Res 12:469-471. 1971 (3) Zeice* E: Effect of metabolism of .V-nitrosammes on their mutagenicity for Salmonella typhimunum in the hostmediated assay. Cancer Res 35:1813-1818. 1975 (4) MallinC HV, Frantz CN. 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' 840. 1979 (21) Weil CS: Tables for convenient calculations of the median effective dose (LD50 or EDS0) and instructions in their um. Biometrics 9:249-263. 1952 (22) Rosenkranz HS, Poirier LA: Evaluauon of the mutagemetn and DNA-modi/ving activity of ardnogens and noncarcinogens in microbial .systems. JNC1 62:873-892. 1979 (23) Simmon VF In vitro muugenicttv assavs of chemical carcino gens and related compounds with Salmonella typhimunum. f h JNd 62:893-899. 1979 V I 1t I i J NATL CANCER INST /4* i VOL. 6*. NO. 4, APRIL 1979 SL 066342