Document Ray3b5GvejGnNnGadMnMn83ga

AR RE _O64E PFOS Genotoxicity - [ 000033 or a OC] IOFN V3IHTRCOOMPMAINCYROBCIOOMLPOOUGNIDCALT-M2U8T1A6GCEoNCICITY ASSAYS Final Report, Revised aren 180 S 8 oJ = By: KDieriescctioern, E.MiMcorrocbeilalmanGse,netPihc.sD. Department Anne Pomeroy,anMdicrobiological Technician Prepared for: MG3ee4dnieCcrOaaMllPANDOYfefpiacretsm,ent3 Center Sc. Paul, Minnesota $3101 Accencion: WT.oxiC.colHoegCyoraSetrcvkices SRI Project LSC-8958 C-- nD Approved: D~avi~d C.. L.2J/onk}s,--Director Toxicology Laboratory WL.ifeA. ScSikeinncneesr,DiEvxiesciuotnive Director 4. 1431353)Ra33v6e.n6s2w0o0odCoAvmse.. SMAeInIloNTPLarMkP,K.Caf+orTnHiXa.349012053731246 000034 smaury SRI International examined 34 Company's Compound T-2816CoC for mutagenic activity wich strains TALS3IS, TALS3?7, TALS3S, TASS, and TAL00 of Salmonella typhimurium in the standard Ames Salmonella/microsome assay and with the yeast Saccharomyces cerevisiae D3. Each assay vas performed in the presence and in the absence of a rat liver metabolic acedvation system. Compound T-2816CoC was not mutagenic or recombinogenic 4n any assay performed. 1 000035 INTRODUCTION SRI International examined 3H Company's Conpound T-2816CoC for mutagentcicy by in vicro microbiological assays with strains TALSIS, TALS37, TALS38, TASS, and TAL00 of the bacterius Salmonella typhimuriun 4n the standard Ames Salsonella/aicrosome assay and with the yeast Saccharosyces cerevisiae D3. An Aroclor 125i-stiaulated, rat liver homogenate metabolic activation system vas included in the assay procedures to provide metabolic steps that the bacteria either are incapable of conducting or do not carry out under the assay conditions. The assay procedure with S. typhimurius has proven to be 80 co 90% reliable in dececting carcinogens as outagens, and it has about the same reliability in idencifying chemicals that are not carcinogenic. The assay procedure with 5. cerevisiae is about 60% reliable in detecting carcinogens as agents thac increase mitotic recosbination. Hovever, because che assay systess do not alvays provide 100% correlation with carcinogenicity investigations in animals, neither a positive nor a negative response conclusively proves chat a chemical is hazardous or nonhazardous to aan. 2 000036 METHODS TSAaIlS3o7n,ellTAaISt3y8,phiTmau9r8i, umanSdtrTaAiLn0s0 TALS3S, The Salmonella typhimurium strains used at SRI are all histidine auxoczophs by virtue of mutations ia the histidine operon. When these hiscidine-dependent cells are grown on mininal medium agar plates containing a trace of histidine, oaly those cells that revert to histidine independence (hisTM) are able to form colonies. The small amouat of histidine allows all the plated bacteria to undergo a few divisions; in may cases, this growth is essencial for mutagenesis to occur. The his Tevertancs are easily visible as colonies against the slight background growth. The spontaneous mutation frequency of each strain is relatively constant, but when a mutagen is added to the agar, the mutation frequency 1s increased, usually in a dose-related mamner. We obtained our S. typhimurium strains from Dr. Bruce Ames of the University of California ac Berkeley. In addition to having mutations 4n the histidine operon, all the indicator strains have a mutation (zfa) chat leads to a defective lipopolysaccharide coat; they also have a delecion that covers genes involved in the synthesis of che vitamin biotin (bio) and in the repair of ultraviolet (uv)-induced DNA damage (vEB). The rfa mutation makes the strains more permeable to many large aromatic molecules, thereby increasing the mutagenic effect of these molecules. The uvrd mutation causes decreased repair of some types of cheaically or physically damaged DNA and thereby enhances the strains' sensicivicy co some mutagenic agents. Scrain TALIS is reverced co his by many aucagens that cause base-pair substitutions. TALOO is derived rom TAIS33 by the introduction of the resistance transfer factor, plasaid pRMI0L. This plasmid is believed to cause an increase in errorProne DNA repair that leads to many more mutations for a given dose of most mutagens. In addiction, plasaid pKMIOL confers resistance to the 3 000037 antibiotic ampicillin, which Of the plasuid in the cell. 1s a convenient The presence of marker to detect this plasaid also the presence makes strain TALOO sensitive to benzo(a)pyrene, aflatoxin some frameshift mucageas 3,, and7,12-dtmechylbenz [e.g., ICR-191, (a)anthracene] Strains TAIS7 Serain 1498 is and TALS3S are reverced by derived from TASI8 by the many frameshift addition of the mutageas. plasmid PRA0L, which makes ic more sensicive to some mutagenic agents. ALL indicator serains are supplenenced wich an excess of kept at 4C on minimal biotin and histidine. agar The places places wich the plasnid-carrying strains ensure stable matacenance of also contatn ampicillin (25 ug/l) to the plasmid pRMIOL. New stock culture bPeleancescheacrekedmafdoerevteheriyr4getnoot6ypwiecekschafrraocntesriinsgtliecscol(obinsy, irsfoal,atwesedt,habtioh)ave farnodmfotrhe tshetocpkresceunltcuereofpltahceespliassmigdr.ownFoorverenaicghhtexapter3i7menCt,inannutirnioecnutlum broch (Oxoid, U7). Aroclor 125-Seimulated Metabolic Activation System the Some carcinogenic chemicals polycyclic hydrocarbon type) (e.g., of the aromatic amino are inactive unless they are type or metabolized : ottohearctoirvgeanfsora(se..g.,Inluanngfmaolrskiadnndeya)an,1s acnapeanbzlyemeofsymsetceamboilniztihneglaivelrargoer nusber of these metabolites are chemicals to carcinogens. very pocenc sutagens in the Some of these intermediate S. typhimurium tesc. Ames has descrived che brief, adult male liver metabolic activation system chat rats (250 to 300 ) are given a single we use. 500-ag/ks In inczapericoneal injection of Aroclor biphenyls). This treatment eahances 1256 (a mixture of polychlorinated the synchesis of enzymes involved in the mecadolic conversion of chemicals. the antzals' food is removed buc drinking Four water days after the is provided ad injection, libitum. On as che 1fch follovs. day, the rats are killed and the liver homogenate is prepared " 000038 The livers are removed aseptically and placed in a preweighed sterile glass beaker. The organ weight is determined, and all subsequent operations are conducted in an ice bath. The livers are washed with ao equal volume of cold, sterile 0.15 M KCL (1 ml/g of wet organ), minced with sterile surgical scissors in three volumes of 0.15 KCl, aad homogenized with a Pocter-Elvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x g, and the supernatant, referred to asthe 5-9 fraction, is quickly frozen in dry ice and stored at -80C. The metabolic activation mixture for each experiments consists of, for 10 a1: + 1.00 ml of $9 fraction + 0.20 ml of MgCl, (0.4 ) and KCl (1.65 ) + 0.05 ml of glucose-6-phosphate (1 ) + 0.40 ul of NADP (0.1%) 5.00 al of sodiun phosphate buffer (0.2 , pH 7.4) + 3.35 al of Hy. Assays in agar To a sterile 13 x 100 =m cesc cube placed in a 43C heating block, we add in the following order: (1) 2.00 81 of 0.62 agar" (2) 0.05 al of indicator organisms (3) 0.50 ml of mecabolic activation mixture (if appropriate) (4) 0.05 al of a solution of the test chemical. This aixcure is stirred gently and then poured onto minimal agar places.' After che top agar has set, che plates are incubated at 37C for 3 days. The number of his' revercant colonies is counted and recorded. "The 0.6% agar concatns 0.05 a histidine, 0.05 a biotin, and 0.6 Nacl. "M0i2nim3alofaMggaSrO.p+l7aHtae0s, c2ognsisotf 35 g of NaHNH.PO.+iHs0. ocfi,tripcer acliidterm,ono1h5ygdraotfe,aga1r0,g 1o0fg KoPfO.g,luaconsde, 5 000039 For negative controls, we use steps (1), (2), and (3) and 0.05 ml of the solvent used for the test chemical. For positive controls, we test each culture by specific mucagens known to revert each strain, using steps (1), (2), (3) and (4). Saccharomyces cerevisiae DI The yeast S. cerevisiae D3 is a diploid microorganism heterozygous for a mutation leading to a defective enzyme in the adeaine-mecabolizing pathuay. When grown on medium containing adenine, cells homozygous for chis mucacion produce a red pigment. These homozygous mutants can be generated from che heterozygotes by mitotic recombination. The frequency of this recombinational event may be increased by incubating the organisms with various carcinogenic or recombinogenic agents. The recombimogenic activity of a compound or its metabolite is determined from the number of red-pigmented colonies appearing on test plates. A stock culture of S. cerevisiae is stored at 4C. For each experiment, broch containing 0.05% MgSO., 0.15% KH.PO., 0.45% (NH.)SO., 0.35% peptone, 0.5% yeast extract, and 2% dextrose is inoculated with a loopful of the stock culture and incubated overnight ac 30C with shaking. The ia vitro yeast mitotic recombination assay in suspension is conducted as follows. The overnight culture is centrifuged and the cells are resuspended at a concentration of 10 cells/ml in 67 aM Pphosphace buffer (pH 7.4). To a sterile cesc cube are added: + 1.00 ml of the resuspended culture * 0.50 al of either the metabolic activation mixture or buffer + 0.20 al of the test chemical + 0.30 31 of buffer. Several doses of the test chemical are tested in each experiment, and appropriate controls are included. The suspension mixture {s incubated at 30C for 4 hours on a roller drum. The sample is then diluted serially fn sterile physiologic 6 000040 : saline, aad 0.2 al of the 103and 107 dilutions is spread on plates containing are spread the same wich the ingredients as the broth plus 2.0% 10~* dilution and three plates are agar; five places spread with the 107 by 2 dilution. days ac 4C The plates to enhance are the incubated for 2 days at 30C, followed development of the red pigment indicative aorfe asdceanninneed-dweifcihciaeadrishsoemcotziynggosimtiyc.roscPolpaeteast co10ntXaimnaignagiftihceat1i0o-n*, dianldutitohen mumber of mitotic recombinants (red The surviving fraction of organisms colonies or red sectors) is recorded. is deternined from the total mumber of colonies appearing on the plates of the 10? dilution. A The number of mitocic recombinants is calculated per 10 survivors. positive response in this assay is indicated by a dose-related increase moifllmiorleitetrhaans 3w-eflolldasiniathetheabsroellauttievenunmubmebrerofofmitmoittoitcicrerceocmobmibniannatnstspeprer 10 survivors. 7 000041 RESULTS AND DISCUSSION Compound T-2616CoC vas tested for mutagenicity in the Ames Salmonella/aicrosome assay and with the yeast Saccharomyces cerevisiae D3 in the presence and in the absence of a metabolic activation system. The compound was tested at least twice on separate days in both assays. The results are presented in Tables 1 through 5. In the Ames Salmonella/microsome assay, T-2816CoC was initially tested in a preliminary assay with strain TAL0D over a wide range of concentrations, from 10 to 5,000 ug/plate. Toxicity was observed at a dose of 5,000 ug/plate (Table 1). Ethanol was used as the solvent in all assays. The results of our tests of T-2816CoC with five strains of S. typhiBurius in the Anes Salmonellalmicrosome assay are presented in Tables 2 and 3. No toxicity or dose-related increase in the nusber of revertants was observed in these assays. The results of the microbiological assays with S. cerevisise D3 on T-2816CoC are presented in Tables 4 and 5. The compound was tested at concencracions from 0.05 to 5.03. No toxicity or significant doserelated increase in the number of mitotic recombinants above background was observed. We therefore conclude that Compound T-2816CoC vas mot mutagenic wich 5. typhimuriup or recombinogenic with S. cerevisiae D3. 8 000042 . g232& Table 1 10 VITBRaOpeArSiSnAeYncStowDWaIotTmeHo:SATL-1Y2D8N1F6EecLbeLrcAuaTryrP19H80RIN Compound ___ Heative Control Etat Posteive Controls 2-hnticantne Sotun tse Gonpound T-2816coc AMcettiavbaotliiocn ;- -5 : ::: `. v.: . AAmdoduentd opferCoBmaptouend i0o m 100 0.5 up 15010002'0.004 15005000000..000 5100.0.00 101s00000ll000 sio000 ReverWtaancttsd.isneer Plate pias nuei a102 ser 12in 15502 ont 1i6m 11n00o8s oxic { Tobe 2 IN VITRO ASScAoYrSeoWuInTDHTS-A2L8Y1O6NE0LcLA TrPDRION Expertaeat Dace: 20 February 1980 -- MAecttaiulnltitoen AAmdoduendt opferCoPmlaptoeund TAIT HIaAtSidIioe ReTveArtSants per oPlAate ion Negative Control Banal -M ++ s50o.001 550000 B BAooao mow owss oonw Bow om ww9 ooww8 8% owAwosoonw oimwp owasmn 6% we de Positive Controls 2-Aathraatne 5 +: 1100 2s a2 os 3oW nsWon owues 5ous SedumArtde : - hd 0.5 moa no 10s : 0 3 50 wus S-tntnoacetdiae, utroivorene, - 50.018 5.0m 0 19 0 40 doz am Cospound T-281608 -: z: 050.008 10500.00 B2moomAms17 3aom9 33 a6 9 s8oaw 1ow o11 3o3%wa oow mom 8 ws ow am 3 4 do :- 1.500000000 E 30EE6 5 1EO3 1 Bow dn asmp 3i8 2 :++ 551.00.000 1 1000 B2 Boo%oM0% 1weooznon 16 ou owwkoonnonaaBoWs5 alwmom oass 19 om 4 5 de aon 5 ++ 1050.0.00 o0no 6 on0 xoeow esoae u moa ns Tote 3 IH VITERxOpeArSiSacAcYoaStmWoDIaTtHesTS-A2L28W51O6NFEceLobLerAuaTrryeu19n8R0ION Compound Negative Control AHcetitvaabtoilolcn _Anmdoduendt opferPColmaptoeund A TRIE IMiAstiFdS ine Revertants perH Plate Tmoor Eehanol i: Postive Concrol sWloOMBNAL16 7 93 W auBNus w oe saoa ZAatheamtne :- 2L5owe now Bow 6d am se = ++ Sodtin Aside - 2L5o 9 16 1m ae 10 4 30 20 ee 10 034g : -Antraacetding - ve 00 am 2.048 37 506 wo 2Meroftuorene Compound Tabac - sou wow su aa 1S 00 OBwowM8 o was oy ow mow :: -: 105500000.1000 10000 B ZBAL oe eos DB wos wn5oonn5 ow koRsm oHaom doa mmHowNw oa Zo g82 ++ `` 15S 0000 OXD? BM ok wee90ows9%wBwooomumnm Goa umss ooaos min e1 ea s aa .` 11a050000:000 BZBwWoo9w wmn wwmooonum wbHooonun ajne yhdn usa "Retented on 6 Maxch; control values of 25 Februncy vere fnvalid. Te 6 IN VITERxOpAeScStAacYnSotmWmDIaTotHne:sTa-c21c031u6Pseeoabmcscuenrsy centvisiar 1980 Conpouns AHecttaubaotliloen Conpceerncternattion Gvorv/) GolsSuperrviavors G0) percent pMitoe Tterinc Rr acosbi inmnto e (10%) evi Negative Control Ethanol :< J Postetve control 6s3s aamm T0o n5.0 | 1.2.3.4-Dieporybucane :- 00.0022s5 wmue 1098m0 11340%0 5 Compound T-2816C0c ::: o0.r05 oj'=s] e33s 57 saom % 137560 21.6 2 z +: so 550 i"7 s7o0 ni 0o.r05 s 54 iaa 3.0 ss +++ oTssoo s 53 am55 52 " ou70 aho aa a1.0 3@ Gospound Netactve Control Ehanal -- 1,2,34-Diegorsbutane Compound T-2816cac Tables I VITERxOpeArSiSmAceYnoStPWoDIuaTmtHeD:sT-cz2o811n6Feecabcreuasry censvisia 1980 AMcettisvbaotliloen ConcPeenrtcernattion (uly or vi CETTESuprevrivtors 1107) recent t Mitotreriuncp Recosbni ssts (210) Servivers -: ssse wamw w0s fi5a s +- o0.z02s5 Ss10 ewwie Lm 1L330%0 -:: : o0o.sr05 To sCEeamam 116 2 m 7n1 : ++ 50 o0.r05 91 im 0 a0 25.00 i2 s2l.o0 u +i+ oTso so F sr e] sin S1lo to ry58 2we 233