Document GKgOdaKqjqgLVo3ZOZrBx6eLm

0 = 1In UpTmHoD IoCmROoBLIOeLtOGeIrCA,L WTITGAGRVICITY ASSATS FR sume 1585 O =C. OHfeetb th Oa obeory lce((t0068), - mek ay Sreparet fort SIFHediielnl Cone A DaTTSd cancer Atvesttons fSanotarst%.eGleason EE.N\ La 3M 2xic8\> -- tepzanet 174 oe Gitasen 5JOwnnseboioynatm,ia,nSaSotcodsy Dizectos nLoesent,avpeirraescotnory 3. A Setamer, Vice Teeaident LTT ESBY <Z% aeonsMorena, 001211 STORY SRI International examined 34 Company's Compound T-3752 for mutagenic activity in the standard Anes Salmonella/microsome assay with strains TAIS35, TALS37, TALS38, TA9S, eyphinuriun. Compound T-3752 and was TALOD of the bacterius Salmonella elso screened for recombinsgenic activ Lty in the yeast Saccharomyces cerevisise D3 assay. Both assays vere perforaed in eystea. ALL the presence and sbsence of a rat-liver tests vere performed in compliance with metabolic activation the United States Pood and Drug Adatnistration (FDA) Good Laboratory Practice Standards. Compound T-3752 was reproducibly nommutagenic and nonrecombinogenic when tested according to these procedures. fey 0iliz CONTENTS QUALITY ASSURANCE STATEMENT....vvsessesessssnnsnns 1V INTRODUCTION.+svssssesesssssnnssnssssssnsssssasnen 1 METHODS. eeeeeerrnnnnsennanesssssssssssnnssannsans 5 RESULTS AND DISCUSSION.eesssssssssesssssessessees 12 TABLES Table Leverssnrsrerreeeesssssssnnnnnnneaaaes 13 Table 2eeeeeeneesrrennnneeesessssssininnnens 10 Table Juurrrrrrrereesenssssssinniesineiiaaies 15 Table eeerenrrrrrrreesessssssnnnnesaesaaaee 16 11 01213 QUALITY ASSURANCE UNIT Final Report Statement SRI International assures the quality and integrity of this study, In Vitro Microbiological Mutagenecity Assays Of Compound T-3752, for the 3H Company. Inspections and audits were performed during different phases of the study, and the Study Director and SRI management were notified of the findings of the Quality Assurance Unit. This report accurately describes the methods and standard operating procedures of the study. Any deviations from the approved protocol were made with the proper authorization and documentation. Any deviations fron standard operating procedures were docunented. 30 so he quatly Adsurance --_-- 1v c0i2ia INTRODUCTION SRI International examined 3M Company's Compound T-3752 for muta~ gentcity in the standard Ames Salmonella/nicroscme assay with strains 7A1535, TALS37, TALS38, TASS, and TALOD of the bacterium Salmonella typhiaurtum. Compound T-3752 vas also tested for recombinogenic activity In the yeast Saccharomyces cerevisiae D3 assay. An Aroclor 1254= stimulated, rat-liver homogenate metabolic activation systes vas included in the assay procedures to provide metabolic steps that the microorganisas either are incapable of conducting or do mot carry out under the assay conditions. The assay procedure with S. typhimurium has proven to be 80 to 90% reliable in detecting carcinogens as mutagens, and it has about the same reliability in identifying chemicals that are not carcinogenic. The assay procedure with S. cerevisise is about 60% reliable in detecting carcino= gens as agents that increase mitotic recosbination. Hovever, because the assay systems do not alvays provide 100% correlation with carcinogenicity vestigations in animals, neither a positive nor a negative response conclusively proves that a chemical 1s carcinogenic or noncarcinogenic to man. Evaluation of experimental results from the Salmonella assay consists of comparing the number of histidine-independent colonies on the treated agar plates vith the number observed on the control plates. Because sll the plated Salmonella indicator organisns undergo a few cell divisions in the presence of the test chemical, the test is semiquantitative in mature. The plate test procedure does not permit quantitative determination of the umber of cells surviving the chemical treatnent. It is the demonstration of a nutagenic dose-response relationship that is important in establishing mutagenicity. 1 01215 The test chemicals are assayed at several dose levels within a mou- toxic dose range----vith the exception of the highest dose level, which sometimes exhibits toxicity. Toxicity is evidenced by several phenomena: clearing of the background bacterial lawn growth, formation of pinpolat colonies consisting of surviving cells, and a decrease in the number of revertant colonies below the spontaneous background. A chemical 1s considered a mutagen in the Salmonella assay if it elicits a reproducible, dose-related increase in the number of histidine revertants per plate in one or more tester strains. The yeast Saccharomyces cerevisiae D3 is a eukaryotic atcroorganisa capable of detecting mitotic recombination, as expressed through a mutation leading to a defective enzyme in the adenine-metabolizing pathvay, result ing ivneraalredc-opnicgeanternatteidoncsoloonfy.theIntestthischaesasiacya,l, tuhesuaylealsytrcaenlglisngarferomexpaosed to se concentration that results in mo killing to one that causes 50% killing. Any concentration that induces 90% killing is considered toxic. When the ausber of genetically altered colonies per milliliter (yield) and the ratio of altered colonies to survivors (frequency) from the treated cells are unequivocally larger than those of the solvent-treated controls, we com~ clude that the exposure of the cells to the compound induces mitotic recom bination. If this event is dose-related, the observation is termed a positive response. The assays vith Compound T-3752 were begua on 24 May 1985 and testing vas completed on 14 June 1985. Copies of the final report will be kept in our files (Building , Room 213) and in SRI's Records Center. The raw data "ill be retained in Building 205, Room 13, for one year after the labora~ cory notebook has been filled and then will be stored in SKI's Records Center. ALL that remains of Compound T-3752 will be kept for six months in our chemical storage room (Building M, Room 217) and then returned to 3M Company. 2 0iR2i6 MATERIALS . o Zest Aretcle - mame: 1-372 - Date Rocetved: 15 Nay 1985 - Description: Asher waxy solid - Storage Conditions: sSteocroenddarayt croonotmaitneemrperature in a - Spectal Testing Condtetons: Nome - Stabilicy: Assured by Sponsor Indicator Organisms ~ Species: Salmonells typhimurium UT2; Saccharomyces cerevisiae - Stratus: $T+AStSyp,hiTmAuLrSi3u7m, TDA3LSfIoSr,S.TAScSe,revaindsiaTeALOO for - Source: Dtrh.e BSraulcaegoeAlmleas;, DUrn.iveF.rsiXKt.yZoifsseCraalsiufao,rniWa., GBeerrakmeyl,ey,forfor the Ssccharoayces Aroclor 1254-tnduced, rat liver S-9; SRI Batch F-5; ~ 31.5 Antal mg/ml protein Supplier: Simonsen Laboratories, Gilroy, California o Negative (Solvent/Diluent) Control Materisl DDaicaeethOyplenoeudl:foxi3deand(DH8SOM)a,y Expiracion Dates 3 and 8 aCnAdS Hay 3Bo.Jun6e7-6189=855 and 3 June 1986, respectively Manufacturer: American Scientific Products, McGraw Park, IL Purity: 0,122-0 (for all) Lot No.: 4948 KY (for all) 3 01217  Positive Control Chemicals 9Ma-nAunftancotaucrreird:ineP,falCtAzS Naon.d B%a0u-e4r5,-9Stamford, CT - 2-Anthranine, Manufacturer! CSAiSgaaNo.Che6m1i3c-a1l3-8Co., St. Louis, MO M2a-nNuiftarcotfulrueorr:eneA,ldrCiAcShNCo.hea6t0c7a-l57-Co8., Milvaukee, WI Sodium azide, Manufacturer: CDiAfScoNo.Lab2o6r6a2t8o-r2i2e-s8, Detroit, MI 1,M2a:n3u,fa4c-tDuireepro:xyPbfuatlatnze,andCASBauNeor., 1S4t6a4m-f5o3r-d5, CT MSatneurfiagcntautroecry:atiAnl,driCcAhS NCoh.emi1c0a0l48C-o1.3,-2Milwaukee, WI Counters Used = NCeowuntBerru,nsMwoidceklScC1i1e1n,tifSiKcI NBoiso.Tra0n03011861A26ut0o0m,ate0d030Col01o5n1y 00, and o012 3318 00 - NC1e1w0,BruSnRIswiNocsk. S0c0i3e0nti1f47i1c B00a,ct0r0o1n2ic310C8ol0o0n,y Caonudnt0e0r1,3 0M7o8d8el00 4 0iRis ETHODS SRalIsoTnellTATsE, typhiTma0u8,riumaSdtraTiAnTsEDTALS3S, The Salmonella typhimurium strains used at SKI ore all histidine auxotzophs by virtue of mutations in the histidine operon. When these Met1dine-dependent cells are grown on minimal medium agar plates con taining a trace of histidine, only those cells that revert to histidine independence (his*) are able to form colonies. The small smount of his= t1dine allovs all the plated bacteria to undergo a fev divisions; in many cases, this growth is essential for mutagenesis to occur. The his* revertants 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 tncreased, usually in a dose-related manner. We obtained our S. typhiaurim strains from Dr. Bruce Ames of the University of California at Berkeley. In addition to having mutations fn the hatidine operon, all the indicator strains have a mutation (fa) that Leads to a defective Lipopolysaccharide coat; they also have a deletion ehat covers genes involved in the synthesis of the vitamin biotin (blo) and in the repair of ultraviolet (uv)-induced DNA damage (uvrB). The rfa mutation makes the strains more persesble to many large molecules, thereby increasing the mutagenic effect of these molecules. The wrB mutation renders the bacteria unable to use the accurate excision repair mechanisn to remove certatn chemically or physically induced DNA lestons and thereby enhances the strains' sensitivity to io reverted to hs by any mutagens some that mutagenic agents. Strain TA1535 cause base-pair substitutions. Strain TAL0O 1s derived fram TALS3S by the introduction of the resistance transfer factor, plasaid pRM10L, This plasmid is believed to cause an increase tn error-prone DNA repair that leads to many more mutations for a given dose of most mutagens. Tn addition, plasaid pRILOL confers Tesis~ tance to the antibiotic smpicillin, which is a convenient marker to detect 5 0i219 the presence of the plasmid in the cell. The presence of this plasaid aloo makes strain TALOO sensitive to some frameshift mutagens [e.g., ICR= 191, benzo(a)pyrene, aflatoxin By, and 7,12-diaethylbenz(a)anthracene.] Strains TALS? and TAISIB are reverted by many frameshift mutagens. Strata TA% 1s derived from TALSIS by the addition of the plasmid pREIOL, Which makes it more sensitive to some mutagenic agents. ALL indicator strains are kept frozen in nutrient broth supplemented with 10% seerile glycerol at -80C fn 1-ul aliquots containing about 10 cells. New frozen stock cultures are made every three months from single colony 1solates that have been checked for their gemotypic characteristics (his, rfa, wrB, bio) md for the presence of the plasaid. For each expertneat, the frozen 1-al cell cultures are alloved to thaw at room temperature before {noculation in 50 ul of glucose minimal liquid mediu supplemented with am excess of biotin and histidine. The cultures are grown at 37C, unshaken for 4 hours, then geatly shaken (100 rpm) for 11 to 14 hours. ALL strains are genetically analyzed whenever experiments are perforaed. Aroclor 1254-Stinulated Metabolic Activation Systen Some carciogente chemicals (e.g, of the aromatic amine type or the polycyclic hydrocarbon type) are inactive unless they are metabolized to active forns. In snissls and man, an enzyme systea in the liver or other organs (e.g., lung or kidney) fs capable of metabolizing a large number of these chemicals to carcinogens. Some of these intermediate metabolites are very potent mutsgens in the 5. typhiauriwm test. Anes has described the liver metabolic activation system that we we. In brief, adult male Sprague-Davley rats (200 to 250 ) are given a single S00-mg/kg intraperitoneal injection of Aroclor 125 (a mixture of polychlorinated biphenyls). This treatmeat enhances the synthesis of enzymes involved ia the metabolic conversion of chemicals. Four days after the infection, the animals' food 1s removed but drinking vater 1s provided ad libitum. On the fifth day, the rats are killed md the liver homogenate is prepared as follows. 6 01220 The livers are removed aseptically and placed in a preweighed, stertle glass beaker. The orgen weight is deterained, and all subsequent operations are conducted dn an ice bath. The livers are vashed with an equal volume of cold, sterile 0.15 KCl, minced with sterile surgicel scissors fn three volumes of 0.15 H KCl (3 ml/g of wet organ), and homogenized with a Potter-Elvehjea apparatus. The homogenate is cem trifuged for 10 minutes at 9000 x g, and the supernatant, referred to as the 5-9 fraction, is quickly frozen on dry ice and stored at -80C. The metabolic sctivation mixture for each experiment consists of, for 50 al: 5.0 ul of 5-9 fraction 1.0 al of MgCl, (0.4 3) and KCL (1.65 ) 0.25 al of glucose-6-phosphate (1 ) 2.0 ul of NDP (0.1K) 25.0 ul of sodium phosphate buffer (0.2 M, PE 7.4) 16.75 ul of sterile HO. The amount of -9 fraction delivered to each plate is 50 kl. Plate Incorporation Assay Prior to testing, the test article is serially diluted fro an ini ttal stock. Generally, a prelintnary experiment is conducted to find a suitable dose Tange for testing. The article is usually tested over a ania of six dose levels, the highest nontoxic dose level being 10 mg/ Plate unless solubility, mutagenicity, or toxicity dictates a lover upper att. When extracts are made, various undiluted aliquots are tested, usually over a dose range of 5 to 100 or 200 yl/plate. When liquids are tested, occasionally the sample Ls not diluted and various aliquots are used. ALL sssays are repeated at least once on a separate day. The plate incorporation assay is performed in the following vay. To a sterile 13 x 100m test tube placed in a 43C heating block ve add: 7 C0i221 (1) 2an.d000.m0l5 oaf 0.h6i%stiadgianrecontaining 0.6% NaCl, 0.05 m biotin, (2) 0.05 nl of indicator organisms (about 10 bacteris) (3) (4) 0.05 al of a solution of the test article 0.50 al of metabolic activation mixture (Af appropriate). This atxture Ls stirred gently and then poured on plates containing sbout 25 al of ainimal glucose sgar. After the top ager has set, the plates are incubated for 48 hours at 37C. The number of his* revertant colonies is counted using a BloTran II automated colony counter when possible. When accurate counts canot be obtained (e.g., because of precipitate), the plates are counted manvally using an electric probe colony counter. Concurrent sterility, negative (solvent/diluent), and positive con- trols are run with every experiment. Sterility controls include plating out separately steps (3) and (4). For negative controls, ve use steps (1), 2, (4), and 0.05 a1 of the solvent/diluent used for the test article, if appropriate. culture using steps (1), For (2), positive (3), and controls, we (4) vith the test each following bacterial mutagens: SanoddiuAm10a0zide for the base-pair substitution mutants TALSIS o S-Amtnoscridine for the frameshift mutant TALS 2-NMitrofluorens for the frameshift muteacs TALSI and TASS m2e-tAanbtohlriacatnaectifvoartiaolnl. tester strains, in the presence of We routinely check for true revertant (bis) colontes by replica Plating fron the parent to minimal glucose agar plates supplemented vith blotta. Criterta for Interpretation Positive. A test erticle is considered a mutagen when it produces a reproducible, dose-related increase in the number of revertants in one of more strains. This increase should occur for at least three dose levels. Negative. A test article is considered a nommutagen when mo doserelated increase in the number of revertants is observed in at least two independent experinents. The maximum dose level tested for nontoxic 5 0122 compounds 1s 10 mg/plate (unless dictated otherwise by the sponsor or by solubility problems). For toxic compounds, only the highest dose level tested should show evidence of toxicity. Inconclusive. When a test article cannot be identified clearly as a autagen or mommutagen in the standard plate assay, the results are Classified as inconclusive. Saccharomyces cerevisiae D3 The yeast S. cerevisiae D3 is a diploid microorganism heterozygous for a mutation leading to a defective enyme in the adenine-metabolizing pathway. When grown on medium containing adenine, cells homozygous for this mutation produce a red pigaeat. These homozygous mutants can be generated fron the heterozygotes by mitotic recombination. The frequency of this Tecombinational event may be increased by incubating the organisms vith various carcinogenic or recombinogenic agents. The recoabinogentc activity of a cospound or its metabolite is deterained from the nusber of red-pigaented colonies appearing on test plates. A culture of 5. cerevisiae 1s stored at 4C. For each experiment, broth containing 0.05% MgS0,, 0.15%KH 0,, 0.45% (NE,)S0,, 0.35% peptone, 0.5% yeast extract, and 2% dextrose 1s fnoculated with a loopful of the stock culture and incubated overnight at 30C, with shaking. The invitro yeast mitotic recombination assay in suspension is con ducted as follows. The overnight cultufe is centrifuged and the cells are resuspended at a concentration of 10% celle/al in 67 mM phosphate buffer (pH 7.4). To a sterile test tube are added: 1.0 nl of the resuspended culture 0.5 ml of either the metabolic activation mixture or buffer 0.2 ul of the test cheatcal 0.3 al of buffer. Several doses of the test chemical are tested (up to 5% w/v or v/v) in each experiment, and appropriate solveat/diluent controls are included. 1,2:3,4-Diepoxybutane without metabolic activation and sterigaatocystin vith activation are used as positive controls. 9 ; COL223 The suspension mixture ls incubated at 30C for 4 hours on a roller drum. The sample is then diluted serially fn sterile physiologic saline, and 0.2 ml of the 1075 and 107 dilutions Ls spread on plates containing the same ingredients as the broth plus 2.0% agar; five plates are spread With the 1073 dilution and three plates are spread vith the 1075 dilution. The plates are incubated for 3 days at 30C, followed by at least 1 day at 4C to enhance the development of the red pigment indicative of adentne-defictent homozygosity. Plates containing the 103 dilution are scanned vith a dissecting microscope at 10x magnification, and the number of mitotic recombinants (red colonies or red sectors) is recorded. The surviving fraction of organisms is deteratned from the total number of colonies appearing on the plates of the 1075 dilution. Criteria for Interpretation Positive. A positive response fn this assay is indicated by a doserelated increase of more than threefold in the absolute mumber of mitotic recombinants per aflliliter and in the relative number of mitotic recombinants per 10% survivors. Negative. When mw reproducible recombinogentc activity is obtained in any of the assays pecforaed, the test results are considered to be negative. Inconclusive. When a test article cannot be identified clearly as causing a positive or a negative response, the results are classified ss taconclustve., Statistical Analysts No statistical analysis io perforaed for any of the assays. The results of the plate incorporation asssy are a tabulation of the nusber of colonies appearing on the plates. The results of the S. cerevisiae D3 assay are tabulated after calculating the number of mitotic recombinants per 105 survivors. All calculations are expressed with two significant dite. 10 coi224 References AansesF,raBm.eshNi.,ftEm.utGa.geGnusr:ney,MetJ.aboAlitMeisllearn,d daenrdivH.atiBvaerstscofh.2-Caacrectiynloagmeinnso UFSlAuo6r9e,ne31a2nd8-3o1t3h2er(s1r97o2m)a.tic mine carcinogens. Proc. Natl. Acad. Sci. mAunteasg,enBs.:K.,A Ws.inEp.leDutressttons,ystE.emYacmoamsbaiknii,nganldiveP.r Dh.omoLgeeen.ateCsarcfiornogaecntsivaarteion aansdm)ba.cteria for detection. Proc. Natl. Acad. Sci. USA10, 2281-2285 asyesst,emB.forN.,theF.deD.tecLteei,onanadndW.clEa.ssDiufrisctaotni.on oAnf miumtpargoevnesd abnadctcearricailnotgeesnts. Proc. Natl. Acad. Sci.USA 10, 782-786 (1973). Agenness', anB.d Nm.u,tagJ.enMscCwaintnh,thaeodSEe.lmYoanmealslaak/im.ammMaeltihaond-snicfrorosodmeetemctuitnaggenciacrictiynotest. Mutat. Res.31, 347-365(1975). Birnugsitcekc,hniD.queJs.,wiatnhd Vy.easWt..MayEeurv.ironN.ewHedaelvtehloPpemresnptescti.n m,uta83g-e8n6ici(t1y973s)c.reen- Kiinerc,igaL.retDt.,e Es.mokYeomcasosnkdie,nsaatneds.B. NP.rocA.nesN.atl.DetAceacdt.ionSeio.f mUuStAa7g1e,nic415a9c-t4i1v6i3ty sre). MgceCnasnna,s mJu.,tagE.ensChoiin,thE.e YSesmlamsoankeil,laa/nndicrB.osoNu.eAmteess.t: DeAtsseacytioofn o30f0cacrhceimnoicals. Proc. Natl. Acad. Sci. USA J2, 5135-5139 (1975). MScaClannonn,elJl.a,/miancdrosB.omNe. tAemste:s. AsDseatyecotfion300ofcchaermciicanlosg:ensDiasscumsustiaogn.ensPrionc.the Tatl. Acad. Sci.USA73, 950-95 (1976). : Mporrotpeelrataynso,f pK.lasE.m,idaRn4d6B.fAr.oDm. itSstocukletrr.avioSleegtr-epgraottieocntionfgtphreompeurttayt.or Mol. Gen. Genet. 167, 317-327 (1979). Zwiimtmhernmiatnrno,usF.aciKd.,, la-ndmetR.hylS-cdh-vnaiietrr.o-l-Innidturcotsioognuaonfidmiinteotiacndgeontheerconavlekryslaito-n isnegna)g.ents in Saccharomyces cerevisime. Hol. Gen. Genet. 100, 63-76 1 oi22s RESULTS AND DISCUSSION 3 Cospany's Compound T-3752 vas screened for mutagenic activity in the Anes Salmonella/microsose in vitro mutagenicity assay using the five standard strains of Salmonella typhimurtus: TALS3S, TALSIY, TALS3S, TASE, and TAL00. The assays were perforaed in duplicate, using three plates per dose, both in the presence and absence of & rat-liver metabolic activation oysten. DUSO vas used as the solvent. The microbial mutagenicity testing of this sample vas performed tuice at dose levels ranging from 10 to 5000 jg/plate (Tables 1 and 2). o increases tn the nusber of revertant colonies over the spontaneous background vere observed under any of the assay conditions used. Background Lawn thinning vas observed in the first assay only vith strain TALSI? on aJl8l/polfatethewitphloautets acattiv5a0t0i0onp.g/pAlatleighatndpronecitpwoitaotfethveasthnroeteedplatate5s000at 1000 J6/plate; therefore, these plates vere hand-counted. The results sre presented in Tables 1 and 2. Compound T-3752 was also screened for Tecombinogentc activity in the yeast Saccharomyces cerevisiae D3 assay for mitotic recosbination. The assays vere perforned twice on separate days, both in the presence and absence of a rat-liver metabolic activation system. DSO was used as the solvent. Since no toxicity vas seen in the range-finding assay, the first experinent vas performed at dose levels of 0.05, 0.1, 0.5, 1.0 and 5.0% T(hwe/v)c.onfWiormaitnocrryeasaesssayinwatshepemrufmboerrmedofunmdietroticconrdietcioomnbsinaindtesntiwcearle observed. to those used tn the initial assay. Agata, no recombinogenic response vas observed. The results of these two experiveats are presented in Tables 3 and 4. nonrecTnoabcionnocgleunsiiocn,wheCnomptoeusntdedT-a3c7c5o2rdiwnags troeptrhoeducpirobcleydunroensmutoaugtelinniecd anfdn this report. =" 01226 Table 1 IN VITRO ASSAYS WITH SALNOVELIA TYPDORIDN conpomp 7-3752 Bxpertnent Date: 28 Hay 1985 Cospound Ne etaboe ltc pCeAodmrpdoemudnad TIDE EI tsrsdine ReRvISEertantspor Plate mo Negastoi.ve Control i- s3ow 1P9R2;ES6 s7 w3 nmm ow29 o15w 1o9 w26nu38 mWI 1m04108 Posies Controls SSomdttmuomscasetidetne -- El1g 972200 36 us 165 100 5 ITuhiaucohfleueomrienes =i- 1s1 162810661032239 12824 08 i- Z5s ammawr uaonn msooau Compound T3752 -z - 0W% a wo L 1w716m2S0os 3 B 37 686 fs2113m18 om12 <- 10%00 000% LSUs M1 ak Las oaat st 62a7 7h oet BWmo as U1W5ooam mo 135 1++ wSoo Wo m 1M2 s o 8 3w6bsaz5 o67s 22%3% 3ow 3 18 % << i1+ W5Soo,000s11301360383 4 757 W3 86 21 33; st oot a 3s 1; oz |8NJT{-- opaapcekcgir-- aoesnstd plae-- wrnfortmheidnnionng.16 June 1985. Frrecipitated at thin dase level; hand-counted. 302 3m 36 61189 8833 0348 97 109 358 23 228013s0 2w0 1w19 wuwzwm uBmayN W mLk 1a9 u1 1162 1106018189 43 %nasow esuwle n 23% 23 %22 ww uEoIsRes8%% 433% mow mez 10 vim assavscownirmonsa7O-W37E5L2LA TrPRDORIOY Rxpectaent Dates 6 June 1985 congo Wteitatboileten peCro"mdpfnolduancde THT ET uactdtae Reverzanes pe Plate [I 9) -- -: sDFow nBwUaD S3 NS3 01 1od5o1 waueo enewum Rontetve Controls sSoidattummonacsriidseine - PF1 se ses a 456.307 330 wo ess 636 x Dhaaitrheeafniienerme :: i53 m5o180w2z6 e1B 0s0 1s0S 0 0136 3o0s6 3% 311 :: IIs omaoowmoaw p88o6n Compound 1-5752 :-:: w0oo% w Go G5BaaRs% 1JE 9ohmABWoByUoBmoou oaon3 o2 muwmsewo Gan $s 8 db Mow ml wm :: wSoot NBoaAn a6u 870B ow7 Bod om13 daooam ouumaemem .ii WISwoo tw Gomw lld oamsoodTrUb 1B%ou1b6 o%31% 24 25 2osp29 Unwakemiese 2e :3: wMGoeooet dGaadsMseds2 wB2 uu7nl6 B7MN om3% %Wboomoonon;a miiome 100 & 8 TTohcEpCueciipnictnatredpersttortsheiss dooase16Tdeuvenles 1h9a85n.i-counted. ote 3 IN VITO ASSAYS ITA SACCIAROHCES CERBVISTAY D3 conromn 13752 Expectacnt Dace: 26 Yay 1985 Compousd Npeetaibvoatliiocn _ComPe(ernuciver)nattion CeSlulrsvivpiencgsl 28 Negative Control miso i- 72.3 Postetve Controls 5 1,2b0u3t,an4e-01epoxySteripmtocyattn -- o.00a0s5 o1n + 0.003 na Compound T9752 -z o0.F05 - ois 7721 :: T50o 7Te ++i 0o.r05 os 7ot8 72 o 1+ 1s0o 5p0e &ETT calculacions re expressed to two stgateicant figures. 81] Survi0)vors 110000 1F05a0 7F 55 10 1080 1050 100 Renceoomtbticnants Epleroal wnss o2n02 75 i5 57 .57 Rceocorormbt1l0ea5sate _stevivers' 1062 1w9o0'2.9 1B0as 1as Ww5.0 n10 0 Table & IN VITRO ASSAYS WITH SACCURONICES CERRVISIAE D3 comm T3752 Bxpectaeic Date: 7 June 1985 Compound Metabolic ComtPeermcternattion CeSlulesvivpianrgal Survivors Reccopomerrbitnaemlnts RMepcoororbit1n0ega,ts pctiwacion _(ul%) OE a Eloth _shrvivers' Negative Conerol so +- Poatesve Controls 5 1,b2u3t,an&e-DteponyStertpmatocyattn -- 00.00s25 + 0.003 67s3 11000 10s 1n 7733 11000 15125 20i 3 100 ae = Compound 1-752 --- oo0i.rs05 -: 1so0 767.s05 1059%0% 675s 190 5s "o1n 15. 1ax73 +i+ [0ox.r05 77726 1011000 75 138523 2g i+ s1o0 7i 110%0 in 1is TRE clculation are expressed to te signiticane furs.