Document 7OzrnZBB7p5Gr1gdBOpp3yk7g

AR R26 1197 FINAL REPORT PMUIRCIRFOISEDO8M-E2 ARLECVOEHROSLE:MUSTALAMTOINOENLALSAS-AEYSCWHIETRHIACHCIOANCFOILRIMMAATMOMRAYLAISASNAY- COVANCE STUDY 22900-0-4090ECD STUDY SCHEDULE: Study Initiation Date: 23 October 2001 - Initial Dose Date: Study Termination Date: Final Report Date: 29 October 2001 10 December 2001 11 February 2002 eg -on a= 388 z 35 STUDY DIRECTOR, SPONSOR. AND TESTING FACILITY STetsutdiyngDiFraeccitloirty:: CLoevoannFc.eStLaabnokroawtsokrii,esJ,r,IncP.hD(Covance) 9Vi2e0n0naL,eeVsAbur2g21P8i2ke s5 OO e Sponsor: Telomer Research Program Consortium a1d7m0i0niMsatienreSdtrbeyetRAND Corp. Santa Monica, California 90407 "This study was conducted in accordance with Covance Standard Operating Procedures; the `Organisation for Economic Cooperation and Development (OECD) Principles of Good ELnavbiorraotnomreyntParlactPircoet,ecEtNioVn/AMgCe/nCcHyE(ME(P9A8-)TS1C7Aa)d,oTpitleed N40ovoeftmhbeerU2S6,Co1d9e9o7;faFneddetrhael Regulations Part 792, issued November 29, 1983 (effective December 29, 1983[revision effective September 18, 1989). `NpTeohrimspiadsrotsciooufnmoietfnmttaheiys tbTheeeltocromannefsrimdiRetentstecidaa,lrrcpehrporPporedorugtcyreado,mfCptuohbenlsTioesrhlteoidum,me.rorReusseedarbcyhoPtrhoergrpaemrsCoonnsswoirtthiouum!. the 000030 ee CPuorviafniecde 8S-t2uAdlyc2oh2o9l00-0-4090ECD 2 TABLE OF CONTENTS PAGE LIST OFTABLES ccc LIST OFAPPENDICES csv SUMMARY .c.crmmmsssssssssmssssmssmssssessssssssssmmsssssssmnmon| 1 OBJECTIVE css 2 21 MATaERIALS ccna.---- 221112 SB HOEOFTEe SSIIcc. ossne-- oeo 10 212.1 2122 FMrIoSzeEnPPRerEmSanc ent SoOCKrS .s...e.....owsmoosrsoosnoesreseeossnoene 1100 213 2131 PTrReOpCaUraItBiOoNnof Overnight CURIE e .....n .....ovoe vosesnsr orornrrror 10 10 2210432 HCoAnVfiErSmationofoTeesttoeireSnIeainnoPmHsEoNsOoLoYsPnESs.o.n.e.s.o.soreneoenonor crore 1110 ZEAL 2142 fPaKMWIaOllLPMIUSOA RACce cr...sses 1]11 2143 215 TCehsatreacrtSertiasitnicMeNdiaumofS..bp...o...n..t.e.ancrous Rieverstanits..s...m....n...m....m...m...o--.. --11 22015512 CMuilntiumrainlgBBOrUoOtMhA.G..A PIAES...........covososonrosrenaensnnmn m-- 12 2153 TopAgarfor Selection OfREVETaNS......oosmrrerr oo 12. 23 223312 2323321 22333312 CVeOhiMclOe CAonTtrCoCs..... cresress-------- 1135 eC SStoeurirlcietyaCnodntGolsr...o.af..P.Od.Siieve COMTONnATtEsSe..m.------ 14 TSEOSUMATCICcose anem-- mmoso-- onsoo-- sneoe-- eoeoe---- e 1 -- 000031 PCuorviafnicede 8S-t2uAdlyc2oh2o9l00-0-4090ECD 3 22441 242 Li$v9eHrOMMOiGcErDoRsIoEmav l Enzyme Reactis on Mixture (59 Me ix). 1153 SOME eons 15 33.1 L EXPERIMENTAL DESIGN c E rn 15 301 a ----T 330132 RaEtviaolnuaaltei.on ofthe eDroesse Rangefrinrding Sturderye... wescone eroe or 1166 33.214 MSeUlAecGtEiRonICofHYthAeSMSaAxYic mum Doseo for the Mur tagenicitym Assay.....s .......... 1176 332212 DFerseigqnu.e..n.c.y...and RutecrrcOrfmsAonAs IRISII--ON....ss.s....vrv..oorm 1177 334a1 BCaOcMteIrNiGaltBHaecPkLgIrEoSundrLarwn ErVARON.. o. 1188 342 Counting REVEHAnt CONES... 18 a421 ADSASLAYPARCCEESPEIANNTCAE v CHETwr rs e 1198 A2Z1LL TfCaSWEaTllSUMA UIOEGNTIYc oo oo or 1199 4212 pRMIOUPSIA ooo 19 42.13 Characteristic Number of Spontaneous Revertants....... nssssesanan 19 422 `Tester Strain Culture Density .. esses nreesana ---- 444222333.21 PPPooosssiihtiiivvveee CCoonnttrrooll Control VVaalluueess.. Values .inthe inthe Ab-- senceof Presenceof $S99 Meriasexs Mix ($9 Mix--Irn-- tegro-- ity)-- .r.. --.2200 44324 CASYSOaOYNIECXGHNYISUOR CFT rrrnee 2211 443312 TTeesstteerrSStrraainnss TTAASISS,3STA0L0O 0d WETAI. STA +o 221] 5. RECORDS TO BE MAINTAINED ccvernnmnnnn 2 6 STUDY RESPONSIBILITIES... 22 000032 Purified 8-2 Alcohol Covance Study 22900-0-4090ECD a 71 72 TDEoSsEeARTaUnCgIeEfHinAdNiAnHgNSGtuo dy (Appendix Ac , Tables 4 and 5r ) o.oo 2223. 73 MTaubtlaegsen7i,c9i-tySUAMsMsaAyL(YADppAeAn)dc ix A, Tar bles 6,a 8 - Indn ividuae l Data,s 23 8. CONCLUSIONS wos 24 9 PROTOCOL DEVIATIONS comms 24 10. LIST OF REFERENCEScosmos 25 000033 Purified 8-2 Alcohol Covance Study 22900-0-4090ECD 5 Table 1. Table2. Table 3. Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 LI OFTS ABLTES PAGE TeSter SHAN GENOYPES...vevrrrcmrsrssmsmsmss POSIiVE COMIOLS vss 14 S9 MIXCOMPONENLS..orrrrsmsmsss 15 Dose Rangefinding St~uTdALOyD.........cocouvrssonrsenn 27 Dose RangefStuidyn=dWPi2UnVPgA. cvs 28 Initial MutaAgsseayRnesuiltsc-Iinditvidyual Plate Counts.................29 Initial Mutagenicity Assay Res-uSUlMMtATsY orn 30. Confirmatory Mutagenicity Assay Results- Individual Plate Counts....... 31 Confirmatory Mutagenicity Assay Res-uSUlMItMAsIY ...... 32 Historical Control Data... fmt 38 000034 Purified 8-2 Alcohol Covance Study 22900-0-4090ECD 6 LIST OF APPENDICES PAGE AppendiAx. EXPErimental Data Tables .......cocvccvevrereronrmsmsmsnsnson 26 Appendix B. DefinitionsofBacterial Background Lawn Evaluation Codes .............. 34 AppendiCx.. QualityAssurance and Compliance SIaIements...................o.wrnron 36 -- 000035 PCuorviafniecde8S-t2uAdlyc2oh2o9l00-0-4090ECD 7 SUMMARY Objective: The objectiveofthis study was to evaluate the abilityofPurified 8-2 Alcohol to induce reverse mutations at the histidine locusof selected Salmonella typhimurium strains, or the tryptophan locusofEscherichia coli strain WP2uvrA, in the presence and absenceofan exogenous mammalian metabolic activation system (S9). Study Design and Parameters: Purified 8-2 Alcohol was tested in the plate incorporation reverse mutation assay using S. typhimurium strains TAOS, TA100, TAIS3S and TAIS37, and E.coli strain WP2uvrA, with and withouat metabolic activation system ($9) containing induced hepatic microsomal enzymes from AroclorTM 1254-treated rat, Initial and confirmatory mutagenicity assays were conducted. Each assay included vehicle and positive controls, and six dose levelsofPurified 8-2 Alcohol, with and without metabolic activation. DosesofPurified 8-2 Alcohol evaluated, based on theresultsof a dose rangefinding assay and selected in conjunction with the Sponsor, were 33.3, 100, 333, 1000, 3330 and 5000 g/plate with and without S9. Each test and control article dose was evaluated in triplicate plates in cach strain. The dimethylsulfoxide (DMSO) vehicle control, as well as the test and positive control articles were administered in a volume of 50 uL. Results: No toxicity was observed in any teste stain i the presence or absence of $9 up to the maximum dose testedof 5000 kg/plate. The mean numberofrevertants per plate did not meet the criteria for a positive response in any tester strain with or without $9. All criteria for a valid study including appropriate vehicle and positive control responses (with the exception ofpositive control values for TALS37 without 9 in Experiment 22900-Cl, see Protocol Deviation), were fulfilled. Conclusion: Purified 8-2 Alcohol was not mutagenic in this test system. 000035 Purified 8-2 Alcohol Covance Study 22900-0-4090ECD 8 1 OBJECTIVE, `The objectiveofthis study was to evaluate the test article for the ability to induce reverse mutations at the histidine locus in several strainsof Salmonella typhimurium (TA98, TA100, TALS35 and TA1537), and at the tryptophan locus in Escherichia col tester strain WP2uvrA, in the presence or absenceofan exogenous metabolic activation system (S9). The assay design was based on OECD Guideline 471, updated and adopted July 21, 1997. 2 MATERIALS 21 Tester Strains, `The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, `TAIS3S and TAIS37 (Ames et al, 1975) and the Escherichia coli tryptophan ausotroph `WP2uvrA (Green and Muriel, 1976). The specific genotypesofthe strains are shown below (Table 1). T--ab_le I. Tester Strain Genotypes Tesersin (hiHsteorry wi Addhiona Muswion"s Plasmid 3 HsD3052 wb 3 MIO Tai iss. wb " pRMIOL Tass his wb oo - Tis hscsors wr " WhrA w wna - -- In addition to a mutation in the histidine or tryptophan operons,thetester strains contain two additional mutations which enhance their sensitivity to some mutagenic compounds. A mutationofthe wvrA gene (Escherichia coli) or the uvrB gene (Salmonella typhimurium), results in a deficient DNA excision repair system which greatly enhances the sensitivity of 000037 PCuorviafniecdeS-t2uAdlyc2oh2o9l00-0-4090ECD 9 these strains to some mutagens. Since the ur deletion extends through the bio gene, the Salmonella typhimurium teste strains containing this deletion also require the vitamin biotin for growth. `The Salmonella typhimurium tester strains also contain the rfa wall mutation which results in the lossof oneofthe enzymes responsible for the synthesis ofpartof the lipopolysaccharide barrier that forms the surfaceof the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classesofchemicals sucha those containing large ring systems (ic., benzo[a]pyrene) that would otherwise be excluded by a normal intact cell wall, Strains TAS and TA100 also contain the pKM101 plasmid, which further increases the sensitivityofthese strains to some mutagens. The mechanism by which this plasmid increases sensitivity to mutagens has been suggested to be by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. Tester strains TAOS and TA1S37 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TAL00, TAIS35 and WP2uvrA are reverted from auxotrophy to prototrophy by base substitution mutagens. 211 SourceofTester Strains "The Salmonella typhimurium ester strains were received from Dr. Bruce Ames, Department of Biochemistry, University ofCalifornia, Berkeley. The Escherichia coli tester stain, WP2uurA, was received from The National CollectionofIndustrial Bacteria, Torrey Research Station, Scotland (United Kingdom). 000038 PCuorviafniecde8S-t2uAdlyc2oh2o9l00-0-4090ECD 10 212 StorageofTester Strain 2121 Frozen Permanent Stocks Frozen permanent stocks were prepared by growing fresh overnight cultures, adding DMSO (0.09 mUimLof culture) and freezing small aliquots at <-70C. 2122 Master Plates Master plates ofthe tester strains were prepared by streaking cach tester srain from a frozen `permanent stock onto minimal agar appropriately supplemented with either histidine and biotin (5. typhimurium) or tryptophan (E. col), and fo strains containing the pKM101 plasmid, ampicillin. Tester srain master plates were stored at 5 & 3C. 213 Preparationof Overnight Cultures 213.1 Inoculation Overnight cultures for use in all esting procedures were inoculated by transferring a colony from the appropriate master plate to a flask containing culture medium. Inoculated flasks were placed in a shaker/incubator which was programmed to begin operation (shaking, 125 225 rpm; incubation, 37 + 2C) so that the overnight cultures were in log phase or late log phase when turbidity monitoring began. 2132 Harvest To ensure that cultures were harvested in late log phase, the lengthofincubation was determined by spectrophotometric monitoringof culture density. Cultures were harvested once a predetermined density was reached, which ensures that cultures have reached a density ofatleast 0.5 x 10 cells/mL and that the cultures have not overgrown. Overgrown of -- 000039 CPuorviafnicede 8S-t2uAdlyc2oh2o9l00-0-4090ECD n stationary cultures may exhibit decreased sensitivity to some mutagens. Cultures were: removed from incubation when the target density was reached and were placed at 5 3C until used inthe assay. 214 ConfirmationofTester Strain Phenotypes Tester strain cultures were checked for the following genetic markers on the dayoftheir use in the mutagenicity assay: 2141 ofa Wall Mutation For the Salmonella tester stcains, the presenceofthe rfa wall mutation was confirmed by demonstrationofthe sensitivityofthe culture o crystal violet. An aliquotof an overnight cultureof ach strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing 10 jigof crystal violet was added. Sensitivity was demonstrated by inhibitionofbacterial growth in a zone immediately surrounding the disk. 2142 PKMI01 Plasmid R-factor The presenceof the pKM101 plasmid was confirmed for culturesofester strains TAS and TAL00 by demonstrationofresistance to ampicillin. An aliquotofan overnight culture of ach stain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing 10 ugof ampicillin was added. Resistance was demonstrated by growth in the zone immediately surrounding the disk. 2143 Characteristic Number of Spontaneous Revertants `The mean numberof spontaneous revertants per plate in the vehicle controls that is characteristicofthe respective strains was demonstrated by plating 100 L aliquotsof each culture along with the appropriate vehicle on selective media. -------------- ee-- 000040 Purified 8-2 Alcohol Covance Study 22900-0-4090ECD 2 21s Tester Strain Media 2151 Culturing Broth `The broth used to grow overnight culturesof the tester strains was Vogel-Bonner salt solution (Vogel and Bonner, 1956) supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2 dry powder). 2152 Minimal Bottom Agar Plates Bottom agar (25 mLper 15 x 100 mm petri dish) was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5% (w/v) agar and 0.2% (wi) glucose. 2153 "Top Agar for Selection ofRevertants Top (overlay) agar was prepared with 0.7% agar (w/v) and 0.5% NaCl (ws) and was supplemented with 10 mL of 1) 0.5 mM histidine/biotin solution per 100mL agar for selectionofhistidine revertants, or 2) 0.5 mM tryptophan solution per 100 mLofagar for selection of tryptophan revertants When $9 mix was required, 2.0 mLofthe supplemented top agar was used in the overlay. However, when 9 mix was not required, water was added to the supplemented top agar (0.5 mLofwater per 2 mLofsupplemented top agar) and the resulting 2.5 mLofdiluted supplemented top agar was used forthe overlay. This dilution ensured thatthe final top agar `and amino acid supplement concentrations remained the same bothin the presence and absence of $9 mix. ------------------ 000041 PCuorviafnicede 8S-t2uAdlyco2h2o9l00-0-4090ECD 3 22 Test Article Test Article: Haskell Number: ~~ Purified 24691 8-2 Alcohol PDhaytseicRaelceDievscerdi:ption: White solid 8 August 2001 The test article, Purified 8-2 Alcohol, was supplied as a white solid. The test article was. stored in a container at ambient temperature. The Sponsor was responsible for the determination and documentation of the analytical purityofthe test article. Any unused test article and/or the original est article container were returned to the Sponsor. 23 Control Articles 231 Vehicle Controls Dimethylsulfoxide (DMSO, CAS 67-68-5, Acros Organics, Lot Nos. AO14387401 and A012097501) was used as the vehicle. Vehicle controls were plated for all tester strains in the presence and absence of $9. The vehicle control was plated on selective agar using a 50 HL aliquot (equal to the maximum aliquotof test article plated), along with 100 uLof the appropriate tester strain and 500 L of9 mix (when necessary). 232 Positive Controls The combinationsofpositive controls, activation condition, and tester strains plated concurrently with the assay are indicated below (Table 2, next page). ------------ 000042 PCuorviafniecde 8S-t2uAdlyc2oh2o9l00-0-4090ECD 1" Table2. Positive Controls esr Sain Somix ---- Dose gpl) TTaaosss + bZeinuaosMlopryeennee TaI00 - 2aminouthracene 2t5o 25 TTaaisn} + So2daimuimnoiudnetcene AIS} : sodium ide 2250 20 TTAhLiSs? Wena -: 2iacmriinoorsntscene - Zamivontracens 22s0 250 -- Wea : Anirogue inoline e Neovide 0 2321 Source and GradeofPositive Control Atticles Benzo{alpyrene (CAS# 50-32-8; purity 297%), 2-nitrofluorene (CASH 607-575; purity 298%), sodium azide (CAS# 26628-22-8; purity 299%), ICR-191 (CAS# 1707-45-0; purity 290%), and 4-nitroquinoline-N-oxide (CAS# 56-575; purity 209%) were obtained from Sigma Chemical Co. All were prepared in DMSO, except for sodium azide, which was dissolved in deionized water 233 Sterility Controls 2331 Test Article The most concentrated test article stock solution was checked for sterility by plating a 50 uL aliquot (the same volume used in the assay) on selective agar. 2332 $9 Mix The S9 mix was checked for sterility by plating 0.5mLon selective agar. -- 000043 PCuorviafnicede 8S-t2uAdlyc2oh2o9l00-0-4090ECD 15 24 Liver Microsomal Enzyme Reaction Misture (59 Mix) 241 9 Homogenate $9 homogenate containing liver microsomal enzymes was purchased from Molecular Toxicology, Inc. [Lot Nos. 1302 (41.3 mg/mL protein) and 1296 (38.9 mg/mL protein). The homogenate was prepared from male Sprague-Dawley rats that had been injected (ip) with AroclorTM 1254 (200mg/mLin com oil) at S00mgkg (Ames et al, 1975). 242 $9 Mix The $9 mix was prepared immediately prior (0 use and contained the components indicated below (Table 3). Table3. $9 Mix Components -- -- CCoommpoenrent hIMoNaH3POYNagHPOS, pi 7.6 01200M2MNGlAcDoPse-Sphosphate 0598HMomKoCgeIns0t2e M Macy --_--m------ 3 EXPERIMENTAL DESIGN qQuumyy o0l7om0m 0i0m2im. 0p4eme. 10m 0m 3 Dose Rangefinding Study The growth inhibitory effect (cytotoxicity)of the test article to the test system was determined in order to allow the selectionof appropriate doses to be tested in the mutagenicity assay. -- 000044 CPuorviafnicede 8S-t2uAdlyc2oh2o9l00-0-4090ECD 16 1 Design The dose rangefinding study was performed using tester srains TAL00 and WP2uvrA in the presence and absence of $9. Ten dosesoftest article, up to 5000 plate, were tested for cytotoxicity (one plate per dose). 312 Rationale "The eytotoxicityofthe test article observed in tester strain TAL0Oi generally representative of that observed on the other Salmonella typhimurium tester strains and becauseof the comparatively high numberof spontaneous revertants per plate observed with this strain, gradationsof cytotoxicity can be readily discerned from routine experimental variation. The Escherichia coli tester strain WP2uvrA does not possess the rfa wall mutation that the Salmonella typhimurium strains have and thus,a different rangeof cytotoxicity may be observed. Also, the cytotoxicity induced by a test article in the presence of $9 may vary greatly from that observed in the absence of $9. Therefore, this would require that different test article dose ranges be tested in the mutagenicity assay based on the presenceorabsence ofthe microsomal enzymes. 313 Evaluationofthe Dose Rangefinding Study Cytotoxicity is detectable as a decrease in the numberofrevertant colonies per plate and/or by a thinning or disappearanceofthe bacterial background lawn. 314 Selectionof the Maximum Dose for the Mutagenicity Assay No cytotoxicity was observed in the dose rangefinding study and the highest dose leveloftest article used in the subsequent mutagenicity assay was that used in the dose rangefinding study (the reduction in background lawn observed in tester strain TA100 without S9 was not accompanied bya decrease in revertant frequency and likely was artifactual), a -------- eS---- EE ------ 000045 PCuorviafniecde8S-t2uAdlye2oh2o9l00-0-4090ECD 17 32 Mutagenicity Assay 321 Design "The assay was performed using tester strains TAS, TA100, TALS3S, TA1537 and WP2uwA in the presence and absenceof $9. Doses of the test article were selected based on the results ofthe dose rangefinding study. The resultsofthe initial mutagenicity assay were confirmed in an independent experiment. 322 Frequency and RouteofAdministration The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames eral. (1975) and Maron and Ames (1983). This methodology has been shown to detecta wide rangeofclassesofchemical mutagens. In the plate incorporation methodology, the test article, the tester strain, and the S9 mix (where appropriate) were combined in molten agar which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All dosesofthe test article, the vehicle controls and the positive conirols were plated in triplicate. 33 Plating Procedures `These procedures were used in the dose rangefinding study and the mutagenicity assay. Each plate was labeled with a code which identified the test article, tes phase, ester strain, activation condition and dose level. The 9 mix and dilutions of the test article were preparedimmediately prior to their use. `When $9 was not required, 100 WL of tester strain and 50 iLoftest or control article were added 10.2.5 mLofmolten selective top agar (maintained at 45 2C), When S9 was required, 500 UL of $9 mix, 100 iL of tester strain and 50 uLoftest or control article were. ee-------- eeeEee------ 000046 PCuorviafnicede 8S-t2uAdlyc2oh2o9l00-0-4090ECD 18 added 10.2.0 mL of molten selective top agar. After the required components had been added, the mixture was vortexed and overlaid onto the surfaceof25 mLofminimal bottom agar contained ina 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and incubated for 52 4 hours at 37 2C. 34 Scoring the Plates Plates which were not evaluated immediately following the incubation period were held at 523C until such time that colony counting and bacterial background lawn evaluation could take place. 341 Bacterial Background Lawn Evaluation The condition of the bacterial background lawn was evaluated macroscopically and. microscopically (using a dissecting microscope) for indicationsofcytotoxicity and test article precipitate. Evidenceofcytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose on the data tables using the code system described below (Appendix B). 342 Counting Revertant Colonics Revertant colonies were counted by automated colony counter 4 DATA a Data Presentation For all replicate platings, the mean revertants per plate and the standard deviation were calculated (Appendix A). A 000047 CPuorviafniecde8S-t2uAdlye2oh2o9l00-0-4090ECD a2 Assay Acceptance Criteria Before assay data were evaluated, the criteria for criteria were used to determine a valid assay: valid assay had to be met. 19 The following 421 Tester Strain Integrity 4211 ofa Wall Mutation All Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet, demonstrating the presenceofthe rfa wall mutation. 4212 PKMI01 Plasmid Tester strains TA98 and TA100 exhibited resistance to ampicillin, demonstrating the presence ofthe pKM101 plasmid. 4213 Characteristic NumberofSpontaneous Revertants All vehicle control cultures exhibited their characteristic number of spontaneous revertans. per plate, demonstrating the requirement for histidine (Salmonella typhimurium) or tryptophan (Escherichia coli). The acceptable ranges for the mean vehicle controls were as follows: Strain TTAaIss TTAAILSS}S WrzrA NumberofReverants s2w00 W2s0s2as -------------------- 0000438 PCuorviafnicede 8S-t2uAdlyc2oh2o9l00-0-4090ECD 20 422 Tester Strain Culture Density The cell densitiesofall tester strain cultures were greater than or equal to 0.5 10" bacteria/m (with the exceptionof TAS in Experiment 22900-B1, 0.4 10" bacteria/mL), or the optical densitiesofthese cultures reached a target value demonstrated to produce cultures with at least 0.5 x 10 bacteria/mL, demonstrating that appropriate numbersofbacteria were plated. 423 Positive Control Values 4231 Positive Control Values in the Absence of $9 Mix `The mean value of the positive control for each tester strain exhibited at leasat 3-fold increase over the mean value of the vehicle control for that strain (with the exceptionoftester strain TA1S37 in Experiment 22900-Cl, see Protocol Deviation), demonstrating that the tester strainswere capableofidentifying a mutagen. 4232 Positive Control Values in the Presenceof$9 Mix (S9 Mix Integrity) "The mean valueofthe positive control for each tester strain exhibited at least a 3-fold increase over the mean valueofthe vehicle control for that train, demonstrating that the S9 mix was capableofmetabolizing a promutagen to its mutagenic form(s) An acceptable positive control in the presence of $9 for a specific strain was evaluated as having demonstrated the integrityof the S9 mix and the abilityof the tester strain to detect a mutagen 000043 PCuorviafnicede 8S-t2udAylc2oh2o9l00-0-4090ECD 2 424 Cytotoxicity A minimumofthree non-toxic doses was used o evaluate assay data, Cylotoxicity can be detected asa decrease in the numberofrevertant colonics per plate and/or by a thinning or disappearance of the bacterial background lawn compared to the appropriate vehicle control, 43 Assay Evaluation Criteria Once the criteria fora valid assay had been met, responses observed in the assay were evaluated as follows: 431 Tester Strains TAS, TAL00 and WP2uwrA Fora test article to be considered positive, it had to produce at least a 2-fod increase in the mean revertants per plateofat least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrationsof the test article. 432 Tester Strains TAIS35 and TAIS37 Fora test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate oaf least one of these tester srains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number ofrevertants per plate had to be accompaniedby a dose response to increasing concentrationsof the test article. 000050 PCuorviafnicede 8S-t2uAdlyc2oh2o9l00-0-4090ECD 2 5. RECORDS TO BE MAINTAINED All raw data, documentation, records, the protocol, and the final report generated as a result of his study will be archived in the storage facilities ofCovance-Viennaforat least one year following submissionofthe final report to the Sponsor. Afier the one year period, the `Sponsor may elect to have the aforementioned materials retained in the storage facilities of Covance-Vienna for an additional periodoftime or sent to a storage facility designated by the Sponsor 6. STUDY RESPONSIBILITIES Function Study Director Laboratory Supervisor Responsible Person(s) Leon F. Stankowski, Jr, PhD Magnus A. Evertson, BS 7 RESULTS 71 Test Article Handling Purified 8-2 Alcohol formed a solution at 100 mg/mL in dimethylformanide after heating. In DMSO at 100 mg/mL, the test article formed a solution that foamed when heated and vortexed. DMSO was sclected as the vehicle. At 100 mg/mL, whichwasthe most concentrated stock prepared for the mutagenicity assay, the test article formed a transparent, colorless solution afer heating to 45C for 3 minutes; it remained freely soluble at all succeeding lower dilutions. 000051 ee PCuorviafnicede 8S-t2uAdlyc2oh2o9l00-0-4090ECD 2 72 Dose Rangefinding Study (Appendix A, Tables 4 and 5) A dose rangefinding assay was conducted on the test article using tester strains TAL00 and WP2uvrA (one plate per dose; Experiment 22900-A1; Tables 4 and 5). Ten doses of test article, from 6.67 to 5000 ug/plate, were evaluated in the presence and absenceof S9. Apparently normal growth was observed in both tester strains at all doses evaluated with and without S9 (the reduction in background lawn observed in tester strain TA100 without S9 was not accompanied by a decrease in revertant frequency and likely was artifactual), However, the test article was found to be incompletely soluble in the aqueous top agar at a doseof 5000 ug/plate with and without $9. 73 9M-utSaugmenmiacriytyDAastsaa)y (Appendix A, Tables 6, 8 - Individual Data, Tables 7, Based upon theresults ofthe dose rangefinding study, Purified 8-2 Alcohol was evaluated in the inital mutagenicity assay in all five tester strains at dosesof 33.3, 100, 333, 1000, 3330 and 5000 pg/plate with and without $9 (Experiment 22900-BI, Tables 6 and 7). All doses of the test article, as well as the concurrent positive and vehicle controls, were evaluated using three plates per dose. Apparently normal growth again was observed in al tester strains at all dosesofPurified 8-2 Alcohol evaluated with and without $9. In addition, the test article: precipitated from solution at doses 21000 ug/plate with and without $9. Revertan. frequencies for all doses of Purified 8-2 Alcohol, in al teste strains with and without $9, approximated or were less than those observed in the concurrent vehicle contol cultures The test article was re-evaluated in an independent confirmatory experiment under identical `conditions, and similar results were observed (Experiment 22900-C1; Tables and 9), Normal growth was again observed in all tester strains at all doses evaluated with and without $9, and the test aticle again precipitated from solution at doses 21000 ug/plate with and without $9. Revertant frequencies for all doses of Purified 8-2 Alcohol, in al tester 000052 Re em -- e PCuorviafnicede 8S-t2uAdlyco2h2o9l00-0-4090ECD. 2 strains with and without $9, again approximated or were less than control values. Except for tester rain TAIS37 without S9 in Experiment 22900-CI (see Protocol Deviation), all positive and vehicle control values were within acceptable ranges in both assays. All eiteia fora valid study were met. 8 CONCLUSIONS "The resultsofthe Salmonella - Escherichia coli/Mammalian- Microsome Reverse Mutation Assay indicate that under the conditions ofthis study, Purified 8-2 Alcohol was not `mutagenic in any ofthe tester trains in the presence or absenceofan exogenous metabolic activation system containing induced hepatic microsomal enzymes from AroclorTM 1254-treated rat. 9 PROTOCOL DEVIATIONS In Experiment 22900-C1, the test article-treated cultures for tester strain TALS37 without $9 were scored even though the concurrent postive control values were below acceptable limits (ll plates hadnorevertants). However, the vehicle control values for tester strain TA1S37 without S9 were within acceptable ranges. In addition, the positive control values for tester strain TAIS37 with $9 also were within acceptable limits, indicating that the tester strain and $9 were functioning properly. Thus, the observed low values were likely due to a technical error. This deviation is not considered to have had an adverse impact upon the integrity of the study or the conclusions derived from it. 000053 --E ---- emeee----e -- `PCuorivfainecde 8S-t2uAdlyc2oh2o9l00-0-4090ECD 2s 10. LIST OF REFERENCES Ames etal, 1975. AthmeeSsalBmNo,neMlclCaa/MnanmJm,aYlaimaans-aMkiicrE.osoMmeethMoudtsagfeonridceitteyctTeisntg. caMructiantoigoennsReasnedamructha1g9e7n5s;with 31:347.64. GGrreeeenn MaHnLd,MaMruireile,l 1976. WJ. Mutagen testing using 7" reversion in Escherichia coli. Mutation Research 1976; 38:3-32 MMaarroonnDaMn,d Ames, 1983. Ames B. Revised methods for the Salmonella Mutagenicity Test. Mutation Research 1983; 113:173-215. VVooggeell HaJn.dBBoonnnneerrD,M1.956A.cstylomithinase of E.coli: Partial purification and some properties. J Biol Chem 1956; 218:97-106 000054 -- Purified 8-2 Alcohol Covance Study 22900-0-4090ECD 2 AppendiAx. _-- Experimental Data Tables 000055 ---------------- Purified 8-2 Alcohol Covance Study 22900-04090ECD 7 T--a_b--le 4 Dose Rangefinding Study ~ TAL00 Test Article 1D: Purified 8-2 Alcohol Date Plated: 29.0001 Experiment ID: Date Counted: Vehicle: DMSO Plating Aliquot: 22900-AT 01-Nov-01 50 TAIOOReverants erPlate: Winso Without59 _T -- ype r PPeer BLaalwinn' P e Ppie enre BEveaLlualawtneion'' 000 (Veihicle) ns N = N Tesi ride 6.67 0 x 100 mn N a3 1% N 7 07 x 10 1 N 3 0 N aw 0 N 1000 106 33% 108 ~ 5000 10s Ne " N Tor N 0 N El N 5 N @ N 08 x x 107 n 103 RP *BNPoamraclikLagRwenO mrvdoaocteiduonnCAobc ndydespsb:eecs E-- S-------- ----------------i---------------- 000058 `PCuorivfainecde 8St2uAdlyc2oh2o9l00-0-4090ECD 28 -- e TTeasblAensite I: DosPueriRfaiendg2efiAnldy cionhoglStudy ~ WPE2xupreai.men 1D: e 2900-41 VDeuhiccPllea:ed: 2D0M.S0O001 DPlaatteinCgouAngutoetd.:__ 5001-Nov-01 [---- wins itn 9 wom --_-- ReTear r PP| e BiLgoonmd| PBou nt ? Rerwer ui[g4od fPeae | SEevwaenr 00ivere) x N Te Avice 667 2 ~ 100 is N 53 ~ 1 v x 100 n N 5 " N i x 1000 z ~ 0 n ~ 5000 uv Nr n x n ~ is x u w i ~ 2 x n ~ w x u ~ 5 xr NFBukgeronnsd LRav0eEvdoanrACyosr:vtps 000057 ------ ---------- -- -- ee ------ a ------ PCuorviafnicede 8S-t2uAdlyc2oh2o9l00-0-4090ECD 2 eTable e6 Test Arie 0: InitPiaulE Mruti5a:g2feAY nilicceoihtdoylIAssayRRoelsulEltxsspe-IrInindmdiievvnIiiDtdh:uaallPPl2la2at5te0e0CC8oo1uunnttss VDehuiccPlieu:ed: D1M3SNOowol DPlaattiengCoAulnieqduo:t: ~~ 5206u:lNov01 Dorie Taos ReverasPe Pic TAD Tass tas wens wSBraecoked. MViechrosseomCeos:nmRltLiver TT 33020 To Tao 0S RS U2 wos 08 Trae 7 70 7s sn TerAnide 31350 pu4sp 33M2 323000303S01d S2Ne9a 0 010S3 0126 91o0H06Sw10s1100 135 B11o0 116s01158hNNN TO0O0 S00 4urs jE MBARS;H 3 as 0 UonSe M1 MBSINNe le 1lB6e05w6 1l8h 0G1s2 09 1502 33S52in15 wNMN PositiveComo' $74 512342 954 108 090 141 165 138 181 148 178 253 260 293 N MVieccrioesCco:nNtovo: R39 06% 0 WU 6s 3d Teas mW Boauw2 1 s8O0 SH 98 9% s0MM 9% 0H 76 3d3soss 2920mro7uhhs NwNn ISW0O0OsM hs B08 e U00 D0%9MMzso%os H mHIsSTlde 6S3 e09 m03 nd0ns oa3omh5 owANp Poiive Commit 208 192.216 1020 1028 S45 664 656 579 1064 867 98 155 128 89 N Nm aRe--nd--Oebonsd Ashi Pps a TTTNAALSDS Dabumembiaoss sd15iuiggppphkeee TAT Zimmer Jip TTTAAABDS hidamdmiemen TAD] RDI fLoruisepinndc p2r0vcim WPA Jimi 20upp WA Sabre ons Toby -- -- ---- -- ---- -- -------- 000058 Purified 8-2 Alcohol Covance Study 22900-0-4090ECD 30 -- TTeasbtlAeri7cle ID: InitPialuMruti5ag2fem AnilicceoihtdoI ylAssay eResnuElhtxspoe-rSiuemmemnmIatDr:y 2290-81 Date Plated: Vehicle: 13-Nov-01 DMSO Date Counted: 26-Nov-01 Plating Aliquot: 50 Dosepive __TaS% Mean Reverants Pe Plate with Standard Deviation TAO TALS TAISY weer sBoauckn.d fan Microsomes: RatLiver an SD Ven SD Mew SB. Mew SB Man Sh Velie Conta os ow o7 mss 2 son ow Teramde WBoaew Sou 2% B 44 a2s 008 08 016 M2 11sws3 3 01 uis 4oo Nxn oN BWooupg 0 we M@ 4odea7 52 16s 35 3d ms sl 4 om9 33 m3 1ie 62 NNpp xn 1 ow Posive Coro SBN 00S ws is i a6 3 N MViicocsiomCeosnt:rNool re I A ] TerAtce WBoawws M43 m2TM 0os mno6s SB ous 6 7% 6 2 2 66 43 1a66 31xN 7 4 of 3 N [00 owyse 21982swom4 0 wg 16s es 2 o9w 3 o4 &735 33 63 1uo 3 ne au 4 ow P-- ositive Control" Ws 9 ww e ewseps 3 N * BiNckogrmoaanddLawnRvaebsdiodn CosO:nobssd Ashik Pegeciin TTTASAASSDS dbDmaiosmolwobernee:nS2SSuuuppgheee CTTAABI dsiamtonees TAI seemanie TAIST Jima: Supe TAS KRISH 2L0ouuggpiiienne 220u05pimue Wea Tamimombocae Ouse WrloeA bamusonetonde |0sgpine -------------------------- 000059 CPuorviafnicede 8S-t2uAdlye2oh2o9l00-0-4090ECD 31 TeTsatble AerSile TDe : CoPnfuirrmaito2fryAilMcueothaodglenicity AssEaxypReersauilmntesn-J0t:pInadivv2ii9do0u0aul-P8P1ilastealCoounnttss DucPlacd: ehice: 20Novol so PlaDtaitnegCoAulnitgeudo:t:_ 0507,-Dec-01, 10-Dec-01 Dems Tam Reva Prite Tw tums mus wets sLBnoatd VMeilcorCoosnoRemaheivse:r Ty Tr To TTT DEE ow wm ow on su ee somos oN Teh WMs3 ow Bow 3HO9M%2 eB UmMeouRswIEmowMSobmnoihon too8wMssow7 80ms 5wsoowwMNxN MWooog So Gpe0omRxeBowmWomOo Wmoso3nw5ndhs onH9ohwmsoo7 uBbob osw oonn ooww a PohcCooiol 98 32 180 1S 2510 18 12 ms 0S6 10 11 4s 3 sen MVeiilcCoondonns MOR os ws 6 ss moa os ow mown Tothnde W3o3 ug Mowe M 2A Am% WROoE sKs % BS oo7 msw Ses 86 27 25 3 7s dnnooom onwow oNx oN BWOgs ABBWWH ow EaHm 000 he BWwo A 8M B 05 0Bosw 64I 824 333 d2boD Wbo4oDmooww a _Po--eCont 27265 20 U0 26 ss 0 0 0 mom msn *BNora oLoverk Renriienr dC|oeOs:mcbiud A=sbent Pepuchinie TTTAAASIS oZiammlomaebrtsioes JS2suugipppliueeee CTTTAAADIS scmLheoannaonsise TAS Sime Sipe TAS KR 2L0oispsiense p20rusipinne Weer Lamimirie 3504p WEA SausinoneNodt |Oboes ---------- Sees ---------- 000060 Purified 8-2 Alcohol Covance Study 22900-0-4090ECD 32 T--a_bl--e 9 Confirmatory Mutag7 enicity Assay Reh sults e - Summy ary Test Article ID; Puri8.f2Ailceohdol Date Plated: 29-Nov-01 ExperimeInDt; 22900-B1 Date Counted: 07-Dec01, 10-Dec-01 Vehicle: DMSO Plating Aliquot: __S0 pL MeanRerunsPee lewith StandardDevistion TTDocte WeTsass Sb WaTnal Sb WeTnassSO WeTmAITSD VMeitucsmCsonotRomalceLisve:r moze 46 1 0 2 Teatice WB3owwg X S3 NmS 60 Waws 61 0ws4 WWOookug X BoSm Wm 7 U 9 3 ss 11 M0oepew3s9%wo2 m m32 omw 33 Posie Ctra aMitcircosoCmoens:tNoelos Wom um em % 9 2 mos mo 6 6 2 6 4 Tian w$3owwe 2w3 s 4sn6ow8 2 67 31 L MoSw E sE s 2 om 4 E S 2 WS00oeks 42% % 164 05 61 36 2 PosieCaml" WM oms moss on 0 0 rBoauckn.d WeWehie$A5 Tawa' ss oN 1osn 62 NN 16 4sxNe aomw 23 we a ow Nn ow 3 x uosw ssnn om sn m 0 33 ooww we 16 n * BNaeocmaklLagwnERrevrosidiucneCdno|dedsO:mobewsd VTTASIO blaummocwmhbncms 22SSuppeppilwee TTAAII dadammiimsocwamebcneennss 2253uubgihwiee WeLoTA 2avisouboccie 250jghie A-abimt Pepesipinis CTTAA%I soadiuvmofildccese TTIAISS soIdCiRAuOmaIide WPharA dnimauindloneNooxide 2100upspiilntec 2200uuggppiiaacc 10 gps ---------- 000061 PCuorviafnicede S-t2uAdlyc2oh2o9l00-0-4090ECD 3 Table 10 Historical Control Data Plate Incorporation Method - Report Period 01 --V -- ei er deVee hC icle Co ontroT lm s withm9 Mib xeweie m M-- x Stain TAS TAO TAINS TAY Whaea `SMteaanndaRredveDreavainaitsipoenrPlate 82 087 2 143 153 104 a4 46 200 6s Maximum Minimum a 1 143 30 6 s 2 2 a 9 CH a o--un_t-- JVeotiiccli2seCCoonntnroollsss Wwitb2h9o4utS59MiEx200 155 = 208 Sm Mean Revertpearnliatse TASS 164 TAI ses TAISS TALS 126 86 Wikwra 190 MSatxaindmaurdmDeviation s4 36 1s 128 as EY 43 2 ss 38 Minimum Count 5 29 a6 x 3 207 | 183 s 194 - -- Swi Positive TA% Convols wih TAO 9 ix =~ TASS TAI -- Weare Mean Revertants per Plc Standard Deviation sss 3 sms aso oma 261 ns 32 sess ma Maximum Minimum oa 146 oss 20 31 7" 21 ki 1055 192 Count 28 30 204 181 198 Ea Posiive Controls withowS9Mies TASS TAO TAINS TAI WemarA Mean RevertantsperPiste StandardDeviation 256 98 90s 169 sa 101 owe 2695 20 m3 MaMixniimmuumm 1w30 isn386 7 3 as 2 1 1 Count 26 am 20 178 190 TTTAANSISS uiammmsiosimoniumeecmosec dluuppee Supe WA amomiee SOU TTAASDS vmsie INST KR Z20ovpninee 20k WEA daoqimlecNorts 10spmiee -- -------- -- -- ee ---------- ee 000062 Purified 8-2 Alcohol Covance Study 22900-0-4090ECD 34 Appendix B. -_-- DefinitionsofBacterial Background Lawn Evaluation Codes 000063 -- -- ---- -- -- A ------------ CPuorviafnicede 8S-t2uAdlyco2h2o9l00-0-4090ECD 3s Bacterial Background Lawn Evaluation Code `mTihcerocsocnodpiitciaolnloyf(tuhseinbgaackdgirssoeucntdinbgacmtiecriraolscloapwen)ifsoervianlduiactaetdiobnostohfmcayctrootsocxoipciitcyalalnydatnesdt article: precipitate a follows: CODE DEFINITION ~ CHARACTERISTICS OF BACKGROUND LAWN N Nomal Ahealthy microcolony lawn. R Reduced A Absent dsiizseotifntchtetmhiicnrnoicnoglooftnihces mciocmrpoacroeldontyo tlhaewnveahnicdleancoinntcrroelaspelaitne.the A complete lackofany microcolony lawn. O Obscuredby Precipitate mTihcerobsaccokpgircoutensdt baratcitcelreiaplrelcaipwintactaen,nmoatcbreosaccocpuircatteelsyteavrtailculaeted due fo precipitate, or plate coloration. E Enhanced Asidziesotfintchtetmhiiccrkoecnoilnogonifesthceommipcarroecdoltoontyhelavwehniaclnedcaonntirnoclrepalsaet.in the Efovlildoewnicnegopfremcaicpritoastceocpoidcettesottahreticcoledeprneucimpbietarteusoendtthoeepvlaaltueaties trheecocrodnedditbiyonoadfditthieonofthe background bacterial lawn. P Precipitate Macroscopic precipitate observed on the plate. -- eee ---- 000064 Purified 8-2 Alcohol Covance Study 22900-0-1090ECD 36 Appendix C. QualityAssurance and Compliance Statements _-- 000065 ------------------------------ CPuorviafnicede 8S-t2uAdlyc2o2ho9l00-0-4090ECD 37 QUALITY ASSURANCE STATEMENT Purified 8-2 Alcohol:MuStaaltmioonenlAlsas-aEyscwhietrhicahCioanCfoirlmia/tMoarmymAaslsiaayn-Microsome Reverse `aTchceorrdepaonrctehwaistbhetehne rGeovoidewLeadbobryatthoeryQuParlaicttyicAesrseugrualnacteioUnnsiatsosfetCofovratnhcien LthaeboErnavtiorroiensmeInnct.alin aPrnodtetchteiOorngAagneiznactyio(nEfPorA-EcToSnCoAm)i,cTiCtoloep4e0roatfitohneaUn.dS.DeCvoedleoopfmeFnetde(raOlERCeDg)ulPartiinocnisplPeasrtof792; GfoololdowLianbgoirnastpoercytiPornasctwiecreeEcNoVnd/uMcCte/dCaHnEdMthe(9f8i)n1d7i;ngasndreapnoyrtaepdpltoictahbeleStaumdeyndDmiernetctso.r aTnhde sCtouvdayndcieremcatonragmaenmaegnetmeanctc.ordWirnigtttoenstsatnadtuasrdreoppoerrtasotifnginpsrpoeccetdiuorness.and findings are issued to Inspection Dates 24-0ct:2001 13-Nov-2001 07-Jan-2002 08-Feb-2002 Phase Protocol Review, 9 Mix Preparation Draft Report Review Final Report Review Dates Reported 10 Study DiDriercetcotrorMaanndagSetmuedyn~t ~ Auditor 24-00-2001 P.Ciceres 14-Nov-2001 J. Howard 07-Jan-2002 C. Smith 08-Feb-2002 C. Smith ig Fe Representative, Quality Assurance Unit UEb P2 Date eee -------- 000068 Purified 8-2 Alcohol 3 Covance Study 22900-0-4090ECD STUDY COMPLIANCE AND CERTIFICATION Except that the test and control article dosing solutions were not analyzed for stability, G(hEooPmoAo-dgTeLnaSebCoiArtay)t,oorrTyaictPlcreuarc4at0iccoyeoftrfhepgerueUlpaaStr,iaotnCisoodnas,e stoehftisFfeosrdtteuhrdayblywRatehsgeuclEoanntvdiiuorcnotsnemPdearnitntac1lo92mP;prloaitneadcntctiehoenwiAtghetnhcey OLragbaonriaztaotriyoPnrafcotriEcecoEnNomVi/cMCCo/opCeHraEtMio(n9a9n1d7Deavndelaonpymeapnptli(cOabElCeDa)mePnridnmceinptlses,ofThGeoroedwere 1af0feoctthtehr deivntieagtriiotnysofftrhoem tshteudayfooretmheenitnitoenreprdetraetgiuolnaotfiotnhseoresttheressiulgtnse.d pTrhoetorcaowl tdhaattawhoauvled `pabrneedesneevnratelevudiaehtweieroednicbnryiettpehrreiaeS.stuednyatDnsiraepcptroorp,rwiahtoe cceornticfliuessitohnatwitthheienvtahleuactoinotneoxfttohfte hteststaurdtiycldeesaisgn Study Direcor % Statsd / LBGeaeoctnneeFrit.aSlatnMaduntkMaoogwlesenkceius,liJasrr.T,Pohxiicology Covance:Vienna Testing Facility Managemen: 2/ufoa `Study CompletDiaotne ian C.NiyhrC,P.RDJ BGaecnteetriicalanMdutMaogleenceusliasrToxiclogy Date2 [ufo Report Reviewed and Accepted for E.L du Pont de Nemours and Co. by: Gerald Kennedy, PhD Date Sponsor Study Monitor VSpona sor Ter chicaia lDProjD ect Mo onitormer we 000067