Document 4vQdyekMxGLO7YG5MKgVk90qG

AR 226 3228 410 AL226_ 3228 FINAL REPORT [-------- 'MICROSOME JsuvvoneLr-sscuericrs COLIIMAMMALIANMUTATION ASSAY WITH A CONFIRMATORY ASSAY COVANCE STUDY 22900-0-4090ECD STUDY SCHEDULE: Study Initiation Date: Initial Dose Date: Study Termination Date: Final Report Date: 23 October 2001 29 October 2001 10 December 2001 11 February 2002 - StudyDirector:(--) STUDY DIRECTOR, SPONSOR, AND TESTING FACILITY i Testing Facility: "ovance Laboratories, Inc. (Covance) 9200 Leesburg Pike | Vienna, VA 22182 Santa Monica, California 90407 "This study was conducted inaccordancewith Covance Standard Operating Procedures; the L`OarbgoarnaitsoatriyoPnrafcotriEcec,onEoNmVi/cMCCo/oCpHerEaMt(io9n8a)n1d7DaedvoepltoepdmNeonvte(mObEeCrD2)6,Pr1i9n9c7i;plaensdofthGeood Environmental Protection Agency (EPA-TSCA), Title 40 of the US Code of Federal " Regulations Part 792, issued November 29, 1983 (effective December 29, 1983 [revision TSiisndeoSnetmetne1c8o,n1i59e0n)al gropeny of + ------) No partof it maya transmitted, reproduced, published, or used by other persons withouthe permission of ~ emtsn TSCACS Company santtized. --- `CovanceStudy 22900-0-4090ECD 2 TABLE OF CONTENTS ~ . PAGE LIST OF TABLES cnr S LIST OFAPPENDICES crams SUMMARY.crarmemmmmmmenrmmmmmmmmsmsmssmssmsmmmmsssssssssssmsasane|s 1 OBJECTIVE crmmsmmmmsmssmmsmssmsmsssssssonss ! 2. 21 MFATOERIALS crams --8 || 211 212 SF torageof TesterStain..S . tmm----------------------=30 2121 2022 FMRROSPIOArTPIREESNo CE SIOGKSr ro n ver BRs: 10 --- 213 2131 Preparation ofOVOright CULES rrr 10 | 221144.1 fCoanfWiarlmlaMtiuontoaftTeisotenr .Sr.ai.n PREROYPeES.r..e..nrrv--ev--vrv--v ----111 2142 2143 pChKaMraIcOteIriPsltaiscmNid uRefaomcftoSrbP.O..nenocoruvs RrEVErants........B ..covovvT vves 11 ! 221155.1 a Culturing Broth rrr en------ 2 2152 20153 TMoipniAmgaalrBfootrtSoemleActgiaornPlOafteRs.EVERServrreem--------1r2l | 2233.21 e VehicleCOMIOS orenrrrnrrrt rssssonssssnsisirn 13 2321 233 Source and GradeofPositiveControlAiles Sterility Controls . A -- 1d mm 14 2331 2332 TSEOSMUAiTXIuCIreororiocrommornsmemns--e--ore--s 4 ld Comppae:ny Saeed. Dove mtcniain TSCA Cor I [ZN : |I~ enm--r) : `Covance Study 22900-0-4090ECD 24 24.1 Liver Microsomal Enzyme Reaction Mixture (S9 Mix) couuureessemissesnes 15 'S9 HOMOERALE crv prensa 3. 31 3.11 3.12 313 3.14 32 321 322 341 342 41 EXPERIMENTAL Dose Rangefinding DESIGN ...cceconsunsmmmmssssssssssssssmsssssssssssssssssnsssssesseseens StAY....cvmrssmmssmssssmsssssssssmsssssssssss 15 1 Design............ PEFR EE CT . 16 Rationale.......... As ---- srs -------- 16 `Eva ofl theu Doa seRt angi efio ndin ng Study ..........coveeerevrssssesssssssssnes 16 SMeUlLeAcZtEiRonIoCfILtYheASMSaAxYicmueunmemDmorssemsfsosretmhsesMmuitsasgmesnsiscsimteyssAsessays.s..m.s..e.s..s.e..s.e..ssens 16 17 Design............ As ---------- Frequency and Route ofAdministration. sssssssserseeeeeesese wll? ---- `Bacterial Background Lawn EVAINAON..........eereeesssimssnines 18 Counting Revertant COlOIEs ............... renee 18 DLS Presentation werwesesmummmmemmenssmemmmemmisssrsemsessessmess 18 4212 4213 422 423 423.1 4232 PKMI01 Plasmid... as -- ---- Characteristic NuomfSpbonte aner ous ReVertants...................... 19 Tester Strain Culture DENStY ........c.comemmsrrmmssssssnssssssssssssssssssssnsssss 20 Positive Control Values. rsp ---- wns 2 Positive Control Values inthe Absence of $9 Mix swimmer Sl Positive Control Values in the Presenceof $9 Mix (S9 Mix Integrity)........ 20 43.1 Tester Strains TA98, TA100 and WP2uvrA crrsa-------------- 21 432 . Tester Strains TA1535and TA1537. RE i"} - 5. RECORDS TO BE MAINTAINED..cccccocuumsmsessssmmsmsmsssessssssssissssssssssssnns 22 6. STUDYRESPONSIBILITIES .ccvvuunmmrrmssmsssmsssessmssssssssssssssssssssssssssesss 22 Company Sanitized, Diane ant anniain TREA CRI - (r--1 4 `CovanceStudy 22900-0-4090ECD 7 RESULY Sussmann 3 71 FE ----. 3 72 Dose Rangefinding Study (Appendix A, Tables 4 nd 5)...v.orurvrs 23 73 TMaubtlaegsen7i,c9i-tSyUAMsMsaAyTY(ADpaptean)dic x A, Tabler s 6, 8- Indm ividual Da: ta, 23 8 CONCLUSIONS nansmsmssssmssssmsssssssssssssssssssnss 34 9 PROTOCOL DEVIATIONS oss 24 10. LIST OFREFERENCESc.sssmsssssssssssssssns 35 | | . Gompany Ssniizad. Doesnotcontaia TSCA CBT -- ( `Covan-- ce Study 22900-0-4090ECD 5 LIST OF TABLES PAGE Table 1 TeSter SUF GEMOIYPES. vrs Table. POSHIVE COMIOIS commen: 14 Tabled, 59MixCOMPONENS.cororrrrnrissnnn 15 Tabled Dose RangefStiudny ding TAL00....crssmmmsssresrne 21 TableS Dose Rangefinding St~WuP2dPAy ovine 28 Table6 Initial Mutagenicity Assay Result-s Individual Plate COUS vr 29 Table7 Initial Mutagenicity Assay RESUS- SUTATY covers: 30 | Table8 Confirmatory Mutagenicity Assay Result-s Individual Plate Counfs........ 31 | Table 9 Confirmatory MutagenicityAssayResults - SUMMATY ............... 2 Table 10 HiStOriCAl CODON DAR rere 33 ~ Company Sanitized. Does not contain TSCA Caf ~- [A --) osozco 6 LIST OF APPENDICES AppendiAx. Experimental Data Tables ......... nn Appendix B. Definitionsof Bacterial Background Lawn Evaluation Codes Appendix C. Quality Assurance and Compliance SIatements PAGE rit 26 34 own: 36 - Company Sanitzed. Does nat contain TSCA C31 - J-- `Covai nce Stud) y 2200-0-4090ECD 7 SUMMARY Objective: The objective ofthis sud was to evalu he ai oJNM induce reverse mutations at the histidine locus of selected Salmonella typhimurium stains, or the tryptophan locusofEscherichia coli strain WP2uvrA, in the presence and absence ofan exogenous mammalian metabolic activation system (S9). Study Design and Parameters: [NERNEY= cs: in te lt incorporation reverse mutation assay using S. yphimurium strains TAS, TAI00, TAIS3S and TAIS37, and | E. coli strain WP2uvrA, with and without a metabolic activation system (59) containing induced hepatic microsomal enzymes from AroclorTM 1254-treated rats. Initial and | confirmatory mutagenicity assays were conducted. Each assay included vehicle and positive controls, and six dose levels J-- with and without metabolic activation. ~ Dosesof Purified 8-2 Alcohol Craluated, based on the results ofa dose rangefinding assay and selected in conjunction withthe Sponsor, were 33.3, 100, 333, 1000, 3330 and 5000 pg/plate with and without $9. Esch test and control article dose was evaluated in triplicate plates in each srain. The dimethylsulfoxide (DMSO) vehicle control, as well as the | test and positive control articles were administered in avolume of 50 L. | Results: No toxicity was observed in any tester stain inthepresence or absence ofS9 up to | the maximum dose tested of 5000 plate. The mean numberofrevertants per plate did not ||| ameveatlidthsetucrd:iyt,eriinaclfourdainpgoaspiptriovperrieastpeovnesheicilneaannydtepsotseirtistvreaicnonwtirtohl roresw;piotnhsoeust($w9i.thAlthlecreixtceerpiatifoonr ofpositive control values for TA1S37 without S9 in Experiment 22900-C, sec Protocol Deviation), were fulfilled. | Concusion{RE = not mutagenic in this test system - Company Sanitzed. Does 101-- 507 - | - | | | : | fe-- . `Covance Study 22900-0-4090ECD 1 OBJECTIVE The objectiveofthis study was to evaluate the test article forthe ability to induce reverse mutations a the histidine locus in several strainsof Salmonella typhimurium (TA98, TA100, TAI535 and TAI 537), and at the tryptophan locus in Escherichia coli tester strain WP2uvr, in the presence or absenceofan exogenous metabolic activation system (9). The assay design was based on OECD Guideline 471, updated and adopted July21, 1997. i : 2 MATERIALS 21 Tester Strains "The teste strains used were theiSalmonella typhignurium histidine auxotrophs TAS, TAL00, TA1535 and TA1537 (Ames ct al., 1975) and the Escherichia coli tryptophan auxotroph WP2uvrA (Green and Muriel, 1976). The specific genotypesofthe strains are shown below (Table 1) Table 1 Tester Strain Genotypes Tesersmin hip Ad wi do Motion nsTr s Plerid Eg = wb 7 RMIT Ta00 pe wp Tass hiss ws # sKMI0! po - wis? HC3076 ws a. - . Wrz ow wn - - In addition to a mutation in the histidine or tryptophan operons, the tester strains contain two additional mutations which enhance theirsensitivity to some mutagenic compounds. A. mutation of the uA gene (Escherichia coli) or the uvrB gene (Salmonella typhimurium), results in a deficient DNA excision repair system which greatly enhances the sensitivity of Gompeny sa5%niizas. Doss82tiain T5048 681 - -- 9 `Covance Study 22950-0-4090ECD these strains to some mutagens. Since the wv deletion extends through the bio gene, the "Salmonella typhimurium tester 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 lossofoneofthe enzymes responsible for the synthesisofpartof the lipopolysaccharide barrier that forms the surfaceofthe bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e. benzo[alpyrene) that would otherwise be excluded by a normal intact cell wall Strains TA98 and TA100 also contain the pKMI01 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 TA98 and TAL537are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TA100, TAIS3 | and WP2uvrA are reverted from auxotrophy to prototrophy by base substitution mutagens. 211 SourceofTester Strains The Salmonella typhimurium tester strains were received from Dr. Bruce Ames, Department . ofBiochemistry, Universityof Califoria, Berkeley. The Escherichia coli ester train, WP2uvrA, was received from The National CollectionofIndustrial Bacteria, Torrey Research Station, Scotland (United Kingdom). _ pt <sohC | compet senniesd 2 - -- ovance Sth udy 22900-0-4090ECD 10 212 StorageofTester Strain 2121 Frozen Permanent Stocks Frozen permanent stocks were prepared by growing fresh overnight cultures, adding DMSO (0.09 mL/mLofculture) and freezing small aliquots at <-70C. 2122 Master Plates Master platesofthe tester strains were prepared by streaking each tester strain from a frozen `permanent stock onto minimal agar appropriately supplemented with cither histidine and biotin (S. typhimurium) or tryptophan (E. coli), and for strains containingthe pKM101 plasmid, ampicillin. Tester strain master plates were stored at 5 3C. - 213 PreparationofOvernight Cultures | 2131 Inoculation Overnight cultures for use in all testing procedures were inoculated by transferring a colony. from the appropriate master plate to aflask containing culture medium. Inoculated flasks were placed in a shaker/incubator which was programmed to begin operation (shaking, 125 25 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 lengthof incubation was | determined by spectrophotometric monitoringofculture density. Cultures wer harvested | once a predetermined density was reached, which ensures that cultures have reached a density ofat least 0.5 x 10" cells/mL and that the cultures have not overgrown. Overgrown of - aotcantsin TSCATE pe anid: -- Dom-- 1 `Covance Study 22 OECD stationary cultures may exhibit decreased sensitivity to some mutagens. Cultures were removed from incubation when the target density was reached and were placed at 3C until used in the assay. 214 ConfirmationofTester Strain Phenotypes. Tester strain cultures were checked for the following genetic markers on the dayoftheir use in the mutagenicity assay: 2141 fa Wall Mutation For the Salmonella tester strains, the presenceofthe rfa wall mutation was confirmed by demonstration of the sensitivityofthe culture to crystal violet. An aliquot of an overnight cultureofeach strain was overlaid onto plates containing selective media and an antibiotic I sensitivity disk containing 10 ugof crystal violet was added. Sensitivity was demonstrated by inhibitionofbacterial growth in a zone immediately surrounding the disk. 2142 pKMIOI Plasmid R-factor `The presenceofthe pKM101 plasmid was confirmed for culturesofteste strains TAS and TA100 by demonstrationofresistance to ampicillin. An aliquotofan overnight culture of ach strain was overlaid onto plates containing selective mediaand an antibiotic sensitivity disk containing 10 ug ofampicillin was added. Resistance was demonstrated by growth in the zone immediately surrounding the disk. . 2143 Characteristic NumberofSpontancous Revertants The mean numberofspontaneous revertants per plate in the vehicle controls that is | characteristicof the respective strains was demonstrated by plating 100 iL aliquotsofeach culture along with the appropriate vehicle on selective media. |= sana (TORTS omany SentiEe78 Pa (--v 12 `Covance Study 22900-0-4090ECD 215 Tester Strain Media 215.1 Culturing Broth "The broth used to grow overnight culturesofthe tester trains 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 (25mLper 15 x 100mm petri dish) was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5% (wiv) agar and 0.2% (w/) glucose. 2153 Top Agar for Selection of Revertants |- Top (overlay) agar was prepared with 0.7% agar (w/v) and 0.5% NaCl (w/v) and was | supplemented with 10 mL of 1) 0.5 mM histidine/biotin solution per 100 mL agar for selectionofhistidine revertants, or 2) 0.5 mM tryptophan solution per 100mLofagar for Selectionoftryptophan revertants. `When $9 mix was required, 2.0 mL ofthe supplemented top agar was used in the overlay. However, when S9 mix was not required, water was added 10 the supplemented top agar (0.5 mL of water per 2 mLofsupplemented top agar) and the resulting 2.5 mLofdiluted. supplemented top agar was used for the overlay. This dilution ensured that the final top agar and amino acid supplement concentrations remained the same both in thepresenceand absenceof S9 mix. oa oresnT --- comes gentes -~ O vance m Study 22900-0-4090ECD 13 22 Test Article TestArticle: Haskell Number Sm) 16 Physical Description: Date Received: ~ White solid 8 August 2001 The test iro =: supplied as awhite solid. The test article was. stored in a container at ambient temperature. The Sponsor was responsible for the determination and documentationofthe analytical purityofthe test article. Any unused test article andlor the original test article container were retumed to the Sponsor: 23 Control Articles 231 Vehicle Controls || as used as the vehicle. Vehi.cle controls were plated for al tester strains in | he presence and absenceof 9. The vehicle control was plated on selective agar using a 50 | ALaliquot (equal to the maximum aliquotoftest article plated), along with 100 uLofthe appropriate tester train and 500 kLof S9 mix (when necessary). 232 Positive Controls The combinationsofpositive controls, activation condition, and tester strains plated : concurcently with the assay are indicatedbelow (Table 2, next page). , ized. 0058 Loonie 750ASE! - compen So - [`rCovmanece Sntundy n22p500--4090ECD . TTaabbllee2d., PPoossiitidvveeColnotrmolwss Ter Sin [on pune cont 14 f-- Taasss Taio :.> s2nmsionleurpeynne 2amnesmircine 32iso snso ais IL! S Sodummaiede t : diamante 33 3 || AaIiSs? :: Siacimnet Wear : Samicantrene 23 0 v Weir : e "niroequmnoinde Noeid dc wo | 2321 Source and Grade of Positive Control Articles Benzo{alpyrene (CASH 50-32-8; purity 297%), 2-itrofluorene (CASH 607-57-8; purity - 229908%%)),, nsodddi-unmitarzoiqduein(oClAiSnHe N2-6o6x2i8d-e22(:C;ASpuHri5t6y-5279-95%;)p,urIiCtRy-219991%()CwAeSr#e 1o7b0t7a-i4n5e-d0f;rpoiu-nrity Sigma Chemftal Co. All were prepared in DMSO, except for sodium azide, which was dissolved in deionized water 233 Sterility Controls || 2331 TestArticle | | "The most concentrated tet article stock solution was checked fo serlity by plating a 50 kL . aliquot (the same volume used in the assay) on selective agar. . 2332 S9Mix The 59 mix was checked for sterility by plating 0.5 mL on selective agar Company Saaltized. Doss not containTSCA Ct --~ (nm--y 1s `ovance Study 22900-0-4090ECD 24 Liver Microsomal Enzyme Reaction Mixture (59 Mix) 241 $9 Homogenate 59 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 (200 mg/mL in com oi) at S00mg/kg (Ames et al, 1975). 242 $9 Mix | | The S9 mix was preparedimmediatelyprior to use and contained the components indicated | below (Table 3). Table3. $9 Mix Components - Component Quay I0M NaH2POYNegHPOR pi 7.4 00100mm. 0021002MMNGlAuDcPose phosphate 00002m8m 05932Ho5mMogKeCnIsie2M MgCly 004m clon ee 100m a. EXPERIMENTAL DESIGN "DoseRangefinding Study . "The growth inhibitory effect (cytotoxicity)ofthe test article to the test system was determined in order to allow the selectionofappropriate doses to be tested in the mutagenicity assay. | | Sempany Saritzed, Dass nat contin TSCA BL | | - (rum vance Study 22900-0-4090ECD 16 L311 Design The dose rangefinding study was performed using tester srains TA100 and WP2urA in the presence and absenceof $9. Ten doses of test article, up to 5000 g/plate, were tested for cytotoxicity (one plate per dose). 312 Rationale The cytotoxicityofthe test article observed in tester train TA100 is generally representative | of that observed on the other Salmonella typhimurium tester strains and becauseofthe | comparatively high numberofspontaneous revertants per plate observed with this strain, gradationsofcytotoxicity 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 steains have and thus, a different rangeofcytotoxicity may be - observed. Also, the cytotoxicity induced by a test article in the presence of $9 may vary greatly from that observed in the absenceof $9. Therefore, this would require that different test article dose ranges be tested in the mutagenicity assay based on the presence or absence | ofthe microsomal enzymes 313 Evaluationofthe Dose Rangefinding Study Cytotoxicity is detectable as a decrease in the number of revertant colonics per plate and/or by a thinning or disappearanceofthe bacterial background lawn. : 314 Selection of the Maximum Dose for the MutagenicityAssay : No cytotoxicity was observed in the dose rangefinding study and the highest dose level of test 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 by a decrease in revertant frequency and likely was artifactual). company Santized. Doss not contain TSCAC --- ----) 1 ovance Study 22900-0-4090ECD 32 Mutagenicity Assay 321 Design The assay was performed using tester sirains TA98, TA100, TA1535, TA1537 and WP2uvrA in the presence and absenceof$9. Dosesof the test article were selected based on the results of the dose rangefinding study. The results ofthe initial mutagenicity assay were confirmed in an independent experiment. | 322 Frequency and RouteofAdministration The tester strains were exposed to the est article via the plate incorporation methodology originally described byAmes ef al. (1975) and Maron and Ames (1983). This methodology as been shown to detect a wide rangeofclassesof chemical 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 colonieswere counted. All dosesofthe test article, the vehicle controls and the positive controlswereplatedin 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, test phase, tester train, activation conditionanddose level. The $9 mix and dilutionsofthe est article were prepared immediately prior 10 their use. When $9 was not required, 100uLoftester srain and 50 iLoftest or control aticle were. added to 2.5 mL of molten selective top agar (maintained at 45 + 2C). When 9 was required, 500 ul. of9 mix, 100 uLoftester strain and 50 ul.oftest or control article were - company Senitized: 0088 7 {ein TSCACE! - (ee 1s "Covance Study 22900-0-4090ECD `added to 2.0 mLof molten selective top agar. After the required components had been added, the mixture was vortexed and overlaid onto the surfaceof25 mLof minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and incubated for 52 + 4 hours at 372C. 9 Sorgen Plates which were not evaluated immediately following the incubation period were held at 5 3C until such time that colony counting and bacterial background lawn evaluation could take place. 34.1 Bacterial Background Lawn Evaluation `The conditionofthe bacterial background lawn was evaluated macroscopically and ~ `microscopically (using a dissecting microscope) for indications ofcytotoxicity and test article precipitate. Evidenceof cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all platesatthat dose on the data tables using the code system described below (Appendix B). 342 Counting Revertant Colonies || Revertant colonies were counted by automated colony counter. 4 DATA . 0 oaasain For allreplicate platings, the mean revertants per plate and the standard deviation were calculated (Appendix A). ~~ rp) 19 `ovance Study 22900-0-4090ECD . 42 Assay Acceptance Criteria Before assay data were evaluated, the criteria for a valid assay had to be met. The following criteria were useto determine a valid assay: 421 Tester Strain Integrity 42.1.1 rfa Wall Mutation All Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet, demonstrating the presenceofthe rfa wall mutation. 4212 pKM101 Plasmid Tester strains TA98 and TA100 exhibited resistance to ampicillin, demonstrating the presence ~ of the pKM101 plasmid. | 4213 Characteristic Number of Spontaneous Revertants All vehicle control cultures exhibited their characteristic numberof spontaneous revertants per plate, demonstrating the requirement for histidine (Salmonella typhimurium) or tryptophan (Escherichia coli). The acceptable ranges for the mean vehicle controls were as follows T sen NumofbReeverrs hTThoois psiew Wfhadr iisa [~ Gamay$= osos wsoace - (e---- 20 `Covance Study 22900-0-4090ECD 422 Tester Strain Culture Density `The cell densitiesofal tester strain cultures were greater than or equal to 0.5 x 10 bacteria/mL (with the exception of TA98 in Experiment 22900-B1, 0.4 x 10 bacteria/mL), or the optical densitiesofthese cultures reachedatarget value demonstrated to produce cultures with at least 0.5 x 10" bacteria/mL, demonstrating that appropriate numbersof bacteria were plated. 423 Positive Control Values | 423.1 Positive Control Values in the Absenceof S9 Mix `The mean valueofthe positive control for each tester strain exhibited at leasta 3-fold increase over the mean value of the vehicle control for that strain (with the exception of tester -- strain TALS37 in Experiment 22900-Cl, see Protocol Deviation), demonstrating that the tester strains were capableofidentifying amutagen. 4232 Positive Control Values in the Presence of S9 Mix (9 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 strain, demonstrating that the S9 mix was capableofmetabolizing a promutagen to its mutagenic form(s). | An acceptable positive control in the presenceof S9 for a specific strain was evaluated as "having demonstrated the integrityof the 9 mix and ihe abiloiftthye tester strain to detecta | mutagen. [= Company Santized. 09587"(cantain TSCACE! - m-- ] 21 ovance Study 229000-4090ECD 424 Cytotoxicity Aminimumof three non-toxic doses was used to evaluate assay data. Cytotoxicity can be detected as a decrease in the numberofrevertant colonies per plate and/or by a thinning or disappearanceofthe bacterial background lawn compared to the appropriate vehicle control. 43 Assay Evaluation Criteria Once the criteria fora valid assay had been me, responses observed in the assay were evaluated as follows: 431 Tester Strains TASS, TA100 and WP2uwrA | Fora test article to be considered positive, it had to produce at least a 2-fold increase in the `mean revertants per plateofat least oneofthese tester strains over the mean revertants per - plate of the appropriate vehicle control. This increase in the mean numberofrevertants per plate had to be accompanied by a dose response to increasing concentrations of the test article ! 432 Tester Strains TA1S3S and TAIS37 | Fora test article to be considered positive, it had to produce at least a 3-fold increase in the meanrevertantsper plateofat least one of these tester strains over the mean revertants per plateof the appropriate vehicle control. This increase in the mean numberof evertants per - plate had to be accompanied by a dose response.to increasing concentrations of the test . article. | ompny Sentized. Das ass cootte"contain TSCA 05 || ~ F`Ceovancne Stundy 22]900-0-4090ECD 2 s. RECORDS TO BE MAINTAINED All raw dats, documentation, records,th protocol, and the final report generated as a result of this study will be archived inthe storage facilities of Covance-Vienna for i least one year following submission ofthe final report to the Sponsor. Afer the one year period, the Sponsor may elect to have the aforementioned materials retained i the storage facilites of Covance-Vienna for an additional periodof ime or sent to a storage facility designated by the Sponsor. 6. STUDY RESPONSIBILITIES | Function Study Director Laboratory Supervisor Responsible Person(s) 2. RESULTS | 7 Test Article Handling em: a solution at 100 mg/mL in dimethylformamide after heating. In DMSO a 100 mg/mL, the test article formed a olution that foamed when heated and vortexed. DMSO was selected asthe vehicle. At 100 mg/mL, which was the m.ost concentrated stock prepared for the mutagenicity assay, the tet article formed a. transparent, colorless solution afer heating to 45C for 3 minutes; it remained frely soluble: at all | `succeeding lower dilutions. : --- iainTORCH compeny asiined, Do13H 72 Dose Rangefinding Study (Appendix A, Tables 4 and 5) A dose rangefinding assay was conducted on the est article using teste szains TA100 and Wu (one plate pe dose; Experiment 22900-A1; Tables 4 and 5). Ten doses of test article, from 6.67 to 5000 pg/plate, were evaluated inthe presence and absenceof$9. Apparently normal growth was observed in both tester stains at all doses evaluated with and without $9 the reduction in background lawn observed in tester rain TA100 without $9 was not accompanied by a decrease in revertant frequency and likely was artifactual). However, the tet article was found to be incompletely soluble i the aqucous op agar at a dose of 5000 pgplate with and without 9. || 3 9M-utSaugmenmiacriytyDAastsaa)y (Appendix A, Tables 6,8- Individual Data, Tables 7, | + Based upon the results of the dose rangefinding uty) evaluated in | he initial mutagenicity assay in all five tester strain at doses of 33.3, 100,333, 1000, 3330 | and 5000 pg/plate with and without S9 (Experiment 22900-B1, Tables 6 and 7). All doses of the test article, as well asthe concurrent positive and vehicle control, were evaluated using thre plats per dose. Apparently normal growth again was observed in all tester trains at all dos fi ith and without $3. Tn addin, the test tice precipiaied fom solution at doses 21000 plate with and without S9. Revertant frequencies for ll doses (i -- all tester srains with and without $9, approximated of were less than those observed in the concurrent vehicle control 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 teste strains at all doses evaluated with and without $9, and the test article again precipitated from solution a doses 21000 pgplate with and without $9. Revertant frequencics for all doses oC ---- in all ester sony Santas: Dost 1ST rscacal - -- 24 `ovance Study 22900-0-4090ECD . strains with and without $9, again approximated or were less than control values. Except for tester train TA1537 without S9 in Experiment 22900-C1 (see Protocol Deviation), all positive and vehicle control values were within acceptable ranges in both assays. All criteria fora valid study were met. 8. CONCLUSIONS "The resultsofthe Salmonella - Escherichia coli/Mammalian- Microsome Reverse Mutation Assay indicate that under the conditions of tis sucNNR+ nt `mutagenic in anyofthe tester strains in the presence or absenceofan exogenous metabolic `activation system containing induced hepatic microsomal enzymes from AroclorTM 1254-treated rats. 9. PROTOCOL DEVIATIONS | WInerEexpsecroiremdenetve2n29t0h0o-uCg1h,ththectoenscturarrteinctlep-otsreiattievde ccuolntturoels vfaolruteesstweerrsetrbaienlToAw1a5c3e7piwaitbheoumtiSt9s (all plates had no revertants). However, the vehicle control values for tester strain TA1537 `without $9 were within acceptable ranges. In addition, the positive control values for tester strain TA1537 with S9 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 integriotfy the study or the conclusions derived from it. - Compan;Senos Boastsrin TEATS) - [-- `Covance Study 22900-0-4090ECD 10. LIST OF REFERENCES Ames BN, McCann J, Yamasaki E. Methods fordetectingcarcinogens and mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research 1975; 31:347-64. ; `Green and Muriel, 1976. Green MHL, Muriel WJ. Mutagen testing using rp" reversion in Escherichia coli. Mutation Research 1976; 38:3-32. : Maron and Ames, 1983. Maron DM, AmesB. Revised methods for the Salmonella Mutagenicity Test. Mutation Research 1983; 113:173-215. | Vogel and Bonner, 1956. Vogel HJ, Bonner DM. Acetylomithinaseof E. coli: Partial purification and some properties. --_-- JBiol Chem 1956; 218:97-106. - Pa ca_ pisSHE - [p-- 26 `ovance Study 22900-0-4090ECD Appendix A. Experimental Data Tables | ne a | oe - prsea ~ [i --) I. `CovanceStudy 22900-0-4090ECD Table 4 Dose Rangefinding Study ~ TA100 TDatoe nPeidoh [9N-00E 1 N] bTDaertpe eCeorumnteen1nd:0t: m29e004n1 01.Naw01 Vebicle: DMSO. Plating Aliquot. S0uL. TAI00ReverPealnise poe voatene winsy Tormeas BeLgnond Pine Bort " ~ Wis 9 Rerevr e Ba5got pwe Evian' ~ TeAricle 667 0 ~ N 100 n x wo ~ ns 1s x 0 ~ | PY " x % x | 100 139 N 8 N - 333 7 N 7 N a x 5 ~ 100 106 x x mo TM N 0~ 500 0s ne 0x BF NurecLiRkveOeBumiaedsaotCAnotpsea:tp =&or" - reoF" |o ps Table 5 Dose Rangefinding Study - WP2uwrA. DuvescicPleact: Du25s-0o101 DPuiengCMoigtrots 0_5010v01 [---- wnso vious) urine RevPeTroteeants TT = Tanne oon BoacPkngerotund N N Revrerrateeanis [ a Background. ort x x | 100 1s N n N | | 33 PY 3 n Ny "15 yN wo uy N - = i ww N uy wo a y ny 0 2x N sow n > 5w | F--en | P=precipitate O =obscbyuprercipeitadte | . - 7 -- yuh) 2 `ovance Study 22900-0-4090ECD Table 6 Initial Mutagenicity Assay Results- Individual Plate Counts TDeutcAPlme[ te:> -- PiaNovol ) DDaotemCnoeund>: 2I6ONoRv-I01 Vetiele Dyiso Pling Aliwor.__50 Dope TW Revers er ic TAO ss mais were sSBoeacmm JrVenice TET rT Tr aT es mm us usm ow sw 7 70 mae sn Tears SWMYahaehsE M32 a22003s30 d MW9oemmNim16s o0r1w6 ww5ws0Mo3s Hb M0e6 ns l1b8 NNN SB0o00 hAME n3nusoDouMN b oUsle ehe Mbu ioeosb alGmssw5 nDtBmienoBa NWoww FosiveConsob $7512.362 9% 108 1090 11 165 138 181 4g 178 21 IN VMeieocnm:mlons m2 0 N68 Wm wR 653 ww N - Teh SG BlhuMeE UnsB osBBooswoomwWM wma wune23w s59a mmmR n mow ooNNN BoWwooMkRi RUdLoEs sBmeomuuoum UbIlS0IoC 3S00e2w3 w bnaonsooBowow oNNWw FostbeCono 208 192 216 101 M28 ME 666 656 9 1064 867898 158138 89 N BaNketrtonBRaenrCotet: ad hesbent Ppa `TTroSsoS DSeioremene iTaeoseoiepc TTTAAHBDDS msdaimriee TSLooorrveeenne HTohn SDiimmiens Bloivgies WTeboNrh. Skmaino S{obreeenp KR& =+oF re - [mm-- 30 "Covance Study 229( 1090ECD Table 7 Initial Mutagenicity Assay Results - Summary TestArT ieID --] Experimen0t; 2290051 - VDeabticcPlh:ied: p13u:sNoow0l PlDaattienCgoAulntieqdu:ot__2064:LN.ov0L DoPise TA Mesa Reser er lt ith Standard Devon TAG TAISN TASS WorswBoatcwk.s e MVehiiccleoC:onReat Liver 03 Viwen oSDS ewwoSDs. WemsoSzO Meam mSDo. eradde wBioghg Mm 2B 44 Bow wdMe@owU+ 6oBw os11 1w80 o35 ww15ooe04 NN Swooo $0 bkkee nooww o54g m6me s4 omads ss3oomomso:so3 oolmm aoo1 wwNw posiveConta wow wn os wos em 3 BN MViecttotseomCeosn:tNro!ne: Bos wo moa os 2 16 2 WN - Tethide WwBoiog dd4se 374 %m m0o6 mm12 2o6s 76oe 443 d110 33o1 NNoN Sm0o0oekk4e od8Ne 552 om4som24 wm5ooss2 6870s23 onomm oeao2wow | PositiveControl' 205 o1e2 999 44 633 47 943 106 ns 3s ON *Bamckgand avnRBerhioannCodeO: chs Mesh Bepris VNTIAADS Saseumcremeso 3Jsiuagpmmee (TITNAAORS)S siJeoeiddvmioumisvuiaeeme S2L0ooLuarppnaee | ThNiT dSiamimmmemiiee S1o5/vgwpwee WTrNeSn dIaRiDnomeNoxde p10rngipivne | & oSie |" otis et -- 5 `ovance Study 22900-0-4090ECD Table 8 Confirmatory Mutagenicity Assay Results - Individual Plate Counts DuTeoPircss. WE oNovaE l ] fDE ateeCp rune:m03e79D0e0cn811, 10-Dec01 vec: ovo PuigAluoe__50 rie mot Seer re mm as er woe pm.oens iVores thr y Hy p 2, a mw mowm www wm su ow ono oN TeaBwk3o3oAwsda3m% Em womm% emmmo o a>onnw hun nmswisa1 woopw oo3 oonmoowoy mwooamemsmExELEE mREiImmS aAsnRaaRnk aArsNi)AonkBBEoAhBoNow Toca 8 0 NE 8 he 0 6 Ww a ores ie wmm ome ens uss momo ow |i - Twice S FmEE3 o wm mu M WAM wNRmW OS W o7 E n3 s8 o77i8ommoommoonH oN || BT-e mmrmErmslzaugmmanoiaennsetlfiliaiizmRoiwEnEs recat 3720 70 Wow a Ew 0 0 0 3 mom x eaiavnEg nmisnncoce ans Asis p=rine aTBnBys I itrm, ei E Dawpge T TTNAEBS dna let sTLooorreeonne I Thon Tomei oPtea DVioon CDamwomitomerioste [Toobroee oope2\ @ oo - ooa - w--) 2 0 `ovance Study 22900-8-4090ECD Tabled Cgnfirmatory MutagenicityAssayResults - Summary Test Arle ID: Experiment 1D: 2290081 Date Plated: 59-Nov-01 Date Counted: 07-Dec.01, 10-Dec-01 Vehicle: pms PlaingAliquot: _ 50 ul. Dore VeMbiiccroasnoeamteisv:er MeasRevers erhcwith SundudDevon pBoacnhde Wemnoe 50 WeTmHO0 WeamssSO WeamisSO Wewmer5aD tae moa ow ow 6 wz ows oN Tae w20omms Bow s3 Moms wo hosw wom somoommow mwoo p 4 w0 55%% w6 was 61 71 oswos3 19 oobu 32 0ow 45 1ws oz6 NNn W 8 o1 w6 s4 oowN wwmo o33 ooam 12 ooww Psi Cont TE Vehice Contr Bos omo6 6 2 6 4 wm 3 0N - Tease w33owks mm3s W%w WBw oom8 21 76 31 1osm 53 xNn Wmooh m6 os3 wos s2mom 43s0s 22 womw 3 NwN SDoowpo 2no:4%wo wswo 61 36 21 wwmm 23 ooww positeCool" wo ws mows om 0 0 ww x BackLagwnrEaotounnCoddes: | VTTAAO abmienolwmibreceoe 1ssiughgwe CTTAAI aZetihvmoalnloiuene ZLoosppuse ATIASSTS ZDmimmesmdeeernse 2ISSiggppuee TTAILSS ICiRoId9uHmaic 2Sov0ppmae WPlrh 2amnoanicese 250g WEA. daimieolonsNaxde 0) once or - nt Goons ~ [-- 3 `Covance Study 22900-0-4090ECD Table 10 HPiistgorIinccaolrCpoonrtartoiolnDpaiteatod Report Period 1 e Sean m Tht Tam lo Tams Ty AI Wher VSaedmaRreDoowisnpewe m722 wuss B4as 0446 06s MVeinenr awawns x> a5 Com wm wm wm wm om Se ouTT n Tas c Talo e TASS TATs Wem SuMnedaReDvoeritnpe ls wsae smess oanse 4sse issso MMeiiom WSowm os3 om] s Cou wm wm ~ Sou Thon TTaY lo TASS TAIT Wed VSaenmdaRDevviwiomnpse unsas mms3s ta1ainm2 smead Mi umm awi owmsmom m ws m Cot mow aw mm Se o Tm T cm Ta mTAv I m TAISS mTAImWw eak MSanedemrRDeovvimiosnpse 2B5es wdeess mueta meess am0s Mviimai _ wmoomw wm w mws a1 coum cw om om mw WWWTIhaSL I pD Temeom m mnme SDhLeoEempIree NNRTaSNmT M amomhme n e eJfSroiileeeenmd - --ectinat cm - Ia 34 ovance Study 22900-0-4090ECD Appendix B. DefinitionsofBacterial Background Lawn Evaluation Codes || | - ct not conttaein TSCACE! Company Sentized: 0 - Om--7 35 `Covance Study 22900-0-4090ECD Bacterial Background Lawn Evaluation Code `The condition of the background bacterial lawn is evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate as follows: CODE DEFINITION CHARACTERISTICS OF BACKGROUND LAWN N Normal Ahealthy microcolony lawn. R Reduced Adistinet thinningof the microcolony lawn and an increase in the sizeofthe microcolonies compared to the vehicle control plate. A Absent A complete lack ofany microcolony lawn. O Obscuredby The background bacterial lawn cannot be accurately evaluated due to Precipitate microscopic test article precipitate, macroscopic test article precipitate, or plate coloration. _ E Enhanced Adistinet thickeningofthe microcolony lawn and an increase in the sizeofthe microcolonies compared to the vehicle control plate. Evidenceofmacroscopic test article precipitate on the plates is recorded by addition of the following precipitate code to the code number used to evaluate the conditionof the background bacterial lawn. P Precipitte Macroscopic precipitate observed on the plate. | Compeny Santigaed: DO notcontain TSCACBE | || - [-- x Covance Study 5] onc Appendix C. Quality Assurance and Compliance Statements. | | - ss 08 - [-- l 3 `Covance Study 2290M-4090ECD QUALITYASSURANCE STATEMENT Comm] uc tation Assac y with a ConCfoirlmiaMtaomrmyaAlsisaany-Microsome Reverse The report has been reviewed by the Quality Assurance Unit of Covance Laboratories Inc. in accordance with the Good Laboratory Practice regulations asset forth in the Environmental ParnodtetchteiOorngAagnieznactyio(nEfPoAr ETcSoCnAom)i,cTiCloloep4e0raotftiohneaUnSd.DeCvoedleopomfeFnetde(raOlERCeDg)ulPartiinocnisplPeasrtof792; `Good Laboratory Practice ENV/MC/CHEM (98)17; and any applicable amendments. The following inspections were conducted and the findings reported o the Study Director and study director management. Written status reports of inspections and findings are issued to Covance management according to standard operating procedures. Dates Reported to Study Inspection Dates Phase Director and Study Auditor r 24:0c1:2001 -- Protocol -- Review ne DI-- 2E4.0cM1:I2S00S1ES-- errP.sCiceres - 13-Nov-2001 89 Mix Preparation 14-Nov-2001 J. Howard 07-Jan-2002 Draft Report Review 07-Jan-2002 C. Smith 08-Feb-2002 Final Report Review 08-Feb-2002 C. smith | peg Fee . Eel 02 : Representative, Quality Assurance Unit Date - ormpany Sat RSp T s ~ [e-- " Covance Study 22900-8%4090ECD STUDY COMPLIANCE AND CERTIFICATION Except that the test and cntrol article dosing solutions were not analyzed for stability, `homogeneity or accuracy ofpreparation, this study was conducted in compliance with the Good Laboratory Practice regulations as set forth by the Environmental Protection Agency (EP-A TSCA), Title 40ofthe U.S. CodeofFederal Regulations Part 792; and the Organization for Economic Cooperation and Development (OECD) PrinciplesofGood. Laboratory Practice ENV/MC/CHEM (98)17; and any applicable amendments. There were no other deviations from the aforementioned regulations or the signed protocol that would affect the integrityofthe study or the interpretationofthe test results. The raw data have { been reviewed by the Study Director, who certifies that the evaluationof the test article as. `presented herein represents an appropriate conclusion within the contextofthe study design and evaluation criteria. Study Director, 2S7 ito] / - `BaLctoenrFi.alStMauntkaogwesnkeis,iJsr, PhD, CGoevnaenticce.aVnideMnonlaecular Toxicology 2/2 "Study Completion Date Testing Facility Management: = C. Yi BBarcitaenriCa.l MMyuhtra,gePnReDsis Genetic and Molecular Toxicology Date 2fufoz Report Reviewed and Acceptedfor E.I du Pont de Nemours and Co. by: GSeproanlsdorKeSntnueddyyM,oPnhiDt.or -- Dae MSpaornisaoDroTnencehrn,icPahlDProject Monitor Date | Company Senikzed. Duss not ontein TSC