Document XzREd8YNMDykGMx5wQmrgEZdK

AR226-3171 TRAPE SECRET Study Title Authors Valentine 0. Wagner, m, M.S. MichelleL.Klug,B.S. ReportCompletion Pate November 16,2000 PerformingLaboratory BioReliance 9630 Medical Center Drive Rockville,MD 20850 for E. I. du Font de Nemours and Cocapaay Stiae Haskell Research Center DuPont Haskell Laboratory P.O. Box 50 1090 Elkton Road Newaric,DE 19714-0050 Peifonning Labogatory Study Number AA34BKL502001.BTL DuPont Project IP DuPont-4670 DuPont-4670 Bleverse Mutation Service Code Pagel of 63 Reverse Mutation DuPont-4670 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Study No. AA34BK.502001.BTL Standards 40 CFR 160 was conducted in compliance with Ifte U.S. EPA GLP and 40 CFR 792, the UK GLP Compliance Programme, Ihe Japanese wGiLthP mSetafnodllaordwianngdexthceepOtiEonCsD: Principles of Good Laboratory Practice in all material aspects Hie identity, strength, purity and composition or other characteristics to define the control substances have not been determined by the testing facility. The control substances have been characterized as per the Certificates of Analysis on file with the testing facility. Analyses to determine the uniformity (as applicable) concentration of Ihe test or control mixtures were not or performed by the testing facility. The Sponsor has not performed these analyses. The stability of me test or control substances in me test or control mixtures has not been determined by the testing facility. The Sponsor has not performed these analyses. Suhmitter/Sponsor: BioReliance Study Director: BioReliance Study Management: DuPont Registration Representative: E. I. du Font de Nemours and Company Wilmington.DE 19898 U.SA. iaw,3ic \fctU^ o, Valentine 0. Wagner, m, M.S. ^? Richard H.C. San, PhJ), \(0 f^o]/3,000 Date f6/^i^sop6 Date Date BioReliance Study No. AA34BK.502001.BTL TRADE SECRET Study Title Authors Valentine 0. Wagner, HI, M.S. MacheIleL.Klug,B.S. Report Completion Pate November 16,2000 Performing Laboratory BioReliance 9630 Medical Center Drive Rockvffie,MD 20850 for . I. du Font de Nemours and Company Sune Haskell Research Center DuPontHaskelS Laboratory P.O. Box 50 1090 Elkton Road Newaric,DE 19714-0050 Performing Labotatory Study Number AA34BK.502001.BTL DuPont Project ID DuPont-4670 DuPont-4670 Reverse Mutation Service Code Page 1 of 63 Company Sanitized. Does nS eontain TCCA CBI Reverse Mutation DuPont-4670 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Study No. AA34BK.50200LBTL was conducted in compliance wilfa the U.S. EPA GLP Standards 40 CPR 160 and 40 CFR 792, the UK GLP Compliance Programme, me Japanese GLP Standard and the OECD Principles of Good Laboratory Practice in all material aspects with the following exceptions: The identity, strength, purity and composition or other characteristics to define the control substances have not been determined by the testing facility. The control substances have been characterized as per the Certificates of Analysis on file with the testing facility. Analyses to determine the uniformity (as applicable) or concentration of me test or control mixtures were not performed by me testing facility. The Sponsor has not performed these analyses. The stability of the test or control substances in the test or control mixtures has not been determined by the testing facility. The St-snnww hac Tint ift.~.<"* *!---- --- -' Suhmitter/Sponsor: BioReliance Study Director: BioReliance Study Management: DuPont Registration Representative: E. I. du Font de Nemours and Company Wamington,DE 19898 U.SA. \Lyj^^ \foiwfct0, Valentine0. Wagner, ffl.M.S. Richard H.C. San, PhJ>. /6?^W<%000 Date /6//r^Sffff6 Date Date BioReliance Study No. AA34BK.50200LBTL Study Title: Quality Assurance Statement Study Number: AA34BK.5Q2001 -BTL Study Director: Valentine 0. Wagner, III. M.S. TOraituayfiastewfanaaedlntoa aerie* of approach, QualifyAssurance monitors each in-prooess phases. Using a random omese phases over ajsories of sampling Procedures, documentation, the study Is performed in equipment records, ate. .are examined; in studies. orderto assure that Reguatfons%CFR accordance with the 58). the U.S. EPA OLPs U.S. PDA Good LaboratoryPractice (40 CFR792and 40 CPRleO). the UK GLP Regulations, Practice and the Japanese to assure that QLP Standard, the study to and the OECD PrtndpteSof Good Laboratory Standard Operating Procedures. conducted according to the protocol and relevant The following are the inspection inspections of this study. dates, phases inspected, and report dates of QA Inspect On Phase 25-Aug-OO - 25-Aug-OO To Study Dir 25-Aug-OO To Mgmt 25-Aug-OO ProtocolReview Inspect On Phase Inspect On Phase 12-Sep-OO - 12-Sep-OO To Study Dfr 12-Sep-OO To Mgmt 14-Sep-OO Preparation of 89 mixture 30-Oct-OO - 30-Oct-OO To Study Dir 30-Oct-OO To Mgmt 01-Nov-OO Draft Report Inspect On Phase 14-New-OO - 14-Nov.OO To Study Dir 14-NovOO To Mgmt 14-Nov-OO Draft to Hna! Report Trehsiusltrsepaocrctudreasteclryibreesflethctetmheetrhaowddsaatnadopf rthoceesdtuudreys. used in the study and the reported / Q ^ / ^ ^ ^ Diane Brecha, B.S. QUALITY ASSURANCE I^AjQ^^JB DATE BioReliance StudyNo.AA34BK.502001.BTL 3 CERTIFICATION DuPoat-4670 We, from this study. ' 1 ; an accurate evaluation of data obtained Issued by Study Director: \[ejU^ 0. W^^'3^ Valentine 0. Wagner, iBfM.S. BioReliance I^^Sc Date Aiq>rovedby Sponsor Study Monitor: r<0^ ^ J-J 9v\ >J Maria Donner.PhD. Senior Research Scientist JSTfJpy ^,000 Date BioReliance Study No. AA34BK.502001 .BTL ial Reverse Mutation DuPont-4670 TABLE OF CONTENTS Page Good Laboratory Practice Compliance Statement.................................................................. 2 s Quality Assurance Stetemeht.................................................................................................. 3 Certification..................................................................................................................4.......... Table of Contents......................................................................................................................5 Study Information .-..................................................................--..............................................7 Summaiy..........................................................................................................................8....... Purpose.............................................................................................................................9....... Characterization of Test and Control Substances................................................................. 9 AteterialsandMethods.................................................................................................1..0........ Results and Discussion........................................................................................................ 15 Conclusion...........................................................................................................................1..5 References...............,................................................................................--........................1..6. Data Tables............................................................................................................................ 17 Table 1: Preliminary Toxicity Test in Salmonella typhifswrwm 17 Table 2: Preliminary Toxicity Test in Salmonella typhimurwm TA98........................... 18 Table 3: Preliminary Toxidty Test in Salmonella typhimwium TA100......................... 19 Table 4: Preliminary Toxicity Test in Salmonella typhimwium TA1535....................... TA1537 Table 5: Preliminary Toxicity Test in Escherichia coli WP2 .......................20 Table 6: Mutagenidty Test in Salmonella typhimwium KwA.................................21 TA98 without Table 7: Mutagenidty Test in Salmonella typhimwium TA98 with S9 S9.....................22 Table 8: Mutagenicity Test in Salmonella typhxmwium TA100 ..........................23 without Table 9: Mutagenicity Test in Salmonella typhtmuman TA100 with S9.............-...24 25 Table 10: Mutagenicity Test in Salmonella typhimwiwn TA1535 S9........................ without Table 11: Mutagenicity Test in Salmonella typhimwiwn TA1535 with S9.................26 Table 12: Mutegenidty Test in Salmonella typhimwium TA1537 S9......................27 without Table 13: Mutagenicity Test in Salmonella typhimwium TA1537 with S9.................28 S9......................29 BioReMance Study No. AA34BK.502001JBTL 5 Reverse Mutation DuPont-4670 Table 14: Mutageoicity Test in Eschericfaa coli WP2 awA without S9 Table 15: MutagenicityTest mEschericfda coli WP2 uwA with S9 ..........................30 Table 16: Mutagenicity Test in Salmonella typhimuriwn TA98 ...............................31 without Table 17: MutagenicityTest in Salmonella typhimuriwn TAPS with S9..................... 32 Table 18: Mutagenieity Test in Salmonella typhimuriv.m TA100 S9..........................33 without Table 19: Mutagenicity Test in Salmonella typhimuriwn TA100 with S9...................34 Table 20: Mutagenicity Test in Salmonella lypSwnwwm TA1535 S9........................35 without 36 Table 21: Mutagenicity Test mSalmonella typhmwriwn TA1535 with S9 S9................. Table 22: Mutagenicity Test mSalmoneila typhimuriwn TA1537 ......................37 without Table 23: Mutagenicity Test in Salmonella typhimuriwn TA1537 with S9.................38 Table 24: Mutagenicity Test in EscherikSaacoK_WP2uwA S9......................39 Tabte^Sy MaE^enicayTesrfi&rcAerfcAfo coli WP2 uwA wnhoutS9........................... with S9 40 Table 26: Salmonella/E. coli Mutagenicity Test- Summary ...............................41 of Results Bl Table 27: Salmonella/E. coli Mutagenicity Test - Sununaly of Results B2 .....................42 .....................43 Appendix A: Historical Control Data.................................................................................... 44 Appendix B: Study Protocol.................................................................................................. 46 Appendix C: Information for Japanese Regulatory Agencies................................................ 57 BioReliance Study No. AA34BK.502001.BTL Bacteria! Reverse Mutation STUDY INFORMATION Substance Tested: i Synonyms/Codes: DuPont.4670 Haskell Number: 24547 c -- | J B ^ Composition: Known Impurities: Not supplied by the sponsor Physical Characteristics: Amber sticky gum Stability: The test substance appeared to be stable under me conditions ofthe study; no evidence of instability was observed. Sponsor: E. I. du Font de Nemours and Company Wilmington, Delaware 19898 U.SA. Study Initiated/Completed:August 24,2000 / (see report cover page) In-Life Initiated/Completed: August 29,2000/October 04,2000 BioReliance Study No. AA34BK.502001.BTL 7 Cempany SaniSiaedl. n@8 TSCA CBI Reverse Mutation DuPont-4670 : SUMMARY f i The test substance, H-24547, was 'Hestedin th$ bacterial reverse mutadon test using 5; typhnnwwm tester WP2 uvrA in Ihe strains TA98, TA100, TA1535, and TA1537, and E. coli tester strain in two phases, uspinregstehneceplaanted ianbcsoernpcoeroaftiAornomcleotrh-oindd. ucTehde rfairtslitvpehraSs9e., tThheeptreeslitmwiansarpyertfooxrimcietdy test, was used to establish the dose range for the mutagemcity assay. The second phase, the DHrtagenicity test (initial and independentrepeat assays), was used to, evaluate the mutagenic potential ofthe test substance. (' Acetone was selected as the compatibility with the target solvent of choice based on solubility of the test substance and 5300 mg/mL. cells. The test substance was a clear solution in acetone at In the preliminary toxicity test, the mgxnnum dose tested was 5000 ug per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 uL plating aliquot Neither precipitate nor toxicity was observed. Based on the findingsof the toxicity test, the maximum dose plated in the mutagemcity test was 5000 ug per plate. m the mutagemcity test, no positive response was observed. In the initial mutagemcity assay, the dose levels tested were 5000, 3333, 1000, 333 and 100 (ig per plate. Neither precipitate nor toxicity was observed. In the independentrepeat assay, me dose levels tested were 5000,1800,600,200 and 75 ug per plate. Neither precipitate nor toxicity was observed. BBII|l|BIIIHIIBII^^BBIHBiacte^a^ --All criteria for a valid study were met as described ja. the protocol. The results of the with an Independent Repeat Assay indicate that, under me Reverse conditions of this Mutation Test study, the substance did not cause a positive mutagenic response with any of the tester strains test in the presence and absence ofAroclor-induced rat liver S9. BioReIiaace Study No. AA34BK.502001.BTL Bacterial Reverse Mutation DuPont-4670 PURPOSE Hie pmpose of this study was to evaluate the mutagenic potential of the test substance by sasuringits ability to induce reverse mutations at selected loci of several strains of Salmonella atybpshenimcewoiufSm9aancdtivaattitohne. ttyptophan locus of Escherichia coli WP2 nvrA in the presence and CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test assigned substance, H-24547, was received by BioReliance on 17 August 2000 aad was the code number AA34BK. The test substance was characterized by the Sponsor as an amber sticky gum that should be stored at ambient temperature. August 2003 An expiration date of 15 was provided. Upon receipt, the test substance was described as a yellow solid and was stored at room temperature, protected from exposure to light and moisture. obtaTinheed vfieohmiclFeisuhseerdStcoiendteifliivce. r H-24547 to the test system was acetone, (CASfr 67-64-1), Positive controls plated concurrently with the mutagenicity test are listed below: Strain All Salmonella Strains WP2wrA TAPS TA100.TA1535 TA1537 WP2wrA S9 Activation + Positive Control 2-aminoarrthracene (Sigma Chemical Co.) 2-nitrofluorene (Aldrich Chemical Co., Inc.) sodium azide (Sigma Chemical Co.) 9-ammoacridine (Sigma Chemical Co.) methyl methanesulfonate (Aldrich Chemical Co., me.) Concentration (u.g/plate) 1.0 10 1.0 1.0 75 1,000 To determine the sterility of the test substance,the highesttest substance dose level used in me mutagenicity test was plated on selective agar with an aliquot volume equal to that used in the assay. BioReliance Stuffy No. AA34BK.502001.BTL C^npany Sanitized. Reverse Mutation DuPont-4670 Test System MATERIALS AND METHODS The tester strains used were the Salmonella typhimwiwn histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Asaes et al. (1975), and Escherichia colt tester strain WP2 ywA. 1998 directly Salmonella tester strains were received on 11 August 1998 and 10 November Scorn Dr. Brace Ames, University of California, Berkeley. E. coli was received on 01 July 1987 from (he National Collection of Industrial and Manse Bacteria, Aberdeen, Scotland. 'Pester sttams TA98 and TA1537 are reverted from histidine hisridiae independence (prototrophy)by franaeshiftmutageos. dependence (auxotrophy)to by mutagens that cause basepair substitutions. Tester strain TTAes1t0er0sitsrarienvTeArte1d53b5yismruetvaegretneds that cause bom frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather man frameshift mutations (Green and Muriel, 1976). Overnight cultures were prepared by inoculating from me appropriate master plate or from the appropriate frozen permanent stock into a vessel containing -50 mL of culture medium. To assure mat cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 372C approximately 12 hours before me anticipatedtime of harvest Each culture was monitored spectrophotometricallyfor turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3xl09 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates, and the data is on file but not presented in mis report. The study was conducted to comply with OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July 1997 and with the PInhtaerrmnaatcioenuatilcaClsonfoferrHenucmeanonUsHea(r1m9o9n6isaantdio1n99o7f).Technical Requirements for Registration of Metabolic Activation System Aroclor 1254-induced rat liver S9 was used as me metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 prepared by BioReliance was batch prepared on 22 June 2000. The S9 purchased from MolTox was batch prepared 17 December 1999. All S9 batches were stored at <-70C until used- Each bulk preparation ofS9 was assayed for its ability to metabolize 2-aminoanmracene and 7,12- dimethylbenz(a)anthraceneto forms mutagenic to Salmonella typlwnuritan TA100. Bion.eiiance Study No. AA34BK.502001.BTL 10 The S9 mix was prepared immediately before its use and contained 10% S9, 5mM glucose-6-phosphate, 4 mM p-mcotinamide-adenine dmucleotide phosphate,8 mM MgCl; and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphatebuffer at pH 7.4, was prepared immediately before its use. To confirm the sterility ofme S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. Solubility Test A solubility test was conducted to select me vehicle. The test was conducted using one or more of the following: water, dimethyl sulfoxide (DMSQ), ethanol and!acetone. The test substance was tested to determine the vehicle, selected in older ofprefereface, that permitted preparation of me highest soluble or workable stock concentration, up to 500'mg/mL. Preliminary Toxidty Test The preliminary toxicity test was used to establish me dose-range over which the test substance would be assayed. Vehicle and ten dose levels of the test substance (6.7,10,33,67, 100,333,667,1000,3333 and 5000 ug per plate) were plated. Each dose level was plated on a single plate, with overnight cultures ofTA98, TA100, TA1535, TA1537 and WP2 uwA on selective minimal agar in both me presence and absence ofrat liver S9 activation. Mntagenicity Test The mutagenicity test initial assay (Experiment Bl) and independent repeat assay (Experiment B2) was used to evaluate the mutagenic potential of me test substance. A minjmimni of five dose levels of test substance along with appropriate vejbicle and positive controls were plated with tester strains TA98, TA100, TA1535, TA1537 and WP2 uwA in the presence and absence of rat Kver S9 activation. All dose levels of test substance, vehicle controls and positive controls were plated in triplicate. Plating and Scoring Procedures In bom the preliminary toxicity .test and the mutagenicity test, the test system was exposed to me test substance via the plate incorporation methodology as described by Ames et al. (1975) and updatedby Maron and Ames (1983). On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCI (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 uM each. Top agar not used with S9 or Sham mix jwassupplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by tine Milli-Q Reagent BioReliance Study No. AA34BK.502001ATL 11 Sanitized, not TSCA CBt Water System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented wim 2.5 % (W/V) Qxoid Nutrient Brolh 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % No. (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Each plate was labeled with a code system that identified the test substance, test phase, dose level, tester strain, and activation, as described in detail in BioReliance's Standard Operating Procedures. S9 Test substance dilutions were prepared immediately before or Shannnix, 100 pt ofiestersfiainanfiT5b uL of use. One-half (0.5) miliiliter of 2.0 mL of molten selective top agar at 452C. vehicle or test After vortexing, substance were added to onto the surface of 25 mL of minimal bottom agar. me mixture When platingthe positive was overlaid substance aliquot was replaced by a 50 uL aliquot of appropriate positive controls, me test overlay had solidified, the plates were inverted and incubated fw control. After approximately48 to 72 the at 372C. Plates mat were not counted immediately following the incubation hours period stored at 2-8C until colony counting could be conducted. were The condition of me bacterial background lawn was evaluated for evidence of test substance toxicny by using a dissectingmicroscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using me codes shown below. BioReliance Study No. AA34BK.502001 .BTL 12 CiMnpanf tSte@A Does nol eortain TSC5A CBI -SM lainiKieiwnaemK ----------^^------ffBacterial Reverse RSHBI^^^^^^^^^ Mutation DuPont-467( Code 1 J 2 Description Normal Slightly Reduced Characteristics Distinguishedby a healthymicrocolony lawn. Distinguishedby a noticeable thinning of the microcolony lawn and possibly a slightincrease in the size of the microcolonies compared to the vehicle control plate. 3 Moderately Distinguished by a marked thinning of the microcolony lawn Reduced resulting in a pronounced increase in the size of the microcolonies compared to the vehicle control plate. Distinguished by an extreme thinning of me microcolony lawn 4 OCAOvV^C^Iwdjlrv wstdtingdn an increase artne size 6T the microcolonies compared to Reduced the vehicle control plate such that the microcolony lawn is visible to the unaided eye as isolated colonies. 5 Absent SD9is0ti%ngoufisthheepdlabtye. a complete lack of any microcolony lawn over 6 OPbrseccuipreitdatbey mThicerobsaccokpgircotuensdt sbuabcstetarniaclelapwrencicpaitnanteo.t be accurately evaluated due to Non- Distinguished by precipitate on the plate that is visible to the naked NP Interfering eye but any precipitate particles detected by the automated colony Precipitate counter total less than 10% of the rcvertant colony count (e.g., <3 particles on a plate with 30 revertants.) Distinguished by precipitate on the plate that is visible to the naked IP Interfering eye and any precipitate particles detected by the automated colony Precipitate cc<ounter exceed 10% of the revertant colony count (e.g., >3 particles oon a plate with 30 revertants.) Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the test was the preliminary tenacity test or the plate exhibited toxicity. Plates with sufficient test substance precipitate to interfere with automated colony counting were counted manually. Evaluation of Results plateFowreereacchalrceupllaicteadteapnldatairnegr,etphoertmede.an and standard deviation of the number of revertants per For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test substance. Date sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TAPS, TA100 and BioReliance Study No. AA34BK.502001.BTL 13 Bacterial Reverse Mutation DuPont-4670 WP2 uvrA were judged positive if (he increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value. Criteria for a Valid Test The following criteria must be met for (he mutagenicity test to be considered valid. All Salmonella tester strain cultures must demonstrate the presence of toe deep rough mutadon (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of toe pKMIOl plasmid R-factor. All WP2 uvrA cultures must demonstrate we deletion in the uvrA gene. AH cultures must demonstrate the characteristic m(1e0an- 5n0u)m; TbAC1T0j)f0^(s8D0o-nt2a4n0e)o;uTsAjB1y5f3ir5ta(n5t-sii4L5&);eTvAeh1i5c3le7 e(o3a- fr2o1ls);aasnfdoWlloPw2su(vinrcAlu(s1i0ve- ):60T).AT9o8 ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10' cells/mL. The mean of each positive three-fold increase in the number of revertants over the mean control must exhibit at least a value of me respective vehicle control A minimum of three non-toxic dose levels are required to evaluate test data. A dose level is considered toxic if one or bom of the following criteria are mefc (1) A >50 % reduction in the mean number of revertants per plate as compared to me mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn. Archives The study raw data, protocol and all reports will be maintained according to Standard Operating Procedure OPQP3040 by the BioReHance RAQA unit headquartered BioReliance, 14920 Broschart Road, Rockvflle, MD 20850. at: all Unused residual dosing solutions were disposed of following administration to the test system and test substance will be disposedof following finalizarion of me report Deviations of thNisosktundoyw. n deviations from me protocol or assay-method SOPs occurred during the conduct BioRsIiance Study No. AA34BK.502001.BTL 14 Company Sanitized. Bws not contain TSCA CB! Reverse Mutation DuPont-4670 Solubility Teat RESULTS AND DISCUSSION Acetone was selected as the compatibility with the target solvent of choice based on solubilityof the test substance and acetone at S300 sag/saL. cells. The test substance was a soluble and clear solution in Preliminary Toxicity Test The results of the preliminary toxicity preliminary toxicity test are presented in Tables 1 through 5. la Hie test, me maximum dose tested was 5000 pg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 uL plating aliquot Neither precipitate nor toxicity was observed. Based on the findingsof the toxicity test, the maximum dose plated in die mutagenicity test was 5000 ug per plate. Mutagenicity Test The results Tables 26 of the mutagenicity test are presented in Tables 6 through 25 and summarized in and 27. These data were generated in Experiments Bl and B2. In the mutagenicity test, no positive response was observed, in the initial mutagenicity assay, the dose levels tested were 5000, 3333, 1000, 333 and 100 u.g per plate. Neither precipitate nor toxicity observed. In me independentrepeat assay, the dose levels tested were 5000,1800,600,200 was 75 ug per plate. Neither precipitate nor toxicity was observed. and No positive responses weae observed with any of me tester strains in the aasbssaeyn.ce of S9 activation in either the initial mutagenicity assay or in the indepepnredseenntcreepaenadt CONCLUSION |tf|BlHacter^ All criteria for a valid study were met as described jn, the protocol. The results of the E an IndependentRepeatAssay indicate that, under Reverse tE? conditions of this Mutation Test study, the substance did not cause a positive mutagenic response with any of the tester strains test in the presence and absence of Aroclor-induced rat liver S. BioReliance Study No. AA34BK.502001.BTL 15 REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the SaImonenafMaasEOsSian Microsome Mutagenicity Test, Mutation Research, 31:347-364. Ames, BX, J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the Scdmonella/MamsaaSiaa Microsome Mutagenicity Test, Mutation Research, 31:347-364. Green, M.H.L. and WJ. Muriel (1976) Mutagen testing using tip^ reversion in EscJwrichia coli, MutationResearch 38:3-32. International Conference on Hannonisation (ICH) of Technical Requirements tor Registration of Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 of the ICH process on July 19,1995. Federal Register 61:18198-18202, April 24,1996. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at step 4 of me ICH process on July 16,1997. Federal Register 62:16026-16030, November 21,1997. Maron, D.M. and B.N. Ames (1983) Revised Methods for the Salmonella Mutagenicity Test, Mutation Research, 113:173-215. OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July 1997. Vogel, HJ. and D.M. Bonner (1956) Acetylornithinase ofE. coli: Partial Purification and Some Properties, J. Biol. Chem., 218:97-106. Study No. AA34BK.50200LBTL 16 iBacterial Reverse Mutation DuPont-4670 Bacterial Mutation Test Preliminary Toxicity Assay Test Substance Id Study Number Strain Experiment Mo. Date Plated Vehicle Plating Aliquot Test Substance Concentration jig per plate Vehicle 54 TA98 Al 29 Aug 2000 acetone 50 Wi'uthL_S9__A_ctivation Revertants Background per plate_____Lawn 21 1 Without S9 Activation Revertants Background per plate Lawn 22 6.7 10 33 67 100 333 667 1000 3333 5000 25 1 29 1 24 1 24 1 19 1 17 1 15 1 20 1 20 1 27 1 ickground Lawn Code 26 1 19 1 17 1 19 1 25 1 10 1 17 1 18 1 22 1 21 1 l=Normal; 2=Slightly reduced; 3=5Severely reduced 4i]=Extre^lely reduced; 5"Absent; 6=0bscured NP=Non-Interfering precipitate; IP= by precipitate C=Contaminated Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 17 Bacterial Reverse Mutation DuPont-4670 Bacterial Mutation Test Preliminary Toxicity Assay Table 2 Test Substance Id Study Number Strain Experiment Mo. Date Plated Vehicle Plating Aliquot Test Substance Concentration pg per plate Vehicle aBSS8-5 02001.BTL TA100 Al 29 Aug 2 000 - ,, acetonLfA&w 50 uL With S9 Reverta nts per pTate Activation Background Lawn 91 1 " Without S9 Activation Revertants Background per plate Lawn 129 1 6.7 10 33 67 100 333 667 1000 3333 5000 108 1 97 I 125 1 117 1 110 1 118 1 132 1 131 1 114 1 121 1 Background Lawn Code 124 1 96 1 87 1 115 1 89 1 00-07 1 134 1 128 1 118 1 161 i j. l=Mormal; 2=Slightly reduced; 3=Severely reduced 4=Extremely reduced; 5Absent; NP=Mon-Interfering precipitate; 6=0bscured by precipitate IP= Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 18 Gompmy Sanitbed. Does not contain T8CA CBl Bacterial Reverse Mutation DuPont-4670 Bacterial Mutation Test Preliminary Toxicity Assay Table 3 Test Substance Id Study Number Strain Experiment Ho. Date Plated Vehicle Plating Aliquot Test Substance Concentration pg per plate Vehicle sA02001.BTL TA153 5 Al 29 Aug2(000 aceto ne 50 U.L With 89 Activation Revertaints Background per platte Lawn 9 1 Without S9 Activation Reyertants Background per plate Lawn 13 1 6.7 10 33 67 100 333 667 1000 3333 5000 10 1 13 1 . 11 1 10 1 15 1 12 1 10 1 16 1 18 1 16 1 16 1 15 1 14 1 1 13 1 11 1 13 l 16 1 9 1 12 1 ackorounc l T.flun f^ria l^Momal; 2=Slightly reduced; 3=Severely reduced 4Bxtremely reduced; 5=Absent; HP=Mon-Interfering precipitate; 6=0bscured by precipitate IP" Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 19 Company SanitfeeA Dws not eosstain TSCA CBS DuPoat-4670 Bacterial Mutation Test Preliminary Toxicity Assay Test Substance Id w Table 4 Study Number Strain Experiment No. Date Plated Vehicle Plating Aliquot Test Substance Concentration TO per plate Vehicle ;; ffAjB^si02001.BTL !; TA1537 ;; Al ;; 29 Aug 21300 :; acetone ;: 50 uL With S9 Activation Revertants Background per plate Lawn 7 1 Without S9 Activation Revertants Background per plate Lawn 6 1 6.7 10 33 67 100 333 667 1000 3333 5000 8 1 5 1 7 1 4 1 6 1 5 1 7 1 11 1 8 1 4 1 7 1 7 1 9 1 4 1 10 1 1 2 1 5 1 2 1 6 1 Baarc*lfk^gT"rro'unrn*/d4 TLaaw.wn Cr*^o./di--e l=Momal; 2=Slightly reduced; 3=Severely reduced N4=PPE=^=xHHtrooennm--IeInnltyerrfeeedrriuinnccgerdop; rree5c=c-iAi_npbH.is,,t-eaa.n--thte.;n;. -- 6 =_0*b<swcuujr.e.du buyf pyfr-wejc-pipmistLaTtiee TIPO=--= -Irn,~t.ie---rf--er:-i--ng-- precipitate BioReliance Study No. AA34BK.502001 .BTL 20 Bacterial Reverse Mutation DuPont-4670 Bacterial Mutation Test Preliminary Toxicity Assay Test Substance Id Table 5 Study Number Strain Experiment Mo. Date Plated Vehicle Plating Aliquot Test Substance Concentration pg per plate Vehicle AA34J BT502001.BTL WP2 uivrR Al 29 Au g 2000 aceto ne 50 uL With S9 Activation Revertants Background per plate Lawn 14 1 Without S9 Activation Revertants Background per plate Lawn 12 1 6.7 10 33 67 100 333 667 1000 3333 5000 16 1 12 1 11 1 14 1 15 1 11 1 10 1 13 1 13 1 15 1 9 1 15 1 11 1 17 1 12 1 10 1 14 1 10 1 13 1 9 1 Background Lawn Code l^Hormal? 2=Slightly reduced; 3a!SSeverely reduced 4Extremely reduced; S^Absent; MP=Non-Interfering precipitate; 6sa0bscured by precipitate IP= Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 21 Company Sanitiml. Reverse Mutation DuPont-4670 Bacterial Mutation Test Table 6 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot T502001.BTL TA98 None acetone 50 pi Experiment Ho Cells Seeded Date Plated Concentration _ug per plate Vehicle Plate Number 01 Revertants Background Average per plate____Code___Revertants 28 02 21 03 44 31 Bl 1.1 X 10" 12 Sep 2000 : oSit-aanndoaarda Deviation 12 100 01 37 02 19 03 25 27 333 01 37 02 34 03 41 37 1000 01 27 02 31 03 28 29 3333 01 28 02 31 03 25 28 5000 01 40 02 21 03 30 30 10 Positive Control 2-nitrofluorene 1.0 pg per plate 01 391 1 02 283 1 ______________03______337 1 Background Lawn Code 337 54 1"Normal; 2=Slightly reduced; 3=Severely reduced 4^Extremely reduced; S^Absent; NP^Non-Interfering precipitate; 60bscured by precipitate IP= Interfering precipitate BioReliance Study No. AA34BK.502001.mi 22 Reverse Mutation DaPoat-4670 Bacterial Mutation Test Test Substance Id Study Muniber Strain Liver Microsomes Vehicle Plating Aliquot A?.- TA98 . _ -, Rat liver S9 acetone 50 pi. Experiment No Cells Seeded Date Plated Concentration __ug per plate Vehicle Plate Number 01 02 03 Revertants Background per plate___Code 27 34 27 Average Revertants 29 Bl 1.1 X 106 12 Sep 2000 Standard Deviation 100 01 27 02 29 03 26 27 333 01 42 02 34 03 37 38 1000 01 38 02 23 03 30 30 3333 01 31 02 34 03 21 29 5000 01 30 02 36 03 25 30 Positive Control 2-0a1ininoanthr3a8c5ene 1.0 1 ug per plate 02 457 1 Background Lawn Code03______313_____1__________385 72 lj.=-Naoornnniaajl.;; 2^=a=-LSxligghhtly reduced; 3s=Severely reduced. 44s=sIE?vxf"f*c"ArTeMn<i-*e'Ily r^e,^d^au.-c--e--d-a?- 5r-SS*'Absent; HP='Hon-Interfering precipitate; 6=0bacured by precipitate IP= Interfering precipitate BioReliance Study No. AA34BK.5Q200LBTL 23 Reverse Mutation DuPont-4670 Bacterial Mutation Test Table 8 Test Substance Id:j Study Number 55353'?. 502001. BTL Strain TA100 Liver Microsollies : Hone Vehicle acetone Plating Aliqu<at ; 50 uL Experinient Mo : B1 Cells Seeded Date Plated : 1.3 X 10 ; 12 Sep 2000 Concentration pg per plate Vehicle Plate Number 01 02 03 Revertants per plate 95 81 iii Background Code 1 1 1 Average R.e- v- e----r--t'--a--n--.l'..wo-r 96 Standard tr^><VfV-!L-C>lL*-i<.U--U 15 100 01 97 1 02 120 1 03 79 1 99 21 333 01 115 1 02 75 1 03 82 1 91 21 1000 01 103 1 02 82 1 03 97 1 94 11 3333 01 83 1 02 91 1 03 93 1 89 5 5000 01 88 1 02 110 1 03 116 1 105 15 Positive Control sodium azide 1.0 ug per plate 01 341 1 02 320 1 Background Lawn 03______426_____1__________362 Code 56 l=Homial; 2=Slightly reduced; 3=Severely reduced 4=Exfcreinely reduced; 5=flbsent; MP-'Mon-Interfering precipitate; 6=0bscured by precipitate IP=' Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 24 Reverse Mutation DuPont-4670 Bacterial Mutation Test Test Substance Id:l Study Number Strain AA34BBA02001. BTL TA100 Liver Micros ones Vehicle : Rat liver S9 acetone Plating Alia'U(at 50"uL" Exper iaent Mo Bl Cells Seeded 1.3 X 108 Date Plated 12 Sep 2000 Concentration ug per plate Vehicle 100 333 Plate Number 01 02 03 01 02 03 01 02 03 Revertants per plate 88 96 107 78 113 96 96 91 81 Background Code 1 1 1 1 1 1 1 1 1 Average Reverfcants 97 yaof 89 Standard Deviation 10 18 8 1000 01 89 1 02 102 1 03 105 1 99 9 3333 01 79 1 02 81 1 03 61 1 74 11 5000 01 86 1 02 87 1 03 82 1 85 3 Positive Control 20-a1minoanth3ra2c1ene 1.0 1 ug per plate 02 971 1 Background Lawn Code03______445_____1__________579 345 l1 =M<E1orma l; 2-- = S- - l i- g h t l y reduced; 3=Severely reduced 4=Extremely reduced; 5=Absent; MP=Non-Interfering precipitate; 6=0bscured by precipitate IP" Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 25 Reverse Mutation DuPont-4670 Bacterial Mutation Test Table 10 Test Substance Zd:J Study Number : aSSSiA 502001. BTL Strain : TA1535 Liver Microsomeis : None Vehicle : acetone Plating Aliquot. : 50 uL Bxper iaent Mo * B1 Cells Seeded 1.7 X 108 Date Plated 12 Sep 2000 Concentration ug per plate Vehicle Plate Number 01 02 63 Revertants per plate 14 18 13 Background Code 1 1 1 Average Revertapi"'8 Standard . knau w -iLo64. <b-.J!L-V"l 15 3 100 01 17 1 02 23 1 03 25 1 22 4 333 01 15 1 02 20 1 03 15 1 17 3 1000 01 17 1 02 2.1 1 03 10 1 16 6 3333 01 13 1 02 16 1 03 1 14 2 5000 01 19 1 02 15 1 03 16 1 17 7 Positive Control sodium azide 1.0 pg per plate 01 401 1 02 262 1 Background Lawn Code03______281_____1__________315 75 l=llonnal; 2==Slightly 44==EE3ttx~rt(r=e>Tmnce>1lyi ^ _, rceodi-uii-cii-e'od^;- r_-e--d-u.--c--ed,,,; 3^ =Ser ve..j.rQejkl_yy r4.ecd:UuLlc^tetfdU 5I;--=ABIb~s--e--n>t.;- '6- =0'-'b- -scured b y precipitate MP^Mon-Interfering precipitate; IP= Interfering precipitate BioReliance Study No. AA34BK.502001.B'IL 26 Bacterial Mutation Test Table 11 Test Substance Id llBBIIBHBll^BHHU Study Number Strain AA34BIK 502001.BTL TA1535 Exxppeeriment No Liver Microsoaies Vehicle Rat liver S9 Cells Seeded Date Plated acetone Plating Aliquot 50 Concentration Plate uL_______________ Revertants Background pg per plate___Number p^"e-Jr- p^l>aJte'_l_t_c_iC-oodde Average Revertanntt,s Vehicle 01 19 l 02 8 1 03 17 1 15 Bl 1.7 X 10" 12 Sep 2000 Standard Deviation 100 01 11 1 02 9 1 03 14 l 1^ 1 333 01 13 l 02 12 1 03 9 1 11 1000 01 17 1 02 11 l 03 14 1 14 3333 01 15 l 02 13 l 03 11 1 13 5000 01 12 1 02 13 1 03 21 1 1l,5, Positive Control 20-a1minoanthra7c9ene 1.0 1 ug per plate 02 69 1 Background Lawn Code03______n______3_ 73 l^Nonnal; 2!=Slightly 4B"EBxEtxrterem^leellyv reducerdi..;. r_e_d_u_c_e--d,; 3,. =^Se..vi-e.4r.ecjl.yy 5'is=sAaKbosoeTni'it-;. rttetduuceda MP=Non-Interfering c6--0<b"s>ic--u--re--d-' b' y precipitate precipitate; IP= Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 27 Bacterial Mutation Test f. Table 12 Test Substance Id Study number 3537Btrf502001. BTL Strain TA:L537 Liver Microsomes * N01ie Vehicle ac<stone Plating Aliquot 50 PL Experiment Nc : Cells Seeded : Date Plated : B1 1.1 X 10" 12 Sep 2000 Concentration ug per plate Vehicle Plate Number 01 02 03 Revertants per plate 5 7 9 Background Code 1 1 1 Average Revertants 7 Standard Deviation 2 100 01 7 1 02 5 1 03 C 6 1 333 01 7 1 02 4 1 03 6 1 6 2 1000 3333 5000 01 9 1 02 9 1 03 3 1 01 9 1 02 10 1 03 5 1 01 9 1 02 6 1 03 2 1 7 3 8 3 6 4 Positive Control 9-aminoacridine 75 ug per plate 01 1070 1 02 978 1 03______691_____1__________913 198 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Severely reduced 4=extremely reduced; 5Absent; 6'=0bscured by precipitate NP=Mon-Interfering precipitate; IP= Interfering precipitate C<'ontaminated BioReliance Study No. AA34BK.50200LBTL 28 Bacterial Mutation Test Table 13 Test Substance I d Study Number Strain Liver Microsomes Vehicle Plating Aliquot aSSBfi'502001. BTL TM537 Rat liver S9 acetone 50 uL Experiment Ho ; B1 Cells Seeded Date Plated : 1.1 X 10' : 12 Sep 2000 Concentration ug per plate Vehicle Plate Number 01 02 03 Revertants per plate 6 4 5 Background Code 1 1 1 Average Revertants 5 Standard Deviation 1 100 01 4 1 02 3 1 . 03 7 1 5 2 333 01 5 1 02 5 1 03 8 1 6 2 1000 01 6 1 02 3 1 03 8 1 6 3 3333 01 2 1 02 6 1 03 5 1 4 2 5000 01 4 1 02 5 1 03 6 1 5 1 Positive Control 2-aniinoanthracene 1.0 pg per plate 01 80 1 02 84 1 _______________03_____113 1 Background Lawn Code 92 18 l==Normal; 2=Slightly reduced; 3=Severely reduced 4Extreaely reduced; 5=Absent; 6=0bscured by precipitate NP=Hon-Interfering precipitate; IP= Interfering precipitate BioReliance Study No. AA34BK.50200LBTL 29 Company Sanitized. Does not contain TSCA CBl Reverse Mutation DuPont-4670 Bacterial Mutation Test Test Substance Id Table 14 Study Number Strain Liver Microsome s Vehicle Plating Aliquot lo2001. BTL WP2 uvri& None acetone 50 uL Experiment Ho ; Bl Cells Seeded : 3.0 X 10" Date Plated ; 12 Sep 2000 Concentration ug per plate Vehicle Plate Number 01 02 03 Reveart ants ' per plate 12 16 19 Background Code 1 1 1 Average Revertants 16 Standard Deviation 4 100 01 11 1 02 15 1 03 10 1 12 3 333 01 12 1 02 16 1 03 22 1 17 5 1000 01 13 1 02 14 1 03 14 1 14 1 3333 01 12 1 02 10 1 03 I? 1 14 5 5000 01 13 1 02 14 1 03 8 1 12 i Positive Control methyl 01 methanesulfonate 73 1 1000 ug per plate 02 69 1 Background Lawn Code03_______72_____1__________ 71 l^Hormal; 2=Slightly reduced; 3s=Severely reduced 4'=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Mon-Interfering precipitate; IPs' Interfering precipitate mce Study No. AA34BK.502001BTL 30 DuPont-4670 Bacterial Mutation Test Table 15 Test Substance Id Study Number Strain Liver Microsome s Vehicle Plating Aliquot ^S9Icyfco2ooi. BTI, WP2 uvrA Rat liver S9 acetone 50 uL Exper inient Ho * B1 Cells Seeded Date Plated 3,0 X 108 12 Sep 2000 Concentration ug per plate Vehicle Plate Number 01 02 03 Revertants per plate 11 16 14 Background Code 1 1 1 Average Standard Revertants . Deviation 14 3 100 01 16 1 02 12 1 03 11 1 13 3 333 01 10 1 02 15 1 03 12 1 12 3 1000 01 12 1 02 9 1 03 10 1 10 2 3333 01 11 1 02 13 1 03 10 1 11 2 5000 01 11 1 02 13 1 03 12 1 12 i Positive Control 2-aminoanthracene 10 pig per plate 01 94 1 02 70 1 Background Lawn 03______171_____1__________112 Code 53 l=Mormal; 2=Slightly reduced? 3=Severely reduced 4s=Extremely reduced; S^Absent; 60bscured by precipitate NP^Non-Interfering precipitate; IP Interfering precipitate BioReIiance Study No. AA34BK.502001.BTL 31 itielwntaSnTSCAcal Reverse Mutation DuPont-4670 Bacterial Mutation Test Test Substance Id:J Table 16 Study NumbeT Strain Liver Micro'someis Vehicle Plating Allquot ^1"SASiBS?502001. BTL TA98 : None k acetone 50 pL Experiment No B2 Cells Seeded Date Plated 2.4 X 10" 20 Sep 2000 Concentratio n ug per plate Vehicle Plate Number 01 02 03 Rev(srtants per plate 16 17 23 Background Code 1 1 1 Average :Standard Revertants Deviation 19 4 75 01 17 1 02 12 1 03 15 1 15 3 200 01 12 1 02 14 1 03 20 1 15 4 600 01 14 1 02 16 1 03 16 1 15 1 1800 01 18 1 02 14 1 03 16 1 16 2 5000 01 17 1 02 16 1 03 18 1 17 1 Positive Control 2-nitrofluorene 01 1.0 pg per plate 191 1 02 190 1 Background Lawn 03______170_____1 Code 184 12 l^Monnal; 2=Slig,, h-t--ly., r_e.,--du--c--e>d.;, 3--=--SueQvvce^r.ecjl.yy r.ceednuuceeod. 41=SBExxttrreemmeelyv reduced; 55s==AAhb.'sBeonn+t-; NP=Mon-Interfering precipitate; <6;=-=0rbn^s~c--u.r--e-d> b'--y- precipitate IP" Interfering precipitate BioReIiance Study No. AA34BK.502001JBTL 32 Company Sanftteed. Does not eontain TSCA CB! 'Bacterial Reverse Mutation DuPont-4670 Bacterial Mutation Test Test Substance Idi Table 17 Study Number Strain Liver Microsome;s Vehicle Plating Aliquot 9SwS^02001. BTL TA98 : Bat liver 39 acetone 50 uL Experiment Ko Cells Seeded Date Plated B2 2,4 X 108 29 Sep 2000 Concentration ug per plate Vehicle 75 200 600 1800 5000 Plate Number 01 02 03 01 02 03 01 02 03 01 02 03 01 02 03 01 02 03 Revertants per plate 19 16 20 18 11 15 14 13 21 14 9 6 13 11 12 13 14 17 Background Code 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Average Revertants 18 15 16 10 12 1R Standard Deviation 2 4 4 4 1 Positive Control 20-a1minoanth1ra1c8 ene 1.0 1 ug per plate 02 191 1 Background Lawn 03______293_____1 Code 201 88 l^Normal; 2=Slightly 4=Extremely reduced; reduced; 3=Severely 5=Absent; reduced MRf-Non-Interfering precipitate; 6=0bscured by precipitate IP= Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 33 Bacterial Mutation Test Test Substance Ids Study Number Strain w*o TAlOO Liver Microsomes Hone Vehicle acetone Plating Aliquot 50 Concentration Plate uL______ Revertants ug per plate Nkuumutubee,Lr per pla te Vehicle 01 124 02 131 03 142 75 01 125 02 120 03 110 200 01 134 02 123 03 88 600 01 123 02 127 03 120 1800 01 146 02 122 03 132 5000 01 138 02 133 03 140 Positive Control sodium azide 1.0 ug 01 573 02 613 Experiment Mo Cells Seeded Date Plated Background Code 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 per plate 1 1 Average Revertants 132 118 115 123 133 '" B2 1.8 X 108 29 Sep 2000 Standard Deviation 9 8 24 4 12 ___ Background L_aw_n__C_o_de0_3_____________5^8_7__________^3___________591_______20 l=Monnal; 2=Slightly reduced; 4=Extremely 3=^Severely reduced MP^Non-Interrfeedruincgedp; re5;c=^ipAbitsaentte; ; fiIsP'O"bsIcnutreerdfebryinpgrepcriepcitiaptietate BioReliance Study No. AA34BK.502001.B-IL 34 Company Sanitted. Does iiwt wlalai TSCA CBI Reveise Mutation DuPont-4670 Bacterial Mutation Test Table 19 Test Substance Idi Study Number Strain Liver Microsome;s Vehicle Plating Aliquot ^15534Sifllo2001.E1TL TA100 ; Rat liver S9 ; acetone ; 50 uL Experiment No : B2 Cells Seeded Date Plated : 1.8 X 108 : 29 Sep 2000 Concentration pg per plate Vehicle Picite Nuniber C H 0 12 0i3 Revertants per plate 98 92 110 Background Code 1 1 1 Average Revertants 100 Standard Deviation 9 75 0 1 85 1 0 2 97 1 0 3 92 1 91 6 200 0 1 121 1 0;2 88 1 0:3 117 1 109 18 600 0:1 102 1 0;2 98 1 0;3 120 1 107 12 . 1800 0]L 118 1 0;> 86 1 0;i 110 1 105 17 5000 03. 124 1 02t 105 1 03 114 1 114 i n Positive Control 20-a1minoanthr6a0c4ene 1.0 1 ug per plate 02 571 1 Background Lawn Code03______520_____1__________565 42 1l^__MMo__r_m_-al; 2-- !!--=-S- lightly 4=Extremely reduced; reduced; 3=Severely 5=Absent; reduced MPsNon-Interfering precipitate; 60bscured by precipitate IP= Interfering precipitate BioReUance Study No. AA34BK.502001.BTL 35 Bacterial Mutation Test Te.st Substance Id Table 20 7 1 Study Number Strain "S02001.BTI. TA1535 &xperiment Wo : B2 Liver Microsomes None Cells Seeded ; 2.5 X 108 Vehicle acetone Date Plated : 29 Sep 2000 _ Plating Aliquot 50 Concentration J^g Plate uL_________________________ Revertants Background Per plate Vehicle N..-u.m^<b-e^r. 01 pyweir: Average ppjl-aactee____CCooddee ___RReevvee rtants ;Standard j Deviation 12 1 /0!2'> 03 10 1 10 75 01 11 1 02 12 1 03 17 1 13 200 01 14 1 02 12 1 03 13 1 13 600 01 11 1 02 12 1 03 12 1 12 1800 01 14 1 02 13 1 03 13 1 13 5000 01 15 1 02 12 1 03 12 1 13 Positive Control sodium 01 azide 1.0 ug per plate 364 1 02 245 1 Background Lawn 03______263_____!_ Code 292 63 l=Nonnal; . 4 2- =_S_l_i3g..h>--tl.yj 4"Extremely reduced; r^e.vd^uu.c^esad.;, 3.3===aSeevveerreely S5=^Absent; reduced NP=Mon-Interfering precipitate; f6e=n0ibisec-.u--r"e^d b'--y p- recipitate IP" Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 36 Bacterial Mutation Test Test Substance Id:J Table 21 Study Humbez'1 Strain Liver Micros ome's Vehicle Plating Aliquot ;'95^1^502001. TA1535 : Rat liver S9 r acetone S 50 uL Concentration ug per plate Vehicle Plate Number 01 02 Revertants per plate 10 8 03 11 75 01 12 02 13 03 16 200 01 8 02 9 03 8 600 01 6 02 8 03 11 1800 01 9 02 7 03 13 5000 01 13 02 9 03 15 BTL Experiment tto ; Cells Seeded : Date Plated : Background Code 1 I 1 1 1 1, 1 1 1 1 1 1 1 1 1 1 1 1 Average Revei-t-a"!'0.< 10 14 8 8 10 1-7 N2 2;. 5 X 108 2i9 Sep 2000 , Standard : .. Li-i>c~v---i-ta-tc-ion 2 2 1 3 3 Positive Control 20-a1ninoanthra9c3ene 1.01 ug per plate 02 133 1 Background Lawn 03______100_____1 Code 109 l=Normal; 2=Slightly 4;=Extre^lely reduced; reduced; 3=;Severely 5'=absent; reduced NP=Non-Interfering precipitate; 6=0bscured by precipitate IP= Interfering precipitate BioReliance Study No. AA34BK.502001.BTL 37 Bacterial Mutation Test Table 22 Test Substance Id: Study Number Strain Liver Microsome's Vehicle Plating Aliquot ^AAsBI?"502001.BTL : TA1537 : None : acetone : 50 pL Exper Cells Date Concentration pg per plate Vehicle Plate Number 01 02 03 Rev<srtants per plate 5 6 5 Background Code 1 1 1 75 01 1 1 02 5 1 03 6 1 200 01 6 1 02 4 1 03 8 1 600 01 4 1 02 4 1 03 5 1 1800 01 4 1 02 3 1 03 4 1 5000 01 7 1 02 6 1 03 1 1 iroent Mo Seeded Plated Average Revert an.(,.0, 5 6 6 4 4 7 B2 0.8 X 108 29 Sep 2000 :Standard Deviation 1 1 2 1 1 1 Positive Control 90-a1 minoacrid21in5 e 75 ug1 per plate 02 196 1 B_a_c_kg_r_ou_n_d__La_w_n__C_od0e3______167_____3_ 193 24 1"Normal; 2=!SSlightly 4=Extreraely reduced; reduced? 3'=Severely 5=Absent; reduced MP=Non-Interfering precipitate; 6=0bscured by precipitate IP" Interfering precipitate BioReliance Study No. AA34BK.502001 .BTL 38 Company Samfeed. Does not contain TSCA CBj  Bacterial Mutation Test Test Sulostance Id Table 23 Study MLunbar Strain Liver MjLcrosomes Vehicle Plating Aliquot R?T02001.BTL TA15 37 Rat liver S9 acet one 50 piL Experiment Mo : B2 Cells Seeded : 0.8 X 10s Date Plated ; 29 Sep 2000 Concentra tion ug per piate Vehic Ie 75 200 600 1800 5000 Pla te Humiber 0 1 0 2 0 3 0 1 0 2 0.3 011 ur\^^> 03 01 02 03 01 02 03 01 02 03 I leverfcants Ewr plate 5 11 7 6 4 6 4 7 7 5 3 6 7 6 6 6 5 2 Background Code 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Average Standard Revertants Deviation 83 51 62 52 61 -i - Positive Control 20-a1minoanthra8c9ene 1.0 1 ug per plate 02 82 1 B_a_ck_g_r_ou_n_d__La_w_n__C_od_e03_______68_____1__________ 80 11 l=Norma_l,; 2- =^S^l.^ig.yh^^tjl-yy 4^="Extremely reduced;/ rse-wdwuicceeda; 3=Severely 5=Absent; reduced NI.TIP-. 'I-*Mon-- -Interfering precipitate; 6=I=PO0=hbBsI/c-n"u1tre-e"rd1feb1--ryin-pg-r-epcriepcitiaptietate BioReIiance Study No. AA34BK.5Q2001 JBTL 39 Reverse Mutation DuPont-4670 Bacterial Mutation Test Table 24 Test Substance Id;| Study Number Strain Liver Microsomes Vehicle Plating Aliquot ^ "ES^?1^12001.BTL : WP2 uvri\ !k None : acetone : 50 uL Exper iment No ; B2 Cells Seeded Date Plated : 3.5 X 10" ; 29 Sep 2000 Concentration pg per plate Vehicle Plate number 01 02 03 Revssrtants per plate 12 14 15 Background Code 1 1 1 Average KReevveerr+caann+t'so 14 Standard Drir-er,v..!i-at'"io-n- 2 75 01 15 1 02 13 1 03 11 1 13 2 200 01 12 1 02 11 1 03 11 1 11 1 600 01 10 1 02 11 1 03 13 1 11 2 1800 01 12 1 02 15 1 03 18 1 15 3 5000 01 12 1 02 16 1 03 16 1 15 2 Positive Control m0e1thyl meth1a2n8esulfona1te 1000 pg per plate 02 94 1 Background Lawn Code03______96_____1__________106 19 4l^=NEoxntrneaml;elvy2rSeldiug.,ch-e-t-dl.y;, r_e--d--u.c-,e^-d^;, 3^--Soe=vvee4r.ee4l.yy 55====&lA->b.'as'e'-ni+t-;" reduced NP^Mon-Interfering precipitate; c6--=='0iib..s--c.u.-r-e--d b*-y" precipitate 1P= Interfering precipitate BioReIiance Study No. AA34BK.502001.BTL 40 Reverse Mutation DuPoat-4670 Bacterial Mutation Test Test Substance Id Table 25 STt;uuday NHumber : AA34B^. ^02001. Strain ; WP2 uvrA Liver Microsomes Vehicle : Rat liver S9 Plating Aliquot : acetone ; 50 ul. Concentration ug per plate Vehicle Plate Number 01 02 03 Rewsrtants per plate 16 12 13 75 01 19 02 10 03 12 200 01 11 02 13 03 11 600 01 11 02 13 03 11 1800 01 14 02 14 03 10 5000 01 15 02 12 03 9 BTL Experiment No * Cells Seeded Date Plated Background Code 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Average RR oeirvae>r-+t a "n*ts" 1A~4Z 14 12 12 13 T-7 B2 3.5 X 108 29 Sep 2000 Standard Deviation 2 5 1 1 2 Positive Control 2-aminoanthracene 01 10 ug per plate 145 1 02 153 1 Background Lawn 03______186_____1 Code 161 22 l'I^M^o-n^n,-a1l;. 2 '^-- _ "-S-->ligg* h^^t l. ^ y ^4=EExxtrtreemBeely reduced; r.ie.wduucueeda;; 3^="sSeevvereliy 5=Absent; reduced MP=Mon-Interfering precipitate; f6eI=sP0n"bhsnI"cn"u't-r'ee--dr1feb'--ryinp-g-r-epcriepcitiaptietate BioReliance Study No. AA34BK.502001 .BTL 41 Reverse Mutation DuPont-4670 Bacterial Mutation Test Summary of Results Test Substance Id :, Table 26 Study Number____: <g!^reI^502001. BTL____Experiment No ; Bl Liver Average Revertants Microsomes: Hone Per Plate Standard Deviation Dose (ug/plate ) TA;98 T.A1 00 Vehicle 100 333 1000 3333 5000 Positive 31 12 27 9 37 4 28 2 28 3 96 15 99 21 91 21 94 11 89 5 30 10 105 15 337 54 362 56 Liver Microsomes:: Rat liver S9 Dose (pg/plate) TA98 Vehicle 100 333 1000 3333 5000 Positive 29 4 27 2 38 4 30 8 29 7 30 6 72 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 50 uL TA100 97 9.6. 89 99 74 85 579 10 18 8 9 11 3 345 TA15i35 15 3 22 4 17 3 16 f 6 14 2 17 2 315 75 TA1535 15 6 11 3 11 2 14 t 3 13 2 15 5 73 5 TAli537 7 2 6 1 6 2 7 3 8 3 6 4 913 198 --- WP2 w /Xa .A 16 4 12 3 17 5 14 1 14 5 12 3 71 j: 2 TA1537 5 1 5 2 6 2 6 3 4 t 2 5 1 92 18 CINiTOft'3t 14 13 12 10 11 12 11?. uv ^rl.xan 3 3 3 2 2 1 53 BioReliance Study No. AA34BK.502001.BTL 42 ial Reverse Mutation DuPont-4670 Bacterial Mutation Test Summary of Results Test Substance Id Table 27 Study Number____:AA3rag" 502001. BTL____Experiment No : B2 Liver A_--v--er^a-vgy^ef R^VCeSVvertants Microsoonsiaeess;: Hone Per Plate Standard Deviation Dose tug/plate; TA98 1 All30 Vehicle 75 19 4 132 9 15 3 118 8 200 600 15 4 115 24 1800 15 16 1 123 + 4 2 133 12 5000 17 1 137 4 Positive 184 12 591 20 Liver Microsomes: Rat liver S9 Dose (ug/plate) TA98 Vehicle 18 2 75 15 4 200 16 4 600 10 4 1800 12 1 5000 15 2 Positive 2!.0-1- - 88 -- Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 50 uL TA100 100 9 91 6 109 18 107 12 105 17 114 10 ^56--5 - 42 -- TAI 535 10 3 13 3 13 1 12 1 13 1 13 2 292 63 TAl^l 10 2 14 2 8 1 8 3 10 3 12 3 -1-0-9 + &21X TJLj&T&TJr*?37 5 1 6 1 6 2 4 1 4 1 7 1 193 24 Kf(CP&3' 14 13 n 11 15 15 106 U-vrA. 2 2 1 2 3 2 19 TifAi^I-J5 37 8 3 5 1 6 2 5 2 6 1 4 2 OOrut J3-. 11 WP2 14 14 12 12 13 12 161 uvrA 2 5 1 1 2 3 22 BioReliance Study No. AA34BK.502001.BTL 43 Company Sanitized. Qsfss not eontain TSCA CB6 Bacterial Reverse Mutation Dupont-4670 APPENDIX A Historical Control Data BioReliance Study No. AA34BK.502001JBTL 44 Historical Nej?ativeand Postorve Control Values 1997-199S9 revertantsperi date Activation Strain TA98 TA100 TA1535 TA1537 WPlwrA Control Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos Mean None SD Min 16 6 4 397- 216 21 128 29 53 592 159 129 11 5 1 424 145 6 6 3 0 819 468 13 16 5 3 190 127 35 Max Mean 59 21 1536- 649 288 144 1920 785 45 12 1024 98 30 7 2786 102 48 17 961 338 Rat Liver SD Min 7 6 330" 56 30 62 300 106 5 1 79 8 3 1 128 2 6 2 282 16 Max 65 2454 258 2871 42 1640 29 1835 115 2632 Substandard deviation; Min=nunimum value; Max^maximum value-, Neg^aegarive control (including but not limited to deionized water, dilmethyl sulfoxide, ethanol and acetone); Postpositive control BioReliance Study No. AA34BK.5020013TL 45 Company Sanffized. noteanSaIn TSCA Q^ (BacterialReverse Mutation APPENDIX B Study Protocol BioReIiance Study No. AA34BK.502001.BTL 46 Company anigte@<. ial Reverse Mutation DuPont-4670 RAWJS^' ^wwd by ^ Sponsor PtagectNumber: --JMSiBtdfiTO. BioReliance Study Number AA34BKJ02001.BT^ Re'veneMutattonTcst 1.0 PURPOSE )^t Tbe purpose of this study measuring its ability is to evxiuate the niutagenic potential of the test substance by to induce reverse mutetioBS fit selected loci of several strains of AS-a.--lm--o--n--e--lla--,,r--yp--h-im--i-Hium ----- the Byptophfffl locus of Egcberriicchlltia coBli WWPP?2uiwwrAA in 2.0 SPONSOR 2.1 Name: 2.2 Address: 2.3 Representadve: 2.4 Sponsor ProjectNo.: 15 WR* Stifle Hastoll Research Center DuPoatHaskell Laboratory P.O. Box 50 1090 ESkton Road Newark, DE 19714-0050 Maria Doaner,PfaJD. DuPont-4670 2.6 Hask-U#: 2.7 Service Code: 3.0 IDEOTTHCA-nONOFTEST AND CONTROL SUBSTANCES 3.1 Test Substance Name: 3.2 Test Substance ID.: 33 Controls: Protocol SPOT50200I 16-AUS-2000 R^eloflO BioReliance Study No. AA34BK.502001.BTL 47 f i l BlORELiANCF ^9^ Formerly Microbiologicj(A>oeiii*c Reverse Mutation DuPont-4670 Sponsor ProjectNumber: BtolteBaaceSmdv Number: p^mt-4670^ AA34BKja20QI.BT^ 3.4 Test Substance Characterization F UatessdtiHateaiTaflj^Deotsaieaude,tbetef^fiK^^tBudlel^^ perfonnanl^ofttedoaingsoliaioa8.T}KSp<M)rwiUbedi^ for deteaDiBation and documentation of the analytic*! purity and composition of the tBasubstanw, (orvehicle). and ttstabiUy and strengthof the test sutetaflce in (be solvent 33 Test Substance Retention Saaiple tfaeSponsor. s tia spoBsi1)UiQfof 4.0 TESTMO PAC'SJTY AND KEYPERSONNEL 4.1 Name: ToxicologyTestingFacility BioReliance 4.2 Address: 963&Medical Center Drive Roclcvflle,MD 20850 43 Study Director: Valentine 0. Wagner in, MS. Phone: 301-610.2152 FBC 301-738-2362 BmaiJ; swsgaetl&biomSiasce.wm 5.0 PROPOSED STUDY DATES 5.1 Experimental Start Date; ^30-Aag-2000 5.2 ExperimenftrfTennination Date: 170ct-2000 5.3 Draft ReportDate: t31-0ct-2000 I '' 5.4 Final ReportDate: 8-NW-2000 6.0 TEST SYSTEM T\i(hse tester sstttnaiiattss will TA1535 anod TA1537 iandducttthe S. ddeltibed Qpliimorium lustidine EWEOtropbsTA98, TA100, as WP2 uwA as described by by Ames et al (1975) aild^ie E. coli tester strain and Model (1976). "'" ^ ftwoBolSroWOMOl lfrAng-2000 BioReliance Study No. AA34BK.502001.BTL P(e2ofl0 48 ABIORELIANCF ~9y railr Microbioiii<icii <i>ocnii SponsorProjectNumber. BioReIiance StudyNumber: _ JSaSaa^SSL M2mSQBlQ. Genotypoefthe Stnam Pied for Muttgen TMtmg I ft^-----^^- Each S. typbunurium tester additional strain contains, in addition to a mutation in the histidine operon, imitations that enhance sensitivity to some mutagens. The rfa mutation results in a cell waU certain classes of chemicals deficiency flat mcteases die penneability of the cell to otherwise be excluded. Hie toch those cootaining deletion in the large ring systems dot would excision-repair system. Tester strains avrB TAPS gene results in a deficient DNA plasmid (canying die R-factor). ft has and TA100 also contain the pKMIOl sensitivity to mutagefls been suggested that by modifying an existing bacterial the plasmid increases complex involved with the mismatch-repair DNA repair polymerase process. TA98 and TA1537 independence are reverted from histidine dependence (auxotrophy) histidine (prototrophy) by fianieduft ftameshift and mutagens. TA100 is to' reverted by both cause base base substitution substitutions. mntagens and TA1535 is reverted only by mutagens that The E. coli tester strain has an AT base pair at die critical gene (Wilcox et aL, 1990). Tester sirain WP2uwA has mutation ate within die ttpE a deletion resulting in a deficient DNA excision-repairsystem. Tryptophaa in die uvrA yeas to a base change at die originally mutated site or by a base revenants can arise due change elsewhere chromosome cansing reversion mechanism die original mutation to be suppressed. Thus, die in specificityof the die 1976). is sensitive to base-pair substitution mutations (Oreen and Muriel, Toe S. typhimurium tester strains University of California, Bcitelcy. were received directly from Dr. Brace Ames, NKaintigodnoaml )C. ollection of Industrial anTdoeMaEr.inceoiBi atcetsetreira,stArabienrdweaesn, reScceoitvleadndfr(oUmnitdeide 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The test substance negative will be tested at a imninuirn of five dose levels along with qpropriate aod positive controls with tester strains TA98, TA100, TA1535, TA1537 and cWoPnt2roulsvraAndwpiothsidanyde without S9 activation. All dose levels of test substance, negative coBtroIs will be elated in trinTii't ft"coisrorazooi ie.Ms.mo BioReIiance Study No. AA34BK.502001 .BTL pge3ono 49 6 ( BlORELIANCE- ^y fBrKlttIr M)cK6iolo(icll 010CHH1 Reverse Mutation DuPoat-4670 Sponsorftoject Number BioR-Uancc StudyNumber DaPmt^lQ AA34BK.S02001.BtL 7.1 SolubilityOeteimioatjoo 7.2 ^3 A solubility determination will be conducted to determine die concentration or wodoMe compat^le wish mispension up to a maximum soluble maximum of 500 rag/mL. Vehicles tlus test systei^mtwier of iwefeience.indude but are not liro to: deionized water (CAS 64-17-5) (CAS 7732-18-5), dimethylsalfoxide (CAS 67-68-5), ethanol solvent, selectedaiiodoardceetroonfep(rCefAeSren6c7e.,6w4-h1i)c.hThe vehicle of choice will be Ac workable/soluble stock acentration, up to permits pfeparation 500 mg/mL. of the highest PreliminaryToxicity Assayto Select Dose Levels aigiiestdoseSevelwinbeseleete!atntteffipttoyiIdprec^tateatonlytne Selection of dose and precipitation levek for the nattagenicityassay wifl be OMed upon the toxicity assay. This profile of the preliminary test substance assessed in a preliminary toxicity assay will be conducted by exposing TAPS, TA100, TA1535, TA1537 and WP2awA to negative concentrations of test substance, o"ple controls and to at feast eight absence of S9 activation. Unless per dose indicated level, ia both the picscncc and wffl be she otherwise highestworiableconcantradon by the Sponsor, the highest dose exceed 5mg/platc. In selectingdose levels in the vehicle of choice but not to fbUowmggudelines win be eo^oyed. Doses for Ac mutagemcity will be selected assay die does not interfere with manual scoring. Wheneiw po^'ble, such that precipitate &e highett dose the aBitagenicityassay wm be selected to give some indicaaoa of for toxidtywitho^ deoxcseeeldeivnegl 5wmflgl /pbleateS.taBga/pr lfaiteee.ly sohAle, ooatoxic test substances, the highest For pfecipitating,noiitoxic test substances,the ow or two dose levels. The SjXHisorwiU be coasultcdregaaling (1) toe maximiim dose levdigaetocied based <mpiBc4rftati<m dose selection if is less than 5mg/plate or (2) the BMximuniaEhievableiiest and mis dose Igvel less than 5 iag/plate and this dose level is noatoxic. substance dose level is Requency and Route of Administration The test system wffl b exposed to methodology originally the test substance via me plate incoipomion and Aroes (1983). described by Ames This et al (1975) sad updated by Maron classes of chemical mutagteensst <syMscteCmanhaaest baeLe, n19s7h5o;wMncQtoudmetaecntd, aAmweidse, 1r9a7n6g)e. of te After the data generated in assay will be repeated. The me first assay have been evaluated, me mutagenicity as those used u 5m dose levels used in the assay unless.me Study second assay will be the same levels should be changeddue to an equivocal Director detennines that the dose excessive precipitate. If the Sponsor is aware response, excessive cytotoxicity or of specific metabolic (e.g., azo compounds), this information will be utilized in requirements designingthe (e.g., activation system or treatment method). This guidance is based assay. OB the Protocol SPOT502001 l6-Ali8-2000 BibReliance StudyNo.AA34BK.502001.BTL P(e4ofl0 ' - 50 fftk ftir)Df:l tAMr'P ^y ormfrr<yMlctfbtoloBica)A*locitpt Reverse Mutation DuPont-4670 Sponsor ProjectNumber. BiolMiaaceStadv Number _I>uPOBt.4<>7Q AA34BK.5020(M.BTT. OECDC^dcliffi1471 (adoptedJuly of RegulatoryGenotoxicity 1997)aadICH Guidance on Specific Aspects Tests for Pharmaceuticals(1997). 7.4 Controls 7.4.1 Positive Controls 7.4.2 jm fcompmanoM with the assay are 01 positive CQI li;sted below: ittds aod (ester stBuos ^i ued coacuneat^ Stnuo SiSIKIDOlite StKXBU W2wA TA98 TA100.TA1535 TA1537 WKuwA AcAntfcm Ru rUi>o*akek lE^UxwCooxid 2-BiaoHaixeaf KidtoiaBicide XBOfl)^fllBBIftIkCrol^Elfll&C CooceotcfixoD OU^teu) 1.0 10 1.0 .1,0 75 1,000 Negative Controls 7.4.3 t o w i,, t h o u.t- --S--9 _.e->. vuuuiB wiu oc PJUKBO ior eaOtt tester activation. The wsuSive control will be the stnuo with and unless there is BO histoiical basis vehicle atone, UK of the selected vehicle. In die latter case, bodiwitteaiedwd vehicle controls will be used. SterilityControls wThilel bmeocsbt ecdoaoecdefmornsfetedrtielistyt .substaocedilutioB and the Sham asd 59 mixes 7.5 Exogenous Metabolic Activation Arocloi'1254-iiMitt^ rat liver S9 will be uacd as the snetabolicactivadoa The S9 bomogeaate win be prepared from male Spragge-Dawleyrats system. induced with a silifte hiti-apenftxiealiBJectjdnof Arodor 1234,500 ingftg, five daysprior to sacrifice. Tbc S9 will be batch pafuxed aod stored fsasea at approximately -70^ unused. Bach batc& of S9boiaoiBBatewill be assayedfor its ability metabolize 2-amiaoaitfBaceoe and 7.12-danethytbeazantluaceae to mutagenic to S. typfwmsnwn TA100. to fonns contain 10% 59 honKUgBBBtB, 5fflM pool to Pp--nMicTotOiDtaomaidnem-ssied-i gdeniiBdimscleotidepfayo,q--ih--a, t-e.yS^^injyMuoMn^., 4*mauMn s 100 roM phosphatebufito at pH mix will be 100 mM phosphate 7.4. This mixture is refiared to as S9'mmiix. Shaimri baffer at pH 7.4. Ptotoc^SPOISOMOl 16-Au-2000 PtaBSoflO fi| BlOREUANCF ^^y fwnRtrly Miciobtoloficjl A(IOCII BioReIiance Study No. AA34BK.502001JBTL 51 Reverse Mutation DuPont-4670 7.6 7.7 Sponsor Project Number BtoltelMBCfe StudyNumber: Preparationof Tester Sfasa DuPQnt-4670 AA34BKJ02001.BTL 1^ Overnight cultures will be inorailitfed from the appropriate master plate the appropriate ftozea stock. To ensure that cultures are harvested in or &om late log phase, the lengthof incubatKm^^ woridng day, inoculated be controlled and momtored. At die end of the each flask will be placed in a Betting ahafcenfecubatorat room ten^ezature. shikei^incubatorwill be pn^ramni^ to te approxaaately 1251pm at 37*2C approximately 12 tune of harvest. hours before die anticipated All cultures tuifoidity will be harvested by spectropbotometricmonitoring of culture rather than by duration of iix^badoo oce oveigrowth of cultures c^ cause loss of sensitivity to some mutuens. incnbatfoa at a densityof approximately 10 Cultures will be removed from cellsAnL. Test System Identification Each piste will be labeled with a code system that identiSes the test substance, phase, dose level, tetter strain and activation type as described in test BioReliaace's Standard OperatingProcedures. 7.8 Test Substance Reparation Unless specified otherwise, prior to All test substance dilutioos will be prepared immediately use. light test substance dosing will be at room temperature under yellow 7.9 7.10 Treatment of Test System 5O0BpeLhoafIfvneihiSiculel,iter(0.5mL)ofS9mixofShasnm10ix0 ,uL of tester strain aad test substance dilation or positive control will be added to 2-0 ML of molten selective top agar at 452C. When necessary to achieve the target ccaKntratioo or elicmute toxic vehicle effects, aliq^Kte of test substaoce/vehicie^positcivoentrol will be plated. The mixture will be voitex mixed and overiaid onto tee aafice of 25 mL of ininimat bottom After the overlay has solidified, the plates war be inverted and agar. incubated for approximately48 to 72 horas at 37*2C Plates that are not counted immediately following the incubation periodwill be stored at 2-8C. Scoring The condidcm of OB bacterial test SBbstancetoxicity bad^round lawn wfll be evaluated for evidence of and precipitate. Evideaw of toxidtywiHbs scored relative to ibe negative control plate and recorded along with the revertant count for that plate. Toxidty will be evaluated as a decrease in the number of revenant colonies per plate and/or a thinningor disappearanceof (he bacterial background lawn. ftotocol SPOWoaXK lfi.Ani.2000 PHae6oflO A| jAMfP ^^P R|f")|?E:j ForHrrly MrcrObfoiOfiCfl Atlacur. BioReliance Study No. AA34BK.502001.BTL 52 Reverse Mutation DuPoat-4670 Sponsor Project Number: BioReliance Study Nilmter DiiPont.4670 AA34BK.502001.BT{, wPritehcoipuittmataiogoniwfiicfal tbioene.valuated after the incubation period by visual examination 7.11 Tester Strain Verification cOhnetdhe^ dfoarythoef ampeproinprtihaetegmeunteatgicemniadritcyeresscsiatye,dainll t6e.s0t.er strain cultures will be 8.0 CRITERIA FOR DETERMINATION OF A VAUD TEST The following criteria must be met for the miltagefiicity assay to be considered valid: 8.1 Tester Strain Integrity w^ To demonstrate the pieseBce cultures exhibit of the f^i imitation, ail f. typhimwium tester soam the must mutation, sensitivityto crystal violet To demonstrate the presence of to ultraviolet light. aUf-o^imuMum tester To SseaoasSMs the straw cultures mu^ exhibit sen$iti^ tester strain cultures must exhibit presence of die uvrA mutation, sensitivityto ultraviolet light. To B coK the presence ofthepKMIOl plasmid R-factor, tester strain cultures demonstrate ofTA98 and TA100 must exhibit resistance to ampicillin. 85 Spontaneous Revertant BackgroundFrequency Based on historical control data, tester strain cultures -'----'-- " ' (vehicle). The must exhibit ..,.-c--nta.n^^ renuauaiuts> ppcejrrfp>luautec imn t(Ahe negative controls (inctaaiw): mean revertants per TA98. 10-5(fc TAlOa pla^te. --mu-s^t 80 jb^e awriit^hhijnn ^fbe ffhbnltnoawnjnqag ran--g--es" WP'2?H'"w.-A* ,1ir0t -6-e0". -240; TA1535, 5-45; TA1537,. 3-21-; 8J Te^erStisainTiteis To ensure dters that appropriatenumbers of bacteria are plated, ail tester strain most be equal to or gnsater than 0.3X109 celts per miliiliter. culture 8.4 Positive Control Values Esch mean positive control . ----- v.--alu-e mujuusMt ecjxuhuiubliut au t lieeaassti a tEhOieKe ftodld inacrreease toe respective meaa negative contro,.1l .v--aliu--e (v--ehi-c..le.) f- or each tester strain. over 8.5 Toxicity A minimum of three non-toxic dose levels will I A dose level is considers ff^k R((^)PC| nuistbel i negative control value (this reduction a_r_e.d--uc--tio^n ^^ io ^vj the witwu backgr ptao ound s e - o e p c .n-_d- e n t - d rop - is th- e revertant count) or lawn. la die event that Jess than three non-toxic Protocol SPOT502001 16-Aug-2000 Pagc7ofl0 lAKIfP ^^y Fofn*rlrMlcrobi6lQxieal AIDPI>-- BioReliance Study No. AA34BK.502001.BTL 53 iBacterialReverse Mutation DuPont-4670 Spocsor Project Number BioRelimceStBdvNnmber DnPont-4670 AA34Bg.5Q200^TL adpopserofperviaetiesachieaancghekiaveddo,stehleevaeflisfe. ct^poitioQof the ass^ will be repeated with an 9.0 EVALUATION OF TEST RESULTS I^r a teat substaiKe to be evtAiated positive, it mat cause a dots-relate iBcssawm cmoenacnenretrvaetriotanastsofpteerspt shu^bcstrafnacteleasasstpOecMifiteedttbeerlsotwra:in over a mininiugi of two mcreaaag 9.1 Strains TA1535 and TA1537 Data sets the dose will be judgedpositive if die increase in meao revertants at the peak of value response (vehicle). is equal to or greater than three times the mean Begadve control 9.2 Strains TA98,TAlOOriWP2wA 10.0 Date sets the dose will be judgedpositive if the increase in mean rcvertants at the peak of vahie response (vehicle). is equal to or greater than two times the mesa negative control REPORT A report of the result* accurately describe of this study win be prepared by the TestingLaboratoryand mil wiilinclnde: aU methods used for generation and analysis of the data. The report Test Subataaee: identificationi and known; paysiflocheinical CAS no., if known; pbygical nature and purity, if stabilityof test substance,ipfrkonpoerwtine.s relevant to the conduct of die study, if known; Ssoulbvsetnatn/cVeehinicsleo:lvjcunstt'ivfiechaitdioen, iffkonr ocwhno.ice of vehicle; solubility and stability of test Strains: strains used; nuiiiber of cena/taL per cidture;stram characteristic Testcondidons:aiQO(mtoftestsub3tanceperp]atewithradoaaIefordoseselectiona number of platesper concentratioo; media used; acdvation system, inchKiuigacceptabili^ type and composition of metabolic criteria;treatnieiitpiocedutes. Results: signs of toxidty, apa of precipitation;individual plate counts; the number of reveitant colonies per plate sad standard deviation; mean dose-response relationship, where possible; statistical analysis, if any; concurrent negative positive control data means and standaid deviations; historical negative and and positive control data with ranges, niffBns and sfswuA rfHinatiiw Discussion of results. Conclusion. I^SPOTM IO-A^X, BioReliance Study No. AA34BIC502001.BTL ^,ono. 54 &BIOREUANCE- ^y FarintlrMjrrobio)ogi<IAf(oe(l> Compans? SanSSSaicS. not enaa(ra CBl M Reverse Mutation DuPottt-4670 11.0 Sponsor Project Number BioReljaace Study Number: Age and mean body weightsofefts used to prepare S9 RECORDS AND ARCHIVES DnPont-4670 AA34BK.S0200t.BTr. All raw data, Operating die protocol and all reports will be maintained according to Standard Procedure OPQP3040 by the BioReIiance RAQA anit headquartered at: BioRcliaflcc, 14920 Broschart Road. Rockville, MD 20850. 12,0 REGIJIATORY REQUIREMENTS/GOODLABORATORY PRACTICE This protocol Chemicals has beea written to comply with OECD Guideline 471 for Testing (Genetic Toxicology:Bacterial Reverse Mutation Assay), Adopted 1997 of and RweitghisttrhaetioInnteorfnPahtaiorrnaaalceCuotoicfiaslcseace on Harmonisation of Technical Requiicineats for for Human Use (1996 and 1997). This study will be performed in compliance OECD, Japanese 04m) and SEC with Ac provisions of die EPA (TSCA), Laboratory Studies. Good LaboratoryPractice Regulations for Noaclisical 13.0 Unless arrangements of following are made to die contrary, unused dosing solutions will be disposed administration to the test system sad all residual test substance will be disposedof followingfinahzation of the report REFERENCES Aiges, and BX, McCaca, J. and Yamasala, E. (1975). Methods for detecting carcinogens mutagens with the Research 31:347-364. Salmtmella/maiBsatvm'mciososmauetagenichy test. Mutation Green, M.H.L., and Muriel, WJ. (1976). Mutagen testing using tip4' &cteri(riteico& Mutation Research 38:3-32. reversion in International Registration Conference oa Hannonisation (ICH) of Technical Requirements of Phannaceuticals for Human Use. Guidance oa for Specify; Aspects of aRdeogputliaotnoraytQesateoptox4iciotyf Tethsets fICorHPhannaceuticals.S2A docomeat recommended for 61:18198-18202,April 24,1996. process OB July 19, 1995. Federal Register International Conference oa HarmonisatioB Registration of Pharmaceuticals (ICH) of Technical Requirements for for Human Use. Genotoxicity;A Standard Batteryfor Genotoxidty Testing of Pharmaccuticals. S2B document step 4 of the ICH process on July 16,1997. Federal recommended for adoption Register 62:16026-16030, at 21,1997. November Protocol SPOI'502001 16-Aug-2000 BioReliance Study No. AA34BK-502001JBTL R^oflO 55 fftk DI/-)DC| |AMr'P~ ^y Fvtm*t}y M!EiafaioloKic( A,6oalei ial Reverse Mutation DuPont-4670 Sponsor ProjectNumber: BioRglimceSfaidv Number DyPont-4670 AA34BK.5020Ql.BTr. McCaan. J. and Araes, BK Saimonella/tsictOSosBS (1976). Detection of carcinogens as mmagens in d USA 73:950-954. test: assay of 300 chemicals: discussion. Pros. Nad. Acad. Sci. McCann. J., maitagens in Choi, E-, Yamasaki, & ad Ames, die Saimonellaimicxaseas B.N. (1975). Detection ofcareinogees as Sci. USA 72^135-5139. test assay of 300 cfiemicals. Proc. Natl. Acad. Maron, D.M. and Ames, Test Mutation Research BX (1983). Revised 113:!73-215. Methods for the Salmonella Mutagcnicity OECD GuideliBe 471 for Testingof Chemicals (Genetic Toxicology:Bacterial Mutation Assay), AdoptedJuly 1997. Reverae Wilcox, P.. Naidoo, A., Wedd, Salmonella lyphmwriwn TA102 DJ. aad Oaieboutc, D.G. (1990). Comparison of 5:285-291. with Escheri^tiacoltWP2 tester strains. Muta^enesis 14.0 APPROVAL R<X^o^ SCpiwownrsnowr RPa.eBMp.1r<e.s^en**^t--a<tivTe^"--/^--^~_--_--_--_--_ fflft.r:^ "t^Mma^- (Print or Type NasBe) \|JZ^^ai^^r --^Z---"Dfait-e^^" 2.COO 2^ Aegaooo Date -^w^T-S^ ftwocolSPOTSOZOOl I6.Ant.2000 BioReliance Study No. AA34BK.502001.BTL P8l0ofl0 BlORELlANCE* ori.rl MlctOHolo,,c.l *>IOM;,M 56 Company SsmftkeS. Does not eontafn 7SCA CB? ial Reverse Mutation DuPont-4670 APPENDIX C BioReliance Study No. AA34BK.502001.BTL 57 ..-..-., Reverse Mutation '--' Repc?rt of Results of Reverse-Mutation Assay in Bacteria 1. General Items Name oflfae new chemical substance (IUPAC nomenclature) DuPont-467 Other name Fluoroalkyi Uremane Molecular weight Appearance at ordinary '1 temperature Structural formula or rational formula (or outline of manufacturing method, in case bom are unknown) ? -Stahiaty 1 Melting point 1 Boiling point Vapor pressure g Partition .S coefficient Purity of the new chemical substance tested Name and concentration of impurities 100.0 wt% Solubility ? Water Degree of s'^ solubility DMSO Acetone Other ( ) 300mg/mL e[Rxteemntaproksss]Bibelec.ause physicochemical properties are reference materials, fill in spaces to 1."Stability"-Fill in the stability for water, other solvents, heat, light, 2. "Vapor pressure''-Fill in the vapor pressure of me test substance at etc. 25C. u3s."ePdafrotirtitohne mcoeeafsfiucrieemnte"n-Ftill in me value, the temperature used and me name 4."Solubility"-Ful in such information as water-soluble, soluble in oil. 5. "Degree ofsolubnity"-FiU in the solubility at 25C for each solvent. of the solvent BioReSiance Study No. AA34BK.502001.BTL 58 , Does no8 contain TSCA CB1 Bacterial Reverse Mutation DuPont-4670 2. Tester Strains (1) Procurement Strain TA98 TA100 TA1535 TA1537 TA1538 TA97 TA102 WP2ywA WP2yvrA (pkMIOl) WP2 (pKMIOl) (2) Storage Freezing method Storage temperature Composition 3.S9Mix Obtained from Date obtained 10 November 1998 Date inspected the strain lot in storage Dr. Bruce Ames University of California, Berkeley 11 August 1998 National Collection of Industrial and Marine Bacteria Aberdeen, Scotland 13 December 1990 14 November 1990 1 July 1987 19 February 1993 The genedc markers for each culture are confirmed on me day of use Large quantity -70C Bacterial suspension DMSO 1.0 mL 0.09 mL (1) Source of S9 Made in-house Purchased fiom: MolTox Prepared on Batch 1030 prepared on 22 June 2000 17 December 1999 (2) Storage Temperature, etc. ofS9 Storage temperature -70C Name and model of So-Low, Model BioReliance Study No. AA34BK.502001 .BTL 59 ( storage apparatus vw-m (3) Preparation ofS9 Animal used Inducing substance Species, Strain Rattus norvegicus, SpragueDawley Name Aroclor 1254 Sex Age (in weeks) Weight Male 9 234 to 252 g Administration method AdnuniiSte&tion period and amount <g^Eg-weigfat) intraperitoneal ; 5 days, 0.5 gin/kg ; body weight 4. Positive Control Substance (1) Positive control_____ Name Manufacture Lot No. 9-Aminoacridine (9AAD) 2-Aminoanthracene (2AA) Methyl metfaanesulfonate (MMS) 2-Nitrofluorene (2NF) Sodium azide (SA) Sterigmatocystin (STM) Sigma Chemical Company Sigma Chemical Company Aldrich Chemical Co., Inc. Aldrich Chemical Co., Inc. Sigma Chemical Company Sigma Chemical Company 106P06681 085H2508 09419LR 11202TF 098H0169 118H4061 Grade Practical Practical Purity (%) >98% >95 % >99% >98% >99% >99% Solvent used DMSO DMSO DMSO DMSO water DMSO (2) Preparation and storage of positive control solution Prepare or Store Store subdivided solutions (Storage temp. -5 to -30C) BioReliance Study No. AA34BK.502001.BTL 60 Cempmy Sanifaed. mt JSC& CBf 5. Preparation of Test Substance Solution Solvent used *Name Acetone 'Manufacture Fisher Scientific *LotNo. 970721; 943596; 971014 'Grade 'Purity (%) Certified ACS Stability of test substance in the solvent * Reason to choose the solvent Unknown Solubility determination Methodof suspension when test substance is difficult to dissolve Not applicable Storage time and temp. from preparation to use for test Conversion by purity <30 minutes at ambient temperature No BioReliance Study No. AA34BK.502001J3'TL 61 6. Conditions ofPrc-culture (^Conditions______ Nutrient broth 7. Agar Plate Medium CQTopagar Agar (2) Minimum Glucose Agar Name Manufacturer Lot No. Made in-house Name Manufacturer Lot No. Volume ofagar plate medium BioReliance Study No. AA34BK.5Q2001.BTL 62 BBLSeIect 'fitonDickiast 1000J3DKSQ BBLSeIect BectonDicldnson 1000J3DKSQ 25 mL ?noteonteSn TSCA CBI 8. Test Results - Judgement of the results Judgement Reason for judgement and referential matters: No positive absence response was observed with any of the tester strains in the ofAroclor-induced rat liver_8S99uuopto 5000 nngo/Wplaat^e. presence and Referential matters The vehicle and positive control values indicate that all tester strains coirectly and characteristic werecapable ofdetectrngamuiagen. The vehicle were functioning controls exhibited numbers offssopoonnttaanneenonues mre ^, vwetr'toa"n*t"s ._ . a--n-d1 ,, t^h- e- .wuvik-uum "test' results c a uj re s v e a xmc lid. the BioReliance Study No. AA34BK.502001.BTL 63