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AR226-3189 DuPont-7326 TRADE SECRET Study Tide H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay Testing Guideline OECD Guidelines for Testing of Chemicals Section 4: Health Effects, No. 471 (1998); OPPTS Guideline 870.5100 Authors Valentine 0. Wagner, ffl, M.S. Trinh T. H. Nguyen, B.S. Study Completion Date 29 October 2001 Performing Laboratory BioReliance 9630 Medical Center Drive Rockville.MD 20850 for E. L du Pent de Nemours and Company DuPont Haskell Laboratory P.O. Box 50,1090 Elkton Road Newark, DE 19714-0050 Performing Laboratory Study Number AA47TV.502001.BTL DuPont Project ID DuPont-7326 Work Request Number Service Code Page 1 of 62 gompany Sanitized. Does not contain TSCACBi H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________DuPont-7326 STATEMENT OF COMPLIANCE Study No. AA47TV.502001.BTL was conducted in compliance with the U.S. PDA GLP Regulations as publishedin 21 CFR 58. the U.S. EPA GLP Standards 40 CTR 160, and 40 CFR 792, the UK GLP Compliance Programme, the 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 test and 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. The stability of the test and control substances has not been determined by the testing facility. Analyses to determine the uniformity (as applicable), or concentration of the test and control mixtures were not performed by the testing facility. The Sponsor has indicated that they have not performed these analyses on the test substance mixtures. The stability of the test and control substances in the test and control mixtures, respectively, has not been determined by the testing facility. The Sponsor has indicated that they have not performed these analyses on the test substance mixtures. ^ y - i /- p \'&^A<, 0. U) Valentine 0. Wagner, ffl, M.S. Study Director BioReliance Study Management 2^ Dc^?oo| Date i^ (ye^f-affo/ Date BioReliance Study No. AA47TV.502001.BTL lEompany Sanitized. Does not contain TSCA CB5 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay____ Quality Assurance Statement DuPont-7326 Study Title: BACTERIAL REVERSE MUTATION ASSAY WITH AN INDEPENDENT REPEAT ASSAY Study Number AA47TV.502001.BTL Study Director: Valentine 0. Wagner, in, M.S. This study has been divided into a series of in-process phases. Using a random sampling approach, Quality Assurance monitors each of these phasesover a series of studies. Procedures, documentation, equipment records, etc., are examined in order to assure that the study is performed in accordance with the U.S. PDA Good Laboratory Practice Regulations (21 CFR 58), the U.S. EPA GLPs (40 CFR 792 and 40 CFR 160), the UK GLP Regulations, the JapaneseGLP Standard, and the OECD Principlesof Good Laboratory Practice and to assure mat the study is conducted according to me protocol and relevant Standard Operating Procedures. The following are the inspection dates, phases inspected, and report dates ofQA inspections of this study. InspectOn Phase 02-Aug-Ol -02-Aug-Ol To Study Dir02-Aug-01 ToMgmt02-Aug-01 Protocol Review Inspect On Phase 03-Aug-Ol - 03-Aug-Ol To Study Dir 03-Aug-Ol To Mgmt 06-Aug-Ol Preparation of S9 mixture Inspect On Phase 17-Aug-Ol - 17-Aug-Ol To Study Dir 17-Aug-Ol To Mgmt 17-Aug-Ol Strain characterization Inspect On Phase 28-Aug-Ol - 28-Aug-01 To Study Dir 28-Aug-01 To Mgmt 29-Aug-O I Weighing the test article InspectOn Phase 29-Aug-Ol - 29-Aug-Ol To Study Dir29-Aug-01 To Mgmt 29-Aug-Ol Preparation of S9 mixture InspectOn Phase 03-Oct-Ol - 04-Oct-Ol To Study Dir 04-Oct-Ol To Mgmt 09-Oct-Ol Draft Report Inspect On Phase 29-Oct-Ol - 29-Oct-Ol To Study Dir 29-Oct-Ol To Mgmt 29-Oct-Ol Draft to Final Report This report describes the methods and procedures used in the study and the reported results accurately reflect the raw data of the study. ^ ^ ^ - Elahe Siadatpour, B.S. QUALITY ASSURANCE OcA. 3R, AOOl DATE BoiioKReuliance Study No. AA47TV.502001 .BTL 3 Sanitized. Does nol containrtTSCACBI pCaonmropannvy' H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________DuPont-7326 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study Director: \<^xJ^tfe &- (j\QuaMSi^,~S^ jZ^d^&of Valentine 0. Wagnerm, M.S.Date Approved by Study Monitor: Q ^A.OL-^ -- ^o0.(_r> Maria Donner, Ph.D. Senior Research Scientist ^1 OCT ZOQ^ Date BioReliance Study No. AA47TV.502001 .BTL 4 company Sanitized. Does not contain TSCA CBi H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________DuPont-7326 TABLE OF CONTENTS Page Good Laboratory Practice Compliance Statement...................................................................^ Quality Assurance Statement....................................................................................................3 Certification...............................................................................................................................4 Study Information.....................................................................................................................7 Summary ...................................................................................................................................8 Purpose.....................................................................................................................................^ Characterization of Test and Control Substances......................................................................9 Materials and Methods............................................................................................................10 Results and Discussion............................................................................................................15 Conclusion....................................................:....................................................................--...15 16 References.............................................................................................................................. DataTables..............................................................................................................................l7 Table Table Table Table Table Table Table Table Table Table Table Table Table 1: Preliminary Toxicity Test in Salmonella typhimurium TA98 .............................17 2: Preliminary Toxicity Test in Salmonella typhimurium TA100 ...........................18 3: Preliminary Toxicity Test in Salmonella typhimurium TA1535 .........................19 4: Preliminary Toxicity Test in Salmonella typhimurium TA1537 .........................20 5: Preliminary Toxicity Test in Escherichia coli WP2 21 uvrA................................... 6: Mutagenicity Test in Salmonella typhimurium TA98 without S9 .....................22 7: Mutagenicity Test in Salmonella typhimurium TA98 with S9 ..........................22 8: Mutagenicity Test in Salmonella typhimurium TA100 without S9 ...................24 9: Mutagenicity Test in Salmonella typhimurium TA100 with S9 ........................25 10: Mutagenicity Test in Salmonella typhimurium TA1535 without S9 .................26 11: Mutagenicity Test in Salmonella typhimurium TA1535 with S9 ......................27 12: Mutagenicity Test in Salmonella typhimurium TA1537 without S9 .................28 13: Mutagenicity Test in Salmonella typhimurium TA1537 with S9 ......................29 Table 14: Mutagenicity Test in Escherichia coliWPl uvrA. without S9...........................30 Table 15: Mutagenicity Test in Escherichia coli WP2 uvrA with S9................................31 Table 16: Mutagenicity Test in Salmonella typhimurium TA98 without S9 .....................32 Table 17: Mutagenicity Test in Salmonella typhimurium TA98 with S9 ..........................33 Table 18: Mutagenicity Test in Salmonella typhimurium TA100 without S9 ...................34 Table 19: Mutagenicity Test in Salmonella typhimurium TA100 with S9 ........................35 Table 20: Mutagenicity Test in Salmonella typhimurium TA1535 without S9 .................36 BioReliance Study No. AA47TV.502001 .BTL 5 """""^""------------------n^ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________DuPont-7326 Table 21:Mutagenicity Test in Salmonella typhimurium TA1535 with S9 ......................37 Table 22: Mutagenicity Test in Salmonella typhimurium TA1537 without S9 .................38 Table 23: Mutagenicity Test in Salmonella typhimurium TA1537 with S9 ......................39 Table 24: Mutagenicity Test in coli WP2 uvrA without S9...............................................40 Table 25: Mutagenicity Test in Escherichia coli WP2 uvrA. with S9................................41 Table 26: SalmonelIa/E. coli Mutagenicity Test - Summary of Results B 1......................42 Table TI-.Salmonella/E. coli Mutagenicity Test - Summary of Results B2......................43 Appendix A: Historical Control Data..................................................................................... 44 Appendix B: Study Protocol................................................................................................... 46 Appendix C: Information for Japanese Regulatory Agencies................................................ 58 BioReliance Study No. AA47TV.502001 .BTL 6 ^aniteed. D<es not contain TSCA CB! H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay 9th Collective Nomenclature Synonyms/Codes: ^ H^25006 Haskell Number: 25006 CAS Registry Number y>5605-58- Composition DuPont-7326 Known Impurities^^^^^BI^L . Physical Characteristics: Amber liquid Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Sponsor: E.L du Font de Nemours and Company Wilmington. Delaware 19898 U.S.A. Study Initiated/Completed: August 2,2001 / (see report cover page) m-Life Initiated/Completed: August 3, 2001 / September 6, 2001 BioReliance Study No. AA47TV.502001.BTL company Sanitized. Does not contain TSCA CB! H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay________________________DuPont-7326 SUMMARY The test substance. H-25006, was tested in the H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay using Salmonella typhirnurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA. in the presence and absence of Aroclor-induced rat liver S9. The test was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay, (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test substance. Tetrahydrofuran (THF) was selected as the solvent of choice based on the Sponsor^ request, compatibility with the target cells and solubility of the test substance. The test substance was soluble and clear in tetrahydroruran (THF) at approximately 500mg/mL, the maximum concentration tested. In the preliminary toxicity test, the maximum dose tested was 5000 ug per plate; this dose was achieved using a concentration of 200 mg/mL and a 25 uL plating aliquot. Dose levels tested were 5000. 3333,1000, 667, 333, 100, 67, 33, 10 and 6.7 ug per plate. Precipitate was observed beginning at 333 ug per plate and no appreciable toxicity was observed. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 ug per plate. In the mutagenicity test, no positive mutagenic response was observed. The dose levels tested were 5000,1500,500,150,50 and 15 ug per plate. Precipitate was observed beginning at 500 ug per plate and no appreciable toxicity was observed. The results of the Bacterial Reverse Mutation Test with an Independent Repeat Assay indicate that, under the conditions of this study, H-25006 did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9. BioReliance Study No. AA47TV.502001.BTL gcrnpany Sanitized. Does not contain TSCA CB! H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 PURPOSE The purpose of this study was to evaluate the mutagenic potential of the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimuriwn and at the tryptophan locus of Escherichia coli strain WP2 MW-A in the presence and absence of Aroclor-induced rat liver S9. CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test substance, H-25006, was received by BioReliance on 27 July 2001 and was assigned the code number AA47TV. The test substance was characterized by the Sponsor as amber liquid that should be stored at ambient temperature, protected from light. An expiration date of 31 August 2004 was provided. Upon receipt, the test substance was described as a pale yellow thick liquid and was stored at room temperature, protected from light The identity, strength, purity, composition or other characteristics to define the test substance have been determined by the Sponsor. The stability of the test substance has been determined by the Sponsor. Results from these analyses have not been provided to BioReliance. The vehicle used to deliver H-25006 to the test system was tetrahydrofiiran (THF, CAS# 109-99-9), Gold Label, 99.9%, purchased from Aldrich Chemical Company. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light. Positive controls plated concurrently with the mutagenicity assay are listed below. All positive controls were diluted with dimethyl sulfoxide (DMSO) except sodium azide, which was diluted with water. AU subdivided solutions of positive control were stored at -5 to -30C. Strain S9 Activation Positive Control Concentration dig/plate) All Salmonella Strains 2-aminoanthracene 1.0 (Sigma Chemical Co.) Rat LotNo.085H2508 WP2wvrA CAS No. 613-13-8 10 Purity >95% BioReliance Study No. AA47TV.502001.BTL 9 japans Sanitized. Does not contain TSGA GB3 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Strain S9 Activation Positive Control Concentration (tig/plate) TA98 TA100, TA1535 TA1537 WP2iwA None 2-nitrofluorcne (Aldrich Chemical Co., Inc.) Lot No. 11202TF CAS No. 607-57-8 Purity >98% Sodium azide (Sigma Chemical Co.) LotNo.085H0476 CAS No. 26628-22-8 Purity >99% 9-aminoacridine (Sigma Chemical Co.) Lot No. 106F06681 CAS No. 9045-9 Purity >98% Methyl methanesulfonate (Aldrich Chemical Co., me.) LotNo.09419LR CAS No. 66-27-3 Purity >99% 1.0 1.0 75 1.000 To confirm the sterility of the test substance, the highest test substance dose level used in the mutagenicity test was plated on selective agar with an aliquot volume equal to that used in the test. These plates were incubated under the same conditions as the assay. MATERIALS AND METHODS Test System The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA. as described by Green and Muriel (1976). Salmonella tester strains were received directly from Dr. Bruce Ames, University of California, Berkeley. E. coli tester strains were received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. Tester strains TA98 and TA1537 arc reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that BioReliance Study No. AA47TV.502001 .BTL 10 ompany Sanitized. Does not contain TSCA CB$ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_____________________DuPont-7326 cause both frameshift and basepair subsdtution mutations. Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976). Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing -50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shakerAncubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 372C approximately 12 hours before the anticipated time of harvest Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a liter of approximately 109 cells perinilliliter. The actual titers were determined by viable count tests on nutrient agar plates, and the data is on file but not presented in this report. The study was conducted to comply with OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July 1997 (published February 1998) and with me International Conference on Harmonisadon of Technical Requirements of Registration of Phannaceuticals for Human Use (1996 and 1997). Metabolic Activation System Aroclor 1254-induced rat liver S9 was used as the 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 batches were prepared and stored at -70C or colder until used. Each bulk preparation of S9 was tested for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared immediately before its use and contained 10% S9, 5mM glucose-6-phosphate, 4 mM 6-nicotinamide-adenine dinucleotide phosphate, 8 mM MgClz and 33 mM Kd in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the 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 the vehicle. The test was conducted using water, dimethyl sulfoxide (DMSO), ethanol (EtOH), acetone, tetrahydrofuran (THF), and N,N-dimethylformamide (DMF). The test substance was tested to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration, up to 50 mg/mL for aqueous solvents and 500 mg/mL for organic solvents. BioReliance Study No. AA47TV.502001.BTL 11 -^w^y Parl^. W ^ ^'^ifT5CA GBi H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________DuPont-7326 Preliminary Toricity Test The preliminary toxicity test was used to establish the dose-range over which the test substance would be tested. Ten dose levels of the test substance were plated, one plate per dose, with overnight cultures of TAPS, TA100, TA1535, TA1537, and WP2 uvrA. on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. Dose levels tested were 5000.3333,1000,667,333,100,67.33.10 and 6.7 ug per plate. Mutagenidty Test The mutagenicity test (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test substance. A minimum of six dose levels of test substance (5000, 1500, 500, 150, 50 and 15 ug per plate) along with appropriate vehicle and positive controls were plated with TAPS, TA100, TA1535, TA1537, and WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test substance, vehicle controls and positive controls were plated in triplicate. Plating and Scoring Procedures The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (19S3). On the day of its use, minimal top agar, containing O.S % 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 was supplemented 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 the Milli-Q Reagent 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 with 2.5 % (W/V) Qxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (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. One-half (0.5) millinter of S9 or Sham mix. 100 uL of tester strain and 25 uL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 452C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 uL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and BioReliance Study No. AA47TV.502001.BTL 12 ^BSpSgpajBltlzedD. oes noi confsSn TSCfi r" H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 incubated for approximately 48 to 72 hours at 372C. Plates that were not counted immediately following the incubation period were stored at 2-8C until colony counting could be conducted (less than 10 days). The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown below. Code 1 2 3 4 5 6 NP IP Description Characteristics Normal Slightly Reduced Moderately Reduced Extremely Reduced Absent Obscured by Precipitate Non-Interfering Precipitate Interfering Precipitate Distinguished by a healthy microcolony lawn. Distinguished by a noticeable thinning of the microcolony lawn and possibly a slight increase in the size of the microcolonies compared to the vehicle control plate. Distinguished by a marked thinning of the microcolony lawn resulting in a pronounced increase in the size of the microcolonies compared to the vehicle control plate. Distinguished by an extreme thinning of the microcolony lawn resulting in an increase in the size of the microcolonies compared to the vehicle control plate such that the microcolony lawn is visible to the unaided eye as isolated colonies. Distinguished by a complete lack of any microcolony lawn over greater than or equal to 90% of the plate. The background bacterial lawn cannot be accurately evaluated due to microscopic test substance precipitate. Distinguished by precipitate on the plate that is visible to the naked eye but any precipitate psubstances detected by the automated colony counter total less than 10% of the revertant colony count (e.g., less than 3 psubstances on a plate with 30 revertants). Distinguished by precipitate on the plate that is visible to the naked eye and any precipitate psubstances detected by the automated colony counter exceed 10% of the revertant colony count (e.g., more than 3 psubstances on a plate with 30 revertants). These plates are counted manually. 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 plate exhibited toxicity. Evaluation of Results For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. BioReliance Study No. AA47TV.502001.BTL 13 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________DuPont-7326 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 over a minimum of two increasing concentrations of test substance. Data sets for tester 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 tester strains TA98, TA100 and WP2 uvrA were judged positive if the 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 the mutagenicity test to be considered valid. All Sabmnella tester strain-cirfturcsTnust-demonstratethe^presence'ofthe deepTough mutation (r;te) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKMIOl plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80-240; TA1535, 5-45; TA1537, 3-21; WP2 uvrA, 10-60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3xl09 cells/mL. The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the 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 both of the following criteria are met' (1) A >50 % reduction in the mean number of revertants per plate as compared to the 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 All raw data, the protocoLand all reports will be maintained according to Standard ! Operating Procedurea----HB^ythe BioReliance RAQA unit headquartered at: BioReliance, J 14920 Broschart Road^ockvlUe,MD 20850. Per this SOP, paper records will be retained for at ; least three years after which time the Sponsor will be contacted for a decision as to the final disposition of the materials. All study materials returned to the Sponsor or destroyed will first be copied and the copy will be retained in the BioReliance archives for a minimum of 10 years. i Unused dosing solutions were disposedof following administration to the test system and all I residual test substance will be disposed of following finalization of the report. j ' Deviations No known deviations from the protocol or assay-method SOPs occurred during the conduct of this study. BioReliance StudyNo.AA47TV.502001.BTL 14 ^^^"TSCACBI H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________DuPont-7326 RESULTS AND DISCUSSION Solubility Test Tetrahydrofuran (THF) was selected as the solvent of choice based on the Sponsor'srequest, compatibility with the target cells and solubility of the test substance. The test substance was soluble and clear in tetrahydrofuran (THF) at approximately 500mg/mL, the maximum concentration tested. Preliminary Toxicity Test The results of the preliminary toxicity test are presented in Tables 1 through 5. These data were generated in Experiment Al. In the preliminary toxicity test, the maximum dose tested was 5000 ug per plate; this dose was achieved using a concentration of 200 mg/mL and a 25 \iL plating aliquot. Dose levels tested were 5000, 3333,1000,667,333,100,67, 33,10 and 6.7 ug per plate. Precipitate was observed beginning at 333 ug per plate and no appreciable toxicity was observed. Based on the findings of the toxicity test, the maximum dose plated in the mutagenicity test was 5000 ug per plate. Mutagenidty Test The results of the mutagenicity test are presented in Tables 6 through 25 and summarized in Tables 26 and 27. These data were generated in Experiments Bl and B2. Dose levels tested were 5000, 1500, 500, 150, 50 and 15 ug per plate. Precipitate was observed beginning at 500 ug per plate and no appreciable toxicity was observed. In Experiment Bl (Initial Mutagenicity Test), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. In Experiment B2 (Independent Repeat Test), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. CONCLUSION All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Test with an Independent Repeat Assay indicate that, under the conditions of this study, H-25006 did not cause a positive mutagenic response in either the presence or absence ofAroclor-induced rat liver S9. BioReliance Study No. AA47TV.502001 .BTL 15 ^P.nySani,ted.Do.sno>o,n,,,nTS>CM H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________DuPont-7326 REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the SalmonellafMacamaXiaa Microsome Mutagenicity Test, Mutation Research, 31:347-364. Green, M.H.L. and WJ. Muriel (1976) Mutagen testing using trp+ reversion in Escherichia coli, Mutation Research 38:3-32. International Conference on Hannonisation (ICH) of Technical Requirements for 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 Hannonisation (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 the ICH process on July 16,1997. FederalRegister 62:16026-16030, November 21,1997. Maron, DM. 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), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February 1998. Vogel, HJ. and DM. Bonner (1956) Acetylomithinase ofE. coli: Partial Purification and Some Properties, J. Biol. Chem., 218:97-106. BioReliance StudyNo.AA47TV.502001.BTL 16 ompany Sanitized. Does not contain TSCA GB5 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 1 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL A1 TA98 3 Aug 2001 tetrahydrofuran 25 uL Test Substance Concentration ug per plate With S9 Activation Etevertants Background Eaer plate Lawn Vehicle 21 1 Without S9 Revertants per plate 31 Activation Background Lawn 1 6.7 27 1 25 1 10 18 1 33 1 33 25 1 17 1 67 21 1 39 1 100 19 1 29 1 333 33 1NP 28 1NP 667 24 IMP 20 IMP 1000 32 1NP 25 1NP 3333 31 1NP 27 1NP 5000 30 IIP 27 IIP Background Lawn Code l=Mormal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate MP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 17 ompanySanitized. Doe not contain TSCA ^BS H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 2 Test Substance Id Study Number Experiment No Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL Al TA100 3 Aug 2001 tetrahydrofuran 25 uL Test Substance Concentration ug per plate With S9 Activation tlevertants Background Eser plate Lawn Vehicle 230 Without S9 Revertants per plate 228 Activation Background Lawn 6.7 192 1 214 1 10 166 1 247 1 33 200 1 239 1 67 212 1 222 1 100 281 1 212 1 333 251 1NP 218 1NP 667 199 1NP 259 1NP 1000 221 1NP 247 1NP 3333 214 1NP 215 1NP 5000 178 IIP 190 IIP Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non~Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 18 ^p^San^.Does.olc.^T^^ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 3 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV..502001.BTL A1 TA1535 3 Aug 2(001 tetrahycirofuran 25 uL Test Substance Concentration ug per plate With S9 Activation Revertants Background per plate Lawn Vehicle 19 1 Without S9 Activation Revertants Background per plate Lawn 12 1 6.7 20 1 16 1 10 21 1 16 1 33 16 1 17 1 67 17 1 23 1 100 20 1 13 1 333 24 1NP 22 1NP 667 16 1NP 24 1NP 1000 25 1NP 19 1NP 3333 26 1NP 16 1NP 5000 14 IIP 18 IIP Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 19 Company Sanitized. Does not contain TSCA CB5 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 4 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL A1 TA1537 3 Aug 2001 tetrahydrofuran 25 uL Test Substance Concentration ug per plate With S9 Activation E^evertants Background E}er plate Lawn Vehicle 6 1 Without S9 Revertants per plate 10 Activation Background Lawn 1 6.7 9 1 9 1 10 4 1 7 1 33 10 1 12 1 67 6 1 7 1 100 6 1 9 1 333 8 1NP 11 1NP 667 16 1NP 8 1NP 1000 9 1NP 13 1NP 3333 7 1NP 7 1NP 5000 17 IIP 6 IIP Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL Company Sanitized. Does not contain TSCA CBI 20 ; H-25006: Bacterial Rev<erse Mutation Test with an Independent Reipeat Assay DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 5 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL Al WP2 uvrA 3 Aug 2001 tetrahydrofuran 25 uL Test Substance Concentration ug per plate With S9 Activation Revertants Background per plate Lawn Vehicle 23 1 Without S9 Activation Revertants Background per plate Lawn 15 1 6.7 ; 10 1 33 21 1 21 1 27 1 17 1 13 1 13 1 67 26 1 13 1 100 20 1 17 1 333 21 1NP 13 1NP 667 19 1NP 16 1NP 1000 16 1NP 17 1NP 3333 18 1NP 23 1NP 5000 18 IIP 15 IIP Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL > ampany Sanitized. Does not contain TSCA C51 21 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 6 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL TA98 None tetrahydrofuran 25 uL Experiment No B1 Cells Seeded Date Plated 2. 2 X 10" ; 17 Aug 2001 Concentration ug per plate Plate Number Revertants per plate Beickground Code Average Revertants Standard Deviation Vehicle 01 02 22 1 23 1 03 26 1 24 2 15 01 18 1 02 24 1 03 22 1 21 3 50 01 26 1 02 23 1 03 23 1 24 2 150 01 25 1 02 25 1 03 24 1 25 1 500 01 18 1NP 02 22 1NP 03 30 1NP 23 6 1500 01 21 1NP 02 22 1NP 03 27 1NP 23 3 5000 01 30 IIP 02 28 IIP 03 20 IIP 26 5 Positive Control 2-nitrofluorene 1.0 ug per plate 01 265 1 02 206 1 03 215 1 229 32 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 22 AaitlTSCACK ^pan, San^d. D^notcon. H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 7 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV. 502001. B"3PL TA98 Rat liver S9 tetrahydrofuran 25 uL Experiment No : Bl Cells Seeded ; 2.2 X 108 Date Plated : 17 Aug 2001 Concentration ug per plate Plat e Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 24 1 31 1 03 20 1 25 6 15 01 40 1 02 30 1 03 27 1 32 7 50 01 24 1 02 37 1 03 30 1 30 7 150 01 22 1 02 38 1 03 35 1 32 9 500 01 30 1NP 02 34 1NP 03 35 1NP 33 3 1500 01 32 1NP 02 27 1NP 03 34 1NP 31 4 5000 01 26 IIP 02 24 IIP 03 28 IIP 26 2 Positive Control 2-aminoanthracene 1.0 ug per plate 01 673 1 02 1051 1 03 816 1 847 191 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interering precipitate BioReliance Study No. AA47TV.502001.BTL 23 p)mpahy Saitea. BQes 1181031^ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 8 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL TA100 None tetrahydrofuran 25 uL Experiment No : Bl Cells Seeded : 1.4 X 108 Date Plated ; 17 Aug 2001 Concentration pg per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 188 1 180 1 03 195 1 188 8 15 01 152 1 02 172 1 03 180 1 168 14 50 01 210 1 02 186 1 03 207 1 201 13 150 01 170 1 02 154 1 03 176 1 167 11 500 01 149 1NP 02 176 1NP 03 180 1NP 168 17 1500 01 170 1NP 02 194 1NP 03 196 1NP 187 14 5000 01 197 IIP 02 211 IIP 03 192 IIP 200 10 Positive Control sodium azide 1.0 ug per plate 01 679 1 02 602 1 03 633 1 638 39 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 24 Company Sanilized. Does not contain TSCA CB5 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 9 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL TA100 Rat liver S9 tetrahydrofuran 25 uL ExperiLinent No : B1 Cells Seeded ; 1.4 X 10" Date Plated ; 17 Aug 2001 Concentration ug per plate Plat e Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 188 1 181 1 03 169 1 179 10 15 01 211 1 02 184 1 03 190 1 195 14 50 01 186 1 02 175 1 03 189 1 183 7 150 01 187 1 02 183 1 03 179 1 183 4 500 01 203 IMP 02 196 1NP 03 165 1NP 188 20 1500 01 220 1NP 02 177 1NP 03 196 1NP 198 22 5000 01 180 IIP 02 198 IIP 03 176 IIP 185 12 Positive Control 2-aminoanthracene 1.0 ug per plate 01 1229 1 02 1438 1 03 1178 1 1282 138 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 25 company Sanitized. Does not contain TSCA CB? H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 10 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL TA1535 None tetrahydrofuran 25 uL Experi ment No : B1 Cells Seeded ; 2.7 X 108 Date Plated : 17 Aug 2001 Concentration ug per plate Plat e Number Revertants per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 24 1 20 1 03 21 1 22 2 15 01 12 1 02 18 1 03 22 1 17 5 50 01 10 1 02 15 1 03 20 1 15 5 150 01 21 1 02 27 1 03 20 1. 500 01 26 1NP 02 13 1NP 03 27 1NP 23 4 22 8 1500 01 24 1NP 02 17 1NP 03 13 1NP 18 6 5000 01 22 IIP 02 22 IIP 03 30 IIP 25 5 Positive Control sodium azide 1.0 ug per plate 01 517 1 02 460 1 03 626 1 534 84 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 26 FoBnpsny BanitizetL Does not contain YSeA | H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 11 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.BlPL TA1535 Rat liver S9 tetrahydrofuran 25 uL Experiment No : Bl Cells Seeded : 2.7 X 10" Date Plated ; 17 Aug 2001 Concentration ug per plate Plat e Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 12 1 15 1 03 13 1 13 2 15 01 14 1 02 12 1 03 11 1 12 2 50 01 22 1 02 11 1 03 14 1 16 6 150 01 23 1 02 11 1 03 11 1 15 7 500 01 21 1NP 02 18 1NP 03 10 1NP 16 6 1500 01 25 1NP 02 20 1NP 03 11 1NP 19 7 5000 01 16 IIP 02 20 IIP 03 14 IIP 17 3 Positive Control 2-aminoantbracene 1.0 ug per plate 01 204 1 02 176 1 03 222 1 201 23 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate "\ BioReliance Study No. AA47TV.502001.BTL 27 Company SaniiizeA Does 8WiteontairaTSCAeBB H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 12 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.B1PL TA1537 None tetrahydrofuran 25 uL Experiment No Bl Cells Seeded Date Plated : 0. 9 X 10" 17 Aug 2001 Concentration ug per plate Picate Nunnber Revertants per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 10 1 6 1 03 7 1 8 2 15 01 8 1 02 9 1 03 5 1 7 2 50 01 6 1 02 8 1 03 8 1 7 1 150 01 6 1 02 10 1 03 5 1 7 3 500 01 11 1NP 02 9 IMP 03 11 1NP 10 1 1500 01 4 1NP 02 6 1NP 03 8 1NP 6 2 5000 01 7 IIP 02 7 IIP 03 6 IIP 7 1 Positive Control 9-aminoacridine 75 ug per plate 01 1634 1 02 1073 1 03 648 1 1118 495 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP==Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 28 ompany Sanitized. Doe? not contain TSCA CBl H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 13 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.5502001.B1rii TA1537 Rat livesr S9 tetrahycirofuran 25 uL Experiment No B1 Cells Seeded 0.9 X 10 Date Plated ' 17 Aug 2001 Concentration ug per plate Plat e Numb er Revesrtants per plate Beickground Code Average Revertants Standard Deviation Vehicle 01 02 6 1 5 1 03 7 1 6 1 15 01 12 1 02 8 1 03 6 1 9 3 50 01 17 1 02 16 1 03 8 1 14 5 150 01 10 1 02 9 1 03 7 1 9 2 500 01 6 1NP 02 12 1NP 03 5 1NP 8 4 1500 01 8 1NP 02 6 1NP 03 4 1NP 6 2 5000 01 7 IIP 02 7 IIP 03 8 IIP 7 1 Positive Control 2-aminoanthracene 1.0 ug per plate 01 242 1 02 115 1 03 231 1 196 70 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extreinely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate -gC* BioReliance Study No. AA47TV.502001.BTL 29 Wpany S^tteed. Does nol contain T5CA ^ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 14 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-2 5006 AA4 7TV.;502001.BTL WP2 uvrl\ Mone tet rahytirofuran 25 uL Experiment No : Bl Cells Seeded Date Plated 5.9 X 108 17 Aug 2001 Concentration ug per plate Plat e Numb>er Revssrtants per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 21 1 15 1 03 14 1 17 4 15 01 18 1 02 18 1 03 11 1 16 4 50 01 18 1 02 12 1 03 18 1 16 3 150 01 9 1 02 11 1 03 23 1 14 8 500 01 14 1NP 02 14 1NP 03 19 1NP 16 3 1500 01 8 1NP 02 18 1NP 03 16 1NP 14 5 5000 01 16 IIP 02 14 IIP 03 12 IIP 14 2 Positive Control methyl methanesulfonate 1000 ug per plate 01 244 1 02 259 1 ________________03______260______1__________254_______ Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 30 CompanySani^. ^es not eon(a,-n TSCA CB.- H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 15 Test Substance Id H-25006 Study Number AA47TV.502001.BTL Experiment No Strain WP2 uvrA Cells Seeded Liver Microsomes Vehicle Rat liver S9 tetrahydrofuran Date Plated Plating Aliquot 25 uL_________ Concentration Plate Revertants Background Average Bl 5.9 X 108 17 Aug 2001 Standard ug per plate Number per plate____Code___Revertants Deviation Vehicle 01 14 02 13 03 14 14 15 01 14 02 15 03 12 14 50 01 17 02 17 03 13 16 150 01 11 02 18 03 11 13 500 01 14 1NP 02 13 1NP 03 9 1NP 12 1500 01 12 1NP 02 17 1NP 03 16 1NP 15 5000 01 16 IIP 02 20 IIP 03 15 IIP 17 Positive Control 2-aroinoanthracene 10 ug per plate 01 515 1 02 556 1 03 694 1 588 94 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interering precipitate BioReliance Study No. AA47TV.502001.BTL 31 Company Sanitized. Does not contain TSCA CB3 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 16 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL TA98 None tetrahydrofuran 25 uL Experiment No B2 Cells Seeded Date Plated * 2.8 X 108 29 Aug 2001 Concentration ug per plate Plate Revertants Baickground Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 23 1 12 1 11 1 15 7 15 01 21 1 02 19 1 03 26 1 22 4 50 01 15 1 02 21 1 03 14 1 17 4 150 01 15 1 02 18 1 03 17 1 17 2 500 01 14 1NP 02 17 1NP 03 16 1NP 16 2 1500 01 23 1NP 02 26 1NP 03 22 1NP 24 2 5000 01 14 IIP 02 21 IIP 03 17 IIP 17 4 Positive Control 2-nitrofluorene 1.0 ug per plate 01 176 1 02 182 1 03 139 1 166 23 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 32 Company Sanitized, Does not contain TSCACBI H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 17 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.50;2001.BTL TA98 Rat liver S9 tetrahydrcafuran 25 uL Experiment No : B2 Cells Seedesd Date Plateca : 2.8 X 108 : 29 Aug 2001 Concentration ug per plate Vehicle Plate Number 01 02 Revertants Background Average per plate___Code____Revertants 26 29 Standard Deviation 03 23 26 15 01 35 02 35 03 24 31 50 01 17 02 26 03 24 22 150 01 22 02 18 03 20 20 500 01 16 1NP 02 26 1NP 03 22 1NP 21 1500 01 12 1NP 02 21 1NP 03 16 1NP 16 5000 01 21 IIP 02 20 IIP 03 21 IIP 21 Positive Control 2-aroinoanthracene 1.0 ug per plate 01 510 1 02 484 1 03 648 1 547 88 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extreinely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 33 companySanitized. Does not contsm TS6A 6!3 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 18 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001^PL TA100 None tetrahydrofuran 25 uL Experiment No : B2 Cells Seeded : 2. 4 X 10" Date Plated : 29 Aug 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 201 1 177 1 173 1 184 15 15 01 167 1 02 205 1 03 219 1 197 27 50 01 184 1 02 161 1 03 184 1 176 13 150 01 170 1 02 198 1 03 186 1 185 14 500 01 160 IMP 02 148 1NP 03 193 1NP 167 23 1500 01 132 1NP 02 154 1NP 03 188 1NP 158 28 5000 01 135 IIP 02 151 IIP 03 179 UP 155 22 Positive Control sodium azide 1.0 ug per plate 01 667 1 02 692 1 03 697 1 685 16 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 34 'eteBtp.sny Ssrtnispd. Dc-s not contain TSCA ^' H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 19 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL TA100 Rat liver S9 tetrahydrofuran 25 uL Experiment No : B2 Cells Seeded Date Plated : 2.4 X 10 : 29 Aug 2001 Concentration ug per plate Plat e Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 170 1 192 1 03 156 1 173 18 15 01 176 1 02 183 1 03 194 1 184 9 50 01 152 1 02 185 1 03 197 1 178 23 150 01 181 1 . 02 153 1 03 157 1 164 15 500 01 165 1NP 02 143 1NP 03 154 1NP 154 11 1500 01 184 1NP 02 176 1NP 03 149 1NP 170 18 5000 01 193 IIP 02 172 IIP 03 178 IIP 181 11 Positive Control 2-aminoanthracene 1.0 ug per plate 01 804 1 02 777 1 03 654 1 745 80 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 35 Company Sanitized. Dees notconl^'nTSCACBJ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 20 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.B1rL TA1535 None t e trahydrofuran 25 uL Experiment No : B2 Cells Seeded : 3.2 X 108 Date Plated : 29 Aug 2001 Concentration p.g per plate Plat e Number Revertants per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 25 1 28 1 03 21 1 25 4 15 01 25 1 02 29 1 03 26 1 27 2 50 01 12 1 02 20 1 03 22 1 18 5 150 01 14 1 02 23 1 03 21 1 19 5 500 01 23 1NP 02 17 1NP 03 19 1NP 20 3 1500 01 18 1NP 02 18 1NP 03 18 1NP 18 0 5000 01 20 IIP 02 24 IIP 03 21 IIP 22 2 Positive Control sodium azide 1.0 ug per plate 01 295 1 02 266 1 03 305 1 289 20 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001 .BTL 36 Company Sanitized. Does not cortamTSOAGBJgl H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 21 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.B1PL TA1535 Rat liver S9 tetrahydrofuran 25 uL Experiment No : B2 Cells Seeded ; 3.2 X 10 Date Plated : 29 Aug 2001 Concentration ug per plate Plat e Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 15 1 12 1 03 8 1 12 4 15 01 14 1 02 19 1 03 17 1 17 3 50 01 10 1 02 14 1 03 8 1 11 3 150 01 22 1 . 02 14 1 03 24 1 20 5 SOO 01 8 1NP 02 14 1NP 03 17 1NP 13 5 1500 01 21 1NP 02 19 1NP 03 14 1NP 18 4 5000 01 21 IIP 02 25 IIP 03 14 IIP 20 6 Positive Control 2-aminoanthracene 1.0 pg per plate 01 105 1 02 192 1 03 196 1 164 51 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 37 Company Saniiized. Does not contain TSCA GB3 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 22 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.ElfL TA1537 None tetrahydrofuran 25 uL Experiment Mo : B2 Cells Seeded ; 1.3 X 10 Date Plated ; 29 Aug 2001 Concentration ug per plate Plat e Revertants Background Average Standard Nunnber per plate Code Revertants Deviation Vehicle 01 02 5 1 7 1 03 7 1 6 1 15 01 4 1 02 10 1 03 4 1 6 3 50 01 5 1 02 2 1 03 8 1 5 3 150 01 5 1 02 3 1 03 6 1 5 2 500 01 6 IMP 02 7 1NP 03 5 1NP 6 1 1500 01 7 1NP 02 7 IMP 03 8 1NP 7 1 5000 01 5 IIP 02 4 IIP 03 6 IIP 5 1 Positive Control 9-aminoacridine 75 ug per plate 01 792 1 02 891 1 03 889 1 857 57 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 38 ^Pany Sanity Doe. ^ comamTSCACgj H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 23 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.5 02001.WPL TA1537 Rat livesr S9 tetrahyd rofuran 25 pL Experiment Mo : B2 Cells Seeded : 1.3 X 108 Date Plated : 29 Aug 2001 Concentration ug per plate Plat e Reve'rtants Baickground Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 6 1 7 1 4 1 6 2 15 01 12 1 02 10 1 03 6 1 9 3 50 01 2 1 02 4 1 03 7 1 4 3 150 01 5 1 02 5 1 03 8 1 6 2 500 01 3 1NP 02 7 1NP 03 5 1NP 5 2 1500 01 2 1NP 02 7 1NP 03 6 1NP 5 3 5000 01 9 IIP 02 8 IIP 03 4 IIP 7 3 Positive Control 2-aminoanthracene 1.0 pg per plate 01 179 1 02 195 1 ________________03______183______1__________186_______ Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 39 Company Sanitized. Does not contain TSCA CBB H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 24 Test Substance Id Study Number Strain Liver Micrpsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL WP2 uvrA None tetrahydrofuran 25 uL Experiment No B2 Cells Seeded Date Plated 5.8 X 10" 29 Aug 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 14 1 15 1 03 13 1 14 1 15 01 12 1 02 20 1 03 8 1 13 6 50 01 16 1 02 11 1 03 18 1 15 4 150 01 12 1 02 9 1 03 9 1 10 2 500 01 13 1NP 02 11 1NP 03 12 1NP 12 1 1500 01 15 1NP 02 17 1NP 03 12 1NP 15 3 5000 01 10 IIP 02 12 IIP 03 11 IIP 11 1 Positive Control methyl methanesulfonate 1000 ug per plate 01 103 1 02 174 1 ________________03______189______1__________155_______46_ Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 40 Company Sanitized. Doss not contain TSCA CB!1 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 25 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25006 AA47TV.502001.BT L WP2 uvrA Rat liver S9 tetrahydrofuran 25 pL Exper iment No Cells Seeded B2 5.8 X 10" Date Plated : 29 Aug 2001 Concentration pg per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 19 1 20 1 17 1 19 2 15 01 17 1 02 11 1 03 16 1 15 3 50 01 13 1 02 21 1 03 20 1 18 4 150 01 11 1 02 14 1 03 18 1 14 4 500 01 18 1NP 02 22 1NP 03 16 1NP 19 3 1500 01 18 1NP 02 17 1NP 03 14 1NP 16 2 5000 01 18 IIP 02 14 IIP 03 11 IIP 14 4 Positive Control 2-aminoanthracene 10 ug per plate 01 469 1 02 500 1 03 544 1 504 38 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 41 s- Does not contafn TSCA CB? H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Summary of Results Table 26 Test Substance I ;d : H -250C16 Study Number : Ai^47T\T. 50200:1.BT1L E'xpe rimeni; 13o : B1 Average Rievert;ants P<sr P:late Sitandard 13e^7iati.on Liver Microsomes: Nonie Dose (ug/plate) TA!98 TA1130 Vehicle 15 50 150 24 21 24 25 2 188 8 3 168 14 2 201 13 1 167 11 500 23 t 6 168 17 1500 23 3 187 14 5000 26 5 200 10 Positive 229 32 638 39 Liver Microsomes: Rat liver S9 Dose (ug/plate) TA98 TA100 Vehicle 15 50 25 32 * 30 6 179 10 7 195 14 7 183 7 150 500 1500 5000 Positive 32 9 183 4 33 3 188 20 31 4 198 t 22 26 2 185 12 847 191 1282 138 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 25 uL TA15 35 22 2 17 5 15 5 23 4 22 8 18 6 25 5 534 84 Tl^rS37 8 2 7 2 7 1 7 3 10 1 6 2 7 t 1 1118 495 TA1535 13 2 12 2 16 6 15 7 16 6 19 7 17 3 201 23 TA1537 6 t 1 9 3 14 5 9 2 8 4 6 2 7 1 196 70 WP2 17 16 16 14 16 14 14 254 UVrA 4 4 3 8 3 5 2 9 WP2 14 14 16 13 12 15 17 588 uvrA 1 2 2 4 3 3 3 94 BioReliance Study No. AA47TV.502001.BTL 42 Company Sanitized. Does not contain TSCA CBB H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Summary of Results Table 27 Test Substance Id H-25006 Study Number____ AA47TV.502001.BTL Experiment No : B2 Average Re^'ert.ants Per PI.ate :t S tand.ard I3evia.ti on Liver Microsomes: None Dose (ug/plate) Vehicle 15 50 150 500 1500 5000 Positive Liver Microsomes: TA98 15 7 22 4 17 4 17 2 16 2 24 2 17 4 166 23 Rat live r TA100 184 15 197 27 176 t 13 185 14 167 t 23 158 28 155 22 685 t 16 S9 TJM5 35 25 4 27 2 18 5 19 5 20 3 18 0 22 2 289 t 20 TJ0537 6 1 6 3 5 3 5 2 6 1 7 t 1 5 1 857 57 WP2 UVTA 14 1 13 t 6 15 4 10 2 12 1 15 3 11 1 155 46 Dose (ug/plate) TA98 Vehicle 15 50 150 500 1500 5000 Positive 26 3 31 6 22 5 20 2 21 5 16 5 21 1 547 88 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 25 uL TA100 173 t 18 184 9 178 23 164 15 154 11 170 18 181 11 745 80 TA1535 12 4 17 3 11 3 20 5 13 5 18 t 4 20 6 164 t 51 TA1537 6 2 9 3 4 3 6 2 5 2 5 3 7 3 186 8 WP2 19 15 18 14 19 16 14 504 uvrA 2 3 4 4 3 2 4 38 BioReliance Study No. AA47TV.502001.BTL 43 company Sanitized. Does not contain TSCA CBB H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________DuPont-7326 APPENDIX A Historical Control Data j^) BioReliance StudyNo.AA47TV.502001.BTL 44 ompany Sanitized. Does not contain TSCA CBS H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Historical Negative and Positive Control Values 1998-2000 revertants per plate Activation Strain TA98 Control Neg Pos Mean 16 425 None SD Min 7 4 206 21 Max 59 1536 Mean 21 592 Rat Liver SD Min 7 7 322 56 Max 58 2454 TA100 Neg Pos 128 31 53 288 568 159 129 1371 138 34 736 301 74 258 198 2871 TA1535 Neg Pos 12 5 378 164 1 45 6 978 12 4 1 42 104 84 18 1640 TA1537 Neg Pos 6 3 708 409 0 30 13 2786 7 3 88 106 1 29 12 2060 WP2MW-A Neg Pos 14 5 4 48 190 138 34 961 16 6 317 299 4 115 22 2632 SD=standard deviation; Min=minunum value; Max=maximum value; Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control BioReliance Study No. AA47TV.502001.BTL 45 Company Sanitized. Does not contain TSCACBIi H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay____ DuPont-7326 APPENDIX B Study Protocol BioReliance Study No. AA47TV.502001.BTL 46 Company Sanitized. Does not contain TSCA C: H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay ______________DuPont-7326 --^edbyRA/OAl^i^300' Sponsor Project Number DuPont-7326 BioReliance Study Number AA47TV.502001.BTL 1.0 PURPOSE H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay The purpose of this study is to evaluate the mutagenic potential of the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimwium and at me tryptophan locus of Escherichia coli WP2 tivrA in the presence and absence of S9 activation. 2.0 SPONSOR 2.1 Name: 2.2 Address: E.I. du Font de Nemouis and Company Stine Haskell Research Center DuPont Haskell Laboratory P.O. Box 50 1090 Elkton Road Newark, DE 19714-0050 2.3 Representative: 2.4 Sponsor Project No.: Maria Dormer, Ph.D. Phone: 302-366-5251 Fax: 302-366-5207 Email: maria.donner^usa.dupont.com DuPont-7326 2.5 WR#: 2.6 Haskell ft 2.7 Service Code: 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance Name: 3.2 Test Substance I.D.: 3.3 Controls: Negative: Positive: Test substance vehicle 9-aminoacridine 2-aminoanthtacene methyl methanesulfonate 2-nitrofluorene sodium azide ZP^t ioy1- Protocol SPGT502001 l-Jan-2001 Page 1 of 11 BioReliance Study No. AA47TV.502001.BTL 47 ^|| BlORELIANCE- Company Sanitized. Does not contain TSCA C. H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Sponsor Project Number: BioReliance Study Number. DuPont-7326 AA47TV.502001 .BTL 3.4 Test Substance Characterization Unless alternate arrangements are n"H<?, the testing facility at BioReliance will not perform analysis of the dosing solutions. The Sponsor mil be directly responsible for determination and documentation of the analytical purity and composition of the test substance, and the stability and strength of UK test substance in me solvent (or vehicle). 3.5 Test Substance Retention Sample The retention of a reserve sample of the test substance will be the responsibilityof the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility BioReliance Address: 9630 Medical Center Drive RockviUe.MD 20850 4.3 Study Director: Valentine O.Wagner HI, M.S. Phone: 301-610-2152 Fax: 301-738-2362 Email: swagnei@bioreliance.com 5.0 PROPOSED STUDY DATES 5.1 Experimental Start Date: 03-Aug-2001 5.2 Experimental Tennination Date: 19-Sep-2001 5.3 Draft Report Date: lO-Oct-2001 5.4 Final Report Date: 6.0 TEST SYSTEM 2 weeks after Sponsor approves draft The tester strains will include the S. typhimwiwn histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and the E. call tester strain WP2MW-A as described by Green and Muriel (1976). Histidine Mutation Tiyptopha n Mutation ___ Additional Mutations teG46 teC3076 /IMD3052 trpE LPS Repair R-factor TA1535 TA1537 - - rfa AOTTB - Protocol SPGT502001 1-Jan-2001 Page 2 of 11 BioReliance Study No. AA47TV.502001.BTL 48 ^HBlORELIANCF Company Sacked. Does not contain TSCA CBl H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Sponsor Project Number BioReliance Study Number PuPont-7326 AA47TV.502001 -BTL Histidine Mutation /ifaG46 TA100 hisC3076 - A&D3052 TA98 - - - Tryptopha n Mutation trpE - WP2ywA Additional Mutations LPS rfa - Repair AwB AKwA R-factor +R - Each S. typhimwium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the wrB gene results in a deficient DNA excision-repair system. Tester strains TA98 and TA100 also contain the pKMIOl plasmid (canying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshifl and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions. The E. coil tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2 iwA has a deletion in me uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of me reversion mechanism is sensitive to base-pair substitution mutations (Green and Muriel, 1976). The S. lyphimurium tester strains were received directly from Dr. Bruce Ames, University of California, Berkeley. The E. coli tester strain was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland (United Kingdom). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The test substance will be tested at a minimum of five dose levels along with appropriate negative and positive controls with tester strains TA98, TA100, TA1535, TA1537 and WP2 uwA with and without S9 activation. All dose levels of test substance, negative controls and positive controls will be plated in triplicate. Protocol SPGT502001 l-Jan-2001 Page 3 of 11 BioReliance Study No. AA47TV.502001.BTL 49 ^ BlORELiANCF ,,-,-- .-"-'"" 6) H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________DuPont-7326 Sponsor Project Number BioReliance Study Number DuPont-7326 AA47TV.502001.BTL 7.1 Solubility Determination Unless the Sponsor has indicated the test substance vehicle, a solubility determination will be conducted to determine the nummiim soluble concentration or workable suspension up to a maximum of 50 mg/mL for aqueous vehicles and 500mg/mL for organic vehicles. Vehicles compatible with mis test system, in order of preference, include but are not limited to deionized water (CAS 7732-18-5), dimethyl sulfoxide (CAS 67-68-5), emanol (CAS 64-17-5) and acetone (CAS 67-64-1). "Ilie vehicle of choice will be the solvent, selected in order of preference, which permits preparation of me highest workable/soluble stock concentration, up to 50 mg/mL for aqueous vehicles and 500 mg/mL for organic vehicles. 7.2 Preliminary Toricity Assay to Select Dose Levels Selection of dose levels for the mutagenicity assay will be based upon me toxicity and precipitation profile of the test substance assessed in a preliminary toxicity assay. This preliminary assay will be conducted by exposing TA98, TA100, TA1535, TA1537 and WP2ywA to negative controls and to at least eight concentrations of test substance, one plate per dose level, in both me presence and absence of S9 activation. Unless indicated otherwise by the Sponsor, the highest dose will be the highestworkable concentration in the vehicle of choice but not to exceed 5 ing/plate. In selecting dose levels for me mutagenicity assay the following guidelines will be employed. Doses will be selected such that precipitate does not interfere with manual scoring. Whenever possible, me highest dose for me mutagenicity assay will be selected to give some indication of toxicity without exceeding 5 mg/plate. For freely soluble, nontoxic test substances, the highest dose level will be 5 mg/plate. For precipitating, nontoxic test substances, the highestdose level will be selected in an attempt to yield precipitate at only the top one or two dose levels. The Sponsor will be consulted regarding dose selection if (1) the maximum dose level is selected based on precipitation and this dose level is less than 5 mg/plate or (2) the mwimiim achievable test substance dose level is less than 5 mg/plate and mis dose level is nontoxic. 7.3 Frequency and Route of Administration The test system will be exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). This test system has been shown to detect a wide range of classes of chemical mutagens (McCann et al., 1975; McCann and Ames, 1976). After the data generated in the Gist assay have been evaluated, the mutagenicity assay will be repeated. The dose levels used in the second assay will be me same as those used in the first assay unless the Study Director detennines that the dose ; levels should be changed due to an equivocal response, excessive cytotoxicity or Protocol SPGT502001 l-Jan-2001 Page 4 of 11 BioReliance Study No. AA47TV.502001 .BTL 50 ^ BlOREUANCE- Company Sanitized. Does not contain TSCA CBi H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Sponsor Project Number BioReliance Study Number DuPont-7326 AA47TV.502001.BTL excessive precipitate. If me Sponsor is aware of specific metabolic requirements (e.g., azo compounds), this information will be utilized in designing the assay. (e.g., activation system or treatment method). This guidance is based on the OECD Guideline 471 (adopted July 1997 and published February 1998) and ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997). 7.4 Controls 7.4.1 Positive Controls All combinations of positive controls and tester strains plated concurrently with the assay are listed below: Strain S9 Activation _______ Positive Control ____ ___ Concentration 4g/plate) Salmonella Strains WP2uwA TA98 TA100, TA1535 TA1537 WP2wrA Rat None 2-aminoanthracene 2-nitrofluorene sodium azide 9-ammoacridine methyl methanesulfonate 1.0 10 1.0 1.0 75 1,000 7.4-2 Negative Controls Appropriate negative controls will be plated for each tester strain with and without S9 activation. The negative control will be the vehicle alone, unless there is no historical basis for use of the selected vehicle. In the latter case, both untreated and vehicle controls will be used. 7.4.3 Sterility Controls The most concentrated test substance dilution and me Sham and S9 mixes will be checked for sterility. 7.5 Exogenous Metabolic Activation Aroclor 1254-induced rat liver S9 will be used as the metabolic activation system. The S9 homogenate will be 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 will be batch prepared and stored frozen at approximately Protocol SPGT502001 l-Jan-2001 Page 5 of 11 BioReliance Study No. AA47TV.502001.BTL 51 ^ BIORELIANCE' companySanitized, Does not contain TSCA CS3 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Sponsor Project Number: BioReliance StudyNumber: DuPont-7326 AA47TV.502001.BTL -70C until used. Each batch of S9 homogenate will be assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenzanthracene to forms mutagenic to S typhimwiwn TA100. Immediately prior to use, the S9 will be thawed and mixed with a cofactor pool to contain 10% S9 homogenate, 5mM glucose-6-phosphate, 4mM R-mcotmamide-adenine dinucleodde phosphate, 8 mM MgCl; and 33 mM KC1 in a 100 mM phosphatebuffer at pH 7.4. This inixture is referred to as S9 mix-Sham mix will be 100 mM phosphatebuffer at pH 7.4. 7.6 Preparation of Tester Strain Overnight cultures will be inoculated from the appropriate master plate or from the appropriate frozen stock. To ensure that cultures are harvested in late log phase, me lengthof incubation will be controlled and monitored. At the end of the working day, each inoculated flask will be placed in a resting shaker/incubator at room temperature. The shaker/incubator will be programmed to begin shaking at approximately 125 rpm at 372C approximately 12 hours before the anticipated time of harvest All cultures will be harvested by spectrophotometric monitoring of culture turbidity rather than by duration of incubation since overgrowth of cultures can cause loss of sensitivity to some mutagens. Cultures will be removed from incubation at a density of approximately 109cells/mL. 7.7 Test System Identification Each plate will be labeled with a code system that identifies the test substance, test phase, dose level, tester strain and activation type as described in BioRetiance's Standard Operating Procedures. 7.8 Test Substance Preparation Unless specified otherwise, test substance dilutions will be prepared immediately prior to use. All test substance dosing will be at room temperature under yellow light 7.9 Treatment of Test System One halfmilliliter (0.5 mL) ofS9 mix or Sham mix, 100 u,L of tester strain and 50 nL of vehicle, test substance dilution or positive control will be added to 2.0 mL of molten selective top agar at 452C. When necessary to achieve the target concentration or eliminate toxic vehicle effects, aliquots of other than 50 u.L of test substance/vehicle/positive control will be plated. The mixture will be vortex mixed and overlaid onto the surface of 25 mL of minipinl bottom agar. Protocol SPGT502001 l-Jan-2001 Page 6 of 11 BioReliance Study No. AA47TV.502001.BTL 52 ^||BlOREUANCE- ewpaW s^.^i^ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay________________________DuPont-7326 Sponsor Project Number BioReliance Study Number DuPont-7326 AA47TV.502001.BTL 7.10 After the overlay has solidified, the plates will be inverted and incubated for approximately 48 to 72 hours at 372C. Plates that are not counted immediately following the incubation period will be stored at 2-8C. Scoring The condition of the bacterial background lawn will be evaluated for evidence of test substance toricity and precipitate. Evidence oftoxicity will be scored relative to the negative control plate and recorded along with the revertant count for that plate. Toxicity will be evaluated as a decrease in the number of revertant colonies per plate and/or a thinning or disappearance of the bacterial background lawn. Precipitation will be evaluated after the incubation period by visual examination without magnification. 7.11 Tester Strain Verification On the day of use in the mutagenicity assay, all tester strain cultures will be checked for the appropriate genetic markers cited in 6.0. 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST The following criteria must be met for the mutagenicity assay to be considered valid: 8.1 Tester Strain Integrity To demonstrate the presence of the rfa mutation, all S. typhimwivm tester strain cultures must exhibit sensitivity to crystal violet To demonstrate me presence of the i/wB mutation, all S. typhimurivm tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate me presence of the nvrA. mutation, all E. coli tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate the presence of the pKMIOl plasmid R-factor, tester strain cultures of TA98 and TA100 must exhibit resistance to ampicillin. 8.2 Spontaneous Revertant Background Frequency Based on historical control data, all tester strain cultures must exhibit characteristic number of spontaneous revertants per plate in me negative controls (vehicle). The mean revertants per plate must be within the following ranges Cmclusive): TA98, 10-50; TA100. 80-240; TA1535, 5-45; TA1537, 3-21; WP2mrA,10-60. 8.3 Tester Strain Titeis To ensure that appropriatenumbers of bacteria arc plated, all tester strain culture titers must be equal to or greater than OJxIO9 cells per milliliter. Protocol SPGT502001 l-Jan-2001 Page 7 of 11 BioReliance Study No. AA47TV.502001 .BTL 53 ^ BlORELIANCF Company Sanitized, Does not contain TSCA CBI H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________DuPont-7326 Sponsor Project Number BioReliance Study Number. DuPont-7326 AA47TV.502001.BTL 8.4 Positive Control Values Each mean positive control value must exhibit at least a 3.0-fold increase over the respective mean negative control value (vehicle) for each tester strain. 8.5 Toricity A minimum of three non-toxic dose levels will be required to evaluate assay data. A dose level is considered toxic if it causes a >50% reduction in the mean number of revertants per plate relative to the mean negative control value (mis reduction must be accompanied by an abrupt dose-dependentdrop in the revertant count) or a reduction in the background lawn. In the event that less man three non-toxic dose levels are achieved, me affected portion of the assay will be repeated with an appropriatechange in dose levels. 9.0 EVALUATION OF TEST RESULTS For a 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 over a minimum of two increasing concentrations of test substance as specified below: 9.1 Strains TA1535 and TA1537 Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater man 3.0-times the mean negative control value (vehicle). 9.2 Strains TA98.TA100 and WP2wrA Data sets will be judged positive if the increase in mean revertants at me peak of the dose response is equal to or greater than 2.0-dmes the mean negative control value (vehicle). 10.0 REPORT A report of the results of this study will be prepared by the Testing Laboratory and will accurately describe all methods used for generation and analysis of the data. The report will include: Test Substance: identification and CAS no., if known; physical nature and purity, if known; physicochemical properties relevant to the conduct of the study, if known; stability of test substance, if known. Solvent/Vehicle: justification for choice of vehicle; solubility and stability of test substance in solvent/vehicle, if known. I Strains: strains used; number of cells/mL per culture; strain characteristics. Protocol SPGT502001 l-Jan-2001 Page 8 of 11 BioReliance Study No. AA47TV.502001.BTL 54 ^| BlORELIANCE- Company Sanitized. Does TidttonlatnTSCACBB H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Sponsor Project Number BioReliance Study Number DuPont-7326 AA47TVJ02001.BTL Test conditions: amount of tea substance per plate with rationale for dose selection and number of plates per concentration; media used; type and composition of metabolic activation system, including acceptability criteria; treatment procedures. Results: signs of toricity; signs of precipitation; individual plate counts; the mean number of revertant colonies per plate and standard deviation; dose-response relationship, where possible; statistical analysis, if any; concurrent negative and positive control data means and standard deviations; historical negative and positive control data. with ranges, means and standard deviation. Discussion of results. Conclusion. 11.0 RECORDS AND ARCHIVES All raw data, the protocolandalL reports will be maintained according to Standard Operating Procedure|UBl9y me BioReliance RAQA unit headquartered at: BioReliance, 14920 fflflSchart RoaCRockville, MD 20850. Per this SOP, paper records will be retained for at least three years after which time the Sponsor will be contacted for a decision as to the final disposition of the materials. All study materials returned to the Sponsor or destroyed will first be copied and the copy will be retained in the BioReliance archives for a minimiim of 10 years. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply with OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Assay), Ninth Addmrtum to me OECD Guidelines for the Testing of Chemicals, publishedby OECD, Paris, February 1998 and with the International Conference on Hannonisarion of Technical Requirements for Registration ofPharmaceuticals for Human Use (1996 and 1997). This study will be performed in compliance with me provisions of the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (GLPs). The protocol, an in-process phase, the raw data, and reports) will be audited per the Standard Operating Procedures (SOPs) of BioReliance by the Quality Assurance Unit of BioReliance for compliance with GLPs, the SOPs of BioReliance and me study protocol. The in-process inspection will be performed to audit the critical assay procedures and systems supporting the assay. A signed QA statement will be included in the final report. This statement will list the system phases inspected during the previous quarter or the study-specific phases, the dates of each inspection, and the dales the results of each inspection were reported to the Study Director and the Study Director's management In addition, a signed GLP compliance statement will be included in the final report. This statement will cite the GLP guidelines) with which the study is compliant and any exceptions to this compliance, if applicable, including me omission of characterization or stability analyses of the test or control substances or their mixtures. Protocol SPGT502001 l-Jan-2001 Page 9 of 11 BioReliance Study No. AA47TV.502001.BTL 55 ft BlORELIANCE- ompany SanitizptS. Doeg nof contain TSCA Ciai? H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Sponsor Project Number BioReliance Study Number DuPont-7326 AA47TV.502001 .BTL Unless arrangements are made to me contrary, unused dosing solutions will be disposed of following administration to the test system and all residual test substance will be disposed of following finalization of the report. 13.0 REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Sa/OTone//a/manunalian-microsome mutagenicity test Mutation Research 31:347-364. Green, M.H.L., and Muriel, W.J. (1976). Mutagen testing using tip* reversion in Escherichia coli. Mutation Research 38:3-32. International Conference on Hannonisation (ICH) of Technical Requirements for Registration of Phannaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Phannaceuticals. 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 Hannonisation (ICH) of Technical Requirements for Registration of Phannaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Phannaceuticals. S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21,1997. McCann, J. and Ames, B.N. (1976). Detection of carcinogens as mutagens in me Salmonellafmcrosome test: assay of 300 chemicals: discussion. Proc. Natl. Acad. Sci. USA 73:950-954. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975). Detection of carcinogens as mutagens in the Salmonella/imcTosome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. USA 72:5135-5139. Maron, D.M. and Ames, B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test. Mutation Research 113:173-215. OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February 1998. Wilcox, P., Naidoo, A., Wedd, D.J. and Gatehouse, D.G. (1990). Comparison of Salmonella typhimwiwn TA102 with Escherichia coli WP2 tester strains. Mutagenesis 5:285-291. Protocol SPGT502001 l-Jan-2001 Page 10 of 11 BioReliance Study No. AA47TV.502001.BTL 56 ^ BlORELIANCF ^.oesotc.n>.^SCACy H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Sponsor Project Number BioReliance Study Number: DuPont-7326 AA47TV.502001.BTL 14.0 APPROVAL R o ^ - ' ^ T f ^ / Sponsor Representative ^0 ^UL 2-OOt Date tAo-r '>' o^ V)op/^^.<^ (Print or Type Name) ^LUC^. 0. iL^^Jt" Studybirector 2 fiaw^2001 Date ? AumJ>^ iy Protocol SPGT502001 l-Jan-2001 Page 11 of 11 BioReliance Study No. AA47TV.502001.BTL 57 ^ BlORELIANCF H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay____ DuPont-7326 APPENDIX C Information for Japanese Regulatory Agencies BioReliance Study No. AA47TV.502001 .BTL 58 ywsse,^.^^^^ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Report of Results of Reverse-Mutation Assay in Bacteria 1. Tester Strains (1) Procurement Strain TA98 TA100 TA1535 TA1537 TA1538 TA97 TA102 WP2vrA WP2vrA (pkMIOl) WP2 (pKMIOl) Obtained from Date obtained 10 November 1998 Date inspected the strain lot in storage Dr. Bruce Ames University of California, Berkeley National Collection of Industrial and Marine Bacteria Aberdeen, Scotland 11 August 1998 13 December 1990 14 November 1990 1 July 1987 19 February 1993 The genetic markers for each culture are confirmed on the day of use (2) Storage Freezing method Storage temperature Composition Large quantity -70C Bacterial suspension DMSO 1.0 mL 0.09 mL 2.S9Mix (1) Source, Storage Temperature, etc. of S9 Made in-house Prepared on 02 May 2001 (Batch R644) 03 July 2001 (Batch R648) Storage temperature -70C or colder Name and model of storage apparatus So-Low, Model PR27-120 BioReliance Study No. AA47TV.502001.BTL 59 Company Sanitized. Does not contain TSCA CB( H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 (2) Preparation of S9 Animal used Species, Strain Rattus norvegicus, Sprague Dawley Sex Male Age (in weeks) Weight 9 (Batch R644) 9 (Batch R648) 209 to 258 g (Batch R644) 224 to 290 g (Batch R648) Inducing substance Name Aroclor 1254 Administration method intraperitoneal Administration period and amount (g/kg-weight) 5 days, 0.5 gm/kg body weight 3. Preparation of Test Substance Solution Solvent used Name Tetrahydrofuran (THF) Manufacturer Aldrich Lot No. Grade Purity (%) JI00352HI Gold Label 99.9% Stability of test substance in the solvent Unknown Reason to choose the solvent Solubility determination was based on the Sponsor's request, compatibility with the target cells and solubility of the test substance Method of suspension when test substance is difficult to dissolve Not applicable Storage time and temp. from preparation to use for test <30 minutes at ambient temperature Conversion by purity No fc) BioReliance Study No. AA47TV.502001.BTL 60 GstTipany Sanitized Does not contain TSCA C?- H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 4. Conditions of Pre-culture Nutrient broth Period of pre-culture Storage time and temp. from inoculation to beginning of shaking culture Storage time and temp. from end of culture to use for test Model and manufacturer of shaker Method of shaking (shaking type, speed, etc.) Culture vessel (shape, capacity) Culture volume Volume of inoculum Name Oxoid Nutrient Broth No. 2 121 hours Manufacturer Oxoid Ltd. Lot No. CH,-B=232622 2 to 5 hours at ambient temperature <8 hours at 2-8C New Brunswick Scientific, model G-24 Rotary (125 rev/min.) shape: cylinder, 200 mL 50 mL 1 colony 5. Agar Plate Medium (1) Top agar Agar Name Manufacturer Lot No. BBL Select Becton Dickinson 1000J3DKSQ (2) Minimum Glucose Agar Name Made in-house Agar Manufacturer Lot No. Volume of agar plate medium BBL Select Becton Dickinson 1000J3DKSQ 25 mL BioReliance Study No. AA47TV.50200LBTL 61 Company Sanitized. Does not contain TSGAC. H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay 6. Test Results - Judgement of the results DuPont-7326 Judgement ' Negative Reason for judgement and referential matters: No positive response was observed with any of the tester strains in the presence and absence ofAroclor-induced rat liver S9. Referential matters The vehicle and positive control values indicate that all tester strains were functioning correctly and were capable of detecting a mutagen. BioReliance StudyNo.AA47TV.502001.BTL 62 .^..----Does"ocon>a..l/."ATFBI