Document gEL5NObw44Z92y7m8dwjJXzGG

ARUM - x i DuPont-8968 TRADE SECRET Study Title H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay Test Substance H-24890 Authors Valentine O. Wagner, m , M.S. Michelle L. Klug, B.S. Study Completion Date 19 March 2002 Performing Laboratory BioReliance 9630 Medical Center Drive Rockville, MD 20850 for E. I. du Pont de Nemours and Company DuPont Haskell Laboratory P.O. Box 50,1090 Elktan Road Newark, DE 19714-0050 Performing Laboratory Study Number AA53CH.502001.BTL Work Request Number Service Code 1 of 64 Company Sanitized. Does not contain T SC A CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 STATEMENT OF COMPLIANCE Study No. AA53CH.502001.BTL was conducted in compliance with the U.S. FDA GLP Regulations as published in 21 CFR 58, the U.S. EPA GLP Standards 40 CFR 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 stability analysis on the test substance mixtures. Study Director BioReliance Study Management Date Date BioReliance Study No. AA53CH.502001.BTL 2 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Quality Assurance Statement Study TMe: H-24890: BACTERIAL REVERSE MUTATION ASSAY WITH AN INDEPENDENT REPEAT ASSAY Study N um ber: AA53CH.502001.BTL Study D irector: Valentine O. Wagner, III, M.S. This study has been divided into a series o f in-process phases. Using a random sampling approach, Quality Assurance monitors each o f these phases over a series o f studies. Procedures, documentation, equipment records, etc., are examined in order to assure that the study is performed in accordance with the U.S. FDA Good Laboratory Practice Regulations (21 CFR 58), the U.S. EPA OLPs (40 CFR 792 and 40 CFR 160), the UK GLP Regulations, the Japanese GLP Standard, and the OECD Principles o f Good Laboratory Practice and to assure the* the study is conducted according to the protocol and relevant Standard Operating Procedures. The following are the inspection dates, phases inspected, and report dates o f QA inspections o f this study. ** Inspect On Phase *+ Inspect On Phase * Inspect On Phase * Inspect On Phase * Inspect On Phase Inspect On Phase Inspect On Phase 18-Dec-01 - 18-Dec-01 To Study Dir 18-Dec-01 To Mgmt 18-Dec-01 Protocol Review 13-Feb-02 - 13-Feb-02 To Study Dir 13-Feb-02 To Mgmt 07-Mar-02 Draft Report I9-Mar-02 - 19-Mar-02 To Study D ir 19-Mar-02 To Mgmt 19-Mar-02 Draft to Final Report 14-Dec-01 - 14-Dec-01 To Study Dir 14-Dec-01 To Mgmt 17-Dec-01 Test and/or control material administration 18-Dec-01 - 18-Dec-01 To Study D ir 18-Dec-01 To Mgmt 18-Dec-01 Entering plate counts into Ames program 04-Jan-02 - 04-Jan-02 To Study Dir 04-Jan-02 To Mgmt 04-Jan-02 Preparation o f S9 mixture 08-Jan-02 - 08-Jan-02 To Study Dir 08-Jan-02 To Mgmt 08-Jan-02 Strain characterization BioReliance Study No. AA53CH.502001.BTL 3 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 * Inspect On Phase * Inspect On Phase * Inspect On Phase * Inspect On Phase * Inspect On Phase * Inspect On Phase * Inspect On Phase 17-Jan-02 -1 7-Jan-02 To Study Dir 17-Jan-02 To Mgmt 18-Jan-02 Dilution o f test and/or control material 22-Jan-02 - 13-Feb-02 To Study D ir 14-Feb-02 To Mgmt 14-Feb-02 Systems Inspection--Administration o f test substance to test system 28-Jan-02 - 28-Jan-02 To Study D ir 28-Jan-02 To Mgmt 29-Jan-02 Entering plate counts into Ames program 06-Feb-02 - 06-Feb-02 To Study D ir 06-Feb-02 To Mgmt 06-Feb-02 Scoring mutagenicity plates 15-Feb-02 - 15-Feb-02 To Study D ir 15-Feb-02 To Mgmt 15-Feb-02 Test and/or control material administration 20-Feb-02 - 01-Mar-02 To Study Dir 01-Mar-02 To Mgmt 01-Mar-02 Systems Inspection - Observation o f Test System/Data Collection and/or Analysis 28-Feb-02 - 28-Feb-02 To Study Dir 28-Feb-02 To Mgmt 28-Feb-02 Strain characterization ** Inspection specific for this study * Systems Inspection This report describes the methods and procedures used in the study and the reported results accurately reflect the raw data o f the study. H `Monaco a_ DATE BioReliance Study No. AA53CH.502001.BTL 4 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Issued by Study Director: Valentine O. Wagner DI, M.S. H (YU tf 2 * 0 2 - Date Approved by Study Monitor: Maria Donner, Ph.D. Senior Research Scientist )3 ~flou - 2oq2- Date BioReliance Study No. AA53CH.50200l.BTL 5 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test TABLE OF CONTENTS Page Certification................................................................................................................................... 5 Study Inform ation.........................................................................................................................8 Summary....................................................................................................................................... 9 Purpose.........................................................................................................................................10 Characterization o f Test and Control Substances...................................................................... 10 Materials and M ethods................................................................................................................12 Results and Discussion................................................................................................................17 Conclusion....................................................................................................................................17 R eferen ces....................................................................................................................................18 Data Tables.................................................................................................................................. 19 Table 1: Preliminary Toxicity Test in Salmonella typhimurium TA 98..............................19 Table 2: Preliminary Toxicity Test in Salmonella typhimurium TA 100........................... 20 Table 3: Preliminary Toxicity Test in Salmonella typhimurium TA1535......................... 21 Table 4: Preliminary Toxicity Test in Salmonella typhimurium TA1537 ......................... 22 Table 5: Preliminary Toxicity Test in Escherichia coli W P2 uvrA ....,.............................. 23 Table 6: Mutagenicity Test in Salmonella typhimurium TA98 without S 9 ..................... 24 Table 7: Mutagenicity Test in Salmonella typhimurium TA98 with S 9 .......................... 25 Table 8: Mutagenicity Test in Salmonella typhimurium TA100 without S 9 ................... 26 Table 9: Mutagenicity Test in Salmonella typhimurium TA100 with S 9 ........................ 27 Table 10: Mutagenicity Test in Salmonella typhimurium TA1535 without S 9 ................. 28 Table 11: Mutagenicity Test in Salmonella typhimurium TA1535 with S 9 ...................... 29 Table 12: Mutagenicity Test in Salmonella typhimurium TA1537 without S 9 ................. 30 Table 13: Mutagenicity Test in Salmonella typhimurium TA1537 with S 9 ...................... 31 Table 14: Mutagenicity Test in Escherichia coli WP2 uvrA without S9........................... 32 Table 15: Mutagenicity Test in Escherichia coli WP2 uvrA with S9.................................33 Table 16: Mutagenicity Test in Salmonella typhimurium TA98 without S 9 ..................... 34 Table 17: Mutagenicity Test in Salmonella typhimurium TA98 with S 9 .......................... 35 Table 18: Mutagenicity Test in Salmonella typhimurium TA100 without S 9 ................... 36 Table 19: Mutagenicity Test in Salmonella typhimurium TA100 with S 9 ........................ 37 Table 20: Mutagenicity Test in Salmonella typhimurium TA1535 without S 9 ................. 38 Table 21: Mutagenicity Test in Salmonella typhimurium TA1535 with S 9 ...................... 39 Table 22: Mutagenicity Test in Salmonella typhimurium TA1537 without S 9 ................. 40 Table 23: M utagenicity Test in Salmonella typhimurium TA1537 with S 9 ...................... 41 BioReliance Study No. AA53CH.502001.BTL 6 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Table 24: Mutagenicity Test in Escherichia coli WP2 uvrA without S9........................... 42 Table 25: Mutagenicity Test in Escherichia coli WP2 wvrA with S9................................ 43 Table 26: Salmonella/E. coli Mutagenicity Test - Summary of Results B 1...................... 44 Table 27: Salmonella/E. coli Mutagenicity Test - Summary of Results B 2...................... 45 Appendix A: Historical Control Data........................................................................................ 46 Appendix B: Study Protocol.......................................................................................................48 Appendix C: Information for Japanese Regulatory Agencies.................................................. 60 BioReliance Study No. AA53CH.50200l.B TL 7 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ STUDY INFORMATION Substance Tested: Svnonvms/Codes: Haskell Number: 24890 Composition! DuPont-8968 Sponsor: E. L du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Tnitiated/Completed: December 17,2001 / (see report cover page) In-Life Initiated/Completed: December 19,2001 / January 24,2002 BioReliance Study No. AA53CH.502001.BTL 8 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 SUMMARY The test substance, H-24890, was tested in the Bacterial Reverse Mutation Test with an Independent Repeat Assay using Salmonella typhimurium tester strains TA98, TA100, TA1S3S and TA1S37 and Escherichia coli testa* 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 test, was used to establish the dose-range for the mutagenicity test The second.phase, the mutagenicity test (initial and independent repeat tests), was used to evaluate the mutagenic potential o f the test substance. Water was selected as the solvent of choice based compatibility with the target cellsl hi the preliminary toxicity test, the maximum dose tested was 5000 pg per plate; this dose was achieved using a concentration o f 50 mg/mL and a 100 pL plating aliquot. Dose levels tested w oe 6 .7 ,1 0 ,3 3 ^ 7 ^ 0 0 ^ 3 3 , 667, 1000,3333 and 5000 pg/plate. Concentrations from ^33 to 50 mg/mL w e r 4 | | | | | | H r t H f l ^ H ^ n d concentrations from 0.67 to 10 mg/mL 3atnerprw ipitate nor appreciable toxicity was observed. Based on the findings of the toxicity test, the maximum dose plated in the mutagenicity test was 5000 pg per plate. In the mutagenicity test, no positive mutagenic response was observed. The dose levels tested were 100, 333, 1000, 3333 and 5000 pg/plate. No precipitate was observed. No reductions in the background lawns were observed. However, reductions in the revertant counts were observed at 5000 pg p a plate with testo* strains TA1535 and TA1537 in the absence of S9 activation in the independent repeat test. The results of the Bacterial Reverse Mutation Test with an Independent Repeat Assay indicate that, under the conditions of this study, H-24890 did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9. BioReliance Study No. AA53CH.502001.BTL 9 Company Sanitized. Does not contain TSC A CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay _____ DuPont-8968 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 typhimurium and at die tryptophan locus of Escherichia cot strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. A copy of the protocol is included in Appendix B. The study was conducted to comply with OECD Guideline 471 (Genetic Toxicology; Bacteria] Reverse Mutation Test), adopted July 1997 (published February 1998) and with the International Conference on Harmonisation of Technical Requirements o f Registration of Pharmaceuticals for Human Use (1996 and 1997). * CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test substance, H-24890, was received by BioReliance on 13 December 2001 and was ngnt. in e laenuty, strengtn, purity, composiuon or other characteristics to denne the test substance have been determined by the Sponsor. The Sponsor indicated that BioReliance would not receive a copy of this report The stability of the test substance has been determined by the Sponsor. The Sponsor indicated that BioReliance would not receive a copy o f the stability report. The vehicle used to deliver H-24890 to the test system was sterile distilled water (CAS No. 7732-18-5), obtained from Invitrogen Corporation, formerly Life Tecbnolojpes, Inc. 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 test are listed below. All positive controls were diluted with dimethyl sulfoxide (DMSO) except sodium azide, which was diluted with water. All subdivided solutions of positive control were stored at -5 to -30C. BioReliance Study No. AA53CH.502001.BTL 10 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Strain S9 Activation Positive Control Concentration (pg/plate) All Salmonella Strains WP2 uvrA TA98 TA100, TA1535 TA1537 WP2 uvrA Rat None 2-aminoanthracene (Aldrich Chemical Co., Inc.) Lot No. 09106PS Exp. Date 14-Sep-2005 CAS No. 613-13-8 Purity >95% 2-nitrofluorene (Aldrich Chemical Co., Inc.) Lot No. 08707HS Exp. Date 08-Mar-2006 CAS No. 607-57-8 Purity >98% Sodium azide (Sigma Chemical Co.) Lot No. 098H0169 Exp. Date 05-Jan-2004 CAS No. 26628-22-8 Purity >99% 9-aminoacridine (Sigma Chemical Co.) Lot No. 106F06681 Exp. Date 18-Nov-2004 CAS No. 90-45-9 Purity >98% Methyl methanesulfonate (Aldrich Chemical Co., Inc.) Lot No. 15526AO Exp. Date 28-Nov-2003 CAS No. 66-27-3 Purity >99% 1.0 10 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 test BioReliance Study No. AA53CH.502001.BTL 11 Company Sanitized. Does not contain T SCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 MATERIALS AND METHODS For submission to Japanese regulatory agencies, additional information is included in Appendix C. 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 and E. coli tester strains were received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. Tester strains TA98 and TA1537 are 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 cause both frameshift and basepair substitution 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 shaker/incubator 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 titer of approximately 109 cells per milliliter. The actual titers were determined by viable count tests on nutrient agar plates, and the data is on fide but not presented in this report. 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 was batch prepared and stored at -70C or colder until used. Each bulk preparation of S9 was tested for its ability to metabolize 2aminoanthracene 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, 5m M glucose-6-phosphate, 4 mM 6-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCfe and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), BioReliance Study No. AA53CH.502001.BTL 12 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 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 and tetrahydrofuran (THF). 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. Preliminary Toxicity Test The preliminary toxicity test was used to establish the dose-range over which the test substance would be tested in the mutagenicity test. Vehicle and ten dose levels of the test substance were plated, one plate per dose, with overnight cultures of TA98, 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 6.7, 10,33,67,100,333,667, 1000,3333 and 5000 pg/plate. Mutagenicity Test The mutagenicity test (initial and independent repeat tests) was used to evaluate the mutagenic potential of the test substance. A minimum of five dose levels of test substance (100, 333, 1000, 3333 and 5000 pg/plate) along with appropriate vehicle and positive controls were plated with TA98, TA100, TA1535, TA1537 and WP2 vrA in the presence and absence of Aroclor-induced rat liver S9. All dose levels o f 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 (1983). On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 jiM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL o f 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 BioReliance Study No. AA53CH.502001.BTL 13 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 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) milliliter of S9 or Sham mix, 100 pL of tester strain and 100 pL 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 pL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and 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 o f precipitation were scored relative to the vehicle control plate using the codes shown below. Code 1 2 3 4 5 6 Description Normal Slightly Reduced M oderately Reduced Extremely Reduced Absent Obscured by Precipitate Characteristics 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. BioReliance Study No. AA53CH.502001.BTL 14 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Code NP IP Description Characteristics Distinguished by precipitate on the plate that is visible to the Non-Interfering naked eye but any precipitate particles detected by the automated Precipitate colony counter total less than 10% of the revertant colony count (e.g., less than 3 particles on a plate with 30 revertants). Distinguished by precipitate on the plate that is visible to the Interfering Precipitate naked eye and any precipitate particles detected by the automated colony counter exceed 10% of the revertant colony count (e.g., more than 3 particles 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 o f ievertants per plate were calculated and are reported. 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 TA1S3S and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-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 2.0-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 Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence o f 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 - SO; TA100, 80 - 240; TA1535, 5 -4 5 ; TA1537, 3 -2 1 ; 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 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels is 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 BioReliance Study No. AA53CH.502001.BTL 15 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 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) At least a moderate reduction in the background lawn (background lawn code 3 ,4 or 5). A copy of the Historical Negative and Positive Control Values is included in Appendix A. Archives All raw data, the protocol and all reports will be maintained according to Standard Operating Procedure OPQP3040 by the BioReliance RAQA unit headquartered at: BioReliance, 14920 Broschart Road, Rockville, 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. Unused dosing solutions were disposed of following administration to the test system and all residual test substance will be disposed of following finalisation of the report. Deviations No known deviations from the protocol or assay-method SOPs occurred during the conduct of this study. BioReliance Study No. AA53CH.50200l.BTL 16 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 RESULTS AND DISCUSSION Solubility Test W ater was selected as the solvent of choice based on compatibility with the target cells. The test substance w Preliminary Toxicity Test The results o f the preliminary toxicity test are presented in Tables 1 through S. These data were generated in Experiment A 1. In the preliminary toxicity test, the maximum dose tested was 5000 pg per plate; this dose was achieved using a concentration o f 50 mg/mL and a 100 pL plating aliquot Dose levels tested were 6 .7,10,33,67,100,333,667,1000,3333 and 5000 pg per plate. Concentrations from 33 to 50 mg/mL w ere^ tE M B M H M M B k oltitions and concentrations from 0.67 to 10 mg/mL w e re jH H H H H fllH lIH ^ ^ c itfie rp re c ip ita te nor appreciable toxicity was observed. Based on the findings of the toxicit^Cest, the maximum dose plated in the mutagenicity test was 5000 pg per plate. Mutagenicity 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 B1 and B2. Dose levels tested were 100,333,1000, 3333 and 5000 pg per plate. No reductions in the background lawns were observed. However, reductions in the revertant counts were observed at 5000 pg per plate with tester strains TA1535 and TA1537 in the absence o f S9 activation in the independent repeat test No precipitate was observed. hi Experiment B1 (Initial Mutagenicity Test), no positive mutagenic responses were observed with any of the tester strains in either die 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 Bacterid Reverse Mutation Test with an Independent Repeat Assay indicate that, under the conditions o f this study, H-24890 did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9. BioReliance Study No. AA53CH.502001.BTL 17 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mmanal\m Microsome Mutagenicity Test, Mutation Research, 31:347-364. Green, M.H.L. and W J. Muriel (1976) Mutagen testing using trp+ reversion in Escherichia cot. Mutation Research 38:3-32. International Conference on Harmonisation (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 Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery fix' Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at step 4 of the ICH process on July 16,1997. Federal Register 62:16026-16030, November 21,1997. Marn, 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), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February 1998. Vogel, H.J. and D.M. Bonner (1956) Acetylomithinase of E. cot: Partial Purification and Some Properties, J. Biol. Chem., 218:97-106. BioReliance Study No. AA53CH.502001.BTL 18 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Preliminary Toxicity Test Table 1 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-24890 AA53CH.502001.BTL Al TA98 19 Dec 2001 water 100 pL Test Substance Concentrt ion pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 18 1 Without S9 Activation Revertants Background per plate Lawn 24 1 6.7 10 33 67 100 333 667 1000 3333 5000 17 1 19 1 22 1 15 1 23 1 18 1 19 1 13 1 22 1 16 1 18 1 21 1 14 1 13 1 13 1 15 1 91 10 1 13 1 11 1 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. AA53CH.502001.BTL 19 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Preliminary Toxicity Test Table 2 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-24890 AA53CH.502001.BTL Al TAI 00 19 Dec 2001 water 100 U.L Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 159 1 Without S9 Activation Revertants Background per plate Lawn 157 1 6.7 10 33 67 100 333 667 1000 3333 5000 167 1 186 1 176 1 168 1 171 1 173 1 178 1 168 1 152 1 147 1 174 1 167 1 156 1 168 1 158 1 188 1 178 1 172 1 204 1 170 1 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 20 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Preliminary Toxicity Test Table 3 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-24890 AA53CH.502001.BTL Al TA1535 19 Dec 2001 water 100 pL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 11 1 Without S9 Activation Revertants Background per plate Lawn 13 1 6.7 10 33 67 100 333 667 1000 3333 5000 10 1 17 1 14 1 24 1 26 1 15 1 13 1 16 1 14 1 10 1 13 1 12 1 12 1 24 1 19 1 19 1 17 1 20 1 15 1 15 1 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. AA53CH.502001.BTL 21 Company Sanitized. Does not contain T SC A CB1 H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Preliminary Toxicity Test Table 4 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-24890 AA53CH.502001. BTL Al TA1537 19 Dec 2001 water 100 pL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 51 Without S9 Activation Revertants Background per plate Lawn 81 6.7 10 33 67 100 333 667 1000 3333 5000 91 71 51 41 91 91 81 31 41 91 91 81 12 1 81 81 91 61 71 51 71 Background Lawn Code 1Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5Absent; 6Obscured by precipitate NP=Non-Interfering precipitate; IP=Interering precipitate BioReliance Study No. AA53CH.502001.BTL 22 Company Sanitized. Does not contain TSCA CBl H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Preliminary Toxicity Test Table 5 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-24890 AA53CH.502001.BTL Al WP2 uvrA 19 Dec 2001 water 100 pL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 13 1 Without S9 Activation Revertants Background per plate Lawn 15 1 6.7 10 33 67 100 333 667 1000 3333 5000 12 1 12 1 12 1 15 1 15 1 16 1 17 1 14 1 10 1 19 1 18 1 16 1 13 1 13 1 17 1 13 1 15 1 19 1 15 1 15 1 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. AA53CH.502001.BTL 23 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 6 Test Substance Id: H-24890 Study Number :AA53CH.502001.BTL Experiment No :B1 Strain :TA98 Cells Seeded :3.0 X 10s Liver Microsomes :None Date Plated :3 Jan 2002 Vehicle :water Plating Aliquot : 100 pL__________________________________________ Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 19 1 15 1 17 1 17 2 100 01 17 1 02 18 1 03 19 1 18 1 333 01 18 1 02 19 1 03 18 1 18 1 1000 01 11 1 02 10 1 03 12 1 11 1 3333 01 20 1 02 12 1 03 16 1 16 4 5000 01 10 1 02 15 1 03 13 1 13 3 Positive Control 2-nitrofluorene 1. 0 ug per plate 01 116 1 02 156 1 03 171 1 148 28 Background Lawn Code l=Normal; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5`Absent ; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 24 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 7 Test Substance Id: H-24890 Study Number : AA53CH.502001.BTL Strain : TA98 Liver Microsomes : Rat liver S9 Vehicle : water 100 pL Experiment No Cells Seeded Date Plated Bl 3.0 X 10* 3 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 31 1 28 1 32 1 30 2 100 01 34 1 02 31 1 03 18 1 28 9 333 01 34 1 02 28 1 03 22 1 28 6 1000 01 24 1 02 21 1 03 22 1 22 2 3333 01 19 1 02 18 1 03 23 1 20 3 5000 01 20 1 02 18 1 03 15 1 18 3 Positive Control 2-aminoanthracene 1.0 pg per plate 01 589 1 02 467 1 03 716 1 591 125 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 25 Company Sanitized. Does not contain TSC A CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 8 Test Substance Id: H-24890 Study Number :AA53CH.502001.BTL Experiment No :B1 Strain :TA100 Cells Seeded :2.9 X 10 Liver Microsomes :None Date Plated :3 Jan 2002 Vehicle :water Plating Aliquot : 100 pL__________________________________________ Concentrtion Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 178 1 147 1 155 1 160 16 100 01 162 1 02 161 1 03 173 1 165 7 333 01 213 1 02 189 1 03 199 1 200 12 1000 01 147 1 02 169 1 03 172 1 163 14 3333 01 167 1 02 154 1 03 143 1 155 12 5000 01 101 1 02 154 1 03 121 1 125 27 Positive Control sodium azide 1.0 pg per plate 01 521 1 02 579 1 03 640 1 580 60 Background Lawn Code 1Normal; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 26 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay__________________________________ DuPont-8968 Bacterial Mutation Test Table 9 Test Substance Id: H-24890 Study Number : AA53CH. 502001.BTL Strain : TA100 Liver Microsomes : Rat liver S9 Vehicle : water Plating Aliquot : 100 UL Experiment No : B1 Cells: Seeded : 2.9 X 10 Date Plated : 3 Jan 2002 Concentration Plate Revertants Background Average Standard yig per plate Number per plate Code Revertants1 Deviation Vehicle 01 02 03 185 1 187 1 172 1 181 8 100 01 173 1 02 154 1 03 203 1 177 25 333 01 191 1 02 165 1 03 206 1 187 21 1000 01 172 1 02 110 1 03 160 1 147 33 3333 01 158 1 02 155 1 03 168 1 160 7 5000 01 100 1 02 159 1 03 162 1 140 35 Positive Control 2-aminoanthracene 1.0 pg per plate 01 886 1 02 914 1 03 780 1 860 71 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitatei; IP=Interfering precipitate BioReliance Study No. AA53CH.50200l.BTL 27 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 10 Test Substance Id: H-24890 Study Number : AA53CH.502001.BTL Strain : TA1535 Liver Microsomes : None Vehicle : water 100 uL Experiment No Cells Seeded Date Plated B1 0.5 X 10 3 Jem 2002 Concentration Plate Revertemts Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 17 1 17 1 15 1 16 1 100 01 17 1 02 13 1 03 18 1 16 3 333 01 12 1 02 18 1 03 16 1 15 3 1000 01 11 1 02 23 1 03 12 1 15 7 3333 01 14 1 02 16 1 03 12 1 14 2 5000 01 19 1 02 11 1 03 16 1 15 4 Positive Control sodium azide 1.0 pg per plate 01 628 1 02 667 1 03 710 1 668 41 Background Lawn Code l=Normal ; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 28 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay__________________________________ DuPont-8968 Bacterial Mutation Test Table 11 Test Substance Id: H-24890 Study Number : AA53CH. 502001.BTL Strain : TA1535 Liver Microsomes : Rat liver S9 Vehicle : water Plating Aliquot : 100 UL Experiment No : B1 Cells Seeded : 0.5 X 10 Date Plated : 3 Jan 2002 Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 17 1 18 1 91 15 5 100 01 18 1 02 13 1 03 14 1 15 3 333 01 21 1 02 10 1 03 13 1 15 6 1000 01 10 1 02 8 1 03 19 1 12 6 3333 01 16 1 02 6 1 03 9 1 10 5 5000 01 17 1 02 9 1 03 10 1 12 4 Positive Control 2-aminoanthracene 1.0 ug per plate 01 108 1 02 114 1 03 101 1 108 Background Lawn Code l=Normal; 2=Slightly reduced; 3-Moderately reduced 4=Extremely reduced; 5=Absent,; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interering precipitate 7 BioReliance Study No. AA53CH.502001JBTL 29 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay__________________________________ DuPont-8968 Bacterial Mutation Test Table 12 Test Substance Id: H-24890 Study Number : AA53CH.502001.BTL Strain : TA1537 Liver Microsomes : None Vehicle : water Plating Aliquot : 100 pL Experiment No : B1 Cells Seeded : 1.1 X 10* Date Plated : 3 Jan 2002 Concentration Plate Eevertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 51 61 10 1 73 100 01 7 1 02 4 1 03 4 1 52 333 01 7 1 02 7 1 03 6 1 71 1000 01 10 1 02 6 1 03 6 1 72 3333 01 11 1 02 11 1 03 4 1 94 5000 01 8 1 02 7 1 03 3 1 63 Positive Control 9-aminoacridine 75 pg per plate 01 801 1 02 979 1 03 939 1 906 93 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent ; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 30 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 13 Test Substance Id: H-24890 Study Number : AA53CH.502001.BTL Strain : TA1537 Liver Microsomes : Rat liver S9 Vehicle : water Plating Aliquot : 100 pL Experiment No : B1 Cells Seeded : 1.1 X 10 Date Plated : 3 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 91 91 61 82 100 01 9 1 02 8 1 03 8 1 81 333 01 10 1 02 7 1 03 10 1 92 1000 01 7 1 02 10 1 03 11 1 92 3333 01 4 1 02 5 1 03 6 1 51 5000 01 4 1 02 6 1 03 7 1 62 Positive Control 2-aminoanthracene 1.0 pg per plate 01 58 1 02 72 1 03 76 1 69 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent ; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate 9 BioReliance Study No. AA53CH.502001.BTL 31 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 14 Test Substance Id: H-24890 Study Number :AA53CH.502001.BTL Experiment No :B1 Strain :WP2 uvrA Cells Seeded :0.9 X 10s Liver Microsomes :None Date Plated :3 Jan 2002 Vehicle :water Plating Aliquot : 100 uL__________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 22 1 19 1 23 1 21 2 100 01 13 1 02 21 1 03 10 1 15 6 333 01 20 1 02 15 1 03 19 1 18 3 1000 01 21 1 02 19 1 03 10 1 17 6 3333 01 13 1 02 10 1 03 17 1 13 4 5000 01 9 1 02 13 1 03 9 1 10 2 Positive Control methyl methanesulfonate 1000 pg per plate 01 90 1 02 96 1 03 90 1 92 Background Lawn Code l=Normal; 2=Slightly reduced; 3^Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate 3 BioReliance Study No. AA53CH.502001.BTL 32 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Teat Table 15 Test Substance Id: H-24890 Study Number : AA53CH.502001.BTL Strain : WP2 uvrA Liver Microsomes : Rat liver S9 Vehicle : water Experiment No Cells Seeded Date Plated B1 0.9 X 10 3 Jan 2002 Concentration lag per plate Vehicle Plate Number 01 02 03 Revertants per plate 18 28 17 Background Code 1 1 1 Average Revertants 21 Standard Deviation 6 100 01 17 1 02 20 1 03 31 1 23 7 333 01 30 1 02 19 1 03 15 1 21 8 1000 01 14 1 02 18 1 03 20 1 17 3 3333 01 19 1 02 21 1 03 34 1 25 8 5000 01 20 1 02 21 1 03 19 1 20 1 Positive Control 2-aminoanthracene 10 usr p e r plate 01 452 1 02 638 1 03 747 1 612 149 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfaring precipitate BioReliance Study No. AA53CH.502001.BTL 33 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repe tt Assay__________________________________ DuPont-8968 Bacterial Mutation Test Table 16 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24890 AA53CH.502001.BTL TA98 None water 100 pL Experiment No : B2 Cells Seeded : 1.7 X 10s Date Plated : 16 Jan 2002 Concentration Platte Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 15 1 61 91 10 5 100 01 8 1 02 6 1 03 7 1 71 333 01 14 1 02 10 1 03 8 1 11 3 1000 01 11 1 02 11 1 03 8 1 10 2 3333 01 6 1 02 8 1 03 9 1 82 5000 01 7 1 02 4 1 03 9 1 73 Positive Control. 2-nitrofluorene 1 .0 pg per plate 01 151 1 02 133 1 03 201 1 162 35 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. AA53CH.50200l.BTL 34 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 17 Test Substance Id: H-24890 Study Number : AA53CH.502001.1BTL Strain : TA98 Liver Microsomes : Rat liver S9 Vehicle : water Plating Aliquot : 100 uL Experiment No : B2 Cells Seeded : 1.7 X 108 Date Plated : 16 Jan 2002 Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 17 1 15 1 29 1 20 8 100 01 20 1 02 24 1 03 19 1 21 3 333 01 17 1 02 15 1 03 19 1 17 2 1000 01 21 1 02 23 1 03 15 1 20 4 3333 01 19 1 02 19 1 03 17 1 18 1 5000 01 28 1 02 14 1 03 17 1 20 7 Positive Control 2-aminoanthracene 1.0 pg per plate 01 689 1 02 514 1 03 654 1 619 93 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 35 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay__________________________________ DuPont-8968 Bacterial Mutation Test Table 18 Test Substance Id: H-24890 Study Number : AA53CH. 502001. BTL Strain : TA100 Liver Microsomes : None Vehicle : water Plating Aliquot : 100 uL Experiment No : B2 Cells Seeded : 2.3 X 10s Date Plated : 16 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 142 1 153 1 163 1 153 11 100 01 196 1 02 129 1 03 152 1 159 34 333 01 162 1 02 162 1 03 192 1 172 17 1000 01 186 1 02 190 1 03 182 1 186 4 3333 01 152 1 02 167 1 03 140 1 153 14 5000 01 185 1 02 123 1 03 130 1 146 34 Positive Control sodium azide 1.0 ug per plate 01 499 1 02 466 1 03 453 1 473 24 Background Lavai Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extreraely reduced; 5=Absent ; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 36 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 19 Test Substance Id: H-24890 Study Number : AA53CH.502001.BTL Strain : TA100 Liver Microsomes : Rat liver S9 Vehicle : water Plating Aliquot : 100 pL Experiment No : B2 Cells Seeded : 2.3 X 10 Date Plated : 16 Jan 2002 Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 175 1 211 1 175 1 187 21 100 01 183 1 02 197 1 03 172 1 184 13 333 01 176 1 02 212 1 03 198 1 195 18 1000 01 178 1 02 185 1 03 126 1 163 32 3333 01 136 1 02 181 1 03 163 1 160 23 5000 01 159 1 02 160 1 03 132 1 150 16 Positive Control 2-aminoanthracene 1.0 pg per plate 01 915 1 02 795 1 03 747 1 819 87 Background Lawn Code l=Normal; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitateif IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 37 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 20 Test Substance Id: H-24890 Study Number :AA53CH.502001.BTL Strain :TA1535 Liver Microsomes :ljfone Vehicle :i^ater Plating Aliquot : .00 UL Experiment No : B2 Cells Seeded :2.6 X10a Date Plated : 16Jan 2002 Concentration Plat$ Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01* 02; 03 ! 23 1 21 1 26 1 23 3 100 01 : 21 1 02 ; 19 1 03 17 1 19 2 333 01 9 1 02 13 1 03 : 13 1 12 2 1000 01 02 03 1 11 1 61 41 74 3333 01 02 : 03 ; 14 1 14 1 11 1 13 2 5000 01 02 : 03 91 11 1 11 1 10 1 Positive Control sodijum azide 1.0 ug per plate 01 ' 336 1 02 356 1 03 369 1 354 17 Background Lawn Code l=Normal; 2=Slidhtly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interferljng precipitate; IP=Interfering precipitate BioReliance Study No. AA53CH.5O2O0l.BTL 38 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 21 Test Substance Id: H-24890 Study Number :AA53CH.502001.BTL Experiment No :B2 Strain :TA1535 Cells Seeded :2.6 X10s Liver Microsomes :Rat liver S9 Date Plated :16Jan 2002 Vehicle :water Plating Aliquot : 100 pL________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 71 22 1 21 1 17 8 100 01 19 1 02 9 1 03 5 1 11 7 333 01 19 1 02 11 1 03 19 1 16 5 1000 01 10 1 02 16 1 03 7 1 11 5 3333 01 9 1 02 18 1 03 6 1 11 6 5000 01 15 1 02 8 1 03 14 1 12 4 Positive Control 2-aminoanthracene 1.0 pg per plate 01 118 1 02 134 1 03 133 1 128 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6-Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate 9 BioReliance Study No. AA53CH.502001 .BTL 39 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 22 Test Substance Id: H-24890 Study Number : AA53CH.502001.BTL Strain : TA1537 Liver Microsomes : None Vehicle : water Plating Aliquot : 100 pL Experiment No Cells Seeded Date Plated B2 1.0 X 10s 16 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 81 51 71 72 100 01 4 1 02 7 1 03 2 1 43 333 01 7 1 02 4 1 03 2 1 43 1000 01 6 1 02 6 1 03 3 1 52 3333 01 7 1 02 6 1 03 6 1 61 5000 01 4 1 02 3 1 03 2 1 31 Positive Control 9-aminoacridine 75 pg per plate 01 799 1 02 575 1 03 749 1 708 118 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 40 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 23 Test Substance Id: H-24890 Study Number :AA53CH.502001.BTL Experiment No :B2 Strain :TA1537 Cells Seeded :1.0 X10a Liver Microsomes :Rat liverS9 Date Plated :16Jan 2002 Vehicle :water Plating Aliquot : 100 pL_____________________________________ _____ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 11 91 81 64 100 01 4 1 02 7 1 03 7 1 62 333 01 6 1 02 7 1 03 10 1 82 1000 01 9 1 02 7 1 03 7 1 81 3333 01 2 1 02 4 1 03 11 1 65 5000 01 8 1 02 5 1 03 5 1 62 Positive Control 2-aminoanthracene 1.0 pg per plate 01 84 1 02 74 1 03 83 1 80 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate 6 BioReliance Study No. AA53CH.502001.BTL 41 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 24 Test Substance Id: H-24890 Study Number :AA53CH.502001.BTL Experiment No :B2 Strain :WP2 uvrA Cells Seeded :8.2 X108 Liver Microsomes :None Date Plated :16Jan 2002 Vehicle :water Plating Aliquot : 100 pL___________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 11 1 81 17 1 12 5 100 01 9 1 02 8 1 03 9 1 91 333 01 7 1 02 7 1 03 6 1 71 1000 01 5 1 02 2 1 03 6 1 42 3333 01 7 1 02 13 1 03 10 1 10 3 5000 01 10 1 02 11 1 03 11 1 11 1 Positive Control methyl methanesulfonate 1000 pg per plate 01 112 1 02 96 1 03 82 1 97 15 Background Lawn Code 1Normal; 2=Slightly reduced; 3-Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 42 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Table 25 Test Substance Id: H-24890 Study Number : AA53CH.502001.BT1> Strain : HP2 uvrA Liver Microsomes : Rat liver S9 Vehicle : water Plating Aliquot : 100 pL Experiment No : B2 Cells Seeded : 8.2 X 108 Date Plated : 16 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 20 1 11 1 12 1 14 5 100 01 12 1 02 9 1 03 12 1 11 2 333 01 8 1 02 12 1 03 11 1 10 2 1000 01 5 1 02 8 1 03 13 1 94 3333 01 12 1 02 13 1 03 11 1 12 1 5000 01 8 1 02 7 1 03 7 1 71 Positive Control 2-aminoanthracene 10 pg per plate 01 673 1 02 748 1 03 850 1 757 89 Background Lawn Code l=Normal; ;Slightly reduced; :Moderately reduced 4=Extremely reduced; 5=Absent; 6Obscured by precipitate NP=Non-Interfering precipitate;; IP=Interfering precipitate BioReliance Study No. AA53CH.502001.BTL 43 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Bacterial Mutation Test Summary of Results Table 26 Test Substance Id : H-24890 Study Number______ : AA53CH.502001.BTL______Experiment No : B1 Average Revertants Per Plate : Standard Deviation Liver Microsomes : None Dose (pg/plate) TA98 TA100 Vehicle 100 333 1000 3333 5000 Positive 17 2 160 16 18 1 165 7 18 1 200 12 11 1 163 14 16 4 155 12 13 3 125 27 148 28 580 60 Liver Microsomes: Rat liver S9 TA1535 16 t 16 15 15 14 15 668 l 1 3 3 7 2 4 41 TA1537 7 5 7 7 9 6 906 3 2 1 2 4 3 93 WP2 uvrA 21 15 18 17 13 10 92 2 6 3 6 4 2 3 Dose (ug/plate) TA98 TA100 Vehicle 100 333 1000 3333 5000 Positive 30 2 28 9 28 6 22 2 20 3 18 3 591 125 181 177 187 147 160 140 860 8 25 21 33 7 35 71 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 100 pL TA1535 15 15 15 12 10 12 108 5 3 6 6 5 4 7 TA1537 8 8 9 9 5 6 69 2 1 2 2 1 2 9 WP2 uvrA 21 6 23 1 7 21 8 17 3 25 8 20 1 612 1 149 BioReliance Study No. AA53CH.502001.BTL 44 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-8968 Bacterial Mutation Test Summary of Results Table 27 Test Substance Id : H-24890 Study Number______ : AA53CH. 502001.BTL______Experiment No ; B2 Average Revertants Per Plate :t Standard Deviation Liver Microsomes: None Dose (pg/plate) TA98 TA100 Vehicle 100 333 1000 3333 5000 Positive 10 5 153 11 7 1 159 34 11 3 172 17 10 2 186 4 8 2 153 14 7 3 146 34 162 35 473 24 Liver Microsomes: Rat liver S9 TA1535 23 19 12 7 13 10 354 t 3 2 2 4 2 1 17 TA1537 7 2 4 3 4 3 5 2 6 1 3 1 708 1 118 WP2 uvrA 12 9 7 41 10 11 97 5 1 1 2 3 1 15 Dose (pg/plate) TA98 TA100 Vehicle 100 333 1000 3333 5000 Positive 20 8 187 21 21 3 184 13 17 2 195 18 20 4 163 32 18 1 160 23 20 7 150 16 619 93 819 87 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 100 pL TA1535 17 11 16 i 11 11 12 128 8 7 5 5 6 4 9 TA1537 6 6 8 8 6 6 80 4 2 2 1 5 2 6 WP2 uvrA 14 11 10 9 12 7 757 5 2 2 4 1 1 89 BioReliance Study No. AA53CH.502001.BTL 45 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse M utation Test with an Independent Repeat Assay______ DuPont-8968 APPENDIX A Historical Control Data BioReliance Study No. AA53CH.50200l.BTL 46 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Historical Negative and Positive Control Values 1998 - 2000 revertants per plate Activation Strain Control None Rat Liver Mean SD Min Max Mean SD Min Max TA98 Neg 16 7 4 59 21 7 7 58 Poi 425 206 21 1536 592 322 56 2454 TA100 N ei 128 31 53 288 138 34 74 258 P oi 568 159 129 1371 736 301 198 2871 TA1535 N ei 12 5 1 45 12 4 1 42 Pol 378 164 6 978 104 84 18 1640 TA1537 N ei 6 3 0 30 7 3 1 29 Pos 708 409 13 2786 88 106 12 2060 Neg 14 5 4 48 16 6 4 115 WP2 uvrA Pos 190 138 34 961 317 299 22 2632 SD=standard deviation; Min=minimum 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. AA53CH.502001.BTL 47 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 APPENDIX B Study Protocol BioReliance Study No. AA53CH.502001 .BTL 48 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 1.0 PURPOSE deceived by Sponsor Prefect Number DuPont-8968 BioRetiaoce Study Number. AA53CH.S02001.BTL H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay 2t%a>.(i*M | P !P i(0 ) f E l The purpose of this study is to evaluate the mutagenic potential of die test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhim uritm and at the tryptophan locus of Escherichia coll WP2 uvrA in the presence and absence of S9 activation. 2.0 SPONSOR 2.1 Name: E.I. du Pont de Nemours and Company 2.2 Address: Stine Haskell Research Center DuPont Haskell Laboratory P.O.Box 50 1090 Elkton Road Newark, DE 19714-0050 2.3 Representative: Maria Donner, PhD. Phone: 302-366-5251 Fax: 302-366-5207 Email: maria.donner(2}usa.dunont.com 2.4 SponsorProject No.: DuPont-8968 1 2.5 WR* 2.6 Haskell* H-24890 2.7 Service Code: 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 TestSubstance Name: 3 2 Test Substance ID.: 3 3 Controls: Negative: Positive: Test substance vehicle 9-aminoacridine ? ^ammnanthrftriTy methyl methanesulfonate 2-oitrofluorene sodium azide Protocol SPGT50200I 1-Jan-2001 Page 1 of 11 BioReliance Study No. AA53CH.50200l.BTL 49 ^ Bio Reliancf Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assa y ___ DuPont-8968 Sponsor Project Number: DuPont-8968 BioRetiance Study Number AA53CH.S02001.BTL 3.4 Test Substance Characterization Unless alternate arrangements are made, die testing facility at BioRetiance will not perforai analysis of the dosing solutions. The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition of die test substance, and die stability and strength of die test substance in the solvent (or vehicle). 3.5 Test Substance Retention Sample The retention of a reserve sample of the test substance will be the responsibility of the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility BioReliance 4.2 Address: 9630 Medical Center Drive Rockville, MD 20850 4.3 Study Director Valentine O. Wagner III, M.S. Phone: 301-610-2152 Fax:301-738-2362 Email: swagner@lxoreliance.com 5.0 PROPOSED STUDY DATES 5.1 Experimental Start Date: 21-Dec-2001 5.2 Experimental Temunadon Date: 06-Feb-2002 5.3 Draft Report Date: 20-Feb-2002 5.4 Final Report Date: 2 weeks after Sponsor approves draft 6.0 TEST SYSTEM The tester strains trill include the S. typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and the K coli tester strain WP2 uvrA as described by Green and Muriel (1976).___________________________ Histidine Mutation Tiyptopha n Mutation Additional Mutations WKH6 WiC3076 J&D3052 trpE LPS Repair R-fector TA1535 TA1537 - - ria t/vrB - Protocol SPGT502001 l-Jan-2001 Page 2 of 11 ^ Bio Reliance- BioReliance Study No. AA53CH.502001.BTL 50 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse M utation Test with an Independent Repeat Assay______ DuPont-8968 Sponsor Project Number: DuPont-8968 BioReliance Study Number: AA53CH.S02001.BTL Histidine Mutation MsG46 TA100 - AC3076 AD3052 - TA98 -- Tryptopha n Mutatimi trpE WP2 uvrA Additional Mutations LPS Repair R-factor rfa uvrB +R - uvrA - Each typhimurmm 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 o f the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system.' Tester strains TA98 and TA100 also contain the pKMIOl plasmid (carrying 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 TA1S37 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both fiameshift and base substitution mutagens and TA1S3S is reverted only by mutagens that cause base substitutions. The E. coli tester strain has an AT base paira t the critical mutation site within the trpE gene (Wilcox el al., 1990). Tester strain WP2 uvrA has a deletion in the 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 tire original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base-pair substitution mutations (Green and Muriel, 1976). The typhimurium 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, TA133S, TA1537 and WP2 uvrA 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. AA53CH.502001.BTL 51 0 Bio Reliancf Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Sponsor Project Number DuPont-8968 BioReliance Study Number AA53CH.502001.BTL 7.1 Solubility Determination Unless the Sponsor has indicated the test substance vehicle, a solubility determination will be conducted to determine the maximum soluble concentration or workable suspension up to a maximum of 50 mg/mL for aqueous vehicles and SOOmg/mL for organic vehicles. Vehicles compatible with this test system, in order of preference, include but are not limited to deionized water (CAS 7732-18-5), dimethyl sulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5) and acetone (CAS 67-64-1). The vehicle of choice will be foe solvent, selected in order of preference, which permits preparation of foe highest wodcable/sohible stock concentration, up to 50 mg/mL for aqueous vehicles and 500 mg/mL for organic vehicles. '7 2 Preliminary Toxicity Assay to Select Dose Levels Selection ofdose levels for foe mutagenicity assay will be based upon foe toxicity and precipitation profile of foe test substance assessed in a preliminary toxicity assay. This preliminary assay will be conducted by exposing TA98, TA100, TA1535, TA1S37 and WP2 uvrA to negative controls and to at least eight concentrations of test substance, one plate per dose level, in both the presence and absence of S9 activation. Unless indicated otherwise by the Sponsor, die highest dose will be the highest workable concentration in the vehicle of choice but not to exceed 5 mg/plate. In selecting dose levels for the mutagenicity assay the following guidelines will be employed. Doses will be selected such that precipitate does not interfere with manual scoring. Whenever possible, the highest dose for the mutagenicity assay will be selected to give some indication oftoxicity without exceeding 5 mg/plate. For freely soluble, nontaxic test substances, foe highest dose level wall be 5 mg/plate. For precipitating, nontaxic test substances, the highest dose level will be selected in an attemptto 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 lewd is less than 5 mg/plate or (2) the maximum achievable test substance dose level is less than 5 mg/plate and this dose level is nontoxic. 7.3 Frequency and Route of Administration The test system will be exposed to the test substance via foe plate incorporation methodology originally described by Ames el at. (1975) and updated by Man and Ames (1983). This test system has been shown to detect a wide range of classes ofchemical mutagens (McCann et a t, 1975; McCann and Ames, 1976). After foe data generated in foe first assay have been evaluated, foe mutagenicity assay will be repeated. The dose levels used in foe second assay will be the same as those used in foe first assay unless foe Study Director determines that the dose levels should be changed due to an equivocal response, excessive cytotoxicity or Protocol SPGT5Q2001 l-Jan-2001 Page 4 of 11 W Bio Reliance- BioReliance Study No. AA53CH.502001.BTL 52 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Sponsor Project Number DuPont-8968 BioReliance Study Number AA53CH.S02001.BTL excessive precipitate. If the Sponsor is aware of specific metabolic requirements (e g., azo compounds), this infonnation 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 (pg'pkte) Salm on ella Strains Rat 2-aminoanthracene 1.0 WP2vrA 10 TA98 2-nitrofluorene 1.0 TA100, TA1535 TA1537 None sodium azide 9-aminoacridine 1.0 75 WP2 uvrA methyl methanesulfonate 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 bass for use of the selected vehicle. In the latter case, both untreated and vehicle controls will be used. 7.43 Sterility Controls The most concentrated test substance dilution and the Sham and S9 mixes will be checked for sterility. 73 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 Sptague-Dtndey rats induced with a single intraperitoneal iryection ofArodor 1254,500 mg/kg, five days prior to sacrifice. The S9 will be batch prepared and stored frozen at approximately Protocol SPGT502001 1-Jan-2001 Page 5 of 11 9 Bio Reliance- BioReliance Study No. AA53CH.502001.BTL 53 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Sponsor Project Number: DuPont-8968 BioReliance Study Number: AA53CH.502001.BTL -70C until used Each botch o f S9 homogenate will be assayed for its ability to metabolize 2-aminoanthracene and 7,12-dunethylbenzanthracene to fonns mutagenic to typhimurium TA100. Immediately prior to use, the S9 will be thawed and mixed with a cofactor pool to contain 10% S9 homogenate, SmM glucose-6-phosphate, 4 mM p-nicotmamide-acknine dinucleotide phosphate, 8 mM MgCl2and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. This mixture is referred to as S9 mix. Sham mix will be 100 mM phosphate buffer at pH 7.4. 7.6 Preparation ofTester 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, the length ofincubation will be controlled and monitored. At the end of the working day, each inoculated flask will be placed in a resting shaket/incubator at room temperature. The shaker/incubator will be programmed to begin shaking at approximately 125 rpm at 372C approximately 12hours before the anticipated time ofharvest. 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 ofapproximately 1O' cells/mL. 7.7 Test System Identification Each plate will be labeled with a code system that identifies foe test substance, test phase, dose level, tester strain and activation type as described in BioReliance'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 ofTest System One half milliliter (0.5 mL) of S9 mix or Sham mix, 100 L of tester strain and SOpL of vehicle, test suhstance 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 ofother than 50 ^L o f test substance/vehicle/positive control will be plated. The mixture will be vortex mixed and overlaid onto the surface of 25 mL of minimal bottom agar. Protocol SPGT502001 1-Jan-2001 Page 6 of 11 W Bio Reliance- BioReliance Study No. AA53CH.502001.BTL 54 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Sponsor Project Number DuPont-8968 BioReliance Study Number AA53CH.502001.BTL Alter 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 foe incubation period will be stored at 2-8C. 7.10 Scoring The condition o f foe bacterial background lawn will be evaluated for evidence of test substance toxicity and precipitate. Evidence of toxicity will be scored relative to the negative control plate and recorded along with foe revertant count for that plate. Toxicity will be evaluated as a decrease in the number ofrevertant colonies per plate and/or a dunning or disappearance of die 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 typhimurium tester strain cultures must exhibit sensitivity to crystal violet To demonstrate the presence of the i/vrB mutation, all X typhimurium tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate die presence o f the uvrA mutation, all . coli tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate the presence of the pKMIOl plasmid R-foctor, tester strain cultures of TA98 and TA100 must exhibit resistance to ampicQlin. 8 2 Spontaneous Revertant Background Frequency Based on historical control data, all tester strain cultures must exhibit characteristic number o f spontaneous revertants per plate in foe negative controls (vehicle). The mean revertants per {date must be within the following ranges (inclusive): TA98, 10- 50; TA100, 80-240; TA1535, 5 - 45; TA1537, 3-21; WP2 wwA, 10 - 60. 8.3 Tester Strain Titers To ensure that appropriate numbers of bacteria are plated, all tester strain culture titers must be equal to or greater than 0.3x1O' cells per milliliter. Protocol SPGT502001 l-Jan-2001 Page 7 of 11 ^ Bio Reliance* BioReliance Study No. AA53CH.502001.BTL 55 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Sponsor Project Number: DuPont-8968 BioReliance Study Number AAS3CH-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 Toxicity A minimum ofthree 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 (tins reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in the background lawn. In the event that less than tim e non-toxic dose levels are achieved, the affected portion of die assay will be repeated with an appropriate change 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 o ftest 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 than 3.0-times the mean negative control value (vehicle). 9.2 Strains TA98, TA100 and WP2 wvrA Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times 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 o f 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 oftest substance, if known. Soivent/Vehide: justification for choice of vehicle; solubility and stability of test substance in soivent/vehicle, if known. Strains: strains used; number ofcells/mL per culture; strain characteristics. Protocol SPGT502001 1-Jan-2001 Page 8 of 11 ^ 9 Bio Reliancf BioReliance Study No. AA53CH.502001.BTL 56 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Sponsor Project Number: DuPont-8968 BioReliance Study Number AA53CH.502001 .BTL Test conditions: amount oftest 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 toxicity; signs of precipitation; individual plate counts; die 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 ofresults. Conclusion. 11.0 RECORDS AND ARCHIVES All raw data, the protocol and all reports will be maintained according to Standard Operating Procedure OPQP3040 by the BioReliance RAQA unit headquartered at: BioReliance, 14920 Broschart Road, Rockville, MD 20850. Per this SOP, paper records will be retained for at least force years after which time the Sponsor will be contacted for a decision as to foe final disposition of the materials. AU 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. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply with OECD Guideline 471 (Generic Toxicology: Bacterial Reverse Mutation Assay), Ninth Addendum to the OECD Guidelines for foe Testing of Chemicals, published by OECD, Paris, February 1998 and with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997). This study will be performed in compliance with the 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 the 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 foe 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 dates the results of each inspection were repotted to the Study Director and the Study Director's management In addition, a signed GLP compliance statement will be included in die final report This statement will cite the GLP guideline^) with which the study is compliant and any exceptions to this compliance, if applicable, including the omission of characterization or stability analyses ofthe test or control substances or their mixtures. Protocol SPGT502001 l-Jan-2001 Page 9 of 11 9 Bio Reliance BioReliance Study No. AA53CH.502001.BTL 57 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Sponsor Project Number: DuPont-8968 BioReliance Study Number AA53CH.502001.BTL Unless arrangements are matte to the 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 &i/fnone//a/mammalian-microsotne mutagenicity test. Mutation Research 31:347-364. Green, M.H.L., and Muriel, W J. (1976). Mutagen testing using tip* reversion in Escherichia cali. Mutation Research 38:3-32. International Conference on Harmonisation (ICH) of Technical Requirements for Registration o f Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 o f the ICH process on July 19, 199S. Federal Register 61:18198-18202, April 24,1996. International Conference on Harmonisation (ICH) of Technical Requirements for Registration o f 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. Federal Register 62:16026-16030, November 21,1997. McCann, J. and Ames, B.N. (1976). Detection of carcinogens as mutagens in the Salmoneita/rmcmsomc test: assay o f 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 Salm onella/m ictosoaa test: assay of 300 chemicals. Proc. Natl. Acad. Sci. USA 72:5135-5139. Marn, 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 o f Chemicals, published by OECD, Paris, February 1998. Wilcox, P., Naidoo, A , Wedd, D J. and Gatehouse, D.G. (1990). Comparison of Salmonella typhinturium TA102 with Escherichia coli WP2 tester strains. Mutagenesis 5:285-291. Protocol SPGT502001 1-Jan-2001 Page 10 of 11 BioReliance Study No. AA53CH.502001.BTL 58 Bio Reliance- Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Sponsor Project Number: DuPont-8968 BioReliance Study Number AA53CH.502001JBTL 14.0 APPROVAL /-*-- Sponsor Representative OR - O e .c - 3 - 0 0 \ Date V V cn-C ' (Print or Type Name) O. BioReliance Study Director BioReliance Study Management H D fio ^U > 0 | Date / / i t e . >o o f Date Protocol SPQT502001 1-Jan-2001 Page 11 o fl 1 BioReliance Study No. AA53CH.502001.BTL 59 ^ Bio Reliancf Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 APPENDIX C Information for Japanese Regulatory Agencies BioReliance Study No. AA53CH.50200l.BTL 60 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 Report of Results of Reverse-Mutation Test in Bacteria 1. Tester Strains (1) Procurement Strain TA98 TA100 TA1535 TA1537 TA1538 TA97 TA102 WP2 uvrA WP2 uvrA (pkM IOl) WP2 (pKM IOl) Obtained from Date obtained 10 November 1998 Dr. Bruce Ames University of California, Berkeley 11 August 1998 13 December 1990 14 November 1990 National Collection of Industrial and Marine Bacteria Aberdeen, Scotland 1 July 1987 19 February 1993 Date inspected the strain lot in storage 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. S9M ix (1) Source, Storage Temperature, etc, of S9 Made in-house Prepared on 26 November 2001 (Batch R636) Storage temperature -70C or colder Name and model of storage apparatus So-Low, Model PR27-120 BioReliance Study No. AA53CH.502001.BTL 61 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 (2) Preparation o f S9 Animal used Inducing substance Species, Strain Rattus norvegicus, Sprague Dawley Name Aroclor 1254 Sex Age (in weeks) W eight Male 9 (Batch R656) 187 to 271 g (Batch R656) A dm inistration method A dm inistration period and amount (g/kg-weight) intraperitoneal 5 days, 0.5 gm/kg body weight 3. Preparation o f Test Substance Solution Solvent used Name M anufacturer Lot No. Grade and/or Purity (%) W ater (CAS No. 7732-18-5) Invitrogen Corporation, formerly 1103072; Life Technologies, Inc. 1111323 Distilled Stability o f test substance in the solvent Unknown Method of suspension when test substance is difficult to dissolve Not applicable BioReliance Study No. AA53CH.502001.BTL 62 Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ -- I--Jfc-- ---- -- I MUMIII! I I>uPont-8968 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 M anufacturer Oxoid Ltd. Lot No. CH,-B=210287 2 to 5 hours at ambient temperature <8 hours at 2-8C New Brunswick Scientific, model G-24 Rotary (125 rev/m in.) shape: cylinder, 200 mL 50 mL 1 colony 5. Agar Plate Medium (!) Top agar________ Agar Name M anufacturer Lot No. BBL Select Becton Dickinson 1000J3DKSQ (2) Minimum Glucose Agar Name Made in-house Agar M anufacturer Lot No. Volume of agar plate medium BioReliance Study No. AA53CH.502001.BTL 63 BBL Select Becton Dickinson 1000J3DKSQ 25 mL Company Sanitized. Does not contain TSCA CBI H-24890: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-8968 6. Test Results - Judgement of the results Judgement Negative Reason for judgement and referential matters: No positive mutagenic response was observed with any o f the tester strains in either the presence or absence of Aroclor-induced rat liver S9. Referential matters The vehicle and positive control values indicate that all tester strains were functioning correctly and were capable o f detecting a mutagen. BioReliance Study No. AA53CH.502001.BTL 64 Company Sanitized. Does not contain TSCA CBI