Document VJ39dkYJR265Rp1nj8KXv8eQw

AR226-3180 DuPont-6405 TRADE SECRET Testing Guidelines 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 and OECD Guidelines for Testing of Chemicals Section 4: Health Effects, No. 471 (Adopted 1997) Study Title H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay Authors Valentine 0. Wagner, DI, M.S. Trinh T. H. Nguyen, B.S. Study Completion Date July 20,2001 Performing Laboratory BioReliance 9630 Medical Center Drive Rockville, MD 20850 for E. L du Font de Nemours and Company Stine Haskell Research Center DuPont Haskell Laboratory P.O. Box 50,1090 Elkton Road Newark, DE 19714-0050 Performing Laboratory Study Number AA44ER.502001.BTL Dupont Project Number DuPont-6405 Worie: Request Number Haskell Number H-24889 Service Code Page 1 of 63 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_____________________________ STATEMENT OF COMPLIANCE Study No. AA44ER.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 Principlesof Good Laboratory Practice in all material aspects with the following exceptions: The identity, strength, purity and characteristics to define the control determined by the testing facility. composition articles have or other not been The stability of the test and control articles 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 article mixtures. The stability of the test and control articles 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 article mixtures. \ ^ ^ o . i j ^ ^ Valentine 0. Wagner, ffl, M.5' Study Director ^ ^ L ^ / ^ BioReliance Study Management '2-*^.. | 2.^1 Date 20 \^fC StOOf Date BioReliance Study No. AA44ER.502001.BTL 2 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay____ Quality Assurance Statement DuPont-6405 Study Title: H 24889 BACTERIAL REVERSE MUTATION ASSAY WITH AN INDEPENDENT REPEAT ASSAY Study Number: AA44ER.502001.BTL Study Director: Valentine 0. Wagner, ffl, 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 phases over 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 Japanese GLP Standard, and the OECD Principles of Good Laboratory Practice and to assure that the study is conducted according to the protocol and relevant Standard Operating Procedures. The following are the inspection dates, phases inspected, and report dates ofQA inspections of this study. Inspect On Phase Inspect On Phase Inspect On Phase Inspect On Phase 14-May-Ol - 14-May-Ol To Study Dir 14-May-Ol To Mgmt 14-May-Ol Protocol Review 30-May-Ol - 30-May-Ol To Study Dir 30-May-Ol To Mgmt 25-Jun-Ol Test and/or control material administration 1 l-Jul-01 - 1 l-Jul-01 To Study Dir 1 l-Jul-01 To Mgmt 13-Jul-Ol Draft Report 24-Jul-Ol - 24-Jul-Ol To Study Dir 24-Jul-Ol To Mgmt 24-Jul-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. S^r^ji^ /B^ /). Becky D. Schreckengost, B.S.I QUALITY ASSURANCE ^ ^u W DATE' BioReliance Study No. AA44ER.502001.BTL 3 ^ Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study Director: ^oQ^ytak 0 - [>^u9^yS- XVa_l1e---nAt"i--ne-- ^0^. TWITa-g_n_ e_r\/mTT,TMK'.SCT. 2o'3/2oof Date Approved by Study Monitor: \\.)^^-G^. <_) ev->,^ Maria Donner, Ph.D. Senior Research Scientist (, ^VL. Z-ool Date BioReliance Study No. AA44ER.502001.BTL 4 Company Sanitized. Does not contain TSCA CBI iD H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay____________________________ TABLE OF CONTENTS Page Certification...................,................................;............,,............................................................4 Study Information .....................................................................................................................7 Summary.................................................................................................................................9 Purpose....................................................................................................................................10 Characterization of Test and Control Articles.........................................................................10 Materials and Methods..............................................................................................--...........11 Results and Discussion............................................................................................................15 Conclusion.........................................:.....................................................................................15 15 References.............................................................................................................................. DataTables..............................................................................................................................l7 Table 1: Preliminary Toxicity Test in Salmonella typhimurium TA98 .............................17 Table 2: Preliminary Toxicity Test in Salmonella typhimurium TA100 ...........................18 Table 3: Preliminary Toxicity Test in Salmonella typhimurium TA1535 .........................19 Table 4: Preliminary Toxicity Test in Salmonella typhimurium TA1537 .........................20 Table 5: Preliminary Toxicity Test in Escherichia coli WP2 iwA...................................21 Table 6: Mutagenicity Test in Salmonella typhimurium TA98 without S9 .....................22 Table 7: Mutagenicity Test in Salmonella typhimurium TA98 with S9 ..........................23 Table 8: Mutagenicity Test in Salmonella typhimurium TA100 without S9 ...................24 Table 9: Mutagenicity Test in Salmonella typhimurium TA100 with S9 ........................25 Table 10: Mutagenicity Test in Salmonella typhimurium TA1535 without S9 .................26 Table 11: Mutagenicity Test in Salmonella typhimurium TA1535 with S9 ......................27 Table 12: Mutagenicity Test in Salmonella typhimurium TA1537 without S9 .................28 Table 13: Mutagenicity Test in Salmonella typhimurium TA1537 with S9 ......................29 Table 14: Mutagenicity Test in Escherichia coli WP2 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 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 Escherichia coli WP2 MvrA without S9...........................40 Table 25: Mutagenicity Test in Escherichia coli WP2 uvrA with S9 ................................41 BioReliance Study No. AA44ER.502001.BTL 5 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_____________________________ Table 26: Salmonella/E. coli Mutagenicity Test - Summary of Results B 1 ......................42 Table 27: 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. AA44ER.502001.BTL 6 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay STUDY INFORMATION ArticleJTestedi^lBBB^HBl Synonyms/Codes: Haskell Number: 4^^^^B t--------i . H-24889 t---- 24889 Compositi DuPont-6405 BioReliance Study No. AA44ER.502001.BTL Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutadon Test DuPont-6405 with an Independent Repeat Assay____________________________ Physical Characterisdcs: Stability: The test article 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: May 11,2001 / (see report cover page) m-Iife Initiated/Completed: May 15,2001 / June 8, 2001 BioReliance Study No. AA44ER.502001.BTL 8 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_____________________________ SUMMARY The test article, H-24889, was tested in the Bacterial Reverse Mutation Test with an Independent Repeat Assay using Salmonella typhimurium 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 toxidty 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 of the test article. Acetone was selected as the solvent of choice based on compatibility with the target cells, and solubility of the test article. The test article was soluble and clear in acetone at a maximum concentration of approximately 500 mg/mL, the highestconcentration tested. In the preliminary toxicity test, the maximum dose tested was 5000 ug per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 uL plating aliquot. Dose levels tested were 5000,3333,1000,667,333,100,67,33,10 and 6.7 ug per plate. Neither precipitate nor 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. m the mutagenicity test, no positive response was observed. The dose levels tested were 5000, 3333, 1000, 333 and 100 ug per plate. Neither precipitate nor appreciable toxicity was observed. All criteria for a valid study were met as described in the protocol. The results of the H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay indicate that, under the conditions of this study, H-24889 did not cause a positive response in the presence and absence of Aroclor-induced rat liver S9. BioReliance Study No. AA44ER.502001.BTL 9 Company Sanitized. Does not contain TSCA CBI t) H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 PURPOSE The pmpose of this study was to evaluate the mutagenic potential of the test article 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 iwA in the presence and absence of Aroclor-induced rat liver S9. CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test article, H-24889, was received by BioReliance on 09 May 2001 and was.assigned ^|H|B the code number AA44ER. The test article was characterized by the Sponsor as should be stored at less than 27C in a ventilated area with no re&igflafion. An lU^Bfhat CTmration.Qate. of.05 April 2003 was provided. Upon receipt, the test article was described as an HIlMliBBBBIBff11^ was stored at room temperature, protected from light The "identity, strength, purity^omposidon or other characteristics to define the test article have been determined by the Sponsor. The stability of the test article has been determined by the Sponsor. The vehicle used to deliver H-24889 to the test system was acetone (CAS# 67-64-1), obtained from Fisher Scientific. Test article 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: Strain S9 Activation Positive Control Concentration (ug/plate) All Salmonella Strains Rat WP2nvrA 2-aminoanthracene (Sigma Chemical Co.) 1.0 10 TA98 TA100, TA1535 TA1537 None 2-nitrofluorene (Aldrich Chemical Co., Inc.) Sodium azide (Sigma Chemical Co.) 9-aminoacridine (Sigma Chemical Co.) 1.0 1.0 75 WP2wvrA Methyl methanesulfonate (Aldrich Chemical Co., Inc.) 1,000 To confirm the sterility of the test article, the highest test article 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. BioReliance Study No. AA44ER.502001.BTL 10 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_________________''__________ MATERIALS AND METHODS Test System The tester strains used were the Salmonella typhimuriwn 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 (see Appendix C). 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 liters 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 the International Conference on Harmonisation 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 (see Appendix C). 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 typhinwrium TA100. The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM B-nicounamide-adenine dinucleotide phosphate, 8 mM MgGb and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 roM phosphate buffer at pH 7.4, was prepared immediately before its use. To BioReliance Study No. AA44ER.502001.BTL 11 Company Sanitized. Does not contain TSCA CBI 6) H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_____________________________ 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), and acetone. The test article 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 Toxichy Test The preliminary toxicity test was used to establish the dose-range over which the test article would be tested. Ten dose levels of the test article 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 5000, 3333,1000,667.333,100,67,33,10 and 6.7 ug per plate. Mutagenicity Test The mutagenicity test (initial and independent repeat tests), was used to evaluate the mutagenic potential of the test article. A minimum of five dose levels of test article (5000,3333, 1000, 333 and 100 ug per plate) along with appropriate vehicle and positive controls were plated with TA98, TA100, TA1535, TA1537, and WP2 uvrA. in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test article, vehicle controls and positive controls were plated in triplicate. Hating and Scoring Procedures The test system was exposed to the test article 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 % 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) Oxoid 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 article, test phase, dose level, tester strain, and activation, as described in detail in BioReliance's Standard Operating Procedures. BioReliance Study No. AA44ER.502001.BTL 12 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 One-half (0.5) milliliter of S9 or Sham mix, 100 uL of tester strain and 50 uL of vehicle or test article 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 article aliquot was replaced by a 50 uL aliquot of appropriate positive control. After the overlay bad 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 article 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 roicrocolony 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 article precipitate. Distinguished by precipitate on the plate that is visible to the naked eye but any precipitate particles detected by the automated 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 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.) 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. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. BioReliance Study No. AA44ER.502001.BTL 13 Company Sanitized. Does not contain TSCA CBI -', ~\ H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_____________________________ Evaluation of Results For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test article 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 article. 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 yvrA 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 mutagenidty 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 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; WP2wrA, 10-60. To ensure that appropriate numbers of bacteria are plated, tester strain culture liters 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, theprotocoLand all reports will be maintained according to Standard Operating Procedure|----lf)y the BioReliance RAQA unit headquartered at BioReliance, 14920 Broschart RoaaJKocRmSM' D 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 article will be disposed of following finalization of the report. Deviations No known deviations from the protocol or assay-method SOPs occurred during the conduct of this study. BioReliance Study No. AA44ER.502001.BTL 14 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay___________________________ RESULTS AND DISCUSSION Solubility Test Acetone was selected as the solvent of choice based on compatibility with the target cells, and solubility of the test article. The test article was soluble and clear in acetone at a maximum concentration of approximately 500 mg/mL, the highestconcentration tested. Preliminary Toxicity Test The results of the preliminary toxicity test are presented in Tables 1 through 5. These data were generated in Experiment A 1. In the preliminary toxicity test, the maximum dose tested was 5000 ug per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 uL plating aliquot. Dose levels tested were 5000, 3333, 1000, 667, 333, 100, 67, 33,10 and 6.7 ug per plate. Neither precipitate nor 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. 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 Bl and B2. Dose levels tested were 5000, 3333, 1000, 333 and 100 ug per plate. Neither precipitate nor appreciable toxicity was observed. For submission to Japanese regulatory agencies, pertinent additional information is included in Appendix C. In Experiment Bl (Initial Mutagenicity Test), no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. In Experiment B2 (Independent Repeat Test), no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. CONCLUSION All criteria for a valid study were met as described in the protocol. The results of the H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay indicate that, under the conditions of this study, H-24889 did not cause a positive response in the presence and absence ofAroclor-induced rat liver S9. REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test, Mutation Research, 31:347-364. BioReliance Study No. AA44ER.502001.BTL 15 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay____ DuPont-6405 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. Federal Register 62:16026-16030, November 21,1997. Maron, D.M. and B.N. Ames (1983) Revised Methods for the Salmonella Mutagenicity Test, Mutation Research, 113:173-215. OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), 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 ofE. coli: Partial Purification and Some Properties, J. Biol, Chem., 218:97-106. BioReliance Study No. AA44ER.502001.BTL 16 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Preliminary Toxicity Test Table 1 Test article Id- Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot Test article Concentration ug per plate Vehicle H-24889 AA44ER.50 200 l.BTL Al TA98 15 May 20 01 acetone 50 uL With S9 Acttivation Revertants Background per plate Lawn 22 1 Without S9 Activation Revertants per plate Background Lawn 11 1 6.7 10 33 67 100 333 667 1000 3333 5000 12 1 23 1 27 1 24 1 26 1 17 1 24 1 14 1 10 1 17 1 10 1 13 1 12 1 15 1 13 1 12 1 14 1 15 1 13 1 15 1 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. AA44ER.502001.BTL 17 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Preliminary Toxicity Test Table 2 Test article Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot Test article Concentration ug per plate Vehicle H-24889 AA44ER.502001.BTL Al TA100 15 May 2001 acetone 50 uL With S9 Activation Revertants Background per plate Lawn 159 1 Without S9 Activation Revertants Background per plate Lawn 128 1 6.7 10 33 67 100 333 667 1000 3333 5000 151 1 156 1 183 1 134 1 145 1 165 1 170 1 154 1 170 1 176 1 129 1 176 1 145 1 153 1 138 1 164 1 178 1 159 1 157 1 166 1 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extreinely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Ihterering precipitate BioReliance Study No. AA44ER.502001.BTL 18 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Preliminary Toxicity Test Table 3 Test article Id Study Number Experiment Mo. Strain Date Plated Vehicle Plating Aliquot Test article Concentration ug per plate Vehicle H-24889 AA44ER.502001.BTL 'Al TA1535 15 May 2001 acetone 50 uL With S9 Activation Revertants Background per plate Lawn 15 1 Without S9 Activation Revertants Background per plate Lawn 15 1 6.7 18 1 13 1 10 16 1 11 1 33 16 1 8 1 67 12 1 10 1 100 14 1 17 1 333 13 1 13 1 667 11 1 10 1 1000 16 1 21 1 3333 15 1 12 1 ^ 5000 11 1 rWj Background Lawn Code 18 1 l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extreinely reduced; 5=Absen.t; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA44ER.502001.BTL 19 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Preliminary Toxicity Test Table 4 Test article Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot Test article Concentration ug per plate Vehicle H-24889 AA44ER.502001.BTL Al TA1537 15 May 2001 acetone 50 uL With S9 Activation Revertants per plate Background Lawn 7 1 Without S9 Activation Revertants Background per plate Lawn 8 1 6.7 10 33 67 100 333 667 1000 3333 5000 8 1 6 1 7 1 C 5 1 7 1 8 1 6 1 8 1 8 1 5 1 9 1 7 1 7 1 8 1 4 1 7 1 9 1 6 1 8 1 Background Lawn Code l=Mormal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate C=Contaminated BioReliance Study No. AA44ER.502001.BTL 20 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Preliminary Toxicity Test Table 5 Test article Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot Test article Concentration ug per plate Vehicle H-24889 AA44ER.50 200 l.BTL Al WP2 uvrA 15 May 20 01 acetone 50 uL With S9 Activation Revertants Background per plate Lawn 11 1 Without S9 Activation Revertantss Background per plate Lawn 17 1 6.7 10 33 67 100 333 667 1000 3333 5000 8 1 8 1 10 1 11 1 8 1 10 1 13 1 10 1 14 1 13 1 12 1 11 1 15 1 12 1 10 1 12 1 13 1 16 1 23 1 14 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. AA44ER.502001.BTL 21 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 6 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER. 502001. BTL TA98 None acetone 50 uL Experiment No : B1 Cells Seeded : 4.9 X 10 Date Plated : 30 May 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 11 1 15 1 03 17 1 14 3 100 01 9 1 02 10 1 03 9 1 9 1 333 01 10 1 02 11 1 03 12 1 11 1 1000 01 15 1 02 12 1 03 9 1 12 3 3333 01 15 1 02 10 1 03 11 1 12 3 5000 01 11 1 02 8 1 03 8 1 9 2 Positive Control 2-nitrofluorene 1.0 ug per plate 01 125 1 02 128 1 03 108 1 120 11 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. AA44ER.502001.BTL 22 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 7 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER.502001.B TL TA98 Rat liver S9 acetone 50 uL Experiment No : B1 Cells Seeded Date Plated ^ 4.9 X 10" ; 30 May 2001 Concentration ug per plate Vehicle Plat e Number 01 02 Revertants per plate 14 22 Background Code 1 1 Average Revertants Standard Deviation 03 15 1 17 4 100 01 11 1 02 13 1 03 15 1 13 2 333 01 12 1 02 15 1 03 8 1 12 4 1000 01 18 1 02 20 1 03 15 1 18 3 3333 01 17 . 1 02 24 1 03 22 1 21 4 5000 01 22 1 02 13 1 03 17 1 17 5 Positive Control 2-aminoanthracene 1.0 ug per plate 01 1052 1 02 952 1 03 1330 1 1111 196 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. AA44ER.502001.BTL 23 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 8 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER. 502001.BTL TA100 None acetone 50 UL Experiment No : B1 Cells Seeded ; 1.3 X 10" Date Plated : 30 May 2001 Concentration ug per plate Plate Number Revertants per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 03 168 1 186 1 176 1 177 9 100 01 176 1 02 153 1 03 142 1 157 17 333 01 150 1 02 183 1 03 196 1 176 24 1000 01 161 1 02 137 1 03 153 1 150 12 %15 3333 01 160 1 02 161 1 03 202 1 174 24 5000 01 200 1 02 167 1 03 140 1 169 30 Positive Control sodium azide 1.0 ug per plate 01 569 1 02 576 1 03 580 1 575 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. AA44ER.502001.BTL 24 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 9 Test article Id Study Number Strain Liver Microsomes' Vehicle Plating Aliquot H-24889 AA44ER.502001.B TL TA100 Rat liver S9 acetone 50 uL Experi ment No : B1 Cells Seeded ; 1.3 X 108 Date Plated : 30 May 2001 Concentration ug per plate Plat e Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 191 1 179 1 130 1 167 32 100 01 144 1 02 198 1 03 190 1 177 29 333 01 140 1 02 135 1 03 136 1 137 3 1000 01 178 1 02 193 1 03 138 1 170 28 3333 01 112 1 02 113 1 03 108 1 111 3 5000 01 226 1 02 130 1 03 127 1 161 56 Positive Control 2-aminoanthracene 1.0 ug per plate 01 1615 1 02 1680 1 03 1994 1 1763 203 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extrenely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA44ER.502001.BTL 25 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 10 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER.502001.BTL TA1535 None acetone 50 uL Experiment No B1 Cells Seeded : 1.5 X 108 Date Plated 30 May 2001 Concentration ug per plate Plate Revtsrtants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 10 1 12 1 11 1 11 1 100 01 6 1 02 9 1 03 11 1 9 3 333 01 16 1 02 18 1 03 13 1 16 3 1000 01 11 1 02 13 1 03 13 1 12 1 3333 01 12 1 02 14 1 03 17 1 14 3 5000 01 13 1 02 16 1 03 10 1 13 3 Positive Control sodium azide 1.0 ug per plate 01 160 1 02 140 1 03 211 1 170 37 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. AA44ER.502001.BTL 26 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 11 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER.-502001.B1PL TA1535 Rat livt31 S9 acetone 50 uL Experiment No ; B1 Cells Seeded Date Plated : 1.5 X 10" : 30 May 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 11 1 9 1 03 8 1 9 2 100 01 10 1 02 13 1 03 8 1 10 3 333 01 11 1 02 8 1 03 9 1 9 2 1000 01 13 1 02 6 1 03 12 1 10 4 3333 01 12 1 02 14 1 03 15 1 14 2 5000 01 16 1 02 10 1 03 7 1 11 5 Positive Control 2-aminoanthracene 1.0 ug per plate 01 131 1 02 115 1 03 89 1 112 21 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. AA44ER.502001.BTL 27 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 12 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER. 502001.BTL TA1537 None acetone 50 uL Experiment No : B1 Cells Seeded Date Plated 4 0.9 X 108 ; 30 May 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 8 1 8 1 03 12 1 9 2 100 01 3 1 02 4 1 03 6 1 4 2 333 01 6 1 02 4 1 03 7 1 6 2 1000 01 6 1 02 5 1 03 9 1 7 2 3333 01 4 .1 02 3 1 03 3 1 3 1 5000 01 6 1 02 5 1 03 7 1 6 1 Positive Control 9-aminoacridine 75 ug per plate 01 819 1 02 1102 1 03 860 1 927 153 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. AA44ER.502001.BTL 28 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 13 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 A A 4 4 E R . .5 0 2 0 0 1 . B TL TA1537 Rat liveer S9 acetone 50 uL Experiment No ; B1 Cells Seeded ; 0.9 X 108 Date Plated ; 30 May 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 6 1 4 1 03 8 1 6 2 100 333 01 9 1 02 4 1 03 6 ,1 01 11 1 02 6 1 03 7 1 6 3 8 3 1000 01 15 1 02 10 1 03 7 1. 11 4 3333 5000 01 5 1 02 13 1 03 6 1 01 7 1 02 9 1 03 9 1 8 4 8 1 Positive Control 2-aminoanthracene 1.0 ug per plate 01 126 1 02 248 1 03 192 1 189 61 Background Lawn Code l=Monnal; 2=Slightly reduced; 3=Moderately reduced 4=Extreinely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance D Study No. AA44ER.502001.BTL 29 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 14 Test article Id Study Number Strain Liver Microsomes' Vehicle Plating Aliquot H-2 4889 AA4 4ER.502001.BTL WP2 uvrA None acetone 50 uL Experiment No : B1 Cells Seeded : 3.4 X 108 Date Plated : 30 May 2001 Concentration ug per plate Plate Number Revertants per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 13 1 13 1 03 11 1 12 1 100 01 10 1 02 11 1 03 14 1 12 2 333 01 9 1 02 13 1 03 11 1 11 2 1000 01 8 1 02 10 1 fo 03 12 1 10 2 !"%/ 3333 01 15 1 02 12 1 03 11 1 13 2 5000 01 14 1 02 11 1 03 16 1 14 3 Positive Control methyl methanesulfonate 1000 ug per plate 01 62 1 02 67 1 _________________03________59______1____________63_______ 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. AA44ER.502001.BTL 30 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 15 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-2 4889 AA4 4ER.502001.1BTL WP2 uvrA Rat liver S9 acetone 50 UL Experiment No ; B1 Cells Seeded Date Plated : 3.4 X 108 : 30 May 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 13 1 12 1 03 14 1 13 1 100 01 12 1 02 11 1 03 12 1 12 1 333 01 11 1 02 15 1 03 8 1 11 4 1000 01 14 1 02 8 1 03 9 1 10 3 3333 ' 01 12 1 . 02 16 1 03 10 1 13 3 5000 01 7 1 02 13 1 03 8 1 9 3 Positive Control 2-aminoanthracene 10 ug per plate 01 163 1 02 176 1 03 154 1 164 11 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extren>ely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA44ER.502001.BTL 31 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 16 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER. 502001.BTL TA98 None acetone 50 uL Experiment No : B2 Cells Seeded ; 15.1 X 108 Date Plated : 5 Jun 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 10 1 15 1 16 1 14 3 100 01 4 1 02 8 1 03 5 1 6 2 333 01 11 1 02 11 1 03 10 1 11 1 1000 01 10 1 02 7 1 03 6 1 8 2 3333 01 6 1 02 18 1 03 5 1 10 7 5000 01 7 1 02 4 1 03 3 1 5 2 Positive Control 2-nitrofluorene 1.0 ug per plate 01 240 1 02 114 1 03 309 1 221 99 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. AA44ER.502001.BTL 32 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 17 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER.!502001.B TL TA98 Rat liver S9 acetone 50 uL Experiment No : B2 Cells Seeded ; 15.1 X 10" Date Plated ; 5 Jun 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 12 1 12 1 03 15 1 13 2 100 01 10 1 02 10 1 03 11 1 10 1 333 01 6 1 02 10 1 03 18 1 11 6 1000 01 7 1 02 18 1 03 12 1 12 6 3333 01 9 1 02 13 1 03 17 1 13 4 5000 01 8 1 02 8 1 03 12 1 9 2 Positive Control 2-aminoanthracene 1.0 ug per plate 01 1380 1 02 960 1 03 790 1 1043 304 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. AA44ER.502001.BTL 33 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 18 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER. 502001.BTL TA100 None acetone 50 uL Experiment No ; B2 Cells Seeded ; 13 .6 X 108 Date Plated ; 5 Jun 2001 Concentration Ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 126 1 150 1 168 1 148 21 100 01 168 1 02 149 1 03 141 1 153 14 333 01 166 1 02 167 1 03 143 1 159 14 1000 01 147 1 02 170 1 03 170 1 162 13 3333 01 152 1 02 197 1 03 144 1 164 29 5000 01 162 1 02 140 1 03 134 1 145 15 Positive Control sodium azide 1.0 ug per plate 01 710 1 02 609 1 03 690 1 670 53 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. AA44ER.502001.BTL 34 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 19 Test article Id Study Number Strain Liver Microscsines' Vehicle Plating Aliqi.lot H-24889 AA44ER. 502001. BTL TA100 Rat liver S9 acetone 50 uL Experiment No B2 Cells Seeded : 13.6 X 10" Date Plated 5 Jun 2001 Concentration ug per plate Plat e Number Revertants per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 165 1 197 1 03 144 1 169 27 100 01 152 1 02 140 1 03 168 1 153 14 333 01 133 1 02 156 1 03 149 1 146 12 1000 01 156 1 02 165 1 03 209 1 177 28 3333 01 149 1 02 159 1 03 165 1 158 8 5000 01 204 1 02 196 1 03 155 1 185 26 Positive Control 2-aminoanthracene 1.0 ug per plate 01 1526 1 02 1628 1 03 1204 1 1453 221 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extreinely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interering precipitate; IP=Interfering precipitate BioReliance D Study No. AA44ER.502001.BTL 35 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 20 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER. 502001. BTL TA1535 None acetone 50 uL Experj ment No : B2 Cells Seeded Date Plated : 3.8 X 10" : 5 Jim 2001 Concentration pg per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 6 1 20 1 03 20 1 . 15 8 100 01 10 1 02 11 1 03 10 1 10 1 333 01 10 1 02 12 1 03 8 1 10 2 1000 01 11 1 02 12 1 03 10 1 11 1 3333 01 13 1 02 17 1 03 12 1 14 3 5000 01 6 1 02 13 1 03 15 1 11 5 Positive Control sodium azide 1.0 ug per plate 01 466 1 02 250 1 03 410 1 375 112 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. AA44ER.502001.BTL 36 Company Sanitized. Does not contain TSCA CB1 % H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 21 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER. 502001.BTL TA1535 Rat liver S9 acetone 50 uL Experiment No Cells Seeded Date Plated : B2 ; . 3.8 X 108 : 5 Jun 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 13 1 7 1 11 1 . 10 3 100 333 1000 3333 01 7 1 02 7 1 03 5 1 01 11 1 02 16 1 03 4 1 01 7 1 02 4 1 03 10 1 01 8 1 02 4 1 03 11 1 6 1 10 6 7 3 8 4 5000 01 16 1 02 8 1 03 3 1 9 7 Positive Control 2-aminoanthracene 1.0 ug per plate 01 274 1 02 250 1 03 120 1 215 83 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. AA44ER.502001.BTL 37 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 22 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER. 502001.BTL TA1537 None acetone 50 pL Experiment No ^ B2 Cells Seeded ; 2.9 X 108 Date Plated ; 5 Jun 2001 Concentration lig per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 3 1 5 1 03 2 1 3 2 100 01 7 1 02 6 1 03 3 1 5 2 333 01 6 1 02 6 1 03 7 1 6 1 1000 01 2 1 02 4 1 03 6 1 4 2 3333 01 5 1 02 4 1 03 8 1 6 2 5000 01 8 1 02 1 1 03 5 1 5 4 Positive Control 9-aminoacridine 75 ug per plate 01 1040 1 02 1252 1 03 1199 1 1164 110 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 D Study No. AA44ER.502001.BTL 38 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 23 Test article Id Study Number Strain Liver Microscsmes Vehicle Plating Aliquot H-24889 AA44ER.502001.BTL TA1537 Rat liver S9 acetone 50 uL Experiment No : B2 Cells Seeded : 2.9 X 10s Date Plated : 5 Jun 2001 Concentration ug per plate Plat e Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 100 01 02 03 333 01 02 03 1000 01 02 6 1 10 1 10 1 10 1 2 1 5 1 7 1 8 1 2 1 10 1 3 1 92 64 63 03 4 1 6 4 3333 01 1 " 1 02 3 1 03 7 1 5000 01 6 1 02 5 1 03 4 1 Positive Control 2-aminoanthracene 1.0 ug per plate 01 190 1 43 51 02 161 1 03 351 1 234 102 Background Lawn Code l=Norrnal; 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. AA44ER.502001.BTL 39 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 24 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-24889 AA44ER. 502001.BTL WP2 uvrA None acetone 50 uL Experiment No : B2 Cells Seeded : 12.2 X 10" Date Plated : 5 Jun 2001 Concentration ug per plate Plate Number Revertants per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 03 14 1 16 1 14 1 15 1 100 01 11 1 02 11 1 03 10 1 11 1 333 01 14 1 02 12 1 03 16 1 14 2 1000 01 17 1 02 15 1 03 13 1 15 2 3333 01 10 1 02 4 1 03 9 1 8 3 5000 01 12 1 02 12 1 03 13 1 12 1 Positive Control methyl methanesulfonate 1000 ug per plate 01 69 1 02 100 1 ________________03_______100______1____________90_______18_ 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. AA44ER.502001.BTL 40 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Table 25 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-2 4889 AA4 4ER.502001.BTL WP2 uvrA Rat liver S9 acetone 50 uL Experiment No : B2 Cells Seeded : 12.2 X 10" Date Plated : 5 Jim 2001 Concentration ug per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 16 1 12 1 03 24 1 17 6 100 01 9 1 02 13 1 03 10 1 11 2 333 01 9 1 02 7 1 03 14 1 10 4 1000 01 10 1 02 12 1 03 8 1 10 2 3333 01 10 1 02 10 1 03 9 1 10 1 5000 01 9 1 02 7 1 03 6 1 7 2 Positive Control 2-aminoanthracene 10 ug per plate 01 729 1 02 585 1 03 490 1 601 120 Background Lawn Code l=Nonnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Mon-Interfering precipitate; IP=Interering precipitate BioReliance Study No. AA44ER.502001.BTL 41 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Summary of Results Table 26 Test article Id Study Number H-24889 AA44ER.502001.BTL Experiment No : Bl Aver-age R(sver\cants P<sr Pi ate :t !tandlard 1^e^n-ati.on Liver Microsomes: Nonf2 Dose (ug/plate) TAS?8 T1M<30 Vehicle 100 333 1000 3333 5000 14 9 11 12 12 9 t 3 177 9 1 157 17 1 176 24 3 150 12 3 174 24 2 169 30 Positive 120 11 575 t 6 Liver Microsoroes: Rat liv<ar S9 TJM5 35 11 1 9 3 16 3 12 1 14 t 3 13 A 3 170 t 37 T1MSi37 9 2 4 2 6 2 7 2 3 1 6 1 927 153 WP2 UVrA ' 12 1 12 2 11 2 10 2 13 t 2 14 t 3 63 4 Dose dig/plate) TA98 TA100 Vehicle 100 333 1000 3333 5000 Positive 17 4 167 32 13 2 177 29 12 4 137 3 18 3 170 28 21 4 111 t 3 17 5 161 56 1111 t 196 1763 203 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 50 p.L TA1535 9 2 10 t 3 9 2 10 4 14 t 2 11 5 112 21 TA1537 6 2 6 3 8 3 11 4 8 * 4 8 1 189 61 WP2 13 12 11 10 13 9 164 uvrA 1 1 4 3 t 3 3 11 BioReliance Study No. AA44ER.502001.BTL 42 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Bacterial Mutation Test Summary of Results Table 27 Test article Id Study Number H -2488 9 AJ^44EB..502<30:L.BTL 1Sxpe;r intentt 1lo : B2 Aver'age RIavert ants P(ar PI ate t ;3tan<Sard I3e^/iati.on Liver Microsomes: Ntonia Dose (ug/plate) TA!98 TJM(30 Vehicle 100 333 1000 3333 5000 Positive 14 3 148 21 6 2 153 14 11 1 159 14 8 2 162 13 10 7 164 29 5 2 145 15 221 99 670 53 Liver Microsomes: Rat liver S9 Dose (ug/plate) TA98 TA100 Vehicle 100 333 1000 3333 5000 Positive 13 2 169 27 10 1 153 14 11 6 146 t 12 12 6 177 28 13 4 158 8 9 2 185 26 1043 304 1453 221 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 50 uL TAli535 15 8 10 1 10 2 11 1 14 t 3 11 5 375 112 TJM!537 3 t 2 5 2 6 1 4 2 6 2 5 4 1164 110 TA1535 10 3 6 1 10 6 7 3 8 4 9 7 215 83 TA1537 9 6 6 6 t 4 5 234 2 4 3 4 3 1 102 WP2 15 11 14 15 8 12 90 U1ITA 1 1 2 2 3 1 18 WP2 17 11 10 10 10 7 601 uvrA 6 2 4 2 1 2 120 BioReliance Study No. AA44ER.502001.BTL 43 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_______________________________ APPENDIX A Historical Control Data BioReliance Study No. AA44ER.502001.BTL 44 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Historical Negative and Positive Control Values 1998-2000 revertants per plate Activation Strain Control Mean None SD Mm Max Mean Rat Liver SD Min Max TA98 Neg Pos 16 7 4 59 425 206 21 1536 21 7 592 322 7 58 56 2454 TA100 Neg Pos 128 31 53 288 568 159 129 1371 138 34 74 258 736 301 198 2871 TA1535 Neg Pos 12 5 378 164 1 45 6 978 12 4 1 42 104 84 18 1640 TA1537 WP2uvrA Neg Pos Neg Pos 6 3 708 409 14 5 190 138 0 30 13 2786 4 48 34 961 7 3 88 106 16 6 317 299 1 29 12 2060 4 115 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. AA44ER.502001.BTL 45 ^ \ \.) Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_______________________________ APPENDIX B Study Protocol BioReliance Study No. AA44ER.502001.BTL 46 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 ^reived by RA/OAAJte^1 Sponsor Project Number, DuPont-6405 BioReliance Study Number AA44ER.502001.BTL H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay 1.0 PURPOSE ofimUR(H- The purpose of this study is to evaluate the mutagenic potential 24X89) by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimwium and at the tryptophan locus of Escherichia coll WP2 ywA in the presence and absence of S9 activation. 2.0 SPONSOR 2.1 Name: 13. Address: 2.3 Representative: 2.4 Sponsor Project No.: E.I. du Font de Nemours and Company Sdne Haskell Research Center DuPont Haskell Laboratory P.O. Box 50 1090 Elkton Road Newark, DE 19714-0050 Maria Dormer, Ph.D. Phone: 302-366-5251 Fax: 302-366-5207 Email: maria.donnei^usa.dupontcom DuPont-6405 2.5 WR#: 2.6 Haskell #: H-24889 2.7 Service Code: 3.0 IDENTIFICATION OF TEST AND CONTROL ARTICLES JHBBHBA 3.1 Test Article Name: fc1--------^^ ^ - l The test article is room temperature and must be heated to 60C each ^ f l and every time. Wbenau material is melted and homogeneously mixed to assure uniformity, an aliquot needs to be taken out for dosing. The mixing should occur around 60C. 32 Test Article ID.: H-24889 (to be used in the report tide and text) 3.3 Controls: Negative: Positive: Test article vehicle 9-aminoacridine Protocol SPGT502001 Ol-May-2001 BioReliance Study No. AA44ER.502001.BTL Page 1 of 11 47 ^ BIORELIANCE' Company Sanitized. Does not contain TSCA C81 H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_______________________________ Sponsor Project Number BioReliance Study Number DuPont-6405 AA44ER.502001.BTL 2-anunoanthracene methyl methanesulfonate 2-nitrofluoreae 3.4 Test Article Characterization Unless alternate arrangements are made, die testing facility at BioReliance will not perform analysis of the dosing solutions. The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition of the test article, and the stability and strength of the test article in the solvent (or vehicle). 3.5 Test Article Retention Sample The retention of a reserve sample of the test article 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 0. Wagner ffl, M.S. Phone: 301-610-2152 Fax: 301-738-2362 Email: swagner@bioreliance.com 5.0 PROPOSED STUDY DATES 5.1 Experimental Start Date: 09-May-2001 5.2 Experimental Termination Date: 28-Jun-2001 5.3 Draft Report Date: 12-Jul-2001 5.4 Final Report Date: 2 weeks after Sponsor approves draft 6.0 TEST SYSTEM The tester strains will include the S. lyphimwium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et at. (1975) and the E. coli tester strain WP2 uvrA. as described by Green and Muriel (1976). Protocol SPGT502001 Ol-May-2001 Page 2 or 11 ^ BlORELIANCE- BioReliance Study No. AA44ER.502001.BTL 48 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Sponsor Project Number BioReliance StudyNumber DuPont-6405 AA44ER.502001.BTL Histidine Mutation hisG46 TA1535 TA100 teC3076 TA1537 - toD3052 - TA98 - - - Tryptopha n Mutation 7>7E - - WP2awA Additional Mutations LPS rfa rfa - Repair AWWB AMW-B AMWA R-fiictor - +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 uwB 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 TA1537 are reverted from histidine dependence (auxotrophy) to histfdine independence (prototrophy) by framestuft mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions. The E. coli tester strain has an AT base pair at the critical mutation site within the frpE gene (Wilcox et al.. 1990). Tester strain WP2 wrA has a deletion in the uwA 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 rainying the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base-pair substitution mutations (Green and Muriel, 1976). The S. typhimwium 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 article 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 uvrA. with and without S9 activation- All dose levels of test article, negative controls and positive controls will be plated in triplicate. Protocol SPGT502001 Ol-May-2001 Page 3 of 11 BioReliance Study No. AA44ER. 502001 .BTL 49 ^HBIORELIANCE- Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_____________________________ Sponsor Project Number BioReliance Study Number DuPont-6405 AA44ERJ02001.BTL 7.1 Solubility Determination Unless the Sponsor has indicated the test article vehicle, a solubility determination will be conducted to determine me maximum soluble concentration or workable suspension up to a maximum of 50 mg/mL for aqueous vehicles and 500 mg/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 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 Toxicity Assay to Select Dose Levels Selection of dose levels for the mutagenicity assay will be based upon the toxicity and precipitation profile of the test article assessed in a preliminary toxicity assay. This preliminaiy assay will be conducted by exposing TA98, TA100, TA1535, TA1537 and WP2 uvrA to negative controls and to at least eight concentrations of test article, one plate per dose level, in both the presence and absence of S9 activation. Unless indicated otherwise by the Sponsor, the 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 of toxicity without exceeding 5 mg/plate. For freely soluble, nontoxic test articles, the highest dose level will be 5 mg/plate. For precipitating, nontoxic test articles, die highest dose level will be selected in an attempt to yield precipitate at only me 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 maximum achievable test article 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 article 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 first assay have been evaluated, the mutagenicity assay will be repeated. The dose levels used in the second assay will be the same as those used in me first assay unless the Study Director determines mat the dose levels should be changed due to an equivocal response, excessive cytotoxicity or excessive precipitate. If the Sponsor is aware of specific metabolic requirements Protocol SPGT502001 Ol-May-2001 Page 4 of 11 6 BlORELIANCE- BioReliance Study No. AA44ER. 50200 l.BTL 50 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Sponsor ProjectNumber: BioReliancc Study Number DuPont-6405 AA44ER.502001 BTL (e.g., azo compounds), tfais infonnation will be utilized in dgrijpiing the assay. (e.g., activation system or treatment method). Tins guidance is based on the OBCD Guideline 471 (adoptedJuly 1997 and published February 1998)and ICH Guidance on Specific Aspects of Regulatory Gcnotoxicity Tests for Pharmaoeudcals (1997). 7.4 Controls 7.4.1 Positive Controls All combinations of positive controls and tester strains plated concurrently with me assay are listed below: Strain S9 Activation Positive Control Concentration (pg/plate) Salmonella Strains WP2uwA TA98 TA100, TA1535 TA1537 WP2uwA Rat None 2-aminoanthracene 2-nitrofluorcnc sodium azide 9-aminoacridine 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 article dilution and the 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-Dawiey rets induced with a single intraperitoneal injection of Aroclor 1254, 500 nig/kg, five days prior to sacrifice. The S9 will be batch prepared and stored frozen at approximately -70C until used. Each batch of S9 homogenate will be assayed for its ability to Protocol SPGTS02001 OI-Mjy-2001 Page 5 of 11 ^ BlORELIANCE' ") BioReliance Study No. AA44ER.502001 .BTL 51 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_______________________________ Sponsor Project Number BioReliance Study Number DuPont-6405 AA44ER.502001.BTL metabolize 2-aminoantmacene and 7,12-dimethylben2anthraccne to fonns mutagenic to S. typhimvrhm TA100. Immediately prior to use, the S9 will be thawed and mixed with a cofactor pool to contain 10% S9 homogenate, 5mM gtucose-6-phosphate, 4mM p-nicotinamide-adeninedinucleodde phosphate, 8 mM MgCl, and 33 mM KC1 in a 100 mM phosphatebuffer at pH 7.4. This mixture 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, the length of incubation will be controlled and monitored. At me 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 10' cells/mL. 7.7 Test System Identification Each plate will be labeled with a code system that identifies the test article, test phase, dose level, tester strain and activation type as described in BioReliance's Standard Operating Procedures. 7.8 Test Article Preparation Unless specified otherwise, test article dilutions will be prepared immediately prior to use. All test article dosing will be at room temperature under yellow light. 7.9 Treatment of Test System One half milliliter (0.5 mL) of S9 mix or Sham mix, 100 nL of tester strain and 50 ^L of vehicle, test article 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 nL of test article/vehicle/positive control will be plated. The mixture will be vortex mixed and overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay has solidified, the plates will be inverted and incubated for approximately Protocol SPGT502001 Ol-Mty-2001 Page 6 of 11 BioReliance Study No. AA44ER.502001.BTL 52 ^ BIORELIANCE' Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_______________________________ Sponsor Project Number BioReliancc Study Number DuPont-6405 AA44ER.502001.BTL 7.10 48 to 72 hours at 372<>C. Plates that are not counted immediately following die incubation period will be stored at 2-8C. Scoring The condition of the bacterial backgroundlawn will be evaluated for evidence of test article toricity and precipitate. Evidence oftoricity will be scored relative to the negative control plate and recorded along with the rcvertant count for that plate. Toricity will be evaluated as a decrease in the number of revertant colonies per plate and/or a thinning or disappearance of me 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. typhimurivm tester strain cultures must exhibit sensitivity to crystal violet To demonstrate the presence of the uwB mutation, all S. typhimuriwn tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate the presence of the wrA mutation, all coli tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate the presence of the pKMIOl plasmid R-fector, tester strain cultures ofTA98 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 the 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; WP2 <wA, 10 - 60. 8.3 Tester Strain Titers To ensure that appropriatenumbers of bacteria are plated, all tester strain culture tilers must be equal to or greater than 0.3x10' cells per milliliter. Protocol SPGT502001 Ol-May-2001 Page 7 of 11 BioReliance Study No. AA44ER.502001.BTL 53 ^ BlORELIANCE- Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay________________________________ Sponsor ProjectNumber BioReliance Study Number DuPont-6405 AA44ER.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 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 ofrevertants per plate relative to me mean negative control value (this reduction must be accompanied by an abruptdose-dependentdrop in me 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 appropriate change in dose levels. 9.0 EVALUATION OF TEST RESULTS 10.0 For a test article 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 article as specified below: 9.1 Strains TA1535 and TA1537 Data sets will be judged positive if me 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 WP2ywA Data sets will be judged positive if the increase in mean revertants at the peak of me dose response is equal to or greater man 2.0-times the mean negative control value (vehicle). 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 Article: 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 article, if known. Solvent/Vehicle: justification for choice of vehicle; solubility and stability of test article in solvent/vehicle, if known. Strains: strains used; number of cells/mL per culture; strain characteristics. Protocol SPGT502001 Ol-May-2001 Page 8 of 11 ^||BlORELIANCE- BioReliance Study No. AA44ER.502001.BTL 54 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay_____________________________ Sponsor Project Number. BioReliance Study Number DuPont-6405 AA44EEL502001BTL Test conditions: amount of test article per plate with rationale fin- 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; 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 protocol and ^11 reports will be maintained according to Standard Operating ProcedurelH^----Hbythe BioReliance RAQA unit headquartered at: BioReliance, 14920 BroscnarffloadR, ockville, 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 me 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. 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 Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February 1998 and with the International Conference on Harmonisadon of Technical Requirements for Registration ofPhannaceuticals 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 me 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. Tne in-process inspection will be performed to audit me 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 dates 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-tie included in die final report. This statement will cite the GLP guidelines) with which the study is compliant and any exceptions to this compliance, if applicable, including the omission of characterization i or stability analyses of the test or control articles or their mixtures. Protocol SPCT502001 Ol-May-2001 Page 9 of II BioReliance Study No. AA44ER. 50200 l.BTL 55 1^9. _ _ BlORELIANCE- Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay______________________________ SponsorProject Number BioReliance Study Number. DuPont-6405 AA44EIL502001BTL Unless arrangements are made to the contrary, unused dosing solutions will be disposed of following administration to die test system and all residual test article will be disposed of following finalization of the report. 13.0 REFERENCES Ames. B.N., McCann, J. and Yamasald, E. (1975). Methods for detecting carcinogens and mutagens with the &ztoione//a/inammalian-microsome mutagenicity test. Mutation Research 31:347-364. Green, M.H.L., and Muriel, WJ. (1976). Mutagen testing using up* reversion in Escherichia coll. Mutation Research 38:3-32. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuricals 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 Phannaceudcals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Phannaceudcals. 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 Sa/OTow//a/microsome test: assay of 300 chemicals: discussion. Proc. Natl. Acad. Sci. USA 73:950-954. McCann, J., Choi, E., Yamasald, E. and Ames, B.N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome 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 typhimurium TA102 with Escherichia coli WP2 tester strains. Mutagenesis 5:285-291. Protocol SPCT502001 Ol-Miy-2001 Page 10 of 11 BioReliance Study No. AA44ER. 50200 l.BTL 56 ^ BlORELIANCE- Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Sponsor ProjectNumber BioReliance Study Number: DuPont-6405 AA44ER.502001.BTL 14.0 APPROVAL V<g^_ "^Tgy^ Sponsor Representative 03 >^/*cM 2^001 Date H-&-<- ^ o- 0 & r ^e<" (Print or Type Name) ^j^st^ g>.(jy^:gr BioReliance Study'Director t ^ L x ^ L v BioRdiance Study Management tfift^ aoc) Date II A^^ aoc'/ --ase--t- ProtocQlSPGTSOMOl Ol-Mjiy-2001 Page 11 of 11 BioReliance Study No. AA44ER.502001.BTL 57 ^ BlORELIANCF Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay____________________________ APPENDIX C Information for Japanese Regulatory Agencies BioReliance Study No. AA44ER.502001.BTL 58 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Report of Results of Reverse-Mutation Assay in Bacteria 1. Tester Strains (1) Procurement Strain TA98 TA100 TA1535 TA1537 TA1538 TA97 TA102 WP2 MvrA WP2nvrA (pkMIOl) WP2 (pKMIOl) Obtained from Date obtained 10 November 1998 Date inspected the strain lot in storage 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 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. ofS9 Made in-house Prepared on Storage temperature -70C or colder 05 February 2001 (Batch R640) 28 March 2001 (Batch R641) Name and model of storage apparatus So-Low, Model PR27-120 BioReliance Study No. AA44ER.502001.BTL 59 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 (2) Preparation of S9 Animal used Species, Strain . Rattus norvegicus, Sprague Dawley Sex Age (in weeks) Weight Male 9(BatchR640) 8(BatchR641) 264 to 293 g (Batch R640) 181 to 232 g (Batch R641) Inducing substance Name Aroclor 1254 Administration method Intraperitoneal Administration period and amount (g/kg-weight) 5 days, 0.5 gin/kg body weight 3. Positive Control Substance (1) Positive control Name Manufacturer 9-Aminoacridine (9AAD) 2-Aminoanthracene (2AA) Methyl methanesulfonate (MMS) 2-Nitrofluorene (2NF) Sodium azide (SA) Sterigmatocystin (STM) Sigma Chemical Company Sigma Chemical Company Aldrich Chemical Co., Inc. Aldrich Chemical Co., Inc. Sigma Chemical Company Sigma Chemical Company Lot No. 106F06681 085H2508 09419LR 11202TF 098H0169 118H4061 Grade Practical Practical Purity (%) >98% >95% >99% >98% >99% >99% Solvent used DMSO DMSO DMSO DMSO water DMSO (2) Preparation and storage of positive control solution Prepare or Store Store subdivided solutions (Storage temp. -5 to -30C) BioReliance Study No. AA44ER.502001.BTL 60 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 4. Preparation of Test article Solution Solvent used Name Acetone Manufacturer Fisher Scientific Lot No. 001064 Grade Purity (%) Certified ACS Stability of test article in the solvent Unknown Reason to choose the solvent Solubility determination Method of suspension when test article is difficult to dissolve Not applicable Storage time and temp. from preparation to use for test <30 minutes at ambient temperature Conversion by purity None BioReliance Study No. AA44ER.502001.BTL 61 Company Sanitized. Does not contain TSCA CBI ) H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 5. 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 CH.-B=228448 2 to 5 hours at ambient temperature <8 hours at 2-8C New Bnmswick Scientific, model G-24 Rotary (125 rev/min.) shape: cylinder, 200 mL 50 mL 1 colony 6. Agar Plate Medium (l)Topagar Agar Name Manufacturer Lot No. (2) Minimum Glucose Agar Name Made in-house Agar Manufacturer Lot No. Volume of agar plate medium BBL Select Becton Dickinson 1000J3DKSQ BBL Select Becton Dickinson 1000J3DKSQ 25 mL BioReliance Study No. AA44ER.502001.BTL 62 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay____ 7. Test Results - Judgement of the results DuPont-6405 Judgement Negative Reason for judgement and referential matters: No positive response was observed with any of the tester strains in the presence and 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 of detecting a mutagen. BioReliance Study No. AA44ER.502001.BTL 63 Company Sanitized. Does not contain TSCA CBI