Document LgjgZQk20GppKvyGwpJ2G9rab

AR226-3180 DuPont-6405 TRADE SECRET Testing Guidelines International Conference on Harmonisation (ICH) of Technical Requirements for Registration o f Pharmaceuticals for Human Use. Guidance on Specific Aspects o f 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 o f Chemicals Section 4: Health Effects, No. 471 (Adopted 1997) Study Title H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay Authors Valentine O. Wagner, DI, M.S. Trinh T. H. Nguyen, B.S. July 20,2001 Performing Laboratory BioReliance 9630 Medical Center Drive Rockville, MD 20850 for E. L du Pont 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 D u P ont-6405 Haskell Number H -24889 Service Code Page 1 o f 63 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 STATEMENT OF COMPLIANCE Study No. AA44ER.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 control articles have not been determined by the testing facility. 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. Study Director BioReliance Study Management Date Date B io R elian ce Study No. AA44ER.502001.BTL 2 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 Quality Assurance Statement Study Tide: H 24889 BACTERIAL REVERSE MUTATION ASSAY WITH AN INDEPENDENT REPEAT ASSAY Study Number: AA44ER.502001.BTL Study Director: Valentine O. Wagner, HI, M.S. This study has been divided into a series of in-process phases. Using a random sampling approach, Quality Assurance monitors each ofthese phases over a series of studies. Procedures, documentation, equipment records, etc., are examined in order to assure that die study is performed in accordance with the U.S. FDA Good Laboratory Practice Regulations (21 CFR 58), die U.S. EPA GLPs (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 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 of QA inspections of this study. Inspect On Phase Inspect On Phase Inspect On Phase Inspect On Phase 14-May-01 - 14-May-01 To Study Dir 14-May-01 To Mgmt 14-May-01 Protocol Review 30-May-01 - 30-May-01 To Study Dir 30-May-01 To Mgmt 25-Jun-01 Test and/or control material administration 1l-Jul-01 - 1l-Jul-01 To Study Dir 1l-Jul-01 To Mgmt 13-Jul-01 Draft Report 24-Jul-01 - 24-Jul-01 To Study Dir 24-Jul-01 To Mgmt 24-Jul-01 Draft to Final Report This report describes the methods and procedures used in the study and the reported results accurately reflect the raw data o f the study. QUALITY ASSURANCE BioReliance Study No. AA44ER.502001.BTL ?</ J lUu 2ool DATE 1 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 o f data obtained from this study. Issued by Study Director: Valentine 0 . Wagner ID, M.S. Z c rfc llC O l Date Approved by Study Monitor: TL) --------- U V L - Z - o o t Maria Donner, Ph.D. Senior Research Scientist Date BioReliance Study No. AA44ER.502001.BTL 4 Company Sanitized. Does not contain TSCA CBI % H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 TABLE OF CONTENTS Certification................... ................................ --........................................................... Study Inform ation....................................................................................................... Sum m ary..................................................................................................................... Purpose......................................................................................................................... Characterization of Test and Control Articles........................................................... Materials and M ethods................................................................................................ Results and Discussion................................................................................................ Conclusion.......................................... ......................................................................... References.................................................................................................... ............... Data Tables................................................................................................................... Table 1: Preliminary Toxicity Test in Salmonella typhimurium T A 98............. Table 2: Preliminary Toxicity Test in Salmonella typhimurium TA 100.......... Table 3: Preliminary Toxicity Test in Salmonella typhimurium TA 1535........ Table 4: Preliminary Toxicity Test in Salmonella typhimurium TA 1537........ Table 5: Preliminary Toxicity Test in Escherichia coli W P2 vrA................... Table 6: Mutagenicity Test in Salmonella typhimurium TA98 without S9 ..... Table 7: Mutagenicity Test in Salmonella typhimurium TA98 with S 9 ......... Table 8: Mutagenicity Test in Salmonella typhimurium TA100 without S 9 ... Table 9: M utagenicity Test in Salmonella typhimurium TA100 with S 9 ...... Table 10: Mutagenicity Test in Salmonella typhimurium TA1535 without S9 Table 11: Mutagenicity Test in Salmonella typhimurium TA1535 with S 9 .... Table 12: M utagenicity Test in Salmonella typhimurium TA1537 without S9 Table 13: M utagenicity Test in Salmonella typhimurium TA1537 with S 9 .... Table 14: Mutagenicity Test in Escherichia coli WP2 uvrA without S9.......... Table 15: Mutagenicity Test in Escherichia coli WP2 uvrA with S 9............... Table 16: Mutagenicity Test in Salmonella typhimurium TA98 without S 9 .... Table 17: Mutagenicity Test in Salmonella typhimurium TA98 with S 9 ........ Table 18: Mutagenicity Test in Salmonella typhimurium TA100 without S 9 .. Table 19: M utagenicity Test in Salmonella typhimurium TA100 with S 9 ...... Table 20: Mutagenicity Test in Salmonella typhimurium TA1535 without S9 Table 21: M utagenicity Test in Salmonella typhimurium TA1535 with S 9 .... Table 22: M utagenicity Test in Salmonella typhimurium TA1537 without S9 Table 23: M utagenicity Test in Salmonella typhimurium TA1537 with S 9 .... Table 24: M utagenicity Test in Escherichia coli WP2 uvrA without S 9......... Table 25: Mutagenicity Test in Escherichia coli WP2 uvrA with S 9.............. BioReliance Study No. AA44ER.502001.BTL 5 Page ..... 4 ..... 7 ..... 9 ....10 ....10 ....11 ....15 .... 15 .... 15 .... 17 .... 17 .... 18 .... 19 .... 20 .... 21 .... 22 .... 23 .... 24 .... 25 .... 26 .... 27 .... 28 .... 29 ..... 30 ..... 31 ..... 32 ..... 33 ..... 34 ..... 35 ..... 36 ..... 37 ..... 38 ..... 39 ......40 ..... 41 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 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 ................... 4 4 ...................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 INFORM ATION Svnonvm s/C odes: DuPont-6405 B io R elian ce Study No. AA44ER.50200l.B T L 7 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 Physical C haracteristics: Stability: The test article appeared to be stable under the conditions of the study; no evidence o f instability was observed. Sponsor: E. I du Pont de Nemours and Company W ilmington, Delaware 19898 U.S.A. study Tnitiateri/Comoleted: May 11,2001 / (see report cover page) Tn-T.ife TnitiatPiH/Cnmpleted: May 15,2001 / June 8, 2001 BioReliance Study No. AA44ER.50200l.BTL Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 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 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 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 highest concentration tested. In the preliminary toxicity test, the maximum dose tested was 5000 |ig per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 pL plating aliquot. Dose levels tested were 5000,3333,1000,667,333,100,67,33,10 and 6.7 pg 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 pg per plate. In the mutagenicity test, no positive response was observed. The dose levels tested were 5000, 3333, 1000, 333 and 100 pg 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 H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-6405 PURPOSE The purpose of this study was to evaluate the mutagenic potential of the test article by measuring its ability to induce reverse mutations at selected loci o f several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA m 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 wa the code number AA44ER. The test article was characterized by the Sponsor as a | ---- t h a t should be stored at less than 27C in a ventilated area with no refrigShtion. An I B g a T o f 05 April 2003 was provided. Upon receipt, the test ' W t t H M t a M i n d was stored at room temperature, protected from light, the or othercharacteristics todefine the test a r tic le ^ b e e n 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 an delivered to the test system at room temperature under yellow light Positive controls piarea concurreimy vvIUI U1WUlWWfevmvfcj - Strain S9 Activation Positive Control - All Salmonella Strains WP2 uvrA Rat 2-aminoanthracene (Sigma Chemical Co.) 2-nitrofluorene TA98 (Aldrich Chemical Co., Inc.) TA100, TA1535 TA1537 None Sodium azide (Sigma Chemical Co.) 9-aminoacridine (Sigma Chemical Co.) WP2 uvrA Methyl methanesulfonate (Aldrich Chemical Co., Inc.) Concentration (pg/plate) 1.0 10 1.0 1.0 75 1,000 To confirm the sterility o f the test article, the highest test article dose level >w*d ta U* 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 with an Independent Repeat Assay____________ ___________ DuPont-6405 MATERIALS AND METHODS Test System The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98 TA100 TA1535, and TA1537 as described by Ames et al. (1975) and Escherichia coll WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were received direcdy from Dr. Bruce Ames, University of California, Berkeley. E. coh tester steams were received from the National Collection of Industrial and M arine Bacteria, Aberdeen, Scotland (see Appendix C). Tester strains TA98 and TA1537 are reverted from histidine dependence (auxoteophy) to histidine independence (piototrophy) by frameshift mutagens. Tester strain TA1535 is averted 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 m utations (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 -5 0 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled an 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 rp at 372C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percenttransmittance 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 file but not presented in this repo . 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 Pharmaceuticals 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 i q r t m i o f Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 batches were prepared and s at -70C or colder until used (see Appendix C). Each bulk preparation of S9 was tested for i ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared immediately before its use and contained 10% S9 5 mM glucose-6-phosphate, 4 mM B-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 an 33 mM K Q in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To BioReliance Study No. AA44ER.502001.BTL Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 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 Toxicity 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 fig per plate. Mutagenicity Test The mutagenicity test (initial and independent repeat tests), was used to evaluate die mutagenic potential of the test article. A minimum of five dose levels of test article (5000,3333, 1000, 333 and 100 fig 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 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 pM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL o f 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 W ater 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 Nutaent Broth N a 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 of Sham mix, 100 pL of tester strain and 50 pL 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 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 article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examinahon 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 NP IP Description Normal Slightly Reduced Characteristics Distinguished by a healthy microcolony lawn. Distinguished by a noticeable thinning of the microcolony lawn and possibly a slight increase in the size o f the microcolonies compared to the vehicle control plate. Distinguished by a marked thinning of the microcolony lawn Moderately resulting in a pronounced increase in the size o f the microcolonies Reduced compared to the vehicle control plate. _ Distinguished by an extreme thinning of the microcolony lawn Extremely Reduced 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 eve as isolated colonies. Absent Distinguished by a complete lack of any microcolony lawn over greater than or equal to 90% of the plate. Obscured by The background bacterial lawn cannot be accurately evaluated due Precipitate to microscopic test article precipitate. Distinguished by precipitate on the plate that is visible to the Non-Interfering nairp/1 eye but any precipitate particles detected by the automated Precipitate colony counter total less than 10% of the revertant colony count fo fT than 1 particles on a plate with 30 revertants.) Distinguished by precipitate on the plate that is visible to me 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.) 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 with an Independent Repeat Assay______ DuPont-6405 Evaluation o f Results For replicate plating, the mean and standard deviation of the number o f 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 o f 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 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value. . Criteria for a Valid Test The following criteria must be met for the mutagenicity test to be considered valid Salmonella tester strain cultures must demonstrate the presence o f 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 m ^ nmmrer of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA10U, 80-240; TA1535, 5 - 45; TA1537, 3 - 21; W P2uvrA, 1 0 -6 0 . To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10? cells/mL. The mean of each positive 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 o f the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count (2) A reduction in the background lawn. Archives All raw data, the protocol and all reports will be maintained according to Standard Operating P r o c e d u r e H M ^ y the BioReliance RAQA unit headquartered a t BioReliance, 14920 Broschart RoI F ro S c 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 w dlfirst 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 with an Independent Repeat Assay______ DuPont-6405 RESULTS AND DISCUSSION Solubility Test Acetone was selected as the solvent of choice based on compatibility with the target cells, and solubility o f the test article. The test article was soluble and clear in acetone at a maximum concentration of approximately 500 mg/mL, the highest concentration tested. Preliminary Toxidty Test The results of the preliminary toxicity test are presented in Tables 1 through 5. These data were generated in Experiment A l. 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 pL plating aliquot. Dose levels tested were 5000, 3333, 1000, 667, 333, 100, 67, 33,10 and 6.7 pg 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 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 5000, 3333, 1000, 333 and 100 pg 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 B1 (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 of Aroclor-induced rat liver S9. REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mananaiian 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 W J . Muriel (1976) Mutagen testing using trp+ reversion in Escherichia coli, 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 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. . 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. 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 IdStudy Number Experiment No. Strain Date Plated Vehicle Plating Aliquot Test article Concentration ug per plate____ Vehicle H-24889 AA44ER.502001.BTL Al TA98 15 May 2001 acetone 50 uL_________ __ With S9 Activation Revertants Background per plate_______Lawn 22 Without S9 Activation Revertants Background per plate______ 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=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipi a NP=Non-Interfering precipitate; IP=Interfering precipi a BioReliance Study No. AA44ER.502001 .BTL 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 H-24889 AA44ER.502001.BTL Al TAI 00 15 May 2001 acetone Tast article Concentration Vehicle With S9 Activation Revertants Background per plate Lawn 159 1 Without S9 Activation Revertants Background per plate Lawn 128 i 67 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 i 176 i 145 i 153 i 138 i 164 1 178 1 159 1 157 1 166 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=Interfermg precipita e 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______ Bacterial Mutation Test Preliminary Toxicity Test rP n K l a DuPont-6405 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 'A l TA1535 15 May 2001 acetone 50 pL____________ With S9 Activation Revertants Background per plate_______Dawn 15 Without S9 Activation Revertants Background per plate______Lawn 15 1 6.7 10 33 67 100 333 667 1000 3333 5000 18 1 16 1 16 1 12 1 14 1 13 1 11 1 16 1 15 1 11 1 13 1 11 1 81 10 1 17 1 13 1 10 1 21 1 12 1 18 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. 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 H-24889 AA44ER.502001.BTL Al TA1537 15 May 2001 acetone 50 uL ------ -- -------- Test article Concentration ug per plate Vehicle Revertants Background per plate Lawn Revertants per plate 8 Background Lawn 6.7 10 33 67 100 333 667 1000 3333 5000 81 61 71 51 71 81 61 81 81 51 91 71 71 81 41 71 91 61 81 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 C=Contaminated BioReliance Study No. AA44ER.502001.BTL 20 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test w u n an niucpcuucin 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.502001.BTL A1 WP2 uvrA 15 May 2001 acetone 50 uL ------------------------- _ ---------------- -- With S9 Activation Revertants Background Without Activation Revertants Background per plate Lawn_______ per plate_______ Lawn 11 17 6.7 10 33 67 100 333 667 1000 3333 5000 81 81 10 1 11 1 81 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=Obscured 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 H-24889 AA44ER.502001.BTL TA98 None Experiment No Cells Seeded Date Plated B1 4.9 X 10B 30 May 2001 Vehicle acetone Plal-ino Aliouot : 50 uL ----- ----------------------- ------------- - Concentration Plate Number Revertants per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 03 11 1 15 1 17 1 14 3 100 01 9 1 02 10 1 03 9 1 91 333 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 92 Positive Control 2--nitrofluorene 1..0 pg per plate 01 125 1 02 128 1 03 108 1 120 il 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=Interfenng 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 H-24889 AA44ER.502001.BTL TA98 Rat liver S9 acetone Experiment No Cells Seeded Date Plated B1 4.9 X 10 30 May 2001 Concentration jiff ppr plate Vehicle Plate Number 01 02 03 Revertants per plate 14 22 15 Background Code 1 1 1 Average Revertants 17 Standard 100 01 11 1 02 13 1 03 15 1 13 333 01 12 1 02 15 1 03 8 1 12 1000 01 18 1 02 20 1 03 15 1 18 3333 01 17 .1 02 24 1 03 22 1 21 5000 01 22 1 02 13 1 03 17 1 17 Positive Control 2 -aminoanthracene 1.0 pg per plate 01 1052 1 02 952 l 03 1330 1 1111 196 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 precipi a e B io R elian ce 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 pL Experiment No : B1 Cells Seeded : 1.3 X 108 Date Plated : 30 May 2001 Concentration Plate Revertants ytg per plate Number per plate Code____ Revertants Deviation Vehicle 01 02 03 168 1 186 1 176 1 177 100 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 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 pg pe 01 569 1 02 576 1 03 580 1 575 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. 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 :H-24889 :AA44ER.502001.BTL :TA100 Experiment.No :B1 Cells Seeded :1.3 X 10 Liver Microsomes-: Rat liver S9 Date Plated :30May 2001 Vehicle : acetone Platina Aliauot : 50 uL Concentration Plate Revertants ua per plate Number per plate _________________________ ____________ Background Average Standard 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 02 113 03 . 108 1 1 1 111 3 5000 01 226 1 02 130 1 03 127 1 161 56 Positive Control 2-aiuinoantliracene 1.0 ug per plate 01 1615 1 02 1680 1 03 1994 1 1763 203 Background Lawn Code l=Norxnal; 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. 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 : H-24889 Study Number : AA44ER.502001.BTL Strain : TA1535 Liver Microsomes : None Experiment No : Bl Cells Seeded : 1.5 X 108 Date Plated : 30 May 2001 Vehicle ; acetone Platina Aliauot : 50 uL Concentration urr ner nlate Plate Revertants 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 93 333 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 pg 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=Obscured 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 H-24889 Study Number ' : AA44ER. 502001.BTL Strain : TA1535 Liver Microsomes ; Rat liver S9 Experiment No Cells Seeded Date Plated Vehicle : acetone Platina Aliauot : 50 pL Concentration Plate Revertants Background Average no ner alate Number per plate Code Revertan Vehicle 01 02 03 11 1 91 81 9 B1 1.5 X 108 30 May 2001 Standard Deviation 100 01 10 1 02 13 1 03 8 1 10 333 01 11 1 02 8 1 03 9 1 9 1000 01 13 1 02 6 1 03 12 1 10 3333 01 12 1 02 14 1 03 15 1 14 5000 01 16 1 02 10 1 03 7 1 11 Positive Control 2 -aminoanthracene 1.0 pg per plate 01 131 1 02 115 l 03 89 1 112 21 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. 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 H-24889 AA44ER.502001.BTL TA1537 None Experiment No Cells Seeded Date Plated B1 0.9 X 10 30 May 2001 Vehicle acetone Plating Aliquot 50 uL________ ___ Concentration Plate Revertants Background Average Standard ug per plate Number per plate______ Code-----RevertantS- Deviation Vehicle 01 02 03 81 81 12 1 100 01 3 1 02 4 1 03 6 1 333 01 6 1 02 4 1 03 7 1 1000 01 6 1 02 5 1 03 9 1 3333 01 4 .1 02 3 1 03 3 1 5000 01 6 1 02 5 1 03 7 1 9-aminoacridine 75 ug i 01 819 i 02 1102 i 03 860 i 927 153 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate N P=N on-Interfering precipitate; IP=Interfenng precipitate BioReliance Study No. AA44ER.502001.BTL Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test W1U1 all uiuopciiucui DuPont-6405 Bacterial Mutation Test Table 13 Test article Id Study Number Strain Liver Microsomes H-24889 AA44ER.502001.BTL TA1537 Rat liver S9 Experiment No : B1 Cells Seeded : 0.9 X 10 Date Plated : 30 May 2001 Vehicle acetone Plating Aliquot 50 uL ------ ------------------------------- Concentration Plate Revertants Background Average iin nei- nlate Number per plate Code Revertants Deviation Vehicle 01 02 03 61 41 81 6 100 01 9 1 02 4 1 03 6 ,1 6 333 01 11 1 02 6 1 03 7 1 8 1000 01 15 1 02 10 1 03 7 1 11 3333 01 5 1 02 13 1 03 6 1 8 5000 01 7 1 02 9 1 03 9 1 8 Positive Control 2-aminoanthracene 1.0 pg per plate 01 126 1 02 248 l 03 192 1 189 61 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 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 MicrosomesVehicle H-24889 AA44ER.502001.BTL WP2 uvrA None acetone Experiment No Cells Seeded Date Plated B1 3.4 X 10s 30 May 2001 i'xacing A-Liquuu ; jv Concentration Plate uo per plate Number - Revertants Background per plate Code Average Revertants Standard Deviation Vehicle 01 02 03 13 1 13 1 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 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 pg per plate 01 62 1 02 67 l 03 59 1 63 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; lP=Interfering precipitate 4 B io R elian ce 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 : H-24889 Study Number : AA44ER.502001.BTL Strain : WP2 uvrA Liver Microsomes ; Rat liver S9 Experiment No : B1 Cells Seeded : 3.4 X 108 Date Plated : 30 May 2001 Vehicle : acetone Platino Aliauot : 50 uL Concentration Plate Revertants Background Average Standard a ner Diate Number Der plate Code Revertants Deviation Vehicle 01 02 03 13 1 12 1 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 93 Positive Control 2 -aminoanthracene 10 pg per plate 01 163 1 02 17 6 l 03 154 1 164 il 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 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 H-24889 AA44ER.502001.BTL TA98 None acetone Experiment No Cells Seeded Date Plated B2 15.1 X 10s 5 Jun 2001 Concentration na Der Diate Vehicle Plate Number 01 02 03 Revertants per plate 10 15 16 Background Code 1 1 1 Average Revertants 14 Standard Deviation 3 100 01 4 1 02 8 1 03 5 1 62 333 01 11 1 02 11 1 03 10 1 11 1 1000 01 10 1 02 7 1 03 6 1 82 3333 01 6 1 02 18 1 03 5 1 10 7 5000 01 7 1 02 4 1 03 3 1 52 Positive Control 2 -nitrofluorene 1..0 pg per plate 01 240 1 02 114 1 03 309 1 221 99 Background Lawn Code l=Norxnal; 2=Slightly reduced? 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate N P=N on-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 H-24889 AA44ER.502001.BTL TA98 Rat liver S9 Experiment No Cells Seeded Date Plated B2 15.1 X 10B 5 Jun 2001 Vehicle acetone Platina Aliauot : 50 UL Concentration Plate Revertants Background Average Standard uer tier nlate Number per plate Code Revertants Deviation Vehicle 01 02 03 12 1 12 1 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 92 Positive Control 2-aminoanthracene 1.0 pg 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=Obscured 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 ; H-24889 Study Number : AA44ER.502001. BTL Strain : TA100 Liver Microsomes : None Experiment No : B2 Cells Seeded : 13.6 X 108 Date Plated : 5 Jun 2001 Vehicle : acetone Plating Aliguot : 50 UL Concentration ua Der 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 3333 01 147 1 02 170 1 03 170 1 01 152 1 02 197 1 03 144 1 162 13 164 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=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. 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 :H-24889 :AA44ER.502001.BTL :TA100 Experiment No :B2 Cells Seeded :13.6 X 108 Liver Microsomes': Rat liver S9 Date Plated :5 Jun2001 Vehicle :acetone r i a t m g Aiiquui. ; __________ Concentration pg per plate Plate Revertants Background Average Standard Number per plate Code Revertants Deviation Vehicle 01 02 03 165 1 197 1 144 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 02 159 03 ' 165 1 1 1 158 8 5000 01 204 1 02 196 1 03 155 1 185 26 Positive Control 2-aminoanthracene 1.0 pg per plate 01 1526 1 02 1628 1 03 1204 1 1453 221 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. 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 : H-24889 Study Number : AA44ER.502001.BTL Strain : TA1535 Liver Microsomes : None Experiment No : B2 g Cells Seeded : 3.8 X 10 Date Plated : 5 Jun 2001 Vehicle : acetone Platina Aliauot : 50 uL Concentration Plate Revertants uo Der Diate Number per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 03 61 20 1 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 pg per plate 01 466 1 02 250 1 03 410 1 375 112 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 36 Company Sanitized. Does not contain TSCA CBI 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 H-24889 AA44ER.502001.BTL TA1535 Rat liver S9 Experiment No Cells Seeded Date Plated B2 3.8 X 10 5 Jun 2001 Vehicle Platina Aliauot acetone UL o tn Concentration Plate Revertants ucr ner Date Number per plate Background Code Average Revertants Standard Deviation Vehicle 01 02 03 13 1 71 11 1 .10 3 100 01 7 1 02 7 1 03 5 1 61 333 01 11 1 02 16 1 03 4 . 1 10 6 1000 01 7 1 02 4 1 03 10 1 73 3333 01 8 1 ' 02 4 1 03 11 1 8 4 5000 01 16 1 02 8 1 03 3 1 97 Positive Control 2-aminoanthracene 1.0 pg per plate 01 274 1 02 250 1 03 120 1 215 83 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. 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 H-24889 AA44ER.502001.BTL TA1537 None Experiment No : B2 Cells Seeded : 2.9 X 108 Date Plated : 5 Jun 2001 Vehicle acetone Plating Aiiquoit : du pu Concentration Plate Revertants Background Average Standard ucr per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 31 51 21 32 100 01 7 1 02 6 1 03 3 1 52 333 01 6 1 02 6 1 03 7 1 61 1000 01 2 1 02 4 1 03 6 1 42 3333 01 5 1 02 4 1 03 8 1 62 5000 01 8 1 02 1 1 03 5 1 54 Positive Control 9-aminoacridine 75 pg 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=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate BioReliance Study No. AA44ER.502001.BTL 38 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test DuPont-6405 with an Independent Repeat Assay________________________ _______________________ Bacterial Mutation Test Table 23 Test article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot :H-24889 :AA44ER.502001.BTL Experiment No :B2 :TA1537 Cells Seeded :2.9 X 108 :Rat liver S9 Date Plated :5 Jun 2001 :acetone : 50 pL________ ______________________ __________________ Concentration Plate Revertants Background Average Standard gg per plate____Number per plate______ Code_____Revertants Deviation____ Vehicle 01 02 03 61 10 1 10 1 92 100 01 10 1 02 2 1 03 5 1 64 333 01 7 1 02 8 1 03 2 1 63 1000 01 10 1 02 3 1 03 4 1 64 3333 01 1 ' 1 02 3 1 03 7 1 43 5000 01 6 1 02 5 1 03 4 1 51 Positive Control 2-aminoanthracene 1..0 pg per plate 01 190 1 02 161 1 03 351 1 234 102 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. 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 :H-24889 Study Number :AA44ER.502001.BTL Strain :WP2 uvrA Experiment No :B2 g Cells Seeded :12.2 X 10 Liver Microsomes :None Date Plated :5 Jun2001 Vehicle : acetone Plating Alicuot : 50 pL ______________ __________________________ _ Concentration ua per plate Plate Reversants Background Average Standard Number per plate Code Revertants 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 83 5000 01 12 1 02 12 1 03 13 1 12 1 Positive Control methyl methanesulfonate 1000 pg 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=Obscured 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-24889 AA44ER.502001.BTL WP2 uvrA Rat liver S9 acetone 50 pL Experiment No : B2 Cells Seeded : 12.2 X 10s Date Plated : 5 Jun 2001 Concentration Plate v>g per plate____Number Vehicle 01 02 03 Revertants per plate 16 12 24 Background Code 1 1 1 Average Revertants 17 Deviation 100 01 9 1 02 13 1 03 10 1 11 333 01 9 1 02 7 1 03 14 1 10 1000 01 10 1 02 12 1 ir\ 03 8 1 10 3333 01 10 1 02 10 1 ' 03 9 1 10 5000 01 9 1 02 7 1 03 6 1 7 Positive Control 2-aminoanthracene 10 us Per plate 01 729 1 02 585 1 03 490 1 601 120 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 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 : H-24889 Study Number______ : AA44ER.502001.BTL______Experiment No ; B1 Average Revertants Per Plate Standard Deviation Liver Microsomes: None Dose (ug/plate) TA98 TA100 Vehicle 100 333 1000 3333 5000 Positive 14 3 177 9 9 1 157 17 11 1 176 24 12 3 150 12 12 3 174 24 9 2 169 30 120 11 575 6 Liver Microsomes: Rat liver S9 TA1535 11 9 16 12 14 13 170 1 3 3 1 3 3 37 TA1537 9 2 4 2 6 2 7 2 3 1 6 1 927 153 WP2 uvrA ' 12 12 11 10 13 14 63 1 2 2 2 2 3 4 Dose (ug/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 3 17 5 161 56 1111 196 1763 203 Vehicle = Vehicle Control Positive .= Positive Control Plating aliquot: 50 u-L TA1535 9 10 9 10 14 11 112 2 3 2 4 2 5 21 TA1537 6 6 8 11 8 8 189 2 3 3 4 4 1 61 WP2 uvrA 13 12 11 10 13 9 164 1 1 4 3 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 : H-24889 Study Number______ ; AA44ER.502001.BTL______Experiment No ; B2 Average Revertants Per Plate Standard Deviation Liver Microsomes: None Dose (ug/plate) TA98______TA100 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 TA1535 TA1537 15 8 3 2 10 1 5 2 10 2 6 1 11 1 4 2 14 3 6 2 11 5 5 4 375 112 1164 110 WP2 uvrA 15 11 14 15 8 12 90 1 1 2 2 3 1 18 Dose (ug/plate) TA98______TA100______ TA1535 Vehicle 100 333 1000 3333 5000 Positive 13 2 169 27 10 1 153 14 11 6 146 12 12 6 177 28 13 4 158 8 9 2 185 26 1043 304 1453 221 10 6 10 7 8 9 215 3 1 6 3 4 7 83 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 50 pL TA1537 WP2 uvrA 9 2 6 4 6 3 6 4 4 3 5 1 234 102 17 6 11 2 10 4 10 2 10 1 7 2 601 120 BioReliance Study No. AA44ER.502001 .BTL 43 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 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 Min Max Mean Rat liver SD Min Max TA98 TA100 TA1535 TA1537 WP2 uvrA Neg I 16 74 59 Pos 425 206 21 1536 Neg 128 31 53 288 Pos 568 159 129 1371 Neg 12 5 1 45 Pos 378 164 6 978 Neg 6 3 0 30 Pos | 708 409 13 2786 Neg | 14 54 48 Pos 1 190 138 34 961 21 7 592 322 138 34 736 301 12 4 104 84 73 88 106 16 6 317 299 7 58 56 2454 74 258 198 2871 1 42 18 1640 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 with an Independent Repeat Assay______ DuPont-6405 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 rmm (Mrtlfl i ) deceived by RA/OAiJitejK^' Sponsor Project Number DuPont-6405 BioReliance Study Number AA44ER.502001.BTL H-24889: Bacterial Reverse M utation Test with an Independent Repeat Assay 1.0 PURPOSE The purpose o f this study is to evaluate the mutagenic potential o f p m |^ H Q ( H - 24889) by its ability to induce reverse mutations at selected loci of several strains of Salm onella typhimurium and at the tryptophan locus of Escherichia coll WP2 i/vrA in the presence and absence o f S9 activation. 2.0 SPONSOR 2.1 Name: 2 2 Address: 2.3 Representative: E.I. du Pont de Nemours and Company Stine Haskell Research Center DuPont Haskell Laboratory P.O. Box 50 1090 Elkton Road Newark, DE 19714-0050 . Maria Donner, Ph.D. Phone: 302-366-5251 Fax: 302-366-5207 Email: maria.donner@usa.dupont.com 2.4 Sponsor Project No.: 2.5 WR#: 2.6 Haskell #: DuPont-6405 1 H-24889 2.7 Service Code: 3.0 IDENTIFICATION OF TEST AND CONTROL ARTICLES 3.1 Test Article Name: The test article is J jH U t room temperature and must be heated to 60C each and every time. 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 Ii).: H-24889 (to be used in die report tide and text) 3.3 Controls: Negative: Test article vehicle Positive: 9-aminoacridine Protocol SPGT502001 Ol-May-ZOOI BioReliance Study No. AA44ER.502001.BTL Page 1 of I t 47 ^ Bio Reliance Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 Sponsor Project Number; DuPont-6405 BioReliance Study Number AA44ER.502001.BTL 2-aminoantbracene methyl methanesulfonate 2-nitrofluorene sodium azide 3.4 Test Article Characterization Unless alternate arrangements are made, the testing facility at BioReliance will not perform analysis of the dosing solutions. The Sponsor vnll be directly responsible for determination and documentation of the analytical purity and mmpngitinn 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 O. Wagner III, 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-M-2001 5.4 Final Report Date: 2 weeks after Sponsor approves draft 6.0 TEST SYSTEM The tester strains will include the S. typhimurium 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 0J-M ay-2001 Page 2 or 11 ^ Bio Reliance" 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 DuPont-6405 BioReliance Study Number AA44ER.S02001-BTL Histidine Mutation Tryptopha n Mutation AfcG46 toC3076 AisD3052 TrpE TA1535 TA100 TA1537 - TA98 - - - - WP2 uvrA Additional Mutations LPS Repair R-factor rfa uvrB - rfa uvrB +R - uvrA - Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, Mitinmil 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 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 TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by ftameshift mutagens. TA100 is reverted by both fiameshifl and base substitution mutagens and TA1535 is reverted only by mutagens Hat cause base substitutions. . The coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox e t 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 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. typhimurium tester strains were received directly from Dr. Bruce Ames, University o f 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 01-M ay-2001 Page 3 of 11 BioReliance Study No. AA44ER.50200l.BTL ^ Bio Reliance- Company Sanitized. Does not contain TSCA CBI % H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 Sponsor Project Number DuPont-6405 BioReliance Study Number AA44ER.502001.BTL 7.1 Solubility Determination Unless the Sponsor has indicated the test article vehicle, a solubility determination will be conducted to determine the maximum soluble concentration or workable c c p g n c m n 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 ofchoice will be the solvent, selected in orderof preference, which permits preparation o ffoe 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 preliminary assay will be conducted by exposing TA98, TA100, TA.1535, 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 foe Sponsor, the highest dose wifi be foe highpgt workable concentration in foe vehicle of choice but not to exceed 5 mg/plate. In selecting dose levels for foe mutagenicity assay the following piiHpiin will be employed. Doses will be selected such that precipitate does not interfere with manual scoring. Whenever possible, foe highest dose for foe mutagenicity assay will be selected to give some indication of toxicity without pypptine 5 mg/plale. For freely soluble, nontoxic test articles, the highest dose level will be 5 mg/plate. For precipitating, nontoxic test articles, foe highest dose level will be selected in an attempt to yield precipitate at only die top one or two dose levels. The Sponsor will be consulted regarding dose selection if (1) foe 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 foe plate incorporation methodology originally described by Ames el al. (1975) and updated by Maron and Ames (1983). This test system has been shown to detect a wide range of classes ofchemical mutagens (McCann el al., 1975; McCann and Ames, 1976). After the dm? generated in the first assay have been evaluated, the mutagenicity assay will be repeated. The dose levels used in the second assay will be foe same as those used in foe first assay unless the Study Director determines that foe 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 01-M y-2001 Page 4 o r 11 ^ Bio Reliancf BioReliance Study No. AA44ER.502001 .BTL Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 Sponsor Project Number: DuPont-6405 BioReliance Study Number AA44ER.5020013TL (e.g., azo compounds), this information will be utilized in designing the assay, (e.g., activation system or treatment method). This guidance is based on the OECD Guideline 471 (adopted July 1997 and published Febniaty 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 tiie assay are listed below: Strain S9 Activation Positive Control Concentration (ng/plate) Salm onella Strains WP2 uvrA TA98 TA100, TA1535 TA1537 WP2 wvrA Rat None 2-aminoanthracene 2-nitrofluorene 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 SterilityControls The most concentrated test article dilution and the Sham and S9 mixes will be checked for sterility. 7.5 ExogenousMetabolicActivation Arcelor 1254-induced rat liver S9 will be used as the metabolic activation system. The S9 homogenate will be prepared from male Sprague-Dawley tats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 will be batch prepared and stored frozen at approximately -70C until used. Each batch of S9 homogenate will be assayed for its ability to Protocol SPGT502001 01-M ay-2001 Page 5 o f 11 ^ Bio Reliance- ' \ BioReliance Study No. AA44ER.502001 .BTL Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 Sponsor Project Number DuPont-6405 BioReliance Study Number AA44ER.502001-BTL nytahnii. 2-aminoantfaracene and 7,12-dimethyIbenzanlhracene to forms mutagenic to S. typhimurhan TA100. Immediately prior to use, the S9 will be thawed and mixed with a cofactor pool to rr>nfrain 10% S9 homogenate, 5mM glucose-6-phosphate, 4mM -nicotinamide-adenine 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 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 pW p die length of incubation will be controlled and monitored. At die end of the working day, each inoculated flask will be placed in a testing 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 ofharvest All cultures will be harvested by spectrophotometric monitoring of culture turbidity ratW 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 10*cells/mL. 7.7 Test System Identification Bach plate will be labeled with a code system that identifies the test article, test ptiaw, 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 ofTest System One half milliliter (0.5 mL) of S9 mix or Sham mix, 100 (L o f tester strain and 50 jiL 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 r/wHihatinn or eliminate toxic vehicle effects, aliquots of other than 50 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 tire overlay has solidified, the plates will be inverted and incubated for approximately Protocol SPGT502001 01-M ay-2001 Page 6 o f 11 BioReliance Study No. AA44ER.502001 .BTL ^ Bio Reliance- Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 Sponsor Project Number DuPont-6405 .... . BioReliance Study Number. AA44ER.502001.BTL 48 to 72 hours at 372C. Plates that are not counted immediately following die incubation period will be stored at 2-8C. 7.10 Scoring The condition of the bacterial background lawn will be evaluated for evidence of test article toxicity and precipitate. Evidence of toxicity will be scored relative to the negative control plate and recorded along with foe revertanl count for that plate. Toxicity will be evaluated as a decrease in foe number of revertant colonies per plate and/or a thinning or disappearance of foe bacterial background lawn. Precipitation will be evaluated after the incubation period by visual examination without magnification. 7.11 Tester Strain Verification On foe 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. typhimurium tester strain cultures must exhibit sensitivity to crystal violet To demonstrate the presence of the uvrB mutation, all S. typhimurium tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate the presence of the wvrA 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 of TA98 and TA100 must exhibit resistance to ampicillin. 8.2 Spontaneous Revertant Background Frequency Based on historical control data, all tester strain cultures must exhibit characteristic number of spontaneous revertants per plate in the negative controls (vehicle). The mean revertants per plate must be within the following ranges (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5-45; TA1537, 3-21; WP2 wvrA, 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 0.3xl09cells per milliliter. Protocol SPGTS02001 01-My-2001 Page 7 of I t ^ Bio Reliancf BioReliance Study No. AA44ER. 502001 ,BTL \S Company Sanitized. Does not contain TSCA CBI % H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 Sponsor Project Number DuPont-6405 BioReliance Study Number AA44ER.502001 .BTL 8.4 Positive Control Values Pa/4i pipan 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 m in im u m of three non-toxic dose levels will be required to evaluate assay data. A dose level is considered toxic if it causes a >50% reduction in the mean number of revertants per plate relative to die mean negative control value (this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in thn background lawn. In the event that less than three non-toxic dose levels are achieved, the affected portion ofthe assay will be repeated with an appropriate change in dose levels. 9.0 EVALUATION OF TEST RESULTS For a test article to be evaluated positive, it must cause a dose-related increase in the m m revertants per plate of at least one tester strain over a minimum of two increasing concentrations o f test article 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 uvrA 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 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 ofcells/mL per culture; strain characteristics. Protocol SPGT502001 O t-M ay-M O l Page 8 of I t 00 Bio Reliance- BioReliance Study No. AA44ER.502001.BTL Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay _____ DuPont-6405 Sponsor Project Number. DuPont-6405 _ BioReliance Study Number AA44ER502001-BTL . Test conditions: amount of test article per plate with rationale for dose sdecticmand m m her 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 ofrevertant colonies per plate and standard deviation; dose-rasponse 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 A ll raw data, the protocol and all reports will be maintained seconding to Standard O o e S L P ^ u r e X i l B r t b y ^ BioReliance RAQA unit headquartered a t S S c T S o S B K k Rockville, MD 20850 Per this SOP,pupern*TMds will be retained for at least three years after which time the Sponsor will becoafccted fo a decision as to the final disposition o f the materials. All study Sponsor or destroyed will first be copied and the copy will be retamed m 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 (A deline Toxicology: Bacterial Reverse Mutation Assay), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, ^ Fblu^ ! ^ T ! with the International Conference on Harmonisation of Technical Requirements for Registration o f 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 proto>l, an in-process phse, the raw data, and reports) will be audited per the Procedures (SOPs) of BioReliance by the Quality Assurance Umt o f BioReliance for compliance with GLPs, the SOPs of BioReliance and the study protocol. ihe inspection will be performed to audit the critical assay procedures and systems ^ S t i n g the assay. A signed QA statement will be included m the final ^ statement will list the system phases inspected during the J J 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 addition, a signed GLP compliance statement will be included rathe final reP01^ 1^ statement w illd te the GLP guideline(s) with which the study is com phm t exceptions to this compliance, if applicable, including the omission o f characterization or stability analyses ofthe test or control articles or their mixtures. Protocol SPGT502001 01-M ay-2001 Page 9 o f 11 * Bio Reliancf BioReliance Study No. AA44ER. 502001.BTL 55 Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 Sponsor Project Number DuPont-6405 , BioReliance Study Number; AA44ER.502001.BTL Unless arrangements are made to the contrary, unused dosing solutions will be disposed of M ow ing administration to the test system and all residual test article will be disposed of following finalization ofthe report 13.0 REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and fpitagiTM with the &rimone//a/mammaan-microsome mutagenicity test Mutation Research 31:347-364. Green, M.H.L., and Muriel, W J. (1976). Mutagen testing using trp* reversion in Escherichia coli. 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. Int/M-narinnal Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at step 4 of 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 Salmonellalmicrosome test: assay of 300 chemicals: discussion. Proc. Natl. Acad. Set. USA 73:950-954. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975). Detection of carcinogens as mutagens in the Salmonella/ncrosom e 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 of Chemicals, published by OECD, Paris, February 1998. Wilcox, P., Naidoo, A., Wedd, D.J. and Gatehouse, D.G. (1990). Comparison of Salm onella typhimurium TA102 with Escherichia coli WP2 tester strains. Mutagenesis 5:285-291. ! Protocol SPGT502001 0I-M ay-2001 Pge 10 of 11 ^ Bio Reliance- BioReliance Study No. A A 44 E R .502001.BTL Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay_____ _ DuPont-6405 Sponsor Project Number DuPont-6405 PipRgiianra Study Number AA44ER-502001.BTL 14.0 APPROVAL Sponsor Representative rV C o-< - Df l r*X .< ' (Print or Type Name) \ic JU ^ r BioReliance StudylMiector i /" \j a r w ' i o w Date II ItIcuj Date " irfv lo o t ) i Protocol SPGT50J001 01-M ay-2001 Pge II of U BioReliance Study No. AA44ER.50200l.BTL ^ Bio Reliance- Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 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______ Report of Results of Reverse-Mutation Assay in Bacteria DuPont-6405 1. Tester Strains (1) Procurement______ Strain Obtained from Date obtained TA98 TA100 TA1535 TA1537 TA1538 TA97 TA102 WP2 uvrA WP2 uvrA (pkMIOl) WP2 (pKMIOl) 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. S9 Mix (1) Source, Storage Tern cerature, etc, or av Made in-house Prepared on Storage temperature -70C or colder 05 February 2001 (Bat<:h R640) 28 March 2001 (Batch R641) Name and model of storage apparatus So-Low, Model PR27-120 BioReliance Study No. AA44ER.50200l.BTL 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 Male Inducing substance Name Aroclor 1254 Administration method Intraperitoneal Age (in weeks) Weight 9 (Batch R640) 8 (Batch R641) 264 to 293 g (Batch R640) 181 to 232 g (Batch R641) Administration period and amount (g/kg-weight) 5 days, 0.5 gm/kg body weight 3. Positive Control Substance (l) rosiuve control Name Manufacturer Lot No. Grade Purity Solvent (%) used 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 106F06681 085H2508 09419LR 11202TF 098H0169 118H4061 Practical Practical >98% >95% >99% >98% >99 % >99% 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. m parauoi1oi 1esi arutic oi Solvent used . Name Acetone Manufacturer Fisher Scientific Lot No. 001064 Grade Purity (%) Certified ACS Stability >ftest article in Unknown the solveiit 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 Conversion by purity <30 minutes at ambient temperature None BioReliance Study No. AA44ER.502001.BTL Company Sanitized. Does not contain TSCA CBI H-24889: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-6405 >. conditions oi rre-cuuure______ Nutrient broth Name Oxoid Nutrient Broth No. 2 Manufacturer Oxoid Ltd. Lot No. CH,-B=232622 CH,-B=228448 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 121 hours 2 to 5 hours at ambient temperature <8 hours at 2-8C New Brunswick Scientific, model G-24 Rotary (125 rev/min.) shape: cylinder, 200 mL 50 mL 1 colony _ 6. Agar Plate Medium (1) lop agar Affar Name Manufacturer Lot No. BBL Select Becton Dickinson 1000J3DKSQ m Minimum Glucose Agar ____ _________ _ _________ ----------------------- Name BBL Select Made in-house Agar Manufacturer Lot No. Becton Dickinson 1000J3DKSQ Volume of agar plate medium 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 o f 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