Document zN5m0vjJ3rVoOgMN5r3K0J20

MU- & o 412 DuPont-8868 TRADE SECRET Study Title H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay Authors Valentine O. Wagner, HI, M.S. Michelle L. Klug, B.S. Study Completion Date 11 February 2002 Performinp Laboratory BioReliance 9630 Medical Center Drive Rockville, MD 20850 for E. L du Pont de Nemours and Company DuPont Haskell Laboratory P.O. Box 50,1090 Elkton Road Newark, DE 19714-0050 Performing Laboratory Study Number AA52XL.502001.BTL Work Request Number Service Code Page 1 o f 62 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 STATEMENT OF COMPLIANCE Study No. AA52XL.502001.BTL was conducted in compliance with the U.S. FDA GLP Regulations as published in 21 CFR 58, the U.S. EPA GLP Standards 40 CFR 160, and 40 CFR 792, the UK GLP Compliance Programme, the Japanese GLP Standard, and the OECD Principles of Good Laboratory Practice in all material aspects with the following exceptions: The identity, strength, purity and composition or other characteristics to define the test and control articles have not been determined by the testing facility. The control articles have been characterized as per the Certificates of Analysis on file with 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 substance 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 stability analysis on the test substance mixtures. Valentine O. Wagner, m , M.S. Study Director I( Rsi 20031 Date BioReliance Study Management Date BioReliance Study No. AA52XL.50200l.BTL 2 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ Quality Assurance Statement DuPont-8868 Study T itle: H -25171: BACTERIAL REVERSE MUTATION ASSAY WITH AN INDEPENDENT REPEAT ASSAY Study N um ber: AA52XL.502001 .BTL S tudy D irector: Valentine O. Wagner, III, M.S. This study has been divided into a series o f in-process phases. Using a random sampling approach, Quality Assurance monitors each o f these phases over a series o f studies. Procedures, documentation, equipment records, etc., are examined in order to assure that the study is performed in accordance with die U.S. FDA 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 o f Good Laboratory Practice and to assure that die study is conducted according to the protocol and relevant Standard Operating Procedures. The following are the inspection dates, phases inspected, and report dates o f QA inspections o f this study. Inspect On 06-Dec-01 - 06-Dec-01 To Study Dir 06-Dec-01 To Mgmt 06-Dec-01 Phase Protocol Review Inspect On Phase 24-Dec-01 - 24-Dec-01 To Study Dir 24-Dec-01 To Mgmt 26-Dec-01 Scoring the plates Inspect On Phase 03-Feb-02 - 03-Feb-02 To Study Dir 04-Feb-02 To Mgmt 07-Feb-02 Draft Report Inspect On Phase 11-Feb-02 - 1l-Feb-02 To Study D ir 1l-Feb-02 To Mgmt 1l-Feb-02 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. AA52XL.502001.BTL DATE 3 Company Sanitized. Does not contain TSCA CBI H-25171 : Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 CERTIFICATION W e, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Issued by Study Director: Valentine O. Wagner IH, M.S. 11 M Z<a Date Approved by Study Monitor: OA Maria Donner, Ph.D. Senior Research Scientist ""Zcoz_ Date BioReliance Study No. AA52XL.502001.BTL 4 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 TABLE OF CONTENTS Page Certification.................................................................................................................................... 4 Study Inform ation..........................................................................................................................7 Sum m ary........................................................................................................................................ 8 Purpose........................................................................................................................................... 9 Characterization of Test and Control Substances........................................................................ 9 M aterials and M ethods................................................................................................................ 11 Results and Discussion.................................................................................................................16 C o n clu sio n .....................................................................................................................................16 R eferen c es.....................................................................................................................................17 Data Tables....................................................................................................................................18 Table 1: Preliminary Toxicity Test in Salmonella typhimurium TA 98..............................18 Table 2: Preliminary Toxicity Test in Salmonella typhimurium TA 1535..........................19 Table 3: Preliminary Toxicity Test in Salmonella typhimurium TA1537 ......................... 20 Table 4: Preliminary Toxicity Test in Escherichia coli WP2 uvrA ....................................21 Table 5: M utagenicity Test in Salmonella typhimurium TA98 without S 9 ..................... 22 Table 6: M utagenicity Test in Salmonella typhimurium TA98 with S 9 .......................... 23 Table 7: M utagenicity Test in Salmonella typhimurium TA100 without S 9 ................... 24 Table 8: M utagenicity Test in Salmonella typhimurium TA100 with S 9 ........................ 25 Table 9: M utagenicity Test in Salmonella typhimurium TA1535 without S 9 ................. 26 Table 10: M utagenicity Test in Salmonella typhimurium TA1535 with S 9 ...................... 27 Table 11: M utagenicity Test in Salmonella typhimurium TA1537 without S 9 ................. 28 Table 12: M utagenicity Test in Salmonella typhimurium TA1537 with S 9 ...................... 29 Table 13: M utagenicity Test in Escherichia coli WP2 uvrA without S 9............................30 Table 14: M utagenicity Test in Escherichia coli WP2 uvrA with S 9.................................31 Table 15: M utagenicity Test in Salmonella typhimurium TA98 without S 9 ..................... 32 Table 16: M utagenicity Test in Salmonella typhimurium TA98 with S 9 ...........................33 Table 17: M utagenicity Test in Salmonella typhimurium TA100 without S 9 ................... 34 Table 18: M utagenicity Test in Salmonella typhimurium TA100 with S 9 ........................ 35 Table 19: M utagenicity Test in Salmonella typhimurium TA1535 without S 9 ................. 36 Table 20: M utagenicity Test in Salmonella typhimurium TA1535 with S 9 ...................... 37 Table 21: Mutagenicity Test in Salmonella typhimurium TA1537 without S 9 ................. 38 Table 22: M utagenicity Test in Salmonella typhimurium TA1537 with S 9 ...................... 39 Table 23: M utagenicity Test in Escherichia coli WP2 uvrA without S9........................... 40 BioReliance Study No. AA52XL.50200l.B TL 5 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Table 24: M utagenicity Test in Escherichia coli WP2 wvrA with S 9.................................41 Table 25: Salmonella/E. coli Mutagenicity Test - Summary of Results B 1...................... 42 Table 26: Salmonella/E. coli Mutagenicity Test - Summary of Results B 2......................43 Appendix A: Historical Control Data......................................................................................... 44 Appendix B: Study Protocol........................................................................................................46 Appendix C: Information for Japanese Regulatory Agencies.................................................. 58 BioReliance Study No. AA52XL.502001.BTL 6 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ STUDY INFORMATION Substance Tested: DuPont-8868 Submitter's Notebook Numberts-): Haskell Number: 25171 Known Impurities Physical Characteristics Stability: The test substance appeared to be stable under the conditions o f the study; no evidence o f instability was observed. Sponsor: E.L du Pont de Nemours and Company W ilmington, Delaware 19898 U.S.A. Study Initiated/Completed: December 05,2001 / (see report cover page) In-Life Initiated/Completed: December 20,2001 / January 22, 2002 BioReliance Study No. AA52XL.50200l.BTL 7 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 SUMMARY The test substance, H-25171, 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 substance. Tetrahydrofuran (THF) was selected as the solvent of choice based on compatibility with the target cells and solubility of the test substance. The test substance was soluble but cloudy in tetrahydrofuran (THF) at a maximum concentration of approximately 200 mg/mL. In the preliminary toxicity test, the maximum dose tested was 5000 pg per plate; this dose was achieved using a concentration of 200 mg/mL and a 25 pL plating aliquot. The test substance was a workable solution from 40 to 200 mg/mL and a soluble and clear solution from 0.27 to 27 mg/mL. Dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 pg per plate. Precipitate was observed beginning at 100 pg per plate. No appreciable toxicity was observed. Based on the findings of the toxicity test, the maximum dose plated in the mutagenicity test was 5000 pg per plate. In the mutagenicity test, no positive mutagenic response was observed. The dose levels tested were 15,50,150, 500,1500 and 5000 pg per plate. Precipitate was observed beginning at 500 pg per plate. No appreciable toxicity was observed. The results of the Bacterial Reverse Mutation Test with an Independent Repeat Assay indicate that, under the conditions of this study, H-25171 did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9. BioReliance Study No. AA52XL.502001.BTL 8 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 PURPOSE The purpose o f this study was to evaluate the mutagenic potential o f the test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. A copy of the protocol is included in Appendix B. This study was conducted in compliance with the testing guidelines o f the ICH (1996 and 1997) and OECD (1998). CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test substance, H-25171, was received by BioReliance on 03 December 2001 and was assim ed th ^x x ie number AA52XL. The test substance was characterized by the SpoUnSsUorI oaos at t f P m m j t h a t should be stored at ambient temperature. An expiration d a te o f 26 November 2004 was provided. Upon receipt, the test substance was described as id was stored at room temperature, protected from light and moisture. The iidoBflfiititt^yP, strengthj*purity, composition or other characteristics to define the test substance and the stability of the test substance have been determined by the Sponsor. The Sponsor indicated that BioReliance would not receive copies of these reports. The vehicle used to deliver H-25171 to the test system was tetrahydrofuran (THF, CAS# 109-99-9), Gold Label, 99.9%, purchased from Aldrich Chemical Company. Test substance dilutions were prepared immediately before use and delivered to the test system under yellow light. To enhance the workability of the test substance, the top concentration was sonicated in a screw-cap tube for approximately 15 minutes at 50C. Subsequent dilutions were maintained at 50C in screw-cap tubes during dosing. Positive controls plated concurrently with the mutagenicity tests are listed below. All positive controls were diluted with dimethyl sulfoxide (DMSO) except sodium azide, which was diluted with water. All subdivided solutions of positive control were stored at -5 to -30C. Strain All Salmonella Strains S9 Activation WP2 uvrA Rat BioReliance Study No. AA52XL.502001.BTL Positive Control 2-aminoanthracene (Aldrich Chemical Co., Inc.) Lot No. 09106PS Exp. Date 14-Sep-2005 CAS No. 613-13-8 Purity >95% 9 Concentration (pg/plate) 1.0 10 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Strain S9 Activation Positive Control Concentration (pg/plate) TA98 TA100, TA1535 TA1537 WP2 uvrA None 2-nitrofluorene (Aldrich Chemical Co., Inc.) Lot No. 08707HS Exp. Date 08-Mar-2006 CAS No. 607-57-8 Purity >98% Sodium azide (Sigma Chemical Co.) Lot No. 098H0169 Exp. Date 05-Jan-2004 CAS No. 26628-22-8 Purity >99% 9-aminoacridine (Sigma Chemical Co.) Lot No. 106F06681 Exp. Date 18-Nov-2004 CAS No. 90-45-9 Purity >98% Methyl methanesulfonate (Aldrich Chemical Co., Inc.) Lot No. 15526AO Exp. Date 28-Nov-2003 CAS No. 66-27-3 Purity >99% 1.0 1.0 75 1,000 To confirm the sterility of the test substance, the highest test substance dose level used in the mutagenicity tests was plated on selective agar with an aliquot volume equal to that used in the test. These plates were incubated under the same conditions as the test. BioReliance Study No. AA52XL.502001.BTL 10 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 MATERIALS AND METHODS For submission to Japanese regulatory agencies, additional information is included in Appendix C. Test System The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were received directly from Dr. Bruce Ames, University of California, Berkeley and E. coli tester strains were received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976). Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing ~50 mL of culture medium. To assure that cultures were harvested in late log phase, the length o f incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 372C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 109 cells per milliliter. The actual titers were determined by viable count tests on nutrient agar plates, and the data is on 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 o f Technical Requirements o f 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 injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at -70C or colder until used. Each bulk preparation of S9 was tested for its ability to metabolize 2aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100. BioReliance Study No. AA52XL.502001.BTL 11 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 The S9 mix was prepared immediately before its use and contained 10% S9, SmM glucose-6-phosphate, 4 mM 6-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar. Solubility Test A solubility test was conducted to select the vehicle. The test was conducted using water, dimethyl sulfoxide (DMSO), ethanol (EtOH), acetone, tetrahydrofuran (THF), acetonitrile, propylene glycol and polyethylene glycol. The test substance was tested to determine tire 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. Prelim inary Toxicity Test The preliminary toxicity test was used to establish the dose-range over which the test substance would be tested. Vehicle and ten dose levels of the test substance were plated, one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and W P2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. Dose levels tested were 6.7,10,33,67,100,333,667,1000,3333 and 5000 pg per plate. M utagenicity Test The mutagenicity test (initial and independent repeat tests) was used to evaluate the mutagenic potential of the test substance. Six dose levels of test substance (15, 50, 150, 500, 1500 and 5000 pg 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 substance, vehicle controls and positive controls were plated in triplicate. H ating and Scoring Procedures The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). On the day o f 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 of water for each 100 mL of minimal top agar. For the preparation of media and BioReliance Study No. AA52XL.50200l.BTL 12 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse M utation Test with an Independent Repeat Assay_______________ DuPont-8868 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) Qxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Each plate was labeled with a code system that identified the test substance, test phase, dose level, tester strain, and activation, as described in detail in BioReliance^ Standard Operating Procedures. One-half (0.5) milliliter of S9 or Sham mix, 100 pL of tester strain and 25 pL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 452C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 pL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 372C. Plates that were not counted immediately following the incubation period were stored at 2-8C until colony counting could be conducted (less than 10 days). The condition o f the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown below. Code 1 2 3 4 5 6 Description Normal Slighdy Reduced M oderately Reduced Extremely Reduced Absent Obscured by Precipitate Characteristics Distinguished by a healthy microcolony lawn. Distinguished by a noticeable thinning of the microcolony lawn and possibly a slight increase in the size o f the microcolonies compared to the vehicle control plate. Distinguished by a marked thinning of the microcolony lawn resulting in a pronounced increase in the size of the microcolonies compared to the vehicle control plate. Distinguished by an extreme thinning of the microcolony lawn resulting in an increase in the size of the microcolonies compared to the vehicle control plate such that the microcolony lawn is visible to the unaided eye as isolated colonies. Distinguished by a complete lack of any microcolony lawn over greater than or equal to 90% of the plate. The background bacterial lawn cannot be accurately evaluated due to microscopic test substance precipitate. BioReliance Study No. AA52XL.502001.BTL 13 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse M utation Test with an Independent Repeat Assay_______________ DuPont-8868 Code NP IP Description Characteristics Distinguished by precipitate on the plate that is visible to the Non-Interfering naked eye but any precipitate particles detected by the automated Precipitate colony counter total less than 10% of the revertant colony count (e.g., less than 3 particles on a plate with 30 revertants). Distinguished by precipitate on the plate that is visible to the Interfering Precipitate naked eye and any precipitate particles detected by the automated colony counter exceed 10% o f the revertant colony count (e.g., more than 3 particles on a plate with 30 revertants). These plates are counted manually. Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Evaluation of Results For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains 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 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value. Criteria for a Valid Test The following criteria must be met for the mutagenicity test to be considered valid. All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the wvrB 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 -2 1 ; WP2 uvrA, 10-60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3xl09 cells/mL. The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels is required to evaluate test data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of BioReliance Study No. AA52XL.502001.BTL 14 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 revertants per plate as compared to die mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A moderate reduction in the background lawn (background code of 3 ,4 or 5). A copy of the Historical Negative and Positive Control Values is included in Appendix A. Archives All raw data, thg protocol, and all reports will be maintained according to Standard Operating P ro ce d u re flB H H flp y the BioReliance RAQA unit headquartered at: BioReliance, 14920 Broschart R oJiR ockviIl^ 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 o f 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 o f following administration to the test system and all residual test substance will be disposed o f 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. AA52XL.502001.BTL 15 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Solubility Test RESULTS AND DISCUSSION Tetrahydrofuran (THF) was selected as the solvent of choice based on compatibility with the target cells and solubility of the test substance. The test substance was soluble but cloudy in tetrahydrofuran (THF) at a maximum concentration of approximately 200 mg/mL. Preliminary Toxidty Test The results of the preliminary toxicity test are presented in Tables 1 through 4. These data were generated in Experiment A 1. In the preliminary toxicity test, the maximum dose tested was 5000 pg per plate; this dose was achieved using a concentration of 200 mg/mL and a 25 pL plating aliquot. The test substance was a workable solution from 40 to 200 mg/mL and a soluble and clear solution from 0.27 to 27 mg/mL. Dose levels tested were 6.7, 10, 33, 67, 100, 333, 667,1000, 3333 and 5000 pg per plate. Precipitate was observed beginning at 100 pg per plate. No appreciable toxicity was observed. Due to tester strain culture contamination, tester strain TA100 in the presence and absence of S9 activation was only evaluated for precipitate and toxicity. 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 o f the mutagenicity test are presented in Tables 5 through 24 and summarized in Tables 25 and 26. These data were generated in Experiments B1 and B2. Dose levels tested were 15, 50, 150, 500, 1500 and 5000 pg per plate. Precipitate was observed beginning at 500 pg per plate. No 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 mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. In Experiment B2 (Independent Repeat Test), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. CONCLUSION All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Test with an Independent Repeat Assay indicate that, under the conditions of this study, H-25171 did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9. BioReliance Study No. AA52XL.50200l.BTL 16 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 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. 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, DAI. 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. AA52XL.502001.BTL 17 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Preliminary Toxicity Test Table 1 Test Substance Id: H-25171 Study Number AA52XL.502001.BTL Experiment No. Al Strain TA98 Date Plated 20 Dec 2001 Vehicle tetrahydrofuran Plating Aliquot 25 uL Test Substance Concentration p.g per plate With S9 Activation Revertants Background per plate Lawn Vehicle 23 1 Without S9 Activation Revertants Background per plate Lawn 23 1 6.7 10 33 67 100 333 667 1000 3333 5000 33 1 22 1 24 1 34 1 29 1NP 23 1NP 22 1NP 24 1NP 22 1NP 30 1NP 14 1 19 1 16 1 14 1 13 1NP 13 1NP 17 1NP 15 1NP 12 1NP 15 1NP 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 BioReiiance Study No. AA52XL.50200l.BTL 18 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Preliminary Toxicity Test Table 2 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25171 AA52XL.502001.BTL Al TA1535 20 Dec 2001 tetrahydrofuran 25 uL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 15 1 Without S9 Activation Revertants Background per plate Lawn 22 1 6.7 10 33 67 100 333 667 1000 3333 5000 17 1 10 1 11 1 17 1 9 1NP 12 1NP 18 1NP 15 1NP 8 1NP 10 1NP 19 13 18 15 11 12 19 C 12 20 1 1 1 1 1NP 1NP 1NP 1NP 1NP 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. AA52XL.50200l.BTL 19 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Preliminary Toxicity Test Table 3 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25171 AA52XL.502001.BTL Al TA1537 20 Dec 2001 tetrahydrofuran 25 pL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 61 Without S9 Activation Revertants Background per plate Lawn 71 6.7 10 33 67 100 333 667 1000 3333 5000 10 1 71 71 51 11 1NP 10 1NP 10 1NP 4 1NP 9 1NP 10 1NP 51 12 1 71 61 6 1NP 8 1NP 12 1NP 8 1NP 7 1NP 6 1NP 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. AA52XL.502001.BTL 20 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Preliminary Toxicity Test Table 4 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25171 AA52XL.502001. BTL Al WP2 uvrA 20 Dec 2001 tetrahydrofuran 25 uL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 22 1 Without S9 Activation Revertants Background per plate Lawn 18 1 6.7 10 33 67 100 333 667 1000 3333 5000 22 1 29 1 13 1 20 1 15 1NP 19 1NP 21 1NP 25 1NP 18 1NP 26 1NP 22 1 14 1 17 1 15 1 21 1NP 13 1NP 17 1NP 31 1NP 11 1NP 18 1NP 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. AA52XL.502001.BTL 21 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Table 5 Test Substance Id: H-25171 Study Number : AA52XL.502001.BTL Strain : TA98 Liver Microsomes : None Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No Cells Seeded Date Plated B1 2.7 X 10 4 Jan 2002 Concentration Plate Revertants Background Average Standard jig per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 15 1 14 1 20 1 16 3 15 01 22 1 02 10 1 03 14 1 15 6 50 01 13 1 02 18 1 03 12 1 14 3 150 01 16 1 02 13 1 03 10 1 13 3 500 01 13 1NP 02 15 1NP 03 10 1NP 13 3 1500 01 14 1NP 02 14 1NP 03 18 1NP 15 2 5000 01 10 1NP 02 12 IIP 03 12 IIP 11 1 Positive Control 2-nitrofluorene 1. 0 jig per plate 01 217 1 02 184 1 03 232 1 211 25 Background Lawn Code l=Norraal; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate BioReliance Study No. AA52XL.502001.BTL 22 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Table 6 Test Substance Id: H-25171 Study Number : AA52XL.502001.BTL Strain : TA98 Liver Microsomes : Rat liver S9 Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : B1 Cells Seeded : 2.7 X 10s Date Plated : 4 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 33 1 20 1 29 1 27 7 15 01 26 1 02 26 1 03 23 1 25 2 50 01 17 1 02 25 1 03 17 1 20 5 150 01 28 1 02 32 1 03 21 1 27 6 500 01 23 1NP 02 25 1NP 03 20 1NP 23 3 1500 01 35 1NP 02 38 1NP 03 25 1NP 33 7 5000 01 18 IIP 02 25 IIP 03 20 IIP 21 4 Positive Control 2-aminoanthracene 1.0 pg per plate 01 480 1 02 483 1 03 338 1 434 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. AA52XL.502001.BTL 23 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________________________ DuPont-8868 Bacterial Mutation Test Table 7 Test Substance Id: H-25171 Study Number : AA52XL.502001.BTL Strain : TA100 Liver Microsomes : None Vehicle : tetrahydrofuran Plating Aliquot : 25 uL Experiment No : B1 Cells Seeded : 1.1 X 10B Date Plated : 4 Jan 2002 Concentration Plate Eevertants Background Average Standard ug per plate Number per plate Code Revertantsi Deviation Vehicle 01 02 03 223 1 190 1 182 1 198 22 15 01 168 1 02 163 1 03 179 1 170 8 50 01 166 1 02 191 1 03 188 1 182 14 150 01 199 1 02 179 1 03 150 1 176 25 500 01 140 1NP 02 145 1NP 03 C 143 4 1500 01 179 1NP 02 202 1NP 03 143 1NP 175 30 5000 01 163 1NP 02 206 1NP 03 192 1NP 187 22 Positive Control sodium azide 1.0 ug per plate 01 672 1 02 618 1 03 851 1 714 122 Background Lawn Code l=Normal; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate C=Contaminated BioReliance Study No. AA52XL.502001.BTL 24 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Table 8 Test Substance Id: H-25171 Study Number :AA52XL.502001.BTL Experiment No :B1 Strain :TA100 Cells Seeded :1.1 X 101 Liver Microsomes :Rat liver S9 Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 pL___________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 151 1 163 1 204 1 173 28 15 01 210 1 02 172 1 03 159 1 180 27 50 01 190 1 02 170 1 03 178 1 179 10 150 01 198 1 02 171 1 03 185 1 185 14 500 01 171 1NP 02 195 1NP 03 165 1NP 177 16 1500 01 214 1NP 02 187 1NP 03 177 1NP 193 19 5000 01 195 1NP 02 183 1NP 03 226 1NP 201 22 Positive Control 2-aminoanthracene 1.0 ug per plate 01 877 1 02 850 1 03 722 1 816 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. AA52XL.502001.BTL 25 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________________________DuPont-8868 Bacterial Mutation Test Table 9 Test Substance Id: H-:25171 Study Number : AA52XL.502001. BTL Strain : TA1535 Liver Microsomes ; None Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : B1 Cells Seeded : 2.6 X 10" Date Plated : 4 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 11 1 15 1 19 1 15 4 15 01 15 1 02 16 1 03 15 1 15 1 50 01 12 1 02 17 1 03 11 1 13 3 150 01 23 1 02 19 1 03 15 1 19 4 500 01 13 1NP 02 22 1NP 03 15 1NP 17 5 1500 01 12 1NP 02 15 1NP 03 8 1NP 12 4 5000 01 18 IIP 02 12 IIP 03 19 1NP 16 4 Positive Control. sodium azide 1.0 jig per plate 01 402 1 02 476 1 03 414 1 431 40 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 BioReliance Study No. AA52XL.502001.BTL 26 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse M utation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Table 10 Test Substance Id: H-25171 Study Number :AA52XL.502001.BTL Experiment No :B1 Strain :TA1535 Cells Seeded :2.6 X 10s Liver Microsomes :Rat liver S9 Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 uL___________________________________________ Concentration Plate Revertants Background Average Standard Ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 15 1 21 1 25 1 20 5 15 01 26 1 02 24 1 03 21 1 24 3 50 01 12 1 02 16 1 03 16 1 15 2 150 01 20 1 02 10 1 03 14 1 15 5 500 01 14 1NP 02 20 1NP 03 15 1NP 16 3 1500 01 14 1NP 02 14 1NP 03 8 1NP 12 3 5000 01 23 1NP 02 13 1NP 03 21 1NP 19 5 Positive Control 2-aminoanthracene 1.0 pg per plate 01 173 1 02 128 1 03 146 1 149 23 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. AA52XL.502001.BTL 27 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Table 11 Test Substance Id: H-25171 Study Number :AA52XL.502001.BTL Experiment No :B1 Strain :TA1537 Cells Seeded :1.3 X 10B Liver Microsomes :None Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 pL___________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 31 71 41 52 15 01 5 1 02 8 1 03 1 1 54 50 01 3 1 02 8 1 03 3 1 53 150 01 11 1 02 5 1 03 6 1 73 500 01 4 1NP 02 5 1NP 03 1NP 51 1500 01 5 1NP 02 3 1NP 03 5 1NP 41 5000 01 9 IIP 02 8 IIP 03 3 IIP 73 Positive Control 9-aminoacridine 75 pg per plate 01 543 1 02 352 1 03 616 1 504 136 Background Lawn Code l=Normal; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA52XL.502001.BTL 28 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Table 12 Test Substance Id: H-25171 Study Number :AA52XL.502001.BTL Experiment No :B1 Strain :TA1537 Cells Seeded :1.3 X 108 Liver Microsomes :Rat liver S9 Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 pL___________________________ ______________ _ Concentration Plate Revertants Background Average Standard Ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 81 13 1 81 10 3 15 01 3 1 02 13 1 03 8 1 85 50 01 7 1 02 6 1 03 10 1 82 150 01 9 1 02 7 1 03 10 1 92 500 01 4 1NP 02 8 1NP 03 9 1NP 73 1500 01 6 1NP 02 8 1NP 03 4 1NP 62 5000 01 9 IIP 02 6 IIP 03 5 1NP 72 Positive Control 2-aminoanthracene 1.0 ug per plate 01 96 1 02 103 1 03 84 1 94 10 Background Lawn Code 1=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. AA52XL.502001 .BTL 29 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Bacterial Mutation Test Table 13 Test Substance Id: H-25171 Study Number :AA52XL.502001.BTL Experiment No :B1 Strain :WP2 uvrA Cells Seeded :2.5 X 10s Liver Microsomes :None Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 p L _____________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 14 1 12 1 15 1 14 2 15 01 17 1 02 14 1 03 13 1 15 2 50 01 16 1 02 12 1 03 11 1 13 3 150 01 16 1 02 14 1 03 10 1 13 3 500 01 11 1NP 02 10 1NP 03 17 1NP 13 4 1500 01 13 1NP 02 18 1NP 03 12 1NP 14 3 5000 01 11 IIP 02 10 IIP 03 8 IIP 10 2 Positive Control methyl methanesulfonate 1000 ug per plate 01 89 1 02 92 1 03 97 1 93 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent ; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate 4 BioReliance Study No. AA52XL.502001.BTL 30 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________________________ DuPont-8868 Bacterial Mutation Test Table 14 Test Substance Id: H-25171 Study Number : AA52XL.502001. BTL Strain : WP2 uvrA Liver Microsomes : Rat liver S9 Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : B1 Cells Seeded : 2. 5 X 108 Date Plated : 4 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 16 1 19 1 12 1 16 4 15 01 18 1 02 16 1 03 11 1 15 4 50 01 21 1 02 16 1 03 14 1 17 4 150 01 10 1 02 16 1 03 13 1 13 3 500 01 14 1NP 02 10 1NP 03 11 1NP 12 2 1500 01 15 1NP 02 16 1NP 03 13 1NP 15 2 5000 01 14 IIP 02 17 1NP 03 9 1NP 13 4 Positive Control 2-aminoanthracene 10 pg per plate 01 965 1 02 584 1 03 749 1 766 191 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. AA52XL.502001.BTL 31 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________________________ DuPont-8868 B a c te ria l M utation T e st T a b le 15 T e s t S u b sta n c e Id : H-25171 S tu d y Number : A A 52XL.5 0 2 0 0 1 .BTL S tra in : TA98 L iv e r M icrosom es : None V ehicle : tetrah y d ro fu ran P la tin g A lig u o t : 25 uL E x p e r im e n t No : B2 C e l l s S e e d e d : 1 . 0 X 10 D ate P la te d : 15 J a n 2002 C o n cen tratio n pg p er p la te P la te R e v e rta n ts Background A verage S tandard Number p e r p la te Code R ev ertan ts D eviation V ehicle 01 02 03 37 1 24 1 21 1 27 9 15 01 24 1 02 16 1 03 29 1 23 7 50 01 29 1 02 16 1 03 27 1 24 7 150 01 29 1 02 27 1 03 27 1 28 1 500 01 19 1NP 02 15 1NP 03 15 1NP 16 2 1500 01 24 IIP 02 32 I I P 03 13 I I P 23 10 5000 01 37 I I P 02 31 I I P 03 14 I I P 27 12 P o s itiv e C o n tro l 2 -n itro flu o re n e 1 .0 pg p e r p la te 01 158 1 02 98 1 03 171 1 142 B ack g ro u n d Lawn Code l= N o r m a l; ;2 = S l i g h t l y r e d u c e d ; 3 = M o d e r a te ly r e d u c e d 4= E xtrem ely reduced;; 5=A bsent; 6=O bscured by p r e c i p i t a t e N P = N on-Interfering p r e c ip ita te ; IP = In te rfe rin g p r e c ip ita te 39 BioReliance Study No. AA52XL.502001.BTL 32 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 B a c te ria l M utation T est T a b le 16 T e st S u b sta n c e Id : H-25171 S tudy Number : AA52XL.5 0 2 0 0 1 .BTL E x p e r im e n t No : B2 S tra in : TA98 C e l l s S e e d e d : 1 . 0 X10 L iv e r M icrosom es : R at l i v e r S9 D ate P la te d : 15Ja n 2002 V ehicle : tetrah y d ro fu ran P la tin g A liq u o t : 25 p L _________________________________________ C o n cen tratio n pg p er p la te P la te R e v e rta n ts B ackground A verage S tandard Number p e r p l a t e Code R ev ertan ts D ev iatio n V ehicle 01 02 03 32 1 16 1 26 1 25 8 15 01 28 1 02 26 1 03 31 1 28 3 50 01 47 1 02 35 1 03 18 1 33 15 150 01 49 1 02 35 1 03 44 1 43 7 500 01 38 1NP 02 35 1NP 03 20 1NP 31 10 1500 01 34 1NP 02 43 1NP 03 27 1NP 35 8 5000 01 24 I I P 02 37 I I P 03 32 I I P 31 7 P o sitiv e C o n tro l 2-am inoanthracene 01 631 02 718 03 852 1 .0 pg p e r p la te 1 1 1 734 111 B ackground Lawn Code l= N orm al; 2 = S lig h tly red u ced ; 3= M oderately red u ced 4= E xtrem ely red u ced ; 5=A bsent; 6=O bscured by p r e c i p i t a t e N P = N on-Interfering p r e c ip ita te ; IP = In te rfe rin g p r e c ip ita te BioReliance Study No. AA52XL.502001.BTL 33 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay __________________________________ DuPont-8868 B a c te ria l M utation T est T a b le 17 T e s t S u b sta n c e Id : H-25171 S tu d y Number : AA52XL.5 0 2 0 0 1 .BTL S tra in : TA100 L iv e r M icrosom es : None V eh icle : tetrah y d ro fu ran P la tin g A liq u o t : 25 uL E x p e r im e n t No : B2 C e lls Seeded : 0 .9 X 108 D ate P la te d : 15 Ja n 2002 C oncentrt i on pig p e r p l a t e P la te R e v e rta n ts Background A verage S tandard Number p e r p l a t e Code R ev ertan ts D ev iatio n V ehicle 01 02 03 140 1 125 1 100 1 122 20 15 01 154 1 02 150 1 03 178 1 161 15 50 01 156 1 02 C 03 178 1 167 16 150 01 148 1 02 126 1 03 167 1 147 21 500 01 178 1NP 02 127 1NP 03 156 1NP 154 26 1500 01 152 1NP 02 131 1NP 03 132 1NP 138 12 5000 01 104 IIP 02 13 6 I I P 03 C 120 23 P o sitiv e C o n tro l s o d iu m 01 02 03 a z id e 1 .0 347 364 387 pig p e r 1 1 1 p la te 366 20 B ackground Lawn Code l = N o r m a l ; :2 = S l i g h t l y r e d u c e d ; :3 = M o d e r a te ly r e d u c e d 4 = E x tre m e ly re d u c e d 5=A b se n t; 6= O b scu red b y p r e c i p i t a t e N P = N on-Interfering p re c ip ita te ,; IP = In te rfe rin g p r e c ip ita te C =C ontam inated BioReliance Study No. AA52XL.502001.BTL 34 Company Sanitized. Does not contain TSCA C8I H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 B a c te ria l M utation T e st T a b le 18 T e s t S u b sta n c e Id : H-25171 S tu d y Number : AA52XL.5 0 2 0 0 1 .BTL E x p e r im e n t No : B2 S tra in : TA100 C e l l s S e e d e d : 0 .9 X1 0 8 L iv e r M icrosom es : R at l i v e r S9 D ate P la te d : 15J a n 2002 V eh icle : tetrah y d ro fu ran P l a t i n g A l i q u o t : 25 pL_____________________________________________ ____ C o n cen tratio n pg p er p la te P la te R e v e rta n ts Background A verage S tandard Number p e r p l a t e Code R ev ertan ts D ev iatio n V ehicle 01 02 03 171 1 152 1 193 1 172 21 15 01 207 1 02 190 1 03 156 1 184 26 50 01 182 1 02 169 1 03 181 1 177 7 150 01 166 1 02 150 1 03 161 1 159 8 500 01 142 1NP 02 168 1NP 03 144 1NP 151 14 1500 01 151 I I P 02 171 I I P 03 116 I I P 146 28 5000 01 187 I I P 02 169 I I P 03 156 I I P 171 16 P o sitiv e C o ntrol 2-am inoanthracene 01 837 02 714 03 664 1 .0 pg p e r p la te 1 1 1 738 B ackground Lawn Code l= N orm al; 2 = S lig h tly red u ced ; 3= M oderately red u ced 4 = E x tre m e ly re d u c e d ; 5=A b s e n t; 6 = O b scu red b y p r e c i p i t a t e N P = N on-Interfering p r e c ip ita te ; IP = In te rfe rin g p r e c ip ita te 89 BioReliance Study No. AA52XL.502001.BTL 35 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 B a c te ria l M utation T e st T a b le 19 T e s t S u b sta n c e Id : H-25171 S tu d y Number : AA52XL.5 0 2 0 0 1 .BTL E x p e r im e n t No : B2 S tra in : TA1535 C e l l s S e e d e d : 0 . 9 X10 L iv e r M icrosom es : None D ate P la te d : 15Ja n 2002 V eh icle : tetrah y d ro fu ran P l a t i n g A l i q u o t : 2 5 uL________________________________________________________ C o n cen trt io n yg p er p la te P la te R e v e rta n ts B ackground A verage S tandard Number p e r p l a t e Code R ev ertan ts D ev iation V ehicle 01 02 03 13 1 81 12 1 11 3 15 01 16 1 02 10 1 03 11 1 12 3 50 01 13 1 02 11 1 03 9 1 11 2 150 01 11 1 02 16 1 03 11 1 13 3 500 01 9 I I P 02 8 I I P 03 11 1NP 92 1500 01 12 I I P 02 18 I I P 03 11 I I P 14 4 5000 01 13 I I P 02 13 I I P 03 16 I I P 14 2 P o s itiv e C o n tro l sodium a z id e 1 .0 yg p e r p la te 01 179 1 02 216 1 03 185 1 193 B ackground Lawn Code l= N orm al; 2 = S lig h tly red u ced ; 3M oderately red u ced 4= E xtrem ely red u ced ; 5=A bsent; 6= 0bscured by p r e c i p i t a t e N P = N o n -In terferin g p r e c ip ita te ; IP = In te r e rin g p r e c ip ita te 20 BioReliance Study No. AA52XL.502001.BTL 36 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 B a c te ria l M utation T est T ab le 20 T e s t S u b sta n c e Id : H-25171 S tu d y Number : A A 52X L.502001.BTL E x p e r im e n t No : B2 S tra in : TA1535 C e l l s S e e d e d : 0 . 9 X10 L iv e r M icrosom es : R a t l i v e r S9 D ate P la te d : 15J a n 2002 V eh icle : tetrah y d ro fu ran P l a t i n g A l i q u o t : 25 pL________________________________________________________ C o n cen tratio n pg p er p la te P la te R ev ertan ts B ackground A verage S tan d ard Number p e r p l a t e Code R ev ertan ts D ev iatio n V ehicle 01 02 03 13 1 81 11 1 11 3 15 01 NC 02 14 1 03 9 1 12 4 50 01 12 1 02 10 1 03 19 1 14 5 150 01 13 1 02 13 1 03 13 1 13 0 500 01 14 1NP 02 18 1NP 03 10 1NP 14 4 1500 01 10 I I P 02 17 I I P 03 10 I I P 12 4 5000 01 19 I I P 02 8 I I P 03 11 I I P 13 6 P o sitiv e C ontrol 2-am inoanthracene 01 142 02 106 03 88 1 .0 pg p e r p la te 1 1 1 112 B ackground Lawn Code l= N orm al; 2= S lig h tly red u ced ; 3= M oderately red u ced 4= E xtrem ely re d u c e d ; 5=A bsent; 6=O bscured by p r e c i p i t a t e N P = N on-Interfering p r e c ip ita te ; IP = In te rfe rin g p r e c ip ita te NC=No c o u n t d u e t o p r o c e d u r a l e r r o r i n w h ic h t h e p l a t e d i d receiv e an a liq u o t of te s te r s tra in 27 not BioReliance Study No. AA52XL.502001.BTL 37 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse M utation Test with an Indepndgnt Repeat Assay_______________ DuPont-8868 B a c te ria l M utation T e st T a b le 21 T e st S u b stan ce Id : H-25171 S tu d y Number : AA52XL.5 0 2 0 0 1 .BTL S tra in : TA1537 L iv e r M icrosom es : None V eh icle : tetrah y d ro fu ran P la tin g A liq u o t : 25 uL E x p e r i m e n t No : B2 C e l l s S e e d e d : 0 .5 X10* D ate P la te d : 15J a n 2002 C o n cen tratio n ug p er p la te P la te R e v e rta n ts Background A verage S tandard Number p e r p la te Code R ev ertan ts D ev iatio n V ehicle 01 02 03 51 21 41 42 15 01 6 1 02 6 1 03 4 1 51 50 01 7 1 02 7 1 03 4 1 62 150 01 5 1 02 2 1 03 6 1 42 500 01 7 1NP 02 4 1NP 03 4 1NP 52 1500 01 8 IIP 02 4 I I P 03 3 I I P 53 5000 01 7 I IP 02 7 I I P 03 4 I I P 62 P o sitiv e C o ntrol 9-am in o acrid in e 01 663 02 342 03 641 75 ug p e r 1 1 1 p la te 549 179 B ack g ro u n d Lawn Cde l= N orm al; 2 = S lig h tly red u ced ; 3= M oderately red u ced 4=E xtrem ely red u c e d ; 5= A bsent; 6=O bscured by p r e c ip ita te N P = N on-Interfering p r e c ip ita te ; IP = In te rfe rin g p r e c ip ita te BioReliance Study No. AA52XL.502001.BTL 38 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________________________ DuPont-8868 B a c te ria l M utation T e st T a b le 22 T e s t S u b sta n c e Id : H-25171 S tu d y Number : AA52XL .5 0 2 0 0 1 .BTL S tra in : TA1537 L iv e r M icrosom es : R a t l i v e r S9 V ehicle : tetrah y d ro fu ran P la tin g A liq u o t : 25 pL E x p e r im e n t No : B2 C e l l s S e e d e d : 0 . 5 X 10 D ate P la te d : 15 Jan 2002 C o n cen tratio n pg p e r p la te P la te R e v e rta n ts B ackground A verage S tandard Number p e r p la te Code R ev ertan ts1 D ev iatio n V ehicle 01 02 03 51 61 81 62 15 01 4 1 02 3 1 03 6 1 42 50 01 4 1 02 5 1 03 12 1 74 150 01 8 1 02 9 1 03 6 1 82 500 01 5 1NP 02 3 1NP 03 7 1NP 52 1500 01 3 I I P 02 6 I I P 03 6 I I P 52 5000 01 3 I I P 02 5 I I P 03 7 I I P 52 P o s itiv e C o n tro l 2-am in o an th racen e 1 .0 pg p e r p la te 01 96 1 02 70 1 03 74 1 80 B ack g ro u n d Lawn Code l= N o rm a l; 2= S l i g h t l y re d u c e d ; 3 M o d e ra te ly re d u c e d 4= E xtrem ely red u ced ; 5=A bsent; 6=O bscured by p r e c ip ita te N P = N on-Interfering p r e c ip ita te ; IP = In te rfe rin g p r e c ip ita te 14 BioReliance Study No. AA52XL.502001.BTL 39 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________________________ DuPont-8868 B a c te ria l M utation T est T a b le 23 T e s t S u b sta n c e Id : H-25171 S tu d y Number : AA52XL.502001.BTL S tra in : WP2 u v rA L iv e r M icrosom es : None V eh icle : tetrah y d ro fu ran P la tin g A liq u o t : 25 pL E x p e r im e n t No : B2 C e lls Seeded : 2 .1 X 108 D ate P la te d : 15 Jan. 2002 C o n cen tratio n ug p er p la te P la te R e v e rta n ts B ackground A verage S tandard Number p e r p l a t e Code R ev ertan ts D ev iatio n V ehicle 01 02 03 11 1 11 1 10 1 11 1 15 01 10 1 02 7 1 03 9 1 92 50 01 15 1 02 12 1 03 7 1 11 4 150 01 4 1 02 9 1 03 11 1 84 500 01 7 1NP 02 17 1NP 03 7 1NP 10 6 1500 01 C 02 12 I I P 03 13 I I P 13 1 5000 01 13 I I P 02 14 I I P 03 14 I I P 14 1 P o sitiv e C o n tro l m ethyl 01 02 03 m ethanesulfo n ate 140 1 81 1 138 1 1000 pg p e r p la te 120 B ack g ro u n d Lawn Code l= N orm al; 2 = S lig h tly red u ced ; 3M oderately red u ced 4= E xtrem ely red u ced ; 5"A bsent ; 6O bscured by p r e c ip ita te N P = N on-Interfering p r e c ip ita te ; IP = In te rfe rin g p r e c ip ita te C =C ontam inated 34 BioReliance Study No. AA52XL.502001.BTL 40 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________________________________ DuPont-8868 B a c te ria l M utation T est ' T a b le 24 T e s t S u b sta n c e Id : H-25171 S tu d y Number : A A52XL.5 0 2 0 0 1 .BTL E x p e r im e n t No : B2 S tra in : WP2 u v rA C e l l s S e e d e d : 2 . 1 X 10 L iv e r M icrosom es : R at l i v e r S9 D ate P la te d : 15 J a n 2002 V ehicle : tetrah y d ro fu ran P l a t i n g A l i q u o t : 25 ytL________________________________________________________ C o n cen tratio n ug p er p la te P la te R e v e rta n ts B ackground A verage S tandard Number p e r p la te Code R ev ertan ts D ev iatio n V ehicle 01 02 03 16 1 22 1 10 1 16 6 15 01 20 1 02 13 1 03 15 1 16 4 50 01 13 1 02 12 1 03 15 1 13 2 150 01 17 1 02 16 1 03 10 1 14 4 500 01 13 1NP 02 8 1NP 03 16 1NP 12 4 1500 01 14 I I P 02 12 I I P 03 13 I I P 13 1 5000 01 13 I I P 02 7 I I P 03 12 I I P 11 3 P o s itiv e C o n tro l 2 -a m in o a n th ra c e n e 10 pg p e r p l a t e 01 816 1 02 1128 1 03 938 1 961 157 B ack g ro u n d Lawn Code l= N orm al; 2 = S lig h tly red u ced ; 3= M oderately red u ced 4 = E x tre m e ly re d u c e d ; 5=A b s e n t; 6 = 0 b sc u re d b y p r e c i p i t a t e N P = N on-Interfering p r e c ip ita te ; IP = In te rfe rin g p r e c ip ita te BioReliance Study No. AA52XL.502001.BTL 41 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 B a c te ria l M utation T est Summary o f R e s u lts T ab le 25 T e st S u b sta n c e Id : H-25171 S t u d y N um ber________; AA52XL.5 0 2 0 0 1 .BTL_______ E x p e r im e n t No : B1 A v e r a g e R e v e r t a n t s P e r P l a t e : S t a n d a r d D e v i a t i o n L iv e r M icrosom es: None D ose ( u g / p l a t e ) TA98 TA100 V ehicle 15 50 150 500 1500 5000 P o sitiv e 16 3 198 22 15 6 170 8 14 3 182 14 13 3 176 25 13 3 143 4 15 2 175 30 11 1 187 22 211 25 714 122 L iv e r M ic ro so m e s: R a t l i v e r S9 TA1535 15 15 13 19 17 12 16 431 t 4 1 3 4 5 4 4 40 TA1537 5 2 5 4 5 3 7 3 5 1 4 1 7 3 504 136 WP2 u v rA 14 15 13 13 13 14 10 93 2 2 3 3 4 3 2 4 D ose ( u g / p l a t e ) TA98 TA100 V ehicle 15 50 150 500 1500 5000 P o sitiv e 27 7 173 28 25 2 180 27 20 5 179 10 27 6 185 14 23 3 177 16 33 7 193 19 21 4 201 22 434 83 816 83 V ehicle = V eh icle C o ntrol P o sitiv e = P o s itiv e C ontrol P la tin g a l i q u o t : 25 uL TA1535 20 24 15 15 5 3 2 5 16 3 12 3 19 5 149 23 TA1537 10 8 8 9 7 6 7 94 3 5 2 2 3 2 2 10 WP2 u v rA 16 4 15 4 17 4 13 3 12 2 15 2 13 4 766 191 BioReliance Study No. AA52XL.502001.BTL 42 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 B a c te ria l M utation T est Summary o f R e s u lts T a b le 26 T e st S u b sta n c e Id : H-25171 S t u d y N um ber________: A A 52XL.5 0 2 0 0 1 .BTL_______ E x p e r im e n t No : B2 A verage R e v e rta n ts Per P la te S tan d ard D ev iatio n L iv e r M icrosom es : None D ose ( u g / p l a t e ) TA98 TA100i V ehicle 15 50 150 500 1500 5000 P o sitiv e 27 9 122 20 23 7 161 15 24 * 7 167 16 28 1 147 21 16 2 154 26 23 10 138 12 27 12 120 23 142 39 366 20 L iv e r M icro so m es: R at l i v e r S9 TA1535 11 12 11 13 9 14 14 193 3 3 2 3 2 4 2 20 TA1537 4t 2 5 1 6 2 4 2 5 2 5 3 6 2 549 179 WP2 u vrA 11 9 11 8 10 13 14 120 1 2 4 4 6 1 1 34 D ose ( u g / p l a t e ) TA98 TA100 V ehicle 15 50 150 500 1500 5000 P o sitiv e 25 8 28 3 33 15 43 7 31 10 35 8 31 7 734 111 172 184 177 159 151 146 171 738 21 26 7 8 14 28 16 89 V ehicle = V eh icle C ontrol P o sitiv e = P o s itiv e C ontrol P l a t i n g a l i q u o t : 25 pL TA1535 11 12 14 13 14 12 13 112 3 4 5 0 4 4 6 27 TA1537 6 4 7 8 5 5 5 80 2 2 4 2 2 2 2 14 WP2 u vrA 16 6 16 4 13 2 14 4 12 4 13 1 11 3 961 157 BioReliance Study No. AA52XL.502001.BTL 43 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 APPENDIX A Historical Control Data BioReliance Study No. AA52XL.502001.BTL 44 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay ___________ DuPont-8868 Historical Negative and Positive Control Values 1998 - 2000 revertants per plate Activation Strain Control None Rat Liver Mean SD Min Max Mean SD Min Max TA98 Neg 16 7 4 59 21 7 7 58 Pos 425 206 21 1536 592 322 56 2454 TA100 Neg 128 31 53 288 138 34 74 258 Pos 568 159 129 1371 736 301 198 2871 TA1535 Neg 12 5 1 45 12 4 1 42 Pos 378 164 6 978 104 84 18 1640 TA1537 Neg 6 3 0 30 73 1 29 Pos 708 409 13 2786 88 106 12 2060 WP2 uvrA Neg Pos 14 5 4 190 138 34 48 961 16 6 317 299 4 115 22 2632 SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negalive control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control BioReliance Study No. AA52XL.502001.BTL 45 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 APPENDIX B Study Protocol BioReliance Study No. AA52XL.502001.BTL 46 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 deceived by R N O Sponsor Project Number: DuPont-8868 BioReliance Study Number: AA52XL502001.BTL 1 H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay 1.0 PURPOSE The purpose of this study is to evaluate the mutagenic potential of die test substance by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA in the presence and absence of S9 activation. 2.0 SPONSOR 2.1 Name: E.I. du Pont de Nemours and Company 2 2 Address: Stine Haskell Research Center DuPont Haskell Laboratory P.O.Box 50 1090 Elkton Road Newark, DE 19714-0050 2.3 Representative: Maria Donner, PhD. Phone: 302-366-5251 Fax: 302-366-5207 Email: maria.donncr@usa.dupont.com 2.4 Sponsor Project No.: DuPont-8868 2.5 WR#: 2.6 Haskell #: 2.7 Service Code: H-25171 (ml 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance Name: 32 Test Substance I.D.: H-25171 (to be used in the report title and text) 3.3 Controls: Negative: Positive: Test substance vehicle 9-aminoacridine 2-aminoanthracene methyl methanesulfonate 2-nhrofluorene sodium azide Protocol SPGT50200I 1-Jan-2001 Page 1 o f 11 BioReliance Study No. AA52XL.502001.BTL 47 ^ Bio Reliancf Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with ,m ^ dependent Repeat Assay_______________ DuPont-8868 Sponsor Project Number DuPont-8868 BioReliance Study Number AA52XL50200I.BTL 3.4 Test Substance Characterization Unless alternate arrangements are made, the testing facility at BioReliance will not perform analysis of foe dosing solutions. The Sponsor will be directly responsible for determination and documentation of foe analytical purity and composition of foe test substance, and the stability and strength of the test substance in foe solvent (or vehicle). 3.5 Test Substance Retention Sample The retention of a reserve sample of foe test substance will be foe responsibility of foe 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: 07-Dec-2001 5.2 Experimental Termination Date: 25-Jan-2002 5.3 Draft Report Date: 08-Feb-2002 5.4 Final Report Date: 2 weeks after Sponsor approves draft TEST SYSTEM The tester strains will include the S. typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames el al. (1975) and the coli tester strain WP2 uvrA as described by Green and Muriel (1976).___________________________ Histidine Mutation Tryptopha n Mutation Additional Mutations hisGA6 hisC3076 A&D3052 trpE LPS Repair R-factor TA1535 TA1537 - - rfa AvrB - Protocol SPGT502001 l-Jan-2001 Page 2 o f 11 ^ Bio Reliancf BioReliance Study No. AA52XL.502001.BTL 48 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Sponsor Project Number DuPont-8868 BioReliance Study Number AA52XL502001 .BTL Histidine Mutation hisG46 TA100 - AC3076 - AisD3052 TA98 - Tryptopha n Mutation trpE WP2 uvrA Additional Mutations LPS Repair R-fector rfa AuvrB +R - yuvrA - Each S. typhimurium tester strain contains, in addition to a mutation in the histidine opcron, 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 avrB gene results in a deficient DNA excision-repair system. Tester strains TA98 and TA100 also contain the pKMIOl plasmid (carrying the R-fector). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with die mismatch-repair process. TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both fiameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions. The K coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2 uvrA has a deletion in die 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 die original mutation to be suppressed. Thus, die 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 of California, Berkeley. The 1 coli tester strain was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland (United Kingdom). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The test substance will be tested at a minimum of five dose levels along with appropriate negative and positive controls with tester strains TA98, TA100, TAI535, TA1537 and WP2 uvrA with and without S9 activation. All dose levels of test substance, negative controls and positive controls will be plated in triplicate. Protocol SPGT502001 l-Jan-2001 Page 3 of 11 BioReliance Study No. AA52XL.502001.BTL 49 ^ Bio Reliancf Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Sponsor Project Number. DuPont-8868 BioReliancc Study Number; AA52XL502001 .BTL 7.1 Solubility Determination Unless the Sponsor has indicated the test substance vehicle, a solubility determination will be conducted to determine the maximum soluble concentration or workable suspension to a maximum of 50 rag/mL for aqueous vehicles and 500 mg/mL for organic vehicles. Vehicles compatible with this test system, in order of preference, include but arc 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 the highest workable/soluble stock concentration, up to 50 mg/mL for aqueous vehicles and 500 mg/mL for organic vehicles. 1 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 substance assessed in a preliminary toxicity assay. This preliminary assay will be conducted by exposing TA98, TA100, TA1535, TA1537 and WP2 uvrA to negative controls and to at least eight concentrations of test substance, one plate per dose level, in both the presence and absence of S9 activation. Unless indicated otherwise by the Sponsor, 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 foe mutagenicity assay foe following guidelines will be employed. Doses will be selected such that precipitate does not interfere with manual scoring. Whenever possible, the highest dose for foe mutagenicity assay mil be selected to give some indication of toxicity without exceeding 5 mg/plate. For freely soluble, nontoxic test substances, foe highest dose level will be 5 mg/plate. For precipitating, nontoxic test substances, the highest dose level will be selected in an attempt to yield precipitate at only foe 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) foe maximum achievable test substance dose level is less than 5 mg/plate and this dose level is nontoxic. 7.3 Frequency and Route ofAdministration The test system will be exposed to foe test substance via foe plate incorporation methodology originally described by Ames el a!, (1975) and updated by Maron and Ames (1983). This test system has been shown to detect a wide range of classes ofchemical mutagens (McCann et a!., 1975; McCann and Ames, 1976). After the data generated in the first assay have been evaluated, the mutagenicity assay mil be repeated. The dose levels used in foe second assay will be foe same as those used in foe first assay unless foe Study Director determines that the dose levels should be changed due to an equivocal response, excessive cytotoxicity or Protocol SPGT502001 l-Jan-2001 Page 4 of 11 ^ Bio Reliancf BioReliance Study No. AA52XL.502001.BTL 50 Company Sanitized. Does not contain TSCA CBI H-2S171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Sponsor Project Number DuPont-8868 BioReliance Study Number: AA52XL502001 ,BTL excessive precipitate. If the Sponsor is aware of specific metabolic requirements (e.g., azo compoundsX this information will be utilized in designing the assay, (e.g., activatitm system or treatment method). This guidance is based on the OECD Guideline 471 (adopted July 1997 and published February 1998) and ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997). 7.4 Controls 7.4.1 Positive Controls All combinations of positive controls and tester strains plated concurrently with the assay are listed below: _________________ ____________ Strain S9 Activation Positive Control Concentration (jig/jplate) Salmonella Strains WP2 irvrA Rat 2-aminoanthracene 1.0 10 TA98 2-nitrofluorene 1.0 TA100, TA1535 TA1537 None sodium azide 9-aminoacridine 1.0 75 WP2 uvrA methyl methanesulfonate 1,000 7.4.2 Negative Controls Appropriate negative controls will be plated for each tester strain with and without S9 activation. The negative control will be the vehicle alone, unless there is no historical basis for use of the selected vehicle. In the latter case, both untreated and vehicle controls will be used. 7.4.3 Sterility Controls The most concentrated test substance dilution and the Sham and S9 mixes will be checked for sterility. 15 Exogenous Metabolic Activation Aroclor 1254-induced rat liver S9 will be used as the metabolic activation system. The S9 homogenate will be prepared from male Sprague-Dawley tats induced with a single intraperitoneal injection of Aroclor 1254,500 tng/kg, five days prior to sacrifice. The S9 will be batch prepared and stored frozen at approximately Protocol SPGT502001 l-Jan-2001 Page 5 of 11 mbBio Reliance BioReliance Study No. AA52XL.50200l.BTL 51 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Sponsor Project Number DuPont-8868 BioReliance Study Number AA52XLS0200I .BTL -70C until used. Each batch of S9 homogenate will be assayed for its ability to metabolize 2-aminoanthraoene and 7,12-dimethylbenzanthracene to forms mutagenic to S. typhimurhm TA100. Immediately prior to use, the S9 will be thawed and mixed with a cofactor pool to contain 10% S9 homogenate, SmM ghicose-6-pbosphate, 4 mM p-nicotinamide-ademne dinucleotide phosphate, 8 mM MgCl2and 33 mM KG 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 phase, the length of incubation will be controlled and monitored. At the end of the working day, each inoculated flask will be placed in a testing shaker/incubalor 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 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 substance, test phase, dose level, tester strain and activation type as described in BioReliance's Standard Operating Procedures. 7.8 Test Substance Preparation Unless specified otherwise, test substance dilutions will be prepared immediately prior to use. All test substance dosing will be at room temperature under yellow light 7.9 Treatment ofTest System One half milliliter (0.5 mL) of S9 mix or Sham mix, 100 of tester strain and 50 |jL of vehicle, test substance dilution or positive control will be added to 2.0 mL of molten selective top agar at 452C. When necessary to achieve die target concentration or eliminate toxic vehicle effects, aliquots of other than 50 of test substance/vehicle/positive control will be plated. The mixture will be vortex mixed and overlaid onto the surface of 25 mL of minimal bottom agar. Protocol SPGT502001 1-Jan-2001 Page 6 of 11 ^ (m Bio Reliance* BioReliance Study No. AA52XL.502001 .BTL 52 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Sponsor Project Number DuPont-8868 BioReliance Study Number AAS2XL502001.BTL After the overlay has solidified, the plates will be inverted and incubated for approximately 48 to 72 hours at 372C. Plates that are not counted immediately following the incubation period will be stored at 2-8C. 7.10 Scoring The condition of the bacterial background lawn will be evaluated for evidence of test substance toxicity and precipitate. Evidence of toxicity will be scored relative to the negative control plate and recorded along with the revertant count for that plate. Toxicity will be evaluated as a decrease in the number of revertant colonies per plate and/or a thinning or disappearance of the bacterial background lawn. Precipitation will be evaluated after the incubation period by visual examination without magnification. 7.11 Tester Strain Verification On the day of use in the mutagenicity assay, all tester strain cultures will be checked for the appropriate genetic markets 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 & typhimurhon tester strain cultures must exhibit sensitivity to crystal violet To demonstrate die presence of the wvrB mutation, all S. typhimurium tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate the presence of the irvrA mutation, all coU tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate the presence of the pKMIOl plasmid R-factor, tester strain cultures of TA98 and TA100 must exhibit resistance to ampicillin. 8.2 Spontaneous Revertant Background Frequency Based on historical control data, all tester strain cultures must exhibit characteristic number of spontaneous revertants per {date 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. 83 Tester Strain Titers To ensure that appropriate numbers of bacteria are plated, all tester strain culture titers must be equal to or greater than 0.3x10*cells pet milliliter. Protocol SPGT502001 l-Jan-2001 Page 7 of 11 BioReliance Study No. AA52XL.502001.BTL 53 (m Bio Reliancf Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse M utation Test with an Independent Repeat Assay_______________ DuPont-8868 Sponsor Project Number DuPont-8868 BioReliance Study Number AAS2XL502001,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) far 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 of revertants per plate relative to the mean negative control value (this reduction must be accompanied by an abrupt dose-dependent drop in the rrvertant count) or a reduction in the background lawn. In the event that less than three non-toxic dose levels are achieved, the affected portion of the assay will be repeated with an appropriate change in dose levels. 9.0 EVALUATION OF TEST RESULTS For a test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per {date of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below: 9.1 Strains TA1535 and TA1537 Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater 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 die 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 Substance: identification and CAS no., if known; physical nature and purity, if known; physicochemical properties relevant to the conduct of tire study, if known; stability oftest substance, if known. Solvent/Vehicle: justification for choice of vehicle; solubility and stability of test substance in solvent/vehicle, if known. Strains: strains used; number of cells/mL per culture; strain characteristics. Protocol SPGT502001 l-Jan-2001 Page 8 of 11 9 Bio Reliance- BioReliance Study No. AA52XL.502001.BTL 54 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Sponsor Project Number DuPont-8868 BioReliance Study Number AA52XL502001 .BTL Test conditions: amount of test substance per plate with rationale for dose selection and number of plates per concentration; media used; type and composition of metabolic activation system, includingacceptability criteria; treatment procedures. Results: signs of toxicity; signs of precipitation; individual plate counts; tibe mean number of revertan! 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 p ra to c o ^ n ^ rf reports will be maintained according to Standard Operating P rocediucfl|j^H H fly the BioReliance RAQA unit headquartered alt BioReliance, 14920 ^m scfil^raR , Rockville, MD 20850. Per this SOP, paper records will be retained for at least three years after which time the Sponsor will be contacted for a decision as to the final disposition of Ok 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 fra- the Testing of Chemicals, published by OECD, Paris, February 1998 and with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997). This study will be performed in compliance with tire provisions of the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (GLPs). The protocol, an in-process phase, the raw data, and reports) will be audited per the Standard-Operating Procedures (SOPs) of BioReliance by the Quality Assurance Unit of BioReliance for compliance with GLPs, tile SOPs of BioReliance and the study protocol. The in-process inspection will be performed to audit the critical assay procedures and systems supporting the assay. A signed QA statement will be included in the final report. This statement will list the system phases inspected during the previous quarter or the study-specific phases, the dales 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 be included in the final report This statement will cite the GLP guiddme(s) with which the study is compliant and any exceptions to this compliance, if applicable, including the omission o f characterization or stability analyses of the test or control substances or their mixtures. Protocol SPGT502001 1-Jan-2001 Page 9 of 11 9 Bio Reliance- BioReliance Study No. AA52XL.502001.BTL 55 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ ________ DuPont-8868 Sponsor Project Number DuPont-8868 BioReliance Study Number AA52XL502001.BTL Unless arrangements are made to the contnuy, unused dosing solutions will be disposed o f following administration to die test system and all residual test substance will be disposed of following finalization of the report 13.0 REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Sa/mone/fo/mammalian-microsome mutagenicity test Mutation Research 31:347-364. Green, M.H.L., and Muriel, W.J. (1976). Mutagen testing using tap* reversion in Escherichia cot. Mutation Research 38:3-32. Internationa) 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. McCann, J. and Ames, B.N. (1976). Detection of carcinogens as mutagens in the Salmonellalvtcrosomt test: assay of 300 chemicals: discussion. Proc. Natl. Acad. Sci. USA 73:950-954. McCann, J., Choi, E., Yamasaki, E. and Ames, BJ4. (1975). Detection of carcinogens as mutagens in the Saimonella/ncrosorac 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, DJ. and Gatehouse, D.G. (1990). Comparison of Salmonella lyphimurium TA102 with Escherichia cot WP2 tester strains. Mutagenesis 5:285-291. Protocol SPGT502001 l-Jan-2001 Page 10 of 11 BioReliance Study No. AA52XL.502001 .BTL 56 H Bio Reliancf Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Sponsor Project Number DuPont-8868 BioReliance Study Number AAS2XL502001JBTL 14.0 APPROVAL k Sponsor Representative 0 'S - O gC - z e oy Date 1 o- Q o A-*- (Print or Type Name) BioReliance Study Director BioReliance Study Management Date tb Aax. z e e ! Date Protocol SPGT502001 1-Jan-2001 Page 11 of 11 BioReliance Study No. AA52XL.50200l.BTL 57 ^ Bio Re l ia n oe Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 APPENDIX C Information for Japanese Regulatory Agencies BioReliance Study No. AA52XL.502001.BTL 58 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 Report o f Results o f Reverse-Mutation Test in Bacteria 1. Tester Strains (1) Procurement Strain TA98 TA100 TA1535 TA1537 TA1538 TA97 TA102 WP2 uvrA W P2vrA (pkM IOl) WP2 (pKM IOl) Obtained from Date obtained 10 November 1998 Dr. Bruce Ames University of California, Berkeley 11 August 1998 13 December 1990 14 November 1990 National Collection of Industrial and Marine Bacteria Aberdeen, Scotland 1 July 1987 19 February 1993 Date inspected the strain lot in storage The genetic markers for each culture are confirmed on the day of use (2) Storage Freezing method Storage temperature C om position Large quantity -70C Bacterial suspension DMSO 1.0 mL 0.09 mL 2. S9 Mix (1) Source, Storage Temperature, etc, of S9 Made in-house Prepared on 26 November 2001 (Batch R656) Storage temperature -70C or colder Name and model of storage apparatus So-Low, Model PR27-120 BioReliance Study No. AA52XL.502001.BTL 59 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 (2) Preparation of S9 Animal used Inducing substance Species, Strain Rattus norvegicus, Sprague Dawley Name Aroclor 1254 Sex Age (in weeks) W eight M ale 9 (Batch R656) 187 to 271 g (Batch R656) A dm inistration m ethod A dm inistration period and amount (g/kg-weight) intraperitoneal 5 days, 0.5 gm/kg body weight 3. Preparation of Test Substance Solution Solvent used Name M anufacturer Lot No. Grade and/or Purity (%) Tetrahydrofuran (CAS No. 109-99-9) Aldrich Chemical Company J100352HI Gold Label, 99.9% Stability of test substance in the solvent Unknown Method of suspension when test substance is difficult to dissolve Sonication and warming to 50C in screw-cap tubes. BioReliance Study No. AA52XL.50200l.BTL 60 Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 4. Conditions o f 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 o f culture to use for test Model and manufacturer of shaker Method of shaking (shaking type, speed, etc.) Culture vessel (shape, capacity) Culture volume Volume of inoculum Name Oxoid Nutrient Broth No. 2 121 hours M anufacturer Lot No. Oxoid Ltd. CH,-B=210287 2 to 5 hours at ambient temperature <8 hours at 2-8C New Brunswick Scientific, model G-24 Rotary (125 rev/m in.) shape: cylinder, 200 mL 50 mL 1 colony 5. Agar Plate Medium (1) Top agar________ Agar Name M anufacturer Lot No. BBL Select Becton Dickinson 1000J3DKSQ (2) Minimum Glucose Agar Name Made in-house Agar M anufacturer Lot No. Volume of agar plate medium BioReliance Study No. AA52XL.502001.BTL 61 BBL Select Becton Dickinson 1000J3DKSQ 25 mL Company Sanitized. Does not contain TSCA CBI H-25171: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8868 6. Test Results - Judgement of the results Judgement Negative Reason for judgement and referential matters: No positive response was observed with any o f 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 o f detecting a mutagen. BioReliance Study No. AA52XL.502001.BTL 62 Company Sanitized. Does not contain TSCA CBI