Document 0ej8OxVJgv11o7Dv08dzxkKM

L _ s a fe H AAS- DuPont-10819 TRADE SECRET Study Title Bacterial Reverse M utation Test w ith an Independent Repeat Assay Authors Valentine O. W agner, ID, M .S. Samuel Atta-Safoh, B.S. Study Completion Date 16 September 2002 Performing Laboratory B io R elian ce 9630 M edical Center D rive Rockville, MD 20850 for E. I du Pont de Nemours and Company DuPont Haskell Laboratory P.O. Box 50 1090 Elkton Road Newark, DE 19714-0050 Performing Laboratory Study Number A A 60E S.502001.B T L W ork Request Number Page 1 o f 65 Company Sanitized. Does not contain TSCA CBI Bacterial Reverse Mutation T e s^ ith ^ in Independent Repeat Assay______________________________ DuPont-10819 STATEMENT OF COMPLIANCE Study M W H S H B H H i 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 U K GLP Compliance Programme, the Japanese GLP Standard, and the OECD Principles o f Good Laboratory Practice in all material aspects with the following exceptions: The stability o f the test substance has not been determined by the testing facility. Analyses to determine the uniformity or concentration o f the test substance mixtures and their stability were not performed by the testing facility or the Sponsor. Valentine O. W agner, HL M.S. Study Director BioReliance Study Management l b S tp a o o a . D ate f t , S e s ji 2 002 D ate B io R e lia n ce 2 Com pan^an^ iBacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Quality Assurance Statement Study Title: BACTERIAL REVERSE MUTATION ASSAY WITH AN INDEPENDENT REPEAT ASSAY ASSAY Study Number Study Director: 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 of these phases over a series o f studies. Procedures, documentation, equipment records, etc., are examined in order to assure that the study is performed in accordance with the U.S. FDA Good Laboratory Practice Regulations (21 CFR 58), the U.S. EPA GLPs (40 CFR 792 and 40 CFR160), the UK GLP Regulations, the Japanese GLP Standard, and the OECD Principles of Good Laboratory Practice and to assure that the study is conducted according to the protocol and relevant Standard Operating Procedures. The following are the inspection dates, phases inspected, and report dates o f QA inspections o f this study. ** Inspect On Phase ** Inspect On Phase ** Inspect On Phase ** Inspect On Phase * Inspect On Phase * Inspect On Phase * Inspect On Phase BioReliance Study No^ 14-Jun-02 - 14-Jun-02 To Study Dir 14-Jun-02 To Mgmt 14-Jun-02 Protocol Review 18-Jun-02 - 18-Jun-02 To Study Dir 18-Jun-02 To Mgmt 20-Jun-02 Preparation of S9 mixture 20-Aug-02 - 20-Aug-02 To Study Dir 20-Aug-02 To Mgmt 29-Aug-02 Draft Report 1l-Sep-02 - 1l-Sep-02 To Study Dir 1l-Sep-02 To Mgmt 16-Sep-02 Draft to Final Report 04-Jun-02 - 04-Jun-02 To Study Dir 04-Jun-02 To Mgmt 05-Jun-02 Preparation of S9 mixture 12-Jun-02 - 12-Jun-02 To Study Dir 12-Jun-02 To Mgmt 13-Jun-02 Dilution o f test and/or control material 27-Jun-02 - 27-Jun-02 To Study Dir 27-Jun-02 To Mgmt 28-Jun-02 Test and/or control material administration 3 CorTT^arr^SanitjzedLJDoes^ iacterial Reverse Mutation fest with an Independent Repeat Assay DuPont-10819 Inspect On Phase 02-M-02 - 02-Ju!-02.To Study Dir 02-Jul-02 To Mgmt 03-Jul-02 Test and/or control material administration Inspect On Phase 09-JuI-02 - 09-M-02 To Study Dir 09-Jul-02 To Mgmt 09-Jul-02 Dilution o f test and/or control material Inspect On Phase 17-M-02 - 17-Jul-02 To Study Dir I7-Jul-02 To Mgmt 17-Jul-02 Strain characterization Inspect On Phase 25-M-02 - 25-Jul-02 To Study Dir 25-Jul-02 To Mgmt 30-Jul-02 Test and/or control material administration Inspect On Phase 3 l-Jul-02 - 3 l-Jul-02 To Study Dir 3l-Jul-02 To Mgmt 01-Aug-02 Scoring the plates Inspect On Phase 07-Aug-02 - 07-Aug-02 To Study Dir 07-Aug-02 To Mgmt 13-Aug-02 Preparation o f S9 mixture Inspect On Phase 21-Aug-02 - 21-Aug-02 To Study Dir21-Aug-02 To Mgmt 28-Aug-02 Strain characterization Inspect On Phase 27-Aug-02 - 27-Aug-02 To Study Dir 27-Aug-02 To Mgmt 28-Aug-02 Test and/or control material administration ** Inspection specific for this study * Systems Inspection This report describes the methods and procedures used in the study and the reported results accurately reflect the raw data of the study. Marcie Bauemschub, B.S. QUALITY ASSURANCE to ? DATE * BioReliance_ Study No. 4 Com gan^ Sanitized^D oesjjo^ grjJg^njrgg^^ g^ Bacterial Reverse Mutation est wii an Independent Repeat Assay DuPont-10819 CERTIFICATION W e, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Issued by Study Director: O - iliValentine O. W agner m , M.S. D ate Approved by Study Monitor: /'~a .... -- M aria Donner, Ph.D. Senior Research Toxicologist Ok S e P l<naZ- D ate BioReliance Study No. 5 iBacterial Reverse Mutation rest with an Independent Repeat Assay DuPont-10819 TABLE OF CONTENTS Page Certification............................................................................................... .....................................5 Study Inform ation....................................................................................... 8 Sum m ary.......................................................................................................................................... 9 P u rp o se ........................................ 10 Characterization o f Test and Control Substances................................... 10 M aterials and M ethods......................................................... 12 Results and Discussion.................................................................................................................. 17 C onclusion.......................... 17 R eferences........................................... 18 D ata T ables............ ........ 19 Table 1: Preliminary Toxicity Test in Salmonella typhimurium T A 98....................... .19 Table 2: Preliminary Toxicity Test in Salmonella typhimurium T A 100.......................20 Table 3: Preliminary Toxicity T est in Salmonella typhimurium T A 1535.................... 21 Table 4: Prelim inary Toxicity Test in Salmonella typhimurium TA 1537..... 22 Table 5: Prelim inary Toxicity Test in Escherichia coli W P2 uvrA............. ..................23 Table 6: M utagenicity T est in Salmonella typhimurium TA98 w ithout S 9 ....................24 Table 7: M utagenicity Test in Salmonella typhimurium TA98 w ith S9 .................... 25 Table 8: M utagenicity Test in Salmonella typhimurium TA100 w ithout S 9 ................. 26 Table 9: M utagenicity Test in Salmonella typhimurium TA100 w ith S 9 .......................27 Table 10: M utagenicity Test in Salmonella typhimurium TA1535 w ithout S 9 ............... 28 Table 11: M utagenicity Test in Salmonella typhimurium TA1535 w ith S 9 .....................29 Table 12: M utagenicity Test in Salmonella typhimurium TA1537 w ithout S 9 ............... 30 Table 13: M utagenicity Test in Salmonella typhimurium TA1537 w ith S 9 .....................31 Table 14: M utagenicity Test in Escherichia coli WP2 wvrA without S 9 ..........................32 Table 15: M utagenicity Test in Escherichia coli WP2 uvrA with S 9 ................................33 Table 16: M utagenicity Test in Salmonella typhimurium TA98 w ithout S 9 ....................34 Table 17: M utagenicity Test in Salmonella typhimurium TA98 w ith S 9 .........................35 Table 18: M utagenicity Test in Salmonella typhimurium TA100 w ithout S 9 ................. 36 Table 19: M utagenicity Test in Salmonella typhimurium TA100 w ith S 9 .......................37 Table 20: M utagenicity Test in Salmonella typhimurium TA1535 w ithout S 9 ............... 38 Table 21: M utagenicity Test in Salmonella typhimurium TA1535 w ith S 9 .................... 39 Table 22: M utagenicity Test in Salmonella typhimurium TA1537 w ithout S 9 ............... 40 Table 23: M utagenicity Test in Salmonella typhimurium TA1537 w ith S 9 .................... 41 Study N o . 4 [ H H j H j H H B Company Sanitized. Does not contain TSCA CBI r Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Table 24: M utagenicity Test in Escherichia coli W P2 wvrA w ithout S 9..........................42 Table 25: M utagenicity Test in Escherichia coli W P2 wvrA w ith S 9 ............................... 43 Table 26: Salmonella/E. coli M utagenicity Test - Summary o f Results B 1.................... 44 Table 27: Salmonella/E. coli M utagenicity Test - Summary o f Results B 2.................... 45 Appendix A: H istorical Control D ata........................................................................................46 Appendix B: Study Protocol...................................................................................................... 48 Appendix C: Inform ation for Japanese Regulatory A gencies................................................60 BioReliance Study No. 7 Company Sanitized. Does not contain TSCA c.Rl p: Bacterial Reverse Mutation Pest with an Independent Repeat Assay STUDY INFORM ATION DuPont-10819 H askell Number: 25339 C om position: Known Impurities: Physical Characteristics: Yellow dispersion in water 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 M tiated/Com pleted: June 13,2002 / (see report cover page) Ih -Iife M tiated/Com pleted: June 18,2002 / July 19,2002 *4 BioReliance Study No. 8 C om g ajTjrS anih^ ^ Bacterial Reverse M utation Test with an Independent Repeat Assay D uP o n t-10819 SUMMARY The test substance, H-25339, was tested in the Bacterial Reverse M utation Test with an Independent Repeat Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia cot tester strain W P2 uvrA in the presence and bsence o f Aroclor-induced rat liver S9. The test was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity test, was used to establish the dose-range for the mutagenicity test. The second phase, the mutagenicity test (initial and independent repeat tests) was used to evaluate the mutagenic potential o f the test substance. W ater was selected as the solvent o f choice based on the Sponsor's request. In the preliminary toxicity test, the maximum dose tested was 5000 fig per plate; this dose was achieved using a concentration o f 50 mg/mL and a 100 fiL plating aliquot. The test substance was soluble but cloudy in water at approximately 50 mg/mL. The dose levels tested were 6 .7 ,1 0 ,3 3 ,6 7 ,1 0 0 , 333,667,1000,3333 and 5000 jig per plate. N either precipitate nor appreciable toxicity was observed. Based on the findings o f the toxicity test, the maximum dose plated in the mutagenicity test was 5000 fig per plate. In the mutagenicity test, no positive mutagenic response was observed. The dose levels tested were 100, 333, 1000, 3333 and 5000 fig per plate. Neither precipitate nor appreciable toxicity was observed. The results o f the Bacterial Reverse M utation Test with an Independent Repeat Assay indicate that, under the conditions o f this study, H-25339 did not cause a positive response in either the presence or absence o f Aroclor-induced rat liver S9. BioReliance Study No. 9 Company Sanitized. Does not contain T s r A r m iBacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 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 o f several strains of Salmonella typhimurium and at the tryptophan locus o f Escherichia coli strain WP2 wvrA in the presence and absence o f Aroclor-induced rat liver S9. A copy o f the protocol is included in Appendix B. The study was conducted to comply w ith OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse M utation Test), adopted July 1997 (published February 1998) and with the International Conference on Harmonisation o f Technical Requirements o f Registration o f Pharmaceuticals for Human Use (1996 and 1997). CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test substance, H-25339, was received by BioReliance on 12 June 2002 and was assigned the code n u m b e rtf H H K r h e test substance was characterized by the Sponsor as yellow dispersion in w a l^ m ^ s n o u ld be stored at ambient temperature. An expiration date of 01 October 2002 was provided. Upon receipt, the test substance was described as a pale yellow cloudy liquid and was stored at room temperature and in the dark. The identity, strength, purity, composition or other characteristics to define the test substance have been determined by the Sponsor. The stability o f the test substance has been determined by the Sponsor but a copy o f the analysis was not provided to BioReliance. The vehicle used to deliver H-25339 to the test system was sterile distilled water (CAS No. 7732-18-5), obtained from Ihvitrogen Corporation. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light. The negative and positive control substances have been characterized as pear the Certificates on file with the testing facility. The stability of the negative and positive control substances and their mixtures was demonstrated by acceptable results that m eet the criteria for a valid test. Positive controls plated concurrently with the mutagenicity test are listed in the following table. All positive controls were diluted w ith dimethyl sulfoxide (DMSO) except sodium azide, which was diluted w ith water. All subdivided solutions o f positive control were stored at -5 to -30C . BioReliance Study No 10 Comganj^SanitizedjJg^ Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Strain All Salmonella Strains WP2 uvrA TA98 TA100, TA1535 TA1537 WP2 uvrA S9 Activation Positive Control Rat None 2-am inoanthracene (Aldrich Chemical Co., Inc.) Lot No. 09106PS Exp. Date 14-Sep-2005 CAS No. 613-13-8 Purity 97.4% 2-nitrofluorene (Aldrich Chemical Co., Inc.) Lot No. 08707HS Exp. D ate 08-Mar-2006 CAS No. 607-57-8 Purity 99.9% Sodium aride (Sigma Chemical Co.) Lot No. 098H0169 Exp. D ate 05-Jan-2004 CAS No. 26628-22-8 Purity 99.8% 9 -am inoacridine (Sigma Chemical Co.) Lot No. 106F06681 Exp. Date 18-Nov-2004 CAS No. 90-45-9 Purity >97% M ethyl methanesulfonate (Aldrich Chemical Co., Inc.) Lot No. 15526AO Exp. Date 28-Nov-2003 CAS No. 66-27-3 Purity 99.9% Concentration (pg/plate) 1.0 10 1.0 1.0 75 1,000 To confirm the sterility of the test substance, the highest test substance dose level used in the mutagenicity test was plated on selective agar w ith an aliquot volume equal to that used in the te st These plates were incubated under the same conditions as the test. BioReliance Study No. 11 Company Sanitized. Does not mntain TSfiA o p i Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 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 W P2 uvrA as described by Green and M uriel (1976). Salmonella tester strains were received directly from Dr. Bruce Ames, University o f California, Berkeley. E. coli tester strains were received from the National Collection o f 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 o f 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 m aster plate or from the appropriate frozen permanent stock into a vessel containing -5 0 mL o f 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 tim e o f harvest Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer o f approximately 109 cells per m illiliter. 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. 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 o f Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was batch prepared by BioReliance or purchased from M oltox and stored at -70C or colder until used. Each bulk preparation o f S9 was tested for its ability to metabolize 2-aminoanthracene and 7,12dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM gIucose-6-phosphate, 4 mM B-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCI2and BioReliance Study No. 12 C om p am /San itized^ acterial Reverse Mutation Test with an Independent Repeat Assay________ _____________________ DuPont-10819 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 o f the S9 and Sham mixes, a 0.5 mL aliquot o f each was plated on selective agar. Solubility Test W ater was selected as the solvent of choice based on the Sponsor's request and compatibility w ith the target cells. Preliminary Toxicity Test The preliminary toxicity test was used to establish the dose-range over w hich the test substance would be tested. Vehicle and ten dose levels o f the test substance were plated, one plate per dose, w ith 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. The dose levels tested were 6 .7 ,10,33,67,100,333,667,1000,3333 and 5000 fig per plate. Mutagenicity Test The mutagenicity test was used to evaluate the mutagenic potential o f the test substance. Five dose levels o f test substance along with appropriate vehicle and positive controls were plated with TA98, TA100, TA1535, TA1537 and WP2 wvrA in the presence and absence of Aroclor-induced rat liver S9. The dose levels tested were 100, 333, 1000, 3333 and 5000 pg per plate. All dose levels o f test substance, vehicle controls and positive controls were plated in triplicate. Plating and Scoring Procedures The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). On the day 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 o f 50 pM each. Top agar not used w ith S9 or Sham mix was supplemented w ith 25 mL o f water for each 100 mL o f minimal top agar. For the preparation o f media and reagents, all references to water imply sterile, deionized w ater produced by the M illi-Q Reagent W ater System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid 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). BioReliance Study No. 13 H H m ^ a c t e r i a l Reverse Mutation x e s ^ ith an Independent Repeat Assay______________________________DuPont-10819 Each plate was labeled with a code system that identified the test substance, test phase, dose level, tester strain, and activation, as described in detail in BioReliance's Standard Operating Procedures. One-half (0.5) m illiliter o f S9 or Sham mix, 100 pL of tester strain and 100 pL o f vehicle or test substance dilution were added to 2.0 mL o f molten selective top agar at 452C. After vortexing, the mixture was overlaid onto the surface o f 25 mL o f minimal bottom agar. W hen 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 o f test substance toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree o f precipitation were scored relative to the vehicle control plate using the codes shown in the following table. Code 1 2 3 4 5 6 NP D escription N orm al Slightly Reduced M oderately Reduced E xtrem ely Reduced Absent Obscured by Precipitate NonInterfering Precipitate C h aracteristics Distinguished by a healthy microcolony lawn. Distinguished by a noticeable thinning o f 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 o f the microcolony lawn resulting in a pronounced increase in the size o f the microcolonies compared to the vehicle control plate. Distinguished by an extreme thinning o f the microcolony lawn resulting in an increase in the size o f 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 o f any microcolony lawn over greater than or equal to 90% o f the plate. The background bacterial lawn cannot be accurately evaluated due to microscopic test substance precipitate. Distinguished by precipitate on the plate that is visible to the naked eye but any precipitate particles detected by the automated colony counter total less than 10% o f the revertant colony count (e.g., less than 3 particles on a plate w ith 30 revertants). B io R elian ce Study No. 14 Company Sanitized. Does not contain TSCA CBI ' Bacterial Reverse Mutation Test wii an Independent Repeat Assay DuPont-10819 Code IP Description Interfering Precipitate Characteristics Distinguished by precipitate on the plate that is visible to the naked eye and any precipitate particles detected by the automated colony counter exceed 10% 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 o f the number o f 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 o f at least one tester strain over a minimum o f two increasing concentrations o f 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 o r greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and W P2 uvrA were judged positive if the increase in mean revertants at the peak o f 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 m ust be met for the mutagenicity test to be considered valid. All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (jfa) and the deletion in the wvrB gene. Cultures o f tester strains TA98 and TA100 must demonstrate the presence o f the pKM IOl plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. A ll cultures must demonstrate the characteristic mean number o f spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA 100,80 - 240; TA 1535,5 - 45; TA 1537,3 - 21; WP2 uvrA, 10 - 60. To ensure that appropriate numbers o f bacteria are plated, tester strain culture titers m ust be greater than or equal to 0.3xl09 cells/mL. The mean o f each positive control must exhibit at least a 3.0-fold increase in the number o f revertants over the mean value o f the respective vehicle control. A minimum o f three non-toxic dose levels is required to evaluate test data. A dose level is considered toxic if one or both o f the following criteria are met: (1) A >50 % reduction in the mean number o f revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count (2) A t least a moderate reduction in die background lawn (background lawn code 3, 4 or 5). A copy o f the Historical Negative and Positive Control Values is included in Appendix A. BioReliance Study No., 15 C om pan^Sj^^  ^ ^ ^ H H p B a c te ria l Reverse Mutation *res^iH m Independent Repeat Assay______________________________ DuPont-10819 Archives A ll raw data, the protocol and all reports will be maintained according to Standard Operating Procedure the BioReliance RAQA unit headquartered at: BioReliance, 14920 Broschart Road, Rockville, MD 20850. Per this SOP, paper records will be retained for at least three years after which time the Sponsor w ill 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 o f the report. D eviatio n s No known deviations from the protocol or test-method SOPs occurred during the conduct o f this study. B io R elian ce Study N o.l 16 Company Sanitized. Does not contain TSCA CBI :Bacterial Reverse Mutation rest with an Independent Repeat Assay DuPont-10819 RESULTS AND DISCUSSION Solubility Test W ater was selected as the solvent of choice based on the Sponsor's request Prelim inary Toxicity Test The results o f the preliminary toxicity test are presented in Tables 1 through 5. These data were generated in Experiment A l. In the preliminary toxicity te s t the maximum dose tested was 5000 |ig per plate; this dose was achieved using a concentration o f 50 mg/iriL and a 100 pL plating aliquot. The test substance was soluble but cloudy in water at approximately 50 mg/mL. The dose levels tested were 6.7,10,3 3 ,6 7 ,1 0 0 ,3 3 3 ,6 6 7 ,1 0 0 0 ,3 3 3 3 and 5000 jig per plate. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the toxicity test, the maximum dose plated in the mutagenicity test was 5000 pg per plate. M utagenicity T est The results o f the mutagenicity test are presented in Tables 6 through 25 and summarized in Tables 26 and 27. These data were generated in Experiments B1 and B2. The dose levels tested were 100, 333, 1000, 3333 and 5000 pg per plate. N either precipitate nor appreciable toxicity was observed. In Experiment B1 (Initial Test), no positive mutagenic responses were observed w ith any o f the tester strains in either the presence or absence of S9 activation. In Experiment B2 (Independent Repeat Assay), no positive mutagenic responses were observed with any o f the tester strains in either the presence or absence o f S9 activation. C O N C L U S IO N All criteria for a valid study were met as described in the protocol. The results o f the Bacterial Reverse M utation Test with an Independent Repeat Assay indicate that, under the conditions o f this study, H-25339 did not cause a positive response in either the presence or absence of Aroclor-induced rat liver S9. BioReliance Study No. 17 Company Sanitized. Does not contain TSCA CSI Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________ DuPont-10819 REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mamaiatan Microsome Mutagenicity Test, Mutation Research, 31:347-364. Green, M.H.L. and W.J. M uriel (1976) M utagen testing using trp+ reversion in Escherichia coli, Mutation Research 38:3-32. International Conference on Harmonisation (ICH) of Technical Requirements for Registration o f Pharmaceuticals for Human Use. Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 o f 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 o f Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing o f Pharmaceuticals. S2B document recommended for adoption at step 4 o f the ICH process on July 16,1997. Federal Register 62:16026-16030, November 21,1997. M arn, D.M. and B.N. Ames (1983) Revised Methods for the Salmonella M utagenicity Test, M utation Research, 113:173-215. OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse M utation Test), Ninth Addendum to the .OECD Guidelines for the Testing o f Chemicals, published by OECD, Paris, February 1998. Vogel, H.J. and D.M. Bonner (1956) Acetylornithinase o f E. cot: Partial Purification and Some Properties, J. Biol. Chem., 218:97-106. BioReliance Study No. 18 Company Sanitized. Does not contain TSCA CBI 'Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Preliminary Toxicity Test Table 1 Test Substance Id H-25339 Study Number s3ttKt Experiment No. Al Strain TA98 Date Plated 18 Jun 2002 Vehicle water Plating Aliquot 100 pL Test Stabstance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 11 1 Without S9 Activation Revertants Background per plate Lawn 13 1 6.7 10 33 67 100 333 667 1000 3333 5000 17 1 17 1 15 1 17 1 18 1 17 1 20 1 19 1 17 1 19 1 13 1 10 1 13 1 10 1 12 1 14 1 14 1 11 1 11 1 13 1 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced? 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study NoJ 19 Com panySam tiz^ acterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Preliminary Toxicity Test Table 2 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25339 Wtb Al TAI00 18 Jun 2002 water 100 pL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 117 1 Without S9 Activation Revertants Background per plate Lawn 171 1 6.7 10 33 67 100 333 667 1000 3333 5000 133 1 146 1 153 1 160 1 152 1 194 1 181 1 182 1 192 1 181 1 169 1 175 1 175 1 200 1 181 1 216 1 160 1 178 1 198 1 212 1 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance. Study No.| 20 Company Sanitized. Does not contain TSCA CBI acterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Preliminary Toxicity Test Table 3 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25339 Al TA1535 18 Jun 2002 water 100 pL Test Substance Concentration Vtg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 16 1 Without S9 Activation Revertants Background per plate Lawn 71 6.7 10 33 67 100 333 667 1000 3333 5000 11 1 11 1 81 10 1 12 1 12 1 12 1 11 1 13 1 11 1 91 10 1 61 11 1 10 1 10 1 10 1 12 1 10 1 12 1 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. ' 21 Company Sanitized. Does not contain TSCA CBI ^ P m ^ B a c t e r i a l Reverse Mutation Test with an Independent Repeat Assay__________________ DuPont-10819 Bacterial Mutation Test Preliminary Toxicity Test Table 4 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25339 Al TA1537 18 Jun 2002 water 100 pL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 10 1 Without S9 Activation Revertants Background per plate Lawn 10 1 6.7 10 33 67 100 333 667 1000 3333 5000 13 1 13 1 11 1 81 71 61 71 61 81 51 81 91 11 1 81 61 81 61 71 81 51 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. 22 Company Sanitized. Does not contain TSCA CBI "Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Preliminary Toxicity Test Table 5 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25339 Al WP2 uvrA 18 Jun 2002 water 100 pL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 12 1 Without S9 Activation Revertants Background per plate Lawn 24 1 6.7 10 33 67 100 333 667 1000 3333 5000 12 1 13 1 15 1 13 1 13 1 14 1 14 1 18 1 16 1 16 1 17 1 16 1 16 1 16 1 16 1 15 1 10 1 13 1 14 1 16 1 Background Lawn Code l=Norxnal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate B io R e lia n ce Study No. 23 Company Sanitized. Does not contain TSCA CBI acterial Reverse Mutation Test with an Independent Repeat Assay D uP ont-10819 Bacterial Mutation Test Table 6 Test Substance Id: H-25339 Study Number Experiment No : B1 Strain : TA98 Cells Seeded : 2.8 X 108 Liver Microsomes : None Date Plated : 2 Jul 2002 Vehicle : water Plating Aliquot : 100 uL_________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 15 1 15 1 14 1 15 1 100 01 16 1 02 13 1 03 17 1 15 2 333 1000 01 12 1 02 14 1 03 17 1 01 12 1 02 14 1 03 12 1 14 3 13 1 3333 01 14 1 02 13 1 03 12 1 13 1 5000 01 13 1 02 14 1 03 15 1 14 1 Positive Control 2-nitrofluorene 1. 0 pg per plate 01 118 1 02 144 1 03 101 1 121 22 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. ; 24 Company Sanitized. Does not contain TSCA CBI Bacterial Reverse Mutation Test with an Independent Repeat Assay D u P o n t-10819 Bacterial Mutation Test Table 7 Test Substance Id: H-25339 Study Number : Experiment No :B1 Strain :T A 9 ^ ^ ^ ^ ^ ^ ^ ^ Cells Seeded :2.8 X 10s Liver Microsomes :Rat liver S9 Date Plated :2 Jul 2002 Vehicle : water Plating Aliquot : 100 pL_________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 13 1 13 1 16 1 14 2 100 01 14 1 02 13 1 03 12 1 13 1 333 01 12 1 02 12 1 03 12 1 12 0 1000 01 14 1 02 13 1 03 12 1 13 1 3333 01 13 1 02 12 1 03 14 1 13 1 5000 01 13 1 02 14 1 03 14 1 14 1 Positive Control 2-aminoanthracene 1.0 pg per plate 01 398 1 02 312 1 03 381 1 364 46 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. 25 Company Sanitized. Does not contain TSCA CBI !Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 8 Test Substance Id: H-25339 Study Number Experiment No : B1 Strain : TA100 Cells Seeded : 1.5 X 10s Liver Microsomes : None Date Plated : 2 Jul 2002 Vehicle : water Plating Aliquot ; 100 pL_________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 150 1 175 1 163 1 163 13 100 01 160 1 02 181 1 03 174 1 172 11 333 01 209 1 02 169 1 03 175 1 184 22 1000 01 154 1 02 193 1 03 155 1 167 22 3333 01 216 1 02 229 1 03 228 1 224 7 5000 01 266 1 02 247 1 03 247 1 253 11 Positive Control sodium azide 1.0 pg per plate 01 665 1 02 693 1 03 696 1 685 17 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. 26 Company Sanitized. Does not contain TSCA CBI iacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 9 Test Substance Id: H - 2 5 3 3 ^ ______ Study Number : Experiment No :B1 Strain :TA100 " Cells Seeded :1.5 X 10 Liver Microsomes :Rat liver S9 Date Plated :2 Jul 2002 Vehicle : water Plating Aliquot : 100 jiL_________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 191 1 174 1 177 1 181 9 100 01 190 1 02 159 1 03 185 1 178 17 333 01 168 1 02 141 1 03 184 1 164 22 1000 01 92 1 02 135 1 03 165 1 131 37 3333 01 210 1 02 194 1 03 150 1 185 31 5000 01 197 1 02 199 1 03 181 1 192 10 Positive Control 2-aminoanthracene 1.0 jig per plate 01 890 1 02 747 1 03 703 1 780 98 Background Lawn Code l=Normal; 2=Slightly.reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; XP=Interfering precipitate BioReliance. Study No. 27 JiuammhSamiim ~s rv IMN*aMMOfifilAaS [Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 10 Test Substance Id: H-25339_________ Study Number : Experiment No :B1 Strain :TA1535 Cells Seeded :2.2 X 10s Liver Microsom.es :None Date Plated :2 Jul 2002 Vehicle : water Plating Aliquot : 100 uL_________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 15 1 10 1 13 1 13 3 100 01 13 1 02 12 1 03 12 1 12 1 333 01 13 1 02 12 1 03 15 1 13 2 1000 01 12 1 02 11 1 03 12 1 12 1 3333 01 10 1 02 10 1 03 10 1 10 0 5000 01 12 1 02 10 1 03 12 1 11 1 Positive Control sodium azide 1.0 ug per plate 01 425 1 02 370 1 03 355 1 383 37 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate * BioReliance Study No. | 28 Company Sanitized, nn ss not mntain t r p a I M Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 11 Test Substance Id: H - 2 5 3 3 9 _____ Study Number : Experiment No :Bl Strain :TA1535 Cells Seeded :2.2 X 108 Liver Microsomes :Rat liver S9 Date Plated :2 Jul 2002 Vehicle : water Plating Aliquot : 100 pL_________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 11 1 10 1 11 1 11 1 100 01 11 1 02 10 1 03 12 1 11 1 333 01 11 1 02 13 1 03 12 1 12 1 1000 01 10 1 02 10 1 03 10 1 10 0 3333 01 14 1 02 10 1 03 10 1 11 2 5000 01 11 1 02 10 1 03 10 1 10 1 Positive Control 2-aminoanthracene 1.0 pg per plate 01 91 1 02 95 1 03 87 1 91 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 BioRelianc Study N o .1 29 Company S a n it i^ nm :Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 12 Test Substance Id: H-25339 Study Number :W t t K K K k Experiment No : Bl Strain : TA1537 Cells Seeded : 1.2' X 10 Liver Microsomes : None Date Plated : 2 Jul 2002 Vehicle : water Plating Aliquot : 100 pL_________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 61 11 12 1 83 100 01 4 1 02 9 1 03 12 1 84 333 01 12 1 02 7 1 03 11 1 10 3 1000 3333 01 10 1 02 6 1 03 13 1 01 10 1 02 10 1 03 11 1 10 4 10 1 5000 01 10 1 02 10 1 03 14 1 11 2 Positive Control 9-aminoacridine 75 pg per plate 01 632 1 02 698 1 03 527 1 619 86 Background Lawn Code l=Normal; 2=Slightly-reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interering precipitate BioReliance Study No. 30 | Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 13 Test Substance Id: H - 2 5 3 3 9 ______ Study Number : Experiment No :B1 Strain :TA1537 Cells Seeded :1.2 X 10s Liver Microsomes :Rat liver S9 Date Plated :2 Jul 2002 Vehicle : water Plating Aliquot : 100 pL_________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 41 41 71 52 100 01 5 1 02 7 1 03 5 1 61 333 01 7 1 02 6 1 03 6 1 61 1000 01 7 1 02 7 1 03 9 1 81 3333 01 7 1 02 7 1 03 8 1 71 5000 01 8 1 02 10 1 03 4 1 73 Positive Control 2-aminoanthracene 1.0 pg per plate 01 54 1 02 52 1 03 50 1 52 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 2 B io R eE an ce Study No. 31 'Bacterial Reverse Mutation Pest with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 14 Test Substance Id: H-25339_____ ____ Study Number :f l B M H H H B M H H R f Experiment No :B1 Strain : WP2 uvrA Cells Seeded :3.7 X 10s Liver Microsomes : None Date Plated :2 Jul 2002 Vehicle : water Plating Aliquot : 100 uL_________________________________________ Concentration Plate Revertants Background Average Standard Vig per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 10 1 10 1 13 1 11 2 100 01 13 1 02 15 1 03 14 1 14 1 333 01 11 1 02 14 1 03 13 1 13 2 1000 01 13 1 02 13 1 03 14 1 13 1 3333 01 14 1 02 16 1 03 12 1 14 2 5000 01 14 1 02 14 1 03 16 1 15 1 Positive Control methyl methanesulfonate 1000 Ug per plate 01 103 1 02 96 1 03 86 1 95 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate 9 BioReliance Study No. 32 Jacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 15 Test Substance Id: H-25339 Study Number : Experiment No :B1 Strain :WP2 uvrA Cells Seeded :3.7 X 108 Liver Microsomes :Rat liver S9 Date Plated :2 Jul 2002 Vehicle : water Plating Aliquot : 100 uL_________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 c 02 14 1 03 15 1 15 1 100 01 12 1 02 12 1 03 11 1 12 1 333 01 14 1 02 14 1 03 12 1 13 1 1000 01 16 1 02 14 1 03 14 1 15 1 3333 5000 01 12 1 02 16 1 03 15 1 01 11 1 02 15 1 03 15 1 14 2 14 2 Positive Control 2-aminoanthracene 10 pg per plate 01 548 1 02 552 1 03 471 1 524 46 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interering precipitate; 1P=Interfering precipitate C=Contaminated BioReliance Study No. 33 Company Sanitari nn> .nnbin r.cr a nn 9 j^ H H |v B a c te r ia l Reverse Mutation T e^w im an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 16 Test Substance Id: H-25339 Study Number : Experiment No : B2 Strain :TA98 Cells Seeded : 2.4 X 10 Liver Microsomes :None Date Plated : 11 Jul 2002 Vehicle : water Plating Aliquot : 100 pL__________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 16 1 14 1 12 1 14 2 100 01 16 1 02 19 1 03 16 1 17 2 333 01 19 1 02 22 1 03 20 1 20 2 1000 01 23 1 02 19 1 03 23 1 22 2 3333 01 18 1 02 17 1 03 20 1 18 2 5000 01 14 1 02 17 1 03 16 1 16 2 Positive Control 2-nitrofluorene 1. 0 pg per plate 01 287 1 02 225 1 03 303 1 272 41 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. M 34 fifflliitfBtti2&iELuB^ABbi&siBtti!ESAEUi Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 17 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25339 Rat liver S9 water 100 pL Coneentrtion Plate Revertants ug per plate Number per plate Vehicle 01 02 03 17 15 16 ; Experiment No : B2 Cells Seeded : 2.4 X 108 Date Plated : 11 Jul 2002 Background Code 1 1 1 Average Revertants 16 Standard Deviation 1 100 01 13 1 02 19 1 03 12 1 15 4 333 01 20 1 02 17 1 03 19 1 19 2 1000 01 19 1 02 20 1 03 17 1 19 2 3333 01 18 1 02 13 1 03 12 1 14 3 5000 01 19 1 02 16 1 03 15 1 17 2 Positive Control 2-aminoanthracene 1.0 ug per plate 01 151 1 02 132 1 03 134 1 139 10 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interering precipitate BioReliance Study No. 35 acterial Reverse Mutation T e st with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 18 Test Substance Id: H-25339 Study Number Strain : TA100 Liver Microsomes : None Vehicle : water Plating Aliquot : 100 pL Concentration Plate Revertants pg per plate Number per plate Vehicle 01 02 03 201 215 167 Experiment No : B2 Cells Seeded : 1.4 X 1081 Date Plated : 11 Jul 2002 Background Code 1 1 1 Average Revertants 194 Standard Deviation 25 100 01 178 1 02 198 1 03 185 1 187 10 333 01 187 1 02 213 1 03 241 1 214 27 1000 3333 5000 01 C 02 192 1 03 192 1 01 208 1 02 210 1 03 201 1 01 201 1 02 215 1 03 217 1 192 0 206 5 211 9 Positive Control sodium azide 1.0 pg per plate 01 574 1 02 671 1 03 563 1 603 59 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 C=Contaminated BioReliance Study No. I 36 [Bacterial Reverse Mutation Test w ith an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 19 Test Substance Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-25339 TA100 Rat liver S9 water 100 uL Concentration Plate Revertants Vig per plate Number per plate Vehicle 01 02 03 164 118 141 i Experiment No : B2 Cells Seeded : 1.4 X 10s Date Plated : 11 Jul 2002 Background Code 1 1 1 Average Revertants 141 Standard Deviation 23 100 01 194 1 02 176 1 03 175 1 182 11 333 01 168 1 02 167 1 03 143 1 159 14 1000 01 148 1 02 197 1 03 188 1 178 26 3333 01 161 1 02 148 1 03 156 1 155 7 5000 01 159 1 02 154 1 03 163 1 159 5 Positive Control 2-aminoanthracene 1.0 gg per plate 01 758 1 02 781 1 03 748 1 762 17 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 B io R elian ce Study No. 37 [Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 20 Test Substance Id: H-25339 Study Number Experiment No : B2 Strain : TA1535 Cells Seeded : 1.9 X 108 Liver Microsomes : None Date Plated : 11 Jul 2002 Vehicle : water Plating Aliquot : 100 yX>__________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 13 1 18 1 18 1 16 3 100 01 11 1 02 14 1 03 10 1 12 2 333 01 12 1 02 12 1 03 13 1 12 1 1000 01 12 1 02 18 1 03 9 1 13 5 3333 01 14 1 02 13 1 03 12 1 13 1 5000 01 12 1 02 12 1 03 13 1 12 1 Positive Control sodium azide 1.0 yig per plate 01 342 1 02 368 1 03 368 1 359 15 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. 38 P .n m n a n u />A O Afa acterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 21 Test Substance Id: H-25339 Study Number : Experiment No :B2 Strain :TA1535 Cells Seeded :1.9 X 10 Liver Microsomes :Rat liver S9 Date Plated :11 Jul 2002 Vehicle : water Plating Aliquot : 100 uL__________________________________________ Concentration Plate Revertants Background Average Standard Ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 11 1 81 81 92 100 01 10 1 02 8 1 03 11 1 10 2 333 01 8 1 02 12 1 03 11 1 10 2 1000 01 11 1 02 13 1 03 6 1 10 4 3333 01 6 1 02 9 1 03 9 1 82 5000 01 10 1 02 9 1 03 12 1 10 Positive Control 2-aminoanthracene 1.0 pg per plate 01 114 1 02 121 1 03 120 1 118 Background Lawn Code l=Normal; 2=Slightly-reduced;3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate 2 4 BioReliance Study No. 39 C n m n a n v ftani+i-roW n. [Bacterial Reverse Mutation fest with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 22 Test Substance Id: H-25339 Study Number : Experiment No :B2 Strain :TM537 ^ ^ ^ ^ ^ Cells Seeded :1.1 X 10s Liver Microsomes :None Date Plated :11 Jul 2002 Vehicle : water Plating Aliquot : 100 pL__________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 51 81 41 62 100 01 6 1 02 10 1 03 3 1 64 333 01 10 1 02 5 1 03 4 1 63 1000 01 8 1 02 10 1 03 6 1 82 3333 01 5 1 02 7 1 03 9 1 72 5000 01 9 1 02 8 1 03 3 1 73 Positive Control 9-aminoacridine 75 pg per plate 01 369 1 02 356 1 03 404 1 376 25 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interering precipitate BioRelian* Study No. 40 liafysifli! [Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 23 Test Substance Id: H-25339 Study Number Experiment No : B2 Strain : TA1537 Cells Seeded : 1.1 X 108 Liver Microsomes : Rat liver S9 Date Plated : 11 Jul 2002 Vehicle : water Plating Aliquot : 100 p.L__________________________________________ Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 31 41 41 41 100 01 4 1 02 7 1 03 3 1 52 333 01 7 1 02 6 1 03 7 1 71 1000 01 7 1 02 S 1 03 7 1 71 3333 01 3 1 02 3 1 03 6 1 42 5000 01 4 1 02 6 1 03 3 1 42 Positive Control 2-aminoanthracene 1.0 pg per plate 01 110 1 02 114 1 03 113 1 112 Background Lawn Code l=Normal; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate 2 B io R e lia n ce. Study No. I 41 ifftfm BflnYifltttefidJa acterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Table 24 Test Substance Id: H-25339________ Study Number Experiment No : B2 Strain : WP2 uvrA Cells Seeded : 4.0 X 10s Liver Microsomes : None Date Plated : 11 Jul 2002 Vehicle : water Plating Aliquot : 100 ytL__________________________________________ Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 16 1 16 1 10 1 14 3 100 01 9 1 02 9 1 03 16 1 11 4 333 01 14 1 02 13 1 03 14 1 14 1 1000 01 14 1 02 18 1 03 12 1 15 3 3333 01 8 1 02 11 1 03 10 1 10 2 5000 01 18 1 02 15 1 03 17 1 17 2 Positive Control methyl methanesulfonate 1000 ug per plate 01 106 1 02 117 1 03 83 1 102 17 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. 42 gamaanYSatiiTirpti .n rr nnUiantaiuafift noi acterial Reverse Mutation Test with an Independent Repeat Assay D uP ont-10819 Bacterial Mutation Test Table 25 Test Substance Id: H-25339 Study Number : Experiment No :B2 Strain :WP2 uvrA Cells Seeded :4.0 X 10s Liver Microsomes :Rat liver S9 Date Plated :11 Jul 2002 Vehicle : water Plating Aliquot : 100 uL__________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 14 1 11 1 13 1 13 2 100 01 15 1 02 13 1 03 18 1 15 3 333 01 15 1 02 12 1 03 14 1 14 2 1000 01 11 1 02 15 1 03 14 1 13 2 3333 01 13 1 02 12 1 03 12 1 12 1 5000 01 14 1 02 10 1 03 17 1 14 4 Positive Control 2-aminoanthracene 10 pg per plate 01 122 1 02 115 1 03 131 1 123 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 8 BioReliance_ Study No. 43 O om n arn /ftsm ifiyori n o o c an ^ t a a T C ^ A p d i acterial Reverse M utation Test with an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Summary of Results Table 26 Test Substance Id : H-25339 Study Number Experiment Ho : B1 Average Revertants Per Plate Standard Deviation Liver Microsomes: None Dose (ug/plate) TA98 TA100 Vehicle 100 333 1000 3333 5000 Positive 15 1 163 13 15 2 172 11 14 3 184 22 13 1 167 22 13 1 224 7 14 1 253 11 121 22 685 17 Liver Microsomes: Rat liver S9 TA1535 TA1537 13 3 8 3 12 13 12 10 11 1 8 2 10 1. 10 0 10 1 11 4 3 4 1 2 383 37 619 86 WP2 uvzA 11 14 13 13 14 15 95 2 1 2 1 2 1 9 Dose (ug/plate) TA98 TA100 Vehicle 100 333 1000 3333 5000 Positive 14 2 181 9 13 1 178 17 12 0 164 22 13 1 131 37 13 1 185 31 14 1 192 10 364 46 780 98 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 100 pL TA1535 11 11 12 10 11 10 91 1 1 1 0 2 1 4 TA1537 5 6 6 8 7 7 52 2 1 1 1 1 3 2 WP2 uvxA 15 12 13 15 14 14 524 1 1 1 1 2 2 46 B io R e lia n ce Study No. 44 C n m n a n v R a n i f i y a r l n i* ia e r>/\t y**. iU.'w TQ^A PPI Bacterial Reverse M utation T est w ith an Independent Repeat Assay DuPont-10819 Bacterial Mutation Test Summary of Results Table 27 Test Substance Id ; H-25339 Study Number______________________________ Experiment No ; B2 Average Revertants Per Plate Standard Deviation Liver Microsomes: None Dose (pg/plate) TA98_____ TA1Q0______TA1535 Vehicle 100 333 1000 3333 5000 Positive 14 2 194 25 16 3 17 2 187 10 12 2 20 2 214 27 12 1 22 2 192 0 13 5 18 2 206 5 13 1 15 2 211 9 12 1 272 ' 41 603 59 359 15 Liver Microsomes: Rat liver S9 TA1537 6 6 6 8 7 7 376 2 4 3 2 2 3 25 WP2 uvrA 14 11 14 15 10 17 102 3 4 1 3 2 2 17 Dose (pg/plate) TA98______TA100_____ TA1535 Vehicle 100 333 1000 3333 5000 Positive 16 1 141 23 9 15 4 182 11 10 19 2 159 14 10 19 2 178 26 10 14 3 155 7 8 17 2 159 5 10 139 10 762 17 118 2 2 2 4 2 2 4 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 100 pL TA1537 4 5 7 7 4 4 112 1 2 1 1 2 2 2 WP2 uvrA 13 15 14 13 12 14 123 2 3 2 2 1 4 8 B io R elian ce. Study No., 45 euinudiiy aufli!iad'"Dt!oes not contain TSCA CBI acterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 APPENDIX A Historical Control Data BioReliance_ Study No. j 46 rinmnrarpi lniiiMtiai imw ain eisn fBacterial Reverse Mutation Fest with an Independent Repeat Assay DuPont-10819 Historical Negative and Positive Control Values 1999-2001 revenants per plate A ctivation Strain C o n tro l None Rat Liver Mean SD Min Max Mean SD Min M ax TA98 Neg 16 6 4 59 21 7 6 58 Pos 310 191 21 1581 680 374 40 2294 TA100 Neg 143 37 53 283 149 39 74 271 Pos 564 160 129 1851 876 438 163 2922 Neg 14 6 1 46 13 5 1 43 TA1535 Pos 335 149 6 978 125 84 11 1640 TA1537 Neg 6 3 0 30 7 3 1 29 Pos 732 391 15 2786 117 130 12 2060 WP2 uvrA Neg Pos 14 4 5 52 168 140 34 990 15 5 322 286 4 115 22 2632 SD=standard deviation; Mm=minimum value; Max=maximum value; Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control BioReliance Study No.J | 47 bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 APPENDIX B Study Protocol B io R elian ce Study No. 48 iaiadi&A fBacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Sponsor Project Number: BioReliance Study Number: DuPont-10819 (Bacterial Reverse Mutation Test with au Independent Repeat Assay 1.0 PURPOSE The purpose o f this study is to evaluate the mutagenic potential o f the test substance by measuring its ability to induce reverse mutations at selected loci o f several strains of Salmonella typhimurium and at the tryptophan locus o f Escherichia coli WP2 uvrA in the presence and absence o f S9 activation. 2.0 SPONSOR 2.1 Name: EX du Pont de Nemours and Company 2.2 Address: DuPont Haskell Laboratory P.O. Box 50 1090 ElktonRoad Newark, DE 19714-0050 2.3 Representative: Maria Donner, Ph.D. Phone: 302-366-5251 Fax: 302-366-5207 Email: maria.donner@usa.dunont.com 2.4 Sponsor Project No.: DuPont-10819 2.5 WR#: 2.6 Haskell#: 2.7 Service Code: H-25339 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance Name: 3.2 Test Substance LD.: H-25339 (to be used in the report title and text) 3.3 Controls: Negative: Positive: Test substance vehicle 9-aminoacridine 2-aminoanthracene methyl methanesulfonate 2-mtrofluorene sodium azide Protocol SPGT502001 06-Jun-2002 Page 1 o f 11 BioReliance* Study No. 49 Bio Re u a n oe mumpmmtf r> itmmmA [Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Sponsor Project Number BioReliance Study Number: DuPont-10819 3.4 Test Substance Characterization Unless alternate arrangements are made, the testing facility at BioReliance will not perform analysis o f the dosing solutions. The Sponsor will be directly responsible for determination and documentation o f the analytical purity and composition o f the test substance, and the stability and strength o f the test substance in the solvent (or vehicle). 3.5 Test Substance Retention Sample The retention o f a reserve sample o f the test substance will be foe responsibility o f the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility BioReliance 4.2 Address: 9630 Medical Carter Drive Rockville, MD 20850 4.3 Study Director Valentine O. Wagner IE, M.S. Phone: 301-610-2152 Fax: 301-738-2362 Email: swagner@bioreliance.com 5.0 PROPOSED STUDY DATES 5.1 Experimental Start Date: 02-Jul-2002 5.2 Experimental Termination Date: 20-Aug-2002 5.3 Draft Report Date: 03-Sep-2002 5.4 Final Report Date: 2 weeks after Sponsor approves draft 6.0 TEST SYSTEM The tester strains will include foe S. typkimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and foe E. coli tester strain WP2 uvrA as described by Green and Muriel (1976). _____________________________ Histidine Mutation Tryptophan Mutation Additional Mutations AsG46 AC3076 teD 3 0 5 2 TrpB LPS Repair . R-factor TA1535 TA1537 - - rfa AuvrB - Protocol SPGT502001 06-Jun-2002 Page 2 o f 11 ^ Bio Reliancf BioReliance Study No. 50 MNMMmU Ml kBacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Sponsor Project N umber BioReliance Study Number: DuPont-10819 Histidine Mutation ArG46 TA100 - isC3076 - hisD3052 TA98 - Tryptophan Mutation TrpB WP2 uvrA Additional Mutations LPS Repair R-factor rfa AuvrB +R - AavrA - Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability o f the cell to certain classes o f chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98 and TA100 also contain the pKMIOl plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshifl mutagens. TA100 is reverted by both fiameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions. The E. coli tester strain has an AT base pair at the critical mutation site within the trpB gene (Wilcox et al., 1990). Tester strain WP2 uvrA has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity o f the reversion mechanism is sensitive to base-pair substitution mutations (Green and Muriel, 1976). The S. typhimurium tester strains were received directly from Dr. Bruce Ames, University o f California, Berkeley. The E. coli tester strain was received from the National Collection o f Industrial and Marine Bacteria, Aberdeen, Scotland (United Kingdom). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The test substance will be tested at a minimum o f five dose levels along with appropriate negative and positive controls with tester strains TA98, TA100, TA1535, TA1537 and WP2 uvrA with and without S9 activation. All dose levels o f test substance, negative controls and positive controls will be plated in triplicate. Protocol SPGT502001 06-Jun-2002 Page 3 o f 11 BioReliance Study No. I 51 ^ BioReliancp Tige rt eeii \Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Sponsor Project Number BioReliance Study Number: DuPont-10819 7.1 Solubility Determination Unless the Sponsor has indicated the test substance vehicle, a solubility determination will be conducted to determine the maximum soluble concentration or workable suspension up to a maximum o f 50 mg/mL for aqueous vehicles and 500 mg/mL for organic vehicles. Vehicles compatible with this test system, in order o f preference, include but are not limited to deionized water (CAS 7732-18-5), dimethyl sulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5) and acetone (CAS 67-64-1). The vehicle o f choice will be fee solvent, selected in order o f preference, which permits preparation o f fee highest workable/soluble stock concentration, up to 50 mg/mL for aqueous vehicles and 500 mg/mL for organic vehicles. 7.2 Preliminary Toxicity Assay to Select Dose Levels Selection o f dose levels for fee mutagenicity assay will be based upon the toxicity and precipitation profile o f fee 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 o f test substance, one plate per dose level, in both fee presence and absence o f S9 activation. Unless indicated otherwise by fee Sponsor, the highest dose will be fee highest workable concentration in the vehicle o f choice but not to exceed 5mg/plate. In selecting dose levels for fee mutagenicity assay the following guidelines will be employed. Doses will be selected such that precipitate does not interfere wife manual scoring. Whenever possible, the highest dose for fee mutagenicity assay will be selected to give some indication o f toxicity without exceeding 5 mg/plate. For freely soluble, nontoxic test substances, fee highest dose level will be 5 mg/plate. For precipitating, nontoxic test substances, fee highest dose level will be selected in an attempt to yield precipitate at only the top one or two dose levels. The Sponsor will be consulted regarding dose selection if (1) the maximum dose level is selected based on precipitation and this dose level is less than 5 mg/plate or (2) the maximum achievable test substance dose level is less than 5 mg/plate and this dose level is nontoxic. 7.3 Frequency and Route o f Administration The test system will be exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). This test system has been shown to detect a wide range of classes o f chemical mutagens (McCann et al., 1975; McCann and Ames, 1976). After the data generated in the first assay have been evaluated, the mutagenicity assay will be repeated. The dose levels used in the second assay will be fee same ') as those used in fee first assay unless the Study Director determines that fee dose levels should be changed due to an equivocal response, excessive cytotoxicity or Protocol SPGT502001 06-Jun-2002 Page 4 o f 11 Bio Reuancf B io R elian ce Study No^ 52 fifineaB i^aattisadU ^^ ^Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Sponsor Project Number: BioReliance Study Number DuPont-10819 excessive precipitate. If the Sponsor is aware o f specific metabolic requirements (e.g., azo compounds), this information will be utilized in designing the assay. (e.g., activation system or treatment method). This guidance is based on the OECD Guideline 471 (adopted July 1997 and published February 1998) and ICH Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals (1997). 7.4 Controls 7.4.1 Positive Controls All combinations o f positive controls and tester strains plated concurrently with the assay are listed below: Strain S9 Activation Positive Control Concentration (pg/plate) Salmonella Strains WP2 uvrA 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 o f 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. 7.5 Exogenous Metabolic Activation Arodor 1254-induced rat liver S9 will be used as the metabolic activation system. The S9 homogenate will be prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection o f Aroclor 1254, 500 mgflcg, five days prior to sacrifice. The S9 will be batch prepared and stored frozen at approximately Protocol SPGT502001 06-Jun-2002 Page 5 o f 11 ^ Bio Reliancf BioReliancj Study No. 53 fln m n a n ii S a n lth ro r f D m nut rnataiftififiAfiat [Bacterial Reverse M utation Test w ith an Independent Repeat Assay DuPont-10819 Sponsor Project Number: BioReliance Study Number: -70C until used. Each batch o f S9 homogenate will be assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenzanthracene to forms mutagenic to S. typhimurium TA100. Immediately prior to use, the S9 will be thawed and mixed with a cofactor pool to contain 10% S9 homogenate, 5m M glucose-6-phosphaie, 4m M p-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCh and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. This mixture is referred to as S9 mix. Sham mix will be 100 mM phosphate buffer at pH 7.4. Preparation o f 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 o f incubation will be controlled and monitored. At the end o f foe working day, each inoculated flask will be placed in a resting shaker/incubator at room temperature. The shaker/incubator will be programmed to begin shaking at approximately 125 tpm at 372C approximately 12 hours before the anticipated time o f harvest All cultures will be harvested by spectrophotometric monitoring o f culture turbidity rather than by duration o f incubation since overgrowth o f cultures can cause loss o f sensitivity to some mutagens. Cultures will be removed from incubation at a density o f approximately 109cells/mL. 7.7 Test System Identification Each plate will be labeled with a code system that identifies foe test substance, test phase, dose level, tester strain and activation type as described in BioReliance's Standard Operating Procedures. 7.8 Test Substance Preparation Unless specified otherwise, test substance dilutions will be prepared immediately prior to use. All test substance dosing will be at room temperature under yellow light 7.9 Treatment o f Test System One half milliliter (0.5 mL) o f S9 mix or Sham mix, 100 pL o f tester strain and 50 pL o f vehicle, test substance dilution or positive control will be added to 2.0 mL o f molten selective top agar at 452C. When necessary to achieve foe target concentration or eliminate toxic vehicle effects, aliquots o f other than 50 pL o f test substance/vehicle/positive control will be plated. The mixture will be vortex mixed and overlaid onto foe surface o f 25 mL o f minimal bottom agar. After foe overlay has solidified, the plates will be inverted and incubated for Protocol SPGT502001 OfWun-2002 Page 6 o f 11 Bio Reliance" BioReliance Study No. 54 !Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Sponsor Project Number: DuPont-10819 BioReliance Study Number 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 o f the bacterial background lawn will be evaluated for evidence o f test substance toxicity and precipitate. Evidence o f 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 o f revertant colonies per plate and/or a thinning or disappearance o f 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 o f use in the mutagenicity assay, all.tester strain cultures will be checked for the appropriate genetic markers cited in 6.0. 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST The following criteria must be met for the mutagenicity assay to be considered valid: 8.1 Tester Strain Integrity To demonstrate the presence o f the rfa mutation, all S. typhimurium tester strain cultures must exhibit sensitivity to crystal violet To demonstrate the presence o f file w rB mutation, all S. typhimurium tester strain cultures must exhibit sensitivity to ultraviolet light To demonstrate the presence o f the uvrA mutation, all E. coli test strain cultures must exhibit sensitivity to ultraviolet light. To demonstrate the presence o f the pKMIOl plasmid R-factor, tester strain cultures o f 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 o f spontaneous revertants per plate in the negative controls (vehicle). The mean revertants per plate must be within (he following ranges (inclusive): TA98, 1 0 -5 0 ; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 -2 1 ; WP2avrA, 10-60. 8.3 Tester Strain Titers To ensure that appropriate numbers o f bacteria are plated, all tester strain culture titers must be equal to or great than 0.3xl09cells per milliliter. Protocol SPGT502001 06-Jun-2002 Page 7 o f 11 BioReliance. Study N o ., 55 ^ Bio Reliance A mm Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________ DuPont-10819 Sponsor Project Number: DuPont-10819 BioReliance Study Number 8.4 Positive Control Values j<* 8.5 Each mean positive control value must exhibit at least a 3.0-fold increase over the respective iqean negative control value (vehicle) for each tester strain. Toxicity A minimum o f 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 o f revertants par plate relative to the mean negative control value (this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in the background lawn. In the event that less than three non-toxic dose levels are achieved, the affected portion o f 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 plate o f at least one tester strain over a minimum o f two increasing concentrations o f test substance as specified below: 9.1 Strains TA1535 and TAI537 Data sets will be judged positive if the increase in mean revertants at the peak o f the dose response is equal to or greater than 3.0-times the mean negative control value (vehicle). 9 2 Strains TA98, TA100 and WP2 uvrA Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean negative control value (vehicle). 10.0 REPORT A report o f the results o f this study will be prepared by the Testing Laboratory and will accurately describe all methods used for generation and analysis o f the data. The report will include: Test Substance: identification and CAS no., if known; physical nature and purity, if known; physicochemical properties relevant to the conduct o f the study, if known; stability o f test substance, if known. Solvent/Vehicle: justification for choice o f vehicle; solubility and stability o f test substance in solvent/vehicle, if known. Strains: strains used; number ofcells/mL per culture; strain characteristics. Protocol SPGT502001 06-Jun-2002 Page 8 o f 11 ^ Bio Reliance' BioReliance Study No] 56 G sm m m ^k inilttPfliPBt ^Bacterial Reverse Mutation Tesjt with an Independent Repeat Assay DuPont-10819 Sponsor Project Number. DuPont-10819 BioReliance Study N um ber Test conditions: amount o f test substance per plate with rationale for dose selection and number o f plates per concentration; media used; type and composition o f metabolic activation system, including acceptability criteria; treatment procedures. Results: signs o f toxicity; signs o f precipitation; individual plate counts; the mean number o f revertant colonies per plate and standard deviation; dose-response relationship, where possible; statistical analysis, if any; concurrent negative and positive control data means and standard deviations; historical negative and positive control data with ranges, means and standard deviation. Discussion o f results. Conclusion. 11.0 RECORDS AND ARCHIVES All raw data, the protocol and all reports will be maintained according to Standard Operating Procedure OPQP3040 by the BioReliance RAQA unit headquartered at: BioReliance, 14920 Broschart Road, Rockville, MD 20850. Per this SOP, paper records will be retained for at least 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 o f 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 (he OECD Guidelines for the Testing o f Chemicals, published by OECD, Paris, February 1998 and with the International Conference on Harmonisation o f Technical Requirements for Registration ofPharmaceuticals for Human Use (1996 and 1997). This study will be performed in compliance with the provisions o f the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (GLPs). The protocol, an in-process phase, the raw data, and repoit(s) will be audited per the Standard Operating Procedures (SOPs) o f BioReliance by the Quality Assurance Unit o f BioReliance for compliance with GLPs, the SOPs o f 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 dates o f each inspection, and the dates the results o f each inspection were reported to the Study Director and the Study Director's management In addition, a signed G IF compliance statement will be included in the final report. This statement will cite the GLP guideline(s) with which the study is compliant and any exceptions to this compliance, i f applicable, including the omission o f characterization or stability analyses o f the test or control substances or their mixtures. Protocol SPGT502001 06-Jun-2002 Page 9 o f 11 ^ Bio Reliance- BioReliance Study No. 57 rinmnanw Rqi iQaei nainirnnturin T ir A bacterial Reverse M utation Test w ith an Independent Repeat Assay D u P o n t-10819 Sponsor Project Number: BioReliance Study Number: DuPont-10819 Unless arrangements are made to the contrary, unused dosing solutions will be disposed o f following administration to the test system and all residual test substance will be disposed o f following finalization o f the report. 13.0 REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Sa/monei/a/mammalian-microsome mutagenicity test. Mutation Research 31:347-364. Green, M.H.L., and Muriel, W J. (1976). Mutagen testing using trp+ reversion in Escherichia coli. Mutation Research 38:3-32. International Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 o f the ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24,1996. International Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing o f Pharmaceuticals. S2B document recommended for adoption at step 4 o f the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21, 1997. McCann, J. and Ames, B.N. (1976). Detection o f carcinogens as mutagens in the Salmonella/mictosoms test: assay o f 300 chemicals: discussion. Proc. Natl. Acad. Sci. USA 73:950-954. McCann, J., Choi, E., Yamasaki, E. and Ames, B.N. (1975). Detection o f carcinogens as mutagens in the Salmonella/microsome test: assay o f 300 chemicals. Proc. Nati. Acad. Sci. USA 72:5135-5139. Marn, D.M. and Ames, B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test. Mutation Research 113:173-215. OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), Ninth Addendum to the OECD Guidelines for the Testing o f Chemicals, published by OECD, Paris, February 1998. Wilcox, P., Naidoo, A .,.W edd, D.J. and Gatehouse, D.G. (1990). Comparison o f Salmonella typhimurium TA102 with Escherichia coli WP2 tester strains. Mutagenesis 5:285-291. Protocol SPGT502001 06-Jun-2002 Page 10 o f 11 B io R elian ce Study No 58 ^ BioReliance- C n m n a n w ^ a n iti-y o z-j r>. -- Tgr>A opl acterial Reverse Mutation Test with an fadependent Repeat Assay DuPont-10819 Sponsor Project Number:. DuPont-10819 BioReliance Study N um ber.. APPROVAL <Sponsor Representative -- | j v r Z o o 2_ Date o-r Co- o n A ? .r (Print o r Type Name) \ledU iltiyi4_ O . BioReliance Study Director BioReliance Study Management 1 3 <3 u n 2 o 0 3 l_ Date f i "(% 2 V 0 Z Date ) Protocol SPGT502001 06-Jun-2002 Page 11 o f 11 B io R e lia n ce Study No. 59 ^ Bio Reliancf H noe nnt Tann nw ^Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 APPENDIX C Information for Japanese Regulatory Agencies BioReliance Study No. 60 C om pany Sanitized. Do* not r n n t e in t s p .a o r . fBacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 Report o f Results of Reverse-M utation Test in Bacteria 1. Tester Strains (1) Procurement Strain TA98 TA100 TA1535 TA1537 TA1538 TA97 TA102 WP2 uvrA W P2 uvrA (pkM IOl) W P2 (pK M IO l) Obtained from Date obtained 10 November 1998 Date inspected the strain lot in storage Dr. Bruce Ames University o f California, Berkeley 11 August 1998 13 Decem ber 1990 14 November 1990 The genetic markers for each culture are confirm ed on the day of use National Collection o f Industrial and M arine Bacteria Aberdeen, Scotland 1 M y 1987 19 February 1993 (2) Storage Freezing m ethod Storage tem perature C om position Large quantity -70C Bacterial suspension DMSO 1.0 mL 0.09 mL B io R elian ce Study No. 61 Company S a n it r) r w not -n n tain o p. [Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 2.S9M X (1) Source, Storage Tem perature, etc, o f S9 Made in-house Prepared on 13 February 2002 (Batch R663) Purchased from M oltox Prepared on Storage temperature -70C or colder 18 A pril 2002 (Lot 1384) 14 M ay 2002 (Lot 1390) Name and model of storage apparatus So-Low, M odel P R 2 7 -1 2 0 (2) Preparation o f S9 Animal used Inducing substance Species, Strain Rattus norvegicus, Sprague D aw ley Name A roclor 1254 Sex Age (in weeks) W eight M ale 9 (Batch R663) unknown (Lot 1384) unknown (Lot 1390) 211 to 251 g (Batch R663) unknown (Lot 1384) unknown (Lot 1390) A dm inistration m ethod A dm inistration period and amount (g/kg-w eight) intraperitoneal single dose at 0.5 gm /kg body w eight, sacrificed after 5 days BioReliance Study No.I 62 omgarr acterial Reverse Mutation Test with an Independent Repeat Assay______________________________ DuPont-10819 3. Preparation o f Test Substance Solution___________________________________________ Solvent used Name M anufacturer Lot No. Grade and/or Purity (%) W ater (CAS No. 7732-18-5) Invitrogen Corporation 1129947 D istilled Stability o f test substance in the solvent Unknown M ethod o f suspension when test substance is difficult to dissolve Not applicable BioReliance Study No. j 63 Com gauj^iari^^ Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-10819 4. Conditions o f Pre-culture N utrient broth Period of pre-culture Storage tim e and temp, from inoculation to beginning of shaking culture Storage tim e and temp, from end o f culture to use for test M odel and m anufacturer o f shaker M ethod o f shaking (shaking type, speed, etc.) Culture vessel (shape, capacity) Culture volume Volume o f inoculum Name Oxoid N utrient Broth No. 2 121 hours M anufacturer Lot No. Oxoid Ltd. CH,-B=246975 2 to 5 hours at am bient temperature <8 hours at 2-8C New Brunswick Scientific, m odel G-24 Rotary (125 rev/m in.) shape: cylinder, 200 mL 50 mL 1 colony BioReliance Study No. 64 Company Sanitized. Does not contain TSCA CBl Bacterial Reverse Mutation est with an Independent Repeat Assay 5. Agar Plate M edium (1) Top agar________ Agar Name M anufacturer Lot No. (2) M inimum Glucose Agar Name M ade in-house Agar M anufacturer Lot No. Volume o f agar plate medium DuPont-10819 BBL Select Becton D ickinson 1000J3DKSQ BBL Select Becton Dickinson 1000J3DKSQ 25 mL 6. Test Results - Judgement o f the results Judgem ent N eg ativ e Reason for judgem ent and referential m atters: No positive response was observed w ith any o f the tester strains in either the presence or absence o f Aroclor-induced rat liver S9. Referential m atters 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.i 65 Company Sanitized. Does not contain TSCA CB1