Document 995ZEYB30v1g1E0n0yaDnjxK6

AR226-3189 DuPont-7326 TRADE SECRET Study Title H-25006: Bacterial Reverse M utation T est w ith an Independent Repeat Assay Testing G uideline OECD G uidelines for Testing o f Chemicals Section 4: H ealth Effects, No. 471 (1998); OPPTS G uideline 870.5100 A u th o rs Valentine O. W agner, HI, M .S. Trinh T. H . Nguyen, B.S. Study Com pletion Date 29 O ctober 2001 Performing Laboratory B io R elian ce 9630 M edical Center D rive Rockville, M D 20850 for E. L du Pont de Nemours and Company DuPont H askell Laboratory P.O. Box 50,1090 Elkton Road Newark, DE 19714-0050 Performing Laboratory Study Number AA47TV.502001JBTL DuPont Project ID DuPont-7326 Work Request Number Service Code Page 1 o f 62 Company Sanitized. Does not contain TSCACBl H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 STATEM ENT O F COM PLIANCE Study No. AA47TV.502001.BTL was conducted in com pliance 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 o f Good Laboratory Practice in all material aspects with the following exceptions: The identity, strength, purity and composition or other characteristics to define the test and control substances have not been determined by the testing facility. The control substances have been characterized as per the Certificates o f Analysis on file w ith the testing facility. The stability of the test and control substances has not been determined by the testing facility. Analyses to determine the uniformity (as applicable), or concentration of the test and control mixtures were not performed by the testing facility. The Sponsor has indicated that they have not performed these analyses on the test substance mixtures. The stability of the test and control substances in the test and control mixtures, respectively, has not been determined by the testing facility. The Sponsor has indicated that they have not performed these analyses on the test substance mixtures. Valentine O. W agner, DI, M .S. Study Director BioReliance Study Management 21 D d jo o \ D ate D ate B io R elian ce Study No. AA47TV.502001 .BTL 2 igompany Sanitized. Does not contain TSC CB! H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ Quality Assurance Statement DuPont-7326 Study Title: BACTERIAL REVERSE MUTATION ASSAY WITH AN INDEPENDENT REPEAT ASSAY Study N um ber AA47TV.502001.BTL Study Director: Valentine O. Wagner, IH, M.S. This study has been divided into a series o f in-process phases. U sing a random sam pling approach, Q uality Assurance m onitors each o f these phases over a series o f studies. Procedures, docum entation, equipment records, etc., are exam ined in order to assure that the study is performed in accordance w ith the U .S. FDA G ood Laboratory Practice R egulations (21 CFR 58), the U .S. EPA GLPs (40 CFR 792 and 40 CFR 160), the U K GLP R egulations, the Japanese GLP Standard, and the OECD Principles o f Good Laboratory Practice and to assure that the study is conducted according to the protocol and relevant Standard Operating Procedures. The follow ing are the inspection dates, phases inspected, and report dates o f QA inspections o f this study. Inspect On Phase 02-A ug-01 - 02-A u g-01 T o Study D ir 02-A ug-01 To Mgmt 02-A ug-01 Protocol R eview Inspect On Phase 03-A ug-01 - 03-A ug-01 To Study Dir 03-A ug-01 To M gmt 06-Aug-01 Preparation o f S9 mixture Inspect On Phase 17-Aug-01 - 17-Aug-01 To Study D ir 17-Aug-01 T o Mgmt 17-Aug-01 Strain characterization Inspect On Phase 28-Aug-O l - 28-A u g-01 T o Study D ir 28-A ug-01 To M gmt 29-A ug-01 W eighing the test article Inspect On Phase 29-A ug-01 - 29-A ug-01 To Study D ir29-A ug-01 To M gmt 29-Aug-01 Preparation o f S9 mixture Inspect On Phase Inspect On Phase 03-O ct-01 - 04-O ct-01 To Study Dir 04-O ct-01 To M gm t 09-O ct-01 Draft Report 29-O ct-O l - 29-O ct-O l To Study D ir 29-O ct-O l To M gm t 29-Oct-Ol Draft to Final Report This report describes the m ethods and procedures used in the study and the reported results accurately reflect the raw data o f the study. Elahe Siadatpour, B .S. QUALITY ASSURANCE DATE BioReliance Study No. AA47TV.50200l.BTL Company Sanitized. Does not contain TSCACBI H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 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 0 . W agnerm , M.S. D ate Approved by Study Monitor: -- M aria Donner, Ph.D. Senior Research Scientist I n O CT ZOOK Date BioReliance Study No. AA47TV.502001.BTL 4 Company Sanitized. Does not contain TSCA CBl H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 TABLE OF CONTENTS , Page Good Laboratory Practice Compliance Statem ent.........................................................................2 Q uality A ssurance Statem ent............................................................................................................3 C ertification.........................................................................................................................................4 Study Inform ation.............................................................................................................................. 7 Sum m ary............................................................................................................................................. 8 P u rp o se ................................................................................................................................................. 9 Characterization o f Test and Control Substances................................ 9 M aterials and M ethods.....................................................................................................................10 Results and D iscussion.....................................................................................................................15 C o n clu sio n ........................................................ 15 R eferences.................................................................... 16 D ata T ables........................................................................................................................................ 12 Table 1: Prelim inary Toxicity Test in Salmonella typhimuriwn T A 98............................... 17 Table 2: Prelim inary Toxicity Test in Salmonella typhimurium T A 100.............................18 Table 3: Prelim inary Toxicity Test in Salmonella typhimurium T A 1535...........................19 Table 4: Prelim inary Toxicity Test in Salmonella typhimurium T A 1537.......................... 20 Table 5: Prelim inary Toxicity Test in Escherichia coli W P2 uvrA..................................... 21 Table 6: M utagenicity Test in Salmonella typhimurium TA98 without S 9 ......................22 Table 7: M utagenicity Test in Salmonella typhimurium TA98 w ith S 9 ............................22 Table 8: M utagenicity Test in Salmonella typhimurium TA100 w ithout S 9 ....................24 Table 9: M utagenicity T est in Salmonella typhimurium TA100 with S 9 ......................... 25 Table 10: M utagenicity T est in Salmonella typhimurium TA1535 w ithout S 9 ..................26 Table 11: M utagenicity Test in Salmonella typhimurium TA1535 w ith S 9 ....................... 27 Table 12: M utagenicity Test in Salmonella typhimurium TA1537 w ithout S 9 ..................28 Table 13: M utagenicity Test in Salmonella typhimurium TA1537 w ith S 9 .......................29 Table 14: M utagenicity Test in Escherichia coli W P2 uvrA without S 9.............................30 Table 15: M utagenicity Test in Escherichia coli W P2 uvrA with S 9 .................................. 31 Table 16: M utagenicity T est in Salmonella typhimurium TA98 without S 9 ...................... 32 Table 17: M utagenicity Test in Salmonella typhimurium TA98 w ith S 9 ............................33 Table 18: M utagenicity Test in Salmonella typhimurium TA100 w ithout S 9 ....................34 Table 19: M utagenicity Test in Salmonella typhimurium TA100 with S 9 ......................... 35 Table 20: M utagenicity Test in Salmonella typhimurium TA1535 w ithout S 9 ..................36 BioReliance Study No. AA47TV.502001.BTL 5 Companysanitfe,ed. Does not contain TSCA CB? H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Table 21: M utagenicity Test in Salmonella typhimurium TA1535 with S 9 ....................... 37 Table 22: M utagenicity T est in Salmonella typhimurium TA1537 without S 9 ..................38 Table 23: M utagenicity T est in Salmonella typhimurium TA1537 w ith S 9 ....................... 39 Table 24: M utagenicity T est in coli W P2 uvrA without S 9 .................................................. 40 Table 25: M utagenicity Test in Escherichia coli WP2 uvrA w ith S 9..................................41 Table 26: Salmonella/E. coli M utagenicity Test - Summary o f Results B 1.......................42 Table 27: Salmonella/E. coli M utagenicity Test - Summary o f Results B 2.......................43 Appendix A: H istorical Control D ata............................................................................................ 44 Appendix B: Study Protocol............................................................................................................46 Appendix C: Inform ation for Japanese Regulatory A gencies.................................................... 58 BioReliance Study No. AA47TV.502001 .BTL 6 Does not contain TSCA CBJ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Haskell Numher: 25006 Physical Characteristics: Amber liquid Stability: The test substance appeared to be stable under the conditions of the study; no evidence o f instability was observed. Sponsor: E.L du Pont de Nemours and Company Wilm inpton. Delaware 19898 U.S.A. Study Tnitiated/Completed: August 2,2001 / (see report cover page) In-Life Initiated/Com pleted: August 3, 2001 / Septem ber 6, 2001 BioReliance Study No. AA47TV.50200l.BTL 7 Company Sanitized. Does not contain TSCA CB1 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 SUMMARY The test substance, H-25006, was tested in the H-25006: Bacterial Reverse M utation Test with an Independent Repeat Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain W P2 uvrA in the presence and absence 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 assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay, (initial and independent repeat assays), was used to evaluate the mutagenic potential o f the test substance. Tetrahydrofuran (THF) was selected as the solvent o f choice based on the Sponsor^ request, compatibility with the target cells and solubility o f the test substance. The test substance was soluble and clear in tetrahydrofuran (THF) at approximately 500 mg/mL, the maximum concentration tested. In the preliminary toxicity test, the maximum dose tested was 5000 |ig per plate; this dose was achieved using a concentration o f 200 mg/mL and a 25 pL plating aliquot. Dose levels tested were 5000, 3333,1000, 667, 333, 100, 67, 33, 10 and 6.7 pg per plate. Precipitate was observed beginning at 333 pg per plate and no appreciable toxicity was observed. Based on the findings o f the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 pg per plate. In the mutagenicity test, no positive mutagenic response was observed. The dose levels tested were 5000,1500,500,150,50 and 15 pg per plate. Precipitate was observed beginning at 500 pg per plate and no appreciable toxicity was observed. The results of the Bacterial Reverse M utation Test with an Independent Repeat Assay indicate that, under the conditions of this study, H-25006 did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9. BioReliance Study No. AA47TV.502001.BTL 8 Gcmpany Sanitized. Does not contain TSCA CB! H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 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 o f Escherichia coli strain W P2 uvrA in the presence and absence o f Aroclor-induced rat liver S9. CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test substance, H-25006, was received by BioReliance on 27 July 2001 and was assigned the code number AA47TV. The test substance was characterized by the Sponsor as amber liquid that should be stored at am bient temperature, protected from light. An expiration date o f 31 August 2004 was provided. Upon receipt, the test substance was described as a pale yellow thick liquid and was stored at room temperature, protected from light. 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. Results from these analyses have not been provided to BioReliance. The vehicle used to deliver H-25006 to the test system was tetrahydrofuran (THF, CAS# 109-99-9), Gold Label, 99.9%, purchased from Aldrich Chemical Company. Test substance Hilntinns were prepared immediately before use and delivered to the test system at room temperature under yellow light. Positive controls plated concurrently with the mutagenicity assay are listed below. All positive controls were diluted with dimethyl sulfoxide (DMSO) except sodium azide, which was diluted with water. All subdivided solutions o f positive control were stored at -5 to -30C. Strain All Salmonella Strains S9 A ctivation WP2 uvrA Rat Positive Control 2-am inoanthracene (Sigma Chemical Co.) Lot No. 085H2508 CAS No. 613-13-8 Purity >95% Concentration (pg/plate) 1.0 10 BioReliance Study No. AA47TV.502001.BTL 9 -g w p . ? Sanitized. Does not contain TSQA OBJ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Strain TA98 TA100, TA1535 TA1537 W P2 uvrA S9 A ctiv atio n None Positive Control 2-nitrofluorene (Aldrich Chemical Co., Inc.) Lot No. 11202TF CAS No. 607-57-8 Purity >98% Sodium azide (Sigma Chemical Co.) Lot No. 085H0476 CAS No. 26628-22-8 Purity >99% 9-am inoacridine (Sigma Chemical Co.) Lot No. 106F06681 CAS No. 90-45-9 Purity >98% Methyl methanesulfonate (Aldrich Chemical Co., Inc.) Lot No. 09419LR CAS No. 66-27-3 Purity >99% Concentration (pg/plate) 1.0 1.0 75 1,000 To confirm the sterility of the test substance, the highest test substance dose level used in the mutagenicity test was plated on selective agar with an aliquot volume equal to that used in the test. These plates were incubated under the same conditions as the assay. MATERIALS AND METHODS Test System The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and M uriel (1976). Salmonella tester strains were received directly from Dr. Brace Ames, University o f California, Berkeley. E. coli tester strains were received from the National Collection of Industrial and M arine Bacteria, Aberdeen, Scotland. Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by fram eshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that BioReliance Study No. AA47TV.502001.BTL 10 Company Sanitized. Does not contain TSCA CB! H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 cause both fram eshift 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 M uriel, 1976). Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing -5 0 mL 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 time of harvest Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer o f approximately 10^ 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 M utation Test), adopted July 1997 (published February 1998) and with die 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 m ale Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 batches were prepared and stored at -70C or colder until used. Each bulk preparation of S9 was tested for its ability to metabolize 9-aminnanthrarftnft and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM 8-nicotinamide-adenine dinucleotide phosphate, 8 mM M gCl2 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 o f each was plated on selective agar. Solubility T est A solubility test was conducted to select the vehicle. The test was conducted using water, dimethyl sulfoxide (DMSO), ethanol (EtOH), acetone, tetrahydrofiiran (THF), and N,N-dimethylformamide (DMF). The test substance was tested to determine the vehicle, selected in order o f preference, that permitted preparation o f the highest soluble or workable stock concentration, up to 50 mg/mL for aqueous solvents and 500 mg/mL for organic solvents. BioReliance Study No. AA47TV.502001.BTL 11 r.rifitii'n {>CA H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ _ DuPont-7326 Preliminary Toxicity Test The preliminary toxicity test was used to establish the dose-range over which the test substance would be tested. Ten dose levels o f 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 o f Aroclor-induced rat liver S9. Dose levels tested were 5 0 0 0 ,3 3 33,1000,667,333,100,67,33,10 and 6.7 pg per plate. Mutagenicity Test The mutagenicity test (initial and independent repeat assays), was used to evaluate the mutagenic potential o f the test substance. A minimum of six dose levels o f test substance (5000, 1500, 500, 150, 50 and 15 pg per plate) along with appropriate vehicle and positive controls were plated with TA98, TA100, TA1535, TA1537, and W P2 uvrA in the presence and absence o f Aroclor-induced rat liver S9. All dose levels o f test substance, vehicle controls and positive controls were plated in triplicate. Plating and Scoring Procedures The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). On the day o f its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W A0, 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 with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water im ply sterile, deionized water produced by the Milli-Q Reagent W ater System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). N utrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Each plate was labeled with a code system that identified the test substance, test phase, dose level, tester strain, and activation, as described in detail in BioReliance's Standard Operating Procedures. One-half (0.5) m illiliter 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 o f minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 pL aliquot of appropriate positive control. A fter the overlay had solidified, the plates were inverted and BioReliance Study No. AA47TV.502001.BTL 12 Sanitized. Does not contain TSC H-25006: Bacterial Reverse M utation Test w ith an Independent Repeat A ssay_______ DuPont-7326 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 o f precipitation were scored relative to the vehicle control plate using the codes shown below. Code 1 2 3 4 5 6 NP IP D escription C h aracteristics N orm al Slightly Reduced Distinguished by a healthy m icrocolony 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 M oderately resulting in a pronounced increase in the size o f the microcolonies Reduced compared to the vehicle control plate. Distinguished by an extreme thinning of the microcolony lawn E xtrem ely Reduced 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. A bsent Distinguished by a complete lack o f any microcolony lawn over greater than or equal to 90% o f the plate. Obscured by The background bacterial lawn cannot be accurately evaluated due Precipitate to microscopic test substance precipitate. Distinguished by precipitate on the plate that is visible to the naked eye but any precipitate psubstances detected by the Non-Interfering automated colony counter total less than 10% of the revertant Precipitate colony count (e.g., less than 3 psubstances on a plate w ith 30 revertants). Interfering Precipitate Distinguished by precipitate on the plate that is visible to the naked eye and any precipitate psubstances detected by the automated colony counter exceed 10% of the revertant colony count (e.g., more than 3 psubstances 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 I the plate exhibited toxicity. i Evaluation of Results For each replicate plating, the mean and standard deviation o f the number of revertants per plate were calculated and are reported. BioReliance V j Study No. AA47TV.502001.BTL 13 )0'6S H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 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 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 o f the dose response is equal to or greater than three times the mean vehicle control value. Data sets for tester strains TA98, TA100 and W P2 ttvrA were judged positive if the increase in mean revertants at the peak o f the dose response is equal to or great: than two times the mean vehicle control value. | Criteria for a Valid Test The following criteria must be met for the mutagenicity test to be considered valid. All Saimone/fa tester strain cultures must demonstrate the presence o f the deep rough mutation (ifd) and the deletion in the ovrB gene. Cultures o f tester strains TA98 and TA100 must demonstrate the presence o f the pKM IOl plasmid R-factor. All W P2 uvrA cultures m ust 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, i 80 - 240; TA1535, 5 - 45; TA1537, 3 -2 1 ; W P2vrA , 1 0 -6 0 . To ensure that appropriate numbers o f bacteria are plated, tester strain culture titers must be greater than or equal to 0.3X109cells/mL. The mean o f each positive control must exhibit at least a three-fold increase in the number o f revertants over the mean value o f the respective vehicle control. A minimum of I three non-toxic dose levels are required to evaluate test data. A dose level is considered toxic if | one or both o f the following criteria are m et (1) A >50 % reduction in the mean number of i revertants per plate as compared to the mean vehicle control value. This reduction must be ' accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the ; background lawn. Archives i j A ll raw data, the protocol and all reports will be maintained according to Standard i Operating P r o c e d u i^ H H f lf lp y the BioReliance RAQA unit headquartered at: BioReliance, ; 14920 Broschart R o aa^ o ciro fle, MD 20850. Per this SOP, paper records w ill be retained for at ! least three years after which rime the Sponsor w ill be contacted for a decision as to the final disposition of the materials. All study materials returned to the Sponsor or destroyed will first be copied and the copy will be retained in the BioReliance archives for a minimum o f 10 years. I Unused dosing solutions were disposed o f following administration to the test system and all I residual test substance will be disposed o f following finalization o f the report, j < Deviations No known deviations from the protocol or assay-method SOPs occurred during the conduct o f this study. B io R elian ce Study No. AA47TV.502001.BTL 14 CompanySanifeed oes ntf contain TscAcfi| H-25006: Bacterial Reverse M utation Test w ith an Independent Repeat Assay______ _ DuPont-7326 RESULTS AND DISCUSSION Solubility Test Tetrahydrofuran (THF) was selected as the solvent o f choice based on the Sponsor's request, compatibility w ith the target cells and solubility o f the test substance. The test substance was soluble and clear in tetrahydrofuran (THF) at approximately 500 mg/mL, the maximum concentration tested. ! Preliminary 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 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 i plating aliquot. D ose levels tested were 5000, 3333, 1000,667,333,100,67, 33,10 and 6.7 pg per plate. Precipitate was observed beginning at 333 pg per plate and no appreciable toxicity was observed. Based on the findings o f the toxicity test, the maximum dose plated in the mutagenicity test w as 5000 pg per plate. Mutagenicity Test 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. Dose levels tested ! were 5000, 1500, 500, 150, 50 and 15 pg per plate. Precipitate was observed beginning at ; 500 pg per plate and no appreciable toxicity was observed. In Experiment B1 (Initial Mutagenicity Test), no positive mutagenic responses were observed with any o f the tester strains in either the presence or absence o f S9 activation. In Experiment B2 (Independent Repeat Test), no positive mutagenic responses were . observed with any o f the tester strains in either the presence or absence of S9 activation. j | CONCLUSION i j All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse M utation Test with an Independent Repeat Assay indicate that, under the conditions of this study, H-25006 did not cause a positive mutagenic response in either the presence or absence o f Aroclor-induced rat liver S9. B io R elian ce ! Study No. AA47TV.502001.BTL ! 15 CompanySanitized. Does no,contain TSM cffl H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the SalmonellaMsramahsn M icrosome 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 of 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. Maron, D M . and B.N. Ames (1983) Revised Methods for the Salmonella Mutagenicity Test, Mutation Research, 113:173-215. OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse 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) Acetylomithinase of E. coli: Partial Purification and Some Properties, J. Biol. Chem., 218:97-106. BioReliance Study No. AA47TV.502001.BTL 16 Company Sanitized. Bees not contain TSCACB5 H-25006: Bacterial Reverse M utation Test with an Independent Repeat Assay_______ DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 1 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL Al TA98 3 Aug 2001 tetrahydrofuran 25 uL___________________________ .------------ Test Substance Concentration With S9 Activation Revertants Background Without S9 Activation Revertants Background ug per plate_____ per plate_______ Lawn_______ per plate_______ Lawn--- Vehicle 21 1 6.7 10 33 67 100 333 667 1000 3333 5000 27 1 18 1 25 1 21 1 19 1 33 1NP 24 1NP 32 1NP 31 1NP 30 IIP 25 1 33 1 17 1 39 1 29 1 28 1NP 20 1NP 25 1NP 27 1NP 27 IIP 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. AA47TV.502001.BTL 17 Company Sanitized. Does not contain TSCA, Zfj H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 2 Test Substance Id Study Number Experiment No Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV.502001. BTL Al TAI 00 3 Aug 2001 tetrahydrofuran 25 pL . Test Substance : Concentration With S9 Activation Revertants Background Without S9 Activation Revertants Background j ug per plate______ per plate_______ Lawn_______ per plate_______ Lawn--- Vehicle 230 1 6.7 10 33 67 100 333 667 1000 3333 5000 192 1 166 1 200 1 212 1 281 1 251 1NP 199 1NP 221 1NP 214 1NP 178 IIP 214 1 247 1 239 1 222 1 212 1 218 1NP 259 1NP 247 1NP 215 1NP 190 IIP Background Lawn Code - i=Nomtal; 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. AA47TV.50200l.BTL 18 Company Sanitized. Does notcontainJSG AG H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ _ DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 3 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL Al TA1535 3 Aug 2001 tetrahydrofuran 25 uL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 19 1 Without S9 Activation Revertants Background per plate Lawn 12 1 6.7 10 33 67 100 333 667 1000 3333 5000 20 1 21 1 16 1 17 1 20 1 24 1NP 16 1NP 25 1NP 26 1NP 14 IIP 16 1 16 1 17 1 23 1 13 1 22 1NP 24 1NP 19 1NP 16 1NP 18 IIP 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. AA47TV.502001 .BTL 19 Company Sanitized. Does not contain TSC CBS H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 4 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL Al TA1537 3 Aug 2001 tetrahydrofuran 25 uL Test Substance Concentration ua ner plate With S9 Activation Revertants Background per plate Lawn Vehicle 61 Without S9 Activation Revertants Background per plate Lawn 10 1 6.7 10 33 67 100 333 667 1000 3333 5000 91 41 10 1 61 61 8 1NP 16 1NP 9 1NP 7 1NP 17 IIP 91 71 12 1 71 91 11 1NP 8 1NP 13 1NP 7 1NP 6 IIP 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. AA47TV.50200l.BTL Company Sanitized. Does not contain TSCA CBI 20 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Preliminary Toxicity Assay Table 5 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25006 AA47TV.502001.BTL A1 WP2 uvrA 3 Aug 2001 tetrahydrofuran 25 pL Test Substance With S9 Activation Without S9 Activation Concentration Revertants Background Revertants Background )ig per plate_____ per plate_______ Lawn_______ per plate_______ Lawn Vehicle 23 1 15 1 6.7 10 33 67 100 333 667 1000 3333 5000 21 1 21 1 27 1 26 1 20 1 21 1NP 19 1NP 16 1NP 18 1NP 18 IIP 17 1 13 1 13 1 13 1 17 1 13 1NP 16 1NP 17 1NP 23 1NP 15 IIP 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. AA47TV.502001.BTL Company Sanitized. Does not contain TSCA CB1 21 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 6 Test Substance Id: H-25006 Study Number : AA47TV.502001.BTL Strain : TA98 Experiment No : B1 Cells Seeded : 2.2 X 10 Liver Microsomes : None Date Plated : 17 Aug 2001 Vehicle : tetrahydrofuran Plating Aliquot : 25 yiL___________________ __________ --____________ Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 22 1 23 1 26 1 24 2 15 01 18 1 02 24 1 03 22 1 21 3 50 01 26 1 02 23 1 03 23 1 24 2 150 01 25 .1 02 25 1 03 24 1 25 1 500 01 18 1NP 02 22 1NP 03 30 1NP 23 6 1500 01 21 1NP 02 22 1NP 03 27 1NP 23 3 5000 01 30 IIP 02 28 IIP 03 20 IIP 26 5 Positive Control 2-nitrofluorene 1 .0 pg per plate 01 265 1 02 206 1 03 215 1 229 32 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 i BioReliance Study No. AA47TV.502001 .BTL 22 not contain TSGACBS Company Sanitized Des H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Bacterial Mutation Test Table 7 Test Substance Id: H-25006 Study Number : AA47TV.502001.BTL Strain : TA98 Liver Microsomes : Rat liver S9 Vehicle : tetrahydrofuran Plating Aliguot : 25 uL Experiment No : B1 Cells Seeded : 2.2 X 108 Date Plated : 17 Aug 2001 Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 24 1 31 1 20 1 25 6 15 01 40 1 02 30 1 03 27 1 32 7 50 01 24 1 02 37 1 03 30 1 30 7 150 01 22 1 02 38 1 03 35 1 32 9 500 01 30 1NP 02 34 1NP 03 35 1NP 33 3 1500 01 32 1NP 02 27 1NP 03 34 1NP 31 4 5000 01 26 IIP 02 24 IIP 03 28 IIP 26 2 Positive Control 2-aminoanthracene 1.0 ug per plate 01 673 1 02 1051 1 03 816 1 847 191 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. AA47TV.502001.BTL 23 phRipahy Shlz. H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay___________________________________ DuPont-7326 Bacterial Mutation Test Table 8 Test Substance Id: H-25006 Study Number : AA47TV.502001.BTL Strain : TA100 Liver Microsomes : None Vehicle : tetrahydrofuran Platina Aliauot : 25 uL Experiment No : B1 Cells Seeded : 1.4 X 108 Date Plated : 17 Aug 2001 Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 188 1 180 1 195 1 188 8 15 01 152 1 02 172 1 03 180 1 168 14 50 01 210 1 02 186 1 03 207 1 201 13 150 01 170 1 02 154 1 03 176 1 167 11 500 01 149 1NP 02 176 1NP 03 180 1NP 168 17 1500 01 170 1NP 02 194 1NP 03 196 1NP 187 14 5000 01 197 IIP 02 211 IIP 03 192 IIP 200 10 Positive Control sodium azide 1.0 pgr per plate 01 679 1 02 602 1 03 633 1 638 39 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. AA47TV.502001 .BTL 24 Company sanitized. Does not contain TSCA CBS H-25006: Bacterial Reverse M utation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 9 Test Substance Id: H-25006 Study Number : AA47TV.502001.BTL Strain : TA100 Liver Microsomes : Rat liver S9 Experiment No : B1 Cells Seeded : 1.4 X 108 Date Plated : 17 Aug 2001 Vehicle : tetrahydrofuran Platincr Aliauot : 25 uL Concentration Plate Revertants Background Average Standard ucr ner plate Number per plate Code Revertants Deviation Vehicle 01 02 03 188 1 181 1 169 1 179 10 15 01 211 1 02 184 1 03 190 1 195 14 50 01 186 1 02 175 1 03 189 1 183 7 150 01 187 1 02 183 1 03 179 1 183 4 500 01 203 1NP 02 196 1NP 03 165 1NP 188 20 1500 01 220 1NP 02 177 1NP 03 196 1NP 198 22 5000 01 180 IIP 02 198 IIP 03 176 IIP 185 12 Positive Control 2-aminoanthracene 1.0 pg per plate 01 1229 1 02 1438 1 03 1178 1 1282 138 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. AA47TV.502001.BTL 25 Company Sanitized. Does not contain TSCA CBf H-25006: Bacterial Reverse M utation Test w ith an Independent Repeat Assay _______________________________ D uP ont-7326 Bacterial Mutation Test Table 10 Test Substance Id: H-25006 Study Number : AA47TV.502001.BTL Strain : TA1535 Liver Microsomes : None Experiment No : B1 Cells Seeded : 2.7 X 10B Date Plated : 17 Aug 2001 Vehicle : tetrahydrofuran Plating Alicruot : 25 uL Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 24 1 20 1 21 1 22 2 15 01 12 1 02 18 1 03 22 1 17 5 50 01 10 1 02 15 1 03 20 1 15 5 150 01 21 1 02 27 1 03 20 1. 23 4 500 01 26 1NP 02 13 1NP 03 27 1NP 22 8 1500 01 24 1NP 02 17 1NP 03 13 1NP 18 6 5000 01 22 IIP 02 22 IIP 03 30 IIP 25 5 Positive Control sodium azide 1.0 pg per plate 01 517 1 02 460 1 03 626 1 534 84 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. AA47TV.502001 .BTL 26 Pempsrsy Sanitized, Does not contain 81 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 11 Test Substance Id Study Number Strain Liver Microsomes H-25006 AA47TV.502001.BTL TA1535 Rat liver S9 Experiment No Cells Seeded Date Plated B1 2.7 X 108 17 Aug 2001 Vehicle tetrahydrofuran Platina Aliauot : 25 uL Concentration Plate Revertants Background Average Standard ua Der Diate Number per plate Code Revertants Deviation Vehicle 01 02 03 12 1 15 1 13 . 1 13 2 15 01 14 1 02 12 1 03 11 1 12 2 50 01 22 1 02 11 1 03 14 1 16 6 150 01 23 1 w 02 11 1 03 11 1 15 7 500 01 21 1NP 02 18 1NP 03 10 1NP 16 6 1500 01 25 1NP 02 20 1NP 03 11 1NP 19 7 5000 01 16 IIP 02 20 IIP 03 14 IIP 17 3 Positive Control 2-aminoanthracene 1.0 pg per plate 01 204 1 02 176 1 03 222 1 201 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. AA47TV.502001.BTL 27 Company Sanitized. Does H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay __________________________ DuPont-7326 Bacterial Mutation Test Table 12 Test Substance Id: H-25006 Study Number :AA47TV.502001.BTL Experiment No : B1 Strain :TA1537 Cells Seeded :0.9X 10s Liver Microsomes :None Date Plated : 17Aug 2001 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 10 1 61 71 82 15 01 8 1 02 9 1 03 5 1 72 50 01 6 1 02 8 1 03 8 1 71 150 01 6 1 02 10 1 03 5 1 73 500 01 11 1NP 02 9 1NP 03 11 1NP 10 1 1500 01 4 1NP 02 6 1NP 03 8 1NP 62 5000 01 7 IIP 02 7 IIP 03 6 IIP 71 Positive Control 9-aminoacridine 75 pg per plate 01 1634 1 02 1073 1 03 648 1 1118 495 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. AA47TV.502001.BTL 28 Company Sanitized. Doe? not contain TSCA CBr. H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 13 Test Substance Id: H-25006 Study Number :AA47TV.502001.BTL Strain :TA1537 Liver Microsomes :Rat liver S9 Experiment No :B1 Cells Seeded :0.9 X10 Date Plated :17Aug 2001 Vehicle :tetrahydrofuran Plating Aliguot : 25 uL Concentration Plate Revertants Background Vig per plate Number per plate Code Average Revertants Standard Deviation Vehicle 01 02 03 61 51 71 61 15 01 12 1 02 8 1 03 6 1 93 50 01 17 1 02 16 1 03 8 1 14 5 150 01 10 1 02 9 1 03 7 1 92 500 01 6 1NP 02 12 1NP 03 5 1NP 84 1500 01 8 1NP 02 6 1NP 03 4 1NP 62 5000 01 7 IIP 02 7 IIP 03 8 IIP 71 Positive Control 2-aminoanthracene 1.0 m 01 242 1 02 115 1 03 231 1 per plate 196 70 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. AA47TV.502001 .BTL 29 not contain TSC ( I Company Sanitized* Does H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 14 Test Substance Id: H-25006 Study Number AA47TV.502001.BTL Strain WP2 u v r A Liver Microsomes None Vehicle tetrahydrofuran Experiment No Cells Seeded Date Plated B1 5.9 X 10s 17 Aug 2001 Concentrt ion pg per plate Vehicle Plate Number 01 02 03 Revertants per plate 21 15 14 Background Code 1 1 1 Average Revertants 17 Standard Deviation 4 15 01 18 1 02 18 1 03 11 1 50 01 18 1 02 12 1 03 18 1 150 01 9 1 02 11 1 03 23 1 16 4 16 3 14 8 500 01 14 1NP 02 14 1NP 03 19 1NP 16 3 1500 01 8 1NP 02 18 1NP 03 16 1NP 14 5 5000 01 16 IIP 02 14 IIP 03 12 IIP 14 2 Positive Control methyl methanesulfonate 1000 ug per plate 01 244 1 02 259 1 03 260 1 254 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. AA47TV.502001.BTL 30 Cempany Sanitized. Does not contain TSCA CB H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay _____ DuPont-7326 Bacterial Mutation Test Table 15 Test Substance Id H-25006 Study Number AA47TV.502001.BTL Strain WP2 uvrA Liver Microsomes : Rat liver S9 Experiment No : B1 Cells Seeded : 5.9 X 10s Date Plated : 17 Aug 2001 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 14 1 13 1 14 1 14 1 15 01 14 1 02 15 1 03 12 1 14 2 50 01 17 1 02 17 1 03 13 1 16 2 150 01 11 1 02 18 1 03 11 1 13 4 500 01 14 1NP 02 13 1NP 03 9 1NP 12 3 1500 01 12 1NP 02 17 1NP 03 16 1NP 15 3 5000 01 16 IIP 02 20 IIP 03 15 IIP 17 3 Positive Control 2-aminoanthracene 10 pg per plate 01 515 1 02 556 1 03 694 1 588 94 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. AA47TV.502001 .BTL 31 Company Sanitized. Does not contain TSCA CBi 1 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_____________________________________ DuPont-7326 Bacterial Mutation Test Table 16 Test Substance Id: H-25006 Study Number :AA47TV.502001.BTL Experiment No :B2 Strain :TA98 Cells Seeded :2.8 X108 Liver Microsomes :None Date Plated :29Auer 2001 Vehicle :tetrahydrofuran Plating Aliquot : 25 yiL________________________________ ________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 23 1 12 1 11 . 1 15 7 15 01 21 1 02 19 1 03 26 1 50 01 15 1 02 21 1 03 14 1 150 01 15 1 02 18 1 03 17 1 22 4 17 4 17 2 500 01 14 1NP 02 17 1NP 03 16 1NP 16 2 1500 5000 01 23 1NP 02 26 1NP 03 22 1NP 01 14 IIP 02 21 IIP 03 17 IIP 24 2 17 4 Positive Control 2-nitrofluorene 1 .0 pg per plate 01 176 1 02 182 1 03 139 1 166 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 B io R elian ce Study No. AA47TV.502001 .BTL 32 Company Sanitized. Does not contain TSC CB3 % m <0 i H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 17 Test Substance Id: H- 25006 Study Number : AA47TV.502001.]BTL Strain : TA98 Liver Microsomes : Rat liver S9 Experiment No : B2 Cells Seeded : 2.8 X 108 Date Plated : 29 Aug 2001 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 26 1 29 1 23 . 1 26 3 15 01 35 1 02 35 1 03 24 1 31 6 50 01 17 1 02 26 1 03 24 1 22 5 150 01 22 1 02 18 1 03 20 1 20 2 500 01 16 1NP 02 26 1NP 03 22 1NP 21 5 1500 01 12 1NP 02 21 1NP 03 16 1NP 16 5 5000 01 21 IIP 02 20 IIP 03 21 IIP 21 1 Positive Control 2-aminoanthracene 1.0 pg per plate 01 510 1 02 484 1 03 648 1 547 88 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. AA47TV.502001.BTL 33 Pofflpany Sanitized. Does not contain TSGA it il I H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 18 Test Substance Id: H-25006 Study Number : AA47TV.502001.BTL Strain : TA100 Liver Microsomes : None Vehicle : tetrahydrofuran Plating Aliquot : 25 uL__________ Experiment No : B2 Cells Seeded : 2.4 X 10s Date Plated : 29 Aug 2001 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 201 1 177 1 173 1 184 15 15 01 167 1 02 205 1 03 219 1 197 27 50 01 184 1 02 161 1 03 184 1 176 13 150 01 170 1 02 198 1 03 186 1 185 14 500 01 160 1NP 02 148 1NP 03 193 1NP 167 23 1500 01 132 1NP 02 154 1NP 03 188 1NP 158 28 5000 01 135 IIP 02 151 IIP 03 179 IIP 155 22 Positive Control sodium azide 1.0 pg per plate 01 667 1 02 692 1 03 697 1 685 16 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. AA47TV.502001.BTL 34 r i Dess not contain TS CA 0^ 1 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 19 Test Substance Id Study Number , Strain Liver Microsomes H-25006 AA47TV.502001.BTL TA100 Rat liver S9 Experiment No Cells Seeded Date Plated B2 2.4 X 108 29 Aug 2001 Vehicle tetrahydrofuran Plating Aliguot : 25 uL Concentrtion Plate Revertants Background Average Standard ua per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 170 1 192 1 156 1 173 18 15 01 176 1 02 183 1 03 194 1 184 9 50 01 152 1 02 185 1 03 197 1 178 23 150 01 181 .1 02 153 1 03 157 1 164 15 500 01 165 1NP 02 143 1NP 03 154 1NP 154 11 1500 01 184 1NP 02 176 1NP 03 149 1NP 170 18 5000 01 193 IIP 02 172 IIP 03 178 IIP 181 11 Positive Control 2-aminoanthracene 1.0 ug per plate 01 804 1 02 777 1 03 654 1 745 80 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent ; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate BioReliance Study No. AA47TV.502001.BTL 35 Company Sanitized. Does not contain TSCACBJ H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______ DuPont-7326 Bacterial Mutation Test Table 20 Test Substance Id: H-25006 Study Number : AA47TV. 502001.BTL Strain : TA1535 Liver Microsomes : None Experiment No : B2 Cells Seeded : 3.2 X 108 Date Plated : 29 Aug 2001 Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Concentration Plate Revertants Background Average Standard uo per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 25 1 28 1 21 . 1 25 4 15 01 25 1 02 29 1 03 26 1 27 2 50 01 12 1 02 20 1 03 22 1 18 5 150 01 14 1 02 23 1 03 21 1 19 5 500 01 23 1NP 02 17 1NP 03 19 1NP 20 3 1500 01 18 1NP 02 18 1NP 03 18 1NP 18 0 5000 01 20 IIP 02 24 IIP 03 21 IIP 22 2 Positive Control sodium azide 1.0 ug per plate 01 295 1 02 266 1 03 305 1 289 20 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. AA47TV.50200l.BTL 36 Company Sanitized. Does not contain TSCACBJ] H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 21 Test Substance Id: H-25006 Study Number : AA47TV.502001.BTL Strain : TA1535 Liver Microsomes : Rat. liver S9 Experiment No : B2 Cells Seeded : 3.2 X 108 Date Plated : 29 Aug 2001 Vehicle : tetrahydrofuran Plating Aliquot : 25 PL Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertantsi Deviation Vehicle 01 02 03 15 1 12 1 81 12 4 15 01 14 1 02 19 1 03 17 1 17 3 50 01 10 1 02 14 1 03 8 1 11 3 150 01 22 .1 02 14 1 03 24 1 20 5 500 01 8 1NP 02 14 1NP 03 17 1NP 13 5 1500 01 21 1NP 02 19 1NP 03 14 1NP 18 4 5000 01 21 IIP 02 25 IIP 03 14 IIP 20 6 Positive Control 2-aminoanthracene 1.0 pg per plate 01 105 1 02 192 1 03 196 1 164 51 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. AA47TV.502001 .BTL 37 Company Sanitized. Does not contain TSCA CB3 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 22 Test Substance Id: H-25006 Study Number : AA47TV.502001.BTL Experiment No : B2 g Strain : TA1537 Cells Seeded : 1.3 X 10 Liver Microsomes : None Date Plated : 29 Aug 2001 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 51 71 71 61 15 01 4 1 02 10 1 03 4 1 63 50 01 5 1 02 2 1 03 8 1 53 150 01 5 1 02 3 1 03 6 1 52 500 01 6 1NP 02 7 1NP 03 5 1NP 61 1500 01 7 1NP 02 7 1NP 03 8 1NP 71 5000 01 5 IIP 02 4 IIP 03 6 IIP 51 Positive Control 9-aminoacridine 75 pg per plate 01 792 1 02 891 1 03 889 1 857 57 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. AA47TV.502001.BTL 38 Company Sanitized, Does H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 23 Test Substance Id Study Number Strain Liver Microsomes Vehicle H-25006 AA47TV.502001.BTL TA1537 Rat liver S9 tetrahydrofuran Experiment No Cells Seeded Date Plated B2 1.3 X 10e 29 Aug 2001 Concentration pg per plate Vehicle Plate Number 01 02 03 Revertants per plate 6 7 4 Background Code 1 1 1 Average Revertants 6 Standard Deviation 2 15 01 12 1 02 10 1 03 6 1 93 50 01 2 1 02 4 1 03 7 1 43 150 01 5 1 02 5 1 03 8 1 500 01 3 1NP 02 7 1NP 03 5 1NP 62 - 52 1500 01 2 1NP 02 7 1NP 03 6 1NP 53 5000 01 9 IIP 02 8 IIP 03 4 IIP 73 Positive Control 2-aminoanthracene 1.0 pg per plate 01 179 1 02 195 1 03 183 1 186 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 i BioReliance Study No. AA47TV.502001 .BTL 39 Company Sanitized. Does not contain TSCA CBB H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 24 Test Substance Id: H-25006 Study Number : AA47TV.502001.BTL Strain : WP2 uvrA Liver Microsomes : None Experiment No : B2 Cells Seeded : 5.8 X 10s Date Plated : 29 Aug 2001 Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Concentration Plate Revertants Background Average Standard Vig per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 14 1 15 1 13 1 14 1 15 01 12 1 02 20 1 03 8 1 13 6 50 01 16 1 02 11 1 03 18 1 15 4 150 01 12 1 02 9 1 03 9 1 10 2 500 01 13 1NP 02 11 1NP 03 12 1NP 12 1 1500 01 15 1NP 02 17 1NP 03 12 1NP 15 3 5000 01 10 IIP 02 12 IIP 03 11 IIP 11 1 Positive Control methyl methanesulfonate 1000 pg per plate 01 103 1 02 174 1 03 189 1 155 46 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. AA47TV.502001.BTL 40 Company Sanitized. Does not contain TSC GBO H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Table 25 Test Substance Id Study Number Strain Liver Microsomes Vehicle H-25006 AA47TV.502001.BTL WP2 uvrA Rat liver S9 tetrahydrofuran Experiment No : B2 Cells Seeded : 5.8 X 108 Date Plated : 29 Aug 2001 Concentration ug per plate Vehicle Plate Number 01 02 03 Revertants per plate 19 20 17 Background Code 1 1 .1 Average Revertants 19 Standard Deviation 2 15 01 17 1 02 11 1 03 16 1 15 3 50 01 13 1 02 21 1 03 20 1 18 4 150 01 11 1 02 14 1 03 18 1 14 . 4 500 01 18 1NP 02 22 1NP 03 16 1NP 19 3 1500 01 18 1NP 02 17 1NP 03 14 1NP 16 2 5000 01 18 IIP 02 14 IIP 03 11 IIP 14 4 Positive Control 2-aminoanthracene 10 jig per plate 01 469 1 02 500 1 03 544 1 504 38 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. AA47TV.502001.BTL 41 ompany Sanitized. Does not contain TSCA Cl? H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Summary of Results Table 26 Test Substance Id : H-25006 Study Nnn^er______ : AA47TV.502001-BTL Experiment No ; B1 Average Revertants Per Plate Standard Deviation Liver Microsomes: None Dose (yig/plate) TA98 TA100 Vehicle 15 50 150 500 1500 5000 Positive 24 21 24 25 23 23 26 229 2 188 3 168 2 201 1 167 6 168 3 187 5 200 32 6 3 8 8 14 13 11 17 14 10 39 Liver Microsomes: Rat liver S9 TA1535_____TA1537 22 17 15 23 22 18 25 534 2 8 2 5 7 2 5 7 1 4 7 3 8 10 1 6 6 2 5 7 1 84 1118 495 WP2 uvrA 17 16 16 14 16 14 14 254 4 4 3 8 3 5 2 9 Dose (ug/plate) TA98_____ TA100______TA1535 Vehicle 15 50 150 500 1500 5000 Positive 25 6 179 10 32 7 195 14 30 7 183 7 32 9 183 4 33 3 188 20 31 4 198 22 26 2 185 12 847 191 1282 138 13 12 16 15 16 19 17 201 2 2 6 7 6 7 3 23 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 25 uL TA1537 6 1 9 3 14 5 9 2 8 4 6 2 7 1 196 70 WP2 uvrA 14 14 16 13 12 15 17 588 1 2 2 4 3 3 3 94 BioReliance Study No. AA47TV.50200l.BTL 42 Company Sanitized. Does not contain TSCA CBS H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Bacterial Mutation Test Summary of Results Table 27 Test Substance Id : H-25006 Study Number______ : AA47TV. 502001.BTL Experiment No : B2 Average Revertants Per Plate Standard Deviation Liver Microsomes: None Dose (pg/plate) TA98 TA100 Vehicle 15 50 150 500 1500 5000 Positive 15 7 184 15 22 4 197 27 17 4 176 13 17 2 185 14 16 2 167 23 24 2 158 28 17 4 155 22 166 23 685 16 Liver Microsomes: Rat liver S9 TA1535 25 27 18 19 20 18 22 289 4 2 5 5 3 0 2 20 TA1537 6 6 5 5 6 7 5 857 1 3 3 2 1 1 1 57 WP2 uvrA 14 13 15 10 12 15 11 155 1 6 4 2 1 3 1 46 Dose (ug/plate) TA98 TA100 Vehicle 15 50 150 500 1500 5000 Positive 26 31 22 20 21 16 21 547 3 173 6 184 5 178 2 164 5 154 5 170 1 181 88 7 4 5 18 9 23 15 11 18 11 80 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 25 pL TA1535 12 17 11 20 13 18 20 164 4 3 3 5 5 4 6 51 TA1537___ WP2 uvrA_ 6 9 4 6 5 5 7 186 2 19 2 3 15 3 3 18 4 2 14 4 2 19 3 3 16 2 3 14 4 8 504 i 38 BioReliance Study No. AA47TV.502001.BTL 43 Company Sanitized. Does not contain TSCA CBS H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 APPENDIX A Historical Control Data BioReliance Study No. AA47TV.502001 .BTL 44 Company Sanitized- Does not contain TSCA CBS H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Historical Negative and Positive Control Values 1998 - 2000 ievertants per plate Activation Strain Control Mean None SD Min Max Mean Rat Liver SD Min Max TA98 TA100 TA1535 TA1537 WP2 uvrA Neg 16 7 4 59 Pos 425 206 21 1536 Neg 128 31 53 288 Pos 568 159 129 1371 Neg 12 5 1 45 Pos 378 164 6 978 Neg 6 3 0 30 Pos 708 409 13 2786 Neg 1 14 54 Pos 1 190 138 34 48 961 21 7 592 322 138 34 736 301 12 4 104 84 73 88 106 16 6 317 299 7 58 56 2454 74 258 198 2871 1 42 18 1640 1 29 12 2060 4 115 22 2632 SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control________________________ ____________ ___________________________ BioReliance Study No. AA47TV.50200l.BTL 45 Company Sanitized. Does not contain TSC CBI ; H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 APPENDIX B Study Protocol BioReliance Study No. AA47TV.502001.BTL 46 Company Sanitized. Does not contain TSCA Ct H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay____________________________________ DuPont-7326 ">vsri by RWOA 3*1~ Sponsor Project N um ber D u P o n t-7 3 2 6 B ioR eliance Study N um ber A A 47TV .502001.BTL H -25006: B acterial R everse M utation T est w ith an Independent R epeat A ssay 1.0 PURPOSE The purpose o f this study is to evaluate the m utagenic potential o f the test substance by m easuring its ability to induce reverse m utations at selected lo ci o f several strains o f Salmonella typhimurium and at the tryptophan locu s o f Escherichia coli W P2 irvrA in the presence and absence o f S9 activation. 2.0 SPONSOR 2.1 Name: E.I. du Pont de N em ours and Company 2.2 Address: Stine H askell R esearch Center DuPont H askell Laboratory P.O . B ox 50 1090 Elkton Road Newark, DE 19714-0050 2.3 Representative: M aria Donner, Ph.D . Phone: 302-366-5251 Fax: 302-366-5207 Email: m aria.donner@ usa.dupont.com 2.4 Sponsor Project N o.: D u P on t-7326 2.5 WR#: 2.6 H askell#: H -25006 2.7 Service Code: 3 .0 IDENTIFICATION OF TEST A N D CONTROL SU BSTA N C ES 3.1 T est Substance Nam e: 3.2 T est Substance I.D.: H -25006 (to b e used in the report title and text) 3.3 Controls: N egative: Positive: T est substance vehicle 9-am inoacridine 2-am inoanthracene m ethyl m ethanesulfonate 2-ntitrofluorene sodium aride ip0' Protocol SPGT502001 l-Jan-2001 Page 1 o f 11 BioReliance Study No. AA47TV.502001.BTL 47 ^ Bio Reliance- Company Sanitized. Does not contain TSCA Ci H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Sponsor Project Number: D u P on t-7326 B ioR eliance Study Num ber. A A 47T V .502001 -BTL 3 .4 T est Substance Characterization U nless alternate arrangements are maria, the testing facility at B ioR eliance w ill not perform analysis o f the dosing solutions. The Sponsor w ill be directly responsible for determ ination and docum entation o f the analytical purity and com position o f the test substance, and the stability and strength o f the test substance in the solvent (or vehicle). 3.3 T est Substance Retention Sam ple The retention o f a reserve sam ple o f the test substance w ill be the responsibility o f the Sponsor. 4 .0 TESTING FACILITY A N D KEY PERSONNEL 4.1 Name: T oxicology T esting Facility BioR eliance 4.2 Address: 9630 M edical Center Drive R ockville, M D 20850 4.3 Study Director: Valentine O. W agner EH, M .S. Phone: 301-610-2152 Fax: 301-738-2362 Email: swagner@ bioreliance.com 5.0 PROPOSED STU D Y DATES 5.1 Experim ental Start Date: 03-A u g-2001 5.2 Experim ental Termination Date: 19-Sep-2001 5.3 Draft Report Date: 10-0ct-2001 5.4 Final Report Date: 2 w eeks after Sponsor approves draft TEST SYSTEM T he tester strains w ill include the S. typhimurium histidine auxotrophs T A 98, TA 100, T A 1535 and T A 1537 as described by A m es et al. (1975) and the K coli tester strain W P2 wvrA as described by Green and M uriel (1976)._________________________________ H istidine M utation Tryptopha n M utation A dditional M utations AwG46 AC3076 /iiD 3 0 5 2 trpE LPS Repair R-factor TA1535 TA1537 - - rfa uvrB - Protocol SPG T 502001 1-Jan-2001 Page 2 o f 11 BioReliance Study No. AA47TV.502001.BTL 48 ^ Bio Reliancf Company Sanitized. Does not contain TSCA CB! H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Sponsor Project N um ber D u P on t-7326 B ioR eliance Study N um ber A A 47T V .502001-BTL H istidine M utation hisG46 isC3076 AD3052 Tryptopha n M utation trpE TA100 - TA98 - - - - W P2 uvrA A dditional M utations LPS Repair R-factor rfe Ai/vrB +R - uvrA - E ach S. typhimurium tester strain contains, in addition to a m utation in the histidine operon, additional m utations that enhance sen sitivity to som e m utagens. The rfa m utation results in a c ell w all deficien cy that increases the perm eability o f the c ell to certain classes o f chem icals such as those containing large ring system s that w ould otherw ise be excluded. The deletion in the uvrB gen e results in a d eficien t D N A excision-repair system . Tester strains T A 98 and T A 100 also contain the pKM IOl plasm id (carrying the R-factor). It has been suggested that the plasm id increases sen sitivity to m utagens by m odifying an existin g bacterial D N A repair polym erase com plex involved w ith the m ism atch-repair process. T A 98 and T A 1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by ftam eshift m utagens. T A 100 is reverted by both fram eshift and base substitution m utagens and TA1S3S is reverted only by m utagens that cause base substitutions. The E. coli tester strain has an A T base pair at the critical m utation site w ithin the trpE gene (W ilcox et al., 1990). Tester strain W P2 uvrA has a deletion in the uvrA gene resulting in a deficien t D N A excision-repair system . Tryptophan revertants can arise due to a base change at the originally m utated site or by a base change elsew here in the chrom osom e causing the original m utation to be suppressed. Thus, the sp ecificity o f die reversion m echanism is sensitive to base-pair substitution m utations (G reen and M uriel, 1976). T he S. typhimurium tester strains w ere received directly from Dr. Bruce A m es, U niversity o f California, B erkeley. The E. coli tester strain w as received from the N ational C ollection o f Industrial and M arine Bacteria, Aberdeen, Scotland (U nited K ingdom ). 7.0 EXPERIM ENTAL DESIGN AND M ETHODOLOGY The test substance w ill be tested at a m inim um o f fiv e d ose lev els along w ith appropriate negative and p ositive controls w ith tester strains T A 98, T A 100, TA 1S3S, T A 1537 and W P2 uvrA w ith and w ithout S9 activation. A ll dose lev els o f test substance, negative controls and positive controls w ill be plated in triplicate. I Protocol SPG T502001 l-Jan-2001 Page 3 o f 11 BioReliance Study No. AA47TV.502001.BTL 49 ^ Bio Relianoe not contati ISGA.GBI Sanitized. 0 <>es ottPnV 1 % H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______ DuPont-7326 Sponsor Project N um ber D u P on t-7326 B ioR eliance Study N um ber A A 47T V .5 0200l.B T L 7.1 Solubility Determ ination U nless the Sponsor has indicated the test substance vehicle, a solubility determ ination w ill be conducted to determine the maximum soluble concentration or workable suspension up to a maximum o f 50 m g/m L for aqueous vehicles and 500m g/m L for organic vehicles. V ehicles com patible w ith this test system , in order o f preference, include but are not lim ited to deionized water (CAS 7732-18-5), dim ethyl sulfoxide (C A S 67-68-5), ethanol (C A S 64-17-5) and acetone (CA S 67-64-1). The vehicle o f choice w ill be the solvent, selected in order o f preference, which perm its preparation o f die highest workable/soluble stock concentration, up to 50 m g/m L for aqueous veh icles and 500 mg/mL for organic vehicles. 7.2 Preliminary T oxicity A ssay to Select D ose L evels Selection o f dose levels for the m utagenicity assay w ill be based upon the toxicity and precipitation profile o f the test substance assessed in a prelim inary toxicity assay. This prelim inary assay w ill be conducted by exposing TA 98, TA100, TA 1535, TA 1537 and WP2 uvrA to negative controls and to at least eight concentrations o f test substance, one plate per dose lev el, in both the presence and absence o f S9 activation. U nless indicated otherwise by the Sponsor, the highest dose w ill be the highest w orkable concentration in the veh icle o f choice but not to exceed 5 m g/plate. In selecting dose levels for the m utagenicity assay the follow ing guidelines w ill b e em ployed. D oses w ill b e selected such that precipitate does not interfere w ith manual scoring. W henever possible, the highest dose for tire m utagenicity assay w ill be selected to giv e som e indication o f toxicity without exceeding 5 m g/plate. For finely soluble, nontoxic test substances, the highest dose lev el w ill be 5 m g/plate. For precipitating, nontoxic test substances, the highest dose lev el w ill be selected in an attempt to yield precipitate at only the top one or tw o dose levels. The Sponsor w ill be consulted regarding dose selection i f (1) the maximum dose level is selected based on precipitation and this dose level is less than 5 m g/plate or (2 ) the maximum achievable test substance dose level is less than 5 m g/plate and tins dose level is nontoxic. 7.3 Frequency and R oute o f Adm inistration The test system w ill be exposed to the test substance via the plate incorporation m ethodology originally described by Am es et al. (1975) and updated by Maron and A m es (1983). This test system has beat show n to detect a w ide range o f classes o f chem ical m utagens (M cCann et al., 1975; M cCann and A m es, 1976). A fier the data generated in the first assay have been evaluated, the m utagenicity assay w ill be repeated. The d ose levels used in the second assay w ill be the same as those used in the first assay unless the Study Director determ ines that the dose lev els should be changed due to an equivocal response, excessive cytotoxicity or Protocol SPG T502001 l-Jan-2001 Page 4 o f 11 BioReliance Study No. AA47TV.50200l.BTL 50 Bio Reliancf Company Sanitized. Does not contain TSCA CBS H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Sponsor Project N um ber D u P on t-7326 B ioR eliance Study N um ber A A 47T V .S02001.BTL excessive precipitate. If d ie Sponsor is aware o f sp ecific m etabolic requirements (e.g ., azo com pounds), this inform ation w ill be utilized in designing the assay, (e.g ., activation system or treatment m ethod). This guidance is based on the OECD G uideline 471 (adopted July 1997 and published February 1998) and ICH Guidance on Specific A spects o f Regulatory G enotoxicity Tests for Pharm aceuticals (1997). 7 .4 Controls 7.4.1 P ositive Controls A ll com binations o f positive controls and tester strains plated concurrently w ith the assay are listed below:_______________________________________ Strain S9 A ctivation P ositive Control C on centration (p g /p la te ) Salm onella Strains W P2 l/vrA Rat 2-am inoanthracene 1.0 10 TA98 2-nitrofhiorene 1.0 TA100, TA1535 TA1537 None sodium azide 9-am inoacridine 1.0 75 W P2 uvrA m ethyl m ethanesulfonate 1,000 7.4.2 N egative Controls Appropriate negative controls w ill be plated for each tester strain w ith and without S9 activation. The negative control w ill 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 w ill be used. 7.4.3 Sterility Controls The m ost concentrated test substance dilution and the Sham and S9 m ixes w ill b e checked for sterility. 7.S Exogenous M etabolic A ctivation Aroclor 1254-induced rat liver S 9 w ill be used as the m etabolic activation system . The S 9 hom ogenate w ill be prepared from m ale Sprague-D awley rats induced w ith a single intraperitoneal injection o f Aroclor 1 2 S 4 ,500 m g/kg, five days prior to sacrifice. The S 9 w ill be batch prepared and stored frozen at approximately Protocol SPG T502001 l-Jan-2001 Page 5 o f 11 BioReliance Study No. AA47TV.502001.BTL 51 M Bio Reliance- Company Sanitized!, Does not contain TSCA CB3 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Sponsor Project N u m b: D uPont-7326 B ioR eiiance Study N u m b: A A 47T V .502001 .BTL -70C until used. Each batch o f S9 hom ogenate w ill be assayed for its ability to m etabolize 2-am inoanthracene and 7,12-dim ethylbenzanthracene to forms m utagenic to typhimurium T A 100. Im m ediately prior to use, the S 9 w ill be thawed and m ixed w ith a cofactor pool to contain 10% S 9 hom ogenate, Sm M glucose-6-phosphate, 4 raM p-nicotinam ide-adenine dinucleotide phosphate, 8 oiM M gCl2 and 33 m M KC1 in a 100 mM phosphate buffer at pH 7 .4 . This mixture is referred to as S 9 m ix. Sham m ix w ill be 100 mM phosphate buffer at pH 7.4. Preparation o f Tester Strain Overnight cultures w ill be inoculated from the appropriate master plate or from the appropriate frozen stock. To ensure that cultures are harvested in late log phase, die length o f incubation w ill b e controlled and m onitored. A t the end o f the working day, each inoculated flask w ill be placed in a resting shaker/incubator at room temperature. The shaker/incubator w ill be programmed to begin shaking at approxim ately 125 rpm at 372C approxim ately 12 hours before the anticipated tim e o f harvest A ll cultures w ill be harvested b y spectrophotom etric m onitoring o f culture turbidity rath than by duration o f incubation since overgrowth o f cultures can cause loss o f sensitivity to som e m utagens. Cultures w ill be rem oved from incubation at a density o f approxim ately 109cells/m L . 7.7 T est Systran Identification Each plate w ill be labeled w ith a code system that identifies the test substance, test phase, dose level, tester strain and activation type as described in BioR eliance's Standard Operating Procedures. 7.8 T est Substance Preparation U nless specified otherw ise, test substance dilutions w ill be prepared im m ediately prior to use. A ll test substance dosing w ill be at room temperature under yellow lig h t 7.9 Treatment o f T est System One h a lf m illiliter (0.5 m L) o f S 9 m ix or Sham m ix, 100 jjL o f tester strain and 50 o f vehicle, test substance dilution or positive control w ill be added to 2 .0 mL o f m olten selective top agar at 452C . W hen necessary to achieve the target concentration or elim inate to x ic vehicle effects, aliquots o f other than 50 o f test substance/vehicle/positive control w ill be plated. The m ixture w ill be vortex m ixed and overlaid onto the surface o f 25 mL o f m inim al bottom agar. Protocol SPG T502001 l-Jan-2001 Page 6 o f 11 BioReiiance Study No. AA47TV.502001.BTL 52 ^ Bio Reliance H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Sponsor Project N um ber D u P on t-7326 B ioR eliance Study Num ber. A A 47T V .502001-BTL After the overlay has solidified, the plates w ill be inverted and incubated for approximately 48 to 72 hours at 372<*C. Plates that are not counted im m ediately follow ing the incubation period w ill be stored at 2-8C . 7.10 Scoring The condition o f the bacterial background lawn w ill be evaluated for evidence o f test substance toxicity and precipitate. Evidence o f toxicity w ill be scored relative to the negative control plate and recorded along w ith the revertant count for that plate. T oxicity w ill 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 w ill be evaluated after the incubation period b y visual examination without m agnification. 7.11 Tester Strain V erification On the day o f use in the m utagenicity assay, a ll tester strain cultures w ill be checked for the appropriate genetic markers cited in 6.0. 8 .0 CRITERIA FOR DETERM INATION OF A VALID TEST The follow in g criteria m ust be m et for the m utagenicity 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 v io le t T o dem onstrate the presence o f the uvrB mutation, all S. typhimurium tester strain cultures m ust exhibit sensitivity to ultraviolet lig h t To demonstrate foe presence o f the uvrA m utation, all K coli tester strain cultures must exhibit sensitivity to ultraviolet lig h t To demonstrate foe presence o f foe pKM IOl plasm id R-fector, tester strain cultures o f TA98 and TA 100 m ust exhibit resistance to am picillin. 8.2 Spontaneous Revertant Background Frequency Based on historical control data, all tester strain cultures must exhibit characteristic number o f spontaneous revertanls per plate in tile negative controls (vehicle). The m ean revertanls per plate m ust be w ithin the follow ing ranges (inclusive): TA 98, 1 0 -5 0 ; TA 100, 80 - 240; T A 1535, 5 - 4 5 ; T A 1537, 3 -2 1 ; WP2 uvrA, 10 - 60. 8.3 Tester Strain Titers T o ensure that appropriate numbers o f bacteria are plated, a ll tester strain culture titers m ust be equal to or greater than 0-3x10' cells per m illiliter. Protocol SPG T502001 l-Jan-2001 Page 7 o f 11 BioReliance Study No. AA47TV.502001.BTL 53 ^ Bio Reliancf Company Sanitized, Does not contain TSCA CBI H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-7326 Sponsor Project N um ber D uPont-7326 B ioR eliance Study Num ber. A A 47T V .502001.BTL 8.4 Positive Control Values Each mean positive control value m ust exhibit at least a 3.0-fold increase over the respective m ean negative control value (vehicle) for each tester strain. 8_5 T oxicity A minimum o f three non-toxic dose lev els w ill be required to evaluate assay data. A dose level is considered toxic i f it causes a >50% reduction in the mean number o f revertants per plate relative to the m ean negative control value (this reduction m ust be accom panied 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 w ill be repeated w ith an appropriate change in dose levels. 9.0 EVALUATIO N OF TEST RESULTS For a test substance to be evaluated p ositive, it m ust cause a dose-related increase in the m ean revertants per plate o f at least on e tester strain over a m inimum o f tw o increasing concentrations o f test substance as sp ecified below : 9.1 Strains T A 1535 and TA1537 Data sets w ill 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-tim es the mean negative control value (vehicle). 9.2 Strains T A 98, TA 100 and W P2 uvrA Data sets w ill be judged positive if the increase in mean revertants at die peak o f the dose response is equal to or greater than 2.0-tim es the m ean negative control value (vehicle). 10.0 REPORT A report o f the results o f th is study w ill be prepared by the T esting Laboratory and w ill accurately describe a ll m ethods used for generation and analysis o f the d ata The report w ill include: T est Substance: identification and CAS no., i f known; physical nature and purity, if known; physicochem ical properties relevant to the conduct o f the study, i f known; stability o f test substance, if known. Solvent/V ehicle: justification for choice o f vehicle; solubility and stability o f test substance in solvent/vehicle, i f known. s Strains: strains used; number o f cells/m L per culture; strain characteristics. Protocol SPG T502001 l-Jan-2001 Page 8 o f 11 BioReliance Study No. AA47TV.502001.BTL 54 ^ Bio Reliancf Company Sanitized. DoesTtoltonfainTSCA CBS H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Sponsor Project N um b: D uPont-7326______ B ioR eliance Study N um ber AA 47TV .502001-BTL T est conditions: amount o f test substance per plate w ith rationale for dose selection and n u m b o f plates per concentration; m edia used; type and com position o f m etabolic activation Systran, including acceptability criteria; treatment procedures. R esults: signs o f toxicity; signs o f precipitation; individual plate counts; the mean n u m b o f revertant colonies per plate and standard deviation; dose-response relationship, where possible; statistical analysis, i f any; concurrent negative and positive control data means and standard deviations; historical negative and positive control data w ith ranges, means and standard deviation. D iscussion o f results. C onclusion. 11.0 RECORDS A N D ARCHIVES A ll raw data, the p ra to co ^ a j^ ^ L reports w ill be m aintained according to Standard O perating P r o c e d u r n ^ B B H V l' d ie B ioR eliance RAQA unit headquartered at: B ioR elian ce, 14920 fflflSchart R o d f R ockville, M D 20850. Per this SO P, p a p records w ill be retained for at least three years after w hich tim e th e Sponsor w ill be contacted for a d ecisio n as to the final disposition o f the m aterials. A ll study m aterials returned to the Sponsor or destroyed w ill first be copied and the copy w ill be retained in the B ioR eliance archives for a minimum o f 10 years. 12.0 REG ULATORY REQUIREM ENTS/GOOD LABORATORY PRACTICE T his protocol has been written to com ply w ith OECD G uideline 471 (G enetic - T oxicology: B acterial Reverse M utation A ssay), N inth Addendum to tire OECD G uidelines for the T esting o f C hem icals, published by OECD, Paris, February 1998 and w ith th e International Conference on H arm onisation o f Technical Requirem ents for R egistration o f Pharm aceuticals for Human U se (1996 and 1997). T his study w ill be perform ed in com pliance w ith d ie provisions o f the G ood Laboratory P ractice R egulations for N onclinical Laboratory Studies (G LPs). The protocol, an in-process phase, the raw data, and rep o rts) w ill be audited per the Standard Operating Procedures (SO Ps) o f BioR eliance by the Q uality Assurance U nit o f B ioR eliance for com pliance w ith G LPs, the SOPs o f B ioR eliance and d ie study protocol. The in-process inspection w ill b e performed to audit the critical assay procedures and system s supporting the assay. A signed QA statem ent w ill be included in foe final report This statem ent w ill list the system phases inspected during foe previous quarter or foe stu d y-sp ecific phases, foe dates o f each inspection, and foe dates foe results o f each insp ection w ere reported to the Study D irector and the Study Director's m anagem ent In addition, a signed GLP com pliance statem ent w ill be included in the final report This statem ent w ill cite foe GLP guideline(s) w ith w hich fo e study is com pliant and any exceptions to this com pliance, i f applicable, including foe om ission o f characterization or stab ility analyses o f the test or control substances or their m ixtures. Protocol SPG T502001 l-Jan-2001 Page 9 o f 11 BioReliance Study No. AA47TV.502001.BTL 55 ^ Bio Reliancf Company Sanitized. Does not contain TSCA Cgf I H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay ' DuPont-7326 Sponsor Project N um ber D u P on t-7326 B ioR eliance Study N um ber A A 47T V .502001.B T L U n less arrangements are m ade to d ie contrary, unused dosing solutions w ill be disposed o f follow in g adm inistration to the test system and a ll residual test substance w ill be disp osed o f follow in g finalization o f th e report 13.0 REFERENCES A m es, B .N ., M cCann, J. and Y am asaki, E. (1975). M ethods for detecting carcinogens and m utagens w ith the & r/m one//<i/m am m alian-tniciosom e m utagenicity te s t M utation R esearch 31:347-364. G reen, M .H .L., and M uriel, W .J. (1976). M utagen testing using tip* reversion in Escherichia coli. M utation Research 38:3-32. International Conference on H arm onisation (ICH) o f T echnical Requirem ents for R egistration o f Pharm aceuticals for Human U se. G uidance on S p ecific A spects o f R egulatory G enotoxicity T ests for Pharm aceuticals. S2A docum ent recom m ended for adoption at step 4 o f the ICH process on July 19, 1995. Federal Register 61:18198-18202, A pril 2 4 ,1 9 9 6 . International Conference on H arm onisation (ICH) o f T echnical Requirem ents for R egistration o f Pharm aceuticals for Human U se. G enotoxicity: A Standard Battery for G en otoxicity Testing o f Pharm aceuticals. S2B docum ent recom m ended for adoption at step 4 o f the ICH process on July 16, 1997. Federal R egister 62:16026-16030, N ovem ber 2 1 ,1 9 9 7 . M cCann, J. and A m es, B .N . (1 9 7 6 ). D etection o f carcinogens as m utagens in the SalmonellaJnncrosome test: assay o f 300 chem icals: discussion. Proc. N atl. Acad. S ci. U SA 73:950-954. M cCann, J., C hoi, E ., Yam asaki, E. and A m es, B .N . (1975). D etection o f carcinogens as m utagens in the Salmonella/nucrosomc test: assay o f 300 chem icals. Proc. N atl. Acad. Sci. U SA 72:5135-5139. M aron, D .M . and A m es, B .N . (1983). R evised M ethods for the Salmonella M utagenicity T est. M utation Research 113:173-215. O ECD G uideline 471 (G enetic T oxicology: Bacterial R everse M utation T est), Ninth Addendum to the OECD G uidelines for the T esting o f C hem icals, published by OECD, Paris, February 1998. W ilcox, P ., N aidoo, A ., W edd, D .J. and G atehouse, D .G . (1 9 9 0 ). Com parison o f Salmonella typhimurium TA 102 w ith Escherichia coli W P2 tester strains. M utagenesis 5:285-291. Protocol SPG T502001 l-Jan-2001 Page 10 o f 11 BioReliance Study No. AA47TV.502001 .BTL 56 ^ Bio Reliancf not contain TSGACEjl H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Sponsor Project N um ber D u P on t-7326 B ioR eliance Study Num ber: A A 47TV .502001.BTL 14.0 APPROVAL Sponsor Representative 3 0 VL 7-OOt D a te V W -r V P o p (Print or T ype N am e) Qce Stud]M>irectr 2 Autf 2.00 1 D ate Protocol SPG T502001 l-Jan-2001 Page 11 o f 11 BioReliance Study No. AA47TV.502001.BTL 57 L-Oc. ^ Bio Reliancf DoesnoisontainTSCAQS) .' H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 APPENDIX C Information for Japanese Regulatory Agencies BioReliance Study No. AA47TV.502001.BTL 58 Does not contain TSCAGB5 H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 Report of Results of Reverse-Mutation Assay in Bacteria 1. Tester Strains (1) Procurement Strain TA98 TA100 TA1535 TA1537 TA1538 TA97 TA102 WP2 uvrA WP2 wvrA (pkMIOl) WP2 (pKMIOl) Obtained from Date obtained 10 November 1998 Dr. Bruce Ames University of California, Berkeley 11 August 1998 13 December 1990 14 November 1990 National Collection of Industrial and Marine Bacteria Aberdeen, Scotland 1 July 1987 19 February 1993 Date inspected the strain lot in storage The genetic markers for each culture are confirmed on the day of use (2) Storage Freezing method Storage temperature Composition Large quantity -70C Bacterial suspension DMSO 1.0 mL 0.09 mL 2. S9 Mix (1) Source, Storage Temperature, etc, of S9 Made in-house Prepared on Storage temperature -70C or colder 02 May 2001 (Batch R644) 03 July 2001 (Batch R648) Name and model of storage apparatus So-Low, Model PR27-120 BioReliance Study No. AA47TV.502001.BTL 59 Company Sanitized. Does not contain TSCA CBI H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 (2) Preparation o f S9 Animal used Species, Strain Rattus norvegicus, Sprague Dawley Sex Male Age (in weeks) Weight 9 (Batch R644) 9 (Batch R648) 209 to 258 g (Batch R644) 224 to 290 g (Batch R648) Inducing substance Name Aroclor 1254 Administration method intraperitoneal Administration period and amount (g/kg-weight) 5 days, 0.5 gm/kg body weight 3. Preparation o f Test Substance Solution Solvent used Name Tetrahydrofuran (THF) Manufacturer Aldrich Lot No. Grade Purity (%) JI00352HI Gold Label 99.9% Stability of test substance Unknown in the solvent Reason to choose the solvent Solubility determination was based on the Sponsor's request, compatibility with the target cells and solubility of the test substance Method of suspension when test substance is difficult to dissolve Not applicable Storage time and temp, from preparation to use for test <30 minutes at ambient temperature Conversion by purity No BioReliance Study No. AA47TV.502001.BTL 60 Company Sanitized. Does not contain TSCA CF' H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ DuPont-7326 4. Conditions of Pre-culture Nutrient broth Period of pre-culture Storage time and temp, from inoculation to beginning of shaking culture Storage time and temp, from end of culture to use for test Model and manufacturer of shaker Method of shaking (shaking type, speed, etc.) Culture vessel (shape, capacity) Culture volume Volume of inoculum Name Oxoid Nutrient Broth No. 2 121 hours Manufacturer Lot No. Oxoid Ltd. CH,-B=232622 2 to 5 hours at ambient temperature <8 hours at 2-8C New Brunswick Scientific, model G-24 Rotary (125 rev/min.) shape: cylinder, 200 mL 50 mL 1 colony 5. Agar Plate Medium (1) Top agar________ Agar Name Manufacturer Lot No. BBL Select Becton Dickinson 1000J3DKSQ (2) Minimum Glucose Agar Name Made in-house Agar Manufacturer Lot No. Volume of agar plate medium BBL Select Becton Dickinson 1000J3DKSQ 25 mL BioReliance Study No. AA47TV.502001.BTL 61 Company Sanitized. Does not contain TSGAC. H-25006: Bacterial Reverse Mutation Test with an Independent Repeat Assay______ 6. Test Results - Judgement of the results DuPont-7326 Judgement ' Negative Reason for judgement and referential matters: No positive response was observed with any of the tester strains in the presence and absence of Aroclor-induced rat liver S9. Referential matters The vehicle and positive control values indicate that all tester strains were functioning conecdy and were capable o f detecting a mutagen. BioReliance Study No. AA47TV.502001.BTL 62 TSCA-CB1 S an t * Ooes not con " Company