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A l z6 - - 2**7 DuPont-8867 TRADE SECRET Study Title H-2S170: Bacterial Reverse M utation Test with an Independent Repeat Assay Authors Valentine O. W agner, HI, M .S. M ichelle L. Klug, B.S. Study Completion Date 11 February 2002 Performing Laboratory B io R elian ce 9630 M edical Center Drive Rockville, M D 20850 for E. I du Pont de Nemours and Company DuPont Haskell Laboratory P.O. Box 50,1090 Elkton Road Newark, DE 19714-0050 Performin p Laboratory Study Number A A 52X K .502001.B TL W ork Request Number Page 1 o f 63 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 STATEMENT OF COMPLIANCE Study No. AA52XK.502001.BTL was conducted in compliance w ith 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 articles have not been determined by the testing facility. The control articles have been characterized as per the Certificates o f Analysis on file with the testing facility. The stability o f the test and control articles has not been determined by the testing facility. Analyses to determine the uniformity (as applicable), or concentration o f 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 o f the test and control articles in the test and control mixtures, respectively, has not been determined by the testing facility. The Sponsor has indicated that they have not performed stability analysis on the test substance mixtures. Valentine O. Wagner, HL M.S. Study Director t| FeJ> D ate BioReliance Study Management D ate BioReliance Study No. AA52XK.502001.BTL 2 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ Quality Assurance Statement DuPont-8867 Study Title: BACTERIAL REVERSE MUTATION ASSAY WITH AN INDEPENDENT REPEAT ASSAY Study Number: AA52XK.502001.BTL Study Director: Valentine O. Wagner, III, M.S. This study has been divided into a series of in-process phases. Using a random sampling approach, Quality Assurance monitors each of these phases over a series o f studies. Procedures, documentation, equipment records, etc., are examined in order to assure that the study is performed in accordance with die U.S. FDA Good Laboratory Practice Regulations (21 CFR 58), the U.S. EPA GLPs (40 CFR 792 and 40 CFR 160), the UK GLP Regulations, the Japanese GLP Standard, and the OECD Principles 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 of QA inspections of this study. Inspect On Phase Inspect On Phase Inspect On Phase Inspect On Phase 06-Dec-01 - 06-Dec-01 To Study Dir 06-Dec-01 To Mgmt 06-Dec-01 Protocol Review 19-Dec-01 - 19-Dec-01 To Study Dir 19-Dec-01 To Mgmt 20-Dec-01 Test and/or control material administration 30-Jan-02 - 01-Feb-02 To Study Dir 01-Feb-02 To Mgmt Q7-Feb-02 Draft Report 1l-Feb-02 -1 l-Feb-02 To Study Dir 1l-Feb-02 To Mgmt 1l-Feb-02 Draft to Final Report This report describes the methods and procedures used in the study and the reported results accurately reflect the raw data o f the study. Elahe Siadatpour, B.S. QUALITY ASSURANCE BioReliance Study No. AA52XK.502001.BTL ___ LI_te ii__doS DATE 3 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-8867 CERTIFICATION W e, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Issued by Study Director: \JoX eStiM ^ ) . Valentine 0 . Wagner m , M.S. 11 P e t `2.<S< D ate Approved by Study Monitor: Maria Dormer, Ph.D. Senior Research Scientist 2. o 2-- D ate BioReliance Study No. AAS2XK.502001.BTL 4 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 TABLE OF CONTENTS Page C e rtific a tio n ......................................................................................................................................... 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.....................................................................................................................11 Results and D iscussion.....................................................................................................................16 C o n clu sio n ................................................................................................. 16 R eferences...................................................................... 17 D ata Tables........................................................................................................................................ 18 Table 1: Prelim inary Toxicity Test in Salmonella typhimurium T A 98...............................18 Table 2: Prelim inary Toxicity Test in Salmonella typhimurium T A 100............................. 19 Table 3: Prelim inary Toxicity Test in Salmonella typhimurium TA 1535...........................20 Table 4: Prelim inary Toxicity Test in Salmonella typhimurium TA1537 .......................... 21 Table 5: Prelim inary Toxicity Test in Escherichia coli W P2 uvrA ..................................... 22 Table 6: M utagenicity T est in Salmonella typhimurium TA98 w ithout S 9 ...................... 23 Table 7: M utagenicity Test in Salmonella typhimurium TA98 w ith S 9 ............................24 Table 8: M utagenicity T est in Salmonella typhimurium TA100 without S 9 .................... 25 Table 9: M utagenicity T est in Salmonella typhimurium TA100 w ith S 9 ......................... 26 Table 10: M utagenicity Test in Salmonella typhimurium TA1535 without S 9 ..................27 Table 11: M utagenicity Test in Salmonella typhimurium TA1535 with S 9 ....................... 28 Table 12: M utagenicity Test in Salmonella typhimurium TA1537 without S 9 ..................29 Table 13: M utagenicity Test in Salmonella typhimurium TA1537 w ith S 9 ....................... 30 Table 14: M utagenicity Test in Escherichia coli W P2 uvrA w ithout S 9............................31 Table 15: M utagenicity Test in Escherichia coli W P2 vrA w ith S 9 ................................. 32 Table 16: M utagenicity Test in Salmonella typhimurium TA98 without S 9 ...................... 33 Table 17: M utagenicity Test in Salmonella typhimurium TA98 with S 9 ............................34 Table 18: M utagenicity Test in Salmonella typhimurium TA100 without S 9 .................... 35 Table 19: M utagenicity Test in Salmonella typhimurium TA100 w ith S 9 ..........................36 Table 20: M utagenicity Test in Salmonella typhimurium TA1535 without S 9 ..................37 Table 21: M utagenicity Test in Salmonella typhimurium TA1535 w ith S 9 ....................... 38 Table 22: M utagenicity Test in Salmonella typhimurium TA1537 without S 9 ..................39 Table 23: M utagenicity Test in Salmonella typhimurium TA1537 with S 9 ....................... 40 BioReliance Study No. AA52XK.502001.BTL 5 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________________ DuPont-8867 Table 24: M utagenicity Test in Escherichia coli W P2 vrA w ithout S 9........................... .41 Table 25: M utagenicity Test in Escherichia coli W P2 uvrA w ith S 9 ..................................42 Table 26: Salmonella/E. coli M utagenicity Test - Summary o f Results B 1.......................43 Table 27: Salmonella/E. coli M utagenicity Test - Summary o f Results B 2.......................44 Appendix A: H istorical Control D ata.............................................................................................45 Appendix B: Study Protocol............................................................................................................47 Appendix C: Information for Japanese Regulatory A gencies.....................................................59 BioReliance Study No. AA52XK.502001 .BTL 6 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ STUDY INFORMATION DuPont-8867 Haskell Numher: 25170 CAS Registry N um berj ) Com position.^1 P u rity iQ Known Im purities^] Physical Characteristics I) J Stability: The test substance appeared to be stable under the conditions o f the study; no evidence o f instability was observed. Sponsor: E.L du Pont de Nemours and Company W ilmington, Delaware 19898 U.S.A. Study Initiated/Completed: December 05, 2001 / (see report cover page) In-Life Initiated/Completed: December 07,2001 / January 10,2002 BioReliance Study No. AA52XK.50200l.BTL 7 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 SUMMARY The test substance, H-25170, 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 coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The test was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity test, was used to establish the dose-range for the mutagenicity test. The second phase, the mutagenicity test, (initial and independent repeat tests), was used to evaluate the mutagenic potential o f the test substance. Tetrahydrofuran (THF) was selected as the solvent o f choice based on com patibility with the target cells and solubility o f the test substance. The test substance was soluble but cloudy in tetrahydrofuran (THF) at a maximum concentration o f approximately 200 mg/mL. In the preliminary toxicity test, the maximum dose tested was 5000 pg per plate; this dose was achieved using a concentration of 200 mg/mL and a 25 pL plating aliquot. The test substance was a soluble but cloudy solution at 200 mg/mL and a soluble and clear solution from 0.27 to 133 mg/mL. Dose levels tested were 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 pg per plate. Precipitate was observed beginning at 67 or 100 pg per plate. No appreciable toxicity was observed. Based on the findings o f the toxicity test, the maximum dose plated in the mutagenicity test was 5000 pg per plate. In the mutagenicity test, no positive mutagenic response was observed. The dose levels tested were 15,50,150,500,1500 and 5000 pg per plate. Precipitate was observed beginning at 500 or 1500 pg per plate. No appreciable toxicity was observed. The results o f the Bacterial Reverse Mutation Test with an Independent Repeat Assay indicate that, under the conditions o f this study, H-25170 did not cause a positive mutagenic response in either the presence or absence o f Aroclor-induced rat liver S9. BioReliance Study No. AA52XK.502001.BTL 8 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 PURPOSE The purpose o f this study was to evaluate the mutagenic potential of the test substance by measuring its ability to induce reverse mutations at selected loci o f several strains o f Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence o f Aroclor-induced rat liver S9. A copy of the protocol is included in Appendix B. The study was conducted in compliance with the testing guidelines of the ICH (1996 and 1997) and OECD ((1998). CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test substance, H-25170, was received by BioReliance on 03 December 2001 and was assignetHhe ax le number AA52XK. The test substance was characterized by the Sponsor as a f t i B M H H j P 3* should be stored at ambient temperature. An expiration d a te o f amber7004 was provided. Upon receipt, the test substance was described as and was stored at room temperature, protected from light and moisture. The identity; purity, composition or other characteristics to define the test substance and the stability o f the test substance have been determined by the Sponsor. The Sponsor indicated that BioReliance would not receive copies o f these reports. The vehicle used to deliver H-25170 to the test system was tetrahydrofuran (THF, CAS No. 109-99-9), Cold Label, 99.9%, purchased from Aldrich Chemical Company. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light. Positive controls plated concurrently with the mutagenicity test are listed below. All positive controls were diluted w ith dimethyl sulfoxide (DMSO) except sodium azide, w hich was diluted with water. All subdivided solutions of positive control were stored at -5 to -30C . Strain All Salmonella Strains WP2 ovrA S9 Activation Rat Positive Control 2-am inoanthracene (Aldrich Chemical Co., Inc.) Lot No. 09106PS Exp. Date 14-Sep-2005 CAS No. 613-13-8 Purity >95% Concentration (jig/plate) 1.0 10 BioReliance Study No. AA52XK.502001.BTL 9 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 Strain TA98 TA100, TA1535 TA1537 WP2 uvrA S9 A ctivation None Positive Control 2-nitrofluorene (Aldrich Chemical Co., Inc.) Lot No. 08707HS Exp. Date 08-M ar-2006 CAS No. 607-57-8 Purity >98% Sodium azide (Sigma Chemical Co.) Lot No. 098H0169 Exp. Date 05-Jan-2004 CAS No. 26628-22-8 Purity >99% 9-am inoacridine (Sigma Chemical Co.) Lot No. 106F06681 Exp. Date 18-Nov-2004 CAS No. 90-45-9 Purity >98% Methyl methanesulfonate (Aldrich Chemical Co., Inc.) Lot No. 15526AO Exp. Dates 08-Jan-2003 and 28-Nov-2003 CAS No. 66-27-3 Purity >99% C oncentration (pg/plate) 1.0 1.0 75 1,000 To confirm the sterility o f 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 te st BioReliance Study No. AA52XK.502001.BTL 10 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 MATERIALS AND METHODS For submission to Japanese regulatory agencies, additional information is included in Appendix C. Test System The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and M uriel (1976). Salmonella tester strains w o e received directly from Dr. Bruce Ames, University o f California, Berkeley and 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 frameshift mutagens. Tester strain TA1535 is reverted by mutagens drat cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976). Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing ~50 mL 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 of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer o f approximately 109 cells per milliliter. The actual titers were determined by viable count tests on nutrient agar plates, and the data is on file but not presented in this report. The study was conducted to comply with OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July 1997 (published February 1998) and with the International Conference on Harmonisation o f Technical Requirements o f Registration o f Pharmaceuticals for Human Use (1996 and 1997). Metabolic Activation System Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at -70C or colder until used. Each bulk preparation o f S9 was tested for its ability to metabolize 2aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100. BioReliance Study No. AA52XK.502001.BTL 11 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 The S9 mix was prepared immediately before its use and contained 10% S9, Sm M glucose-6-phosphate, 4 mM B-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility o f the S9 and Sham mixes, a 0.5 mL aliquot o f each was plated on selective agar. Solubility Test A solubility test was conducted to select the vehicle. The test was conducted using water, dimethyl sulfoxide (DMSO), ethanol (EtOH), acetone, acetonitrile, tetrahydrofuran (THF), propylene glycol and polyethylene glycol. The test substance was tested to determine the vehicle, selected in order of 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. Preliminary Toxidty Test The preliminary toxicity test was used to establish the dose-range over which the test substance would be tested. Vehicle and ten dose levels o f the test substance were plated, one plate per dose, with overnight cultures o f TA98, TA100, TA1535, TA1537 and W P2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. Dose levels tested were 6 .7 ,1 0 ,3 3 ,6 7 ,100,333,667,1000,3333 and 5000 pg per plate. Mutagenicity Test The mutagenicity test (initial and independent repeat tests) was used to evaluate the mutagenic potential o f the test substance. Six dose levels o f test substance (15, 50, 150, 500, 1500 and 5000 pg per plate) along with appropriate vehicle and positive controls were plated with TA98, TA100, TA1535, TA1537 and 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 of 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 o f water for each 100 mL of minimal top agar. For the preparation o f media and BioReliance Study No. AA52XK.502001.BTL 12 Company Sanitized. Does not contain TSCA CBi H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 reagents, all references to water imply sterile, deionized water 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 w ith 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). N utrient Broth was Vogel-Bonner salt solution supplemented w ith 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 o f tester strain and 25 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. When plating the positive controls, the test substance aliquot was replaced by a 50 pL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 372C. Plates that were not counted immediately following the incubation period were stored at 2-8C until colony counting could be conducted (less than 10 days). The condition o f the bacterial background lawn was evaluated for evidence 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 below. Code 1 2 3 4 5 6 D escription N orm al Slightly Reduced M oderately Reduced E xtrem ely Reduced Absent Obscured by 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. BioReliance Study No. AA52XK.502001.BTL 13 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Code NP IP D escription C h aracteristics Distinguished by precipitate on the plate that is visible to the Non-Interfering naked eye but any precipitate particles detected by the automated Precipitate colony counter total less than 10% o f the revertant colony count (e.g., less than 3 particles on a plate w ith 30 revertants). Distinguished by precipitate on the plate that is visible to the Interfering Precipitate naked eye and any precipitate particles detected by the automated colony counter exceed 10% o f the revertant colony count (e.g., more than 3 particles on a plate with 30 revertants). These plates are counted manually. Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Evaluation of Results For each replicate plating, the mean and standard deviation of the number 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 o f the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak 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 must be met for the mutagenicity test to be considered valid. All Salmonella tester strain cultures must demonstrate the presence o f die deep rough mutation (ifa) and the deletion in the uvrB gene. Cultures o f tester strains TA98 and TA100 m ust demonstrate the presence o f the pKM IOl plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 -4 5 ; 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 0.3x1c)9 cells/mL. The mean o f each positive control must exhibit at least a 3.0-fold increase in the number of 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 of the following criteria are met: (1) A >50 % reduction in the mean number of BioReliance Study No. AA52XK.502001.BTL 14 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 revertants per plate as compared to the mean vehicle control val. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count (2) A moderate reduction in the background lawn (background code o f 3 ,4 or 5). A copy o f the Historical Negative and Positive Control Values is included in Appendix A. Archives All raw data, th^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 will be contacted for a decision as to the final disposition o f the materials. All study materials returned to the Sponsor or destroyed w ill first be copied and the copy w ill be retained in the BioReliance archives for a minimum o f 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. Deviations No known deviations from the protocol or assay-method SOPs occurred during the conduct o f this study. BioReliance Study No. AA52XK.502001JBTL 15 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 RESULTS AND DISCUSSION Solubility Test Tetrahydrofuran (THF) was selected as the solvent o f choice based on compatibility with the target cells and solubility o f the test substance. The test substance was soluble but cloudy in tetrahydrofuran (THF) at a maximum concentration o f approximately 200 mg/mL. 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 o f 200 mg/mL and a 25 pL plating aliquot. The test substance was a soluble but cloudy solution at 200 mg/mL and a soluble and clear solution from 0.27 to 133 mg/mL. Dose levels tested were 6.7, 10, 33, 67, 100, 333, 667,1000,3333 and 5000 pg per plate. Precipitate was observed beginning at 67 or 100 pg per plate. No appreciable toxicity was observed. Based on the findings o f the toxicity test, the maximum dose plated in the mutagenicity test was 5000 pg per plate. Mutagenicity Test The results o f the mutagenicity test are presented in Tables 6 through 25 and summarized in Tables 26 and 27. These data were generated in Experiments B1 and B2. Dose levels tested were 15, 5 0 ,1 5 0 ,5 0 0 ,1 5 0 0 and 5000 pg per plate. Precipitate was observed beginning at 500 or 1500 pg per plate. No appreciable toxicity was observed. In Experiment B1 (Initial M utagenicity 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 of the tester strains in either the presence or absence o f S9 activation. CONCLUSION All criteria for a valid study were m et 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-25170 did not cause a positive mutagenic response in either the presence or absence o f Aroclor-induced rat liver S9. BioReliance Study No. AA52XK.502001.BTL 16 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 REFERENCES Ames, B.N., J. McCann and E. Yamasaki (1975) M ethods for Detecting Carcinogens and M utagens with the Salmonella/Maxamaiian M icrosome M utagenicity Test, Mutation Research, 31:347-364. Green, M .H.L. and W J. Muriel (1976) M utagen testing using trp+ reversion in Escherichia coli, M utation 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) 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. Marn, D.M. and B.N. Ames (1983) Revised M ethods for the Salmonella Mutagenicity Test, M utation 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. 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. AA52XK.502001.BTL 17 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 Bacterial Mutation Test Preliminary Toxicity Test Table 1 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25170 AA52XK.502001.BTL Al TA98 7 Dec 2001 tetrahydrofuran 25 pL Test Substance Concentrt ion pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 13 1 Without S9 Activation Revertants Background per plate Lawn 21 1 6.7 10 33 67 100 333 667 1000 3333 5000 91 91 10 1 10 1NP 19 1NP 17 1NP 12 1NP 10 1NP 16 1NP 10 1NP 91 17 1 12 1 12 1NP 16 1NP 9 1NP 8 1NP 10 1NP 11 1NP 13 1NP Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA52XK.502001.BTL 18 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 Bacterial Mutation Test Preliminary Toxicity Test Table 2 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25170 AA52XK.502001.BTL Al TA100 7 Dec 2001 tetrahydrofuran 25 uL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 148 1 Without S9 Activation Revertants Background per plate Lawn 129 1 6.7 10 33 67 100 333 667 1000 3333 5000 121 1 102 1 142 1 134 1NP 147 1NP 144 1NP 134 1NP 124 1NP 113 1NP 109 1NP 152 1 114 1 139 1 150 1NP 138 1NP 132 1NP 123 1NP 131 1NP 127 1NP 142 1NP Background Lawn Code 1Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA52XK.502001.BTL 19 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Preliminary Toxicity Test Table 3 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25170 AA52XK.502001.BTL Al TA1535 7 Dec 2001 tetrahydrofuran 25 UL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 17 1 Without S9 Activation Revertants Background per plate Lawn 12 1 6.7 10 33 67 100 333 667 1000 3333 5000 91 71 11 1 18 1NP 12 1NP 11 1NP 14 1NP 13 1NP 13 1NP 12 1NP 12 1 10 1 10 1 13 1NP 15 1NP 18 1NP 11 1NP 13 1NP 13 1NP 19 1NP Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA52XK.502001.BTL 20 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Preliminary Toxicity Test Table 4 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25170 AA52XK.502001.BTL Al TA1537 7 Dec 2001 tetrahydrofuran 25 pL Test Substance Concentration pg per plate With S9 Activation Revertants Background per plate Lawn Vehicle 41 Without S9 Activation Revertants Background per plate Lawn 51 6.7 10 33 67 100 333 667 1000 3333 5000 51 71 51 8 1NP 5 1NP 2 1NP 4 1NP 4 1NP 8 1NP 5 1NP 51 51 31 7 1NP 5 1NP 3 1NP 4 1NP 5 1NP 4 1NP 3 1NP Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA52XK.50200l.BTL 21 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Preliminary Toxicity Test Table 5 Test Substance Id Study Number Experiment No. Strain Date Plated Vehicle Plating Aliquot H-25170 AA52XK.502001.BTL Al WP2 uvrA 7 Dec 2001 tetrahydrofuran 25 pL Test Substance Concentration ug per plate With S9 Activation Revertants Background per plate Lawn Vehicle 14 1 Without S9 Activation Revertants Background per plate Lawn 14 1 6.7 10 33 67 100 333 667 1000 3333 5000 14 1 14 1 10 1 12 1NP 13 1NP 12 1NP 10 1NP 16 1NP 9 1NP 10 1NP 10 1 12 1 11 1 81 9 1NP 16 1NP 12 1NP 17 1NP 13 1NP 10 1NP Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA52XK.502001.BTL 22 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________________ DuPont-8867 Bacterial Mutation Test Table 6 Test Substance Id: H-25170 Study Number : AA52XK.502001.BTL Strain : TA98 Liver Microsomes : None Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : Bl Cells Seeded : 2.4 X 108 Date Plated : 19 Dec 2001 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 10 1 10 1 10 1 10 0 15 01 14 1 02 12 1 03 12 1 13 1 50 01 10 1 02 10 1 03 10 1 10 0 150 01 12 1 02 8 1 03 10 1 10 2 500 01 12 1NP 02 10 1NP 03 9 1NP 10 2 1500 01 10 1NP 02 10 1NP 03 16 1NP 12 3 5000 01 12 1NP 02 8 1NP 03 12 1NP 11 2 Positive Control 2-nitrofluorene 1.0 pg per plate 01 157 1 02 178 1 03 203 1 179 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. AA52XK.502W1.BTL 23 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________________ DuPont-8867 Bacterial Mutation Test Table 7 Test Substance Id: H-25170 Study Number : AA52XK.502001.BTL Strain : TA98 Liver Microsomes : Rat liver S9 Vehicle : tetrahydrofuran Plating Aliquot : 25 jiL Experiment No : B1 Cells Seeded : 2.4 X 10s Date Plated : 19 Dec 2001 Concentrtion Plate Revertants Background Average Standard jig per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 16 1 10 1 23 1 16 7 15 01 17 1 02 14 1 03 13 1 15 2 50 01 12 1 02 17 1 03 18 1 16 3 150 01 17 1 02 18 1 03 19 1 18 1 500 01 10 1NP 02 24 1NP 03 12 1NP 15 8 1500 01 14 1NP 02 14 1NP 03 15 1NP 14 1 5000 01 17 1NP 02 16 1NP 03 14 1NP 16 2 Positive Control 2-aminoanthracene 1 .0 jig per plate 01 467 1 02 462 1 03 408 1 446 33 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. AA52XK.502001.BTL 24 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Table 8 Test Substance Id: H-25170 Study Number :AA52XK.502001.BTL Experiment No :B1 Strain :TA100 Cells Seeded :4.1 X10 Liver Microsomes :None Date Plated :19Dec 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 03 160 1 154 1 164 1 159 5 15 01 187 1 02 150 1 03 141 1 159 24 50 01 140 1 02 162 1 03 148 1 150 11 150 01 146 1 02 144 1 03 128 1 139 10 500 01 156 1NP 02 133 1NP 03 141 1NP 143 12 1500 01 68 1NP 02 151 1NP 03 154 1NP 124 49 5000 01 193 1NP 02 153 1NP 03 153 1NP 166 23 Positive Control sodium azide 1.0 pg per plate 01 491 1 02 503 1 03 516 1 503 13 Background Lawn Code l=Normal; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA52XK.502001.BTL 25 Company Sanitized. Does not contain TSCA CBI H-2S170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________________ DuPont-8867 Bacterial Mutation Test Table 9 Test Substance Id: H-25170 Study Number : AA52XK.502001.BTL Strain : TA100 Liver Microsomes : Rat liver S9 Vehicle : tetrahydrofuran Plating Aliquot : 25 v*L Experiment No : B1 Cells Seeded : 4. 1 X 108 Date Plated : 19 Dec 2001 Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 134 1 163 1 191 1 163 29 15 01 180 1 02 165 1 03 167 1 171 8 50 01 147 1 02 141 1 03 164 1 151 12 150 01 235 1 02 164 1 03 169 1 189 40 500 01 153 1NP 02 181 1NP 03 163 1NP 166 14 1500 01 193 1NP 02 187 1NP 03 147 1NP 176 25 5000 01 196 1NP 02 171 1NP 03 165 1NP 177 16 Positive Control 2-aminoanthracene 1 .0 ug per plate 01 763 1 02 692 1 03 515 1 657 128 Background Lawn Code l=Normal; t!=Slightly reduced; 3=Moderately reduced 4=Extremely reduced ; 5=Absent; 6=Obscured by precipitate NP=Non-Interering precipitate; IP=Interfering precipitate BioReliance Study No. AA52XK.502001.BTL 26 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________________ DuPont-8867 Bacterial Mutation Test Table 10 Test Substance Id: H-25170 Study Number : AA52XK.502001.BTL Strain : TA1535 Liver Microsomes : None Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : B1 Cells Seeded : 2.7 X 10s Date Plated : 19 Dec: 2001 Concentration Plate Revertants Background Average Standard jig per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 12 1 15 1 13 1 13 2 15 01 18 1 02 14 1 03 10 1 14 4 50 01 10 1 02 12 1 03 12 1 11 1 150 01 11 1 02 15 1 03 11 1 12 2 500 01 10 1NP 02 10 1NP 03 8 1NP 91 1500 01 10 1NP 02 12 1NP 03 12 1NP 11 1 5000 01 12 1NP 02 13 1NP 03 13 1NP 13 1 Positive Control sodium azide 1.0 jig per plate 01 297 1 02 312 1 03 230 1 280 44 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfaring precipitate; IP=Interfering precipitate >i U JJ A H BioReliance Study No. AA52XK.502001.BTL 27 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________________ DuPont-8867 Bacterial Mutation Test Table 11 Test Substance Id: H-25170 Study Number : AA52XK.502001.BTL Strain : TA1535 Liver Microsomes : Rat liver S9 Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : B1 Cells Seeded : 2.7 X 108 Date Plated : 19 Dec 2001 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 91 91 13 1 10 2 15 01 12 1 02 9 1 03 10 1 10 2 50 01 9 1 02 14 1 03 12 1 12 3 150 01 12 1 02 5 1 03 8 1 84 500 01 20 1NP 02 14 1NP 03 15 1NP 16 3 1500 01 11 1NP 02 9 1NP 03 6 1NP 93 5000 01 14 1NP 02 5 1NP 03 12 1NP 10 5 Positive Control 2-aminoanthracene 1 .0 pg per plate 01 121 1 02 85 1 03 128 1 111 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. AA52XK.502001 .BTL 28 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Table 12 Test Substance Id: H-25170 Study Number :AA52XK.502001.BTL Experiment No : B1 Strain :TA1537 Cells Seeded :1.6 X10s Liver Microsomes :None Date Plated : 19Dec 2001 Vehicle :tetrahydrofuran Plating Aliquot : 25 uL___________________________________________ Concentration Plate Revertanta Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 51 21 41 42 15 01 2 1 02 6 1 03 4 1 42 50 01 8 1 02 3 1 03 4 1 53 150 01 6 1 02 3 1 03 3 1 42 500 01 4 1NP 02 5 1NP 03 6 1NP 51 1500 01 8 1NP 02 7 1NP 03 4 1NP 62 5000 01 6 1NP 02 3 1NP 03 5 1NP 52 Positive Control 9-aminoacridine 75 pg per plate 01 605 1 02 837 1 03 635 1 692 126 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. AA52XK.502001.BTL 29 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________________ DuPont-8867 Bacterial Mutation Test Table 13 Test Substance Id: H-25170 Study Number : AA52XK.502001.BTL Strain : TA1537 Liver Microsomes : Rat liver S9 Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : B1 Cells Seeded : 1.6 X 10* Date Plated : 19 Dec 2001 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 51 31 91 63 15 01 12 1 02 6 1 03 7 1 83 50 01 4 1 02 5 1 03 5 1 51 150 01 10 1 02 5 1 03 5 1 73 500 01 9 1NP 02 5 1NP 03 5 1NP 62 1500 01 5 1NP 02 5 1NP 03 7 1NP 61 5000 01 5 1NP 02 8 1NP 03 6 1NP 62 Positive Control 2-aminoanthracene 1 .0 pg per plate 01 101 1 02 96 1 03 86 1 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 8 BioReliance Study No. AA52XK.50200l.BTL 30 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Table 14 Test Substance Id: H-25170 Study Number : AA52XK.502001.BTL Strain : WP2 uvrA Liver Microsomes : None Vehicle tetrahydro furan Experiment No Cells Seeded Date Plated B1 11.2 X 10" 19 Dec 2001 Concentration pg per plate Vehicle Plate Number 01 02 03 Revertants per plate 15 10 13 Background Code 1 1 1 Average Revertants 13 Standard Deviation 3 15 01 10 1 02 10 1 03 10 1 10 0 50 01 12 1 02 12 1 03 12 1 12 0 150 01 14 1 02 14 1 03 12 1 13 1 500 01 12 1NP 02 12 1NP 03 12 1NP 12 0 1500 01 13 1NP 02 10 1NP 03 13 1NP 12 2 5000 01 17 1NP 02 11 1NP 03 13 1NP 14 3 Positive Control methyl methanesulfonate 1000 ug per plate 01 113 1 02 96 1 03 77 1 95 18 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate BioReliance Study No. AA52XK.502001.BTL 31 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 Bacterial Mutation Test Table 15 Test Substance Id: H-25170 Study Number :AA52XK.502001.BTL Experiment No :B1 Strain :WP2 uvrA Cells Seeded :11.2 X 10 Liver Microsomes :Rat liver S9 Date Plated :19 Dec 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 10 1 10 1 16 1 12 3 15 01 11 1 02 12 1 03 10 1 11 1 50 01 11 1 02 10 1 03 10 1 10 1 150 01 10 1 02 10 1 03 10 1 10 0 500 01 11 1NP 02 14 1NP 03 13 1NP 13 2 1500 01 5 1NP 02 10 1NP 03 13 1NP 94 5000 01 17 1NP 02 14 1NP 03 14 1NP 15 2 Positive Control 2-aminoanthracene 10 ug per plate 01 713 1 02 413 1 03 420 1 515 171 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. AA52XK.502001.BTL 32 Company Sanitized. Does not contain TSCA CBI H-2S170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________________ DuPont-8867 Bacterial Mutation Test Table 16 Test Substance Id: H-25170 Study Number : AA52XK. 502001.:BTL Strain : TA98 Liver Microsomes : None Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : B2 Cells Seeded : 2.7 X 108 Date Plated : 4 Jem 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 22 1 19 1 15 1 19 4 15 01 9 1 02 18 1 03 20 1 16 6 50 01 11 1 02 17 1 03 8 1 12 5 150 01 9 1 02 10 1 03 10 1 10 1 500 01 10 IIP 02 17 1NP 03 8 IIP 12 5 1500 01 8 1NP 02 2 1NP 03 5 1NP 53 5000 01 11 IIP 02 6 IIP 03 9 IIP 93 Positive Control 2-nitrofluorene 1 .0 pg per plate 01 247 1 02 239 1 03 228 1 238 10 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. AA52XK.50200l.BTL 33 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Table 17 Test Substance Id: H-25170 Study Number :AA52XK.502001.BTI> Experiment No :B2 Strain :TA98 Cells Seeded :2.7 X 10* Liver Microsomes :Rat liver S9 Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 pL__________________________________________ Concentrt ion Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 22 1 24 1 25 1 24 2 15 01 28 1 02 11 1 03 18 1 19 9 50 01 23 1 02 20 1 03 26 1 23 3 150 01 15 1 02 20 1 03 15 1 17 3 500 01 17 1NP 02 16 1NP 03 26 1NP 20 6 1500 01 29 IIP 02 16 IIP 03 25 IIP 23 7 5000 01 17 IIP 02 16 1NP 03 24 IIP 19 4 Positive Control 2-aminoanthracene 1.0 pg per plate 01 1011 1 02 975 1 03 1046 1 1011 36 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. AA52XK.50200l.BTL 34 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 Bacterial Mutation Test Table 18 Test Substance Id: H-25170 Study Number :AA52XK.502001.BTL Experiment No :B2 Strain :TA100 Cells Seeded :1.1 X 10 Liver Microsomes :None Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 pL__________________________________________ Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 191 1 217 1 174 1 194 22 15 01 182 1 02 188 1 03 175 1 182 7 50 01 186 1 02 184 1 03 200 1 190 9 150 01 194 1 02 160 1 03 197 1 184 21 500 01 221 1NP 02 169 1NP 03 163 1NP 184 32 1500 01 178 1NP 02 215 1NP 03 176 1NP 190 22 5000 01 181 1NP 02 183 1NP 03 195 1NP 186 8 Positive Control sodium azide 1.0 pg per plate 01 764 1 02 775 1 03 775 1 771 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced1; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; IP=Interfering precipitate 6 BioReliance Study No. AA52XK.502001.BTL 35 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________________________________ DuPont-8867 Bacterial Mutation Test Table 19 Test Substance Id: H-25170 Study Number : AA52XK.502001.BTL Strain : TA100 Liver Microsomes : Rat liver S9 Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : B2 Cells Seeded : 1.1 X 10 Date Plated : 4 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 163 1 201 1 199 1 188 21 15 01 204 1 02 185 1 03 183 1 191 12 50 01 176 1 02 182 1 03 208 1 189 17 150 01 151 1 02 162 1 03 165 1 159 7 500 01 179 1NP 02 221 1NP 03 166 1 189 29 1500 01 197 1NP 02 193 1NP 03 166 1NP 185 17 5000 01 192 1NP 02 189 1NP 03 204 1NP 195 8 Positive Control 2-aminoanthracene 1 .0 pg per plate 01 907 1 02 1002 1 03 1084 1 998 89 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. AA52XK.502001.BTL 36 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Table 20 Test Substance Id: H-25170 Study Number AA52XK.502001.BTL Strain TA1535 Liver Microsomes None Vehicle tetrahydrofuran Experiment No Cells Seeded Date Plated B2 2.6 X 10s 4 Jan 2002 Concentration pg per plate Vehicle Plate Number 01 02 03 Revertants per plate 18 18 18 Background Code 1 1 1 Average Revertants 18 Standard Deviation 0 15 01 10 1 02 20 1 03 19 1 16 6 50 01 23 1 02 21 1 03 15 1 20 4 150 01 12 1 02 13 1 03 19 1 15 4 500 01 20 1NP 02 21 1NP 03 28 1 23 4 1500 01 17 1NP 02 13 IIP 03 18 1NP 16 3 5000 01 16 IIP 02 14 IIP 03 18 IIP 16 2 Positive Control sodium azide 1.0 pg per plate 01 420 1 02 484 1 03 408 1 437 41 Background Lawn Code l=Normal; 2=Slightly reduced; 3Moderately reduced 4=Extremely reduced; 5Absent; 6Obscured by precipitate NP=Non-Interfering precipitate; IPInterfering precipitate BioReliance Study No. AA52XK.502001 .BTL 37 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Table 21 Test Substance Id: H-25170 Study Number :AA52XK.502001.BTL Ebqperiment No :B2 Strain :TA1535 Cells Seeded :2.6 X 10s Liver Microsomes :Rat liver S9 Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 pL__________________________________________ Concentrt ion Plate Revertants Background Average Standard lig per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 12 1 14 1 18 1 15 3 15 01 19 1 02 14 1 03 17 1 17 3 50 01 20 1 02 16 1 03 11 1 16 5 150 01 13 1 02 14 1 03 10 1 12 2 500 01 8 1NP 02 20 1NP 03 14 1NP 14 6 1500 01 15 1NP 02 18 IIP 03 15 1NP 16 2 5000 01 13 IIP 02 6 IIP 03 11 IIP 10 4 Positive Control 2-aminoanthracene 1.0 pg per plate 01 168 1 02 173 1 03 158 1 166 Background Lawn Code 1Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate 8 B io R elian ce Study No. AA52XK.502001 .BTL 38 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Table 22 Test Substance Id: H-25170 Study Number :AA52XK.502001.BTL Experiment No :B2 Strain :TA1537 Cells Seeded :1.3 X 10 Liver Microsomes :None Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 uL__________________________________________ Concentrtion Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 81 61 51 62 15 01 7 1 02 5 1 03 5 1 61 50 01 10 1 02 3 1 03 8 1 74 150 01 10 1 02 5 1 03 4 1 63 500 01 6 IIP 02 7 IIP 03 3 1NP 52 1500 01 9 1NP 02 4 IIP 03 4 IIP 63 5000 01 3 1NP 02 3 IIP 03 2 1NP 31 Positive Control 9-aminoacridine 75 UST per plate 01 696 1 02 629 1 03 956 1 760 173 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. AA52XK.502001.BTL 39 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Table 23 Test Substance Id: H-25170 Study Number :AA52XK.502001.BTL Experiment No :B2 Strain :TA1537 Cells Seeded :1.3 X 10 Liver Microsomes :Rat liver S9 Date Plated :4 Jan 2002 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 81 91 10 1 91 15 01 12 1 02 4 1 03 10 1 94 50 01 8 1 02 4 1 03 1 1 44 150 01 5 1 02 10 1 03 11 1 93 500 01 3 1NP 02 2 1NP 03 9 1NP 54 1500 01 8 1NP 02 7 IIP 03 9 IIP 81 5000 01 9 IIP 02 6 1NP 03 3 1NP 63 Positive Control 2-aminoanthracene 1.0 pg per plate 01 216 1 02 113 1 03 178 1 169 52 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reducedl; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate ; IP=Interfering precipitate BioReliance Study No. AA52XK.502001.BTL 40 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Table 24 Test Substance Id: H-25170 Study Number :AA52XK.502001.BTL Experiment No :B2 Strain :WP2 uvrA Cells Seeded :2.5 X 10s Liver Microsomes :None Date Plated :4 Jan 2002 Vehicle :tetrahydrofuran Plating Aliquot : 25 uL__________________________________________ Concentration Plate Revertants Background Average Standard ug per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 10 1 91 12 1 10 2 15 01 14 1 02 14 1 03 13 1 14 1 50 01 12 1 02 9 1 03 16 1 12 4 150 01 21 1 02 14 1 03 6 1 14 8 500 01 10 1 02 12 1 03 17 1 13 4 1500 01 5 1NP 02 12 1NP 03 13 1NP 10 4 5000 01 9 1NP 02 12 1NP 03 8 1NP 10 2 Positive Control methyl methanesulfonate 1000 ug per plate 01 108 1 02 121 1 03 122 1 117 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. AA52XK.502001.BTL 41 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse M utation Test with DuPont-8867 Bacterial Mutation Test Table 25 Test Substance Id: H-25170 Study Number : AA52XK.502001.BTL Strain : WP2 uvrA Liver Microsomes : Rat liver S9 Vehicle : tetrahydrofuran Plating Aliquot : 25 pL Experiment No : B2 Cells Seeded : 2. 5 X 108 Date Plated : 4 Jan 2002 Concentration Plate Revertants Background Average Standard pg per plate Number per plate Code Revertants Deviation Vehicle 01 02 03 91 91 12 1 10 2 15 01 10 1 02 5 1 03 7 1 73 50 01 9 1 02 11 1 03 13 1 11 2 150 01 10 1 02 10 1 03 10 1 10 0 500 01 14 1 02 8 1 03 11 1 11 3 1500 01 14 1NP 02 12 1NP 03 17 1 14 3 5000 01 11 1NP 02 16 1NP 03 9 1NP 12 4 Positive Control 2-aminoanthracene 10 pg per plate 01 972 1 02 1164 1 03 844 1 993 161 Background Lawn Code l=Normal; 2=Slightly reduced; 3=Moderately reduced 4=Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfaring precipitate ; IP=Interfering precipitate BioReiiance Study No. AA52XK.502001.BTL 42 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Bacterial Mutation Test Summary of Results Table 26 Test Substance Id : H-25170 Study Number______ : AA52XK.502001.BTL_____ Experiment No ; B1 Average Revertants Per Plate Standard Deviation Liver Microsomes: None Dose (pg/plate) TA98 TA100 Vehicle 15 50 150 500 1500 5000 Positive 10 0 159 5 13 1 159 24 10 0 150 11 10 2 139 10 10 2 143 12 12 3 124 49 11 2 166 23 179 23 503 13 Liver Microsomes: Rat liver S9 TA1535 13 14 11 12 9 11 13 280 * 2 4 1 2 1 1 1 44 TA1537 4 2 4 2 5 3 4 2 5 1 6 2 5 2 692 126 WP2 uvrA 13 10 12 13 12 12 1 14 95 3 0 0 1 0 2 3 18 Dose (pg/plate) TA98 Vehicle 15 50 150 500 1500 5000 Positive 16 15 16 18 15 14 16 446 7 2 3 1 8 1 2 33 TA100 163 t 29 171 8 151 12 189 40 166 14 176 25 177 16 657 128 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 25 )iL TA1535 10 10 12 8 16 9 10 111 2 2 3 4 3 3 5 23 TA1537 6 8 5 7 6 6 6 94 3 3 1 3 2 1 2 8 WP2 uvrA 12 3 11 1 10 t 1 10 t 0 13 t 2 9 4 15 2 515 171 BioReliance Study No. AA52XK.502001 .BTL 43 Company Sanitized. Does not contain TSCA CBI H-2S170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 Bacterial Mutation Test Summary of Results Table 27 Test Substance Id : H-25170 Study Number______ : AA52XK.502001 .BTL______Experiment No ; B2 Average Revertants Pei: Plate :t Standard Deviation Liver Microsomes : None Dose (jig/plate) TA98 TA100 Vehicle 15 50 150 500 1500 5000 Positive 19 4 194 22 16 6 182 7 12 5 190 9 10 1 184 21 12 5 184 32 5 3 190 22 9 3 186 8 238 10 771 6 Liver Microsomes: Rat liver S9 TA1535 18 16 20 15 23 16 16 437 0 6 4 4 4 3 2 41 TA1537 6 2 6 1 7 4 6 3 5 2 6 3 3 1 760 173 WP2 uvrA 10 14 12 14 13 10 10 db 117 2 1 4 8 4 4 2 8 Dose (pg/plate) TA98 TA100 Vehicle 15 50 150 500 1500 5000 Positive 24 2 188 21 19 9 191 12 23 3 189 17 17 3 159 7 20 6 189 29 23 7 185 17 19 4 195 8 1011 36 998 89 Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 25 pL TA1535 15 17 16 12 14 16 10 166 3 3 5 2 6 2 4 8 TA1537 9t 9 4 9 5 8 6t 169 1 4 4 3 4 1 3 52 WP2 uvrA 10 2 7 3 11 2 10 0 11 3 14 3 12 4 993 161 BioReliance Study No. AA52XK.502001 .BTL 44 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 APPENDIX A Historical Control Data BioReliance Study No. AA52XK.502001.BTL 45 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 Historical Negative and Positive Control Values 1998 - 2000 revertants per plate A ctivation Strain Control None Rat Liver Mean SD Min Max Mean SD Min Max TA98 Neg 16 7 4 59 21 7 7 58 Pos 425 206 21 1536 592 322 56 2454 TA100 Neg 128 31 53 288 138 34 74 258 Pos 568 159 129 1371 736 301 198 2871 TA1535 Neg 12 5 1 45 12 4 1 42 Pos 378 164 6 978 104 84 18 1640 TA1537 Neg 6 3 0 30 7 3 1 29 Pos 708 409 13 2786 88 106 12 2060 WP2 uvrA Neg Pos 14 5 4 190 138 34 48 961 16 6 317 299 4 115 22 2632 SD=standard deviation; Min=nmnimum 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. AA52XK.502001.BTL 46 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 APPENDIX B Study Protocol BioReliance Study No. AA52XK.502001.BTL 47 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_________ d eceiv ed b y SponsorProjectNumber: D uP ont-8867 pSTTl---- DuPont-8867 fimmao. BioRelianceStudyNumber AA52XK.S02001JBTL H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay 1.0 PURPOSE The purposeofthisstudyistoevaluatethemutagenicpotentialofthetestsubstance by measuring itsabilityto induce reversemutationsatselected lociofseveral strainsof S a lm o n e lla ty p h im u riu m and attiretryptophan locusofE s c h e ric h ia c o li WP2 uvrA in thepresenceandabsenceofS9activation. 2.0 SPONSOR 2.1 Name: 22 Address: 2 3 Representative: E.l.du PontdeNemours andCompany StineHaskellResearchCenter DuPontHaskellLaboratory P.O.Box 50 1090ElktonRoad Newark,DE 19714-0050 MariaDonner,PhD. Phone: 302-366-5251 Fax: 302-366-5207 Email: maria-donncr@usa-dupont.com 2.4 SponsorProjectNo.: DuPont-8867 2.5 WR#: 16 Haskell#: 2.7 ServiceCode: 3.0 IDENTIFICATIONOF TESTAND C( (TROLSUBSTANCES 3.1 TestSubstanceName: 3 2 TestSubstanceID.: H-25170(tobeusedinthereporttitleandtext) 3.3 Controls: Negative: Positive: Testsubstancevehicle 9-aminoacndmc 2-amrooanthraeenc methylmethanesulfonatc 2-nitrofluorene sodiumazide Protocol SPGTS02001 l-Jan-2001 Page l of 11 ^ BioReliancf BioReliance Study No. AA52XK.502001JBTL 48 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 SponsorProjectNumber DuPont-8867 BioRelianceStudyNumber: AA52XK.502001,BTL 3.4 TestSubstanceCharacterization Unlessalternatearrangements aremade, thetestingfacilityatBioReliance will not perform analysis of the dosing solutions. The Sponsor will be directly responsible for determination and documentation of the analytical purity and composition of the test substance, and die stability and strength of the test substanceinthesolvent(orvehicle). 3.5 TestSubstanceRetentionSample Theretentionofareservesampleofthetestsubstancewillbetheresponsibilityof theSponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: ToxicologyTestingFatality BioReliance 4.2 Address: 9630MedicalCenterDrive Rockville,M D 20850 4.3 StudyDirector ValentineO.WagnerHI,M.S. Phone:301-610-2152 Fax:301-738-2362 Email:swagner@lnoreliance.com 5.0 PROPOSED STUDY DATES 5.1 ExperimentalStartDate: 07-Dec-200l 5.2 ExperimentalTerminationDate: 25-Jan-2002 5.3 DraftReportDate: 08-Feb-2002 5.4 FinalReportDate: 2weeksafterSponsorapprovesdraft 6.0 TESTSYSTEM The testerstrainswillincludethe& typ h im u riu m histidineauxotrophs TA98, TA100, TAI535 and TA1537 asdescribedby Ames e t a l. (1975) and the c o lt testerstrain WP2 u v rA asdescribedbyGreenandMuriel(1976)._____________________ HistidineMutation Tryptopha n Mutation AdditionalMutations h isQ 4 6 A&C3076 AD3052 trp E LPS Repair R-factor TA1535 TA1537 - - rfa & u v rB - ProtocolSPGT502001 l-Jan-2001 Page2of11 |p BioReliancf BioReliance Study No. AA52XK.502001.BTL 49 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay___________ __ DuPont-8867 SponsorProjectNumber DuPont-8867 BioRelianceStudyNumber AAS2XK.502001,BTL HistidineMutation Tryptopha n Mutation h isQ 4 6 AC3076 ID3052 trp E TAUX) - TA98 - - - - WP2 uvrA AdditionalMutations LPS Repair R-factor rfa AUvrB +R - uvrA - Each S . typ h im u riu m testerstraincontains, in addition to a mutation inthe histidine operon, additional mutations that enhance sensitivity to some mutagens. The r fa mutationresultsina cellwall deficiencythatincreasesthepermeabilityofdiecellto certainclasses of chemicals such as those containing large ring systems thatwould 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 bacteria] DNA repair polymerase complexinvolvedwiththemismatch-repairprocess. TA98 and TA1S37 are reverted from histidinedependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshiftand base substitutionmutagens and TA1S35 isrevertedonly by mutagens thatcausebasesubstitutions. The c o li testerstrainhasanAT basepairatthecriticalmutationsitewithinthetrp E gene (Wilcox e t a l., 1990). TesterstrainWP2 uvrA has a deletion inthe uvrA gene resultinginadeficientDNA excision-repairsystem.Tryptophanrevertantscanarisedue toa basechange attheoriginallymutated siteor by a base change elsewhere inthe chromosome causingtheoriginalmutationtobe suppressed.Thus,thespecificityofthe reversionmechanism issensitivetobase-pairsubstitutionmutations(GreenandMuriel, 1976). The S . typ h im u riu m tester strains were received directly from Dr. Bruce Ames, University of California, Berkeley. The . c o li tester strain was received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland (United Kingdom). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The testsubstancewillbetestedataminimum offivedoselevelsalongwithappropriate negativeandpositivecontrolswithtesterstrainsTA98, TA100, TA1S3S, TA1S37 and WP2 uvrA withand without S9 activation.All dose levelsoftestsubstance,negative controlsandpositivecontrolswillbeplatedintriplicate. ProtocolSPGT50200I l-Jan-2001 Page3of11 BioReliance Study No. AA52XK-502001.BTL 50 | | BioReliancf Company Sanitized. Does not contain TSCA CB1 H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 SponsorProjectNumber: DuPont-8867 BioRelianceStudyNumber: AA52XK.502001,BTL 7.1 SolubilityDetermination Unless the Sponsor has indicated the test substance vehicle, a solubility determinationwillbeconductedtodeterminethemaximum solubleconcentration orworkablesuspensionuptoamaximum ofSOmg/mL foraqueousvehiclesand 500mg/mL fororganic vehicles.Vehicles compatible with thistestsystem, in order of preference, include but are not limited to deionized water (CAS 7732-18-5), dimethyl sulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5) and acetone (CAS 67-64-1). The vehicle ofchoice will be the solvent,selected in orderofpreference,which permits preparation ofdiehighestwotkable/soluble stockconcentration,up to 50mg/mL foraqueous vehiclesand 500mg/mL for organicvehicles. 7.2 PreliminaryToxicityAssaytoSelectDoseLevels Selectionofdoselevelsforthemutagenicityassaywillbebaseduponthetoxicity andprecipitationprofileofthetestsubstanceassessedina preliminarytoxicity assay. This preliminary assay will be conducted by exposing TA98, TA100, TA1535, TA1537 and WP2 i/vrA to negative controls and to at least eight concentrationsoftestsubstance,oneplateperdoselevel,inboththepresenceand absenceofS9 activation.Unlessindicatedotherwiseby theSponsor,thehighest dosewillbethehighestworkableconcentrationinthevehicleofchoicebutnotto exceed 5mg/plate. In selecting dose levels for die mutagenicity assay the following guidelines will be employed. Doses will be selected such that precipitatedoesnotinterfoewithmanualscoring.Wheneverpossible,thehighest dosefixthemutagenicityassaywillbeselectedtogivesomeindicationoftoxicity without exceeding 5mg/plate. For freelysoluble,nonloxic testsubstances,the highestdose levelwillbe 5mg/plate. Forprecipitating,nontoxictestsubstances, thehighestdoselevelwillbeselectedinanattempttoyieldprecipitateatonlythe toponeortwodoselevels.TheSponsorwillbeconsultedregardingdoseselection if(1)themaximum dose levelisselectedbased on precipitationand thisdose levelislessthan5mg/plateor(2)diemaximum achievabletestsubstancedose levelislessthan5mg/plateandthisdoselevelisnontoxic. 7.3 FrequencyandRouteofAdministration Thetestsystemwillbe exposedtothetestsubstanceviathe{dateincorporation methodologyoriginallydescribedby Ames e t a l. (1975)and updatedby Maron and Ames (1983).This testsystem has been shown todetecta wide range of classesofchemicalmutagens(McCannet a l., 1975;McCannandAmes, 1976). Afterthedatageneratedinthefirstassayhavebeenevaluated,themutagenicity assaywillberepeated.The doselevelsusedinthesecondassaywillbe thesame asthoseusedinthefirstassayunlesstheStudyDirectordeterminesthatthedose levelsshouldbechangedduetoanequivocalresponse,excessivecytotoxicityor ProtocolSPGT502001 l-Jan-2001 Page4of11 ^ Bio Reliance B io R elian ce Study No. AA52XK.502001.BTL 51 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 SponsorProjectNumber. DuPont-8867 BioRdianceStudyNumber AA52XK.50200I,BTL excessiveprecipitate.IftheSponsor isaware ofspecificmetabolicrequirements (e.g.,azo compounds),thisinfonnationwillbe utilizedindesigningtireassay, (e.g.,activation system or treatment method). This guidance isbased on the OECD Guideline471 (adoptedJuly 1997andpublishedFebruary 1998)andICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals(1997). 7.4 Controls 7.4.1 PositiveControls Allcombinationsofpositivecontrolsandtesterstrainsplatedconcurrently withtheassayarelistedbelow._________________________ Strain S9 Activation PositiveControl Concentration (pgfrtate) S a lm o n e lla Strains WP2 uvrA Rat 2-aminoanthracene 1.0 10 TA98 2-nitrofluorene 1.0 TA100, TA1535 TA1537 None sodiumazide 9-aminoacridme 1.0 75 WP2 uvrA methyl methanesulfonate 1,000 7.4.2 NegativeControls Appropriatenegativecontrolswillbeplatedforeachtesterstrainwithand without S9 activation. The negative control will be the vehicle alone, unlessthereisno historicalbasisforuseoftheselectedvehicle.Inthe lattercase,bothuntreatedandvehiclecontrolswillbeused. 7.4.3 SterilityControls ThemostconcentratedtestsubstancedilutionandtheSham andS9mixes willbecheckedforsterility. 7.5 ExogenousMetabolicActivation Aroclor1254-inducedratliverS9willbeusedasthemetabolicactivationsystem. The S9 homogenate willbe prepared from male Sprague-Dawley tatsinduced withasingleintraperitonealinjectionofAroclor1254,500mg/kg,fivedaysprior tosacrifice.The S9 willbe hatchprepared and stored frozenatapproximately ProtocolSPGT502001 1-Jan-2001 Page5of11 0Bio Reliancf BioReliance Study No. AA52XK.50200l.BTL 52 Company Sanitized. Does not contain TSCA C8I H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 SponsorProjectNumber DuPont-8867 BioRelianceStudyNumber AA52XK.S02001.BTL -70Cuntilused.EachbatchofS9 homogenate willbeassayedforitsabilityto metabolize 2-aminoanthracene and 7,12-dimethylbenzanthracene to forms mutagenicto typ h im u riu m TA100. Immediatelypriortouse,theS9 willbethawedandmixed withacofactorpoolto contain 10% S9 homogenate, Sm M glucose-6-phosphate, 4 m M ^-nicotinamide-adeninedinucleotidephosphate,8m M MgCljand33m M KC1 in a 100m M phosphatebufferatpH 7.4.ThismixtureisreferredtoasS9mix.Sham mixwillbe 100m M phosphatebufferatpH 7.4. 7.6 PreparationofTesterStrain Overnightcultureswillbe inoculatedfromtheappropriatemasterplateorfrom the appropriate frozen stock. To ensure thatculturesare harvested in latelog phase,thelengthofincubationwillbecontrolledandmonitored.Atfoeendoffoe workingday,eachinoculatedflaskwillbeplacedinarestingshaker/incuhatorat room temperature.Theshaker/incubatorwillbeprogrammedtobeginshakingat approximately 125rpm at372C approximately 12hoursbeforetheanticipated timeofharvest All cultures will be harvested by spectrophotometric monitoring of culture turbidityratherthanby durationofincubationsinceovergrowthofculturescan cause loss of sensitivity to some mutagens. Cultures will be removed from incubationatadensityofapproximately10*cells/mL. 7.7 TestSystemIdentification Eachplatewillbelabeledwithacodesystemthatidentifiesthetestsubstance,test phase,dose level,testerstrainandactivationtypeasdescribedinBioRcliancc's StandardOperatingProcedures. 7.8 TestSubstancePreparation Unlessspecifiedotherwise,testsubstancedilutionswillbepreparedimmediately priortouse.Alltestsubstancedosingwillbeatroom temperatureunderyellow light 7.9 TreatmentofTestSystem One halfmilliliter(0.5mL) ofS9 mix orSham mix, 100 oftesterstrainand 50pL ofvehicle, testsubstance dilution or positive control will be added to 2.0 mL ofmoltenselectivetopagarat452C. When necessaryto achievethe targetconcentrationoreliminatetoxicvehicleeffects,aliquotsofotherthan50 of testsubstance/vchicle/positive control will be plated. The mixture will be vortexmixed and overlaidontofoesurfaceof25mL ofminimal bottom agar. ProtocolSPGT502001 l-Jan-2001 Page6of11 m Bio Reliance- BioReliance Study No. AA52XK.502001.BTL 53 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay DuPont-8867 SponsorProjectNumber DuPont-8867 BioRelianceStudyNumber: AA52XK.S02001,BTL After the overlay has solidified, the plates will be invertedand incubated for approximately48to72hoursat372C. Platesthatarenotcountedimmediately followingtheincubationperiodwillbestoredat2-8C. 7.10 Scoring The conditionofthebacterialbackgroundlawn willbeevaluatedforevidenceof testsubstancetoxicityandprecipitate.Evidenceoftoxicitywillbescoredrelative tothenegativecontrolplateandrecordedalongwiththerevertantcountforthat plate.Toxicitywillbeevaluatedasadecreaseinthenumberofrevertantcolonies perplateand/ora dunning or disappearanceoffoebacterialbackground lawn. Precipitationwillbeevaluatedaftertheincubationperiodby visualexamination withoutmagnification. 7.11 TesterStrainVerification On foe day ofuse infoemutagenicity assay, alltesterstraincultures will be checkedfortheappropriategeneticmarkerscitedin$6.0. 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST The followingcriteriamustbemet forfoemutagenicityassaytobeconsideredvalid: 8.1 TesterStrainIntegrity To demonstratefoepresenceofther/omutation,allS . typ h im u riu m testerstrain culturesmustexhibitsensitivitytocrystalvioletTo demonstratefoepresenceof theuvrBmutation,all typ h im u riu m testerstrainculturesmustexhibitsensitivity toultravioletlightTo demonstratefoepresenceoffoeuvrAmutation,all c o li testerstrainculturesmust exhibitsensitivitytoultravioletlightTo demonstrate foepresenceofthepKMIOt plasmidR-factor,testerstrainculturesofTA98 and TA100 mustexhibitresistancetoampidllin. 8.2 SpontaneousRevertantBackgroundFrequency Based on historical control data, all tester strain cultures must exhibit characteristicnumberofspontaneousrevertantsperplateinfoenegativecontrols (vehicle). The mean revertantsper platemust be within foe following ranges (inclusive): TA98, 10-50; TA100, 80-240; TA1535, 5-45; TA1537, 3-21; WP2vrA, 10-60. 8.3 TesterStrainTiters To ensurethatappropriatenumbersofbacteriaare{dated,alltesterstrainculture titersmustbeequaltoorgreaterthan0.3x10*cellspermilliliter. ProtocolSPGT502001 1-Jan-2001 Page7of11 ^ (m Bio Reliancf BioReliance Study No. AA52XK.502001.BTL 54 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat A ssay______________ DuPont-8867 SponsorProjectNumber: DuPont-8867 BioRelianceStudyNumber: AA52XK.5Q2001.BTL 8.4 PositiveControlValues Eachmeanpositivecontrolvaluemustexhibitatleasta3.0-foldincreaseoverthe respectivemeannegativecontrolvalue(vehicle)foreachtesterstrain. 8.5 Toxicity A minimumofthreenon-toxicdoselevelswillberequiredtoevaluateassaydata. A doselevelisconsideredtoxicifitcausesa>50%reductioninthemeannumber ofrevertantsperplaterelativetothemean negativecontrolvalue(thisreduction mustbeaccompaniedbyanabruptdose-dependentdropinthercvertantcount)or a reductioninthebackground lawn. Indieeventthatlessthanthreenon-toxic doselevelsareachieved,theaffectedportionoftheassaywillberepeatedwithan appropriatechangeindoselevels. 9.0 EVALUATION OF TEST RESULTS Fora testsubstancetobeevaluatedpositive,itmust causea dose-relatedincreaseinthe mean revertantsperplateofatleastonetesterstrainoveraminimum oftwo increasing concentrationsoftestsubstanceasspecifiedbelow: 9.1 StrainsTA1535andTA1537 Datasetswillbejudgedpositiveiftheincreaseinmean revertantsatthepeakof thedoseresponseisequal toorgreaterthan 3.0-timesdiemean negativecontrol value(vehicle). 9.2 StrainsTA98.TA100andWP2*vrA Datasetswillbejudgedpositiveiftheincreaseinmean revertantsatthepeakof diedoseresponseisequaltoorgreaterthan2.(Mimesthemean negativecontrol value(vehicle). 10.0 REPORT A reportoftheresultsofthisstudywillbepreparedbytbeTestingLaboratoryandwill - accuratelydescribeallmethodsusedforgenerationandanalysisofdiedata.The report willinclude: TestSubstance:identificationand CAS no.,ifknown: physical natureandpurity,if known; physicochemical propertiesrelevanttotheconductofthestudy,ifknown; stabilityoftestsubstance,ifknown. Solvent/Vehicle: justification for choice of vehicle; solubility and stabilityof test substanceinsolvent/vehicle,ifknown Strains: strainsused;numberofcells/mLperculture;straincharacteristics. ProtocolSPGT502001 l-Jan-2001 Page8of11 m Bio Reliancf BioReliance Study No. AA52XK502001.BTL 55 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 SponsorProjectNumber DuPont-8867 BioRelianceStudyNumber AA52XK.502001,BTL Testconditions:amountoftestsubstanceperplatewithrationalefordoseselectionand number ofplatesperconcentration;media used;typeand compositionofmetabolic activationsystem,includingacceptabilitycriteria;treatmentprocedures. Results: signsoftoxicity;signsofprecipitation; individualplatecounts;themean number of revertant colonies per plate and standard deviation; dose-response relationship, where possible; statistical analysis, if any, concurrent negative and positivecontroldatameans and standarddeviations;historicalnegativeand positive controldatawithranges,meansandstandarddeviation. Discussionofresults. . Conclusion. 11.0 RECORDS AND ARCHIVES All raw data, the protoco^n^dl reports will be maintained according to Standard Operating Proceduiefl|HHj^ny die BioReliance RAQA unit headquartered at: BioReliance,14920BrosSa^KRm, Rockville,M D 20850. PerthisSOP,paperrecords willberetainedforatleastthreeyearsafterwhichtimetheSponsorwillbecontactedfor a decisionastothefinaldispositionofthematerials.Allstudymaterialsreturnedtothe Sponsor or destroyed will first be copied and the copy will be retained in the BioReliancearchivesforaminimum of10years. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE Hus protocol has been written to comply with OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Assay), Ninth Addendum to the OECD GuidelinesfortheTestingofChemicals,publishedby OECD, Paris,February 1998and with the International Conference on Harmonisation of Technical Requirements for RegistrationofPharmaceuticalsforHuman Use (1996and 1997). ThisstudywillbeperformedincompliancewiththeprovisionsoftheGood Laboratory Practice Regulations for Nonclinical Laboratory Studies (GLPs). The protocol, an in-processphase,theraw data,and reports)willbe auditedpertireStandardOperating Procedures (SOPs) ofBioReliance by theQuality Assurance Unit ofBioReliance for compliancewithGLPs, theSOPs ofBioRelianceandthestudyprotocol.The in-process inspection will be performed to audit the critical assay procedures and systems supportingtheassay.A signedQA statementwillbe includedinthefinalreportThis statement will list the system phases inspected during the previous quarter or the study-specificphases, thedatesofeach inspection, and the dates the resultsofeach inspectionwerereportedtotheStudyDirectorandtheStudyDirector'smanagement In addition,a signedGLP compliancestatementwillbeincludedinthefinalreport Ibis statement will cite the GLP guideline(s)with which the study iscompliant and any exceptionstothiscompliance,ifapplicable,includingtheomission ofcharacterization orstabilityanalysesofthetestorcontrolsubstancesortheirmixtures. ProtocolSPGT502001 1-Jan-2001 Page9of11 ^ 9Bio Reliance- BioReliance Study No. AA52XK.502001.BTL 56 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 SponsorProjectNumber: DuPont-8867 BioRelianceStudyNumber: AAS2XK.502001.BTL Unlessarrangementsaremade tothecontrary,unuseddosingsolutionswillbe disposed offollowing administrationtothe testsystem and allresidual testsubstancewill be disposedoffollowingfinalizationofthereport 13.0 REFERENCES Ames, B.N.,McCann, J.and Yamasaki, E. (1975). Methods fordetectingcarcinogens and mutagens withtheSa/mone/fa/mammalian-microsome mutagenicitytestMutation Research31:347-364. Green, M.H.L., and Muriel, WJ. (1976). Mutagen testing using trp+ reversion in E s c h e ric h ia co/i.MutationResearch38:3-32. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity TestsforPharmaceuticals. S2A document recommended for adoption at step 4 of the ICH process on July 19, 1995. Federal Register 61:18198-18202,April24,1996. International Conference on Harmonisation (ICH) of Technical Requirements for RegistrationofPharmaceuticalsforHuman Use. Genotoxicity: A StandardBatteryfor GenotoxicityTestingofPharmaceuticals.S2B documentrecommended foradoptionat step 4 of the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21,1997. McCann, J. and Ames, B.N. (1976). Detection of carcinogens as mutagens in the S a lm o n eU a ltm cm sa m e. test:assayof300 chemicals:discussion.Proc.Natl. Acad. Sci. USA 73:950-954. McCann,J.,Choi,IL,Yamasaki,E.andAmes, B.N.(1975).Detectionofcarcinogensas mutagensintheS a lm o n e lla lmicrosometest:assayof300 chemicals. Proc.Natl.Acad. Sci.USA 72:5135-5139. Marn,D.M.andAmes,B.N.(1983).RevisedMethodsfortheS a lm o n e lla Mutagenicity Test MutationResearch 113:173-215. OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), Ninth AddendumtotheOECD GuidelinesfortheTestingofChemicals,publishedbyOECD, Paris,February 1998. Wilcox, P., Naidoo, A., Wedd, DJ. and Gatehouse, D.G. (1990). Comparison of S a lm o n e lla typ h im u riu m TA102 withE s c h e ric h ia c o f WP2 testerstrains.Mutagenesis 5:285-29!. ProtocolSPGT502001 l-Jan-2001 Page10of11 BioReliance Study No. AA52XK.502001.BTL 57 ^ Bio Reliancf Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 SponsorProjectNumber. DuPont-8867 BioRelianceStudyNumber. AA52XK.5Q2001.BTL .14.0 APPROVAL c v ., SponsorRepresentative QO - Qgc- - Z-o o V Date K O-r C Qc/%z\-C (PrintorTypeName) 5'TbCc3<*>l Date of, A4LX- zeei ' Date ProtocolSPGT502001 l-Jan-2001 Page11of11 BioReliance Study No. AA52XK.502001.BTL 58 Bio Reliance* Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 APPENDIX C Information for Japanese Regulatory Agencies BioReliance Study No. AA52XK.502001.BTL 59 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay______________ DuPont-8867 Report o f Results of Reverse-M utation Test in Bacteria 1. Tester Strains (1) Procurement Strain TA98 TA100 TA1535 TA1537 TA1538 TA97 TA102 W P2 uvrA W P2 uvrA (pkM IOl) WP2 (pKM IOl) Obtained from Date obtained 10 November 1998 Dr. Bruce Ames U niversity o f California, Berkeley 11 August 1998 13 December 1990 14 November 1990 N ational Collection of Industrial and M arine Bacteria Aberdeen, Scodand 1 July 1987 19 February 1993 Date inspected the strain lot in storage The genetic markers for each culture are confirm ed on the day of use (2) Storage Freezing method Storage temperature C om position Large quantity -70C Bacterial suspension DMSO 1.0 mL 0.09 mL 2. S9 M ix (1) Source, Storage Temperature, etc, o f S9 M ade in-house Prepared on 17 October 2001 (Batch R654) 26 November 2001 (Batch R656) Storage tem perature -70C or colder Name and model o f B io R elian ce Study No. AA52XK.502001.BTL 60 So-Low, Model Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ storage apparatus DuPont-8867 PR27-120 (2) Preparation o f S9 Animal used Inducing substance Species, Strain Rattus norvegicus, Sprague D aw ley Name Aroclor 1254 Sex M ale Adm inistration m ethod intraperitoneal Age (in weeks) W eight 8 (Batch R654) 9 (Batch R656) 181 to 198 g (Batch R654) 187 to 271 g (Batch R656) Adm inistration period and amount (g/kg-w eight) 5 days, 0.5 gm/kg body w eight 3. Preparation o f Test Substance Solution Solvent used Name M anufacturer Lot No. Tetrahydrofuran (CAS No. 109-99-9) Stability o f test substance in the solvent M ethod o f suspension when test substance is difficult to dissolve Aldrich Chemical Company J100352H1 Unknown N ot applicable Grade and/or Purity (%) Gold Label, 99.9% BioReliance Study No. AA52XK.502001.BTL 61 Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 4. Conditions o f Pre-culture N utrient broth Period of pre-culture Storage tim e and temp, from inoculation to beginning o f shaking culture Storage tim e and temp, from end o f culture to use for test M odel and m anufacturer of 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 Oxoid Ltd. Lot No. C H ,-B = 232622 C H ,-B = 210287 2 to 5 hours at ambient tem perature <8 hours at 2-8C New Brunswick Scientific, model G-24 Rotary (125 rev/m in.) shape: cylinder, 200 mL 50 mL 1 colony 5. Agar Plate M edium (1) Top agar Agar Name M anufacturer Lot No. BBL Select Becton Dickinson 1000J3DKSQ (2) M inimum Glucose Agar Name M ade in-house Agar M anufacturer Lot No. Volume of agar plate medium BioReliance Study No. AA52XK.502001.BTL 62 BBL Select Becton Dickinson 1000J3DKSQ 25 mL Company Sanitized. Does not contain TSCA CBI H-25170: Bacterial Reverse Mutation Test with an Independent Repeat Assay_______________ DuPont-8867 6. Test Results - Judgement o f the results Judgem ent N egative Reason for judgem ent and referential matters: No positive response was observed with any o f the tester strains in the presence and absence o f Aroclor-induced rat liver S9. Referential matters The vehicle and positive control values indicate that all tester strains were functioning correctly and were capable o f detecting a mutagen. BioReliance Study No. AA52XK.502001.BTL 63 Company Sanitized. Does not contain TSCA CBI