Document bB200Qo9n6XaarGx5M6y6yR8k

Final Report AR226-3072 DiiPoi)IHl.O-IW7-(KK135 TRADE SECRET Study Title H-22051: MUTAGEN1CITY TESTING IN THE ^Al.MONI-'J.I.A /YI'HlMdfWIM AND 1-:SCHI:RU'HIA COI.I PLATE INCORPORATION ASSAY Laboratory Project ID Haskell Laboratory Repor* No. 1997-00035 Authors Valentine 0 Wagner, III, M.S John D Reece, B S Study Completed on 04-02-97 Performing Laboratory- E 1 du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P. 0 Box 50 Newark, Delaware 19714 Microbiological Associates, inc. Study No. G96CF43.5020! I Medical Research Project P.-itic I of 54 Company Sanitized. Does not contain TSCA CBI H-220S1; Mutagenicity Testing in the Salmonella lyphiinuriwn and rjicherichia coll Plate Incorporation Assay DuPonf HLO-1997-0003 5 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with EPA FIFRA (40 CFR 160) EPA TSCA (40 CFR 792) Good Laboratory Practice Standards. OECD Principles of Good Laboratory Practice (C(81)30(Final), Annex 2), and MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850) with the following exceptions: The identity, strength, purity and composition or other characteristics to define the test or control article were not determined by the testing facility. Analyses to determine the uniformity, concentration, or stability of the test or control mixtures were not performed by the testing facility. The stability of the test or control article under the test conditions was not determined by the testing facility. Treatment solutions or suspensions were not analyzed for identity, composition, uniformity, or stability of the test and control articles. The procedures used by trained personnel to prepare the treatment solutions are intended to ensure: A. The accuracy of concentration because the test and control articles were weighed on an analytical balance accurate to 3 decimal places and the vehicle in which the test and control substances were dissolved was accurately measured with graduated pipets or flasks, B. Uniformity because all treatment solutions or suspensions were mixed prior to administration to the test system, and C. Stability because treatment solutions or suspensions were prepared just prior to administration to the test system. These deviations did not affect the validity or the integrity of the study. Submitter. Sponsor: E. I. du Pont de Nemours und Company Si ^-.;.? DuPont SpecialtyChemicals Study Director: Study Monitor: /-7 \/JuwAtA j>. UL,<upu4..3S'____ Valentine 0. Wagner, III, M.S. Microbiological Associates, Inc. ^ / J ^ BriariH. Mathison, Ph.D. Research Toxicologist 2. 4fr.< f*}^ Date " w ^ 'ate MA .Study No. G96CF43.5020 II - 2- Company Sanitized. Does not contain TSCA CBI H-2'>U^ 1 Muliigcnicin Testing in ihc Stilmiinelitt tvphwiiinwi and Kschmchia wli Plate IpcorporaHon Assay GENERAL INFORMATION r _ /J^^BB Test Substance. &a^ f-ndes/Svnonvms: H-2205: DuPont HLO-1997-(XM)35 Stability: Test substance appeared to be stable under the condition of the study; no evidence of instability was observed. Composition: Physical State.. l.iquid I MA .Study No G%C'Fl1.502()l 1 "! - Company Sanitized. Does not contain TSCA CBI H-22l51 Miiiaycnicuy Testing in the Salniimt'llii nphiiiiiiriinn and Hxcfiutichiiic-ilt Plate Incorporation Assay_______PuPont HLO-1997-0003 5 Major Impurities: . Unknown, none considered to be of lexicological significance at this time. Sponsor: DuPont Specialty Chemicals E. I. du Pont de Nemours and Company ; Wilmington, Delaware 19898 Study Initiated/Completed :\ November 27, 1996/April 2, 1997 In-Life Initiated/Completed: 'December 3, 1996/January 6, 1997 '^ Archives ^; * All raw data, and reports will'tie maintained in the archives of Microbiological Associates, Inc., located in Rockville, Maryland A sample of the test substance will be archived at E. I. du Pont de Nemours and Company, Haskell Laboratory for Toxicology and Industrial Medicine, Newark, Delaware r - MA Studv No GWCF43.5()2(> II - 4- Company Sanitized. Does not contain TSCA CBI H-22051 Mutagenicity Testing in the Salmonella lyphimiirium and Escherichia cull Plate IncorporationAssay PuPont HLO-1997.00035 QUALITY ASSURANCE STATEMENT Study Title; Study Number: Study Director: H-22051: MUTAGEMICITY TESTING IN THE SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI PLATE INCORPORATION ASSAY G96CF43.502011 Valentine 0. 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 of studies. Procedures, documentation, equipment records, etc., are examined in order to assure that the study is performed in accordance with the U.S. PDA Good Laboratory 'Practice Regulations (21 CFR 58), the U.S. EPA GLPs (40 CFR 792 and 40 CPR 160), the JK GLP Compliance Programme, the Japanese GLP Standard, ar-i the OECD Principles of Good Laboratory Practice and to assure that che study is conducted according to the prof; sol and relevant Stardar'. Operating Procedures. The following are the inspection dates, phases inspected, nd .-eport dates of QA inspections of this study. IN3PECT OM 02 DEC 96, TO STUDY DIR 02 DEC 96. TO MGMT 02 DEC 96 PHASE: Protocol Review INSPECT OM 03 DEC 96, TO STUDY DIR 06 DEC 96, TO MGMT 06 DEC 96 PHASE: Scrain characterization INSPECT OM 12 DEC 96, TO STUDY DIR 12 DEC 96, TO MGMT 13 DEC 96 PHASE: Preparation of S9 mixture INSPECT OM 13 FEB 97, TO STUDY DIR 13 FEB 97, TO MSMT 20 PEB 97 PHASE: Draft Report INSPECT OM 04 APR 97, TO STUDY DIR 04 APR 37. TO MGMT 04 APR 97 PHASE: Draft to Final Report This report describes the methods and procedures used in the study and the reported results accurately reflect the raw data of the study. C4ouuu. ^ GQ^) kmoueM Claire L. Cour'i:emanche, B.S. QUALITY ASSURANCE O^i ^97 DATE MA Study No. G96CF43.502011 - 5 Company Sanitized. Dnpsn^^.,^^ ^. H-22051: Mutagenicily Testing in the Salmonella typhimuriwn and Eicherichia coii Plate Incorporation A&say OuPont HLO-1997-00035 9 TABLE OF CONTENTS Page Sponsor-supplied Information ................................... 2 Summary .............................................. 7 Purpose ................................................ 8 Characterization of Test and Control Articles ......................... 8 Materials and Methods Test System ...................................... 9 9 Metabolic Solubility .Acti Test . . .vatio n . .S.ys.te.m. . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .......... 9 10 MPruetlaimgeinnaicriytyToAxsis.cai.yty..A.ss.a.y..................................................................... 10 10 EPvlaatliunagtiaonndoSfcRoersinugltsP.ro.c.ed.u.re.s................................................................. 10 12 Criteria for a Valid .. Test ........................................................................ 12 Results and Discussion Solubility Test ...................................... 13 13 Preliminary Mutagenesis . . . Toxicity Assays . . . . Assay . .. ..................;. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ................ 13 13 ........................................ Conclusion ............................................. 13 References ............................................. 14 Data Tables ............................................. 15 Appendix I: Historical Control Data .............................. 42 Appendix II: Study Protocol ................................... 44 x MA Study No. G96CF43.502011 - 6 ComspagnyaSia^nai oita^^awai^^u^im^^ H-22051: Mutagenicity Testing in the Salmonella typhimwiwn and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-00035 SUMMARY The test article, H-22051, was tested in the bacterial reverse mutation assay using S. typhimwiwn tester strains TA98, TA100, TA1535 and TA97a and E. coli tester strain WP2 uvrA (pKMIOl) in the presence and absence of Aroclor-induced rat liver S9. The assay 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 mutagenicityassay (initial and independentrepeat assays), v.^s used to evaluate the mutagenic potential of the test article. Water was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in water at a concentration of approximately 500 mg/ml, the maximum concentration tested. In the preliminary toxicity assay, the maximum dose tested was 5000 ^g per plate; this dose was achieved using a concentration of 100 mg/ml and a 50 id plating aliquot. Neither precipitate nor appreciable toxicity was observed. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 ^g per plate. In the mutagenicity assay, no positive response was observed. Neither precipitate nor appreciable toxicity was observed. The overall evaluation and dose ranges tested are as follows: S9 AClivilioo TA91 .Low High None - 100 sooo Rat 100 5000 OvecO EviliutiM^uid Doif BudTt*ted4/pl.t) .. TA100 Low Hitfa TA 1135 Low Higt. TAWa Low Hish . WImrA ^Wt Ww ''. ; - - 100 SOOO 100 SOOO 100 5000 100 5000 1. 100 5000 100 5000 100 5000 100 5000 '-=ncglivc. + "porilive (nuximum fold incrcju) Under the conditions of this study, test article H-22051 was concluded to be negative in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay. MA Study No. G96CF43.502011 7- Com^pa^nvSi.iUtigfUtetf^BWUtfi^^ H-22051: Mutagenicity Testing b the Salmonella typhimurium and Escherichia coil Plate Incorporation Auay DuPont HLO-1997-OOQ35 PURPOSE The purpose of this study was to evaluate the mutagenic potential of the test article (or its metabolites) by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimwium and one strain off. coli in the presence and absence of S9 activation. CHARACTERIZATION OF TEST AND CONTROL ARTICLES The test article, H-22051, was received by Microbiological Associates, Inc. on 11/22/96 and was assignedthe code number 96CF43. The test article was characterized by the Sponsor as a yellow liquid that should be stored at <30C. An expiration date was not provided. Upon receipt, the test article was described as a yellow liquid and was stored at room temperature, protected from exposure to light. The vehicle used to deliver H-22051 to the test system was sterile distilled water, (CAS# 7732-18-5), obtained from Life Technologies, Inc. Positive controls plated concurrently with the mutagenicity assay are listed below; Strain S9 Activation Positive Control TA98, TA100, TA1535 1.0 TA97a WP2uwA (pKMIOl) + 2-aminoanthracene (Sigma Chemical Co.) 2.0 10 TA98 2-nitrofluorene (Aldrich Chemical Co, lac.) TA100, TA153S TA97a sodium azide (Sigma Chemical Co.) 9-aminoacridine (Sigma Chemical Co.) 1.0 1.0 75 WP2uwA (pKMIOl) methyl methanesutfonate (Aldrich Chemical Co., Inc.) 1.GOO To determine the sterility of the test article, the highest test article dose level used in the mutagenicity assay was plated on selective agar with an aliquot volume equal to that used in the assay. MA Study No. G96CF43.502011 -8- Companv Sanit^d Hn. ^c&^ H-22051: Mutagenicity Testing in the Salmonella typhimwium and Escherichia coll Plate Incorporation Assay DuPont HLO-1997-00035 MATERIALS AND METHODS Test System The tester strains used were the Salmonella typhimwium histidine auxotropbs TA98, TA100, TA1535 and TA97a as described by Amcs et al. (1975) and Levin et al, (1982) and the . coli tester strain WP2 uvrA (pKMIOl) as described by Green and Muriel (1976). Salmonella tester strains were received on 11/26/90 and 11/10/92 directly from Dr. Bruce Ames. University of California, Berkeley. E. coli was received on 07/01/87 from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. Tester strains TA98 and TA97a are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976; Brusick et al., 1980). Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing -50 ml of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 372C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 109 cells per milliliter. The actual tilers were determined by viable count aw's on nutrient agar plates. 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 on 08/07/96,09/25/96 and 11/25/96 and stored at ^-70C until used. Each bulk preparation of S9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimwium TA100. The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate,4 mMj3-mcotinamide*adeninedinucleotidephosphate,8 mMMgCl; and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 ml aliquot of each was plated on selective agar. MA Study No. G96CF43.502011 - 9- Company Sanitized. Does not contain TSCA CBI H-22051: Mutegenicity Testing in the Salmonella typhimunwn and Escherichia colt Plate Incorporation Assay DuPont HLO-1997-00035 Solubility Test A solubility test was conducted to select the vehicle. The test was conducted using one or more of the following solvents in the order of preference as listed: purified water, dimethylsulfoxide, ethanol and acetone. The test article was tested to determine we vehicle, selected in order of preference, that permitted preparation of the highestsoluble or workable stock concentration, up to 500 rag/mi. Preliminary Toxiclty Assay The preliminary toxidty assay was used to establish the dose-range over which the test article would be assayed. Ten dose levels of the test article were plated, one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA97a and WP2 wrA (pKMIOl) on selective minimal agar in both the presence and absence of rat liver S9 activation. Mutageniclty Assay The mutagenicity assay (initial and independent repeat assays)was used to evaluate the mutagenic potential of the test article. A minimum of five dose levels of test article along with appropriate vehicle and positive controls were plated with tester strains TAPS, TA100, TA1535, TA97a and WP2 wrA (pKMIOl) in the presence and absence of rat liver S9 activation. All dose levels of test article, vehicle controls and positive controls were plated in triplicate. Plating and Scoring Procedures The test system was exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975)and updated by Maron and Ames (1983). On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and Oj % NaCI (w/v), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 /*M 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 imply sterile, deionized water produced by the Milli-Q Reagent Water System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/v) Oxoid Nutrient Broth No. 2 (dry powder). MA Study No. G96CF43.502011 - 10 Company Sanitized. Does. not contain TSCA CBI H-22051: Mutagenidty Testing in the Salmonella lyphlmurium and Eseherichhi coB Plate Incorporation Assay DuPont HLO.1997-00035 Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain, and activation, as described in detail in Microbiological Associates, Inc.'s Standard Operating Procedures. Test article dilutions were prepared immediately before use. One-half (0.5) milliliter of S9 or Sham mix, 100 fil of tester strain and 50 fd of vehicle or test article were added to 10 ml of molten selective top agar at 452C. After vortexing, the mixture was overlaid onto the surface of 25 ml of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 or 100 y\ 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 42C until colony counting could be conducted. The condition of the bacterial background lawn was evaluated for evidence of test article toxidty by using a dissectingmicroscope. Precipitate was evaluated by visual examination without magnification. Toxidty and degree of predpitation were scored relative to the vehicle control plate using the codes shown below._______ ::;::-:.: 'y'4vLKnM^.B^QSn^psy^0.^9.-'"' ^..jj,^,,.,^,ij.^.Aiii,,.^^^gy^uMyj^^^^^^^^ "w1 Nonnal Distinguishedby a healthy microcniony lawn. Distinmiished by a noticeable thinninz of the niicrocolony lawn and 2 Slightly Reduced possibly a slight increase in the size of the microcolonies compared to the vehicle control plate. Moderately Distinguished by a marked thinning of the niicrocolony lawn resulting in 3 Reduced a pronounced increase in the size of the microcolonies compared to the vehicle control olate. nuftin^uufhftd by qn extreme thinning of the microwlwy a" 'resulting 4 Severely in an increase in the size of the microcolonies compared to the vehicle Reduced control plate such that the microcdony lawn is visible to the unaided eye as isolated colonies. 5 Absent Distinguishedby a complete lack of any microcolony lawn over >90% of the plate. 6 Obscured by The background bacterial lawn cannot be accurately evaluated due to Precipitate microscopic test article precipitate. Distinguishedby precipitate on the plate that is visible to the naked eye NP Non-Interfering nit any precipitate particles detected by the automated colony counter Precipitate total less than 10% of the revcrtant colony count (e.g., <3 particles on a date with 30 rcvertants.) Distinguished by precipitate on the plate that is visible to the naked eye IP Interfering and any precipitate particles detected by the automated colony counter Precipitate exceed 10% of the revertant colony count (e.g., >3 particles on a plate with 30 revertants.) MA Study No. G96CF43.502011 11 Company Sanitized. Does not contain TSCA CR^ H-22051: Mutagcnidty Testing in the Salmonella typhunwium and Escherichia coll Plate Incorporation Auay DuPont HLO-W7-OQ035 Revertant colonies for a given tester strain and activation condition were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually. Evaluation of Results For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article. Data sets for strains TA1535 was judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA98, TA100, TA97a and WP2 wrA (pKMIOl) were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value. Criteria for a Valid Test The following criteria must be met for the mutagenicity assay to be considered valid. All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and ihe deletion in the uvrB gene. Cultures of tester strains TA98, TA100, TA97a and WP2 wrA (pKMIOl) must demonstrate the presence of the pKM 101 plasmid R-factor. All WP2 uvrA (pKMIOl) cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100,80. 240; TA1535,5 - 45; TA97a, 80 240; WP2 uvrA (pKMIOl), 150 - 380. To ensure that appropriate numbers of bacteria are plated, tester strain culture liters must be greater than or equal to 0.3xl09 cells/ml. The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels are required to evaluate assay data. A dose level is considered toxic if cue or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn. MA Study No. G96CF43.502011 - 12 - Company Sanitized. Does not contain TSCA CBI H-22Q51: Mutagenidty Testing b the Salmonella typhimwium and Eschaichia aril Plate Incorporation Auay DuPont H? 0-1997-00035 RESULTS AND DISCUSSION Solubility Test Water was selected as the solvent of choice based on solubilityof the test article and compatibility with the target cells. The test article was soluble in water at a concentration of approximately 500 mg/ml, the maximum concentration tested. Preliminary Toxiclty Assay The results of the preliminary toxicity assay are presented in Tables 1 through 5. In the preliminary toxidty assay, the maximum dose tested was 5000 fig per plate; this dose was achieved using a concentration of 100 mg/ml and a 50 fil plating aliquot. Neither precipitate nor appreciable toxidty was observed. Based on the findingsof the toxidty assay, the maximum dose plated in the mutagenidty assay was 5000 fig per plate. Mutagenicity Assay The results of. the mutagenidty assay are presented in Tables 6 through 25 and summarized in Tables 26 and 27. These data were generated in Experiments Bl and B2. Neither predpitate nor appredable. toxicity was observed. In Experiment Bl, the initial mutagenidty assay, no positiveresponses were observed with any of the tester strains in the presence and absence of S9 activation. In Experiment B2, the independentrepeat assay, no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. CONCLUSION All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay with an Independent Repeat Assay indicate that, under the conditions of this study, H-22051 did not cause a positive response with any of the tester strains in the presence and absence of Arodor-induced rat liver S9. MA Study No. G96CF43.502011 13 ConipanySan.tized.Doesnotcon^^^.. I H-22051: MutagpnidtyTesting in the Salmonella typhimwium mad Bscheridw coll Plate Incorporation Assay____DuPont HLO-1997-00035 REFERENCES Ames, B.N.. J. McCann and E. Yamasald (1975)Methods for Detecting Carcinogens and Mutagens with the Salmonella/MaaaaaSiw Microsome Mutagenicity Test, Mutation Research, 31:347-364. Brusick, DJ., Simmon, V.F., Rosenkranz, H.S., Ray, W.A.. and R.S. Stafford (1980) An Evaluation of the Eschericfua coli WP2 and WPA uvrA Reverse Mutation Assay. Mutation Research, 76:169-190. Green, M.H.L. and WJ. Muriel (1976) Mutagen testing using trp4' reversion in Eschericfua coli. Mutation Research 38:3-32. Levin, D.E., Yamasald, E. and B.N. Ames (1982)A new Salmonella tester strain, TA97, for the detection of firameshift mutagens. A run of cytosines as a mutational hot-spot. Mutation Research, 94:315-330. Maron, DM and B.N. Ames (1983) Revised Methods for the Salmonella Mutagenicity Test, Mutation Research, 113:173-215. Vogel, HJ. and D.M. Bonner (1956) Acetylornithinase otE. coli: Partial Purification and Some Properties. J. Biol. Chem., 218:97-106. MA Study No. G96CF43.502011 - t4 - _ompan^anmze_Doe_Tot_ontair^BCA^BI H-22051: Mutagenidty Testing u the Salmoiutta fyphi^wiwn and Eschctichia coll Plate Incorporation Auay DuPont HLO-1997400SS Salmonella Hutagenicity Assay Preliminary Toxieity Aay Table 1 Test Article Id Study Number Experiment No. Date Plated Counted by Vehicle Plating Aliquot H-22051 G96CF43.502011 Al 12/03/96 hand water 50 pi Test Article Concentration /jg per plate TA98 Vtth S9 Aeration Revcrtants Background per plate Code* Without Activation Revertanf Background per plate Code* Vehicle 22 11 6.7 10 33 67 100 333 667 1000 3333 5000 16 1 16 1 19 1 14 1 12 1 23 1 20 1 16 1 8 1 17 1 10 1 15 1 9 1 15 1 8 1 10 1 10 1 13 1 12 1 14 1 'Bickcrouad baefri*! valuation coda 1-HoioaL 2SU(btl.y raducad *Eztruly raducad S>Aiirnt BP-S6n-Intar(ring Pracipifcat* Mkd*rataly raduead (Hibicurad by praciplbata IP'Infrtarlni Ptaclpiff MA Study No. G96CF43.5020 II 15Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing b the Salmomlla lyphSmwium and EscheHchlu cod Plate Incorporation Auay DuPont HLO-1997-00035 Salmonella Mutageniclty Assay Preliminary Toxicity Assay Table 2 Teat Article Id Study Number Experiment No. Date Plated Counted by Vehicle Placing Aliquot H-22051 G96CF43.5 02011 A1 12/03/96 machine water 50 pi Teat Article Concentration jig per plate TA ^00 With S9 Activation Revertants Background per plate Code* Without Activation Revertants Background per plate Code" Vehicle 167 1 165 1 6.7 10 33 " 67 100 333 667 1000 3333 5000 162 1 198 1 162 1 185 1 191 1 169 1 154 1 172 1 162 1 134 1 143 1 149 1 167 1 147 1 163 1 156 1 165 1 155 1 160 1 159 1 'Background bct*xll valuation cod* rionul. ^Extcfly r*ducd 2>SUhtly i*ducd )>Abeafc HP-lIoo-IntTfTlir Frclplft* 3^kduafLy rduod 6'Obicufd by priclpitat* IP-InfctTini Pfeipltaf MA Study No. G96CF43.502011 16Company Sanitizec H-22051: Mutagenidty Telling in the Salmonella typhlmurium and Escherichia call Plate Incorporation Aasay DuPont HLp.1997-00035 Salmonella Hutagenicity Assay Preliminary Toxicity Assay Table 3 Test Article Id Study Number Experiment No. Date Plated Counted by Vehicle Plating Aliquot H-22051 G96CF43.502011 Al 12/03/96 hand water 50 fit Test Article Concentration jig per plate TA1535 V4th g9 Ae^vatt9n Revertants Background per plate Code* Without Activation Revertants Background per plate Code* Vehicle 14 13 6.7 10 33 67 100 333 667 1000 333^ 5000 12 1 11 1 10 1 8 1 8 1 8 1 7 1 10 1 10 1 8 1 11 1 7 1 12 1 7 1 7 1 8 1 4 1 11 1 10 1 9 1 *B*ckBxouad b*ot*xlal. vkluakloo cod* l-Koaial 2-SUghtly i*ducd *>Extfa*ly r*duc*d S^Urut IIP>Hon-Int*rt*tlnc Pcflpltat* 3^1od*ffly xdued e*4bcui*d by prcipift* XP-Infttdlir Prclpl.tat* MA Study No. G96CF43.502011 17- Company Sanitized. Does not contain TSCA C& H-22051; Mutifonldty Tcrting b the Salmwulla OpMrnurium ad Escherichia coll Plate Incorporation A--ay DuPont HLO-1997-00035 Salmonella Hutagenicity Assay Preliminary Toxicity Assay Table 4 Test Arcicle Id Study Number Experiment No. Date Plated Counted by Vehicle Plating Aliquot H-22051 G96CF43.502011 Al 12/03/96 machine water 50 pi Test Article Concentration jig per plate TA97a With S9 Activation Revertants Background per plate Code* Without Activation RevertaT'ts Background per pla. - Code* Vehicle 179 130 6.7 10 33 67 100 333 667 1000 3333 5000 137 1 ISO 1 148 1 151 1 168 1 152 1 172 1 149 1 160 1 121 1 131 1 109 1 121 1 131 1 104 1 127 1 104 1 124 1 132 1 110 1 "Background bacterial valuation cod* l-Koiaal. 2-SU(htly xaducad 4ExtrMly raducad S'Abcnfc BP-Bon-Intartatiir Precipitate 3'Modeiately reducad f^Obtcurad by ptecipift* IP-Interterim Prectplttt* MA Study No. G96CF43.502011 18- ,...1.1,,,.,.,,,^^ H-22Q51: Mutogenidty Testing in the Salmonella typhimuriwn and EicheHchIa cott Plate Incorporatioa Amy DuPou HLO-1997-OOD35 ^alaonella Hutageniclty Asaay Prelialnary Toxtclty Asaay Table S Test Article Id Study Number Experlaent No. Date Plated Counted by Vehicle Plating Aliquot H-22051 G96CF43.502011 Al 12/03/96 machine water 50 fil Test Article Concentration /jg per plate WP2 uvrA With S9 Activation Revertants Background per place Code* /nIMlOn Without Activation Revertants Background per plate Code* Vehicle 160 135 6.7 10 33 67 100 333 667 1000 3333 5000 140 1 153 1 148 1 155 1 113 1 168 1 153 1 173 1 136 1 ' 144 1 152 1 152 1 170 1 135 1 117 1 171 1 145 1 160 1 170 1 140 1 'Btcktxound bacfccritL valiutioo cod* l-Hoia*L 2-SlichtLy reduced 4>Exfcra*ly fduccd S"Abuit HP-Ban-InfcTfTinf Frcipift* ' 3^ted*rat*ly r*due*d O'Obieufd by pfcipitabi IP>IntTfrinc Precipitate MA Study No. G96CF43.502011 19. Company Sanitized. Does not contain TSCA CBI H-22051: Mulafenlcity Testing in the Salmonella typhimwium aad Escherichia eoU Plate Incorporation Auay DuPont HUM997-OOQ35 Salmonella Mufgenlcity Aasay Table 6 Test Article Id Study Nuaber Strain Liver Hicrosomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA98 None water 50 pi Experiment No : Bl Cella Seeded Date Plated : 1.0 X l0* : 12/12/96 Counted by : hand Concentration MB Pr plate Plate Revertanf Background Average Standard Nuaber per plate Code* Revertants Deviation Vehicle 01 02 03 27 1 29 1 18 1 25 6 100 01 26 1 02 32 1 03 46 1 . 35 , 10 333 01 16 1 02 20 1 03 12 1 16 4 1000 01 25 1 02 22 1 03 15 1 21 5 3333 01 20 1 02 12 1 03 20 1 17 5 5000 01 20 1 02 20 1 03 19 1 20 1 Positive Control 2-nitrofluorene 1.0 ^<g per plate11 01 100 1 02 123 1 03 172 1 132 37 *B*ck|round bactri*l valuation cod* l-Hornrl Z-Sllthtly 4"ExtcB*ly reduced S>Abftit NP"Hon-Infttrlnt PrcclpitaL* ^otitiv control pliL** MT* oichin* counttd r*ducd 3^tedratly rduod 6^&curd by prcipib*fc* IP-Intrfrln Pr*clpit*&* MA Study No. G96CF43.502011 20- ^saaatthSaataa^auu^wM^w^ H-22Q51: Mufgenicity Tciting in the Salmomlta lyphimwium and Escherichia coS Plate Incorporation Auay DuPoni HLO-1997-00035 Salmonella MuCagenlclty Aay Table 7 Tet Article Id Study Number Strain Liver Hicrosomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA98 Rat liver S9 water SO itl Experiment No Cells Seeded Date Plated Counted by Bl 1.0 X 10" 12/12/96 hand Concentration fig per plate Vehicle Plate Number 01 02 03 Reverfcanf per plate 25 17 15 Background Average Code* Revertants 19 Standard Deviation 100 01 28 02 24 03 27 26 333 01 26 02 27 03 29 27 1000 01 33 02 29 03 35 32 3333 01 33 02 25 03 39 32 5000 01 24 02 31 03 25 27 Positive Control 2-amlnoanthracene 1.0 fig per plate1' 01 719 1 02 702 1 03 782 1 734 42 'Background b*ctrll (valuation cod* 1-Horarl 2>Sll(hfcly 4~Ex&r*orXy reduced i'Abriit NP-llon-Inftfrin Prcipitf ''Potlttv control pL*tc wr urchin* eountd reduced 3'ttodr*t*ly reduced 6"0bicur*d by pfdplffc* IPIntrfTlng Pfdplfcif MA Study No. G96CF43.502011 21 - Company Sanitized. Does not contain TSCA C81 H-22Q51: Muugenidty Testing b the SalmoMUa typhimwium and Escherichia colt Plate Incorporation Amy DuPont HLO-1997-00035 Sfilaonella Hutageniclty Aaaay Table 8 Teifc Article Id Study Number Strain Liver Hicrosoaes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA100 None water 50 jil Experiasnt No Cell* Seeded Date Plated Counted by Bl 0.8 X 10 12/12/96 machine Concentration fig per plate Vehicle Plate Number 01 02 03 Revertants per plate 144 159 131 Background Average Code* Revertanf U'} Standard Deviation 14 100 01 116 02 156 03 127 133 21 333 01 128 02 150 03 124 134 14 1000 3333 01 147 02 154 03 126 01 121 02 128 03 138 142 15 129 5000 01 114 02 135 03 118 122 11 Positive Control sodium azide 1.0 /ig per plate 01 736 1 02 762 1 03 743 1 747 13 'Backcroimd b*ct*ri*l valuation cod* 1-Honul 4-Ectr----l.y reduced 231ihtly rduod i'Abcnfc KP-Roa-Inkerfrin( rrclpltct* 3>4fadr*fly reduced 6'Obicurad by preclpifcat* IP-Intrfriir Pr*clpibf MA .Study No. G96CF43.502011 22Company Sanitized. Does nnt r.nnt.in T.^A PR. H-2205I: Mutagenicity Testing in the Salmonella typhmwrium and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-00035 Salmonella Mutagenicity Assay Table 9 Test Article Id Study Number Strain Liver Microsooes Vehicle Plating Aliquot H.22051 G96CF43.S02011 TA100 Rat liver S9 water SO ^1 Experiment No : Bl Cells Seeded : 0.8 X 108 Date Plated : 12/12/96 Counted by : machine Concentration ^g per place Vehicle Plate Number 01 02 03 Revertants per plate 153 124 148 Background Average Code* Revertants 1 1 1 142 Standard Deviation 16 100 01 167 1 02 129 1 03 142 1 146 19 333 01 160 1 02 141 1 03 154 1 152 10 1000 01 148 1 02 166 1 03 141 1 152 13 3333 01 136 1 02 122 1 03 150 1 136 14 5000 01 148 1 02 161 1 03 147 1 152 8 Positive Control 2-aminoanthracene 1.0 "ig per plate 01 761 1 02 778 1 03 806 1 7a2 23 'Background b*c&*ri*l. valuation cod* l-Hc'nn*! 2-Sl.lght.ly *-Extrnrly reduced S"Abnfc HP-Hon-Infr firing Precipitate rduod 3-ModrBtl.y rducd 6*0b*curd ay precipitate IP-Infcrtrlng Pnclpttal* MA Study No. G96CF43.S02011 23 Company Sanitized. Does not contain TSCA CB1 H-22051: Mutagenidty Tecdog in (he Salmonella typhimurtum and Escherichia coti Plate Incorporation Auay DuPont HLO-1997-00035 Salmonella Hutagenlcity Assay Table 10 Test Article Id Study Number Strain Liver Hicrosomes Vehicle Plating Aliquot H-22051 G96CF43 .502011 TA1535 None water 50 pi Experiment No : Bl Cells Seeded Date Plated : 3.0 X 10' : 12/12/96 Counted by : hand Concentration Plate /ig per plate Number Vehicle 01 02 03 Revertants per plate 8 13 11 Background Average Code* Revertants 1 1 1 11 Standard Deviation 3 100 01 12 1 02 11 1 03 6 1 10 3 333 01 17 1 02 6 1 03 11 1 11 6 1000 01 13 1 02 10 1 03 8 1 10 3 3333 01 11 1 02 11 1 03 12 1 11 1 5000 01 11 1 02 13 1 03 11 1 12 1 Positive Control sodium azide 1.0 /ig per plate1' 01 556 I 02 535 1 03 559 1 550 13 *BBck|round brcfriil vl.uBtion cod* 1-lfofl 4-Extxjokly rdde*d Z-SUghtly S-Abnt HP-llon-IntTfTint PfcipUt ^Potltiv control plat** r machine countad reduced 3^tod*rafly reduced 6^}llicurd by precipifL* IP-Intrtrin Pxeclpltat* MA Study No. G96CF43.502011 24. Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenidty Testing in the Salmonella lyphunwium and Escherichia coli Plate Incorporation Aay____DuPont HLO-1997-00035 Salmonella Mutagenicity Asaay Table 11 Test Article Id Study Number Strain Liver Hicrosoaes Vehicle Plating Aliquot H-22051 G96CF43.S02011 TA1S35 Rat liver S9 water 50 pi Experiment No : Bl Celli Seeded Date Plated : 3.0 X 10' : 12/12/96 Counted by : hand Concentration /jg per plate Plate Number Revertants per plate Background Average Code* Revertants Standard Deviation Vehicle 01 02 03 14 1 13 I 12 1 13 1 100 01 17 1 02 10 1 03 11 1 13 4 333 01 21 I 02 16 1 03 14 1 17 4 xOOO 01 17 1 02 25 1 03 20 1 21 4 3333 01 13 1 02 11 1 03 12 1 12 1 5000 01 16 1 02 14 1 03 16 1 15 1 Positive Control 2-aminoanthracene 1.0 /*g per plate*" 01 91 1 02 116 1 03 128 1 112 19 *BckBround bacterial valuation cod* wf rMonrl 2-Sllihtly 4-Ixtrxuly reduced 5-Abnt HP-on-Intttrln Pfdplft* ''Poiltiv control plat** oachin* eounfd reduced 3^odttly reduced 6-0bcurd by prciplff IP-Inttttln Pfcipiff MA Study. No. G96CF43.502011 - 25 - Company Sanitized. Does not con^.n T.^A pm | H-22051: Mutagenicity Testing in the Salmonella typhimurium and Escherichia coK Plate Incorporation AsMy DuPont HLO.1997-00035 Salmonella Hutagenicity Assay Table 12 Test Article Id Study Number Strain Liver Hicrosomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA97a None water 50 pi Experiment No Cells Seeded Date Plated Counted by Bl 3.1 X 10 12/12/96 machine Concentration ^g per plate Plate Revertants Background Average Standard Number per plate Code* Revertants Deviation Vehicle 01 135 02 150 03 157 147 11 100 01 135 02 136 03 129 133 333 01 190 02 139 03 163 164 26 1000 01 162 02 166 03 173 167 3333 01 196 02 160 03 192 183 20 5000 01 180 02 188 03 193 187 Positive Control 9-aminoacridine 100 /Jg per plate 01 1934 I 02 2118 1 03 1682 1 1911 219 "Background bacfrl*! valuifclon cod* 1-Normal 2-SUghtly r*ducd 4-Extrtoely nductd S-Abrnt HP-Hon-IntTfirIng Prclpitf 3ModrItaly reduced 6"0b*cufd by pr*clpibat IP-IntTtrlng Pr*clplfc*t* MA Study No. G96CF43.502011 26Company Sanitized. Does not contain TSCA CBI H.22Q51: Mutagenidly Tectiog in Ike Salmonella typhinwrium and EicAeHchia coli Plate Incorporation Auay DuPont HLO.1997-00035 Salmonella Hutagenicity Asaay Table 13 Test Article Id Study Number Strain Liver Hicrosomes Vehicle Plating Aliquot H.22051 G96CF43 .502011 TA97a Rat liver S9 water SO /il Experiment No : B1 Cells Seeded Date Plated 3.1 X 108 12/12/96 Counted by machine Concentration /<g per plate Plate Number ReverbcUlf per plate Background Average Code* Revertants Standard Deviation Vehicle 01 02 03 235 1 230 1 205 I 223 16 100 01 220 1 02 240 1 03 254 1 238 17 333 01 206 1 02 233 1 03 146 1 194 44 1000 01 215 1 02 213 1 03 198 1 209 9 3333 01 192 1 02 221 1 03 219 1 211 16 5000 01 217 1 02 229 1 03 234 1 227 9 Positive Control 2-aminoanthracene 2.0 /ig per plate 01 1230 1 02 1481 1 03 1662 1 1458 217 "B*ck(round b*etil*l valuation cod* rHonul 2-Sllghtl.y reduced 4-Eltr-Btly rduc*d 5-Abnt HP--Bon-IntTtTl.nt Pnclpltif 3'*fadr*t*ly rducd 6-0bicur*d by prciplbat* IP-Infrfrln Prclplttt* MA Study No. G96CF43.502011 27- Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella lyphimwium and EscheHcUa coll Plate Incorporation Assay DuPont HLO.1997-00035 E. coli Hutagenicity Aray Table 16 Test Article Id Study Number Strain Liver Hicrosomes Vehicle Plating Aliquot H-22051 G96CF43 .502011 WP2 uvrA (pKMIOl) None water 50 fil Experiment No : B1 Cell* Seeded : 7.6 X 10' Date Plated : 12/12/96 Counted by : machine Concentration ftg per plate Vehicle Plate Number 01 02 03 Rev ertantfi per plate 232 201 194 Baekground Average Code* Revertants 1 1 1 209 Standard Deviation 20 100 01 258 1 02 235 1 03 212 1 235 23 333 01 267 1 02 250 1 03 251 1 256 10 . 1000 01 236 1 02 279 1 03 271 1 262 23 3333 01 244 1 02 256 1 03 305 1 268 32 5000 01 273 1 02 313 1 03 272 1 286 23 Positive Control methyl methanesulfonate 1000 /ig per plate 01 1829 1 02 1771 1 03 1792 1 1797 29 "Backfround bcfc*rl*l valuation cod* 1'Honul 2-31lhtly fducad 4>Extcaly fducad !"Abmfc HP-Bon-Infcrfrln< Pr*cipitf . 3^k>drfly r*duc*d . 6^&lcurd by pfdpltaf IP-Intrfrin Pocipltjt* H-22051; Mutagenicity Testing in the Salmonella typhimurium and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-OOOa5 E. coli Mutagenicity Assay Table 15 Test Article Id Study {lumber Strain Liver Microsomes Vehicle Plating Aliquot H-22051 G96CF43.502011 WP2 uvrA (pKMIOl) Rat liver S9 water 50 pi Experiment No Cells Seeded Date Plated Counted by Bl 7.6 X 10" 12/12/96 machine Concentration ft6 per plate Vehicle Plate Number 01 02 03 Revertants per plate 242 276 222 Background Average Code* Revertants 247 Standard Deviation 27 100 01 254 02 294 03 263 270 21 333 01 315 02 268 03 265 283 28 1000 01 305 02 286 03 310 300 13 3333 01 285 02 303 03 313 300 14 5000 01 296 02 303 03 293 297 Positive Control 2-aminoanthracene 10 fig per plate 01 1664 1 02 1.591 1 03 1410 1 1555 131 'Background bacterial (valuation cod* 1-Hormal 2-Slightly caducad 4-Exbmialy raducad S-Abimt NP-Hon-Intarfarini Pracipitata 3'Modarataly raducad 6-0bcurad by pracipitata IMntartaring Pracipitata MA Study No. G96CF43.502011 29Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella lyphimunum and Eschenchia coti Plate Incorporation Assay DuPont HLO-1997-OOQ35 Salmonella Kutagenlcity Assay Table 16 Test Article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-22051 G96CF43.S02011 TA98 None water SO pi Experiment No Cells Seeded Date Plated Counted by B2 1.1 X 10" 01/03/97 hand Concentration Itg per plate Vehicle Plate Number 01 02 03 Revertants per plate 14 15 16 Background Average Code* Revertants 15 Standard Deviation 100 01 20 02 18 03 16 18 333 01 11 02 11 03 5 1000 01 7 02 10 03 16 11 3333 01 7 02 6 03 11 5000 01 4 02 2 03 11 Positive C>ntrol 2-nitrofluorene 1.0 /g per plate1' 01 138 1 02 152 1 03 122 1 137 15 'Background bacterial valuation cod* 1-Honnal 2-Slightly 4'Extramal.y raducad S'Abiant HP'Hon-Intartailna Pxacipltata ''Foaitlva control platas wara aachina counted raducad 3^todarataly raducad 6-Obicurad by pracipitaLe IP-IntartarIng Pracipltata MA Study No. G96CF43.502011 30Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenidty Testing in the Salmonella lyphlmuriwn and EscheHclua coli Plate Incorporation Assay DuPont HLO-1997.00035 Salmonella Hutagenicity Assay Table 17 Test Article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA98 Rat liver S9 water 50 ill Experiment No Cells Seeded Date Plated Counted by B2 1.1 X 108 01/03/97 hand Concentration /ig per plate Vehicle Plate Number 01 02 03 Revertants per plate 19 16 25 Background Average Code* Revertants 20 Standard Deviation 100 01 18 02 25 03 21 21 4 333 01 26 02 19 03 35 27 1000 01 20 02 13 03 16 16 3333 01 18 02 13 03 23 18 5000 01 6 02 7 03 14 4 Positive Control 2-aminoanthracene 1.0 tig per plate1' 01 746 1 02 824 1 03 656 1 742 84 'Background bicfrlal valuation cod* 1-KonuL 2-Slithtly *"Extraly reduced l"Abcnt wr HP-Bon-Intrfrln frclpift* 'PotUiv control pl*&* chin* counttd r*duc*d 34<od>r*fc*l.y r*duc*d 6-0bcur*d by pfdpiff IP-Intrfrlng Praclplft* MA Study No. G96CF43.502011 31 - Company Sanitized Do^ nnf r-nn^in T^A r^. H-22051: Mutagenicity Testing in the Salmonella typhmwrium and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-00035 Salmonella Mutagenicity Assay Table 18 Test Article Id Study Number Strain Liver Hicrosomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA100 None water 50 1*1 Experiment No : B2 Cells Seeded : 1.5 X 10" Date Plated : 01/03/97 Counted by : machine Concentration fig per plate Vehicle .Plate Number 01 02 03 Revertants per plate 134 134 93 Background Average Code* Revertants I 1 I 120 Standard Deviation 24 100 01 121 I 02 131 1 03 82 1 111 26 333 01 121 1 02 116 1 03 112 I 116 5 1000 01 104 I 02 100 I 03 107 I 104 4 3333 01 134 1 02 117 1 03 125 1 125 9 5000 01 103 1 02 87 1 03 99 1 96 8 Positive Control sodium azide 1.0 tig per plate 01 699 1 02 696 1 03 660 1 685 22 'Background b*cfrl*l vuluatlon code 1-Hormal 2'SlighLLy reduced t-Eztriwly r*duc*d S'Absmfc HP-Hon-lBtTtrlng Precipitate 3-ttodstabel.y reduced 6-Obscufd by preclpitat* IP-InftfMing Prclpitaf MA Study No. G96CF43.502011 32 - Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella lyphimumm and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-00035 Salmonella Mutageni.ci.ty Assay Table 19 Test Article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA100 Rat liver S9 water 50 /il Experiment No : B2 Cells Seeded Date Placed : 1.5 X 106 : 01/03/97 Counted by ; machine Concentration fig per plate Plate Number Reverfcanfcs per plate Background Average Code* Revertants Standard Deviation Vehicle 01 02 03 139 1 117 1 121 1 126 12 100 01 129 1 02 142 1 03 114 1 128 14 333 01 150 1 02 117 1 03 132 1 133 17 1000 01 147 1 02 133 1 03 136 1 139 7 3333 01 141 1 02 132 1 03 127 1 133 7 5000 01 134 1 02 137 1 03 156 1 142 12 Positive Control 2-aminoanthracene 1.0 ftg per plate 01 1183 1 02 1112 1 03 1019 1 1105 82 Background bacterial avaLuafclon coda 1-Korreal 2-SUBhtly 4'Extramaly raducad 5-Ablant HP-Hon-Intarfaring FraclpiLaLa raducad 3-Modratly raducad 6-Obacurad by pracipitata Ir-Intarfaring Pracipitat* MA Study No. G96CF43.502011 33Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella typhimwium and Escherichia coli Plate Incorporation Assay____DuPont HLO-1997-00035 Salmonella Hutagenicity Assay Table 20 Test Article Id Study Number Strain Liver Microsomes Vehicle Placing Aliquot H-22051 G96CF43.502011 TA1535 None water 50 fil Experiment No : B2 Cells Seeded Date Plated : 2.8 X 10" : 01/03/97 Counted by : hand Concentration ^ig per plate Plate Revertants Background Average .Standard Number per plate Code* Revertants Deviation Vehicle 01 02 03 11 1 15 1 14 1 13 2 100 01 11 1 02 18 1 03 14 1 333 1000 3333 5000 Positive 01 02 03 01 02 03 01 02 03 01 02 03 Control sodium 01 02 03 8 1 11 1 8811 10 1 9 1 11 1 6211 11 1 9 1 6 1 azide 1.0 ^g per 552 1 549 1 524 1 plate1' 14 4 9 2 9 1 6 5 9 3 542 15 "Background trcfc*il*l vhrtlon cod* l"Honul 2-SUht.ly (Extremely rduc*d S"Abrnfc HP-Hon-IntTfTing Precipltat* ^oslttv control plaf --f machin* countid reduced 3-ModTtttl.y fduod 6-0bcurd by pncipitaf IP-InfrtTtng Prcipltat MA Study No. G96CF43.502011 - 34 Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in (he Salmonella typhimuriwn and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-00035 Salmonella Hutagenicicy Assay Table 21 Test Article Id Study Number Strain Liver Nicrosoaes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA1S3S Rat liver S9 water 50 /il Experiment No : B2 Cells Seeded : 2.8 X 10" Date Plated : 01/03/97 Counted by : hand Concentration jig per plate Vehicle Plate Number 01 02 03 Revertants per plate 12 11 9 Background Average Code* Revertants 1 1 1 11 Standard Deviation 2 100 01 15 1 02 11 1 9 1 03 333 01 12 1 02 20 1 03 17 1 12 3 16 4 1000 01 18 1 02 10 1 03 13 1 14 4 3333 5000 01 11 1 8 1 02 03 16 1 01 12 1 02 18 1 03 10 1 12 4 1.3 4 Positive Control 2-amlnoanthracene 1.0 ^g per plate** 01 97 1 02 102 1 03 103 1 101 3 "Background bacterial valuation cod* 1-Norarl 2-SUghtly 4-Eztreooly reduced 5-Abient HP-Bon-Interfering Precipitate *'Poltlve control plate* were aechlne counted reduced 3-Modrtly reduced 6-0bcurd by precipitate IP-Interfering Precipitate MA Study No. G96CF43.502011 - 35 - Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella typhimiuium cad Escherichia coli Plate Incorporation Assay DuPont HLO.1997-00035 Salmonella Mutagenicity Asaay Table 22 Tesc Article I d Study Number Strain Liver Hicrosomes Vehicle Plating Aliquot H-22051 G96CF43.502011 TA97a None water 50 ^1 Experiment No : B2 Cells Seeded : 1.3 X 10' Date Plated : 01/03/97 Counted by : machine Concentration Itg per plate Plate Number Revertants per plate Background Average Code* Revertants Standard Deviation Vehicle 01 02 03 116 1 104 1 98 1 106 9 100 01 146 1 02 126 1 03 114 1 129 16 333 01 125 1 02 154 1 03 112 1 130 22 1000 01 127 1 02 132 1 03 121 1 127 6 3333 01 109 1 02 132 1. 03 130 1 124 13 5000 01 127 1 02 130 1 03 142 1 133 8 Positive Control 9-ami.noacrldine 100 ^g per plate 01 2130 I 02 2283 I 03 876 1 1763 772 'Backfiound bccfflil valuation cod* rHonnal. 2-Sl.lghtl.y r*duc*d i-Extfnrl.y fducd 5-Abfnt HPHon-InfcfTing Prcipl.tkf 3-tlodratl.y r*duc*d S^lbicund by prccipiff IPIntrrlng Pr*clpit*f MA Study No. G96CF43.502011 .36. Company Sanitized. Dc ^IC^^^ H-22051; Mutagenidty Testing in the Salmonella typhlmuriwn and Escherichia coil Plate Incorporation Assay DuPont HLO-1997-00035 Salmonella Mutagenidty Assay Table 23 Test Article Id Study Number Strain Liver Microsomes Vehicle Plating Aliquot H-22051 G96CF43 .502011 TA97a Flat liver S9 water 50 pi Experiment (Io B2 Cells Seeded . 1.3 X 10" Date Placed ^ 01/03/97 Counted by : machine Concentration lig per plate Plate Revertants Background Average Standard Number per plate Code* Revertants Deviation Vehicle 01 02 03 147 1 155 1 142 1 148 7 100 01 146 1 02 146 1 03 159 1 150 8 333 01 140 1 02 168 1 03 162 1 157 15 1000 01 163 1 02 163 1 03 180 1 169 10 3333 01 154 1 02 155 1 03 160 1 156 3 5000 01 145 1 02 146 1 03 165 1 152 11 Positive Control 2-amlnoanthracene 2.0 fig per plate 01 2068 1 02 2052 1 03 2126 1 2082 39 'Background bacterial valuation cod* l-floriBJl Z'Slifhtly r*diicd 4>ExtrwLy r*ducd i'Abcnt Hi'-Hon-InfrtTint Pfcipiff 3"Modrfly reduced 6-0beurd by prclpltt It"IntTf*rin Pfclplffc* MA Study No. G96CF43.502011 37Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella typhimwium and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-00035 E. coli Hutagenlcifcy Assay Table 24 Test Article Id Study Number Strain Liver Microsomes Vehicle Placing Aliquot H-22051 G96CF43.502011 VP2 uvrA (pKMIOl) None water 50 ftl Experiment No Cells Seeded Date Plated Counted by B2 4.5 X 10 01/03/97 machine Concentration ^g per plate Plate Revertants Background Average Standard Number per plate Code* Revertants Deviation Vehicle 01 02 03 164 1 145 1 150 1 153 10 100 01 192 1 02 144 1 03 175 1 170 24 333 01 182 1 02 194 1 03 193 1 190 ^ 1000 01 169 1 02 158 1 03 199 1 175 21 3333 01 158 1 02 180 1 03 212 1 183 27 5000 01 205 1 02 146 1 03 170 1 174 30 Positive Control methyl me thanesulfonate 1000 /ig per plate 01 1501 1 02 1585 1 03 1500 1 1529 49 'Background b*ct*rlX vluation cod* 1-Horaal 2-SUht.ly rduccd *Extifly r*duc*d S-Abcnt HP-!lon-lDt,rTin Pr*clpltat* J-Modtffly rducd 6"0bcufd by pr*cipiL*L IP-IntTfTing Pnclpltaf MA Study No. G96CF43.502011 .38- Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenidty Testing in the Salmonella typhlmwium und Escherichia coli Plate Incorporation Assay DuPont HLO.1997-00035 E. coli Mutagenidty Assay Table 25 Test Article Id Study Number Strain Liver Hicrosomes Vehicle Plating Aliquot H-22051 G96CF43.502011 WP2 uvrA (pKMIOl) Rat liver S9 water 50 MI Experiment No Cells Seeded Date Plated Counted by B2 4.5 X 10' 01/03/97 machine Concentration ng per plate Plate Revertanfcs Background Average Standard Number per plate Code* Revertants Deviation Vehicle 01 02 03 164 1 171 1 160 1 165 6 100 01 182 1 02 165 1 03 171 1 173 9 333 01 203 1 02 185 1 03 229 1 206 22 1000 01 174 1 02 173 1 03 207 1 185 19 3333 01 187 1 02 199 1 03 212 1 199 13 5000 01 251 1 02 202 1 03 192 1 215 32 Positive Control 2-aminoanthracene 10 /ig per plate 01 1668 1 02 1690 1 03 1714 1 1691 23 ^ckftound bacfriil vluitlon cod* 1-Hororl Z-SUghtly rducgd 4-ExfcrwrXy reduced 5-Abnt NF-Ilon-IntKtTing Pfclplbaf 3-ttodrafly raduod 6-Obicund by pr*clplt*t* IP-Infcrtring Prjcipi&af MA Study No. G96CF43.502011 39- Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella typhimurium and Eschenchia coS Plate Incorporation Assay DuPont HLO-IW7-00035 Salmonella/E. coli Mutagenicity Assay Summary of Results Table 26 Test Article Id Study Number H-22051 C96CF43.502011 Experiment No : Bl Average Revertants Per Liver Hicrosomes: None Dose (^g) TA98 TA100 Plate t Standard Deviation TA1535 TA97a UP2 uvrA (pKMIOl) 0.0 100 333 1000 3333 5000 Pos 25 6 145 14 11 3 147 11 209 20 35 10 133 t 21 10 3 133 4 235 23 16 4 134 14 11 6 164 26 256 10 21 5 142 15 10 3 167 6 262 23 17 5 129 9 11 1 183 20 268 32 20 1 122 11 12 1 187 7 286 23 132 37 747 t 13 550 13 1911 219 1797 29 Liver Hicrosomes: Dose Dig) Rat liver S9 TA98 TA100 TA1535 TA97a WP2 uvrA (pKMIOl) 0.0 100 333 1000 3333 5000 Pos 19 5 142 t 16 13 1 223 16 247 27 26 2 146 19 13 4 238 17 270 21 27 2 152 10 17 4 194 44 283 28 32 3 152 13 21 ^ 4 209 9 300 13 32 7 136 14 12 1 211 16 300 14 27 4 152 8 15 1 227 + 9 297 5 734 42 782 23 112 19 1458 + 217 1555 131 0.0 - Vchlcl* pitting aliquot of 50 I Po Poiitiv Control concm&catiOM picltitd in Hafcuiala and Mtthoda fctlon. MA Study No. G96CF43.502011 40Company Sanitized. Does not contain TSCA CBI H-22U51: Mulugcniuily Testing in the SMmonella lyphiinurium and Eschenchiu call Plate Incorporation Assay DuPont HLO-1997-00035 Salmonella/E. coll Mutageniclty tssay Summary of Results Table 27 Tea*, Article Id Study Number H-22051 G96CF43.502011 Experiment No : B2 Average Revertants Per Liver Microsoroes: None Dose (ng) TA98 TAlOO Plate Standard Deviation TA1535 TA97a WP2 uvrA (pKMIOl) 0.0 100 333 1000 32:,3 5000 Pos 15 1 120 24 13 2 106 9 153 10 18 2 111 26 14 4 129 16 170 24 9 3 116 5 11 5 104 4 9 2 i30 t 22 190 7 9 1 127 1 6 1.75 21 8 3 125 9 6 5 124 13 183 27 6 5 96 8 9 3 133 t 8 174 30 137 15 685 22 542 15 1763 772 1529 49 Liver Microsomes : Dose (*g) Rat liver S9 TA98 TAIUO TA1535 TA97a WP2 uvrA (pKMIOl) 0.0 100 333 1000 3333 5000 Pos 20 3 126 12 11 21 t 4 128 14 12 27 8 133 17 16 16 4 139 ^ 7 14 , 18 t 5 133 ^ 7 12 9 t 4 142 12 13 742 84 1105 82 101 2 148 7 165 6 3 150 8 173 t 9 4 157 15 206 22 4 169 t 10 185 19 4 156 3 199 13 4 152 .+ 11 215 32 3 2082 39 1^91 t 23 0.0 - Vthlcl* pLktIna iLiq-iot of SO nl Poa - Poiltiv Control conc*n&r*tlon pcl{lod in HaLirlaLi and Hithod* rction. MA Study No. G96GF43.5020;. 41 - Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella typliimwium and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-00035 APPENDIX I Historical Control Data MA Study No. G96CF43.5U,-01 i - 42 - Company Sanitized. Does not contain TSCA CBI H-22051: Mutagcnicity Testing in ihe Salmonella typhimuiiuw and Escherichia coli Plate Incorporation Assay DuPoni HLO-1997-00035 Historical Negative and Positive Control Values 1993 -1995 revertants per plate Activation Strain Control Mean None SD Mb Max Mean Rat Liver SD Min Max TA98 Nsg Pos 20 7 316 185 4 52 17 3045 27 8 1091 538 4 65 94 3506 TA100 Neg Pos 137 25 67 268 672 194 100 2054 156 27 1133 510 50 323 136 3682 TA1535 Neg Pos 12 5 503 206 1 53 17 3704 13 5 128 130 0 46 18 2153 TA97 and TA97a Neg Pos 131 24 67 176 1019 534 382 2541 174 35 101 2(>2 1024 4()() 376 1S42 WP2 uvrA (pKMIOl) Neg Pos 242 57 91 420 1918 506 557 3072 268 01 1469 4<i9 113 425 540 2668 SD=slandard deviation; Min= minimum value; Max= maximum value; Neg = negative control (including but not limited to dcionizcd water, dimethylsulfoxidc, elhiinol and acetone); Pos= positive control MA Study No. G%CF43.502011 43- Company Sanitized. Does not contain TSCA CBI ll-;2l)51: Mnl.ii;L'nn-ily'IV-sliny in llic .^iiliiinm-Uu lyiiliiiiiitriuin ;ind Esi-lu'rirliiti ciili Flalc Incorpor.iliiin Assay Dul'niil lll.(-l"l^-l)lHll!i APPENDIX II Study Protocol M \ SliiiK N'.i (;'M.C1.H.5()L'IH I - 44 Company Sanitized. Does not contain TSCA CBI H-22051; Mutagenicity Testing in the Salmonella typhimurium and Escherichw coli Plate Incorporation Assay____DuPont HLO-1997-OOU35 .^2/^ '01^... . . /i^PfP^ffW.^fn) MA sludv Numlw: ^-13-?' a" B.iclrriul MrttTM' Mulaliun Assay uiih an Independent Rrpeal Assay io n-Rposi; The purpose of this stuJ\ is 10 evaluate the mutagenii: poienlial of llic Icsl arliclr b> measuring its ahilii> in inJuLC revcrw: muluiions at sclcclcd loci ol' several sir.iins ol Sdlmiineitu lyph.iuiirium and at the ir\piiiphan IIKUS of E.\<:hi'rii-lwi rn/i ^'1'; IIITA (pKMIOl) in the presence and absence of SO aciivjtion. 2.0 SPONSOR 2.1 Name: E.I. du Poni de Ncmours and Company 2.2 Address: Iliiskfll Laboraton- for ToMcolo(!y and InJuslriul Medicine PO Dox SO. Elklcm Road Ncrark,DE 19714 2.3 Represenialivc Brian H. Malhison. Ph.D. 2.4 Sponsor Project c: MR-10850 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 TcslAnicIc: ^^ -H-W3W-- r-Z<o?l "|.i|^ To O^ATi'- '/ft-<'< iof~-"'--"*^ 3.2 Controls: SL'naiivc: Test an.cle vehicle Positive: 9'aminoacridine 2-aminoanihrjccntf mcihyl meihancsulfonate 3-nitrofluorene sodium azide 3.3 Determination ol' Strength. Purity, etc. The Sponsor ui|l he directly responsible for determination and documenialion of the analuical purity and composition of the Icsl article and (he stability and 5trcn|:lh of the dpsin;: solutions. '' 4 Test Article Reicnuon Sample The retention o(".i reserve sample ol'lhe IL-SI nrlicle uill be the responsibility of the Sponsor. $ ^R^^gG^L MA Study No. C96CF43.502011 - 45 Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella typhimwium and Eschefichia coS Plate Incorporation Assay DuPont HLO-1997-00035 4(1 ILST1NG FACILITY AND KEY PERSONNEL Name: Toxicology Tesiini; Facility Microhioloyical As-.nci.ilcs. Inc. 4.; Address: %30 Medical Center Drive Rocfcville. MD 20850 4.3 Study Director: Valentine 0. Wanner III. M.S. 5.0 TEST SCHEDULE 5.1 Proposed Experimental Initiation Date: 11.13'l^t* 5.2 Proposed Experimental Completion Date: lli"<^"7 5.3 Proposed Report Date: l^tVll 6.0 TEST SYSTEM The tester strains will include the S. typhimiirium hisiidine auxotrophs TA98. TA100. TA1535 and TA97A as described by Ames el al. (1975) and Levin cl al. (1983) and the . wli tester strain WP2 mvA (pKMIOl) as described by Green and Muriel (1976). Genoupe of tht Sirairu Died for MulJfen Twine_____________ Hisiidine Mutelion Tiyptophan Mumion Additional Mutations JmC46 AuD}05: luiMt'V IrpE LPS Repair R-bcior TAIS3? . r/a ^mrB TA100 TAM TAWA . r/tf Aiif'B R - - WP2 m-rA - AuwA (pKMIOl) l-.ach S. lyphimiiritini lestcr Strain contains, in addition to a mutation in the histidine opcron addiiionjl mutations that enhance sensitivity to some mutagens. The rfa mutation resiilis in a cell uall dd'iciency that increases the permeability of the cell to certain classes at chemicals such as those containing large ring systems thai would otherwise be excluded. The deletion in the KITO gene results in a deficient DNA excision-repair system. Tester strains TA8. TA100 and TA97A also coniain the pKMIOl plasmid (carrying the R.faclor) 11 has hern sugvcsicd lhai the plasmid increases scnsilivi'.y to mulagens by modify ing an >:\islini; bacterial DNA repair polymeriisc complex involved with the mismatch-repair process. 1"A98 anil TA17A ari: rescued from hisliilinc di-pcnilcncc (au\oiri)ph>) to hisiidine inji;pcnjeni;c (prnliiirophv) by Ir.imeshill -nulayens TA100 is reiencd by both framcshill I'rnlncnl Slt;T?0:ni I 1 1 IH l|i : of 10 ^^> MICROBIOLOGICAL ASSOCIATES. INC. MA Study No. G96CF43.502011 46Company Sanitized. Does not contain TSCA CBI H-22051: Mutagcnicily Testing in the Salmonella typhimurium and Esclierichia coll Plate Incorporation Assay DuPoni HLO-1997-00035 .inj bj-c Mih-.lilulii'n iniii.igcns and TA15?5 is mcrted >'nl\ by mulagens thai cause I'-isc MiritlllUlliUl.s I hi; /: n'/ teskT slum h. .111 A I base pjir .il ihr i.nlic.il mutation site within the /'."// :r\ [\\ ilct'x > .<;'. I1'")!!! Feslcr slr:iiii V \'l i.\r.\ (pKMIOl I lus .1 delL-lim lit ihc m-rA uene rcsiilnng in .1 deficient U\'A excision-repair system. Tester sir.iin \M'; iiiyA (pKMKlli :'lso coniains ihc pKMIOl plasnnJ Jcscribcil .ihoic Trvpiophan rL-ieri.inis i;;in arise line lo a base i-lvini:r at ihe orii;inall\ injtalfil sue or b> a base chani;>; ciscwiicre in ihc chronios-oinc raiisin,; Ihtf orminal mul.ilion in he supprL'sscJ. I'hus. ihe spccificily ol'lhi; r>;\crsn'n mechanism is scnsiiivc 10 hasc-p.nr .suh-.iiiulion nnilalions. rather lhan Iramcshil't mulaiioni (Green and Muriel. l^Td) The .^ typliiiiiiirium lesler sirains were rt'eeivcJ ilirecily ftom Dr. Bruce Ames. l^nncrsily o I" California. Bcrkflcv. I'hc E cu/' lesler strain uas receivcil Irom ihc National Collcclinn ol' Industrial and Marine Bacieria. Aberdeen. Scotland (United Kingdom). 7.0 EXPERIMENTAL DESIGS" A\D METHODOLOGY The lest article will he iL'sied at a minimum of rue dose levels along uilh appropriate nL-eativc and posiliM: conlrols with lesler strains TA98. TA100. TA1535. TA97A and \VP2 tivr.\ (pKMIOli with anrf vsithoul S9 activation. All dose levels of lesi article, negative i;ontrols and posimc crnlrols will be plaicd in triplicate. 7 I Srluhilil\ Dclcrn-.iiiatit.in Unless Ihr Sponsol has indicated the lest article vehicle, a solubility determination nill he condiirlcil to determine the ma\imum soluble concentration or workable suspension up to a maximum of 500 mp nil. Vehicles compatible iviih this test system, in order ol" preference, include but are not limiled to: dcioni/cd water (CAS 77.';:.1S-5). diniL-lhylsulfoxidc (CAS 67-61^.i'l. elhanol (CAS 6.4-17.5> and acetone (CAS 67-64-1) The vehicle of choice will he the solvent, selectrd in order of preference, that permits preparation of the highest workable'soliiblr stock concentration, up to 500 mg'ml 7 ; t'rcliniinary To\icil> Assay to Select Dose l.cicis Sclecimn ol' ursr levels for the minagenicili. ;iss;i> will be based upon the loxicily .ind prfcipii.umn profile of the lest article assessed in a preliminary lo\icit> assay I Ins prcliiiiin.irv ;iss.i> will be condiiclcJ b> e\posing TA'/K. TA100. TA15.''5. [.\''"-\ .inil ^'1'? M'-A (pls.MIOI) ii> ncr.iiiVL' controls and to al least eii;hl i.oiH'L'nir.iiiiins ol' lest article, one pl.-'ic pi'r il.ise level. IP both the presence and iib'.cin.c rl S1' .icin'aiion Unless indic.ncd ('.hern isc by the Sponsor, ihe highest dose \M|I he ihc hiL-licsl workable coni-enlr.iln'n in ihc vehicle ol'choice but not to exceed ' iiir pl.iif In\icii\ will be cvalu.iiei) ;i.< ;i decrease in the number of revenani riilrnies per "l.ilc .ind cr a thinning f'r disappearance of the bacterial background l.iwn I'rci.'iril'11"''! "ill be r'.;ilii:iird liilli'wing ihe incubalion period In the event I'ul llu- ICNI .inijc ...innti'. he Ji'livi.-rril .11 .i high enough concentration in ;in Jppiri'n.iiL- i.-'iiji.' lo he IDMC rr il lr\i ,i."'ili.- prccipil.ilc i< present on the plates ,, .,..,isi.(.r-;(i:nii iru..,, .'ndn ^^M> IACSRSOOBCIIOATLEOSG. IICNAC.L MA Study No. G96CF43,502011 - 47 - Company Sanitized. Does not contain TSCA CBI H-22051; Mutagenidty Testing in the Salmonella typhbnwwm and Escherichia coli Plate Incorporation Assay DuPontHU).1997.00035 alter incubation. the Sponsor will tic consulted prior to selection of dose levels for; the mutauenicitv assay. In selecting Jose leiels lor the mutagenicity as-i.n.^ihc lolloping uuiikTincs will be employed. Whenever possible, the highest dose tor4.hi: w: mulayenicily assav v'.ill be selected lo some indication or loxicily without exceeding 5 mg pl-nc. For freely soluble, nontoxie lest articles, the highest dose level will be 5 mg'plaie. For precipitating. nomoxic tesi anicles. ihe highest do.se level will be selected in an aitrmpi to yield precipitale at only the lop one or two dase levclfi. The precipitate will be evaluated al'icr the incubaiion period by Msii.i] examination vviihout magnification. Doses M be selected such thai preeipiiuie does not interfere with scoring. . ^ 7.3 Frequency and Route of Administration The test system will be exposed lo ihe lea anicle via ihe plale incorporation, methodology originally described by Ames vi ul. (1975) and updated by Naron'arid':: Ames (1983). This methodology has been shown to detect a wide raniiepf .<:1as<ieS: or chemical muuigens (McCann n ul.. 1975; McCann and Ames. 197^);:: " : ,; After the data generated in the first assay have been evaluated, the mulagcriicily assay will be repealed. Thcdosr levels used in (he second assay will.bc the same asihpsc used in the first assay unless the Study Director determines th.il the ' N: range should be changed due (o such parameters as cxcccssive cytoioxicity or pi. jpitaie. : . 7.4 Controls ^":'^': 7.4.1 Positive Controls -,' ' - All combinations of positive controls and tester strains plaicd concurrently uilh the assay arc listed below: :: Strain Poiilie Controls S9 Activation Pdtilive Control __ ;_ Coitccniraiioh {ns/vlau^.,..,, TA98.TA100.TAI535 . i;o.; .J TA9'A - U'P; KfA (pKMIOl) TA9g . ^-aminoanlhraccne 2-nitrolluarene is"'. ''' i : 1:0 TA100. TAI?3$ lodium azidc 10 TAr'A . 9-aminojcnJmc 7} WP; u>rA IpKMIOl) - mrthy) meihanesuironale 1.000 7.42 NccaliM: Controls Appropriate ncualive controls will he plated for each lester strain with and Mihiiu( Sl .iciivation. The negative control will be the vehicle rrm..coisr<;T:;(i:iiii ii'n.r> 4 ,,f i<i ^ ^ MICROBIOLOGICAL ASSOCIATES, INC. MA Study No. G96CF43.502011 48- Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella typhimurium and Escherichia coli Plate Incorporation Assay . DuPont HLO-1997-00035 alone, unless (here is no historical basis lor use of:he selected vehicle. In the l.mer case. both untreated and vehicle controls \\\\\ be used. 7.4.? Sterility Controls The most crneeniroted lesi anicle'dilution :md the Sham anil S1) mixes will be checked lor sterility. 7.5 Exogenous Metabolic Activation Aroclor 1254-induced rat liver S9 will be used as the metabolic activation system. The S9 homogenalc will be prepared from male Sprague-Dawley rats induced uiih a single inirapenioncal injection of Aroclor 1254. 500 mg/kg. rive days prior to sacrifice. The S9 will be batch prepared and stored frozen at approximately -70C until used. Each batch ofS9 homogenaic will be assayed for its ability to metabolize 2-aminoanihracene and 7.12-dimethylbcrrzanihraccne to forms mulagenic 10 X. lyphimwium TA100. Immediately prior to use. the S9 will be thawed and mixed with a cofacior pool to contain 10% S9 homogcnale. 5 mM glucosc-6-phosphaie. 4 mM p-nicoiinamideadcnine dinucleoiide phosphate. 8 mM MgCI: and 33 mM KCI in a 100 mM phosphate buffer at pH 7.4. This mixture is referred to as S9 mix. Sham mix will be 100 rnM phosphate buffer at pH 7.4. 7.6 Preparation of Tester Strain Overnight cultures will be inoculated from the appropriate master plate or from the appropriate frozen stock. To ensure thai cultures are harvested in late loi: phase, the Icnyiri of incubation will be controlled and monitored. At the end of the working day. each inoculated flask will be placed in a resting shakcr/incubalor at room temperature. The shakei/incubator will b'e programmed to begin shaking at approximately 125 .rpm at 372''C approximately 12 hours before the anticipated lime of harvest. All cultures will be harvested by spccirophoiomeiric monitoring of culture turbidity rather than by duration of incubation since overgrowth of cultures can cause loss of scnsitn iiy to sonic rnuiagens. Cultures will be removed from incubation ai a density ol" approximately 10'cells/nil. 7.7 Test System Idrntiricaiion L:ich plule uill he l.'ibcled uilh a code s'slcm llial identifies the test article, lest phase, dose level, tester strain and activation type as described in Microbiological Asscciaies' Microhial Mulagenesis Sl:iml;iril Opernling Procedures. MA Study No. G96CF43.502011 $ %%O^OG^t - 49 . Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella typhimunum and Escherichia colt Plate Incorporation Assay DuPont HLO-1997-00035 7.8 Test Article Prcparalion Unless iipccified olherinsc. leal article Jiltilion.s will btf prcpjrcd immediately prior id use. All les! article Josiny will he .11 roiiin iempi:rulurc under ycllou liuhl. 7.9 Treatment ol'Tesi System One halt'milliliiLT (0.5 nil) ol'S9 mix or Sham mix. 100 pi of tester sir.iin and 50 il of vehicle, ic.si article diluiion or positive control will be added to 2.0 ml of molim seleciive lop agar at 452C. When necciisary lo achieve the target concentralion or eliminate toxic vehicle eftccis. aliquois or other than 50 pi of icsi anicle/venicle/posjiive control will be plaied. The mixture will be vortex mixed and overbid omo the surface of 25 ml of minimal bottom agar. After (he overlay has solidified, the' plates will be invened and incubated for approxiinaicly 48 to 72 hours at 372C. Plalcs (hat are not counted immediately fbllowiny the incubation period wilt be stored at 42<>C. 7.10 Colony Counting The condition of ihc bacterial background lawn will be evaluated 'for evidence of lest article toxicily and precipitate. Evidence of loxicily will be scored relative to the negative control plate and recorded along with the revcrtani count for thai plate. 7.11 Tester Strain Verification On the day of use in the mutayenicily assay, all S. i\piiiinuriiim tester strain cultures uill be checked for the following genetic markers: The presence of the rfn wall mutation will be confirmed'for all tester strains by demonstrating sensitifily to crystal violet. The presence of the urrB mutation will be confirmed for tester strains TA8. TAIOO. TA1535 and TA97A by drmonstraiing sensitivity to ultraviolet light. The presence of 'he pKMIOl plasmid will be confirmed for tester strains TA98. TAIOO and TA97A by dcmonsiraiing resistance 10 ampicillin. On the day of use in the mulagenicily assay, the E colt tester sirain cultures will be' checked for the presence of (he urrA mulaiion by demonstrating sensilivily to ultraviolet light. The presence of the pKMIOl plasmid will be confirmed for lesier strain \VP2 firrA (pKMIOl) by demonstrating resistance to ampicillin. s o c RI rnRiA FOR DEITRMINATION' OF A VALID TEST 'I he lollc.winu criicri.i nuisl he mel for ihc mulauenicily assay to be considered valid: I'rnnici.l SI'(;T511:ni I IIWW MA Study No. G96CF43.502011 fc of 10 - 50 - ^^> MICROBIOLOGICAL ASSOCIATES, INC. Company Sanitized. Does not contain TSCA CBI H-22051: Mutagcnicily Testing in the Salmonella typhimurium and Escherichia ' " Plate Incorporation Assay DuPont HLO.1997-00035 S. 1 Tester Strain Integrity To demonstrate the presence of the r/ii mutation. all S lyphimuriiim lesier sirain cultures must exhibit sensitivity 10 cry st.il ^ iottfl. To demonstrate the presence of the iivrO muuilion. all A' n-phimiiriiim tester sirain cultures musi exhibit sensilivily |o ultraviolet light. To demonstrate the presence of the KITA mutation. nil E. viili tesicr strain cullures must c.\hibil sensitivity to ultraviolet light. To dcmonsirale the presence of ihe pKMIOl plasmid R-1'acior. lesier strain cultures of TA8. TA100. TA97A and WP2 iivr.\ (pKMIOl) must exhibit resistance to ampicillin. 8.2 Spontaneous Revenant Background Frequency Based on historical control data. all tester strain cultures must exhibit characteristic number of spontaneous revertanis per plate in the negative controls (vehicle). The mean revertanis per plate musi be within the following ranges (inclusive): TA98. wA 10 50; TA100, 80 - 240; TA1535.5 - 45; TA97A. 80 - 240; WP2 150 - 380. (pKMIOl). 8.3 Tester Strain Tilers To ensure that approprialc numbers of bacteria are plated, all tester strain culture liters must be equal to or greater than 0.3x10* cells per milliliier. 8.4 Positive Control Values Each mean positive control value musi exhibit at least a three fold increase over the respective mean negative control value (vehicle) for each tester strain. 8.5 Toxicity A minimum of three non-toxic dose levels will be required to evaluate assay data. A dose level is considered toxic if it causes a >50% reduction in the mean number of revertanis per plate relative to the mean negative control value (this reduction must be accompanied by an abrupt dose-dependent drop in the rcvertani count) or a reduction in the background lawn. In the event that fewer than three non-toxic dose levels are achieved, the affected portion of the assay will be repeated with an appropriate change in dose levels. 9,0 EVALUATION OF TEST RESULTS For a test article to he evaluated positive, it must cause a dose-related increase in the mean rcu'nams per plaic of ai Icasi one lesier sirain over a minimum of iwo increasing concenlralions of lesi anicic as speciCicd below rr,,n>coisp(:T?o:nn i i/(u it, - of in ^ MICROBIOLOGICAL ^> ASSOCIATES, INC. I MA Study, No. G96CF43.502011 - 51 - Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in the Salmonella typhimuriwn and Escherichia coli Plate Incorporation Assay OuPont HLO-1997-00035 O.I SirainTAl535 Data sets will be judged positive if the increase in mean revertants al the peak oflhi; dose response is equal 10 or greater than three limes the mean negative control value (vehicle). 9.2 Strains TA9S. TA 100. TA97A and \VP; mrA (pKM 101 > Data sets will be judged positive if the increase in mean revenants at the peak of the dose response is equal to or greater than two times the mean. negative coniro! value (vehicle). In consultation with the Sponsor, negative results may be confirmed as needed and equivoc.il results may be clarified by further testing using modified experimental conditions. 10.0 REPORT A repon of the results of ihis study will be prepared by the Testing Laboratory and, will accurately describe all methods used for generation and analysis of the data. The repon will include: Test substance: identification and CAS no.. if known; physical nature and purity, if known; physicochemical properties relevant to the conduct of the study, if known; stability of test article, if know. Solvent/Vehicle: justification for choice of vehicle; solubility and stability of lest article in solvent/vehicle, if known. Strains: strains used; number of cclls/ml per culture; strain characteristics. Test conditions: amount of lest substance per plate with rationale for dose selection and number pf plates per concentration; media used; type and composition of neiabo^c activation system, including acceptability criteria; treatment procedures. Results: signs of loxicity; signs of precipitation; individual plate founts: the mean number of revertant colonies per plate and standard deviation: dose-response rdiiiionship. where possible; statistical analysis, if any; concurrent negative and positive control data means and standard deviations; historiciil negative and positive cor.irol data with ranges, means and standard deviation. Discussion of results Conclusion I'riiiMiiKi'r.Tfii:^! ii/ii.iK* MA Study No. G')6CF43.502011 .Hnfii) ^MICROBIOLOGICAL --^ ASSOCIATES INC - 52 - Company Sanitized. Does not contain TSCA CBI H-22051: Mutagenicity Testing in (he Salmonella typhimurium and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-00035 11.0 RECORDS AND ARCHIVES Upon conipleiion of (he Final report, all raw data and reports will be maintained by the Quality Assurance Unit of Microbiological Associates. Rockville. MD in accordance with the rclcvani Good Laboralory Practices Rcyulaiions. HO REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply with OECD Guidelines 471 and 473 (Genetic Toxicology: Bacterial Reverse Mutation Assay). Revised Draft Document. Sept-.'mber 1995 and with the International Conference on Harmonisaiion of Technical Requirements for Registration of Phannaceuticats for Human Use. Gcnoloxicily: Guidance on Specific Aspects of Regulatory Gcnotoxicily Tests for Pharmaccuiicals. Step 4 Final Draft. July 18. 1995. This study will be performed in compliance with the provisions of the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. Will this jiudy be submitted to a regulatory agency? yes agencies? EPA/TSCA. OECD: MAFF If so. :o which agency or Unless arrangements are made 10 the contrary, unused dosing solutions will be disposed of following administration to he test system and all residual lest article wilt be disposed of following Hnalization of the report. 13.0 REFERENCES Amcs. B.N.. McCann. J. and Yamasaki. E. (1975). Methods for dete'ling carcinogens ind mutagcns with the 5u/mom//o/mammalian'microsome mulagenicily lest. Mutation Research J/:347-364. Green. M.H.L.. and Muriel. '.'-'.J. (1975). Mulayen lesting using lrp' reversion in Escherichw coli. Mutation Research 3S:3-32. Intcrnalional Conference on Harmonisaiion of Technical Requirements for Registration of Pharmaccuiicals for Human Use. Genoloxicity: Guidance on Specific Aspects of Regulatory Gcnoloxicily Tests for Pharmaceuticals. Step 4 Final Draft. July (8, 1995. Levin. DC.. Yamasaki. E. and Ames. B.N. (1982). 11 new Sutmowlla tester strain. TA97. for ihe uclrclinn of frarneshifl mulagens. A run of cylosines as a mutational hot-spot. Mutalion Research 94:315-330. McCann. J. and Ames. B.N. (1976). Dclrriion of carcinogens as mulagens in the Sniinwvllii'microstfmt lest: assay of 300 chemicals: discussion. Proc. Nail. Acad. Sci. USA 73:950-954 friilr>fi>ISPr.T'll;nil 11 (I.I 1(> MA Study No. G96CF43 502011 of 10 - 53 ^^MAICSRSOOCBIIAOTLEOSG, IICNCA.L Company Sanitized. Does not contain TSCA CBI <^ H-22051: Mutagenidty Testing in the Salmonella typhimurium and Escherichia coli Plate Incorporation Assay DuPont HLO-1997-00035 McCann. J.. Choi. E.. Yamasaki. E. and Amcs. B.N. (1975). Detection of carcinogens as iiiuljycns in the Sulniunrllii'micmscimc lest: assa\ ol'?00 chemicals. Proc. Nail. AcaJ. Sci. L'SA 72:5135-5139. Maron. D.M. and Ames. B.N. (19S3). Revised Methods lor the Siilmumltti Mulagcnicily Tesi. Mutation Research 113:173-215. OECD Guidelines 471 and 472 (Genclie Toxicoloey: Bacterial Reverse Mutation Assay). Revised Drat'l Document. September 1995. Wilcox. P.. Naidoo. A.. Wedd. DJ. and Gatehouse, D.G. (1990). Comparison of SulmunrHu tvphmuriiiin TA102 with Esclwrichia coli WP2 tester Strains. Mulagenesis 5:285-29). 14.0 APPROVAL / < - / S^PONSOR-RSPRfesaCf^TA^T-lV_E_________1^DA,1TE/V<. ___ (Prim o ____ r rype Na m' 3 e) ^ . / y //. ^Ju.^.xC^. ik<u>^. "nr STUDY DIRECTOR itmr>f/t"^)______ ii(n/?fa DATE rniii.cni sr(~.TMi:iii i 11'nj'q<> MA Study No. G96CF43.502011 in iif in - 54 - ^^> MICROBIOLOGICAL ASSOCIATES. INC. Company Sanitized. Does not contain TSCA CBI