Document ZJxgbaD4bRZBxZOvw3jdDba6p

TRADE SECRET DuPont HL-1997-00676 AR226-3078 Study Title SalmonHe-l2la27ty6p2h: imMuurtiuagmenPilcaittey Testing in the Incorporation Assay Laboratory Project ID: HL-1997-00676 AUTHOR: N. Lawrence Gladnick, B.A. Study Completed on: August 26,1997 Performing Laboratory: HE.aIs.kdeullPLoanbtodraetNoreymfooruTrsoaxnidcoCloogmypaanndyIndustrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Medical Research No.: Page 1 of 16 Company Sanitized. Does not contain TSCA CB\ H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPont HL-1997-00676 Haskell Number: 22762 Known Impurities Physical Characteristics i Stability: Test substance appeared to be stable under the conditions of the study; no evidence of instability was observed Sponsor: E. L du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Smdv Tnitiated/Completed: July 16,1997 / (see report cover page) Tn-T.ife Tnitiated/Completed: July 16,1997 / July 25,1997 All study records, raw data, and the final report are retained at Haskell Laboratory or at Iron Mountain, 200 Todds Lane, Wilmington, Delaware 19802 (formerly known as the E. I. du Pont de Nemours and Company Records Management Center). Company Sanitized. Does not contain TSCA CBf H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPont HL-1997-00676 TABLE OF CONTENTS GENERAL INFORM ATION..................................... 2 SUMMARY..................................................................................................................................................... 4 S IG N A T U R E S ................................................................................................................................................ 4 STUDY PERSONNEL................................................................................................................................. 5 INTRODUCTION......................................................................................................................................... 6 MATERIALS AND METHODS...............................................................................................................6 STTEUSTDMY APRTOERTOIACLOSL..................................................................................................................................................................................................................................................66 Test Substance and Negative Control.....................................................................................6 Positive Controls......................................................................................................................... 6 TESTER STRAIN CHARACTERIZATION, STORAGE, AND CULTURE......................7 Tester Strain Characterization.................................................................................................. 7 Tester Strain Storage and Culture............................................................................................7 METABOLIC ACTIVATION SYSTEM.....................................................................................8 DOSE SELECTION..........................................................................................................................8 STABILITY AND CONCENTRATION VERIFICATION.....................................................8 BACTERIAL MUTAGENICITY A SSAY S............................................................................... 8 STATISTICAL ANALYSIS............................................................................................................9 ACCEPTABILITY CRITERIA......................................................................................................9 RESULCTLSAASNSIDFICCOATNICOLNUGSUIOIDNE..L...I..N...E...S.................................................................................................................................190 RESULTS.......................................................................................................................................................10 CONCLUSION.............................................................................................................................................10 REFERENCES.............................................................................................................................................11 ABBREVIATIONS FOR TABLES....................................................................................................... 12 T A B L E S ............................. 13 TABLE 1 - T A 100................................................................................................................... 13 TABLE 2 - TA 1535........................................................................................................................ 14 TTAABBLLEE 43 -- TTAA9978a........................................................................................................................................................................................................................................................1165 - 3 - Company Sanitized. Bees nut cornait t &c a v-a. H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPont HL-1997-00676 SUMMARY The test substance, H-22762, was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 with and without an exogenous metabolic activation system (S9). Concentrations o f 10, 50,100,500,1000,2500, and 5000 pg/plate were evaluated in comparison to negative (solvent) controls. SEavlimdeonnceellfaortytpehsitmsuubrisutamncstertaoinxsic, iwtyitthoatnhde bacteria was observed and documented without S9, in concentrations as low as in all 1000 pg/plate. SUanldmeornthelelacotynpdhitimionursioumf thstirsasitnuidny,thneoaebvsiednecneceoropfrmesuetnacgeenoifctahcetievxiotygewnaosudsemteecttaebdoilnicaancytivation system. The test substance was negative. Reported and Approved for c Issue by Study Director: __ Toxicology Associate -4- M fipany Sanitized Does not contain TSCA CBf SHa-l2m2o7n62el:laMtyupthaigmenuirciiutmy TPelasttienIgnicnortphoeration Assay STUDY PERSONNEL DuPont HL-1997-00676 Manager: Matthew S. Bogdanffy, Ph.D., DABT The following individuals were responsible for conducting the in-life portion of the assay: Study Director: N. Lawrence Gladnick, B.A. Toxicology Associate Primary Technicianfsl: N. Lawrence Gladnick, B.A. The following individuals were responsible for the results generation: Study Director: N. Lawrence Gladnick, B.A. Report Preparation: Debra L. Dacey N. Lawrence Gladnick, B.A. Report Reviewed bv: Brian H. Mathison, Ph.D. Research Toxicologist - 5 - (Compatty Saniuzad, Does not contain TSCA cf H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPont HL-1997-00676 INTRODUCTION TtyhphisimstuudriyumevastlruaaitnesdTthAe1m00u,tTagAe1n5ic3p5,oTteAnt9ia7lao, afnthdeTteAs9t8s.ubTshtaenScae,lmHo-n2e2l7la62s,trianinSsalamreounnelalable to synthesize histidine, an essential amino acid, because of mutations in the genes coding for histidine. biosynthetic enzymes. Additional mutations in the defective genes can result in individual Salmonella bacteria regaining the ability to synthesize histidine.(1) A trace of histidine in the top agar permits several generations of auxotrophic cell division to fix pro-mutagenic lesions. This results in the formation of a microscopic "lawn" of bacteria. However, only those bacteria that revert to a prototrophic state can continue to divide and form macroscopic colonies. These revertants can be scored by their ability to grow on agar plates deficient in histidine. By comparing the number o f chemically induced revertants to the number of spontaneous revertants, the mutagenicity o f the test substance can be assessed. MATERIALS AND METHODS A. STUDY PROTOCOL The protocol consisted ofcthe Short Term Study Protocol Sheet and the Haskell General Testing P roced u rejH H B H ft'B acterial Mutagenicity Testing of Solids, Liquids, and Gases", effective 571/97). B. TEST MATERIALS 1. Test Substance and Negative Control The test substance is H-22762. Additional information regarding the test substance is found on the general information page of this report. The test substance was assumed to be stable during this assay and no evidence of instability was observed. Based on information supplied by the sponsor, dimethyl sulfoxide (DMSO) was chosen as the test ssuubbssttaannccee saotl5v0enmtga/nmdLasretqhueinreedgastoinveiccaotinotnrotlo. mPareinptaariantihoonmoofgtheenesittoyckansdolsuotliuobniloitfyt.heAtdedstitional dilutions of the test substance were performed at room temperature in DMSO. The negative control was assumed to be stable during this study, and no evidence of instability was observed. Any impurities were not expected to have interfered with the study. 2. Positive Controls Positive controls included the following: 2-aminoanthracene (2AA), 2-nitrofluorene (2NF), sodium azide (NAAZ), ICR 191 Acridine (ICR 191). Deionized water was the solvent for NAAZ and ICR 191. The solvent for the other positive controls was DMSO. The positive controls were assumed to be stable in this study and no evidence of instability was observed. Any impurities were not expected to have interfered with the study. -6- empany Sanitized. Does not contain TSCA CBf SHa-l2m2o7n62el:laMtyupthaigmenuirciiutmy TPelsattienIgnicnorthpeoration Assay DuPont HL-1997-00676 C. TESTER STRAIN CHARACTERIZATION, STORAGE, AND CULTURE 1. Tester Strain Characterization pS.hteynpohtiympuicricuhmartaecstteerrissttircasinasnwd emruetoabtitoaninaeldsefnrosimtivDitri.esBorufctheeASm. teysp,hBimerukreiluemy, CA. The strains are as follows: Strain TA100 TA1535 TA97a TA98 Histidine Gene Locus Affected hisG46 hisG46 hisD6610 hisD3052 DNA Repair AuvrB AuvrB AuvrB AuvrB Additional Characteristics R-factor Mutational Sensitivity LPS Plasmid rfa Present Base pair substitution rfa Absent Base pair substitution rfa Present Frameshift rfa Present Frameshift The deletion (A) in uvrB (a gene which codes for DNA excision repair) increases the bacterial sensitivity to mutagens.(3) The uvrB trait is confirmed by demonstrating an increased bacterial mbpseaioonrtltsieiinactliubvlloiietosysssyitsionnitunhtlhcetrerseaiisanv,sitteoehdlgeert(biAtlaiygmcohteetfs.rtiheaBtereaelciqlpa,uoui1psr9eoe7ltey3hxs)iao.scgdTceehnhlaoeertupiiosdrnebesi(aeoLlntsPiocneSef)oxocftreetgnlhlrdeowswRatth-lhflra.socoutTogthrhheapatrlafpgaseemnrmmeidunet(aaepbetlidiaoleisntmdycfaitdoourslaersgae pKMIOl which carries ampicillin resistance as a marker gene) results in an enhancement of an error-prone DNA repair system which is endogenous to these bacteria.(4) Although the testing guidelines cited require S. typhimurium strain TA1537, the more recently developed TA97a strain was used. TA97 was the recommended replacement for TA1537 and has been demonstrated to be more sensitive to frameshift mutagens.(1,5) However, the reconstructed strain, TA97a, is now routinely used in place of TA97 due to its improved growth properties (personal communication with Bruce Ames and associates). 2. Tester Strain Storage and Culture A-7ll0bCac.teOrivaelrsntrigahintscwuletureresstowreedrefrporzeepnariend8b%y iDnoMcuSOlatiinngO2x0omidLnuotfriOenxtobidrontuhtariteanptpbrrooxthimwaittehly 0.1 m L of thawed bacterial suspension and incubating at 37C with shaking. Overnight cultures were then stored on ice until used for mutagenesis assays. -7 - Sttipanf Saitiraea. Qam mi contain TSUACi H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPont HL-1997-00676 D. METABOLIC ACTIVATION SYSTEM Because the tester strains lack many of the enzymes required to convert various promutagens to a reactive state, the assay was performed with and without a rat liver homogenate activation system (S9 Mix) similar to the method of Maron and Ames (1983). The S9 Mix consisted o f the following: 8 33 mM mM MgC KC1 l2 5 mM glucose-6-phosphate 1040 mmMM NsoAdiDuPm+phosphate pH 7.4 1.6 mg S9 protein /1 .0 mL S9 Mix The S9 (purchased from MOLTOXTM) was the 9000 x g supernatant of liver homogenate 0 g wet liver: 3.0 mL KC1). Livers were from young male Sprague Dawley rats injected i.p. with Aroclor 1254 (500 mg/kg) five days before sacrifice. E. DOSE SELECTION In accordance with EPA and OECD test guidelines, the highest concentration evaluated in this study was a nominal concentration of 5000 pg/plate. Solubility information was confirmed empirically as needed during this study. Concentrations were calculated with the assumption that addition of the test substance to the solvent did not change the volume of the resulting solution. ` F. STABILITY AND CONCENTRATION VERIFICATION Solutions of the test substance were prepared immediately prior to treatment and were presumed to be stable under the conditions o f the study. Treatment and control dosing solutions were not analyzed for concentration, uniformity, or stability. Top agar was not assayed for stability or concentration of the test substance or control articles since this assessment was not considered necessary to achieve the objectives of the study. G. BACTERIAL MUTAGENICITY ASSAYS This study consisted o f one trial with and without activation. Two replicates were plated for each tester strain, test concentration, and condition. Positive and negative controls were run concurrently for all assays. Treatments with activation were conducted by adding 0.1 mL of negative control, positive control, or test substance solution, 0.5 mL of S9 mix, and 0.1 mL of an overnight culture containing approximately 1 xlO8bacteria, to 2 mL of top agar [0.6% agar (w/v) and 0.6% NaCl (w/v)] supplemented with 0.05 mM L-histidine and 0.05 mM D-biotin. These components were mixed and poured onto a minimal glucose agar plate (approximately 25 mL, purchased from MOLTOXTM). Treatments without activation were the same as those with activation with the exception that the S9 mix was replaced with 0.5 mL of sterile phosphate buffered saline. Revertant colonies were counted after the individually labeled plates were incubated at approximately 37C for about 48 hours. When necessary, plates were refrigerated prior to counting. - 8 - Company Sanitized. Does mu contain TSCA CB1 H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPont HL-1997-00676 H. STATISTICAL ANALYSIS For each tester strain, the average number o f revertants and the standard deviation at each concentration with and without S9 activation were calculated. I. ACCEPTABILITY CRITERIA An individual trial must have included at least five test concentrations (of which at least four must have been acceptable), a negative control, and a positive indicator for each selected tester strain. Data acceptability criteria were as follows: A single data point may have been rejected if contamination or excessive toxicity was seen on a treatment plate. A single data point may also have been rejected if excessive precipitate on the plate prevented accurate colony counting. A negative control data point may have been rejected if it fell outside the acceptable spontaneous mutation range. A concentration level was rejected if there were less than two data points at the treatment level or if the data point values were judged by the study director to be too divergent. A trial for the affected strain was rejected if the negative control was rejected or if there was no evidence of mutagenic activity on any positive indicator plate. Only those trials that met the criteria of acceptability were included in this report. J. CLASSIFICATION GUIDELINES A test substance was classified as POSITIVE when: (1) the average number of revertants in any strain at any test substance concentration studied was at least two times greater than the average number of revertants in the negative control; and (2) there was a positive dose-response relationship in that same strain. A test substance was classified as NEGATIVE when either: (1) there were no test substance concentrations with an average number of revertants which was at least two times greater than the average number of revertants in the negative control; and (2) there was no positive doseresponse relationship. Results not meeting these criteria for positive or negative assessments were evaluated using scientific judgment. *9* CompanySanitized*Do*'nvcontain oOwt-fo H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPont HL-1997-00676 RESULTS AND CONCLUSION RESULTS The test substance, H-22762, was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 with and without an exogenous metabolic activation system (S9). Concentrations of 10,50,100,500,1000,2500, and 5000 jig/plate were evaluated in comparison to negative (solvent) controls. CONCLUSION USanldmeornthelelacotynpdhitimiounrsiuomf thstirsasitnuidnyt,hneoaebvsiednecneceoropfrmesuetnacgeenoifctahceteivxiotygewnaosudsemteecttaebdoilnicaancytivation system. The test substance was negative. -10 - Company Sanitized. Does not contain TSCA CBI H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPont HL-1997-00676 REFERENCES 1. Maron, D. M. and B. N. Ames (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research 113,173-215. 2. Ames, B. N., F. D. Lee, and W. E. Durston (1973). An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nad. Acad. Sci. USA 70,782-786. 3. McCann, J., N . E. Springam, J. Kobori, and B. N. Ames (1975). Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids. Proc. Natl. Acad. Sci. USA 72,979-983. - 4. Levin, D. E,, E. Yamasaki, and B. N. Ames (1982). A new Salmonella tester strain, TA97a, for the detection of frameshift mutagens. A run of cytosines as a mutational hot spot. Mutation Research 94.315-330. -11 S a n M . Dees net contain TBCA CBf HSa-l2m27o6n2e:llaMtyupthaigmenuirciuitmy TPelasttienIgnicnortphoeration Assay DuPont HL-1997-00676 ABBREVIATIONS FOR TABLES Evidence for test substance toxicity to the bacteria was documented by recording the appearance of the plates and background lawn using the following key: TO Normal, background microcolony lawn appears normal. T1 Slightly reduced, background microcolony lawn is noticeably thinner. T2 M oderately reduced, background lawn is markedly thinner resulting in an increase in the size of microcolonies compared to the vehicle control plate(s). T3 Severely reduced, background lawn is distinguished by an extreme thinning of the microcolony lawn resulting in an increase in the size o f the microcolonies compared to the vehicle control plate(s). Microcolonies may be seen readily by T4 Athbesuennati,dpeldateey(se)aanrde darisetignrgeuatislyheednlbayrgaedcormelpatleivtee ltaocckonotfraonlsy. microcolony lawn over a majority of the area of the plate(s) Formation of a precipitate by the test substance will be documented using the following key: PPOI NMoicprroesccipopitiactpe,rnecoippriteactipe,itparteecoipbistaertevepdresent which does not interfere with background lawn evaluation or automated colony counting P2 Non-interfering precipitate, precipitate present that is visible to the naked eye which does not interfere with automated colony counting P3 Interfering precipitate, precipitate present which requires plate to be P4 Hcoeuanvtyedinbteyrhfearnidng precipitate, precipitate present that prevents accurate colony counting and obscures the background lawn requiring plate rejection Other appropriate observations relevant to the appearance of the plates: N None R Plate rejected - 12- Sompany Sanitized. Does not contain TSCA C H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation AssayDuPont TABLE 1 MUTAGENIC ACTIVITY OF H-22762 IN STRAIN TA100 Concentration H-22762 ftig/plate) Revertants Plate 1 Plate 2 A. WITHOUT ACTIVATION 0.0 5100..00 100.0 500.0 1000.0 2500.0 5000.0 NAAZ 2 pg/plate B. WITH ACTIVATION 0.0 10.0 50.0 510000..00 1000.0 2500.0 5000.0 2AA 1 pg/plate 101 112 111 113 110120 91 70 915 134 115 118 117 124 133 128 30 449 110 111122 124 80 123 14116 872 134 111256 137 113 124 11116 457 Average (S.D.) 106 112 112 119 91 (6) (0) ((18)) (16) 117 101 ((91)4) 58 (17) 894 (30) 134 121 117 ((08)) (2) 127 119 129 122 21 (14) (8) (6) ((81)3) 453 (6) DuPont HL-1997-00676 Observations T0,P0 T0,P0 T0,P0 T0,P0 T0.P0 T1,P0 T1,P0 T2,P0 'N T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T1.P0 T3,P0 N - 13- BoW piSf la lltlze iL Boss Ito!contain TSCA CB! H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation AssayDuPont TABLE2 MUTAGENIC ACTIVITY OF H-22762 IN STRAIN TA1535 DuPont HL-1997-00676 Concentration H-22762 Qig/plate) Revertants Plate 1 Plate 2 A. WITHOUT ACTIVATION 100..00 50.0 100.0 500.0 1000.0 2500.0 5000.0 NAAZ 2 (ig/plate B. WITH ACTIVATION 0.0 10.0 50.0 510000..00 1000.0 2500.0 5000.0 2AA 2 |ig/plate 18 17 19 1154 1116 7 632 21 16 17 2117 15 186 626 15 14 17 14 1153 13 9 593 16 22 23 20 15 19 21 5 582 Average (S.D.) 1176 18 15 15 1152 8 (2) (2) (1) (1) ((12)) (1) (1) 613 (28) 19 2109 21 16 17 179 (4) ((44)) (1) ((31)) (4) (2) 604 (31) Observations T0,P0 TT00,.PP00 T0,P0 T0.P0 T1.P0 T1,P0 T2,P0 N T0,P0 TT00,,PP00 T0,P0 T0,P0 T0,P0 TT12,,PP00 N -14- Coppanjf Sanitized. Does notcontain 7SC CBf H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation AssayDuPont TABLE 3 MUTAGEINNICSTARCATIINVITTAY97OaF H-22762 DuPont HL-1997-00676 Concentration H-22762 (fig/plate) Revertants Plate 1 Plate 2 A. WITHOUT ACTIVATION 100..00 50.0 100.0 500.0 21500000..00 5000.0 ICR 191 2 p.g/plate B. WITH ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 25500000..00 2AA 1 (ig/plate 100 103 110134 122 102 78 0 2493 102 126 123 112259 120 103 113 509 107 107 102 96 19195 68 0 2536 112 131 135 131 123 128 100 103 653 Average (S.D.) 104 105 (5) (3) 108 100 110119 73 0 ((68)) ((52)) (7) (0) 2515 (30) 107 129 129 130 124 124 102 108 (7) (4) (8) (1) ((16)) (2) (7) (102) Observations TTQ0,,PP00 T0,P0 T0JP0 T0,P0 TT01,.PP00 T2.P0 N T0,P0 TT00,,PP00 T0,P0 T0,P0 T1.P0 TT11..PP00 N 10--0M - 15- gompttig S a sllz e d Does m l contain TSCA CBS H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation AssayDuPont TABLE4 MUTAGENINICSATRCATIIVNITTAY9O8 F H-22762 DuPont HL-1997-00676 Concentration H-22762 (pg/plate) Revertants Plate 1 Plate 2 A. WITHOUT ACTIVATION 0.0 10.0 50.0 100.0 1500000..00 2500.0 5000.0 2NF 25 |Ag/plate B. WITH ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 2AA 2 jig/plate 26 25 25 27 22 20 16 18 1614 18 28 28 30 31 28 21 15 1404 20 25 2239 22 14 16 13 1411 2255 24 23 27 2238 20 1325 Average (S.D.) Observations 23 25 27 25 2127 16 16 (4) (0) (3) (3) (0) (4) (0) (4) 1513 (144) 22 27 26 ((52)) (3) 27 29 28 22 18 (5) (3) (0) ((14)) 1365 (56) T0,P0 T0,P0 TT00.,PP00 T0,P0 T0,P0 TT11J.PP00 N T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T0,P0 T1,P0 T1.P0 N e w p lS f Sanitized. Bees notcontain ISCA CBt