Document 2Jv1YKp0yVq9qeYYprYvm2L9R

TRADE SECRET AR226-3078 Study Title H-22762: Mutagenicity Testing in the Salmonella typhimuriwn Plate Incorporation Assay LABORATORY PROJECT ID: HL-1997-00676 AUTHOR: N. Lawrence Gladnick, B.A. STUDY COMPLETED ON; August 26,1997 PERFORMING LABORATORY: E. L du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 MEDICAL RESEARCH No.: & ^ Page 1 of 16 GENERAL INFORMATION Substance Tested: JJ Synonyms/Codes: Haskell Number: 22762 Known Impurities:|m| Physical j m | Characteristics 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. Study Initiated/Completed: July 16,1997 / (see report cover page) In-Life Initiated/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). -2- Does not eonteii H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPont HL-1997-00676 TABLE OF CONTENTS GENERAL INFORMATION......................................................................................................2 SUMMARY....................................................................................................................................4 SIGNATURES...............................................................................................................................4 STUDY PERSONNEL..................................................................................................................5 INTRODUCTION.........................................................................................................................6 MATERIALS AND METHODS..................................................................................................6 STUDYPROTOCOL..........................................................................................................6 TESTMATERIALS............................................................................................................6 Test Substance and Negative Control........................................................................... 6 Positive Controls........................................................................................................... 6 TESTER STRAIN CHARACTERIZATION, STORAGE, AND CULTURE...................? Tester Strain Characterization.......................................................................................? Tester Strain Storage and Culture................................................................................. 7 METABOLIC ACTIVATION SYSTEM........................................................................... 8 DOSESELECTION............................................................................................................8 STABILITY AND CONCENTRATION VERIFICATION............................................... 8 BACTERIAL MUTAGENICITY ASSAYS ...................................................................... 8 STATISTICAL ANALYSIS...............................................................................................9 ACCEPTABILITY CRITERIA ..........................................................................................9 CLASSIFICATION GUIDELINES..............................,....................................................^ RESULTS AND CONCLUSION............................................................................................... 10 10 RESULTS..................................................................................................................................... CONCLUSION............................................................................................................................ 10 11 REFERENCES............................................................................................................................ ABBREVIATIONS FOR TABLES........................................................................................... 12 13 TABLES....................................................................................................................................... TABLE 1 - 13 TA100............................................................................................................ TABLE 2 - TA1535 .......................................................................................................... 14 TABLE 3 - 15 TA97a............................................................................................................. TABLE 4 - TA98 .............................................................................................................. 16 3 - - ReasBanv Does o^ ^omam TSC.".... H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay______________ _______DuPontHL-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 of 10,50,100,500,1000,2500, and 5000 u,g/plate were evaluated in comparison to negative (solvent) controls. Evidence for test substance toxicity to the bacteria was observed and documented in all Salmonella typhimurium strains, with and without S9, in concentrations as low as 1000 ug/plate. Under the conditions of this study, no evidence ofmutagenic activity was detected in any Salmonella typhimurium strain in the absence or presence of the exogenous metabolic activation system. The test substance was negative. Reported and Approved for Issue by Study Director: /^ X/' ^^ (.^ y --s^- C^Ad^- fi^^gfc^^ N. Lawrence Gladnick, B.A. Toxicology Associate S^'//oKa/ ^^ Date -4- ^ampany Sanitized. Does not onSaIn TSCA CBI H-22762: Mutagenicity Tesdng in the Salmonella typhimurium Plate Incorporation 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 Technician(s): 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 by: Brian H. Mathison, Ph.D. Research Toxicologist - 5- Dyes not TSCA C8? INTRODUCTION This study evaluated the mutagenic potential of the test substance, H-22762, in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98. The Salmonella strains are unable to synthesize histidine, an essential ammo 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.^ 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 of chemically induced revertants to the number of spontaneous revertants, the mutagenicity of the test substance can be assessed. MATERIALS AND METHODS A. STUDY PROTOCOL The protocol consisted o^the Short Term Study Protocol Sheet and the Haskell General Testing ProcedureJfBHBI)'Bacterial 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 substance solvent and as the negative control. Preparation of the stock solution of the test substance at 50 mg/mL required sonication to maintain homogeneity and solubility. Additional 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- ompany 'Sanitized, not contain CB? H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation Assay DuPontHL-1997-00676 C. TESTER STRAIN CHARACTERIZATION, STORAGE, AND CULTURE 1. Tester Strain Characterization S. typhimurium tester strains were obtained from Dr. Bruce Ames, Berkeley, CA. The phenotypic characteristics and mutational sensitivities of the S. typhimurium strains are as follows: Strain TA100 TA1535 TA97a TA98 Histidine Gene Locus Affected hisG46 hisG46 hisD6610 hisD3052 DNA Repair AMvrB AyvrB AMvrB AyvrB 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.^ The mrB trait is confirmed by demonstrating an increased bacterial sensitivity to ultraviolet light. Because this deletion also extends through a gene needed for biotin biosynthesis, the bacteria require exogenous biotin for growth. The rfa mutation causes a partial loss in the integrity of the lipopolysaccharide (LPS) cell wall so that permeability to large molecules is increased (Ames et al., 1973). The presence of the R-factor plasmid (plasmid 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.^ 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'^ 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 All bacterial strains were stored frozen in 8% DMSO in Oxoid nutrient broth at approximately -70C. Overnight cultures were prepared by inoculating 20 mL of Oxoid nutrient broth with 0.1 mL of thawed bacterial suspension and incubating at 37C with shaking. Overnight cultures were then stored on ice until used for mutagenesis assays. -7- not TSCA C6I H-22762: Mutagenicily 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 of the following: 8 mM MgCh 33 mM KC1 5 mM glucose-6-phosphate 4 mM NADP"1' 100 mM sodium 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 (1 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 ug/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 of 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 of 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 xl08 bacteria, to 2 mL of top agar [0.6% agar (w/v) and 0.6% NaCI (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. 0 _ _ .Company SaniiKed. Does noi contain TSCA CBI 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 ofrevertants 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 ofmutagenic 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 ofrevertants 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 ofrevertants which was at least two times greater than the average number ofrevertants 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 - 9o"s n'x TSCA Cb, 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 typhimuriian 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 p,g/plate were evaluated in comparison to negative (solvent) controls. CONCLUSION Under the conditions of this study, no evidence ofmutagenic activity was detected in any Salmonella typhimurium strain in the absence or presence of the exogenous metabolic activation system. The test substance was negative. -10 - .Company Sanitized. Does not contain TSCA CBI 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. 'Sompaw not T0^ 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. T 1 Slightly reduced, background microcolony lawn is noticeably thinner. T2 Moderately 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 of the microcolonies compared to the vehicle control plate(s), Microcolonies may be seen readily by the unaided eye and are greatly enlarged relative to controls. T4 Absent, plate(s) are distinguished by a complete lack of any 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: PO No precipitate, no precipitate observed PI Microscopic precipitate, precipitate present 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 counted by hand P4 Heavy interfering 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- ,,C.,ompany Sanitized. Does not contain TSCA < H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation AssayDuPont TABLE 1 MUTAGENIC ACTIVITY OF H-22762 IN STRAIN TA100 Concentration H-22762 (Hg/plate) Revertants Plate 1 Plate 2 A. WITHOUT ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 NAAZ 2 (Xg/plate B. WITH ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 2AA 1 p,g/plate 101 112 111 113 102 110 91 70 915 134 115 118 117 124 133 128 30 449 110 112 112 124 80 123 111 46 872 134 126 115 137 113 124 116 11 457 Average (S.D.) 106 (6) 112 (0) 112 (1) 119 (8) 91 (16) 117 (9) 101 (14) 58 (17) 894 (30) 134 (0) 121 (8) 117 (2) 127 (14) 119 (8) 129 (6) 122 (8) 21 (13) 453 (6) DuPont HL-1997-00676 Observations TO,PO TO,PO TO,PO TO,PO TO,PO T1.PO T1,PO T2.PO N TO.PO TO.PO TO,PO TO.PO TO,PO TO,PO T1,PO T3,PO N -13. H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation AssayDuPont TABLE 2 MUTAGENIC ACTIVITY OF H-22762 IN STRAIN TA1535 DuPontHL-1997-00676 Concentration H-22762 (ng/plate) Revertants Plate 1 Plate 2 A. WITHOUT ACTIVATIO]^ 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 NAAZ 2 fig/plate B. WITH ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 2AA 2 pg/plate 18 17 19 15 14 16 11 7 632 21 16 17 21 17 15 16 8 626 15 14 17 14 15 13 13 9 593 16 22 23 20 15 19 21 5 582 Average (S.D.) 17 (2) 16 (2) 18 (1) 15 (1) 15 (1) 15 (2) 12 (1) 8 (1) 613 (28) 19 (4) 19 (4) 20 (4) 21 (1) 16 (1) 17 (3) 19 (4) 7 (2) 604 (31) Observations TO,PO TO,PO TO.PO TO.PO TO,PO T1.PO T1,PO T2,PO N TO,PO TO,PO TO,PO TO.PO TO,PO TO,PO T1,PO T2,PO N -14- Seized. Ooes nofeonfain TSCA CBt H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation AssayDuPont TABLES MUTAGENIC ACTIVITY OF H-22762 IN STRAIN TA97a DuPont HL-1997-00676 Concentration H-22762 (Hg/plate) Revertants Plate 1 Plate 2 A. WITHOUT ACTTVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 ICR 191 2 p-g/plate B. WITH ACTIVATION 0.0 10.0 50.0 100.0 500.0 1000.0 2500.0 5000.0 2AA 1 p,g/plate 100 103 113 104 122 102 78 0 2493 102 126 123 129 125 120 103 113 509 107 107 102 96 115 99 68 0 2536 112 131 135 131 123 128 100 103 653 Average (S.D.) 104 (5) 105 (3) 108 (8) 100 (6) 119 (5) 101 (2) 73 (7) 0 (0) 2515 (30) 107 (7) 129 (4) 129 (8) 130 (1) 124 (1) 124 (6) 102 (2) 108 (7) 581 (102) Observations TO.PO TO.PO TO,PO TO.PO TO.PO TO.PO T1.PO T2,PO N TO.PO TO.PO TO,PO TO.PO TO,PO T1,PO T1,PO T1.PO N 15 H-22762: Mutagenicity Testing in the Salmonella typhimurium Plate Incorporation AssayDuPont TABLE 4 MUTAGENIC ACTIVITY OF H-22762 IN STRAIN TA98 DuPont HL-1997-00676 Concentration H-22762 (p,g/plate) Revertants Plate 1 Plate 2 A. WITHOUT ACTIVATI01N 0.0 10.0 50.0 100.0 500.0 1000.0 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 p-g/plate 26 25 25 27 22 20 16 18 1614 18 28 28 30 31 28 21 15 1404 20 25 29 23 22 14 16 13 1411 25 25 24 23 27 28 23 20 1325 Average (S.D.) Observations 23 (4) 25 (0) 27 (3) 25 (3) 22 (0) 17 (4) 16 (0) 16 (4) 1513 (144) 22 27 26 27 29 28 22 18 1365 (5) (2) (3) (5) (3) (0) (1) (4) (56) TO.PO TO.PO TO,PO TO,PO TO,PO TO,PO T1.PO T1.PO N TO,PO TO.PO TO,PO TO,PO TO.PO TO,PO T1.PO T1,PO N -16- Does not certain TSCA CBt