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AR226-3094 D u P o n t H L O - 1998-01211 TRADE s e c r e t FINAL REPORT Study Title H-23005: IN VITRO MAMMALIAN CELL GENE MUTATION TEST WITH AN INDEPENDENT REPEAT ASSAY Laboratory Project ID: HLO-1998-01211 Authors: Richard H.C. San, Ph.D. Jane J. Clarke, B.S. Study Completed on: March 19,1998 Performing Laboratory: MA BioServices, Inc. 9630 Medical Center Drive, Rockville, MD 20850 for E. I. du Pont de Nemours and Company Haskell Laboratoiy for Toxicology and Industrial Medicine Elkton Road, P. O. Box 50 Newark, Delaware 19714-0050 Performing Laboratory Study No.: G97CK07.782001 Page 1 o f31 Bwnpany Sanitized. Does no! contain TSCA CBS H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-OI211 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with EPA FIFRA (40 CFR 160) and EPA TSCA (40 CFR 792) Good Laboratory Practice Standards, and OECD Principles of Good Laboratory Practice (C(81)30(Final), Annex 2) with the following exception: 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 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 pipettes 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. This deviation did not affect the validity or the integrity of the study. Submitter/Submitter: E. I. du Pont de Nemours and Company Study Director: Study Monitor: Richard H.C. San, Ph.D. Scientific Director :.m < lison, Ph.D. Y / ? / ? 8 Date ate H-23005: In Vitro Mammalian Ceil Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-012II CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Reported Approved and Issued by Study Director: Richard H.C. San, Ph.D. Scientific Director Date -3Sompany Does noi eonfafn TSCA CBS ---- - x/. , ^ iuwmuaium v-cu ociie mutation lest (CHO/HGPRT) with an Independent Repeat Assay STUDY INFORMATION Substance Tested: Synonyms/Codes: H-23005 Physical Characteristics: Tan liquid Stability: The test substance appeared to be stable under the conditions of the study; no evidence o f instability was observed. Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.. '..... -...................... Study Initiated/Completed: December 11,1997/ March 19, 1998 In-Life Initiated/Completed: December 16, 1997 / February 13, 1998 -4- H-230Q5: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 Study Title: Study Number: Study Director: QUALITY ASSURANCE STATEMENT IN VITRO MAMMALIAN CELL GENE MUTATION TEST WITH AN INDEPENDENT REPEAT ASSAY G97CK07.782001 Richard H. C. San, Ph.D. This study has been divided into a series of in-process phases. Using a random s a m p l i n g _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. FDA Good Laboratory Practice Regulations (21 CFR 58), the U.S. E P A GLPs (40 CFR 792 and 40 CFR 160), the UK GLP Compliance Programme, the Japanese GLP Standard, and the OECD Principles of Good Laboratory Practice and to assure that the study is conducted according to the protocol and relevant Standard Operating Procedures. The following are the inspection dates, phases inspected, and report dates of QA inspections of this study. * INSPECT ON 11 DEC 97, TO STUDY DIR 11 DEC 97, TO MGMT 11 DEC 97 PHASE: Protocol Review INSPECT ON 23 JAN 98-27 FEB 98, TO STUDY DIR 27 FEB 98, TO MGMT 27 FEB 98 PHASE: Scoring toxicity plates INSPECT ON 23 FEB 98-24 FEB 98, TO STUDY DIR 24 FEB 98, TO MGMT 27 FEB 98 PHASE: Draft Report INSPECT ON 19 MAR 98, TO STUDY DIR 19 MAR 98, TO MGMT 19 MAR 98 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. DAT 'Company S a" *5-'* n w . of TSCA OB! H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 TABLE OF CONTENTS . Page Good Laboratory Practice ComplianceStatement ..................................................................2 C e rtific a tio n .................................................................................................................................. Study Information ................................................................................................................. 4 Quality Assurance Statement ................................................................................................ 5 Table of C ontents...................................................................................................................... 6 Summary ................................................................................................................................... 7 Purpose..................................................................................................................................... .... Characterization of Test and Control A rtic le s ........................................................................8 Materials and Methods .............................................................................................................8 Results and Discussion ....................................................................................................... ] l Conclusion ........................................................................................................................... 12 Records and Sample Storage ........................................................................................... . .13 References.............................................................................................................................. 13 Data T a b le s........................................................................................................................... 14 Appendix I: Historical Control Data ...................... 21 Appendix II: Study Protocol...................................................................................................23 -6- npany Sanitized. Does nc contain r'SCAC H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 SUMMARY The test article, H-23005, was tested in the CHO/HGPRT Mutation Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the dose range for the initial mutagenesis assay. The initial and independent repeat mutagenesis assays were used to evaluate the mutagenic potential of the test article. The dosing solutions were adjusted to compensate for the purity of the test article. Sterile distilled water was determined to be the solvent of choice based on solubility information from the Sponsor and compatibility with the target cells. The test article was soluble in sterile distilled water at a concentration of 50 mg/mL, the maximum concentration prepared in the preliminary toxicity assay. In the preliminary toxicity assay, the maximum concentration of H-23005 tested was 5000 pg/mL. There was visible precipitate in the treatment medium at concentrations --1500 pg/mL at the end of treatment. Selection of dose levels for the mutagenesis assay was based on the cloning efficiency relative to the solvent control and the solubility limitations. Substantial toxicity, i.e., cloning efficiency <50% of the solvent control, was observed at >50 pg/mL without activation and at 5000 pg/mL with S9 activation. Based on these findings, the doses chosen for the initial mutagenesis assay ranged from 15 to 2500 pg/mL for both the non-activated and S9-activated cultures. In the initial mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 10s clonable cells, were observed. Visible precipitate was observed in treatment medium at concentrations >1500 pg/mL at the end o f treatment. Concentrations of <500 pg/mL were soluble in treatment medium. Toxicity, i.e., cloning efficiency <50% of the solvent control, was observed at doses o f >1500 pg/mL without S9 activation and at 2500 pg/mL with S9 activation. Based on these findings, the doses chosen for the independent repeat assay ranged from 50 to 1500 pg/mL for the non-activated cultures and from 50 to 2500 pg/mL for the S9-activated cultures. In the independent repeat assay, no positive responses were observed. Visible precipitate was observed in treatment medium at concentrations >750 pg/mL at the end o f treatment. Concentrations of <500 pg/mL were soluble in treatment medium. No toxicity was observed at any dose levels, with or without S9 activation. Under the conditions of this study, test article H-23005 was concluded to be negative in the CHO/HGPRT Mutation Assay. -7Company Satirized. Does not contain TSC CB! H-23005: in Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 PURPOSE The purpose of this study is to evaluate the mutagenic potential of the test article based on quantitation o f forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus of Chinese hamster ovary (CHO) cells. CHARACTERIZATION OF TEST AND CONTROL ARTICLES The test article, H-23005, was received by MA BioServices, Inc. (MA) on December 9, 1997 and was assigned the code number 97CK07. The test article was characterized by the Sponsor .^ ^ le a i^ ta rn iq u id ^ h ic h s h o u ld be stored at room temperature. Its purity was given ^ H p H B I j B I H H B Q l n l J p o n receipt, the test article was described as pale yellow liquid with very tiny, dark particles and was stored at room temperature, protected from exposure to light. The vehicle used to deliver H-23005 to the test system was sterile distilled water (CAS 7732 18-5) obtained from Life Technologies. The dosing solutions were adjusted to compensate for the purity of the test article. Ethyl methanesulfonate (EMS), CAS 62-50-0, lot 04625BR, expiration 3/98, was obtained from Aldrich Chemical Company and was used at a stock concentration of 20 pl/mL as the positive control for the non-activated test system. Benzo(a)pyrene (B(a)P), CAS 50-32-8, lot 96H2600, expiration 9/98, was obtained from Sigma Chemical Company and was used at a stock concentration of 400 pg/mL as the positive control for the S9-activated test system. Test System MATERIALS AND METHODS CHO-K,-BH4 cells were obtained from Dr. Abraham Hsie, Oak Ridge National Laboratories, Oak Ridge, TN. CHO cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT) then frozen. The freeze lot o f cells was tested and found to be free o f mycoplasma contamination. Cells used in the mutation assay were within four subpassages from frozen stock in order to assure karyotypic stability. Metabolic Activation System , Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor-1254, 500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at < -70C until used. Each bulk preparation o f S9 was assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethyl-benz(a)anthracene to forms mutagenic - 8- H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 to Salmonella typhimurium TA100. Immediately prior to use, the S9 reaction mixture was prepared by mixing S9 and 10 mM calcium chloride (CaCI2) with a filter-sterilized cofactor pool to contain 100 pi S9/mL cofactor pool, 4 mM nicotinamide adenine dinucleotide phosphate (NADP), 5 mM glucose-6-phosphate, 30 mM potassium chloride (KC1), 10 mM magnesium chloride (MgCl2), and 50 mM sodium phosphate buffer, pH 8.0. The S9 reaction mixture was stored on ice until used. The S9 reaction mixture for the preliminary toxicity assay contained only 2.5 mM glucose-6-phosphate due to a calculation error. Since the data from the preliminary toxicity assay provided a toxicity profile that was consistent with that in the initial mutation assay, this protocol deviation was not considered to have adversely impacted the integrity or conclusions of this study. Preliminary Toxicity Assay The preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay and consisted of evaluation of test article effect on colony-forming efficiency. CHO cells were exposed for 5 hours at 371C to the vehicle alone and nine concentrations o f test article ranging from 0.5 to 5000 pg/mL in both the absence and presence of S9-activation. Mutagenesis Assays The initial and independent repeat mutagenesis assays were used to evaluate the mutagenic potential o f the test article. CHO cells were exposed for 5 hours at 371C to the vehicle alone, appropriate positive controls and at least five concentrations of test article in duplicate in both the absence and presence of S9. Treatm ent of the Target Cells The mutagenesis assay was performed according to a protocol developed from published methodologies (Hsie et al., 1981; and O'Neill et al., 1977). Exponentially growing CHO-K,-BH4 cells were seeded in F12FBS5-Hx at a density of 5xl05 cells/25 cm2 flask and were incubated at 371C in a humidified atmosphere o f 51% CO, in air for 18-24 hours. The cells for the preliminary toxicity assay were seeded at 5.6x105 cells/25 cm2 flask due to a calculation error. Since the data from the preliminary toxicity assay provided a toxicity profile that was consistent with that in the initial mutation assay, this protocol deviation was not considered to have adversely impacted the integrity or conclusions o f this study. F12FBS5-Hx is Ham's F12 medium without hypoxanthine supplemented with 5% dialyzed FBS, 100 units penicillin/mL, 100 pg streptomycin/mL and 2mM L-glutamine/mL. The time o f initiation of chemical treatment was designated as day 0. Treatment was carried out by refeeding the treatment flasks with 4.5 mL F12FBS5-Hx/25 cm2 flask for the non activated study and 3.5 mL F12FBS5-Hx and 1 mL S9 reaction mixture/25 cm2 flask for the -9Company Sanitized. Does not contain TSCA CBS H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 S9-activated study, to which was added 500 ill dosing solution of test article in vehicle or vehicle alone. The positive control cultures were treated with 50 pL of EMS or B(a)P in a total of 5 mL per 25 cm2 flask. Duplicate flasks of cells were exposed to at least five concentrations of the test article for 5 hours at 371C. After the treatment period, all media were aspirated, the cells washed with Ca++- and Mg++-free Hanks' balanced salt solution (CMF-HBSS) and cultured in F12FBS5-Hx for an additional 18-24 hours at 371C. In the preliminary toxicity assay, the cultures treated with 5000 and 1500 pg/mL were rinsed twice with CMF-HBSS instead of once. This is a deviation from the Standard Operating Procedures of the testing facility; however, the Study Director has determined that this deviation did not affect the integrity or conclusions of the study. At this time, the cells were subcultured to assess cytotoxicity and to initiate the phenotypic expression period. Evaluation of Cytotoxicity For evaluation o f cytotoxicity, the replicates from each treatment condition were detached using trypsin and subcultured independently in F12FBS5-Hx, in triplicate, at a density of 100 cells/60 mm dish. After 7-10 days incubation, the colonies were rinsed with HBSS, fixed with methanol, stained with 10% aqueous Giemsa, counted and cloning efficiency determined. Expression of the M utant Phenotype For expression of the mutant phenotype, the replicates from each treatment condition were trypsinized and subcultured independently in F12FBS5-Hx, in duplicate, at a density no greater than 106 cells/100 mm dish. Subculturing by trypsinizing at 2-3 day intervals was employed for the 7-9 day expression period. At the end of the expression period, selection for the mutant phenotype was performed. Selection of the M utant Phenotype For selection of the TG-resistant phenotype, the replicates from each treatment condition were trypsinized and replated, in quintuplicate, at a density of 2xl05 cells/100 mm dish in F 12FBS5-Hx containing 10 pM 6-thioguanine (TG, 2-amino-6-mercaptopurine). For cloning efficiency determinations at the time o f selection, 100 cells/60 mm dish were plated in triplicate. After 7-10 days of incubation, the colonies were fixed, stained and counted for both cloning efficiency and mutant selection. Evaluation of Test Results The cytotoxic effects of each treatment condition were expressed relative to the solventtreated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2xl06 cells: 10 plates at 2x1011cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 106 clonable cells. For experimental conditions in which no mutant colonies were observed, H-23005: In Vitro Mammalian Ceil Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-012II mutant frequencies were expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses giving < 10% relative survival are presented in the data but were not considered as valid data points. Because spontaneous mutant frequencies are very low for the CHO/HGPRT assay, calculation of mutagenic response in terms of fold increase in mutant frequency above the background rate does not provide a reliable indication of the significance of the observed response. The wide acceptable range in spontaneous mutant frequency also suggests the need to set a minimum mutant frequency for a response to be considered positive. Hsie et al. (1981) refer to a level of 50 mutants per 106 clonable cells. In this laboratory, a more conservative approach is used which sets the minimum level at >40 mutants per 106 clonable cells. All conclusions were based on sound scientific judgement; however, the following criteria are presented as a guide to interpretation of the data: The test article was considered to induce a positive response if there was a concentrationrelated increase in mutant frequencies with at least two consecutive doses showing mutant frequencies o f >40 mutants per 106 clonable cells. If a single point above 40 mutants per 106 clonable cells was observed at the highest dose, the assay was considered suspect. If no culture exhibited a mutant frequency of >40 mutants per 106clonable cells, the test article was considered negative. Criteria for a Valid Test The cloning efficiency of the solvent control must be greater than 50%. The spontaneous mutant frequency in the solvent control must fall within the range of 0-25 mutants per 106 clonable cells. The positive control must induce a mutant frequency at least three times that of the solvent control and must exceed 40 mutants per 106 clonable cells. There must be at least four analyzable test article concentrations with mutant frequency data. RESULTS AND DISCUSSION Solubility Sterile distilled water was determined to be the solvent of choice based on solubility information from the Sponsor and compatibility with the target cells. The test article was soluble in sterile distilled water at a concentration of 50 mg/mL, the maximum concentration prepared in the preliminary toxicity assay. - 11 - e o m p a "1' 0 . . TSCA C B i H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 Preliminary Toxicity Assay The results o f the preliminary toxicity assay are presented in Table 1. CHO cells were exposed to solvent alone and nine concentrations o f test article ranging from 0.5 to 5000 pg/mL in the absence and presence o f S9 reaction mixture. There was visible precipitate in the treatment medium at concentrations >1500 pg/mL at the end of treatment. The osmolality o f the solvent control was 319 mmol/kg and the osmolality of the top dose, 5000 pg/mL (which was non-precipitating at the beginning of treatment), was 307 mmol/kg. Cloning efficiency relative to the solvent controls (RCE) was 0% at 5000 pg/mL without activation and 1% at 5000 pg/mL with S9 activation. Based on the results of the toxicity test, the doses chosen for the initial mutagenesis assay ranged from 15 to 2500 pg/mL for both the non-activated and S9-activated cultures. Mutagenesis Assays The cytotoxic effects of the test article (concurrent cytotoxicity) from the initial mutagenesis assay are presented in Table 2. Mutagenicity data are presented in Tables 3 and 4. In both the non-activated and S9-activated systems, cultures treated with concentrations of 15, 50, 150, 500, 1500 and 2500 pg/mL were cloned. Visible precipitate was observed in treatment medium at concentrations >1500 pg/mL at the end of treatment. Concentrations of <500 pg/mL were soluble in treatment medium. Relative cloning efficiency was 0% and 24% at the highest dose tested in the non-activated and S9-activated systems, respectively. The non-activated cultures treated with 2500 pg/mL were not seeded for mutant selection due to toxicity. None o f the treated cultures that were cloned for mutant selection exhibited mutant frequencies of greater than 40 mutants per 106 clonable cells. The cytotoxic effects of the test article from the independent repeat assay are presented in Table 5. Mutagenicity data are presented in Tables 6 and 7. In the non-activated system, cultures treated with concentrations of 50, 150, 500, 750, 1000 and 1500 pg/mL were cloned. In the S9-activated system, cultures treated with concentrations of 50, 150, 500, 1000, 2000 and 2500 pg/mL were cloned. Visible precipitate was observed in treatment medium at concentrations >750 pg/mL at the end of treatment. Concentrations of <500 pg/mL were soluble in treatment medium. Relative cloning efficiency was 68% and 73% at the highest dose tested in the non-activated and S9-activated systems, respectively. None o f the treated cultures exhibited mutant frequencies of greater than 40 mutants per 10s clonable cells. CONCLUSION All criteria for a valid study were met as described in the protocol. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions o f this study, H-23005 did not cause a positive response in the non-activated and S9-activated systems and was concluded to be negative. - 12 - g 'PrtjQi Sg 4 am H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 RECORDS AND SAMPLE STORAGE Upon completion of the final report, all raw data and reports are maintained by the Quality Assurance Unit of MA BioServices, Inc., Rockville, Maryland, in accordance with the relevant Good Laboratory Practice Regulations. REFERENCES Hsie, A.W., D.A. Casciano, D.B. Couch, B.F. Krahn, J.P. O'Neill, and B.L. Whitfield (1981) The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity o f chemicals. A report of the Gene-Tox Program, Mutation Research 86:193-214. O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch, and A.W. Hsie (1977) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system, Mutation Research 45:91-101. - 13 - Company Sanded. Does not contain TSCA CBI H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 TABLE 1 CHO/HGPRT MUTATION ASSAY Preliminary Toxicity Assay Using H-23005 -S9 Treatment1 Colonies per dish 123 Cloning Efficiency2 Relative Cloning Efficiency3 <%> +S9 Treatment* Colonies per dish CPg/ml) 123 Relative Cloning Cloning Efficiency3 Efficiency2 <%) Uater (100 pl/mL): 82 77 H-23005 C/Jg/mL): 0.5 80 89 1.5 57 62 5.0 74 67 15 58 76 50 32 28 150 33 45 500 18 23 1500' 27 22 5000' 00 87 60 77 64 56 52 38 25 11 0 0.82 0.76 0.65 0.68 0.63 0.37 0.39 0.22 0.20 0.00 100 83 82 73 0.79 93 0.5 60 59 70 0.63 80 1.5 59 72 64 0.65 83 5.0 78 79 52 0.70 77 15 78 52 52 0.61 46 50 61 85 76 0.74 47 150 49 77 59 0.62 27 500 48 56 35 0.46 24 1500' 46 57 44 0.49 0 5000' 0 1 1 0.01 100 79 82 88 76 93 78 58 62 1 | Cells were exposed to the test article for 5 hours at 371C. Cloning efficiency = totai colonies counted__________ number of dishes x 100 cells/dish Relative cloning efficiency = cloning efficiency of treatment group X 100 . cloning efficiency of solvent group visible precipitation, as observed at the end of the treatment period - 14 - Cw'g.srv c'armfeac. -,r" H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 TABLE 2 CHO/HGPRT MUTATION ASSAY Concurrent Cytotoxicity Test Using H-23005 Initial Assay __________________________ ;S9____________________________ Relative Cloning Treatment Colonies per dish Cloning Efficiency3 1 2 3 Efficiency2 C%) ___________________________+S9__________________________ Relative Cloning Treatment* Colonies per dish Cloning Efficiency3 12 3 Efficiency2 (%) Water (100 pL/mL): A 47 75 B 56 65 EMS (0.2 fjL/mL): A 23 31 B 27 34 H-23005 (Pg/mL): 15 A 65 88 B 50 50 50 A 78 62 B 46 43 150 A 57 62 B 36 39 500 A 52 61 B 46 42 1500' A B 9 12 15 2500' A 0 0 B0 0 63 36 38 25 45 49 54 42 46 40 46 28 12 2 0 0 0.57 0.30 0.58 0.54 0.47 0.46 0.07 0.00 A 80 86 81 100 B 81 92 82 B(a)P (4.0 pg/mL): A 10 17 28 52 B 14 12 19 0.84 0.17 15 A 87 93 77 101 B 89 96 73 0.86 50 A 91 78 76 95 B 80 86 76 0.81 150 A 110 113 109 82 B 76 85 99 0.99 500 A 91 73 98 80 B 80 77 82 0.84 1500' A 60 70 58 12 B 66 77 65 0.66 2500' A 11 13 16 0 B 27 30 23 0.20 100 20 103 97 118 100 79 24 A and B are duplicate cultures * Cells were exposed to the test article for 5 hours at 371C. Cloning efficiency = total colonies counted__________ number of dishes x 100 cells/dish Relative cloning efficiency = cloning efficiency of treatment group X 100 . cloning efficiency of solvent group visible precipitation, as observed at the end of the treatment period - 15 - Company Sanitized. Does noi contain TSCA CBi H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 TABLE 3 CHO/HGPRT MUTATION ASSAY Non-activated (-S9) Study Using H-23005 Treatment1 Cloning Effici encv at Selection Colonies per Dish Total Cloning 1 2 3 Colonies Efficiency2 Mutant Colonies/Selection Dish 1 2 34 5 Total Mutants/10 Mutant Clonable Colonies Celts3 Water <100 pL/mL): A 43 B 56 EMS (0.2 pL/mL): A 65 B 75 H-23005 (pg/mL): 15 A B 64 57 50 A B 47 47 150 A B 43 58 500 A B 70 66 1500* A B 56 71 40 57 71 68 72 63 61 49 33 68 72 79 58 81 59 63 318 57 77 413 78 73 407 63 46 313 43 62 307 71 58 416 47 68 381 0.53 0.69 0.68 0.52 0.51 0.69 0.64 4 2 a a2 0 1 3 2 1 15 17.7 32 44 57 56 37 41 37 43 41 35 423 307.3 0 1 0 00 0 2 2 10 6 0 0 0 00 3 4 3 6 1 17 0 0 0 02 0 0 0 10 3 1 1 a 00 0 0 0 11 4 4 2 4 42 0 0 1 0 0 17 4.4 16.3 2.9 3.2 13.4 A and B are duplicate cultures 1 Celts were exposed to the test article for 5 hours at 371C. 2 Cloning efficiency = _______ total colonies counted_______ number of dishes x 100 cells/dish 3 Mutants/106 clonable cells = __________________ total mutant colonies_______________________x 10 number selection dishes X cloning efficiency X 2 x 10s cells ^ plate tost to contamination visible precipitation, as observed at the end of the treatment period - 16 - H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 TABLE 4 CHO/HGPRT MUTATION ASSAY Activated (+S9) Study Using H-23005 T reatment1 Clonino Effici encv at Selection Colonies oer Dish Total Cloning 1 2 3 Colonies Efficiency2 Total Mutants/10 Mutant Colonies/Selection Dish Mutant Clonable 1 2 3 4 5 Colonies Cells3 Water (100 /JL/mL): A 78 B 63 B(a)P (4.0 /Jg/mL): A 51 B 54 H-23005 U l g / mL): 15 A B 78 67 50 A B 86 62 150 A B 71 72 500 A B 63 54 1500* A B 60 98 2500* A B 67 60 84 67 71 78 84 55 74 51 76 76 66 75 63 88 63 63 70 75 437 53 60 367 69 47 400 72 59 404 67 72 434 65 64 387 69 53 431 67 49 369 0.73 0.61 0.67 0.67 0.72 0.65 0.72 0.62 1 2 0 31 0 0 0 00 7 4.8 24 26 22 30 23 30 22 36 27 23 263 215.0 0 0 0 00 0 1 0 10 2 1 1 1 41 2 4 2 3 1 20 0 0 0 00 0 0 1 12 4 0 1 0 00 0 13 23 10 1 0 0 00 0 0 0 00 1 0 0 0 00 0 0 0 00 0 1.5 14.9 2.8 7.8 0.7 <0.84 A and B are duplicate cultures 1 Cells were exposed to the test article for 5 hours at 371C. 2 Cloning efficiency = total colonies counted_________ number of dishes x 100 cells/dish 3 Mutants/10 clonable cells = __________________ total mutant colonies_______________________x 10s number selection dishes X cloning efficiency X 2 x IQ3 cells Calculated on the basis of <1 mutant colony observed in the total number of dishes prepared, visible precipitation, as observed at the end of the treatment period - 17 Company Sanitized. Does not contain T S C A CBt H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 TABLE 5 CHO/HGPRT MUTATION ASSAY Concurrent Cytotoxicity Test Using H-23005 Independent Repeat Assay -S9____________________________ Relative Cloning T reatmeiit1 Colonies per dish Cloning Efficiency3 1 2 3 Efficiency2 (%) ___________________________ +S9__________________________ Relative Cloning Treatment1 Colonies per dish Cloning Efficiency3 12 3 Efficiency"! (%> Water (100 pL/mL): A 64 54 B 52 72 EMS (0.2 pL/mL): A 33 34 B 24 42 H-23005 (pg/mL): 50 A 63 75 B 80 80 150 A 36 48 B 39 43 500 A 56 54 B 59 48 750' A 57 73 B 58 35 1000' A 39 44 B 50 42 1500' A 38 36 B 38 39 60 56 46 37 46 61 29 63 62 55 52 45 53 64 51 42 0.60 0.36 0.68 0.43 0.56 0.53 0.49 0.41 A 65 57 74 100 B 61 69 71 B(a)P (4.0 pg/mL): A 28 35 29 60 B 18 19 12 0.66 0.24 50 A 55 59 82 113 B 62 65 58 0.64 150 A 80 81 65 72 B 49 67 46 0.65 500 A 76 75 79 93 B 56 66 65 0.70 1000* A 52 68 60 89 B 46 60 51 0.56 2000' A 61 73 46 82 B 60 48 58 0.58 2500' A 55 65 59 68 B 28 42 39 0.48 100 36 96 98 105 85 87 73 A and B are duplicate cultures ` Celts were exposed to the test article for 5 hours at 371C. Cloning efficiency = total colonies counted___________ number of dishes x 100 cells/dish 3 Relative cloning efficiency = cloning efficiency of treatment group X 100 ,, cloning efficiency of solvent group visible precipitation, as observed at the end of the treatment period - 18 - H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-012II TABLE 6 CHO/HGPRT MUTATION ASSAY Non-activated (-S9) Study Using H-23005 Independent Repeat Assay Treatment1 Cloning Effici ency at Selection Colonies cer Dish Total Cloning 1 2 3 Colonies Efficiency2 Mutant Colonies/Seleetion Dish 1 2 34 5 Total Mutants/10' Mutant Clonable Colonies Cells3 Water (100 /it/mL): A 63 60 B 36 66 EMS (0.2 fjL/ml): A 36 46 B 31 36 H-23005 Ci/g/mL): 50 A B 52 76 56 50 150 A 8 60 96 60 56 500 A B 48 50 55 49 750' A B 57 69 54 61 < o o o 63 44 B 84 61 1500' A B 68 63 49 60 58 65 348 51 32 232 56 74 364 69 59 400 46 73 321 73 67 381 63 73 388 82 53 375 0.58 0.39 0.61 0.67 0.54 0.64 0.65 0.63 2 0 0 10 0 0 0 00 3 2.6 84 75 79 76 85 56 54 49 61 54 67 870.3 1 0 0 00 1 0 1 10 4 1 1 0 01 0 1 0 00 4 0 0 0 00 0 0 0 00 0 1 0 0 12 0 0 0 00 4 0 0 0 00 0 0 0 00 0 000 000 10 00 1 3.3 3.0 <0.9J 3.1 <0.8J 0.8 A^ and B are duplicate cultures 2 Celts were exposed to the test article for 5 hours at 371C. Cloning efficiency = _______ total colonies counted_______ number of dishes x 100 cells/dish 3 Mutants/105 clonable celts = ___________________total mutant colonies__________________ _ x 10s number selection dishes X ctoning efficiency X 2 x 10s cells / . . Calculated on the basis of <1 mutant colony observed in the total number of dishes prepared, visible precipitation, as observed at the end of the treatment period - 19 Company Sanitized. Does not contain TSCA CB1 H-23005: In Vitro Mammalian Ceil Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-012il TABLE 7 CHO/HGPRT MUTATION ASSAY Activated (+S9) Study Using H-23005 Independent Repeat Assay Treatment1 Clonino Effici encv at Selection Colonies tier Dish Total Cloning 1 2 3 Colonies Efficiency Total Mutants/10 Mutant Colonies/Selection Dish Mutant Clonable 1 2 3 4 5 Colonies Cells3 Water (100 pL/mL): A 108 127 B 75 82 B(a)P (4.0 pg/mL): A 121 81 B 72 96 H-23005 (pg/mL>: 50 A B 84 115 76 92 150 A B 97 110 75 99 500 A B 87 79 76 89 1000* A S 76 80 79 81 2000* A B 86 91 86 71 2500* A B 55 77 104 91 118 64 574 103 82 555 109 82 558 115 100 596 115 96 542 98 65 479 95 79 508 66 111 504 0.96 0.93 0.93 0.99 0.90 0.80 0.85 0.84 0 0 0 00 0 0 1 01 2 1.0 33 26 19 24 30 15 17 21 21 19 225 121.6 2 1 4 41 0 0 0 1 1 14 0 2 1 20 0 0 0 00 5 3 3 2 22 0 0 0 00 12 0 0 0 00 10 1 11 4 0 0 0 00 0 3 0 00 3 0 0 0 00 0 0 0 00 0 7.5 2.5 6.6 2.5 1.8 <0.64 A and B are duplicate cultures 1 Cells were exposed to the test article for 5 hours at 371C. 2 Cloning efficiency = total colonies counted________ number of dishes x 100 cells/dish 3 Mutants/10 clonabte cells = ___________________total mutant colonies______________________x 10 number selection dishes X cloning efficiency X 2 x 10 cells . Calculated on the basis of <1 mutant colony observed in the total number of dishes prepared, visible precipitation, as observed at the end of the treatment period - 20 - H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 APPENDIX I Historical Control Data -21 - -- -------- -- ------- Company Sanitized. Does not contain TSCA CBI / H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 CHO/HGPRT Assay Historical Control Data 1995 - 1997 Mean MF SD Maximum Minimum Non-activated Solvent Control 0.2pL/mL EMS 7.0 312.3 6 .2 97.3 22.9 725.7 0.0 144.0 S9-activated Solvent Control 4.0pg/mL B(a)P 6.1 180.3 6.0 63.0 23.1 349.0 0.5 38.1 Solvent control (culture medium, distilled water, saline, DMSO, ethanol, acetone or vehicle supplied by Sponsor) EMS B(a)P MF SD Ethyl methanesulfonate Benzo(a)pyrene Mutant frequency per 106 clonable cells Standard deviation - 77 - Sanivz'i H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 APPENDIX II Study Protocol - 23 Company Sanitized. Does no! contain TSCA CBS H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 Received by RklQh^ zttfi.( MA Study Number: C- K 0 7 . 72X0 I 1.0 PURPOSE In Vitro Mammalian Cell Gene Mutation Test with an Independent Repeat Assay The purpose o f this study is to assess the mutagenic potential o f a test article based on quantitation o f forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus o f Chinese hamster ovary (CHO) cells. 2.0 SPONSOR 2.1 Name: E. I. du Pont de Nemours and Company 2.2 Address: Haskell Laboratory for Toxicology and Industrial Medicine P.O . Box 50, Elkton Road Newark, DE 19714 2.3 Representative: Brian H .'Mathison, Ph.D. 2.4 Sponsor Project #: 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Article: H-23005 3.2 Controls: Negative: Positive: Test article solvent (or vehicle) Ethyl methanesulfonate (EMS) Benzo(a)pyrene (BaP) 3.3 Determination o f Strength, Purity, etc. - The Sponsor w ill be directly responsible for determination and documentation o f the analytical purity and composition o f the test article and the stability and strength o f the dosing solutions. 3.4 Test Article Retention Sample The retention o f a reserve sample o f the test article will be the responsibility o f the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility MA BioServices, Inc. Protocol No. S P GT782001 12/02/97 ^ M A Bio Services* - 24 - Sanftize. miti, r r-tst H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 4.2 Address: 9630 Medical Center Drive Rockville, MD 20850 4.3 Study Director: Richard H. C. San, Ph.D. 5.0 TEST SCHEDULE 5.1 Proposed Experimental Initiation Date: 5.2 Proposed Experimental Completion Date: ' */'* / ? 7 */ f a /? 8 5.3 Proposed Report Date: 6.0 TEST SYSTEM */ *7/> 8 The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number o f 20, a population doubling time o f 12-14 hours, and a cloning efficiency o f usually greater than 80% (Li et al., 1987). This subclone (D l) was derived by Dr. Abraham Hsie, Oak Ridge National Laboratories, Oak Ridge, TN. CHO cells were cleansed in medium supplemented with HAT (hypoxanthine, aminopterin and thymidine) then frozen. Cells used in the mutation assay will not exceed four subpassages from frozen stock. Each freeze lot o f cells has been tested and found to be free o f mycoplasma contamination. The CHO/HGPRT assay was designed to select for mutant cells which have become resistant to such purine analogues as 6-thioguanine (TG) and 8-azaguanine as a result o f mutation at the X-chromosome-linked HGPRT locus (O'N eill et al., 1977; Hsie et at., 1981; Machanoff et al., 1981; Li et al., 1987). This system has been demonstrated to be sensitive to the mutagenic action o f a variety o f chemicals (Hsie et al., 1981). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The assay will be performed by exposing CHO cells for 5 hours to five concentrations o f test article as well as positive and the solvent controls in the presence and absence o f an exogenous source o f metabolic activation. After a seven to nine day expression period, the treated cells w ill be cultured in the presence o f 10 iM TG for selection o f mutant colonies. The mutagenic potential o f a test article will be determined by its ability to induce a dose-related increase in the number o f TG-resistant mutant colonies when compared to the solvent control. 7.1 Selection o f Solvent Unless the Sponsor has indicated the test article solvent, a solubility determination will be conducted to measure the maximum soluble concentration in a variety o f solvents. Solvents compatible with this test system, in order o f preference, include, but are not limited to, culture medium or distilled water (CAS 7732-18-5), dimethylsulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5) and acetone (CAS 67 64-1). The solvent o f choice will be that solvent, selected in order o f preference. Protocol No. SPGT7S200I 12/02/97 2 of 8 ^ M A Bio Services - 25 - Eompany no! contain T S C A CB! H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 that permits preparation o f the highest soluble stock concentration, up to a maximum o f 500 mg/ml. 7.2 Dose Selection The optimal dose levels for the mutation assay will be selected following a preliminary toxicity test based upon colony-forming efficiency. Approximately 5 x 10s CHO cells will be seeded into 25 cm2 flasks and incubated at 371C in a humidified atmosphere o f 51% CO, in air. Eighteen to 24 hours later, cells w ill be exposed to solvent alone and to no less than nine concentrations o f test article, the highest concentration being the lowest insoluble dose in treatment medium not to exceed 5000 pg/ml. The pH o f the treatment medium will be adjusted, i f necessary, to maintain a neutral pH in the treatment medium. The osmolality o f the highest soluble treatment condition will also be measured. Exposure w ill be for 5 hours at 371C in a humidified atmosphere o f 51% C 0 2 in air in the presence and absence o f S9 activation. After the treatment period, all media will be aspirated, the cells washed, and cultures given fresh culture medium. Eighteen to 24 hours later, the treated cells will be trypsinized and reseeded at a density o f 100 cells/60 mm dish. After 7-10 days incubation at 371C in a humidified atmosphere o f 51% CO, in air, colonies will be fixed with 95% methanol, stained with 10% aqueous Giemsa, and counted. The cell survival o f the test article-treated groups will be expressed relative to the solvent control (relative cloning efficiency). Whenever possible, the high dose will be selected to give a cell survival o f 10 20%. Four lower doses will be selected, at least one o f which will be non-toxic. For freely soluble, non-toxic test articles, the highest concentration will be 5000 pg/ml. For relatively insoluble, non-toxic test articles, the highest concentration will be the lowest insoluble dose in treatment medium but not to exceed 5000 pg/ml. If dose-related cytotoxicity or mutagenicity is noted, irrespective o f solubility, then the top concentration will be based on toxicity as described above. In all cases, precipitation will be evaluated at the beginning and at the end o f the treatment period using the naked eye (ICH, 1995). 7.3 Frequency and Route o f Administration Cell cultures w ill be treated for 5 hours by way o f a vehicle compatible with the system, both in the presence and absence o f metabolic activation. This technique o f administration has been demonstrated to be effective in the detection o f chemical mutagens in this system. 7.4 Exogenous Metabolic Activation Aroclor 1254-induced rat liver S9 will be used as the metabolic activation system. The source o f S9 will be adult male Sprague-Dawley rats induced by a single injection o f Aroclor 1254 at a dose level o f 500 mg/kg body weight five days prior to sacrifice. The S9 will be batch prepared and stored frozen approximately -70C until used. Protocol No. SPGT78200I 12/02/97 3 of 8 -26 - Xi i/i.rsFtiz& d . OSes, n o t c H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 Immediately prior to use. the S9 will be thawed and mixed with a cofactor pool to contain 100 pi S9/ml reaction mixture o f approximately 4 mM NADP. 5 mM glucose-6-phosphate, 10 mM MgCI,, 30 mM K.C1. 10 mM CaCl,, and 50 mM sodium phosphate buffer, pH 8.0 (Machanoff et al,, 1981). The S9 reaction mixture will be stored on ice until used. Controls 7.5.1 Negative Control The solvent (or vehicle) for the test article will be used as the negative control. 7.5.2 Positive Control Ethyl methanesulfonate (EMS) will be used at a concentration o f 0.2 pl/ml as the positive control for the non-activated study. Benzo(a)pyrene (BaP) will be used at a concentration o f 4 pg/ml as the positive control for the S9 activated study. 7.6 Preparation o f Target Cells Exponentially growing CHO-K1-BH4 cells will be seeded in F12 medium without hypoxanthine, supplemented with 5% dialyzed serum, 2mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 pg/ml) (F12FBS5-Hx), at a density o f 5 x 10s 1 ' ceils/25 cm2 surface area and w ill be incubated at 371C in a humidified atmosphere o f 51% CO, in air for 18-24 hours. 7.7 Identification o f the Test System Using a permanent marking pen, the treatment flasks will be identified by the study number and a code system to designate the treatment condition and test phase. 7.8 Treatment o f Target Cells The time o f initiation o f chemical treatment will be designated as day 0. Cells will be exposed, in duplicate cultures, to five concentrations o f test article for 5 hours at 371C in a humidified atmosphere o f 51% C 0 2 in air. For each 25 cm2 o f surface area treated, the treatment medium will consist o f 5 ml F12FBS5-Hx and 50 pi o f control or test article diluted to the appropriate concentration in solvent for the non-activated study, or 4 ml F12FBS5-Hx, 1 ml S9 reaction mixture, and 50 pi o f control or test article diluted to the appropriate concentration in solvent for the activated study. After the treatment period, all media will be aspirated, the cells washed with Hank's Balanced Salt Solution (CMF-HBSS) and cultured in F12FBS5-Hx at 371C in a humidified atmosphere o f 51% CO, in air. After 18-24 hours incubation, the cells will be subcultured to assess cytotoxicity and to continue the phenotypic expression period. Protocol No. SPGT782001 12/02/97 4 of 8 - 27 Company Sanitized. Does not contain TSCA CBP H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 7.9 Estimation o f Toxicity For evaluation o f cytotoxicity, the replicate cultures from each treatment condition . will be subcultured independently in F 12FBS5-H.X. in triplicate, at a density o f 100 cells/60 mm dish. After 7-10 days incubation at 371C in 5 1% CO, in air. colonies will be fixed with 95% methanol, stained with 10% aqueous Giemsa, and counted. Cytotoxicity will be expressed relative to the solvent-treated control cultures. 7.10 Expression o f the Mutant Phenotype For expression o f the mutant phenotype, the replicates from each treatment condition will be subcultured independently in F12FBS5-Hx, in duplicate, at a density o f no greater than 106 cells/100 mm dish. Subculture as above at 2-3 day intervals will be performed for the 7-9 day expression period. At this time, selection for the mutant phenotype will be performed. 7.11 Selection o f the Mutant Phenotype For selection o f the TG-resistant phenotype, cells from each treatment condition will be plated into a maximum o f five dishes at a density o f 2 x 10s cells/100 mm dish in F12FBS5-Hx containing 10 pM TG. For cloning efficiency at the time o f selection, 100 cells/60 mm dish will be plated in triplicate in medium free o f TG. After 7-10 days o f incubation, the colonies w ill be fixed, stained and counted for both cloning efficiency at selection and mutant selection. 7.12 Independent Repeat Assay An independent repeat mutation assay, with and without a metabolic activation system, will be conducted. 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST 8.1 Negative Controls The cloning efficiency o f the solvent (or vehicle) control must be greater than 50%. The spontaneous mutant frequency in the solvent (or vehicle) control must fall within the range o f 0-25 mutants per 106 clonable cells. 8.2 Positive Controls The positive control must induce a mutant frequency at least three times that o f the solvent control and must exceed 40 mutants per 106 clonable cells. 8.3 Test Article-Treated Cultures A minimum o f four analyzable concentrations with mutant frequency data will be required. Protocol No. SPGT782001 12/02/97 5 of 8 & M A Bio Services- -28 - turn ' l"Ji i' i->2ESCi }BvA H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 9.0 e v a l u a t io n o f t e s t r e s u l t s The cytotoxic effects o f each treatment condition are expressed relative to the solvent treated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition is calculated by dividing the total number o f mutant colonies by the number o f cells selected, corrected for the cloning efficiency o f cells at the time o f mutant selection, and is expressed as TG-resistant mutants per 106 cionable cells. For experimental conditions in which no mutant colonies are observed, mutant frequencies will be expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses giving <10% relative survival are not considered as valid data points and w ill not be included in the data analysis. Spontaneous mutant frequencies in this assay range from 0 to 25 mutants per 106 cionable cells. A s a result, calculation o f mutagenic response in terms o f fold increase in mutant frequency above the background rate does not provide a reliable indication o f the significance o f the observed response. The wide acceptable range in spontaneous mutant frequency also suggests the need to set a minimum mutant frequency for a response to be considered positive. Hsie et al, (1981) refer to a level o f 50 mutants per 106 cionable cells. In this laboratory, a more conservative approach is used which sets the minimum significant level at >40 mutants per 106 cionable cells. All conclusions will be based on sound scientific judgement; however, the following will be used as a guide to interpretation o f the data. The test article will be considered to induce a positive response if there is a concentration-related increase in mutant frequencies with at least two consecutive doses showing mutant frequencies o f >40 mutants per 106 cionable cells. If a single point above 40 mutants per IO6 cionable cells is observed at the highest dose, the assay will be considered suspect If no culture exhibits a mutant frequency o f >40 mutants per 10s cionable cells, the test article will be considered negative. 10.0 REPORT A report o f the results o f this study will be prepared by the Testing Laboratory and will accurately describe all methods used for generation and analysis o f data. Results presented will include, but not be limited to: test substance: identification and CAS no., i f known; physical nature and purity, if known; physicochemical properties relevant to the conduct o f the study, if known; stability o f test article, if known. solvent/vehicle: justification for choice o f vehicle; solubility and stability o f test article in solvent/vehicle, i f known. cell type used, number o f cultures, methods for maintenance o f cell cultures rationale for selection o f concentrations and number o f cultures Protocol No. SPGT782001 12/02/97 6 of 8 - 29 - Company S a n d e d . Ones no! contain TSC CBF H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont HLO-1998-01211 test conditions: composition o f media. CCX concentration, concentration o f test substance, vehicle, incubation temperature, incubation time, duration o f treatment, cell density during treatment, type o f metabolic activation system, positive and negative - controls, length o f expression period, selective agent method used to enumerate numbers o f viable and mutant cells dose-response relationship, if applicable positive and solvent control historical data 11.0 RECORDS AND ARCHIVES Upon completion o f the final report, all raw data and reports will be maintained by the Quality Assurance Unit o f MA BioServices, Inc., Rockville, MD in accordance with the relevant Good Laboratory Practice Regulations. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply with EPA Health Effects Testing Guidelines, Draft Document OPPTS 870.5300 (Detection o f Gene Mutations in Somatic Cells in Culture), Fed. Register, vol. 61, June 1996, with OECD Guideline for the Testing o f Chemicals, Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test), Proposal for Updating Guideline 476, February 1997, and with the International Conference on Harmonisation o f Technical Requirements for Registration o f Pharmaceuticals for Human Use, Genotoxicity: Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals, Step 4 Final Draft, July 18, 1995. This study will be performed in compliance with the provisions o f the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. Will this study be submitted to a regulatory agency? If so, to which agency or agencies? f f t f { tscjJ g y .rcg? Unless arrangements are made to the contrary, unused dosing solutions will be disposed o f following administration to the test system and all residual test article will be disposed o f following finalization o f the report. 13.0 REFERENCES EPA Health Effects Testing Guidelines, Draft Document OPPTS 870.5300 (Detection o f Gene Mutations in Somatic Cells in Culture), Fed. Register, vol. 61, June 1996. Hsie, A.W., Casciano, D.A., Couch, D.B., Krahn, B.F., O'Neill, J.P. and Whitfield, B.L. (1981). The use o f Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity o f chemicals. A report o f the Gen-Tox Program. Mutation Research 86: 193-214. Protocol No. SPGT782001 12/02/97 7 of 8 M A Bio Services' -30- mitlzed, H-23005: In Vitro Mammalian Cell Gene Mutation Test (CHO/HGPRT) with an Independent Repeat Assay DuPont H LO-1998-01211 International Conference on Harmonisation o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicitv: Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals, Step 4 Final Draft. July 18, 1995. Li, A. P., Carver, J.H., Choy, W.N., Hsie, A.W ., Gupta, R.S., Loveday. K..S., O'Neill, J.P., Riddle, J.C., Stankowski. L.F. and Yang, L.L. (1987). A guide for the performance o f Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase gene mutation assay. Mutation Research 189: 135-141. Machanoff, R., O'Neill, J.P. and Hsie, A.W. (1981). Quantitative analysis o f cytotoxicity and mutagenicity o f benzo(a)pyrene in mammalian cells (CHO/HGPRT). Chem. Biol. Interactions. 34: 1-10. O'Neill, J.P., Brimer, P.A., Machanoff, R , Hirsch, J.P. and Hsie. A.W. (1977). A quantitative assay o f mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition o f the system. Mutation Research 45: 91-101. OECD Guideline for the Testing o f Chemicals, Guideline 476 {In Vitro Mammalian Cell Gene Mutation Test), Proposal for Updating Guideline 476, February 1997. 14.0 APPROVAL y' ^___ ^ S P O N S O R RERSENTATIVE (Print or Type Name) MA STUDY DIRECTOR DATE * /" /9 7 DATE Protocol No. SPGT782001 12/02/97 8 of 8 - 31 - 9 M A Bio Services" Company c-mfafrt TSC4 f:
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