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M U - 3 30*1 /V/U ai- 230J D u p on t-13542 FINAL REPORT Study Title H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Authors Ramadevi Gudi, Ph.D. Meena Rao, B.S. Study Completion Date 24 November 2003 Testing Facility BioReliance 9630 Medical Center Drive Rockville, MD 20850 for E. I. duPont de Nemours and Company DuPont Haskell Laboratory P.O.Box 50 1090 Elkton Road Newark, DE 19714-0050 BioReliance Study Number AA79YX.341.BTL Page 1 o f 39 Company Sanitized. B o w not contain TSCA C8i H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________ Dupont-13542 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards except for the item documented below. The items listed do not impact the validity o f the study. The test substance was characterized by the Sponsor prior to the initiation o f this study. Although characterization was not performed under Good Laboratory Practice Standards, the accuracy o f the data is considered sufficient for the purposes of this study. Analyses to determine the uniformity or concentration o f the test mixtures and their stability were not performed by the testing facility or the Sponsor. BioReliance Study Director: BioReliance Study Management Ramadevi Gudi, Ph.D. t $ lf //6UJ$oq3 Date * 4 N<rv* 003 Date BioReliance Study No. AA79YX.341.BTL 2 Company Sanitized. Does not contain TSCA CBS H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 Quality Assurance Statement Study Title: H-24768: IN VITRO MAMMALIAN CHROMOSOME ABERRATION STUDY IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES Study Number: AA79YX.34LBTL Study Director: Ramadevi Gudi, PhD. This study has been divided into a series of in-process phases. Using a random sampling approach, Quality Assurance monitors each of these phases over a series of studies. Procedures, documentation, equipment records, etc., are examined in order to assure that the study is performed in accordance with the U.S. FDA Good Laboratory Practice Regulations (21 CFR 58), the U.S. EPA GLPs (40 CFR 792 and 40 CFR 160), the UK GLP Regulations, the Japanese GLP Standard, and the OECD Principles of Good Laboratory Practice and to assure that the study is conducted according to the protocol and relevant Standard Operating Procedures. The following are the inspection dates, phases inspected, and report dates of QA inspections of this study. Inspect On: 18-Sep-03 - 18-Sep-03 To Study Dir 18-Sep-03 To Mgmt 18-Sep-03 Phase: Protocol Review Inspect On: 16-Oct-03 - 16-Oct-03 To Study Dir 16-Oct-03 To Mgmt 16-Oct-03 Phase: Coding of slides Inspect On: 09-Nov-03 - 09-Nov-03 To Study Dir 10-Nov-03 To Mgmt ll-Nov-03 Phase: Draft Report and data audit Inspect On: 17-Nov-03 - 21-Nov-03 To Study Dir 21-Nov-03 To Mgmt 24-Nov-03 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. QUALITY ASSURANCE DATE BioReliance Study No. AA79YX .341 .BTL 3 Company Sanitized. Does not contain TSCA CSI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes CERTIFICATION Dupont-13542 We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study Director: Ramadevi Gudi, Ph.D. BioReliance Study Director Date Approved by Study Monitor: E. Maria Donner, PhJD. Senior Research Toxicologist 13 - N o^ - Oo Z Date BioReliance Study No. AA79YX.341.BTL 4 Company Sanitized. Does not contain TSCA CBl H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes TABLE OF CONTENTS Dupont-13542 PAGE G O O D L A B O R A T O R Y P R A C T IC E C O M P L IA N C E S T A T E M E N T ......................................................................... 2 Q U A L IT Y A S S U R A N C E S T A T E M E N T .....................................................................................................................................3 C E R T IF IC A T IO N ............................................................................................................ 4 S T U D Y IN F O R M A T IO N ..................................................................................................................................................................... 7 S U M M A R Y ................................................................................................................................................... 8 P U R P O S E ......................................................................................................................................................... 10 C H A R A C T E R IZ A T IO N O F T E S T A N D C O N T R O L S U B S T A N C E S ...................................................................... 10 M A T E R IA L S A N D M E T H O D S .....................................................................................................................................................10 Testing G u id e l in e s .........................................................................................................................................................................10 T est Sy s t e m ........................................................................................................................................................................................ 11 a c t iv a t io n Sy s t e m .........................................................................................................................................................................1 1 S olubility Te s t .................................................................................................................................................................................11 P relim inary t o x ic it y a s s a y .....................................................................................................................................................11 C h ro m o so m e A be r r a t io n A s s a y ............................................................................................................................................ 12 C ollection o f M et a ph a se Cel ls............................................................................................................................................. 13 S lide P re pa r a t io n ........................................................................................................................................................................... 13 S election of D o s e L ev els fo r A n a l y s is ............................................................................................................................. 13 EVALUATION OF METAPHASE CELLS............................................................................................................................................ 14 C o ntr o ls.............................................................................................................................................................................................. 14 Ev a lu a t io n o f T e s t R e s u l t s .................................................. 14 C riteria fo r d e t e r m in a t io n o f a V alid Te s t ....................................................................................................................15 D e v ia t io n s.......................................................................................................................................................................................... 15 A r c h iv e s ............................................................................................................................... 15 R E S U L T S A N D D I S C U S S I O N ....................................................................................................................................................... 16 S olubility T e s t ............................................................................................................... ;.............................................................. 16 P relim inary T o xicity A s s a y ..................................................................................................................................................... 16 C h ro m o so m e Ab e r r a t io n A s s a y ............................................................................................................................................ 16 C O N C L U S I O N ........................................................................................................................................................................................ 17 R E F E R E N C E S ........................................................................................................................................................................................ 18 BioReliance Study No. AA79YX.341.BTL 5 Company Sanitized. Does not contain TSCA CBl H-24768: In Vitro Mammalian Chromosome Aberration Study TABLE 1 PRELIMINARY TOXICITY TEST USING H-24768 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT.........................................................19 TABLE 2 PRELIMINARY TOXICITY TEST USING H -24768 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT....................................................... 20 TABLE 3 PRELIMINARY TOXICITY TEST USING H -24768 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION.................................................................................................... 21 TABLE 4 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24768 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST......................................................22 TABLE 5 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24768 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST.....................................................23 TABLE 6 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24768 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 20 HOUR TREATMENT, 20 HOUR HARVEST.................................................. 24 TABLE 7 SUM M ARY................................................................................... 25 APPENDIX A HISTORICAL CONTROL DATA...............................................................................................26 APPENDIX B STUDY PROTOCOL................................ 29 BioReliance Study No. AA79YX.341.BTL 6 Company Sausuxed Uoes not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________ STUDY INFORMATION Dupont-13542 . H-24768 Haskell Number: 24768 CAS Registry Number: Physical Characteristics: Tan to light brown paste 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.A. Study Initiated/Completed: September 15,2003 / (see report cover page) In-Life Initiated/Completed: September 17,2003 / October 26, 2003 BioReliance Study No. AA79YX.341.BTL 7 Company Samuzea. Does not contain 5CA CBf H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 SUMMARY The test substance, H-24768, was tested in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL) in both the absence and presence o f an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for testing in die cytogenetic test. The chromosome aberration assay was used to evaluate the clastogenic potential o f the test substance. Dimethyl sulfoxide (DMSO) was determined to be the solvent o f choice based on the solubility o f the test substance and compatibility with die target cells. The test substance was soluble in DMSO at a concentration of 100 mg/mL, the maximum concentration prepared in the assay. In the preHminary toxicity assay, the maximum dose tested was 1000 pg/mL. Human peripheral blood lymphocytes were treated in the absence and presence o f an Aroclor-induced S9 activation system for 4 hours, and continuously for 20 hours in the absence o f S9 activation. The test substance was soluble in treatment medium at all concentrations tested. Selection o f dose levels for the chromosome aberration assay was based on a reduction in the mitotic index relative to the solvent control. Substantial toxicity, i.e., at least a 50% reduction in mitotic index, was observed at doses o f 30 pg/mL in all three exposure groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 2.5 to 100 pg/mL for all three exposure groups. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system. All cells were harvested at 20 hours after treatment initiation. The test substance was soluble in treatment medium at all concentrations tested. Selection o f doses for microscopic analysis was based on mitotic inhibition (the lowest dose with at least 50% reduction in mitotic index, relative to the solvent control and two lower doses) in all harvests. The results o f the assay are summarized in the following table: Treatment Time (hours) Recovery Time (hours) Harvest Time (hours) Mitotic Index Reduction* at S9 highest dose LED for Structural scored (25 pg/mL) Aberrations (pg/mL) LED for Numerical Aberrations (pg/mL) 4 16 20 - 52% None None 20 0 20 - 58% None None 4 16 20 + 52% None None * relative to solvent control at high dose evaluated for chromosome aberrations LED = Lowest Effective Dose BioReliance Study No. AA79YX.341.BTL 8 Company Sanitized. Does not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 Based on the findings o f this study, H-24768 was concluded to be negative for the induction o f structural and numerical chromosome aberrations in the non-activated and S9 activated test systems in the in vitro mammalian chromosome aberration test using human peripheral lymphocytes. BioReliance Study No. AA79YX.341.BTL 9 Company Sanitized. Does not contain TSC CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 PURPOSE The purpose o f this study was to evaluate the clastogenic potential o f a test substance based upon its ability to induce chromosome aberrations in human peripheral lymphocytes. A copy of the study protocol is included in Appendix B. CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test substance, H-24768, was received by BioReliance on September 9, 2003 and was assigned the code number AA79YX. The test substance was characterized by the Sponsor as a tan to light brown paste that should be stored at room temperature. Upon receipt, the test substance was described as a brown pasty substance and was stored at room temperature, protected from exposure to light. The Sponsor has determined the identity, strength, purity composition or other characteristics to define the test substance and the stability o f the test substance. The solvent used to deliver H-24768 to the test system was dimethyl sulfoxide (DMSO, CAS No.: 67-68-5) obtained from Fisher Scientific. Mitomycin C (MMC; CAS No.: 50-07-7), was obtained from the Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations of 30 and 60 pg/mL for use as the positive control in the non-activated test system. Cyclophosphamide (CP; CAS No.: 6055-19-2), was obtained from Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations o f 2 and 4 mg/mL for use as the positive control in the S9 activated test system. For each positive control, one dose with sufficient scorable metaphase cells was selected for analysis. The solvent for the test substance was used as the solvent control at the same concentration as that found in the test substance-treated groups. The negative and positive control substances have been characterized as per the Certificates o f Analysis on file with the testing facility. The stability o f the negative and positive control substances and their mixtures was demonstrated by acceptable results that m et the criteria for a valid test. MATERIALS AND METHODS Testing Guidelines This study was conducted in compliance with OECD Guideline 473 {In Vitro Mammalian Chromosome Aberration Test), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February 1998 and with the International Conference on Harmonization o f Technical Requirements for Registration o f Pharmaceuticals for Human Use (1996 and 1997). BioReliance Study No. AA79YX.341.BTL 10 Company Sanitized. Does not contain TSC C: H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 Test System Peripheral blood lymphocytes were obtained from a healthy non-smoking 27 year old adult male on 15 September 2003 for the preliminary toxicity assay, and on 29 September 2003 for the definitive assay. The donor had no recent history o f radiotherapy, viral infection or the administration o f drugs. This test system has been demonstrated to be sensitive to the clastogenic activity o f a variety o f chemicals (Preston et al., 1981). 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 o f 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 its ability to metabolize 2-aminoanthracene and 7,12-dimethyl-benz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100. Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 iL S9 per milliliter medium (RPMI 1640 serum-free medium supplemented with 100 units penicillin and 100 pg streptomycin/mL, and 2 mM L-glutamine). Solubility Test A solubility test was conducted to select the solvent. The test was conducted using one or more o f the following solvents in the order o f preference as listed: dimethyl sulfoxide (DMSO), ethanol, and acetone. The test substance was tested to determine the solvent, selected in order o f preference, that permitted preparation o f the highest soluble or workable stock concentration, up to 500 mg/mL. Preliminary Toxicity Assay The toxicity test was performed -for the purpose o f selecting concentrations for the chromosome aberration assay and consisted of an evaluation o f test substance effect on mitotic index. Approximately 0.6 mL heparinized blood was inoculated into centrifuge tubes containing 9.4 mL RPM I-1640 complete medium supplemented w ith 1% phytohaemoagglutinin (PHA). The tubes were incubated at 371C in a humidified atmosphere o f 51% CO2 in air for 44-48 hours. The pH and osmolality o f the highest treatment condition were measured, and the pH was adjusted, if necessary, in order to maintain a neutral pH in the treatment medium. At the time o f test substance treatment the culture tubes were centrifuged, the supernatant was aspirated, and the cells were resuspended in either 10 mL o f fresh RPM I-1640 complete medium containing 1% PHA for the - BioReliance Study No. AA79YX.341.BTL 11 Company Sanitized. Does not contain TSCA CBi H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 non-activated study or 10 m L S9 reaction mixture (8 mL serum free medium containing 1% PHA + 2 mL o f S9 cofactor pool), to which was added 0.1 mL test substance dosing solution in solvent or solvent alone. The cells were exposed to solvent alone and to nine concentrations o f the test substance for 4 hours in both the presence and absence o f S9 activation, and for 20 hours continuously in the absence o f S9 activation. The cells were incubated at 371C in a humidified atmosphere o f 51% CO2 in air. At the completion o f the 4 hour exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with RPMI-1640 complete medium and returned to the incubator for an additional 16 hours. Two hours prior to the scheduled cell harvest, Colcemid was added to the cultures at a final concentration o f 0.1 pg/mL and the cultures were returned to the incubator until cell collection. Cells were collected by centrifugation, treated with hypotonic potassium chloride (0.075M KC1), fixed, stained and the number of cells in mitosis per 500 cells scored was determined in order to evaluate test substance effect on mitotic index. Chromosome Aberration Assay The chromosome aberration assay was performed using standard procedures (Evans, 1976; Evans and O'Riordan, 1975) by exposing duplicate cultures o f human peripheral blood lymphocytes (HPBL) to at least 4 concentrations of the test substance as well as positive and solvent controls. The dividing cells were harvested at approximately 20 hours from the initiation o f treatment. For the chromosome aberration assays, 0.6 mL heparinized blood was inoculated into centrifuge tubes containing 9.4 mL complete medium supplemented with 1% PHA. The tubes were incubated at 371C in a humidified atmosphere o f 51% CO2 in air for 44-48 hours. Treatment was carried out by refeeding with approximately 10 mL fresh complete medium or S9 reaction mixture to which was added 0.1 mL o f dosing solution o f test or control substance in solvent or solvent alone. In the non-activated study, the cells were exposed for 4 or 20 hours at 371C in a humidified atmosphere o f 51% CO2 in air. In the 4 hour exposure group, after the exposure period, the treatment medium was removed, the cells washed with calcium and magnesiumfree phosphate buffered saline (CMF-PBS), refed with complete medium containing 1% PHA and returned to the incubator for an additional 16 hours. Two hours prior to the scheduled cell harvest at 20 hours after treatment initiation, Colcemid was added to the cultures at a final concentration o f 0.1 pg/mL. In the 20 hour exposure group treatment was continuous until the time o f cell collection. Two hours prior to the scheduled cell harvest at 20 hours after treatment initiation, Colcemid was added to the cultures at a final concentration of 0.1 pg/mL. BioReliance Study No. AA79YX.341.BTL 12 Company Sanitized. Does not contain f SCA CB1 H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 In the S9 activated study, the cells were exposed for 4 hours at 371C in a humidified atmosphere o f 51% CO2 in air. After the exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with complete medium containing 1% PHA and returned to the incubator for an additional 16 hours. Two hours prior to the scheduled cell harvest at 20 hours after treatment initiation, Colcemid was added to the cultures at a final concentration o f 0.1 pg/mL. Collection o f M etaphase Cells Two hours after the addition o f Colcemid, metaphase cells were harvested for both the activated and non-activated studies by centrifugation. The cells were collected by centrifugation at approximately 1200 rpm for about 5 minutes. The cell pellet was resuspended in 5 mL 0.075 M KC1 and incubated at 371C for 20 minutes. A t the end o f the KC1 treatment and immediately prior to centrifuging, the cells were gently mixed and approximately 0.5 mL o f fixative (methanolrglacial acetic acid, 3:1 v/v) was added to each tube. The cells were collected by centrifugation, the supernatant aspirated, and the cells were fixed with two washes with approximately 3-5 mL o f fixative and stored in fixative overnight or longer at approximately 2-8C. Slide Preparation To prepare slides, the fixed cells were centrifuged at approximately 1200 rpm for 5 minutes, the supernatant was aspirated, and the cells were resuspended in 1 mL cold fresh fixative. The cells were collected by centrifugation and the supernatant aspirated, leaving 0.1 to 0.3 mL fixative above the cell pellet. An aliquot o f the cell suspension was dropped onto a glass slide and allowed to air dry overnight. Slides were identified by the study number, dose level, activation condition, harvest time, replicate tube designation and date prepared. The dried slides were stained with 5% Giemsa, air dried and permanently mounted. Selection o f Dose Levels for Analysis The selection o f dose levels for analysis o f chromosome aberrations in HPBL was based upon toxicity, in the following order. The highest dose level selected for evaluation was the dose which induced at least 50% toxicity, as measured by mitotic inhibition, relative to the solvent control, with a sufficient number o f scorable metaphase cells. A t least two additional lower dose levels were included in the evaluation. BioReliance Study No. AA79YX.341.BTL 13 Company s.,,i!te l D o not contain TSCA CM H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 Evaluation of M etaphase Cells Slides were coded using random numbers by an individual not involved with the scoring process. Initially, die non-activated and S9 activated 4 hour exposure groups were evaluated for chromosome aberrations and if a positive result was obtained in the non-activated 4 hour exposure group, the non-activated 20 hour continuous exposure group was not necessarily evaluated for chromosome aberrations. Metaphase cells with 46 centromeres were examined under oil immersion without prior knowledge o f treatment groups. Whenever possible, a minimum o f 200 metaphase spreads (100 per duplicate treatment condition) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate flask was reduced if the percentage o f aberrant cells reached a statistically significant level before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence o f any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part o f the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (> 10 aberrations) also were recorded. Chromatid gaps (an aligned achromatic region in one chromatid, the size of which is equal to or smaller than the width o f the chromatid) and isochromatid gaps (an aligned, achromatic region in both chromatids, the size o f which is equal to or smaller than the width o f the chromatids) were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted. The percent polyploid and endoreduplicated cells was evaluated per 100 cells. Controls MMC was used as the positive control in the non-activated study at final concentrations o f 0.3 and 0.6 pg/mL. CP was used as the positive control in the S9 activated study at final concentrations o f 20 and 40_ pg/mL. For both positive controls one dose level exhibiting a sufficient number o f scorable metaphase cells was selected for analysis. The solvent vehicle for the test substance was used as the solvent control at the same concentration as that found in the test substance-treated groups. Evaluation of Test Results The toxic effects o f treatment are based upon mitotic inhibition relative to the solvent-treated control and are presented for the preliminary toxicity test and the chromosome aberration assay. The number and types o f aberrations per cell, the percentage o f structurally and numerically damaged cells (percent aberrant cells), and the frequency o f structural BioReliance Study No. AA79YX.341.BTL 14 Company Sanitized. Does not contain TSCA C81 H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 aberrations per cell (mean aberrations per cell) in the total population o f cells examined was calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage o f cells with one or more aberrations or in the frequency o f structural aberrations per cell. Statistical analysis o f the percent aberrant cells was performed using the Fisher's exact test. Fisher's exact test was used to compare pairwise the percent aberrant cells o f each treatment group with that o f the solvent control. In the event o f a positive Fisher's exact test at any test substance dose level, the Cochran-Armitage test was used to measure dose-responsiveness. All conclusions were based on sound scientific judgement; however, a s 1a guide to interpretation o f the data, the test substance was considered to induce a positive response when the percentages o f cells with aberrations were increased in a dose-responsive manner with one or more concentrations being statistically elevated relative to the solvent control group (p<0.05). A reproducible significant increase at the high dose only with no dose response or a reproducible significant increase at one dose level other than the high dose with no dose response will be considered positive. However, values that are statistically significant but do not exceed the range o f historic solvent controls may be judged as not biologically significant. The test substance was concluded to be negative if no statistically significant increase was observed relative to the solvent control. Criteria for Determination of a Valid Test The frequency o f cells with structural chromosome aberrations in the untreated and solvent controls m ust be no greater than 6% within the historical range for negative solvent controls. The percentage o f cells with chromosome aberrations in the positive control must be statistically increased (p<0.05, Fisher's exact test) relative to the solvent control. D eviation s No known deviations from the protocol or assay-method SOPs occurred during the conduct o f this study. Archives All raw data, the protocol, all reports, and stained and coded slides will be maintained according to Standard Operating Procedure OPQP3040 by the BioReliance RAQA unit headquartered at: BioReliance, 14920 Broschart Road, Rockville, MD 20850. Paper records will be retained for at least three years after which time the Sponsor will be contacted for a decision as to the final disposition o f the materials. All study materials returned to the Sponsor or destroyed will first be copied and the copy will be retained in the BioReliance archives for a minimum o f 10 years. BioReliance Study No. AA79YX.341.BTL 15 Company Sanitized. Does not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 RESULTS AND DISCUSSION Solubility Test DMSO was determined to be the solvent o f choice based on the solubility o f the test substance, and compatibility with the target cells. The test substance was soluble in DMSO at a concentration o f 100 mg/mL, the maximum concentration prepared in the assay. Preliminary Toxicity Assay Dose levels for the chromosome aberration assay were selected following a preliminary toxicity test and were based upon a reduction in mitotic index relative to the solvent control. The results o f the evaluation o f mitotic inhibition are presented in Tables 1 ,2 , and 3. HPBL cells were first exposed to nine concentrations o f H-24768 ranging from 0.1 pg/mL to 1000 pg/mL, as well as solvent controls, in both the absence and presence o f an Aroclorinduced S9 activation system for 4 hours, or continuously for 20 hours in the absence o f S9 activation. The test substance was soluble in treatment medium at all concentrations tested. The osmolality in treatment medium of die highest concentration tested, 1000 pg/mL, was 407 mmol/kg. The osmolality o f the solvent (DMSO) in treatment medium was 404 mmol/kg. The pH o f the highest concentration o f test article in treatment medium was approximately 7.0. Toxicity (mitotic inhibition) in excess o f 50%, relative to the solvent control, was observed at 30 pg/mL in all three treatment groups. Based on the results o f the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows: Treatment Condition Non-activated S9 activated Treatment Time (hr) 4 Recovery Time (hr) Dose levels (pg/mL) 16 2.5,5, 10,2 5 ,5 0 ,7 5 ,1 0 0 20 0 2.5, 5 ,1 0 ,2 5 , 50, 75, 100 4 16 2.5, 5 ,1 0 ,2 5 , 50, 75, 100 Chromosome Aberration Assay hi the chromosome aberration assay, the test substance was soluble in treatment medium at all concentrations tested. The osmolality in treatment medium o f the highest concentration tested, 100 pg/mL, was 417 mmol/kg. The osmolality o f the solvent (DMSO) in treatment BioReliance Study No. AA79YX.341.BTL 16 Company Sanitized. Does not contain TSCA CB1 H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 medium was 403 mmol/kg. The pH o f the highest concentration o f test article in treatment medium was approximately 7.0. The findings o f the cytogenetic analysis o f the non-activated 4 hour exposure group are presented by treatment flask in Table 4 and summarized by group in Table 7. A t the highest test concentration evaluated microscopically for chromosome aberrations, 25 pg/mL, mitotic inhibition was 52%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 5, 10 and 25 pg/mL. The percentage o f cells w ith structural and numerical aberrations in the test substance-treated groups was not significantly increased above that o f the solvent control (p>0.05, Fisher's exact test). The percentage of cells with structurally damaged chromosomes in the MMC (positive control) treatment group (29.0%) was statistically significant. The findings o f the cytogenetic analysis o f the S9 activated group are presented by treatment flask in Table 5 and summarized by group in Table 7. A t the highest test concentration evaluated microscopically for chromosome aberrations, 25 pg/mL, mitotic inhibition was 52%, relative to the solvent control. The dose levels selected for analysis o f chromosome aberrations were 5, 10 and 25 pg/mL. The percentage o f cells with structural and numerical aberrations in the test substance-treated groups was not significantly increased above that o f the solvent control (p>0.05, Fisher's exact test). The percentage o f cells with structurally damaged chromosomes in the CP (positive control) treatment group (15.5%) was statistically significant. The findings o f the cytogenetic analysis o f the non-activated 20 hour exposure group are presented by treatment flask in Table 6 and summarized by group in Table 7. A t the highest test concentration evaluated microscopically for chromosome aberrations, 25 pg/mL, mitotic inhibition was 58%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 5, 10 and 25 pg/mL. The percentage o f cells with structural and numerical aberrations in the test substance-treated groups was not significantly increased above that o f the solvent control (p>0.05, Fisher's exact test). The percentage o f cells with structurally damaged chromosomes in the MMC (positive control) treatment group (33.0%) was statistically significant. CONCLUSION The positive and solvent controls fulfilled the requirements for a valid test. Under the conditions o f the assay described in this report, H-24768 was concluded to be negative for the induction o f structural and numerical chromosome aberrations in the non-activated and S9 activated test systems in the in vitro mammalian chromosome aberration test using human peripheral lymphocytes. BioReliance Study No. AA79YX.341.BTL Compan, S a m **. Doe no. contain T8 C A C * H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 REFERENCES Evans, H J. (1976) Cytological methods for detecting chemical mutagens, in: A Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, vol 4. Plenum Press, New York. Evans, H J. and M.L. CRiordan. 1975. Human peripheral blood lymphocytes for the analysis o f chromosome aberrations in mutagen tests. Mutation Res. 31:135-148. Galloway, S.M., M J. Aardema, M. Mdate Jr., J.L. Ivett, D.J. Kirkland, T. Morita, P. Mosesso and T. Sofuni (1994) Report from working group on in vitro tests for chromosomal aberrations, Mutation Research 312(3):241-261. International Conference on Harmonization (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicity: Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 o f the ICH process on July 19,1995. Federal Register 61:18198-18202, April 24,1996. International Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing o f Pharmaceuticals. S2B document recommended for adoption at step 4 o f the ICH process on July 16,1997. Federal Register 62:16026-16030, November 21,1997. OECD Guideline for the Testing of Chemicals, Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), Revised Draft Document, Ninth Addendum to the OECD Guidelines for the Testing o f Chemicals, published by OECD, Paris, February 1998. Preston, R.J., W. Au, M.A. Bender, J.G. Brewen, A.Y. Cairano, J.A. Heddle, A.F. McFee, S. W olff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenetic assays: a report o f the Gene-Tox Program, Mutation Research, 87:143-188. Scott, D., N.D. Danford, B.J. Dean, and D.J. Kirkland. 1990. Metaphase Chromosome Aberration Assays In Vitro. In: Basic Mutagenicity Tests: UKEMS Recommended Procedures. D.J. Kirkland (ed.) Cambridge University Press, New York, NY. Swierenga S.H.H., J.A. Heddle, E.A. Sigal, J.P.W. Gilman, R.L. Brillinger, G.R. Douglas and E.R. Nestmann (1991) Recommended protocols based on a survey o f current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovaiy, V79 Chinese lung and human lymphocyte cultures, Mutation Research 246:301-322. BioReliance Study No. AA79YX.341.BTL 18 not contetn TSCACa Company Sanitized. 0@s H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 TABLE 1 PRELIMINARY TOXICITY TEST USING H -24768 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT TREATMENT -S9 (Hg/mL) DMSO H-24768 0.1 0.3 1 3 10 30 100 300 1000 MITOTIC INDEX (%) 8.8 PERCENT CHANGE (%) 8.0 -9 8.2 -7 7.6 -14 7.2 -18 7.4 -16 4.2 -52 2.8 -68 1.6 -82 0.6 -93 Treatm ent: Human peripheral blood lymphocyte cells were treated in the absence o f an exogenous source o f metabolic activation for 4 hours at 371C. Metaphase cells were collected following a 16 hour recovery period. M itotic Index = (cells in m itosis/500 cells scored) x 100. Percent Change = (treatment mitotic index - control m itotic index)/control mitotic index, expressed as a percentage. BioReliance Study No. AA79YX.341BTL 19 Company . 0 , * n o t w 1'" H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________ Dupont-13542 TABLE 2 PRELIMINARY TOXICITY TEST USING H -24768 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT TREATMENT +S9 Oig/mL) DMSO H-24768 0.1 0.3 1 3 10 30 100 300 1000 MITOTIC INDEX (%) 9.2 PERCENT CHANGE (%) 8.4 -9 8.2 -11 8.0 -13 7.8 -15 7.8 -15 4.4 -52 3.4 -63 2.0 -78 1.2 -87 Treatm ent: Human peripheral blood lymphocyte cells were treated in the presence o f an exogenous source o f metabolic activation for 4 hours at 371C. Metaphase cells were collected follow ing a 16 hour recovery period. M itotic Index = (cells inm itosis/500 cells scored) x 100. Percent Change = (treatment mitotic index - control mitotic index)/control m itotic indny^ expressed as a percentage. BioReliance Study No. AA79YX.341.BTL 20 Company SafffteecL Does not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 TABLE 3 PRELIMINARY TOXICITY TEST USING H -24768 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 20 HOUR TREATMENT TREATMENT -S9 (jig/mL) MITOTIC INDEX (%) PERCENT CHANGE (%) DMSO 8.0 H-24768 0.1 0.3 1 3 10 30 100 300 1000 8.0 0 7.6 -5 7.4 -8 6.0 -25 4.2 -48 3.0 -63 2.0 -75 1.4 -83 0.4 -95 Treatm ent: Human peripheral blood lymphocyte cells were treated in the absence o f an exogenous source o f metabolic activation for 20 hours at 371C. Metaphase cells were collected follow ing a 16 hour recovery period. M itotic Index = (cells in m itosis/500 cells scored) x 100. Percent Change = (treatment mitotic index - control mitotic index)/control m itotic index, expressed as a percentage. BioReliance Study No. AA79YX.341.BTL 21 Company Sa*itted. Does not contain TSCA CBi H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 TABLE 4 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24768 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST Treatment (pg/mL) Mitotic % Aberrant Cells Flask Index Cells (%) Scored Numerical Structural Total Number of Structural Aberrations Gaps Chromatid Chromosome Br Ex Br Die Ring Severely Damaged Cells Average Aberrations Per Cell DMSO A 9.6 100 B 8.8 100 0 0 0 30 0 00 0 0 0 00 0 00 0 0 0.000 0.000 H-24768 5 A 8.4 100 B 8.0 100 0 0 0 00 0 00 0 0 0 00 0 00 0 0 0.000 0.000 10 A 8.2 100 0 0 00 0 00 0 0 0.000 B 7.6 100 0 0 00 0 00 0 0 0.000 25 A 4.8 100 0 0 00 0 00 0 0 0.000 B 4.0 100 0 1 0 1 0 00 0 0 0.010 MMC, 0.6 A 7.2 50* B 7.8 50* 0 0 26 1 12 4 0 0 0 0 32 3 11 6 0 0 0 0 0.320 0.340 * Numerical aberrations are out o f 100 cells scored. Treatment: Human peripheral blood lymphocytes were treated for 4 hours at 37 1C in the absence o f an exogenous source o f metabolic activation. An additional dose level o f 2.5 pg/mL was tested as a safeguard against excessive toxicity at higher dose levels but was not required for microscopic examination. Dose levels 5 0 ,7 5 and 100 pg/mL were not analyzed due to excessive toxicity. M itotic index = number mitotic figures x 100/500 cells counted. % A berrant Cells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps. Chromatid breaks include chromatid and isochromatid breaks and fragments (Br); chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements. Chromosome B reaks include breaks and acentric fragments (Br); D ie, dicentric chromosome. Severely Damaged Cells includes cells with one or more pulverized chromosome and cells with 10 or more structural aberrations. Average Aberrations Per Cell: severely damaged cells and pulverizations were counted as 10 aberrations. BioReliance Study No. AA79YX.341.BTL 22 Company S a g g e d . Does not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________ Dupont-13542 TABLE 5 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H -24768 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST Treatment (pg/mL) DMSO H-24768 5 10 25 CP, 20 Mitotic % Aberrant Cells Flask Index Cells (%) Scored Numerical Structural Total Number o f Structural Aberrations Gaps Chromatid Chromosome Br Ex Br Die Ring Severely Damaged Cells Average Aberrations Per Cell A 9.6 100 B 9.6 100 0 0 0 0 0 0 00 0 0 0 00 0 00 0 0 0.000 0.000 A 8.4 100 B 8.2 100 A 8.0 100 B 8.4 100 A 4.8 100 B 4.4 100 A 7.8 100 B 8.0 100 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 00 0 0 1 01 0 00 0 0 0 10 0 00 0 0 0 0 0 0 00 0 0 0 10 0 00 0 0 16 0 12 5 1 0 0 15 3 13 5 0 0 0 0 0 0.000 0.000 0.010 0.000 0.000 0.000 0.180 0.180 Treatment: Human peripheral blood lymphocytes were treated for 4 hours at 37 1C in the presence o f an exogenous source o f metabolic activation. An additional dose level o f 2.5 jig/mL was tested as a safeguard against excessive toxicity at higher dose levels but was not required for microscopic examination. D ose levels 5 0 ,7 5 and 100 pg/mL were not analyzed due to excessive toxicity. M itotic index = number mitotic figures x 100/500 cells counted. % Aberrant Cells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps. Chromatid Breaks include chromatid and isochromatid breaks and fragments (Br); chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements. Chromosome Breaks include breaks and acentric fragments (Br); Die, dicentric chromosome. Severely Damaged Cells includes cells with one or more pulverized chromosome and cells with 10 or more structural aberrations. Average Aberrations Per Cell: severely damaged cells and pulverizations were counted as 10 aberrations. BioReliance Study No. AA79YX.341.BTL 23 Company Sa*itized. Does not contain TSCA CBf H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 TABLE 6 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H -24768 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 20 HOUR TREATMENT, 20 HOUR HARVEST Treatment (fig/mL) Mitotic % Aberrant Cells Flask Index Cells (%) Scored Numerical Structural Total Number of Structural Aberrations Gaps Chromatid Chromosome Br Ex Br Die Ring Severely Damaged Cells Average Aberrations Per Cell DMSO A 9.8 100 B 9.2 100 0 0 0 0 0 0 00 0 0 0 2 0 0 00 0 0 0.000 0.000 H-24768 5 A 8.2 100 B 8.0 100 0 0 0 10 0 00 0 1, 0 1 0 0 0 0 0 0 0.000 0.010 10 A 7.8 100 0 0 20 0 00 0 0 0.000 B 8.0 100 0 0 00 0 00 0 0 0.000 25 A 4.4 100 0 1 0 1 0 00 0 0 0.010 B 3.6 100 0 1 1 1 0 00 0 0 0.010 MMC, 0.3 A 7.8 50* B 8.0 50* 0 0 34 2 13 6 1 0 0 32 3 12 7 0 0 0 0 0 0.400 0.380 * Numerical aberrations are out o f 100 cells scored. Treatment: Human peripheral blood lymphocytes were treated for 20 hours at 37 1C in the absence o f an exogenous source o f metabolic activation. An additional dose level o f 2.5 pg/mL was tested as a safeguard against excessive toxicity at higher dose levels but was not required for microscopic examination. D ose levels 50, 75 and 100 pg/mL was not analyzed due to excessive toxicity. M itotic index = number mitotic figures x 100/500 cells counted. % Aberrant Cells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps. Chromatid Breaks include chromatid and isochromatid breaks and fragments (Br); chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements. Chromosome Breaks include breaks and acentric fragments (Br); Die, dicentric chromosome. Severely Damaged Cells includes cells with one or more pulverized chromosome and cells with 10 or more structural aberrations. Average Aberrations P er Cell: severely damaged cells and pulverizations were counted as 10 aberrations. BioReliance Study No. AA79YX.341.BTL 24 Company Sanitized. Does not contain TSCACBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________ Dupont-13542 TABLE 7 SUMMARY Treatment (Hg/mL) S9 Activation Mean Treatment Mitotic Cells Time Index Scored Aberrations Cells With Aberrations Per Cell Numerical Structural (Mean +/- SD) (%) (%) DMSO - 4 9.2 200 0.000 0.000 0.0 0.0 H-24768 5 10 25 - - 4 8.2 200 0.000 0.000 0.0 4 7.9 200 0.000 0.000 0.0 4 4.4 200 0.005 0.071 0.0 0.0 0.0 0.5 MMC, - 4 7.5 100} 0.330 0.551 0.0 29.0** 0.6 DMSO H-24768 5 10 25 CP, 20 + + + + + 4 9.6 200 0.000 0.000 0.0 0.0 4 8.3 200 0.000 0.000 0.0 4 8.2 200 0.005 0.071 0.0 4 4.6 200 0.000 0.000 0.0 0.0 0.5 0.0 4 7.9 200 0.180 0.457 0.0 15.5** DMSO H-24768 5 10 25 MMC, 0.3 - - - - 20 9.5 200 0.000 0.000 0.0 0.0 20 8.1 200 0.005 0.071 0.0 20 7.9 200 0.000 0.000 0.0 20 4.0 200 0.010 0.100 0.0 0.5 0.0 1.0 20 7.9 100} 0.390 0.601 0.0 33.0** } Numerical aberrations are out 200 cells scored. Treatment: Cells from all treatment conditions were harvested at 2 0 hours after the initiation o f the treatments. Aberrations per Cell: Severely damaged cells were counted as 10 aberrations. Percent Aberrant Cells: *, p<0.05; **, psO.Ol; using Fisher's exact test. BioReliance Study No. AA79YX.341.BTL 25 Compart* H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 APPENDIX A Historical Control Data BioReliance Study No. AA79YX.341.BTL 26 TSCACBI 0-- H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________ Dupont-13542 IN VITRO MAMMALIAN CYTOGENETIC TEST USING HUMAN PERIPHERAL BLOOD LYMPOCYTES HISTORICAL CONTROL VALUES STRUCTURAL CHROMOSOME ABERRATIONS 2000-2002 NON-ACTIVATED TEST SYSTEM Historical Values Mean Standard Deviation Range Percent Aberrant Cells (%) Solvent Control1 Positive Control2 0.1 16.9 0.3 8.1 0.0-1.5 6.5-50.0 S9 ACTIVATED TEST SYSTEM Historical. Values Mean Standard Deviation Range Percent Aberrant Cells (%) 2 Solvent Control1 Positive Control 0.1 13.6 0.4 5.4 0.0-2.0 7.0-34.0 1Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor-supplied vehicles. 2 Positive control for non-activated studies is mitomycin C (MMC). 3Positive control for S9 activated studies is cyclophosphamide (CP). BioReliance Study No. AA79YX.341.BTL 27 [SGACBi Satutiart- Does notcontain Company H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________ Dupont-13542 IN VITRO MAMMALIAN CYTOGENETIC TEST USING HUMAN PERIPHERAL BLOOD LYMPOCYTES HISTORICAL CONTROL VALUES NUMERICAL CHROMOSOME ABERRATIONS 2000-2002 NON-ACTIVATED TEST SYSTEM Historical Values Mean Standard Deviation Range Percent Aberrant Cells (%) Solvent Control1 0.0 Positive Control2 0.1 0.2 0.3 0.0-1.5 1.5 S9 ACTIVATED TEST SYSTEM Historical Values Mean Standard Deviation Range Percent Aberrant Cells (%) Solvent Control1 0.1 0.2 Positive Control2 0.1 0.3 0.0-0.5 0.0-1.5 Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor-supplied vehicles. P ositive control for non-activated studies is mitomycin C (MMC). P o sitiv e control for S9 activated studies is cyclophosphamide (CP). BioReliance Study No. AA79YX.34I.BTL 28 CoaspaRf Saatis$L Does not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes APPENDIX B Study Protocol Dupont-13542 BioReliance Study No. AA79YX.341 .BTL 29 Company SanmzetL Does not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes___________________ deceived by Dupont-13542 Sponsor Project Number DuPont - 13542 BioReliance Study Num ber AA79YX.341.BTL H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes 1.0 PURPOSE The purpose of this study is to evaluate the clastogenic potential o f the test substance based upon its ability to induce chromosome aberrations in human peripheral blood lymphocytes (HPBL). 2.0 SPONSOR 2.1 Name: E.L du Pont de Nemours and Company 2.2 Address: Stine Haskell Research Center DuPont Haskell Laboratory P.O. Box 50 1090 Elkton Road Newark, DE 19714-0050 2.3 Representative: E. Maria Donner, Ph.D. Phone: 302-366-5251 Psck 302-366-5207 E-mil: mariadonner@usa.dupont.com 2.4 Sponsor Project#: DuPont-13542 2.5 2.6 Haskell#: H-24768 2.7 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance Name:! 3.2 Test Substance ID .: H-24768 (to be used in the report and text) 3.3 Controls: Solvent: Positive: Test Substance Solvent or Vehicle Mitomycin C (MMC) Cyclophosphamide (CP) Protocol No. SPGT341 04 Jun 2003 Page 1 o f 10 BioReliance Study No. AA79YX.341.BTL 30 CQIft|&3!8T|f Bio Reliancf OCMH? i*IOf CSDUiflsilH T IS C A 0 0 1 H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________ Dupont-13542 Sponsor Project Number: DuPont -1 3 5 4 2 BioReliance Study Number: AA79YX.341.BTL 3.4 Test Substance Characterization Unless alternate arrangements are made, the testing facility at BioReliance will not perform analysis o f the dosing solutions. The Sponsor will be directly responsible for determination and documentation o f the analytical purity and composition of the test substance, and the stability and strength o f the test substance in the solvent (or vehicle). 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility BioReliance 4.2 Address: 9630 Medical Center Drive Rockville, M D 20850 4.3 Study Director: Ramadevi Gudi, P hD . Phone: 301-610-2169 Fax: 301-738-2362 E-mail: rgudi@bioreliance.com 5.0 PROPOSED STUDY DATES 5.1 Experimental Start Date: 17 Sep 2003 5.2 Experimental Termination Date: 31 Oct 2003 5.3 Draft Report Date: 14 Nov 2003 5.4 Final Report: 2 weeks after Sponsor approves draft 6.0 TEST SYSTEM Peripheral blood lymphocytes w ill be obtained from healthy adults without a recent history o f either radiotherapy, viral infections or the administration o f drugs. Ib is system has been demonstrated to be sensitive to the clastogenic activity o f a variety o f chemicals (Preston et al,, 1981). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The assay w ill be conducted using standard procedures, by exposing human lymphocytes to a minimum o f four concentrations of the test substance as well as to positive and solvent '\ controls. In the non-activated test system, treatment w ill be for 4 hours and for 20 hours; in the S9 activated test system, exposure w ill be for 4 hours (Swierenga et al., 1991). The dividing cells w ill be arrested in metaphase and harvested for microscopic evaluation of Protocol No. SPGT34I 04 Jun 2003 Page 2 o f 10 ^ Bio Reliance' BioReliance Study No. AA79YX.341.BTL 31 Company Sanitized. Does not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________ Dupont-13542 Sponsor Project Number DuPont -1 3 5 4 2 BioReliance Study Number. AA79YX.341.BTL chromosome aberrations at approximately 20 hours (1.5 normal cell cycles) after the initiation o f treatment in order to ensure evaluation of first-division metaphase cells (Galloway et al., 1994). The clastogenic potential o f the test substance w ill be measured by its ability to increase chromosome aberrations in a dose-responsive manner when compared to a control group. The 4 hour non-activated and S9-activated studies w ill be scored initially, hi the event o f a positive response in the 4 hour non-activated study, the prolonged exposure non-activated study may not be scored. The test substance w ill also be assessed for its ability to induce numerical chromosome aberrations. 7.1 Solvent Selection 7.1.1 Solubility Determination Unless the Sponsor has indicated the test substance solvent, a solubility determination w ill be conducted to determine the solvent and the maximum soluble concentration up to a maximum o f 500 mg/ml. Solvents compatible with this test system, in order o f preference, include but are not limited to sterile water (CAS 7732-18-5), dimethyl sulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5), and acetone (CAS 67-64-1). The solvent w ill be die test substance solvent, selected in order o f preference, that permits preparation o f the highest soluble stock concentration, up to 500 mg/ml. 7.2 Preliminary Toxicity Test for Selection o f D ose Levels Selection o f the dose levels for the cytogenetics assay w ill be based upon inhibition o f m itosis after treatment as determined in a cytotoxicity study. Cells will be exposed to solvent alone and to at least nine concentrations o f test substance, the highest concentration being 5000 Pg/ml or 10 mM whichever is lower. The pH of the highest test substance dosing solution will be measured, and will be adjusted, if necessary, in order to maintain a neutral pH in the treatment medium. The osm olality o f the highest dosing solution, lowest precipitating dose level (where applicable) arid the highest soluble dose level (where applicable) w ill also be measured. Peripheral blood cells w ill be cultured in RPMI-1640 containing 15% fetal bovine serum, 2m M L-glutamine, 100 units penicillin and 100 ug streptomycin/ml and 1% phytohemagglutinin. Cells seeded approximately 46 hours earlier w ill be exposed for 4 hours in the absence and presence o f S9 and for 20 hours in the absence o f S9. After exposure the cultures w ill be grown in complete medium for 16 hours. Eighteen hours after treatment initiation Colcemid (0.1 /rg/ml) will be added to the cultures. Cells w ill be collected at 20 hours after the initiation o f treatment by centrifugation, treated with hypotonic KC1 solution and fixed with methanol-glacial acetic acid. Metaphase preparations w ill be made and stained. The percentage o f cells in mitosis per 500 cells scored (mitotic index) will be determined for each treatment group. Protocol No. SPGT341 BioReliance Study No. AA79YX.341.BTL 04 Jun 2003 Page 3 o f 10 ^ Bio Reliance- 32 Company Sanitized. Does not contain TSC CBi H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes______________' Dupont-13542 Sponsor Project Number: DuPont -1 3 5 4 2 BioReliance Study N um ber AA79YX.341.BTL Whenever possible, the high dose for the chromosome aberration assay will be selected to give at least 50% toxicity (mitotic inhibition relative to the solvent control). At least two additional dose levels, demonstrating minimal or no toxicity, w ill be evaluated in the chromosome aberration assay. In the event the test substance cannot be dissolved at a high enough concentration in an appropriate solvent to be toxic, then the highest dose to be tested w ill be the concentration resulting in minimum precipitation in test medium. Precipitation w ill be determined with the unaided eye. hi the event the test substance demonstrates a dose-responsive increase in toxicity (mitotic inhibition relative to the solvent control) at concentrations that exceed solubility in treatment medium, then the highest dose to be evaluated for chromosome aberrations w ill be the concentration resulting in minimum precipitation in test medium. In the event that neither cytotoxicity nor insolubility is observed in the preliminary test, the highest dose in the chromosome aberration assay will be 5 mg/ml or 10 mM whichever is lower. If excessive precipitation o f the test substance solvent solution occurs upon addition to treatment medium, or if the osmolality o f the treatment medium is excessive, the Sponsor w ill be consulted. 7.3 Frequency and Route o f Administration Target cells w ill be treated for 4 hours in the absence and presence o f S9, and for 20 hours in the absence o f S9, by incorporation o f the test substance -solvent mixture into the treatment medium. This technique has demonstrated to be an effective method o f detection o f chemical clastogens in this test system (Evans, 1975). If the Sponsor is aware o f specific metabolic requirements, then this information w ill be utilized in the preparation o f the study design. Verification o f a clear positive response is not required Negative results w ill not be confirmed when justification can be provided Equivocal results may be confirmed, upon consultation with the Sponsor, and may employ a modification o f the study design. This guidance is based on the OECD Guideline 473 (adopted July 1997) and ICH Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals (1997). 7.4 M etabolic Activation system Aroclor 1254-induced rat liver S9 w ill be used as the metabolic activation system. The S9 w ill be prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection o f Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 w ill be batch prepared and stored frozen at approximately -70C until used. Each batch preparation o f S9 w ill be assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(oQanthracene to forms J mutagenic to Salmonella typhimurium TA100. Protocol No. SPGT341 BioReliance Study No. AA79YX.341.BTL 04 Jun 2003 Page 4 o f 10 Bio Reliance- 33 Company Sanitized. Does not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes___________ Dupont-13542 Sponsor Project Number: DuPont -1 3 5 4 2 ,'j BioReliance Study Number: A A 79Y X .341.BTL Immediately prior to use, the S9 w ill be thawed and mixed with a cofactor pool. The final concentration o f the cofactors and S9 in the reaction vessel w ill be 2 mM MgCk, 6 mM KC1, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate and 20 fil S9 per ml RPMI-1640 serum-free medium. 7.5 Controls 7.5.1 Solvent (or Vehicle) Control The solvent for the test substance w ill be used as the solvent control. For solvents other than water, physiological buffer, or medium, the final concentration in treatment medium w ill not exceed 1%. 7.5.2 Positive Controls Mitomycin C w ill be used at two concentrations within the range o f 0.1-0.50 Pg/ml as the positive control for the non-activated test system. For the S9activated system, cyclophosphamide w ill be used at two concentrations within the range o f 10-75 Pg/ml. One dose level o f each positive control w ill be evaluated microscopically for chromosome damage. 7.6 Preparation o f Target Cells Peripheral blood lymphocytes w ill be cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin and 100 fig streptomycin/ml, and 1% phytohemagglutinin) by adding 0.6 ml heparinized blood to a centrifuge tube containing 9.4 ml complete medium. The tubes w ill be incubated upright at 37 1C in a humidified atmosphere o f 5 1% CO2 in air for 44-48 hours. 7.7 Identification o f Test System Using a permanent marking pen, the test system w ill be identified by the BioReliance study number, treatment condition and date. 7.8 Treatment o f Target Cells Forty-four to 48 hours after culture initiation, duplicate centrifuge tubes will be refed with approximately 10 ml complete medium for the non-activated exposure or 10 ml S9 reaction mixture for the activated exposure to which w ill be added 100 fil o f dosing solution o f test or control substance in solvent. Larger volumes o f dosing solution may be used if water or medium is used as the solvent. ) Protocol No. SPGT341 BioReliance Study N o. AA79YX.341.BTL 04 Jun 2003 Page 5 o f 10 ^ BioReliance- 34 CoHjpafispS taffzsiiL Etassotcanltfa ISCJ&CBS H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 Sponsor Project Number: DuPont - 13542 BioReliance Study Number: AA79YX.34LBTL For the S9-activated exposure, the cells w ill be treated for 4 hours in the presence of an S9 reaction mixture, washed free o f chemical and cultured for an additional 16 hours with Colcemid (0.1 pg/ml) present for the last 2 hours. For the nonactivated exposure, treatment w ill be for 4 hours followed by a 16 hour recovery period and for 20 hours continuously with Colcemid (0.1 /zg/ml) present for the last 2 hours. 7.9 Collection o f Metaphase Cells Cells w ill be collected approximately 20 hours after initiation of treatment (about 64-68 hours after culture initiation). This time is selected to represent the firstdivision metaphase after initiation of test substance treatment. Two hours prior to harvest, Colcemid w ill be added to the cultures at a final concentration of 0.1 pg/m l. The cells w ill be collected by centrifugation, treated with 0.075M KC1, washed with two changes o f fixative (methanohglacial acetic acid, 3:1 v/v), capped and stored overnight or longer at approximately 2-8C. To prepare slides, the cells w ill be collected by centrifugation and resuspended in fresh fixative. An aliquot o f fixed cells w ill be applied dropwise onto a microscope slide and air-dried. The slide w ill be identified by the experiment number, treatment condition and date. At least two slides w ill be prepared from each treatment tube. The slides w ill be stained with Giemsa and permanently mounted. 7.10 Scoring for Metaphase Aberrations Slides w ill be coded using random numbers by an individual not involved with the scoring process. At least 3 dose levels in each harvest w ill be evaluated in the aberration assay and w ill be selected according to the criteria described in. section 7.2. The 4 hour non-activated and S9-activated studies w ill be scored initially, hi the event o f a positive response in 4 hour non-activated study, slides from the extended non-activated exposure may not be scored. Metaphase cells will be examined under o il immersion without prior knowledge o f treatment groups. Whenever possible, a minimum o f 200 metaphase spreads containing 46 centromeres from each dose level (100 per duplicate treatment tube) will be examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number o f metaphase spreads that will be examined and scored per duplicate flask m ay'be reduced if the percentage o f aberrant cells reaches a statistically significant level before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence o f any exchange figure w ill be scored as a break (chromatid or chromosome). Fragments observed with an exchange figure w ill not be scored as an aberration but w ill be considered part o f the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (>10 aberrations) will Protocol No. SPGT341 04 Jun 2003 Page 6 o f 10 Bio Reliance' B ioR elian ce Study N o. A A 79Y X .341.BTL 35 ' Company Sanitized. Does not contain TSCA CBS H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 Sponsor Project Number: DuPont -1 3 5 4 2 BioReliance Study Number: AA79YX.341.BTL also be recorded. Chromatid and isochromatid gaps w ill be recorded but not included in the analysis. The XY coordinates for each cell with a structural aberration or gap will be recorded using a calibrated microscope stage. The m itotic index w ill be recorded as the percentage o f cells in mitosis per 500 cells counted. The percent polyploid and endoreduplicated cells w ill be evaluated per 100 metaphase cells for each dose level analyzed for structural aberrations. 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST 8.1 Solvent Controls The frequency of cells with structural chromosome aberrations in the solvent controls must be within the range of the historical solvent control. 8.2 Positive Controls The percentage of cells with aberrations must be statistically increased (p<0.05, Fisher's exact test) in the positive control relative to the solvent control. 9.0 EVALUATION OF TEST RESULTS The toxic effects o f treatment are based upon inhibition o f mitosis and w ill be reported for the cytotoxicity and chromosome aberration study. The number and types o f aberrations (structural and numerical) found, the percentage o f structurally damaged cells in the total population o f cells examined (percent aberrant cells), the percentage o f numerically damaged cells in the total population o f cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) w ill be calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in tire average number o f aberrations percell. Statistical analysis o f the percentage o f aberrant cells w ill be performed using the Fisher's exact test. The Fisher's test will be used to compare pairwise the percent aberrant cells o f each treatment group with that o f the solvent control. In the event o f a positive Fisher's exact test at any test substance dose level, the Cochran-Armitage test w ill be used to measure dose-responsiveness. A ll conclusions will be based on sound scientific judgement; however, as a guide to interpretation o f the data, the test substance w ill be considered to induce a positive response if the percent aberrant cells is increased in a dose-responsive manner with one or more concentrations being statistically significant (p<0.05). A reproducible significant increase at the high dose only with no dose response or a reproducible significant increase at one dose level other than the high dose with no dose response w ill be considered positive. Test .) substances not demonstrating a statistically significant increase in aberrations will be concluded to be negative. Protocol No. SPGT341 04 Jun 2003 Page 7 o f 10 ^ Bio Reliancf BioReliance Study No. AA79YX.341JBTL 36 Company Sanitized. Does not cordasi*TSCA CM H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 Sponsor Project Number: DuPont - 13542 ^ BioReliance Study Number AA79YX.341.BTL 10.0 REPORT A report o f the results o f this study w ill be prepared by the Testing Facility and will accurately describe all methods used for generation and analysis of the data. Results presented will include, but not be limited to: Test substance: identification and CAS no., if known; physical nature and purity, if known; physicochemical properties relevant to the conduct o f the study, if known; stability o f test substance, if known. Solvent/Vehicle: justification for choice o f vehicle; solubility and stability of test substance in solvent/vehicle, if known. source o f cells and time the cells were obtained, karyotype features (modal chromosome number) and suitability o f the cell type used. test conditions: composition o f medium; CO2 concentration; incubation time; solvent and solvent selection rationale; concentration o f test substance and concentration selection rationale; composition and acceptability criteria for the metabolic activation (S9) system; '^ duration o f treatment; duration o f treatment with and concentration of Colcemid , type of metabolic activation system used; positive and solvent controls; methods of slide preparation; number o f cell cultures; criteria for scoring aberrations and criteria for considering studies positve, negative or equivocal. results: description of precipitation; pH and osmolarity o f the treatment medium; mitotic inhibition relative to the solvent control; mitotic index and number o f metaphases analyzed; type and number o f aberrations (structural and numerical) given separately for each treated and control culture; concentration-response relationship; statistical analysis; historical control data 11.0 RECORDS AND ARCHIVES All raw data, the protocol and all reports w ill be maintained according to Standard Operating Procedure OPQP3040 by the BioReliance RAQA unit headquartered at: BioReliance, 14920 Broschart Road, R ockville, MD 20850. Per this SOP, paper records w ill be retained for at least three years after which time the Sponsor w ill be contacted for a decision as to the final disposition o f the materials. A ll study materials returned to the Sponsor or destroyed w ill first be copied and the copy w ill be retained in the BioReliance archives for a minimum o f 10 years. >. ) Protocol No. SPGT341 BioReliance Study No. AA79YX.341.BTL 04 Jun 2003 Page 8 o f 10 || Bio Reliance- 37 Company Sanitized. Does not contain TSC CBi H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 Sponsor Project Number. DuPont -1 3 5 4 2 BioReliance Study Number: AA79YX.341.BTL 12.0 REGULATORY REQUBREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply with OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), adopted July 1997 and with the International Conference on Harmonization o f Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997). This study w ill be performed in compliance with the provisions o f the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (GLPs). The protocol, an in-process phase, the raw data, and report(s) w ill be audited per the Standard Operating Procedures (SOPs) o f BioReliance by the Quality Assurance Unit o f BioReliance for compliance with GLPs, the SOPs of BioReliance and the study protocol. The in-process inspection w ill be performed to audit the critical assay procedures and systems supporting the assay. A signed QA statement will be included in the final report. This statement w ill list the system phases inspected during the previous quarter or the study-specific phases, the dates o f each inspection, and the dates the results o f each inspection were reported to the Study Director and the Study Director's management In addition, a signed GLP compliance statement will be included in the final report This statement w ill cite the GLP guideline(s) with which the study is compliant and any exceptions to this compliance, if applicable, including the omission o f characterization or stability analyses o f the test or control substances or their mixtures. Unless arrangements are made to the contrary, unused, dosing solutions w ill be disposed o f follow ing administration to the test system and all residual test substance w ill be disposed o f follow ing finalization of the report. 13.0 REFERENCES Evans, H.J. and M X. ORiordan. 1975. Human peripheral blood lymphocytes for the analysis o f chromosome aberrations in mutagen tests. Mutation Res. 31:135-148. Galloway, S.M ., M J. Aardema, M. Ishidate Jr., J.X Ivett, D.J. Kirkland, T. Morita, P. M osesso and T. Sofuni (1994) Report from working group on in vitro tests for chromosomal aberrations, Mutation Research 312(3):241-261. International' Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 o f the ICH process on July 19,1995. Federal Register 61:18198-18202, April 24,1996. International Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing o f Pharmaceuticals. S2B document recommended for adoption at step 4 o f the ICH process on July 16, 1997. Federal Register 62:16026-16030, November 21, 1997. ^ Protocol No. SPGT341 04Jun2003 Page 9 o f 10 Bio Reliance- BioReliance Study No. AA79YX.341.BTL 38 Company Sanitized. Does not contain TSCA CBI H-24768: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Dupont-13542 Sponsor Project Number: DuPont - 13542 ~ \ BioReliance Study Number A A79YX.341 -BTL OECD Guideline 473 (Genetic Toxicology: In Vitro Mammalian Chromosome Aberration Test), Ninth Addendum to die OECD Guidelines for the Testing o f Chemicals, published by OECD, Paris, February 1998. Preston, R J., W . Au, M.A. Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F. McFee, S. W olff and J.S. Wassom. 1981. Mammalian in vivo and in vitro cytogenetics assays: A report o f the US EPA's Gene-Tox Program. Mutation Res. 87:143-188. Scott, D ., NJ>. Danford, B J. Dean and D J. Kirkland. 1990. Metaphase Chromosome Aberration Assays hi Vitro. In: Basic Mutagenicity Tests: UKEMS Recommended Procedures. D J Kirkland (ed). Cambridge University Press, New York, NY. Swierenga S.H.H., J.A. Heddle, E.A. Sigai, J.P.W. Gilman, R.L. Brillinger, G.R. Douglas and E.R. Nestmann (1991) Recommended protocols based on a survey o f current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V 79 Chinese lung and human lymphocyte cultures, Mutation Research 246:301-322. 14.0 APPROVAL fio v A : _ E. Maria Donner, Ph.D. Sponsor Representative -- f a c tu z jd o ju ' BioReliance Study D irector^--s. ST BioReliance Study Management 0 Date AT- Date / S S-Ssp 2 0 S Date ) Protocol No. SPGT341 BioReliance Study No. AA79YX.341.BTL 04 Jun 2003 Page 10 of 10 % BioReuanoe 39 Company Sanitized, Does not contain TSCA GB!
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