Document pBV4KDN4eJ5GbxLnqwrDrGoJw

DownloadViewSend us a messageRandom document
AR226-3181 TRADE SECRET Testing Guidelines International Conference on Harmonisation (ICH) of Technical Requirements for Registration ofPharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory GenotoxicityTests for Phannaceuticals. S2A document recommended for adoption at step 4 of the ICH process on July 19,1995 and OECD Guidelines for Testing of Chemicals Section 4: Health Effects, No. 473 (Adopted 1997) Study Title H:24889: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Authors Ramadevi Gudi, Ph.D. Caren Brown, M.S. Study Completion Date July 24,2001 Performing Laboratory BioReliance 9630 Medical Center Drive RockviUe.MD 20850 for E. I. du Font de Nemours and Company Stine Haskell Research Center DuPont Haskell Laboratory P.O. Box 50,1090 Elkton Road Newark, DE 19714-0050 Performing Laboratory Study Number AA44ER.341.BTL DuPont Project Number DuPont-6406 Page 1 of 40 SofflpfiJV.IoiMkadi OooBitolectttaIn TSC& 6B8 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes_________________________DuPont-6406 STATEMENT OF COMPLIANCE Study No. AA44ERJ41.BTL was conducted in compliance with the U.S. FDA GLP Regulations as published in 21 CTR 58, the U.S. EPA GLP Standards 40 CFR 160, and 40 CFR 792, the UK. GLP Compliance Programme, the Japanese GLP Standard, and me OECD Principles of Good Laboratory Practice in all material aspects with the following exceptions: The identity, strength, purity and composition or other characteristics to define the control articles have not been determined by the testing facility. The control articles have been characterized as per the Certificates of Analysis on file with the testing facility. The stability of the test and control articles has not been determined by me testing facility. Analyses to determine me uniformity (as applicable), or concentration of the test and control mixtures were not performed by the testing facility. The Sponsor has indicated mat they have not performed these analyses on me test article mixtures. The stabilityof the test and control articles in the test and control mixtures, respectively, has not been determined by the testing facility. The Sponsor has indicated that they have not performed these analyses on me test article mixtures. Ka^Ajdec^ ^<_^' Ramadevi Gudi, Ph.D. Study Director f>^ BioReliance Study Management ^7^7^V Date jt 4 yf^yiooi Date -:' BioReliance Study No. AA44ER.3413TL 2 ,^~ K j H-24889: In Vitro Chnnnosome Aberration Study in Human PeripheralBlood Lymphocytes____ Quality Assurance Statement DuPont-6406 Study TMe: H 24889 IN VITRO MAMMALIAN CHROMOSOME ABERRATION STUDY W HUMAN PERIPHERAL BLOOD LYMPHOCYTES Study Number: AA44ER.341.BTL Study Director: Ramadevi Gudi, Ph.D. 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 me 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 ofQA inspections of this study. Inspect On Phase Inspect On Phase Inspect On Phase Inspect On Phase 10-May-Ol - 10-May-Ol To Study Dir 10-May-Ol To Mgmt 10-May-Ol Protocol Review 07-Jun-Ol - 07-Jun-Ol To Study Dir 07-Jun-Ol To Mgmt 08-Jun-Ol Dilution of test article and control material 18-Jul-Ol - 19-Jul-Ol To Study Dir 20-Jul-Ol To Mgmt 24-Jul-Ol Draft Report 24-Jul-Ol - 24-Jul-Ol To Study Dir 24-Jul-Ol To Mgmt 24-Jul-Ol 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. ^ ^ . ^ ^ ^ Becky D. Schreckengost, B.S. ' QUALITY ASSURANCE o ^ ^ W DATE BioReliance Study No. AA44ER.341.BTL 3 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes CERTIFICATION DuPont-6406 We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by StudyDirector: KcU^wkeCf.' ^LLC^' L Ramadevi Gudi BioReliance Study Director >4 y^/t^oo/ Date Approved by Study Monitor: Ko^^ 737^, Maria Donner, Ph.D. Senior Research Scientist a^^<- 2,001 Date BioReliance Study No. AA44ER-341.BTL H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____ STUDY INFORMATION Substance Teste< Synonyms/Code! DuPont-6406 Physical Characteristics: Yellow to amber solid Stability: The test article appeared to be stable under the conditions of the study; no evidence of instability was observed. BioReliance Study No. AA44ER.341 .BTL 5 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes___________DuPont-6406 Sponsor: E. L du Pont de Nemours and Company Wilnrington, Delaware 19898 U.S.A. Study Initiated/Completed: May 10,2001 / (see report cover page) In-Life Initiated/Completed: May 24,2001 / June 19,2001 BioReliance Study No. AA44ER.341.BTL 6 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____ TABLE OF CONTENTS GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT, QUALITY ASSURANCE STATEMENT.......................................__.., DuPont-6406 PAGE 9 .,,..... PURPOSE 11 .......... CHARACTERIZATION OF TEST AND CONTROL ARTICLES....... 11 ......... MATERIALS AND METHODS ...................^.........................................................--...............----.......... 11 TEST SYSTEM ..................................................................................................................................................11 ACTIVATION SYSTEM...................................................................................................................................... 12 SOLUBILITY TEST............................................................................................................................................ 12 PRELIMINARY TOMCITY ASSAY...................................................................................................................... 12 CHROMOSOME ABERRATION ASSAY............................................................................................................... 13 COLLECTION OP METAPHASE CELLS 14 ............................................................................................................... SLIDE PREPARATION........................................................................................................................................ 14 SELECTION OF DOSE LEVELS FOR ANALYSIS................................................................................................... 14 EVALUATION OF METAPHASE CELLS............................................................................................................... 14 CONTROLS 15 ....................................................................................................................................................... EVALUATION OF TEST RESULTS...................................................................................................................... 15 CRITERIA FOR DETERMINATION OF A VALID TEST .......................................................................................... 16 16 DEVIATIONS.................................................................................................................................................. 16 ARCHIVES........................................................................................................................................................ RESULTS AND DISCUSSION...........................................................................--....................................... 16 SOLUBILITY TEST 16 ............................................................................................................................................ PRELIMINARY TOMCITY ASSAY...................................................................................................................... 16 CHROMOSOME ABERRATION ASSAY............................................................................................................... 17 ^" BioReliance Study No. AA44ER-341 STL 7 I contain TSCACBil H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes___________________________DuPont-6406 TIAVA TAUT 1S*C ^H TABLE 1 PRELIMINARY TOXIdTY TEST USING H-24889 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT........................................................................... 20 TABLE 2 PRELIMINARY TOXICTTY TEST USING H-24889 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT........................................................................... 21 TABLE 3 PRELIMINARY TOXICTTY TEST USING H-24889 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 20 HOUR TREATMENT......................................................................... 22 TABLE 4 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24889 IN THE ABSENCE OP EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST................................................. 23 TABLE 5 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24889 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST................................................ 24 TABLE 6 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24889 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 20 HOUR TREATMENT, 20 HOUR HARVEST............................................... 25 TABLE? 26 SUMMARY.............................................................................................................................. APPENDIX A HISTORICAL CONTROL DATA ...................................:................^.................^........... 27 APPENDIX B STUDY PROTOCOL.....__......................,,..............,,....................,,,,.....,,........,,.._..... 30 BioReliance Study No. AA44ER.341 .BTL 8 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes___DuPont-6406 SUMMARY The test article, H-24889, was tested in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. The chromosome aberration assay was used to evaluate the clastogenic potential of the test article. Acetone was determined to be the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in acetone at a maximum concentration of 500 mg/mL. In the preliminary toxicity assay, the maximum dose tested was 5000 ug/mL. Human peripheral blood lymphocytes were treated in the absence and presence of an Aroclor-induced S9 activation system for 4 hours, and continuously for 20 hours in the absence of S9 activation. Visible precipitate was observed in treatment medium at concentrations of 1500 and 5000 ug/mL. Concentrations of <500 ug/mL were soluble in treatment medium. Selection of 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 dose level 5000 Pg/mL in the non-activated 4 hour and the 20 hour exposure groups. Substantial toxicity was observed at dose levels 1500 and 5000 pg/mL in the 89-activated 4 hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 125 to 3000 pg/mL for both the non-activated exposure groups, and from 125 to 2000 pg/mL for the 89-activated 4 hour exposure group. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the nonactivated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatment initiation. Visible precipitate was observed in treatment medium at concentrations of 1500 to 3000 (ig/mL in the non-activated 4 hour and the 20 hour exposure groups and at 1500 and 2000 pg/mL in the 89-activated 40 hour exposure group. Concentrations of $1250 pg/mL were soluble in treatment medium. Toxicity (mitotic inhibition) was approximately 36% and 15% at the highest dose level evaluated for chromosome aberrations, 1500 pg/mL, in the non-activated 4 hour and 20 hour exposure groups, respectively. Toxicity (mitotic inhibition) was approximately 57% at the highest dose level evaluated for chromosome aberrations, 1250 pg/mL, in the 89-activated 4 hour exposure group. Highest dose levels were selected based on the lowest precipitating dose levels. Initially, the non-activated and S9 activated 4 hour exposure groups were scored for structural and numerical chromosome aberrations. No statistically significant increases in structural and numerical chromosome aberrations were observed in the non-activated or S9 activated 4 hour exposure groups relative to the solvent control group, regardless of dose level (p>0.05. Fisher's exact test), fa the absence of a positive response in the non-activated 4 hour exposure group, the non-activated 20 hour continuous exposure group was evaluated for BioReliance Study No. AA44ER.341 .BTL 9 BQeontefn TSQ& e8& H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 structural and numerical chromosome aberrations. No statistically significant increases in structural and numerical chromosome aberrations were observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, regardless of dose level (p>0.05. Fisher's exact test). Based on the findings of this study, H-24889 was concluded to be negative for the induction of structural and numerical chromosome aberrations in the in vitro mammalian chromosome aberration test using human peripheral lymphocytes. BioReliance Study No. AA44ER.341.BTL 10 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes______^_____________DuPont-6406 PURPOSE The purpose of this study was to evaluate the clastogenic potential of a test article based upon its ability to induce chromosome aberrations in human peripheral lymphocytes. CHARACTERIZATION OF TEST AND CONTROL ARTICLES The test article, H-24889, was received by BioReliance on May 9 2001 and was assigned the code number AA44ER. The test article was characterized by the Sponsor as a yellow to amber solid that should be stored at <27C in a ventilated area, protected from exposure to light, with an expiration date of April 5,2003. Upon receipt, the test article was described as off-white jelly material and was stored at room temperature, protected from exposure to light and moisture. The solvent used to deliver H-24889 to the test system was acetone (CAS No.: 67-64-1), obtained from the Fisher Scientific Company. The test article was heated to 60C and mixed well before each weighing. 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 25 and 50 ug/mL for use as the positive control in the non-activated test system. Cyclophosphamide (CP), CAS No.: 6055-19-2, was obtained from the Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations of 2 and 4 mg/mL for use as the positive control in the S9 activated test system. For each positive control, one dose level exhibiting a sufficient number of scorable metaphase cells was selected for analysis. The solvent for the test article was used as the solvent control at the same concentration as that found in the test article-treated groups. MATERIALS AND METHODS Test System Peripheral blood lymphocytes were obtained from a healthy non-smoking 26 year old adult male on May 22, 2001 for the preliminary toxicity assay, and from a healthy non smoking 26 year old adult male on June 5,2001 for the definitive assay. Neither donor had a recent history of radiotherapy, viral infection or the administration of drugs. This test system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals (Preston et al., 1981). BioReliance Study No. AA44ER.341.BTL 11 H-24889; In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________DuPont-6406 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 of 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 lucotmamide adenine dinucleotide phosphate (NADP) and 20 uL S9 per milliliter medium (RPMI 1640 semm-free medium supplemented with 100 units penicillin and 100 ug streptomycin/mL, and 2 mM L-glutamine). Solubility Test A solubihty test was conducted to select the solvent. The test was conducted using one or more of the following solvents in the order of preference as listed: purified water, dimethyl sulfoxide (DMSO), ethanol, and acetone. The test article was tested to determine the solvent, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration, up to 500 mg/mL. Preliminary Toxicity Assay The toxicity test was performed for the purpose of selecting concentrations for the chromosome aberration assay and consisted of an evaluation of test article effect on mitotic index. Approximately 0.6 mL heparinized blood was inoculated into centrifuge tubes containing 9.4 mL RPMI-1640 complete medium supplemented with 1% phytohaemoagglutinin (PHA). The tubes were incubated at 371C in a humidified atmosphere of 51% C02 in air for 44-48 hours. The pH and osmolality of 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 of test article treatment the culture tubes were centrifuged, the supernatant was aspirated, and the cells were resuspended in either 10 mL of fresh RPMI-1640 complete medium containing 1% PHA for the nonactivated study or 10 mL S9 reaction mixture (8 mL serum free medium containing 1% PHA + 2 mL of S9 cofactor pool), to which was added 100 uL test article dosing solution in solvent or solvent alone. The cells were exposed to solvent alone and to nine concentrations of the test article for 4 hours in both the presence and absence of S9 activation, and for 20 hours continuously in the absence ofS9 activation. The cells were incubated at 371C in a humidified atmosphere of 51% C02 in air. At the completion of the 4 hour exposure period, the treatment medium BioReliance Study No. AA44ER.341.BTL 12 IP H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 was removed, the ceUs 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 of 0.1 ug/mL and me 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 article effect on mitotic index. Chromosome Aberration Assay The chromosome aberration assay was performed using standard procedures (Evans, 1976; Evans and ORiordan, 1975) by exposing duplicate cultures of human peripheral blood lymphocytes (HPBL) to at least 4 concentrations of the test article as well as positive and solvent controls. The dividing cells were harvested at approximately 20 hours from the initiation of 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 of 51% C02 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 100 uL of dosing solution of test or control article 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 of 51% C02 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 of 0.1 ug/mL. In the 20 hour exposure group treatment was continuous until the time of 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 tig/mL. In the S9 activated study, the cells were exposed for 4 hours at 371C in a humidified atmosphere of 51% COi 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 of 0.1 ug/mL. BioReliance Study No. AA44ER.341 .BTL 13 'aapany SHba& Oosa noS eontein TBCA C6\ H-24889: In Vitro ChromosQme Aberration Study in Human Peripheral Blood Lymphocytes____________________________DuPont-6406 Collection ofMetaphase Cells Two hours after the addition of 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. At the end of the KC1 treatment and immediately prior to centrifuging, the cells were gently mixed and approximately 0.5 mL of fixative (methanohglacial 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 of 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 of 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 of Dose Levels for Analysis The selection of dose levels for analysis of chromosome aberrations in HPBL was based upon precipitation of the test article in treatment medium. In the presence of test article precipitation in the treatment medium, the highest dose level evaluated was the lowest precipitating dose level, regardless of toxicity. At least two additional lower dose levels were included in the evaluation. Evaluation of Metaphase Cells Slides were coded using random numbers by an individual not involved with the scoring process. Initially, the 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 of treatment groups. Whenever possible, a minimum of 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 metapbase spreads that were examined and scored per duplicate flask was reduced if the percentage of aberrant cells reached a statistically significant level before 100 cells are scored. Chromatid-type aberrations include chromaud and isochromatid breaks and exchange 0 BioReliance Study No. AA44ER.341.BTL 14 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____________________________DuPont-6406 figures such as quadriradials (symmetrical and asymmetrical interchanges), triiadials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromarid or acentric) observed in the absence of any exchange figure were scored as a break (cbromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (SlO 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 of the chromatid) and isochromatid gaps (an aligned, achromatic region in both chromatids, the size of which is equal to or smaller than the width of 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. index was recorded as the percentage of cells in mitosis per 500 cells counted. polyploid and endoreduplicated cells was evaluated per 100 cells. The mitotic The percent Controls MMC was used as the positive control in the non-activated study at final concentrations of 0.25 and 0.5 ug/mL. CP was used as the positive control in the S9 activated study at final concentrations of 20 and 40 ug/mL. For both positive controls one dose level exhibiting a sufficient number of scorable metaphase cells was selected for analysis. The solvent vehicle for the test article was used as the solvent control at the same concentration as that found in the test article-treated groups. Evaluation of Test Results The toxic effects of treatment are based upon mitotic inhibition relative to the solvent-treated control and arc presented for the preliminary toxicity test and the chromosome. aberration assay. The number and types of aberrations per cell, the percentage of structurally and numerically damaged cells (percent aberrant cells), and the frequency of structural aberrations per cell (mean aberrations per cell) in the total population of cells examined was calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but arc not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell. Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-rcsponsiveness. All conclusions were based on sound scientific judgement; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner with BioReliance Study No. AA44ER.341 .BTL 15 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____________________________DuPont-6406 one or more concentrations being statistically elevated relative to the solvent control group (p<0.05). A reproducible significant increase at the highdose 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 of historic solvent controls may be judged as not biologically significant. The test article 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 of cells with structural chromosome aberrations in the solvent controls must be within the historical range for solvent controls. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (pSO.05, Fisher's exact test) relative to the solvent control. Deviations No known deviations from the protocol or assay-method SOPs occurred during the conduct of this study. Archives All raw data, the protocol, all reports, and stained and coded slides will be maintained according to Standard Operating ProceduruHHIUoy the BioReIiance RAQA unit headquartered at: BioReIiance, 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 of the materials. All study materials returned to the Sponsor or destroyed will first be copied and the copy will be retained in the BioReIiance archives for a Tninimiirn of 10 years. RESULTS AND DISCUSSION Solubility Test Acetone was determined to be the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in acetone at a maximum concentration of 500 mg/mL. Preliminary Toricity 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 of the evaluation of mitotic inhibition are presented in Tables 1, 2, and 3. HPBL BioReIiance StudyNo.AA44ER.341.BTL 16 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 cells were first exposed to nine concentrations of H-24889 ranging from 0.5 ug/mL to 5000 ug/mL, as well as solvent controls, in both the absence and presence of an Aroclor-induced S9 activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. Visible precipitate was observed in treatment medium at concentrations of 1500 and 5000 ug/mL. Concentrations of $500 ug/mL were soluble in treatment medium. The osmolality and pH of the highest concentration tested, 5000 pg/mL, were 258 mmol/kg and approximately 7.5, respectively. The osmolality of the lowest precipitating dose level, 1500 Pg/mL, was 256 mmol/kg. The osmolality of the highest soluble dose level, 500 |ig/mL, was 261 mmol/kg. The osmolality of the solvent (acetone) in the treatment medium was 265 mmol/kg. Toxicity (mitotic inhibition) in excess of 50%, relative to the solvent control, was observed at 5000 ug/mL in the non-activated 4 hour and the 20 hour exposure groups and at 1500 and 5000 ug/mL in the S9-activated 4 hour exposure group. Based on the results of 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 20 4 Recovery Time (hr) 16 0 . 16 Dose levels (ug/mL) 125,250,500,1000.1500,1750, 2000,2500 and 3000 125,250,500,1000,1500,1750. 2000,2500 and 3000 125,250,500,1000.1250,1500 and 2000 Chromosome Aberration Assay In the chromosome aberration assay, visible precipitate was observed in treatment medium at concentrations of 1500,1750,2000,2500 and 3000 ug/mL in the non-activated 4 hour and the 20 hour exposure groups. Concentrations <1000 ug/mL were soluble in treatment medium. Visible precipitate was observed in treatment medium at concentrations of 1500 and 2000 ug/mL in the S9-activated 4 hour exposure group. Concentrations ^1250 Pfi/mL were soluble in treatment medium. The osmolality in treatment medium of the highest concentration tested, 3000 (ig/mL, was 380 mmol/kg. The osmolality of the lowest precipitating dose level, 1500 Pg/mL, was 379 mmol/kg. The osmolality of the highest soluble dose level, 1250 Hg/mL, was 375 mmol/kg.The osmolality of the solvent (acetone) in treatment medium was 382 mmol/kg. The pH of the highest concentration of test article in treatment medium was approximately 7.5. BioReliance Study No. AA44ER.341.BTL 17 ompwy SffHSaswS. PGQC no9 eontain TSCA C@!l H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 The findings of the cytogenetic analysis of the non-activated 4 hour exposure group are presented by treatment flask in Table 4 and summarized by group in Table 7. At the highest test concentration evaluated microscopically for chromosome aberrations, 1500ug/mL, mitotic inhibition was 36%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 125, 500 and 1500 ug/mL. The highest dose was selected to score chromosome aberrations was the lowest precipitating dose level in the culture medium. The percentage of cells with structural and numerical aberrations in the test article-treated groups was not significantly increased above that of 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 (6.5%) was found to be statistically significant. The findings of the cytogenetic analysis of the S9 activated group are presented by treatment flask in Table 5 and summarized by group in Table 7. At the highest test concentration evaluated microscopically for chromosome aberrations, 1250 ug/mL, mitotic inhibition was 57%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 125, 500, and 1250 pg/mL. The percentage of cells with structural and numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p>0.05. Fisher's exact test). The percentage of cells with structurally damaged chromosomes in the CP (positive control) treatment group (8.0%) was found to be statistically significant. In the absence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20 hour exposure group were evaluated for chromosome aberrations. The findings of the cytogenetic analysis of the non-activated 20 hour exposure group are presented by treatment flask in Table 6 and summarized by group in Table 7. At the highest test concentration evaluated microscopically for chromosome aberrations, 1500 ug/mL, mitotic inhibition was 15%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 125,500, and 1500 ug/mL. The percentage of cells with structural and numerical aberrations in the test article-treated groups was not significantly increased above that of 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 (10.5%) was found to be statistically significant. CONCLUSION The positive and solvent controls fulfilled the requirements for a valid test. Under the conditions of the assay described in this report, H-24889 was concluded to be negative for the induction of structural and numerical chromosome aberrations in the nonactivated and S9 activated test systems in the in vitro mammalian chromosome aberration test using human peripheral lymphocytes. 1 BioReliance Study No. AA44ER.341 .BTL 18 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes_______________________DuPont-6406 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. ORiordan. 1975. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Res. 31:135-148. Galloway, S.M., MJ. Aardema, M. Ishidate 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 Hannonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 of the ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24,1996. International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at step 4 of 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 of Chemicals, published by OECD, Paris, February 1998. Preston, R.J., W. Au, M.A. Bender, J.G. Brewen, A.V. Canano, J.A. Heddle, A.P. McPee, S. Wolff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenetic assays: a report of 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 of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese lung and human lymphocyte cultures, Mutation Research 246:301-322. ^ BioReliance Study No. AA44ER.341.BTL 19 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes___________________________DuPont-6406 TABLE 1 PRELIMINARY TOXICITY TEST USING H-24889 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT MITOTIC PERCENT TREATMENT INDEX CHANGE (-S9) (%) (%) ug/mL Acetone ________4._0 __________ H-24889 0.5 1.5 5 15 50 150 500 1500 5000 3.4 -15 3.0 -25 3.4 -15 3.6 -10 3.6 -10 3.4 -15 3.2 -20 2.4 -40 0.4 -90 Treatment: Human peripheral blood lymphocyte cells were treated in the absence of an exogenous source of metabolic activation for 4 hours at 371C. Metaphase cells were collected following a 16 hour recovery period. Mitotic Index = (cells in mitosis/500 cells scored) x 100. Percent Change = (treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage. BioReliance Study No. AA44ER.341 .BTL 20 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes________________________DuPont-6406 TABLE 2 PRELIMINARY TOXICITY TEST USING H-24889 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION TREATMENT (+S9) ug/mL Acetone 4 HOUR TREATMENT MITOTIC INDEX (%) PERCENT CHANGE (%) 4.4 _____ H-24889 0.5 4.6 5 1.5 5.4 23 5 4.0 -9 15 5.0 14 50 4.8 9 150 5.6 27 II 500 1500 3.2 -27 1.0 -77 5000 0.4 -91 Treatment: Human peripheral blood lymphocyte cells were treated in the presence of an exogenous source of metabolic activation for 4 hours at 371C. Metaphase cells were collected following a 16 hour recovery period. Mitotic Index = (cells in mitosis/500 cells scored) x 100. Percent Change = (treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage. <~-\ BioReliance Study No. AA44ER.341.BTL 21 @mg3eBSysniBteaa. Boss not eontein TSGA 081 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____________________________DuPont-6406 TABLE 3 PRELIMINARY TOXICITY TEST USING H-24889 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION TREATMENT (-S9) ug/mL Acetone H-24889 0.5 1.5 5 15 50 150 500 1500 5000 20 HOUR TREATMENT MITOTIC PERCENT INDEX CHANGE (%) (%) 4.6 ___ ____ 3.8 -17 3.6 -22 4.2 -9 4.2 -9 4.6 0 5.0 9 4.0 -13 4.0 -13 0.8 -83 Treatment: Human peripheral blood lymphocyte cells were treated in the absence of an exogenous source of metabolic activation for 20 hours at 371C. Metaphase cells were collected following a 16 hour recovery period. Mitotic Index = (cells in mitosis/500 cells scored) x 100. Percent Change = (treatment mitotic index - control mitotic index)/control mitotic index, expressed as a percentage. BioReliance StudyNo.AA44ER.341.BTL 22 i TCA CB H-24889: In Vitro Chromosome Aberration Study in Human Peripheral.Blood Lymphocytes DuPont-6406 TABLE 4 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24889 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION Treatment ug/mL Acetone H-24889 125 500 1500 MMC 0.5 DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST Flask Mitotic Index (%) Cells Scored %Abcnant Cells Numerical Structural A 4.4 100 0 0 B 4.0 100 0 0 Total Number ofStnidturalAlicnations Gaps Chroinfltid Br Ex Chromasome Br Die Ring 0 0 0 0 0 0 0 0 0 00 0 Severely Damaged Cells 0 0 A 3.6 100 0 0 0 0 0 00 0 0 B 4.4 100 0 0 0 0 0 0 0 0 0 A 3.0 100 0 0 0 0 0 0 0 0 0 B 2.8 100 0 0 0 0 0 0 0 0 0 A 2.8 100 0 0 0 0 0 00 0 0 B 2.6 100 0 0 0 0 0 0 0 0 0 A 1.6 100 0 7 0 4 3 1 0 0 0 B 2.6 100 0 6 0 4 0 1 ' 0 0 Avenge Aberrations Per Cell 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.080 0.060 Treatment: Human peripheral blood lymphocytes were treated for 4 hours at 37 1C in the absence of an exogenous source of metabolic activation. Additional dose levels of 250 and 1000 (ig/mL were tested as a safeguardagainst excessive toxicity at higher dose levels but were not required for microscopic examination. Dose levels 1750,2000, 2500 and 3000 ug/rnL were not analyzed due to excessive toxicity. MHotic 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 Aberratioos Per Cell: severely damaged cells and pulverizations were counted as 10 aberrations. BioReliance Study No. AA44ER.341 .BTL 23 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____ DuPont-6406 TABLE 5 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24889 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION Treauneiil ug/mL Acetone 125 500 1250 CP 20 DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST Flask Mitotic Index (%) Cells Scored % Aberrant Cells Numerical SUuctural 3.6 100 3.4 100 A 3.4 100 0 0 B 2.8 100 0 0 A 2.6 100 0 0 B 2.6 100 0 1 A 1.6 100 0 0 B 1.4 100 0 1 A 1.6 100 0 7 B 1.0 100 0 9 Total Number of Structural Aberrations Gaps Chromatid Br Ex 0 0 0 0 0 0 Chromosome Br Die Ring 000000 00 0 0 0 0 0 0 0 0 1 0 00 0 0 0 0 0 0 0 0 1 0 00 0 0 0 0 0 7 0 0 7 1 00 0 11 0 Severely Damaged Cells 0 0 0 0 0 0 0 0 Avenge Aberrations Per Cell 0.000 0.000 0.000 0.000 0.000 0.010 0.000 0.010 0.070 0.100 Treatment: Human peripheral blood lymphocytes were treated for 4 hours at 37 1C in the presence of an exogenous source of metabolic activation. Additional dose levels of 250 and 1000 ug/mL were tested as a safeguard against excessive toxicity at higher dose levels but were not required for microscopic examination. Dose levels 1500, and 2000 ug/mL were not analyzed due to excessive toxicity. Mitotfc 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 Celb 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. AA44ERJ41 .BTL 24 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____ DuPont-6406 FABLE I5 iCYTOCiENEl1C ANALYSIS OF ITOMANI'ERIPHIiRALlBLOOI)LYAPIV.3CYTESTREATED VTTHIi[-24889 [NTHEAB!SENCEOFEXOGENOl(SME1ABOLIC'J\cn\NATION 'Crefltment 4g/rnL Acetone H-24889 125 500 1500 MMC 025 E)EFINITIVEASSA'Y'.20HOVIRTREAJMHff,20]TOUIIHA]RVEST Flask Mitotic Index (%) Cells Scored %AberraBt Cells Numerical !Structural A 4.8 100 0 0 B 4.4 100 0 1 TomrNumber of StructuralAt>erraticms Gaps Chroinatid Br Ex ChtowDSIline Br Die 1Ung 0 0 0 0 1 0 0 0 0 00 0 Severely Damaged Cells 0 0 A 3.8 100 0 0 B 3.6 100 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 A 3.4 100 0 0 B 4.8 100 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 A 3.8 100 0 0 B 4.0 100 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 A 4.0 100 0 12 0 8 4 01 0 0 B 4.8 100 0 9 0 6 2 0 2 0 0 Average Aberrations Per Cell 0.000 0.010 0.000 0.000 0.000 0.000 0.000 0.010 0.130 0.100 Treatment: Human peripheral blood lymphocytes were treated for 20 hours at 37 1C in the absence of an exogenous source of metabolic activation. Additional dose levels of 250 and 1000 ug/mL were tested as a safeguard against excessive toxicity at higher dose levels but were not required for microscopic examination. Dose levels 1750, 2000,2500 and 3000 ug/mL were not analyzed due to excessive toxicity. Mitotic index = number mitotic figures x 100/500 cells counted. % Aberrant Celb: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps. Cbromadd Breaks include cbromatid 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. il) BioReliance Study No. AA44ER.341.BTL 25 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____ DuPont-6406 Treatment ug/mL Acetone H-24889 125 500 1500 MMC 0.5 TAI3LE7 SUMMARY S9 Activation Treatment Time Mean Mitotic Index Cells Scored Abe[rations PCrCell (MeaiI+/-SD) Cells With Aberrations Numerical (%) Structural (%) 4 4.2 200 0.000 0.000 0.0 0.0 4 4.0 200 0.000 0.000 0.0 0.0 4 2.9 200 0.000 0.000 0.0 0.0 4 2.7 200 0.000 0.000 0.0 0.0 4 2.1 200 0.070 0.275 0.0 6.5** Acetone + 4 3.5 200 0.000 0.000 0.0 0.0 H-24889 125 + 4 3.1 200 0.000 0.000 0.0 0.0 500 1250 + 4 2.6 200 0.005 0.071 0.0 0.5 + 4 1.5 200 0.005 0.071 0.0 0.5 CP + 4 1.3 200 0.085 0.313 0.0 8.0** 20 Acetone H-24889 125 500 1500 MMC 0.25 20 4.6 200 0.005 0.071 0.0 20 3.7 200 0.000 0.000 0.0 20 4.1 200 0.000 0.000 0.0 20 3.9 200 0.005 0.071 0.0 20 4.4 200 0.115 0.350 0.0 - 0.5 0.0 0.0 0.5 10.5** Treatment: Cells from all treatment conditions were harvested at 20 hours after the initiation of the treatments. Aberrations per Cell: Severely damaged cells were counted as 10 aberrations. Percent Aberrant Cells: **, p^O.Ol; using Fisher's exact test BioReliance Study No, AA44ER-341.BTL 26 SBBa^BSRSSsMa^&.l H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____ APPENDIX A Historical Control Data DuPont-6406 BioReliance Study No. AA44ER.341 .BTL 27 epB^8aBlBs@&0e*a contain TSCAB8 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 IN VITRO MAMMALIAN CYTOGENETIC TEST USING HUMAN PERIPHERAL BLOOD LYMPHOCYTES HISTORICAL CONTROL VALUES STRUCTURAL CHROMOSOME ABERRATIONS 1997-1999 NON-ACTIVATED TEST SYSTEM Historical Values Mean Standard Deviation Range Percent Aberrant Cells (%) Untreated Control Solvent Control' Positive Control2 0.2 0.3 16.1 0.3 0.5 9.5 0.0-1.5 0.0-2.0 6.5-48.0 S9 ACTIVATED TEST SYSTEM Historical Values Mean Standard Deviation Range Percent Aberrant Cells (%) Untreated Control Solvent Control1 Positive Control3 0.2 0.4 15.7 0.4 0.7 7.5 0.0-1.5 0.0-3.5 7.0-39.0 Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor-supplied vehicles. Positive control for non-activated studies is mitomycin C (MMC). Positive control for S9 activated studies is cyclophosphamide (CP). D BioReliance Study No. AA44ER.341.BTL 28 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 IN VITRO MAMMALIAN CYTOGENETIC TEST USING HUMAN PERIPHERAL BLOOD LYMPHOCYTES HISTORICAL CONTROL VALUES NUMERICAL CHROMOSOME ABERRATIONS 1997-1999 NON-ACTIVATED TEST SYSTEM Historical Values Mean Standard Deviation Range Percent Aberrant Cells (%) Untreated Control Solvent Control' Positive Control2 0.2 0.1 0.1 0.3 0.2 0.3 0.0-1.0 0.0-1.5 0.0-1.5 S9 ACTIVATED TEST SYSTEM Historical Values Mean Standard Deviation Range Percent Aberrant Cells (%) Untreated Control Solvent Control1 Positive Control3 0.2 0.1 0.2 0.3 0.3 0.4 0.0-1.0 0.0-1.0 0.0-1.5 Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor-supplied vehicles. Tositive control for non-activated studies is mitomycin C (MMC). Positive control for S9 activated studies is cyclophosphamide (CP). t) BioReliance Study No. AA44ER.341.BTL 29 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes APPENDIX B Study Protocol DuPont-6406 D BioReliance Study No. AA44ER.341.BTL 30 H-24889: In Vitro Chromosome Aberration Study PA/WieS^ in Human Peripheral Blood Lymphocytes__________________________DuPont-6406 d-e-c--e:i.v.-e.-di ib*yuDAffi& tK~yfK^y*^~~~' Sponsor Project Number DuPont-6406 BioReliance Study Number AA44ER.341.BTL H-24889: 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 oj^^--^-- 24889) based upon its ability to induce chromosome aberrations in human peripheral1 lymphocytes (HPBL). 2.0 SPONSOR 2.1 Name: E.I-OU Font deNemours and Company 11 Address: Stine Haskell Research Center DuPont Haskell Laboratory P.O. Box 50 1090 Elkton Road Newaik.DE 19714-0050 i 23 Representative: Maria Donner.Ph-D. Phone: 302-366-5251 Fax: 302-366-5207 E-mail: maria.donner@usa-dupont.coin 2.4 Sponsor Project ff: DuPont-6406 2-5 WR#: 2.6 Haskell fc H-2488? 2.7 Service Code: ^A 3.0 IDENTIFICATION OF TEST AND CONTROL ARTICLES 3.1 Test Article Name: The test article is a solid at room temperature and must be heated to 60C each and eveiy time. When all material is melted and homogeneously mixed to assure uniformity, an aliquot needs to be taken out for dosing. The mixing should occur around 60C. , 33. Test Article I.D.: H-24889 (to be used in the ieport and text) Protocol No. SPGT341 Ol-May-2001 Page I of 10 ^6^. BlORELlANCE- BioReliance Study No. AA44ER.341 -BTL 31 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 33 Controls: Sponsor Project Number BioReliance Study Number DuPont-6406 AA44ER.341.BTL Solvent Positive: Test Article Solvent or Vehicle MitomydnC(MMC) Cyclophosphamide (CP) 3.4 Test Article Characterization Unless alternate arrangements are made, the testing facility at BioReliance will not perform analysis of me dosing solutions. The Sponsor will be directly responsible for detennination and documentation of me analytical purify and composition of the test article, and me stability and strength of the test article in the solvent (or vehicle). 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility BioReliance 4.2 Address: 43 Study Director 9630 Medical Center Drive Rockville.MD 20850 Ramadevi Gudi, Ph-D. Phone: 301-610-2169 Fax: 301-738-2362 E-mail: igudi@bioreliance.com. 5.0 PROPOSED STUDY DATES 5.1 Experimental Start Date: 23-May-2001 (Selection of dose levels for me chromosome aberration study and any possible repeats will be done in consultation with the Sponsor.) 5.2 Experimental Termination Date: 53 Draft Report Date: 20-Jul-2001 30-Jul-2001 5.4 Final Report: 6.0 TEST SYSTEM 2 weeks after Sponsor approves draft Peripheral blood lymphocytes will be obtained from healthy adults without a recent history of either radiotherapy, viral infections or the administration of drugs. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals (Preston et al., 1981). Protocol No. SPGT341 Ol-May-2001 Page 2 of 10 t) BioReliance Study No. AA44ER.341.BTL 32 ^ BlORELIANCF ogs^waySsnRbad. Pose ned eostiafn TSCA CBS H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes___________________DuPont-6406 Sponsor Project Number BioReliance Study Number DuPont-6406 AA44ERJ41JBTL 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The assay wiU be coixlurted using staolard procedures, by exposing human lymphocytes to a minimum of four concentrations of the test article as well as to positive and solvent controls. In the non-activated test system, treatment will be for 4 hours and for 20 hoins; in the S9 activated test system, exposure will be for 4 hours (Swierenga et al., 1991). The dividing cells will be arrested in metaphase and harvested for microscopic evaluation of chromosome aberrations at approximately 20 hours (1.5 normal cell cycles) after me initiation of treatment in order to ensure evaluation of first-division metaphase cells (Galloway etal, 1994). Tte clastogemc potential of the test article will be measured by its abmty to increase chromosome aberrations m a dose-responsive maimer when compared to a control group. The 4 hour non-activated and S9-activated studies will be scored inmally. In the event of a positive response in the 4 hour non-activated study, the prolonged exposure non-activated study may not be scored. The test article will also be assessed for its ability to induce numerical chromosome aberrations. 7.1 Solvent Selection 7.1.1 Solubility Determination Unless me Sponsor has indicated the test article solvent, a solubility determination '; will be conducted to detennine the solvent and the maximum soluble concentration up to a maximum of 500 mg/ml. Solvents compatible with this test system, in order of 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 will be the test article solvent, selected in order of preference, that permits preparation of the highestsoluble stock concentration, up to 500 mg/ml. 7.2 Preliminary Toxicity Test for Selection of Dose Levels Selection of the dose levels for the cytogenetics assay will be based upon inhibition of mitosis after treatment as determined in a cytotoxicity study. Cells will be exposed to solvent alone and to at least nine concentrations of test article, me highest concentration being 5000 pg/ml or 10 mM whichever b lower. The pH of me highest test article dosing solution will be measured, and will be adjusted, if necessary, in order to maintain a neutral pH in the treatment medium. The osmolality of the highest dosing solution, lowest precipitating dose level (where applicable) and the highest soluble dose level (where applicable) will also be measured. Peripheral blood cells will be cultured in RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin and 100 ug streptomycin/ml and 1% phytoheinagglurinin- Cells seeded approximately 46 hours earlier will be exposed for 4 hours in the absence and presence of S9 and for 20 hours in the absence of S9. After exposure the cultures will be grown in complete ) medium for 16 hours. Eighteen hours after treatment initiation Colcemid*' (0.1 ug/ml) will be added to the cultures- Cells will be collected at 20 hours after the Protocol No SPGT341 Ol-May-2001 Pee3ofl0 ^6^ _ _ BlORELIANCE- BioReliance StudyNo.AA44ER.341.BTL 33 wsa^asagsarates'a Boss no! soirtain TSCA CBl H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 Sponsor Project Number. BioReliance Study Number. DuPont-6406 AA44ERJ4LBTL initiation of treatment by centrifugation, treated with hypotonic KC1 solution and fixed mm methanol-glacial acetic acid. Metaphase preparations will be made and stained. The percentage of cells in mitosis per 500 cells scored (mitotic index) will be detennmed for each treatment group. Whenever possible, the high dose for me chromosome aberration assay will be selected to give at least 50% toxkaty (mitotic inhibition relative to me solvent control). At feast two addmonal dose levels, demonstrating minimal or no toxicity, will be evaluated in the chromosome aberration assay. In the event the test article cannot be dissolved at a high enough concentration in an appropriate solvent to be toxic, men the highest dose to be tested will be me concentration resulting in minimum precipitation in test medium. Precipitation will be determined with the unaidedeye. In the event the test article demonstrates a dose-responsive increase in toxicity (mitotic inhibition relative to (he solvent control) at concentrations that exceed solubility in treatment medium, then the highest dose to be evaluated for chromosome aberrations will be the concentration resulting in minmniim precipitation in test medium. In the event that neither cytotoxicity nor insolubility is observed in the preliminary test, the highest dose in me chromosome aberration assay will be 5 mg/ml or 10 mM whichever is lower. If excessive precipitation of the test article solvent solution occurs upon addition to treatment medium, or if the osmolality of me treatment medium is excessive, the Sponsor will be consulted. ';i 7.3 Frequency and Route of Administration Target cells will be treated for 4 hours in me absence and presence ofS9, and for 20 hours m the absence of S9, by incorporation of the test article-solvent mixture into the treatment medium. This technique has demonstrated to be an effective method of detection of chemical clastogens in mis test system (Evans, 1975). If me Sponsor is aware of specific metabolic requirements, then mis information will be utilized in the preparation of the study design. Verification of a clear positive response is not required. Negative results will not be confirmed when justification can be provided. Equivocal results may be confirmed, upon consultation with the Sponsor, and may employ a modification of the study design. This guidance is based on the OECD Guideline 473 (adopted July 1997) and ICH Guidance on Specific Aspects of Regulatory Genotoxunty Tests for Pharmaceuticals (1997). 7.4 Metabolic Activation system Aroclor 1254-induced rat liver S9 will be used as the metabolic activation system. The S9 will be prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 ing/kg, five days prior to sacrifice. ') The S9 will be batch prepared and stored frozen at approximately-70K: until used. Each batch preparation of S9 will be assayed for sterility and its ability to Protocol No. SPGTMl Ol-May-2001 Page 4 of 10 <H 6r^ _ B l O _ R E L I A N C F BioReliance Study No. AA44ER.341.BTL 34 QMparayanSS!z9s8. Doss not contain TSCA CB! H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes__________________DuPont-6406 Sponsor Project Number BioReliance StudyNumber DuPont-6406 AA44ERJ41BTL metabolize 2-flminoanthracene and 7,12-diinethylben2(a)antnracene to fonns nssaagewto Salmonella typhimuriwn'TA.lW. Immediately prior to use, tteS9wiU be thawed and mixed with a cofactor pool. The final concentration of the cofactors and S9 in the reaction vessel will be 2 mM MgCli, 6 mM KQ, 1 mM glucosc-o-phosphale, 1 mM nicotmamide adeoine dinucleotide phosphateand 20 pi S9 per ml RPMI-1640 serum-fiee medium. 75 Controls 75.1 Solvent (or Vehicle) Control The solvent for me test article will be used as the solvent control For solvents other than water, physiological buffer, or medium, me final concentration in treatment medium win not exceed 1%. 7.5.2 Positive Controls Mitomycin C will be used at two concentrations within the range of 0.1-050 pg/ml as the positive control for the non-activated test system. For the S9- activated system, cyclophosphamide will be used at two concentrations '. within the range of 10-75 pg/ml. One dose level of each positive control will be evaluated microscopically for chromosome damage. 7.6 Preparation of Target Cells Peripheral blood lymphocytes will be cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin and 100 ug streptomycin/mi, and 1% phytohemagglutinin) by adding 0.6 ml heparinized blood to a centrifuge tube cpntaming 9A ml complete medium. The tubes will be incubated upright at 37 1C in a humidified annosphere of 5 1% CO, in air for 44-48 hours. 7.7 Identification of Test System Using a permanent marking pen, the test system will be identified by the BioReliance study number, treatment condition and date. 7.8 Treatment of 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 Of 10 ml S9 reaction mixture for the activated exposure to which will be added 100 pi I . of dosing solution of test or control article in solvent Larger volumes of dosing solution may be used if water or medium is used as me solvent Protocol No. SPGT341 Ol-May-2001 Pgc5ofl0 <6^9. _ BlORELIANCE- 1 j BioReliance StudyNo.AA44ER.341.BTL 35 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 Sponsor Project Number BioReliance Study Number DuPont-6406 AA44ER341.BTL For the S^-activated exposure, the celb will be treated for 4 hours in the presence of an S9 reaction mixture, washed free of chemical and cultured for an additional 16 hours with Colccmid* (0.1 (tg/inl) present for the last 2 hours. For the non-activated : exposure, treatment will be for 4 hours followed by a 16 hour recovery period and for 20 hours continuously with Colccmid* (0.1 tig/mi) present for the last 2 hours. 7.9 Collection ofMetaphase Cells Cells will be collected approximately 20 hours after initiation of treatment (about 64-68 houns after culture initiation). This time is selected to represent the firstdivision metaphase after initiation of test article treatment Two hours prior to harvest, Cotcemid* will be added to the cultures at a final concentration of 0.1 ug/ml. The cells will be collected by ccntrifugation, treated with 0.075M KC1, washed with two changes of fixative (methanokglacial acetic add, 3:1 v/v), capped and stored overnight or longer at approximately 2-8<>C. To prepare slides, the cells will be collected by centrifiigarion and resuspended in fresh fixative. An aliquot of fixed cells will be applied dropwise onto a microscope slide and air-dried-The slide will be identified by the experiment number, treatment condition and date. At least two slides will be prepared from each treatment tube. The slides will be stained with Giemsa and permanently mounted. \ 7.10 Scoring for Metaphase Aberrations Slides will be coded using random numbers by an individual not involved with the scoring process. At least 3 dose levels in each harvest will be evaluated in me abenation assay and will be selected according to the criteria described in section 7.2- The 4 hour non-activated and 89-activated studies will be scored initially. In the event of 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 oil immersion without prior knowledge of treatment groups. Whenever possible, a mhnmiim of 200 metaphase spreads containing 46 centromeres from each dose level (100 per duplicate treatment tube) will.be examined and scored for chromarid-type and chromosome-type abenations (Scott et aL, 1990). Tte number of metaphase spreads that will be examined and scored per duplicate flask may be reduced if the percentage of abenant 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 of any exchange figure will be scored as a break (chromatid or chromosome). Fragments observed with an exchange figure will not be scored as ) an aberration but will be considered part of me incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (^10 aberrations) will Protocol N&SPGT341 Ol-May-2001 Pge6ofl0 1S9^ _ _ BlORELIANCE- BioReliance StudyNo.AA44ER.341.BTL 36 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 Sponsor Project Number BioReliance Study Number DuPont-6406 AA44ER341.BTL also be recorded. Chromalid aad isochromatid gaps will be recorded but not included in the analysis. The XY coordinates for each cell with a structural abenatKm or gap wm be recorded using a calibrated microscope stage. Themitotic index will be recotded as the percentage of cells in mitosis per 500 cells counted. The percent polyploid and cndoreduplicated cells will be evaluated per 100 metaphasecells for each dose level analyzed for structural aberrations. 8.0 CRITERIA FOR DETERMINATION OF A VAUD 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. 92 Positive Controls The percentage of cells with aberrations must be statistically increased (p^O.05, Fisher's exact test) in the positive control relative to the solvent control. 9.0 EVALUATION OF TEST RESULTS , The toxic effects of treatment are based upon inhibition of mitosis and will be reported for the cytotoxicity and chromosome aberration study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in me total population of cells examined (percent aberrant cells), me percentage of numerically damaged cells in the total population of cells examined, and me average number of structural abemations per ceU (mean abenanons per ceU)wiU be caloilated and reported for each treatment group. Chromatid and isochromatid gaps are presented in me data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per celL Statistical analysisof me percentage of aberrant cells will be performed using the Fisher's exact test. The Fisher's test will be used to compare pairwise the percent aberrant cells of each treatment group with that of me solvent control. In the event of a positive Fisher's exact test at any test article dose level, the Cochran-Annitage test will be used to measure dose-responsiveness. All conclusions will be based on sound scientific judgement; however, as a guide to interpretation of me data, the test article will be considered to induce a positive response if me percent aberrant cells is increased in a dose-responsive manner with one or more concentrations being statistically significant (p^O.05). A reproducible significant increase at the high dose only with no dose response or a reproducible significant increase at one dose level other man the high dose with no dose response will be considered positive. Test ) articles not demonstratinga statisticaUy significant increase m aberrations will be concluded to be negative. Protocol No. SPGT341 Ol-Mjy-2001 Page 7 of 10 1J9^ B_ lOR_ ELIANCE- BioReliance Study No. AA44ER.341 .BTL 37 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____________________________DuPont-6406 Sponsor Project Number BioReliance Study Number. DuPont-6406 AA44ERJ41.BTL 10.0 REPORT A report of the results of this study will be prepared by the Testing Facility and will accurately describe all methods used for generation and analysis of the data. Results presentedwill include, but not be limited to: Test article: identification and CAS no., if known; physical nature and purity, if known; physicochemkal properties relevant to me conduct of me study, if known; stabilityof test article, if known- Solvent/Vehicle: justification for choice of vehicle; solubility and stability of test article in solvent/vehicle, if known. source of cells and time the cells were obtained, karyotype features (modal chromosome number) and suitability of die cell type used. test conditions: composition of medium; CO; concentration; incubation time; solvent and solvent selection rationale; concentration of test article and concentration selection rationale; composition and acceptability criteria for the metabolic activation (S9) system; duration of treatment; duration of treatment with and concentration ofColcemid*; type of , metabolic activation system used; positive and solvent controls; methods of slide preparation; number of cell cultures; criteria Sac scoring aberrations and criteria for considering studies positve, negative or equivocal results: description of precipitation; pH and osmolarity of the treatment medium; mitotic inhibition relative to me solvent control; mitotic index and number of metaphases . analyzed; type and number of 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 will be maintained according to Standard Operating Procedure OPQP3040 by me BioReliance RAQA unit headquartered at BioReliance, 14920 Broschart Road, Rockville, MD 20850. Per mis SOP, 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 of the matt-rial; AH study materials returoed to the Sponsor or destroyed will first be copied and the copy will be retained in me BioReliance archives for a minimum of 10 years. Protocol N&SPGT341 01-My-2001 BioReliance Study No. AA44ER.341 .BTL Page 8 of 10 ^ BlORELIANCF 38 aapessySanlBgaA eg y^t contain TSCA C^i I D H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes______________DuPont-6406 Sponsor Project Number: BioReliance StudyNumber. DuPont - 6406 AA44ER341.BTL ' 110 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This protocol has been written to comply wimOECD Guideline 473 (2n TOro Mammalian Chromosome Abcnation Test), adoptedJuly 1997 and with the International Conference on Hannonizarion of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997). Thisstudy will be perfonned in compliance with the povisions of the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies (GLPs). The protocol, an in-process phase, the raw data, and report(s) will be audited per (he Standard Operating Procedures (SOPs) of BioReliance by the Quality Assurance Unit of BioReliance for compliance with GLPs, the SOPs of BioReliance and the study protocol. The in-process inqiection will be performed to audit the critical assay procedures and systenis supporting me assay. A signedQA statement will be included in the final report. This statement will list the system phases inspected during the previous quarter or me study-specific phases, the dates of each inspection, and the dates the results of 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 will cite the GLP guideline(s) with which the study is compliant and any exceptions to this compliance, if applicable, including me omission of characterization or stability analyses of the test or control articles or weir mixtures. ) Unless arrangements are made to the contrary, unused dosing solutions will be disposed of following administration to die test system and all residual test article will be disposed of following fualization of the report. 13.0 REFERENCES Evans, HJ. and MJL- 0'Riordan- 1975. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mutation Res. 31:135-148. Galloway, SM, MJ. Aardema, M. Ishidate Jr., J.L. Ivett, DJ. Kirkland, T. Morita, P. Mosesso and T. Sofimi (1994) Report from working group on in vitro tests for chromosomal atxarations. Mutation Research 312(3):241-261. International Conference on Harmooisatioo (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Geootoxkity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 of the ICH process on July 19,1995. Federal Register 61:18198-18202, April 24,1996. International Conference on Hannonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for ^ Genotoxicity Testing of Pharmaceuticals. S2B document recommended for adoption at ; step 4 of the ICH process on July 16,1997. Federal Register 62:16026-16030, November 21,1997. Protocol No. SPGT341 ei-My-2001 Pge9ofIO (69^. B_ I O _ R E L I A N C E - BioReliance StudyNo.AA44ER.341.BTL 39 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes____________________________DuPont-6406 Sponsor Project Number. BioReliance Study Number DuPont-6406 AA44ER.341.BTL i OECD Guideline 473 (Genetic Toxicology: In Vitro Mammalian Chromosome Aberration Test), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, Febniaiy 1998. Preston, RJ, W. All, MA. Bender, J.G. Brewen, A.V. Canano, JA. Hoodie, AJ. McFee, S. WolfiFand J.S. Wassom. 1981. Mammalian in vno and in vitro cytogenetics assays: A report ofthe US EPA's Gene-ToxProgram. MutationRes-87:143-188. Scott, D., NJ). Danforo, BJ. Dean and DJ. Kiridand- 1990. Metaphase Chromosome Aberration Assays In Vitro. In: Basic Mutagenicity Tests: UKEMS Recommended Procedures. DJ Kiridand (ed). Cambridge Univeisity Press, New York, NY. Swierenga SJLH-, JA. Heddle, EA. Sigat, JJ'.W. Guman, ILL. BriUinger, G.R- Douglas and EJLNesunaim (1991) Re<nunended protocols based on a survey of current practice in genotoxkaty testing laboratories, IV. Chromosome aberration and sister-chromarid exchange in Chinese hamster ovary, V79 Chinese lung and human lymphocyte cultures, Mutation Research 246:301-322. 14.0 APPROVAL ) K ~ - CL^g-- i~\ ^-^<SVN^g^-^________03 MA^f SPONSOR REPRESENTATIVE'DATE 2-00 TY<sc-c- ^><rvs/^t-<' yrint or Type Name) /?^^cgZ^^-/^^-_________JO ^ Qoo\ BIORELIANCE STUDY DIRECTORDATE ^7C^L^^~^ irl^f^s.ool BIORELIANCE STUDY MANAGEMENT DATE Protocol No. SPGTMl Ol-May-2001 Page 10 of 10 BioReliance Study No. AA44ER.341 .BTL 40 ^ BlORELIANCF
Contact UsLoginSign Up
CUNY - The Graduate CenterColumbia University Mailman School of Public Health - Sociomedical Sciences