Document EvvQR5EkqqgKvvBK6M7GqXgXj

DownloadViewSend us a messageRandom document
H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes AR226-3181 DuPont-6406 TRADE SECRET Testing Guidelines International Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. 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 and OECD Guidelines for Testing o f Chemicals Section 4: Health Effects, N o. 473 (Adopted 1997) Study Title H:24889: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes A uthors Ramadevi Gudi, Ph.D. Caren Brown, M.S. Study Completion Date July 24,2001 Performing Laboratory BioReliance 9630 M edical Center Drive Rockville, MD 20850 for E. I. du Pont 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 o f 40 H-24889: In Vitro Chromosome Abenation Study in Human Peripheral Blood Lymphocytes DuPont-6406 STATEM ENT O F COM PLIANCE Study No. AA44ER.341.BTL was conducted in compliance with the U.S. FDA GLP Regulations as published in 21 CFR 58, the U.S. EPA GLP Standards 40 CFR 160, and 40 CFR 792, the UK GLP Compliance Programme, the Japanese GLP Standard, and the OECD Principles o f 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 o f Analysis on file w ith the testing facility. The stability o f the test and control articles has not been determined by die testing facility. Analyses to determine die uniformity (as applicable), or concentration o f the test and control mixtures were not performed by the testing facility. The Sponsor has indicated that they have not performed these analyses on die test article mixtures. The stability o f 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 die test article mixtures. Ramadevi Gudi, Ph.D. Study Director Date BioReliance Study Management D ate B ioR eliance S tudy N o. A A 44E R .3413 T L 2 O agpes# SanSSssd 0 net contain TSA CBI H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes______ Quality Assurance Statement DuPont-6406 S tu d y T itle : H 24889 IN VITRO M AM M ALIAN CH RO M O SO M E A B ER R A TIO N STUDY IN HUM AN PERIPH ERA L BLOOD LYM PHOCYTES S tu d y N u m b er: A A 44ER.341 .BTL S tu d y D ire c to r: R am adevi G udi, P h D . T his study has been divided in to a series o f in-process phases. U sing a random sam pling approach, Q uality A ssurance m onitors each o f these phases over a series o f studies. Procedures, docum entation, equipm ent records, etc., are exam ined in order to assure th a t th e study is perform ed in accordance w ith d ie U .S. FD A G ood L aboratory P ractice R egulations (21 CFR 58), th e U .S. E PA G LPs (40 C FR 792 and 40 C FR 160), the U K GLP R egulations, the Japanese GLP Standard, and the OECD P rinciples o f G ood L aboratory P ractice an d to assure th at th e study is conducted according to the protocol and relev an t Standard O perating Procedures. The follow ing are th e inspection dates, phases inspected, and report dates o f QA inspections o f th is study. Inspect On Phase 10-M ay-01 - 10-M ay-01 To S tudy D ir 10-M ay-01 T o M gm t 10-M ay-01 Protocol R eview Inspect O n Phase 07-Jun-01 - 07-Jun-01 To Study D ir 07-Jun-01 T o M gm t 08-Jun-01 D ilution o f te st article and control m aterial Inspect O n Phase 18-Jul-01 - 19-Jul-01 To Study D ir 20-Jul-01 T o M gm t 24-Jul-01 D raft R eport Inspect O n Phase 24-Jul-01 - 24-Jul-01 To Study D ir 24-Jul-01 T o M gm t 24-Jul-01 D raft to Final R eport T his rep o rt describes th e m ethods and procedures used in the study and th e reported resu lts accurately reflect th e raw d ata o f th e study. QU A LITY ASSURANCE BioReliance Study No. AA44ER.341.BTL DATE 3 Sal&s, Does not contain TSC CBl % l H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes C E R T IFIC A T IO N DuPont-6406 We, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Issued by Study D irector Ramadevi Gudi BioReliance Study Director 3M "JculhJ-oo/ Date17 Approved by Study Monitor: ^ -- <J-S) O o f -- Maria Donner, Ph.D. Senior Research Scientist 5 3 y u - 2.00 1 Date # BioReliance Study No. AA44ER.341JBTL 4 gffljgMwy SartSEss S e e s msScontain YSC A BJ H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes STUDY INFORM ATION S vnonvm s/C odei H -24889 H ask ell N um ber: 24889 DuPont-6406 P h y sical C h a ra c te ristic s: Y ellow to am b er so lid S ta b ility : T he te s t a rtic le ap p eared to b e sta b le u n d e r th e co n d itio n s o f th e stu d y ; no ev id en ce o f in sta b ility w as o b serv ed . BioReliance Study No. AA44ER.341.BTL 5 % H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes Sponsor: E. L du Pont de N em ours and Company W ilm ington, D elaw are 19898 U .S.A . Study Initiated/C om oleted : M ay 10,2001 / (see rep o rt cover page) In-L ife Initiated/C om oleted: M ay 2 4 ,2 0 0 1 / June 19,2001 DuPont-6406 V BioReliance Study No. AA44ER.341.BTL 6 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 TABLE OF CONTENTS PAGE GOOD LABORATORY PRACTICE COM PLIANCE STATEM ENT. .2 QUALITY ASSURANCE STA TEM EN T. .3 C E R T IF IC A T IO N ----- .4 STUDY INFORM ATION. .5 SUM M ARY. 9 PU R PO SE... a 11 CHARACTERIZATION OF TEST AND CONTROL ARTICLES.... 11 M ATERIALS AND METHODS Test System ......................................................... activation System ............................................. Solubility T est................................................... Preliminary Toxicity Assay............................ Chromosome Aberration Assay ..................... Collection of Metaphase Cells..................... Slide Preparation............................................... Selection of Dose Levels for Analysis......... Evaluation of metaphase Cells..................... Controls.............................................................. Evaluation of Test Results............................ Criteria for Determination o f a Valid Test Deviations........................................................... Archives............................................................... RESULTS AND DISCUSSION 11! 11 12 12 12 13 14 14 14 14 15 15 16 16 16 16 Solubility Test................................................................................................................................................ 1 Preliminary Toxicity Assay......................................................................................................................... Chromosome Aberration Assay.................................................................................................................. 17 C O N C L U SIO N 18 R EFER E N C ES.... 19 BioReliance Study No. AA44ER.341.BTL 7 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 D A T A T A B L E S ____________________________________________________________________________________ " TA B LE 1 PRELIM IN A RY TO X IC ITY T E ST U SIN G H -24889 IN T H E A BSEN CE O F EXOGENOUS M ETA B O LIC A C T IV A TIO N 4 H OU R T R E A T M E N T ....................................................................................... 20 TA B LE 2 PRELIM IN A RY T O X IC ITY T E ST U SIN G H -24889 IN T H E PRESEN CE O F EXO GEN OU S M ETA B O LIC A C T IV A T IO N 4 H O U R T R E A TM EN T ....................................................................................... 21 TA B LE 3 PRELIM IN A RY TO X IC ITY T EST U SIN G H -24889 IN T H E A BSEN CE O F EXOGENOUS M ETA B O LIC A C TIV A TIO N 20 H O U R T R E A T M E N T .................................................................................... 22 TA B LE 4 C Y TO G EN ETIC A N A LY SIS O F H UM A N PER IPH ER A L BLO O D LYM PHOCYTES TR EA TED W IT H H -24889 IN T H E A BSEN CE O F EX O G EN O U S M ETA B O LIC A CTIV A TIO N D E FIN IT IV E A SSAY : 4 H O U R TR EA TM EN T, 2 0 H O U R H A R V E ST ........................................................23 TA B LE 5 CY TO G EN ETIC A N A LY SIS O F H U M A N PE R IPH E R A L BLO O D LY M PHOCYTES TREA TED W ITH H -24889 IN T H E PRESEN CE O F EX O G EN O U S M ETA B O LIC A CTIV A TIO N D EFIN IT IV E A SSAY : 4 H O U R TR EA TM EN T, 20 H O U R H A R V E ST ...................................................... 24 TA BLE 6 C Y TO G EN ETIC A NA LY SIS O F H U M A N PER IPH ER A L BLOOD LYM PHOCYTES TREA TED W IT H H -24889 IN T H E A BSEN CE O F EX O G EN O U S M ETA B O LIC A CTIV A TIO N D EFIN IT IV E A SSAY : 2 0 H O U R TREA TM EN T, 20 H O U R H A R V E ST ..................................................... 25 TA B LE 7 SU M M A R Y ................................................................................................................................................. 26 A PPEN D IX A H IST O R IC A L C O N T R O L DATA A P P E N D IX B STU D Y P R O T O C O L ..___________ BioReliance Study No. AA44ER.341.BTL 8 ampanySanHsaiL 0e* neSeontalm TSCA CBH 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 o f choice based on solubility o f the test article and compatibility with the target cells. The test article was soluble in acetone at a maximum concentration o f 500 mg/mL. In the preliminary toxicity assay, the maximum dose tested was 5000 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 of S9 activation. Visible precipitate was observed in treatment medium at concentrations of 1500 and 5000 pg/mL. Concentrations o f <500 pg/mL were soluble in treatm ent 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 S9-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 S9-activated 4 hour exposure group. 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, and all cells were harvested at 20 hours after treatment initiation. Visible precipitate was observed in treatment medium at concentrations o f 1500 to 3000 pg/mL in the non-activated 4 hour and the 20 hour exposure groups and at 1500 and 2000 pg/mL in the S9-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 S9-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). In 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 H -24889: In Vitro C hrom osom e A berration Study in H um an P erip h eral B lood L ym phocytes 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 o f dose level (p>0.05, Fisher's exact test). Based on the findings o f this study, H-24889 was concluded to be negative for the induction o f structural and numerical chromosome aberrations in the in vitro m a m m a lia n chromosome aberration test using human peripheral lymphocytes. m B ioR eliance Study N o. A A 44E R .341.BTL 10 tpr--ymwySesriflsosS. 08S neontaln TTSCACBS H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 PURPOSE The purpose o f this study was to evaluate the clastogenic potential o f a test article based upon its ability to induce chromosome aberrations in human peripheral lymphocytes. CHARACTERIZATION O F TEST AND CONTROL ARTICLES The test article, H-24889, was received by BioReliance on M ay 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, w ith 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 pg/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. M ATERIALS AND METHODS Test System Peripheral blood lymphocytes were obtained from a healthy non-smoking 26 year old adult malp. 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 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 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 A ctivation System Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced w ith 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 typhimunum 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 pL S9 per m illiliter medium (RPMI 1640 serum-fiee 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: purified water, dimethyl sulfoxide (DMSO), ethanol, and acetone. The test article 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. Prelim inary Toxicity Assay The toxicity test was performed for the purpose o f selecting concentrations for the chromosome aberration assay and consisted o f an evaluation o f test article 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 with 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 of the highest treatment condition were measured, and the pH was adjusted, if necessary, in order to m a in ta in a neutral pH in the treatment medium. At the tim e of test article 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 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 100 pL test article dosing solution in solvent or solvent alone. The cells were exposed to solvent alone and to nine concentrations o f the test article 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 of 51% CO2 in air. At the completion of the 4 hour exposure period, the treatment medium BioReliance Study No. AA44ER.341.BTL 12 0069 not contain TSQ C8 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 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 o f cells in mitosis per 500 cells scored was determined in order to evaluate test article effect on mitotic index. Chromosome A berration 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 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 of 51% CO2 in air for 44-48 hours. Treatment was carried out by refeeding w ith approximately 10 mL fresh complete medium or S9 reaction m ixture to which was added 100 pL o f dosing solution o f 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 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 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. 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 o f 0.1 Pg/mL. 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. BioReliance Study No. AA44ER.341.BTL 13 BBpwy SanStbadL 0s* no8contain T8GA C H-24889: In Vitro Chromosqme Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 Collection o f M etaphase 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 (methanolrglacial acetic acid, 3:1 v/v) was added to each tube. The cells were collected by centrifugation, the supernatant aspirated, and die cells were fixed with two washes w ith approximately 3-5 mL of fixative and stored in fixative overnight or longer at approximately 2-8C. Slide P reparation 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 w ith 5% Giemsa, air dried and permanently mounted. Selection of Dose Levels fo r Analysis . The selection o f 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 treatm ent medium, the highest dose level evaluated was the lowest precipitating dose level, regardless of toxicity. A t least two additional lower dose levels were included in the evaluation. Evaluation o f M etaphase 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 valuated 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. M etaphase cells w ith 46 centromeres were examined under oil immersion without prior knowledge o f treatm ent groups. W henever possible, a m i n i m u m 0f 200 metaphase spreads (100 per duplicate treatm ent 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 isochrom atid breaks and exchange B ioR eliance Study N o. A A 44E R .341.B TL 14 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 figures such as quadriradials (symmetrical and asymmetrical interchanges), tm adials, 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 were scored as a break (chromatid or chromosome). Fragments observed w ith 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 o f 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 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. The mitotic index was recorded as the percentage o f cells in mitosis per 500 cells counted. The percent polyploid and endoreduplicated cells was evaluated per 100 cells. C o n tro ls MMC was used as the positive control in the non-activated study at final concentrations o f 0.25 and 0.5 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 article was used as the solvent control at the same concentration as that found in the test article-treated groups. Evaluation of T est Results The toxic effects of treatment are based upon m itotic inhibition relative to the solvent-treated control and are 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 o f structural 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 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 o f each treatment group with that o f the solvent control. In the event o f a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness. 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 o f 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 high dose only w ith 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 article was concluded to be negative if no statistically significant increase was observed relative to the solvent control. C riteria fo r D eterm ination o f a V alid T est The frequency of cells w ith 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 (p-- > Fisher's exact test) relative to the solvent control. D eviations No known deviations from the protocol or assay-method SOPs occurred during the conduct of this study. A rchives All raw data, the protocol, all reports, ancH tam ec^nd coded slides will be maintained according to Standard Operating P r o c e d u r f lp |J ||||B J > y 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 of 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 m in im u m of 10 years. RESULTS AND DISCUSSION Solubility Test Acetone was determined to be the solvent o f choice based on solubility o f the test article and com patibility with the target cells. The test article was soluble in acetone at a maximum concentration o f 500 mg/mL. Prelim inary 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 of mitotic inhibition are presented in Tables 1, 2, and 3. HPBL B ioR eliance Study N o. A A 44E R .341.B TL 16 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 cells were first exposed to nine concentrations o f H-24889 ranging from 0.5 pg/mL to 5000 Hg/mT.j as well as solvent controls, in both the absence and presence o f 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 treatm ent medium at concentrations of 1500 and 5000 jig/mT- Concentrations o f <500 pg/mL were soluble in treatm ent medium. The osmolality and pH o f the highest concentration tested, 5000 pg/mL, were 258 mmol/kg and approximately 7.5, respectively. The osmolality o f the lowest precipitating dose level, 1500 Pg/mL, was 256 mmol/kg. The osmolality o f the highest soluble dose level, 500 Hg'mL, was 261 mmol/kg. The osmolality o f 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 pg/mL in the non-activated 4 hour and the 20 hour exposure groups and at 1500 and 5000 pg/mL in the S9-activated 4 hour exposure group. 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 20 4 Recovery Time (hr) 16 0. 16 Dose levels (pg/mL) 1 2 5 ,2 5 0 ,5 0 0 ,1 0 0 0 ,1 5 0 0 ,1 7 5 0 , 2000,2500 and 3000 1 2 5 ,2 5 0 ,5 0 0 ,1 0 0 0 ,1 5 0 0 ,1 7 5 0 , 2000,2500 and 3000 125,250,500,1000,1250,1500 and 2000 Chromosome A berration Assay In the chromosome aberration assay, visible precipitate was observed in treatment medium at concentrations o f 1500, 1750,2000,2500 and 3000 pg/mL in the non-activated 4 hour and the 20 hour exposure groups. Concentrations 1000 pg/mL were soluble in treatment medium. Visible precipitate was observed in treatm ent medium at concentrations of 1500 and 2000 pg/mL in the S9-activated 4 hour exposure group. Concentrations 1250 pg/mT, were soluble in treatm ent medium. The osmolality in treatment medium o f the highest concentration tested, 3000 pg/mL, was 380 mmol/kg. The osmolality of the lowest precipitating dose level, 1500 Pg/mL, was 379 mmol/kg. The osmolality o f the highest soluble dose level, 1250 Pg/mL, was 375 mmol/kg.The osmolality of the solvent (acetone) in treatment medium was 382 mmol/kg. The pH o f the highest concentration o f test article in treatment medium was approximately 7.5. BioReliance Study No. AA44ER.341.BTL 17 omipaiy SanM sed. 0@e# no8 eo ntaln TSCA CB8 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 The findings o f 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, 1500pg/mL, mitotic inhibition was 36%, relative to the solvent control. The dose levels selected for analysis o f chromosome aberrations were 125, 500 and 1500 pg/mL. The highest dose was selected to score chromosome aberrations was the low est precipitating dose level in the culture medium. The percentage o f 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 o f 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 pg/mL, mitotic inhibition was 57%, relative to the solvent control. The dose levels selected for analysis o f chromosome aberrations were 125, 500, and 1250 pg/mL. The percentage o f 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 fin d in g s o f 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 pg/mL, mitotic inhibition was 15%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 125,500, and 1500 pg/mL. The percentage o f 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, Fishers exact test). The percentage o f cells with structurally damaged chromosomes in the MMC (positive control) treatment group (10.5%) was found to be statistically significant. C O N C L U S IO N The positive and solvent controls fulfilled the requirements for a valid test. Under the conditions o f the assay described in this report, H-24889 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. AA44ER.341.BTL 18 fflpc3i^ S a ttig s e d 0 o e # nod eo n tain TSCA CfE& 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., M J. 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 Harmonization (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. Carrano, J.A. Heddle, A.F. McFee, S. W olff 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 0 es not eonfialn TSCA CSJ % 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 TREATMENT (-S9) pg/m L Acetone M ITO TIC INDEX (% ) 4.0 PERCENT CHANGE (%) 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 T reatm 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. P ercent Change = (treatment m itotic index - control m itotic index)/control mitotic index, expressed as a percentage. BioReliance Study No. AA44ER.341.BTL 20 oofflpemj?SanRIxed. Does not contain TSC CB % H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 TABLE2 PRELIMINARY TOXICITY TEST USING H-24889 IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT TREATMENT MITOTIC PERCENT INDEX CHANGE (+S9) pg/mL Acetone (%) (%) 4.4 H-24889 0.5 1.5 5 15 50 150 500 1500 5000 4.6 5 5.4 23 4.0 -9 5.0 14 4.8 9 5.6 27 3.2 -27 1.0 -77 0.4 -91 T reatm 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 following a 16 hour recovery period. M itotic Index = (cells in mitosis/500 cells scored) x 100. P ercent C hange = (treatment mitotic index - control m itotic index)/control mitotic index, expressed as a percentage. i BioReliance Study No. AA44ER.341.BTL 21 SssnSfea. Sees not eonteln YSC CESI 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) pg/m L Acetone 20 HOUR TREATMENT MITOTIC PERCENT INDEX CHANGE (%) (%) 4.6 H-24889 0.5 1.5 5 15 50 150 500 1500 5000 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 T reatm 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 following a 16 hour recovery period. M itotic Index = (cells in m itosis/500 cells scored) x 100. P ercent C hange = (treatm ent mitotic index - control m itotic index)/control m itotic index, expressed as a percentage. B ioR eliance S tudy N o. A A 44E R .341.BTL 22 eragsaray Does net onan TSCA CBS % H-24889: In Vitro Chromosome Aberration Study in Human Peripheral. Blood Lymphocytes DuPont-6406 TABLE4 CYTOGENETIC ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES TREATED WITH H-24889 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST T reatm ent pg/mL Acetone H-24889 125 500 1500 MMC 0.5 Mitotic % Aberrant Cells Index Cells Flask (%) Scored Numerical Structural A 4.4 B 4.0 100 100 0 0 0 0 A 3.6 B 4.4 100 100 A 3.0 B 2.8 100 100 A 2.8 B 2.6 100 10O A 1.6 100 B 2.6 10O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 6 Total Number o f Structural Aberrations Gaps Chromatid Br Ex 00 00 0 0 Chromosome B r Die Ring 00 00 0 0 Severely Damaged Cells 0 0 00 00 00 00 00 00 04 04 0 0 0 0 0 0 3 0 00 0 00 0 00 00 0 0 00 00 0 0 10 0 11 0 0 0 0 0 0 0 0 0 Average Aberrations Per Cell 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.080 0.060 T reatm ent: Human peripheral blood lymphocytes were treated for 4 hours at 37 1C in the absence o f an exogenous source o f metabolic activation. A dditional dose levels o f250 and 1000 pg/m L were tested as a safeguard against excessive toxicity at higher dose levels but were not required for m icroscopic examination. Dose levels 1750,2000, 2500 and 3000 pg/mL were not analyzed due to excessive toxicity. M itotic index =number m itotic figures x 100/500 cells counted. % A b erran t C ells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps. C hrom atid breaks include chromatid and isochromatid breaks and fragments (Br); chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements. Chrom osom e B reaks include breaks and acentric fragments (Br); Die, dicentric chromosome. Severely Dam aged Cells includes cells with one or more pulverized chromosome and cells w ith 10 or more structural aberrations. A verage A berrations P er C ell: severely damaged cells and pulverizations were counted as 10 aberrations. B ioR eliance S tudy N o. A A 44E R .341.BTL 23 OfflspatiySanRhed. Dees no! eonSalra TSG CB8 % 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 DEFINITIVE ASSAY: 4 HOUR TREATMENT, 20 HOUR HARVEST Treatment pg/mL Acetone H-24889 125 500 1250 CP 20 M itotic % A benant Cells Index Cells Flask (%) Scored Numerical Structural A 3.6 B 3.4 100 100 0 0 0 0 A 3.4 B 2.8 100 100 A 2.6 B 2.6 100 100 A 1.6 100 B 1.4 100 A 1.6 100 B 1.0 100 0 0 0 0 0 0 0 0 0 0 0 1 0 1 7 9 Total Number o f Structural Aberrations Gaps Chromatid Br Ex 00 0 00 0 Chromosome Br Die Ring 00 00 0 0 Severely Damaged Cells 0 0 00 00 0 0 00 0 010 00 0 010 07 07 0 1 00 0 00 0 00 00 0 0 00 0 00 0 00 0 11 0 0 0 0 0 0 0 0 0 Average Aberrations Per Cell 0.000 0.000 0.000 0.000 0.000 0.010 0.000 0.010 0.070 0.100 T reatm ent: Human peripheral Wood lymphocytes were treated for 4 hours at 37 1C in the presence o f an exogenous source o f metabolic activation. A dditional dose levels o f 250 and 1000 pg/mL were tested as a safeguard against excessive toxicity at higher dose levels but w o e not required for m icroscopic examination. Dose levels 1500, and 2000 pg/mL were not analyzed due to excessive toxicity. M itotic index = number m itotic figures x 100/500 cells counted. % A berran t C ells: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps. C h ro m atid B reak s include chrom atid and isochrom atid breaks and fragm ents (B r); chrom atid exchange figures (Ex) include quadriradials, trirad ials and com plex rearrangem ents. Chrom osom e B reaks include breaks and acentric fragments (Br); Die, dicentric chromosome. Severely Dam aged Cells includes cells w ith one or more pulverized chromosome and cells with 10 or more structural aberrations. A verage A berrations P er C ell: severely damaged cells and pulverizations were counted as 10 aberrations. B ioR eliance S tudy N o . A A 44E R .341J3TL 24 Offlpai SssffisodL Dees me eensfa TSCA CBS H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes * DuPont-6406 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 Treatment (ig/mL Acetone H-24889 125 500 1500 MMC 0.25 M itotic % Aberrant Cells Index Cells Flask (%) Scored Numerical Structural A 4.8 B 4.4 100 100 0 0 0 1 A 3.8 B 3.6 A 3.4 B 4.8 A 3.8 B 4.0 A 4.0 B 4.8 to o 100 to o too 100 100 10O 100 0 0 0 0 0 0 0 0 0 0 0 0 0 1 12 9 Total Number o f Structural Aberrations Gaps Chromatid Br Ex 00 0 010 Chromosome B r Die Ring 00 00 0 0 00 0 00 0 00 00 0 0 00 0 010 08 4 06 2 00 00 0 0 00 0 00 0 00 0 00 0 01 0 02 0 Severely Damaged Cells 0 0 0 0 0 0 0 0 0 0 Average Aberrations 0.000 0.010 _ 0.000 0.000 0.000 0.000 0.000 0.010 0.130 0.100 T reatm ent: Human peripheral blood lymphocytes were treated for 20 hours at 37 1C in the absence o f an exogenous source o f metabolic activation. A dditional dose levels o f230 and 1000 gg/mL were tested as a safeguard against excessive toxicity at higher dose levels but were not required for microscopic examination. Dose levels 1750, 2000,2300 and 3000 pg/mL w ere not analyzed due to excessive toxicity. M itotic index --number m itotic figures x 100/500 cells counted. . % A b erran t C elb: numerical cells include polyploid and endoreduplicated cells; structural cells exclude cells with only gaps. C hrom atid B reaks include chrom atid and isochromatid breaks and fragm ents (Br); chromatid exchange figures (Ex) include quadriradials, triradials and complex rearrangements. Chrom osom e B reaks include breaks and acentric fragments (Br); Die, dicentric chromosome. Severely D am aged C ells includes cells with one or more pulverized chromosome and cells with 10 or more structural aberrations. A verage A berrations P e r C ell: severely damaged cells and pulverizations were counted as 10 aberrations. BioReliance Study No. AA44ER.341.BTL 25 B ^3 an!ffe8& 09Bn8entafes TSGA GB! % H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 T reatm ent pg/m L A cetone H -24889 125 500 1500 MMC 0.5 A cetone H -24889 125 500 1250 CP 20 A cetone H -24889 125 500 1500 MMC 0.25 TABLE 7 SUMMARY M ean S9 Treatment M itotic Cells Activation Time Index Scored A berrations Per Cell (M ean+/-SD ) Cells W ith 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** + 4 3.5 200 0.000 0.000 0.0 0.0 + 4 3.1 200 0.000 0.000 0.0 0.0 + 4 2.6 200 0.005 0.071 0.0 0.5 + 4 1.5 200 0.005 0.071 0.0 0.5 + 4 1.3 200 0.085 0.313 0.0 8.0** - 20 4.6 200 0.005 0.071 0.0 0.5 __ . 20 3.7 200 0.000 0.000 0.0 0.0 20 4.1 200 0.000 0.000 0.0 0.0 - 20 3.9 200 0.005 0.071 0.0 0.5 - 20 4.4 200 0.115 0.350 0.0 10.5** T reatm ent: Cells from all treatment conditions were harvested at 20 houre after the initiation o f the treatm ents. A berrations p er C ell: Severely damaged cells were counted as 10 aberrations. P ercen t A b erran t C ells: *, p^O.Ol; using Fisher's exact te s t BioReliance Study No. AA44ER.341.BTL 26 H-24889: In Vitro Chromosome Aberration Study % in Human Peripheral Blood Lymphocytes APPENDIX A H istorical C ontrol D ata DuPont-6406 BioReliance # Study No. AA44ER.341.BTL 27 (Bow pafl 8 miBk a& Boa nel contain TSCA CB8 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 H isto rical V alues Mean Standard Deviation Range Percent Aberrant Cells (%) Untreated Control Solvent Control1 Positive Control"4 0.2 0.3 0.0-1.5 0.3 0.5 0.0-2.0 . 16.1 9.5 6.5-48.0 S9 ACTIVATED TEST SYSTEM H istorical V alues Mean Standard Deviation Range Percent Aberrant Cells (%) Untreated Control 0.2 0.4 0.0-1.5 Solvent Control1 0.4 0.7 0.0-3.5 Positive Control* 15.7 7.5 7.0-39.0 'Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor-supplied vehicles. 2Positive control for non-activated studies is mitomycin C (MMC). 3Positive control for S9 activated studies is cyclophosphamide (CP). 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 H isto rical V alues M ean Standard Deviation Range Percent Aberrant Cells (%) Untreated Control 0.2 0.3 0.0-1.0 Solvent Control1 0.1 0.2 0.0-1.5 Positive Control4 0.1 0.3 0.0-1.5 S9 ACTIVATED TEST SYSTEM H isto rical Values Mean Standard Deviation Range Percent Aberrant Cells (%) Untreated Control 0.2 0.3 0.0-1.0 Solvent Control1 0.1 0.3 0.0-1.0 Positive Control* 0.2 0.4 0.0-1.5 S olvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor-supplied vehicles. P o sitiv e control for non-activated studies is mitomycin C (MMC). P o sitiv e control for S9 activated studies is cyclophosphamide (CP). 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 m BioReliance Study No. AA44ER.341.BTL 30 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 deceivedbyRWOftJsas^ 1 Sponsor Project N um ber: rD' u"P on*t-6*4A0ni6L B ioR eliance Study N u m b er A A44ER.341 -BTL IpIPifOlfiDi H-24889: In Vitro Mammalian Chromosome Aberration Study in Human Peripheral Blood Lymphocytes 1.0 PURPOSE T he purpose o f tins study is to evaluate the clastogenic potential 24889) upon its ability to induce chrom osom e aberrations in hum an peripheral olood lym phocytes (H PBL). 2.0 SPONSOR 2.1 N am e: 2.2 A ddress: F l du Pont de Nem ours and Com pany Stine H askell R esearch C enter D uPont H askell Laboratory P.O .B ox 50 1090 Elkton R oad N ewark, DE 19714-0050 23 R epresentative: M aria D onner, Ph.D . Phone: 302-366-5251 Fax: 302-366-5207 E-m ail: m aria.donner@ usa.dupont.com 2.4 Sponsor Project #: DuPont-6406 25 W R#: |j B ^ \ 2.6 H askell#: H -24889 2.7 Service Code: W 3.0 IDENTIFICATION O F TEST AND CONTROL ARTICLES 3.1 T est A rticle N am e: T he test article is a solid a t room tem perature and m ust be heated to 60C each and every tim e. W hen all m aterial is m elted and hom ogeneously m ixed to assure uniform ity, a n aliquot needs to be taken out fo r dosing. T he m ixing should occur around 60C. 32 T est A rticle I.D .: H-24889 (to be used in the report and text) Protocol No. SPGT341 01-May-2001 Page I of 10 ^ Bio Relianoe BioReliance Study No. AA44ER.341 .BTL 31 es bo?contain TSC GB % % H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 Sponsor Project N u m b er D uP ont-6 4 0 6 B ioR eliance Study N u m b er A A 44E R .341J3TL 33 C ontrols: S olvent P o sitiv e : T est A rticle Solvent o r V ehicle M itom ycin C (M M C) C yclophospham ide (C P) 3.4 T est A rticle Characterization U nless alternate arrangem ents are m ade, the testing facility at B ioR eliance w ill n o t perform analysis o f tire dosing solutions. T he Sponsor w ill b e directly responsible fo r determ ination and docum entation o f die analytical purity and com position o f the test article, and th e stability and strength o f the test article in th e solvent (or vehicle). 4.0 TESTIN G FA CILITY AND KEY PERSONNEL 4.1 N am e: T oxicology T esting Facility B ioR eliance 42 A ddress: 9630 M edical C enter D rive R ockville, M D 20850 43 Study D irector: Ram adevi G udi, Ph.D . Phone: 301-610-2169 Fax: 301V738-2362 E-m ail: rgudi@ bioteliance.com . 5.0 PROPO SED STUDY DATES 5.1 Experim ental S tart D ate: 23-M ay-2001 (S election o f dose levels fo r the chrom osom e aberration study and any possible repeats w ill be done in consultation w ith the Sponsor.) 5.2 Experim ental Term ination D ate: 20-Jul-2001 5 3 D raft Report D ate: 30-M -2001 5.4 F inal R eport: 2 w eeks after Sponsor approves draft 6.0 T EST SYSTEM P eripheral blood lym phocytes w ill be obtained from healthy adults w ithout a recent history o f eith er radiotherapy, viral infections or th e adm inistration o f drugs. T his system has been dem onstrated to b e sensitive to the clastogenic activity o f a variety o f chem icals (P reston et al., 1981). Protocol No. SFGT341 01-May-2001 P *gc2ofl0 ^ Bio Reliancf BioReliance Study No. AA44ER.341 .BTL 32 ggapes^Ssnlssd Ps* nononaffi fBGA CB5 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 S ponsor P roject N u m b er D uP ont--6406 B ioR eliance Study N u m b er AA44ER-341JBTL 7.0 EXPERIM ENTAL DESIGN A ND M ETHODOLOGY T he assay w ill b e conducted using standard procedures, by exposing hum an lym phocytes to a m inim um o f four concentrations o f th e te st article as w ell as to positive and solvent controls. In the non-activated test system , treatm ent w ill be fo r 4 hours and fo r 20 hours; in the S9 activated test system , exposure w ill be fo r 4 hours (Sw ierenga e t a l., 1991). T he dividing cells w ill be arrested in m etaphase an d harvested for m icroscopic evaluation o f chrom osom e aberrations at approxim ately 20 hours (1.S norm al cell cy cles) after the initiation o f treatm ent in order to ensure evaluation o f first-division m etaphase cells (G allow ay e t al., 1994). The clastogenic poten tial o f th e test article w ill be m easured by its ability to increase chrom osom e aberrations in a dose-responsive m an n a w hen com pared to a control group. The 4 hour non-activated and S9-activated studies w ill be scored initially. In th e event o f a positive response in th e 4 hour non-activated study, th e prolonged exposure non-activated study m ay no t be scored. T he test article w ill also be assessed fo r its ability to induce num erical chrom osom e aberrations. 7.1 Solvent Selection 7.1.1 Solubility D eterm ination U nless tiie Sponsor has indicated th e test article solvent, a solubility determ ination w ill be conducted to determ ine the solvent and th e m axim um soluble concentration up to a m axim um o f 500 m g/m l. S olvents com patible w ith this test system , in order o f preference, include but are not lim ited to sterile w ater (CA S 7732-18-5), dim ethyl sulfoxide (CAS 67-68-5), ethanol (C A S 64-17-5), and acetone (CA S 67-64-1). The solvent w ill be th e test article solvent, selected in order o f preference, th at perm its preparation o f the highest soluble stock concentration, up to 500 m g/m l. 7.2 Prelim inary Toxicity T est fo r Selection o f D ose Levels Selection o f the dose levels fo r the cytogenetics assay w ill be based upon inhibition o f m itosis after treatm ent as determ ined in a cytotoxicity study. C ells w ill be exposed to solvent alone and to a t least nine concentrations o f test article, die highest concentration being 5000 pg/m l o r 10 mM w hichever b low er. T he pH o f tiie highest test article dosing solution w ill be m easured, and w ill b e adjusted, if necessary, in order to m ain tain a neutral pH in the treatm ent m edium . The osm olality o f the highest dosing solution, low est precipitating dose level (w here applicable) and the highest soluble dose level (w here applicable) w ill also be m easured. Peripheral blood cells w ill be cultured in RPM I-1640 containing 15% fetal bovine serum , 2 mM L -glutam ine, 100 units penicillin and 100 ug streptom ycin/m l and 1% phytohem agglutinin. C ells seeded approxim ately 46 hours earlier w ill be exposed for 4 hours in th e absence and presence o f S9 and for 20 hours in th e absence o f S9. A fter exposure th e cultures w ill be grow n in com plete ) m edium fo r 16 hours. Eighteen hours after treatm ent initiation Colcenud (0.1 pg/m l) w ill be added to the cultures. C ells w ill be collected at 20 hours after the Protocol No.SPGT341 01-May-2001 P a g e 3 o fl0 ^ Bio Reliance- BioReliance Study No. AA44ER.341.BTL 33 Ssra!!8fea& 0es roofeorttaln TSCA CBS H -24889: In Vitro C hrom osom e A berration Study in H um an P eripheral B lood L ym phocytes DuPont-6406 Sponsor Project N um ber. D uPont -6 4 0 6 B ioR eliance S tudy N u m b er A A 44E R 341.BTL initiation o f treatm ent by centrifugation, treated w ith hypotonic KC1 solution and fixed w ith m ethanol-glacial acetic acid. M etaphase preparations w ill b e m ade and stain ed T he percentage o f cells in m itosis p er 500 cells scored (m itotic index) w ill be determ ined for each treatm ent group. W henever possible, th e high dose for th e chrom osom e aberration assay w ill be selected to give a t least 50% toxicity (m itotic inhibition relative to th e solvent control). A t least tw o additional dose levels, dem onstrating m inim al o r no toxicity, w ill be evaluated in the chrom osom e aberration assay. In th e event th e te st article cannot be dissolved at a high enough concentration in an appropriate solvent to be toxic, then th e highest dose to be tested w ill b e die concentration resulting in m in im um precipitation in te st m edium . P recipitation w ill b e determ ined w ith the unaided eye. In th e event the te st article dem onstrates a dose-responsive increase in toxicity (m itotic inhibition relative to the solvent control) a t concentrations that exceed solubility in treatm ent m edium , then th e highest dose to be evaluated fo r chrom osom e aberrations w ill be th e concentration resulting in m inim um precipitation in te st m ed iu m . In the event th at neither cytotoxicity n o r insolubility is observed in the prelim inary test, th e highest dose in the chrom osom e aberration assay w ill be 5 m g/m l o r 10 m M w hichever is low er. I f excessive precipitation o f the test article solvent solution occurs upon addition to treatm ent m edium , o r if the osm olality o f the treatm ent m edium is excessive, th e Sponsor w ill be consulted. 7.3 Frequency and Route o f A dm inistration T arget cells w ill be treated for 4 hours in d ie absence and presence o f S 9, and for 20 hours in the absence o f S9, by incorporation o f the test article -solvent m ixture into the treatm ent m edium . T his technique has dem onstrated to be an effective m ethod o f detection o f chem ical clastogens in this test system (E vans, 1975). I f d ie Sponsor is aw are o f specific m etabolic requirem ents, then th is inform ation w ill be utilized in the preparation o f th e study design. V erification o f a clear positive response is no t required. N egative results w ill not be confirm ed w hen justification can be provided. E quivocal results m ay be confirm ed, upon consultation w ith the Sponsor, and m ay em ploy a m odification o f th e study design. T his guidance is based on the O ECD G uideline 473 (adopted July 1997) and ICH G uidance on Specific A spects o f R egulatory G enotoxicity T ests for Pharm aceuticals (1997). 7.4 M etabolic A ctivation system A roclor 1254-induced rat liver S 9 w ill be used as the m etabolic activation system . T he S9 w ill be prepared from m ale Sprague-D aw ley rats induced w ith a single intraperitoneal injection o f A roclor 1254, 500 m g/kg, five days prior to sacrifice. T he S9 w ill b e batch prepared and stored frozen a t approxim ately -70C until used. E ach batch preparation o f S9 w ill be assayed fix' sterility and its ability to Protocol No. SPGT341 01-M ay-2001 Page 4 o f 10 ^ Bio Reliancf B ioR eliance S tudy N o. A A 44E R .341.B T L 34 OBBpasajr SanKizod. Doss not contain TSGA CBi H -24889: In Vitro C hrom osom e A berration Study in H um an P eripheral B lood Lym phocytes DuPont-6406 Sponsor Project N um ber D uP ont-6 4 0 6 B ioR sliance Study N u m b er A A 44E R .341.B TL m etabolize 2-am inoanthiacene and 7,12-dim ethylbenz(a)antbxacene to form s mutagenic to Salmonella typhimurium TA100. Im m ediately prior to use, the S9 w ill b e thaw ed and m ixed w ith a cofactor pool. The final concentration o f th e cofactors and S 9 in th e reaction vessel w ill b e 2 mM M gC lj, 6 mM K.CI, 1 mM glucosc-6-phosphalc, 1 m M nicotinam ide adenine rfinnrlenfide phosphate and 20 p i S9 p er m l R PM I-1640 serum -fiee m edium . 1 5 C ontrols 1 5 .\ Solvent (or V ehicle) C ontrol The solvent fo r die test article w ill be used a s the solvent control. For solvents o ther than w ater, physiological buffer, o r m edium , th e final concentration in treatm ent m edium w ill not exceed 1%. . 1 5 2 Positive Controls M itom ycin C w ill be used a t tw o concentrations w ithin the range o f 0.1-0.50 p g /tn l as the positive control fo r th e non-activated test system . For th e S9activated system , cyclophospham ide w ill be used a t tw o concentrations w ithin the range o f 10-75 pg/m l. One dose level o f each positive control w ill be evaluated m icroscopically fo r chrom osom e dam age. 7 .6 Preparation o f T arget C ells Peripheral blood lym phocytes w ill be cultured in com plete m edium (RPM I-1640 containing 15% fetal bovine serum , 2m M L-glutam ine, 100 units penicillin and 100 p g streptom ycin/m L and 1% phytoheraagglutinin) by adding 0 .6 m l heparinized blood to a centrifuge tube containing 9A m l com plete medium. T he tubes w ill be incubated upright a t 37 1C in a hum idified atm osphere o f 5 1% C O j in a ir for 44-48 hours. 7.7 Identification o f T est System U sin g a perm anent m arking p en , the test system w ill be identified by the B ioR eliance study num ber, treatm ent condition and date. 7.8 Treatm ent ofT arget C ells Forty-four to 48 hours after culture initiation, duplicate centrifuge tubes w ill be refed w ith approxim ately 10 m l com plete m edium for th e non-activated exposure o r 10 m l S9 reaction m ixture fo r the activated exposure to w hich w ill be added 100 p i o f dosing solution o f te st o r control article in so lv en t Larger volum es o f dosing solution m ay be used i f w ater or m edium is used as the so lv en t Protocol N o. SPG T 34I 01-M ay-2001 P agcS oflO ^ Bio Reliance" BioReliance Study No. AA44ER.341.BTL 35 Goapany SanfHsed Does noeonteim? CB % % w H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 Sponsor Project N u m ber D uP ont--6406 B ioR eliance Study N u m b er A A 44ER 341 .BTL For th e S9-activaied exposure, the cells w ill be treated for 4 hours in th e presence o f an S9 reaction m ixture, w ashed free o f chem ical and cultured fo r an additional 16 hours w iftC oIcem id* (0.1 pg/m l) present for th e last 2 hours. F or th e non-activated exposure, reatnvnt w ill be for 4 hours follow ed by a 16 hour recovery period and for 20 hours continuously w ith Colcem id* (0.1 pg/m l) present fo r th e last 2 horns. 7.9 C ollection o f M etaphase C ells C ells w ill be collected approxim ately 20 hours after initiation o f treatm ent (about 64-68 hours after culture initiation). T his tim e is selected to represent the first division m etaphase after initiation o f test article treatm ent Tw o hours prior to harvest, Colcemid w ill be added to th e cultures a t a final concentration o f 0.1 pg/m l. T he cells u n ii be collected by centrifugation, treated w ith 0.075M KC1, w ashed w ith tw o changes o f fixative (m ethanol.glacial acetic acid, 3:1 v/v), capped and stored overnight o r longer a t approxim ately 2-8C . To prepare slides, the cells w ill be collected b y centrifugation and resuspended in fresh fixative. A n aliquot p f fixed cells w ill be applied dropw ise onto a m icroscope slide and air-dried. T he slide w ill be identified by th e experim ent num ber, treatm ent condition and date. A t least tw o slides w ill be prepared from each treatm ent tube. T he slides w ill be stained w ith G iem sa and perm anently m ounted. . 7.10 Scoring fo r M etaphase A berrations Slides w ill be coded using random num bers by an individual no t involved w ith the % scoring process. A t least 3 dose levels in each harvest w ill be evaluated in die aberration assay and w ill b e selected according to the criteria described in section 7.2. The 4 hour non-acdvated and S9-acdvated studies w ill be scored initially. In the event o f a positive response in 4 hour non-achvated study, slides from the extended non-activated exposure m ay no t be- scored. M etaphase cells m il be exam ined under o il im m ersion w ithout prim know ledge o f treatm ent groups. W henever possible, a m in im u m o f 200 m etaphase spreads containing 46 centrom eres from each dose level (100 per duplicate treatm ent tube) w iU .be exam ined and scored fo r chrom atid-type and chrom osom e-type aberrations (S cott et a l, 1990). T he num ber o f m etaphase spreads th a t w ill be exam ined and scored per duplicate < bdr m ay b e reduced i f the percentage o f aberrant cells reaches a statistically significant level before 100 cells are scored. Chrom atid-type aberrations include chrom atid and isochrom atid breaks' and exchange figures such as quadriradials (sym m etrical and asym m etrical interchanges), triradials and com plex rearrangem ents. Chrom osom e-type aberrations include chrom osom e breaks and exchange figures such as dicentrics and rings. Fragm ents (chrom atid o r acentric) observed in the.absence o f any exchange figure w ill be scored as a break (chrom atid o r chrom osom e). Fragm ents observed w ith an exchange figure w ill not be scored as ) an aberration b u t w ill be considered part o f die incom plete exchange. Pulverized chrom osom e(s), pulverized cells and severely dam aged cells (>10 aberrations) w ill Protocol No. SPGT341 O l-M ay-2001 P ag e6 o fl0 ^ Bio Reliance BioReliance Study No. AA44ER.341.BTL 36 fam S& sd Does m contain YSCA C& % H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 Sponsor Project N um ber D uP ont-6 4 0 6 B ioR eliance Study N um ber: A A 44ER .341.B TL atq i he recorded. C hrom atid and isochrom atid gaps w ill be recorded but not tnrtnrtw t in die analysis. T he X Y coordinates for each cell w ith a structural aberration o r gap w ill be recorded using a calibrated m icroscope stage. T he m itotic index w ill b e recorded a s the percentage o f cells in m itosis per 500 cells counted. T he percent polyploid and endoreduplicated cells w ill b e evaluated p er 100 m etaphase cells fo r each dose level analyzed for structural aberrations. 8.0 CRITERIA FO R DETERM INATION O F A VALID TEST 8.1 Solvent C ontrols T he frequency o f cells w ith structural chrom osom e aberrations in_ the solvent controls m ust be w ithin th e range o f the historical solvent control. 8.2 Positive C ontrols T he percentage o f cells w ith aberrations m ust be statistically increased (p<P-05, Fisher's exact test) in foe positive control relative to foe solvent control. 9.0 EVALUATION O F TEST RESULTS T he toxic effects o f treatm ent are based upon inhibition o f m itosis and w ill be reported for foe cytotoxicity and chrom osom e aberration study. The num ber and types o f aberrations (structural and num erical) found, foe percentage o f structurally dam aged cells in the total population o f cells exam ined (percent aberrant cells), foe percentage o f num erically dam aged cells in 'fo e to tal population o f cells exam ined, and foe average num ber o f structural aberrations per cell (m ean aberrations p er cell) w ill be calculated and reported for each treatm ent group. C hrom atid and isochrom atid gaps are presented in foe data b u t are not included in.the total percentage o f cells w ith one o r m ore aberrations o r in foe average num ber o f aberrations p er cell. . S tatistical analysis o f foe percentage o f aberrant cells w ill be perform ed using the Fisher's exact test. T he Fisher's test w ill be used to com pare pairw ise the percent aberrant cells o f each treatm ent group w ith that o f foe solvent control. In the event o f a positive Fisher's exact te st at any test article dose level, the C ochran-A nnitage test w ill be used to m easure dose-responsiveness. - A ll conclusions w ill be based on sound scientific judgem ent; how ever, a s a guide to interpretation o f the data, foe test article w ill be considered to induce a positive response if foe percent aberrant cells is increased in a dose-responsive m a n n a w ith one o r m ote concentrations being statistically significant (p^0.05). A reproducible significant increase a t the high dose only w ith no dose response o r a reproducible significant increase a t one dose level other than foe high dose w ith no dose response w ill be considered positive. T est 1 articles n o t dem onstrating a statistically significant increase in aberrations w ill be concluded ' to be negative. Protocol No. SPGT341 01-M ay-2001 P age 7 o f 10 ^ Bio Reliance- B ioR eliance Study N o. A A 44E R .341.B T L 37 gesti&se, Dem mi sensata TSC CBS H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes DuPont-6406 Sponsor Project N um ber D uP ont-6 4 0 6 B ioR eliance Study N u m b er A A 44E R 341JBTL 10.0 REPORT A report o f die results o f this study w ill b e prepared by the T esting F acility and w in accurately describe all m ethods used for generation and analysis o f the data. R esults presented w ill include, b u t not b e lim ited to: * T est article: identification and CAS no., i f known; physical nature and purity, if know n; physicochem ical properties relevant to the conduct o f die study, i f know n; stability o f test article, if know n. * S olvent/V ehkle: justification for choice o f vehicle; solubility an d stability o f te st article in solvent/vehicle, if know n. * source o f cells and tim e the cells w ere obtained, karyotype features (m odal chrom osom e num ber) and suitability o fth e cell type u sed * test conditions: com position o f m edium ; C 0 2 concentration; incubation tim e; solvent and solvent selection rationale; concentration o f test article and concentration selection rationale; com position and acceptability criteria for die m etabolic activation (S9) system ; duration o f treatm ent; duration o f treatm ent w ith and concentration o f Colcem id*; type o f m etabolic activation system used; positive and solvent controls; m ethods o f slide preparation; num ber o f cell cultures; criteria for scoring aberrations and criteria for considering studies positve, negative o r equivocal * results: description o f precipitation; pH and osm olarity o f the treatm ent m edium ; m itotic inhibition relative to th e solvent control; m itotic index and num ber o f m etaphases , analyzed; type and num ber o f aberrations (structural and num erical) given separately for each treated and control culture; concentration-response relationship; statistical analysis; historical control d ata 11.0 RECORDS AND ARCHIVES A ll raw data, the protocol and a ll reports w ill be m aintained according to Standard O perating Procedure O PQ P3040 by die B ioR eliance RAQA u n it headquartered at: B ioR eliance, 14920 B roschart R oad, R ockville, M D 208S0. P er th is SO P, paper records w ill be retained for a t least th ree years after w hich tim e tire Sponsor w ill b e contacted for a decision as to the final disposition o f the m aterials A ll study m aterials returned to the . S ponsor o r destroyed w ill first be copied and th e copy w ill be retained in d ie B ioR eliance archives for a m inim um o f 10 years. Protocol NO.SPGT341 01-M*y-2001 BioReliance Study No. AA44ER.341.BTL Page 8 o f 10 ^ Bio Reliancf 38 QBSPeSasifteeA 0esm i coniata TSAC0 H-24889: In Vitro Chromosome Aberration Study in Human Peripheral Blood Lymphocytes_______ DuPont-6406 Sponsor Project N u m b er D uP ont--6406 B ioR eliance Study N um ber. A A 44E R 341.B T L / 1 1 0 REGULATORY REQUIREM ENTS/GOOD LABORATORY PRACTICE T his protocol has been w ritten to com ply w ith OECD Guideline 473 (fit Vitro M am m alian Chrom osom e A berration T est), adopted July 1997 and w ith th e International Conference on H arm onization o f Technical R equirem ents fo r R egistration o f Pharm aceuticals fo r Human U se (1996 and 1997). . T h is study w ill be perform ed in com pliance w ith th e provisions o f the G ood Laboratory P ractice R egulations for N o nd in ical L aboratory S tudies (G L Ps). T he protocol, an in-process phase, the raw d ata, and ieport(s) w ill be audited p er th e Standard O perating Procedures (SO Ps) o f B ioR eliance by th e Q uality A ssurance U nit o f B ioR eliance for com pliance w ith G LPs, th e SO Ps o f B ioR eliance and the study protocol. T he in-process inspection w ill be perform ed to audit th e critical assay procedures and system s supporting th e assay. A signed Q A statem ent nil b e included in th e fin al re p o rt T his statem ent w ill list the system phases inspected during the previous quarter o r th e study-specific phases, th e datiy o f each inspection, and th e dates th e results o f each inspection w ere reported to th e Study D irector and th e Study D irector's m anagem ent In addition, a signed GLP . com pliance statem ent w ill be included in th e fin al re p o rt T h is statem ent w ill cite the G LP guideline(s) w ith w hich the study is com pliant and any exceptions to this com pliance, if applicable, including th e om ission o f characterization or stability analyses o f th e te st o r control articles o r th eir m ixtures. ) U nless arrangem ents are m ade to the contrary, unused dosing solutions w ill be disposed o f follow ing adm inistration to the test system and all residual te st article w ill be disposed o f follow ing finalization o f the rep o rt 13.0 REFERENCES Evans, H J. and MJL. G R iordan. 1975. H um an peripheral Wood lym phocytes for the analysis o f chrom osom e aberrations in m utagen tests. M utation R es. 31:135-148. G allow ay, SA L, M J. A ardem a, M . Ishidate Jr., JA . Ivett, D J. K irkland, T . M onta, P. M osesso and T . Sofuni (1994) R eport from w orking group on in vitro tests for chrom osom al aberrations. M utation R esearch 312(3):241-261. In tern atio nal Conference o n H arm onisation (IC H ) o f T echnical R equirem ents for R egistration o f Pharm aceuticals fo r Hum an U se. G uidance on Specific A spects o f R egulatory G enotoxicity T ests for Pharm aceuticals. S2A docum ent recom m ended for adoption at step 4 o f file ICH process on July 19,1995. Federal R egister 61:18198-18202, A pril 24,1996. International Conference on H arm onisation (IC H ) o f T echnical R equirem ents for R egistration o f Pharm aceuticals for Hum an U se. G enotoxicity: A Standard B attery for j G enotoxicity Testing o f Pharm aceuticals. S2B docum ent recom m ended for adoption at step 4 o f the ICH process on July 16,1997. Federal R egister 62:16026-16030, Novem ber 2 1 ,1 9 9 7 . Protocol No. SFGT341 O l-M iy-2001 P ag e9 o ftO ^ Bio Reliance" BioReliance Study No. AA44ER.341.BTL 39 Sasffeed Bess EtesconSalraTS0CBS H -24889: In Vitro C hrom osom e A berration Study in H um an P eripheral B lood L ym phocytes DuPont-6406 Sponsor Project N u m b er D uPont -6 4 0 6 B ioR eliance Study N u m b er A A44ER.341.BTL OECD G uideline 473 (G enetic Toxicology: In Vitro M am m alian Chrom osom e A berration T est), N inth Addendum to th e OECD G uidelines fo r th e T esting o f Chem icals, published by OECD , Paris, February 1998. Preston, R .J., W. A u, M A . B ender, J.G . B rew en, A .V . C airano, J A . H eddle, A T . M cFee, S. W olff and J.S. W assom. 1981. M am m alian in vivo and in vitro cytogenetics assays: A report o f th e US EPA 's Gene-Tox Program . M utation R es. 87:143-188. S cott, D ., N .D . D anfoid, B J . D ean and D J . K irkland. 1990. M etaphase Chrom osom e A berration A ssays In V itro. In; B asic M idagenicity T ests: UKEM S Recomm ended Procedures. DJ K irkland (ed). Cam bridge U niversity Press, N ew Y ork, N Y . Sw ierenga S A H ., J A . H eddle, E A . Sigal, JJ*.W . G ilm an, R .L. B rillinger, G .R. D ouglas and E.R . N estm ann (1991) Recom m ended protocols based on a survey o f current practice in genotoxidty testing laboratories, IV . Chrom osom e aberration and sister-chrom atid exchange in C hinese ham ster ovary, V 79 C hinese lung and hum an lym phocyte cultures, M utation R esearch 246:301-322. 14.0 APPROVAL -- _______________ Q~3 SPONSOR REPRESENTATIVE DATE 2 ,o o > K o - r C e~ (Print or Type Nam e) BIORELIANCE STUDY DIRECTOR 0 to y 0 o o \ DATE to z o o / BIORELIANCE STUDY MANAGEMENT DATE Protocol No. SPC T34J 01-May-2001 BioReliance Study No. AA44ER.341.BTL Page 10 o f 10 Bio Reliancf 40 <S0BSpBS^ SastSlsafiL 0008 BQfrioTMTS 6B8
Contact UsLoginSign Up
CUNY - The Graduate CenterColumbia University Mailman School of Public Health - Sociomedical Sciences