Document Ex5XGkX0621N1L2q067yr9nX0

AR226-3149 DuPont-3871 TRADE SECRET Study Title H-23960; In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells Authors Ramadevi Gudi, Ph.D. Elizabeth H. Schadly, B.S. Report Completion Date May 4,2000 Performing Laboratory BioReliance 9630 Medical Center Drive RockvUle.MD 20850 for E. L du Font de Nemouis and Company Haskell Laboratory for Toxicology and Industrial Medicine P.O. Box 50, Elkton Road Newark, DE 19714-0050 Performing Laboratory Study Number AA26XN.331JBTL DuPont Project H) DuPont-3871 Work Request Number Page 1 of 40 Company Sanitized. Does not contain TSCA CBI H-23960; In Vitro Mammalian Chromosome Aberration Test to Chinese Hamster Ovary (CHO) Cells____________________DuPont-3871 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. BioReliance Study Director: ^^^i^gdjgg^USLUL 0^/of/C^ Ramadevi G<u2di^, P_h_.D_. _______ Date ACTrovedby Study Monitor. k<T\^ 0^^: cs- .7^ Maria Donner, Ph.D. Senior Research Scientist OS'{Qt{oo Date B ioReliance Study No. AA26XN .331 .BTL 2 - Company Sanitized. Does not contain TSCA CBI H-23960; In Vttro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Celb___________________DuPont-3871 TABLE OF CONTENTS Page 2 Certification............................................................................................................................ Study Infommdon................................................................................................................... 5 7 Summary................................................................................................................................. 9 Purpose........--...,...............................................................................................--"................. Characterization of Test and Control Substances.................................................................... 9 Materials and Methods............................................................................................................. 9 Results and Discussion..................................................................................................... 14 16 Conclusion............................................................................................................................. References.............................................................................................................................. 17 Data Tables.............................................................................................................................18 Tablel: Preliminary Toxicity Assay with H-24335 in CHO Cells in the Absence of Exogenous Metabolic Activation. 4 Hour Treatment....................................................... 18 Table 2: Preliminary Toxicity Assay with H-24335 in CHO Cells in the Presence of Exogenous Metabolic Activation. 4 Hour Treatment....................................................... 19 Table 3: Preliminary Toxicily Assay with H-24335 in CHO Cells in the Absence of Exogenous Metabolic Activation. 20 Hour Treatment..................................................... 20 Table 4: Concurrent Toxicity Assay with H-24335 in CHO Cells in <he Absence of Exogenous Metabolic Activation. 4 Hour Treatment........................................................21 Table 5: Cytogenetic Analysis of CHO Cells Treated with H-24335 in me Absence of Exogenous S9 Metabolic Activation. 4Hour Treatment, 16 Hour Recovery Period.........22 Table 6: Concurrent Toxicity Assay with H-24335 in CHO Cells in me Presence of Exogenous Metabolic Activation. 4 Hour Treatment........................................................23 Table 7: Cytogenetic Analysis of CHO Cells Treated with H-24335 in the Presence of Exogenous S9 Metabolic Activation. 4Hour Treatment, 16 Hour Recovery Period.........24 BioReliance Study No. AA26XN.331 .BTL - 3- Company Sanitized. Does not contain TSCA CBI H-23960; IH Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells___________________DnPont-3871 Table 8: Concurrent Toxidty Assay with H-24335 in CHO Cells in the Absence of Exogenous Metabolic Activation. 20 Hour Trcatinent......................................................25 Table 9: Cytogenetic Analysis of CHO Cells Treated with H-24335 in the Absence of Exogenous S9 Metabolic Activation. 4Hour Treatment, 16 Hour Recovery Period......... 26 Table 10: Summary: Cytogenetic Analysis of CHO Cells Treated with H-24335............27 Appendix A: Historical Control Data.................................................................................... 28 Appendix B: Study Protocol.................................................................................................. 31 BioRelianceStudyNo.AA26XN.331.BtL -4- Company Sanitized. Does not contain TSCA CBI H-23960; Is Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells STUDY INFORMATION Substance Tested:jjUUjf) Synonyms/Codea H-23960 Haskell Number: 23960 DuPont-3871 Physical Characteristics: Stability: The test substance appeared to be stable under the conditions offihe study; no evidence of instability was observed. Solubility: Aquatics: soluble but cloudy in water at concentrations ;Sl5mg/mL. BioReliance Study No. AA26XN.331 .BTL - 5- Company Sanitized. Does not contain TSCA CBI H-23960: in Mfro Mammalian Chromosome Aberration Test In Chinese Hamster Ovary (CHO) Cells_______________DnPont-3871 Sponsor: E. I. du Font deNemours and Company Haskell Laboratory for Toxicology and Industrial Medicine P.O. Box 50, EBcton Road Newaik.DE 19714-0050 Study ImtiatoVCompleted: February 25,2000/(see report cover page) to-Life Initiated/Completed: February 29,2000 /March 27,2000 Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells___________________DnPont-3871 SUMMARY The test substance, H-23960, was tested in die in vitro chromosome aberration test using Chinese hamster ovary (CHO) cells in both die absence and presence of an Aroctor-induced exogenous S9 metabolic activation system. A preliminary toxicity assay was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenicpotential of the test substance. Water was determined to be Hie solvent of choice based on information provided by the Sponsor, the solubility of the test substance, and compatibility with the target cells. The test substance was soluble but cloudy in water at a concentration of 50 mg/mL, the maximum concentration tested. In the preliminary toxidty assay, me maximum dose level tested was 5000 ug/mL. The test substance was soluble in the treatment medium at all dose levels tested in the non-activated studies. Visible precipitates were observed in the treatment medium at dose levels S:1500. ug/mL in me S9 activated study. Dose levels of $500 pg/mL were soluble in me treatment medium in the S9 activated study. Selection of dose levels for the chromosome aberration assay was based on total cell growth inhibition relative to me solvent control. No substantial toxidty, Le., at least 50% cell growth inhibition, was observed at any dose level tested in either me non-activated or the S9 activated treatment groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 625 to 5000 ug/mL for both me nonactivated and the S9 activated treatment groups. In me chromosome aberration assay, the cells were treated for 4 and 20 hours in the nonactivated test system and far 4 hcnirs m me S9 activated test system, and aU celb were harvester at 20 hours after treatment initiation. The test substance was soluble in treatment medium at all dose levels tested in the non-activated studies. Visible precipitates were observed in the treatment medium at the dose levels S2500 u.g/mL in the S9 activated study. The dose levels of $1250 ug/mL were soluble in treatment medium in the S9 activated study. No substantial toxicity (at least 50% cell growth inhibition) was observed at the highest dose level evaluated for chromosome aberrations, 5000 jig/mL, in the non-activated 4 hour and 20 hour exposure groups, respectively. No substantial toxicity (at least 50% cell growth inhibition) was observed at the highest dose level evaluated for chromosome aberrations in the S9 activated study, 2500 ug/mL. The highest dose level selected for analysis of chromosome aberrations the nonactivated studies, 5000 ug/mL, was based upon the absence of at least 50% toxidty and the lack of test substance predpitation in the treatment medium. The highest dose level selected for analysis of chromosome aberrations in the S9 activated study, 2500 pg/mL, was based upon the absence of at least 50% toxicity and the presence of test substance predpitation in the treatment medium (the lowest precipitating dose was selected as me highestdose to evaluate). The non- activated and S9 activated 4 hour treatment groups were scored for structural and numerical chromosome aberrations. No statistically significant increases in structural and numerical chromosome aberrations were observed in me non-activated or S9 activated 4 hour treatment BioReliance Study No. AA26XNJ3 l.BTL - 7- Company Sanitized. Does not contain TSCA CBI H-23960; In Vitro Mammalian Chromosome Aberration Test to Chinese Hamster Ovary (CHO) Cells___________________DuPont-3871 groups relative to the solvent control group, regardless of dose level (pX).05, Fisher's exact test). In the absence of a positive response in the non-activated 4 hour treatment group, the non-activated 20 hour 'continuous treatment group was evaluated for structural and numerical chromosome aberrations. A statistically significant increase in structural chromosome aberrations -was observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, at the dose level 5000 u.g/mL (p<0.05. Fisher's exact test). The CochranAmiitage test was also positive for a dose response (p$0.05). However, the percentage of structurally aberrant cells observed at the dose level 5000 u.g/mL (2.5%) was within the range of structurally aberrant cells observed with me historical solvent control (0%-6%). Therefore, the statistically significant increase in the percentage of structurally aberrant cells observed at 5000 u.g/mL was not considered biologicallysignificant No statisticallysignificant increases in 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). The positive and solvent controls fulfilled the requirements for a valid test. Under the conditions described in this report, H-23960 was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9 activated in vitro mammalian chromosome aberration test in Chinese hamster ovary (CHO) cells. BioReHance Study No. AA26XN.331JBTL - 8 Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells___________ DuPont-3871 _____ PURPOSE The purpose of this study -was to evaluate the clastogenic potential of the test substance, H23960, based upon its ability to induce in vitro chromosome aberrations in the Chinese banister (miry (CHO) cells. CHARACTERIZATION OF TEST AND CONTROL ARTICLES The test substance, H-23960, was received by BioRfiliance on 15 February 2000 and was " " H H B I J ^ [the code number AA26XN. The test substance was characterized by the Sponsor as an should I1 stored in a well-ventilated place at An expirado&date for the test^ubstance was not provided, aJ|IBHBH|B|Qfid Upon receipt, the test substance was described as was stored ~ at room temperature, in a well-ventilated area, protected from exposure to light. Based on information provided by Ihe Sponsor, sterile distilled water (CAS 7732-18-5), obtained from the Life Technologies Company, was the solvent used to deliver H-24335 to ifae test system. Mitomycin C (MMC; CAS No.: 50-07-7), was obtained fiom me Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations of 1 and 2 pg/mL for use as the positive control in the non-activated test system. Cyclophosphamide (CP; CAS No.: 6055-19-2), was obtained fiom Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations of 100 and 200 Hg/mL for use as the positive control in the S9 activated test system. For each positive control one dose with sufficient scorable metaphase cells was selected for analysis. The solvent for the test substance was used as me solvent control at die same concentration, as that found in the test substance-treated groups. MATERIALS AND METHODS Test System Chinese hamster ovary (CHO-K,) cells (repository number CCL 61) were obtained fiom the American Type Culture Collection, Manassas, VA, on May 29,1997. m order to assure the karyotypic stability of me cell line, working cell stocks were not used beyond passage 20. CHQ wUs at passage 6 were used for the preliminary toxicity and CHO cells at passage 12 were used for the chromosome aberration assay. The freeze lot of cells was tested using we Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston etal., 1981). BioReliance Study No. AA26XN.331 STL - 9 Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test to Chinese Hamster Ovary (CHO) Cells___________________DuPont-3871 Metabolic Activation System Aroclor 1254-induced rat liver S9 was used as the exogenous metabolic activation system. The 89 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 S-70C until used. Each bulk preparation of S9 was assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella fyphimwium 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 glucoses-phosphate, 1 mM nicotinamide adenine dinucleodde phosphate (NADP) and 20 uL S9 per miUiliter medium (McCo/s 5A serum-fiee medium supplemented with, 100 units penicillin and 100 ug streptomycin/mL, and 2 mM L-glutamine). Preliminary Toxicity Assay The preliminary toxidty assay was performed for the purpose of selecting dose levels for the chromosome aberration assay and consisted of an evaluation of test substance effect on cell growth. CHO cells were seeded for each treatment condition at approximately 5 x 105 cells/25 cm2 flask and were incubated at 371C in a humidified atmosphere of 51% CO; in air for 16-24 hours. Treatment was carried out by refeeding the flasks with 4.5 mL complete medium (McCoy's 5A medium supplementedwith 10% fetal bovine serum (FBS), 100 units penicillin and 100 ug streptomycin/mL, and 2 mM L-glutamine) for the non-activated study or S9 reaction mixture (3.5 mL sennn-fiee medium plus I mL of 5X S9 mix) for the activated study, to which 500 uL dosing solution of test substance in solvent or solvent alone was added. The osmolality of the highest concentration of the dosing solution in the treatment medium was measured. The pH of the highest concentration of the dosing solution in the treatment medium was measured using test tape. The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9. At completion of the 4 hour exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with 5 mL complete medium and returned to the incubator for a total time period of 20 hours from the initiation of the treatment At 20 hours after me initiation ofthe treatment the cells were harvested by trypsinization and counted using a Coulter counter. The presence of test substance precipitate was assessed using the unaided eye. Cell viability was determined by tiypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to me solvent control. Chromosome Aberration Assay The chromosome aberration assay was performed using standard procedures (Evans, 1976), by exposing duplicate cultures of CHO cells to the test substance as well as positive and solvent BfoReliance Study No. AA26XN.331 .BTL - 10 - Company Sanitized. Does not contain TSCA CBI H-23960; In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Celb___________________DuPont-3871 controls. For the chromosome aberration assay, CHO cells were seeded at approximately 5 x 105 cells/25 cm2 flask aod were incubated at 371C in a humidified atmosphere of 51% CO, in air for 16-24 hours. Treatment was earned out by refeeding duplicate flasks with 4,5 mL complete medium (McCoy's 5A medium supplementedwith 10% FBS, 100 units penicillin and 100 ng streptomycin/mL, and 2 mM L-glutamine) for we non-activated study or 4.5 mL S9 reaction mixture for me S9 activated study, to which 500 pL of dosing solution of test or control article in solvent or solvent alone was added. The osmolality of the highest concentration of the dosing solution in the treatment medium was measured. The pH of me highest concentration of the dosing solution in me treatment medium was measured using test tape. m the non-activated study, me cells were exposed to me test substance for 4 hours or continuously for 20 hours up to the cell harvest at 371C in a humidified atmosphere of 51% CO; in air (Swierenga et aL, 1991). In the 4 hour exposure group, the treatment medium was removed after me exposure period, and the cells washed with CMF-PBS, refed wim complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition at a final concentration of 0.1 pg/mL and the flasks returned to the incubator until cell collection. m me S9 activated study, the cells were exposed for 4 hours at 371C in a humidified atmosphere of 5l% CO, in air (Swierenga et al., 1991). After the exposure period, the treatment medium was removed, the cells washed wim CMF-PBS, refed with complete medium and returned to the incubator. Two hours prior to the scheduled cell harvest, Colcemid9 was added to duplicate flasks for each treatment condition at a final concentration of 0.1 pg/mL and the flasks were returned to the incubator until cell collection. A concuirent toxicity assay was conducted in both the non-activated and the S9 activated assay systems. After cell harvest an aliquot of the cell suspension was removed ftom each culture and counted using a Coulter counter. The presence of test substance precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control. Cell Harvest Two hours after the addition of Colcemid, metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinization. Cells were collected approximately 20 hours after initiation of treatment (Galloway et al., 1994). The cells were collected by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 2-4 mL 0.075 M potassium chloride (KC1) and allowed to stand at room temperature for 4-8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed wim two washes of approximately 2 mL Camoy's fixative (methanohglacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2-8C. BioReliance Study No. AA26XNJ31 .BTL -11- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells__________________DnPont-3871 Slide Preparation To prepare sHdes, the fixed celk were centrifi%ed at approximately 800 rpm for 5 minutes, the supernatant was aspirated, and 1 mL fresh fixative was added. After additional centrifagation (at approximately 800 ipm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative. A sufficient amount of cell suspension was droppedonto the center of a glass slide and allowed to air dry. Slides were identified by the study number, date prepared and the treatment condition. The dried slides were stained with 5% Giemsa, air dried and permanently mounted. Evaluation of Metaphase Cells Slides were coded using random numbers by an individual not involved with the scoring process. To ensure dial a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase celts with 202 centromeres were examined under oil immersion without prior knowledge of treatment groups. 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 evaluated for chromosome aberrations. Whenever possible, a mininnnn of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et at., 1990). The number of metaphase spreads that are examined and scored per duplicate flask may be reduced if the percentage of aberrant cells reaches a statistically significant level before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatidor acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (S;10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. Polyploid and endoreduplicated cells were evaluated ftom each treatment flask per 100 metaphase cells scored. Controls Mitomycin C was used as the positive control in the non-activated study at final concentrations of 0.1 and 0.2 pg/mL. Cyclophosphamide was used as the positive control in the S9 activated study at final concentrations of 10 and 20 yig/mL. For both positive controls one dose level exhibiting a sufficient number of scorable metaphase cells was selected for BioReliance Study No. AA26XN.331 .BTL -12 - Company Sanitized. Does not contain TSCA CBI H-23960; In Vitro Mammalian Chromosome Aberration Teat in Chinese Hamster Ovary (CHO) Cells___________________DuPont-3871 analysis. The solvent vehicle for the test substance was used as the solvent concentration as that found m the test substance-treated groups. DuPontsti were conducted simultaneously and thus the same solvent and positive between these two studies. Evaluation of Test Results The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per ceH was calculated and rq)brtea for eacn group. Chromatid and isochromatid gaps are presented in the data but are not included in die total percentage of cells with one or more aberrations or m the frequency of structural aberrations per c^U. Statistical analysis of me percent aberrant cells was performed using we 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, m the event of a positive Fisher's test at any test substance dose level, the Cochran-Annitage test was used to measure dose-responsiveness. All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test substance was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p$0.05). Test substances not demonstrating a statistically significant increase in aberrations will be concluded to be negative. Negative results with metabolic activation may need to be confirmed on a case-by-case basis, m those cases where confirmation ofnegative results is not necessary, justification will be provided. Criteria for a Valid Test The frequency of cells with structural chromosome aberrations in the solvent control must be within the range of the historical solvent control The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p<0.05. Fisher's exact test) relative to the solvent control. Deviations No known deviations from me 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 ProcedureM----------the^bByioReliance Regulatory BioReliance Study No. AA26XN331.BTL - 13 - Company Sanitized. Does not contain TSCA CBI H-2396D: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (QUO) Celb DuPont-3871 Affairs/Quality Assurance Unit headquartered at: BioReliance, 14920 Broschart Road, Rockville,MD, 20850. RESULTS AND DISCUSSION SoIubiKty Water was determined to be the solvent of choice based on information provided by the Sponsor, the solubility of the test substance, and compatibility with the target cells. The test substance was soluble but cloudy in water at a concentration of 50 mg/mL, the maxiTmim concentration tested. Preliminary Toxicity Assay Dose levels for the chromosome aberration assay were selected following a preliminary toxicity assay and were based upon a reduction of total cell growth (cell growth inhibition) relative to the solvent control. The results of the evaluation of cell growth inhibition are presented in Tables 1-3. CHO cells were exposed to solvent alone and to nine concentrations of test substance ranging from 0.5 pg/mL to 5000 ng/mL in the absence and presence of an S9 reaction mixture. The test substance was soluble in treatment medium at all dose levels tested in the non-activated studies. Visible precipitates were observed in the treatment medium at me dose levels S1500 pg/mL in the S9 activated study. The dose levels S500 pg/mL were soluble in treatment medium in me S9 activated study. The osmolality of the solvent (water) in treatment medium was 314 mmol/kg. The osmolality in treatment medium of we highest concentration tested, 5000 pg/mL, was 315 mmol/kg, basically identical to the osmolality of the solvent. The pH of the highest concentration of test substance in treatment medium was approximately 7.0. No substantial cell growth inhibition (S50%) relative to the solvent control was observed at any dose level tested in both me non-activated and S9 activated treatment groups. Based upon the results of me toxicity study, the dose levels selected for testing in the chromosome aberration assay were as follows: Treatment Condition -S9 +S9 Treatment Time(hr) 4 Recovery Time(hr) 16 Dose levels (ug/mL) 625,1250,2500,5000 20 0 625,1250,2500,5000 4 16 625,1250,2500,5000 Chromosome Aberration Assay In the chromosome aberration assay, the test substance was soluble in treatment medium at all dose levels tested in the non-activated studies. Visible precipitates were observed in the BioReliance Study No. AA26XN.331.BTL -14- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test In Chinese Hamster Ovary (CHO) Cells__________________DuPont-3871 treatment medium at the dose levels >2500 )Jg/mL in the S9 activated study. The dose levels <1250 pg/mL were soluble in treatment medium in me S9 activated study. The osmolality in treatment medium of the highest concentration tested, 5000 ug/mL, ms 226 mmol/kg, exactly the same as the osmolality of the solvent (water) in treatment medium. The pH of the highest concentration of test substance in treatment medium was approximately 7.0. CeU growth inhibition relative to me solvent control in CHO cells was 25% after treatment with H-23960 at 5000 ug/mL for 4 hours in the absence of S9 activation (Table 4). This was the highestdose level evaluated for chromosome aberrations. The ability of H-23960 to induce chromosome aberrations is presented by treatment flask in Table 5, and summarized by treatment group in Table 10. The mitotic index was 7% reduced relative to me solvent control at the highest dose level evaluated for chromosome aberrations, 5000 ug/mL. The dose levels selected for microscopic analysis were 1250,2500, and 5000 pg/mL. The percentage of cells with structural and numerical aberrations in the test substance-treated' groups was not significantly increased above that of me solvent control (p>0.05. Fisher's exact test). The percentage (18.5%) of structurally damaged cells in the MMC group was found to be statistically significant Cell growth inhibition relative to the solvent control in CHO cells wasl3% after treatment with H-23960 at 2500 ug/mL for 4 hours in the presence of S9 activation (Table 6). This was the highestdose level evaluated for chromosome aberrations. The ability of H-23960 to induce chromosome aberrations is presented by treatment flask in Table 7, and summarized by treatment group in Table 10. The mitotic index at the highest dose level evaluated for chromosome aberrations, 2500 pg/mL, was 25% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 625,1250, and 2500 pg/mL, in we absence of at least 50% cell growth inhibition. The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased above that of me solvent control (p>0.05. Fisher's exact test). The percentage (26.5%) of structurally damaged cells in the CP group was found to be statistically significant In the absence of a positive response in we non-activated 4 hour exposure group, slides from the non-activated 20 hour exposure group were evaluated for chromosome aberrations. Cell growth inhibition relative to the solvent control was 19% at 5000 pg/mL (Table 8), the highest dose level evaluated for chromosome aberrations in the non-activated 20 hour continuous exposure group. The ability of H-23960 to induce chromosome aberrations is presented by treatment flask in Table 9, and summarized by treatment group in Table 10. The mitotic index at the highest dose level evaluated for chromosome aberrations, 5000 (ig/mL, was 14% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 1250, 2500, and 5000 ug/mL. The percentage of cells with structural aberrations in me test substance-treated groups was significantly increased above that of the solvent control only at dose level 5000 pg/mL (p<0.05, Fisher's exact test). The Cochran-Annitage test was also positive for a dose response (p<0.05). However, the percentage of structurally aberrant cells observed at dose level 5000 ug/mL (2.5%) was within the range of structurally aberrant cells BioReliance Study No. AA26XN.331.BTL -15 - Company Sanitized. Does not contain TSCA CBI H-23960; In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (310) CelB______________ DuPont-3871 observed with the historical solvent control (0%-6%). Therefore, die statistically significant increase in the percentage of structurally aberrant cells observed at 5000 pg/mL -was not considered biologically significant The percentage of cells 'with numerical aberrations in the test substance-treated groups was not significantly increased above that of the solvent control (pX).05, Fisher's exact test). The percentage (28.5%) of structurally damaged ecus in the MMC group was found to be statisticallysignificant The study was concluded to be negative. An independent repeat assay was not required because no unique metabolic requirements were known about me test substance and because no equivocal responses were observed. CONCLUSION The positive and solvent controls fulfilled die requirements for a valid test Under the conditions described in this report, H-23960 was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9 activated in vitro tngnimailian chromosome aberration test in Chinese hamster ovary (CHO) cells. BioReUanceStudyNo.AA26XN.331.BTL .16. Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells__________________DuPont-3871 KEFERENCES Evans, HJ. (1976) Cytological methods for detectingchemical mutageas, in: A. Hollaeoder (Ed.), Chemical Mutagens, Principles and Methods for their Detection, vol 4. Plenum Press, New York. Galloway, SJM., MJ. Aardema, M. Ishidate Jr., J.L. Ivett, DJ. Kiridand, 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 Regisfaation 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 My 19, 1995. Federal Register 61:18198-18202, April 24,1996. International Conference on Hannomsation (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 (he ICH process on Juty 16, 1997. Federal Register 62:16026-16030, November 21,1997. OECD Guideline for the Testing of Chemicals, Guideline 473 (In Vitro Mammalian Chromosome Abectanon Test), adopted July 1997. Preston, RJ., W. Au, MA. Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F. McPee, S. Wolffand J.S. Wassom (1981) Mammalian in vh>o and in vitro cytogenetic assays: a report ofthe Gene-Tox Program, Mutation Research, 87:143-188. Scott, D., N.D. Danford, BJ. Dean and DJ. Kiridand. 1990. Metaphase Chromosome Aberration Assays In Vitro, m: Basic Mutagenicity Tests: UKEMS Recommended Procedures. DJ Kiridand (ed). Cambridge University Press, New Yoric, NY. Swierenga S.H.H., JA. Heddle, E.A. Sigal, J.P.W. Gilman, RJL Brillinger, GJEL Douglas and E.R. Nestmann (1991) Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sisterchromatid exchange in Chinese hamster ovary, V79 Chinese lung and human lymphocyte cultures. Mutation Research 246:301-322. BioReliance Study No. AA26XN.331BTL -17 - Company Sanitized. Does not contain TSCA CBI H-23960: In VUro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Ceils DuPont-3871 TABLE1 PREUMII<ARYTOKIISnYASSAYVMTH WK1960 IN CHO CELLS IN ' THE/B8ENCEOFEXOOEN01JSMETAEK3UCACTVKTION 4HIOURTREATMENT.ieKI3URREO3VERYPER1OD Treatment4 ((igftiiL) CeD Count (xlO8) Cell WMy1 (%) Mean Cella/ Flask' OrtO') Celt Growth Index* (%) Cell Growth iRhilftkNr (%) Water H-23960 0.5 1.5 5 15 50 150 500 1500 5000 2.50 89% 2.47 100% 2.32 2.94 2.88 2.87 2.85 2.83 2.88 2.71 2.53 09% 2.30 98% 2.88 98% 2.84 99% 2,85 99% 2.83 97% 2.74 98% 2.82 98% 2.65 97% 2.45 93% 117% 115% 115% 114% 111% 114% 107% 99% ---- 7% -17% -15% -15% -14% -11% -14% -7% 1% ' CHO cells were treated in the absence of an exogenous source of metabolic activation for 4 houre t 371C. 2 Viability detenmined by trypn blue dye exclusion. ' Viable clisfflaskcea count x% viable cells 4 Growth index (celte per flask treated group/cell* per flask control group), expressed as a percentage. ' Cell growth inhibition " 100% - % cell growth Index; not calculated for negative controls. BioReliance Study No. AA26XN.331.BTL - 18 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells_________________DuPoat-3871 TABLE 2 PREUMINARYTQXICITYASSAYVffIH H-23960 IN CHO CELLS IN THE PRESENCE OF EXOGENOUS METABOUC ACTIVATION HOUR TREATMENT, 16 HOUR RECOVERY PERIOD Treatment' (ug/mL) Water H-23960 0.5 1.5 5 15 50 150 500 1500' " 5000" Cell Count (xlcC) Cell Viability2 {%) Mean Cdsl Flask* (xlOl Cel Growth Index* (%) Cell Growth InhbBon' (%) 2.51 95% 2.38 100% 2.57 2.63 2.62 2.68 2.40 2.32 2.33 2.28 2.65 99% 09% 98% 100% 96% 99% 98% 95% 97% 2.54 2.60 2.57 2.68 2.30 2.30 2.28 2.17 2.57 107% 109% . 108% 113% 97% 96% 96% 91% 108% -7% -9% -8% -13% 3% 4% 4% 9% -8% ' CHO cells were treated hi the presence of an exogenous source of metaboSc activation for 4 hours at 371C. 2 ViabHitydetetrnirod by bypan blue dya exclusion. * ViablBcelis/flask-cat! count x%viabl8 cells 4 Growth Index "(calla par flask treated group/cells per flask cntro)Broup), expressed as a percentage. ' CeUgrawlh Inhibition" 100%-% cell growth index; not calculated for negative controte, * Visible precipitates observed. BioReliance Study No. AA26XN.331.BTL " 19 - Company Sanitized. Does not contain TSCA CBI H-23960: III Vitro Mammalian Chromosome Aberration Teat la Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABI E3 PREUMIINAWTOiXKary ASSAY'IMTHH.23B601NCHO CELLS IN THE,ABSENCE 0 FEXOGENOUSMETAEIOUCACTIVAT10N 20HOIJRCONT1NUOUSTRE/IkTMENT Treatment1 (MS/mL) Call Count (x10) Cell Viability2 <%) Mean wast Flask' (xlO") Cell Growth !n<tex4 (%) Cell Growth inhibition5 (%) Water H-23960 0.5 1.5 5 15 50 150 500 1500 5000 2.99 2.94 3.32 2.78 2.79 3.09 2.90 3.07 3.03 2.89 98% 96% 98% 95% 93% 97% 97% 95% 98% 91% 2.93 2.82 3.19 2.64 2.59 2.99 2.81 2.92 2.97 2.45 100% 96% 109% 90% 88% 102% 96% 100% 101% 84% -- 4% -9% 10% 12% .2% 4% 0% ^1% 16% ' CHO cte WOT treated continuously In the absence of an exogenous source of metabolic acthmfion for 20 hours at 371C. 2 Viabllfty determlnad by tiypan bins dye exclusion. 1 Viable cells/flask c call count x% viable ceBs < Growth Index "(caltepw flask trBatadgTOUp/calls per flaBk control group), expressed as percentage. 5 Cell growth InhMUon" 100%-% cell growth Index; not calculated for negative controls. BioReliance Study No. AA26XN.331 .BTL - 20 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Teat in Chinese Hamster Ovary (CHO) Cells___________________DuPont-3871 TABLE 4 CONCURRENTTOX1CITYASSAYWITH H-23960 IN CHO CELLS IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOW TREATMENT, 16 HOUR RECOVERY PERIOD Treatment1 (tig/nil.) Coll Count Averages Flask (xlO^ Cea Viability1 Mean Cells par Flask' (xKfl Cell Growth index* (%) Cell Growth IntilbHonS (%) Water A 2.52 B 3.10 H-23960 A 2.85 B 2.79 1250 A 2.49 B 2.85 2500 A 2.43 B 2.48 5000 A 1.99 B 2.21 MMC.0.1 A 1.95 B 2.05 MMC.0.2 A 2.07 B 2.03 96% 98% 96% 99% 97% 95% 98% 95% 98% 97% 98% 97% 97% 95% 2.73 2.74 2.56 2.37 2.04 1.95 . 1.97 100% 101% 94% 87% 75% 71% 72% -1% 6% 13% 25% 29% 28% * CHO eels were treated In the absence of an exogenous source of metabolic activation for 4 hours at 371C. 2 VlablNty determined by trypan blue dye exclusion. 1 Viable cells/flask "cell count x% viable cells, naported as mean of Rasks A and B. 4 Growth index (moan cells per flask treated group/mean cete per flask control group), expressed as a percentage. 8 Cell growth Inhibition ' 100% % call growth index; not calculated for negative controls. BioReHanceStudyNo.AA26XN.331.BTL -21- Company Sanitized. Does not contain TSCA CBI H-23960; In Vtfro Mammalian Chromosome Aberration Test to Chinese Hamster Ovary (CHO) Cells TABLES CYTOGENET1CANALYSISOF CHO CELLS TREATED WITH H-23960 IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD DuPont-3871 Treatment1-* (MS/mL) Flask Mitotte Index* (%) Cafe Scored %Abemint Cells' Numerical Structural Water A 11.0 100 2 0 B 11.4 100 3 1 H-23960 1250 A 10.8 100 4 1 B 0.8 100 0 1 2500 A 8.8 100 4 1 B 7.8 100 2 1 5000 A 10.8 100 2 1 B 10.0 100 3 1 MMC, 0.2 A 9.6 100 2 16 B 10.6 100 5 21 Total H ofStradimilAI Qapa Chronatid* Br Ex ChrOfflO)tOnW Br Dte Ring Severely Damaged Cells' 1 0 0 00 0 0 0 0 0 01 0 0 1 0 0 01 0 0 0 1 0 00 0 0 0 0 0 01 0 0 0 1 0 0 0 0 0 1 1 0 00 0 0 1 1 0 00 0 0 1 14 11 40 0 0 0 19 7 13 0 0 Average Abenaflons PwCeff 0.000 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.290 0.300 CHO calls were treated for 4 hour* at 37A1C In the absence of an exogenous source of metabolic activation. Mttollc indax number mitotic figures x 100/500 oeto counted. Numerical: Includes polyplold and endoredupficated ceis.; Structural; exclude* cells wKh only gaps. Chromatid breaks include cnromaUd and isochromtid breaks and fragments; chromatid exchange figures (Exch) Include quadrtadiate, triradlais and complex reanangement*. Chramosome bieala Inchj^ bmaks ami acentric Iragments; dte, dicentric chromoaome. Severely damaged celte Includes cells with one or more pulverized dironiounies and cells with 10 or more aberrations. Severely damaged eels and pulverizatlom were counted as 10 aberrations. An additional dose level of 625 (igAnL was tested as a safeguard against excessive taxicity at higher dose levels but was not required for microscopic examination. BioReliance Study No. AA26XN.331.BTL - 22 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Teat in Chinese Hamster Ovary (CHO) Celb DuPont-3871 TABLE 8 CONCURRENT TOXICtTYASSAYWITH H-23960 IN CHO CELLS IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOUR TREATMENT, 16 HOUR RECOVERY PEH10D Treatment1 (MS/mL) Cell Count Averages Flask (xlO") Cel ViabiSty2 Mean Cells per Flask* 0(10") Celt Growth Index4 W) Call Growth Inhibition' W Water H-23960 625 1250 2500' 5000' CP,10 CP.20 A 2.84 8 2.35 A 2.40 B 2.56 A 2.11 B 2.08 A 2.03 B 2.12 A 1.68 B 2.04 A 1.62 B 1.71 A 1.38 B 1.50 98% 98% 98% 87% 98% 97% 98% 97% 98% 97% 97% 99% 98% 98% 2.55 2.42 2.04 2.02 1.81 1.63 1.38 100% 95% 80% 79% 71% 64% 54% ^^ 5% 20% 21% 29% 36% 46% ' CHO cefls were treated in the presence of an exogenous source of metabolic activation for 4 hours at 371'C. 2 Viabffiy determined by trypan blue dye exclusion. ' Viable cells/Bask B cell count x% viable ceis, reported as mean of Flasks A and B. 4 Growth Index " (mean cells per flask treated group/mean calls per flask control group), expressed as a percentage. ' Cell growth Inhibition 100% - % call growth Index; not calculated for negative controls. 8 Visible preclpltatw observed. BioRsliance Study No. AA26XN.331 .BTL - 23 Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro MammaliaQ Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABIE7 CYTOGENET1G ANALYSIS OF GHO CELLS TREATED WITH H-23960 INTHE PRESET OF EXOGENOUS METABOLIC ACTIVATION 4 HOURTREATMENT. 16 HOUR RECOVERY PERiOD Treatment1-' (MAIL) Flask Mttoflc Index1 (%) Cells Scored %Abemmt Cells' Numerical Structural Water A 12.0 100 3 2 B 11.2 100 4 3 H-23960 625 A 9.4 100 4 3 B 10.4 100 4 4 1250 A 8.6 100 4 6 B 9.8 100 5 S 2500* A 8.4 100 8 6 B 9.0 100 7 5 CP.10 A 7.6 100 2 25 B 8.2 100 1 28 Total Number ofStnicturalj"Lbemitions Gaps ChronfiaBd4 Br Ex Ch romosome* Br Die Ring Severely Damaged Cells* 1 2 0 0 0 0 0 0 2 0 0 1 0 0 2 1 2 0 0 0 0 0 4 0 0 0 0 0 0 5 1 0 0 0 0 0 S 0 0 0 0 0 1 6 0 0 0 0 0 2 4 1 0 0 0 0 5 35 6 0 0 0 0 2 39 3 0 0 0 0 Avenge Aberrations PwCelP 0.020 0.030 0.030 0.040 0.060 0.060 0.060 0.050 0.410 0.420 1 CHO cells were treated for 4 how at 371 C in the presence of an exogenous source of mettbollc activation. 2 Mttotic index - number mitotte figures x 100600 cells counted. 3 Nurneri<;:lndudespo)yploid and andofedupUcated cells.; Structural: excludes cells with only gaps. 4 Chromatid breaks include chromatid and Isochromatid breaks and fragments; chromatid exchange figures (Exch) Include quadriradlals, triradials and complex rearrangements. ' Chiomosornebrealalndude breaks and acertnc fragments; die, dicentrto chromosome. ' Severely damaged cells includes cell* with one or more pulverized chromosomes and eels with 10 or more aberrations. 7 Severely damaged cells and pulverizations were counted as 10 aberrations. ' Dose level 5000 ugAnL was not analyzed due to test substance precipitation In the treatment medium. ' Lowest precipitatingdose level. BioReIiance Study No. AA26XN.331.BTL -24- Compgny Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Teat In Chinese Hamster Ovary (CHO) Celfa DuPont-3871 TABLES CONCURRENTTOXICrTYASSAYVWTH H-23960 IN CHO CELLS IN TTC ABSENCE OF EXOGENOUS METABOUCACTWAT10N 20 HOUR CONTINUOUS TREATMENT Treatment1 (US/mL) Water H-23960 625 1250 2500 5000 MMC. 0.1 MMC, 0.2 Cell Count Avenges Flask OriO") A 2.34 B 2,79 A 2.52 B 2.82 A 2.59 B 2.85 A 2,20 B 2.38 A 2.30 B 1.84 A 2.43 B 2.06 A 1.79 B 1.66 CeH Viability' 97% 99% 95% 99% 99% 97% 100% 97% 96% 97% 99% 95% 98% 96% MeanCete per Flask1 (xlO") 2.51 2.59 2.66 2.25 2.04 2.18 1.67 eel Growth Index* (%) 100% 103% 106% 90% 81% 87% 67% Cell Growth Inhibition* (%) -- -3% -6% 10% 19% 13% 33% ' CHO cells were treated In the absence of an exogenous source of metabolic activation for 20 hours at 371C. 2 VIabllrtydetamilned by tiypan blue dye exclusion. , 1 Viabtecall^aak "cell court x% viable wife, reported as mean of Flasks A and B. 4 Growth Index (mean cells per flask treated group/mean cells per flask control group), expressed as a percentage. 8 Cell growth inhibition - 100% - % cell growth Index; not calculated for negative controls. BioReliance Study No. AA26XN.331 .BTL - 25 - Company Sanitized. Does not contain TSCA CBI H-23960; In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLES CYTOGENET1CANALYSIS OF CHO CELLS mEATEDVUITH H^39601NTHE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION 20 HOUR CONTINUOUS TREATMENT Treatment1-' (PBtet) Nilotic Index2 Rask (%) Cefe Scored %Abetffint Cells1 Numerical Structural Water A 10.6 100 0 0 B 9.6 100 0 0 H-239SO 1250 A 7.6 100 2 1 B 7.8 100 1 0 2500 A 10.0 100 2 1 B 9.2 100 0 0 5000 A 8.4 100 2 3 B 9.0 100 0 2 MMC.0.1 A 11.6 100 2 30 B 10.2 100 1 27 Total Number ofStroctural^kbarrcAtom Gap* CtirornaUd* Br Ex Ch roniosome1 Br Dte Ring Severely Damaged CeBs* 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 1 0 0 1 0 0 1 0 0 4 27 18 5 3 2 0 0 14 16 4 2 2 0 Avenge Aberrations PerCeB7 0.000 0.000 0.010 0.000 0.010 0.000 0.030 0.020 0.550 0.380 ' CHO calls ware treated for 20 hours at 371C In the absence of an exogenous source of metabolic activation. 2 MHofclmiex" number rnltoBc figures x 100/500 cells counted. ' Numerha!:lndudepolyploid and endoredupNcatedceHa.iStnieturafcexciudescalla with only gap*. 4 Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Exch) Include quadrlradlato, trindlate and complex rearrangements. ' Chromosome breaks Include bleaks and acentric fragments; die, dicentric chromosome. Sevarely<lamaged cells ineUidescelte with one or mow pulvartted chromosomes and <lls with 10 or more aberratton^^ 7 Severely damaged cete and pulverizationswe counted as 10 aberrations. ' An addBonal dose [eve! of 625 ug/rnt was tested aa a sa^iMrd against ex<sshrato)dcity at higher dose levelt but was not required for microscopic examination. BioReliance Study No. AA26XN.331 .BTL - 26 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test In Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLE 10 SUMMARY: CYTOGENEnC ANALYSIS OF CHO CELLS TREATED WITH H-23960 Treatment (uaftnU Water H-23960 1250 2500 5000 MMC, 0.2 S9 Activation Treatment Time Mean Mitotic Cells Index Scored Aberrations Per Cell (Mean+/-SD) Calls With Aberrations Numerical Structural (%) (%) 11.2 200 0.005 0.071 2.5 0.5 10.2 200 0.010 0.100 2.0 8.3 200 0.010 0.100 3.0 10.4 200 0.010 0.100 2.5 10.1 200 0.285 *0.722 3.5 1.0 1.0 1.0 18.5"* Water H-23960 625 1250 2500 CP.10 11.6 200 0.025 10.157 3.5 9.9 200 0.035 0.184 4.0 9.2 200 0.060 0.258 4.5 8.7 200 0.055 0.229 7.5 7.9 200 0.415 0.785 1.5 2.5 3.5 5.5 5.5 26.5** Water H-23960 1250 2500 5000 MMC, 0.1 20 10.1 200 0.000 0.000 0.0 20 7.7 200 0.005 0.071 1.5 20 9.6 200 0.005 0.071 1.0 20 8.7 200 0.025 0.157 1.0 20 10.9 200 0.465 0.924 1.5 0.0 0.5 0.5 2.5* 28.5** 1 Cells from all treatment conditions wen harvested at 20 hours after the initiation of the treatments. 2 Severely damaged cells were counted as 10 aberrations. ' * p^O.05; **, pfiO.01; Fisher's exact test BioReliance Study No. AA26XN.331 .BTL - 27 Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 APPENDIX A Historical Control Data BioReliance Study No.: AA26XN.331.BTL - 28 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST USING CHINESE HAMSTER OVARY (CHO) CELLS HISTORICAL CONTROL VALUES STRUCTURAL CHROMOSOME ABERRATIONS 1996-1998 Historical Values Mean Standard Deviation Range NON-ACTIVATED TEST SYSTEM Percent Aberrant Cells (%) Untreated Control 1.3 1.3 0.0 to 6.0 Solvent Control1 1.4 1.3 0.0 to 6.0 Positive Control2 25.9 19.3 6.5 to 100.0 Historical Values Mean Standard Deviation Range S9-ACTIVATED TEST SYSTEM Percent Aberrant Cells (%) Untreated Control 1.5 1.3 0.0 to 6.0 Solvent Control' 1.6 1.4 0.0 to 6.5 Positive Control3 34.0 18.0 6.5 to 100.0 Solvents include water, saline, dimethyl sulfoxide, ethanol, acetone, non-standard solvents and Sponsor-supplied vehicles. Positive control for non-activated studies, N-methyl-N'-mtro-N-mtrosogiiamdme (MNNG, 0.75-2 ug/ml), and Mitomycm C (MMC, 0.08-0.15 ug/ml). Positive control for S9-activated studies, cyclophosphamide (CP, 10-50 ug/ml), and benzo(a)pyrene, (B[a]P, 30 ug/ml). BioReliance Study No.: AA26XN.331.BTL -29. Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 IN VITRO MAMMALIAN CHROMOSOME ABERRATION USING CHINESE HAMSTER OVARY (CHO) CELLS HISTORICAL CONTROL VALUES NUMERICAL CHROMOSOME ABERRATIONS 1996-1998 Historical Values Mean Standard Deviation Range NON-ACTIVATED TEST SYSTEM Percent Aberrant Cells (%) Untreated Control 2.4 1.7 0.0 to 9.5 Solvent Control' 2.3 1.3 0.0 to 8.0 Positive Control2 32 1.9 0.0 to 9.5 Historical Values Mean Standard Deviation Range SO-ACTIVATED TEST SYSTEM Percent Aberrant Cells (%) Untreated Control 2.4 1.7 0.0 to 7.0 Solvent Control' 3.1 2.1 0.0 to 13.5 Positive Control3 3.5 23. 0.0 to 10.5 Solvents include water, saline, dimethyl sirifoxide, ethanol, acetone, and other nonstandard solvents and Sponsor-supplied vehicles. Positive control for non-activated studies, N-memyl-N'-mtro-N-mtrosoguanidine (MNNG, 0.75-2 ug/ml), and Mitomycin C (MMC, 0.08-0.15 (ig/ml). Positive control for S9-activated studies, cyclophosphamide (CP, 10-50 fig/mi), and benzo(a)pyrene (B[a]P, 30 pg/ml). BioReliance Study No.: AA26XN.331BTL -30- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian ChromoMime Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 APPENDIX B Study Protocol BioReliance Study No.: AA26XN.331.BTL -31- Company Sanitized. Does not contain TSCA CBI H-23960; In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 IUMZ^*W;AHMMMiinteiaireiMiuuAbantlMiT(<tfci ailBCt8HaattrOw)r(CHO)Cdl OdPafr3S71 KiritdbactSlxdyNnibcnAAZWLBLBTL 1.0 PURPOSE In Vitro MxmmdfMDChromoaomeAbemticBTertiBCliiBese Hamster Ovary (CHO) Crib The puipose of tins study is to evaluate the clastogcnicpotential of a test substance based upon itsability to induce chromosome abcnanons in Oimese hamster ovary (CHO) cells. 2.0 SPONSOR 2.1 Name: EJ.dii Font deNemours and Company 2.2 Address: HastceU Laboratory for Toxicology and Industrial Medicine P.O. Box 50, Elision Road Newaric.DE 19714-0050 2.3 Study Monitor; Maria Donner, Ph.D. 2.4 SponsorEmieetfe DuPont-3871 Service CodeC----fl 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance ffllB^H|3 32 Test Substance Name to be used in the Report: H-23960 3.3 Controls: Solvent: Positive: Water Mitomycin C (MMC) Cyclophosphamide(CP) 3.4 Detsmination of Strength,Purity, etc. Unless alternate arrangements are made, the testing facility at BioReliance will not perfonn analysis of the dosing solutions. The Sponsor will be directly responsible for determination and documentation of me analyticalpurityand composition of me test substance, and the stabilityand strength of me test substance in me solvent (or vehicle). Protocol No. SPGT331 February 23,2000 1 of9 BioReliance Study No.: AA26XN.331BTL-32- ^9 BIORELIANCE' ^y ForfrfyMlcfoblolo{]e*IAiieeifiei Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 Cktatt Hw Mi--dim Cknauot Abontin Ten ta. '(CHOXMs ,..... '.... -. IWotUCT KoReIiMcx Said; NrkKAAiaCNJSIJTL 3.5 Test Substance Retention Sample The retention of a reserve sample of the test substance wiU be the responsibilityof the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility BioReiiance 4.2 Address: 9630 Medical Center Drive Rockville,MD 20850 43 StudyDirector Ramadevi Gudi, PhJ). Phone: (301)610.2169 Fax: (301)738-2362 E-mail; i5odi@bioreliance.com 5.0 TEST SCHEDULE 5.1 Proposed Experimental Start Date: 2/29/2000 5.2 Proposed Experimental Tenninadon Date: 3/31/2000 5.3 ProposedReport Date: 4/I4QOOO 6.0 TEST SYSTEM The CHO-K) cell line is a prolise auxotroph with a modal chromosome number of 20 and a population doublingtime of 10-14 houre. CHO-K, cells were obtained 6om the American Type Culture Collection (repositorynumber CCL 61), Manassas, VA. The stability of the modal (Aroreosome number ofthe cell line is routmely checked and the cell line is routinely tested and determined to be fiee fiom mycoplasnaa contamination. This system has been demonstrated to be sensitive to die clastogenic activity of a variety of chemicals (Preston et aL,1981). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The chromosome aberration test will be conducted using standard procedures (Evans, 1976), by treatment culnws of CHO cells to a minimum of four concentrations of the test article as well as to positive and solvent controls. DuPont Studies 3870 and 3871 mil be conducted simultaneously and thus the same solvent and positive controls will be used between these two studies. In the non-activated test system, treaanent will be for 4 hours and for 20 hours; in the S9 activated test system, treatment will be for 4 hoins (Swierenga et ProtoeotNo.SPG'raBI Febnnly 23,2000 BioReliance Study No.: AA26XN.331.BTL 2 of 9 - 33 - 6l ^y BlORELIANCE- FofrattlyMlcr9bi9la|icltAnoc*tei Company Sanitized. Does not contain TSCA CBt H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 CaHiiU-2tsttT6ilMMBmHB^SBUt-O^ATR(ytiio'l(lCMtHO*O)CutBi)nCteKiMHAben*lllTc(tla DiP<MI.M71 Bk>Rtl>waSai<lrNirha-jmXNJ3lJTI. at., 1991). To ensure evaluation of first division metaphasecells me dividing cells will be arrested in metaphaseand harvested for microscopic evaluation of chromosome aberrations at approximately 20 BOWS (IS nonnal cell cycles) after me initiation of treatment (Galloway etal^ 1994). The clastogenic potential of the test article will be measured by its ability to induce structural chromosome aberrations in a dose-responsive manner wfaen compared to me solvent, control group. In me event of a positive response in the 4 hour non-activated study, the prolongedtreatment non-activated study may not be scored. The test article will also be assessed for its ability to induce numerical chromosome aberrations. 7.1 Solubility Detenninanon Water will be used as the test article solvent based on the solubility data available with the Sponsor. 7.2 Preliminary Toxicity Test for Selection of Dose Levels Selection of the dose levels for the cytogenetics assay will be done in consultation with the Sponsor, and added to the protocol by an amendment based upon post- treatment toxicity (cell growth inhibition relative to me solvent control) and solubility of me test substance. CHO cells will be treated to solvent alone and to at least nine concentrations of test substance. The highestconcentration tested will be 5 ing/ml or 10 mM whichever is lower for freely soluble test substances, or the maximum concentration resulting in a workable suspension for pooriy soluble test substances not to exceed 5 mg/mL The pH will be measured at the highest test substance treatment condition and will be adjusted, if necessaiy, in older to maint^n a neutral pH in me treatment medium. The osmolality of the highestdose level, lowest precipitatingdose level (where applicable)and me highestsoluble dose level (where applicable)in treatment medium will also be measured. Cells seeded 16-24 hours earlier will be treated for 4 hours in the absence and presence of S9 and for 20 hours in me absence of S9. Just prior to bypsuuzation Ac cell cultures will be visually inspected for the extent of monolayer confluency relative to me solvent control. Twenty hours after treatment initiation me cells will be harvested by trypsinizatioD and counted using:an automatic cell c(xi2tcm^Jh^ell viability will ^BHHtf be assessed using oypan blue dye exclusion (SOP The wn "cunts and percent viability will be used to detennine cell growth inhibition relative to me solvent control (SOP l U ^ ^ B j v^^^^^^^^^^s^p Whenever possible, the high dose to evaluate chromosome aberrations will be selected to give at least 50% cytotoxichy observed as (cell growth inhibition relative to the solvent control) irrespective of solubility. The highestdose will not to exceed 5 mg/ml or 10 mM. At least two additional dose levels, demonstrating minimal or no toxicity, will be included. In the event the test substance cannot be dissolved at a high enough concentration in an appropriate solvent to be toxic, then me highest dose to be tested in the chromosome aberration assay will be me concentration resulting in niinimuin precipitationin test medium. Precipitation will be determined Protocol No. SPGT331 February 23,2000 3of9 BioReIiance Study No.: AA26XN.331.BTL-34- 1f^9 B_ IOR_,ELIANCE- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 CUMW MM MiiinulluClmHUKafAbanilloaTtXiB '(CHOCtH ffttwn BteHtgMctSnriyNlMttr^AMXPWIOTL by direct visual inspection. In me event (be test substance demonstrates a doseresponsive increase in toricity at concentrations that exceed solubility in treatment medium, then the highestdose to be tested wffl be the maximim concentration that results in at least 50% toxicity. In me event that neither cytotoxicitynor insotubility is observed m (he preliminary test. the highest dose in me chromosome abeoation assay will be 5 sag/mi or 10 raM whichever is lower. If excessive precipitation of me test substance-solvent solution occurs upon addition to treatment medium, or if the osmolality of the treatment medium is considered excessive, the Sponsorwill be consulted. 7.3 Frequency and Route of Administration Target cells will be treated for 4 hours in me absence and presence of S9, and for 20 hours in the absence ofS9, by incorporation of the test substance-solvent mixture into the treatment medium. This techoiQue has been demonstrated to be an effective method of detection of chemical clastogensm mis test system (Evans, 1976). No repeats ofthe chromosome aberration tests will be done. 7,4 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\^ be batch preparedand stored frozen at approximately .70C until used. Each batch preparation of S9 will be assayed for sterility and its ability to metabolize 2-amiaoantBtacene and 7,12-dinicthylbenz(a)anthraceBe to fonns mutagenic to Salmonella typhimwivm TA100. Immediately prior to use, the S9 will be thawed and mixed with cofectors to contain 2 mM magnesium chloride (MgCl^) 6 mM potassium chloride (KC1), ImM glucose-6-phospnate, 1 mM nicotinamide aaenine dinucleotide phosphate(NADP) and 20 pi S9 per ml serum free medium. 7.5 Controls 7.5.1 Solvent (or Vehicle) Control The solvent for me test substance will be used as the solvent control. For solvents other than water, physiological buffer, or medium, the final concentration in treatment medium will not exceed 1%. 7.5.2 Positive Controls Mitomycin C will be used at a concentration within 0.05-0.3 tig/ml Protocol NB.STGTOI February 23,2000 4 of 9 BioRfiliance Study No.: AA26XN.331.BTL - 35 - fit BlORELIANCE- "y ForR>crlrMi(rotlielo{JeilAl(ocij|l Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 VSm MH--dluCtniwweAbanUlMTatlii (CHO>Ctfil Mvuwn amanttSMs Hwbcrjuiwmwjm. as die positive coatrol in the non-activated stady. Cyctophosphamidewill be used at a concentration within 10-50 ug/mi as the positive control in the So-activated stud/. 7.6 Preparation of Target Cells Exponentially growing CHO-K, cells will be seeded in complete medium (McCo/s 5A medium containing 10% fetal bovine serum, 2 ioM L-glutamine, 100 units penicillin/ml and 100 }ig streptomycin/ml) for each treatment condition at approximately 5 x 105ceBs/25 cm2 flask. The flasks will be incubated at 37 1C in a humidified atmosphereof 5 1% CO; in air for 16-24 hours. 7.7 Identification of Test System Using a pennanent marking pen, the treatment flasks will be identified by the BioReIiancc study number and a code system to designate me treatment condition 7.8 Treatment of Target Cells Treatment will be carried out in duplicate by refeeding the flasks with 5 ml complete medium for die non-activated treatment or 5 ml S9 reaction mixture for v^ the S9-acuvated treatment, to which be added SO ul of dosing solution of test or control substance in solvent or solvent alone. Larger volumes of dosing solution may be used if water, physiologicalbuffer, or medium is used as the solvent. In me non-activated study, me cells will be treated for 4 hours and fin- 20 hours; in . the So-activated study flie cells will be treated fbr 4 hours. Treatment will be carried outat37lCmahumidifiedatni03phereof5l%CO,niair. Afterme4hour treatment period in the non-activated and me S9-activated studies, the treatment medium will be aspirated, me cells washed with phosphate buffered saline, refed with complete medium and returned to the incubator, 7.9 Cell Harvest Cells will be collected approximately 20 hours after initiation of treatment. This post-treatment harvest time represents approximately 1.5 nonnal cell cycles and was selected to ensure that me cells are analyzed in me first division metaphase after initiation of treatment. Two hours prior to cell harvest, Colcemid* will be added to the cultures at a final concentration of 0.1 ugtol. Cells will be harvested by trypsinization, collected by centrifiigarion and an aliquot will be removed for counting using an automatic cell counter and trypan blue dye exclusion. The remainder of the cells will be swollen with 0.075M KCl. washed Protocol No. SPGT331 Febnury 23,2(100 fof9 BioReliance Study No.: AA26XN.331 .BTL - 36 - fi| BlOREUANCE" -5y FiMrlrMicrob(oloKirl Miacitlei Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 ^l-^2'M"W--<--^q^S^--^l^1^--^"^fv--la--v --MU--MU|lAlnamamnuAbtnill--Teilli CStSlBMtimWSSBStrrfVlnS#S<wCvHO)Cdl IMaaSfn Bi>RtllstrSta>dyNatwAA2(XjN.ULB7L with two consecutive changes of fixative (methanol:glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at approximately 2-8C. The cell counts and percent viability will be used to determine cell growth inhibition relative to the solvent control (% toxicity). To prepare slides, the cells will be collected by centrifugation and resuspeadedin fresh fixative. The suspension of fixed cells will be appliedto glass microscopeslides and air-dried. The slides will be identified by the experiment number, treatment condition and date. The slides will be stained with Gieinsa and pemianently mounted. 7.10 Scoring for Metaphase Aberrations To ensure that a sufficient number ofmetaphase cells are present on the slides, the percentageof(lbmnutosisper50<)cellsscorcd(mitoticindex)willbedeterniined and recorded for each coded treatment group selected for scoring chromosome aberrations. Slides will be coded using random numbers by an individual not involved with the scoring process. In the event of a positive response in the 4 hour non-activated study, the prolongedtreataientnon-activated study may not be scored. Metaphase cells with 202 centromeres will be examined under oil immersion withoat prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads from each dose level (100 per duplicate flask) will be exammed and scored for chromarid-typcand chromosome-type aberrations (Scott et aL, 1990). The number of metaphflsespreadsmat 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 iacludechromatid and isochromatid breaks and exchangefigures sucfaas 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 wiU 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 the incomplete exchange. Pulverized chromosome(s), pulverizedcells and severely damaged cells (210 aberrations) will also be recorded. Chromadd and isocbroraarid gaps will be recorded but not included in me analysis. The XY coordinates for each cell with a structural aberration will be recorded using a calibrated microscope stage. The percent polyploidand endoreduplicated cells will be evaluated per 100 cells for each dose level analyzed tat structural aberrations. 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST 8.1 Solvent Control The frequency of cells with structural chromosome aberrations in the solvent control must be within me range of the historical solvent control and not exceed 6%. Protocol No. SFGT331 February 23,2000 6 of 9 BioReliance Study No.: AA26XN.331 .BTL-37- 1^s^ ^^y B_ lOR_ ELlANCE" FerarrlyMfcroblatflgicjl Aliacutet Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Maromaliaa Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 VIM MnnHlluanuiMitAlmnllaaTltthi (CHO>Ct)b 8.2 Positive Control DrftK-BTI BtoRiiteaet Sbdy NniheKAA2fXm3UTL The percentage of cells with aberrations must be statistically increased (psO.05, Fisher's exact test) relative to the solvent control. 9.0 EVALUATION OF TEST RESULTS The cytotoxic effects of the treatments are based upon cell growth inhibition relative to the solvent control and will be presented for the toricity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent abenant cells) in the total population of cells examined, and the ineaii aberrations per cell will be calculated and reported for each treatment group. Chromand and isochromatidgqs are presentedin the data but are not included in fee total percentage of cells with one or more aberrations or in die frequency of structural aberrations per cell. Statistical analysisof tile percentage of abenant cells win be pctfonncd using the Fishers exact test The Fisher's test win be used to compare pairwisc the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's exact test at any test substance dose level, toe Cochran-Annitage test will be used to measure dose-responsiveness. All conclusions will be based on sound scientific basis; however, as a guide to interpretation of the data, the test substance will be considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (pSO.05). Test substances not demonstrating a statistically significant increase in aberrations will be concluded to be negative. 10.0 REPORT A report of the results of this study will be prepared by BioReliance and will accurately describe all methods used for generationand analysisofthe data. Results presentedwill include, but not be limited to: Test substance: identification and CAS no, if known; physical nature and purity, if known; physicochemicalpropertiesrelevant to the conduct of the study, if known; stability oftest substance, ifknown. Solvent/Vehicle: justification fan choice of vehicle; solubility and stability of test substance in solvent/vehicle, iflawwn. Source ofcells, karyotypefeatures (modal chromosome number) and suitability of the cell type used, absence ofmycoplasma, cell cycle length, passage number. Test conditions: composition of medium; CO; concentration; incubation time; cell seeding density; solvent and solvent selection rationale; concentration of test substance and concentration selection rationale; composition and acceptability criteria for the metabolic PnrtotoINo-SPGTMl Febnary 23,2009 7 of 9 BioReliance Study No.: AA26XN.331.BTL -38- fit BlOREUANCF <y raimflrlr Miciabliitosicil Awcifm Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (310) Cells DuPont-3871 IU.w3Ms^3^JH^B^HHfatl^vMu>tau\ dtaainniMKAImliMTt(tli cCuMaHaKt--W--SKrwrOSaSif<fvCaHsOa)nCdb IWsaWIt BMItllntcSli^Nrb(KAA2CXK3.BTL activation (S9) system; duration of treatment; duration of treatment with and concentration of Colcemid*; type of metabolic activation system used; positive and solvent controls; methods of slide preparation; number of cell cultures; criteria for scoring aberrations and criteria for consideringstudies positive, negative. * Results: descriptionof precipitation; pH and osmolality of the treatment medium; cell growth inhibition relative to the solvent control; robotic index and number ofmetaphases analyzed; type and number of aberration (structural and numerical) given separately for each treated and control culture; concentration-response relationship; statistical analysis; historical comrol data. 11.0 RECORDS AND AROfiVES All raw date, me protocol and all reports will be maintained according to Standard Operating Procedurfl----B^bythe BioReliance RAQA unit headquartered at: BioReliance, 14920 Brbschart RoaS,Rockville, MD 20850. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This Non-GLP study will be performed using the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies as a general guideline. This protocol has been written to comply with OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), February 1998 and with the International Conference on Hannonization of Technical Requirements for Registration ofPhannaceutfcals for Human Use (1996 and 1997). Will this study be submitted to a regulatory agency? NO If so, to which agency or agencies? NA Unless arrangements are made to we contrary, unused dosingsolutions will be disposedof following administration to the test system and all residual test substance will be disposed of following finalization of the report. 13.0 REFERENCES Evans, HJ. (1976) Cytological methods for detecting chemical mutagens, in: A. Hollaender (Ed.), Chemical Mutagens, Principlesand Methods for tneir Detection, vol. 4. Plenum Press, New Yolk, NY. Galloway, SM, MJ. Aaidema, M. Ishidate Jr., J.L. Ivett, DJ, Kiridand, T. Morita, P. Mbsesso and T. Softmi (1994) Report from working group on in vitro tests for chromosomal aberrations. Mutation Research 312(3):24I-261. Protocol No. SPGTOl February 23> 2000 gof BioReliance Study No.: AA26XN.331 .BTL-39- 0jg1^ -- -- BlORELIANCF T^ ^y formerly Mltroliiolo(ic*lAiioeit(* Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test io Chinese Hamster Ovary (CHO) Cells :49AM:OuPonT Pfmiia i30Z 4S1 *27 DuPont-3871 T/ T TMI* rHirtiii.MXumun. fflteniatiooal Conference on VxaiaaSuSaa (ICH) of Technical Requirements far RegiitratKm of PhamwraiiiMh for Human Vie. Guidance on Specific Aspects of Repitatoty Geootoxiefty Tests for Phannacfuticalt. S2A documeat iBcomiseoded for idoptioattep4offlieICHiacesson July 19.1995. FedefslRsguter 61:18198-18^)2, April 24,1996. btemattoiul Conference on HsniKxusatkm (KM) of Tecbafcd Reqaireraeiits for Registration of Ptannceuiied for HUZBIH Uw. Genotoxicty: A Sttudaid Battoy fior GenotoKiciiyTeirtingofPhannaccudcal*. SZBdociaieatiBcomnnidfdfisradoptioaBtKep 4oftheICH procest oa July 16,1997. Federal RegiKer 62:16026-16030, November 21, 1997. ' OECD Guideline for &e Tetting of Chenacato. Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), Febiuny 1998. Preston, RJ, W. Au. MJL Bender, J.O. Brewen, A.V. Cmmo, SJL Heddle. AJF. McFte, S.WolfftndJAWtsaom (1981) Mmmriim m vivo and m vitro cytogenedc mays: s report of die Geae-Tos Program, Mutadoa Refeach, 87:143-188. Scott. D^ NJ). Dtnfbtd, BJ. Dean aad DJ. KiffciBBtL 1990. Meffbase Cluomosoffle Abettarion Assays m Vitro. In: Basic Mutapaicity Tests: UKEMS Recommended Procedures. D JKirUand(cd). Cambridge Univenity Press, New Yotk,NY. SwiercntaSJUt.JA.Heddie.EA.Sigal.Ji'.W.Ganm^RJ.BriIliager.GJI. Douglas ami EJR. Nestmaim (1991) ReconmieBded protocolsbased on ft sifrvey of cgnent practice in geootoxidty testing Sabotatoriet, W. Chromosome aberration ud xister'dBOioadd exchange m Chinese hamster ovary, V79 Chinese loaf aad banian lympfaocytecultures, MuUtioo Researeh 246:301-322. 14.0 APPROVAL l<0^:-- ^cv^^t^ STUDY MONITOR ^1^1^ DATE Maria Domr.PhJ3. (Print or Type Name) f/mtzt^:^^' BIORBLJANCE STUDY DIRECTOR ^S'^ DATE BIOREUANCE STUDY MANAGEMENT DATE Proto8rfNc.SPGT331 Fibruary 23,2036 BioReliance Study No.: AA26XN.33! .BTL f9 - 40 - 6 ^9 BtORELIANCE f'w(<vMICfb)l*a)CBlA|keum Company Sanitized. Does not contain TSCA CBI