Document KG09xdv8g20qGdZyMp9aJpGOo

AR226-3148 DuPont-3870 TRADE SECRET Study Title H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells Ramadevi Gudi, Ph.D. Elizabeth H. Schadly, B.S. Report Completion Date May 4. 2000 Performing Laboratory BioReliance 9630 Medical Center Drive Rockville, MD 20850 for E. I. du Font de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine P.O. Box 50, Elkton Road Newark, DE 19714-0050 Performing Laboratory Study Number AA26XP.33LBTL DuPont Project ID DuPont-3870 Work Request Number Company Sanitized. Does not contain TSCA CBI Page 1 of 39 H-24335; In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary ^CHO)Cells DuPont-3870 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. BioReliance Study Director: A^Z^a^fe^ C^t^t.^ Ramadevi Gudi, Ph.D. Otfjo^/OD Approved by Study Monitor: rto^c^ .0-0''^' Maria Donner. Ph.D. Senior Research Scientist O^o^oo Date Company Sanitized. Doss not contain TSCA CB1 BioReliance Study No. AA26XP.331.BTL -2- H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells_________________DuPont-3870 TABLE OF CONTENTS Page 2 Certification............................................................................................................................ Study Information................................................................................................................... 5 6 Summary............................................................................................................................... 8 Purpose.................................................................................................................................... Characterization of Test and Control Substances................................................................... 8 Materials and Methods............................................................................................................. 8 Results and Discussion........................................................................................................ 13 15 Conclusion............................................................................................................................. 16 References.............................................................................................--..........--................. Data Tables.................?............,.......................................,......................................................17 Tablel: Preliminary Toxicity Assay with H-24335 in CHO Cells in the Absence of Exogenous Metabolic Activation. 4 Hour Treatment....................................................... 17 Table 2: Preliminary Toxicity Assay with H-24335 in CHO Cells in the Presence of Exogenous Metabolic Activation. 4 Hour Treatment....................................................... 18 Table 3: Preliminary Toxicity Assay with H-24335 in CHO Cells in the Absence of Exogenous Metabolic Activation. 20 Hour Treatment................................................... 19 Table 4; Concurrent Toxicity Assay with H-24335 in CHO Cells in the Absence of Exogenous Metabolic Activation. 4 Hour Treatment.................................................... 20 Table 5: Cytogenetic Analysis of CHO Cells Treated with H-24335 in the Absence of Exogenous S9 Metabolic Activation, 4Hour Treatment, 16 Hour Recovery Period......... 21 Table 6: Concurrent Toxicity Assay with H-24335 in CHO Cells in the Presence of Exogenous Metabolic Activation. 4 Hour Treatment........................................................22 Company Sanitized. Does not contain TSCA CBi BioReliance Study No. AA26XP.331 .BTL - 3 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells____________________DuPont-3870 Table 7: Cytogenetic Analysis of CHO Cells Treated with H-24335 in (he Presence of Exogenous S9 Metabolic Activation. 4Hour Treatment, 16 Hour Recovery Pedod.........23 Table 8; Concurrent Toricity Assay with H-24335 in CHO Cells in the Absence of Exogenous Metabolic Activation. 20 Hour Treatment.....................................................^ 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.........25 Table 10: Summary: Cytogenetic Analysis of CHO Cells Treated with H-24335 ............26 Appendix A: Historical Control Data.................................................................................... 27 Appendix B: Study Protocol ..................................................,,......................................."..... 30 BioReUanceStudyNo.AA26XP.331.BTL Company Sanitized. Does not contain TSCA CB1 .4- H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Celts STUDY INFORMATION Substance 11 I 11 IBBB1U| \ Synonyms/Codes:* H-24335 DuPont-3870 Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Solubility: Aquatics: Soluble but cloudy in water at concentrations of ^15 mg/mL. Sponsor: E. I. du Pent de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine P.O. Box 50, Elklon Road Newark, DE 19714-0050 Study Initiated/Completed: February 25,2000 / (see report cover page) In-Life Initiated/Completed: February 29, 2000 / March 29,2000 BioReliance Study No. AA26XP.331.BTL Company Sanitized. Does na! contain TSCA CB[ -5- H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells____ DuPont-3870 SUMMARY The test substance, H-24335, was tested in the fn vitro mammalian chromosome aberration test using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced exogenous S9 metabolic activation system. A preliminary toxicity assay was performed to establish the dose range for the chromosome aberration assay. Tbs chromosome aberration assay was used to evaluate the clastogenicpotential of the test substance. Water was determined to be the solvent of choice based on information provided by me Sponsor, the solubilityof the test substance, and compatibility with the taiget cells. The test substance was soluble but cloudy in water at a concentration of 50 mg/mL, the maximum concentration tested. In the preliminary toxicity assay, the maximum dose level tested was 5000 ^ig/mL. Visible precipitateswere observed in the treatment medium at dose levels ^1500 ug/mL. Dose levels ^500 ug/mL were soluble in the treatment medium. Selection of dose levels for the chromosome aberration assay was based on total cell growm inhibition relative to the solvent control. No substantial toxicity, i.e., at least 50% cell growm inhibition, was observed at any dose level tested in neither the non-activated nor the S9 activated 4 hour treatment groups. Substantial toxicity was observed at the dose level 5000 pg/mL in the non-activated 20 hour continuous treatment group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 625 to 5000 ug/mL for the non-activated and the S9 activated exposure groups. m the chromosome aberration assay, the cells were treated for 4 and 20 hours in the nonactivated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatment initiation. Visible precipitates were observed in (he treatment medium at the dose levels >2500 pg/mL. The dose levels ^1250 p,g/mL were soluble in treatment medium. No substantial toxicity (>50% cell growth inhibition) was observed at the highestdose level evaluated for chromosome aberrations, 2500 ug/mL, in the non-activated and S9 activated exposure groups. Substantial toxicity (2:50% cell growth inhibition) was observed in the non-activated 20 hour exposure study at the dose levels 3500 and 5000 ug/mL. Excessive mitotic inhibition was also observed at these dose levels. Therefore, for the non- activated and S9 activated 4 hour exposure groups, in (he absence of at least 50% cell growth inhibition, dose selection was based upon test substance precipitation in the treatment medium (the lowest precipitating dose was the highest dose evaluated). In the non-activated 20 hour exposure group, the highest dose level evaluated was the dose level with at least a 50% reduction in mitotic index, relative to the solvent control. Initially, 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 the non-activated or S9 activated 4 hour treatment groups relative to the solvent control group, regardless of dose level (p>0.05. Fisher's exact test), m me Company Sanitized. Does not contain TSCA CB BioReliance Study No. AA26XP.33i.BTL - 6- H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells________________PuPont-3870 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. No statistically significant increases in structural and numerical chromosome aberrations were observed in the non-activated 20 hour continuous treatment group relative to the solvent control group, regardless of dose level (pX).05, Fisher's exact test). Under me conditions described in this report, H-24335 v/as 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 using Chinese hamster ovary (CHO) cells. BioRclianceStudyNo.AA26XP.331.BTL Company Sanitized. Does not contain TSCA CF -7- H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cella__________ _________DuPont-3870 PURPOSE The purpose of this study was to evaluate the clastogenic potential of the test substance based upon its ability to induce in vitro chromosome aberrations in Chinese hamster ovary (CHO) cells. CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test substance, H-24335, was received by BioReliance on 15 February 2000 and was ii^ed the code ninnberAA26XP. The test substance was characterized by the Sponsor as an _SKSffHHSHflffwsat should be stored in a well ventilated place at temperatures below 120F. An expiration date for the test substance was not provided. sajan^sj^Ul^lUf^f^ifmf^f^fif^^f^nyld Upon receipt, the test substance was describbeed as was stored at room temperature in a well-ventilated area,, protectetrfrom exposure to light, iin a tightly closed container. Based on information provided by the Sponsor, sterile distilled water (CAS 7732-18-5), obtained from the Life Technologies Company, was the solvent used to deliver H-24335 to the test system. 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 1 and 2 ugAnL for use as the positive control in the non-activated test system. Cyclophosphamide (CP; CAS No.: 6055-19-2), was obtained from Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations of 100 and 200 pg/mLforuseasthe positive control in the S9 activated test system. For each positive control one dose with sufficient scorable metaphase cells was selected for analysis. The solvent for the test substance was used as the solvent control at me same concentration as that found in the test substance-treated groups. MATERIALS AND METHODS Test System Chinese hamster ovary (CHO-Ki) cells (repository number CCL 61) were obtained fiom American Type Culture Collection, Manassas, VA, on May 29, 1997. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 20. CHO cells at passage 6 were used for the preliminary toxicity assay and CHO cells at passage 12 were used for the chromosome aberration assay. The freeze lot of cells was tested using the 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 BioReliance Study No. ____ AA26XP.331 .BTL Company Sanitized. Does not contain TSCA CBS - 8- H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells____________________DuPont-3870 clastogens(Preston et al., 1981). Metabolic Activation System Aroclor 1254-induced tat liver 89 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254,500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at ^-70C until used. Each bulk preparation of S9 was assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to SalmoneSa typhimuriwn TA100. Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 (iL S9 per milliliter medium (McCoy's 5A serum-free medium supplemented with, 100 units penicillin and 100 pg streptomycin/mL, and 2 mM L-glutamine). Preliminary Toxicity Assay The preliminary toxichy 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 10s 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 (McCo/s 5A medium supplemented with 10% fetal bovine serum (PBS), 100 units penicillin and 100 pg streptomycin/mL, and 2 mM L-glutamine) for the non-activated study or S9 reaction mixture (3.5 mL serum-free medium plus 1 mL of 5X S9 mix) for the activated study, to which 500 pL dosing solution of test substance in solvent or solvent alone was added. The osmolality of the highestconcentration of the dosing solution in the treatment medium was measured. The pH of the highestconcentration 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-nee phosphate buffered saline (CMF-PBS), refed with 5 mL complete medium and returned to the incubator for a total time period of 20 hows from the initiation of me treatment At 20 .hours after the initiation of the 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 trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control. BioReliancc Study Not AA26XP.331.BTL "~~-~~9" company Sanitized. Does not contain TSCA CBI H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells____________DuPont-3870 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 controls. For the chromosome aberration assay, CHO cells were seeded at approximately 5 x 105 cells/25 cm2 flask and were incubated at 371C in a humidified atmosphereot5l% CO, in air for 16-24 hours. Treatment was carried out by refeeding duplicateflasks with 4.5 mL complete medium (McCoy's 5A medium supplementedwith 10% FBS, 100 units penicillin and 100 pg streptomycin/mL, and 2 mM L-glutamine) for the non-activated study or 4.5 mL S9 reaction mixture for the S9 activated study, to which 500 uL of dosing solution of test or control substance in solvent or solvent alone was added. The osmolality of me highest concentration of the dosing solution in the treatment medium was measured. The pH of the highestconcentration of the dosing solution in the treatment medium was measured using test tape. In 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 of5l% COj in air (Swierenga et al, 1991). In the 4 hour exposure group, tile treatment medium was removed after the exposure period, and the cells washed with CMF-PBS, refed with 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 me incubator until cell collection. m the S9 activated study, me cells were exposed for 4 hours at 371"C in a humidified atmosphere of 51% COy, in air (Swierenga et al., 1991). After tile exposure period, the treatment medium was removed, the cells washed with CMF-PBS, refed with 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 were returned to the incubator until cell collection. A concurrent toxidty 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 from 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. Ceil 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 'ompany Sanitized. Does not contain TSCA CB5 BioReliance Study No. AA26XP.331.BTL -10 - H-24335: In Vitro Mammalian Chromosome Apa-ratioa Test in Chinese Hamster Ovary (CHO) Cells __ DuPont-3870 centri&gation at approximately800 rpm for 5 minutes. The cell pellet was resuspendedin 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 centrifagation, the supernatant aspiratedand the cells fixed with two washes of approximately2 mL Camoy's fixative (methanokglacialacetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately2-8C. Slide Preparation To prepare slides, the fixed cells were centrifuged at approximately800 rpm for 5 minutes, the supernatant was aspirated, and 1 mL fresh fixative was added. After additional centrifagation (at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted aod me cells resuspended to opalescence in fiesh fixative. A sufficient amount of cell suspension was dropped onto me 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 that a sufficient number of metaphase cells were present on the slides, me percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells 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, me non-activated 20 hour continuous exposure group was not evaluated for chromosome aberrations. Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 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 (chromatid or acentric) observed in me 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 (5:10 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 from each treatment flask per 100 metaphase cells scored. BioRfiliance Study No. AA26XP.331 .BTL -11 - j^mpany Sanitized. Does not contain TSCA CBS H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 Controls Mitomycin C was used as die positive control in the non-acdvated study at final concentrations of 0.1 and 02 pgtoL. Cyclophosphamidewas used as the positive control in the S9 activated study at final concentrations of 10 and 20 pg/mL. For both positive controls one dose level exhibiting a sufficient number of scorable metaphase cells was selected for analysis. The solvent vehicle for the test substance was used as the solvent control at the same concentration as that found in the test substance-treated groups. The solvent vehicle for the test substance was used as the solvent controLatJhg same.oncentration as that found in the test substance-treated groups. DuPont stiKUespJ^na|jQvereconducted simultaneously and thus the same solvent and positive controls were%sedbetweenthese 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 me toxicity and aberration studies. The number and types of aberrations found, me percentage of structurally and numerically damaged cells (percent aberrant cells) in the total populationof cells examined, and me mean aberrations per cell was calculated and reported for each 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 of each treatment group with that of me solvent control. In the event of a positive Fisher's test at any test substance dose level, the Cochran-Anmtage test was used to measure dose-responsiveness. All conclusions were based on sound scientific basis; however, as a guide to interpretation of the date, 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 of negative 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. BioReliance Study No. AA26XP.331.BTL-Tl- Company Sanitized. Does not contain TSCA CB1 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells____________________DuPont-3870 Deviations No known deviations &om the protocol or assay method SOPs occurred during the conduct of this study. Archives lUH^by All raw data, the protocol, all reports, and stained and coded slides will be maintained according to Standard Operating Procedure the BioRellance Regulatory Affairs/Quality Assurance Unit headquartereS' at: BioKflliance, 14920 Broschart Road, Rockville, MD, 20850. RESULTS AND DISCUSSION SolubUity 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 ceils. The test substance was soluble but cloudy in water at a concentration of 50 mg/mL, the maximum concentration tested. Preliminary Tosdeuy Assay Dose levels for tha 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 presentedin Tables 1-3. CHO cells were exposedto solvent alone and to nine concentrations of test substance ranging from 0.5 ug/mL to 5000 ug/mL in the absence and presence of an S9 reaction mixture. Visible precipitate was observed in treatment medium at dose levels ^1500 ug/mL. Dose levels ^500 pg/mL were soluble in treatment medium. The osmolalhy of me solvent (water) in treatment medium was 314 mmol/kg, and not considered significantly altered. The osmolality in treatment medium of me highestconcentration tested, 5000 pg/mL, was 274 mmol/kg. The pHofthe highest concentration of test substance in treatment medium was approximately 7.0. Cell growth inhibition relative to the solvent control was 17% and 12% at 5000 (ig/mL, the highest concentration tested in both the non-activated and S9 activated 4 hour groups, respectively (Table 1 and 2). Cell growth inhibition relative to the solvent control was 70% at 5000 pg/mL, me highest concentration tested in the non-activated 20 hour continuous treatment group (Table 3). Based upon the results of the toxicity study, me dose levels selected for testing in the chromosome aberration assay were as follows: BioReliance Study No. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CBI -13 - H-24335: In Vitro Mammalian Chromosome Abenation Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 Treatment Condition -S9 +S9 Treatment Time(hr) 4 Recovery Tune(hr) 16 Dose levels (ug/mL) 625,1250,2500,5000 20 0 625,1250,2500,3500.5000 4 16 625,1250,2500,5000 Chromosome Aberration Assay In the chromosome aberration assay, visible precipitates were observed in treatment medium at dose levels ^2500 ug/mL. Dose levels <1250 pg/mL were soluble in treatment medium. The osmolality of the solvent (water) in treatment medium was 293 mmol/kg. The osmolality in treatment medium of the highest concentration tested, 5000 pg/mL, was 329 mmol/kg, and not considered significantly altered. The pH of me highest concentration of test substance in treatment medium was approximately 7.0. Cell growth inhibition relative to the solvent control in CHO cells was 20% after treatment with H-24335 at 2500 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-24335 to induce chromosome aberrations is presented by treatment flask in Table 5, and summarized by treatment group in Table 10. The mitotic index at the highest dose level evaluated for chromosome aberrations, 2500 pg/mL, was 63% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 625,1250, and 2500 pg/mL. m the absence of at least 50% cell growth inhibition, the highest dose level selected for evaluation was the lowest precipitating dose. The percentage of cells with structural and numerical aberrations in me test substance-treated groups was not significantly increased above that of the 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 statisticallysignificant Cell growth inhibition relative to the solvent control in CHO cells was 17% after treatment with H-24335 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-24335 to induce chromosome aberrations is presented .by treatment flask in Table 7, and summarized by treatment group in Table 10. The mitotic index was 61% reduced relative to the solvent control at the highest dose level evaluated for chromosome aberrations, 2500 ug/mL. The dose levels selected for microscopic analysis were 625,1250, and 2500 ng/mL. In the absence of at least 50% cell growth inhibition, me highest dose level selected for evaluation was me lowest precipitating dose. The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not statistically increased above that of the solvent control (p>0.05. Fisher's exact test). The percentage (26.5%) of structurally damaged cells in the CP Sanitized. Does not contain TSCA CBI _Bi_oR_e_lia_nc_e _St_ud_y_No_._A_A2_6X_P_.3_31_.B_T_L____-_1_4 -__Company H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovaiy (CHO) Cells DuPont-3870 group was found to be statisticallysignificant. 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. Cell growth inhibition relative to the solvent control was 14% at 2500 ug/mL (Table 8), the highest dose level of H-24335 evaluated for chromosome aberrations in the non-activated 20 hour continuous exposure group. The ability of H-24335 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 highestdose level evaluated for chromosome aberrations, 2500 pg/mL, was 62% reduced relative to me solvent control. The dose levels selected for microscopic analysis were 625,1250, and 2500 pg/mL. Due to excessive mitotic inhibition at dose levels with ^50% cell growth inhibition, the highest dose level selected for evaluation was the dose level with at least 50% mitotic inhibition. The percentage of cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased above that of the solvent control (p>0.05, Fisher's exact test). The percentage (28.5%) of structurally damaged cells 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 the requirements for a valid test Under the conditions described in this report, H-24335 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 using Chinese hamster ovary (CHO) cells. BioReliance Study No. AA26XP.331.BTL -15 - Company Sanitized. Does not contain TSCA CBB H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Celb____________________DuPont-3870 REFERENCES Evans, H.J. (1976) Cytological methods for detectingchemical mutagens, in: A. Hollaender (Ed.), Chemical Mutagens, Principlesand Methods for their Detection, vol 4. Plenum Press, New York. Galloway, SM, MJ. Aardema, M. Ishidate Jr, J.L. Ivett, D.J. Kiridand, T. Morita, P. Mosesso and T. Sofani (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 ofPhannaceuticals for Human Use. Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoricity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 of me ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24,1996. International Conference on Hannonisation (ICH) of Technical Requirements for Registration ofPhannaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing ofPhannaceuticals. 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 me Testing of Chemicals, Guideline 473 {In Vitro Mammalian Chromosome Aberration Test), adopted, July 1997. Preston, RJ., W. Au, MA. Bender, J.G. Brewen, A.V. Carrano, JA. Heddle, A.F. McFee, S. Wblffand J.S. Wassom (1981) Mammalian in viva and in vitro cytogenetic assays: a report of the Gene-Tox Program, Mutation Research, 87:143-188. Scott, D., N.D. Danfoid, B.J. Dean and DJ. Kiridand. 1990. Metaphase Chromosome Aberration Assays In Vitro, m: Basic Mutagenicity Tests: UKEMS Recommended Procedures. D.J Kiridand (ed). Cambridge University Press, New York, NY. Swierenga S.H.H., J.A. Heddle. EA. Sigal, J.P.W. Gilman, RJL. BriUinger, 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 sisterchromatid exchange in Chinese hamster ovary, V79 Chinese lung and human lymphocyte cultures. Mutation Research 246:301-322. BioReliance Study No. AA26XP.33 l.BTL - 16 - Company Sanitized. Does not contain TSCA CBS H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells____________________DuPont-3870 TABLE 1 PREUMIMARYTOXICITYASSAYWTH H543351NCHOCELLSIN THE ABSENCE OF EXOGENOUS S9 METABOLIC ACTIVATION 4 HOUR TREATMENT, 16 HOW RECOVERY PERIOD Treatment1 (tigftnL) Ceil Count (xlO") Cell Viabffity3 (%) Mean Calls/ Flask* (x10) Cell Growth Index4 (%) Cell Growth Inhibition* (%) Watar 2.50 99% 2.47 100% H-24335 0.5 1.5 5 15 50 150 500 1500' 5000* 3.60 2.95 2.38 2.79 2.80 2.55 3.02 2.51 2.12 99% 100% 99% 98% 98% 99% 100% 98% 97% 3.S7 2.95 2.35 2.73 2.75 2.52 3.02 2.46 2.05 144% 119% 95% 110% 111% 102% 122% 100% 83% -44% -19% fi% -11% -2% -22% 0% 17% 4 CHO calls were treated In the absence of an exogenous source of metabolic activation for 4 hours at 371 C. 2 Viability datonnlned by trypan blue dye exclusion. ' Viable celteffiask-call count x% viable oelto 4 Growth index " (cells per flask treated group/cell* per flask control group). expressed as a percentage. ' CeH growth Inhliiaon" 100%-%ceU growth Index; not calculated far negative controls. BioReliance Study No. AA26XP.331 -BTL - 17 - Company Sanitized. Does not contain TSCA CBI H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 TABLE 2 PREUM1NARYTOXICITYASSAYWITH H-24335 IN CHO CELLS !N THE PRESENCE OF EXOGENOUS S9MCTABOUC ACTIVATION 4 HOUR TREATMBO-, 16 HOUR RECOVERY PERIOD Treatment1 (lig/mL) Cell Count (x10") Coll Viability1 {%) Mean Cels/ Flagk' (x10) Cel Growth Index4 (%) Coll Growth Inhibition' (%) Water 2.51 95% 2.38 100% H.2433S 0.5 1.5 5 IS 50 -r 150 500 1500* 5000' 2.42 2.68 3.00 3.14 2.64 2.76 2.99 2.40 2.13 100% 99% 99% 98% 98% 99% 97% 98% 98% 2.42 2.66 3.03 3.08 2.59 2.73 2.90 2.35 2.09 102% 111% 127% 129% 109% 115% 122% 99% 88% .2% -11% -27% -29% -9% -15% -22% 1% 12% 4 CHO cells ware treated In the presence of an exogenous source of metabolic activation for 4 hours at 371C, 2 Viabilitydetermined by trypan blue dye exclusion. 1 Viable cete/(lk cell count x%viabte cell* 4 Growth Index " (cells per flask treated group/cells per flask control group), expressed as a percentage. 1 Celt growth inhibition -100% % cell growth Index; not calculated for negative controls. ' Visible precipitates obasrved, BioReliance Study No. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CBI .18. H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 TABLES PREUM1NARYTOX1CITYASSAYWTH H-24335 IN CHO CEU.S IN THE ABSENCE OF EXOGENOUS S9METABOUC ACTIVATION 20 HOUR CONTINUOUS TREATMENT TiBttmBnt1 (USftnL) Water H-24335 0.5 1.5 5 15 50 150 500 1500' 5000" Call Count (x106) Coll Viability2 (%) Mem Cells/ Flask* (x106) Gill Growth Index4 (%) CeB Growth Inhibition* (%) 2.99 98% 2.93 100% -- 2.54 2.43 2.18 2.25 1.79 2.21 1.77 1.95 0.90 98% 96% 99% 95% 94% 95% 100% 97% 98% 2.49 2.34 2.16 2.14 1.68 2.10 1,77 1.89 0.88 85% 15% 80% 20% 74% 26% 73% 27% 57% 43% 72% 28% 60% 40% ^ 65% 35% 30% 70% ' CHO cells ware treated continuously In the absence of an exogenous source of metabolic activation for 20 hours at 371C. 2 Viability determined by bypan blue dye exclusion. 3 Vteblecatentask "call count x%viabto cells * Growth Index (cells per flask treated group/cells per fliok control group), expressed as a percentage. * Cgl! growth Inhibition" 100%-% ceil growth index; not calculated tor negative controls. * Visible precipitates observed. BioReliance Study No. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CBI 19- H-24335:AFaroMamnilalianC:hromosofflc> Aberration Test in Chine!leHamster Ovary (CHO)Celb DuPont-387( TAB LE4 CONCHIRRENTTOXIICITY ASSAY WTTH H-24335IINCHOCEU5 IN WE.t\BSENCEOF'EXOGENOl1SS8METABO!L1CACTVATK3K1 4mOURTRE/i11MENT.161tOURRECOVERY PERIOD Treatment' (MBtoL) Water H-24335 625 1250 2500 S000 MMC. 0.1 MMC. 0.2 Cell Count Averages Flask (x106) A 2.52 B 3.10 A 2,05 B 2.31 A 2.03 B 1.90 A 1.88 B 2,54 A 1.86 B 1.51 A 1.95 B 2,05 A 2.07 B 2.03 Cell Viabffity2 Mean Cells per Flask* (xm) 98% 98% 2.73 100% 98% 98% 99% 99% 98% 98% 98% 98% 97% 87% 95% 2.16 1.94 2.18 1.66 1.95 1.97 Ceil Growth Indwc' (% 100% 79% 71% 80% 61% 71% 72% CeHQrowth Inhibition' (%) ,,,-,-, 21% 29% 20% 39% 29% 28% ' CHOcetewera treated In the absence of an exogenous source of rmtaboilc activaUon for 4 hours at 37i1C. 1 Vlabttitydetennlned by taypan blue dye exclusion. 3 Viable cellsfflask cell count x % viable cells, reported as mean of Flasks A and B. 4 Growth Index " (mean cells per flask treated group/mean ceUs per flask control group), expressed as a percentage. ' Cell growth inhibition -100% - % cell growth Index; not calculated for negative controls. Visible precipitates observed. ;ompany Sanitized. Does not contain TSCA CB1 r BioReliance Study No. AA26XP.331 .BTL .20- H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovaly (CHO) Cells DuPont-3870 TABLES CYTOGENCTCANALYSlSOFCHOCELLSTREATEDWnM H343351NTHE ABSENCE OF EXOGENOUS S9MrABOUCACTIVAT10N 4 HOUR TREATMENT, 18 HOW RECOVERY PERIOD Treabrnent'-' (ug/ml) Flask Mitotte index2 (%) Calls Scored % Aberrant Calls' Numerical Structural Water A 11.0 100 2 0 B 11.4 100 3 1 H-243.35 625 A 6.2 100 3 1 8 5.6 100 4 1 1250 A 5.2 100 0 1 B 4.8 100 4 2 2500" A 4.6 100 4 2 B 3.8 100 3 4 MMC. 0.2 A 9.6 100 2 16 B 10.6 100 5 21 Total Number of Structural Aberrations Gaps Chromatid4 Br Ex Chromosome8 Br Die Ring Severely Damaged Cells' 010000010000 0 0 311000000100 0 0 010100000110 0 0 300111 010110 0 0 1 14 11 40 0 0 0 19 7 130 0 Avenge Aberrations PerCelF 0.000 0,010 0.010 0.010 0.010 0.020 0.020 0.040 0.290 0.300 1 CHO ceBs ware treatadfof 4 hours at 371C(n the absence of ai!) exogenous source of metabolic activation. Mitotte Index-number mitoBc figures x 100/500 cells counted. Numerical: Includes polypioldand endorodupllcated cells.; Structural: excludes calls with only gaps. Chromatid breaks include chnxnatid and Isochromatid breaks and fragments; chromatid exchange figures (Exch) include quadrindfate, triradlals and complex rearrangements. Chromosome breaks include break* and acentric fragments; die, dteentrte chromosome. Severely damaged cells Includes calls with one or more pulverized chromosomes and cells wtth 10 or more aberrations, Severely damaged cells and pulverizations were counted as 10 aberrations. Dose level 5000 iig/mL was not analyzed due to test substance precipitation in the medium. Lowest precipitating dose level. BioReliance Study No. AA26XP.331 .BTL Company Sanitized. Does not contain TSCA CBI - 21 - H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPoat-3870 TABLES CONCURRENT TOXiCtTY ASSAY WITH H-24335 IN THE PRESENCE OF EXOGENOUS S9 METABOLIC ACTIVATION 4 HOUR TREATMENT. 18 HOUR RECOVERY PERIOD Treatment4 Wl-? Water H-24335 825 1250 2500 5000' CP.10 CP.20 Cell Count Averages Flask (x10*6) A 2.84 B 2.35 A 2.08 B 1.88 A 2.17 B 2.27 A 2.25 B 2.11 A 1.95 B 1.95 A 1.82 B 1.71 A 1.38 B 1.50 Cett Viability* Mean Colls perFlaBk1 (x10l>e) 98% 98% 99% 100% 100% 99% 98% 97% 98% 97% 97% 99% 98% 96% 2.55 1.98 2.21 2.13 1.90 1.83 1.38 Cell Growth Index* (%) 100% 77% 87% 83% 75% 84% 54% Cell Growth Inhibition* (%) 23% 13% 17% 25% 38% 48% 1 CHO cells were treated In the presence o{ an exogenous source of metabolic activation (or 4 hours at 371C. 2 Viability determined by tiypan blue dye exclusion. 3 Viable celtefflask " cell count x % viable cells, reported as mean of Flasks A and B. 4 Growth index (mean eels per flask treated group/mean oeMs per flask control group), expressed as a percentage. * Cell growth inhibition 100% - % cell growth Index; not calculated for negative controls. 9 Visible precipitates obseived. BioReliance Study No. AA26XP.331 .BTL -22- Company Sanitized. Does not contain TSCA CB1 H-24335: In Vitro Mammalian Chromosome Aberration Test to Chinese Hamster Ovary (CHO) Cells DuPont-3870 TABLE 7 CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH H-24335 !N THE PRESENCE OF EXOGENOUS S9 METABOLIC ACTIVATION 4 HOUR TREATMENT, 18 HOUR RECOVERY PERIOD Treatment1-' (MB/mL) Flask Mitotto Index* (%) Cells Scored %Abemmt Cells' Numerical Structural Water A 12.0 100 3 2 B 11.2 100 4 3 H-24335 625 A 7.0 100 3 4 B 6.4 100 1 7 1250 A 8.0 100 4 5 B 6.2 100 3 6 2BOO' A 5.2 100 3 7 B 3.8 100 2 5 CP.10 A 7.6 100 2 2S B 8.2 100 1 28 Total Numbm ofStructural/ikbemittons Gaps Chnxinatid4 Br Ex Chrornosome' Br Die Ring Severely Damaged Cells* 1 2 0 0 0 0 0 0 2 0 0 1 0 0 2 4 0 0 1 0 0 1 8 1 0 1 0 0 4 5 1 0 0 0 0 0 5 0 0 1 0 0 3 7 0 0 0 0 0 0 5 0 0 0 0 0 5 35 6 0 0 0 0 2 39 3. 0 0 0 0 Average Aberrations Per CriP 0.020 0.030 0.050 0.080 0.080 0.060 0.070 0.050 0.410 0.420 CHO cells were treated for 4 hours at 371 C In the presence of an exogenous source of metabolic activation. Mitotte Index" number rnltotlcflguiwx 100/500 Mils counted. Numerical: Includes polyploidand endoredupllcated cells.; Structural: excludes cells with only gaps. Chromatid breaks Include chromafld and IsochromatM breaks and fragments; chromatid exchange figures (Exch) Include quadrimdialStInitials and complex rearrangements. Chromosome breaks indude bmato and aoentric ftagimnts; dl(^ dicenlric chromosome. Severely damaged cells ln<^ctoscall with OTM or more pu^erizedchromoaomes and celts vrith 10 or more aberrti<ms. 7 Severely damaged cells and pulverizations were counted as 10 aberrations. Dose level 5000 pg/mL was/ not analyzed due to test substance precipitation In the medium. Lowest precipitating dose level. BioReliance Study No. AA26XP.331.BTL -23- Company Sanitized. Doss not conra'n TSCA CB? H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 TABLES CONCURRENTTOXICITYASSAYVUTH ?34335 IN THE ABSENCE OF EXOGENOUS SS^ABOLIC ACTIVATION 20 HOUR CONTINUOUS TREATMENT Treatment1 (Ufl/mL) Water H.24336 925 1250 2500' 3500' SOOO" MMC, 0.1 MMC.0.2 Cell Count Average* Flask (x10) Cell Viability2 MenCei!s perFtesk* OdO') A 2.34 B 2.79 A 2.10 B 2.18 A 2.20 B 2.82 A 2.38 B 2.04 A 1.32 B 1.24 A 0.98 B 1.10 A 2.43 B 2.08 A 1.79 B 1.88 97% 99% 100% 100% 99% 100% 99% 98% 100% 98% 97% 98% 99% 95% 98% 98% 2.51 2.14 2.50 2.18 1.27 6.99 2.18 1.87 Cell Growth index* (%) Cell Growth Inhibition5 (%) 100% - 85% 99% 88% 51% 40% 87% 87% 15% 1% 14% 49% 80% 13% 33% *' rC'MHOnrcrtellKleUwHeMifiOtrlneautietdMIinlntdhue iaabtMsemncneknofiaiHn &exvongMeunuouusiasfonuiirncwe nofnmuettaalbwoillilcf activation for 20 hours at 371C, 2 Viability determined by trypan blue dye exclusion. ' Viable ceNsfflaskB coN countx % viable celto, reported as mean of Flasks A and B. 4 Growth Index " (mean cells per flask treated group/mean celts per flask control group), expressed as a percentage, 8 Cell growth Inhibition 100% - % call growth Index; not calculated for negative controls. Visible precipitates observed. BioReliance Study No. AA26XP.331.BTL -24- Company Sanitized. Does not contain TSCA CB! H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 TABLE 9 CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH H-24335 IN THE ABSENCE OF EXOGENOUS S9 METABOLIC ACTIVATION 20 HOUR CONTINUOUS TREATMENT Treatment1-' (va/mL) Flask MKotfe Index' (%) Cafe Soared %AbemmtCete* Numerical Structural Water A 10.S 100 0 0 B 9.6 100 0 0 H-24335 625 A 7.4 100 1 0 e 6.2 100 0 0 1250 A 5.8 100 2 0 B 5.2 100 0 0 2500 A 4.0 100 2 0 B 3.6 100 2 0 3500 A 0.2 ,, -- -- B 0.4 - - - 5000 A 0.0 B 0.0 - - - MMC, 0.1 A 11.6 100 2 30 B 10.2 100 1 27 Total INumber of StructhaalAbomithms Gaps Chronnatid* Br Ex ChlVMMtome' Br Oto Rtag Severely Damaged Cete* 0 0 0 00 0 0 0 0 0 00 0 0 0 0 0 00 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 00 0 0 0 0 0 00 0 0 ,, ,, -- - - ^ - - - - ,, w ^ ^ -- - - " - - - - 4 27 18 53 2 0 0 14 16 42 2 0 Average Aberrations Per Call7 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 ,, - - 0.550 0.380 ^ ' ' CHO celb were treated for 20 hours at 371C In the absence of an exogenous source of metabolic activation. 2 Mttotte Index" number mitotteflgunnx 100/500 calls counted. 1 Numerical: Includes polyploidand endoredupflcatad cells.; Structural: excludes calls with only gaps. 4 Chromatid breaks Include diromafid and isochnxnatid breaks and fragments; ohromadd exchange figures (Excri) Include quadrlradtals, Mradiate and complex (earmngements. ' Chromosome breaks Include breaks and acentric fragments; die, dicentric chromosome. 9 Severely damaged cetts Includes cells with one or more pulverized chromosomes and colls with 10 or more aberrations. 7 Severely damaged oeRs and pulverizations were counted as 10 aberrations. ' Dose levels 3500 and 5000 ugrmL were not analyzed for chromosome aberrations due to excessive toxicity. BioReliance Study No. AA26XP.331 .BTL Company Sanitized. Does not contain TSCA CBI - 25 - H-24335: AlFfliro MlunmalianCturoinosome Aberration Test in CtlineseHamsiha-<Ovary (CiHO)CelIs DuPont-387C TAtSLE10 SUMMARY:CYTO(^ iEFCTICANWLYSIS OFCHO CELLS TREATEDWITHH-2i4335 Treatment* (VBArt.) Water H.24335 628 1250 2500 MMC. 0.2 39 Activation Treatment Time Mean MItofic Calls Index Scoped Abarrafions PerCelP (Mean +/- SD) Cans With/iibwrattonB* Numerical Structural (%) (%) - 4 11.2 200 0.005 0.071 2.5 0.5 4 5.9 200 0.010 0.100 3.5 1.0 4 5.0 200 0.015 0.122 2.0 1.5 4 4.2 200 0.030 0.171 3.5 3.0 4 10.1 200 0.295 10.722 3.5 18.5** Water * 4 11.6 200 0.025 *0.157 3.5 2.5 H-24335 625 1250 + 4 6.7 200 0.065 0.267 2.0 5.5 + 4 6.1 200 0.060 0.258 3.5 5.5 2500 + 4 4.5 200 0.060 0.238 2.5 6.0 CP.10 + 4 7.9 200 0.415 0.785 1.5 26.5** Water - H-24335 625 1250 2500 - 35004 50004 MMC. 0.1 - 20 10.1 200 0.000 0.000 0.0 20 7.8 200 0.000 AO.OOO 0.5 20 5.5 200 0.000 0.000 1.0 20 ' 3.8 200 0.000 0.000 2.0 20 0.3 20 0.0 20 10.9 200 0.465 10.924 1.5 0.0 0.0 0.0 0.0 28.5** 1 CeKs from all treatnwit conditions were harvested at 20 hours alter the Initiation of the treatments. 2 Severely damaged cells ware counted as 10 berrationa. 1 *, p^).D5;", iKO.01; Flahaft exact teat, 4 Doae level not evaluated for clastogenicity due to excessive toxicity. BioReliance Study No. AA26XP.331.BTL -26- Company Sanitized. Does not contain TSCA CB3 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Celb APPENDIX A Historical Control Data DuPont-3870 BioReliance Study No.: AA26XP.331.BTL -27- Company Sanitized. Does noi contain TSCA CB1 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST USINO 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 Control' 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. 2 Positive control for non-activated studies, N-methyl-N'-nitro-N-nittosoguanidine (MNNG, 0.75-2 ug/ml), and Mitomycin C (MMC, 0.08-0.15 pg/ml). 3 Positive control for S9-activated studies, cyclophosphamide (CP, 10-50 ug/nu), and benzo(a)pyrene, (B[a]P, 30 pg/ml). Company Sanitized. Does noi contain TSCA CB1 BioReliance Study No.: AA26XP.331.BTL -2g- H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST 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 3.2 1.9 0.0 to 9.5 Historical Values Mean Standard Deviation Range S9-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 2.2 0.0 to 10.5 Solvents include water, saline, dimethyl sulfoxide, ethanol, acetone, and other nonstandard solvents and Sponsor-supplied vehicles. Positive control for non-activated studies, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.75-2 |Ag/ml),and Mitomycin C (MMC, 0.08-0.15 ug/ml). Positive control for S9-activated studies, cyclophosphamide (CP, 10-50 ^ig/ml), and benzo(a)pyrene (B[a]P, 30 ug/ml). BioReliance Study No.: AA26XP.331.BTL .29- Company Sanitized. Does no? contain TSCA CBf H-24335; In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells____ _______________DuPont-3870 APPENDIX B Study Protocol BioReliance Study No.: AA26XP.331.BTL - 30 - Company Sanitized. Does not contain T8CA CBS H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 0P(l-J*70 BlolWiMctSwiyNiH.ttn AAKXMMJ'n. 1.0 PURPOSE Vitro MmimaHm Chromosome Aberration Tut in Chinese Hanuter Ovary (CHO) Cdb The purpose of this study is to evaluate the clastogenic potential of a test substance based upon its ability to induce chromosome aberrations in Chinese hamster ovaiy (CHO) cells. 2.0 SPONSOR 2.1 Name: 2.2 Address: E.I. du Pant de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine P.O. Box 50, Elkton Road Newaric.DE19714.0050 2.3 Study Monitor: 2.4 Sp Maria Donner, Ph.D. DuPont-3870 Service Code^H | 3,0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance lUBHBl 3.2 Test Substance Name to be used in the Report: H-24335 3.3 Controls: SolventPositive: Water Mitomycin C (MMC) Cyclophosphamidc(CP) 3.4 Determination of Strength, Purity, etc. Unless alternate arrangements are made, the testing facility at BioRcliance will not perform analysisof Ac dosing solutions. The Sponsor will be directly responsible for determination and documentation of the analytical purity and compositionof the test substance, and the stability and strength of the test substance in the solvent (or vehicle). Protocol No. SPCT331 February 23,2000 I of 9 BioReliance Study No.: AA26XP.331BTL -31- 6 BlORELIANCE" Company Sanitized. Does not contain T9C.' H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 ovfwit-svn BlilRtilunSartyNMtxr; AAlfXTJMJTL 3.5 Test Substance Retention Sample The retentionof a reserve sampleof the test substance will be the responsibilityof the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility BioReliance 4.2 Address: 4.3 Study Director 9630 Medical Center Drive Rockville.MD 20850 Raanadevi Gudi, Ph.D, Phone: (301)610-2169 Fax; (301)738-2362 E-mail: rgudi@bioreliance.com 5.0 TEST SCHEDULE 5.1 ProposedExperimentalStart Date: 2/29/2000 52 Proposed Experimental Termination Date: 3/31/2000 5.3 Proposed Report Date: 4/14/2000 6.0 TEST SYSTEM The CHO-KI cell line is a proline auxotroph with a modal chromosome number of 20 and a population doublingtime of 10-14 hours. CHO-K| cells were obtained from me American Type Culture Collection (repositorynumber CCL 61), Manassas, VA. The stability of the modal chromosome number of me cell line is routinely checked and me cell line is routinely tested and determined to be fiee from mycoplasma contamination. This system has been demonstrated to be sensitive to the 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 treating cultures of CHO cells to a minimum of four concentradonsofthe test article as well as to positive and solvent controls. DuPont Studie^----^2K|pBbgewiil conducted simultaneously and thus the same solvent and positive controls will be used between these two studies. In me non-activated test system, treatment will be for 4 hours and for 20 hours; in me S9 activated test system, treatment will be for 4 hours (Swierenga et Protocol No. SPGTOl Febnrry 23,2000 BioReliance Study No.: AA26XP.331.BTL 2 of 9 -32- l l~y BIORELIANCE" FormrlrMlcrob<BlD|iccllllAAtoc<al Company Sanitized. Does not contain TSCA CB1 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells___________________DuPont-3870 WastWW BloRtUllKtStMlyNMBbtt; AAKXPJIUTL al., 1991). To ensure evaluation of fast division metaphasecells die dividing cells will be anested in metaphaseand harvested for microscopicevaluation of chromosome abeoations at approximately .20 hours (1.5 normal cell cycles) after the initiation of treatment (Galloway et al., 1994). The clastogenicpotentialof die test article will be measured by its ability to induce structural chromosome aberrations in & dose-responsive manner when compared to the solvent, control group. In the event of a positive response in die 4 hour non-activated study, die 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 Determination Water will be used as the test article solvent based on the solubility data available with uie Sponsor. 73, PrelinunaryToMcity Test for Selection ofDose Levels Selection of the dose levels for the cytogeneticsassay will be done in consultation with die Sponsor, and added to the protocol by an amendment based upon post- treatinent toricity (cell growth inhibition relative to the solvent control) and solubilityof the test substance. CHO cells will be treated to solvent alone and to at least nine concentrations of test substance. The highest concentration tested will be 5 ing/ml or 10 mM whichever is lower for freely soluble test substances, or die maximum concentration resulting in a workable suspensionfor poorly soluble test substances not to exceed 5 mg/ml. The pH will be measured at die highest test substance treatment condition and will be adjusted, if necessary, in order to maintain a neutral pH to the treatment medium. The osmolality of the highest dose level, lowest precipitatingdose level (where applicable)and the 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 hows in die absence of S9. Just prior to trypsiniaanon the cell cultures will be visually inspected for the extent of monolaycr confluency relative to me solvent control. Twenty hours after treatment initiation the cells will be harvested by trypsinizationand counted using an automatic cell counter and the cell viability will be assessed using trypan bloc dye exclusion (SOP The cell counts ^l--iq and percent viability wwUgll be used tjgo determine cell growth inhibition relative to the ^iiii--qy solvent control (SOP Whenever possible, die high dose to evaluate chromosome aberrations will be selected to give at least 50% cytotoxicity observed as (cell growth inhibition relative to me 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 appropriatesolvent to be toxic, then die highest dose to be tested in die chromosome aberration assay will be the concentration resulting in minimum precipitation in test medium. Precipitation will be determined Protocol No. SPGT331 Fcbnrry 23,2000 3 of 9 BioReliance Study No: AA26XP.331BTE"33"""" 19 BlORELIANCE- Sc^. r^. r- ^P ForinrlyMicroblolo|lcjl Aiiaciit Company Sanitized. Does not contain TSCA CBfr H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 sufwmn WUBMWSIlrijrNMktf: AA2iXr.i31.Brn. by direct visual inspection. In the cvenFshe test substance demonstrates a doseresponsive increase in toxicity at concemrations that exceed solubility in treatment medium, then the highestdose to be tested will be the maximum concentration that results in at least 50% toxichy. to me event that neither cytotoxicitynor insolubility is observed in the preliminary test, toe highestdose in die chromosome aberration assay will be 5 mg/ml or 10 mM whichever is lower. If excessive precipitation of the test substance-solvent solution occurs upon addition to treatment medium, or if me osmolality of die treatment medium is considered excessive, the Sponsor will be consulted. 7.3 Frequency and Route of Administration Target cells will be treated for 4 hours in the absence and presence of S9, and for 20 hoursinthcabsenceofS9, by incorporation of da test substance-solvent mixture into die treatment medium. This techniquehas been demonstrated to be an effective method of detection of chemical clastogensin this test system (Evans, 1976). No repeats ofme chromosome aberration tests will be done. 7.4 Activation System Aroclor 1254-induced rat liver S9 will be used as me metabolic activation system. The S9 will be 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 will be batch prepared and stored frozen at approximately-PO'C until used. Each batch preparation of S9 will be assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethylbcn2(o)anthraccne to forms mutagenic to Salmonella lypfiimurium TA100. Immediately prior to use, the S9 will be thawed and mixed with cofactors to contain 2 mM magnesium chloride (MgCl,,) 6 mM potassium chloride (KC1), ImM glucosc-6-phosphatc,1 mM mcotinamide adenine dinucleodde phosphate(NADP) and 20 pi S9 per ml seruin free medium. 7.5 Controls 7.5.1 Solvent (or Vehicle) Control The solvent for the 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 p~-..^, ^^ BioReliance Study No.: AA26XP.331.BTL ..,, .34- A. BIORELIANCF Company Sanitized. Does noi contain TSCA CB1 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 wfiwwa BMtdluceSlrirNilbCR AAKXPJIIJTL as tile positive control in the non-activated study. Cyclophosphamidwe ill be used at a wacecSzaSom within 10-50 Hg/ml as the positive control in the S9-activated study. 7.6 Preparationof Target Cells Exponentially growing CHO-K, cells will be seeded in complete medium (McCo/s 5A medium containing 10% fetal bovine senan, 2 mM L-ghttamine, 100 units penicillin/ml and 100 ug streptomycin/ml) for each treatment condition at approximately 5 x 10' cells/25 cm' flask. The flasks will be incubated at 37 1C in a humidified atmosphereof5l% CO; in air for 16-24 hours. 7.7 Identification of Test System Using a permanent marking pen, the treatment flasks will be identified by the BioReliance study number and a code system to designateme treatment condition and test phase. 7.8 Treatment of Target Cells Treatment will be carried out in duplicate by refeeding the flasks with 5 ml complete medium for me non-activated treatment or 5 ml S9 reaction mixture for the S9-activated treatment, to which will be added 50 u! of dosingsolution 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 me solvent In the non-activated study, the cells will be treated for 4 hours and for 20 hours; in the S9-activated stiuly the cells will be treated for 4 hours. Treatment wiU be carried outat37lCmahuniidiHedatmosphercof5 l%CO;inair. After the 4 hour 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 approximately20 hours after initiation of treatment. This post-treatment harvest tune represents approximately1.5 normal cell cycles and was selected to ensure mat the cells are analyzed in toe first division metaphase after initiation of treatment Two hours prior to cell harvest, Colccmid* will be added to the cultures at a final concentration of 0.1 us/mi. Cells will be harvested by nypsuuzation, collected by centrifagation and an aliqujt 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 KC1, washed with two consecutive changes of fixative (memanokglacial acetic acid, 3:1 v/v), Protocol No. SPGT331 February 23,2000 BioReliance Study No.: AA26XP.331.BTL S of 9 -35- fit BlORELIANCE- 'y Fofmrrlr MfrrobiolBfcl A*otijtii Company Sanitized. Does no! contain TSCA CB2 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 F 8.0 ] Wtwawn MdMImeSOriyNKKtmt AAKXT.MI.BTL cappedand stored overnight or longer at approximately2-8C, 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 centrifiigadonand resuspendedin fresh fixative. The suspensionof fixed cells will be appliedto glassmicroscopeslides and air-dried. The slides will be identified by the experiment number, treatment condition and date. The slides will be stained with Giemsa and permanentlymounted. 7.10 Scoringfor Metaphasc Aberrations To ensure that a sufficient number of metaphase cells are present on tne slides, the percentage of cells in mitosis per 500 cells scored (mitotie index) will be detennined and recorded for each coded treatment group selected tor scoring chromosome aberrations. Slides win be coded using random numbers by an individual not involved with the scoring process. Enthe event of a positive response in the 4 hour non-activated study, the prolongedtreatment non-activated study may not be scored. Metaphase cells with 202 cennomeres will be examined under oil immersion without prior knowledge of treatment groups. Whenever possible,a minimum of 200 metaphase spreads Horn each dose level (100 per duplicate flask) will be examined and scored tor chromatid-lypc and chromosome-type aberrations (Scott et al,, 1990). The number of metaphase spreadsthat will be 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. Chroniarid-type aberrations include chromadd and isocbromadd breaks and exchange figures such as qusdriradtels(symmetrical and asymmetrical interchanges), triiadials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromadd or acentric) observed in the absence of any exchange figure will be scored as a break (chromatid or chromosome). Fragments observed with an exchange figure will not be scored as an aberration but will be considered part of die incomplete exchange. Pulverized chromosomes), pulverized cells and severely damaged cells (2 10 aberrations) will also be recorded. Chromadd and isochromatid gaps will be recorded but not included in the analysis. The XY coordinates for each cell with a structural aberration will be recorded using a calibrated microscope stage. The percent polyploidand endoreduplicatedcells will be evaluated per 100 cells for each dose level analyzedisa structural aberrations. CRITERIA FOR DETERMINATION OF A VAUD TEST 8.1 Solvent Control The frequencyof cells with structural chromosome aberrations in the solvent control must be within me range of the historical solvent control and not exceed 6%. 8-2 Positive Control Protocol No. SPGT331 Ftbniiry 23,2000 BioReUance Study No.: AA26XP.33 l.BTL 6 Of 9 -36- fit BlORELIANCE- ^? FlfMilyMictobtDlOBttllA*wa'> Company Sanitized. Does not contain TSCA CB1 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells_____________________DuPont-3870 & i) Sufwawn NoBtillMeStwIyNuebffi AAMXMIi.BTL The percentage of cells with aberrations must be statistically increased (pSO.05, Fisher's exact test) relative to die solvent control. 9.0 EVALUATION OF TEST RESULTS The cytotoxic effects of the treatments are based upon cell growth inhibition relative to me solvent control and will be presentedfor the toricity and aberration studies. The number and types of aberrations found, we percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean abcn-ations per cell will be calculated and reported for each treatment group. Chromatid and isochromarid gaps are presentedin 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 analysisof the percentage of aberrant cells will be performedusing the Fisher's exact test The Fisher's test will be used to compare pairwise the percent aberrant cells of each treaanent group with that of the solvent control. In die event of a positive Fisher's exact test at any test substance dose level, the Cochran-Annitage test will be used to measure dose-responsiveness. All conclusions will be based oa sound scientific basis; however, as a guide to interpretation of me data, the tost 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 (pSQ.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 BioRcliance and will accurately describe all methods used for generation and analysis of the data, Results presentedwill include, but not be limited to: Test substance; identification and CAS no., if known; physical nature and purity, if known; physicochcmicalproperties relevant to the conduct of the study, if known; stability of test substance, if known. Solvent/Vehicle: justification for choice of vehicle; solubility and stability of test substance in solvent/vehicle, ifknown. Source of cells, karyotype features (modal chromosome number} and suitabilityof the cell type used, absence ofmycoplasma, cell cycle length,passage number. Test conditions; compositionof 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 activation (S9) system; duration of treatment; duration of treatment with and concentration Protocol No. SPGT33I February 23,2000 7or9 BioReliance Study No.: AA26XP.331 .BTL - 37 - fi| BlORELIANCE- ^y foimvitf MicfObiolOticil Aitflciji^ Company Sanitized. Doas no? contain TSCA CBS H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 I ihipwM-aw BitRritoBCtSttdyN.iatw WSaSStSTL 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 fin- consideringstudies positive,negative. Results: descriptionof precipitation;pH and osmolality of the treatment medium; cell growth inhibition relative to the solvent control; mitoric index and number ofaietaphascs analyzed; type and number of aberration (structural and numerical) given separately for each treated and control culture; concentration-responserelationship; statistical analysis; historical control data. 11.0 RECORDS AND ARCHIVES All raw data, me pa Operating Procedure) BioRcliancc, 14920 B reports will be maintained according to Standard [bythe BioReliance RAQA unit headquartered at: Rockville.MD 20850. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This Non-GLP study will be performedusingthe Good Laboratory Practice Regulations for Noncliaical Laboratory Studies as a generalguideline. 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 Harmonization of Technical Requirements for Registration ofPhannaceuticals for Human Use (1996 and 1997). Wll this study be submitted to a regulatory ageacy? NO If so, to which agency or agencies? NA Unless an-angements are made to the contrary, unused dosingsolutions will be disposedof following administration to the test system and all residual tost substance will be disposed of following finalization of me report. 13.0 REFERENCES Evans, HJ. (1976) Cytological methods for detecting chemical mutagens, in: A. Hollaender (Ed.), Chemical Mutagens, Principlesand Methods for their Detection, vol. 4. Plenum Press, New York, NY. Galloway, S.M., M.J. Aardema, M. Ishidate Jr., J.L, Ivett, D.J. Kiridand, T. Morita, P. Moscsso and T. Sofilm (1994) Report from working group on in vitro tests for chromosomal aberrations, Mutation Research 312(3):241-261. International Conference on Harmonisarion (ICH) of Technical Requirements for Protocol No. SPGT331 Februwy 23,2000 BioReliance Study No.: AA26XP.331 .BTL 8 of 9 -38- ^ SpMUAKSF Company Sanitized.Doesnot'conlaTiSnCA CBl H-24335; In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovaiy (CHO) Cells 2-2-00| e : 9AM:OuPonT PnTffi ;302 <5i a27 DuPont-3870 BifMMRW fa KrtMi MXP TL Regutntion of PIttroueeotialc for Humas Use. Guidfoce oa Specific Afpects of Regulffixy Geaotosidty Tots for Phamuceuticais. S2A document recommended for adoption it rtep 4 of the ICHproccts on July 19.1995. FedecatRegifisr 61:18198-18202, April 24,1996. btenutioiial Confisreace oa HaononiMticm (KH) of Tecfaaical Beqaiiemeate fir Regisotioit of Pinmucea&ab for fioinaa Ute. GeBaUiwaXy, A Studttd Bittoy for OcBotoxicity Testinf ofPhinmcfflitiolg. S2B docoffleut lecomiocaded for idoptloo at step 4 of the ICH proccw on July 16,1997. Federal Register 62:16026-16030, November 21. 1997. OECD Onideime for the Testing of Cheaiictds, Guideline 473 (In Vitro Msmnuliaa Chromosome Atxaration Test), February 1998. I^ttoa.iU.,W.Au.MABeodK,J.G.BIwen,A.V.C^^lK,JAHBd(^AJ^.McFce. S. Wblffmd ?.S. Wassom (1981) Mammalies ia vivo and in vitro cytogecetic ussys: a report of the Gene-ToxProgmn, Mutation Research, 87:143-188. Scott, D., N,D. Danford, BJ. Dem aad OJ. Kufciand. 1990. Metqduie ChromiwoiDe Abenation Assays &> Vitro, bu Basic Mutagenicity Teat*: UKEMS Recommeoded Procedures. DJKiridaBd(ed).CatabridgcUniveisityPBest,NewYoric,NY. Swierenga SJLH., JA. Heddle, E.A. Sigal, JP.W. Gilsnan, fLL. BriIIiagef, GJt Douglas and EJ^Netmann(!99l)Reconiineodi protocols based on urv(y of curreatpncdee in geaotoxicity teing tobomories, IV. Chromosome aberration and siater-ehromatid exchange io Chinese hamster ovary, V79 Chinese lung and bumao lymphocytecultum, Motttioa Researcb 246:301.321 14.0 APPROVAL H^&^j.^ '^-a STUDY MONITOR 2'to/oo DATE Maria poBner.Ph.D. (Print or Type Name) ^a^a^'/^^' BIORELIANCB STUDY DIRECTOR P^/W^00 DATE BIOREUANCE STUDY MANAGEMENT DATE Pmtoco(No.SPGT331 FtbroBry 23,2000 BioReliance Study No.: AA26XP.331 .BTL 9of9 -39- ^ &OREL!ANCF Company Sanitized. Does not contain TSCA Cffl