Document 5kzgQaq7jr3dy56oE09zeqgVJ

AR226-3148 D uPont-3870 TRADE SECRET Study Title H-24335: In Vitro M am m alian Chrom osom e A berration Test in C hinese H am ster O vary (CH O ) Cells A uthors Ramadevi Gudi, Ph.D. Elizabeth H. Schadly, B.S. May 4, 2000 Performing Laboratory B io R elian ce 9630 M edical Center Drive Rockville, MD 20850 for E, I. du Pont 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 A A 26X P .331.BTL DuPont Project ID D uP ont-3870 Work Request Number Company Sanitized. Does not contain TSCA CBI Page ! of 39 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 CERTIFICATION W e, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. BioReliance Study Director: Ramadevi Gudi, PhD . Approved by Study Monitor: k M aria Donner, P hD . Senior Research Scientist S 01(00 Date Company Sanitized. Does 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 C ertification.......... Study Inform ation S um m ary. Purpose................... Characterization o f Test and Control Substances.............. M aterials and .................... Results and ^Discussion ......................... ...................... *** 8 8 .a 1* 3 C onclusion............. . R eferen ces.............. D ata T ables............... r.... > 16 ................., 1 7 Table1: Prelim inary Toxicity Assay w ith H-24335 in CHO Cells in flu; Absence o f Exogenous M etabolic A ctivation. 4 Hour Treatm ent..........................................................17 Table 2: Prelim inary Toxicity Assay w ith H-24335 in CHO Cells in the Presence o f Exogenous Mietabohc A ctivation. 4 Hour Treatm ent 18 Table 3: Prelim inary Toxicity Assay w ith H-24335 in CHO C ells in the Absence o f Exogenous M etabolic A ctivation. 20 Hour Treatm ent ***a********************19 Table 4: Concurrent Toxicity A ssay w ith H-24335 in CHO Cells in the Absence o f Exogenous M etabolic A ctivation. 4 Hour Treatm ent........................................................... 20 Table 5: Cytogenetic Analysis o f CHO Cells Treated w ith H-24335 in the Absence o f Exogenous S9 M etabolic A ctivation. 4Hour Treatm ent, 16 Hour Recovery Period.........21 Table 6: Concurrent Toxicity Assay w ith H-24335 in CHO Cells in the Presence o f Exogenous M etabolic A ctivation. 4 Hour Treatm ent........................................................... 22 Company Sanitized. Does not contain TSCA CB1 BioReliance Study No. AA26XP.331.BTL -3 - H-24335: In Vitro M ammalian Chromosome A berration Test in Chinese Hamster Ovary (CHO) Cells D uPont-3870 Table 7: Cytogenetic Analysis o f CHO Ceils Treated w ith H-24335 in the Presence o f Exogenous S9 M etabolic Activation. 4Hour Treatm ent, 16 Hour Recovery Period.........23 Table 8: Concurrent Toxicity Assay w ith H-24335 in CHO C ells in the Absence o f Exogenous M etabolic Activation. 20 Hour Treatm ent.............................................. .......... 24 Table 9: Cytogenetic Analysis o f CHO C ells Treated w ith H-24335 in the Absence o f Exogenous S9 M etabolic A ctivation. 4Hour Treatm ent, 16 H our Recovery Period.........25 Table 10: Summary: Cytogenetic Analysis o f CHO C ells Treated w ith H-24335............26 Appendix A : H istorical Control D ata.........................................................................................27 Appendix B: Study Protocol.................................................................................. ......................30 BioReliance Study N o. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CB1 4 - H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells STUDY INFORM ATION Substance Tested: \ Haskell Number: 24335 DuPont-3870 Stability: The test substance appeared to be stable under the conditions o f the study; no evidence o f instability was observed. Solubility: Aquatics: Soluble but cloudy in water at concentrations of >15 mg/mL. Sponsor: E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine P.O. Box 50, Elkton Road Newark, DE 19714-0050 Study Initiated/Com pleted: February 25,2000 / (see report cover page) In-Life Initiated/Completed: February 29, 2000 / M arch 29,2000 BioReliance Study No. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CB: -5 - H-24335: In Vitro M ammalian Chromosome Aberration T est in Chinese H am ster Ovary (CHO) Cells D uPont-3870 SUMMARY H ie test substance, H-24335, was tested in the in vitro mammalian chmmnsnny. aberration test using Chinese hamster ovary (CHO) cells in both the absence and presence o f an Aroclor-mduced exogenous S9 metabolic activation system. A prelim inary toxicity assay was performed to establish the dose range for the chromosome aberration assay. The nhmwmsnmi aberration assay w as used to evaluate the clastogenic potential o fthe test substance. W ater w as determined to be the solvent o f choice based on inform ation provided by the Sponsor, the solubility o f the test substance, and compatibility with the target cells. The test substance w as soluble but cloudy in water at a concentration o f 50 mg/mL, the mawnwim concentration tested. In the prelim inary toxicity assay, the maximum dose level tested was 5000 pg/mL. Visible precipitates were observed in the treatm ent medium at dose levels >1500 pg/mL. Dose levels <500 pg/mL were soluble in the treatm ent medium. Selection o f dose levels for the chromosome aberration assay was based on total cell growth inhibition relative to the solvent control. No substantial toxicity, i.e., at least 50% cell growth inhibition, w as observed at any dose level tested in neither the non-activated nor the S9 activated 4 hour treatm ent groups. Substantial toxicity was observed at Ore dose level 5000 pg/mL in the non-activated 20 hour continuous treatm ent group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 625 to 5000 pg/mL for the non-activated and the S9 activated exposure groups. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non activated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatm ent initiation. Visible precipitates were observed in the treatm ent medium at the dose levels >2500 pg/mL. The dose levels 1250 pg/mL were soluble in treatment medium. No substantial toxicity Q>50% oe il growth inhibition) was observed at the highest dose level evaluated for chromosome aberrations, 2500 pg/mL, in the non-activated and S9 activated exposure groups. Substantial toxicity (>50% ceil growth inhibition) was observed in the non-activated 20 hour exposure study at the dose levels 3500 and 5000 pg/mL. Excessive m itotic inhibition was also observed at these dose levels. Therefore, for the non activated and S9 activated 4 hour exposure groups, in the absence o f at least 50% cell growth inhibition, dose selection was based upon test substance precipitation in the treatm ent 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 w ith at least a 50% reduction in m itotic index, relative to the solvent control. Initially, the non-activated and S9 activated 4 hour treatm ent groups were scored for structural and numerical chromosome aberrations. N o statistically significant increases in structural and numerical chromosome aberrations w ere observed in the non-activated or S9 activated 4 hour treatm ent groups relative to the solvent control group, regardless o f dose level (p>0.05, Fisher's exact test). In the Company Sanitized. Does not contain TSCA CB BioReliance Study No. AA26XP.331.BTL -6 - H-24335: In Vitro M ammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells D uPont-3870 nfrrenre o f a positive response in the non-activated 4 hour treatm ent group, die non-activated 20 hour continuous treatm ent group was evaluated for structural and numerical chromosome aberrations. N o statistically significant inmeases in structural and numerical chromosome aberrations w ere observed in the non-activated 20 hour continuous treatm ent group relative to the solvent control group, regardless o f dose level (jji>0.05, Fisher's exact test). Under the conditions described in this report, H-24335 was concluded to be negative for the induction o f structural and num erical chromosome aberrations in the non-activated and S9 activated in vitro mammalian chromosome aberration test using Chinese hamster ovary (CHO) cells. BioReliance Study N o. AA26XP.331.BTL Company Sanitized. Does not contain TSCA Ci: -7 - H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Celia DuPont-3870 PURPOSE The purpose o f this study was to evaluate the clastogenic potential o f the test gnhgfeinf* based upon its ability to induce in vitro chromosome aberrations in Chinese ham ster ovary (CHO) cells. 3 CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES The test substance, H-24335, was received by BioReliance on 15 February 20(H) and was E^jSjg2*jj^b^jod^M im be^A A 2ffiQ ^The test substance was characterized by the Sponsor as an H H B H H H H b a t should be stored in a well ventilated place at temperatures below 120F. An expiration date for the test substance was not provided. Upon receipt, the test substance was described as at room temperature in a well-ventilated area, closed container. exposure to 1was stored in a tightly 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. M itomycin C (MMC; CAS No.: 50-07-7), was obtained from the Sigma Chemical Company, and was dissolved and diluted in sterile distilled w ater to stock concentrations o f 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 from Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations o f 100 and 200 jig/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 tire test gnhstann was used as the solvent control at the same concentration as that found in the test substance-treated groups. T est System MATERIALS AND METHODS Chinese ham ster ovary (CHO-K,) cells (repository num ber CCL 61) were obtained from American Type Culture Collection, Manassas, VA, on M ay 29, 1997. In order to assure the karyotypic stability o f 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 w o used for the chromosome aberration assay. The freeze lot o f cells was tested using the Hoechst staining procedure and found to be free o f mycoplasma contamination. This cell line has an average cell cycle tim e o f 10-14 hours with a m odal chromosome rwimbw o f 20. The use o f CHO cells has been demonstrated to be an effective method o f detection o f chemical BioReliance Study No. AA26XP.33l.BTL Company Sanitized. Does not contain TSCA CBI -8 - H-24335: In Vitro M ammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells DuPont-3870 clastogens (Preston et aL, 1981). M etabolic A ctivation System Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from m ale Sprague-Dawley rats induced with a single intraperitoneal injection o f Aroclor 1254,500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at -70C until used. Each bulk preparation o f S9 was assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100. Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 pL S9 per m illiliter medium (McCoy's 5A serum-free medium supplemented with, 100 units penicillin and 100 fig streptomycin/mL, and 2 mM L-glutamine). Preliminary Toxicity Assay The prelim inary toxicity assay was performed for the purpose o f selecting dose levels for the chromosome aberration assay and consisted o f an evaluation o f test substance effect on cell growth. CHO cells were seeded for each treatm ent condition at approxim ately 5 x 10s cells/25 cm2 flask and were incubated at 371C in a hum idified atmosphere o f 51% C 0 2 in air for 16-24 hours. Treatment was carried out by refeeding the flasks w ith 4.5 mL complete medium (McCoy's 5A medium supplemented w ith 10% fetal bovine serum (FBS), 100 units penicillin and 100 pg streptomycin/mL, and 2 mM L-glutamine) for the non-activated study or S9 reaction m ixture (3.5 mL serum-free medium plus 1 mL o f 5X S9 m ix) for the activated study, to which 500 pL dosing solution o f test substance in solvent or solvent alone was added. The osmolality o f the highest concentration o f the dosing solution in the treatm ent medium was measured. The pH o f the highest concentration o f the dosing solution in the treatm ent medium was measured using test tape. The cells were treated for 4 hours w ith and without S9, and continuously for 20 hours w ithout S9. A t com pletion o f the 4 hour exposure period, the treatment m edium was removed, the cells washed w ith calcium and magnesium-free phosphate buffered saline (CMF-PBS), refed with 5 mL complete medium and returned to tire incubator for a total tim e period o f 20 hours from the initiation o f the treatm ent A t 20 .hours after the in itia tio n o f the treatment the cells were harvested by trypsinization and counted using a Coulter counter. The presence o f 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. Company Sanitized. Does not contain TSCA CBS BioReliance Study N o. AA26XP.331.BTL -9 - H-24335: In Vitro M ammalian Chromosome Aberration T est 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 o f CHO cells to the test substance as well as positive and solvent controls. For the chromosome aberration assay, CHO cells were seeded a t approximately S x 10s cells/25 cm2 flask and were incubated at 371C in a humidified atmosphere o f 51% C 02 in air for 16-24 hours. Treatment was carried out by refeeding duplicate flasks with 4.5 mL complete m edium (McCoy's 5A medium supplemented w ith 10% FBS, 100 units penicillin and 100 pg streptomyein/mL, mid 2 mM L-glutamine) for the non-activated study or 4.5 mL S9 reaction m ixture for the S9 activated study, to which 500 pL o f dosing solution o f test or control substance in solvent or solvent alone w as added. The osm olality o f foe highest concentration o f foe dosing solution in foe treatm ent medium was measured. The pH o f foe highest concentration o f the dosing solution in the treatm ent medium was measured using test tape. In the non-activated study, foe cells were exposed to the test substance for 4 hours or continuously for 20 hours up to foe cell harvest at 371C in a hum idified atmosphere o f 51% C 02 in air (Swierenga et al., 1991). h i foe 4 hour exposure group, foe treatm ent medium was removed after the exposure period, and foe cells washed w ith CMF-PBS, refed w ith complete medium and returned to the incubator. Two hours prior to foe scheduled cell harvest, Colcemid w as added to duplicate flasks for each treatm ent condition at a final concentration o f 0.1 pg/m L and foe flasks returned to the incubator until cell collection. In foe S9 activated study, foe cells were exposed for 4 hours at 371C in a humidified atmosphere o f 51% C 02 in air (Swierenga et al., 1991). After foe exposure period, the treatm ent m edium was removed, the cells washed w ith CMF-PBS, refed w ith complete medium and returned to tire incubator. Two hours prior to the scheduled cell harvest, Colcemid w as added to duplicate flasks for each treatm ent condition at a final concentration of 0.1 pg/mL and foe flasks were returned to the incubator until cell collection. A concurrent toxicity assay was conducted in both the non-activated and foe S9 activated assay system s. After cell harvest an aliquot o f foe cell suspension w as removed from each culture and counted using a Coulter counter. The presence o f test substance precipitate was assessed using foe unaided eye. Cel! viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to foe solvent control. Ceil H arvest Two hours after the addition o f Colcemid, metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinization. Cells were collected approximately 20 hours after initiation o f treatm ent (Galloway et al., 1994). The cells were collected by ' o m p a n y Sanitized. Does no! contain TSCA CBI BioReliance Study No. AA26XP.331.BTL - 10- H-24335: In Vitro M ammalian Chromosome Aberration Teat in Chinese Hamster Ovary (CHO) Cells D uPont-3870 centrifugation a t approximately 800 rpm for 5 minutes. The ceil 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 with tw o trashes o f approximately 2 mL Camoy's fixative (methanol:glacial acetic add, 3:1, v/v). Tire cells were stored overnight or longer in fixative at approximately 2-8C. Slide Preparation To prepare slides, tire fixed cells were centrifuged at approximately 800 rpm for 5 minutes, tire supernatant was aspirated, and 1 mL fresh fixative was added. After additional centrifugation (at approximately 800 rpm for 5 minutes) tire supernatant fluid was decanted and the ceils resuspended to opalescence in fresh fixative. A sufficient amount o f cell suspension was dropped onto tire center o f a glass slide and allowed to air dry. Slides were identified by tire study num ber, dato prepared mid the treatm ent condition. The dried slides were stained with 5% Giemsa, air dried and permanently mounted. . Evaluation o f M etaphase Cells Slides w ere coded using random numbers by an individual not involved with the scoring process. To ensure that a sufficient number o f metaphase cells were present on tire slides, the percentage o f cells in m itosis per 500 cells scored (mitotic index) was determined for each treatment group. M etaphase cells with 202 centromeres were examined under oil immersion without prior knowledge o f 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 minimum o f 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et a l, 1990). The number o f metaphase spreads that are examined and scored per duplicate flask m ay be reduced if the percentage o f aberrant cells reaches a statistically significant level before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence o f any exchange figure were scored as a break (chromatid or chromosome). Fragments observed w ith an exchange figure were not scored as an aberration but instead were considered part o f the incom plete 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 w ere recorded using a calibrated microscope stage. Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored. BioReliance Study No. AA26XP.331.BTL -11- company Sanitized. Poes not contain T SCA CBS H-24335: In Vitro M ammalian Chromosome Aberration Test in Chinese H am ster Ovary (CHO) Cells____________________________________ DuPont-3870 Controls M itomycin C was used as the positive control in the non-aetivated study at final concentrations o f 0.1 and 0.2 pg/mL. Cyclophosphamide was used as the positive control in the S9 activated study at final concentrations o f 10 and 20 pg/m L, For both positive controls one dose level exhibiting a sufficient number o f scorable metaphase cells was selected for analysis. The solvent vehicle for the test substance was used as the solvent control at the same concentration as that found in the test substance-treated groups. The solvent vehicle for the test substance w as used as the solvent controlayfag same concentration as that found in die test substance-treated groups. DuPont s t u d i e s f l |K m f |^ v e r e conducted simultaneously and thus die same solvent and positive controls were usedhetw een these two studies. Evaluation o f T est Results The toxic effects o f treatm ent were based upon cell growth inhibition rotative to the solvent-treated control and are prerented for die toxicity and aberration gmHip? The numtwr and types o f aberrations found, the percentage o f structurally and numerically damaged cells (percent aberrant cells) in the total population o f cells examined, and tire mean aberrations per cell was calculated and reported for each group. Chromatid and isochromatid gap are presented in the data but are not included in the total percentage o f cells with one or more aberrations o r in the frequency o f structural aberrations per cell. Statistical analysis o f the percent aberrant cells was performed using the Fisher's exact te st Fisher's test w as used to compare pairwise die percent aberrant cells o f each treatment group with that o f the solvent control. In the event o f a positive Fisher's test at any test substance dose level, the Cochran-Armitage test was used to measure dose-responsiveness. All conclusions were based on sound scientific basis; however, as a guide to interpretation o f the date, the test substance was considered to induce a positive response when the percentage o f cells w ith aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p<0.05). Test substances not ffem/wetrating a statistically significant increase in aberrations will be concluded to be negative. Negative results with m etabolic activation may need to be confirmed on a case-by-case basis. In those cases where confirm ation o f negative results is not necessary, justification w ill be provided. Criteria for a V alid Test The frequency o f cells with structural chromosome aberrations in the solvent control m ust be within the range o f the historical solvent control. The percentage o f cells with chromosome aberrations in the positive control must be statistically increased (p<0.Q5, Fisher's exact test) relative to the solvent control. BioReliance Study N o. AA26XP.331.BTL -12- Company Sanitized. Does not contain TSCA CBI H-24335: In Vitro Mammalian Chromosome A benation T est in Chinese Hamster Ovary (CHO) Cells DuPont-3870 D ev iatio n s No known deviations from the protocol or assay method SOPs occurred during the conduct o f this study. Archives All raw data, the protocol, all according to Standard Operating Afifirs/Quality Assurance Unit Rockville, M D, 20850. coded slides will be maintained by the BioReliance Regulatory 14920 Broschart Road, Solubility RESULTS AND DISCUSSION W ater was determined to be the solvent o f choice based on information provided by the Sponsor, the solubility o f the test substance, and com patibility w ith the target cells. The test substance w as soluble but cloudy in water at a concentration o f 50 mg/mL, the maximum concentration tested. Prelim inary Toxicity Assay Dose levels for the chromosome aberration assay were selected follow ing a preliminary toxicity assay and ware based upon a reduction o f total cell growth (cell growth inhibition) relative to the solvent control. The results o f die evaluation o f cell growth inhibition are presented in Tables 1-3. CHO cells were exposed to solvent alone and to nine of test substance ranging from 0.5 pg/mL to 5000 pg/mL in the absence and presence o f an S9 reaction m ixture. Visible precipitate was observed in treatm ent medium at dose levels >1500 pg/mL. Dose levels <500 pg/mL were soluble in treatm ent medium. The osmolality o f the solvent (w ater) in treatm ent medium was 314 mmol/kg, and not considered significantly altered. The osm olality in treatm ent medium o f the highest concentration tested, 5000 pg/mT. was 274 mmol/kg. The pH o f the highest concentration o f test substance in treatment medium was approxim ately 7.0. Cell growth inhibition relative to the solvent control was 17% and 12% at 5000 pg/m L, the highest concentration tested in both die non-activated and S9 activated 4 hour groups, respectively (Table 1 and 2). O il growth inhibition relative to the solvent control was 70% a t 5000 pg/m L, die highest concentration tested in the non-activated 20 hour continuous treatm ent group (Table 3). Based upon the results o f the toxicity study, die dose levels selected for testing in the chromosome aberration assay were as follows: BioReliance Study N o. AA26XP.331.BTL -13 - Company Sanitized. Does not contain TSCA CBI H-24335: In Vitro M ammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 T reatm ent Condition -S9 +S9 T reatm ent Time (hr) 4 Recovery Time (hr) 16 Dose levels (pg/m L ) 625,1250,2500,5000 20 0 6 2 5 ,1 2 5 0 ,2 5 0 0 ,3 5 0 0 ,5 0 0 0 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 pg/mL. Dose levels <1250 pg/mL were soluble in treatment medium. The osm olality o f the solvent (water) in treatm ent medium was 293 mmol/kg. The osmolality in treatm ent medium o f the highest concentration tested, 5000 pg/mL, was 329 mmol/kg, and not considered significantly altered. The pH o f the highest concentration o f test substance in treatm ent 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 pg/mL for 4 hours in the absence o f S9 activation (Table 4). This was the highest dose level evaluated for chromosome aberrations. The ability o f H-24335 to induce chromosome aberrations is presented by treatm ent flask in Table 5, and summarized by treatment group in Table 10. The m itotic index at the highest dose level evaluated for chromosome aberrations, 2500 pg/inL, was 63% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 625,1250, and 2500 pg/mL. In the absence o f at least 50% cell growth inhibition, the highest dose level selected for evaluation was the lowest precipitating dose. The percentage o f cells w ith structural and numerical aberrations in the test substance-treated groups was not significantly increased above that o f the solvent control (p>0.05, Fisher's exact test). The percentage (18.5%) o f structurally damaged cells in the MMC group w as found to be statistically significant Cell grow th inhibition relative to the solvent control in CHO cells was 17% after treatment with H-24335 at 2500 pg/mL for 4 hours in the presence o f S9 activation (Table 6). This was the highest dose level evaluated for chromosome aberrations. The ability o f H-24335 to induce chromosome aberrations is presented by treatm ent flask in Table 7, and summarized by treatm ent group in Table 10. The m itotic index was 61% reduced relative to the solvent control at the highest dose level evaluated for chromosome aberrations, 2500 pg/mL. The dose levels selected for m icroscopic analysis were 625,1250, and 2500 pg/m L. In the absence o f at least 50% cell grow th inhibition, the highest dose level selected for evaluation was the lowest precipitating dose. The percentage o f cells with structural or numerical aberrations in the test substance-treated groups was not statistically increased above that o f the solvent control (p>0.05, Fisher's exact test). The percentage (26.5%) o f structurally damaged cells in the CP Company Sanitized. Does not contain TSCA CBI BioReliance Study N o. AA26XP.331.BTL -14 - H-24335: In Vitro M ammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells____________ _______________________DuPont-3870 group was found to be statistically significant. In the absence o f 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 foe solvent control was 14% at 2500 pg/mL (Table 8), foe highest dose level o f H-24335 evaluated for chromosome aberrations in foe non-activated 20 hour continuous exposure group. The ability o f H-24335 to induce chromosome aberrations is presented by treatm ent flask in Table 9, ami summarized by treatm ent group in Table 10. The m itotic index at foe highest dose level evaluated for chromosome aberrations, 2500 pg/mL, was 62% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 625,1250, and 2500 pg/mL. Due to excessive m itotic inhibition at dose levels w ith >50% cell growth inhibition, foe highest dose level selected for evaluation was the dose level with at least 50% m itotic inhibition. The percentage o f cells with structural o r numerical aberrations in foe test substance-treated groups was not significantly increased above that o f the solvent control (p>0.05, Fisher's exact test). The percentage (28.5% ) o f structurally damaged cells in foe MMC group was found to be statistically significant. The study was concluded to be negative. An independent repeat assay was not required because no unique metabolic requirements were known about foe test substance and because no equivocal responses were observed. CONCLUSION The positive and solvent controls fulfilled foe requirements for a valid te st Under the conditions described in this report, H-24335 w as concluded to be negative for the induction o f structural and num erical chromosome aberrations in foe non-activated and S9 activated in vitro mammalian chromosome aberration test using Chinese hamster ovary (CHO) cells. BioReliance Study N o. AA26XP.331.BTL -15- Company Sanitized. Does not contain TSCA CBI H-24335: In Vitro M ammalian Chromosome Aberratimi T est in Chinese Hamster Ovary (CHO> C ells____________________________________ DuPont-3g70 REFERENCES Evans, H J . (1976) Cytological methods for detecting chemical m utagen^ in; a . Hollaender (Ed ). Chemical Mutagens, Principles and Methods for their Detection, vol 4. Plenum Press, New Y ork Galloway, S.M ., M J. Aardema, M. Ishidate Jr., J.L. Ivett, D.J. Kiridand, T. M onte, P. M osesso and T. Sofuni (1994) Report from working group on in vitro tests for chromosomal aberrations, M utation Research 312(3):241-261. International Conference on Harmonization (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicity: Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 o f the ICH process on July 19, 1995. Federal Register 61:18198-18202, April 24,1996. International Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing o f Pharmaceuticals. S2B document recommended for adoption at step 4 o f the ICH process on July 16, 1997. Federal Register 62:16026-16030, Novem ber 21,1997. , OECD Guideline for die Testing o f Chemicals, Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), adopted, July 1997. Preston, R .J., W. Au, M.A. Bender, J.G. Brewen, A.V. Carrano, J.A. Heddle, A.F. McFee, S. W olff and J.S. Wassom (1981) M ammalian in vivo and in vitro cytogenetic assays: a report o f the Gene-Tox Program, M utation Research, 87:143-188. Scott, D ., N.D . Danford, B.J. Dean and D.J. Kiridand. 1990. M etaphase Chromosome Aberration Assays In Vitro. In: Basic M utagenicity Tests: UKEMS Recommended Procedures. D.J Kiridand (ed). Cambridge University Press, N ew York, NY. Swierenga S.H.H., J.A. Heddle, E.A. Sigal, J.P.W . Gilman, R X . Brillinger, G.R. Douglas and E.R. Nestmann (1991) Recommended protocols based on a survey o f current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sisterchrom atid exchange in Chinese ham ster ovary, V79 Chinese lung and human lymphocyte cultures, M utation Research 246:301-322. BioReliance Study N o. AA26XP.33 l.BTL -16- Company Sanitized. Does not contain TSCA CB1 H-24335: In Vitro M ammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells D uPont-3870 TABLE 1 PRELIMINARYTOXICITYASSAY WITH H-24335 INCHOCELLS IN THE ABSENCE OF EXOGENOUS S9 METABOLICACTIVATION 4 HOURTREATMENT, 16 HOUR RECOVERY PERIOD ill! Treatment12 (pgftnL) Cell Count (x10*) Cell Viability3 <%) Cell Growth Index45 (%> Cell Growth Inhibition* (%) W ater 2.50 99% 2.47 100% -- H-24335 0.5 3.60 99% 3.57 144% -44% 1.5 2.% 100% 2.95 119% -19% 5 2.38 99% 2.35 95% 5% 15 2.79 98% 2.73 110% -10% 50 2.80 98% 2.75 111% -11% 150 2.55 99% 2.52 102% -2% 500 3.02 100% 3.02 122% -22% 1500* 2.51 98% 2.46 100% 0% 5000* 2.12 97% 2.05 83% 17% 1 CHO calls ware treated In the absence of an exogenous source of metabolic activation for 4 hours at 371C. 2 Viability determined by trypan blue dye exclusion. * Viable cells/flask * cell count x % viable cells 4 Growth index * (cells per flask treated group/eells per flask control group), expressed as a percentage. 5 Cell growth Inhibition 100% - % cell growth Index; not calculated for negative controls. ` Visible precipitates observed. BioReliance Study N o. AA26XP.331.BTL -17- Company Sanitized. Does not contain TSCA CBI H-24335: In Vitro M ammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) C ells D uPont-3870 TABLE 2 Prelim inary toxicity a ssa y w ith h-24335 in c h o c ells in THE PRESENCE OF EXOGENOUS S9 METABOLICACTIVATION 4 HOURTREATMENT, 18 HOUR RECOVERY PERIOD Treatment' (pg/mL) Cell Count (xIO8) Cell Viability* (%) Mean C els/ Flask* (X108) C el Growth Index4 (%) Cell Growth Inhibition8 (%) W ater H-24335 0.5 2.51 95% 2.38 100% 2.42 100% 2.42 102% "" 2% 1.5 2.S8 99% 2.66 111% -11% 5 3.06 99% 3.03 127% -27% 15 3.14 98% 3.08 129% 29% 50 2.64 98% 2.59 109% -9% 150 2.76 09% 2.73 115% -15% 500 2.99 97% 2.90 122% -22% 1500* 2.40 98% 2.35 99% 1% 50008 2.13 98% 2.09 88% 12% 1 CHO cells ware treated In the presence of an exogenous source of metabolic activation for 4 hours a t 371"C. 2 Viability determined by trypan blue dye exclusion. 2 Viable cells/flask cell countx % viable cells 4 Growth index (cells per flask treated group/cells per flask control group), expressed as a percentage. 8 C el growth inhibition -100% - % cell growth index; not calculated for negative controls. 8 Visible predpitatas observed. BioReliance StudyNo. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CB! " _^ H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells D uPont-3870 TABLES PRELIMINARYTOXICITYASSAYWfTH H-24335 INCHO CELLS IN THEABSENCE OF EXOGENOUS S9 METABOLICACTIVATION 20 HOUR CONTINUOUSTREATMENT Treatment1 mm Cell Count <x106) Cell Viability2 (%) Celts/ Flask* (x106) Cell Growth Index4 <%) C ei Growth Inhibition* (%) W ater 2.99 98% 2.93 100% __ H-24335 0.5 2.54 98% 2.49 85% 15% 1.5 2.43 96% 2.34 60% 20% 5 2.13 99% 2.16 74% 28% 15 2.25 95% 2.14 73% 27% 50 1.79 94% 1.68 57% 43% 150 2.21 95% 2.10 72% 28% 500 1.77 100% 1.77 60% 40% 1500 1.95 97% 1.89 65% 35% 50004 0.90 98% 0.88 30% 70% 1 CHO cells ware treated continuously in the absence of an exogenous source of metabolic activation for 20 hours at 371C. 2 Viability determined by trypan blue dye exclusion. Viable ceHsfflask cell count x % viable cells 4 Growth index * (cells per flask treated group/cells per flask control group), ' expressed as a percentage. * Cell growth Inhibition = 100% - % cell growth Index; not calculated for negative controls. * Visible precipitates observed. B ioR eliance Study N o. A A 26X P.331.BTL -19 - Company Sanitized. Does not contain TSCA CBI '^ H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells DuPont-3870 TABLE4 CONCURRENTTOXICITYASSAYWITH H-24335 IN CHO CELLS IN THEABSENCE OF EXOGENOUS S9 METABOLICACTIVATION 4 HOURTREATMENT, 16 HOUR RECOVERY PERIOD T reatm ent1 (pg/mL) Cell Count Averages W1Mnw.|lvf 1x10*6) W ater A 2.52 B 3.10 H-24335 625 A 2.05 B 2.31 1250 A 2.03 B 1.90 2500s A 1.88 B 2.54 5000* A 1.86 B 1.51 MMC, 0.1 A 1.95 B 2.05 MMC, 0.2 A 2.07 B 2.03 Cell Viability2 Mean Cells per Flask1 (X ltW ) 98% 98% 2.73 100% 98% 98% 99% 99% 98% 98% 98% 98% 97% 97% 95% 2.16 1.94 2.18 1.66 1.95 1.97 Celt Growth Index* (%) 100% 79% 71% 80% 61% 71% 72% Ceil Growth InHMBon* <%) 21% 29% 20% 39% 29% 28% 1 CHO celts were treated In the absence of an exogenous source of metabolic actuation for 4 hours at 371"C. ' 2 Viability determined by tiypan blue dye exclusion. 1 Viable cellsfflask cell count x % viable cells, reported as mean of Flasks A and B. * Growth Index * (mean ceils per flask treated group/moan cells per flask control group), expressed as a percentage. 9 Cell growth inhibition 100% - % cell growth Index; not calculated for negative controls. Visible precipitates observed. BioReliance Study N o. AA26XP.331.BTL -20- Company Sanitized. Does not contain TSCA CBJ H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) C ells DuPont-3870 TABLE 5 CYTOGENETICANALYSISOF CHO CELLS TREATED WITH H-24335 IN THE ABSENCE OF EXOGENOUS S9 METABOLICACTIVATION 4 HOURTREATMENT, 18 HOUR RECOVERY PERIOD Mitotic % Aberrant Ceils* mowr Cells Treatment'-* rlBSK <%) Scored Numerical Structural (pg/mL) W ater H-24335 625 1250 2500* MMC.0.2 A 11.0 100 B 11.4 100 S^ 8t" A 6.2 B 5.6 A 5.2 B 4.8 100 100 A 4.6 100 B 3.8 100 A 9.6 100 B 10.6 100 2 3 3 4 0 4 4 3 2 5 0 1 1 1 1 2 2 4 16 21 Total Number of Structural Aberrations Gaps Chromatid4 Br Ex Chromosome* Br Dio Ring Severely Dam aged Cells* 10 00 0 0 00 0 01 0 0 0 310 10 0 00 11 0 0 30 01 1 1 1 14 11 0 19 7 00 01 01 00 00 11 40 13 0 i 1 0 0 0 OO 0 0 0 0 0 0 0 0 Average Aberrations Per Cell7 0.000 0.010 0.010 Q.010 0.010 0.020 0.020 0.040 0.290 0.300 ' CHO ceRe were treated for4 hours at 371C In the absence of as exogenous source of metabolic activation. 2 Mitotic Index * number mitotic figures x 100/500 cell counted. * Numerical: includes polyploid and endoredupllcated cells.; Structural: excludes cells with only gaps. 4 Chromatid breaks include chromatid and Isochromatid breaks and fragments; chromatid exchange figures (Exch) include quadriradMs, trlradlals and complex rearrangements. ; * Chromosome breaks Include breaks and acentric fragments; die, dicentric chromosome. 1 Severely damaged cells Includes cells with one or more pulverized chromosomes and cells with 10 or more aberrations. 7 Severely damaged cells and pulverizations were counted as 10 aberrations. * Dose level 5000 pgftnL was not analyzed due to test substance precipitation in Uw medium. * Lowest precipitating dose level. BioReliance Study N o. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CB! -21- H-24335: In Vitro M ammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells D uPont-3870 TABLES CONCURRENTTOXICITYASSAYWITH H-24335 IN THE PRESENCE OF EXOGENOUS S9 METABOLICACTIVATION 4 HOURTREATMENT, 16 HOUR RECOVERY PERIOD Treatment4 (Mfl/mL) Cell Count Averages Flask (x10*6) swa aa- af--er A 2.84 B 2.35 H-24335 625 A 2.06 B 1.88 1250 A 2.17 B 2.27 2500 A 2.25 B 2.11 5000 A 1.95 B 1.95 CP, 10 A 1.62 B 1.71 CP, 20 A 1.36 B 1.50 C el Viability* 98% 98% 99% 100% 100% 99% 98% 97% 98% 97% 97% 99% 98% 96% Mean Cells per Flask3 (x10*6) 2.55 1.96 2.21 2.13 1.90 1.63 1.38 Cell Growth Index4 (%) 100% 77% 87% 83% 75% 64% 54% Cell Growth inhibition* (%> 23% 13% 17% 25% 36% 48% 1 CHO cells were treated In the presence ot an exogenous source of metabolic activation for 4 hours at 371C. 2 Viability determined by trypan blue dye exclusion. 3 Viable ceNsfflask cell count x % viable cells, reported a s mean of Flasks A and B. 4 Growth index * (mean cels per flask treated group/mean c e ls per flask control group), expressed a s a percentage. 3 Cell growth inhibition * 100% - % cell growth Index; not calculated for negative controls. 3 Visible precipitates observed. BioReliance Study N o. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CB1 -22 H-24335: In Vitro M ammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Ceils D uPont-3870 TABLE 7 CYTOGENETICANALYSIS OF CHOCELLS TREATEDWITH H-24335 INTHE PRESENCE OF EXOGENOUS S0 METABOLICACTIVATION 4 HOURTREATMENT, 16 HOUR RECOVERY PERIOD Mitotic % Aberrant Celia8 Index1 Cells *Tfr.eautlm.. eiinnti*l'f*t Flask (%) Scored Numerical Structural (ugMiL) W ater A 12.0 100 B 11.2 100 3 4 2 3 H-24335 625 1250 A 7.0 B 6.4 A 6.0 B 6.2 100 100 100 100 3 1 4 3 4 7 5 6 2500* A 5.2 B 3.8 100 100 3 2 7 5 CP, 10 A 7.6 B 8.2 100 100 2 1 25 28 Total Number of Structural Aberrations Gap Chromatid4 Br Ex Chromosome5 Br Die Ring Severely Damaged . Celts* 12 0 00 0 0 2 0 01 0 0 0 2 4 0 01 0 1 8 1 01 0 45 1 05 0 00 0 01 0 37 05 0 0 00 0 00 0 5 35 6 00 0 2 39 3 . 0 0 0 0 0 0 0 0 0 0 0 Average Aberrations Per Cell7 0.020 0.030 0.050 0.080 0.060 0.060 0.070 0.050 0.410 0.420 1 CHO cells were treated for 4 hours at 371C in the presence of an exogenous source of m etabolc activation. * Mitotic Index number mitotic figures x 100/500 cells counted. 1 Numerical: Includes polyploid and endoredupUcated cells.; Structural: excludes cells with only gaps. * Chromatid breaks include chromatid and Isochromatid breaks and fragments; chromatid exchange figures (Exch) Include quadrlradlala, triradlais and complex rearrangements. ' 5 Chromosome breaks indude breaks and acentric fragments; die, dicentric chromosome. * Severely damaged calls includes calls with one or more pulverized chromosomes and cells with 10 or more aberrations. 7 Severely damaged cells and pulverizations were counted as 10 aberrations. * Dose leva) 5000 pgfmLw as/ not analyzed due to test substance precipitation In the medium. * Lowest precipitating dose level. BioReliance Study N o. AA26XP.33i.BTL -23- Company Sanitized. Does not confa'n TSCA CBf H-2433S: In Vitro M ammalian Chromosome DuPont-3870 TABLES CONCURRENTTOXICITYASSAYWITH H-24335IN THE ABSENCE OF EXOGENOUS S9 METABOLICACTIVATION 20 HOUR CONTINUOUS TREATMENT Treatment1 (pg/mL) Cell Count Averages Cell Flask (x10*) Viability2 twflfantteerr A 2.34 B 2,79 97% 99% H-24335 S2S A 2.10 B 2.18 100% 100% 1250 A 2.20 99% B 2.82 100% 2500s A 2.36 B 2.04 99% 98% 3500s A 1.32 B 1.24 100% 98% 5000s A 0.98 B 1.10 97% 96% MMC, 0.1 A 2.43 B 2.08 99% 95% MMC, 0.2 A 1.79 B 1.66 98% 96% Mean C els per Flask* 0*10s) 2.51 2.14 2.50 2.16 1.27 0.99 2.18 1.67 Cell Growth Index4 (%> Cell Growth Inhibition5 (%) 100% 85% 99% 86% 51% 40% 87% 67% -- 15% 1% 14% 49% 60% 13% 33% ' CHO cells were treated In the absence of an exogenous source of metabolic activation for 20 hours at 371C. 2 viability determined by trypan blue dye exclusion. ' Viable cefls/flask * cell count x % viable cells, reported a 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, 4 Cell growth inhibition 100% - % cell growth Indue; not calculated for negative controls, 9 Visible precipitates observed. BioReiiance Study N o. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CBI -24- H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells D uPont-3870 TABUE 9 CYTOGENETICANALYSIS OF CHO CELLS TREATEDWITH H-24335 NTHE ABSENCEOF EXOGENOUS S9 METABOLICACTIVATION 20 HOURCONTINUOUS TREATMENT Mitotic % Aberrant CeHs* Index3 C els Treatment7-* ErrlautsnKl* (% ) Scored Numerical Structural (pgftnL) W ater A 10.6 100 B 9.6 too 0 0 0 0 H-24335 625 A 7.4 100 B 8.2 100 1 0 0 0 1250 A 5.8 100 B 5.2 100 2 0 0 0 2500 3500 5000 A 4.0 B 3.8 A 0.2 B 0.4 A 0.0 B 0.0 100 100 - 2 2 - - 0 0 - - MMC, 0.1 A 11.6 100 B 10.2 100 2 1 30 27 Tota! Numberof Structural Aberrations Gaps Chromatid4 Chromosome4 Br Ex Br Die Ring Severely Damaged CeHs* 00 0 00 0 00 0 00 0 0 0 00 00 0 0 00 0 00 0 00 00 0 0 --- - -_ --- 4 27 18 0 14 16 00 0 00 0 00 0 00 0 00 0 00 0 --- ---- - - ------ - 53 2 42 2 0 0 0 0 0 0 -- -- 0 0 Average Aberrations Per Cel!7 0.000 0 . 0.000 0.000 0 .0 0 0 0.000 0.000 0.000 -- - 0.550 0.380 ' CHO cells were treated for 20 hours at 371C In the absence of an exogenous source of metabolic activation. * Mitotic Index number mitotic figures x 100/500 cells counted. 1 Numerical: Includes polyploid and endoreduplicated cells.; Structural: exdudes ceils with only gaps. 4 Chromatid breaks Include chromatid and isochromatid breaks and fragments; chromatid exchange figures (Exeh) Include quadriredials, triradials and complex rearrangements. * Chromosome breaks Include breaks and acentric fragments; die, dicentric chromosome. 8 Severely damaged celts Indudes cells with one or more pulverized chromosomes and cells with 10 or more aberrations. 7 Severely damaged cels and pulverizations were counted as 10 aberrations. * Dose levels 3500 and 5000 pg/mL were not analyzed for chromosome aberrations due to excessive toxicity. BioReliance Study N o. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CBI - 25- H-24335: In Vitro M ammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) C ells DuPont-3870 TABLE 10 SUMMARY: CYTOGENETICANALYSIS OFCHO CELLSTREATEDWITH H-24335 Treatment* (Mfl/rttU Mean Aberrations CeltsWith Aberrations1 S9 Treatment Mitotic Cells Per Cell2 Numerical Structural Activation Time Index Scored (Mean +/- SD) (%) (%) Water - 4 11.2 200 0.005 0.071 2.5 0.5 H-24335 625 1250 2500 m 4 5.9 200 0.010 0.100 30 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 MMC, 0.2 - 4 10.1 200 0.295 0.722 3.5 18.5** Water H-24335 625 1250 2500 CP, 10 4 11.6 200 0.025 0.157 3.5 2.5 + 4 6.7 200 0.065 0.267 2.0 5.5 4 8.1 200 0.060 0.258 3.5 5.5 4 4.5 200 0.060 0.238 2.5 6.0 *t* 4 7.9 200 .415 0.785 1.5 26.5** Water - 20 10.1 200 0.000 0.000 0.0 0.0 H-24335 625 1250 2500 35004 50004 20 7.8 200 0.000 0.000 0.5 0.0 - 20 5.5 200 0.000 0.000 1.0 0.0 - 20 * 3.8 200 0.000 0.000 2.0 0.0 20 0.3 ---- .. - 20 0.0 - - - - - MMC. 0.1 20 10.9 200 0.465 0.924 1.5 28.5** 1 Celts from all treatment conditions were harvested at 20 hours after the initiation ofthe treatments. 2 Severely damaged cells were counted as 10 aberrations. * *, psO.05; " , psfl.01; Fisher's exact te s t 4 Dose level not evaluated for clastogenldty due to excessive toxicity. BioReliance Study No. AA26XP.331.BTL Company Sanitized. Does not contain TSCA CB1 -26- H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells APPENDIX A H istorical Control Data DuPont-3870 BioReliance Study No.: AA26XP.33.BTL -27- Company Sanitized. Does not contain TSCA CB1 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster O vary (CHO) C ells D uPont-3870 IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST USING CHINESE HAM STER OVARY (CHO) CELLS HISTORICAL CONTROL VALUES STRUCTURAL CHROMOSOME ABERRATIONS 1996-1998 H istorical V alu es M ean Statuiard Deviation Range NO]M-ACHVATED TEST SYSTEM Percent Aberrant Cells (%) U ntreated Control 1.3 1.3 0.0 to 6.0 Solvent C o n tro l' 1.4 1.3 0.0 to 6.0 Positive Control2 25.9 19.3 6.5 to 100.0 1 H istorical V alues M ean Standard Deviation Range S9-ACTTVATED TEST SYSTEM Percent Aberrant Cells (%) Untreated . C o n tro l 1.5 1.3 0.0 to 6.0 Solvent C o n tro l1 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-m ethyl-N '-nitro-N-nitrosoguanidine (M NNG, 0.75-2 pg/m l), and M itomycin C (MMC, 0.08-0.15 pg/m l). Positive control for 39-activated studies, cyclophospham ide (CP, 10-50 jig/m l), and benzo(a)pyrene, (B[a]P, 30 pg/ml). Company Sanitized. Does not contain TSCA CB1 BsoReliance Study No.: AA26XP.331.BTL -28- 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 H istorical V alu es M ean Standard Deviation Range NON-ACTTVATED TEST SYSTEM Percent Aberrant Cells (% ) 1 U n treated C o n tro l 2.4 1.7 0.0 to 9.5 Solvent C o n tro l1 2.3 1.3 0.0 to 8.0 Positive Control2 3.2 1.9 0.0 to 9.5 H istorical V alues M ean Standard Deviation Range S9-ACTIVATED TEST SYSTEM Percent Aberrant Cells (%) U ntreated Control 2.4 1.7 0.0 to 7.0 Solvent C o n tro l1 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 non standard solvents and Sponsor-supplied vehicles. Positive control for non-activated studies, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.75-2 pg/m l), and M itomycin C (MMC, 0.08-0.15 pg/m l). Positive control for S9-activated studies, cyclophosphamide (CP, 10-50 pg/m l), and benzo(a)pyrene (B[a]P, 30 pg/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 TSC CBI H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 opo-jrro IM M wceSM tjrNiwk; AAJ6X PJJIJTL H-2433: 1.0 PURPOSE V n t Mammalian Chromosome A berration Test in Chinese H am ster O vary (CHO) Cells The purpose o f tins study is to evaluate the clastogenic potential o f a test <n.h<*mw. upon its ability to induce chromosome abenations in Chinese ham ster ovary (CHO) cells. 2.0 SPONSOR 2.1 Name: 2.2 Address: EX du Pont de Nem ours and Company H askell Laboratory fo r Toxicology and Industrial M edicine P.O . Box SO, Elkton Road Newark, DE 19714-0050 1 3 Study M onitor: M aria Dormer, P hD . 2.4 SpomrorPmjfgUf DuPont-3870 dPSSiWR#; H aske Service Cod l 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance 3.2 T est Substance Nam e to be used in the Report: H-24335 3.3 Controls: SolventPositive: W ater M itomycin C (M M C) Cyclophospham ide (CP) 3.4 Determ ination o f Strength, Purity, etc. U nless alternate arrangements are made, the testing facility at BioReliance w ill not perform analysis o f the dosing solutions. The Sponsor w ill be directly responsible for determ ination and documentation o f the analytical purity and com position o f the test substance, and the stability and strength o f the test substance in the solvent (or vehicle). Protocol No. SPGT331 February 23,2000 1of9 BioReliance Study No.: AA26XP.331DTL -31 - # Bio Reliance" formerly M ittobiolojreal Avsocui* Company Sanitized. Does not contain TSC.' H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells DuPont-3870 DkFomJSTS BioRtSaiceStudy AAJiXFJJWTI, 3.5 Test Substance Retention Sample The retortion o f a reserve sample o f the test substance w ill be the responsibility o f the Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name: Toxicology Testing Facility BioReliance 4 2 Address: 4.3 Study D irector 5.0 TEST SCHEDULE 9630 M edical C arter Drive Rockville, M D 20850 Ramadevi Gudi, Ph.D. Phone: (301)610-2169 Fax: (301)738-2362 E-mail: rgudi@ taoreliance.com 5.1 Proposed Experim ental Start Date: 2/29/2000 5 2 Proposed Experim ental Term ination Date: 3/31/2000 5.3 Proposed Report Date: 4/14/2000 6.0 TEST SYSTEM The CHO-K, cell line is a proline auxotroph with a m odal chromosome num ber o f 20 and a popuM on doubling tim e o f 10-14 horns. CHO-K, cells were obtained from the American Type C ulture Collection (repository num ber CCL 61), M anassas, VA. The stability o f the m odal chromosome num ber o f the cell line is routinely checked and die cell line is routinely tested and determ ined to be fiee fiom mycoplasma contam ination. This system has been dem onstrated to be sensitive to the clastogenic activity o f a variety o f chem icals (Preston et al., 1981). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY T he chrom osome aberration test w ill be conducted using <anfigp} procedures (Evans, 1976), by treating cultures o f CHO cells to a minim um o f four concentrations o fth e test article as m il as to positive and solvent controls. DuPont S tu d ie f M h n J p M w ill be conducted sim ultaneously and thus the sam e solvent and positive controls w ill be used betw een these two studies. In the non-activated test system , treatm ent w ill be for 4 hours and fo r 20 hours; in tire S9 activated test system, treatm ent w ill be for 4 hours (Swierenga et Protocol No. SPGT33! Febra.ry 23,2000 BioReliance Study No.: AA26XP.331.BTL 2 of9 - 32- ) Bio Reliance* formerly Mtcrobolo|icil Associates Company Sanitized. Does not contain TSCA CBt H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells DuPont-3870 *SttntrNiMsbr AA2SXPJ31.BTL al., 1991). To ensure evaluation o f first division m etaphase cells die dividing cells w ill be arrested inm etaphase and harvested for microscopic evaluation o f chrom osome aberrations a t approxim ately ,20 hours (1.5 normal cell cycles) after the initiation o f treatm ent (G allow ay et al,, 1994). The clastogenic potential o f die test article w ill be measured by its ability to induce structural chromosome aberrations in a dose-responsive m m w com pared to the solvent, control group. In die event o f a positive response in die 4 hour non-activated study, die prolonged treatm ent non-activated study m ay not be scored. The test article w ill also be assessed for its ability to induce num erical chromosome 7.1 Solubility Determination W ater w ill be used as the test article solvent based on the solubility data available w ith die Sponsor. 7 2 Preliminary Toxicity Test for Selection o fD ose Levels Selection o f the dose levels fo r die cytogenetics assay w ill be done in consultation w ith die S ponsor, and added to the protocol by an amendment based upon post treatm ent toxicity (cell grow th inhibition relative to the solvent control) and solubility o f the test substance. CHO cells w ill be treated to solvent alone and to at least nine concentrations o f test substance. The highest concentration tested w ill be 5 mg/ml o r 10 mM whichever is lower fo r freely soluble test substances, or die maximum concentration resulting in a w orkable suspension for poorly soluble test substances not to exceed 5 mg/ml. The pH w ill be m easured at the test substance treatm ent condition and w ill be adjusted, if necessary, in order to m aintain a neutral pH in the treatm ent medium. The osm olality o f die higK r dose level, lowest precipitating dose level (where applicable) and the highest soluble dose level (where applicable) in treatm ent medium w ill also be m easured. C ells seeded 16-24 hours earlier w ill be treated for 4 hours in th e absence and presence o f S9 and for 20 hours in the absence o f S9. Just prior to trypsinization th e cell cultures w ill be visually inspected for tire extent o f m onolayer confiuency relative to tire solvent control Twenty hours after treatm ent initiation the cells w ill be harvested by trypsinization and counted using an autom atic cell counter and th ece ll viability w ill be assessed using trypan blue dye exclusion (SO P The cell counts and percent viability \flll be used to determ ine cell grow th inhibition relative to the solvent control (SOP W henever possible, the high dose to evaluate chromosome aberrations w ill be selected to give at least 50% cytotoxicity observed as (cell grow th inhihitm q relative to the solvent control) irrespective o f solubility. T he highest dose w ill not to exceed 5 mg/ml or 10 mM. At least tw o additional dose levels, dem onstrating minimal or no toxicity, w ill be included, to the event the test substance cannot be dissolved at a high enough concentration in an appropriate solvent to be toxic, then tire dose to be tested in the chrom osome aberration assay w ill be toe concentration resulting in minimum precipitation in test m edium . Precipitation w ill be <fotmiinwt Protocol No. SPGT331 February 23,2000 3 of9 BioReliance Study No.: AA26XP.331.BTL - 33- % Bi o Re l ia n c ffo rm erly M ic ro b io lo g ical Assoc m e t Company Sanitized. Does not contain TSCA C B t H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells DuPont-3870 DotaMS MUBMKtSMrNnktn AAXMJI.OTL by direct visual inspection. In the event die test substance dem onstrates a dosetespoosive increase in toxicity at concentrations that m eed solubility in medium, then the highest dose to be tested w ill be the yim um concentration that resultem at least 50% toxicity. In the event that neither cytotoxicity nor insolubility is observed in the prelim inary test, the highest dose in the chrom osome aberration assay w ill be 5 mg/ml o r 10 mM whichever is lower. I f excessive precipitation o f the test substance-solvent solution occurs upon addition to treatm ent m edium , o r if die osm olality o f die treatm ent medium is considered excessive, the Sponsor w ill be consulted. 7.3 Frequency and Route o fA dm inistration Target cells w ill be treated for 4 hours in the absence and presence o f S9, and for 20 hours in die absence o f S9, by incorporation o f die tost substance-solvent m ixture into the treatm ent m edium . This technique has been dem onstrated to be an effective method o f detection o fchemical clastogens in this test system (Evans, 1976). N o repeats o f die chromosome aberration tests w ill be done. 7.4 A ctivation System A roclor 1254-induced rat liver S9 w ill be used as the m etabolic activation system. The S9 w ill be prepared from m ale Sprague-Dawley rats induced w ith a single intrapentoneal injection o f A roclor 1254, 500 m g/kg, five days prior to sacrifice, p S9 w ill be batch prepared and stored frozen a t approxim ately -70C until used. Each batch preparation o f S9 w ill be assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dim ethylbenz(a)anthracene to form s mutagenic to Salmonella typhhmrtum TA100. Immediately prior to use, the S9 w ill be thaw ed and m ixed w ith cofactors to contain 2 mM magnesium chloride (M gClj.) 6 mM potassium chloride (KC1), Im M glucose-6-phosphate, 1 mM nicotinam ide adenine dinucleotide phosphate (NADP) and 20 p i S9 per m l serum free medium. ^ 7.5 Controls 7.5.1 Solvent (or Vehicle) Control The solvent fo r the test substance w ill be used as the solvent control. F or solvents other than w ater, physiological buffer, o r m edium , the final concentration in treatm ent medium w ill not exceed 1%. 7.5.2 Positive C ontrols M itomycin C w ill be used at a concentration w ithin 0.05-0.3 pg/m l Protocol No. SPGT331 February 23,2000 BioReliance Study No.: AA26XP.331.BTL Company Sanitized. Does not contain TSCA CB1 34 H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Ceils DuPont-3870 DufoM-CTO BtoRcOuctStBdjrNmbcr: AAKXPJUJTL as the positive control in the non-activated study. Cyclophosphamide w ill be usJ a t a concentration w ithin 10-50 pg/m l as the positive control in the S9-activated study. 7.6 Preparation o f Target Cells Exponentially growing CHO-K, cells w ill be seeded in com plete medium (M cCoy's 5A medium containing 10% fetal bovine sentm , 2 mM L-glutam ine, 100 units pemcillm/ml and 100 pg streptom ycin/m l) for each treatm ent condition at approxim ately 5 x 105cells/25 cm* flask. The flasks w ill be incubated at 37 1C m a hum idified atmosphere o f 5 1% C 0 2in a ir for 16-24 hours. 7.7 Identification o f Test System U sing a perm anent marking pen, the treatm ent flasks w ill be identified by the B toSeliance study number and a code system to designate tire treatm ent condition and test phase. 7.8 Treatm ent o f Target Ceils Treatm ent w ill be earned out in duplicate by refeeding the flasks w ife 5 m l com plete medium for fee non-activated treatm ent or 5 m l S9 reaction m ixture for tire S9-activated treatm ent, to which wifi be added 50 p i o f dosing solution o f test or control substance in solvent or solvent alone. Larger volum es o f dosing solution m ay be used if w ater, physiological buffer, o r m edium is used as tire solvent In fee non-activated study, fee cells w ill be treated fo r 4 hours and for 20 hours; in fee S9-activated study fee cells w ill be treated for 4 hours. Treatm ent w ill be carried o u ta t3 7 l C in a humidified atm osphere o f 5 1% C 0 2 in air. A fter the 4 hour treatm ent period in fee non-activated and fee S9-activated studies, fee m -a w n t m edium w ill be aspirated, tire cells washed w ith phosphate buffered m TM refed w ith complete medium and returned to the incubator. 7.9 C ell Harvest C ells wall be collected approximately 20 hours after initiation o f treatm ent This post-treatm ent harvest tim e represents approxim ately 1.5 norm al cell cycles and was selroted to ensure that tire cells are analyzed in tire first division m etapho r after initiation o f treatm ent Two hours prior to ceil harv est Colcemid w ill be " 'H to tire cultures at a final concentration o f 0.1 pg/m l. C ells w ill be harvested by trypsinization, collected by centrifugation and an aliquot w ill be removed for counting using an autom atic cell counter a id trypan blue dye exclusion. The rem ainder o f tire cells w ill be sw ollen w ith 0.075M KC1, washed w ith two consecutive changes o f fixative (m ethanohglacial acetic ad d , 3:1 v/v), Protocol No. SPGT331 February 23,2000 S of 9 BioReliance Study No.: AA26XP.331.BTL " T 3 5 T Company Sanitized. Does no! contain TSCA CBf H-24335: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3870 M ly N n * i AA26XFJJI.BTL caPPed^ overnight or longer at approximately 2-8C. The cell counts and percent viability w ill be used to determine cell grow * inhibition relative to toe so lv e control (% toxicity). To prepare slides, die cells w ill be collected by cratafugation and resuspended in fresh fixative. The suspension o f fixed cells w ill be applied to glass microscope slides and air-dried. The slides w ill be identified bv the experim ent num b, treatm ent condition and date. The slides w ill be ^ w ith Ciem sa and perm anently mounted. 7.10 Scoring for M etaphase Aberrations T o ensure that a sufficient num ber o f m etaphase cells are present on die slides, die penw sage o f cells in m itosis per 500 cells scored (m itotic index) w ill be determ ined and recorded for each coded treatm ent group selected for scoring chromosome aberrations. Slides w ill be coded using random numbers by an individual not involved w ith the scoring process. In the event o f a positive response in the 4 hour iron-activated study, die prolonged treatm ent non-activated study m ay not be scored. M etaphase cells w ith 20 2 centromeres w ill be cammed under oil w ithout prior knowledge o f treatm ent groups. W henever possible, a minimum o f 200 m etaphase spreads from each dose level (100 per duplicate flask) w ill be cammed and sew ed for chrom atid-type ami chromosome-type aberrations (Scott et al.,1990). The number o f metqphase spreads that w ill be examined and scored per duplicate flask may be reduced if the percentage o f abenant cells re a - w a statistically significant level before 100 cells are scored. Cbromatid-tvne aberrations include chrom atid and isochrom atid breaks and exchange figures such as quadriradtsls (symmetrical and asymmetrical interchanges), trim riink att rearrangements. Chromosome-type aberrations include chromosome hretk; and "" ^ e figures such as dicentrics and rings. Fragments (chrom atid o r acentric) observed in the absence o f any exchange figure w ill be scored as a break (chromatid o r chromosome). Fragments observed with an exchange figure w ill not be scored as an aberration but w ill be considered part o f the incom plete exchange. Pulverized chrom osom e , pulverized cells and severely damaged cells (2 10 aberrations) w ill also be recorded. Chromatid and isochrom atid gaps w ill be recorded but not included in the analysis. The XY coordinates for each cell w ith a structural aberrauon w ill be recorded using a calibrated microscope stage. The percent polyploid and endoreduplicated cells w ill be evaluated p er 100 cells for each dose level analyzed fra structural aberrations. 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST 8.1 Solvent Control The frequency o f cells w ith structural chromosome aberrations in the solvent control m ust be w ithin die range o f the historical solvent control and not exceed 6%. 8.2 Positive Control Protocol No. SPGT331 Fobrury 23,2000 BioReliance Study No.: AA26XP.33 l.BTL 6 of9 -36- # BIORELIANCE' form erly M icrobiological A ssociates Company Sanitized. Does not contain TSCA CB1 H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells DuPont-3870 AA4WJJ.BTL 9.0 EVALUATION OF TEST RESULTS T he cytotoxic effects o f the treatments are based upon cell grow * inhibition relative to toe so lv m t control and w ill be presented for the toxicity and aberration studies. The num ber and types o f aberrations found, the percentage o f structurally and numerically damaced tolls (percent aberrant cells) in the total population o f cells exam ined, and th em ean aberrations per cell w ill be calculated and reported for each treatm ent group. Chromatid and isochrom atid gaps are presented in the data but are not included in the total percentage o f cells w ith one or m ore aberrations o r in toe fiequency o f structural aberrations per cell S tatistical m a ly a sp f toe percentage o f aberrant cells w ill be perform ed using toe S h e * exact te s t The Fisher's test w ill be used to com pare pairw ise toe percent aberrant cells o f each treatm ent group w ith to o f toe solvent control. In toe event o f a positive Fisher's exact test at any test substance dose level, tire Cochran-A nnitage test w ill be used to m easure dose-responsiveness. A ll conclusions w ill be based on mH h ^ ,. how ever, as a guide to interpretation o f the data, the test substance w ill be considered to m duce a positive response when the percentage o f cells w ith aberrations is increased in a ^ ^ n a v e m anner w ith one o r more concentrations being statistically significant G fTM .p ' Test substances not dem onstrating a statistically significant increase in aberrations w ill be concluded to be negative. 10.0 REPORT A report o f toe results o f this study w ill be prepared by BioReliance and will accurately describe all m ethods used for generation and analysis o f toe data. R esults presented wifi include, but not be limit to: ' Test identification and CAS no., i f known; physical nature and purity, if t f S l r Pe,tieS relevant to lhB conduct oftfae study, if known; stability SolventAfchicle: justification for choice o f vehicle; solubility and stability o f test substance in solvent/vehicle, if known. Source o f cells, karyotype features (m odal chromosome num ber) mid suitability o f the cell type used, absence o f mycoplasma, cell cycle length, num ber. T est conditions: composition o f medium; C 02concentration; incubation tim e; cell density; solvent red solvent selection rationale; concentration o f test substance and concentration selection rationale; composition and acceptability criteria for toe m etabolic activation (S9) system; duration o f treatm ent; duration o f treatm ent w ith and Protocol No. SPGT33I Febreory 23,2000 7of 9 BioReliance Study No.: AA26XP.331.BTL 37 Bio Reliance- Fotm efly M icrobiological A ito c u tr i Company Sanitized. Does not contain TSCA CBI H-24335: In Vitro M ammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells DuPont-3870 D*Poal-T*7S B M bgaa Stwiy N,mken AA26XTJ3I.BTL o f Colcemid; type o f metabolic activation system used; positive and solvent controls; m ethods o f slide preparation; num ber o f ceil cultures; criteria for scoring aberrations and criteria fo r considering studies positive, negative. R esults: description o f precipitation; pH and osm olality o f the treatm ent medium; cell grow th inhibition relative to the solvent control; m itotic index ami m itn h r o f m etaphases analysed; type and num ber o f aberration (structural and num erical) given separately for each treated and control culture; concentration-response relationship; statistical analysis* historical control data. 11.0 RECORDS AND ARCHIVES A ll raw data, the protocol and all reports w ill be m aintained according_ to Standard O perating P r o c e d u te p H B f tg b y the B ioReliance RAQA unit hheeaaddqquuaartered at: B ioR eliance, 14920 B roschart R offl, R ockville, M D 20850. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE T ins N on-GLP study w ill be perform ed using th e Good Laboratory Practice R egulations fo r N onclinical Laboratory Studies as a general gmVfriin. D u s protocol has been w ritten to com ply w ith OECD G uideline 473 (In Vitro M ammalian Chrom osom e A berration Test), February 1998 and w ith the International Conference on H am w m zation o f Technical Requirem ents for Registration o f Pharm aceuticals for Human U se (1996 ami 1997). W ill this study be subm itted to a regulatory agency? NO I f so, to which agency o r agencies? NA U nless arrangem ents are made to the contrary, unused dosing solutions w ill be disoosed o f follow ing adm inistration to the test system and all residual test substance will be disposed o f follow ing finalization o f fl report 13.0 REFERENCES Evmis, H J. (1976) Cytological m ethods for detecting chem ical mutagens, in: A. H oilaender (Ed.), Chemical M utagens, Principles and M ethods for their Detection, vol 4 Plenum Press, New York, NY. G allow ay, S.M ., M .J. Aardema, M. Ishidate Ir., J.L. Ivett, D J. Kirkland T. M onta, P. M osesso and T. Sofuni (1994) Report from w orking group on in vitro tests for chrom osom al aberrations, M utation Research 3 12(3):241-261. International Conference on Harmonisation (ICH) o f Technical Requirem ents for Protocol No. SPGT331 February 23,2000 BioReliance Study No.: AA26XP.331.BTL 8 of? -38- ) Bio Reliance' ` form erly M icrobiologie^ I A iiociat Company Sanitized. Does not contain TSCA CBf H-24335: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells 2 - 2 6 - 0 0 j e : g A M iO u P o n t P n rn 302 4$1 **27 DuPont-3870 'NwtmA u a m m J r f H w maceaticals ft Human U se. Guidance on Specific A spects o f G eno to nd ^r Tests for Pham uceutkals, S2A document recommended for adoption i t step 4 o f the ICH process on July 19,1995. Federal Register 61:18198-18202, A p n l2 4 ,1996. _. . ^ 00 Harmonisation (ICH) o f Technical Requirements for Ge"ot0*icity: A Standaid Baaery for G enotoxidty Testing o f Ph*imceuticals. S2B document recommended for adoption at step 4 o f the K H process on M y 16, 1997. Federal Register 62:16026-16030, November 21, 1997. O ECD G uideline fo r foe Testing o f Chem icals, G uideline 473 (In (Taro M ammalian Chrom osom e A berration Test), February 1998. 7L' * * * Beadsr' J G - Brewen>A.V. C anano, J A Meddle, A F . M cFee, S . W olff and 3S. W asson (1981) M ammalian in vivo and in vitro cytogenetic assays: a report o f the Gene-Tox Program, M utation Research, 87:143-188. S cott, D.. N.D. Daaford, B J. Dean and D J. K irkland. 1990. M etephiae Chromosome A te re tio n t e * w fo Vitro. In: Basic M utagenicity Teats: UKEMS Recommended Procedure*. DJ Rutland (ed). Cambridge University Press, NewYork,NY. Sw ierenga S.H .H ., J A Meddle, E A Sigal, JP .W . G ilm an, R L . Brillinger, G i t Douglas and B i t N estm ann (1991) Recommended protocols based on a survey o f current practice in genotoxidty testing laboratories, IV . Chromosome aberration and sister-chrom atid exchange in C hinese hamster ovary, V79 Chinese lung and human lymphocyte cultures. M utation R esearch 246:301-322. 14.0 APPROVAL STU D Y MONITOR M aria D onner. Pfa.D. (P rint o r ty p e Name) X IZSfoo DATE BIORELIANCE STUDY DIRECTOR BIORELIANCE STUDY MANAGEMENT DATE DATE Ptatecol N e. SPGT33I F tb ra *ry 23,2000 9o f BioReliance Study No.: AA26XP.331.BTL -3 9 - BjOREUANCr Company Sanitized. Does not contain TSCA CB1