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AR226-3149 DuPont-3871 TRADE SECRET Study Title H-23960: In Vitro M am m alian Chrom osom e A berration T est In Chinese H am ster O vary (CHO) Cells Authors Ramadevi Gudi, Ph.D. Elizabeth H. Schadly, B.S. Report Completion Date May 4,2000 Perfnrminp Laboratory BioReliance 9630 Medical Center Drive Rockville, MD 20S50 for E. L 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 AA26XN.331JBTL DuPont Protect ID DuPont-3871 Work Reouest Number Page 1 of 40 Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 C E R T IF IC A T IO N W e, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. BioReiiance Study Director: Ramadevi Gudi, PbJD. Date Approved by Study Monitor. M aria Donner, PhD . Senior Research Scientist o rjo iJ o o Date BioReiiance Study No. AA26XN.331.BTL Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Ceils DuPont-3871 TABLE OF CONTENTS Page C ertific a tio n ............................................................ Study Inform ation....................................*............. Sum m ary................................................................. Purpose................................................................... Characterization o f Test and Control Substances M aterials and M ethods....................................... . Results and D iscussion........................................ C o n clu sio n ............................................................ R eferen ces............................................................. Data T ables........................................................... 2 5 7 9 9 9 14 16 17 18 T ablel: Prelim inary Toxicity Assay with H-24335 in CHO Cells in fire Absence o f ^ Exogenous M etabolic Activation. 4 Hour Treatm ent......................................................... Table 2: Prelim inary Toxicity Assay w ith H-24335 in CHO Cells in the Presence o f Exogenous M etabolic Activation. 4 Hour Treatm ent...................... ............................. 19 Table 3: Prelim inary Toxicity Assay w ith H-24335 in CHO Cells in the Absence o f Exogenous M etabolic Activation. 20 Hour Treatm ent................................................. 20 Table 4: Concurrent Toxicity Assay with H-24335 in CHO Cells in the Absence o f Exogenous M etabolic Activation. 4 Hour Treatm ent.............................................. ..... 21 Table 5: Cytogenetic Analysis o f CHO Cells Treated w ith H-24335 in the Absence o f Exogenous S9 M etabolic Activation. 4Hour Treatment, 16 Hour Recovery Period........ .22 Table 6: Concurrent Toxicity Assay with H-24335 in CHO Cells in the Presence o f Exogenous M etabolic Activation. 4 Hour Treatm ent.............................*.......................... .23 Table 7: Cytogenetic Analysis of CHO Cells Treated with H-24335 in the Presence o f Exogenous S9 M etabolic Activation. 4Hour Treatment, 16 Hour Recovery Period........ .24 BioReliance Study No. AA26XN.331.BTL Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test In Chinese Hamster Ovary (CHO) Cells DuPont-3871 Table 8: Concurrent Toxicity Assay with H-24335 in CHO Cells in the Absence o f Exogenous M etabolic Activation. 20 Hour Treatm ent.................................................... . Table 9: Cytogenetic Analysis o f CHO Cells Treated w ith H-24335 in the Absence o f Exogenous S9 Metabolic Activation. 4Hour Treatment, 16 Hour Recovery Period------ 26 Table 10: Summary: Cytogenetic Analysis o f CHO Cells Treated with H-24335........... 27 Appendix A: Historical Control D ata....... ........................................... *................................. 28 . 31 Appendix B: Study Protocol............................................ *........................................................ BioReliance Study No. AA26XN.331.BTL Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro M am m alian Chromosome A berration T est in Chinese H am ster O vary (CHO) Cells STUDY INFORM ATION DuPont-3871 Haskell Number: 23960 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 >15 mg/mL. BioReliance Study No. AA26XN.331.BTL -5- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test In Chinese Hamster Ovary (CHO) Cells DuPont-3871 Sponsor: E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial M edicine P.O. Box 50, Elkton Road Newark, DE 19714-0050 TnitiatpH/r,omnleted: February 25,2000 / (see report cover page) In-LifeTnitiated/Completed: February29,2000/March27,2000 BioReliance Study N o. AA26XN.331.BTL Company Sanitized. Does not contain TSCA CBI SUMMARY The test s u b s .* , H-23960. was tetoed to t o t . * chromosome abgrrto n lest usmg CUnese horister ovav (CHO) calls to both t o a b ^ a o d p ^ ^ a n A ^ ^ K r i exogenous S9 metabolic activation system. A preliminary toxicity assay establish the dose range for the chromosome aberration assay . The chromosome aberrah assay was used to evaluate the clastogenic potential o f toe test substance. toWater was determined be the solvento f tS Snonsor the solubility o f the test substance, and compatibility with the target ceils, itm test ^ ^ t a T S b but cloudy to water at a c o n c e to a ta o f 50 m gfaL , t o nuunmum concentration tested. In 1he preliminary toxicity assay, the maximum dose level tested was 5000 pg/mL. The test substance was soluble in the treatment medium at all d o s e le v e k te s t^ in tte studies. Visible precipitates were observed in the treatment medium atctose lw eb ^ S O O pg/mL in the S 9a*ivated study. Dose levels o f 5500 pg/mL were * M k medium in the S9 activated study. Selection o f dose levefc for the assay was based on total cell growth inhibition relative to the solvent control. N o s u h to to d toxidty, Le., at least 50% cell growth inhibition, was observed at any dose I n M n m t e the non-activated or the S9 activated treatment groups. Based on these fiitom p, the doses aberration assay ranged from 625 to 5000 pg/mL for both the non activated and the S9 activated treatment groups. In flie chromosome aberration assay, the cells were Heated for 4 and20 3 :- - <1250 ugAnL were soluble to tteatoMUi medium to t o S9 actuated study. No s"*>"t o | d d ty ( to least 50% cell gruw li inhibition) was o b a e r te d a t t o t o ^ ^ t o ^ ^ t o e d t o chromosome aberrations, 5000 |ig faL , in t o ncm^*vnted 4 hum m d * * ^ " "2 ^ 0 ^ 8 respectively. No substantial toxicity (at least 50% cell growth "^ bl^ on2 j ^ bs? ! ! : K b S S T d o s J l e v e l evaluated t o ctoomosome aberradonsin t o ngtaL . The highest dose level selected fcr a n a l) o i aS vated studies, 5000 irg/mL, was based u p t o absence r f a t least 50% to n m q " J J * " j o f test substance precipitation in the treatment medium. The highest dose level seieciea ror ^ i s ^ S o m S aberrations inthe S9 activated study, 2500 pg/mL, was based upon the S ^ L e o f at least 50% toxicity and the presence o f test substance precipitation m the M m o tt medium (the lowest precipitating dose was selected as the W ghejdoseto e v a iu ^ . activated and S9 activated 4 hour treatment groups were scored foistivctotsl aad num encj chromosome aberrations. No statistically significant increases m J u c te a l and chromosome aberrations were observed in the non-activated or S9 activated 4 hour treatment Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 groups relative to the solvent control group, regardless o fd o selev el Snon-arctivat*eda2b0sehnocuerocfoantPin0u8o^us trreeastPmoennset g"roup was evaluated for structural and^nuSmiecriacall chromosome aberrations. A statistically significant increase in structural ctom osom e aberrations was observed in foe non-activated 20 hour continuousexposure group solvent control group, at foe dose level 5000 pgtoL Annitage test was also positive for a dose response <pD.G5). However, foe^pwcmtege o f structurally aberrant ceils observed at foe dose level 5000 m AL o f structurally aberrant cells observed with foe historical solvent control (0%-6-*). Therefore, foe statistically significant increase in foe p e rc e n t^ o f^ "S S u S S f i ^ in 5000 pg/mL was not considered biologically significant No numerical chromosome aberrations were observed m 20 ^ exposure group relative to foe solvent control group, regardless o f dose level e S c t t S t f S positive and solvent controls fulfilled foe requirements for a ^ d te s t Under foe conditions d S b e d in tins report, H-23960 was concluded to be n ^ v e fOTthe r n i ^ o n o f structural and numerical chromosome aberrations in foe non-activatedm d S9 activated in vitro mammalian chromosome aberration test in Chinese hamster ovary (CHO) cells. BioReliance Study N o. AA26XN.331.BTL Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 PURPOSE The purpose o f this study was to evaluate the clastogenic potential o f the test substance, H23960, based upon its ability to induce in vitro chromosome aberrations in the Chinese hamster ovary (CHO) cells. CHARACTERIZATION OF TEST AND CONTROL ARTICLES The test substance, H-23960, was received by BioReliance on 15 February 2000 and was l the code number AA26XN. X?ietest substance was characterized by the Sponsor as an ____ _ should be stored in a well-ventilated place at Anexpirationdate for thetest^bstancew asnotprovided. Upon receipt, the test substance was described as at room temperature, in a well-ventilated area, exposure to lig h t was stored Based on information provided by the Sponsor, sterile distilled water (CAS 7732-18-5), obtained from the Life Technologies Company, was die solvent used to deliver H-24335 to the test system. Mitomycin C (MMC; CAS No.: 50-07-7), was obtained from die Sigma Chemical Company, and was dissolved and diluted in sterile distilled water to stock concentrations o f 1 and 2 pg/snL 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 pg/mL for use as the positive control in the S9 activated test system. For each positive control one dose with sufficient scorable metaphase cells was selected for analysis. The solvent for the test substance was used as the solvent control at the same concentration,as that found in the test substance-treated groups. MATERIALS AND M ETHODS Test System Chinese hamster ovary (CHO-K,) cells (repository number CCL 61) were obtained from the American Type Culture Collection, Manassas, VA, on May 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 and CHO cells at passage 12 were used for the Rhmmnsr>nrie 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 modal chromosome number o f 20. The use of CHO cells has been demonstrated to be an effective method o f detection o f chemical clastogens (Preston et al., 1981). BioReliance Study No. AA26XN.3313 T L -9- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Trat in Chinese Hamster Ovary (CHO) Ceils DuPont-3871 M etabolic A ctivation System A rador 1254-induced rat liver S9 was used as the exogenous metabolic activation system. The S9 was prepared fiom male Sprague-Dawley rats induced with a single m ttapm toneal injection o f A rador 1254,500 mg/kg, five days prior to sacrifice. The S9 was batch prepared and stored at S-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)anihracene to forms mutagenic to Salmonella typhimurium TA100. Immediately prior to use, the S9 was thawed and mixed with a cofector pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM ^ u M s e -6 -p h o ^ ^ , l ^ i n i c o t i n e adenine dinucleotide phosphate (NADP) and 20 pL S9 P (McCoy's 5A serum-free medium supplemented with, 100 units penicillin and 100 pg streptomycin/mL, a id 2 mM L-glutamine). Prelim inary Toxicity Assay The preliminary toxicity assay was performed for the purpose o f sel^ting the chromosome aberration assay and consisted o f an evaluation o f test substance effect on cell growth. CHO cells were seeded for each treatment condition at approximate y 5 x l(T ceUs/25 cm2 flask and were incubated at 371C in a humidified atmosphere o f 51% C 02 m an for 16-24 hours. Treatment was carried out by refeeding the flasks with 4 5 mL complete medium (McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS), 100 units penicillin and 100 pg streptomycin/mL, and 2 mM L-glutamine) for the non-activaied study or S9 reaction m ixture (3.5 mL serum-fiee medium plus 1 mL o f 5X S9 mix) for the a c tiv a d sto^r, 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 treatment medium was measured. The pH o f the highest concentration o f the dosing solution in the treatment medium was measured using test tape. The cells were treated for 4 horns with and without S9, and continuously for 20 hours without S9. At completion o f tire 4 hour exposure penod, lira treatment medium was removed, the ceils washed with calcium and magnesium-ftee phosphate buffered saline (CMF-PBS), refed with 5 mL complete medium and returned to the meubator for a total tim e period o f 2Q hours fiom the initiation o f the treatm ent A t 20 hours after foe initiation o ffoe treatment foe cells were harvested by trypsinization and counted using a Coulter counter. The presence o f test substance precipitate was assessed using foe unaidedeye. 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. Chromosome A berration 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 BioReliance Study No. AA26XN.331.BTL - 10 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test In Chinese Hamster Ovary (CHO) Cells DuPont-3871 controls. For the chromosome aberration assay, CHO cells were seeded at approximately 5 x 105cells/25 cm2flask and were incubated at 371C in a humidified atmosphere o f 51% C 02 in air for 16-24 to u rs. Treatment was carried out by refeeding duplicate flasks with 4.5 mL complete medium (McCoy's 5A medium supplemented w ith 10% FBS, 100 units penicillin and 100 pg streptam ydn/m L, and 2 mM L-glutamine) for foe non-activated study or 4.5 mL 39 reaction mixture for foe S9 activated study, to which 500 pL o f dosing solution o f test or control article in solvent or solvent alone was added. The osmolality o f the highest concentration o f the dosing solution in the treatment medium was measured. The pH o f foe highest concentration o f foe dosing solution in the treatment medium was measured using test tape. ' In the non-activated study, foe cells were exposed to foe test substance for 4 to u rs or continuously for 20 hours up to the cell harvest at 371C in a humidified atmosphere o f 51% C 02 in air (Swierenga et a!., 1991). In the 4 hour exposure group, the treatment medium was removed after the exposure period, and foe cells washed with CMF-PBS, refed with complete medium and returned to foe incubator. Two to u rs prior to the scheduled cell harvest, Colcemid was to duplicate flasks for each treatment condition at a final concentration o f 0.1 pg/mL and the flasks returned to foe incubator until cell collection. In foe S9 activated study, foe cells were exposed for 4 to u rs at 371C in a humidified atmosphere o f 51% C 02 in air (Swierenga et al., 1991). After foe exposure period, foe treatm ent was removed, foe cells washed with CMF-PBS, refed with complete medium and returned to foe incubator. Two to u rs prior to the scheduled cell harvest, Colcemid was added to duplicate flasks for each treatment condition at a final concentration o f 0.1 pg/mL and foe flasks were returned to foe incubator until cell collection. A concurrent toxicity assay was conducted in both foe non-activated and the S9 activated assay systems. After cell harvest an aliquot o f foe cell suspension was removed from each culture and counted using a Coulter counter. The presence o f test substance precipitate was assessed using foe 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 foe solvent control. Cell H arvest Two hours after foe addition o f Colcemid, metaphase cells were harvested for both foe non-activated and S9 activated studies by trypsinization. Cells were collected approximately 20 hours after initiation o f treatment (Galloway et al., 1994). The cells were collected by centrifugation at approximately 800 xpm for 5 minutes. The cell pellet was resuspended in 2-4 mL 0.075 M potassium chloride (KC1) and allowed to stand at room temperature for 4-8 minntfg The cells were collected by centrifugation, foe supernatant aspirated and the cells fixed with two washes o f approximately 2 mL Camoy's fixative (methanokglacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2-8C. BioReliance Study No. AA26XN33 l.BTL -11- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Ham ster Ovary (CHO) Cells DuPont-3871 Slide P reparation To prepare slides, tire fixed cells were centrifuged at approximately 800 ipm for 5 minutes, the supernatant was aspirated, and 1 mL fresh fixative was added. After additional centrifugation (at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative. A sufficient amount o f cell suspension was dropped onto the center o f 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 o f M etaphase Cells Slides were 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 the slides, the percentage o f cells in m itosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 2Q2 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 al., 1990). The number o f mofapbacg spreads that are examined a id scored per duplicate flask may 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 gynhangi figure were scored as a break (chromatid or chromosome). Fragments observed with an mmhawge figure were not scored as an aberration but instead were considered part o f the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely AnrmaeA cells (StlO 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 evaluate! from each treatment flask per 100 metaphase cells scored. C ontrols Mitomycin C was used as the positive control in the non-activated study at final fyMny.ntratirmB 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/mL. For both positive controls one dose level exhibiting a sufficient number o f scorable metaphase cells was selected for BioReliance Study No. AA26XN.331.BTL -12 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHQ) Cells DuPont-3871 analysis. The solvent vehicle for the test substance was used as the solvent control at mi concentration as that found in the test substance-treated groups. DuPont s tu d ie a H H d S jB A were conducted simultaneously and thus the same solvent and positive rantro^vw reused between these two studies. Evaluation o f T est Results . The toxic effects o f treatment were based upon ceil growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types o f aberrations found, tire percentage o f structurally and numerically damaged (percent aberrant cells) in the total population o f cells examined, and the mean aherratinnc per cell was calculated and reported for each group. Chromatid and isochromatid gtps are presented in tire data but are not included in fire total percentage o f cells with one or more aberrations or in the frequency o f structural aberrations per cell. Statistical analysis o f fire percent aberrant cells was performed using the Fisher's <y* test Fisher's test was used to compare pairwise fire 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, fire 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 fire data, fire test substance was considered to induce a positive response when fire percentage o f 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. In those cares where confirmation o fnegative results is not necessary,justification will be provided. C riteria fo r a V alid Test ^ The frequency o f cells with structural chromosome aberrations in fire solvent control must 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<0.05, Fisher's pyot test) relative to the solvent control D eviations No known deviations from the protocol or assay method SOPs occurred during the conduct o f this study. A rch iv es AH raw data, the protocol all reports, and^stained ang coded slides will be maintain.*! according to Standard Operating fro c e d u re M B H ^ B lb y the BioReliance Regulatory BioReliance Study No. AA26XN331.BTL - 13 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHQ) Cells DuPont-3871 Affairs/Quality Assurance Unit headquartered at: BioReliance, 14920 Broschart Road, Rockville, MD, 20850. RESULTS AND DISCUSSION Solubility Water was determined to be die solvent o f choice based on information provided by the Sponsor, the solubility o f the test substance, mid compatibility with the target cells. The test gnhstanra was soluble but cloudy in water at a concentration o f SO mg/mL, the maximum concentration tested. Prelim inary Toxicity Assay Dose levels for the chromosome aberration assay were selected following a preliminary toxicity assay and were based upon, a reduction o f total cell growth (cell growth inhibition) relative to the solvent control. The results o f the evaluation o f cell growth inhibition are presented in Tables 1-3. CHO cells were exposed to solvent alone and to nine concentrations o f to t gfogtanri ranging from 0.5 pg/mL to 5000 pg/mL in the absence and presence o f an S9 reaction mixture. The test substance was soluble in treatment medium at all dose levels tested in the non-activated studies. Visible precipitates were observed in the treatment medium at tire dose levels >1500 pg/mL in the S9 activated study. The dose levels <500 pg/mL were soluble in treatment medium in the S9 activated study. The osmolality o f the solvent (water) in treatment medium was 314 mmol/kg. The osmolality in treatm ent medium o f the highest concentration tested, 5000 pg/mL, was 315 mmol/kg, basically identical to the osmolality o f the solvent The pH o f the highest concentration o f test substance in treatment medium was approximately 7.0. No substantial cell growth inhibition (>50%) relative to the solvent control was observed a t any dose level tested in both the non-activated and S9 activated treatment groups. Based upon the results o f the toxicity study, the dose levels selected for testing in the chromosome aberration assay were as follows: Treatment Condition -S9 +S9 Treatment Time (hr) 4 Recovery Time (hr) 16 Dose levels (pg/m L ) 625,1250,2500,5000 2Q 0 625,1250,2500,5000 4 16 625,1250,2500,5000 Chromosome A berration Assay In the chromosome aberration assay, the test substance was soluble in treatment medium at all dose levels tested in the non-activated studies. Visible prcipittes were observed in the BioReliance Study No. AA26XN.33 l.BTL - 14 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 treatment medium at the dose levels >2500 pg/mL in the S9 activated study. The dose levels <1250 pg/inL were soluble in treatment medium in the S9 activated study. The osmolality in treatment medium o f the highest concentration tested, 5000Tpg/mL, was 226 mmol/kg, exactly the same as the osmolality o f the solvent (water) in treatment medium. The pH o f the highest concentration o f test substance in treatment medium was approximately 7.0. Cell growth inhibition relative to the solvent control in CHO cells was 25% after treatment with H-23960 at 5000 pgfoiL 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-23960 to induce nhrnmnsnm aberrations is presented by treatment flask in Table 5, and summarized by treatment group in Table 10. The mitotic index was 7% reduced relative to the solvent control at the highest dose level evaluated for chromosome aberrations, 5000 pg/mL. The dose levels selected for microscopic analysis were 1250,2500, and 5000 pg/mL. The percentage o f cells with structural and numerical aberrations in the test substance-treated' groups was not significantly increased above that o f the solvent control (p>0.Q5, Fisher's exact test). The percentage (18.5%) o f structurally damaged cells in the MMC got?) was found to be statistically significant Cell growth inhibition relative to the solvent control in CHO cells w asl3% after treatment with H-23960 at 2500 pg/mL for 4 hours in tire presence o f S9 activation (Table 6). This was the highest dose level evaluated for chromosome aberrations. The ability o f H-23960 to induce chromosome aberrations is presented by treatment flask in Table 7, and summarized by treatment group in Table 10. The mitotic index at tire highest dose level evaluated for chromosome aberrations, 2500 pg/mL, was 25% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 625,1250, and 2500 pg/mL, in tire absence o f at least 50% cell growth inhibition. The percentage o f cells with structural or numerical aberrations in the test substance-treated groups was not significantly increased above that o f tire solvent control (p>0.05, Fisher's exact test). The percentage (26.5%) o f structurally damaged cells in the CP group was found to be statistically significant In tire absence o f a positive response in tire non-activated 4 hour exposure group, slides from the non-activated 20 hour exposure group were evaluated for chromosome aberrations. Cell growth inhibition relative to the solvent control was 19% at 5000 pg/mL (Table 8), the highest dose level evaluated for chromosome aberrations in the non-activated 20 hour continuous exposure group. The ability o f H-23960 to induce chromosome aberrations is presented by treatm ent flask in Table 9, and summarized by treatment group in Table 10. The mitotic index at the highest dose level evaluated for chromosome aberrations, 5000 pg/mL, was 14% reduced relative to tire solvent control. The dose levels selected for microscopic analysis were 1250, 2500, and 5000 pg/mL. The percentage o f cells with structural aberrations in tire test substance-treated groups was significantly increased above that o f the solvent control only at dose level 5000 pg/mL (p<0.05, Fisher's exact test). The Cochran-Armitage test was also positive for a dose response (p<0.05). However, the percentage o f structurally aberrant cells observed at dose level 5000 pg/mL (2.5%) was within the range o f structurally aberrant cells BioReliance Study No. AA26XN.331.BTL -15 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 observed w ith the historical solvent control (0%-6%). Therefore, die statistically significant increase in the percentage o f structurally aberrant cells observed at 5000 pg/mL was not considered biologically significant The percentage o f cells with 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 (28.5%) o f structurally damaged cells in the 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 the test substance and because no equivocal responses were observed. CONCLUSION The positive and solvent controls fulfilled the requirements for a valid te st Under the conditions described in this report, H-23960 was concluded to be negative for the induction o f structural and numerical chromosome aberrations in the non-activated and S9 activated in vitro mammalian chromosome aberration test in Chinese hamster ovary (CHO) cells. BioReUance Study No. AA26XN.331.BTL -16- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 , REFERENCES Evans, HJ . (1976) Cytological methods for detecting chemical mutagens, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, voi 4. Plenum Press, New York. Galloway, S.M., M J. Aardema, M. Ishidate Jr., J.L. Ivett, D.J. Kirkland, T. M erita, P. Mosesso and T. Solimi (1994) Report from working group on in vitro tests for chromosomal aberrations, Mutation 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 Battety for Genotoxicity Testing o f Pharmaceuticals. S2B document recommended for adoption at step 4 o f the ICH process oh July 16, 1997. Federal Register 62:16026-16030, November 21,1997. OECD Guideline for the 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, JA . Heddle, A.F. McFee, S. W olff and J.S. Wassom (1981) Mammalian in vivo and in vitro cytogenetic assays: a report o f the Gene-Tox Program, Mutation Research, 87:143-188. Scott, D., N.D. Danford, B.J. Dean and D.J. Kirkland. 1990. Metaphase Chromosome Aberration Assays In Vitro. In: Basic Mutagenicity Tests: UKEMS Recommended Procedures. D J Kirkland (ed). Cambridge University Press, New York, NY. Swierenga S.H.H., JA . Heddle, E.A. Sigai, J.P.W . Gilman, RJL Brillinger, GJL Douglas and E.R. Nestmann (1991) Recommended protocols based on a survey o f current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sisterchromatid exchange in Chinese hamster ovary, V79 Chinese lung and human lymphocyte cultures, Mutation Research 246:301-322. BioReliance Study No. AA26XN.331.BTL -17- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLE 1 PRELIMINARYTOXICITYASSAY WITH H-23960 INCHO CELLS IN ' THE ABSENCE OF EXOGENOUS METABOLICACTIVATION 4 HOURTREATMENT. 16 HOUR RECOVERY PERIOD Treatm ent1 (pg/mL) W ater Cell Count (*1QP) CM VlabHty2* <%) M ean C ells/ Flask' (x10*) Cell Growth Index4 (%> 2.50 89% 2.47 100% Call Growth InhlbHon* <%> -- H-23960 0.5 2.32 89% 2.30 93% 7% 1.5 2.94 98% 2.88 117% -17% 5 2.88 88% 2.84 115% -15% 15 2.87 89% 2.85 115% -15% 50 2.85 99% 2.83 114% -14% 150 2.83 97% 2.74 111% -11% 500 2.88 98% 2.82 114% -14% 1500 2.71 98% - 2.65 107% -7% 5000 2.53 97% 2.45 99% 1% 1 CHO cells were treated In the absence of an exogenous source of m etabolic activation for 4 hours a t 371C. 2 Viability determined by trypan blue dye exclusion. 2 Viable cells/flask = cell count x % viable cells 4 Growth index * (cell* per flask treated group/cells per flask control group), expressed a s a percentage. 8 Cell growth M M o n 100% - % cell growth Index; not calculated for negative controls. BioReliance Study No. AA26XN.331.BTL - 18 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test la Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLE 2 PRELIMINARY TOXICITYASSAY WITH H-23960 IN CHO CELLS IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION 4 HOURTREATMENT, 16 HOUR RECOVERY PERIOD T reatm ent1 (pg/mL) Cell Count (x10*) Cell Viability2*5 {%> liii C el Growth Index* <%) Cell Growth IniiMHon* (%) W ater 2.51 95% 2.38 100% -- H-23960 0.5 2.57 99% 2.54 107% -7% 1.5 2.63 99% 2.60 109% . -9% 5 2.62 98% 2.57 108% -8% 15 2.68 100% 2.68 113% -13% 50 2.40 96% 2.30 97% 3% 150 2.32 99% 2.30 96% 4% 500 2.33 98% 2.28 96% 4% 1500* " 2.28 95% 2.17 91% 9% 5000* 2.65 97% 2.57 108% -8% 1 CHO cells wore boated hi the presence of an exogenous source of metabolic activation for 4 hours a t 371C. 2 Viability determined by trypan blue dye exclusion. 2 Viable celis/flask *ceU count x % viable cells * Growth Index (cells per flask treated group/cells per flask control group), expressed a s a percentage. 5 CeU growth Inhibition = 100% - % cell growth index; not calculated for negative control*. 8 Visible precipitates observed. BioReliance Study No. AA26XN.331.BTL - 19 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test In Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLES PRELIMINARY TOXICITYASSAYWITH H-23960 !N CHO CELLS IN THE ABSENCE OF EXOGENOUS METABOUCACTIVATION 20 HOURCONTINUOUS TREATMENT T reatm ent1 (pg/hiL) Cell Count (X1*) Cell Viability2* <%) Mean C ells/ Flask9 <x10") Cell Growth Index4* (%) Cell Growth InhlbUon9 (%) W ater 2.99 98% 2.93 100% -- H-23960 0.S 2.94 96% 2.82 96% 4% 1.5 3.32 98% 3.19 109% -9% 5 2.78 95% 2.64 90% 10% 15 2.79 93% 2.59 88% 12% 50 3.09 97% 2.99 102% -2% 160 2.90 97% 2.81 98% 4% 500 3.07 95% 2.92 100% 0% 1500 3:03 98% 2.97 101% -1% 5000 2.59 91% 2.45 84% 18% 1 CHO cefls w ere treated continuously in th e absence o f an exogenous source of metabolic activation for 20 hours at 371C. 2 Viabltfty determined by trypan blue dye exclusion. 2 Viable celts/flask = cell count x % viable ceBs 4 Growth Index = (ceil* per flask treated group/cells per flaBk control group), expressed a s a percentage. 9 Cell growth Inhibition * 100% - % cell growth Index; not calculated for . negative controls. BioReliance Study No. AA26XN.331.BTL - 20 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Teat in Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLE 4 CONCURRENTTOXICITYASSAYWITH H-23960 IN CHO CELLS IN THE ABSENCE OF EXOGENOUSMETABOLICACTIVATION 4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD Treatm ent1 Qjg/mL) Cell Count A v erag es Flask (x10*) Cell Viability1 Mean Cells par Flask* (x10*) Cell Growth Index4 <%) Cell Growth InhibKonS (* ) W ater H -23960 625 1250 2500 5000 MMC, 0.1 MMC, 0.2 A 2.52 B 3.10 A 2.85 B 2.79 A 2.49 B 2.85 A 2.43 B 2.48 A 1.99 B 2.21 A 1.95 B 2.05 A 2.07 B 2.03 96% 98% 96% 99% 97% 95% 98% 95% 98% 97% 98% 87% 97% 95% 2.73 2.74 2.56 2.37 2.04 1.95 . 1.97 100% 101% 94% 87% 75% 71% 72% -- -1% 6% 13% 25% 29% 26% * CHO cells were treated In the absence of an exogenous source of metabolic activation for 4 hours at 371C. 1 Viability determined by trypan blue dye exclusion. ` 3 Viable cells/Rask * cell count x % viable cells, reported a s m ean of Flasks A and B. 4 Growth index (mean calls per flask treated groupftnean ceSs per flask control group), expressed a s a percentage. 5 Cell growth inhfoltlon -100% - % cell growth Index; not calculated for negative controls. BioReliance Study No. AA26XN.331.BTL -21- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLES CYTOGENETICANALYSISOF CHO CELLS TREATEDWITH H-23960 INTHE ABSENCE OF EXOGENOUS METABOLICACTIVATION 4 HOURTREATMENT. 16 HOUR RECOVERY PERIOD Mitotic % Aberrant Ceils5* Index2* Cefis Treatment1-* Flask (%) Scored Numerical Structural (MO/mL) W ater A 11.0 100 B 11.4 11 2 3 0 1 H-23960 1250 2500 A 10.6 to o B 9.8 100 A 8.8 B 7.8 100 100 4 0 4 2 1 1 1 1 5000 A 10.8 100 B 10.0 100 2 3 1 1 MMC, 0.2 A 9.6 100 B 10.6 100 2 5 16 21 Total Number of Structural Aberrations G aps Chromatid4 Br Ex Chromosome" Br Die Ring Severely Damaged Celia* 1 0 0 00 0 0 0 0 01 0 0 0 10 01 0 0 00 01 0 0 11 0 11 0 1 14 11 0 19 7 01 00 01 00 00 00 40 13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Average Aberrations PerCeR7 0.000 0.010 0.010 0.010 0.010 0.010 0.010 0.010 0.290 0.300 1 CHO calls w are treated for 4 hours a t 371C in the absence of an exogenous source of metabolic activation. 2 Mitotic index o num ber mitotic figures x 100/500 cefls counted. 5 Numerical: Includes polyploid and endoredupficatad ceRs.; Structural: excludes cells wtth only gaps. 4 Chromatid breaks include chromatid and isochromatid breaks and fragm ents; chromatid exchange figures (Exeh) Include quadriradials, triradtato and complex rearrangem ents. 1 Chromosome breaks Include breaks ami acentric fragments; die, dicentric chromosome. * 4 Severely dam aged cells Includes cells with one or more pulverized chromosomes and cells with 10 or more aberrations. 7 Severely dam aged ceRs and pulverizations were counted a s 10 aberrations. * An additional dose level o f625 pgfrnLwas tested a s a safeguard against excessive toxicity at higher dose levels but was not required for m icroscopic examination. BioReliance Study No. AA26XN.33 l.BTL - 22 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test In Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLES CONCURRENTTOXICITYASSAYWITH H-23960 IN CHO CELLS IN THE PRESENCE OF EXOGENOUS METABOLICACTIVATION 4 HOUR TREATMENT, 16 HOUR RECOVERY PERIOD T reatm ent1 (pg/mL) Cell Count A verages Flask (x10") Mean Cells C el p er Flask9 Viability2*45 (x10") Cell Growth index4 <%) Celt Growth Inhibition5 (%) W ater H-23960 62S 1250 2500* 5000* CP, 10 CP, 20 A 2.84 B 2.35 A 2.40 B 2.56 A Z11 B 2.08 A 2.03 B 2.12 A 1.68 B 2.04 A 1.62 B 1.71 A 1.36 B 1.50 98% 98% 98% 97% 98% 97% 98% 97% 98% 97% 97% 99% 98% 96% 2.55 2.42 2.04 2.02 1.81 1.63 1.38 100% 95% 80% 79% 71% 64% 54% -- 5% 20% 21% 29% 36% 46% 1 CHO cads were treated In the presence of an exogenous source of metabolic activation for 4 hours at 3 7 1 '0 . 2 VlabiRy determined by trypan blue dye exclusion. 9 Viable cells/Raak = cell count x % viable ceBs, reported a s m ean of Flasks A and B. 4 Growth Index **(m ean cells per flask treated groupfmean cells per flask control group), expressed a s a percentage. 5 Celt growth inhibition 100% - % cell growth Index; not calculated for negative controls. 9 Visible precipitates observed. BioReiiance Study No. AA26XN.331.BIT, - 23 Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLE 7 CYTOGENETICANALYSIS OF CHO CEILS TREATED WITH H-23960 INTHE PRESENCE OF EXOGENOUS METABOLICACTIVATION 4 HOURTREATMENT, 16 HOUR RECOVERY PERIOD Mitotic % Aberrant CeUs* Index2 Cells Treatment1-4 Flask <%) Scored Numerical Structural (pg/rnL) W ater A 1 i0 100 B 11.2 100 3 4 2 3 H-23960 625 A 9.4 B 10.4 100 100 4 4 3 4 1250 A 8.6 B 9.B 100 100 4 5 6 5 2500* CP. 10 A 8.4 B 9.0 A 7.6 B 8.2 100 100 100 100 8 7 2 t 6 5 25 28 Total Number of Structural Aberrations Caps Chromatid4 Br Ex Chromosome9 Br Die Ring Severely Damaged Cells8 12 02 0 0 00 O1 0 0 0 0 21 04 05 06 16 24 5 35 2 39 2 0 1 0 0 1 6 3 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Average Aberrations P erC eS 1 0.020 0.030 0.030 0.040 0.060 0 .0 ) 0 .0 ) 0.050 0.410 0.420 1 CHO cells were treated for 4 hours a t 371C in the presence of an exogenous source of metabolic activation. 2 Mitotic Index num ber mitotic figures x 100/500 cells counted. 3 Numerical: Includes polyploid and endoreduplicated cells.; Structural: exdudes cells with only gaps. 4 Chromatid breaks include chromatid and Isochromatid breaks and fragm ents; chromatid exchange figures (Exch) Include quadriradlals, triradials and complex rearrangem ents. 9 Chromosome breaks include breaks and acentric fragm ents; die, dicentric chromosome. 9 Severely dam aged cells includes cells with one or more pulverized chromosomes and c e ls with 10 o r more aberrations. 7 Severely dam aged cells and pulverizations were counted a s 10 aberrations. Dose level 5000 pg/mL w as not analyzed due to te st substance precipitation In the treatm ent medium. * Lowest precipitating dose level. BioReliance Study No. AA26XN.331.BTL - 24 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLES CONCURRENT TOXICITYASSAYWITH H-23960 IN CHO CELLS IN THE ABSENCE OF EXOGENOUS METABOLICACTIVATION 20 HOURCONTINUOUS TREATMENT Treatm ent12 (pg/mL) Celt Count A verages Flask OdO*) Cd! Viability3* Mean C els per Flask5 (X10*) W ater A 2.34 B 2.79 97% 99% 2.51 H -23960 625 A 2.52 B 2.82 95% 99% 2.59 1250 A 2.59 B 2.85 2500 A 2.20 B 2.38 5000 A 2.30 B 1.94 MMC, 0.1 A 2.43 B 2.08 MMC, 0.2 A 1.79 B 1.66 99% 97% 100% 97% 96% 97% 99% 95% 98% 96% 2.66 2.25 2.04 2.18 1.67 C el Growth Index* (%) Cell Growth Inhibition* (%) 100% - -- 103% -3% 106% -6% 90% 10% 81% ' 19% 87% 13% 87% 33% 1 CHO cells were treated In the absence of an exogenous source of metabolic activation for 20 hours a t 3711C. 2 Viability determined by trypan blue dye mcdusion. , * Viable ceUs/Rask= cell count x % viable cells, reported a s m ean of Flasks A and B. * Growth Index *>(mean ceils per flask treated group/mean cells per flask control group), expressed a s a percentage. 5 Cell growth inhibition -100% - % cell growth Index; not calculated for negative controls. BioReliance Study N o. AA26XN.331.BTL - 25 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLE 9 CYTOGENETICANALYSIS OF CHO CELLS TREATED WITH H-23960 IN THE ABSENCE OF EXOGENOUS METABOUCACTIVATION 20 HOUR CONTINUOUS TREATMENT Mftotic iimnaUewx*2 C els % Aberrant Cells13 Treatment1* Flask <%) Scored Numerical Structural (pg/m l) W ater A 10.6 100 B 9.6 100 0 0 0 0 H-23960 1250 A 7.6 100 B 7.8 100 2 1 1 0 2500 A 10.0 100 B 9.2 100 2 0 1 0 5000 A 8.4 100 B 9.0 100 2 0 3 2 MMC.0.1 A 11.6 100 B 10.2 100 2 1 30 27 Total Number of Structurai Aberrations Severely G aps Chromatid4 Chromosome1* Damaged 8 r Ex Br Die Ring Cels 00 00 0 0 00 00 0 0 0 0 00 1 00 0 010 00 0 02 0 010 4 27 18 0 14 16 00 00 00 00 00 01 53 42 0 0 0 0 1 0 2 2 0 0 0 0 0 0 0 0 Average P er Cell7 0.000 0.000 0.010 0.000 0.010 0.000 0.030 0.020 0.550 0.380 1 CHO cells w ere treated for 20 hours a t 371C In the absence of an exogenous source of metabolic activation. 2 Mitotic Index number mitotic figures x 100/500 cells counted. 3 Ntimoricsl! fndudos polyploid and andoredupScatad caHs.; Structural! excludes calls with only jp p f 4 Chromatid breaks include chromatid and isochromatid breaks and fragm ents; chromatid exchange figures (Exch) include quadriradlals, trkadlals and complex rearrangem ents. 5 Chromosome breaks Include breaks and acentric fragm ents; die, dicentric chromosome. 8 Severely-dam aged c e ls includes cefls with one or more pulverized chromosomes and cells with 10 or more aberration. 7 Severely dam aged c e ls and pulverizations were counted a s 10 aberrations. * An additional dose level o f625 pg/mL w as tested a s a safeguard against excessive toxicity a t higher dose levels but was not required for microscopic examination. BioReliance Study N o. AA26XN.331 ,BTL - 26 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 TABLE 10 SUMMARY: CYTOGENETIC ANALYSIS OF CHO CELLS TREATED WITH H-23960 T reatm ent (usftnL) W ater H-23960 1250 2500 5000 MMC, 0.2 Mean Aberrations Celts With Aberrations S9 Treatment Mitotic Ceils Per Cell Numerical Structural Activation Time Index Scored (Mean +/- SD) (%) (%) - 4 112 200 0.005 0.071 2.5 0.5 _ 4 10.2 200 0.010 0.100 2.0 1.0 4 8.3 200 0.010 0.100 3.0 1.0 - 4 10.4 200 0.010 0.100 2.5 1.0 - 4 10.1 200 0.295 0.722 3.5 18.5** W ater + 4 11.6 200 0.025 0.157 3.5 2.5 H-23960 625 + 4 9.9 200 0.035 0.184 4.0 3.5 1250 + 4 9.2 200 0.060 0.258 4.5 5.5 2500 4 8.7 200 0.055 0.229 7.5 5.5 CP, 10 + 4 7.9 200 0.415 0.785 1.5 26.5** W ater - 20 10.1. 200 0.000 0.000 0.0 0.0 H-23960 1250 2500 5000 . ,, 20 7.7 200 0.005 0.071 1.5 20 9.6 200 0.005 0.071 1.0 _ 0.5 0.5 - 20 8.7 200 0.025 0.157 1.0 2.5* MMC, 0.1 - 20 10.9 200 0.465 0.924 1.5 ' 28.5** 1 Cells from all treatm ent conditions were harvested a t 20 hours after the initiation of the treatm ents. 2 Severely dam aged cells were counted a s 10 aberrations. 2 *, psO.05; **, psO.01; Fisher's exact te s t BioReliance Study N o. AA26XN.331.BTL - 27 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 APPENDIX A H istorical C ontrol D ata BioReliance Study No.: AA26XN.331.BTL - 28 - Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Ceils DuPont-3871 IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST USING CHINESE HAMSTER OVARY (CHO) CELLS HISTORICAL CONTROL VALUES STRUCTURAL CHROMOSOME ABERRATIONS 1996-1998 H isto rical V alu es M ean Standard Deviation J Range NO N-ACTIVATED TEST SYSTEM Percent Aberrant Cells (%) U ntreated Control 1.3 1.3 0.0 to 6.0 Solvent Control1 1.4 1.3 0.0 to 6.0 Positive Control2 25.9 19.3 6.5 to 100.0 H isto rical V alu es M ean Standard Deviation Range S9-ACTTVATED TEST SYSTEM Percent Aberrant Cells (%) U n treated 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 1 1 1 Solvents include water, saline, dimethyl sulfoxide, ethanol, acetone, non-standard solvents and Sponsor-supplied vehicles. Positive control for non-activated studies, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 0.75-2 pg/ml), and M itomycin C (MMC, 0.08-0.15 pg/ml). Positive control for S9-activated studies, cyclophosphamide (CP, 10-50 pg/ml), and benzo(a)pyrene, (B[a]P, 30 pg/ml). BioRelianee Study No.: AA26XN.331.BTL -29- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 IN VITRO MAMMALIAN CHROMOSOME ABERRATION USING CHINESE HAMSTER OVARY (CHO) CELLS HISTORICAL CONTROL VALUES NUMERICAL CHROMOSOME ABERRATIONS 1996-1998 H isto rical V alues M ean Standard Deviation Range NON-ACTTVATED TEST SYSTEM Percent Aberrant Cells (%) j U n treated Control 2.4 1.7 0.0 to 9.5 Solvent Control1 2.3 1.3 0.0 to 8.0 Positive Control2 32 1.9 0.0 to 9.5 H isto rical V alu es M ean Standard Deviation Range S9-ACTTVATED TEST SYSTEM Percent Aberrant Cells (%) Untreated Control 2.4 1.7 0.0 to 7.0 Solvent Control1 3.1 2.1 0.0 to 13.5 Positive Control3 3.5 22 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 Mitomycin C (MMC, 0.08-0.15 pg/ml). Positive control for S9-activated studies, cyclophosphamide (CP, 10-50 pg/ml), and benzo(et)pyrene (B[a]P, 30 pg/ml). BioReliance Study No.: AA26XN.331.BTL -30- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells DuPont-3871 APPENDIX B Study Protocol BioReliance Study No.: AA26XN.33 l.BTL -31- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro M ammalian Chromosome A berration Test in Chinese H am ster O vary (CHO) Cells . DuPont-3871 H-2J960ZooyJ TWO: Ik Vitro MaaiaxHsiChrottOMBcAbtmtioTetttm Cklnae H au ler Onry(CHO>Ct!l> DcJPofc3*71 B M M niSldr W rebtnAAKXRJHJTL H-; 1.0 PURPOSE In Vitro Mammalian Chromosome Aberration Test in Chinese 1 HamsterOvary (CHO) Cells . The purpose o f this study is to evaluate the ciastogenic potential o f a test substance based 2.0 SPONSOR 2.1 Name; E I, du Pont de Nemours and Company 2.2 Address: 2.3 Study Monitor : Haskell Laboratory Sir Toxicology and Industrial Medicine P.O. Box 50, Elkton Road Newark, DE 19714-0050 Maria Donner, PhD. 2.4 Spons w r #4 DuPont-3871 i# :239 _ _ Service Code 3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES 3.1 Test Substance 3 32 Test Substance Name to be used in die Report H-23960 3.3 Controls: Solvent: Water Positive: Mitomycin C (MMC) Cyclophosphamide (CP) 3.4 Determination o f Strength, Purity, etc. Unless alternate arrangements ate made, the testing facility at BioReliance will not perform analysis o f the dosing solutions. The Sponsor will be directly responsible test substance, and the stability and strength o f die test substance in d ie solvent (or vehicle). Protocol No. SPGT331 February 23,2000 i of ? BioReliance Study No.: AA26XN.331.BTL - 32 - 6) Bio Reliancefo rm erly M icrobiological A ssociate* Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test In Chinese Ham ster Ovary (CHO) Cells DuPont-3871 CUM* M ir a r ti CkivMaonAbcmliM T ot is. (CHOCUb ... DuPonUsn ' W W IieceSaxb NteratA26XNJ31.BTL 3.5 Test Substance Retention Sample The retention o f a reserve sample o f the test substance will be the responsibility of die Sponsor. 4.0 TESTING FACILITY AND KEY PERSONNEL 4.1 Name; Toxicology Testing Facility BioReliance 4.2 Address: 43 Study Director 9630 Medical Cotter Drive Rockville, MD 20850 Ramadevi Gudi, PhD. Phone: (301)610-2169 Fax: (301)738-2362 E-mail: igudi@bioreliance.com TEST SCHEDULE 5.1 ProposedExperimental Start Date: 2/2912000 5.2 Proposed Experimental Termination 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 modal chromosome number o f 20 and a population doubling time o f 10-14 hours. CHO-K, cells were obtained from the American Type Culture Collection (repository number CCL 61), Manassas, VA. The stability o f the modal chromosome number o fthe cell line is routinely checked and the cell line is routinely tested and determined to be free from mycoplasma contamination. This system has been demonstrated to be sensitive to the clastogenic activity o fa variety o f chemicals (Preston et aL, 1981). 7.0 EXPERIMENTAL DESIGN AND METHODOLOGY The chromosome aberration test will be conducted using standard procedures (Evans, 1976), by treatment cultures o f CHO cells to a minimum o f four concentrations o f die test article as well as to positive and solvent controls. DuPont Studies 3870 and 3871 will be conducted simultaneously and thus the same solvent and positive controls will be used between these two studies. In the nan-activated test system, treatment will be for 4 hours and for 20 hours; intheS9 activated test system, treatment will be for 4 hours (Swierenga et Protocol No. SPGT331 February 23,2000 2 of9 BioReliance Study No.: AA26XN.331.BTL - 33 - Bio Reliance^ Form erly M icrobiological A troci ste t Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Ceils DuPont-3871 OaFoaWCT al., 1991). To ensure evaluation o f first division metaphase cells die dividing cells will be arrested in metaphase and harvested for microscopic evaluation o f chromosome aHermrin^e at approximately 20 horns (15 normal cell cycles) after the initiation o f treatment (Galloway et al,, 1994). The clastogenic potential o f the test article wiH be measured by its ability to induce structural chromosome aberrations in a dose-responsive m m , when conqrared to the solvent control group. In tire event o f a positive response in tire 4 hour non-activated study, tire prolonged treatment 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 tire test article solvent based on tire solubility data available with tire Sponsor. 7.2 Preliminary Toxicity Test for Selection o f Dose Levels Selection o f tire dose levels for the cytogenetics assay will be dotre in consultation with tire Sponsor, and added to the protocol by an am endm ent based upon post treatment toxicity (cell growth inhibition relative to tire solvent control) md solubility o f tile 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 5mg/ml or 10 mM whichever is lower for fieely soluble test substances, or the maximum concentration resulting in a workable suspension for poorly soluble test substances not to exceed 5 mg/ml. The pH will be measured at the hiEw test substance treatment condition and will be adjusted, if necessary, in order to maintain a neutral pH in tire treatment medium. The osmolality oftire highest dose,level, lowest precipitating dose level (where applicable) and tire highest soluble 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 o f S9 and for 20 hours in the absence o f S9. Just prior to ttypsinization tire cell cultures will be visually inspected for tire extent o f monolayer confluency relative to tire solvent control. Twenty hours after treatment initiation tire cells will be harvested by ttypsinization and counted using an automatic cell arenJm uxyirereell viability will be assessed using trypan bit dye exclusion (SOP The cell mt and percent viability will be used to determine cell growth inhibition relative tn the solvent control (SOP I Whenever possible, tire high dose" to evaluate chromosome aberrations will be selected to give at least 50%cytotoxicity observed as (cell growth inhibition relative to the solvent control) irrespective o f solubility. The highest dose will not to exceed 5 mg/ml or 10 mM. At least two additional dose levels, demonstrating minimal or no toxicity, will be included. In the event the test substance cannot be dissolved at a high enough concentration in an appropriate solvent to be toxic, then tire highest dose to be tested in tire chromosome aberration assay will be tire concentration resulting in minimum precipitation in test medium. Precipitation will be determined Protocol No. SPGT331 F ebruary 23,2000 3 of BioReliance Study No.: AA26XN.331.BTL - 34 - Bio Reliancf form erly M icrobiological A tto c ta m Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Ceils DuPont-3871 CbiNsel M B * MW Mlnuui C tm jraom j AbcmtlooTM in v8?y (CHO) Cell* D a ta ta n 3to8e*M S a 4 r NBtanAA26XNJ31HTL by direct visual inspection. In the event the test substance demonstrates a dose responsive increase in toxicity at concentrations that w i solubility in medium, dien the highest dose to be tested will bethe maximum concentration that results in at east 50% toxicity, hi the event that nafflwifMnwfmriritv nnr inmhiMu*, is observed in the preliminary test, the highest dose in t e S L r e a b e t r a t i S assay will be 5 mg/inl or 10 mM whichever is lower. If excessive pw e-tpe^n 0f die test substance-solvent solution occurs upon addition to mtment or f the osmolality o fthe treatment medium is considered excessive, the Sponsorwill be consulted. 7.3 Frequency and Route o fAdministration Target cells will be treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence o f S9, by incorporation o f the test substance-solvent mixture into the treatmentmedium. This technique has been demonstrated to be an effective method o f detection o fchemical clastogens in this test system (Evans, 1976). No repeats ofthe chromosome aberration tests will be done. 7.4 Activation System Aroclor 1254-induced rat liver S9 will be used as the metabolic activation system. The S9 will be prepared from male Sprague-Dawley rate induced with a intraperitoneal injection of Aroclor 1254,500 rog/kg, five days prior to sacrifice. The S9 will be batch prepared and stored frozen at approximately -70C until used. Each batch preparation o f S9 will be assayed for sterility and its ability to metabolize 2-ammoanthiacene and 7,12-dimethylbatz(a)anthracen<s to forms mutagenic to Salmonellatyphimurium TA100. Immediately prior to use, the S9 will be thawed and mixed with cofectors to mntem 2 mM magnesium chloride (MgO*) 6 mM potassium chloride (KCI), ImM glucose-6-phosphate, I mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 pi S9 per ml serum free medium. 7.5 Controls 7.5.1 Solvent (or Vehicle) Control The solvent for ti test substance will be used as the solvent control. For solvents other than water, physiological buffar, 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 pg/ml Protocol No. SPGT331 F ebruary 23,2000 < 0f9 BioReliance Study No.: AA26XN.331.BTL - 35 - Bio Reliance" ^ 5 * fo rm erly M icro b io lo g ical A u o c ia te i Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Ham ster Ovary (CHO) Cells DuPont-3871 H- **" Mm w Uu i CtnwojMncA birrtdoa TetfI D tfM *J871 tHOCeKl . BfaSrihuieeSOKfy Ni*bcr-.AA2SXN.331.m. as the positive control in the non-activated study. Cyclophosphamide will be used at a concentration within 10-50 pg/ml as fl positive control in the S9-activated study. 7.6 Preparation o fTarget Cells Exponentially growing CHO-K, cells will be seeded in complete medium (McCoy's 5A medium containing 10% feta! bovine serum, 2 mM L-glutamine, 100 units pemcillintol and 100 pg streptomycin/ml) for each treatment mditirm at approximately 5 x 10s cells/25 cm3flask. The flasks will be incubated at 37 l c in a hmmdified atmosphere o f 5 1%CO* in air for 16-24 horns. 7.7 Identification o f Test System . Using a pennanent marking pen, the treatment flasks will be by the BioReliance study number and a code system to designate the treatment condition and test phase. 7.8 Treatment of Target Cells Treatment will be carried out in duplicate by refeeding the with 5 ml ccnpplete medium for the non-activated treatment or 5 ml S9 reaction mbnm for the S9-achvated treatment, to which will be added SOpi o f dosing solution o ftest or control substance in solvent or solvent alone. Larger volumes o f dosing solution may be used if water, physiological buff, or medium is used as the solvent. to the non-activated study, the cells will be treated for 4 hours and for 20 hours- in the S9-actrvafed study the cells will be treated for 4 hours. Treatment will be carried out at 37 1C in a humidified atmosphere o f 5 1% CP2in air. After the 4 hour treatment period in the non-activated and foe S9-activated studies, foe treatment medium will be aspirated, foe cells washed with phosphate buffered TMi;TM- refed with complete medium and returned to the incubator. 7.9 Cell Harvest Cells will be collected approximately 20 hours after initiation o f treatment This post-treatment harvest time represents approximately 1.5 normal cell cycles and was selected to ensure that foe cells are analyzed in foe first division merapbts* alter initiation o f treatment Two hours prior to cell harvest, Colcemid* will be added to the cultures at a final concentration o f0.1 pg/ml. Cells will be harvested by trypsimzation, collected by centrifugation urn) an aliquot will be removed for counting using an automatic cell counter and trypan blue dye exclusion. The remainder o f foe cells will be swollen with 0.075M KC1, Protocol No. SPGT331 February a ,2 0 0 0 Sof9 BioReliance Study No.: AA26XN.331,BTL - 36 - Bio Reliancf Tarmorlr M icrobiological A necate Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in C h in ee H am ster Ovary (CHO) Cells DuPont-3871 Tedia ' BioRetlaacrStady NaUrAA2iXNJJUTL with two consecutive changes o f fixative (metbanokelacial acetic acid 3-1 v/vk caqpped and stored overnight or longer at approximately 2-8C. The cell counts and Percent viability will be used to determine cell growth inhibition relative to the solvent control (% toxicity). To prepare slides, the cells wifi be collected by centrifugation and resuspended in fiesh fixative. The suspension o f fixed cells will be applied to glass microscope slides and air-dried. The slides will be identified by the experiment number, treatment condition and date. The slides will be with Giemsa and permanently mounted. 7.10 Scoring for Metaphase Aberrations = To ensure that a sufficient number o f metaphase cells are present on the Me, the percentage o f cells in mitosis per 500cells scored (mitotic index) will be determined and recorded fin each coded treatment group selected for scoring chromosome aberrations. Slides will be coded using random numbers by an individual not involved with the scoring process. In the event o f a positive response in the 4 hour non-activated study, tire prolonged treataentnon-activated study may not be scored. Metaphase cells with 20 2 centromeres will be examined under oil without prior knowledge of treatment groups. Whenever possible, a m inim um o f 200 metaphase spreads fiom each dose level (100 per duplicate flask) will be examined and scored for chromatid-lype and chromosome-type aberrations (Scott et aL, 1990). The number of metapbase spreads that will be examined and scored per d elicate flask may 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 rh as V ^ ^ fa ^ C iQ T O ie e M .e n d asymmetrical mterchanges), trimdials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed m the absence ofany 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 o f the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (2 10 aberrations) will also be recorded. Chromatid and isochromatid gaps will be recorded but not included in the analysis. The XY coordinates for each cell with a .m l aberration will be recorded using a calibrated microscope stage. The percent polyploid and endoreduplicated cells will be evaluated per 100 cells for each dose level analyzed fin structural aberrations. 8.0 CRITERIA FOR DETERMINATION OF A VALID TEST 8.1 Solvent Control . Tire frequency o fcells with structural chromosome aberrations in the solvent control must be within fire range ofthe historical solvent control and not exceed 6%. Protocol No. SPGT331 F ebruery 23,2900 6 of9 BioReliance Study No.: AA26XN.331.BTL -37- Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration T est in Chinese Hamster Ovary (CHO) Cells DuPont-3871 f(CHO>Cdti 82 Positive Control Do?m-3S71 BioRrihoctStedy NnfcenAA2iXiU3IJTTL The percentage o f cells with aberrations must be statistically increased (psO.OS, Fisher's exact test) relative to the solvent control. 9.0 EVALUATION OF TEST RESULTS 11 cytotoxic effects o f the treatments are based upon cell growth inhibition relative to the solvent control and will be presented for the toxicity and aberration studies. The m im i^ mid types o f aberrations found, the percentage o f structurally and numerically damaged cells (percent aberrant cells) in the total population o f ceils examined, and the mean aberrations per cell will be calculated and reported for each treatment group. Chromatid and isochromatid g^ss are presented in the data but are not included in the total percentage o f cells w ith one or more aberrations or in fee frequency o f structural aberrations per cell. Statistical analysis o f the percentage o f aberrant cells will be performed using fee Fisher's exact te st The Fisher's test will be used to compass pairwise fee percent aberrant cells of each treatment group with feat a t fee solvent control. In fee event o f a positive Fisher's exact test at any test substance dose level, fee Cochran-Axmitage test will be used to measure dose-responsiveness. All conclusions will be based on sound scientific baas; however, as a guide to interpretation o f fee data, fee test substance will be considered to induce a positive response when the percentage o f cells wife aberrations is increased in a dose-responsive manner wife one or more concentrations being statistically significant (p0.05). Test substances not demonstrating a statistically significant increase in aberrations will be concluded to be negative. 10.0 REPORT A report o f fee results o f this study will be prepared by BioReliance and will accurately describe all methods used for generation and analysis o fthe data. Results presented will include, but not be limited to: Test substance: identification and CAS n o , if known; physical nature and purity, if known; physicochemical properties relevant to fee conduct o f fee study, if known; stability o ftest substance, if known. Solvent/Vehicie: justification for choice o f vehicle; solubility and stability o f test substance in solvcnt/vebicle, if known. Source o fcells, karyotype features (modal chromosome number) and suitability o fthe cell type used, absence o fmycoplasma, cell cycle length, passage number. Test conditions: composition o fmedium; C 02concentration; incubation time; cell reeding density: solvent and solvent selection rationale; concentration o f test substance and concentration selection rationale; composition and acceptability criteria for fee metabolic Protocol No. SPG T33! F ebrutry 23,2000 7 of9 BioReliance Study No.: AA26XN.331.BTL -38- e, m Bio Reliance" . Form erly M icrobiological A tted ile C o m p an y S an itize d . D o es n o t co n tain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHQ) Cells DuPont-3871 . CUatse K ftw M i n J^ C k rD a tQ * c e A b O T a a iT e * tia (CHO)CUU Ite fta tO T ] B fadlute SM y NbtKAA2XNJ3I.BTL Coiceoud; type o f metabolic activation system used; positive and solvent controls; methods o f slide preparation; number o f cell cultures; criteria for scoring aberrations and criteria for considering studies positive, negative. * Results: description o f precipitation; pH and osmolality of the treatment medium; cell growth inhibition relative to foe solvent control; mitotic index and number o f metaphases analyzed; type and number of aberration (structural and numerical) given separately for each treated and control culture; concentration-response relationship; statistical analysis; historical control data 11.0 RECORDS AND ARCHIVES All raw data, foe protocol and all reports will be maintained according to Standard Operating P ro c e d u n V M ^ A b y foe BioRcliance RAQA unit headquartered at: BioReliance, 14920 BfbschartRoao, Rockville, MD 208S0. 12.0 REGULATORY REQUIREMENTS/GOOD LABORATORY PRACTICE This Non-GLP study will be performed using foe Good Laboratory Practice Regulations for Nonclinical Laboratory Studies as a genera! guideline. This protocol has been written to comply with OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test), February 1998 and with foe International Conference on Harmonization o f Technical Requirements for Registration o f Pharmaceuticals for Human Use (1996 and 1997). Will this study be submitted to a regulatory agency? NO If so, to which agency or agencies? NA Unless arrangements are made to foe contrary, unused dosing solutions will be disposed o f following administration to foe tea system and all residual test substance will be disposed o ffollowing finalization o f foe report 13.0 REFERENCES Evans, H J. (1976) Cytological methods for detecting chemical mutagens, in: A. Hollaender (E d), Chemical Mutagens, Principles and Methods for their Detection, vol. 4. Plenum Press, New York,NY. Galloway, S.M., M J. Aardema, M. Ishidate J r, J.L. Ivett, D J. Kirkland T. Morita, P. Mosesso and T. Sofuni (1994) Report from working group on in vitro tests for chromosomal aberrations, Mutation Research 312(3):241-261. Protocol No. SPG T33I February 23,2000 g 0f BioReliance Study No.: AA26XN.33f.BTL -39- BioReuan cf Form erly M icrobiological A ssociates Company Sanitized. Does not contain TSCA CBI H-23960: In Vitro Mammalian Chromosome Aberration Test in Chinese Hamster Ovary (CHO) Cells 2-28-00; :49AM:ouPon`t P r u r m t DuPont-3871 7/ hrterattional Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for B u n n Use, Guidaase on Specific Aspects of Regulatory Geaotoxicity Tests for Pharmaceuticals. S2A document recommended for option at step 4 o f the ICH process on July 19,1995. Federal Register 61:18198-18202, April 24,1996. btem athm al Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Geaotoxicity: A Standard Battery for Geaotoxicity Testing ofPhannaceuticalj. S2B document recommendedfor adoption at step 4 o f the ICH process on July 16,1997. Federal Register 62:16020-16030, November 21, 1997. OECD Guideline for tire Testing o f Chemicals, Guideline 473 (in Vitro Mammalian Chromosome Aberration Test), February 1998. Pteeton, R Jn W . Au, M A . Bender, J.G. Brewea, A.V. C am s, LA. Heddle, AJF. McFee, S. W olif and J.S. Wasaom (1981) Mammalian in vivo and in vitro cytogenetic assays: s report o f the Geae-Tcx Program, Mutation Research, 87:143-188. Scott. D., N.D. Danfbrd, B J. Dean and D J. Kirkland. 1990. Metapbase Chromosome Aberration Assays in Vitro. In: Bade Mutagenicity Tests: UKEMS Reconunended Procedures. DJ Kirkland (ed). Cambridge University Press, New York, NY. Swierenga SMIL, LA. Heddie, E.A. Sigal, J.P.W. Gfiman, ILL Briffinger, GJL Douglas ami B i t Nestmann (1991) Recommendedprotocols bared on a survey ofcurrentpractice in genom xidty testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese lung and human lymphocyte cultures, Mutation Research 246:301-322. 14.0 APPROVAL STUDY MONITOR 2/2S-/PO DATE (Print or Type Name) STUDY DIRECTOR BIORELIANCE STUDY MANAGEMENT DATE Protocol No. SPGT331 F ebruary 2 3 ,2MS J o t9 BioReliance Study No.: AA26XN.331.BTL - 40 - # Bio Reliance fleraneftrlivMUtennkklleelleaitciiliAAiHteteMeu*t Company Sanitized. Does not contain TSCA CBI
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