Document 6w3Q3197VGLyJDdz1yg5031gg

; ARDE-2 120 CORNING Hazleton EET MUTAGENICITY TEST ON T-6295 1 MAY 27 1996 y Co, My IN AN IN VIVO MOUSE MICRONUCLEUS ASSAY "<U/COLS EINALREPORT AUTHOR Hemalatha Murli, Ph.D. PERFORMINGLABORATORY. Coming Hazleton Inc. (CHV) 9200 Leesburg Pike Vienna, Virginia 22182 LABOPRR OJEA CTIT DENTO IFIR CATY ION CHV Study No.: 17403-0455 SUBMITTTEDO 3M 3M Center, Building 220-2E-02 St. Paul, Minnesota 55144-1000 STUDY COMPLETIONDATE May 23,1996 CHV Study No.: 17403-0455 1of26 01507 CORNING Hazleton QUALITY ASSURANCE STATEMENT Project Title: In Vivo Mouse Micronucleus Assay Project No.: 20996 Assay No.: 17403 Protocol No.: 455 Edition No.: 17 Quality Assurance inspectionsofthe study and reviewofth final reportofthe above referenced project were conducted according to the Standard Operating Proceduresofthe Quality Assurance Unit and according to the general requirementsofthe appropriate Good Laboratory Practice regulations. Findings from the inspections and final report review were reported to management and to the study director on the following dates: _ Findings R " Harvest/03/21/1996 Draft Report Review/05/15,16/1996 Final Report Review/05/23/1996 03/21/1996 05/17/1996 05/23/1996 C. Smith C. Orantes C. Orantes C alles 4 AL `Quality Assurance Unit ate Kel CHV Study No.: 17403-0-455 : 2 01503 `CORNING Hazleton STUDY COMPLIANCE AND CERTIFICATION The described study was conducted in compliance with the Good Laboratory Practice regulations as set forth in the Foodand Drug Administration (FDA) Title 21ofthe U.S. Codeof Federal Regulations Part 58, issued December 22, 1978, (effective June 20, 1979) with any applicable amendments. There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrity ofthe study or the interpretationofthe test results. `The raw data have been reviewed by the Study Director, who certifiesthatthe evaluationofthe test article as presented herein represents an appropriate conclusion within the context of the study design and evaluation criteria. All test and control results in this report are supported by an experimental data recordandthis record has been reviewed by the Study Director. All raw data, documentation, records, protocol and a copyofthe final report generated as a resultofthis study will be archived in the storage facilities of Corning Hazleton Inc. for at least one year following submission of the final report to the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned `materials retained in the storage facilities of Corning Hazleton Inc. for an additional period of time, or sent to a storage facility designated by the Sponsor. Submitted By: Study Director: Nowstat, Mui: `Hemalatha Murli, Ph.D. `Mammalian Cytogenetics Department ofGenetic and Cellular Toxicology 5 Jaz)9e Study Completion Date CHV Study No.: 17403-0-455 3 01809 CORNINGHazleton TABLE OF CONTENTS PageNo. SUMMARY .....onnnninnneannnneaaainseeasssiisseessiinneeesnnnnnees6 10 SPONSOR ....ooeeiiiieiiieieseeeisie T 20 MATERIAL(TeStATClE) .....ooroneoeirenniineeeeniiineeeee 21 Clients ldentification 22 DateReceived 23 PhysicalDescription 24 GeneticsAssayNo. nine7 30 TYPEOFASSAY .....ccooiiiiiiiiiiiiiiienineninenineeineae7n 40 PROTOCOLNO........eeeeeiiiieeaniiieeessiiieeeeeeenniine7e 50 STUDYDATES......coeoivummeaaninnnnaanninnnnssnninennssnninnneessT 5.1 Initiation Date 52 ExperimSetanrttDaatle 53 Experimental Termination Date 60 SUPERVISORY PERSONNEL 6.1 StudyDirector 62 Laboratory Supervisor ............ccceiiiimremeniiinnesnnnnineees? 70 OBJECTIVE .........ooiieiiiinieiiiieeeaiineeeeniineeeennineees 1 80 MATERIALS ........oiiiiiiiiniiinniinsninsineeieeeieene8e 90 SOLUBANIDSLTABIILTITYY ....0...iiieeenniiineannineneenineeee 8 100 DOSERANGEFISTNUDDYITNG .....oeviiiinreniiineeanniinnneeneenn 10.1 Dose Selection 102 Dosing Information 103 ResanduIntelrprtetatsion 104 Conclusion CHV Study No.: 17403-0455 : 4 01310 `CORNINGHazleton 110 DOSERANGEFINDING STUDYII.......vevveeeniiinnnneninneeen1n 11.1 Dose Selection 112 Dosing Information 113 ResanduIntelrprtetatsion 114 Conclusion 120 MICRSO TUDN Y..U ...C .oovL virE eniiU iieS essnininessnniinnennnn13 12.1 DoseSelection 122 Micronucleus Assay Dosing Information 13.0 BONEMARROWHARVEST,SLIDE PREPARATIONAND ANALYSIS ........14 140 EVALUATION CRITERIA ......oooviiiniinaniiianieniniinniiennnnennn 15 141 General 142 DataPreseanndtInatetrprietoatinon 150 RESULTSANDINTERPRETATION ..........oooriiniiinnnniiinnnnnn1nSn HO CORCUUBION 11000ceccosssrssermsivssssmmorissssssinsssasssonssnesssld 178 REFERENOER .vs00riieosnsriserssstressnttpsrresorsoprrssessonsansanatl 180 DEVFI ROMA THT ESII GNEDOPRONTOCS OL .........oivienniennnne 17 19.0 EXPE DATR ATAI BLEM S...E ....N ....T ... ee 1B CHV Study No.: 17403-0-455 5 01811 CORNING Hazleton SUMMARY Mutagenicity Test on T-6295 in an Jn Vivo Mouse Micronucleus Assay `The objectiveofthis in vivo assay was to evaluate the abilityofthe test article, T-6295, to induce `micronuclei in bone marrow polychromatic erythrocytesof Crl:CD-1%(ICR) BR mice. Inthedose selection study, the test article was suspended in deionized water and dosed by oral gavage at 500, 1630, 2750, 3880 and 5000 mg/kg, andthe dose selection study was repeated testing dose levelsof700, 1000, and 1300 mg/kg. Six animals (three males and three females) were assigned to each dose group. Animals were observedforthree days afer dosing for toxic signs and/or mortality. Based on the resultsofthe dose rangefinding study, the maximurn tolerated dose was estimated as 950 mg/kg. In the micronucleus assay, the test article was suspended in deionized water and dosed by oral gavage at 237.5, 475, and 950 mg/kg. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups, euthanized approximately 24 hours afier dosing, were included in the assay. The animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extractionofthe bone marrow. The test material, T-6295, did not induce a significant increase in micronuclei in bone marrow `polychromatic erythrocytes under the conditionsofthis assay and is considered negative in the mouse bone marrow micronucleus test. CHV Study No.: 17403-0455 6 01512 `CORNINGHazleton Mutagenicity Test on T-6295 in an I Vivo Mouse Micronucleus Assay 10 SPONSOR: 3M 20 MATERIAL (Test Article) 21 Clients Identification: T-6295 22 Date Received: January 22, 1996 23 Physical Description: Off-white mixtureofpowder and flakes 24 Genetics Assay No.: 17403 30 TYPEOF ASSAY: In Vivo Mouse Micronucleus Assay 40 PROTOCOL NO.: 455, Edition 17 50 STUDY DATES 5.1 Initiation Date: January 22, 1996 52 Experimental Start Date: March 13, 1996 53 Experimental Termination Date: April 18, 1996 60 SUPERVISORY PERSONNEL 6.1 Study Director: Hemalatha Murli, PhD. 62 Laboratory Supervisor: Monica Vegarra, B.S. 70 OBJECTIVE The objectiveofthis in vivo assay was to evaluate the ability of the test article, T-6295, to induce micronuclei in bone marrow polychromatic erythrocytesof Crl:CD-1%(CR) BR mice. This study was conducted using modificationsofthe procedures suggested by Heddle etal. (1983). CHV Study No.: 17403-0455 7 01513 `CORNINGHazleton 80 MATERIALS Adult male and female mice, strain Crl:CD-1%(ICR) BR, were purchased from Charles River Laboratories, Portage MI. This healthy, random bred strain was selected to `maximize genetic heterogeneity andatthe same time assure access to a common source. `The protocol for this study was approved by the CHV-ACUCpriorto the initiation of dosing. Animals were housed sevenpercage during quarantine, and housed fivepercage at randomization. The temperature and relative humidity were maintained at 72 + 6 F and 55+ 15%, respectively, except on March 10 and 16, 1996,forthe second dose rangefinding study, when the relative humidity was recorded as 36.1% and 39.0%, respectively. A 12-hour light/12-hour dark cycle was maintained. A commercial diet (Purina Certified Laboratory Pellets # 5002) and water were available ad libitum for the durationofthe study. The feed was analyzed by the manufacturer for concentrations ofspecified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed on a retrospective basis for specified `microorganisms, pesticides, alkalinity, heavy metals, and halogens. Sanitized caging was used for housing the animals. Personnel handling animals or working within the animal facilities were required to wear suitable protective garments and equipment. Animals were quarantinedforseven days before being placed on study. Animals were randomly assigned to study groups and were individually weighed prior to dosing. All `animals were dosed based upon the individual body weights. Animals were uniquely identified by ear tag. Dose or treatment groups wereidentifiedby cage card/label. Attheterminationof the study all surviving animals were euthanized by CO, inhalation, followed by penetrationofthe thorax. Any extra animals not used for the study were euthanized by CO, inhalation, followed by penetrationofthe thorax. 90 SOLUBILITY AND STABILITY `Thetest article, T-6295, was supplied as aoff-white mixtureofpowder and flakes. The solubilityofthe test article was evaluated in com oil, 0.5% high viscosity carboxymethyl cellulose, and acetone: com oil, 20%:80%, v:v. Suspensions suitable for dosing was not obtained with these vehicles. The test article was crushed in a mortar and pestle and a translucent, off-white suspension was obtained in deionized water, that passed easily through an 18G needle after mixing in a Tissumizer. Deionized waterwasthe vehicle ofchoice for this assay. The stabilityofthe test materialunderthe dosing conditions of this assay is the responsibilityofthe sponsor. CHV Study No.: 17403-0455 8 001514 CORNING Hazleton 100 DOSE SELECTION STUDIY 10.1 Dose Selection Dose levels of 500, 1630, 2750, 3880 and 5000 mg/kg were administered by oral gavage for the first dose rangefinding study. 102 Dosing Information `Theanimals used in the first doserangefindingassaywere dosed on March 13, 1996. The weight rangeoftheanimals used in thefirstdose range finding assay was 30.8- 40.3 and 24.4 - 30.8 grams, forthe malesand females, respectively. Dosing solutions were preparedjust prior to dosing and were prepared by making 2250 mg/ml stock for the high dose (5000 mg/kg ). This was prepared by adding deionized water (Lot # 19, prepareadt CHV) 0 3.5025 g ofT-6295 up to a volumeof 14.0 mi, resulting in a cloudy cream colored suspension. This was `mixed for =2 minutes with a Tissumizer. Dilutions ofthis stock were prepared for the 3880, 2750, 1630 and 500 mg/kg dose levels. All dosing stocks were placed on magnetic stir plates during the dilution and the dosing procedure. Dosing was achieved using 2 20.0 ml/kg dosing volume. All animals were nine `weeks and two days old at the tiofdmosieng. An outlineofthe dosing scheme is found in the following table. Dosing Scheme for Dose Rangefinding Assay I -- T T6295reamme Malen Femat le 500 mg/kg 33 1630 mg/kg 303 2750 mghkg. 33 3880 mghkg 303 5000 mgrkg 33 A total of 30 animals was used in this assay. All doses given wereonan acute (one-time only) basis. 103 Results and Interpretation Al animals were examined after dosing and daily throughout the durationofthe study (three days) for toxic effects and/or mortalities. All animals appeared CHV Study No.: 17403-0455 9 01815 `CORNING Hazleton normal immediately after dosing. The toxicity observations are shown in the following table: Animal Observations for Toxicity for Dose Rangefinding Assay I Time Afler Dosing (hows) _ = ig Lg 2 = Dose Level (mg/kg) 500 1630 2750 3880 5000 500 1630 2750 3880 5000 500 1630 2750 S00 1630 _ Observations Normal Slightly hypoactive Hypoactive with dyspnea Hypoactive with dyspnea _Hypoactive with dyspnea, lacrimation and tremors Normal Hypoactive Allanimals found dead, except female # 6978,whichwas hypoactive, cold to the touch, pale and had tremors All animals found dead _All animals found dead Normal Males# 6963 and 6967, and femal#e 6984 found dead, all other animals appeared normal Female# 6978 found dead Nomul All remaining animals appeared normal `The mortality data for this assay are summarized in the following table: Summary of Mortalities Within 3 Days in Mice Dosed Acutely with T-6295 for Dose Rangefinding Assay I -- 500 mT g/kg ream M[a7le e n Fem[t 3a]le 1630 mg/kg 2 173 2750 mg/kg. 33 33 3880 mg/kg 13 33 5000 mg/kg 33 33 CHV Study No.: 17403-0455 10 : 01316 CORNING Hazleton 104 Conclusion Based on these results, the maximum tolerated dose could not be determined. 110 DOSE RANGEFINDING STUDY It 111 Dose Selection Dose levels of 700, 1000 and 1300 mg/kg were administered by oral gavage for the second dose rangefinding study. 112 Dosing Information `The animals used in the second dose rangefinding assay weredosedon March 16, 1996. The weight rangeofthe animals used in the second dose range finding assay was 32.7 - 39.8 and 25.0 - 31.3 grams, for the males and females, respectively. Dosing solutions were preparedjust prior to dosing andwereprepared by `making a 65.0 mg/ml stock for the highdose (1300 mg/kg ). This was prepared. by adding deionized water (Lo#t 19, prepared at CHV) to 780.0 mgofT-6295 up toa volume of 12.0 ml, resulting in a cloudy cream colored suspension. Dilutions ofthis stock were prepared for the 1000, and 700 mg/kg dos levels. All dosing stocks were placed on magnetic stir platesduringthe dilution and the dosing procedure. Dosing was achieved usia2n0.g0 ml/kg dosing volume. All animals were nine `weeks and five days old at the timeofdosing. An outlineofthe dosing scheme is found in the following table. Dosing Scheme for Dose Rangefinding Assay II --T6T2r9e5atment 700 mg/kg 1000 mg/kg 1300 mg/kg MaleFemale 33 33 33 A totalof 18 animals was used in this assay. All doses given were on an acute (one-time only) basis. CHV Study No.: 17403-0455 n 01517 CORNINGHazleton 113 Results and Interpretation Al animals were examined after dosing and daily throughout the durationofthe study (three days) for toxic effects and/or mortalities. All animals appeared normal immediatelyafterdosing. The toxicity observations are shown in the following table: Animal Observations for Toxicity for Dose Rangefinding Assay IT Time After Dosing (hors) _ =1 4 43 =70 Dose Level (mg/kg) 700 1000 1300 700 1000 1300 700 1000 1300 700 1000 1300 Observations Normal Slightly hypoactive _Hypoactive and hunched Normal Nomal Male # 7259 found dead, all remaining appeared hypoactive and hunched Normal Female # 7255 found dead, al others appeared normal All remaining males and females #'s 7263 and 7261 found dead, one surviving female appeared normal Normal All remaining appeared normal _All remaining appeared normal `The mortality data for this assay are summarized in the following table: Summary of Mortalities Within 3 Days in Mice Dosed Acutely with T-6295 for Dose Rangefinding Assay IT -- 700 mT g/kg rcam Ma0l3een Fem0t a3le 1000 mg/kg 3 13 1300 mg 33 2 CHV Study No.: 17403-0455 : 2 01813 CORNING Hazleton 114 Conclusion Based on these results, the maximunn tolerated dose was determined to be 950 mg/kg. 120 MICRONUCLEUS STUDY 12.1 Dose Selection Based on results from the dose rangefinding study, dose levels 0f237.5, 475, and 950 mg/kg were selected for testing in this study. 122 Micronucleus Assay Dosing Information `The animals used in the micronucleus assay were dosed on March 19, 1996. Cyclophosphamide (CAS # 6055-19-2; Sigma, Lot # 44H046), the positive control, was solubilized in sterile deionized water (Lot # 19, prepared at CHV) `and was administered by oral gavage at 80.0 mg/kg. The vehicle control, deionized water (Lot # 19, prepared at CHV), was administered concurrently with the test article at a volumeof 20.0 mUkg. The weight rangeofthe animals used in the micronucleus assay was 29.9-37.0 and 23.1-29.2 gramsforthe males and females, respectively. The dosing solutions for the assay were prepared by `making a 47.5 mg/ml stock for the high dose (950 mg/kg ). This was preparbeyd adding the vehicle 10 2.3751 gofground up T-6295upto avolumeof50.0 ml, stirring with a spatula and a cloudy cream colored suspension was obtained. Dilutionsofthis stock were prepared for the remaining dose levels. All dosing stockswereplaced on magnetic sir plates during the dilution and the dosing procedure. A second groupofanimals (designated Secondary Dose Group) was also assigned to the study and was dosedwiththe high doseofthe test article. These animals were only used in the assay as replacements for any which died in the primary dose group. Ten animals (five males and five females)wererandomly assignteod each dose/harvest time group. Vehicle and positive control groups, euthanized approximately 24 hours afier dosing, were included in the assay. The animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extractionofthe bone marrow. An outlineofthe dosing scheme is found in the following table: CHV Study No.: 17403-0455 13 01519 CORNINGHazleton Dosing Scheme for Micronucleus Assay Treatment T-6295 237.5 mg/kg 475.0 mgikg 950.0 mg/kg Vehicle Control,deionized water, 200mlkg ~~ Positive Control, Cyclophosphamide, 80.0 mghkg NumberofAnimals Assigned Primary Dose Groups Secondary Dose 24Hr 48Hr 72H Growp* MF MF MF MF 55 55 55 a. 55 55 55 - 55 55 55 5s 55 -- -- - 55 - - - - - * Theanimalsassignedtothesecondarydosegroups weredosedandwereonlyusedto replace animals which died in the primary dose group at the high dose level. All extra animals not used as replacementswereeuthanized at the completionofthe trial. A total of 120 animals was usedinthis assay. The ageoftheanimalsat the time of dosing was nineweeksandoneday. Volumes dosed were 20.0 ml/kg (except positive control) and were based upon individual animal weights. Volumes dosed to positive control were 10.0 mi/kg. 130 BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS At the appropriate harvest time, the animals were euthanized by CO, inhalation followed. by penetrationofthe thorax. The adhering soft tissue and epiphysesofboth femora were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 - 5 mlbovine serum (one tube for each animal). Following centrifugation to pellet the tissue, the superatant was removed by aspiration and portionsofthe pellet `were spread on slides and air dried. The slides were fixed in methanol, and stained in May-Grunwald solution followed by Giemsa (Schmid, 1975). The air-dried slides were coverslipped using Depex mounting medium. The slides were coded for analysis, and scored for micronucleiandthe polychromatic erythrocyte (PCE)tonormochromatic erythrocyte (NCE) cell ratio. Standard forms were used to record these data. One thousand PCES per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleatedcells based on the total PCESs present in the scored optic field. The normal frequencyof micronuclei in this Crl:CD-1%(ICR) BR strain is about 0.0 - 0.4%. CHV Study No.: 17403-0455 : 14 01320 `CORNING Hazleton `The frequency of PCE versus NCEs was determined by scoring the number ofPCES and NCE observed in the optic fields while scoring the first 1000 erythrocytes. 140 EVALUATION CRITERIA 141 General The criteria for the identificationofmicronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the sizeofthe PCE. The unitofscoring. was the micronucleated cell,notthe micronucleus; thus the occasional cell with `more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color `ofPCEs and NCES (bluish-grey and red, respectively). 142 Data Presentation and Interpretation Data are summarized by sex and dose groupsforthe different time points. Individual animal data are also presented. The analysisofthese data was performed using an analysisofvariance (Winer, 1971) on either untransformed (when variances are homogeneous) and rank transformed (when variances are. heterogeneous) proportionsofcells with micronuclei per animal.Ifthe analysis ofvariance was significant (p<0.05), a Dunnett' t-test (Dunnett, 1955; 1964) was used to determine which dose groups,ifany, were significantly different from the negative control. Analyses were performed separately for each harvest time and sex combination. The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCES, or the detection ofa reproducible and statistically significant positive response for at leastone dose level. A test article that induced neithear statistically significant dose response nor a statistically significant and reproducible increase at one dose. level was considered negative. In either case, the final decision was based on scientific judgment. 15.0 RESULTS AND INTERPRETATION Al animals were observed immediately after dosing and periodically throughout the durationofthe assay for toxic symptoms and/or mortalities. All animals in the vehicle and positive control groups appeared normalafterdosing and remained healthy until the appropriate harvest times. All test article dosed groups appeared normal immediately and CHV Study No.: 17403-0455 15 601321 `CORNINGHazleton `about one hour after dosing. The toxicity and mortality observationsofthe test article dosedanimalsare shown in the following table: Animal Observations for Toxicity/Mortality Time After Dosing (hours) =2 wit 1 Dose Level (mg/kg) 237.5 475.0 950.0 2375 475.0 950.0 237.5 4750 950.0 Observations Normal Normal Males #7304 (24 hour harvest), 7314 (48 hour harvest), and 7305 (secondary); and females # 7369 (48 hour harvest), al 72 hour harvest, and 7332 (secondary) were found dead. Two males went into convulsions upon opening of their cages but recovered after a few minutes. All others remaining appeared normal. Normal Female #7343 (48 hour harvest) found dead, all remaining appeared normal Males # 7300 (48 hour harvest) and 7286 (secondary), and females #7368 (48 hour harvest) and 7379 (secondary) were found dead. All remaining appeared normal. Normal All remaining appeared normal Male # 7282 (secondary) found dead, all remaining appeared normal `The test article, T-6295, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observedinthe vehicle controls in either sex or at anyofthe harvest times. Bone marrow toxicity was manifested by the significant reduction in the PCE/NCE ratiosofthe 48 and 72 hour males from the 237.5 and 950 mg/kg dose groups, 72 hour males from the 475 mg/kg dose group, 48 hour females from the 475 mg/kg dose group, 72 hour females from the 950 mg/kg dose group, and the positive control females. The positive control, cyclophosphamide, induced significant . increases in micronucleated PCE in both seasxcomeparsed to the vehicle controls, with `means and standard errors of3.36% 0.97% and 4.52+% 0.72%forthe males and females, respectively. The data summarized by dose group are presented in Table 1 and individual animaldataare found in Tables 2 through 7. Historical control data are presented in Table 8. CHV Study No.: 17403-0455 16 01322 CORNING Hazleton 160 CONCLUSION The test material, T-6295, did not inducea significant increase in micronuclei in bone `marrow polychromatic erythrocytes under the conditionsofthis assay and is considered negative in the mouse micronucleus assay. 17.0 REFERENCES Dunnett, C.W.: A multiple comparisons procedure for comparing several treatments with acontrol. J. Am. Statist. Assoc., 50:1096-1121, 1955. Dunnett, C.W.: New tables for multiple comparisons with a control. Biometrics, 20:482491, 1964. Heddle, J.A., Hite, M,, Kirkhart, B., Larsen, K., MacGregor, J.T, Newell, G.W. and Salamone, M.F.: The inductionofmicronuclei as a measureofgenotoxicity. Mutation Res, 123:61-118, 1983. Schmid, W.: The micronucleus test. Mutation Res., 31:9-15, 1975. Schmid, W.: The micronucleus testforcytogenetic analysis. Chemical Mutagens: Principles and Methods for Their Detection, Vol. 4 (A. Hollaender, ed). Plenpp.u31m-5,3, 1976. Winer, BJ: Statistical Principles in Experimental Design, McGraw-Hill, New York, Second Edition, 1971 18.0 DEVIATIONS FROMTHE SIGNED PROTOCOL 1. On March 10and 16, 1996,forthe second dose rangefinding study, the relative `humidity was recorded as 36.1%and 39.0%, respectively. This did not affect the `animals or the integrityofthe study. 2. The actual dose levels achieved for the dose rangefinding study were 3880 and 1630 mg/kg and not 3875 and 1625 mg/kg, respectively. These doselevelsare very close to the dose levels (<0.05% difference) mentioned in the protocol `amendment and hence there was no impact on the integrityofthe study. CHV Study No.: 17403-0-455 | 1" 01323 `CORNINGHazleton 19.0 EXPERIMENT DATA TABLES CHV Study No.: 17403-0455 18 01824 CORNINGHazleton `SPONSO3RM: TEST ARTICLE: T-6295 ASSAY: 17403 TREATMENT DOSE conTRoLS VEHICLE POSITIVE War CP8OOmghy TABL1E MICRONUCLEUS DATA SUMMARY TABLE HTAIRMVEEST ME9A%NMOIF C1R00O0NPUERCALNEAIPTMCE2EADSsLE. (HR) MALES FEMALES TOTAL 2h 0200 0144006 008400 | 20h 3364091 42407 3942060| RATMIEOAPNCESNE.CE MALES FEMALES 02008 038s00S 0774008 0554000 TESTARTICLE 27Smpke 24h Ah Th Some 2h "th 7h 900mpks 2h ath Th 0064004 0164007 004002 0162005 00400 0144005 02400 0200 0oa0n 010005 0me0R 2E00s 004004 005400 omsos 0@s0@ 0ma00 0ma0m 0082002 | 009400 | 008a0m | 00940m | 000m| ON+0ss | 002400 | 0064003 | 00ss0% | 057401 O4sa00Re 039s0n 072011 OTa005 0200 0562013 0sesourv 017soss 0s24010 0802010 000M 059008 0374007 040s0R 05+008 owsoln 037sossee *+*SiSginginfiifciacnatnltylgyresastertthhaannthetchorcroersrpeosnpdoindnigvneghviechlieclceocnotnrtoo,lp,<p0<.00055. Ouftouraonimfals Outof threeanimals : CP=Cyclophosphamide CHVStudy No.: 17403-0-455., 19 01325 CORNING Hazleton TABLE2 'MICRONUCLEUS TEST- INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO: 17403 TREATMENT NAUNMIBMEARL | oPTCBNCS PRCAETNICOE 24 HOUR HARVEST MALE VEHICLE CONTROL POSTIVE CONTROL. TESTARTICLE Water Pao mpg 75 moe 4150 more 9500mpg aas mo' 0os6ts [[II o1e0 no 099 am s 3wosost m mmo eow 0osm nse om mnneso2 oosns 551% o1 00931 i 0 03 annM 2' 01136 m moo o3 o07s3 0 ' 0s m nee o0 oossss w 795 o1 023 mo 026 C+ PSe=coCnydcalroyphdoossephgarmoiudpeanimal PNCNE ==PMoilcyrcohnurcolmeautsic eryhrocyte #`NMCEN=PNComEm=ocMhircotmoaniuccieartyetdhrPoCcyEtSe CHV Study No.: 17403-0455 : 20 01826 (CORNING Hazleton TABLE3 MICRONUCLEUSTEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M TEST ARTICLE: T-6295 ASSAY NO: 17403 TREATMENT NAUNMIMBAELR | pPTCNB<S PRCAETNICOE HOUR HARVEST FEMALE VEHICLE CONTROL POSITIVE CONTROL. TEST ARTICLE ater cPa0 mors ws mpg 50mys 5500 mpte m m2 o 0o3r6 wnooo 23 0o8s6s noo Los m mo a2% o0ms m ne ee3% oeme ne 0 ow nmses 2! 0o7r mn0 eo o0&n3 we 08 a [Eo Y o0ss nneeo2 0o5s3s wo oi 7m3s 0: 00366 n nel oo0 0oas nso 03s CMNP= =CMyicclroopnhuocslpehuasmide P# CMEN=PPClEysch=roMmicartoincuceriyetahtreodeyPieCES NCE = Nommochromatic erythrocyte CHV Study No.: 17403-0455 21 01827 `CORNINGHazleton TABL4E MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M . `TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT NAUNMIMBAELR 2PTICE4S RPACTENICOE 48 HOURBARVEST MALE TEST ARTICLE 275 mete 250 mpg 9500 mots [mIe o0s3s5 [BiIz i 004870 mo oss 7m 306 o1 0o6ss n io ro1 0o8 a mo on n weo o 00537 7m0e1 e 2i 0o3n6 mise ***ASneicmoanldfaoruynddosdeegardoup animal MPCNE==MPilcyrcohnruocmlaetuisc eythrocyte #NCMENP= CNEomsmo=cMhircormoantuiccleeactyethdroPcCytEes: CHV Study No.: 17403-0455 2 01328 `CORNING Hazleton TABLES MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M `TEST ARTICLE: T6295 ASSAY NO: 17403 TREATMENT NAUNMIBMEARL CPTCEN0y pReEA:TNICOE 48HOURHARVEST FEMALE TEST ARTICLE 275 mgs 150 mors 9500make mnseo1 001396 n my mo o o0ns nso 100 m 41 o' 0o3s1 mBsaee ' 02 wo 030 7[3A ' oosat nTeer mo oa *MNAni=mMailcfroounnucdldeeuasd P# CMEN=PPCoElsyc=hrMoimcariocnuecyitahioecdyPtCeES NCE = Normochromati erythrocyte CHV Study No.: 17403-0455 : 23 91829 `CORNINGHazleton TABLE MICRONUCLEUSTEST - INDIVIDUAL ANIMAL DATA SPONSOR: 3M `TEST ARTICLE: T-6295 ASSAY NO.: 17403 TREATMENT ANNUIMMBAELR |PTPCVEBS CREANTICOE 72 HOUR HARVEST MALE TEST ARTICLE 275 mye 150mya 9500 mas ne15 !' oonn nBm oo0 o05n2 ms oo oor a1ms 2' o0i2ss n9e% 3i o0i4s0 neo oa n meso2 0o2n6 m70e5 01 0o3o1s i ' o10 PCMNE ==PMoilcyrcohnruocmleaitsic erythrocyte N#MCEN=PNCoEmSo=cMhircormoanutcclratyerdocPyCtEeS CHV Study No.: 17403-0455 . 24 01330 CORNING Hazleton TABLE? MICRONUCLEUS TEST- INDIVIDUAL ANIMAL DATA SPONSOR: 3M `TEST ARTICLE: T-6295 ASSAY NO: 17403 TREATMENT TT aANeIMAL NUMBER qPCTEeNY aRAmTIoO PENCE 72HOURHARVEST FEMALE TEST ARTICLE 315 mere 750 meg 9500 mys Enta 3' ooozr nBsmi I' 009020 nooo 04s 3m 46 o! 0o3n m no oo2 0023s8 ms ' oi mms ao0 oonr m nee o1 o0n3 nw 0 ols M+ SNec=onMdiacrryonduocsleeugsroup animal #PMCEN=PCPoElsyc=hMriocmartoincuceiryattherdocyPtCeES 'NCE = Normochromatic erythrocyte: CHV Study No.: 17403-0455 : 25 01831 CORNING Hazleton TABLES MOUSE MICRONUCLEUS HISTORICAL CONTROL DATA 7/95 THROUGH 12/95 FOOLEDVEICLECONTROLS My MAAVXG N eMICRONUCLEATED PCEs PER1000 PCE MEANOF 1000 PER ANIMAL 2 SE. MALES FEMALES TOTAL RATIO PENCE MEAN +E. MALES FEMALES 000 0o7oax00m " om 008102240008 @ oat 031 00802107005| 0o5s0s00 Pl " ox 0sw17z0o0s Pl POCSreIoTpIhoVsEpChaOnNiTdeR,O0L.S0 mye MwAyX ANVG 2s0e0 361094020 613560 310210245 s24a ooann 342612014| 057i4o000 001090 058i0o 02 P`CNCEE==PoNloymcohcrhormoatmiacteircytnhyrotchyotceyte CHV Study No.: 17403-0-455 . 2 01332 CPHRVOTOSCTOULDYNoN.O. 435E,DITION 17 CORNINGc:Haz won IN VIVO HOUSE MICRONUCLEUS ASSAY CLoarbnoirnagtorHyazlPertaocnticIenc.(GL(PC)HV)Regwuillalticoonnsd.uctThtihsisprsottuodcyoli,n ccroimtpilciaalncephwaiset(hs)Gooodf Atshesuwcaonrckeininpraocgcroersdsancaendwitthhe fSOiPnsslatreCpoorritniwinlglHabzelestuobnjecItnc.to aThueditstubdyyQwuialllitybe conducted by CHV at 9200 Leesburg Pike, Vienna, Virginia 22162. PART 1. SPONSOR INFORMATION AND APPROVALS I. SPONSOR IDENTIFICATION Company Name: __BIN address: St. Rud, mA 11. est asmieus somvipreatron: _7 - 6295 II. TEST ARTICLE ANALYSTS CDheatrearcmtienraitsitoincsofasthedeftienstedarintictlhee GsLtPabrileigtuylatainodnstheistetshte arretsipcolnesibility of the Sponsor. IV. NOTIFICATION OF REGULATORY SUBMISSION mIunstordbeernottoifcioemdplyifwiatllh torhepaGrLtP orfegaulasttiuodnys,iscoinnstuelndteidngfolraborreagtuolraiteosry rSuebgmuilsastioorny. revCiHeVw.mainPtlaeiansse ainmdaisctaetre wshcihcehdulaegenocfy,stuifdieasny,whmiicghhtfalrlecuenidveer the results of this study: -- = perermin-- et 2 mn 3 -- mama CO mam wer C0 vow 53 oso omm 41s : 108 10 01833 otoco. Fo. 433, Eoreron 37 -- `CORNINGHazleton Proposed Expecioantal Termination paces [-- stots Disastors TLR 0 _-- - Stun CLs sacer 2/145 01834 PROTOCOL HO. 455, EDITION 17 CORNINGHaz! cron PART 2 - STUDY PROTOCOL IN VIVO HOUSE MICRONUCLEDS ASSAY I. omerrve cpTlhoaelsyccoohbgejeoenmciatctiivceacteoifrvyittthhyrisocsynstdteuddiysstrieusmptctieololnesvaofliunatmthoeeums&iettoebtsointce amarapttpiracrolawetuifsmorviinvo. 1m. peemarions | cMhircornoonsuocmlee(uss): aandsomrallofcahcreonmtartiicn cbohdryo,moscoomnssifsrtaignhgenotf(se)n,tiwrehich lcahgrsanobsechnien(ds)atanmditoftriacgneannta(psh)asem.ay nAoftterbeteilnocplhuadseed,inthetshee daughter ncuyctloepil,asma.nd may form single or multiple micronuclei in the 11. mariomss aThgeentmsicraondnuctleesutsartteisctlecsanwhsiecrhve inasteraferraepiwditshcrneoernmaflormictloatsitcogecneilcl dFiovrimesdionfro(mscchmhirdo,mos1o9m75e:s Hoerddclheroemocsoalm.e, fr19a8g3a)e.ntsMilcerftomubcelheiindardeuring a(nSacphmhiads,e a1n9d75)c.an Ibne tshciosreadssdauyr,ingpoliynctherropnhaasteicbeercyatuhsreoctyhteeyspe(rFsCEiss)t Diunrithneg mbaotneurmatairornowfarroem esscyotrherdobfloarstthetoperreystehnrcoecyotfemitcheronnuucclleeiu.s is Dexettreucdteido,n wohfilmeicrmoincruocnluecileiin,noinf-npurcelseenatt,edrceemlalisn iisn tthheuscyoplasa. ifnacitlrietaatteedd,celalndpotpinuelatiinovnoslveids ienlimsienaartcehdi.ng Tfeosrtmaerttaipchlaesse spreads Calfafsetcotgienngicspaignednltesficbaenrbefudnectteicotnedor thfroorumgahtimoincraosnuwcellleusas induction (schnia, 1975). . warms A saimls Y1o0unwgeekasdulotldmaaltethaendtifmeemaloef mdoisciengo.f wtihlelICbeR psutrracihna,sed8fSprroamguCeh-aDralveiseyR.iveIrnc.LaboTrhiastorsitersa,infhca.s. beorenHasrellaencted to pr 3 of 20 C01835 PROTOCOL NO. 455, EDITION 17 CORNINGHazleton meanxsiurneizeaccgeesnsetitco &hetceormomgonenesioturycea.nd at the same tine 5. control articles a3C0nyccnlwoiipklhelo)spbehwiaalnldindiebnei(suCtsPe,erde8da0smbgytlhkeogr:sp)odsgoiaseviienvsges.cvoolnutTnrheoelvoefahritcilcele UCCshoeendtrsoaFlmoer atcrhoteuitcelteesatsw,ilaalrntdcicalncesoinscatunrdroewfinlttllhyebwesietalhdv,emnitntihesotretrevesedthicbiye vaaodrlntuiinnceilsewtiealrnelddnionttoametohxeucneteesdxpe2er0qiuamalelnkttgaolftonhreinrnaaelxsli.sguamvTahvgeoeludamonesdsing12 Sassdosmlauiytniiosantr.reatoviarotencrso,.fn0o.Ti5hi7e. vaeqnuiecoluesscgaernbeorrarlmleyihuysiecdeliisnlocshee v. Eemnmo seston A anima) sesbanery vASiLeiLvienapbpepleriLcoacoballgaeeteddCuAVrbiynSgOsePxqs.uevcialAnlntiimbnaeel,sfowlsilnlodlwewdib.lslhAobenusiehmdaolussuepdto ZSupeemtphoeorcFaaitsvudee,upnrdi7eo2:rF tv+hoe SfxFop:lelrhoiuwmmieinnggtiscIyln,iimteiSea5i7tico+nc.o15n%d;iscniiLooinagsni:es wc(ayPtculezeri,fwais1l2lCohrboeeuirasfviaalidilgihLbtal/bedoararakdt.orLyi&biCtchuoomm.wmer1c5Ti0h0ae2l)feaeaidnedistap RSaypndeaccloiycfzaieredbdobnhyse,atvhyeargmmaeancmoauplfnsao,cstpeahrfaiesarecsof,roirna,ncdocnhcsieponertcirinafsitceiesodns of npfieestttreioicseipndececast,ivehTehaebvayswiasmteetrfaolris.sapanelackliafylisieenddisabyii,esrmaomnoadriglpeyanliooognnesnas. Rbeiivnaglsplawcileld oben qsuewaerra:ntined for at ease 7 days before APanrcoiccmoearcldusirnsgws.ilclo ACbnoeinmiasnsigssigHwnaiezldllettoboenswtSeutidagyrhdeagdrrdouPprSsipoerartattoirnagsnodsoimng. wTCeheiesgyhtwsiT.lrleatbmaeennidtnoaslgesrdouwbpiaslslewdibleulpounnbeiqtuhieedleynitnidifidvieiencdtuiafbliyedcaangibemyalear iaberscasa. ass cot 10 001836 PROTOCOL NO. 455, EDITION 17 CORNINGHazleton aSnainmiatlasryorcawgoerskwiinlglwibtehiusnedt.he aPneirmsaolnneflacihlaintdileisngwill ebqeuirpemqeunitr.ed to wear suitable protective garments and B. Dose Selection Tmhaexihziugnhtodolseeratgeednerdoaslel.y wiTlhle hbeighsedloescetedshoasuld80Zproofductehe osfomeratiinodicoaftiPoCnEsoftotnooxmimcoicthyro(mea.tg.i.c deeraytthh,rocdyetpersession w(iNlClEs)n.ormaOlnley-hablefusaendd oasne-tqheuaritnetreromfeditahtise hanidghldoowsedose ltheevellsi,kelriehsopoecdtitvhealty.a wUeseek ocflaasthoiggehn wdiolsle ibencrdeeatseecsted, and is therefore recommended. Iffinndoingappsrtoupdyriactaen rbeangpeerffoirnmdeidn.g daThtea atroep adovsaeilatbelset,edainrantghee dose wrialnlgefbiendiisnsguedstausdyanwilalmenbdeme5n0t0.0 mg/kg. The dose levels tested DOSE RANGEFINDING STUDY Tghreoupdso.se Eraacnhgeoffindtihnegfisvteudygrwoiulpls wbiellcocnodnuscitsetd uofsin3gmaflieveantdrea3tment female mice. Group Designation and Treatment Regimens Group No. MNaulseber ofFeMmilcee Route DurDaavtsion 1 3 3 0 3 2 3 3 0 3 3 3 3 0 3 " 3 3 0 3 5 3 3 0 3 rss 5 of 10 01837 PROTOCOL G. NO. 455, EDITION 17 CORNING en Ttihhneajtecrtoviuaotsnee waoirfltliacdlbmeeinecimhspatlrroaayctetideo.rniswtiTihlcelsssbeprceoocsraitleusdegaovfoargaaeld.agaaivIsancgreti,hceiIoenvenhtave bsnoeitem.nineixssceteleredacttie20odnmblef/ockragustehfiosrheovyraasltarepgraovtcehegedeumroaens:dt cIPoTmhaemdomndionsriiosnuigtreasvtoilooefnmse.wilOlther mCiaontstetrraaismaslocfuwlisaldrlm,ingiessnuteber-aactluiltoyennsboetuhsastolamudbmyiilniibzesetdruarteidinonrosneeofoifnbtyrtahevfeenefdoo:ulsl,owTihneg test Ssioonllduviteviniotdnsu,:]woarbtoedcryo,rwne0.oii5gl%h.ss.aAlLinLDeo,saeni0s.La5el7vselasiqulweliolulsbecbeduorsabesessximgbmeaeestdehdybluycpeolanlutose protocal amendment. WBpiorldelypawzbeeeidgphartnesdpawhrieelldld jbauetstatpbarbkiieoenrntprtoiroermdopsetiornagtd.iorsiensbga.smiingDiossiodnlogusitnifgoonrsm(u0wl-i2altlionbe Bvoiurnsg). tomAeLrL animals will be euttanized 3 days after ceceiving a Tih0ne:halatahnteiiaoadnlusraFwtoiillollnovebodef otbhybesepresvnteasddcyr.adtaiiAolnnyimofafolrsthtweoixlitlchorsbaeixg.nesutahnandizmeodrtablyitCoyr wVTihTleLtdabCiehleynusebodebsaetsrosvaietgsintoeindmsatfeoorfththeoexiMcasxuibsmsyeumqmputoeTmnostleraesnvtdve/edogrenDmoesroeirctsa(lHiIatDs)i.ees. dDaotsaes sicomcizus stop Dosing Schedule and Route of administration NaHdaomrrivmneaislstltyrvaiatlniloancb)eutwseiplpdlroossibienagsutsreeeldgyim(e3sn4e,e (T4s8ai,bnlgel7e2behloouwr)s. after aSCodonmptirrnooislsitnraaatrtetilitoylne2so.fhotuAhrestottaeafsltteraofrtaid1cm3l0ien,iansitamrnaadltsiatonwilolf btehe SSStnnieoedaaclhs'Sacteoranelsaitsmmteeinmntbgergsroofuopf5.-m1a0lAenmsalaedasneditainedomnss3il1e0sgrwofiuelpmlaloefbse used may boSefoxidtchoeisteydsesaisss amsaxtpseaerccitoaanldd.araytThdtiohsseegbrigogruhopudpwoiwvlielthsnbdethedtohseneidgahniimfdaolsse aISnneycotwnhhdiiascrhygradooiusepewpigrlriloorupomtwoiiylslubtehbaewneaedsdaitase.ermirneTehpedlacsbeyeemetnhOtefs sthtFeuordy Srervery Precis peeperen solutes wii) be ass : of 20 01838 PROTOCOL NO. 455, EDITION 17 CORNING(GHHaazz]leton etmopxliocyesdi.gnsThaendamnoirmtaallsitwyi.ll be observed daily for NOMIER OF ANIMALS USED FOR MICRONUGLEUS ASSAY Harvest Times After Treatment Group No. Irestment 2(4MaHloeusrsand 4F8emHaoluerss) 72 Hours 21 VpeohsiictlieveCoCnotnrtorlol 55 ++35 ---- I 43 LMoewdiuDmoseDose 554455 5s4e5s 5s4i5s 5 High Dose 545 ses 54s Total sseess 1as5s4e1ass 1543s oral 25425 15415 15415 Tthhee reovuetnet otfhatadmtienstistarrattiicolne wcihlalracbeteroirsalticgsavapgree.cludIen doorsalinggavvaogleu,meIwililnjneocttieoxnceweidll 20be mle/mkpgl.oyedT.heseTheroutes otfheamdomsitnisctormamtoinonrouhtaevse bofeeandmsienliecstterdatiboencaufsoer tthhiesy atreest Pursoecdedaurree.intrOatvheenrourso,uteisntroafmuasdcmuilnairs,trastuibo-ncuttahnateoumsay be administrations or by feed: 54s D. Extraction of Bone Marrow Etuhtorhaaxn,asiaandwihlilndbeLiwmibthboCneOs, wiflolllobweedrebmyovpeedneftorratmiaornroowf the etxrtarnascfteirorne.d toThecemntarrirfouwgewiltlubebse fclounsthaeidninfgrom3-5thmel bbonoevianned . serun (one tube for each animal). 5. Preparation of Slides sFuolpleorwniantgantcewnitlrlifubgeatrieomnovetdo bpyelalestpirtahteiotnissauned, pothretions of the pellet will be spread on slides and sir-dried. MTahey-GsrluindvesaldwilSlolutthieonnbaendfiGxieedmsian, meatndhanporlo,tecstteadinebdy in msoluindetsingarewictohdecdovefrosrliapnsa.lysiFso.r control of bias, all ars . 7 of 10 01839 PROTOCOL F. NO. 55, EDITION 17 CORNINGNINHazlon Scoring the Slides Aannimaatlt.emptThewilflreqbeuemnacdyeoftomisccroorneucolneea-ttehdouscaenldlsPwCiEsllpebre neuxmpbreersseofd aPsCEsperacneanlytzemdi.cronTuheclensotremdalceblalcskgrboausnedd on the arroeuqnudenc0y.0-o0f.4m7i.cronuclei in the ICR mouse strain is T(hNeCEf)rewqiulelncybeofdetCeErsminveedrsubsy msactourrinegerthyethrnoucmybteersof FsCcEosriangndthNeCEsfirosbtser1v0e0d0 eirnytthhreocoyptteisc ofnieltdhse wshliildee. VI. Dama TSchhemicdrit(e1r9i76a).forMitchreoniudcelnetiifairceatdiaornkloyf msitcarionneudclaenid agreenertahlolsye of roocucnudr,. aMlitchroounguhclaelimonhdaveandsharripng-bosrhdaepresd manidcroanreuclgeeineroaclclaysiobentawleleyn m1i/c20roannudcle1a/5tedthecelsli,zenootf Tthhee mPiCEc.ronuTchleeuusn;itthofusscthoerinogccaissiotnhael cmeilclrowniutclhemaotreed 2tChEa,n onnoet mtiwocro(nourclmeourse)imsiccroounnutceldeia.s one Tphoelycshtraoimnaitnigc pranodcendourrmeocpherrommiattsictheerdyitfhfroecryetnetsiat(ibolnuibsyh-cgorleoyr anodf red, respectively). Data Presentation The data reported will include the number of ECEs scored, the nPuCEmsb,eraonfd mtihcerornautciloeaotfedpolPyCEcsh,romtahteicpertcoenntoargmeochorfommaitcirconucleated erythrocytes for each experimental animal. Evaluation Criteria Tshiegncirfiitcearnitadofsoer-raelpaotseidtiviencrreesaspeonsien misicraonsutcalteiastteidcalPlCyEs, or the dreestpeocntsieonfoorfaatreleparsotduconiebledosaendlesvtealt.istAicatlelsyt asritgincilfeicatnhtatpoisnidtuicvees nsetiatthiesrtiacalsltyatissitginciaflilcyantsiganndifirceapnrtodduocsiebleresipnocnrseeasenorataone dose Liseveblaseids ucpoonnsidsecrieedntinfeigcatijvued.gemeInnt.either case, the final decision als 8 of 10 01340 PROTOCOL NO. 455, EDITION 17 CORNINGHazl oR VII. IIENSTTERPRETATION vTahreiaanncaelys(iWsineorf, th1i9s71)datoan weiiltlherbeupnetrrfaonrsmfeodrmeudsin(gwhesnn vaanrailaynsciessofare hhoemtoegreongeeonueso)us)orprroapnokrttiroannssfoofrmecedll(svhweinthvamriicsrnocnesuclaerle per animal. Itf-tecshte a(nDaulnynestits, of195v5a;ria1n9c64e) iswilslignbeifiucsaendtto(pd<e0t.e0r5)m,ine& wDhuincnhettd'osse gcroonutprso,l. ifAnaanyl,yseasrewislilgnbieficpaenrtfloyrmeddiffseerpeanrtateflryomfotrheeancehgathiavrevest tine and sex combination. VIII. REFERENCES Dturnenaettmte,ntsC.Ww.i:th Aa mcounlttriopll.e coJ.mpaAmr.isoSntsatipsrto.ceAdsusroec.f.or50c:o1m0p9a6r-i1n1g21s,eve19r5a5l. DBuinonmeetttr,icsC,.W2.0::48N2e-w49t1a,ble1s964f.or multiple comparisons with a control. HNeewdedllle,, JG..AW..,'anHditeS,alMa.m,oneK,irMk.hFa.r:e, T5h.,e Lianrdsuecnt,ionK.,ofMamciGcrreognourc,ielJ.1a.s, a measure of genotoxicity. Mutation Res., 123:61-118, 1983. Schmid, W.: The micronucleus test. Mutation Res., 31:9-15, 1975. CShcehmiidc,alW.M:utagTehnes:micrPorniunccliepulses taensdt MfeotrhocdystogfeonretTihceiranDeelytseicst.ion,In, Vol. 4 (A. Hollaender, ed.). Plenum, pp. 31-53, 1976. WMicnGerra,w-HBi.lJl.,: NeSwtatYoirskt,icaSlecoPnrdinEcdiiptlieosn,in19E7x1p.erimental Design, IX. REPORT FORMAT CfHiVnalemprleopyosrtawisltlandparrodvidreepotrhte ffoorlmlaotwinfgorinefaocrhmaatsisoany. design. The Sponsor identification. . QSutaaltietmyentAssofuraGnLcPeCosmtpaltieamnecnet.. STeisgtnataurrteicloef isdtuednytifdiirceacttioorn. ind CHV Study Number. A prehcyesiipctalwidlelscrbeiptiinocnludoefdthien ttehsits asretcitciolne. and date of Type of asssy and protocol number. . SDtautdeys dofirescttuodry ainnditisaetniioonr atnedchnciocmipalne.tion. o Methods. 01841 ales : 9 of 10 PROTOCOL NO. 455, EDITION 17 CORNINGHazl = eton IEvnatleuraptrieotnaticorinteorfiar.esults. RCeofnecrleunscieosn.s. Test results presented in tabular form. X. CHAORNREGVIESIOSNS AsniygnecdhanbgyestheorStruedvyisDiiornesctoofr,thidsateadp,praonvdedmapirnottaocionledwwililthbethidsocupmreonttoecdo,l. XI. ANTMALCAREANDUSESTATEMENT aIpnprtohperioaptien,iontheofsttuhedySdtouedsy nDoitrecdtuoprl,icantoealatneyrnparteivvieoustewsotrikngwmietthhotdhsisare mpartoetroicaoll,wialnld bteherneuvmibeewredanbdy tshpeeciCeHsV-IsAeClUeCctefdoracreompalpiparnocperiawtiet.h This croemgpulliaatnocrey, gauimdoedliifneiscatcioonncewrinlilngbetherecqaurireeda.nd Aunsye cohfanagneismalosr. revIifsinoontsin rofevitehwi.s approved protocol will be sent to the CHV-IACUC for their III. RECORDS TO BE MAINTATNED gALeLnerraatweddataas,'adorceusmuelnttaotfiotnh,is resctourddys,wilplrotboecoalrsc,hivaendd itnhethfeinasltorraegpeort sfaucbimliistsiieosn ooff CtoheninfginaHlazlreetpoorntIntco. thfeorspaotnsloera.st Aofnteeryeatrhefoonlelowyienagr arpdeedrtiiaotidin,oendatlhienpecsrhpeioondssotroofrmaagtyeimefelaoecfcitlistetionetshatovoefaCtohsertnoiranafggoereHmafezanlcteiitlooinnteydIndcme.astiegfrnoiaratleasdn by the sponsor. 01842 ares 10 of 20 AMENDMENT TO THE STUDY PROTOCOL STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455, Edition 17 STUDY NO.: 17403-0-455 Page 1of 1 `Amendment #1 Section 2, Part V.B. Based on the solubility test, the test article will be suspended in water for the study. Inthedose selection study, dose levels of 500, 1625, 2750, 3875, and 5000 mg/kg will be tested. STUDY DIRECTOR hata ts: Hemalatha Murli, Ph.D. Mammalian Cytogenetics Departmentof Genetic and Cellular Toxicology _3/e/e6 Date 01543 AMENDMENT TO THE STUDY PROTOCOL~~ Page 1 of1 STUDY TITLE: IN VIVO MOUSE MICRONUCLEUS ASSAY PROTOCOL NO.: 455, Edition 17 STUDY NO.: 17403-0455 `Amendment #2 Section 2, Part V.B. The dose selection study will be repeated testing dose levels of700, 1000, and 1300 mg/kg. STUDY DIRECTOR \ . a Hemalatha Murli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology 3)15/9 Date 01844 AMENDMENT TO THE STUDY PROTOCOL STUDY TITLE: PROTOCOL NO.: IN VIVO MOUSE MICRONUCLEUS ASSAY 455, Edition 17 STUDY NO.: 17403-0-455 Page 1 of1 Amendment #3 Section 2, Part V.B. Based on the resultsofthe dose selection study, the mouse micronucleus assay will be conducted testing dose levels of 237.5, 475, and 950 mg/kg. A secondary dose group will be used. STUDY DIRECTOR Pr: Hemalatha Muli, Ph.D. Mammalian Cytogenetics Department of Genetic and Cellular Toxicology _3isje Date 01845