Document wrqpY0VOKL3dp6KknY9BOeLQB

'"tiAZLEMON W A S H IN G T 0 N 9200 LEESBURG PIKE VIENNA. VA.221 82-1699 ANALYSIS OF T-5877 IN A CELL PROLIFERATION ASSAY IN RAT LIVER CELLS FINAL REPORT AUTHOR Maria A. Cifone, Ph.D. PERFORMING LABORATORY Hazleton Washington, Inc. 9200 Leesburg Pike Vienna, Virginia 22182 LABORATORY PROJECT ID HWA Study No.: 154-208 SUBMITTED TO 3M Corporation Building 220-2E-02 3M Center St. Paul, MN 55144-1000 STUDY COMPLETION DATE November 1, 1994 154-208 1 of 35 HAZLETON WASHINGTON QUALITYASSURANCESTATEMENT PROJECTTITLE: Analysis of T-5877 in a Cell ProliferationAssay in Rat Liver Cells PROJECT NO.: 20991 PROTOCOL NO.: 493 HWA STUDY NO.: 154-208 EDITION NO.: 1, Modified for 3M Corporation Quality Assurance inspection(s)of the study and review of the final report of the above referenced project were conducted according to the Standard Operating Procedures of the Quality Assurance Unit and according to the general requirements of the 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: Inspection/Date Dosing and pump implantation (surgery) Draft report review/ 7-25,26-94 Final report review/ 11-1-94 Findings Reported 2-7-94 7-26-94 11-1-94 Auditor B. Mullett B. Mullett B. Mullett 154-208 juality AssuranceUnit Date Released 2 HA?=Lr;mtCN WASHINGTON COMPLIANCEAND CERTIFICATIOSNTATEMENT The describedstudywas conductedin compliancewith the Good Laboratory Practice Regulations as set forth in the Code of Federal Regulations (21 CFR 58, 40 CFR 792, and 40 CFR 160). There were no significant deviations from the aforementionedregulations or the signed protocol that would affect the integrityof the study or the interpretationof the test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation of the test article as presented herein represents an appropriate conclusionwithin the context of the study design and evaluationcriteria. All raw data, documentation, records, protocols, specimens and final reports generated as a result of this study will be archived by Hazleton for a period of at least one year following'submissionof the final report to the sponsor. After the one year period, the sponsor may elect to have these materials retained in the storage facilitiesof Hazleton for an additionalperiod of time or sent to a storage facility designated by the sponsor. SUBMITTED BY: Andrea L. Ham, M.S. Associate Scientist Study Director: Date Mai@'i'Aa. Cifone Ph. D. st dy Director Geneticand CellularToxicology Study Completion Date* 154-208 3 HAZLF=OCCN W A S H IN G T 0 N TABLE OF CONTEKTS PAGE NUMBER ................................ 6 ABSTRACT I. SPONSOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 II. MATERIAL TESTED . . . . . . . . . . . . . . . . . . . . . . . . . . 7 A. Genetics Assay No. B. Identification C. Physical Description D. Date Received III. TYPE OF ASSAYS . . . . . . . . . . . . . . . . . . . . . . . . . . 7 IV. PROTOCOL NUMBER . . . . . . . . . . . . . . . . . . . . . . . . . . 7 7 V. STUDY DATES A. Study ini:tl:a'ti*on*D@t@ B. Experimental Start Date C. Experimental Termination Date VI. SUPERVISORY PERSONNEL . . . . . . . . . . . . . . . . . . . . . . . 7 A. Study Director B. Associate Scientist VII. OBJECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 VIII. DEFINITION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 IX. MATERIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 A. Indicator Cells B. Osmotic Pumps and Label for Cell Proliferation Analysis C. Control Articles X. EXPERIMENT DESIGN . . . . . . . . . . . . . . . . . . . . . . . . . 9 A. Dosing Procedure B. Implantation of Osmotic Pumps C. Tissue Collection and Preparation D. Immunohistochemical Staining E. Assessment of Cell Proliferation 4 154-208 ligtZLF=ECCN W A S H IN G T 0 N TABLE OF CONTENTS (CONTINUED) XI. ASSAY EVALUATION CRITERIA . . . . . . . . . . . . . . . . . . . . . 11 XII. INTERPRETATION OF RESULTS . . . . . . . . . . . . . . . . . . . . . 12 A. General Observations B. Summary of Labeled Cell Counts for the Liver XIII. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 XIV. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 XV. EXPERIMENTAL DATA TABLE ... . . . . . . . . . . . . . . . . . . . . is APPENDIX A Individual Animal and Slide Labeling Indices . . . . . . . . 17 APPENDIX B Individual Animal Body and Liver Weights and Liver to .............. ... .... 24 Body Weight Ratios APPENDIX C Histopathology R(?port. . . . . . . . . . . . . . . . . . . . 29 APPENDIX D Statistical Analysis of Labeling Indices . . . . . . . . . . 32 154-208 5 HAZLF=MCCDN W A S H IN G T 0 N ABSTRACT The purpose of this study was to determine the hepatotoxicity of T-5877 by measuring cell proliferation(CP) assayed as S-phase induction in rat liver cells after jn vivo treatment. The doses chosen for the study were 100, 200, 400 and 800 mg/kg. Dimethylnitrosamine (DMM) at 15 mg/kg was included as a positive control. In the cell proliferationassay, a single oral dose of the test material was administered and five animals per condition were labeled with BrdU for 72 hours using ALZETO osmotic pumps. No histomorphological alterations were observed at 100 and 200 mg/kg but minimal to moderate vacuolization was observed at 400 and 800 mg/kg.' Treatment-related changes were also observed in the dimethylnitrosamine(DMN) positive control animals. Following determination that there were no treatment-related lobular differencesin the labeling indices, sections from the left lateral lobe of the livers, as well as samples from the duodenum, were processed for immunohistochemistry. Each slide was prepared with sections from both liver and duodenum. The duodenum (a rapidly proliferating organ) was used as an internal control for delivery of label and immunohistochemicalstaining. The percentage of nuclei incorporating label in the liver was determined microscopically. Only hepatocyte nuclei were enumerated. The control animals had a labeling index of 1.34 and treated animals had labeling indices that ranged from 1.40 to 4.42. When comparing vehicle and treated groups, there were no significantincreases in the labeling index in any of the treated groups and no positive trend was observed. The high dose (Group 5) animals did have an elevated mean labeling index but it was not significant because the average value showed heterogeneous variance. Significant increases in cell proliferationwere observed in the positive control animals. T-5877 was therefore considered negative for the induction of cell proliferationin rat livercells. 6 154-208 [IAZLR;OCCN WASHINGTON Analysis Of T-5877 in a Cell ProliferationAssay in Rat Liver Cells I. SPONSOR: 3M Corporation II. MATERIAL TESTED: A. Genetics Assay No.: 154-208 B. Identification:T-5877 C. Physical Description: waxy amber solid D. Date Received: January 19, 1994 III. TYPE OF ASSAYS: Analysis of Cell Proliferationin Rat Liver Cells IV. PROTOCOL NUMBER: 493, Edition 3, Modified for 3M Corporation V. STUDY DATES: A. Study InitiationDate: January 10, 1994 B. Experimental Start Date: February 7, 1994 C. Experimental Termination Date: June 14, 1994 VI. SUPERVISORY PERSONNEL: A. Study Director: Maria A. Cifone, Ph.D. B. Associate Scientist: Andrea L. Ham, M.S. VII. OBJECTIVE: The objective of this assay was to measure hepatotoxicity caused by T-5877 by measuring cell proliferation (CP) measured as S-phase induction induced in rat liver cells after in vivo treatment. 7 154-208 HAZLCMCN WASHINGTON Cell proliferationmeasured the fraction of cells undergoing cell replication in rat liver using an immunohistochemicaltechnique (1,2) to detect bromodeoxyuridine (BrdU) incorporated during DNA synthesis. Animals were given a single oral dose of the test material and the livers were isolated following administrationof BrdU for 72 hours in vivo with an ALZETO osmotic pump implanted subcutaneously. Quantification of cells that have incorporated DNA precursors over the 72-hourperiod indicatesincreasedcell proliferationin the liver (3). VIII. DEFINITION: Hepatotoxicants such as carbon tetrachloride and dinitrotoluene induce an increase in cell proliferationto replace necrotictissue (2,4). These proliferating cells may be detected during S-phase analysis. Other chemicals may induce S-phase in the absence of hepatotoxicity. It is not apparent how cell proliferationmay act in the carcinogenic process but there are numerous mechanisms which can be affected during replication(5-8). Chemically induced cell proliferationmay increase the probability of spontaneous mutations as well as increase the probability of converting unrepaired DNA-adducts into mutations. Unscheduled cell proliferationmay also play a role in the expansion of preneoplastic populations leading to the emergence of a fully transformed clone of cells. Some of these examples act by a nongenotoxic mechanism and it is theoretically possible to detect nongenotoxic carcinogens as well as genotoxic carcinogens using this technique. IX. MATERIALS: A. Indicator Cells Young adult male rats of the Sprague-Dawley strain, 10-12 weeks old at the time of dosing, were purchased from Charles River Laboratories, Raleigh, NC (Crl:CDOBR). This healthy random bred strain was selected to maximize genetic heterogeneity and assure access to a common source. Animals scheduled for this study were housed according to standard operating procedures and were fed Purina CertifiedO Rodent Chow (Fomula 5002) and water Ad libitum. Animals were quarantined a minimum of 7 days prior to random assignment to study groups and identificationby implantable microidentificationdevice for the cell proliferationassay. Animals were anesthetized prior to surgery for preparation of cell cultures, using about 60 mg/kg sodium pentobarbital,and were exsanguinated during the harvest procedure. The cell proliferationassay was initiatedwith rats that ranged from 306 to 355 grams. Approximately 2 hours after dosing, the animals were anesthetized using Metofane's(methoxyflurane, PitmanMoore, Inc.) inhalation anesthesia and one ALZETO pump per animal was aseptically inserted subcutaneously (dorsal surface). 8 154-208 HAZLCMCON WASHINGTON Seventy-twohours later, animals were anesthetizedwith CO. prior to removal of the livers and duodenum. B. Osmotic Pumps and Label for Cell ProliferationAnalysis ALZETO osmotic pumps (ALZA Corporation, Palo Also, CA), Model 2ML1 were used. A single lot (#042301)was used throughoutthe study. The pump has a 2000 Al capacity with a pump rate of 10 Al/hour. The pumps were pre-filledwith BrdU at a concentrationof 20 mg/ml. C. ControlArticles 1. Vehicle control A vehicle controlconsistingfive rats was dosed by oral gavage (P.O.)with the vehicle,corn oil (Duke'sCorn Oil, Lot 2L290833). Tissues from vehiclecontrol animalswere subjected to the same manipulationsused for the tissues derived from treated animals. The dosing volume of the vehicle control animalsdid not exceed 10 ml/kg. 2. Positivecontrolarticle The positive control compound is known to induce S-phase in rat hepatocytesjn vivo. The positivecontrol, dimethylnitrosamine (DMN, CAS# 62-75-9, Sigma Chemical Co., Lot# 82HO365) was dosed at 15.0 mg/kg. Five rats were treated P.O.. D. Test Article For the preparationof the dosing solutionsof the test article, the test articlewas suspendedin corn oil at concentrationsof 10, 20, 40 and 80 mg/ml prior to dosing. The maximum dosing volumes for the test articledid not exceed 10 ml/kg. X. EXPERIMENT DESIGN: A. Dosing Procedure Five rats per conditionwere treatedby oral gavage with T-5877 for the cell proliferationassay. Deliveryvolumeswere calculatedon the basis of the most recent animalweight and the target dose. The maximum volume df the test article suspensions administereddid not exceed 10 ml/kg. Fresh preparationsof test article in vehiclewere used for any testingpurpose. 9 154-208 HAZLF=OCCN W A S H IN G T 0 N B. C. D. E. 154-208 Confirmationof the concentrationof the test materialunder conditionsof preparationand dosing of the assay was not determinedin conjunctionwith this study. Implantationof Osmotic Pumps For the cell proliferation assay, ALZETO Model 2ML1 osmotic pumps (Lot #042301) were preloaded with 2000 141of BrdU at a concentrationof 20 mg/ml. The animals were anesthetizedusing Metofanes according to standard procedures and one pump per animal was asepticallyinsertedsubcutaneously(dorsalsurface) approximately2 hours after dosing. The incisionwas closed with wound clips and the animalsmonitoreduntil the time of sacrifice to ensure that therewere no clinicalsigns of infection. The osmotic pumps were'implantedthree days prior to sacrifice. Tissue Collection and Preparation 'Each animal was anesthetizedprior to removal of organs for analysis. The thoracic cavity was opened and the liver removed and fixed in neutralbufferedformalin. A cross sectionof duodenum,a tissue with high cell turnover,was also removed from each animal and fixed. The duodenum was included as an indicator that label was administeredcorrectlyto each animal. For the livers from high dose (Group5) animals, 5 u paraffin embedded sectionswere taken from the left lateral,median and right anteriorlobes. Once it was determinedthat no treatment-related lobulardifferenceswere present,slidesfrom the left lateral lobe were prepared from each animal. Sections of the duodenum were also made and a section of the duodenum was mounted on each slide containinga liver section. Slideswere also prepared accordingto standard proceduresfor examinationby a pathologist to determine if any abnormalitieswere present. ImmunohistochemicalStaining The slides were deparaffinizedand rehydratedprior to staining. The slideswere stainedfor determinationof cell proliferationas measuredby incorporationof BrdU into DNA usingBiogenix antibodieswith peroxidase-conjugatedstreptavidinand a 3,3-diaminobenzidinetetrahydrochloride(DAB) chromogen and hematoxylincounterstain. Assessmentof Cell Proliferation The section of the duodenum was microscopicallyexamined to ensure that the label was properly administeredto the animal. Once label deliverywas confirmed,slidesfrom the differentlobes of the high dose animals were examined for lobular differences. Labelingwas similar among the lobes thereforecell countingwas 10 HAZLETON W A S H IN G T 0 N performedwith sectionsfrom the left laterallobe from all animals. The percentageof nuclei incorporatinglabel in the liver was determinedmicroscopically. The areas to be counted were randomly generated by computer. A 1.0 mm square indexed ocular grid divided into 10 x 10 squareswas used to define the counting area. At least 2000 nuclei were examined per animal with a minimum of 3 sectionsand 6 fields per section. Any nuclei that were blue were consideredunlabeled and any nuclei containing any brown chromogenic hue were considered labeled unless a clear artifactwas present. Only hepatocytenuclei were enumerated. Fields that containedareas of necrosiswere not includedin the evaluation. The slideswere coded for (blind) evaluation as to treatmentgroup. S-phase nuclei labeling indices for each animal were calculatedas follows: Labeled S-phase nuclei (LI)=no.of labeled hepatocytenuclei-X 100 total no. of hepatocytescounted XI. ASSAY EVALUATION CRITERIA The proportionsof the number of cells labeledto the number of cells counted were analyzed by repeated measures analysis of variance (ANOVA) techniquesto determineany slide, and related interactioneffects. The sphericitytest was also utilizedto test variancehomogeneity. Additionally.theaveragevalue from the three slides of each animal was calculated to conduct one-way ANOVA, Dunnett's t-test, Terpsa-Jonkheere test, and regressiontests for trend using both untransformedand ranked data. See AppendixC for statisticalanalysisof labelingindices. For the terminalwhole body weights,liver weights,and liver to terminal body weight ratios, a mean and standard deviation were calculatedfor each treatmentgroup using the individualanimal mean S-phasevalues. Statisticalanalysisof labelingindex was performed using one-way analysis of variancetechniques. Control versus treatment group comparisonswere done with Dunnet's t-test and control versus positive group comparisonswere done using the Student'st-test. In the case of varianceheterogeneity,rank transformationsof the data were performedprior to analysisof varianceand Dunnet'st-test. A labelingindex,terminalbody weight,terminalliverweight and liver to body weight ratio in a dose group that deviatesfrom the values in the concurrentcontrol group at a significancelevel of p:50.05was consideredsignificantlydifferentthan the controlgroup. 154-208 11 'OHAZLEE:C:N WASHINGTON XII. INTERPRETATION OF RESULTS A. General Observations All animals survived treatment. No histomorphologicalalterations were observed at 100 and 200 mg/kg but minimal to moderate vacuolization was observed at 400 and 800 mg/kg. Treatmentrelated changes were also observed in the dimethylnitrosamine (DMN) positive control animals. Details of the histopathologyare in Appendix A. Cells stained with the brown DAB chromogen were observed in the duodenum from all of the animals used in the study. The presence of label in all the animals indicated proper delivery of the BrdU label and acceptabte immunohistochemical staining. There was no apparent preferential labeling in any of the lobes and the label was random within the lobes. None of the livers of the treated animals showed a dose-related increase in weight compared to control animals. The mean liver weight of the positive control was not significantlyelevated even though large increases in DNA synthesis (and subsequent cell proliferation)were induced. However, the 400 mg/kg (Group 4) and 800 mg/kg (Group 5) animals had mean terminal body weights that were less than the Group 1 control value (p :50.01). When the liver to body weight ratios were determined, there were significantincreasesin the liver to terminal body weight ratios with a p value of between 0.01 and 0.05 for Group 3 (200 mg/kg) and increases at 400 mg/kg (Group 4) and 800 mg/kg (Group 5)(p:50.01). B. Summary of Labeled Cell Counts for the Liver A summary of the labeled cell counts for each group is shown in Table 1. Individual animal counts are shown in Appendix A. The mean labeling index (LI) for each group is presented in the third column in Table 1. The mean background labeling index (Group 1) was 1.34 which indicates that less than 2% of the nuclei had undergone DNA synthesis during the 72-hour labeling period. The labeling indices of the test article-treatedcells ranged from 1.40 to 4.42. None were considered significantlyelevated and there was 154-208 12 HAZLF."CCN W A S H IN G T 0 N no indicationof a significantpositivetrenddue to treatment. The high dose (800mg/kg) had the highestlabelingindexof 4.42, but becauseof a heterogeneousresponse,there was a lack of significance.If the resultsfrom individualanimalsare compared two of the five animalsresponded,with labeling (see AppendixA) indicesof 8.00 a'nd 9.05,while the remainingthree animalshad labelingindicesclose to backgroundlevels. This may indicatea weak response,but also may be the resultof animalvariability. See AppendixD for statisticalanalysis. The mean labelingindex of the DMN positivecontrolanimalswas 34.96 which is significantleylevated(p <-0.01). These resultsdemonstratethat T-5877did not inducesignificant dose-relatedincreasesin the LI in the liverin male rats after treatmentwith singleoral doses at concentrationsof 100 mg/kg to 800 mg/kg. Large increasesin the labelingindexwere observedin the DMN positivecontrolanimals. The mean labelingindex in the DMN-treatedpositivecontrolanimalswas 34.96 (p <-0.01). XIII.CONCLUSIONS The test material,T-5877,did not inducesignificantchangesin the number S-phasecells followinga singleoral dose of 100 mg/kg to 800 mg/kg. The animalswere labeledfor 72 hours and no significant dose-relatedtrend in the mean labelingindex was observedin the treatedgroups. T-5877was thereforeevaluatedas negativefor the inductionof DNA synthesisin rat livercells. 13 154-208 HA7,LIEOCCN WASH INGTO N XIV. REFERENCES DeFazio, A., Leary, J.A., Hedley, D.W. and Tattersall, M.H.N. 1. Immunohistochemicaldetection of proliferatingcells in (1987) vivo. 5. Histochem. Cytochem. 35, 571-577. 2. Lanier, T.L., Berger E.K., and Eacho, P.I. (1989). Comparison of 5-bromodeoxyuridine a'nd 3H-thymidine in rodent hepatocellular proliferationstudies. Toxicologist9, 64. 3. Butterworth, B.E., Ashby, J., Bermudez, E., Casciano, D., Mirsalis,J., Probst, G., and G. Williams: A protocol and guide for the in vivo rat hepatocyte DNA-repair assay. Mutation Res., 189:123-133, 1987.' 4. Mirsalis, J.C. and Butterworth, B.E.: Induction of unscheduled DNA synthesis in rat hepatocytes followingjn vivo treatment with dinitrotoluene. Carcinogenesis, 3:241-245, 1982. 5. Marsman, D.S., Cattley, R.C., Conway, J.G., and Popp, J.A. (1988). Relationshipof hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferatorsdi(2-ethylhexyl)phthalataend [4-chloro-6-(2,3xylidino)-2-pyrimidinylthioa]cetic acid (Wy-14,643)in rats. Cancer Res. 48, 6739-6744. 6. Craddock, V.M. (1976). Cell proliferationand experimentalliver cancer. In: "liver Cell Cancer", Cameron, H.M., Linsell, C.A. and Warwick, G.P., Elsevier, North Holland Biomedical Press, Amsterdam. 7. Columbano, A., Rajalaksmi, S., and Sarma, D.S.R @1981). Requirementof cell proliferationfor the initi@tionof liver carcinogenesis as assayed by three different procedures. Cancer Res. 41, 2079-2083. S. Glinos, A.D., Butcher, N.L. R., and Aub, J.C. (1951). The effect of liver regeneration on tumor formation in rats fed 4-diaminobenzene. J. Exp. Med. 933, 313-324. 9. Ham, A. and Cifone, M.A. (1991). Use of cell proliferationto study liver effects induced by a single dose of DMN. Environmental and Molecular Mutagenesis 17(19), 16. 14 154-208 dahHAZLCTON W W A S H IN G T 0 N XV. EXPERIMENTAL DATA TABLE 15 154-208 HAZLF=OCCN W A S H IN G T 0 N Client: 3M Corporation Client Code: T-5877 Table I Cell Proliferation Summary HWA Assay No.: 154-208 Trial InitiationDate: February7, 1994 Group/Sexa Dose Labeling Index Level (mg/kg) Liver Weight (grams) Terminal Body Liver/Body Weight (grams) Weight (%) im OC 1.34 0.66 13.58 1.09 344.2 12.9 3.94 0.20 2M 100 2.98 1.61 14.32 1.81 348.1 17.3 4.11 0.36 3M 200 1.40 0.92 15.00 2.24 335.6 20.5 4.45 0.38*t 4M 400 2.26 1.26 14.76 1.02 303.6 13.0**l 4.86 0.25**t 5M 800 4.42 3.60 15.38 1.05 300.0 15.0**l 5.13 0.19**t 6Md 15d 34.96 14.86**t 12.73 1.61 319.6 22.5 3.97 0.29 afive animals per group bpercentage of labeled hepatocyte nuclei per total number of hepatocytes counted (at least 2000) cvehicle control, Corn oil dpositive control, 15 mg/kg of DMN Significant at 0.01 :5p :50.05 Significant at p :50.01 t Increasein the mean 4 Decrease in the mean 16 154-208 w--H-A'ZLE-ZON W A S H IN G T 0 N APPENDIX A Individual Animal and Slide Labeling Indices 154-208 17 HAZLR;OCCN WASHINGTON Slid.#e A-nmia.l ID 16 47733 17 47733 18 47733 GROUD im im im Llibeled 10 21 5 Counted % Labeled 700 1.43 700 3.00 700 0.71 Mean 1.71 SD 1.17 19 47734 iimm 89 770000 11..2194 700 1.00 20 47734 21 47734 im 7 Mean 1.14 SD 0.14 22 47735 iimm 54 770000 00..7517 0.57 23 47735 24 47735 im 4 700 Mean 0.62 SD 0.08 25 47736 im 11 26 47736 im 10 27 47736 im 15 700 1.57 700 1.43 700 2.14 Mean 1.71 SD 0.38 28 47737 im 7 29 47737 im 13 30 47737 im 12 700 1.00 700 1.86 700 1.71 Mean 1.52 SD 0.46 GROUP MEAN GROUP SO 1.34 0.66 18 154-208 HAZLE"CON WASHINGTON slid-e# AnimaIlD Group 46 47738 2M 47 47738 2M 48 47738 2M Labeled 21 16 29 Counted Lla-b-e-leA 700 3.00 700 2.29 700 4.14 Mean 3.14 SD 0.94 49 47739 2M 22 50 47739 2M 19 51 47739 2M 16 700 3.14 700 2.71 700 2.29 Mean 2.71 SD 0.43 52 47740 2M 14 53 47740 2M 26 54 47740 2M 8 700 2.00 700 3.71 700 1.14 Mean 2.29 SD 1.31 55 47741 2M 37 56 47741 2M 22 57 47741 2M 49 700 5.29 700 3.14 700 7.00 Mean 5.14 SD 1.93 58 47742 2M 12 59 47742 2M 4 60 47742 2M 18 700 1.71 700 0.57 700 2.57 Mean 1.62 so 1.00 GROUP MEAN GROUP SD 2.98 1.61 19 154-208 4aHAZLEMON WASHINGTON Slide # Animal ID GrouD 61 47743 3M 62 47743 3M 63 47743 3M Labeled 6 2 6 Counted % Labeled 700 0.86 700 0.29 700 0.86 Mean 0.67 SD 0.33 64 47744 3M 14 65 47744 3M 9 66 47744 3M 11 700 2.00 700 1.29 700 1.57 Mean 1.62 SD 0.36 67 47745 3M 16 68 47745 3M 11 69 47745 3M 27 700 2.29 700 1.57 700 3.86 Mean 2.57 SD 1.17 70 47746 3M 71 47746 3M 72 47746 3M 4 700 0.57 6 700 0.86 7 700 1.00 Mean 0.81 SD 0.22 73 47747 3M 9 74 47747 3M 3 75 47747 3M 16 700 1.29 700 0.43 700 2.29 Mean 1.33 SD 0.93 GROUP MEAN GROUP SD 1.40 0.92 154-208 20 HAZLAMON WASHINGTON Slide # Animal ID Group L_Lgbel_ed 31 47748 4M 11 32 47748 4M 14 33 47748 4M 15 Counted % Labeled 700 1.57 700 2.00 700 2.14 Mean 1.90 SD 0.30 34 47749 4M 7 35 47749 4M 19 36 47749 4M 10 700 1.00 700 2.71 700 1.43 Mean 1.71 SD 0.89 37 47750 4M 19 38 47750 4M 39 39 47750 4M 16 700 2.71 700 5.57 700 2.29 Mean 3.52 SD 1.79 40 47751 4M 4 41 47751 4M 13 42 47751 4M 9 700 0.57 700 1.86 700 1.29 Mean 1.24 SD 0.64 43 47752 4M 14 44 47752 4M 17 45 47752 4M 30 700 2.00 700 2.43 700 4.29 Mean 2.90 SD 1.21 GROUP MEAN GROUP SD 2.26 1.26 21 154-208 w HWAASHZILNGETTONON Slide # Anim_alID GrouD 1 47753 5M 2 47753 5M 3 47753 5M Labeled 23 8 18 Counted % Labeled 700 3.29 700 1.14 700 2.57 Mean 2.33 SD 1.09 4 47754 5M 62 5 47754 5M 60 6 47754 5M 46 700 8.86 700 8.57 700 6.57 Mean 8.00 SD 1.25 7 47755 5m 12 8 47755 5M 5 9 47755 5M 5 700 1.71 700 0.71 700 0.71 Mean 1.05 SD 0.58 10 47756 5M 63 11 47756 5M 56 12 47756 5M 71 700 9.00 700 8.00 700 10.14 Mean 9.05 SD 1.07 13 47757 5m 15 14 47757 5M 10 15 47757 5M 10 700 2.14 700 1.43 700 1.43 Mean 1.67 SD 0.41 GROUP MEAN GROUP SD 4.42 3.60 22 154-208 "AZLV;NC(ON W A S H IN G T 0 N Slide # Animal ID GrouD 76 47758 6M 77 47758 6M 78 47758 6M Labeled 260 364 435 79 47759 6M 265 80 47759 6M 251 81 47759 6M 171 82 47760 6M 155 83 47760 6M 246 84 47760 6M 369 85 47761 6M 270 86 47761 6M 292 87 47761 6M 285 88 47762 6M 54 89 47762 6M 165 90 47762 6M 89 Counted % Labeled 700 37.14 700 52.00 700 62.14 Mean 50.43 SD 12.57 700 37.86 700 35.86 700 24.43 Mean 32.71 SD 7.24 700 22.14 700 35.14 700 52.71 Mean 36.67 SD 15.34 700 38.57 700 41.71 700 40.71 Mean 40.33 SD 1.61 700 7.71 700 23.57 700 12.71 Mean 14.67 SD 8.11 GROUP MEAN GROUP SD 34.96 14.86 154-208 23 'w" HWAASZHILNrGTmO-N NCCN I APPENDIX B Individual Animal Body and Liver Weights and Liver to Body Weight Ratios 154-208 24 tiAFLFOCCN WASH IN GTO N APPENDIX B ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS *DRAFT* ABSOLUTE ORGAN WEIGHTS (9) *DRAFT* STUDY NUMBER: 154208 ---------------------------------------------------------------------------------------------------------- ORGAN ABBREVIATION: Ll - LIVER SEX DOSE ANIMAL GROUP NUMBER TERMINAL BODY WT (g) Ll ----------------------------------------------------------------------------------------------------------- m 1 847733 344.0 14.24 M 1 B47734 350.0 13.77 m 1 B47735 322.0 11.66 K I B47736 352.0 14.19 m I B47737 353.0 14.11 -- -- -- - -- -- -- - -- -- -- - -- -- -- - -- -- -- -- -- - -- - -- -- - -- -- - - NUMBER IN GROUP: MEAN: STANDARD DEV: 5 344.2 12.9 5 13.59 1.09 ------------------------------------------------------------------------------------------------------- --- -- - -- - -- M 2 B47738 371.0 16.35 m 2 B47739 350.0 14.31 N 2 B47740 340.0 12.12 N 2 B47741 355.0 15.67 M 2 847742 324.6 12.98 - -- -- - -- -- - -- -- -- - -- -- -- -- -- - -- -- - -- -- - -- NUMBER IN GROUP: MEAN: STANDARD DEV: 5 348.1 17.3 5 14.32 1.81 ---------------------------------------------------------------------------------------------------------- m 3 B47743 368.0 18.79 m 3 B47744 330.0 14.2B m 3 B47745 327.0 14.33 14 3 B47746 313.0 12.87 N 3 847747 340.0 14.70 ----------------------------------------------------- NUMBER IN GROUP: MEAN: STANDARD DEV: 5 335.6 20.5 5 15.00 2.24 -------------------------------------------------------------------------------------------------------- 25 154-208 HAZLrm-MON WASHINGTON APPENDIX B ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS *DRAFT* ABSOLUTE ORGAN WEIGHTS (g) *DRAFT* STUDY NUMBER: 154208 ------------------------------------------------------------------------------------------------------- ORGAN ABBREVIATION: Ll - LIVER SEX DOSE ANIMAL GROUP NUMBER TERMINAL BODY WT (g) LI ----------------------------------------------------------------------------------------------------- M 4 B47748 300.0 15.02 M 4 B47749 308.0 13.74 M 4 B47750 324.0 16.31 M 4 847751 291.9 13.96 M 4 B47752 294.0 14.77 - - -- -- - -- - -- - -- -- -- - -- -- - -- -- -- - -- -- -- -- - -- -- - -- - -- - - NUMBER IN GROUP: MEAN: STANDARD DEV: 5 303.6 13.0 5 14.76 1.02 ------------------------------------------------------------------------------------------------------ M 5 B47753 314.0 16.26 M 5 B47754 293.0 14.30 K 5 B47755 278.6 14.19 M 5 B47756 314.0 15.89 M 5 B47757 300.2 16.25 --- - -- -- - - -- -- - -- -- - -- -- - -- - --- - --- - -- -- -- - -- -- -- - -- - NUMBER IN GROUP: MEAN: STANDARD DEV: 5 300.0 15.0 5 15.38 1.05 ------------------------------------------------------------------------------------------------------ N 6 B47758 336.0 14.50 M 6 B47759 294.0 11.08 M 6 847760 334.0 14.20 N 6 847761 296.0 11.21 N 6 B47762 338.0 12.64 -- -- -- - -- -- - -- -- -- - -- -- - -- -- - -- -- -- -- -- - -- -- -- - -- -- - - NUMBER IN GROUP: MEAN: STANDARD DEV: 5 319.6 22.5 5 12.73 1.61 ------------------------------------------------------------------------------------------------------ 26 154-208 HAZLCTON W A S H IN G T 0 N APPENDIX B ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS *DRAFT* ORGAN-TO-TERMINAL BODY WEIGHT RATIOS (%) *DRAFT* STUDY NUMBER: 1542DB ------------------------------------------------------------------------------------------------------- ORGAN ABBREVIATION: Ll - LIVER SEX DOSE GROUP ANIMAL NUMBER TERMINAL BODY WT (9) RATIO ------------------------------------------------------------------------------------------------------ - -- - m 1 B47733 344.0 4.138 m 1* B47734 350.0 3.933 N 1 B47735 322.0 3.622 m 1 847736 352.0 4.03D M I B47737 353.0 3.998 - -- - -- - --- -- - -- -- -- - -- - -- -- - -- -- -- -- - -- - -- -- - -- - NUMBER IN GROUP: MEAN: STANDARD DEV: 5 344.2 12.9 5 3.944 0.195 ------------------------------------------------------------------------------------------------------ m 2 B47738 371.0 4.407 M 2 B47739 350.0 4.087 M 2 B47740 340.0 3.564 m 2 B47741 355.0 4.470 M 2 847742 324.6 3.999 -- - -- - -- -- -- - -- - --- -- - -- - -- - -- -- -- -- -- -- -- - -- - -- - -- -- NUMBER IN GROUP: MEAN: STANDARD DEV: 5 348.1 17.3 5 4.106 0.363 ------------------------------------------------------------------------------------------------------ M 3 B47743 368.0 5.107 M 3 B47744 330.0 4.326 N 3 847745 327.0 4.382 m 3 B47746 313.0 4.112 N 3 B47747 340.0 4.325 - -- -- -- - -- -- - -- -- -- -- - -- -- -- - -- - --- -- -- - -- -- -- - -- - -- - NUMBER IN GROUP: MEAN: STANDARD DEV: 5 335.6 20.5 5 4.450 0.381 ----------------------------------------------------------------------------------------------------- 27 154-208 -**HAZLEOCON WASHINGTON APPENDIX B ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS *DRAFT* ORGAN-TO-TERMINAL BODY WEIGHT RATIOS (%) *DRAFTSTUDY NUMBER: 154208 ------------------------------------------------------------------------------------------------ ORGAN ABBREVIATION: Ll - LIVER SEX DOSE ANIMAL GROUP NUMBER TERMINAL BODY 'dT(g) RATIO ------------------------------------------------------------------------------------------------ m 4 B47748 300.0 5.006 N 4 B47749 308.0 4.460 N 4 B47750 324.0 5.033 M 4 847751 291.9 4.782 N 4 B47752 294.0 5.024 -- -- - -- -- - -- - -- -- - - --- -- - -- -- -- -- - -- -- - -- -- - -- -- - -- -- NUMBER IN GROUP: MEAN: STANDARD DEV: 5 303.6 13.0 5 4.861 0.247 ------------------------------------------------------------------------------------------------ M 5 B47753 M 5 B47754 M 5 B47755 m 5 B47756 314.0 293.0 278.6 314.0 5.179 4.879 5.093 5.060 M 5 B47757 300.2 5.412 -- -- - -- -- - -- - -- - -- -- - --- -- - -- - --- - -- -- - -- -- - -- -- - -- -- NL14BER IN GROUP: MEAN: STANDARD DEV: 5 300.0 15.0 5 5.125 0.194 ------------------------------------------------------------------------------------------------ m 6 B47758 M 6 B47759 M 6 847760 N 6 B47761 336.0 294.0 334.0 296.0 4.315 3.768 4.251 3.789 N 6 B47762 338.0 3.739 - --- - -- -- - -- -- - -- - -- -- -- -- - -- -- -- -- -- - -- - -- -- - -- -- -- - NUMBER IN GROUP: MEAN: STANDARD DEV: 5 319.6 22.5 5 3.972 0.285 ----------------------------------------------------------------------------------------------- 154-208 28 w HAZLCItCON WASHINGTON APPENDIX C Histopathology Report 154-208 29 VIAZLV;MCN WASH IN GTON Pathology Report Analysis of Cell Proliferationin Rat Liver Cells Project No. 154-208 General Irotocol Thirty, young adult, male Sprague-Dawley rats were placed in six groups of five rats/group. Group 1 served as the vehicle control. Groups 2, 3, 4, and 5 served as the low-, low-mid-, high-mid-, and high-dose groups, respectively, receiving 100, 200, 400, and 800 mg/kg of the test material, T-5877, via oral gavage. Group 6 served as the positive control, receiving 15 mg/kg of dimethylnitrosamine (DMN) via oral gavage. After dosing, an ALZET'sModel 2ML1 osmotic pump containing 20 mg/mL of bromodeoxyuridine (BrdU) was implanted subcutaneously in each rat while it was under MetofaneO anesthesia. Seventy-two hours after pump implantation, all rats were anesthetized, exsanguinated, and necropsied. Liver, duodenum, and all gross lesions from each rat were placed in 10% neutral-buffered formalin and processed as per HWA SOPS. These tissues from all rats were evaluated microscopically by a board- certified veterinary pathologist. Histopatholoqy Treatment-related change in the liver consisted of minimal to moderate vacuolization in Group 4 (400 mg/kg) and Group 5 (800 mg/kg) rats. Group 6 (DMN treated) rats had varying severities of centrilobularnecrosis, hepatocellular hypertrophy, chronic inflammation,peliosis, and increased mitoses. The single cases of pelvic dilatation in the kidney and degeneration with mineralization of the testis in Group 5 are considered to be spontaneous and without relation to treatment. 30 154-208 w HAZLCtC:N WASHINGTON Summary The test material, T-5877, when administered to male Sprague-Dawley rats in single oral gavage doses of 100, 200, 400, and 800 mg/kg, produced vacuolization in the liver of rats dosed at 400 and 800 mg/kg. No treatmentrelated histomorphologicchanges were noted in the liver of rats dosed at loo and 200 mg/kg. Pathologist: Samuel V. Machotka, D.V.M..,D.A.B.T., Diplomate, American College of Veterinary Pathologists Department of Pathology Date 154-208 31 -**HAZLamCON I-1w@ W A S H I N G T 0 N APPENDIX D Statistical Analysis of Labeling Indices 154-208 32 HAZLETON WASHINGTON Methods: The proportions of the number of cells labeled to the number of cells counted were analyzed by repeated measures analysis of variance (ANOVA) techniques to determine any significant dose, slide, and related interaction effects. The sphericitytest was also utilized to test variance homogeneity. The model used was: proportion - ;t+ dose + slide + slidexdose+ Additionally, since the study did not show any significant between-slide variation, the average value from the 3 slides of each animal was then calculated to conduct one-way ANOVA, Terpstra-Jonckheere test [1], and regression tests for trend using both untransformed-and ranked data. Results: Since the sphericity tests rejected variance homogeneity for both vehicle vs positive control and vehicle vs treated groups (p - 0.0215 and 0.0000, respectively), the Greenhouse-Geisserprobabilities were used for the significance evaluation. As Text Table 1 indicates,there is no significant finding in comparing vehicle with treated groups. Within- and between-group slide-to-slidevariationswere not significantin either case. Only the positive control showed highly significantelevation in labellingover vehicle control. The data based on the average values showed extremely heterogeneous variance (p - 0.0000) in comparing vehicle vs treated groups so that the rank transformation was used to conduct one-way ANOVA. As Text Tables 2 and 3 indicate, there are no significantdifferences between vehicle and treated groups for both untransformed and transformed cases (p - 0.0953 and p - 0.0692, respectively). Furthermore, Terpstra-Jonckheeretest and regression of ranktransformed data did not show any significant positive trend as indicated in Text Table 4 even though the regression based on the untransfomed data showed marginally significanttrend over doses (p - 0.0240). There was no significant lack of fit for both regressions(p - 0.075 and 0.378). The followingnotations are used to denote direction and statistical significance: - significantat p :50.05 significant at p :50.01 t - effect in the positive direction Text Table I - Univariate ANOVA Vehicle vs Positive Control Vehicle vs Treated Groups Dose p Slide p SlidexDose p 0.0004 0.2269 0.2499 0.0963 0.4920 0.1360 154-208 33 dWabHW AAZSLCH MICN NG T 0 N Text Table 2 - The DescriDtive Statistics Untransformed Dose(mg/kg) Mean SD Median Rank-Transformed Mean SD Vehicle vs Positive Control Vehicle Positive 1.3400 34.9620 0.4649 13.1132 1.5200 36.6700 3.0000 8.0000 1.5411 1.5811 Vehicle vs Treated Groups Vehicle 100 200 400 800 1.3400 2.9800 1.4000 2.2540 4.4200 0.4649 1.3313 0.7588 0.9313 3.7928 1.5200 2.7100 1.3300 1.9000 2.3300 8.0000 17.7000 7.9000 15.2000 16.2000 5.1962 5.2631 6.4070 6.3008 8.8713 34 154-208 HAZLETON WASH IN GTO N Treatment Text Table 3 - One-Way ANOVA_ Vehicle vs-PositiveControl Vehicle-vs Treated Groups Untransformed Transformed Untransfomed Transformed .0004 ** .0010 ** .0953 .0692 Text Table 4 - Test for Trend for Vehicle vs Treated Groups Terpstra-JonckheereTest Regression of Untransformed Data Regression of Rank Transformed Data .0865 t .0240 *t .1870 t Discussion: The resultsof the present study indicatethat therewas no statistically significantincreasein cell proliferationover controldue to treatmentby the chemical. Moreover,there was no indicationof a significantpositivetrend due to treatment. References: (1] Ajit K. Thakur, A Fortran Program to Perform the Nonparametric TerpstraJonckheere Test, Computer Programs in Biomedicine 18: 235-240, 1984. [2] SAS (StatisticalAnalysisSystem),SAS Institute,Cary, NC, 1991. 154-208 35 HAZLEOCON W A S H IN G T 0 N HWA Study No. Protocol No. 493, Edition 3 ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS Hazleton Washington, Inc. (HWA) will conduct this study in compliance with EPA and FDA Good Laboratory Practice (GLP) Regulations. This protocol, critical phase(s) of the work in progress and the final report will be subject to audit by Quality Assurance in accordance with SOPs at Hazleton Washington, Inc. This study will be conducted by HWA at 9200 Leesburg Pike, Vienna, Virginia 22182. PART 1. SPONSOR INFORMATION AND APPROVALS I. SPONSOR IDENTIFICAT ON Company Name: 3H Corporation Address: Building 220-2E-02, 3M Center, St. Paul, MN 55144-1000 ii. T-59-77 TESTARTICLEIDENTIFICATION: III. TEST ARTICLE ANALYSIS Determination of the test article stability and the test article characteristics as defined in the GLP regulations of FDA (21 CFR 58.105), EPA-TSCA (40 CFR 792.105), and EPA-FIFRA (40 CFR 160.105) is the responsibility of the Sponsor. IV. NOTIFICATION OF REGULATORY SUBMISSION In order to comply with U.S. federal regulation codes (FDA, 21 CFR 58.10; EPA-TSCA, 40 CFR 792.10; EPA-FIFRA, 40 CFR 160.10) and certain foreign agencies, consulting laboratories must be notified if all or part of a study is to be submitted to the agency. HWA maintains a master schedule of studies which fall under regulatory review. Please indicatewhich agency,if any, might receive the resultsof this study: Undetermined rl-.z FDA 1= MAFF F=7 MOHW OECD EPA-TSCA 1= OTHER EPA-FIFRA 10/93 Page 1 HAZLEOCON W A S H IN G T 0 N V. STUDY DATES Proposed Experimental Start Date: Proposed Experimental Termination Date: VI. APPROVAL OF STUDY PROTOCO Study Director: Maria A. Cifone, Ph.D. Sponsor's Authorized Representative: Protocol No. 493, Edition 3 Date: Date: 10/93 Page 2 '",HAZLE-CC:N W A S H IN G T 0 N Protocol No. 493, Edition 3 1. OBJECTIVE PART 2. STUDY PROTOCOL ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS The objective of this assay is to detect hepatotoxicity caused by the test material by measuring cell proliferation (CF) measured as S-phase induction induced in rat liver cells after i-nvi-votreatment. Cell proliferation is designed to measure the fraction of cells undergoing cell replication in rat liver using an immunohistochemical technique (1,2). Animals are given a single oral dose of the chemical and the livers are isolated following administration of bromodeoxyuridine (BrdU) for 72 hours in vivo with an ALZETO osmotic pump implanted subcutaneously. Quantification of cells that have incorporated DNA precursors over the 72-hour period has been shown to be useful for the evaluation of chemicals that may cause increased cell proliferationin the liver (3). ii. DEFINITION Hepatotoxicants such as carbon tetrachloride and dinitrotoluene induce an increase in cell proliferation to replace necrotic tissue (2,4). These proliferating cells may be detected during S-phase analysis. Other compounds may induce S-phase synthesis in the absence of hepatotoxicity. It is not apparent how cell proliferation acts in the carcinogenic process, but there are numerous processes that can be affected during replication (5-8). Chemically induced cell proliferation may increase the probability of spontaneous mutations as well as increase the probability of converting DNA adducts into mutations prior to a repair process. Unscheduled cell proliferation may also play a role in the expansion of preneoplastic cells leading to the emergence of a fully transformed clone of cells. Some of these examples act by a nongenotoxic mechanism. It is therefore possible to detect nongenotoxic carcinogens as well as genotoxic carcinogens using this technique, III. MATERIALS A. Animals 10/93 Young adult male rats of the Sprague-Dawley strain, 10-12 weeks old at the time of dosing, will be purchased from Harlan Sprague Dawley, Inc. (HSD:Sprague-DawleYO(SDO)BR) or Charles River Laboratories, Inc. (Crl:CDOBR). This healthy random bred strain has been selected to maximize genetic heterogeneity and at the same time assure access to a common source. Page 3 tiAZLaOCC:N W A S H IN G T 0 N Protocol No. 493, Edition 3 The animals will be housed according to standard operating procedures and will be fed Purina Certified4ORodent Chow (formula 5002) and water Ad libitum. They will be quarantined a minimum of seven days prior to use and will be randomly assigned to study groups. The rats to be used for the assay will be anesthetized before surgery. The animals will be anesthetized using Metofanes (methoxyflurane, Pitman-Moore, Inc.) inhalation anesthesia and one pump per animal will be aseptically inserted subcutaneously (dorsal surface). Seventy-two hours later, animals will be anesthetized with C02 prior to removal of the livers and duodenum (control organ). B. Osmotic Pumps and Label for Cell Proliferation Analysis ALZEI'O osmotic pumps (ALZA Corporation, Palo Alto, CA), Model 2ML1 will be used. A single lot will be used throughout the study. The ALZEI'O Model 2ML1 osmotic pump has a 2000 pl capacity with a pump rate of 10 pl/hr. The pumps will be filled with bromodeoxyuridine (BrdU) at a concentration of 20 mg/ml. C. Control Articles 1. Vehicle control A vehicle negative control consisting of a minimum of five rats for cell proliferation. They will be treated with the vehicle or solvent selected for the test material. The same dosing methods (usually oral gavage) used for the test material treatments will be employed for the vehicle control. Where possible dosing volumes for oral gavage will not exceed about 10 ml/kg body weight. 2. Positive control article The positive control articles used are known to induce S-phase in rat hepatocytes in vivo. The positive control for cell proliferation will be 15 mg/kg of DMN. At least five rats will be treated by per os. D. Test Article Unless specified by the sponsor, the test article will normally be tested as supplied. Any operations performed on the test article such as grinding, extraction, or solvent-exchange must be specified by the sponsor prior to the initiation of testing. All operations performed on the test article will be described in the final report. 10/93 Page 4 '"-'*HAZLE'C(ON W A S H IN G T 0 N Protocol No. 493, Edition 3 IV. EXPERIMENTAL DESIGN A. Dosing Procedure A preliminary test will be performed to determine vehicle/solvent selection for the test article unless a vehicle/solvent is specified by the Sponsor. Materials which may be selected include water, methylcellulose, carboxymethylcellulose, corn oil or another suitable vehicle/solvent. Rats will be treated by oral gavage with the test article in volumes that will not exceed about 10 ml/kg body weight. Alternate routes.of exposure may be requested by the Sponsor. DMN will be di@ssolvedin sterile deionized water. Fresh preparations of the test article and positive controls in the solvent or vehicle will be used for any testing purpose. Stability of the test material under conditions of preparation and dosing will be the responsibility of the Sponsor. B. Dose Selection Unless specified otherwise, the highest dose selected will usually be 1 g/kg or half the IJ)50,whichever is less. Four doses will be selected using approximately two-fold dilution steps. Five animals from each dose level and control group will be used to analyze cell proliferation at 72-hours. C. Im2lantation of Osmotic Pumps ALZEI'O Model 2ML1 osmotic pumps will be preloaded with 2000 Al of BrdU at a concentration of 20 mg/ml. Following dosing with the test material, the animals will be anesthetized using MetofaneO (methoxyflurane, Pitman-Moore, Inc.) inhalation anesthesia and one pump per animal will be aseptically inserted subcutaneously (dorsal surface). The incision will be closed with wound clips and the animals monitored until the time of sacrifice to ensure that there are no clinical signs of infection. The osmotic pumps will be kept in the rats for three days prior to sacrifice. D. Tissue Collection and Preparation Each animal will be anesthetized prior to removal of organs for analysis. The thoracic cavity will be opened and the liver removed and fixedin neutralbufferefdormalin,A cross section of duodenum, a tissue with high cell turnover, will also be removed 10/93 Page 5 '"-'@'HAZLCNZC:N W A S H IN G T 0 N Protocol No. 493, Edition 3 from each animal and fixed. The duodenum will be included as an indicator that the label was administered correctly to the animal. For the liver, 3-5 p paraffin embedded sections will be taken. In the high dose animals, three slides from the left lateral lobe and one slide from the median and right anterior lobes will be prepared. Three slides from the remaining animals will also be prepared following qualitative analysis of the high dose slides. If labeling is similar among the lobes, all three slides will be prepared from the left lateral lobe. If labeling is different among the lobes, a slide from each lobe will be prepared. 3-5 p sections of the duodenum will also be made. The liver sections will be mounted on slides and a sample from the duodenum will be included on each slide. One slide each from the left lateral, median and right anterior lobes of the livers will also be prepared for analysis by a pathologist. Liver sections from all animals will be analyzed for histopathology, including gross lesions. E. Immunohistochemical Stainin The slides will be deparaffinized and rehydrated prior to staining using 1) Biogenix primary and secondary antibodies with peroxidaseconjugated streptavidin, 3,3-diaminobenzidine tetrahydrochloride (DAB) chromogen and hematoxylin counterstain. Separate slides for histopathology will be stained with hematoxylin and eosin. F. Assessment of Cell Proliferation Rates The section of the intestine will be microscopically examined to ensure that the label was properly administered to the animal. If adequate labeling is not observed, slides from the particular animal will not be analyzed. Once label distribution has been confirmed, a sampling of liver slides from the different lobes from the high dose animals will be examined to determine if differences in labeling are observed. If a qualitative difference in labeling among the liver lobes is observed, all the lobes will be counted. If no differences are observed, labeled hepatocytes in the left lateral lobe will be determined. At least 2000 nuclei will be examined per animal with a minimum of 6 fields per section analyzed. Counting will be confined to hepatocyte nuclei but other cell types such as inflammatory cells may be counted (separately) if the data appears relevant. The coverslips will be coded to prevent bias in counting. 10/93 Page 6 k@,w@ HAZLETON W A S H IN G T 0 N Protocol No. 493, Edition 3 V. DATA PRESENTATION The final report will include the following information in tabular form for each timepoint, for the negative control, positive control, and each analyzed treatment: The calculated %S-phase standard deviation among the three slides for each animal analyzed for S-phase. vi. ASSAY EVALUATION CRITERIA A mean and standard deviation for the percentage of S-phase cells will be calculated for each treatment group using the individual animal mean S-phase values. Statistical analysis of labeling index will be performed using one-way analysis of variance techniques (9). Control versus treatment group comparisons will be done with Dunnet's t-test (10,11). In the case of variance heterogeneity, rank transformation of the data will be performed prior to analysis of variance and Dunnet's t-test. Student's t-test will be used for comparison of the positive control versus the vehicle control. An S-phase percentage in a dose group that deviates from the S-phase percentage in the concurrent control group at a significance level of p--;0.0w5ill be considered significantly different than the control group. VII. REFERENCES 1. DeFazio, A., Leary, J.A., Hedley, D.W. and Tattersall, M.H.N. (1987). Immunohistochemical detection of proliferating cell in vivo. J. Histochem. Cytochem. 35, 571-577. 2. Lanier, T.L., Berger, E.K. and Eacho, P.I. (1989) Comparison of 5bromodeoxyuridine and 3H-thymidine in rodent hepatocellular proliferation studies. Toxicologist 9, 64. 3. Butterworth, B.E., Ashby, J., Bermudez, E., Casciano, D., Mirsalis, J., Probst, G., and G. Williams: A protocol and guide for the in vivo rat hepatocyte DNA repair assay. Mutation Research, 189:123133, 1987. 4. Mirsalis, J.C. and Butterworth, B.E.: Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene. Carcinogenesis, 3:241-245, 1982. 5. Marsman, D.S., Cattley, R.C., Conway, J.G. and Popp, J.A. (1988). Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalateand (4-chloro-6-(2,3xylidino)-2-pyrimidinylthio]aceticacid (Wy-14,643) in rats. Cancer Res. 48, 6739-6744. 10/93 Page 7 '"-'@'HAZLE-EC:N W A S H IN G T 0 N Protocol No. 493, Edition 3 6. Craddock, V.M. (1976). Cell proliferation and experimental liver cancer. In: "Liver Cell Cancer", Cameron,H.M., Linsell,C.A. and Warwick,G.P., Elsevier, North Holland Biomedical Press, Amsterdam. 7. Columbano, A., Rajalaksmi, S. and Sarma, D.S.R. (1981). Requirement of cell proliferation for the initiation of liver carcinogenesis as assayed by three different procedures. Cancer Res. 41, 2079-2083. 8. Glinos, A.D., Butcher, N.L.R. and Aub, J.C. (1951) The effect of liver regeneration on tumor formation in rats fed 4-diaminobenzene. J. Exp. Med. 933, 313-324. 9. Winer, B.J. (1971). Statistical Principles in Experimental Desizn, McGraw-Hill, New York, 2nd Edition, pp. 149-220. 10. Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 10961121. 11. Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482-491. Viii. REPORT FORMAT The final report will provide the following information. Sponsor identification. Test material identification and Assay Number. A physical description of the test material and date of receipt will be included in this section. Type of assay and protocol number. Dates of study initiation and completion. Names of Study Director, Senior Technician, Scientist Interpretation of results. Conclusions. Historical control data for negative and positive control cultures. Signatures of Study Supervisor and Study Director. Test results presented in tabular forms. Methods. o Evaluation criteria. References. Quality Assurance statement. 10/93 Page 8 HAZLE=CCDN W A S H IN G T 0 N Protocol No. 493, Edition 3 ix. CHANGES OR REVISIONS Any changes or revisions of this approved protocol will be documented, signed by the study director, dated, and maintained with this protocol. The sponsor will be notified of any change or revisions. X. RECORDS-TO BE MAINTAINED All raw data, documentation, records, protocols, and final reports generated as a result of this study will be archived in the storage facilities of Hazleton 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 Hazleton for an additional period of time or sent to a storage facility designated by the sponsor. 10/93 Page 9 HAZLCTON WASHINGTON AMENDMENT TO STUDY PROTOCOL STUDY TITLE: ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS HWA PROTOCOL: 493, EDITION 4 EFFECTIVE DATE: FEBRUARY 8, 1994 ASSAY NO.: 154-208 AMENDMENT NO.: 1 The following changes are made to the study protocol: The second paragraph of Section III. A. Animals is changed from: "The animals will be housed according to standard operating procedures and will be fed Purina CertifiedS Rodent Chow (formula 5002) and water &d libit They will be quarantined a minimum of seven days prior to use and will be randomly assigned to study groups. The rats to be used for surgery will be anesthetized before surgery." to: "The animals will be housed according to standard operating procedures and will be fed Purina Certified@ Rodent Chow (formula 5002) and water Ad libit No contaminants are known to be present in the diet or water at levels which might interfere with the study. The animals will be quarantined a minimum of seven days prior to use and will be randomly assigned to study groups. Randomization of the animals and animal identification will be performed according to standard operating procedures of the Mammalian Toxicology Section. The rats to be used for surgery will be anesthetized before surgery." The first two sentences of Section IV. D. Tissue Collection and Preparation is changed from: "Each animal will be anesthetized prior to removal of organs for analysis. The thoracic cavity will be opened and the liver removed and fixed in neutral buffered formalin.1' to: "Each animal will be weighed and anesthetized prior to removal of organs for analysis. The thoracic cavity will be opened and the liver removed, weighed and fixed in neutral buffered formalin.11 Reaso Information inadvertently left out of protocol. HAZLENCON W A S H IN G T 0 N -PAGE 2 - AMENDMENT TO STUDY PROTOCOL STUDY TITLE: ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS AMENDMENT NO.: 1 ASSAY NO.: 154-208 STUDY DIRECTOR'S SICNATURE: DATE: 3M InternaCIorrespondence cc: To: From: Subject: Date: R- G. Perkins-220-2E-10 B. C. Copley -53-3S-02 R. D. Howells - 53-3S-02 Key toFC AlcoholTox Samples September 14,1995 T5877 T5710 T5711 T5794 T5795 T5878 Wide Range ETFOSE FM @3924Lot 547 Retainfrom 2 yearfeedingstudy AnalyticalRequest 41220 L-13203 Narrow Range ETFOSE Lot 884 TypicalRaw Material forFC-807 AnalyticalRequest 41220 L-10059 Wide Range MEFOSE Lot 555 TypicalRaw Material forFX-845 AnalyticalRequest 41220 L-1276 Narrow Range MEFOSE Notebook 97900-107-2 Lab PreparedSample AnalyticalRequest 41343 L-13097 Wide Rana0e ETFOSE -\-Otebook97900-112-2 Lab PreparedSample AnalyticalRequest 41343 L-1@J098 Wide Range MEFOSE Lab Preparedfrom Washed POSF AnalyticalRequest 42607 RD@@ Attachments 41220 R. Howells GC/MS analysesoftheseswnpleswere accomplishedusinga 25 m X 0.32mm BP- I GC column to introducethesamplesintotheFinniganSSQ-70 mass spectrometerT.he samplecomponents were ionized usingchemicalionizatiownithmethane asthereagentgas.The GC column was operatedfrom 40 to300 C ata rateof10 degreesperniinuteT.he resultosftheseanalysescombined withtheircorrespondingGC/FID area percentsshow thefollowing: Component I.D. N-Ethyl-carboxamid(eRsf-C(O)N(Et)H) N-ETFOS Amide (CsFl7SO2N(Et)H) L-10059 N.R. N-ETFOSE Lot 0.22% 0.04% W.R. N-ETFOSE Lot547 1.76% 0.13% C2F5SO2N(Et) OH C3F7SO2N(Et)CH2CH20H C4F9SO2N(Et)CH2CH20H C8Fl7SO2N(Et)2 C5FiiSO2N(Et)CH2CH20H C6Fl3SO2N(Et)CH2CH20H C8Fl7SO2N(Et)CH2CH2CI C7Fl5SO2N(Et)CH2CH20H N-ETFOSE C8Fl7SO2N(Et)CH2CH20H C8Fl5SO2N(Et)CH2CH20H 0.01% 0.13% 0.11% 0.03% 0.03% 0.51% -.0.08% 0.82% 96.09% 0.78% 2.12% 1.17% -1 1.52% 0.04% 1.34% 3.52% 0.16% 1.40% 87.16% --0.50% C8Fl7SO2N(Et)(CH2CH20)2H C8Hl7SO2N(Et)H C8Fl7SO2N(Et)CH2CH2OCO2CH2CH3 (orsimilar) 0.17% 0.16% -0.21% 0.37% 0.22% OtherHigh Boilers 0.28% 0.59% ComponentI.D. N-Methyl-carboxamide(sRf-C(O)N(Me)H) C2F5S02N(Me)CH2CH20H C8Fl7SO2N(Me)2 C3F7SO2N(Me)CH2CH20H C8Fl7SO2N(Me)H C4F9SO2N(Me)CH2CH20H C5FiiSO2N(Me)CH2CH20H C6Fl3SO2N(Me)CMCH20H C8Fl7SO2N(Me)CH2CH2CI C7Fl5SO2N(Me)CH2CH20H N-MEFOSE C8Fl7SO2N(Me)CH2CH20H CgF19SO2N(Me)CH2CH20H C8Fl7SO2N(Me)(CH2CH20)2H C8Hl7SO2N(Me)H OtherHig Boilers L-1276 N-MEFOSE Lot 1.46% 0.24 % trace 1.15% trace 1.62% 1.34% 5.05% 0.13% 1.69% 83.88% 0.87% 0.34% 0.54% 1.55% Furtherwork has been done by GC on thesesampleswhich involvedserivatizatioofnthealcoholswith trifluoroaceatnihcydride(TFAA) and againwithBSA (togivethetrimethylsileytlhers)T.hiswork was designedtoinvestigattehepotentiaplroblems thatcouldbe overlookedby any one method of analysis. Preffininarryesultshow thatanalyzingtheETFOSE underivatizecdouldhidea significanatmount of N-ETFOS Amide undertheC-3 alcoholpeak inwide rangematerial.However, analyzingthe same materialthathas been derivatizewdith TFAA shows thatany EtFOSE-chloridethatispresentin thesample isnow completelymasked by thederivatizeCd-8 alcohol.The BSA derivativheas not been evaluatedyet,but similarproblems are expectedbecauseof thenumber of differenctomponents inthesample. The same sortofproblems willmost likelyexistwithMEFOSE and willbe even more complicatedintheanalysisofMEFOSEA. 7/2/93 REQ 41343 J.Grant GC/MS analyseosfthesseamplewsereaccomplishuesdinga 25m X 0.32mm HP-1GC columnto introducethesamples intotheFinniganSSQ-70 mass spectrometer.The sample components were ionized usingchemicalionizatiownith methane as thereagentgas.The GC column was operatedfrom 40 to300 C ata rateoflo degreesper minute.The resultosf theseanalysescombined with theircorrespondingGC/FIID area percentsshow the following: Component I.D. N-Ethyl-carboxamide(sRf-C(O)N(Et)H) C6Fl3SO2N(Et)H C2F5SO2N(Et)CH2MOH C7Fl5SO2N(Et)H N-ETFOS Amide (C8Fl7SO2N(Et)H) C6Fl3SO2N(Et)2 C3F7SO2N(Et)CH2CH20H C4F9SO2N(Et)CH2CH20H C5Fl iS02N(Et)CH2CH20H C6Fl3SO2N(Et)CH2CH20H C7Fl5SO2N(Et)CH2CH20H N-ETFOSE C8Fl7SO2N(Et)CH2CH20H W.R. N-ETFOSE Precut 97900-112-1 68.34% 1.28% trace trace 0.41% 1.41% 1.53% 0.62% 1.48% 20.16% 3.52% 1.14% W.R. N-ETFOSE main cut 97900-112-2 1.27% 0.32% trace trace trace trace 0.51% 0.42% 2.16% 60.86% 22.55% 11.92% Component I.D. N.R. N-MEFOSE B.P. 132 97900-107-2 ----------- mw 137possibl-ySO2N(Me)CIbCH20C7Fl5SO2N(Me)CH2CH20H N-MEFOSE C8Fl7SO2N(Nle)CH2CIi2OH CgF19SO2N(Me)CH2CH20H C8Fl7SO2N(Me)(CH2CH20)2H C8Hl7SO2N(Me)H OtherIfig Boilers 0.58% 0.41% 98.19% 0.59% trace trace 0.23% Component I.D. N-Ethyl-carboxamide(sRf@-C(O)N(Et)H) C3F7SO2N(Et)H C4F9SO2N(Et)H C5FiIS02N(Et)H C6Fl3SO2N(Et)H C7Fl5SO2N(Et)H N-ETFOS Amide (C8Fl7SO2N(Et)H) C8Fi5SO2N(Et)H numerous otherimpuritieosf most homologs thatincludehydrides,chlorineinthebackbone,and unidentifiehdighboilers W.R. N-ETFOS Amide 97900-111 10.23% trace 0.71% 3.58% 52.50% 20.06% 12.93% trace trace To: From: Subject: Date: 1.Muggli R. M. Payfer 53-6S-02 I 236-2B-11 (612)733-4212 SA&C AnalyticalRequest No. 42607 Dec. 21, 1993 GC/MS analysisofthissample was accomplished using a 25 m X 0.32 mm HP-1 GC column to introducethe sample intothe FinniganSSQ-70 mass spectrometer.The sample components were ionizedusingchemical ionizatiownith methane as the reagentgas. The GC column was operated from 40 to 300 C at a rateof 10 degrees per minute. GC analysiswithflame ionizatiodnetectionwas alsodone, and the area percent valuesfrom thiswork were appliedto the peak identitiefsrom the mass spec work. The resultsofthese analyses (whichare not necessarilyquantitatives)how the following: Mol. Weight Component I.D. L-13202 N-MEFOSE 427 N-Methyl-carboxamidesC7F,5-C(O)N(Me)H 0.06% 527 CsFl7SO2N(Me)2 0.12% 513 CsFl7SO2N(Me)H 0.25% 357 C4F9SO2N(Me)CH2CH20H 0.03% 407 C5FilSO2N(Me)CH2CH20H 0.52% 457 C6Fl3SO2N(Me)CH2CH20H 507 C7Fl5SO2N(Me)CH2CH20H 3.38% 2.16% 557 N-MEFOSE C8Fl7SO2N(Me)CH2CH20H 89.48% 519 C8FiSS02N(Me)CH2CH20H 0.84% 607 CgFl9SO2N(Me)CH2CH20H 0.55% 573 C8Fl6CIS02N(Me)CH2CH20H 0.67% 665 C8Fl6SFS-SO2N(Me)CH2CH20H 0.29% 4 601 C8Fl7SO2N(Me)(CH2CH20)2H Other High Boilers 0.54% 1.05%