Document LgbBKxNQDyOQqmXM5Mqp3n3xQ

HAZLETON WASHINGTON 9 2 0 0 LE E S B U R G PIKE V I E N N A . VA. 2 2 1 8 2 - 1 6 9 9 154-208 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 10 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 ( 0u^>34 /HU2-O270 1 of 35 HAZLETON WASHINGTON QUALITY ASSURANCE STATEMENT PROJECT TITLE: Analysis of T-5877 in a Cell Proliferation Assay 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: InsDection/Date Dosing and pump implantation (surgery) Draft report review/ 7-25,26-94 Final report review/ 11-1-94 Findinas Reoorted 2-7-94 7-26-94 11-1-94 Auditor B. Mullett B. Mullett B. Mullett 154-208 7U u&_i f Quality Assurance Unit oosaz Date7 (efeased 2 HAZLETON WASHINGTON COMPLIANCE AND CERTIFICATION STATEMENT The described study was conducted in compliance with 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 aforementioned regulations or the signed protocol that would affect the integrity of the study or the interpretation of 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 conclusion within the context of the study design and evaluation criteria. 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 submission of the final report to the sponsor. After the one year period, the sponsor may elect to have these materials retained in the storage facilities of Hazleton for an additional period of time or sent to a storage facility designated by the sponsor. SUBMITTED BY: vLuzs- ^ Andrea L. Ham, M.S. Associate Scientist Study Director: ip * /* n a A. C'ifone./'Ph. D. Stjidy Director V Genetic and Cellular Toxicology Date //- /- <?.-4 Study Completion Date 154-208 C0353G 3 HAZLETON WASHINGTON TABLE OF CONTENTS PAGE NUMBER ABSTRACT ............................................................... I. S P O N S O R ........................................................... II. MATERIAL TESTED ................................................... A. Genetics Assay No. B. Identification C. Physical Description D. Date Received III. TYPE OF A S S A Y S ................................................... IV. PROTOCOL NUMBER ................................................... V. STUDY DATES ....................................................... A. Study Initiation Date B. Experimental Start Date C. Experimental Termination Date VI. SUPERVISORY PERSONNEL ............................................. A. Study Director B. Associate Scientist VII. OBJECTIVE......................................................... VIII. D E F I N I T I O N ....................................................... IX. MATERIALS......................................................... A. Indicator Cells B. Osmotic Pumps and Label for Cell Proliferation Analysis C. Control Articles X. EXPERIMENT DESIGN ................................................. A. Dosing Procedure B. Implantation of Osmotic Pumps C. Tissue Collection and Preparation D. Immunohistochemical Staining E. Assessment of Cell Proliferation 6 7 7 7 7 7 7 7 8 8 9 154-208 r t0^337 4 HAZLETON WASHINGTON TABLE OF CONTENTS (CONTINUED) XI. ASSAY EVALUATION CRITERIA ........................................ XII. INTERPRETATION OF RESULTS ......................................... A. General Observations B. Summary of Labeled Cell Counts for the Liver XIII. CONCLUSIONS....................................................... XIV. R E F E R E N C E S ....................................................... XV. EXPERIMENTAL DATA TABLE ........................................... APPENDIX A APPENDIX B APPENDIX C APPENDIX D Individual Animal and Slide Labeling Indices .............. Individual Animal Body and Liver Weightsand Liver to Body Weight Ratios ......... Histopathology Report ...................................... Statistical Analysis of Labeling Indices ................ 11 12 13 14 15 17 24 29 32 154-208 C0as38 5 HAZLETON WASHINGTON 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 in vivo treatment. The doses chosen for the study were 100, 200, 400 and 800 mg/kg. Dimethylnitrosamine (DMN) at 15 mg/kg was included as a positive control. In the cell proliferation assay, a single oral dose of the test material was administered and five animals per condition were labeled with BrdU for 72 hours using ALZET 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 differences in 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 immunohistochemical staining. 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 significant increases 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 proliferation were observed in the positive control animals. T-5877 was therefore considered negative for the induction of cell proliferation in rat liver cells. 154-208 C-0 * 3 5 3 3 6 HAZLETON WASHINGTON Analysis of T-5877 in a Cell Proliferation Assay 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 Proliferation in Rat Liver Cells IV. PROTOCOL NUMBER: 493, Edition 3, Modified for 3M Corporation V. STUDY DATES: A. Study Initiation Date: 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. 154-208 03540 7 HAZLETON WASHINGTON Cell proliferation measured the fraction of cells undergoing cell replication in rat liver using an immunohistochemical technique (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 administration of BrdU for 72 hours in vivo with an ALZET osmotic pump implanted subcutaneously. Quantification of cells that have incorporated DNA precursors over the 72-hour period indicates increased cell proliferation in the liver (3). VIII. 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 chemicals may induce S-phase in the absence of hepatotoxicity. It is not apparent how cell proliferation may act in the carcinogenic process but there are numerous mechanisms which 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 unrepaired DNA adducts into mutations. Unscheduled cell proliferation may 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:CDBR). 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 Certified Rodent Chow (Formula 5002) and water a libitum. Animals were quarantined a minimum of 7 days prior to random assignment to study groups and identification by implantable microidentification device for the cell proliferation assay. 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. 154-208 The cell proliferation assay was initiated with rats that ranged from 306 to 355 grams. Approximately 2 hours after dosing, the animals were anesthetized using Metofane (methoxyflurane, PitmanMoore, Inc.) inhalation anesthesia and one ALZET pump per animal was aseptically inserted subcutaneously (dorsal surface). 8 HAZLETON WASHINGTON Seventy-two hours later, animals were anesthetized with C02 prior to removal of the livers and duodenum. B. Osmotic Pumps and Label for Cell Proliferation Analysis ALZET osmotic pumps (ALZA Corporation, Palo Also, CA), Model 2ML1 were used. A single lot (#042301) was used throughout the study. The pump has a 2000 il capacity with a pump rate of 10 /xl/hour. The pumps were pre-filled with BrdU at a concentration of 20 mg/ml. C. Control Articles 1. Vehicle control A vehicle control consisting five rats was dosed by oral gavage (P.0.) with the vehicle, corn oil (Duke's Corn Oil, Lot 2L290833). Tissues from vehicle control animals were subjected to the same manipulations used for the tissues derived from treated animals. The dosing volume of the vehicle control animals did not exceed 10 ml/kg. 2. Positive control article The positive control compound is known to induce S-phase in rat hepatocytes in vivo. The positive control, dimethylnitrosamine (DMN, CAS# 62-75-9, Sigma Chemical Co., Lot# 82H0365) was dosed at 15.0 mg/kg. Five rats were treated P.O.. D. Test Article For the preparation of the dosing solutions of the test article, the test article was suspended in corn oil at concentrations of 10, 20, 40 and 80 mg/ml prior to dosing. The maximum dosing volumes for the test article did not exceed 10 ml/kg. X. EXPERIMENT DESIGN: A. Dosing Procedure Five rats per condition were treated by oral gavage with T-5877 for the cell proliferation assay. Delivery volumes were calculated on the basis of the most recent animal weight and the target dose. The maximum volume of the test article suspensions administered did not exceed 10 ml/kg. Fresh preparations of test article in vehicle were used for any testing purpose. 154-208 9 HAZLETON WASHINGTON B. C. D. E. 154-208 Confirmation of the concentration of the test material under conditions of preparation and dosing of the assay was not determined in conjunction with this study. Implantation of Osmotic Pumps For the cell proliferation assay, ALZET Model 2ML1 osmotic pumps (Lot #042301) were preloaded with 2000 jzl of BrdU at a concentration of 20 mg/ml. The animals were anesthetized using Metofane according to standard procedures and one pump per animal was aseptically inserted subcutaneously (dorsal surface) approximately 2 hours after dosing. The incision was closed with wound clips and the animals monitored until the time of sacrifice to ensure that there were no clinical signs of infection. The osmotic pumps were implanted three days prior to sacrifice. Tissue Collection and Preparation Each animal was anesthetized prior to removal of organs for analysis. The thoracic cavity was opened and the liver removed and fixed in neutral buffered formalin. A cross section of 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 administered correctly to each animal. For the livers from high dose (Group 5) animals, 5 /z paraffin embedded sections were taken from the left lateral, median and right anterior lobes. Once it was determined that no treatment-related lobular differences were present, slides from 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 containing a liver section. Slides were also prepared according to standard procedures for examination by a pathologist to determine if any abnormalities were present. Immunohistochemical Staining The slides were deparaffinized and rehydrated prior to staining. The slides were stained for determination of cell proliferation as measured by incorporation of BrdU into DNA using Biogenix antibodies with peroxidase-conjugated streptavidin and a 3,3-diaminobenzidine tetrahydrochloride (DAB) chromogen and hematoxylin counterstain. Assessment of Cell Proliferation The section of the duodenum was microscopically examined to ensure that the label was properly administered to the animal. Once label delivery was confirmed, slides from the different lobes of the high dose animals were examined for lobular differences. Labeling was similar among the lobes therefore cell counting was 10 603543 HAZLETON WASHINGTON performed with sections from the left lateral lobe from all animals. The percentage of nuclei incorporating label in the liver was determined microscopically. The areas to be counted were randomly generated by computer. A 1.0 mm square indexed ocular grid divided into 10 x 10 squares was used to define the counting area. At least 2000 nuclei were examined per animal with a minimum of 3 sections and 6 fields per section. Any nuclei that were blue were considered unlabeled and any nuclei containing any brown chromogenic hue were considered labeled unless a clear artifact was present. Only hepatocyte nuclei were enumerated. Fields that contained areas of necrosis were not included in the evaluation. The slides were coded for (blind) evaluation as to treatment group. S-phase nuclei labeling indices for each animal were calculated as fol1ows: Labeled S-phase nuclei ( L n = no. of labeled hepatocyte nuclei X 100 total no. of hepatocytes counted XI. ASSAY EVALUATION CRITERIA The proportions of the number of cells labeled to the number of cells counted were analyzed by repeated measures analysis of variance (AN0VA) techniques to determine any slide, and related interaction effects. The sphericity test was also utilized to test variance homogeneity. Additionally.the average value from the three slides of each animal was calculated to conduct one-way AN0VA, Dunnett's t-test, Terpsa-Jonkheere test, and regression tests for trend using both untransformed and ranked data. See Appendix C for statistical analysis of labeling indices. For the terminal whole body weights, liver weights, and liver to terminal body weight ratios, a mean and standard deviation were calculated for each treatment group using the individual animal mean S-phase values. Statistical analysis of labeling index was performed using one-way analysis of variance techniques. Control versus treatment group comparisons were done with Dunnet's t-test and control versus positive group comparisons were done using the Student's t-test. In the case of variance heterogeneity, rank transformations of the data were performed prior to analysis of variance and Dunnet's t-test. A labeling index, terminal body weight, terminal liver weight and liver to body weight ratio in a dose group that deviates from the values in the concurrent control group at a significance level of p<0.05 was considered significantly different than the control group. 154-208 003544 11 HAZLETON WASHINGTON XII. INTERPRETATION OF RESULTS A. General Observations All animals survived treatment. No histomorphological alterations 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 histopathology are 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 acceptable 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 significantly elevated 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 < 0.01). When the liver to body weight ratios were determined, there were significant increases in 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<0.01). 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-treated cells ranged from 1.40 to 4.42. None were considered significantly elevated and there was 154-208 C3545 12 HAZLETON WASHINGTON no indication of a significant positive trend due to treatment. The high dose (800 mg/kg) had the highest labeling index of 4.42, but because of a heterogeneous response, there was a lack of significance. If the results from individual animals are compared (see Appendix A), two of the five animals responded, with labeling indices of 8.00 and 9.05, while the remaining three animals had labeling indices close to background levels. This may indicate a weak response, but also may be the result of animal variability. See Appendix D for statistical analysis. The mean labeling index of the DMN positive control animals was 34.96 which is significantly elevated (p < 0.01). These results demonstrate that T-5877 did not induce significant dose-related increases in the LI in the liver in male rats after treatment with single oral doses at concentrations of 100 mg/kg to 800 mg/kg. Large increases in the labeling index were observed in the DMN positive control animals. The mean labeling index in the DMN-treated positive control animals was 34.96 (p < 0.01). XIII. CONCLUSIONS The test material, T-5877, did not induce significant changes in the number S-phase cells following a single oral dose of 100 mg/kg to 800 mg/kg. The animals were labeled for 72 hours and no significant dose-related trend in the mean labeling index was observed in the treated groups. T-5877 was therefore evaluated as negative for the induction of DNA synthesis in rat liver cells. 154-208 13 HAZLETON WASHINGTON XIV. REFERENCES 1. DeFazio, A., Leary, J.A., Hedley, D.W. and Tattersall, M.H.N. (1987). Immunohistochemical detection of proliferating cells in vivo. J. Histochem. Cytochem. 35, 571-577. 2. Lanier, T.L., Berger, E.K., and Eacho, P.I. (1989). Comparison of 5-bromodeoxyuridine 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 Res., 182:123-133, 1987. 4. Mirsalis, J.C. and Butterworth, B.E.: Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene. Carcinogenesis, 2:241-245, 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)phthalate and [4-chloro-6-(2,3xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643) in rats. Cancer Res. 48, 6739-6744. 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-diami nobenzene. J. Exp. Med. 933, 313-324. 9. Ham, A. and Cifone, M.A. (1991). Use of cell proliferation to study liver effects induced by a single dose of DMN. Environmental and Molecular Mutagenesis 17(19), 16. 154-208 e o a a "*7*ii 14 HAZLETON WASHINGTON XV. EXPERIMENTAL DATA TABLE HAZLETON WASHINGTON Client: 3M Corporation Client Code T-5877 Table 1 Cell Proliferation Summary HWA Assay No.: 154-208 Trial Initiation Date: February 7, 1994 Group/Sexa Dose Labeling Indexb Level (%) (mg/kg) Liver Weight Terminal Body Liver/Body (grams) Weight (grams) Weight (%) 1M 0C 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**4. 4.86 0.25**t 5M 800 4.42 3.60 15.38 1.05 300.0 15.0**4 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 Percentage of labeled hepatocyte nuclei per total number of hepatocytes counted (at least 2000) ^Vehicle control, Corn oil Positive control, 15 mg/kg of DMN * Significant at 0.01 < p < 0.05 ** Significant at p < 0.01 t Increase in the mean i Decrease in the mean 154-208 000549 16 HAZLETON WASHINGTON APPENDIX A Individual Animal and Slide Labeling Indices 154-208 C 0550 17 HAZLETON WASHINGTON Slide # Animal ID GrouD # Labeled 16 47733 17 47733 18 47733 1M 1M IN 10 21 5 # Counted % Labeled 700 1.43 700 3.00 700 0.71 Mean SD 1.71 1.17 19 47734 1M 8 700 1.14 20 47734 1M 9 700 1.29 21 47734 1M 7 700 1.00 Mean SD 1.14 0.14 22 47735 1M 5 700 0.71 23 47735 1M 4 700 0.57 24 47735 1M 4 700 0.57 Mean SD 0.62 0.08 25 47736 26 47736 27 47736 1M 1M 1M 11 10 15 700 700 700 Mean SD 1.57 1.43 2.14 1.71 0.38 28 47737 29 47737 30 47737 1M 1M IN 7 13 12 700 700 700 Mean SD 1.00 1.86 1.71 1.52 0.46 GROUP MEAN GROUP SD 1.34 0.66 154-208 ^0^551 18 HAZLETON WASHINGTON Slide if Animal ID Group # Labeled # Counted % Labeled 46 47738 47 47738 48 47738 2M 2M 2M 21 16 29 700 700 700 Mean SD 3.00 2.29 4.14 3.14 0.94 49 47739 50 47739 51 47739 2M 2M 2M 22 19 16 700 700 700 Mean SD 3.14 2.71 2.29 2.71 0.43 52 47740 53 47740 54 47740 2M 2M 2M 14 26 8 700 700 700 Mean SD 2.00 3.71 1.14 2.29 1.31 55 47741 56 47741 57 47741 2M 2M 2M 37 22 49 700 700 700 Mean SD 5.29 3.14 7.00 5.14 1.93 58 47742 59 47742 60 47742 2M 2M 2M 12 700 4 700 18 700 Mean SD GROUP MEAN GROUP SD 1.71 0.57 2.57 1.62 1.00 2.98 1.61 154-208 03552 19 HAZLETON WASHINGTON Slide # Animal ID G rouD # Labeled # Counted % Labeled 61 47743 3M 6 700 0.86 62 47743 3M 2 700 0.29 63 47743 3M 6 700 0.86 Mean SD 0.67 0.33 64 47744 65 47744 66 47744 3M 3M 3M 14 9 11 700 700 700 Mean SD 2.00 1.29 1.57 1.62 0.36 67 47745 68 47745 69 47745 3M 3M 3M 16 11 27 700 700 700 Mean SD 2.29 1.57 3.86 2.57 1.17 70 47746 3M 4 700 0.57 71 47746 3M 6 700 0.86 72 47746 3M 7 700 1.00 Mean SD 0.81 0.22 73 47747 74 47747 75 47747 3M 3M 3M 9 3 16 700 700 700 Mean SD 1.29 0.43 2.29 1.33 0.93 GROUP MEAN GROUP SD 1.40 0.92 154-208 003553 20 HAZLETON WASHINGTON ! # Animal ID GrotiD # Labeled # Counted % Labeled 31 47748 32 47748 33 47748 4M 4M 4M 11 14 15 700 700 700 Mean SD 1.57 2.00 2.14 1.90 0.30 34 47749 35 47749 36 47749 4M 4M 4M 7 19 10 700 700 700 Mean SD 1.00 2.71 1.43 1.71 0.89 37 47750 38 47750 39 47750 4M 4M 4M 19 39 16 700 700 700 Mean SD 2.71 5.57 2.29 3.52 1.79 40 47751 41 47751 42 47751 4M 4M 4M 4 13 9 700 700 700 Mean SD 0.57 1.86 1.29 1.24 0.64 43 47752 44 47752 45 47752 4M 4M 4M 14 17 30 700 700 700 Mean SD 2.00 2.43 4.29 2.90 1.21 GROUP MEAN GROUP SD 2.26 1.26 154-208 003554 21 HAZLETON WASHINGTON Slide # Animal ID GrouD # Labeled 1 47753 2 47753 3 47753 5M 5N 5M 23 8 18 # Counted % Labeled 700 3.29 700 1.14 700 2.57 Mean SD 2.33 1.09 4 47754 5 47754 6 47754 5M 5N 5M 62 60 46 700 700 700 Mean SD 8.86 8.57 6.57 8.00 1.25 7 47755 8 47755 9 47755 5N 5M 5M 12 5 5 700 700 700 Mean SD 1.71 0.71 0.71 1.05 0.58 10 47756 11 47756 12 47756 5M 5M 5M 63 56 71 700 9.00 700 8.00 700 10.14 Mean SD 9.05 1.07 13 47757 14 47757 15 47757 5M 5N 5M 15 10 10 700 700 700 Mean SD 2.14 1.43 1.43 1.67 0.41 GROUP MEAN GROUP SD 4.42 3.60 154-208 C03555 22 HAZLETON WASHINGTON Slide # Animal ID GrouD # Labeled 76 47758 6M 260 77 47758 6M 364 78 47758 6M 435 # Counted % Labeled 700 37.14 700 52.00 700 62.14 Mean SD 50.43 12.57 79 47759 6M 265 80 47759 6M 251 81 47759 6M 171 700 700 700 Mean SD 37.86 35.86 24.43 32.71 7.24 82 47760 6M 155 83 47760 6M 246 84 47760 6M 369 700 700 700 Mean SD 22.14 35.14 52.71 36.67 15.34 85 47761 6M 270 86 47761 6M 292 87 47761 6N 285 700 700 700 Mean SD 38.57 41.71 40.71 40.33 1.61 88 47762 6M 54 89 47762 6M 165 90 47762 6N 89 700 700 700 Mean SD 7.71 23.57 12.71 14.67 8.11 GROUP MEAN GROUP SD 34.96 14.86 154-208 G0,3o 56 23 HAZLETON WASHINGTON APPENDIX B Individual Animal Body and Liver Weights and Liver to Body Weight Ratios 154-208 C0oo57 24 HAZLETON WASHINGTON 154-208 APPENDIX B ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS DRAFT* ABSOLUTE ORGAN WEIGHTS (g) *DRAFT* STUDY NUMBER: 154208 ORGAN ABBREVIATION: LI - LIVER SEX DOSE ANIMAL GROUP NIMBER TERMINAL BODY WT (g) LI M 1 B47733 M 1 B47734 M 1 B47735 M 1 B47736 M 1 B47737 344.0 350.0 322.0 352.0 353.0 14.24 13.77 11.66 14.19 14.11 NUMBER IN GROUP: MEAN: STANDARD DEV: 5 344.2 12.9 5 13.59 1.09 M 2 B47738 M 2 B47739 M 2 B47740 M 2 B47741 M 2 B47742 NUMBER IN GROUP: MEAN: STANOARD DEV: 371.0 350.0 340.0 355.0 324.6 5 348.1 17.3 16.35 14.31 12.12 15.87 12.98 5 14.32 1.81 M 3 B47743 M 3 B47744 M 3 B47745 M 3 B47746 M 3 B47747 NUMBER IN GROUP: MEAN: STANDARD DEV: 368.0 330.0 327.0 313.0 340.0 5 335.6 20.5 18.79 14.28 14.33 12.87 14.70 5 15.00 2.24 C0355S 25 HAZLETON WASHINGTON APPENDIX B ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS DRAFT* ABSOLUTE ORGAN WEIGHTS (g) `DRAFT* STUDY NUMBER: 154208 ORGAN ABBREVIATION: LI - LIVER SEX DOSE ANIMAL TERMINAL GROUP NUMBER BODY WT (g) LI M 4 B47748 M 4 B47749 M 4 B47750 M 4 B47751 M 4 B47752 NUMBER IN GROUP: MEAN: STANDARD DEV: 300.0 308.0 324.0 291.9 294.0 5 303.6 13.0 15.02 13.74 16.31 13.96 14.77 5 14.76 1.02 M 5 B47753 M 5 B47754 M 5 B47755 M 5 B47756 M 5 B47757 NUMBER IN GROUP: MEAN: STANDARD DEV: 314.0 293.0 278.6 314.0 300.2 5 300.0 15.0 16.26 14.30 14.19 15.89 16.25 5 15.38 1.05 M 6 B47758 M 6 B47759 M 6 B47760 M 6 B47761 M 6 B47762 NUMBER IN GROUP: MEAN: STANDARD DEV: 336.0 294.0 334.0 296.0 338.0 5 319.6 22.5 14.50 11.08 14.20 11.21 12.64 5 12.73 1.61 154-208 C03559 26 HAZLETON WASHINGTON APPENDIX B ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS DRAFT* 0R6AN-T0-TERMINAL BODY WEIGHT RATIOS (X) `DRAFT* STUDY NUMBER: 154208 ORGAN ABBREVIATION: LI - LIVER SEX DOSE ANIMAL GROUP NUMBER TERMINAL BODY WT (g) RATIO M 1 B47733 M 1 B47734 M 1 B47735 M 1 B47736 M 1 B47737 NUMBER IN GROUP: MEAN: STANDARD DEV: 344.0 350.0 322.0 352.0 353.0 5 344.2 12.9 4.138 3.933 3.622 4.030 3.998 5 3.944 0.195 M 2 B47738 M 2 B47739 M 2 B47740 M 2 B47741 M 2 B47742 371.0 350.0 340.0 355.0 324.6 NUMBER IN GROUP: MEAN: STANDARD DEV: 5 348.1 17.3 4.407 4.087 3.564 4.470 3.999 5 4.106 0.363 M 3 B47743 M 3 B47744 M 3 B47745 M 3 B47746 M 3 B47747 NUMBER IN GROUP: MEAN: STANDARD DEV: 368.0 330.0 327.0 313.0 340.0 5 335.6 20.5 5.107 4.326 4.382 4.112 4.325 5 4.450 0.381 154-208 C0vi5S0 27 HAZLETON WASHINGTON APPENDIX B ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS DRAFT* ORGAN-TO-TERMINAL BODY WEIGHT RATIOS (X) DRAFT* STUDY NUMBER: 154208 ORGAN ABBREVIATION: LI - LIVER SEX DOSE ANIMAL GROUP NUMBER TERMINAL BODY VT (9 ) RATIO M 4 B47748 M 4 B47749 M 4 B47750 M 4 B47751 M 4 B47752 300.0 308.0 324.0 291.9 294.0 NUMBER IN GROUP: MEAN: STANDARD DEV: 5 303.6 13.0 5.006 4.460 5.033 4.782 5.024 5 4.861 0.247 M 5 B47753 M 5 B47754 M 5 B47755 M 5 B47756 M 5 B47757 NUMBER IN GROUP: MEAN: STANDARD DEV: 314.0 293.0 278.6 314.0 300.2 5 300.0 15.0 5.179 4.879 5.093 5.060 5.412 5 5.125 0.194 M 6 B47758 M 6 B47759 M 6 B47760 M 6 B47761 M 6 B47762 336.0 294.0 334.0 296.0 338.0 NUMBER IN GROUP: MEAN: STANDARD DEV: 5 319.6 22.5 4.315 3.768 4.251 3.789 3.739 5 3.972 0.285 154-208 28 HAZLETON WASHINGTON APPENDIX C Histopathology Report HAZLETON WASHINGTON Pathology Report Analysis of Cell Proliferation in Rat Liver Cells Project No. 154-208 General Protocol 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 Model 2ML1 osmotic pump containing 20 mg/mL of bromodeoxyuridine (Brdll) was implanted subcutaneously in each rat while it was under Metofane 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 boardcertified veterinary pathologist. Histopatholoav 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 centrilobular necrosis, 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. 154-208 30 HAZLETON 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 histomorphologic changes were noted in the liver of rats dosed at 100 and 200 mg/kg. Pathologist: Samuel V. Machotka, D.V.M., D.A.B.T., Dipl ornate, American College of Veterinary Pathologists Department of Pathology Date 154-208 003564 31 HAZLETON WASHINGTON APPENDIX D Statistical Analysis of Labeling Indices 154-208 C0*i5S5 32 HAZLETON m 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 sphericity test was also utilized to test variance homogeneity. The model used was: proportion - n + dose + slide + slidexdose + 8 . 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-Geisser probabilities 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 siide-to-sl ide variations were not significant in either case. Only the positive control showed highly significant elevation in labelling over 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 significant differences between vehicle and treated groups for both untransformed and transformed cases (p - 0.0953 and p 0.0692, respectively). Furthermore, Terpstra-Jonckheere test 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 untransformed data showed marginally significant trend over doses (p - 0.0240). There was no significant lack of fit for both regressions (p - 0.075 and 0.378). The following notations are used to denote direction and statistical significance: * significant at p < 0.05 ** - significant at p < 0.01 t * effect in the positive direction Text Table 1 - 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 003566 33 HAZLETON WASHINGTON Text Table 2 - The Descriptive Statistics Untransformed Dose(mg/kg) Mean SD Median Rank-Transformed Mean SD Vehicle vs Positive Control Vehicle 1.3400 0.4649 Positive 34.9620 13.1132 1.5200 36.6700 3.0000 8.0000 1.5411 1.5811 Vehicle vs Treated Groups Vehicle 1.3400 0.4649 100 2.9800 1.3313 200 1.4000 0.7588 400 2.2540 0.9313 800 4.4200 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 154-208 C035S7 34 HAZLETON WASHINGTON __________________ Text Table 3 - One-Way ANOVA___________________________ Vehicle vs Positive Control Vehicle vs Treated Groups ____________ Untransformed Transformed Untransformed Transformed Treatment .0004 ** .0010 ** .0953 .0692 Text Table 4 - Test for Trend for Vehicle vs Treated Groups Terpstra-Jonckheere Test .0865 t Regression of Untransformed Data .0240 *t Regression of Rank Transformed Data_______ .1870 t______ Discussion: The results of the present study indicate that there was no statistically significant increase in cell proliferation over control due to treatment by the chemical. Moreover, there was no indication of a significant positive trend 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 (Statistical Analysis System), SAS Institute, Cary, NC, 1991. 154-208 C03568 35 HAZLETON WASHINGTON 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 IDENTIFICATION Company Name : 3M Corporation___________________________________ Address: Building 220-2E-02. 3M Center. St. Paul. MN 55144-1000 TII. TEST ARTICLE IDENTIFICATION: -5 8 7 7 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 indicate which agency, if any, might receive the results of this study: Undetermined FDA EPA-TSCA l!--- i EPA-FIFRA MAFF = J M0HW EZ 3 OECD L==J OTHER 10/93 coasss Page 1 HAZLETON WASHINGTON V. STUDY DATES Protocol No. 493, Edition 3 Proposed Experimental Start Date: Proposed Experimental Termination Date: VI. APPROVAL OF STUDY PROTOCOL Study Director: _____________________________________ Maria A. Cifone, Ph.D. Date : 10/93 6 0 ,3 5 7 0 Page 2 HAZLETON WASHINGTON Protocol No. 493, Edition 3 PART 2. STUDY PROTOCOL ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS I. OBJECTIVE The objective of this assay is to detect hepatotoxicity caused by the test material by measuring cell proliferation (CP) measured as S-phase induction induced in rat liver cells after in vivo treatment. 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 1 3 vivo with an ALZET 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 proliferation in 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 hepato toxicity. 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-Dawley(SD)BR) or Charles River Laboratories, Inc. (Crl:CDBR). 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 C03571 HAZLETOFM WASHINGTON Protocol No. 493, Edition 3 The animals will be housed according to standard operating proce dures and will be fed Purina Certified Rodent 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 Metofane (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 ALZET osmotic pumps (ALZA Corporation, Palo Alto, CA) , Model 2ML1 will be used. A single lot will be used throughout the study. The ALZET Model 2ML1 osmotic pump has a 2000 n l capacity with a pump rate of 10 /il/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 003572 Page 4 HAZLETON WASHINGTON 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 dissolved in 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 LD50. 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. Implantation of Osmotic Pumps ALZET Model 2ML1 osmotic pumps will be preloaded with 2000 pi of BrdU at a concentration of 20 mg/ml. Following dosing with the test material, the animals will be anesthetized using Metofane (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 fixed in neutral buffered formalin. A cross section of duodenum, a tissue with high cell turnover, will also be removed coas10/93 Page 5 ?3 HAZLETON WASHINGTON 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 n 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 Staining The slides will be deparaffinized and rehydrated prior to staining using 1) Biogenix primary and secondary antibodies with peroxidase conjugated 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 003574 Page 6 HAZLETON WASHINGTON 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 ZS-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^0.05 will 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, 1:241-245, 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)phthalate and [4-chloro-6-(2,3xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) in rats. Cancer Res. 48, 6739-6744. 10/93 Page 7 t UuJ ( HAZLETON WASHINGTON 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 Design. 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, 1096- 1121. 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. Evaluation criteria. References. Quality Assurance statement. 10/93 Page 8 HAZLETON WASHINGTON 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 HAZLETON WASHINGTON AMENDMENT TO STUDY PROTOCOL STUDY TITLE: ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS HWA PROTOCOL: 493, EDITION 4 ASSAY N O . : 154-208 EFFECTIVE DATE: FEBRUARY 8, 1994 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 Certified Rodent 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 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 libitum. 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." 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." Reason: Information inadvertently left out of protocol. C03578 HAZLETON WASHINGTON 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 f' SIGNATURE: AAJ3LS DATE: 003S79 3 M Internal Correspondence cc: To: From: Subject: Date: R.G. Perkins-220-2E-10 B. C. Copley -53-3S-02 R. D. Howells -53-3S-02 Key to F C Alcohol Tox Samples September 14,1995 T5877 T5710 T5711 T5794 T5795 T5878 Wide Range EtFOSE F M 3924 Lot 547 Retain from 2 year feeding study Analytical Request 41220 L-13203 Narrow Range EtFOSE Lot 884 Typical Ra w Material forFC-807 Analytical Request 41220 L-10059 Wide Range MeFOSE Lot 555 Typical Raw Material forFX-845 Analytical Request 41220 L-1276 Narrow Range MeFOSE Notebook 97900-107-2 Lab Prepared Sample Analytical Request 41343 L-13097 Wide Range EtFOSE Notebook 97900-112-2 Lab Prepared Sample Analytical Request 41343 L-13098 Wide Range MeFOSE Lab Prepared from Washed POSF Analytical Request 42607 RDH/j}d V --\ ^ f Attachments REQ 41220 R. Howells GC/MS analyses ofthese samples were accomplished using a 25 m X 0.32 m m HP-1 G C colu m n to introduce the samples into the Finnigan SSQ-70 mass spectrometer. The sample components were ionized using chemical ionization with methane as the reagent gas. The G C column was operated from 40 to 300 C ata rate of10 degrees per minute. The resultsofthese analyses combined with theircorresponding GC/FtD area percents show the following: Component I.D. L-10059 N.R. N-EtFOSE Lot W.R. N-EtFOSE Lot 547 N-Ethy1-carboxamides (Rf-C(O)NCEt)H) 0.22% 1.76 % N-EtFOS Amide (C8Fi7S02N(Et)H) 0.04% 0.13% C2F5SO2N(E0CH2CH2OH 0.01 % 0.12% C3F7S02N(Et)CH2CH20H 0.13% 1.17% C4F9S02N(Et)CH2CH20H 0.11 % 1.52% C8Fl7S02N(Et)2 0.03% 0.04% CsFi iS02N(Et)CH2CH20H C 6F 13S O 2NCEOCH2C H 2O H C8Fl7SO 2N (Et)CH2CH2Cl 0.03 % 0.51% 0.08% 1.34 % 3.52% 0.16% C7Fl5S0 2 N (Et)CH2CH20H 0.82% 1.40% N-EtFOSE C8Fi7S02N(Et)CH2CH20H 96.09% 87.16% C8Fi5SO2N(Et)CH2CH20H 0.78% 0.50 % CaFi7S02N(Et)(CH2CH20)2H CsHi7SO 2N (Et)H CsF17S O 2N(EOCH2C H 2O C O 2C H 2C H 3 (or similar) 0.17% 0.16% 0.21 % 0.37 % 0.22 % Other High Boilers 0.28% 0.59 % C035S1 Component I.D. N-M ethyl-carboxamides (Rf-C(0)N(M e)H) C 2F5S02N (M e)C H 2C H 20H CsFi7S02N(M e)2 C 3F7S02N (M e)C H 2C H 20H C8Fi7S02N(M e)H C 4F9S02N (M e)C H 2C H 20H CsFiiS02N(M e)CH2CH20H C 6F i3S02N (M e)C H 2C H 20H C 8Fi7S02N (M e)C H 2C H 2C l C 7Fi5S02N (M e)C H 2C H 20H N-MeFOSE C8Fi7S02N(Me)CH2CH20H C 9Fi9S02N (M e)C H 2C H 20H C 8FnS02N (M e)(C H 2C H 20)2H C8Hi7S02N(M e)H Other High Boilers L-1276 N -M eFO SE 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% Further work has been done by G C on these samples which involves derivatization ofthe alcohols with trifluoroacetic anhydride (TFAA) and again with B S A (to give the trimethylsilyl ethers). This work was designed to investigate the potentialproblems thatcould be overlooked by any one method of analysis. Preliminary results show that analyzing the EtFOSE underivatized could hide a significantamount ofN-EtFOS Amide under the C-3 alcohol peak in wide range material. However, analyzing the same material thathas been derivatized with T F A A shows thatany EtFOSE-chloride that ispresent in the sample isnow completely masked by the derivatized C-8 alcohol. The B S A derivative has not been evaluated yet, but similarproblems are expected because of the number ofdifferent components in thesample. The same sortofproblems willmost likelyexistwith Me F O S E and willbe even more complicated inthe analysis ofMeFOSEA. 7/2/93 003532 REQ 41343 J. Grant GC/MS analyses ofthese samples were accomplished using a 25 m X 0.32 m m HP-1 G C column to introduce the samples intothe Finnigan SSQ-70 mass spectrometer. The sample components were ionized using chemical ionization with methane as thereagent gas. The G C column was operated from 40 to 300 C ata rate oflO degrees per minute. The resultsofthese analyses combined with theircorresponding GC/FID area percents show the following: Component I.D. N-Ethyl-carboxamides (Rf-C(0)N(Et)H) C6Ft3S02N(Et)H C2F5S02N(Et)CH2CH20H C7Fi5S02N(Et)H N-EtFOS Amide (CsFi7S02N(Et)H) C6Fl3S02N(Et)2 C 3F7S O 2NCEOCH2C H 2O H C4F9S02N(Et)CH2CH20H C 5F1iS02N(Et)CH2CH20H C6Fi3S02N(Et)CH2CH20H C7Fi5S02N(Et)CH2CH20H N-EtFOSE CsFi7SO 2N (EOCH2C H 2O H 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 C-03533 mw 137 possibly -S02N(M e)CH2CH20C7Fi5S02N(M e)CH2CH20H N-MeFOSE C8Fi7S02N(Me)CH2CH20H C9Fi9S02N(M e)CH2CH20H C8FnS02N(M e)(CH2CH20)2H CsHi7S02N(M e)H Other High Boilers Component I.D. N-Ethyl-carboxamides (Rf-C(0)N(Et)H) C3F7S02N(Et)H C4F9S02N(Et)H C5FnS02NCEt)H C6Fi3S02N(Et)H C7Fi5S02N(Et)H N-EtFOS Amide ( CsFi7S02N(Et)H) C sF i5S 02N (E t)H numerous other impurities o f most hom ologs that include hydrides, chlorine in the backbone, and unidentified high boilers 0.58 % 0.41 % 98.19 % 0.59 % trace trace 0.23% W.R. N-EtFOS A m id e 97900-111 10.23 % trace 0.71 % 3.58% 52.50 % 20.06% 12.93 % trace trace 003584 To: From: Subject: Date: I. Muggli 53-6S-02 R. M. Payfer 236-2B-11 (612)733-4212 SA&C Analytical Request No. 42607 Dec. 21, 1993 GInCtro/MduSceanthaelyssiasmopf ltehiisntsoatmhepleFiwnnaisgaanccSoSmQp-l7is0hemdausssinsgpeact2r5ommeXter0..3T2hme msamHPp-le1 cGoCmcpoolnuemnntstowere faiorlosnmoized4do0nuteos,in3ag0n0dchCtheemataicaraerlaaiotpeneiozrcfae1tino0tndvewagliutrheeesmsferpothemar ntmheiinsauswteoth.rekGwrCeearageneaanpltypgslaiisesdw. tiTtohhtfehlaeGmpCeeaciooknluiidzmeanntiotiwtnieadssefotreopcmetrioathnteedwas mass spec work. The results of these analyses (which are not necessarily quantitative) show the following: Mol. Weight Component I.D. NL--1M3e2F0O2 SE 427 N-Methyl-carboxamides C7Fis-C(0)N(Me)H 0.06 % 527 C8Fi7S02N(Me)2 0.12% 513 C8Fi7S02N(Me)H 0.25 % 357 C4F9S02N(Me)CH2CH20H 0.03 % 407 CsFi 1S02N(Me)CH2CH20H 0.52 % 457 C6Fi3S02N(Me)CH2CH20H 3.38 % 507 C7Fi5S02N(Me)CH2CH20H 2.16% 557 . N-MeFOSE CaFi7S02N(Me)CH2CH20H 89.48 % 519 C8FisS02N(Me)CH2CH20H 0.84 % 607 C9Fi9S02N(Me)CH2CH20H 0.55 % 573 CsFi6CIS02N(Me)CH2CH20H 0.67 % 665 CaFi6SF5-S02N(Me)CH2CH20H 0.29 % 003585 601 C8Fi7S02N(Me)(CH2CH20)2H Other High Boilers 0.54 % 1.05% C.*>586