Document g2jz0mBvKeeBxmooooBEbZ71e
HAZLE-CON
WASHINGTON
9200
LEESBURG
PI6KE
VIENNA.
VA.22 182 1 99
ANALYSIS OF T-5878
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.: 154-209
SUBMITTED TO 3M Corporation Building 220-2E-02
3M Center St. Paul, MN 55144-1000
STUDY COMPLETION DATE November 1, 1994
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EHAZLEMON
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QUALITYASSURANCESTATEMENT
PROJECTTITLE:
Analysis of T-5878 in a Cell Proliferation Assay in Rat Liver Cells
PROJECT NO.: 20991 PROTOCOL NO.: 493
HWA STUDY NO.: 154-209 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)/ 2-7-94
Draft report review/ 7-12,13,14-94
Final report review/ 11-1-94
Findings Reported
2-8-94 7-14-94 11-1-94
Auditor B. Mullett B. Mullett B. Mullett
154-209
Quality Assurance Unit
Date Relea 2
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COMPLIANCAEND CERTIFICATIOSNTATEMENT
The describedstudywas conductedin compliancweith 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 aforementionedregulationsor 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 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 submissionof the final report tcythe 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:
-/
)@ c,,,,
Andrea L. Ham, M.S. Associate Scientist
Study Director:
I(// / 7 Al
Date
Ma',r,"Ai.a Cifone, P D. Stpdy Director Genetic and Cellular Toxicology
Study Completion Date
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TABLE OF CONTENTS
PAGE NUMBER
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
I. SPONSOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
II. MATERIAL TESTED . . .
7
A. Genetics Assay N;.*
B. Identification
C. PhysicalDescription
D. Date Received
III. TYPE OF ASSAYS . . . . . . . . . . . . . . . . . . . . . . . . . . 7
IV. PROTOCOL NUMBER . . . . . . . . . . . . . . . . . . . . . . . . . . 7
V. STUDY DATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 A. Study InitiationDate B. ExperimentalStart Date C. Experimental Termination Date
VI. SUPERVISORY PERSONNEL . . . . . . . . . . . . . . . . . . . . . . . 7 A. Study Director
B. AssociateScientist
VII. OBJECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
VIII.DEFINITION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
IX. MATERIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 A. IndicatorCells B. Osmotic Pumps and Label for Cell ProliferationAnalysis C. ControlArticles D. Test Article
X. EXPERIMENT DESIGN . . . . . . . . . . . . . . . . . . . . . . . . . 9 A. Dosing Procedure B. Implantation of Osmotic Pumps C. Tissue Collectionand Preparation D. ImmunohistochemicalStaining E. Assessmentof Cell Proliferation
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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 . . . . . . . . . . . . . . . . . . . . . . . 15
APPENDIX A IndividualAnimal and Slide LabelingIndices :
17
APPENDIX B IndividualAnimal Body and Liver Weights and Liv;r.t.o . . . .
Body Weight Ratios . . . . . . . . . . . . . . . . . . . . . 24
APPENDIX C HistopathologyReport . . . . . . . . . . . . . . . . . . . . 29
APPENDIX D StatisticalAnalysisof LabelingIndices . . . . . . . . . . 32
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ABSTRACT
The purpose of this study was to determine the hepatotoxicityof T-5878 by measuringcell proliferation(CP) assayedas S-phaseinductionin rat liver cells after in vivo treatment. The doses chosen for the study were 500, 1000, 2000, and 30b-Omg/kg. Dimethylnitrosamine(DMN) at 15 mg/kg was included as a positivecontrol.
In the cell proliferationassay,a singleoral dose of the test materialwas administeredand five animals per conditionwere labeled with BrdU for 72 hours using ALZETIDosmotic pumps. No histomorphologicalalterationswere observed at 500, 1000 and 2000 mg/kg but increasedmitoses were observed at 3000 mg/kg. Treatment-relatedchanges were also observed in the dimethylnitrosamine(DMN) positivecontrol animals.
Followingdeterminationthat there were no treatment-relatedlobular differencesin the labelingindices,sectionsfrom the left laterallobe of the livers, as well as samples from the duodenum,were processed for immunohistochemistry.Each slide was preparedwith sectionsfrom both liver and duodenum. The duodenum (a rapidly proliferatingorgan) was used as an internalcontrol for deliveryof label and immunohistochemicasltaining. The percentageof nuclei incorporatinglabel in the liver was determined microscopically. Only hepatocytenuclei were enumerated. The control animals had a labeling index of 1.46 and treatedanimalshad labelingindicesthat ranged from 6.73 to 7.83. When comparing vehicle and treated groups and there were significantincreasesin the labelingindex at 2000 and 3000 mg/kg (0.01:5p:50.0a5n)d a positive trend was observed. The average values showed heterogeneousvariancewhen comparingvehicle and treated groups. Significant increasesin cell proliferationwere also observedin the positivecontrol animals.
T-5878 was thereforeconsideredpositivefor the inductionof cell proliferatioinn rat livercells.
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Analysis of T-5878 in a Cell Proliferation Assay
in Rat Liver Cells
I. SPONSOR: 3M Corporation
II.
MATERIAL TESTED:
A. Genetics Assay No.: 154-209
B.
Identification: T-5878
C.
Physical Description: waxy cream colored solid
D.
Date Received: January 19, 1994
Ill. 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: May 26, 1994
VI. SUPERVISORY PERSONNEL:
A.
Study Director: Maria A. Cifone, Ph.D.
B.
Associate Scientist: Andrea Ham, M.S.
VII. OBJECTIVE:
The objective of this assay was to measure hepatotoxicity caused by T-5878 by measuring cell proliferation (CP) measured as S-phase induction induced in rat liver cells after in vivo treatment.
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Cell proliferation measured 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 administration of BrdU for 72 hours i-n vivo with an ALZETO 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-pha.sein 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: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 Certified,&Rodent Chow (Formula 5002) and water ad libitum. Animals were quarantined a minimum of 7 days prior to random assignment to study groups and identification by ear tag for the dose rangefinding assay and by implantable microidentification device for the cell proliferation assay.
The cell proliferation assay was initiated with rats that ranged from 289 to 355 grams. Within 2 hours after dosing, the animals were anesthetized using Metofane@ (methoxyflurane, Pitman-Moore, Inc.) inhalation anesthesia and one ALZETO pump per animal was aseptically inserted subcutaneously (dorsal surface). Seventy-two hours later, animals were anesthetized with C02 prior to removal of the livers and duodenum.
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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 capacitywith a pump rate of 10 lil/hour. The pumps were pre-filledwith BrdU at a concentrationof 20 mg/ml.
C. ControlArticles
1. Vehicle control
A vehiclecontrolconsistingfive rats was dosed by oral gavage (P.O.)with the vehicle,0.5% high viscosity carboxymethylcellulose(CMC) (Sigma, Lot 121F-0544;CAS 9004-32-4). Tissuesfrom vehiclecontrolanimalswere subjected to the same manipulationsused for the tissues derived from treated animals. The dosing volume of the vehicle control animals did not exceed 10 ml/kg.
2. Positivecontrolarticle
The positive control compound is known to induce S-phase in rat hepatocytesin 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 suspended in CMC at concentrationsof 50, 100, 200 and 300 mg/ml prior to dosing. The maximum dosing volumesfor the test articledid not exceed 10 ml/kg.
X. EXPERIMENT DESIGN:
A. Dosing Procedure
Five rats per conditionwere treated by oral gavage with T-5878 for the cell proliferationassay. Deliveryvolumeswere calculatedon the basis of the most recent animalweight and the target dose. The maximum volume of the test article suspensions administereddid not exceed 10 ml/kg. Fresh preparationsof test article in vehiclewere used for any testingpurpose. Confirmationof the concentrationof the test material under conditionsof preparationand dosing of the assay was not determinedin conjunctionwith this study.
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B. C.
D. E. 154-209
Implantation of Osmotic Pumps
For the cell proliferation assay, ALZETO Model 2MLI osmotic pumps (Lot #042301) were preloaded with 2000 lil of BrdU at a concentration of 20 mg/ml. The animals were anesthetized using MetofaneO 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 A 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 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
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ocular grid dividedinto 10 x 10 squareswas used to define the counting area. At least 2000 nucleiwere examinedper animal with a minimum of 3 sectionsand 6 fields per section.
Any nuclei that were blue were consideredunlabeled and any nuclei containing any brown chromogenichue 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) evaluationas to treatmentgroup.
S-phase nuclei labelingindicesfor each animal were calculatedas follows:
Labeled S-phasenuclei (LI)=no.of labeled he2atocytenuclei 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.theaverage value from the three slidesof 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,liverweights,and liver to terminal body weight ratios, a mean and standard deviationwere calculatedfor each treatmentgroup using the individualanimal mean S-phasevalues. Statisticalanalysisof labelingindex was performed using one-way analysisof variancetechniques. Control versus treatment group comparisonswere done with Dunnet's t-test and control versus positivegroup 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,terminalliver weight and liver to body weight ratio in a dose group that deviates from the values in the concurrentcontrol group at a significancelevel of p:50.05was consideredsignificantlydifferentthan the controlgroup.
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XII. INTERPRETATION OF RESULTS
A. General Observations
All animalssurvivedtreatment. No treatment-related histomorphologicalalterationswere observed at 500, 1000 and 2000 mg/kg but increased mitoses were observed at 3000 mg/kg. Treatment-relatedchanges were also observed in the dimethylnitrosamin(eDMN)positivecontrol 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 animalsindicatedproperdeliveryof the BrdU label and acceptable immunohistochemicalstaining. There was no apparentpreferentiallabelingin any of the lobes and the label was random within the lobes.
Two of the dose groups (Groups3 and 5) had mean liver weights that were significantlyelevated (p:50.01a)bove the Group 1 controlliver weights but the increaseswere not dose-related. The mean liver weight of the positivecontrolwas not significantlyelevatedeven though large increases in DNA synthesis(and subsequentcell proliferation)were induced. The 500 mg/kg (Group 2) animals also had a mean terminal body weight that was less than the Group 1 controlvalue (0.01<p<0.05). No dose-relatedtrend in body weights or liver weights was observed. However,when the liver to body weight ratios were determined, therewere significantincreasesin the liver to terminalbody weight ratios with p:50.01Groups 3 through 5.
B., Summary of Labeled Cell Counts-for the Liver
A summary of the labeled cell counts for each group is shown in Table 1. Individualanimal counts are shown in AppendixA. The mean labelingindex (LI) for each group is presentedin the third column in Table 1.
The mean background labeling index (Group 1) was 1.46 which indicatesthat less than 2% of the nuclei had undergone DNA synthesisduring the 72-hourlabelingperiod. Significant increasesin the labeling index were induced by T-5878 in dosed Groups 4 and 5 (2000 mg/kg and 3000 mg/kg; 0.01 :5p :50.05). The labeling indices at 400 and 800 mg/kg were 6.92% and 7.83% respectivelywhich represent4.7- to 5.4-foldincreasesover background. Large increases were observed at 500 mg/kg and 1000 mg/kg, but the heterogeneousnature of the response resulted in a lack of significance. There was also indicationof a
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XIII. CONCLUSIONS The test material, T-5878, induced significant changes in the number S-phase cells following a single oral dose of 2000 mg/kg and 3000 mg/kg. The animals were labeled for 72 hours and a significant dose-related trend in the mean labeling index was observed in the treated groups. T-5878 was therefore evaluated as active in the induction of DNA synthesis in rat liver cells.
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XIV.
REFERENCES
1. DeFazio, A., Leary, J.A., Hedley, D.W. and Tattersall, M.H.N. (1987). Immunohistochemicaldetection of proliferating cells i-n vivo. J. Histochem. Cytochem. 35, 571-577.
2. Lanier, T.L., Berger, E.K., and Eacho, P.I. (1989). Comparison of 5-bromodeoxyuridine and 3 H-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.,
189:123-133, 1987.
4. Mirsalis, J.C. and Butterworth, B.E.: Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with
dinitrotoluene. Carcinogenesis, 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]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 proliferationfor 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. 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.
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XV. EXPERIMENTAL DATA TABLE
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Client: 3M Corporation Client Code: T-5878
Table I Cell ProliferationSummary
HWA Assay No.: 154-209
Trial InitiationDate: February7, 1994
Group/Sexa Eose, La,beli?RIndexb eve
(mg/kg)
Liver Weight (grams)
Terminal Body Liver/Body Weight (grams) Weight
im
oc
1.46 1.23 12.92 1.06 333.5 14.3 3.87 0.20
2M
500
6.91 7.04 14.56 0.75 291.2 21.0**4 5.01 0.25
3M
1000
6.73 5.11 16.09 0.58**t 307.8 11.3 5.23 0.09*t
4M
2000
6.92 3.49*t 14.15 1.15 309.6 16.8 4.57 0.32**t
5M
3000
7.83 5.04*t 15.80 1.96**t354.3 22.5 4.44 0.29**t
6Md
15d 34.96 14.86 13.13 0.73 318.4 7.7 4.12 0.19
afive animals per group bpercentageof labeled hepatocytenuclei per total number of hepatocytes counted (at least2000) CVehicle control, Carboxymethylcellulose dpositive control, 15 mg/kg of DMN
Significantat 0.01 :5p :50.05 Significantat p :50.01
t Increase in the mean I Decrease in the mean
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APPENDIX A IndividualAnimal and Slide Labeling Indices
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Slide# Animal ID GrouD
16
47773
im
17
47773
im
18
47773
im
Labeled
34 10 9
Counted % Labeled
700
4.86
700
1.43
700
1.29
Mean
2.52
SD
2.02
19
47774
im
20
47774
im
21
47774
im
5
700
0.71
7
700
1.00
4
700
0.57
Mean
0.76
SD
0.22
22
47775
im
23
47775
im
24
47775
im
2
700
0.29
5
700
0.71
2
700
0.29
Mean
0.43
SD
0.25
25
47776
im
26
47776
im
27
47776
im
11
700
1.57
3
700
0.43
8
700
1.14
Mean
1.05
SD
0.58
28
47777
im
29
47777
im
30
47777
im
16
700
2.29
18
700
2.57
19
700
2.71
Mean
2.52
SD
0.22
GROUP MEAN
1.46
GROUP SD
1.23
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Slide # Animal ID Grou2
46
47778
2M
47
47778
2M
48
47778
2M
Labeled
124
159
140
Counted % Labeled
700
17.71
700
22.71
700
20.00
Mean
20.14
SD
2.50
49
47779
2M
so
47779
2M
51
47779
2M
47
700
6.71
20
700
2.86
39
700
5.57
Mean
5.05
SD
1.98
52
47780
2M
53
47780
2M
54
47780
2M
21
700
3.00
35
700
5.00
13
700
1.86
Mean
3.29
SD
1.59
55
47781
2M
56
47781
2M
57
47781
2M
19
700
2.71
18
700
2.57
23
700
3.29
Mean
2.86
SD
0.38
58
47782
2M
59
47782
2M
60
47782
2M
32
700
4.57
22
700
3.14
14
700
2.00
Mean
3.24
SD
1.29
GROUP MEAN
6.91
GROUP SD
7.04
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Slide
61 62 63
Animal ID GrouD
47783
3M
47783
3M
47783
3M
Labeled
10 12 22
Counted % Labeled
700
1.43
700
1.71
700
3.14
Mean
2.10
SD
0.92
64
47784
3M
65
47784
3M
66
47784
3M
99
700
14.14
98
700
14.00
125
700
17.86
Mean
15.33
SD
2.19
67
47785
3M
68
47785
3M
69
47785
3M
45
700
6.43
38
700
5.43
38
700
5.43
Mean
5.76
SD
0.58
70
47786
3M
71
47786
3M
72
47786
3M
58
700
8.29
47
700
6.71
63
700
9.00
Mean
8.00
SD
1.17
73
47787
3M
74
47787
3M
75
47787
3M
26
700
3.71
12
700
1.71
14
700
2.00
Mean
2.48
SD
1.08
GROUP MEAN GROUP SD
6.73 5.11
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Slide # Animal ID Groul
31
47788
4M
32
47788
4M
33
47788
4M
Labeled
71 73 55
Counted % Labeled
700
10.14
700
10.43
700
7.86
Mean
9.48
SD
1.41
34
47789
4M
35
47789
4M
36
47789
4M
37
47790
4M
38
47790
4M
39
47790
4M
40
47791
4M
41
47791
4M
42
47791
4M
43
47792
4M
44
47792
4M
45
47792
4M
23
700
3.29
38
700
5.43
49
700
7.00
Mean
5.24
SD
1.86
61
700
8.71
89
700
12.71
95
700
13.57
Mean
11.67
SD
2.59
21
700
3.00
33
700
4.71
23
700
3.29
Mean
3.67
SD
0.92
28
700
4.00
31
700
4.43
37
700
5.29
Mean
4.57
SD
0.65
GROUP MEAN GROUP SD
6.92 3.49
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HAZLE-CC:N
W A S H IN G T 0 N
Slide
1 2 3
Animal ID Grou2
47793
5M
47793
5M
47793
5M
Labeled # Counted % Labeled
84
715
11.75
66
700
9.43
71
700
10.14
Mean
10.44
SD
1.19
4
47794
sm
5
47794
5M
6
47794
5M
24
700
3.43
32
700
4.57
17
700
2.43
Mean
3.48
SD
1.07
7
47795
5M
8
47795
5M
9
47795
5M
10
47796
5M
11
47796
5M
12
47796
5M
66
700
9.43
60
700
8.57
67
700
9.57
Mean
9.19
SD
0.54
109
700
15.57
112
700
16.00
85
700
12.14
.Mean SD
14.57 2.11
13
47797
5M
14
47797
5M
15
47797
5M
8 10 13
GROUP GROUP
700 700 700
Mean SD
MEAN SD
1.14 1.43 1.86
1.48 0.36
7.83 5.04
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'"-@'HAZLENCON
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Slide # Animal ID GrouR
76
47758
6M
77
47758
6M
78
47758
6M
Labeled
260 364 435
Counted % Labeled
700
37.14
700
52.00
700
62.14
Mean
50.43
SD
12.57
79
47759
6M
80
47759
6M
81
47759
6M
82
47760
6M
83
47760
6M
84
47760
6M
265
700
37.86
251
700
35.86
171
700
24.43
Mean
32.71
SD
7.24
155
700
22.14
246
700
35.14
369
700
52.71
Mean
36.67
SD
15.34
85
47761
6M
86
47761
6M
87
47761
6M
270
700
38.57
292
700
41.71
285
700
40.71
Mean
40.33
SD
1.61
88
47762
6M
89
47762
6M
90
47762
6M
54
700
165
700
89
700
Mean SD
GROUP MEAN GROUP SD
7.71 23.57 12.71
14.67 8.11
34.96 14.86
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- @ _-w
'w" HAZLENZCON W A S H IN G T 0 N
APPENDIX B Individual Animal Body and Liver Weights and Liver to Body Weight Ratios
154-209
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HAIZLF.-CC:N
W A S H IN G T 0 N
ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS
STUDY NUMBER: 154209
*DRAFT* ABSOLUTE ORGAN WEIGHTS (g) *DRAFT*
------------------------------------------------------------------------------------------------------
ORGAN ABBREVIATION: Ll - LIVER
SEX DOSE ANIMAL GROUP NUMBER
TERMINAL BODY WEIGHT (g) Ll
------------------------------------------------------------------------------------------------------
m 1 B47773
327.7
13.25
m I B47774
341.0
13.77
m 1 B47775
313.0
11.32
m 1 B47776
334.9
12.40
m 1 B47777
351.0
13.84
- - --- -- -- -- -- -- --- -- -- -- -- --- -- -- --- - -- -- -- -- -- -- -- --
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 333.5 14.3
5 12.92 1.06
------------------------------------------------------------------------------------------------------
M 2 B47778
299.0
15.04
m 2 B47779
316.0
15.37
m 2 B47780
273.0
14.68
m 2 B47781
302.0
14.25
m 2 B47782
266.0
13.45
- -- -- -- -- -- --- -- -- --- - --- -- -- -- --- -- -- -- -- -- -- -- -- - --
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 291.2 21.0
5 14.56 0.75
------------------------------------------------------------------------------------------------------
m 3 B47783
294.8
15.31
M 3 B47784
312.0
16.02
M 3 B47785
324.0
16.94
m 3 B47786
308.0
16.06
M 3 B47787
300.0
16.14
-- -- -- -- -- -- -- --- -- -- --- -- -- -- -- -- -- -- -- --- - -- -- -- -- -
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 307.8
11.3
5 16.09 0.58
------------------------------------------------------------------------------------------------------
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25
HAZLV;NCC:N
WASHINGTON
ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS
STUDY NUMBER: 154209
*DRAFT* ABSOLUTE ORGAN WEIGHTS (g) *DRAFT*
------------------------------------------------------------------------------------------------------
ORGAN ABBREVIATION: Ll - LIVER
SEX DOSE ANIMAL TERMINAL
GROUP NUMBER BODY WT (g)
Li
------------------------------------------------------------------------------------------------------
M 4 B47788
285.0
12.44
m 4 B47789
322.0
14.56
m 4 B47790
299.0
14.98
m 4 B47791
322.0
13.58
M 4 B47792
320.0
15.22
- - -- -- -- --- -- -- -- -- -- -- -- -- -- --- -- -- -- -- -- -- -- -- -- -- -
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 309.6
16.8
5
14.15 1.15
-----------------------------------------------------------------------------------------------------
m 5 B47793
350.8
14.83
m 5 B47794
379.0
17.67
M 5 B47795
350.0
15.60
m 5 B47796
370.9
17.76
M 5 B47797
321.0
13.13
- -- - -- -- --- -- -- -- --- - --- - --- -- -- --- -- --
- - --- -
- -- -- -
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 354.3 22.5
5 15.80 1.96
-----------------------------------------------------------------------------------------------------
M 6 B47798
322.9
13.60
M 6 B47799
317.0
12.39
M 6 B47800
306.0
12.91
m 6 B47801
320.0
12.58
M 6 B47802
326.0
14.14
- -- --- - -- -- --- -- -- -- -- -- --- - -- --- -- -- -- -- -- -- -- -- -- - -
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 318.4
7.7
5 13.13
0.73
-----------------------------------------------------------------------------------------------------
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HAZLFgt(=N
WASHINGTON
ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS
*DRAFT* ORGAN-TO-TERMINAL BODY WEIGHT RATIOS (%) -DRAFT* STUDY NUMBER: 154209 ------------------------------------------------------------------------------------------------------
ORGAN ABBREVIATION: LI - LIVER
SEX DOSE ANIMAL TERMINAL
GROUP NUMBER BODY WT (g)
Ll
------------------------------------------------------------------------------------------------------
m 1 B47773
327.7
4.044
m I B47774
341.0
4.039
m I B47775
313.0
3.617
m I B47776
334.9
3.701
m 1 B47777
351.0
3.944
- -- -- -- -- --- - --- -- -- --- -- -- -- -- --- - --- -- -- -- -- - -- -- --
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5
333.5 14.3
5 3.869 0.198
------------------------------------------------------------------------------------------------------
M 2 B47778
299.0
5.031
M 2 B47779
316.0
4.865
m 2 B47780
273.0
5.377
M 2 B47781
302.0
4.717
M 2 B47782
266.0
5.055
-- - -- --- -- -- --- -- -- -- -- --- - --- -- -- -- --- -- -- -- - --- - -- -
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 291.2
21.0
5 5.009 0.247
------------------------------------------------------------------------------------------------------
m 3 B47783
294.8
5.192
M 3 B47784
312.0
5.136
m 3 B47785
324.0
5.229
M 3 B47786
308.0
5.214
m 3 B47787
300.0
5.379
-- --- -- -- -- --- - --- -- -- -- -- --- -- - --- -- -- -- -- -- -- -- -- --
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 307.8 11.3
5 5.230 0.091
------------------------------------------------------------------------------------------------------
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'"-**'HAZLE-CC:N
WASHINGTON
ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS
STUDY NUMBER: 154209
*DRAFT* ORGAN-TO-TERMINAL BODY WEIGHT RATIOS (%) -DRAFT*
------------------------------------------------------------------------------------------------------
ORGAN ABBREVIATION: LI - LIVER
SEX DOSE ANIMAL
TERMINAL
GROUP NUMBER
BODY WT (g) LI
------------------------------------------------------------------------------------------------------
m 4 B47788
285.0
4.363
M 4 B47789
322.0
4.522
m 4 B47790
299.0
5.DO9
m 4 B47791
322.0
4.216
M 4 B47792
320.0
4.757
-- -- -- -- -- --- -- -- -- --- -- -- - --- -- -- --- -- -- -- - -- -- - -- --
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 309.6
16.8
5 4.574 0.315
------------------------------------------------------------------------------------------------------
m 5 B47793
350.8
4.226
m 5 B47794
379.0
4.662
m 5 B47795
350.0
4.456
m 5 B47796
370.9
4.787
m 5 B47797
321.0
4.090
- - --- -- -- -- --- -- -- -- -- -- -- --- -- -- -- -- --- - -- -- -- -- -- --
NUMBER IN GROUP: MEAN:
STANDARD DEV:
5 354.3
22.5
5 4.444 0.291
------------------------------------------------------------------------------------------------------
M 6 B47798
322.9
4.212
M 6 B47799
317.0
3.910
M 6 B47800
306.0
4.219
m 6 B47801
320.0
3.931
m 6 847802
326.0
4.339
-- -- - --- -- -- -- --- -- -- -- -- -- -- -- --- -- -- -- -- -- -- -- -- -- -
NUMBER IN GROUP: MEAN,
STANDARD DEV:
5 318.4
7.7
5 4.122 0.191
------------------------------------------------------------------------------------------------------
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w'"HAZLV;ECC:N WASHINGTON
APPENDIX C Histopathology Report
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29
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Pathology Report Analysis of Cell Proliferationin Rat Liver Cells ProjectNo. 154-209
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 500, 1000, 2000, and 3000 mg/kg of the test material, T-5878, via oral gavage. Group 6 served as the positive control, receiving 15 mg/kg of dimethylnitrosamine (DMN) via oral gavage. After dosing, an ALZETO Model 2ML1 osmotic pump containing 20 mg/mL of bromodeoxyuridine (BrdU) was implanted subcutaneously in each rat while it was under Metofanel&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 veterinarypathologist.
Hi stopathol ogy Treatment-relatedchange in the liver consisted of minimally to slightly increased mitoses in Group 5 (3000 mg/kg) rats. Group 6 (DMN treated) rats had varying severitiesof centrilobularnecrosis, hepatocellularhypertrophy, chronic inflammation,peliosis, and increased mitoses. Various spontaneous disease lesions and incidental findings, including chronic inflammationof the spleen capsule in a Group 3 rat and renal pelvis dilatationin three rats, are unrelatedto treatment. The chronic active inflammationwith acanthosis of the nonglandular stomach of a Group 2 rat is probably an effect of gavage trauma.
154-209
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HAZLEWCCDN
W A 6 H IN G T 0 N
Summary The test material, T-5878, when administered to male Sprague-Dawley rats in single oral gavage doses of 500, 1000, 2000, and 3000 mg/kg, produced increased mitoses in the liver of rats dosed at 3000 mg/kg. No treatmentrelated histomorphologicchanges were noted in the liver of rats dosed at 500, 1000, and 2000 mg/kg.
Pathologist:
Samuel V. Machotka, D.V.M., D.A.B.T.,
Date
Diplomate, American College of Veterinary
Pathol ogi sts
Department of Pathology
154-209
31
w" HAZLEOCCDN WASHINGTON
APPENDIX D Statistical Analysis of Labeling Indices
154-209
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HAZLEOCCDN
W A S H IN G T 0 N
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 significantdose, slide, and related interaction
effects. The sphericitytest was also utilized to test variance homogeneity.
The model used was:
e
proportion =,p + 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, Dunnett's t-tests, Terpstra-Jonckheere test [1], and regression tests for trend using both untransformedand ranked
data.
Results: Since the sphericity test rejected variance homogeneity in comparing vehicle vs positive control (p = .0227), the Greenhouse-Geisserprobabilitieswere used for this significanceevaluation. There was no significantheterogeneity in comparing vehicle vs treated groups (p = .7922). As Text Table 1 indicates,there is no significantfinding in comparing vehicle with treated groups. Within- and between-groupslide-to-slidevariations were not significantin either case. Only the positive control showed highly significantelevation in labellingover vehicle control. The data based on the average values showed homogeneous variance (p = .1449) in comparing vehicle vs treated groups for the untransformeddata. However, closer examination of the individual values in each group along with the means and standard deviations (Text Table 2) indicates that the analyses based on the rank-transformed data may be necessary. As Text Tables 2 and 3 indicate, there is significantdifference between vehicle and treated groups in transformed data (p = .0314). Dose 2000 and 3000 showed significant increase in labellingover control. Furthermore,there was a significantpositive trend as indicated in Text Table 4 using Terpstra-Jonckheeretest (p = .0049) and regression of rank-transformeddata (p = .0090) even though the regression based on the untransformed data did not show any significance. There was no significantlack of fit for both regressions(p = 0.252 and 0.473). The following notations are used to denote direction and statisticalsignificance:
= significant at p :50.05 Significantat p :50.01
t = effect in the positivedirection
Text Table I - Univariate ANOVA
Vehicle vs Positive ControlVehicle vs Treated Groups
Dose p
0.0005 **0.3029
Slide pO.27920.9965
SlidexDose
pO.20530.2266
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HAZLE'TCDN
WASHINGTON
Text Table 2 The Descriptive Statistics
Untransformed
Dose(mg/kg) Mean
SD
Median
Rank-Transformed
Mean
SD
Vehicle vs Positive Control
Vehicle
1.4560
0.9958
Positive
13.1132
1.0500 36.6700
3.0000 8.0000
1.5411 1.5811
Vehicle vs Treated Groups
Vehicle
1.4560
0.9958
500
6.9160
7.4409
1000
6.7340
5.3855
2000
6.9260
3.4649
3000
7.8320
5.3229
1.0500 3.2900 5.7600 5.2400 9.1900
4.2000 14.0000 14.0000 17.0000 15.8000
3.0944 6.5574 8.2158 3.8730 7.7910
Text Table 3 - One-Way ANOVA and Dunnett's T-Test
Vehicle vs Positive Control
Vehicle vs Treated Groups
Untransformed
Transformed Untransformed
Transformed
Treatment .0005
.0010
.3022
.0314 *
Vehicle vs 500 Vehicle vs 1000 Vehicle vs 2000 Vehicle vs 3000
.0817 t .0826 t .0151 *t .0315 *t
Text Table 4 - Test for Trend for Vehicle vs Treated Groups
Terpstra-Jonckheere Test Regression of Untransformed Data Regression of Rank Transformed Data
.0049 **t .1170 t
.0090 **t
Discussion: The results of the present study based on the rank-transformed data
indicatethat there was significant increase in cell proliferationat Dose 2000 and 3000 compared to control due to treatment by the chemical. There was also indicationof a significantpositive 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 (StatisticalAnalysis System), SAS Institute, Cary, NC, 1991.
154-209
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HAZLEIC(MN
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 IDENTIFICATION
Company Name:
3H Cor2oration
Address: Building 220-2E-02, 3M Center, St. Paul, MN
55144-1000
ii. TEST ARTICLE IDENTIFICATION: - T-
(A& u--ilsw
5-S7
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:
r-77U7ndetermined
NIAFF
MOHW
FDA OECD
EPA-TSCA
F= OTHER
EPA-FIFRA
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WASHINGTON
V. STUDY DATES Proposed Experimental Start Date: Proposed Experimental Termination Date:
VI. APPROVAL OF STUDY PROTOCO Study Director:
Maria A. Cifone, Ph.D. s5pgnp.o@r's Au thoTi-r.,,eRdepreessentative:
Protocol No. 493, Edition 3
Date: Date:
10/93
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W A S H IN G T 0 N
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 in 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 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. KATERIALS
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(SD6')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
HAIZLEOCON
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 CertifiedO 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 MetofaneO (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'* 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 lAl/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 @e 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.
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'"-'@'HAZLCNCC:N
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 g*avage 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 diissolvedin 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 Selectio
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. IMRIantation 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 Preparatio
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
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HAZLENC(ON
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 A 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 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.
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HAZLE-CC:N
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 %S-phase standard deviation among the three slides for each animal analyzed for S-phase.
vi. ASSAY EVALUATION CRITERI
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:50.05will 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. Careinogenesis, 3:241-245, 1982.
5. 10/93
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.
Page 7
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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 carcinogenesisas 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 PrinciRles in Ex-perimentalDesi 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 FORKAT
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
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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
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3M InternalCorrespondence
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 *4Wg'73
Wide Range ETFOSE F.LM3924 Lot 547 Retainfrom 2 yearfeedingstudy
Narrow Range ETFOSE Lot 884 TypicalRaw Material forFC-807
Wide Range MEFOSE Lot 555 TypicalRaw Material forFX-845
Narrow Range MEFOSE ,Notebook97900-107-2 Lab PreparedSample
Wide Range ETFOSE '\-oteboo9k7900-112-2 Lab PreparedSample
Wide Range MEFOSE Lab Preparedfrom Washed POSF
AnalyticaRlequest41220 L-13203 AnalyticaRlequest41220 L-10059
AnalyticaRlequest41220 L-1276
AnalyticaRlequest41343 L-13097 AnalyticaRlequest41343 L-I'>098 AnalyticaRlequest42607
Attachments
REQ 41220
R. Howells
GC/MS analysesofthesesampleswere accomplishedusinga 25 m X 0.32mm BP- 1 GC column to introducethesamples intotheFinniganSSQ-70 mass spectrometer.The sample components were ionized usingchemicalionizationwithmethane as thereagentgas. The GC column was operatedfrom 40 to300 C ata rateof10 degreesperminute. The resultosf theseanalysescombined withtheircorrespondingGC/FIID area percentsshow thefollowing:
Component I.D.
L-10059 N.R. N-ETFOSE Lot
W.R. N-ETFOSE Lot547
N-Ethyl-carboxamide(sRf-C(O)N(Et)H)
0.22%
1.76%
N-ETFOS Amide (C8Fl7SO2N(Et)H)
0.04%
0.13%
C2F5SO2N(Et)CH2CH20H
0.01%
0.12%
C3F7SO2N(Et)CH2CH20H
0.13%
1.17%
C4F9SO2N(Et)CH2CH20H
0.11%
1.52%
C8Fl7SO2N(Et)2
0.03%
0.04%
C5FilSO2N(Et)CH2CH20H
0.03%
1.34%
C6Fl3SO2N(Et)CH2CH20H
0.51%
3.52%
C8Fl7SO2N(Et)CH2CH2CI
0.08%
0.16%
C7Fl5SO2N(Et)CH2CH20H
1 0.82%
1.40%
N-ETFOSE C8Fl7SO2N(Et)CH2CH20H
96.09%
87.16%
C8Fl5SO2N(Et)CH2CH20H
0.78%
0.50%
C8Fl7SO2N(Et)(CH2CH20)2H
0.17%
C8Hl7SO2N(Et)H
0.16%
0.37%
C8Fl7SO2N(Et)CH2CH2OCO2CH2CH3 (orsimilar)
0.21%
0.22%
Other High Boilers
0.28%
0.59%
ComponentI.D.
N-Methyl-carboxamide(sRf-C(O)N(Me)H) C2F5SO2N(Me)CH2CH20H C8Fl7SO2N(Me)2 C3F7SO2N(Me)CH2CH20H C8Fl7SO2N(Me)H C4F9SO2N(Me)CH2CH20H C5FiiSO2N(Me)CH2CH20H C6Fl3SO2N(Me)CH2CH20H C8Fl7SO2N(Me)CH2CH2CI C7Fl5SO2N(Me)CH2CIbOH N-MEFOSE C8Fl7SO2N(Me)CH2CH20H CgF19SO2N(Me)CH2CH20H C8Fl7SO2N(Me)(CH2CH20)2H C8Hl7SO2N(Me)H OtherHigh 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 involvesderivatizatioofnthealcoholswith trifluoroaceatnihcydride(TFAA) and againwithBSA (togivethetrimethylsileytlhers)T.hiswork was designedtoinvestigattehepotentiaplroblems thatcould be overlookedby any one method of analysis. Preliminaryresultshow thatanalyzingtheETFOSE underivatizecdouldhidea significanatmount of N-ETFOS Amide undertheC-3 alcoholpeak inwide rangematerial.However, analyzingthesame materialthathas been derivatizewdithTFAA shows thatany EtFOSE-chloridethatispresentinthesample isnow completelyma-,;ked by thederivatizeCd-8 alcohol.The BSA derivativheas notbeen evaluatedyet,but similarproblems are expectedbecauseof thenumber of differenctomponents in thesample. The same sortof problems wW most likelyexistwithMEFOSE and willbe even more complicatedintheanalysisofMEFOSEA.
7/2/93
REQ 41343
J.Grant
GC/MS analysesof thesesampleswere accomplishedusinga 25 m X 0.32mm HP-1 GC colunm to introducethesamples intotheFinniganSSQ-70 mass spectrometer.The sample components were ionized usingchemicalionizatiownith methane asthereagentgas. The GC column was operatedfrom 40 to 300 C ata rateoflo degreesperniinute.The resultosf theseanalysescombined withtheircorrespondingGC/FBD area percentsshow thefollowing:
Component I.D.
N-Ethyl-carboxamide(sgf@-C(O)N(F-t)H) C6Fl3SO2N(Et)H C2F5SO2N(Et)CH2CH20H C7Fi5SO2N(Et)H N-ETFOS Amide (C8Fl7SO2N(Et)H)
C6Fl3SO2N(Et)2
C3F7SO2N(Et)CH2CH20H C4F9SO2N(Et)CH2CH20H C5Fi iS02N(Et)CH2CH20H C6Fl3SO2N(Et)CH2CH20H C7Fl5SO2N(Et)CH2CH20H IN-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 137 possibly -SO2N(Me)CH2CH20C7Fi5SO2N(Me)CH2CH20H N-MEFOSE C8Fl7SO2N(Me)CH2CH20H CgF19SO2N(Me)CH2CH20H C8Fl7SO2N(Me)(CH2CH20)2H C8Hl7SO2N(Me)H OtherHig Boilers
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 Aniide C8Fl7SO2N(Et)H) C8Fl5SO2N(Et)H numerous otherimpuritiesof most homologs thatincludehydrides,chlorineinthebackbone, and unidentifiehdigh boilers
0.58% 0.41% 98.19% 0.59% trace trace 0.23%
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
53-6S-02
R. M. Payfer
236-2B-1 1 (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 FinniganSSO-70 mass spectrometer.The sample components were ionizedusingchemical ionizatiownithmethane as the reagentgas. The GO column was operated from 40 to 300 C ata rateof 10 degrees per minute. GC analysiswithflameionizatiodnetectionwas alsodone, and the area percentvalues from thiswork were appliedto the peak identftiefsrom the mass spec work. The resultsofthese analyses (which are not necessarilyquantitativseh)ow the
following:
Mol. Weight Component I.D.
L-13202 N-MEFOSE
427
N-Methyl-carboxamidesC7Fl5-C(O)N(Me)H 0.06%
527
C8Fl7SO2N(M8)2
0.12%
513
C8Fl7SO2N(Me)H
0.25%
357
C4FgSO2N(Me)CH2CH20H
0.03%
407
C5Fl lS02N(Me)CH2CH20H
0.52%
457
C6Fl3SO2N(Me)CH2CH20H
3.38%
507
C7Fl5SO2N(Me)CH2CH20H
2.16%
557
N-MEFOSE C8Fl7SO2N(Me)CH2CH20H
89.48%
519
C8Fl5S02N(Me)CH2CH20H
0.84%
607
CgFi9SO2N(Me)CH2CH20H
0.55%
573
C8Fi6CIS02N(Me)CH2CH20H
0.67%
665
C8Fl6SF5-SO2N(Me)CH2CH20H
0.29%
- .. I I I
A
-. -
w
- W- @
601
C8Fl7SO2N(Me)(CH2CH20)2H
Other High Boilers
0.54% 1.05%