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Pathology Associates International
A Company of Science Applications International Corporation
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Sponsor: 3M
St. Paul, Minnesota
PROTOCOL
Study Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluoroctanesulfonamide
(PFOSA 3M T-7091.1) in Rats
Date: January 12, 1999
Performing Laboratory R.O.W. Sciences 15 Firstfield Road
Gaithersburg, Maryland 20878
Laboratory Study Identification:
Study Number: (1132-100)
PAI Project Num ber: (Histology number to be assigned by PAI by protocol amendment)
15 Worman's Mill Court, Suite I * Frederick, Maryland 21701 * (301) 663-1644 * (301) 663-8994 FAX
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Study Number 1132-100 Page 2
Study Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluoroctanesulfonamide (PFOSA 3M T-7091.1) in Rats
Purpose To assess cell proliferation and peroxisome proliferation in rats administered test material in the diet.
Sponsor 3M Corporate Toxicology Building 220-2E-02,3M Center St. Paul, MN 55144-1000
Study Representative Marvin T. Case, D.V.M, Ph.D. 3M Corporate Toxicology Phone No.: 651 733-5180 Fax No.: 651 733-1773 Email: mtcase@mmm.com
Alternative Study Representative Andrew M. Seacat, Ph.D. 3M Corporate Toxicology Phone No.: 651 575-3161 Fax No.: 651 733-1773 Email: amseacat@mmm.com
Study Location R.O.W. Sciences 15 Firstfield Road Gaithersburg, Maryland 20878
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Study Number 1132-100 Page 3
Study Monitor Sandra R. Eldridge, Ph.D. Pathology Associates International Phone No. 301 624-2036 Fax No. 301 663-8994 Email: srepaisaic@aol.com
Study Director Gary W. Wolfe, Ph.D., D.A.B.T. R.O.W. Sciences Phone No.: 301 330-3723 Fax. No.: 301 330-3738 Email: gwolfe@lab.row.com
Principal Investigator Sandra R. Eldridge, Ph.D. Pathology Associates International Phone No. 301 624-2036 Fax No. 301 663-8994 Email: SREPAISAIC@aol.com
Study Pathologist Carolyn Moyer, D.V.M., Diplomate, A.C.V.P. Pathology Associates International Phone No. 301 624-2928 Fax No. 301 663-8994
Proposed Study Timetable In-life Start Date: To be added by protocol amendment; Day 0 In life End Date: To be added by protocol amendment Audited Draft Report Date: To be added by protocol amendment
Regulatory Compliance This study will be conducted in the spirit of Good Laboratory Practice (GLP) regulations.
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Animal Care and Use Statement All procedures in this protocol are in compliance with the Animal Welfare Act Regulations, 9 CFR 1-4. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work.
Quality Assurance Not applicable.
Test Materials
Test Material:
N-EtFOSE (completed by 3M)
Identification: N-EtFOSE
Lot Number: FM 3929
Purity:
99.2%
Stability:
> 5 years
Storage
room temp.
Conditions:
Characteristics: waxy solid
PFOS (completed by 3M)
PFOS
217
99%
>5 years
room temp.
white powder
PFOSA (to be added by protocol amendment)
PFOSA
Wy-14,643 (to be added by protocol amendment)
Wy
> 5 years room temp. amber waxy solid
Reserve (Archive) Samples
A reserve sample (approximately 5 g) of each lot will be taken and stored.at room temperature. These samples will be transferred to the Sponsor after completion of the in-life phase to be retained in accordance with 40 CFR 792.195.
Disposition of Test Material
After authorization from the Sponsor, any remaining test material will be returned to: Marvin Case, D.V.M., Ph.D. 3M Corporate Toxicology Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 Phone No.: 651-733-5180 Fax No.: 651-733-1773
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Animals
Species: Strain:
Rat Crl:CD(SD) IGS BR
Source:
Charles River Laboratories, Inc., Raleigh, NC
Age at Initiation of Treatment: Preferable 6 weeks of age, but not more than 8 weeks of age
Weight at Initiation of Treatment: 150 to 300 g
Number and Gender:
males
Identification:
unique identification by individual car tags and cage cards
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Husbandry Housing:
Single housed in hanging stainless steel wire cages
Diet: Teklad 7012 Certified Rodent Diet. Fresh food will be provided weekly.
Feed is analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinate hydrocarbons, organophosphates, and specified nutrients. Specified nutrients analyses are on file at R.O.W. Sciences.
Water:
Tap water, provided M libitum via an automatic watering system or water bottles. The water is analyzed at least two times per year for contaminants and specific microbes. The results of these analyses are on file at R.O.W. Sciences.
Contaminants:
The study director and/or the Sponsor have considered possible interfering substances potentially present in animal feed and water, including the test material itself or possible structurally related materials as well as the items listed in (2) and (3) above. None of these contaminants are reasonably expected to be present in animal feed or water at levels sufficient to interfere with this study.
Environment:
The targeted temperatures are between 64 and 79F with a relative humidity between 30% and 70%. Temperature and humidity are monitored continuously. A 12-hour light/12-hour dark cycle will be maintained. Tenor greater air changes/hour will be maintained.
Acclimation:
Animals will be acclimated to the facility for a minimum of 7 days prior to the start of dosing. Animals will be observed for general health and suitability for testing during this period. Animals that are diseased or unsuitable for testing will be removed from the study.
Randomization:
Using computer-generated random numbers wit assignment to groups, At the time of randomization, the weight variation of the animals of each sex used should not exceed 2 S.D. of the mean weight, and the mean body weights for each group of each sex will not be statistically different.
Justification:
Rats will be used because of the extensive historical data base, and the FDA requirements for a rodent species.
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Group Designations, Dietary Levels and Scheduled Sacrifice Time Points Number of Male Rats
Group Number Control
N-EtFOSE
PFOS PFOSA W y-14,643 Total No.
(Time Point) (Oppm) 300 100 30 ppm 20 PPm 100 ppm 1000 ppm of Animals
1 (48 hrs)
10 10 10 10 10
10
5
65
2 (7 days)
10 5 5 5 5
5
5
40
3 (14 days)
10 5 5 5 5
5
5
40
4 (1 wk recovery'
10
55 5
5
5
5
40
5 (4 wk recovery]
Total No. of Animals
10 50
55 5 30 30 30
5 30
5 30
5 25
40 225
Dosing Procedures
Method of Administration Dietary. Animals in Groups 1 through 3 will receive test diet for 48 hours, 7 days, and 14 days, respectively.
Animals in Groups 4 and 5 will receive test diet for 14 days followed by a 1 or 4 week recovery period, respectively.
Reason for Dosing Route The potential human exposure is by the oral route.
Dose Preparation Before initiation of treatment, dose preparation of each test material will be mixed. All dose preparations will be stored at room temperature. Dose preparation will be documented and reported. See Attachment I for test diet preparation procedures.
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Retention Sample Samples (approximately 100 g) will be taken from the dose preparation and stored at room temperature. Unless used for analyses, these samples will be discarded at least 1 month after completion of the in-life phase.
Observation of Animals
Clinical Observations Each animal will be observed twice daily (a.m. and p.m.) for mortality and moribundity; findings will be recorded as they are observed.
Body W eights Prior to treatment (at randomization), weekly for Week 1 through 4 weeks of recovery.
Food Consumption Weekly for Week 1 through 4 weeks of recovery.
Clinical Chemistry Animals will be fasted overnight before animal's scheduled necropsy; blood will be collected from a jugular vein into an EDTA-coated tube. Serum enzyme levels of alanine aminotransferase (ALT), alkaline phosphatase, aspartate aminotransferase (AST), cholesterol and triglycerides will be determined.
Term ination Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with C 0 2, weighed, and exsanguinated.
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Scheduled Sacrifices
Interim Sacrifices At 48 hrs, 7 days, and 14 days, animals will be fasted overnight, bled for serum samples, anesthetized with COz, weighed, and exsanguinated.
NOTE: Two serum samples will be needed, (1) a 0.5 ml sample for clinical chemistry and (2) a 1.5 ml sample for compound level analysis.
The abdominal cavity of each animal will be opened, the liver will be removed and weighed, and liver samples will be collected. Animals will be discarded after liver collection.
Terminal Sacrifices After 1 and 4 weeks of recovery, animals will be fasted overnight, bled for serum samples, anesthetized with C 02, weighed, exsanguinated, and necropsied.
NOTE: Two serum samples will be needed, (1) a 0.5 ml sample for clinical chemistry and (2) a 1.5 ml sample for compound level analysis.
Postmortem Procedures
Necropsy The necropsy will include an examination of the external features of the carcass; all external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues.
Cell Proliferation Tissue Collection and Immunohistochemical Evaluation Representative samples of the left lateral lobe of the liver and any macroscopic lesions of the liver will be collected and preserved in zinc formalin.
After fixation, each sample of liver will be delivered to: Sandra R. Eldridge, Ph.D. Pathology Associates International 15 Worman's Mill Court, Suite I Frederick, Maryland 21701
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Proliferation cell nuclear antigen (PCNA) evaluation will be done on the samples. In addition, liver sections prepared from the same tissue block will be stained with hematoxylin and eosin and examined microscopically.
Palmitoyl-CoA Oxidase Tissue Collection and Analyses A sample (approximately 500 mg) of the right lateral lobe of the liver will also be collected from select animals and flash-frozen in liquid nitrogen. See Attachment II for procedure. The liver tissue will be stored in a freezer set to maintain -60 to -80 C until analyzed by Covance for palmitoyl-CoA Oxidase activity. The liver samples to be analyzed will include all study animals, EXCEPT for the Wy-14,643 animals and all animals from the 4-week recovery groups. In addition to this study, samples from a previous 3M study will be analyzed for palmitoyl-CoA Oxidase activity; these samples consist of liver samples from 35 rats and 35 guinea pigs.
Tissue Collection for Electron Microscopic Evaluation Sections of liver from all animals will be collected, minced to approximately one millimeter cubes and placed in a fixative appropriate for electron microscopy. The containers and fixative will be provided by PAL Electron microscopy will be performed on one animal per treatment group exhibiting the highest cell proliferative response as well as one control animal at the discretion of the Sponsor, from one time point as well as the 4-week recovery. Thus, EM will be performed on one animal from the control, N-EtFOSE (one dose only to be determined), PFOS, PFOSA, and Wy groups at one of the time points, as well as the 4 week recovery, for a total of 10 animals.
Remaining Liver Tissue The remaining liver tissue will be frozen and stored at -60 to -80 C for possible future analysis.
Organ Weights At the scheduled sacrifices, the liver will be weighed.
H istopath ology Liver from each animal that is examined for cell proliferation will be stained with hematoxylin and eosin, and examined microscopically for histopathologic changes.
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Reports One copy of the draft report will be sent to the Sponsor. The report will include the following information:
Experimental Design and Methods
R esu lts dose analyses mortality clinical observations body weights body weight changes food consumption test material consumption clinical pathology results palmitoyl-CoA oxidase activities macroscopic observations microscopic observations ultrastructural observations cell proliferation assessments
Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a result of this study will be archived in the storage facilities of PAI for a period of 1 year following submission of the final report to the Sponsor. One year after submission of the final report, all of the aforementioned materials will be sent to the Sponsor and a return fee will be charged. All raw data stored on magnetic media will be retain by PAI.
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PROTOCOL APPROVAL Marvin Case, D.V.M., Ph.D. Study Representative 3M Corporate Toxicology
Pathology Associates International
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Attachment: I Test Diet Preparation Procedures 1) Determine the amount of test diet (feed) that is to be prepared and weigh out that amount of feed. 2) Calculate the amount of test article that is needed to prepare the test diet at the desired concentration. 3) Accurately weigh out the necessary amount of test article. 4) Transfer the weighed test article to a container and add a small volume of acetone to container. Manually mix to dissolve the test material adding acetone as necessary (typical ratio of test material to acetone is 1 g: 15-20 ml acetone). Visually inspect test material/acetone for solubility of test material. 5) Prepare a pre-mix by transferring the dissolved test material into 4 kg of feed in a Hobart mixing bowl. Mix for 10 minutes. Transfer the premix to a larger mixer, add remaining amount of weighed diet, mix for 30 minutes.
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Attachment: II Collection of Tissue Samples for Biochemical and M olecular Analysis Because of the extreme instability of certain enzymes and biomolecules, it is essential that tissues be harvested as soon after death as possible and flash frozen immediately in liquid nitrogen. Failure to follow these procedures may lead to loss of the entire sample and all the energies and resources that were invested into generating the samples. Therefore, make every effort to comply with the following: 1) Harvest the tissue samples as soon as possible after death. Delays may allow for biodegradation and/or inactivation of the desired endpoint. 2) Immediately submerse the tissue sample directly into liquid nitrogen. Dry ice or other alternatives will not suffice. It is important that the tissue be immersed directly in liquid nitrogen Transferring it to a dry vessel (or sample container) suspended in liquid nitrogen will not suffice. The tissue may freeze to the vessel wall and will then be impossible to remove without completely destroying the vessel (or sample container). 3) Be absolutely sure to maintain the tissue frozen. It should be stored in a sealed container at 70 C and shipped or transferred on dry ice. If needed, the frozen sample can be fractured (broken into portions for different applications) by placing in a crucible which contains liquid nitrogen to keep the sample frozen while grinding/fracturing.
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