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THE DEVELOPMENT SERVICES COMPANY
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Sponsors: APME Ad-Hoc APFO Toxicology Working Group
PROTOCOL
Study Title:
26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys
Date: September 23,1998
Performing Laboratory:
Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595
Laboratory Study Identification:
Proposal No. w6806a
Covance 6329-231
T- 3
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Study 26-Week Capsule Toxicity Study with Ammonium Perfluorooctanoate (APFO) in Cynomolgus Monkeys
Purpose To assess the effect of the test material on critical enzyme levels, hormones, and other selected biochemical parameters when administered daily by capsule to cynomolgus monkeys for at least 26 weeks
Sponsors APME Ad-Hoc APFO Toxicology Working Group
Study Monitor Paul Lieder, PhD, DABT 3M Toxicology Services Building 220-2E-02,3M Center St. Paul, Minnesota, 55144-1000 Telephone No.: 612.737.2678 Facsimile No.: 612.733.1773
Alternate Study Monitor John Butenhoff, PhD, DABT 3M Telephone No.: 612.733.1962 Facsimile No.: 612.733.1733
Sponsor's Representative David Farrar, PhD ICI Chemicals & Polymers Limited Occupational Health The Heath, Runcorn, PO Box 13 Cheshire, WA7 4QF Telephone No.: 01928.513953 Facsimile No.: 01928.515146
Study Location Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704
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Mailing Address: PO Box 7545-2595 Madison, Wisconsin 53707-7545
Study Director Peter J. Thomford, PhD Covance Laboratories Inc. Telephone No.: 608.241.7207 Facsimile No.: 608.242.2736
Toxicologist Dale Aldridge, BS Covance Laboratories Inc.
Proposed Study Timetable Experimental Start Date: September 29,1998 In-Life Start Date: September 29,1998 In-Life End Date: M y 2, 1999 Audited Draft Report Date: October 22, 1999 Experimental Termination Date: To be determined
Regulatory Compliance This study will be conducted in compliance with the Environmental Protection Agency Good Laboratory Practice Regulations as set forth in Title 40 of the US Code of Federal Regulations, Part 792, issued November 29,1983 (effective December 29, 1983), and with any applicable amendments except that bile acid determinations done by University of Dundee will not be done in compliance with GLPs.
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 The protocol, study conduct, and final report will be audited by the Covance Quality Assurance Unit (QAU). The proliferation cell nuclear antigen evaluation data and report will be audited by the QAU of Pathology Associates International. The serum, urine, feces, and liver APFO analysis and report will be audited by the QAU of 3M Environmental Laboratories. The blood hormone determinations and report will be audited by the QAU of Ani Lytics Inc and DuPont, as appropriate. The receptor level determinations and report will be audited by the QAU of DuPont. Bile acid determinations done by the University of Dundee will not be audited.
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Test Material
Identification Ammonium Perfluorooctanoate (APFO)
Lot Number The lot number will be maintained in the raw data.
Purity Responsibility of the Sponsor
Stability Responsibility of the Sponsor
Storage Conditions At room temperature
Characteristics Information on synthesis methods, composition, or other characteristics that define the test material is on file with the Sponsor.
Gelatin Capsules Capsules (Size No. 2) obtained from Torpac, Inc. (Fairfield, New Jersey); lot number will be supplied by the manufacturer. Gelatin capsules will be stored at room temperature. A copy of the Certificate of Analysis provided by the manufacturer will be maintained in the data.
Reserve (Archive) Samples A reserve sample of each lot of test material (1 g each) will be taken and stored at room temperature. These samples will be transferred to the Sponsor after completion of the in-life phase.
Disposition of Test Material Any remaining test material will be returned after authorization from the Sponsor.
Animals
Species Cynomolgus monkey
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Source Covance Research Products Inc.
Age at Initiation of Treatment Young adult/adult
Weight at Initiation of Treatment 3 to 5 kg
Number and Sex 22 males
Identification Collar tag
Husbandry
Housing Individual; animals will be housed in suspended, stainless steel cages.
Diet Certified primate diet (#8726C, Harlan Teklad) once or twice daily. The diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Results of specified nutrient and contaminant analyses are on file at Covance-Madison. Fruit, vegetables, or other supplements may be provided but will not require analysis.
Water Ad libitum. Samples of the water are routinely analyzed for specified microorganisms and environmental contaminants. The results are on file at Covance-Madison.
Contaminants There are no known contaminants in the diet or water at levels that might interfere with this study.
Environment Environmental controls for the animal room will be set to maintain 18 to 29 C, a relative humidity of 30 to 70%, and a 12-hour light/12-hour dark cycle.
Acclimation Minimum of 4 weeks
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Randomization Animals will be weighed, stratified by body weight, and allocated to the number of blocks equal to the number of animals to be selected for each group. Animals in each block will then be assigned to groups using computer-generated random numbers.
Justification APFO is a known hepatic peroxisome proliferator (PP) in the rat. When exposed to PP, nonhuman primates (such as the cynomolgus monkey) respond similarly to humans (i.e., low to no hepatic response) and therefore are an appropriate human surrogate species.
Group Designations and Dosage Levels
Group
Dose Level (mg/kg/day)
Number of Males
1 (control) 2 (low-dose) 3 (mid-dose) 4 (high-dose)
Oa 3 10 30
6b 4 6b 6b
a The control group (Group 1) will receive empty gelatin capsules. b Two animals in Groups 1, 3, and 4 designated as recovery animals will be
treated for 26 weeks, then treatment will be discontinued, and the animals will be observed for reversibility, persistence, or delayed occurrence of toxic effects for at least 13 weeks posttreatment. Based on plasma test material levels and clinical pathology findings, the recovery may be reduced or extended at the discretion of the study director after consultation with the Sponsors.
Dosing Procedures
Method of Administration Orally by gelatin capsules, daily (7 days/week) for at least 26 weeks
Reason for Dosing Route To compare with previously conducted toxicology studies using the oral route
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Dose Preparation Capsules will be prepared at least weekly. Tire size and number of capsules will depend on the physical characteristics of the test material, the dose level, and the weight of the monkey. Dose levels will be based on the test material as supplied. Individual daily doses will be based on the most recent individual body weights, with the exception of body weight collection days when the previous body weight will be used. All dose preparations will be stored at room temperature until dosed.
Dose Analysis Because the test material is not being mixed with a vehicle, dose analyses are not required.
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.
Each animal will be observed daily and food consumption will be assessed qualitatively; abnormal findings will be recorded. Once weekly, each animal will be observed; abnormal findings, or an indication that the animal is normal will be recorded.
Additional findings will be recorded as they are observed.
Body W eights Weekly before initiation of treatment, Day -1 (for the Day 1 dose calculations),on the first day of treatment, and weekly thereafter
Ophthalmic Examinations Before initiation of treatment and before each scheduled sacrifice. The anterior portion of the eye, optic media, and ocular fundus will be examined using an indirect ophthalmoscope.
Clinical Pathology
Frequency
Unscheduled Collections When possible, blood will be collected for clinical pathology tests from animals sacrificed at unscheduled intervals
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Scheduled Collections Before initiation of treatment; and (before the daily dose) after at least 30, 60, 90, and 180 days of treatment; and after at least 30, 60, and 90 days of recovery
Number of Animals All
Method of Collection Animals will be fasted overnight; blood will be collected from a femoral vein. Anticoagulants will be sodium citrate for coagulation tests and potassium EDTA for hematology tests. No anticoagulant is required for clinical chemistry tests. Urine will be collected on wet ice overnight (approximately 16 hours).
Tests
Hematology
red blood cell (erythrocyte) count
platelet count
hemoglobin
white blood cell (leukocyte) count
hematocrit
differential blood cell count
mean corpuscular volume
blood cell morphology
mean corpuscular hemoglobin
reticulocyte count
mean corpuscular hemoglobin
concentration
Coagulation
activated partial thromboplastin time
prothrombin time
fibrinogen
Clinical Chemistry
glucose
aspartate aminotransferase
urea nitrogen
gamma glutamyltransferase
creatinine
sorbitol dehydrogenase
total protein
creatine kinase
albumin
calcium
globulin
inorganic phosphorus
total bilirubin
sodium
direct bilirubin (if total bilirubin is
potassium
greater than 2.0 mg/dL)
chloride
cholesterol
bile acids
triglycerides
amylase
alanine aminotransferase
lipase
alkaline phosphatase
pancreatic-specific amylase
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Urinalysis
appearance
glucose
volume (approximately 16 hours)
ketones
specific gravity
bilirubin
PH blood
protein
microscopic examination of sediment
urobilinogen
Blood Hormone Determination
Frequency Three times before initiation of treatment; after at least 30,60,90, and 180 days of treatment; and after at least 30, 60, and 90 days of recovery
Number of Animals All
Method of Collection Animals will not be fasted; blood will be collected from a femoral vein (an alternative vein may be used if necessary). Approximately 6 mL of blood for plasma samples will be collected into tubes with potassium EDTA as the anticoagulant Approximately 6 mL of blood for serum samples will be collected without anticoagulant and allowed to clot Blood samples will be maintained at room temperature until serum is harvested. Blood samples will be maintained chilled until plasma is harvested.
Serum Sample Handling Samples for serum will be centrifuged within 1 hour after collection, and serum will be harvested. Serum will be divided into two approximately equal aliquots and stored in a freezer set to maintain -60 to -80 C until packed on dry ice and shipped to:
Dr. Saroj Das Ani Lytics Inc. 200 Girard Street, Suite 200 Gaithersburg, Maryland 20877 Telephone No.: 301.921.0168 Facsimile No.: 301.977.0433
Ani Lytics Inc. will be notified regarding shipment of samples.
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Serum Tests Samples will be analyzed by Ani Lytics Inc., for estradiol, estrone, estriol, thyroid stimulating hormone, total and free triiodothyronine (T3), and total and free thyroxin (T4). Results will be provided for inclusion in the final report.
Plasma Sample Handling Samples for plasma will be centrifuged within 1 hour after collection. Plasma will be harvested and stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped to:
Jon C. Cook, PhD, DABT E. I. du Pont de Nemours & Company, Inc. Haskell Laboratory for Toxicology and Industrial Medicine Building 11 Room 1325 1090 Elkton Road Newark, Delaware 19711-0500 Telephone No.: 302.366.5495 Facsimile No.: 302.366.5003
DuPont will be notified regarding shipment of samples.
Plasma Analysis Samples will be analyzed by DuPont Haskell Company for cholecystokinin. Results will be provided for inclusion in the final report.
Serum APFO Level Determination
Frequency After 7 days of treatment and every 2 weeks thereafter during treatment and recovery
Number of Animals All
Method of Collection Animals will not be fasted; blood (approximately 2 mL) will be collected from a femoral vein (an alternative vein may be used if necessary) without an anticoagulant.
Sample Handling Samples will be centrifuged within 1 hour after collection, and serum will be harvested. Serum samples will be stored in a freezer set to maintain -10 to -30C.
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Sample Shipping Serum samples will be packed on dry ice and shipped to:
Kris J. Hansen, PhD 3M E.T. & S Bldg. 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55106
Kris J. Hansen, PhD or her alternate will be notified regarding the shipment of the samples. Analysis of serum for APFO will be done on the samples. Results will be provided for inclusion in the final report.
Additional Blood Collection At scheduled and unscheduled sacrifices, blood (as much as possible, up to 20 mL) will be collected from animals at the time of exsanguination. Approximately equally sized samples of serum (collected without anticoagulant) and whole blood and plasma (both collected using EDTA as an anticoagulant) from each animal will be transferred into containers (approximately 5 mL each). Whole blood, serum, and plasma samples will be stored in a freezer set to maintain -60 to -80C until shipped to 3M (Kris Hansen) for possible future analysis.
Urine and Feces APFO Level Determination
Frequency After 7 days of treatment and every 2 weeks thereafter during treatment and recovery (concurrent with serum APFO sample collection)
Number of Animals
ah
Method of Collection Animals will not be fasted; urine (collected on wet ice) and feces will be collected overnight.
Sample Handling Samples of urine (at least 2 mL) and feces (at least 5 grams) will be stored in a freezer set to maintain -10 to -30C.
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Sample Shipping Samples will be packed on dry ice and shipped to:
Kris J. Hansen, PhD 3M E.T. & S Bldg. 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55106
Kris J. Hansen, PhD or her alternate will be notified regarding the shipment of the samples. Analysis of urine and feces for APFO will be done on the samples. Results will be provided for inclusion in the final report.
Termination
Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with ketamine and xylazine, weighed, and exsanguinated.
Scheduled Sacrifices After at least 26 weeks of treatment, four animals/sex/group will be fasted overnight, then anesthetized with ketamine and xylazine, weighed, exsanguinated, and necropsied.
After at least 26 weeks of treatment and at least 13 weeks without treatment, the remaining animals will be fasted overnight, then anesthetized with ketamine and xylazine, weighed, exsanguinated, and necropsied.
Postmortem Procedures
Necropsy The necropsy will include examination of:
all orifices cranial cavity external surface of the brain; the external surface of the spinal cord and cut surfaces of the brain and spinal cord will be examined whenever tissue trimming is performed cervical tissues and organs thoracic, abdominal, and pelvic cavities and viscera external surface of the body nasal cavity and paranasal sinuses
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Organ Weights From each animal at scheduled and unscheduled sacrifices, the following organs (when present) will be weighed; paired organs will be weighed separately:
adrenal (2) brain epididymis (2) kidney (2)
liver pancreas testis (2) thyroid (2) with parathyroid
Organ-to-body weight percentages and organ-to-brain weight ratios will be calculated.
Palmitoyl CoA Oxidase Determinations The right lateral lobe of liver will be collected from each animal at the scheduled and unscheduled sacrifices, weighed, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C until disposition of the samples is determined by the Sponsor.
Cell Proliferation Evaluation Representative samples of left lateral lobe of the liver, left and right testes, and pancreas will be collected from each animal at the scheduled and unscheduled sacrifices, and preserved in zinc formalin.
After fixation, samples for proliferation cell nuclear antigen (PCNA) evaluation will be embedded in paraffin maintained at ambient temperature until disposition of the samples is determined by the Sponsor.
Bile Add Determination All available bile (up to 5 mL) will be collected from each animal at the scheduled and unscheduled sacrifices, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C. Bile samples will be packed on dry ice and shipped to:
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Biomedical Research Centre Level 5 Ninewells Hospital and Medical School University of Dundee Dundee DD1 9SY UK Dr. Cliff Elcombe Biomedical Research Centre Ninewells Hospital and Medical School University of Dundee Dundee DD1 9SY, UK Telephone: +44 (0)1382-632641 Fax: +44 (0)1382-425586
Results of bile acid determinations analyses will be provided for inclusion in the final report.
Receptor Level Determination Samples (approximately 2 g) of liver and pancreas will be collected from each animal at the scheduled and unscheduled sacrifices, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C. Liver and pancreas samples will be packed on dry ice and shipped to:
Jon C. Cook, PhD, DABT E. I. du Pont de Nemours & Company, Inc. Haskell Laboratory for Toxicology and Industrial Medicine Building 11 Room 1325 1090 Elkton Road Newark, Delaware 19711-0500 Telephone No.: 302.366.5495 Facsimile No.: 302.366.5003
DuPont will be notified regarding shipment of samples. Results of receptor level determinations will be provided for inclusion in the final report.
Tissue Preservation Three samples (approximately 5 g each) of the liver and all remaining pancreas and left and right testes tissue, if any, (divided into three approximately equal samples) will be collected from each animal at the scheduled and unscheduled sacrifices, weighed, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C for possible future analysis.
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The following tissues (when present) from each animal will be preserved in 10% neutral-buffered formalin, unless otherwise specified, for possible future microscopic examination:
adrenal (2) aorta brain cecum colon duodenum epididymis (2) esophagus eye [(2) to be preserved in Davidson's
fixative from sacrificed animals only] femur with bone marrow (articular
surface of the distal end) gallbladder heart ileum jejunum kidney (2) lesions liver lung mammary gland
mesenteric lymph node pancreas pituitary prostate rectum salivary gland [mandibular (2)] sciatic nerve seminal vesicle (2) skeletal muscle (thigh) skin spinal cord (cervical, thoracic, and
lumbar) spleen sternum with bone marrow stomach testis [(2) to be preserved in Bourns'
solution from sacrificed animals only] thymus thyroid (2) with parathyroid trachea urinary bladder
Bone Marrow Smear From the sternum of each animal at unscheduled and scheduled sacrifices (made and held for possible future examination)
Liver APFO Determination A sample of liver (any non-formalin treated liver remaining after sampling for histopathology) will be collected from each animal at the scheduled and unscheduled sacrifices, weighed, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80 C until shipped with serum samples to 3M (Kris Hansen) for analysis. Analysis of liver for APFO will be done on the samples. Results will be provided for inclusion in the final report.
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Histopathology Tissues from each animal in the control and high-dose groups and each animal that dies or is sacrificed at unscheduled interval will be embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.
The adrenals, pancreas, liver, spleen, and testes from each animal in the low- and mid-dose groups will be embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.
Suspected target organs noted at the high-dose will be embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically from all animals (at the Sponsor's request and added by amendment).
Reports A draft report which includes the following information will be prepared and submitted:
Experimental Design and Methods
Results mortality clinical observations body weights body weight gains food consumption ophthalmic examination results clinical pathology results hormone analyses (provided by Ani Lytics, Inc. and the DuPont Haskell
Company) serum, urine, feces, and liver APFO levels (provided by 3M) organ weights organ-to-body weight percentages organ-to-brain weight ratios macroscopic observations microscopic observations palmitoyl CoA oxidase activities (provided by the Sponsor's designee) cell proliferation evaluations (provided by Pathology Associates International) bile acid determinations (provided by University of Dundee) receptor determination (provided by DuPont)
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Statistical Evaluation Levene's test will be done to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, transformations will be used to stabilize the variance. Comparison tests will take variance heterogeneity into consideration.
One-way analysis of variance (ANOVA) will be used (if applicable) to analyze initial body weights, body weight gains, continuous clinical pathology values, palmitoyl CoA oxidase activities, and organ weight data. If the ANOVA is significant, Dunnett's t-test will be used for control versus treated group comparisons.
One-way analysis of covariance (ANCOVA) will be used to analyze body weights, with initial body weights as the covariate. If the ANCOVA is significant, covariate-adjusted means will be used for control versus treated group comparisons.
Group comparisons (Groups 2 through 4 versus Group 1) will be evaluated at the 5.0% two-tailed probability level. Only data collected on or after the first day of treatment will be analyzed statistically. Data collected before the first day of treatment or during recovery will not be analyzed statistically.
At the end of 1 year after issuance of the audited draft report, if no requested revisions or instructions to finalize have been communicated by the Sponsor, then the audited draft report will be considered 'final' and issued as the final report, signed by the study director, and submitted to the Sponsor.
Any modifications or changes to the audited draft report requested 1 year after issuance will be performed at additional cost to the Sponsor.
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 Covance-Madison 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. The Sponsor may elect to have the materials retained in the Covance archives for an additional period of time, and Covance will charge a storage fee. If the Sponsor chooses to have Covance dispose of the materials, a disposal fee will be charged. All raw data stored on magnetic media will be retained by Covance.
Within one year after submission of the final report, all of the aforementioned materials from the Sponsor's designees (Ani Lytics Inc., E. I. du Pont de Nemours & Company, Inc., 3M E.T. & S, Pathology Associates International, and University of Dundee) will be sent to the Sponsor (Paul Lieder, PhD, DABT, 3M) to be maintained.
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PROTOCOL APPROVAL
Covance 6329-231 _________ Page 18
__ ___________________________________ Paul Lieder, PhD Diplomate, ABT Study Monitor 3M
David Farrar, PhD ICI Chemicals & Polymers Limited
Covance Laboratories Inc.
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