Document n9MBbQDvq1DOozBoZzQeEJZqX

Attachments to Letter to C. Auer dated May 4, 2000 Perfluoroctane Sulfonate Studies Ongoing Research/Study Protocols 104-week Dietary Chronic Study and Carcinogenicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS: T-6295) in Rats, Covance Laboratories Inc., Study Number 6329-183. In progress. Interim data provided. 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS: T-6295) in Cynomolgus Monkeys, Covance Laboratories Inc., Study Number 6329-223. In progress. Interim data provided. Protocol for Study: Low Level PFOS Dose versus Rat Serum and Liver PFOS, 3M Medical Department, Corporate Toxicology, Study No. T-6295.16 DT31, October 29, 1998. (Study in progress.) Protocol for Study: Pharmacokinetic Study of POSF in Rats, 3M Medical Department, Corporate Toxicology, Protocol for Study No. T-7098.1, January 7, 1999. (Study in Progress). Study Plan, ST-43: Standard Procedure for Liver Subellular Fractionation, undated, 3M Toxicology Laboratory Plan for Study Nos. T-6295.23; ST-46, Exploratory In-Vitro Pervutaneous Absorption Study of Theophylline, Salicylic Acid, Perfluorooctylsulfonate, and Ammonium Perfluorooctanoate in SkinEthic Reconstituted Epidermis Model, May 4, 2000, 3M Toxicology Laboratory 7) Study Plan, Luebker, Perfluorooctane Sulfonic Acid Induced HMG-CoA Reductase Inhibition in Pregnant Rats and Rat Pups, January 21, 2000 004411 10/29/98 T-6295.16; DT-31 Low Level PFOS 3M M EDICAL DEPARTMENT, CORPORATE TOXICOLOGY Protocol for Study No. T-6295.16 DT-31 Low Level PFOS Dose versus R at Serum and Liver PFOS /i-J Projected Start Date: October 29th, 1998 Study Objective'. The objective o f this study is to obtain data from which to construct standard curves o f single oral dose versus serum and liver PFOS concentration in male and female rats. These curves will be used to interpret future oral absorption, distribution, metabolism and excretion (ADME) studies on POSF-based polymeric fluorochemicals (FCs) which may contain low-level contamination with PFOS. These curves will be used to compare serum PFOS concentration after a single oral dose o f POSF-based polymeric FCs containing known quantities o f PFOS to expected serum level and liver level increases predicted by the standard curve based on an equimolar dose o f the residual. These curves will help evaluate whether an increase in serum and liver concentration o f PFOS is a result o f metabolic degradation o f the polymer or absorption o f residuals. Research Client: 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul MN 55133-3220 Sponsor: 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul MN 55133-3220 Study Location: 3M Strategic Alternative Toxicology Laboratory 3M Center, Building 270-SB-181 Saint Paul, MN 55133-3220 Study Director: Andrew M. Seacat Ph.D. Sr. Research Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center Building 220-2E-02 Saint Paul, MN 55133-3220 Ph.: 651-575-3161, FAX: 651-733-1773 Study Toxicologist. Deanna Nabbefeld, MS Advanced Research Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center Building 220-2E-02 Saint Paul, MN 55133-3220 Ph.: 651-737-1374, FAX: 651-733-1773 Proposed Study Timeline: In-Life S ta rt D ate: October 29th, 1998 In-Life E n d D ate: November 19th, 1998 004412 10/29/98 T-6295.16; DT-31 Low Level PFOS Regulatory Compliance: This is a rangefinder study and thus non-GLP. Test Material: The sponsor will provide a sample of PFOS. Analytical documentation o f the starting material will be the responsibility o f the sponsor. A chemical composition specification sheet will be kept on file. Identification: Name: Perfluorooctane Sulfonic Acid, Potassium Salt; CAS # 2795-39-3 M olecular Form ula: C*Fi70S0 2~K+ L o t N um ber: The lot numbers will be maintained in the raw data. Purity: Documentation will be kept in on file. Stability: Documentation will be kept on file. Storage Conditions: Upon receipt, test material will be stored tightly sealed at room temperature. C h aracteristics: Information on synthesis methods, composition or other characteristics that define the test material will be kept on file. Animals: Species: Rat Strain: Sprague Dawley Source: Harlan Laboratories, Inc. Age at initiation of treatm ent: 6-8 weeks old W eight at initiation of treatm ent: approximately 150-200g N um ber an d sex: 75 males, 75 females GROUP 1: control - 15 male, 15 female GROUP 2: 3pg/kg - 15 male, 15 female GROUP 3: 30pg/kg - 15 male, 15 female GROUP 4: lOOgg/kg - 15 male, 15 female GROUP 5: 300pg/kg - 15 male, 15 female Identification: ear tag Husbandry: Housing: All rats will be group housed in standard cages. Diet/W ater: All rats will be provided tap water and Harlan Teklad LM-485 Mouse/Rat Sterilizable Diet (Harlan Teklad, Madison, WI) ad libitum throughout the study. 004413 2 10/29/98 T-6295.16; DT-31 Low Level PFOS E n vironm ent: Environmental controls for the animal room will be set to maintain a temperature o f 72 3F, humidity o f 30-70%, a minimum o f 10 exchanges o f room air per hour and a 12 hour light/dark cycle. Dose and Dosing Procedures; A lmg/mL stock solution o f PFOS in 2% Tween 80 will be prepared immediately prior to dosing. Serial dilutions o f the stock solution will be performed to reach the appropriate concentration for each dose level. A single dose o f compound, using a dose volume o f 5 ml dosing solution / kg body weight, will be administered via oral gavage to rats in groups 2-5 on day zero o f the study. The control rats will receive no treatment. Dose Selection Justification: The doses selected for this low dose PFOS study are designed to yield serum PFOS levels that are at or near the limit o f quantification (15 ppb). Based on previous data, 1 mg o f ingested PFOS yields approximately 5 ppm PFOS in the blood. Therefore, using linear extrapolation, 3 pg/kg will theoretically yield about 15 ppb PFOS in the blood. Three pg/kg was thus selected as the low dose. Mixing procedure: A 1 mg/ml stock suspension o f PFOS will be prepared by homogenizing 10 mg PFOS in 10 ml 2% Tween 80 using a 15 ml glass tissue grinder. A serial dilution will be performed based on the following calculations: Assume a dosing volume o f (5 ml/kg) ( 0.3 kg) = 1.5 ml/ rat 0.3 m g/ K g dose group: (0.3 mg/kg dose)(0. 3 kg rat)/ 1.5 ml/ rat = 0.06 mg/ml PFOS solution in 2% Tween 80 needed. Dilute 6 ml o f the 1 mg/ml solution to 100 ml in 2% Tween 80. 0.1 m g/kg dose group: (0.1 mg/kg) (0.3 kg rat)/ 1.5 ml/rat = 0.02 mg/ml PFOS solution in 2% Tween 80 needed. Dilute 33.333 ml o f 0.06 mg/ml solution up to 100 ml in 2% Tween 80. 0.03 mg/kg dose group (0.03mg/kg) (0.3 k g ) /1.5 ml/rat = 0.006 mg/ml PFOS solution in 2% Tween 80 needed. Dilute 30 ml o f the 0.02 mg/ml solution up to 100 ml in 2% Tween 80. 0.003 mg/kg dose group (0.003) (0.3 kg)/1.5 ml / rat = 0.0006 mg/ml PFOS solution in 2 % Tween 80 needed. Dilute 10 ml o f the 0.006 mg/ml solution up to 100 ml in 2% Tween 80. Dose Verification: Samples o f the concentrated 1 mg/ ml PFOS stock, the 0.06 mg/ml stock (for the 0.3 mg/kg dose group) and the 0.02 mg/ml stock (for the 0.1 mg/kg dose group) will be analyzed by LCMS for dose verification by Kris Hansen, 3M Environmental Lab. It will not be possible to sufficiently dilute the 0.006 and 0.0006 mg/ml dose solutions and still quantify the PFOS. Extrapolation to the lower dose levels will 004414 3 10/29/98 T-6295.16; DT-31 Low Level PFOS therefore, be performed based on the quantification o f the more concentrated dose solutions. All doses will also be confirmed by total organic fluorine analysis performed by Dr. Ventakaswarlu Pothapragada (Dr. V.), 3M SCD Lab. Observation o fAnimals: Clinical Observations: Each animal will be observed daily for mortality and morbidity and notable findings will be recorded. Weekly, each animal will be removed from its cage and a record o f abnormal findings or normality will be made. Additional findings will be recorded as they are observed. Body Weights: Each animal will be weighed immediately prior to dosing, weekly thereafter and immediately prior to euthanasia. Sample Collection: Frequency and Number of Animals: Scheduled sacrifices: Five males and five females from each group will be euthanized on days 1, 4 and 14 post dose. See Table 1 - schedule. 004415 4 TABLE 1 - SCHEDULE Novi Nov 2 day 4 post dose NECROPSY 5 M, 5 F each, gips 2 & 3 (20) Nov 8 Nov 15 Nov 9 day 4 post dose NECROPSY 5 M, 5 F each, grps 1, 4 & 5 (30) Nov 16 Nov 3 NovlO Nov 17 Nov 4 Nov 11 Oct 29 day 0 DOSE grps 2 & 3 (60) Nov 5 day 0 DOSE grps 1,4 &5 (90) Nov 12 day 14 post dose NECROPSY 5 M, 5 F each, grps 2 & 3 (20) Oct 30 day 1 post dose NECROPSY 5M.5F each, grps 2 & 3 (20) Nov 6 day 1 post dose NECROPSY 5 M, 5 F each, grps 1,4 & 5 (30) Nov 13 Nov 18 Nov 19 day 14 post dose NECROPSY 5 M, 5 F each, grps 1, 4 & 5 (30) Nov 12 10/29/98 T-6295.16; DT-31 Low Level PFOS Oct 31 Nov 7 Nov 14 Nov 21 Method of Sample Collection: At all designated times, animals will be euthanized by C 0 2 and gross necropsy performed. During necropsy, systemic blood ( 5 ml) will be collected via the abdominal aorta and transferred to blood collection tubes without anticoagulant. All blood samples will be allowed to clot for a period o f 15 to 30 minutes at room temperature and the clot will be spun down in a centrifuge at 1100 x g for 5 minutes. The serum will be transferred to labeled 1.5 ml microfuge tubes and centrifuged again at 2000 x g to remove any remaining red blood cells. The serum will then be transferred to labeled polypropylene microfuge tubes and flash-frozen in liquid nitrogen. Livers will be removed, weighed, placed individually into labeled tubes and flash frozen in liquid nitrogen. Sample Handling: Samples will temporarily be stored in a freezer set to maintain -60 to 80C. For analysis, these samples will be packed in dry ice and shipped to: 004416 5 10/29/98 T-6295.16; DT-31 Low Level PFOS Kris Hansen, Ph.D. 3M Environmental Technology and Safety Services 935 Bush Avenue St. Paul, MN 55133-3331 Telephone N o.: 651 -778-6081, Facsimile N o .: 651 -778-6176. The number, type and date o f samples to be generated for analysis are as follows: TABLE 2 - SAMPLES Sanrole Oct 30 N ov 2 Serum 20 20 Liver 20 20 Nov 6 30 30 Nov 9 30 30 N ov 12 20 20 N ov 19 30 30 Total 150 150 Analysis: Samples will be analyzed by 3M Environmental for PFOS concentration. Results will be provided for inclusion in the final report. Report: Best-fit curves will be obtained by linear regression based on mean serum and liver PFOS concentrations for each group. Responsibilities: Andrew M. Seacat: Study Director, Deanna Nabbefeld: Study Toxicologist Deanna Nabbefeld and Andrew Seacat are responsible for dosing the animals, performing the necropsy and collecting and sending tissue samples for analysis. Kris Hansen, 3M Environmental, will be responsible for the analytical analysis. Andrew Seacat will draft a report and ensure that it receives appropriate 3M review before a final draft is issued. Signatures: Study Director Deanna Nabbereld, MS Advanced Toxicologist Study Toxicologist Date 004417 6 11/5/98 T-6295.16; DT-31 Low Level PFOS Protocol Amendment # 1 Study No. T-6295.16, DT-31 Low Level PFOS Dose versus Rat Serum and Liver PFOS Sponsor: 3M Specialty Chemicals Division Study Location: 3M Strategic Alternative Toxicology Laboratory Study Director:______Andrew M. Seacat Ph.D.___________ This amendment modifies the following portions o f the protocol: Effective November 5,1998 1. Page 2. Animals, Number and Sex: To strengthen the low-dose portion o f the dose response curve, Dose Group 5 was replaced with a 10 ug/kg dose group. The dose groups will now be as follows: GROUP 1: control - 15 male, 15 female GROUP 2: 3jig/kg -1 5 male, 15 female GROUP 3: 30pg/kg - 15 male, 15 female GROUP 4: lOOgg/kg - 15 male, 15 female GROUP 5: 10 pg/kg -1 5 male, 15 female 2. Page 3. Mixing Procedure: The 0.010 mg/kg dosing solution will be made by making a 10 fold dilution o f the 0.1 mg/kg dosing solution. Therefore, 10 ml o f the 0.02 mg/ml PFOS solution in 2% Tween 80 will be diluted to 100 ml in 2% Tween 80 to yield the 0.002 mg/ ml PFOS solution in 2 % Tween 80 needed. 3. Page 4. Dose Verification: The 0.002 mg/ ml PFOS solution in 2 % Tween 80 will be confirmed by total organic fluorine analysis performed by Dr. Ventakaswarlu Pothapragada (Dr. V.), 3M SCD Lab. 4. Page 6. Analysis: Serum and liver samples will be analyzed by Battelle Laboratories for PFOS concentration. Results will be provided for inclusion in the final report. Shipping Address: Battelle Laboratories Attention: Angela Capers 505 King Avenue Columbus, Ohio 43201 Phone: (614) 424-5588, Fax: (614) 424 -3268 00441S 7 Amendment Approval: Signatures: Study Director Deanna Nabbefeld, MS Advanced Toxicologist Study Toxicologist 11/5/98 T-6295.16; DT-31 Low Level PFOS Date 004419 8 04/20/99 T-6295.16; DT-31 Low Level PFOS Amendment #2 Protocol Amendment # 2 Study No. T-6295.16, DT-31 Low Level PFOS Dose versus Rat Serum and Liver PFOS Sponsor: 3M Specialty Chemicals Division Study Location: 3M Strategic Alternative Toxicology Laboratory Study Director:______Andrew M. Seacat Ph D,____________________ Effective April 26th, 1999 The purpose o f this amendment is to reflect the decision o f the Sponsor to add a dose group o f 5mg/kg body weight and a necropsy date of 28 days post dose to the overall study design. All modifications are additions to the original protocol and Amendment #1. The modifications are as follows: 1. Page 1 Protocol, Proposed Study Timeline: Additional Proposed Timeline specific to Amendment #2: In-Life Start Date: 3/2/99 In-Life End Date: 5/24/99 2. Page 2 Protocol / Page 1 Amendment #1, Animals, Number and Sex: The following 2 dose groups will be added to the protocol: GROUP 6: vehicle control - 12 male, 12 female GROUP 7: 5mg/kg - 20 male, 20 female 3. Page 3 Protocol / Page 1 Amendment #1, Mixing Procedure: A single 5mg/kg dose o f compound will be administered via oral gavage to rats in groups 7 on day zero o f the study. The compound will be prepared as a 1% (1 mg/mL) uniform suspension in 2% Tween 80 using a 15 ml tissue grinder. A volume o f 5 ml suspension / kg body weight will be administered. Re-suspension o f solids will be performed with 5 strokes o f the tissue grinder pestle before each sample is drawn-up in the syringe for dosing. The 10 control rats (group 6) will be dosed with 5 ml/kg 2% Tween 80 (vehicle control). 4. Page 4 Protocol / Page 1 Amendment #1, Dose Verification: The vehicle control sample (2% Tween 80) and the 1 mg/ ml PFOS solution in 2 % Tween 80 will be confirmed by total organic fluorine analysis performed by Dr. Ventakaswarlu Pothapragada (Dr. V.), 3M SCD Lab. 5. Page 4 Protocol. Sample Collection, Frequency and Number of Animals: Frequency and Number of Animals: Scheduled sacrifices: Five males and five females from group 7 and 3 males and 3 females from group 6 will be euthanized on days 1, 4, 14 and 28 post dose. See Table 1 - i schedule. Page 1 o f 3 004420 04/20/99 T-6295.16; DT-31 Low Level PFOS Amendment #2 TABLE 1 - SCHEDULE April 26 April 27 day 0 DOSE grps6 & 7 (6 4 ) day 1 post dose NECROPSY (16) May 2 May 3 May 4 April 28 May 5 April 29 April 30 day 4 post dose NECROPSY (16) May 6 May 7 May 1 May 8 May 9 May 10 day 14 post dose NECROPSY (16) May 16 May 17 May 23 May 24 Day 28 post dose NECROPSY ___ SL May 11 May 18 May 12 May 19 May 13 May 20 May 14 May 21 May 15 May 22 k. 004421 Page 2 o f 3 Amendment Approval: 04/20/99 T-6295.16; DT-31 Low Level PFOS Amendment #2 Signatures: ^ **1____ j/A Andrew M. Seacat, Ph.D. Senior Research Toxicologist Study Director Date Deanna Nabbefeld, MS Advanced Toxicologist Study Toxicologist Date Page 3 of 3 004422