Document 9930Dn3byrm450Qd6aO9LbwNV

ARZ&-02IH 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Protocol for Study No. T-7132.2; ST-39 PHARMACOKINETIC STUDY OF PERFLUOROOCTANE SULFONAMIDE IN RATS Study O bjective. The objective o f this study is to assess the potential for oral absorption, urinary and fecal clearance and biological persistence of Perfluorooctane Sulfonamide (PFOSA) in male Sprague Dawley rats after a single oral dose. Analysis o f the serum, liver, urine and feces for potential metabolites of PFOSA will be performed by LCMS and perhaps other methods. Previous studies o f the N-ethyl derivative o f PFOSA have concluded that PFOSA is the ultimate metabolite in rats (1,2); however, the analytical technique used in these studies was unable to detect all potential metabolites, including perfluorooctanesulfonate (PFOS). No pharmacokinetic studies have yet been done on PFOSA to resolve this issue and search for other possible metabolites in the urine and feces. Recently, validated methods have been developed for the quantitation o f PFOSA and its potential metabolite, PFOS, in serum and liver down to the low part per billion level (3). One goal o f this study is to determine the potential for, if occurring, the extent o f conversion o f PFOSA to PFOS. This information will help to explain the pharmacokinetics o f NEtFOSE and its metabolites and provide data for proper risk characterization. Research Client: 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul, MN 55144 Sponsor: Stittfy Location: 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul, MN 55144 3M Strategic Toxicology Laboratory 3M Center, Building 270-3S-06 room SB314 Saint Paul, MN 55144 Study Director: Andrew M. Seacat, Ph.D. Toxicology Specialist 3M Medical Dept. / Corporate Toxicology 3M Center, Building 220-2E-02 Saint Paul, MN 55144 Ph.: 651-575-3161 FAX: 651-733-1773 Study Toxicologist: Deanna Luebker, MS Advanced Research Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center, Building 220-2E-02 Saint Paul, MN 55144 000153 Ph: 651-737-1374 FAX: 651-733-1773 T-7132.2; ST-39 PFOSAPK Proposed Study Tim eline In-Life S tart Date: October 4th, 1999 In-Life End Date: November 2nd, 1999 Analytical Com pletion Date: TBA Final R eport Completion Date: TBA Regulatory Compliance: This study will be performed in the 3M Strategic Toxicology Laboratory under a defined protocol and classified as a "Class B Study" as explained in TOX SOP 0950, Strategic Toxicology Lab GLP Program Procedure. Test M ateriali Dan Hakes, Product Responsibility Liaison 3M Chemicals Division, will furnish high-purity PFOSA. < Identification: Name: Perfluorooctanesulfonamide M olecular Formula: C8F17SO2NH2 Lot Num ber: L -10009 (Prepared by George Moore, 3M SMD Lab, Bldg 236, April 1996) Purity: Analysis by GCMS determined that the staring material was over 99% pure (4). Qualitative and quantitative compositional results that were derived from the single trial 'H /^ -N M R cross-integration analysis revealed that the composition was 65.8% CF3(CF2)x-S02-NH2 (Normal chain), 18.7% CF3(CF2)x-CF(CF3)-(CF2)y-S02-NH2 (Internal monomethyl branch), 11.2% (CF3)2CF-(CF2)x-S02-NH2 (Isopropyl branch), 3.5% CxF2x+l-CF(CF3)-S02-NH2 (Alpha branch) and 0.28% (CF3)3C-(CF2)*-S02-NH2 (t-Butyl branchX5). HPLC/MS characterization o f the PFOSA sample revealed 9,600 ppm o f PFOS, 1,100 ppm o f C7F15S02NH2, 510 ppm o f QF^SCfeNH* 6,600 ppm o f C*F16HSO2NH2, 24,000 ppm o f C1F3HSO2NH2, 1,200 ppm o f C8F1SH2SO2NH2 and lower concentrations o f several other amides. Based on the sum o f the impurities, the purity o f the PFOSA sample would be approximately 96 % (6 & appendix I). Stability: Documentation will be kept on file with the Sponsor. 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 with the Sponsor. 000154 2 A nim als: Species: Rat Strain: Sprague Dawley Source: Harlan Age at initiation of treatm ent: 6-8 weeks W eight a t initiation of treatm ent: approximately 150-250g Num ber and sex: 30 males T-7132.2; ST-39 PFOSAPK Table 1 - Dose Groups Group Dose 1 0 mg/kg 2 5 mg/kg N 15 15 Euthanasia 5 each on days 1,4 and 29 post dose 5 each on days 1,4 and 29 post dose Identification: AUA Num ber: ear tag with animal number or unique tail mark. 2246 H usbandry: H o u sin g : All rats from groups 1 and 2, which are to be sacrificed on day 29 post dose, will be housed individually in metabolism cages for portions o f the study (see Table 2). When not in metabolism cages, these rats will be group housed in standard cages. All other rats will be group housed in standard cages throughout the study. Diet/W ater: Harlan Teklad LM-485 Mouse/Rat Sterilizable Diet, supplied by Harlan Teklad, Madison, WI, and tap water will be provided to all rats ad libitum throughout the study. Environment: 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. D ose and D osing Procedures: M ethod of administration/Dose preparation: A single 5mg/kg dose of PFOSA will be administered via oral gavage to rats in group 2 on day zero o f the study. The PFOSA will be prepared from a stock solution of lOOmg/ml PFOSA in acetone. A final 0.1% (lmg/ml) uniform suspension (or emulsion) o f PFOSA in 2% Tween 80 and 1% acetone will be prepared using a 15 ml dounce tissue grinder. A volume o f 5 ml suspension / kg body weight will be administered to each rat. Re suspension of solids will be performed with 5 strokes of the tissue grinder pestel before each sample is drawn-up in the syringe for dosing. A single 5 000155 3 T-7132.2; ST-39 PFOSAPK ml / kg body weight dose o f vehicle, 2% Tween 80 and 1% acetone, will be administered via oral gavage to rats in group 1 on day zero o f the study. Observation o fA nim als: Clinical O bservations: Each animal will be observed daily (excluding weekends and holidays) for mortality and morbidity and notable findings will be recorded. Additional findings will be recorded as they are observed. Body W eights: Each animal will be weighed immediately prior to treatment, weekly thereafter and immediately prior to euthanasia. Specim en Collection: Frequency (See table 2): Urine and feces collections will be made on days 1 - 4 post dose. Necropsies will be performed on days 1, 4 and 29 post dose. Table 2 - Schedule Sun Mon Oct 3 Oct 4 day 0 DOSING O ct 10 day 6 PD O ct 17 day 13 PD O ct 11 day 7 PD O ct 18 day 14 PD Tues Oct 5 day 1 PD Collection, Dy 1 PD sac O ct 12 day 8 PD O ct 19 day 15 PD W ed Oct 6 day 2 PD Collection Oct 13 day 9 PD Oct 20 day 16 PD Thurs Oct 7 day 3 PD Collection O ct 14 day 10 PD O ct 21 day 17 PD Fri Oct 8 day 4 PD Collection Switch to reg cages. Dy 4PD sac. O ct 15 day 11 PD Oct 22 day 18 PD Sat Oct 9 day 5 PD O ct 16 day 12 PD O ct 23 day 19 PD O ct 24 day 20 PD O ct 31 day 27 PD O ct 25 day 21 PD N ovi day 28 PD O ct 26 day 22 PD Nov 2 day 29 PD Dy 29 PD sac. Oct 27 day 23 PD Oct 28 day 24 PD Oct 29 day 25 PD Oct 30 day 26 PD M ethod of Specimen Collection: Urine and feces will be collected from each metabolism cage at the designated times. The initial volume o f urine will be recorded, the sides of the urine collection apparatus will be washed with approximately 5 -10ml deionized water and the final volume of urine will be brought to 15 ml with additional deionized water. Daily feces weight will be recorded for each animal. At the designated times, animals will be euthanized by CO2 and gross necropsy performed. During necropsy, blood (* 6 ml) will be collected via the abdominal aorta and transferred to blood collection tubes without anticoagulant. Blood samples will be allowed to clot for a period 000156 4 T-7132.2; ST-39 PFOSAPK 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. Each sera sample will then be transferred to a separate labeled polypropylene microfuge tube and frozen in dry ice. Livers will be rem oval, weighed, placed individually into labeled sterile sample bags and flash frozen in liquid nitrogen then maintained on dry ice. Specimen Handling: Specimens will temporarily be stored in a freezer set to maintain -60 to 80C. For metabolite analysis, these specimens will be packed in dry ice and shipped to: Kris Hansen, Ph.D. 3M Environmental Technology and Safety Services 935 Bush Avenue St. Paul, MN 55133-3331 Ph: 612-778-6081, FAX: 612-778-6176. All results will be provided for inclusion in the final report. The number, type and date o f collection o f specimens to be generated for analysis are as follows: Table 3 - Specimens Soecimen day 1 post dose Serum 10 (5/group/day) Liver 10 (5/group/day) Urine 10 (5/group/day) Feces 10 (5/group/day) day 2 post dose 10 10 Day 3 post dose 10 10 day 4 post dose 10 10 10 10 day 29 post dose 10 Total 30 10 30 40 40 Data Analysis: Data collected on parent compound and identifiable metabolites will be analyzed for toxicokinetic parameters and for statistically significant differences between groups using ANOVA and/or Students T-test. 000157 5 T-7132.2; ST-39 PFOSAPK R esp o n sib ilities: Deanna Luebker and Andrew Seacat will be responsible for dosing the animals, collecting in-life specimens, performing the necropsies and collecting and sending tissue specimens for analysis. Kris Hansen, 3M Environmental, will be responsible for analytical evaluation o f the Andrew Seacat will draft a final report and ensure the report receives appropriate 3M review before a final report is issued. 000158 6 Signatures: h u k to O O J j, Andrew M. Seacat Ph.D. Toxicology Specialist Study Director Deanna Luebker, MS Advanced Research Toxicologist Study Toxicologist T-7132.2; ST-39 PFOSAPK -------------J--pr-/'-X---7--- '-A-----f1------------------Date j O /> 7M 9 D ate 000159 7 References: T-7132,2; ST-39 PFOSAPK 1. Grossman M.R. and Bowen J.M. (1990) Tissue analysis o f fluorinated sulfonamide pesticide: an evaluation o f distribution, elimination, and potential for bioaccumulation in orally exposed rats. M S. Thesis, Univ. of Georgia, Athens, G A (also possibly published as: Grossman Marie R. and Bowen J.M. (1990) Tissue distribution and elimination o f a fluorinated sulfonamide pesticide in rats. Fundam. Appl. Toxicol., but notfound). 2. Grossman M.R., Mispagel, M.E. and Bowen J.M. (1992) Distribution and tissue in rats during and after prolonged dietary exposure to a highly fluorinated sulfonamide pesticide. J. Agrie. FoodChem. 40, 2505 - 2509. 3. K.J. Hansen, L .A Clemen, M.E. Ellefson, H.O. Johnsoa (1999). Compound Specific Characterization o f Organic Fluorochemicals in General Population Human Sera Samples. 3M Environmental Lab, St. Paul, MN 55133. 4. Payfer R.M. GC/MS analyses of PFOSA (L-10009). SA&C Analytical Request No. 59426. Report 9/24/99. 3M SA&C Lab Building 236-2B-11. 5. Tom Kestner Chemical Characterization o f PFOSA L-10009, by 1H and 19FNMR Spectroscopy Requests # 59426. 3M Specialty Adhesives & Chemicals Analytical Laboratory / SMMD-236-2B-11, September 25,1999. 6. DeRoos F.L. Characterization of PFOSA Samples, T-7132-1 (L-10009) and TNA-1584. Request # A-151254. Report 10/7/99. Corporate Analytical Technology Center, Building 201-1-29, CATC - Chromatography Group. 000160 8 .\ Appendix I: Tel: 736-0665 201-1W -29 T-7132.2; ST-39 PFOSAPK Corporate Analytical Technology Center To: Larry A . W endling/US-Corporate/3M /US cc: Andrew Seacat/U S-C orporate/3M /U S Subject: Characterization of PFOSA Sam ple for Toxicology Larry, I have completed the H PLC /M S characterization of the PFO SA sample (T -7132-, L-10009 prepared by G . M oore 4 /9 6 ) that is proposed to be used for the animial feeding study. I found 9,600 ppm o f P FO S , 1,100 ppm o f C7F 15SO2NH2, 510 ppm o f C9F 19S O 2NH2, 5 2 26,600 ppm o f CgF-i H S O NH , 24,000 ppm o f C 'j8F36H S 2 NH2> 1 2 0 0 PP*n o f 5 2 2 2C^F^ H S O N H an d lo w er concentrations o f several o th er am ides. B ased on th e sum o f th e im p u ritie s , th e purity o f th e PFO SA sam ple w ould be ap p ro xim ately 96 %. I've thought a little m ore about apparent presence of C 18F36HSO2NH2 in the sam ple. The identification o f this compound was based primarily on the observed molecular weight and the relative elution order in the chromatogram. Rather than C i8 F36HS 2 NH2 ' it seem s 7 02 2more probable that this compound is actually (C g F i S > NH. Confirmation of this identification will require additional analyses. I also need to think more about w hether the other partially hydrogenated perfluoroamides may actually be of this The PFO S was quantified using a standard curve prepared by analyzing PFO S standards so its concentration should be accurate. The am ide impurities, however, including the partially hydrogenated am ides, were all quantified using a PFOSA standard curve assuming that they had the sam e mass spectral response factor as did PFO SA. W hile this assumption will introduce some error to the quantitative data, it is the best we can do since w e do not have standards for each of these amides. Also, this calculated purity assumes that all o f the impurities are am enable to HPLC and are detected by the analysis. W hile not always true, this sam ple w as analyzed by Rick Payfer using G C /M S and by CATC using our G C method. Neither o f the analyses found volatile or sem ivolatile impurities, e.g., N-m ethyi FO S E , N ethyl FO SE, etc. at concentrations greater than 50 ppm. I also carried out a semiquantitative assessment of the purity o f the PFO SA by comparing the PFO SA response to a PFOSA standard curve. Using this technique, the purity of the PFO SA sam ple was found to be 118 %. These analyses were carried out in duplicate, with triplicate injections o f each solution, so they should be relatively accurate as analytical variation would be averaged. In general, however, it is not highly accurate to use a chromatographic method to quantify a relatively pure material due to the extrem ely large dilution factor, in this case >100,000, that must be applied. It is not expected that instrumental variation would bias the purity high. It is possible that the purity o f the sample is actually higher than the purity of the PFO SA that we are using as a standard! If this were true, the purity would be determined to be > 100%. Andrew Seacat has reviewed the concentrations of the impurities that were determ ined and calculated that the concentration of PFO S will not adversely affect the study. At 9,600 ppm, he calculates that the rats receiving 3 m g/kg/day would ingest app. 0.20 mg PFO S total in 000161 9 T-7132.2; ST-39 PFOSAFK Appendix I Continued: 28 days assuming a 0.25 kg rat. if we further assume that 30 % of that is deposited in the liver (based on previous feeding studies) and liver is approximately 12 gram s, then the predicted concentration in the liver from the residual PFO S would be 200 ug P FO S x 0.3 /1 2 g liver weight = 5 ug/gram , or 5 ppm, which in itself should not impose any toxicological consequences. Any PFO S measured significantly over that could be attributed to metabolism o f PFO SA to PFO S. H e believes that the 1-H and 2-H perfluoro am ides are interesting as they m ay be subject to metabolic attack, however these am ides should be analytically distinct from the Perfluorinated species on down the line, and would not amount to a foreseeable toxic concentration and therefore would be acceptable if no other substitute can be prepared. Andrew is still thinking about whether the (CgF<|ySO ^N H will have a negative im pact on the study. Please give m e a call at 6-0665 if you have any questions concerning our analyses or if I can provide additional information. Fred L. DeRoos CATC 000162 10