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T-7211.1 FR ST-37 Title Adipate (T-7211) Metabolite Identification in Rats Final Report Date: 2-24-04 3M Medical Department Study Number: T-7211.1 3M Strategic Toxicology Laboratory Protocol Study No. ST-37 3M Environmental Laboratory Report No.: FACT Tox-141 Sponsor: 3M Specialty Chemicals Division (PCDP) Saint Paul, MN 55133-3220 Sponsors Representative: Bill Reagen 3M Environmental Lab2-3E-09 Saint Paul MN Request #W-1507-1 Study Location: 3M Strategic Toxicology Laboratory 3M Center, Building 270-SB-314 Saint Paul, MN 55133-3220 Study Director: Andrew M. Seacat Ph.D. Toxicology Specialist 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 Deanna Nabbefeld, MS Toxicologist: 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 C00004 T-7211.1 FR ST-37 Summary The objective was to provide liver and sera from rats dosed with the Adipate for method development and qualitative metabolite identification. A single five mg/kg dose was administered via oral gavage to two male and two female Sprague Dawley rats. One male and one female rat were euthanized on day 1 and day 4 post dose. The fluorochemicals quantified in liver were perfluorooctanesulfonate (PFOS), perfluorooctanesulfonamide (FOSA), perfluorooctanesulfonamido acetate (M556), N-methyl perfluorooctanesulfonamido acetate (M570), and MeFOSE-Epi-1-alcohol. The apparent cumulative amount of these monomers present in the liver exceeded the cumulative amount of these residual monomers present in the dose in both the liver and in the serum. For example, the percent of residuals that were present in the dose that were present in the liver ranged from 128% on day one post-dose to 825% on day 4 post dose in males. Thus these data suggest that the Adipate polymer was apparently metabolized to some extent by the rat following a single oral dose. However, this study was designed to provide qualitative data only, therefore, a definitive conclusion cannot be made based on these data. Test Material: Name: The "Adipate". (Lot Number: LB-1000-1546-8; L15468, 118103-594 ("Adipate"); R-l 1483-9, Lot 3, Stripped. Originator: M. Burleigh , 2/16/98. 3M Environmental Lab: TN-A-2432. 3M Medical Department: T-7211). Structure: The adipate is synthesized by the addition of 2 moles epichlorohydrin (epi) to N-MeFOSE and subsequent esterification with adipic acid. The ideal structure of the product should be: [C8F17S02N-(CH3)-CH2-CH2-0-(CH2-CH-(CH2Cl)-0)2-C0CH2CH2-]2 However other major N-MeFOSE-epi-adipate products exist consisting of at least three primary ester types. These structures are elucidated in an NMR Analysis of the Adipate report completed by Richard A. Newmark, 3M Corporate Analytical Tech Center (Request C147746, 4/5/99). Purity: The overall purity of the Adipate as identified by NMR (Newmark R. M. Request C l47746), and was reported to be: Table 1: NMR analysis of the Adipate, L-15468, Lot 3, Stripped. Component mole % wt% All N-MeFOSE-epi-adipate products MIBK Toluene Probable triethyl ammonium salt 89.5% 9.6% 0.050% 0.8% 95.1% 4.4% 0.024% 0.5% Analytical characterization of this same lot of the FC-Adipate for residual FC alcohols was conducted by HPLC Analysis by Joel W. Miller of the 3M SMMD 2 coooo^ T-7211.1 FR ST-37 Analytical (Request: 58175,4/ 22/1999). These levels are based on the weight percent analyte in the sample as received (Table 2).. Table II. Residual FC alcohol levels in the Adipate as determined by HPLC Sample Prep 1 2 3 AverageSD Wt % N-MeFOSE 0.0445% 0.0446% 0.0438% 0.0443 0.0004% Wt % NMeFOSE-EPI 0.269% 0.267% 0.273% 0.269% 0.003% As described in the method, the N-MeFOSE-EPI level is the total concentration of all the N-MeFOSE-EPI oligomers in the sample. Further analysis was performed by LC/MS revealed that there was 0.0029% perfluorooctanesulfonate (PFOS) in this sample of Adipate (SMMD Analytical Report 58175, In FACT Tox 141). Procedures: Dose and Dosing Procedures: A single dose of adipate, five mg/kg body weight, was administered via oral gavage to two male and two female Sprague Dawley rats from Harlan Laboratories, Inc., 6-8 weeks old, weighing approximately 310 - 480 g. The dose was be prepared by forming a 5 mg/ml solution of the Adipate in acetone. The adipate/acetone solution was then be suspended in com oil to a final concentration of 1 mg Adipate per ml of 20% acetone/80% com oil. A volume of 5 ml/kg of the suspension was be administered to all rats. One male and one female rat were euthanized on day 1 and day 4 post dose according to the schedule in Table 3. Table 3 Schedule Group 1 2 Dose 5 mg/kg Adipate 5 mg/kg Adipate N 2 (1M, IF) 2 (1M, IF) Sacrifice Day Day 1 post dose Day 4 post dose Sample Collection: During necropsy, systemic blood (approximately 6 ml) was collected via heart puncture and transferred to blood collection tubes without anticoagulant. All blood samples were allowed to clot for a period of 15 to 30 minutes at room temperature, then centrifuged at 1100 x g for 5 minutes. The serum was transferred to labeled 1.5 ml microfuge tubes and centrifuged again at 2000 x g to remove any remaining red blood cells. The serum was transferred to labeled polypropylene microfuge tubes and frozen on dry ice. Livers were removed, C 00006 T-7211.1 FR ST-37 placed separately into labeled tubes and frozen on dry ice. Samples (liver and serum) were stored in a freezer set to maintain -60 to -80C then packed in dry ice and shipped to: Kris Hansen, 3M Environmental Technology and Safety Services \ Analysis: Liver and sera samples were analyzed by the 3M Environmental for the parent compound, methyl FOSE and metabolites all N-MeFOSE-epi-adipate products. An aliquot of the dosing solution of 1 mg/ml adipate suspended in acetone/com oil (20:80) is included for analytical verification of dosing material (Amended report FACT Tox-141). Results and Discussion Body weight, liver weight and dose amount are shown in Table 4. No effects of treatment were noted during the in-life phase of the study or by gross observation of internal organs at necropsy. The residual fractions (Wt/Wt) and amount (pg) of PFOS, N-MeFOSE, N-MeFOSE Epi-1 Alcohol and the Total residuals in dose are shown in Table 5. The analytical results for fluorochemical concentrations in the liver are shown in Table 6, and the fluorochemical concentrations in the serum are shown in Table 7. The fluorochemicals quantified in liver were perfluorooctanesulfonate (PFOS), perfluorooctanesulfonamide (FOSA), perfluorooctanesulfonamido acetate (CgFi7S02N(H)CH2CH2OH; M556), Nmethyl perfluorooctanesulfonamido acetate (CgFi7S02N(CH3)CH2CH2OH; M570), and MeFOSE-Epi-1-alcohol (CgFi7S02N(CH3)CH2CH2O(CH2C(H)(CH2Cl)O)H; Epi-l-OH). The cumulative amount of these monomers present in the liver exceeded the cumulative amount of these residual monomers present in the dose in both the liver and in the serum. For example, the percent of residuals that were present in the dose that were present in the liver ranged from 128% to 825%. These analyses support the conclusion stated in the 3M Environmental Lab report ( FACT TOX 141) that " the levels of (MeFOSE-OH metabolites) detected (in the liver) greatly exceeded theoretical levels predicted based on MeFOSE-OH residuals in the dose material" . However, one should use caution not to over interpret the quantitative aspect of this study as it was not designed as a definitive study to quantify the metabolites of Adipate. Thus, these data suggest that the Adipate polymer was apparently metabolized to some extent by the rat following a single oral dose. However, a definitive conclusion can not be made based on these data alone. 4 T-7211,1 FR ST-37 Signatures: Andrew M. Seacat Toxicology Specialist Studv Director fV l> ____ '2- /*z i / / o y Date C0Q008 T-7211.1 FR ST-37 References Altman PL and Dittmer DS, Blood and Other Body Fluids. Bethesda, Maryland. Federation of the American Societies for experimental Biology (FASEB), 1971, p5 Hansen K. Amended Analytical Lab Report. Data for metabolite identification o f L-15468 Adipate (Reference number 118103-59A) in rat liver and sera samples - A pilot study. FACT TOX-141, LRNW2430, 12/20/00 6C 0 0 0 0 9 T-7211.1 FR ST-37 Tables Table 4 Biological Parameters ST-37; T-7211.1 Adipate Pilot Study in Rats - BW & Liver Wt Data Dose animal # sex necropsy Day 0 Adipate terminal serum liver wt 8R03- XXX date Body Dose w t(g) (mg) Body wt volume/rat (g) (9) (ml)1 5mg/kg adipate 823 male dy 1 post dose 478 2.39 453 14.6 15.8 5mg/kg adipate 872 female dy 1 post dose 315 1.57 313 10.1 8.7 5mg/kg adipate 5mg/kg adipate 824 male 871 female dy 4 post dose dy 4 post dose 463 2.31 449 14.5 17.6 311 1.55 314 10.1 10.4 1. There are 32.3 mL Plasma per Kg of rat.(Alman and Dittmer, Blood and 0 ther Body Fluids. FASEB, 1971, p5) 7 c o o o io T-7211.1 FR ST-37 Table 5 Fluorochemicals in the Dose ST-37; T-7211.1 Adipate Dose data Dose anim al # Adipate 8R03-XX* Dose (mg) Residual fraction P F O S in dose (wt/wt) fraction of Fraction Total Amount of residual N- residual residual N- MeFOSE Fraction in (MeFOSE MeFOSE Epi-1 dose + Epi-1 - In dose Alcohol (wt/wt) OH) in (wt/wt)<1> residual in dose (ug) dose (wt/wt) 5mg/kg adipate 823 2.39 0.00003 0.00044 0.00269 0.00316 7.56 5mg/kg adipate 872 1.575 0.00003 0.00044 0.00269 0.00316 4.98 5mg/kg adipate 824 2.315 0.00003 0.00044 0.00269 0.00316 7.32 5mg/kg adipate 871 1.555 0.00003 0.00044 0.00269 0.00316 4.92 1) from Miller J. W. SMMD Analytical report Request # 5'75 , April 22, 1999 HPLC-UV analysis of FC- Adipate for residual FC alcohols coon T-7211.1 FR ST-37 Table 6: Liver Fluorochemical Concentration Amount and Percent of Dose. ST-37; T-7211.1 Adipate Pilot Study in Rats - BW & Liver Wt Data D ose o f a n im al Sex Necropsy PFOS PFOSA M556 M570 Epi-1- A d ip ate # day in liver in liver in liver in liver OH in (m g/K g) 8R03- XXX S pm> !i?pm) r m > liver jppm) Total Total Total Total Total P F O S P F O S A M556 M570 Epi-1in liver in liver in liver in liver OH in (ug) (ug) (ug) (ug) liver (ug) Total Percent residual of in liver residua (ug) I dosed in liver (2) 5 823 M d1 0.19 0.11 0.00 0.31 0.00 3.02 1.75 0.00 4.90 0.00 9.67 128% 5 872 F d1 0.27 0.17 0.26 0.64 0.00 2.37 1.49 2.23 5.55 0.00 11.63 234% 5 824 M d4 1.55 0.26 0.36 1.27 0.00 27.28 4.51 6.28 22.35 0.00 60.42 825% 5 871 F d4 0.80 0.25 0.30 0.84 0.00 8.33 2.61 3.14 8.77 0.00 22.85 465% 1.) Analytical Laboratory Report on Data for Metabolite Identification of L-15468 Adipate. (3M Medical Department Number: T-7211.1; 3M Environmental Lab Number: FA C TTO X 141).________________________________________________________________________________________________ 2.)Percent of residual dosed in liver = (Total PFOS residual in liver (ug))/(Amount of residual (MeFOSE + Epi-1-OH) in dose (ug))*100 TOOOD 9 T-7211.1 FR ST-37 Table 6: Serum Fluorochemical Concentration Amount and Percent of Dose. ST-37; T-7211.1 Adi pate Pilot Study - Serum data D ose anim al sex PFOS PFOSA M556 in M570 in Epi-1- Total Total Total Total Total Total Percent % of Adipate # 8R03- in in serum serum OH in PFOS PFOSA M556 in M570 in Epi-1- metabolit of total (mg/Kg) X X X serum serum (ppm) (ppm) serum in in serum serum OH in e in residual dose in (ppm) (ppm) (1) (1) (ppm) serum serum (ug)(2) (ug)(2) serum serum dosed serum (1) (1) (1) (ug)(2) (ug)(2) (ug)(2) (ug) (3) in (5) serum (4) 5 823 M 0.011 0.006 0.056 0.221 0.018 0.16 0.09 0.83 3.23 0.26 4.58 61% 0.19 5 872 F 0.105 0.021 0.214 0.574 0.040 1.06 0.22 2.16 5.80 0.40 9.65 194% 0.61 5 824 M 5 871 F 0.093 0.010 0.073 0.560 0.010 1.34 0.14 1.06 8.12 0.15 0.393 0.026 0.251 0.665 0.000 3.99 0.27 2.55 6.74 0.00 10.81 148% 13.54 275% 0.47 0.87 Note: There are 32.3 mL Plasma per Kg of rat.(Alman and Dittmer, Blood and Other Body Fluids. FASEB, 1971, p5) 1.) Analytical Laboratory Report on Data for Metabolite Identification of L-15468 Adipate. (3M Medical Department Number: T-7211.1; 3M Environmental Lab Number: FACT TOX 141). 2) PFOS in serm (ug) = Serum volume (ml) x{PFO S in serum (ppm) 3.) Total residual in serum (ug) = sum of residuals serum (ppm) x serum volume (ml) 4) Fraction of residual dosed in serum = (Total PFOS residual in serum (ug)) x (Amount of residual (MeFOSE + Epi-1-OH) in dose (ug)) 5)% of total dose in serum = (Total PFOS residual in serum (ug)/ residual in dose (ug))*100 000013 10