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FINAL REPORT /t 3 2 -o ? < ? 3 Study Title: Toxicokinetic Study of Perfluorooctane Sulfonamide (PFOSA; T-7132.2) in Rats Authors: Andrew M. Seacat Ph.D. Toxicology Specialist 3M Medical Department, Corporate Toxicology Deanna J. Luebker M.S. Advanced Toxicologist 3M Medical Department, Corporate Toxicology Date: August 11, 2000 Testing Facility 3M Strategic Toxicology Laboratory 3M Center 270-3S-05 Saint Paul, MN 55144 -u r-j CO CO H 3M Strategic Toxicology ST39 T -7132.2 PFOSA PK. study in rats Summary: Purpose: The purpose of this study was to assess the relative absorption, metabolism, and elimination kinetics of Perfluorooctane Sulfonamide (PFOSA) in the rat following a single oral dose. Methods: Two groups of fifteen male rats each received a single dose of either 2% Tween 80, as the vehicle control, or 5 mg/kg PFOSA suspended in 2% Tween 80 by oral gavage. Perfluorooctanoate (POAA) was found at a level of 2.5 ng/'ml in the dosing solution. The PFOSA was found to be 96 % pure Five rats from each dose group were sacrificed on days l, 4, and 29 post dose. Liver and sera were analyzed for PFOSA and its predicted metabolites using highpressure liquid chromatography/electrospray tandum mass spectrometry (HPLC/'ESMSMS). Results: The vehicle control group had no detectable levels of PFOSA or perfluorooctanesulfonate (PFOS) in liver or sera. The PFOSA dose group had decreasing levels of PFOSA, with evidence of conversion of PFOSA to PFOS in liver and sera throughout the study. Liver concentrations of PFOSA averaged 7.0 ppm, 3.7 ppm, and 0.1 ppm on days 1,4, and 29 post-dose, respectively, with an apparent liver half-life of elimination of 5.2 days. Serum concentrations of PFOSA averaged 0.3 ppm and 0.1 ppm on days 1 and 4 post dose respectively, and were undetectable by day 29 post-dose, with an apparent serum half-life of elimination estimated to be less than 4 days. Liver PFOS concentrations were 28.2 ppm, 37.4 ppm and 23.8 ppm, representing 22.0%, 32.3%, and 25.2% of the PFOSA dosed, on days 1,4 and 29 post-dose respectively. Sera PFOS averaged 8.2 ppm, 8.6 ppm, and 4.6 ppm on days 1, 4 and day 29 post dose respectively. The average total concentrations of PFOS equivalents (PFOS - PFOSA) in liver were 35.2, 41.1, and 23.9 ppm on days 1, 4. and 29 post-dose, respectively. The average concentrations of PFOS equivalents in the sera increased from 8.5 ppm on day 1 post dose to 8.7 ppm on day 4 post dose and decreased to 4.6 ppm on day 29 post dose. The liver to sera PFOS concentration ratio increased throughout the study with values of 4.2. 4.8, and 5.4 on days 1, 4, and 29 post dose, respectively. Total PFOS equivalents in the liver and sera peaked on day 4 post dose at 35.4% and 6.0 % of the PFOS equivalents dosed, respectively. An average of 0.1 ppm POAA was found in the sera on day 1 and day 4 post dose, and equaled 31.2% and 24.1%of the POAA residual present in the dose, respectively. Conclusions: Perfluorooctanesulfonamide was readily absorbed from the gut and found in the liver and sera following an oral exposure. PFOSA was progressively metabolized to PFOS, which accumulated in the liver as evidenced by the high concentration of PFOS detected in the liver, representing 22.0% 32.3% and 25.2% of the PFOSA dosed, on days 1,4 and 29 post dose respectively. The apparent half-live of elimination of PFOSA in the liver was approximately 5.2 days. The POAA present in the sera on days 1 and 4 post-dose could be accounted for by the residual POAA in the dosing solution, therefore, no direct evidence for metabolism of PFOSA to POAA was found in this study. Page 2 of 18 3M Strategic Toxicology ST39 T-7132.2 PFOSA PK study in rats Introduction The objective of this study was to assess the potential for oral absorption, urinary and fecal clearance, and biological persistence of Perfluorooctanesulfonamide (PFOSA) in male Sprague Dawley rats after a single oral dose. Analysis of the serum and liver for PFOSA and potential metabolites of PFOSA was performed by LCMS. Urine and feces were also collected and may be analyzed by LCMS and perhaps other methods as deemed n ec e s s a ry . Previous studies onN-ethylperfluoroctanesulfonamide (1), theN-ethyl derivative of PFOSA, mistakenly concluded that PFOSA was the ultimate metabolite in rats (1,2); however, the gas chromatography analytical technique used in those studies was unable to detect PFOS. Dietary administration ofN-ethylperfluoroctanesulfonamidoethanol (N-EtFOSE) in rats, and in-vi(ro metabolism by hepatocytes results primarily in the formation of PFOS with a minor amount of PFOSA (3, 4, and 5). Furthermore, PFOSA has been identified as a potent direct uncoupler of mitochondrial respiration in-vuro (6). Prior to the cun-ent study, direct metabolism of PFOSA to PFOS had not been demonstrated and the relative toxicokinetics of PFOSA were unknown. Methods were recently validated for the quantitation of PFOSA and its potential metabolite, PFOS, in serum and liver down to the low part per billion level (7). In-vivo studies were, thus, warranted to investigate the toxicokinetics of PFOSA. Materials The test material was identified as Perfluorooctanesulfonamide (PFOSA); Lot number L-10009. Initial analysis by GCMS determined that tne-staring material was over 99% pure (8). Qualitative and quantitative compositional results derived from lH and 19F-NMR analysis revealed that the isomer distribution was approximately 65.8% CF3 (CF2) X-S02-NH2 (Normal chain), 18.7% internal monomethyl branch and 11.2% Isopropyl branch (9). HPLC/MS characterization revealed low-level impurities of 9,600 ppm PFOS (0.96 %), and lower concentrations of several other amides (10). Perfluorooctanoate (POAA) was found in the dosing solution at a level of 2.5 jig/ml (0.25% of the nominal dose) (12). Based on the sum of the impurities, the purity of the PFOSA sample was determined to be approximately 96 % (8, 9 & 10). Methods The protocol (11) and analytical methods (12) used in this study are briefly described below. Dose and Dosing Procedures: A single 5mg/kg dose of PFOSA was administered via oral gavage to 15 male Sprague Dawley rats (obtained from Harlan Laboratories) on day zero of the study. The PFOSA was prepared as a 1% (1 mg/ml) uniform suspension in 2% Tween 80. A volume of 5 ml suspension / kg body weight was administered to each rat. The vehicle control group, consisting of 15 male Sprague Dawley rats (also obtained from Harlan Laboratories), received 2% Tween 80 in deionized water at a volume of 5 ml/kg on day zero of the study. Page 3 of 18 u 3M Strategic Toxicology ST39 T -7132.2 PFOSA PK study in rats Specimen Collection: Urine and feces collections were made on days 1 - 4 post dose from the animals designated for sacrifice on day 29 post dose group. Five animals from each dose group were euthanized by CO2 on days 1, 4, and 29 post dose and gross necropsy was performed. Sera and liver were collected from each animal and flash-frozen in liquid nitrogen. Specimen Handling: Specimens (urine, feces, liver, and serum) were maintained at -70C and sent to Kris Hansen, Ph.D., 3M Environmental Technology and Safety Services, for analysis. Liver and sera samples were analyzed by 3M Environmental for the parent compound and metabolites. The limits of detection (LODs) for PFOSA were 3.5 ng/ml in the liver and 1.51 ng/ml in the sera. The LODs for PFOS were 8.45 ng/ml in liver and 1.74 ng/ml in the sera (12). Urine and feces were retained for possible future analysis as appropriate. Results Control Groups: Bodv and Liver Weight The average initial body weight SD of the fifteen vehicle control male rats was 273.7 7.3 g. These rats lost an average of 2.2 g body weight from day zero to day 1 post dose . From day zero to day 4 post' dose, the average body weight gain was 24.2 g and from day zero to day 29 post dose, body weight increased an average of 114.1 g . Liver weights and relative liver to body weight percentages are shown in Table 1A. Liver and Sera Concentrations Control animals had no detectable levels of PFOSA or PFOS in liver or sera at any time during this study (data not shown). PFOSA Dose Groups: Bodv and Liver Weight The average initial body weight SD of the fifteen PFOSA dosed male rats was 270.0 3.6 g. These rats lost an average of 4.5 g body weight from day zero to day 1 post dose . From day zero to day 4 post dose, the average body weight gain was 20.0 g and from day zero to day 29 post dose, body weight increased an average of 100.9 g . Liver weights and relative liver to body weight percentages were equivalent to control values (Table IB). Liver and Sera Concentrations PFOSA and PFOS were detected in the liver and sera of all animals dosed with PFOSA. Liver and sera levels of PFOSA were highest on day 1 post dose and dropped significantly between Page 4 of 18 7 3M Strategic Toxicology ST39 T -7132.2 PFOSA PK srudy in rats days 1 and 4 post dose and again between days 4 and 29 post dose (Tables 2 and 3). The average liver concentrations of PFOSA decreased from approximately 7.0 ppm to 3.7 ppm to 0.1 ppm from days 1 to 4, to 29 post dose, respectively (Table 2). The half-life of elimination of PFOSA in the liver was calculated from a log linear regression of the liver PFOSA concentrations to be 5.2 days. The sera concentrations of PFOSA were 0.3 0.1 ppm, 0.1 0.0 and <LOD (1.74 ng/ml) on days 1, 4, and 29 post dose, respectively (Table 3). The half-life of elimination of PFOSA in the serum cannot be calculated from this data, but is estimated to be less than 4 days. Average PFOS liver and sera levels peaked on day 4 post dose and declined by day 29 post dose to lower levels than found on day one. Average SD liver PFOS levels increased significantly from 28.2 6.1 ppm on day 1 post dose to 37.4 3.6 ppm on day 4 post dose. Levels then decreased significantly to 23.8 4.1 ppm by day 29 post dose (Table 2). Average sera PFOS values were approximately 8 ppm on day 1 and day 4 post dose, then decreased significantly to approximately 4.6 ppm on day 29 post dose (Table 3). Liver and Sera Concentrations as a Percentage of Dose The percentage of PFOSA dosed detected as PFOSA in the liver and sera peaked on day one post dose at 5.4% and 0.2 % respectively. By day 4 post dose, these levels had dropped to 3.2% and 0.0 % and by day 29 post dose the liver levels dropped to 0.1 % and the sera levels remained at 0.0% (Table 6). The percent PFOS equivalents dosed detected as PFOS in the liver and sera was 22.0% and 5.2 % on day 1 post dose and increased to 32.3% and 6.0% by day 4 post dose. On day 29 post dose, these levels had dropped to 25.2% and 4.1%, respectively (Table 6). Residual PFOS in the dose could account for 3.5%, 2.5%. and 5.6% of the PFOS found in sera, and 3.6%, 2.7%, and 3.9 % of the PFOS found in liver on days 1, 4, and 29 post dose, respectively (Tables 4 and 5). The percent PFOS equivalents dosed detected as PFOS equivalents in the liver and sera was 27.2 % and 5.3 % n day 1 post dose and increased to 35.4 % and 6.0 % by day 4 post dose. On day 29 post dose these levels decreased to 25.4 % and 4.1 % in the liver and sera respectively (Table 6). Sera POAA Concentrations Perfluorooctanoate (POAA) was found at a level of 2.5 ng/ml in the dosing solution (12). Sera samples in the PFOSA dose group contained POAA at average values of 0.1 and 0.8 ppm POAA on days 1 and 4 post-dose respectively. These values equal 30.8% and 23.5%, respectively, of the POAA dosed (Table 7). Conclusions: No compound related effects were observed on body weight, liver weight, or liver/body weight ratios under treatment conditions. Both PFOSA and PFOS were found at high concentrations in the liver and sera of rats one day following an ora! dose. Approximately 38% of the PFOSA dosed was converted to PFOS four Page 5 of 18 B 3M Strategic Toxicology ST39 T-7132.2 PFOSA PK study in rats days post-dose. PFOSA levels diminished in both sera and liver over time, with a liver half-life of approximately 5.2 days, and a much shorter residence period in the sera. Significantly more PFOS was found in the liver and sera at all time points than were present in the dose, therefore metabolic conversion of PFOSA to PFOS occurred. No direct evidence for metabolism of PFOSA to POAA was found in this study. The route of elimination of PFOSA in this study was not determined. No glucuronide or other conjugates were analyzed for, and further investigation of possible conjugates in the urine and feces is warranted. It is possible that some of the PFOSA was eliminated through the lung via expiration, but this has not been determined. The present results suggest that the PFOSA from the dose is progressively converted to PFOS and that the resulting PFOS is accumulated in the liver. Page 6 of 18 9 3M Strategic Toxicology 5T39 T-7132.2 PFOSA PKstudy in rats Report Prepared by: Signatures Deanna Luebker, MS^ Advanced Research Toxicologist ft] , Andrew M. Seacat, Ph.D. Toxicology Specialist Report Reviewed by: John L. Butenhoff, Ph.D., DABT, CIH Senior Laboratory Manager :Ot//(p (9-00! . Date O f / Up C l Date /& jy y o / Date Page 7 of 18 /0 3M Strategic Toxicology ST39 T -7132.2 PFOSA PK study in rats References: 1. Grossman M.R. and Bowen J.M. (1990) Tissue analysis of fluorinated sulfonamide pesticide: an evaluation of distribution, elimination, and potential for bioaccumulation in orally exposed rats. M.S. Thesis, U n iv . of Georgia, Athens, GA. (also possibly published as: Grossman Mark R. and Bov/en 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. Agric. Food Chem. 40, 2505 - 2509. 3. Mulvana D.E. and Henion J. 1996. Qualitative investigation of the in-vitro metabolism o f T6292, T-6293, T-6294 and T-6295 by rat and human hepatocytes using ion spray LC/MS and LC/MS/MS. Analytical Report: 96ADEM01.3M, Advanced Bioanalytical Services, Inc. 45pp. 4. Poon G.K., Lowes S. 1998. Additional Characterization of metabolites ofT-6292, T-6293 and T-6294 from rat and human hepatocytes by TurboIonSpray LC/MS and LC/MS/MS. Semi-quantitative analysis of T-6295 in rat and human hepatocytes incubated with T-6292, T-6293 and T-6294 by LC/MS/MS. Analytical Report: 96AGKP01.3M, Advanced Bioanalytical Services, Inc. 69pp. 5. Seacat A.M., Thomford P.J.. Hansen K.J. and Butenhoff J.L. Sub-Chronic Dietary N-Ethyl Perfluorooctanesulfonamido Ethanol Toxicity in Rats, (in preparation, 8/11/2000). 6. Wallace K.B. and Starkov A. 1998. The effect of periluorinated arylalkylsulfonamides on bioenergetics of rat liver mitochondria. Dept, of Biochemistry and Molecular Biology, University of MN School of Medicine. Duluth, MN 55812, USA. Supported by a grant from 3M Company 7. K.J. Hansen, L.A. Clemen, M.E. Ellefson, H.O. Johnson. (1999). Compound Specific Characterization of Organic Fluorochemicals in General Population Human Sera Samples. 3M Environmental Lab, St. Paul. MN 55133. 8. Pavfer R.M. GC/MS analysis of PFOSA (L-10009). SA&C Analytical Request No. 59426. Report 9/24/99. 3M SA&C Lab Building 236-2B-11. 9. Tom Kestner. Chemical Characterization of PFOSA, 1-10009, by 1H and 19F-NMR Spectroscopy Requests # 59426. 3M Specialty Adhesives & Chemicals Analytical Laboratory /SMMD-236-2B-11, September 25, 1999. 10. DeRoos F.L. Characterization of PFOSA Samples, T-7132-1 (L-10009) and TN-A-1584. Request# A-151254. Report 10/7/99. Corporate Analytical Technology Center, Building 201-1-29, CATC - Chromatography Group. 'Page 8 of 18 II cn 3M Strategic Toxicology ST39 T-7132.2 PFOSA PK study in rats 11. Seacat & Luebker. Protocol for Study No. T-7132.2; ST-39. PHARMACOKINETIC STUDY OF PFOSA IN RATS. 3M Medical Department, Corporate Toxicology. October 1999. 12. Laboratory Report: Analytical Report of Data for PFOSA (T-7132.2) Pharmacokinetic Study in Rats (Sera and Liver). Laboratory Report No. FACT-TOX-145 (W2814). Testing Laboratory 3M Environmental Technology & Safety Services, 3M Environmental Laboratory, Fluorine Analytical Chemistry Team. Laboratory Contact: Kris Hansen, Ph.D. Alman and Dittmer, Blood and Other Body Fluids. FASEB, 1971, p5 Page 9 of 18 3M Strategic Toxicology ST39 T -7132.2 PFOSA PK study in rats List of Tables: Table 1: Biological Parameters 1A: Control Group IB: PFOSA Dose Group. Table 2: Liver PFOSA and metabolites in the PFOSA Group. Table 3: Serum PFOSA and metabolites in the PFOSA Group Table 4: Percent Dose: Liver PFOS equivalents in PFOSA Group. Table 5: Percent Dose: Serum PFOS equivalents in PFOSA Group. Table 6: Summary: Percent of dose in sera and liver. Table 7: Serum POAA in the PFOSA Group. /3 Page 10 of 18 3M Strategic Toxicology ST39 T -7132.2 PFOSA PK study in rats Table 1A: Biological Parameters. Control Group Time Dose Day 1 C o n tro l Day 1 C o n tro l Day 1 C o n tro l Day 1 C o n tro l Day 1 C o n tro l Avg SD Day 4 C o n tro l Day 4 C o n tro l Day 4 Day 4 Day 4 C o n tro l C o n tro l C o n tro l Avg SD Day 29 C o n tro l Day 29 C ontrol Day 29 C o ntrol Day 29 C ontrol Day 29 C o ntrol Avg SD animal # Initial Terminal BW Gain Liver Liver wt as 9R03-xxx BW (g) BW (g) 230 282.5 262.0 231 272.1 268.4 (9) wt (g) % of BW -0.5 12.0 4.3% -3.7 11.2 4.2% 232 277.4 277.1 -0.3 12,4 4.5% 233 274.1 274.8 0.7 11.5 4.2% 234 267.6 260.5 -7.1 11.2 4.3% 274.7 272,6 -2.2 11.7 4.3% 5.6 235 266.9 8.3 294.5 3.2 27~g 0.5 11.6 0.1% 3.9% 236 275.8 301.8 26.0 11.4 3.8% 237 261.9 283.4 21.5 11.9 4.2% 238 279.0 302.7 23.7 11.3 3.7% 239 268.3 291.2 22 9 10.7 3.7% 270.4 294.7 24.3 11.4 3.9% 6.9 8.0 2.4 0.4 0.2% 240 273.5 371.8 98.3 13.9 3.7% 241 270.5 380.5 110.0 14.4 3.8% 242 280.2 389.4 109.2 14.2 3.6% 243 289.9 444.0 154.1 17.5 3.9% 244 266.3 365.2 96.9 14.3 3.9% 276.1 390.2 114.1 14.9 3.8% 9.2 31.4 23.0 1.5 0.1% Page ll of 18 3M Strategic Toxicology ST39 T -7 132.2 PFOSA PK study in rats Table 1B: Biological Parameters. PFOSA Dose Group Time Dose animal # Initial Terminal BW Gain Liver 9R03-xxx BW(g) BW|g) (9) wt (g) Day 1 i5 mq/kq 245 263.2 257.8 -5.4 10.4 Day 1 5 mq/kq 246 266.1 259.2 -6.9 10.2 Day 1 5 mq/kq 247 266.5 264.1 -2.4 10.9 Day 1 5 mq/kq 248 267.2 264.0 -3.2 10.1 Day 1 5 mq/kq 249 268.7 264.3 -4.4 10.7 Avg | 266.3 261.9 -4.5 10.5 SD I 2.0 3.1 1.8 0.3 Day 4 5 mq/kq 250 266.6 286.3 19.7 11.3 Day 4 5 mq/kq 251 272.1 294.6 22.5 11.7 Day 4 5 mq/kq 252 271.7 294.5 22.8 12.1 Day 4 5 mq/kq 253 271.8 291.4 19.6 11.3 Day 4 5 mq/kq 254 272.9 288.4 15.5 11.7 Avg 271.0 291.0 20.0 11.6 SD 2.5 3,7 2.9 0.3 Day 29 5 mq/kq 255 275.2 385.4 110.2 16.7 Day 29 5 mq/kq 256 273.6 380.3 106.7 14.4 Day 29 5 mq/kq 257 271.4 373.9 102.5 13.9 Day 29 5 mq/kq 258 274.5 371.4 96.9, 14.6 Day 29 5 mg/kg 259 268.7 356.7 88.0 12.7 Avg 272.7 373.5 100.9 14.5 |SD 2.6 10.9| 8.7 1.5 Liver wt as % of BW 4.0% 3.9% 4.1% 3.8% 4.0% 4.0% 0.1% 3.9% 4.0% . 4.1% 3.9% 4.1% 4.0% 0.1% 4.3% 3.8% 3.7% 3.9% 3.6% 3.9% 0.3% Page 12 of 18 3M Strategic Toxicology ST39 T -7132.2 PFOSA PK. study in rats Table 2: Liver PFOSA and metabolites in tbe PFOSA dose Group animal # 9R03- Liver (PFOS] Liver [PFOSA] Liver PFOSA PFOS Time Dose XXX ppm ppm eq ppm1 Day 1 5 mg/kg 245 23.7 6.02 6.02 Day 1 5 mg/kg 246 32.3 7.02 7.02 Day 1 5 mg/kg 247 36.2 6.24 6.24 Day 1 5 mg/kg 248 21.2 6.66 6.66 Day 1 5 mg/kg 249 27.8 8.81 8.81 Avg 2 8.2 6.95 6.95 SD 6.1 1.11 1.11 Range Min 21.2 6.02 6.02 Max 36.2 8.81 8.81 n 55 5 5 Day 4 5 mg/kg 250 36.8 3.52 3.52 Day 4 5 mg/kg 251 32.6 3.47 3.47 Day 4 5 mg/kg 252 42.6 3.96 3.96 Day 4 5 mg/kg 253 36.9 3.45 3.45 Day a 5 mg/kg Avg SD 254 38.3 37.4 * 3.6 4.11 3 .70 * 0.31 4.11 3.70 0.3 1 Range Min 32.6 3.45 3.45 Max 42.6 4.11 4.11 n 55 5 5 Day 29 5 mg/kg 255 20.3 0.09 0.09 Day 29 5 mg/kg 256 20.6 0.02 0.02 Day 29 5 mg/kg 257 22.0 0.10 0.10 Day 29 5 mg/kg 258 26.2 0.14 0.14 Day 29 5 mg/kg Avg 259 29.9 23.8 ** 0.30 0.13 * f m# 0.30 0.13 SD 4.1 0 .1 1 0.11 Range Min 20.3 0.02 0.02 Max 29.9 0.30 0.30 n 55 5 5 1 Assuming a 1:1 ratio of PFOSA:PFOS equivalents; Liver PFOSA PFOS equivalents =Liver PFOSa * = significantly different from day 1 values by a two tailed T-test (p<0.05) '*= significantly different from day 4 values by a two tailed T-test (p<0.05) PQL = practical quantitation limit in liver (PFOS = 30 ng/g; PFOSA 10ng) MDL = method detection limit mliver: (PFOS = 15 ng/g, PFOSA = 5 ng/g) Page 13 of 18 3M Strategic Toxicology ST39 T -7132.2 PFOSA PK study in rats Table 3: Serum PFOSA and m eta b o lites in the PFOSA Group . Time Dose animal 9R03- Sera [PFOS] XXX ppm Sera [PFOSA] ppm Sera PFOSA PFOS eq ppm1 bay 1 5 mg/kg 6.9 JOT o Day 1 Day 1 Day 1 5 mg/kg 5 mg/kg 5 mg/kg 246 10.8 247 9.4 248 6.7 0.44 0.44 0.26 5 1 6 0.33 0.33 Day 1 5 mg/kg 249 7.3 0.28 0.28 Avg 8.2 0.31 0.31 SD 1.8 0.08 0.08 Range Min Max 6.7 10.8 0.26 0.26 0.44 0.44 n 5 5 55 Day 4 5 mg/kg 250 7.8 0.06 0.06 Day 4 5 mg/kg 251 7.5 0.06 0.06 Day 4 5 mg/kg 252 11.3 0.08 0.08 Day 4 5 mg/kg 253 8.6 0.06 0.06 Day 4 5 mg/kg Avg 254 7.9 8.6 0.06 0.06 V 0.06 0.06 Day 29 Day 29 SD Range n 5 mg/kg 5 mg/kg Min Max 5 255 256 1.6 7.5 11J 5 3.6 4.0 0.01 0.06 CTOS' 5 <LOD <LOD 0.01 0.06 -------------m 5 Day 29 5 mg/kg Day 29 5 mg/kg 257 4.0 258 4.1 <LOD -dLOD Day 29 5 mg/kg 259 7.4 0.0 Avg 4.6 * / ** 0.0 SD Range Min 1.5 3.S9 Max 7.36 n5 55 1. Assuming a 1:1 ratio of PFOSA :PFOS equivalents, Sera PFOSA PFOS equivalents =Sera PFOSA concentration *= significantly different from day 1 values by a two tailed T-test (p<0.05) " = significantly different from day 4 values by a two tailed T-test (p<0.Q5) MDL = method detection limit (PFOS = 4.75 ng/ml; PFOSA = 2.89 ng/ml) LOG = limit of detection (PFOS = 1.74 nq/ml: PFOSA = 1 51 nq/ml) Page 14 of 18 17 TM Strategic Toxicology ST39 T-7132.2 PFOSA PK simly in rals Table 4: Percent Dose Liver PFOS equivalents in PFOSA Group Time Dose Day 1 Day 1 Day 1 Day 1 Day 1 5 mg/kg 5 rng/kg 5 mg/kg 5 mg/kg 5 mg/kg Avg SD Range Day 4 Day 4 Day 4 Day 4 Day 4 n 5 rnglkg 5 rnglkg 5 mg/kg 5 mg/kg 5 mg/kg Avg SD Range Day 29 Day 29 Day 29 Day 29 Day 29 n 5 mg/kg 5 mg/kg 5 mg/kg 5 mgiky 5 mg/kg Avg SD Range n animal ff 9R03-XX* 245 246 247 240 249 Min Max 5 250 251 252 253 254 Min Max 5 255 256 257 258 259 Min Max PFOS eq Total PFOS eg in concentration in whole liver (ug)2 liver (p p m )1 29.7 309.1 39.3 401.1 42.4 462.6 27.9 281.4 36.6 391.7 35.2 369.2 6.2 73.4 27.9 281 4 42.4 462.6 55 40.3 455.6 36.1 422.0 46.6 563 4 40.4 456 0 42.4 41.1 496.2 478.6 3.0 54.2 36 1 422.0 46.6 563.4 5.0 5.0 20.4 340.4 20.6 296.9 22.1 307.2 26.3 304.6 30.2 23.9 44 383.6 342.5 * 4.2 41.2 20 4 296.9 30.2 384.6 55 PFOS eq in dose (ug) 3 % PFOS eq Residual % of PFOS in liver dosed found in PFOS in dose from residual liver 4 (ug)s PFOS in dose {%)* 1316.0 23.5 12.6 4.09% 1330.5 30.1 12.8 3.18% 1332.5 34.7 12.8 2.77% 1336.0 21 1 12.8 4 56% 1343 5 29.2 12.9 3.29% 1331.7 27.7 12.8 3.58% 10.1 5 5 0.1 0.73% 1316.0 21 1 12.6 2.77% 1343.5 34.7 12.9 4.56% 55 5 5 1333.0 34 2 12.8 2.81% 1360.5 31.0 13.1 3.09% 1358.5 41.5 13.0 2.31% 1359.0 33.6 13.0 2.86% 1364.5 1355.1 36 4 35.3 4 13.1 13.0 2.64% 2.74% 12.6 3.9 0.1 0.29% 1333.0 31.0 12.8 2.31% 1364.5 41.5 13.1 3.09% 5.0 5.0 5 5 1376.0 24.7 13.2 3.88% 1368.0 21.7 13.1 4 42% 1357.0 22.6 13.0 4.24% 1372.5 28.0 13 2 3.43% 1343.5 1363.4 28.6 25.1 *-* 12 9 13.1 3.36% 3.87% 13.2 3.1 0.1 0.47% 1343.5 21 7 12.9 3.36% 1376.0 28.6 13.2 4.42% 55 5 5 1. PFOS equivalents in liver (ppm) = Liver PFOS (ppm) + Liver PFOSA PFOS equivalents (ppm); 2. Total PFOS equivalents in whole liver (ug) = PFOS equivalent concentration in liver (ppm) * liver weight (g); 3. Assuming a 1:1 ratio of PFOSA:PFOS equivalents. PFOS equivalents in dose (ug) = Initial BW (g) ` dose (mg/kg) = BW`5; 4. % PFOS eq dosed as PFOSA in liver =(PFOS equivalents in whole liver (ug)/ PFOS equivalents in dose)*100; 5. Residual PFOS in dose (ug) = dose`0.96% = (5mg/kg*BW)0.0096; 6. % o fl'FOS in liver from residual PFOS in dose = (residual t'FOS in dose / total PFOS eq in liver+VI4)*100, * Significantly different from day 1 values by a two tailed T-lest (p <0.05); ** Significanlly different from day 4 values by a two tailed T-lest (p<0.05); PQL - practical quantitation limit (PFOS = 30 ng/g; PFOSA 10n g )_____________ 3M Strategic Toxicology ST39 T 7132.2 FOSA PK study in rats Table 5: Percent Dose; Serum PFOS equivalents in PFOSA Group Time Day 1 Dose animal PFOS eq cone PFOS q Liver/serurn Total PFOS # 9RD3- in Serum cone in PFOS ratio Eq in whole XX X (p p m )1 liver (ppm) (ppm/ppm) liver (ug) 5 mg/kg 24b 72 29 7 4.1 309 3 Serum Total PFOS Liver/ Serum PFOS eq % PFOS eq Residual % of PFOS in volume Eq in Serum PFOS Eq in dose dosed found PFOS in serum from (ml) 2 (ug)5 ratio4 (ug)5 in serum4 dose (ug)7 residual PFOS in dose (%)* 8 3 60.0 5.2 1316.0 4.6 12 6 21.1% Day 1 5 mg/kg 246 11.2 39.3 3.5 401.4 84 94.1 4.3 1330.5 7.1 12.8 13 6% Day 1 5 mg/kg 247 96 42.5 4 4 462 9 8.5 81.9 5.6 1332.5 6.1 12.8 15.6% Day 1 5 mg/kg 248 70 27 9 4.0 281.7 8.5 59.6 4.7 1338.0 4 5 12.8 21.5% Day 1 5 mg/kg 249 7.6 36.6 4.8 392 1 8.5 65.0 6.0 1343.5 4.8 12.9 19.8% Avg 8.5 35.2 4.2 369.5 8.5 72.1 5.2 1331.7 5.4 12.8 17.7% SO 1.8 6 2 0.5 73.4 0 .1 15.3 0.7 10.1 1.1 0 . 1 3.5% Range Min 7.0 27.9 3 5 281.7 8.3 59.6 4.3 1316.0 4.5 12.6 13.6% Max 11.2 42.5 4.8 462 9 8 5 94.1 6.0 1343 5 7.1 12.9 21.5% N 5 5 5.00 5 55 5 55 55 5 Day 4 5 mg/kg 250 78 40.3 5.1 455.8 9.2 72.5 6.3 1333.0 5.4 12.8 17.6% Day 4 5 mg/kg 251 7.5 36.1 4.8 422.2 9.5 71.7 5.9 1360.5 5.3 13.1 18.2% Day 4 5 mg/kg 252 11.4 4G.6 4 1 563.6 9.5 10B.2 5 2 1358.5 8.0 13.0 12.1% Day 4 5 mg/kg 253 87 40.4 4.7 456.1 9.4 81.6 5.6 1359.0 6.0 13.0 16.0% Day 4 5 mg/kg 254 8.0 42.4 5.3 496.4 9.3 74.2 6.7 1364.5 5.4 13.11 17.6% Avg 8.7 41.2 4.8 478.8 9.4 81.7 6.9 1355.1 6 .0 13.01 15.9% SD 1.6 3.8 0.5 54.2 0.1 15.3 0.6 12.6 1.1 0.1 2.5% Range Min 7.5 36.1 4.1 422.2 9.2 71.7 5.2 1333.0 5.3 12.8 12.1% Max 11.4 46.6 5.3 563.6 9.5 108.2 6.7 1364.5 8.0 13.1 18.2% N 55 5 5 55 5 55 55 5 Day 29 5 mg/kg 255 3.6 20.4 5.7 340.4 12.4 44.7 7.6 1376.0 3.2 13.2 29.6% Day 29 5 mg/kg 256 4.0 20.6 5? 296.9 12.3 48.9 6.1 1368.0 3 6 13.1 26.9% Day 29 5 mg/kg 257 4.0 22 1 5.5 307.3 12.1 48.8 6.3 1357.0 3.6 13.0 26.7% Day 29 5 mg/kg 258 4.1 26.3 6.4 384.6 12.0 49.5 7.8 1372.5 3.6 13.2 26.6% Day 29 5 mg/kg Avg 259 7A 4.6 * * 30.2 23.9 4.1 383.6 11.5 5.4 342.6 12,1 84 8 55.3 4.5 1343.5 6.5 1363.4 6.3 12.9 4.1 13.1 15.2% 23.7% SD 1.5 4.2 0.8 41.2 0.4 16.6 1.3 13.2 1.3 0.1 5.6% Range Min 3.6 20.4 4.1 296.9 11.5 44.7 4.5 1343.5 3 2 12.9 15.2% Max 7.4 30.2 6.4 384.6 12.4 84.8 7 8 1376.0 6.3 13.2 29.G% N 55 5 55 5 55 55 5 1. PFOS equivalents in sera (ppm) = sera PFOS (ppm) + sera PFOSA PFOS equivalents (ppm); 2 There are 32.3 mL Plasma per Kg of rat. (9); 3 Total PFOS equivalents in sera (ug) = PFOS equivalent concentration in sera (ppm) * serum volume (ml); 4. Liver/ Serum total PFOS Eq ratio = total PFOS equivalents in whole liver (ug)/ total PFOS equivalents in serum (ug); 5. Assuming a 1.1 ratio ot PFOSA'.PFOSA PFOS equivalents, PFOS equivalents in dose (ug) = Inilial BW (g) * dose (mg/kg) = BVl/*5; 6 % PFOS equivalents dosed In serum - (PFOS equivalents In sera (ug)/ PFOS equivalents in dose (Ug)) "tOO; 6. Residual PFOS in dose (ug) - dose'0,96% = (5mgfhg'BW)0.009f>; 6. % oI PFOS in sera from residual PFOS in dose = (residual PFOS in dose/ioial PFOS eq in sera)* 100;*" Sign, different from dy 4 values by a two tailed T-lest (p<0.05); MOL - method detection limit in sera: (PFOS = 4.75 ng/rol; PFOSA = 2.89 ng/ml) Page 16 o f 18 3M Strategic Toxicology ST39 T -7132.2 PI OSA PK. study in rats Table 6 - Summary: % + SD of Dose or of PFOS Equivalents Dosed Detected in Liver & Sera Day 1 post dose day 4 post dose day 29 post dose % PFO SA dosed detected as PFO SA in: liver sera liver & sera 5.4 + 0.9 0.2 + 0.1 5.6 + 0.9 3.2 + 03 00 + 0.0 32 + 0.3 0.1 + 0.1 0 0 + 0.0 0.1 + 0.1 % P FO S E q u ivalen ts d o s e d d e te c te d as PFOS in: liver 22.0 + 5.2 32.3 + 3.7 25.2 +_3.0 sera liver & sera 5.2 + 1 1 27.1 +6.2 6.0 +.1 1 38.3 + 4.8 4.1 + 1.2 29.3 + 3.8 NA = not analyzed PFOS equivalents (j>pm) - PFOS (ppm ) + PFOSA PFOS' equivalents (ppm) 3M Strategic Toxicology ST39 T-7I2.2 f'FOSA PK study in rats Table 7: Serum POAA in th e PFOSA Group. lO ia l 7o I'U A A POAA in dosed an im al H Sera ( olal PO A A found us 9 R 0 3 - POAA Serum in dose POAA in Ti me Dose XXX (ppm ) (ug) (ug) sera Day 1 5 mg/kg 24 5 O i l 0.90 3.29 27.3% Da7 1 Day 1 5 mg/kg 5 mg/kg 246 0.15 247 0.13 1.23 1.09 3.33 3.33 37.0% 32.8% Day 1 5 mg/kg 248 0.10 0.88 3 34 26.3% Day 1 5 mg/kg Avg SD 249 0.t2 J .02 3.36 30.5% 0 .1 2 1.02 3 .3 3 3 0 .8 % 0 .0 2 0 .15 0 .0 3 4 .3 % Range M in 0.10 0.88 3.29 26.3% Max 0.15 1.23 3.36 37.0% Y 5 5 5 5 5 Day 4 5 mg/kg 250 0.07 0.63 3.33 18.9% Day 4 5 mg/kg 251 0.07 0.64 3.40 18.9% Da y 4 5 mg/kg 252 0.11 1.00 3.40 29.4% Da y 4 5 mg/kg 253 0.10 0.95 3.40 28.0% Day 4 5 mg/kg 254 0.08 0.76 3.41 22.2% Avg 0 .0 8 0 .8 0 3.39 23.5% SD Range M in 0 .0 2 0.07 0 .17 0.63 0 .0 3 3.33 5 .0 % (8.9% Max 0.11 1.00 3.41 29.4% n 5 55 55 Limil of detection (LOD): POAA 24.7 ng/ml NA. Not analyzed * based on work by Kris Hansen finding 2.5 ug/ml P O A A in dosing solution ' There are 32.3 mL Plasma per Kg of rat.(13) i Page 18 o f 18