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T-7132.1 FR DTI 5 A e z z b -173/ Title: Comparative Molecular Biology o f Perfluorooctanesulfonamide (FOSA, T- 7132) in Rats following four consecutive days o f dosing. Final Report February 18, 2004 Study Number: DT 15 Protocol Amendment Number*: 2. 3M Medical Department Study Number: T-7132.1 Study Director: Andrew Seacat Ph.D. Analytical laboratories: 1. Kendall B.Wallace, Ph.D. D.A.B.T. Professor, Dept of Biochemistry and Molecular Biology School of Medicine 10 University Drive Duluth, MN 55812-2496 2. Dr. Xin Lu Department of Pharmacology and Physiology 601 Elmwood Ave, box 711 Rochester, New York 14642 Original Study Protocol Title: Comparative Molecular Biology of Peroxisome Proliferation in Rats and Guinea Pigs Original Study Initiated: In-Life Start Date: November 16, 1998 In-Life End Date: December 21, 1998 Z\ AV iWOBZ : o -:> CD T-7132.1 FR DT15 Purpose: The purpose of this study was to treat three male rats treated with either no compound (vehicle control) or 40 mg/kg/day perfluorooctanesulfonamide: C8F17S02NH2 (FOSA, PFOSA, FOSAmide, T-7132). The study was designed to evaluate the metabolism and certain toxicological effects, such as hepatic peroxisome proliferation, of this compound and to compare these effects to other compounds dosed at the same concentration.. Methods Test Materials: Perfluorooctanesulfonamide (FOSA, PFOSA, FOSAmide, C8F17S02NH2 NB # 120067-108, G. Moore 10/28/00, T-7132) was investigated. The vehicle control was propylene glycol. Dose Groups: Three male rats received propylene glycol as the vehicle control by oral gavage at a volume o f 5 ml/kg body weight. Three male rats received a dose of 40 mg FOSA/Kg body weight, via oral gavage on days one through four of the study, and were euthanized on day five. A suspension of 8 mg/ml of FOSA in propylene glycol was prepared, and a volume of 5 ml/kg was administered. This dosing regiment achieved a cumulative dose of 160 mg/kg after four successive days of dosing. This dose was the same as the dose of PFOS administered under amendment 1 of this protocol and is comparable to effective dose levels for hepatic peroxisome proliferation in rats of PFOS found in the literature. The LD50 for FOSA is not known, but the cumulative dose of FOSA administered under this protocol is below the LD50 for PFOS 251 mg/kg for PFOS in com oil, as a point of reference. Method o f Specimen Collection: The liver was removed as rapidly as possible after euthanasia. The livers were divided into ~ 1 g pieces and flash-frozen directly in liquid nitrogen in tared polypropylene (Nalgene) containers. The containers were moved to dry ice and weighed after all liquid nitrogen had evaporated. The tissue was stored at -70 C and shipped on dry ice. Sera: Up to 10 ml of blood was collected from each animal into glass serum tubes. Following clotting, the blood was centrifuged for 10 minutes at 1100 x g at 4 C. the sera was transferred to new tubes and be centrifuged again for 10 minutes at 1100 x g at 4 C. Two aliquots of the serum sample (~ 0.75 ml) were saved for possible metabolite analysis. T-7132.1 FR DTI 5 Specimen Handling: A 1-2 gram aliquot of the liver samples that were flash frozen in liquid nitrogen were shipped in dry ice to the analytical laboratories listed according to the livers sample identification chart below to: Kendall B.Wallace, Ph.D. D.A.B.T. Professor, Dept of Biochemistry and Molecular Biology School of Medicine University of Minnesota 10 University Drive Duluth, MN 55812-2496 Liver samples were analyzed by previously published methods for P450 content, Lauroyl CoA oxidase activity (Poosch and Yamazaki 1986) and protein content (Bradford 1976) in the laboratory of Ken Wallace Dept of Biochemistry and Molecular Biology at the University of MN. Another 1-2 gram aliquot of the liver sample was sent to: Dr. Michael Wempe Laboratory of M. W. Anders Department of Pharmacology and Physiology 601 Elmwood Ave, box 711 Rochester, New York 14642 Dr. Lin Xu performed the analysis of these samples and provided a brief summary of the analysis of liver samples from rats given a range of fluorocarbons. The parent and metabolites of the fluorocarbons were determined in liver samples by LC-MS/MS using previously published methods (Hansen et al. 2001). Liver Sample Identification Chartfo r T-7132.1 Sample # Animal # Species Sex Dose Group 1 1R00742 Rat M control 2 1R00743 Rat M control 3 1R00744 Rat M control 4 1R00745 Rat M 40 mg/kg/day FOSA 5 1R00746 Rat M 40 mg/kg/day FOSA 6 1R00747 Rat M 40 mg/kg/day FOSA T-7132.1 FR DTI 5 Results and Discussion Average body weights of the FOSA treatment group were significantly lower than the control group on days four and five (Table 1). Liver weights were not significantly different between the treated and control groups, but the liver weight as a percentage of body weight was significantly increased in the FOSA treatment group The results of P450 content (Figure 1) and Acyl CoA oxidase activity (Figure 2) for the liver samples indicated that FOSA induced the expression of these proteins and FOSA is therefore a hepatic peroxisome proliferator in rats. The concentrations of the parent compound and metabolites in livers were measured (Table 2). The data show that PFOS was the major metabolite found in the livers of rats given FOSA and FOSA A-glucuronide was identified as a minor metabolite. Approximately 0.3 percent of the cumulative dose was present in the liver as either the parent compound, FOSA, or as the metabolite PFOS, one day after the last dose (Table 3). The low percentage of FOSA in the liver and the apparent low conversion of FOSA to PFOS has also been noted in in-vitro microsomal and liver slice metabolism studies (Xu et al. 2003) which observed that FOSA was converted to PFOS in-vitro by liver slices at a low rate, but not by microsomes or cytosol. It is noteworthy that the control animals (1R00742, 1R00743, and 1R00744) contained significant concentrations of FOSA. Furthermore, no PFOS or FOSA A-glucuronide were found in the control samples with a high background of FOSA. A parallel analysis of livers from Fischer 344 rats maintained in the University of Rochester Vivarium did not show detectable concentrations of FOSA. These results suggest that the control liver samples for this study were contaminated at some point time ex-vivo by trace quantities of FOSA, as no metabolism to PFOS or FOSA A-glucuronide had occurred in the control samples. T-7I32.1 FR DTI 5 Signatures: Report prepared by, l'-x ' . Andrew M. Seacat, PhD, DABT Study Director zLU&t" __ a /'ifaf Date T-7132.1 FR DTI 5 References: Bradford, M. M. (1976). A rapid and sensitive method for the quantitation o f microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem TL, 248-54. Hansen, K. J., Clemen, L. A., Ellefson, M. E., and Johnson, H. O. (2001). Compound-specific, quantitative characterization o f organic fluorochemicals in biological matrices. Environmental Science and Technology 35, 766-770. Poosch, M. S., and Yamazaki, R. K. (1986). Determination of peroxisomal fatty acyl-CoA oxidase activity using a lauroyl-CoA-based fluorometric assay. Biochim Biophys Acta 884, 585-93. Xu, L., Seacat, A. M., Butenhoff, J. L., and Anders, M. W. (2003). Biotransformation of N-Ethyl-N-(2hydroxyethyl)perfluorooctanesulfonamide (N-EtFOSE) by rat liver microsomes, cytosol and slices. In Toxicological Sciences, Suppl., Vol. 72, p. 314. T-7132.1 FR DTI 5 Tables Table 1 Biological Parameters DT15, Amendment #2 Comparative Molecular Biology of Perfluorooctanesulfonamide (FOSA, T-7132) in Rats following four consecutive days of dosing. DT15 Body Weight (BW), Liver Weight (LW), Thyroid Weight (TW) Date 2/19/01 2/19/01 2/21/01 2/22/01 2/23/01 2/23/01 Sample Animal # Dose Group # BW BW BW day 1 day 2 day 3 ia )____| M ___ (9) BW day 4 (9) BW day 5 (g) LW (g) LW/BW TW (%) (g) 1 1R00742 control 2 1R00743 control 3 1R00744 control Avg SD 250 259 260 264 272 10.73 3.9% 10.62 269 278 285 292 295 10.82 3.7% 10.87 256 260 267 268 276 10.70 3.9% 10.65 258 266 271 275 281 10.75 3.8% 10.71 10 11 13 15 12 0.06 0.14% 0.14 4 1R00745 40 mg/kg/day FOSA 5 1R00746 40 mg/kg/day FOSA 6 1R00747 40 mg/kg/day FOSA Avg SD P-value T-Test* 250 254 250 239 227 10.77 4.7% 10.78 249 245 243 233 225 10.81 4.8% 10.66 259 265 263 255 254 10.64 4.2% 10.84 253 255 252 242 235 10.74 4.6% 10.76 6 10 10 11 16 0.09 0.34% 0.09 0.21 0.13 0.06 0.02 0.01 0.44 0.01 0.32 *T-Test (unpaired, one tailed, equal variance). A P-value of < 0.05 was considered significantly different from control. T-7132.I FR DTI 5 Table 2 Liver Fluorocarbon Concentration Hepatic Concentrations of Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given FOSA (40 mg/kg/day) Sample # Animal # Sex 1 1R00742 M 2 1R00743 M 3 1R00744 M 4 1R00745 M 5 1R00746 M 6 1R00747 M Note: n.m. = not measured. Treatment Control Weight of liver sample (g) 0.4600 Control 0.9236 Control 1.1287 FOSA 0.2402 FOSA 0.7905 FOSA 0.8305 PFOS (ppm) 0.0 0.0 0.0 163.2 214.3 202.8 FOSA (ppm) FOSAA (ppm) FOSA Nglucuronide (ppm) 154.1 0.0 0.00 113.2 0.0 0.00 273.2 0.0 0.00 195.7 n.m. 0.44 174.1 n.m. 0.39 163.9 n.m. 0.34 T-7132.1 FR DTI 5 Table 3 Percent Fluorocarbon Dose in Liver Hepatic Percent of Dosed Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given FOSA (40 mg/kg/day) for Four Days Percent Dose in Liver Sample Animal # Dose # Group 1 1R00742 control 2 1R00743 control 3 1R00744 control Avg SD 4 1R00745 FOSA 5 1R00746 FOSA 6 1R00747 FOSA Avg SD Total Dose (mg) PFOS Liver Liver % dose Liver in PFOS PFOS as FOSA dose** (ppm) (mg) PFOS (PPm) in liver (%) Liver % dose as FOSA FOSA in (mg) liver (%) 0.00 0.00 0.00 0 0 0 154.1 113.2 273.2 180.17 83.12 644.84 644.84 163.2 660.19 660.19 214.3 614.34 614.34 202.8 193.43 26.81 1.76 2.32 2.16 2.08 0.29 0.27 0.35 0.35 0.32 0.05 195.7 174,1 163.9 177.90 16.24 2.1 1.9 1.7 1.91 0.18 0.33 0.29 0.28 0.30 0.02 T-7132.1 FR DTI 5 Figures Figure 1 Cytochrome P450 content in Liver Total CYTP450 Content in Rat Liver Following Treatmiant With 0 .2 0 0 0 FOSA 0 1500 - on E 8 *a. 0 .1000 o tf> O o c 0 .0500 1 A verage of n=3; Error bars represent the standard error o f the m ean T Control FOSA T-7132.1 FR DT15 Figure 2: Lauroyl CoA oxidase activity in liver LCoA-Oxidase Activity in Rat Liver Homogenate Following Treatment With FO SA __ 8.00 o> - i 7.80 'C NJ 7.60 o JN 7.40 08 7.20 1 7.00 6.80 6.60 T%J 6.40 O 6.20 i. O 6.00 5.80 i - Average of n=3; Error bars represent the Standard error of the mean. Control FOSA T-7132.1 FR DTI 5 Appendix 1 Cytochrome P450 content Absorbance Values Sample # Treatment 1 Control 2 Control 3 Control 4 FOSA 5 FOSA 6 FOSA 450 -0.0366 0,02542 -0.0144 -0.0404 0.13601 0.00594 500 -0.0312 0.02209 -0.0172 -0.0456 0.10083 -0.013 protein cone, (ug/ul) 8.20 8.79 9.25 9.79 10.48 9.86 mg protein added 5 5 5 5 5 5 450 - 500 0.0054 0.00333 0.0028 0.0052 0.03518 0.01894 uM volum e CYTP450 (ml) 0.059 3 0.037 3 0.031 3 0.057 3 0.387 3 0.208 3 nm oles total CYTP450/ nm ole mg 0.178 0.0356 0.110 0.0220 0.092 0.0185 0.171 0.0343 1.160 0.2320 0.624 0.1249 Ttest (unpaired, one tailed, unequal variance) n=1 n=2 n=3 Average Stdev Sterrar Varriance ftest p value Control 0.0356 0.0220 0.0185 0.0253 0.0091 0.0052 0.0001 1 0.50 Statistics FOSA 0.0343 0.2320 0.1249 0.1304 0.0989 0.0571 0.0098 0.02 0.10 T-7132.1 FR DTI 5 Protein Determination Standard Curve ul BSA 5 20 40 60 80 ul Lysis Buffer 95 80 60 40 20 Absorbance 0.4645 0.6167 0.7992 0.9413 1.0379 0 20 40 60 80 100 T-7132.1 FR DTI 5 s a m p le 1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B Treatment Control Control Control Control Control Control FOSA FOSA FOSA FOSA FOSA FOSA ul 5 5 5 5 5 5 5 5 5 5 5 5 Absorbance 0.7799 0.7635 0.8004 0.7885 0.8155 0.8086 0.844 0.8217 0.8493 0.8695 0.8305 0.8404 ug protein mg/ml average 42.064935 8.412987 8.2 39.935065 7.987013 44.727273 8.945455 8.790909 43.181818 8.636364 46.688312 9.337662 9.248052 45.792208 9.158442 50.38961 10.07792 9.788312 47.493506 9.498701 slope 0.0077 0.0077 0.0077 0.0077 0.0077 0.0077 0.0077 0.0077 intercept 0.456 0.456 0.456 0.456 0.456 0.456 0.456 0.456 51.077922 10.21558 10.47792 53.701299 10.74026 48.636364 9.727273 9.855844 49.922078 9.984416 0.0077 0.0077 0.0077 0.0077 0.456 0.456 0.456 0.456 sa m p le 1 2 3 4 5 6 m g /m l 8.20 8.79 9.25 9.79 10.48 9.86 ul needed to get 5 mg 609.8 568.8 540.7 510.8 477.2 507.3 T-7132.1 FR DTI 5 Appendix 2: Lauroyl CoA Oxidase Activity Sample # average stdev %dev average stdev %dev 1 206.3 207.5 209.6 207.8 1.7 0.8% 1 123 120.3 120.2 121.2 1.6 1.3% 2 205.4 205 206.9 205.8 1.0 0.5% 2 122.9 121.7 121.5 122.0 0.8 0.6% 3 209.8 216.1 210.3 212.1 3.5 1.7% 3 126.3 125.9 124.4 125.5 1.0 0.8% 4 216.8 218.5 216.5 217.3 1.1 0.5% 4 124.3 121.9 121.7 122.6 1.4 1.2% 5 227.4 229.4 228.9 228.6 1.0 0.5% 5 124.3 123 123.3 123.5 0.7 0.6% 6 218.6 218.7 219.5 218.9 0.5 0.2% 6 131.1 129.3 125.4 128.6 2.9 2.3% nmoles H 2 0 2 produced/ 30 min: Sam ple Control D iffe re n c e 1 4.02 0.03 3.99 2 3.93 0.07 3.86 3 4.22 0.23 3.99 4 4.46 0.10 4.36 5 4.98 0.14 4.84 6 4.54 0.37 4.16 slope 21.692 21.692 21.692 21.692 21.692 21.692 intercept 120.5 120.5 120.5 120.5 120.5 120.5 T-7132.1 FR DTI 5 1 1 nm oles H 2 0 2 produced/m in/m g protein: nm oles H 2 0 2 tim e (min) ug protein Control 1 3.99 30 2 3.86 30 3 3.99 30 FOSA 4 4.36 30 5 4.84 30 6 4.16 30 20 20 20 20 20 20 average (n=3) 6.66 Control 6.43 6.6 6.65 7.27 FOSA 8.07 7.4 6.94 n=1 n=2 n=3 Average _____ Stdev Sterror Control FOSA (nmoles H 2 0 2 produced/min/mg protein) 6.66 7.27 6.43 8.07 6.65 6.58 0.13 0.07 6.94 7.43 0.58 0.34 T-7132.1 FR DTI 5 ! H2O 2 Standard Curve Data. H2O2 Stock was 8.6 M nmnoles H 202 average SD % dev 0 109.7 112 113.8 111.8 2.1 1.8% 1.72 151.5 153.2 154.9 153.2 1.7 1.1% 3.44 202.4 206.3 199.3 202.7 3.5 1.7% 6.88 280.7 288.6 282.2 283.8 4.2 1.5% 13.76 412.9 410.7 408.3 410.6 2.3 0.6% H202 Standard Curve 450 , 0 . ---0 --, - 5 -- , ----10 -n 15 nmoles H202 T-7132.1 FR DTI 5 Protein Standard Curve ui BSA ul Lysis Buffer 5 95 20 80 40 60 60 40 80 20 Absorbance 0.4464 0.6769 0.854 1.0135 1.037 sample ul Absorbance ug protein ug/ul average slope intercept 1 5 0.7814 34.435644 6.887129 7.054455 0.0101 0.4336 1 5 0.7983 36.108911 7.221782 0.0101 0.4336 sam p le ug/ul protein ul needed to g et 2 0 ug 2 5 0.7956 35.841584 7.168317 7.617822 0.0101 0.4336 1 7.05 2.84 25 0.841 40.336634 8.067327 0.0101 0.4336 2 7.62 2.63 3 5 0.7963 35.910891 7.182178 7.369307 0.0101 0.4336 3 7.37 2.71 3 5 0.8152 37.782178 7.556436 0.0101 0.4336 4 7.88 2.54 4 5 0.8225 38.50495 7.70099 7.882178 0.0101 0.4336 5 8.38 2.39 4 5 0.8408 40.316832 8.063366 0.0101 0.4336 6 8.61 2.32 5 5 0.8592 42.138614 8.427723 8.379208 0.0101 0.4336 5 5 0.8543 41.653465 8.330693 0.0101 0.4336 6 5 0.8501 41.237624 8.247525 8.610891 0.0101 0.4336 6 5 0.8868 44.871287 8.974257 0.0101 0.4336