Document 37NvLXB7V5ERonKMLkqRbj1vE

T-7132.1 FR DT15 Title: Comparative Molecular Biology of Perfluorooctanesulfonamide (FOSA, T- 7132) in Rats following four consecutive days of 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. A nalytical 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 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: C8F 17SO2NH2 (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, C8F17SO2NH2 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 of 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 corn oil, as a point of reference. Method of 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 1I00 x g at 4 C. Two aliquots of the serum sample (- 0.75 ml) were saved for possible metabolite analysis. T-7132.1 FR DT15 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 School of Medicine University of Minnesota 10 University Drive Duluth, MN 55812-2496 Biology 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 601 Elmwood Ave, box 711 Rochester, New York 14642 and Physiology Dr. Lin Xu performed the analysis of these samples and provided analysis of liver samples from rats given a range of fluorocarbons. metabolites of the fluorocarbons were determined in liver samples previously published methods (Hansen et al. 2001). a brief summary of the The parent and by LC-MS/MS using Liver Sample Identification Chart for T- 7132.1 Sample# Animal # Species Sex DoseGroup 1 1R00742 Rat M control 2 1R00743 Rat M control 3 1R00744 Rat M control 4 1R00745 Rat M 40 mg/kg/dayFOSA 5 1R00746 Rat M 40 mg/kg/dayFOSA 6 1R00747 Rat M 40mg/kg/dayFOSA T-7132.1 FR DT15 Results and Discussion Average body weights of the FOSA treatment group were significantly lower than the control group on days four and five (Table I). 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 N-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 (1 R00742, 1R00743, and 1R00744) contained significant concentrations of FOSA. Furthermore, no PFOS or FOSA N-glucuronide were found in the control samples with a high background ofFOSA. 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 N-glucuronide had occurred in the control samples. T-7132.1 FP, DT15 Sign (l[ll res: Report prepared by, ........ x .. _ _,ud_"_'_ .... Andrew M. Seacat, PhD, DABT Study Director ... _c- _': Reviewed by, Daniel C. Hakes Sponsor representative Date I / Date / T-7132.1 FR DT15 References: Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72,248-54. Hansen, K. J., Clemen, L. A., Ellefson, M. E., and Johnson, H. O. (2001). Compound-specific, quantitative characterization of organic fluorochemicals in biological matrices. Enviromnental 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 DT15 Tables Table 1 Biological Parameters IDT15, Amendment #2 i! ,I i Ir IComparative Molecular Biology of Perfluorooctanesulfonamide (FOSA, T-7132) in Rats following Ifour consecutive days of dosing. iDT15 Body Weight (BW), Liver Weight (LW), Thyroid Weight (TW) ! i '] I I J Date i ' : 2/19/01 2/19/01 J2/21/01i2/22/01 J2/23/012/23/01i ISampleAnimal# Dose Group I# ! BW day1 BW day2 [BW iBW rBW iLW(g) LW/BWTW Jday3 )day4 ._,,,y5 I(%) !(g) ; ........................ 111R00742 control 2 1R00743 control 3 1R00744 control Avg SD I J ._ ; ...... 4 -, 250 259[ 260r 264 r 272[ 10.73i 3.9%10.621 269 278J 285] 292i 295 i 10.82! 3.7%10.87[ 256 260 267 i 2681 276! 10.70', 3.9%!10.65 258 266 271 2751 2811 10.75i 3.8%10.71i 10 11 13 15l 121 0.06 0.14%[ 0.141 , b , 411R00745 4F0OSmAg/kg/day 5'1R00746 40 mg/kg/day FOSA 6 1R0074740mg/kg/day FOSA Avg so i P-valueT-Test* 250 254 250 239_ 2271 10.771i 4.7%;i10.7_ I 2491 245 2431 233 225 10.81 4.8%!10.61_ ' r 259: 265 263 255 2541 10.64j 4.2% 10.841 ] ] ] I 253 255 252 24r2 235[10._i 44.6O10/o.7e 6 0,21; 10 0,13 0,1. 000,01261r 10,011161o0,4.401 090.L,3014000,3.2/09 *T-Test (unpaired, one tailed, equal variance). A P-value of-< 0.05 was considered significantly different from control. T-7132.1 FR DT15 Table 2 Liver FluorocarbonConcentration Hepatic Concentrations of Fluorocarbons of Rats Given FOSA (40 mg/kg/day) and Fluorocarbon Metabolites in Livers I 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. Weight of 1 t FOSA N- Treatment Control Control Control FOSA FOSA FOSA liver(gsa)mple 0.4600 0.9236 1.1287 0.2402 0.7905 0.8305 P(pFpOmS) 0.0 F(pOpSmA) F(OppSmA)A I glu(cpuprmon) ide 154.1 0.t3 0.0C 0.13 1! 3.2 0.13 0.0C 0.13 273.2 0.13 0.0 163.2 195.7 n.m. 0.44 214.3 174.1 n.m. 0.39 202.8 163.9 n.m. 0.34 T-7132.1 FR DT15 Table 3 Percent Fluorocarbon Dose in Liver Hepatic Percent of Dosed Fluorocarbons and Fluorocarbon Rats Given FOSA (40 mg/kg/day) for Four Days Metabolites in Livers of 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 6ilR00747 FOSA Avg SD Total Dose (mg) PFOS in dose** Liver Liver PFOS PFOS (ppm)(rag) % dose as PFOS Jnliver (%) Liver FOSA (ppm) Liver % dose as FOSAtFOSA in (rag) liver(%) 0.00 0 0.00 0 0.00 0 154.1 113.2 273.2 180.17 83.12 644.84 644.84 163.2 1.76 660.19 660.19 214.3 2.32 !614.34 614.34 202.8 2.16 1193.43 2.08 26.81 0.29 0.27 0.35 0.35 0.32 0.05i 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 DT15 Figures Figure 1 Cytochrome P450 content in Liver Total CYTP450ContenitnRatLiverFollowingTreatmenWt ith 0,2000 FOSA 0,1500o} E O u_ a. _- 0.1000 Averageof n=3; Errorbarsrepresenthe standarderror of the mean O E = 0.0500 ! I 0.0000...... T Control FOSA T-7132.1 FR DT15 Figure 2: Lauroyl CoA oxidase activity in liver LCoA-OxidasAectivityin RatLiverHomogenatFeollowing TreatmenWt ithFOSA 8.00 "_ : .EE 7.80 . .E .EE 7.60 _ O 7.40, ,_ 7.20 o Average of n=3; Error bars represent the Standard errorof the mean. ,_c 7.00 _ 6.80 6.60: "6.40 _ 6.206.00 5.80 T Control I /T FOSA geez|e|||a1e77e7] [sig|e[1g/7e11e7]y| eH TH ||gem| |e|]]e|[[e[]e]| { fl Tf ham i e 111 2 by san TTT 3 | amie [aki aEn fF LLEHR-MTTTTITTT I| HHH tt LmLm i TI n ll TT|?. 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