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SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 A g 2 t > - / 3 / DT15-B E. Technical Report: Liver Fluorocarbon Metabolites - University of Rochester. Title: Summary of Quantitative Analysis of Fluorocarbon Metabolites in Rat Liver Samples Lin Xu and M. W. Anders This is 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. The results are presented below. I Three groups of liver samples were analyzed: The first group (Group 1) consisted of livers from control rats, from rats given 40 mg/kg/day FOSA orally, and from rats given 160 mg/kg/day FOSAA (M556) orally. The concentrations of parent compounds and their metabolites in livers were measured (Table 1). The data show that PFOS was the major metabolite found in the livers of rats given FOSA; FOSA A'-glucuronide was identified as a minor metabolite. Two metabolites were identified in the livers of rats given FOSAA (M556): PFOS and FOSA. It is noteworthy that the control animals (1R00742, 1R00743, and 1R00743) contained significant concentrations 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. The second group (Group 2) of rats was treated in November, 1998. Group 2 contained livers from control rats, from rats given 40 mg/kg/day PFOS orally, from rats given 40 mg/kg/day A-EtFOSE alcohol orally, and from rats given 40 mg/kg/day A-EtFOSA orally. The concentrations of parent compounds and identified metabolites are shown in Table 2. The data show that livers from rats given PFOS contained parent compound PFOS; FOSA was also found in these samples. The livers from rats given A-EtFOSE alcohol contained several metabolites. The major metabolites were A-EtFOSAA, PFOS, and FOSAA (M556). The minor metabolites were FOSA, FOSE alcohol, A-EtFOSE alcohol glucuronide, and FOSA A-glucuronide. PFOS was the major metabolite in livers from rats given A-EtFOSA; FOSA and FOSA A-glucuronide were identified as minor metabolites. In contrast to Group 1, the livers from control rats did not contain detectable concentrations of parent compounds or metabolites. The third group (Group 3) was treated in March, 1999. Group 3 contained livers from control rats, from rats given 40 mg/kg/day PFOS orally, and from rats given 160 mg/kg/day A-EtFOSE alcohol orally. The concentrations of the chemicals and metabolites are shown in Table 3. As with Group 2, PFOS and low concentrations of FOSA were identified in livers from rats given PFOS. In the livers of rats given A-EtFOSE alcohol, A-EtFOSAA, PFOS, and FOSAA (M556) were identified as major metabolites, and the minor metabolites identified were FOSA, FOSE alcohol, A-EtFOSE alcohol glucuronide, and FOSA Aglucuronide. Similar to Group 1, control animals showed detectable hepatic concentrations of FOSA. Discussion: These data allow conclusions about the overall metabolic fate of the fluorocarbons studied (see Scheme), but do not allow inferences about the exact routes of metabolite formation. FOSA was consistently identified as a metabolite of PFOS, whether PFOS was administered directly or formed as a metabolite, but the source of the amino group is not readily apparent. Whatever its route of formation, FOSA was metabolized to FOSA A-glucuronide. A-EtFOSE alcohol gives rise to a range of major and minor metabolites. FOSE alcohol could arise from the A-deethylation of A-EtFOSE alcohol, and A-EtFOSAA could arise by the oxidation of the alcohol to the carboxylic acid. Glucuronidation of the parent A-EtFOSE alcohol would give the observed A-EtFOSE alcohol glucuronides. FOSAA could be formed by the A-deethylation of A-EtFOSAA or by the oxidation of FOSE alcohol, or both. FOSA could be formed by the A-deethylation of A-EtFOSA or by the removal of the carboxymethyl group of FOSAA as glyoxylate. FOSA A-glucuronide may be formed by the glucuronidation of FOSA. 59C C 0 O 7 2 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B E. Technical Report: Liver Fluorocarbon Metabolites - University of Rochester. Title: Summary of Quantitative Analysis of Fluorocarbon Metabolites in Rat Liver Samples Lin Xu and M. W. Anders This is 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. The results are presented below. Three groups of liver samples were analyzed: The first group (Group 1) consisted of livers from control rats, from rats given 40 mg/kg/day FOSA orally, and from rats given 160 mg/kg/day FOSAA (M556) orally. The concentrations of parent compounds and their metabolites in livers were measured (Table 1). The data show that PFOS was the major metabolite found in the livers of rats given FOSA; FOSA A-glucuronide was identified as a minor metabolite. Two metabolites were identified in the livers of rats given FOSAA (M556): PFOS and FOSA. It is noteworthy that the control animals (1R00742, 1R00743, and 1R00743) contained significant concentrations 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. The second group (Group 2) of rats was treated in November, 1998. Group 2 contained livers from control rats, from rats given 40 mg/kg/day PFOS orally, from rats given 40 mg/kg/day /V-EtFOSE alcohol orally, and from rats given 40 mg/kg/day A-EtFOSA orally. The concentrations of parent compounds and identified metabolites are shown in Table 2. The data show that livers from rats given PFOS contained parent compound PFOS; FOSA was also found in these samples. The livers from rats given A-EtFOSE alcohol contained several metabolites. The major metabolites were A-EtFOSAA, PFOS, and FOSAA (M556). The minor metabolites were FOSA, FOSE alcohol, A-EtFOSE alcohol glucuronide, and FOSA A-glucuronide. PFOS was the major metabolite in livers from rats given A-EtFOSA; FOSA and FOSA A-glucuronide were identified as minor metabolites. In contrast to Group 1, the livers from control rats did not contain detectable concentrations of parent compounds or metabolites. The third group (Group 3) was treated in March, 1999. Group 3 contained livers from control rats, from rats given 40 mg/kg/day PFOS orally, and from rats given 160 mg/kg/day A-EtFOSE alcohol orally. The concentrations of the chemicals and metabolites are shown in Table 3. As with Group 2, PFOS and low concentrations of FOSA were identified in livers from rats given PFOS. In the livers of rats given AEtFOSE alcohol, A-EtFOSAA, PFOS, and FOSAA (M556) were identified as major metabolites, and the minor metabolites identified were FOSA, FOSE alcohol, A-EtFOSE alcohol glucuronide, and FOSA Aglucuronide. Similar to Group 1, control animals showed detectable hepatic concentrations of FOSA. Discussion: These data allow conclusions about the overall metabolic fate of the fluorocarbons studied (see Scheme), but do not allow inferences about the exact routes of metabolite formation. FOSA was consistently identified as a metabolite of PFOS, but the source of the amino group is not readily apparent. Whatever its route of formation, FOSA was metabolized to FOSA A-glucuronide. A-EtFOSE alcohol gives rise to a range of major and minor metabolites. FOSE alcohol could arise from the A-deethylation of A-EtFOSE alcohol, and A-EtFOSAA could arise by the oxidation of the alcohol to the carboxylic acid. Glucuronidation of the parent A-EtFOSE alcohol would give the observed A-EtFOSE alcohol glucuronides. FOSAA could be formed by the A-deethylation of A-EtFOSAA or by the oxidation of FOSE alcohol, or both. FOSA could be formed by the A-deethylation of A-EtFOSA or by the removal of the carboxymethyl group of FOSAA as glyoxylate. FOSA A-glucuronide may be formed by the glucuronidation of FOSA. Revised page 59 C r-0073 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B The fate of FOSAA appears relatively straightforward. Loss of the carboxymethyl group would give FOSA or loss of the glycine moiety would give PFOS directly. 60 C90974 T-7071.1 FR DTI 5 Table 1. Hepatic Concentrations of Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given FOSA (40 mg/kg/day) or FOSAA (M556) (160 mg/kg/day) (Group 1) Sample # Animal # 1 1R00742 2 1R00743 3 1R00744 4 1R00745 5 1R00746 6 1R00747 7 1R00748 8 1R00749 9 1R00750 Weight of liver sample Sex Treatment (g) M Control 0.4600 M Control 0.9236 M Control 1.1287 M FOSA 0.2402 M FOSA 0.7905 M FOSA 0.8305 M FOSAA 0.5520 M FOSAA 0.9373 M FOSAA 0.5320 Note: n.m. = not measured. PFOS (ppm) 0.0 0.0 0.0 163.2 214.3 202.8 238.4 81.9 100.7 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. 0.34 447.3 614.4 0.00 235.7 424.0 0.00 267.4 627.1 0.00 00 O 3 61 T-7071.1 FR DTI 5 Table 2: Hepatic Concentrations of Fluorocarbons and Fluorocarbon Metabolites in Livers from Rats Given PFOS (40 mg/kg/day), /V-EtFOSE alcohol (40 mg/kg/day). or /V-EtFOSA (40 mg/kg/day) (Group 2) Sample # Animal # Sex Treatment 1 8R04032 M Control 2 8R04033 M Control 3 8R04041 F Control 4 8G01477 M Control 5 8G01478 M Control 6 8G01485 F Control 7 8G01486 F Control 8 8G01483 M PFOS 9 8G01484 M PFOS 10 8G01491 F PFOS 11 8G01479 M /V-EtFOSE alcohol 12 8G01480 M /V-EtFOSE alcohol 13 8G01487 F /V-EtFOSE alcohol 14 8G01489 F /V-EtFOSE alcohol 15 8G01481 M /V-EtFOSA 16 8G01482 M /V-EtFOSA 17 8G01488 F /V-EtFOSA 18 8G01490 F /V-EtFOSA Note: n.m. = not measured. Weight N- of liver N- FOSE EtFOSE /V-EtFOSE alcohol FOSA N- samples PFOS FOSA FOSAA EtFOSAA alcohol alcohol glucuronide glucuronide (g) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) 0.7516 0.0 0.0 0.0 0.0 0.0 n.m. (ppm) (ppm) 0.00 0.00 1.1 0.0 0.0 0.0 0.0 0.0 n.m. 0.00 0.00 0.413 0.0 0.0 0.0 0.0 0.0 n.m. 0.00 0.00 1.081 0.0 0.0 0.0 0.0 0.0 n.m. 0.00 0.00 0.6009 0.0 0.0 0.0 0.0 0.0 n.m. 0.00 0.00 1.206 0.0 0.0 0.0 0.0 0.0 n.m. 0.00 0.00 0.8228 0.0 0.0 0.0 0.0 0.0 n.m. 0.00 0.00 0.7756 304.0 69.8 n.m. n.m. n.m. n.m. n.m. n.m. 0.789 90.8 71.0 n.m. n.m. n.m. n.m. n.m. n.m. 1.1795 127.5 67.6 n.m. n.m. n.m. n.m. n.m. n.m. 0.7712 29.0 5.1 34.2 52.9 2.4 0.96 0.31 0.36 0.7744 147.8 4.7 22.1 55.1 4.6 1.67 0.47 0.40 0.7137 76.7 4.3 26.0 85.5 4.2 0.82 1.02 0.25 0.4796 12.7 9.8 34.8 124.4 2.2 0.96 1.35 0.29 0.707 112.5 0.3 0.0 0.0 0.0 n.m. 0.00 0.12 0.993 80.0 0.6 0.0 0.0 0.0 n.m. 0.00 0.16 0.6395 125.8 1.1 0.0 0.0 0.0 n.m. 0.00 0.26 0.754 83.3 0.0 0.0 0.0 0.0 n.m. 0.00 0.23 9^0003 62 T-707I.1 FR DTI 5 Table 3. Hepatic Concentrations of Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given A'-EtFOSE alcohol (160 mg/kg/day) or PFOS (40 mg/kg/day) (Group 3) Sample # Animal # Sex Treatment 19 9R00463 M Control 20 9R00464 M Control 21 9R00469 F Control 22 9R00470 F Control 23 9R00465 M A'-EtFOSE alcohol 24 9R00466 M A'-EtFOSE alcohol 25 9R00471 F A'-EtFOSE alcohol 26 9R00472 F A'-EtFOSE alcohol 27 9R00467 M PFOS 28 9R00468 M PFOS 29 9R00473 F PFOS 30 9R00474 F PFOS 31 9G00045 M Control 32 9G00046 M Control 33 9G00051 F Control 34 9G00052 F Control 35 9G00047 M A'-EtFOSE alcohol 36 9G00048 M A'-EtFOSE alcohol 37 9G00053 F A'-EtFOSE alcohol 38 9G00054 F A'-EtFOSE alcohol 39 9G00049 M PFOS 40 9G00050 M PFOS 41 9G00055 F PFOS 42 9G00056 F PFOS Weight of PFOS liver (ppm) samples (g) FOSA (ppm) FOSAA N- (ppm) EtFOSAA (ppm) FOSE alcohol (ppm) A'EtFOSE alcohol (ppm) A'-EtFOSE alcohol glucuronide (ppm) PFOSA glucuronide (ppm) 0.4459 0.903 0.8444 0.5802 0.1707 0.885 0.304 0.2791 0.5589 0.411 0.6576 0.4673 0.1608 0.3882 0.712 0.59 0.7258 0.713 0.9192 0.44 0.787 0.43 0.9398 0.5605 0.0 0.0 0.0 0.0 1241.7 1006.8 865.3 918.5 907.5 1032.3 967.5 765.3 0.0 0.0 0.0 0.0 530.8 446.3 590.5 109.8 312.8 70.5 37.5 257.3 2.6 n.m. 2.8 n.m. 4.6 n.m. 4.1 n.m. 25.2 111.0 11.7 77.5 17.3 63.6 18.8 71.4 3.7 n.m. 31.8 n.m. 21.2 n.m. 15.3 n.m. 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 11.4 169.3 4.9 89.5 11.2 290.7 21.1 145.0 14.5 n.m. 11.6 n.m. 3.0 n.m. 2.2 n.m. n.m. n.m. n.m. n.m. 376.4 212.7 178.2 225.5 n.m. n.m. n.m. n.m. 0.0 0.0 0.0 0.0 342.1 181.1 457.6 318.8 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. 13.9 10.3 29.8 64.6 n.m. n.m. n.m. n.m. 0.0 0.0 0.0 0.0 21.8 10.1 28.1 29.2 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. 2.92 4.66 7.45 5.34 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. 2.46 2.13 1.43 2.37 n.m. n.m. n.m. n.m. 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.24 0.09 0.29 0.08 0.11 0.23 0.28 0.24 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. 0.00 0.00 0.00 0.00 0.00 0.00 2.75 0.49 1.26 0.48 8.57 0.25 3.58 0.35 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. . G O O D 63 8 c .G O v 5 T-7071.1 FR DTI 5 Note: n.m. = not measured. Metabolism of Fluorocarbons in Rats o R -S -N o^ --------------------- R - S - N Oii R--Sn--NH-Glucuronide O FOSA N-glucuronide PFOS 64 T-7071.1 FR DT15 Appendix 7: Clinical Chemistry. Individual and Summary Clinical Chemistry Data. SPEC SEX DOSE GRP ID GP F CONT 8G0148 8G0148 9G0005 9G0005 z N M ean SD NE tF O S A 40mkd 8G0148 8 8G0149 N M ean SD NE tF O S E 40mkd 8G0148 7 800148 N M ean SD PFOS 40mkd 8G 0I49 1 CHOL m g/dL1 63 CA m g/dL 13.1 PHOS m g/dL 9.0 TBIL m g/dL 0.2 ALB g/dL 2.7 TP g/dL 5.6 BUN m g/dL 14 GLU m g/dL 416 ALKP U /L 156 AST U /L 46 ALT U /L 50 LDH U /L 257 Na+ mm ol/L K+ m m ol/L 141 N R Clm m ol/L CREA m g/dl TRIG m g/dL 103 0.5 124 62 11.9 9.7 0.1 2.4 5.0 13 202 117 45 46 118 144 12.0 102 0.4 132 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 23 NR N R 0.2 1.9 4.3 14 310 202 37 42 133 141 12.9 104 0.3 78 3 2 2 3 3 33 3 3 33 3 3 2 3 33 49.2 12.50 9.35 0.17 2.33 4 .9 7 13.7 309.3 158.3 42.7 46.0 169. 142.0 12.45 103.0 0.40 111.3 23.1 0.85 0.49 0.06 0.40 0.65 0.6 107.0 42.5 4.9 4.0 76.3 1.7 0.64 1.0 0.10 29.1 67 11.6 8.7 0.2 2 .6 5.7 17 198 126 38 49 160 138 5.8 100 0.5 107 116 2 91.5 34.6 70 12.6 8.6 2 12.10 2 8.65 0.71 0.07 11.7 9.5 0.2 2 0.20 0.00 0.1 2 .9 6.3 13 2 22 2.75 6.00 15.0 0.21 0.42 2.8 2.5 5.5 17 263 2 230.5 46.0 182 122 38 56 158 2 124.0 2 38.0 2 52.5 2 159. 2.8 0.0 4.9 1.4 121 32 34 117 138 2 138.0 0.0 141 6.6 2 6.20 0.57 11.0 100 2 100.0 0.0 99 0.5 2 0.50 0.00 0.6 143 2 125.0 25.5 96 23 2 46.3 33.6 75 12.7 9.2 2 12.20 2 9.35 0.71 0.21 12.4 10.2 0.2 2 0.15 0.07 0.1 2.4 5.0 10 2 2.45 2 5.25 2 13.5 0.07 0.35 4.9 2.6 5.6 15 286 2 234.0 73.5 326 125 32 43 166 2 123.0 222 32.0 38.5 141. 2.8 0.0 6.4 34.6 163 77 60 286 141 2 141.0 0.0 133 13.2 2 12.10 1.56 6.1 104 2 101.5 3.5 100 0.4 2 0.50 0.14 0.5 117 2 106.5 14.8 127 65 T-7071.1 FR DTI 5 8G0149 2 9G0005 5 9G0005 6 N M ean 71 NR NR 2 73.0 11.9 7.4 0.4 2.6 5.6 NR NR NR NR NR NR NR NR NR NR 2 2 2 22 12.15 8.80 0.23 2.60 5.60 SD 2.8 0.35 1.98 0.25 0.00 0.00 M CONT 8G0147 23 7 8G 0I47 48 8 9G 0004 23 5 9G0004 NR 6 N3 M ean 31.0 11.2 8.3 11.7 9.2 NR NR NR NR 2 11.45 2 8.75 0.3 0.1 0.1 NR 3 0.15 2.5 5.3 2.4 5.1 1.7 4 .0 NR NR 33 2.20 4.80 SD 14.7 0.35 0.64 0.13 0.44 0.70 NE tF O S A 40mkd 8G0148 1 8G0148 2 N M ean SD NE tF O S E 40mkd 8G0147 9 8G0148 0 N M ean SD 55 49 2 52.0 4.2 23 23 2 22.5 0.0 12.8 9.7 0.1 2.6 5.5 11.8 8.9 2 12.30 2 9.30 0.71 0.57 12.8 9.0 0.1 2 0.10 0.00 0.3 2.6 5.5 2 2.60 2 5.50 0.00 0.00 2.6 5.4 12.4 9.6 2 12.60 2 9.30 0.28 0.42 0.1 2 0.18 0.18 2.3 4.8 22 2.45 5.10 0.21 0.42 coooso 10 NR NR 2 12.5 3.5 15 16 16 NR 3 15.7 0.6 12 315 110 44 55 344 134 NR NR NR NR NR NR NR NR NR NR NR NR 2 320.5 7.8 2 136.5 37.5 22 2 60.5 57.5 315. 0 23.3 3.5 41.0 2 133.5 0.7 154 110 64 45 303 144 211 194 49 52 113 140 275 3 2 9 36 52 141 141 NR NR NR NR NR NR 3 213.3 3 211.0 333 49.7 49.7 185. 3 141.7 60.5 110.5 14.0 4.0 102. 6 2.1 485 147 54 51 2 0 8 135 4.1 NR NR 2 5.10 1.41 12.1 NR 14.2 NR 2 13.15 1.48 7.5 100 NR NR 2 100.0 0.0 103 103 107 NR 3 104.3 2.3 97 0.5 NR NR 2 0.50 0.00 0.5 0.4 0.3 NR 3 0.40 0.10 0.6 101 NR NR 2 114.0 18.4 168 183 84 NR 3 145.0 53.4 201 16 2 14.0 2.8 13 194 2 339.5 205.8 449 128 2 137.5 13.4 163 47 57 218 2 50.5 4.9 2 54.0 4.2 2 213. 0 7.1 117 62 514 136 2 135.5 0.7 138 6.7 2 7.10 0.57 12.7 101 2 99.0 2.8 98 0.7 103 22 0.65 152.0 0.07 69.3 0.5 154 16 2 14.5 2.1 372 2 410.5 54.4 108 2 135.5 38.9 41 47 151 2 79.0 2 54.5 2 332. 53.7 10.6 256. 7 140 2 139.0 1.4 11.9 2 12.30 0.57 103 2 100.5 3.5 0.5 2 0.50 0.00 126 2 140.0 19.8 66 T-7071.1 FR DTI 5 PFOS 40mkd 8G0148 3 8G0148 4 9G0004 9 9G0005 0 N M ean SD 52 54 NR 23 3 42.8 17.6 12.6 9.1 11.6 7.6 NR NR NR NR 2 12.10 2 8.35 0.71 1.06 0.1 0.2 NR 0.3 3 0.20 0.10 3.0 6.2 2.9 6.2 NR NR 1.8 4.5 33 2.57 5.63 0.67 0.98 CONT 8R04040 8R04041 9R00469 9R00470 N M ean 96 77 86 81 4 85.0 SD 8.2 NEtFO SA 40mkd 8R04044 8R04045 N M ean 82 88 2 85.0 SD 4.2 NE tF O S E 160mkd 9R00471 NR 9R00472 N M ean SD NR 0 NR NR NE tF O S E 40mkd 8R04042 8R04043 70 89 12.9 12.1 NR NR 2 12.50 10.5 10.7 NR NR 2 10.60 0.4 0.3 0.4 0.4 4 0.38 0.57 0.14 0.05 11.8 8.2 0.3 4.3 3.8 3.1 3.6 4 3.70 7.1 6.7 5.9 6.5 4 6.55 0.50 0.50 3.8 7.0 12.2 2 12.00 8.3 2 8.25 0.28 0.07 NR NR 0.3 2 0.30 0.00 0.4 4.1 7.4 22 3.95 7.20 0.21 0.28 NR NR NR 0 NR NR 11.3 NR 0 NR NR 9.5 NR 1 0.40 NR 0.2 NR NR 00 NR NR NR NR 3.5 6.7 12.0 10.1 0.3 3.7 6.8 16 17 NR 16 3 16.3 0.6 15 13 20 19 4 16.8 3.3 14 342 131 NR 248 3 240.3 105.7 426 351 188 215 4 295.0 112.8 328 102 115 NR 270 3 162.3 93.5 267 214 253 274 4 252.0 26.8 283 49 57 131 47 62 155 NR NR NR 70 66 348 3 55.3 12.7 3 61.7 4.5 3 211. T D 119. 0 86 84 80 94 4 86.0 5.9 76 69 72 78 4 73.8 4.0 374 343 416 307 4 360. 0 46.3 69 66 139 133 138 NR 134 3 135.0 2.6 149 149 148 148 4 148.5 0.6 151 4.1 2.7 NR 6.4 3 4.40 1.87 6.8 5.7 7.2 7.4 4 6.78 0.76 4.5 95 101 NR 103 3 99.7 4.2 0.8 0.7 NR 0.3 3 0.60 0.26 118 94 NR 85 3 99.0 17.1 98 98 105 104 4 101.3 3.8 106 0.6 0.5 0.4 0.5 4 0.50 0.08 0.7 75 57 81 62 4 68.8 11.1 53 19 2 16.5 3.5 24 315 2 321.5 9.2 NR 234 2 258.5 34.6 184 80 2 74.5 7.8 70 2 68.0 2.8 180 2 159. C J 29.0 60 77 NR 150 2 150.5 0.7 NR 4.6 2 4.55 0.07 NR 102 2 104.0 2.8 NR 0.6 2 0.65 0.07 NR 70 2 61.5 12.0 35 NR NR NR NR NR NR NR NR NR NR NR 10 1 110 0 0 0 01 24.0 NR 184.0 60.0 77.0 NR NR NR NR NR 35.0 NR NR NR NR NR NR NR NR NR NR NR 14 248 2 3 6 71 56 2 1 9 147 5.1 105 0.6 40 18 318 229 77 87 222 147 5.7 100 0.5 105 67 T-7071.1 FR DT15 PFOS 40mkd N M ean 2 79.5 SD 13.4 8R04046 57 8R04047 9R00473 9R00474 N M ean 23 NR NR 2 39.8 SD 24.4 2 11.65 2 9.80 0.49 0.42 11.9 8.1 12.4 NR NR 2 12.15 8.1 NR NR 2 8.10 0.35 0.00 2 0.25 0.07 0.3 0.4 NR NR 2 0.35 0.07 2 3.60 2 6.75 0.14 0.07 4.1 7.5 4.6 NR NR 2 4.35 8.0 NR NR 2 7.75 0.35 0.35 CONT 8R04032 8R04033 9R00463 9R00464 N M ean 88 73 92 86 4 84.8 SD 8.2 12.0 11.6 NR NR 2 11.80 11.0 11.1 NR NR 2 11.05 0.4 0.4 0.4 0.4 4 0.40 0.28 0.07 0.00 3.6 6.5 3.7 6.8 3.7 6.6 3.1 6.0 44 3.53 6.48 0.29 0.34 NE tF O S A 40mkd 8R04036 8R04037 N M ean 75 79 2 77.0 SD 2.8 NE tF O S E 160mkd 9R00465 9R00466 N M ean 50 NR 1 50.0 SD NR N- 8R04034 63 11.8 9.4 0.2 3.4 6.4 11.9 2 11.85 10.5 2 9.95 0.07 0.78 NR NR 0.3 2 0.25 0.07 0.4 3.5 6.6 22 3.45 6.50 0.07 0.14 3.1 6.0 NR 0 NR NR 12.2 NR 0 NR NR 11.0 0.4 2 0.40 0.00 0.4 3.4 6.4 22 3.25 6.20 0.21 0.28 3.7 7.0 2 16.0 . 2 283.0 2.8 49.5 2 232.5 4.9 2 74.0 4.2 2 71.5 21.9 2 220. D 2.1 2 147.0 0.0 16 303 165 58 66 197 147 20 NR NR 2 18.0 2.8 345 NR NR 2 324.0 29.7 153 NR NR 2 159.0 8.5 66 NR NR 2 62.0 77 NR NR 2 71.5 5.7 7.8 443 NR NR 2 320. 0 173. 9 146 NR NR 2 146.5 0.7 18 15 25 25 4 20.8 5.1 376 216 327 249 4 292.0 72.8 239 301 325 308 4 293.3 37.5 80 87 72 130 4 92.3 25.9 71 70 65 85 4 72.8 8.6 330 746 310 1291 4 669. J 460. 7 150 150 153 148 4 150.3 2.1 14 300 341 68 54 179 - 152 2 5.40 0.42 3.2 3.3 NR NR 2 3.25 0.07 7.5 5.4 5.9 8.5 4 6.83 1.43 4.0 2 102.5 3.5 102 104 NR NR 2 103.0 1.4 2 0.55 0.07 0.6 0.7 NR NR 2 0.65 0.07 2 72.5 46.0 43 44 NR NR 2 43.5 0.7 99 99 101 101 4 100.0 1.2 0.6 0.5 0.5 0.4 4 0.50 0.08 82 132 105 137 4 114.0 25.5 104 0.6 85 17 2 15.5 2.1 19 347 2 323.5 33.2 178 335 2 338.0 4.2 238 84 2 76.0 60 2 57.0 178 2 178. 11.3 4.2 0.7 63 94 227 151 2 151.5 0.7 143 5.0 2 4.50 0.71 6.6 102 2 103.0 1.4 104 0.6 2 0.60 0.00 0.6 86 2 85.5 0.7 39 23 2 21.0 2.8 16 244 2 211.0 46.7 330 160 2 199.0 55.2 68 2 65.5 89 2 91.5 3.5 3.5 241 2 f2t34. 9.9 266 69 67 422 148 2 145.5 3.5 153 4.4 2 5.50 1.56 5.2 104 2 104.0 0.0 99 0.5 2 0.55 0.07 0.5 24 2 31.5 10.6 114 68 T-7071.1 FR DTI 5 E tF O S E 40mkd 8R04035 N M ean 70 2 66.5 SD 4.9 PFOS 40mkd 8R04038 63 8R04039 9R00467 9R00468 N M ean 49 59 NR 3 57.0 SD 7.2 11.0 2 11.60 9.8 2 10.40 0.2 2 0.30 0.85 0.85 0.14 12.3 10.6 0.6 12.2 NR NR 2 12.25 9.6 NR NR 2 10.10 0.4 0.4 NR 3 0.47 0.07 0.71 0.12 3.4 6.6 19 2 22 3.55 6.80 17.5 0.21 0.28 2.1 4.6 7.8 21 4.5 4.0 NR 3 4.37 7.8 7.1 NR 3 7.57 20 17 NR 3 19.3 0.32 0 .40 2.1 243 2 286.5 61.5 365 346 379 NR 3 363.3 16.6 258 2 262.0 5.7 68 66 22 68.5 66.5 0.7 0.7 272 2 347. 0 106. 1 203 71 90 527 160 227 NR 3 196.7 77 59 NR 3 69.0 93 83 NR 3 88.7 482 274 NR 3 427. 33.9 9.2 5.1 135. 0 150 2 151.5 2.1 148 150 147 NR 3 148.3 1.5 4.8 2 5.00 0.28 3.1 3.0 4.6 NR 3 3.57 0.90 101 0.6 70 2 22 100.0 0.55 92.0 1.4 0.07 31.1 100 103 101 NR 3 101.3 1.5 0.8 0.7 0.6 NR 3 0.70 0.10 55 54 42 NR 3 50.3 7.2 1. The LOD for CHOL was 45 mg/dL. When CHOL < LOD, half of LOD (22.5 mg/dL) was entered to perform statistics C 80G 0 3 69 T-7071.1 FR DTI 5 r> o o 00 > 70 T-7071.1 FR DTI 5 A. Statistics on Clinical Chemistries for rats, male and female values combined. CHOL mg/dL By Dose Group (mg/Kg/day) Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or SumWgts) 0.701781 0.631612 10.64141 72.52273 22 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 4530.1636 17 1925.0750 21 6455.2386 Mean Square 1132.54 113.24 307.39 F Ratio 10.0013 Prob>F 0.0002 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 1 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 84.8750 81.0000 50.0000 73.0000 50.1000 Std Error 3.762 5.321 10.641 5.321 4.759 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 84.8750 7.6052 N-EtFOSA40mkd 4 81.0000 5.4772 N-EtFOSE160mkd 1 50.0000 ? N-EtFOSE40mkd 4 73.0000 11.1654 PFOS40mkd 5 50.1000 16.2496 Std Err Mean 2.6888 2.7386 ? 5.5827 7.2670 Dif=Mean[i]-Mean[j] CONT N-EtFOSA40mkd CONT 0.0000 -3.8750 Means Comparisons N-EtFOSA40mkd 3.8750 0.0000 N-EtFOSE40mkd 11.8750 8.0000 PFOS40mkd 34.7750 30.9000 N-EtFOSE160mkd 34.8750 31.0000 71 C009S5 T-7071.1 FR DTI 5 N-EtFOSE40mkd PFOS40mkd N-EtFOSE 160mkd -11.8750 -34.7750 -34.8750 -8.0000 -30.9000 -31.0000 0.0000 -22.9000 -23.0000 22.9000 0.0000 -0.1000 23.0000 0.1000 0.0000 Alpha= Abs(Dif)-LSD CONT N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd N-EtFOSE 160mkd 0.05 Comparisons for each pair using Student's t t 2.10980 CONT N-EtFOSA40mkd N-EtFOSE40mkd -11.2256 -9.8736 -1.8736 -9.8736 -15.8755 -7.8755 -1.8736 -7.8755 -15.8755 21.9758 15.8392 7.8392 11.0618 5.8987 -2.1013 PFOS40mkd 21.9758 15.8392 7.8392 -14.1994 -24.4942 N-EtFOSE160mkd 11.0618 5.8987 -2.1013 -24.4942 -31.7509 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.74189 Abs(Dif)-LSD CONT CONT -14.5888 N-EtFOSA40mkd N-EtFOSE40mkd -13.9926 -5.9926 PFOS40mkd 18.1412 N-EtFOSE 160mkd 3.9275 Positive values show pairs of means that are significantly different. Cholesterol in rats male and female combined for DT15. Sig reduction by 40 mkd PFIOS and 160 mkd N-EtFOSE. 72 CC00S6 T-7071.1 FR DTI 5 Ca mg/dL By Dose Group (mg/Kg/day) C^00S7 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.28157 0.101962 0.418828 11.975 16 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 0.8250000 12 2.1050000 15 2.9300000 Mean Square 0.275000 0.175417 0.195333 F Ratio 1.5677 Prob>F 0.2484 Means for Oneway Anova Level Number CONT 4 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 4 Mean 12.1500 11.9250 11.6250 12.2000 Std Error 0.20941 0.20941 0.20941 0.20941 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 12.1500 0.544671 N-EtFOSA40mkd 4 11.9250 0.189297 N-EtFOSE40mkd 4 11.6250 0.567891 PFOS40mkd 4 12.2000 0.216025 Std Err Mean 0.27234 0.09465 0.28395 0.10801 Dif=Mean[i]-Mean[j] PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd Means Comparisons PFOS40mkd CONT 0.000000 0.050000 -0.05 0.000000 -0.275 -0.225 -0.575 -0.525 N-EtFOSA40mkd 0.275000 0.225000 0.000000 -0.3 N-EtFOSE40mkd 0.575000 0.525000 0.300000 0.000000 Alpha= Abs(Dif)-LSD PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.17882 PFOS40mkd CONT N-EtFOSA40mkd -0.64527 -0.59527 -0.37027 -0.59527 -0.64527 -0.42027 -0.37027 -0.42027 -0.64527 -0.07027 -0.12027 -0.34527 N-EtFOSE40mkd -0.07027 -0.12027 -0.34527 -0.64527 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.68294 Abs(Dif)-LSD PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd CONT -0.74457 -0.79457 -0.56957 -0.26957 Positive values show pairs of means that are significantly different. PHOS mg/dL By Dose Group (mg/Kg/day) 74 ONDOSO T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or SumWgts) 0.471871 0.339839 0.889171 9.78125 16 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 8.476875 12 9.487500 15 17.964375 Mean Square 2.82562 0.79063 1.19763 F Ratio 3.5739 Prob>F 0.0470 Means for Oneway Anova Level Number CONT 4 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 4 Mean 10.8250 9.1000 10.1000 9.1000 Std Error 0.44459 0.44459 0.44459 0.44459 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 10.8250 0.27538 N-EtFOSA40mkd 4 9.1000 1.08012 N-EtFOSE40mkd 4 10.1000 0.64807 PFOS40mkd 4 9.1000 1.22474 Std Err Mean 0.13769 0.54006 0.32404 0.61237 Dif=Mean[i]-Mean[j] CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd Means Comparisons CONT N-EtFOSE40mkd 0.00000 0.72500 -0.72500 0.00000 -1.72500 -1.00000 -1.72500 -1.00000 N-EtFOSA40mkd 1.72500 1.00000 0.00000 0.00000 PFOS40mkd 1.72500 1.00000 0.00000 0.00000 Alpha= Abs(Dif)-LSD CONT 0.05 Comparisons for each pair using Student's t t 2.17882 CONT N-EtFOSE40mkd N-EtFOSA40mkd -1.36991 -0.64491 0.35509 PFOS40mkd 0.35509 0^0999 T-7071.1 FR DT15 N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd -0.64491 0.35509 0.35509 -1.36991 -0.36991 -0.36991 -0.36991 -1.36991 -1.36991 Positive values show pairs of means that are significantly different. Male and female rats combined Ca and Phos clinical chemistries. No sig change from control. -0.36991 -1.36991 -1.36991 76 CC0090 T-7071.1 FR DTI 5 7C C 0 0 9 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.485514 0.377201 0.069491 0.358333 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 0.08658333 19 0.09175000 23 0.17833333 Mean Square 0.021646 0.004829 0.007754 F Ratio 4.4825 Prob>F 0.0102 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 0.387500 0.275000 0.400000 0.275000 0.420000 Std Error 0.02457 0.03475 0.04012 0.03475 0.03108 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 0.387500 0.035355 N-EtFOSA40mkd 4 0.275000 0.050000 N-EtFOSE160mkd 3 0.400000 0.000000 N-EtFOSE40mkd 4 0.275000 0.095743 PFOS40mkd 5 0.420000 0.109545 Std Err Mean 0.01250 0.02500 0.00000 0.04787 0.04899 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSE160mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd Means Comparisons PFOS40mkd N-EtFOSE160mkd 0.000000 0.020000 -0.02 0.000000 -0.0325 -0.0125 -0.145 -0.125 -0.145 -0.125 CONT 0.032500 0.012500 0.000000 -0.1125 -0.1125 N-EtFOSA40mkd 0.145000 0.125000 0.112500 0.000000 0.000000 N-EtFOSE40mkd 0.145000 0.125000 0.112500 0.000000 0.000000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSE160mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.09301 PFOS40mkd N-EtFOSE160mkd CONT -0.09199 -0.08622 -0.05042 -0.08622 -0.11875 -0.08597 -0.05042 -0.08597 -0.07272 0.047433 0.013915 0.023434 0.047433 0.013915 0.023434 N-EtFOSA40mkd 0.047433 0.013915 0.023434 -0.10284 -0.10284 N-EtFOSE40mkd 0.047433 0.013915 0.023434 -0.10284 -0.10284 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT PFOS40mkd -0.07464 N-EtFOSE160mkd -0.11473 CONT -0.09396 N-EtFOSA40mkd -0.00258 N-EtFOSE40mkd -0.00258 78C ^ 0 0 9 2 T-7071.1 FR DT15 Positive values show pairs of means that are significantly different. Above is for rat TBIL, male and femal combined data, no sig changes. 79 CC0093 T-7071.1 FR DTI 5 80 C00094 T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.595877 0.506072 0.315634 3.752174 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 2.6441413 18 1.7932500 22 4.4373913 Mean Square 0.661035 0.099625 0.201700 F Ratio 6.6352 Prob>F 0.0018 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 3.61250 3.70000 3.25000 3.57500 4.36000 Std Error 0.11159 0.15782 0.22319 0.15782 0.14116 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 3.61250 0.387068 N-EtFOSA40mkd 4 3.70000 0.316228 N-EtFOSE160mkd 2 3.25000 0.212132 N-EtFOSE40mkd 4 3.57500 0.150000 PFOS40mkd 5 4.36000 0.288097 Std Err Mean 0.13685 0.15811 0.15000 0.07500 0.12884 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.00000 0.66000 -0.66000 0.00000 -0.74750 -0.08750 -0.78500 -0.12500 -1.11000 -0.45000 CONT 0.74750 0.08750 0.00000 -0.03750 -0.36250 N-EtFOSE40mkd 0.78500 0.12500 0.03750 0.00000 -0.32500 N-EtFOSE160mkd 1.11000 0.45000 0.36250 0.32500 0.00000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd CONT -0.41939 0.215166 0.369464 0.215166 -0.4689 -0.31858 0.369464 -0.31858 -0.33156 0.340166 -0.3439 -0.36858 0.555195 -0.12428 -0.16174 N-EtFOSE40mkd 0.340166 -0.3439 -0.36858 -0.4689 -0.24928 N-EtFOSE160mkd 0.555195 -0.12428 -0.16174 -0.24928 -0.66312 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd CONT 0.257735 -0.43859 -0.42955 -0.48859 -0.31668 81 0^0095 T-7071.1 FR DTI 5 Positive values show pairs of means that are significantly Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.674822 0.60256 0.361958 6.834783 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 4.8939239 18 2.3582500 22 7.2521739 Mean Square 1.22348 0.13101 0.32964 F Ratio 9.3386 Prob>F 0.0003 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 6.51250 6.85000 6.20000 6.77500 7.64000 Std Error 0.12797 0.18098 0.25594 0.18098 0.16187 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 6.51250 0.397986 N-EtFOSA40mkd 4 6.85000 0.443471 N-EtFOSE160mkd 2 6.20000 0.282843 N-EtFOSE40mkd 4 6.77500 0.170783 PFOS40mkd 5 7.64000 0.350714 Std Err Mean 0.14071 0.22174 0.20000 0.08539 0.15684 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.00000 0.79000 -0.79000 0.00000 -0.86500 -0.07500 -1.12750 -0.33750 -1.44000 -0.65000 N-EtFOSE40mkd 0.86500 0.07500 0.00000 -0.26250 -0.57500 CONT 1.12750 0.33750 0.26250 0.00000 -0.31250 N-EtFOSE160mkd 1.44000 0.65000 0.57500 0.31250 0.00000 C0096 T-7071.1 FR DTI 5 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd -0.48095 0.279880 0.354880 0.279880 -0.53771 -0.46271 0.354880 -0.46271 -0.53771 0.693982 -0.12817 -0.20317 0.803769 -0.00856 -0.08356 CONT 0.693982 -0.12817 -0.20317 -0.38022 -0.28868 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT N-EtFOSE160mkd CONT 0.565854 -0.2658 -0.3408 -0.4926 -0.46636 Positive values show pairs of means that are significantly different. GLU mg/dL By Dose Group (mg/Kg/day) N-EtFOSE160mkd 0.803769 -0.00856 -0.08356 -0.28868 -0.76044 400 - 350 - -j 1) 300 ~ r> _j o x \ A .n / _i. \ i __ __ 4 \ A A r* \ / \ A / \ \ */ \_/ w ;/ ' { / ; - .....' ' \ \ ///V ' ' \ '\\\ s^ 250 - 'vV ' J \ \ a/ O' 200 - ;' i \I i CONT 1L J 1 1 N-EtFOSf)hWaFOSE40mkd ' Each Pair 4fetFOSE160ffl84 ^Student's t Dose Group (mg/Kg/day) 0.06 ......... , ' With Control , 7 Dunnett's ' \ 0.05 Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.312227 0.159389 60.99861 301.6087 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 30404.528 18 66974.950 22 97379.478 Mean Square 7601.13 3720.83 4426.34 F Ratio 2.0429 Prob>F 0.1312 Level CONT Means for Oneway Anova Number Mean 8 293.500 Std Error 21.566 83 GC0997 T-7071.1 FR DTI 5 N-EtFO$A40mkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd 4 322.500 2 211.000 4 284.750 5 347.600 30.499 43.133 30.499 27.279 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 293.500 87.8879 N-EtFOSA40mkd 4 322.500 19.9416 N-EtFOSE160mkd 2 211.000 46.6690 N-EtFOSE40mkd 4 284.750 45.6317 PFOS40mkd 5 347.600 28.6671 Std Err Mean 31.073 9.971 33.000 22.816 12.820 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0 .0 0 0 25.100 -25.100 0 .0 0 0 -54.100 -29.000 -62.850 -37.750 -136.600 -111.500 CONT 54.100 29.000 0.000 -8.750 -82.500 N-EtFOSE40mkd 62.850 37.750 8.750 0 .0 0 0 -73.750 N-EtFOSE160mkd 136.600 111.500 82.500 73.750 0 .0 0 0 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t 4 PFOS40mkd -81.051 -60.867 -18.958 -23.117 29.380 2.10091 N-EtFOSA40mkd -60.867 -90.617 -49.477 -52.867 0.517 CONT -18.958 -49.477 -64.076 -69.727 -18.813 N-EtFOSE40mkd -23.117 -52.867 -69.727 -90.617 -37.233 N-EtFOSE160mkd 29.380 0.517 -18.813 -37.233 -128.152 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Idl 2.72184 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd CONT -40.5507 -72.6711 -83.0141 -92.9211 -48.7569 Positive values show pairs of means that are significantly different. 84 GC0098 T-7071.1 FR DT15 4.5 4.0 - *5) E 3.5 - W - wA Alb mg/dL By Dose Group (mg/Kg/day) /f\ o oA A W 0!VV. /f. ii t\ --// ' .... \ / / > --A v \ .. i_\ V I/ \ ./ 3.0 ' ------------------------- --------------------- CONI N-EFOS4$OMFOSE40mkd N-ElFOSEl6B?aSi Dose Group (mg/Kg/day) Student's! 0.05 -Dunnett's 0.05 85 "0099 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.595877 0.506072 0.315634 3.752174 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 2.6441413 18 1.7932500 22 4.4373913 Mean Square 0.661035 0.099625 0.201700 F Ratio 6.6352 Prob>F 0.0018 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 3.61250 3.70000 3.25000 3.57500 4.36000 Std Error 0.11159 0.15782 0.22319 0.15782 0.14116 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 3.61250 0.387068 N-EtFOSA40mkd 4 3.70000 0.316228 N-EtFOSE160mkd 2 3.25000 0.212132 N-EtFOSE40mkd 4 3.57500 0.150000 PFOS40mkd 5 4.36000 0.288097 Std Err Mean 0.13685 0.15811 0.15000 0.07500 0.12884 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.00000 0.66000 -0.66000 0.00000 -0.74750 -0.08750 -0.78500 -0.12500 -1.11000 -0.45000 CONT 0.74750 0.08750 0.00000 -0.03750 -0.36250 N-EtFOSE40mkd 0.78500 0.12500 0.03750 0.00000 -0.32500 N-EtFOSE160mkd 1.11000 0.45000 0.36250 0.32500 0.00000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd CONT -0.41939 0.215166 0.369464 0.215166 -0.4689 -0.31858 0.369464 -0.31858 -0.33156 0.340166 -0.3439 -0.36858 0.555195 -0.12428 -0.16174 N-EtFOSE40mkd 0.340166 -0.3439 -0.36858 -0.4689 -0.24928 N-EtFOSE 160mkd 0.555195 -0.12428 -0.16174 -0.24928 -0.66312 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Idi 2.72184 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd CONT 0.257735 -0.43859 -0.42955 -0.48859 -0.31668 C C O lO O T-7071.1 FR DTI 5 Positive values show pairs of means that are significantly Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.674822 0.60256 0.361958 6.834783 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 4.8939239 18 2.3582500 22 7.2521739 Mean Square 1.22348 0.13101 0.32964 F Ratio 9.3386 Prob>F 0.0003 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 6.51250 6.85000 6.20000 6.77500 7.64000 Std Error 0.12797 0.18098 0.25594 0.18098 0.16187 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 6.51250 0.397986 N-EtFOSA40mkd 4 6.85000 0.443471 N-EtFOSE160mkd 2 6.20000 0.282843 N-EtFOSE40mkd 4 6.77500 0.170783 PFOS40mkd 5 7.64000 0.350714 Std Err Mean 0.14071 0.22174 0.20000 0.08539 0.15684 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.00000 0.79000 -0.79000 0.00000 -0.86500 -0.07500 -1.12750 -0.33750 -1.44000 -0.65000 N-EtFOSE40mkd 0.86500 0.07500 0.00000 -0.26250 -0.57500 CONT 1.12750 0.33750 0.26250 0.00000 -0.31250 N-EtFOSE160mkd 1.44000 0.65000 0.57500 0.31250 0.00000 87C O 0101 T-7071.1 FR DTI 5 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd -0.48095 0.279880 0.354880 0.279880 -0.53771 -0.46271 0.354880 -0.46271 -0.53771 0.693982 -0.12817 -0.20317 0.803769 -0.00856 -0.08356 CONT 0.693982 -0.12817 -0.20317 -0.38022 -0.28868 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT 0.565854 -0.2658 -0.3408 CONT N-EtFOSE160mkd -0.4926 -0.46636 Positive values show pairs of means that are significantly different. N-EtFOSE 160mkd 0.803769 -0.00856 -0.08356 -0.28868 -0.76044 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or SumWgts) 0.312227 0.159389 60.99861 301.6087 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 30404.528 18 66974.950 22 97379.478 Mean Square 7601.13 3720.83 4426.34 F Ratio 2.0429 Prob>F 0.1312 Level CONT Means for Oneway Anova Number Mean 8 293.500 Std Error 21.566 88 C00102 T-7071.1 FR DTI 5 N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd 4 322.500 2 211.000 4 284.750 5 347.600 30.499 43.133 30.499 27.279 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 293.500 87.8879 N-EtFOSA40mkd 4 322.500 19.9416 N-EtFOSE160mkd 2 211.000 46.6690 N-EtFOSE40mkd 4 284.750 45.6317 PFOS40mkd 5 347.600 28.6671 Std Err Mean 31.073 9.971 33.000 22.816 12.820 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40hnkd 0.000 25.100 -25.100 0.000 -54.100 -29.000 -62.850 -37.750 -136.600 -111.500 CONT 54.100 29.000 0.000 -8.750 -82.500 N-EtFOSE40mkd 62.850 37.750 8.750 0.000 -73.750 N-EtFOSE160mkd 136.600 111.500 82.500 73.750 0.000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t * PFOS40mkd -81.051 -60.867 -18.958 -23.117 29.380 2.10091 N-EtFOSA40mkd -60.867 -90.617 -49.477 -52.867 0.517 CONT -18.958 -49.477 -64.076 -69.727 -18.813 N-EtFOSE40mkd -23.117 -52.867 -69.727 -90.617 -37.233 N-EtFOSE160mkd 29.380 0.517 -18.813 -37.233 -128.152 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd CONT -40.5507 -72.6711 -83.0141 -92.9211 -48.7569 Positive values show pairs of means that are significantly different. 89 C09103 T-7071.1 FR DT15 90 CC0104 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.264802 0.110024 3.301116 18.375 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 74.57500 19 207.05000 23 281.62500 Mean Square 18.6438 10.8974 12.2446 F Ratio 1.7108 Prob>F 0.1892 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 18.7500 16.0000 22.0000 16.7500 18.8000 Std Error 1.1671 1.6506 1.9059 1.6506 1.4763 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 18.7500 4.49603 N-EtFOSA40mkd 4 16.0000 2.44949 N-EtFOSE160mkd 3 22.0000 2.64575 N-EtFOSE40mkd 4 16.7500 2.21736 PFOS40mkd 5 18.8000 2.16795 Std Err Mean 1.5896 1.2247 1.5275 1.1087 0.9695 Dif=Mean[i]-Mean[j] N-EtFOSE 160mkd PFOS40mkd CONT N-EtFOSE40mkd N-EtFOSA40mkd Means Comparisons N-EtFOSE 160mkd PFOS40mkd 0.00000 3.20000 -3.20000 0.00000 -3.25000 -0.05000 -5.25000 -2.05000 -6.00000 -2.80000 CONT 3.25000 0.05000 0.00000 -2.00000 -2.75000 N-EtFOSE40mkd 5.25000 2.05000 2.00000 0.00000 -0.75000 N-EtFOSA40mkd 6.00000 2.80000 2.75000 0.75000 0.00000 Alpha= Abs(Dif)-LSD N-EtFOSE 160mkd PFOS40mkd CONT N-EtFOSE40mkd N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t t 2.09301 N-EtFOSE160mkd PFOS40mkd CONT -5.64139 -1.84581 -1.42760 -1.84581 -4.36980 -3.88889 -1.42760 -3.88889 -3.45463 -0.02704 -2.58488 -2.23104 0.72296 -1.83488 -1.48104 N-EtFOSE40mkd -0.02704 -2.58488 -2.23104 -4.88559 -4.13559 N-EtFOSA40mkd 0.72296 -1.83488 -1.48104 -4.13559 -4.88559 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT N-EtFOSE160mkd -2.79392 PFOS40mkd -5.03944 CONT -4.46373 N-EtFOSE40mkd -3.46693 N-EtFOSA40mkd -2.71693 91 croios T-7071.1 FR DTI 5 Positive values show pairs of means that are significantly Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.63982 0.563993 36.65097 243.875 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 45338.050 19 25522.575 23 70860.625 Mean Square 11334.5 1343.3 3080.9 F Ratio 8.4379 Prob>F 0.0004 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 272.625 298.250 194.000 247.250 181.600 Std Error 12.958 18.325 21.160 18.325 16.391 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 272.625 37.3896 N-EtFOSA40mkd 4 298.250 50.1290 N-EtFOSE160mkd 3 194.000 39.9500 N-EtFOSE40mkd 4 247.250 17.5760 PFOS40mkd 5 181.600 31.9343 Std Err Mean 13.219 25.065 23.065 8.788 14.281 Dif=Mean[i]-Mean[il N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd PFOS40mkd Means Comparisons N-EtFOSA40mkd CONT 0.000 25.625 -25.625 0.000 -51.000 -25.375 -104.250 -78.625 -116.650 -91.025 N-EtFOSE40mkd 51.000 25.375 0.000 -53.250 -65.650 N-EtFOSE160mkd 104.250 78.625 53.250 0.000 -12.400 PFOS40mkd 116.650 91.025 65.650 12.400 0.000 9C C 0 3 . 0 6 T-7071.1 FR DTI 5 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.09301 N-EtFOSA40mkd CONT N-EtFOSE40mkd -54.2427 -21.3506 -3.2427 -21.3506 -38.3554 -21.6006 -3.2427 -21.6006 -54.2427 45.6612 26.6915 -5.3388 65.1908 47.2931 14.1908 N-EtFOSE160mkd 45.6612 26.6915 -5.3388 -62.6341 -43.6217 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT N-EtFOSA40mkd -35.0721 CONT -49.5590 N-EtFOSE40mkd -35.3221 N-EtFOSE160mkd 11.5218 PFOS40mkd 34.5190 Positive values show pairs of means that are significantly different. PFOS40mkd 65.1908 47.2931 14.1908 -43.6217 -48.5162 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.468919 0.357112 11.9777 75.875 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 2406.7833 19 2725.8417 23 5132.6250 Mean Square 601.696 143.465 223.158 F Ratio 4.1940 Prob>F 0.0134 Level CONT Means for Oneway Anova Number Mean 8 89.1250 Std Error 4.2348 93 C00107 T-7071.1 FR DTI 5 N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd 4 75.2500 3 63.6667 4 71.2500 5 66.2000 5.9889 6.9153 5.9889 5.3566 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 89.1250 17.7074 N-EtFOSA40mkd 4 75.2500 7.9739 N-EtFOSE160mkd 3 63.6667 4.0415 N-EtFOSE40mkd 4 71.2500 4.0311 PFOS40mkd 5 66.2000 8.0436 Std Err Mean 6.2605 3.9870 2.3333 2.0156 3.5972 Dif=Mean[l]-MeanO] CONT N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd CONT 0.0000 -13.8750 -17.8750 -22.9250 -25.4583 Means Comparisons N-EtFOSA40mkd 13.8750 0.0000 -4.0000 -9.0500 -11.5833 N--EtFOSE40mkd 17.8750 4.0000 0.0000 -5.0500 -7.5833 PFOS40mkd 22.9250 9.0500 5.0500 0.0000 -2.5333 N-EtFOSE160mkd 25.4583 11.5833 7.5833 2.5333 0.0000 Alpha= Abs(Dif)-LSD CONT N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd N-EtFOSE 160mkd 0.05 Comparisons for each pair using Student's t t 2.09301 CONT N-EtFOSA40mkd N-EtFOSE40mkd -12.5347 -1.4768 2.5232 -1.4768 -17.7268 -13.7268 2.5232 -13.7268 -17.7268 8.6332 -7.7671 -11.7671 8.4862 -7.5638 -11.5638 PFOS40mkd 8.6332 -7.7671 -11.7671 -15.8553 -15.7748 N-EtFOSE160mkd 8.4862 -7.5638 -11.5638 -15.7748 -20.4691 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd CONT -16.1961 -5.9611 -1.9611 4.4586 3.5287 Positive values show pairs of means that are significantly different. ALT U/L By Dose Group (mg/Kg/day) 94 CO0 1 0 8 T-7071.1 FR DTI 5 RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.478735 0.368994 9.014511 74.20833 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 1417.9917 19 1543.9667 23 2961.9583 Mean Square 354.498 81.261 128.781 F Ratio 4.3624 Prob>F 0.0114 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 73.2500 62.5000 86.6667 69.0000 81.8000 Std Error 3.1871 4.5073 5.2045 4.5073 4.0314 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 73.2500 6.2278 N-EtFOSA40mkd 4 62.5000 7.0000 N-EtFOSE160mkd 3 86.6667 8.7369 N-EtFOSE40mkd 4 69.0000 12.9872 PFOS40mkd 5 81.8000 10.8028 Std Err Mean 2.2019 3.5000 5.0442 6.4936 4.8311 Dif=Mean[i]-Mean[j] N-EtFOSE160mkd PFOS40mkd CONT N-EtFOSE40mkd N-EtFOSA40mkd Means Comparisons N-EtFOSE 160mkd PFOS40mkd 0.0000 4.8667 -4.8667 0.0000 -13.4167 -8.5500 -17.6667 -12.8000 -24.1667 -19.3000 CONT 13.4167 8.5500 0.0000 -4.2500 -10.7500 N-EtFOSE40mkd 17.6667 12.8000 4.2500 0.0000 -6.5000 Alpha= 0.05 Comparisons for each pair using Student's t t N-EtFOSA40mkd 24.1667 19.3000 10.7500 6.5000 0.0000 95 cn0109 T-7071.1 FR DTI 5 Abs(Dif)-LSD N-EtFOSE160mkd PFOS40mkd CONT N-EtFOSE40mkd N-EtFOSA40mkd N-EtFOSE160mkd -15.4052 -8.9122 0.6433 3.2564 9.7564 2.09301 PFOS40mkd -8.9122 -11.9328 -2.2061 0.1433 6.6433 CONT 0.6433 -2.2061 -9.4337 -7.3039 -0.8039 N-EtFOSE40mkd 3.2564 0.1433 -7.3039 -13.3413 -6.8413 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD N-EtFOSE160mkd PFOS40mkd CONT N-EtFOSE40mkd N-EtFOSA40mkd CONT -3.0877 -5.3480 -12.1893 -10.6788 -4.1788 Positive values show pairs of means that are significantly different. N-EtFOSA40mkd 9.7564 6.6433 -0.8039 -6.8413 -13.3413 RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.502305 0.397527 23.94016 70.41667 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 10990.342 19 10889.492 23 21879.833 Mean Square 2747.59 573.13 951.30 F Ratio 4.7940 Prob>F 0.0076 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 Mean 91.3750 73.5000 32.6667 82.2500 Std Error 8.464 11.970 13.822 11.970 96 C C O ilO T-7071.1 FR DTI 5 PFOS40mkd 47.6000 10.706 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 91.3750 30.2982 N-EtFOSA40mkd 4 73.5000 15.5027 N-EtFOSE160mkd 3 32.6667 7.7675 N-EtFOSE40mkd 4 82.2500 33.9644 PFOS40mkd 5 47.6000 6.3482 Std Err Mean 10.712 7.751 4.485 16.982 2.839 Dif=Mean[i]-Mean[j] CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd N-EtFOSE 160mkd CONT 0.0000 -9.1250 -17.8750 -43.7750 -58.7083 Means Comparisons N-EtFOSE40mkd 9.1250 0.0000 -8.7500 -34.6500 -49.5833 N-EtFOSA40mkd 17.8750 8.7500 0.0000 -25.9000 -40.8333 PFOS40mkd 43.7750 34.6500 25.9000 0.0000 -14.9333 N-EtFOSE 160mkd 58.7083 49.5833 40.8333 14.9333 0.0000 Alpha= Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.09301 CONT N-EtFOSE40mkd N-EtFOSA40mkd -25.0535 -21.5591 -12.8091 -21.5591 -35.4310 -26.6810 -12.8091 -26.6810 -35.4310 15.2096 1.0372 -7.7128 24.7858 11.3135 2.5635 PFOS40mkd 15.2096 1.0372 -7.7128 -31.6904 -21.6596 N-EtFOSE160mkd 24.7858 11.3135 2.5635 -21.6596 -40.9122 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd N-EtFOSE160mkd CONT -32.3716 -30.5220 -21.7720 6.8657 14.8770 Positive values show pairs of means that are significantly different. Combined rat data for males and females. Sig decreases in ALKP, AST and TRIG by 40 mkd PFOS and 160 mkd N-EtFOSE in rats. 97 c r o ili T-7071.1 FR DT15 GLU mg/dL By Dose Group (mg/Kg/day) 98 COOi T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or SumWgts) 0.312227 0.159389 60.99861 301.6087 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 30404.528 18 66974.950 22 97379.478 Mean Square 7601.13 3720.83 4426.34 F Ratio 2.0429 Prob>F 0.1312 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 293.500 322.500 211.000 284.750 347.600 Std Error 21.566 30.499 43.133 30.499 27.279 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 293.500 87.8879 N-EtFOSA40mkd 4 322.500 19.9416 N-EtFOSE160mkd 2 211.000 46.6690 N-EtFOSE40mkd 4 284.750 45.6317 PFOS40mkd 5 347.600 28.6671 Std Err Mean 31.073 9.971 33.000 22.816 12.820 Dif=Mean[i]-MeanQ] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE 160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.000 25.100 -25.100 0.000 -54.100 -29.000 -62.850 -37.750 -136.600 -111.500 CONT 54.100 29.000 0.000 -8.750 -82.500 N-EtFOSE40mkd 62.850 37.750 8.750 0.000 -73.750 N-EtFOSE160mkd 136.600 111.500 82.500 73.750 0.000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE 160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd CONT -81.051 -60.867 -18.958 -60.867 -90.617 -49.477 -18.958 -49.477 -64.076 -23.117 -52.867 -69.727 29.380 0.517 -18.813 N-EtFOSE40mkd -23.117 -52.867 -69.727 -90.617 -37.233 N-EtFOSE160mkd 29.380 0.517 -18.813 -37.233 -128.152 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT PFOS40mkd -40.5507 N-EtFOSA40mkd -72.6711 CONT -83.0141 N-EtFOSE40mkd -92.9211 N-EtFOSE160mkd -48.7569 99 C 0013.3 T-7071.1 FR DTI 5 Positive values show pairs of means that are significantly different. LDH U/L By Dose Group (mg/Kg/day) 1200 - 1100 - 1000 - 900 - 800 - =5 700 - o 600 - 500 - K * /sk y / .\ 400 - \ . / n t~ s / \ 300 200 - a \ 1 \/ \\ / / \f \ ./; / jL / XT' 1 ( ^ CONT \ i\ i K -J 1\ / 1\ ! 1 y 1 N-^tEOSAf3l*FOSE40m kd JElch Pair .. ' With Control , / / V NpFOSE16WieS4 Dose Group (mg/Kg/day) StudW s t 0.05 .......... ' Dunnetfs - 0.05 .. .........- y y ' Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.295198 0.138575 228.9437 361.6957 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 395163.0 18 943473.8 22 1338636.9 Mean Square 98790.8 52415.2 60847.1 F Ratio 1.8848 Prob>F 0.1569 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 514.625 169.000 234.000 283.750 384.600 Std Error 80.94 114.47 161.89 114.47 102.39 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 514.625 345.236 N-EtFOSA40mkd 4 169.000 20.017 N-EtFOSE160mkd 2 234.000 9.899 N-EtFOSE40mkd 4 283.750 95.318 PFOS40mkd 5 384.600 141.952 Std Err Mean 122.06 10.01 7.00 47.66 63.48 Dif=Mean[i]-Mean[j] CONT PFOS40mkd N-EtFOSE40mkd N-EtFOSE160mkd N-EtFOSA40mkd CONT 0.000 -130.025 -230.875 -280.625 -345.625 Means Comparisons PFOS40mkd N-EtFOSE40mkd 130.025 230.875 0.000 100.850 -100.850 0.000 -150.600 -49.750 -215.600 -114.750 N-EtFOSE160mkd 280.625 150.600 49.750 0.000 -65.000 N-EtFOSA40mkd 345.625 215.600 114.750 65.000 0.000 100 C 0 0 1 5 .4 T-7071.1 FR DTI 5 Alpha= Abs(Dif)-LSD CONT PFOS40mkd N-EtFOSE40mkd N-EtFOSE160mkd N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t t 2.10091 CONT PFOS40mkd N-EtFOSE40mkd -240.495 -144.181 -63.670 -144.181 -304.205 -221.808 -63.670 -221.808 -340.111 -99.631 -251.825 -366.799 51.080 -107.058 -225.361 N-EtFOSE 160mkd -99.631 -251.825 -366.799 -480.990 -351.549 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT CONT -311.574 PFOS40mkd -225.224 N-EtFOSE40mkd -150.723 N-EtFOSE160mkd N-EtFOSA40mkd -212.016 -35.973 Positive values show pairs of means that are significantly different. N-EtFOSA40mkd 51.080 -107.058 -225.361 -351.549 -340.111 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.429799 0.303087 1.94829 148.913 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 51.50109 18 68.32500 22 119.82609 Mean Square 12.8753 3.7958 5.4466 F Ratio 3.3919 Prob>F 0.0310 Level CONT Means for Oneway Anova Number 8 Mean 149.375 Std Error 0.6888 I01 CO0115 T-7071.1 FR DT15 N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd 4 151.000 2 145.500 4 149.250 5 147.600 0.9741 1.3776 0.9741 0.8713 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 149.375 1.68502 N-EtFOSA40mkd 4 151.000 0.81650 N-EtFOSE160mkd 2 145.500 3.53553 N-EtFOSE40mkd 4 149.250 2.87228 PFOS40mkd 5 147.600 1.51658 Std Err Mean 0.5957 0.4082 2.5000 1.4361 0.6782 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd CONT N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd Means Comparisons N-EtFOSA40mkd CONT 0.00000 1.62500 -1.62500 0.00000 -1.75000 -0.12500 -3.40000 -1.77500 -5.50000 -3.87500 N-EtFOSE40mkd 1.75000 0.12500 0.00000 -1.65000 -3.75000 PFOS40mkd 3.40000 1.77500 1.65000 0.00000 -2.10000 N-EtFOSE160mkd 5.50000 3.87500 3.75000 2.10000 0.00000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd CONT N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 N-EtFOSA40mkd CONT N-EtFOSE40mkd -2.89431 -0.88155 -1.14431 -0.88155 -2.04659 -2.38155 -1.14431 -2.38155 -2.89431 0.65421 -0.55847 -1.09579 1.95520 0.63906 0.20520 PFOS40mkd 0.65421 -0.55847 -1.09579 -2.58875 -1.32460 N-EtFOSE 160mkd 1.95520 0.63906 0.20520 -1.32460 -4.09318 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD N-EtFOSA40mkd CONT N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd CONT -1.62237 -2.65146 -3.12237 -1.24813 -0.31733 Positive values show pairs of means that are significantly different. K+ mmol/L By Dose Group (mg/Kg/day) 102 C ^ O i l G T-7071.1 FR DTI 5 9- . 8- () 6- /\ A /^ \ ` f-- i 5- A \ ix ~ -r-~ 7 \ y ~ _s\ l P i - > V V 4- \y -- A 3N f/ CONT 1 II 1 N -E tF O S A 40 M fflF O S E 40 Each Pair N-EtFOSE169?filQS Student's t Dose Group (mg/Kg/day) 0.05 /"~ -N /( / f T --y 1 \ ry Vy With Control Dunnett's 0.05 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.744756 0.688035 0.848512 5.282609 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 37.813543 18 12.959500 22 50.773043 Mean Square 9.45339 0.71997 2.30787 F Ratio 13.1302 Prob>F <.0001 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 6.80000 4.52500 5.50000 5.20000 3.44000 Std Error 0.29999 0.42426 0.59999 0.42426 0.37947 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 6.80000 1.06100 N-EtFOSA40mkd 4 4.52500 0.41130 N-EtFOSE160mkd 2 5.50000 1.55563 N-EtFOSE40mkd 4 5.20000 0.37417 PFOS40mkd 5 3.44000 0.65803 Std Err Mean 0.3751 0.2056 1.1000 0.1871 0.2943 Dif=Mean[i]-Mean[j] CONT N-EtFOSE160mkd N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT 0.00000 -1.30000 -1.60000 -2.27500 -3.36000 Means Comparisons N-EtFOSE160mkd 1.30000 0.00000 -0.30000 -0.97500 -2.06000 IN-EtFOSE40mkd 1.60000 0.30000 0.00000 -0.67500 -1.76000 N-EtFOSA40mkd 2.27500 0.97500 0.67500 0.00000 -1.08500 Alpha= 0.05 Comparisons for each pair using Student's t t PFOS40mkd 3.36000 2.06000 1.76000 1.08500 0.00000 " ^ 0 1 1 7 T-7071.1 FR DTI 5 Abs(Dif)-LSD CONT N-EtFOSE 160mkd N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT -0.89132 -0.10930 0.50836 1.18336 2.34374 2.10091 N-EtFOSE160mkd -0.10930 -1.78265 -1.24382 -0.56882 0.56853 N-EtFOSE40mkd 0.50836 -1.24382 -1.26052 -0.58552 0.56417 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT CONT -1.15476 N-EtFOSE160mkd -0.52583 N-EtFOSE40mkd ' 0.18572 N-EtFOSA40mkd 0.86072 PFOS40mkd 2.04338 N-EtFOSA40mkd 1.18336 -0.56882 -0.58552 -1.26052 -0.11083 Positive values show pairs of means that are significantly different. PFOS40mkd 2.34374 0.56853 0.56417 -0.11083 -1.12744 RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.26878 0.106287 2.256164 101.8261 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 33.67935 18 91.62500 22 125.30435 Mean Square 8.41984 5.09028 5.69565 F Ratio 1.6541 Prob>F 0.2043 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 Mean 100.625 103.500 104.000 101.250 Std Error 0.7977 1.1281 1.5953 1.1281 104 c n e iia T-7071.1 FR DTI 5 PFOS40mkd 5 102.000 1.0090 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 100.625 2.66927 N-EtFOSA40mkd 4 103.500 1.91485 N-EtFOSE160mkd 2 104.000 0.00000 N-EtFOSE40mkd 4 101.250 2.62996 PFOS40mkd 5 102.000 1.58114 Std Err Mean 0.9437 0.9574 0.0000 1.3150 0.7071 Dif=Mean[i]-Mean[j] N-EtFOSE160mkd N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT Means Comparisons N-EtFOSE160mkd N-EtFOSA40mkd 0.00000 0.50000 -0.50000 0.00000 -2.00000 -1.50000 -2.75000 -2.25000 -3.37500 -2.87500 PFOS40mkd 2.00000 1.50000 ' 0.00000 -0.75000 -1.37500 N-EtFOSE40mkd 2.75000 2.25000 0.75000 0.00000 -0.62500 CONT 3.37500 2.87500 1.37500 0.62500 0.00000 Alpha= Abs(Dif)-LSD N-EtFOSE 160mkd N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT 0.05 Comparisons for each pair using Student's t t 2.10091 N-EtFOSE160mkd N-EtFOSA40mkd PFOS40mkd -4.73999 -3.60496 -1.96576 -3.60496 -3.35168 -1.67968 -1.96576 -1.67968 -2.99784 -1.35496 -1.10168 -2.42968 -0.37229 -0.02764 -1.32721 N-EtFOSE40mkd -1.35496 -1.10168 -2.42968 -3.35168 -2.27764 CONT -0.37229 -0.02764 -1.32721 -2.27764 -2.37000 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT N-EtFOSE160mkd -1.47982 N-EtFOSA40mkd -0.88552 PFOS40mkd -2.12586 N-EtFOSE40mkd -3.13552 CONT -3.07046 Positive values show pairs of means that are significantly different. CREA mg/dL By Dose Group (mg/Kg/day) 105 G C 0 1 5 ..9 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.557128 0.458712 0.070907 0.573913 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 0.11384783 18 0.09050000 22 0.20434783 Mean Square 0.028462 0.005028 0.009289 F Ratio 5.6609 Prob>F 0.0039 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 0.500000 0.625000 0.550000 0.550000 0.680000 Std Error 0.02507 0.03545 0.05014 0.03545 0.03171 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 0.500000 0.075593 N-EtFOSA40mkd 4 0.625000 0.050000 N-EtFOSE160mkd 2 0.550000 0.070711 N-EtFOSE40mkd 4 0.550000 0.057735 PFOS40mkd 5 0.680000 0.083666 Std Err Mean 0.02673 0.02500 0.05000 0.02887 0.03742 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd CONT Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.000000 0.055000 -0.055 0.000000 -0.13 -0.075 -0.13 -0.075 -0.18 -0.125 N-EtFOSE160mkd 0.130000 0.075000 0.000000 0.000000 -0.05 N-EtFOSE40mkd 0.130000 0.075000 0.000000 0.000000 -0.05 Alpha= 0.05 Comparisons for each pair using Student's t t CONT 0.180000 0.125000 0.050000 0.050000 0.000000 106 T-7071.1 FR DTI 5 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd N-EtFOSE 160mkd N-EtFOSE40mkd CONT PFOS40mkd -0.09422 -0.04493 0.005364 0.030069 0.095075 2.10091 N-EtFOSA40mkd -0.04493 -0.10534 -0.05401 -0.03034 0.033776 N-EtFOSE160mkd 0.005364 -0.05401 -0.14897 -0.12901 -0.06777 N-EtFOSE40mkd 0.030069 -0.03034 -0.12901 -0.10534 -0.04122 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd CONT CONT 0.069975 0.006814 -0.10258 -0.06819 -0.0965 Positive values show pairs of means that are significantly different. Clinical chemistries for all rats (males and females combined in DT15B. Shows significant decrease in potassium by 40 mkd PFOS, N-EtFOSE and N-EtFOSA, with the most significant changes occurring in the 40 mkd PFOS dose group. Creatinine (CREAT) was significantly increased in the 40 mkd PFOS and 40 mkd N-EtFOSA dose groups. CONT 0.095075 0.033776 -0.06777 -0.04122 -0.07448 107 f / y f M 04! T-7071.1 FR DT15 B. Statistics on Clinical Chemistries for Guinea Pigs, male and female values combined. 108 fV-1 *>*? T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.303785 0.164542 23.365 49.44737 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 3573.102 15 8188.846 18 11761.947 Mean Square 1191.03 545.92 653.44 F Ratio 2.1817 Prob>F 0.1327 Means for Oneway Ahova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 40.0833 71.7500 34.3750 54.9000 Std Error 9.539 11.682 11.682 10.449 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 40.0833 19.9785 N-EtFOSA40mkd 4 71.7500 30.4344 N-EtFOSE40mkd 4 34.3750 23.7500 PFOS40mkd 5 54.9000 20.7497 Std Err Mean 8.156 15.217 11.875 9.280 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd PFOS40mkd CONT N-EtFOSE40mkd Means Comparisons N-EtFOSA40mkd PFOS40mkd 0.0000 16.8500 -16.8500 0.0000 -31.6667 -14.8167 -37.3750 -20.5250 CONT 31.6667 14.8167 0.0000 -5.7083 N-EtFOSE40mkd 37.3750 20.5250 5.7083 0.0000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd PFOS40mkd CONT N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSA40mkd PFOS40mkd -35.2147 -16.5576 -16.5576 -31.4970 -0.4798 -15.3394 2.1603 -12.8826 CONT -0.4798 -15.3394 -28.7527 -26.4382 N-EtFOSE40mkd 2.1603 -12.8826 -26.4382 -35.2147 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD N-EtFOSA40mkd CONT -7.9838 PFOS40mkd CONT N-EtFOSE40mkd -22.3787 -35.4644 -33.9421 Positive values show pairs of means that are significantly different. Cholesterol, Male and female guinea pig combined. 109 C0 0 1 2 3 T-7071.1 FR DTI 5 110 C 0 0 2 4 T-7071.1 FR DT15 111C 0 9 1 2 5 T-7071. FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.079281 -0.1509 0.602426 12.175 16 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 0.3750000 12 4.3550000 15 4.7300000 Mean Square 0.125000 0.362917 0.315333 F Ratio 0.3444 Prob>F 0.7938 Means for Oneway Anova Level Number CONT 4 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 4 Mean 11.9750 12.2000 12.4000 12.1250 Std Error 0.30121 0.30121 0.30121 0.30121 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 11.9750 0.805709 N-EtFOSA40mkd 4 12.2000 0.588784 N-EtFOSE40mkd 4 12.4000 0.496655 PFOS40mkd 4 12.1250 0.457347 Std Err Mean 0.40285 0.29439 0.24833 0.22867 Dif=Mean[i]-Mean[j] N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT Means Comparisons N-EtFOSE40mkd N-EtFOSA40mkd 0.000000 0.200000 -0.2 0.000000 -0.275 -0.075 -0.425 -0.225 PFOS40mkd 0.275000 0.075000 0.000000 -0.15 CONT 0.425000 0.225000 0.150000 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT 0.05 Comparisons for each pair using Student's t t 2.17882 N-EtFOSE40mkd N-EtFOSA40mkd -0.92813 -0.72813 -0.72813 -0.92813 -0.65313 -0.85313 -0.50313 -0.70313 PFOS40mkd -0.65313 -0.85313 -0.92813 -0.77813 CONT -0.50313 -0.70313 -0.77813 -0.92813 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.68294 Abs(Dif)-LSD N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT CONT -0.71788 -0.91788 -0.99288 -1.14288 Positive values show pairs of means that are significantly different. PHOS mg/dL By Dose Group (mg/Kg/day) 112C 0 0 1 2 6 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.137384 -0.07827 0.776343 8.98125 16 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 1.1518750 12 7.2325000 15 8.3843750 Mean Square 0.383958 0.602708 0.558958 F Ratio 0.6371 Prob>F 0.6054 Means for Oneway Anova Level Number CONT 4 N-EtFO SA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 4 Mean 9.05000 8.97500 9.32500 8.57500 Std Error 0.38817 0.38817 0.38817 0.38817 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 9.05000 0.58023 N-EtFOSA40mkd 4 8.97500 0.49917 N-EtFOSE40mkd 4 9.32500 0.27538 PFOS40mkd 4 8.57500 1.32256 Std Err Mean 0.29011 0.24958 0.13769 0.66128 Dif=Mean[i]-Mean[j] N-EtFOSE40mkd CONT N-EtFOSA40mkd PFOS40mkd Means Comparisons N-EtFOSE40mkd CONT 0.000000 0.275000 -0.275 0.000000 -0.35 -0.075 -0.75 -0.475 N-EtFOSA40mkd 0.350000 0.075000 0.000000 -0.4 PFOS40mkd 0.750000 0.475000 0.400000 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.17882 N-EtFOSE40mkd CONT N-EtFOSA40mkd -1.19608 -0.92108 -0.84608 PFOS40mkd -0.44608 113 CC0127 T-7071.1 FR DTI 5 CONT N-EtFOSA40mkd PFOS40mkd -0.92108 -0.84608 -0.44608 -1.19608 -1.12108 -0.72108 -1.12108 -1.19608 -0.79608 -0.72108 -0.79608 -1.19608 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.68294 Abs(Dif)-LSD CONT N-EtFOSE40mkd -1.19782 CONT N-EtFOSA40mkd -1.47282 -1.39782 PFOS40mkd -0.99782 Positive values show pairs of means that are significantly different. the two analyses directly Above are for guinea pig Ca and PHOS, male and female combined. No sig changes occured 11c4 coins T-7071.1 FR DTI 5 15 y' x. X'1' 00 00 OD y_ 5- CONT N-EtFOSA40mkd PFOS40 N-EtFOSE40mkd Dose Group (mg/Kg/day) Student's t 0.05 Dunnett's 0.05 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.926687 0.909768 1.140108 8.823529 17 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 213.59259 13 16.89800 16 230.49059 Mean Square 71.1975 1.2998 14.4057 F Ratio 54.7738 Prob>F <.0001 Means for Oneway Anova Level Number CONT 4 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 12.8000 6.6500 12.2000 4.6800 Std Error 0.57005 0.57005 0.57005 0.50987 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 12.8000 1.01653 N-EtFOSA40mkd 4 6.6500 0.69522 N-EtFOSE40mkd 4 12.2000 0.96264 PFOS40mkd 5 4.6800 1.54661 Std Err Mean 0.50827 0.34761 0.48132 0.69166 Dif=Mean[i]-Mean[j] CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd Means Comparisons CONT N-EtFOSE40mkd 0.00000 0.60000 -0.60000 0.00000 -6.15000 -5.55000 -8.12000 -7.52000 N-EtFOSA40mkd 6.15000 5.55000 0.00000 -1.97000 PFOS40mkd 8.12000 7.52000 1.97000 0.00000 Alpha= Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.16037 CONT N-EtFOSE40mkd N-EtFOSA40mkd -1.74164 -1.14164 4.40836 -1.14164 -1.74164 3.80836 4.40836 3.80836 -1.74164 6.46773 5.86773 0.31773 PFOS40mkd 6.46773 5.86773 0.31773 -1.55777 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.64945 Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT -2.13593 -1.53593 4.01407 6.09368 Positive values show pairs of means that are significantly different. Above is for guinea pig combined male and femal potasium levels. Sig reduced by PFOS and N-etFOSA at 40 mg/Kg/day. 116 C00130 T-7071.1 FR DT15 TBIL mg/dL By Dose Group (mg/Kg/day) 117 C00131 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.05849 -0.12981 0.106758 0.171053 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 0.01062061 15 0.17095833 18 0.18157895 Mean Square 0.003540 0.011397 0.010088 F Ratio 0.3106 Prob>F 0.8174 Means for Oneway Ahova Level Number Mean CONT 6 0.158333 N-EtFOSA40mkd 4 0.150000 N-EtFOSE40mkd 4 0.162500 PFOS40mkd 5 0.210000 Std Error 0.04358 0.05338 0.05338 0.04774 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 0.158333 0.091742 N-EtFOSA40mkd 4 0.150000 0.057735 N-EtFOSE40mkd 4 0.162500 0.110868 PFOS40mkd 5 0.210000 0.143178 Std Err Mean 0.03745 0.02887 0.05543 0.06403 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd Means Comparisons PFOS40mkd N-EtFOSE40mkd 0.000000 0.047500 -0.0475 0.000000 -0.05167 -0.00417 -0.06 -0.0125 CONT 0.051667 0.004167 0.000000 -0.00833 N-EtFOSA40mkd 0.060000 0.012500 0.008333 0.000000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t * 2.13144 PFOS40mkd N-EtFOSE40mkd -0.14391 -0.10514 -0.10514 -0.1609 -0.08612 -0.14271 -0.09264 -0.1484 CONT -0.08612 -0.14271 -0.13137 -0.13855 N-EtFOSA40mkd -0.09264 -0.1484 -0.13855 -0.1609 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd CONT -0.11828 -0.177 -0.16204 -0.17283 Positive values show pairs of means that are significantly different. Alb mg/dL By Dose Group (mg/Kg/day) 118 c00132 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or SumWgts) 0.215626 0.058751 0.340474 2.473684 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 0.4780088 15 1.7388333 18 2.2168421 Mean Square 0.159336 0.115922 0.123158 F Ratio 1.3745 Prob>F 0.2887 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 2.26667 2.67500 2.45000 2.58000 Std Error 0.13900 0.17024 0.17024 0.15226 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 2.26667 0.382971 N-EtFOSA40mkd 4 2.67500 0.150000 N-EtFOSE40mkd 4 2.45000 0.129099 PFOS40mkd 5 2.58000 0.471169 Std Err Mean 0.15635 0.07500 0.06455 0.21071 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT Means Comparisons N-EtFOSA40mkd PFOS40mkd 0.000000 0.095000 -0.095 0.000000 -0.225 -0.13 -0.40833 -0.31333 N-EtFOSE40mkd 0.225000 0.130000 0.000000 -0.18333 CONT 0.408333 0.313333 0.183333 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd -0.51315 -0.39181 -0.28815 CONT -0.0601 119 G99133 T-7071.1 FR DTI 5 PFOS40mkd N-EtFOSE40mkd CONT -0.39181 -0.28815 -0.0601 -0.45897 -0.35681 -0.1261 -0.35681 -0.51315 -0.2851 -0.1261 -0.2851 -0.41898 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT -0.16945 -0.22868 -0.39445 -0.51679 Positive values show pairs of means that are significantly Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.3468 0.21616 0.550989 5.321053 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 2.4177456 15 4.5538333 18 6.9715789 Mean Square 0.805915 0.303589 0.387310 F Ratio 2.6546 Prob>F 0.0863 Means for Oneway Anova Level Number CONT 6 N-EtF OSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 4.88333 5.75000 5.17500 5.62000 Std Error 0.22494 0.27549 0.27549 0.24641 Level CONT Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev 6 4.88333 0.611283 Std Err Mean 0.24956 120 0^0134 T-7071.1 FR DTI 5 N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd 4 5.75000 0.378594 4 5.17500 0.330404 5 5.62000 0.694262 0.18930 0.16520 0.31048 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT Means Comparisons N-EtFOSA40mkd PFOS40mkd 0.000000 0.130000 -0.13 0.000000 -0.575 -0.445 -0.86667 -0.73667 N-EtFOSE40mkd 0.575000 0.445000 0.000000 -0.29167 CONT 0.866667 0.736667 0.291667 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd -0.83043 -0.65781 -0.25543 -0.65781 -0.74276 -0.34281 -0.25543 -0.34281 -0.83043 0.108594 0.025532 -0.46641 CONT 0.108594 0.025532 -0.46641 -0.67804 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT CONT -0.06836 -0.14047 -0.64336 -0.83632 Positive values show pairs of means that are significantly different. BUN mg/dL By Dose Group (mg/Kg/day) 17- -A- A A 16- A A /A /A / '\ A. / '\ 15 - / .V \ / \ i -- .-- ^ 141) E 13z 3 12- 11 - A/ W ^7 " \ / \ X T/ \/ A7 __ 10- o CONT iiI N-EtFOSA40mkd PFOS40 Each Pair N-EtFOSE40mkd Student's t Dose Group (mg/Kg/day) 0.05 :;A ' A With Control Dunnett's 0.05 121 0^0135 T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.018487 -0.17782 2.382342 14.52632 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 1.603509 15 85.133333 18 86.736842 Mean Square 0.53450 5.67556 4.81871 F Ratio 0.0942 Prob>F 0.9621 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 14.6667 14.5000 14.0000 14.8000 Std Error 0.9726 1.1912 1.1912 1.0654 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 14.6667 1.21106 N-EtFOSA40mkd 4 14.5000 2.38048 N-EtFOSE40mkd 4 14.0000 3.16228 PFOS40mkd 5 14.8000 2.77489 Std Err Mean 0.4944 1.1902 1.5811 1.2410 Dif=Mean[i]-Mean[j] PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd Means Comparisons PFOS40mkd CONT 0.000000 0.133333 -0.13333 0.000000 -0.3 -0.16667 -0.8 -0.66667 N-EtFOSA40mkd 0.300000 0.166667 0.000000 -0.5 N-EtFOSE40mkd 0.800000 0.666667 0.500000 0.000000 Alpha= Abs(Dif)-LSD PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 PFOS40mkd CONT N-EtFOSA40mkd -3.21150 -2.94144 -3.10631 -2.94144 -2.93168 -3.11105 -3.10631 -3.11105 -3.59056 -2.60631 -2.61105 -3.09056 N-EtFOSE40mkd -2.60631 -2.61105 -3.09056 -3.59056 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd CONT -3.65919 -3.61602 -3.87617 -3.37617 Positive values show pairs of means that are significantly different. GLU mg/dL By Dose Group (mg/Kg/day) 122 e r o in e T-7071. FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.053125 -0.13625 106.4679 282.0526 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 9539.66 15 170031.28 18 179570.95 Mean Square 3179.9 11335.4 9976.2 F Ratio 0.2805 Prob>F 0.8386 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 261.333 285.000 322.250 272.400 Std Error 43.465 53.234 53.234 47.614 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 261.333 93.863 N-EtFOSA40mkd 4 285.000 137.033 N-EtFOSE40mkd 4 322.250 114.782 PFOS40mkd 5 272.400 86.777 Std Err Mean 38.319 68.516 57.391 38.808 Dif=Mean[i]-Mean[j] N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT Means Comparisons N-EtFOSE40mkd N-EtFOSA40mkd 0.0000 37.2500 -37.2500 0.0000 -49.8500 -12.6000 -60.9167 -23.6667 PFOS40mkd 49.8500 12.6000 0.0000 -11.0667 CONT 60.9167 23.6667 11.0667 0.0000 Alpha= Abs(Dif)-LSD N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSE40mkd N-EtFOSA40mkd -160.464 -123.214 PFOS40mkd -102.379 CONT -85.566 'if'-,0137 T-7071.1 FR DTI 5 N-EtFOSA40mkd PFOS40mkd CONT -123.214 -102.379 -85.566 -160.464 -139.629 -122.816 -139.629 -143.523 -126.346 -122.816 -126.346 -131.018 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT CONT -119.760 -157.010 -158.423 -161.602 Positive values show pairs of means that are significantly different. ALKP U/L By Dose Group (mg/Kg/day) 300 - 250 " 3 Q_ 55 < 200 - / Z2\ ;\ \ \ _ // 150 - V/ j. /\ M / \ / A S / / \\ \ f\L// j . \ j ~ \\ / O \? / \ jV - A.________A___V \ JM _____ ______ - A . CONT N -EN f|^S A40rW PFOS4Ca feach Pair / \ With Control , / V N-EtKoSE40mkd Dose Group (mg/Kg/day) Studentls.i,.,. . . . . ^Bunnett's 0.05 0.05 ^ Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.159836 -0.0082 59.956 153.0526 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 10258.114 15 53920.833 18 64178.947 Mean Square 3419.37 3594.72 3565.50 F Ratio 0.9512 Prob>F 0.4409 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 184.667 130.750 129.250 152.000 Std Error 24.477 29.978 29.978 26.813 Level CONT Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev 6 184.667 80.2438 Std Err Mean 32.759 124 c 00138 T-7071.1 FR DTI 5 N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd 4 130.750 11.1168 4 129.250 23.6414 5 152.000 70.1391 5.558 11.821 31.367 Dif=Mean[i]-Mean[j] CONT PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd Means Comparisons CONT PFOS40mkd 0.0000 32.6667 -32.6667 0.0000 -53.9167 -21.2500 -55.4167 -22.7500 N-EtFOSA40mkd 53.9167 21.2500 0.0000 -1.5000 N-EtFOSE40mkd 55.4167 22.7500 1.5000 0.0000 Alpha= Abs(Dif)-LSD CONT PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 CONT PFOS40mkd N-EtFOSA40mkd -73.7812 -44.7157 -28.5732 -44.7157 -80.8232 -64.4760 -28.5732 -64.4760 -90.3631 -27.0732 -62.9760 -88.8631 N-EtFOSE40mkd -27.0732 -62.9760 -88.8631 -90.3631 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT -91.0039 -62.7790 -47.8288 -46.3288 Positive values show pairs of means that are significantly 125 C00139 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or SumWgts) 0.083057 -0.10033 21.12468 50.68421 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 606.3219 15 6693.7833 18 7300.1053 Mean Square 202.107 446.252 405.561 F Ratio 0.4529 Prob>F 0.7191 Means for Oneway Ahova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 46.1667 44.2500 55.5000 57.4000 Std Error 8.624 10.562 10.562 9.447 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 46.1667 10.1472 N-EtFOSA40mkd 4 44.2500 7.7621 N-EtFOSE40mkd 4 55.5000 41.2189 PFOS40mkd 5 57.4000 15.0100 Std Err Mean 4.143 3.881 20.609 6.713 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd Means Comparisons PFOS40mkd N-EtFOSE40mkd 0.0000 1.9000 -1.9000 0.0000 -11.2333 -9.3333 -13.1500 -11.2500 CONT 11.2333 9.3333 0.0000 -1.9167 N-EtFOSA40mkd 13.1500 11.2500 1.9167 0.0000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 PFOS40mkd N-EtFOSE40mkd CONT -28.4770 -28.3044 -16.0313 -28.3044 -31.8382 -19.7308 -16.0313 -19.7308 -25.9958 -17.0544 -20.5882 -27.1475 N-EtFOSA40mkd -17.0544 -20.5882 -27.1475 -31.8382 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd CONT -22.3956 -26.5153 -32.0640 -33.9319 Positive values show pairs of means that are significantly different. ALT U/L By Dose Group (mg/Kg/day) 126 C~940 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or SumWgts) 0.47363 0.368357 6.390531 51.89474 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 551.2061 15 612.5833 18 1163.7895 Mean Square 183.735 40.839 64.655 F Ratio 4.4990 Prob>F 0.0192 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 47.8333 53.2500 46.5000 60.0000 Std Error 2.6089 3.1953 3.1953 2.8579 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 47.8333 4.1191 N-EtFOSA40mkd 4 53.2500 3.8622 N-EtFOSE40mkd 4 46.5000 11.6762 PFOS40mkd 5 60.0000 4.3012 Std Err Mean 1.6816 1.9311 5.8381 1.9235 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.0000 6.7500 -6.7500 0.0000 -12.1667 -5.4167 -13.5000 -6.7500 CONT 12.1667 5.4167 0.0000 -1.3333 N-EtFOSE40mkd 13.5000 6.7500 1.3333 0.0000 Alpha= Abs(Dif)-LSD PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 PFOS40mkd N-EtFOSA40mkd -8.61470 -2.38727 CONT 3.91872 N-EtFOSE40mkd 4.36273 127 r -f.4! v' ,> H IM .. J k T-7071.1 FR DTI 5 N-EtFOSA40mkd CONT N-EtFOSE40mkd -2.38727 3.91872 4.36273 -9.63153 -3.37568 -2.88153 -3.37568 -7.86411 -7.45901 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT PFOS40mkd 1.99340 N-EtFOSA40mkd -5.42807 CONT -9.69983 N-EtFOSE40mkd -9.51141 -2.88153 -7.45901 -9.63153 Positive values show pairs of means that are significantly different. RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.10172 -0.07794 110.3027 211.6316 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 20666.12 15 182500.30 18 203166.42 Mean Square 6888.7 12166.7 11287.0 F Ratio 0.5662 Prob>F 0.6457 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 177.500 186.000 237.000 252.800 Std Error 45.031 55.151 55.151 49.329 Level CONT Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev 6 177.500 81.343 Std Err Mean 33.208 ^ 001^ 2 T-7071.1 FR DTI 5 N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd 4 186.000 31.454 4 237.000 185.801 5 252.800 103.541 15.727 92.900 46.305 Dif=Mean[i)-Mean[j] PFOS40mkd N-EtFOSE40mkd N-EtFOSA40mkd CONT Means Comparisons PFOS40mkd N-EtFOSE40mkd 0.0000 15.8000 -15.8000 0.0000 -66.8000 -51.0000 -75.3000 -59.5000 N-EtFOSA40mkd 66.8000 51.0000 0.0000 -8.5000 CONT 75.3000 59.5000 8.5000 0.0000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSE40mkd N-EtFOSA40mkd CONT 0.05 Comparisons for each pair using Student's t t 2.13144 PFOS40mkd N-EtFOSE40mkd N-EtFOSA40mkd -148.693 -141.912 -90.912 -141.912 -166.243 -115.243 -90.912 -115.243 -166.243 -67.062 -92.259 -143.259 CONT -67.062 -92.259 -143.259 -135.737 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT PFOS40mkd -100.294 N-EtFOSE40mkd -127.684 N-EtFOSA40mkd -178.684 CONT -167.422 Positive values show pairs of means that are significantly different. Na+ mmol/L By Dose Group (mg/Kg/day)____________________ 129 COOi-'l3 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.793274 0.751929 1.727876 138.4211 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 171.84825 15 44.78333 18 216.63158 Mean Square 57.2827 2.9856 12.0351 F Ratio 19.1866 Prob>F <.0001 Means for Oneway Ahova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 141.833 136.750 140.000 134.400 Std Error 0.70540 0.86394 0.86394 0.77273 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 141.833 1.72240 N-EtFOSA40mkd 4 136.750 1.50000 N-EtFOSE40mkd 4 140.000 1.41421 PFOS40mkd 5 134.400 2.07364 Std Err Mean 0.70317 0.75000 0.70711 0.92736 Dif=Mean[i]-Mean[j] CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd Means Comparisons CONT N-EtFOSE40mkd 0.00000 1.83333 -1.83333 0.00000 -5.08333 -3.25000 -7.43333 -5.60000 N-EtFOSA40mkd 5.08333 3.25000 0.00000 -2.35000 PFOS40mkd 7.43333 5.60000 2.35000 0.00000 Alpha= Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 CONT N-EtFOSE40mkd N-EtFOSA40mkd -2.12630 -0.54395 2.70605 -0.54395 -2.60418 0.64582 2.70605 0.64582 -2.60418 5.20325 3.12946 -0.12054 PFOS40mkd 5.20325 3.12946 -0.12054 -2.32925 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT -2.62265 -1.09887 2.15113 4.68268 Positive values show pairs of means that are significantly different. Cl- mmol/L By Dose Group (mg/Kg/day) l3fcC0i-14 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.405319 0.286382 2.382342 101.2105 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 58.02456 15 85.13333 18 143.15789 Mean Square 19.3415 5.6756 7.9532 F Ratio 3.4079 Prob>F 0.0452 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 103.667 99.500 101.000 99.800 Std Error 0.9726 1.1912 1.1912 1.0654 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 103.667 1.75119 N-EtFOSA40mkd 4 99.500 1.73205 N-EtFOSE40mkd 4 101.000 2.94392 PFOS40mkd 5 99.800 2.94958 Std Err Mean 0.7149 0.8660 1.4720 1.3191 Dif=Mean[i]-Mean[j] CONT N-EtFOSE40mkd PFOS40mkd N-EtFOSA40mkd Means Comparisons CONT N-EtFOSE40mkd 0.00000 2.66667 -2.66667 0.00000 -3.86667 -1.20000 -4.16667 -1.50000 PFOS40mkd 3.86667 1.20000 0.00000 -0.30000 N-EtFOSA40mkd 4.16667 1.50000 0.30000 0.00000 Alpha= Abs(Dif)-LSD CONT 0.05 Comparisons for each pair using Student's t t 2.13144 CONT N-EtFOSE40mkd PFOS40mkd -2.93168 -0.61105 0.79189 N-EtFOSA40mkd 0.88895 131 C O O l 45 T-7071.1 FR DTI 5 N-EtFOSE40mkd PFOS40mkd N-EtFOSA40mkd -0.61105 0.79189 0.88895 -3.59056 -2.20631 -2.09056 -2.20631 -3.21150 -3.10631 -2.09056 -3.10631 -3.59056 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Idi 2.62898 Abs(Dif)-LSD CONT CONT -3.61602 N-EtFOSE40mkd -1.37617 PFOS40mkd 0.07415 N-EtFOSA40mkd 0.12383 Positive values show pairs of means that are significantly Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.295588 0.154706 0.126359 0.5 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 0.10050000 15 0.23950000 18 0.34000000 Mean Square 0.033500 0.015967 0.018889 F Ratio 2.0981 Prob>F 0.1434 Means for Oneway Anova Level Number Mean CONT 6 0.400000 N-EtFOSA40mkd 4 0.575000 N-EtFOSE40mkd 4 0.500000 PFOS40mkd 5 0.560000 Std Error 0.05159 0.06318 0.06318 0.05651 Level CONT Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev 6 0.400000 0.089443 Std Err Mean 0.03651 132 CC0146 T-7071.1 FR DTI 5 N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd 4 0.575000 0.095743 4 0.500000 0.081650 5 0.560000 0.194936 0.04787 0.04082 0.08718 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT Means Comparisons N-EtFOSA40mkd PFOS40mkd 0.000000 0.015000 -0.015 0.000000 -0.075 -0.06 -0.175 -0.16 N-EtFOSE40mkd 0.075000 0.060000 0.000000 -0.1 CONT 0.175000 0.160000 0.100000 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd -0.19044 -0.16567 -0.11544 -0.16567 -0.17034 -0.12067 -0.11544 -0.12067 -0.19044 0.001150 -0.00309 -0.07385 CONT 0.001150 -0.00309 -0.07385 -0.1555 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT CONT -0.03943 -0.04115 -0.11443 -0.19179 Positive values show pairs of means that are significantly different. 133 000147 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.130895 -0.04293 34.82966 123.2105 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 2740.575 15 18196.583 18 20937.158 Mean Square 913.52 1213.11 1163.18 F Ratio 0.7530 Prob>F 0.5375 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 128.167 138.500 123.250 105.000 Std Error 14.219 17.415 17.415 15.576 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 128.167 42.6447 N-EtFOSA40mkd 4 138.500 45.3836 N-EtFOSE40mkd 4 123.250 24.0468 PFOS40mkd 5 105.000 17.2482 Std Err Mean 17.410 22.692 12.023 7.714 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd CONT N-EtFOSE40mkd PFOS40mkd Means Comparisons N-EtFOSA40mkd CONT 0.0000 10.3333 -10.3333 0.0000 -15.2500 -4.9167 -33.5000 -23.1667 N-EtFOSE40mkd 15.2500 4.9167 0.0000 -18.2500 PFOS40mkd 33.5000 23.1667 18.2500 0.0000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd CONT N-EtFOSE40mkd PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSA40mkd CONT N-EtFOSE40mkd -52.4938 -37.5867 -37.2438 -37.5867 -42.8610 -43.0034 -37.2438 -43.0034 -52.4938 -16.3000 -21.7863 -31.5500 PFOS40mkd -16.3000 -21.7863 -31.5500 -46.9518 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT N-EtFOSA40mkd -48.7727 CONT -52.8660 N-EtFOSE40mkd -54.1893 PFOS40mkd -32.2797 Positive values show pairs of means that are significantly different. Above is for the male and female guinea pigs combined clinical chemistry statistics. 134 CCO 8 T-7071.1 FR DTI 5 Appendix 8. Hepatic Palmitoyl Co-A oxidase activity SPECIES GP SEX F M DOSE GRO CONT N-EtF0SA4 Omkd N-EtFOSE160mkd N-EtF0SE4 Omkd PF0S4 Omkd CONT N-EtF0SA4 Omkd N-EtFOSE16Omkd N-EtF0SE4 Omkd ID 8G01485 8G01486 9G00051 9G00052 N Mean SD PCOAO 2.00 1.00 3.00 3.00 4 2.250 0.957 U 8G01488 8G01490 N Mean SD 3.00 1.00 2 ' 2.000 1.414 9G00053 9G00054 N Mean SD 3.00 3.00 2 3.000 0.000 8G01487 8G01489 N Mean SD 2.00 2.00 2 2.000 0.000 8G01491 8G01492 9G00055 9G00056 N Mean SD 2.00 Missing 3.00 6.00 3 3.667 2.082 8G01477 8G01478 9G00045 9G00046 N Mean SD 1.00 1.00 2.00 2.00 4 1.500 0.577 8G01481 8G01482 N Mean SD 1.00 1.00 2 1.000 0.000 9G00047 9G00048 N Mean SD 2.00 Missing 1 2.000 Missing 8G01479 8G01480 1.00 1.00 135 CO 0 3 .^ 9 T-7071.1 FR DTI 5 SPECIES R SPECIES SEX F M SEX DOSE GRO PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE4 Omkd PFOS4 Omkd M556-160mkd DOSE GRO CONT ID N Mean SD 8G01483 8G01484 9G00049 9G00050 N Mean SD 8R04040 8R04041 9R00469 9R00470 N Mean SD 8R04044 8R04045 N Mean SD 9R00471 9R00472 N Mean SD 8R04042 8R04043 N Mean SD 8R04046 8R04047 9R00473 9R00474 N Mean SD 1R00748 1R00749 1R00750 N Mean SD ID 1R00742 1R00743 1R00744 8R04032 8R04033 9R00463 PCOAO 2 1.000 0.000 U 1.00 2.00 3.00 3.00 4 2.250 0.957 Missing 4.00 6.00 7.00 3 5.667 1.528 Missing Missing 0 Missing Missing 5.00 8.00 2 6.500 2.121 Missing Missing 0 Missing Missing Missing Missing 10.00 18.00 2 14.000 5.657 Missing Missing Missing 0 Missing Missing PCOAO U Missing Missing Missing 0.00 4.00 9.00 136 CC0150 T-7071.1 FR DTI 5 FOSA4Omkg N-EtF0SA4 Omkd N-EtFOSE160mkd N-EtF0SE4 Omkd PF0S4 Omkd 9R00464 N Mean SD 9.00 4 5.500 4.359 1R00745 1R00746 1R00747 N Mean SD Missing Missing Missing 0 Missing Missing 8R04036 8R04037 N Mean SD Missing Missing ,0 Missing Missing 9R00465 9R00466 N Mean SD 19.00 20.00 2 19.500 0.707 8R04034 8R04035 N Mean SD Missing Missing 0 Missing Missing 8R04038 8R04039 9R00467 9R00468 N Mean SD Missing 11.00 21.00 20.00 3 17.333 5.508 137 C O 0151 T-7071.1 FR DTI 5 138 V f-w T-7071.1 FR DTI 5 Appendix 9. Effect of acute FC administration on catalase and acylCoA oxidase expression The following report summarizes the effects of in vivo administration of N-EtFOSE or PFOS on catalase and acylCoA oxidase gene expression and enzyme activity in liver tissue from exposed rats and guinea pigs, and was presented as a poster at the 2001 Society of Toxicology meeting (Wallace e t al. 2001). Tissues analyzed in this study were derived from approximately 1 g of frozen liver from each of two male and two female rats and guinea pigs that received either 40 mg/kg/day PFOS or 160 mg/kg/day 2(N-ethylperfluorooctanesulfonamido)-ethyl alcohol (N-EtFOSE) orally for 4 days, or an equivalent volume of 2% tween 80 (vehicle control) from March 1, 1999 to March 4, 1999. All animals were killed on March 5, one day after the last consecutive dose. Portions of the respective livers were quick frozen in liquid nitrogen and shipped on dry ice to Duluth for analysis. E n zym e S a m p le P rep a ra tio n - The enzyme fraction consisted of the 6,000 g supernatant of a 10% (wt/vol) homogenate of 0.5-1.0 g frozen liver tissue in 300 mM mannitol-10 mM HEPES-1 mM EGTA (pH 7.2). Protein concentration was estimated according to the method of Bradford using commercial bovine serum albumin as standard. L -C o A O xidase A s sa y - The equivalent of ca., 5 pg/ml tissue homogenate was suspended in 60 mM KH2PO4-O.O2 % Triton X I00 (pH 7.4) containing 1 mM p hydroxyphenylacetate (PHPA), 4 units/ml peroxidase, 20 pM FAD, and 60 pM laurylCoA (LCoA). The reactions were allowed to incubate at 37C for 30 min in a shaking water bath and terminated by adding 3 volumes of 2 mM KCN in 100 mM sodium carbonate (pH 10.5). The concentration of H2O2 generated during the reaction was estimated from the fluorescence of PHAP as measured with an excitation wavelength of 317 nm and emission at 405 nm. The fluorescence was calibrated with commercial H2O2 and the results are expressed as nmol peroxide generated/min/mg mitochondrial protein (Table 1). Protein was quantitated by the Bradford method. C atalase A s s a y - The activity of catalase was estimated by a modification of the original method published by Claiborne and Fridovich (J. Biol. Chem. 254, 4245-52, 1979), which is based on the direct measurement of H2O2 disappearance as quantified spectrophotometrically at 240 nm. In this procedure, the tissue sample was diluted in 50 mM potassium phosphate (pH 7.0). The medium was warmed to 27C and the reaction initiated by adding 10.3 mM H2O2. The progress of the reaction was monitored at 240 nm for 5 min. Catalase activity was estimated from the initial linear rate ( E240=43.6 mM' `cm"1) and expressed as units/mg protein (Table 1). One unit of activity is defined as that amount of enzyme which catalyzes the decomposition of 1 pmole of H2O2 per min. 139 c 0 0 1 5 3 T-7071.1 FR DTI 5 N o rth e rn B lo t A n a ly se s - Quantitation of mRNA for both acylCoA oxidase (ACoAO) and catalase were performed by Northern blot analysis of quick frozen liver samples from treated rats and guinea pigs. Approximately 1 g of frozen liver was powderized in liquid nitrogen using a mortar/pestle. Total RNA was recovered using the PERFECT RNATM isolation kit and the concentration quantified spectrophotometrically at 260nm. The RNA was electrophoresed on a 1% agarose gel, blot transferred to a cellulose membrane and hybridized to the corresponding randomly [32P] labeled oligonucleotides that were PCR amplified from primers to ca., 350 base sequence of the respective rat liver gene. mRNA band density was quantified autoradiographically using phospho-imaging software. RESULTS: R a ts - Both male and female rats express LCoAO and catalase activities, and there is no substantial difference in enzyme activities between the sexes. The same is true for the constitutive expression of mRNA for both enzymes, with the exception that unexposed male rats do not express the message for PCoAO. Acute exposure to N-Et-FOSE causes a doubling of the specific activity of LCoAO in liver from both male and female rats, but catalase activity is unchanged. Associated with this is a proportionate 2-fold increase in the concentration of mRNA encoding for PCoAO, but no change in catalase mRNA, in livers from both sexes. Again, there is no remarkable sex difference in response to N-Et-FOSE exposure, at-least with this limited number of animals tested (n=2 ). Exposure of rats to PFOS elicits a similar doubling of LCoAO activity for both sexes, and possibly a slight increase in catalase activity in liver from male rats. Hepatic catalase activity in female rats does not appear to be affected by PFOS exposure. Unlike N-EtFOSE, which caused a doubling of mRNA band intensity for pCoAO, acute exposure to PFOS caused a 3-6 fold increase in PCoAO mRNA expression. This enhanced expression of message was more pronounced in female compared to male rats, and in both sexes resulted in no greater LCoAO enzyme activity than was observed at half the mRNA level for rats exposed to N-Et-FOSE. G u in ea P ig s - The response to guinea pigs to exposure to N-Et-FOSE and PFOS was dramatically different from that observed for rats. The constitutive activity of LCoAO in unexposed guinea pigs of either sex was very low (near LOD values) whereas catalase activity was 1.5-3 times higher. However, the activity of neither enzyme was stimulated following acute exposure to N-Et-FOSE or PFOS in vivo. Perhaps the most dramatic difference between species is the fact that guinea pig mRNA encoding for PCoAO was undetectable, even following exposure to N-Et-FOSE or PFOS. Part of the explanation for this observation may be that we were using hybridization probes developed against 140 CC0154 T-7071.1 FR DTI 5 the sequence for rat liver ACoAO because the sequence of the guinea pig enzyme is not known at this time. It may be that the guinea pig sequence is sufficiently different that the PCR product lacks complementarity for adequate hybridization to the probe for the rat sequence. Although this may indeed be an artifact, the absence of constitutively expressed message and the inability to stimulate expression of PCoAO mRNA are consistent with the low and uninducible enzyme activity in guinea pigs. CONCLUSIONS: These data provide strong evidence that: 1) N-Et-FOSE and PFOS stimulate both the transcriptional and translational expression of acylCoA oxidase in rats in vivo , and 2 ) there is a marked difference in the response of rats and guinea pigs to in vivo exposure to these two fluorochemicals. These results are very consistent with the suggestion that these fluorochemical compounds are "peroxisome proliferators" in rats and, much like what has been demonstrated for the classical "peroxisome proliferator" chemicals, guinea pigs are resistant to this effect of fluorochemical exposures. We were not given the data for organ weights or necropsy findings and thus cannot comment on whether exposure to either agent caused hepatomegaly or other indications that are associated with peroxisome proliferation. However, based on our data, we suspect that the evidence will reveal the classical signs of peroxisome proliferation in rats, but not guinea pigs, caused by these acute exposures. 141 C0 9 1 5 5 T-7071.1 FR DTI 5 Summary of Screen for Hepatic Peroxisome Proliferation Animal Specie Se Treatment Body Liver Liver P450 LaurylCoA Catalase ug pCoAO Catalase sX wt wt Protein content Oxidase activity RNA/u mRNA mRNA (g) (g) (mg/g) (nmol/mg) Activity 1 (% (% (nmol/min/mg) (Units/mg Control Control protein) )) 9R00463 Rat M Veh 9R00464 Rat M Veh 9R00469 Rat F Veh 9R00470 Rat F Veh 298 12 151.8 313 13.1 145.6 210 7.6 188.3 201 7.1 153.3 0.086 0.067 0.154 0.055 4 219.9 2.9 nd 126.7 5.3 158.5 2.65 nd 73.3 9.2 141.1 2.19 100 91.5 10.2 204.7 3.38 100 108.5 9R00465 Rat M N-EtFOSE 9R00466 Rat M N-EtFOSE 9R00471 Rat F N-EtFOSE 9R00472 Rat F N-EtFOSE 272 13 157.5 231 11.1 134.9 174 7.5 165.8 175 7.3 215.8 0.331 0.284 0.04 0.188 12 219.5 3.5 290 140.1 11.7 204.3 2.6 292 97.8 17.8 186.1 1.93 253 117.8 14.2 171.9 1.74 320 147.3 9R00467 Rat M PFOS 9R00468 Rat M PFOS 9R00473 Rat F PFOS 9R00474 Rat F PFOS 257 11.9 115.4 229 12.5 153 175 8.3 143.1 153 7.3 238.6 0.423 0.241 0.077 0.099 13.9 252 3.9 482 155.1 12 236.9 2.08 277 115.7 13.7 204.1 1.98 630 180.4 19.9 155.6 2 404 147.3 9G00045 9G00046 9G00051 9G00052 9G00047 9G00048 9G00053 9G00054 9G00049 9G00050 9G00055 9G00056 Gpig Gpig Gpig Gpig M Veh M Veh F Veh F Veh 296 11.3 175.2 298 11.9 252.7 274 11.9 180.3 289 14.2 222.1 0.123 0.089 0.188 0.17 Gpig Gpig Gpig Gpig M N-EtFOSE M N-EtFOSE F N-EtFOSE F N-EtFOSE 240 8.7 212.7 - - 226 8.5 269.1 0.013 230 9.6 176.4 0.126 236 8.9 218.3 0.195 Gpig M PFOS 215 7.2 Gpig M PFOS 240 10 Gpig F PFOS 221 8.4 Gpig F PFOS 237 7.4 (-) insufficient sample for analysis (nd) not detectable 153.2 222.1 127.8 171.6 . 0.074 0.031 0.016 1.1 314.7 1.19 nd 1.3 390.7 1.63 nd 1.2 550.6 1.65 nd 0.9 488 2.3 nd 349.3 - nd 0.7 379.3 1.71 nd 0.7 416.8 1.77 nd 1.5 478.1 1.28 nd 413 - nd 0.9 460.8 1.27 nd 0.9 672.9 1.65 nd 1.4 531.6 1.52 nd 103 97 91 109 - 92 105 104 _ 97 103 96 142 "0 1 5 6 T-7071.1 FR DTI 5 Appendix 10: Final Report for FOSA (T-7132.1). Title: Comparative Molecular Biology of 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 Study Protocol Title: Comparative Molecular Biology of Peroxisome Proliferation in Rats and Guinea Pigs Study Initiated: In-Life Start Date: November 16, 1998 In-Life End Date: December 21, 1998 T-7071.1 FR DTI 5 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 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 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. 144 CC0158 T-7071.1 FR DTI 5 Two aliquots of the serum sample (~ 0.75 ml) were saved for possible metabolite analysis. 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 e t al. 2001). Liver Sample Identification Chartfor T-7132.1 Sample # Animal # Species Sex Dose Group 1 1R00742 Rat M control 2 1R 00743 Rat M control 3 1R 00744 Rat M control 4 1R00745 Rat M 40 m g/kg/day FO SA 5 1R00746 Rat M 40 m g/kg/day FO SA 145 C09159 T-7071.1 FR DTI 5 6 1R00747 Rat M 40 m g/kg/day FO SA 146 CO0 1 6 0 T-7071.1 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 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 e t 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 yV-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 V-glucuronide had occurred in the control samples. 147 T-7071.1 FR DTI 5 Signatures: Report prepared by, Andrew M. Seacat, PhD, DABT Study Director Reviewed by, Daniel C. Hakes Sponsor representative Date Date 148 0^0162 T-7071.1 FR DTI 5 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. A n a l B io ch em 72, 24854. Hansen, K. J., Clemen, L. A., Ellefson, M. E., and Johnson, H. O. (2001). Compoundspecific, quantitative characterization of organic fluorochemicals in biological matrices. E n v ir o n m e n ta l S c ie n c e a n d T e c h n o lo g y 35, 766-770. Lazarow, P. B. (1981). Assay of peroxisomal beta-oxidation of fatty acids. M eth o d s E n zy m o l 72, 315-9. Poosch, M. S., and Yamazaki, R. K. (1986). Determination of peroxisomal fatty acylCoA oxidase activity using a lauroyl-CoA-based fluorometric assay. B io ch im B io p h ys A c ta 884,585-93. Wallace, K. B., Luebker, D. J., Butenhoff, J. L., and Seacat, A. M. (2001). Perfluorooctane sulfonate and 2-(N-ethylperfluorooctanesulfonamido)-ethyl alcohol are peroxisome proliferators in rats, but not guinea pigs. T o xico lo g ist 60, 348 Abstract ID: 1657. Xu, L., Seacat, A. M., Butenhoff, J. L., and Anders, M. W. (2003). Biotransformation of N-Ethyl-N-(2-hydroxyethyl)perfluorooctanesulfonamide (N-EtFOSE) by rat liver microsomes, cytosol and slices. In Toxicological Sciences, Suppl., Vol. 72, p. 314. 149 c 00163 T-7071.1 FR DTI 5 Tables Table1BiologicalParameters D T 1 5, A m e n d m e n t #2 C o m p a ra tiv e M o le c u la r B io lo g y o f P e rflu o ro o c ta n e s u lfo n a m id e (F O S A , T -7 1 3 2 ) in R a ts fo llo w in g fo u r consecutive days o f dosing. D T 15 B ody W e ig h t (B W ), Live r W e ig h t (LW ), T hyro id W e ig h t (TW)1 D a te 2/19/01 2/19/01 2/21/01 2/22/01 2/23/01 2/23/01 S am ple A nim al # Dose G roup # BW BW day 1 day 2 is)___ (9) BW day 3 (g) BW day 4 (g) BW day 5 (g) L W (g ) LW /B T W W (% ) (g) Avg SD 1 1R0074 2 2 1R0074 3 3 1R0074 4 control control control 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 1R0074 5 5 1R0074 6 6 1R0074 7 Avg SD P-value T-Test* 40 m g/kg/day FOSA 40 m g/kg/day FOSA 40 m g/kg/day FOSA | 250 249 259 253 6 0.21 254 245 265 255 10 0.13 250 243 263 252 10 0.06 239 233 255 242 11 0.02 227 10.77 4.7% 10.78 225 10.81 4.8% 10.66 254 10.64 4.2% 10.84 235 10.74 4.6% 10.76 16 0.09 0.34% 0.09 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. 150 T-7071.1 FR DTI 5 Table2LiverFluorocarbonConcentration Hepatic Concentrations of Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given FOSA (40 Sample # Animal # 1 1R00742 2 1R00743 3 1R00744 4 1R00745 5 1R00746 6 1R00747 mg/kg/day) Weight of liver sample PFOS Sex Treatment (g) (ppm) FOSA (ppm) FOSAA (ppm) FOSA Nglucuronide (ppm) M Control 0.4600 0.0 154.1 0.0 0.00 M Control 0.9236 0.0 113.2 0.0 0.00 M Control 1.1287 0.0 273.2 0.0 0.00 M FOSA 0.2402 163.2 195.7 n.m. 0.44 M FOSA 0.7905 214.3 174.1 n.m. 0.39 M FOSA 0.8305 202.8 163.9 n.m. 0.34 Note: n.m. = not measured. 151 T-7071.1 FR DTI 5 Table3PercentFluorocarbonDoseinLiver Hepatic Percent of Dosed Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given FOSA (40 mg/kg/day) for Four Days P e rce n t D ose in Liver S am ple Anim al # D ose Total # G roup Dose (m g) PFOS in dose** L iv e r PFOS (ppm ) L iv r PFOS (m g) % dose as PFOS in liver (% ) L iv e r FOSA (PPm) L iv e r FOSA (m g) % dose as FOSA in liver (% ) Avg SD. 1 1R00742 2 1R00743 3 1R00744 c o n tro I contro I c o n tro I 0.00 0.00 0.00 0 0 0 154.1 113.2 273.2 180.17 83.12 Avg SD 4 1R00745 5 1R00746 6 1R00747 FOSA FOSA FOSA 644.8 660.2 614.3 644.8 660.2 614.3 163.2 214.3 202.8 193.43 26.81 1.76 2.32 2.16 2.08 0.29 0.27 195.7 0.35 174.1 0.35 163.9 0.32 177.90 0.05 16.24 2.1 1.9 1.7 1.91 0.18 0.33 0.29 0.28 0.30 0.02 152 G r,01<SG T-7071.1 FR DTI 5 Figures Figure 1 Cytochrome P450 content in Liver Total CYTP450 Content in Rat Liver Following Treatment With 0.2000 FOSA Ui 0.1500 in CL 0.1000 o a1>0 o Ec 0.0500 Average of n=3; Error bars represent the standard error of the mean 0.0000 Control FOSA 153 rV ~- a** .^LV(O7 T-7071.1 FR DTI 5 Figure 2: Lauroyl CoA oxidase activity in liver LCoA-Oxidase Activity in Rat Liver Homogenate Following Treatment With FOSA 8.00 o> 7.80 eJ 7.60 - O CM X 7.40 - <0/>> 7.20 - o Ec 7.00 & 6.80 J> U < 6.60 - 4> w "COO 6.40 - *x O 6.20 <o o_ l 6.00 5.80 - Average of n=3; Error bars represent the Standard error of the mean. Control * FOSA 154 CC01G8 Appendix 1 Cytochrome P450 content Absorbance Values Sam Treat pie # ment 450 500 Contro 1 I -0.0366 -0.0312 Contro 0.0254 0.0220 2l 2 9 Contro 3 l -0.0144 -0.0172 4 FOSA -0.0404 -0.0456 0.1360 0.1008 5 FOSA 1 3 0.0059 6 FOSA 4 -0.013 protein cone. (ug/ul) 8.20 8.79 9.25 9.79 10.48 9.86 mg protein added nmole s uM volu total CYTP CYTP me nmol 450/m 450 - 500 450 (ml) e 9 5 0.0054 0.059 3 0.178 0.0356 5 0.00333 0.037 3 0.110 0.0220 5 0.0028 0.031 3 0.092 0.0185 5 0.0052 0.057 3 0.171 0.0343 5 0.03518 0.387 3 1.160 0.2320 5 0.01894 0.208 3 0.624 0.1249 Statisti cs n=1 n=2 n=3 Averag e Stdev Sterror Varria nee ftest Ttest (unpaired, one tailed, unequal variance) P value Control 0.0356 0.0220 0.0185 0.0253 0.0091 0.0052 0.0001 1 0.50 FOSA 0.0343 0.2320 0.1249 0.1304 0.0989 0.0571 0.0098 0.02 0.10 - 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 n nV = M 7 7 Y + 0 20 40 60 80 100 sample ul Absorbanc ug mg/ml average slope' intercep Treatment e protein t 1A Control 5 0.7799 42.064935 8.412987 8.2 0.0077 0.456 1B Control 5 0.7635 39.935065 7.987013 0.0077 0.456 2A Control 5 0.8004 44.727273 8.945455 8.790909 0.0077 0.456 2B Control 5 0.7885 43.181818 8.636364 0.0077 0.456 3A Control 5 0.8155 46.688312 9.337662 9.248052 0.0077 0.456 3B Control 5 0.8086 45.792208 9.158442 0.0077 0.456 4A FOSA 5 0.844 50.38961 10.07792 9.788312 0.0077 0.456 4B FOSA 5 0.8217 47.493506 9.498701 0.0077 0.456 sample mg/m ul 1 neede 5A FOSA 5B FOSA 6A FOSA 6B FOSA dto get5 mg 5 0.8493 51.077922 10.21558 10.47792 0.0077 0.456 1 8.20 609.8 5 0.8695 53.701299 10.74026 0.0077 0.456 2 8.79 568.8 5 0.8305 48.636364 9.727273 9.855844 0.0077 0.456 3 9.25 540.7 5 0.8404 49.922078 9.984416 0.0077 0.456 4 9.79 510.8 5 10.48 477.2 6 9.86 507.3 Appendix 2: Lauroyl CoA Oxidase A c tiv ity 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 H202 produced/ 30 min: Sample Control Difference 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 ii nmoles H202 produced/min/mg protein: Control FOSA 1 2 3 4 5 6 nmoles H202 time (min) ug protein . 3.99 3.86 3.99 4.36 4.84 4.16 30 30 30 30 30 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 H202 produced/min/mg protein) 6.66 7.27 6.43 8.07 6.65 6.94 6.58 7.43 0.13 0.58 0.07 0.34 -- H2O2 Standard Curve Data. H2O2 Stock was 8.6 M nmnoles H202 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% H 202 Standard Curve Fluorescence nmoles H202 Protein Standard Curve ul BSA ul Lysis Buffer 5 95 20 80 40 60 60 40 80 20 Absorbanc e 0.4464 0.6769 0.854 1.0135 1.037 sample 1 1 ul 5 5 25 25 35 35 45 45 55 55 65 65 Absorbanc ug ug/ul average slope intercep e protein t 0.7814 34.435644 6.887129 7.054455 0.0101 0.4336 0.7983 36.108911 7.221782 0.0101 0.4336 0.7956 0.841 0.7963 0.8152 0.8225 0.8408 0.8592 0.8543 0.8501 0.8868 35.841584 7.168317 7.617822 0.0101 0.4336 40.336634 8.067327 0.0101 0.4336 35.910891 7.182178 7.369307 0.0101 0.4336 37.782178 7.556436 0.0101 0.4336 38.50495 7.70099 7.882178 0.0101 0.4336 40.316832 8.063366 0.0101 0.4336 42.138614 8.427723 8.379208 0.0101 0.4336 41.653465 8.330693 0.0101 0.4336 41.237624 8.247525 8.610891 0.0101 0.4336 44.871287 8.974257 0.0101 0.4336 sample ug/ul protein 1 7.05 2 7.62 3 7.37 4 7.88 5 8.38 6 8.61 ul needed to get 20 ug___ 2.84 2.63 2.71 2.54 2.39 2.32 Appendix 11: Final Report for FOSAA (T-7071.1) Final Report Title: Comparative Molecular Biology of Peroxisome Proliferation in Rats following four consecutive days of Perfluorooctanesulfonamidoacetate: (FOSAA, T-7071) Study Number: DT15 Protocol Amendment Number*: 2. 3M Medical Department Study Number: T-7071.1 Study Director. Andrew M. Seacat Ph.D., DABT 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 Study Protocol Title: Comparative Molecular Biology of Peroxisome Proliferation in Rats and Guinea Pigs Study Initiated: In-Life Start Date: November 16, 1998 In-Life End Date: December 21, 1998 uc, to Purpose The purpose of this study was to treat three male rats treated with either no compound (vehicle control) or 160 mg/kg/day perfluorooctanesulfonamidoacetate: C8F17S02NHCH2C00- (FOSAA, M556, T-7071). 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.. Test Materials Perfluorooctanesulfonamidoacetate: (FOSAA, M556, C8F17S02NHCH2C00- NB# 12837-84/30, from G. Moore 12/2000. 3M Medical department # T-7071) 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 160 mg FOSAA/Kg/day, via oral gavage on days one through four of the study, and were euthanized on day five. A suspension of 32 mg/ml M556 in propylene glycol was prepared, and a volume of 5 ml/kg was administered. This dosing regiment achieved a cumulative dose of 640 mg/kg M556 after four successive days of dosing. This dose was comparable to the cumulative dose of NEtFOSE which induced peroxisomal palmitoyl Co-A oxidase (PCOAO) activity in the rat in a 4 week feeding study with 300 ppm N-EtFOSE. 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 1100 x g at 4 C. Two aliquots of the serum sample (~ 0.75 ml) were saved for possible metabolite analysis. 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 e t al. 2001). O .2) 0 1 vl G3 Results and Discussion Average body weights of the FOSAA treatment group were significantly lower than the control group on day 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 FOSAA treatment group The results of P450 content (Figure 1) and Acyl CoA oxidase activity (Figure 2) for the liver samples indicated that FOSAA induced the expression of these genes and is therefore a good peroxisome proliferator in rats. The concentrations of parent compound and metabolites in livers were measured (Table 1). The concentration of the parent compound FOSAA in the liver was approximately 555 ppm, which is equivalent to about 0.24 % of the cumulative dose of FOSAA (Table 3). The data show that PFOS and FOSA (apparent) were the major metabolites found in the livers of rats given FOSAA. The amount of PFOS and FOSA in the liver represented approximately 0.07 and 0.14 percent of the cumulative dose, respectively, if one subtracts the background levels of FOSA in the control lives from the FOSA levels found in the FOSAA treatment group, the concentrations are decreased by approximately 50%. Therefore a quantitative interpretation of the amount of metabolism of the parent compound to FOSA cannot be provided however the data qualitatively suggest that such metabolism occurred. It is noteworthy that the control animals (1R00742, 1R00743, and 1R00744) contained significant concentrations of FOSA. No PFOS was found in the control samples with a high background of FOSA. These results suggest that the control liver samples were contaminated at some point time ex-vivo by trace quantities of FOSA, as no metabolism to PFOS had occurred in the control samples. A parallel analysis of livers from Fischer 344 rats maintained in the University of Rochester Vivarium did not show detectable concentrations of FOSA O "> o h* to Signatures: Report prepared by, Andrew M. Seacat, PhD, DABT Study Director Reviewed by, Daniel C. Hakes Sponsor representative Date Date o 00 Tables Table 1 Biological Parameters D T15 Body W eight (BW ), Liver W eig h t(L W ) D a te 2/19/01 2/19/01 2/21/01 2/22/01 2/23/01 2/23/01 Sam ple A nim al # D ose G roup BW BW BW BW BW L W (g ) LW /BW (g) (g) (g)# day 1 day 2 day 3 day 4 day 5 (% ) (9) (9) Avg SD 1 1R0074 2 2 1R0074 3 3 1R0074 4 control control control 250 259 260 264 272 10.73 3.9% 269 278 285 292 295 10.82 3.7% 256 260 267 268 276 10.70 3.9% 258 266 271 275 281 10.75 3.8% 10 11 13 15 12 0.06 0.14% 7 1R0074 8 8 1R0074 9 9 1R0075 0 Avg SD T-Test* 160 m g/kg/day M556 160 m g/kg/day M556 160 m g/kg/day M556 254 263 264 258 250 10.74 4.3% 254 262 265 263 262 10.77 4.1% 267 270 267 247 227 10.79 4.8% 258 265 265 256 246 10.77 4.4% 8 4 2 8 18 0.03 0.33% 0.50 0.46 0.26 0.07 0.02 0.35 0.03 *T -T est (unpaired, one tailed, equal variance, P < 0.05 considered significantly different from control). Table 2 Liver Fluorocarbon Concentration Hepatic Concentrations of Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given FOSAA (160 mg/kg/day) Sample # Animal # 1 1R00742 2 1R00743 3 1R00744 7 1R00748 8 1R00749 9 1R00750 Weight of liver sample PFOS Sex Treatment (R) (ppm) FOSA FOSAA (ppm) (ppm) M Control 0.4600 0.0 154.1 0.0 M Control 0.9236 0.0 113.2 0.0 M Control 1.1287 0.0 273.2 0.0 M FOSAA 0.5520 238.4 447.3 614.4 M FOSAA 0.9373 81.9 235.7 424.0 M FOSAA 0.5320 100.7 267.4 627.1 i auic j r crucili r luurocaiuun i^usc ni i^ivci Hepatic Percent of Dosed Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given FOSAA (160 mg/kg/day) for Four Days Sam ple Anim al ## Dose G roup Total PFOS Dos in . e(mg) d * o s e * (m g) Liver Liver % Liver Liver % FO SAA Liver % dose PFOS PFOS dose FOSA FOSA dose (ppm ) FOSAA as (PPm) (m g) as (PPm ) (mg) as (mg) FO SAA PFOS FOSA in liver in liv e r in liv e r (% ) (%) (%) Avg SD 1 1R0074 2 2 1R0074 3 3 1R0074 4 control control control 0.00 0.00 0.00 0 0 0 154.1 113.2 273.2 180.2 83.1 0 0 0 Avg SD 7 1R0074 8 8 1R0074 9 9 1R0075 0 FOSAA 2465 FOSAA 2453 FOSAA 2439 2452 13 2212 238.4 2201 81.9 2189 100.7 2201 140.3 12 85.4 2.56 0.88 1.09 1.51 0.92 0.12 447.3 0.04 235.7 0.05 267.4 0.07 316.8 0.04 114.1 4.8 0.19 614.4 2.5 0.10 424.0 2.9 0.12 627.1 3.4 0.14 555.2 1.2 0.05 113.8 6.6 0.27 4.6 0.19 6.8 0.28 6.0 0.24 1.2 0.05 ** M ole fraction P -O S , e.g. for M 556, D ose (m g) M 556 * (499 M W o f PFO S / M W M 556) = 2464.5 * (499 /5 5 6 ) = 2212 mg P FO S in dose. O o CQ CO Figures Figure 1 Cytochrome P450 content in Liver Total C YTP450 C ontent in Rat Liver Follow ing Treatm ent W ith FOSAA nmoles CYTP450/mg 0.2000 0.1500 0.1000 0.0500 0.0000 Average of n=3; Error bars represent the standard error of the mean Control FOSAA Figure 2: Lauroyl CoA oxidase activity in live r LCoA-Oxidase Activity in Rat Liver Homogenate Following Treatment With FOSAA o> 14.00 "e E 12.00 O CM X 10.00 <<AD O E 8.00- _c >% > 6.00 - o < <tnD 4.00 - m o 'x O 2.00 - < o o _l 0.00 Averageof n=3; Error bars represent tie Standard error of the mean. Control * FOSAA T-7071.1 FR DT15 172 c r o i SG T-7071.1 FR DTI 5 Appendix 1 Cytochrome P450 content Values 450 1 -0.0366 2 0.02542 3 -0.0144 7 -0.0355 B -0.0231 9 -0.0586 protein cone, (uq/ul) 1 8.20 2 8.79 3 9.25 7 10.96 B 10.59 9 9.58 Absorbance 500 -0.0312 0.02209 -0.0172 -0.0298 -0.022 -0.0262 mg protein added 5 5 5 5 5 5 450 - 500 0.0054 0.00333 0.0028 0.0057 0.0011 0.0324 uM CYTP450 volume (ml) 0.059 3 total nmole 0.178 0.037 0.031 0.063 0.012 0.356 3 0.110 3 0.092 3 0.188 3 0.036 3 1.068 nmoles CYTP450/mg 0.0356 0.0220 0.0185" 0.0376 0.0073 0.2136 average (n=3) Contra 1 0.0253 FOSAA 0.0862 Statistics Control n=1 0.0356 n=2 0.0220 n=3 0.0185 FOSAA 0.0376 0.0073 0.2136 Average 0.0253 0.0862 O o f** T-7071.1 FR DTI 5 Stdev 0.0091 Sterror 0.0052 Varriance 0.0001 ftest 1 Rest (unpaired, one tailed, unequal variance) p value 0.50 | 0.1114 0.0643 0.0124 0.01 0.22 O *C) H* Ci) GO 174 rru ie m L/cicnm ium uii 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 O <') 0 'Jj co sample ul Absorbanc ug e protein mg/ml average slope intercep t 1A 5 0.7799 42.064935 8.412987 8.2 0.0077 0.456 1B 5 0.7635 39.935065 7.987013 0.0077 0.456 2A 5 0.8004 44.727273 8.945455 8.790909 0.0077 0.456 2B 5 0.7885 43.181818 8.636364 0.0077 0.456 3A 5 0.8155 46.688312 9.337662 9.248052 0.0077 0.456 3B 5 0.8086 45.792208 9.158442 0.0077 0.456 7A 5 0.8816 55.272727 11.05455 10.95584 0.0077 0.456 7B 5 0.874 54.285714 10.85714 0.0077 0.456 sample mg/m 1 8A 5 8B 5 0.8679 0.8597 53.493506 10.6987 10.59221 0.0077 52.428571 10.48571 0.0077 0.456 0.456 1 8.20 2 8.79 9A 5 0.8187 47.103896 9.420779 9.581818 0.0077 0.456 3 9.25 9B 5 0.8311 48.714286 9.742857 0.0077 0.456 7 10.96 8 10.59 9 9.58 ul needed to get 5 mg 609.8 568.8 540.7 456.4 472.0 521.8 average stdev %dev average stdev %dev 12 206.3 205.4 207.5 205 209.6 206.9 207.8 205.8 1.7 1.0 0.8% 0.5% 12 123 122.9 120.3 121.7 120.2 121.5 121.2 122.0 1.6 0.8 1.3% 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% 78 309.6 249.1 308.3 252.1 308.5 248.4 308.8 249.9 0.7 2.0 0.2% 0.8% 78 136 130.5 131 133.2 128.2 128.1 131.7 130.6 4.0 2.6 3.0% 2.0% 9 283.9 283.4 280.2 282.5 2.0 0.7% 9 128.7 131.9 125.6 128.7 3.2 2.4% nmoles H202 pro< uced/ 30 min: Sample Control Differenc e 1 4.02 0.03 3.99 2 3.93 0.07 3.86 3 4.22 0.23 3.99 7 8.68 0.52 8.16 8 5.96 0.47 5.50 9 7.47 0.38 7.09 slope 21.692 21.692 intercep t 120.5 120.5 21.692 21.692 21.692 21.692 120.5 120.5 120.5 120.5 nmoles H202 produced/min/mg protein: Control 1 2 3 FOSAA 7 8 9 ugnmoles time (min) H202 protein 3.99 30 20 3.86 30 20 3.99 30 20 8.16 30 20 5.50 30 20 7.09 30 20 average (n=3) 6.66 Control 6.43 6.6 6.65 13.60 FOSAA 9.16- 11.5 11.81 n=1 n=2 n=3 Average Control FOSAA (nmoles H202 produced/min/mg protein) 6.66 13.60 6.43 9.16 6.65 11.81 6.58 11.53 - 0 ^0192 II H2O2 Standard Curve Data. H2O2 Stock was 8.6 M nmnoles H202 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% H 202 Standard Curve Q> C 0o> C0O) o 3 0 5 10 nmoles H202 15 C C 0194 Protein Standard Curve ul BSA ul Lysis Buffer 5 95 20 80 40 60 60 40 80 20 Absorbanc e 0.4464 0.6769 0.854 1.0135 1.037 sample 1 1 ul 5 5 25 25 35 35 75 75 85 85 95 95 Absorbanc ug ug/ul average slope intercep e protein t 0.7814 34.435644 6.887129 7.054455 0.0101 0.4336 0.7983 36.108911 7.221782 0.0101 0.4336 sample ug/ul ul needed protein to get 20 0.7956 35.841584 7.168317 7.617822 0.0101 0.4336 1 7.05 ug 2.84 0.841 40.336634 8.067327 0.0101 0.4336 2 7.62 2.63 0.7963 35.910891 7.182178 7.369307 0.0101 0.4336 3 7.37 2.71 0.8152 37.782178 7.556436 0.0101 0.4336 7 8.75 2.29 0.8708 43.287129 8.657426 8.748515 0.0101 0.4336 8 7.88 2.54 0.88 44.19802 8.839604 0.0101 0.4336 9 8.54 2.34 0.8226 38.514851 7.70297 7.878218 0.0101 0.4336 0.8403 40.267327 8.053465 0.0101 0.4336 0.8562 41.841584 8.368317 8.542574 0.0101 0.4336 0.8738 43.584158 8.716832 0.0101 0.4336 K+ mmol/L By PFOS (ROC) (ug/g) 1*5 CV T*1 V> ' ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT -- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSA40mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE40mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) K+mmol/L Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 0.025 0.05 0.918156 12.8 1.01653 Bivariate Dose Group (mg/Kg/da=N-EtFOSA40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 100.4 22.36172 -0.24593 K+ mmol/L 6.65 0.695222 Bivariate Dose Group (mg/Kg/da=N-EtFOSE40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 66.55 60.59266 -0.62394 K+ mmol/L 12.2 0.962635 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 148.2 106.5116 -0.25046 K+mmol/L 4.825 1.746186 Signif. Prob Number 0.0818 4 Signif. Prob Number 0.7541 4 Signif. Prob Number 0.3761 4 Signif. Prob Number 0.7495 4 ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSA40mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE40mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) CHOL mg/dL Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 0.016667 0.040825 -0.43116 40.08333 19.97853 Bivariate Dose Group (mg/Kg/da=N-EtFOSA40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 100.4 22.36172 -0.30166 CHOL mg/dL 71.75 30.43436 Bivariate Dose Group (mg/Kg/da=N-EtFOSE40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 66.55 60.59266 0.111675 CHOL mg/dL 34.375 23.75 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 148.2 106.5116 0.24662 CHOL mg/dL 50.875 21.58848 Signif. Prob Number 0.3933 6 Signif. Prob Number 0.6983 4 Signif. Prob Number 0.8883 4 Signif. Prob Number 0.7534 4 n p p c u u iA i ^ n u j j u i i i i v_ u i I c i a i i u u i u L / i v c i i r t / k 3 A. Correlation of effects by different treatment groups to liver PFOS in rats, Correlation of effects to liver PFOS (ug/g) in Rats % BWdO By PFOS (ROC) (ug/g) S> cv T*1 O c w ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=M556-160mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT -- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=FOSA40mkg ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE160mkd Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) % BW dO Mean 140.3333 95.53118 Std Dev 85.44685 9.405515 Correlation 0.158821 Variable PFOS (ROC) (ug/g) % BW dO Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 00 0 106.2098 2.660886 Bivariate Dose Group (mg/Kg/da=FOSA40mkg Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 193.4333 26.80678 0.253937 % BW dO 93.07698 4.329203 Bivariate Dose Group (mg/Kg/da=N-EtFOSE160mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 1008.075 166.3236 0.662735 % BW dO 87.44432 4.602872 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 918.15 113.933 0.3006 % BW dO 85.28738 3.742806 Correllation of BW to liver PFOS concentrations. Signif. Prob Number 0.8985 3 Signif. Prob Number 1.0000 6 Signif. Prob Number 0.8365 3 Signif. Prob Number 0.3373 4 Signif. Prob Number 0.6994 4 CC098 ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=M556-160mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=FOSA40mkg ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE160mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Bivariate Dose Group (mg/Kg/da=M556-160mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 140.3333 85.44685 -0.12908 LW/BW ratio 0.043867 0.003308 Variable PFOS (ROC) (ug/g) LW/BW ratio Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 00 0 0.03818 0.002552 Bivariate Dose Group (mg/Kg/da=FOSA40mkg Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 193.4333 26.80678 -0.21723 LW/BW ratio 0.045793 0.003394 Bivariate Dose Group (mg/Kg/da=N-EtFOSE160mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 1008.075 166.3236 0.751977 LW/BW ratio 0.045166 0.003235 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 918.15 113.933 0.609934 LW/BW ratio 0.049007 0.003768 Signif. Prob Number 0.9176 3 Signif. Prob Number 1.0000 6 Signif. Prob Number 0.8606 3 Signif. Prob Number 0.2480 4 Signif. Prob Number 0.3901 4 Figs, males and female combined, Correlation of effects to liver PFOS (ug/g) in Guinea Pigs Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSA40mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE160mkd -- ~ Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE40mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) % BW dO Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 0.014286 0.037796 0.592756 107.5171 5.69689 Bivariate Dose Group (mg/Kg/da=N-EtFOSA40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 100.4 22.36172 -0.20567 % BW dO 94.66277 0.909529 Bivariate Dose Group (mg/Kg/da=N-EtFOSE160mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 419.35 214.6788 -0.92559 % BWdO 91.83908 2.210568 Bivariate Dose Group (mg/Kg/da=N-EtFOSE40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 66.55 60.59266 0.63895 % BWdO 99.38514 0.513448 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 171.4857 116.5397 -0.30285 % BW dO 92.95562 5.748654 Signif. Prob Number 0.1607 7 Signif. Prob Number 0.7943 4 Signif. Prob Number 0.0744 4 Signif. Prob Number 0.3611 4 Signif. Prob Number 0.5091 7 LW/BW ratio By PFOS (ROC) (ug/g) Gf*'- coo ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSA40mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE160mkd ----- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE40mkd --- Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) LW/BW ratio Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 0.014286 0.037796 -0.27546 0.041022 0.004556 Bivariate Dose Group (mg/Kg/da=N-EtFOSA40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 100.4 22.36172 0.032225 LW/BW ratio 0.034365 0.003385 Bivariate Dose Group (mg/Kg/da=N-EtFOSE160mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 419.35 214.6788 0.343072 LW/BW ratio 0.038328 0.00237 Bivariate Dose Group (mg/Kg/da=N-EtFOSE40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 66.55 60.59266 0.214886 LW/BW ratio 0.03445 0.002243 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 171.4857 116.5397 -0.21072 LW/BW ratio 0.036088 0.004271 Signif. Prob Number 0.5499 7 Signif. Prob Number 0.9678 4 Signif. Prob Number 0.6569 4 Signif. Prob Number 0.7851 4 Signif. Prob Number 0.6502 7
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