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AR226-3205 DuPont-2927 Supplement No. 1 TRADE SECRET Study Title H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Supplement No. 1 Laboratory Project ED: DuPont-2927 :g:iW:a Sfir AUTHOR: Carol Finlay, B.A. STUDY COMPLETED ON: August 3,2000 SUPPLEMENT No. 1 COMPLETED ON: December 12,2001 PERFORMING LABORATORY: E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences EUrton Road, P.O. Box 50 Newark, Delaware 19714-0050 WORK REQUEST NUMBER: [j^^I^B^--.j SERVICE CODE NUMBER: y m Page 1 of 40 Compansf Sanitized. H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Reviewed by: /Paul M. Ifindertter, Ph-D. Postdoctoral Fellow C^g^gC Reviewed by; <'. ^^g^g-^- /fJudith C. Sladfer. Fh.D-. D.A.B.T. ^/ Director Issued by Study Director; QL^f^ ( ^UifrC Carol fvday.B.A. Q Staff Scientist it-J^e-^ Date Q^-^e.c'^Q/ Date n^M. Date ^\ -2- Company SanESized. Does not contain TSCA CBJ H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rate DuPont-2927 Supplement No. 1 TABLE OF CONTENTS Page CERTIFICATION ........................................................................................................................2 LIST OFTABLES........................................................................................................................ 4 LIST OFFIGURES...................................................................................................................... 4 LIST OF APPENDICES..............................................................................................................^ STUDYINFORMATION.............................................................................................................5 STUDY PERSONNEL..................................................................................................................? REASON FOR SUPPLEMENT NO. 1....................................................................................... 8 SUMMARY....................................................................................................................................9 MATERIALS AND METHODS ............................................................................................... 10 RESULTS AND DISCUSSION................................................................................................. 10 A. Liver Weights................................................................................................................... 10 1. TestSubstance.....................................................................................,..................................................10 2. Positive Controls....................................................................................................................................10 B. Liver Fluorine Data.......................................................................................................... 11 1. Positive Controls....................................................................................................................................11 2. Test Substance........................................................................................................................................ 11 CONCLUSIONS......................................................................................................................... 11 TABLES....................................................................................................................................... 12 FIGURES..................................................................................................................................... 16 APPENDICES............................................................................................................................. 22 Company Sanitized. Does not contain TSCA CB8 H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 LIST OF TABLES Page 1. MEAN BODY AND LIVER WEIGHTS............................................................................................................. 13 2. MEANLIVERFLUORINELEVELS..................................................................................................................15 3. MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE.................................................. 15 LIST OF FIGURES Page 1. COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND NEGATIVE CONTROLS .........................................................................................................................................................17 2. COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND POSITIVE CONTROLS.........................................................................................................................................................18 3. COMPARISON OF RELATIVE LIVER WEIGHT AND MEAN LIVER FLUORINE CONCENTRATION FOR H-24018 AND NEGATIVE CONTROLS................................................................ 19 4. MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE..................................................20 5. COMPARISON OF MEAN BLOOD AND MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE ..................................................................................................................................21 LIST OF APPENDICES Page A. INDIVIDUAL FINAL BODY WEIGHTS, LIVER WEIGHTS, AND RELATIVE LIVER TO BODY WEIGHT.............................................................................................................................................................^ B. INDIVIDUAL FLUORINE LEVELS..................................................................................................................34 -4- Company Sanitized. Do8 not contain TSCACBI H-24018: B iopersistence Screening 10-Dose Oral Gavage Study in Rats STUDY INFORMATION TEST SUBSTANCE; Substance Tested: Synonyms/Codes: H-24018 Haskell Number: 24018 DuPont-2927 Supplement No. 1 Known Impurities: POSITIVE CONTROL; Substance Tested: Potassium perfluoroalkyi sulfonate Synonyms/Codes: H-24019 .5- Sanitized. Does not contain TSCA CB1 H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 STUDY INFORMATION (Continued) POSITIVE CONTROL: Substance Tested: Octanoic acid, pentadecafluoro-, ammonium salt Synonyms/Codes: H-24020 Perfluorooctanoate, ammonium salt APFO Ammonium perfluorooctanoate Haskell Number: 24020 Composition Sponsor: E. I. du Font de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: April 30,1999 / (see report cover page) m-Life Initiated/Completed: June 13,1999 / September 14,1999 -6- Company Sanstaed. Ooes noi contain 'TSCA CB6 H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 STUDY PERSONNEL Study Director: Management: Primary Technician: Carol Finlay, B.A. Judith C. Stadler, Ph.D., D.A.B.T. Richard P. Mathena Liver Fluorine Data Analysis: Paul M. Hmderliter, Ph.D. Management: Matthew S. Bogdanffy, Ph.D., D.A.B.T. Toxicology Report Preparation: Wanda F. Dinbokowitz Laboratory Veterinarian: Wanda L. West, D.V.M., A.C.L.A.M. -7- Conipany Sanitized. Does noi contain TSCA CBI H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 REASON FOR SUPPLEMENT NO. 1 The purpose of this supplement is to present the results from analysis of selected liver samples from rats treated with the test substance (H-24018), 2 positive controls (H-24019 and H-24020), and 2 negative controls (corn oil and corn oil/acetone). R - - Company Sanitized. Does not contain TSCA CBB H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 SUMMARY The objective of this study was to evaluate the potential for H-24018, when administered by gavage, to be absorbed and to accumulate in a mammalian system. Six groups of 5 male Crl:CD(SD)IGSBR rats each were given 1000 mg/kg/day of H-24018. The test substance was administered to one group of 5 rats for 5 consecutive days and to 5 groups for 10 days. On selected days (5,10,13,24, 52,94) 5 rats per group were euthanized and the blood and livers were collected. Livers were weighed and those collected on study days 10 and 94 were analyzed for total fluorine. This supplement includes liver weight data and the results of total fluorine analyses in liver. Additionally, 2 negative controls, corn oil and corn oil/acetone (80:20), and 2 positive controls, H-24019 (10 mg/kg/day) and H-24020 (20 mg/kg/day), were tested as described for H-24018. The mean relative liver weight (liver/body weight) of rats dosed with the test substance, H-24018, was up to approximately 27% higher than negative controls on day 10. On day 94, the weights were similar. On day 10, the mean relative liver weight of rats dosed with the positive control H-24019 was up to 27% higher than the negative controls. By day 94, the mean relative liver weight was up to 28% higher than the negative controls. In rats given H-24020, the mean relative liver weight was up to 75% higher at day 10 than the negative controls. By day 94, the weights were similar. The concentration of fluorine in the livers from rats dosed with the test substance, H-24018, was 77.12 iiM equivalents on day 10 and 8.59 ^.M equivalents on day 94. On day 10, the mean ^M. equivalent concentrations of fluorine in the livers from rats dosed with the positive control materials were approximately 65-fold (H-24019) and 15-fold (H-24020) greater than the fluorine concentration in livers from rats treated with the test substance. By day 94, the concentration for H-24019 was approximately 140x the fluorine concentration in rats treated with H-24018. The concentrations were similar for H-24018 and H-24020 at day 94. The pM equivalents of fluorine in livers from rats dosed with the test substance, H-24018, were higher than levels in the blood. Under the conditions of this study, there was some absorption and retention of fluorine in the liver from rats dosed with H-24018. Fluorine levels in the livers from rats dosed with the test substance were lower than levels in rats dosed with the positive control, H-24019. Fluorine levels in the livers from rats dosed with the test substance were lower on day 10 than levels in rats dosed with the positive control H-24020. On day 94, the fluorine levels were similar. Liver weights were elevated in rats dosed with the test substance, H-24018, at the end of the dosing period, but not at the end of the recovery period. -9- Com^.y ^amt^d. uo^s nu. ^.ua.n SCA CBi H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 MATERIALS AND METHODS Blood and livers were collected on days 5,10,13,24, 52, and 94. The livers were weighed. Only the test day 10 and 94 livers were analyzed for total fluorine. These time points provided sufficient data to determine the accumulation and persistence of fluorine in the liver. See the original report for details of the analysis. RESULTS AND DISCUSSION A. Liver Weights (Table 1, Figures 1-3, Appendix A) As with body weights, comparison of absolute liver weights was complicated by the fact that there was a difference in age on test day 1 between rats dosed with the test substance and those dosed with the positive or negative controls. The rats dosed with the test substance, H-24018, were older and heavier in weight than the corn oil negative control and H-24020 positive control rats, but younger and lighter in weight than the corn oil/acetone negative control and H-24019 positive control rats. Therefore, liver weights relative to body weight are presented for comparison. 1. Test Substance The mean relative liver weight of rats dosed with the test substance, H-24018, was up to approximately 27% higher than negative controls on day 10. On day 13, the weight was up to approximately 26% higher than the negative controls. The weights were similar throughout the remainder of the recovery period. 2. Positive Controls The mean relative liver weight of rats dosed with one of the positive controls, H-24019, was similar on day 10 to the liver weight of rats dosed with the test substance, H-24018. On days 13, 24, and 52 (recovery), the mean relative liver weights of rats dosed with H-24019 were 10 or 20% higher than the liver weights of rats dosed with H-24018. By test day 94 (end of recovery), the mean relative liver weight of rats dosed with H-24019 was 17% higher than the liver weight of rats dosed with H-24018. The mean relative liver weight of rats dosed with H-24020, the other positive control, was 38% higher on day 10 than the liver weight of rats dosed with H-24018. On days 13 and 24 (recovery), the mean relative liver weights of rats dosed with H-24020 were 29 and 22% higher than the liver weights of rats dosed with H-24018. On days 52 and 94, the weights were similar. The mean relative liver weight of rats dosed with H-24019 was up to 27% higher at day 10 than the negative control groups. By day 94, the mean relative liver weight of rats dosed with -10- CO^ "^LBI ^^------ Comp^anaynS^adni.tized. Does noteontam TSCACB1 H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 H-24019 was up to 28% higher than the negative controls. The mean relative liver weight of rats dosed with H-24020 was up to 75% higher at day 10 than the negative controls. The mean relative liver weights of rats dosed with H-24020 began to decrease during the recovery period and were similar by test day 94 to the negative controls. Therefore/liver weights of rats dosed with the test substance, H-24018, were elevated at the end of the 10-day dosing period. The mean relative liver weight by the end of recovery in rats dosed with the test substance was higher than in rats dosed with the positive control H-24019 but similar to rats dosed with the positive control H-24020. B. Liver Fluorine Data (Tables 2 and 3, Figures 3 through 5, Appendix B) 1. Positive Controls The positive controls were H-24019 and H-24020. The concentrations of fluorine in the livers on day 10 from rats dosed with the positive control materials were 5045.85 and 1127.25 ^M equivalents for H-24019 and H-24020, respectively. By day 94, the concentrations were 1190.15 and 10.72 fJiM equivalents. 2. Test Substance Levels of total fluorine in livers from rats dosed with the test substance, H-24018, were generally lower than the levels in livers from rats dosed with the positive control materials. The total fluorine concentration in the liver from rats dosed with H-24018 was 77.12 juM equivalents at day 10 and 8.59 pM equivalents at day 94. For the positive control H-24019, the liver concentrations were approximately 65x higher (day 10) and approximately 140x higher (end of study) than H-24018. For H-24020, the liver concentration was approximately 14x higher at the end of dosing. The concentrations were similar by the end of the study. The jtiM equivalents of fluorine in the livers from rats dosed with the test substance, H-24018, were higher than levels in the blood. CONCLUSIONS Under the conditions of this study, there was some absorption and retention of fluorine in the liver from rats dosed with H-24018. Fluorine levels in the livers from rats dosed with the test substance were lower than levels in rats dosed with the positive control, H-24019. Fluorine levels in the livers from rats dosed with the test substance were lower on day 10 than levels in rats dosed with the positive control H-24020. On day 94, the fluorine levels were similar. Liver weights were elevated in rats dosed with the test substance, H-24018, at the end of the dosing period, but not at the end of the recovery period. Company Sanitized. Does not contain TSCA CBI H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 TABLES 12. CrfMUBiigrfi-t-ijO,' d(uli&u uuca >iw& b>u(ii>ai i^fwj^wdl Company Sanitized. Does not eontaij) TSCA CBI H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 Test Days 5 10 13 24 52 94 TABLE 1 MEAN BODY AND LIVER WEIGHTS (g) CORN OIL (NEGATIVE CONTROL) Absolute________ Body Weight Liver Weight 259.3 12.620 311.1 15.861 322.9 14.906 391.2 16.635 464.7 17.240 557.8 20.118 Mean Relative Liver Weight(% of Body Weight) 0.049 0.051 0.046 0.043 0.037 0.036 Test Days 5 10 13 24 52 94 CORN ODLACETONE (NEGATIVE CONTROL) Absolute Body Weight Liver Weight 313.4 13.632 356.5 15.597 377.6 17.715 423.5 17.172 527.6 21.275 567.8 18.447 Mean Relative Liver Weight(% of Body Weight) 0.043 0.044 0.047 0.041 0.040 0.032 Test Days 5 10 13 24 52 94 H-24019 (POSITIVE CONTROL) Absolute________ Body Weight Liver Weight 306.6 15.456 310.1 17.336 344.8 22.049 387.9 21.313 438.1 19.826 597.9 24.455 Mean Relative Liver Weight(% of Body Weight) 0.050 0.056 0.064 0.055 0.045 0.041 13Company Sanitized, i H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 Test Days 5 10 13 24 52 94 TABLE 1 (Continued) MEAN BODY AND LIVER WEIGHTS (g) H-24020 (POSITIVE CONTROL) Absolute BodyWei^ght Liver Weight 244.8 17.052 284.4 21.944 289.1 21.780 370.3 20.619 456.7 17.713 538.7 18.205 Mean Relative Liver Weight(% of Body Weight) 0.070 0.077 0.075 0.056 0.039 0.034 Test Days 5 10 13 24 52 94 H-24018 (TEST SUBSTANCE) Absolute BodyWei^sht Liver Weight 265.5 14.828 296.5 16.650 333.8 19.479 406.6 18.642 488.9 20.056 596.5 20.595 Mean Relative Liver Weight(% of Body Weight) 0.056 0.056 0.058 0.046 0.041 0.035 -14Comp'any Sanitized. Does not contain TSCA CBI H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rate DuPont-2927 Supplement No. 1 TABLE 2 MEAN LIVER FLUORINE LEVELS Negative Controls Positive Controls Test Substance Test Corn Oil Corn OihAcetone H-24019 H-24020 H-24018 K)0.2<0.5328.18 Days 94 (ppm)_____(ppm)______(ppm) ____(ppm)______(ppm) (19.3)"155.76 (29.2)131.3 (30.1) 0.6"______I3______77.56 (10.1) 1.68 (0.3) 14.8 (7.9) a One value. Four of the values were below the level of detection (LOD). b Standard deviation is in parentheses. TABLE 3 MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE Test Days 10 94 Positive Controls Test Substance H-24019 H-24020 H-24018 fiM F Equivalents 5045.85 (296.91)' fiM F Equivalents___juM F Equivalents 1127.25 (211.37) 77.12 (17.7) 1190.15 (156.05) 10.72 (2.14)______8.59 (4.7) a Standard deviation is in parentheses. -15- Company Sanitized. Does not contain TSCACBI H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 FIGURES -16- Company Sanitized. Does not contain TSCACB1 H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURE 1 COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND NEGATIVE CONTROLS _i S -17- H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURE 2 COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND POSITIVE CONTROLS Test Day -18- H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURES COMPARISON OF RELATIVE LIVER WEIGHT AND MEAN LIVER FLUORINE CONCENTRA FOR H-24018 AND NEGATIVE CONTROLS Day 10 DayW Day 10 D H Corn Oil (Negative Control) Corn OiLAcetone (Negative Control) D H-24018 (Test Substance) -19- H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURE 4 MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE 6000 5000 4000 ss B" 3000 a |X| S 2000 1000 Day 10 Day 94 .20. H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURE 5 COMPARISON OF MEAN BLOOD AND MEAN LIVER FLUORINE CONCENTRATION NORMALIZE 6000 5000 4000 Sg. 3000 a. 2000 1000 Blood / Liver Blood / Liver Blood / Liver -21- H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 APPENDICES Sanitized. Does not contain TSCA CBS Comply ^.^u. uo- .u^..^> .^Au6t H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 APPENDIX A Individual Final Body Weights, Liver Weights, and Relative Liver to Body Weight '"Con.p^San^.O-no.c.^TSCACB, H-24018: Biopersistaice Screening 10-Dose Oral Gavage!Study in Rats DuPont-2927 Supplement No. 1 CORKr OIL (NEGATIVE CONTROL) INDI^iTDUAL FINAL BCIDY WEIGHTS (g), LIVER WEIGHTS (cr), AND RELATIVE LFi/ER TO BODY WEI GHT OF MALE RATS Animal Number 627789 627801 627805 627810 627818 Final Body Wt. 247.7 240.1 283.7 265.0 260.2 GROUP I Liver Wt. 11.428 12.216 14.081 13.395 11.981 Relative Liver to Body Wt. 0.046 0.051 0.050 0.051 0.046 Test Day 5 5 5 5 5 Animal Number 627793 627797 627799 627800 627808 Final Body Wt. 283.5 324.2 294.7 331.3 321.8 GROUP II Liver Wt. 15.292 15.803 14.285 16.344 17.580 Relative Liver to Body Wt. 0.054 0.049 0.048 0.049 0.055 Test Day 10 10 10 10 10 Animal Number 627796 627806 627807 627809 627816 Final Body Wt. 322.7 320.3 305.2 336.2 330.2 GROUP III Liver Wt. 13.404 15.413 14.935 14.967 15.811 Relative Liver to Body Wt. 0.042 0.048 0.049 0.045 0.048 Test Day 13 13 13 13 13 -24. Company Sanitized. Does not contain TSCA CBI H-24018: Biopersistelice Screening 10-Dose Oral Gavage!Study in Rats DuPont-2927 Supplement No. 1 CORN" OIL (NEGATIVE CONTROL) INDI^TDUAL FINAL BO'DY WEIGHTS (g) ,, LIVER WEIGHTS (cr), AND RELATIVE LBrER TO BODY WEI:GHT OF MALE RATS - Animal Number 627791 627794 627804 627812 627817 Final Body Wt. 365.6 416.7 378.1 388.4 407.3 GROUP IV Liver Wt. 15.371 19.105 15.326 16.775 16.600 Relative Liver to Body Wt. 0.042 0.046 0.041 0.043 0.041 Test Day 24 24 24 24 24 Animal Number 627792 627798 627813 627814 627815 Final Body Wt. 496.8 517.3 416.8 386.9 505.9 GROUP V Liver Wt. 16.359 19.293 15.234 14.358 20.958 Relative Liver to Body Wt. 0.033 0.037 0.037 0.037 0.041 Test Day 52 52 52 52 52 Animal Number 627795 627811 627819 627820 627821 Final Body Wt. 485.0 511.6 653.1 618.0 521.5 GROUP VI Liver Wt. 16.649 17.405 21.828 26.766 17.943 Relative Liver to Body Wt. 0.034 0.034 0.033 0.043 0.034 Test Day 94 94 94 94 94 -25- Company Sanitized. Does not eontain TSCA CBj H-24018: Biopersistelice Screening 10-Dose Oral Gavage!Study in Rats DuPont-2927 Supplement No. 1 CORN OIL.:ACETONE (NEGA1IVE CONTROL) INDI\riDUAL FINAL BODY WEIGHTS (g), LIVER WEIGHTS (g]1 , AND RELATIVE LI^/ER TO BODY WEK3HT OF MALE RATS . Animal Number 625361 625363 625364 625380 625383 Final Body Wt. 349.7 286.7 312.0 305.5 313.1 GROUP I Liver Wt. 16.800 11.236 .13.845 13.253 13.026 Relative Liver to Body Wt. 0.048 0.039 0.044 0.043 0.042 Test Day 5 5 5 5 5 Animal Number 625355 625362 625367 625368 625378 Final Body Wt. 333.6 332.9 363.8 386.7 365.4 GROUP II Liver Wt. 14.409 14.363 16.139 17.745 15.328 Relative Liver to Body Wt. 0.043 0.043 0.044 0.046 0.042 Test Day 10 10 10 10 10 Animal Number 625357 625358 625370 625384 625385 Final Body Wt. 405.6 340.7 407.0 357.1 377.6 GROUP III Liver Wt. 20.091 12.954 21.654 16.520 17.355 Relative Liver to Body Wt. 0.050 0.038 0.053 0.046 0.046 Test Day 13 13 13 13 13 -26- Company Sanitized. Does not contain TSCA CBI H-24018: Biopersistelice Screening 10-Dose Oral Gavage!Study in Rats DuPont-2927 Supplement No. 1 CORN OIL,:ACETONE (NEGA'riVE CONTROL) INDIVTDUAL FINAL BCIDY WEIGHTS (g) ,, LIVER WEIGHTS (gr), AND RELATIVE LIii7ER TO BODY WEIGHT OF MALE RATS Animal Number 625354 625365 625371 625376 625381 Final Body Wt. 396.1 459.6 447.3 432.9 381.8 GROUP IV Liver Wt. 16.383 18.313 17.859 19.168 14.135 Relative Liver to Body Wt. 0.041 0.040 0.040 0.044 0.037 Test Day 24 24 24 24 24 Animal Number 625356 625359 625360 625372 625374 Animal Number 625353 625369 625373 625375 625377 Final Body Wt. 533.7 530.3 555.4 506.2 512.3 Final Body Wt. 565.3 514.8 673.7 588.8 496.6 GROUP V Liver Wt. 20.498 22.901 23.312 21.287 18.378 Relative Liver to'Body Wt. 0.038 0.043 0.042 0.042 0.036 GROUP VI Liver Wt. 18.655 17.379 20.073 18.869 17.261 * Relative Liver to Body Wt. 0.033 0.034 0.030 0.032 0.035 Test Day 52 52 52 52 52 Test Day 94 94 94 94 94 -27- Company Sanitized. Does not contain TSCACBB H-24018: Biopersistaice Screening 10-Dose Oral Gavage;Study in Rats DuPont-2927 Supplement No. 1 H-2.4019 (POSITIVE CONTROL) 1NDI\TDUAL FINAL BC)DY WEIGHTS (g), LIVER WEIGHTS (gr), AND RELATIVE LFi/ER TO BODY WEI GHT OF MALE RATS Animal Number 625322 625323 625336 625343 625349 Final Body Wt. 329.0 298.0 318.1 294.0 293.7 GROUP I Liver Wt. 16.409 14.830 16.545 13.809 15.685 Relative Liver to Body Wt. 0.050 0.050 0.052 0.047 0.053 Test Day 5 5 5 5 5 Animal Number 625320 625330 625332 625341 625350 Final Body Wt. 312.1 310.1 318.3 325.6 284.3 GROUP II Liver Wt. 18.845 16.343 17.528 18.254 15.709 Relative Liver to Body Wt. 0.060 0.053 0.055 0.056 0.055 Test Day 10 10 10 10 10 Animal Number 625321 625327 625342 625345 625348 Final Body Wt. 371.2 355.3 324.4 336.9 336.4 GROUP III Liver Wt. 22.322 22.259 20.718 24.403 20.541 Relative Liver to Body Wt. 0.060 0.063 0.064 0.072 0.061 Test Day 13 13 13 13 13 -28Company Sanitized. Does not contain TSCA CBI H-24018: Biopersistelice Screening 10-Dose Oral Gavage;Study in Rats DuPont-2927 Supplement No. 1 H-2.4019 (POSITIVE CONTROL) INDUi'TDUAL FINAL BCIDY WEIGHTS (g) , LIVER WEIGHTS (gf), AND RELATIVE LFi/ER TO BODY WE:IGHT OF MALE RATS Animal Number 625325 625328 625338 625340 625352 Final Body Wt. 381.3 423.9 339.9 419.0 375.3 GROUP IV Liver Wt. 21.479 22.100 18.018 23.131 21.835 Relative Liver to Body Wt. 0.056 0.052 0.053 0.055 0.058 Test Day 24 24 24 24 24 Animal Number 625324 625329 625333 625334 625344 Final Body Wt. 447.4 498.1 427.4 426.5 391.0 GROUP V Liver Wt. 20.900 20.911 20.508 19.144 17.666 Relative Liver to Body Wt. 0.047 0.042 0.048 0.045 0.045 Test Day 52 52 52 52 52 Animal Number 625326 625335 625346 626347 625351 Final Body Wt. 577.5 579.5 556.9 638.9 636.8 GROUP VI . Liver Wt. 23.749 21.548 23.945 27.416 25.615 Relative Liver to Body Wt. 0.041 0.037 0.043 0.043 0.040 Test Day 94 94 94 94 94 -29Company Sanitized. Does not contain TSCAGBI H-24018: Biopersistelace Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 H-2.4020 (POSITIVE 'CONTROL) iironODUAL FINAL BCIDY WEIGHTS (g), LIVER WEIGHTS (ar), AMD RELATIVE LPi^ER TO BODY WEFGHT OF MALE RATS Aniinal Number 627756 627767 627771 627773 627777 Final Body Wt. 237.5 248.6 239.6 262.3 236.0 GROUP I Liver Wt. 15.360 19.033 16.968 17.629 16.268 Relative Liver to Body Wt. 0.065 0.077 0.071 0.067 0.069 Test Day 5 5 5 5 5 Animal Number 627768 627774 627776 627782 627785 Final Body Wt. 293.5 338.2 254.4 268.2 267.9 GROUP II Liver Wt. 22.536 27.332 18.821 20.172 20.857 Relative Liver to Body Wt. 0.077 0.081 0.074 0.075 0.078 Test Day 10 10 10 10 10 Animal Number 627758 627763 627764 627787 627788 Final Body Wt. 308.7 285.8 306.7 234.9 309.4 GROUP III Liver Wt. 26.021 22.123 21.526 16.498 22.730 Relative Liver to Body Wt. 0.084 0.077 0.070 0.070 0.073 Test Day 13 13 13 13 13 -30- Company Sanitized. Does not contain TSCACB1 H-24018: Biopersiste:ace Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 H-2-4020 (POSITIVE CONTROL) iND:nTTOUAL FINAL BCIDY WEIGHTS (g) , LIVER WEIGHTS (g ) , AND RELATIVE LFi/ER TO BODY WE!:GHT OP MALE RATS Ariimal Number 627759 627761 627770 627779 627786 Final Body Wt. 380.1 359.4 379.8 406.2 326.1 GROUP IV Liver Wt. 22.707 19.579 19.540 24.858 16.410 Relative Liver to Body Wt. 0.060 0.054 0.051 0.061 0.050 Test Day 24 24 24 24 24 Animal Number 627757 627760 627765 627772 627781 Final Body Wt. 436.7 456.5 455.6 513.5 421.0 GROUP V Liver Wt. 16.899 18.240 16.505 20.225 16.695 Relative Liver to Body Wt. 0.039 0.040 0.036 0.039 0.040 Test Day 52 52 52 52 52 Animal Number 627762 627769 627775 627778 627783 Final Body Wt. 528.6 609.6 552.8 445.3 557.2 GROUP VI Liver Wt. 17.732 19.324 17.296 15.628 21.044 Relative Liver to Body Wt. 0.034 0.032 0.031 0.035 0.038 Test Day 94 94 94 94 94 -31Company Sanitized. Does not contain TSCA CBI H-24018: Biopersist<ince Screening 10-Dose Oral Gavage; Study in Rats DuPont-2927 Supplement No. 1 H-:24018 (TEST SUB STANCE) INDI VIDUAL FINAL BC>DY WEIGHTS (g), LIVER WEIGHTS (gr), AND RELATIVE LFi/ER TO BODY WEI GHT OF MALE RATS - Animal Number 627252 627253 627260 627273 627274 Final Body Wt. 247.9 271.4 265.0 293.7 249.3 GROUP I Liver Wt. 14.099 15.371 15.086 16.354 13.228 Relative Liver to Body Wt. 0.057 0.057 0.057 0.056 0.053 Test Day 5 5 5 5 5 Animal Number 627246 627258 627261 627268 627269 Final Body Wt. 283.0 272.7 314.3 338.6 273.7 GROUP II Liver Wt. 14.068 15.101 17.363 20.561 16.156 Relative Liver to Body Wt. 0.050 0.055 0.055 0.061 0.059 Test Day 10 10 10 10 10 Animal Number 627249 627262 627265 627266 627277 Final Body Wt. 357.2 322.7 312.7 309.1 367.3 GROUP III Liver Wt. 22.366 19.749 17.171 17.390 20.717 Relative Liver to Body Wt. 0.063 0.061 0.055 0.056 0.056 Test Day 13 13 13 13 13 -32-Companif Sanifaed. Does not contain TSCA CBS H-24018: Biopersistelice Screening 10-Dose Oral Gavage!Study in Rats DuPont-2927 Supplement No. 1 H-;24018 (TEST SUE1STANCE) INDIVTDUAL FINAL BC1DY WEIGHTS (g) , LIVER WEIGHTS (cr), AND RELATIVE DPi/ER TO BODY WEI:GHT OP MALE RATS - Animal Nuinber 627247 627250 627255 627257 627259 Final Body Wt. 397.3 419.9 430.7 378.3 -- GROUP IV Liver Wt. 17.316 21.643 20.139 15.468 -- Relative Liver to Body Wt. 0.044 0.052 0.047 0.041 -- Test Day 24 24 24 24 24 Animal Number 627256 627264 627271 627275 627278 Animal Number 627248 627251 627254 627263 627270 Final Body Wt. 468.5 489.0 495.9 455.6 535.5 Final Body Wt. 591.0 651.5 628.7 531.5 579.6 GROUP V Liver Wt. 18.735 20.115 19.999 18.417 23.014 Relative Liver to Body Wt. 0.040 0.041 0.040 0.040 0.043 Test Day 52 52 52 52 52 GROUP VI Liver Wt. 19.084 23.777 20.844 18.753 20.515 Relative Liver to Body Wt. 0.032 0.036 0.033 0.035 0.035 Test Day 94 94 94 94 94 Rat found dead on test day 9 -33-Company Sanitized. Does not contain TSCA CBl H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 APPENDIX B Individual Fluorine Levels -34- TSCA CBS Company Sanifeed. Does not contain H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 Terms: Active % Active Mol Wt Active Formulation Dose % F in Active Mol Wt F TERMS AND CALCULATIONS Fluorine containing compound The % of formulation that is made up of fluorine containing components The molecular weight of the fluorine containing components (g/mole) The mg of formulation given per kg of animal body weight The % fluorine in the fluorine containing components of the formulation (weight basis) The molecular weight of fluorine g/mol Compound Calculations: Dose Active (mg/kg) The mg of fluorine containing compound administered per kg of animal body weight. ^ (% active/I 00) x Formulation Dose Dose Active (mmole/kg) The mmole of fluorine containing compound administered per kg of animal body weight = dose [mg/kg] / Mol Wt Active [mg/mmol] Dose F (mg/kg) The mg Fluorine administered per kg of animal body weight = (% F in active/I 00) x Dose Active [mg/kg] Dose F (mmol/kg) The mmole of fluorine administered per kg of animal body weight = Dose F [mg/kg] / Mol Wt F [mg/mmol] Molar Ratio (Active/F) The moles of fluorine containing compound per mole of fluorine = Dose Active [mmol/kg] / Dose F [mmol/kg] Formulation Dose Normalization Factor The formulation dose that would be required to administer the amount of active needed to achieve the normalized dose = (Normalized dose of Active [mmol/kg] / Dose Active [mmol/kg]) x Formulation Dose -35- Company Sanitized. Does not contain TSCA CB1 H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 TERMS AND CALCULATIONS Individual Animal Measurement: ppm F in liver The ppm fluoride measured in liver Individual Animal Calculations: ppm F in Liver minus Bkg 0.2 ppm ppm F in Liver normalized to 0.1 mmol/kg Dose The ppm fluoride measured in liver minus the background fluoride measured in control animal liver. In this case the value was established at 0.2 ppm. The ppm fluoride minus background that would be expected if the active dose was 0.1 mmol/kg instead of the actual active dose. This assumes linearity between administered dose and liver fluorine levels, but is needed because different doses of active were used in the study. == ( 0.1 [mmol/kg] / Active dose [mmol/kg]) x (ppm F in liver minus background) /imolar equivalents of active in liver The ^imolar [/nnol/L] concentration of fluorine containing compound in liver based on the ppm fluorine in liver normalized to 0,1 mmol/kg active dose. This assumes that all liver fluorine is derived from the fluorine-containing component in the formulation. Note: 1 ppm - Img/L = (Normalized ppm [mg/L] fluorine in liver / Mol Wt F [mg/mmol]) x molar ratio active/F [mmol active/mmol F] x 1000 /miol/mmol -36Company Sanitized. Does not contain TSCA CBI H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2927 Supplement No. 1 _____FACTORS INFLUENCING INTERPRETATION OF KINETIC ANALYSIS_____ Considerations: - The data used for kinetic'analysis was from a limited screen and extrapolation should be done cautiously. - Sample size is low - Analytical data used without validation - Steady-state not reached - Terminal phase may not be reached - Some compounds are mixtures of fluorinated compounds - Different active and formulation doses were used - Different vehicles were used to deliver formulations - Each compound may have very different potency for producing toxicity Assumptions: (May or may not be justified in all cases) - Liver fluorine concentrations are linear with respect to dose - Analytical method is appropriate for all types of compounds - Elimination kinetics can be determined based on total fluorine rather than on concentrations of individual components - Background Fluorine is 0.2 ppm - % F data is the % Fluorine of the active (Fluorine containing components) in the formulation) - Molecular weight is the molecular weight of the active component in the formulation -37Corepany Sanitized. Does not contain TSCACBI H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H- 24019 DuPont-2927 Supplement No. 1 Given: Mol Wt. Active (g/mole): 497 Formulation Dose (mg/kg): 10 % Active (F Containing) in Formulation: 100 %F in Adive: 65 Mol Wt.F (g/mol): 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Formulation Dose Normalization Factor: 10 0.020 49.7 Dose F (mmol/kg): 0.342 Molar Ratio (Active/F): 0.059 Dose F (mg/kg):_____6.5 Rat Number Test Day Sample ppm F in Liver Group II 625320 10 319.4 625330 10 355.4 625332 10 338.4 625341 10 305.0 625350 10 322.7 ppm Fin Liver Minus Bkg 0.2 ppm 319.2 355.2 338.2 304.8 322.5 Group VI 625326 94 625335 94 625346 94 625347 94 625351 94 79.8 89.6 73.8 62.5 82.1 79.6 89.4 73.6 62.3 81.9 ppm F in Liver Normalized to 0.1 mmoles/kg Dose 1586.42 1765.34 1680.85 1514.86 1602.83 395.61 444.32 365.79 309.63 407.04 jxmolar Equivalents of Active in Liver 4910.77 5464.62 5203.08 4689.23 4961.54 1224.62 1375.38 1132.31 958.46 1260.00 .38Company Sanitized. Does not contain TSCACBI H-24018: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H- 24020 DuPont-2927 Supplement No. 1 Given: Mol Wt. Active (g/mole): 426 Formulation Dose (mg/kg): 20 % Active (F Containing) in Formulation: 100 %Fin Active: 69 MolW't. F (g/mol): 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Formulation Dose Normalization Factor: 20 0.047 42.6 Dose F (mmol/kg): 0.726 Molar Ratio (Active/P): 0.065 Dose F (mg/kg):_____13.8 Rat Number Test Day Sample ppmF in Liver Group 11 627768 10 627774 10 627776 10 627782 10 627785 10 182.5 109.0 174.0 147.5 165.8 ppmF in Liver Minus Bkg 0.2 ppm 182.3 108.8 173.8 147.3 165.6 Group VI 627778 94 1.9 1.7 627783 94 1.2 1.0 627762 94 1.9 1.7 627775 94 1.6 1.4 627769 94 1.8 1.6 ppm F in Liver Normalized to 0.1 mmoles/kg Dose ptmolar Equivalents of Active in Liver 388.30 231.74 370.19 313.75 352.73 1321.01 788.41 1259.42 1067.39 1200.00 3.62 2.13 3.62 2.98 3.41 12.32 7.25 12.32 10.14 11.59 -39Compansf Sanitized. Does nol contain TSCACBI H-24018: Biopersistence Screening 10-Dose Oral Gavage Study m Rats Data for H-24018 DuPont-2927 Supplement No. 1 Given: Mol Wt. Active (g/mole): 845 % Fin Active: 50 Formulation Dose (mg/kg): 1000 MolWt.F(g/mol): 19 % Active (F Containing) in Formulation: 34 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Formulation Dose Normalization Factor: 340 0.402 248.5 Dose F (mmol/kg): 8.947 Molar Ratio (Active/F): 0.045 Dose F (mg/kg):_____170 Rat Number Test Day Sample ppmF in Liver Group II 627246 10 126.4 627258 10 130.6 627261 10 169.4 627268 10 86.6 627269 10 143.5 ppmF in Liver Minus Bkg 0.2 ppm 126.2 130.4 169.2 86.4 143.3 Group VI 627248 94 627251 94 627254 94 627263 94 627270 94 10.7 8.9 27.6 17.4 9.4 10.5 8.7 27.4 17.2 9.2 ppm F in Liver Normalized to 0.1 mmoles/kg Dose ^tmolar Equivalents of Active in Liver 31.36 32.41 42.05 21.47 35.61 74.24 76.71 99.53 50.82 84.29 2.61 2.16 6.81 4.27 2.29 6.18 5.12 16,12 10.12 5.41 -40Cwpaa^ Sanitized. Does not eonSain TSCA CBI