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AR226-3209 TRADE SECRET Study Title H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Supplement No. 1 Laboratory Project ID: DuPont-2920 AUTHOR: Carol Finlay, B.A. STUDY COMPLETED ON: August 29,2000 SUPPLEMENT No. 1 COMPLETED ON: December 13,2001 PERFORMING LABORATORY: E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 L 1I WORK REQUEST NUMBER: ^r (--> ^j r ^ i SERVICE CODE NUMBER: i -- i Page 1 of 40 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Reviewed by; Paul M. Hiluieriiter, PhJ). Postdoctoral Fellow Reviewed b^: CL^W J^^/. ^ HJudith C. Stadler/PhJ)., D.A.B.T. ^ Director JJL Issned by Study Director: ____<f ^M.K- h^lAu^ Carc/FmIay.B.A.fl Staff Scientist /3'P^-^' Date ^-h^-^OO I Date 13_l^^_^0f Date 2- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 TABLE OF CONTENTS Page CERTIFICATION.......................................................................................................................^ LIST OF TABLES........................................................................................................................4 LIST OF FIGURES......................................................................................................................4 LISTOFAPPENDICES...............................................................................................................4 STUDY INFORMATION............................................................................................................. 5 STUDY PERSONNEL.................................................................................................................. 7 REASON FOR SUPPLEMENT NO. 1....................................................................................... 8 SUMMARY.................................................................................................................................... 9 MATERIALS AND METHODS ............................................................................................... 10 RESULTS AND DISCUSSION................................................................................................. 10 A. Liver Weights.................................................................................................................... 10 1. Test Substance........................................................................................................................................ 10 2. Positive Controls....................................................................................................................................10 B. Liver Fluorine Data...........................................................................................................II 1. Positive Controls ....................................................................................................................................11 2. Test Substance........................................................................................................................................11 11 CONCLUSIONS......................................................................................................................... 12 TABLES....................................................................................................................................... FIGURES..................................................................................................................................... 16 APPENDICES.............................................................................................................................22 -3- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 LIST OF TABLES Page 1. MEAN BODY AND LIVER WEIGHTS.............................................................................................................13 2. MEAN LIVER FLUORINE LEVELS.................................................................................................................. 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-23926 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- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats STUDY INFORMATION TEST SUBSTANCE: Synonyms/Codes: H-23926 Haskell Number: 23926 Composition: Known Impurities: None POSITIVE CONTROL: DuPont-2920 Supplement No. 1 Known Impurities: Unknown 5- cowy sanM^. Does "ot eortata TSCA CBI STUDY INFORMATION (Continued) POSITIVE CONTROL; Substance Test Known Impurities: Unknown Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: April 30,1999 / (see report cover page) In-Life Initiated/Completed: May 16, 1999 / August 17, 1999 -6- H-23926: Biopersistence Screening ^ 10-Dose Oral Gavage Study in Rats____________________________ DuPont-2920 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. Hinderliter, 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- H-23926: Biopersistence Screening DuPont-2920 10-Dose Oral Gavage Study in Rats_________________________________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-23926), 2 positive controls (H-24019 and H-24020), and 2 negative controls (corn oil and corn oil/acetone). 8- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 SUMMARY The objective of this study was to evaluate the potential for H-23926, when administered by gavage, to be absorbed and to accumulate in a mammalian system. Six groups of 5 male Crl:CD(SD)IGS BR rats each were given 1000 mg/kg/day of H-23926. 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, 13, and 94 were analyzed for total fluorine. This supplement includes liver weight data and the results of total fluorine analyses in livers. 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-23926. The mean relative liver weights (liver/body weight) of rats dosed with the test substance, H-23926, were similar to the negative controls throughout the dosing and recovery periods. 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. On 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-23926, was 8.68 (JtM equivalents on day 10 and 5.36 ^M equivalents on day 94. On day 10, mean ^M equivalent concentrations of fluorine in the livers from rats dosed with the positive control materials were approximately 580-fold (H-24019) and 130-fold (H-24020) greater than the ' fluorine concentration in livers from rats treated with the test substance. By day 94, the concentrations were approximately 220x and 2x the fluorine concentration in rats treated with H-23926. The fiM equivalents of fluorine in the livers from animals dosed with the test substance 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-23926. However, fluorine levels in the liver were significantly lower than levels in rats dosed with the positive control materials, H-24019 and H-24020. Liver weights were not affected in rats dosed with the test substance, H-23926. .9- ffiniipair8aayiRffi2<ES.Bs Rfi eesuSaEtiiTSCA C5 H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 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 10,13, 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 1,2, and 3; Appendix A) As with the 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-23926, 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 total body weight are presented for comparison. 1. Test Substance The mean relative liver weights of rats dosed with the test substance, H-23926, were similar to the negative controls throughout the dosing and recovery periods. 2. Positive Controls The mean relative liver weight of rats dosed with one of the positive controls, H-24019, was 24% higher on day 10 than the liver weight of rats dosed with the test substance, H-23926. On days 13 and 24 (recovery), the mean relative liver weights of rats dosed with H-24019 were 42 or 45% higher than the liver weights of rats dosed with H-23926. By test day 94 (end of recovery), the mean relative liver weight of rats dosed with H-24019 was 21% higher than the liver weight of rats dosed with H-23926. The mean relative liver weight of rats dosed with H-24020, the other positive control, was 71% higher on day 10 than the liver weight of rats dosed with H-23926. The mean relative liver weights of rats dosed with H-24020 began to decrease during the recovery period and were similar by days 52 and 94 to the liver weights of rats dosed with H-23926. 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 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 -10- Cotnpsny SanKteed. Does not certain TSCA CB H-23926: Biopersistence Screening - DuPont-2920 10-Dose Oral Gavage Study in Rats_______T________________________Supplement No. 1 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-23926, were not affected. B. Liver Fluorine Data (Tables 2-3, Figures 3-5, 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. At day 13, the liver concentrations were 4135.69 and 334.49 [JM equivalents. By day 94, the concentrations were 1190.15 and 10.72 fiM equivalents. 2. Test Substance Levels of total fluorine in livers from rats dosed with the test substance, H-23926, were 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-23926 was 8.68 /xM equivalents at day 10, 11.70 {iM at day 13, and 5.36 ^M equivalents at day 94. For the positive control H-24019, the liver concentrations were approximately 580x higher (day 10) and approximately 220x higher (end of study) than H-23926. For H-24020, the liver concentrations were approximately 130x higher (end of dosing) and 2x higher (end of study) than H-23926. The ^M equivalents of fluorine in the livers from animals dosed with the test substance 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 following dosing with H-23926. However, fluorine levels in the liver in rats dosed with H-23926 were significantly lower than the levels in rats dosed with the positive control materials, H-24019 and H-24020. Liver weights were not affected in rats dosed with the test substance. -11- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 TABLES 12S. Does noB eanSmIn H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 Test Days 5 10 13 24 52 94 TABLE 1 MEAN BODY AND LIVER WEIGHTS (g) CORN OIL (NEGATIVE CONTROL) Absc>lute 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 13- Cteipaisy ganEBbed. DO ng QQ^^ TSCA H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats TABLE 1 (Continued) DuPont-2920 Supplement No. 1 MEAN BODY AND LIVER WEIGHTS (g) Test Days 5 10 13 24 52 94 H-24020 (POSITIVE CONTROL) Abs olute Body Weight Lh^er 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 H-23926 (TEST SUBSTANCE) Test Days 5 10 13 24 52 94 __B_o_d_y_W_e_igAhbt_s_o_l_uLtivee_r_W_e_ig_h_t __ 304.4 13.704 329.8 14.832 349.4 15.748 401.4 15.395 509.8 19.756 574.9 19.587 Mean R.elativs Liver Weight(% of Body Weight) 0.045 0.045 0.045 0.038 0.039 0.034 -14- Company SanRIzed. Does not contain TSCA CBI H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 TABLE 2 MEAN LIVER FLUORINE LEVELS Negative Controls Positive Controls Test Substance Test Corn Oil Corn OiLAcetone H-24019 H-24020 H-23926 K)<02OJ328.18 Days 13 (ppm)_____(ppm)_______(ppm) (ppm)_______(ppm) (19.3)"155.76 (29.2)8.88 (1.3) - - 269.02 (10.1) 46.36 (10.7) 11.70 (1.4) 94 0.63_____1^_____77.56 (10.1) 1.68 (0.3) 5.56 (2.1) a One value. Four of the values were below the level of detection (LOD). b Standard deviation is in parentheses. Indicates no value for this day. TABLE 3 MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE Test Days 10 13 94 Positive Controls Test Substance H-24019. H-24020 H-23926 iiM F Equivalents 5045.85 (296.91)3 jttM F Equivalents___fiM F Equivalents 1127.25 (211.37) 8.68 (1.27) 4135.69 (155.38) 334.49 (77.70) 11.50 (1.42) 1190.15 (156.05) 10.72 (2.14)______5.36 (2.13) a Standard deviation is in parentheses. -15- Compsmy Sanftfzed. Does not contain TSCA CBt H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats___________'__________ DuPont-2920 Supplement No. 1 FIGURES -16- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats 0.060 FIGURE 1 COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND NEGATIVE CONTROLS 0.0504 0.040 ^SIS ^ <s ^ ^ S S 0.030 0.020 0.010 0.000 Test Day 17CCTgnpatey^anretewd. D H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURE 2 COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND POSITIVE CONTROLS -18- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURES COMPARISON OF RELATIVE LIVER WEIGHT AND MEAN LIVER FLUORINE CONCENTR FOR H-23926 AND NEGATIVE CONTROLS Day 10 Day 94 E3 Corn Oil (Negative Control) H Corn Oil:Acetone (Negative Control) D H-23926 (Test Substance) 19Coinnipany Sainitteed. Does not co H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURE 4 MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE 6000 5000 4000 .2; a er M to s 2000 1000 Day 10 D a y. .1 3 Day 94 -20- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURES COMPARISON OF MEAN BLOOD AND MEAN LIVER FLUORINE CONCENTRATION NORMALI 6000 5000 4000 3000 u te. S a. 2000 1000 Blood / Liver Blood / Liver Blood / L -21- GwcspsenySanilteed. Does no H-23926: ^BiopersistenceScreening DuPont-2920 10-Dose Oral Gavage Study in Rats_________________________________Supplement No. 1 APPENDICES .22- CiassspissSvafnSfzwS. Qwss noS eoniato TSCA CBI H-23926: Biopersistence Screening , DuPont-2920 10-Dose Oral Gavage Study in Rats________________________________Supplement No. 1 APPENDIX A Individual Final Body Weights, Liver Weights, and Relative Liver to Body Weight -23- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 t Supplement No. 1 CORKI OIL (NEGATIVE CONTROL) INDI\?IDUAL FINAL BC)DY WEIGHTS (g), LIVER WEIGHTS (gf), AND RELATIVE LI'VER TO BODY WEHGHT 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- CoiffilpamySanKized. Does not eonts'n TSCA CB H-23926: Biopersistence Screening 10-Dose Oral Gavage Study'in Rats DuPont-2920 Supplement No. 1 CORKF OIL (NEGATIVE CONTROL) INDF/IDUAL FINAL BC>DY WEIGHTS (g),, LIVER WEIGHTS (cr), AND ,RELATIVE LF/ER TO BODY WE I 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- H-23926; Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 CORN OH,: ACETONE (NEGA1IVE CONTROL) INDI\riDUKL FINAL BCDY WEIGHTS (g), LIVER WEIGHTS (cI), AND RELATIVE LF/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 16.1-39 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- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 CORN 01 L:ACETONE (NEGA'TIVE CONTROL) INDP/IDUAL FINAL B.ODY WEIGHTS (g) , LIVER WEIGHTS (gr), AND RELATIVE L:EVER TO BODY WEI:GHT 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 Final 'Body Wt. 533.7 530.3 555.4 506.2 512.3 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 Test Day 52 52 52 52 52 Animal Number 625353 625369 625373 625375 625377 Final Body Wt. 565.3 514.8 673.7 588.8 496.6 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 94 94 94 94 94 -27- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 H-24'019 (POSITIVE (CONTROL) IND IVIDUAL FINAL BOH)Y WEIGHTS (g), LIVER WEIGHTS (g ) , AND RELATIVE LIV.1ER TO BODY WEK3HT 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 -28- Company Sanitized. Does not contain TSCA CB1 H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 H-2^1019 (POSITIVE (CONTROL) IVSD1\^DUAL FINAL BOCY WEIGHTS (g), LIVER WEIGHTS (g'), AND RELATIVE m /ER TO BODY WEK3HT 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 Number625326 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 -29- Compeny Sanitized. Does not contain TSCA CB1 H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ; DuPont-2920 Supplement No. 1 H-24'020 (POSITIVE iCONTROL) IND3:VIDUAL FINAL BOE)Y WEIGHTS (g). LIVER WEIGHTS ( g), AND RELATIVE LIV:ER TO BODY WEIl3HT OF MALE RATS Animal 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- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 H-240 20 (POSITIVE CONTROL) INDI VIDUAL FINAL BOD1!' WEIGHTS (g), LIVER WEIGHTS (gr), AND RELATIVE LIVE,R TO BODY WEI GHT OF MALE RATS Animal Nuinber 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 -31- H-23926: Biopersistence Screening f 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 H-:23926 (TEST SUB;STANCE) INDIV^IDUAL FINAL BOIDY WEIGHTS (g), LIVER WEIGHTS (g ) , AND RELATIVE LP/ER TO BODY WEK3HT OF MALE RATS Animal Number 626448 626450 626453 626457 - 626460 Final Body Wt. 318.6 285.7 311.4 283.4 323.1 GROUP I Liver Wt. 13.999 12.368 14.869 11.552 15.734 Relative Liver to Body Wt. 0.044 0.043 0.048 0.041 0.049 Test Day 5 5 5 5 5 Animal Number 626430 626433 626444 626449 626452 Final Body Wt. 320.7 344.4 307.8 356.6 319.7 GROUP II Liver Wt. 14.500 15.356 13.668 16.219 14.419 Relative Liver to Body Wt. 0.045 0.045 0.044 0.045 0.045 Test Day 10 10 10 10 10 Animal Number 626435 626436 626445 626459 626472 Final Body Wt. 301.4 348.4 392.4 358.0 346.7 GROUP III Liver Wt. 12.439 14.497 20.528 15.015 16.261 Relative Liver to Body Wt. 0.041 0.042 0.052 0.042 0.047 Test Day 13 13 13 13 13 32- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 H-:23926 (TEST SUB.STANCE) INDIV]:DUAL FINAL BCIDY WEIGHTS (g) ,, LIVER WEIGHTS (c?), AND RELATIVE LI17ER TO BODY WEI GHT OF MALE RATS Animal Number 626437 626438 626443 626456 626458 - Final Body Wt. 366.5 410.1 399.5 445.2 385.9 GROUP IV Liver Wt. 15.057 15.879 15.002 15.075 15.963 Relative Liver to Body Wt. 0.041 0.039 0.038 0.034 0.041 Test Day 24 24 24 24 24 Animal Number 626429 626432 626439 626442 626461 Final Body Wt. 513.4 522.8 547.9 459.7 505.0 GROUP V Liver Wt. 18.960 19.234 22.369 18.616 19.603 Relative Liver to Body Wt. 0.037 0.037 0.041 0.040 0.039 Test Day 52 52 52 52 52 Animal Number 626434 626441 626446 626447 626455 Final Body Wt. 601.3 532.3 571.8 575.3 593.6 GROUP VI Liver Wt. 19.471 17.986 19.983 20.851 19.643 Relative Liver to Body Wt. 0.032 0.034 0.035 0.036 0.033 Test Day 94 94 94 94 94 33- Cosnpany Sanitized. Does not contain TSCA CBt H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 APPENDIX B Individual Fluorine Levels -34- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 Supplement No. 1 Terms: Active % Active Mol Wt Active Formulation Dose % Fin 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- CoCTiparBySanifiteesS. Does not contain TSCA CBI H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 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) jiimolar equivalents of active in liver The /zmolar [/xmol/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 36- H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-2920 ' 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 offluorinated 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 component(s) in the formulation) - Molecular weight is the molecular weight of the active component in the formulation -37- CoBiBpany Sanitized. Does not contain TSCA CBl H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H- 24019 DuPont-2920 Supplement No. 1 Given: Mol Wt. Active (g/mole): 497 Formulation Dose (mg/kg): 10 % Active (F Containing) in Formulation: 100 %F in A.ctive: 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^_ Rat Number Test Day Sample ppmF in Liver Group II 625320 10 319.4 625330 10 355.4 625332 10 338.4 625341 10 305.0 625350 10 322.7 Group III 625321 13 625327 13 625342 13 625345 13 625348 13 255.9 275.3 277.9 260.4 275.6 Group VI 625326 94 625335 94 625346 94 625347 94 625351 94 79.8 89.6 73.8 62.5 82.1 ppm F in Liver Minus Bkg 0.2 ppm 319.2 355.2 338.2 304.8 322.5 255.7 275.1 277.7 260.2 275.4 79.6 89.4 73.6 62.3 81.9 ppm F in Liver Normalized to 0.1 mmoles/kg Dose /imolar Equivalents of Active in Liver 1586.42 1765.34 1680.85 1514.86 1602.83 4910.77 5464.62 5203.08 4689.23 4961.54 1270.83 1367.25 1380.17 1293.19 1368.74 3933.85 4232.31 4272.31 4003.08 4236.92 395.61 444.32 365.79 309.63 407.04 1224.62 1375.38 1132.31 958.46 1260.00 .38- ffiiiipareByanSBBzed, BI(S>@n8 eonfialit TSCA CBP H-23926: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H- 24020 DuPont-2920 Supplement No. 1 Given: Mol Wt. Active (g/mole): 426 % F in Active: 69 . Formulation Dose (mg/kg): 20 Mol Wt. F (g/mol): 19 % Active (F Containing) in Formulation: 100 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/F): 0.065 Dose F (mg/kg):_____13.8 Rat Number Test Day Sample ppmF in Liver Group II 627768 10 627774 10 627776 10 627782 10 627785 10 182.5 109.0 174.0 147.5 165.8 Group III 627758 13 627764 13 627787 13 627763 13 627788 13 61.5 40.5 33.3 51.3 45.2 Group VI 627778 94 1.9 627783 . 94 1.2 627762 94 1.9 627775 94 1.6 627769 94 1.8 ppm F in Liver Minus Bkg 0.2 ppm 182.3 108.8 173.8 147.3 165.6 61.3 40.3 33.1 51.1 45.0 1.7 1.0 1.7 1.4 1.6 ppm F in Liver Normalized to 0,1 mmoles/kg Dose /zmolar Equivalents of Active in Liver 388.30 231.74 370.19 313.75 352.73 1321.01 788.41 1259.42 1067.39 1200.00 130.57 85.84 70.50 108.84 95.85 444.20 292.03 239.86 370.29 326.09 3.62 2.13 3.62 2.98 3.41 12.32 7.25 12.32 10.14 11.59 .39- Cofflipany Sanitized. Qees nof contain TSCA CBI H-23926: Biopersistence Screening, 10-Dose Oral Gavage Study in Rats Data for H-23926 DuPont-2920 Supplement No. 1 Given: Mol Wt. Active (g/mole): 2500 % F in Active: 40 Formulation Dose (mg/kg): 1000 Mol Wt. F (g/mol): 19 % Active (F Containing) in Formulation: 25 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Formulation Dose Normalization Factor: 250 0.100 1000.0 Dose F (mmol/kg): 5.263 Molar Ratio (Active/F): 0.019 Dose F (mg/kg):_____100 Rat Number Test Day Sample ppmF in Liver Group II 626430 10 8.6 626433 10 10.3 626444 10 6.9 626449 10 9.1 626452 10 9.5 Group III 626435 13 11.3 626436 13 10.8 626445 13 10.2 626459 13 13.8 626572 13 12.4 Group VI 626434 94 8.4 626441 94 7.3 626446 94 3.8 626447 94 4.0 626455 94 4.3 ppm F in Liver Minus Bkg 0.2 ppm 8.4 10.1 6.7 8.9 9.3 11.1 10.6 10.0 13.6 12.2 8.2 7.1 3.6 3.8 4.1 ppm F in Liver Normalized to 0.1 mmoles/kg Dose 8.40 10.10 6.70 8.90 9.30 11.10 10.60 10.00 13.60 12.20 8.20 7.10 3.60 3.80 4.10 /imolar Equivalents of Active in Liver 8.40 10.10 6.70 8.90 9.30 11.10 10.60 10.00 13.60 12.20 8.20 7.10 3.60 3.80 4.10 .40-