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AR226-3192 DuPont-6541 TRADE SECRET Study Title H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Laboratory Project ID: DuPont-6541 Author: Carol Finlay, B.A. Study Completed on: November 26,2001 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 Work Request Number Service Code Number Page 1 of 84 Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Reviewed by: ________________ / Paul Hinderliter, PhD. Postdoctoral Fellow Z~ - A /* * ~ f f 9' Date Reviewed by: O d { u j jt-- 0 . / } Judith C. Stadler, P hD ., D.A.B.T. ( / Director J /k ~ A J d t/-Z O e > f Date Issued bv Study Director: t L A b C 1 .'fa t. A L , ' CaroiFinlay, B A . % Staff Scientist . ifc , fim i l>atc -2Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 TABLE OF CONTENTS Page CERTIFICATION......................................... 2 LIST OF TABLES........................................................................................................................ 4 LIST OF FIGURES...................................................................................................................... 4 LIST OF APPENDICES.............................................................................................................. 5 STUDY INFORMATION.............................................................................................................6 STUDY PERSONNEL...................................................................................................................8 SUMMARY................................................................................................................................... 9 INTRODUCTION............................................................ *..........................................................11 MATERIALS AND METHODS................................................................................................11 A. Test Substance and Positive Controls...............................................................................11 B. Test Species......................................................................................................................H C. Animal Husbandry............................................................................................................^ D. Quarantine and Pretest......................................................................................................12 E. Study Design.....................................................................................................................^3 F. Assignment to Groups and Study Start....;...................... ;................................................. 13 G. Dosing Material Preparation and Administration.............................................................. 13 H. Body Weights.....................................................................................................................I4 I. Mortality and Clinical Observations..... !............................................................................I4 J. Collection and Analysis of Blood, Livers, and Fat............................................................ 14 K. Treatment of Fluorine Data.............................................................................................. 15 L. Statistical Methods............................................................................................................1^ RESULTS AND DISCUSSION.......................................................................................... 17 A. In-Life Toxicology............................................................................................................. 17 B. Liver Weights..................................................................................................................... 17 C. Fluorine D ata..................................................................................................................... 18 CONCLUSIONS.......................................................................... RECORDS AND SAMPLE STORAGE....................................................................................20 TABLES........................................................................................................................................21 FIGURES......................................................................................................................................28 APPENDICES..............................................................................................................................39 -3Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 LIST OF TABLES Page 1. MEAN BODY WEIGHTS..................................................................................................................................... 22 2. MEAN BODY WEIGHT GAINS.......................................................................................................................... 23 3. MEAN BODY AND LIVER WEIGHTS............................................................................................................... 24 4. MEAN BLOOD FLUORINE LEVELS.................................................................................................................. 25 5. MEAN BLOOD FLUORINE CONCENTRATION NORMALIZED TO DOSE............................................... 25 6. MEAN LIVER FLUORINE LEVELS.................................................................................................................... 26 7. MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE............................................... --26 8. MEAN FAT FLUORINE LEVELS........................................................................................................................ 22 9. MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE....................................................... 27 LIST OF FIGURES Page 1. MEAN BODY WEIGHTS ................................................................................................... 29 2. COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND NEGATIVE CONTROL............................................................................... 30 3. COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND POSITIVE CONTROLS.............................................................................................................................................................31 4. MICROMOLAR EQUIVALENTS IN RAT BLOOD............................................................................................ 32 5. NORMALIZED H-24951 AND POSITIVE CONTROL BLOOD AUCINF/D RESULTING FROM A 10-DAY ORAL GAVAGE IN RATS..................................................................................................................... 34 6. MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE...................................................35 7. COMPARISON OF RELATIVE LIVER WEIGHT AND MEAN LIVER FLUORINE CONCENTRATION FOR H-24951 AND NEGATIVE CONTROL................................................................... 36 8. MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE....................................................... 37 9. COMPARISON OF MEAN BLOOD, MEAN LIVER, AND MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE.................................................................................................. 38 -4Company Sanitized. Does not contain TSCA CBi H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 LIST OF APPENDICES Page A. INDIVIDUAL BODY WEIGHTS......................................................................................................................... 40 B. INDIVIDUAL CLINICAL OBSERVATIONS......................................................................................................50 C. TERMS AND CALCULATIONS FACTORS INFLUENCING INTERPRETATION OF KINETIC ANALYSIS.............................................................................................................................................................. 66 D. INDIVIDUAL FLUORINE LEVELS IN BLOOD................................................................................................. 70 E. INDIVIDUAL FLUORINE LEVELS IN LIVER................................................................................................... 77 F. INDIVIDUAL FLUORINE LEVELS IN FAT....................................................................................................... 81 -5Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats STUDY INFORMATION TEST SUBSTANCE: 9th Collective Nomenclaturej Svnonvms/Codesl c Haskell Number: 24951 CAS Registry Number^J Composition! Known Impurities:' POSITIVE CONTROL: I ) Substance Tested: Potassium perfluoroalkyl sulfonate Svnonvms/Codes: H-24019 DuPont-6541 Haskell Number: 24019 Compositio Known Impurities Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 STUDY INFORMATION (Continued) POSITIVE CONTROL: Substance Tested: Octanoic acid, pentadecafluoro-, ammonium salt Perfluorooctanoate, ammonium salt APFO Ammonium perfluorooctanoate Haskell Number: 24020 Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: May 22, 2001 / (see report cover page) In-Life Initiated/Completed: May 22, 2001 / August 23, 2001 -7Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 STUDY PERSONNEL Study Director: Carol Finlay, B.A. Management: Judith C. Stadler, Ph.D., D.A.B.T. Primary Technician: James C. Mackay II 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: William Singleton, D.V.M., A.C.L.A.M -8 Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 SUMMARY The objective of this study was to evaluate the potential for H-24951, when administered by gavage, to be absorbed and to accumulate in a mammalian system. Two groups of 5 male Crl:CD(SD)IGS BR rats each were exposed to 1000 mg/kg/day of H-24951 for 10 consecutive days. Blood was collected from the orbital sinus of 5 rats (group I) approximately 2 hours after dosing on test days 1 and 5. Approximately 2 hours after the last dose, these rats were euthanized and blood, livers, and fat were collected. Blood was collected from the orbital sinus of the remaining 5 rats (group DI) on test days 13, 24, and 52. These rats were euthanized on test day 94 and blood, livers, and fat were collected. Body weights and clinical signs were recorded on each day of dosing and then weekly during the recovery period. Additionally, a negative control, deionized water, and 2 positive controls, H-24019 (10 mg/kg/day) and H-24020 (20 mg/kg/day), were tested as described for H-24951. No deaths occurred. One rat dosed with the test substance, H-24951, exhibited nasal discharge during the dosing period. Comparison of body weights was complicated by the fact that there was a difference in age on test day 1 between the rats dosed with the test substance and those dosed with the positive controls or negative control. This difference resulted in differences in mean body weights on test day 1. Accounting for the age difference at study start and the expected rate of body weight gain, the mean body weights and mean body weight gains of the rats dosed with H-24951 were comparable to the negative control and equal to or greater than the positive controls. The mean relative liver weight (liver/body weight) of rats dosed with the test substance, H-24951, was 29% higher at day 10 than the liver weight of the negative control rats. By day 94, the weights were similar. The mean relative liver weight of rats dosed with the positive control H-24019 was 38% higher at day 10 than the negative control group. The mean relative liver weight of rats dosed with the positive control H-24020 was 88% higher on day 10 than the negative control group. By day 94, the mean relative liver weights of rats dosed with H-24019 and H-24020 were similar to the negative control group. A steady-state for fluorine levels in whole blood was not achieved during 10 consecutive days of dosing with 1000 mg/kg H-24951. An area under the curve (estimated to infinity) was calculated and normalized for fluorine content for the test substance and each positive control. The AUCINF/D for the fluorine component of the test substance, H-24951, was 4.03xl02compared to AUCINF/D values of 5.22x10s and 8.15xl04 for H-24019 and H-24020, respectively. The concentration of fluorine in the livers from rats dosed with the test substance, H-24951, was 19.85 jU.Mequivalents on day 10 and 1.21 /xM equivalents on day 94. On day 10, mean /xM equivalent concentrations of fluorine in the livers from rats dosed with the positive control materials were approximately 240-fold (H-24019) and 45-fold (H-24020) greater than the fluorine concentration in livers from rats treated with the test substance. By day 94, the -9Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 concentrations were approximately 1070x and 14x the fluorine concentration in rats treated with H-24951. The fluorine concentration in the fat from rats dosed with the test substance was 20.94 /xM equivalents on day 10. On day 94, the concentration was 1.60 /xM equivalents. The fluorine concentrations of the positive controls were approximately 9x or 3x higher than H-24951 on day 10. By day 94, the /xM equivalent concentration of fluorine in the fat from rats dosed with the positive control H-24019 was approximately lOx higher than H-24951. There was no detectable fluorine by day 94 in the fat from rats dosed with H-24020. The /xM equivalents of fluorine in the liver and fat of animals dosed with the test substance were higher than levels in the blood. Under the conditions of this study, there was minimal absorption and retention of fluorine in the blood in rats dosed with H-24951. Administration of the test substance to male rats for 10 consecutive days resulted in some absorption and retention of fluorine in the liver and fat. However, fluorine levels in blood and liver were significantly lower than levels in rats dosed with the positive control materials, H-24019 and H-24020. Some retention of fluorine in the fat was evident. Fluorine levels in fat from rats dosed with the test substance were lower than the levels in rats dosed with the positive control H-24019 and were slightly lower on day 10 than the levels in fat from rats dosed with the positive control H-24020, but higher on day 94. Liver weights were elevated in rats dosed with the test substance at the end of dosing but were similar to negative controls following the recovery period. P -10Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 INTRODUCTION The objective of this study was to define the potential of H-24951 to be absorbed and to bioaccumulate in a mammalian system, as indicated by analytical determination of total fluorine in blood, liver, and fat. The test substance was compared to 2 positive controls that were materials previously shown to bioaccumulate in mammals. The daily dosage of 1000 mg/kg for the test substance was selected based on available toxicity data. The dosage of 1000 mg/kg was selected for the limit dosage for this project. The limit dosage of 1000 mg/kg was chosen for the study and was expected to produce less than a 10% difference in mean body weight over 10 days when compared to the negative control. MATERIALS AND METHODS A. Test Substance and Positive Controls The test substance, H-24951, was supplied by the sponsor as a pale pink waxy solid. The positive controls, H-24019 and H-24020, were supplied by the sponsor as white solids. The test substance and positive controls appeared to be stable under the conditions of the study. No evidence of instability, such as a change in color or physical state, was observed. B. Test Species Male Crl:CD(SD)IGS BR rats were received from Charles River Laboratories, Inc., Raleigh, North Carolina. The Crl:CD(SD)IGS BR rat was selected on the basis of extensive experience with this strain and its suitability with respect to longevity, hardiness, sensitivity, and low incidence of spontaneous diseases. C, Animal Husbandry 1. Housing Environment Rats were housed singly in stainless steel, wire-mesh cages suspended above cage boards. Animal rooms were maintained on an approximate 12-hour light/dark cycle (fluorescent light) and at a temperature of 23 1C and a relative humidity of 50 10%. 2. Feed and Water Tap water was provided ad libitum. All rats were fed PMI Nutrition International, Inc. Certified Rodent LabDiet 5002 chow. The feed is guaranteed by the manufacturer to meet specified nutritional requirements and to be free of specified contaminants. -11 Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 3. Identification Each rat was assigned a unique identification number which was recorded on a card affixed to the cage. The last 3 digits of the number were tattooed on the tail of each rat. 4. Animal Health Monitoring Program As specified in the Haskell Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity of the study: Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Feed samples are analyzed for total bacterial, spore and fungal counts. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluation of these data did not indicate any conditions that affected the validity of the study. D. Quarantine and Pretest Upon arrival at Haskell Laboratory, the rats were removed from shipping cartons and quarantined for 6 days. The rats were weighed 3 times during the pretest period and examined daily for any clinically apparent signs of disease or injury. The rats were observed daily for mortality and signs of illness, injury, or abnormal behavior. On the bases of acceptable body weight gains and freedom from clinically apparent signs of disease or injury, the rats were released from quarantine by the designee of the laboratory animal veterinarian. - 12Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats E. Study Design Substance Negative Control Deionized water Positive Controls H-24019 H-24020 Test Substance H-24951 DuPont-6541 Vehicle Not applicable Acetone/Com Oil Acetone/Com Oil 0.5 Aqueous Methylcellulose Dosage Number of (mg/kg) Animals 0 10 10 10 20 10 1000 10 F. Assignment to Groups and Study Start After the quarantine period, the rats were selected on the bases of adequate body weight gain and freedom from any clinical signs of disease or injury. The selected rats were arbitrarily assigned to each group. After assignment to groups, each rat was housed individually. The rats were 7or 8 weeks of age at the time of dosing. Dosing began on test day 1. Rats that were not assigned to the study were released for other laboratory purposes, or were sacrificed by carbon dioxide asphyxiation and discarded without pathology evaluation. G. Dosing Material Preparation and Administration 1. Test Substance H-24951 was heated in a 60-75C waterbath until liquefied and then stirred for at least 30 minutes until homogenous. The test substance was then aliquoted into 10 containers. On each day of dosing, one container was similarly heated and stirred. The liquefied test substance was suspended in 0.5% aqueous methylcellulose. The amount of the suspension each rat received was based on the body weight collected on each day of dosing and the suspension concentration. The rats were dosed at a volume of 1 mL/100 g of body weight. The suspension was stirred on a magnetic stir plate at least 30 minutes before dosing and throughout the dosing procedure to maintain homogeneity. 2. Positive Controls It was necessary to dissolve H-24019 and H-24020 in acetone before suspending them in com oil. The ratio of acetone to com oil was 20:80. The amount each rat received was based on the body weight collected on each day of dosing and the suspension concentration. The rats were dosed at a volume of 1 mL/100 g of body weight. The dosing preparations were stirred on a magnetic stir plate throughout the dosing procedure to maintain homogeneity. -13Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 3. Negative Controls Deionized water was chosen as the negative control. The rats were dosed at a volume of 1 mL/100 g of body weight. These rats were dosed in a separate room from the rats dosed with the test substance or positive controls. H. Body Weights All rats were weighed on each day of dosing and weekly during the recovery period. I. Mortality and Clinical Observations Cage-site examinations to detect moribund or dead rats and abnormal behavior and appearance among rats were conducted at least once daily throughout the study. At every weighing, each rat was individually handled and examined for abnormal behavior and appearance. J. Collection and Analysis of Blood, Livers, and Fat At time points selected for blood sampling other than sacrifice days, approximately 1 mL of blood was collected into EDTA tubes from the orbital sinus of each rat. Rats designated for sacrifice were euthanized by carbon dioxide anesthesia and exsanguination and blood, livers, and fat were collected according to the following schedules: Group I I I ffl in m in Dosing Days 1-10 1-10 1-10 1-10 1-10 1-10 1-10 Tissue Collected Blood Blood Blood, Liver, and Fat Blood Blood Blood Blood, Liver, and Fat Sampling Time Test day 1 (2 hours post dosing) Test day 5 (2 hours post dosing) Test day 10 at sacrifice (2 hours post dosing) Test day 13 Test day 24 Test day 52 Test day 94 As much blood as possible was collected into EDTA tubes at sacrifice. The livers and fat were weighed. The fat weights were used only for the calculation of fluorine levels. The liver weights were used for the calculation of fluorine levels and for the calculation of liver weight relative to total body weight. The blood from all rats was refrigerated and the livers were frozen. The livers and fat were appropriately packaged and shipped refrigerated to Jackson Laboratory, Deepwater, New Jersey where they were analyzed for total fluorine. The liver and fat samples remained frozen while shipped. The total fluorine content of the samples was determined by using a Wickbold torch combustion method, followed by analysis with a fluoride ion selective electrode. The samples were decomposed or volatilized in the presence of wet oxygen and swept through an oxy-hydrogen - 14Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 flame in a closed quartz apparatus. The combustion products were collected in an aqueous absorbing solution and analyzed with a fluoride ion selective electrode. Kinetic analysis of the data received (ppm F in each sample) was performed by Haskell Laboratory personnel for evaluation of fluorine biopersistence. K. Treatment of Fluorine Data Since the test substance and positive control materials had different toxicity profiles, it was not possible to administer a uniform mg/kg dose for all test materials. Properly conducted kinetic comparisons in situations with varied doses required the use of a normalized dose. The dosenormalization was conducted in pmolar units to accommodate test material molecular weight differences and was adjusted to an arbitrary normalized dose of 0.1 mmoles/kg. H-24019 and H-24020 were used as positive controls and a dosing diluent was used as the negative control. The percent of fluorine and molecular weight of the test substance and positive controls were used as provided by the sponsor. The measured total fluorine values were used as received (ppm F) from Jackson Laboratory. For the Wickbold torch method, the background fluorine level is 0.2 ppm. This is the limit of detection (LOD) of this method and was subtracted from each sample. The limit of quantification (LOQ) for this method is 0.5 ppm, and any values listed as less than 0.5 ppm were excluded from further treatment. Since the dosages and molecular weight for positive controls and test material differed, the data were converted from a mass to a molar basis. In addition, the data were standardized to a single reference dose to allow comparison between compounds of differing fluorine content. The doses were first converted from a mg of test material basis to millimoles of fluorine. Raw fluoride ion data (ppm F) were then normalized to a 0.1 millimole dose of active component. Finally the molar dosage and normalized concentration were combined to yield the /xmolar (/xM) equivalents of active component in the tissue. Detailed calculations can be found in Appendix C. The /xM equivalents can be compared across compounds provided the considerations listed in Appendix C are observed. Noncompartmental analysis was conducted on fluorine data derived from rats dosed with H-24951 and the positive controls using WinNonlin Version 3.1 software (Pharsight Corp, Mountain View, CA). WinNonlin software provided a means of computing derived pharmacokinetic parameters from experimental data. All analysis was calculated using the fiM equivalent in the data. The maximum observed concentration in blood was Cmax (/xM equivalent). Biopersistence was assessed by quantifying terminal elimination blood half-life (Ty2, days). The points included in determination of the T>/2were selected manually and included only points after apparent log-linear elimination was achieved. Internal exposure was determined by calculating the blood areaunder-the-curve (AUC). AUC, which is simply the integral of blood concentration over time, is the most common means for expressing internal dose. With the calculated half-life, the AUC can be extrapolated to infinity (AUCINF) to reflect the elimination of the compound. AUCINF - 15Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 normalized to dose (AUCENF/D) can be used to compare the relative exposures of different compounds and dosages. L. Statistical Methods Descriptive statistics (e.g. mean, standard deviation) were used. - 16Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 RESULTS AND DISCUSSION A. In-Life Toxicology (Tables 1-2, Figure 1, Appendices A-B) No deaths occurred. One rat dosed with the test substance, H-24951, exhibited black nasal discharge during the dosing period. Hair loss observed in one rat during the dosing period was considered spurious. A swollen mouth was observed during the dosing period in a negative control rat. One negative control rat exhibited black nasal discharge during the recovery period. A rat dosed with H-24019 exhibited red ocular discharge during the dosing period. Hair loss was observed in rats dosed with H-24019 and H-24020, and a wound was observed in a rat dosed with H-24020. Ocular discharge, dark eyes, comeal opacity, enophthalmus, and exophthalmus observed in several rats are considered a result of orbital sinus bleeding. Comparison of body weights was complicated by the fact that there was a difference in age on test day 1 between the rats dosed with the test substance and those dosed with the positive controls or negative control. This difference resulted in differences in mean body weights on test day 1. The rats dosed with the test substance, H-24951, were older and heavier in weight than the positive and negative control rats. Accounting for the age difference at study start and the expected rate of body weight gain, the mean body weights and mean body weight gains of the rats dosed with H-24951 were comparable to the negative control and equal to or greater than the positive controls. B. Liver Weights (Table 3, Figures 2, 3, and 7) 1. Test Substance The mean relative liver weight (liver/body weight) of rats dosed with the test substance, H-24951, was 29% higher at day 10 than the liver weight of the negative control group. The weights were similar by day 94. 2. Positive Controls The mean relative liver weights of rats dosed with one of the positive controls, H-24019, were similar on day 10 and day 94 to the liver weights of rats dosed with the test substance, H-24951. The mean relative liver weight of rats dosed with the other positive control, H-24020, was 46% higher at day 10 than the liver weight of rats dosed with H-24951. By day 94, the weights were similar. The mean relative liver weight of rats dosed with H-24019 was 38% higher at day 10 than the negative control group. By day 94, the mean relative liver weight of rats dosed with H-24019 - 17 Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 was similar to the negative control group. The mean relative liver weight of rats dosed with H-24020 was 88% higher on day 10 than the negative control group. By day 94, the weights were similar. Therefore, the relative liver weight of rats dosed with the test substance, H-24951, was 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 similar to the weights in rats dosed with the positive control materials, H-24019 and H-24020. C. Fluorine Data (Tables 4-9, Figures 4-9, Appendices C-F) 1. Factors Influencing Interpretation of Analysis The data used in the kinetic analysis were derived from a limited screen, and therefore several caveats and considerations are important. A couple of considerations of particular importance are (1) a single dosage was used and kinetics may or may not be linear, (2) the kinetics apply only to blood, (3) steady-state may not have been achieved, and (4) the sample size is low and may impact calculation of the terminal half-life. A more complete list of considerations is shown in Appendix C. 2. Positive Controls The positive controls were H-24019 and H-24020. The H-24019 and H-24020 normalized fiM equivalents in rat blood continued to rise throughout the dosing period and may not have reached steady-state (Figures 4A and 4B). The Cmax for H-24019 was 541.45 50.37 /xM equivalents (Mean SD) with a terminal half-life of 42.2 days. The Cmax for H-24020 was ' 1043.08 54.57 /xM equivalents (Mean SD) with a terminal half-life of 15.1 days. For each of the positive controls, blood was sampled at seven time points throughout the study, with only four of them occurring post-dose. The small sample size and analytical variability should be taken into account when using the derived terminal half-life for comparative purposes. The total internal exposure resulting from a normalized dose was described by AUCINF/D and was the basis for comparison between positive controls and the test substance. The AUCINF/D for the fluorine component was 5.22xl05for H-24019 and 8.15xl04 for H-24020. The concentrations of fluorine in the livers on day 10 from rats dosed with the positive control materials were 4802.15 and 866.67 jiM equivalents for H-24019 and H-24020, respectively. By day 94, the concentrations were 1292.92 and 17.10 /xM equivalents. The concentrations of fluorine in the fat on day 10 from rats dosed with the positive control materials were 194.46 and 57.25 pM equivalents for H-24019 and H-24020, respectively. By day 94, the concentration of fluorine in the fat from rats dosed with H-24019 was 15.38 /xM equivalents. There was no detectable fluorine by day 94 in the fat from rats dosed with H-24020. -18Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 3. Test Substance The H-24951 normalized /xM equivalents in rat blood rose rapidly and did not reach steady-state (Figure 4C). The Cmax for H-24951was 1.43 0.20 /xM equivalents (Mean SD) with a terminal half-life of 46.1 days. Blood was sampled at seven time points throughout the study, with only four of them occurring post-dose. The small sample size and analytical variability should be taken into account when using the derived terminal half-life for comparative purposes. The total internal exposure resulting from a normalized dose was described by AUCINF/D and was the basis for comparison between H-24951 and the positive controls. The AUCINF/D for the fluorine component of H-24951 was 4.03 xlO2as compared to AUCINF/D values of 5.22x10s and 8.15xl04 for H-24019 and H-24020, respectively. Levels of total fluorine in livers from rats dosed with the test substance, H-24951, 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-24951 was 19.85 /xM equivalents at day 10 and 1.21 /xM equivalents at day 94. For the positive control H-24019, the liver concentrations were approximately 240x higher (day 10) and approximately 1070x higher (end of study) than H-24951. For H-24020, the liver concentrations were approximately 45x higher (end of dosing) and 14x higher (end of study) than H-24951. Levels of total fluorine in fat from rats dosed with the test substance were generally lower than the levels in fat from rats dosed with the positive control materials. The fluorine concentration in the fat from rats dosed with the test substance was 20.94 /xM equivalents on day 10 and 1.60 /xM equivalents on day 94. The fluorine concentration in the fat from rats dosed with the positive controls were approximately 9x or 3x higher than H-24951 on day 10. By day 94, the /xM equivalent concentration of fluorine in the fat from rats dosed with the positive control H-24019 was approximately lOx higher than H-24951. There was no detectable fluorine by day 94 in the fat from rats dosed with H-24020. The /xM equivalents of fluorine in the liver and fat of animals dosed with the test substance were higher than levels in the blood. CONCLUSIONS Rats dosed for 10 consecutive days with 1000 mg/kg H-24951 exhibited no mortality or body weight effects. Clinical signs were observed, and liver weights were elevated at the end of dosing in rats dosed with the test substance. A steady-state for fluorine in the blood was not achieved during the 10-day dosing period with the test substance. Dose-adjusted areas under the curve (AUCINF/D) for positive controls, H-24019 and H-24020, were approximately 1300x and 200x the AUCINF/D for the test substance. Under the conditions of this study, there was minimal absorption and retention of fluorine in the blood following dosing with H-24951. Administration of the test substance, H-24951, to male rats for 10 consecutive days resulted in some absorption and retention of fluorine in the liver and fat. However, levels in blood and liver in rats dosed with H-24951 were significantly lower than -19Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 the fluorine levels in rats dosed with the positive control materials, H-24019 and H-24020. Some retention of fluorine in the fat was evident. Fluorine levels in fat were significantly lower than levels in rats dosed with the positive control material H-24019 and were slightly lower on day 10 than the levels in fat from rats dosed with the positive control H-24020, but higher on day 94. RECORDS AND SAMPLE STORAGE All original records will be retained at Haskell Laboratory, E. I. du Pont de Nemours and Company, Newark, Delaware or at Iron Mountain Records Management, 200 Todds Lane, Wilmington, Delaware. Laboratory specific or site-specific raw data such as personnel files and equipment records will be retained at the facility where the work was done. - 20Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 TABLES -21 Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 TABLE 1 MEAN BODY WEIGHTS (g) Test Days 1 2 3 4 5 6 7 8 9 10 17 24 31 38 45 52 59 66 67 73 80 87 94 Negative Control Deionized Water 189.6 195.5 204.6 211.7 222.1 225.0 235.2 242.3 248.3 257.0 293.3 325.5 359.3 380.9 407.4 420.5 436.4 - 452.8 472.6 489.6 497.7 511.7 Positive Controls H-24019 H-24020 184.4 184.1 189.2 187.8 199.5 206.4 197.8 204.8 216.2 212.5 222.0 216.0 229.4 233.7 223.6 226.9 240.4 246.5 290.1 313.3 234.2 243.0 297.4 338.6 348.4 370.8 381.9 404.9 403.2 434.4 422.8 439.6 460.6 483.1 -- 455.6 480.1 502.7 525.6 496.5 512.2 542.1 546.2 524.6 570.5 - Indicates the animals were not weighed. Test Substance H-24951 253.7 260.0 271.4 278.8 286.6 292.7 300.7 307.1 314.5 320.8 359.3 390.3 416.5 440.2 454.0 489.1 505.0 527.2 532.1 550.6 562.4 573.5 - 22Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 TABLE 2 MEAN BODY WEIGHT GAINS (g) Test Days 1-5 5-10 1-10 Negative Control Deionized Water 32.5 34.9 67.4 Dosing Positive Controls H-24019 H-24020 31.8 28.4 30.3 30.5 62.1 58.9 Test Substance H-24951 32.9 34.2 67.1 Recovery Test Negative Control Positive Controls Test Substance Days_____Deionized Water______ H-24019_________ H-24020________. H-24951 10-17 17-24 36.3 32.2 43.6 54.4 38.5 23.2 41.2 31.0 24-52 52-94 95.0 91.2 109.5 101.8 122.0 109.9 98.8 84.4 10-94 254.7 278.1 327.5 252.7 - 23Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 % TABLE 3 Test Days 10 94 MEAN BODY AND LIVER WEIGHTS (g) DEIONIZED WATER (NEGATIVE CONTROL) Absolute Body Weight Liver Weight 258.8 511.7 10.814 17.868 Mean Relative Liver Weight (Liver/Body Weight) 0.042 0.035 Test Days 10 94 H-24019 (POSITIVE CONTROL) Absolute Body Weight Liver Weight 243.9 14.205 524.6 19.296 Mean Relative Liver Weight (Liver/Body Weight) 0.058 0.037 . Test Days 10 94 H-24020 (POSITIVE CONTROL) Absolute Body Weight Liver Weight 243.1 19.174 570.5 19.590 Mean Relative Liver Weight (Liver/Body Weight) 0.079 0.034 Test Days 10 94 H-24951 (TEST SUBSTANCE) Absolute Body Weight Liver Weight 319.9 17.159 573.5 20.090 Mean Relative Liver Weight (Liver/Body Weight) 0.054 0.035 -24Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 TABLE4 MEAN BLOOD FLUORINE LEVELS Negative Controls Positive Controls Test Substance Test Deionized Water H-24019 H-24020 H-24951 Days________(ppm)_____________ (ppm) ________ (ppm)____________ (PPTM) 1 0.60a 5C 10 1.10a 13 c 24 0.5a 52 c 94 c 2.60 (0.1) 31.32 (1.1) 68.00 (3.5) 53.98 (1.2) 39.62 (3.4) 23.56 (2.1) 12.60 (1.2) 9.40 (2.4) 74.92 (7.0) 61.76 (5.5) 29.52 (4.9) 11.18 (2.9) 2.26 (1.1) 0.85d (0.1) 2.76 (0.8) 4.96 (0.8) 8.20 (L I) 4.16 (0.6) 2.14 (0.5) 1.40 (0.2) 0 .9 0 a a One of 5 values. Four of the values were below the limit of quantification (LOQ) or non-detectable. b Standard deviation is in parentheses, c All values were below the LOQ or non-detectable. d Mean of 2 of the 5 values. Three of the values were below the LOQ. TABLE 5 MEAN BLOOD FLUORINE CONCENTRATION NORMALIZED TO DOSE Test Days 1 5 10 13 24 52 94 Positive Controls H-24019 H-24020 /M F Equivalents M F Equivalents 36.92 (l.l)a 66.67 (17.5) 478.77 (17.6) 541.45 (50.4) 1043.08 (54.6) 446.09 (39.7) 827.38 (19.0) 212.46 (35.6) 606.46 (53.1) 79.57 (20.8) 359.38 (32.7) 14.93 (7.7) 190.77 (19.2) 4.71b (0.5) Test Substance H-24951 M F Equivalents 0.46 (0.1) 0.85 (0.1) 1.43 (0.2) 0.71 (0.1) 0.35 (0.1) 0.21 (0.0) 0.12C a Standard deviation is in parentheses. b Mean of 2 of the 5 values. Three of the values were below the limit of quantification (LOQ). c One of 5 values. Four of the values were below the LOQ or non-detectable. - 25Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 TABLE MEAN LIVER FLUORINE LEVELS Negative Control Positive Controls Test Substance Deionized Test Water H-24019 H-24020 H-24951 Days_______ (ppm)____________ (ppm)___________ (ppm)_____________(PPm) 10 0.90 (0.2)a 312.34 (19.7) 119.80 (3.5) 111.62 (11.1) 94_______0.78 (0.0)______ 84.24 (7.4)_________ 2.56 (1.3)__________ 6.98 (0.4) a Standard deviation is in parentheses. TABLE 7 MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE Positive Controls Test Substance Test H-24019 H-24020 H-24951 Pays_______ ixM Equivalents /xM Equivalents_________ /M Equivalents 10 4802.15 (303.8)a 866.67 (25.5) 19.85 (2.0) 94________ 1292.92 (114.1)________ 17.10 (9.8)_____________ 1-21 (0.1) a Standard deviation is in parentheses. - 26Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats TABLE 8 MEAN FAT FLUORINE LEVELS Negative Control Deionized Test Water Days________ (ppm) 10 -a 94 -a Positive Controls H-24019 H-24020 (ppm) _________(ppm) 12.84 (1.7)b 8.10 (1.1) 1.20 (0.3)__________ -a a All values were below the limit of quantification (LOQ) or non-detectable. b Standard deviation is in parentheses. DuPont-6541 Test Substance H-24951 (PPm) 117.78 (19.9) 9.20 (1.1) TABLE 9 MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE Positive Controls Test Substance Test H-24019 H-24020 H-24951 Days /M Equivalents _____ /xM Equivalents_______ nM Equivalents 10 194.46 (26.8)a 57.25 (7.8) 20.94 (3.5) 94__________ 15.38 (4.1)______________ ^ _______________ 160 (0.2) a Standard deviation is in parentheses, b All values were non-detectable. - 27Company Sanitized. Does not contain TSCA CBI t H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 FIGURES 0 ' - 28Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats w _____________________________________________DuPont-6541 FIGURE 1 MEAN BODY WEIGHTS (g) Company Sanitized. Does not contain TSCA CBI - 29- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats_____________________ __________________________________ FIGURE 2 COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND NEGATIVE CONTROL DuPont-6541 Company Sanitized. Does not contain TSCA C8I - 30- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats______________________________________________________________________________________________ DuPont-6541 0.08 0.07 0.06 u ^ 0.05 * S 0 04 20> o aa "2 FIGURE 3 COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND POSITIVE CONTROLS EDH-24019 (Positive C ontrol) H-24020 (Positive Control) DH-24951 (Test Substance) Company Sanitized. Does not contain TSCA CBI Test Days -31 - H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats__________________________ _________________________ DuPont-6541 FIGURE 4 MICROMOLAR EQUIVALENTS IN RAT BLOOD A. N or ma li z ed Rat Blood H- 24019 t M Equi val ent s Resulting from a 10-Day Or al Ga va ge Micromolar (/xM) equivalents of H-24019 (positive control) in rat blood resulting from a 10-day oral gavage exposure. Values are means and error bars are standard deviation. B. N o r ma l i z ed Rat Blood H-24020 itM Equival ent s Resulting from a 10-Day Oral Gavage Micromolar (f iM ) equivalents of H-24020 (positive control) in rat blood resulting from a 10-day oral gavage exposure. Values are means and error bars are standard deviation. - 32Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 C. Normalized Rat Blood H-24951 /jM Equivalents Resulting from a 10-Day Oral Gavage Micromolar (/iM) equivalents of H-24951 (test substance) in rat blood resulting from a 10-day oral gavage exposure. Values are means and error bars are standard deviation. Only one sample was above the limit of quantification on day 94. -33Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats_________________________________________________________ FIGURE 5 NORMALIZED H-24951 AND POSITIVE CONTROL BLOOD AUCINF/D RESULTING FROM A 10-DAY ORAL GAYAGE IN RATS 600000 -| 500000 - DuPont-6541 z 3 oo CD CO CC I1. Company Sanitized. Does not contain TSCA CBI 0|e i a u c i n f /d H-24019 5.22E +05 H -24020 8.1 5E +04 -34- H-24951 4.03E +02 % H-24951: B iopersistence Screening 10-Dose Oral Gavage Study in Rats_____________________________________________________ FIGURE 6 MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE DuPont-6541 H1H-24019 (Positive Control) H-24020 (Positive Control) H-24951 (Test Substance) 'EId 2500 - s% 2000 Company Sanitized. Does not contain TSCA CBI Day 10 - 35 - Day 94 H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 FIGURE 7 COMPARISON OF RELATIVE LIVER WEIGHT AND MEAN LIVER FLUORINE CONCENTRATION FOR H-24951 AND NEGATIVE CONTROL Company Sanitized. Does not contain TSCA CBI D ay 10 D ay 94 D a y 10 Day 94 El Deionized Water (Negative Control) H-24951 (Test Substance) -36- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in R w a ts ____________________________________________ DuPont-6541 FIGURE 8 MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE Company Sanitized. Does not contain TSCA CBI - 37- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURE 9 COMPARISON OF MEAN BLOOD, MEAN LIVER, AND MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE DuPont-6541 Company Sanitized. Does not contain TSCA CBI -38 - H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats_______________________________________ ______________ DuPont-6541 APPENDICES -39Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 APPENDIX A Individual Body Weights - 40Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats INDIVIDUAL BODY WEIGHTS EXPLANATORY NOTES ABBREVIATIONS: SD - sacrificed by design DuPont-6541 -41 Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 646932 646933 646934 646935 646936 Day 1 197.1 191.9 182.6 203.0 196.8 Day 2 202.1 195.4 188.9 208.9 201.4 DEIONIZED WATER (NEGATIVE CONTROL) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS GROUP I Day 3 TEST DAY Day 4 Day 5 Day 6 212.9 205.0 195.4 218.0 208.6 221.5 213.2 207.0 228.1 204.0 233.6 226.5 213.9 239.7 216.7 234.1 219.3 215.9 243.0 218.6 Day 7 242.4 232.9 224.2 257.2 229.9 ANIMAL NUMBER Day 10 646932 646933 646934 646935 646936 264.7 254.4 248.6 278.0 248.1 SD test day 10 SD test day 10 SD test day 10 SD test day 10 SD test day 10 TEST DAY DuPont-6541 Day 8 251.1 239.5 230.9 265.7 234.'9 Day 9 256.1 246.4 238.1 270.5 240.3 Company Sanitized. Does not contain TSCA O CD H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 646937 646938 646939 646940 646941 Day 1 184.5 163.7 193.3 191.5 191.6 ANIMAL NUMBER Day 10 646937 646938 646939 646940 646941 249.8 237.2 277.9 244.5 266.3 ANIMAL NUMBER Day 73 646937 646938 646939 646940 646941 504.3 417.4 540.1 407.3 493.7 Day 2 186.8 173.8 204.2 197.1 195.9 Day 17 282.6 270.4 329.7 271.5 312.4 Day 80 520.5 431.0 568.9 424.0 503.5 _____________________________ DuPont-6541^ DEIONIZED WATER (NEGATIVE^ CONTROL) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS Day 3 GROUP III TEST DAY Day. 4 Day 5 Day 6 Day 7 Day 8 Day 9 199.0 180.9 214.6 203.8 207.9 205.9 189.3 221.1 209.8 216.8 213.6 200.6 234.4 218.5 223.1 221.0 204.1 240.7 222.1 231.2 229.6 215.9 248.9 229.3 241.3 238.7 221.8 259.2 232.0 249.1 241.4 228.3 267.3 237.5 257.0 Day 24 322.3 296.5 372.8 291.3 344.8 TEST DAY Day 31 Day 38 366.1 325.8 415.2 317.9 371.6 395.0 342.9 443.2 332.8 390.7 Day 45 425.0 367.5 475.2 356.3 412.8 Day 52 443.9 373.7 484.1 367.0 433.6 Day 59 461.7 378.7 505.7 380.3 455.8 Day 67 478.7 401.3 516.4 392.5 475.3 Day 87 533.7 432.1 578.1 430.8 513.8 TEST DAY Day 94 545.7 450.6 586.9 449.4 525.8 SD test day 94 SD test day 94 SD test day 94 SD test day 94 SD test day 94 Company Sanitized. Does not contain TSCA CBI -43 - 9% # H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats_________________________ _______________________________________________________ DuPont-6541 H-24019 (POSITIVE CONTROL) ANIMAL NUMBER Day 1 Day 2 INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS GROUP I Day 3 TEST DAY Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 646910 646911 646912 646913 646914 184.3 184.4 180.5 186.4 185.5 185.3 187.1 185.9 189.7 189.6 197.3 195.7 195.9 198.1 198.8 204.8 202.0 202.5 205.7 203.8 212.7 . 211.7 211.9 221.8 215.4 216.3 217.2 217.7 225.0 222.7 226.4 225.2 227.2 228.6 226.7 232.3 225.6 232.4 238.4 231.5 237.5 231.9 238.5 245.8 238.8 ANIMAL NUMBER Day 10 646910 646911 646912 646913 646914 241.1 236.6 241.9 257.0 243.0 SD test day 10 SD test day 10 SD test day 10 SD test day 10 SD test day 10 TEST DAY Company Sanitized. Does not contain TSCA CBI - 44- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER Day 1 646915 646916 646917 646918 646919 . 182.7 185.0 187.8 175.5 192.0 Day 2 188.3 192.7 194.3 181.9 196.9 H-24019 (POSITIVE CONTROL) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS GROUP III Day 3 TEST DAY Day 4 Day 5 Day 6 200.1 205.5 205.8 189.8 207.9 206.3 211.6 210.4 197.6 218.9 211.1 . 220.0 221.8 207.0 228.3 220.6 226.9 227.5 212.4 234.0 Day 7 225.8 239.5 233.9 220.2 240.8 DuPont-6541 Day 8 231.1 240.1 235.8 224.1 245.8 Day 9 236.8 249.3 244.3 228.2 252.9 ANIMAL NUMBER Day 10 646915 646916 646917 646918 646919 240.4 257.0 250.2 237.1 260.2 Day 17 283.4 310.0 280.3 270.6 306.1 Day 24 302.9 334.4 308.2 292.7 328.5 TEST DAY Day 31 Day 38 326.7 383.2 339.7 323.2 369.2 337.9 411.9 362.8 350.8 390.4 Day 45 371.1 450.3 387.9 379.4 427.3 Day 52 399.7 469.8 409.0 396.7 439.0 Day 59 412.5 494.8 420.0 418.8 451.7 Day 67 433.1 511.8 441.5 433.8 457.6 ANIMAL NUMBER Day 73 646915 646916 646917 646918 646919 461.2 544.2 457.5 448.1 489.5 Day 80 475.0 565.5 464.7 472.5 504.8 Day 87 498.1 578.5 479.5 480.8 524.2 TEST DAY Day 94 513.3 590.9 495.7 500.3 522.9 SD test day 94 SD test day .94 SD test day 94 SD test day 94 SD test day 94 Company Sanitized. Does not contain TSCA CBI -45 - H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 646921 646922 646923 646924 646925 Day 1 204.5 178.3 185.9 188.7 172.2 Day 2 204.9 180.9 185.1 194.6 176.0 H-24020 (POSITIVE CONTROL) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS GROUP I Day 3 TEST DAY Day 4 Day 5 Day 6 215.7 188.9 196.0 209.5 183.9 222.2 197.4 202.3 212.6 193.4 233.3 - 197.7 203.5 230.4 202.7 238.8 202.4 204.9 225.9 202.7 ANIMAL NUMBER Day 10 646921 646922 646923 646924 646925 262.6 218.6 240.6 259.1 234.8 SD test day 10 SD test day 10 SD test day 10 SD test day 10 SD test day 10 TEST DAY Day 7 244.3 202.4 219.1 236.2 215.1 DuPont-6541 Day 8 244.9 204.0 224.9 236.5 221.5 Day 9 252.2 207.7 231.7 250.9 228.7 Company Sanitized. Does not contain TSCA CBI - 46- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 646926 646927 646928 646929 646930 Day 1 182.0 172.2 190.4 178.6 187.9 ANIMAL NUMBER Day 10 646926 646927 646928 646929 646930 250.8 243.3 254.1 225.5 240.3 ANIMAL NUMBER Day 73 646926 646927 646928 646929 646930 535.3 551.2 540.3 502.6 498.5 Day 2 187.8 178.8 195.3 182.5 191.6 Day 17 297.2 306.2 310.6 280.9 292.3 Day 80 551.9 574.5 557.0 509.6 517.6 H-24020 (POSITIVE CONTROL) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS Day 3 GROUP. Ill TEST DAY Day 4 Day 5 Day 6 197.0 191.1 204.5 190.7 201.0 203.6 195.1 212.3 198.3 210.4 206.0 206.6 222.1 203.5 219.6 213.2 213.2 230.1 204.2 224.4 Day 7 222.9 218.2 236.1 214.6 226.7 Day 24 349.3 355.1 349.4 323.2 316.2 TEST DAY Day 31 Day 38 383.8 404.0 392.8 376.0 352.7 412.1 424.8, 422.4 396.8 368.4 Day 45 432.1 463.2 443.5 426.4 406.8 Day 52 467.8 484.0 464.8 450.2 436.0 Day 87 564.4 541.3 568.3 526.7 530.3 TEST DAY Day 94 580.6 602.5 581.9 544.0 543.4 SD test day 94 SD test day 94 SD test day 94 SD test day 94 SD test day 94 DuPont-6541 Day 8 229.9 228.7 235.8 210.3 232.6 Day 9 239.7 233.1 243.4 219.2 235.3 Day 59 493.2 511.7 492.2 463.8 454.7 Day 67 509.5 527.2 518.6 484.2 474.2 Company Sanitized. Does not contain TSCA CBI -47 - H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 648894 648895 648896 648897 648898 Day 1 230.2 281.2 235.2 256.4 274.5 Day 2 238.2 281.4 239.1 259.8 276.8 H-24951 (TEST SUBSTANCE) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS GROUP I Day 3 TEST DAY Day 4 Day 5 Day 6 249.3 294.6 249.5 277.7 297.3 253.4 310.5 257.9 275.4 304.9 260.1 . 314.9 263.1 283.2 312.2 266.7 317.7 261.9 291.3 316.1 Day 7 274.0 322.8 266.0 301.3 327.1 ANIMAL NUMBER Day 10 648894 648895 648896 648897 648898 293.8 341.8 283.3 325.2 355.3 SD test day 10 SD test day 10 SD test day 10 SD test day 10 SD test day 10 TEST DAY DuPont-6541 Day 8 284.1 325.2 272.6 307.6 337.9 Day 9 289.8 332.8 271.4 316.2 350.8 Company Sanitized. Does not contain TSCA CBI -48- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 648899 648900 648901 648902 648903 Day 1 243.4 239.8 261.0 251.2 264.4 ANIMAL NUMBER Day 10 648899 648900 648901 648902 648903 311.3 313.1 328.1 314.0 342.3 ANIMAL NUMBER Day 73 648899 648900 648901 648902 648903 502.5 528.1 572.2 485.2 572.5 Day 2 248.7 250.2 266.7 263.7 275.1 Day 17 345.6 350.7 373.9 336.2 389.9 Day 80 518.0 548.8 591.7 502.8 591.9 H-24951 (TEST SUBSTANCE) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS GROUP III Day 3 TEST DAY Day 4 Day 5 Day 6 260.3 260.0 277.5 265.4 282.5 264.9 265.4 289.8 277.5 288.5 271.2 279.5 293.0 286.9 302.3 282.1 280.6 307.7 291.0 311.4 Day 7 289.8 290.5 313.3 300.5 321.8 Day 24 370.6 388.3 411.9 356.2 424.4 TEST DAY Day 31 Day 38 395.7 417.2 445.0 375.1 449.3 421.1 441.6 465.2 393.2 480.1 Day 45 446.0 406.1 490.0 420.7 507.1 Day 52 469.0 474.1 521.0 445.7 535.8 Day 87 529.6 562.5 599.7 519.0 601.3 TEST DAY Day 94 534.5 574.7' 614.9 531.9 611.7 SD test day 94 SD test day 94 SD test day 94 SD test day 94 SD test day 94 DuPont-6541 Day 8 296.1 294.8 320.5 302.8 328.9 Day 9 302.8 304.8 332.1 308.4 336.2 Day 59 479.2 497.2 537.0 459.5 552.3 Day 66 497.4 522.4 563.5 481.9 570.6 Company Sanitized. Does not contain TSCA CBI -49- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 APPENDIX B Individual Clinical Observations -50Company Sanitized. Does not contain TSCA CBI Company Sanitized. Does not contain TSCA CBI % H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DEIONIZED WATER (NEGATIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I Animal Observation Days 646932 General observation, No Abnormality Detected Eye Observations, Exophthalmus, Left Eye Observations, Bled via Orbital for Clin Path, Left 1 2-9 1,5 Discharge, Eye left, Black 10 646933 Sacrificed by design General observation. No Abnormality Detected 10 1-5 Eye Observations, Bled via Orbital for Clin Path, Left 1,5 Eye Observations, Dark, Left 6-10 Discharge, Eye left, Black 6-7 646934 Swollen Observations, Mouth Sacrificed by design General observation, No Abnormality Detected 10 10 1-5,10 Eye Observations, Bled via Orbital for Clin Path, Left 1,5 Discharge, Eye left. Black 0-9 Sacrificed by design 10 -51 - DuPont-6541 H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DEIONIZED WATER (NEGATIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Animal Observation GROUP I (Continued) Days 646935 General observation, No Abnormality Detected 1-10 Eye Observations, Bled via Orbital for Clin Path, Left 1,5 Sacrificed by design 10 646936 General observation, No Abnormality Detected 1-10 Eye Observations, Bled via Orbital for Clin Path, Left 1,5 Sacrificed by design 10 DuPont-6541 Company Sanitized. Does not contain TSCA CBI -52- Company Sanitized. Does not contain TSCA C8I H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DEIONIZED WATER (NEGATIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Animal 646937 646938 GROUP III Observation General observation, No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design General observation, No Abnormality Detected Eye Observations, Enophthalmus, Right Eye Observations, Exophthalmus, Right Eye Observations, Bled via Orbital for Clin Path, Bilateral Days 1-10,17-94 13 24,52 94 1-10,17-45 80-94 52-73 13,52 Eye Observations, Bled via Orbital for Clin Path, Right Eye Observations, Corneal Opacity, Right 24 80-94 646939 Discharge, Eye right. Black Sacrificed by design General observation, No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 59 94 1-10,17-94 13 24,52 94 -53 - DuPont-6541 Company Sanitized. H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DEIONIZED WATER (NEGATIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP III (Continued) Animal Observation Days 646940 General observation, No Abnormality Detected 1-10,17-80,94 Eye Observations, Bled via Orbital for Clin Path, Left 13 Eye Observations, Bled via Orbital for Clin Path, Right 24,52 Discharge, Nose, Black 87 Sacrificed by design 94 646941 General observation, No Abnormality Detected 1-10,17-94 Eye Observations, Bled viaOrbital for Clin Path, Bilateral 52 Eye Observations, Bled via Orbital for Clin Path, Left 13 Eye Observations, Bled via Orbital for Clin Path, Right 24 Sacrificed by design 94 DuPont-6541 H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats H-24019 (POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I Animal Observation Days 646910 General observation, No Abnormality Detected 1-10 646911 Eye Observations, Bled via Orbital for Clin Path, Left Sacrificed by design General observation, No Abnormality Detected 1,5 10 1,10 646912 646913 646914 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Dark, Left Sacrificed by design General observation, No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design General observation, No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Sacrificed by design General observation, No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Sacrificed by design 1,5 2-9 10 1-10 1 5 10 1-10 1,5 10 1-10 1,5 10 Company Sanitized. Does not contain TSCA CBI -55- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Animal 646915 646916 646917 H-24019 (POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS ! GROUP III Observation Days General observation, No Abnormality- Detected 1,17-38,73 Eye Observations, Bled via Orbital for Clin Path, Left 13,24,52 Discharge, Eye right, Red 2-10 Hair Loss, Forelimb, Right 80 Hair Loss, Forepaw, Right 45-67,87-94 Sacrificed by design 94 General observation. No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin Path, Bilateral 24 Eye Observations, Bled via Orbital for Clin Path, Left 13 Eye Observations, Bled via Orbital for Clin Path, Right 52 Sacrificed by design 94 General observation, No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin.Path, Left 13,24 Eye Observations, Bled via Orbital for Clin Path, Right 52 Sacrificed by design 94 DuPont-6541 Company Sanitized. Does not contain TSCA CBI -56- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats H-24019 (POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP III (Continued) Animal Observation Days 646918 General observation, No Abnormality Detected 1-10,17-94 Eye Observations, Bledvia Orbital for Clin Path, Left Eye Observations, Bledvia Orbital for Clin Path, Right Sacrificed by design 13,24 52 94 646919 General observation, No Abnormality Detected Eye Observations, Bledvia Orbital for Clin Path, Left 1-10,17-94 13,24 Eye Observations, Bledvia Orbital for Clin Path, Right 52 Sacrificed by design 94 DuPont-6541 Company Sanitized. Does not contain TSCA CBI -57 - Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats H-24020 (POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I Animal Observation Days 646921 General observation, No Abnormality Detected 1,5-10 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Dark, Left 1,5 2-4 Sacrificed by design 10 646922 General observation, No Abnormality Detected 1-10 Eye Observations,Bled via Orbital for Clin Path, Left 1 Eye Observations,Bled via Orbital for Clin Path, Right 5 646923 Sacrificed by design General observation, No Abnormality Detected Eye Observations, Exophthalmus, Left 10 1,7-10 4-6 Eye Observations,Bled via Orbital for Clin Path, Left 1 Eye Observations,Bled via Orbital for Clin Path, Right Eye Observations, Dark, Left 5 2-6 Sacrificed by design 10 -58- DuPont-6541 Company Sanitized. H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Animal 646924 646925 H-24020 (POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I (Continued) Observation Days General observation. No Abnormality Detected 1-10 Eye Observations, Bledvia Orbital for Clin Path, Left 1 Eye Observations, Bledvia Orbital for Clin Path, Right 5 Sacrificed by design 10 General observation. No Abnormality Detected 1-10 Eye Observations, Bledvia Orbital for Clin Path, Left 1 Eye Observations, Bledvia Orbital for Clin Path, Right 5 Sacrificed by design 10 coooorno oo3 >CoO oCJ DuPont-6541 H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats H-24020(POSITIVE CONTROL) Animal 646926 646927 646928 INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP III Observation Days General observation, No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin Path, Bilateral 52 Eye Observations, Bled via Orbital for Clin Path, Left 13,24 Sacrificed by design 94 General observation. No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin Path, Bilateral 52 Eye Observations, Bled via Orbital for Clin Path, Left 13,24 Sacrificed by design 94 General observation, No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin Path, Left 13,24,52 Sacrificed by design 94 DuPont-6541 Company Sanitized. Does not contain TSCA o 03 H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats H-24020(POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Animal 646929 GROUP III (Continued) Observation General observation, No Abnormality Detected Eye Observations, Bledvia Orbital for Clin Path, Left Eye Observations, Bledvia Orbital for Clin Path, Right Hair Loss, Forelimb, Bilateral Hair Loss, Neck, Left Hair Loss, Neck, Ventral Days 1-10,17-31 13,24 52 52-73 73 38-67,80-94 646930 Wound, Superficial, Face Sacrificed by design General observation, No Abnormality Detected Eye Observations, Bledvia Orbital for Clin Path, Left 59 94 1-10,17-94 13,24 Eye Observations, Bledvia Orbital for Clin Path, Right 52 Sacrificed by design 94 DuPont-6541 Company Sanitized. Does not contain TSCA CBI -61 - H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats_____________________________________________ _____________ _______ H-24951 (TEST SUBSTANCE) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Animal 648894 GROUP I Observation General observation, No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled viaOrbital for Clin Path, Right Days 1-10 5 1 648895 648896 Sacrificed by design General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Discharge, Eye right, Black Sacrificed by design General observation, No Abnormality Detected 10 1-4 5 1 5-10 10 1-10 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 5 1 10 DuPont-6541 Company Sanitized. Does not contain TSCA CBI - 62- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats______________________________________________________________________________________________ DuPont-6541 H-24951 (TEST SUBSTANCE) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I (Continued) Animal Observation Days 648897 General observation, No Abnormality Detected 1,3-4,7-10 Eye Observations, Bledvia Orbital for Clin Path, Bilateral 1 Eye Observations, Bledvia Orbital for Clin Path, Left 5 Discharge, Nose, Black 5-6 Discharge, Eye left, Red 2 Sacrificed by design 10 648898 General observation. No Abnormality Detected 1-10 Eye Observations, Bledvia Orbital for Clin Path, Left 5 Eye Observations, Bledvia Orbital for Clin Path, Right 1 Sacrificed by design 10 Company Sanitized. Does not contain TSCA CBI - 63- Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats H-24951 (TEST SUBSTANCE) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP III Animal Observation Days 648899 General observation, No Abnormality Detected 1-10,17-94 Eye Observations, Bledvia Orbital for Clin Path, Left 13,24,52 Eye Observations, Bledvia Orbital for Clin Path, Right 24,52 648900 Sacrificed by design General observation. No Abnormality Detected Eye Observations, Bledvia Orbital for Clin Path, Left Eye Observations, Bledvia Orbital for Clin Path, Right 94 1-10,17-94 13,24,52 24,52 Sacrificed by design 94 648901 General observation, No Abnormality Detected Eye Observations, Bledvia Orbital for Clin Path, Left Eye Observations, Bledvia Orbital for Clin Path, Right 1-6 13,24,52 24,52 Hair Loss, Abdomen, Right Hair Loss, Forelimb, Bilateral Hair Loss, Forepaw, Bilateral 80-94 38-94 7-10,17-94 Hair Loss, Hindlimb, Bilateral 73-94 Hair Loss, Hindlimb, Left 59-66 Sacrificed by design 94 -64- DuPont-6541 H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats H-24951 (TEST'SUBSTANCE) INDIVIDUAL. CLINICAL OBSERVATIONS IN MALE RATS GROUP III (Continued) Animal 648902 648903 Observation General observation, No Abnormality Detected Eye Observations, Bledvia Orbital for Clin Path, Left Eye Observations, Bledvia Orbital for Clin Path, Right Sacrificed by design General observation, No Abnormality Detected Eye Observations, Bledvia Orbital for Clin Path, Left Eye Observations, Bledvia Orbital for Clin Path, Right Sacrificed by design Days 1-10,17-94 13,24,52 24,52 94 1-10,17-94 13,24,52 24,52 94 DuPont-6541 Company Sanitized. Does not contain TSCA CBI -65 - H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 APPENDIX C Terms and Calculations Factors Influencing Interpretation of Kinetic Analysis - 66Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats________________________________________ DuPont-6541 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/100) 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/100) 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 - 67Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 TERMS AND CALCULATIONS Individual Animal Measurement: ppm F The ppm fluoride measured Individual Animal Calculations: ppm F minus Bkg 0.2 ppm The ppm fluoride measured minus the background fluoride measured in control animal. In this case the value was established at 0.2 ppm. ppm F normalized to 0.1 mmol/kg Dose 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 blood 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 blood minus background) /xmolar equivalents of active The /xmolar [/xmol/L] concentration of fluorine containing compound based on the ppm fluorine normalized to 0.1 mmol/kg active dose. This assumes that all fluorine is derived from the fluorine-containing component in the formulation. Note: 1 ppm - 1 mg/L = (Normalized ppm [mg/L] fluorine / Mol Wt F [mg/mmol]) x molar ratio active/F [mmol active/mmol F] x 1000 /xmol/mmol - 68Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 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 bejustified in all cases) - 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 - 69Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 APPENDIX D Individual Fluorine Levels in Blood - 70Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24019 DuPont-6541 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): %Active (F Containing) in Formulation: 497 10 100 %F in Active: Mol Wt. F (g/mol): 65 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): 10 0.020 0.342 Molar Ratio (Active/F): 0.059 Dose F (mg/kg): 6.5 Rat Number Test ppm F Day in Sample Blood Group I 646910 646911 646912 646913 646914 1 1 1 1 1 2.6 2.7 2.5 2.6 2.6 ppm F in Blood Minus Bkg 0.2 ppm 2.4 2.5 2.3 2.4 2.4 ppm F in Blood Normalized to 0.1 mmoles/kg Dose 11.93 12.43 11.43 11.93 11.93 /molar Equivalents of Active in Blood 36.92 38.46 35.38 36.92 36.92 Group I 646910 646911 646912 646913 646914 5 5 5 5 5 30.1 30.3 31.3 32.2 32.7 29.9 30.1 31.1 32.0 32.5 148.60 149.60 154.57 159.04 161.53 460.00 463.08 478.46 492.31 500.00 Group I 646910 10 71.5 646911 10 70.5 646912 10 66.9 646913 10 62.5 646914 10 68.6 71.3 70.3 66.7 62.3 68.4 354.36 349.39 331.50 309.63 339.95 1096.92 1081.54 1026.15 958.46 1052.31 -71 Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Rat Number Test ppmF Day in Sample Blood Group m 646915 646916 646917 646918 646919 13 13 13 13 13 55.0 55.3 52.2 53.8 53.6 ppm F in Blood Minus Bkg 0.2 ppm 54.8 55.1 52.0 53.6 53.4 ppm F in Blood Normalized to 0.1 mmoles/kg Dose DuPont-6541 /molar Equivalents of Active in Blood 272.36 273.85 258.44 266.39 265.40 843.08 847.69 800.00 824.62 821.54 Group III 646915 646916 646917 646918 646919 Group III 646915 646916 646917 646918 646919 24 24 24 24 24 52 52 52 52 52 38.9 43.8 37.6 35.4 42.4 23.2 20.3 25.9 24.9 23.5 38.7 43.6 37.4 35.2 42.2 23.0 20.1 25.7 24.7 23.3 192.34 216.69 185.88 174.94 209.73 114.31 99.90 127.73 122.76 115.80 595.38 670.77 575.38 541.54 649.23 353.85 309.23 395.38 380.00 358.46 Group III 646915 646916 646917 646918 646919 94 94 94 94 94 13.3 11.7 12.9 11.0 14.1 13.1 11.5 12.7 10.8 13.9 65.11 57.16 63.12 53.68 69.08 201.54 176.92 195.38 166.15 213.85 - 72Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24020 DuPont-6541 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): %Active (F Containing) in Formulation: 426 20 100 %F in Active: Mol Wt. F (g/mol): 69 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): 20 0.047 0.726 Molar Ratio (Active/F): 0.065 Dose F (mg/kg): 13.8 Rat Number Test ppm F Day in Sample Blood Group I 646921 1 10.6 646922 1 10.7 646923 1 9.2 646924 1 5.3 646925 1 11.2 ppm F in Blood Minus Bkg 0.2 ppm 10.4 10.5 9.0 5.1 11.0 ppm F in Blood Normalized to 0.1 mmoles/kg Dose /molar Equivalents of Active in Blood 22.15 22.37 19.17 10.86 23.43 75.36 76.09 65.22 36.96 79.71 Group I 646921 646922 646923 646924 646925 5 5 5 5 5 78.7 83.2 77.0 66.1 69.6 78.5 83.0 76.8 65.9 69.4 167.21 176.79 163.58 140.37 147.82 568.84 601.45 556.52 477.54 502.90 Group I 646921 10 63.0 646922 10 69.1 646923 10 60.3 646924 10 62.5 646925 10 53.9 62.8 68.9 60.1 62.3 53.7 133.76 146.76 128.01 132.70 114.38 455.07 499.28 435.51 451.45 389.13 - 73Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats_______________________________________________________ DuPont-6541 Rat Number Test ppm F Day in Sample Blood ppm F in Blood Minus Bkg 0.2 ppm ppm F in Blood Normalized to 0.1 mmoles/kg Dose /Imolar Equivalents of Active in Blood Group III 646926 646927 646928 646929 646930 13 13 13 13 13 34.8 30.0 23.7 33.7 25.4 34.6 29.8 23.5 33.5 25.2 73.70 63.47 50.06 71.36 53.68 250.72 215.94 170.29 242.75 182.61 Group III 646926 646927 646928 646929 646930 24 24 24 24 24 11.9 10.4 8.1 15.7 9.8 11.7 10.2 7.9 15.5 9.6 24.92 21.73 16.83 33.02 20.45 84.78 73.91 57.25 112.32 69.57 Group III 646926 646927 646928 646929 646930 52 52 52 52 52 2.5 1.7 1.3 4.0 1.8 2.3 1.5 1.1 3.8 1.6 4.90 16.67 3.20 10.87 2.34 7.97 8.09 27.54 3.41 11.59 Group III 646926 646927 646928 646929 646930 94 94 94 94 94 0.8 <0.5 <0.5 0.9 <0.5 0.6 * * 0.7 * * Below LOQ (Limit of Quantification) 1.28 4.35 ** ** 1.49 5.07 ** - 74Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24951 DuPont-6541 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): % Active (F Containing) in Formulation: 594 1000 100 %F in Active: Mol Wt. F (g/mol): 56.14 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): 1000 1.684 29.547 Molar Ratio (Active/F): 0.057 Dose F (mg/kg): 561.4 Rat Number Test ppm F Day in Sample Blood Group I 648894 648895 648896 648897 648898 1 1 1 1 1 3.8 2.3 1.9 3.4 2.4 ppm F in Blood Minus Bkg 0.2 ppm 3.6 2.1 1.7 3.2 2.2 ppm F in Blood Normalized to 0.1 mmoles/kg Dose 0.21 0.12 0.10 0.19 0.13 /tmolar Equivalents of Active in Blood 0.64 0.37 0.30 0.57 0.39 Group I 648894 648895 648896 648897 648898 5 5 5 5 5 5.7 5.2 5.6 4.1 4.2 5.5 5.0 5.4 3.9 4.0 0.33 0.98 0.30 0.89 0.32 0.96 0.23 0.69 0.24 0.71 Group I 648894 10 8.8 8.6 0.51 1.53 648895 10 8.8 8.6 0.51 1.53 648896 10 9.3 9.1 0.54 1.62 648897 10 6.7 6.5 0.39 1.16 648898 10 7.4 7.2 0.43 1.28 - 75Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Rat Number Test ppm F Day in Sample Blood Group III 648899 648900 648901 648902 648903 13 13 13 13 13 5.2 3.6 4.3 3.7 4.0 ppm F in Blood Minus Bkg 0.2 ppm 5.0 3.4 4.1 3.5 3.8 ppm F in Blood Normalized to 0.1 mmoles/kg Dose 0.30 0.20 0.24 0.21 0.23 DuPont-6541 /imolar Equivalents of Active in Blood 0.89 0.61 0.73 0.62 0.68 Group III 648899 648900 648901 648902 648903 24 24 24 24 24 2.9 1.7 2.1 2.3 1.7 2.7 1.5 1.9 2.1 1.5 0.16 0.48 0.09 0.27 0.11 0.34 0.12 0.37 0.09 0.27 Group III 648899 648900 648901 648902 648903 52 52 52 52 52 1.6 1.2 1.5 1.5 1.2 1.4 1.0 1.3 1.3 1.0 0.08 0.25 0.06 0.18 0.08 0.23 0.08 0.23 0.06 0.18 Group III 648899 648900 648901 648902 648903 94 94 94 94 94 0.9 <0.5 <0.5 <0.5 <0.5 * Below LOQ (Limit of Quantification) 0.7 * * * * 0.04 0.12 ** ** ** ** -76Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 APPENDIX E Individual Fluorine Levels in Liver - 77Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24019 DuPont-6541 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): %Active (F Containing) in Formulation: 497 10 100 %F in Active: Mol Wt. F (g/mol): 65 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): _______________ 10 0.020 0.342 Molar Ratio (Active/F): 0.059 Dose F (mg/kg): 6.5 __________________ Rat Number Test ppm F Day in Sample Liver Group I 646910 646911 646912 646913 646914 10 320.9 10 334.0 10 318.6 10 281.6 10 306.6 ppm F in Liver Minus Bkg 0.2 ppm 320.7 333.8 318.4 281.4 306.4 ppm F in Liver Normalized to 0.1 mmoles/kg Dose /molar Equivalents of Active in Liver 1593.88 1658.99 1582.45 1398.56 1522.81 4933.85 5135.38 4898.46 4329.23 4713.85 Group III 646915 646916 646917 646918 646919 94 94 94 94 94 85.6 75.9 87.5 78.0 94.2 85.4 75.7 87.3 77.8 94.0 424.44 376.23 433.88 386.67 467.18 1313.85 1164.62 1343.08 1196.92 1446.15 - 78Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24020 DuPont-6541 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): %Active (F Containing) in Formulation: 426 20 100 %F in Active: Mol Wt. F (g/mol): 69 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): 20 0.047 0.726 Molar Ratio (Active/F): 0.065 Dose F (mg/kg): 13.8 Rat Number Test ppm F Day in Sample Liver Group I 646921 10 118.3 646922 10 122.7 646923 10 114.3 646924 10 121.6 646925 10 122.1 ppm F in Liver Minus Bkg 0.2 ppm 118.1 122.5 114.1 121.4 121.9 ppm F in Liver Normalized to 0.1 mmoles/kg Dose /imolar Equivalents of Active in Liver 251.55 260.93 243.03 258.58 259.65 855.80 887.68 826.81 879.71 883.33 Group III 646926 646927 646928 646929 646930 94 94 94 94 94 1.8 1.2 2.2 4.7 2.9 1.6 1.0 2.0 4.5 2.7 3.41 11.59 2.13 7.25 4.26 14.49 9.59 32.61 5.75 19.57 - 79Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24951 DuPont-6541 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): %Active (F Containing) in Formulation: 594 1000 100 %F in Active: Mol Wt. F (g/mol): 56.14 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): 1000 1.684 29.547 Molar Ratio (Active/F): 0.057 Dose F (mg/kg): 561.4 Rat Number Test ppm F Day in Sample Liver Group I 648894 10 126.9 648895 10 115.1 648896 10 109.8 648897 10 96.0 648898 10 110.3 Group III 648899 648900 648901 648902 648903 94 94 94 94 94 6.9 6.8 6.6 7.6 7.0 ppm F in Liver Minus Bkg 0.2 ppm 126.7 114.9 109.6 95.8 110.1 6.7 6.6 6.4 7.4 6.8 ppm F in Liver Normalized to 0.1 mmoles/kg Dose 7.53 6.83 6.51 5.69 6.54 0.40 0.39 0.38 0.44 0.40 /tmolar Equivalents of Active in Liver 22.57 20.47 19.52 17.06 19.61 1.19 1.18 1.14 1.32 1.21 - 80Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-6541 APPENDIX F Individual Fluorine Levels in Fat -81 Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24019 DuPont-6541 Given: Mol Wt. Active (g/mole): 497 Formulation Dose (mg/kg): 10 % Active (F Containing) in Formulation: 100 % F in Active: Mol Wt. F (g/mol): 65 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): 10 0.020 0.342 Molar Ratio (Active/F): 0.059 Dose F (mg/kg): 6.5 Rat Number Test ppm F Day in Sample Fat Group 1 646910 646911 646912 646913 646914 10 10 10 10 10 14.4 14.9 10.9 11.6 12.4 ppm F in Fat Minus Bkg 0.2 ppm 14.2 14.7 10.7 11.4 12.2 ppm F in Fat Normalized to 0.1 mmoles/kg Dose 70.57 73.06 53.18 56.66 60.63 molar Equivalents of Active in Fat 218.46 226.15 164.62 175.38 187.69 Group III 646915 646916 646917 646918 646919 94 94 94 94 94 1.3 1.1 1.3 0.8 1.5 1.1 0.9 1.1 0.6 1.3 5.47 16.92 4.47 13.85 5.47 16.92 2.98 9.23 6.46 20.00 - 82Company Sanitized. Does not contain TSCA CBI H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24020 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): % Active (F Containing) in Formulation: 426 20 100 Calculated Values: Dose Active (mg/kg): 20 Dose Active (mmole/kg): 0.047 Dose F (mmol/kg):____________________0726 Rat Number Test ppm F Day in Sample Fat Group I 646921 646922 646923 646924 646925 10 10 10 10 10 6.2 8.5 8.7 8.8 8.3 ppm F in Fat Minus Bkg 0.2 ppm 6.0 8.3 8.5 8.6 8.1 P Ni 0. Group III 646926 646927 646928 646929 646930 94 94 94 94 94 ND Non-detectable. ND ND ND ND ND ND ND ND ND ND Company Sanitized. Does not contain TSCA CBI - 83- H-24951: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24951 DuPont-6541 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): % Active (F Containing) in Formulation: 594 1000 100 %F in Active: Mol Wt. F (g/mol): 56.14 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg):_________________ 1000 1.684 29.547 Molar Ratio (Active/F): 0.057 Dose F (mg/kg): 561.4 Rat Number Test ppm F Day in Sample Fat Group I 648894 10 151.0 648895 10 111.7 648896 10 111.9 648897 10 116.7 648898 10 97.6 Group III 648899 648900 648901 648902 648903 94 94 94 94 94 9.5 8.5 9.6 10.6 7.8 ppm F in Fat Minus Bkg 0.2 ppm 150.8 111.5 111.7 116.5 97.4 9.3 8.3 9.4 10.4 7.6 ppm F in Fat Normalized to 0.1 mmoles/kg Dose 8.96 6.62 6.63 6.92 5.79 0.55 0.49 0.56 0.62 0.45 ftmolar Equivalents of Active in Fat 26.86 19.86 19.90 20.75 17.35 1.66 1.48 1.67 1.85 1.35 - 84Company Sanitized. Does not contain TSCA CBI