Document jyXY74K1evrGNrbeO7Qvky40Q

TRADE SECRET Study Title H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Laboratory Project ED: DuPont-5591 AR226-3190 DuPont-5591 AUTHOR: Carol Finlay, B.A, STUDY COMPLETED ON: November 20, 2001 PERFORMING LABORATORY: E.I. du Font de Nemours and Company Haskell Laboratory for Health and Environmental Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 WORK REQUEST NUMBER ^ Sciences SERVICE CODE ^ NUMBER; 3 Page 1 of 83 Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 CERTinCATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Reviewed by: ' Ami Hinderliter, Ph-D. ' Posidoctoral Fellow Reviewed by; ^ L Q j ^ ^ / ^ Judith C. Stadlfr, Ph.D., D.AS.T. Directw Issoed by Study Director: J^iff^ f^AK.Carol pnlaayy,,BB-AA. ( Staff Scientist 7fi-/J1,.,^ .. ao* i Date <^>'A/C>U-t.60f Date ^. h^ Date ^ ^oi -2- Company Sanitized. Does not contain TSCA CBI I -a"^ H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 TABLE OF CONTENTS Page CERTIFICATION ........................................................................................................................2 LIST OF TABLES........................................................................................................................4 LISTOFFIGURES.......................................................................................................................4 LISTOFAPPENDICES...............................................................................................................5 STUDY INFORMATION.............................................................................................................6 STl]DPERSONNEi...................................................................................................................8 SLnVIMARY....................................................................................................................................9 INTRODUCTION....................................................................................................................... 11 MATERIALS AND METHODS................................................................................................ 11 A. Test Substance and Positive Controls ............................................................................... 11 B. Test Species....................................................................................................................... 11 C. Animal Husbandry............................................................................................................. 11 D. Quarantine and Pretest....................................................................................................... 12 E. Study Design 13 ..................................................................................................................... F. Assignment to Groups and Study Start............................................................................. 13 G. Dosing Material Preparation and Administration ............................................................. 13 H. Body Weights.................................................................................................................... 14 I. Mortality and Clinical Observations................................................................................. 14 J. Collection and Analysis of Blood, Livers, and Fat............................................................ 14 K. Treatment of Fluorine Data............................................................................................... 15 L. Statistical Methods............................................................................................................ 16 RESULTS AND DISCUSSION..................................................................................................17 A. In-Life Toxicology ............................................................................................................17 B. Liver Weights.................................................................................................................... 17 C. Fluorine Data..................................................................................................................... 18 CONCLUSIONS.......................................................................................................................... 19 RECORDS AND SAMPLE STORAGE.................................................................................... 20 TABLES...................................................................................................................................... 21 FIGURES..................................................................................................................................... 28 APPENDICES.............................................................................................................................39 3Company Sanitized. Does not contain TSCA CBI LIST OF TABLES Page 1. MEANBODY WEIGHTS...................................................................................................................................22 2. MEAN BODY WEIGHT GAINS ........................................................................................................................23 3. MEAN BODY AND LIVER WEIGHTS .............................................................................................................24 4. MEANBLOOD FLUORINE LEVELS...............................................................................................................^ 5. MEAN BLOOD FLUORINE CONCENTRATION NORMALIZED TO DOSE................................................25 6. MEAN UVER FLUORINE LEVELS.................................................................................................................^ 7. MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE..................................................26 8. MEAN FAT FLUORINE LEVELS...........................................................-.......................................................^? 9. MEAN PAT FLUORINE CONCENTRATION NORMALIZED TO DOSE......................................................27 LIST OF FIGURES Page 1. MEANBODY WEIGHTS...................................................................................................................................29 2. COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND NEGATIVE CONTROL........................................................................................................................................................... 30 3. COMPARISON OF MEAN RELATIVE UVER WEIGHTS: TEST SUBSTANCE AND POSITIVE CONTROLS........................................................................................................................................... '..............31 4. MICROMOLAR EQUIVALENTS IN RAT BLOOD..........................................................................................32 5. NORMALIZED H-24761 AND POSITIVE CONTROL BLOOD AUCINF/D RESULTING FROM A 10-DAY ORAL GAVAGE...................................................................................................................................34 6. MEAN UVER FLUORINE CONCENTRATION NORMALIZED TO DOSE..................................................35 7. COMPARISON OF RELATIVE LIVER WEIGHT AND MEAN UVER FLUORINE CONCENTRATION FOR H-24761 AND NEGATIVE CONTROL ..................................................................36 8. MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE......................................................37 9. COMPARISON OF MEAN BLOOD, MEAN UVER, AND MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE................................................................................................38 -4- Company Sanitized. Does not contain TSCA CBI H-24761; Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 LIST OF APPENDICES Page A. INDIVIDUAL BODY WEIGHTS 40 ....................................................................................................................... B. INDIVIDUAL CLINICAL OBSERVATIONS ....................................................................................................50 C. TERMS AND CALCULATIONS FACTORS INFLUENCING INTERPRETATION OP KINETIC ANALYSIS...........................................................................................................................----....................^ D. INDIVIDUAL FLUORINE LEVELS IN BLOOD..........................................................................-..................69 E. INDIVIDUAL FLUORINE LEVELS IN UVER.................................................................................................76 F. APPENDDC F INDIVIDUAL FLUORINE LEVELS IN FAT.............................................................................80 -5- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats TEST SUBSTANCE; STUDY INFORMATION Substance Tested: Synonyms/Codes:] H-24761 Haskell Number: 24761 Composition: B^H B T Purity, ^s^^^^^ Known Impurities: ^^^^^ POSITIVE CONTROL; Substance Tested: Potassium perfluoroalkyi sulfonate Synonyms/Codes: H-24019 DuPont-5591 Haskell Number: 24019 Composidor Known Impurities: .6- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 STUDY INFORMATION (Continued) POSITIVE CONTROL; Substance Tested: Octanoic acid, pentadecafluoro-, ammonium salt Synonyms/Codes: H-24020 C-8 Perfluorooctanoate, ammonium salt APFO Ammonium perfluorooctanoate Haslcell Number: 24020 Known Impurities: Sponsor: Corporate New Business Development Study Initiated/Completed: January 26, 2001 / (see report cover page) In-Life Initiated/Completed: January 28, 2001 /May 1, 2001 -7- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 STUDY PERSONNEL Study Director: Management: Primary Technician: Carol Finlay, B.A. Judith C. Stadler, Ph.D., D.A.B.T. Nita B. Baker 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 -8- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 SUMMARY The objective of this study was to evaluate the potential for H-24761, when administered by gavage, to be absorbed and to accumulate in a mammalian system. Two groups of 5 male Crl:CD(SD)IGSBR rats each were exposed to 1000 mg/kg/day of H-24761 for 9 consecutive days. On the 10th day, the rats were exposed to 700 mg/kg of H-24761. 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 ffl) 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-24761. No deaths occurred. Two rats dosed with the test substance, H-24761, exhibited nasal discharge during the dosing period and another exhibited nasal discharge during the recovery 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-24761 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-24761, was 52% higher at day 10 than the liver weight of the negative control group. "By day 94, the mean relative liver weight of rats dosed with the test substance was 40% higher than the negative control. 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-24761: 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-2476f, was 384, compared to AUCINF/D values of 5.22xl05 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-24761, was 22.71 iiM equivalents on day 10 and 3.10 P.M equivalents on day 94. On day 10, mean /iM equivalent concentrations of fluorine in the livers from rats dosed with the positive control materials were approximately 210-fold (H-24019) and 40-fold (H-24020) greater than the -9- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 fluorine concentration in livers from rats treated with the test substance. By day 94, the concentrations were approximately 420x and 6x the fluorine concentration in rats treated with H-24761. The fluorine concentration in the fat from rats dosed with the test substance was 0.19 ^M equivalents on day 10. On day 94, the concentration was 0.34 /iM equivalents. The fluorine concentrations of the positive controls were approximately 1025x or 300x higher than H-24761 on day 10. By day 94, the /iM equivalent concentration of fluorine in the fat from rats dosed with the positive control H-24019 was approximately 45x higher than H-24761. There was no detectable fluorine by day 94 in the fat from rats dosed with H-24020. Levels of total fluorine in livers from rats dosed with the test substance, H-24761, were higher than levels in the blood. In the fat, the fiM equivalents of fluorine were lower than the blood levels at day 10 but higher at day 94. Under the conditions of this study, there was minimal absorption and retention of fluorine in the blood in rats dosed with H-24761. 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. 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 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. 10Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 INTRODUCTION The objective of this study was to define the potential of H-24761 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 the results of a rangefinding study. In the rangefinding study, a group of 5 male rats was dosed by oral gavage for 5 consecutive days with H-24761 at a dosage of 1000 mg/kg. The rats had an overall weight gain of 29 grams. 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-24761, was supplied by the sponsor as a clear/slightly amber viscous liquid. The positive controls, H-24019 and H-24020, were supplied by DuPont Chemical Solutions 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%. 11Company Sanitized. Does not contain TSCA CBI H-24761; Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 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. 3. Identification Prior to assignment to groups, each rat was temporarily identified by cage identification. After assignment to groups, an individual identification number was tattooed on the tail of each rat. The information on the cage labels included the unique 6-digit Haskell animal number assigned to 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. 12Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 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. E. Study Design Substance Negative Control Deionized water Positive Controls H-24019 H-24020 Test Substance H-24761 Vehicle Not applicable Acetone/Corn Oil Acetone/Corn Oil Not applicable Dosage (mg/kg) 0 10 20 1000* Number of Animals 10 10 10 10 * The rats were dosed at 700 mg/kg on test day 10 because there was insufficient test substance to dose at 1000 mg/kg. 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 last 3 digits of the animal number was tattooed on the tail of each rat. The rats were 7 or 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-24761 was dosed as received. The amount of test substance each rat received was based on the body weight collected on each day of dosing and the test substance density of 1773 mg/mL. The mixture was stirred on a magnetic stir plate 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 corn oil. The ratio of acetone to corn oil was 20:80. The amount each rat received was based on the 13Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 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. 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 ffl ffl ffl 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. Total organic and inorganic fluorine were measured in the whole blood and tissues. The blood, livers, and fat were refrigerated (blood) or frozen (livers and fat) until analysis. The total fluorine content of the blood and tissue 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 14Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 oxy-hydrogen 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 controls 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 dose-normalization was conducted in j^molar 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 or suppliers. The measured total fluorine values were used as received (ppm F). 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 j^molar (/AM) equivalents of active component in the tissue. Detailed calculations can be found in Appendix C. The p.M 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-24761 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 p.M equivalent in blood data. The maximum observed concentration in blood was Cmax QiM equivalent). Biopersistence was assessed by quantifying terminal elimination blood half-life (T%, days)'. The points included in determination of the T.^ were 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-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 normalized to dose (AUCINF/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-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 RESULTS AND DISCUSSION A. In-Life Toxicology (Tables 1-2, Figure 1, Appendices A-B) No deaths occurred. Two rats dosed with the test substance, H-24761, exhibited black or red nasal discharge during the dosing period and another exhibited black nasal discharge during the recovery period. 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 were observed in several rats. These clinical signs are considered to be 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-24761, 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-24761 were comparable to the negative control and equal to or greater than the positive controls. B. Liver Weights (Table 3, Figures 2-3) 1. Test Substance The mean relative liver weight of rats dosed with the test substance, H-24761, was 52% higher at day 10 than the liver weight of the negative control group. By day 94, the mean relative liver weight of rats dosed with the test substance was 40% higher than the negative control. 2. Positive Controls The mean relative liver weight of rats dosed with one of the positive cpntrols, H-24019, was 9% lower at day 10 than the liver weight of rats dosed with H-24761. By day 94, the mean relative liver weight of rats dosed with H-24019 was 24% lower than the liver weight of rats dosed with H-24761. The mean relative liver weight of rats dosed with the other positive control, H-24020, was 23% higher at day 10 than the liver weight of rats dosed with H-24761. By day 94, the mean relative 17Company Sanitized. Does not contain TSCA CBI H-24761; Biopersistence Screening 10-Dose Oral Gavage Study in Rats__________________________________DuPont-5591 liver weight of rats dosed with H-24020 was 31% lower than the liver weight of rats dosed with H-24761. 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 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, liver weights of rats dosed with the test substance, H-24761, were elevated at the end of the 10-day dosing period. The mean relative liver weight by the end of recovery in rats dosed with the test substance was higher than in rats dosed with the positive control 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 jaM 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 /iM equivalents (Mean SD) with a terminal half-life of 42.2 days. The Cmax for H-24020 was 1043.08 54.57 /AM 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 5.22xl05 for 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 fiM equivalents for H-24019 and H-24020, respectively. By day 94, the concentrations were 1292.92 and 17.10 fi,M 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 /zM equivalents for H-24019 and H-24020, respectively. By 18Company Sanitized. Does not contain TSCA CBI fesy H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 day 94, the concentration of fluorine in the fat from rats dosed with H-24019 was 15.38 p.M equivalents. There was no detectable fluorine by day 94 in the fat from rats dosed with H-24020. 3. Test Substance The H-24761 normalized fiM equivalents in rat blood rose rapidly and did not reach steady-state (Figure 4C). The Cmax for H-24761was 0.92 0.08 /AM equivalents (Mean SD) with a terminal half-life of 24.9 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-24761 and the positive controls. The AUCINF/D for the fluorine component of H-24761 was 3,84xl02 as compared to AUCINF/D values of 5.22xl05 and 8.15xl04 for H-24019 and H-24020, respectively. Levels of total fluorine in livers from rats dosed with the test substance, H-24761, 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-24761 was 22.71 itU equivalents at day 10 and 3.10 fiM equivalents at day 94. For the positive control H-24019, the liver concentrations were approximately 21 Ox higher (day 10) and approximately 420x higher (end of study) than H-24761. For H-24020, the liver concentrations were approximately 40x higher (end of dosing) and 6x higher (end of study) than H-24761. 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 0.19 on day 10 and 0.34 ftM equivalents on day 94. The fluorine concentration in the fat from rats dosed with the positive controls were approximately 1025x or 300x higher than H-24761 on day 10. By day 94, the /zM equivalent concentration of fluorine in the fat from rats dosed with the positive control H-24019 was approximately 45x higher than H-24761. Levels of total fluorine in livers from rats dosed with the test substance, H-24761, were higher than levels in the blood, hi the fat, the /AM equivalents of fluorine were lower than the blood levels at day 10 but higher at day 94. CONCLUSIONS Rats dosed for 10 consecutive days with 1000 mg/kg H-24761 exhibited no mortality or body weight effects. Clinical signs were observed, and liver weights were elevated 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 1360x and 210x the AUCINF/D for the test substance. 19Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 Under the conditions of this study, there was minimal absorption and retention of fluorine in the blood in rats dosed with H-24761. 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. Fluorine levels in fat from rats dosed with the test substance were lower than the levels in fat from rats dosed with the positive control H-24019 and were 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. RECORDS AND SAMPLE STORAGE All original records will be retained at Haskell Laboratory, E. L du Pont de Nemours and Company, Newark, Delaware or at Iron Mountain Records Management, 200 Todds Lane, Wilmington, Delaware. -20- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 TABLES -21Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats TABLE 1 MEAN BODY WEIGHTS Test Days 1 2 3 4 5 6 7 8 9 10 17 19 24 26 31 33 38 40 45 47 52 54 59 61 67 68 73 75 80 82 87 89 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 184.4 184.1 189.2 187.8 199.5 197.8 206.4 204.8 216.2 212.5 222.0 216.0 229.4 223.6 233.7 226.9 240.4 234.2 246.5 243.0 290.1 297.4 - - 313.3 338.6 - - 348.4 381.9 - - 370.8 404.9 - 403.2 434.4 - - 422.8 - 460.6 439.6 483.1 - - 455.6 502.7 - - 480.1 - 525.6 496.5 542.1 - 512.2 - 546.2"' - - 524.6 570.5 Indicates the animals were not weighed. DuPont-5591 Test Substance H-24761 274.5 279.7 287.5 303.7 304.2 304.7 311.3 315.4 312.4 316.8 - 318.7 - 342.8 - 378.5 - 413.0 - 442.8 - 463.7 - 483.9 - 500.2 - 526.8 - 534.4 - 551.3 566.1 -22Company Sanitized. Does not contain TSCA CB1 H-24761; Biopersistence Screening 10-Dose Oral Gavage Study in Rats TABLE 2 MEAN BODY WEIGHT GAINS (g) Test Days 1-5 5-10 1-10 Negative Control Deiomzed 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 DuPont-5591 Test Substance H-24761 29.7 12.6 42.3 Test Days 10-17/19 17/19-24/26 24/26-52/54 52/54-94 Negative Control Deionized Water 36.3 32.2 95.0 91.2 10-94 254.7 Recovery Positive Controls H-24019 H-24020 43.6 54.4 23.2 41.2 109.5 122.0 101.8 109.9 278.1 327.5 Test Substance H-24761 1.9 24.1 120.9 102.4 249.3 "VSiS .23Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 TABLES MEAN BODY AND LIVER WEIGHTS (g) Test Days 10 94 DEIONEED WATER (NEGATIVE CONTROL) Mean Relative ___Bo_d_y_W_e_igAhtbsoluLtieve_r_W_e_ig_h_t __ Liver Weight (Liver/Body Weight) 258.8 10.814 0.042 511.7 17.868 , 0.035 Test Days 10 94 H-24019 (POSITIVE CONTROL) ___Bo_d_y_W_e_igAhtbsoluLtieve_r_W_e_ig_h_t __ 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-24761 (TEST SUBSTANCE) Absolute Body Weight___Liver Weight 306.9 19.638 566.1 , 27.641 Mean Relative Liver Weight (Liver/Body Weight) 0.064 0.049 .24Company Sanitized. Does not contain TSCA CBI fe H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 TABLE 4 MEAN BLOOD FLUORINE LEVELS Test Days 1 5 10 13 24 52 94 Negative Control Deionized Water (ppm) 0.602 c 1.10" c 0.5" c c Positive Controls H-24019 H-24020 (ppm) (ppm) 2.60 (0.1)" 9.40 (2.4) 31.32 (1.1) 74.92 (7.0) 68.00 (3.5) 61.76 (5.5) 53.98 (1.2) 29.52 (4.9) 39.62 (3.4) 11.18 (2.9) 23.56 (2.1) 12.60 (1.2) 2.26 (1.1) 0.85'1 (O.I) Test Substance H-24761 (ppm) 1.24 (0.1) 3.28 (0.5) 6.16 (0.5) 6.14 (0.6) 4.48 (1.0) 1.50 (0.2) 0.86 (0.2) 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 Positive Controls Test Substance Test H-24019 H-24020 H-24761 Days____j^M F Equivalents___^M F Equivalents____p.M F Equivalents 1 36.92 (I.I)2 66.67 (17.5) 0.16 (0.0) 5 478.77 (17.6) 541.45 (50.4) 0.48 (0.1) 10 1043.08 (54.6) 446.09 (39.7) 0.92 (0.1) 13 827.38 (19.0) 212.46 (35.6) 0.92 (0.1) 24 606.46 (53.1) 79.57 (20.8) 0.66 (0.1) 52 359.38 (32.7) 14.93 (7.7) 0.20 (0.0) 94 190.77 (19.2) 4.71*' (0.5) 0.10 (0,0) 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). -25Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 TABLE 6 MEAN LIVER FLUORINE LEVELS Test Negative Control Deionized Water Positive Controls H-24019 H-24020 Test Substance H-24761 P1a0ys____0_.9(0pp(0m.2))"_____3_1_2(.3p4pm(19)._7)_____11_9(.p80pm(3).5_)_______14(p7.p34m()10.9) 94____0.78 (0.0)____84.24 (7.4)_____2.56 (1.3)______20.26 (4.2) 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-24761 Days____/zM Equivalents_____jaM Equivalents 10 4802.15 (303.8)3 866.67 (25.5) jxM Equivalents 22.71 (1.7) 94_____1292.92 (114.1)_____17.10 (9.8)________3.10 (0.7) a Standard deviation is in parentheses. -26- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats TABLES MEAN FAT FLUORINE LEVELS Test Days 10 94 Negative Control Deionized Water (ppm) a a Positive Controls H-24019 . (Ppm) 12.84 (1.7)" 1.20 (0.3) H-24020 (Ppm) 8.10 (1.1) a All values were below the limit of quantification (LOQ) or non-detectable. b Standard deviation is in parentheses. DuPont-5591 Test Substance H-24761 (ppm) 1.46 (0.6) 2.42 (0.8) TABLE 9 MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE Test Positive Controls H-24019 H-24020 Test Substance H-24761 Days_____^tM Equivalents____^tM Equivalents_____fiM Equivalents 10 194.46 (26.8)2 57.25 (7.8) 0.19 (0.1) 94______15.38 (4.1)_________^__________0.34 (0.1) a Standard deviation is in parentheses. b All values were non-detectable. s)' -27Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 FIGURES -28- Company Sanitized. Does not contain TSCA CBI i Q 60 N--^ w >> 0 Q fe I >-! Q 0 II a.s ^ >->! M /"^ tJ 0 a .a.. 0 3.. 2^ r?- i9o S (8) sq3i3M -(pog neai\[ Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats v^y FIGURE 2 COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND NEGATIVE CONTROL Test Days -30- H-24761: Biopcrsistence Screening 10-Dose Oral Gavage Study in Rats FIGURES COMPARISON OF MEAN RELATIVE LIVER WEIGHTS: TEST SUBSTANCE AND POSITIVE CONTROLS Test Days -31- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 FIGURE 4 MICROMOLAR EQUIVALENTS IN RAT BLOOD A. Normalized Rat Blood H-24019 uM Equivalent Reiulling from a 10-Day Oral Qavage 1200 Micromolar (^M) 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. Normalized RatBloodH-24020(jM Equivalents Resulting from a 10-Day Oral Gavage Micromolar (/iM) equivalents ofH-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-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats C. Normalized Rat Blood H-24761 i/M Equivalents Resultin from a 10-Day Oral Gavage DuPont-5591 Micromolar (/xM) equivalents of H-24761 (test substance) in rat blood resulting from a 10-day oral gavage exposure. Values are means and error bars are standard deviation. -33Company Sanitized. Does not contain TSCA CBI Q ^ ;=> g 2^ CT^ rf^ Q i . 0 0 iri ug>^ ||E2 o E S^ ^E ^ 0 sr~o~ 'Hz, ^ <t (S F^:-:i a Q 0 yss ^ .Sa OSco ^^ g8 2 oo g s o m % op; *' CO g.0 ^a <o S^ r- o ?,Q a2 o/dNionv pooig leu Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURE 6 MEAN LIVER FLUORINE CONCENTRATION NORMALIZED TO DOSE Day 10 -35- Day 94 0\ U-l in ^ Q I 2 0 U 0 g ^ rt 5^ 0U l> ^ g S<3 slugj >--i \0 s>- 1^-1 3 ^ ^"g {-I 0 a.s gff! M) en ^ C *-1 0 -- Cu w ^^ g^ U 3 b ^ a, ^ios ? U vo 2^ r^ o ^ CM )Q-< a 0 (mdil) uo|)&lfua3uo3 ja(jonu JUn omw g a o U u li ^'"- *M^ & S OT I IU .a r'ot a^s Cl/A Ipg:mfli0) '1AI "n HIHI'II unaM Company Sanitized. Does not contain TSCA CBI H-24761: B iopersistence Screening 10-Dose Oral Gavage Study in Rats FIGURES MEAN FAT FLUORINE CONCENTRATION NORMALIZED TO DOSE -37- H-24761: 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 6000 5000-f " 4000 E 3000 o' a a. 2000 1000 Blood / Liver / Fat Blood / Liver/ Fat Blood / Liver -38- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 APPENDICES .39- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 APPENDIX A Individual Body Weights .40- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats INDIVIDUAL BODY WEIGHTS ABBREVIATIONS: SD - sacrificed by design EXPLANATORY NOTES DuPont-5591 -41- Company Sanitized. Does not contain TSCA C81 H-24761: Biopcrsistence 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 ANIMAL NUMBER 646932 646933 646934 646935 646936 Day 10 264.7 254.4 248.6 278.0 248.1 - 3D test day 10 SD test day 10 SD test day 10 SD test day 10 SD test day 10 TEST DAY Day 7 242.4 232.9 224.2 257.2 229.9 -42- H-24761: 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 Day 2 186.8 173.8 204.2 197.1 195.9 ANIMAL NUMBER 646937 646938 646939 646940 646941 Day 10 249 .8 237 .2 277 .9 244 .5 266 .3 Day 17 282.6 270.4 329.7 271.5 312.4 ANIMAL NUMBER 646937 646938 646939 646940 646941 Day 73 504 .3 417 .4 540 .1 407 .3 493 .7 Day 80 , ' 520. 5 431. 0 568. 9 424. 0 503. 5 DEIONIZED WATER (NEGATIVE CONTROL) INDIVIDUAL BODY WEIGHTS (g) OP MALE RATS Day 3 GROUP III TEST DAY Day 4 Day 5 Day 6 199.0 180.9 214.6 203.8 207.9 205 .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 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 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 Day 7 229 .6 215 .9 248 .9 229 .3 241 .3 Day 52 443 .9 373 .7 484 .1 367 .0 433 .6 -43- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 646910 646911 646912 646913 646914 Day 1 184.3 184.4 180.5 186.4 185.5 Day 2 185.3 187.1 185.9 189.7 189.6 H-24019 (POSITIVE CONTROL) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS GROUP I Day 3 TEST DAY Day 4 Day 5 Day 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 ANIMAL NUMBER 646910 646911 646912 646913 646914 Day 10 241.1 236.6 241.9 257.0 243.0 SD test day 10 .SD test day 10 SO test day 10 SD test day 10 SD test day 10 TEST DAY Day 7 226.4 225.2 227.2 228.6 226.7 -44- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 646915 646916 646917 646918 646919 Day 1 182.7 185.0 187.8 175.5 192.0 Day 2 188.3 192.7 194.3 181.9 196.9 H-24019 (POSITIVE CONTROL) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS Day 3 GROUP III T EST 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 ANIMAL NUMBER 646915 646916 646917 646918 646919 Day 10 240.4 257.0 250.2 237.1 260.2 Day 17 283.4 310.0 280.3 270.6 306.1 Day 24 302.9 334.4 308.2 292.7 328.5 TES'r 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 ANIMAL NUMBER 646915 646916 646917 646918 646919 Day 73 461.2 544.2 457.5 448.1 489.5 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 -45- H-24761: 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 TE,ST 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 646921 646922 646923 646924 646925 Day 10 262 .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 -46- H-24761: 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 Day 2 187.8 178.8 195.3 182.5 191.6 ANIMAL NUMBER 646926 646927 646928 646929 646930 Day 10 250.8 243.3 . 254.1 225.5 240.3 Day 17 297 .2 306 .2 310 .6 280 .9 292 .3 ANIMAL NUMBER 646926 646927 646928 646929 646930 Day 73 535 .3 551 .2 540 .3 502 .6 498 .5 Day 80 t. 551 .9 574 .5 557 .0 509 .6 517 .6 H-24020 (POSITIVE CONTROL) INDIVIDUAL BODY WEIGHTS (g) OP MALE RATS Day 3 GROUP III TE ST 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 24 349.3 355.1 349.4 323.2 316.2 TE ST 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 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 Day 7 222.9 218.2 236.1 214.6 226.7 Day 52 467 .8 484 .0 464 .8 450 .2 436 .0 -47- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 645243 645244 645245 645246 645255 Day 1 270. 4 271. 3 274. 1 285. 8 274. 9 Day 2 267.4 264.2 270.7 288.7 288.6 H-24761 (TEST SUBSTANCE) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS GROUP I Day 3 TE ST DAY Day 4 Day 5 Day 6 283.0 271.5 290.6 296.2 292.6 287.1 294.0 302.9 315.9 309.3 292. 3 296. 9 299. 8 317. 5 312. 1 293.0 293.9 303.8 314.1 281.5 Day 7 295. 5 296. 4 309. 8 325. 3 290. 0 ANIMAL NUMBER 645243 645244 645245 645246 645255 Day 10 299.9 293.3 319.3 334.3 287.7 SD test day 10 SD test day 10 SD test day 10 SD test day 10 SD test day 10 TEST DAY -48- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats ANIMAL NUMBER 645256 645257 645261 645263 645267 Day 1 271.8 272.6 277.5 274.1 272.9 Day 2 280.2 284.0 284.8 279.8 288.4 ANXMAL NUMBER 645256 645257 645261 645263 645267 Day 10 312.4 328.1 316.6 327.9 348.0 Day 19 282.6 308.1 293.7 331.2 377.8 ANIMAL NUMBER 645256 645257 645261 645263 645267 Day 75 485 .1 505 .8 510 .6 552 .4 580 .0 Day 82 ' 484 .1 518 .3 521 .6 556 .9 591 .0 H-24761 (TEST SUBSTANCE) INDIVIDUAL BODY WEIGHTS (g) OF MALE RATS Day 3 GROUP III TEST DAY Day 4 Day 5 Day 6 283.4 288.6 289.5 286.3 293.6 301.5 308.9 305.5 302.1 309.6 300 .7 306 .9 304 .2 301 .5 310 .3 306.3 315.5 311.1 309.4 318.7 Day 7 308.1 321.0 316.1 317.5 333.7 Day 26 312.4 301.7 326.8 358.0 415.2 Day 89 495.9 536.9 539.3 577.9 606.3 TEST DAY Day 33 Day 40 ' 339.7 331.5 378.0 394.0 449.3 368 .8 375 .5 414 .6 433 .0 473 .2 Day 47 396.9 413.6 437.5 459.5 506.5 TEST DAY Day 94 508. 1 549. 3 549. 4 602. 7 621. 0 SD test day 94 SD test day 94 SD test day 94 SD test day 94 SD test day 94 Day 54 423.8 447.5 449.1 483.4 514.5 -49- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 APPENDIX B Individual Clinical Observations .50- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DEIONIZED WATER (NEGATIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I Animal 646932 646933 646934 Observation General observation, No Abnormality Detected Eye Observations, Exophthalmus, Left Eye Observations, Bled via Orbital for Clin Path, Left Discharge, Eye left, Black Sacrificed by design General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Dark, Left Discharge, Eye left. Black Swollen Observations, Mouth Sacrificed by design General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Discharge, Eye left, Black Sacrificed by design Days 1 2-9 1,5 10 10 1-5 1,5 6-10 6-7 10 10 1-5.10 1,5 6-9 10 -51 H-24761: Biopcrsistence Screening 10-Dose Oral Gavage Study in Rats Animal 646935 646936 DEIONIZED WATER (NEGATIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I (Continued) Observation Days 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 1-10 1,5 10 1-10 1,5 10 -52- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DEIONIZED WATER (NEGATIVE CONTROL) Animal 646937 646938 646939 INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Observation GROUP III Days 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 1-10,17-94 13 24,52 94 General observation. No Abnormality Detected Eye Observations, Enophthalmus, Right 1-10,17-45 80-94 Eye Observations, Exophthalmus, Right 52-73 Eye Observations, Bled via Orbital for Clin Path, Bilateral Eye Observations, Bled via Orbital for Clin Path, Right 13.52 24 Eye Observations, Corneal Opacity, Right Discharge, Eye right. Black Sacrificed by design ^ General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left 80-94 59 94 1-10,17-94 13 Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 24.52 94 -53- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DEIONIZED WATER (NEGATIVE CONTROL) Animal 646940 646941 INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Observation GROUP III (Continued) Days 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 General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Bilateral Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 1-10,17-94 52 13 24 94 -54- H-24761: B iopersistence Screening 10-Dose Oral Gavage Study in Rats H-24019 (POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I Animal 646910 646911 646912 646913 646914 Observation 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 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 Days 1-10 1,5 10 1,10 1,5 2-9 10 1-10 1 5 10 1-10 1,5 10 1-10 1,5 10 -55- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Animal 646915 646916 646917 H-24019 (POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Observation GROUP III Days General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left 1,17-38.73 13,24,52 Discharge, Eye right. Red Hair Loss, Porelimb, Right 2-10 80 Hair Loss, Forepaw, Right Sacrificed by design 45-67,87-94 94 General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Bilateral Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 1-10,17-94 24 13 52 94 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 1-10,17-94 13,24 52 94 -56- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Animal 646918 646919 H-24019 (POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Observation GROUP III (Continued) Days General observation. No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right 13,24 52 Sacrificed by design 94 General observation. No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 13,24 52 94 -57- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats H-24020 (POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I Animal 646921 646922 646923 Observation General observation, No Abnormality Detected 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 Sacrifi-ced by design General observation. No Abnormality Detected Eye Observations, Exophthalmus, Left Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Eye Observations, Dark, Left Sacrificed by design Days 1,5-10 1,5 2-4 10 1-10 1 5 10 1,7-10 4-6 1 5 2-6 10 -58- H-24761: 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, Bled via Orbital for Clin Path, Left 1 Eye Observations, Bled via Orbital for Clin Path, Right 5 Sacrificed by design 10 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 1-10 1 5 10 -59- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Animal 646926 646927 646928 H-24020(POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Observation GROUP Ill- Days General observation. No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin Path, Bilateral Eye Observations, Bled via Orbital for Clin Path, Left Sacrificed by design 52 13,24 94 General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Bilateral Eye Observations, Bled via Orbital for Clin Path, Left Sacrificed by design 1-10,17-94 52 13,24 94 General observation. No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin Path, Left Sacrificed by design 13,24,52 94 -60- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Animal 646929 646930 H-24020(POSITIVE CONTROL) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Observation GROUP III (Continued) Days General observation. No Abnormality Detected 1-10,17-31 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Hair Loss, Forelimb, Bilateral Hair Loss, Neck, Left 13,24 52 52-73 73 Hair Loss, Neck, Ventral Wound, Superficial, Face Sacrificed by design 38-67,80-94 59 94 General observation. No Abnormality Detected 1-10,17-94 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 13,24 52 94 -61- H-24761: Biopersistence Screening 10-Pose Oral Gavage Study in Rats H-24761 (TEST SUBSTANCE) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS GROUP I Animal 645243 645244 645245 645246 645255 Observation General observation, No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path. Left General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Bilateral Eye Observations, Bled via Orbital for Clin Path, Left General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Discharge, Nose, Red ' Discharge, Eye left. Red Days 1-10 1,5 1-10 1,5 1-10 5 1 1-10 1,5 1-9 1,5 10 10 -62- H-24761: Biopersistence Screening 10-Pose Oral Gavage Study in Rats Animal 645256 645257 645261 H-24761 (TEST SUBSTANCE) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Observation GROUP III Days General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Discharge, Nose, Black 1-10,26-47,54-94 13 24,52 19 Sacrificed by design 94 General observation. No Abnormality Detected Eye Observations, Bled via Orbital for Clin Path, Bilateral Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 1-10,19,26-47,54- 52 13 24 94 General observation. No Abnormality Detected 1-4,10,19,26-47,5 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Discharge, Nose, Black 13,52 24 5-9 Sacrificed by design 94 -63- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Animal 645263 645267 H-24761 (TEST SUBSTANCE) INDIVIDUAL CLINICAL OBSERVATIONS IN MALE RATS Observation GROUP III (Continued) Days General observation. No Abnormality Detected 1-10,19,26-47,54-9 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 13,52 24 94 General observation. No Abnormality Detected 1-10,19,26-47,54-9 Eye Observations, Bled via Orbital for Clin Path, Left Eye Observations, Bled via Orbital for Clin Path, Right Sacrificed by design 13,52 24 94 -64- H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 APPENDIX C Terms and Calculations Factors Influencing Interpretation of Kinetic Analysis .65- Company Sanitized. Does not contain TSCA CBI ,<5"'\ w H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 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 compoundj)er 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 66Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 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, m this case the value was established at 0.2 ppm. ppmF 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) {i molar equivalents of active The ^molar [/imol/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 ^mol/mmol .67- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 FACTORS INFLUENCING INTERPRETATION OF KINETIC ANALYSIS Considerations: - The data used for kinetic analysis was from a limited screen and extrapolation should be done cautiously. - Sample size is low - Analytical data used without validation - Steady-state not reached - Terminal phase may not be reached - Some compounds are mixtures of fluorinated compounds - Different active and formulation doses were used - Different vehicles were used to deliver formulations - Each compound may have very different potency for producing toxicity Assumptions: (May or may not be justified in all cases) - 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 -68Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 fc) <A- APPENDK D Individual Fluorine Levels in Blood -69Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24019 DuPont-5591 Given: Mol Wt. Active (g/mole): 497 Formuladon Dose (mg/kg): 10 % Active (F Containing) in Formulation: 100 % Fin Active: 65 Mol Wt. F (g/mol): 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): 10 0.020 0.342 Molar Ratio (Acl;ive/F): Dose F (mg/kg): 0.059 6.5 Rat Number Test Day Sample ppm F in Blood Group I 646910 1 2.6 646911 1 2.7 646912 1 2.5 646913 1 2.6 646914 1 2.6 Group I 646910 5 646911 5 646912 5 646913 5 646914 5 30.1 30.3 31.3 32.2 32.7 Group I 646910 10 71.5 646911 10 70.5 646912 10 66.9 646913 10 62.5 646914 10 68.6 ppm F in Blood Minus Bkg 0.2 ppm 2.4 2.5 2.3 2.4 2.4 29.9 30.1 31.1 32.0 32.5 71.3 70.3 66.7 62.3 68.4 ppm F in Blood Normalized to 0.1 mmoles/kg Dose 11.93 12.43 11.43 11.93 11.93 148.60 149.60 154.57 159.04 161.53 354.36 349.39 ,. 331.50 309.63 339.95 limolar Equivalents of Active in Blood 36.92 38.46 35.38 36.92 36.92 460.00 463.08 478.46 492.31 500.00 1096.92 1081.54 1026.15 958.46 1052.31 .70- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Rat Number Test Day Sample ppmF in Blood Group in 646915 646916 646917 646918 646919 13 13 13 13 13 ' 55.0 55.3 52.2 53.8 53.6 Group III 646915 24 646916 24 646917 24 646918 24 646919 24 Group III 646915 52 646916 52 646917 52 646918 52 646919 52 38.9 43.8 37.6 35.4 42.4 23.2 20.3 25.9 24.9 23.5 ppm F in Blood Minus Bkg 0.2 ppm 54.8 55.1 52.0 53.6 53.4 38.7 43.6 37.4 35.2 42.2 23.0 20.1 25.7 24.7 23.3 Group III 646915 94 13.3 13.1 646916 94 11.7 11.5 646917 94 12.9 12.7 646918 94 11.0 10.8 646919 94 14.1 13.9 ppm F in Blood Normalized to 0.1 mmoles/kg Dose 272.36 273.85 258.44 266.39 265.40 192.34 216.69 185.88 174.94 209.73 114.31 99.90 127.73 122.76 115.80 65.11 57.16 63.12 53.68 69.08 DuPont-5591 /tmolar Equivalerits of Active in Blood 843.08 847.69 800.00 824.62 821.54 595.38 670.77 575.38 541.54 649.23 353.85 309.23 395.38 380.00 358.46 201.54 176.92 195.38 166.15 213.85 71Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-240:20 DuPont-5591 Given: MoPiVt. Active (g/mole): 426 Formulation Dose (ing/kg): 20 %Acfive (F Contai ning) in Formulation: 100 % F in Active: 69 MolWt.F(g/mc!): 19 Calculat(ed Values: Dose Active (mg/kj?): Dose Active (mmol[e/kg): Dose F (mmol/kg): 20 0.047 0.726 Molar Ratio (Acltive/F): Dose F (mg/kg): 0.065 13.8 Rat Number Test Day Sample ppmF in 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 Group I 646921 5 646922 5 646923 5 646924 5 646925 5 78.7 83.2 77.0 66.1 69.6 78.5 83.0 76.8 65.9 69.4 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 ppm F in Blood Normalized to 0.1 mmoles/kg Dose 22.15 22.37 19.17 10.86 23.43 167.21 176.79 163.58 140.37 147.82 133.76 146.76 ' 128.01 132.70 114.38 {imolar Equivalents of Active in Blood 75.36 76.09 65.22 36.96 79.71 568.84 601.45 556.52 477.54 502.90 455.07 499.28 435.51 451.45 389.13 .72Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Rat Number Test Day Sample ppmF in Blood Group III 646926 13 646927 13 646928 13 646929 13 646930 13 34.8 30.0 23.7 33.7 25.4 ppmF in Blood Minus Bkg 0.2 ppm 34.6 29.8 23.5 33.5 25.2 ppm F in B lood Normalize dto 0.1 nunoles/kg Dose 73.70 63.47 50.06 71.36 53.68 DuPont-5591 /{molar Equivalents of Active in Blood 250.72 215.94 170.29 242.75 182.61 Group III 646926 24 11.9 11.7 646927 24 10.4 10.2 646928 24 8.1 7.9 646929 24 15.7 15.5 646930 24 9.8 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 52 2.5 2.3 646927 52 1.7 1.5 646928 52 1.3 1.1 646929 52 4.0 3.8 646930 52 1.8 1.6 4.90 3.20 2.34 8.09 3.41 16.67 10.87 7.97 27.54 - 11.59. Group III 646926 94 0.8 0.6 646927 94 <0.5 * 646928 94 <0.5 * 646929 94 0.9 0.7 646930 94 <0.5 * Below LOQ (Limit of Quantification) 1.28 * * 1.49 * 4.35 * * 5.07 * -73- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-247(S1 DuPont-5591 Given: MoUNt. Active (g/imole): Form illation Dose (nig/kg): %Active (F Containing) in Fonmulation: 2757 1000 100 % F in Acth/e: Mol Wt. F (jg/mc>1): 64.8 19 Calculat<id Values: Dose Active (mg/kj?): Dose Active (mmol e/kg): Dose F (mmol/kg): 1000 0.363 34.105 Molar Ratio (Acl:ive/F): Dose F (mg/kg): 0.011 648 Rat Number Test Day Sample ppmF in Blood Group I 645243 1 1.2 645244 1 1.2 645245 1 1.3 645246 1 1.2 645255 1 1.3 ppm F in Blood Minus Bkg 0.2 ppm 1.0 1.0 1.1 1.0 1.1 ppm F in Blood Normalized to 0.1 mmoles/kg Dose 0.28 0.28 0.30 0.28 0.30 /(molar Equivalents of Active in Blood 0.15 0.15 0.17 0.15 0.17 Group I 645243 5 2.9 2.7 , 645244 5 2.9 2.7 645245 5 3.5 3.3 645246 5 3.0 2.8 645255 5 4.1 3.9 0.74 0.74 0.91 0.77 1.08 0.42 0.42 0.51 0.43 0.60 Group I 645243 10 6.2 6.0 645244 10 6.6 6.4 645245 10 5.5 5.3 645246 10 5.8 5.6 645255 10 6.7 6.5 1.65 1.76 .,, 1.46 1.54 1.79 0.93 0.99 0.82 0.86 1.00 -74Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Rat Number Test Day Sample ppmF in Blood Group III 645256 13 6.0 645257 13 5.2 645261 13 6.5 645263 13 6.4 645267 13 6.6 ppmF in Blood Minus Bkg 0.2 ppm 5.8 5.0 6.3 6.2 6.4 Group in 645256 24 3.9 3.7 645257 24 6.0 5.8 645261 24 3.5 3.3 645263 24 4.4 4.2 645267 24 4.6 4.4 Group III 645256 52 1.3 1.1 645257 52 1.5 1.3 645261 52 1.3 1.1 645263 52 1.6 1.4 645267 52 1.8 1.6 Group III 645256 94 1.0 0.8 645257 94 1.1 0.9 645261 94 0.7 0.5 645263 94 0.9 0.7 645267 94 0.6 0.4 ppm F in EHood Normalizeid to 0.1 mmoles/kg Dose 1.60 1.38 1.74 1.71 1.76 1.02 1.60 0.91 1.16 1.21 0.30 0.36 0.30 0.39 0.44 0.22 0.25 0.14 0.19 0.11 DuPont-5591 /tmolar Equivalents of Active in Blood 0.90 0.77 0.97 0.96 0.99 0.57 0.90 0.51 0.65 0.68 0.17 0.20 0.17 0.22 0.25 0.12 0.14 0.08 0.11 0.06 -75Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 APPENDIX E Individual Fluorine Levels in Liver ) .76Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24019 DuPont-5591 Given: Mol Wt. Active (g/mole): 497 Formulation Dose (mg/kg): 10 % Active (F Containing) in Formulation: 100 % F in Active: 65 Mol Wt. F (g/mol): 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose P (mmol/kg): 10 0.020 0.342 Molar Ratio (Active/F): Dose F (mg/kg): 0.059 6.5 Rat Number Test Day Sample ppm F in 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 Group III 646915 94 646916 94 646917 94 646918 94 646919 94 85.6 75.9 87.5 78.0 94.2 85.4 75.7 87.3 77.8 94.0 ppm F in Liver Normalized to 0.1 nunoles/kg Dose 1593.88 1658.99 1582.45 1398.56 1522.81 424.44 376.23 433.88 386.67 467.18 /(molar Equivalents of Active in Liver 4933.85 5135.38 4898.46 4329.23 4713.85 1313.85 1164.62 1343.08 1196.92 1446.15 -77- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Datafcr H-24(20 DuPont-5591 Given: MolWt:. Active (g;'nlole): 426 Formuliation Dose (rng/kg): 20 % Acthie (F Containling) in Formulation: 100 % F in Active: 69 MolWt.F(g/n[iol): 19 Calculated Values: Dose Aictive (mg/kS.): Dose A(stive (mmo Ie'/kg): Dose F (mmol/kg): 20 0.047 0.726 Molar Ratio (A.ctive/F): Dose F (mg/kg): 0.065 13.8 Rat Number Test Day Sample ppmF in 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 Group III 646926 94 1.8 1.6 646927 94 1.2 1.0 646928 94 2.2 2.0 646929 94 4.7 4.5 646930 94 2.9 2.7 ppm F in Liver Normalized to 0.1 mmoles/kg Dose /(molar 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 3.41 2.13 4.26 9.59 5.75 11.59 7.25 14.49 32.61 19.57 -78- Company Sanitized. Does not contain TSCA.CBI H-24761: B iopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24761 DuPont-5591 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): % Active (F Containing) in Formulation: 2757 1000 100 % F in Active: Mol Wt. F (g/mol): 64.8 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): 1000 0.363 34.105 Molar Ratio (Active/F): Dose F (mg/kg): 0.011 648 Rat Number Test Day Sample ppm F in Liver Group I 645243 10 155.2 645244 10 134.2 645245 10 144:4 645246 10 141.4 645255 10 161.5 Group III 645256 94 645257 94 645261 94 645263 94 645267 94 22.6 25 17.7 21.7 14.3 ppm Fin Liver Minus Bkg 0.2 ppm 155.0 134.0 144.2 141.2 161.3 22.4 24.8 17.5 21.5 14.1 ppm F in Liver Normalized to 0.1 mmoles/kg Dose /(molar Equivalents of Active in Liver 42.73 36.94 39.76 38.93 44.47 23.92 20.68 22.25 21.79 24.89 6.18 6.84 4.82 5.93 3.89 3.46 3.83 2.70 3.32 2.18 79Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats DuPont-5591 APPENDIX F Individual Fluorine Levels in Fat 80Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24019 DuPont-5591 Given: Mol Wt. Active (g/mole): 497 Formulation Dose (mg/kg): 10 % Active (F Containing) in Formulation: 100 % F in Active: 65 Mol Wt. P (g/mol): 19 Calculated Values: Dose Active (mg/kg): Dose Active (mmole/kg): Dose F (mmol/kg): i- 10 0.020 0.342 Molar Ratio (Active/F): Dose F (mg/kg): 0.059 6.5 Rat Number Test Day Sample ppm F in Fat Group I 646910 10 646911 10 646912 10 646913 10 646914 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 Group III 646915 94 1.3 1.1 646916 94 1.1 0.9 646917 94 1.3 1.1 646918 94 0.8 0.6 646919 94 1.5 1.3 ppm F in Fat Normalized to 0.1 mmoles/kg Dose 70.57 73.06 53.18 56.66 60.63 5.47 4.47 5.47 2.98 6.46 /tmolar Equivalents of Active in Fat 218.46 226.15 164.62 175.38 187.69 16.92 13.85 16.92 9.23 20.00 -81- Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-240;10 DuPont-5591 Given: Mol Wt. Active (g/;mole): 426 Formulation Dose (mg/kg): 20 % Active (F Containjng) in Fcirmularion: 100 % F in Acth/e: 69 MolWt.F(,g/mc>1): 19 Calculated Values: Dose Active (mg/kj?): Dose Active (mmol e/kg): Dose F (mmol/kg): 20 0.047 0.726 Molar Ratio (Acl;ive/F): Dose F (mg/kg): 0.065 13.8 Rat Number Test Day Sample ppmF in Fat Group I 646921 10 6.2 646922 10 8.5 646923 10 8.7 646924 10 8.8 646925 10 8.3 ppmF in Fat Minus Bkg 0.2 ppm 6.0 8.3 8.5 8.6 8.1 Group III 646926 94 ND ND 646927 94 ND ND 646928 94 ND ND 646929 94 ND ND 646930 94 ND ND ND Non-detectable. ppm F in Fat Normalized to 0.1 mmoles/kg Dose 12.78 17.68 18.11 18.32 17.25 ND ND ND ND ND ^molar Equivalents of Active in Fat 43.48 60.14 61.59 62.32 58.70 ND ND ND ND ND 82Company Sanitized. Does not contain TSCA CBI H-24761: Biopersistence Screening 10-Dose Oral Gavage Study in Rats Data for H-24761 DuPont-5591 Given: Mol Wt. Active (g/mole): Formulation Dose (mg/kg): % Active (F Containing) in Formulation: 2757 1000 100 % F in Active: Mol Wt. F (g/mol): 64.8 19 Calculated Values: Dose Aictive (mg/k S): Dose A<stive (mmoile/kg): DoseF (mmoVkg): 1000 0.363t 34.1C15 --------^---------- Molar Ratio (Aictive/F): Dose I ? (mg/kg:): 0.011 648 Rat Number Test Day Sample ppmF in Fat Group I 645243 10 0.6 645244 10 2.1 645245 10 1.7 645246 10 1.3 645255 10 1.6 ppmF in Fat Minus Bkg 0.2 ppm 0.4 1.9 1.5 1.1 1.4 Group III 645256 94 2.2 2.0 645257 94 3.6 3.4 645261 94 2.1 1.9 645263 94 2.7 2.5 645267 94 1.5 1.3 ppm F in Fat Normalizfd to 0.1 mmoless/kg Dose /xmolar Equivalents of Active in Fat 0.11 0.52 0.41 0.30 0.39 0.06 0.29 0.23 0.17 0.22 0.55 0.94 0.52 0.69 0.36 0.31 0.52 0.29 0.39 0.20 .83Company Sanitized. Does not contain TSCA CBI