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Home My WebLink AboutCH-FW-DEQ-0008648 DuPont-18405-849 Page 1 of 118 TRADE SECRET Unpublished Work Copyright ©2011 E.I. du Pont de Nemours and Company STUDY TITLE: H-28548: Toxicokinetic Study in Pregnant Rats AUTHOR: Susan M. Munley, M.A. STUDY COMPLETED ON: March 29, 2011 PERFORMING LABORATORY: E.I. du Pont de Nemours and Company DuPont Haskell Global Centers for Health & Environmental Sciences P.O. Box 50 Newark, Delaware 19714 U.S.A. LABORATORY PROJECT ID: DuPont-18405-849 WORK REQUEST NUMBER: 18405 SERVICE CODE NUMBER: 849 SPONSOR: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. CH-FW-DEQ-0008648 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 2 - GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are compatible with current OECD Good Laboratory Practices. CH-FW-DEQ-0008649 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 3 - QUALITY ASSURANCE STATEMENT Work Request Number: 18405 Service Code Number: 849 Key inspections for the above referenced study were completed by the Quality Assurance Unit of DuPont Haskell and the findings were submitted on the following dates: Audit Dates Date Reported to Principal Investigator Date Reported to PI Management Date Reported to Study Director Date Reported to SD Management Protocol: June 04, 2010 June 04, 2010 June 04, 2010 Conduct: June 14, 2010 June 14, 2010 June 14, 2010 July 07, 2010 July 07, 2010 July 07, 2010 Report/Records: August 05,06, 2010 August 06, 2010 August 06, 2010 August 06, 2010 August 06, 2010 August 18-20,23, 2010 August 23, 2010 August 23, 2010 October 01, 04, 2010 October 04, 2010 November 01, 2010 October 28, 29, 2010 October 29, 2010 March 07, 2011 February 25, 2011 February 25, 2011 February 25, 2011 CH-FW-DEQ-0008650 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 4 - CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. CH-FW-DEQ-0008651 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 5 - TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT...................................2 QUALITY ASSURANCE STATEMENT...................................................................................3 CERTIFICATION.........................................................................................................................4 TABLE OF CONTENTS ..............................................................................................................5 LIST OF TABLES.........................................................................................................................7 LIST OF APPENDICES...............................................................................................................7 STUDY INFORMATION.............................................................................................................8 SUMMARY....................................................................................................................................9 OBJECTIVE ................................................................................................................................10 ANIMAL WELFARE ACT COMPLIANCE ...........................................................................10 STUDY DESIGN..........................................................................................................................11 A. Treatment Groups and Daily Dosage.................................................................................11 B. Dosing and Sacrifice Schedule..........................................................................................11 C. Selection of Route of Administration and Dose Levels.....................................................11 MATERIALS AND METHODS................................................................................................12 A. Vehicle...............................................................................................................................12 B. Test Substance ...................................................................................................................12 C. Dosing Formulations..........................................................................................................12 1. Preparation.............................................................................................................................................12 2. Sampling and Analysis...........................................................................................................................12 D. Test System........................................................................................................................12 E. Justification for Animal Model..........................................................................................13 F. Animal Husbandry.............................................................................................................13 1. Animal Health and Environmental Monitoring Program.......................................................................14 G. Quarantine and Pretest Period............................................................................................14 H. Assignment of Animals to Groups.....................................................................................14 I. In-life Observations ...........................................................................................................14 J. Animal Euthanasia.............................................................................................................15 K. Postmortem Evaluations....................................................................................................15 1. Females Surviving to Scheduled Euthanasia..........................................................................................15 L. Blood Collection and Plasma Analysis..............................................................................16 M. Serum Preparation and Storage..........................................................................................16 N. Ovary/Uterus Collection and Storage................................................................................16 STATISTICAL ANALYSES......................................................................................................16 RESULTS AND DISCUSSION..................................................................................................17 Formulation Analytical Evaluation............................................................................................17 CH-FW-DEQ-0008652 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 6 - A. Dosing Formulations Analysis...........................................................................................17 In-Life, Maternal Gross, and Caesarian Section Data.............................................................17 A. Maternal Mortality and Clinical Observations ..................................................................17 B. Maternal Body Weights, Body Weight Changes, Food Consumption..............................17 C. Maternal Gross Postmortem Findings ...............................................................................17 D. Reproductive Outcome and Litter Data.............................................................................18 E. Maternal Liver and Kidney Weights..................................................................................18 Toxicokinetics Conclusions.........................................................................................................18 A. Plasma Analysis.................................................................................................................18 RECORDS AND SAMPLE STORAGE....................................................................................19 REFERENCES.............................................................................................................................19 TABLES........................................................................................................................................20 APPENDICES..............................................................................................................................33 CH-FW-DEQ-0008653 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 7 - LIST OF TABLES Page Table 1 Summary of Maternal Clinical Observations...........................................................................22 Table 2 Mean Maternal Body Weights.................................................................................................23 Table 3 Mean Maternal Body Weight Gains ........................................................................................26 Table 4 Mean Maternal Food Consumption.........................................................................................28 Table 5 Summary of Maternal Gross Observations..............................................................................30 Table 6 Reproductive Outcome ............................................................................................................31 Table 7 Mean Absolute and Relative Organ Weights in Female Rats..................................................32 LIST OF APPENDICES Page Appendix A Certificate of Analysis.............................................................................................................35 Appendix B Protocol....................................................................................................................................37 Appendix C Analysis for H-28548 in Dose Samples...................................................................................51 Appendix D Individual Maternal Clinical Observations..............................................................................58 Appendix E Individual Maternal Body Weights..........................................................................................65 Appendix F Individual Maternal Body Weight Gains.................................................................................76 Appendix G Individual Maternal Food Consumption..................................................................................84 Appendix H Individual Maternal Gross Observations .................................................................................87 Appendix I Individual Reproductive Data..................................................................................................91 Appendix J Individual Animal Organ Weight Data....................................................................................96 Appendix K Quantitation of Plasma Samples for H-28548: Toxicokinetic Study in Pregnant Rats.........100 CH-FW-DEQ-0008654 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 8 - STUDY INFORMATION Substance Tested: • HFPO Dimer Acid Ammonium Salt • 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid, ammonium salt • 62037-80-3 (CAS Number) • H-28548 Haskell Number: 28548 Composition: Proprietary Purity: 84% Physical Characteristics: Clear and colorless liquid Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Study Initiated/Completed: June 4, 2010 / (see report cover page) Experimental Start/Termination: June 7, 2010 / July 8, 2010 In-Life Initiated/Completed: June 7, 2010 / June 21, 2010 Notebook Number(s): E-113111-BZ CH-FW-DEQ-0008655 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 9 - SUMMARY Groups of presumed pregnant Crl:CD(SD) rats (5 per group) were administered solutions of the test substance, H-28548, in deionized water once daily via oral gavage on gestation days 6 through 20. The dose levels tested were 0, 5, 10, 100, and 1000 mg/kg/day, and doses were administered at a volume of 10 mL/kg. The dose formulations were analyzed and confirmed to be at target concentrations and stable under the conditions of the experiment. Blood was collected from all animals at euthanasia on gestation day 20 approximately 2 hours following the last dose. Trunk blood was collected from all fetuses and pooled by litter. An additional group of rats at 1000 mg/kg/day was bled via the tail vein on gestation day 6, as well as at the terminal blood collection time point. This group was added to provide toxicokinetic data following a single dose, as compared with after 15 consecutive doses on gestation day 20. The measured plasma concentrations are summarized below: Summary of Plasma Concentrations for Parent Compound H-28548 Concentration (ng/mL) Dams Pooled Pups Dose Day 6 Day 20 Day 20 Pup:Dam Group (mg/kg/day) Mean SD Mean SD Mean SD Plasma Ratio 1 0 33 16 19 23 2 5 3984 469 1134 175 0.3 3 10 9312 1710 2458 465 0.3 4 100 85560 10092 18320 9128 0.2 5 1000 338400 160168 99800 26482 0.3 6 1000 430600 162712 348400 130362 102240 28295 0.3 The toxicokinetic data indicated that the dose response curve was linear between 5 and 100 mg/kg/day. At 1000 mg/kg/day the concentration was less than what would be predicted if the dose response curve was linear through 1000 mg/kg/day. The mean plasma concentration on day 6 was less than the mean plasma concentration on day 20. This implies that steady state was achieved by day 6 and that there is no accumulation in the dams between day 6 and day 20. The concentration in plasma pooled from pups was approximately one-third of the concentration in plasma from the dam at the same time point. CH-FW-DEQ-0008656 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 10 - OBJECTIVE The objective of this study was to measure levels of the test substance, H-28548, in plasma prepared from blood collected from pregnant rats on gestation day (GD) 20 and from GD 20 fetuses. Plasma samples from pregnant rats at the highest dose level of 1000 mg/kg/day on GD 6 were also collected and analyzed. ANIMAL WELFARE ACT COMPLIANCE This study complied with all applicable sections of the Final Rules of the Animal Welfare Act regulations (9 CFR) and the Guidelines from the Guide for the Care and Use of Laboratory Animals (NRC 1996). All studies conducted by or for DuPont Haskell adhere to the following principles: • The sponsor and/or the study director ensure that the study described in this report does not unnecessarily duplicate previous experiments, and is in compliance with the DuPont Policy on Animal Testing. • Whenever possible, procedures used in this study have been designed to implement a reduction, replacement, and/or refinement in the use of animals in an effort to avoid or minimize discomfort, distress or pain to animals. All methods are described in this study report or in written laboratory standard operating procedures. • DuPont Haskell policy is that animals experiencing severe pain or distress that cannot be relieved are painlessly euthanized, as deemed appropriate by the veterinary staff and study director or appropriate designee. The sponsor was advised by the study director of all circumstances that could lead to this action in as timely a manner as possible. • Methods of euthanasia used during this study were in conformance with the above referenced regulation and the recommendations of the American Veterinary Medical Association (AVMA), 2007 Guidelines on Euthanasia. • Animals were provided with species-appropriate environmental enrichment. • DuPont Haskell is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. CH-FW-DEQ-0008657 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 11 - STUDY DESIGN A. Treatment Groups and Daily Dosage Group Dosage (mg/kg/day)a Formulation Concentration (mg/mL)b Number of Timed Mated Females 1 0c 0 5 2 5 0.5 5 3 10 1 5 4 100 10 5 5 1000 100 5 6 1000 100 5 a Formulations of test substance in deionized water were administered once daily by gavage on GD 6-20 at a dosing volume of 10 mL/kg. b To achieve these concentrations of active ingredient, the formulations were adjusted for sample purity. c The control group animals received vehicle, deionized water, only at a dosing volume of 10 mL/kg. B. Dosing and Sacrifice Schedule Rats were dosed once daily by oral gavage on GD 6 to 20 at the dose levels listed in the table above. The volume administered (10 mL/kg) was based on the most recent body weight. Rats assigned to group 6 were bled via tail vein on GD 6 two hours (± 5 minutes) following dosing. Rats assigned to groups 1 through 6 were euthanized on GD 20 two hours (± 5 minutes) following dosing; blood was collected at sacrifice and selected tissues were weighed (livers, kidneys) and retained (livers, kidneys, ovaries, uterus). A maximal volume of blood was collected from each rat at sacrifice and split for preparation of plasma and serum samples. In addition, trunk blood from fetuses was also collected and pooled by litter for preparation of plasma samples. C. Selection of Route of Administration and Dose Levels The test substance formulations were administered orally because previous developmental and reproductive toxicity studies(1,2,3) were conducted using the same route of exposure. The doses selected for the current study are 0, 5, 10, 100, and 1000 mg/kg/day. These doses have been tested in the previously cited studies and the current study was intended to provide information regarding the relative internal doses relevant to the previous work. CH-FW-DEQ-0008658 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 12 - MATERIALS AND METHODS A. Vehicle The vehicle was deionized water. There were no known contaminants in the vehicle that would be expected to have had any adverse impact on the integrity of the study. The vehicle was assumed to be stable under the conditions of the study. The vehicle was stored at room temperature. B. Test Substance (Appendix A) The test substance, H-28548, was supplied by the sponsor as a clear and colorless liquid with a purity of 84%. The test batch used for this study was assigned Haskell number 28548. C. Dosing Formulations 1. Preparation Formulations of the test substance in the vehicle were prepared and used within the period of established stability. Formulation stability was established previously in a separate study(4) and demonstrated that the test substance formulations were stable at room temperature for up to 12 days at concentrations ranging from 0.01 to 100 mg/mL. Dosing formulations were stored at room temperature until used. The method of mixing the test substance with the vehicle was documented in the study records. 2. Sampling and Analysis (Appendix B) Samples of each formulation were taken 2 times; near the beginning and end of the study. Analyses addressed concentration verification Samples were analyzed by the DuPont Haskell Analytical Chemistry Group on the day the samples were collected. The Analytical Method used was documented in the study records and is included in this report in Appendix B. D. Test System Species/Strain: Crl:CD(SD) rat Sex: Female (nulliparous, timed mated, GD 0 = day mating confirmed) Supplier: Charles River Laboratories, Inc., Raleigh, North Carolina Gestation Day at Arrival: GD 2 Number Received: 30 received on June 3, 2010 CH-FW-DEQ-0008659 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 13 - Age at Arrival: Approximately 86 days Age at Start of Study: Approximately 91 days Weight at Arrival: Approximately 225-253 grams Identification: Each animal was identified by the supplier prior to shipping by marking the tail in indelible ink with a unique number from a list provided by DuPont Haskell. The tail marks were verified routinely and darkened, if necessary, to ensure tracking of animal identity. GD 0 body weights were supplied by the vendor and included in the study records. E. Justification for Animal Model The rat was selected for this study because it is a preferred species for developmental toxicity testing as recommended by test guidelines. The Crl:CD(SD) strain was chosen because extensive background information is available from the literature, the supplier, and previous studies conducted at DuPont Haskell. This strain is also considered suitable relative to hardiness and incidence of spontaneous disease. F. Animal Husbandry Housing: individually in solid bottom caging with bedding, and nestlets as enrichment Cage Rack Positioning: Cage racks were not relocated within the animal room. Climate: Temperature of 18-26C (64-79ºF) Relative humidity of 30%-70% Any excursions outside of these ranges were of insufficient magnitude and/or duration to have adversely affected the validity of the study. The relative humidity and temperature values in the housing rooms were monitored continuously and recorded automatically at regular intervals each day. The temperature and humidity records were reviewed by the laboratory veterinarian and/or designee; a copy of the animal room accountability records, which contain manually recorded temperature and humidity checks (once or twice daily during study), are retained with the study records. Illumination: Artificial (fluorescent light) on an approximate 12-hour light/dark cycle. Water: Tap water ad libitum Feed: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 (pellets) ad libitum. CH-FW-DEQ-0008660 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 14 - 1. Animal Health and Environmental Monitoring Program As specified in the DuPont Haskell 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. • 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. G. Quarantine and Pretest Period Rats were quarantined for at least 3 days and then released for the study based on acceptable body weights and freedom from any adverse clinical observations. H. Assignment of Animals to Groups Before dosing began, animals were randomly assigned to control or experimental groups using a computerized randomization procedure designed to produce a homogeneous distribution of body weights across groups within each breeding lot. I. In-life Observations Procedure Frequency Quarantine and Pretest Mortality/Moribundity At least once daily Clinical Observations GD 4 Body Weights GD 4 Food Consumption GD 4 CH-FW-DEQ-0008661 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 15 - Procedure Frequency Testing Period Mortality/Moribundity Twice daily (AM and PM) Blood Collection For animals in group 6, once 2 hours (± 5 minutes) post-dosing on GD 6 Clinical Observations Twice daily on GD 6-19 (during weighing and at least 2 hours post- dosing) Once on GD 20 Body Weights Daily on GD 6-20 Food Consumption GD 6, 8, 10, 12, 14, 16, 18, and 20 J. Animal Euthanasia Adult females were euthanized by carbon dioxide asphyxiation and exsanguination; a maximal volume of blood was collected. The whole blood was divided so that both plasma and serum could be prepared. Fetuses were euthanized by decapitation and trunk blood was collected and pooled by litter. Plasma was prepared from the whole blood samples collected for each litter. K. Postmortem Evaluations 1. Females Surviving to Scheduled Euthanasia Gross external and a visceral examinations were performed immediately after euthanasia. The uterine horns from uteri with no visible implantation sites were placed in a 10% aqueous solution of ammonium sulfide(5) to detect very early resorptions. The uterine body was frozen for possible future evaluation. For each animal, blood was collected and prepared to plasma and serum. In addition, the weight of the liver and kidneys were recorded. For each female, kidneys, a portion of the liver, and one ovary were placed in formalin. The remainder of the liver, uterus, and the remaining ovary were flash frozen in liquid nitrogen and stored between -60ºC and -80ºC. These tissues were retained for possible future histopathologic examination. For each female with visible implantation sites, the types of implantations (live and dead fetuses, early and late resorptions) and their relative positions in the uterus were recorded. Live fetuses were decapitated to collect trunk blood which was pooled by litter and prepared to plasma. The types of implantations were classified as follows: live fetus: fully formed and responds to stimuli dead fetus: fully formed with little or no evidence of maceration late resorption: identifiable structures (i.e. digital rays) early resorption: no visible fetal structures CH-FW-DEQ-0008662 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 16 - L. Blood Collection and Plasma Analysis Whole blood was collected from rats via tail vein in Group 6 two hours (± 5 minutes) after dosing on GD 6. The actual time of blood collection was recorded in the study records. Whole blood from the animals in group 6 as well as from all other groups 1 through 5 was collected from the vena cava at sacrifice. Whole blood from fetuses was collected from the trunk following decapitation and pooled by litter. Whole blood samples were collected using EDTA or heparin as an anticoagulant and processed to plasma by centrifugation. The red blood cell fraction was discarded after separation. Plasma was stored frozen at  20ºC until delivery to the DuPont Haskell Analytical Chemistry Group. Samples were analyzed for the test substance using LC-MS-MS. The analytical method used can be found in Appendix K. M. Serum Preparation and Storage A minimum of 0.6 mL of whole blood was placed in a serum separator tube on ice until the serum was prepared. Serum was stored between -60ºC and -80ºC for possible future analysis. N. Ovary/Uterus Collection and Storage For each rat, one ovary and the uterus were collected and flash frozen in liquid nitrogen and stored between -60ºC and -80ºC for possible future analysis. STATISTICAL ANALYSES Descriptive statistics were performed on endpoints evaluated for this study. CH-FW-DEQ-0008663 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 17 - RESULTS AND DISCUSSION Formulation Analytical Evaluation A. Dosing Formulations Analysis (Appendix C) Data from the analysis of the formulation samples indicated that the test substance was at the targeted concentrations for the study. Test substance was not found in the 0 mg/mL samples (control). In-Life, Maternal Gross, and Caesarian Section Data Data collected during the in-life period of the study were collected for the purpose of monitoring the health of the animals and for calculating individual dose volumes. A. Maternal Mortality and Clinical Observations (Table 1, Appendix D) There was no test substance-related mortality at any level tested; all animals on study survived until scheduled euthanasia. There were no test substance-related clinical observations at any level tested; the observations that were recorded were unremarkable and occurred infrequently. B. Maternal Body Weights, Body Weight Changes, Food Consumption (Tables 2-4, Appendices E-G) Data for body weights, weight changes, and food consumption were collected for the purpose of monitoring the health of the animals and for calculating the daily dose volumes. Evaluation for potential dose-related effects evident in these endpoints in pregnant rats at the same dose levels has previously been conducted in the developmental toxicity study with the test substance (18405-841). Therefore, these data are provided in the current report primarily to confirm the health status of the animals. C. Maternal Gross Postmortem Findings (Table 5, Appendix H) All females appeared grossly normal at necropsy with the exception of a single adult female at 1000 mg/kg/day that had liver discoloration. CH-FW-DEQ-0008664 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 18 - D. Reproductive Outcome and Litter Data (Table 6, Appendix I) All females were pregnant with the exception of a single control group female. Data for litter sizes and incidences of resorptions were comparable across all groups tested. E. Maternal Liver and Kidney Weights (Table 7, Appendix J) Mean liver weights were 12 to 18% higher than controls at 100 and 1000 mg/kg/day. Otherwise, mean liver weights at the lower levels and mean kidney weights at all dose levels were generally comparable. Toxicokinetics Conclusions A. Plasma Analysis (Appendix K) The measured plasma concentrations for dams and fetuses are provided in Text Table 1: Text Table 1: Summary of Plasma Concentrations for Parent Compound H-28548 Concentration (ng/mL) Dams Pooled Pups Dose Day 6 Day 20 Day 20 Pup:Dam Group (mg/kg/day) Mean SD Mean SD Mean SD Plasma Ratio 1 0 33 16 19 23 2 5 3984 469 1134 175 0.3 3 10 9312 1710 2458 465 0.3 4 100 85560 10092 18320 9128 0.2 5 1000 338400 160168 99800 26482 0.3 6 1000 430600 162712 348400 130362 102240 28295 0.3 The dose response curve was linear between 5 and 100 mg/kg/day. At 1000 mg/kg/day the concentration was less than what would be predicted if the dose response curve was linear through 1000 mg/kg/day. The mean plasma concentration on day 6 was less than the mean plasma concentration on day 20. This implies that steady state was achieved by day 6 and that there is no accumulation in the dams between day 6 and day 20. CH-FW-DEQ-0008665 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 19 - The concentration in plasma pooled from pups was approximately one-third of the concentration in plasma from the dam at the same time point. RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, the protocol, amendments (if any), and the final report will be retained at DuPont Haskell, Newark, Delaware, Iron Mountain Records Management, Wilmington, Delaware, or Quality Associates Incorporated, Fulton, Maryland. REFERENCES 1. WIL Research Laboratories, LLC (in progress). An Oral (Gavage) Prenatal Developmental Toxicity Study of H-28548 in Rats. Unpublished report, DuPont-18405-841. 2. DuPont Haskell (in progress). H-28548: Follow-Up Developmental Toxicity Study in Rats. Unpublished report, DuPont-18405-840. 3. WIL Research Laboratories, LLC (in progress). An Oral (Gavage) Reproduction/Developmental Toxicity Screening Study of H-28548 in Mice. Unpublished report, DuPont-18405-1037. 4. WIL Research Laboratories, LLC (in progress). A 90-Day Oral (Gavage) Study of H-28548 in Rats with a 28-Day Recovery. Unpublished report, WIL-189216. 5. Salewski, E. (1964). Farbemethode zum makroskopischen Nachweis von Implantationstellen am Uterus der Ratte. Archiv. Path. Exp. Pharmakol. 247, 367. CH-FW-DEQ-0008666 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 20 - TABLES CH-FW-DEQ-0008667 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 21 - TABLES EXPLANATORY NOTES ABBREVIATIONS: -/---/./…/Blank space - no data/data could not be calculated kg - milogram mg - milligram N/n - number in group/number of values used in calculation of mean Ma - mating NOTES: Due to rounding differences, values in tables may be slightly different than appendices. CH-FW-DEQ-0008668 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 22 - Table 1 Summary of Maternal Clinical Observations -------------------------------------------------------------------------------------------------------------------------------------- Day numbers relative to Mating Date Sex: Female 0 5 10 100 1000 1000 mg/kg/day mg/kg/day mg/kg/day mg/kg/day mg/kg/day mg/kg/day --------------------------------------------------------------------------------------------------------------------------------- Scheduled sacrifice Number of Observations 5 5 5 5 5 5 Number of Animals 5 5 5 5 5 5 Days from - to 20 20 20 20 20 20 20 20 20 20 20 20 Hair loss Number of Observations 12 . . . . . Number of Animals 1 . . . . . Days from - to 9 20 . . . . . Mass Number of Observations . . 6 . . . Number of Animals . . 1 . . . Days from - to . . 15 20 . . . Wet fur Number of Observations . . . 1 7 . Number of Animals . . . 1 2 . Days from - to . . . 14 14 8 17 . CH-FW-DEQ-0008669 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 23 - Table 2 Mean Maternal Body Weights CH-FW-DEQ-0008670 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 24 - Table 2 Mean Maternal Body Weights (Continued) CH-FW-DEQ-0008671 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 25 - Table 2 Mean Maternal Body Weights (Continued) CH-FW-DEQ-0008672 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 26 - Table 3 Mean Maternal Body Weight Gains CH-FW-DEQ-0008673 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 27 - Table 3 Mean Maternal Body Weight Gains (Continued) CH-FW-DEQ-0008674 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 28 - Table 4 Mean Maternal Food Consumption CH-FW-DEQ-0008675 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 29 - Table 4 Mean Maternal Food Consumption (Continued) CH-FW-DEQ-0008676 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 30 - Table 5 Summary of Maternal Gross Observations -------------------------------------------------------------------------------------------------------------------------------------- 0 5 10 100 1000 1000 mg/kg/day mg/kg/day mg/kg/day mg/kg/day mg/kg/day mg/kg/day Number of Animals on Study : 5 5 5 5 5 5 Number of Animals Completed: (5) (5) (5) (5) (5) (5) -------------------------------------------------------------------------------------------------------------------------------------- WHOLE BODY; Submitted........................................................ (0) (0) (0) (0) (0) (0) No Visible Lesions............................................... 5 5 5 5 4 5 LIVER; Submitted........................................................ (0) (0) (0) (0) (0) (0) No Visible Lesions............................................... 0 0 0 0 0 0 Discoloration; tan; left ........................................ 0 0 0 0 1 0 CH-FW-DEQ-0008677 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 31 - Table 6 Reproductive Outcome --------------------------------------------------------------------------------------------------------------------------- 0 5 10 100 1000 mg/kg/day mg/kg/day mg/kg/day mg/kg/day mg/kg/day ------------------------------------------------------------------------------------------------------------------------- Number of females with live foetuses at scheduled kill 5 5 5 5 5 Number of implantations 57 64 63 64 66 Number of implantations per female Mean 11.4 12.8 12.6 12.8 13.1 Number of Intra-Uterine Deaths - Early Resorption 0 0 1 0 0 Number per female Including total resorptions Mean 0.0 0.0 0.2 0.0 0.0 Number per female Excluding total resorptions Mean 0.0 0.0 0.2 0.0 0.0 Post-implantation loss 0 0 1 0 0 Rate per female Including total resorptions Mean 0.00 0.00 0.02 0.00 0.00 Rate per female Excluding total resorptions Mean 0.00 0.00 0.02 0.00 0.00 % loss Including total resorptions 0.00 0.00 1.59 0.00 0.00 Excluding total resorptions 0.00 0.00 1.59 0.00 0.00 Number of live foetuses 57 64 62 64 66 Number per female Including total resorptions Mean 11.3 12.8 12.4 12.8 13.1 Number per female Excluding total resorptions Mean 11.4 12.8 12.4 12.8 13.1 % of implantations Including total resorptions 100.00 100.00 98.41 100.00 100.00 Excluding total resorptions 100.00 100.00 98.41 100.00 100.00 -------------------------------------------------------------------------------------------------------------------------------------- Statistical analysis not performed - Arithmetic mean values presented Calculated values do not include animals which either, were not pregnant, did not survive to the scheduled kill, aborted or are marked for exclusion CH-FW-DEQ-0008678 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 32 - Table 7 Mean Absolute and Relative Organ Weights in Female Rats CH-FW-DEQ-0008679 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 33 - APPENDICES CH-FW-DEQ-0008680 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 34 - APPENDICES EXPLANATORY NOTES ABBREVIATIONS: -/---/./…/Blank space - no data/data could not be calculated GD - gestation day kg - kilogram mg - milligram N/n - number in group/Number of values used in calculation of mean Wt - weight NOTES: Due to rounding differences, values in tables may be slightly different than appendices. CH-FW-DEQ-0008681 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 35 - Appendix A Certificate of Analysis CH-FW-DEQ-0008682 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 36 - CH-FW-DEQ-0008683 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 37 - Appendix B Protocol CH-FW-DEQ-0008684 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 38 - CH-FW-DEQ-0008685 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 39 - CH-FW-DEQ-0008686 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 40 - CH-FW-DEQ-0008687 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 41 - CH-FW-DEQ-0008688 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 42 - CH-FW-DEQ-0008689 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 43 - CH-FW-DEQ-0008690 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 44 - CH-FW-DEQ-0008691 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 45 - CH-FW-DEQ-0008692 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 46 - CH-FW-DEQ-0008693 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 47 - CH-FW-DEQ-0008694 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 48 - CH-FW-DEQ-0008695 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 49 - CH-FW-DEQ-0008696 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 50 - CH-FW-DEQ-0008697 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 51 - Appendix C Analysis for H-28548 in Dose Samples CH-FW-DEQ-0008698 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 52 - CH-FW-DEQ-0008699 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 53 - CH-FW-DEQ-0008700 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 54 - CH-FW-DEQ-0008701 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 55 - CH-FW-DEQ-0008702 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 56 - CH-FW-DEQ-0008703 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 57 - CH-FW-DEQ-0008704 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 58 - Appendix D Individual Maternal Clinical Observations CH-FW-DEQ-0008705 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 59 - INDIVIDUAL MATERNAL CLINICAL OBSERVATIONS EXPLANATORY NOTES ABBREVIATIONS: X - present NOTES: Clinical observations and fates are recorded in the following time slots: A - careful (with body weights or pre-treatment) and mode of death B - careful post-treatment CH-FW-DEQ-0008706 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 60 - Individual Maternal Clinical Observations ---------------------------------------------------------------------------------------------------------------------------------------------------------- Day numbers relative to Mating Date 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 Group Sex Animal Clinical Sign Site A B A B A B A B A B A B A B A B A B ---------------------------------------------------------------------------------------------------------------------------------------------------------- 1 f 101 No Abnormalities Detected X X X X X X . . . . . . . . . . . . Scheduled sacrifice . . . . . . . . . . . . . . . . . . Hair loss Forelimb bilateral . . . . . . . . . . . . . . X X X X Hair loss Inguen . . . . . . X X X X X X X X X X X X 102 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 103 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 104 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 105 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 2 f 201 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 202 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 203 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 204 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 205 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 3 f 301 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . Mass Abdomen . . . . . . . . . . . . . . . . . . Comment Present . . . . . . . . . . . . . . . . . . 302 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 303 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 304 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 305 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . ---------------------------------------------------------------------------------------------------------------------------------------------------------- Group 1 - 0 mg/kg/day Group 2 - 5 mg/kg/day Group 3 - 10 mg/kg/day Group 4 - 100 mg/kg/day Group 5 - 1000 mg/kg/day Group 6 - 1000 mg/kg/day CH-FW-DEQ-0008707 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 61 - Individual Maternal Clinical Observations ---------------------------------------------------------------------------------------------------------------------------------------------------------- Day numbers relative to Mating Date 15 15 16 16 17 17 18 18 19 19 20 Group Sex Animal Clinical Sign Site A B A B A B A B A B A ------------------------------------------------------------------------------------------------------------------------- 1 f 101 No Abnormalities Detected . . . . . . . . . . . Scheduled sacrifice . . . . . . . . . . X Hair loss Forelimb bilateral X X X X X X X X X X X Hair loss Inguen X X X X X X X X X X X 102 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 103 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 104 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 105 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 2 f 201 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 202 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 203 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 204 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 205 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 3 f 301 No Abnormalities Detected X . . . . . . . . . . Scheduled sacrifice . . . . . . . . . . X Mass Abdomen . X X X X X X X X X X Comment Present . * * * * * * * * * * 302 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 303 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 304 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 305 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X ---------------------------------------------------------------------------------------------------------------------------------------------------------- Group 1 - 0 mg/kg/day Group 2 - 5 mg/kg/day Group 3 - 10 mg/kg/day Group 4 - 100 mg/kg/day Group 5 - 1000 mg/kg/day Group 6 - 1000 mg/kg/day CH-FW-DEQ-0008708 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 62 - Individual Maternal Clinical Observations ---------------------------------------------------------------------------------------------------------------------------------------------------------- Day numbers relative to Mating Date 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 Group Sex Animal Clinical Sign Site A B A B A B A B A B A B A B A B A B ---------------------------------------------------------------------------------------------------------------------------------------------------------- 4 f 401 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 402 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 403 No Abnormalities Detected X X X X X X X X X X X X X X X X X . Scheduled sacrifice . . . . . . . . . . . . . . . . . . Wet fur Perineum . . . . . . . . . . . . . . . . . X 404 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 405 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 5 f 501 No Abnormalities Detected X X X X X . . . . X X X X X X . X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . Wet fur Perineum . . . . . X X X X . . . . . . X . . 502 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 503 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . Wet fur Perineum . . . . . . . . . . . . . . . . . . 504 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 505 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 6 f 601 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 602 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 603 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 604 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . 605 No Abnormalities Detected X X X X X X X X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . . . . . . . . . ---------------------------------------------------------------------------------------------------------------------------------------------------------- Group 1 - 0 mg/kg/day Group 2 - 5 mg/kg/day Group 3 - 10 mg/kg/day Group 4 - 100 mg/kg/day Group 5 - 1000 mg/kg/day Group 6 - 1000 mg/kg/day CH-FW-DEQ-0008709 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 63 - Individual Maternal Clinical Observations ---------------------------------------------------------------------------------------------------------------------------------------------------------- Day numbers relative to Mating Date 15 15 16 16 17 17 18 18 19 19 20 Group Sex Animal Clinical Sign Site A B A B A B A B A B A ------------------------------------------------------------------------------------------------------------------------- 4 f 401 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 402 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 403 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X Wet fur Perineum . . . . . . . . . . . 404 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 405 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 5 f 501 No Abnormalities Detected X X X . X X X X X X X Scheduled sacrifice . . . . . . . . . . X Wet fur Perineum . . . X . . . . . . . 502 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 503 No Abnormalities Detected X X X . . X X X X X X Scheduled sacrifice . . . . . . . . . . X Wet fur Perineum . . . X X . . . . . . 504 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 505 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 6 f 601 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 602 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 603 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 604 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X 605 No Abnormalities Detected X X X X X X X X X X X Scheduled sacrifice . . . . . . . . . . X ---------------------------------------------------------------------------------------------------------------------------------------------------------- Group 1 - 0 mg/kg/day Group 2 - 5 mg/kg/day Group 3 - 10 mg/kg/day Group 4 - 100 mg/kg/day Group 5 - 1000 mg/kg/day Group 6 - 1000 mg/kg/day CH-FW-DEQ-0008710 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 64 - Individual Maternal Clinical Observations ---------------------------------------------------------------------------------------------------------------------------------------------------------- Comments -------- Day Time Group Sex Animal Number Slot Comment ---------------------------------------------------------------------------------------------------------------------------------------------------------- 3 f 301 15 B firm, 5 mm dia. 16 A firm, 5 mm dia. B firm, 5 mm dia. 17 A firm, 5 mm dia. B firm, 5 mm dia. 18 A firm, 5 mm dia. B firm, 5 mm dia. 19 A firm, 5 mm dia. B firm, 5 mm dia. 20 A firm, 5 mm dia. CH-FW-DEQ-0008711 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 65 - Appendix E Individual Maternal Body Weights CH-FW-DEQ-0008712 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 66 - INDIVIDUAL MATERNAL BODY WEIGHTS EXPLANATORY NOTES ABBREVIATIONS: N - number of values used in calculation SD - standard deviation CH-FW-DEQ-0008713 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 67 - Individual Maternal Body Weights (g) CH-FW-DEQ-0008714 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 68 - Individual Maternal Body Weights (g) CH-FW-DEQ-0008715 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 69 - Individual Maternal Body Weights (g) CH-FW-DEQ-0008716 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 70 - Individual Maternal Body Weights (g) CH-FW-DEQ-0008717 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 71 - Individual Maternal Body Weights (g) CH-FW-DEQ-0008718 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 72 - Individual Maternal Body Weights (g) CH-FW-DEQ-0008719 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 73 - Individual Maternal Body Weights (g) CH-FW-DEQ-0008720 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 74 - Individual Maternal Body Weights (g) CH-FW-DEQ-0008721 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 75 - Individual Maternal Body Weights (g) CH-FW-DEQ-0008722 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 76 - Appendix F Individual Maternal Body Weight Gains CH-FW-DEQ-0008723 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 77 - INDIVIDUAL MATERNAL BODY WEIGHT GAINS EXPLANATORY NOTES ABBREVIATIONS: N - number of values used in calculation SD - standard deviation CH-FW-DEQ-0008724 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 78 - Individual Maternal Body Weight Gains (g) CH-FW-DEQ-0008725 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 79 - Individual Maternal Body Weight Gains (g) CH-FW-DEQ-0008726 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 80 - Individual Maternal Body Weight Gains (g) CH-FW-DEQ-0008727 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 81 - Individual Maternal Body Weight Gains (g) CH-FW-DEQ-0008728 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 82 - Individual Maternal Body Weight Gains (g) CH-FW-DEQ-0008729 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 83 - Individual Maternal Body Weight Gains (g) CH-FW-DEQ-0008730 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 84 - Appendix G Individual Maternal Food Consumption CH-FW-DEQ-0008731 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 85 - Individual Maternal Food Consumption (g/animal/day) CH-FW-DEQ-0008732 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 86 - Individual Maternal Food Consumption (g/animal/day) CH-FW-DEQ-0008733 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 87 - Appendix H Individual Maternal Gross Observations CH-FW-DEQ-0008734 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 88 - Individual Maternal Gross Observations -------------------------------------------------------------------------------------------------------------------------------------- Group: 1 Dose: 0 mg/kg/day Sex: Female Animal Death Ref. Mode Of Death Day (Week) Observation(s) ------- ------------------------------ ------------ ------------------------------------------------------------------------------- 101 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 102 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 103 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 104 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 105 SCHEDULED SACRIFICE 20 (2) No Visible Lesions -------------------------------------------------------------------------------------------------------------------------------------- Group: 2 Dose: 5 mg/kg/day Sex: Female Animal Death Ref. Mode Of Death Day (Week) Observation(s) ------- ------------------------------ ------------ ------------------------------------------------------------------------------- 201 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 202 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 203 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 204 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 205 SCHEDULED SACRIFICE 20 (2) No Visible Lesions CH-FW-DEQ-0008735 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 89 - Individual Maternal Gross Observations -------------------------------------------------------------------------------------------------------------------------------------- Group: 3 Dose: 10 mg/kg/day Sex: Female Animal Death Ref. Mode Of Death Day (Week) Observation(s) ------- ------------------------------ ------------ ------------------------------------------------------------------------------- 301 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 302 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 303 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 304 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 305 SCHEDULED SACRIFICE 20 (2) No Visible Lesions -------------------------------------------------------------------------------------------------------------------------------------- Group: 4 Dose: 100 mg/kg/day Sex: Female Animal Death Ref. Mode Of Death Day (Week) Observation(s) ------- ------------------------------ ------------ ------------------------------------------------------------------------------- 401 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 402 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 403 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 404 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 405 SCHEDULED SACRIFICE 20 (2) No Visible Lesions CH-FW-DEQ-0008736 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 90 - Individual Maternal Gross Observations -------------------------------------------------------------------------------------------------------------------------------------- Group: 5 Dose: 1000 mg/kg/day Sex: Female Animal Death Ref. Mode Of Death Day (Week) Observation(s) ------- ------------------------------ ------------ ------------------------------------------------------------------------------- 501 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 502 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 503 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 504 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 505 SCHEDULED SACRIFICE 20 (2) LIVER (2); left; Discoloration; tan; 0.5 cm or less >0.5 Any remaining protocol required tissues, which have been examined, have no visible lesions -------------------------------------------------------------------------------------------------------------------------------------- Group: 6 Dose: 1000 mg/kg/day Sex: Female Animal Death Ref. Mode Of Death Day (Week) Observation(s) ------- ------------------------------ ------------ ------------------------------------------------------------------------------- 601 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 602 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 603 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 604 SCHEDULED SACRIFICE 20 (2) No Visible Lesions 605 SCHEDULED SACRIFICE 20 (2) No Visible Lesions CH-FW-DEQ-0008737 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 91 - Appendix I Individual Reproductive Data CH-FW-DEQ-0008738 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 92 - INDIVIDUAL REPRODUCTIVE DATA EXPLANATORY NOTES ABBREVIATIONS: % / Mean - percentage or mean of the individual values for the group A - abortion E - excluded N - number of values used in calculation NP - not pregnant S.D. - standard deviation TLL - total litter loss U - unscheduled death NOTES: All A, E, NP, TLL, and U dams are excluded from all group summary calculations. A column showing late resorptions appears in this appendix only if at least one late resorption occurred in the study. Indices, which compare Implantations with Corpora Lutea, do not include animals, with an Implantation count > Corpora Lutea Count. Indices, which compare Foetuses with Implantations, do not include animals, with a Foetus count > Implantation Count. CH-FW-DEQ-0008739 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 93 - Individual Reproductive Data -------------------------------------------------------------------------------------------------------------------------------------- Group: 1 0 mg/kg/day - Number of Intra-Uterine Deaths - % Post- Live Foetuses Dam Number of Early Total implantation Number of as % of Number Implantations Resorption Deaths Loss live foetuses implantations Left Right L R L R Left Right ----------------------------------------------------------------------------------------------------------------------- 101 10 3 0 0 0 0 0.0 10 3 100.0 102 1 7 0 0 0 0 0.0 1 7 100.0 103 5 6 0 0 0 0 0.0 5 6 100.0 104 7 5 0 0 0 0 0.0 7 5 100.00 105 5 8 0 0 0 0 0.0 5 8 100.0 -------------- --------- --------- ------------ ------------- ------------- 21 24 0 0 0 0 21 24 TOTAL 45 0 0 45 Litter Mean 11.3 0.0 11.3 100.0 % / Mean 0.0 100.0 S.D. 2.4 2.4 N 4 4 4 4 Group: 2 5 mg/kg/day - Number of Intra-Uterine Deaths - % Post- Live Foetuses Dam Number of Early Total implantation Number of as % of Number Implantations Resorption Deaths Loss live foetuses implantations Left Right L R L R Left Right ----------------------------------------------------------------------------------------------------------------------- 201 7 4 0 0 0 0 0.0 7 4 100.0 202 9 6 0 0 0 0 0.0 9 6 100.0 203 6 7 0 0 0 0 0.0 6 7 100.0 204 7 7 0 0 0 0 0.0 7 7 100.0 205 3 8 0 0 0 0 0.0 3 8 100.0 -------------- --------- --------- ------------ ------------- ------------- 32 32 0 0 0 0 32 32 TOTAL 64 0 0 64 Litter Mean 12.8 0.0 12.8 100.0 % / Mean 0.0 100.0 S.D. 1.8 1.8 N 5 5 5 5 CH-FW-DEQ-0008740 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 94 - Individual Reproductive Data -------------------------------------------------------------------------------------------------------------------------------------- Group: 3 10 mg/kg/day - Number of Intra-Uterine Deaths - % Post- Live Foetuses Dam Number of Early Total implantation Number of as % of Number Implantations Resorption Deaths Loss live foetuses implantations Left Right L R L R Left Right ----------------------------------------------------------------------------------------------------------------------- 301 6 7 0 0 0 0 0.0 6 7 100.0 302 9 6 0 0 0 0 0.0 9 6 100.0 303 8 5 0 0 0 0 0.0 8 5 100.0 304 5 6 0 0 0 0 0.0 5 6 100.0 305 4 7 1 0 1 0 9.1 3 7 90.9 -------------- --------- --------- ------------ ------------- ------------- 32 31 1 0 1 0 31 31 TOTAL 63 1 1 62 Litter Mean 12.6 0.2 0.2 1.8 12.4 98.2 % / Mean 1.6 98.4 S.D. 1.7 0.4 0.4 1.9 N 5 5 5 5 Group: 4 100 mg/kg/day - Number of Intra-Uterine Deaths - % Post- Live Foetuses Dam Number of Early Total implantation Number of as % of Number Implantations Resorption Deaths Loss live foetuses implantations Left Right L R L R Left Right ----------------------------------------------------------------------------------------------------------------------- 401 7 5 0 0 0 0 0.0 7 5 100.0 402 5 8 0 0 0 0 0.0 5 8 100.0 403 6 5 0 0 0 0 0.0 6 5 100.0 404 5 8 0 0 0 0 0.0 5 8 100.0 405 6 9 0 0 0 0 0.0 6 9 100.0 -------------- --------- --------- ------------ ------------- ------------- 29 35 0 0 0 0 29 35 TOTAL 64 0 0 64 Litter Mean 12.8 0.0 12.8 100.0 % / Mean 0.0 100.0 S.D. 1.5 1.5 N 5 5 5 5 CH-FW-DEQ-0008741 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 95 - Individual Reproductive Data -------------------------------------------------------------------------------------------------------------------------------------- Group: 5 1000 mg/kg/day - Number of Intra-Uterine Deaths - % Post- Live Foetuses Dam Number of Early Total implantation Number of as % of Number Implantations Resorption Deaths Loss live foetuses implantations Left Right L R L R Left Right ----------------------------------------------------------------------------------------------------------------------- 501 8 5 0 0 0 0 0.0 8 5 100.0 502 5 7 0 0 0 0 0.0 5 7 100.0 503 9 6 0 0 0 0 0.0 9 6 100.0 504 4 8 0 0 0 0 0.0 4 8 100.0 505 6 7 0 0 0 0 0.0 6 7 100.0 -------------- --------- --------- ------------ ------------- ------------- 32 33 0 0 0 0 32 33 TOTAL 65 0 0 65 Litter Mean 13.0 0.0 13.0 100.0 % / Mean 0.0 100.0 S.D. 1.2 1.2 N 5 5 5 5 Group: 6 1000 mg/kg/day - Number of Intra-Uterine Deaths - % Post- Live Foetuses Dam Number of Early Total implantation Number of as % of Number Implantations Resorption Deaths Loss live foetuses implantations Left Right L R L R Left Right ----------------------------------------------------------------------------------------------------------------------- 601 5 8 0 0 0 0 0.0 5 8 100.0 602 6 5 0 0 0 0 0.0 6 5 100.0 603 3 13 0 0 0 0 0.0 3 13 100.0 604 5 8 0 0 0 0 0.0 5 8 100.0 605 5 8 0 0 0 0 0.0 5 8 100.0 -------------- --------- --------- ------------ ------------- ------------- 24 42 0 0 0 0 24 42 TOTAL 66 0 0 66 Litter Mean 13.2 0.0 13.2 100.0 % / Mean 0.0 100.0 S.D. 1.8 1.8 N 5 5 5 5 CH-FW-DEQ-0008742 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 96 - Appendix J Individual Animal Organ Weight Data CH-FW-DEQ-0008743 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 97 - INDIVIDUAL ANIMAL ORGAN WEIGHTS KEY TO APPENDIX CALCULATED MEASUREMENT FORMULAE: Organ/Body Weight = Organ Weight (g)/Body Weight (g) x 100 ABBREVIATIONS: g - gram NOTES: Due to rounding differences, values in tables may be slightly different than appendices. CH-FW-DEQ-0008744 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 98 - Individual Animal Organ Weight Data CH-FW-DEQ-0008745 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 99 - Individual Animal Organ Weight Data CH-FW-DEQ-0008746 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 100 - Appendix K Quantitation of Plasma Samples for H-28548: Toxicokinetic Study in Pregnant Rats CH-FW-DEQ-0008747 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 101 - CH-FW-DEQ-0008748 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 102 - CH-FW-DEQ-0008749 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 103 - CH-FW-DEQ-0008750 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 104 - CH-FW-DEQ-0008751 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 105 - CH-FW-DEQ-0008752 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 106 - CH-FW-DEQ-0008753 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 107 - CH-FW-DEQ-0008754 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 108 - CH-FW-DEQ-0008755 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 109 - CH-FW-DEQ-0008756 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 110 - CH-FW-DEQ-0008757 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 111 - CH-FW-DEQ-0008758 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 112 - CH-FW-DEQ-0008759 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 113 - CH-FW-DEQ-0008760 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 114 - CH-FW-DEQ-0008761 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 115 - CH-FW-DEQ-0008762 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 116 - CH-FW-DEQ-0008763 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 117 - CH-FW-DEQ-0008764 H-28548: Toxicokinetic Study in Pregnant Rats DuPont-18405-849 - 118 - CH-FW-DEQ-0008765