Document Qk9Qra1GL8Xqk34DJ11Edxxy7

_ 14fe D u P on t-13309 Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rats Work Request Number 14787 Service Code 1569 Protocol Haskell Animal W elfare Committee Number: <> -1- RECEIVED 0PPTNC1C 2803SEP -* AH 8*1* 5 Perfluorooctanoic Acid: Lactational and Placental Transport Phannacokinetic Study in Rats D u P on t-13309 TABLE OF CONTENTS Page INTRODUCTION...............................................................................................................................3 OBJECTIVE........................................................................................................................................ 3 SPONSOR AND TEST FACILITY............. ....................................................................................3 REGULATORY COMPLIANCE.....................................................................................................3 STUDY DESIGN................................................................................................................................4 A. Dose Group Subsets and Dosing Schedules................................................ 4 B. Selection o f Dose Levels......................................................................................... ;...........4 C. Gestation Subset Sacrifice Schedule And Sample Collection....... ......... 5 D. Lactation Subset Sample Collection And Sacrifice Schedule...............................................5 MATERIALS AND METHODS........... ........................................................................................... 6 A. Test Substance.................. 6 B. V ehicle....................................................................... 6 C. Test Substance Administration, Preparation, and Sampling.................................................6 D. A nim als............................................................................................... 7 E. Animal Husbandry......................................................................................................... 8 F. Random ization....................................................................................................................... 9 G. Adult Female R ats.,........................................ 9 H. Gestation Procedures...................... 10 I. Lactation Procedures........................................... ^.10 SAMPLE ANALYSES........................................................ .......................................................... 11 STATISTICAL ANALYSES............................................................... .......................................... 11 SAFETY PRECAUTIONS AND DISPOSAL OF WASTE MATERIAL............. ..................... 11 RECORDS AND SAMPLE STORAGE........................................................................................ 11 STUDY PERSONNEL.............................. .................................................................. .................. 11 STUDY DATES................................................................................................................................ 12 REFERENCES.................................................................................................................................. 12 SIGN A TU RES.................................................................................................................................. 13 -2 - Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rate D u P on t-13309 INTRODUCTION The test substance, Perfluorooctanoic acid, is a surfactant in industrial processes. In rat metabolism studies with PFOA, whole body elimination o f the compound was found to be much more rapid in females compared to males following oral dosing, however, available data on the transport concentrations o f the test material when administered to pregnant females is limited in scope and there is no existing data on the concentration o f the test material transported via the milk from lactating fem ales/1'2'1 OBJECTIVE The objective o f this study is to evaluate the concentrations of PFOA in maternal m ilk and blood plasma, placental, embryonic, fetal, and neonatal tissue(s) following repeated oral dosing o f the dam at three concentrations. SPONSOR AND TEST FACILITY This study is co-sponsored by 3M, St. Paul, Minnesota, and E.I. du Pont de Nemours and Company, Wilmington, Delaware. The sponsor's approval was effective the date the sponsor authorized the work on the contract. The study will be conducted at Haskell Laboratory for Health and Environmental Sciences, E.I. du Pont de Nemours and Company, Newark, Delaware. REGULATORY COMPLIANCE This study will be conducted in compliance with all applicable Good Laboratory Practice Standards, which are consistent with the OECD Principles of Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 and MAFF Japan Good Laboratory Practice Standards (59 NohSan Number 3850.(3,4,5) -3 - Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rats D uPont-13309 STUDY DESIGN The study design is as follows: Dose Suspension Concentration Time-Mated Group (mg/kg/day)8 (mg/mL)b Females I 0 0 20 II 3 0.6 20 m 10 2.0 20 IV 30 6.0 20 a Formulations o f test substance in deionized water w ill be administered once daily by oral gavage at a dosing volume o f 3 mL/kg. b To achieve these concentrations o f active ingredient, the formulations w ill be adjusted for sample purity (%). c The control group animals w ill receive vehicle deionized water only at 5 mL/kg. A. Dose Group Subsets and Dosing Schedules Fem ale Subset A Dose Days Group 4 - 10G" I5 n5 in 5 IV 5 a Gestation b Postpartum Fem ale Subset B Dose Days 4-15G 5 5 5 5 Fem ale Subset C Dose Days 4-21G 5 5 5 5 Fem ale Subset D D ose Days 4G -21P P b 5 5 5 5 The first 5 animals in each group will be designated as Subset A, the second 5 as Subset B, the third 5 as subset C, and the remaining 5 as Subset D. B. Selection o f Dose Levels Dose levels selected for this study were based upon the results o f a two-generation reproduction study in rats.(6) Groups o f 30 male and 30 female rats were dosed by oral gavage at 0 ,1 ,3 ,1 0 , or 30 mg/kg/day for approximately 70 days before cohabitation. Dosing o f the P0 females continued throughout mating, gestation, and lactation or approximately 112 days total. W ith the exception o f significantly reduced kidney weight and kidney weight to terminal body weight and to brain weight ratios, no test substance related maternal toxicity was observed at doses up to 30 mg/kg/day. Offspring mortality, attributed to failure to thrive, was observed in Fi generation weanlings at 30 mg/kg/day. -4 - Perfluorooctanoic Acid: Lactational and Placental Transport Phannacokinetic Study in Rats D u P on t-13309 C. Gestation Subset Sacrifice Schedule And Sample Collection Subset A B C Terminal Sacrifice 10G after dosing 15G after dosing 21G after dosing Samples Collected Maternal Blood, Amniotic Fluid, Placentas, Whole Embryos Maternal Blood, Amniotic Fluid, Placentas, Whole Embryos Maternal Blood, Amniotic Fluid, Placentas, Whole fetuses, Fetal Blood Maternal blood sample volume 0.5 mL per animal collected in tubes containing heparin Amniotic Fluid sample minimum volume approximately 100 pL collected in glass tubes Fetal blood sample minimum volume approximately 100 pL collected in tubes containing heparin All embryos and fetuses collected in cryotox tubes D. Lactation Subset Sample Collection And Sacrifice Schedule Subset D Number of Maternal Females/Litters Samples Number o f Pups for Terminal Sacrifice Pup Samples Day 3pp after dosing Day 7pp after dosing Day 14pp after dosing Day 21pp-after dosingb 5/group 5/group 5/group 5/group Milk, Blood Milk, Blood Milk, Blood Milk, Blood Minimum o f 1 per litter8 Minimum o f 1 per litter Minimum of 1 per litter Minimum o f 1 per litter Blood Blood Blood Blood a At the discretion o f the study director, the number o f pups sacrificed may be increased to ensure sufficient sample volum es for analysis, b Maternal Terminal Sacrifice Maternal milk sample minimum volume approximately 100 pL collected in glass tubes Maternal blood sample volume 0.5 mL per animal collected in tubes containing heparin Fetal blood sample minimum volume approximately 100 pL collected in tubes containing heparin -5 - Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rats MATERIALS AND METHODS D u P on t-13309 A. Test Substance 1. Identification The test substance will be obtained from 3M, St. Paul, Minnesota, and was assigned Haskell Laboratory Number H-24921 upon receipt Available information on the purity, composition, contaminants, synonyms, hazards, and hazardous material classification(s) will be provided by the vendor and documented in the study records and report. 2. Purity The vendor reported purity was 95.2 %. 3. Test Substance Stability The stability o f the test substance over the course o f the study will be confirmed by purity analyses conducted near the beginning and end of the study. B. Vehicle The vehicle will be Nano-Pure water. C. Test Substance Administration, Preparation, and Sampling 1. Administration The test substance will be administered by oral gavage once daily at a dose volume o f 5 mL/kg. Animals assigned to gestation subsets A, B, and C, will be dosed for 7,12, and 18 days respectively. Animals assigned to the lactation subset (D) will be dosed for approximately 47 days. Females in the process o f delivery or showing signs o f delivery will not be dosed. Dose volumes will be adjusted daily based on body weights. 2. Preparation Solutions o f the test substance in the vehicle will be prepared daily and stored at room temperature until used. The method o f mixing the test substance with the vehicle will be documented in the study records. 3. Sampling Samples of each test solution (approximately 3 mL) will be taken 3 times during the study. If necessary, additional samples may be taken at the discretion of the study director or designee. -6 - Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rats D aP on t-13309 Analyses will address concentration, homogeneity, and stability. Samples will be submitted shortly after preparation to the Analytical Group at Haskell. * Analysis o f the first sampling will be taken from dose preparation near the beginning o f study and will address uniformity o f the mixture, concentration, and 5-hour room temperature stability. Three independent samples from each test solution will be collected. One sample o f the vehicle and 2 samples from each test suspension will be analyzed immediately after submission. The third sample will be held for 5 hours at room temperature, then analyzed for stability. For the second and third samplings taken from dose preparation near the middle and end o f the study, analysis will address concentration. Samples o f the vehicle and 2 samples from each concentration o f the solutions will be analyzed immediately following submission to verify concentration. If samples cannot be analyzed at the specified times, they will be frozen until analyses can be conducted. On days samples are taken, the solutions remaining after dosing will be stored in the refrigerator for additional analysis. The analytical method used will be documented in the Analytical study records. D. Animals 1. Species (Strain) Crl:CD(SD)IGS BR rats 2. Reason for Selection The rat was selected for this study as it is a preferred species for reproductive toxicity testing as recommended by the guidelines. The Crl:CD(SD)IGS BR strain was selected based on consistently acceptable health status and on extensive experience with this strain at Haskell Laboratory. 3. Description Eighty nulliparous, time-mated females, will be received on July 8,2003 (60 females) arid July 11,2003 (20 females) from Charles River Laboratories, Iric., Raleigh, North Carolina. The location o f the supplier (city/state) will be documented in the study records and final report. The rats for this study were requested to be 63 days old and be at 1 day o f gestation (day 0G) upon arrival. This age corresponds to an estimated weight range o f201-225 grams according to the vendor; however, the weight range is not a factor for the purposes o f this study and, therefore, deviations outside o f this range are expected not to have any impact on the study. Body weights -7 - Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rats D u P on t-13309 on the day the rats are mated, day OG, will be supplied by the vendor. The day OG body weights will be documented in the study records and provided in the final report. Each rat is assigned a unique number and identified with an AVID Microchip implant by the supplier prior to shipping. Upon receipt, each animal will be assigned a Haskell animal number. Both the Haskell animal number and unique vendor animal number will be recorded on each cage card. A master list o f unique vendor numbers, Haskell animal numbers, and corresponding unique AVID Microchip implant numbers will be maintained with the study records. E. Animal Husbandry 1. Environmental Conditions Animal rooms will be maintained at a temperature o f 18-26C (targeted to 22-24C) and a relative humidity o f 30-70% (targeted to 40-60%). Animal rooms will be artificially illuminated (fluorescent light) on an approximate 12 hour light/dark cycle. Unless judged by the study director or the laboratory veterinarian to have significantly affected the results o f the study, the relative humidity and temperature ranges in the housing rooms will be recorded but will not be included in the final report. 2. Housing All rats will be housed individually in stainless steel, wire-mesh cages, suspended above cageboard until sacrifice or day 19G. Females selected for the lactation subset (D) will be housed in polycarbonate pans containing bedding material on day 20G through day 21pp. 3. Food All rats will be provided tap water and pelleted PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum. 4. Water Water from United W ater Delaware will be available ad libitum. 5. Animal Health Monitoring As specified in the DuPont 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 file scientific integrity o f the study. W ater 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. 8- Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rats D u P on t-13309 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 o f key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence o f these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity o f the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Data are maintained separately from study records and may be included in the final report at the discretion of the study director. 6. Quarantine Rats will be quarantined according to procedures outlined in Haskell Laboratory SOP LA003-P, and then released for the study upon approval o f the Laboratory Animal Veterinarian or a designee. Rats that die or are sacrificed in extremis during the quarantine period will be necropsied to check for the presence o f disease. Dependent upon these findings, further diagnostic procedures m aybe employed at the discretion o f the study director, the pathologist assigned to the study, or the laboratory veterinarian. F. Randomization Upon arrival, dams will be assigned to lots according to their gestation day. Then, they w ill be ranked within their respective lots on the basis o f day OGbody weights and assigned to control and experimental groups by random sampling from the ranked list. The distribution should result in mean body weights for all groups that are not statistically different (p > 0.05). Dams that lose excessive weight or appear ill prior to die start o f dosing will be removed from the study. G. Adult Female Rats 1. In-life Observations o f Females a. Body Weight Body weights w ill be recorded on the day after arrival and daily until the end o f the study. 2. Clinical Observations Clinical observations will be recorded on the day after arrival and daily until the end o f the study. -9 - Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rate D u P on t-13309 H. Gestation Procedures On day 10,15, and 21G, animals assigned to Subset A, B, and C, respectively, will be euthanized by carbon dioxide inhalation. For each female with visible implantation sites, the intrauterine location o f each embryo (Day 10 and 15 G) or fetus, and implantation type (live or early resorption) will be recorded. A gross examination o f maternal viscera will be done and lesions noted will be retained for further examination at the discretion o f the study director. Blood samples from each dam will be collected from the vena cava approximately 2 hours (30 minutes) post-dose and processed to obtain plasma samples. Amniotic fluid from each implantation site, and all placentas and embryos/fetuses will be collected. On day 21G, fetuses will be euthanized with an overdose injection (ip) o f sodium pentobarbital and fetal blood samples will be taken from a transverse cut made to the carotid artery. Females that die or are sacrificed in extremis during the gestation or period will be necropsied and a gross postmortem examination performed. Lesions will be retained at the discretion o f the study director, the pathologist assigned to the study, or the laboratory veterinarian. Dam carcasses w ill be discarded. I. Lactation Procedures The day when delivery is complete is designated day 0 postpartum. At each examination period (days 0,3 ,7 ,1 4 , and 21 postpartum), offspring will be individually handled and examined for abnormal behavior and appearance; any dead, missing, or abnormal pups will be recorded. Live and dead pups in each litter will be counted by sex as soon as possible after delivery is completed and litter weights will be recorded. Dead pups will be discarded. On days 3,7, and 14PP, animals assigned to Subset D, will be anesthetized by Isoflorane inhalation. During administration o f anesthesia, milk and blood samples (orbital sinus) will be collected. Blood samples will be collected approximately 2 hours ( 30 minutes) post-dose. Dams will be removed from their litters approximately 1-2 hours before the milking procedure begins. Day 3 litters will be kept warm during the absence o f the dam by the use o f either a circulating hot water pad, heated gel packs, or heat lamp. Dams will be returned to their litters after recovery from anesthesia. Randomly selected pups from these dams will be euthanized on the same postpartum days with an overdose injection (ip) of sodium pentobarbital. Pup blood samples will be taken from a transverse cut made to the carotid artery on days 3 and 7 or from the vena cava on day 14PP. Maternal and pup blood samples will be processed to obtain plasma samples and pups will be discarded. On day 21PP, animals will be anesthetized by Isoflorane inhalation. During administration o f anesthesia, m ilk samples will be collected. After milk collection but before recovery, dams will be euthanized by exsanguination. Blood samples will be taken from the vena cava approximately 2 hours (30 minutes) post-dose and the dam will be discarded. Randomly selected pups from these dams will be euthanized with an overdose injection (ip) o f sodium pentobarbital. Pup -10- Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rats D u P on t-13309 blood samples will be taken from the vena cava. Maternal and pup blood samples will be processed to obtain plasma samples and dams and pups will be discarded. SAMPLE ANALYSES Samples will be analyzed for test material concentration by the Haskell Toxicology group. Samples not requiring processing will be stored at approximately -15C as soon as possible after collection and until analysis, otherwise, samples will be stored under the same conditions until processing and analysis. HPLC-MS or another suitable analytical method will be used to analyze samples. The method used will be documented in the study records. STATISTICAL ANALYSES Group means and concentration data will be represented as Mean SD. Other statistical evaluations may be performed if necessary. SAFETY PRECAUTIONS AND DISPOSAL OF WASTE MATERIAL Good housekeeping procedures will be practiced to avoid potential health hazards and contamination o f dosing solution preparation facilities. To avoid skin contact, gloves will be worn when handling either the test substance or dosing solutions. In addition, the test substance will be handled in a chemical hood. Dosing solutions will be prepared in properly ventilated areas. Animal carcasses, feces, and unused dosing solutions and diet will be incinerated. RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. STUDY PERSONNELimmwi] Study Director: SD SDTitle Analytical Evaluation: <> o PP -11 - Perfluorooctanoic Acid: Lactational and Placental Transport Phannacokmetic Study in Rats PPTitle D u P on t-13309 STUDY DATES Initiation o f Test Substance Administration July 11,2003 In-Life Completion Date: August 22,2003 (Approximate) REFERENCES 1. Vanden Heuvel, J.P., Kuslikis, B.I., Van Rafelghem, M.J. and Peterson, Re. E. (1991). Tissue Distribution, Metabolism, and Elimination o f Perfluorooctanoic Acid in Male and Female Rats. J. Biochem. Toxicol 6920, 83-92. 2. DuPont Haskell Laboratory (2003). Perfluorooctanoic Acid: Toxicokinetics in the Rat. Unpublished report, DuPont-7473. 3. EPA/FIFRA Good Laboratory Practice Standards (40 CFR 160). (1989). 4. OECD Principles o f Good Laboratory Practice (as revised in 1997, Published in EN V /M C /CH EM (98)17). 5. MAFF Japan Good Laboratory Practice Standards (59 NohSan Number 3850). 6. Argus Laboratories (2002). Oral (gavage) 2-generation (o new-litter per generation) reproduction study o f ammonium perfluorooctanoic (APFO) in rats. Unpublished report. - 12- Perfluorooctanoic Acid: Lactational and Placental Transport Pharmacokinetic Study in Rats SIGNATURES Approved by: Eve Mylchreest Management sd Study Director cc: <Q.A. Contact> D.M. Hoban <P.&S. Contacts> K. McHhatton <Primary Technidan> S.C. Craven J.W. Green D u P on t-13309 D ate D ate - 13-