Document KGDG3xzmEEmdde1BJk5GvZYvo

AR226-2812 TRADE SECRET Study Title Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat AUTHOR: Paul M. Hinderliter, Ph.D. STUDY COMPLETED ON: November 5,2003 PERFORMING LABORATORY: E.I. du Font de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 LABORATORY PROJECT ID: DuPont-12944 WORK REQUEST NUMBER: UBP OT^^^^n SERVICE CODE NUMBER: I^^^^^J1R^ SPONSOR: Association of Plastics Manufacturers of Europe (APME) Fluoropolymers Committee Avenue E. Van Nieuwenhuyse 4 Box 3, B-1160 Brussels Belgium Page 1 of 56 Company Sanitized. Does not contain TSCA CBt 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 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) except for the item documented below. The item listed does not impact the validity of the study. The test substance is commercially available and was characterized by the supplier prior to the initiation of this study. A certificate of analysis was provided. Although the characterization 'was not performed under Good Laboratory Practice Standards, based on our monitoring, we have no reason to doubt the validity of the analysis. Ph.D.Date f^ly^^^^L^^^________ Study Director: ' PsfolM. Hinderliter, Research Toxicologist oS-Hof Zoo3 E.L du Pont de Nemours and Company -2- Company Sanitized. Does not contain TSCA CBI Al) QUALITY ASSURANCE STATEMENT HaskeH Sample Number(s): 25811 Dates of Inspections: i Protocol: June 10,2003 Conduct: June 13,17,20, 2003 Records, Reports: September 16-18, 25, 28, 2003 Dates Findings Reported to: Study Director: September 29, 2003 Management: September 29, 2003; October 6, 2003 Reported by: ^T^ ^j> cJoseph Z^ 7C. //-. C . H a i m i r D a t e Staff Quality Assurance Auditor 0$^j/^w3 -3- Company Sanitized. Does not contain TSCA CBI CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Inhalation Data Evaluation by: -/- 7-T/ ^ U . f ,ff U t U /? '' f / F\ /) C^/^^Lg l^Cchael P. DeLLoorri&nc,.PPhh.D. Research Toxicologist ^o^ro-^ Date Approved by: Gary W. Jcpson, Ph.D. Principal Research Toxicologist and Manager <?^ - A/i?(/-2<?<5r Date Approved by: ^^^^^(^^l '^^W^t^S' Matfliew S. BogdAf^P, h-D.jfo/^.'D' ~ Research Manager and Director A3-/Vw-^->a^ Date .. Issued by Study Director: PaulM.H5ndertiter.PlLD. Research Toxicologist <D^-A/o^^ZjCW3 Date -4- Company Sanitized. Does not contain TSCA CBI TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT.................................. 2 QUALITY ASSURANCE STATEMENT...................................................................................3 CERTIFICATION ........................................................................................................................4 LIST OF TABLES........................................................................................................................ 6 LIST OF FIGURES ......................................................................................................................7 LIST OF APPENDICES............................................................................................................... 7 STUDY INFORMATION.............................................................................................................8 STUDY PERSONNEL..................................................................................................................9 10 SUMMARY.................................................................................................................................. INTRODUCTION....................................................................................................................... 11 MATERIALS AND METHODS ............................................................................................... 11 A. Test Substance................................................................................................................... 11 4||| ^^ B. Test System .......................................................................................................................11 C. Animal Husbandry............................................................................................................ 12 1., Animal Housing.......................................................................................................... 12 2. Environmental Conditions........................................................................................... 12 3. Feed and Water............................................................................................................ 12 4. Animal Health and Environmental Monitoring Program............................................ 12 D. Inhalation Exposure System.............................................................................................. 13 1. Atmosphere Generation.............................................................................................. 13 2. Chamber Construction and Design............................................................................. 13 3. Exposure Mode........................................................................................................... 13 E. Characterization of Chamber Atmosphere........................................................................ 14 1. Test Substance Sampling and Analysis.....................................................;................. 14 2. Preliminary Air Sampling Studies .............................................................................. 14 3. Particle Size Determination......................................................................................... 14 4. Environmental Monitoring.......................................................................................... 14 F. Inhalation Exposure........................................................................................................... 15 1. Phase I (Single Exposure)........................................................................................... 15 2. Phase n (Repeated Exposure)..................................................................................... 15 G. Analytical Methods........................................................................................................... 15 1. Extraction of PFOA from Plasma for LC/MS Analysis.............................................. 15 2. 16 Instrumentation............................................................................................................ 3_ _._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Chromatographic Methods.......................................................................................... 17 -5Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat __ _________DuPont-12944 H. Statistical Analysis............................................................................................................ 17 RESULTS AND DISCUSSION.................................................................................................. 17 A. Phase I (Single Exposure)................................................................................................. 17 1. Chamber Concentrations of Test Substance................................................................ 17 2. Chamber Environmental Conditions.......................................................................... 18 3. Blood Collection......................................................................................................... 18 4. 18 Pharmacoldnetics........................................................................................................ B. Phase n (Repeated Exposure)........................................................................................... 19 1. Chamber Concentrations of Test Substance................................................................ 19 2. Chamber Environmental Conditions.......................................................................... 19 3. Blood Collection......................................................................................................... 19 4. 19 Pharmacoldnetics........................................................................................................ CONCLUSIONS.........................................................................................................................20 RECORDS AND SAMPLE STORAGE................................................................................... 20 REFERENCES............................................................................................................................ 20 TABLES....................................................................................................................................... 21 FIGURES .....................................................................................................................................31 APPENDICES.......................................................................................................................... 42 LIST OF TABLES Page 1. PHASE I (SINGLE EXPOSURE) CHAMBER CONCENTRATIONS OF PFOA............................................23 2. PHASE II (REPEATED EXPOSURE) CHAMBER CONCENTRATIONS OF PFOA......................................23 3. PHASE n (SINGLE EXPOSURE) PARTICLE SIZE DISTRIBUTION OF PFOA ...........................................24 4. PHASE I (SINGLE EXPOSURE) CHAMBER ENVIRONMENTAL CONDITIONS......................................^ 5. PHASE n (REPEATED EXPOSURE) CHAMBER ENVIRONMENTAL CONDITIONS ...............................26 6. PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION DATA IN MALE RATS ...........................27 7. PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION DATA IN FEMALE RATS.......................28 8. PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION DATA IN MALE RATS ....................29 9. PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION DATA IN FEMALE RATS ...............30 -6- Company Sanitized. Does not contain TSCA CBI LIST OF FIGURES Page 1. SCHEMATIC OF EXPOSURE SYSTEM..................................................-.........................-..........................32 2. PHASE n (SINGLE EXPOSURE) MEAN DAILY CHAMBER CONCENTRATIONS ...................................33 3. PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION ON A LINEAR SCALE ............................34 4. PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION ON A LOGARITHMIC SCALE ...............35 5. PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION IN MALE RATS WITH ' STANDARD DEVIATION BARS......................................................................................................................36 6. PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION IN FEMALE RATS WITH STANDARD DEVIATION BARS...................................................................................................................... 37 7. PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION IN MALE RATS ................................38 8. PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION IN MALE RATS WITH STANDARD DEVIATION BARS...................................................................................................................... 39 9. PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION IN FEMALE RATS ...........................40 10. PHASE II (REPEATED EXPOSURE) PLASMA CONCENTRATION IN FEMALE RATS WITH STANDARD DEVIATION BARS......................................................................................................................41 LIST OF APPENDICES Page A. CERTIFICATE OF ANALYSIS..........................................................................................................................44 B. PHASE n (REPEATED EXPOSURE) DAILY CHAMBER CONCENTRATIONS .........................................46 C. PHASE n (REPEATED EXPOSURE) DAILY CHAMBER ENVIRONMENTAL CONDITIONS ..................48 D. PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION DATA ........................................................52 E. PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION DATA.................................................54 -7- Company Sanitized. Does not contain TSCA CBI STUDY INFORMATION 9th Collective Nomenclature: Synonyms/Codes: Haskell Number: 25811 CAS Registry Number: Purity:1 Physical Characteristics: White powder Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Sponsor: Association of Plastics Manufacturers of Europe (APME) Fluoropolymers Committee Avenue E. Van Nieuwenhuyse 4 Box 3, B-1160 Brussels Belgium Study Initiated/Completed: April 22,2003 / (see report cover page) -8- Company Sanitized. Does not contain TSCA CBI ,-' STUDY PERSONNEL Study Director: Primary Technicians: Management: Paul M. Hinderliter, Ph.D. Shawn A. Gannon, B.S. Brian P. Shertz, A.A. Matthew S. Bogdanffy, Ph.D., D.A.B.T. Gary W. Jepson, Ph.D. Inhalation lexicologist: Michael P. DeLorme, Ph.D. Management: Arthur J. 0'Neill, B.S. Toxicology Report Preparation: Management: Lisa G. Burchfield, A.A. Maryanne M. Wilford, B.A. Nancy S. Selzer, M.S. Laboratory Veterinarian: Thomas W. Mayer, D.V.M., Diplomate A.C.L.A.M. Management: Janice L. Connell, M.S., B.A., C.I.H -9- Company Sanitized. Does not contain TSCA CBI SUMMARY A large pharmacokinetic database exists describing the behavior ofperfluorooctanoic acid (PFOA) following oral exposure. In rat studies, whole body elimination ofPFOA was found to be much more rapid in female rats compared with male rats following oral dosing. The objective of this study was to quantify plasma PFOA concentrations in rats, following single and repeated inhalation exposures at three airborne concentrations, for the purpose of bridging the oral and inhalation exposure data sets. The study was comprised of two separate experiments: Phase I consisted of a single 6-hour nose-only exposure while Phase n consisted of repeated exposures (6 hours per day, 5 days per week for 3 weeks), hi both experiments, male and female rats were exposed nose only to an aerosol ofO, 1, 10, or 25 mg/m3PFOA. Levels were selected to produce plasma concentrations similar to those observed in previous oral gavage studies. In Phase I, blood was drawn via the tail vein pre-exposure, during exposure at 0.5,1, 3 and 6 hours, and post-exposure at 1, 3, 6,12, 18 and 24 hours. Rats were not removed from the exposure chamber during the exposure period and blood was taken from the tail vein while the animals remained in the nose-only restrainers attached to the exposure chamber. For Phase n, blood was collected immediately before and after the daily inhalation exposure period three days per week. Plasma derived from the whole blood samples was analyzed by liquid chromatography-mass spectrometry (LC-MS). PFOA appears rapidly in the blood of both male and female rats exposed via the inhalation route. PFOA elimination is sex-dependent, with female rats eliminating PFOA from the plasma much more efficiently than male rats. PFOA plasma concentrations are proportional to the inhalation exposure concentrations from 1 to 25 mg/m3. Repeated daily inhalation exposures produce little plasma carryover in female rats, but significant carryover in male rats. Male rats reach a steady state plasma concentration by three weeks with plasma concentrations of 8, 21, and 36 ftg/mL respectively for the 1,10, and 25 mg/m3 exposures. Female rats reached post-exposure plasma concentrations of 1,2, and 4 /Ag/mL respectively for the 1,10, and 25 mg/m3 exposures, but returned to baseline levels for the pre-exposure time points. -10- Company Sanitized. Does not contain TSCA CBI INTRODUCTION A large pharmacokinetic database exists describing the behavior ofperfluorooctanoic acid (PFOA) following oral exposure. In rat studies, whole body elimination ofPFOA was found to be much more rapid in female rats compared v/ith male rats following oral dosing. The objective of this study was to quantify plasma PFOA concentrations in rats, following single and repeated inhalation exposures at three airborne concentrations, for the purpose of bridging the oral and inhalation exposure data sets. MATERIALS AND METHODS A. Test Substance ------fFOA was obtained from Aldrich Chemicals (Milwaukee, Wisconsin) and assigned Haskell Laboratory Number 25811 upon receipt. Available information on the purity, composition, and contaminants were provided by the vendor and documented in the study records and report. Since this study utilized the inhalation route of exposure, no attempt was made to establish the actual dose each rat received. All results were therefore reported as a function of the group exposure concentrations) rather than a function of dose. Molecular Weight: Molecular Formula: Structure: 414.1 Purity: B. Test System Male and female Crl:CD(SD)IGSBR rats were obtained from Charles River Laboratories, me., Raleigh, North Carolina. The Sprague-Dawley rat was chosen for tms study because of the extensive experience with this strain and its suitability with respect to longevity, sensitivity, and low incidence of spontaneous diseases. Furthermore, the Sprague-Dawley rat has been used previously for toxicokinetic testing of PFOA and other fluorinated test materials.0 '^ Upon arrival at Haskell Laboratory, all rats were housed singly, sexes separate, in quarantine. The rats were: quarantined for at least 5 days. -11- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat___________________DuPont-12944 identified temporarily by cage identification. weighed twice during the quarantine procedure. observed with respect to weight gain and any gross signs of disease or injury. The rats were released from quarantine by the laboratory animal veterinarian or designee on the basis of body weights and clinical signs. At the time of exposure, rats were approximately 6-8 weeks of age and the weight variation did not exceed 20% of the mean weight by exposure group. Each animal was assigned an identification number that was used throughout the study. The last 3 digits of the animal identification number was marked on the tail of each animal in indelible ink. C. Animal Husbandry 1. Animal Housing Except during exposure, rats were housed singly in stainless steel, wire-mesh cages suspended above cage boards. Each cage rack contained only rats of one sex. 2. Environmental Conditions Rats were housed in proximity to the inhalation chambers. Animal room(s) were maintained at a temperature of 18-26C (targeted to 22-24C) and a relative humidity of 30-70% (targeted to 40-60%). Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle, except on blood collection days where lights were turned on as needed up to one hour extra per day. The relative humidity and temperature ranges in the housing rooms were recorded but were not included in this report. 3. Feed and Water Except during exposure, PMI Nutrition International, LLC Certified Rodent LabDiet 5002 and tap water was available ad libitum. 4. Animal Health and Environmental 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 ofcoliforms, lead, and other contaminants. Feed samples are analyzed for total bacterial, spore, and fungal counts. -12- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat___________________DuPont-12944 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. Inhalation Exposure System (Figure 1) 1. Atmosphere Generation For this study, a 5% (weight/weight) solution was prepared by diluting the powdered test substance in deionized water. Concentrated ammonium hydroxide solution was added to achieve a test substance solution pH of approximately 6.0 to 8.0. Chamber atmospheres were generated by aerosolization of the 5% test substance solution in air with a Spraying Systems nebulizer. The test substance was metered into the nebulizer with a Harvard Apparatus model 22 syringe infusion pump. Filtered, high-pressure air, metered into the nebulizer by a Brooks model 5850E mass flow controller, carried the resulting atmosphere into the exposure chamber. The pump and flow controller were controlled and monitored by the Camile Inhalation Toxicology Automated Data System (CITADS). Chamber concentrations of test substance were controlled by varying the syringe infusion rate to the nebulizer except for the 1 mg/m3 chamber where dilutional air was also used to achieve the desired atmosphere concentration. See Figure 1 for a schematic of the inhalation exposure setup. Test atmospheres were exhausted through an MSA charcoal/HEPA filter cartridge prior to discharge into the fume hood. Air from the control chamber was exhausted directly into the fume hood. 2. Chamber Construction and Design All exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 150 L. A dispersion plate inside the chamber promoted uniform chamber distribution of the test atmosphere. 3. Exposure Mode During exposure, animals were individually restrained in polycarbonate (Phase I) or perforated stainless steel cylinders (Phase II) with conical nose pieces. The restrainers were inserted into a polymethylmethacrylate faceplate which was attached to the exposure chamber so that the nose of each animal extended into the exposure chamber. Before the start of the exposure phase, 13Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat DuPont-12944 animals were placed in restrainers on 2 separate days for approximately 15 minutes each day to acclimate the animals to the restrainers. During exposure, the animals from all exposure levels were kept warm with a heat lamp placed behind the animals (see Figure 1). E. Characterization of Chamber Atmosphere 1. Test Substance Sampling and Analysis The atmospheric concentration of PFOA was determined by gravimetric analysis at approximately 60-minute intervals during each exposure. Known volumes of chamber atmosphere were drawn from the sampling port through a 25 mm filter cassette containing a preweighed Gelman glass fiber (Type A/E) filter. The filters were weighed on a Cahn model C-31 Microbalance. The filter weights were automatically transferred to CITADS, which calculated the chamber concentrations based on the difference in pre- and post-sampling filter weights divided by the volume of chamber atmosphere sampled. Gravimetric sample start- and stoptimes for each sample were controlled and recorded by CITADS. 2. Preliminary Air Sampling Studies For this study, the test substance, PFOA, was diluted to 5% (w/w) in deionized water and subsequently aerosolized into the exposure chambers. In order to determine if the gravimetric analytical method used to quantitate the airborne test substance concentration indicated the mass ofjust the test substance or test substance plus water, a preliminary desiccation study was || performed. In this study, air samples were taken from chamber atmospheres that ranged from r 1 to 25 mg/m3 test substance as described in Materials and Methods Section El. Following the determination of the filter weight immediately post-sampling, the filters were placed in a desiccation chamber for 24 hours to remove any water that was on the filter. After desiccation, the filters were re-weighed, the mass recorded and compared to the values obtained immediately following air sampling. Results demonstrated that the filters lost 0.03% of their mass following 24 hours of desiccation, indicating that the material collected on the filter reflected the total mass of suspended aerosol in the exposure chambers. 3. Particle Size Determination A sample to determine particle size distribution (mass median aerodynamic diameter and percent particles less than 1,3, and 10 p,m diameter) was taken from each test exposure chamber at least once per week during the Phase II study with a Sierra Series 210 cyclone preseparator/Cascade impactor and Sierra series 110 constant flow air sampler.^ 4. Environmental Monitoring Chamber airflow was set at the beginning of each exposure to achieve at least 10 air changes per hour and was monitored continually with a calibrated Brooks model 5850E mass flow controller. Chamber temperature was targeted at 22 3C. Chamber relative humidity was targeted at 40 to 60%. Airflow, temperature, and relative humidity were monitored continuously with a CAMILE Inhalation Toxicology Automated Data System (CITADS) and recorded at approximately 15- minute intervals during each exposure. Chamber oxygen concentration was targeted to at least -14- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat___________________DuPont-12944 19%. Chamber oxygen concentration was measured with a Biosystems model 3100R oxygen analyzer and was recorded twice during each exposure F. Inhalation Exposure 1. Phase I (Single Exposure) To determine the proper exposure concentrations for Phase n, four groups of 3 male and 3 female rats each were exposed nose only for a single, 6-hour period to an aerosol of the test substance. The test groups were exposed to an atmospheric concentration of 1 mg/m , 10 mg/m3, or 25 mg/m3. A control group of rats was exposed to air only. Blood was collected via the tail vein and was drawn pre-exposure, during exposure at 0.5,1, 3 and 6 hours, and post-exposure at 1,3,6, 12, 18 and 24 hours. Individual animal exposure times were staggered by five minutes to allow time for blood collection. Rats were not removed from the exposure chamber during the exposure time. Blood was taken from the tail vein while the animals remained in the nose-only restrainers in the exposure chamber. Blood was collected in heparinized tubes and stored on wet ice. Plasma was separated by centrifugation. Rats were sacrificed following the final blood collection. Plasma derived from the whole blood samples was analyzed by LC-MS as described in Section H. The exposure concentrations selected for Phase II were levels that produced plasma concentrations similar to plasma concentrations observed in a previous study of oral gavage dosing in rats.^ 2. Phase II (Repeated Exposure) After the dose levels were selected, four groups of 5 male and 5 female rats each were exposed (nose only) for a 6-hour period, 5 days per week (weekends excluded), for 3 weeks to an aerosol of the test substance. The starting time of the exposure is defined as the time when the nose only restrainers containing the animals are loaded into the exposure chamber. The exposure end time is defined as the time when the nose only restrainers are removed from the chamber. After each exposure, rats were returned to their cages. The control group of rats was exposed to air only. Exposure start times were staggered by 10-30 minutes by exposure concentration group to allow time for blood collection. As in Phase I, blood was collected via the tail vein. Blood was collected before and after the daily inhalation exposure period three days per week. Rats were sacrificed following the final blood collection. G. Analytical Methods 1. Extraction of PFOA from Plasma for LC/MS Analysis Plasma samples were processed by protein precipitation (PPT) using Isolute Array protein precipitationcolumns (Argonaut Technologies, Foster City, CA); A 0.5 /Ag/ml solution of fIBHUUIfr-^rich Chemicals, Milwaukee, WI) in ACN was used as an internal standard. Plasma samples were thawed, and a 20 ^L aliquot of each sample was applied to the PPT array. The plasma samples were precipitated by adding appropriate dilution rate volumes of -15- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid:- -Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat DuPoni-12944 ACN/mtemal standard solution. Dilution rates, ranging from 1:4 (60 pL of internal standard solution) to 1:50 (980 /zL of internal standard solution), were utilized in order to capture the sample concentrations within the range of the standard curve concentrations. The array was slowly eluted under vacuum into a 96-well receiver plate, centrifuged at ~3000 rpm for 5 minutes, and the extracts were analyzed by LC/MS. 2. Instrumentation System: Detector: Mode: Source: LM 1 resolution: HM 1 resolution Ion energy 1: Entrance: Collision: Exit: LM 2 resolution: HM 2 resolution: Ion energy 2: Multiplier (V): Capillary (kV): Cone(V): Extractor (V): RF lens (V): Source temperature: Desolvation temperature: MS Method: Mode: Time: Chl: Ch2: Limit ofQuantitation: Waters 2790 Liquid Chromatograph, equipped heater, and autosampler Quattro Micro Mass Spectrometer Multiple Reaction Monitoring (MRM) Negative Electrospray with quaternary pump, column 10.0 10.0 1.0 5 2 .5 14 14 2.0 650 2.0 15 0 0 130C 350C MRM, 2 transitions O-lLOmin 413.00-> 369.00 Dwell: Collision energy: 469.00-> 419.00 Dwell: Collision energy: 0.1/ig/mL-PhaseI 0.05 ^g/mL - Phase II 0.25 sec 15 eV 0.25 sec 15 eV -16- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat 3. Chromatographic Methods Method: Column: Column temperature: Mobile phases: Plasma concentration analysis Waters Xten-a MS CIS, 2.1x30mm, 2.5 lim Ambient A: 50 mM Ammonium Acetate B: Acetonitrile Gradient: Time (min) %B 0.0 10 0.5 10 10.0 100 10.9 100 11.0 10 Flow rate: Stop time: Injection volume: 0.25 mL/nun ll.Onun 5/iL DuPont-12944 n H. Statistical Analysis Parameter Exposure Concentration Data Environmental Data Plasma Concentration Preliminary Test None Method of Statistical Analysis If preliminary test is not If preliminary test is significant significant Descriptive statistics (e.g., mean, standard deviation) RESULTS AND DISCUSSION A. Phase I (Single Exposure) (Tables 1, 3-4, and 6-7, Figures 3-6, Appendix D) 1. Chamber Concentrations of Test Substance The mean chamber atmosphere concentrations for the three levels were all within 20% of the targeted concentrations (Table 1). Due to the logistics of performing venipunctures on the animals during the exposure, aerosol size analysis could not be performed during Phase I. However, aerosol size data obtained during the preliminary rangefinding work demonstrated aerosol sizes were 1.8-2.0 ^m mass median aerodynamic diameter (MMAD) with geometric standard deviations that ranged from 1.9-2.1 (im for atmosphere concentrations that were similar to those used in the Phase I study. Aerosols were also shown to be respirable under identical generation conditions used in the Phase II studies (Table 3). Therefore, the test substance aerosols were considered to be respirable in Phase I. -17- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat________ _________DuPont-12944 2. Chamber Environmental Conditions The mean temperature in all the exposure chambers ranged from 24-25C except for the control chamber, where the mean temperature was 26 C, which is outside of the targeted range of 22 3C (Table 4). This temperature was out of range due to heat lamps that were utilized to keep the animals warm to facilitate the tail venipuncture procedure during the exposures. This temperature deviation (control only) is not expected to adversely affect the results of this study. The mean relative humidity in all the exposure chambers during the single 6 hour exposure ranged from 34-40%, which was within the targeted range of 50 20%. The airflow into the chambers ranged from 27-70 L/min, which was adequate to achieve at least 10 air changes per hour. The mean oxygen concentration in all the exposure chambers was 21% which was within the targeted oxygen concentration of>19%. Overall, the environmental conditions in the Phase I study were within acceptable comfort ranges for the animals and was considered adequate for the conduct of the study. 3. Blood Collection Almost all blood collections occurred within five minutes of the described sample time; the maximum difference was seven minutes which occurred on only two samples. This difference was not judged to impact the quality of the data since the animals were not removed from the exposure chamber during blood sampling. 4. Pharmacokinetics For both sexes, PFOA plasma concentrations rose during the 6 hour inhalation exposure (Tables 6-7). A proportional relationship was produced in both the male and female rat maximum plasma concentrations (Cmax) between 1 and 25 mg/m3 (Figure 3). The male Cmax values were approximately 2-3 times higher than the female Cmax. The female Cmax occurred at the end of the exposure period or 1 hour post exposure while the male Cmax occurred at the end of the exposure period up to 6 hours post exposure. Elimination of PFOA from female rat plasma was rapid and complete at all exposure levels in Phase I. Plasma concentrations dropped below the analytical limit of quantification (0.1 /Ag/mL) by 12 hours post exposure (Figure 4). Plasma elimination was much slower in male rats than female rats. After 24 hours of post exposure recovery, the plasma concentrations were approximately 90% of the peak concentrations at all tested exposure levels. The male and female elimination kinetics are consistent with the previously reported elimination kinetics of PFOA following oral dosing.^ In addition, there did not appear to be a concentration dependence of the elimination rate in either male.or female rats as the plasma curves are parallel on a log scale. Figures 5 and 6 also show the Phase I plasma concentrations with standard deviation bars indicating the significant differences between exposure groups. For the setting of exposure levels for Phase II of the study, male and female Cmax values were compared to plasma Cmax from an oral gavage study of PFOA/'0 This study examined single oral gavage doses from 0.1 to 25 mg PFOA/kg body weight. The Cmax values for this study ranged from 0.598 to 160.00 pg/mL (male) and 0.67 to 132.65 (female). Because the Cmax 18Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat___________________DuPont-12944 values from Phase I fell within the middle of the existing range and oral gavage data, the inhalation exposure concentrations were not changed for Phase II. B. Phase II (Repeated Exposure) (Tables 2-3, 5, and 8-9, Figures 2 and 7-10, Appendices B-C and E) 1. Chamber Concentrations of Test Substance The mean chamber atmosphere concentrations for the three levels were all within 10% of the targeted concentrations (Table 2, Figure 2). Aerosol size analysis of all three exposure levels indicated an aerosol size distribution of 1.3-1.9 ^m MMAD with geometric standard deviations of 1.5-2.1, which is considered to be respirable to rats (Table 3). Therefore, these exposure methods were considered acceptable for the purpose of this study. 2. Chamber Environmental Conditions The mean temperature in all the exposure chambers ranged from 24-25C, which was within the targeted range of 22 3 C (Table 5). The mean relative humidity in all the exposure chambers during the 3 week exposure period ranged from 33 -39%, which was within the targeted range of 50 20%. The airflow into the chambers ranged from 28-70 L/min, which was sufficient to achieve at least 10 air changes per hour. The mean oxygen concentration in all the exposure chambers was 21% which was within the targeted oxygen concentration of>19%. Overall, the environmental conditions in the Phase 11 study were within acceptable comfort ranges for the animals and considered adequate for the conduct of the study. 3. Blood Collection No clinical signs were noted among rats during Phase II. Blood collections occurred within thirty minutes of the described sample time, samples were taken before and after the 6 hour exposure periods. Blood was collected on the first, third, and fifth exposure day for all three weeks. 4. Pharmacokinetics Phase II plasma concentrations are shown in Tables 8-9 and in Figures 5-8. Exposures were conducted five days per week, with plasma samples taken before and after exposure on the first, third and fifth exposure day per week. The male (Figure 7) and female (Figure 9) concentration curves demonstrate the effects of the differing elimination kinetics on plasma levels following repeated exposure. In the female rats, there was little day-to-day carryover of PFOA in the plasma. There is no significant difference between the peak heights of the sampled exposure days. For the male rats, there is carryover between test days as expected from Phase I, Steady state was not achieved until week 3. This steady state value is approximately 2-3 times as high as the mean single dose Cmax. Figures 8 and 10 show the mean plasma concentrations with standard deviation bars to demonstrate the differences by exposure level. As in Phase I, repeated exposures demonstrate a proportional relationship between exposure concentration and plasma concentrations in male and female rats. Male rats reach a steady state plasma concentration by -19- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat___________________DuPont-12944 three weeks with plasma concentrations of 8, 21, and 36 ^ig/mL respectively for the 1,10, and 25 mg/m3 exposures. Female rats reached post-exposure plasma concentrations of 1,2, and 4 fJLg/mLrespectively for the 1,10, and 25 mg/m3exposures, but returned to baseline levels for the pre-exposure time points. CONCLUSIONS PFOA appears rapidly in the blood of both male and female rats exposed via the inhalation route. PFOA elimination is sex-dependent, with female rats eliminating PFOA from the plasma much more efficiently than male rats. At the tested concentrations from 1 to 25 mg/m3, PFOA plasma concentrations relate proportionally to the inhalation exposure concentration. Repeated daily inhalation exposures produce little plasma carryover in female rats, but significant carryover in male rats. Male rats reach a steady state plasma concentration by three weeks with plasma concentrations of 8, 21, and 36 /^g/rnL respectively for the 1,10, and 25 mg/m3 exposures. Female rats reached post-exposure plasma concentrations of 1, 2, and 4 /^g/rnL respectively for the 1,10, and 25 mg/m3 exposures, but returned to baseline levels for the pre-exposure time points. 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. REFERENCES 1. Vanden Heuvel, J.P., Kuslikis, B.I., Van Rafelghem, M.J. and Peterson, R.E. (1991) Tissue Distribution, Metabolism, and Elimination of Perfluorooctanoic Acid in Male and Female Rats. J. Biochem. Toxicol. 6(2): 83-92. 2. Biegel,L.B. (1997) Hazard Characterization for Human Health C8 Exposure. DuPont Haskell Laboratory. 3. Vanden Heuvel, J.P., Davis, J.W., Sommers, R., and Peterson, R.E. (1992). Renal Excretion of Perfluorooctanoic Acid in Male Rats: Inhibitory Effect of Testosterone. J Biochem Toxicology, 7(1): 31-36. 4. DuPont Haskell Laboratory (2003). Perfluorooctanoic Acid: Toxicokinetics in the Rat. 5. Calculation described in Sierra Instruments, Inc., Bulletin 7-79-219IM, Instruction Manual: Series 210 Ambient Cascade mipactors and Cyclone Preseparators. -20- Company Sanitized. Does not contain TSCA CBI TABLES -21 Company Sanitized. Does not contain TSCA CBI TABLES ABBREVIATIONS: EXPLANATORY NOTES n S.D. S.E.M. NOTES: number of samples standard deviation standard error of the mean Tables 1-5: Values are reported to 2 significant figures. Calculations were performed prior to rounding values. -22- Company Sanitized. Does not contain TSCA CBI TABLE 1 PHASE I (SINGLE EXPOSURE) CHAMBER CONCENTRATIONS OF PFOA Design Measured Concentration Concentration ____________(mg/m3)___________ (mg1 /m3)_________M1e.2an____S0..3D8.______0R.a66n-g1e.7_,____n6_ 10 9.8 0.58 9.0-11 6 25 27 3.1 24-32 6 a Values represent the mean, standard deviation (S.D.), and range of the values obtained from 1 exposure. TABLE 2 PHASE II (REPEATED EXPOSURE) CHAMBER CONCENTRATIONS OF PFOA Design Concentration (mg/m3) 1 10 25 Measured Concentration _____(mg/m3)_____ M1e.1an_____S.0E.0.M4 ._______R0.8a1n-g1e.3______n1_5 10 0.11 9.4-11 15 25 0.43 21-27 15 a Values represent the mean, standard error of the mean (S.E.M.), and range of the daily mean values obtained from n exposures. .23- Company Sanitized. Does not contain TSCA CBI TABLES PHASE n (SINGLE EXPOSURE) PARTICLE SIZE DISTRIBUTION OF PFOA Design Concentration (mg/m3) 1 10 25 Exposure 2 4 7 12 3 8 13 5 9 14 Mass Median Aerodynamic Diameter (fim) 1.7 1.7 1.8 1.3 1.3 1.5 1.8 1.7 1.8 1.9 Geometric Standard Deviation 1.6 1.6 1.6 1.9 1.9 2.1 1.5 2.0 1.8 1.5 <1 fim 12 13 14 33 30 29 10 24 15 6 % Particle by Mass <3 inm 90 <10 fim 100 90 100 85 100 87 100 92 100 84 100 84 100 80 100 85 100 84 100 -24- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat TABLE 4 PHASE I (SINGLE EXPOSURE) CHAMBER ENVIRONMENTAL CONDITIONS Design Concentration Temperature ro8 Relative Humidity w Airflow (L/min)" (mg/m3) Mean S.D. Range n Mean S.D. Range n Mean S.D. Range 0 26 0.62 24-27 27 37 1.1 36-40 27 28 0.025 28 1 25 1.2 22-27 28 34 4.5 31-48 16 70 0.050 70 10 25 1.5 22-26 30 40 1.7 38-45 30 28 0.040 28 25 24 0.93 21 - 25 32 34 2.5 30-38 32 27 0.60 26-28 a Values represent the mean, standard deviation (S.D.), and range of the values obtained from 1 exposure. -25- luorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat t) TABLE 5 PHASE H (REPEATED EXPOSURE) CHAMBER ENVIRONMENTAL CONDITIONS Design Concentration (mg/m3) 0 1 10 25 Temperature w Mean 25 25 25 24 S.E.M. 0.070 0.16 0.090 0.080 Range 25 - 26 24-26 24 - 26 23 - 24 Relative Humidity (%)8 Mean 39 35 38 33 S.E.M. 0.39 1.6 0.57 0.58 Range 36 - 42 27-47 33-41 29 - 37 Mean 28 70 28 28 Airflow (L/min)8 S.E.M. R 0.066 0.014 0.031 0.090 27 a Values represent the mean, standard error of the mean (S.E.M.), and range of the daily mean values obtained from -26- Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat TABLE 6 PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION DATA IN MALE RATS Ch amber Concentration (m)!/m3) 0 1 10 # # # Hours Mean S.D. >LOQ Mean S.D. >LOQ Mean S.D. >LOQ M Exposure Predose <LOQ * 0 <LOQ * 0 <LOQ * 0 0 0.5 <LOQ * 0 <LOQ * 0 <LOQ * 0 1 <LOQ * 0 <LOQ * 0 0.98 * 2 2 3 <LOQ * 0 1.05 0.32 3 3.04 0.53 3 5 6 <LOQ * 0 2.41 0.91 3 5.85 1.09 3 1 Post-Exposure 1 <LOQ * 0 3 <LOQ * 0 6 <LOQ * 0 12 <LOQ * 0 18 <LOQ * 0 24 <LOQ * 0 \ 2.16 0.42 3 2.37 * 2 1.81 0.92 3 2.25 1.13 3 2.03 0.77 3 2.09 0.57 3 Data expressed as /ig/mL. *Standard deviation not calculated for 2 or fewer samples >LOQ. 7.39 1.29 3 1 7.89 0.97 3 1 8.57 * 2 1 7.43 1.32 3 1 6.97 1.19 3 6.63 1.33 3 1 -27- Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat TABLE? PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION DATA IN FEMALE RATS Ch amber Conce ntration (mg /m3) 0 1 10 # # # Hours Mean S.D. >LOQ Mean S.D. >LOQ Mean S.D. >LOQ M Exposure Predose <LOQ * 0 <LOQ * 0 <LOQ * 0 < 0.5 <LOQ * 0 <LOQ * 0 0.43 * 2 1 <LOQ * 0 <LOQ * 0 1.2 0.18 3 3 <LOQ * 0 0.83 * 2 2.62 * 2 6 <LOQ * 0 0.88 0.14 3 3.68 0.53 3 Post-Exposure 1 <LOQ * 0 0.75 0.14 3 3.69 0.66 3 3 <LOQ * 0 0.67 0.21 3 2.54 * 1 6 <LOQ * 0 <LOQ * 0 0.54 0.07 3 12 <LOQ * 0 <LOQ * 0 <LOQ * 0 < 18 <LOQ * 0 <LOQ * 0 <LOQ * 0 < 24 <LOQ * 0 <LOQ * 0 <LOQ * 0 < i, Data expressed as ^g/mL. *Standard deviation not calculated for 2 or fewer samples >LOQ. -28- TABLES PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION DATA IN MALE RATS 0 # Hours Mean S.D. >LOQ 0 <LOQ a 0 6 <LOQ a 0 48 <LOQ a 0 54 <LOQ a 0 96 <LOQ a 0 102 <LOQ a 0 168 0.83 a 1 174 0.49 a 1 216 <LOQ a 0 222 <LOQ a 0 264 <LOQ a 0 270 <LOQ a 0 336 <LOQ a 0 342 <LOQ a 0 384 <LOQ a 0 390 <LOQ a 0 432 <LOQ a 0 438 <LOQ a 0 Steady State" 0 Mean Chambier Con centration (rog/m3) 1 10 # # S.D. > LOQ Mean S.D. > LOQ 0.58 a 1 1.35 0.51 5 2.20 0.86 4 4.38 11.62 5 4.65 11.33 5 6.18 11.58 5 4.90 1.36 5 6.29 1.64 5 6.58 11.40 5 7.32 11.90 5 7.58 11.81 5 8.15 2.14 5 6.82 2.06 5 7.65 1.78 5 7.35 1.48 5 8.11 1.84 5 8.33 1.84 5 9.25 2.07 5 <LOQ a 0 3.69 1.32 5 7.39 2.78 5 11.22 5.10 5 13.73 6.19 5 15.81 6.24 5 13.97 5.23 5 16.70 5.55 5 18.81 6.31 5 19.50 6.42 5 20.33 7.73 5 21.73 8.17 5 . 20.86 7.76 5 19.43 6.85 5 20.16 7.18 5 21.48 7.85 5 21.87 6.89 5 24.13 6.90 5 8 21 Data expressed as fig/mL. 'Standard deviation not calculated for 2 or fewer samples >LOQ. ""Steadystate values calculated as the mean of 336-438 hour samples. 25 # Mean S.D. >LOQ <LOQ a 0 7.73 3.08 5 17.68 5.25 5 29.26 7.04 4 30.04 5.65 5 40.33 9.97 5 30.10 5.26 5 35.06 6.83 5 32.09 11.10 5 35.67 6.54 5 36.36 10.42 5 39.58 11.63 5 30.88 10.29 5 32.64 10.88 5 31.89 9.19 5 34.79 10.89 5 39.58 17.78 5 46.32 23.74 5 36 -29- Company Sanitized. Does not contain TSCA CBI TABLE 9 PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION DATA IN FEMALE RATS "Chamber Concentration (mg/m3) 10 25 Hours Mean S.D. >LOQ 0' 1.71 a 1 6 <LOQ a 0 48 <LOQ a 0 54 <LOQ a 0 96 0.74 a 1 102 <LOQ a 0 168 <LOQ a 0 174 <LOQ a 0 216 <LOQ a 0 222 <LOQ a 0 264 <LOQ a 0 270 <LOQ a 0 336 <LOQ a 0 342 <LOQ a 0 384 <LOQ a 0 390 <LOQ a 0 432 <LOQ a 0 438 <LOQ a 0 Steady State0 0 Mean SJ). >LOQ <LOQ a 0 0.86 0.41 4 <LOQ a 0 0.72 0.20 5 <LOQ a 0 0.84 0.11 5 1.18 a 1 0.77 0.28 5 <LOQ a 0 0.73 0.22 5 <LOQ a 0 0.60 0.17 5 <LOQ a 0 0.75 0.15 5 <LOQ a 0 0.66 0.14 5 <LOQ a 0 0.81 0.17 5 1 Mean SJ). >LOQ <LOQ a 0 2.12 1.07 5 0.33 a 1 2.46 0.68 5 0.27 a 2 1.51 0.99 3 <LOQ a 0 2.14 1.18 4 <LOQ a 0 2.03 0.24 4 <LOQ a 0 2.41 0.85 4 <LOQ a 0 1.01 a 2 <LOQ a 0 1.73 0.61 4 <LOQ a 0 2.02 1.29 4 2 Mean S.D. >LOQ <LOQ a 0 2.97 1.00 5 0.62 0.27 4 3.53 1.75 5 0.45 0.20 4 4.18 2.38 5 <LOQ a 0 3.30 2.28 5 0.38 a 2 5.71 1.34 5 0.38 0.21 3 3.95 2.78 5 <LOQ a 0 3.45 1.37 5 0.37 0.13 3 4.15 4.32 5 0.41 a 2 6.47 3.94 5 4 Data expressed as fig/mL. 'Standard deviation not calculated for 2 or fewer samples >LOQ. ""Steadsytate values calculated as the mean of all post-exposure samples. -30- Company Sanitized. Does not contain TSCA CBI FIGURES -31- Company Sanitized. Does not contain TSCA CBI Houseline Air FIGURE 1: SCHEMATIC OF EXPOSURE SYSTEM Spraying Systems Nebulizer n ^ ^ Baffle Dilution Air (Low Level Only) ------ ^ Syringe Drive (Low, Intermediate & High only) Nose-Only ^ Restrainers Exposure Chamber Heat Lamp exhaust -32- Company Sanitized. Does not contain TSCA CBI FIGURE 2: PHASE n (SINGLE EXPOSURE) MEAN DAILY CHAMBER CONCENTRATIONS -33- Company Sanitized. Does not contain TSCA CBI FIGURES: PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION ON A LINEAR SCALE end of exposure -34- Company Sanitized. Does not contain TSCA CBI FIGURE 4: PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION ON A LOGARITHMIC SCALE end of exposure is Study Hours 0 -25 lug/in" male ----25 mg/m* female -0- -10 mg/m'mate ----10 ing/m* female --A--1 mg/m'male --A--1 ins/in* female M-'Oingftn'male -0 ing/in' female .35- Company Sanitized. Does not contain TSCA CBI FIGURE 5: PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION IN MALE RATS WITH STANDARD DEVIATION BARS 20 |10 e ."'a-' y^- ^ 15 Study Hours - 0 -25 ing/in' male I - 0- lOmg/m'male A--1iiigftn'nial9 <t -0 mginf male -36- Company Sanitized. Does not contain TSCA CBI FIGURE 6: PHASE I (SINGLE EXPOSURE) PLASMA CONCENTRATION IN FEMALE RATS WITH STANDARD DEVIATION BARS 15 Study Houre -25 nig/Hi" female "lOiiiu/ni* femslfi ~1 fflflrtu* female l-K-Oingftii'female -37- Comp^ny Sanitized. Does not contain TSCA CBI FIGURE 7: PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION IN MALE RATS 200 250 Study Hours -38- Company Sanitized. Does not contain TSCA CBI FIGURE 8: PHASE H (REPEATED EXPOSURE) PLASMA CONCENTRATION IN MALE RATS WITH STANDARD DEVIATION BARS -39- Company Sanitized. Does not contain TSCA CBI FIGURE 9: PHASE n (REPEATED EXPOSURE) PLASMA CONCENTRATION IN FEMALE RATS 6 200 250 Study Hours -40- Company Sanitized. Does not contain TSCA CBI FIGURE 10: PHASE H (REPEATED EXPOSURE) PLASMA CONCENTRATION IN FEMALE RATS WITH STANDARD DEVIATION BARS 200 250 Study Hours -41- Company Sanitized. Does not contain TSCA CBI APPENDICES -42- Company Sanitized. Does not contain TSCA C81 ^ APPENDICES ABBREVIATIONS: EXPLANATORY NOTES LOQ n NS S.D. NOTES: limit of quantitation number of samples no sample standard deviation Appendices A-B: Values are reported to 2 significant figures. Calculations were performed prior to rounding values. -43- Company Sanitized. Does not contain TSCA CBI APPENDIX A Certificate of Analysis -44- Company Sanitized. Does not contain TSCA CBt SIGMA-ALDRICH 3050 Spruce Street Saw Louis. Missouri 63105 USA Telephone (800) 521-8356 . (SM) 771-5765 Fax (BOO3) 254052 (314) 77-S757 Vfett Us At wwMj.SgnwiWich.com Certffteate of Analysis PRODUCT KOMB: PRODUCT NAME FORMOIA APPEARANCE INFRARED SPECTRUM TITRATION GAS LIQUID CHROMATOGRAPHY TITRATION QUALITY CONTROL ACCEPTANCE DATE WHITE POWDER CONFORMS TO STRUCTURE AMD STANDARD APRHi, 2003 / ALDRICH CHEMICAL COMPANY RONNIE MARTIN APRIL 24, 2003 We are Committed b> the success 'of OUT Customers, Employes and Shareholders through leadershipm Life Science, Sigh Technology unit Service. -45- Company Sanitized. Does not contain TSCA CBI APPENDIX B Phase II (Repeated Exposure) Daily Chamber Concentrations -46- Company Sanitized. Does not contain TSCA CBI Phase II Daily Chamber Concentrations (mg/m3) Exposure Mean 1 mg/m' S.D. Range 10 mg/m3 25 ing/in3 n Mean S.D. Range n Mean S.D. Range n 1.1-1.6 1 1.0 0.44 0.39 - 1.4 6 10 1.5 9.1 - 13 6 26 2.0 23-29 6 2 1.0 0.31 0.51 - 1.4 6 9.4 1.2 7.2 - 10 6 24 5.0 20-33 6 3 1.1 0.46 0.25 - 1.5 6 9.8 0.73 8.6 - 10 6 27 3.3 21-31 6 4 1.1 0.29 0.67 - 1.5 6 11 1.1 8.6 - 12 6 26 18 8.9 - 68 9 5 1.2 0.35 0.60 - 1.6 6 9.9 0.58 9.1 - 11 6 27 6.1 21-36 6 6 1.2 0.32 0.77 - 1.7 6 11 0.98 9.2 - 12 6 24 2.9 21-27 6 7 1.2 0.092 1.1 - 1.3 6 11 1.7 8.3 - 13 6 26 2.5 23-30 6 8 1.1 0.48 0.36 - 1.7 6 9.6 0.72 8.6 - 11 6 27 1.7 24 - 29 6 9 1.3 0.22 6 10 0.77 8.9 - 11 6 26 7.1 22 - 42 7 10 1.0 0.24 0.65 - 1.3 6 11 0.41 10-11 6 24 5.4 15-32 6 11 1.2 0.55 0.83 - 2.2 5 10 2.2 8.7 - 14 6 26 2.4 23-29 6 12 0.95 0.46 0.56 - 1.7 6 9.4 0.79 8.0 - 10 6 23 5.6 18-34 6 13 1.1 0.60 0.38 - 2.1 6 10 0.92 9.1 - 11 6 21 3.1 18 - 26 7 14 0.81 0.34 0.25 - 1.2 6 9.8 1.3 8.4 - 12 6 25 2.5 21-28 6 15 0.82 0.50 0.33 - 1.6 6 10 2.07 9.0 - 15 6 24 3.2 18-27 6 -47- Company Sanitized. Does not contain TSCA CBI s APPENDIX C Phase n (Repeated Exposure) Daily Chamber Environmental Conditions -48- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat Exposure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean 25 25 25 25 25 25 25 25 25 25 25 2-6 25 25 26 Phase II Daily Chamber Environmental Conditions - Temperature (C) 0 nig/in3 S.D. Range 1 mg/m3 n Mean S.D. Range 10 mg/m3 n Mean S.D. Range n Mean 0.65 23 - 26 24 24 0.49 23 - 25 24 25 0.56 23 - 25 24 24 0.52 24 - 26 24 25 0.51 23 - 26 24 25 0.91 23 - 25 24 24 0.63 23 - 26 24 24 0.46 23 - 25 24 25 1.0 22 - 26 24 24 0.34 24 - 25 24 24 0.45 23 - 25 24 25 0.87 23 - 26 24 23 0.96 22 - 26 24 24 0.48 23 - 24 24 25 0.77 23 - 26 24 23 0.79 22 - 26 24 24 0.45 22 - 24 24 25 0.88 22 - 26 24 23 0.69 23 - 26 24 25 0.48 23 - 25 24 25 0.72 23 - 26 24 23 0.69 23 - 26 24 25 0.45 23 - 25 24 25 0.66 22 - 25 24 24 0.78 24 - 26 24 25 0.55 23 - 25 24 25 0.70 23 - 26 24 23 0.48 24 - 26 24 24 0.51 23 - 25 24 25 1.0 22 - 26 24 24 0.70 23 - 26 24 25 0.76 23 - 26 24 24 0.66 23 - 25 24 24 0.56 24 - 27 24 26 0.71 24 - 27 24 26 0.88 23 - 27 24 24 0.69 23 - 26 24 26 0.40 25 - 26 24 25 0.76 22 - 26 24 24 0.35 24 - 25 24 26 0.53 24 - 26 24 25 0.73 23 - 26 24 24 0.70 23 - 27 24 25 0.39 24 - 26 24 25 0.74 23 - 26 24 24 25 mg/m3 S.D. Ran 0.38 23 0.67 22 0.52 22 0.45 22 0.36 22 0.31 22 0.37 22 0.36 23 0.53 22 0.20 23 0.54 22 0.52 22 0.39 23 0.43 22 0.32 23 -49 'erfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat Phase II Daily Chamber Environmental Conditions - Relative Humidity (%) 0 mg/m3 Exposure .Mean S.D. Range 1 mg/m3 n Mean S.D. Range 10 mg/m3 n Mean S.D. Range 25 mg/m3 n Mean S.D. Ran 31-38 1 36 1.2 34 - 40 24 28 0.82 26 - 30 24 38 1.6 36 - 43 24 32 1.6 30 - 2 38 1.1 36 - 42 24 27 0.99 25 - 29 24 38 1.6 35 - 43 24 33 2.4 30 - 3 37 0.89 36 - 40 24 28 1.6 26 - 31 24 36 1.4 33 - 41 24 31 2.6 29 - 4 40 0.89 38 - 42 24 31 1.2 29 - 33 24 39 1.4 37 - 44 24 30 ..3.1 26 - 5 38 1.4 36 - 42 24 32 0.94 30 - 34 24 37 1.6 36 - 44 24 30 1.6 27 - 6 38 1.3 37 - 43 24 35 1.4 24 39 1.2 37 - 42 24 33 2.2 29 - 7 40 1.4 38 - 43 24 34 1.1 32 - 36 24 41 1.4 39 - 45 24 35 2.0 32 - 8 40 1.0 38 - 41 24 32 1.5 30 - 37 24 40 1.1 38 - 42 24 35 0.95 33 - 9 39 1.3 37 - 42 24 30 0.93 29 - 32 24 39 0.93 38 - 42 24 33 1.9 32 - 10 40 0.91 38 - 42 24 35 1.2 32 - 37 24 40 1.2 38 - 42 24 37 0.87 35 - 11 42 0.59 41 - 43 24 47 4.2 40 - 53 24 40 2.0 38 - 48 24 36 1.5 34 - 12 40 1.0 38 - 44 24 37 3.3 32 - 42 24 41 1.7 39 - 46 24 32 2.2 29 - 13 38 0.82 36 - 39 24 43 3.3 40 - 51 24 39 1.1 37 - 42 24 29 0.68 28 - 14 40 1.1 38 - 43 24 42 4.5 24 - 45 24 33 4.1 18 - 36 24 32 1.6 30 - 15 37 1.3 35 - 41 24 44 1.5 40 - 47 24 36 2.4 34 - 47 24 30 1.7 28 - -50 luorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat Exposure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean 28 28 28 28 28 28 28 28 28 28 28 2B 28 28 28 Phase II Daily Chamber Environmental Conditions - Airflow (L/min) 0 ing/in3 S.D. Range 1 nig/in' 10 mg/m3 25 ing/in' n Mean S.D. Range n Mean S.D. Range n Mean S.D. Ran 0.15 27 - 28 24 70 0.042 70 24 28 0.050 28 24 28 0.42 28 0.016 28 24 70 0.044 70 24 28 0.028 28 24 27 0.39 26 0.11 27 - 28 24 70 0.052 70 24 28 0.067 28 24 29 0.44 28 0.023 28 24 70 0.027 70 24 28 0.021 28 24 28 0.016 2 0.14 27 - 28 24 70 0.052 70 24 28 0.063 28 24 28 0.022 2 0.15 27 - 28 24 70 0.052 70 24 28 0.063 28 24 28 0.021 2 0.022 28 24 70 0.036 70 24 28 0.023 28 24 28 0.027 2 0.11 27 - 28 24 70 0.049 70 24 28 0.064 28 24 28 0.020 2 0.024 28 24 70 0.058 70 24 28 0.024 28 24 28 0.027 2 0.11 27 - 28 24 70 0.052 70 24 28 0.055 28 24 28 0.023 2 0.11 27 - 28 24 70 0.067 70 24 28 0.053 28 24 28 0.032 2 0.022 28 24 70 0.047 70 24 28 0.043 28 24 28 0.029 2 0.15 27 - 28 24 70 0.036 70 24 28 0.072 28 24 28 0.020 2 0.027 28 24 70 0.036 70 24 28 0.032 28 24 28 0.023 2 0.10 28 24 70 0.035 70 24 28 0.059 28 24 28 0.036 2 51 APPENDIX D Phase I (Single Exposure) Plasma Concentration Data -52- Company Sanitized. Does not contain TSCA CBI Predose Males, 0 mg/m3 101 <LOQ 102 <LOQ 103 <LOQ Hours During Exposure 0.5 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Hours Post-Exposure 12 18 24 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Females, 0 mg/m3 201 <LOQ 202 <LOQ . 203 <LOQ Males, 1 mg/m3 301 <LOQ 302 <LOQ 303 <LOQ Females, 401 402 403 1 mg/m3 <LOQ <LOQ <LOQ Males, 10 mg/m3 501 <LOQ 502 <LOQ 503 <LOQ Females, 601 602 2001 10 mg/m3 <LOQ <LOQ <LOQ Males, 25 mg/m3 701 0.64 702 <LOQ 703 <LOQ Females, 25 mg/m3 801 <LOQ 802 <LOQ 803 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ NS <LOQ <LOQ NS <LOQ <LOQ NS <LOQ <LOQ NS <LOQ <LOQ <LOQ 0.92 <LOQ <LOQ 1.42 <LOQ <LOQ 0.82 2.07 3.45 1.72 2.03 2.63 1.81 1.87 2.87 NS 1.69 2.79 0.96 1.61 3.55 1.58 1.94 2.84 1.31 1.94 2.72 1.60 <LOQ <LOQ 1.01 0.94 <LOQ <LOQ 0.64 0.99 <LOQ <LOQ <LOQ 0.72 0.83 0.72 <LOQ <LOQ <LOQ <LOQ 0.83 0.84 <LOQ <LOQ <LOQ <LOQ 0.59 0.44 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 1.18 <LOQ 0.77 3.50 2.46 3.15 6.70 4.62 6.22 8.29 5.91 7.98 8.86 6.92 7.88 9.91 NS 7.23 8.26 5.91 8.12 7.97 5.65 7.30 7.82 5.20 6.88 0.46 1.16 0.40 1.40 <LOQ 1.05 NS 2.69 2.55 4.01 3.97 3.07 3.81 <LOQ 0.52 <LOQ <LOQ <LOQ 4.28 2.54 0.48 <LOQ <LOQ <LOQ 2.97 <LOQ 0.61 <LOQ <LOQ <LOQ 1.51 <LOQ 0.84 2.86 NS 2.24 7.36 4.04 6.27 12.84 10.58 15.20 15.26 11.39 19.29 17.68 13.04 22.61 17.24 13.04 22.47 16.25 12.84 21.13 15.75 12.72 19.82 15.42 12.17 18.44 1.14 1.52 <LOQ 3.73 2.65 1.83 6.48 5.83 5.42 8.23 8.03 4.36 7.87 6.63 3.33 <LOQ <LOQ <LOQ 8.07 6.65 2.61 <LOQ <LOQ <LOQ 5.49 5.78 "3.42 <LOQ <LOQ <LOQ Data expressed as /ig/mL. -53- Company Sanitized. Does not contain TSCA CBI Perfluorooctanoic Acid: Relationship Between Repeated Inhalation Exposures and Plasma PFOA Concentration in the Rat____ ____________EhiPont-12944 APPENDIX E Phase II (Repeated Exposure) Plasma Concentration Data -54- Company Sanitized. Does not contain TSCA CBI 48 54 96_102 168 _1_74___21H6 ou2r2s2 __2_64___2_70___33_6___34_2__3_8_4 __3_90___4_32___43_8_ Males, O mg/m3 101 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 102 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.83 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 103 <LOQ <LOQ <LOQ <:LOQ <LOQ <LOQ <LOQ 0.49 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 104 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 105 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Males, 1 ing/m3 301 0.58 302 <LOQ 303 <LOQ 304 <LOQ 305 <LOQ 1.87 1.39 1.05 0.64 1.79 3.25 2.24 2.15 1.15 NS 5.52 3.49 4.06 2.37 6.46 5.16 4.44 4.27 2.87 6.50 7.67 4.94 5.83 4.48 7.96 6.18 4.10 4.89 3.08 6.24 7.27 6.73 5.84 3.69 7.93 7.59 5.59 6.82 4.76 8.14 8.53 5.95 8.00 4.77 9.34 7.58 6.84 8.64 5.02 9.80 10.06 6.71 9.41 5.10 9.45 8.73 5.40 8.42 3.97 7.57 9.60 7.36 8.58 4.84 7.89 8.74 6.99 8.11 4.94 7.95 10.07 6.95 9.69 5.69 8.13 10.00 7.99 8.88 5.31 9.46 10.77 8.45 10.57 5,95 10.52 Males, 10 m g/m3 501 <LOQ 502 <LOQ 503 <LOQ 504 <LOQ 505 <LOQ 2.15 3.59 4.23 2.87 5.60 6.01 7.90 8.86 3.44 10.72 8.79 11.57 14.92 3.97 16.86 12.23 15.14 20.43 4.01 16.86 15.61 19.88 13.36 6.98 23.24 13.19 17.50 17.52 5.16 16.47 16.76 18.16 21.31 7.27 20.00 17.39 22.58 9.12 25.81 19.16 17.80 24.20 26.01 9.62 19.86 15.39 25.06 29.87 10.29 21.03 15.36 27.48 31.46 11.82 22.51 29.79 21.94 24.84 9.06 18.69 14.91 23.72 27.73 10.54 20.25 15.37 23.79 28.88 10.67 22.10 16.04 25.62 31.35 11.47 22.92 16.12 24.80 31.57 14.54 22.32 18.43 26.50 33.14 16.05 26.53 Males, 25 m g/m3 701 <LOQ 702 <LOQ 703 <LOQ 704 <LOQ 705 <LOQ 10.44 5.67 10.27 3.42 8.87 23.60 12.02 22.95 14.93 14.91 NS 27.54 39.31 27.30 22.87 37.73 31.80 30.59 27.80 22.28 51.07 49.36 38.84 35.11 27.25 38.17 30.75 29.79 28.05 23.72 45.28 37.27 35.00 29.57 28.19 47.69 37.55 18.13 28.51 28.57 45.84 38.23 30.19 30.47 33.61 40.66 52.07 24.81 31.87 32.38 49.34 54.76 29.07 31.72 33.00 42.11 40.94 19.19 23.50 28.65 45.23 43.18 21.09 25.47 28.24 43.12 40.21 22.16 26.83 27.15 47.39 45.59 23.64 28.68 28.65 54.23 62.72 22.58 27.06 31.30 68.00 75.42 22.41 31.26 34.49 Data expressed as pg/mL. -55- Company Sanitized. Does not contain TSCA CBI _4_8___5_4___9_6___1_02___1_68___1_74___2H16ou2r2s2___26_4___27_0___33_6___34_2___38_4___39_0___43_2___43_8_ Females, 201 202 203 204 205 O mg/m3 <LOQ <LOQ <LOQ <LOQ 1.71 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.74 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Females, 401 402 403 404 405 1 mg/m3 <LOQ <LOQ <LOQ <LOQ <LOQ 0.48 <LOQ 0.60 <LOQ 0.96 <LOQ <LOQ <LOQ 1.39<LOQ 0.77 <LOQ 0.81 <LOQ 0.46 <LOQ 0.59 <LOQ 0.98 <LOQ 0.93 <LOQ 0.75 1.18 0.89 <LOQ 0.93 <LOQ 0.69 <LOQ 0.95 <LOQ 0.90 <LOQ 0.28 <LOQ 0.94 <LOQ 0.80 <LOQ 0.93 <LOQ 0.51 <LOQ 0.74 <LOQ 0.98 <LOQ 0.51 <LOQ 0.77 <LOQ 0.69 <LOQ 0.37 <LOQ 0.69 <LOQ 0.46 <LOQ 0.64 <LOQ 0.59 <LOQ 0.82 <LOQ 0.74 <LOQ 0.97 <LOQ 0.59 <LOQ 0.79 <LOQ 0.74 <LOQ 0.75 <LOQ 0.45 <LOQ 0.73 1.03 0.90 0.79 0.59 Females, 10 mg/m3 601 <LOQ 602 <LOQ 603 <LOQ 604 <LOQ 605 <LOQ 1.99<LOQ 1.43<LOQ 2.90 0.33 0.81 <LOQ 3.46 <LOQ 1.97 0.22 2.60 <LOQ 3.11 NS NS NS 2.87 <LOQ 1.25<LOQ 1.53 0.32<LOQ<LOQ 2.82 <LOQ 0.67 <LOQ 2.84 <LOQ NS NS 1.49<LOQ 0.83 <LOQ 3.38 <LOQ 2.28 <LOQ NS NS 2.19<LOQ 1.82<LOQ 1.83<LOQ 2.24 <LOQ 0.90 <LOQ NS NS NS NS 3.66 <LOQ <LOQ <LOQ 1.91<LOQ<LOQ<LOQ 1.83<LOQ 1.1KLOQ 2.01 <LOQ NS NS 2.38 <LOQ 1.59<LOQ 0.95 <LOQ 2.73 NS 3.42 1.29 0.62 Females, 25 mg/m3 801 <LOQ 802 <LOQ 803 <LOQ 804 <LOQ 805 <LOQ 2.24-SLOQ 1.59 0.32 3.84 0.74 3.58 0.93 3.61 0.48 2.98 0.74 1.44<LOQ 3.72 0.29 3.24 0.38 6.27 0.38 7.07 <LOQ 1.74<LOQ 2.54 <LOQ 3.19<LOQ 6.36 <LOQ 7.00 0.51 1.67<LOQ 4.02 <LOQ 1.98<LOQ 1.81 0.24 7.72 0.62 6.33 0.26 4.34 <LOQ 4.98 <LOQ 5.16 0.25 8.37 <LOQ 2.10 <LOQ 2.1KLOQ 2.14 <LOQ 5.04 <LOQ 5.42 0.52 4.31 <LOQ 2.26 <LOQ 2.34 0.27 2.92 0.33 11.33 0.58 2.67 <LOQ 0.94 <LOQ 0.96 <LOQ 4.87 0.24 13.03 6.42 5.34 2.52 5.03 Data expressed as pg/mL. -56- Company Sanitized. Does not contain TSCA CBI