Document dQeVzV6yY8KoO1z6mz9dKdrm0

r -- --------------------------------------------------------------------- -- -- -- *------------ AtM- 503 TRADE SECRET Study Title H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Test Guidelines: U.S. EPA Health Effects Test Guidelines OPPTS 870.3200 (1998) OECD Guideline for the Testing o f Chemicals Section 4: Health Effects, Number 410 (1981) Author: Carol Finlay, B.A. Study Completed on: November 3,2004 Performing Laboratory: E.I. du Pont de Nemours and Company HaskellSMLaboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Laboratory Project ID: DuPont-13830 Work Request Number: Service Code Number: Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Page 1 o f 231 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 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 1997) published in ENV/MC/CHEM(98)17, except for the item documented below. Test substance characterization and stability analyses were performed at Regional Analytical Services (RAS), a non-GLP laboratory. The test substance analyses performed were in compliance with regulatory guidelines. None o f the aforementioned analyses were performed under Good Laboratory Practice Standards; however, the analyses were conducted in compliance with IS09002 regulations. All o f the analyses are considered valid and sufficient for the purposes o f this study. Applicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Director: Staff Toxicologist Date Applicant / Sponsor: DuPont Representative Date -2 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ QUALITY ASSURANCE DOCUMENTATION DuPont-13830 Work Request Number: Study Code Number: The conduct o f this study has been subjected to periodic Quality Assurance inspections. The dates o f inspection are indicated below. Phase Audited Conduct: Evaluation Tables and Appendices Tables and Appendices Tables and Appendices Report/Records: Audit Dates 1 Dec 2003 16, 18,23 Feb 2004 23-26 Feb 2004 9-12, 16 Mar 2004 6-8 Oct 2004 Dates Reported to Study Director and Management 1 Dec 2003 23 Feb 2004 26 Feb 2004 16 M ar 2004 8 Oct 2004 Reported by: Donna M. Johnston Quality Assurance Auditor to '/ Date -3 Company Sanitized. Does not contain TSCA C8I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Nancy^E. Everds, D.V.M. Diplomats A.C.V.P. Principal ResearchScientist Anatomic Pathology Evaluation by: " GregSykes, V.tfEb. Diplomate A.C.V.P., A.C.L.A.M., A.B.T. VeterinaryPathologist Date 0 3 ' Ha/'^COCf Date Anatomic Pathology EvaluationPeer Review by: StevenR, Frame, D.VJM., Ph.D. Diplomate A.C.VJP. VeterinaryPathologist Date Report Peer Review by: SusanA. MacKenzie, V.MJD., PhD., D.A.B.T. Senior Research Toxicologist Date Approved by: /t* ScottE. Loveless, Fh.D. Research ManagerandDirector 02-Ajou-2c>'t Date Issued by StudyDirector: tA&L At: h.1 ttu -______ _ Carol ^nlay,B.A. (J StaffToxicologist 0<V Date -4 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 TABLE OF CONTENTS Page GOOD LA BO RATO RY PR A C TIC E C O M PLIA N CE ST A T E M E N T ..................................... 2 Q UALITY ASSURANCE D O CU M EN TA TIO N .............................................................................3 C E R T IF IC A T IO N ..................................................................................................................................4 LIST O F T A B L S ..................................................................................................................................7 LIST O F F IG U R E S ................................................................................................................................8 LIST O F A PPEN D IC ES........................................................................................................................8 STUDY IN FO R M A TIO N ......................................................................................................................9 SUM MARY.............................................................................................................................................10 IN TR O D U C TIO N ................................................................................................................... STUDY D ESIG N ................................................................................................................................... 11 M ATERIA LS AND M E T H O D S .......................................................................................................11 A. Test Guidelines............................................. 11 B. Test Substance............................................................................................................................ 12 C. Test Species............................................. 12 D. Animal H usbandry.....................................................................................................................12 E. Quarantine and P retest.............................................................. 13 F. Assignment to Groups and Study S tart....................................................................................13 G. Test Substance Preparation and Administration........................ 13 H. Body W eights............................................................ 14 I. Food Consumption and Food Efficiency................................................................................. 14 J. Observations................................................................ 14 K. Total Fluorine and Perfluorooctanoic Acid Level Evaluations............................... L. Clinical Pathology Evaluation...................................................................................................15 M. Anatomic Pathology Evaluation............................................................................................... 17 N. Statistical Analyses......................................................................................... 17 RESULTS AND D ISC U SSIO N ......................................................................................................... 19 In-Life Toxicology.................................................................................................................................19 A. Mean Body Weights and Body Weight Gains.........................................................................19 B. Food Consumption and Food Efficiency................................................................................. 19 C. Clinical Observations, Dermal Effects, and M ortality...........................................................19 D. Perfluorooctanoic Acid (PFOA) and Total Fluorine Evaluations........................................ 20 E. In-Life Conclusions....................................................................................................................20 Clinical Pathology E valuation..................................................................................... A. Hematology................................................................................................................................20 11 14 20 -5 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 B. Coagulation................................................................................... 21 C. Clinical Chemistry........................................................................ 21 D. Urinalyses.................................................................................................................................... 21 E. Urine and Plasma Fluoride...................................................... 22 F. Clinical Pathology Conclusions................................................. 22 Anatomic Pathology Evaluation........................................................................................................22 A. Cause o f D eath............................................................................. 22 B. Organ Weights.............................................................................. 22 C. Gross Observations................................................................................................................... 22 D. Microscopic Findings................................................................................................................ 23 E. Anatomic Pathology Conclusions............................... 23 C O N C L U S IO N S ..................................................................................... 24 RECORDS AND SAMPLE STORAGE............................................ 24 R E F E R E N C E S ........................................................................................ 25 TABLES.................................................................................................................................................. 26 FIG URE................................................................................................. 52 APPENDICES....................................................................................................................................... 54 -6 Compny Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________ ____ DuPont-13830 LIST OF TABLES Page 1. MEAN BODY WEIGHTS OF MALE RATS...............................................................................................................29 2. MEAN BODY WEIGHT GAINS OF MALE RATS.....................................................................................................30 3. MEAN DAILY FOOD CONSUMPTION BY MALE RATS......................................................................................31 4. MEAN DAILY FOOD EFFICIENCY OF MALE RATS.............................................................................................32 5. SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - PREDOSING............................................ 33 6. SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - POSTDOSING.........................................35 7. PERCENT SURVIVAL OF MALE RATS.....................................................................................................................37 8. MEAN PFOA CONCENTRATIONS IN MALE RATS.............................................................................................. 38 9. MEASURED CONCENTRATION (PPB) WHOLE BLOOD FLUORINE............................................................. 39 10. SUMMARY OF HEMATOLOGY VALUES FOR MALE RATS............................................................................. 40 11. SUMMARY OF COAGULATION VALUES FOR MALE RATS ............................................................................ 42 12. SUMMARY OF CLINICAL CHEMISTRY VALUES FOR MALE RATS..............................................................43 13. SUMMARY OF URINALYSIS VALUES FOR MALE RATS...................................................................................45 14. MEAN FINAL BODY AND ORGAN WEIGHTS FOR MALE RATS................................................................... 46 15. INCIDENCES OF GROSS OBSERVATIONS IN MALE RATS..............................................................................47 16. INCIDENCES AND LESION GRADES OF MICROSCOPIC OBSERVATIONS IN MALE RATS................ 49 -7 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 LIST OF FIGURES Page 1. MEAN BODY WEIGHTS OF MALE RATS............................................................................................................... 53 LIST OF APPENDICES Page A. INDIVIDUAL BODY WEIGHTS....................................................................................................................................55 B. INDIVIDUAL FOOD CONSUMPTION........................................................................................................................ 64 C. INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY RECORDS....................................................70 D. INDIVIDUAL CLINICAL OBSERVATIONS - POSTDOSING............................................................................... 80 E. INDIVIDUAL EDEMA AND ERYTHEMA SCORES................................................................................................ 93 F. PFOA ANALYTICAL REPORT....................................................................................................................................119 G. INDIVIDUAL BLOOD FLUORINE LEVELS........................................................................................................... 185 _ H. INDIVIDUAL ANIMAL CLINICAL PATHOLOGY D A T A .................................................................................. 187 I. INDIVIDUAL ANIMAL BODY AND ORGAN WEIGHTS....................................................................................209 J. INDIVIDUAL ANIMAL GROSS AND MICROSCOPIC OBSERVATIONS....................................................... 213 -8 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats__________ ___ STUDY INFORMATION DuPont-13830 Haskell Number: 25425 CAS Registry Number: Purity: Known Impurities: Physical Characteristics: Study Initiated/Completed: November 18, 2003 / (see report cover page) In-life Initiated/Completed: November 25, 2003 / December 23, 2003 -9 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats................ ...... DuPont-13830 SUMMARY The objective o f this study was to determine the potential toxicity o f H-25425 following repeated dermal exposure. The test substance was applied to the shaved, intact skin o f male Crl:CD(SD)IGS BR rats for 6 hours/day for 28 consecutive days. Three groups o f 10 male rats were treated dermally with 10,100, or 1000 mg/kg/day H-25425. A control group o f 10 male rats was similarly treated with deionized water. The rats were observed for mortality, clinical signs, dermal effects, and body weight effects during the study. Food consumption was determined weekly during the study. Blood samples for total fluorine were collected on test days 0, 7 ,1 4 ,2 1 , and 27. The day 21 samples were analyzed. Blood and urine samples were collected for clinical pathology prior to sacrifice on day 28. Blood samples were collected at necropsy for analysis o f concentration o f perfluorooctanoic acid (PFOA). All rats underwent necropsy for gross and microscopic pathological evaluation. No deaths occurred, and no test substance-related effects on body weight or nutritional parameters were observed. No clinical signs attributable to systemic toxicity occurred. No dermal irritation was observed during the study. PFOA concentrations in plasma from rats in the control group and the 10 mg/kg/day group were below the lower limit o f quantitation (10 ppb). The mean concentrations were 13 ppb in the 100 mg/kg/day group and 105 ppb in the 1000 mg/kg/day group. Fluorine levels in blood from treated and control rats were either not detectable or below the limit o f quantification (500 ppb). No adverse effects were observed on any clinical pathology parameters (hematology, clinical chemistry, coagulation parameters, urinalysis), organ weights, or gross or microscopic pathology parameters. The no-observed-effect level (NOEL)8 for male rats was 1000 mg/kg/day, the highest dosage tested. The NOEL is based on a lack o f adverse test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameter in male rats treated dermally at dosages up to 1000 mg/kg/day. The no-observed-effect (NOEL) for this study is defined as the highest dose at which toxicologically important effects attributable to the test substance were not detected. Thus, for this study, the NOEL is equivalent to the NOEL as defined by the United States Environmental Protection Agency (1985), and the no-observed-adverseeffect level (NOAEL) as defined by the European Union (1994). - 10Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 INTRODUCTION The substance, H-25425, is belongs to the group o f compounds known previously conducted 90-day oral gavage study with the test substance, male and female rats were dosed at 0 (control), 10, 100, or 1000 mg/kg for 90 consecutive days.(1) No adverse test substance-related effects on body weight, body weight gain, food consumption, food efficiency, or clinical pathology parameters were observed in any male or female dose group. Reversible, minimal to mild thyroid follicular hypertrophy was observed in the 1000 mg/kg/day male and female groups. The no-observed- effect level (NOEL) for subchronic toxicity endpoints in male and female rats was 100 mg/kg/day. Dose levels for the present study were selected to duplicate the dose levels o f the oral gavage study. Group DC XI xm XV STUDY DESIGN Number/Group 10 10 10 10 Dosage (mg/kg) 0 (Control) 10 100 1000 MATERIALS AND METHODS A. Test Guidelines The shady design complies with the following test guidelines except as noted below: Office of Prevention, Pesticides and Toxic Substances (OPPTS) U.S. Environmental Protection Agency (EPA) (1998). OPPTS 870.3200 21/28-Day Dermal Toxicity. Health Effects Test Guidelines. Organisation for Economic Co-Operation and Development (OECD) (1981). 410 Repeated Dose Dermal Toxicity: 21/28-Day Study. Guidelinefo r the Testing o f Chemicals. Exception - The wrappings o f 7 rats became dislodged during the exposure period. Therefore, on these occasions they may not have been exposed to the test substance for 6 hours. Four o f these rats were in the control group, and three rats were in different test groups. This exception did not affect the objectives or the validity of the study because, considering that no significant toxicological effects were observed at any dosage level, it is not likely that an occasional exposure period of less than 6 hours for some animals would have changed the study results. -11Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male R a ts______ _______ Exception - Only male rats were treated because in previous studies with other males were more sensitive to the toxicological effects than females. DuPont-13830 B. Test Substance The test substance, H-25425, was supplied by the sponsor as an! The test substance appeared to be stable under the conditions o f the * tdy. No evidence of insti ility, such as a change in color or physical state, was observed. C. Test Species On November 18, 2003, 44 male Crl:CD(SD)IGS BR rats, with an assigned birth date o f October 6,2003, were received from Charles River Laboratories, Inc., Raleigh, North Carolina. The Crl:CD(SD)IGS BR rat was selected based on consistently acceptable health status and on extensive experience with the strain at Haskell Laboratory. D. Animal Husbandry 1. Housing Rats were housed singly in stainless steel, wire-mesh cages suspended above cage boards. 2. Environmental Conditions Animal rooms were maintained at a temperature o f 18-26C and a relative humidity of 30-70%. Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. 3. Feed and Water Tap water was provided ad libitum. PMI Nutrition International, LLC Certified Rodent LabDiet 5002 meal was available ad libitum except when the rats were fasted. 4. Identification Prior to assignment to groups, each rat was temporarily identified by a cage card affixed to the outside o f its cage. After assignment to groups, each rat was assigned an animal number which was tattooed on the tail o f each rat and included on the cage labels. 5. 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 o f the study: - 12Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-13830 Water samples are analyzed for total bacterial counts, and the presence o f coliforms, lead, and other contaminants. Feed samples are analyzed for total bacterial, spore, and fungal counts. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations o f 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 o f the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluation o f these data did not indicate any conditions that affected the validity o f the study. E. Quarantine and Pretest Upon arrival at Haskell Laboratory, the rats were removed from shipping cartons and quarantined for 6 days. The rats were weighed 3 times during the quarantine period and examined daily for any clinically apparent signs o f disease or injury. The rats were observed daily for mortality and signs o f illness, injury, or abnormal behavior. On the bases o f acceptable body weight gains and freedom from clinically apparent signs of disease or injury, the rats were released from quarantine on test day -1 by the laboratory animal veterinarian. F. Assignment to Groups and Study Start Forty rats were selected for study use on the bases o f adequate body weight gain, freedom from clinical signs o f disease or injury, and a body weight within 20% o f the mean. The selected rats were divided by computerized, stratified randomization into 4 groups o f 10 rats, so that there were no statistically significant differences among group body weight means. Dose administration with H-25425 began on test day 0 when the rats were approximately 7 weeks o f age. G. Test Substance Preparation and Administration Approximately 24 hours prior to the first treatment, the fur of each rat was closely shaved to expose the skin from the back and trunk. The area to be treated was marked on the back o f each rat with a water-insoluable marker. The rats were treated for 28 consecutive days at approximately the same time each day. The exposure period was approximately 6 hours per day. - 13Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 The test substance was moistened with deionized water to form a thick paste and applied as evenly and thinly as possible to the test site. The amount o f test substance needed to treat each rat was based on the most recently determined body weight measurement. The control rats were treated with deionized water. The test site was covered with a 2-ply porous gauze dressing followed by successive layers o f stretch gauze (no more than 8 layers) and self-adhesive bandage. After the exposure period, the wrappings were removed and excess test substance was washed off with warm tap water. Each rat was then gently patted dry and returned to the cage. The control animals received the same washing technique as the treated animals. It was not possible to cover an area o f 10% o f the total body surface for any dose group without excessively diluting the test substance. The approximate total body surface area covered with the thick paste for the 10,100, and 1000 mg/kg/day groups was 1%, 1%, and 3.3%, respectively. Following an evaluation rats were reshaved as needed during the study. The entire area that was originally shaved (including the untreated control skin) was reshaved. H. Body Weights The rats were weighed at 3-4 day intervals throughout the study. I. Food Consumption and Food Efficiency The amount o f food consumed by each rat was determined once per week throughout the study. Individual food consumption was determined by weighing the amount o f diet in each feeder at the beginning and end o f the week and subtracting the final weight and the amount o f spillage from the feeder during the week from the initial weight. From these measurements, mean daily food consumption was determined. From the food consumption and body weight data, mean daily food efficiency was calculated. J. Observations The rats were observed for clinical signs and dermal effects after washing. The rats were also observed for clinical signs at each weighing. The Draize Scale was used to score skin irritation. Adjacent areas o f untreated skin were used for comparison. The rats were checked twice daily for mortality and for signs o f illness, injury, or abnormal behavior. K. Total Fluorine and Perfluorooctanoic Acid Level Evaluations Blood was collected into EDTA tubes from the orbital sinus o f all rats on test days 0, 7,14, 21, and 27 approximately one hour after removal of the test substance. The blood was refrigerated. The total fluorine content o f the day 21 blood samples was determined by using a Wickbold torch combustion method followed by analysis with a fluoride ion selective electrode.(2) The limit o f - 14Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats............... ....... DuPont-13830 quantification (LOQ) for this method is 500 ppb. Fluorine blood analysis was performed at Stereochem, Newark, Delaware. Additional blood was collected from the vena cava o f all rats at necropsy into a tube containing EDTA. Plasma was prepared and stored frozen at -80C to -20C until analyzed for concentration o f perfluorooctanoic acid (PFOA). PFOA was extracted from the plasma by protein precipitation in acetonitrile. The compound was quantified by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using selected reaction monitoring (SRM). Quantification was performed using extracted calibration standards containing an internal standard. The lower limit o f quantitation (LLOQ) for this method is 10 ppb. Plasma samples were analyzed for PFOA at Exygen Research, 3058 Research Drive, State College, PA 16801. See Appendix F for additional details o f this analysis. L. Clinical Pathology Evaluation A clinical pathology evaluation was conducted on all animals 28 days after initiation o f the study. The day before collection o f samples for the clinical pathology evaluation animals were placed in metabolism cages. Animals were fasted overnight (approximately 16 hours) and urine collected from each animal. Blood samples for hematology and clinical chemistry measurements were collected from the orbital sinus o f each animal while the animal was under carbon dioxide anesthesia. Blood samples for plasma fluoride, PFOA, and coagulation parameters were collected at sacrifice from the abdominal vena cava of each animal while the animal was under carbon dioxide anesthesia. Additional blood collected from the vena cava was placed in a serum tube, processed to serum, and frozen at -80C. Serum was discarded without analysis because further tests were not required to support experimental findings. Bone marrow smears were inadvertently not collected at sacrifice. All blood samples were evaluated for quality by visual examination prior to analysis. Results were maintained in the study records and reported only if the sample was analyzed. 1. Hematology and Coagulation Complete blood counts, including reticulocytes, were determined on a Bayer Advia 120 hematology analyzer or determined from microscopic evaluation o f the blood smear. WrightGiemsa stained blood smears from all animals were examined microscopically for confirmation of automated results and evaluation of cellular morphology. Blood smears, stained with new methylene blue, were prepared from each animal undergoing a hematology evaluation, but were not needed for examination. Coagulation times were determined on a Sysmex CA-1000 Coagulation Analyzer. The following parameters were determined: red blood cell count hemoglobin hematocrit mean corpuscular volume platelet count white blood cell count differential white blood cell count microscopic blood smear examination - 15Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ mean corpuscular hemoglobin mean corpuscular hemoglobin concentration red cell distribution width absolute reticulocyte count DuPont-13830 prothrombin time activated partial thromboplastin time 2. Clinical Chemistry Serum clinical chemistry parameters were determined on an Hitachi 717 clinical chemistry analyzer. Plasma fluoride concentrations were determined using a Beckman <t>12 digital pH/ISE meter and a fluoride-selective electrode. The following parameters were determined: aspartate aminotransferase alanine aminotransferase sorbitol dehydrogenase alkaline phosphatase total bilirubin urea nitrogen creatinine cholesterol triglycerides glucose total protein albumin globulin calcium inorganic phosphorus sodium potassium chloride fluoridea 3. Urinalysis Urine volume and appearance (quality, color, and clarity) were measured and evaluated visually, respectively. Urine constituents were semi-quantitatively measured on a Bayer Clinitek AtlasTM Automated Urine Chemistry analyzer. Urine protein was measured on an Hitachi 717 clinical chemistry analyzer. Urine osmolality was determined using an Advanced Osmometer 3900. Urine fluoride concentrations were determined using a Beckman 0 1 2 digital pH/ISE meter and a fluoride-selective electrode, and total urine fluorides were calculated (volume x concentration). Sediments from all urine specimens were evaluated microscopically. The following parameters were determined: quality color clarity volume osmolality ketone bilirubin blood urobilinogen fluoride Fluoride determination was analyzed on EDTA plasma. - 16Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ pH glucose protein microscopic urine sediment examination DuPont-13830 M. Anatomic Pathology Evaluation After 28 days o f exposure to the test substance (test day 28), groups o f 10 male rats from the 0, 10, 100, and 1000 mg/kg/day groups were sacrificed and necropsied. The rats were fasted overnight on the afternoon before their scheduled sacrifice. The order o f sacrifice for scheduled deaths was random among all treatment groups. Rats were euthanized by carbon dioxide anesthesia and exsanguination. Gross examinations were performed on all rats. The following tissues were collected from all rats. liver skin (treated) kidneys skin (untreated) thyroid gland__________________________ gross observations a________________________ a. Gross observations made at necropsy for which histopathology was not appropriate (e.g., fluid, ruffled fur, and missing anatomic parts) were generally not collected. The following tissues were weighed: liver, kidneys, and thyroid gland (after fixation). Organ weight/fmal body weight ratios were calculated. Gross lesions for which a microscopic diagnosis would not be additive (e.g., osteoarthritis, pododermatitis, chronic dermatitis o f the tail, urinary calculi, and deformity o f the teeth, toe, tail, or pinna) were saved but were generally not processed for microscopic evaluation. All tissues were fixed in 10% neutral buffered formalin. Processed tissues were embedded in paraffin, cut at a nominal thickness o f 5 micrometers, stained with hematoxylin and eosin (H&E), and examined microscopically. All collected tissues from rats in the control and 1000 mg/kg/day groups were processed to slides and received a full histopathological examination. N. Statistical Analyses Significance was judged at p < 0.05. Parameter Body Weight Body Weight Gain Food Consumption Food Efficiency Preliminary Test Test for lack o f trend(3) Method o f Statistical Analysis If preliminary test is not If preliminary test is significant significant Sequential application<4) o f the Jonckheere-Terpstra Preliminary tests for trend test pairwise comparison ORa - 17Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Parameter Organ Weight Clinical Pathology0 Survival Incidence o f Clinical Observations Preliminary Test Levene's test for homogeneity and Shapiro-Wilk test for normality11 Levene's test for homogeneity and Shapiro-Wilk test for normality11 None DuPont-13830 Method o f Statistical Analysis If preliminary test is not If preliminary test is significant significant One-way analysis o f variance followed with Dunnett's test10,10 Kruskal-Wallis test( followed with Dunn's test(13) One-way analysis o f variance followed with Dunnett's test(9,10'U) Kruskal-Wallis test(12) followed with Dunn's test( Cochran-Armitage test for trendd a Pairwise comparisons and associated preliminary tests were only conducted if the test for lack o f trend was significant. b If the Shapiro-Wilk test was not significant but Levene's test was significant, a robust version o f Dunnett's test was used. If the Shapiro-Wilk test was significant, Kruskal-Wallis test was followed by Dunn's test, c When an individual observation was recorded as being less than a certain value, calculations were performed on half the recorded value. For example, if bilirubin was reported as <0.1, 0.05 was used for any calculations performed with that bilirubin data. d If the incidence was not significant, but a significant lack o f fit occurred, then Fisher's Exact test(14>with a Bonferroni correction was used. -18Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 RESULTS AND DISCUSSION In-Life Toxicology A. Mean Body Weights and Body Weight Gains (Tables 1-2, Figure 1, Appendix A) No test substance-related effects on mean body weights or mean body weight gains were observed in any group o f male rats. No statistically significant differences in mean body weights were observed. Mean body weight gains in all groups were greater than control over some intervals (not statistically significant). Mean body weight gains in all test groups were statistically decreased compared to control in the interval days 10-14. The differences were not considered test substance-related because they did not exhibit a monotonic dose-response. The mean overall body weight gains in all test groups were lower than control. The differences were not considered to be test substance-related because they were not statistically significant and did not exhibit a monotonic dose-response. B. Food Consumption and Food Efficiency (Tables 3-4, Appendix B) No effects on food consumption were observed. Mean overall food consumption (test days 0-27) in the 1000 mg/kg/day group was 101% o f control (not statistically significant). No test substance-related effects on food efficiency were observed. Mean food efficiency was statistically decreased in the interval days 7-14 at all dosages. These differences were not considered test substance-related because they did not exhibit a monotonic dose response. Mean overall food efficiency was statistically decreased in the 100 and 1000 mg/kg/day groups. These differences were not considered test substance-related because they were 10% or less than control, mean food efficiency in these groups was greater than control over 1 or 2 weekly intervals (not statistically significant), and the decreased food efficiency did not result in any reduction in final body weight or overall body weight gain. C. Clinical Observations, Dermal Effects, and Mortality (Tables 5-7, Appendices C-E) No clinical signs resulting from systemic toxicity occurred. Ocular discharge and nasal discharge were not considered test substance-related as they were observed in both treated and control animals. Dark eyes, corneal opacity, or enophthalmus were observed in several rats during the study. These clinical signs were not considered test substance-related because the incidence did not exhibit a monotonic dose response. These signs may be a result of orbital sinus bleeding. - 19Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats _________ DuPont-13830 Hair loss, superficial wounds, swollen face, and staining o f the fur/skin were present in both treated and control animals and were not considered test substance-related. No dermal irritation was observed during the study. No deaths occurred. D. Perfluorooctanoic Acid (PFOA) and Total Fluorine Evaluations (Tables 8-9, Appendices F-G) PFOA concentrations in plasma from rats in the control group and the 10 mg/kg/day group were below the lower limit o f quantitation (LLOQ or 10 ppb). The mean concentrations were 13 ppb in the 100 mg/kg/day group and 105 ppb in the 1000 mg/kg/day group. These levels were considerably lower than those observed in the oral study with this test substance.(1) The source o f the PFOA in the plasma, whether from direct application or metabolism of chemical in the test substance, is unknown. Fluorine levels in blood from treated and control rats were either not detectable or below the limit o f quantification (LOQ or 500 ppb). This is in contrast with the measurable total fluorine evident in animals dosed at all levels (10 mg/kg/day and above) in the oral study. E. In-Life Conclusions No adverse effects were observed on mean body weight, nutritional parameters, mortality, or clinical signs in male rats treated up to and including 1000 mg/kg/day. PFOA was present in plasma from rats treated at 100 and 1000 mg/kg/day. Blood fluorine levels were not detectable at levels above the LOQ o f 500 ppb in any dose group. Clinical Pathology Evaluation A. Hematology (Table 10, Appendix H) There were no adverse changes in hematologic parameters in male rats. The following statistically significant changes in mean hematologic parameters were not related to treatment: Mean corpuscular hemoglobin concentration was minimally but statistically increased in males treated with 10 or 1000 mg/kg (both 102% o f control mean). These changes did not occur in a dose-related pattern, and were therefore considered to be unrelated to treatment. - 20Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 B. Coagulation (Table 11, Appendix H) There were no treatment-related or statistically significant changes in coagulation parameters in male rats. C. Clinical Chemistry (Table 12, Appendix H) There were no adverse changes in clinical chemistry parameters in male rats. The following statistically significant changes in mean clinical chemistry parameters were either treatmentrelated and not adverse, or not related to exposure to compound: Minimally decreased urea nitrogen and creatinine in males treated with 100 or 1000 mg/kg were considered to be treatment-related. Mean concentrations o f urea nitrogen were 83% of controls in both groups, while mean creatinine concentrations were 83% and 76% o f controls in rats treated with 100 or 1000 mg/kg, respectively. In addition, with the exception o f one rat treated with 10 mg/kg, creatinine concentrations o f this group were also decreased. Generally, increases in urea nitrogen and creatinine are considered adverse; minimal decreases in these parameters have no toxicologic significance. The cause o f minimally decreased urea nitrogen and creatinine was not addressed in this study. Cholesterol and triglyceride concentrations were minimally decreased in males treated with 1000 mg/kg (statistically significant only for cholesterol; means were 81% and 72% of respective controls). These decreases were likely to be treatment-related based on the distribution o f the individual animal data in comparison to control rats. However, because the range of individual values was similar between control and 1000 mg/kg groups, these changes were considered to be non-adverse. Potassium was minimally decreased (statistically significant) in males treated with 1000 mg/kg (90% o f control group mean). This decrease was likely to be treatment-related based on the consistency o f the individual animal data. However, in comparison to historical laboratory data, individual animal potassium concentrations in both the control and 100 mg/kg groups o f rats were within the historical range for individual animal values (5.1-7.3 mmol/L), with the exception o f one outlier in the control group (#902) with a potassium concentration o f 7.72 mmol/L. In addition, means for both groups were within the historical range of means (5.7-6.5 mmol/L). Therefore, this change, although treatmentrelated, was not considered to be adverse. D. Urinalyses (Table 13, Appendix H) There were no adverse changes in urinalysis parameters in male rats. The following statistically significant change was not related to treatment: -21Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13 830 Urine protein was increased in males treated with 10 mg/kg. This change occurred only in the low dose group, and therefore was considered to be unrelated to compound. E. Urine and Plasma Fluoride (Tables 12-13, Appendix H) There were no statistically significant or treatment-related changes in plasma fluoride or urine fluoride at the end o f the dosing period. F. Clinical Pathology Conclusions Treating male rats dermally with H-25425 at dosages up to 1000 mg/kg for 28 days resulted in minimally decreased urea nitrogen and creatinine at 100 and 1000 mg/kg/day and minimally decreased cholesteral, triglycerides, and potassium at 1000 mg/kg/day. None o f these changes were considered to be adverse. Therefore, there were no adverse effects on hematology, coagulation, clinical chemistry, or urinalysis parameters in the dermal study which is consistent with the oral study. Anatomic Pathology Evaluation A. Cause of Death (Appendix J) There were no test substance-related deaths. All 40 rats survived until the scheduled 28-day sacrifice. B. Organ Weights (Table 14, Appendix I) There were no dermal exposure-related effects on absolute or relative organ weights. All individual and mean organ weight differences were considered to be spurious and unrelated to test substance administration. C. Gross Observations (Table 15, Appendix J) There were no test substance-related gross observations. The three gross observations made at necropsy were interpreted to be incidental lesions unrelated to test substance administration. - 22 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 D. Microscopic Findings (Table 16, Appendix J) There were no test substance-related microscopic findings. All microscopic findings in this study are known to occur naturally in rats o f this stock and age and were not present in a doseresponse fashion in either incidence or severity. E. Anatomic Pathology Conclusions No adverse effects were observed on mortality, organ weights, gross pathology, or histopathology in male rats treated dermally up to and including 1000 mg/kg/day. This is in contrast with the reversible, minimal to mild thyroid follicular hypertrophy observed at 1000 mg/kg/day in the oral study. - 23Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 CONCLUSIONS Under the conditions o f this study, the no-observed-effect level (NOEL)a for H-25425 in male rats was 1000 mg/kg/day, the highest dosage tested. The NOEL is based on a lack o f adverse test substance-related effects on any in-life, clinical pathology, or anatomic pathology parameter in male rats treated dermally at dosages up to 1000 mg/kg/day. RECORDS AND SAMPLE STORAGE All data and records for analytical characterizations conducted by or for the sponsor will be retained by the sponsor. Laboratory-specific or site-specific raw data, such as personnel files and equipment records will be retained by the facility where the work was done. A sample o f the test substance will be collected for archive purposes and retained at Haskell Laboratory, Newark, Delaware. 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. The NOEL for this study is defined as the highest dose at which toxicologically important effects attributable to the test substance were not detected. Thus, for this study, the NOEL is equivalent to the NOEL as defined by the United States Environmental Protection Agency05) and to the no-observed-adverse-effect level (NOAEL) as defined by the European Union.06' - 24Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ DuPont-13830 REFERENCES 1. DuPont Haskell Laboratory (2003). H-25425: Subchronic Toxicity, 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations. Unpublished report, 2. Capka V., Bowers, C., Narvesen, J.N., and Rossi, R.F. Determination o f total fluorine in blood at trace concentration levels by the Wickbold decomposition method with direct potentiometric detection. Talanta 2004 accepted for publication. 3. Draper, N.R. and Smith, H. (1981). Applied Regression Analysis, 2nd edition, pp 266-273. Wiley, New York. 4. Selwyn, M.R. (1995). The use o f trend tests to determine a no-observable-effect level in animal safety studies. Journal o f the American College o f Toxicology 14(2), 158-168. 5. Jonckheere, A.R. (1954). A distribution-free K-sample test against ordered alternatives. Biometrika 41,133-145. 6. Levene, H. (1960). Robust test for equality o f variances. Contributions to Probability and Statistics (J. Olkin, ed.), pp 278-292. Stanford University Press, Palo Alto. 7. Shapiro, S.S. and Wilk., M.B. (1965). An analysis o f variance test for normality (complete samples). Biometrika 52, 591-611. 8. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th edition, pp 246-248 and 349-352. The Iowa State University Press, Iowa. 9. Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482-491. 10. Dunnett, C.W. (1980). Pairwise multiple comparisons in the unequal variance case. J. Amer. Statist. Assoc. 75, 796-800. 11. Tamhane, A.C. (1979). A comparison o f procedures for multiple comparison o f means with unequal variances. J. Amer. Statist. Assoc. 74,471-480. 12. Rruskal, W.H. and Wallis, W.A. (1952). Use o f ranks in one-criterion analysis o f variance. J. Amer. Statist. Assoc. 47, 583-621. 13. Dunn, O.J. (1964). Multiple contrasts using rank sums. Technometrics 6, 241-252. 14. Fisher, R.A. (1985). Statistical Methodsfo r Research Workers, 13th edition. Hafftier, New York. 15. Hazard Evaluation Division, Standard Evaluation Procedure, Toxicity Potential: Guidance for Analysis and Evaluation o f Subchronic and Chronic Exposure Studies Paynter, O. E. et al., United States Environmental Protection Agency, Office o f Pesticide Programs, Washington, D.C., 20406. EPA-540/9-85-020. (June 1985). 16. Risk Assessment o f Notified New Substances. Technical Guidance Document (XE283/94-EN), Chapter I, Sections 2.24 and 2.25. 1994. - 25Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 TABLES -26Company Sanitized. Does not contain TSCA CBJ H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______ _______ TABLES Abbreviations EXPLANATORY NOTES mg/kg = mg/kg/day g/anm/day = grams/animal/day Summary of Hematology Values RBC - red blood cell count HGB - hemoglobin HOT - hematocrit MCV - mean corpuscular volume MCH - mean corpuscular hemoglobin MCHC - mean corpuscular hemoglobin concentration RDW - red cell distribution width ARET - absolute reticulocyte count PLT - platelet count WBC - white blood cell count ANEU - absolute neutrophil (all forms) ALYM - absolute lymphocyte AMON - absolute monocyte AEOS - absolute eosinophil ABAS - absolute basophil ALUC - absolute large unstained cell -- - no data Summary of Coagulation Values PT - prothrombin time APTT - activated partial thromboplastin time -- - no data DuPont-13830 - 27Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 TABLES EXPLANATORY NOTES Summary of Clinical Chemistry Values AST - aspartate aminotransferase ALT - alanine aminotransferase SDH - sorbitol dehydrogenase ALKP - alkaline phosphatase BILI - total bilirubin BUN - urea nitrogen CREA - creatinine CHOL - cholesterol TRIG - triglycerides GLUC - glucose TP - total protein ALB - albumin GLOB - globulin CALC - calcium IPHS - inorganic phosphorous NA - sodium K - potassium CL - chloride PFLU - plasma fluoride -- - no data Summary of Urinalysis Values VOL - volume UOSM - urine osmolality pH - the logarithm o f the reciprocal of the hydrogen ion concentration URO - urobilinogen UFLU - urine fluoride UMTP - urine protein -- - no data Clinical Pathology Note When an individual observation was recorded as being less than a certain value, calculations were performed on half the recorded value. For example, if bilirubin was reported as <0.1, 0.05 was used for any calculations performed with that bilirubin data. -28Company Sanitized. Does not contain TSCA CBI Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ Group : Dosage(mg/kg) Test DAYO DAY3 DAY7 D A Y 10 DAY14 D A Y 17 DAY21 DAY24 DAY27 DAY28 TABLE 1 MEAN BODY WEIGHTS OF MALE RATS (g) IX XI XIII 0 10 100 206.8 9.0(10) 219.0 13.9(10) 248.2 12.8(10) 271.3 16.7(10) 299.1 17.5(10) 310.0 13.3(10) 324.2 16.4(10) 339.9 16.3(10) 350.3 21.6(10) 332.6 21.0(10) 202.4 9.7(10) 220.9 10.7(10) 255.2 11.8(10) 277.4 14.4(10) 300.9 14.9(10) 309.5 18.5(10) 319.9 20.5(10) 337.5 19.0(10) 344.0 24.4(10) 326.4 22.6(10) 204.5 8.4(10) 218.6 9.1(10) 247.9 12.8(10) 267.3 15.2(10) 283.6 23.5(10) 294.5 24.0(10) 309.4 21.3(10) 322.9 20.8(10) 331.4 24.6(10) 312.8 25.5(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) There were no statistically significant differences from control at p < 0.05. XV 1000 207.0 6.6(10) 222.4 8.9(10) 253.5 10.0(10) 274.1 10.0(10) 297.5 8.6(10) 307.3 16.2(10) 321.4 13.0(10) 333.6 15.0(10) 337.3 23.7(10) 320.3 21.6(10) -29- DuPont-13830 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Group: Dosage(mg/kg) Test DAYO -DAY3 DAY3 -DAY7 DAY7 -DAY10 DAY10 - DAY14 DAY14 -DAY17 DAY17 -DAY21 DAY21 -DAY24 DAY24 -DAY27 DAY27 -DAY28 DAYO -DAY28 TABLE 2 MEAN BODY WEIGHT GAINS OF MALE RATS (g) IX XI XIII 0 10 100 XV 1000 12.2 6.4(10) 29.2 6.3(10) 23.1 6.0(10) 27.9 3.6(10) 10.8 6.9(10) 14.3 8.8(10) 15.7 8.4(10) 10.4 10.1(10) -17.7 3.3(10) 125.7 16.3(10) 18.6 5.8(10) 34.3 3.6(10) 22.2 4.7(10) 23.6# 4.1(10) 8.5 10.6(10) 10.5 15.7(10) 17.6 7.6(10) 6.5 16.3(10) -17.6 4.5(10) 124.0 19.8(10) 14.1 2.9(10) 29.3 5.7(10) 19.4 5.0(10) 16.3# 10.7(10) 10.9 7.0(10) 14.9 7.5(10) 13.5 5.4(10) 8.5 8.7(10) -18.6 3.3(10) 108.3 21.8(10) 15.4 5.1(10) 31.1 2.1(10) 20.7 1.9(10) 23.4# 5.1(10) 9.8 10.0(10) 14.1 8.1(10) 12.2 6.5(10) 3.7 13.5(10) -17.1 4.1(10) 113.3 19.8(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) # Statistically significant difference at p < 0.05 by Jonckheere-Terpstra trend test -30- DuPont-13830 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 3 Group : Dosage(mg/kg) Test DAY0-DAY7 DAY7-DAY14 DAY14-DAY21 DAY21-DAY27 DAY0-DAY27 MEAN DAILY FOOD CONSUMPTION BY MALE RATS (g/day) IX XI XIII 0 10 100 21.7 1.7(10) 25.8 2.0(10) 24.9 0.9(10) 27.1 1.8(10) 24.792 1.230(10) 23.3 1.1(10) 26.4 1.6(10) 24.7 1.9(10) 27.0 2.0(10) 25.293 1.331(10) 22.0 1.3(10) 24.2 2.6(10) 23.9 2.4(10) 25.7 2.9(10) 23.863 1.974(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) There were no statistically significant differences from control at p < 0.05. DuPont-13830 XV 1000 22.9 1.5(10) 26.1 1.2(10) 25.1 1.0(10) 26.3 2.8(10) 25.053 1.303(10) Company Sanitized. Does not contain TSCA CBI -31 - H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Group: Dosage(mg/kg) Test DAY0-DAY7 DAY7-DAY14 DAY14-DAY21 DAY21-DAY27 DAY0-DAY27 TABLE 4 MEAN DAILY FOOD EFFICIENCY OF MALE RATS (g body weight gain/g food consumed) IX XI XIII 0 10 100 0.272 0.042(10) 0.281 0.023(10) 0.144 0 . 042 (10) 0.156 0.080(10) 0.214 0 . 020 (10) 0.324 0.055(10) 0.248# 0.038(10) 0.106 0.084(10) 0.145 0.090(10) 0.207 0.024(10) 0.281 0.034(10) 0.206# 0.075(10) 0.156 0.050(10) 0.137 0.075(10) 0.196# 0.022(10) Data arranged as: Mean Standard deviation (Number of values included in calculation) # Statistically significant difference at p < 0.05 by Jonckheere-Terpstra trend test DuPont-13830 XV 1000 0.289 0.028(10) 0.241# 0.020(10) 0.136 0.043(10) 0.092 0.099(10) 0.192# 0.025(10) Company Sanitized. Does not contain TSCA CBI -32- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 5 SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - PREDOSING Treatment Group Dose Animal Count Eye Observations Dark Incidence Mean onset (Days) Corneal Opacity Incidence Mean onset (Days) Opaque Incidence Mean onset (Days) Enophthalmus Incidence Mean onset (Days) Discharge Eye incidence Mean onset (Days) Nose Incidence Mean onset (Days) Hair Loss Incidence Mean onset (Days) IX 0 mg/kg (Control) 10 XI 10 mg/kg 10 2 ( 20%) 17 0 0 0 1 ( 10%) 28 0 0 0 4 ( 40%) 15 2 ( 20%) 19 2 ( 20%) 21 2 ( 20%) 3 1 ( 10%) 17 1 ( 10%) 17 XIII 100 mg/kg 10 3 ( 30%) 14 1 ( 10%) 17 0 1 ( 10%) 21 2 ( 20%) 7 3 ( 30%) 15 1 ( 10%) 24 XV 1000 mg/kg 10 1 ( 10%) 28 0 0 0 2 ( 20%) 7 3 ( 30%) 22 0 DuPont-13830 Company Sanitized. Does not contain TSCA CBI - 33- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 5 (Continued) SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - PREDOSING Treatment Group Dose Animal Count IX 0 mg/kg (Control) 10 XI 10 mg/kg 10 XIII 100 mg/kg 10 Wound Superficial Incidence Mean onset (Days) 2 ( 20%) 19 3 ( 30%) 21 2 ( 20%) 18 Incidence - The number of animgls for which an observation was recorded. Mean onset (Days) - The mean of the first test day an observation was recorded for that group. There were no statistically significant differences at p < 0.05 by Cochran-Armitage trend test. XV 1000 mg/kg 10 1 ( 10%) 14 DuPont-13830 Company Sanitized. Does not contain TSCA CBI -34- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE Treatment Group Dose Animal Count SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - POSTDOSING IX 0 mg/kg (control) 10 XI 10 mg/kg 10 XIII 100 mg/kg 10 XV 1000 mg/kg 10 Eye Observations Dark Incidence Mean onset (Days) Corneal Opacity Incidence Mean onset (Days) Opaque Incidence Mean onset (Days) Enophthalmus Incidence Mean onset (Days) Discharge Eye Incidence Mean onset (Days) Nose Incidence Mean onset (Days) Hair Loss Incidence Mean onset (Days) 3 ( 30%) 17 0 1 ( 10%) 27 0 10 (100%) 0 10 (100%) 11 2 ( 20%) 21 1 ( 10%) 8 0 0 0 .10 (100%) 0 10 (100%) 12 1 ( 10%) 17 4 ( 40%) 13 1 ( 10%) 15 0 1 ( 10%) 18 10 (100%) 0 10 (100%) 12 1 ( 10%) 24 0 0 0 0 10 (100%) 0 10 (100%) 12 0 DuPont-13830 Company Sanitized. Does not contain TSCA CBI - 35- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 6 (Continued) Treatment Group Dose Animal Count SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - POSTDOSING IX 0 mg/kg (Control) 10 XI 10 mg/kg 10 XIII 100 mg/kg 10 XV 1000 mg/kg 10 Wound Superficial Incidence Mean onset (Days) 2 i( 20%) 17 3 ! 30%) 18 2 ( 20%) 15 Stain Fur/Skin Incidence Mean onset (Days) 2 i1 20%) 19 1 -( 10%) 11 1 ( 10%) 11 Swollen Observations Face Incidence Mean onset (Days) 2 1[ 20%) 1 2 i( 20%) 2 1 ( 10%) 0 Incidence - The number of animals for which an observation was recorded. Mean onset (Days) - The mean of the first test day an observation was recorded for that group. There were no statistically significant differences at p < 0.05 by Cochran-Armitage trend test. 1 ( 10%) 11 2 ( 20%) 19 0 DuPont-13830 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 TABLE 7 PERCENT SURVIVAL OF MALE RATS Group: Dosage (mg/kg) IX 0 XI x m XV 10 100 1000 Davs on Test 0 7 14 21 28 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Number at study start Alive on test day 28 10 10 10 10 10 10 10 10 Percent Survival = (Number o f rats alive/Number o f rats at risk)* 100 Number o f rats at risk = Number at study start. /-"V There were no statistically significant decreases in survival. - 37Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ TABLE 8 MEAN PFOA CONCENTRATIONS IN MALE RATS Dosage (mg/kg/day) PLASMA PFOA (ppb)a Mean S.D. n 0 10 100 1000 < 10 < 10 13b 105 0.0 0.0 2 24 10 10 10 10 n = number o f samples S.D. = Standard Deviation a Lower limit o f quantitation (LLOQ) = 1 0 ppb b Value o f 10 was used in the calculation when the concentration was below LLOQ (clOppb) DuPont-13830 - 38- Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______ _______ TABLE 9 MEASURED CONCENTRATION (ppb) WHOLE BLOOD FLUORINE Dosage (mg/kg/day)________________Mean___________________ n 0 10 100 1000 < 500 ppb or ND ND <500 ppb or ND < 500 ppb or ND n = number o f samples ND = not detectable = values < 200 ppb Values "less than 500 ppb" are > 200 ppb and < 500 ppb. 500 ppb = Limit o f quantification (LOQ). 5 5 5 5 DuPont-13830 - 39Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 TABLE 10 SUMMARY OF HEMATOLOGY VALUES FOR MALE RATS Group DC 0 mg/kg Group XI 10 mg/kg Group x n r 100 mg/kg Group XV 1000 mg/kg RBC (xlO%tL) DAY 28 ' HGB (g/dL) DAY 28 HCT (%) DAY 28 MCV (fL) DAY 28 MCH (pg) DAY 28 MCHC (g/dL) DAY 28 RDW (%) DAY 28 ARET (x l0 3/gL) DAY 28 PLT (xlOV^tL) DAY 28 WBC (xlOVjiiL) DAY 28 ANEU (x l0 3/ju.L) DAY 28 ALYM (x l0 3/juL) DAY 28 7.31 0.15(7) 14.6 0.4(7) 47.3 1.5(7) 64.8 2.0(7) 20.0 0.7(7) 30.9 0.6(7) 11.5 0.6(7) 321.9 95.3(7) 1264 260(2) 14.85 3.11(7) 2.49 0.95(7) 11.50 2.93(7) 7.16 0.28(9) 14.7 0.4(9) 46.7 1.4(9) 65.3 1.5(9) 20.6 0.6(9) 31.5* 0.4(9) 11.6 0.4(9) 264.4 58.4(9) 1142 117(7) 13.80 2.50(9) 2.15 0.46(9) 10.86 2.06(9) 7.07 0.26(9) 14.3 0.5(9) 45.7 1.7(9) 64.6 1.1(9) 20.3 0.5(9) 31.4 0.4(9) 11.7 0.9(9) 302.0 140.7(9) 1210 81(8) 14.31 2.41(9) 2.25 1.02(9) 11.32 1.64(9) 7.18 0.36(9) 14.5 0.4(9) 45.7 1.4(9) 63.7 1.6(9) 20.1 0.6(9) 31.6* 0.3(9) 11.2 0.3(9) 251.0 55.3(9) 1242 112(8) 13.84 2.23(9) 1.97 0.40(9) 11.05 1.99(9) - 40Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 TABLE 10 (Continued) SUMMARY OF HEMATOLOGY VALUES FOR MALE RATS Group DC 0 mg/kg Group XI 10 mg/kg Group XD! 100 mg/kg Group XV 1000 mg/kg AMON (xlOVjuL) DAY 28 AJEOS (xlOVjuL) DAY 28 ABAS (x l0 3//xL) DAY 28 ALUC (xlOVjuL) DAY 28 0.39 0.08(7) 0.20 0.10(7) 0.08 0.04(7) 0.20 0.14(7) 0.32 0.13(9) 0.15 0.06(9) 0.09 0.05(9) 0.23 0.12(9) 0.36 0.11(9) 0.13 0.07(9) 0.07 0.04(9) 0.17 0.10(9) 0.42 0.11(9) 0.15 0.04(9) 0.06 0.02(9) 0.19 0.05(9) Data arranged as: Mean Standard deviation (Number o f values included in calculation) * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test. -41 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 TABLE 11 SUMMARY OF COAGULATION VALUES FOR MALE RATS Group IX Group XI Group XDI Group XV 0 mg/kg________ 10 mg/kg_______ 100 mg/kg______ 1000 mg/kg PT (sec) DAY 28 APTT (sec) DAY 28 15.5 0.5(10) 18.0 1.6( 10) 15.9 0.6(10) 18.7 2.2( 10) 15.6 0.5(10) 17.5 1.2 ( 10) 16.1 0.8(10) 18.8 1.9(10) Data arranged as: Mean Standard deviation (Number o f values included in calculation) There were no statistically significant differences from control at p < 0.05. -42- Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 TABLE 12 SUM M ARY OF CLINICAL CHEMISTRY VALUES FOR MALE RATS Group IX 0 mg/kg Group XI 10 mg/kg Group XEt 100 mg/kg Group XV 1000 mg/kg AST (U/L) DAY 28 ALT (U/L) DAY 28 SDH (U/L) DAY 28 ALKP (U/L) DAY 28 BILI (mg/dL) DAY 28 BUN (mg/dL) DAY 28 CREA (mg/dL) DAY 28 CHOL (mg/dL) DAY 28 TRIG (mg/dL) DAY 28 GLUC (mg/dL) DAY 28 TP (g/dL) DAY 28 ALB (g/dL) DAY 28 122 30(10) 52 7(10) 18.6 7.6(10) 212 45(10) 0.05 0.00(10) 18 2(10) 0.29 0.05(10) 48 11(10) 43 18(10) 113 28(10) 6.1 0.3(10) 4.1 0.2(10) 122 22(10) 51 9(10) 15.0 7.0(10) 220 47(10) 0.06 0.02(10) 17 2(10) 0.26 0.05(10) 49 7(10) 48 24(10) 104 12(10) 6.1 0.4(10) 4.1 0.3(10) 106 20(10) 44 9(10) 11.9 2.4(10) 205 43(10) 0.05 0.00(10) 15* 2(10) 0.24* 0.02(10) 48 5(10) 36 13(10) 101 10(10) 6.0 0.3(10) 4.1 0.1(10) 108 23(10) 47 11(10) 12.9 4.6(10) 244 51(10) 0.06 0.02(10) 15* 2(10) 0.22* 0.03(10) 39* 6(10) 31 13(10) 107 18(10) 6.0 0.3(10) 4.1 0.2(10) - 43Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats............... ..... DuPont-13830 TABLE 12 (Continued) SUMMARY OF CLINICAL CHEMISTRY VALUES FOR MALE RATS Group IX 0 mg/kg Group XI 10mg/kg Group X m 100 mg/kg Group XV 1000 mg/kg GLOB (g/dL) DAY 28 CALC (mg/dL) DAY 28 IPHS (mg/dL) DAY 28 NA (mmol/L) DAY 28 K (mmol/L) DAY 28 CL (mmol/L) DAY 28 PFLU (jU.g/mL) DAY 28 2.0 0.3(10) 11.4 0.7(10) 11.9 2.2(10) 147.2 1.7(10) 6.41 0.65(10) 102.3 2.1(10) 0.1 0.0(10) 2.1 0.2(10) 11.2 0.6(10) 11.2 1.5(10) 147.6 2.3(10) 6.25 0.42(10) 102.6 1.7(10) 0.1 0.0(10) 2.0 0.2(10) 11.1 0.3(10) 10.4 0.7(10) 147.7 1.6(10) 6.05 0.45(10) 103.2 1.3(10) 0.1 0.0(10) 1.9 0.2(10) 11.2 0.2(10) 10.7 0.6(10) 148.2 1.8(10) 5.80* 0.30(10) 104.2 2.5(10) 0.1 0.0(10) Data arranged as: Mean Standard deviation (Number o f values included in calculation) * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test. -44- Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 TABLE 13 SUMMARY OF URINALYSIS VALUES FOR MALE RATS Group IX 0 mg/kg Group XI 10 mg/kg Group X m 100 mg/kg Group XV 1000 mg/kg VOL (mL) DAY 28 UOSM (mOsm) DAY 28 pH DAY 28 URO (EU/dL) DAY 28 UMTP (mg/dL) DAY 28 UFLU (fig) DAY 28 9.0 5.0(10) 1069 691(10) 7.5 0.4(9) 0.2 0.0(9) 62 35(10) 8.0 2.9(10) 7.2 4.5(10) 1461 662(10) 7.4 0.8(10) 0.2 0.0(10) 111* 59(9) 7.1 2.0(10) 11.2 5.0(10) 778 407(10) 7.6 0.5(10) 0.2 0.0(10) 48 33(10) 8.1 1.6(10) 8.5 3.4(10) 924 346(10) 7.4 0.5(10) 0.2 0.0(10) 59 33(10) 8.0 1.6(10) Data arranged as: Mean Standard deviation (Number o f values included in calculation) * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test. - 45Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 14 MEAN FINAL BODY AND ORGAN WEIGHTS FOR MALE RATS Group : Dosage(mg/kg) Test LIVER KIDNEYS THYROID GLAND FINAL BODY WEIGHT LIVER/ FINAL BODY * 100 KIDNEYS/ FINAL BODY * 100 THYROID GLAND/ FINAL BODY * 100 MEAN FINAL BODY, ABSOLUTE AND RELATIVE ORGAN WEIGHTS (GRAMS) IX XI 0 10 XIII 100 XV 1000 10.405 1.287(10) 2.843 0.264(10) 0.016 0.003(10) 10.571 1.087(10) 2.890 0.294(10) 0.017 0.004(10) 9.923 1.142(10) 2.818 0.246(10) 0.017 0.003(10) 10.292 0.744(10) 2.815 0.146(10) 0.017 0.004(10) 332.550 20.986(10) 326.380 22.630(10) 312.830 25.545(10) 320.270 21.593(10) 3.120 0.215(10) 3.236 0.199(10) 3.166 0.170(10) 3.218 0 . 2 0 2 (10) 0.855 0.052(10) 0.886 0 . 0 7 3 (10) 0.901 0 . 0 4 2 (TO) 0.881 0.047(10) 0.005 0.001(10) 0.005 0.001(10) 0.005 0.001(10) 0.005 0.001(10) Data summarized as: Mean Standard Deviation (n) Statistical Methods: Trend test (Jonckheere-Terpstra). There were no statistically significant differences at p < 0.05. DuPont-13830 Company Sanitized. Does not contain TSCA CBI -46- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 LESIONS TABLE 15 INCIDENCES OF GROSS OBSERVATIONS IN MALE RATS TREATMENT LESION INCIDENCE (Numeric) Males 0 mg/kg IX 10 mg/kg XI 100 mg /kg XIII 1000 mg /kg XV LIVER NO ABNORMALITY DETECTED DISCOLORATION, BROWN. KIDNEYS NO ABNORMALITY DETECTED DILATATION, RIGHT, PELVIS. THYROID GLAND NO ABNORMALITY DETECTED ORAL CAVITY ULCER/EROSION. GO) 10 (10) 9 1 (10) 10 (10) 9 1 GO) 10 (10) 10 GO) 10 GO) 10 GO) 10 (1) 1 (10) 10 GO) 10 GO) 10 Fi g u r e s in p a r e n t h e s e s are the n u m b e r of a n i m a l s g r o s s l y e x a m i n e d for this' tissue The absence of a number indicates the finding specified was not identified Company Sanitized. Does not contain TSCA CBI -47- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 LESIONS TABLE 15 (Continued) INCIDENCES OF GROSS OBSERVATIONS IN MALE RATS TREATMENT LESION INCIDENCE (Numeric) Males 0 mg/kg IX 10 mg/kg XI 100 mg /kg XIII 1000 mg/kg XV SKIN, TREATED NO ABNORMALITY DETECTED SKIN, UNTREATED NO ABNORMALITY DETECTED (10) 10 (10) 10 (10) 10 (10) 10 (10) 10 (10) 10 GO) 10 GO) 10 Figures in parentheses are the number of animals grossly examined for this tissue The absence of a number indicates the finding specified was not identified Company Sanitized. Does not contain TSCA CBI -48- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 TABLE 16 INCIDENCES AND LESION GRADES OF MICROSCOPIC OBSERVATIONS IN MALE RATS LESIONS TREATMENT LESION INCIDENCE (NUMERIC) Males 0 10 100 1000 mg/kg mg /kg mg/kg mg/kg IX XI XIII XV LIVER NECROSIS, FOCAL. minimal Total observations per lesion INFLAMMATION, SUBACUTE/CHRONIC. minimal mild Total observations per lesion HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED. minimal Total observations per lesion FIBROSIS, FOCAL, CENTRILOBULAR. mild Total observations per lesion KIDNEYS NO ABNORMALITY DETECTED CHRONIC PROGRESSIVE NEPHROPATHY. minimal (10) 1 1 7 3 10 2 2 (10) 1 5 (1) 1 1 (10) 1 1 6 4 10 (10) 71 3 Figures in parentheses are number of animals microscopically examined for this tissue The absence of a number indicates the lesion specified was not identified Company Sanitized. Does not contain TSCA CBI -49- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ) ) DuPont-13830 TABLE 16 (Continued) INCIDENCES AND LESION GRADES OF MICROSCOPIC OBSERVATIONS IN MALE RATS LESIONS LESION INCIDENCE (NUMERIC) Males TREATMENT o 10 100 1000 1 mg/kg mg/kg mg/kg mg/kg j IX XI XIII XV KIDNEYS CHRONIC PROGRESSIVE NEPHROPATHY. Total observations per lesion HYDRONEPHROSIS, UNILATERAL. minimal Total observations per lesion AGGREGATES, LYMPHOID. minimal Total observations per lesion THYROID GLAND NO ABNORMALITY DETECTED HYPERTROPHY, FOLLICULAR CELL. minimal Total observations per lesion ECTOPIC THYMUS TISSUE. (10) 5 1 1 5 5 (10) 8 2 2 1 Figures in parentheses are number of animals microscopically examined for this tissue The absence of a number indicates the lesion specified was not identified (10) 3 1 1 (10) 8 2 2 Company Sanitized. Does not contain TSCA CBI -50- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ) DuPont-13830 TABLE 16 (Continued) INCIDENCES AND LESION GRADES OF MICROSCOPIC OBSERVATIONS IN MALE RATS LESIONS TREATMENT LESION INCIDENCE (NUMERIC) Males 0 10 100 1000 mg/kg| mg/kg mg/kg mg/kg IX XI XIII XV SKIN, TREATED NO ABNORMALITY DETECTED INFLAMMATION, FOCAL, ACUTE. minimal Total observations per lesion SKIN, UNTREATED NO ABNORMALITY DETECTED INFLAMMATION, FOCAL, SUBACUTE/CHRONIC. minimal Total observations per lesion (10) 10 (10) 10 Figures in parentheses are number of animals microscopically examined for this tissue The absence of a number indicates the lesion specified was not identified (10) 9 1 1 (10) 8 2 2 Company Sanitized. Does not contain TSCA CBI -51 - H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 FIGURE -52Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats FIGURE 1 MEAN BODY WEIGHTS OF MALE RATS DuPont-13830 0 mg/kg/day 10 mg/kg/day 100 mg/kg/day 1000 mg/kg/day Company Sanitized. Does not contain TSCA CBI -53- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDICES -54Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDIX A INDIVIDUAL BODY W EIGHTS -55Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Study, Print Date: 14-Jan-2004 Print Time: 15:02:40 Individual Body Weights Body Weight Body Weight 9 Day 0 9 Day 3 Male, IX 0 mg/kg (Control) Body Weight 9 Day 7 Body Weight 9 Day 10 Body Weight 9 Day 14 Body Weight 9 Day 17 Body Weight 9 Day 21 Body Weight 9 Day 24 Body Weight 9 Day 27 901 214.8 902 203.8 903 203.9 904 203.6 905 195.7 906 206.9 907 220.9 908 193.4 909 218.1 910 207.1 225.3 209.0 220.3 218.6 202.8 214.7 243.7 197.2 231.7 226.8 - 259.9 241.9 249.9 250.1 230.5 243.4 264.1 228.0 249.0 265.2 281.5 262.5 266.7 272.8 249.3 263.1 295.2 246.1 284.1 291.2 310.3 284.1 297.0 298.2 276.5 288.1 320.2 277.8 315.0 323.8 323.7 296.6 312.5 308.9 288.5 310.0 326.6 293.7 315.0 324.0 337.2 315.3 309.6 327.5 308.6 320.0 346.8 298.2 342.8 336.2 340.9 321.6 338.5 337.7 333.2 343.6 359.2 308.8 360.0 355.6 357.9 331.5 352.6 337.7 344.2 352.1 356.9 309.9 371.8 388.4 Company Sanitized. Does not contain TSCA CBI 56 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study:! II Body Weight g Day 28 Male, IX 0 mg/kg (Control) 901 340.8 902 309.3 903 337.4 904 321.5 905 323.1 906 331.8 907 340.9 908 296.8 909 357.3 910 366.6 Individual Body Weights Company Sanitized. Does not contain TSCA CBI -57- DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:40 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study) Body Weight Body weight Body Weight ggg Day 0 Day 3 Day 7 Male, XI 10 mg/kg Individual Body Weights Body Weight Body Weight Body Weight ggg Day 10 Day 14 Day 17 Body Weight g Day 21 DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:40 Body Weight Body Weight gg Day 24 Day 27 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 186.5 203.6 206.7 198.7 192.7 202.2 217.6 194.7 215.2 205.6 217.5 219.2 228.8 211.1 202.6 221.9 237.0 211.6 232.6 226.7 , 256.0 254.3 266.3 244.2 236.3 260.8 268.1 238.5 267.2 260.4 283.8 273.4 286.9 264.6 253.1 281.3 295.7 261.9 296.6 276.4 309.2 297.1 300.9 288.1 274.6 305.6 323.0 291.1 321.7 297.9 331.4 309.3 306.8 282.9 293.0 306.7 329.6 308.9 337.3 288.7 341.5 323.7 316.4 309.8 305.1 306.9 317.8 294.0 365.8 318.1 367.9 336.8 328.3 323.0 322.7 325.0 342.3 325.6 375.1 328.0 367.0 328.2 354.2 331.8 324.7 341.4 378.1 305.1 377.7 331.5 Company Sanitized. Does not contain TSCA O CD H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Body Weight g Day 28 Male, XI 10 mg/: 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 349.2 313.8 338.7 317.7 301.8 326.3 352.1 293.8 359.9 310.5 Individual Body Weights Company Sanitized. Does not contain TSCA CBI -59- DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:40 ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ) DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:40 Individual Body Weights Body Weight Body Weight gg Day 0 Day 3 Male, XIII 100 mg/kg Body Weight g Day 7 Body Weight g Day 10 Body Weight g Day 14 Body Weight g Day 17 Body Weight g Day 21 Body Weight g Day 24 Body Weight g Day 27 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 213.5 205.1 201.9 195.4 198.2 200.9 219.5 192.9 212.1 205.4 230.8 219.9 213.9 208.0 213.1 220.5 233.4 204.7 221.7 220.1 . 261.2 257.2 234.2 233.0 241.1 256.4 266.3 231.8 255.2 242.6 279.8 274.1 246.1 246.3 267.6 280.8 285.5 252.3 281.2 259.6 300.1 301.1 234.7 260.7 280.9 306.8 306.7 269.8 297.4 278.1 307.0 315.2 249.8 269.8 302.6 314.6 325.8 272.1 297.2 291.0 325.5 327.6 279.0 281.4 302.5 328.0 339.7 290.5 317.3 302.8 338.2 338.9 290.2 294.8 320.8 348.0 341.2 304.7 337.0 315.1 345.3 348.3 301.2 297.3 342.5 366.5 337.4 303.8 354.6 317.1 Compny Sanitized. Does not contain TSCA CBI 60 ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats____________ Study Body Weight g Day 28 Male, XIII 100 mg/kg 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 329.0 327.8 280.7 278.4 319.5 346.2 325.2 283.0 340.3 298.2 ) _____ _______________ Individual Body Weights Company Sanitized. Does not contain TSCA CBI -61 - DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:40 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study Body Weight Body Weight Body Weight ggg Day 0 Day 3 Day 7 Male, XV 1000 mg/kg Individual Body Weights Body Weight Body Weight Body Weight ggg Day 10 Day 14 Day 17 Body Weight g Day 21 DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:40 Body weight Body Weight gg Day 24 Day 27 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 212.5 203.6 211.9 201.8 200.0 202.3 217.1 198.2 213.7 208.8 226.3 220.8 237.9 213.4 215.3 210.7 226.2 214.4 231.8 226.7 . 254.3 249.3 272.1 245.0 246.4 239.5 259.7 246.3 263.9 258.3 277.7 270.0 290.7 268.7 264.0 259.8 280.2 266.0 285.3 279.0 293.1 289.2 315.2 295.2 294.6 284.4 300.5 296.5 303.5 302.9 305.6 290.4 338.4 298.2 307.8 294.9 311.4 285.2 323.8 317.6 312.4 319.2 340.4 306.6 319.8 306.2 331.4 309.1 337.5 331.6 329.3 332.1 358.8 317.6 336.1 314.4 340.8 316.7 337.1 353.5 330.6 325.2 384.7 305.7 356.0 308.1 347.7 335.0 327.8 352.5 Company Sanitized. Does not contain TSCA O CO H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Body Weight g Day 28 Male, XV 1000 mg/kg 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 311.7 306.0 362.2 294.8 337.9 294.7 332.9 311.3 314.4 336.8 Individual Body Weights Company Sanitized. Does not contain TSCA CBI -63- DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:40 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDIX B INDIVIDUAL FOOD CONSUMPTION -64Com pany Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ INDIVIDUAL FOOD CONSUMPTION EXPLANATORY NOTES Abbreviation g/anm/day = grams/animal/day DuPont-13830 - 65 Com pany Sanitized. Does not contain T SC A C B I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Food Cons. Food Cons. Food Cons g/anm/day g/anm/day g/anm/day Day 7 Day 14 Day 21 Individual Food Consumption Food Cons, g/anm/day Day 27 Male, IX 0 mg/kg (Control) 901 21.5 25.9 902 20.4 23.6 903 21.3 23.8 904 23.5 27.2 905 18.9 23.6 906 22.3 24.5 907 23.2 27.1 908 19.5 24.6 . 909 22.5 28.4 910 23.7 28.9 25.6 23.4 23.5 25.9 24.8 25.5 24.8 24.8 26.2 24.6 25.1 25.0 27.5 25.7 28.6 28.4 25.7 26.0 29.3 30.1 DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:49 Company Sanitized. Does not contain TSCA CBI -66- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Study: Individual Food Consumption Food Cons. Food Cons. Food Cons. Food Cons g/anra/day g/anm/day g/anm/day g/anm/day Day 7 Day 14 Day 21 Day 27 Male, XI 10 mg/kg 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 21.7 22.1 24.3 22.9 24.1 24.5 24.5 21.8 24.0 23.6 27.7 24.8 28.2 23.8 25.7 27.3 27.3 25.6 28.2 25.1 26.3 24.3 26.0 23.5 25.4 25.2 23.2 22.0 28.4 23.0 28.5 25.5 28.0 23.9 26.2 28.8 30.1 26.1 28.3 24.5 ) DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:49 Company Sanitized. Does not contain TSCA CBI -67- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ) Individual Food Consumption Food Cons, Food Cons, Food Cons. Food Cons, g/anm/day g/anm/day g/anm/day g/anm/day Day 7 Day 14 Day 21 Day 27 Male, XIII 100 mg/kg 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 23.7 22.9 19.3 20.4 22.1 23.0 22.8 22.5 21.9 21.4 27.5 25.3 18.7 21.7 24.2 26.2 24.3 23.5 26.7 23.4 26.8 26.2 22.5 21.5 24.3 24.2 24.3 19.0 26.1 23.8 28.6 26.0 22.5 24.0 28.6 29.1 22.9 22.0 28.7 24.5 DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:49 Company Sanitized. D o e s not con tain TSCA CBI -68- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Food Consumption Food Cons. Food Cons. Food Cons. Food Cons g/anm/day g/anm/day g/anm/day g/anm/day Day 7 Day 14 Day 21 Day 27 Male, XV 1000 mg/kg 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 22.4 22.2 24.6 22.4 23.2 20.7 21.7 26.0 23.6 22.7 26.2 24.8 27.1 26.6 27.3 24.5 25.5 28.0 25.5 24.9 25.4 24.9 25.7 23.1 27.1 24.1 25.1 25.3 24.9 24.9 27.3 25.8 29.7 23.3 30.0 24.1 25.4 28.2 21.6 28.0 DuPont-13830 Print Date: 14-Jan-2004 Print Time: 15:02:49 Company Sanitized. Does not contain TSCA CBI - 69- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDIX C INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY RECORDS -70Company Sanitized. Does not contain TSC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY RECORDS EXPLANATORY NOTES Note Clinical observations reported in this appendix were recorded during body weight collection. Clinical observations recorded post-dosing are not included but are reported in Appendix D. -71 Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study:, Individual Clinical Observations Sex Group Animal Observation Days M IX 901 General observation. No Abnormality Detected 0-21 Eye Observations, Dark, Right 24-28 Discharge, Eye right. Red 24 Sacrificed by design 28 M IX 902 General observation, No Abnormality Detected 0-17,24-28 Wound, Superficial, Shoulder, Left 21 Sacrificed by design 28 M IX 903 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 M IX 904 General observation, No Abnormality Detected 0-7,17-24 Eye Observations, Dark, Right 10-14 Eye Observations, Opaque, Right 28 Sacrificed by design 28 M IX 905 General observation. No Abnormality Detected 0,7-14,21-28 Discharge, Eye bilateral, Red 3 Wound, Superficial, Dorsal Body 17 Sacrificed by design 28 M IX 906 General observation. No Abnormality Detected 0-17 Hair Loss, Forepaw, Bilateral 21-28 Sacrificed by design 28 ) DuPont-13830 Print Date: 19-Feb-2004 Print Time: 08:01:56 Company Sanitized. Does not contain TSCA CBI -72- fi-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats . Study M IX 907 M IX 908 M IX 909 M IX 910 Individual Clinical Observations General observation, No Abnormality Detected 0-21 Discharge, Eye bilateral, Red 24 Discharge, Nose, Red 28 Sacrificed by design 28 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 General observation, No Abnormality Detected 0-7,14-28 Discharge, Eye bilateral. Red 10 Discharge, Nose, Red 10 Sacrificed by design 28 General observation. No Abnormality Detected 0-17 Hair Loss, Forepaw, Bilateral 21-28 Sacrificed by design 28 ) DuPont-13830 Print Date: 19-Feb-2004 Print Time: 08:01:56 Company Sanitized. Does not contain TSCA CBI - 73- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats study M XI M XI M XI M XI M XI M XI M XI 1101 1102 1103 1104 1105 1106 1107 Individual Clinical Observations General observation, No Abnormality Detected 0-28 Sacrificed by design 28 General observation, No Abnormality Detected 0-14 Hair Loss, Forepaw, Bilateral 21-28 Hair Loss, Forepaw, Left 17 Wound, Superficial, Forepaw, Left 21-24 Sacrificed by design 28 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 General observation, No Abnormality Detected 0,7-17,24-28 Discharge, Eye bilateral, Red 3 Wound, Superficial, Neck 21 Sacrificed by design 28 General observation, No Abnormality Detected 0,7-28 Discharge, Eye bilateral, Red 3 Sacrificed by design 28 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 General observation. No Abnormality Detected 0-14,21-28 Discharge, Nose, Red 17 Sacrificed by design 28 ) DuPont-13830 Print Date: 19-Feb-2004 Print Time: 08:01:56 Company Sanitized. Does not contain TSCA CBI 7 4- - )> H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________________________ M XI M XI M XI 1108 1109 1110 Individual Clinical Observations General observation, No Abnormality Detected 0-17,28 Wound, Superficial, Shoulder, Left 21-24 Sacrificed by design 28 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 } DuPont-13830 Print Date: 19-Feb-2004 Print Time: 08:01:56 Company Sanitized. Does not contain TSCA C8I -75- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats 4 $Study! M XIII 1301 M XIII 1302 M XIII 1303 M XIII 1304 M XIII 1305 M XIII 1306 M XIII 1307 Individual Clinical Observations General observation, No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Black Sacrificed by design General observation. No Abnormality Detected Hair Loss, Forepaw, Bilateral Sacrificed by design General observation, No Abnormality Detected Eye Observations, Enophthalmus, Right Eye Observations, Dark, Right Sacrificed by design General observation. No Abnormality Detected Wound, Superficial, Shoulder, Bilateral Sacrificed by design General observation, No Abnormality Detected Eye Observations, Dark, Right Sacrificed by design General observation. No Abnormality Detected Sacrificed by design 0-28 28 0-7,17-28 10 10-14 28 0-21 24-28 28 0-3 21-28 7-28 28 0-10,21-28 14-17 28 0-21 24-28 28 0-28 28 DuPont-13830 Print Date: 19-Feb-2004 Print Time': 08:01:56 Company Sanitized. Does not contain TSCA CBI -76- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study- M XIII 1308 M XIII 1309 M XIII 1310 Individual Clinical Observations General observation. No Abnormality Detected Eye Observations, Corneal Opacity, Right Discharge, Eye bilateral. Red Discharge, Nose, Red Comments,wound superficial right eye Sacrificed by design General observation. No Abnormality Detected Eye Observations, Dark, Right Sacrificed by design General observation, No Abnormality Detected Discharge, Nose, Red Sacrificed by design 0,7-14 17,24-28 3,17 17-21 21-28 28 0-7,14-28 10 28 0-14,21-28 17 28 ) DuPont-13830 Print Date: 19-Feb-2004 Print Time: 08:01:56 Company Sanitized. Does not contain TSCA CBI - 77- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study u Individual Clinical Observations M XV M XV M XV M XV M XV M XV M XV M XV 1501 1502 1503 1504 1505 1506 1507 1508 General observation, No Abnormality Detected Discharge, Eye bilateral. Red Discharge, Nose, Red Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Discharge,' Nose, Red Sacrificed by design General observation. No Abnormality Detected Wound, Superficial, Dorsal Body Sacrificed by design General observation. No Abnormality Detected Discharge, Eye bilateral, Red Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Discharge, Nose, Red Sacrificed by design General observation, No Abnormality Detected Sacrificed by design 0,7-24 3 28 28 0-28 28 0-17,24-28 21 28 0-10,21-28 14-17 28 0-7,14-28 10 28 0-28 28 0-14,21-28 17 28 0-28 28 ) DuPont-13830 Print Date: 19-Feb-2004 Print Time: 08:01:56 Company Sanitized. Does not contain TSCA CBI -78- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats StudyJ Individual Clinical Observations M XV M XV 1509 1510 General observation. No Abnormality Detected Eye Observations, Dark, Right Sacrificed by design General observation, No Abnormality Detected Sacrificed by design 0-24 28 28 0-28 28 DuPont-13830 Print Date: 19-Feb-2004 Print Time: 08:01:56 Company Sanitized. Does not contain TSCA CBI -79- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDIX D INDIVIDUAL CLINICAL OBSERVATIONS - POSTDOSING -80Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 INDIVIDUAL CLINICAL OBSERVATIONS- POSTDOSING EXPLANATORY NOTES Clinical observations reported in this appendix were recorded daily after removal o f the test substance. Clinical observations recorded during body weight collection are not included but are reported in Appendix C. -81 Com pany Sanitized. Does not contain T SC A CBI immmjH-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Sex Group Animal M IX 901 Individual Clinical Observations - Po Observation General observation, No Abnormality Detected Eye Observations, Dark, Right Discharge, Eye bilateral, Red Discharge, Nose, Red M IX 902 Discharge, Eye right, Red Stain Fur/Skin, Thoracic, Red General observation. No Abnormality Detected Discharge, Eye bilateral, Red M IX 903 Discharge, Nose, Red Wound, Superficial, Shoulder, Left General observation, No Abnormality Detected Discharge, Eye bilateral, Red M IX 904 Discharge, Nose, Red Swollen Observations, Face General observation, No Abnormality Detected Eye Observations, Opaque, Right Eye Observations, Dark, Right Discharge, Eye bilateral. Red Discharge, Nose, Red Company Sanitized. Does not contain TSCA CBI -82- st Dosing Days 3,8,10-11,20 22- 27 0-2,4-7,9, 14-16,21-23 12-15,17-19, 21-23 24-27 19 13,26 0-12,14-16, 23- 25,27 11-12,21-23 17-23 20- 21,25 0-19,22-24, 27 11-19,23,26-27 2 20,24-26 27 8-13 0-12,14-19, 21- 23,27 11-12,14-19 DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study:! Individual Clinical observations - Po Sex Group Animal Observation M IX 904 Swollen Observations, Face M IX 905 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Wound, Superficial, Dorsal Body M IX 906 General observation. No Abnormality Detected Discharge,' Eye bilateral, Red M IX 907 Discharge, Nose, Red Hair Loss, Forepaw, Bilateral General observation, No Abnormality Detected Eye Observations, Dark, Right Discharge, Eye bilateral, Red Discharge, Nose, Red M IX 908 General observation, No Abnormality Detected Discharge, Eye bilateral. Red M IX 909 Discharge, Nose, Red Stain Fur/Skin, Thoracic, Red General observation. No Abnormality Detected Company Sanitized. Does not contain TSCA CBI -83- st Dosing Days 0 11,13,18-27 0-10,15-16 12,14-16 16- 17 0-1,4,10-14, 17- 20 2-3,5-9, 15-16,21-23 15-16 21- 27 13,16,21,25-26 22- 23 0- 12,14-15, 22-24,27 11-12,14-15, 17-20,23,27 0,11-14,20,24-26 1- 10,15-19, 22-23,27 15-19,21 19 13,20,25-26 DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: ) Sex Group Animal M IX 909 Individual Clinical Observations Observation Discharge, Eye bilateral, Red Discharge, Nose, Red M IX 910 General observation, No Abnormality Detected Discharge, E?ye bilateral, Red Discharge, Nose, Red Hair Loss, Forepaw, Bilateral Company Sanitized. Does not contain TSCA CBI -84- - Post Dosing Days 0-12,14-16, 19,21-24,27 9-12,15-19, 21- 23 13,20 0-12,14-16, 22- 23 7,11-12,15-19, 23 21-27 DuPont-13830 Print Date: 20-Jan-2Q04 Print Time: 12:55:22 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: ) Sex Group Animal M XI 1101 Individual Clinical Observations Observation General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red M XI 1102 Swollen Observations, Face General observation. No Abnormality Detected Discharge, Eye bilateral. Red M XI 1103 Discharge, Nose, Red Hair Loss, Forepaw, Bilateral Hair Loss, Forepaw, Left Wound, Superficial, Forepaw, Left Swollen Observations, Face 'General observation, No Abnormality Detected Discharge, Eye bilateral, Red M XI 1104 Discharge, Nose, Red General observation. No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Wound, Superficial, Neck Post Dosing Days 3.10.14.20.24- 27 0-2,4-9, 11-12,16,23 11-13,15-19, 21-23 1 13 0-12,14-19, 23 11-12,14-23 21-27 17- 20 18- 24 2 5.8.13.15.20.24- 0-4,6-7, 10- 12,14,16-19, 21- 23 9,11-12,14 13.24- 26 0-10,12,14-19, 22- 23,27 11-12,14-19, 21-23,27 18-23 DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 27 Company Sanitized. Does not contain TSCA CBI -85- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study > Individual Clinical Observations Sex Group Animal Observation M XI 1105 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Rec M XI 1106 General observation, No Abnormality Detected Discharge, Eye bilateral. Red Discharge,' Nose, Rec M XI 1107 General observation, No Abnormality Detected Discharge, Eye bilateral, Red M XI 1108 Discharge, Nose, Rec General observation. No Abnormality Detected Eye Observations, Dark, Right Discharge, Eye bilateral. Red M XI 1109 Discharge, Nose, Rec Wound, Superficial, Shoulder, Left General observation. No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Rec Company Sanitized. Does not contain TSCA CBI -86- - Post Dosing Days 11,13,20,25-26 . 0-10,12,14-16, 21-24,27 8-9,12,14-19, 21-24,27 0-1,3,13,20,24-27 2,4-12,14-16, 21-23 11-12,17-19, 21- 23 6,8,10-13,24-26 0-5,7,9,14-20, 22- 23 15-23,27 3,11-14,25-27 8 0-2,4-10, 15-19,22-23 16,18-19 19-24 5,11,13,20,24-26 0-4,6-10,12, 14-16,21-23, 27 12,15-19,21-23 ') DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Clinical Observations - Sex Group Animal Observation M XI 1110 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Stain Fur/Skin, Neck, Red Company Sanitized. Does not contain TSCA CBI -87- Post Dosing Days 13,17-27 0-12,15-16 14 11-12 ) DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study Sex Group Animal M XIII 1301 M XIII 1302 M XIII 1303 M XIII 1304 Individual Clinical Observations Observation General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Discharge, Eye bilateral, Red Discharge, Nose, Black Discharge, Nose, Red General observation, No Abnormality Detected Eye Observations, Dark, Right Discharge, Eye bilateral, Red Discharge, Nose, Red Hair Loss, Forepaw, Bilateral Stain Fur/Skin, Forelimb, Bilateral, Red Stain Fur/Skin, Shoulder, Bilateral, Red Stain Fur/Skin, Neck, Red General observation. No Abnormality Detected Eye Observations, Enophthalmus, Right Eye Observations, Dark, Right Discharge, Eye bilateral, Red Post Dosing Days 6,11-14,17-21, 24-27 0-5,7-10, 15- 16,22-23 23 0-12,15-16, 18-19,21-25, 27 9-16 8,17-23,25-27 13,15,20 14 0-12,22-23 16- 19,21-23 24-27 11 11 11 3,6 18-27 7- 27 0-2,4-5, 8- 10,16 DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 Company Sanitized. Does not contain TSCA CBI -88- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Sex Group Animal M XIII 1304 Individual Clinical Observations - Po Observation Discharge, Nose, Red M XIII 1305 M XIII 1306 General observation. No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Wound, Superficial, Shoulder, Bilateral General observation, No Abnormality Detected Eye Observations, Dark, Right Discharge, Eye bilateral, Red M XIII 1307 Discharge, Nose, Red General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red M XIII 1308 M x i i i 1309 Swollen Observations, Face General observation, No Abnormality Detected Eye Observations, Corneal Opacity, Right Discharge, Eye bilateral. Red Discharge, Nose, Red Comments, wound superficial right eye General observation, No Abnormality Detected Company Sanitized. Does not contain TSCA CBI - 89- st Dosing Days 14-16,21-23, 27 4,10,21-22:,24-26 0-3,5-9,15-16 3,15-16,23,27 11-20 3.8.11- 13,17-21 22-27 0-2,4-7, 9-10,22-23,27 14- 16 13,20,22,24-26 0-12,14-19, 21,23 11-12,14-19, 21,23,27 0-1 10 15- 27 0-9,11-20 9.11- 23,27 18-27 20-21,24-26 DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male R a t s _______ Study- Individual Clinical Observations Sex Group Animal Observation M XIII 1309 Eye Observations, Dark, Right Discharge, Eye bilateral, Red Discharge, Nose, Red M XIII 1310 General observation, No Abnormality Detected Discharge, Eye bilateral. Red Discharge, Nose, Red Company Sanitized. Does not contain TSCA CBI - 90- - Post Dosing Days 9-13 0-9,15-16, 22-23,27 11-12,14-19, 23,27 5,10,13,21,24-27 0-4,6-9,16, 22-23 8,11-12,14-20, 23 DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study:, 7 Individual Clinical Observations - Post Dosing Sex Group Animal Observation Days M XV 1501 General observation, No Abnormality Detected 10-13,20,24-26 Discharge, Eye bilateral, Red 0-9,14-16 Discharge, Nose, Red 15-19,21-23, 27 M XV 1502 General observation, No Abnormality Detected 2,13,20,24-27 Discharge, Eye bilateral, Red 0-1,3-12, 15-19,22-23 Discharge, Nose, Red 11-12,14-19, 21-23 M XV 1503 General observation. No Abnormality Detected 5-6,10,22,24-26 Discharge, Eye bilateral, Red 0-4,7-9 Discharge, Nose, Red 11-21,23,27 M XV 1504 General observation, No Abnormality Detected 2-4,6-7,10, 24,26 Discharge, Eye bilateral, Red 0-1,5,8-9, 15-16 Discharge, Nose, Red 14-23,27 Wound, Superficial, Dorsal Body 11-16 Wound, Superficial, Shoulder, Left 25 Stain Fur/Skin, Thoracic, Red 19-20 M XV 1505 General observation, No Abnormality Detected 13,17,20,24-26 Discharge, Eye bilateral, Red 0-10,15-16 Discharge, Nose, Red 11-12,14-16, 18-19,21-23, 27 ) DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 Company Sanitized. Does not contain TSCA CBI -91- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: Individual Clinical Observations - Sex Group Animal Observation M XV 1506 General observation, No Abnormality Detected Discharge, Eye bilateral, Red M XV 1507 Discharge, Nose, Red General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge: Nose, Red M XV 1508 General observation, No Abnormality Detected Discharge, Eye bilateral. Red Discharge, Nose, Red M XV 1509 Stain Fur/Skin, Thoracic, Red General observation. No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red M XV 1510 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Company Sanitized. Does not contain TSCA CBI -92- - Post Dosing Days 6,11,13-14,20, 25-27 0-5,7-10,12, 15-19,23-24 12,15-19,21-23 2-6,22,24-25 0-1,7-21,23, 26-27 11-12,16-21, 23,27 3,5,8,13,22,24,26-27 0-2,4,6-7, 9-12,15-16. 11-12,14-21, 23,25 19 2,5,9-10,13,16, 20.22.24- 26 0-1,3-4,6-8 11-12,14-15, 17-19,21,23,27 10.13.24- 26 0-9,11-12, 14-20,27 9,11-12,15-16, -18-23 DuPont-13830 Print Date: 20-Jan-2004 Print Time: 12:55:22 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDIX E INDIVIDUAL EDEMA AND ERYTHEMA SCORES -93Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 INDIVIDUAL EDEMA AND ERYTHEMA SCORES EXPLANATORY NOTES DRAIZE3 SCALE FOR SCORING SKIN IRRITATION Evaluation of Skin Reactions Erythema and eschar formation: No erythema........................................................................................................................ Very slight erythema (barely perceptible)....................................................................... Well-defined erythema............................ .......................................................................... Moderate to severe erythem a............................................................................................ Severe erythema (beet redness) to slight eschar formation (injuries in dep th )........... Edema formation: No edem a........................................................................................................................... Very slight edema (barely perceptible).......................................................................... Slight edema (edges o f area well defined by definite raising)................ ................... Moderate edema (raised approximately 1.0 mm )......................................................... Severe edema (raised more than 1.0 mm extending beyond the area o f exposure) .. Score 0 1 2 3 4 0 1 2 3 4 a Draize, J. H., "Dermal Toxicity." Appraisal o f the Safety o f Chemicals in Foods. Drugs and Cosmetics. The Editorial Committee o f the Association o f Food and Drug Officials o f the United States, Austin, Texas, 1959, pp. 46-59. - 94- Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity mm 728-Day Study in Male Rats Study: Edema Edema Edema Day 0 Day 1 Day 2 Male, IX 0 mg/kg (Control) 901 0 0 0 902 0 0 0 903 0 0 0 904 0 0 0 905 0 0 0 906 0 0 0 907 0 0 0 908 0 0 0 909 0 0 0 910 0 0 0 Edema Day 3 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 4 Day 5 Day 6 Day 7 Day 8 0 00 00 0 0000 0 000 0 00000 00000 0 00 00 00000 0 00 0 0 0 0000 00000 Edema Day 9 0 0 0 0 0 0 0 0 0 0 DuPont-13830 Print Date: 15Print Time: 12 Edema Day 10 Edema Day 11 00 0. 0 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA CBI -95- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study:! Edema Edema Edema Day 12 Day 13 Day 14 Male, IX 0 mg/kg (Control) 901 0 0 0 902 0 0 0 903 0 0 0 904 0 0 0 905 0 0 0 906 0 0 0 907 0 0 0 908 0 0 0 909 0 0 0 910 0 0 0 Edema Day 15 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 16 Day 17 Day 18 Day 19 Day 20 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 Edema Day 21 0 0 0 0 0 0 0 0 0 0 DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:21 Edema Day 22 Edema Day 23 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA CBI -96- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study / Edema Edema Edema Day 24 Day 25 Day 26 Male, IX 0 mg/kg (Control) 901 0 0 0 902 0 0 0 903 0 0 0 904 0 0 0 905 0 0 0 906 0 0 0 907 0 0 0 908 0 0 0 909 0 0 0 910 0 0 ' 0 Edema Day 27 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores ) DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:21 Company Sanitized. Does not contain TSCA CBI -97- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rate______________ Edema Edema Day 0 Day 1 Male, XI 10 mg/kg 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Edema Day 2 0 0 0 0 0 0 0 0 0 .0 Edema Day 3 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 4 Day 5 Day 6 Day 7 Day 8 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 Edema Day 9 0 0 0 0 0 0 0 0 0 0 ) DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:21 Edema Day 10 Edema Day 11 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA CBI -98- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: Edema Edema Day 12 Day 13 Male, XI 10 mg/kg 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Edema Day 14 0 0 0 0 0 0 0 0 0 0 Edema Day 15 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 16 Day 17 Day 18 Day 19 Day 20 000 0 0 000 0 0 000 0 0 000 0 0 000 0 0 000 00 000 0 0 000 00 000 00 000 0 0 Edema Day 21 0 0 0 0 0 0 0 0 0 0 DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:21 Edema Day 22 Edema Day 23 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA CBI -99- H-25425: Repeated-Dose Dermal Toxicity 28-Day Studyin Male Rats_______ Edema Edema Day 24 Day 25 Male, XI 10 mg/kg 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Edema Day 26 Edema Day 27 00 00 00 00 00 00 00 00 00 0 0 Individual Edema and Erythema Scores DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:21 Company Sanitized. Does not contain TSCA CBI - 100- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study Edema Edema Day 0 Day 1 Male, XIII 100 mg/kg 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Edema Day 2 0 0 0 0 0 0 0 0 0 0 Edema Day 3 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 4 Day 5 Day 6 Day 7 Day 8 00000 00000 0 0 .0 0 0 00000 00000 00000 00000 00000 00000 00000 Edema Day 9 0 0 0 0 0 0 0 0 0 0 DuPont-13830 Print Date: lb-Jan-2004 Print Time: 12:04:21 Edema Day 10 Edema Day 11 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA CBI - 101- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study! Edema Edema Day 12 Day 13 Male, XIII 100 mg/kg 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Edema Day 14 0 0 0 0 0 0 0 0 0 0 Edema Day 15 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 16 Day 17 Day 18 Day 19 Day 20 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 Edema Day 21 0 0 0 0 0 0 0 0 0 0 DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:21 Edema Day 22 Edema Day 23 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA CBI - 102- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study Edema Edema Day 24 Day 25 Male, XIII 100 mg/kg 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Edema Day 26 0 0 0 0 0 0 0 0 0 0 Edema Day 27 0 0 0 0 0 0 0 0 0 0 individual Edema and Erythema Scores DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:21 Company Sanitized. Does not contain TSCA CBI -103- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Edema Edema Day 0 Day 1 Male, XV 1000 mg/kg 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Edema Day 2 0 0 0 0 0 0 0 0 0 '0 Edema Day 3 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 4 Day 5 Day 6 Day 7 Day 8 00000 00000 00000 00000 00000 00000 00000 00000 00000 00000 Edema Day 9 0 0 0 0 0 0 0 0 0 0 ) DuPont-13830 Print Date: 15-Jan-2004 Print Time:' 12:04:21 Edema Day 10 Edema Day 11 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA CBI -104- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: Edema Edema Day 12 Day 13 Male, XV 1000 mg/kg 1501 1502 1503 1504 1505 1505 1507 1508 1509 1510 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Edema Day 14 0 0 0 0 0 0 0 0 0 0 Edema Day 15 0 0 0 0 0 0 0 0 0 0 ) Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 16 Day 17 Day 18 Day 19 Day 20 00000 00000 00000 0 00 00 0 00 00 00000 0 00 00 0 00 00 0 00 00 00000 Edema Day 21 0 0 0 0 0 0 0 0 0 0 DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:21 Edema Day 22 Edema Day 23 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA C8I -105- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Edema Edema Day 24 Day 25 Male, XV 1000 mg/kg 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Edema Day 26 0 0 0 0 0 0 0 0 0 '0 Edema Day 27 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores ) DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:21 Company Sanitized. Does not contain TSCA CBI -106- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Individual Edema and Erythema Scores Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Male, XX 0 mg/kg (Control) 901 0 0 0 0 0 0 0 0 0 0 0 0 902 0 0 0 0 0 0 0 0 0 0 0 0 903 0 0 0 0 0 0 0 0 0 0 0 0 904 0 0 0 0 0 0 0 0 0 0 0 0 905 0 0 0 0 0 0 0 0 0 0 0 0 906 0 0 0 0 0 0 0 0 0 0 0 0 907 0 0 0 0 0 0 0 0 0 0 0 0 908 0 0 0 0 0 0 0 0 0 0 0 0 909 0 0 0 0 0 0 0 0 0 0 0 0 910 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI -107- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Edema and Erythema Scores ______________ DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Day 12 Day 13 Day 14 Male, IX 0 mg/kg (Control) Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 Day 21 22 Day 23 901 902 903 904 905 906 907 908 909 910 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 0 -0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI -108- H-25425: Repeated-Dose Dennal Toxicity 28-Day Study in Male Rats Study: Erythema Erythema Erythema Erythema Day 24 Day 25 Day 26 Day 27 Male, IX 0 mg/kg (Control) 901 0 0 0 0 902 0 0 0 0 903 0 0 0 0 904 0 0 0 0 905 0 0 0 0 906 0 0 0 0 907 0 0 0 0 908 0 0 0 0 909 0 0 0 0 910 0 0 0 0 Individual Edema and Erythema Scores ) DuPont-13830 Print Date: 15-Jan-2004 Print Time-.12:04:45 Company Sanitized. Does not contain TSCA CBI -109- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Individual Edema and Erythema Scores Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Male, XI 10 mg/kg 1101 0 0 0 0 0 0 0 0 0 0 0 0 1102 0 0 0 0 0 0 0 0 0 0 0 0 1103 0 0 0 0 0 0 0 0 0 0 0 0 1104 0 0 0 0 0 0 0 0 0 0 0 0 1105 0 0 0 0 0 0 0 0 0 00 0 1106 0 0 0 0 0 0 0 0 0 0 0 0 1107 0 0 0 0 0 0 0 0 0 0 0 0 1108 0 0 0 0 0 0 0 0 0 0 0 0 1109 0 0 0 0 0 0 0 0 0 0 0 0 1110 0 0 -0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 110- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Individual Edema and Erythema Scores Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 Day 21 Day 22 Day 23 Male, XI 10 mg/kg 1101 0 0 0 0 0 0 0 0 0 0 0 0 1102 0 0 0 0 0 0 0 0 0 0 0 0 1103 0 0 0 0 0 0 0 0 0 0 0 0 1104 0 0 0 0 0 0 0 0 0 0 0 0 1105 0 0 0 0 0 0 0 0 0 0 0 0 1106 0 0 0 0 0 0 0 0 0 0 0 0 1107 0 0 0 0 0 0 0 0 0 0 0 0 1108 0 0 0 0 0 0 0 0 0 0 0 0 1109 0 0 0 0 0 0 0 0 0 0 0 0 1110 0 0 -0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 111- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study Erythema Erythema Erythema Erythema Day 24 Day 25 Day 26 Day 27 Male, XI 10 mg/kg 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Company Sanitized. Does not contain TSCA CBI 112 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Individual Edema and Erythema Scores Erythema Erythema Erythema Erythema Erythema Erythema Eryth ema Erythema Erythema Erythema Erythema Erythema Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Male, XIII 100 mg/kg 1301 0 0 0 0 0 0 0 0 0 0 0 0 1302 0 0 0 0 0 0 0 0 0 0 0 0 1303 0 0 0 0 0 0 0 0 0 0 0 0 1304 0 0 0 0 0 0 :0 0 0 0 0 0 1305 0 0 0 0 0 0 0 0 0 0 0 0 1306 0 0 0 0 0 0 0 0 0 0 0 0 1307 0 0 0 0 0 !0 0 0 0 0 0 1308 0 0 0 0 0 0 !o 0 0 0 0 0 1309 0 0 0 0 0 0 !o 0 0 0 0 0 1310 0 0 0 0 0 0 0 0 0 0 0 0 i Company Sanitized. Does not contain TSCA i O03 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rate______________ DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Individual Edema and Erythema Scores Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 Day 21 Day 22 Day 23 Male, XIII 100 mg/kg 1301 0 0 0 0 0 0 0 0 0 0 0 0 1302 0 0 0 0 0 0 0 0 0 0 0 0 1303 0 0 0 0 0 0 .0 0 0 0 0 0 1304 0 0 0 0 0 0 0 0 0 0 0 0 1305 0 0 0 0 0 0 0 0 0 0 0 0 1306 0 0 0 0 0 0 0 0 0 0 0 0 1307 0 0 0 0 0 0 0 0 0 0 0 0 1308 0 0 0 0 0 0 0 0 0 0 0 0 1309 0 0 0 0 0 0 0 0 0 0 0 0 1310 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI -114- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study Erythema Erythema Erythema Erythema Day 24 Day 25 Day 26 Day 27 Male, XIII 100 mg/kg 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores ) DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Company Sanitized. Does not contain TSCA CBI -115- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Studya 7 DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Individual Edema and Erythema Scores Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Male, XV 1000 mg/kg 1501 0 0 0 0 0 0 0 0 0 o- 0 0 1502 0 0 0 0 0 0 0 0 0 0 0 0 1503 0 0 0 0 0 0 0 0 0 0 0 0 1504 0 0 0 0 0 0 0 0 0 0 0 0 1505 0 0 0 0 0 0 0 0 0 0 0 0 1506 0 0 0 0 0 0 0 0 0 0 0 0 1507 0 0 0 0 0 0 0 0 0 0 0 0 1508 0 0 0 0 0 0 0 0 0 0 0 0 1509 0 0 0 0 0 0 0 0 0 0 0 0 1510 0 0 .0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI -116- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study/ DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Individual Edema and Erythema Scores Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Erythema Day 12 Day 13 Day 14 Day 15 Day 16 Day 17 Day 18 Day 19 Day 20 Day 21 Day 22 Day 23 Male, XV 1000 mg/kg 1501 0 0 0 0 0 0 0 0 0 0 0 0 1502 0 0 0 0 0 0 0 0 0 0 0 0 1503 0 0 0 0 0 0 0 0 0 0 0 0 1504 0 0 0 0 0 0 0 0 0 0 0 0 1505 0 0 0 0 0 0 0 0 0 0 0 0 1506 0 0 0 0 0 0 0 0 0 0 0 0 1507 0 0 0 0 0 0 0 0 0 0 0 0 1508 0 0 0 0 0 0 0 0 0 0 0 0 1509 0 0 0 0 0 0 0 0 0 0 0 0 1510 0 0 .0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI -117- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Erythema Erythema Erythema Erythema Day 24 Day 25 Day 26 Day 27 Male, XV 1000 mg/kg 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 0 0 2 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 0 0 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores ) DuPont-13830 Print Date: 15-Jan-2004 Print Time: 12:04:45 Company Sanitized. Does not contain TSCA CBI -118- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDIX F PFOA ANALYTICAL REPORT - 119Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 STUDY TITLE H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Carol Finlay ANALYTICALPHASE COMPLETED ON March 10,2004 PERFORMING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR E. Ldu Pont de Nemours andCompany (DuPont) P.O.Box 50 Newark. DE 19714-0050 USA PROJECT Exygen Study Number: 008-406 Sponsor StudyNo.: DuPont-13830 Total Pages: 65 -120Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number 008-406, entitled "H-25425: Repeated-Dose Dermal Toxicity 28Day Study in Male Rats," was conductedfor DuPont in compliance with US EPA TSCA (40 CFRpart 792) GoodLaboratoryPractice Standards, which are consistent with OECD Principles of Good Laboratory Practice (as revised in 1997), published in ENV7MS/CHEM(98)17. Principal Investigator Exygen Research Study Director DuPont SponsorRepresentative DuPont Exygen Research Date Date /6-A /K - l-ooy Date Page 2 of 65 - 121 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen Study No.: 008-406 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number 008-406, entitled, "H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats". All phases were reviewed for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director andto management. Phase Fortification, and Extraction Raw Data &Draft Analytical Report Review Final Analytical Report Review Date Inspected DateRepeated DateReportedto toExygen DateReported to Principal Management StudyDirector and Investisrator Sponsor Management 01/13/04 01/15/04 01/19/04 02/23/04 01/26/04 01/29/04 02/02/04 02/23/04 02/23/04 02/23/04 02/23/04 02/23/04 Miwa Nabetani Quality AssuranceAuditor Date ExygenResearch Page 3 of 65 -122 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo.: 008-406 CERTIFICATION OFAUTHENTICITY This report, for Exygen Study Number 008-406, is a true and complete representation of the rawdata for the study. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: EaR. Decker Scientist/Principal Investigator ExygenResearch 3/4 * Date Exygen Research Facility Management: Exygen Research StudyDirector, DuPont ilL A b C Carol Finlay ( DuPont f SponsorRepresentative, DuPont: S. MarkKennedy DuPont ExygenResearch -'UQH Date Date Page4 of 65 - 123Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 EXYGEN STUDY IDENTIFICATION H-25425: Repeated-DoseDermal Toxicity 28-Day Study in Male Rats SPONSOR STUDY NUMBER: DuPont-13830 EXYGEN STUDY NUMBER: 008-406 TYPE OFANALYTICALPHASE: Residue SAMPLE MATRIX: Rat Plasma TEST SUBSTANCE: Pentadecafluorooctanoic acid (PFOA) SPONSOR: E. I. du Pont de Nemours andCompany P.O. Box 50 Newark, DE 19714-0050 Phone: (302) 366-5255 STUDY DIRECTOR: Carol Finlay DuPont PERFORMINGLABORATORY: ExygenResearch 3058 Research Drive State College, PA 16801 ANALYTICALPHASE TIMETABLE: StudyInitiation Date: 11/18/03 Analytical StartDate: 01/13/04 Analytical Termination Date: 01/15/04 Analytical Report Completion Date: 03/10/04 Exygen Research Page 5 of 65 -124Company Sanitized. Does not contain TSCA C8I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 PROJECT PERSONNEL Hie Study Director fra:Otisproject at DuPont was Carol Finlay. The following personnel fromExygen Research were associated withvarious phases of the study: Name Paul Connolly Emily Decker Karen Risha Mark Ammetman Shawn Robb Tide Technical Lead-LCMS Scientist Scientist Sample Custodian Sample Custodian Exygen Research Page 6 of 65 - 125Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats__________________________ DuPont-13830 Exygen Study No.: 008-406 TABLE OF CONTENTS Page TITLEPAGE............................................................................................................ 1 GOODLABORATORY PRACTICECOMPLIANCE STATEMENT...........................2 QUALITY ASSURANCE STATEMENT................................................................... 3 CERTIFICATIONOF AUTHENTICITY.................................................................... 4 STUDY IDENTIFICATION.......................................................................................5 PROJECTPERSONNEL...........................................................................................6 TABLE OF CONTENTS............................................................................ 7 LIST OFTABLES..................................................................................................... 7 LIST OFFIGURES................................................................................................... 8 LIST OFAPPENDICES............................................................................................ 8 1.0 SUMMARY.......................................................................................................9 2.0 OBJECTIVE.......................................................................................................9 3.0 INTRODUCTION........... ................................................................................... 9 4.0 TEST SYSTEM.................................................................................................. 9 5.0 REFERENCEMATERIAL................................................................................ 10 6.0 DESCRIPTIONOF ANALYTICALMETHOD...................................................11 6.1 Extraction Procedure........................................................................................11 6.2 Preparation of Standards andFortification Solutions.......................................... 11 6.3 Chromatography................ 12 6.4 Instrument Sensitivity......................................................................................12 6.5 Description of Instrument and Operating Conditions.......................................... 12 6.6 Quantitation andExample Calculation..............................................................13 7.0 EXPERIMENTALDESIGN...............................................................................15 8.0 RESULTS.........................................................................................................15 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTEDTHE QUALITY OR INTEGRITY OFTHEDATA............................................ 15 10.0 CONCLUSIONS............................................................................................ 15 11.0 RETENTION OFDATA AND SAMPLES........................................................15 LIST OF TABLES Page Table I. Summary of PFOA (ppb) in Rat Plasma Control Samples.......................... 17 Table H. Summaryof Recoveries of PFOA (%)in Rat Plasma Samples................... 18 Table HL Summaryof Residue Found for PFOA (ppb) in Rat PlasmaSamples 19 -126Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 LIST OF FIGURES Figure 1. Typical CalibrationCurve forPFOA ...................................................21 Figure 2. ChromatogramRepresenting aCalibration Standardfor PFOA at JOppb with - 5 ng/mLof^C-PFOA...................................................................22 Figure 3. ChromatogramRepresenting Control RatPlasmafor PFOA with - 5 ng/mLof l3C-PFOA (Exygen ID: 0302238 Control A, SponsorID: 669160, Set- 011404A).................................................... 23 Figure 4. ChromatogramRepresenting Control Rat PlasmaFortified at 10 ppb with FFOA and- 5 ng/mLof "C-FFOA (Exygen ID: 0302238 Spk A, SponsorID: 669160, Set: 011404A).......... 24 Figure 3. ChromatogramRepresenting Rat Plasma Sample for PFOA with - 5 ng/mL of ,3C-PFOA (Exygen ID: L0001573-0021, Sponsor ID: 1301, Set: 011304B)........................................................................................ 25 LIST OFAPPENDICES Appendix A StudyProtocol DuPont-13830 (Exygen Study No. 008-406) and Amendments andDeviation.............................................................. 26 Appendix B Analytical MethodExM-008-276 Revision 2, "Method of Analysis for the Determination ofPentadecafluorooctanoic Acid (PFOA) in Serum by LC/MS/MS" .................................................................................... 45 ExygenResearch Page 8 of 65 -127 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Exygen StudyNo.: 008-406 1.0 SUMMARY Exygen Research (Exygen) analyzed samples of rat plasma for residues of PFOA in according to protocol DuPont-13830 (Appendix A) using the analytical method ExM008-276 Revision 2 (AppendixB). The limit of quantitation ffoorr.this method 10 ppb, which was established at Exygen in the method validationstud; Residues ofPFOAin foe ratplasmasamples rangedfrom< 10.0 ppbto 134ppb. The average recovery standarddeviation for PFOA in fortified ratplasma samples was 97%13.3%. 2.0 OBJECTIVE The objective of this analytical phase was to analyze rat plasma samples received at Exygen using foe method ExM-008-276 Revision 2 entitled "Method of Analysis for the Determination ofPentadecafluorooctanoicAdd (PFOA) in SerumbyLC/MS/MS". 3.0 INTRODUCTION This report details foe results of foe analysis of rat plasma samples for PFOA using foe analytical method ExM-008-276 Revision2. The study was initiated on November 18, 2003, when the study director signed protocol numberDuPont-13830. Theanalytical start datewas January 13,2004, andfoe analytical terminationdate was January IS, 2004. 4.0 TEST SYSTEM The control rat plasma used for control samples and laboratory control fortified samples was received from DuPont on 10/01/03. Forty rat plasma samples were received at Exygen on January 06, 2004. All samples were logged in by Exygen personnel upon receipt and placedin frozen storage( -20C). Sample login and chain of custody information can be found in foe raw data package associated with this study. Storage records will be kept at Exygen Research and a true copy of foe storagerecords will be furnish! upon request. Exygen Research Page 9 of65 - 128Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 5,0 REFERENCE MATERIAL The analytical standardpentadecafluorooctanoic acid (PFOA) was received at Exygen on March 05, 2003 from Critical Path Services. The control article (internal standard) nperfluorooctanoic acid (1,2 di-1JC) was received at Exygen on February 20, 2003 from DuPont The available information for the reference material is listed below. The analytical standard was stored in a refrigerator and the control article was stored at room temperature. Compound PFOA 13C-PFOA Exygen InventoryNo. SP2516 SP2492 Lot No, Purity (%) SspijratiQuPate 03/05/04 01/31/05 The structure of perfluorooctanoic acid (PFOA) and the internal standard are given below. PFOA Chemical Name = Molecularweight = perfluorooctanoic acid 414 13c - pfoa Chemical Name = Molecular weight = perfluorooctanoic acid, [l,2-di-l3C] 416 Exygen Research Page 10 of65 - 129Company Sanitized. Does not contain TSCA CB1 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________ DuPont-13830 Exygen Study No.: 008-406 6.0 DESCRIPTION OFANALYTICAL METHOD 6.1 Extraction Procedure 1. Measure 0.1 mL of sample into l mL eppendorf centrifuge tubes (fortify with analyte as needed, close lid, and vortex - 10 seconds). 2. Add enough acetonitrile containing internal standard at 0.005 pg/mL (accounting for fortification volume) to make extraction volume 500 pL andvortex for ~ 10 seconds. 3. Centrifuge for ~ 10 minutes at ~ 14,000 rpm. 4. Analyze samples using electrosprayLC/MSflVIS. 6.2 Preparation of Standards and Fortification Solutions The stock standard solution of PFOA was prepared on September 17, 2003, at a concentration of 100 pg/mL by dissolving approximately 10 mg of the standard (corrected for purity) in methanol. The stock standard solution of l3C-PFOA was prepared on September 17, 2003, at a concentration of 100 pg/mL by dissolving approximately 10 mg of the standard (corrected for purity) in methanol. A fortification solution of PFOA at 10 pg/mL was prepared by taking 10 mL of the stock standard solution and bringing the volume up to 100 in acetonitrile. A 1.0 pg/mL fortification solution of ,3C-PFOA was prepared by taking 1.0 mL of the stock and bringing the volume up to 100 mLwith acetonitrile. A set of standards containing PFOA with internal standards was prepared by dilution of the 10.0 pg/mL PFOA solution and the 1.0 pg/mL 13C-PFOA solution in the following manner. These solutions were used to fortify the samples and the extracted calibration standards. Initial Cone. Volume (mL) Diluted to (mL) Final Cone. (uz/mL)1 10.0 10.0 - xP (pgtaL)1 1.0 10.0 1.0 1002 0.1 10....0.-------- 0.1 1Q02 0.01 20.5 mLof the 1.0 pg/mL ,3C-PFOA solution was addedpriorto making final volume to give a concentration of 0.005 pg/mL of ,3C-PFOAin each solution. The calibration standards are processed through the extraction procedure, identical to the samples. The extracted standards are assigned a two-week expiration date from the date of extraction based on refrigerator stability data obtained during the method validation study (Exygen study: 008-277). The fortification of the standards before extraction is done accordingto the following table: ExygenResearch Page 11 of 65 - 130Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 Cone. Of Fortification Volume of PFOA Volume (pL) Control Fortification Sample (mL) Solution (u/mL) 0.01 100 0.1 0.01 200 0.1 0.1 50 0.1 0.1 100 0.1 0.1 200 0.1 Cone, of Extracted Calibration Standard (ppb) 10 20 50 100 200 The extraction solution was prepared by taking 0.5 mL of the 1.0 pg/xnL ,3C-FFOA solution and bringing the volume up to 100 mL with acetonitrile. The dilution solution was prepared by taking 0.5 mL of the 1.0 pg/mL ,3C-PFOA solution and bringing the volume up to 100 mLwith 50:50 methanokwater. The stock standard solution andall fortification/calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation can be found in the rawdata associated with this report. ( 3 Chromatography Quantification of PFOA was accomplished by analysis using electrospray LC-MS/MS. The retention time of PFOA was ~ 2.0 min., with no significant interfering peaks (< 20% ofthe LOQ standard)in the control samples correspondingto the analyte retention times. 6.4 Instrument Sensitivity The smallest amount of PFOA injected during the chromatographic run was equivalent to 10ppb. 6 S Description of Instrument and Operating Conditions A PE Sciex LC-MS/MS coupled to a Hewlett Packard HPLC system was used. Data acquisition andprocessing were performed using Analyst 1.2 software. Detailed operatingconditions are listed below: Mass Spec: Interface: Computer: Software: HPLC: PE SCIEXAPI4000 Biomolecular Mass Analyzer SCIEXTurbo Ion SprayLiquidIntroduction Interface TurboIon Spraytemperature = 350 C with N2 at ~7 L/min Dell Optiplex GX110 PE Sciex Analyst: Version 1.2 Hewlett Packard (HP) Series 1100 Exygen Research Page 12of 65 -131 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 HP Quat Pump HPVacuum Degasser HP Autoinjector HPColumn Compartment MiterCartridge(s): 2 Keystone Hypercarbs in tandem, exiting pump HPLCColumn: Genesis C8(Jones Chromatography), 2.1 mmx 50 mm, 4p ColumnTemp.: 30 C Mobile Phase (A): 2 mMAmmoniumAcetatein water Mobile Phase (B): Methanol Time 0 3.0 3.5 3.7 7.0 7.5 9.0 9.5 12.0 Injection Vol.: 5 pL Ions monitored: %A 40 40 0 0 0 40 40 40 40 %B Flnw fmiymint 60 0.3 60 0.3 100 0.3 100 0.5 100 0.5 60 0.5 60 0.5 60 0.3 60 0.3 Analvte PFOA 13C-PFOA Mode Transition Monitored Negative 413 --369 Negative 415 -> 370 6.6 Quantitation and Example Calculation Five microliters of sample or calibration standard were injectedinto the LC-MS/MS. The peak area was measured and the standard curve was generated by linear regression using 1/x weighting of the ratio analyte peak area/intemal standardpeak area versus theratio of the concentration of analyte/concentration of internal standard using Analyst 1.2 (or equivalent) software system. The residue concentration for PFOA was determined from the following equations: Equation 1 was used to calculate the amount of analyte found (in ppb or ng/mL, based on ratio of analyte peak area to internal standard peak area) using the standard curve (linear regression parameters) generatedby the Analyst software program. Analyte found (ppb) = ((AnalvtePeakarea/ISpeakarea) - intexceptY)x IS cone. (ngtaL) x DF slope Exygen Research Page 13 of 65 -132 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ ______ DuPont-13830 ExygenStudyNo.: 008-406 For samples fortified with known amounts of analyte prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2? Recovery (%)= Analvte found tppbl - ave. Analvte found in control (bob) x 100 AmountAnalyte added (ppb) Forplasma samples fortified with known amounts of analyte prior to extraction, Equation 3 was usedto calculate the percent recovery. Recovery (%) - Analvte found (nob)- ave. Analvte found in sample (nob) x 100 Amount Analyte added (ppb) An example of a calculation using an actual sample follows: Rat plasma sample Exygen ID 0302238 Spk A (Set: 011404A), fortified at 10 ppb with PFOA. Where: analyte peakarea = 216030 IS peakarea = 535991 IS cone. (ng/mL) as 4.167 ng/mL intercept as 0.000435159 slope k 0.173906 dilution factor (DF) as 1 ppb added (fort level) = 10 amtfoundin control = ND From equation 1: Analyte found (ppb) = 1(216030/5359911 - 0.0004351591 x 4.167 x 1 0.173906 = 9.65 ppb From equation 2: %Recovery = 19.65 ppb-0 ) x 100 10 ppb = 97% Note: This example calculation was done using rounded numbers, andtherefore maybe slightly different fromthe values shown in the RAWDATA. ExygenResearch Page 14of 65 -133 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo.: 008-406 7.0 EXPER AL DESIGN Each analytical set was extracted according to method ExM-008-276 Revision 2 and consisted of a reagent blank, one matrix control, two laboratory matrix controls fortified at known concentrations, one field sample fortified at aknown concentration, 20 plasma samples, andone field sample extractedin duplicate. 8.0 RESULTS The PFOAfound in the control ratplasmasamples arelisted in Table I. Individual recoveries for PFOAin the laboratoryfortified ratplasma samples are detailed in Table n . The average percent recoveries standard deviation for PFOA in foe rat plasmasamples was 97% 13.3%. Individual results for foe PFOA in foe rat plasma samples are given in Table III. Residues ofPFOAin foe ratplasma samples rangedfrom< 10.0 ppbto 134 ppb. A typical calibration curve for PFOA and representative chromatograms are given in Figures 1-5. 9.0 CIRCUMSTANCES THAT MAYHAVEAFFECTED THE QUALITY OR INTEGRITY OF THE DATA There areno known circumstancesthathave affectedfoe qualityor integrityoffoe data. 10-0 CONCLUSIONS The rat plasma samples were successfully analyzed according to method ExM-008-276 Revision 2. 11.0 RETENTION OF DATAAND SAMPLES When foe final report is complete, all original papa: data generated by Exygen Research will be shipped to foe sponsor. This does not include facility-specific raw data such as instrument logs. Exact copies of all raw data, as well as a signed copy of foe final analytical report and all original facility-specific raw data, will be retained in foe Exygen Research's archives for foe period of time specified in 40 CFR Part 792. Retained samples ofreference substances arearchivedbyfoesponsor. Exygen Research Page 15 of 65 - 134Company Sanitized. Does not contain TSCA CBi H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 T A U T 17C JL A B - L / iL ExygenResearch Page 16 of 65 -135 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 Table I. Summary of PFOA (ppb) in Rat Plasma Control Samples Sponsor PFOA _____________ ID__________________ ;__________ (ng/mL) 669160 Control A 01/13/04 NO 669119 Control A 01/14/04 ND ND = Not Detected (less than 10.0 ppb) Exygen Research Page 17 of 65 - 136 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-13830 Exygen StudyNo.: 008-406 Table II. Summary of Recoveries of PFOA (%) in Rat Plasma Samples Sportto r ID 669160 (L C S A) 01/13AW 869160 (L C S B ) 01/13/0* 1301 (Matrix Spk) 01/13/04 66911 (LC S A) 01/14AM 669119 (LC S B ) 01/14/04 901 (Matrix SUM 01/14/04 LCS = LaboratoryControl Spike PFOA <PPb) 1 0 j* 881 778 9.65 1090 1100 Amount ForttHad < *) 10.0 1000 1000 10.0 1400 1000 AVERAGE: STD DEV: % BSD: Racovaty <*> 104 88 78 96 109 110 97 13.3 13.7 ExygenResearch Page 18 of 65 -137 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo.: 008*406 Table III. Summary of Residue Found for FFOA (ppb) in Rat Plasma Samples Sponsor ID 901 901 Dup 902 903 904 90S 906 907 90S 909 910 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1301 1301 Dup 1302 1303 1304 1305 1306 1307 1308 1309 1310 1501 1502 1503 1504 1505 1506 1507 1508 1609 1510 PFOA (ppb) <10.0 <10.0 <10.0 <10.0 < 10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <m o <10.0 11.6 11.4 13.6 14.0 16.2 11.0 <10.0 14.5 13.2 13.1 13.1 92.9 124 75.4 134 72.1 119 133 75.2 114 115 ExygenResearch Page 19 of 65 -138 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 BxygenStudyNo.: 008-406 FIGURES Exygen Research Page 20 of 65 -139 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 Figure 1. Typical Calibration Curve for FFOA a 011404AJdb (PFQA): 'Unoaf Ragrwaion (*1 waightlng): y * 0.173908 x +0000435159 (r 0.9997871) Exygen Research Page21 of 65 -140Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen Study No.: 008-406 Figure 2. Chromatogram Representing a Calibration Standard for PFOA at 10 ppb with - 5 ng/mL of 13C-PFOA Exygen Research Page22 of 65 - 141 Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 Figure 3. Chromatogram Representing Control Rat Plasma for PFOA with - 5 ng/mL of uC-PFOA (Exygen ID: 0302238 Control A, Sponsor ID: 669160, Set: 011404A) Exygen Research Page23 of 65 - 142Com pany Sanitized. Does not contain TSC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats____________ DuPont-13830 ExygenStudyNo.: 008*406 Figure 4. Chromatogram Representing Control Rat Plasma Fortified at 10 ppb with PFOA and ~ 5 og/mL of 13C-PFOA (Exygen ID: 0302238 Spk A, Sponsor ID: 669160, Set: 011404A) ExygenResearch Page24 of 65 - 143Compfany Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________ DuPont-13830 ExygenStudyNo.; 008-406 Figure 5. Chromatogram Representing Rat Plasma Sample for PFOA with ~ 5 ng/mL of 13C-PFOA (Exygen ID: L0001573-0021, Sponsor ID: 1301, Set: 011304B) M M1 I* " M U II i't Exygen Research Page 25 of 65 - 144Company Sanitized. Does not contain T S C A CB I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.; 008-406 APPENDIX A Study Protocol DuPont-13830 (Exygen Study No. 008-406) and Amendments and Deviation ExygenResearch Page 26 of 63 -145Com pany Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 DuPont-13830 j H-25425: Repeated-Dose Dermal Toxicity 1 28-Day Study ia Male Rats i 1 ji 1 Exygen Research *]Page 27 of 65 -146Com pany Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats D uPont-13 830 ExygenStudy No.: 008-406 /<^ae^s\ H-25425: Repe*ted-Dce DermalToxicity 28-Dty Stody in M ite Ra DuPod-13830 TABLE OF CONTENTS Page IN TRO DUCTIO N ,. _3 O B JEC T IV E.. -3 SPONSOR AND TEST FA C ILIT Y -3 REG U LATO RY CO M PLIAN CE.. .3 STU D Y D ESIGN - .4 M A TERIA LS AND M ETHODS.. A . Test Substance. B . Test Species. : C. Animal Husbandly D . Pretest Period E . Assignment to Groups, F . Administration ofTest Substance.. A A .4 3 .S ,6 6 0 . Body Weights- .7 H. Food Consumption and Food Efficiency,. 1. Clinical Observations and M ortality.. 7 7 J. Total Fluorine and Ferfluorooctaiioic Acid Level Evaluations. .7 K . Clinical Pathology Evaluation........................................................... 8 1. Hematology.. 8 2. C linical Chemistry.. .9 j 3. UrinalysisL . AnatomicalPathology.. IIIHUflMlIHMMIIimllllM*IIMMIItM*<i 9 9 1. Pretest- 9 2. Dosing Phase.. .9 STA TISTIC A L A N A LY SES., 11 SA FETY AND H O U SEKEEPIN G ., RECORDS AND SAM PLE STO RAGE, a .12 R EFER EN C ES., 13 PROTOCOL A PPEN D IX... SIG N A TU RE- 14 15 ! j 1 Exygen Research -2- i P*^8 of 65 -147Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 H-25425: Repeated-Dose DcmulToxicity ! 28-Day Study in M ile Rus DuPont-13830 iNmoDuenoN The test substance, H-25425, is a l a previously conducted SMTdayoraTgavagestudy with the testsnbstance,maiea_____________ _ were dosed at 0 (control), 10,100, ot 1000 mg/kg for 90 consecutivedays,(l> No adverse test substance-related effects on body weight, body weight gain, food consumption, foodefficiency, or clinical pathology parameter were observedin any male or female dose group. Reversible, minimal to mild thyroid follicular hypertrophy was observed m the 1000 mg/kg/day male and female groups. The no-observed-effectlevel (N O EL) for subchronic toxicity endpoints in male "i and female rats was 100 mg/kg/day. ! Dose levels for foepresent study were selected to duplicate the dose levels o fthe oral gavage study. . O BJEC TIV E The objective o fthis study is to evaluate foe potential toxicity of H-25425 when administered dennaliy to male tats for 28 days. The derma! route ofadministration was selected because it is a potential route ofhuman exposure. SPONSOR AND T E S T FA C ILIT Y This study is sponsoredfay E i du Pont deNemours and Company, Wilmington, Delaware. The sponsor's approval was effective die date the sponsor authorized the work on the Work j Authorization Fbrm. The study will'be conducted at Haskell Laboratory for Health and Environmental Sciences, E .I. da Pont de Nemours and Company, Newark, Delaware. REGULATORYCOMPLIANCE Tins study w ill be conductedin compliance with U .S. EPA TSC A (40 C FR part792) Good LaboratoryPractice Standards, which are consistent with theOECD Principles o fGood LaboratoryPractice (as revised in 1997) published in ENV/MCVCHEM(98)17.P'3> The study design is based on U .S. Environmental Protection Agency (EPA ) O ffice o fPrevention,- Pesticides, and Toxic Substances (OPPTS),(<>Organization for Economic Cooperation and Development (O ECD ),^ test guidelines. Areas o fnoncomptiance w ill be documented in the 3 final report `. ? " , i I- I I Exygen Research Page 29 of 65 -148Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dennal Toxicity 28-Day Study in Male Rats DuPont-13830 ExygenStudyNo.: 008-406 H-23425: Repeated-DowsDennalToxicity 2B-DayStudyin MaleRats DuFonb.13830 ... | STU D Y DESIGN The study design is as M ow s: V G ro w I 'm V " . V II NumbctfGrouD 10 io . 10 10 Dosage (tng/kg) 0 (Control) 10 .100 1000 Study Parameters BodyWeight Food Consumption Clinical Observations Observations for Dermal Effects Mortality/Moribnndity Checks Blood Collection for Total Fluorine Analysis Clinical Pathology PPOA Blood Analysis Necropsy Frequency Test day 0 and at 3-4 day Intervals thereafter Test day 0 and weekly thereafter D aily My Twice dally Test days 0 ,7 ,1 4 ,2 1 ,2 Week 4 Week 4 Week 4 M A TER IA LS AND M ETHODS A . Test Substance The test substance was supplied by the sponsor and was assigned die unique Haskell Laboratory Number 25425. B , Test Species Male Crl:CD (SD)IGSBR rats w ill be obtained from Charles River Laboratories, Inc. The location o fdie supplier (cityVstate) w ill be documented indie study records and final report. The C rl:CD *(SD )IG S B R rat has been selected on dm bases o f extensive experience with this strain at Haskell Laboratory and its suitability with respectto hardiness, longevity, sensitivity, and low incidence of spontaneous diseases. ExygenResearch ' -4- \ Page 30 of 65 -149 Com pany Sanitized. Does not contain T S C A CB I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______ . DuPont-13830 Exygen Study No.: 008-406 H-25425: Repoate4-DowDmil Toxicity 28-Day Study ia Male Rat*___________________________________ ________________ DuPont-13830 C . Animal Husbandly - ij A ll rats w ill b housed in stainless steel, wire-mesh cages suspended above cage boards. Animal rooms w ill be maintained at a temperature of22 3C and 'relative humidify o f50 20%. Animal roomsw ill be artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. i 'It A ll rats w ill be provided tap waterad libitum. A ll rats w ill be fed PM1Nutrition International, L L C Certified Rodent LabDiet 5002 ad libitum. 'i j A s specified in the Haskell Laboratory animal health and environmental monitoringprogram, the following procedures am performed periodically to ensure that contaminant levels are below :i those that would be expected to impact the scientific integrity of dmstudy: Water samples am analyzed for total bacterial counts, and the'presence o fcoliforms, lead, and other contaminants. Feed samples are analyzed for total bacterial, spore, and fimgat counts. - Samples from freshly wasted cages and cage racks are analyzedta ensure adequate sanitation by the cagewashcra. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations o fkey contaminants, including specified heavy metals, aflatoxm, chlorinated hydrocarbons, and organophospbates. The presence ofthese contaminants below themaximum concentration stated by the manufacturer would not be expected to impact the integrity o fthe study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Data bos maintained separately from study records and may be included in the final report at the discretion o fthe study director. D. Pretest Period I Upon arrival at Haskell Laboratory, a ll rats w ill be housed oneper cage in quarantine. The rots w ill be: - quarantined for a minimum o f5 days. 1 identified temporarily by cage identification. weighed at least 3 rimes during quarantine. observedwith respect to weight gain and any gross signs o fdisease or injury. The rats w ilt be releaseclfrom quarantine by the laboratory animal veterinarian ordesignee on the bases of bodyweights and:clinical signs. . .* I Bxygen Research Page31 of 65 - 150Com pany Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Exygcn StudyNo.: 008-406 H-2S425: Repeated-Dose Derma) Toxicity 28-Day Study in MaleRate____________ DuPont-13830 Rais that aie accidentally killed or removed from study duringthe pretest period w ill be discarded without necropsy, -Sets thatare found dead or sacrificed in extremis during the pretest period w ill be sentto Pathology and given a gross examination to check for the presence of disease. Dependentupon these findings, ftaher diagnosticproceduresmay be employed at foe discretion of the study director, a pathologist, or foe laboratory animai veterinarian. The results w ill not be reported in fob final report unless considered significant to foeevaluation o f foe study. E Assignment to Groups' The rats, selected on foe bases o fadequate body weight gain and freedom from any clinical signs of disease or injury, w ill be distributed by computerized, stratified randomization into study groups as designated in the Study Design, so that there are no statistically significant differences among group body weightmeans! The weightvariation o f selected rats w ifi not exceed 20% of themean weight- Each rat m il be housed individually and assigned an animal number. Prim to assignment to groups, each rat w ill be temporarily identified by cage identification.. After assignment to groups, foe animat numberw ill be tattooed on foe tail ofeach rat and included on the cage label. At study start (test day 0) therats w ill be 8 weeks old. On test day 0, when possible, rats with body weights that are notwithin 20% o ffoe mean w ill be removed from study and replaced' with rats having body weights within that range (subject to the same selection criteria as the original rats). Rats that have notbeen assigned to a test group or which have been removed from study on test dayO, for out-of-rangebody weight, w ill be released for otherlaboratory purposes, or be sacrificed by carbon dioxide asphyxiation and discarded withoutpathological evaluation, at foe discretion o f the study director. F . Administration o fTest Substance % Approximately 24 hours prior to foe first treatment, tile fur ofeach rat w ill be closely shaved to expose the skin firoptthe back and trunk. The target area to be treated is 5 cm x 7,4 cm -3 7 cm2. The 37 cm1treatment area ts approximately equal to 10% o f foe total body surface area for rats in the 200 to 300 g range ofbody weights. The testsubstance w ill be moistenedwith deionized water to form a thick paste aad applied in a thin anduniform layer to cover as much surface of the target area as possible. For some doses, the volume o f test substance may be too small to cover the entire target area. The approximate area to be covered w ill be marked on the-backo f each ratwith a water insoluble marker and foe percentage o fthe marked area covered w ill be documented in foe study records. The test substance w ilt be covered with 8 2-ply gauzepad followed by successive layers o fstretch gauze and self-adhesive bandage. The controlrats w ill . receive deionized water at the',same volume as foe high-dose rids. The rats maybe fitted with plastic collars during the exposure period to prevent ingestion o fthe tost substance and disruption ofthe wrappings. -6- Exygen Research Page32 of 65 - 151 Company Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.; 008-406 H-25425: RepoM-DoM DermalToxicity 28-Day Study in Mil Rate DuPont-13830 The rats w ill be treated for 28 consecutive days. The amount o ftest substance needed to treat each rat w ill be based on themost recently determined body weightmeasurement The exposure period w ill be approximately 6 hours. Alter the exposure period, the wrappings and collar if used w ill be removed and excess test substancefrill be trashed o ffwith warm tap water. Soap w ill beused if necessary. The test site of each rat w ill then gently be patted dry and the rat returned to its cage. Control animalsw ill receive the same washing technique as the treated animals. ' Tim animals w ill bereshaved during the study to facilitate the evaluation o fdermaleffects. The entire area that was originally shaved (including theuntreated control skin) w ill be reshaved. The animals w ill bereshaved only after an evaluation. G . .Body Weights The rats w ill be weighed at 3-4 day intervals during the study. H . Food Consumption andFood E fficiency ' The amount o f food consumed by each rat w ill he determined weeklyby weighing each feeder at fee beginning and end o f die week and subtracting the final weight and the amount o fspillage from die feeder during the week from the initial weight From these measurements, mean daily food consumption over die week w ill be determined. From the food consumption and body weight data, the mean daily food efficiency w ill be calculated. I. Clinical Observations and Mortality The rats w ill be observed for clinical signs and dermal effects after removal o fthe test substance. The rats w ill also be observed for clinical signs at each weighing. The Drarze Seale w ill be used to score skin irritation. 'Adjacent areas o funtreated skin w ill be used for comparison. Rats w ill be checked twice daily for mortality and for signs o fillness, injury, or abnormal behavior. J . Total Fluorine and Perihrarooctanoic Acid Level Evaluations Blood (approximately-i m L) w ill be collected into ED TA tubes from the orbital sinus o fall rats ontestdays 0 ,7 ,1 4 ,2 1 , and 28. The blood w ill be collected from the animals approximately one hour after removal of the test substance. The blood w ill be refrigerated. The total fluorine contort of the blood samples from the first five animals in each group w ill be determinedby using a Wickbold torch combustion method followed by analysis.with a fluoride ion selective electrode. The analysis w ill be done at Jackson Laboratory, Deepwater. N J 08023. initially, only the day 21 samples w ill 1 analyzed. Additional analyses w ill be requestedby foe study director based on foe results o fthe day 21 analysis. Exygen Research Page33 of 65 -152 Company Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 ExygeaStudyNo.: 008-406 H-25425: Repeated-Dose Demnl Toxicity 25-DaySlaty in Male Ran DuPtmt-138M) Additional blood (approximately 2 m L) w ill be collected from die vena cava ofall.tats at i necropsy into a tube containingED TA . Plasma w ill be prepared and stored frozen at -B0C to -20C until analyzed far concentration ofperfluotooctanoic acid (PFO A), Plasma w ill be evaluated from all rats. Plasma samples w ill be extracted by organic solventprotein precipitation and then analyzed for PFOA by an appropriate method at Exygea Research (3058 Research Drive, Siate College, PA 16801). , i ij | 1 K . Clinical Pathology Evaluation | A clinical pathology evaluation w ill be conducted on all rats the day after the last dose. The day - before collection ofblood samples for the clinical pathology evaluation, the animals w ill be placed in metabolism cages. These animals w ill be fasted overnight (approximately 16 bouts) and urine w ill be collected from each animal. Blood samples'forhematology and clinical f chemistry measurements w ill be collected from the orbital sinus o feach animal while the animal is under light carbon dioxide anesthesia. Blood samples for plasma fluoride, PFOA, and ; coagulation measurementsw ilt be collected at sacrifice from the abdominal vena cava o feach i animal while the animal is under carbon dioxide anesthesia. Additional blood collected from the vena cava w ill be placed in a serum tube, processed to serum, and frozen at -80C to -20C. Bone marrow smears w ill he prepared at the final sacrifice from all surviving animals and w ill he j evaluated i f warranted by experimental findings. A t the discretion o fthe study director or clinical pathologist, additional samples for selected clinical pathology tests w ill be collected from animals showing clinical evidence o f toxicity or sacrificed in extremis. i 1. Hematology Blood samples w ill be evaluated for quality by visual examination prior to analysis. J The following hematologyparametersw ill be determined: red bloodcell count hemoglobin hematocrit mean corpuscular volume mean corpuscular hemoglobin mean corpuscular hemoglobin concentration red cell distribution width absolutereticulocyte count platelet count white blood cell count differentialwhite blood cell count microscopic blood smear examination I prothrombin time activated partial thromboplastin time 1 In addition, blood smears, stained with new methylene-blue, w ill be prepared from all hematology samples and w ill be' evaluated, if required, to substantiateor clarify the results o f hematology findings. >, - |, 1\i 1 ! | i [ ExygenResearch Page34 of 65 -153 Company Sanitized. Does not contain T SC A C 8I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________ DuPont-13830 ExygcnStudyNo.: 008-406 H-25425: Repeated-DoseDeaml Toxicity Z8-DayStudy In Male Ran DuPooc-13830 2. Clinical Chemistry 1 The M owing serum chemistry parameters win be determined: aspartate aminotransferase glucose i alanine aminotransferase. ' total protein '' sorbitol dehydrogenase albumin alkaline phosphatase globulin total bilirubin calcium urea nitrogen inorganic phosphorus. creatinine sodium cholesterol potassium triglycerides chloride fluoride* 3. Urinalysis The M owing urinalysis parameters win be determined: quality A color clarity volume osmolality pH glucose ketone bilirubin blood urobilinogen fluoride protein microscopic urine sediment examination Anatomical Pathology i 1. Pretest 4 See Materials andMethods, Section D . Pretest Period. 2. Dosing Phase A ll rats found dead*accidentally killed, sacrificed in extremis, or sacrificed by design w ill undergo a gross and microscopic evaluation. A ll rats removed from study (except for out-of- range body weight on test day 0} w ill be sent to Pathology for gross evaluation and collection of tissues. Rats w ill be euthanatized by carbon dioxide anesthesia and exsangumation. Rats sacrificed by design w ill be fasted after 3 p,m. on the afternoon before their scheduled, sacrifice. A final sacrifice w ill be performed on surviving rats followingthe final clinical pathology evaluation. The order of sacrifice for scheduled deaths w ill be random amonga ll treatment groups. t Fluorde-dstcmrnation will be analyzed from EDTA plasma. Exygen Research -9 - Page35 of 65 - 154 Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo.: 008-406 H-25425: Repcatcd-Do Dermal Toxicity 2>-DyStudy in Mult Bate______________________________________________________ DuPont-13830 The following tissues w ill be collected from rata which are found dead or accidentally killed ; (tissue integrity permitting), sacrificed in extremis, removed tram study (except out-of-rangc ^ body weight on test day 0), or sacrificed by design: liv e r* ' kidneys * , thyroid gland (alter fixation) * skin (treated and untreated) . gross observations*__________`_______________________________ ___________________________ a Organs tobe weighed. . b Grewobservationsmadatnecropsyforwhichhlstopathologylanotappropriate (c.g.. fluid, ruffledflu,and missinganatomicpans) willgenerallynmbeElected. A ll tissues w ill be placed in the appropriate fixative. Rats sacrificed by design w ill have the following organs weighed: -liver, kidneys, and thyroid. Relative organ weights (percent o ffinal body weight) w ill be calculated. Final bodyweights determinedjust prior to necropsyw ill be used in the assessment oforganweight changes. Organs from rats found dead, sacrificed in extremis, or accidentally killed may beweighed at the discretion of the pathologist or study director. Tissues collected from rats sacrificed by design in die high-dose and control group, and from rats that are found dead or accidentally lolled (tissue integrity permitting), or are sacrificed in extremis, w ill be further processed to slides, stained with hematoxylin and eoain, and examined microscopically. Most gross lesions and any additional target organs from.rats In the low- and intermediate-dosage groups w ill be evaluated microscopically. Selected gross observations for which a microscopic: diagnosis would not be additive (e .g , osteoarthritis, \ pododermatitis, chronic tail dermatitis, calculus, and deformities of the teeth, toe, tail, or ear pinna) w ill be saved, but w ill generally notbe processed for microscopic evaluation. Tissues i from rats removed fiorn study (for other than out-of-range bodyweighton test day 0), w ill not be a processed for microscopic evaluation unless considered necessary by the study director or i pathologist. 1 tAdditional procedures to identify and/or clarify histologic features o f lesions may be performed at the discretion ofthe pathologist and w ill be documented in die final report. . ai Exygen Research -10Page 36 of 65 -155Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo.: 008-406 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in M ile Rata_______________________ ________ ___________________ PuP<jpt-l3&3Q 1 STA TIST IC A L A N ALYSES Significance wQi bejudged atp< 0,05. Parameter Preliminary Test Body Weight Body Weight Gain Test far lack o f trend Food Consumption FoodEfficiency Levene's test for Organ-Weight homogeneity and Shapiro-Wilk test*10* far normality1 Levene's test for Clinical Pathology1 homogeneity and Shapiro-Wilk test? for normality* Survival Incidence ofClinical Observations None Incidence ofDermal Effects Methodo f Statistical Analysis If preliminary test is not If preliminary test is significant significant. Sequential application1'' of the loncHieere- Preliminary tests for Temstra trend test pairwise comparison OR' One-way analysis of variance1'11* followed with Dunnetfs teat1*.31 Kroskal-W allis test1131 followed with Dunn's test041 One-way analysis of; variance'1'* followed" with Dunnetfs test1 Kruskal-Wailis. test?13* followed with Dunn's test4* Cuchrun-Armitage test for trend111,4 a Pairwise companions and associated prelimiiury tests are onJy conducted ifthe lest for tack o ftrend U ignificajiL b Ifthe Shapiro-Wilk test is aot significantbut Lovenc's test ii lignifteaoi, a robustversionofDumtett's test win boused. c Whenan individualobservation la recorded asbeing teas than a certain value, calculations arc performed on halfthe recorded value. For example, if bilirubin Is reported as <0.\, 0.05 Is used for anycateuhtioas performed with that hUmibin data. d If the incidence is trotsignificant, but a significant lack o ffit occurs,then Fisher's Exact test^9 with a Bonfecroui correction is used. Other methods w ill be used, if appropriate, at the time o fanalysis. The statistical methods used w ill be described in the final report S A FE TY AND HO USEKEEPING Good housekeeping procedures w ill be practiced to avoid contamination o f die test substance and to avoid potential health hazards. To avoid skin contact, loves w ill be worn when handling the test substance. Animal carcasses and feces w ill be incinerated. -ll - t ExygenResearch . Page 37 of 65 -156Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats____________ DuPont-13830 Exygea Study No.: 008-406 H-2S425: Repeated-Dose Dennil Toxicity 28-Day Study in Male Rats DuPont-13830 RECORDS AND SAMPLE STORAGE 1 A ll data and records foranalytical characterizations conducted by the sponsorw ill be archived by the sponsor. Laboratory-specific or site-specific raw data such as personnel files, instrument, '] equipment, refrigerator and/or freezer raw data w ill be retained at the fhcility wherethe work wasdone. "" t A sample o fthe test substancew ill be collected for archive purposes and retained at Haskell Laboratory,Newark, Delaware. Specimens (if applicable),taw data, and the final report w ill be retained at Haskell Laboratory, Newark, Delaware, or at bon Mountain Records Management, i Wilmington, Delaware. - j I ij. !1l s t Exygen Research If"; -n- Page38 of 65 -157 Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats....................... DuPont-13830 Exygen StudyNo.: 008-406 H-25425: Rfipertetf-Dosc Dermal Toxicity 28-DayStudy in Mile Rats__________ DuPont-12838 REFEREN CES 1. DuPont Haskell Laboratory (2003). H-25425: Subdnonio Toxicity, 90--DDaayyj^avaBgStud^-in Eats with One-Generation Reproduction Evaluations, ttepublishcdi 2. EPA/TSCA Good Laboratory Practice Standards (40 CFR792), (1989). 3. OECD Principles-ofGood Laboratory Practice (as revised in 1997, published in ENV/MC/CHEM(98)17 (OCDE'GD(92)32). 4 . United Statea Environmental Protection Agency (EPA ), Office o f Prevention, Pesticides, and Toxic Substances ( PPTS) Health Effects Test Guidelines, QPPTS870.3200,21/28-Day Dermal Toxicity (AUG-IS&8). 5. Organisation for Economic Cooperation,and Development. Guidelines for Testing of Chemicals, Section 4 (Part 410); Health Effects (1981). 6. Draper,N JL and Smith, H . (1981). Applied Regression Analysts, Z" 1edition, pp 266-273. W iley, New York. 7. Selwyn, M R . (1995). The use of trend tests to determine a no-observable-effect level in animal safety studies. Journalo ftheAmerican CoUege o fToxicology 14(2), 158-168. 8. Jonekfeeere, A JL (1954), A dajtabution-fiee K-sample testagainst ordered alternatives. Biometrika 41, 133-145. 9. Levene, H . (1960). Robust test for equality of variances. Contributions to Probability and Statistics (J. OHrin, ed.), pp 278-292. Stanford University Press, Palo Alto. 10. Shapiro, S.S. and W ilk., M B . (1965). An analysis of variance test for normality (complete samples). Biometrika 52,591-611. 11. Scedecor, G.W . and Cochran,W .G. (1967). Statistical Methods, 6th edition, pp 246-248 and 349-352. The Iowa State University Press, Iowa 12. Dunnett, C.W . (1955). A multiple comparison procedure for comparmgseveral treatments with a control J . Amor. Statist Assoc. 50,1096-1121. 13. Kruskal, W .H . and W allis, W .A . (1952). Use o franks in one-criterion analysis o fvariance. J . Amor. Statist Assoc. 47,583-621. . 14. Dunn, O J. (1964), Multiple contrasts using rank sums. Tec/uiometrlas 6 ,241-252. 15. Fisher, R-A. (1985). Statistical Methodsfo r Research Workers, 13 edition. Haflher, N ew York. '1 ExygenResearch Page39 of 65 -158 Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 H-25425: Repeated-Dose Dermal Toxicity 28-DayStudy io Male Rats ______________ DuPont-13830 I f Study Fonction I Study Director PROTOCOLAPPEN D IX Study Pmsonnel Carol Finlay StaffToxicologist Clinical PathologyEvaluation Nancy E . Everds Principal Research Clinical Pathologist and Manager ; Anatomic Pathology Evaluation Greg P.Sykes Veterinary Pathologist StudE-Patsa Initiation o fTest Substance Administration November 23,2003 Collection of Blood for Fluorine Analysis November 25,2003 December 2,2003 December 9,2003 December 16,2003 December 23,2003 Clinical Pathology Evaluations December 23,2003 Anatomic Pathology Evaluation December 23,2003 i Scheduled Sacrifice 'mI December 23,2003 I Exygen Research -14- i Page40 of 65 -159Company Sanitized. Does not contain T SC A CB I H-25425: Repeated-Dose Dennal Toxicity 28-Day Study in Male Rats DuPont-13830 Exygen StudyNo.: 008-406 H-25425: Repeated-Dot DecmalToxicity 28-Pay Studyin M ile Rats ____________________________ j 3 SIGNATURE DuPont-13330 Approvedb y:-----------f .A A & L j c M L t-u ., CarolF to liy StudyDirector D its I Haskell Exygen Research \ -ts. Page41 of 65 -160Company Sanitized. Does not contain T S C A CBt H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 H-25425: Repeated-DoseD enial Toxicity 28-DayStgdym Male Rea____________ Work Request Ni Service Coi DcPont-13820 Protocol Amendment 1 .4 The protocol is amended as follows: Page 4, StudyDesign, change groupnumbers w IX , X I, X E I, andX V . i I*tr Rationale: To avoid duplicating animal numbers with another studyhoused in die sameanimal room. Approvedb y f A M L fo fttfc c . C sW Enlay T Study Director 5jayi2fii>3 Date i j I cc: Haskell 1 ExygenResearch 1 1 it - 1- Page42 of 65 - 161 Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo,: 008-406 H-25425: Repeated-Don:DermalToxwity 28-DayStudyIn Male Rate______________________________________________________ DcJont-13830 Work RequestN um bea^ ^ ^ H I Service C o d ^ ^ ^ B V Protocol Amendment 2 I The protocol is amended as follows: I' t 1. Page 4, Study Design, andPage 7, section J . Total Fluorine andPcffluorooctanolc Acid Level Evaluations, changethe last blood collection for fluorine analysis from test day 28 to test day27. Rationale: The daywas incorrect, 2, Page 14, StudyDates, change the last date ofblood collection for fluorine analysis form December 23,2003 to December 22,2003. i Rationale: The daywas incorrect ii towafcy-. Z /L A .1 1i Date Exygen Research Page43 of 65 - 162Com pany Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 sen RES EARCH .P red st__ \ Proven P R O TO CO L DEVIATION Deviation Number: 1____ Date gf Occurrence: 01/14/04 Page _1_ of 1 ExygehStudy Number. 006-406 Protocol Number. DuPont-13630 kmyij ; irai ? 1. Section 1. Analytical Method Section 4.5.4.e~accepted recovery of 76* for Exygen sample ID 10001573-0001 Spk C a t 1000 ppb. 1. Protocol Deviation Issued. ACTIONS TAKEN - deVaBonlMuad. SOP raViton. tte. Recorded Bye. Date; I fa ,, IMPACT ON STUDY1. No negative Impart because the LCS'pt the llO Q and 1000 ppb levels were within the acceptable range for this analytical set. \ Ffrjp&p^tn^ p to r Signature <- & h .( Study Director SIfinatnre -- Management Signature 9 "y / J fffy H a l______________ :_____________ \ \ \ \\ Datm 1 I'lrjkk.'U tV Date t f? - (**&--ivptf Date \ Date Exygen QAU Review-. I'T h .'V t/ 3-k/ * ' / UnFOTMtPROTOCOC.DVIA'nN.oc \ aiisasam Exygen Research g : 800J8U2M \ h B14J72.1019 exjgcn.com Page44 of 65 -163 Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen Study No,: 008-406 APPENDIX B Analytical Method ExM-008-276, Revision 2 "Method of Analysis for the Determination of Pentadecafluorooctanoic Acid (PFOA) in Serum by LC/MS/MS" ExygenResearch Page45 of 65 - 164 Com pany Sanitized. Does not contain T S C A C 8I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 ExygenStudyNo.: 008-406 m Pr e s e a r c h I own Results. mu *\ RJethod of Analysis for the Determination ofperfluorocctanoic A dd (PFO A ) in Serum \ ' by LO M S/M S - Revision 2......................... m sfii E m ily Decker \ D A T E ISSUkiM January 8,2004 \ SPONSOR \ \ DuPont Haskell laboratory for Health & Environmental Sciences \ P .O .B o x 50 Newark. D B 19714-0050 U SA DuPont'Study Number. DuPont-13090 \ \ PERFORMING JABORATOKE ExygeU Research 3058 Research D rive State Colleg& PA 16801 M ETHOD NUM BER \. ExM-008-276 Revistop 2 Exygen Research Page46 of 65 -165 Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo.: 008-406 Managem ent approval S g iw fllV B/peckcr ; ScientistExygen Research . y ___ m Date ^ /io h a Flaherty Vice President Exygen Research / //S4> Date . S. Math Kennedy ^ Sponsor Representative DuPont ? -J^'j ~2doc Date Elygen Research ExygenResearch Pige 2 g (20 Page 47 of 65 - 166Company Sanitized. Does not contain TSCA CB1 H -25425: Repeated-Dose Dermal Toxicity 28-D ay Study in M ale Rats D u P on t-13830 ExygenStudyNo.: 008-406 j TABLE OFCONTENTS T T T LE ................................ - ................................ M AN AGEM EN T A P PR O V A L.................. ! T A B L E O F C O N TEN TS ...,......................... L IS T O F T A B L E S .......... ........................ ....... L IS T O F FIG U R ES......................................... 1. SU M M ARY............................................ 2. EX P ER IM EN TA L CO M PO UN DS... 3. C H EM IC ALS AND S U P P LIES ......... 3 .1 . C h e m ic a l s..................................... 3 3 . S t a n d a r d s ....................................... 3 3 . E q uipm ent a n d S u p p l ie s........... 3.4. S o l u t io n s. ..... ................................ 3 3 . Preparation o f S tandards a n d F ortification 3 3 .1 . St o c k so l u t io n ......................- .................... 3 .5 3 . F o r u h c a u o n S o lutio ns 3 3 .3 . C a l ib rat io n St a n d a r d s ... 4. M ETHO D..................................- .................... 4 .1 . FLOWDIAGRAM-- ............................... 4.2. S am ple Pr o c e s sin g ..... ................... 4 3 . S am ple P r e pa r a tio n ....................... 4 .4 . E x t r a c t io n ......................................... 4 3 . Q u a n t it a t io n ____ 4.5.1. LCYMS/MS System and Operating Conditions. 4 3 3 . T u n e F e e PARAMBIERS.......... .................................. 4 . 5 3 . c a u b r a t io n P r o c e d u r e s ., 4 .5 .4 . S am ple A n a l y s is ............ . . . 4 .6 . A cceptance C riteria---- ------- ---4 .7 . P erfo rm anc e C r it e r ia .................. 4 .8 . T im e R eq u ir e d for A n a l y s i s ..... 5 . C A LC U LA TIO N S....................................... 6. S A F E T Y .......................................................... f!- Exygen Research Exyg en Research ..1 2 ..3-. .4 ,4 ..5 ..6 ..7 ..7 ..7 ..8 ..8 .9 ...9 ...9 .10 .10 .10 .10 .11 .11 ..u .11 ..12 ..12 ..1 3 ..1 4 ..1 4 ..1 5 ..1 5 ..1 5 Pages of 20 f i ? f it ; Page48 of 65 - 167Company Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13 830 ExygenStudyNo.: 008-406 LIST OFTABLES Table L Recovery ofPFO A from Fortifications In Monkey Seram ................................. 16 LIST OFFIGURES Figure 1. Calibration curye for PFO A in Control Monkey Seram.........................................17 Figure 2. Representative Chromatogram of aControl Monkey Seram Sample for PFO A with - Sng/m L o f^ C -FFO A ............................................................ ~.............. IS Figure 3 . Representative Chromatogram o f a 10 ppb Standard for PFO A in Control Monkey Seram with ~ 5 n g taL of *' C-PFO A ...............................................-- 19 Figure 4. Representative Chromatogram of Control Monkey Serum Fortified at 10 ppb with PFO A and with - 5 ngftnL of '^C-PFOA................... ........................................ 20 I S Eaygeo Research Exygen Research l }i f Page 4 of70 Page49 of 65 -168Com pany Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo.: 008-406 1. SUMMARY. This report details a method of analysis for perfiuocooctanoic add (PFO A) in serum. PFO A is extracted from serum by protein precipitation in acetonitrile. Quantification of PFO A is accomplished by liquid chromatography/tandem mass spectrometry (LC/M S/M S) analysis using selected reaction monitoring (SRM ). The chemical formula of PFO A Jb given in section 2 of this method. The lower lim it of quantitation (LX.OQ) for this method is 10 ppb for PFO A in serum. Quantification is performed using extracted calibration standards containing an internal standard. Th is methodwas developed using monkey serum. The overall percent recovery standard deviation for PFO A in monkey serum at 10,100, and 1000 ppb was 94% 65% (Table I). A representative calibration curve for PFO A in monkey serum is shown in Figure 1. Representative chromatograms for PFO A in monkey serum are shown in Figures 2 to 4. D 'g ; j i i j | I Bxygon Research Exygen Research Page 5 a t20 Page 50 of 65 - 169Com pany Sanitized. Does not contain T SC A C8I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo.: 008-406 2. EXPERIMENTALCOMPOUNDS The structure o f pcruorooctanoic add (PFO A) and theinternal standard are given below. PFO A Chemical Name . = Molecular weight = perflucnooctanoiQ acid 414 " C -PFO A Chemical Name Molecular weight a = perfluorooctanoic add, [l^ -d i-,3C ] 416 it I Exygen Research ExygenResearch Page 6 o f20 Ii Page 51 of 65 -170Com pany Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 2 8 -D a y Study in M ale Rats................. ...................................... D u P on t-13830 Exygen Study No.: 008-406 3. CHEMICALSAND SUPPLIES 3.1. CHBMICALS___________________________ ' Chtndeal G rad e Source: Methanol (MeOH) HPLC Acetonitrile (A CN ) HPLC Ammonium Acetate ' Reagent OmniSolv Water____________H PLC EM Science EM Science Sigma-Aldnch EM Science ____________ Catalog No. JT9093-2 AX014S-1 A-7330 W X0004-1 3X Standards Standard L o t Number perfluorooctanoic acid (PFO A) perfluorooctannic add, fU -d i-n C i____________ Pu rity J*L Source Oakwood Products DuPont j I | | jj j ; Exygen Rticwch ExygenResearch ' Pxge 7 of20 Page 52 of 65 - 171 Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________ DuPont-13830 Exygen Study No.: 008-406 3.3. E q u ipm en t and S u p p lib s Equipm ent S u p p lie r j Balance, analytical (display at least 0.0001 g) M ettier L Centrifuge ` 2 triC. eppendorfeentrifuge tubes Eppendorf : : VWR 1 | Disposable micropipets (50-100uL, 100-20M L) Drummond (V W R) ) Class A pipetS and volumetric flasks various suppliers : [ Hypercatb drop-in guard column (4 mm) Keystone ! (part if 844017-400) | Standalone drop-ln guard cartridge holder Keystone I 125-xnL LD P E namow-mduth botdes Nalgene 2 m L clear H P LC vial k it (cat # 5181-3400) HP Standard lab equipment (graduated cylinders, various suppliers disposable tubes etc.) LCVMS/MS and H PLC systems A sd escrib e d in | section 4J . ! Note: Equivalent materials may be substituted for those specified in this method t if they can be shown to produce satisfactory results. j Notes: 1. In order to avoid contamination, the use o f disposable iabware is highly recommended (tubes, pipets, etc.). 2. P T FE or P T F E lined containers or equipment, including PTFE-lined H P LC vials for the H PLC autosampler must pet be used. 3 . It is necessary to check the solvents (methanol) for the presence of contaminants by LQ M S/M S before use. Certain lot numbers have been found to be unsuitable for use. 4. Use disposable micropipets or pipets to aliquot standard solutions to make calibration standards and sample fortifications. 5 . The hypercart) cartridges should be changed when the system contaminants (elevated baseline) move to witbin 1 minute from the elution ofPFO A . . - \ | | p | I 3.4. Solutions , (1 ) 2 tnM ammonium acetate solution is prepared b weighing 0.1S g of ammonium acetate and dissolving in 1 L o f OmniSolv W ater, Jr N ote; The aforementioned example is'proviSed for guidance, alternative volumes may he prepared as long as the ratios of the solvent to solute are maintained. f1 j i ; r Exygcn Research Exygen Research Rage8af20 Page 53 of 65 - 172Company Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 J&cygen Study No.: 008-406 ' 1 j ; i | j | ; j ; ! i j | ! ' '" i ! =-- 3.5. P reparation o f Stan dar ds a n d F ortification S olutions Analytical standards are used for three purposes: 1. Calibration Standards - These standards are prepared in control monkey seta and are used to calibrate the response o f the detector used in th e ,. analysis. 2. laboratory Control Spikes - These fortifications are usually prepared at concentrations corresponding to the LLO Q and lOx LLO Q and are used to determine analytical recovery. Laboratory control spikes are prepared in control monkey serum. 3 . M atrix Spites - These fortifications are prepared by spiking into the field samples at known concentrations. M atrix spites are used to evaluate the effect of the sample m atrix on analytical recovery. The absolute volumes o f the standards may be varied by the analyst as long as the correct proportions of solute to solvent are maintained. Alternate concentrations may be prepared as long as the preparation and concentration are accurately recorded in the raw data. Note also that additional concentrations may be prepared if necessary. 3.5.1. Stock solution Prepare individual stock solutions of - ICO pg/mL of PFO A and 13CFFO A by weighing out - 10 mg of analytical standard (corrected for purity) and dilute to 100 m L with methanol in a 100-mL volumetric flask, Each stock solution (in 125-mL LD PB bottles) is to be', stored in a refrigerator at 2C to 6"C and is stable for a maximum period of 1 year from the date o f preparation. 3.5.Z Fortification Solutions a. Prepare a fortification standard of 10.0 pg/mL far PFO A by diluting 10.0 m L o f the PPOA solution described in 3.3.1 to 100 m L with . acetonitrile in a volumetric flask. b. Prepare a fortification standard of 1.0 pg/mL-, for UC-PFQ A by diluting 1.0 m L of the 13C-PFOA solution described in 3.5.1 to 100 m L with acetonitrile in a volumetric flask. c, Prepare a fortification standard of 1.0 pg/mL. for PFO A containing 0.005 pgfcnL of I3C-PFO A by diluting 10.0 m L of the PFO A solution described in 3.5.2.a and 0.5 m L of flip ,J C-PFO A solution described in 3.5.2.b to 100 m L with acetonitrile in a volumetric flask. d .. Prepare a fortification atandardjrf 0.1 pg/mL for PFO A containing ' 0.0G5 pgfrnL of u C-PFO A by luting 1.0 m L o f the PFO A solution ... described in 3.5.2.a and 0.5 m L of tho "C -PFO A solution described in 3.5.Zb to 100 m L with acetonitrile in a volumetric flask. - e. Prepare a fortification standard of .8.01 pg/mL for PFO A containing . 0,005 pgfm L of 13C-PFO A by diluting 0.1 m L of the PFO A solution ExygenResearch Page 9 of 20 ExygenResearch Page 54 of 65 - 173 Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen StudyNo.: 008-406 described in 3.5.2.a and 0.5 m L o f the 13C-PFOA solution described in 3.5.2.b to 100 m L with acetonitrile in a volumetric flask. I Store a ll fortification standard solutions in a refrigerator (ha 125-m LLD PE bottles) at aC to 6C for a maximum period of 1 year from the date o f preparation*after which time it is necessary to make new standards using, the stock solution. i.J .3 . Calibration Standardr I The calibration standards are processed through the extraction procedure, identical to the samples. The fortification of the standards before extraction is done according to the following table: Cone. O f Fortification Volume of Cone, of M ixed Volume (p L) Control Extracted Fortification Sample (m L) Calibration Solution Standard (p g fin L ) (ppb) 0.01 100 0.1 10 0.01 200 0.1 20 ] 0.1 50 0.1 50 0.1 100 0.1 100 0.1 200 0.1 200 4. METHOD 4 .1 . FLOW DIAGRAM The flow diagram of the method is given below, followed by a detailed description of each step. Method Flow-Diagram Measure 0.1 m L o f sample (using disposable micropipette) t (fortify samples when designated) 4 i Protein precipitation in acetonitrile 4 i, LQ M 5/M S analysis *. ..W* 4.2. S am ple P ro cessing ` No sample processing is needed for serum sajnples. However, frozen samples must be allowed to completely thaw, un-aided, at room temperature. Samples Exygen Restateli Page 10 of 20 ExygenResearch Page55 of 65 -174Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13 830 Exygen Study No.: 008-406 I stored,refrigerated should also be allowed to equilibrate to room temperature. A il samples must be thoroughly mixed before faring sampled forextftfction. * ! 4 3 . Sam ple P reparation : ij a. Each batch of samples extracted (typically 30 or less) must include at least jj fanejeagent control (acetonitrile blank), one matrix control (method blank), | and two m atrix controls fortified at known concentrations to verify procedural recovery for the batch. ; I b. A t least onesample per batch should beextracted in doplicate ' I\ c. A t least one sample extracted should be separately fortified at a known concentration and carried' through the procedure to verify recovery. ; 1 Additional m atrix spikes may be performed at the sponsor's request j 4.4. Extr action 1. Measure 0.1 m L o f sample into 2 mL, eppendorf centrifuge tubes (fortify with analyte as needed, close lid , and vortex ~ 10 seconds). 2 . Add enough acetonitrile containing internai standard at O.OOS pg/mL (accounting for fortification volume) to make extraction volume 500 p L and vortex for - 10 seconds. 3. Centrifuge for - 10 minutes at -14,000 ipm. 4. Analyze samples using electrospray LC/M S/M S. j, j ! j 4.5. Quantitation I 4.5.1. LC/MS/MS System and Operating Conditions 1 jI.Instrument: P E S C IE X A P I 4000 BiomolecularMass Analyzer SC1EX Turbo Ion Spray liq u id Introduction Interface Computer: D ell OptiPlex Q X 110 Software; P E S cie x Analyst 1.2 H P1C Equipment: Hewlett Packard (H P) Scries 1100 Quatpump G1311A Vacuum Degasser Q1322A AutoirtjectorG1313A " Column Compartment G1316A * `jr Note: Two 4 9< 10 mm hypereaib drop-in guard cartridges (Keystone, part # 844017-400) are attached in-line after the purge valve and before the sample injector port to hap any residue contaminants that may be in the mobile phase and/orH PLC system. , tl Exygen Research Page 11 of20 Exygen Research Page56 of 65 -175Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 Exygen Study No.: 008-406 HPLCColum n: Generis Ca (lanes Chromatography), 2.1 mm x 50 mm, 4 )i Column Temperature: 30 C Injection Volume:' 5 p L Mobile Phase (A ): 2 mM Ammonium Acetate in OmniSolv Water Mobile Phase (B ):: Methanol I 1 rim s %A % B - B qw. M i 0.0 40 60 0.3 3 40 60 0,3 3.5 0 100 0.3 3.7 0 100 0.5 70 7.5 40 100 60 0.5 0.5 9 40 60 0.5 9.5 40 60 0.3 12 40 60 0.3 It may be necessary to adjust the H PLG gradient in d e r to optimize instrument performance. Ions monitored: Anaivte PFOA ,3C-PFO A Mode Negative Negative Transition M ou'tcral 413 -> 369 415-370 Approximate RetentionTim? -2 .8 min. - 2 .8 min. pH t F The retention times may vary, on a day-to-day basis, depending on the batch of mobile phase etc. D rift in retention times is acceptable within an analytical run. as long as the drift continues through the entire analysis and the standards are interspersed throughout the analytical run. Note: An alternative LC/M S/M S system may be used once demonstrated to be equivalent. 4.5.2. Tune F ile Parameters . The mass spectrometer is tuned for tire analyte by infusing a - 1 p.g/mL standard solution of PFO A (at 10 pl/m m , using an infusion pump) via a "T " into a stream o f mobile phase containing 60% methanol and 40% 2mM ammonium acetate at 0.3 mL/min flow rate. The analyte is initially toned fo r the parent ion and then tuned for the product ion. Once the instrument is totted, the optimized parameters - ore saved as a tune file . Th is tune file .is then used during routine analysis. 4.5.5. Calibration Procedures a. Inject the same aliquot (5 p L ) of each calibration standard into (be LC/M S/M S. i \ Exygen Sesetreh Page 1 2 o fI 0 Exyg en Research Page 57 of 65 - 176Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats____________ DuPont-13830 ExygenStudyNo.: 008-406 b. Use linear standard curves for quantitation. lin e a r standard curves are generated for each analyte by linear regression using 1/x weighting of the Tatio analyte pedk area/intemal standard peak area versus the ratio o f the concentration of analyte/concentratrcm of internal standard using Analyst 1.2 (or equivalent) software system. A ny: calibration standard found to be a atatistfcnLoutlier by using the appropriate outlier test (e g. Huge Outlier Test}, may be excluded ram the calibration curve. However, the total number o f calibration standards dial could be excluded must not exceed 20% o f the total number of standards injected and at least one calibration standard at the LLO Q must be retained. c. The correlation coefficient (r) for calibration curves generated must be 0,9925 (r* 0.985). I f calibration results tail outside these lim its, then appropriate steps must be taken to adjust instrument operation, and die standards or the relevant set of samples should be reanalyzed. 4.5.4. Sample Analysis a. Inject the same aliquot (S p L ) of each standard, sample, recovery, control, etc. into the LC/M S/M S system. b. Standards corresponding to at least five or more concentration levels must be included in an analytical set. c . An entire set of calibration standards should be injected at the beginning of a set followed by calibration standards interspersed every 5-10 samples (to account fo r a second set o f standards). As an alternative, an endre set o f calibration standards may be included at the beginning and at the end of a sample set In either case, calibration standards must be the first and last injection in a sample set d. The concentration of each sampk/fortification/control is determined from the standard curve, based on the peak area of each analyte. The standard responses should bracket responses of the residue found in each sample set If necessary, dilute die samples in 50:50 methauol:water-.to give a response within the standard curve range. e. Fortification recoveries falling within 85 to 115% (80 to 120% for levels at the IX O Q ) are considered acceptable. f. Extracted samples must be stored refrigerated between 2G to 6C until analysis. Jr g. Samples in'w hich no peaks sre detected (i.e. sig nal: noise ratio < 3 :1 ) at the corresponding analyte retention times w ill be reported as NO (not detected). Samples in which peaks are detected at t&e corresponding analyte retention Exygen Research Page 13 of20 ExygenResearch Page 58 of65 - 177 Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 2 8 -D a y Study in M ale Rats________________ D u P on t-13830 ExygenStudyNo.: 008-406 tiipes but are less than the lowest concentration of the calibration standards (Id ppb whipe LLO Q = 10 ppb) w ill be reported as <10 ppb." * 4.6. A c c e p t a n c e C r it e r ia i The fallowing criteria must be met to ensure the presence of FFO A and 13C- t FFO A: ' ,, I 1. The chromatograms must show the foilowing peaks: FFO A : a daughter ion at 369 anni from a parent of 413 amu 13C-PFO A: a daughterion at 370 amu from a parent of 415 amu | ! ! 2. Any'analyte present in method blanks must be at least 5-fold lower than the LLO Q . Any analyte present in the reagent blank must be at least 5fold lower than die LLO Q . 3. Recoveries, o f tab control spikes and m atrix spikes must be between 85115% (80-120% for levels at the LLO Q ) o f their known values. Any method fortification Gab control spike) falling ontside the acceptable lim its,warrants re-extraction of the entire analytical set. Any m atrix spike outside the acceptable range 'should be evaluated by the analyst to determine if re-extraction is warranted. i. L | s j 4. Any calibration standard found to be a statistica] outlier by using as appropriate outlier test, may be excluded from the curve. However, the total number of calibration standards that could be excluded must not exceed 20% of the total number of standards injected and-at least one calibration standard at the LLO Q must beretained. ; A 5. The correlation coefficient (r) for calibration curves generated must be j i 0.9925 (i3 0.985). I f calibration results fall outside these lim its, then 1 appropriate steps must be takeh to adjust instrument operation, and the I standards or the relevant set o f samples should be reanalyzed. f 4 .7 . P e r f o r m a n c e C r it e r ia t t The following two criteria must be performed as a system suitability test, before the commencement of analysis when using an instrumentation set-up that has not been used for this method. p ra t Criterion: . Run a standard solution on LC/M S/M S corresponding to the estimated LLO Q and obtain, a signal-to-noiae ratio o f at leasbg:1, compared to a reagent blank. If this criterion pannot be met, optimize and change instrument operating parameters. ! i ! .. \ j | ExygenResearch Exygen Research Page 14 of 20 Page59 of 65 -178 Company Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 : Second Criterion: | Run a set o f standards of rive or more concentration levels, spanning a range 1 starting at or below the LLO Q up to the highest concentration level to be included I 1 in the analysis. Generate a calibration curve for each analyte and obtain a linear { . regression with a coefficient of determination (r2) o f at least 0.985. Once this i I criterion is ije t, samples may be analyzed with standards imerspereed- _ g 4.8. T im e R e q u ir e d f o b An a l y sis A set o f 35 samples <1 reagent control, 1 matrix control, 1 laboratory spike, 2 I laboratory control spikes, and 30 samples) can be taken through foe extraction I procedure ip approximately 8 hours by one person. The LC/M S/M S analysis ' (standards and 35 samples) w illtake approximately 9 hours. 5. CALCULATIONS a. Use Equation 1 to calculate the amount of analyte found (in ng/nCL, based on peak area) using the standard curve (linoar regression parameters) generated by the Analyst software program. ; E quation!; ' A nalyte found (ppb) = fAnalytePunk anuflS peak rest - intercept x IS cone. (ng/mL) slops b. For samples fortified with known amounts of analyte prior to extraction, use Equation 2 to calculate the percent recovery. j ; j Recovery (96) = : i Analvte found Inobl - avg. Analyte found in control fnobl x 100% j. 1 Amount Analyte added(ppb) j 6. SAFETY The analyst should read the material safety data sheets for all standards and reagents before performing this method. Use universal precautions when handling standards and reagents. Including working in fume hoods and wearing laboratory coats, safety glasses, and gloves. Use blood-bome pathogen handling precautions whan handbag serum. Exygen Research Exygen Research Page IS of 20 Page60 of 65 - 179- Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 ExygenStudyNo.: 008-406 Table LRecovery ofPFO from Fortifications In Monkey Serum Summary ofl*FOA Recoveries for 10 ppb FortiScatiQ* in Monkey Serum Eygw Sponsor Extraction Analysis Fact. Lerci % Recovery ID ID Date Date ' CB*> ! 0300808 SpIt - LtK 40X 7400 03/30/03 02/3M& 10 95 ____ I 0100808 SpfcB Lo i 40X 7400 o s tm 02/30/03 to 94 r { OSOOSOSSOkC Lo t40X7400 a a o m 02/30/03 10 98 - AVERAGE: H STANDARD DEVIATIO N : 2.1 < 1 R ELA T IV E STANDARD DEVIATIO N : Z3 I Su m m ery o f F F O R e c o w ie s fo r 100 p pb E a rtific e U e n In M onkey Seru m * Exygwj 3D 0300808 SpkD 0300808 SpieB 300808 Si& F Sponsor ID Lo t40X7400 LO140K 7400 Lot 40X7400 Extraction Analysis Fort.Level Dae Date CwW 05/30/03 05/3003 100 05/30/03 05/3M 100 csao A 05/30/03 100 AVERAGE: STANDARD OBVIATIO N : R ELA T IV E STANDARD D EVIATIO N : Recovery 92 90 93 92 13 1.7 Summary ofFFOA Recoveriesfor 1000 ppb Fortlflcetioa iu Monkey Serum Exygen ID 0300608 SpkG 0300808 SpkH 0300808 Spkt Spoator ID Lot 40X7400 U t 40X 7400 Lot 40X 7400 Extraction Analysis F o il Level Dale Date 03/30/03 03/30/03 1000 05/30/03 S/30/D3 1000 05/30/03 0S/3Q/03 1000 A VERA O E: STANDARD D EVIA TIO N : R ELA T IV E STANDARD D EVIATIO N : Recovery 107 95 81 94 19. 1X 8 OVERALL AVERAGE: OVERALL STANDARD DEVIATION: OVERALLRELATIVESTANDARD DEVIATION: 94 49 7.3 Exygen RcMirch ExygenResearch l Pge IS of 20 Page61 of 65 -180 Company Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 ExygenStudyNo.: 008-406 Figure 1. Calibration curve forPFOAin Control Monkey.Serum OKJCOiAJTJ(PTO^: X'rm m ' f t /*" y. 034232U >. ail5CS83 {p. 0.BWS6; 1 I I \ 1 i ^SSW au^ Exygen Research ExygenResearch Pag 17 o f 20 Page 62 of 65 -181Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Exygen StudyNo.: 008-406 Figure 2. Representative Chromatogramof a Control Monkey Serum Samplefor PFOAwith - 5ng/mL of uC-PFOA i I f 1 .i Research ExygenResearch f Page 18 of 20 Page63 of 65 -182 Company Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Exygen Study No.: 008-406 Figure 3. Representative Chromatogramof a 10 ppb Standard for EFOA in Control Monkey Serumwith ~ S ng/mL'of 13CPFOA Etygetl Research Exygen Research Pago 19 o f 20 Page64 of65 -183 Com pany Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 BxygenStudy No.: 008-406 Figure 4. Representative Chromatogramof ControlMonkey Serum Fortified at 10 ppb with PFOAand with ~ 5 ng/mL of 13CFFOA K l I. i f Bxygen Research Bxygen Research Paje 20 of 20 K f 1 I Page65 of 65 -184Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDIX G INDIVIDUAL BLOOD FLUORINE LEVELS -185 - Company Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______ _______ DuPont-13830 Group IX IX IX IX DC XI XI XI XI XI xm xm xm xm xm XV XV XV XV XV INDIVIDUAL BLOOD FLUORINE LEVELS Dosage (mg/kg/day) 0 0 0 0 0 10 10 10 10 10 100 100 100 100 100 1000 1000 1000 1000 1000 Animal Number 906 907 908 909 910 1106 1107 1108 1109 1110 1306 1307 1308 1309 1310 1506 1507 1508 1509 1510 ppb Fluorine less than 500 ND ND ND ND ND ND ND ND ND less than 500 ND ND ND ND less than 500 less than 500 less than 500 ND less than 500 ND = not detectable = values < 200 ppb Values "less than 500 ppb" are > 200 ppb and < 500 ppb. 500 ppb = limit of quantification (LOQ). - 186Com pany Sanitized. Does not contain T SC A C 8 I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDIX H INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA - 187 Company Sanitized. Does not contain T S C A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13 830 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTES ABBREVIATIONS : General: Adeq adequate CLOT - saitple clotted NP - not taken or not performed OK - sample condition OK for testing QNS - quantity not sufficient for testing UTD - unable to determine individual Hematology valuesi COND - sample condition RBC - red blood cell count HGB _ hemoglobin HCT - hematocrit MCV - mean corpuscular volume MCH - mean corpuscular hemoglobin MCHC - mean corpuscular hemoglobin concentration RDW - red cell distribution width ARET - absolute reticulocyte count PLT - platelet count WBC - white blood cell count ANEU - absolute neutrophil (all forms) ALYM - absolute lymphocyte AMON _ absolute monocyte AEOS - absolute eosinophil ABAS - absolute basophil ALUC - absolute large unstained cell Individual Red Blood Cell Morphology Values: ANIS - anisocytosis MIC - microcytes MAC - macrocytes POLY - polychromasia HYPO - hypochromasia ECHI - echinocytes ACAN - acanthocytes TARG - Target cells RX - rouleaux HJB - Howell-Jolly body Mod - moderate - - not observed or not examined; see whole blood sample condition Individual White Blood cell / Platelet Morphology Values: SM - Smudge white blood cells TOX - toxic neutrophils DB - Dohle bodies VC - vacuolated cytoplasm BC - basophilic cytoplasm PCE - platelet clumps / estimate GP - giant platelets BP - bizarre platelets Mod - moderate - - not observed or not examined; see whole blood sample condition Individual Coagulation values: PHEM - plasma hemolysis PLIP - plasma lipemia PICT - plasma icterus PT - prothrombin time APTT - activated partial thromboplastin time -188 Com pany Sanitized. Does not contain T SC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats................ ..... DuPont-13830 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTES (Continued) ABBREVIATIONS: (Continued) Individual Clinical Chemistry Values: SHEM - serum hemolysis SLIP - serum lipemia SICT - serum icterus AST - aspartate aminotransferase ALT - alanine aminotransferase SDH - sorbitol dehydrogenase ALKP - alkaline phosphatase BILI - total bilirubin BUN - urea nitrogen CREA - creatinine CHOL - cholesterol TRIG - triglycerides GLUC - glucose TP - total protein ALB - albumin GLOB - globulin CALC - calcium IPHS - inorganic phosphorous NA - sodium K - potassium CL - chloride FHEM - plasma hemolysis from blood sample for fluoride determination FLIP ~ plasma lipemia from blood sample for fluoride determination FICT - plasma icterus from blood sample for fluoride determination PFLU - plasma fluoride Individual Urinalysis Values: QUAL - quality (modifies color) COL - color CLAR - clarity VOL - volume UOSM - urine osmolality pH - the logarithm of the reciprocal of the hydrogen ion concentration UGLU - urine glucose KET - ketone UBIL - urine bilirubin BLD - blood URO - urobilinogen UMTP - urine protein FLU - urine fluoride Individual Urine Microscopic Examination Values: EPIT - epithelial cells UWBC - urine white blood cells URBC - urine red blood cells NCRY - normal crystals MICR - mi croorgani sms SPER - sperm -189 Com pany Sanitized. Does not contain TSC A CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTES (Continued) NOTES : When individual animal data are not reported, it may be due to one of the following reasons or other reasons, all of which are explained in the study records: the sample was clotted (CLOT) there was insufficient sample for testing (QNS) a valid result could not be obtained CRNV) the sample was not suitable for testing the animal died, prior to sample collection no sample was available for testing (NSR) - 190Com pany Sanitized. Does not contain T S C A CB I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 901 902 903 904 905 906 907 908 909 910 Group COND OK OK CLOT CLOT OK OK OK OK CLOT OK IX - 0 mg/kg - RBC HGB xlO6/ult g/dL 7.50 7.49 NP NP 7.30 7.12 7.32 7.32 NP 7.13 14.3 15.0 NP NP 15.0 14.3 14.1 14.6 'NP 15.0 Day HCT % 47.2 49.3 NP NP 47.4 45.0 45.7 48.3 NP 48.5 Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group XI - 10 mg/kg - COND RBC xl06/fiL HGB g/dL CLOT OK OK OK OK OK OK OK OK OK NP 6.80 7.27 7.27 7.02 7.36 7.11 6.81 7.06 7.70 NP 14.5 14.9 14.5 14.8 15.1 14.0 14.4 14.9 15.5 Day HCT % NP 46.0 47.3 45.7 46.8 47.9 45.5 45.2 46.4 49.8 28 MCV fL 63.0 65.8 NP NP 64.9 63.2 62.4 66.0 NP 68.1 28 MCV fL NP 67.7 65.1 62.8 66.7 65.0 64.0 66.4 65.8 64.6 MCH pg 19.1 20.0 NP NP 20.5 20.1 19.2 19.9 NP 21.1 MCH pg NP 21.4 20.5 19.9 21.1 20.5 19.7 21.1 21.1 20.1 MCHC g/dL 30.4 30.4 NP NP 31.6 31.8 30.8 30.2 NP 30.9 MCHC g/dL NP 31.5 31.4 31.8 31.6 31.5 30.8 31.8 32.1 31.1 RDW % 12.5 11.8 NP NP 11.0 11.5 12.0 10.8 NP 11.2 ARET PLT xlO3/h x 103/m L 496.9 404.3 NP NP 314.3 280.8 269.9 230.8 NP 256.3 1447 NP NP NP NP NP 1080 NP NP NP RDW % NP 11.6 11.8 11.7 11.3 10.8 12.4 11.8 11.9 11.4. ARET PLT x 103/L x 103/m L NP 261.4 258.1 379.7 246.7 208.9 238.9 340.5 241.4 204.4 NP 1103 1193 NP 1036 1143 1377 1094 NP 1048 DuPont-13830 Company Sanitized. Does not contain TSCA CBI -191- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Individual Animal Male, Animal 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 Group COND OK OK CLOT OK OK OK OK OK OK OK XIII - RBC xioV ml 7.02 7.25 NP 6.97 7.15 6.96 6.88 6.65 7.21 7.56 100 HGB g/dL 14.0 14.8 NP 14.3 14.8 13.6 14.1 14.0 14.4 15.1 mg/kg HCT % 44.6 47.0 NP 45.1 47.0 44.0 44.0 44.3 46.0 49.1 Male, Animal 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 Group COND CLOT OK OK OK OK OK OK OK OK OK XV - RBC Xl06/ML NP 7.34 7.68 6.78 6.95 7.74 7.23 7.20 6.79 6.89 1000 HGB g/dL NP 14.4 15.2 14.2 14.1 14.8 14.7 14.7 13.8 14.2 mg/kg HCT % NP 45.7 47.8 44.4 44.7 47.3 46.1 46.4 43.3 45.5 Day MCV fL 63.5 64.8 NP 64.7 65.7 63.3 64.0 66.7 63.7 64.9 Day MCV L NP 62.3 62.3 65.5 64.3 61.1 63.8 64.5 63.7 66.0 28 MCH pg 19.9 20.4 NP 20.5 20.7 19.5 20.5 21.0 20.0 20.0 28 MCH pg NP 19.6 19.7 20.9 20.2 19.1 20.3 20.4 20.3 20.6 Company Sanitized. Does not contain TSCA O CD Clinical Pathology Data MCHC g/dL 31.5 31.4 NP 31.7 31.4 30.8 32.0 31.5 31.3 30.8 RDW % 11.4 11.3 NP 11.5 11.2 11.8 11.0 13.9 11.9 11.3 ARET PLT x 103//xL x 103/m L 123.4 291.2 NP 307.5 277.5 363.0 246.7 632.6 265.1 211.3 1092 1144 NP 1148 1292 1261 1250 NP 1175 1317 MCHC g/dL NP 31.5 31.7 31.9 31.4 31.3 31.8 31.6 31.9 31.2 RDW % NP 11.4 11.3 11.5 11.0 10.9 11.0 11.3 11.7 11.0 ARET PLT xl03//iL x 103/m L NP 264.8 117.0 301.7 261.7 245.1 307.6 241.5 269.4 249.9 NP 1344 1286 1219 1160 1231 1420 NP 1053 1219 DuPont-13830 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Individual Animal Clinical Pathology Data Male, Animal 901 902 903 904 905 906 907 908 909 910 Group WBC x 1 0 3/ mL 21.31 13.98 NP NP 11.55 14.77 13.96 13.00 NP 15.40 IX 0 mg/kg Day 28 ANEU ALYM AMON AEOS ABAS ALUC xl03/L xIOVtfL x 103/ mL x I O V m L x 1 0 3/ L x 1 0 3/ mL 2.23 2.67 NP NP 1.68 4.43 1.85 1.83 NP 2.77 17.84 10.46 NP NP 8.97 9.90 11.12 10.54 NP 11.69 0.52 0.37 NP NP 0.37 0.44 0.39 0.28 NP 0.34 0.22 0.29 NP NP 0.31 0.00 0.15 0.18 NP 0.24 0.09 0.04 NP NP 0.09 0.00 0.13 0.08 NP 0.10 0.42 0.16 NP NP 0.13 0.00 0.31 0.10 NP 0.26 Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group WBC x 1 0 3 / mL NP 14.12 17.03 12.38 13.18 14.21 18.00 13.54 9.82 11.94 XI ANEU x 1 0 3 / mL NP 2.00 2.41 1.73 1.67 2.12 2.96 2.64 1.62 2.18 10 mg/kg - Day ALYM AMON AEOS x 1 0 3 / mL x1 0 3/#iL xloVftL NP 11.15 13.70 10.53 10.79 11.01 14.03 10.12 7.55 8.86 NP 0.45 0.32 0.00 0.32 0.39 0.33 0.29 0.38 0.44 NP 0.12 0.25 0.12 0.14 0.17 0.21 0.05 0.16 0.14 28 ABAS x 1 0 3 / mL NP 0.09 0.10 0.00 0.07 0.16 0.11 0.14 0.03 0.08 ALUC x 10 3/ L NP 0.31 0.25 0.00 0.19 0.36 0.36 0.30 0.09 0.23 DuPont-13830 Company Sanitized. Does not contain TSCA CBI 193- Company Sanitized. Does not contain TSCA H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Individual Animal Clinical Pathology Data Male, Animal 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 Group XIII- 1 0 0 mg/kg- Day WBC ANEU ALYM AMON AEOS xl03/fiL x 1 0 3/ i L xl03///L xlOViiL x 1 0 3/ mL 17.08 12.72 NP 13.14 10.84 14.19 14.19 15.15 18.74 12.71 4.06 1.47 NP 1.32 1.17 2 .1 1 1.87 1.97 3.70 2.59 12.30 1 0 .1 1 NP 11.17 9.20 11.31 11.53 1 2 .8 8 14.14 9.27 0.34 0.56 NP 0.29 0.19 0.37 0.34 0.30 0.38 0.46 0 .2 2 0.13 NP 0.13 0.05 0.15 0.13 0.0 0 0.17 0 .2 1 28 ABAS x 1 0 3/ iL 0.06 0.08 NP 0 .1 0 0.04 0.08 0 .1 1 0 .0 0 0 .1 1 0.05 ALUC xlQ1/L 0.1 2 0.37 NP 0.1 2 0.18 0.16 0.2 0 0.0 0 0.23 0.13 Male, Animal 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 Group XV 1000 mg/kg - Day 28 WBC ANEU ALYM AMON AEOS ABAS ALUC x 103/ L xl03/L x 103/ L x 1 0 3/ mL x 103/ mL x 1 0 3/ mL x 1 0 3/ L NP 12.76 19.14 13.30 14.92 12.65 13.68 12.38 14.19 11.55 NP 1.97 2.34 1.98 2.02 2.14 2.47 1.12 2.01 1.64 NP 9.96 15.88 10.30 11.98 9.85 10.36 10.62 11.36 9.14 NP 0.45 0.44 0.58 0.47 0.30 0.52 0.23 0.48 0.35 NP 0.19 0.20 0.11 0.13 0.16 0.14 0.15 0.07 0.17 NP 0.05 0.04 0.05 0.09 0.05 0.06 0.08 0.05 0.06 NP 0.14 0.24 0.28 0.24 0.14 0.12 0.19 0.21 0.19 O CO DuPont-13830 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 901 902 903 904 905 906 907 908 909 910 Group ANIS _ NP NP - NP - IX - MIC _ - NP NP - - NP - 0 MAC _ - NP NP - - NP *- mg/kg POLY Few Trace NP NP Few Trace Trace Trace NP Trace Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group XI 10 mg/kg ANIS MIC MAC NP NP NP -- -- --- --_ ---- Day28 POLY NP Few Trace Few Trace Trace Trace Few Trace Trace Day HYPO _ - NP NP - _ NP - HYPO NP _ - 28 ECHI Trace Trace NP NP - Trace NP - ACAN _ - NP NP - Trace Trace - NP - ECHI NP Trace - Trace Trace - Trace Trace Mod - ACAN NP _ - Trace Trace - TARG _ - NP N-P - - _ NP - TARG NP _ RX _ _ NP NP _ - N_P RX NP _ _ _ - HJB _ NP N-P _ _ N_P HJB NP _ _ _ _ _ _ _ DuPont-13830 Company Sanitized. Does not contain TSCA CBI -195- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ) Individual Animal Clinical Pathology Data Male, Animal 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 Group ANIS _ NP - Male, Animal 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 Group ANIS NP - _ - XIII - MIC _ - NP - _ - 100 MAC _ NP - - '- mg/kg - POLY Trace Trace NP Few Trace Few Trace Mod Trace Trace Day HYPO _ NP - - - XV - MIC NP - _ - 1000 MAC NP - - - - _ - mg/kg - POLY NP Trace Trace Few Trace Trace Trace Trace Few Few Day HYPO NP - - - 28 ECHI _ Trace NP Trace - Trace Mod Trace Trace - 28 ECHI NP Trace - - - - Few Trace * ACAN _ NP Trace - Few Trace Trace Trace - ACAN NP Trace - - - Trace Trace TARG _ - NP - - - - - TARG NP - _ - - - - - - RX - NP - - - _ - _ RX NP - _ _ _ - - HJB NP _ _ ,, _ _ HJB NP - _ _ _ _ _ - DuPont-13830 Company Sanitized. Does not contain TSCA CBI 196 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Individual Animal Clinical Pathology Data Male, Animal 901 902 903 904 905 906 907 908 909 910 Group SM NP NP NP - Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group SM NP ~ - - IX - TOX _ NP NP NP - XI - TOX NP - - - 0 mg/kg - Day DB VC BC __ --NP NP NP NP NP NP -------- NP NP NP '- - - 10 mg/kg - Day DB VC BC NP NP NP --- --- -- - - -- --- - - - --- -- - -- 28 PCE UTD NP NP UTD UTD _ Adeq NP Adeq 28 PCE NP - UTD UTD - GP _ NP NP - - _ _ NP - GP NP '_ - '_ _ - - BP _ NP NP _ _ _ NP - BP NP _ _ _ - _ _ _ _ DuPont-13830 Company Sanitized. Does not contain TSCA CBI -197- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Individual Animal Clinical Pathology Data Male, Animal 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 Group SM _ NP - - - XIII - TOX _ NP - - - 100 DB _ - NP - - - _ - mg/kg - VC _ NP - ~ - Day BC _ NP - - - - Male, Animal 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 Group SM NP - - - - _ XV - TOX NP - - - 1000 DB NP - ,, - - - mg/kg - VC NP _ - - - Day BC NP - - - - 28 PCB _ NP - UTD - 28 PCE NP - - UTD - GP _ NP - - - - - GP NP - - - BP - NP - - BP NP _ - DuPont-13830 Company Sanitized. Does not contain TSCA CBI -198- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Individual Animal Clinical Pathology Data Male, Group IX- Animal 901 902 903 904 905 906 907 908 909 910 PHEM None None None None None None None None None None PLIP None None None None None None None None None None 0 PICT None None None None None None None None None None mg/kg- PT Sec 14.5 16.2 15.1 15.5 15.6 15.7 15.9 15.9 14.8 15.5 Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group XI - 10 mg/kg - PHEM None None None None None None None None None None PLIP None None None None None None None None None None PICT None None None None None None None None None None Day PT Sec 15.6 16.4 16.5 15.2 16.0 15.7 16.3 16.7 15.0 15.7 Day APTT Sec 15.4 17.2 17.1 20.0 19.0 18.2 19.6 16.2 17.2 19.7 28 APTT Sec 14.2 21.3 21.8 17.0 18.3 18.3 19.4 20.3 17.2 18.7 DuPont-13830 Company Sanitized. Does not contain TSCA C8I -199- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Animal Clinical Pathology Data Male, Group XIII Animal 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 PHEM None None None None None None None None None None PLIP None None None None None None None None None None Male, Group XV Animal 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 PHEM None None None None None None None None None None PLIP None None None None None None None None None None 100 PICT None None None None None None None None None None mg/kg - PT Sec 15.0 15.7 15.4 15.7 15.2 16.0 15.8 16.5 15.1 15.5 Day APTT Sec 17.1 15.8 17.1 16.6 18.3 19.5 19.6 16.9 16.9 17.4 1000 PICT None None None None None None None None None None mg/kg - PT Sec 15.2 17.1 16.1 17.1 16.0 14.9 15.5 15.5 16.7 16.7 Day APTT Sec 19.3 20.4 18.5 16.1 22.2 19.3 19.3 15.9 18.5 18.1 DuPont-13830 Company Sanitized. Does not contain TSCA CBI -200- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ Male, Group IX Animal 901 902 903 904 905 906 907 908 909 910 SHEM None None None None None None None None None Trace Male, Group SLIP None None None None None None None None None None XI Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 SHEM None None None Trace None None None None Trace None SLIP None None None None None None None None None None - SICT None None None None None None None None None None - SICT None None None None None None None None None None Individual Animal 0 mg/kg - AST ALT SDH U/L U/L U/L 165 44 9.2 115 51 20.2 139 54 24.7 114 59 20.2 105 51 16.0 144 49 9.9 53 40 17.0 142 49 10.1 125 60 27.7 118 64 30.6 10 mg/kg - AST ALT SDH U/L U/L U/L 115 52 17.6 107 42 14.5 109 53 17.7 148 45 6.4 120 38 13.3 132 71 31.5 98 57 16.4 104 53 11.0 169 46 7.5 117 51 14.0 Company Sanitized. Does not contain TSCA O 00 DuPont-13830 Clinical Pathology Data Day ALKP U/L 144 257 192 281 232 246 145 203 223 195 Day ALKP U/L 244 163 286 264 214 190 266 152 246 179 28 BILI mg/dL 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 28 BILI mg/dL 0.05 0.05 0.05 0.10 0.05 0.05 0.05 0.05 0.05 0.05 BUN mg/dL 16 20 15 21 20 18 18 17 17 15 BUN mg/dL 17 20 16 19 17 15 16 18 16 16 CREA mg/dL 0.34 0.35 0.23 0.32 0.29 0.27 0.21 0.25 0.36 0.30 CREA mg/dL 0.30 0.27 0.22 0.38 0.24 0.26 0.25 0.26 0.25 0.20 CHOL mg/dL 48 51 41 39 51 61 38 34 68 47 CHOL mg/dL 47 51 52 54 43 37 50 39 55 60 TRIG mg/dL 16 46 33 42 85 40 49 29 51 37 TRIG mg/dL 86 28 61 64 38 28 80 21 48 21 -201 - H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Animal Male, Group Animal 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 SHEM None None None None None None None Trace None None Male, Group Animal 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 SHEM None Trace None None None None None None None None XIII SLIP None None None None None None None None None None XV SLIP None None None None None None None None None None - SICT None None None None None None None None None None - SICT None None None None None None None None None None 100 AST U/L 89 110 124 96 93 86 105 151 88 116 1000 AST U/L 83 151 100 84 90 135 91 124 100 121 mg/kg ALT U/L 37 58 43 40 35 33 54 51 37 47 mg/kg ALT U/L 30 50 47 29 48 68 43 48 48 56 - SDH U/L 12.7 16.4 12.3 11.0 11.1 9.3 12.2 7.4 12.8 13.3 - SDH U/L 10.9 23.1 12.1 9.3 9.0 17.7 11.1 13.2 7.9 14.3 Company Sanitized. Does not contain TSCA O at DuPont-13830 Clinical Pathology Data Day ALKP U/L 227 241 157 171 223 266 200 244 133 184 Day ALKP U/L 220 234 208 184 235 329 182 296 244 310 28 BILI mg/dL 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 28 BILI mg/dL 0.05 0.05 0.05 0.12 0.05 0.05 0.05 0.05 0.05 0.05 BUN mg/dL 14 15 15 16 15 17 13 14 15 19 BUN mg/dL 15 17 14 20 12 15 15 14 17 15 CREA mg/dL 0.24 0.23 0.27 0.23 0.22 0.23 0.20 0.24 0.23 0.28 CREA mg/dL 0.19 0.28 0.19 0.22 0.18 0.20 0.21 0.24 0.23 0.24 CHOL mg/dL 41 52 46 48 55 45 49 42 43 55 CHOL mg/dL 34 35 43 53 40 34 40 32 42 39 TRIG mg/dL 28 37 23 26 36 37 60 16 49 45 TRIG mg/dL 30 15 35 25 31 14 58 38 40 26 -202- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Animal Male, Animal 901 902 903 904 905 906 907 908 909 910 Group GLUC mg/dL 159 . 108 89 149 92 90 86 92 134 128 Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group GLUC mg/dL 107 93 111 126 103 106 118 95 93 89 IX - 0 mg/kg TP g/dL ALB g/dL 6.2 4.2 6.7 4.3 5.6 3.9 6.1 4.1 6.2 4.3 6.0 4.2 5.9 4.0 6.0 4.4 6.1 -4.1 6.3 3.9 Day28 GLOB g/dL 2.0 2.4 1.7 2.0 1.9 1.8 1.9 1.6 2.0 2.4 CALC mg/dL 11.5 12.5 10.5 11.9 11.4 10.9 11.1 11.0 12.5 11.0 XI - TP g/dL 6.6 5.5 6.1 6.2 6.1 6.6 6.3 5.5 6.2 6.3 10 ALB g/dL 4.4 3.7 4.2 4.1 4.1 4.2 4.1 3.6 4.1 4.4 mg/kg - Day GLOB g/dL CALC mg/dL 2.2 12.6 1.8 11.0 1.9 10.6 2.1 11.1 2.0 11.6 2.4 11.1 2i2 10.7 1.9 10.8 2.1 11.2 1.9 11.2 IPHS mg/dL 11.6 13.7 9.6 13.0 10.9 11.2 10.0 10.9 16.9 11.0 28 IPHS mg/dL 13.8 11.0 9.6 12.8 11.4 9.6 10.1 10.2 13.0 10.3 Company Sanitized. Does not contain TSCA 0O0 Clinical Pathology Data DuPont-13830 NA mmol/L 143.9 146.4 146.5 146.7 147.1 147.7 148.3 145.9 148.9 150.2 K mmol/L 6.01 7.72 5.69 5.55 6.51 6.54 6.34 6.35 7.16 6.23 CL mmol/L 102.5 103.6 100.5 101.2 99.3 105.2 99.6 102.4 103.6 104.8 FHEM None None None None None None None None None None FLIP None None None None None None None None None None FICT None None None None None None None None None None PFLU ig/mL 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 NA mmol/L 149.3 147.5 144.8 144.3 146.8 147.9 145.0 150.2 149.1 151.0 K mmol/L 6.59 5.88 6.18 7.21 5.89 6.12 6.22 5.75 6.47 6.23 CL mmol/L 104.4 102.1 99.5 101.9 103.9 102.9 101.5 101.4 105.4 103.3 FHEM None None None None None None None None None None FLIP None None None None None None None None None None FICT None None None None None None None None None None PFLU Mg/mL 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 -203- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Animal Male, Animal 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 Group GLUC mg/dL 112 95 118 81 93 99 97 103 106 102 XIII - TP g/dL 6.1 5.7 5.9 5.9 6.3 6.1 5.9 6.0 5.9 6.6 100 ALB g/dL 4.1 4.0 4.0 4.1 4.0 4.1 3.9 4.0 4.0 4.4 mg/kg - GLOB g/dL 2.0 1.7 1.9 1.8 2.3 2.0 2.0 2.0 1.9 2.2 Day CALC mg/dL 11.2 11.6 11.0 11.2 10.9 11.2 11.1 10.6 10.6 11.5 Male, Animal 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 Group GLUC mg/dL 91 155 105 99 115 109 101 101 104 93 XV - TP g/dL 6.0 6.0 5.7 5.6 6.1 6.7 5.8 5.9 5.9 6.0 1000 ALB g/dL 4.2 4.4 3.9 3.7 4.1 4.3 4.0 4.1 4.0 4.2 mg/kg - GLOB g/dL 1.8 1.6 1.8 1.9 2.0 2.4 1.8 1.8 1.9 1.8 Day CALC mg/dL 11.2 11.4 10.9 11.0 11.1 11.3 11.2 11.2 11.2 11.6 28 IPHS mg/dL 9.2 10.7 11.0 11.2 10.1 10.5 10.1 11.0 9.3 10.9 28 IPHS mg/dL 11.0 11.8 10.0 11.1 10.1 10.9 10.8 10.2 10.2 11.2 Company Sanitized. Does not contain TSCA O CO Clinical Pathology Data DuPont-13830 NA mmol/L 148.0 149.2 146.5 146.7 146.1 149.4 147.2 145.6 147.8 150.7 K mmol/L 5.89 6.26 5.26 6.23 6.55 6.04 5.53 6.11 5.89 6.77 CL mmol/L 102.3 102.1 103.9 100.9 103.2 104.1 101.8 104.7 104.6 103.9 FHEM None None None None None None None None None None FLIP None None None None None None None None None None FICT None None None None None None None None None None PFLU ig/mL 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 NA mmol/L 148.3 148.9 145.0 145.5 147.6 149.7 148.4 148.3 149.5 150.8 K mmol/L 5.90 5.78 6.21 5.78 5.45 5.71 5.85 5.94 5.23 6.17 CL mmol/L 106.8 106.0 102.1 101.0 102.3 105.1 104.5 104.4 101.4 108.5 FHEM None None None None None None None None None None FLIP None None None None None None None None None None FICT None None None None None None None None None None PFLU Mg/mL 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 -204 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ Individual Animal Male, Animal 901 902 903 904 905 906 907 908 909 910 Group IX - 0 mg/kg - Day VOL QUAL COL CLAR mL Medium Light Light Medium Light Light Light Light Light Light Yellow Yellow Yellow Yellow Brown Yellow Yellow Yellow Yellow Yellow HazyClear Hazy Hazy Cloudy Hazy Hazy Hazy Hazy Hazy 6.4 12.2 5.3 1.3 10.8 5.0 11.0 12.8 18.4 6.5 28 UOSM mOsm/kg 1547 695 1362 2767 938 1032 664 531 526 627 Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group QUAL Medium Medium Medium Light Light Medium Light Medium Light Light XI - COL Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow 10 mg/kg CLAR VOL mL Hazy Hazy Hazy Cloudy Hazy Hazy Hazy Hazy Hazy Hazy 10.0 3.5 3.9 5.8 13.2 2.6 5.4 2.0 13.0 12.2 Day UOSM mOsm/kg 860 1859 2215 975 512 2126 1936 1901 1668 556 pH 7.5 8.0 7.0 NP 7.5 7.0 7.5 7.0 8.0 8.0 28 pH 7.5 6.5 8.5 8.0 7.5 7.0 8.0 6.0 7.0 8.0 Company Sanitized. Does not contain TSCA Oro Clinical Pathology Data DuPont-13830 UGLU mg/dL KET mg/dL UBIL URO BLD EU/dL NEGATIVE NEGATIVE NEGATIVE NP NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE TRACE TRACE 15 NP 15 15 TRACE TRACE TRACE 15 NEGATIVE NEGATIVE NEGATIVE NP NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE SMALL MODERATE NP MODERATE TRACE TRACE MODERATE SMALL NEGATIVE 0.2 0.2 0.2 NP 0.2 0.2 0.2 0.2 0.2 0.2 UMTP mg/dL 74 29 99 124 75 90 34 21 33 40 UFLU Mg 10.4 8.8 6.6 2.8 7.8 7.2 8.6 8.6 13.5 5.2 UGLU mg/dL KET mg/dL UBIL BLD NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE 15 15 15 15 TRACE TRACE TRACE TRACE TRACE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE SMALL NEGATIVE NEGATIVE MODERATE SMALL NEGATIVE MODERATE TRACE NEGATIVE NEGATIVE SMALL TRACE NEGATIVE URO EU/dL 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 UMTP mg/dL 98 140 180 106 22 180 148 NP 97 25 UFLU MU 7.1 5.2 8.7 7.5 9.5 5.2 8.5 3.1 8.9 6.8 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Animal Male, Animal 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 Group QUAL Light Light Light Light Light Light Light Light Light Light XIII - 100 COL Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow CLAR HazyClear Hazy Hazy Hazy Hazy Hazy Hazy Hazy Hazy mg/kg - Day VOL UOSM mL mOsm/kg 5.0 18.0 14.6 13.0 12.4 8.9 5.5 15.8 15.0 4.0 1581 478 476 522 565 879 970 470 486 1351 Male, Animal 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 Group QUAL Light Light Light Light Light Light Light Light Medium Light XV - COL Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow 1000 mg/kg - Day CLAR VOL UOSM mL mOsm/kg Hazy Hazy Cloudy Hazy Hazy Clear Hazy Hazy Hazy Hazy 12.4 11.0 10.8 4.5 8.4 8.0 9.0 5.0 3.1 12.8 652 692 823 987 1022 726 803 1382 1632 520 28 pH 7.0 7.5 7.5 8.0 8.0 7.0 7.0 8.0 8.0 8.0 28 pH 7.5 8.0 8.0 7.0 7.5 6.5 7.5 7.5 6.5 7.5 Company Sanitized. Does not contain TSCA O CO Clinical Pathology Data DuPont-13830 UGLU mg/dL KET mg/dL UBIL BLD NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE TRACE TRACE NEGATIVE TRACE NEGATIVE 15 TRACE NEGATIVE NEGATIVE TRACE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE LARGE LARGE LARGE MODERATE TRACE MODERATE NEGATIVE SMALL TRACE NEGATIVE URO EU/dL 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 UMTP mg/dL 101 26 25 25 23 53 76 22 23 101 UFLU ms 7.3 10.5 8.5 8.5 8.7 7.7 6.1 9.1 9.3 5.2 UGLU mg/dL KET mg/dL UBIL URO BLD EU/dL NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE TRACE TRACE TRACE TRACE TRACE TRACE TRACE TRACE TRACE TRACE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE NEGATIVE TRACE TRACE MODERATE SMALL SMALL TRACE TRACE NEGATIVE LARGE MODERATE 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 UMTP mg/dL 29 42 60 45 61 56 56 61 145 32 UFLU MS 9.8 9.4 7.1 6.9 9.8 8.3 9.3 6.5 5.3 7.5 -206- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Animal Clinical Pathology Data Male, Animal 901 902 903 904 905 906 907 908 909 910 Group EPIT None None None QNS Few None None None None None Male, Animal 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 Group EPIT Few Few None Few None QNS None QNS None None IX UWBC None None Few NP Few None None None None None XI - UWBC Few None Few Few None NP None NP None None 0 URBC None None None NP None None None None None None mg/kg NCRY Few Few Few NP Few Few Few Few Few Few Day MICR Few Few Few NP Many Few Few Few Few None 10 URBC None None None None None NP None NP None None mg/kg - NCRY Few Few Few Few Few NP None NP Few Few Day MICR Few Few Few Mod Few NP Few NP Few Few 28 SPER Few None None NP Few Mod Mod Few None None 28 SPER Few None Few Mod None NP None NP None Few DuPont-13830 Company Sanitized. Does not contain TSCA CBI -207- Company Sanitized. Does not contain TSCA H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________ ' Individual Animal Clinical Pathology Data Male, Animal 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 Group EPIT None None None None None Few None None None Few XIII - UWBC None None None Few Few None None None None None 100 URBC Few None None None None None None None None None mg/kg - NCRY Few Few Few Few Few None Few Few Few Few Day MICR Few Few Few Mod Few Few Few Few Few Few Male, Animal 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 Group EPIT None None Few None None None None None None Few XV UWBC Few Few Few None Few None None Few None Few 1000 URBC None None Few None None None None None None None mg/kg - NCRY Few Few Mod Few None None None None Few Mod Day MICR Mod Few Mod Few Few Few None None Few Mod 28 SPER None Few Few Few Mod Few Mod Few None None 28 SPER None Mod None None Few None None Few None None O ro DuPont-13830 H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 APPENDIX I INDIVIDUAL ANIMAL BODY AND ORGAN WEIGHTS - 209 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ INDIVIDUAL ANIMAL BODY AND ORGAN WEIGHTS EXPLANATORY NOTES Note mg/kg = mg/kg/day DuPont-13830 - 210Company Sanitized. Does not contain TSCA C8I H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ _________ Organ,Weight Listing % DuPont-13830 Individual Animal & Summary Stats Report p l a c e s 2 0 0 0 vi.400 Group : IX 0 mg/kg (Control) Treatment : 0 mg/kg IX Sex :MALES 1 ANIMAL 1 FBW (Gms) 901 340.80 902 309.30 903 337.40 904 321.50 905 323.10 906 331.80 907 340.90 908 296.80 909 357.30 910 366.60 KIDNEYS (Gms) %FBW 2.842 0.8339 2.484 0.8031 2.641 0.7828 2.939 0.9142 2.834 0.8771 2.652 0.7993 3.175 0.9314 2.587 0.8716 2.962 0.8290 3.314 0.9040 (Gms) LIVER %FBW 10.916 3.2031 9.819 3.1746 9.813 2.9084 9.571 2.9770 9.716 3.0071 10.253 3.0901 11.271 3.3062 8.367 2.8191 11.252 3.1492 13.076 3.5668 THYROID GLAND (Gms) %FBW 0.016 0.0047 0.015 0.0048 0.012 0.0036 0.018 0.0056 0.014 0.0043 0.012 0.0036 0.018 0.0053 0.015 0.0051 0.019 0.0053 0.018 0.0049 Mean S.D. 332.55 20.986 2.843 0.8546 0.264 0.0523 10.405 3.1202 1.287 0.2146 0.016 0.0047 0.003 0.0007 Group : XI 10 mg/kg Treatment : 10 mg/kg XI Sex -.MALES J ANIMAL FBW KIDNEYS (Gms) (Gms) %FBW LIVER (Gms) %FBW 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 349.20 313.80 338.70 317.70 301.80 326.30 352.10 293.80 359.90 310.50 2.861 0.8193 2.666 0.8496 2.842 0.8391 2.503 0.7879 2.907 0.9632 3.044 0.9329 2.868 0.8145 2.739 0.9323 3.616 1.0047 2.849 0.9176 10.871 3.1131 8.825 2.8123 11.477 3.3885 10.116 3.1841 9.635 3.1925 10.793 3.3077 12.537 3.5606 9.933 3.3809 11.533 3.2045 9.989 3.2171 Mean S.D. 326.38 22.630 2.890 0.8861 0.294 0.0731 10.571 3.2361 1.087 0.1987 THYROID GLAND (Gms) %FBW 0.016 0.0046 0:016 0.0051 0.012 0.0035 0.013 0.0041 0.014 0.0046 0.020 0.0061 0.019 0.0054 0.018 0.0061 0.023 0.0064 0.022 0.0071 0.017 .0053 0.004 0.0011 I 1 I 1 FBW - Final Body Weight Company Sanitized. Does not contain TSC A CBI -211- ) H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats__________________ __________ ) ) DuPont-13830 Organ, Study Group : XIII 100 mg/kg Treatment : 100 mg/kg XIII Sex :MALES | ANIMAL FBW KIDNEYS (Gms) (Gms) %FBW (Gms) LIVER %FBW THYROID GLAND (Gms) %FBW 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 329.00 327.80 280.70 278.40 319.50 346.20 325.20 283.00 340.30 298.20 3.100 0.9422 2.843 0.8673 2.629 0.9366 2.711 0.9738 2.840 0.8889 3.193 0.9223 2.852 0.8770 2.357 0.8329 3.004 0.8828 2.652 0.8893 10.853 3.2988 9.868 3.0104 8.880 3.1635 9.022 3.2407 10.094 3.1593 11.686 3.3755 10.697 3.2894 7.852 2.7746 10.795 3.1722 9.483 3.1801 0.016 0.0049 0.014 0.0043 0.013 0.0046 0.019 0.0068 0.018 0.0056 0.020 0.0058 0.021 0.0065 0.014 0.0049 0.017 0.0050 0.015 0.0050 Mean S.D. 312.83 25.545 2.818 0.9013 0.246 0.0418 9.923 3.1664 1.142 0.1700 0.017 0.0053 0.003 0.0008 Group : XV 1000 mg/kg Treatment : 1000 mg/kg XV Sex :MALES I ANIMAL 1 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 FBW (Gms) 311.70 306.00 362.20 294.80 337.90 294.70 332.90 311.30 314.40 336.80 Mean S.D. 320.27 21.593 KIDNEYS (Gms) %FBW 2.675 0.8582 2.879 0.9408 2.881 0.7954 2.677 0.9081 3.049 0.9023 2.777 0.9423 3.007 0.9033 2.725 0.8754 2.612 0.8308 2.869 0.8518 2.815 0.8808 0.146 0.0475 (Gms) LIVER %FBW 10.061 3.2278 10.345 3.3807 10.946 3.0221 9.789 3.3206 11.708 3.4649 10.338 3.5080 10.772 3.2358 9.414 3.0241 9.182 2.9205 10.367 3.0781 10.292 3.2183 0.744 0.2018 *** Listing Complete *** THYROID GLAND (Gms) %FBW 0.017 0.0055 0.014 0.0046 0.016 0.0044 0.018 0.0061 0.019 0.0056 0.013 0.0044 0.017 0.0051 0.016 0.0051 0.012 0.0038 0.026 0.0077 0.017 0.0052 0.004 0.0011 Individual Animal & Summary Stats Report PLACES 2000 vl.400 1 1 | j FBW - Final Body Weight Company Sanitized. Does not contain TSCA CBI - 212- H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________ ___ DuPont-13830 APPENDIX J INDIVIDUAL ANIMAL GROSS AND MICROSCOPIC OBSERVATIONS -213Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 INDIVIDUAL ANIMAL GROSS AND MICROSCOPIC OBSERVATIONS EXPLANATORY NOTES Note mg/kg = mg/kg/day Lesion Grading Histopathological changes are described according to their morphologic character, distribution and severity. The distribution (extent o f tissue involvement) is indicated, where appropriate, by modifiers such as focal, multifocal, diffuse, unilateral, bilateral, etc.; A severity score, if appropriate, is also assigned as follows: Minimal: the amount o f change present barely exceeds that which is considered to be within normal limits. Mild: ingenerai, the lesion is easily identified but o f limited severity. The lesion probably does not produce any functional impairment. Moderate: the lesion is prominent but there is significant potential for increased severity. Limited tissue or organ dysfunction is possible. Severe: the degree o f change is either as complete as considered possible or great enough in intensity or extent to expect significant tissue or organ dysfunction. Comment: grades minimal through severe represent progressive involvement/severity along a continuum with minimal lesions being the least severe and severe lesions being the most severe. While the grades refer to the morphological characteristics of lesions, they also indicate their relative biological significance. Gross observations listing multiple masses for a tissue are distinguished with letters (i.e., a, b, c, d, etc.) -214Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________________________________________ DuPont-13830 Individual Animal Listing animal data for individual animals S T U D Y '- I p H H t f l H H j i H j * A P P R O V E D P R O T O C O L *) Dose G r o u p T l X ^ ^ m ^ n c ^ t ^ o n t r o l ) Treatment: 0 mg/kg IX Sex: 1 D a t e :2 0 - F E B - 04 Males Animal Ref Microscopic & Macroscopic Findings 901 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. KIDNEYS : CHRONIC PROGRESSIVE NEPHROPATHY, minimal. No Microscopic Abnormality Observed : THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 902 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. KIDNEYS : AGGREGATES, LYMPHOID, minimal. THYROID GLAND : ECTOPIC THYMUS TISSUE. No Microscopic Abnormality Observed : THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED - 215 Compny Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 STUDY : Do s e Gr Individual Animal Listing tg a n i m a l d a t a f o r i n d i v i d u a l a n i m a l s U * A P P R O V E D P R O T O C O L *) Control) Treatment: 0 mg/kg IX Sex: 2 Date:20-FEB-04 Males Animal Ref Microscopic & Macroscopic Findings 903 Terminal Sacrifice K illed on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUB A C U T E /C H R O N I C , minimal. KIDNEYS : CHRONIC PROGRESSIVE NEPHROPATHY, minimal. No Microscopic Abnormality Observed : THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 904 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. KIDNEYS : CHRONIC PROGRESSIVE NEPHROPATHY, minimal. No Microscopic Abnormality Observed : THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -216Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 STUDY Dose Group Individual Animal Listing Ihowir a n i m a l d a t a for i n d i v i d u a l a n i m a l s * A P P R O V E D P R O T O C O L *) Control) Treatment: 0 mg/kg IX Sex: 3 D a t e :2 0 - F E B - 0 4 Males Animal Ref Microscopic & Macroscopic Findings 905 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, KIDNEYS : AGGREGATES, LYMPHOID, minimal. minimal. No Microscopic Abnormality Observed : THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 906 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, INFLAMMATION, SUBACUTE/CHRONIC, minimal. KIDNEYS : AGGREGATES, LYMPHOID, minimal. minimal. No Microscopic Abnormality Observed : THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -217Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 STUDY Dose Group Individual Animal Listing owi: g a n i m a l d a t a f o r i n d i v i d u a l a n i m a l s A * A P P R O V E D P R O T O C O L *) Control) Treatment: 0 mg/kg IX Sex: 4 D a t e :2 0 - F E B - 0 4 Males Animal Ref Microscopic & Macroscopic Findings 907 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathoiogy : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. THYROID GLAND : HYPERTROPHY, FOLLICULAR CELL, minimal. No Microscopic Abnormality Observed : KIDNEYS, SKIN, TREATED, SKIN, UNTREATED 908 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathoiogy : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. NECROSIS, FOCAL, minimal. KIDNEYS : AGGREGATES, LYMPHOID, minimal. THYROID GLAND : HYPERTROPHY, FOLLICULAR CELL, minimal. No Microscopic Abnormality Observed : SKIN, TREATED, SKIN, UNTREATED -218Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats............... ..... DuPont-13830 Individual Animal Listing animal data for individual animals STUDY A P P R O V E D P R O T O C O L *) Dose G i m i ^ ^ ^ L ^ ^ ^ ^ ^ ^ ^ ^ f c o n t r o l ) Treatment: 0 mg/kg IX Sex: 5 D a t e :2 0 - F E B - 0 4 Males Animal Ref Microscopic & Macroscopic Findings 909 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : KIDNEYS : DILATATION, RIGHT, PELVIS. No Macroscopic Abnormality Observed : LIVER, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. HEMATOPOIESIS, EXTRAMEDULLARY, INCREASED, KIDNEYS : CHRONIC PROGRESSIVE NEPHROPATHY, minimal. HYDRONEPHROSIS, UNILATERAL, minimal. minimal. No Microscopic Abnormality Observed : THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 910 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necr o p s y Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. KIDNEYS : AGGREGATES, LYMPHOID, minimal. CHRONIC PROGRESSIVE NEPHROPATHY, minimal. 910 C o n t i n u e d o n t he n e x t p a g e .... -219Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 STUDY Dose Group Individual Animal Listing animal data for individual animals * A P P R O V E D P R O T O C O L *) ontrol) Treatment: 0 mg/kg IX Sex: 6 D a t e :2 0 - F E B - 0 4 Males Animal Ref Microscopic & Macroscopic Findings 910 Continued from previous page Histopathology : No Microscopic Abnormality Observed : THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED - 220Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male R ats_____________ DuPont-13830 Individual Animal Listing ^ ^ ^ ^ ^ ^ ^ h o w i n g animal data for individual animals STUDY J J H H A P P R O V E D P R O T O C O L *) Dose G r ^ ^ * T r e a t m e n t : 10 m g /kg XI Sex: Males 7 D a t e :2 0 - F E B - 0 4 Animal Ref Microscopic & Macroscopic Findings 1101 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1102 Terminal Sacrifice K i lled on D a y : 28 Animal is sign e d off fr o m n ecropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1103 Terminal Sacrifice Kill e d on D a y : 28 Animal is s i gned off from ne c r o p s y Gross Pathology No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1104 Terminal Sacrifice K illed on D a y : 28 Animal is signed off from necropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, Un t r e a t e d -221 Compny Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 Individual Animal Listing animal data for individual animals STUDY :y||[ A P P R O V E D P R O T O C O L *) Dose Group ^ X ^ ^ ^ ^ m ^ ^ y ^ T r e a t m e n t : 10 mg/ k g XI Sex: Males 8 Date:20-FEB-04 Animal Ref Microscopic & Macroscopic Findings 1105 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology- No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1106 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy . Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1107 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology LIVER : DISCOLORATION, BROWN, RIGHT DORSAL LOBE. No Macroscopic Abnormality Observed : KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology LIVER : FIBROSIS, FOCAL, CENTRILOBULAR, mild, with inflammation. - 222Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____ _ DuPont-13830 S T U D Y ____ Dose Group Individual Animal Listing g animal data for individual animals (* A P P R O V E D P R O T O C O L *) Treatment: 10 mg/ k g XI Sex: Males 9 D a t e :2 0 - F E B - 04 Animal Ref Microscopic & Macroscopic Findings 1108 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1109 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1110 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -223Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ __ DuPont-13830 STUDY Dose Group Individual Animal Listing animal data for individual animals * APPROVED PROTOCOL * Treatment: 100 mg/kg XIII Sex: 10 D a t e :20- F E B - 04 Males Animal Ref Microscopic & Macroscopic Findings 1301 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology- ORAL CAVITY : U L C E R /EROSION, BETWEEN UPPER INCISORS <2MM DIA. No Macroscopic Abnormality Observed : LIVER, K IDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1302 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1303 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1304 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy Gross Pathology No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -224Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13830 S T U D Y :l D o s e Gri Showi; Individual Animal Listing f animal data for individual animals * A P P R O V E D P R O T O C O L *) :g T r e a t m e n t : 100 m g / k g X I I I S ex: 11 D a t e :2 0 - F E B - 04 Males Animal Ref M i c r o s c o p i c Sc M a c r o s c o p i c F i n d i n g s 1305 Terminal Sacrifice K i l l e d on D a y : 28 A n i m a l is signed off from n e c r o p s y Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1306 Terminal Sacrifice K i l l e d on D ay : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1307 Terminal Sacrifice K i l l e d on D a y : 28 A n imal is signed off from n e c r o p s y Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1308 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -225Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 STUDY D o s e Gri Individual Animal Listing r animal data for individual animals * A P P R O V E D P R O T O C O L *) :g T r e a t m e n t : 100 m g / k g X I I I Sex: 12 D a t e :2 0 - F E B - 0 4 Males Animal Ref Microscopic & Macroscopic Findings 1309 Terminal Sacrifice K i l l e d o n D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1310 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -226Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 STUDY D o s e Gr' Individual Animal Listing animal data for individual A P P R O V E D P R O T O C O L *) Treatment: 1000 mg/kg XV 13 D a t e :2 0 - F E B - 0 4 animals Sex: Males Animal Ref Microscopic & Macroscopic Findings 1501 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : I N F L A M M A T I O N , S U B A C U T E /C H R O N I C , mild. No Microscopic Abnormality Observed : KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1502 Terminal Sacrifice K i l l e d on D ay : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. No Microscopic Abnormality Observed : KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1503 Terminal Sacrifice K illed on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED - 227 Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________________ ________________ DuPont-13830 STUDY Dose Grou)^^X\^^0^^^^3w3 Individual Animal Listing animal data for individual A P P R O V E D P R O T O C O L *) Treatment: 1000 mg/kg XV 14 D a t e :2 0 - F E B - 0 4 animals Sex: Males Animal Ref Microscopic & Macroscopic Findings 1503 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. KIDNEYS : CHRONIC PROGRESSIVE NEPHROPATHY, minimal. AGGREGATES, LYMPHOID, minimal. THYROID GLAND : HYPERTROPHY, FOLLICULAR CELL, minimal. No Microscopic Abnormality Observed : SKIN, TREATED, SKIN, UNTREATED 1504 Terminal Sacrifice Killed on D ay : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. No Microscopic Abnormality Observed : KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1505 Terminal Sacrifice K illed on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, UNTREATED 1505 Continued on the next page- TREATED, SKIN, -228Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 STUDY Dose Individual Animal Listing animal data for individual A P P R O V E D P R O T O C O L *) Treatment: 1000 mg/kg XV animals Sex: Males 15 D a t e : 2 0 -FEB-04 Animal Ref Microscopic & Macroscopic Findings 1505 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. No Microscopic Abnormality Observed : KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1506 Terminal Sacrifice K i l l e d o n D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC-, minimal. No Microscopic Abnormality Observed : KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1507 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from ne c r o p s y Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. 1507 C o n t i n u e d o n t h e n e x t page- .... -229Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_______ __________________________________ DuPont-13830 STUDY D o s e G: Individual Animal Listing animal data for individual * A P P R O V E D P R O T O C O L *) Treatment: 1000 mg/Xg XV 16 D a t e :2 0 - F E B - 0 4 animals Sex: Males Animal Ref Microscopic & Macroscopic Findings 1507 Continued from previous page Histopathology : THYROID GLAND : HYPERTROPHY, FOLLICULAR CELL, minimal. SKIN, UNTREATED : INFLAMMATION, FOCAL, SUBACUTE/CHRONIC, minimal, dermal. No Microscopic Abnormality Observed : KIDNEYS, SKIN, TREATED 1508 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED H i s t o p a t h o l o g y .: LIVER : INFLAMMATION, SUBACUTE /C H R O N I C , mild. KIDNEYS : CHRONIC PROGRESSIVE NEPHROPATHY, minimal. No Microscopic Abnormality Observed : THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 1509 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, UNTREATED 1509 C o n t i n u e d on the n e x t p age .... TREATED, SKIN, -230Company Sanitized. Does not contain TSCA CBI H-25425: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________ DuPont-13830 STUDY Dose Gr Individual Animal Listing animal data for individual * A P P R O V E D P R O T O C O L *) Treatment: 1000 mg/kg XV 17 Date:20-FEB-04 animals Sex: Males Animal Ref Microscopic & Macroscopic Findings 1509 Continued from previous page Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, minimal. NECROSIS, FOCAL, minimal. SKIN, UNTREATED : INFLAMMATION, FOCAL, SUBACUTE/CHRONIC, minimal, muscular. No Microscopic Abnormality Observed : KIDNEYS, THYROID GLAND, SKIN, TREATED 1510 Terminal Sacrifice K i l l e d on D a y : 28 Animal is signed off from necropsy Gross Pathology : No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED Histopathology : LIVER : INFLAMMATION, SUBACUTE/CHRONIC, mild. KIDNEYS : CHRONIC PROGRESSIVE NEPHROPATHY, minimal. SKIN, TREATED : INFLAMMATION, FOCAL, ACUTE, minimal, epidermal. No Microscopic Abnormality Observed : THYROID GLAND, SKIN, UNTREATED *** L i s t i n g C o m p l e t e **-* -231 Company Sanitized. Does not contain TSCA CBI