Document mpXYavaOmnYye384RjB0LR614

b -- --------------------------------------------- ------------------------------------------------------------------------------ 498 fa ite 3 3 DuPont-13882 TRADE SECRET Study Title H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats Test Guidelines: U.S, EPA Health Effects Test Guidelines OPPTS 870.3200 (1998) OECD Guideline for the Testing of Chemicals Section 4: Health Effects, Number 410(1981) Author: Carol Finlay, B.A. Study Completed on: May 10,2004 Performing Laboratory: EJ. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Laboratory Project ID: DuPont-13882 Work Request Number: Service Code Number: Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 'U.S.A. Page 1 of 187 Company Sanitized. Does not contain TSCA C8I H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles of Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 except for the item documented below. The item listed does not impact the validity of the study. The test fabrics were characterized prior to the initiation o f this study by determining the quantity of the melt additive incorporated into the fabric layers. Although the characterization was not performed under Good Laboratory Practice Standards, the accuracy of the composition o f the melt additive at concentrations documented in this report is considered sufficient for the purpose of this study. Applicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Director: fa v ifri ic h i U f Carni FIfnhlay, B.A(J Staff Toxicologist \b -Y h A jL'-'lbht/ Dat-1 Applicant / Sponsor:_________________________________________ __________ DuPont Representative Date Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 QUALITY ASSURANCE DOCUMENTATION Haskell Sample Number(s): 26136,26137, 26138,26139 Dates of Inspections: Protocol: November 17,19, 2003 Conduct: November 21, 2003 Records, Reports: February 6,11, 2004; April 19-20, 2004 Dates Findings Reported to: Study Director: November 21,2003; February 6,11, 2004; April 20,2004 Management: November 21, 2003 ; February 6,11,2004; April 20, 2004 Reported by: StaffQuality Assurance Auditor -3 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats__________________ _______________ DuPont-13882 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Clinical Pathology Evaluation by: Nancy E. Evets, D.V.M. Diplomate A.CVP. Principal Research Scientist Date ' ' JL r ^ Q ps GregP. Sykes, VJMD. Diplomate A.C.V.P., ACJLAJM., A.B.T. Veterinary Pathobgist 1 ~ ' ^ "`'Z ^ Date Asatelc Pathalagy StevenR. Frame, D.V.M., PhD. Diplomate A.C.VJP. Principal Research Pathobgist and Research Manag: Daft Approved by: JL rl*' A, Scott E. Loveless, PhD. Management Date Issued by Study Director: Card! Finlay, B A f Staff Toxicologist / 6 ' / K>o,j Date (J -4 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26I39: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 TABLE OF CONTENTS Page GOOD LABORATORY PR A C TIC E COMPLIANCE STATEM ENT................................... 2 QUALITY ASSURANCE DOCUM ENTATION........................................................................... 3 C ER TIFIC A TIO N ............... >...............................................................................................................4 LIST OF TABLES.............................................................................................................................. .7 LIST OF FIG U R ES............................................................................................................................. 7 L IST OF A PPEN D IC ES......................................................................... ....................................... ...7 STUDY IN FO R M A TIO N .......... ............................................................ ...........................................8 STUDY PERSONNEL............................................................................. .........................................10 SUM M ARY................................................................. 11 IN T R O D U C T IO N ........................................ 12 STUDY DESIGN............................................. 12 M ATERIALS AND M ETH O D S.............................. 12 A. Test Guidelines...................................................................... 12 B. Test Substances...................................... 13 C. Test Species..............................................................................................................................13 D. Animal Husbandry.... .............................................................................................................. 13 E. Quarantine and Pretest....................................................... :............................ ........ ..............14 -F. Assignment to Groups and Study Start..................................................................................15 G. Dosing Procedure...... ................................ .................................... ....................................... 15 H. Body Weights.......................... 15 I. Food Consumption and Food Efficiency..............................................................................15 J. Observations................................... 16 K. Perfluorooctanoic Acid Level Evaluation...... .............................................................. '........ 16 L. Clinical Pathology Evaluation .............................................................................. 16 M. Anatomic Pathology................................................................................................................. 17 N. Statistical Analyses.................................................................................................................. 18 RESULTS AND D ISCUSSION............................................................... 19 In-Life Toxicology............................................ 19 A. Mean Body Weights and Body Weight G ains......................................................................19 B. Food Consumption and Food Efficiency...............................................................................19 C. Clinical Observations, Dermal Effects, and Mortality......................................................... 19 D . In-Life Conclusions............... 19 Clinical Pathology E v alu atio n ............. 20 A. Clinical Chemistry.................................................................................................................. 20 -5 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 B. Urinalysis............... 20 C. Urine Fluoride.......................................................................................................................... 20 D. Clinical Pathology Conclusions..............................................................................................20 Anatomic Pathology Evaluation............................................................................................ 20 A. Mortality:...................................................................................................................................20 B. Organ W eights..... ....... 21 C. Gross Observations................................................................................................................. 21 D. Microscopic Findings.................. 21 E. Anatomic Pathology Conclusions.......................................................................................... 21 PFOA ANALYSIS..............................................................................................................................21 COMPARISON TO STUDY WITH THE MELT ADDITIVE........................... 22 CONCLUSIONS....................... 22 RECORDS AND SAMPLE STORAGE............................................... 23 REFERENCES................................................................................................................... 23 TABLES....................................................................................................... 24 FIGURES.................................................................................................... 40 APPENDICES............................................................................................ 42 -6 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 LIST OF TABLES Page TABLE 1 MEAN BODY WEIGHTS OF MALE RATS......................................................................... ......................26 TABLE 2 MEAN BODY WEIGHT GAINS OF MALE RATS...............................................................................-..,,27 TABLE 3 MEAN DAILY FOOD CONSUMPTION BY MALE RATS......................................................................28 TABLE 4 MEAN DAILY FOOD EFFICIENCY OF MALE RA TS........................ .........:......................................... 29 TABLE 5 SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - PREDOSING..............................30 TABLE 6 SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - POST DOSING ..................31 TABLE 7 M E A N jU p E V E L S IN MALE RATS.................................................................................................... 33 TABLE 8 SUMMARY OF CLINICAL CHEMISTRY VALUES FOR MALE RATS ....................................... 34 . TABLE 9 SUMMARY OF URINALYSIS VALUES FOR MALE RATS.... .............................................................. 35 TABLE 10 PERCENT SURVIVAL OF MALE RATS................................................ ................................................ 36 TABLE 11 MEAN FINAL BODY AND ORGAN WEIGHTS FOR MALE R A TS.................................................. 37 TABLE 12 INCIDENCES OF GROSS OBSERVATIONS IN MALE RATS............................................................39 LIST OF FIGURES Page FIGURE 1 MEAN BODY WEIGHTS OF MALE RATS.............................................................................................. 41 LIST OF APPENDICES Page APPENDIX A INDIVIDUAL BODY WEIGHTS..... ................................................................................................... 43 APPENDIX B INDIVIDUAL FOOD CONSUMPTION.............................................................................................. 48 APPENDIX C INDIVIDUAL CLENICALOBSERVATIONS AND MORTALITY RECORDS............................. 54 APPENDIX D INDIVIDUAL CLINICAL OBSERVATIONS - POST-DOSING......... .............................................62 APPENDIX E INDIVIDUAL EDEMA AND ERYTHEMA SCORES....................... ............................................ . 75 APPENDIX J g H ANALYTICAL REPORT......................................................................................................... 101 APPENDIX G INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA............................................................ 165 APPENDIX H INDIVIDUAL ANIMAL FINAL BODY AND ORGAN WEIGHTS............................................... 171 APPENDIX I INDIVIDUAL ANIMAL GROSS OBSERVATIONS......................................................................... 175 -7- Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dennal Toxicity 28-Day Study in Male Rats ____ Control Fabric STUDY INFORMATION . H-26136 DuPont-13882 Known Impurities: Physical Characteristics: Off-white to bluish-white fabric Test Fabric #1 Known Impurities:] Physical Characteristics: Off-white to bluish-white fabric -8 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Submitter's Notebook Numberfs): Haskell Number: 26138 Composition: Known Impurities: Physical Characteristics: Off-white to bluish-white Test Fabric #3 Submitter's Notebook Numberfs): Haskell Number: 26139 Composition: Known Impurities: None Physical Characteristics: Off-white to bluish-whit^ Study Initiated/Completed: November 18, 2003 / (see report cover page) In-Life Initiated/Completed: November 24, 2003 / December 22, 2003 Company Sanitized. Does not contain TSCA CBI H-26137. H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________________ DuPont-13882 STUDY PERSONNEL Study Director: Carol Finlay, B.A. Management: Jeanette M. Erhardt, Ph.D. Primary Technician: James C. Mackay Management: Janice L. Connell, M.S., B.A., C.I.H. Clinical Pathologist: Nancy E. Everds, D.V.M., Diplomate A.C.V.P. Management: Jeanette M. Erhardt, Ph.D. Anatomic Pathologist: Greg P. Sykes, V.M.D., Diplomate A.C.V.P., .C.L.A.M., A.B.T. Management: Scott E. Loveless, Ph.D. Toxicology Report Preparation: Sean M. Callaghan, B.A. Management: Em D, Birkmeyer, M.S. Laboratory Veterinarian: Thomas W. Mayer, D.V.M., Diplomate A.C.L.A.M. Management: Janice L. Connell, M.S., B.A., C.I.H. - 10Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ________________________ DuPont-13882 SUMMARY The objective of this study was to determine if the effects that occurred in a previously conducted 28-day dermal toxicity study with the active ingredient would be observed when the active material was incorporated into a polypropylene fabric. O f specific interest were dermal and clinical chemistry effects and the levels of blood plasma. The fabric samples were identified as H-26137, H-26138, and H-26139. Each test fabric was applied to the shaved, intact skin o f 10 male Crl:CD(SD)IGS BR rats for 6 hours/day for 28 consecutive days. A group of 10 male rats was similarly treated with the control fabric H-26136, 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 and urine samples were collected for clinical pathology prior to sacrifice on day 28. Blood samples were collected at necropsy for analysis' o f plasma concentration o f perfluorooctanoic acid. A ll rats underwent gross necropsy and select tissues were weighed and/or collected. No microscopic pathology evaluations were conducted. There were no effects on body weight, food consumption, food efficiency, or mortality. No clinical signs attributable to systemic toxicity occurred. No dermal irritation was observed during the study. There were no test substance-related or adverse effects on organ weights or gross observations at necropsy. The clinical chemistry effects seen previously with the active ingredient were not confirmed in this study. However, rats treated with H-26138 and H-26139 had minimally decreased cholesterol and minimally increased urine flouride; these two effects were o f uncertain relationship to treatment, but were similar to effects observed during the previous dermal study with this compound. The mean levels o f l l B m plasma were 35.7 ppb, 98.3 ppb, and 159.3 ppb for rats exposed to test fabrics H-26137, H-26138, and H-26139, respectively. The levels o t f B b the control group H-25136 were below the limit o f quantitation (10 ppb). The source o f the the plasma, whether from direct application or metabolism o f chemical in the test material, is unknown. Under the conditions of this study, application o f the test fabrics, H-26137, H-26138, and H-25139, caused no adverse effects in male rats. - 11 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 INTRODUCTION The objective o f this study was to determine if the effects that occurred in a previously conducted 28-day dermal toxicity study with the active ingredient (a fluorotelomer melt additive) would be observed when the active material was incorporated into a polypropylene fabric. Three fabrics were tested in this study, H-26137, H-26138, and H-26139. In the previous study, mildly decreased cholesterol and triglycerides occurred in rats treated at 500 mg/kg/day. Urine fluoride was increased in rats treated at 50 and 500 mg/kg/day and fluorine was present in the day 27 blood. was present in the plasma of rats treated with the melt additive. No test substance-related dermal irritation occurred.^ O f specific interest in conducting the present study were dermal and clinical chemistry effects and the levels o f| in blood plasma. STUDY DESIGN Group I m V vn Number/Group 10 10 10 10 Haskell Laboratory Number H-26136 (Control) H-26137 H-26138 H-26139 Study Parameters Frequency Body Weight Test day 0 and weekly thereafter Food Consumption Test day 0 and weekly thereafter Clinical Observations Daily Observations for dermal effects Daily Mortality/Moribundity Checks Twice daily Clinical Pathology Week 4 tflflh jb lo o d analysis Week 4 Necropsy Week 4 MATERIALS AND METHODS A. Test Guidelines The study design complies with the following test guidelines except as noted below: - 12Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Office of Prevention, Pesticides and Toxic Substances (OPPTS) U.S. Environmental Protection Agency (EPA) (1998). OPPTS 870.3200 2 1/28-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. - The wrappings of some rats became dislodged during the exposure period. Therefore, on these occasions they may not have been exposed for a full 6 hours. This exception did not affect the objectives or the validity of the study because, considering that no significant toxicological effects were observed, it is not likely that an occasional exposure period1of less than 6 hours for some animals would have changed the study results. - Only male rats were treated. This exception did not affect the objectives or validity of the study because in previous studies with other fluorotelomers males are more sensitive to the toxicological effects compared to females. In addition, the comparison 28-day dermal study with the melt additive was conducted with male rats. B. Test Substances The test substances were supplied by the sponsor as off-white to bluish-white fabrics. H-26136 contained 0.03% melt additive and served as control. The test fabrics, H-26137, H-26138, and H-26139, contained 0.05%, 0.78%, and 1.46% melt additive, respectively. The sponsor marked the side of the fabric not treated with melt additive to distinguish it from the side to be placed against the animal. The test substances appeared to be stable under the conditions of the study. No evidence o f instability, such as a change in color, 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. - 13Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 2. Environmental Conditions Animal rooms were maintained at a temperature of 19-25C and a relative humidity of 30-70%. Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. Excursions outside o f these ranges were o f insufficient magnitude and/or duration to have adversely affected the validity o f the study. 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 of its cage. After assignment to groups, each rat was assigned an animal number which was tattooed on the tail o f each rat. The information on the cage labels included the animal number assigned to each rat. 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 re below those that would be expected to impact the scientific integrity of the study: Water samples are analyzed for total bacterial counts, and the presence 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 of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence o f these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity o f the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluation o f these data did not indicate any conditions that affected the validity of 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 - 14Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 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 0 (prior to dosing) by the designee o f the laboratory animal veterinarian. F. Assignment to Groups and Study Start Forty rats were selected for study use on the bases of adequate body weight gain, freedom from clinical signs of 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 of 10 rats, so that there were no statistically significant differences among group body weight means. Dosing of the control and test fabrics began on test day 0 when the rats were approximately 8 weeks of age. G, Dosing Procedure On the day prior to the first treatment, the fur of each rat was closely shaved to expose the skin from the back and trunk. Ota each day o f treatment, the rats wore plastic collars during the exposure period to prevent oral exposure to the fabrics. The rats were treated at approximately the same time each day. The rats were treated for 28 consecutive days. The exposure period was approximately 6 hours per day. The control and test fabrics were slightly moistened with deionized water, wrapped around the animal, and secured. The side o f the fabric treated with melt additive was placed against the skin. The approximate shaved total body surface area covered was 20%. After the exposure period, the collars and fabrics were removed and the shaved area was washed with warm tap water. Each rat was then gently patted dry and returned to the cage. The rats were reshaved as needed during the study. The entire area that was originally shaved was reshaved. The animals were shaved only after an evaluation. EL Body Weights All rats were weighed weekly throughout the study. Additionally, they were weighed on the day o f sacrifice (test day 28). 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 of spillage from the feeder during the week from the initial weight. From these measurements, mean daily - 15Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 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 removal o f the fabrics. The rats were also observed for clinical signs at each weighing. The Draize Scale was used to score skin irritation. The rats were checked twice daily for mortality and for signs o f illness, injury, or abnormal behavior. K. !Level Evaluation Blood was collected from the vena cava of all rats at necropsy into a tube containing EDTA. Plasma was prepared and stored frozen at -80C to -20C until analyzed. A H f ^ w a s 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. 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, the animals were placed in metabolism cages. These animals were fasted after 3 p.m. (overnight) and urine was collected from each animal. Blood samples for clinical chemistry measurements were collected from the orbitalsinus o f each animal while the animal was under carbon dioxide anesthesia. Blood for^JJjj^analysis was collected from the vena cava. Additional blood collected from the vena cava was placed in a serum tube, processed to serum, and frozen at -80C. Samples for clinical chemistry and for frozen serum 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. Clinical Chemistry Serum clinical chemistry parameters were determined on an Olympus AU640 clinical chemistry analyzer. The following parameters were determined: cholesterol triglycerides -16Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____ ________ ___________________ DuPont-13882 2. Urinalysis Urine volume was measured. Urine fluoride concentrations were determined using a Beckman 01 2 digital pH/ISE meter and a fluoride-selective electrode, and total urine fluorides were calculated (volume x concentration). The following parameters were determined: volume fluoride M. Anatomic Pathology After 28 days o f exposure to the control and three test fabrics (test day 28), groups o f 10 male rats from Groups I, HI, V, and VII were sacrificed and necropsied. The rats were fasted overnight on the afternoon before their scheduled sacrifice. On the day of necropsy, the fur was shaved from an untreated area and the untreated area was outlined. The order of 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________________________ The following tissues were weighed from all rats: liver, kidneys, and thyroid gland (after fixation). Organ weight/fmal body weight ratios were calculated. Gross lesions which were diagnosed at necropsy and for which microscopic examination was not appropriate (e.g., fluid accumulation, ruffled fur, missing anatomic parts) were generally not collected. 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. Tissues were not processed to slides and were not evaluated microscopically. - 17Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 N. Statistical Analyses Significance was judged at p < 0.05. Parameter Body Weight Body Weight Gain Food Consumption Food Efficiency Organ Weight Clinical Pathology' Survival Incidence of Clinical Observations Preliminary Test Test for lack of trend Levene's test for homogeneity and ShapiroWilk test for nprmalityb Levene's test for homogeneity and ShapiroWilk test for normality1' Method of Statistical Analysis If preliminary test is not I f preliminary test is significant significant Sequential application131 of the JonckheereTerpstra trend test Preliminary tests for pairwise comparison OR* One-way analysis of Kruskal-Wallis variance followed with test(U) followed with Dunnett's test9' Dunn's test(12) One-way analysis of Kruskal-Wallis variance^followed with test*'11'1followed with Dunnett's test9' Dunn's test1 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. - 18Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 RESULTS AND DISCUSSION In-Life Toxicology A. Mean Body Weights and Body W eight Gains (Tables 1-2, Figure 1, Appendix A) No effects on body weights or body weight gains occurred in any group o f rats. B. Food Consumption and Food Efficiency (Tiables 3-4, Appendix B) No effects on food consumption or food efficiency occurred in any group o f rats. C. Clinical Observations, Dermal Effects, and M ortality (Tables 5-6, Appendices C-D) No clinical signs attributable to systemic toxicity occurred. Ocular and nasal discharge were observed in most rats treated with the control and test fabrics. These clinical signs are considered to be due to the wrapping/coilar application procedure. One rat treated with the control fabric, H-26136, exhibited swollen face. This finding was considered due to the collar being too tight. One rat treated with test fabric H-26137 and three rats treated with test fabric H-26138 exhibited swollen forelimbs. This finding was probably a result o f the wrapping procedure. Hair loss present in one rat treated with test fabric H-26137 was considered incidental and not due to the test fabric. Rats treated with both the control and test fabrics exhibited superficial wounds during the study. Stained perineum observed in one rat treated with test fabric H-26137 was not considered due to the fabric because it was only observed in one rat. No dermal irritation was observed during the study. No deaths occurred. D. In-Life Conclusions Under the conditions of this study and for the in-life parameters evaluated, there were no adverse effects on in-life parameters for any o f the test substances. - 19Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ Clinical Pathology Evaluation DuPont-13882 A. Clinical Chemistry (Table 8, Appendix G) There were no statistically significant or adverse changes in clinical chemistry parameters in the male rats. Cholesterol was minimally lower in animals treated with H-26138 and H-26139 (means were 80% and 86% of control group means, respectively). However, these changes were not statistically significant and were very small compared to the variability of the parameter; thus they were of uncertain relationship to treatment. B. Urinalysis (Table 9, Appendix G) There were no statistically significant or adverse changes in urinalysis parameters in the male rats. Urine volume was mildly increased in four treated rats (two treated with H-26137 and two treated with H-26138). These changes were considered to be unrelated to treatment due to the variability o f urine volumes. C. Urine Fluoride (Table 9, Appendix G) There were no statistically significant changes in mean excreted fluoride (urine fluoride) at the end o f the dosing period. A few individual rats treated with H-26138 and H -26139 had minimally higher urine fluoride amounts compared to rats in other groups. However, these changes were very small and of uncertain relationship to treatment. D. Clinical Pathology Conclusions Under the conditions of this study and for the clinical pathology parameters measured, there were no adverse effects on clinical pathology parameters for any of the test substances. Anatomic Pathology Evaluation A, Cause of Death (Appendix I) There were no deaths. All 40 rats survived until the scheduled 28-day sacrifice. - 20Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________________ DuPont-13882 B. Organ Weights (Table 11, Appendix H) There were no test substance-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 12, Appendix I) 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 exposure. D. Microscopic Findings Microscopic examination o f tissues was not performed. E. Anatomic Pathology Conclusions Under the conditions o f this study and for anatomic pathology parameters measured, there were no test substance-related effects on pathology parameters for any of the substances. (Appendix F, Table 7) j^pM P^as present in plasma from rats treated with the test fabrics. The mean levels in plasma collected at terminal sacrifice were 35.7 ppb, 98.3 ppb, and 159.3 ppb for test fabrics H-26137, H-26138, and H-26139, respectively. The levels o f !( 0 0 f c n the plasma from control rats were below the limit o f quantitation (10 ppb). It was not determined if the source of the in the blood came from a H 0 k source in the test fabrics or resulted from metabolism of a chemical ingredient in the test material. -21 Company Sanitized. Does not contain TSCA CB H-26137, H-26138, and H:26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Comparison To Study With The Melt Additive A 28-day dermal toxicity study was previously conducted with the neat melt additive at dosages of 0, 5, 50, and 500 mg/kg/day.(1) There were no adverse effects on body weights, clinical observations, nutritional parameters, pathology parameters, or hematologic and coagulation parameters. (P fe v e ls in plasma were 61.4 ppb, 155.5 ppb, 1315.9 ppb, and 14,176 ppb for dosages of 0, 5, 50, and 500 mg/kg/day, respectively. Mildly decreased cholesterol in rats treated at 500 mg/kg/day was considered treatment-related and potentially adverse. Mildly decreased triglycerides in rats treated at 500 mg/kg/day was considered treatment-related but not adverse. Increased urine fluoride at 50 or 500 mg/kg/day and the presence of fluorine in day 27 blood indicated exposure to and metabolism of a fluoride-containing test substance and was considered treatment-related but not adverse. The no-observed-effect level was 50 mg/kg/day based on the presence o f decreased cholesterol at 500 mg/kg/day. In the present study, cholesterol was minimally lower (although not statistically significant) in rats treated with test fabrics H-26138 and H-26139 (means were 80 and 86% o f control group means, respectively). Urine fluorides were increased in a few rats treated with test fabrics H-26138 and H-26139. These two effects were o f uncertain relationship to treatment. There were no effects on triglycerides. f W e v e l s in plasma were 35.7 ppb, 98.3 ppb, and 159.3 ppb for test fabrics H-26137, H-26138, and H-26139, respectively. Levels o f ((BMhin the plasma from control rats were below the limit of quantitation. CONCLUSIONS No deaths occurred during the study. No test substance-related effects on body weight, food consumption, food efficiency, clinical signs, or dermal irritation were seen in male rats that received 28 applications of the test and control fabrics applied daily to the shaved skin. WHKRH was present in a dose response fashion in the plasma from fats treated with the test fabrics. There were no test substance-related or adverse effects on gross pathology, organ weights, or triglyceride parameters. In rats treated with test fabrics H-26138 and H-26139, minimally decreased cholesterol and minimally increased urine fluoride in a few rats were o f uncertain relationship to treatment. Under the conditions o f this study, application o f the test fabrics, H-26137, H-26138, and H-25139, caused no adverse effects'in male rats. - 22Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 RECORDS AND SAMPLE STORAGE All data and records for analytical characterizations conducted by the sponsor will be retained by the sponsor. A sample o f each fabric was 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. REFERENCES 1. DuPont Haskell Laboratory (2003). H-25509: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats. Unpublished report, DuPont-11763. 2. Draper, N.R. and Smith, H. (1981). Applied Regression Analysis, 2nd edition, pp 266-273. Wiley, New York. 3. Selwyn, M.R. (1995). The use of 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. 4. Jonckheere, A.R. (1954). A distribution-free K-sample test against ordered alternatives. Biometrika 41, 133-145. 5. Levene, H. (1960). Robust test for equality of variances. Contributions to Probability and Statistics (J. Olkin, ed.), pp 278-292. Stanford University Press, Palo Alto. 6. Shapiro, S.S. and Wilk., M.B. (1965). An analysis o f variance test for normality (complete samples). Biometrika 52, 591-611. 7. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th edition, pp 246-248 and 349-352. The Iowa State University Press, Iowa. 8. Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482-491. 9. Dunnett, C.W. (1980). Pairwise multiple comparisons in the unequal variance case. J. Amer. Statist. Assoc. 75, 796-800. 10. Tamhane, A.C. (1979). A comparison o f procedures for multiple comparison of means with unequal variances. J. Amer. Statist. Assoc. 74, 471-480. 11. Kruskal, 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. 12. Dunn, O.J. (1964). Multiple contrasts using rank sums. Technometrics 6,241-252. - 23Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____ DuPont-13882 TABLES - 24Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 TABLES ABBREVIATIONS: EXPLANATORY NOTES Summary of Clinical Chemistry Values CHOL - cholesterol TRIG - triglycerides Summary of Urinalysis Values UVOL - volume UFLU - urine fluoride NOTES: Summary of Urinalysis Values Groups with identical values may vary in statistical significance, because tabulated statistics are rounded to fewer decimal places than the values used for statistical determination. -25 Company Sanitized. Does not contain TSCA CBI ti-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 1 Group : M aterial Test DAYO DAY7 DAY14 DAY21 DAY27 DAY2 8 MEAN BODY WEIGHTS OF MALE RATS (g) I H-26136 III H-26137 V H-26138 193.0 10.1(10) 244.8 14.4 (10) 293.6 18.3(10) 329.7 22.1 (10) 345.8 25.8 (10) ' 327.0 24.9(10) 191.5 8.6(10) 241.2 12.0 (10) 293.3 11.1(10) 327.7 14.0(10) 347.6 16.9(10) 328.8 16.7(10) 192.8 10.9(10) 245.6 13.0 (10) 288.8 17.0 (10) 326.1 17.1 (10) 338.3 17.7(10) 320.1 18.6(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-13882 VII H-26139 193.0 10.6(10) 245.7 11.3(10) 294.5 13.8 (10) 333.2 22.7(10) 350.7 29.5(10) 331.1 27.1(10) Company Sanitized. Does not contain TSCA CBI - 26- H-26137, Til-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ' ) Group: M aterial Test DAY0 -DAY7 DAY7 -DAY14- DAY14 -DAY21 DAY21 -DAY27 DAY27 -DAY28 DAY0 -DAY28 TABLE 2 MEAN BODY WEIGHT GAINS OF MALE RATS (g) I H-26136 III H-26137 V H-26138 51.9 8.1 (10) 48.8 6.3(10) 36.1 ,7.4(10) 16.1 9.6(10) -18:7 2.4(10) 134.1 18.7(10) 49.6 10.1(10) 52.1 13.0 (10) 34.5 4.5(10) 19.9 7.6(10) -18.9 2 .3 (10) 137.2 18.6(10) 52.7 7.0(10) 43.2 7.9(10) 37.4 6.3 (10) 12.2 7.2 (10) -18.2 3.2(10) 127.3 16.1(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-13882 VII H-26139 52.7 6.5(10) 48.7 8.9(10) 38.7 10.6(10) 17.5 9.0(10) -19.6 3.1(10) 138.1 26.2 (10) Company Sanitized. Does not contain TSCA OCO fl-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ) TABLE 3 Group: M aterial Test DAY0-DAY7 DAY7-DAY14 DAY14-DAY21 DAY21-DAY27 DAY0-DAY27 MEAN DAILY FOOD CONSUMPTION BY MALE RATS (g/day) I H-26136 III H -26137 V H -26138 23.1 1.9(10) 26.0 1.9(10) 26.8 2 .1 (10) 27.8 2.3(10) 25.879 1.941(10) 22.8 1.0(10) 26.8 1.1 (10) 27.2 1 .5 (10) 28.4 1.3(10) 26.232 0.845(10) 23.3 1.3(10) 26.0 1.7(10) 27.3 1.8(10) 27.3 1.5(10) 25.936 1.272(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-13882 VII H-26139 24.0 2 .0 (10) 26.7 1.8 (10) 27.7 1.9(10) 28.1 1.5(10) 26.568 1.588 (10) Company Sanitized. Does not contain TSCA CBI - 28- H-26137, H-26I38, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 4 Group: M aterial T est DAY0-DAY7 DAY7-DAY14 DAY14-DAY21 DAY21-DAY27 DAY0-DAY27 MEAN DAILY FOOD EFFICIENCY OF MALE RATS I H -26136 III H -2 6 1 3 7 V H -26138 0.319 0.030 (10) 0.267 0.025(10) 0.192 0.032(10) 0.094 0.052 (10) 0.218 0.014 (10) 0.311 0.065(10) 0.276 0.057(10) 0.180 0.017 (10) 0.116 0.042 (10) 0.220 0.024 (10) 0.323 0.039(10) 0.237 0.040(10) 0.195 0.025 (10) 0.074 0.043(10) 0.208 0;. 018 (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-13882 VII H-26139 0.315 0.034(10) 0.261 0.046(10) 0.198 0.047(10) 0.102 0.047(10) 0.219 0.032(10) Company Sanitized. Does not contain TSCA CBI - 29- ) fl-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 5 SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - PREDOSING Treatment Group Material Animal Count Discharge Nose Incidence Mean onset (Days) Eye bilateral Incidence Mean onset (Days) Hair Loss. Shoulder Incidence Mean onset (Days) Wound Superficial Incidence Mean onset (Days) I H-26136 10 0 1 ( 10%) 7 0 0 III. H-26137 10 0 0 1 ( 10%) 21 2 ( 20%) 11 V H-26138 10 0 0 0 2 ( 20%) 21 VII H-26139 10 1 ( 10%) 14 1 ( 10%) 14 0 2 ( 20%) 14 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 from control at p < 0.05. ) DuPont-13882 Company Sanitized. Does not contain TSCA CBI - 30- m H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ) ____________ _____________ DuPont-13882 TABLE 6 SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - POST DOSING Treatment Group Material Animal Count Discharge Nose Incidence Mean onset (Days) Eye bilateral Incidence Mean onset (Days) Hair Loss Shoulder Incidence Mean onset (Days) Wound Superficial Incidence Mean onset (Days) Stain Fur/Skin Perineum Incidence Mean onset (Days) I H-26136 10 8 ( 80%) 13 9 ( 90%) 0 0 1 ( 10%) 19 0 III H-26137 10 9 ( 90%) 15 10 (100%) 0 1 ( 10%) 21 3 ( 30%) 9 1 ( 10%) 26 V H-26138 10 9 ( 90%) 13 10 (100%) 1 0 3 ( 30%) 6 0 VII H-26139 10 9 ( 90%) 13 10 (100%) 3 0 3 ( 30%) 7 0 Company Sanitized. Does not contain TSCA CBI -31- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 6 (CONTINUED) SUMMARY OF CLINICAL OBSERVATIONS FOR MALE RATS - POST DOSING Treatment Group Material Animal Count Swollen Observations Face Incidence Mean onset (Days) Forelimb Incidence Mean onset (Days) I H-26136 10 1 ( 10%) ' 16 0 III H-26137 10 0 1 ( 10%) 8 V H-26138 10 0 3 ( 30%) 5 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 from control at p < 0.05. VII H-26139 10 0 0 ) DuPont-13882 Company Sanitized. Does not contain TSCA CBI - 32- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats TABLE 7 MEAN M B i LEVELS IN MALE RATS Fabric PLASMA MEAN S.D. H-26136 < 1 0 0.0 H-26137 35.7 5.4 H-26138 98.3 26.6 H-26139 159.3 53.1 -N--- --mimbepof-samples- S.D. = standard deviation a limit of quantitation = 10 ppb (ppb)a N 10 10 10 10 DuPont-13882 -33Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 TABLE 8 SUMMARY OF CLINICAL CHEMISTRY VALUES FOR MALE RATS Group I H-26136 Group HI . H-26137 Group V H-26138 Group VU H-26139 CHOL (mg/dL) DAY 28 TRIG (mg/dL) DAY 28 44 9(10) 32 11(10) 47 12(10) 30 10(10) 35 9(10) 28 12(10) 38 8(10) 27 6(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. -34Company Sanitized. Does not contain TSCA CBI H-26.137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________ DuPont-13882 TABLE 9 SUMMARY OF URINALYSIS VALUES FOR MALE RATS Group I H-26136 Group HI H-26137 Group V H-26138 Group VR H-26139 UVOL (mL) DAY 28 UFLUOxg) DAY 28 8.9 4.7(10) 10.1 2.7(10) 13.0 7.1(10) i 11.5 1.9(10) 11.2 9.5(10) 13.6 4.4(10) 7.9 2.8(10) 13.2 4.1(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. -35Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ TABLE 10 PERCENT SURVIVAL OF MALE RATS Group: I Material: H-26136 Days on Test 0 7 14 21 28 100 100 100 100 100 in H-26137 100 100 100 100 100 V vn H-26138 H-26139 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. There were no statistically significant decreases in survival. DuPont-13882 -36Company Sanitized. Does not contain TSCA CBI ) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ) DuPont-13882 TABLE 11 MEAN FINAL BODY AND ORGAN WEIGHTS FOR MALE RATS (GRAMS) Group: LIVER KIDNEYS THYROID GLAND FINAL BODY WEIGHT I H-26136 III H-26137 V H-26138 MEAN FINAL BODY AND ABSOLUTE ORGAN WEIGHTS 10.879 0.865(10) 11.051 0.946(10) 10.003 0.614 (10) 3.064 0.279 (10) 3.037 0.275(10) 2.968 0.159(10) 0.021 0.003(10) 0.021 0.004 (10) 0.019 0.003(10) 327.040 24.920(10) 328.760 16.747 (10) 320.090 18.562 (10) VII H-26139 11.106 1.624(10) 3,046 0 . 2 9 5 (-10) 0.019 0.003(10) 331.110 27.091 (10) : Company Sanitized. Does not contain TSCA CBI - 37- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 TABLE 11 (CONTINUED) MEAN FINAL BODY AND ORGAN WEIGHTS FOR MALE RATS (GRAMS) Group: LIVER/ FINAL BODY * 100 KIDNEYS/ , FINAL BODY * 100 THYROID GLAND/ FINAL BODY * 100. I H-26136 III H-26137 V H-26138 MEAN RELATIVE ORGAN WEIGHT (% ORGAN TO BODY) 3.329 0.154(10) 3.363 0.244(10) 3.1270.130(10) 0.937 0.046(10) 0.923 0.057 (10) 0.930 0.067(10) 0.006 0.001(10) 0.006 0.001(10) 0.006 0.001(10) Data summarized as: Mean Standard Deviation (n) There were no statistically significant differences at p < 0.05. VII H-26139 3.341 0.246(10) 0.919 0.032(10) 0.006 0.001(10) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_______ ____________________________________________________________ _________________________ DuPont-13882 TABLE 12 INCIDENCES OF GROSS OBSERVATIONS IN MALE RATS LESION INCIDENCE (Numeric) Males LESIONS | TREATMENT 1 1 1 I 11 1 1 11 ! III 1 V 11 1 1 1 VII 1 1 LIVER NO ABNORMALITY DETECTED 11 1 (10) 1 (10) 1 10 1 10 1 (10) 1 (10) 10 10 KIDNEYS 1 (10) (10) (10) (10) NO ABNORMALITY DETECTED DILATATION, RIGHT, PELVIS. CYST, BILATERAL, FEW, <2MM. THYROID GLAND 1 10 1 11 11 9 1 i UO) 1 (10) 91 1 1i 9 1 (10) 1 10) NO ABNORMALITY DETECTED 1 10 10 10 1 10 SKIN, TREATED 1 (10) (10) (10) 1 (10) NO ABNORMALITY DETECTED 1 10 1 10 10 1 10 SKIN, UNTREATED NO ABNORMALITY DETECTED 1 (10) 1 (10) 1 10 1 10 11 (10) ! (10) 10 1 10 Figures in parentheses is 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 - 39- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 FIGURES - 40Company Sanitized. Does not contain TSCA CBI ) )' H-26137, H-26138, and H-26139: 28-Day Repeated Dose Dermal Toxicity Study in Rats FIGURE 1 MEAN BODY WEIGHTS OF MALE RATS DuPont-13882 -- i h ~ . Group IH -26136 -B -^G roup III H-26137 - A - G ro u p V H-26138 --K --Group VII H-26139 Company Sanitized. Does not contain TSCA CBI -41 - H-26137, H-26138, ndH-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male R ats________ __________ ___________ _________________________DuPont-13882 APPENDICES -42Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats__________________________ _______ DuPont-13882 APPENDIX A Individual Body Weights -43Company Sanitized. Does not contain TSCA CBI l 41-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Body Weight 9 Day 0 Body Weight g Day 7 Body Weight g Day 14 Individual Body Weights Body Weight Body Weight Body Weight g gg Day 21 Day 27 Day 28 Male, I H-26136 101 172.9 102 198.1 103 194.7 104 178.8 105 201.2 106 192.0 107 192.8 108 197.3 109 195.1 110 206.6 217.8 246.4 258.6 221.9 251.3 256.8 241.0 244.4 256.2 253.7 264.0 300.5 301.3 262.2 304.2 313.9 279.9 297.7 308.4 303.6 294.8 335.7 349.7 294.9 334.4 345.2 312.6 328.3 358.3 342.9 300.1 340.3. 364.9 307.1 363.8 376.1 331.2 353.5 367.3 353.2 283.3 323.2 342.4 291.8 343.1 359.6 309.8 333.7 348.5 335.0 ) DuPont-13882 Print Date: 14-Jan-2004 Print Time: 15:11:16 Company Sanitized. Does not contain TSCA O 00 H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____ Study: 14992 1012 Individual Body Weights Body Weight Body Weight Body Weight Body Weight Body Weight Body Weight g Day 0 g Day 7 g Day 14 g Day 21 g' Day 27 g Day 28 Male, III H-26137 301 176.7 302 197.4 303 192.1 304 180.8 305 195.3 306 186.3 307 197.1 308 198.0 309 187.2 310 204.5 229.0 250.3 216.7 234.3 240.9 241.9 256.0 242.4 246.-2 253.9 277.1 298.6 299.2 279.9 289.2 303.1 311.1 283.8 301.5 289.1 ` 303.6 331.4 340.3 317.2 325.7 339.6 345.8 312.1 339.7 321.8 329.7 351.8 371.2 336.6 345.9 370.8 357.2 322.0 357.2 333.9 307.8 331.5 351.6 316.9 324.9 353.3 336.5 306.7 340.4 318.0 ) DuPont-13882 Print Date: 14-Jan-2004 Print Time: 15:11:16 Company Sanitized. Does not contain TSCA CB1 45 [-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Individual Body Weights Body Weight 9 Day 0 Body Weight 9 Day 7 Body Weight g Day 14. Body Weight g Day 21 Body Weight 9. Day 27 Body Weight g Day 28 Male, V H-26138 501 502 503 504, 505 506 507 508 509 510 177.6 196.2 192.7 179.7 203.8 183.4 198.2 197.5 187.5 211.7 220.9 251.8 249.8 236.5 249.3 237.0 242.2 263.4 240.-9 263.8 261.5 298.3 274.3 284.8 292.9 275.4 285.9 317.9 285.8 310.9 293.5 334.3 322.2 332.5 325.1 311.6 319.7 357.4 324.7 340.2 307.7 344.0 349.1 344.7 346.7 318.4 324.7 369.5 333.6 344.6 286.0 327.6 334.0 328.7 321.8 298.0 309.5 350.6 316.4 328.3 DuPont-13882 Print Date: 14-Jan-2004 Print Time: 15:11:16 Company Sanitized. Does not contain TSCA Otn H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 ) Individual Body Weights Male, VII H-26139 701 171.8 702 201.3 703 188.9 704 183.5 705 200.2 706 189.9 707 198.7 708 193.2 709 192.6 710 210.0 226.8 261.1 246.9 228.8 252.5 246.9 240.6 242.9 253:3 257.4 266.9 308.5 309.3 278.5 293.6 305.5 292.4 293.2 306,5 290.1 292.0 363.1 358.0 318.0 322.5 349.8 336.6 328.2 351.4 312.3 302.7 392.7 371.6 327.8 330.4 38,2.6 352.0 339.9 378.6 328.7 287.2 368.8 349.5 313.0 310.2 360.0 329.9 322.6 359.6 310.3 DuPont-13882 Print Date: 14-Jan-2004 Print Time: 15:11:16 Company Sanitized. Does not contain TSCA 001 H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats _____ ___ DuPont-13882 APPENDIX B Individual Food Consumption -48Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats INDIVIDUAL FOOD CONSUMPTION EXPLANATORY NOTES Abbreviation g/artm/day = grams/animal/day DuPont-13882 - 49Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ______ Study: 14992 1012 ) Individual Food Consumption Food Cons. Food Cons. Food Cons. Food Cons g/anm/day g/anm/day g/anm/day g/anra/day Day 7 Day 14 Day 21 Day 27 Male, X H-26136 101 20.6 102 23.1 103 25.4 104 19.7 105 23.8 106 25.5 107 22.1 108 23.6 109 24.3' 110 23.3 23.9 26.6 27.3 23.7 28.1 29.0 23.4 25.2 26.8 26.3 24.0 27.0 29.1 23.8 28.9 28.1 23.9 27.2 28.7 27.9 24.5 27.0 27.9 23.8 30.5 29.8 26.2 29.8 29.0 29.0 ) DuPont-13882 Print Date: 14-Jan-2004 Print Time: 15:11:55 Company Sanitized. Does not contain TSCA CBI -50 ) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats__________ ___________ Study. 14992 1012 ) 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,, III H-26137 301 22.2 302 23.4 303 22.1 304 21.2 305 23.6 306 21.9 307 24.0 308 23.2 309 22.5 310 24.2 26.2 26.2 29.0 25.8 26.8 26.9 28.1 25.4 27.4 26.1 26.7 26.8 28.8 28.1 27.9 26.7 28.3 23.8 28.4 26.7 29.2 27.6 30.0 28.5 28.9 28.8 28.2 25.3 29.0 28.6 ) DuPont-13882 Print Date: 14-Jan-2004 Print Time: 15:11:55 Company Sanitized. Does not contain TSCA CBI -51 - ) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 ) Food Cons. g/anm/day Day 7 Food Cons. g/anm/day Day 14 Food Cons. g/anm/day Day 21 Individual Food Consumption Food Cons, g/anm/day Day 27 Male, V H-26138 501 21.3 502 25.4 503 23.4 504 22.4 505 24.2 506 23.1 507 21.9 508 23.7 509 22.7 510. 24.9 23.6 29.1 24.5 25.9 25.7 25.9 24.6 26.6 25 . 28.3 24.7 28.9 30.3 . 28.9 26.3 28.4 24.7 27.0 26.8 27.3 25.2 28.6 27.1 29.3 28.9 27.2 25.0 27.8 27.5 26.2 )' Print Date: 14-Jan-2004 Print Time: 15:11:55 Company Sanitized. Does not contain TSCA CBI - 52- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_______________________________________________________________________________________________ DuPont-13882 Study: 14992 1012 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 Print Date: 14-Jan-2004 Print Time: 15:11:55 Maie,, VII H-26139 701 24.1 702 25.0 703 22.5 704 21.1 705 28.0 70S 25.4 707 21.9 ' 708 23.1 709 24.7 710 23.9 25.5 28.9 26.7 24.4 28.5 29.0 25.2 25.7 28.3 24.9 26.7 30.4 28.3 26.3 28.8 29.0 26.8 26.6 30.2 24.2 26.1 30.8 28.0 27.3 27.5 29.9 28.0 27.7 '29.7 26.2 Company Sanitized. Does not contain TSCA CBI 53- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 APPENDIX C Individual Clinical Observations and M ortality Records - 54Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 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. -55Company Sanitized. Does not contain TSCA CBI Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Sex Group Animal MI 101 MI 102 MI 103 MI 104 MI 105 MI 106 MI 107 MI 108 MI 109 MI 110 Individual Clincal Observations and Mortality Records Observation Days General observation, No Abnormality Detected 0,14-28 Discharge, Eye bilateral, Red 7 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 General observation, No Abnormality Detected 0-28 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 General observation. No Abnormality Detected 0-28 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 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 - 56- ) DuPont-13882 Print Date: 30-Jan-2004 Print Time: 10:16:36 li-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Individual Clincal Observations and Mortality Records Sex Group Animal .Observation . Days M III 301 General observation, No Abnormality Detected 1 0-28 Sacrificed by design 28 M III 302 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 M III 303 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 M III 304 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 M III 305 General observation, No Abnormality Detected 0-28 Sacrificed by design 28 .M III 306 General observation, No Abnormality Detected 0-7,28 Hair Loss, Shoulder, Left 21 Wound, Superficial, Shoulder* Left 14 Sacrificed by design 28 M III 307 General observation. No Abnormality Detected 0-28 Sacrificed by design 28 M III 308 - General observation, No Abnormality Detected 0-28 Sacrificed by design 28 M III 309 General observation,. No Abnormality Detected 0-28 Sacrificed by design 28 M III 310 General observation, No Abnormality Detected 0,14-28 ) DuPont-13882 Print Date: 30-Jan-2004 Print Time: 10:16:36 Company Sanitized. Does not contain TSCA CBI - 57- -26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 ) Individual Clincal Observations and Mortality Records Sex Group Animal Observation Days M III 310 Wound, Superficial, Shoulder, Right 7 Sacrificed by design 28 DuPont-13882 Print Date: 30-Jan-2004 Print Time: 10:16:36 Company Sanitized. Does not contain TSCA CBI - 58- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 ) Sex Group Animal MV 501 MV '502 MV 503 MV 504 MV 505 MV 506 MV 507 MV 508 MV 509 MV 510 Individual Clincal Observations and Observation General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Wound, Superficial, Dorsal Body Sacrificed by design General observation. No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Wound, Superficial, Shoulder, Left Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design Company Sanitized. Does not contain TSCA C8I - 59- Mortality Records Days 0-28 28 0-21 28 28 0-28 28 0-28 28 0-28 28 0-28 28 0-7,21-28 14 28 0-28 28 0-28 28 0-28 28 DuPont-13882 Print Date: 30-Jan-2004 Print Time: 10:16:36 (H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________ Study: 14992 1012 ) Sex Group Animai Observation Individual Clincal Observations and Mortality Records Days M VII 701 M VII 702 M VII 703 M VII 704 M VII 70S. M VII 706 M VII 707 M VII 708 General observation, Ho Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation. No Abnormality Detected Sacrificed by design General'observation, No Abnormality Detected 1 Sacrificed by design General observation, No Abnormality Detected Wound, Superficial, Shoulder, Bilateral Wound, Superficial, Shoulder, Left Sacrificed by design General observation, No Abnormality Detected Sacrificed by design General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Sacrificed by design 0-28 28 0-28 28 0-28 28 0-28 28 0-28 28 0-7 21-28 14 28 0-28 28 0-7,21-28, 14 14 28 ) DuPont-13882 Print Date: 30-Jan-2004 Print Time: 10:16:36 Company Sanitized. Does not contain TSCA CBI - 60- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study : 14992 1012 Sex Group- Animal M VII 709 M VII 710 Individual Clincal Observations and Mortality Records Observation Days General observation, No Abnormality Detected 0-28 Sacrificed by design 28 General observation, No Abnormality Detected 0-7,21-28 Wound, Superficial, Shoulder, Left 14 Sacrificed by design 28 DuPont-13882 Print Date: 30-Jan-2004 Print Time: 10:16:36 Company Sanitized. Does not contain TSCA CBI - 61- \ H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ DuPont-13882 APPENDIX D Individual Ciinicai Observations - Post-Dosing -62Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ____ __________________ DuPont-13882 INDIVIDUAL CLINICAL OBSERVATIONS- POST-DOSING EXPLANATORY NOTES Clinical observations reported in this appendix were recorded daily after removal o f the material (fabric). Clinical observations recorded during body weight collection are not included. -63 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________________________________________________________________ _____ Study: 14992 1012 Individual Clincal Observations - Post Dosing Sex Group Animal Observation Days M I 101 General observation, No Abnormality Detected 7-13,14-19, 23,26-27 Discharge, Eye bilateral, Red 0-6,20,21-22, 24 Discharge, Nose, Red 20,22,24-2$ M I 102 General observation, No Abnormality Detected 0-6,7-13, 14-20,21-27 M I 103 General observation, No Abnormality Detected 3-5,7-8,11, 14,21,23-26 Discharge, Eye bilateral. Red 0-2,6,9-10,, 12-13,15-20, 22,27 Discharge, Nose, Red 12-13,15-20, 22 M I 104 General observation, No Abnormality Detected 6,7-9,14,21,23 Discharge, Eye bilateral, Red 0-5,10-13, 15-20,22,24-27 . Discharge, Nose, Red 12-13 Swollen Observations, Face 16 M I 105 General observation, No Abnormality Detected 5-6,8,10,21-26 Discharge, Eye bilateral, Red '0-4,7,9,11-13,. 15-20 Discharge, Hose, P-ed 12-13,14-20, 27 M I 106 General observation, No Abnormality Detected 2-4,7-8, 12-13,14-18,21 23-27 DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 Company Sanitized. Does not contain TSCA CBI - 64- )' H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 ) Individual Clincal Observations Sex Group Animal Observation MI 106 Discharge,.Eye bilateral. Red Discharge, Hose, Red M I 107 General observation, No Abnormality Detected Discharge, Eye bilateral. Red MX 108 Discharge, Hose, Red Wound, .Superficial, Shoulder, Left General observation, No Abnormality Detected Discharge, Eye bilateral, Red M I 109 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Hose, Red M I 110 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Hose, Red Company Sanitized. Does not contain TSCA CBI - 65- - Post Dosing Days 0- 1,5-6, 9-11,20 19-20,22 0,4-6', 7-9, 11,14-15,18,21-27 1- 3,10,12-13, 16-17,20 12-13 19 0-1,3-4,6, 8-9,12-13, 14-20,21-27 2,5,7,10-11 0,2-6,7,12-13, 14-17,21-23, 27 1 8-11,18-20, 24-26 3,14,23,27 0-2,4-6, 7-13,15-20, 21-22,24-26 12-13,16-20, 22,25 DuPont-13882 Print Date: 20-Jan-2004 Print Tinle: 12:58:37 ) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 ) Sex Group Animal M III 301 Individual Clincal Observations - Po Observation General observation, No Abnormality Detected M III 302 Discharge, Eye bilateral, Red Discharge, Nose, Red Wound, Superficial, Dorsal Body General observation, No Abnormality Detected M III 303 Discharge, Eye bilateral, Red Swollen Observations, Forelimb, Left General observation, No Abnormality Detected M III 304 M III 305 Discharge, Eye bilateral. Red Discharge, Nose, Red General observation, No Abnormality Detected Discharge, Eye bilateral. Red Discharge, Nose, Red General observation, No Abnormality Detected M III 3Q6 Discharge, Eye bilateral, Red Discharge, Nose, Red Stain Fur/Skin, Perineum, Yellow General observation, No Abnormality Detected Company Sanitized. Does not contain TSCA CBI -66- st Dosing Days 0,2-4,6,7-8, 12-13,14,21-24 1.5.9- 11,15-20 16- 20,25-27 10-11 3,5-6,7,9-13, 14-15,21-27 0-2,4,16-20 8 0-1,3-6, 7-10,12-13,14, 17- 18,21-27 2,11,15-16,19-20 19-20 7.9- 10,12-13, 14-15,17,21-26 0-6,8,11,16,18-20 16,18-20,27 2,4-6,7-13, 14-18,21-25, 27 0-1,3 19-20 26 5-6,9,26-27 DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study : 14992 1012 Sex Group Animal M hi 306 M III 307 M hi 308 M hi 309 M hi 310 Individual Clincal Observations - Post Dosing Observation Days Discharge, Eye. bilateral. Red 0-4,7-8, 10-13,15 Discharge, Nose, Red 12-13,15,18-20, 22 Hair Loss, Shoulder, Left 21-25 Wound, Superficial, Shoulder, Left 12-13,14-20 General observation, No Abnormality Detected 0,2,4,7,10-11, 14,21-27 Discharge, Eye bilateral, Red Discharge, Eye bilateral, Red, , Discharge, Nose, Red 1,3,5-6,15-20 8-9 12-13,15-20 General observation, No Abnormality Detected 3-5,8-10, 12-13,14,17,21, 27 Discharge, Eye bilateral, Red 0-2,6,7,11, 15-16,18-20,22, 24-26 Discharge, Nose, Red 16,18-20,22,25 Discharge, Eye bilateral. Red 0-6,7-13, 14-20,21-2:7 Discharge, Nose, Red 12-13,15-2,0, 22-27 General observation, No Abnormality Detected 5,7-8 Discharge, Eye bilateral, Red 0-4,6,9-13:, 15,21-27 Discharge, Nose, Red 12-13,15,1:7-20 21-27 ) DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 Company Sanitized. Does not contain TSCA CBI - 67- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxieity 28-Day Study in Male Rats______ Study: 14992 1012 Individual Clincal Observations Sex Group Animal Observation M III 310 Wound, Superficial, Dorsal Body Wound, Superficial, Shoulder, Right Company Sanitized. Does not contain TSCA CBI - 68- - Post Dosing Days 14-20 DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________ ' ________ __________ Study: 14992 1012 Sex Group Animal MV 501 Individual Clincal Observations - Po Observation General observation, No Abnormality Detected Discharge, Eye bilateral, Red. Discharge, Nose, Red M V 502 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Wound, Superficial, Dorsal Body M V 503 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Swollen Observations, Forelimb, Right M V 504 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red M V 505 General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red Company Sanitized. Does not contain TSCA CBI - 69- st Dosing Days 6,7-9,11-13, 14,25-27 0- 5,10,15-20, 21-24 16-20,22-2/4 0,5-6,14-15, 21,23-24 1- 4,8-13, 16-20,22 12-13,17-20, 22 7-10,25-27 1- 3, 5-6, 7-9,14-16,21-27 0,10-11,13>18-20 12-13,17-20 4 12,14,21 0-6,7-11,13, 15-20,22-27 13 0-1,5,9-10, 12 2- 4,6,7-8, 11,13,14-20,21-27 13,15,17-20,22-2.4 DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 )rf 26137, H' -26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 ) Sex Group Animal MV 506 MV 507 MV 508 MV 509 ' ^ MV 510 Individual Clincal Observations - Post Dosing Observation. Days General observation, No Abnormality Detected 0,3-6,8,10-13 14-15,21-27 Discharge, Eye bilateral, Red 1-2,7,9,16-20 General observation, No Abnormality Detected 0,2-4,7,21-24 26-27 Discharge, Eye bilateral, Red 1,5-6,11-13, 15-20,25 Discharge, Nose, Red 12-13,16-20 Wound, Superficial, Shoulder, Left 9-13,14-19 Swollen Observations, Forelimb, Left 8 General observation. No Abnormality Detected Discharge, Eye bilateral. Red 3-5,7,9,13,14, 21-23,27 0-2,6,8,10-12, 15-20,24-26' Discharge, Nose, Red 12,17-20,24-25 General observation, No Abnormality Detected 2, 6,7-10,12-13 14-15,17-20, 21-27 Discharge, Eye bilateral, Red 0- 1,11 Discharge, Nose, Red 16 Wound, Superficial, Dorsal Body 3-5 Comments, wound is from staple clip 3-5 General observation, No Abnormality Detected 5,14,18,21-22, 25-27 ) DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 Company Sanitized. Does not contain TSCA CBI - 70- ) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 11i.t4-992 1012 Sex Group Animal MV 510 Individual Clincal Observations Observation Discharge, .Eye bilateral, Red Discharge, Nose, Red & Swollen Observations, Forelimb, Right Company Sanitized. Does not contain TSCA CBI -71 - - Post Dosing Days 0-4,6,7-13, 15-17,19-20, 23-24 8,16-17,24 2 DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 H-26137, H-26138, and H-26139: Repeated-Dose Dennal Toxicity 28-Day Study in Male Rats _______________________ ________ ________________ Study: 14992 1012 Individual Clincal Observations - Post Dosing Sex Group Animal Observation Days M VII 701 General observation, No Abnormality Detected 5,7,14,21,27 Discharge, Eye bilateral, Red 0-4,6, 8-13'y 15-20,22-24 M VII 702 Discharge, Nose, Red General observation. No Abnormality Detected 12-13,17-20, 24-26 5-6,7-9,23, 26-27 Discharge, Eye bilateral, Red 0-4,10-13, 14-20,21-22, 24-25 M VII 703 Discharge, Nose, Red General observation, No Abnormality Detected 12-13,17-20, 22,24-25 4-6,12-13, 14-20,21-27 Discharge, Eye bilateral, Red 0-3,7-11 M VII 704 General observation, No Abnormality Detected 0-6,7-13,21-27 Discharge, Eye bilateral, Red 16-20 Discharge, Nose, Red 17-20 Wound, Superficial, Dorsal Body 14-17 M VII 705 General observation. No Abnormality Detected 0,4,8-13,14, 17-19,21,23,26-27 Discharge, Eye bilateral, Red 1-3,5-6,7, 15-16,20,22,24-25 Discharge, Nose, Red 16 M VII 706 General observation, No Abnormality Detected 7,12 DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 Company Sanitized. Does not contain TSCA CBI - 72- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ Study: 14992 1012 Sex Group Animal M VII 706 Individual Clincal Observations Observation Discharge, Eye bilateral, Red M VII 707 Discharge, Nose, Red Wound, Superficial, Dorsal Body Wound, Superficial, Shoulder, Bilateral Wound, Superficial, Shoulder, Left Comments, wound is from staple clip General observation, No Abnormality Detected Discharge, Eye bilateral, Red Discharge, Nose, Red M VII 708 General observation, No Abnormality Detected Discharge, Eye bilateral, Red M VII 709 Discharge, Nose, Red .General observation, No Abnormality Detected M VII 710 Discharge, Eye bilateral, Red Discharge, Nose, Red General observation. No Abnormality Detected Discharge, Eye bilateral, Red Company Sanitized. Does not contain TSCA CBI -73- - Post Dosing - Days 0-3,9-11,13, 15-20 13,15-20,22-25 0- 6, 8-10 19-20,21-27 14- 18 0-6 5.8.14.21.25- 27 0-4,6,7,9-13, 15- 20,22-24 12-13,15-20, 24 0-2,5-6,7, 10.14.21.25- 27 3-4,8-9, 11- 13,15-20,22-24 12- 13,15-20 0-6,7-10, 12-13,14,21-23*, 25-27 11 15-20,24 0,2-6,7-11, 21-22,25-27 1,23-24 DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ Study: 14992 1012 Sex Group Animal M VII 710 Individual Clincal Observations Observation Discharge, Nose, Red Wound, Superficial, Shoulder, Left Company Sanitized. Does not contain TSCA CBI - 74- - Post Dosing DuPont-13882 Print Date: 20-Jan-2004 Print Time: 12:58:37 H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________ ' __________________ DuPont-13882 APPENDIX E Individual Edema and Erythema Scores - 75Company Sanitized. Does not contain TSCA CBI i^ * \ , H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats INDIVIDUAL EDEMA AND ERYTHEMA SCORES EXPLANATORY NOTES DuPont-13882 DRAIZE3 SCALE FOR SCORING SKIN IRRITATION Evaluation o f Skin Reactions '________ Score Erythema and eschar formation: No-eryfeema.....:.,^.....,.,....,............................................................................................. Very slight erythema (barely perceptible)...................................................................... Well-defined erythema..................................................................................................... Moderate to severe erythema.......................................................................................... Severe erythema (beet redness) to slight eschar formation (injuries in depth)........... 0 1 2 3 4 Edema formation: No edema......................................................................................................................... Very slight edema (barely perceptible)......................................................................... Slight edema (edges o f area well defined by definite raising).................................... Moderate edema (raised approximately 1.0 m m )........................................................ Severe edema (raised more than 1.0 mm extending beyond the area o f exposure)... 0 1 2 3 4 a Draize, J. H., "Dermal Toxicity." Appraisal of the Safety of Chemicals in Foods. Drugs and Cosmetics. The Editorial Committee of the Association o f Food and Drug Officials of the United States, Austin, Texas, 1959, pp. 46-59. -76Company Sanitized. Does not contain TSCA CBI > H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______ Study: 14992 1012 ) Edema Day 0 Edema Day 1 Male, I H-26136 101 0 102 0 103 0 104 0 105 0 106 0 107 0 108 0 109 0 110 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 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 4 Day 5 Day 6 Day 7 Day 8 000 0 0 0 00 0 0 0 0 000 0 0 0 000 0 0 0 000 0 0 0 000 0 0 0 000 0 0 0 000 0 0 0 000 0 0 0 000 0 0 0 Edema Day 9 ) DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Edema Day 10 Edema Day 11 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA O00 ) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats _________________ . ___________ _ Study: 14992 1012 Edema Day 12 Edema Day 13 Edema Day 14 Edema Day 15 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 16 Day 17 Day' 18 Day 19 Day 20 Male, I H-26136 101 0 0 0 0 0 0 0 0 0 102 0 0 0 0 0 0 0 0 0 103 0 0 0 0 0 0 0 o 0 104 0 0 0 0 0 0 0 0 0 105 0 0 0 0 0 0 0 0 0 106 0 0 0 0 0 0 0 0 0 107 0 0 0 0 0 0 0 0 0 108 0 0 0 0 0 0 0 0 0 109 0 0 , 0 0 0 0 0 0 0 110 0 0 0 0 0 0 0 0 0 Edema Day 21 0 0 0 0 0 0 0 0 0 0 DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Edema Day 22 Edema Day 23 00 00 00 00 00 00 00 00 00 00 Company Sanitized. Does not contain TSCA CBI -78- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats _ _______________ ___________________________________ DuPont-13882 Study: 14992 1012 Print Date: 15-Jan-2004 Print Time: 12:06:26 Individual Edema and Erythema Scores Edema Edema Edema Edema Day 24 Day 25 Day 26. Day 27 Male, I H-26136 101 0 102 0 103 0 104 0 105 0 106 0 107 0 108 0 109 0 110 0 00 00 00 00 00 00 00 00 0 ,0 00 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 79- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_________________________________ Study: 14992 1012 Edema Day 0 Edema Day 1 Edema Day 2 Edema Day 3 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 4 Day 5 Day 6 Day 7 Day 8 Edema Day 9 DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Edema Day 10 Edema Day 11 Male, III H-26137 301 0 0 0 0 302 0 0 0 0 303 0 0 O ' 0 304 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 0 305 0 0 0 0 0 0 0 0 0 0 0 0 306 0 0 0 0 0 0 0 0 0 0 0 0 307 0 0 0 0 0 0 0 0 0 0 0 0 308 0 0 0 0 0 0 0 0 0 0 0 0 309 0 0 0 0 0 0 0 0 0 0 0 0 310 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA o CD H-26137, H-26138, andH-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats _____ Study: 14992 1012 . Edema Day 12 Edema Day 13 Edema Day 14 Edema Day 15 Individual Edema and Erythema Scores Edema Edema Edema Edema 1 Edema Day 16 Day 17 Day 18 Day 19 Day 20 ______ ______________ DuPont-13882 Print Date: 15-Jan-2004 Print' Time: 12:06:26 Edema Day 21 Edema Day 22 Edema Day 23 Male, III H-26137 301 0 0 0 0 0 0 0 0 0 0 0 0 302 0 0 0 0 0 0 0 0 0 0 0 0 303 0 0 0 0 0 0 0 0 0 0 0 0 304 0 0 0 0 0 0 0 0 0 0 0 0 305 0 0 0 0 0 0 0 0 0 0 0 0 306 0 0 0 0 0 0 0 0 0 0 0 0 307 0 0 0 0 0 0 0 0 0 0 0 0 308 0 0 0 0 0 0 0 0 0 0 0 0 309 0 0 0 0 0 0 0 0 0 0 0 0 310 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 81- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Edema Day 24 Edema Day 25 Edema Day 26 Edema Day 27 Individual Edema and Erythema Scores Male, III H-26137 301 0 0 0 0 302 0 0 0 0 303 0 0 0 0 304 0 0 0 0 305 0 0 0 0 306 0 0 0 0 307 0 0 0 0 308 0 0 0 0 309 0 0 0 0 310 0 0 0 0 ) DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Company Sanitized. Does not contain TSCA O H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Edema Day 0 Edema Day 1 Edema Day 2 Edema Day 3 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 4 Day 5 Day 6 Day 7 Day 8 Edema Day 9 DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Edema Day 10 Edema Day 11 Male, V H-26138 501 0 0 0 0 0 0 0 0 0 0 0 0 502 0 0 0 0 0 0 0 0 0 0 0 0 503 0 0 00 0 0 0 0 0 0 0 0 504 0 0 0 0 0 0 0 0 0 0 0 0 505 0 0 0 0 0 0 0 0 0 0 0 0 506 0 0 0 0 0 0 0 0 0 0 0 0 507 0 0 0 0 0 0 0 0 0 0 0 , 0 508 0 0 0 0 0 0 0 0 0 0 0 0 509 0 0 0 0 0 0 0 0 0 , 0 0 0 510 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA O CD ) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Edema Day 12 Edema Day 13 Edema Day 14 Edema Day 15 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 16 Day 17 Day 18 Day 19 Day 20 Edema Day 21 DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Edema Day 22 Edema Day 23 Male, V H-26138 501 0 0 0 0 0 0 0 0 0 0 . 0 0 502 0 0 0 0 0 0 0 0 0 0 0 0 503 0 0 0 0 0 0 0 0 0 0 0 0 504 0 0 0 0 0 0 0 0 0 0 0 0 505 0 0 0 0 0 0 0 0 0 0 0 0 506 0 0 0 0 0 0 0 0 0 0 0 0 507 0 0 0 0 0 0 0 0 0 0 0 0 508 0 0 0 0 0 0 0 0 0 0 0 0 509 0 0 0 0 0 0 0 0 0 0 0 0 510 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 84- ri-26137, H-26138, and H-26139: Repeated-Dose Dennal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Edema Day 24 Edema Day 25 Edema Day 26 Edema Day 27 Individual Edema and Erythema Scores Male, V H-26138 501 0 502 0 503 0 504 0 505 0 506 '0 507 0 508 0 509 0 510 0 0 0 0 0 0 0 0 0 0 0 00 00 00 00 0. 0 00 00 00 00 00 ) . DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Company Sanitized. Does not contain TSCA CBI - 85- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Edema Day 0 Edema Day 1 Edema Day 2. Edema Day 3 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 4 Day 5 Day 6 Day 7 Day 8 Edema Day 9 DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Edema Day 10 Edema Day 11 Male, VII H-26139 701 0 0 0 0 0 0 0 0 0 0 0 0 702 0 0 0 0 0 0 0 0 0 0 0 0 703 0 0 0 0 0 0 0 0 0 0 0 0 704 0 0 0 0 0 0 0 0 0 0 0 0 705 0 0 0 0 0 0 0 0 0 0 0 0 706 0 0 0 0 0 0 0 0 0 0 0 0 707 0 0 0 0 0 0 0 0 0 0 0 0 708 0 0 0 0 0 0 0 0 0 0 0 0 709 0 0 0 0 0 0 0 0 0 0 0 0 710 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 86- ) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Edema Day 12 Edema Day 13 Edema Day 14 Edema Day 15 Individual Edema and Erythema Scores Edema Edema Edema Edema Edema Day 16 Day 17 Day 18 Day 19 Day 20 Edema Day 21 DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Edema Day 22 Edema Day 23 Male, VII H-26139 701 0 0 C 0 0 0 0 0 0 0 0 0 702 0 0 C 0 0 0 0 0 0 '0 0 0 703 0 0 0 0 0 0 0 0 0 0 0 0 704 0 0 a 0 0 0 0 0 0 0 0 0 705 0 0 0 0 0 0 0 0 0 0 0 0 706 0 0 0 0 0 0 0 0 0 0 0 . 0 707 0 0 0 0 0 0 0 0 0 0 0 0 708 0 0 0 0 0 0 0 0 0 0 0 0 709 0 0 0 0 0 0 0 0 0 0 0 0 710 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 87- it-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Edema Day 24 Edema Day 25 Edema Day 26 Edema Day 27 Male, VII H-26139 701 0 0 0 0 702 0 0 0 0 703 0 0 0 0 704 . 0 0 0 0 705 0 0 0 0 706 0 0 0 0 707 0 0 0 0 708 0 0 0 0 709 0 0 0 0 710 0 0 0 0 Individual Edema and Erythema Scores DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:06:26 Company Sanitized: Does not contain TSCA O DO ii-2 6 137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Study: 14992 1012 Print Date: 15-Jan-2004 Print Time:,12:07:19 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, I H-26136 101 0 0 0 0 0 0 0 0 0 0 0 0 102 0 0 0 0 0 0 0 0 0 0 0 0 103 0 0 0 0 0 0 0 0 0 0 0 0 104 0 0 0 0 0 0 0 0 0. 0 0 0 105 0 0 0 0 0 0 0 0 000 0 106 0 0 0 0 0 0 0 0 0 0 0 0 107 0 0 0 0 0 0 0 0 0 0 0 0 108 0 0 0 0 0 0 0 0 0 0 0 0 109 0 0 0 0 0 0 0 0 0 0 0 0 110 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI -89- .1-26137, II-26J38, and 11-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats _______ ________________________________________________________D uPont-13882 Study: 14992 1012 Print Date: 15-Jan-2004 Print Time: 12:07:19 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, I H-26136 101 0 0 0 0 0 0 0 0 0 0 0 0 102 0 0 0 0 0 0 0 0 0 0 0 . 0 103 0 0 0 0 0 0 0 0 0 0 0 0 104 0 0 0 0 0 0 0 0 0 0 0 0 105 0 0 0 0 0 0 0 0 0 ' 0 0 0 106 0 0 0 0 0 0 0 0 0 0 0 0 107 0 0 0 0 0 0 0 0 0 0 0 0 108 0 0 0 0 0 0 0 0 0 0 0 0 109 0 0 0 0 0 0 0 0 0 0 0 0 110 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 90- ))) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study m Male R a ts _____________________________________________ DuPont-13882 Study: 14992 1012 Print Date: lS-Jan-2004 Print Time: 12:07:19 Individual Edema and Erythema Scores Erythema Erythema Erythema Erythema Day 2.4 Day 25 Day 26 Day 27 Male, I H-26136 101 0 0 0 0 102 0 0 0 0 103 0 0 0 0 104 0 0 0 0 105 0 0 0 0 106 0 0 0 0 107 0 0 0 0 108 0 0 0 0 109 0 0 0 0 110 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 91- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:07:19 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, III H-26137 301 0 0 0 0 0 0 0 0 0 0 0 0 302 0 0 a 00 00 0 0 0 .0 0 303 0 304 0 0 0 c0 00 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 305 0 0 0 0 0 0 0 0 0 0 0 0 306 0 0 0 0 0 0 0 0 0 0 0 0 307 0 0 0 0 0 0 0 0 0 0 0 0 308 0 0 0 0 0 0 0 0 0 0 0 0 309 0 0 0 0 0 0 0 0 0 0 0 0 310 0 0 ' 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA O CD ) H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 )" DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:07:19 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, III H-26137 301 0 0 0 0 0 0 0 0 0 0 0 0 302 0 0 0 0 0 0 0 0 0 0 0 ' 0 303 0 0 0 0 0 0 0 0 0 0 0 0 304 0 0 0 0 0 0 0 0 0 0 0 0 305 0 0 0 0 0 0 0 0 0 0 0 0 306 0 0 0 0 0 0 0 0 0 0 0 0 307 0 0 0 0 0 0 0 0 0 0 0 0 308 0 0 0 0 0 0 0 0 0 0 0 0 309 0 0 0 0 0 0 0 0 0 0 0 0 310 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 93- rl-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Erythema Erythema Erythema Erythema Day 24 Day 25 Day 26 Day 27 Individual Edema and Erythema Scores Male, III H-26137 301 0 0 0 0 302 0 0 0 0 303 0 0 0 0 304 0 0 0 0 305 0 0 0 0 306 0 0 0 0 307 0 0 0 0 308 0 0 0 0 309 0 0 0 0 310 0 0 0 0 ) DuPont-13882 Print Date: 15-Jan~2004 Print Time: 12:07:19 Company Sanitized. Does not contain TSCA CBI - 94- ) H-26137, H-26138, and H-26139: Repeated-Dcse Dermal Toxicity 28-Day Study in Male Rats ________ Study: 14992 1012 ) ) DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:07:19 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, V H-26138 501 0 502 0 503 0 504 0 505 0 506 0 507 0 508 0 509 0 510 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 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 - 95- fi-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats __________________________ DuPont-13882 Study: 14992 1012 Print Date: 15-Jan-2Q04 Print Time: 12:07:19 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, V H-26138 501 0 0 0 0 0 C 0 0 0 0 0 0 502 0 0 0 0 0 C 0 0 0 0 0 0 503 0 0 0 0 0 C 0 0 0 0 0 0 504 0 0 0 0 0 C 0 0 0 0 0 0 505' 0 0 0 0 0 C 0 0 0 0 0 0 506 0 0 0 0 0 C 0 0 0 ' 0 0 0 507 0 0 0 0 0 C 0 0 0 0 0 0 508 0 0 0 0 0 C 0 0 0 0 0 0 509 0 0 0 0 0 c 0 000 0 0 510 0 0 0 0 0 c 0 0 00 0 0 Company Sanitized. Does not contain TSCA CBi - 96- ri-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 ' Erythema Erythema Erythema Erythema Day 24 Day 25 Day 26 Day 27 Individual Edema and Erythema Scores Male, V H-26138 501 0 502 0 503 0 504 0 505 0 506 0 507 0 508 0 509 0 510 0 0 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 DuPont-13882 Print Date: 1_5-Jan-2004 Print Time: 12:07:19 Company Sanitized. Does not contain TSCA CBI -97- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Study: 14992 1012 Print Date: 15-Jan-2004 Print Time: 12:07:19 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,, VII H-26139 701 0 0 0 0 0 0 0 0 0 0 0 0 702 0 0 0 0 0 0 0 0 0 0 0 0 703 0 0 0 0 0 0 0 0 0 0 0 0 704 0 0 0 0 0 0 0 0 0 0 0 0 705 0 0 0 0 0 0 0 0 0 0 0 0 706 0 0 0 0 0 0 0 0 0 0 0 0 707 0 0 0 0 0 0 0 0 0 0 0 0 708 0 0 0 0 0 0 0 0 0 0 0 0 709 0 0 , 0 0 0 0 0 0 0 0 0 0 710 ' 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 98- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Study: 14992 1012 Print Date: 15-Jan-2004 Print Time: 12:07:19 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 D a y 118 Day 19 Day 20 Day 21 Day 22 Day 23 Male, VII H-26139 701 0 0 0 0 0 0 0 0 0 0 0 0 702 0 0 0 0 0 0 0 0 0 0 0 0 703 0 0 0 0 0 0 0 0 0 0 0 0 704 0 0 0 0 0 0 0 0 0 0 0 0 '705 0 0 0 0 0 0 0 0 0 0 0 0 706 0 0 0 0 0 0 0 0 0 0 0 0 707 0 0 0 0. 0 0 0 0 0 0 0 0 708 0 0 0 0 0 0 0 0 0 0 0 0 709 0 0 . 0 0 0 0 0 0 0 0 0 0 710 0 0 0 0 0 0 0 0 0 0 0 0 Company Sanitized. Does not contain TSCA CBI - 99- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Study: 14992 1012 Erythema Erythema Erythema Erythema Day 24 Day 25 Day 2.6 Day 27 Male, VII H-26139 701 0 702 0 703 0 704 0 0 0 0 0 0 0 0 0 0 0 0 0 705 0 0 0 0 706 0 0 0 0 707 0 708 0 709 0 710 0 0 0 0 0 0 0 0 0 0 0 0 0 Individual Edema and Erythema Scores ____________ DuPont-13882 Print Date: 15-Jan-2004 Print Time: 12:07:19 Company Sanitized. Does not contain TSCA mo H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 APPENDIX F Analytical Report - 101 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 STUDY TITLE H-26137, H26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Carol Finlay ANALYTICAL PHASE COMPLETED ON March 10,2004 PERFORM ING LABORATORY Exygen Research 3058 Research Drive State-College, P 16801 Phone: 814-272-1039 STUDY SPONSOR E. L du Pont de Nemours and Company (DuPont) P.O .B ox 50 Newark, DE 19714-0050 USA PROTECT Exygen Study Nu Sponsor Study No.: DuPont-13882 - 102Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study N GOOD LABORATORYPRACTICE COMPLIANCE STATEMENT Exygen Study Number flB pM fentitL ed "H-26137, H26138, and H-26139: RepeatedDose Dermal Toxicity 28-Day Study in Male Rats," was conducted for DuPont in compliance with US EPA TSCA (40 CFR part 792} Good Laboratory Practice Standards, which are consistent with OECD Principles of Good Laboratory Practice (as revised in 1997), published in ENV/MS/CHEM(98)17, except for the item documented below. This item does not impact the validity of the study. 1. There was no in 4 a i^ u d it for this study ^ J B P fW io w e v e r, an in-lab audit was conducted for study d M W h e same procedures performed by the same personnel were observed for the same client within the last six months. There were no technical problems observed. D ie in-lab audit fo4M M B W ill serve as a process-based inspection for this study. Principal Investigator Exygen Research Study Director DuPont S. M ark jsmmeay Sponsor Representative DuPont Exygen Research Date D ate Page 2 of 63 -103Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________________ DuPont-13882 Exygen Study No QUALITYASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number analytical phase of the study entitled, "H-26137, H26138, and H-26139: RepeatetLDose Eternai 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 and to management. Phase Date Inspected Date Reported Date Reported to to Exygen P rin c ip a l Management Investigator Date Reported to Study Director and Sponsor Management *1. Standard Preparation, Fortification, and Extraction 01/13/04 01/15/04 01/19/04 02/23/04 2. Raw Data & Draft 01/23,26/04 Analytical Report Review 01/28/04 01/29/04 02/23/04 Final Analytical Report Review 02/23/04 02/23/04 02/23/04 02/23/04 A O 'fr& zts*' M iwaNabetani Quality Assurance Auditor ? / t o /o ,. Date * Exygen QA observed an in-lab procedure for study the above date. This procedure was also used in the current study and was performed by the same personnel for the same client within a six-month period o f the original lab inspection. Since no technical problems were observed during the origina] in-lab inspection, a process-based inspection for this procedure was issued for this study Exygen Research Page 3 of 63 - 104Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study N o.: CERTIFICATION OFAUTHENTICITY This report. for Exygen Study Number the raw data far the study. is a true and complete representation of Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814)272-1039 Principal Investigator, Exygen: sly R. Decker Seientist/Principal Investigator Exygen Research ..j y g f . Date Exygen Research Facility Management M. Flaherty Vice President Exygen Research Study Director, DuPont: Carol Frminlay DuPont <- { l L , m / U r ' 4H Date SponsorRepresentative, DuPont S. M ark Kennedy DuPont Exygen Research ? >o>-j Date Page 4 of 63 -105Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study No.:' EXYGEN STUDY IDENTIFICATION H-26137, H26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats SPONSOR STUDY NUMBER: DuPont-13882 EXYGEN STUDY NUMBER: TYPE OF ANALYTICAL PHASE: Residue SAMPLE MATRIX- Rat Plasma TEST SUBSTANCE: SPONSOR: E. I. du Pont de Nemours and Company P.O. Box 50 Newark, DE 19714-0050 Phone: (302)366-5255 STUDY DIRECTOR: Carol Finlay DuPont PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 11/18/03 Analytical Start Date: 01/12/04 Analytical Termination Date: 01/13/04 Analytical Report Completion Date: 03/10/04 Exygen Research Page 5 of 63 - 106Comparty Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study iri Male Rats________________________________ DuPont-13882 Bxygea Study No. PRO.TECT PERSONNEL The Study Director for this project at DuPont was Carol Finlay. The following personnel from Exygen Research were associated with various phases of the study: Name Paul Connolly Emily D eck Karen Risha Mark Neeley Shawn Robb T itle Technical Lead-LC/MS Scientist Scientist Scientist * Sample Custodian B xygea Research Page 6 of 63 - 107Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study No. TABLE OF CONTENTS TITLE PA G E.:.......................................................................................... GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT. QUALITY ASSURANCE STATEMENT.................... ......................... CERTIFICATION OF AUTHENTICITY................................................ EXYGEN STUDY IDENTIFICATION.................................................. PROJECT PERSONNEL........................................... ............................. TABLE OF CONTENTS.......... ............................ .................................. LISTOFTABLES................ :-- ................................ ........................... LI&TOEHGDRES.................................................... . LIST OF APPENDICES........................................... . 1.0 SUM M ARY.................................................. ..... 2.0 OBJECTIVE................................- ............-- 3.0 INTRODUCTION............................................... 4.0 TEST SYSTEM.......... ....................................... 5.0 REFERENCE MATERIAL................................ . 6.0 DESCRIPTION OF ANALYTICAL METHOD 6.1 Extraction Procedure.................. .................................................................... 6.2 Preparation of Standards and Fortification Solutions..................................... 6.3 Chromatography.................................................. ............................................. 6.4 Instrument Sensitivity....................................................................................... 6.5 Description of Instrument and Operating Conditions..................................... 6.6 Quantitation and Example Calculation............................................................ 7.0 EXPERIMENTALDESIGN............................................................................... 8.0 RESULTS............................................................................................................ 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE QUALITY OR INTEGRITY OF THE DATA...............................................- ........................... 10.0 CONCLUSIONS........................................ 11.0 RETENTION OF DATA AND SAMPLES LISTOFTABLES .1 .2 ,,3 ..4 ,,5 .6 ,,7 .7 .8 .8 .9 .9 ,,9 .9 10 11 11 11 12 12 12 13 15 15 15 15 15 Table L Summary of M H Bfppb) in Rat Plasma Control Samples............................. 17 Table II. Summary of Recoveries in R at Plasma Samples...................... 18 Table TEL Summary of Residue Found for in Rat Plasma Samples.......... 19 Exygen Research rage 7 of 63 - 108Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________ DuPont-33882 Exygen Study No,: LIST OFFIGURES Figure I. Typical Calibration Curve for 21 Figure 2. Chromatogram Representinga Calibration Standardfi with ~ 5,ng/mL of I3C' it XOppb 22 Figure 3. Chromatogram Representing Control Rat Plasma fo| ~ 5 ng/mL of (with 23 Figure 4. Chromatogram Representing Control Rat Plasma Fortified at 10 ppb with Figure 5. Chromatogram Re - 5 ng/mL of 24 1301, .25 LIST OFAPPENDICES Appendix A Study Protocol DuPont-13882 (Exygen Study N o.SH B V and Amendment........... ...................................... ................. -................. 26 Appendix B AnalyticalMethod ExM-008-276 Revision 2,"Method of Analysis for the Determination of i Serum by LC/MS/MS" ..... .............................. ................................................43 Exygen Research Page 8 Of 63 - 109Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dennal Toxicity 28-Day' Study in Male Rats _____________________________ DuPont-13882 Exygen Study No. 1,0 SUMMARY Exygen Research (Exygen) analyzed samples of rat plasma for residues of lm according to protocol DuPont-13882 (Appendix A) using die analytical method, sion 2 (Appendix B). The limit of quantitation for this method is 10 ppb, which was established at Exygen in the method validation study 008-277. Residues o fl the rat plasma samples ranged from < 10.0 ppb to 230 ppb. The average recovery standard deviation foM H M n fortified rat plasma samples was 89% 11.1%. 2.0 OBJECTIVE The objective of this analytical phase was to analyze rat plasma samples received at Exygen using the entitled "Method of Analysis for the Determination of fQ H H H B H IH H H b B S M M I Serum by LC/MS/MS". 3.0 INTRODUCTION This report details the results of the analysis of rat plasma samples for PPOA using the analytical method! The study was initiated on November 18, 2003, when the study director signed protocol number DuPont-13882. The analytical start date was January 12,2004, and the analytical termination date was January 13,2004. 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 placed in frozen storage (< -20C). Sample login and chain of custody information can be found in the raw data package associated with this study. Storage records will be kept at Exygen Research and a true copy of the storage records will be furnished upon request. Exygen Research Page 9 o f 63 - 110Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study No.ri 5.0 REFERENCE MATERIAL The analytical stan d ax d C H H H H H H H H H H H H P ) was roce>ve<l st Exygen on Match 05, 2003 from Critical Path Services. The control article (internal standard) nperfluorooctanoic acid (1,2 di-13C) 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 Exvgen Lot No. InventoryNo._____ A Purity (% \ Expiration Date 97 96.35 03/05/04 01/31/05 The structure of below. and the internal standard are given tienne Molecular weight Chemical Name Molecular weight Exygen Research Page 10 of 63 - Ill Company Sanitized. Does not contain TSCA CBI H-26137, E-26I38, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study No. 6.0 DESCRIPTION OFANALYTICALMETHOD 6.1 Extraction Procedure 1. Measure 0.1 mL of sample into 1 inL eppendorf centrifuge tubes (fortify with analyte as needed, close lid, and vortex ~ 10 seconds). 2. Add enough acetonitrile containing internal standard at 0.005 pgfrnL (accounting for fortification volume) to make extraction volume 500 pL and vortex for - 10 seconds. 3. Centrifuge for ~ 10minutes at - 14,000 rpm. 4. Analyze Samples using electrosprayLC/MS/MS. 6.2 Preparation of Standards and Fortification Solutions The stock standard solution oftH H R w as prepared on September 17, 2003, at a concentration of 100 pg/tnL by dissolving approximately 10 mg of the standard (corrected for purity) in methanol. The stock standard solution of 13C H H ^w as 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 ofC m 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 l3C M m w a s prepared by taking 1.0 mL of the stock and bringing the volume up to 100 mL with acetonitrile. A set of standards containing MHIwirh internal standards was prepared by dilution of the 10.0 pg/mLfM H kiution and the 1.0 pg/raL 13c M B so lu tio n in the following manner. These solutions were used to fortify the samples and the extracted calibration standards. Initial Cone. (pg/mL)1 10.0 10.0 1 10.0 ----- Volume (mL) Diluted to (mL) 10.0 icxF L.O 1002 0.1 1002 Final Cone. (up/mL)1 1.0 0.1 0.01 20.5 mL of the 1.0gg/mL !3C-CBBColution was added prior to making final volume to give a concentration of 0.005 pg/reL of 13C | ^ | in 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 oh refrigerator stability data obtained during the method validation study (Exygen study: C M V )- The fortification of the standards before extraction is done according to the following table: Exygen Research Page 11 of 63 - 112Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study No. Fortification Volume of Volume (pL) Control Fortification Sample (mL) Solution (pg/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 O.S mL of the 1.0 pg/mL solution and bringing the volume up to 100 mL with acetonitrile. The dilution solution was prepared by taking O.S mL of the 1.0 pg/inL 13C fH P . solution and bringing the volume up to 100 mL with 50:50 methanol:water. The stock standard solution and all fortification/calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation can be found in the raw data associated with this report. &3 Chromatography Quantification of was accomplished by analysis using electrospray LC-MS/MS. The retention time o ^ H lw a s ~ 2.0 min., with no significant interfering peaks (< 20% of the LOQ standard) in the control samples corresponding to the analyte retention times. 6A Instrument Sensitivity The smallest amount of flH H bjected during the chromatographic run was equivalent to 10 ppb. 6S Description o f Instrument and Operating Conditions A PE Sciex LC-MS/MS coupled to a Hewlett Packard HFLC system was used. Data acquisition and processing were performed using Analyst .2 software. Detailed operating conditions are listed below: Mass Spec: Interface: Computer.Software: HPLC: PE SCIEX AH 4000 Biomolecular Mass Analyzer SCIEX Turbo Ion Spray Liquid Introduction Interface Turi Ion Spray temperature = 350 C with N2 at -7 L/min Dell Optiplex GXl 10 EESciex Analyst: Version 1.2 Hewlett Packard (HP) Series 1100 Exygen Research Page 12 of 63 - 113Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats __________ DuPont-13882 Exygen Study No.: 008-405 HP QuatPump HP VacuumDegasser HP Autoinjector HP Column Compartment Filter Cartridge(s): 2 Keystone Hypercarbs in tandem, exiting pump HPLC Column: Genesis Cj (Jones Chromatography), 2.1 mm x 50 mm, 4p Column Temp.: 30 C Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B):Methanol ' Time 0 3.0 3.5 3.7 7.0 7.5 9.0 9.5 12.0 Injection Voi.: 5 pL Ions monitored: %-A 40 40 0 0 0 40 40 40 40 How-ln/saiit) 60 0.3 60 0.3 100 0.3 100 0.5 100 0.5 60 0.5 60 0.5 60 03 60 0.3 Analvte Mode Transition Monitored Negative 413 - 369 Negative 415 370 6.6 Quantitation and Example Calculation Five microliters of sample or calibration standard were injected into 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 standard peak area versus die ratio of the concentration of analyte/concentration of internal standardusing Analyst 1.2 (or equivalent) software system. The residue concentration f o r ^ B 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) generated by die Analyst software program. Equation 1: Analyte found (ppb) = ((AnalytePeakarea/IS peakareal-intercentUx IS cone. (ng/mL) x DF slope ExygenResearch Page 13 of 63 - 114Company Sanitized. Does not contain TSCA CSI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study N o.:| 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 fppbl - ave. Analvte found in control fopbl x 100 Amount Analyte added (ppb) Fewplasipa samples fortified with known amounts of analyte prior to extraction, Equation 3 was used to calculate the percent recovery. Equation 3: , Recovery (%) = Analvte found (ppb) - avp. Analvte found in sample fppht x 100 Amount Analyte added(ppb) An example of a calculation using an actual sample follows: Rat plasma sample Exygen fortified at 10 ppb witi Where: analyte peak area = 170017 IS peak area = 413671 IS cone. (ngrinL) 4.167 ngftnL intercept = 0.0146455 slope = 0.1712 dilution factor (DF) = 1 - ppb added (fort level) = 10 amt found in control = ND From equation 1: Analyte found (ppb) = ra70017/4136711 - 0.01464551 x 4.167 x 1 0.1712 = 9.65 ppb From equation. 2: % Recovery = (95nob - 01 x 100 10 ppb = 97% Note: This example calculation was done using rounded numbers, and therefore may be slightly Efferentfrom the values shown in the RAW DATA. Exygen Research Page 14 of 63 - 115Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Exygen Study No. DuPont-13882 7.0 EXPERIMENTALDESIGN Each analytical set was extracted according to m e t h o d ^ ^ ^ ^ g g ^ p H H i and consisted of a reagent blank, one matrix control, two laboratory' matrix controls fortified at known concentrations, one field sample fortified at a known concentration, 20 plasma samples, and one field sample extractedin duplicate. 8.0 RESULTS The ^ m fo u n d in the control rat plasma samples are listed in Table I. Individual recoveries f o r ^ 0 i in the laboratory fortified rat plasma samples are detailed in Table IL The average percent recoveries standard deviation fo rflH B in the rat plasma samples was 89% 11.1%. Individual results for the ffH B in the rat plasma samples ate given in Table IH. Residues o f ^ H tin the rat plasma samples ranged from < 10.0 ppb to 230 ppb. A typical calibration curve Figures 1-5. and representative chromatograms are given in 9.0 CIRCUMSTANCES THATMAYHAVEAFFECTED THE QUALITY ORINTEGRITY OF THE DATA There are no known circumstances that have affected the quality or integrity of the data. 10.0 CONCLUSIONS The rat plasma samples were successfully analyzed according to method\ 11.0 RETENTION OFDATAAND SAMPLES When tire final report is complete, all original paper data generated by Exygen Research will be shipped to the sponsor. Hus does not include facility-specific raw data such as instrument logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research's archives for the period of time specified in 40 CFR Part 792. Retained samples ofreference substances are archived by the sponsor. Exygen Research Page 15 of 63 -116Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygeit Study No TABLES Exygen Research Page 16 of 63 - 117Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________________________________________________________ DuPont-13882 Table I. Summary ol Bxygen Study Na Ftppb) in Rat Plasma Control Samples Sponsor ID 670127 Control A 01/12/04 670127 Control A2 01/12/04 ND = Not Detected (less than 10.0 ppb) ND ND Exygen Research Page 17 of 63 -118Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study No Table II. Summary ofRecoveries Samples ) in Rat Plasma i*o r i A) 01/12/04 01/12/04 t o 01/12/04 01/12/04 P)01/12/D 4 t o 01/12/04 (PPb) 0.6$ 1030 697 8.61 8SS 1030 j) Amount Fortified <PPb> 10.0 1000 1000 10.0 1000 1000 AVERAGE: STD DEV: % BSD: R e co v e ry <%> 96 103 70 86 90 91 80 11.1 12.9 LCS = Laboratory Control Spike Exygen Research Page 18 of 63 -119Company Sanitized. Does not contain TSCA CBI H-26137, H-26138. and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study No Table m . Summary of Residue Found for Samples Sponsor in 101 101 Dup 102 103 104 1 105 106 107 103 108 110 301 302 303 304 305 306 307 308 308 310 501 501 Dup 502 503 504 505 506 507 50B 509 510 701 702 703 704 70S 706 707 708 709 710 in Rat Plasma PFOA <PP&> <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 < 10.0 <10.0 <10.0 25.2 36.7 36.0 39.4 34.1 34.6 40.9 37.3 29.2 43.6 114 116 79.9 72.8 103 75.2 102 158 70.2 112 85.3 196 73.9 98.6 229 144 149 230 170 115 187 Exygen Research Page 19 of 63 - 120Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study No.l FIGURES Exygen Research Page 20 of 63 - 121 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________________________ DuPont-13882 Figure 1. Typical Calibration Curve for| Exygen Study N o| U M M m "Linear* Regraraton H / * - weighting): y 0.1712 x +0.014645S (r = 0^998873) Exygen Research Page 21 of 63 - 122Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study NoJ Exygen Research Page 22 of 63 - 123 Company Sanitized. Does not contain TSCA C8I H-26137, H-26138, and Hr-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats _______________________ DuPont-13882 Exygen Research Page 23 of 63 - 124 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Research Page 24 of 63 - 125 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Figure 5. Chromatogram Represent w ith ~ S ng/m L o f I DuPont-13882 Exygen StudyNo.; Exygen Research Page25 of 63 -126Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male R a t s ___________ _______________ DuPont-13882 Exygen Study No.:l APPENDIX A Study Protocol DuPont-13882 (Exygen Study No. and Amendment and Deviation Exygen Research Page 26 of 63 -127Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study No.J DiiPogt-13882 H-26137, H-26138, tad H-26139: Repeated-Dose Dennal Toxicity i 28-Day Studyin Male Rats WorkRequestN u m be^^H H K Service C o d e^ H H jj^ Protocol Haskell Animal Welfare CommitteeNumber: 3 Exygen Research l. Page 27 of 63 -128Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study No. H-26137, H-26I3E, and H-26139: RepeatecLDott! Dermal Toxicity 28-Pay Study ia Male Rate ____________ DuPont-13882 TABLE OF CONTENTS INTRODUCTION.. OBJECTIVESPONSOR ANDTEST FACILITY.. REGULATORY COMPLIANCE,, STUDY DESIGN- MATERIALS ANDMETHODSA. .Materials.. B. Test Species_____ C. Annul Husbandry.. D. Pretest Period E . Assignments Groups F. Administration o fTest Substance, G. BodyWeights. H. Food ConsumptionandFood Efficiency L Clinical Observations endMortality J. PeifluorooctanoicAcid Level Evaluation. K. Clinical Pathology Evaluation 1. Clinical Chemistry 2. Urinalysis-- ......... L. AnatomicalPathology. 1. Pretesi. 2. DosingPhase. STATISTICAL ANALYSES SAFETY AND HOUSEKEEPING RECORDS AND SAMPLE STORAGE REFERENCES PROTOCOLAPPENDIX, SIGNATURE Page __O ...3 _3 ...J ...4 -.4 ,,4 ..4 ..5 -6 ,,6 -6 ...7 -.7 -.7 ..7 ,,8 ..10 ..10 ..II -12 -13 -1 4 ! I Exygen Research i. Page 28 of 63 -129Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, andH-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study No.: K-26D7, H-2613S, and H-2S139: RepcttK-Do*cD enral Tcxidiy Xg-DayStadymM iteRaO ________________ DuPopM3882 l INTRODUCTION The materials to be tested. H-26137, H-26138, andH-26139,,are fabrics containingvarying i amountsofrneUadditiYt ',Ihe results o fthiB studym il be compared to the results of a | previously conducted studywiththe neat melt additive. ' i,, | . OBJECTIVE . | The objective ofthis jfudy is to evaluatethe potential toxicity ofH-26137, H-26138, and H-26139 when administered dennally to malerats for 28 days. The dennatroute of : sdmiiustiatioiiwasseiectedbecauseitisapotentialrouteofliumsncxposure. SPONSORAND TEST FACILITY This study is sponsored by E.L durout deNemours andCompany, Wilmington, Delaware. The sponsor's approval was effective the datethe sponsorauthorized du;wart on die Work Authorization Forai The studywill be conducted atHaskell laboratory Ibr Health andEnvironmental Sciences, E.I. duPont de Nemours and Company,Newark, Delaware. REGULATORY COMPLIANCE HisstudywllI becondnctedin compliancewitirthS. PA'TSCA(40'CER part 792) Good LaboratoryPractice Standards, which are consistentwiththe OECD Principles ofGood 1 LaboratoryPractice (as-revised in 1997)published in ENWMC/CHEM(98)!7.S-'1 The study designis based onUB. EnvironmentalProtection Agency (EPA) Office ofPrevention, ! Pesticides, sad Toxic Substances (OPFTS),*** Organization for Economic Cooperation and Development (OHCD)j^ test guidelines. Areas ofnoncompliance will be documented in the i final report .. - Exygen Research I1 Page 29 of 63 -130Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen StudyNo.i . H-26137,H-2613B, and H-26139: Sepeated-Doje Dtrma] Toxicity 28-PayStudyinMMtRte_________________ _____________________________ DuPonf-13882 STUDY DESIGN The study design is as follows: Group Number/Gtouc HaskettLaboratory Number ,i 10 H-26136 (Control) HI 10 H-26137 V 10 H-2613S VD 10 H-26139 Study Parametern Frequency Body Wright Test day 0 andweeklythereafter Food Consnmpticai Testday 0 andweeklythereafter Clinical Observations Daily Observations for dermal effects Dally t Morulity/Moribundity Checks Twice dally Clinical Pathology Week 4 PFOA blood analysis WeeM Necropsy Week 4 MATERIALS ANDMETHODS A. Materials The raateriais'(&rics} were supplied by the sponsor andassigned foe uniqueHaskell Laboratory Numbers H-26136 (forfoe control) andH-26137,H-26138, andK-26139 (for thetestxnsteiiais}. \ B. Test Species Msle Cri:CD(SD)lGS BR ratewill be obtained from Charles RiverLaboratories, Inc. The location of fixesupplier(city/aate) will be documentedin tbe studyteootds andfinal report. The Cri:CD(SD)IGS BR rathas beenselected on the bases ofextensive experience with fins strain I at HaskellLaboratoryand its suitabilitywith respectto hardiness, longevity, sensitivity, and low I incidence ofspontaneous diseases. C. AnimalHusbandly ' 1 All rats will be boused in stairilesssteel, wire-mesh cages suspendedabove cageboards. Animal rooms will be maintained at a temperatureo f 22 3C and a relative humidity of SO4 20%. ! li Exygen Research t. Page 30 of 63 -131 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________ DuPont-13882 Exygen Study No.: H -26137,H -a6!38,aH M fl39: Repted-Do*eEternal Toxicity 2R-DayStudy m Mate Rat DuPoaM3ES2 Animal rooms will be artificially illuminated (fluorescent light) on an spproximate.l2-hour iigfat/daik cycle. All ratswill beprovided tap watered fiMfum, All rats will bp fed PM Nutrition International, LLC Certified RodentLabDiet* 5002 adltbitum , As specified in file Haskell Laboratoryanimal health andenvironmentalmonitoringprogram, the following procedures areperformedperiodically to ensure that contaminant levels are below thosethat would be expectedto im p^Jbc scientific integrity ofthe study; Water samples are analyzed fortotal bacterial counts, and the presenceofcoliforms, lead, andother contaminants. Feed samples are analyzed for totalbacterial, spore, and fungal counts. Samples from fleshly washed cagesandean racks are analyzed to ensureadequate sanitation by the cagewashets. Certified animal feed is used, guaranteedbythe manufacturerto meet specified nutritional requirements andnotto exceed stated maximumconcentrations ofkey contaminants, including specified heavy metals, aflataxin, chlorinated hydrocarbons, and orgpacphosphates. The presence ofthese contaminants belowtic maximum concentration statedby the manufacturer would not be expectedto impactthe integrity ofthe study. The animalhealth and environmentalmonitoring program is administered by the attending laboratoryanimal veterinarian. Dstaaieroamtainedseparatelyfiomstudyiecocdsandniaybc included in the final report at file discretion ofthe study director. D. Pretest Period Uponarrival at Haskell Laboratory,allrats wiilbe housed onepar cage in quarantine. Therats will be: quarantined for a minimumof5 days. identified temporarilyby cageidentification. weighed atleast,3 times during quarantine. . observed withreflect to weightgainand any gross signs ofdisease or injury. The rats wiUbe reltascxl from quarantiiie by the laboratory-anirnal veterinarian or designee on the bases ofbody weights and clinical signs. Rats that are accidentally killed or removedfiom studyduring the pretestperiodwill be discarded withoutnecropsy. Ratsthat are found deador sacrificed in extrem is dnringthe pretest . period will be aent to Pathology andgiven a gross examinatwgito cheek forthepresence of disease. Dependent uponthese finding1:, further diagnosticprocedures maybe employee at the discretionoffile study director, a pathologist, orthe laboratory animal veterinarian. The results will notbe reported in the final report unless consideredsignificantto file evaluation ofthe study.' -v III--. it L i | p t | f ! | i Exygen Research Page 31 of 63 -132Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, andH-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats______________________________ DuPont-13882 Exygen Study N o.:| H416137, H'251SBt SQdH-261S9: liepeafcd-DreeDemulToxieiiy 28-Day Study in M tlc Bao DuPont-13882 E. Assignmentto Groups The rats, selected on diebases ofadequate body weight gain and freedom from anyclinical signs ofdisease or injury, will be distributed by computerized, stratified randomization into study groups as designatedin the StudyResign,so thatthere ate no statistically significant differences among group body weight means. The weight variation ofselected ratswifi notexceed 20% of the mean weight ' i 1 | | 5 Each ratwillbe houscit intSviduaBvandassigned an animal number. Eriortoaasienmeittto groups, each ratwill be temporarilyidentified by cage identification. Afisr asaignment to groups, the animal numberwill be tattooed on the tail o f each rat andincluded onthe cage label. 1 At study start (test day0) tire rats will be 3 weeks old. On test day 0; when possible, rats with body weights thatare not within 20% ofthe mean wfii be removed from studyand replaced with rats havingbody weights within that range (subjectto the sameselection criteria, asthe original rats). ' Rats that have notbeen assignedto a test groupor winch have beenremoved from studyon test day0, forout-of-rangebodyweight, will be released for other laboratorypurposes, or be sacrificedby carbon dioxide asphyxiation and discardedwithoutpathological evaluation, atthe discretionofthe study director. n r j; .* F. Administration ofTest Substance Approximately24 hoursprior to the first treatment, the fttr ofeach ratwillbe closely shavedto erqrose the skin fromthe backand hunk. The fabric will be slightly moistened with deionized water, wrapped aroundthe animal in a single layer, and secured. Theapproximate area of ddn: coveredwitheach fabric will be documentedin the study records. Therats willbe fitted with plastic collars duringthe exposureperiod toprevent oral exposure to thewrapping. The rats will be tceatedibr28 consecutivedays. The exposureperiodwill be approximately 6 hours cash day. After the exposureperiod, the fabrics and collars will be removed andthe shavedareawill bewashedwithwarmup water. The shaved area ofeach ratwill then gentlybe patted thy andthe rat returned to its cage. The animals will be reshavedriutnrgthe study as needed. The entire area (hat was originally shaved willbe reshaved. The animals will be reshaved only after an evaluation. On the day of necropsy, the furwill be shaved from anuntreated area and outlined. G. BodyWrights ' The rats will be weighed atweekly intervals duringtee study. t i' Exygen Research Page 32 ofJ -133Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________ DuPont-13882 Exygen StudyNo.:I H-2il37,H-26138,rurdB.26139: RepealedUoae D e ra il Toxicity 28-Pay Studyin Mule B a t a _______________________________ D uP ont-m H. Food Consumption andFood Efficiency The amount of food consumedby eachrat willbedeteimined weeklyby weighing each feederat the beginning andend ofthe week andsubtracting fee final weight Badfee amount of pillage from the feederduringthe weekfrom the initialweight Fnnh these measurements, mean daily food consumptionoverthe weekwillbe determined. From the food.consumption andbody weight data, the meandaily food efficiencywill be calculated. I Clinical ObservationsandMortality The rats mill be observed for clinical signs anddermal effects afterremoval offee test fabric. Therats will also be observedfor clinical signs at each weighing. The Drarze Seale winbe used to score sldn irritation. Rats will bechecked twice dailyfor mortality and for signs ofillness, injury, or abnormal behavior. I. Perfluorooctaitoic Acid level Evaluation Blood (approximately 2 mL) will be collect! feom thevena cava of?all rats at necropsy into a tribe containingEDTA. Plasmawill be prepared andstored frozen at -80Cto -20"C until analyzed for conccntm tiaitxrfM dgU BM BM M ^H M Plasmawill be evaluated from nl! rats. Plasma sampleswillbe ramactedby organic solvent protein precipitation andthen analyzed forfl000by an appropriate methodat ExygenResearch (3058 ResearchDrive, State College, PA 16801). K. Clinical PathologyEvaluation A clinicalpathology evaluation willbe conducted on all rats the dayafterthe last dose. The day be&ae collection of'HoodiampIeirfbrthe-clinreal-pathology'evaluation; the animals wiUhe placed in metabolism cages. Theseanimals will be fasted ovcnrtgbt(approximately 16 hours) andurine will be collected rum each animal. Blood samples forclinical chemistry measurements will'be collected from the orbital sinus ofeach animal while the animal is under light carbon dioxide anesthesia. Blood samples for FFOA will be collected at sacrifice from the ' abdominal vena cava ofeachanimal while the animal is undercarbondioxide anesthesia. Additional blood collected from thevara cava will be placed in a serum tube, processed to serum, and frozen at -80"C to -20"C Atthe discretion ofthestudy directoror clinical pathologist, additional samples for selected clinical pathologytests.will be collected ftom animats showingclinical evidence o f toxicity or sacrificed in extremis. Exygen Research Page 33 of 63 -134Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________ i_______________________ DuPont-13882 Exygsn. Study NoJ| H-26137, H-26138, and H-26139; Sepestcd-Dose Dermal T o a d y 28-Day Study in Mile EaU__________________________ ____________________________ DuPoal-13882 1. Clinical Chemistry i ^ Tne following seram chemistryparameters will be dctcnnined: j chofcslSlDl ] triglycerides - '- ' . 2. Urinalysis T ie following urinalysis psrariieter will be determined: fluoride J ! L. -AnatomicalPathology 1. Pretest See Materials and Methods, Sectiont>. PretestPeriod. - i 2. Dosing Phase All tats fotmd dead, accidentallytrilled, sacrificed in extremis, or sacrificed by design will undergo a gross evaluation. AHrats removedfrom study (except for out-of-range bodyweight on test-day 0) will be sent to Pathology for gross evaluation andcollection oftissues. Eats will be euthanatized by carbon dioxideanesthesiaandexsanguination. Rats sacrificed by designwiQ be fasted after3 pan. onthe afternoonbefore theirscheduled sacrifice. A final sacrifice will be: performed on surviving rats followingthe final clinieil pathology evaluation. The order of sacrifice for scheduled deathswillberandomamongall treatment groups. The followingtissueswill be collected from rats which are found dead or accidentally hilled (tissue integrity permitting), sacrificed in extremis, removed, from study (except out-of-range body weight on test day 0), or sacrificed by design: liver* ~ ` kidneys* * thyroid glend (oiler fixation) * skin (treated andrmtrdsied) " gross observations* '_______________ .______________ ._______________ i Organs to be weighed 6 Gross observations made at necropsy-fur which histqpsthology is not appropriate (eg., fluid, rutiled fur, and missing anatomic parts) will generally not be colleeted. All tissueswillbe placed inthe appropriate fixative. - -8 - i f r; Exygen Research Page 34 of 63 - 135 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, andH-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ________________ DuPont-13882 Exygen Study No H-26137.H-26138. s a l H-2613S: Kspeaied-DnstD eam l Toxicity 2S-DayStudy in MaleRata DuPoat-338g2 Rats sacrificed by design will havethe following organsweighed: liver, kidneys,,andthyroid. Relativeorgan weights (percent offinal bodyweight) will be calculated. Final body weights ii determinedjust prior to necropsywill be used m the assessmento f organ weight changes. Organs from, rats founddead, sacrificed in extremis, or accidentally killed may beweighedatfoe \ discretion ofthe pathologist or study director. v Tissuescollected from rats sacrificed by design, andfrom nils font are founddead or accidentally killed (tissue mtegrity,peimittmg), orarc sacrificed in extremis, may be forth'processedto slides, stainedwi&hicznatogyKnandeosnu and^aarinfiriiaiciMCohicaBv.mfo&ji{cret3rm-nf.foft study director orpathologist. Selected grossobservations forwhich a microscopic rHagno,* wouldsotbe additive (e.g;, osteoarthritis, pododennatitis, chronic tail deematitu, calculus,and deformities offoe teeth,foe, tail, or earpinna) will be saved,butwiil generally notbe processed fornuoroscopiccv&loafioa Tissues fromrats removed fromstudy (for otherthan out-of-range body weight on test day 0), will sot beprocessed formicroscopic evaluationunless considered necessary by the study director orpathologist. Additional procedures to identifyand/or clarify histologic features o flesions may be performed at the discretion offoe pathologistandwill be documentedin foe finalreport 1: | Exygen Research Page 35 of 63 - 136Company Sanitized. Does not contain TSCA C8I /0m^\ H -26137, H -26138, and H -26139: R epeated-Dose Dermal Toxicity 28-Day- S tudy in M ale R ats________________________________________ Exygen Study No D u P o n t-1 3 8 8 2 H-26l37,H-261J8,andR.2613fc Repeatai-DoseDirmi!Toxicity 28-Day Study m Male Rato____________________________________ ________________________ DuPpnt-13882 STATISTICAL ANALYSES i Significance will bejudged atp < 0.05. Parameter Preliminary Test Body Weight BodyWeight Gain Test for lack of trend*8* Food Consumption Food Efficiency Orgaa'Weight Levate'* test fbr homogeneityt5) and Shapiro-Wilk test*10* fbr normalit Levene's test for Clinical Pathology" hoxnogenhy*9* and Shapiro-Wilk test**'5 for narmality15 Survival Incidence ofClinical Observation None Incidence o fDamai Effects Method of StatisticalAnalysis Ifpreliminarytest is not Ifpreliminary testis significant significant XeqnentiTppH{rattaWJ ofthe Jonckheere- Terpstra trendtest*** Preliminary tests for pairwise campanson OR" . One-wayanalysis of variance1" 1followed with Dunnetfs test!!* Kmskal-Wallis test*1* followed with Dunn's test11* One-wayanalysis of. variance*111followed, with Dumtetfs test!1* Kruskal-Wallis test*13* followed wife Duns's test*" * Cochran-Armitagetest fortrend0' 11 a Pam riie comparison* aod ajsociated preliminaiy testa are only conducted if the test for lack o f trend is i significant. i> I f tbs SbqHro-Wilk test is not rigniScant but Lev* test is gnificant, u robust Ycnrion o f D aosetft test will tensed. C When an individual obser vation u recorded as being les* than a certain value, calculations are performed on halftbe recorded value. For example, i f bSirobui is reported as *0.1, 0.05 is used for any eilcufetioiu 3 performed with fbst blliniVna data. d I f the incidence is not aigoifictnt, but a significant lack o f fit occurs, then Fisher's E a s t test<ts>with a i Bonfenod correction is used. Othermethods -Rill be.used, if appropriate, the time of analysis. The statistical methods used will be described in tKe final report. SAFETY AND HOUSEKEEPING .. Good housekeepingprocedures will be practicedto avoid contamination o fdie test substance andto avoid potential health Hazards. To avoid shin contact, gloves will be wont when handling the test substance. Animal carcasses andfeces 111be inaircrated. I I Exygen Research Page 36 of 63 -137Com pany S a n itized . Does not contain T S C A C B I H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study NoI H-26]37,H-26138,and H-26139: Bejwatod-Dost D m m i Toxkaty 28-Dty Study in M ile f a n __________________________________ DuPort-13882 RECORDS AND SAMPLE STORAGE All dataand records for analytical characterizations conductedtrythe sponsorwill be archived by thesponsor. Laboratory-specificor rite-specific raw datasuch as personnel files,instrument, equipment, re&iseratarand/orfreezer rawdatawill be retained at the facility where filework was done. A sampleof the test substancewill be collected for archivepurposes andretained at Haskell Laboratpiy, Newark, Delaware. Specimens (if applicable), raw data, andthe final report wilt be retained at HaskclfLaboratory, Newart, Dekware, or"atIron Mountain Records'MaSgement, Wilmington, Delaware. Exygen Research Page 37 of 63 - -138Company Sanitized. Does not contain TSCA CBl H -26137, H -26138, and H -26139: Repeated-Dose Dermal Toxicity 2 8 -D a y Study in M ale Rats_________________________________________ D u P on t-13882 E xygen S tu d y N o . H-26137, H-26138, and H-26139: Rcpesfcd-Do* Dermal Toxicity 28-Day Stadyin Male Rate _____________ DnPogt-13882 REFERENCES i 3. DuPont Haskell Laboratory (2003). H-25509: RcpestediDoaeDermal Toxicity, Z8-Day Study in MaleRata. ''Unpublishedreport, DuPont-11763, 2. EPA/TSCA GoodLaboratoryPractice Standards (40 CFR.792). (1989). 3. OECDPrinciples of Good Laboratory Practice (as revisedin 19S7, publishedin ENVMCT2KE(v(y8)Hi;0(33E/GD(92)32); 4. .United States Environmental Protection Agency (EPA), Office ofPrevention,Pesticides, and Toxic Substances (OPPTS) Health Effects Test Guidelines, OPPTS'8703200,21/28-Day CermalToxicity (AUG-1998). 5. Organisation for Economic Cooperation and Development. Guidelines for Testing of Chemicals, Section 4 (Part410): HealthEffects (1981). 6. Draper, N.R, and Smith, H, (1981). Applied RegressionAnalysis,2"' editioD,pj 266-273, Wiley, Hew York. . 7. Selwyn, M X (1995). The use oftrend tests to determine a no-observable-effect level in artimal safety studies. Journal o fthe America* College o f Toxicology 14(2), 158-168. 8. Jonekheere, AJR. (1954). A distribution-free K-sampletsst against ordered alternatives. B iom etriia 41, 133-145. 9. Levene, H. (I960). Robust test for equality ofvariances. Contributions to Probability and Statistics (J. Ottan, ed.), pp278-292. Stanford UniversityPress, Palo Alto. 10. Shapiro, S.S. andW ife, M 3. (1965). An analysis o f variance test for normality (complete samples). Biom etrika 52,591-611. 11. Snedccor, G.W. and Cochran, W.G. (1967): Statistical Methods, 6" edition, pp246-248 and 349-352. The Iowa StateUnrversity Press, Iowa. 12. Dunnea;G.W.(1955). A riuiifiplecomparisonprocedure for comparingseveraltreatments with a control. J . Ajner. S ta tist issu e. 50,1096-1121. 13. Kiuskal, W.H,and Wallis,W A (1952). Use ofranks in.one-criterion analysis ofvariance. J. in te r. S ta tist Assoc. 47,583-621. 14. Dunn.OJ. (1964). Multiple contrasts usingrank sums. Techoometrics 6,241-252., 15. Fisher, RA. (1985). Statistical Methodsfo r Research Workers, 13 edition. Hafiner, New York. Exygen R esearch P ag e 38 of 63 - 139Com pariy Sanitized. Does not contain T SC A CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study Nol ZMteyShujyiaMalclUuH-26137,H-26138, sn4H-2SX3!>: Reprated-Dmt Dermal Toxicity Do?ODt-13&S2 j Study Function PROTOCOL APPENDIX Study rcrsonti"! Study Director Carol Finlay StaffToxicologist CHiSraTPhljlogy fivfluificttr ffancypf Events Principal Research Clinical Pathologist andManager Anatomic PathologyEvaluation GregP. Sykes Veterinary Pathologist StudyDfltefl Initiation ofTest Substance Administration Clinical PathologyEvaluations Anatomic Pathology Evaluation Scheduled Sacrifice November24,2003' December 22,2003 December 22,2003 December 22,2003 Exygen Research - 11- Page 39 of 63 -140Company Sanitized. Does not contain TSCA CBI H -26137, H -26138, and H -26139: R epeated-D ose D erm al T oxicity 2 8 - D a y S tu d y in M a le R a ts___________________________________________ D uPont-13882 Exygen Study No.: H-26137, H-26138, rad H-26139; Repeated-Dose Dermal Toxicity 23-Day Study to M ils R a t a _______________________________ SIGNATURE DuPom-13882 f./approvedby:______ SLAa L . i' icf lni ljttb. y----( CarolKnjiy Stcdy Director = Haskstl Exygen Research Page 40 of 63 -141 - Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Bxygen Study No. K-25l37j H-26138, and K-26139: Repeatsd-Dose Dermal Tcodcity 28-Psy Study in M ile Rais_________________________________ WorkRequest Ninsl ) Service Ci Duiom-m2 Protocol AmendmentNo. 1 Page 8, Z UrinalysisAdd: "Urinevolume** to list ofnrioaiysisparameters to be determined. Reason: Urinevolume is needed to calculatethe amountof fluoride in fee urine. Approvedby_______ L iL M L .' i C h l j U ^ . I CarolFiipay ,=c Hasten Exygen Research If Page41 of 63 - 142Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study N o.| RESEARCH ____ seaich. .. Prwen Resets. Exygeo Research Page42 of 63 - 143 Company Sanitized. Does not contain TSCA CBi H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 Exygen Study No.: Analytical Method _ _ _ _ ^ pM ethod of Analysis the Determination of ~" in Serum by LC/MS/MS" Exygen Research Page 43 of 63 - 144Compariy Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Exygen Study No. DuPont-13882 RESEARCH i Precise Research, fro ra i Results. T IT L E ji Method of Analysisior theDetermination oil \ by LC/MS/MS -Ttevinon 2 jin Serum ATHOR Emily Decker DATE ISSUED \ January8,2004 SPONSOR \ \ DuPont Haskell Babaiatory for Health & Environmental Sciences \ P.O.Box 50 Newark, DB 19714-0050 USA DuPont'StndyNumber DuPont-13090 '\ ir PERFORMING LABORATORY \ BxygrisReseaich 3058 Research Drive State CoI)ege\PA 16801 Exygen Research 20 1 3056 Rasoadl * \S B te CeS^e, PA16801, USA IT: 8082813219 \ fatty81452n7coZ1l019 Page 44 of 63 -145Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________ _______________ DuPont-13882 Exygen Study No.: MANAGEMENTAPPROVAL it L Da rzL /''fa h n F A7 la h /fa A e rty 't f ' - 'M f Da Vice-PrtsideatI Exygen Research j *c~________ f - T M 'Zeo'j i S. Mad: Kennedy t Da i SponsorRepresentative I DriPont iI I Exygen Research Exygen Research F i Page2.o f 20 it. Ii i Page45 of 63 - 146Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study No TABLE OF CONTENTS MANAGEMENT APPROVAL--------------------- .....----------------------- ------ -------- 2 t a b l e o f c o N rrm ra _____________________ -- ............................. ......... ,,3 . LET OF TABLES---------------------------- ------------------------- ---------------------- 4 LIST OFHGURES____ ,,______ ,,___ ________________ _____ ____________ 4 I-. -StffcM A RY :.-----.------------........ ....... .... .......................... .......................... 5 2. EXPERIMENTAL COMPOUNDS............... ...................... ...................... ............ 6 3. CHEMICALS AND SUPPLIES......................................................... .....................7 3.1. Chemicals_____________ 2......... ............ .............-- -- .............. ...........7 33. Standards__ ____________________-- ------ ----------------- ------------- 7 . 3 3 . Equipmentand Supplies---------------- ------------------------ --------------------8 35. PREPARATIONOFSTANDAKDSANDPCe.TffICATIONSolutions.................... .... ? 35.1. Stocksolution.................. ,,. ......................................................... 9 3 5 3 . FORTTHCATIQNSOLUTIONS----------- ------------------ ----- ----------- ----9 3 5 5 . CalibrationStandards-- __ _________ __- ..................... ....... ,,10 4. METHOD________________________ 10 4.1. FlowDiagram........................ ........ ...... ......................... ....... ..... ...... ,,.10 43. SampleProcess ...................... -.1 0 4.3. SamplePreparation_____ _____ ------------ ----------------- --- ................ n 4.4. Extraction_______ _________ ----------- ------ ------- ---------------------n 45. Quantitation-- ..... ........................... ...... -- --------------- ---- .----------- U 45.1. LC/MS/MS System andOperating Conditions - .......... ........ .__ ____ 11 4 5 2 . TuneFejeParameiers... ---- ------ -----------................ ........... 12 4 5 3 ..C aubraiionErocedures-------- ------------- --.12 4.5.4. SampleAnalysis- __________________ ______ __ ______13 4.6. Acceptance Criteria---------------------------- ---------- ------ ------ ------- ,,14 4.7. P erformanceCriteria.............. ................... ............. ..... .......................... 14 4.8. time Required fur Analysis..---------- --------------------- --------- ---------- 15 5. CALCULATIONS----------- 15 Exygen Research Exygen Research Page3 of20 Page 46 of 63 -147Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ Exygen Study N DuPont-13882 LIST OFTABLES i Table I Recovery " S ' FoxtiSsations In Monkey Semm............ ................... is . ' w LKTF FIGURES Figure 1. Calibration curve is Control Monkey Seram------------------ --------17 Figure 2. Representative Chrom ato^rM ^fa&ntrot Monkey Serum Sample for 5 | Figure 3. Representative Chromatogram of a lO p^^m dand 'a Control Monkey Serum safe - 5 ng/mL of ..................................... .......19 1l Hgure 4. Representative Chromatogramof Control Monkey Serum Fortified at 10 ppb w ife- 5 ng/mLof ,34 H I B ....... ...............................20 Exvgjn Research Exygen Research Page* of i [ !UI Page 47 o f 63 - 148Company Sanitized. Does not contain TSCA C81 H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ `DuPont-13882 Exygen Study No 1. SUMMARY. This report details a method of analysis for i1 serum. m V i s extracted from serum by protein, precipitation in acetonitrile. Quantification 0fBiMfe_asiirofohed-by-Uqm<idiroriffltQsaphytaiidcm-nra55 spectrometry (LC/MS/MS) analysis using selected reaction monitoring (SRM). The chemical formula of PFOA is given in section 2 of this method. The.lower finrit of qosntitatioii (IXOQ) for this method is 10 ppb fori serum. in Quantification is performed using extracted calibration standards containing an internal standard. This method was developed using monkey serum. The overall percent recovery standard deviation fo j^ | ^ | in monkey serum at 10,100, and 1000 ppb was 94% 6.9% (Table 1). A representativecalibration carve f o r ^ m ^ r monkey serum is shown in Figure 1. Representative chromatograms for M M g n monkey serum are shown in F igu res2to4. i ! s. ]t I ExyBeo Research Exygen Research Pages o f20 Page48 of 63 -149Company Sanitized. Does not containTSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________________________ DuPont-13882 Exygen Study No.: 2. EXPERIMENTAL COMPOUNDS Eiygen Ratareh Exygen Research Page 6 of20 Page 49 of 63 - 150Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_____________________________ __ DuPont-13882 Exygen Study No.: i 3. CHEMICAIiS AND SUPPLIES 3JL Chemicals Chnfleal . Methanol {McOH) Acetonitrile (ACN) AnnnomnnrAcetaieOmniSolv Water Grade HPLC HPLC Reagent HPLC Sourds'- EM Science EM Science Sigjna-AlririehEM Science Calaloe No. -- JT9093-2 AXD14M --A-733Q-- W X0004-1 Standards Standard Lot Number Purity (% ) Source Exygen Research Exygen Research Page 7 o f ZO Page 50 of 63 -151 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Da.y Study in Male Rats___________________________ ____ DuPont-13882 Exygen Study No.: 3 .3 . E q u ipm e n t an d S u p pl ie s Eauietneni Supplier Balance, analytical (display at least 0.0001 g) Mcttlcr 1 Centrifuge ' Eppeodoif i 2 m f. eppendorfcentrifuge tabes - VWR Disposablemiaropipets (50-100uL, 100-200tiL) X>rummond(VWR) Class A pipett and volumetricflasks various suppliera Hyptacariirirop-in^uaideolumnf't mm) Keystone- (part if 844017-400) Stand-alone drop-in guard cartridge holder Keystone 125-rriLIXiPE narrow-mouthbottles Nsdgene 2 tnL clearHPLC vial kit (cat# 5181-3400) HP i Standard lab equipment (graduated cylinders, disposabletubes etc.) various suppliers I LC/MS/MS andHPXjC system s As described in section 4.5. Note: Equivalent materials may be substituted for those specifies in this method if theycan be shown to produce satisfactory results. | j Notes: f j 1. In other to avoid contamination, the use of disposable lahwere is highly ; recommended (tubes, pipes, etc.). 2. F IF E or F IE E lined containers or equipment, including PTEB-lined HPLC vials for theHPtCautosamplermastBjg beused. X It is necessary to check the solvents (methanol) for the presence of contaminants by LC/MSfMS before use. Certain lot numbers have been found tobcunsuitable for use. : I1 4. Use disposable micropipets or pipets to aliquot standard solutions to make 1 calibration standards and sample fortifications. | X The hypeicaib cartridges should be changed- when ftie system f contaminants (elevated baseline) move to within l minute from the elution [ ofHFOA. ;! 3 A SOUOTIONS (1) 2 mM ammonium acetate solution is prepared by weighing 0.15 g of ammonium acetate and dissolving in 1 L of OmniSolv Water, Note: Th aforementionedexample is'proviSed for guidance, alternative volumes may be prepared as long as the ratios of die solvent to solute arc maintained. Exygen Research Exygen R esearch P8C Bo f 20 E Page 5 i of 63 -152Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, andH-26139: Repeated-Dose Dermal Toxicity 28-Day Study ip Male Rats_____ __________________________ DuPont-13882 Exygen Study No fj * * * i\ j 3 5 . P r e pa r a t io n o f St a n d a r d s a n d F o r t if ic a t io n S o l u t io n s Analytical standards are used for three purposes: 1. Calibration Standards - These standards are prepared in control monkey serum and are used to calibrate the response of the detector used in the analysis. laboratory Control Spikes - These fortifications are usually prepared at concentrations corresponding to the IXOQ and 10s IiLOQ and are used \ to' determine anaiyScat recovery! `Laboratory cantrol spiiccs aie prepared in control monkey serum. 3. Matrix Spites - These fortifications are prepared by spiking into the field samples at known concentrations. Matrix, spikes are used to evaluate the effect of the sample matrix on analytical recovery. The absolute volumes of 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. 3.5.7. Slock solution Prepare individual stock solutions of - 100 pg/mL of i^0M k id n Cweighing out - 10 mg of analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 100-mL volumetric flask. Each stock solution (in 125-mL LDFE bottles) is to be; stored in a refrigerator at 2 "C to 6"C and is stable for a maximum period of 1 year from the date ofpreparation. 3.5.2. F ortification Solutions i a. Prepare a fortification standard of 10.0 jig/mL for PFOA by diluting i. 10.0 mL of tb e M g solution described in 3.5.1 to 100 mL with ! acetonitrile a a volumetric flask. b. Prepare a fortification standard of L0 pgfaL; for by diluting 1.0 mL of the uO (M B solution described in 35.1 to 100 mL with acetonitrile in a volumetric flask. c. Prepare a fortificatioi^tnjKtad of 1.0 pg/mL foJJH B ^on tain in g i 0.005 pg/mL of ,3d B B R > y diluting 10.0 mL of th e^B K o lu tio n described in 35 .2 .a and 0.5 mL of the 13C m k o lu d o n 'described in 3.5.2.b to 100 mL with acetonitrile in a volumetric flask. j d . . Prepare a fortification standa r d ^ 0.1 pg/mL for )J^^h ntaiiu ng ' 0.005 pg/mL of 13C ^ 0 > y diluting 1.0 mL of th ^ H R o lu tio n described in 35.2.S and 0.5 rrX of th e'3CPFOA solution described in 3.5.2.b to 10Q mL with acetonitrile in a volumetric fla s lc ^ ^ e. Prepare a fortification standard of M l pg/mL for flftn ta im n g 0.005 pg/mL of `' H P -b y diluting 0.1 mL of th e fjjito lu tio n Exygen Research P sje9 of 20 f. Exygen Research Page 52 o f 63 -153Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_______________________ ________ DuPont-13882 Exygen Study No.: described in 3.5.2.a and 0 5 mL of the described in ! 35-?.b to 100 mL with acetonitrile in a volumetric {last. 5` ! 1 Store aS fortification standard soludons in a refrigerator (in I25-mL LDPE : , bottles) at d"C to 6C for a maximum period of 1 year from the date of I 1 'preparation,.after which time it is necessary to make new standards using. \ : the stock solution. f j 3.J.3 . C alibration Standar d s ______ _________________ _ [ ! The calibration standards are processed through die extraction procedure. ; : identical to the samples. The fortilication o f the standards before : - extriction is done according to the following table: Cone. O f Mixed Fortification Solution (pgfaL) 0.01 0.01 0.1 0.1 0.1 Fortification Volume (pL) 100 200 50 100 200 Volume of Control Sample (mL) 0.1 0.1 0.1 0.1 0.1 Cone, of Extracted Calibration Standard (pph) 10 20 50 100 200 4. METHOD 4 4 . F l o w D ia g r a m The Sow diagram of the method is given below, followed by a detailed description of each step. Method Flow'Diaetam M easure 0 .1 m L o f sam p le (u sin g disp o sab le m icropipet!) (fortify samples when designated) Protein precipitation in acetonitrile 4LC/MS/MS analysis rr 4.2. Sample Processing No sample processing is needed for serum samples. However, frozen samples must be ailpwed to completely (haw, un-aided, at room temperature. Samples B rysen Research Page 10 of 20 Exygen Research Page 53 of 63 -154Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, andH-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_______________________ __ DuPont-13882 Exygen Study No.: stored refiigerated should also be allowed to equilibrate to room temperature. AS samples must be thoroughlymixed before being sampled for exffitetion. \ 4.3. Sample Pebtasation i: 1 .. . ' . .... .. t a. Each batch of samples extracted (typically 30 or less) most include at least p 1 one,reagem control (acetonitrile blank), one matrix control (method blank), | ij and two matrix controls fortified at known concentrations to verify procedural f. recover forthebBich. i | b. At leastone sample per batch should be extracted in duplicate ; i c. At least one sample extracted should be separately fortified at a known i concentration and tamed' through the procedure to verify recovery. ! Additional matrix spikes may be performed at the sponsor's request. 4.4, Extraction ; 1. Measure 0.1 mL of sample into 2 mL eppendorf centrifuge tubes (fortify with analyte as needed, close lid, and vortex -1 0 seconds). 2. Add enough acetonitrile containing internal standard at 0.005 pg/mL (accounting for fortxficstioa volume) to make extraction volume 500 pL and vortex for -1 0 seconds. 3. Centrifuge f o r - 10minutes a t - 14,000rpm. 4 . Analyzesamples using electrospray LOMS/MS. >, ;r ; | j 4 3 . Quantitation | 4.5.1. LC/M S/M S System and O perating C onditions 1 Instrument: PE SCIEX API 4000 BiomolecularMass Analyzer 1 Sd E X Turbo Ion Spray liquid Introduction Interface , Computer: Dell OptiHex G X 110 Software: PE Sciex Analyst 1 2 HPLC Equipment Hewlett Packard (3P ) Series 1100 QuatpumpGl311A Vacuum Degasser G1322A Autoinjector G1313 " Column Compartment G1316A ' JSNote: Two 4 pc 10 mm hypercarb drop-in guard cartridges. (Keystone, part # 844017-400) ar attached In-line after the purge valve and before the sample injector port to trap any residue contaminants a t may be in the mobile phase and/or HPLC system. ** v ft Exygen Research Page 11 o f20 t Exygen Research Page 54 o f 63 -155Compahy Sanitized. Does not contain TSC CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_______________________ ________ DuPont-13882 Exygeo StudyNoJ HELC Column: Genesis Cj (Tones Chromatography), 2.1 mm x 50 mm, 4(1 ColumnTemperature;30 C Injection Volume:- 5 pL Mobile Phase (A): 2 mM Ammonium Acetate m OmniSolv Water Mobile Phase (B): :. Methanol 1 1 ** Time SA Flow fmL/mmt - - 1 0.0 40 60 03 3 40 3.5 O' 60 TOO 03 03 1 f 3.7 0 100 0.5 \ '; 7 0 100 03 7.5 40 60 03 9 40 60 03 ' 9.5 12 40 40 60 60 03 03 . It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. fans monitored: Mode Negative Negative Transition Monitored 4 1 3 --369 415 -- 370 Approximate Retention Time -2.8 min. -2 .8 min. The retention times may vary, on a day-to-day basis, depending cat'the batti of mobile pba etc. Drift in retention times is acceptable within an analytical nm. as long as the drift continues,through the entire analysis and the standards are interspersed throughoutthe analyticalrun. Nate: An alternative LC/MSMS system may be used once demonstrated to be equivalent 4.5.2. Tune F ile Param eters t The mass spectrometer is toned for the analyte by infosing a --1 lig/mL standard solution o j ^ l ^ a t 10 pL/min, 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 tuned for the parent ion and then tuned for the product ion. Once the instrument is tutted, the optimized parameters - are saved as a tunefile. Tifo tune file rs thenused during routine analysis. 4.5.3. CaUbratian Procedures a. Inject the same aliquot (5 pL) of each calibration standard into the LCfM SlM S.- ` Heygen Research E xygen R esearch . PigeloCM P age 55 of 63 -156Company Sanitized. Does not contain TSCA CBI H -26137, H -26138, and H-26139: Repeated-Dose Dermal Toxicity 28-D ay Study in M ale Rats D u P on t-13882 Exygen Study No! b. Use linear standard curves for quantitation. Linear standard carves ate generated for each analyte by linear regression using lix weighting of the ratio analyte pedk arearintemal standard peak area versos the ratio of the concentration of anaLyte/concentrarion of internal standard using Analyst 1.2 (or equivalent) software system. Any;calibration standard found to be a statisticsioutlier by using the appropriate outlier test (e.g. gugc OutlierTest), may be excluded from the calibration curve. However, rite total number of calibration standards drat could be excluded must not exceed 20% of the total j number of standards injected and at least one calibration standard at the EDOQ mustberStainedT............................ c. The correlation coefficient (r) for calibration curves generated must .be >0.9325 (y* R0.9S5). If calibtatian results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set of samples should be reanalyzed. 4.5.4. Sam ple A nalysis a. Inject the same aliquot (5 jiL ) o f each standard, sample, recovery, control, etc. into fireLC/MS/MS 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 for a second act of standards). As an alternative, an'.entire set of calibration standards may be included at she beginning and at the end of a sample set In either case, calibration standards must be the first and last j injection in a sample set d. The concentration of each sample/fbrtiScation/controJ is determined from the standard curve, based on the peak area of each analyte. The standard responses should bracket responses of the residue found is each sample set If necessary, dilute due samples in 50:50 metbanoUwater-.to give a response within the standard curverange. e. Fortification recoveries failing within 85 to 115% (80 to 120% for levels at theILOQ) are considered acceptable. I f. Extracted samples must be stored refrigerated between 2C to 6C until analysis. 3! g. Samples in which no peaks are detected (i.e. signal: noise ratio < 3:1) at the corresponding analyte retention times will be reported as HD (not detected). Samples in which peaks are detected at % ! corresponding analyte retention Exygen Research I . . Page 13 of 20 j Exygen Research Page56 of 63 - 157 - Com pany Sanitized. Does not contain T SC A CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats _____________________________ DuPont-13882 Exygen Study No.: ticpes but are less than the lowest concentration of the calibration standards (10 pjib wh^re LLOQ = 10 ppb) will bereported as <10 ppb.` * 4.6. Ac c e p t a n c e Cr it e r ia The following criteria must be met toensm e the presence o d H H I and a C- JTOA: ' -p > .. 1. ^ledupmatograrnB must show the following peaks: W p B K ^ toighte-ion at 369 amn from a parent of 413 amu daughterion at 370 amu form a patent of415 amu Z Anyanalyte present in method blanks must be at least 5-fold lower than the LLOQ. Any analyte present in the reagent blank must be at least 5fold lower thanthe ILOQ. 3. Recoveries of lab control spikes and matrix spikes must be between 85- 115 (80-120 for levels at the LLOQ) of their known values. Any method fortification (lab control spike) falling outside the acceptable limits warrants re-extraction of die entire analytical set. Any matrix spike outside the acceptable range 'should be evaluated by the analyst to determineif re-extraction is warranted. 4. Any calibration standard found to be & statistical outlier by using an appropriate outlier test, may be excluded from the curve. However, foe 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 LLOQ mustbe retained. 5. The correlation coefficient (r) for calibration curves generated must be 0.9925 (i5 0.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or foe relevant set of samples shouldhe reanalyzed. 4.7. P e r fo r m a n c e C r it e r ia 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. grpt Criterion: : Rim a standard solution tm LC/MS/MS corresponding to the estimated LLOQ -and obtain.a;signal-to-naise ratio of at ieasb:l, compared to a reagent blank. If this criterion yannot be met, optimize- and change instalment operating parameters. i Exygen R esearch Exygen Research .Page 14 of 20 Page 57 of63 -158Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, andH-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygsn Study N o.l ! Ssaaw4.Cgtwmwip j Rum a set of standards of five or mote concentration levels, spanning a range l starting at or below the LLOQ up to the highest concentration level to be included in die analysis. Generate a calibration curve for each analyte and obtain a linear ! regression with a coefficient of determination (r2) of at least 0.9SS. Once this E criterion ii njet, samplesmaybe analyzed with standards mterspersed. i 4.8.- Tim e Required for Analysis A set of 35 samples (1 reagent' control, 1 matrix consol, I laboratory spite; 2 laboratory control spikes, and 30 samples) can be taken through the extraction procedure ip approximately 8 hours by one person. The LC/MS/MS analysis (standards and 35 samples) will-take approximately 9 horns. a fi f j : 5. CALCOLAMONS a. Use Equation 1 to calculate the amount of analyte found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. : Equation 1: ............. Analyte found (ppb) = fXeahtePeaharea/tS real:areal-intercentlt XIS eone.(ngML) dope b. For samples fortified with known amounts of analyte prior to extraction, use Equation 2 to calculate die percent recovery. Equation.2: Recovery (%) = i ; Analyte found fnpb^--mre. Analyte foundin control fonbl >' 100% AmountAnalyte added (ppb) | 6. SAFETY .. | | [ lb s analyst should read the material safety data sheets for all standards and reagents before petfomnrig 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-borne pathogen handling precautions when handling serum. | f : i Exygeu Research Hxygen Research Pige IS of20 \ I Page 58 of 63 -159 Company Sanitized. Does not contain TSCA CBI H -26137, H -26138, and H -26139: R epeated-D ose Dermal Toxicity 2 8 -D a y Study in M ale Rats_______________________________________ _ D u P on t-13882 Exygen Study No.: Table I . Recovery ol m Fortications Ih Monkey.Serum iTcriesfor 10 ppbFoHtOektioninMonkey Serum Eacyca fittnetitts Ase^sb Pert.Leve! fc Recovery ID Dade Dite ' (m 050003 050MB 0500/03 050003 10 V . 10 95 _T .. 94 IP I m 050003 050003 10 98 ' 1 J AVERAGE: 96 1 -t .111 RELATIVE STANDARDDEVIATION: XT 22 ! tveriesfor 100ppbFortification in Monkey Scrum Spossar Extnettan Aujtfs KotlIawI g> Date 5Recovery : 0500*03 050003 100 92 i 050003 050003 100 90 OS003 050003 100 93 AVERAGE: STANDARD DEVIATION: RELATIVE STANDARD OBVIATION: 92 1 L7 Summary o l w ie s fo r IODOppb Fortification in M onkey Serum Extraction Analysis Pert.Lewi S>Recovery ID Dt Date to 050003 1000 107 H h o m b 050003 1000 95 ^ 0 5 0 0 0 3 050003 1000 81 AVERAGE: 94 STANDARD DEVIATION: m RELATIVE STANDARDDEVIATION: IAS O V ERA LL AVERA G E; OVERALLSTANDARD DEVIATION* OVERALL RELATIVE STANDARD DEVIATION* M 62 7.3 t i l Biygm'Research Esygen Research Pige 16o f20 r Page59 of 63 -160- Com pany San itized . D oes not contain T S C A C B I H -26137, H -26138, and H -26139: R epeated-D ose Derm al T oxicity 2 8 - D a y S tu d y in M a le R a ts___________________________________________ Exygea Study No. D uPont-13882 Figure 1. Calibration curve fo) Control MonkeySerum I J1 ] I '\ Exygea Research Exygen Research iv 1 Pbc 17 o f 20 f Page60 of 63 -161 - Company Sanitized. Does not contain TSCA CBI H -26137, H -26138, and H -26139: Repeated-Dose Dermal Toxicity 2 8 -D a y Study in M ale R ats________________________________________ D u P on t-13882 Exygen Study No.: ; I E iv ftn Research Exygen Research f i Fuge38 of20 \ Page 61 of 63 -162- Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats_______ DuPont-13882 Ejcygen Study No. Figure 3. Representative Chromatogram of a 10 ppb Standard for i Control Monkey Seram "with- 5 ng/mT.~ofBC- i i 1 Exygcn Rcwaxch Exygen Research Page 19 o f 20 Page62.of 63 -163Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Exygen Study No.: Figure 4. Representative Chromatogram of ControlMonkey Serum Fortified at 10 ppb 'w ithflH B > d vrith - Sng/mL ofUC- ExygtaRracaicii Exygen Research' Page20of20 Page 63 of 63 -164Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________________ V DuPont-13882 APPENDIX G INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA -165Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 INDIVIDUAL ANIMAL CLINICAL PATHOLOGY DATA EXPLANATORY NOTES ABBREVIATIONS: Individual Clinical Chemistry Values: CHOL - cholesterol TRIG - triglycerides Individual Urinalysis Values: UVOL - volume UFLU - urine fluoride N O TESr When individual animal data are not reported, it may be due to one of the following reasons or other reasons, al-1 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 (RNV) the sample was not suitable for testing the animal died prior to sample collection no sample was available for testing (NSR) - 166Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats Individual Animal Clinical Pathology Data Male, Group i H-2613 Animal 101 102 103 104 105 106 107 108 109 110 Male, SHEM Trace None Non None None None None None None None SLIP None None None None None None None None None None SICT None None None None None None None None None None Group III - H-26137 Animal 301 302 303 304 305 306 307 308 309 310 SHEM None None None None None None None None None None SLIP None None None None None None None None None None SICT None None None None None None None None None None Day CHOL mg/dL 35 35 40 51 38 46 39 62 54 37 Day CHOL mg/dL 40 38 69 65 50 49 44 32 39 39 28 TRIG mg/dL 27 24 23 32 57 29 24 42 35 27 28 TRIG mg/dL 49 28 23 36 25 40 35 23 25 18 DuPont-13882 -167Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats ______________________ Individual Animal Clinical Pathology Data Male, Animal 501 502 503 504 505 506 507 508 509 510 Group V - H--26138 SHEM None None None None None None None None None None SLIP None None None None None None None None None None SICT None None None. None None None None None None None Day CHOL mg/dL 50 21 44 28 41 32 31 41 29 35 28 TRIG mg/dL 21 15 43 27 54 27 20 21 23 28 Male, Group VII - H-26139 Animal SHEM 701 None 702 None 703 None 704 None 705 None 706 Trace 707 None 708 None 709 None 710 - None SLIP None None None None None None None None None None SICT None None None None None None None None None None - Day CHOL mg/dL 27 52 38 43 31 42 43 36 36 29 28 . TRIG mg/dL 24 41 27 26 29 29 24 23 31 19 DuPont-13882 -168Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ Individual Animal Clinical Pathology Data Male, Group I - H-26136 - UVOL OFLU Animal mL pg Day 28 101 102 103 104 105 106 107 108 109 110 13.0 5.0 13.8 10.6 4 . 017.0 8.2 4.0 9.0 4.0 11.8 9.5 12.5 8.4 9.7 13.6 7.7 6.6 1 4 '. 1 7.6 Male, Animal 301 302 303 304 305 306 307 308 309 310 Group III - H-26137 - Day 28 UVOL mL 6.6 6.8 17.0 7.6 8.2 16.6 25.0 7.0 11.6 23.6 UFLU pg 10.2 9.7 13.6 10.4 9.8 12.6 15.0 9.3 12.3 11.6 DuPont-13882 - 169Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats___________ Individual Animal Clinical Pathology Data Male, Animal 501 502 503 504 505 506 507 508 509 510 Group UVOL mL 11.8 4.0 5.4 30.2 13.0 8.2 2.0 3.0 25.0 9.0 V - H-26138 - Day 28 UFLU psr 14.7 18.0 10.4 20.7 13.6 12.0 9.4 7.0 18.2 11.5 Male, Group VII - H-26139 - Day 28 Animal UVOL mL UFLU ng 701 4.6 702 9.0 703 7.4 704 8.4 705 6.8 706 7.0 707 8.0 708 4.8 709 8.2 710 - 14.6 11.8 21.6 11.6 13.6 13.4 10.0 12.5 6.1 15.9 15.5 DuPont-13882 -170Company Sanitized. Does not contain TSCA CB1 H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 APPENDIX H Individual Animal Final Body and Organ Weights _171 _ Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated-Dose Dennal Toxicity 28-Day Study in Male Rats ) DuPont-13882 Organ Weigh, .istina Study:; Individual Animal & Summary Stats Report PLACES 2000 vl.400 Group : I H-26136 Treatment : I Sex -.MALES i ANIMAL 1 FBW KIDNEYS i 1 (Gms} (Gras) %FBW i LIVER 1(Gras) %FBW i 101 1283.30 2.694 0.9509 i 102 1323.20 3.028 0.9369 i 103 1342.40 3.328 0.9720 i 104 1291.80 2.693 0.9229 i 105 1343.10 2.934 0.8551 i 106 1359.60 1 3.411 0.9486 i 107 1309.80 1 2.895 0.9345 i 108 1333.70 1 2.960 t).8870 i 109 1348.50 1 3.253 0.9334 i 110 1335.00 1 3.441 1.0272 I! ! Mean 1327.04 I 3.064 0.9368 i S .D , 124.920 1 0.279 0.0461 1 9.721 3.4313 1 11.295 3.4947 1 10.882 3.1782 1 9.582 3.2838 l 10.733 3.1282 1 12.121 3.3707 1 10.145 3.2747 1 11.533 3.4561 1 10.898 3.1271 1 11.884 3.5475 1 10.879 3.3292 1 0.865 0.1536 Group : III H-26137 Treatment : III Sex :MALES 1 ANIMAL 1 FBW 1 KIDNEYS 1 1 (Gms) 1(Gms) %FBW i LIVER 1(Gms) %FBW 1 301 1307.80 1 2.915 0.9470 1 302 1331.50 1 2.928 0.8833 1 303 1351.60 1 3.378 0.9608 1 304. 1316.90 1 2.858 0.9019 1 305 1324.90 1 2.561 0.7882 1 306 1353.30 3.507 0.9926 1 307 1336.50 1 3.054 0.9076 ! 308. 306.70 1 2.939 0.9583 1 11.221 3.6455 1 10.250 3.0920 1 12.466 3.5455 1 11.661 3.6797 1 10.152 3.1247 1 12.579 3.5604 1 11.324 3.3652 1 10.422 3.3981 1 THYROID GLAND 1Gms) %FBW 1 0 1017 0.0060 1 0.025 0.0077 1 0.020 0.0058 1 0.018 0.0062 1 0.02}. 0.0061 1 0.01 0.0050 1 0.015 0.0048 1 0.025 0.0075 1 0.023 0.0066 1 0.023 0.0069 I 1 0.021 0.0063 1 0.003 0.0009 1 THYROID GLAND 1(Gms) %FBW 1 0.025 0.0081 1 0.018 0.0054 1 0.027 .0.0077 1 0.017 0.0054 1 0.015 0.0046 1 0.024 0.0068 1 0.024 0.0071 1 0.016 0.0052 | 1 1 1 1 1 ) 1 1 FBW - Final Body Weight Company Sanitized. Does not contain TSCA CBI -172- ) H-26I37, H-26138, and H-26139: Repeated-Dose, Dermal Toxicity 28-Day Study in Male Rats _________________ DuPont-13882 Organ Weight Li Study: ] Group : III H-26137 Treatment : III Sex MALES 1 ANIMAL 1 FBW 1 KIDNEYS 1 1 (Gms) 1(Gms) %FBW 1 309 1340.40 1 3.253 0.9556 1 310 1318.00 1 2.975 0.9355 I Mean 1328.76 1 3.037 0.9231 1 S.D. 116.747 | . 0.275 0.0575 1 LIVER 1(Gms) %FBW 1 10.238 3.0076 1 10.196 3.2063 1 11.051 3.3625 i 0.946 0.2438 1 THYROID GLAND i(Gms) %FBW 1 0.020 0.0059 | 0.021 0.0066 1 | 0.021 0.0063 | 0.004 0.0012 Group : V H-26138 Treatment : V Sex -.MALES 1 ANIMAL 1 FBW 1 KIDNEYS 1 1 (Gms) 1(Gms) %FBW 1 501 1286,00 1 2.901 1.0143 1 502 1327.60 1 3.159 0.9643 1 503 1334.00 1 2.825 0.8458 1 504 1328.70 1 3.034 0.9230 1 505 1321.80 1 3.032 0.9422 1 506 1298.00 1 2.766 0.9282 1 507 1309.50 1 3.258 1.0527 1 508 1350.60 1 3.009 0.8582 1 509 1316.40 1 2.841 0.8979 1 510 1328.30 1 2.851 0.8684 1 j Mean 1320.09 j 2.968 0.9295 1 S.D. 118.562 1 0.159 0.0672 1 LIVER 1(Gms) %FBW 1 9.213 3.2213 1 10.632 3.2454 1 10.240 3.0659 1 10.799 3.2854 1 10.581 3.2881 1 9.292 3.1181 1 9.411 3.0407 1 10.268 2.9287 1 9.382 2.9652 1 10.207 3.1090 1 j 10.003 3.1268 1 0.614 0.1295 1 THYROID GLAND 1(Gms) %FBW 1 0.016 0.0056 1 0.018 0.0055 1 0.020 0.0060 1 0,022 0.0067 1 0,018 0.0056 1 0,014 0.0047 1 0.019 0.0061 1 0.018 0.0051 1 0.018 0.0057 1 0.023 0.0070 1 0.019 0.0058 1 0.003 0.0007 Individual Animal & Summary Stats Report PLACES 2000 vl.400' 1 | | | 1 I | 1 1 1 1 1 1 1 1 1 1 1 1 1 1 FBW - Final Body Weight Company Sanitized. Does not contain TSCA CBI -173- ) H-26137, H-26138, and H-26139; Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats _______ DuPont-13882 Organ Weight Listin' Study: If ''' Group : VII H-26139 Treatment : VII Sex :MALES ! ANIMAL 1 FBW 1 KIDNEYS 1 1 (Gms) 1(Gms) %FBK 1 LIVER 1(Gms) %FBW 1 701. 1287.20 1 2.697 0.9391 1 702 1368.80 1 3.466 0.9398 1 703 1349.50 1 3.258 0.9322 1 704 1313.00 1 2.792 0.8920 1 705 1310.20 1 2.844 0.9168 1 706 1360.00 1 3.154 0.8761 1 707 329.90 1 2.976 0.9021 1 708 1322.60 1 3.041 0.9427 1 709 1359.60 1 3.507 -0.9753 1 710 1310.30 1 2.720 0.8766 Mean 1 S,D. I 1331.11 1 3.046 0.9193 127.091 1 0.295 0.0323 1 9.428 3.2827 1 13.359 3.6223 1 11.525 3.2976 1 9.930 3.1725 1 10.251 3.3046 1 12.566 3.4906 1 10-.651 3.2286 1 9.914 3.0732 1 13.845 3.8501 1 9.591 3.0909 J 11.106 3.3413 1 1.624 0.2455 * * * Listing Complete *** I THYROID GLAND i(Gms) %FBW 1 0.016 0.0056 . 1 0,013 0.0035 1 0.018 0.0052 1 0.022 0.0070 1 0.024 0.0077 1 0.020 0.0056 1 0.019 0.0058 1 0.017 0.0053 1 0.022 0.0061 0.021 0.0068 1 0.019 0.0058 1. 0.003 0.0012 Individual Animal 5 Summary Stats Report PLACES 2000 vl.400 | t 1 1 1 1 1 1 1 t 1 1 j 1 Company Sanitized. Does not contain TSCA CBI FBW - Final Body Weight -174- H-26137, H-26138, and H-26139: Repeated-Dose Dermal Toxicity 28-Day Study in Male Rats__________ _____________________ DuPont-13882 APPENDIX I Individual Animal Gross Observations -175Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats______ _____ _______________________________ DuPont-13882 Individual Animal Listing Showing animal data for individual animals STUDY : 0 | A H I (* APPROVED PROTOCOL *) Dose Group :: I H-26136 Treatment: I Sex: Males 1 Date: 3-FEB-04 Animal Ref Macroscopic Findings Only 101 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 102 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 103 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 104 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED - 176 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 Individual Animal Listing Showing animal data for individual animals STUDY : T f B k (* APPROVED PROTOCOL *) Dose Group : I H-26136 Treatment: I Sex: Males 2 Date: 3-FEB-04 'Animal Ref Macroscopic Findings Only 105 Terminal Sacrifice Killed on Day .: 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 106 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 107 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 108 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -177Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ DuPont-13882 STUDY : Dose Group Individual Animal Listing Showing animal data for individual animals (* APPROVED PROTOCOL *) : I H-26136 Treatment: I Sex: Males 3 Date: 3-FEB-04 Animal Ref Macroscopic Findings Only 109 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 110 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -178Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats____________________ DuPont-13882 Individual Animal Listing Showing animal data for individual animals STUDY : j p H (* APPROVED PROTOCOL *) Dose Group :: III H-26137 Treatment: III Sex: Males 4 Date: 3-FEB-04 Animal Ref Macroscopic Findings Only 301 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, ; UNTREATED 302 Terminal Sacrifice Ki.lled on .Day : .28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, .THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 303 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS,-THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 304 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -179 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 STUDY : Dose Group Individual Animal Listing Showing animal data for individual animals (* APPROVED PROTOCOL *> :: III H-26137 Treatment: III Sex: Males 5 Date: 3-FEB-04 Animal Ref Macroscopic Findings Only 305 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 306 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 307 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 308 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy KIDNEYS : DILATATION, RIGHT, PELVIS. No Macroscopic Abnormality Observed : LIVER, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED. -180Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 STUDY : Dose Group Individual Animal Listing Showing animal data for individual animals (* APPROVED PROTOCOL *) : III H-26137 Treatment: III Sex: Males 6 Date: 3-FEB-04 Animal Ref Macroscopic Findings Only 309 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED , 310 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -181Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats ______ _______________________ DuPont-13882 STUDY : Dose Group Individual Animal Listing Showing animal data for individual B i {* APPROVED PROTOCOL *) : V H-26138 Treatment: V Sex: Males animals 7 Date: 3-FEB-04 Animal Ref Macroscopic Findings Only 501 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 502 Terminal Sacrifice Killed, b n .J2ay : 2.8 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 503 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 504 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED - 182 Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats_________________________ ~ DuPont-13882 STUDY Dose Group Individual Animal Listing Showing animal data for individual animals (* APPROVED PROTOCOL *) : V H-26138 Treatment: V Sex: Males Date: 3-FEB-04 Animal Ref Macroscopic Findings Only 505 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 506 Terminal Sacrifice Killed on. Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 507 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy KIDNEYS : CYST, BILATERAL, FEW, <2MM. - No Macroscopic Abnormality Observed : LIVER, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 508 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED - 183Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats________________________________ D uPont-13882 STUDY Dose Gr<? 'Animal Ref 509 510 Individual Animal Listing Showing animal data for individual animals (* APPROVED PROTOCOL *) '26138 Treatment : V Sex: Males 9 Date: 3-FEB-04 Macroscopic Findings Only Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, , UNTREATED Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED ____ Company Sanitized. Does not contain TSCA CBI H-26137, H-26I38, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 STUDY Dose Group Individual Animal Listing Showing animal data for individual animals H I H K (* APPROVED PROTOCOL *) : VII H-26139 Treatment: VII Sex: Males 10 Date: 3-FEB-04 Animal Ref Macroscopic Findings Only 701 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 702 Terminal Sacrifice .Killad. on Day : 28 Animal is signed off from necropsy KIDNEYS : DILATATION, RIGHT, PELVIS. No Macroscopic Abnormality Observed : LIVER, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 703 ' Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 704 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED -185Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats DuPont-13882 STUDY : Dose Group Individual Animal Listing Showing animal data for individual animals H <* APPROVED PROTOCOL *) : VII H-26139 Treatment: VII Sex: Males 11 Date: 3-FEB-04 'Animal Ref Macroscopic Findings Only 705 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 706 Terminal Sacrifice Killed o n Day. : 28 Animal is signed off from necropsy No Macroscopic'Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 707 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 708 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED - 186- Company Sanitized. Does not contain TSCA CBI H-26137, H-26138, and H-26139: Repeated Dose Dermal Toxicity 28-Day Study in Male Rats _____________________________ DuPont-13882 Individual Animal Listing Showing animal data for individual animals STUDY : f g l ^ 1 ^ (* APPROVED PROTOCOL *) Dose Group ^ V I ^ H - 2 6139 Treatment: VII Sex: Males 12 Date: 3-FEB-04 Animal Ref Macroscopic Findings Only 709 Terminal Sacrifice Killed on Day : 28 Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED 710 ' Terminal Sacrifice Killed on Day : 28 . Animal is signed off from necropsy No Macroscopic Abnormality Observed : LIVER, KIDNEYS, THYROID GLAND, SKIN, TREATED, SKIN, UNTREATED ** * Listing Complete *** - 187 Company Sanitized. Does not contain TSCA CBI