Document r6d5JVXJQ044xZXo2QY75NDke

P* M AA6 - Z l t f TRADE SECRET DuPont-8837 Study Title H-24616: In Vitro Dermal Penetration Through Rat Skin Laboratory Project ID: DuPont-8837 Test Guidelines: None were applicable Author: William J. Fasano, Sr., B.S. Study Completed on: July 26,2002 Performing Laboratory: E. I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Company Sanitized. Does not contain TSCA CBf Page 1 of 20 H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles of Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 and MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850) except for the item documented below. The item listed does not impact the validity of the study. The test substance was characterized by the sponsor prior to the initiation of this study. Although the characterization was not performed under Good Laboratory Practice Standards, the accuracy of the data is considered sufficient for the purposes of this study. Applicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Director: William J. Fasano, Sn B.S. Research Scientist 2.> ' T u ! - 2sQ 0i _ Date Applicant / Sponsor:__________________________________________ __________ DuPont Representative Date * Company Sanitized. Does not contain TSCA CBI -2- H-24616: In Vitro Dermal Penetration Through Rat Skin QUALITY ASSURANCE STATEMENT Haskell Sample Number(s): 24616 Dates of Inspections: Protocol: March 11,2002 Conduct: March 11,12,21,2002 Records, Reports: July 10-12,15-18, 2002 Dates Findings Reported to: Study Director: July 22, 2002 Management: July 25, 2002 DuPont-8837 Reported by: ----- K. i c ' / iA u ______________________________ Wonda KCKclly Quality Assurance Auditor M - f o t M IA , bate Company Sanitized. Doss not contain TSCA CB! -3- H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study Director: William J. Fasano, Sr., B.S. Research Scientist 2-6 - <TU.<- - -- Approved by: Matthew S. Bogdanffy, Ph.QifOjAB.T. Management fi-JtcL Date Company Samtfzed. Decs no! contain TSCA CB H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEM ENT............................................. 2 QUALITY ASSURANCE STA TEM EN T..............................................................................................3 C E R T IF IC A T IO N ....................................................................................................................................... 4 STUDY INFORM ATION.........................................................................................................................6 STUDY PERSONNEL.............................................................................................................................. 7 SUM MARY................................................................................................................................................. 8 INTRODUCTION....................................................................................................................................... 9 MATERIALS AND M ETHODS.............................................................................................................. 9 A. Regulatory Test Guidelines...........................................................................................................9 B. Test Substance............................................................................................................................... 9 C. Test S pecies....................................................................................................................................9 D. Animal Husbandry......................................................................................................................... 9 E. Animal Health M onitoring.......................................................................................................... 10 F. Preparation of Skin Membranes................................................................................................. 10 G. 7n Vitro Diffusion C ells.............................................................................................................. 11 H. Assessment of Membrane Integrity and Membrane Equilibration....................................... 11 I. In Vitro Penetration of H -24616................................................................................................ 11 J. Total Fluorine D eterm ination.....................................................................................................12 K. Data Presentation......................................................................................................................... 12 RESULTS AND DISCUSSION............................................................................................................. 13 A. Positive Control D ata...................................................................................................................13 B . Negative Control Group D a ta .....................................................................................................13 C. Exposure Group D ata...................................................................................................................13 CONCLUSIONS....................................................................................................................................... 13 RECORDS AND SAMPLE STO RA G E............................................................................................... 13 REFEREN CES..........................................................................................................................................14 T A B L E S ...................................................................................................................................................... 15 1. Positive Control D a ta .................................................................................................................... 16 2. Negative Control G roup................................................................................................................17 3. Exposure Group D a ta .................................................................................................................... 18 FIG U R E......................................................................................................................................................19 1. Static Diffusion C e ll................................................................................................................... 20 Company Sanitized. Does no*contain TSCA H-24616: In Vitro Dermal Penetration Through Rat Skin STUDY INFORMATION Substance Tested: Haskell Number: 24616 Composition: Known Impurities in Total Solids: DuPont-8837 Physical Characteristics: Opaque tan liquid Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: March 6, 2002 / (see report cover page) In-Life Initiated/Completed: March 11, 2002 / March 12, 2002 Company Sanitized. Does not contain TSCA CB1 -6- H-24616: In Vitro Dermal Penetration Through Rat Skin STUDY PERSONNEL Study Director: William J. Fasano, Sr., B.S. Management: Matthew S. Bogdanffy, Ph.D., D.A.B.T. Primary Technician: Brian P. Shertz, A.A. Analytical Associate: Richard F. Rossi, B.S. Analytical Chemist: Vladimir Capka, Ph.D. Management: S. Mark Kennedy, Ph.D. Toxicology Report Preparation: Lisa G. Burchfield, A.A. Management: Nancy S. Selzer, M.S. DuPont-8837 Company Sanitized Doss not contain TSCA CBI -7- H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 SUMMARY The dermal penetration potential of H-24616 has been evaluated using rat skin mounted in an in vitro static diffusion cell systenr H-24616 is an aqueous suspension containing an was applied undiluted to rat epidermal membranes at a 'rate of 20 /Licmz. T h^esm atSflral, a total of 1233.6 fig F or 82.4 ppm, remained in contact with the skin for 6 hours. At the end of the exposure period, the receptor fluid was removed from the static cell system and combusted using a Wickbold Torch followed by quantitative analysis for total fluorine using a fluoride ion selective electrode. The results presented in this study show that following a 6-hour dermal exposure to H-24616 applied to rat epidermal membranes mounted in an in vitro static diffusion cell system, receptor fluid total fluorine levels were found to be below the limit of detection (<0.052 ppm). Under the conditions of this study, H-24616 did not penetrate rat epidermal membranes. Company Sanitised. Does not contain TSCA CBI H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 INTRODUCTION H-24616 is an aqueous dispersion product containing a fluorinated active ingredient currently under investigation by the sponsor. The objective of this study was to determine the dermal penetration of H-24616, based on total fluorine, following a 6-hour exposure to a single, finite dermal application o f H-24616 to rat epidermal membranes using an in vitro static diffusion cell test system. MATERIALS AND METHODS A. Regulatory Test Guidelines This study was not designed to meet any specific regulatory guideline requirement. There are, however, guidelines on percutaneous absorption from the Organization for Economic Co-operation and Development (OECD) that provided information on the practical use of the in vitro dermal absorption m ethod/11 B. Test Substance The test material, H-24616, was an opaque liquid containing was the fluorinated active ingredient. Characterization of theTest substance was the responsibility of Regional Analytical Services (RAS), Jackson Laboratories, Deepwater, New Jersey. C. Test Species Female rats of the Sprague-Dawley strain, Crl:CD(SD)IGS BR, were obtained from Charles River Laboratories, Inc., Raleigh, North Carolina. Rats were approximately 28 days old at the time of sacrifice for the collection of skin. Dermal contamination is a potential route of human exposure. The rat was the species of choice for use in the in vitro test system as this species has been used in toxicological evaluations of H-24616. In vitro dermal techniques employing glass (static) diffusion cells (Figure 1) have been shown to predict percutaneous absorption of various chemicals in vivo. (2,3,4) D. Animal Husbandry Upon arrival at Haskell Laboratory, animals were removed fro>m shipping cartons and housed in appropriate cages, according to Standard Operating Procedure: less specified in the study records. Animals were maintained under quarantine for an ea st 6 da,yyss unless approved Company Sanffizecf. Doss not contain TSCA CB' -9- H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 otherwise by the site veterinarian, had 3 recorded weight gains, and no abnormalities detected. After the quarantine period, animals were selected for study. Animal rooms were targeted at a temperature of 23 1C and a relative humidity of 40-60%. Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. E. Animal Health Monitoring As specified in the Haskell Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity of the study: Water samples are analyzed for total bacterial counts, and the presence of 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. n Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Data are maintained separately from study records and may be included in the final report at the discretion of the study director. F. Preparation of Skin Membranes Following quarantine, rats were euthanized by carbon dioxide asphyxiation, and the fur from the dorsal region was carefully shaved using clippers. Any animals showing obvious abrasion within the region of the test skin area were considered unsuitable and discarded. The shaved area was excised, held briefly on wet ice, and frozen at <-10C until processed. Frozen samples of skin were allowed to thaw at room temperature before treatment with sodium bromide. Full thickness skin was soaked for approximately 18 hours in 1.5M sodium bromide. The skin sample was then removed, rinsed in distilled water, the epidermis carefully peeled from the dermis, floated onto an aluminum pan, and allowed to air-dry. Each epidermal membrane was identified using the Haskell animal number and stored refrigerated at Q-10C until readied for use. Company Sanitized. Doss not contain TSCA CB1 - 10- H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 G. In Vitro Diffusion Ceils Glass [static] diffusion cells (Figure 1) were used in this study. The in vitro cells had an exposure area of 0.64 cm2. H. Assessment of Membrane Integrity and Membrane Equilibration The integrity of each membrane was assessed by measurement of electrical resistance prior to application of test substance. Membranes were removed from refrigeration storage and hydrated in 0.9% saline for approximately 15 minutes. Following hydration, the membrane was mounted onto the top of the receptor chamber, which was filled with 0.9% saline. The donor chamber was then clamped in place and filled with 0.9% saline. The membrane was then allowed to equilibrate for approximately 30 minutes. During equilibration, the water-jacketed cells were maintained at approximately 32C using a recirculating water bath system. Following equilibration, a resistance measurement of each skin membrane was taken. Membranes with a resistance of >5.8 ki2 were considered intact and retained for use on study. Membranes not meeting this criteria were replaced, and electrical resistance confirmed following equilibration. This procedure was followed until a minimum of 16 skin preparations represented by at least 3 individual rats was achieved. Following electrical resistance measurement, saline in the donor chamber was removed and discarded. Saline in the receptor chamber was reduced to approximately half of the total volume, and cells with acceptable membranes maintained at approximately 32C overnight, without occlusion of the donor chamber, prior to dose application. The receptor chamber sampling arm remained occluded with Parafilm. I. In Vitro Penetration of H-24616 1. Experimental Groups This study was composed of the following groups. Group A B Designation Exposure group Negative controls Number of replicates 8 8 Exposed to H-24616 Deionized water 2. Pretreatment Procedures Following overnight equilibration, the contents of the receptor chamber were removed and 'discarded and refilled with 0.9% saline. Company Sanitized. Does not contain TSCA CBS H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 3. Application o f the Test Substance The test substance was applied to the skin surface of group A, via the donor chamber, as a single application at a rate of approximately 20 pUcm2 (i.e., 12.8 ph). Group B received a dose of deionized water at 20 juL/cm2 (i.e., 12.8 pL). The donor chambers remained unoccluded for the duration of the exposure period. 4. Exposure Period The exposure period for groups A and B was 6 hours. 5. Terminal Procedures At the end of the 6-hour exposure phase, the contents of the receptor chamber for each replicate in Groups A and B was removed and each placed separately into a glass container. No other samples were collected and skin samples were discarded. Samples of receptor fluid not immediately processed for analysis were stored refrigerated at 0-10C. 6. Positive Controls A set of 6 replicates was prepared by spiking deionized water with 12.8 pL of H-24616. These samples were used to determine average fluorine applied to the test skins (Group A). J. Total Fluorine Determination Receptor fluid samples (Groups A and B) and positive controls were combusted using a Wickbold Torch followed by analysis for total fluorine using a fluoride ion selective electrode. K. Data Presentation Calculated values in tables were computer generated and rounded appropriately for inclusion in the report. As a consequence, calculation of mean data will, in some instances, yield a minor variation from an aesthetic calculation. company Sanitized. Does not contain TSCA CB1 H-24616: In Vitro Dermal Penetration Through Rat Skin RESULTS AND DISCUSSION DuPont-8837 A. Positive Control Data (Table 1) The mean weight of H-24616'for the six replicates of 12.8 piL each was established to be 12.3 mg (%CV = 3.71). The mean amount of total fluorine (F) contained in each 12.8 ptL of H-24616, as determined by Wickbold Torch combustion, was 1233.6 ju.g F (82.4 ppm). B. Negative Control Group Data (Table 2) Total fluorine in receptor fluid from the negative control group, dosed with 12.8 /xL of deionized water to establish background fluorine levels associated with the test system, ranged from not detected (<LOD of 0.052 ppm) to 0.105 ppm. C. Exposure Group Data (Table 3) Following a 6-hour dermal exposure to H-24616, applied at a rate of 20 /xL/cm2 (12.8 /xL), total fluorine levels in receptor fluid samples were found to be below the limit of detection (<0.052 ppm). CONCLUSIONS The results presented in this study show that following a 6-hour dermal exposure to H-24616 applied to rat epidermal membranes mounted in an in vitro static diffusion cell system, receptor fluid fluorine levels were found to be below the limit of detection (<0.052 ppm). Under the conditions of this study, H-24616 did not penetrate rat epidermal membranes. RECORDS AND SAMPLE STORAGE Laboratory-specific or site-specific raw data, such as personnel files and equipment records will be retained by the facility where the work was done. Raw data and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. Characterization data (percent purity, composition, and known impurities) will be stored at Regional Analytical Services, Jackson Laboratories, Deepwater, New Jersey. Company Sanitized. Does not contain TSCA CBI - 13- H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 REFERENCES 1. OECD Guideline for the Testing of Chemicals (Draft Guideline 428), Skin Absorption: in vitro Method (December 2000). 2. Scott R.C., Batten P.L., Clowes H.M., Jones B.K. and Ramsey J.D. (1992). Further Validation of an In Vitro Method to Reduce the Need for In Vivo Studies for Measuring the Absorption of Chemicals through Rat Skin. Fundamental and Applied Toxicology 19,484492. 3. Ramsey J.D., Woollen B.H., Auton T.R. and Scott R.C. (1994). The Predictive Accuracy of In Vitro Measurements for Dermal Absorption of a Lipophilic Penetrant (Fluazifop-Butyl) through Rat and Human Skin. Fundamental and Applied Toxicology 23, 230-236. 4. Scott R.C., W alter M. and Dugard .P.H. (1986). A comparison of the in vitro permeability properties of human and some laboratory animal skins. International Journal o f Cosmetic Science 8, 189-194. Company Sanitfe*. D o no! contain TSCA CBI - 14- H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 TABLES Company Sanitized Doss re' eona'n TSC CB1 H-24616: In Vitro Dermal Penetration Through Rat Skin Table 1: Positive Control Data DuPont-8837 Replicate Weight of H-24616 (mg) Weight Aliquot weight of a of H-24616 1:10 dilution ofA plus water (A) for combustion (g) (g) ppm fluorine, A Gig F/g) Total H-24616 fluorine, A Percent fluorine OigF) (%F) 1 12.2 14.7909 0.5006 91.9 1359.3 11.1 2 12.3 14.9951 0.5005 73.7 1104.5 8.98 3 12.0 15.0190 0.5002 75.4 1132.1 9.44 4 13.0 15.0207 0.5013 87.4 1313.2 10.1 5 12.6 15.0192 0.5013 84.4 1267.4 10.1 6 11.7 15.0204 0.5007 81.5 1224.3 10.5 Mean SD %CV 12.3 0.46 3.71 14.9776 0.0920 0.61 0.5008 0.0004 0.09 82.4 1233.6 7.00 100.1 8.49 8.11 10.0 0.76 7.56 Company Sanitized. Does net contain T5CA CB1 H-24616: In Vitro Dermal Penetration Through Rat Skin Table 2: Negative Control Group Replicate B1 B2 B3 B4 B5 B6 B7 B8 Weight of receptor fluid (g) 5.0054 4.8118 4.7837 4.7907 5.1710 5.1669 5.1647 4.8344 Aliquot weight for combustion (g) 3.0009 3.0005 3.0010 3.0004 3.0003 3.0012 3.0006 3.0014 aLOD = 0.052 n g F/g (ppm); LOQ = 0.103 pig F/g (ppm) bNot detected; below the LOD DuPont-8837 ppm, fluorine receptor fluid (Hg F/g)a NDb ND ND 0.105 <0.103 <0.103 <0.103 ND Company Sanitized, Goss net contain TSC OBI H-24616: In Vitro Dermal Penetration Through Rat Skin Table 3: Exposure Group Data Replicate Al A2 A3 A4 A5 A6 A7 A8 Weight of receptor fluid (g) 4.8725 4.7954 5.1263 5.0250 5.0181 4.9800 5.1295 4.8289 Aliquot weight for combustion (g) 3.0013 3.0013 3.0006 3.0009 3.0007 3.0014 3.0005 3.0009 aLOD = 0.052 g F/g (ppm); LOQ = 0.103 g F/g (ppm) bNot detected; below the LOD DuPont-8837 ppm, fluorine receptor fluid OigF/g)' NDb ND ND ND ND ND ND ND Company Sanitized. Does not contain TSCA CB1 H-24616: In Vitro Dermal Penetration Through Rat Skin DuPont-8837 FIGURES Company Sanitized. Does net contain TSCA CB1 H-24616: In Vitro Dermal Penetration Through Rat Skin FIGURE 1 - STATIC DIFFUSION CELL DuPont-8837 donor compartment pinch clamp sampling port for analysis receptor compartment V______ J -mater jacket Company Sanitized. Doss net contain TSCA CBI -20-