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AR 226 _ 324.9 "1 - . TRADE SECRET Raa- 3249 pyesw 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 Saniizod. Doss not contain TSCA cat H24616: In Vitro Dermal Penetration Through Rat Skin Dupont8837 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT `This study was conducted in compliance with U.S. EPA TSCA (40 CER 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. Appli/ cSpaonnsotr: EL du Pont de Nemours and Company Wilmington, Delaware 19898 USA. -- Study Director: pide A `WillRiaemsJe.arFcahsaScnioe,ntis$tf, B.S. 26-Tul- 200 Date Applican/t Sponsor: DuPont Representative Date Company Sanitized. Does nt contain TSCA CBI -_-- HL24616: In Vitro Dermal Penetration Through Rat Skin QUALITY ASSURANCE STATEMENT Haskell Sample Number(s): 24616 Dates of Inspections: Protocol: Conduct: Records, Reports: March 11,2002 March 11,12 21, 2002 July 10-12,15-18, 2002 Dates Findings Reported to: Study Director: July 22,2002 Management: July 25, 2002 Reportedby: onda) KX. als `Wonda K(Kely Quality Assurance Auditor DuPont $837 4 ful Company Saniized. Docs not contain TSCA cB1 EE E -- 1.24616: In Vio DermalPenetration Thiough Rat Skin Duron 8837 CERTIFICATION `We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issuedby StudyDirector: We 2 or Wil ReseJa.rFcahsaScnioe,ntSirs.,t B.S. 26-Juco Approvedby: _`_M"aPotttehnec5hw.onBosgSda:n.tiy, Fh/8 ABT. Management 2se/aety l c2te0r0 ~ `Company Sanilized. Dozs not contain TSCA CB! _-- H-24616: In Vitro DermalPenetration ThroughRatSkin . DuPont8837 TAOFBCOL NTEE NTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT... QUALITY ASSURANCESTATEMENT wv A I CERTIFICATION. .....sssssssssssssssssssssssssssndd STUDY TPORMATION wcll STUDY PERSONNEL cemmsseimmmmmammmmmmmmmmmmmms--------------niell SUMMARY....... - --------------"3 INTRODUCTION. " A --------------------) MATERIALS AND METHODS........... ans---- nnd A. ReguTelstaGuitdeolinres.y.. ---- nnd B. TestSUbSIANCEvv ------ nnd C. TestSpecies.. wn ------ nnd D. Animal HUSA ---- nd E. AnimalHealth MOORING... " nn10 F. PAEDATAtiON OF SKIN MEMBIANES. vrs 10 Gu [0 Vitro DIRUSION CElIS sss LL H. Assessment of Membrane Integrity and Membrane EQUIlIDIGtON veer 11 1 TeV Pension cETACIE ummm ~ J, Tol EInrns DARI R mmmamemmmmm------ nil RB. DsPSD mmm mpm sell RESANUDDILSCUTSSISON cccnmmmmmmmsenammimmmmsmmmeiaannts re 3 A, PO CARDOESL wmmmmmmmmmammmmmm------------------"1b B: NeComg molCu rop ss t ammo C. ExposureGroup Data.... ------ nmi CONCLUSIONS. mn13 REACNDSO AMPR LESD TORSAGE... ' msi REFERENCES sss ES 1. PositiveControl Data ansr---------- 2. NegativeControl Group. - m--r 3. Exposure GroupData pms--------_8 BUTORE: copiers mm------------ 1. StaticDiffusionCell ssms---- - `Company Sanitized. Dos not contain Tsca car H24616: I Viro Dermal Pentrion Though Rt Sin rm STUDY INFORMATION Substance Testes: NID Synonyms/Codes: H-24616 n Haskell Number: 24616 " "I Dupo as Physical Characteristics: Opaque tan liquid Sponsor: E. I du Pont de Nemours and Company Wilmington, Delaware 19898 US.A. `Study Initiated/Completed: March 6, 2002/ (see report cover page) In-Life Initiated/Completed: March 11, 2002/ March 12, 2002 Company Saniizoc. Does not contain TSCA 0B: EE 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.ABT. Primary Technician: Brian P. Shertz, A.A. Analytical Associate: Richard F. Rossi, B.S. `Analytical Chemist: Vladimir Capk, Ph.D. Management: S. Mark Kennedy, Ph.D. Toxicology ReportMaPrneapgaermateinotn::LiNsaanGc.Sy.BuSreclhzfeire,ldM,.SA..A. DuPont 8837 --~ -- coPan Sanilzed. Doesnet contain sea ops E-- T 'H:24616: In Viro Dermal Penetration Through Rat Skin DuPont8837 SUMMARY The dermal penetration potential of H-24616 has been evaluated using rat skin mounted in an rin vitro static diffusion cell systeg, hFi-c2h4w6a1s6aipspalnieadquunedoiulsutseudsptoenrsaitoenpicdoenrmtaalinmieanmnbgranes at a Twaittehotfh2e0skpiln/efmo'r.6 hTouhres.tesAttmatheefefnad,ofattohtealeoxfpo1s2u3r3e.6pejriigoFdo,rth8e2.4recpepptmo,rrfleumiadiwnaeds irnecmoonvteadct 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 applied to rat epidermal mtheims bsrtaudnyesshmoowunthtaetdfionllaonwiinngviatr6o-hsotautricddeirfmfaulsieoxnpcoesllurseystoteHm-,2r4e6c1ep6tor cflouniddittiotoanlsfolfuotrhiinsestluevdeyl,sHw-e2r4e61f6oudniddtnoobtepebneeltorwatteheratliempiitdoefrmdeatlecmteimobnr(a<n0e.s0.52 ppm). Under the Company Sanitized. Doss not contain TSCA CBI ra H24616: In Vitro Derma Penstration Through Rat Skin -- INTRODUCTION Duponi$537 uHn.d2e4r61in6veisstaingaatqiuoneobuystdhiessppeorsnisoonr.prTohdeucotbcjeocnttiavienionfgtahifslsutouridnyawteadsatcotidveeteinrgmriendeietnhte cduerrrmeanltly dpeenremtarlataipopnliocfaHti-o2n4o6f16H,-2b4a6se1d6otno aottaelpfildueorrminael,mfeomllborwainnegsau6s-ihnoguranexipnovsiutrroesttoataicsidnigflfeu,sifionnitceell test system. MATERIALS AND METHODS A. Regulatory Test Guidelines h"Tohwiesvsetru,dyguwiadselniontesdeosnigpneerdcuttoamnceoeutsaanbysosrppetciifoincfrreogmultahteorOyrggauniidzelaitnieonrefqouriErecmoennot.micThere are, iCnov-iotpreoradteiromnalanadbsDoervpetiloonpmmeenttho(dO.E)CD) that provided information on the practical use of the B. Test Substance --_ `The was ttehset fmlautoerriinaalt,eHd-a2c4ti6v1e6,inwgraesdiaennto.paCqhuarealcitqeuriidczaotionn aofnthe test substance was the NesepwonsJeirbsielyi.ty of Regional Analytical Services (RAS), Jackson Laboratories, Deepwater, C. Test Species RFievmearleLarbaotrsaotofrtihees,SpIrnac.guRea-lDeiagwhl,eyNosrttrahinC,arCorlli:naC.D%R(aStDs)wIeGrSe BaRp,prwoexriemaotbetlayin2e8d dfaryosmoClhdaartltehse time of sacrifice for the collection of skin fDoerrumsaelicnotnhteamiinnvaittiroonteisstasypsotteenmtiaasltohuistsepoecfiheusmhaans beexpeonsuursee.d iTnhteoxriactowloagsictahleesvpaelcuiaetsioonfschoafice F-24616. In vitro dermal techniques employing glass (static) diffusion cells (Figure 1) have been shown to predict percutaneous absorption of various chemicals in vivo, 3% D. Animal Husbandry Upon arival at Haskell Laboratory, animals were removed from shipping cartons and housed in satpuprdoyprreicaotredsc.ageAsn,iamcaclosrdwienrgetmoaSitnatnadianredd Oupnedreartqiunagraontcince for AICaSt 6 nSayscunlesspsecaipfpierdoviendthe Company Sanitizod. Deas not contain TSCA C3 TT m------ 24616: In Vitro Dermal Penetration Through Rat Skin DuPont837 -- 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 analyzedfortotal 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 analyzedto ensure adequate sanitation by the cagewashers. ~~ Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. `The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. 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 0-10C until readied for use. `Company Sanitized. Doos not contain TSCA CBI -_-- H24616: In VitroDermalPenetration ThroughRatSkin -- G. In Vitro Diffusion Cells DuPont8837 Glass [static] diffusion cells (Figure 1) were used in this study. The in vitro cells had an exposure area of 0.64 cm'. H. Assessment of Membrane Integrity and Membrane Equilibration The integrityofeach membrane was assessed by measurement of electrical resistancepriorto 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 arecirculating water bath system. Following equilibration, a resistance measurement of each skin membrane was taken. Membranes witharesistance of 25.8 KE 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 least3 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 o 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. "Grow BA Designation NEexgpatoisvuerecognrtoruolps Number of replicaies 55 Exposed fo DeioHn2i4ze6d1w6ater 2. Pretreatment Procedures . Following overnight equilibration, the contents of the receptor chamber were removed and discarded and refilled with 0.9% saline. Company Sanitized. Docs not contain TSCA CBI -_-- H24616: In Vitro Dermal Penetration Through Rat Skin --~ 3. Applicationof the Test Substance DuPont.8837 "The test substance was appliedto the skin surface of group A, via the donor chamber, as a single application at a rate of approximately 20 uL/cm' (i.e., 12.8 uL). Group B received a dose of deionized water at 20 uL/em? (i.e., 12.8 uL). 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 LL 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 thereport. As a consequence, calculation of mean data will, in some instances, yield a minor variation from an aesthetic calculation. --~ ompany Sanitized. Does nol contain TSCA CBI wv H-24616: In Vitro Dermal Penetration Through Rat Skin ~ RESULTS AND DISCUSSION DuPont 8837 A. Positive Control Data (Table 1) `The mean weight ofH-24616for the six replicates of 12.8 uL each was established to be 12.3 mg (%CV = 3.71). The mean amount of total fluorine (F) contained in cach 12.8uL. of H-24616, as determined by Wickbold Torch combustion, was 1233.6 ug F (824 ppm). B. Negative Control Group Data (Table 2) Total fluorine in receptor fluid from the negative control group, dosed with 12.8 KL of deionized `water 10 establish background fluorine levels associated with the test system, ranged from not detected (LOD of 0.052 ppm) 10 0.105 ppm. C. Exposure Group Data (Table 3) Following a 6-hour dermal exposure to H-24616, applied at a rate of 20 wien" (12.8 KL), 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 limitofdetection (<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 Tron 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. Doos not contain TSCA CBI HL24616: In Vitro Dermal Pencration ThroughRatSkin DuPont853 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 HM., 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 andApplied Toxicology 19, 484 492. 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., Walter M. and Dugard .P.H. (1986). A comparison of the in vitro permeability `properties of human and some laboratory animal skins. International Journal of Cosmetic Science 8, 189-194. Company Sanitized. Doas net contain sc cay 5 H24616: InVioDermal PenetrationThroughRat Skin Dupont.8837 TABLES --_ Company Saniized. Doss nc? contain TSCA BY = --_-- -- H24616: InVitroDemslPenetThrroaugthRiatSoknin Tablel: Positive Control Data Dupon:8537 Weight ofWHe2i4gh6t16 AliLqiu0odtiwuetiigohntooffAa ppm Tol B24616 Replicate of H-(m2g4616 pluswater (A) forcombustion fluorineA, MuorineA,_PercentMuorine BEF) (gh) (%F) 1 22 147909 05006 919 13593 111 2 23 149951 05005 7 11045 898 3 120 15.0190 os002 754 1321 944 4 130 15.0207 os013 14 B12 101 5 126 150192 os013 44 12674 101 6 17 15.0204 05007 sis 12243 105 Men 123 149776 0.5008 sD 046 00520 00004 524 2336 100 700 1001 076 ~ av am [Xe 009 849 811 756 --_ `Company Sanitized. Does net contain TSCA CBY ee H24616: In Vitro Dermal Penetration Through Rat Skin --_ Table2: Negative Control Group TT Replicate Bi B 5 Be BS B6 B71 Bs reWceepmtowrfolurid I) soos asus 47837 007 S170 5.1660 sear 4304 Alfoqrucootmbwuesgtikotn 30009 3.0005 30010 3.0004 30003 30012 30006 30014 "NLoODde=t0e.c0t5e2d ubeglFoiwgt(hpepLm)O;DLOQ= 0.103 ig Fig (ppm) DuPont8837 preocmeptforufrlusid pg Fi) Np no wD 010s <0103 103 0103 ~ Company Sanitized. Doss n2t contain TSCA CBI Bg H-24616: InVitroDermalPenetration Through Rat Skin --~ Table3: Exposure Group Data Replicate al an a I as a6 a as `Weight of receptor fluid @ 48725 47954 5.1263 5.0250 s0181 49800 5.1095 48289 Aliquot weight for combustion 30013 30013 3.0006 30009 30007 30014 3.0005 30009 "LOD = 0.052 ug Fg (ppm); LOQ = 0.103 pg Fi (ppm) "Not detected: below the LOD Dupont8837 ppm, fluorine receptor fluid (ug Fle) ND* ND ND ND ND ND ND ND --~ Company Sanitized. Doss rot contain TSCA CBI Tse H24616: In Vitro Dermal Penetration Through Rat Skin DuPon-8837 FIGURES --_ -_-- Company Sanitized. Doss nct contain TSCA CBI HL24616: in VinDem PentrTonuighon Sin _ FIGURE 1 - STATIC DIFFUSION CELL Dupon 857 donor --| compartment pinch clamp ------ membrane sampli)n|g port for analysis recepptsor compPartment |e ~ ------e Company Sanftzed. Doss nc! contain TSCA CB eee