Document 7RM8yO7an5raMejBVQYVZEGZj

AR3RG. 4705 27 Arar. 2708 Duron 14365 `TRADE SECRET Study Title Hydrolytic Stability of H-25435 as a Functionof pH TEST GUIDELINES: OECD Guideline 111 AUTHOR: Bogdan Szostek, Ph.D. STUDY COMPLETOEND: October 15, 2004 "PERFORMING LABORATORY: E.I du Pont de Nemours and Company HaskellTM Laboratory for Health and Environmental Sciences -- Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 LABORATORY PROJECT ID: DuPont-14965 SPONSOR: E.L du Pont de Nemours and Company `Wilmington, Delaware 19898 USA. `WORK REQUEST NUMBER: 15028 SERVICE CODE NUMBER: 392 EE Cumin SeitzTTfeseNeooni TSCA CI1 -- Hydrol Stability of H-25435 ss a Fncion ofpt Dupont14965 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT "LTahbiosrsattuodryywParsacctoinceduScttaenddairndsc,omwphliicahncareewciotmhpUa.tSi.blEePwAitThSthCeAO(E4C0 DCFPRrinpcairptl7e9so2)fGGoooodd LJaabpoarnaGtooroydPLraabctoircaeto(rasyrPervaicsteidce1S9t97a)n,daErNdVs/(M11C/NCoHhESMan(9N8u)m1b7e,rO6E28C3D),, ePxacreip,t 1f9o98t,heanidteMmAFF documented below. The item listed does not impact the validoiftthye study. "`TAhlethtoeustghsutbhsetcahnacreacwtaesriczhaatriaocntweraiszendotbypetrhfeosrpmoendsuonrdperrioGrotoodthLeanbiotraattioornyoPfratcitsicestSudtya.ndards, the cchoanrsaicdteerreidzastuifofnicwiaenstdfoonrethbeypaunrpIoSsOes9o0ft0h0icsersttiufdiye.d laboratory, and the accuracy of the data is Applican/t Sponsor: EW.i.lmdiungPtoonnt,dDeelNaevmioaruers19a8n9d8Company USA. Study Director: b DO u SeBnoigodraRnesSezaorsicehk,ChPehmDi.st 1c$o-m0ecrT- 2004 Dae Applicant Sponsor: DuPont Representative _Da_e _ -------------- Compa-- ny Sa--iz.--Dross-- eNot C--omin--TSCA--CBI ------------ yl Silty oH2a5s F5oci5nof pt Dupon-14965 QUALITY ASSURANCE STATEMENT `Work Request Number: Study Code Number: 15028 392 `The conduct of this studyhasbeen subjected to periodic Quality Assurance inspections. The dates of inspection are indicated below. Pheasee Audited Conduct ReportRecords: Audit Dates 06 May 2004 09-12 Aug 2004 DirectboruanRe MeaneagSemaent 07 May 2004 24 Aug 2004 Reported by:Loe Ql Quality AssuraJnochensAtuodnitor 150d2mf Date Company Sti. Droek Conia TSCACPL TT Hye Subity ofH-2543 a Faction i Duponeaases CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluationof data obtained from this study. Appeored-- irs Ga Nie d 15-e0e200 LovedbyStayDistE or: Eb Sn----a Sen Rear Cams IS ecT20-m _-- Company Suis. DpsrNeetCom TSCACB. Hydrolytic StaobfiH2l54i35taya Functionof il DuPont14965 TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT crs QUALITY ASSURANCE STATEMENT evs CERTIFICATION cosmid LIST OF TABLES coms LIST OF FIGURES css] STUDY INFORMATION cvs SUMMARYmss INTRODUCTION cms sss d0 STUDYDESIGN crm10 MATERIALS AND METHODS css10 A. Materials ,-- -------- 21.. TResteSusb8s2itTBadAnicuneH.2a.54.l35 ----------------------------rconlndll --~ 3. Prepoafrbufafetrsoilutoionns. __.___.......... ------------ n2 Be W1. BPrSelimiirnawry1mst.smmmmmmmm--m-------------------- 122 23.. pAH nCaAolSf eUystsIsaubCistTaTsEnMcIeP.E.TE.AINE ! FE rere 212 45.. CAOnOaNSlo.fy..s2TiBAs. reer swernma--_ap0 RESULTS AND DISCUSSION ccm21 CONCLUSIONSwns32 RECORDS AND SAMPLE STORAGE oc 33 USY FIGURES commissions TT `CompanySunized.DAosRs NsotCoTs SCAaCBI. ---- Hydrol Stabilityof 25435 a aFncionof il. Duron 14965 LI OFTSABLTES Page 1. A(0N3A-LJUYNTEI-2C0A0L4)RAENSDUL5T(S08F-OJURNAE-Q2U00E4O.US STABI-LITY OF H-254r 35, pH-- 12, 37C, DAYS0 2s 2. A(1N3A-LMYAYT-I2C0A04L)RAENSDULT(S18F-OMARY-A2Q0U04E)O,US STABILITY OF H-25435,pH 40, 50C, DAYS0 2 3. A(NA0LY6TI-CAMLARAENYSDU5-LT(2S1-F0MOARY0-A2Q40U0)E8O)US STAcoBILITY OF H-254i3i57.,0, 50C, DAYS 0mmmnndl 4 A(NA1LY3TI-CAML RAAENSYDU5L-T(SI2BF-OM0ARY0A-2Q08U0E9))OUS Sc TABILITY OF H-2c 5435, pi 0, 50C,o DAYS0 38 5. OMETASUEREDDRCONCEmNTRsATmIONmS OmF 8m:2 TmBAmFO--Rp--i 12--, 37--C--, D--AYS--0--(3---J--UN--E---20A0N4D) 6. DMERASNUREEDECONCENTRATmIOmNSeOF8m.2TmBAmFOnR pnH 4m0, 5m0Cm, DAmYSm0 (--13---MAY---20--04)--AN--D 5--r 7. MAEASEUREAID CONCENTRArTImONmS OmF 8.m2 TrBA mFORmpHm7.0r, 50mC,eDA--YS --0(0--6-M--AY-2004) AND 5 3 8 (M1E8AMSAUY-R2E0D08C)ONCENTRATIONS OF 6:2 TBAFORpi 9.0, 50C, D0A(13-Y MAY-2S 004) AND 5 2 - 9. DAILYTEMPERATUREREADINGS OF THE SHAKING WATERBATH... 33 10. pH MEASUREMENFTOSR D0A ANDDY AY5 34 _-- CompanySeize.DocsNotConTsSCiACBnI. Ame Hydrolytic Stability ofH-25435 as a Functionof pH DuPont14965 LIST OF FIGURES Page 1. EXAMPLE CALIBRATION CURVES OBTAINED FOR MONITORED Jounnddl 2. BEXLAAMNPKLSEAMCPHLREOMFAORTOpGHR7.A0MBSUFOFFEMROONNITDOARYE0D. i. JOBTAINED FOR A n 3. CEAXLAIMBPRLAETICOHNRSOTMAANTDOAGRRDACMOSNOTFAIMNOINNIGT$.3ORmEgD/L H-25435. J OBTAINED FOR A 38 4. DEXAAYM0P, LpEH 7C.H0 RHOYDMRAOTLOYGTRIACMSSTAOBFILMIOTNYISTAMOPRLEE.D|.. JOBTAINED FOR 39 5. EDXAAYM5P, LpHE C7.H0 RHOYDMRAOTLOYGTRIACMSSTAOBFILMIOTNYISTAOMRPLEED{... J OBTAINED FOR 6. REPRESENTATIVE CALIBRATION CURVE FOR 8:2 TBA. c a 7. ARENPDR5E1S5EpNgT/ALTDI-8V-E2CTHBRAOINMTAETRONGARLASMTSANODFA1R0D4.ug/..L...82 TBA CALIiBRATION STANDARD a2 5. REPRESENTATIVE CHROMATOGRAMS FOR DAY 5, pH 7.0 BUFFER BLANK SAMPLE. ...........43 9. `RSEAPMRPELSEEANTNADT5I1V5EpCg/HLRDO-8M-A2TTOBAGRIANMTESRFNOARLDSATYAN5,DpAHRD7..0 HYc DROLYTICc STABILITY s ~ 10. `RSEAPMRPELSEEANTNADT1I5VEpCg/HLRDO-8M:A2TTOBAGRIANMTESRFNOARLDSATAYN0D,ApRHD7...0 .HYDROLYTIC STABILITY as Company Santized. DFoSss NotCanainTSCACBI. AR Hydrolyic Stability of25435 153 Funcion of pif DuPont-14965 STUDY INFORMATION `SubstanceTested: [ l Synonyms/Codes: ++ [H25435 ] 1 1 HaskellNumber: 25435 Composition: [ ] { 1 r ] r 1 [ 1 ( 1 KnownImpurities: None identified _ Physical Characteristics: Amberbrown liquid Stability: The test substance appeared to be stable under the conditionsofthe study; no evidenceofinstability was observed. Study Initiated/Completed: May 4, 2004/(see report cover page) IInn-Liitfieated/Completed: May 6, 2004/July 6, 2004 -_-- Company Suitzed. Docs NotConTtSCAiCBnI Re Hydrolyic Stability ofH-25435 as a FunctionofpH. DuPon-14965 SUMMARY The hydrolytic stability ofH-25435 was tested in buffered aqueous solutions at pH 4.0, 7.0, 9.0 at 50C and pH 1.2.at 37C for five days. [ representing major chemical species present in H-25435 were monitored during the hydrolytic stability study. Measured concentrations and recoveries of individual [ J reported as average concentrations and recoveries for Day 0 and Day 5 replicate samples, were used to evaluate the hydrolytic stability ofH-25435. The average percent recoveries for the Day 0 and Day 5, pH 1.2 replicate samples were 76: 5.9% and 63 + 28%, respectively. The average percent recoveries for the Day 0 and Day S, pH 4.0 buffer replicate samples were 97 3.0% and 95 44.4%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 7.0 replicate samples were 92: 1.1% and 90 + 0.9%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 9.0 replicate samples were 96 0.6% and 95 9.3%, respectively. `The H-25435 was demonstratedtobe hydrolytically stable at pH 9.0, pH 7.0, and pH 4.0. The data obtained for hydrolytic stability of H-25435 at pH 1.2 show the differenceofaverage recoveries for monitored [ between Day 0 and Day $ samples is larger than 10 percent. However, a precipitate was observed after additionofthe test substance to the pH 1.2 buffer. The precipitate was difficult to completely dissolve even after addition of acetonitrile and extended sonication, especially for the Day 5 samples. This lowered the: observed recoveries, especially for the bis esters. Therefore, the observed change in the average --- recoveries between the Day 0 and Day 5 samples is most likely because ofobserved solubility. problems rather than hydrolysis. `The measured concentration of 8-2 TBA did not indicate any hydrolytic degradationof H-25435 108-2 TBA for any of the investigated pHs. The observed concentrations of 8-2 TBA can be explained by the residual 8-2 TBA present in the test substance (H-25435). There was no significant increase of 8-2 TBA concentration observed between Day 0 and Day 5 samples for any of the investigated pHs. -- p ep p `Compaay Seized.DoAsRs2N2o6t ContiTSCACBI Hydrolytic SsbiltyofH-25435 as a Funcionof pH DuPont.14965 INTRODUCTION `The purpose of the study was to investigate the hydrolytic stability of H-25435 in pH 1.2, 4.0, 7.0, and 9.0 aqueous buffer solutions. The study was conducted according to the protocol: "Hydrolytic stability ofH-25435 as a functionofpH", which was pattemed after the OECD Guideline 111.0" Additionally, the concentration of the 8-2 TelomeBr Alcohol (8-2 TBA) was monitored to investigateif 8-2 TBA is released from the test substance during the test. STUDY DESIGN A preliminary test was performed on the test substance at 50 0.1C at eachof the pH 4.0, 7.0, 9.0.and at 37 0.1C for pH 1.2. Ifless than 10% ofthe test substance hydrolysis is observed. after 5 days, the test substance was considered hydrolytically stable and no additional testing was. performed. In addition to the test substance determination during the preliminary test, the: samples were analyzed for 8-2 TBA. MATERIALS AND METHODS A. Materials 1. Name: Test Substance Hasiss Composition: ( ( 11 ({ 1 1 ( 1 Haskell number: t ) 25035 2. Residual 8-2 TBAin H-25435 Residual 8-2 TBA content in the test substance (H-25435)was determined by LC/MS using the same analytical method as described in Analysis of 8-2 TBA in Methods. Five replicate samples of H-25435 (approximately 45 mg) were introduced to glass vials, weighed, 10 mL ofTHF (tetrahydrofurane) was added, vials crimped, and sonicated for 1.5 hours.A 0.1 mL aliquot of dissolved sample was transferred to a glass LC vial and 0.9 mL acetonitrile and intemal standard - (raDn-g8e-2fTroBmA)10w0atsoa1d0d2e0d0. nCga/limbLra8t-i2onTBsAta.ndards were made in 10/90% THF/acetonitrile in the Company Saniized. DAoRczsN2o6tContinTSCACBI. 3. Preparationofbuffer solutions "The following buffer solutions were prepared as described in the Annex to OECD guideline 111.0 a pHI2 "The 0.2 M potassium chloride (KCI) was prepared by dissolving 3.73 g of KCI (purity 99.0%, Sigma) in 250 mLof water. The 0.2 M hydrochloric acid (HCI) was prepared by diluting 4.5 mL. `of hydrochloric acid (36.5-38%, Sigma) in 250 mL water. The pH 1.2 buffer solution was prepared by placing 125 mL of 0.2 MKCland [ ] mL ofa0.2 M HCI solution into a 500-mL ask and bringing the flask to volume with water. The pHofthe buffer was adjusted to 1.2 with sodium hydroxide. bo pH4 "The 0.1 M solution ofpotassium hydrogen phihalate was prepared by dissolving 5.1 g of potassium hydrogen phthalate (purity 99-+%, Sigma) in 250 mLof water. The pH 4.0 buffer solutionwas prepared by placin2gmL of2 0.1 M sodium hydroxide solution and 250 mLof a 0.1 M potassium hydrogen phthalate solution into a 500-mL flask and bringing the flask to volume with water. The pHofthe buffer was adjusted to 4.0 with hydrochloric acid. e pHT: - `Tphheos0p.h1atMe, smoolnuotiboansoifcp(oKtHazsPsOisu,mppuhroistpyh9a9t.e5w%a,sSpirgempaa)reidn 2by50dimssLoolfviwnagt3e.r4.gTohfepoptHas7sbiuufmfer Solutionwas prepared by placing 148 mLof a 0.1 M sodium hydroxide solution and 250 mL of the 0.1 M potassium phosphate solution into a 500-mL flask and bringing the flak to volume with water. The pH of the buffer was adjusted to 7.0 with hydrochloric acid a pH: "The 0.1 M boric acid (HBO) solutionin 0.1 M potassium chloride (KCI) waspreparedby dissolving 1.90 g of KCI (purity 99.0%, Sigma) and 1.55 g ofHyBO (purity 99.5%, Sigma) in 250 mLof water. The pH 9.0 buffer solution was prepared by placing 250mL of a 0.1 M H;BOs solution in 0.1 MKCl and 106.5 mL oaf 0.1 M sodium hydroxide solution into a 500-mL flask and bringing the flask to volume with water. The pH of the buffer was adjusted t0 9.0 with hydrochloric acid. `Water used for buffer preparation was obtained from a Banstead NANOpure Diamond water purification system. The war resistivity was > 18.2 megaohmeem. All buffer solutions were sterilized prior to use by filtering through Corning Sterilization Filter systems with 0.22-pm cellulose acetate filter. The buffer solutions were stored at room temperature. e Company Saiz. Dosse Nt Cons TSCA CBI ARz26 Hydrolyic Stability ofH-25435 as a Functionof pit DuPont 14965 4. Equipment pWHatMeerteBra:th OPrreicoinsiMoondReelci2p5r0ocAal Shaking Water Bath, Model 50, Precision AAuuttoocmlaatviec: Pipers: AReMscSaCrcOh MPorode1l003,0210300, 5000; Eppendorf CBoanltainfcuege: MSeotrtvlaelrLAeEg-e1n0d0RT VoGrltaesxserVi:als: M1u0lmi-Ltbuobreosviolritceaxteer,SeVrWuRm.Type Reaction vials (Supelco) with 20 mumBari Septa (3F0omiilnSibleifcoornees),e0.1" thick (Supelco). Thevialsandsepta were autoclaved at 121C for B. Methods 1. Preliminary test `The preliminary test was performed on H-25435 at 50C at each of the pH 4.0, 7.0, 9.0 and at 37C for pH 1.2. The same sample preparation procedure and study design was followed for ach of the pHs tested. Approximately 45mgof the H-25435 was weighed into sixteen 10-mL. autoclaved glass vials and 3 mL of appropriate buffer solution was added. The vials were crimp capped with the aluminum foil covering the partof the septa facing the inside of the vial. Eight ofthe vials were processed foranalysisas the Day 0 samples. The remaining eight sampleswere wrapped with the aluminum foil, submerged in the water bath, and shaken at 100 rpm in the -- water bath for five days at the appropriate temperature. Eight vials containing only 3 mL of appropriate buffer were prepared to serve as the blanks. Four of them were used as the Day 0 blanks and the other four were wrapped in aluminum foil and placedinthe water bath to be the Day 5 blanks. Additionally, six vials containing 3 mLof appropriate buffer were spiked with 8-2 TBA stock solution (4.5 uL of 1040 mg/L 8-2 TBA). The vials were crimp capped with the aluminum foil covering thepartof the septa facing the inside of the vial. Threeofthe vials were: used as the QC samples for 8-2 TBA analysis on Day 0. The other three vials were wrapped in aluminum foil and placed in the water bath together with eight ofthe test substance vials. These serveda role of QC samples for 8-2 TBA analysis on Day 5. Fourofthe Day 0 or fourofthe. Day 5vials and two blank vials were processed for the H-25435 determination. The other four ofthe Day 0 or Day 5 vials, two blanks vials, and 8-2 TBA QC vials were processed for the 8-2 TBA determination. 2. pH measurement and temperature `The pH was measured on Day 0 and Day $ in the vials containing the bufferandthe test siunbtsrtoadnuccee,d dteostihgenavtiaeldsfaonrdthteheH-p2H54re3a5diannaglwysaiss.taTkehne. vTiahlessweevriealusnwceaprpeefdu,rtthheerppHroecleescsterdodfeorwas. H-25435 analysis. The temperatureofthe water bath was monitored daily. The water bath has. an automatic temperature control built in. The readingsofthe temperature control were checked and recorded daily. 3. Analysis oftest substance H-25435 is amixture of[ - present in H-25435 - Company Sniize. DAocseNotContainTSCA CBI. 1 The 1 10 Hydrolytic Stabilityof H-25435 as a Function ofpH. { DuPont14965 1 -_-- Company Seaitized. DpocrsNeotContain TSCA CBI Hydrolytc Stability of H-25435 as Function ofpH 1 DuPont14965 1 Company Santaed. DoEScs NotConiaTSCA CBI. pre Hydrolytic Stability ofH-25435 as a Function ofpH. [ DuPont 14965 1 -- --Company Snized. DoesNotContain TSCA CBI ARs Hydrolyic Stabilityof H-25435as a Functionof pH ( DuPont-14965 1 `Company Sanized. DoseseNot ConanTSCA CB ren Hydrolytic Stability of25435 a5 a Functionofpit DuPont.14965 The test substance H-25435 has not been characterized to determine the actual contentsofthe. cchheemmiiccaall ssppeecciieess tthhaatt wweerree mmoonniittoorreedd dduurriinngg tthhee ssttuuddyy. weTrhee rceopnrceesnetnrtaetdioansstohfeitnodtailvidual concentration of the H-25435. Vials designated for H-25435 analysis were uncapped, 6 mL of acetonitrile was added, vials waceerteonrietcrailpep:ewdataenrdbevfoorrteexaenadlyfsoirs.30 Tmhineutbelsa.nkTshaemopblteasiwneerdeexsturbajcetctweadstdoitlhuetesd1a:m1e0p0r0ocweidtuhre2,:1 except the pH measurement. "The stock solutionof H-25435 was made by dissolving 0.01 g of H-25435 in 10 mL of2 mL. isopropanol:8 mL water. Calibration standards for the H-25435 analysis were made by appropriate dilutionofthe stock. The calibration standards ranged from 1 10 10 mg/L of H-25435. Fresh calibration standards were prepared for each instanceofanalysis. Linearor quadratic regression of the peak area vs. standard concentration was used to establish the: calibration curves. _-- Company Sania.Dos NotContTSaCAiCBnI AR26 Hydrolytc StabilityofH-25435 ss Function ofpH DuPont 14965 Instrumentationand conditions: HMPSLICnsItnrsutmreunmtent MQoudaetlroAgMiilcernot, 1W1a0t0e,rsAgilent CLoClPuamrnam:eters: Mobile Phase: XterraAeMS0.C1158%,(3v0vm)macexti2c.1acmkmd/,102.mMumtriethylamine in water GradiBc-at:0.15% (vi) aceticacid/10mM ricthylamine in methanol Tim0e0(min) %Bs0 Flow (0m2m5in) 2800 110000 002255 81101 110000 003500 110520 5500 002255 CInojelcutimonTVemopleuramt:ure: souolc. MToSniPzaartiaomnetmeordse:: Electrospray, negative ions CCaopniellvaorlyavgoel:tage: 232v1v _ SDoeusroclevaTteomnpeTreamtpuerrea:ture: ~~ 415200CC Data Acquistion Function: CC68-:P mmoonnoo eestteerr fioonn:: SSIIRRooff#$4433 mmizz;; 00--1122 mmiinn C(C1o0--PP-mIoPAnomiesxteedreiosnt:SonI:R oSfI6R4o3f4m8iz5;m0z-1;20m1i2n min ((CCIB0P-PI-PIAPAmmiixxeeddsesteriojno:n:SISRIoRfof568855mmzi;z;0-01-212mimnin (CPCC6B-bbiiPssee-ssteeCtrfiEoonn::SSIIRRooff788899mmiizz:;00.-1122 mmiinn `CC1B0--PP--CCB1I0CEb-isP-sCtIeO biions:esIteRr ioofn:10S8R9 wozf;9809-1m2izm;i0n-12 min. 4. Analysis of 8-2 TBA Samples designated for 8-2 TBA analysis were processed by injecting 3 mL methyl-tertbutyl ether (TBE) through the vial septum using a glass syringe. The pH 1.2 samples required neutralization before the extraction. Neutralization was done by injecting 0.27 mL of IN sodium hydroxide to the sample vial before the MTBE addition. Neither Day 0 nor Day 5 samples were uncapped before MTBE injection to avoid any losses of 8-2 TBA. The vials containing injected MTBE were vortexed for 20 or 30 minutes on a multitube vortexer, centrifuged for 20 minutes at 4150 rpm at room temperature. Occasionally, the MTBE and aqueous layers would not separate. even after centrifugation. In these cases, samples were be placed in a freezer at -20C for overnight storage, removed from the freezer, allowed to come to room temperature and centrifuged for 20 minutes. Subsequently, the sample vials were uncapped and 0.1 mL of the `Company Sized. DTosisNo ContainTSCA CBI. AR26 Hydrol Stability ofH-25435 3s a Functionofpt DuPont.14965 MTBE extract was transferred toa glass LC vial, 0.9 mL acetonitrile added, the extract was spiked with internal standard, crimped capped, and subjected to LC/MS analysis for 8-2 TBA. `uTsheed 1aHs,th1eH,an2aHly,ti2cHa-lpesrtfalnudoarroddoefca8n--21T-oBlA.(97T.h6e%,1OD,ak1wD,oo2dD,Pr2oDd,uc3t-s1,3CW-ehsetptCaodleucmabfilau,orSoCd)ecwaansol (M+5) (abbreviated as D-8-2 TBA) was used as the internal standard. Stock solutions (w1e0r0e0smtogr/eLd)reoffritgheeraatneadl.ytiTchael sctaalinbdraartdioanndsttahnedairndtesrnwaelrsetparnedpaarrdewdefrreesphrleypfaorreedaicnhmceatlhiabnraotlioanndin 10/90% MTBEacetonitile. The calibration standards were made in the range from 100 to 10,000 pg/L 8-2 TBA. A constant levelof internal standard was used: 515 ug/L D-8-2 TBA. `The calibration curves were constructed using the ratio of the peak area for ions m/z monitored for 8-2 TBA and peak area for ions monitored for D-8-2 TBA and the concentrations of 82TBA. Company Saized. DTochsNotCons TSCACBI pre Hydrol StabilityofH-25435 ss a Funcion ofp - instrumentation and conditions Dupont14965 MHSPLlCnsItnustmreunmtent 7Mo0d,eWlat2e7r95s, Waters CoLlCuPmarnameters: Mobile Phase: CInojelcmtiaonTVeomtpeurmaet:ure: Column Switch AtewraaeMrS CI,30mmx 2.1mm, 2.5ym BGr-aTdmiieetmnheta:(nmoiln) un us o2v EFfoy 10000 ai 0 Iro} 3200C4 5 0 wo Zand min Flow 0(2ms m) 0023ss 0023ss 02s MToSniPzaartaomnetmoedres: Blctospy, negative ons CCSoaonpeiulvroTyleumvgpoele:traagteu:res 2o125v0kC - DDeastoalAvcaqtuiiosniTtieomnpFuenrctsio:n: $D302-8TC2HAT:BAS:ISRoIfR4o6f34,657.23280.6mmiisn0.6 min 5. Calculations "mTohneitfoolrleodwfionrgHc-a2lc5u4l3at5io(nTsabwleerse1u-4s)e.d tFoordeetxearmmpilnee,tihfe re0.c0o45vegroifesHo.f2i5n4d3i5viwduaaslwcehiegmihceadlisnpfoectihees veiaaclhoanfdt3hemcLheomficbauflfseprewcaiessawdadse:d(t0o.0t4h5e vga*l1t0h0e0nmogm/ign)al/0c.o0n0c3enLtr=ati1o5n,0t0h0atmwga/sL.asTshigenendomtional wcoanscecnatlrcautliaotendubsyeddifovridriencgotvheerymecaalscuurleatdicoonnwcaesntarantaivoenrbaygetohfefnooumrinraeplliccoantcese.ntrTahtieon%,rteicmoevsery 100%. "fTohleloewxsp:ecttheed4(.5noimiLnaolf)1c0o4n0cemngt/rLat8i-o2n oTfB8A-2sTpiBkAe rienptrheessepnitksed4.5 sLampxle1s04w0asngc/aplLc=ul4a6t8ae0dsng o8-f28.T2BAT.BAThiinstahemoMuTnBtEwaesxterxatcrtaicst:e4d6w8i0thng3/m3 LmLo=f1M5T6B0En.g/TmhLe. eT xpec%th erdecnooe vmeirnayliscocnaclecnutlraatteidon by dividing the measured concentration by the nominal concentration, times 100%. d"TehreivHe.d25fr4o3m5 rceosnitdauianls8r-e2siTdBuaAli8n-t2hTeBAM.TBTEheexetxrpaecctt3ed,mLm)a,xaismsuummicnogncceonmtprlaettioeneoxftr8a-c2tiTonBAfoar 4(159m,4g00saumgp/Ll)e.ofH-25435 is: 45 mg of H-25435 x (0.129%/100)/0.003 L= 19.4 mg/L S-- e---- Company Sai-- . DaogeeNAo--t C--on-- ti--nT--SCA--CB--. ---- ------------ Hydrolytic Stbilty ofH-25435 as a FunctionofpH DuPon-14965 RESULTS AND DISCUSSION TJambelaessur1e-4dpfroersHen-t25th4e35resaunldtsafvoerramgeeascuonrceedntcroantcieonntsraatnidonrseaconvderrieecsovfeorrieDsayof0inadnidviDdauayl5[ replicate ScaalmcpulleastefdoraspaHn 1.a2, v4e.r0ag,e7o.f0,co9n.c0,enrtersapteicotnisvealny.d rTehceovaevrieersagoefconcentration and recoveries w1 ere `monitored for H-25435. The average percent recoveries for the Day 0 and Day 5, pH 1.2 replicate samples were 76: 5.9% and 63 + 28%, respectively. The average percent recoveries for the Day 0 and Day 5, pH 4.0 buffer replicate samples were 97 + 3.0% and 95 + 4.4%, rweesrpeec9ti2v4ely1..1T%ahnedav9e0rage0.p9e%r,cernetspreecctoivveerliye.s TfohretaheveDraagye0pearncdenDtaryec$,ovpeHrie7s.0forreptlhiecaDtaeysa0mapnldes Day 5, pH 9.0 replicate samples were 96:= 0.6% and 95 + 9.3%, respectively. The difference of aavnedrpagHe9r.e0cobvufefreiress. beTthwiseeinndtihceatDeasyth5atatnhdeDHa-y25043sa5mipslheysdwroalsytliecsaslltyhasnta1bl0e%atfotrhepsHesp4H.s0, pH 7.0, (12 >one year). Similarly, the difference in recoveries 1 for Day 0 and Day 5 samples at pH 4.0, pH 7.0, and pH 9.0 buffers were less than 10%, except the C10-P-C10 bis ester at pH 4.0 and pH 9.0 buffer. The average recoveries for Day 0 and Day 5 samplesofthe pH 1.2 bufferdifferby more than 10%. However, in case of pH 1.2 buffer a precipitate was observed after additionof the test substance to the buffer. The precipitate was difficult to completely dissolve even after addition of acetonitrile and extended sonication, especially for the Day 5 samples. This lowered the observed recoveries, especially for the bis - esters. Therefore, the observed change in the average recoveries between the Day0 and Day 5 samples is most likely because of observed solubility problems rather than hydrolysis. Figure 1 shows example calibration curves for [ J monitored during the hydrolytic stability study. Figures 2-5 show example chromatograms obtained for the monitored [ 1 fora pH 7.0, Day 0 blank sample, calibration standard containing 5.3 mg/L. H-25435, pH 7.0, Day 0 test sample, and pH 7.0, Day 5 test sample. The contentofresidual 8-2 TBA in H-25435 was measured using the procedure described in Section A.2. Based on the resultsoffive replicatesofthe H-25435, the content ofresidual 82 TBA in H-25435 was calculated to be 0.129+0.0048%. Assuming that the buffer extracts all the residual 8-2 TBA from the test substance during the time of hydrolytic stability experiment, t(hseeeex5pCeacltceudlactoinocnse)n.traTtaibolneosf48--82prTeBseAntinthtehemeteasstusryesdtecmonwcoenutlrdatbieon1s9.o4f m8-g2/LTB(1A9,f4o0r0Dpagy/L)0.and Day5 replicate samples for pH 1.2, 4.0, 7.0, 9.0, respectively. The concentrationsof 8-2 TBA `measured for the Day 0 samples are slightly higher than the expected concentrationof8-2 TBA originating from residual 8-2 TBA. This may be due to different extraction methods used for determinationofresidual 8-2 TBA in the test substance and the method used for analysis of hydrolytic stability samples. The Day 5 concentrationsof8-2 TBA are comparable with these observed for Day 0 samples. The observed differences in concentrationof8-2 TBA between the Day 0 and Day $ samples for pH 7.0, 9.0 buffersare in the range of error for the analytical `method used (+15%). A decreaseof 8-2 TBA concentration between Day 0.and Day 5 may be --- e8x-p2lTaiBnAedcboyncaednstorrapttiioonnsobfet8w-2eeTnBDAayon0tahnedprDeacyipi$taftoer pobHse4r.v0esdafmoprlethsisisplHa.rgTerhethdainfcfaernebnec.e of Company Saiized. Doi csNotConia TSCA CBI Ame Hydrolytic Stability ofH-25435 as a Function ofpH. Dupont. 14965 explained by the analytical method error (Table 6). However, the observed QC sample recoveries for Day 5 samples indicate higher than expected bias of the analytical method for this Set of samples. Thus, the increase of 8-2 TBA concentration for Day 5 samples is most likely due to analytical method performance rather than the test substance hydrolysis. Figure 6 shows a representative calibration curve obtained for 8-2 TBA. Figures 7-10 show. chromatograms for 8-2 TBA calibration standard; Day 5, pH 7.0 blank sample; Day 5, pH 7.0 test sample; and Day 0, pH 7.0 test sample, respectively. `Table 9 summarizes the water bath temperature recording for pH 1.2, 4.0, 7.0, and 9.0. In all cases, the water bath temperature was maintained at the specified level with deviation not exceeding + 0.1C. Table 10 summarizes results of the pH measurements for pH 1.2, 4.0, 7.0, and 9.0. The measured pHof Day 0 and Day 5 replicate samples for allofthe investigated pHs was at the specified nominal level with the deviation not exceeding 0.1. CONCLUSIONS The hydrolytic stability ofH-25435 was tested in buffered aqueous solutions at pH 4.0, 7.0, 9.0.21 50C and pH 1.2 at 37C for five days. The H-25435 was demonstrated to be -~ hydrolytically stable at pH 9.0, pH 7.0,and pH 4.0 with ty >one year. The data obtained for hydrolytic stabilityof H-25435 at pH 1.2 show the differenceof average recoveries for `monitored [ J between Day 0 and Day 5 samples is larger than 10 percent. However, in case of pH 1.2 buffer a precipitate was observed after additionofthe test substance. to the buffer. The precipitate was difficult to completely dissolve even after addition of acetonitrileandextended sonication, especially for the Day 5 samples. This lowered the observed recoveries, especially for the [ ) Therefore, the observed change in the average recoveries between the Day 0 and Day 5 samples is most likely becauseofobserved solubility problems rather than hydrolysis. `The measured concentration of 8-2 TBA did not indicate any hydrolytic degradationof H-25435 108-2 TBA for anyofthe investigated pHs. The observed concentrations of 8-2 TBA can be explained by the residual 8-2 TBA present in the test substance (H-25435). There was no significant increase of 8-2 TBA concentration observed between Day 0 and Day 5 samples for anyofthe investigated pHs. = -_-- Company Saniized. Docs NtContinTSCA CBI. ARz26 Hydrolytic Stability ofH-25435 as a Functionofpil DuPont14965 RECORDS AND SAMPLE STORAGE `Specimens(ifapplicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. REFERENCES 1. Organisation for Economic Cooperation and Development (OECD). May 12, 1981. OECD Guidelines for Testing of Chemicals, Hydrolysis as a Functionof pH, OECD Guideline No. 111. EE `Company Saiized. Dpoers NeotContainTSCACBI Hydrolytic Stabilityof H-25435 as a FunctoifopHn. DuPont-14965 TABLES -_-- Company Seized. Docs NotContainTSCA CBI Anz TiBg [BL] | ee] Hf Je] re oe 3ILE] ol] el 3id[He] fe] Jo 3H A | CA PEE | 2 H[ El fe | : [PP Ee fgP| Jf [IPE BL Ee | IF [LR ie ie P| 3 401% [[3EEReeE] el] [PFeE] ee| i dF [EErE(ell EERE el| IER EEIEEEERNMHAEREE : -# & BE Bg L(A el] Ee [ee 3 Fl PL ele] ol 3) Pl] Pe ee gAREI=|IC1E8 EC8RCER" RECEEN Eg RE 3i [BL] el] el ||: 8< L==HLi ] euil] ee =* || {* 3 --- 32i$0cH]] fl ol Pl fo Bl | 13g |i | 1 FH [|[fee ole B|l |5 I SEINCECNCENEEEERERs I WEINCCECRERRNCCEENERE oF03 E EEEREE(TT|al)eld) FEREEREER (rel 8g LAER EERE 2& TtBg [I [Fe 2 ef 3) Eel es 2 3 LL] fl] ef ef EBg I=LE] El | | &[Al [EP P| |5 2 I Be | ee BE EI 3 [LE PL Ff PL 8 I QE ECEER EEN EEEN Cet < 3 1& 2 [31 [EE] ee oo 5% [El EaRE 30 F a8g3 ERR (elf [AD[ElE=] 3 EEE FLlaEleA (held 3 |& TgBg 1El] 1 120d 3] Pl PE eee [ee 3 |e AREICCCCRCEENEEEEREER 3{7H ol] || 8H]a> g fe 3 |g 8 o|go] eg2 |]3 2 TE ofl] oo] ee ef $1140 PE PLE EE EI 1 feed 2 J Ib EJ | PeJo jAE LE ee] 3 8][-]-]-[+] [-[-T] 3 Eee [I-11] i dg Lol ERRE ~- i i Ra iSara -- lm `Table 5: M5e(a0s8u-rJeund-c2o0n0c4e)n.trationsof8-2 TBA for pH 1.2, 37C, Days 0 (03-June-2004) and coma| gm FPC er TE wT ] |-- 1 ET ----] C--r E--t re e--e a a ] FCiieeTTaear o ne----T --m--T] --w] ~ T = --rTTT ] me--]-- CCCEee T Tw e a] ] EC S rT --T ---- ] -- | C T rTrr Toe e e ae] ] Fa E e rTo so a ] EC E TT -- T-- e-- T ---- cesi 5cinc Hydeolyio Sublliy of H-25433saFunctionotpll _____ DPontid%6s `Table 6: Measured concentrationsof8-2 TBA for pH 4.0, 50C, Days 0 (13-May-2004) and CoT ]ee n [ m| 5 (18-May-2004). rr 1 [oo TT ww 1 [eo Tom aw 11 [oe Tos 1 awe 177 [eo ToleTwa7} TT CT1 wee Tm | [ ovis|| ow | 1 1 1 [oo Todeerl so Te|w ws | [eo Toleal woTew "7 | [oo[oeo] weTw Tw | rrr rT - [These we | LT sebwioeisin | 20 | rr rT [ooTs 1 wm 1 1 [ooTso 1 ow |] [oo Tw [os wm e rrr rT LT hee| wT w [ omtinoiien || rT [eo TsToa] weTo[o ww | [ooTsfea Two 1a0 [7m | 1 [IT 7 Awe[wr | LTTsoT wie [55] EE Table7: M5e(a1s1u-rMaeyd-c2o0n0c4e)n.trationsof8-2 TBA for pH 7.0, 50C, Days 0 (06-May-2004) and LL=] rTTT [oe Tol1mm [roTo = TT ow || ee ST Taw [oe oT om 1 TTT EE E T --] T1711 [oo eerTwo | ww | we | [oo ea Two | we | wm | [oe ToesTwo Tw | wm | TTT Teme | | CC Sootiponmn | 10 | rr rT [ooTs mew 1 = Tw| oe ss| [oo sm 1 aw | [ 1 rT Es ET 111 [oe [ser we | ww | ww | [0[51ea | we | we | | [oe[se| we | ww | wn | rr 1 [TaT w | ww] meets CT somttomiin|5] me EEE T 2 e eS ------------ Table 8: LL es Measured concentrationsof8-2 TBA for pH 9.0, 50C, Days 0 (13-May-2004) and 5 (18-May-2004). hi + Concentration (ug/L) [r oo Lo r Tr 1 [ooJo em [ooLolo Pm foofol TT rrr 1 TE |=1] | 1 TT r T [oo [oTeaTm oeTw1o wr| [oo JoleaPoe1 we 1 ww | [ooJoleo mseTw Tw | - TTT TT ew |ow | FT somtimes [50 | rr 1 Foo sem [ooIs = Tm [ooTs TT ew es sm rrr 1 eosmerTew m 11 --TT [oo Tea Twa1 wo 1 | --------- [ooIs[eo ae Tw To w | rrr C1 eww | CT somites | 56 | = Hydrol Subilyof 25435asaFunctionofpH Dubon1d96s Table 9: Daily temperature readings of the shaking water bath. [mom |woT w [em] 11 Ee omen [m0 | e oT e oommeenn || 00 || Fe weno [0 | ee omer[0] se omen [0| ------ rT Fe so wma [00 | ow oman[00 | [w omao n | m0 sw emo| 0 | Fe mao[00 | ~ Fs man[a0 | ---- 7 1 [om Fo oman | 0 | oman| 00 | Fe oman| + Tw oman || FT sT T oe mmaann|[000 || [1 1 eTe oma | 00 | oe man | 00 | ee oman | so | [ToToman | so | C[ sT To we maannT | | ssooo [E-- TT `CompanySized.Does NoConTtSCiACrnB. ------------ AR2 HydrolytcStabilityofH-25435 as a Functionofpi DuPont14965 `Table 10: pH measurements for Day 0 and Day 5. [-- -- onThT e |Tm om-- a7nT|seamrapn] ee |ee [e e e 1m e e a ee |Tee e r e T e se e s a e e ae -- 71 e Taa e] eT ae | [rT Tae| ---~ Tw| sm Taw | ss Tam -- v --| ------ e rT 1Tw | 1 me Te | 15 me Te | Tw | [Te T r e T ow s s se ee| -- T -- r e r 1 | eT Se on e T m e ]| ee ow s (----1 e 1 1om Te om Cs Toa CTTe | ow | A ------ `CompanyS-- anitzed.Doese -- NotCoe nTtSa-- Ce AiCBn.e-- ------ -- -- AR226 Hydrolytic StabilityofH-25435 1s a Function of ptt DuPont14965 - FIGURES _-- Eo `Company Sanitzed. DAoeRs Nsot ConsinTSCA CB. Hydrolytic Stability ofH-25435 as a Functionof pit Figure 1: Example calibration curves obtained for monitored] DuPont.14965 I -- Company Sized. Docs oTtConTsin TSCA CBI ARz2 A ------ Figure 2: Example chromatograms of monitored | sample for pH 7.0 buffer on Day 0. EE = EE owrasos Jobtained for a blank ---- ee 2 mH FE fi Ce EX Compa Sen cotToon Hydrolyic ability of25435 ssa Functionofpit Figure 3: cEaxlaimbrpalteiocnhsrtoamnadtaorgdrcaomnstaoifnmiongni5t.o3rmegd/L|. H-25435. Dupont14965 obtained for a Er ue Az o BE Al mg) N --_-- CompanySniied.DoesNotConTSCtACaB. ae Hydrol SabiltyofH25435 as o Functionofpif Figure 4: Ehyxdarmopllyteicchsrtaobmilaittoygsraammpsleo.f monitore|d EE Te EE Dupont. 14965 Iobtained for Day 0, pH 7.0 re] 1] LL : EE =a re LE Ll --- CompanySuze.DocsNot ConTStCAiCBn. Ava Hydrol abilityof 254358 a Functionofpit Figure 5: EPxHam7.p0lehycdrhorloymtaietostgarbailmistoyfsmamopnliet.ored | rEe E 0: Te od E[FF E DuPont14965 Iobtained for Day 5, = ree]t Fes EaN TE [4E%P os x Te]4 --_-- CompanySuze.Docs NotConTsSCiACBnI. wn Hydrolytic Stability of125435 as a Function ofpH. Figure 6: Representative calibration curve for 8-2 TBA. `ompound name: 8-2TBA. orrelation coefficient r = 0.999939, 2 = 0.999878. alibrationcurve: 1.14*x9+7128.1779 Response type: Intemal Std (Ref 2), Area * (IS Conc. /1S Area ) urve type: Linear, Origin: Exclude, Weighting: 1/x, Axs trans: None 12045 DuPont14965 Response: 12 0 2000 4000 6000 8000 10000 ppb) E CompanySaniized. DoesE NotContTSaCAiCBnI AR Hydrolytic Stability of H-25435 as Function ofpi DuPont 14965 Figure 7: R5e1p5ruesge/nLtaDt-i8v-e2 cThBrAomianttoergnraalmsstaonfd1a0r4d.pg/L 8-2 TBA calibration standard and 051104015 Smooth(n,1:3) stat 100. 82TBA 3.50 167187 F1SIR of2 channels ES7.4496030,+050243 % o 1.00 200 3.00 400 5.00 min _ 05110415 Smooth(vn,1:3) stat 1S-62TBA _ F2SIR of2 channels S- 348 467,528 100. 783782 333904005 % 0 1.00 200 3.00 4.00 5.00 min _-- Company Suniized. DrroN.oteConstin TSCA CBI AR Hydrolytc Stabilityof25435ss FunctionofpH DuPont. 14965 Figure 8: Representative chromatograms for Day 5, pH 7.0 buffer blank sample. b0l5a1n1k014:5p0H7 S7;moDaoyth5(Mn 1x3) 100 F1:SIR of 2channels,ES6.8496830,4502033 % o 1.00 2.00 300 400 5.00 min -- b0l5a1n1k041;5p07H S7;moDoathy5(Mn,1x3) 100 F2:SIR of 2 chann4e6l7s,,5E2S8196604003 % o 100 200 300 400 5.00 min --_--nmm CompanySniiae.DoHesoNotContTSaCAiCBnL aR Hydrolytc StabilityofH.25435 ss FunctionofpH DuPont 14965 Figure 9: Representative chromatograms for Day 515 ug/L D-8-2 TBA internal standard. 5, pH 7.0 hydrolytic stability sample and r0e5p111;04p6H387, SDmaoyo5th(Mn,1x3) 0 8:2TBA _ 3.49 338073.0 FA:SIR of 2 channels,ES13142603,4050263 * 0 1.00 200 300 400 5.00 min ~ r0e5p111;04p5H378;SDmaoo5yth(Mn 1x3) F2:SIRof 2 chan4n6e7l,s5E2S8- [100 1-38.246TBA_ 2.392+005 635915 % 0 1.00 200 300 400 5.00 min Company Sniized. Docs Noet CronseincTStCA-- CBI -- pre Hydrol Stability of H25435 a Function ofpil. Dupont. 14965 Figure 10: aRenpdre$s1e5nptagt/iLveD-c8h-r2oTmBatAoginrtaemrsnaflosrtDaandyar0d,.pH 7.0 hydrolytic stability sample 0re1p291:0p4H5377: SDmaoyo0th(Mn, 16) 100 8-A27BA 3534105 F1:SIRof 2 chann4e6l3s,E5S2-3 140604006 * 100 200 30 400 500 min - r0e1p291;04p0H377; DSamoyo0th(Mn,1x3) F2.SIR of 2 chan4n6e7ls,5E2S8- 4 or 15-8278 3.49 28334005 7501.1 * o 100 200 300 400 500 min -_-- Company Suze. DAocns sNokContin TSCA CB.