Document jy3YrO3xyz2o01OXON3njyVep

423 -- Haskell Laboratory for Toxicology and Industrial Medicine April 5, 2002 M226 _ 324 TO: Study in Rats Ca G. A. Ladics FROM: J. C. Maslanka Analytical Chemist epeated-Dose Oral Toxicity Gavage Range Finder ANALYSIS FOR H-24691 IN DOSING SUSPENSIONS MIXING AND STABILITY STUDY MHaesdkielclaSRameplsesNupmboere: Norbr: Analytical Reference: Analytical Report Number: SSetruvdiycNeuCmobdee:r: Notebook References: (246Y 91 24691 HA-2001-037, report to satisfy protocol requirements for toxicology studies with ut may be used for other purposes. - atconain TSCA on Company Sanitized. 0% motor - peated-Dose OralToxicity avage Range Finding Study in Rats ANALYSIS FOR H-24691 IN DOSING SUSPENSIONS MIXING AND STABILITY STUDY HMaesdkieclallSRaemspelaercNhuPmrboejrec:t Number: Analytical Report Number: SUMMARY [o24y 691 ] HA-2001-037 2401 hs ports iv nin 1) In preparationforbi-weekly mixing i this study and a 90-Day Study with H-24691, a `amnidxi2n5g.0anmdg/stmaLbiloitfyHs-t2u4d6y9w1aswedroenep.reDpaorseidn,gcsolulsepcetnesdioannsdastucbomnictetnetdraftoirons of 1.0, 5.0, homogencity/concentration verification and stability analysis (5-hour room temperature) on Test day4. Dosing suspensions from the same preparation were collected and submitted afer 7 daysofrefrigeration for homogeneity (1.0 mg/mL level only), concentration verification and refrigerated stability along with a 5-hour room temperature. _ stability. In addition, the 0mg/mL (control) samples was collected and submitted for aannailmyasliss iwnitthheesatcuhdsyetanodfsbaim-pwleeesk.lyTmhiexirnefgrwigaesrantoetd isnaimtpialteesd winertehensottuaddymniniilsttehreeddattoathe was available from this work. ConcentrationsofH-24691 in separate dosing suspensions were measured by gas chromatography (GC). "The suspension vehicle for the study was 0.5% aqueous methylcellulose. Results from analysisofthe dosing suspensions collected for homogeneity/concentration verification and stability on Test Day 4 indicated that test substance was homogeneously mixed, at the expected concentrations (+ 11.2%) and stable in the vehicle for5 hours at room temperature. Results from analysisofthe dosing suspensions collected aftr 7 days of refrigeration for homogeneity and/or concentration verification and stability on Test Day 11 indicated that test substance was re-dispersed properly, at the expected concentrations (212.6%) in all levels and was stable inthe vehicle for $ hours a room temperature. H-24516 was not detected in the 0 mg/mL (control) sample. Sot ein sanitized. Duss het corn TSCA GB HA2001-037; Page 2 SAMPLE SUBMITTAL Samples containing H-24691 at the concentrationsof 0, 1.0, 5.0, and, 25.0 mg/mL were collected on Test Day 4. These samples were analyzed to determine homogeneity/concentration verification and -room temperature stability. Samples from the same preparation were collected on Test Day 11 after 7 daysofrefrigeration. These samples were analyzed to determine homogeneity (1.0 mg/mL level only), concentration verification and refrigerated stability along with 5-hour room temperature stability aftr refrigeration. All dosing suspension samples were collected on the same day the suspensions were prepared or at the established stability time for analysis. They were analyzed when received. `The suspension vehicle was 0.5% methylcellulose. METHODS 1. Analytical Methods a. Dosing Suspension Treatment Each dosing sample (3 mL) was dillted to 100 mL with methanol and mixed todissolvethe - H-24691 in the suspension. The dosing samples were analyzed without further dilution or were further diluted with the 0 mg/mL sample (initial dilution) to an expected concentration of approximately 0.03mg/mL (a.i) prior to analysis. Before all final dilutions, the intemal standard diluted solution (refer to Calibration and Quantitation Section)wasadded to each test `sample to give a final concentrationofapproximately 0.04 mg/mL. Each sample (final dilution) had an equivalent amount matrix (methylcellulose in methanol) when analyzed. `Samples submitted for analysis were analyzed the day the suspensions were received by the testing group. b. Chromatographic Conditions CoInlsturmum:ent HJeWwl,et-DPBa-c1k7a0r1d, M3o0dmel, 603.3920mGaCm 1, 1um film thickness DIentjeeccttoorr FSpIlDi;t225500CC Carrier Gas: Split vent: H2e2l3iummm(2i.1nmin) OInvjeenctiPornogVroalmu:me: G2rmaidcireonltiter IInniittiiaall TTeimmpeerature: 110000mCin LLeevveell 1| TReamtep:erature: 226000CCimin = LToetvaell r1uTntiimmee: 09.0000 mmiinn.. r= HA-2001.037; Page 3 c. CalibanrdaQuatntiitaotinon SAtoscekpasroaltuetisonamwpalseporfepHa-r2e4d6i9n1mewtahasnoolb.taiTnheids tsotoucske saoslutthieonanwaalsytsiocnailcraetfeedrteoncaesssutraendthaartd.allA `material was in solution. Before analysis, appropriate aliquots ofthe stock were diluted with the 0 mg/mL sample (initial dilution) to make calibration standards, which bracketed the target concentration ofthe diluted dosing samples. A stock solutionofinterna standard (1H, 1H, 2H, 2H-perfluoro-9-methyldecan-1-ol, 98% pure) was prepared in methanol and added to cach calibration standard and test sample to give a final concentration of0.04mg/mL. The ratioofthe iunsteedrn0alcsotnasntdraurcdt aancdalHi-b2ra4t6i9o1n cpueravkehbeyiglhetasstfsrqoumarreesplriecgarteesGsiCona(nsaeleysFiisgoufrtehIsfeorsaolruetpiroenssenwtearteive curve). Measured concentrations for the dosing samples were determined by applying the peak height ratios from replicate injectionsofeach sample to the calibration curve. Company sanitized. Doesnot contain TSCA CBI HA-2001-037; Page 4 ANALYTICAL RESULTS A. Chromatography H-24691 eluted from the GC colasuresmolvned peak with a retention time of approximately 3.5 minutes. The intemal standard eluted from the GC column as a resolved peak with a retention time ofapproximately 3.9 minutes. Representative GC chromatograms are shown in Figures 2(a-c). Test substance was not detected in the 0 mg/mL control. B. Mixing and Stability Study Analytical results from dosing suspensions collected on Test Da4y and Test Day 11 and analyzed for homogeneity and/or concentration verification and stability are shown in Table I and Summary Table 1. `The following table summarizes the results for all homogeneity and/or concentration verification and stability analyses TepslteDTayyp Nomml MeawcmdlT.MB M9e%aNno(miTnMalB) CV. Sabiliy 8) %Nominal - HomToesgtenDeaityy 0 Not -- -- 10 0944,0884,0836 50 aaa3,a7 898082 46 s9s5a0 TestDay 11 3 nannu2 20 5 960 7 Day SRteafbriilgietryated' 0 Np -- -- 5100 09004,4068,6444,40859 887.04 30 084248 2s 242,239 964 1 sat ab VSiaemapnlersesheulsdf$orhothuersanaatlrysoiosmoftetmhpeer(aotpur(e1.), middle (V) and bottom (B) samples. d@ MDeenaontersesnulotneofadletlecatneadl.yses reported. Mean result ofall analyses reported except for one duplicate repeat sample which was low due to aliquot & MDuepalnicraetseulctoonfcednutprlaitciaotnevre-rsifaimcpaltiionngfsraommpltehs anoarilgyizneadledxicluetpetdfsoarmtphlee.1.0Ormiggi/nmalLalneavleyls(ihsowmoagselneoiwteyr).than expecteddue fo aliquot errorand not reported. - omens $m oss otsentain TSCACE! HA2001-037: Page 5 `The data for samples collected on Test Day 4 indicates that the test substance was homogeneously mixed in the vehicle at all levels (C.V.'s = 6, 4 and 5, respectively). The test substance was at the targeted concentration in the samples ( 11.2% of nominal) and was stable in the vehicle when held $ hours at room temperature. `The data for samples collected on Test Day 11 after 7 daysofrefrigeration indicates that the test substance was homogeneously re-suspended in the vehicle at all levels (C.V.'s = 3,0and 1, respectively). The test substance was at the targeted concentration (+ 12.6% of nominal) and stable in the vehicle when held refrigerated for 7 days followed by 5 hours at room temperature. The S-hour sample for the 25 mg/mL level (84.8% of nominal) appeared to be less stable than expected but this can be attributedto sampling. and analytical variability and not stabilityofthe test material in the vehicle. Test substance was not found in the 0 mg/mL samples. C. Conclusions Data from the analysisofthe samples during the mixing study indicate that the test substance could be mixed homogeneously, was at the targeted levels and stable under the conditions necessary for mixing the dosing suspensions twice weekly for any study within the range of the concentrations tested. Test substance was not foundinthe 0 mg/mL samples. a201.057 Page 6 ACKNOWLEGEMENTS `Sample analysis by Sheila A. Riley, (Chemistry Associate). SIGNATURES sionATRES Reports: opa a rPt alas, t w5wsMapoaron vets Goi mar CompanySazed. Does notcontain Ta cy HA2001.037; Page 7 "Table Homogeneity and Silty ofH-24691 in Dosing Suspensions Tse mgmLHZM Pee e T Type Teowd M Nomme al ared Nominal HCoomnoTgernosLiy 000 NDA) -- MTDoLrE 1100 0089844 4844 Boron 110010 00886009 880690 102 mean(): 005.84306:0.05 840 3.6% Mean): 0.888:0.05 (58.8%) cron MiTooores 5500 a437 887846 Borrow 5500.01 4a1n6 48s2 502 mean): 45722310.56 o1e05s.4) - Mean): 4513019 02%) ca% MiTooLr e 225500 2272 80888 Boron 250 212 965 Mean(): 2c3.s05s10 02.0%) `suabilidP) 100 0818 818 11004214 00888933 858933 1028 0822 822 50 mean(): 04.785s40.04 (8955.40%) Denes mo Gece 20 240 960 ((8)) TMheeanerreasuglet omfeahseuraeidincaonlcaelniiqruaotton0,)avaenradgdeuppelirccaetnetolfiqnuomrsifro(m1hpearoernitghneaslesi),usetadndsaarmdpdleevi1at,io2n,.snd ) ScaoeiTlicenstaomfplveasrihseilodnofhoopur,smirdodolem,enmdperbaottutroem.(mean resul, =) cepfo 26 mgm. vel btom (v1). (E) aMenan)e,al aforigin (0) sliguo, on duplicate 1) aliquot and dplcate re-analysisof thesecond aliquot a oo coniein TSCAC HA-2001-037; Page `Table I. Homogeneity and Stability ofH-24691 in Dosing Suspeasions (continued) Tm T gm Tewbay L _ E241__ Pew_ em Sample Type Nominal Messed Nominal femur `HomoCgeonnciTtyR/oStLability 000 NDA) - Tor MmDLE BotToM `ConcentrationVer#ification/Subility " # " 10 0900 10 0864 10 0859 Mean): 0.874:0.02 cn 50 446 50 444 Mean():4.4520.01 cv.o% 250 242 250 29 Mean():24.13021 cv% 90.0 864 859 (7.4%) 892 8838 (9.0%) 968 95.6 96.4%) Stability --_ 7-DAY REFRIGERATED/S HOUR RT 10 0955 955 10 0852 852 Mean): 0.904+0.07 0.4%) crn 7-DAY REFRIGERATED/S HOUR RT 50 418 836 50 an 846 Mean(D):4.21:0.04 42%) cvi% 7-DAY REFRIGERATED/S HOUR RT 250 205 820 250 28 87.2 Mean(D):21.2:0.92 (54.8%) cian W(B) DTheeotaveesrragoermdeeatseueredd concentration, averagepercentofnominal (in parentheses), standard deviation, and coefficient ()oTfhvearaivaetriaogneofmetaospu,rmeidddcloen,ceanntdrabtoitotno,ma.verage percent of nominal (in parentheses) standard deviation, and coefFcient (D) oTfhve aaverraigoeafmedtuapsiluiroceadntecosnacmepnltersa.tion, average percentof nominal in parentheses),standarddeviation, and coefficient o`fwavsalroiawteirotnohafndeupxlpieccatteedsnamoplreesp.orteMde.an resultofduplicate re-analysisofthe original sample. Original sampleanalysis HA2001037; Page 9 - Representative AnalyFtiigcuarleC1alibration Curve 12 Equation for the fitted line: 109 Y=0.012550+23.0624 * X 08+ R-Squared = 0.9968 S 2 os 53 IxT 04 &3 02 - 00 0.00 0.01 0.02 0.03 0.04 0.05 Concentration, mg/mL Figure 1: (Csaqluiabrreast)iofnorcucralviebrsahtoiwoinnsgolluitnieoanrsofift lHi-n2e4)6t9o1redpilliuctaetdeopveesrkahecioghntcraetinotmeraarsautnrgieemooenfnts 0.0156 to 0.0468 mg/mL. The 0mg/mL control diluted sample was used as the zero. - any Santed. Dogs TAcontalGn ES TSCA CBI pam - Representative High PerformanceFiLgauird2eChromatogeaphs Chromatozeams |Fire a: Re ect LC ge f mg (eslsieikr sd | EF AT pe eo ss. - ||:2 ; |a i L T Frere 2s a ReeT seBPSe drut5mn124] 9 rgloitesemi | :> ig | |= -- TE YT FP -- Le ee el e------ Sanit aesnot con1tSaLAlLnS: HA-2001-037; Page 11 --_ TABL1E SUMMARY OF DOSING MIXING AND STABILITY ANALYSES Dosing Concentration of H-24691 (mg/mL. HomogeNnoemiitnyalS:amples 10 50 50 TestToDpay 4 0944 443 22 044)" (586) (8) Middle 0884 437 27 (88.4) 14) 008) Bottom 0836" am 22 3.6) (044) 068) Average Measured Cone. 0388 as1 230 `Average Percent Nominal ) 003) 20 Standard Deviation 005 2019 10 CoefficientofVariation % % 5% Stability Samples $Hour Room Temperature 054% 475 20 (85.4) 50) 960) "Test Day 11 (7-TDoapy Refrigerated) 0500 4460 22 00" 92) 068) Middle 0864 aaa 29 ~ (364) (s88) 56) Bottom 0859 -- - #59) Average Measured Cone. 0874 44s 21 `Average Percent Nominal `Standard Deviation 10092 20.0)1 06042)1 CoefficientofVariation 3% 03% 1% 5Hour RoomTemperature 00900.44)" (a8242') (281428) *+MeNuamnpbesaruTlortofethenrtiinhalreealhiesquroeteaspnesdcduplpiceateealiqruootmroam vhaelvoersiginaldilutedsample. StMaetiasntriecssublatsferdoininthaelaavleirqauogte,menaesudurpeldiccoantceeanlriaqtuioatnndrrme-)aonafltyhsiestoofpt,hiesdelceoandnddubploitctaotmeoaflieqxuocth.dosing evel MDeupalnicarteseulctoonfcednterlatiiconerv-erainflicyastiiosnosfatmhpeloersigrienpaolrtseadmfpoleifort ofmixing.