Document nmp0dyLR63Lr6NrbEQQ9Ynr3X
6295.23; ST-46 Protocol.
AR6-9182,
050400
3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Protocol for Study Nos. T-6295.23; ST-46
ExploratoryIn-VitroPercutaneousAbsorptionStudyofTheophylline,Salicylic
os R--6
`Reconstituted EpidermisModel
Study Objective:
M`TohdiselstbudyyciosnddeuscitginnegdatsoigmapilnefaabmsiolriaprtiitoynwsittuhdythoenStkwinoEptrheivciRoeucsolnyscthiatruatcetderEipzieddermis
cfloumoproouchnedmsic(atlhecoophmyploluinneda(npdersfalluicoyrloiocctayclisdulifnoanaptaep,eKr*)b.y DSouupcpeotr,ticnt.gaa)na,lyatsicwaelllwoasrkonfeor
tphairstsotfumdeytwhiolldbaenddoinnestirnutmheentnqeuwalCiofircpaotriaont.e TAoxMicToTloagsysaLyabwoirllatboerycoinndBuucitleddinagt 2th3e6eansd
of the absorption study the test compounds.
to
determine
if
SkinEthic
viability
was
affectedbyapplication
of
Research Client:
3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul, MN 55144
Sponsor:
3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul, MN 55144
Study Location:
3M Strategic Toxicology Laboratory 3M Center, Building 270-35-06 room SB314 Saint Paul, MN 55144
Study Director:
John Butenhoff, Ph.D. Senior Laboratory Manager 3M Medical Dept. / Corporate Toxicology 3M Centr, Building 220-2E-02 Saint Paul, MN 55144 Ph.: 651-733-1962 FAX: 651-733-1773
Study Toxicologist:
Kathy Thompson, MP.H. Senior. Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center, Building 220-2E-02 Saint Paul, MN 55144 Ph: 651-733-9578 FAX: 651-733-1773
Proposed Study Timeline
Start Date: Wednesday, April 12%, 2000
EAnnadlyDtaitcea:l CFroimdpayl,etAiproinlD1a4t%e,:2T00B0D Final Report Completion Date: TBD
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Regulatory Compliance: p`Trhoitsosctoluadyndwiclllasbseifpieerdfoasrmaed"CilnastsheC3SMtuSdtyr"ataesgiecxpTloaxiinceodliongyTLOaXborSaOtPor0y9u5n0d,eSrtraatdeefgiinced. Toxicology Lab GLP Program Procedure.
Test Material: `LTihaeisfolnu,o3roMchCehmeimcialcatlesst Dmiavtiesriioanl.wTihllebedrpurgovtiesdtedmabteyrDiaalnwHilalkebse,suPprpoldiuecdtbRyesSpiongsmiab.ility
Identification: Names: TPehrefolpuhoyrloloicnteyl(suTlHfEo)nate, K* (PFOS, K* salt) Salicylic Acid (SA)
Molecular Formulas:
CiFnSOsK*
(PFOS)
C7-H8-N4-02 (THE)
C7-H6-03
(SA)
Lot Number: Documentation will be kept on file by the Sponsor for PFOS (Lot 217) and by the Corporate Toxicology Analytical Laboratory for THE and SA. Purity: CDoorcpuomreantteaTtoixoincwoillolgbyeAkneapltyotincfailleLabbyortahteoSrpyofnosroTrHfoEr aPnFdOSS,A.andbythe Stability: CDoorcpuomreantteatTiooxnicwoillolgbyeAkneapltytoincaflilLeawbiotrhattoherySpfoornTsoHrEfoarnPdFSOAS., and by the Storage Conditions: Upon receipt, test material will be stored tightly sealed at room temperature. Characteristics: Information on synthesis methods, composition or other characteristics that definethetest material will be kept on file with the Sponsor for PFOS, andbythe Corporate Toxicology Analytical LaboratoryforTHE and SA.
SkinEthic Preparation Procedure:
aTshepeSrklihneEtmhaincufRaecctounrsetristuitnesdtrEupcitdioenrsmi(sscweiAllttbaecphrmeepnatreOdnei)m.meAdbisaotrepltyiounpotnesatrirnigval
will begin on April 13, 2000. using aseptic techniques.
All
handling
of SkinEthic
material
will
be
done
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T-6295.23; ST-46 Protocol.
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Materials:
a
Twelve
filters
SkinEthic
Human
Reconstituted
Epidermis,
Size
0.63
cm',
and
twelve
blank
@ a
ASdkdiintEitohniaclMeamipnttyen1a2ncweelMlecdulituumre(psluaptpelsi(edBebcytomnanDuifcakcitnusroenr))
a Heating Block (37C)
a Incubator (37C, S%CO, saturated humidity)
Reagents:
R1e0c2e7p7t8o1r),M1e.d5i%uBmo-viPnheosSpehartuemBAulfbfuermeidnS(aSliignmea(,GAi-b8c0o2B2RLLoCta#t3#91H40260901-)07a5d,deLdofot#r
FC compound
@ Compound application vehicle~ Milli-Q Water
@ @
T1r%ypSsionap(SSioglumtai,olnot(T#w3e9eHn092502,,Maclalii#nTc-k7r4o1d8t), lot #H285 N13H32)
Q MTT (Sigma, lot #39H5076, cat #M-2128)
Procedure:
@ bUlnapnakcfkilatenrdwemlalisntaacicnorSdkiinngetthoicmaRneucfoancsttuirteurt'esd iEnpsitdreurctmiiosns(RinEpc)ontsrkoinllceedlltceumlptuarneds and humidity incubator.
@ eBnevfiorroenpmreondtuacltcaopnpdliitciaotniosnf, oe2xrpohsouerRsEtp0 eaqnudilbiblraantkefwiilttehr waemlblisentot sattamnodsaprhderreo.om
a Place 0.6 ml receptor medium in each well of 12-well culture plates.
Transfer block.
REp
and
blank
filters
gently
with
forceps
to
each
well
and
place
on
heating
2 Evenly apply 200 ul ofTHE and SA compound solutions to REp and blank filter
surface in triplicate, of blank application
200 ul of PFOS vehicle (water)
compound solution to the remainder and
in quadruplicate, start timer:
and
200
ul
1. THE applied as saturated water solutionof 6 mg/ml
2. SA applied as saturated water solution of2 mg/ml
3. PFOS applied asasolution of 0.025 mg/ml (50 uM)
a TmreadnisfuemraRfEtepr 0a.n2d5bhlra,n0k.5fihltre,r1gehnrt,l2yhwri,t4hbfro,racenpds6tohrn.ew well containing receptor
Atendofexperiment, polypropylene vial for
place later
the receptor analysis.
medium
of
each
plate
well
into
a
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T-6295.23; ST-46 Protocol.
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@ rWiansseh3thteimReEspwiatnhdsbalmaenkvofillutemresuorffawcaete3r.tiGmeenstwliytwhi2p0e0culeLanoefd1s9%ursfoaacepwsiotlhutiaocn,otatnodn tip. Save washing fluids and tips in a polypropylene vial for later analysis.
sCaomnpdluecstfMoTr TneRgeadtuivceticoonntraosls(awyaotneroanpeplsiacmaptlieonfoonrleya)cthotdeesttecrommipneouSnEdvaianbdiltitwyo. @ dCiugtesrtemfaorin4i8nghosukrisn wmietmhbTrraynpesisnaantd4b0laC.nk fAifltteerrshionmtoogsemnailzlaptiieocnesanwditchenstcriisfsuorgsatainodn
p(o1l0y0p0rgo)pyfloern1e0vimailn,fofrilltaetrersuapnealrynsaitsa.nt through a0.45 jum filter and save in a
Numberofanalytical samples generated : total =182:
AS.urfPaFcOeSW:ashing FluiBdas:tt=6hrs
B. THE:
6att=6hrs
C sa:
6att=6hrs
D. Water:
4att=6hrs
Receptor Medium:
FE.. TPFHOES:: Gceacah actth=0a.2t5hthrrs=s,,000..552hhrr5ss,, 11..00hhrrss,, 22.00hhrsr,44..s00hhr,rss,, 66..00hhrrss.
G. SA: H. Water:
Geachatt=025hrs, 4cachatt=025 hrs,
0.5 0.5
hrs, hrs,
1.0 1.0
hrs, hrs,
2.0hrs, 2.0 hrs,
4.0 4.0
hrs, hrs,
6.0 6.0
hrs hrs
1DigePsFtOFSi:ltered Supern6aatratnt=:6hrs
J. THE:
4att=6hrs
K SA:
4att=6hrs
`Ship samples on dry ice to: Dave Ehresman, BS Toxicology Specialist 33MM CMeendtiecra,lBDueipltd.in/gCo2r3p6o-rCat1e46Toxicology PSahi:n6t5P1a-ul7,36M-8N41505144
tforrimaentahlyylsuirsiocfatchide,teasntdc1o,m3p-doiumnetdhyalnudritcheacpirdesfourmTedHEm,etgaebnotliistiecsa(c3i-dmfeotrhySlAx,anntohnienef,or1,3,7-
PROS).
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T-6295.23; ST-46 Protocol.
[ZT
Data Analysis:
Analytical results will be used to derive maximal flux rate in jig/hour/cm'. The flux rate.
obtained for THE and SA will be comparedtothat reported in the paper byDoucet,et. al. Mass balance recovery should be 90+ 10%. MTT results will be used to determine if skin viability was affected (as compared to negative control) by application of the test compounds. Responsibilities: Kathy Thompson will be responsible for performing the experimeandt sending
specimens for analysis; Dave Ehresman will be responsible for analytical evaluation of the samples. Kathy Thompsonwil draft afinal report and ensure the report receives appropriate.
3M review befor a final reporit issued, to be signed by Kathy Thompson and Dave Ehresman. References: Doucet, O., N. Garcia and L. Zastrow. Skin Culture Model: a Possible Alternative to the UseofExcised Human Skin for Assessing In Vitro Percutaneous Absorption. Toxicology in Vitro 12 (1998) 423-430.
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.
T-6295.23; ST-46 Protocol.
Signatures:
05/0400 |
John Butenhoff, Ph.D.
Date
Senior Laboratory Manager
Study Director
Kathy Thompson, MP.H.
Date
Senior Toxicologist
Study Toxicologist
Sponsor Representative.
Date
004440
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