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Availableonlineatwww.
ijpcr.
comInternationalJournalofPharmaceuticalandClinicalResearch2015;7(1):102-104ISSN-09751556ResearchArticle*AuthorforCorrespondenceIn-VivoAcuteDermalToxicityStudyofFormulationsonRabbits*ShetkarM.
A.
,MoreR.
R.
,KumbharS.
P.
,AuteA.
A.
,PoulB.
NDepartmentofPharmaceutics,MaharashtraCollegeofPharmacy,Nilanga,Dist.
Latur(MS)413521,IndiaAvailableOnline:1stJanuary,2015ABSTRACTPurpose:TodevelopanAceclofenactransdermalgelwithacapabilityfortopicaldrugdeliveryandThepurposeofthisstudyistoobtainscientificinformationregardinghealthhazardsthatmayresultfromaacutedermalexposuretotheagriculturalchemicalforprovidingthebasisoflayingdownthemeasureofsafetyprotectioninapplication.
Methods:Aceclofenacgelformulations,incorporatingvariouspermeationenhancers,werepreparedusingcarbomerasagellingagent.
Theformulationswereexaminedfortheirinvitrocharacteristicsincludingviscosity,pHanddrugreleaseaswellasin-vivopharmacologicalactivities.
Results:Theformulationscontaining5%ofeitherPropyleneglycolaspermeationenhancersgavedrugreleasepatternscomparabletothatofthereferenceproduct.
Propanolincreasedtheapparentviscosityofthetestgelstothesameextentasthatofthereference.
DrugreleasefromtheformulationsfittedbesttotheHiguchimodel.
Asignificantinvivoanalgesiceffectwasproducedbythetestformulationsandtheeffectwassuperiortothatobtainedwiththereferenceproduct.
Conclusion:Aceclofenacgelpreparationscontainingpropyleneglycolrespectively,exhibitedpronouncedanalgesicactivityandcouldbefurtherdevelopedfortopicaldeliveryofAceclofenac.
Keywords:Transdermalgel,toxicitystudy,PrimarydermalirritationstudyAceclofenac,PenetrationenhancerINTRODUCTIONThetestsubstanceisappliedtotheskinofthetestanimalsindifferentdosesandtheanimalsareobservedforeffectsandmortality.
Animalsshowingseveresignsoftoxicity/distressarehumanelykilled.
Attheendofthetestallsurvivinganimalsaretobesacrificed.
Necropsyisperformedonalldead,killedandsacrificedanimals.
TotesttheirritancypotentialofexperimentalAceclofenacgels,rabbitswereused,theselectedgelswereappliedtotheshavedskinontherabbitsback.
Theselectionofrabbitsforthisstudyisdescribedasfollows.
MATERIALSANDMETHODSPreparationofAceclofenacgelsRequiredquantitiesofCarbomer940,971,974weresoakedinsomeamountofdistilledwaterfor2to3hrs.
Thenitwasneutralizedbyaddingsufficientquantityoftriethanolaminewhilestirringwithhelpofoverheadstirrer(PhaseI).
Aceclofenacwasdissolvedinthemixtureofethanol(99.
9%),eitherofthePEG200,400,600andpropyleneglycol(PhaseII),Transcutol&DMSOwereaddedinsomeformulationsaspenetrationenhancers.
(PhaseII)wasaddedslowlytothe(phaseI)whilestirringwithhelpofoverheadstirrer,theremainingquantityofdistilledwaterwasthenaddedtomakeupthefinal100gmweight.
pHofallformulationswasmaintainedintherangeof6.
8to7.
4.
DeterminationofgelviscosityTheviscosityoftheformulationsandreferencewasperformedusingaBrookfielddigitalviscometer(ModelDV-II,USA)equippedwithspindleS27.
Theapparentviscositywasmeasuredat17seconds1shearrate(50rpm)androomtemperature,aftera3-minresttime.
EvaluationofinvitroibuprofenreleaseAsynthetichydrophilicmembranewasmountedonaFranzdiffusioncell(PermeGear,Riegelsville,PA,USA).
Thereceptorcompartmentcontained6.
5mLofphosphatebuffer(pH7.
4).
Onegramofthetestformulationorreferencewasappliedtothemembraneoveranareaof1.
131cm2areaacrossthedonorcompartment.
Thedonorcellwasexposedtoambienttemperatureandcoveredwithparafilmtopreventevaporation.
Thetemperatureofthereceptorcompartmentwasmaintainedat32CwhilethebuffersolutionwasstirredcontinuouslywithaTeflon-coatedmagneticbar.
Samples(0.
5ml)werewithdrawnfromthereleasemediumat0,0.
5,1,1.
5,2,3,4,6,and8handreplacedwithanequalvolumeoffreshbuffersolutiontomaintainsinkconditions.
Thesampleswereanalyzedbyhighperformanceliquidchromatography(HPLC)usingtheoperatingparametersdescribedbelow(see'HPLCanalysis').
Cumulativeamountofthedrugpermeatedversustime(Zeroorder)andalsoversussquarerootoftime(Higuchimodel)Housingandfeedingconditions:Temperature-22±3Cforrodentsand20°(±3°)forrabbitsRelativeHumidity–50-60%(However,humidityshouldnotbebelow30%orexceed70%atanygivenpointoftime).
Lighting–12hourslightanddarkcycleDietandwater–Standardlaboratorydietspecifictothespeciesandfilteredwaterfreefromcontamination.
Selectionandrandomization:Animalswererandomlyplacedincagesuponreceiptandthenrandomizedaccordingtothebodyweights,AnimalsShetkaret.
al.
/InVitroAcuteDermal…IJPCR,January2015–February2015,Volume7,Issue1,102-104Page103consideredunsuitablebecauseofoutlayingbodyweightswereexcludedfromthestudy.
Theanimalbackswerecarefullyshavedusingsterilizedshavingbladetoremovethefur.
Theselectedanimalsweregroupedinto4groups.
Andweremarkedwithidentificationcodesusingdilutedpicricacidsolution(1.
2%w/v).
Circularareasof2.
54cm(1inchdiameter)weremarkedonthebackofeachanimalusingmarkerinkonespotonrightsideandonespotonleftsideofvertebralcolumn.
Thespotonrightsidewasabradedusingshavingbladeandthespotonleftsidewaskeptintact1gmquantityoftheselectedgelswasappliedonmarkedspotsusingabsorbentcottonwool.
Thetoxicmanifestationsifanyontheskinwerethenassessedbyobservingtheskinatpre-selectedtimeintervalsaftertreatmente.
g.
1hrs,4hrs,12hrs,24hrs,48hrsand72hours,Theobservationswererecordedasnumericalscoresasforeachanimalasfollows0=novisiblereaction1=milderythema2=intenseerythema3=intenseerythemawithedema4=intenseerythemawithedemaandvesicularerosionThescorefortreatmentgroupandcontrolgroupwerethencomparedTable1:CompositionoftheAceclofenacgelformulationsNameofingredientWeight(gm)F1F2F3F4F5F6F7F8F9Aceclofenac111111111Ethanol(99.
9%)23.
9423.
9423.
9423.
9423.
9423.
9423.
9423.
9423.
94PEG40022.
4022.
4022.
4022.
4022.
4022.
4022.
4022.
4022.
40Carbopol9740.
512Carbopol971---0.
512---Carbopol9400.
512Propyleneglycol15.
5215.
5215.
5215.
5215.
5215.
5215.
5215.
5215.
52Triethanolamineq.
sq.
sq.
sq.
sq.
sq.
sq.
sq.
sq.
sDistilledwater(upto100gm\)100100100100100100100100100Table2:TheselectionofrabbitsforthisstudywithDosefortopicalApplication.
TestanimalHealthyRabbitsStrainNewZealandwhiterabbitsAge22weeksSexMaleWeight2-2.
5kgNoofanimalspergroups3animalsDoses1gmpersquareinchtopicallyNo.
ofGroupsGroupI=F1GelbasetreatedcontrolGroupII=1gmF3Formulation(withoutpenetrationenhancer)GroupIII=1gmF6Formulation(withpenetrationenhancer)DMSO1%w/w.
GroupIV=1gmF9Formulation(withpenetrationenhancer)Transcutol1%w/w,G-Gelbase,PEG200,400&Transcutol-penetrationenhancer,Carbopol974,940&971-asGellingAgentTable3:NumericalscoreforirritancypotentialforvehicletreatedcontrolGroup–I(F1Formulation)Sr.
No.
Animalcode1hrs4hrs12hrs24hrs48hrs72hrsIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbraded1A0000000000002B0000000000003C000000000000Shetkaret.
al.
/InVitroAcuteDermal…IJPCR,January2015–February2015,Volume7,Issue1,102-104Page104Table4:NumericalscoreforirritancypotentialforGroup-IItreatedwith[F3Formulation]whichcontainingnopenetrationenhancerSr.
No.
Animalcode1hrs4hrs12hrs24hrs48hrs72hrsIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbraded1D0000000000002E0000000000003F000000000000Table5:NumericalscoreforirritancypotentialforGroup-IIItreatedwith[F6Formulation]whichcontainingDMSOaspenetrationenhancerSr.
No.
Animalcode1hrs4hrs12hrs24hrs48hrs72hrsIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbraded1G0000000000002H0000000000003I000000000000Table6:NumericalscoreforirritancypotentialforGroup-IVtreatedwith[F9Formulation]whichcontainingTranscutolaspenetrationenhancerSr.
NoAnimalcode1hrs4hrs12hrs24hrs48hrs72hrsIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbraded1J0000000000002K0000000000003L000000000000RESULTSTableno1,2,3,4&5givesthesescoresforGroupI,GroupII,GroupIIIandGroupIVrespectivelyObservationsandExamination:Thefollowingfor72hrs.
Forvehiclecontrolgelbaseanimalsthosetreatedwithselectedformulationsdidn'tindicateanymanifestationofskinirritationsuchasrednessofskinorinflammationatsiteofapplication.
Boththeabradedareas&intactareaswhenappliedwithvehicleorselectedformulationwerefoundtobefreeofanysignofirritation.
Thusitcanbeconcludedthatalloftheselectedformulaearesafefortopicalapplication.
HoweverthisstudywasperformedusingasingleapplicationofgelproductsHence,itisneededtoconfirmsafetyafterrepeatedapplicationsalso.
Finding:Onlyaverysilenterythemareactionwasnotedonanimalsatexposureobservationtime.
Nodermalirritationwasnotedatthe24,48or72hourexamination.
REFERENCES1.
SethS.
D.
andMaulikM.
G.
,In;Analgesicsanti-pyreticsandanti-inflammatorydrugs,Eds;TextbookofPharmacology,1998;165.
2.
SatoskarandBhandarkar,In;Analgesic-AntipyreticsandNon-steroidalanti-inflammatorydrugs(NSAIDS)Eds;PharmacologyandPharmacoeherapeutics",1991;1(12),139-158.
3.
PalT,GanesanM.
BioavailabilityandBioequivalenceinPharmaceuticalTechnology.
India:CBSPublishers;2004.
4.
JagerW,BuchbauerG,JirovetzL,FritzerM.
PercutaneousAbsorptionofLavenderOilfromMassageOil.
JournaloftheSocietyofCosmeticChemists1992;43:49-54.
5.
PednekarPP,VakilBV,SaneRT,DatarAG.
AntimicrobialActivityofEssentialOilofBlumeaerianthaDCagainstSkinPathogens.
IntJPharmPharmSci2012;4:296-299.
6.
OrganizationforEconomicCo-operationandDevelopmentGuidelineforTestingofChemicals:RepeatedDoseDermalToxicity:21/28-DayStudy.
OECD.
1981;p.
410.
7.
SaiduYLS,BilbisML,IsezuoSA,HassanSW,AbbasAY.
AcuteAndSub-ChronicToxicityStudiesofCrudeAqueousExtractofAlbizziachevalieriharms(Leguminosae).
AsianJournalofBiochemistry2007;2:224–236.
8.
FallerC.
,BracherM.
,DamiN.
,RoguetR.
.
Predictiveabilityofreconstructedhumanepidermisequivalentsfortheassessmentofskinirritationofcosmetics.
ToxicologyinVitro2002;16,557-572.
9.
Fentem,J.
H.
,Briggs,D.
,Chesné,C.
,Elliott,G.
R.
,Harbell,J.
W.
,Heylings,J.
R.
,Portes,P.
,Roguet,R.
,vandeSandt,J.
J.
M.
&Botham,P.
A.
Aprevalidationstudyoninvitrotestsforacuteskinirritation:resultsandevaluationbytheManagementTeam.
ToxicologyinVitro2001;15,57-93.
10.
PortesP.
,GrandidierM.
-H.
,CohenC.
,RoguetR.
RefinementoftheEPISKINprotocolfortheassessmentofacuteskinirritationofchemicals:follow-uptotheECVAMprevalidationstudy.
ToxicologyinVitro2002;16,765-770
ijpcr.
comInternationalJournalofPharmaceuticalandClinicalResearch2015;7(1):102-104ISSN-09751556ResearchArticle*AuthorforCorrespondenceIn-VivoAcuteDermalToxicityStudyofFormulationsonRabbits*ShetkarM.
A.
,MoreR.
R.
,KumbharS.
P.
,AuteA.
A.
,PoulB.
NDepartmentofPharmaceutics,MaharashtraCollegeofPharmacy,Nilanga,Dist.
Latur(MS)413521,IndiaAvailableOnline:1stJanuary,2015ABSTRACTPurpose:TodevelopanAceclofenactransdermalgelwithacapabilityfortopicaldrugdeliveryandThepurposeofthisstudyistoobtainscientificinformationregardinghealthhazardsthatmayresultfromaacutedermalexposuretotheagriculturalchemicalforprovidingthebasisoflayingdownthemeasureofsafetyprotectioninapplication.
Methods:Aceclofenacgelformulations,incorporatingvariouspermeationenhancers,werepreparedusingcarbomerasagellingagent.
Theformulationswereexaminedfortheirinvitrocharacteristicsincludingviscosity,pHanddrugreleaseaswellasin-vivopharmacologicalactivities.
Results:Theformulationscontaining5%ofeitherPropyleneglycolaspermeationenhancersgavedrugreleasepatternscomparabletothatofthereferenceproduct.
Propanolincreasedtheapparentviscosityofthetestgelstothesameextentasthatofthereference.
DrugreleasefromtheformulationsfittedbesttotheHiguchimodel.
Asignificantinvivoanalgesiceffectwasproducedbythetestformulationsandtheeffectwassuperiortothatobtainedwiththereferenceproduct.
Conclusion:Aceclofenacgelpreparationscontainingpropyleneglycolrespectively,exhibitedpronouncedanalgesicactivityandcouldbefurtherdevelopedfortopicaldeliveryofAceclofenac.
Keywords:Transdermalgel,toxicitystudy,PrimarydermalirritationstudyAceclofenac,PenetrationenhancerINTRODUCTIONThetestsubstanceisappliedtotheskinofthetestanimalsindifferentdosesandtheanimalsareobservedforeffectsandmortality.
Animalsshowingseveresignsoftoxicity/distressarehumanelykilled.
Attheendofthetestallsurvivinganimalsaretobesacrificed.
Necropsyisperformedonalldead,killedandsacrificedanimals.
TotesttheirritancypotentialofexperimentalAceclofenacgels,rabbitswereused,theselectedgelswereappliedtotheshavedskinontherabbitsback.
Theselectionofrabbitsforthisstudyisdescribedasfollows.
MATERIALSANDMETHODSPreparationofAceclofenacgelsRequiredquantitiesofCarbomer940,971,974weresoakedinsomeamountofdistilledwaterfor2to3hrs.
Thenitwasneutralizedbyaddingsufficientquantityoftriethanolaminewhilestirringwithhelpofoverheadstirrer(PhaseI).
Aceclofenacwasdissolvedinthemixtureofethanol(99.
9%),eitherofthePEG200,400,600andpropyleneglycol(PhaseII),Transcutol&DMSOwereaddedinsomeformulationsaspenetrationenhancers.
(PhaseII)wasaddedslowlytothe(phaseI)whilestirringwithhelpofoverheadstirrer,theremainingquantityofdistilledwaterwasthenaddedtomakeupthefinal100gmweight.
pHofallformulationswasmaintainedintherangeof6.
8to7.
4.
DeterminationofgelviscosityTheviscosityoftheformulationsandreferencewasperformedusingaBrookfielddigitalviscometer(ModelDV-II,USA)equippedwithspindleS27.
Theapparentviscositywasmeasuredat17seconds1shearrate(50rpm)androomtemperature,aftera3-minresttime.
EvaluationofinvitroibuprofenreleaseAsynthetichydrophilicmembranewasmountedonaFranzdiffusioncell(PermeGear,Riegelsville,PA,USA).
Thereceptorcompartmentcontained6.
5mLofphosphatebuffer(pH7.
4).
Onegramofthetestformulationorreferencewasappliedtothemembraneoveranareaof1.
131cm2areaacrossthedonorcompartment.
Thedonorcellwasexposedtoambienttemperatureandcoveredwithparafilmtopreventevaporation.
Thetemperatureofthereceptorcompartmentwasmaintainedat32CwhilethebuffersolutionwasstirredcontinuouslywithaTeflon-coatedmagneticbar.
Samples(0.
5ml)werewithdrawnfromthereleasemediumat0,0.
5,1,1.
5,2,3,4,6,and8handreplacedwithanequalvolumeoffreshbuffersolutiontomaintainsinkconditions.
Thesampleswereanalyzedbyhighperformanceliquidchromatography(HPLC)usingtheoperatingparametersdescribedbelow(see'HPLCanalysis').
Cumulativeamountofthedrugpermeatedversustime(Zeroorder)andalsoversussquarerootoftime(Higuchimodel)Housingandfeedingconditions:Temperature-22±3Cforrodentsand20°(±3°)forrabbitsRelativeHumidity–50-60%(However,humidityshouldnotbebelow30%orexceed70%atanygivenpointoftime).
Lighting–12hourslightanddarkcycleDietandwater–Standardlaboratorydietspecifictothespeciesandfilteredwaterfreefromcontamination.
Selectionandrandomization:Animalswererandomlyplacedincagesuponreceiptandthenrandomizedaccordingtothebodyweights,AnimalsShetkaret.
al.
/InVitroAcuteDermal…IJPCR,January2015–February2015,Volume7,Issue1,102-104Page103consideredunsuitablebecauseofoutlayingbodyweightswereexcludedfromthestudy.
Theanimalbackswerecarefullyshavedusingsterilizedshavingbladetoremovethefur.
Theselectedanimalsweregroupedinto4groups.
Andweremarkedwithidentificationcodesusingdilutedpicricacidsolution(1.
2%w/v).
Circularareasof2.
54cm(1inchdiameter)weremarkedonthebackofeachanimalusingmarkerinkonespotonrightsideandonespotonleftsideofvertebralcolumn.
Thespotonrightsidewasabradedusingshavingbladeandthespotonleftsidewaskeptintact1gmquantityoftheselectedgelswasappliedonmarkedspotsusingabsorbentcottonwool.
Thetoxicmanifestationsifanyontheskinwerethenassessedbyobservingtheskinatpre-selectedtimeintervalsaftertreatmente.
g.
1hrs,4hrs,12hrs,24hrs,48hrsand72hours,Theobservationswererecordedasnumericalscoresasforeachanimalasfollows0=novisiblereaction1=milderythema2=intenseerythema3=intenseerythemawithedema4=intenseerythemawithedemaandvesicularerosionThescorefortreatmentgroupandcontrolgroupwerethencomparedTable1:CompositionoftheAceclofenacgelformulationsNameofingredientWeight(gm)F1F2F3F4F5F6F7F8F9Aceclofenac111111111Ethanol(99.
9%)23.
9423.
9423.
9423.
9423.
9423.
9423.
9423.
9423.
94PEG40022.
4022.
4022.
4022.
4022.
4022.
4022.
4022.
4022.
40Carbopol9740.
512Carbopol971---0.
512---Carbopol9400.
512Propyleneglycol15.
5215.
5215.
5215.
5215.
5215.
5215.
5215.
5215.
52Triethanolamineq.
sq.
sq.
sq.
sq.
sq.
sq.
sq.
sq.
sDistilledwater(upto100gm\)100100100100100100100100100Table2:TheselectionofrabbitsforthisstudywithDosefortopicalApplication.
TestanimalHealthyRabbitsStrainNewZealandwhiterabbitsAge22weeksSexMaleWeight2-2.
5kgNoofanimalspergroups3animalsDoses1gmpersquareinchtopicallyNo.
ofGroupsGroupI=F1GelbasetreatedcontrolGroupII=1gmF3Formulation(withoutpenetrationenhancer)GroupIII=1gmF6Formulation(withpenetrationenhancer)DMSO1%w/w.
GroupIV=1gmF9Formulation(withpenetrationenhancer)Transcutol1%w/w,G-Gelbase,PEG200,400&Transcutol-penetrationenhancer,Carbopol974,940&971-asGellingAgentTable3:NumericalscoreforirritancypotentialforvehicletreatedcontrolGroup–I(F1Formulation)Sr.
No.
Animalcode1hrs4hrs12hrs24hrs48hrs72hrsIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbraded1A0000000000002B0000000000003C000000000000Shetkaret.
al.
/InVitroAcuteDermal…IJPCR,January2015–February2015,Volume7,Issue1,102-104Page104Table4:NumericalscoreforirritancypotentialforGroup-IItreatedwith[F3Formulation]whichcontainingnopenetrationenhancerSr.
No.
Animalcode1hrs4hrs12hrs24hrs48hrs72hrsIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbraded1D0000000000002E0000000000003F000000000000Table5:NumericalscoreforirritancypotentialforGroup-IIItreatedwith[F6Formulation]whichcontainingDMSOaspenetrationenhancerSr.
No.
Animalcode1hrs4hrs12hrs24hrs48hrs72hrsIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbraded1G0000000000002H0000000000003I000000000000Table6:NumericalscoreforirritancypotentialforGroup-IVtreatedwith[F9Formulation]whichcontainingTranscutolaspenetrationenhancerSr.
NoAnimalcode1hrs4hrs12hrs24hrs48hrs72hrsIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbradedIntactAbraded1J0000000000002K0000000000003L000000000000RESULTSTableno1,2,3,4&5givesthesescoresforGroupI,GroupII,GroupIIIandGroupIVrespectivelyObservationsandExamination:Thefollowingfor72hrs.
Forvehiclecontrolgelbaseanimalsthosetreatedwithselectedformulationsdidn'tindicateanymanifestationofskinirritationsuchasrednessofskinorinflammationatsiteofapplication.
Boththeabradedareas&intactareaswhenappliedwithvehicleorselectedformulationwerefoundtobefreeofanysignofirritation.
Thusitcanbeconcludedthatalloftheselectedformulaearesafefortopicalapplication.
HoweverthisstudywasperformedusingasingleapplicationofgelproductsHence,itisneededtoconfirmsafetyafterrepeatedapplicationsalso.
Finding:Onlyaverysilenterythemareactionwasnotedonanimalsatexposureobservationtime.
Nodermalirritationwasnotedatthe24,48or72hourexamination.
REFERENCES1.
SethS.
D.
andMaulikM.
G.
,In;Analgesicsanti-pyreticsandanti-inflammatorydrugs,Eds;TextbookofPharmacology,1998;165.
2.
SatoskarandBhandarkar,In;Analgesic-AntipyreticsandNon-steroidalanti-inflammatorydrugs(NSAIDS)Eds;PharmacologyandPharmacoeherapeutics",1991;1(12),139-158.
3.
PalT,GanesanM.
BioavailabilityandBioequivalenceinPharmaceuticalTechnology.
India:CBSPublishers;2004.
4.
JagerW,BuchbauerG,JirovetzL,FritzerM.
PercutaneousAbsorptionofLavenderOilfromMassageOil.
JournaloftheSocietyofCosmeticChemists1992;43:49-54.
5.
PednekarPP,VakilBV,SaneRT,DatarAG.
AntimicrobialActivityofEssentialOilofBlumeaerianthaDCagainstSkinPathogens.
IntJPharmPharmSci2012;4:296-299.
6.
OrganizationforEconomicCo-operationandDevelopmentGuidelineforTestingofChemicals:RepeatedDoseDermalToxicity:21/28-DayStudy.
OECD.
1981;p.
410.
7.
SaiduYLS,BilbisML,IsezuoSA,HassanSW,AbbasAY.
AcuteAndSub-ChronicToxicityStudiesofCrudeAqueousExtractofAlbizziachevalieriharms(Leguminosae).
AsianJournalofBiochemistry2007;2:224–236.
8.
FallerC.
,BracherM.
,DamiN.
,RoguetR.
.
Predictiveabilityofreconstructedhumanepidermisequivalentsfortheassessmentofskinirritationofcosmetics.
ToxicologyinVitro2002;16,557-572.
9.
Fentem,J.
H.
,Briggs,D.
,Chesné,C.
,Elliott,G.
R.
,Harbell,J.
W.
,Heylings,J.
R.
,Portes,P.
,Roguet,R.
,vandeSandt,J.
J.
M.
&Botham,P.
A.
Aprevalidationstudyoninvitrotestsforacuteskinirritation:resultsandevaluationbytheManagementTeam.
ToxicologyinVitro2001;15,57-93.
10.
PortesP.
,GrandidierM.
-H.
,CohenC.
,RoguetR.
RefinementoftheEPISKINprotocolfortheassessmentofacuteskinirritationofchemicals:follow-uptotheECVAMprevalidationstudy.
ToxicologyinVitro2002;16,765-770
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SoftShellWeb是一家2019年成立的国外主机商，商家在英格兰注册，提供的产品包括虚拟主机和VPS，其中VPS基于KVM架构，采用SSD硬盘，提供IPv4+IPv6，可选美国(圣何塞)、荷兰(阿姆斯特丹)和台湾(台北)等机房。商家近期推出台湾和荷兰年付特价VPS主机，其中台湾VPS最低年付49美元，荷兰VPS年付24美元起。台湾VPSCPU：1core内存：2GB硬盘：20GB SSD流量...
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