in vitro and in silico - edoc sohn.pdf · having welcomed me in the institute of pharma technology...
TRANSCRIPT
InvitroBiorelevantandinsilicoSunscreenPerformance
EvaluationontheBasisofFilmThicknessFrequency
DistributionofFormulationsandUVFilterRepartition
Inauguraldissertationzur
ErlangungderWürdeeinesDoktorsderPhilosophievorgelegtder
Philosophisch‐NaturwissenschaftlichenFakultätderUniversitätBasel
von
MYRIAMSOHN
ausRosenau,Frankreich
Basel,2016
Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel Edoc.unibas.ch
GenehmigtvonderPhilosophisch‐NaturwissenschaftlichenFakultätaufAntragvonProf.Dr.GeorgiosImanidis,FakultätsverantwortlicherProf.Dr.JörgHuwyler,KorreferentPDDr.BerndHerzog,KorreferentBasel,den21.April2015
Prof.Dr.JörgSchiblerDekan
Acknowledgements
Firstandforemost,IwouldliketoexpressmygratitudetoProf.Dr.GeorgiosImanidisfor
havingwelcomedmeintheInstituteofPharmaTechnologyattheSchoolofLifeSciences
FHNW for my Ph.D. studies. I would like to thank him for his great supervision, the
valuableandproductivediscussions,thedeepexplanations,hispatience,hisadvicesin
writingthepapers;helearnedmynevergiveup,especiallywiththeconvolutionapproach
andhelpedinthereflectionofaddressinganewtopic.
IwouldliketothankGebertRüfStiftungforthefundingofthisresearchwork.
Furthermore,IwouldliketodeeplythankDr.BerndHerzogfromBASFforhisprecious
support during my entire Ph.D. work, his expertise and our fruitful discussions on
simulations,andforhisavailability.
Iwould like to thankProf.Dr. JörgHuwyler of theUniversity ofBasel, department of
PharmaceuticalSciencesforbeingco‐refereeforthisthesis.
SpecialthankstoUliOsterwalderfromBASFforhishelpfulcontributiontothiswork,his
ideas,andpositivethinking.
IwouldliketoprofoundlythankTheodorBühlerforhishugesupportonconfocalRaman
microspectroscopy measurements, his helpfulness, his time, and our dynamic
discussions.
ToFabienneThoenenabigthankforhergreathelpinthelaboratoryandpreparationof
pigearskin,herkindness,andhelpfulness.
I thank themasterstudentsVerenaKorn fromtheFHNWandAdelineHêche fromthe
University of Basel for their great help in the advancement of the laboratory work
regarding the development of the methods for SPF in vitro on pig ear skin and film
thicknessdistributionmeasurements.ThankstoVerenaforherdynamismanddailygood
mood.
IwouldliketothankPatriceBelinandDenisGeorgesfromAltimetfortheirsupportinthe
developmentofthefilmthicknessassessmentmethod.
My gratitude also goes to Marcel Schnyder from BASF for having supported me and
believedinmewhenIdecidedtopursuedoctoralstudies,AndreaZamponiandNathalie
BouillotohavemadeitpossiblewithinBASF.
AwarmThankYougoestoEmilieRoggforherinvaluablefriendship,herendlesssupport,
advices,andencouragementsduringmyentirethesisandbefore.
I keep my deepest gratitude to my Dad and Mom for their unconditional love, they
encouraged me throughout my entire life, endlessly supported me in my academic
studies,todayIamwhatIamthankstoyou.IdeeplythankMamama,withoutherInever
couldhavepursuedacademicstudies.Atenderthoughtfor Joël, forthe15yearsspent
together,withthehopethatwhatwelivedtogetherwastrue.AwarmthanktoStaszek
forhisattentiveness,tenderness,andhissupportsinceoneyear,“bardzocielubie”.Tomy
children,mojeaniołky,AymericandMaël,allmyloveforyou.
Abstract
Exposure to ultraviolet (UV) radiation is known to cause various damages to human
health.Topicallyappliedsunscreensarewidelyusedby thepopulation topreventsun
damages and are an efficient, simple, and convenient means of photoprotection. The
active ingredientsofsunscreensare theUV filters thatareable toabsorbselectivelya
wavelengthrangeintheUVspectrum.ThelevelofUVprotectionaffordedbyasunscreen
primarily depends on the UV filters contained in the product according to their
concentration, absorbance profile, and photostability properties, along with the
compositionoftheUVfiltersystem.However,sunscreenscontainingthesameUVfilter
mixturewerereportedtoproducedifferentlevelofphotoprotection.Hence,expectedUV
performanceofa sunscreencannotbe solelypredictedbasedon theUV filter system
contained in the product. Therefore, the present work aims at understanding the
mechanisms of UV performance by evaluating the behavior of a sunscreen after
applicationontheskinintermsoffilmformationandUVfilterrepartition.Theimpactof
sunscreen film thickness and UV filter repartition on the photoprotection was
investigated independenceof thesunscreenvehicle.Toevaluatetheperformanceofa
sunscreen, a methodology was developed for the determination of the in vitro UV
protectionthatisafurtherpartofthepresentwork.
Thepresentthesisconsistsoffourstudies,whichaimedatimprovingtheunderstanding
ofthemechanismsofsunprotectionforeffectiveproductdevelopment.Tothisend, in
vitrotestsalongwithinsilicoapproachwereemployedforevaluatingsunscreenefficacy.
Thefindingsmayimprovethepredictabilityoftheperformanceofsunscreensduringthe
developmentstagetooptimizetheirefficacy.
i
Abstract ii
Inthefirststudy,weexaminedtheuseofpigearskinasabiologicalsubstrateforSPFin
vitrodeterminationwithdiffusetransmissionspectroscopy.Thepolymethylmethacrylate
(PMMA)platescurrentlyemployedtothispurposemostlyfailinyieldingasatisfactory
correlationbetweensunprotectionfactor(SPF)invitroandinvivo,theSPFinvivobeing
thegoldstandardand,todate,theonlyapprovedmethodbyregulatorybodies.Trypsin‐
separated stratum corneum and heat‐separated epidermis of pig ear showed a lower
roughnessthanfullthicknessskinandPMMAplatesbuttheskinpreparationsubstrate
yieldedSPFinvitrovaluesthatmoreaccuratelyreflectedtheSPFinvivothanthePMMA
plates.Thisstudyrevealedthatbesidesroughness,theimprovedaffinityofthesunscreen
totheskinsubstratecomparedtoPMMAplatesmayexplainthebetterinvivoprediction
ofSPFachievedwiththeuseofthebiologicalsubstrate.
In the second study, we aimed at understanding the relationship between thickness
frequency distribution of a sunscreen film formed upon application and sunscreen
efficacysincesunscreenformulationswiththesameUVfiltersystemwerereportedto
produce different SPFs.We developed a method tomeasure the film thickness of an
applied sunscreen on pig skin substrate based on topographical measurements and
investigated the influence of sunscreen vehicle and of sunscreen application on the
averagemean film thickness (Smean) and SPF in vitro. Five sunscreen vehicles were
investigated including an oil‐in‐water cream, an oil‐in‐water spray, a water‐in‐oil
emulsion,agel, andaclearalcoholic spray.Thisworkevidencedastrong influenceof
vehicleandapplicationconditiononsunscreenefficacyarising fromdifferences in the
filmthickness.LowvehicleviscosityresultedinsmallerSmeanandlowerSPFinvitrothan
highvehicleviscosity;continuousoilphaseformulationsproducedthelargestSmeanand
SPFvalues.Long spreading time reducedSmeanandSPF; increasedpressure reduced
SPF.Theseresultsareofhighpracticalimportanceintherouteofunderstandingwhich
parametersimpactsunprotectionandsubsequentlyhowsunscreenswork.
Thethirdstudyreliesonthesecond;thepurposewastoquantitativelyassesstheroleof
film thickness frequency distribution for sunscreen efficacy. We developed a
computationalmethod for calculating theSPF in silico usingbesides the spectroscopic
propertiesoftheusedUVfiltermixturethecompletethicknessdistributionofasunscreen
filmobtainedfromtopographicalmeasurements.
Abstract iii
TheinvestigatedformulationscontainingthesameUVfiltermixturedifferedintheirSPF
invitroandfilmthicknessdistribution.WefoundaverygoodagreementbetweenSPFin
silicoandSPFinvitrodemonstratingthehighrelevanceoffilmthicknessdistributionfor
theinterpretationofsunscreenefficacy.Integratingvehicle‐dependentfilmparameters
intotoolsforinsilicopredictionofsunscreenperformanceis,therefore,ofhighinterest
toimproveUVefficacypredictions.
Finally,thefourthstudyfocusedontheevaluationoftherepartitionofanoilmiscibleand
awatersolubleUVfilterintheappliedsunscreenfilm;theUVfiltersshouldbeuniformly
distributedthroughoutthesunscreenlayerforoptimumefficacy.Weemployedconfocal
Ramanmicrospectroscopy(CRM)asahighlysensitiveanalyticaltechniquetoprecisely
detectthespatialdistributionofthetwoinvestigatedUVfiltersthroughoutthesunscreen
filmappliedonapigearsubstrate in threedifferent formulations.Thisworkrevealed
noticeabledifferencesintherepartitionofthetwostudiedUVfiltersdependingonthe
sunscreenvehicle,clearalcoholicspraydifferedfromothertestedoil‐in‐waterandwater‐
in‐oilformulations.ThetwoUVfiltersappearedcompletelydisjointedinthefilmformed
bytheclearalcoholicsprayformulationindicatinganon‐homogeneousdistributionofthe
two UV filters in the sunscreen film. This result is of high significance as a worse
repartitionofUVfiltersintheappliedfilmwouldleadtoreducedphotoprotectionwhen
theUVfiltersshowadifferentabsorbanceprofilewhichiscommonlythecase.
This thesis provides novel insights into the understanding of the mechanisms that
influenceUVefficacy.Theknowledgeofthebehaviorofsunscreenswithrespecttofilm
thicknessdistributionandrepartitionofUVfiltersisfundamentalinformationthatallows
the optimization of a sunscreen formulation during early development stage helping
expeditedevelopment.Thisadvancedunderstandingincombinationwithinvitroandin
silico methodologies may improve the ability to accurately predict SPF in vivo
performancewiththeobjectiveofreducingclinicaltrialsinhumansandinthelongrunin
theestablishmentofavalidatedinvitromethod.
Contents
Abstract i
1 Introduction 1
1.1. Background 1
1.2. Objectives 3
2 Theoreticalsection:anoverview 5
2.1. Solarradiation 5
2.1.1.Sunlight 5
2.1.2.Effectsofsunlightexposure 6
2.1.2.1. Benefitsofsunexposure 7
2.1.2.2. AdverseeffectsattributedtoUVBradiation 7
2.1.2.3. AdverseeffectsattributedtoUVAradiation 8
2.1.2.4. Skincancers 9
2.2. Naturalphotoprotection. 11
2.2.1.Propertiesofhumanskin 11
2.2.2.Constitutiveskincolor 12
2.2.3.Facultativeskincolor 13
2.3. Artificialphotoprotection 14
2.3.1.Historyofsunscreens 14
2.3.2.RequirementsforgoodUVprotection 15
2.3.2.1. Technology 16
2.3.2.2. Assessmentandmeasurementmethods 24
2.3.2.3. Normsandstandards 33
2.3.2.4. Compliance 34
iv
Contents v
2.3.3.Theidealsunscreen,outlookinthefutureofphotoprotection 35
2.3.3.1. HomeostasicUVprotection 35
2.3.3.2. Benefitsofdailyphotoprotection 36
3 Porcine ear skin as a biological substrate for in vitro testing of sunscreen
performance 37
3.1. Abstract 37
3.2. Introduction 38
3.3. Materialsandmethods 40
3.3.1.Chemicalsandequipment 40
3.3.2.Preparationofbiologicalsubstrate 41
3.3.2.1. Method1–isolationofstratumcorneumbytrypsintreatment42
3.3.2.2. Method2–isolationofepidermalmembranebyheat
treatment 42
3.3.3.Skintissuethicknessmeasurement 43
3.3.4.Polymethylmethacrylateplates 43
3.3.5.Surfacetopographicalassessment 44
3.3.6.Sunscreenformulations 45
3.3.7.MeasurementofSPFinvitrousingspectraltransmissionofultraviolet 46
3.3.8.Statisticalanalysis 47
3.4. Resultsanddiscussion 47
3.4.1.Skinthickness 47
3.4.2.Surfacetopographicalassessment 50
3.4.3.Measurementofsunprotectionfactor 53
3.5. Conclusion 59
4 Film thickness frequency distribution of different vehicles determines
sunscreenefficacy 60
4.1. Abstract 60
4.2. Introduction 61
4.3. Materialsandmethods 63
4.3.1.Chemicalsandequipment 63
4.3.2.Preparationofskinsubstrate 64
4.3.3.Characterizationofsunscreenformulations 64
Contents vi
4.3.4.Applicationofsunscreens 66
4.3.5.Measurementofsunprotectionfactorinvitrousingspectral
transmissionofultraviolet 67
4.3.6.Assessmentofsunscreenfilm 68
4.3.7.Statisticalanalysis 70
4.4. Results 71
4.4.1.Filmassessment 71
4.4.2.ImpactofvehicleonfilmparametervaluesandSPFinvitro 73
4.4.3.Impactofpressureandspreadingprocedureonfilmparameter
valuesandSPFinvitro 77
4.5. Discussion 80
4.6. Conclusion 84
5 InsilicocalculationofSPFwithdifferentsunscreenvehiclesusingmeasuredfilm
thicknessdistribution‐comparisonwithSPFinvitro 85
5.1. Abstract 85
5.2. Introduction 86
5.3. Materialsandmethods 88
5.3.1.Chemicalsandequipment 88
5.3.2.Preparationofskinsubstrate 89
5.3.3.Sunscreenvehicles 89
5.3.4.Measurementofthesunprotectionfactorinvitro 89
5.3.5.Assessmentofthefilmthicknessdistributionofanappliedsunscreen 90
5.3.6.Determinationofthecorrectedfilmthicknessfrequencydistribution
ofanappliedsunscreenusingconvolutionapproach 67
5.3.7.Calculationofthesunprotectionfactorinsilico 92
5.4. Resultsanddiscussion 94
5.4.1.Measurementerroroffilmthickness 94
5.4.2.Filmthicknessdistributionofsunscreens 96
5.4.3.Sunprotectionfactorinsilicoandinvitro 100
5.4.4.Modelingfilmthicknessfrequencydistribution 103
5.5. Conclusion 105
Contents vii
6 RepartitionofanoilmiscibleandawatersolubleUVfilterinanapplied
sunscreenfilmusingconfocalRamanmicrospectroscopy 106
6.1. Abstract 106
6.2. Introduction 107
6.3. Materialsandmethods 109
6.3.1.Chemicalsandequipment 109
6.3.2.Preparationofskinsubstrate 110
6.3.3.Sunscreenvehicles 110
6.3.4.Measurementofthesunprotectionfactorinvitro 112
6.3.5.ConfocalRamanmicrospectroscopymeasurements 112
6.3.5.1. Linedepthscanassessment 113
6.3.5.2. Surfacedepthscanassessment 114
6.3.5.3. ControlexperimentforcorrectionofRamansignal
attenuation115
6.4. Resultsanddiscussion 116
6.4.1.RamanspectraofEHMCandPBSA 116
6.4.2.CorrectionforRamansignalattenuation 118
6.4.3.Linedepthscan 119
6.4.4.Surfacedepthscan 121
6.4.5.Consequencesforsunprotection 127
6.4.6.Invitrosunprotectionfactor 128
6.5. Conclusion 128
7 Conclusionandoutlook 130
Bibliography 132
ListofAbbreviations 155
ListofSymbols 157
ListofFigures 158
ListofTables 161
CurriculumVitae 163
Chapter1
Introduction
1.1.Background
Overthepastdecades,thebehaviorofpeopletowardsunexposurehaschangedalotwith
amarkedtrendforoutsiderecreationaloccupations,ortravellingincountrieswherethe
sunlightintensitymightnotbeadaptedfortheirskin.Thishasledtogenerallyhigherand
uncontrolled exposure of people to solar radiation. Although ultraviolet (UV) sun
radiation is prerequisite for life on Earth needed for photosynthesis, and shows vital
biologicalbenefits1,itisalsorecognizedthatexcessiveexposuretosolarradiationcauses
detrimentalhealthdamages2‐5.Thetypeofphotodamageisdependentonthewavelength
range;somebeingassociatedrathertotheexposuretoUVBortoUVAradiation.
Themainmeansofphotoprotectionareavoidingsunexposure,seekingshade,wearing
clothesandhats,andapplyingtopicalsunscreens.Thelatterisanefficient,convenient,
andsimplemeansofsunprotection6,7.TheactiveingredientsofsunscreensaretheUV
filters thatareable toabsorbselectivelyawavelengthrange intheUVspectrum8.The
protectionabilityofasunscreenprincipallydependsontheUVfiltersystemcontainedin
the product with respect to the absorbing, photostability and photocompatibility
properties of the UV filters, along with their concentration. The performance of a
sunscreenislargelydescribedbythesunprotectionfactor(SPF)whosedetermination
takesintoaccountthehumansensitivitytoerythema.
1
Chapter1.Introduction 2
SPFcanbedeterminedbyinvivo9,invitro10,orinsilico11methodologies,butonlythein
vivo basedmethod is currentlyapprovedby regulatorybodies. Invivo approachbeing
timeconsuming,laborious,expensiveandethicallyquestionable,thereisaconsiderable
interestfromallplayersintheindustryindevelopinganinvitrotechniqueabletodeliver
SPFinvitrovaluesmatchingclinicalSPFinvivovalues.ThedeterminationoftheSPFin
vitro is based on themeasurement of the UV light transmitted through a suitable UV
transparentsubstratebeforeandaftersunscreenapplication12,13.Amajorissueforthe
establishmentofaninvitrostandardremains,mostcertainly,thechoiceofthesubstrate
forsunscreenapplicationthatwouldbeabletosimulatehumanskinatbestwithrespect
toroughnessandsurfaceproperties.SincethebeginningsofSPFinvitrotesting,different
biological and synthetic substrate types have been employed 10,12,14‐17,
polymethylmethacrylate (PMMA) plates being the currently favourite substrate.
However,despitetheavailabilityofPMMAplateswithdifferentroughnesscharacteristics
including a type developed especially to reproduce human skin roughness 18, none of
theseplatessucceedinyieldingSPFinvitrovaluesinanaccurateandreproduciblefashion
19correlatingwiththeclinicalSPF.Asaresult,thereisstillmissingapropersubstrateto
succeedintheestablishmentofavalidatedinvitromethod.
Further,anotherunclearaspectinmeasuringsunscreenperformanceistheexperimental
variabilityofSPFvaluesobtainedforsunscreenscontainingthesameUVfiltermixture
20,21despiteusingthesamemethodologyforSPFdetermination.BeyondUVfiltersystem,
other factors must play a role for sun performance. Some studies reported that the
application procedure impacted performance and cream thickness; a more rubbed
applicationledtoasmallerSPFinvivo22andacrudecomparedtoacarefulapplicationto
asmallercreamthickness23.Theeffectofcarefulversuscrudespreadingofsunscreenon
themagnitudeoferythemaoccurrencewassimulated,andunderlinedthe“importanceof
homogeneityofspreadingonthelevelofdeliveredprotection”24.Theidealconditionfor
optimumperformanceistheachievementofanuniformsunscreenlayerwithconstant
film thickness resembling the perfectly homogeneous distribution of UV filters into a
solution state 25,26. This can, however, neverbe attainedundernormalmanual invivo
applicationsinceskinsurfaceisnotflatandprecludestheachievementofanuniformfilm.
The importance of homogeneity of distribution of the sunscreen on SPF efficacy was
reported27.Nevertheless,theexacteffectofvehicleonfilmformationremainsunclear.
Chapter1.Introduction 3
Asaresult,thereisstillanincompleteunderstandingofthemechanismsthatinfluence
sunprotectionofsunscreensonceappliedonasubstratewithanunclearsituationonthe
parametersthatarerelevantforUVefficacybesidesthemereUVfiltercompositionand
UVfilterconcentration.
1.2.Objectives
Thegeneralaimofthisthesisistoimprovetheunderstandingofthemechanismsofsun
protectionbyasunscreenappliedonasubstratewiththe identificationof factors that
may influence sunscreen efficacy. This work is subdivided into five chapters, which
address analytical‐methodological and computational aspects of the performance
evaluationofasunscreenappliedonpigearskinsubstrate.
ThetheoreticalsectioninChapter2aimsatreviewingononehandthesolarradiationand
itseffectonhumanhealth,andontheotherhandthephotoprotection, fromnaturalto
artificial,thelatterfocusingontheuseofsunscreens.ItgivesareviewontheUVfilters,
UVtestmethods,sunscreennorms,andconsumercompliances.
Chapter 3 focuses on the use of skin from porcine ear as a substrate for SPF in vitro
measurement.Theaimistoexaminetherelevanceofusingabiologicalpreparationfor
SPFinvitromeasurementwiththeinvestigationifabiorelevantsubstratemayproduce
SPFinvitrovaluescorrelatingbetterwithSPFinvivovaluescomparedtothecurrently
usedsyntheticPMMAplates.
ThepurposeinChapter4isthedeterminationofthefilmthicknessfrequencydistribution
ofdifferentsunscreenformulations.Theaimistoinvestigateifthedivergenceofefficacy
betweensunscreenvehiclescontainingthesameUVfiltercompositionmayarisefromthe
differenceinthefilmthicknessofanappliedsunscreenonpigskinsubstrate.
Chapter1.Introduction 4
Chapter5followsthestudyinChapter4andaimsatquantitativelyassessingtheroleof
film thickness frequencydistribution for sunscreen efficacy.Weused a computational
method for calculating the SPF in silico by making use besides the spectroscopic
properties of the UV filter system of the complete thickness distribution of a spread
sunscreenfilm.SPFinsilicowascomparedtotheSPFinvitrotoinvestigatetherelevance
offilmthicknessdistributionforUVefficacy.
Finally,theobjectiveinChapter6istheinvestigationoftherepartitionofanoilmiscible
andawatersolubleUVfilterintheappliedsunscreenfilm.Thepurposeistoassessthe
influenceofthesunscreenvehicleontheUVfilterdistributionandsubsequentlyonthe
deliveredphotoprotection.
Chapter2
Theoreticalsection
2.1.Solarradiation
2.1.1.Sunlight
ThesunemitstotheEarthaportionofelectromagneticenergyintheformofradiation.
Thesolarspectrumisconstituted fromultraviolet (UV),visible (VIS)and infrared(IR)
radiation.UV radiation encompasseswavelengths between290‐400nmand is divided
intoUVC(200‐290nm),UVB(290‐320nm)andUVA(320‐400nm)part;UVAbeingfurther
subdividedintoUVAIIbandextendingfrom320to340nmandUVAIbandextendingfrom
340to400nm.Thevisiblepartrangesfrom400to780nm,followedbytheinfraredpart
from 780 to 3000nm. UV, VIS, and IR differentiate themselves with their energy and
penetration depth ability into the skin; the longer the wavelength, the deeper the
penetrationintotheskinlayers.TheshortwavelengthandhighenergeticUVCraysare
absorbed through the stratospheric ozone layer by O2 and O3 molecules present at
altitudesbetween10and50kmthatsubsequentlypreventsthemfrompassingthrough
thestratosphereandreachingtheEarthsurfaceandtheskin28.Theenergyabsorbedby
theozonelayeristhenreleasedinformofheatresponsibleforthehighertemperatureof
thestratosphere.Also,alargepartoftheshort‐waveUVBraysareblocked.
5
Chapter2.Theory:anoverview 6
There is a significant environmental and health issue concerning the depletion of the
stratospheric ozone layer by chlorine compound contained in the emission of
Cholorofluorocarbons;adepletionoftheozonelayerresultinginanincreasedamountof
carcinogenic UV radiation reaching the Earth surface and an increase in skin cancer
incidences28,29.TheresidualUVBandUVAraysreachhumanskin,UVBradiationislargely
captured by the upper skin layers, whereas UVA radiation penetrates more deeply
throughtheepidermisanddermis,attainingtheconnectivetissueofthedermis30,31.In
total,theUVregionrepresentsonly5%ofthesolarspectrum,butwasshowntoproduce
acuteandchronicharmfulhealthdamages.UVarecomposedfromaround3.5%UVBand
96.5% UVA on a summer day 32; both show an irradiance peak maximum between
11.30amand1.30pm30,althoughUVAirradianceremainsmorestablethroughouttheday
and the year compared toUVB irradiance that varies, UVA irradiance being higher in
summerthaninwinter,atmiddaythaninthemorningorevening,athighaltitudes,and
accentuatedinsomegeographicalzones33.
IncomparisontoUV,VISlightandIRareregardedaslessharmful,whilsttheeffectsof
IRAdrewsomeattentionrecently34,35.Infra‐redradiationrepresents30%ofsolarrays,
theywereshowntoengenderalterationofgenesexpressionofskincells36,acceleration
ofskinageing37,andcontributiontothedevelopmentofcancers38.
2.1.2.Effectsofsunlightexposure
Withrespecttoitseffectsonhumanthesunshowsadualbehaviorsinceitexhibitsboth
positiveandnegativeeffects.Positivepropertiesofsunexposureembracepsychologically
and physically effects, but excessive exposure to solar radiation leads to detrimental
healthissues39.
SundamagemightbeexpressedbyfollowingequationproposedbyCripps40
undamage=UVintensityxdurationofexposurenatureofdefenseagainstdamage
2.1
where, the received UV intensity varies depending on geo‐orbital and environmental
factors32;principallyontheseason33,timeduringtheday41,latitude33,surfacereflection
42,andweather;thedurationofexposuredependsprincipallyontheamount
Chapter2.Theory:anoverview 7
ofexposuretime,occupation,andareaofexposedbodysites41;andthenatureofdefense
refers principally to the individual natural protection factor, reinforcedwith artificial
protectionmeanssuchassunscreens.
2.1.2.1.Benefitsfromsunexposure
AnimportantvitalbeneficialbiologicaleffectisthesynthesisofvitaminDproducedinthe
skinafterexposuretosunlight43.VitaminDshowsanactionspectrumwithamaximum
(max)at295nmandis, therefore,builtprincipallyunderUVBexposure.VitaminD is
furthermetabolizedtoproducethebiologicallyactivevitaminDmetaboliteinvolvedin
thesupportofcalciumhomeostasisbyinteractingwithspecificreceptorsinthebonesand
intestine, and is, therefore, essential to develop and maintain a healthy mineralized
skeleton 1. Besides calcium fixation, active vitamin D was also involved in
immunoregulation,protectionagainstoxidativestress44,andagainstinfectiousagents.A
deficiencyinvitaminDwasshowntobeinvolvedinmultipletypesofcancers45,46,and
riskofincidenthypertension47.BesidesvitaminDformation,sunlightisalsousedtotreat
skindiseasessuchaspsoriasis48andisalsowellknowntopromotefeelingofwell‐being.
Currently,moreoftentheadverseeffectsofthesunareputforwardasexcessiveexposure
tosunlightwasshowntoinducediverseimmediateandlong‐termphoto‐damages.The
effect on skin and health is highly wavelength dependent; some photo‐damages are
inducedmorebyUVBandothersmorebyUVAradiation.
2.1.2.2AdverseeffectsattributedmainlytoUVBexposure
AsingleacuteexposuretoUVBradiationresultsintheimmediateandfamiliarcutaneous
response called erythema, or more commonly known as sunburn. Sunburn is
characterizedbyaskinredness,sensationofburning,withpotentiallytheformationof
oedema and is due to the liberation of inflammatory mediators resulting in a
vasodilatation.TheUVdoserequiredtoinduceanerythemalresponseisdependenton
thewavelength.
Chapter2.Theory:anoverview 8
Thewavelengthatwhicherythemaformationismaximal(max)isapproximately308nm
49.Histological andbiochemical changes after inductionof an erythema reactionwere
studied 50. Major histological alterations were the formation of altered keratinocytes
(sunburn cells) and disappearance of Langerhans cells in the epidermis, and vascular
changesinthedermis.Atabiochemicallevel,Histaminecontentroseinducingtheearly
phaseofsunburnthroughvasodilation,whileProstaglandinE2roseprogressively.Ata
molecularlevel,amajoradverseeffectofUVBirradiationisDNAdamage51;UVBraysare
directly absorbed by cellular DNA leading to DNA lesions such as the formation of
cyclobutanepyridiminedimers(CPD)andpyrimidinephotoproducts (6‐4PP), theUVB
signature,whichmaximuminductioninhumanskinwasshowntobearound300nm52‐
54.TheseUVB‐inducedDNAdamageswereshowntoberesponsibleforgenemutation,
e.g.inducingthedysfunctionoftumorsuppressorgenessuchasp53proteininhumans
andmice51,55‐58thatwasshowntocontributetoskincancerdevelopmentinhumansand
inanimals59.Mutationsinp53tumorsuppressiongenearisebeforetheappearanceof
skin tumors, theyweredetected insun‐exposedskin fromnormalpatientsandactinic
keratoses,suggestingthatp53mutationsearlybiologicalindicatorofskincancerrisk.
2.1.2.3.AdverseeffectsmainlyattributedtoUVAradiation
ExposuretoUVAradiationleadstoanimmediateandweakskinpigmentationknownas
immediatepigmentdarkening(IPD)believedtobeduetoaphoto‐oxidationofexisting
melanin60;IPDisweakanddifficulttomeasureasitisunstableandfadesrapidlywithin
minutestoabouttwohoursdependingontheUVAirradiationdose61.Itisreplacedby
the persistent pigment darkening (PPD) which lasts for 24h under sufficient UVA
irradiation 62. UVA‐induced skin pigmentation is not protective 63. IPD is driven by
exposureatwavelengthsintheUVAtoVISregion(320‐700nm)64,whilePPDmayresult
eitherfromUVC,UVB,orUVAexposureandleadstoanincreaseofmelanin,thenatural
UVfilteroftheskin.UVAradiationismostlyresponsibleforchronicphoto‐damagessuch
asskinpigmentation (agespots), inductionofoxidativestress 65, andvisibleeffectsof
prematureskinageing throughthegenerationofreactiveoxygenspecies(ROS)66and
inductionofMatrixmetalloproteinase(MMP)67,68.
Chapter2.Theory:anoverview 9
Photo‐agedskinischaracterizedamongothersbyskindryness,wrinkles,elastosis69‐71,
irregular pigmentation particularly in Asians 72, immunosuppression 2,73, and actinic
keratose 74‐76 since UVA rays penetrate more deeply into the dermis achieving the
connectivetissuescomparedtoUVB66,77.Areviewonphotoaging, itsmechanismsand
repairopportunitiesisgivenbyRabeet.al78.
ExposuretoaveryhighUVAdoseisalsoabletoinduceanerythema,about1000times
higherthanrequiredforUVB79.
Atamolecularlevel,UVAraysarealsoabletoinduceCPDs80‐82,andtogenerateROSvia
theabsorptionthroughendogenousphotosensitizercompounds35,83.ROSarethenable
to produce diverse adverse effects such as photo‐ageing 74, immunosuppression in
animals and humans 84, mutation in mitochondrial DNA 66, and skin cancers 85 by
damagingDNAbyanindirectmechanism86.ItwasshownthatUVAinducedmelanomas
andmelanomaprecursorsintwoanimalmodels87.
2.1.2.4.Skincancers
Typesofskincancers
ExposuretoUVlightisthemostimportantfactorresponsibleforskincanceroccurrence,
itisalsoonethatcanbecontrolledbyourselves.BothUVBandUVAradiationisclassified
ashumancarcinogen88.Threemaintypesofskincancerwithrespecttotheinvolvedcells
exist,thetwonon‐melanomaskincancersincludingsquamouscellcarcinoma(SCC)and
basalcellcarcinoma(BCC)andmalignantmelanomathemostlethalformofskincancer
89.SCCmainlyoccursinsun‐exposedareasanditsoccurrenceisassociatedwithchronic
exposuretoUVradiationduringlifetime90;BCCandmelanomaareratherassociatedwith
intermittent sun exposure e.g. sunburning. Mutation of p53 gene was shown to be
involvedinsquamouscellcarcinoma(SCC)andbasalcellcarcinoma(BCC).Skincancer
representsasignificantandgrowingpublichealthconcernworldwideasincidenceshave
steadilyincreasedinrecentdecades91.Thismayberelatedtoachangeinlifestylehabits
withanincreaseofoutsiderecreationaloccupationsandofvacationtocountrieswhere
thesunlightmaynotbesuitedtotheskintype.
Chapter2.Theory:anoverview 10
Incidencesandsunscreenuse
Itis,thus,notsurprisingthatAustraliashowsthehighestincidencerateofskincancer92
most probably in connectionwith the fair skin of Australian population and the high
intensity of the sun; in the opposite Japan and China show the lowest melanoma
incidencesmostprobablyduetotheculturalattitudedifferenceofJapaneseandChinese
people towards sun exposure. The incidences of malignant melanoma in the D‐A‐CH
(Germany,Austria,andSwitzerland)regionareaboutquarter tohalf the incidences in
Australiabutare,however,muchhigherthaninJapan93.Theroleofsunscreensinskin
cancerpreventionisstilldiscussedcontroversiallyassomestudieshaveshowneitherno
association or even a positive association between sunscreen use and skin cancer 94.
However,anexplanationforthisparadoxistheuseofsunscreenswithsmallSPFvalues,
inaninadequateamount,andthatwereUVBbiasedatthetimeofmostconductedstudies;
also the lackof consideringpositiveandnegative confoundingwereproblematic fora
correctinterpretationofthestudydata95.However,anAustralianstudyconductedinthe
1990sbyGreenconsistingofafiveyearslongrandomizedtrial,“theGreenstudy”showed
theprotectivebenefitsof regularapplicationofa sunscreenwithSPF16 inprolonged
preventionofSCCandreductionof incidenceofnewprimarymelanomas forup to10
years after trial cessation 4,96. Based on this outcome, US‐FDA (Food and Drug
Administration) is the first authority to officially consider sunscreens as a means to
reducetheriskofskincancerandtoallowadirectclaimofskincancerriskprevention
for sunscreens with a labeled SPF of at least 15 and complying with the UVA
recommendation as reported in the finalmonograph for SunscreenDrugProducts for
Over‐the‐CounterHumanUsepublishedin201197.Developingefficientsunscreenswith
modernUV filters isessential inhelping toreduce thecontinuousgrowthofnewskin
cancersrelatedtosunexposure.
Epidemiologyandskincancers
Some studies reported the positive relationship between frequency of occurrence of
erythema in childhood till 15 to 20 years of age and increased risk ofmelanomas in
adulthood98,99.Thisissupportedbythefactthatteenagersandyoungadultmoreoften
geterythemadue to theirpoorprotectionbehavior100, less than40%usesunscreens.
Roughly,25%oflifetimesunexposureoccursbefore18yearsofage101.
Similarly, a positive relationship was found between incidence of skin cancers and
increasingamountofambientUVradiation,e.g.higherlatitudewherethesunirradiance
isgreater98,102.
Chapter2.Theory:anoverview 11
Further, fair skinned individuals are more disposed to develop non‐melanoma skin
cancersthandarkskinnedindividuals103,104,duetothedifferencesintheamountofUV‐
induced free radicals and better prevention of DNA damage for heavily pigmented
melanocytesthantheirlightercounterparts105,106.
2.2.Naturalphotoprotection
2.2.1Propertiesofhumanskin
Whensunlighthitstheskin,itcanbeabsorbedinthedifferentcelllayers,transmittedtill
dissipated,orscatteredback107.Skinbeinganheterogeneousmaterialisabletoscatter
incidentlightbeamasaresultofabruptchangesintherefractiveindex(RI)ofair(RIof
air=1.00)andthatofstratumcorneum(RIofSCisofaround1.52108);theRIofskinis
independentofskintypeandageofhumansubjects109.Approximately4%of incident
radiationisscatteredbackwhenattainingtheskinsurface110.Theintensityofscattering
withinthedermisisinverselyproportionaltothewavelength;therefore,attenuationof
light through scattering decreases with increased wavelength resulting in deeper
penetration depth for greater wavelength. Human epidermis shows a minimal
transmission in wavelengths around 275nm since it contains natural UV absorbing
chromophores that absorb in this range. These include aromatic amino acids (max
=275nm),nucleicacids(max=260nm),urocanicacid(max=277nm).Peptidebonds
areresponsibleforthelightabsorptionofskinofwavelengthssmallerthan240nm111.
AfterUV irradiation,oneof themechanismsofskinprotection is the thickeningof the
stratumcorneumwithanincreaseofnumberofcelllayersinstratumcorneum112.Itwas
reportedthatthicknessofthestratumcorneumaccountsfor2/3ofthephotoprotection
ofnormalskin,whereasthicknessofepidermiswasnotimportant113.Further,another
naturalprotectionmechanismisthesynthesisandredistributionofmelanin,thenatural
UVfilteroftheskin.
Chapter2.Theory:anoverview 12
2.2.2Constitutiveskincolor
Humanskincolorisconsideredeitherasconstitutiveorfacultative114.Constitutiveskin
colorreferstothebaseornaturalskincolorwithoutanysolarexposure,inthecontrary
offacultativeskincolorcorrespondingtoatanninginducedbysolarexposure63.
HumanskinpossessesitsownlineofdefenseagainstUVlightirradiation,theproduction
ofmelanin,anaturalUVfilter115presentintwoforms,thebrownish‐blackeumelaninand
the reddish‐yellow pheomelanin116. Difference in human skin color most probably is
relatedtothebalancebetweenthesetwoformsofmelanin117.Fitzpatrickclassification
givessixskinphototypeswithrespecttoskincolorasgiveninTable2.1118.
Table2.1.SkinphototypesaccordingtoFitzpatrickclassification
Skintype Characteristics
I Whiteskin,reddishhaircolor
Alwaysburnseasily,nevertans
II Whiteskin,blondhaircolor
Alwaysburnseasily,tansminimally
III Burnsmoderately,tansgradually
IV Burnsminimally,tanswell
V Rarelyburns,tansprofusely
VI Almostneverburns,deeplypigmented
Thereareconsiderabledifferencesinmelanincontentandcompositionintheskinofthe
differenthumanethnicities119.ThenaturallevelofprotectionagainstUVirradiationis
different among human races and is related to the amount of eumelanin versus
pheomelanin:Pheomelaninbeingpredominantinfair‐skinnedpeopleisalsoresponsible
for the weak capacity of photoprotection of this people 120. Human skin color is not
randombuthasevolvedwithmigrationofpeople toadapt to sunlight intensity in the
differentworldzones121,122.Naturalselectionofskincolorallowsabalancebetweenthe
protectionofbody´s folatebeingdestructedunderUV irradiation that isnecessary for
DNA synthesis and cell division, and the production of vitaminD under UV exposure
necessaryamongothersforthedevelopmentoftheskeleton123.
Chapter2.Theory:anoverview 13
Constitutivemelaninshowsanabsorptionspectrumwithamaximumaround335nmand
whichextendsoverthewholeUVandVISrange115,124‐126,theabsorbancespectrumofthe
twoformsofmelaninbeingquitesimilar,butdiffersafterUVAirradiation.Differencesin
theamountofmelaninproducedinthemelanocytes,inthetransferanddistributionof
melanosomes to the keratinocytes, are responsible for the differences in natural UV
protection between humans with different skin colors 127. This relative natural
photoprotectionfactorormelaninprotectionfactoragainsterythemavariesfromafactor
normalizedto1forskintype1tonearly10forskintype6accordingtoCripps40,meaning
thatskintype6ownsduetoitsdarkerskincoloranaturalphotoprotectionthatisten
timesgreaterthanskintype1.KaidbeyreportedaUVtransmissionthroughepidermis
five times higher for Caucasians than for black skin 128. Further, the constitutive
pigmentationwasshowntoaffordaDNAprotectionfactorof2and4forfairandblack
skinnedpeople129.
AbsorptionspectraofUV inducedmelanogenesisanderythemaare similar suggesting
that the two endpoints have a common chromophore most probably in the same
epidermalsite120,130.AlsoerythemalspectrumwasreportedtobesimilartothatofDNA
photodamageintheformofcyclobutanepyrimidinedimers,andbyspectralassociation
formelanogenesis.
2.2.3.Facultativeskincolor
FacultativeskincolorreferstoanUV‐inducedpigmentation.Delayedskinpigmentation
i.e.tanningoccurswithinfewdaysafterUVexposureandlastsformonthsandisreferred
to as melanogenesis, a natural protection. The size and number of melanocytes,
melanosomes andmelanin increase to reinforcenatural defense againstUV exposure;
suntanenhancingthenaturalprotectionfactorbyafactorbetween2and3forskintypes
IItoIV40,131,meaningthatthereisnocorrelationbetweentheleveloftanandprotection
against erythema. UV‐induced tanning means that skin was exposed to UV radiation
inducingaprotectionreaction.
Chapter2.Theory:anoverview 14
Tanningappearstobeanearlyreactionoftheskintosignalthatlong‐termdamagesare
beinginduced77,however,tanningisstillassociatedbypeopletobeatrendy,attractive,
andhealthy looking.Ameanstoachievethisdesirablefashiontanningistheexposure
underartificialsourcee.g.tanningbed.Sincetheearly1970ssunbedindustrywasborn
anduseofsunbedsiswidespreadtoday.Sunbedusersmainlyincludeyoungpeople132.
Tanning beds predominantly emit UVA radiation, although a small amount of UVB
radiation133.TheintensityofUVAradiationoftanninglampscanbe10to15timeshigher
than thatof themidday sun.ThesehighUVAdosesmightbe responsibleof erythema
occurrence reported by some sunbed users 133, their danger on human skin was
addressed 77. An association between the incidence of cutaneousmelanoma and non‐
melanomaskincancerswithsunbeduseespeciallywheninitiationoccursearlyinlifewas
established134‐138.Basedonrisingevidenceaboutthecarcinogenicityofartificialtanning
lamps, regulation on sunbed industrywas strengthen over past decade, especially for
young people; sunbed use is banned for people under 18 in UK and several other
European countries, Australia, parts of Canada and USA, and is completely banned in
Brazil139.
2.3.Artificialphotoprotection
2.3.1.Historyofsunscreens
Besides sun avoidance, shade seeking, clothes and hat wearing, topically applied
sunscreensareasimple,suitable,andefficientmeanstoprotectagainstharmfulphoto‐
damages95.Sunscreenscontainspecialactiveingredients,UVabsorbingcompoundsalso
referredtoasUVfilters,toprovideprotectionagainstUVirradiation.Inthe1950s‐1960s
atthebeginningsofsunprotectionwithtopicalsunscreens,theprimeobjectivewasto
protectagainsterythema,theimmediateandvisiblesundamage.Sinceerythemamainly
originatesfromUVBradiation,thefirstdevelopedUVfilterswereabsorbingintheUVB
range. In1956, theconceptofSPF for rating theprotectionabilityofa sunscreenwas
inventedbySchulze,andallowedadirectcomparisonofperformancebetweensunscreen
products140.Firstly,sunscreensshowedverylowSPFsattainedvaluesupto4.
Chapter2.Theory:anoverview 15
Tremendouschangesinlifestylehabitsduetohigherincomesandpaidholidayledtoan
increase of outside recreational occupations and of vacation to countries where the
sunlightmay not be suited to the skin type. This led to the increase of the sunscreen
marketwiththedevelopmentofproductswithgrowingSPFsattainingvaluesashighas
20inthe1980s.Inthebeginningsofthe1990s,itwasrecognizedthatUVAirradiation
wasnotasharmlessasthought,andratherresultsinlong‐termhealthdamage,especially
withrespecttoprematureageingofphoto‐exposedskin.
Furthermore, over exposure to sunlight owing to sun‐seeking practice of white
Caucasianstogettannedortothetendencyofspendingleisuretimeoutsidealsoledto
long‐termdamage such as skin cancer. This conducted to a slow change in consumer
attitudestowardsunexposure.Over lastdecade, theplacementonthemarketofnew,
photostable,broad‐spectrumandUVAfiltersallowedaconsiderableimprovementofthe
sunprotectionprofileofsunscreensclaimingnowadaysSPFvaluesupto50+141.
2.3.2Requirementsforgoodphotoprotection
Osterwalder&HerzogidentifiedfourkeyrequirementsforagoodUVprotection142:
Technology
Assessmentandmeasurementmethods
Normsandstandards
Compliance
Theserequirementsinterlinkandareinfluencedbydifferentstakeholders;achievingonly
one of these aspects is not sufficient for delivering an adequate photoprotection, e.g.
developingasunscreenwithefficientUVfiltersisnotenoughtoguaranteesatisfactory
photo‐protection. The performance of the sunscreen product has to be evaluated and
characterizedaccordingtostandards,andconsumersmustultimatelyapplytheproduct
inasufficientmannertogettheexpectedUVprotection.
Chapter2.Theory:anoverview 16
2.3.2.1.Technology
UV filters are the core ingredients of sunscreen products; they are able to reduce the
intensityofUVlightreachingtheskin.UVfiltersaregenerallyclassifiedinorganicand
inorganicparticulateUVfilters; theorganicclassbeingfurthersubdividedintosoluble
andparticulatecompounds.ThesolubleorganicUVfiltersactbyabsorption,whereasthe
mechanism of action of particulate UV filters includes absorption, scattering and
reflection143,144.ParticulateUVfiltersareabletoincreasetheopticalpathlengthofUV
radiationduetotheirinherentscatteringproperties,therebyincreasingthelikelihoodof
UVradiationtomeetadissolvedUVfiltermoleculebeforereachingtheskinsurface.They
areabletoamplifytheUVperformanceoftheusedfilteringsystemresultinginaboosting
oftheUVprotection145,146.
OrganicUVfilters
UVfilterscontainsuitablechromophors,agroupofatoms,abletoabsorbwavelengths
greaterthan200nmthatispossiblewithconjugatedπ–electronsystems.Asagenerality,
alargechromophorenablesastrongerabsorption,andagreaternumberofconjugated
double bonds in the molecule shifts the absorption maximum towards longer
wavelengths 147. UV filtersmay be in the form of a liquid, a powder, or a particulate
dispersion. The two commercialized particulate organic UV filters, MBBT (INCI,
Methylene Bis‐Benzotriazolyl Tetramethylbutylphenol) and TBPT (INCI, Tris Biphenyl
Triazine), are obtained from a milling process that results in a water dispersion of
particles whose average size approximates 160nm and 120nm for MBBT and TBPT,
respectively148,149.Theoriginalparticlesizeisreducedtoachievemaximumabsorbance
efficacy;theabsorbingpropertiesbeingdirectlydependentontheparticlesize150.This
type of UV filter combines the advantages of soluble organic UV filters as well as of
particulate inorganicUV filters. Forward andbackward scattering contribute to about
10%oftheoveralleffectintheregionofabsorptionbandforMBBT150.
Chapter2.Theory:anoverview 17
InorganicUVfilters
Micronized titanium dioxide and zinc oxide are the two main representatives of the
categoryofinorganicUVfilters143,151,152;someauthorsproposedceriumoxideasanew
innovativeinorganicfilter153,154,whichis,however,notyetallowedforuseasaUVfilter.
Titaniumdioxideusedforsuncareapplicationshowsaprimaryparticlesizerangingfrom
10to30nm,butformsaggregatesintodispersionresultinginasizeashighas100nmin
formulations.Withthisparticlesize,titaniumdioxideisquitetransparentonskin,tothe
oppositeoftitaniumdioxidegradesusedfordecorativecosmeticswhosesizeisrather
closeto200nmtoprovidedesiredopacityonskinforfoundationforexample.Thepartof
lightattenuated throughabsorptionversus scatteringphenomenonhighlydependson
the size of the particle; in the grades used for sunscreens, absorption is the major
mechanism of action. Titanium dioxide being highly photo‐catalytic 155, the cosmetic
gradesoftitaniumdioxideare,therefore,coatedtopreventtheformationoffreeradicals
under lightexposure;several typesofcoatingexist includingstearicacidandalumina,
silica,dimethicone,oraluminumhydroxideandstearicacid.Regardingzincoxide,itcan
beusedcoatedornon‐coated.
Tobefullyusableinsunscreens,Osterwalder&Herzogdefinedfourbasicrequisitesfor
UVfilters142:
Efficacy:UVfiltersarecharacterizedbyanddifferintheirabsorbanceprofile,E1,1,and
photostabilityprofile
Safety
Registration
Patentfreedom
The lack of one of these requirements highly compromises the chances of
commercializationand/ormarketsuccess;safetyandregistrationbeingamust.
Efficacy
UVfiltersarecharacterizedbytheirabsorbanceproperties.UVB,UVA,orbroad‐spectrum
UVfiltersareavailable,nowadaysitis,therefore,technologicallypossiblebycombining
severalUVfilterswithcomplementaryabsorbanceprofilestocoverthewholeUVrange
for achieving optimum photo‐protection. First UV filters mainly consisted of UVB
absorbingcompoundstoprotectagainsterythema.
Chapter2.Theory:anoverview 18
Overlastdecade,severalUVAandbroad‐spectrumfiltersweredevelopedandplacedon
themarketenablingabreakthroughinsunprotection156,157.
Figure2.1. illustratestheabsorbanceprofileoftwosunscreenswiththesamenominal
SPFvalueof30butdifferentUVAprotection.Thetypicalabsorbanceprofileofan“old”
sunscreen (black line) is UVB biasedwhereas the absorbance of a “today” sunscreen
(dashedline)showsamorebalancedabsorbancewithintheUVAprotectionrange.
Figure 2.1. Absorbance profile of an “old” sunscreen (black line; 10% ethylhexyl
methoxycinnamate, 5% titanium dioxide, 5% zinc oxide) and of a “today” sunscreen
(dashed line; 1.5% ethylhexyl triazone, 2% bis‐ethylhexyloxyphenol methoxyphenyl
triazine,7%methylenebis‐benzotriazolyltetramethylbutylphenol)
Generally, a smaller concentration ofUV filters is necessary forUV filtermixture that
showsabalancedabsorbanceprofile in comparison toaUV filtermixturewithaUVB
biasedUVabsorbance;infigure2.1.aconcentrationof20%ofUVfiltersisrequiredfor
the “old” sunscreen type to achieve the same SPF value as for the “today” sunscreen
requiringaconcentrationofUVfiltersof10.5%only.
Chapter2.Theory:anoverview 19
Besidestheir intrinsicabsorbanceproperties,UVfiltersarecharacterizedalsobytheir
intrinsic photo‐stability and photo‐compatibility with other UV filters 158. The two
worldwideacceptedUVAfilterBMDBM(INCI,butylmethoxydibenzoylmethane)andUVB
filterEHMC(INCI,ethylhexylmethoxycinnamate)areknowntobeveryphoto‐unstable
underUVexposure,thus,resultinginalossofperformance159‐161.Thephotostabilitywas
showntobeimpactedbythesolventused162,163.Moreover,theircombinationleadstoan
increasedphotochemical instabilitydue toa2+2‐hetero‐photocycloadditionproducing
non‐UV absorbing cyclobutylketone photoproducts 162,164. This photo‐incompatibility
finally results in a lower UV protection as expected from the mere spectroscopic
characteristicsoftheUVfilters165.Thisissueoftenobligedsunscreenmanufacturersto
useeithertheoneortheotherfilterintheirsunscreendevelopment.
Molecules that absorbenergy fromUV radiationmove fromaground state (S0) to an
excited singlet state (S1) by a delocalization of an electron. This excited state being
instable, several processes to dissipate the absorbed energy exist either through
emissions or through radiationless pathways as depicted in the Jablonski diagram in
figure2.2.Inthecaseofthephoto‐unstableUVfilterBMDBM,themoleculecanperform
anintersystemcrossingfromthesingletexitedstatetothetripletexcitedstate,thelatter
showingalongerlifetimeand,therefore,promotingphoto‐degradationofthemolecule
166.Asa consequence, thestabilizationofphoto‐unstableUV filters suchasBMDBM is
possibleeitherbyquenchingtheexcitedsingletstate167,168toavoidtheformationofthe
tripletexcitedstateorbyquenchingtheformedtripletexcitedstate169‐171.Triplet‐triplet
energytransferfromthephoto‐unstablemoleculetothequenchermoleculeisthemost
common energy transfer mechanism for photo‐stabilization. To make this process
working, thequenchingmoleculemustshowanequalorslightly lowerenergy levelto
that of the photo‐excited state of the photo‐unstablemolecule in order to absorb the
excitationenergy166,172.
For photostableUV filters the dissipation of absorbed energy occurs through internal
conversion, the absorbed in then released into harmless heat via energy transfer by
collisiontosurroundingmolecules173.
Chapter2.Theory:anoverview 20
Figure2.2. Jablonskidiagramforelectronictransitionsanddissipationpathwaysafter
excitationofamolecule
Safety
UVmoleculesmustatfirstbeapprovedtobeallowedforuseinsunscreens.Arequisite
forapprovalissafety,irrespectivelyoftheregulatoryenvironment.Adossiercontaining
thedatarelatedtoaseriesoftoxicologicalteststoensurehumansafetymustbeprepared
andsubmittedtotherelevantauthority.OnlyUVmoleculesthatareirreproachablewith
respecttotheirtoxicologicalprofilecanbeapproved.Asageneralrule,tests including
skin irritation, skin corrosion, eye irritation, skin sensitization, mutagenicity, toxicity,
carcinogenicity,reproductivetoxicity,andpercutaneousabsorptionarerequiredforthe
human safety and health risk assessment. In Europe, toxicological assessment is
performedaccordingtotheSCCS(ScientificCommitteeonConsumerSafety)guidelines
requirements.ThesafetyisthenevaluatedbytheSCCSpublishingascientificopinionthat
mustbepositivesothattheEuropeancommissionfinallyvotestheofficialadditionofthe
newUVmoleculeontotheannexVIoftheEuropeanCosmeticRegulation1223/2009/EU
listingthepermittedUVfiltersinEurope.Thisisaverystructuredapprovalprocess.
Chapter2.Theory:anoverview 21
Since2013,thereisananimaltestingbanforanynewcosmeticingredientthatleadsto
anunclearsituationregardingtheregistrationofnewUVfiltersasnoinvitroreplacement
existsforallrequestedhumansafetytests.
Registrationstatus
Theregulationofsunscreensstronglydiffersbetweenthemaingeographicalregions174;
sunscreensareregulatedeitherascosmeticsinEurope,Over‐The‐CounterinUS,orquasi‐
drugsinJapan.Tobeallowedforbeingused,UVfiltersmustbelistedonapositivelist
givingallpermittedUVfilterswiththeirmaximumallowedconcentration,e.g.onannex
VIoftheEuropeanCosmeticRegulationforEUorintheFDAover‐the‐countersunscreen
monographforUS.Othersimilarpositivelistingexistsformostcountries.Asageneral
observation, the requirements for registering a newUV filter becomemore andmore
stringent,aswith theexampleof the“nano issue” inEuroperecently.TBPT, the latest
approvedUVfilterinEurope,isanorganicnanoparticulateUVfilterthatwassubmitted
for safety evaluation to the SCCS in 2005 and placed finally on the Annex VI on the
EuropeanRegulationon cosmeticproducts inAugust2014only.Thisdelayof several
yearsintheexpectedregistrationdatewasdirectlylinkedtoconsequencesofthenano‐
relatedconcerntopicandthenewrequirementsofregisteringthenanoformoftheUV
molecule requiring new tests. New UV filters usually are developed, approved and
commercializedatfirstinEuropefollowedveryrapidlybyotherregionssuchasSouth
America,Korea,Japan,andAsean.Totheopposite,intheUSAtheregistrationofanewUV
filterisaverylongprocessthatisverycomplex.TheapprovalofthelastUVfilteronthe
sunscreenmonographintheUSAdatesfrom1998;itwasBMDBMapprovedinEurope
alreadyin1978.ThecreationoftheTEA(TimeandExtentApplication)procedurewas
aimedtoeasetheapprovalofnewfiltersintheUSA.However,thisroutewasnotyetvery
successfulassixUVfiltersincludingEHT(INCI,ethylhexyltriazone),IMC(INCI,isoamyl
p‐methoxycinnamate), BEMT (INCI, bis‐ethylhexyloxyphenol methoxyphenyl triazine),
MBBT (INCI, methylene bis‐benzotriazolyl tetramethylbutylphenol), TDSA (INCI,
terephthalidenedicamphorsulfonicacid),andDTS(INCI,drometrizoletrisiloxane)arein
theTEApipeline;someawaitingforapprovalsince2003.Thismissingregistrationofthe
newestUVfiltersintheUSAlocksthedevelopmentofglobalworldwideformulationsfor
sunscreenmanufacturers and prevents the accessibility of latest technologies already
availableoutsidetheUSAforAmericanconsumers.
Chapter2.Theory:anoverview 22
Patentfreedom
The UV filter molecule and its combination with other UV filters and formulation
excipients should be intellectually protected as largely as possible by the UV filter
manufacturer.ThisisnecessarytoensurefreedomofuseoftheUVfilteringredientby
anysunscreenmanufacturer.Theriskofweakpatentprotectionofanewcompoundis
that the new molecule is blocked from third party patents in specific ingredient
combination or application claims that hinder other sunscreen players to use the UV
compound in the specific patented claims. This can be a very strong limitation of the
concernedUVfilter,itsuse,marketpenetrationandgrowth,aswellasfinallyfortheend
consumerwhoinsomecasescannotbenefitfromthenewesttechnologies.
Besidestraditionalpatentfilling,itisnowadayspossibletostrategicallyquicklypublish
on the internet information related to thenew ingredient e.g. combinationsor claims,
enabling creation of prior art in the form of technical disclosure to prevent blocking
patentsfromthirdparties(e.g.www.ip.com).
AsummaryofthemainUVfilterswiththewavelengthoftheirhighestabsorbance(max),
their E1,1, (absorption corresponding to a concentration of 1% (w/v) solution at an
opticalthicknessof1cm),andregistrationstatusisgiveninTable2.2.
Chapter2.Theory:anoverview 23
Table2.2.MainUVfilterswiththeirspecificcharacteristics
UVfilter
(INCIabbr.)
max
(nm)*
E1,1* Registration
status
Physicalform
BEMT 310&343 736&819 Worldexcept
USA,inTEA
Powder,oilsoluble
MBBT 305&360 419&519 Worldexcept
USA,inTEA
Particulatewater
dispersion
DHHB 354 900 Worldexcept
USA
Powder,oilsoluble
BMDBM 357 1120 World Powder,oilsoluble
TBPT 310 1170 Europe Particulatewater
dispersion
EHT 314 1448 Worldexcept
USA,inTEA
Powder,oilsoluble
EHMC 311 803 world Liquid,oilmiscible
OCR 303 355 world Liquid,oilmiscible
PBSA 303 927 world Water soluble, to be
neutralized
EHS 305 196 world Liquid,oilmiscible
TiO2 290 500 world Particle, powder or in
dispersion
*ThedataofthemaxandE1,1wereprovidedbyBASF
The values for TiO2 depend on the commercial grade; here the values correspond to
EusolexT‐2000fromMerck.
Chapter2.Theory:anoverview 24
2.3.2.2.Assessmentandmeasurementmethods
The efficacy of sunscreens is largely expressed by the SPF value and level of UVA
protection. Methods to measure these two parameters are, therefore, necessary to
characterizethelevelofprotectionofasunscreenwithrespecttothesetwocriteria.Test
methods can be based on in vivo, in vitro, or in silico methodologies. As a general
statement, in vivo methods show the drawbacks of being costly, time consuming and
ethicallyquestionable. Therefore, thedevelopment of invitromethods that are faster,
simpler,andcheaperisofgeneralinterest.
Sunprotectionfactor(SPF)
The SPF value gives the degree of protection afforded by a topical sunscreen against
erythema;itwasthefirstcriterionintroducedfordescribingthelevelofprotectionofa
sunscreen.Itremainsavery‐wellknownprotection‐relatedindicationfortheconsumer
and also a purchase criterion 175. It can be tested in vivo, in vitro, or even in silico;
nevertheless, only the invivoprocedurehasbeenvalidated and is approved so farby
regulatorybodies9.
Figure2.3.illustratestheerythemaeffectivenessspectrumshowingthewavelengthrange
responsibleforerythemaformation(blackline)176.Theerythemaeffectivenessspectrum
is the product of the erythema action spectrum 9,176 that gives human sensitivity to
erythema(grayline)andthespectralirradianceofterrestrialsunlight(dashedgrayline)
givenhereformiddaymidsummersunlightforSouthernEurope(latitude40°N)15.Itis
clearfromfigure2.3.thaterythemaoriginatesprimarilyfromUVBradiation,butfigure
2.3.alsorevealsthatUVAII(320‐340nm)radiationcontributestoacertainextendtothe
erythemadevelopmentaswell177,178.
Chapter2.Theory:anoverview 25
Figure2.3.Erythemaeffectivenessspectrum(black line)expressingtheoccurrenceof
erythemadependentonwavelengthbeingtheproductoftheerythemaactionspectrum
(grayline)9,176andtheterrestrialsolarspectrum(dashedgrayline)15
o SPFinvivo
SPFinvivoisthegoldstandardfortheevaluationofsunscreenefficacy;itisdefinedasthe
ratio of minimal erythemal dose (MED) on sunscreen protected skin (MEDp) and
unprotectedskin(MEDup)andisexpressedbyEquation(2.2.):
SPFinvivo=MEDpMED up
(2.2)
The MED describes the minimal UV energy required to initiate the first perceptible
erythema,orminimalerythemalresponse.Erythemaresponseismaximum6to24hafter
irradiationdependingontheapplieddose179.Itisevaluatedbyapplyingincrementally
increasingUVdosesfromanartificiallightsourcewithasolar‐simulatedspectrum9on
areasofhumanvolunteers`backonsunscreenprotectedandunprotectedzones.Asthe
MEDp and MEDup are determined on the same human volunteers, skin type is not
impactingthedeterminationoftheSPFinvivo.
Chapter2.Theory:anoverview 26
Thedeterminationofthismerebiologicalendpointdoesnotprovideanyinformationon
theabsorbanceprofileofthestudiedsunscreen,meaningthattwosunscreensmayexhibit
thesamenominalSPFvaluebutmayhighlydifferintheirUVAprotectionasexplained
previously(section2.3.2.1,figure2.1.).
o SPFinvitro
Because of the drawbacks of in vivo testing, much effort has been placed into the
development of an in vitro methodology for SPF determination. However, up to now,
despitecosmetic,pharmacologicalandchemicallaboratories,institutes,andtaskforces
putalotofeffortsindevelopinganinvitroSPFtechniquecorrelatingwiththeclinicalin
vivoSPF,noundertakenattemptledtoreproducible,repeatableandreliableoutcomes.
Manychallengesremain19,amajorissuemostprobablyisthesubstrateusedtoapplythe
sunscreen.SPFinvitrodeterminationisbasedonthemeasurementofUVtransmittance
through a layer of sunscreen applied on a suitable UV transparent substrate 13. UV
transmittance represents the inverse of an UV attenuation factor of a protecting
sunscreenfilmdescribedbythefollowingrelationship12:
SPF ∑ ser λ . Ss λ
∑ ser λ . Ss λ . T λ 2.3.
where, the inverse transmittance (1/T) in theUV spectral range isweightedwith the
erythemaactionspectrum9,ser(λ),andthespectralirradianceoftheUVsource9,Ss(λ).As
dataforser(λ)andSs(λ)areavailablefromliterature,theSPFinvitroisdeterminedfrom
UVtransmittancebetween290and400nmbeforeandafterapplicationofasunscreen
appliedonsuitableUVtransparentsubstrate13.Manydifferentkindsofsubstrateshave
been used since the beginnings of invitro SPF including either biological or synthetic
substrates. Biological skin substrates used for testing sunscreen performance include
epidermisofhuman14,180ofpigear181,182,andofhairlessmouse12,183.
Chapter2.Theory:anoverview 27
Syntheticsources includematerialssuchasone‐sideroughenedquartzplates, surgical
adhesivetape(transporetape)fixedona flatquartzplate14,15,184,syntheticskin(vitro
skin)185,andPMMAplates10,16,17,thelatterarepresentlyfavored.PMMAplatesareeither
sand‐blastedormoldedononesidetocreateacertainroughnessvaryingbetween5to
17mdependingontheplatetype,supposedtosimulateroughnessofhumanskin18,186.
However, none of these substrates succeed in achieving a reproducible method that
furthercorrelatedwiththeclinicalstandardSPF19.Severalfactorswereshowntoimpact
theSPFinvitromeasurement187,188,onemajorfactormostprobablybeingthesurface
properties189.Toproducerelevantdata,thesubstrateshouldatbestsimulatehumanskin
characteristicswithrespecttoroughnessandmoreparticularlywithrespecttosurface
energypropertiestoreproduceatbesttheapplicationoftheinvivosituation.However,
surface freeenergyofcurrentlyemployedPMMAplatesdonotreproducehumanskin
surface properties; different solutions were proposed to increase the product‐to‐
substrate affinity 189,190.However, none of these attemptswere verypromising as not
applicableforallformulations.
o SPFinsilico
Someauthorsintroducedaninsilicoapproachforthecalculationoftheperformanceof
sunscreens191,192.TheevaluationoftheSPFinsilicomakesuseofthesamealgorithmas
for thedeterminationof theSPF invitro (Equation(2.3.)).However, themeasuredUV
transmittanceusedfortheinvitromethodissubstitutedbyacalculatedtransmittancein
theinsilicoapproach.ThecalculationoftheUVtransmittancerequiresthespectroscopic
performances (spectral average molar absorption coefficient and the molar
concentration)ofthestudiedUVfiltermixture193,theamountoftheusedUVfilters193,
andthepropertiesoftheappliedfilmmeaningthenominalaveragefilmthicknessalong
withamathematicalmodeltodescribesunscreenfilmirregularityprofile.Severalmodels
forexpressingfilmthicknessdistributionweredescribedstartingfromthe“two‐stepfilm
model”byO`Neil in198425,the"four‐stepfilmmodel"byTunstall194, the"calibrated
two‐stepfilmmodel"byHerzog193tothe“continuousheightdistributionmodel”usinga
GammafunctionbyFerrero191,195andthecalibratedquasi‐continuousstepfilmmodelby
Herzog196.The“sunscreensimulator”calculationtoolfromBASF,freelyavailableonthe
internet(www.basf.com/sunscreen‐simulator)allowsthecalculationoftheSPFandUVA
indices.
Chapter2.Theory:anoverview 28
FormoreprecisionandcorrelationofpredictedSPFinsilicovaluestotheinvivovalues,
thistoolfurtherconsidersthephoto‐instabilitiesoftheindividualUVfilters,thephoto‐
incompatibilitiesbetweenUVfilters,thephoto‐stabilizationeffectofsomeUVfilterson
others197aswellasthesynergisticeffectobtainedfromthedistributionofUVfiltersin
theoilandwaterphaseofanemulsion192.Thepotentialeffectoftheformulationbaseis,
however,notyettakenintoaccount.
o MeaningofSPF
AwidespreadmisconceptionisthatSPF60isnottwiceaseffectiveasSPF30duetothe
smalldifferenceinpercentageoffilteredUVradiationbetweenthesetwoSPFs,96.7%and
98.3%forSPF30andSPF60,respectively.However,amuchmorerelevantcriterionfor
UVprotectionishowmuchUVradiationistransmittedtotheskin,e.,g.3.3%and1.7%for
SPF30andSPF60, respectivelymeaning thatonlyhalfofphotonswill reach the skin
whenusingaSPF60comparedtoaSPF30.Thisisafactor2differenceconfirmingthat
SPF60istwiceaseffectiveasSPF30intheamountoflighttransmitted198.
UVAprotection
ComparedtoUVBirradiation,UVAirradiationratherresultsinlong‐termsundamages,
and, therefore,wasconsidered fora long time,wrongly,asharmlessregardinghuman
health.EvidenceonUVA‐relatedhealthdamagesconductedtothedevelopmentofUVA
andbroad‐spectrumfilters173.Several invitroand invivobasedmethodsforassessing
theperformanceofasunscreenagainstUVAexposurewereintroducedinthedifferent
worldregions.DifferentmethodsbecamestandardforUVAtestingindifferentregions;
UK,Japan,andAustraliawerethepioneersinUVAprotectiontesting.
o 1992‐UK
In 1992, Boots introduced in the UK the Boots star rating system based on the
determinationoftheUVA:UVBratio invitro, thatistheaverageabsorbanceintheUVA
dividedbytheaverageabsorbanceinUVBrange.Themethodwasrevisedin2008andin
2011withtheintroductionofanUVexposurestepwithafixeddoseof17.5J/cm²and
representsstillthestandardforUVAtestinginUK199.Theprotectionisexpressedasa
numberofstars,accordingtothevalueoftheratiobeforeandafterirradiation.
Chapter2.Theory:anoverview 29
o 1995‐Japan
TheinvivodeterminationoftheUVA‐PFcorrespondingtothemeasurementofthePPD
wasthefirstofficialstandardforUVAtestingestablishedinJapanbytheJapanCosmetic
IndustryAssociation(JCIA)in1995.UVA‐PFismeasuredsimilarlytotheSPFinvivo,but
usinganUVAlampforirradiationexcludingUVBradiationtoproduceUVA‐relatedskin
persistentpigmentdarkening.UVA‐PFinvivoistheratioofminimalpersistantpigment
darkeningdose(MPPDD)onsunscreenprotectedskin(MPPDDp)andunprotectedskin
(MPPDDup).TheMPPDDisdefinedasthelowestUVAdoseneededtoinducedemarcated
andeasilyidentifiedpersistentskinpigmentation.AccordingtothevalueoftheUVA‐PF,
differentlevelsofUVAprotectioncanbeclaimed.Since2011,thisprocedureisanofficial
ISOmethodandisstillusedinJapanastheofficialUVAprotectiontesting200.
o 1998‐Australia
In1998AustraliaestablishedtheinvitroAustralianStandardbasedonUVtransmittance
measurementof tested sunscreen in anoptical cell of 8mthickness.Broad‐spectrum
claimswereallowedwhenthetransmissionatanywavelengthbetween320and360nm
waslowerthan10%.Thisparameterisapass/failcriterionthatwasveryweakasitwas
achievedveryeasily,especiallywithlargeSPFvalues.
o 2006‐Europe
Theeffortofsuncarestakeholders,taskforces,suncaremanufacturers,institutesfora
globalharmonizationresultedinthevalidationandpublicationofanofficialISOmethod
in2010forUVAtestingthatis,nowadays,usedasastandardinmanyregionsincluding
Europe,Australia,China,SouthAmerica201.Itisbasedonacombinationofinvitroand
invivomeasurements.TheUVA‐PF(UVAprotectionfactor)iscalculatedfrominvitro
absorbancemeasurementfrom320to400nmbeforeandafterirradiationaccordingto
Equation(2.4.):
Chapter2.Theory:anoverview 30
CPPDUVA
UVA
TSs
Ss
PFUVA
PPD
invitro
400
320
400
320 (2.4)
where, SUVA is the spectral irradiance for the UVA source 201, SPPD is the persistence
pigmentdarkeningactionspectrum201,andCanadjustableparametertoadjustthe in
vitro spectruminsuchawaythatSPF invitroequalsSPF invivovalue.UVA‐PF is first
calculatedfromthetransmittancecurveofunexposedsampleafteradjustmenttothein
vivoSPFbymultiplyingtheabsorbancevalueswiththescalingfactorC.TheUVA‐PFvalue
beforeirradiation,UVA‐PF0,isusedfordeterminingtheirradiationdosecorresponding
to1.2xUVA‐PF0inJ/cm2.TheUVA‐PFafterirradiationiscalculatedaspreviouslyafter
mathematicaladjustmentoftheabsorbancecurveusingthesamevalueforfactorC.The
UVA‐PFmustbeatleastonethirdoftheSPFinvivotoclaimanUVAprotection.
o 2011‐US
In theUSA, theFDAadopted in the final sunscreenmonographpublished in2011 the
criticalwavelength(c) invitromethodbasedontheapproachintroducedbyDiffeyin
1994202withtheadditionofafixedpre‐irradiationstep.Itconsistsofdeterminingthe
wavelengthatwhichthespectralabsorbancecurvereaches90%oftheintegraloverthe
UV spectrum from 290 to 400nm; the larger the c, the greater should be the UVA
protection.Testedsunscreenmust reachat leastacvalueof370nmtobeallowed to
claimbroad‐spectrumprotection.Thismethod,however,appearstobeaweakcriterion
that is reached easily especially with larger SPFs and that does not allow huge
differentiationbetweensunscreenswithrespecttoUVAprotection203,204,moreover,the
fixedirradiationdoseismodestconsideringthehighestallowedSPFclaimof50+.
Factorsthatimpactperformanceofsunscreens
o Amountofappliedsunscreen
Majorinfluencingfactorfordeliveredprotectionistheamountofsunscreenapplied.The
observedrelationshipbetweenSPFandapplicationamountisquasi‐linear19,205.
Chapter2.Theory:anoverview 31
Though,forUVBbiasedsunscreens,thisrelationshipshowsasaturation‐likeeffectofthe
SPFwithincreasedapplicationamount,indeedtheUVBloadedsunscreenwillcontinue
to transmit theerythemallyactiveUVAII radiation independently fromtheapplication
amount.Indeed,amere“UVBsunscreen”wouldreachinprincipleamaximumSPFof11
only206.Ontheotherhand,homeostasicsunscreens,withsimilarUVBandUVAprotection
willrathershowanexponentialbehaviorindependenceontheapplicationamount.Yet,
mostsunscreensonthemarketshowalinearrelationship.ClinicalSPFismeasuredusing
adefinedapplicationamountofsunscreenof2mg/cm²,however,consumersgenerally
usemuchless.Only18%ofrespondentsofaninterviewaboutsuncareknowledgeinNew
Jersey know about the right amount of sunscreen to apply 175. Methods employed to
estimatetheapplicationamountofpeopleunderreallifeareoftenbasedonweighingthe
sunscreenbottlebeforeandafterusebynaïvevolunteersandconvertingintoanamount
inmg/cm²207‐210.Otherauthorsusedanapproachbasedonfluorescencespectroscopy211
oratechniqueusingswab212.Thesestudiesrevealedthatconsumersusuallyapplyonly
aquartertohalfoftheamountusedforofficialinvivoSPFdeterminationmeaningthat
thedeliveredSPFishalfashighasclaimed.
o Spectralsource
Thespectralirradianceofthelightsourceusedforinvivoperformanceevaluationdiffers
from spectral irradiance of terrestrial sunlight as depicted in figure 2.4. The solar‐
simulatedlightsourceusedforinvivotestingisUVBbiasedandisfilteredintheUVAIand
visiblerange9comparedtotheterrestrialsunspectrum15.Thesedifferenceshavenearly
noconsequencesonthepredictionoftherealprotectiontonaturalterrestrialsunlight
exposureforsunscreensshowingabroad‐spectrumabsorbanceprofileastheprotection
providedisuniformindependentlyfromthewavelength.Ontheotherhand,theSPFis
overestimated forUVBbiasedsunscreensundersolar‐simulated lightsourceexposure
comparedtotherealsunprotectionundernaturalterrestrialsunlightexposure26,213,214.
Chapter2.Theory:anoverview 32
Figure2.4.Terrestrialsolarspectrum15versussolar‐simulated9spectrum
o Impactofskinstatusonerythemasensitivity
Thestatusoftheskine.g.dryversuswetpriorUVexposurewasreportedtoimpactlight
transmissionthroughskin.Pre‐immersionofskinintoliquidmediapriorUVirradiation
leadstoanincreaseinlighttransmissionduetothereductionofreflectionandscattering
onto the skin surface and to the reductionof internal scattering in the cell layers and
intercellular material, the skin becoming more transparent 215. The transmission
increasesoverallwavelengthsastherefractiveindex(RI)oftheliquidinwhichtheskin
isimmersedapproachestheRIofskin(RIofstratumcorneum=1.52107).Immersingskin
into liquids with RI greater than water (RI of water=1.33) such as emollients that
generallyshowRI>1.45resultsinagreaterlighttransmittancethroughskinthanwater
does.Thiswasshowninhumanvolunteersaftertheapplicationofanoil‐in‐water(OW)
formulationthatincreasedUVlighttransmissionthroughtheepidermisby20%between
300and410nm216.Thiseffectisresponsiblefortheincreasedsensitivitytoerythemafor
wet skin e.g. during swimming or sweating, resulting in a reduced MED and a more
erythematousskinforwetcomparedtodryskin.Thiswastestedinhumans217aswellin
hairlessmiceandalbinosrabbitsskin218.
Chapter2.Theory:anoverview 33
o Sunscreenvehicleandapplication
SomeauthorsreportedthatsunscreenscontainingthesameUVfiltermixtureproduced
different SPF values 20,21 and the homogeneity of the spread sunscreen film was of
importanceforperformance24,27.
2.3.2.3.Normsandstandards
ThesettingofnormsandstandardsisessentialforcharacterizingagoodUVprotection.
Regarding SPF measurement, there is more or less an harmonization in the SPF
measurementandclaimaspublishedbytheOfficialJournalofEuropeanUnionin2006
219.
For UVA protection, the global picture is much more complicated than for the SPF
criterion since a variety of methods and parameters are available to express UVA
protection differing between the regions. Some methods are based on a pass / fail
criterion,someonaratingsystem.Sincetheproceduresoftestinge.g.invivoorinvitro,
theirradiationstep,andtheclaimsdifferbetweenthemethods,acomparisonofthelevel
ofprotectionagainstUVAexposurebetweenthemethodsisdifficult.
Table2.3.summariestheUVAtestmethodsandassociatedallowedclaims.
Table2.3.SummaryofUVAstandardsandassociatedUVAprotectionclaims
Region EuropeAustraliaMercosur
UK Japan USA
Method ISO24443 Bootsstarrating
ISO24442 Final rulesunscreenmonograph
UVAfactorinvitroinvivo
UVA‐PF&cUVA‐PF(PPD)
UVA:UVBratio‐
‐UVA‐PF(PPD)
c‐
UVA Claimandconditions
UVA‐PF/SPF1/3andc370nm
from three tofivestars
PA+(UVA‐PF:2‐4)PA++(UVA‐PF:4‐8)PA+++(UVA‐PF:8‐16)PA++++(UVA‐PF16)
Broad‐spectrumwhenc370nm
Chapter2.Theory:anoverview 34
2.3.2.4.Compliance
AsunscreenexhibitingagreatSPFandgoodUVAprotection,havinggoodphotostability,
and being water resistant can only be fully effective and provide the expected
photoprotectionifthefinaluserappliesitintheamountusedintheperformancetesting
procedureandasuniformlyaspossible.Thisisknownasconsumercompliance.Lackof
compliancehasdifferentreasons,technological,UVknowledge‐related,awareness,and
variedmessagesthroughpubliceducation.
Technologicalreasons
Amongthementionedtechnologicalreasons,thebadsensorialaspectofsunscreense.g.
tackiness,greasiness,difficultyofapplication,isamajorfactor220‐222.Aestheticsappears
tobeakeycriterionfortheamountappliedbyvolunteers223.Aconsumerstudywithfour
distinctsunscreenshasshownastrongcorrelationbetweenthedistributionproperties
andthewillingnesstousethesunscreen93.Itis,thus,theultimateobjectiveforsunscreen
manufacturerstodevelopformulationsthatimproveconsumercompliancebyproposing
productsthatconsumersarewillingtoapplyproperlytoachievethepromisedprotection
thatis,therightamountinauniformway.
UVknowledge‐relatedreasons
Merely50%oftherespondentsknowthemeaningofSPF,however,only18%knowabout
therightamounttoapply175.
Awareness
Quiteahighpercentageofindividuals,86%,70%,and64%ofrespondentsofastudyon
sunscreenknowledgeknowthatsunscreencanpreventsunburn,skincancer,andsigns
ofskinaging,respectively175.DespitethespreadknowledgeofUV‐induceddamages,83%
ofyoungadultsreportedatleastonesunburnduringthesummer.
Chapter2.Theory:anoverview 35
Publiceducation
Increaseawarenessofsun‐safetybehaviorsisprimordial.Wangsummarizedtheaspects
of public education in photoprotection 224. There are twomainmotivation factors to
increase awareness of people on UV‐induced photodamage aiming at increasing
compliance.Thesearehealth‐basedandappearance‐based225‐227;health‐basedmessages
focusingonskincancerrisksandappearance‐basedonskinaging.Messagesshouldcome
from health care providers, or media and organization; they should be simple,
straightforward,andappealtopeople´sintellectualandemotionalreceptivity.
2.3.3. The ideal sunscreen, outlook in the future of
photoprotection
2.3.3.1.HomeostasicUVprotection
TherearetwobasicdimensionsinUVprotection,thequalityandquantityofprotection.
The ideal sunscreen shouldprotect against thedifferent knownphoto‐damages, short
termaswellaslong‐term,particularlysunburn,skinphoto‐aging,andskincancer,coming
rather from theoneor theotherwavelength range.Duringevolution,humanskinhas
evolvedandadaptedtobeinharmonywiththeterrestrialsolarspectrum122.Thismeans
thatinavoidingsunorseekingnaturalshadethequantityofsunlightreachingourskinis
quantitativelyreducedwhileonlyminimallyqualitativelymodified.asanexample,fabrics
areanefficientmeansofhomeostasicUVprotectionasfabricabsorbslightuniformlyover
thewholeUVrange228,229.Further,protectingskinbywearingfabricduringUVexposure
wasshowntoreducephotoaging,skinpigmentation,andskindehydration230.
The ultimate goal is, therefore, the development of innovative sunscreens thatwould
protectsimilarlytotextile.Theidealsunscreenshouldprovideuniformprotectionover
theentireUVrangeinordertoattenuatetheintensityofsunlightwithoutmodifyingthe
qualityofthisnaturalsolarspectrumtowhichhumanhasadaptedandevolved.
Chapter2.Theory:anoverview 36
2.3.2.2.Benefitsofdailyphotoprotection
TherearemoreandmoredailycareproductscontainingUVprotectiononthemarket
withSPFreachingvaluesupto30.ThereisnorecommendationontheUVAprotectiona
dailycareshouldafford;however,itismeaningfulthatdaycareproductsprovideabroad
protectionovertheUVrange, ideallyattaininghomeostasis.Exposuremeasurementto
solar UV radiation in an urban environment during typical outdoor activities e.g.
shopping,walking,sittinginacafé,cycling,oratanopenairpoolrevealedthatthereare
somerisksituationsandUVprotectionshouldbeappliedforcertainactivitiesevenina
city231.AdailyUVprotectionwasshowntoreducesignificantlyUV‐inducedhistologic
damageinhumanskincomparedtotheprotectionaffordedbysunscreenswithequalor
higherSPFvalueappliedinanintermittentmanner232.Adaycarewithaphotostableand
broad‐spectrum protection was shown to prevent major alterations connected with
photoaging233,234,abalancedabsorbancespectruminUVAachievingbetterprotection
againstfibroblastalterationandMMP‐1release,higherSPFdonotcompensateforlow
UVAprotection235,dailyuseofabroad‐spectrumsunscreenwasalsoshowntoreduce
solarkeratoseaprecursorofSCC236andtoprovideabetterprotectionagainstUVinduced
suppressionofcontacthypersensitivity237.Itis,therefore,highlyrecommendedtoapply
adailyUVprotection,moreparticularlywithabroad‐spectrumabsorbanceprofile.
Chapter3
Porcineearskinasa
biologicalsubstratefor
invitrotestingofsunscreen
performance
3.1.Abstract
Thepurposeofthestudywastoexaminetheuseofskinfromporcineearasabiological
substrateforinvitrotestingofsunscreensinordertoovercometheshortcomingsofthe
presently used polymethylmethacrylate (PMMA) plates that generally fail to yield a
satisfactorycorrelationbetweensunprotectionfactor(SPF)invitroandinvivo.Trypsin‐
separated stratum corneum and heat‐separated epidermis provided UV transparent
substrates that were laid on quartz or on PMMA plates and were used to determine
surface roughness by chromatic confocal imaging and measure SPF in vitro of two
sunscreensbydiffusetransmissionspectroscopy.
M.Sohnetal.,“Porcineearskinasabiologicalsubstrateforinvitrotestingofsunscreenperformance,”SkinPharmacol.Physiol.28(1),31–41(2015).
37
Chapter3.Pigskinforinvitrotesting 38
Therecoveredskinlayersshowedalowerroughnessthanfullthicknessskinbutyielded
SPF in vitro values thatmore accurately reflected the SPF determined by a validated
procedure invivo thanPMMAplates, although the latterhad inpart roughnessvalues
identicaltothoseofintactskin.
CombinationofskintissuewithahighroughnessPMMAplatealsoprovidedaccurateSPF
invitro.Besidesroughness,theimprovedaffinityofthesunscreentotheskinsubstrate
comparedtoPMMAplatesmayexplainthebetterinvitropredictionofSPFachievedwith
theuseofbiologicalsubstrate.
3.2.Introduction
Overthepastdecades,lifestylehabitshaveundergonesubstantialchangeswithamarked
trend for outside recreational occupations that have led to generally higher and
uncontrolled exposure of people to solar radiation. Although ultraviolet (UV) sun
radiation isvitalwithbiologicalbenefitssuchas thesynthesisofvitaminD1, it isalso
recognizedthatexcessiveexposuretosolarradiationcausesdetrimentalhealthissues.
UVAandpartlyUVBraysreachhumanepidermisanddermisatanintensitythatenables
themtoproducediverseimmediateorlong‐termphoto‐damages,asthoroughlycompiled
bySeitéandMatsumura238.
BesidestheappearanceoftheknownerythemamainlyasanimmediateresponsetoUVB
exposure,amajoradverseeffectisDNAdamages51thatcan,onthelongrun,leadtoskin
cancer. UVA radiation is mostly responsible for chronic photo‐damages such as skin
pigmentation (age spots), inductionofoxidative stress 65, photoimmunosuppression 2,
visibleeffectsofprematureskinageing76andcontributiontoskincancerbygeneration
ofradicaloxygenspecies35.
Topicallyappliedsunscreensconstituteasuitableandcommonlyemployedmeasureto
protectskinfromsundamages.Todate,theSPFisstillthepredominantcriterionusedto
describethedegreeofphoto‐protectionaffordedbyatopicalsunscreen.
Chapter3.Pigskinforinvitrotesting 39
TheonlyvalidatedprocedureforSPFdeterminationisaninvivomeasurementonhuman
volunteers9basedonerythemalresponse,abiologicalendpointmainlyattributedtoUVB
radiation. In vivo methods have the drawbacks of being costly, time consuming and
ethically questionable. Therefore, there is considerable interest from the industry in
developinganinvitroapproachtoSPFtesting.
AlthoughindustryplayershaveputalotofeffortindevelopinganSPFinvitrotechnique
thatcorrelateswiththeclinicalSPF invivo,noundertakenattempthasbeenvalidated,
manyissuesstillremaining19.
OnemajorinfluencefactorforsuccessfulestablishmentofastandardmethodforSPFin
vitrotestingisthechoiceofasubstrateforsunscreenapplicationthatbestmimicshuman
skin. The current use of roughened polymethylmethacrylate (PMMA) plates for this
purposefailedtoyieldsatisfactoryresults19.Thereasonofthepersistingdiscrepancies
between in vivo and in vitro datamight be that PMMAplates do not properly imitate
humanskin.
Attemptstousealternativesubstratestobetterimitateskinsurfacehavebeenreported.
VeryearlystudieswithhairlessmouseepidermisforSPFinvitromeasurementswitha
scanning spectrophotometer provided encouraging results 12,183. Other workers used
humanepidermisassubstrateanddemonstratedagoodcorrelationbetweeninvitroand
invivoprotectionfactorthatwasmeasured,however,onlyatonewavelength180.
The aimof thepresentworkwas to investigate theuse of skin fromporcine ear as a
biologicalsubstratefor invitrotestingofsunscreenperformance.Thepigearskinwas
compared to PMMA plates that are currently the industry standard for SPF in vitro
measurement. Porcine skin is already extensively employed in pharmacological and
toxicologicalresearchasaninvitromodelofhumanskinbecauseofthehighdegreeof
similaritybetweenthetwotissues239,240.Anumberofstudiesemployingpigasinvitro
modelofhumantissuehavebeensummarizedbySimon241.Thesestudiesreportofthe
overallanatomicalandphysiologicalresemblancebetweenpigandman.Thelikenessof
stratumcorneum(SC)betweenskinofporcineearandhumanskinencompassesseveral
aspects.Corneocytesofpigskinhaveapolygonalshape242,243andsize243,244whichare
close to the morphological examinations reported for human corneocytes 243,245.
Moreover,thickness239,240,242,243,barrierfunction239,andpenetrationproperties246ofthe
SChavebeenfoundtobeanalogousinpigandinhuman.
Chapter3.Pigskinforinvitrotesting 40
AUVtransparentsubstrate,whichisaprerequisitefortransmittancemeasurement,was
obtainedbyisolatingonlytheupperskinlayersofpigearsusingtwodifferentpreparation
methods.Inafirststep,wecharacterizedtherecoveredupperskinlayerswithrespectto
thicknessandroughnessandcomparedtheresultstodataavailableforhumanskin.Ina
secondstep,wemeasuredtheSPFinvitrooftwodistinctivesunscreensusingthedifferent
porcine skin substrates and a standardized solar irradiance profile. The results were
compared to SPF in vitro obtained with PMMA plates and to the SPF in vivo of the
individual sunscreens and evaluated with respect to substrate properties that are
relevantforproperpredictionofSPF.
3.3.Materialsandmethods
3.3.1.Chemicalsandequipment
Thefollowingreagentswereused:Trypsin2.5%(10X)liquid(Gibco,Zug,Switzerland);
sodium chloride, sodium hydroxide 1 M, sodium phosphate monobasic and trypsin
inhibitor from glycine max (Soybean) 10000 U/mg (Sigma‐Aldrich, St Gallen,
Switzerland); Tinosorb S, Tinosorb M, Uvinul T150, Uvinul A Plus, Uvinul MC80
abbreviatedasBEMT,MBBT,EHT,DHHB,EHMC,respectively(BASFAG,Ludwigshafen,
Germany);Eusolex232abbreviatedasPBSA(Merck,Darmstadt,Germany).
Quartz plates were obtained from Helma Analytics (Zumikon, Switzerland),
polymethylmethacrylate (PMMA) plates from HelioScreen Labs (Marseille, France),
SchönbergKunststoffe(Hamburg,Germany)andShiseidoIricatechnology(Kyoto,Japan),
andpetridishesfromNunc(Roskild,Denmark).
Chapter3.Pigskinforinvitrotesting 41
The following equipment was used: Electric shaver (Favorita II GT104, Aesculap,
Germany), epilator (Silk‐épil7 Xpressive Pro, Braun, Germany), dermatome (Air
Dermatome,ZimmerInc.,UnitedKingdom),waterpurificationequipment(Arium61215,
Sartorius, Goettingen, Germany), Raman confocal laser scanning microspectrometer
(Alpha500R,WITec,Ulm,Germany),surfacetextureanalysis instrument(Altisurf500,
Altimet SAS, Thonon‐les‐Bains, France), UV transmittance analyzer (Labsphere UV‐
2000S,LabsphereInc.,NorthSutton,NH,USA).
3.3.2.Preparationofbiologicalsubstrate
Ears of freshly slaughtered pigs were obtained from the local slaughterhouse (Basel,
Switzerland) no more than a few hours postmortem. The study did not require the
approvaloftheethicscommitteeofanimalresearchastheearsweretakenfrompigsnot
specifically slaughtered for the purpose of this study. The ears were washed under
runningtapwater,shaved,andepilated.
Thefullthicknessskinofthedorsalsidewasremovedfromtheunderlyingcartilageusing
a scalpel and served as the starting material for further preparation. Two different
methodswereusedfortissuepreparation.Themethodsandtheusedsupportmaterials
aresummarizedinTable3.1.
Table3.1.Skinsampletypesusedinthestudy
Skin
preparation
Materialfordeposition Analysis
Trypsin‐
separatedSC
Quartz UVTransmittancemeasurement
PMMASPFMasterPA‐01 UVTransmittancemeasurement
Petridish Thicknessmeasurement
Heat‐
separated
Quartz UVTransmittancemeasurement
epidermal
membrane
Petridish Thicknessmeasurement
Chapter3.Pigskinforinvitrotesting 42
3.3.2.1. Method 1 ‐ Isolation of stratum corneum (SC) by trypsin treatment
(modifiedmethodafterKligman247)
Sheets of full thickness skinwere dermatomed to a thickness of around500µm. This
tissuewasimmediatelyusedorstoredat‐20°Cuntilfurtheruse.Afterwashingwithwater
purifiedbyreverseosmosis,thedermatomedskinwaslaidflatwiththestratumcorneum
facingupwardonfilterpaperssaturatedwithtrypsinsolution(0.5%inphosphatebuffer
atpH7.4)inaglasspetridishandstoredfor4hat37°Cinasaturatedvaporatmosphere.
The digestion occurred from the dermis end of the tissue, ensuring that SC remained
undamaged.Thetoplayerrepresentingstratumcorneumwascarefullyremovedusing
forcepsandwashedwithpurifiedwater.ComparedtoKligman247,therecoveredSCslice
wasadditionallyimmersedintrypsininhibitorsolution(0.01%inphosphatebufferatpH
7.4) for2hat37°C to stop theenzymatic reaction.The tissuewaswashedagainwith
purifiedwaterandkeptinphosphatebuffer.Finally,piecesofSCwereplacedflateither
onquartzplatesoronPMMASPFMasterPA‐01platesforSPFinvitromeasurement,or
onpolystyrenepetridishes for thicknessanalysis.WhenSCwas laidonPMMAplates,
vacuumwasappliedtopreventairenclosurebetweentheSCandtheplate.Theplates
withtheSCwerestoredat4°Cinadesiccatoroversaturatedsodiumchloridesolution
(relativehumidityof80%)untiluse.
3.3.2.2.Method2‐Isolationofepidermalmembranebyheattreatment
Thesheetoffullthicknessskinwasimmediatelyusedorstoredat‐20°Cuntilfurtheruse.
Theskinwasthawedifnecessaryatroomtemperatureandimmersedinawaterbathat
60°Cfor60s.Subsequently,theepidermalmembranewasseparatedfromthedermisby
gentlepeelingoff248.Theisolatedepidermalmembranewasthenlaidonquartzplatesfor
SPF in vitro measurement or on polystyrene petri dishes for thickness analysis. The
prepared samples were stored at 4°C in a desiccator over saturated sodium chloride
solutionuntiluse.
Chapter3.Pigskinforinvitrotesting 43
3.3.3.Skintissuethicknessmeasurement
Ramanconfocallaserscanningmicrospectroscopy(Alpha500R,WITec,Ulm,Germany)
wasemployedfortissuethicknessmeasurement.Ramanspectrawererecordedfrom0to
4000cm‐1(spectralgratingof600g/mm,spectralresolutionof3cm‐1perpixel)usinga
532nm excitation laser source, a Nikon EPI plan 100x 0.95 numerical aperture (NA)
objectiveandan integration timeof1s.Theequipmentpermittedanx‐y resolutionof
340nmandazresolutionof500nm.ThistechniquecombinesRamanspectroscopywith
confocalmicroscopyallowingadepthanalysisofthesample.
Thethicknessoftheisolatedtissuewasassessedbyscanningthesamplesoveralineof
40µminthexdirection(with120pointsperline)andoveradepthof40µminzdirection
(with240linesperimage).Themeasurementswereconductedinclusteranalysismodus
withtheWITeccontrolsoftware.TherawdatawereevaluatedwithWITecProjectPlus
2.04software.
3.3.4.Polymethylmethacrylateplates
ThreetypesofPMMAplatesservedassyntheticUVtransparentsubstrate.Theplatesare
roughenedononesidetomimicskinsurfaceanddifferintheirmanufacturingprocess
andtopographicalproperty(Table3.2).
Chapter3.Pigskinforinvitrotesting 44
Table 3.2. CharacteristicsofPMMAplates
HelioplateHD6 Schönberg SPFMaster
PA‐01
Manufacturer HelioScreenLabs Schönberg ShiseidoIrica
technology
Manufacturingprocess Moldinjected Sand‐blasted Moldinjected
Surfacesizeassupplied 4.7cmx4.7cm 5.0cmx5.0cm 5.0cmx
5.0cm
Surfacesizeadjustedfor
thestudy
2.0cmx2.0cm 2.0cmx2.0cm 2.0cmx
2.0cm
RoughnessRaorSa
givenbythesupplier
Ra=4.5µm Ra=5.9µm Sa=17.8µm
Appliedamountof
sunscreenforSPFinvitro
testing
1.3mg/cm² 1.3mg/cm² 2.0mg/cm²
3.3.5.Surfacetopographicalassessment
Wecarriedoutsurfacetopographicalmeasurementsofskinspecimenslaidonquartzor
PMMAplatesandofthePMMAplatesbychromaticconfocalimagingbasedonwhitelight
chromaticaberrationsprincipleusing theAltisurf®500 instrument.Thisallowednon‐
contact surface topography measurement and analysis. The employed optical sensor
allowed an axial resolution (z) of 5nm and a lateral resolution (x‐y) of 1.1µm. The
motorizedx‐ytablepermittedscanningofsamplesinthemmrangebasedonwhichthe
three‐dimensional microtopographical surface structure of the samples was
reconstructed.
In thisstudy,anareaof5mm×5mmwasscanned in10µmincrementstepsandthe
arithmeticalmeanheightoveranarea,Sa(Equation3.1.,ISO25178guideline249),was
selectedasarepresentativemeasureofskinsurfacetopography250.Thismeasurewas
alsousedforthePMMAplates.
Chapter3.Pigskinforinvitrotesting 45
1| , | 3.1.
where,LxandLyisthelengthinthexandydirection,respectively.Z(x,y)isthealtitude
ofthesamplingpointmeasuredfromthesamplingsurface.Useofanarealparametersuch
asSafordescribingsurfacetexturebetterservestheneedsofthepresentstudycompared
to,forexample,theroughnessoveraprofile(e.g.RainISO4287guideline251)whichhas
alsobeenusedtodescribeskinsurface.
3.3.6.Sunscreenformulations
WetestedtheinvitroandinvivoperformanceoftwoOil‐in‐Water(OW)sunscreens.The
filter system and the SPF in vivo of the sunscreens measured in accordance with
ISO24444:2010guidelines9,aregiveninTable3.3.
Table3.3.Testedsunscreens
Sunscreen
designation
InvivoSPF
meanSDa
UVfilter(%)asactiveingredientb
EHMC BEMT MBBT DHHB EHT PBSA
OWNr.1 27.57.6 5 2 4 ‐ ‐ ‐
OWNr.2 19.95.8 ‐ 2 ‐ 4.5 3 2
aSPFinvivoevaluatedinaccordancewithISO24444:2010guidelines,withn=5
b abbreviation: EHMC, Ethylhexyl Methoxycinnamate; BEMT, Bis‐Ethylhexyloxyphenol
MethoxyphenylTriazine;MBBT,MethyleneBis‐BenzotriazolylTetramethylbutylphenol,
DHHB, Diethylamino Hydroxy Hexyl Benzoate; EHT, Ethylhexyl Triazone; PBSA,
PhenylbenzimidazoleSulfonicAcid
Chapter3.Pigskinforinvitrotesting 46
3.3.7. Measurement of the sun protection factor in
vitro using spectral transmission of ultraviolet
radiation
SPFinvitro isderivedfromdiffusetransmissionspectroscopymeasurementsbasedon
themodelproposedbySayre12.
SPF ∑ ser λ . Ss λ
∑ ser λ . Ss λ . T λ 3.2.
where,ser(λ)istheerythemaactionspectrumasafunctionofwavelengthλ9,Ss(λ)isthe
spectral irradiance received from the UV source at wavelength λ 9, and T(λ) is the
measuredtransmittanceofthelightthroughasunscreenfilmappliedonasuitableUV
transparentsubstrate13.
ThespectralUVtransmittancewasrecordedfrom290to400nmin1nmincrementsteps
throughasubstratebeforeandafterapplicationofasunscreenusingtheLabsphereUV‐
2000S. The linear range of the device was checked by measuring the absorbance of
increasing concentrations of the UVB filter ethylhexyl methoxycinnamate in ethanol
solutionsandplottingthemeasuredabsorbancedataagainsttheexpectedabsorbance.
Theblanktransmittancespectrumbeforeapplicationofthesunscreenwasrecordedfor
thePMMAplatesusingtheplatescoveredwithglycerinandfortheskinsubstratesusing
thebare skin specimensonquartzorPMMASPFMasterPA‐01plateswithout further
treatment248.ForthePMMAplates,asingleblanktransmittancespectrumwasmeasured
inthecenterofaplateandusedfortheevaluationoftheSPFvaluesofallplatesofthe
sametype.Fortheskinsamples,ablanktransmittancespectrumwasrecordedineach
singlemeasurementposition.Weapplied1.8mg/cm²ofsunscreenontheskinsamples;
theamountofsunscreenappliedonthePMMAplatesisgiveninTable3.2.Theapplication
ofthesunscreenandtheequilibrationstepwereconductedaspreviouslyreported223.
Chapter3.Pigskinforinvitrotesting 47
Asurfaceareaofsubstrateof2cm×2cmwasusedintheSPFmeasurements.Thisarea
was chosen because skin specimens of this dimension could be easily prepared. The
impactof thesurfaceareaof thesubstrateonSPF invitrowasassessedbycomparing
PMMAplateswithasizeofabout5.0cm×5.0cmwhichareroutinelyused,withplates
cutto2.0cm×2.0cm.
3.3.8.Statisticalanalysis
Statistical analysis was performed using Statgraphics centurion XVI (Statpoint
Technologies,Inc.,Warrenton,VA,USA)software.Thestatisticalsignificanceat5%
confidence levelof thedifferencebetween twogroupswasevaluatedusingMann‐
Whitneytest.
3.4.Resultsanddiscussion
3.4.1.Skinthickness
Thicknessofhumanandporcineearskiniscommonlymeasuredbylightmicroscopyof
histological sections of stained skin biopsies using formalin‐paraffin or freezing
preparation 240,252. Thickness of SC was measured by tape stripping requiring
determinationoftheamountofremovedcorneocytes253.Suchproceduresaregenerally
timeconsumingandmayintroduceartifactsduetopreparationordataevaluation.Anon‐
invasive method based on confocal Raman spectroscopy that required no tissue
preparationwasintroducedformeasuringSCthicknessinvivoonhumanvolunteers254.
Thiswasbasedonthefactthatwatercontentremainsconstantintheviableepidermis
255.
In the present investigation,we employed a procedure for assessing the thickness of
trypsin‐separated and of heat‐separated skin using also confocal Raman
microspectroscopy.TheRamanspectraacquired for theskinsamplesandpolystyrene
petridishareshowninfigure3.1.
Chapter3.Pigskinforinvitrotesting 48
TheRamanspectrumof theskinwas identical for the trypsinseparationand theheat
separationprocedure.Ramanprofilesofskinandthepolystyreneofthepetridishdiffered
noticeably (figure 3.1.). As an example, a peak that is characteristic to the skin is
detectableat1650‐1690cm‐1correspondingtotheamideIband256.ThisamideIband
isabsentinthepolystyreneofthepetridish.
Figure3.1.Ramanspectraofskinspecimen(black)andpolystyrenepetridish(grey)
Aclusteranalysiswasperformedbythesoftwareinwhichthenumberofclusterswasset
equal to three. This analysis detected spectral differences between thematerials as a
function of depth. Figure 3.2. shows the result of this analysis. A clear differentiation
betweenair,skintissueandpolystyreneisevident.Fromthisrepresentation,estimation
ofthicknessoftheskinspecimenswaspossibleaftercorrectionbymultiplyingtheextent
oftheopticalskinlayerwiththeratioofrefractiveindexofstratumcorneumtoair,being
equalto1.55215,257.
1650‐1690cm
‐1
Chapter3.Pigskinforinvitrotesting 49
Figure3.2.a. Figure3.2.b.
Figure3.2.VisualizationofclusterevaluationobtainedfromRamanspectradifferences
correspondingtoair(topblackzone),skintissue(whitezone)andpolystyrene(bottom
blackzone).VerticalcoordinatecorrespondstodepthinZdirection(40µm).
Figure3.2.a.trypsin‐separatedskin,Figure3.2.bheat‐separatedskin.
Thetrypsinseparationandtheheatseparationproceduregaveskinlayerthicknessesof
about 5.9µm (n=2) and 14µm (n=2), respectively. Both procedures allowed the
separation of an upper skin layer from the full thickness skin, the heat separation,
however,ledtotherecoveryofathickertissuelayerthanthetrypsinseparation,which
wasconsistentwithresultsofotherauthors 247.This isbecause the trypsinprocedure
enablesrecoveringtheSCexclusively,whereastheheatprocedureleadstotherecovery
ofalmosttheentireepidermis.
Thethicknessobtainedviatrypsinseparationwassmallerthanpreviouslypublisheddata
on SC thickness of porcine ears using two photon microscopy 242, quantitative tape
strippingprocedure253,cryo‐scanningelectronmicroscopy258,orcommonhistological
examination 240.Thisdifferencemaybeexplainedby thewater evaporationoccurring
during the storage and equilibration of the skin specimens over salt solution in our
experiment.ThisstepwasrequiredforthesubsequentSPFmeasurements.Theseresults
demonstrate that the developedmethodmakes possible to measure the thickness of
isolatedskinlayersatmultiplelocationsinafast,exactandconvenientmannerrequiring
nospecialpreparation.
Chapter3.Pigskinforinvitrotesting 50
3.4.2.Surfacetopographicalassessment
Differentmethodshavebeenused for roughnessmeasurementofhumanskinsuchas
topographicalanalysisusingdigitalstripeprojectiontechnique250,astylusprofilometry
onskinreplica259,3Dopticalinvivotopographyanalysis260andconfocalscanninglaser
microscopy 261. A non‐exhaustive list of the invasive, semi‐invasive and non‐invasive
methodsisgivenin262.Theidealsystemtoassesstherealtopographyofskinshouldallow
a non‐contact measurement, a spatial resolution in themicrometer range, a range of
measurement covering the amplitude of the skin relief, a three‐dimensional
reconstruction and the collection of the data in a reasonable time. Most of these
recommendationswerefulfilledbythewhitelightaberrationsprincipleofmeasurement
usedinthepresentstudy.
SaroughnessparametervaluesofthedifferentsubstratesarereportedinTable3.4.
Table3.4.Saarithmeticalmeanoverasurfaceofselectedsubstrates
Selectedsubstrates Sa(µm)measured
Fullthicknesspigskina 21.7
Humanskinb22onbackforearm261
17.4onback18
Heat‐separatedpigskinfixedonquartzplatec 2.560.74
Trypsin‐separatedpigskinfixedonquartzplated 1.260.20
Trypsin‐separatedpigskinfixedonSPFMasterPA‐01
PMMAplatea19.2
PMMAHelioplateHD6e 6.070.03
PMMASchönbergplatee 6.050.51
PMMASPFMasterPA‐01e 22.231.90
an=1ear,baccordingtopaper,cn=13ears(61singlemeasurements),dn=3ears,en=3
plates
Chapter3.Pigskinforinvitrotesting 51
Sa of full thickness skin of porcine ear had a value of about 22µm. This result is in
accordancewiththedataavailableforhumanskinroughness261.Anillustrationofthe
surfaceoffullthicknesspigearskinisgiveninfigure3.3.
Figure 3.3. illustrates the differences in altitude (µm range on scale) and the highly
organizedarchitectureoftheskinsurfaceincludingthev‐shapedfurrows.Thispatternis
characteristic also for human skin as shown using optical laser profilometry 262 or
scanningelectronmicroscopy255.Theseresultshenceconfirmthatfullthicknessskinof
porcineearpresentsthesamesurfacearchitectureashumanskin.
Figure3.3.Threedimensionalviewoffullthicknessskinfromporcineear
The roughness parameter Sa of the isolated tissue layers decreased compared to full
thickness tissue to 1.26µm and 2.56µm for trypsin‐separated and heat‐separated
porcineskin,respectively.Athree‐dimensionalrepresentationofthesurfaceofaheat‐
separated sample is shown in figure 3.4. This Figure illustrates that the typical
topographicalreliefoffullthicknessskinwaslostasaresultofthepreparationprocedure.
Thetopographicalreliefofthefullthicknessskinisprincipallycharacterizedbyclusters
separated by invaginations resembling valleys also referred to as furrows which are
extensionsoftheSCintotheepidermisandcanreachdownintothebasallayer242.
Chapter3.Pigskinforinvitrotesting 52
Byremovingtheconnectivetissue(dermis)thesevalleysdisappearresultinginamore
flatskinsurfaceandalossofskinroughness.Additionally,thetrypsin‐separatedSCwhich
isthinnerthantheheat‐separatedepidermalmembrane(about6µmcomparedto14µm)
showedaconsiderablysmallerSavaluethantheheat‐separatedskinlayer.Theseresults
indicatethatthethicknessoftheskinsampleaffectsitsroughness.
Figure3.4.Threedimensionalviewofheat‐separatedepidermalmembranefromporcine
ear
Two of the PMMA plates (Helioplate HD6 and Schönberg) exhibited a Sa value of
approximately6µm.Thisvalueissmallerthantheoneoffull thicknessskinbutlarger
than thoseof the twoskinpreparations.Bycomparison, theSPFMasterPA‐01PMMA
plates,whichweredevelopedtomimicthetopographyofhumanskin18,hadaSavalueof
roughly22µm,whichwascomparabletothatoffullthicknessskin.
TheSameasuresofallPMMAplateswereinlinewiththedataprovidedbythesuppliers
fortheusedbatches.
Finally,toobtainaUVtransparentskin‐surfacedsubstratehavingtheSaoffullthickness
skin,trypsin‐separatedSCwaslaidontheSPFMasterPA‐01PMMAplates.Themeasured
Saofthiscombinedsubstratewasapproximately19µm(Table3.4).
TheeffectofthedifferentsubstratesandtheirSavaluesonSPFinvitroisdiscussedbelow.
Chapter3.Pigskinforinvitrotesting 53
3.4.3.Measurementofsunprotectionfactor
ThreetypesofskinpreparationswereusedforSPFmeasurements, i.e.,heat‐separated
epidermalmembraneonquartzplates,trypsin‐separatedSConquartzplatesandtrypsin‐
separated SC on PMMA plates (SPF Master PA‐01). Directly after the preparation
procedure,theskinsampleslookedtranslucentandbecametransparentduringstorage
under controlled temperature and humidity conditions. The time required to reach
sufficient transparency forUV transmittancemeasurementswith trypsin‐separatedSC
and heat‐separated epidermal membrane samples was 24 hours and four days,
respectively,afterpreparation.Thisisrelatedtothicknessoftheobtainedtissuelayer.
TheimpactofthesizeoftheplateonSPFinvitrowasfirstlydeterminedwiththethree
typesofPMMAplatesusingsunscreenOWNr.1.Themeasurementswerecarriedoutby
twooperators.TheSPFinvitrovaluesof5.0cm×5.0cm(n=3)plateswerenotfoundto
be significantly different from SPF in vitro values of 2.0cm × 2.0cm (n=3) plates
independentlyofplatetypeandoperator(Mann‐Whitney,p>0.05;resultsnotshown).As
aresult,asampleareaof2.0cm×2.0cmwasusedforfurtherstudies.
SPFinvitrodatameasuredonPMMAplatesandonskinpreparationswerecomparedto
theSPFinvivo.TheresultsforeachsubstratewithsunscreenOWNr.1andOWNr.2are
showninFigure3.5.andFigure3.6.,respectively.RelativestandarddeviationforOWNr.1
andoperator1was32‐38%forPMMAplatesand38%forskinsubstrate;foroperator2,
21‐43%forPMMAplatesand32‐57%forskinsubstrate.Relativestandarddeviationfor
OWNr.2andoperator1wassimilartoOWNr.1.Astatisticalanalysisofthedifference
betweenSPFinvivoandSPFinvitroforeachsubstrateissummarizedinTable3.5.
Chapter3.Pigskinforinvitrotesting 54
Figure3.5.AverageSPF invitro(columns)andstandarddeviation(bars)ofsunscreen
OWNr.1measuredonthreetypesofPMMAplates(HelioplateHD6,Schönberg,andSPF
MasterPA‐01eachn=9)andthreetypesofskinpreparation(heat‐separatedepidermal
membraneonquartz,trypsin‐separatedSConquartz,andtrypsin‐separatedSConSPF
MasterPA‐01plate,eachn=63).TheSPFinvivo(drawnhorizontalline)isequalto27.5
(standarddeviation7.6).
Chapter3.Pigskinforinvitrotesting 55
Figure3.6.AverageSPF invitro(columns)andstandarddeviation(bars)ofsunscreen
OWNr.2measuredonthreetypesofPMMAplates(HelioplateHD6,SchönbergandSPF
MasterPA‐01eachn=12)andonheat‐separatedepidermalmembraneonquartz,n=33.
TheSPFinvivo(drawnhorizontalline)isequalto19.9(standarddeviation5.8).
Chapter3.Pigskinforinvitrotesting 56
Table3.5.DifferencebetweenSPFinvitroandSPFinvivoforevaluatedsubstrates
Sunscreen Substrate
Statisticalsignificanceat5%
confidencelevelofthe
differencebetweenSPFinvitro
andSPFinvivo(Mann‐Whitney)
OWNr.1 Trypsin‐separatedSConquartz Yes(p<0.05)
Trypsin‐separatedSConSPF
MasterPA‐01
No(p>0.05)
Heat‐separatedepidermal
membraneonquartz
No(p>0.05)
HelioplateHD6 Yes(p<0.05)
Schönberg Yes(p<0.05)
SPFMasterPA‐01 Yes(p<0.05)
OWNr.2 Heat‐separatedepidermal
membraneonquartz
No(p>0.05)
HelioplateHD6 Yes(p<0.05)
Schönberg Yes(p<0.05)
SPFMasterPA‐01 No(p>0.05)
ThereproducibilitywasassessedusingsunscreenOWNr.1.Itisworthpointingoutthat
SPFinvitroobtainedwithPMMAplatesmaydependamongotherfactorsontheoperator
19.Inthepresentstudy,therewasastatisticallysignificantdifferencebetweenSPFinvitro
values measured by the two operators for all types of PMMA plates (Mann‐Whitney,
p<0.05)butnot for theheat‐separatedepidermalmembrane(Mann‐Whitney,p>0.05).
This result might suggest that the biological substrate possibly provides more
reproducible data than the habitually used synthetic plates even though standard
deviationoftheresultsbythetwooperatorsmayvary.
ForsunscreenOWNr.1,trypsin‐separatedSConPMMASPFMasterPA‐01plateandheat‐
separatedepidermalmembraneonquartzplateyieldedSPFinvitroresultsthatwerenot
statisticallysignificantlydifferentfromtheSPFinvivovalue.Oftheskin‐basedsubstrates,
onlytheresultoftrypsin‐separatedSConquartzplatewassignificantlydifferentfromthe
SPFinvivo,althoughthissignificancewasbarelyreached(p=0.036).
Chapter3.Pigskinforinvitrotesting 57
Inthecontrary,SPFinvitrovaluesobtainedwiththePMMAplateswereaboutfourtofive
timeslargerthanandsignificantlydifferentfromtheSPF invivo(Figure3.5.andTable
3.5.)
TheseresultsdemonstratethatsyntheticplateswerenotadequatefortestingtheSPFof
thissunscreensincenoneofthePMMAplatesapproachedtheSPFinvivo.Withtwoout
ofthethreeskinpreparationsontheotherhand,theSPFinvivovaluewasalsoobtained
invitro.
TheSPF invitroof sunscreenOWNr.2wasevaluatedbyoneoperatorusing the three
PMMAplatetypesandtheheat‐separatedepidermalmembraneonquartz.Alsowiththis
sunscreen,theskinpreparationproducednosignificantlysignificantdifferencefromthe
in vivo reference. Of the PMMA plates, the ones manufactured by mold injection
(HelioplateHD6&andSPFMasterPA‐01)gavelowerandtheonemanufacturedbysand
blasting (Schönberg) higher SPF in vitro values than the in vivo reference. This was
different fromtheresultobtainedwithsunscreenOWNr.1.Nevertheless, theresultof
PMMASPFMasterPA‐01showednostatisticallysignificantdifferencetotheSPFinvivo
whiletheothertwoPMMAplatesdid.Interestingly,thePMMASPFMasterPA‐01plate
hasthegreaterroughness(Sa=22.23µm)ofthetwomoldinjectedplates.Generally,an
impact of substrate roughness on efficacy, reproducibility and repeatability of invitro
sunscreensmeasurementshasalsobeenreportedbyFageonetal.andFerreroetal.10,16.
TheoutcomeoftheSPF invitroassessmentwithtwodifferentOWsunscreensshowed
thattheusedbiologicalsubstrateyieldedresultsreachinginmostcasestheSPFinvivo
valuewhilethePMMAplatesgenerallydidnot.Roughnessdifferedconsiderablybetween
theskin‐basedpreparations(Sa2.56versus19.2),this,however,didnotseemtoaffect
the determined SPF in vitro. Only trypsin‐separated SC on quartz having the smallest
roughness(Sa1.26)didnotreachthereferenceSPFvalue.Thispreparationmaytherefore
not be suitable for SPF in vitro testing suggesting that a minimal roughness of the
substratemayberequired.Conversely,noneofthesyntheticsubstrateshavingdifferent
roughness characteristics achieved a satisfactory SPF in vitro with the two OW
sunscreens.EventheSPFMasterPA‐01platewhichhasaSavalueandasurfacepattern
similar to that of human skin did not always yield accurate results. This implies that
roughness may not be the sole critical surface characteristic of the substrate to be
consideredforachievingaccurateSPFinvitromeasurements.
Chapter3.Pigskinforinvitrotesting 58
Besidessurfacetopography,theaffinityofthesunscreenforthesubstrateseemstobe
equallyimportant.Affinityreferstothepropensityofthesunscreentobedistributedand
adheretothesubstrateuponspreading.Invitromeasurementscarriedoutonskin‐based
substratesareassumedtobettersimulatetheproduct‐to‐substrateaffinitythatapplies
to the in vivo situation. This may explain why these substrates, including the heat‐
separatedepidermalmembraneonquartzandthetrypsin‐separatedSConPMMASPF
MasterPA‐01plates,resultedinmoreaccurateSPFinvitrovaluescomparedtothePMMA
plates.
ItshouldbepointedoutthatthepresentdatawerecollectedwithOWformulations.Ana
prioritransferoftheresultstoothertypesofformulationscannotbemadeatthispoint.
Forexample,WOorsinglephaseformulationsmightexhibitadifferentaffinityand/or
spreading behavior. Therefore, additional investigations are required for generalizing
theseobservations.
Theaffinityaspectwas further invoked inconnectionwith thepoorSPF invitro value
obtainedwithhighlyhydrophobicmoldinjectedplatesthatcausedalackofadherenceof
the product and consequently a non‐uniform protection film for some sunscreen
formulations190.ToincreasetheadherenceofthesunscreenonmoldedPMMAplatesthe
authorsproposedtheuseofachemicalpretreatmentoftheplatepriortheapplicationof
thesunscreen.Chemicalpretreatment,however,mightalterthestructureoftheapplied
emulsionandseems,therefore,nottobeanidealsolution.Alternatively,modificationof
interfacialpropertiesof themoldedPMMAplatesbyplasmatreatment to improve the
product‐to‐substrateaffinitywasproposed263.However,inthisevaluationtherequired
plasma treatmentproducingdifferent levelsof surfaceenergywasproductdependent
andnosingletreatmentwassuitablefortheentiresetofproducts.
Inadditiontoroughnessandaffinity,theinfluenceofotherexperimentalfactorsonSPF
invitrohasalreadybeenexaminedsuchastheapplicationprocess10,thespectrumofthe
lampsource214ortheamountofproductapplied18,180,248.Otherpossibleinfluencefactors
suchas the impactofpressureor the formationofthe filmduringproductapplication
havenotbeenfullyexploredyetconstitutingastillopenareaofresearchinthefield.
Chapter3.Pigskinforinvitrotesting 59
The approach introduced in this study provided interesting insights in the in vitro
methodology forpredictingSPF invivo. Itcouldbeuseful in the finalstageofproduct
development fordeterminingabsoluteSPFvalueprior to carryingout clinical studies.
Also,useofthemethodmayberecommendedforsunscreenperformanceverification,for
example,incaseofchangeofrawmaterial,vehiclecompositionormanufacturingprocess.
Since,however,themethodisratherlaborious,itmaynotbeappropriateforscreeningor
largescaleproductcomparisontests.
3.5.Conclusion
SubstratesforSPFinvitromeasurementthatinvolvetheuseofskintissuelayersappear
toprovideabetterpredictionofSPFforthetestedOWformulationsthanconventionally
usedPMMAplates.Aminimalsurfaceroughnessoftheskin‐basedsubstrateseemstobe
required.However,reproducingthenaturalroughnessofskininaPMMAplatealonewas
not sufficient to achieve accurate SPF in vitro values. Instead, improved affinity of
sunscreenforthesubstrateimpartedbytheuseofskintissueisconcludedtobecritically
important.Significantly,despitethelossoftheoriginalreliefoffullthicknessskinduring
preparation, the use of tissue as a substrate was adequate for in vitro testing of the
performanceofthesunscreens.
Chapter4
Filmthicknessfrequency
distributionofdifferent
vehiclesdetermines
sunscreenefficacy
4.1.Abstract
SunscreenefficacydependsprimarilyontheabsorbancepropertiesofthecontainedUV
filters.However,sunprotectionfactor(SPF)frequentlydiffersbetweensunscreenswith
the same filter composition. We tested here the hypothesis that thickness frequency
distributionofthesunscreenfilmisalsoresponsibleforandcanexplainthedivergence
in SPF values. For this, we developed a method to measure film thickness from the
differenceoftopographybeforeandafterapplicationof2mg/cm²ofsunscreenonpigear
epidermalmembrane.
M.Sohnetal.,“Filmthicknessfrequencydistributionofdifferentvehiclesdeterminessunscreenefficacy,”J.Biomed.Opt.19(11),115005(2014)..
60
Chapter4.Filmthicknessdistribution 61
Theinfluenceoffivevehicleformulationsandofapplicationpressureandspreadingtime
onfilmthicknessfrequencydistribution,meanthickness(Smean)andSPF invitrowas
investigated. The vehicle had a significant impact, low vehicle viscosity resulting in
smallerSmeanandlowerSPFinvitrothanhighviscosity;continuousoilphaseproduced
the largest Smean and SPF values. Long spreading time reduced Smean and SPF and
increasedpressurereducedSPF.TherewasapositivecorrelationbetweenSmeanand
SPF invitro,underlining therelevanceof film thickness for interpretingUVprotection
differencesofformulationswiththesamefiltercomposition.Thisworkdemonstrateda
stronginfluenceofvehicleandapplicationconditiononsunscreenefficacyarisingfrom
differencesinfilmthicknessdistribution.
4.2.Introduction
Topicallyappliedsunscreensconstituteasuitableandcommonlyemployedmeasureto
protectskinfromsundamages6,7.Efficacyofsunscreensintermsofsunprotectionfactor
(SPF),UVAprotection,photostability,andbalancedabsorbancedependsprimarilyonthe
intrinsicabsorbanceandphotostabilitypropertiesofUVfilterscontainedintheproduct
inconjunctionwiththeusedconcentration20,264.Theidealsunscreenachievesbalanced
protectionby attenuating equallyUVB andUVA radiations, similarly to theprotection
affordedbyclothingandshade228,229.Therefore,anappropriateUVfiltersystemshould
combineUVBandUVAfilterstoachieveoptimizedUVshield141.Reasonably,theamount
ofproductappliedalsoaffectsprotection180,265‐268.However,theSPFfrequentlydiffers
between sunscreens with different vehicle formulations containing the same filter
composition20,21yetthecauseofthisdifferencehasnotbeeninvestigated.Also,invitro
inter‐laboratorytrialswiththesamesunscreenhaveproducedvariableresults19andthe
applicationprocedurewasfurtherfoundtoinfluencethemeasuredSPF22,269.Inaddition
to the absorbing property of the UV filters and the amount of applied product,
homogeneityofdistributionofthesunscreenwasfoundtoplayanimportantrolewith
respecttoSPFinvivo27.Theidealsituationforoptimalperformanceistoachieveafilm
withuniformthickness,resemblingtheperfectlyhomogeneousdistributionofasolution
ofUVfiltersinanopticalcell.
Chapter4.Filmthicknessdistribution 62
Understandably, this condition can never be reached under in vivo condition of
applicationduetotheskinsurfacetopography.Skinreliefshowsridgesandfurrowsthat
preclude the formation of an even sunscreen film 270. In addition,manual application
makes it practically impossible to achieve a uniform film. This irregularity of the film
thickness isprobably a causeof the reported experimental variability of SPF andwas
suggested to be responsible for the divergence of orders of magnitude between
predictionsbasedonUVtransmissionofdilutetransparent filtersolutionsandclinical
studyresults25.
Theaimofthepresentworkwastounderstandtherelationshipbetweenfilmthickness
frequencydistributionandefficacyofsunscreens.Tothisend,wedevelopedamethodfor
determining theprecise thickness distribution of the applied sunscreen filmbasedon
topographicalmeasurementswithhighspatialresolution.Weusedepidermalmembrane
ofpigearskinasabiologicalsubstrateforinvitrosunscreenapplicationaswerecently
showedthatusingthissubstrateforSPFinvitrotestingprovidedbetterpredictionofSPF
invivothanconventionallyusedsyntheticsubstrates.Substrate‐to‐productaffinityrather
thantopographywasdiscussedtoberesponsibleforthisbetterpredictionofSPFinvivo
(section3.4.3.).SkinofpigearhasalsobeenusedforinvitroassessmentofUV‐induced
damagesonDNA271,UVfilterpenetration181,272,andsunscreenphotostabilitytests.182
Usingthedevelopedfilmassessmentmethodweinvestigatedthesunscreenfilmresidue
in termsof thickness andhomogeneity of distribution for five sunscreen vehicles and
different application conditions. In parallel, we measured SPF in vitro on the same
preparationstodetermineUVprotectionefficacy.Theimpactofvehicleswiththesame
UV filtercombinationandof theapplicationconditionson filmparametersandSPF in
vitro as well as the correlation between film parameters and SPF in vitro was then
assessed. Identificationof formulationandapplicationrelated factors thatmay impact
filmcharacteristicsandUVprotectionwasafurthergoalofthepresentwork.Thisisput
forthasfundamentalaspectforunderstandingthemechanismofsunscreenefficacy.
Chapter4.Filmthicknessdistribution 63
4.3.Materialsandmethods
4.3.1.Chemicalsandequipment
Thefollowingreagentswereused:PotassiumCarbonatefromSigma‐Aldrich,StGallen,
Switzerland; Tinosorb S abbreviated BEMT (INCI, Bis‐ethylhexyloxyphenol
MethoxyphenylTriazine),UvinulN539TabbreviatedOCR(INCI,Octocrylene),SalcareSC
91, Cetiol AB, Lanette O, Dehymuls LE, Edeta BD, all from BASF SE, Ludwigshafen,
Germany;Eusolex232abbreviatedPBSA(INCI,PhenylbenzimidazolSulfonicAcid)from
Merck, Darmstadt, Germany; Parsol 1789 abbreviated BMDBM (INCI, Butyl
Methoxydibenzoylmethane), Amphisol K from DSM, Kaiseraugst, Switzerland; Neo
HeliopanOS abbreviated EHS (INCI, Ethylhexyl Salicylate) from Symrise, Holzminden,
Germany;Arlacel 165 fromCroda,EastYorkshire,England;KeltrolRD fromCPKelco.
Atlanta,GA,USA;CarbopolUltrez10,CarbopolUltrez21fromLubrizol,Brussels,Belgium;
Tegin OV from Evonik Industries, Essen, Germany; Paracera M from Paramelt,
Heerhugowaard, The Netherlands; Beeswax white from Koster Keunen, Bladel, The
Netherlands;GlycerinfromSigma‐Aldrich,StGallen,Switzerland;TrisAminoUltra‐Pure
fromAngus,BuffaloGrove,IL,USA;PhenonipfromClariant,Muttenz,Switzerland.
Quartz plates with a size of 4.2cm 4.2cm were obtained from Hellma Analytics,
Zumikon,Switzerland.
The following equipment was used: Electric shaver (Favorita II GT104, Aesculap,
Germany),epilator(Silk‐épil7XpressivePro,Braun,Germany);waterpurificationdevice
(Arium61215,Sartorius,Goettingen,Germany);precisionbalances (XS105Dual range
and XA3001S, Mettler‐Toledo, Columbus, OH, USA); surface metrology instrument
(Altisurf500,Altimet,Thonon‐les‐Bains,France);UVtransmittanceanalyzer(Labsphere
UV‐2000S,LabsphereInc.,NorthSutton,NH,USA).
Thefollowingsoftwarepackageswereused:BalanceLink(MettlerToledo,Columbus,OH,
USA)withbalanceXA3001Sfortherecordingofpressureduringspreadingofsunscreen;
Phenix and Altimap (Altimet, France) for topographicalmeasurement and evaluation,
respectively; UV‐2000 (Labsphere Inc., USA) for UV transmittance measurement;
StatgraphicscenturionXVIsoftware(StatpointTechnologies,Inc.,Warrenton,VA,USA)
forstatisticalevaluation.
Chapter4.Filmthicknessdistribution 64
4.3.2.Preparationofskinsubstrate
We used epidermal membrane of pig ears as a biological substrate for sunscreen
applicationasdescribedinsection3.3.2.,method2(3.3.2.2.).Earsoffreshlyslaughtered
pigswereobtainedfromthelocalslaughterhouse(Basel,Switzerland)nomorethanfew
hourspostmortem.The studydidnot require the approval of the ethics committeeof
animal research as the earswere taken from pigs not specifically slaughtered for the
purpose of this study. The epidermalmembranewas isolated using a heat separation
procedure.Thefullskinwasimmersedinawater‐bathat60°Cfor90s.Theepidermal
membranewasseparatedfromthedermisbygentlepeelingoff,cuttoadimensionof2cm
2cm,laidflatonquartzcarrierplates,andstoredat4°Cinadesiccatoroversaturated
potassiumcarbonatesolutionuntiluse.
4.3.3.Characterizationofsunscreenformulations
WeassessedSPFinvitroandfilmthicknessdistributionoffivedifferentsunscreens.The
formulations included an oil‐in‐water cream (OW‐C), an oil‐in‐water spray (OW‐S), a
water‐in‐oil emulsion (WO), a gel (GEL) and a clear lipo‐alcoholic spray (CAS). They
containedthesameUVfiltercombinationandemollient.Thefiltersystemwascomposed
of8w‐%OCR,5w‐%EHS,2w‐%BMDBM,1w‐%BEMT,and1w‐%PBSA.Basedonthis
UV filter composition a SPF in silico of 25 was calculated with the BASF sunscreen
simulator273.ThedetailedcompositionofthesunscreensandtheirrespectiveSPFinvivo
values are given in Table 4.1. SPF in vivo values were measured in accordance with
ISO24444:2010guidelines9.
The sunscreens showeddifferent rheological characteristics (figure 4.1.). GELhad the
highestshearviscosityfollowedbyOW‐CandWO,whereasOW‐SandCASweremuchless
viscous. Viscosity of all sunscreens decreased with increasing shear rate whereas
hysteresisdependedontheformulation.
Chapter4.Filmthicknessdistribution 65
Table4.1.Composition(w‐%)andSPFinvivoofinvestigatedsunscreens
Sunscreendesignation OW‐C OW‐S GEL WO CAS
SPFinvivoSDa38.8
8
24
5
19.4
5
19.5
3.1
17.8
2.2
Ingredient
typeTradename Composition(w‐%)
Emulsifier
systemArlacel165
AmphisolK
DehymulsLE
TeginOV
1.5
1.5
‐
‐
‐
2.5
‐
‐
‐
‐
‐
‐
‐
‐
1.0
2.0
‐
‐
‐
‐
Thickener
system
LanetteO
KeltrolRD
SalcareSC91
CarbopolUltrez10
CarbopolUltrez21
ParaceraM
Beeswax
0.5
0.15
1.8
‐
‐
‐
‐
‐
‐
‐
‐
‐
‐
‐
‐
0.15
1.8
0.2
0.15
‐
‐
0.5
‐
‐
‐
‐
0.5
1.0
‐
‐
‐
‐
‐
‐
‐
Emollient CetiolAB 5.0 5.0 5.0 5.0 5.0
Filtersystem MixtureofUVfilters 17.0 17.0 17.0 17.0 17.0
Neutralizing TrisAminoUltraPure qs qs qs qs ‐
agent NeutrolTE ‐ ‐ ‐ ‐ qs
Additional Glycerin 3.0 3.0 3.0 3.0 ‐
ingredients EdetaBD
Phenonip
Water
Ethanol
0.2
1.0
qs
100%
‐
0.2
1.0
qs
100%
‐
0.2
1.0
qs
100%
‐
0.2
1.0
qs
100%
‐
‐
‐
‐
qs100%
aSPFinvivoandstandarddeviationevaluatedinaccordancewithISO24444:2010
guidelineswithn=5
Chapter4.Filmthicknessdistribution 66
Figure4.1. Rheological behavior of sunscreensmeasuredwith AR‐G2 rheometer (TA
instrument),CP4°/40mm,Gap100µm,T=23°C
4.3.4.Applicationofsunscreen
Weapplied2.0mg/cm²ofsunscreennominallycorrespondingtoafilmthicknessof20
µm.Thesunscreenwasappliedinformof20to30smalldropsevenlydistributedover
theskinsurfaceandspreadmanuallywiththefingertipusingapre‐saturatedfingercoat.
Twospreadingprocedureswereemployed.Inthefirst,thesunscreenwasspreadonthe
specimenwithlightcircularmovementsfollowedbyleft‐to‐rightlinearstrokesfromtop
tobottomstartingateachsideofthespecimen(designatedspreading1);inthesecond,
thecompletelinearstrokestepwasrepeatedfourtimes(designatedspreading2).The
spreadingprocedure2resultedinalongerapplicationtime.Furthermore,thepressure
used to distribute the product was varied for spreading 1 between low and high,
corresponding to a force of 10014g and 28135g, respectively. These values
representextremesusedintheauthors’laboratorywiththissubstratepreparation.
Chapter4.Filmthicknessdistribution 67
The twopressureandspreadingconditionswereusedsolelywith thegel formulation
(GEL).Allothersunscreenformulationswereappliedwithhighpressureandspreading
procedure1.
4.3.5.Measurementofthesunprotectionfactorin
vitrousingspectraltransmissionofultraviolet
radiation
MeasurementofSPFinvitroisbasedondiffuseUVtransmissionspectroscopyaccording
totheapproachproposedbySayre12,
SPF ∑ ser λ . Ss λ
∑ ser λ . Ss λ . T λ 4.1.
where,ser(λ)istheerythemaactionspectrumasafunctionofwavelengthλ9,Ss(λ)isthe
spectral irradiance of the UV source at wavelength λ9 ,and T(λ) is the measured
transmittanceofthelightthroughasunscreenfilmappliedonasuitableUVtransparent
substrateatwavelengthλ13.
ForSPFdetermination,thespectralUVtransmittancewasregisteredfrom290to400nm
in1nmincrementstepsthroughskinsubstratepreparationsbeforeandafterapplication
ofasunscreenusingLabsphereUV‐2000S.TheUVtransmittanceof fourpositionsper
2cm2cmskinsubstratewasmeasuredtocovervirtuallythecompletesurfaceareaof
thepreparation.
Theblanktransmittancespectrumwasrecordedatfirstforeachsinglepositionbefore
sunscreen application followed by topographical measurement of the bare skin (see
section4.3.6.).Subsequently,sunscreenwasappliedandtopographicalmeasurementwas
performed again. After completion of topographical measurement which lasted
approximately4h,UVtransmissionthroughthesunscreen‐coveredskinsubstratewas
measured. Stability of SPF in vitro values over 4hwas checked and confirmed for all
sunscreens.
Chapter4.Filmthicknessdistribution 68
4.3.6.Assessmentofthesunscreenfilm
Thelayerofsunscreenappliedonpigskinsubstratewasinvestigatedusingtopographical
measurementswithanopticalprobebasedonwhitelightchromaticaberrationprinciple
(Altisurf 500 instrumentation). The instrumentation allowed non‐contact surface
topographymeasurement and analysis. The employed optical sensor yielded an axial
resolution(z)of5nmandalateralresolution(x‐y)of1.1µm.Themotorizedx‐ystage
permittedscanningofsamplesinthemmrange.Skinpreparationsonquartzplateswere
fixedonthestageusingacustommadeholder.
Surfacetopographyofbareskinandskincoveredwithsunscreenwasmeasuredinorder
toassessthesunscreenfilm.Skinpreparationswereremovedfromthedesiccatorand
allowed to equilibrate for 12 h next to the device at room conditions before starting
topographical measurements. Repeated measurements on bare skin using the "loop"
optionoftheoperatingsoftwarerevealedthatthisequilibrationwasnecessarytoallow
stabilizationofthesurfacepositionalongthezaxis(datanotshown).Aftermeasuringthe
surfacetopographyofbareskinsunscreenwasapplied,equilibratedfor15min,andthe
sameareawasscannedagain.
Figure4.2.illustratestheareaoftopographicalandUVtransmittancemeasurements.
Figure4.2.IllustrationofareasfortopographicalandUVtransmittancemeasurements;
thebigsquarecorrespondstothecarrierquartzplate,thedottedsmallsquaretotheskin
surfaceareawithadimensionof2cmx2cm,thefourcirclescorrespondtotheareasof
UVtransmittancemeasurements(SPF)withadiameterof1cmandthetworectanglesto
thetwoareasoftopographicalmeasurements.
Chapter4.Filmthicknessdistribution 69
Topographicalmeasurementswereperformedontworectangularareas(approximately
23mm8mm)perspecimen(figure4.2.).Apartoftherectangulararea(about5mm
8mmonlefthandside)correspondedtoquartzwithoutskinandservedasreference.The
skinarea(righthandsideoftherectangle)measuredabout18mm8mm.Topography
wasrecordedinlineseachextendingoverthequartzandtheskinpartoftherectangle
withanincrementstepof10µm.Therectangularareaswerescannedwithlinesin10µm
intervalsresultingin1840000singlemeasurementpointsperrectangle.
The raw data of the topographical measurements were redressed by a line‐by‐line
levelingcorrectionofeachrectangularsurfacetothesamex‐yplaneusingthequartzpart
ofeachmeasuredline(leftsideofrectangle,figure4.2.)Thisredressingprocedurewas
carriedoutwiththedataofbareskinandskincoveredwithsunscreenandwasessential
in order to correct for variation due to positioning and due to environmental factors
changinginthecourseoftheexperiment.Eachrectangularsurfaceareawasdividedinto
twozonesof8mm8mmcoincidingwiththefourpositions(circles)ofUVtransmittance
measurementsfigure4.2.).
Thefilmthicknessofappliedsunscreenwasobtainedasthedifferenceoftheredressed
skintopographydatawithandwithoutsunscreencomputedforeachsinglemeasurement
point.Theresultwasexpressedasadistributionoffrequenciesoffilmthicknessoverthe
measuredsurfaceareanormalizedto100%andisreferredtoasthicknessdistribution
curve.Athresholdof0.5%ofareaundthecurvewasappliedtoremoveextremevalues
atbothendsofthefilmthicknessdistribution.
Tovalidate thismeasurementandcalculationmethod,a surfaceareaofbareskinwas
measuredtwiceandfilmthicknesswascomputed.Theresultwasfoundtobecentered
around 0m (n=8), confirming the validity of the method for measurement of the
sunscreenfilmthicknessdistributiononskin.
Chapter4.Filmthicknessdistribution 70
Data extracted from the distribution curve and serving to characterize the applied
sunscreenfilmaregiveninTable4.2.
Table4.2.Dataextractedfromthethicknessdistributioncurveofappliedproduct
Parameter Meaning
Smean (m) Average of film thickness over the measured area
Smean to median ratio Indicator of film homogeneity
Abbott-Firestone curve Cumulative frequency of occurrence of film thickness
Smean is the frequencyweightedaveragethickness.TheSmeantomedianratioof the
thickness distribution is a measure of skewness of distribution and is used as an
expressionoffilmhomogeneity;thesmallerthisratiothegreaterthehomogeneityofthe
film. The Abbott‐Firestone curve is commonly used in surface metrology 274 and is
employedheretodepicttheexperimentallydeterminedthicknessdistribution,indicating
thicknessanduniformityofappliedproductlayer.
4.3.7.Statisticalanalysis
StatisticalanalysiswasperformedusingStatgraphicscenturionXVIsoftware(Statpoint
Technologies,Inc.,Warrenton,VA,USA).TheimpactofformulationvehicleonSPFinvitro
andonfilmparameterswasassessedwithKruskal‐Wallisnon‐parametrictest,andthe
impact of application conditionswas assessedwithMann‐WhitneyU test, bothwith a
statistical significance at 5% confidence level. In case Kruskal‐Wallis test revealed a
statistically significant difference among sunscreens for an investigated parameter, a
multiplepairwisecomparisontestusingBonferroniapproachwasperformedtoidentify
which sunscreens differed significantly from which other. Correlations between film
parameters and SPF in vitro values within each formulation were assessed using
Spearman`srankcorrelationcoefficienttest.
Chapter4.Filmthicknessdistribution 71
4.4.Results
4.4.1.Filmassessment
The film thickness distribution of sunscreen, extracted from the topographical
measurements,isvisualizedthree‐dimensionallyforqualitativeassessmentinfigure4.3.,
andisdisplayedquantitativelyasadistributioncurveofthicknessfrequency.Fromthe
distribution curve, the Abbott‐Firestone curve (cumulative frequency) was deduced
(figure4.4.).
Figure4.3.Exampleofthree‐dimensionalvisualizationoffilmthicknessdistributionofOW‐Csunscreen
Chapter4.Filmthicknessdistribution 72
Figure4.4. Example of distribution of film thickness frequency and Abbott‐Firestone
curveofOW‐C sunscreen
Thickness distributionwas always positively skewed, the degree of skewness varying
between the different sunscreens. In the example of figure 4.4., the most frequently
occurringfilmthicknesswasintherangeof2to4µmwhileathicknessaslargeas10to
13µmwasrecorded.Asmallpercentageofareaunderthethicknessdistributioncurve
laybelowafilmthicknessof0m,whichwaslikelyduetoexperimentalerror.Thiswas
includedinthecalculationoftheSmeanvalue.
Chapter4.Filmthicknessdistribution 73
4.4.2.Impactofvehicleonfilmparametervaluesand
SPFinvitro
Figure4.5.givestheaverageofAbbott‐Firestonecurvesofallmeasurementswitheach
investigatedsunscreenusinghighpressureandspreading1conditionsofapplication.
Figure 4.5. Abbott‐Firestone profiles of investigated sunscreens applied with high
pressureandspreading1
TheAbbott‐Firestoneprofilesdifferedconsiderablybetweenthesunscreens(figure4.5.).
Filmthicknesswasdifferentforthedifferentvehiclesanddecreasedroughlyintheorder
WO>GEL>OW‐C>CAS>OW‐S. For WO for example, a film thickness of 2.41m
corresponds to 50% of cumulative thickness frequency meaning that 50% of the
measuredsurfaceareaofthesampleexhibitedafilmthicknessgreaterthan2.41µm.As
a comparison, 50% of themeasured area of OW‐S exhibited a thickness greater than
merely1.20m.Moreover,theshapeofthecurvedifferedbetweentheusedvehicles,the
WO,forexample,showedamoreflat‐shapedprofilecomparedtoCAS(figure4.5.).
Chapter4.Filmthicknessdistribution 74
These differences between the vehicles are reflected by the calculated film thickness
parametersSmeanandSmeantomedianratioofdistribution.
Table4.3.givesthevaluesofthemedianandinterquartilerangeforthefilmparameters
ofallindividualmeasurementsofeachinvestigatedsunscreen.Also,SPFinvitrovaluesof
thesesunscreensaregiveninTable4.3.
Table 4.3.Medians of SPF in vitro, Smean, and Smean to median ratio of thickness
distributionwith interquartilerangeQ1–Q3(inbrackets) for investigatedsunscreens
withhighpressureandspreading1.
Sunscreen SPFinvitro Smean(m) Smeantomedianratio
OW‐C(n=27) 33(30–48) 2.3(2.0–2.7) 1.30(1.25–1.44)
OW‐S(n=20) 16(13–26) 1.6(1.2–2.0) 1.41(1.30–1.96)
GEL(n=28) 28(20–34) 2.6(2.4–3.1) 1.19(1.16–1.23)
WO(n=24) 72(55–85) 2.9(2.6–3.2) 1.19(1.17–1.21)
CAS(n=20) 14(7–20) 2.2(1.7–2.6) 1.71(1.44–1.99)
SPFinvitrovariedmarkedlybetweentheinvestigatedsunscreenformulationsattaining
valuesfrom14forCASto72forWO.SPFinvitrovaluesarecomparedtotheSPFinvivo
in figure4.6.SPF invitrovaluesgenerallyapproachedSPF invivoand,consideringthe
declaredvariationrange,asatisfactoryagreementbetweenSPF invitroand invivo for
spreading1andhighpressureconditionisfound.WOsunscreenwasanexception,with
surprisinglylowandhighSPFinvivoandSPFinvitro,respectively.Insilicoestimationof
SPFgaveavalueof25(figure4.6.).Thiscomputationalapproachtakesintoaccountthe
absorbancespectrumofeachUVfilter,theirphotostabilityandmutualstabilizationorde‐
stabilization, and their distribution in the phases of the vehicle and uses the Gamma
distributionfunctiontodescribefilmirregularity.275Theestimatedvaluelaywithinthe
rangeoftheexperimentalvaluesofallvehicles,yettheinsilicocalculationcannotpredict
the effect of formulation on SPF. In figure 4.6. the Smean of the formulations is also
visualized.
Chapter4.Filmthicknessdistribution 75
Figure4.6.SPFinvivo(whitecolumns)withstandarddeviation(bars),mediansofSPFin
vitro(graycolumns)withinterquartilevalues(bars)withOW‐Cn=27,OW‐Sn=20,GEL
n=28,WOn=24,CASn=20,SPFinsilico(blackline),andmediansofSmeanvalues(square)
ofsunscreensappliedwithhighpressureandspreading1
TheimpactofvehicleonSPF invitroandfilmparameterswasevaluatedwithKruskal‐
Wallistest(Table4.4.).
Table 4.4. Impact of vehicle on SPF in vitro, Smean, and Smean to median ratio of
thicknessdistribution
ParameterStatisticallysignificantdifferencea
SPFinvitro
Smean
Smeantomedianratio
Yes(p<0.05)
Yes(p<0.05)
Yes(p<0.05)
abetweenthedifferentformulationsonSPFinvitro,Smean,andSmeantomedianratioof
thicknessdistributionat5%confidencelevel(Kruskal‐Wallis)
Chapter4.Filmthicknessdistribution 76
This statistical test revealeda significanteffectofvehicleonall testedparameters.To
identify which sunscreens differed significantly from each other with respect to the
studiedparameters,amultiplepairwisecomparisontestbasedonBonferroniapproach
wasemployed.TheresultsaregiveninTable4.5.,4.6.,and4.7.
Table4.5.MultiplepairwisecomparisontestusingBonferroniapproachforSPFinvitro
Group
classificationa
WO OW‐C GEL CAS OW‐S
Group1 X ‐ ‐ ‐ ‐
Group2 ‐ X X ‐ ‐
Group3 ‐ ‐ X X X
asunscreensthatwerenon‐significantlydifferentfromeachotherwithrespecttoSPFin
vitrowereassignedtothesamegroup
Table4.6.MultiplepairwisecomparisontestusingBonferroniapproachforSmean
Group
classificationa
WO GEL OW‐C CAS OW‐S
Group1 X X ‐ ‐ ‐
Group2 ‐ X X ‐ ‐
Group3 ‐ ‐ X X X
Groupe4 ‐ ‐ ‐ X X
asunscreensthatwerenon‐significantlydifferentfromeachotherwithrespecttoSmean
wereassignedtothesamegroup
Table4.7.Multiplepairwisecomparison testusingBonferroniapproach forSmean to
medianratio
Group
classificationa
WO GEL OW‐C CAS OW‐S
Group1 X X X ‐ ‐
Group2 ‐ ‐ ‐ X X
asunscreensthatwerenon‐significantlydifferentfromeachotherwithrespecttoSmean
tomedianratiowereassignedtothesamegroup
Chapter4.Filmthicknessdistribution 77
This multiple comparison test resulted in a group classification of the investigated
sunscreens.Formulationsofonegroupdiffer statistically fromthoseofanothergroup
while formulationsthatbelongtothesamegroupdonotdiffersignificantly fromeach
otherwithrespecttotheconsideredparameter.Whenthesameformulationiscontained
in twodifferent groups it does not differ significantly from the formulations of either
group.Thenumberofgroupswasdifferentforthetestedparameters;two,three,andfour
groupswere foundforSmeantomedianratio,SPF invitro,andSmean,respectively.A
smallnumberofgroupsmeanslessdifferencebetweenthesunscreens.
WOyieldedasignificantlyhigherSPFinvitrothanallothersunscreens,agreaterSmean
thanOW‐C,CASandOW‐SandasmallerSmeantomedianratiothanCASandOW‐S.OW‐
CgaveahigherSPFinvitroandasmallerSmeantomedianratiothanCASandOW‐S.The
GELandOW‐Cformulationsdidnotdiffersignificantlyfromeachotherwithrespectto
any of the criteria. Also, CAS and OW‐S did not differ from each other. OW‐C yielded
greaterSPF invitro,agreaterSmeanandasmallerSmeantomedianratio thanOW‐S,
whichwas interestingbeing that these two sunscreensvariedonly in their contentof
thickeners,hencetheirviscositycharacteristic.
Finally, thecorrelationof theSPF invitrowithbothfilmparameters for the individual
measurementswithin each sunscreenwas evaluatedusing Spearman rank correlation
test.AsignificantpositivecorrelationbetweenSPFinvitroandSmeanwasfoundforevery
sunscreenformulation(p<0.05).AnegativecorrelationwasfoundbetweenSPFinvitro
andSmeantomedianratioforWO;OW‐S,andCAS(p<0.05),whereasnocorrelationwas
foundforOW‐CandGEL.
4.4.3.Impactofpressureandspreadingprocedureon
filmparametervaluesandSPFinvitro
In addition to the vehicle, the impact of the application conditions, i.e., spreading
procedureandpressureonfilmparametersandSPFinvitrowasstudiedusingtheGEL
sunscreen. Intotal, threeconditionsofapplicationwereinvestigated,spreading1with
highpressure,spreading1withlowpressure,andspreading2withhighpressure.
Chapter4.Filmthicknessdistribution 78
Figure4.7.showstheaverageofAbbott‐FirestonecurvesoftheGELsunscreenforeach
applicationconditionandTable4.8.givesthemedianandinterquartilerangevaluesof
SPFinvitroandthefilmparametersfortheinvestigatedconditions.
Figure4.7.Abbott‐FirestoneprofilesofGELsunscreenappliedwithtwopressure(high
andlow)andspreading(1and2)conditions
Itisevidentfromfigure4.7.thattheshapeofAbbott‐FirestonecurveofGELsunscreenis
differentbetweenspreading2andspreading1,whilenodifferencewasfoundbetween
lowandhighpressureusingspreading1.ThedifferencesoftheAbbott‐Firestonecurves
arereflectedintheSmeanandSmeantomedianratio.
Chapter4.Filmthicknessdistribution 79
Table 4.8.Medians of SPF in vitro, Smean, and Smean to median ratio of thickness
distributionwithinterquartilerangeQ1–Q3(inbrackets)forinvestigatedconditionsof
applicationforGELsunscreen
Application of GEL
sunscreen
SPFinvitro Smean(m) Smean to median
ratio
Spreading 1, high
pressure,n=28
28(20–34) 2.6(2.4–3.1) 1.19(1.16–1.23)
Spreading 1, low
pressure,n=24
39(30–54) 2.7(2.4–3.1) 1.19(1.17–1.21)
Spreading 2, high
pressure,n=24
20(15–25) 1.9(1.5–2.3) 1.57(1.50–1.91)
SPFinvitrodatameasuredforeachconditionofapplicationwerecomparedtotheSPFin
vivoforGELsunscreen(figure4.8.).Fromthisevaluation,spreading2withhighpressure
seemstogiveabetterapproximationoftheSPFinvivo.However,asthisconditioncould
notbepracticallyappliedtoalltypesofformulation,spreading1withhighpressurewas
usedasanalternativeintheinvestigationofthedifferentvehicles.
Figure4.8. SPF in vivo (white columns),medians of SPF in vitro (gray columns)with
interquartilevalues(bars),andmediansofSmeanvalues(square)ofGELsunscreen.
Chapter4.Filmthicknessdistribution 80
Theimpactofspreading(procedure1versus2)andpressure(lowversushigh)onSPFin
vitroandfilmparameterswereevaluatedusingMann‐WhitneyUtest(Table4.9.).
Table4.9.ImpactofapplicationconditionsonSPFinvitro,SmeanandSmeantomedian
ratioofthicknessdistributionofGELsunscreen
Applicationcondition ParameterStatistically significant
differencea
Spreading
(1versus2)
SPFinvitro
Smean
Yes(p<0.05)
Yes(p<0.05)
Smeantomedianratio Yes(p<0.05)
Pressure SPFinvitro Yes(p<0.05)
(lowversushigh) Smean No(p>0.05)
Smeantomedianratio No(p>0.05)
abetweentestedapplicationcondition(eitherspreadingorpressure)andSPF invitro,
Smean,Smeantomedianratioat5%confidencelevel(Mann‐WhitneyUtest)
Spreading2showedasignificantlysmallerfilmthickness(Smean)andalargerSmeanto
medianratiocomparedtospreading1(Table4.8.and4.9.).Both,spreadingandpressure
hadasignificanteffectonSPFinvitro.Forspreading2comparedtospreading1andhigh
comparedtolowpressureareductionofSPFinvitrowasfound.Filmparameterswere
notinfluencedsignificantlybypressure.
4.5.Discussion
Thiswork tests the hypothesis that film thicknessdistribution canbeused to explain
variationofSPFbetweensunscreenvehiclesandapplicationconditions.Forthispurpose,
accuratemeasurementoffilmthicknesswasnecessary.
Manytechniques forassessingthefilmdistributionofanappliedsunscreenhavebeen
usedprovidingmerelyqualitativeorsomequantitativeinformationaboutitsdistribution.
Chapter4.Filmthicknessdistribution 81
Forqualitativeassessment,fluorescenceresultingeitherfromaUVfilterorfromanadded
fluorescentmarkerwasusedtovisualizethehomogeneityofdistributionoftheapplied
product.Sunscreendistributionwasevaluatedinvivousinganappropriateillumination
sourceoptionallycombinedwithphotography220,276,277ormultiphotontomography246;
forinvivoandontapestripsevaluationtheuseoflaserscanningmicroscopy27wasalso
reported. Alternatively, for sunscreens containing titanium dioxide as UV filter, light
microscopyoncrosssectionsofskinbiopsies20wasusedthatgavearoughestimationof
the thickness layer based on the visualization of titanium dioxide particles; optical
coherent tomography278wasalsousedon intactskinthatdetectedthedistributionof
titaniumdioxideparticleswithinthesunscreen layer.Forquantitativeassessment, the
use of in vivo fluorescence spectroscopy gave indirect information about the film
thickness by converting the fluorescence intensity into an equivalent thickness of an
appliedproduct23,269.Whensunscreensarenotintrinsicallyfluorescent,thistechnique
requirestheadditionofafluorescentagentwhich,however,oftenproducedinconclusive
results because of immiscibility or interference issues 248. An alternative approach
reported theuseof an invivo skin swabbing technique in conjunctionwith sunscreen
quantificationbyUVspectroscopytoevaluatethethicknessofthefilm212.Noneofabove
mentionedmethods,however,provideda fullcharacterizationofthesunscreenfilmin
termsofthicknessandhomogeneityofdistribution.
Inourwork,westartedfromanapproachbasedontopographicalmeasurements.This
methodwasusedbeforeonskinreplicatesandprovidedasemi‐quantitativeassessment
of film thickness 279. In contrast to that work, we used a biological substrate for the
application of sunscreen to reproduce as closely as possible the product‐to‐substrate
adherence relevant for the in vivo situation. In addition, by developing a reference‐
correctedmeasurementprotocolandquantitativedataevaluationthecompletethickness
distribution could be determined. Topographical evaluation was combined with
measurement of SPF invitro both ofwhichwereperformed in the sameposition and
nearlythesamesurfaceareamakingitpossibletorevealexistingcorrelations.
The composition of the five studied vehicles principally differed in the thickener and
emulsifiersystem,theUVfiltercombinationremainingthesame.Theformulationofthe
vehicleshadasignificanteffectonSmeanandSmeantomedianratio(Table4.6.andTable
4.7.).
Chapter4.Filmthicknessdistribution 82
OfthetwosunscreensOW‐CandOW‐Swhichdifferedmainlyintheirthickenersystem,
OW‐SshowedasignificantlysmallerSmeanandgreaterSmeantomedianratiothanOW‐
C.ThethickenersLanetteO,KeltrolRD,andSalcareSC91containedinOW‐Cbutnotin
OW‐Scorrespondedtoarelativeweightdifferenceofonly10%intheremainingfilmon
theskinsurfaceofOW‐SversusOW‐C,buttheyappeartoberesponsibleforthesignificant
differenceoffilmthicknessandhomogeneitybetweenthetwosunscreens.Thisindicates
thatthickeners,whichenabletheformationofafirmfilmuponspreadingleadalsotoa
thicker andmorehomogeneous film.OW‐S andCASdidnotdiffer in their Smeanand
SmeantomedianratiobothyieldingasmallerSmeanandlargerSmeantomedianratio
thantheothervehicles.Thisalsoseemstoberelatedtotheabsenceofthickenersinboth
formulations.Theemulsifier,thatwaspresentintheOW‐Semulsion,butnotinCASwhich
wasamono‐phase,seemstoplayaminorrolefortheSmeanandtheSmeantomedian
ratio. The same observation is true for OW‐C and GEL sunscreens that did not differ
statisticallyinSmeanandSmeantomedianratiobothcontainingthickenersbutonlyOW‐
Ccontainingemulsifiers.WOhadastatisticallylargerSmeanthanOW‐C,OW‐SandCAS
which might be related to its continuous oil phase; yet it did not show a significant
differencetoGEL.WithrespecttoSmeantomedianratio,thelowviscosityvehiclesCAS
andOW‐Sshowedahigherpositivelyskewedthicknessdistribution,henceagreaternon‐
homogeneityof film thanthehighviscosityvehiclesWO,OW‐CandGEL. It shouldbe
pointedoutthatSmeandifferencesbetweenthevehicleswerenotduetodifferencesin
masslossduringapplication.
The formulation of the vehicles had a significant effect on SPF in vitro (Table 4.5.). It
appearsthatlargeandsmallSmeanvaluesamongvehiclescorrespondedrespectivelyto
high and low SPF in vitro. Therefore, the differences in SPF between vehiclesmay be
discussed in relation to the film parameter Smean. For this we consider that smaller
Smean is connected to a greater occurrence of small film thicknesses and that light
transmittance, which is inversely proportional to SPF, increases exponentially with
decreasingfilmthickness.OW‐SandCASforinstance,exhibitedthesmallestSmeanvalues
andyieldedalsothelowestSPF.Thesetwosunscreenswhichlackedthickenersandhad
the lowest viscosity compared to the rest may leave larger areas of ridges virtually
uncoveredwhile accumulating in the furrows thus leading to low SPF. Therefore, the
presenceof thickeners inthe formulationseemstobeaprevailingprerequisite forUV
efficacy.Further,WOexhibitedboth,thelargestSmeanvalueandthehighestSPF.
Chapter4.Filmthicknessdistribution 83
Thisisconsistentwithminimalsurfaceareawithverysmallfilmthicknessthatwouldbe
virtuallyunprotected.Furthermoreandincontrasttotheothersunscreens,theUVfilters
of WO are distributed in the continuous phase which does not evaporate, forming a
uniformprotectingfilmwiththehelpofthethickeners.Anincreaseofabout45%ofSPF
invitrowasfoundfortheWOsunscreencomparedtoOW‐C,whichisinlinewithdata
reportedpreviouslyonsunscreenswithsmallerSPFvalues20.CASandOW‐Saswellas
OW‐C and GEL did not differ with respect to any of the tested criteria and can be
considered as very similar in terms of film forming ability and SPF efficacy. Taken
together, the SPF variation observed between sunscreens containing the same filter
compositionisproposedtoarisefromthedifferenceintheirfilmthicknessdistribution.
Withineverysunscreen,SmeancorrelatedpositivelywithSPFinvitro.Further,Smeanto
median ratio showed a negative correlation with SPF in vitro for three of the five
sunscreens.Thisdemonstratesthesignificantconnectionbetweenthefilmformationand
sun protection efficacy and supports the observation discussed above about the
differences between sunscreens. The present data addressing film formation and
thickness distribution go beyond previous studies that showed that film thickness
resultingfromdifferentapplicationamountofsunscreenstrongly impactsSPFefficacy
180,265.
Besidesthevehicleformulation,thisworkdemonstratedusingtheGELthatapplication
conditions can significantly impact sunscreen performance. We found that a longer
spreadingtimeresultedinalargerSmeantomedianratio,asmallerSmeanandsmaller
SPF in vitro values (Table 4.8.) further corroborating the correlation between film
characteristicsandsunscreenefficacy;also, increaseofpressureby180gresultedina
significantdecreaseinSPFvalues.Interestingly,thiseffectofprolongedandhighpressure
applicationwasanalogoustothatelicitedbylowviscosityformulations,whichmightbe
related to a thinning of the GEL under these application conditions. The effect of
application conditions on the performance of the other vehicles still needs to be
investigated.Someauthorsreportedthatevenachangeinpressureof50gledtodifferent
SPFinvitrowhenusingsyntheticplatesassubstrate188.Formerstudiesreportedthata
morerubbedapplicationledtoasmallerSPFinvivo22andacrudecomparedtoacareful
applicationtoasmallercreamthickness23.
Chapter4.Filmthicknessdistribution 84
Finally,morerecently,theeffectofcarefulversuscrudespreadingofsunscreenonthe
magnitude of erythemaoccurrencewas simulated, andunderlined the "importance of
homogeneityofspreadingonthelevelofdeliveredprotection"24.
Figure4.9.summarizestheconnectionbetweentheinfluencingfactors, i.e.,application
conditionandvehicle,thefilmdistributionandthemeasuredSPFinvitroofsunscreens.
Figure4.9.Connectionsbetweeninfluencingfactors,filmdistribution,andSPFefficacy
4.6.Conclusion
Thetypeandtheviscosityofsunscreenvehiclesandapplicationconditionsplayarolefor
the film thicknessparameters that finally influenced theSPFefficacy.Highapplication
pressure,longspreadingtime,lowviscosityofformulationand/orabsenceofthickeners
were shown to impact unfavorably UV protection. As application condition can in
principlebe fixed, the impactof a vehicleon the formed filmcannowbe investigated
during the product development step. Sunscreen composition might be optimized
accordinglytoachievealargefilmthicknesswithuniformdistribution;minimizationof
thesmallthicknessfractionofthefilmbeingcrucialforultimatesunscreenperformance.
Developmentofamethodtoquantifythefilmthicknessdistributionofsunscreenonskin
wasshowntobeessentialforunderstandingthemechanisminfluencingUVefficacy.
Chapter5
CalculationoftheSPFof
sunscreenswithdifferent
vehiclesusingmeasured
filmthicknessdistribution–
comparisonwithSPFinvitro
5.1.Abstract
Thesunprotectionfactor(SPF)dependsonUVfiltercomposition,andamountandtype
ofvehicleoftheappliedsunscreen.Inchapter4,weshowedthatthevehicleaffectedthe
averagethicknessofsunscreenfilmthatisformeduponapplicationtoaskinsubstrate
andthatfilmthicknesscorrelatedsignificantlywithSPFinvitro.
M.Sohnetal.,“CalculationoftheSPFofsunscreenswithdifferentvehiclesusingmeasuredfilmthicknessdistribution–comparisonwithSPFinvitro”J.Photochem.Photobiol.B.159(2016)74‐81.
85
Chapter5.SPFinsilico 86
Here, we quantitatively assess the role for sunscreen efficacy of the complete film
thicknessfrequencydistributionofsunscreenmeasuredwithanoil‐in‐watercream,an
oil‐in‐water spray, a gel, a water‐in‐oil, and an alcoholic spray formulation. A
computational method is employed to determine SPF in silico from calculated UV
transmittance based on experimental film thickness and thickness distribution, and
concentration and spectral properties of the UV filters. The investigated formulations
exhibiteddifferentSPFinvitroanddifferentfilmthicknessdistributionespeciallyinthe
smallthicknessrange.WefoundaverygoodagreementbetweenSPFinsilicoandSPFin
vitroforallsunscreens.Thisresultestablishestherelationshipbetweensunprotection
and the film thickness distribution actually formed by the applied sunscreen and
demonstratesthatvariationinSPFbetweenformulationsisprimarilyduetotheirfilm
formingproperties.Italsoopensthepossibilitytointegratetheinfluenceofvehicleinto
toolsforinsilicopredictionoftheperformanceofsunscreenformulations.Forthis,useof
the Gamma distribution was found to be appropriate for describing film thickness
distribution.
5.2.Introduction
Topicalsunscreensrepresentasimple,practicalandefficientmeans7ofprotectingskin
from damages inflicted by solar radiation 2‐5. The evaluation of the performance of
sunscreenproducts is carriedoutbyan invivomethodologyrequiringclinical trials 9.
Although this is a laborious, time consuming, expensive and ethically questionable
method,itremainstodatetheonlyvalidatedmethodfordeterminingthesunprotection
factor(SPF)that isapprovedbyregulatorybodies.Therefore,alternative invitroor in
silicomethodsareurgentlyneeded.Alotofefforthasbeenputintothedevelopmentof
an in vitro method for SPF determination. Current in vitro methodology utilizes
measurementofspectraltransmittanceofUVlightthroughalayerofsunscreenapplied
toasuitablesubstrateandtakes intoaccount theerythemaleffectivenessspectrumto
determineSPF.Yet,noattempthasbeenfullysuccessfulsofarinreproducingtheinvivo
results ina repeatable fashion,many issuesstill remaining17‐19,189,190 concerning fore‐
mostly(i)thechoiceofasubstratethatbestmimicshumanskinand(ii)controlofthe
processofapplication10,23.Notably,substratesofPMMAroutinelyusedintheindustrydo
notprovidesatisfactoryresults10,189,190.
Chapter5.SPFinsilico 87
Some authors introduced an in silico approach for an a priori calculation of the
performanceofsunscreens25,191,192.InanalogytoSPFinvitro,SPFinsilicoisbasedonUV
spectraltransmittancewhichiscombinedwitherythemaleffectiveness.
ASpectraltransmittanceiscalculatedinthiscasebasedontheabsorptionpropertiesof
the UV filters, their concentration and the product layer thickness. Since thickness
uniformitywasfoundtodependonsubstrateroughnessandtheapplicationprocess195
and,moreover,toplayaroleforsunscreenperformance24,25,246,areliableestimateoffilm
thicknessdistributionisrequired.Forthis,acalibratedstepfilmmodelatfirst193,197and
lateracontinuousthicknessdistributionmodel11,191,195wereused.Thelatterapproach
utilizedthegammadistribution,astatisticalprobabilitydistributionfunctioncontaining
oneadjustableshapeparameter,todescribethecumulativethicknessdistributionofthe
sunscreen film. By deducing the value of the shape parameter through fitting to
experimentaldataofinvitrospectralabsorption191,195orinvivoSPF192,193,195thismodel
wasshowntodescribetheresultsaccuratelyandtherefore,providethepossibility for
theoreticalinsilicopredictionoftheperformanceofsunscreens.Yettheneedtoassume
afilmthicknessdistributionmodelandthechoiceofexperimentaldata,i.e.,formulation
vehicle,applicationamountandprocess,substrateroughness,thatareusedasreference
fordefiningitsshaperemainalimitation.
Inchapter3,wedemonstratedthattheuseofasubstrateforSPFinvitromeasurement
consistingofisolatedepidermisfrompigearskinlaidonquartzplatesprovidedresults
thatdidnotdiffersignificantlyfromSPFdeterminedclinically.Moreover,wedevelopeda
methodbasedontopographicalmeasurementstodeterminetheaccuratefilmthickness
frequency distribution of sunscreens applied to this substrate (chapter 4). Using five
differentsunscreenformulationscontainingthesameUVfiltercombinationindifferent
vehicle types we examined the hypothesis that divergence of efficacy between the
sunscreensisrelatedtodifferencesinthicknessoftheappliedproductlayer.Thetypeand
theviscosityofthesunscreenformulationandtheprocedureofapplicationwerefound
to affect theweighted average film thicknesswhich exhibited a significant correlation
withthemeasuredSPFinvitro.Thissupportedthevalidityofthetestedhypothesisand
explained to a large extent the differences of sun protection performance between
sunscreenformulations.
The objective here is to quantitatively assess the role of film thickness frequency
distributionfortheperformanceofsunscreensandutilizethistoelucidatetheoriginof
variationofSPFobservedbetweendifferentsunscreenformulations.
Chapter5.SPFinsilico 88
For this purpose, a computationalmethodwas employedmakinguse of the complete
experimentalthicknessfrequencydistributionofsunscreenfilmforcalculatingtheSPF
value.Thismethod isbasedonUVspectral transmittance taking intoconsideration in
addition to film thickness distribution the UV filter absorption spectrum and
concentration.UVtransmittanceiscombinedwiththeerythemaleffectivenessspectrum
forastandardizedsolarradiationspectrumyieldingfinallyanintegralSPFoverasurface
area ofmeasurement roughly corresponding to that of invitro and invivo conditions.
Compared to previous studies this work employs in the calculation measured film
thickness data instead of an assumed thickness distribution making it possible to
investigatedifferencesbetweentheusedformulations.
ThecalculatedSPFvalueiscomparedwithSPFmeasuredinvitroinordertoestablish
thevalidityofthecomputationalapproachinvolvingfilmthicknessfrequency
distributionand,ultimately,proposeamethodologyforcalculatingSPFinorderto
predicttheefficacyofsunscreenproducts.For this methodology, the possibility to
express the film thickness frequency distribution by a model function for routine
application is explored.
5.3.Materialsandmethods
5.3.1.Chemicalsandequipment
The following UV filters were used: Tinosorb S abbreviated BEMT (INCI, bis‐
ethylhexyloxyphenol methoxyphenyl triazine), Uvinul N539T abbreviated OCR (INCI,
octocrylene)fromBASFSE,
Ludwigshafen, Germany; Eusolex 232 abbreviated PBSA (INCI, phenylbenzimidazol
sulfonicacid)fromMerck,Darmstadt,Germany;Parsol1789abbreviatedBMDBM(INCI,
butylmethoxydibenzoylmethane)fromDSM,Kaiseraugst,Switzerland;NeoHeliopanOS
abbreviatedEHS(INCI,ethylhexylsalicylate)fromSymrise,Holzminden,Germany.
Following equipment was used: Surface metrology instrument (Altisurf 500, Altimet,
Thonon‐lesBains,France);UVtransmittanceanalyzer(LabsphereUV‐2000S,Labsphere
Inc.,NorthSutton,NH,USA).
Chapter5.SPFinsilico 89
Following software packages were used: Phenix and Altimap (Altimet, France) for
topographicalmeasurementandevaluation,respectively;UV‐2000(LabsphereInc.,USA)
forUVtransmittancemeasurement;IgorPro6.32A(WaveMetrics,Inc.,Portland,OR,USA)
forthedatafittingandconvolutionoperation.
5.3.2.Preparationofskinsubstrate
We used epidermalmembrane of pig ear for sunscreen application prepared by heat
separationasdescribedinsection3.3.2.,method2(3.3.2.2.).
5.3.3.Sunscreenvehicles
Fivedifferentsunscreenvehiclesincludinganoil‐in‐watercream(OW‐C),anoil‐in‐water
spray(OWS),awater‐in‐oilemulsion(WO),agel(GEL)andaclearlipo‐alcoholicspray
(CAS)wereused.TheycontainedthesameUVfiltercompositionandemollient.Thefilter
systemwascomposedof8w‐%OCR,5w‐%EHS,2w‐%BMDBM,1w‐%BEMT,and1w‐
%PBSA.Thefullcompositionofinvestigatedsunscreenformulationsisgiveninsection
4.3.3.,Table4.1.
5.3.4. Measurement of the sun protection factor in
vitro
SPFinvitromeasurementwasbasedonUVtransmittance,whichdenotestheinverseof
theUVintensityattenuationfactormeasuredwithaprotectingsunscreenfilm12:
SPF ∑ ser λ . Ss λ
∑ ser λ . Ss λ . T λ 5.1.
Chapter5.SPFinsilico 90
where, the inverse transmittance (1/T) in theUV spectral range isweightedwith the
erythemalactionspectrum,ser(λ)9,andthespectralirradianceoftheUVsource,Ss(λ)9.
Ablanktransmittancespectrumrecording,topographicalmeasurementofbareskin(see
section 5.3.5.), sunscreen application, new topographical measurement, and UV
transmittance recording throughsunscreencoveredskin substratewereperformed in
sequence.
A sunscreen amount of 2.0 mg/cm² corresponding to a theoretical film thickness of
approximately 0.002 cm before water evaporation was applied. This experimental
procedurewasdescribedinchapter4.
5.3.5.Assessmentofthefilmthicknessdistributionof
anappliedsunscreen
Thesunscreenfilmwasinvestigatedusingtopographicalmeasurementswithaconfocal
opticalprobebasedonwhitelightchromaticaberrationprinciple.Thefilmthicknesswas
obtainedasthedifferenceofskintopographydatawithandwithoutsunscreen.Theresult
was expressed as a histogram of frequencies of film thicknesses over the measured
surfacearea and is referred tohereas film thicknessdistribution curve.The filmwas
assessed in an area coincidingwith that of the SPF in vitrodetermination. A detailed
descriptionofthismethodwasgiveninsection4.3.6..
5.3.6.Determination of the corrected film thickness
frequencydistributionofanappliedsunscreenusinga
convolutionapproach
The error of the method employed for measuring film thickness based on surface
topographywasestimatedbyrepeateddeterminationof the topographyof thesame
surface and calculation of the difference between consecutive topography
determinationsinapoint‐by‐pointfashionwiththesameprocedureastheoneusedto
calculatefilmthickness.
Chapter5.SPFinsilico 91
Thedeviationofthisdifferencefromzerosignifiedtheamplitudeofnoiseandfolloweda
frequencydistributionthatwasusedasmeasurementerror.Thiserrorestimationwas
performedwiththebaresubstrateconsistingofepidermalmembraneonquartzandwith
sunscreenappliedtothesubstrate.Theerrordistributioncurvesalsoreferredtoasblank
curveswereapproximatedbyEquation(5.2.).
1
2 exp 3 2 exp 4 2 15exp 6 2
(5.2.)
where,disnoiseamplitudeoffilmthicknessmeasurement,andthesixcoefficientsB1to
B6were deduced by least square fitting to the determined error data for each tested
condition.Themeasuredfilmthicknessdistribution,M,representstheconvolutionofthe
"true"distribution,q,withthedistributionofthemeasurementerror,B(Eq.5.3.).
∗ (5.3.)
In order, therefore, to obtain the true, i.e., corrected thickness distribution function, a
deconvolution operation must be performed. Since however deconvolution of two
functionsiscomputationallydifficult,thecorrectedfilmthicknessfrequencydistribution,
q,wasobtainedfromthemeasureddistribution,M,andtherespectiveblankcurve,B,by
aconvolutionoperationofqwithB(Eq.5.3.)andsimultaneousfittingtotheexperimental
resultsofMbyleastsquareanalysistodeterminethecoefficientsoffunctionq.Forthis
purpose,BofEq.(5.2.)withknowncoefficientsB1toB6andqdescribedbyEquation
(5.4.)wereused.
1
2 3 4 q5 4 1
67 8 ² 1
9
(5.4.)
where, d is the film thickness, and the nine coefficients q1 to q9 were treated as
adjustableparametersthatwerededucedfromtheleastsquareoptimization.Theform
offunctionqwasdefinedmakinguseoftheassumptionthatthisfunctionshouldalso
provide an adequate fit of the measured film thickness distribution, M. For the
convolution operation the error distribution, B,was centered on the zero point. The
convolve/AoptionoftheIgorPro6softwarewasemployed.
Chapter5.SPFinsilico 92
The determined function given by Eq. (4) was then used to build the corrected film
thickness distribution for each investigated sunscreen in a discrete form in 0.058 µm
incrementsteps.Asmallpercentageofareaunderthecorrectedthicknessdistribution
curvewas below a film thickness of 0 µm for each sunscreen. The percentage of film
thicknesswithavaluesmallerthan0µmwasdeletedandthethicknessdistributionwas
adjustedto100%givingtheqadjdistribution.Usingthisqadjdistributionmadepossibleto
calculate the SPF of every sunscreen as outlined below and to compare this to the
measuredSPFinvitrovalue.
5.3.7.Calculationofthesunprotectionfactorinsilico
ThemethodologyforcalculatingtheSPFinsilicoisbasedonthealgorithmusedwithSPF
invitromeasurements(Equation5.1.).However,themeasuredUVtransmittanceinthein
vitromethodisreplacedbyacalculatedUVtransmittanceaccordingtoEquation5.5.:
10 5.5.
where, ε(λ) is the average molar absorption coefficient (in L/(mol.cm)), c the molar
concentrationoftheUVfiltermixtureintheformulation(inmol/L),disfilmthickness
andgisequalto0.0015/Smean.Thefactorgisusedtoadjustthefilmthicknesstothevalue
correspondingtotheconcentrationofUVfiltersintheappliedsunscreenandaccounts,
therefore, for the evaporation of volatile components of the vehicle upon application.
Smeanistheaveragefilmthicknessincmobtainedfromtheqadjdistribution.Thevalueof
0.0015cmistheaveragefilmthicknessbeforeevaporation.Thisvalueratherthan0.002
cmisusedfortheapplied2mg/cm2consideringthatapproximately25%ofsunscreen
remained on the finger coat and beyond the edge of the substrate in the process of
spreadingasdeterminedgravimetrically.ForasunscreencontainingseveralUVfilters,as
itisgenerallythecase,thetransmittanceiscalculatedusingtheaveragemolarabsorption
coefficient of the UV filter mixture and molar concentration based on the average
molecularweightoftheusedUVfilters.
Chapter5.SPFinsilico 93
Consequently, to generate relevant calculated transmittance data, mixed absorbance
spectraarecalculatedfromtheUVspectroscopicperformancesandamountoftheused
UV filters 193.AsEq. (5.5.) shows, the global transmittancedataof a sunscreen film is
obtained as the sum of the transmittance through each thickness fraction of the film.
Transmittancewascalculatedatwavelengthsfrom290to400nmat5nmintervals.Using
thetransmittancevaluesobtainedfromEq.(5.5.),theSPFinsilicowascalculatedwithEq.
(5.1.).
Asummaryofthestepsfollowedtodeterminetheunknowncoefficientsoffunctionqto
obtainthecorrectedfilmthicknessdistributionandtocalculatetheSPFinsilicooftested
sunscreensisgiveninfigure5.1
Figure5.1.Stepsforthedeterminationofthecorrectedfilmthicknessdistributionand
SPFinsilicoofanappliedsunscreen
Chapter5.SPFinsilico 94
5.4.Resultsanddiscussion
5.4.1.Measurementerroroffilmthickness
Theexperimentalresultsoferrorestimationforthebaresubstrateandeachofthefive
sunscreenformulationsappliedonthesubstrateareshowninFigure5.2.
Figure5.2.Errordistributioncurvesforthebaresubstrateandeachofthefivesunscreen
formulationsappliedonthesubstrate
Theerrordistributioncurveforthebaresubstratewassymmetricalaroundthezeropoint
andhadamplituderangingapproximatelybetween‐1and1µm.Theerrordistribution
curves for the different sunscreen formulationswere also symmetrical but somewhat
widerthanthatofthebaresubstrateandthepositionoftheirapexwasshiftedintothe
negativevaluerange.
Chapter5.SPFinsilico 95
Theaveragefilmthickness,Smean,computedasthemeanofthefrequencydistributionwas
‐222nm,‐215nm,‐175nm,‐169nm,‐165nm,and‐113nmforthewater‐inoil(WO),
clear lipo‐alcoholicspray(CAS),oil‐in‐watercream(OW‐C),oil‐in‐waterspray(OW‐S),
andgel(GEL)formulation,respectively.Thissuggestsaslightrecessionofthesurfaceof
thesunscreenbetweenthetwomeasurementsprobablyduetoevaporationofvolatile
componentsoftheformulation.Themagnitudeofthisevaporationwastoosmalltobe
detectedbyanalyticalweightmeasurement(datanotshown).Bycomparison,Smeanfor
thebaresubstratewas‐12nm.
TheseexperimentalerrordistributionsweredescribedbyEquation(5.2.).Thefunction
givenbyEq.(5.2.)wasdeterminedempiricallybystartingfromtheLorentzianfunction
andaddingexponentialtermsinthedenominatorandaGauss‐liketermtoimprovethe
approximation.ThesixcoefficientsB1toB6deducedbyfittingforeachtestedcondition
aregiveninTable5.1.Theestimateswereaccurateasevidencedbytheirrathersmall
standarderror.
Table5.1.EstimatedcoefficientsinEquation(5.2.)fortheerrordistributioncurveBfor
thebareskinandeachofthefiveinvestigatedsunscreens
Coefficient BareskinSkincoveredwithsunscreen
OW‐C OW‐S GEL WO CAS
B1 20.490.21 45.691.86 49.943.53 11.440.33 16.201.6 25.320.75
B2 ‐0.030.00 0.340.01 ‐0.230.00 ‐0.100.00 ‐0.310.00 ‐0.300.00
B3 8.680.14 9.750.07 9.360.17 3.750.07 5.130.17 4.860.07
B4 9.340.17 6.950.16 8.490.24 4.660.09 5.090.17 4.540.06
B5 1.060.08 ‐9.260.58 ‐10.191.15 2.450.11 0.420.53 ‐3.290.24
B6 2.420.15 33.370.72 40.750.91 40.032.78 13.382.97 5.490.31
TheblankfunctionB(Eq.(5.2.))foreachsunscreenformulationwasusedtocorrectthe
measured film thickness frequency distribution of the respective formulation by the
convolutionoperation.For thispurposedistributionBwascenteredon thezeropoint
becauseashiftofthesunscreensurfaceisnotapplicableinfilmthicknessmeasurement
asthefirsttopographicalmeasurementiscarriedoutonbaresubstrate.
Chapter5.SPFinsilico 96
5.4.2.Filmthicknessdistributionofsunscreens
Figure5.3.a.showsanexampleoffittingoftheresultofconvolutionoffunctionsqwithB
tothemeasuredthicknessdistributionMfortheGELsunscreen.Thecorrespondingerror
distribution,B,isalsoshownonadifferentscaleforcomparison.Anexcellentagreement
between the fitted and themeasured film thickness frequency distribution curvewas
obtained.Thecorrectedfilmthicknessdistribution,q,wasslightlynarrowerespeciallyin
theleftflankofthecurvecomparedtothemeasuredcurve(Fig.5.3.b.).
Chapter5.SPFinsilico 97
Figure5.3.Exampleoffittingtheresultoftheconvolutionq∗BfortheGELsunscreen;(a)
Fitted(blackline)andmeasured(grayline)filmthicknessfrequencydistributions(left
axis); dashed line is error distribution (right axis). (b) Corrected film thickness
distribution(dottedline,rightaxis)comparedtothedistributionfittedtothemeasured
data(solidline,leftaxis).
Thevaluesoftheninecoefficientsq1toq9offunctionqdeducedfromthefittingaregiven
foralltestedsunscreenformulationsinTable5.2..Thestandarderrorsoftheestimated
coefficientswererathersmallevidencingthegoodnessoftheapproximation.
Chapter5.SPFinsilico 98
Table5.2.Estimated coefficients standarderrorofdistributionq (Eq.5.4.) for each
investigatedsunscreen
Coefficient OW‐C OW‐S GEL WO CAS
q1 0.02330.0005 0.01910.0003 0.01550.0002 0.02010.0003 0.02550.0003
q2 0.1300.050 0.0440.014 3.1e‐64.9e‐6 0.0210.009 0.0340.012
q3 3.9570.152 3.4120.141 7.1240.836 3.7350.214 7.9650.473
q4 0.4930.060 0.0630.036 1.3400.016 1.4640.031 ‐0.0370.042
q5 0.3550.017 0.3880.018 0.3030.014 0.5010.026 0.3620.014
q6 0.00430.0002 0.00510.0002 0.00600.0003 0.00600.0002 0.00250.0001
q7 0.2250.012 0.1870.007 0.20.012 0.3030.012 0.1170.009
q8 2.6200.068 2.2460.041 3.3110.052 3.2500.034 3.1260.077
q9 ‐4e‐42.8e‐5 ‐3e‐42e‐5 ‐8e‐45.7e‐5 ‐4e‐42.4e‐5 ‐3e‐42.4e‐5
The form of function q given by Eq. (5.4.) was defined empirically based on the
assumptionthatthisfunctionshouldalsofitthemeasuredfilmthicknessdistribution,M
(fitnotshown).Eq.(5.4.)consistsoftwooverlappingLorentzianfunctionsoneofwhich
containsanexponentialfunctioninthedenominatortoaccountfortheasymmetryofthe
distribution.Frequencyvaluescorrespondingtofilmthicknesssmallerthan0µmwere
deleted as they probably originated from application andmeasurement artifacts. For
example, sharp ridges on the skin surface may be crushed during spreading of the
sunscreenduetotheappliedpressure.Theseridgesarepresentandmeasuredinthefirst
topographical measurement on bare skin but not in the second measurement on
sunscreencoveredskinresultinginalowerrecordedsurfaceheightandhence,negative
thicknessvalues.Also sharp ridgesmay lead toerroneous focuswith theusedoptical
technique280.Thefinalfilmthicknessdistributionwasadjustedtoatotalfrequency(area
underthecurve)of100%forfurtherconsideration.
Figure5.4.displaysthecorrectedandadjustedfilmthicknessfrequencydistributionofall
investigated sunscreens. It is evident that thedistribution curvediffered considerably
between the sunscreens. All sunscreens exhibited a certain percentage of film with
thicknessequalto0µmreflectinganunprotectedskinsurfaceareaintermsofUVlight
exposure;thispercentagedifferedbetweenthesunscreens.
Chapter5.SPFinsilico 99
Figure 5.4. Corrected and adjusted film thickness frequency distribution of all
investigatedsunscreens
Forexample,CASandOW‐Sexhibitedmorethan2%and1.6%,respectively,offilmwith
thickness=0µmandthelargestpercentageofsmall filmthicknessescomparedtothe
otherformulations.WO,bycomparison,showedlessthan0.3%offilmwiththickness=0
µmandthesmallestpercentageofsmallfilmthicknesses.Furthermore,WOexhibitedthe
thickest film, themaximumfilmthickness frequencyoccurringatapproximately2µm,
closelyfollowedbyGEL.Thestudiedformulationsexhibitedthemaximum(peak)oftheir
thickness frequency distribution at decreasing thickness in the order WO>GEL>OW‐
C>OW‐S>CAS. No differentiation between the formulations was found above 8 µm.
Notably,CASandOW‐Shad the lowestviscosityof all formulations.WO,on theother
hand,wastheonlyformulationconsistingofanon‐evaporatingcontinuousphase.These
characteristicsmayberesponsibleforthedifferentfilmformingpropertiesofthevehicles
andwerediscussedindetailchapter4.Filmthicknessfrequencydistributionreflectsfilm
irregularityoverthesurfaceareaofapplicationwhichisfoundtodependstronglyonthe
usedformulation.Itshouldbepointedoutthatthesubstrateusedinthepresentstudy
consistedofheatseparatedepidermis.
Chapter5.SPFinsilico 100
Theroughnessofthissubstrateismuchsmallerthattheroughnessoffullthicknessskin
andalsosmallerthantheroughnessofthePMMAplatesroutinelyusedinindustryfor
SPFinvitrodetermination.Yettheheatseparatedepidermissubstratewasfoundinan
earlierstudytoprovideSPFinvitroresultsmuchbettermatchingtheSPFdeterminedin
vivo(chapter3).Thiswasattributedtothebetterproduct‐to‐substrateaffinityafforded
by the heat separated epidermis compared to the artificial plates. Therefore, this
substratewasusedinsubsequentstudies.ItisworthmentioningthatSPFinvitrocannot
bemeasuredwithfullthicknessskinbecauseoftheopticalnon‐transparencyofthetissue.
Further,filmthicknessfrequencydistributiononfullthicknessskinoronPMMAplates
hasnotbeendetermined.Therefore,theeffectofsubstrateroughnessandnatureonfilm
irregularity cannot be ascertained although literature reports have suggested that
increasedsubstrateroughnessmaypromotefilmirregularity.Finally,theinfluenceofthe
totalamountofappliedsunscreenonfilmthicknessdistributionwasnotinvestigated.
5.4.3.Sunprotectionfactorinsilicoandinvitro
ForcalculatingSPF insilicoofasunscreenwithEq.(5.1.),UVspectraltransmittanceis
required.ThiswascalculatedwithEq.(5.5.)usingthecorrectedfilmthicknessfrequency
distribution data shown in Fig. 5.4. Further, the spectral average molar absorption
coefficientandthemolarconcentrationoftheemployedUVfiltermixture,aswellasthe
averagefilmthicknesswereused.TheconcentrationoftheUVfiltersinthesunscreens
was determined prior to application by HPLC (data not shown). As explained under
methods(5.3.7),anaveragefilmthicknessofthesunscreenbeforeevaporationof15µm
wasusedfortheapplicationamountof2mg/cm².
TheobtainedcalculationresultsareshowninFig.5.5.togetherwithSPFinvitrovalues
measuredonthesamepreparationsasthoseusedforfilmthicknessmeasurement.Asthe
calculationofSPFinsilicowascarriedoutwiththeaveragefilmthicknessdistributionof
allmeasurements,thereportedvariationofSPFinsilicovalueswasbasedonthevariation
of thickness frequencies between individual measurements for each sunscreen. Also,
percentagevaluesofsunscreenfilmexhibitingathicknessof0µmarereported.
Chapter5.SPFinsilico 101
SPFvaluesdifferedconsiderablybetweenthedifferentsunscreen formulations.Figure
5.5. reveals a very good agreement between SPF in silico and SPF in vitro for every
sunscreen;theagreementwasperfectforWOwhilethedifferencebetweenthetwoSPF
valueswasbetween6and7%forthethreesunscreensOW‐C,OW‐SandCAS,thegreatest
differenceof21%beingfoundfortheGELsunscreen.
Figure5.5.CalculatedSPFinsilico(whitecolumns)withvariationrange(bars),medians
ofmeasuredSPFinvitro(graycolumns)withinterquartilevalues(bars),andpercentage
valuesofsunscreenfilmexhibitingathicknessof0µmfortheinvestigatedsunscreens;
n=27forOW‐C,n=20forOW‐S,n=28forGEL,n=24forWO,n=20forCAS.
Thus, using the measured film thickness frequency distribution for calculating SPF
resultedforallsunscreensinverygoodagreementwithSPFinvitrodataobtainedfrom
UVtransmittancemeasurementsperformedonthesamepreparations.Thisagreement
supportsthevalidityofthecalculationprocedureinvolvingfilmthicknessmeasurement
for prediction of SPF. The procedure offers the possibility to take quantitatively into
accountfilmirregularityfortheperformanceofsunscreens.
Chapter5.SPFinsilico 102
Therefore,itisproposedasavalidfirstlineoptionfortheinsilicopredictionofUVlight
protectionefficacyofsunscreens.Fortheusedformulationexamples,thedeterminedSPF
invitrowasinrathergoodagreementwithclinicalSPFvalues(Fig.4.6.insection4.4.2.).
Before, however, the claim of the present procedure for in silico prediction of SPF is
definitelyestablished,additionalvalidationwithclinicalSPFdatawillhavetotakeplace
using further formulations of the same type, different types of formulation, e.g. oils,
lotions,siliconbased,anddifferentUVfiltercompositions.
TheagreementofthecalculatedSPFforallsunscreensbasedontheprocedureusingfilm
thicknessdistributionwiththeSPFinvitrofurtherstronglyindicatesthatthedifference
in SPF between sunscreen formulations with the same UV filter composition is fore‐
mostlybecauseof thedifference in film formingpropertiesbetween the formulations.
Hence, theempiricalcorrelationbetweenaverage filmthicknessofsunscreenandSPF
established in chapter 4 is confirmed by the present exact evaluation. The inverse
correlationofthepercentageoffilmwithathickness=0µmwithSPFimpliedbyFig.5.5.
pointsouttherelevanceofunprotectedsubstrateareasfortheresultingSPFgiventhat
lighttransmittanceincreasesexponentiallywithdecreasingfilmthickness.Theseresults
demonstratetheadvantageofvehiclesformingcontinuousregularlayersontheskinfor
optimizedUVlightprotection.
TheformulationmayadditionallyaffectsunscreenperformancebymodifyingUVfilter
repartitionuponvehicletransformationduetoevaporationofvolatilecomponentsonthe
skinsurface.Ongoingworkofthisgrouponthisquestionistobereportedinthenear
future.
Finally, photolability was not of concern in the SPF in vitro measurement with the
Labsphereequipmentbecauseoftheveryshortexposuretimeusedandwasnottaken
intoaccountintheSPF insilicocalculation.Hence,thecomparabilityofthetwovalues
was assured. However, taking into account photolability is possible in the model
calculationasshown11,275.
Chapter5.SPFinsilico 103
5.4.4.Modelingfilmthicknessfrequencydistribution
The measurement of film thickness distribution elucidated the marked differences
between formulations with the same UV filter composition with respect to SPF. Film
thicknessdistributionofeachsunscreenreflectsthefilmirregularityoverthesurfacearea
of application. Existing methodologies for SPF prediction taking into account film
irregularityrelyontheuseofamodelfunctiontodescribefilmthicknessdistributionof
theappliedproduct 11,25,191,197 . TheGammadistributionhasbeenusedasamodel to
describe thehighly asymmetric film thicknessdistributionusingone adjustable shape
parameter191,195.Amodelfunctiongenerallyismoreconvenientforroutineusebecause
itcircumventslaboriousfilmthicknessmeasurement.However,theuseofamodelentails
theassumptionthatthemodeladequatelydescribesthefilmthicknessdistributionand,
moreover, itneedstobecalibratedwithexperimentaldata.Nosuchcalibrationtaking
intoaccounttheeffectofformulationorapplicationprocedureoftheproductexistsso
far.
In order to test whether the Gamma distribution can be adopted to describe the
experimental film thickness results, the probability density function of the Gamma
distributiongiveninEq.(5.6.)wasfittedtothefilmthicknessfrequencydistributiondata
oftheusedsunscreens.
0 0
5.6.
where,disthickness,bandcarescaleandshapeparameters,respectively,(c)isGamma
functionwithargumentcandd0istheshiftofthethicknessaxistoaccountforafinite
frequencyofzerothickness.Allthree,c,bandd0weretreatedasadjustableparameters
inthefitting.
TheresultsareshowninFig.5.6.Anexcellentapproximationoftheexperimentalresults
bythefunctionoftheGammadistributionwasfound.Theseresultsprovideevidencethat
theGammadistributioncanindeeddescribefilmirregularityofappliedsunscreeninan
adequatemanner.
Chapter5.SPFinsilico 104
Furthermore,byadjustingthevalueofitsparametersthisdistributioncanbeadaptedto
reflectexperimentaldifferencesbetweenformulations.Hence,thisworkprovidesforthe
firsttimeindicationthattheGammadistributioncanbeuniversallyusedtodescribefilm
irregularity.
For different formulations and application procedures the model will have to be
recalibratedandvalidatedbasedonadditionalinvitroandclinicalexperimentaldata.
Chapter5.SPFinsilico 105
Figure5.6.Experimentalcorrectedandadjustedfilmthicknessfrequencydistributionof
sunscreens(stars)andfittedprobabilitydensityfunctionoftheGammadistributiongiven
byEq. 6.6. (continuous (red) curve). Sunscreen formulations anddeducedparameter
valuesofEq.(6.6.): Topleft:GEL,c=2.515,b=1.377,d0=0.64.Topright:OW‐C,c=2.26,
b=1.25,d0=0.733.Middleleft:OW‐S,c=1.49,b=1.92,d0=1.07. Middleright:WO,c=2.8,
b=1.08,d0=0.275.Bottomleft:CAS,c=1.0,b=2.51,d0=1.45.
5.5.Conclusion
Thedifferencebetweensunprotectionefficaciesofdifferentsunscreenformulationswith
thesameUVfiltercompositionisshowntobebecauseofdifferencesinfilmthicknessand
thicknessfrequencydistributionyieldedbythesesunscreens.Thepresenceofverysmall
filmthicknessesisparticularlycrucialinthisrespect.Emulsiontypeandviscosityappear
tobethedominantcharacteristicsforfilmformingpropertiesoftheformulation.Hence,
the vehicle is shown to substantially impact sunscreen performance. This study
demonstrates that use of the film thickness frequency distributionwith the proposed
computationalmethodprovidesaccurateresultsandis,therefore,ofhighrelevancefor
thepredictionofsunprotectionefficacy.Giventheinadequacyofcurrentinvitromethods
foraccurateSPFdetermination,insilicotoolsrepresentavalidalternative.Thepresent
resultsmayservetofurtherimprovethepowerofcurrentlyavailabletoolsfortheinsilico
predictionofsunscreenperformance273bydevelopingmethodologytointegratevehicle
relatedparameters.ThiscouldbeachievedbasedontheGammadistributionbydefining
parameter values that reflect vehicle related effects. This is of high interest for the
developmentofsunscreenproducts.
Chapter6
Repartitionofoilmiscible
andwatersolubleUVfilters
inanappliedsunscreenfilm
determinedbyconfocal
Ramanmicrospectroscopy
6.1.Abstract
Photoprotectionprovidedbytopicalsunscreensisexpressedbythesunprotectionfactor
(SPF)whichdependsprimarilyontheUVfilterscontainedintheproductandtheapplied
sunscreenamount.Recently,thevehiclewasshowntosignificantlyimpactfilmthickness
distributionofanappliedsunscreenandsunscreenefficacy.
M.Sohnetal.,“RepartitionofoilmiscibleandwatersolubleUVfiltersinanappliedsunscreenfilmdeterminedbyconfocalRamanmicrospectroscopy”Photochem.Photobiol.Sci.15(2016)861‐71.
106
Chapter6.RepartitionofUVfilters 107
In the present work, repartition of the UV filters within the sunscreen film upon
application is investigated for its role to affect sun protection efficacy. The spatial
repartitionofanoil‐miscibleandawater‐solubleUVfilterwithinthesunscreenfilmwas
studiedusingconfocalRamanmicrospectroscopy.Epidermisofpigearskinwasusedas
substrate for application of three different sunscreen formulations, an oil‐in‐water
emulsion,awater‐in‐oilemulsion,andaclearlipo‐alcoholicspray(CAS)andSPFinvitro
was measured. Considerable differences in the repartition of the UV filters upon
application and evaporation of volatile ingredients were found between the tested
formulations.Anearlycontinuousphaseoflipid‐miscibleUVfilterwasformedonlyfor
theWOformulationwithdispersedaggregatesofwater‐solubleUVfilter.OWemulsion
andCASexhibitedinterspersedpatchesofthetwoUVfilters,whereasthesegregatedUV
filterdomainsofthelatterformulationwerebycomparisonofamuchlargerscaleand
spannedtheentirethicknessofthesunscreenfilm.CASthereforedifferedmarkedlyfrom
theother two formulationswithrespect to filterrepartition.Thisdifferenceshouldbe
reflectedinSPFwhentheabsorptionspectraoftheemployedUVfiltersarenotthesame.
ConfocalRamanmicrospectroscopywasshowntobeapowerfultechniqueforstudying
thismechanismofsunprotectionperformanceofsunscreens.
6.2.Introduction
TheperformanceofasunscreendependsmainlyontheabsorptionpropertiesoftheUV
filterscontainedintheproduct281.Thisperformanceisprincipallycharacterizedbythe
sunprotectionfactor(SPF)whosedeterminationisbasedonthesensitivityofhumanskin
to erythema caused primarily by UVB radiationwhile protection fromhealth damage
induced by UVA radiation is addressed by UVA‐PF 2,3,67,201. In vivo9, in vitro10, or in
silico11,282methodologiesareavailablefordeterminingSPF,butonlytheinvivomethodis
currentlyapprovedbyregulatorybodies. Initialdatahave indicatedthatSPFvaluesof
sunscreenswiththesameUVfiltercompositionmaydifferwhendifferentvehiclesare
used to formulate the sunscreen 20,21. Furthermore, uniformity of distribution of the
sunscreenontheskinwasfoundtoplayaroleforSPFinvivo27.Hence,knowledgeofthe
factors besides UV filter composition that affect performance is essential for
understandingthemechanismofactionofsunscreens.
Chapter6.RepartitionofUVfilters 108
Inchapter4,wedevelopedamethodtodeterminetheprecisethicknessdistributionof
theappliedsunscreenfilmbasedontopographicalmeasurementsinordertoexaminethe
relationshipbetweensunscreenfilmthicknessandefficacy.
UsingfivedifferentsunscreenformulationscontainingthesameUVfiltercombinationwe
showedthatthevehiclesignificantlyimpactedtheaveragefilmthicknessandtheSPFin
vitrovalueandthatapositivecorrelationexistedbetweenaveragefilmthicknessandSPF.
Wevalidatedthisfindingwithanewlydevelopedcomputationalmethodologymakinguse
ofthecompletethicknessfrequencydistributionofasunscreenfilmforcalculatingSPF.
Thedivergenceofefficacybetweendifferentsunscreenvehicleswasdemonstratedwith
thismethodtobestronglyrelatedtodifferencesintheaveragethicknessandthickness
distributionoftheappliedsunscreenfilm(chapter5).However,beyondthebehaviorof
the complete sunscreen formulationwith respect to film formation upon application,
repartition of UV filters within the applied film needs to be considered. Commonly,
mixtures of UV filters are used in order to cover the UVA and the UVB range of the
spectrumand toattainphoto‐stability 157,161.For thesereasons thedifferentUV filters
mustbehomogeneouslydistributedinthesunscreen.AsUVfilterscanbelipid‐orwater‐
solubleormiscible,theemployedformulationtypemayinfluencefilterrepartitionwithin
the applied film and therefore sunscreen performance. Only isolated reports on the
distributionofaparticulateUVfiltercanbefoundintheliterature283.
Theaimofthepresentworkwastoinvestigatethespatialrepartitionofanoil‐miscible
andawater‐solubleUVfilterwithinthesunscreenfilmuponapplicationofthreedifferent
types of vehicle. For this purpose, we developed a method using confocal Raman
microspectroscopy.Ramanspectroscopyprovides thepossibility to identifymolecules
based on their characteristic vibrational spectrum and in combination with confocal
microscopyallowsaspatialanalysisinx,y,andzdirectionofthestudiedsample.Thisis
apowerfultechniquewhichrequiresnotissuepreparation,isnon‐invasive,worksinreal‐
time,andislabelfree.Ithasbeenpreviouslyemployedtostudymolecularcomposition
andconformationalnatureofhumanskin,nail,andhair284,tomeasurestratumcorneum
thicknessinhumansinvivo254oronporcineearskinexvivo(chapter3)andtodetermine
skinconstituentsandtheirdistributionthroughouttheskin285,286.
Chapter6.RepartitionofUVfilters 109
Furtherusesincludeddetectionofmolecularabnormalitiesinbenignandmalignantskin
lesions forcancerdiagnosis 287, followingofdrugpermeation throughtheskinbarrier
286,288‐290,monitoringofchangesinproteinstructureandlipidcompositionofhumanskin
for the development of anti‐ageing formulations 291, determination of water
concentration254andhydrationstatusoftheskin285,andassessmentofthedistribution
ofnaturalskinantioxidants292,293.
Inthisstudyweusedepidermalmembraneofpigearskinasabiologicalsubstratefor
sunscreen application which in a previous study provided better in vitro predictive
results of SPF than other substrates (chapter 3) and therefore continued to be used
(chapter 4 & 5). First, a line scan as a function of depth analysiswas carried out for
assessingthethicknessandtherepartitionofthetwoUVfiltersalongthedepthofthe
sunscreen layer and secondly, a surface scan as a function of depth analysis was
performed for assessing the lateral repartition of the UV filters within the applied
sunscreen. Spatial complementarity or co‐localization of the employed UV filters was
assessedandtheeffectoftransformationofthethreedifferentvehiclesuponapplication
on repartition was evaluated. Finally, the SPF in vitro of the three sunscreens was
measuredinordertorelateUVfilterrepartitionwithsunprotectionefficacy.
6.3.Materialsandmethods
6.3.1.Chemicalsandequipment
Following chemicals were used: Potassium carbonate from Sigma‐Aldrich, St Gallen,
Switzerland; Uvinul MC80 abbreviated EHMC (INCI, ethylhexyl methoxycinnamate),
Neutrol TE (INCI, tetrahydroxypropyl ethylenediamine), Eumulgin VL75 (INCI, lauryl
glucoside (and) polyglyceryl‐2 dipolyhydroxystearate (and) glycerin), Dehymuls LE
(INCI, PEG‐30 dipolyhydroxystearate), isopropyl palmitate, Lanette O (INCI, cetearyl
alcohol) fromBASFSE,Ludwigshafen,Germany;Eusolex232abbreviatedPBSA (INCI,
phenylbenzimidazolsulfonicacid)fromMerck,Darmstadt,Germany;Arlacel165(INCI,
glycerylstearate(and)PEG‐100stearate)fromCrodaEastYorkshire,England;KeltrolRD
Chapter6.RepartitionofUVfilters 110
(INCI,xanthangum)fromCPKelco,Atlanta,Georgia;Sepigel305(INCI,polyacrylamide
(and)C13‐14isoparaffin(and)laureth‐7)fromSeppic,Puteaux,France.
Quartzplateswithasizeof2cm2cmwereobtainedfromHellmaAnalytics(Zumikon,
Switzerland).
Followingequipmentwasused:Balance(XS104,Mettler‐Toledo,Columbus,OH,USA);UV
transmittanceanalyzer (LabsphereUV‐2000S,Labsphere Inc.,NorthSutton,NH,USA);
confocalRamanmicrospectrometer(Alpha500R,WITec,Ulm,Germany).
Followingsoftwarepackageswereused:WITecControlandWITecProjectFour(WITec,
Germany) for the acquisition and evaluation of Raman measurements, respectively;
StreamMotion(Olympus,Tokyo,Japan)forimageprocessing;UV‐2000(LabsphereInc.,
USA)forUVtransmittancemeasurement;IgorPro6.32A(WaveMetrics,Inc.,Portland,OR,
USA)fordatafitting.
6.3.2.Preparationofskinsubstrate
Weusedepidermalmembraneofpigearsforcreamapplicationasdescribedearlierin
section3.3.2.,method2 (3.3.2.2.).Theepidermalmembranewas isolatedusingaheat
separationprocedure,cuttoadimensionof2cm2cm,laidflatoncarrierquartzplates,
andstoredat4°Cinadesiccatoroversaturatedpotassiumcarbonatesolutionuntiluse.
6.3.3.Sunscreenvehicles
WeselectedEHMCasoilmiscibleandPBSAaswatersolubleUVfilter.EHMCwasusedat
aconcentrationof10wt%andPBSAataconcentrationof6wt%.BothUVfilterswere
incorporatedinthreedifferentvehicles,i.e.,anoil‐in‐wateremulsion(OW),awater‐in‐oil
emulsion(WO),andaclearlipo‐alcoholicspray(CAS).PBSAwasneutralizedwithNeutrol
TE inwater for the OW andWO vehicles and in ethanol for the CAS vehicle. The full
compositionofthesunscreenformulationsisgiveninTable6.1.
Chapter6.RepartitionofUVfilters 111
Placeboformulations(withoutUVfilters)oftheOWandWOsunscreenswereprepared
forRamanmeasurements.Intheplaceboformulations,theamountofEHMCwasreplaced
byisopropylpalmitateandtheamountofPBSAandNeutrolTEbywater.
Table6.1.Composition(w‐%)ofinvestigatedformulations
Sunscreendesignation OW WO CAS
Ingredient
typeTradename Composition(w‐%)
Emulsifier
system
Arlacel165
EumulginVL75
DehymulsLE
TeginOV
2.0
5.0
‐
‐
‐
‐
1.0
2.0
‐
‐
‐
‐
Emollient Isopropylpalmitate 11.0 11.0 13.0
Thickening
LanetteO
KeltrolRD
Sepigel305
1.5
0.3
3.0
1.5
‐
‐
‐
‐
‐
Filtersystem EHMC
PBSA
10.0
6.0
Neutralizing
agent
NeutrolTE QstopH7
Additional Glycerin 3.0 3.0 ‐
ingredients Water
Ethanol
Magnesiumsulfate
Qsp100%
2.5
‐
Qsp100%
2.5
0.8
‐
Qsp100%
‐
Chapter6.RepartitionofUVfilters 112
6.3.4. Measurement of the sun protection factor in
vitro
Measurement of SPF in vitro was based on diffuse UV transmission spectroscopy as
proposedbySayre12:
SPF ∑ ser λ . Ss λ
∑ ser λ . Ss λ . T λ 6.1.
where, the inverse transmittance (1/T) in theUV spectral range isweightedwith the
erythemaactionspectrum,ser(λ)9,andthespectralirradianceoftheUVsource,Ss(λ)9.As
dataforser(λ)andSs(λ)areavailablefromliterature,theSPFinvitrocanbedetermined
only from UV transmittance measurements registered from 290 to 400nm in 1nm
incrementstepsthroughskinsubstratepreparationsaftersunscreenapplication.TheUV
transmittanceof fourpositionsper2cm 2cmskin substrateplatewasmeasured to
cover virtually the complete surface area of the skin preparation. In total, four skin
substrateplatespersunscreenwereused.Ablanktransmittancespectrumwasrecorded
atfirstforeachsingleposition.Subsequently,2.0mg/cm²ofsunscreenwasappliedwith
the fingertip using a pre‐saturated finger coat in form of 20 to 30 small drops. The
sunscreen was spread using light circular movements followed by left‐to‐right linear
strokesfromtoptobottomstartingateachsideoftheskinpreparation.Transmittance
measurement was carried out 15 minutes after sunscreen application to allow for
equilibrationwithenvironmentalconditions.
6.3.5.ConfocalRamanmicrospectroscopy
measurements
ConfocalRamanlaserscanningmicrospectroscopywasperformedonsunscreensapplied
to skin substratepreparations asdescribed for SPF invitromeasurements.Datawere
collectedwithaWITecAlpha500RinstrumentequippedwithaEMCCDhighintensitylow
noisecameraandahighprecisionpiezoelectricscanningstage.
Chapter6.RepartitionofUVfilters 113
Raman spectra were recorded from 0 to 4000cm‐1 (spectral grating of 600g/mm,
spectralresolutionof3cm‐1perpixel)usinga532nmexcitationgreenlasersource.As
pinhole a glass fiberwith a diameter of 50mwas used. Thepower of the laserwas
adjustedtoanintensityof6mWforallmeasurements.Allmeasuredspectraweretreated
withtheCRR(CosmicRayRemoval)optionintheWITecProjectFoursoftwareandwere
backgroundcorrectedbybaselinesubtractionusingapolynomialfunctionof5thorder.
The Raman spectra of neat EHMC and of PBSA at a concentration of 37.5% inwater
neutralizedwithNeutrolTEwererecorded.ApeakthatwasuniqueforeachUVfilterwas
selectedtodetectanddifferentiatetheUVfiltersinthesamples.Thefiltermanageroption
available in theWITecProject Fourdata treatment softwarewasused to identify and
visualizetheUVfiltersbasedontheirspectralpeakcharacteristic.
The combination of Raman spectroscopy with confocal microscopy allowed a depth‐
resolvedanalysis.Twodifferentkindsofmeasurementwereperformed,alinedepthscan
analysisforassessmentofthesunscreenlayerthicknessandasurfacedepthscananalysis
forassessingthelateralrepartitionoftheUVfiltersatdifferentdepthsinthesunscreen
layer.
6.3.5.1.Linedepthscan
Thesunscreenwasscannedoveralineof100µminxdirectionat225pointsperlineand
overadepthof30µminzdirectionwith50linesperimageresultingtoatotalof11250
recorded individualspectra.Themeasurementswereperformedusinga50×objective
(NikonEPIplan)withanumericapertureNAof0.80permittinganx‐y(lateral)resolution
of405nmaccordingtotheRayleighcriterionandadiffractionlimitedz(axial)resolution
ofaround1.2µmassumingarefractiveindexofthesample=1. Anintegrationtimeof
0.05s was used. Six individual depth line scan measurements were performed per
sunscreen at different locations of the sample. This measurement provided two‐
dimensional(2D)imagesinthex‐zplaneshowingtheUVfilterlocationinthesunscreen
film.
Chapter6.RepartitionofUVfilters 114
6.3.5.2.Surfacedepthscan
Scans of a surface area of the sunscreen with dimension 100µm × 100µm were
performed in the x‐y plane with 180 points per line and 180 lines per image, thus,
acquiringatotalof32400individualspectrapersurface.A50×objective(NikonEPIplan)
wasusedwithanumericapertureNAof0.55permittinganx‐yresolutionof590nmand
azresolutionofapproximately2.5m,withanintegrationtimeof0.05s.
Surface scans were repeated at 1m steps in the z direction along the depth of the
sunscreenfilmstartingwiththefirstsurfacemeasurementclearly in theairabovethe
sunscreenfilmasillustratedbysurfaceS1inFig.6.1.andendingwellbelowthesunscreen
filmintotheskinasillustratedbysurfaceSxinFig.6.1.Thesemeasurementsprovided2D
imagesinthex‐yplaneshowingthelaterallocationoftheUVfiltersatdifferentzpositions
inacolor‐codedfashionusingthecombinedpictureoptionofthesoftware.Percentageof
surfaceoftheimagescorrespondingtoeachcolorwascalculatedwiththestreammotion
software.Theseimageswerefurthersuperimposedforallzpositionstoproduceasingle
2DimageillustratingtheabundanceoftheUVfiltersthroughoutthecompletesunscreen
layer.
Figure6.1.Three‐dimensionalvisualizationofskinwithaschematizationofthesurface
scanmeasurementsperformedthroughoutthesunscreenfilm,S1correspondingtothe
firstsurfacemeasurementonthetopoutsidethesunscreenfilmintotheairandSxtothe
lastmeasurementendingon thebottominto theskin,eachsurfacescanmeasurement
beingspacedby1minthezaxis
Chapter6.RepartitionofUVfilters 115
SinceintensityoftheRamansignaldecreasedwithincreasingdepthofmeasurementin
thesample,acorrectionofeachindividualsurfacescanforRamansignalattenuationhad
tobeperformedas explainedbelow. The surface scandatawere corrected for signal
attenuation,CRRtreatedandbackgroundsubtractedbeforeuseforfurtherevaluation.
6.3.5.3.ControlexperimentforcorrectionofRamansignalattenuation
ThedecreaseofintensityofthemeasuredRamansignalatincreasingdepthduetolight
scatteringwasdeterminedforeachsunscreenformulation.Forthispurpose,twocover
slipswerepositionedonaglassslideandthegapbetweenthemwasfilledfirstwithan
excessofsunscreenusingapipette;thenathirdcoverslipwasglidedoverthesunscreen
toattainafilmthicknessequaltothethicknessofthecoverslips.Theamountofsunscreen
appliedontheglassslidethusproducedanestimatedfilmthicknessofroughly60µmor
moreafterevaporationofvolatilecomponentsoftheformulationandwasmuchlarger
thanthethicknessobtainedbytheusualapplicationof2mg/cm2ofsunscreen.Raman
measurementsalongalineof4µminthexdirectionwith8pointsperlineandinadepth
of60µminzdirectionwith120linesperimagewereperformed.Measurementsstarted
intheairabovethesunscreen.Ramanspectrawereacquiredwiththe50×objective,NA
0.55andanintegrationtimeof1s.TheintensityofthespectralbandataRamanshiftof
1613cm1wasmeasured andaveragedover the8pointsper line. Thedataof signal
intensityasafunctionofdepthweretreatedmathematicallyasdescribedearlier294and
wereusedtocorrectfortheRamansignalattenuationoccurringatincreasingdepthin
thesurface‐depthscanningexperiments.
Chapter6.RepartitionofUVfilters 116
6.4.Resultsanddiscussion
6.4.1.RamanspectraofEHMCandPBSA
Figure6.2.givestheRamanspectraofthepureUVfiltersusedinthisstudy.EHMCisan
oilyliquidthatwasmeasuredneatwhilePBSAwasmeasuredina37%w/vwatersolution
thatwastitratedtopH7withNeutrolTE.EHMCandPBSAshowpeaksat1170cm1and
1545cm1,respectively,whichareuniquetothesecompounds.Thepeakat1170cm‐1of
theRamanspectrumofEHMCwasalsopreviouslyreported295andattributedtotheC‐H
bend in themoleculewhile noprevious studywas found for PBSA. Thesepeaks also
appearintheRamanspectrumofthesunscreenformulationscontainingbothUVfilters
(Fig.6.3.).Therefore,theywereusedtodetecttheUVfiltersandassesstheirlocationin
thepreparationsofsunscreenappliedtoskinsubstrate.Theplaceboformulationswere
shownnottointerferewiththisdetection(Fig.6.3.).Thepeakat1613cm1,ontheother
hand, was present in the Raman spectrum of both UV filters and was used for the
correctionoftheRamanintensityattenuation.
Figure6.2.RamanspectraofUVfilterEHMC(grayline)andUVfilterPBSA(blackline)
Chapter6.RepartitionofUVfilters 117
Figure6.3.RamanspectraofsunscreenformulationscontainingUVfilters(blackline)
and placebo formulations without UV filters (gray line). Top, for OW; bottom, WO
sunscreen
Chapter6.RepartitionofUVfilters 118
6.4.2.CorrectionforRamansignalattenuation
To assure that signal intensity in the depth scan experiments provided an accurate
representation of the abundance ofUV filter in the produced images, a correction for
Ramansignalattenuationduetolightscatteringasafunctionofdepthwasperformed.
For this purpose, signal intensity as a function of depth was calibrated with control
experiments. Figure 6.4. displays the change of measured intensity of the peak at
1613cm1 for each sunscreen with increasing depth. For the CAS formulation, the
intensityoftheRamansignalremainedunchangeduptoadepthofapproximately10µm.
This depth is larger than the film thickness of the applied sunscreen inwhichRaman
measurementwascarriedout,takingintoaccountthicknessreductionduetoevaporation
ofvolatilecomponents. Therefore,nocorrectionofsignal intensitywasperformedfor
thisformulation.
FortheOWandWOsunscreens,theintensityofRamansignaldecreasedmarkedlywithin
adepthof10µm.ThesignalattenuationofthesesunscreenswasdescribedwithEq.(6.2.)
andEq.(6.3.),respectively.32
∙ 1 6.2.
∙ 1 6.3.
where,IisintensityofRamansignal,zisdepthwithz0andz=0correspondingtothe
surfaceofthesunscreen.
ThecoefficientsaandbwerededucedbyfittingEq.(6.2.)andEq.(6.3.)totheintensity
data of Fig. 6.4. whereas forWO only data from 0 to 12.5µm depthwere used. The
intensitywasnormalizedtoamaximumvalueofI(z=0)=1. FortheOWsunscreena=
0.9924andb=10.78andfortheWOsunscreena=0.064wasobtained.
ThemeasuredintensityoftheRamansignalinthesurface‐depthscanningexperiments
wascorrectedfortheattenuationofthesignalasafunctionofdepthby
6.4.
where,I(z)wasobtainedforthecorrespondingdepthfromEq.(6.2.)andEq.(6.3.)forthe
OWandtheWOformulation,respectivelyusingthededucedcoefficientvalues.
Chapter6.RepartitionofUVfilters 119
Thiscorrectionwasappliedtoeachacquiredsurfacescanmeasurementforproducing
thecorrespondingimage.
Figure6.4.Ramansignalintensityofthepeakat1613cm‐1asafunctionofdepthintothe
formulationfortheOW,WOandCASsunscreens.Eachcurverepresentstheaverageof
threemeasurements
6.4.3.Linedepthscan
Figure 6.5. gives an example of 2D images (x‐z plane) of the line‐depth scan
measurements.UV filtersEHMCandPBSAwere identified basedon spectral bands at
Raman shift 1170 cm1 and 1545cm1, depicted in green and red color, respectively.
Yellowcolorresultedfromsuperpositionofgreenandred.
AcontinuouslayerofUVfiltersofthesunscreensisevidentwithanapparentthicknessof
2to5µm.Black‐depictedregionslocatedaboveandbelowthesunscreenlayerprovided
nosignalatthespecificRamanshiftsandareattributedtoairandskin,respectively.
Chapter6.RepartitionofUVfilters 120
Figure6.5.Two‐dimensionalimagesinthex‐zplaneresultingfromline‐depthconfocal
Ramanscan.Fullscalelength=100µm;fullscaleheightofeachimage=30µm.Green
colorrepresentsEHMC,redcolorrepresentsPBSA.Blackregionsaboveandbelowthe
UV filters correspond to air and skin, respectively. Top, OW sunscreen; middle, WO
sunscreen;bottom,CASsunscreen.
The images demonstrate that confocal Raman scanning microscopy allows the
localizationanddetectionofrepartitionofindividualUVfiltersintheappliedsunscreen
film.FortheOWandtheWOformulations,atightinterspersionalongthex‐axisofsmall
areas of the oilmiscible EHMC and thewater soluble PBSAwas observed. Some co‐
localization(yellowspots)oftheUVfilterswasseenintheWOformulation. However,
distributionoftheUVfiltersalongthezaxiscouldnotbeaccuratelyascertainedinthis
visualrepresentation.
Notably,ratherlargedomainsofEHMCandPBSAwereobservedalongthexaxisinthe
CASformulation,whichappeartospantheentirethicknessofthesunscreenfilm.Hence,
a large difference of lateral repartition of theUV filters after sunscreen application is
evidentbetweentheusedformulations.Thisisdiscussedinmoredetailinthesurface‐
depthscanresults.
Chapter6.RepartitionofUVfilters 121
6.4.4.Surfacedepthscan
Allsurfacescanmeasurementswerefirstcorrectedforsignalattenuation,CRRtreated
and background subtracted. The detection of theUV filterswas performed as above.
RamansignalofEHMCandofPBSA fromsurfacescanswerecombinedwithinone2D
image(x‐yplane)foreachmeasuredpositionalongthez(depth)axis.InFigures6.6.a.,
6.6.b.,and6.6.c.Ramanspectralmapscorrespondingtoindividualsurfacescansthatwere
recorded in depth intervals of 1m are pasted together for the three investigated
sunscreens.ThetopleftandthebottomrightimageineachoftheFigs.6.6.a.,6.6.b.,and
6.6.c.correspondtoairandskinaboveandbelowthesunscreen,respectively,andappear
blackreflectingtheabsenceofthespecificRamanspectralbands. Assurfacescanning
progressesalongthezaxis,thesunscreenlayeristraversedwhichisevidencedbythe
detectionofRamansignaloftheUVfilters.Thesunscreensappeartospanathicknessof
approximately 8 µm along the depth axis which, given the worse z resolution of the
surface‐depthscanmeasurementscomparedtotheline‐depthscan,isconsistentwiththe
resultsofFig.6.5.Thepositionalpatternofsignaldetectioninthesuccessionofoptical
sectionsimpliesthatthesunscreenlayerwasnotperfectlyhorizontaland/orhadnota
uniformthickness.
Fig.6.6.a.
Chapter6.RepartitionofUVfilters 122
Fig.6.6.b
Fig.6.6.c.
Figure6.6. Pasting of all images from individual confocal Raman surface scans (x‐y
plane)recordedinintervalsof1malongthezcoordinate. Sequencestartsattopleft
andendsatbottomrightintheorderfromlefttorightandtoptobottom.Whitebarin
everyimage=10µm.GreencolorrepresentsEHMC,redcolorrepresentsPBSA.a,OW
sunscreen;b,WOsunscreen;c,CASsunscreen.
Chapter6.RepartitionofUVfilters 123
GreencolorcorrespondstoEHMC,whichisalipid‐miscibleUVfilter.IntheOWsunscreen
thisrepresentedtogetherwiththeemollientthedispersedphaseoftheformulation.Fig.
6.6.a. (detail in Fig. 6.7.) shows that although discrete green spots of < 5 µm were
discernible,aggregationandpossiblysomecoalescenceofthedispersedphasehavetaken
placeuponapplicationandevaporationofvolatilecomponentsofthecontinuousphase
oftheformulation.TheaveragedropletsizeofthefreshlymadeOWemulsionwasaround
2µm.Redcolorcorresponds toPBSAwhich isawater‐solubleUV filter. Independent
experimentshaveverifiedthatuponwaterevaporationPBSAdoesnotcrystallizewhenit
isneutralizedwithtetrahydroxypropylethylenediamine(NeutrolTE)insteadforminga
viscousmass.ThisisdetectedasredspotsinterspersedamongEHMCoftheoilphase.
However,nocontinuousphasewasevident.Also,noco‐localization(yellowcolor)was
detectedattheusedresolution.
IntheWOsunscreen(Fig.6.6.b.anddetail inFig.6.7.),EHMCisdetectedascontinuous
greencolorreflecting theexternalphaseof the formulation. Rather largespotsofred
colorwerefoundindicatingclusteringofPBSAcontainedinthedispersedphase.
TheCASformulation(Fig.6.6.c.anddetailinFig.6.7.)producedsegregateddomainsof
EHMC and PBSA that were much larger than the spots observed in the other two
formulations. Hence, repartition of UV filters on the surface of skin upon sunscreen
applicationisdemonstratedtostronglydependonthetypeofformulationinuse.
TheproportionofgreenandredcolorcorrespondingtoEHMCandPBSAwasquantified
intheimagesofFig.6.7.
Chapter6.RepartitionofUVfilters 124
Fig.6.7.a. Fig.6.7.b.
Fig.6.7.c.
Figures6.7.Combined2Dpicture (x‐yplane) for investigatedsunscreens inasurface
scanmeasuredatonezcoordinateillustratingthelocationatwhichRamansignalwas
detectedforEHMC(greenzones)andPBSA(redzones),a.OW;b.WO;c.CASsunscreen
EHMC occupied 33%, 36%, and 48% and PBSA occupied 16%, 15%, and 52% of the
surfaceareaoftheimageoftheOWsunscreen,theWOsunscreen,andtheCASsunscreen,
respectively.Thedifferenceofthesumofthesenumbersto100%reflectstheblackarea
(nosignal)oftheimages.
Chapter6.RepartitionofUVfilters 125
TheratioofEHMCtoPBSAabundanceinthesectionsoftheOWandtheWOsunscreenis
in perfect agreement with the amount of these UV filters in the formulations. The
abundance of PBSA in the CAS image was probably overestimated because a clear
distinctionoftheredcolorfromblackwasnotpossibleinthisimage.
Hence,thepresentworkallowstheidentificationandlocalizationoftheUVfiltersinthe
applied sunscreen film. The two UV filters were found to be mutually interspersed
occupyingadjacentareaswithintheopticalsectionstakenat1µmintervalswithlittleor
noco‐localizationbeingdetectedattheapplicableresolution.Thescalepatternoftheir
repartitionisinfluencedbythephasespreexistingintheappliedformulations.Thus,the
oilandthewaterphasesoftheOWandtheWOsunscreenscontainingEHMCandPBSA,
respectively,couldbedistinguishedalthoughaggregationandprobablysomecoalescence
tookplaceuponevaporationofthewaterphase.TheCASsunscreenconsistedofasingle
phaseinwhichbothUVfiltersweredissolved.Uponevaporationoftheethanol,theUV
filtersrepartitionedformingratherlargedomains.Thismightberelatedtotheabsence
ofemulsifierinthisformulation.
Togetacompleteviewofthesunscreenfilm,superpositionofallindividualsurfacescans
was performed (Fig. 6.8.). Since the intensity of the surface scanmeasurementswas
correctedforsignalattenuationasafunctionofdepth,thesignal(color)intensityinthe
finalimageisproportionaltothetotalabundanceoftheUVfiltersandconsequentlyto
filmthickness.
LargeyellowareaswerefoundfortheOWandtheWOsunscreens(Fig.6.8.)indicating
overlapoftheEHMCandthePBSAspecificsignalsattherespectivex‐ycoordinates.This
suggestsanoverlapoftheUVfiltersalongthez(depth)axis.However,alsoareascovered
solelybyEHMCorPBSAareobserved.FortheCASsunscreen,ontheotherhand,very
littleoverlapwasdetected,mostoftheimageareabeingcoveredbyratherlargedomains
ofeitherEHMCorPBSA.ThisshowsthatthedomainsoftheUVfiltersdetectedinthe
Ramanspectralmapsofthesurfacescans(Fig.6.6.c.)extendedthroughtheentirefilm
thicknessof this sunscreen. These findings fromthesuperimposed imagesof theCAS
sunscreen are congruentwith those of the line‐depth scanning experiment (Fig. 6.5.).
EHMCandPBSA,hence,areshowntoformcomparatively largesegregatedpoolsafter
applicationofthisformulation.Theseresultsunderscoretherelevanceofformulationfor
UV filter repartition. The bright and dark areas of the images on Fig. 6.8. reflect
fluctuationsofsunscreenfilmthickness.
Chapter6.RepartitionofUVfilters 126
Fig.6.8.a. Fig.6.8.b.
Fig.6.8.c.
Figure6.8.Combined2Dpicture(x‐yplane)fromthesuperimpositionofallindividual
surface scanmeasurements for investigated sunscreens to visualize the presence and
locationofthetwoUVfiltersinthecompletesunscreenfilm;detectedRamansignalfor
EHMCingreen,forPBSAinred,overlappingofEHMCandPBSAinyellow;a.forOW,b.for
WO,c.forCASsunscreen
This work underscores the advantages of confocal Raman microspectroscopy for
obtaining3Dlocation‐specificmolecularandstructuralinformationontheinvestigated
sample as demonstrated in the biological 284‐286, the pharmaceutical 286‐290 and the
cosmetic254,285,292,293fieldbefore.
Chapter6.RepartitionofUVfilters 127
6.4.5.Consequencesforsunprotection
Foreffectivesunprotection,acompletecoverageoftheskinbysunscreen isessential.
However,manualapplicationdefiesstandardizationsothatauniformfilmthicknessof
sunscreencannotbepossiblyattained.Undertheconditionsemployedinthisworkthe
skin was mostly covered by UV filters although some locations may have remained
exposed,i.e.,poorlyprotectedasshowninFig.6.8..Adetailedquantitativestudyonthe
relationship between film thickness frequency distribution and sun protection factor
illustratingthedramaticeffectofarelativelysmalluncoveredskinsurfaceareaonsun
protectionweregiveninchapter4and5.
Inaddition,theabsorptionspectrumoftheUVprotectionsystemshouldideallybethe
samethroughoutthecoveredskinarea.SinceUVfiltercombinationsarenormallyused
inordertoguaranteeabsorptionthroughouttheentirespectrumofterrestrialsunlight,
this entails that the UV filters should be homogeneously distributed in the sunscreen
layer. For sunscreen vehicles consisting of an oil and awater phase it is considered
essentialthatbothphasescontainUVfilterinordertoassureanuninterruptedcoverage
oftheskin192,196.
The optical sections of the present study (Figs. 6.6. and 6.7.) demonstrate a
complementarity of EHMC and PBSA in the x‐y plane. These UV filters aremutually
immiscibleandwerefoundtoformdistinctphasesatthelateralresolutionof590nm.In
thezdimension,however,anoverlapoftheseUVfilterswasobservedfortheOWandthe
WOformulations(Fig.6.8.)assuringacombinedUVabsorptionspectruminfairlylarge
areasoftheappliedsunscreen.Yet,itshouldbepointedoutthattherewerestillareasin
whicheitheroneoftheUVfiltersdominated.Thismightbeduetoasmallthicknessof
the sunscreen film in those areas. Film thickness frequency distributions reported in
chapter4and5support thisview. FortheCASformulation,poolsofEHMCandPBSA
werefullysegregatedthroughoutthethicknessofthesunscreenfilm.Thiswouldresult
inanon‐uniformUVabsorptionacrossthecoveredareaand,hence,acompromisedsun
protectionefficacy.WiththeusedUVfilters,therefore,theCASformulationappearstobe
inferiortotheothertwoformulationsintermsofUVfilterrepartitionandconsequently
sunprotectionwhentheUVfiltershavedifferentabsorptionspectra.
Chapter6.RepartitionofUVfilters 128
Thus,thisworkrevealsamechanismbywhichthetypeoftheusedvehiclemayinfluence
sunprotectionefficacyofasunscreeninadditiontotheroleofthevehicle forthefilm
formingpropertiesoftheproductthatwereshowntoalsoinfluenceperformance.
6.4.6.Invitrosunprotectionfactor
ToevaluatetheeffectofUVfilterrepartitiononsunprotectionaffordedbythedifferent
formulations,theSPFinvitrowasdetermined.TheOWsunscreen,theWOsunscreenand
theCASsunscreenyieldedanSPFinvitrovalueof20,21and18,respectively,showing
thattherewasnoconsiderabledifferenceintheUVprotectionefficacybetweenthethree
sunscreen formulations. This is probably because the absorption spectrum and the
maximumabsorbanceofEHMCandPBSAareverysimilar142,158.Therefore,theobserved
difference of repartition of the twoUV filters between the sunscreens did not elicit a
differenceinlightabsorptionbetweenthedifferentskinareas. However,thesituation
maychangeformarketproductscontainingacombinationofseveralUVfiltersexhibiting
differentabsorptionproperties.Infuturework,combinationsofUVfilterswithdifferent
absorptionpropertiesappliedoverlargesurfaceareawillbeusedtomorecloselystudy
therelationshipbetweenfilterrepartitionandsunlightprotectionefficacy.
6.5.Conclusion
Thetypeofvehiclestronglyinfluencesrepartitionofawater‐solubleandalipid‐miscible
UVfilterinthesunscreenfilmuponapplicationtoskin.Followingevaporationofvolatile
componentsof the formulation,anearlycontinuousphaseof lipid‐miscibleUVfilter is
formedonlyfortheWOemulsionvehiclewithdispersedaggregatesofwater‐solubleUV
filter.OWemulsionandclearlipo‐alcoholicformulation(CAS)ontheotherhand,exhibit
interspersedpatchesofthetwoUVfilters,whereasthesegregatedUVfilterdomainsof
the latter formulation are by comparison of a much larger scale and span the entire
thicknessofthesunscreenfilm.
Chapter6.RepartitionofUVfilters 129
SinceUVfiltercombinationsarealwaysusedinsunscreenproductsinordertocoverthe
wavelength range of terrestrial sunlight and achieve filter photo‐stability, repartition
behaviorofUVfiltersandpotentialsegregationmayinfluencephotoprotectionefficacy.
This mechanism of contribution to the performance of sunscreens has not been
investigatedbefore.ConfocalRamanmicrospectroscopyisshowntodeliverprecisedata
at micrometer resolution about the location of the investigated compounds on skin
surface.
Chapter7
Conclusionandoutlook
The invivo predictionof sunscreenefficacy is of great interest for a fast andeffective
developmentofnewsunscreenformulations.However,predictionsofsunscreenefficacy
lackaccuracy.Thepresentthesisaimedatimprovingtheunderstandingoftheworking
mechanismof sunscreenswith the identificationof factors thatmay influenceefficacy
usinginvitroandinsilicomethodologies,advancedanalyticalmeans,andmathematical
modelingtoultimatelyimproveinvivopredictionsoftheperformanceofsunscreens.
The in vitro assessment of sunscreen performance with themeasurement of the sun
protection factor requires an adequate substrate for sunscreen application to give
reproducible results. We selected skin of pig ear as biological substrate to better
reproduce the product‐to‐substrate affinity relevant for the in vivo situation. We
identified filmthicknessdistributionofanappliedsunscreenasasignificant factor for
sunscreenefficacy.Wefoundastronginfluenceofvehicleonsunscreenefficacyarising
fromdifferencesinthefilmthicknessassumedtooriginatefromthedifferenceinsomeof
theformulationexcipients.Further,weinvestigatedtherepartitionoftwoUVfiltersinan
appliedsunscreenfilmandfoundconsiderabledifferencesbetweensunscreenvehiclesas
well.
130
Chapter7.Conclusionandoutlook 131
However,adirectrelationshipbetweenUVfilterrepartitionandSPFcouldnotbedrawn
duetothesimilarityoftheabsorbancepropertiesofthetwostudiedUVcompounds.Ina
futurework,onemayconfirmthisobservationinalargersurfaceareausingUVfilters
thatshowdifferentabsorbancecharacteristicstoinvestigatetherelationshipbetweenUV
filterrepartitionandUVefficacymoreextensively.
Anoutlookisthefullunderstandingofthemodificationofasunscreenformulationupon
application with the core questions being how the sunscreen layer looks like after
applicationoftheformulationonskinthatisrelatedtothefilmthicknessdistributionand
howtheUVfiltersre‐distributeonskinafterapplication.Wedevelopedmethodologiesto
assessthesetwoaspectsandshowedaneffectofvehiclefromdifferentformulationtypes.
However, a futureworkmay focusmore indetailof the impactofdifferent functional
excipients in a sunscreen formulationon theUVefficacybyexamining the connection
between film formation and UV filter repartition with SPF. The achievement of an
homogeneous film and an homogeneous UV filter repartition upon application for an
improvedperformancethroughanoptimizedingredientcompositionisthegoalofnext
generationofsunscreens.
The advancements in the knowledge of the factors influencing sunscreen efficacy put
forwardinthisworkmaymarkedly improvethepredictionofsunscreenperformance.
Thepresent thesisallowedagreat step forward towardanaccuratepredictionof sun
protectionprovidedbyatopicalsunscreen.
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ListofAbbreviations
2D Two‐dimensional
BCC Basalcellcarcinoma
BMDBM Butylmethoxydibenzoylmethane
BEMT Bis‐ethylhexyloxyphenolmethoxyphenyltriazine
CAS Clearalcoholicspray
CPD Cyclobutanepyridiminedimers
CRM ConfocalRamanmicrospectroscopy
CRR Cosmicrayremoval
EHMC Ethylhexylmethoxycinnamate
EHT Ethylhexyltriazone
EMCCD Electronmultiplyingcharge‐coupleddevice
DTS Drometrizoletrisiloxane
FDA Foodanddrugadministration
IMC Isoamylp‐methoxycinnamate
INCI Ingredientnomenclatureofcosmeticingredients
IPD Immediatepigmentdarkening
IR Infrared
JCIA Japanesecosmeticindustryassociation
MBBT Methylenebis‐benzotriazolyltetramethylbutylphenol
155
Abbreviations 156
MED Minimalerythemaldose
MMP Matrixmetalloproteinase
MPPDD Minimalpersistentpigmentdarkeningdose
NA Numericalaperture
OCR Octocrylene
OW Oil‐inWater
PBSA Phenylbenzimidazolsulfonicacid
PMMA Polymethylmethacrylate
PPD Persistentpigmentdarkening
RI Refractiveindex
ROS Reactiveoxygenspecies
SC Stratumcorneum
SCC Squamouscellcarcinoma
SCCS Scientificcommiteeonconsumersafety
SPF Sunprotectionfactor
TBPT Trisbiphenyltriazine
TDSA Terephthalylidenedicamphorsulfonicacid
TEA Timeandextentapplication
TiO2 Titaniumdioxide
UV Ultraviolet
UVA‐PF UVAprotectionfactor
VIS Visible
WO Water‐in‐Oil
ZnO Zincoxide
ListofSymbols
E1,1 Absorptionataconcentrationof1%(w/v)solutionatanopticalthicknessof
1cm
λc Criticalwavelength
λmax Wavelengthwiththemaximumabsorbance
Sa Arithmeticalmeanheight
Ser(λ) Erythemaactionspectrumatwavelengthλ
Smean Averageoffilmthicknessoverameasuredarea
SPPD(λ) Persistencepigmentdarkeningactionspectrumatwavelengthλ
Ss(λ) SpectralirradianceoftheUVsourceatwavelengthλ
SUVA(λ) SpectralirradianceoftheUVAsourceatwavelengthλ
T(λ) Transmittanceatwavelengthλ
157
ListofFigures
Chapter 2
2.1. Absorbanceprofileofan"old"sunscreenandofa"today"sunscreen 18
2.2. Jablonskidiagramforelectronictransitionsanddissipationpathwaysafter
excitationofamolecule 20
2.3. Erythemaeffectivenessspectrum 25
2.4. Terrestrialsolarspectrumversussolar‐simulatedspectrum 32
Chapter3
3.1. Ramanspectraofskinspecimenandpolystyrenepetridish 48
3.2. VisualizationofclusterevaluationobtainedfromRamanspectradifferences
forair,skintissue,andpolystyrene 49
3.3. Threedimensionalviewoffullthicknessporcineearskin 51
3.4. Threedimensionalviewofheat‐separatedepidermalmembranefrom
porcineear 52
3.5. AverageSPFinvitroandstandarddeviationofsunscreenOWNr.1
measuredonthreetypesofPMMAplatesandthreetypesofskin
preparation 54
3.6. AverageSPFinvitroandstandarddeviationofsunscreenOWNr.2
measuredonthreetypesofPMMAplatesandonheat‐separated
epidermalmembraneonquartz 55
158
ListofFigures 159
Chapter4
4.1. RheologicalbehaviorofsunscreensmeasuredwithAR‐G2rheometer 66
4.2. IllustrationofareasfortopographicalandUVtransmittancemeasurements 68
4.3. Exampleofthreedimensionalvisualizationoffilmthicknessdistribution
ofOW‐Csunscreen 71
4.4. ExampleofdistributionoffilmthicknessfrequencyandAbbott‐Firestone
curveofOW‐Csunscreen 72
4.5. Abbott‐Firestoneprofilesofinvestigatedsunscreensappliedwithhigh
pressureandspreading1 73
4.6. SPFinvivowithstandarddeviation,mediansofSPFinvitrowith
interquartilevalues,SPFinsilico,andmediansofSmeanvaluesof
sunscreensappliedwithhighpressureandspreading1 75
4.7. Abbott‐FirestoneprofilesofGELsunscreenappliedwithtwopressure
andspreadingconditions 78
4.8. SPFinvivo,mediansofSPFinvitrowithinterquartilevalues,and
mediansofSmeanvaluesofGELsunscreen 79
4.9. Connectionsbetweeninfluencingfactors,filmdistribution,andSPFefficacy 84
Chapter5
5.1. Stepsforthedeterminationofthecorrectedfilmthicknessdistributionand
SPFinsilicoofanappliedsunscreen 93
5.2. Errordistributioncurvesforbaresubstrateandeachofthefivesunscreen
formulationsappliedonthesubstrate 94
5.3. Exampleoffittingtheresultsoftheconvolutionq*BfortheGELsunscreen 97
5.4. Corrected and adjusted film thickness frequency distribution of all
investigatedsunscreens 99
5.5. Calculated SPF in silicowith variation range,mediansofmeasuredSPF in
vitro with interquartile values, and percentage values of sunscreen film
exhibitingathicknessof0mforinvestigatedsunscreens 101
5.6. Experimentalcorrectedandadjustedfilmthicknessfrequencydistribution
ofsunscreensandfittedprobabilitydensityfunctionoftheGammaexhibiting
distribution 105
ListofFigures 160
Chapter6
6.1. Three‐dimensionalvisualizationofskinwithaschematizationofthe
surfacescanmeasurementsperformedthroughoutthesunscreenfilm 114
6.2. RamanspectraofoilmiscibleUVfilterEHMCandwatersolubleUVfilter
PBSA 116
6.3. Ramanspectraofsunscreenformulationsandplaceboformulations
withoutUVfilters 117
6.4. Ramansignalintensityattenuationofthepeakat1613cm‐1asa
functionofpenetrationdepthforeachsunscreen 119
6.5. Two‐dimensionalimagesinthex‐zplaneresultingfromlinedepthconfocal
Ramanscansforinvestigatedsunscreens 120
6.6. Pasting of all images from individual confocal Raman surface scans for
investigatedsunscreens 122
6.7. Combined2Dpictureforinvestigatedsunscreensinasurfacescan
measuredatonezcoordinate 124
6.8. Combined2Dpicturefromthesuperimpositionofallindividual
surfacescanmeasurementsforinvestigatedsunscreens 126
ListofTables
Chapter 2
2.1. SkinphototypesaccordingtoFitzpatrickclassification 12
2.2. MainUVfilterswiththeirspecificcharacteristics 23
2.3. SummaryofUVAstandardsandassociatedUVAprotectionclaims 33
Chapter 3
3.1. Skinsampletypesusedinthestudy 41
3.2. CharacteristicsofPMMAplates 44
3.3. Testedsunscreens 45
3.4. Saarithmeticalmeanoverasurfaceofselectedsubstrates 50
3.5. DifferencebetweenSPFinvitroandSPFinvivoforevaluatedsubstrates 56
Chapter4
4.1. Composition(w‐%)andSPFinvivoofinvestigatedsunscreens 65
4.2. Dataextractedfromthethicknessdistributioncurveofappliedproduct 70
4.3. MediansofSPFinvitro,Smean,andSmeantomedianratioofthickness
distributionwithinterquartilerangeQ1‐Q3forinvestigatedsunscreenswith
highpressureandspreading1 74
4.4. ImpactofvehicleonSPFinvitro,Smean,andSmeantomedianratioof
thicknessdistribution 75
4.5. MultiplepairwisecomparisontestusingBonferroniapproachfor
SPFinvitro 76
4.6. MultiplepairwisecomparisontestusingBonferroniapproachforSmean 76
161
ListofTables 162
4.7. MultiplepairwisecomparisontestusingBonferroniapproachforSmean
tomedianratio 76
4.8. MediansofSPFinvitro,Smean,andSmeantomedianratioofthickness
distributionwithinterquartilerangeQ1‐Q3forinvestigatedconditions
ofapplicationforGELsunscreen 79
4.9. ImpactofapplicationconditionsonSPFinvitro,Smean,andSmeanto
medianratioofthicknessdistributionofGELsunscreen 80
Chapter5
5.1. EstimatedcoefficientsfortheerrordistributioncurveBforthebareskin
andeachofthefiveinvestigatedsunscreens 95
5.2. Estimatedcoefficientsofdistributionqforeachinvestigatedsunscreen 98
Chapter6
6.1. Composition(w‐%)ofinvestigatedformulations 111
CurriculumVitae
MyriamSohn
9DrueSavigneux
68128Rosenau,France
Tel:0033663614697
BorninStrasbourg,France
October26th,1977
Education
2011–2015 Ph.D.studiesinPharmaceuticalSciences
University of Basel and University of Applied Sciences and Arts
Northwestern,Switzerland
2001–2002 Master’sDegreeM2inCosmetology,MinorinManagementand
Marketingfield
UniversityofLyon1,facultyofPharmacy,France
1997–1999 Master’s DegreeM1 in Health Engineering, Formulation and
ControlofCosmeticProducts,passedwithhonors
UniversityofMontpellier1,facultyofPharmacy,France
163
CurriculumVitae 164
Workexperience
2009–today BASFGrenzach,GmbH,Grenzach‐Whylen,Germany
(afteracquisitionofCibaSpecialtyChemicalsbyBASF)
2012‐today TechnicalmanagerofkeyaccountcustomersinSun
Careapplication
2009‐2012 SeniorLaboratoryHeadoftheTechnicalServicefor
BASFUVfilters
2002–2009 CibaSpecialtyChemicals,Grenzach‐Whylen,Germany
LaboratoryHeadTechnicalServiceforCibacosmeticrawmaterials
2000–2001 STRANDCosmeticsEUROPE,France
(subcontractingcompanyforcustommadecosmeticformulations)
Formulationscientistofdecorativecosmeticproducts
Listofpublications
Sohn M, Buehler T, Imanidis G. (2016) “Repartition of oil miscible and water
soluble UV filters in an applied sunscreen film determined by confocal Raman
microspectroscopy”.Photochem.Photobiol.Sci.15:861‐871.
SohnM,HerzogB,OsterwalderU,ImanidisG.(2016)“Calculationofsunprotection
factor of sunscreens with different vehicles using measured film thickness
distribution–comparisonwithSPFinvitro”.J.Photochem.Photobiol.B159:74‐81.
CurriculumVitae 165
Sohn M. (2016) UV booster and photoprotection in Principles and Practice of
Photoprotection, Ed. Steven Q. Wang and Henry W. Lim, Springer Publisher
Chapter13:227‐245
SohnM,KornV,HerzogB, ImanidisG. (2015) “Porcine ear skin as a biological
substrateforinvitrotestingofsunscreenperformance”.SkinPharmacol.Physiol.
28:31‐41.
Sohn M, Heche A, Herzog B, Imanidis G. (2014) “Film thickness frequency
distributionofdifferent vehiclesdetermines sunscreenefficacy”. J.Biomed.Opt.
19:115005.
Osterwalder U, SohnM, Herzog B. (2014) “Global state of sunscreens”, review
article.Photodermatol.Photoimmunol.Photomed.30:62‐80.
Listofpostersandoralpresentations
SohnM,HerzogB, ImanidisG. (2015) “Filmthickness frequencydistributionof
differentvehiclesdeterminessunscreenefficacy”(oralpresentation)
SunCareConference,London,UK,June2015.
CurriculumVitae 166
SohnM,HerzogB, ImanidisG. (2015) “Filmthickness frequencydistributionof
differentvehiclesdeterminessunscreenefficacy”(oralpresentation)
DGK (Deutsche Gesellschaft für Wissenschafltiche und Angewandte Kosmetik)
SymposiumMenschundSonne,Darmstadt,Germany,May2015.
SohnM,HerzogB, ImanidisG. (2015) “Filmthickness frequencydistributionof
differentvehiclesdeterminessunscreenefficacy”(oralpresentation)
AnnualResearchMeeting,DepartmentofPharmaceuticalSciences,Universityof
Basel,Switzerland,February2015.
SohnM,HerzogB, ImanidisG. (2014) “Filmthickness frequencydistributionof
differentvehiclesdeterminessunscreenefficacy”(oralpresentation)
GPEN(TheGlobalizationofPharmaceuticsEducationNetwork)meeting,Helsinki,
Finland,August2014.
SohnM,HerzogB, ImanidisG. (2014) “Impact of vehicle on film thickness and
performanceofsunscreens”(posterpresentation)
IFSCC(InternationalFederationofSocietiesofCosmeticChemists)congress,Paris,
France,October2014.
SohnM,ImanidisG.(2013)“Filmlayerthicknessandhomogeneityofdistribution
ofappliedsunscreens”(posterpresentation)
JournéesJean‐PaulMarty,Paris,France,December2013.