In vitro and in silico - edoc Sohn.pdf · having welcomed me in the Institute of Pharma Technology at the School of Life Sciences FHNW for my Ph.D. studies. I would like to thank

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




4.3.2.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





5.3.3.Sunscreenvehicles 89

5.3.4.Measurementofthesunprotectionfactorinvitro 89

5.3.5.Assessmentofthefilmthicknessdistributionofanappliedsunscreen 90

5.3.6.Determinationofthecorrectedfilmthicknessfrequencydistribution

ofanappliedsunscreenusingconvolutionapproach 67

5.3.7.Calculationofthesunprotectionfactorinsilico 92


5.4.1.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





6.3.3.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


6.4.1.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.


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.


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


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.):


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.


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.


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.


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


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.


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.


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.


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.


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).


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


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.


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).


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.


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


3.3.7. Measurement of the sun protection factor in

vitro using spectral transmission of ultraviolet

radiation

SPFinvitro isderivedfromdiffusetransmissionspectroscopymeasurementsbasedon

themodelproposedbySayre12.


∑ 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.


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.


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


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.


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


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.


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.


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.


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).


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).


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).


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.


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.


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.


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.




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.


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.


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


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.


The twopressureandspreadingconditionswereusedsolelywith thegel formulation

(GEL).Allothersunscreenformulationswereappliedwithhighpressureandspreading

procedure1.

4.3.5.Measurementofthesunprotectionfactorin

vitrousingspectraltransmissionofultraviolet

radiation

MeasurementofSPFinvitroisbasedondiffuseUVtransmissionspectroscopyaccording

totheapproachproposedbySayre12,


∑ 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.


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.


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.


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.


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


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.


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.).


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.


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)


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


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.


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.


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.


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.


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.).


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.


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.


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.


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.


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.



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).


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.


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.


vitro

SPFinvitromeasurementwasbasedonUVtransmittance,whichdenotestheinverseof

theUVintensityattenuationfactormeasuredwithaprotectingsunscreenfilm12:


∑ ser λ . Ss λ . T λ 5.1.



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.


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.


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.


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



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.


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.


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.).


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.


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.


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.


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.


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.


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.


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.


Furthermore,byadjustingthevalueofitsparametersthisdistributioncanbeadaptedto

reflectexperimentaldifferencesbetweenformulations.Hence,thisworkprovidesforthe

firsttimeindicationthattheGammadistributioncanbeuniversallyusedtodescribefilm

irregularity.

For different formulations and application procedures the model will have to be

recalibratedandvalidatedbasedonadditionalinvitroandclinicalexperimentaldata.


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.


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.


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.



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


(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.


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.


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%

‐



vitro

Measurement of SPF in vitro was based on diffuse UV transmission spectroscopy as

proposedbySayre12:


∑ ser λ . Ss λ . T λ 6.1.


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.


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.


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


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.



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)


Figure6.3.RamanspectraofsunscreenformulationscontainingUVfilters(blackline)

and placebo formulations without UV filters (gray line). Top, for OW; bottom, WO

sunscreen


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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.


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



DGK (Deutsche Gesellschaft für Wissenschafltiche und Angewandte Kosmetik)

SymposiumMenschundSonne,Darmstadt,Germany,May2015.



AnnualResearchMeeting,DepartmentofPharmaceuticalSciences,Universityof

Basel,Switzerland,February2015.



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.

Documents

In vitro and in silico - edoc Sohn.pdf · having welcomed me in the Institute of Pharma Technology at the School of Life Sciences FHNW for my Ph.D. studies. I would like to thank