11

THINKING SMALL: NANOPARTICLES INTHINKING SMALL: NANOPARTICLES IN

COSMETIC PREPARATIONS - A CONSUMERCOSMETIC PREPARATIONS - A CONSUMER

OR OCCUPATIONAL HEALTH RISK?OR OCCUPATIONAL HEALTH RISK?

Gerhard J. Nohynek, M.Sc., Gerhard J. Nohynek, M.Sc., Ph.D.Ph.D., D.A.B.T., D.A.B.T.

LL’’OREAL GLOBAL SAFETY EVALUATIONOREAL GLOBAL SAFETY EVALUATION

gnohynecgnohynec@@rd.loreal.comrd.loreal.com

ECOPA WORKSHOPECOPA WORKSHOP

DecemberDecember 17, 2005 17, 2005

22

THE CHALLENGE OF UNDERSTANDINGTHE CHALLENGE OF UNDERSTANDING

NANOSCALE IS NANOSCALE IS SIZESIZE……

NanomaterialsNanomaterials have have atat leastleast oneone dimension dimension atat <100 nm <100 nm

1 dimension: 1 dimension: layerslayers (films, (films, coatingscoatings))

2 dimensions: 2 dimensions: nanotubesnanotubes, , nanowiresnanowires

3 dimensions: nanoparticles (3 dimensions: nanoparticles (NPsNPs))

100 m 10-1 m 10-2 m 10-3 m 10-4 m 10-5 m 10-6 m 10-7 m 10-8 m 10-9 m 10-10 m

(1 m) (1 mm) (1 m) (1 nm)(100 nm)

hair (80 m) RBC (7 m)Football (30 cm) Flea (1 mm) Herpes virus (100 nm)Fullerene, C60, buckyball

(0.7 nm)

GN/ED: 10/2005

Salicylic acid

(1 nm)

TiO2 nanoparticles (40

to 200 nm

33

SOURCES OF NANOMATERIALSSOURCES OF NANOMATERIALS

BiologicalBiological::FerritinFerritin

ATP ATP synthasesynthase

VirusesViruses, , nanobacteriananobacteria

NaturalNatural::

VulcanicVulcanic ashash

SeaspraySeaspray

Forest Forest firesfires

ErosionErosion

Man-madeMan-made::

Diesel Diesel exhaustexhaust

FiresFires / toasters / toasters

ManufacturedManufactured nanoparticles nanoparticles

OneOne cm cm33 ofof urbanurban air air containscontains>10.000 nanoparticles>10.000 nanoparticles

44

WORLD-WIDE PRODUCTION OF WORLD-WIDE PRODUCTION OF NPsNPs ((mtmt//yearyear) *) *

* Source: ECETOC, 2005

10103 - 3 - 101044

1010221010NanofiltrationNanofiltration, membranes, membranesEnvironmentalEnvironmental

101011<1<1NanocompositesNanocomposites andand encapsulatesencapsulates,,

targetedtargeted drugdrug deliverydelivery, diagnostic, diagnostic

markers, markers, biosensorsbiosensorsBiotechnologyBiotechnology

>10>1033101022

1010NanoelectronicNanoelectronic andand optoelectronicoptoelectronic

materialsmaterials, , organicorganic light light emittersemitters,,

nanophosphorsnanophosphors

Information,Information,

communicationcommunication

technologiestechnologies

101033101033101033MetalMetal oxidesoxides (ZnO, TiO (ZnO, TiO22))Skin careSkin care

10104 4 - 10- 1055

1010441010CeramicsCeramics, , catalystscatalysts, films,, films,

coatingscoatings, , metalsmetalsStructuralStructural

20202020201020102003/042003/04MATERIAL / DEVICEMATERIAL / DEVICEAPPLICATIONAPPLICATION

GN/ED: 10/200

Woldwide research spending on NMs has tripled between1997 and 2002 (700 to >2000 M$)

55

TWO PRINCIPAL FEATURES MAY AFFECT PHYSICAL ANDTWO PRINCIPAL FEATURES MAY AFFECT PHYSICAL AND

TOXICOLOGICAL PROPERTIES OF TOXICOLOGICAL PROPERTIES OF NPsNPs//NMsNMs

Quantum Quantum effectseffects

important important atat thethe lowlow endend ofof

nanoscalenanoscale

maymay produceproduce changes in changes in opticaloptical,,

magneticmagnetic, thermal or, thermal or

conductivityconductivity propertiesproperties

IncreasedIncreased surface area surface area perper

unit unit massmass

1 mL 1 mL ofof nanoparticles (2.5 nm; 5 nanoparticles (2.5 nm; 5

g/cmg/cm33) ) hashas a surface a surface ofof 240 m240 m

Surface Surface maymay affect dissolution affect dissolution

kineticskinetics / / bioavailabilitybioavailability or or

increasedincreased surface surface activityactivity

GN/ED: 10/200

Supergel: nano-SiOnano-SiO22 + +

waterwater

66

NANO-TOXICOLOGY:NANO-TOXICOLOGY: TYPICAL TOXICOLOGICALTYPICAL TOXICOLOGICAL

PROPERTIES OF PROPERTIES OF NPsNPs DO NOT EXIST DO NOT EXIST

NOTE:NOTE: ToxicologicalToxicological profiles profiles ofof substances, substances, bulkbulk, micro, nano,, micro, nano,vapourvapour or solution, tend to or solution, tend to bebe thethe samesame or or similarsimilar

ParticleParticle sizesize ofof a substance a substance hashas littlelittle impact on impact on itsits toxicologicaltoxicological profile, profile,UNLESSUNLESS::

SizeSize effecteffect: absorption or absorption : absorption or absorption kineticskinetics maymay bebe affectedaffected by by particleparticlesizesize

Surface Surface effectseffects: surface : surface activityactivity playsplays a a rolerole (relative surface (relative surface increasesincreaseswithwith particleparticle sizesize))

EfectEfect on on externalexternal exposureexposure: : smallersmaller particlesparticles longer longer timetime ofofsettlementsettlement increasedincreased inhalation inhalation exposureexposure

Relative Relative particleparticle surface surface: : mm-particlesmm-particles << << microparticlesmicroparticles

<< nanoparticles << solutions or << nanoparticles << solutions or vapoursvapours ( (individualindividual moleculesmolecules))

GN/ED: 10/200

77

INHALATION EXPOSURE: INHALATION EXPOSURE: NPsNPs TEND TO AGGREGATE / TEND TO AGGREGATE /

AGGLOMERATE IN THE AIR *AGGLOMERATE IN THE AIR *

GN/ED: 11/200* A. Maynard, NIOSH, June 2005

NB.: particle size increases by a factor of 104 within 1 minute!(Major technical challenge for inhalation toxicity studies)

ZnO

88

HUMAN RESPIRATORY DEPOSITION OF PARTICLESHUMAN RESPIRATORY DEPOSITION OF PARTICLES

DepositionDeposition mainlymainlydependsdepends on on particleparticle sizesize

NanoparticleNanoparticle depositiondepositionprimarilyprimarily by diffusion by diffusion

EffectEffect ofof shapeshape andandagglomerationagglomeration: : unclearunclear

5-30 5-30 mm: : extrathoracicextrathoracic

regionregion::

impactionimpaction

1-5 m: thoracic region

bronchial, bronchiolar: sedimentation,

11 mm: : aveolaraveolar regionregion::

diffusiondiffusion

GN/ED: 11/200

99

INHALATION EXPOSURE TO SMALLINHALATION EXPOSURE TO SMALL

PARTICLES: PARTICLE SIZE DETERMINESPARTICLES: PARTICLE SIZE DETERMINES

REGION OF DEPOSITION *REGION OF DEPOSITION *

* ECETOC, 2005GN/ED: 10/2005

0

10

20

30

40

50

60

70

80

1 10 100 1000

Diameter, nm

De

po

sit

ion

, %

ET

Bb

AI

Mouth breathing

Nose breathing

NB: at <100 nm, ET (nose, upper airways) deposition increases!

Regional deposition of inhaled NP with

diameters between 1 nm and 1000 nm for

nose and for mouth breathing in the

extrathoracic airways (ET), the bronchial

airways (Bb) and the alveolar region (AI)

during breathing at rest, as predicted by ICRP

66 model (ICRP, 1994)

Silicosis

1010

PHYSIOPATHOLOGY OF LUNG OVERLOADPHYSIOPATHOLOGY OF LUNG OVERLOAD((InertInert, insoluble , insoluble particlesparticles: TiO: TiO22 andand CB) CB)

Macrophage Macrophage overloadoverload persistent inflammation persistent inflammation cellcell injuryinjury necrosisnecrosis, , cellcell proliferationproliferation

MechanismMechanism ( (rodsentrodsent): inflammation ): inflammation cellcell proliferationproliferation fibrosisfibrosis, , andand eventuallyeventually lunglungtumourstumours

WhenWhen particlesparticles cannotcannot bebe removedremoved by macrophages by macrophages (fibres)(fibres) increasedincreased rate rate ofof cellcell deathdeath,,inflammation inflammation andand adverse adverse effectseffects fibrosisfibrosis tumourstumours ( (asbestosasbestos))

Rats are Rats are thethe mostmost sensitive sensitive speciesspecies for for lunglung overloadoverload ( (highhigh breathingbreathing rate / rate / reducedreduced particleparticleclearance)clearance)

GN/ED: 11/200

Alveolar macrophagemacrophage and

asbestos fiber

Phagocyte migration Macrophage

phagocytosis andmigration – Fe NPs

(Dr. D. Warheit,Nov. 2004)

1111

EXAMPLE 1: MICROFINE (EXAMPLE 1: MICROFINE (mfmf) AND ULTRAFINE () AND ULTRAFINE (ufuf) TiO) TiO22::

COMPARATIVE INHALATION TOXICITY *COMPARATIVE INHALATION TOXICITY *

No qualitative differences between uf- and mf-TiO2 toxicity ((nono new newhazardshazards withwith uf-TiO uf-TiO2))

EffectsEffects typicaltypical for for particleparticle overloadoverload withwith associatedassociated changes changes

SpeciesSpecies sensitivitysensitivity: r>m>h: r>m>h

SimilarSimilar pulmonarypulmonary effectseffects atat 50 mg/m 50 mg/m33 mf-TiO mf-TiO2 andand 10 mg/m 10 mg/m33 uf- uf-TiO2TiO2

PulmonaryPulmonary effectseffects//lunglung burdenburden correlatecorrelate bestbest withwith surface area surface area ofoftotal total particleparticle exposureexposure

ComparisonComparison ofof lung-associatedlung-associated lymphlymph nodenode burdenburden suggestssuggests sinimalsinimaltranslocation translocation ofof ufuf- - andand mf-TiO2 mf-TiO2 underunder non-overloadnon-overload conditions conditions

CONCLUSION: CONCLUSION: ufuf TiO TiO22 maymay requirerequire a a somewhatsomewhat lowerlower TLV value TLV value thanthanpresentpresent inertinert dustdust value (5-times value (5-times lowerlower?)?)

GN/ED: 10/200

* CIIT, 2003/2004

1212

EXAMPLE 2: INHALATION OF SIOEXAMPLE 2: INHALATION OF SIO2 2 DUSTS PRODUCESDUSTS PRODUCES

FIBROSIS: FIBROSIS: NPsNPs APPEAR LESS POTENT THAN SIO APPEAR LESS POTENT THAN SIO22 MPsMPs

Intra-trachealIntra-tracheal (IT) instillation wih 20 mg SiO (IT) instillation wih 20 mg SiO22, , nano-sizenano-size

(10(10±5 nm) or micro-size (1-5 m)±5 nm) or micro-size (1-5 m)

FibroticFibrotic grade of rat lungs grade of rat lungs

+ = cellular nodules; ++ = + = cellular nodules; ++ = fibroticfibrotic cellular nodules, +++ = large cellular cellular nodules, +++ = large cellular fibroticfibrotic nodules nodules

GN/ED: 10/200* * ChenChen et et alal.., , ToxTox IndInd HealthHealth (2005) (2005)

++++NanoNano

SiOSiO22

++/+++++/++++/+++/++MicroMicro

SiOSiO22

0000SalineSaline

2 2 monthsmonths1 1 monthmonth

1313

EXAMPLE 3: SINGLE-WALL CARBON NANOTUBESEXAMPLE 3: SINGLE-WALL CARBON NANOTUBES

PRODUCED GRANULOMA FOLLOWING INTRATRACHEALPRODUCED GRANULOMA FOLLOWING INTRATRACHEAL

INSTILLATION IN RODENTS *INSTILLATION IN RODENTS *

Intra-trachealIntra-tracheal instillation instillation ofof differentdifferent

grades grades ofof CNTsCNTs vs. vs. carboncarbon black black andand quartz quartz

positive control groupspositive control groups

No No effectseffects in CB groups, in CB groups, inflammatoryinflammatory reactionsreactions / /

cytotoxicity cytotoxicity followingfollowing quartz quartz exposureexposure ( (expectedexpected))

DeathsDeaths + + interstitialinterstitial epitheloidepitheloid granulomasgranulomas ( (foreignforeign

body body reactionreaction) in ) in CNT-groupsCNT-groups

DeathsDeaths relatedrelated to « to «bolusbolus» administration» administration

(IT instillation)(IT instillation)

New inhalation New inhalation toxicitytoxicity studiesstudies ongoingongoing (US (US

NTP)NTP)

GN/ED: 10/200

* * WarheitWarheit et et alal., ., Lam Lam et et alal., Inhalation ., Inhalation ToxicologyToxicology, , 20042004

1414

LUNG GRANULOMA (RAT) 1 MONTH AFTER SWCNT IT-LUNG GRANULOMA (RAT) 1 MONTH AFTER SWCNT IT-

INSTILLATION: FIBER-, BUT NOT NANO-RELATEDINSTILLATION: FIBER-, BUT NOT NANO-RELATED

TOXICITY *TOXICITY *

* Slide by the courtesy of Dr. D. Warheit / DuPont, USA

ReactionReaction ofof thethe lunglung to to foreignforeign bodies bodies thatthat cancan notnot bebe removedremoved

((typicaltypical for fibres, for fibres, nano-unrelatednano-unrelated))

1515GN/ED: 11/200

Transport Transport ofof inhaledinhaled NPsNPs to tothethe olfactoryolfactory bulb bulb

cerebrumcerebrum cerebellumcerebellum.. (?).. (?)

needsneeds to to bebe confirmedconfirmed

((1414C-study C-study ongoingongoing))

EXAMPLE 4: TRANSLOCATION OF INHALED 13C NP INTOTHE OLFACTORY BULB AND THE BRAIN? (Oberdörster,

2003)

1616

OCCUPATIONAL EXPOSURE DURING MANUFACTURINGOCCUPATIONAL EXPOSURE DURING MANUFACTURING

OF SWCNT OR CARBON BLACKOF SWCNT OR CARBON BLACK

Concentrations during handling materialConcentrations during handling material

were very low:were very low:

A. Maynard (US NIOSH): always < 53 A. Maynard (US NIOSH): always < 53 μμ

g/mg/m33

DuPont (US) DuPont (US) studystudy: : exposureexposure levelslevels

belowbelow limitlimit ofof detectiondetection

EuropeanEuropean studiesstudies in CB in CB manufacturingmanufacturing

plants: plants: veryvery lowlow exposureexposure, , mainlymainly due due

to to externalexternal sources, sources, suchsuch as as forkfork lifts, lifts,

gasgas heatingheating or or neighbouringneighbouring traffictraffic

((KuhlbuschKuhlbusch et et alal., 2004)., 2004)

InternalInternal cosmeticcosmetic industryindustry data data suggestsuggest

negligiblenegligible inhalation inhalation riskrisk fromfrom or n or n

TiOTiO22 (<10% (<10% ofof max max dustdust value + value +

respiratoryrespiratory protection) protection)

GN/ED: 11/2005

Raw SWCNTs during

handling (DuPont, US)

Carbon Black Manufacture

(DEGUSSA, D)

1717

HUMAN ORAL EXPOSURE TO HUMAN ORAL EXPOSURE TO NPsNPs: PLASMA KINETICS OF: PLASMA KINETICS OF

A POORLY SOLUBLE ORAL DRUG (PHENACETIN): ROLEA POORLY SOLUBLE ORAL DRUG (PHENACETIN): ROLE

OF PARTICLE SIZEOF PARTICLE SIZE

GN/ED: 10/200

1818

HUMAN ORAL EXPOSURE TO NANOPARTICLESHUMAN ORAL EXPOSURE TO NANOPARTICLES

10101212 to 10 to 101414 micro or nanoparticles (0.1 to 3 micro or nanoparticles (0.1 to 3 mm) ) ingestedingested dailydaily((mainlymainly silicates silicates andand titaniumtitanium dioxidedioxide). ). UnclearUnclear whetherwhether man-man-mademade NP NP presentpresent an an additionaladditional burdenburden..

SystemicSystemic absorption absorption studiesstudies on on NPsNPs hadhad mixedmixed resultsresults::

SomeSome studiesstudies report report slightslight systemicsystemic exposureexposure ( (liverliver, spleen, , spleen, lymphlymphnodesnodes, , bloodblood, , GI-mucosaGI-mucosa))

OtherOther studiesstudies reportedreported nono systemicsystemic uptakeuptake by by thethe GI-systemGI-system

No No evidenceevidence for for targettarget organorgan toxicitytoxicity

CONCLUSIONCONCLUSION: : atat presentpresent, , humanhuman systemicsystemic exposureexposure to tonanoparticles nanoparticles afterafter oral oral uptakeuptake isis unclearunclear; ; nono evidenceevidence for foradverse adverse effectseffects or NP or NP storagestorage in in thethe humanhuman body body

* ECETOC, 2005; ** Böckmann et al., 2000; *** Jani et

al., 1992; Jani et al., 1994; **** Kanapilly and Diel,1980; Kreyling et al., 2002

GN/ED: 11/200

1919

HUMAN DERMAL EXPOSURE TO HUMAN DERMAL EXPOSURE TO NPsNPs::

COSMETICSCOSMETICS

GN/ED: 10/200

Use / exposure: NPs used incosmetics consist mainly of ZnO orTiO2 (sunscreens)

Systemic exposure: penetration ofNPs into / through the skin, systemicexposure?

Hazard: does nano-size increase thereactivity / toxicity of cosmetic NPs,such as ZnO or TiO2?

Risk management: can a chemical /photo-chemical / biological activity ofZnO or TiO2 be modified (coating)?

2020

PUBLISHED IN VIVO STUDIES ON DERMAL ABSORPTIONPUBLISHED IN VIVO STUDIES ON DERMAL ABSORPTION

OF NANOPARTICLES SHOW NO PENETRATIONOF NANOPARTICLES SHOW NO PENETRATION

No No penetrationpenetration intointo epidermisepidermis / / dermisdermis in in

vivo, vivo, manmanFluorescent Fluorescent polymericpolymeric NPsNPsHoward, P, 2005Howard, P, 2005

((SoTSoT Meeting, 2005) Meeting, 2005)

No No evidenceevidence for for penetrationpenetration intointo living living

skin (skin (preliminarypreliminary data, ECETOC, 11/2005) data, ECETOC, 11/2005)Fluorescent Fluorescent particlesparticlesEU EU NanodermNanoderm projectproject

((TilmannTilmann ButzButz))

No No penetrationpenetration intointo epidermisepidermis / / dermisdermis,,

accumulation in accumulation in thethe folliclefollicle orifice, but orifice, but nono

penetrationpenetration ibtoibto living skin ( living skin (pigpig))

PolystyrenePolystyrene NPsNPs, 20 , 20 andand

200 nm200 nmAlvarez-RomanAlvarez-Roman et et alal.,.,

20042004

TiOTiO2 , 2 , 10 10 andand 100 nm 100 nmPflückerPflücker et et alal., 2001., 2001

No No penetrationpenetration intointo epidermisepidermis / / dermisdermis in in

vivo, vivo, manmanMicrofine TiOMicrofine TiO22LademannLademann, 1999, 1999

Microfine TiOMicrofine TiO22Tan et Tan et alal., 1996., 1996

RESULTSRESULTSMATERIALMATERIALSTUDYSTUDY

CONCLUSION:CONCLUSION: NO EVIDENCE THAT TOPICALLY APPLIED NO EVIDENCE THAT TOPICALLY APPLIED

NANOPARTICLES PENETRATE INTO NORMAL SKINNANOPARTICLES PENETRATE INTO NORMAL SKIN

((NPsNPs willwill alwaysalways penetratepenetrate lessless thanthan a compound in solution!) a compound in solution!) GN/ED: 10/200

2121

IN VIVO PERCUTANEOUS ABSORPTION OF TiOIN VIVO PERCUTANEOUS ABSORPTION OF TiO22

NANOPARTICLES: RECOVERY FROM THE STRATUMNANOPARTICLES: RECOVERY FROM THE STRATUM

CORNEUM OF HUMAN SKIN *CORNEUM OF HUMAN SKIN *

RELATIVE

HORNY

LAYER

THICKNESS

[%]

0

100

TITANIUM CONCENTRATION [ g/square centimetre tape]

5

4

14

NUMBER

OF

TAPE

STRIPPING

1

5

10

15

40

50

60

78

0,05

0,2

0,4

0,4

0,3

1

2

3

Conclusion: some absorption into the upper layers of the stratum corneum,

but no penetrationGN/ED: 10/200* Lademann et al., 1999

2222

PERCUTANEOUS PENETRATION (PIG SKIN) OF ZnO ANDPERCUTANEOUS PENETRATION (PIG SKIN) OF ZnO AND

TiOTiO22––CONTAINING SUNSCREEN-GRADE CONTAINING SUNSCREEN-GRADE NPsNPs IN IN

COSMETIC FORMULATIONS *COSMETIC FORMULATIONS *

* Gamer et al., Toxicol. In Vitro, 2005, BASF GN/ED: 10/200

ZnO (10%), mean particle size: 80 nm TiOTiO22 (10%), (10%), meanmean particleparticle sizesize: 30-60 x: 30-60 x

10 nm10 nm

2323

IN VITRO PERCUTANEOUS PENETRATION OF TiOIN VITRO PERCUTANEOUS PENETRATION OF TiO22 ANDAND

ZnO IN PIG SKIN (4 mg/cm , 24-hrs): ABSORBED DOSEZnO IN PIG SKIN (4 mg/cm , 24-hrs): ABSORBED DOSE

= ZERO *= ZERO *

0.00.0

0.00.0

0.00.0

ABSORBEDABSORBED

DOSEDOSE

(%)(%)

0.8 to 1.4 **0.8 to 1.4 **

0.00.0

0.00.0

RECEPT.RECEPT.

FLUIDFLUID

(%)(%)

102.3 to102.3 to

106.8106.81.4 to 1.5 **1.4 to 1.5 **

98.6 to98.6 to

102.3102.3NPNP

ZnO, 80 nm, O/WZnO, 80 nm, O/W

emulsionemulsion (10.3%) (10.3%)

86.1 to86.1 to

93.093.00.1 to 0.50.1 to 0.50.0 to 0.30.0 to 0.385.4 to 92.985.4 to 92.9

TiOTiO22, 30-60 x 10 nm,, 30-60 x 10 nm,

methicone-coatedmethicone-coated O/W O/W

emulsionemulsion (10%) (10%)

98.2 to98.2 to

100.4100.40.1 to 0.30.1 to 0.30.1 to 0.20.1 to 0.297.7 to 100.297.7 to 100.2

TiOTiO22, 30-60 x 10 nm, , 30-60 x 10 nm, s ilicasilica

/ / methicone-coatedmethicone-coated O/W O/W

emulsionemulsion (10%) (10%)

RECOV.RECOV.

(%)(%)

SKINSKIN

(%)(%)

TAPETAPE

STRIPSSTRIPS

(%)(%)

SKINSKIN

WASHWASH

(%)(%)

TEST MATERIALTEST MATERIAL

Gamer et al., Toxicol. In Vitro, BASF, 2005; ** values at or below background levelsGN/ED: 10/2005

CONCLUSION: CONCLUSION: NO EVIDENCE FOR PERCUTANEOUS PENENTRATIONNO EVIDENCE FOR PERCUTANEOUS PENENTRATION

2424

Liposomes

100-300 nm

(Caffeine)

Water

Lipid

Nanocapsule

100-600 nm

(Vitamin A, E)

Polymer

Nanoemulsion

50 nm

(transparent)

Oleosomes

150-500 nm

Examples of nanomaterials

NANO-SIZED COSMETIC FORMULATIONSNANO-SIZED COSMETIC FORMULATIONS

Oil

2525

A complete review on liposomes in cosmetics and drugs concluded that liposomes do

not penetrate through the intact stratum cormeum (SC) or enhance penetration of active

ingredients (1995)

Joke Bouwstra (University of Leiden) published more than 30 articles on the

percutaneous penetration of liposomes or similar formulations using 14C-labelled capsule

membranes.

It was concluded that lipids from soft capsules penetrate into the deep layers of the

SC, but were absent in the living skin. Lipids form hard capsules were found only in / on

the superficial leyers of the SC.

Intact capsules – hard or soft - were only found on the surface of the SC

1. Imbert D and Wickett R: Topical delivery with liposomes. Cosmetics and Toiletries magazine. 1995; 111:32-45.

2. Honeywell-Nguyen P et al.: Quantitative assessment of the transport of elastic and rigid vesicle components and a model drug from these vesicle formulations

into human skin in vivo. Journal of Investigative Dermatology. 2004; 123(5):902-10.

3. Van den Bergh B et al.: Interactions of elastic and rigid vesicles with human skin in vitro: electron microscopy and two-photon excitation microscopy.

Biochimica and Biophysica Acta. 1999; 1461:155-173.

NANO-SIZED COSMETIC FORMULATIONS DO NOT

PRODUCE PENETRATION INTO OR THROUGH THE LIVING

SKIN

2626

SKIN PENETRATION OF SMALL MOLECULES INSKIN PENETRATION OF SMALL MOLECULES IN

SOLUTION VS. INSOLUBLE SOLUTION VS. INSOLUBLE NPsNPs**

DIFFUSION OF MOLECULES INTO THE

SKIN IS LIKE A BREAKING DAM

* Prof. T. Butz, Chairman Nanoderm Task Force, 11/2005

NPs MOVE BY MECHANICAL FORCE:WHY SHOULD A ROCK MOVE ONLY

IN ONE DIRECTION? (NOMECHANISM TO DRIVE ACTIVE

PENETRATION)

?

?

2727

GENOTOXICITY / PHOTO-GENOTOXICITY / PHOTO-

GENOTOXICITY OF TiOGENOTOXICITY OF TiO22 AND ZnO AND ZnO

NANOPARTICLESNANOPARTICLES

GN/ED: 11/200

2828

PHOTO-GENOTOXICITY OF 10 TiOPHOTO-GENOTOXICITY OF 10 TiO22-POWDERS-POWDERS

USED IN COSMETICS *USED IN COSMETICS *

UponUpon requestrequest ofof thethe SCCNFP (1999), an SCCNFP (1999), an industryindustryconsortium (TiOconsortium (TiO22 producersproducers andand cosmeticcosmeticindustryindustry) ) investigatedinvestigated

CytotoxicityCytotoxicity

Genotoxicity Genotoxicity andand photo-genotoxicityphoto-genotoxicity ( (AmesAmes, CHO), CHO)

Test Test materialmaterial: TiO: TiO22

NPsNPs andand microparticlesmicroparticles

Rutile Rutile andand anatase ( anatase (photo-activephoto-active) TiO) TiO22-crystalline -crystalline formsforms

CoatedCoated andand non-coatednon-coated particlesparticles

ProgramProgram performedperformed by COVANCE / UK by COVANCE / UK

GN/ED: 10/200

* SCCNFP, 24 October, 2000

2929

GENO- AND PHOTO-GENOTOXICITY OF TiOGENO- AND PHOTO-GENOTOXICITY OF TiO22 PARTICLES PARTICLES

(AMES TEST, CHO CELLS)(AMES TEST, CHO CELLS)

negativenegativenegativenegativeAlAl22OO33//StearicStearic acidacid1515RutileRutile88

negativenegative

negativenegative

negativenegative

negativenegative

negativenegative

negativenegative

negativenegative

negativenegative

negativenegative

AmesAmes / / Photo-Photo-

AmesAmes

11-2811-28

1515

2020

1717

2020

200.000200.000

6060

6060

1414

MEANMEAN

PARTICLE SIZEPARTICLE SIZE

(nm)(nm)

negativenegativeAlAl22OO33/SiO/SiO22RutileRutile1010

negativenegativeUncoatedUncoatedRutileRutile99

negativenegativeUncoatedUncoatedRutileRutile77

negativenegativeAlAl22OO33//StearicStearic acidacidRutileRutile66

negativenegativeAlAl22OO33//DimethiconeDimethiconeRutileRutile55

negativenegative * *UncoatedUncoatedAnataseAnatase44

negativenegativeUncoatedUncoatedAnataseAnatase33

negativenegativeAlAl22OO33/SiO/SiO22AnataseAnatase22

negativenegativeAlAl22OO33//DimethiconeDimethiconeRutileRutile11

CHO /CHO /

Photo-CHOPhoto-CHOCOATINGCOATINGCRYSTALLINECRYSTALLINE

FORMFORMNAMENAME

* Some inconclusive / positive results at high cytotoxic levels; overall rated negativeGN/ED: 10/200

3030

CYTOTOXICITY AND PHOTO-GENOTOXICITY OF TiOCYTOTOXICITY AND PHOTO-GENOTOXICITY OF TiO22

(NANO/MICRO/COATED/UNCOATED/RUTILE/ANATASE)(NANO/MICRO/COATED/UNCOATED/RUTILE/ANATASE)

PARTICLES *PARTICLES *

AllAll TiO TiO2 2 materialsmaterials showedshowed minimal cytotoxicity in minimal cytotoxicity in thethe absence absence andandpresencepresence ofof UV UV

AllAll TiO TiO22 materialsmaterials werewere negativenegative in in thethe AmesAmes-, -, photo-Amesphoto-Ames andand CHO-, CHO-,andand photo-CHOphoto-CHO tests tests

UV, UV, particleparticle sizesize or or crystallinecrystalline formform hadhad nono effecteffect on on cytoxiccytoxic or genotoxic or genotoxicpotentialpotential ofof TiO TiO2 2 materialsmaterials

In In vivovivo ( (hairlesshairless micemice), TiO), TiO22 protectsprotects againstagainst genotoxic genotoxic andand carcinogeniccarcinogenicactivityactivity ofof UV light UV light

Conclusion: Conclusion: fine fine particleparticle TiO TiO22 isis notnot expectedexpected to to presentpresent a genotoxic or a genotoxic orphoto-genotoxicphoto-genotoxic riskrisk for for humanshumans underunder normal conditions normal conditions ofof use use

* Expert report, Dr. David * Expert report, Dr. David KirklandKirkland, COVANCE, 16 , COVANCE, 16 SeptemberSeptember, 1999; , 1999; alsoalso seesee opnionopnion ofof thethe SCCNFP, 24 SCCNFP, 24 OctoberOctober 2000, 2000,

http://http://europa.eu.inteuropa.eu.int//commcomm//healthhealth//ph_riskph_risk//committeescommittees//sccpsccp//sccp_opinions_en.htmsccp_opinions_en.htm

GN/ED: 10/200GN/ED: 10/200

3131

GENOTOXICITY / PHOTO-GENOTOXICITY OF ZINC OXIDEGENOTOXICITY / PHOTO-GENOTOXICITY OF ZINC OXIDE

(ZnO) (ZnO) NPsNPs: RESULTS OF GLP STUDIES: RESULTS OF GLP STUDIES

Photo-clastogenicPhoto-clastogenic??Positive Positive atat 195195

g/mLg/mLCHOCHOPhoto-Photo-

clastogenesisclastogenesis * *

Photo-clastogenicPhoto-clastogenic??Positive Positive atat 2.5 or 3.0 2.5 or 3.0

g/mLg/mLV-79V-79Photo-Photo-

clastogenesisclastogenesis * *

EquivocalEquivocalNegativeNegative ( (kckc),),

slightlyslightly positive (V- positive (V-

79)79)

HumanHuman

keratinocytes, V-keratinocytes, V-

79 79 cellscells

CometComet test / test / photo-photo-

cometcomet test test

ClastogenicClastogenicPositive Positive atat 10.0 or 10.0 or

20.0 g/mL20.0 g/mLV-79V-79In In vitrovitro

clastogenesisclastogenesis

ClastogenicClastogenicPositive Positive atat 814814

g/mLg/mLCHOCHOIn In vitrovitro

clastogenesisclastogenesis

Non-photo-genotoxicNon-photo-genotoxicNegativeNegativeS. typhimuriumS. typhimurium

TA98, 100, 1537TA98, 100, 1537

Photo-AmesPhoto-Ames test test

Non-photo-genotoxicNon-photo-genotoxicNegativeNegativeS. typhimuriumS. typhimuriumAmesAmes test test

COMMENTCOMMENTRESULTRESULTTEST ORGANISMTEST ORGANISMTESTTEST

GN/ED: 10/200

4-FOLD INCREASE IN CLASTOGENIC POTENCY: « PHOTO-CLASTOGENIC »?

3232

«« PHOTO-GENOTOXIC RISK » OF ZnO PHOTO-GENOTOXIC RISK » OF ZnO NPsNPs

Microfine ZnO Microfine ZnO doesdoes notnot penetratepenetrate intointo or orthroughthrough thethe living skin living skin

ZnO ZnO isis clastogenic (chromosome breaks) clastogenic (chromosome breaks)in vitroin vitro –– nono evidenceevidence for clastogenicity for clastogenicityin vivoin vivo

ZnO ZnO isis non-photo-reactivenon-photo-reactive, , non-photo-non-photo-toxictoxic andand non-photo-sensitisingnon-photo-sensitising

In In vivovivo, ZnO , ZnO preventedprevented photo-damagephoto-damage in inthethe skin skin ofof hairlesshairless micemice

ZnZn isis an essential an essential elementelement for DNA for DNApolymerasespolymerases andand DNA DNA stabilitystability ––genotoxic?genotoxic?

New data New data suggestsuggest thatthat ZnO ZnO isis non-photo-non-photo-genotoxicgenotoxic

GN/ED: 10/200

3333

Most insoluble Most insoluble NPsNPs do do notnot penetratepenetrate intointo thethe

interiorinterior ofof mammalianmammalian cellscells * *

Fluorescent (Sodium Green, Texas Red) polyacrylamide NPs incubated with

CHO cells

GN/ED: 10/200* Pictures by curtesy of the University of Jena, DE,

2005

3434

NANO-HYPE NANO-HYPE andand NANO-FACTS NANO-FACTS

SWCNTsSWCNTs are are -sized-sized, insoluble fibres , insoluble fibres andand produceproduce

toxicitytoxicity typicaltypical for fibres for fibresInhalation Inhalation ofof NPsNPs producesproduces asbestos-asbestos-

likelike pulmonarypulmonary toxicitytoxicity

SomeSome evidenceevidence for syst. absorption ( for syst. absorption (lunglung overloadoverload),),

nono evidenceevidence for adverse for adverse systemicsystemic effectseffects..

Translocation to Translocation to thethe brainbrain needsneeds to to bebe confirmedconfirmed..

InhaledInhaled NPsNPs are are systemicallysystemically absorbedabsorbed

andand affect affect thethe CV CV systemsystem andand thethe

brainbrain..

No No evidenceevidence for for penetrationpenetration intointo or or throughthrough thethe

living skin. living skin. DamagedDamaged skin skin needsneeds confirmation, but confirmation, but

nono mechanismmechanism suggestingsuggesting active active penetrationpenetration..

NPsNPs penetratepenetrate throughthrough thethe skin skin andand

produceproduce systemicsystemic exposureexposure

ConflictingConflicting evidenceevidence: possible : possible minorminor syst. syst. exposureexposure

afterafter oral oral uptakeuptake ofof somesome NPsNPs. No . No evidenceevidence for for

adverse adverse effectseffects. Adverse . Adverse effectseffects on CV or CNS on CV or CNS

systemssystems isis a a hypothesishypothesis onlyonly..

Oral Oral uptakeuptake ofof NPsNPs producesproduces systemicsystemic

exposureexposure ofof thethe organismorganism andand,,

possiblypossibly, adverse , adverse effectseffects on on thethe

cardiovascularcardiovascular or or CNSsCNSs

SomeSome NPsNPs (TiO (TiO22, CB) , CB) werewere somewhatsomewhat more more toxictoxic

thanthan MPsMPs, , othersothers (SiO (SiO22, ZnO) , ZnO) equallyequally or or lessless toxictoxicInhalation Inhalation ofof NPsNPs producesproduces new new

toxicitiestoxicities. . NPsNPs more more toxictoxic thanthan MPsMPs

InertInert NPsNPs (TiO (TiO22, CB) , CB) werewere carcinogeniccarcinogenic in rats in rats afterafter

chronicchronic lunglung overloadoverload: : irrelevantirrelevant for for manman underunder

normal normal exposureexposure conditions conditions

NPsNPs are are carcinogeniccarcinogenic afterafter inhalation inhalation

FACTFACTHYPEHYPE

3535

USE OF NANOPARTICLES IN COSMETICS - ISUSE OF NANOPARTICLES IN COSMETICS - IS

THERE A HEALTH RISK: CONCLUSIONTHERE A HEALTH RISK: CONCLUSION

AvailableAvailable data data suggestsuggest thatthat use use ofof NPsNPs in in cosmeticcosmetic preparationspreparations

poses poses nono healthhealth riskrisk to to thethe consumer consumer

This This viewview isis consistent consistent withwith thethe conclusion conclusion ofof thethe recentrecent ECETOC ECETOC

ConferenceConference, 7-9 Nov., 2005 (Chairman Prof. Helmut Greim):, 7-9 Nov., 2005 (Chairman Prof. Helmut Greim):

ConcernConcern levellevel = Inhalation > oral = Inhalation > oral uptakeuptake >> >> dermaldermal exposureexposure

NB: NB: insufficientinsufficient bioavailabilitybioavailability isis thethe major obstacle for major obstacle for thethe failurefailure ofof new new drugsdrugs ––

thethe pharmaceuticalpharmaceutical industryindustry wouldwould paypay billionsbillions for for NPsNPs thatthat are are systemicallysystemically

availableavailable afterafter inhalation, oral or inhalation, oral or topicaltopical adminsitrationadminsitration –– BUT BUT todaytoday, , wewe hardlyhardly have have

intravenousintravenous NP NP drugdrug formulations. formulations.

3636

Use Use ofof alternative alternative methodsmethods for for determinationdetermination ofof humanhuman healthhealth risksrisks

ofof NPsNPs

GeneralGeneral principlesprinciples::

Tests must Tests must includeinclude a standard substance in a standard substance in orderorder to to distiguishdistiguish substance-substance-relatedrelated (ZnO) (ZnO) fromfrom particle-size-relatedparticle-size-related effectseffects ( (egeg TiO TiO22 vs nTiO vs nTiO22))

NOTE:NOTE:hazardhazard studiesstudies (in vitro / in vivo (in vitro / in vivo toxicologytoxicology) ) onlyonly, , whenwhen penetrationpenetrationintointo living tissue has been living tissue has been shownshown

InhalationInhalationTierTier 1: 1: intra-trachealintra-tracheal instillation, rodent instillation, rodent

TierTier 2: inhalation 2: inhalation studystudy, body distribution, rodent, body distribution, rodent

No alternative No alternative methodmethod availableavailable

Oral Oral exposureexposureBody distribution, rodentBody distribution, rodent

PenetrationPenetration ofof NPsNPs intointo or or throughthrough GI tract GI tract epithelialepithelial cellscells ( (screenscreen))

In In vivovivo toxicitytoxicity studiesstudies if if bioavailabilitybioavailability ofof NPsNPs >> standard substance? >> standard substance?

DermalDermal exposureexposureStudiesStudies in in humanhuman subjectssubjects (EM / skin biopsies) (EM / skin biopsies)

HumanHuman or or pigpig skin in vitro ( skin in vitro (penetrationpenetration ofof insoluble insoluble NPsNPs unlikelyunlikely –– Franz Franz cellcell maymay bebeunsuitedunsuited for for measuringmeasuring NP NP penetrationpenetration (T. (T. ButzButz))

MicroscopicalMicroscopical methodsmethods (EU (EU NanodermNanoderm projectproject))

CellCell cultures? ( cultures? (problematicproblematic, due to absent or , due to absent or compromisedcompromised stratum corneum) stratum corneum)

3737

Barber saucers Hair jacks

GN/ED: 11/200

COSMETIC APPLICATIONS OF NANOBOTSCOSMETIC APPLICATIONS OF NANOBOTS……

T

H

THANK YOU FOR YOUR ATTENTION!THANK YOU FOR YOUR ATTENTION!