SCCS/1489/12
Revision of 11 December 2012

Scientific Committee on Consumer Safety
SCCS

OPINION ON
Zinc oxide (nano form)
COLIPA S 76

The SCCS adopted this opinion at its 16th plenary meetingof 18 September 2012

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About the Scientific Committees
Three independent non-food Scientific Committees provide the Commission with thescientific advice it needs when preparing policy and proposals relating to consumer safety,
public health and the environment. The Committees also draw the Commission's attention
to the new or emerging problems which may pose an actual or potential threat.
They are: the Scientific Committee on Consumer Safety (SCCS), the Scientific Committee
on Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging and
Newly Identified Health Risks (SCENIHR) and are made up of external experts.
In addition, the Commission relies upon the work of the European Food Safety Authority(EFSA), the European Medicines Agency (EMA), the European Centre for Disease prevention
and Control (ECDC) and the European Chemicals Agency (ECHA).
SCCSThe Committee shall provide opinions on questions concerning all types of health and safety
risks (notably chemical, biological, mechanical and other physical risks) of non-foodconsumer products (for example: cosmetic products and their ingredients, toys, textiles,
clothing, personal care and household products such as detergents, etc.) and services (forexample: tattooing, artificial sun tanning, etc.).
Scientific Committee members
Jürgen Angerer, Ulrike Bernauer, Claire Chambers, Qasim Chaudhry, Gisela Degen, Elsa
Nielsen, Thomas Platzek, Suresh Chandra Rastogi, Vera Rogiers, Christophe Rousselle, Tore
Sanner, Jan van Benthem, Jacqueline van Engelen, Maria Pilar Vinardell, Rosemary Waring,
Ian R. White.
Contact
European Commission
Health & Consumers
Directorate D: Health Systems and Products
Unit D3 - Risk Assessment
Office: B232
B-1049 Brussels
Sanco-SCCS-Secretariat@ec.europa.eu© European Union, 2012ISSN 1831-4767

ISBN 978-92-79-30777-5

Doi:10.2772/88660

ND-AQ-12-027-EN-N

The opinions of the Scientific Committees present the views of the independent scientists
who are members of the committees. They do not necessarily reflect the views of theEuropean Commission. The opinions are published by the European Commission in their
original language only.
http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm

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Opinion on zinc oxide (nano form)___________________________________________________________________________________________ACKNOWLEDGMENTSSCCS members
Dr. Q. Chaudhry (chairman)
Prof. T. SannerDr. J. van Engelen
SCENIHR members
Prof. K. Dawson
Dr. W. de Jong (rapporteur)
Prof. P. Hoet
Dr. T. Jung
External experts
Dr. W. Kreyling, Helmholtz Zentrum München, Germany
Dr. R. Schins, IUF - Leibniz Research Institut für umweltmedizinische Forschung, Germany

Keywords: SCCS, scientific opinion, ZnO (nano form), directive 76/768/ECC, CAS 1314-132, EC 215-222-5

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion on ZnO(nano form), 18 September 2012
This opinion has been subject to a commenting period of four weeks after its initialpublication. Comments received during this time have been considered by the SCCS anddiscussed in the subsequent plenary meeting. Where appropriate, the text of the relevant
sections of the opinion has been modified or explanations have been added. In the cases
where the SCCS after consideration and discussion of the comments, has decided tomaintain its initial views, the opinion (or the section concerned) has remained unchanged.
Revised opinions carry the date of revision.

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SCCS/1489/12
Opinion on zinc oxide (nano form)___________________________________________________________________________________________TABLE OF CONTENTS
1.

BACKGROUND ............................................................................................. 5

2.

TERMS OF REFERENCE.................................................................................. 6

3.

OPINION..................................................................................................... 7
3.1

Chemical and physical specifications ....................................................... 7

3.1.1
Chemical identity .................................................................... 7
3.1.2
Physical form ......................................................................... 8
3.1.3
Molecular weight .................................................................... 8
3.1.4
Purity, composition and substance codes.................................... 9
3.1.5
Impurities/accompanying contaminants ..................................... 9
3.1.6
Solubility ............................................................................... 9
3.1.7
Partition coefficient (Log Pow).................................................. 11
3.1.8
Additional physical and chemical specifications.......................... 11
3.1.9
Particle size ......................................................................... 12
3.1.10Microscopy .......................................................................... 15
3.1.11
Crystal structures ................................................................. 17
3.1.12
UV absorption ...................................................................... 18
3.1.13
Zeta potential ...................................................................... 21
3.1.14Droplet size in formulations.................................................... 22
3.1.15
Homogeneity and stability...................................................... 25
3.1.16
Summary on supplementary physicochemical characterization .... 26
3.2
Function and uses .............................................................................. 27
3.3

Toxicological evaluation ...................................................................... 27

4.

3.3.1
Acute toxicity ....................................................................... 27
3.3.2
Irritation and corrosivity ........................................................ 32
3.3.3
Skin sensitization.................................................................. 37
3.3.4
Dermal/percutaneous absorption ............................................ 40
3.3.5
Repeated dose toxicity .......................................................... 55
3.3.6
Mutagenicity/genotoxicity ...................................................... 59
3.3.7
Carcinogenicity..................................................................... 68
3.3.8
Reproductive toxicity............................................................. 68
3.3.9
Toxicokinetics ...................................................................... 68
3.3.10
Photo-induced toxicity ........................................................... 69
3.3.11
Human data ......................................................................... 79
3.3.12
Special investigations ............................................................ 82
3.3.13
Safety evaluation (including calculation of the MoS)................... 88
3.3.14
Discussion ........................................................................... 91
CONCLUSION ............................................................................................ 95

5.

MINORITY OPINION.................................................................................... 98

6.

REFERENCES ............................................................................................. 98

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1. BACKGROUND
Zinc oxide (CAS No. 1314-13-2; EC No. 215-222-5) has widespread use in cosmetic
products with the following functions: bulking, skin protection and as a UV absorber,besides its authorized use in all cosmetics as a cosmetic colorant with the Colour Index No.
CI 77947 in Annex IV.
Two former submissions on zinc oxide in pigmentary form as well as in the form of ananomaterial were submitted in order to have zinc oxide approved as a UV-filter in cosmetic
sunscreen products at a maximum level of 25%.
The first scientific opinion (SCCNFP/0649/03, final) on zinc oxide was adopted by the
SCCNFP during its 24th plenary meeting of 24-25th June 2003 with the following conclusion:"A considerable part of the investigations and their results submitted have been performed
15 or more years ago and consequently cannot fulfil modern requirements. However, there
is a broad basic knowledge on Zn2+ and its compounds, e.g. ZnO.
The physicochemical specifications of ZnO used in many of the studies are incomplete, thepurity/impurities not specified. On the other hand, ZnO is practically insoluble in water.
Thus, in general, ZnO may be considered as a non-toxic substance, including when used incosmetic products.
The main concern of the present evaluation is related to the risk assessment of micronized(approximately 0.2 μm) ZnO, which may be coated by other compounds, and which is used
as an ingredient in sunscreen formulations.
Micronized ZnO has been demonstrated to be photoclastogenic, possibly photo-aneugenic,
and a photo-DNA damaging agent in mammalian cells cultured in vitro. The relevance of
these findings needs to be clarified by appropriate investigations in vivo.
There is a lack of reliable data on the percutaneous absorption of micronized ZnO and thepotential for absorption by inhalation has not been considered.
As to a safety assessment of a use of UV-filters by children over the age of 1 year, theSCCNFP issued a position statement (SCCNFP/0557/02)."
In the statement (SCCP/0932/05) on zinc oxide adopted by the SCCP on 20th September
2005, the request for a safety dossier was repeated for microfine zinc oxide.
In the general opinion (SCCP/1147/07) on the use of nanomaterials in cosmetics adopted
by the SCCP on 18th December 2007 it was concluded that, "A complete safety dossier on
micron-sized and nano-sized ZnO was requested by the SCCNFP in its opinion on ZnO in
2003 (SCCNFP/0649/03). An opinion on the safety of such materials will be dependent on
the availability of an adequate dossier."
Finally, a clarification (SCCP/1215/09) on the statement (SCCP/09328/05) on zinc oxide
was adopted by the SCCP on 21st January 2009 with the clarification that:"The SCCP considers that on the basis of the dossier reviewed in 2003 the use of ZnO in its
non-nano form (pigment grade, with particle sizes above 100 nm) is considered safe. The
concern expressed in the SCCNFP opinion 0693/03 with regard to phototoxicity is not
relevant for this form of ZnO due to the absence of dermal penetration."
According to COLIPA, the current submission III contains the information contained in theprevious submissions I and II. Submission III also presents the new information that
became available since the last submission and provides an overall safety assessment forthis ingredient, which takes into account the entire available information.

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2. TERMS OF REFERENCE
1. Does the SCCS consider zinc oxide in its nano-form safe for use as a UV-filter witha concentration up to 25% in cosmetic products taking into account the scientific
data provided?
2. Does the SCCS confirm that zinc oxide in its non-nano form is safe for use as a UVfilter with a concentration up to 25% as stated in the SCCP clarification(SCCP/1215/09)?
3. And/or does the SCCS have any further scientific concern with regard to the use ofzinc oxide in cosmetic products?

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3. OPINION

3.1

Chemical and physical specifications

3.1.1

Chemical identity

3.1.1.1

Primary name and/or INCI name

Zinc oxide (INCI name)3.1.1.2

Chemical names

Zinc oxide CI 77947; CI pigment white 4
Colipa No. S76
3.1.1.3

Trade names and abbreviations

Information has been provided on nano-sized ZnO raw materials (source materials as
powder for sunscreen formulations) and some final cosmetic products containing some of
the nano-sized ZnO raw materials. Information has also been provided on selected raw
materials from various manufacturers, in the form of both coated and uncoatednanomaterials and products.
The following materials/products were included in the submitted dossier:
Examples of nano-sized ZnO raw materials (powder):
Z-COTE® (ZnO)

BASF SE

Nanox (ZnO)

Elementis

Nano TEC® 50 (ZnO)

Grillo Zinkoxid GmbH

Nano TEC® 60 (ZnO)

Grillo Zinkoxid GmbHFINEX-50 (ZnO)

Sakai Chemical

MZ 30 (ZnO)

Tayca

Zinc Oxide Neutral (ZnO)

Symrise GmbH

Zano® 10 (former Zano®, ZnO)

UmicoreZ-COTE® HP1 (ZnO coated with triethoxycaprylylsilane)

BASF SE

Z-COTE® MAX (ZnO coated with dimethoxydiphenylsilanetriethoxycaprylylsilane
cross-polymer)
BASF SE
Zinc Oxide NDM (ZnO coated with dimethicone)

Symrise GmbH

Zano® 10 Plus (former Zano® Plus, ZnO coated with octyl triethoxy
silane)UmicoreExamples of final cosmetic products with nano-sized ZnO raw materials:
W/O emulsion, uncoated ZnO (Sun Defense SPF 30/PA++,
Ponds, 8.4% ZnO)
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W/O emulsion, coated ZnO (20% ZnO)

UmicoreO/W emulsion, coated ZnO (9% ZnO)

UmicoreO/W emulsion, coated ZnO (Perfect results SPF 15/PA++,
Ponds, 2.2% (Z-COTE® HP)

UnileverO/W emulsion, coated ZnO (all day moisture Lotion SPF 15,
Olay, ZnO (Z-COTE® HP1)

Procter & Gamble

Comment
The data provided on the safety evaluation are mainly on uncoated ZnO. According to theinformation in submission II, the currently used coating materials are not UV absorbers andare common cosmetic ingredients that are widely used for different purposes in cosmetic
products. The coatings consist of organic and inorganic materials such as silica,
dimethicone, dimethoxydiphenylsilane and triethoxycaprylylsilane.
(References: 3, 4, 15, 16, 17, 18, 21, 24, 41, 51, 52, 84, 102, 103, 111, 112, 117)
3.1.1.4

CAS/EC number

CAS No.:1314-13-2

EC No.:

215-222-5

3.1.1.5

Structural formula

Formula:ZnO

Crystallinity:

Wurtzite

3.1.1.6

Empirical formula

Formula:
3.1.2

ZnO

Physical form

Solid, white odorless powder.
3.1.3

Molecular weight

Molecular weight:

81.38 g/mol.

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3.1.4

Purity, composition and substance codes

The purity has been reported to be ≥99.0% of zinc oxide for the various nanomaterials
included in the dossier.
(References: 10, 11, 51, 54, 55, 108, 112)
Comment
Two other references (52, 109) indicated for evidence of purity do not provide informationon the purity of the nanomaterials.
A number of tests have been performed with a nano zinc oxide (Finex-50) with an indicatedpurity of ≥96%. However, in the description of the physico-chemical characteristics of theZnO materials information on the purity has not been provided.
3.1.5

Impurities/accompanying contaminants

No information has been provided on the presence of impurities/contaminants.
Comment
As a purity of ≥99% was
impurities/contaminants are ≤1%.

demonstrated

in

the

references

below,

the(References: 10, 11, 51, 54, 55, 108, 112).
Two other references (52, 109) indicated do not provide information on the purity of thenanomaterials.
3.1.6

Solubility

Solubility was determined by mixing 10 grams of zinc oxide in 100 ml of demineralized
water for 30 minutes at room temperature. After mixing, the suspension was centrifuged for6 hours to separate the (undissolved) zinc oxide particles from the solution. Aftercentrifugation the solutions were not completely clear optically, and a blue haze wasnoticeable. A sample of the solution was used to analyze the Zn content using Inductively
Coupled Plasma (ICP) spectroscopy. Only uncoated preparations of zinc oxide wereanalyzed. The possible effect of coating on solubility was not determined.
The zinc concentration in the aqueous solution was:Zinc oxide A:

Zano 10 (uncoated, Umicore), batch: Z08009

42 mg/L

Zinc oxide B:

NanoTec 50 (uncoated, Grillo), batch: MZ6362

20 mg/L

Zinc oxide C:

Z-COTE (uncoated, BASF), batch: EHHG1407

47 mg/L

Zinc oxide D:

FINEX-50 (uncoated, Sakai Chemicals, Japan), batch: 9Z22

27 mg/L(Reference: 115)
The solubility of pigmentary, coated and uncoated ZnO was also determined in water andtissue culture medium. In these assays, atom absorption spectroscopy was used for thedetermination of the Zn content.
The following results were obtained:
SampleMediumpH

ZnO (pigmentary, Fluka)

Aqua dest.

6.5–7
9

Solubility basedon Zn concentration<0.1 mg/L

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Opinion on zinc oxide (nano form)___________________________________________________________________________________________ZnO (pigmentary, Fluka)

DMEM/FCS**

About 7.435 mg/LZnO (uncoated, Z-COTE, BASF)

Aqua dest.

6.5–7

1.5 mg/L

ZnO (uncoated, Z-COTE, BASF)

M4 medium*

About 7.4

1.2 mg/L

ZnO (uncoated, Z-COTE, BASF

DMEM/FCS

About 7.4

37 mg/LZnO (coated, Z-COTE HP1, BASF) M4 medium

About 7.4

2.4 mg/L* M4 medium: reconstituted water containing salts and trace elements (water hardness: 2.5mmol/L; alkalinity: 0.9 mmol/L).
** DMEM/FCS: Dulbecco/Vogt modified Eagle’s minimal essential medium/fetal calf serum.
(References: 8, 9)At low pH simulating stomach environment at pH 2.7, the solubility of particles ranged from
89.6% (particles >3 mm) to 98.5% (particles <1 mm).
(Reference: 57)
Comment
For the tests with uncoated ZnO (Reference: 115) some haze remained visible after
centrifugation indicating that some non-dissolved ZnO particles remained in dispersion. The
visible haze is very likely to be due to agglomerates/aggregates since dispersed
nanoparticles would not be visible. These were also measured by ICP spectrometry. Thus,
there is an overestimation of the solubility as determined by the method used. The data
provided strongly suggest that equilibrium solubility had been reached, although it remains
unclear if the solution was saturated, or if the particles remained undissolved because of thepresence of some surface layer.
The data provided suggest that:• the solubility of the nano-sized formulation in water is about 10× that of thepigmentary grade,• in tissue culture medium the solubility of nano-sized and pigmentary grade is similar.
• the solubility of the coated and uncoated Z-COTE ZnO shows only a twofold difference,the coated ZnO being slightly more soluble. It should be noted that for the solubilityof Z-COTE, two different solubility values (1.5 mg/L and 47 mg/L) have been
provided and no explanation of this difference is given.
Additional information was provided on the effect of particle size on solubility in a simulated
stomach environment (pH 2.7). Particles with a size <1 mm showed 98.5% dissolution. As
the information on size was limited to <1 mm, no relevant information was provided
specifically on nano-sized ZnO. This high solubility at low pH of both nano- and micron-sized
ZnO particles was also recently demonstrated by Li et al. (Reference: AR16). In view of this,the SCCS considers that the solubility of ZnO in a stomach environment can be assumed tobe high.
In conclusion, the data provided on solubility are inadequate to draw conclusions on the
dissolution of ZnO nanoparticles. It is the view of the SCCS that the solubility data aspresented are insufficient. Furthermore, to enable proper estimation of consumer exposure
to any partially dissolved and/or insoluble fractions, the information for nanomaterials to be
used as cosmetic ingredients should be provided not only on solubility, but also on the
dissolution rate since equilibrium solubility is normally not achieved in the body (see SCCS
Guidance on the Safety Assessment of Nanomaterials in Cosmetics SCCS/1484/12).

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However, some additional information on solubility of ZnO nanoparticles in general isavailable in the open literature. The literature indicates that solubility of ZnO nanoparticles
is in the same range as reported in the dossier (References: AR15, AR16, AR17, AR19,
AR21). Some of the data indicate that the solubility of uncoated and coated ZnO(Reference: AR15), or ZnO either as nanoparticles or micron-sized particles, is similar andpH dependent (References: AR16, AR17). Also the solvent itself (water, tissue culture
medium, presence of citric acid) influences the dissolution of ZnO nanoparticles (Reference:
AR15, AR19, AR21). Reed et al. (Reference: AR21) compared dissolution rates between
nano- and bulk-sized ZnO, and showed that the nano-form has a higher dissolution rate.
In view of the available information on the solubility of ZnO in aqueous media, it can be
expected that very small concentrations of ZnO (e.g. <50 mg/L) will be in the form of acompletely dissolved solution under static equilibrium conditions. This means that ZnO
in a formulation will be simultaneously present as a mixture of solubilized Zn ions and amajor part as undissolved ZnO particles - both with different diffusion and uptake
characteristics.
The dissolution rate, also applicable to nanoparticles, describes the solubilization of ZnO
nanoparticles in a certain fluid matrix over time, and will differ greatly depending on thefluid composition. In particular, the dissolution rate will depend critically on the fractional
saturation of the matrix with Zn ions and the potential mechanisms for diffusion or active
transport of Zn and O (and other) ions typical for biological processes. The dynamic natureof dissolution rate of any solubilization process implies that any mass fraction of Zn ionsremoved or washed away will lead to further dissolution and eventually complete
solubilization of the particulate fraction of ZnO. Thus, under non-static conditions, e.g. inbiological environments, ZnO nanoparticles will keep on releasing a certain mass fraction of
Zn ions until they are completely solubilized.
(References: 8, 9, 57, 115)
3.1.7

Partition coefficient (Log Pow)

Log Pow:

According to the applicant, measurement of the partition coefficient is notapplicable as ZnO is practically insoluble in water.

Comment
This statement cannot be accepted in view of the partial solubility as presented in section
3.1.6.
Note: depending on the coating on the particles, a partitioning of particles between aqueousand octanol phases may also be expected.
3.1.8

Additional physical and chemical specifications

Melting point:

1,975°C

Boiling point:-

Flash point:-

Vapour pressure:-

Density:

5.47 g/cm2

Viscosity:

-

pKa:

-

Refractive index:-

UV/vis spectrum (200–800 nm): -(Reference: 87)
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3.1.9

Particle size

The particle size distribution was provided for four uncoated ZnO nanoparticles designated
A, B, C, D:
Zinc oxide A:

Zano 10 (uncoated, Umicore), batch: Z08009

Zinc oxide B:

NanoTec 50 (uncoated, Grillo), batch: MZ6362

Zinc oxide C:

Z-COTE (uncoated, BASF), batch: EHHG1407

Zinc oxide D:

FINEX-50 (uncoated, Sakai Chemicals, Japan), batch: 9Z22

To determine particle size distribution, 30 w% zinc oxide dispersions were prepared byadding zinc oxide to a mixture of water and dispersing agent. The dispersion was composedof ZnO (150 g), Tego Dispers 752W1 (30 g) and water (320 g). The dispersion waspremixed in a dissolver and transferred to Dispermat SL-12 bead mill thereafter. The beadmill was operated using 0.3 mm YTZ beads at a filling ratio of 85% and a rotor speed of
4,500 rpm. The zinc oxide suspensions were circulated through the mill over a period of 60
minutes. After bead milling, a sample of the suspension was collected for particle size
analysis.
The particle size distribution was measured using a CPS Disc Centrifuge (model DC20000)2,
operated at a constant rotation of 10.000 rpm. 0.1 g of the 30 w% suspension was diluted
in 30 ml water, after which 0.15 ml of the dilution was injected into a rotating disc andparticle size measurements were recorded. The results are expressed as weight averageand number average particle size.
The applicant claims that the particle size distribution (PSD) determination in aqueous
environments can only be done for uncoated ZnO and considers it not relevant to determine
the PSD for coated ZnO. It is argued in the submission that the coating would not change
the particle size of the ZnO particle. This is supported by electron microscopy images where
no differences in particle size and morphology can be identified between coated anduncoated materials. The applicant recognized that in cosmetic formulations the particle size
distribution of coated and uncoated ZnO may be of interest, but the methodology will only
provide information very close to the primary particle size. It is nevertheless unlikely that
normal coatings will bring any significant change to particle size distribution of ananomaterial.

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Average particle size distribution based on weight measured using a CPS Disc Centrifuge isshown below.

The corresponding percentile values are as follows for particle size distribution based onweight.

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Average particle size distribution based on number of particles measured using a CPS Disc
Centrifuge is shown below.

The corresponding percentile values are as follows for particle size distribution based on
number of particles.

Comment
For all ZnO preparations, the number distribution results show that 90% of the particles
have a size below 100 nm, whereas for the weight distribution the percentage of particles
with a size below 100 nm varies (ZnO A d75, ZnO B d75, ZnO C d25, ZnO D d90).
The median particle size distribution (the D50 indicates that 50% of the particles have asize below that measurement) of the four evaluated ZnO nanomaterials ranged from 30 nm(ZnO D) to 55 nm (ZnO C).
Additional parameters of the ZnO nanomaterials such as number of particles per mass unit,
and surface area per mass unit were not provided.
(Reference: 114)
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3.1.10 Microscopy
The ZnO nanoparticle distribution of the formulations was investigated with two different
electron microscopy techniques: Cryo-SEM and ambient TEM. Several examples of both EM
and TEM evaluations are presented.
3.1.10.1

TEM evaluations of uncoated ZnOA) Z-COTE®, uncoated ZnO (BASF, left image, TEM, Reference: 13)
ZnO particles have different shapes (rod-like and isometric) and a broad size distribution
range of 30–200 nm. The ZnO particles were generally present as clusters.
B) Z-COTE® HP1, coated ZnO (BASF, right image, TEM, Reference: 13)
ZnO nanoparticles have different shapes (rod-like and isometric) and a broad size
distribution range of 30–200 nm. The ZnO particles were generally present as clusters.

Comment
Both uncoated and coated Z-COTE® ZnO particles show similar morphology in the TEM
images.
3.1.10.2

TEM evaluation of ZnO in formulationsA) W/O emulsion, coated ZnO (20% ZnO, Umicore, left image, TEM, Reference:
18)
ZnO nanoparticles have different shapes (rod-like, star-like and isometric) and a broad size
distribution range of 30–150 nm. The ZnO particles were present mainly as clusters.
B) O/W emulsion, coated ZnO (9% ZnO, Umicore, right image, TEM, Reference:
25)
ZnO nanoparticles have different shapes (rod-like, star-like and isometric) and a broad size
distribution range of 30–150 nm. The ZnO particles were present mainly as clusters. In
addition, spherical particles with a size between 200 nm and 1 μm were present.

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Comment
The TEM image of O/W emulsion (right image, above) as presented in the dossier could not
be found in the cited reference (Reference: 24) but it is contained in Reference: 25. There isa similar discrepancy in Reference: 24 which refers to SEM images, whereas the image
presented above shows TEM results.
The various formulations presented all showed the presence of ZnO particulates mainly asclusters, with size ranges as indicated for the raw materials. These clusters may be present
in the original formulation, but it is also possible that it is due to the excessive load of the
EM sample. Z-COTE® and Z-COTE®HP1 with a size of 30–200 nm show clusters with rodlike, star-like and isometric forms. Umicore ZnO with a size of 30–150 nm also showsclusters with rod-like, star-like and isometric forms. Similar results are shown for both O/W
and W/O emulsions.
The star-like forms present in the formulations are not observed in the raw material for ZCOTE®. Both ZnO nanomaterials contain rod-shaped particles for which the Z-COTE® ZnO
nanoparticles show more irregular shapes compared to the Umicore ZnO nanomaterial.
(References: 13, 18, 25)
3.1.10.3

SEM evaluation of uncoated ZnO nanomaterials

A) Zano® 10 (uncoated, Umicore, left image, Reference: 110)B) Nano Tec® 50 (uncoated, Grillo, right image, Reference: 58)

C) Nano Tec® 50 (uncoated, Grillo, Reference: 109)
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Comment
The SEM images of the three examples presented show similar morphology of the uncoated
Nano Tec® 50 and Zano® 10 nanomaterials.
SEM images of ZnO nanomaterial in various formulations were included in the dossier
submitted by the applicant. As these are relevant for final cosmetic products they have been
considered in this opinion as additional background information but have not been further
evaluated.
(References: 58, 109, 110)3.1.11 Crystal structures
X-ray diffraction (XRD) analysis was used for determination of the crystal structure. Rietveld
refinement characterization was used for analysis of crystallite size.
An example of the XRD spectrum is presented below.

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The crystal structure for all samples was identified as purely hexagonal zinc oxide. For zincoxide this is often referred to as a “Wurtzite” structure (see also section 3.1.1.5). The
crystallite sizes determined via Rietveld refinement characterization were as follows:
Zinc oxide A:

Zano 10 (Umicore), batch: Z08009

48 nm

Zinc oxide B:

NanoTec 50 (Grillo), batch: MZ6362

36 nm

Zinc oxide C:

Z-COTE (BASF), batch: EHHG1407

67 nm

Zinc oxide D:

FINEX-50 (Sakai Chemicals, Japan), batch: 9Z22 17 nm(References: 53, 116)
3.1.12 UV absorption
UV absorption was determined for four uncoated ZnO nanomaterials. A suspension of zincoxide samples was prepared using a mild bead mill operation to break up agglomerates andstabilize the individual particles as much as possible. These dispersions were then used tomeasure the UV/vis absorbance and reflection spectra. The 30 w% dispersions were diluted
to a concentration of 0.1 g/L ZnO and the UV/vis absorption spectrum was measured using
a Genesys 6 UV/vis spectrometer.
The following ZnO preparations were analysed:
Zinc oxide A:

Zano 10 (uncoated, Umicore), batch: Z08009

Zinc oxide B:

NanoTec 50 (uncoated, Grillo), batch: MZ6362

Zinc oxide C:

Z-COTE (uncoated, BASF), batch: EHHG1407

Zinc oxide D:

FINEX-50 (uncoated, Sakai Chemicals, Japan), batch: 9Z22

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Opinion on zinc oxide (nano form)___________________________________________________________________________________________The optical properties in the UV/vis region from approximately 200 to 900 nm were also
determined by generating absorption and reflection spectra using the so-called integrating
sphere.

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Opinion on zinc oxide (nano form)___________________________________________________________________________________________(References: 56, 114)

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Opinion on zinc oxide (nano form)___________________________________________________________________________________________

Comment
A similar pattern was observed regarding the UV absorption for the four evaluated ZnOnanomaterials. However, only uncoated ZnO materials were analysed.
3.1.13 Zeta potential
Zeta potential was determined using electrophoresis (Zetasizer Nano from Malvern). The pH
was adjusted with NaOH.
The following samples were investigated:
FINEX-50 (Sakai Chemicals, Japan, supplied by Umicore)
Zano 10 (Umicore)
NanoTec 50 (Grillo)
Z-COTE (BASF, batch: EHH/0201)
There was no significant difference between the samples in terms of the charge.
The curve shapes are characteristic for ZnO powders. The iso electric point (IEP) was in therange of pH 9.5–10.
(Reference: 31)

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3.1.14 Droplet size in formulations
The applicant provided additional information on the effects of various formulation andtechnical parameters (sunscreen type, nanomaterial concentration, viscosity, propellant gas,
actuator and valve type) on size and size distribution of droplets generated when asunscreen is used as an emulsion for spray application. The information was provided as theresults of an investigative study (Reference: 38).
Different types of emulsion; water in silicone (W/Si) and water in oil (W/O), were preparedusing high concentrations of organic and inorganic UV-filters (ZnO as Z-COTE MAX, 1% in
W/Si emulsion, 1% in W/O emulsion, among others). These formulations were incorporated
in aerosol cans with gas (a blend of butane, propane and isobutane).

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SCCS/1489/12
Opinion on zinc oxide (nano form)___________________________________________________________________________________________The size and size distribution of the droplets were analyzed by laser diffraction using a
Malvern® Spraytec.
The measuring distance of the droplets was set at 11 cm for all the experiments. In general,
assessments for these types of evaluations and measurements are carried out at a distanceof 30 cm from the laser beam. However, the authors of the paper considered that 11 cm
was a more common distance for normal use of sunscreen sprays.
A size distribution curve obtained with 22% of gas propellant for the W/Si emulsion at a–1
cm and b–11 cm is shown below.

The figure below shows the influence of concentration of inorganic filters on the droplet sizein the W/O emulsion.

The results showed that the sprayability of the formulation and the particle size
characteristics of the emitted sprays are dependent on the physicochemical properties ofthe formulations. Sprayable waterproof sunscreen emulsions with a high sun protection
factor and a low (<5%) percentage of emitted droplets below 30 μm could be developed by
optimizing formulation parameters and using appropriate actuators and valves. For all
experiments, the percentage of droplets below 10 μm was less than 0.1%. Droplet size and23

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size distribution of the emitted droplets were highly influenced by all the parameters studied
and especially by propellant concentration, viscosity, presence or absence of inorganic filters(powder) and the design of valves and actuators. Considering the aspect of the final productand droplet size, the best products (in terms of optimal droplet size) were obtained with thelower gas concentration (22%), higher concentrations of inorganic sunscreen filter andhigher viscosities. The final choice of the valve and actuator was made based on the aspectof the final product after spraying. Therefore, when sunscreen-containing aerosols areformulated it is important to consider each technical and formulation parameter in order toreach the optimum size and size distribution of droplets.
(Reference: 38)
According to the applicant, no sunscreen spray products containing nano-sized ZnO areavailable on the EU market. It was stated that if such sprayable sunscreen products were to
be produced and marketed they would comply with current standards and requirements
according to the relevant guidance documents as referred to below.
- The Technical Guidance Document on Risk Assessment of the European Chemical
Council emphasized that aerosols with a Mass Medium Aerodynamic Diameter (MMAD)
greater than 10–15 μm are not respirable for humans, i.e. the aerosol particle sizes
above this cut-off will deposit in the upper regions of the lungs due to their large
particle sizes. Only particles with a size less than 10–15 μm reach the gas-exchange
region of the lung.
- The U.S. Silicones Environmental, Health and Safety Council (SEHSC) recommends
that, “When considering a consumer aerosol application for any silicone-based material,

regardless of the method of aerosol generation, the particle size MMAD should be at least
30 μm with no more than 1% of the particles having an aerodynamic diameter of 10 μm or
less. Following this guidance ensures that virtually all aerosol particles will be trapped in
the nasopharyngeal region and very few if any particles will be deposited in the
tracheobronchial region.”(References: 43, 45, 86)Comment
The results indicate that mainly larger droplets were present when spraying a water- or
silicone-based nanoparticle containing test aerosol at a distance of 11 cm. The small fractionof droplets with a diameter less than 30 μm was based on droplet size distribution
measured by weight. It should be noted that even a low fraction based on aerosol weight isrelevant as it contains a high number of droplets. At larger distances such as 30 cm which isnormally used for such investigations, a short time lapse may occur allowing for evaporationof aerosol liquid components resulting in smaller droplet sizes than measured at 11 cm.
These dried residual particles spreading in the air also need to be taken into account forpossible inhalation exposure. Notably, this mechanism is even more relevant when highvapour pressure fluids such as alcohols are used in the generation of the aerosol, compared
to when water or oil are used as dispersion fluids.
The report indicates that in certain tests, an inorganic filler such as TiO2 or ZnO (Z-COTEmax) was used. However, it was not indicated in the results section which inorganic filler
was used in the experiments for which the data were presented. Therefore, the data cannot
be exclusively ascribed to ZnO nanomaterial.
In view of the presented data, the possibility that the dried residual aerosol particles maylead to lung exposure after inhalation needs to be taken into account. Therefore themeasurement of the droplet size distribution needs to be complemented by measurementsof the size distribution of the dried residual aerosol particles as well.

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Opinion on zinc oxide (nano form)___________________________________________________________________________________________(References: 30, 71)
3.1.15 Homogeneity and stability
Photostability
MethodPhotostability was measured by UV absorption according to a BASF internal method: PMMA
plates (Helioscreen HD6, 6 μm), 1.3 mg/cm2 application rate, four plates and 11
scans/plate, measurements with Labsphere 1000S.
Samples
O/W emulsions with uncoated and coated ZnO
Formulation A:

Z-COTE (10%, uncoated, BASF):

UV10037-1-1 (SPF 6.6)

Formulation B:

Z-COTE HP1 (10%, coated, BASF):

UV 10037-2-2 (SPF 6.6)

Formulation C:

Zano 10 Plus (20%, coated, Umicore): UM08-ZP20-OW (SPF 31.3)

Light source
Atlas CPS+ with Atlas filter solar standard according to DIN 67501.
Results/Remarks
The photostability of the uncoated ZnO is presented below.

The O/W formulations were photostable over the whole UV range (from 290 to 400 nm).
However, according to the applicant, few absorbance curves were higher following
irradiation as it was technically not possible to measure exactly the same point on a PMMA
plate before and after irradiation, and also in case the formulation spreading of the plate
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was not homogenous. This is known to result in slightly different absorbance behaviour. The
observed minimal differences could be considered as an indication of variation in themeasurement technique and not as variation in the photostability of the nanomaterials.
It was concluded by the applicant that all three model O/W formulations with uncoated (ZCOTE) and coated (Z-COTE HP1, Zano 10 Plus) ZnO were photostable over the whole UV
range between 290 and 400 nm.
(Reference: 29)
3.1.16 Summary on supplementary physicochemical characterization
The comparative analysis and presentation of the physicochemical characteristics of therepresentative selected nano-sized ZnO materials shows that all nano-sized ZnO materials
as described in the dossier can be considered to be broadly similar, irrespective of the
manufacturing processes used (wet chemical or pyrolytic). The crystal structure of all of theZnO nanomaterials included in the dossier is the same (wurtzite/hexagonal) and the particle
shapes are roughly similar. The particle size distributions for all of the products fall within
the same range and so do all other parameters tested such as solubility, zeta potential andphotostability. According to the applicant, the set of physicochemical data on representative
nano-sized ZnO raw materials, as well as on examples of additional nano-sized ZnO raw
materials found on the EU market, indicates that the nano-sized ZnO products can beconsidered to be similar overall.
Comment
It should be noted that it is technically possible to produce nano-dispersed ZnO withdifferent physicochemical properties to those described in this dossier. Therefore, thisopinion specifically relates to those coated and uncoated ZnO nanomaterials for which data
have been submitted in this dossier. It is required that manufacturers carefully analyse
the raw materials which they obtain, particularly if they come in a finally stabilized
formulation in fluids which may contain different forms/surface modifications of thenanomaterials. Thus, this assessment applies to ZnO nanoparticles similar to those includedin this dossier, with the following characteristics:• ZnO nanoparticles of purity ≥99%, with wurtzite crystalline structure and physicalappearance as described in the dossier, i.e. clusters that are rod-like, star-like and/or
isometric shapes.
• Median diameter (D50: 50% of the number below this diameter) of the particle
number size distribution is between 30 nm and 55 nm, whereas the D1 (1% below thissize) is above 20 nm.
• ZnO nanoparticles that are either uncoated or coated (with triethoxycaprylylsilane,
dimethicone, dimethoxydiphenylsilanetriethoxycaprylylsilane cross-polymer, or octyl
triethoxy silane).
• ZnO nanoparticles that have a comparable solubility to that reported in the dossier,i.e. below 50 mg/L (approximately the maximum solubility of the ZnO nanomaterials
for which data are provided in the dossier).
Regarding the characterization of the ZnO nanomaterials, product parameters such as thenumber of particles per mass unit and surface area per mass unit were not provided. It is at
the moment under consideration that mass (weight) alone may not be the best parameterfor the description of a dose response relationship in toxicity studies. Therefore, it is theview of the SCCS that additional parameters, such as number of particles per mass andsurface area per mass, are usefull additions to the characterization of the nanomaterials.
Where such parameters are available, it is possible to evaluate dose response relationships

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for toxicity by these alternative dose parameters (number of particles, total surface areaadministered) as well.

3.2

Function and uses

According to the European Union Risk Assessment Report (Reference: 44), zinc oxide isused in industrial settings as a rubber compounding material, and in glass and ceramic
products. Zinc oxide acts as a catalyst in alkylation, oxidation, hydrogenation anddesulphurisation reactions.
In paints, zinc oxide is mainly used as a corrosion inhibitor and to a lesser extent as amildew control agent.
As zinc is an essential trace element, zinc oxide is added to fertilizers, animal feeds andhuman vitamin supplements. It is used in medical plasters, baby creams, calamine lotion
and in dental cement (in combination with eugenol). Zinc oxide absorbs ultraviolet light,
and is used as a UV-filter in pharmaceutical and cosmetic products. No specific informationon the uses of nano-sized ZnO is available.
ZnO is also used as colorant according to the Cosmetics Directive 76/768/EEC, Annex IV,
but no information of ZnO particle sizes used in such applications is available, and it is notclear whether it contains a nanofraction.
The present submission applies for the use of ZnO nanomaterial as a UV-filter in cosmetic
sunscreen products up to 25%.

3.3

Toxicological evaluation

3.3.1

Acute toxicity

3.3.1.1

Acute oral toxicity

The acute oral toxicity of one ZnO nanoparticle product (FINEX-50) was investigated with adose of 2,000 mg/kg.
Study Design
Guideline/method:
Species/strain:
Group size:
Test substance:
Batch:
Purity:Dose levels:
Dose volume:
Vehicle:Route:
Exposure:
Observation period:
GLP:
Published:
Reference:OECD Guideline 423 (2001).
Rat/Sprague-Dawley (Crl:CD(SD)Three males
FINEX-50 ZnO (supplied by Sakai Chemical Industry Co., primaryparticle size: 20 nm, non-coated).
OZ52≥96% as indicated in the submission
2,000 mg/kg bw as 20% (W/V) aqueous suspension.
10 mL/kg bw
Water for injection.
Oral (gavage)
Single application
14 days
No
No
99
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Study period
Date of report:

Eight days
March 2006

The maximum dose (2,000 mg/kg body weight) as specified in OECD Guideline 423 foracute oral testing was used. The dose was administered as a 25% aqueous suspension byoral gavage. The animals were observed prior to dosing and for treatment-related effects
after 2 hours and on day 1, and then at least once a day for a total of one week. Body
weights were determined on days 1, 2, 3, 4, 6 and 8 after application. Gross pathology was
performed in all rats at two weeks after oral administration.
Results
No animals died during the course of the study. On day 3 of observation, decreases in bodyweight and fecal excretion were observed in one animal, but these findings disappeared fourdays after dosing. There were no abnormal findings at necropsy at two weeks after oral
administration.
Conclusion
The approximate acute oral toxicity (LD50) was >2,000 mg/kg bw for male Sprague-Dawley
rats.
(Reference: 99)
Comment
For the ZnO used, information on surface area and number of particles per mass was notprovided. Also, information on the dose expressed as surface area and number of particles
was not provided.
The purity of the FINEX-50 used was indicated in the submission to be ≥96%. However,data on the purity is not provided in the physico-chemical characterization of the FINEX-50(section 3.1.4), and was also not provided in the study reference 99.
The SCCS agrees that the acute oral toxicity of the ZnO materials tested in rat is >2,000
mg/kg body weight.
Additional study
An exploratory acute oral toxicological investigation comparing the effects of nano-scale (20nm) or pigmentary (submicron-scale 120 nm) ZnO (Produced by Jiangsu Haitai
Nanomaterials Co. Ltd. Jiangsu Province, China) was performed in mice as reported by
Wang et al. (Reference: 118). The primary mean size as determined by TEM was 20 ± 5 nm
and 120 ± 20 nm, respectively. Surface area was 4.4×105 cm2/g and 9.1×104 cm2/g,
respectively. The particle number concentration was 2.2×1016/g and 2.0×1014/g,
respectively. When administered in a 1% sodium carboxymethylcellulose solution the size
range of these materials was between 30 nm and 70 nm, and between 186 nm and 190 nm,
respectively. Male (n=5) and female (n=5) CD-ICR mice received a single oral application of
20 nm ZnO or 120 nm ZnO powder at dose levels of 1,000, 2,000, 3,000, 4,000 and 5,000
mg/kg bw suspended in 1% sodium carboxymethylcellulose. Doses expressed as surface
area were: 4.4-8.8-13.2-17.6-22x105 cm2/kg bw for the 20 nm ZnO; and 9.1-18.2-27.336.4-45.5x104 cm2/kg bw for the 120 nm ZnO. Doses expressed as number of particles
were: 2.2-4.4-6.6-8.8-11x1016 particles/kg bw for the 20 nm ZnO; and 2-4-6-8-10x1014
particles/kg bw for the 120 nm ZnO. The control animals received the vehicle only. The
animals were observed for 14 days and at termination blood was sampled for selected
hematological and clinical pathology examinations. Several organs were collected forhistopathological investigations and ZnO content analysis. For the 120 nm sized ZnO the28

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highest dose (5,000 mg/kg) induced the highest mortality; three out of five females andtwo out of five males died, whereas one out five females died in the 4,000 mg/kg group,and one out of five females died in the 2,000 mg/kg group. For the 20 nm sized ZnO, one
out of five males died in the 5,000 mg/kg group, and one out of five females died in the
2,000 mg/kg group. In addition, effects were reported showing alterations in individual
hematology, clinical pathology parameters, and pathological findings (stomach, liver, heart,spleen). Indications of Zn accumulation in selected tissues (bone, pancreas and kidney)were observed for both 20 nm and 120 nm ZnO.
(Reference: 118)
Comment
In view of the size ranges reported in Reference 118, the larger ZnO cannot be consideredas pigment grade. The published paper describes the particles as submicron-sized with amean size of 120 nm. Although not all effects showed a clear dose response relationship,
many effects were noted at the highest dose administered that should be considered dose
related even when they only occur at the highest dose. The pathological findings indicate adose response relationship with the highest dose inducing the most severe alterations. The
lowest dose investigated (1,000 mg/kg) also induced pathological effects in the animals.
Most effects showed some minor differences between the 20 and 120 nm sized ZnO. Target
organs for toxicity were liver, heart, spleen, pancreas and bone. A no observed adverse
effect level (NOAEL) was not dentified because alterations were also observed at the lowest
dose investigated (1,000 mg/kg).
No deaths were observed in a similar study using a single oral dose of 5 mg/kg in mice
(Reference: AR16). ZnO nanoparticles were approximately 50 nm in size (TEM evaluation),
which were compared to ZnO microparticles showing at least one diameter >100 nm (TEM
evaluation). DLS evaluation showed an average hydrodynamic diameter of 1,226 ± 120 nm
for the ZnO microparticles, and an average hydrodynamic diameter of 93 ± 14 nm for the
ZnO nanoparticles. After oral administration for both ZnO nanoparticles and microparticles,
Zn could be observed in serum indicating uptake from the GI–tract, either as particulatematerial, dissolved Zn ions, or in both forms. In liver, spleen and lung, ZnO nanoparticles
showed higher Zn distribution compared to ZnO microparticles. Serum liver enzyme analysis(aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate
dehydrogenase (LDH)) indicated liver toxicity due to both ZnO nanoparticles and ZnO
microparticles which was confirmed by histopathology (Reference AR16). Histopathologicallesions were only observed for ZnO nanoparticles in the liver (Reference 118).
Additional studies in the open literature
An exploratory study of acute oral toxicity with a commercial ZnO nanomaterial issummarized below.
Study Design
Guideline/method:

OECD Guideline 423 (2001)

Species/strain:

Rats, Sprague-Dawley, in house animal facility.

Group size:

Five males and five females per group.

Test substance:

ZnO nanoparticles (20 nm, 63 nm in SEM) from Nanostructured andAmorphous Materials Inc., USA), surface area 50 m2/g.
Micro- sized ZnO from Sigma- – Aldrich (product no. ZO385).

Batch:

Stock no. 5180HT

Purity:Not stated29

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Dose levels:

5, 50, 300, 1,000, 2,000 mg/kg bw as 20% (W/V) aqueous
suspension, dose as surface area 0.25, 2.5, 15, 50, and 100 m2/kg
bw.

Dose volume:

10 mL/kg bw

Vehicle:Distilled water

Route:

Oral (gavage)

Exposure:Single application

Observation period:

14 days

GLP:

No

Published:

Yes

Study period:

14 days

Date of report:

2011

In this study, the acute oral toxicity of ZnO nanomaterial was compared to a ZnO
preparation from Sigma-Aldrich. The study was performed according to the OECD guideline
for acute oral toxicity OECD Guideline 423 (2001). Toxicity and animals were evaluated up
to 14 days after a single administration by oral gavage.
Results
There was an inverse relationship with serum levels of liver enzymes (aspartate
aminotransferase (AST) and alanine aminotransferase (ALT), the lower doses inducing thehighest increase. On a weight basis the nano-sized material induced serum alterations in
AST and ALT that were not observed with the commercial non-nano ZnO (Sigma-Aldrich).
The incidences of microscopic lesions in the liver, pancreas, heart and stomach were higher
at lower doses of nano-size zinc oxide compared to higher doses. It was concluded thatnano-sized zinc oxide exhibited toxicity at lower doses probably due to lower aggregation of
the material at low concentrations. No measurements of Zn ions (or particles) in blood werereported. Thus on a worst-case basis, it can be assumed that the translocating ZnO was in
nanoparticulate form (SCCS Guidance on Nanomaterials in Cosmetics SCCS/1484/12). The
nano-ZnO induced histopathological lesions in the liver, pancreas, stomach and heart at all
the doses investigated (lowest dose 5 mg/kg), whereas the commercial ZnO did so only at
the highest dose investigated (2,000 mg/kg).
Conclusion
The nano-sized ZnO induced toxicity at the lowest dose investigated (5 mg/kg bodyweight).
(Reference AR20)
Comment
In this study, the size of the commercial micro-ZnO is not reported. Also for the ZnO
information on number of particles per mass was not provided. Information on dose
expressed as number of particles was not provided. The acute toxicity evaluation was
performed based on blood parameters and histopathology. Body weights were not reported.
Exploratory study

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Study Design
Guideline/method:

No

Species/strain:

ICR mice

Group size:

Five males and five females.

Test substance:

ZnO nanoparticles (50 nm, TEM evaluation) and ZnO microparticles(1,226i nm, DLS measurement) from Top Nanotechnology Co. Ltd.,
Taiwan.

Batch:

Not stated

Purity:Not stated

Dose levels:

1.25, 2.5, 5 g/kg bw.

Dose volume:

10 mL/kg bw

Vehicle:Sterile water plus 1% hydroxypropyl methyl cellulose.

Route:

Oral (gavage) and intraperitoneal administration.

Exposure:Single application

Observation period:

14 days

GLP:

No

Published:

Yes

Study period:

14 days

Date of report:

2011

In this study, ZnO nanoparticles used were approximately 50 nm in size (TEM evaluation),
which were compared to ZnO microparticles showing at least one diameter >100 nm (TEM
evaluation). DLS evaluation showed an average hydrodynamic diameter of 1,226 ± 120 nm
for the ZnO microparticles and an average hydrodynamic diameter of 93 ± 14 nm for the
ZnO nanoparticles. Toxicity was evaluated by analysing serum biochemistry parameters andhistopathology. Genotoxicity was evaluated using the Ames test and the micronucleus test.
Zinc absorption and tissue distribution was evaluated by ICP-MS determination of elemental
Zn. In addition an in vitro cytotoxicity assay was performed.
Results
All animals survived a single oral or intraperitoneal dose of 5 g/kg body weight of bothnano-sized ZnO (50 nm) and micro-sized ZnO (1,226 nm), although the large ZnO particles
induced a body weight reduction in both male and female animals. After oral andintraperitoneal administration of a single dose of 2.5 g/kg bw for both ZnO nanoparticles
and microparticles, Zn could be observed in serum indicating uptake from the GI-tract. For
ZnO nanoparticles, the systemic availability was significantly higher (approximately 24% to50%) at 2, 4 and 6 hours after administration compared to that of ZnO microparticles as
indicated by Zn measurements by ICP-MS. In the liver, spleen and lung, treatment with
ZnO nanoparticles showed a higher distribution of Zn compared to treatment with ZnO
microparticles. Serum liver enzyme analysis (aspartate aminotransferase (AST), alanine
aminotransferase (ALT) and lactate dehydrogenase (LDH)) indicated liver toxicity at 24, 48
and 72 hours after treatment, both for ZnO nanoparticles and ZnO microparticles. This was
confirmed by histopathology. Histopathological lesions were only observed for ZnO
nanoparticles in the liver.

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SCCS/1489/12
Opinion on zinc oxide (nano form)___________________________________________________________________________________________The in vitro studies revealed a dose related induction of cytotoxicity for both nano-ZnO andmicro-ZnO up to a concentration of 100 μg/mL. The Ames test (in vitro) and the in vivo
micronucleus test were negative for both nano-ZnO and micro-ZnO.
(Reference: AR16)

Comment
For the ZnO nanoparticles and microparticles, information on surface area and number ofparticles per mass was not provided. Information on dose expressed as surface area andnumber of particles was not provided.
The results indicate a similar uptake, tissue distribution, and toxicity of ZnO nanoparticles
and ZnO microparticles. As Zn was measured by ICP-MS, it cannot be concluded whether it
was present in particulate form or as dissolved ionic Zn. Although serum levels of aspartate
aminotransferase, alanine aminotransferase and lactyate dehydrogenase indicated similarliver toxicity for both ZnO nanoparticles and ZnO microparticles, histopathological lesions
were only observed for ZnO nanoparticles in the liver.
3.3.1.2

Acute dermal toxicity

No information provided.
3.3.1.3

Acute inhalation toxicity

No information provided.
3.3.2

Irritation and corrosivity

Skin Irritation
The skin irritation potential was determined for one ZnO nanomaterial (FINEX-50) according
to Japanese guidelines.
Study Design
Guideline/method:

Guide to marketing and manufacturing of cosmetics and quasi drugs
in Japan.

Species/strain:

Guinea pig/HartleyGroup size:

Three males per concentration.

Test substance:

FINEX-50 ZnO (supplied by Sakai Chemical Industry Co., primaryparticle size: 20 nm, non-coated).

Batch:

OZ52

Purity:≥96% as indicated in the submission

Vehicle:Ethanol

Dose level:

25% or 40% dispersion in ethanol.

Dose volume:

0.05 mL, once a day for three consecutive days.

Exposure:

Intact skin (open)

GLP:

No

Study period:

Up to three days

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Date of report:

February 2009

The cumulative acute dermal irritation potential was investigated in individually housed
Hartley Guinea pigs. The hair was clipped on the dorsal area of the trunk one day prior toapplication and prior to examination on day 3. An amount of 0.05 mL of the test substanceas a 25% or 40% ethanolic dispersion was applied to the test site (2 × 2 cm) on the rightback of each of the six animals and left exposed without dressing. Percutaneousadministration was used for the substance. The administration was conducted once daily forthree consecutive days. The animals were examined for erythema, incrustation and edemaon each day of administration.
Results
There were no signs of systemic toxicity and no mortality. Slight erythema was observed in
one of three animals in the 40% test substance group on day 3 of the administration. No
other abnormal dermal changes were observed at any time during the administration andobservation periods. No abnormal dermal changes were observed at any time during theadministration and observation periods in the 25% test substance group.
Conclusion
The three day consecutive skin irritation study in male Guinea pigs led to skin irritation
scores of 0.1 and 0.0 for the 40% and 25% test substance dispersions indicating a weak
irritation according to the Japanese guideline used.
(Reference: 101)
Comment
For the ZnO information on surface area and number of particles per mass was notprovided. Information on dose expressed as surface area and number of particles was notprovided.
The purity of the FINEX-50 used was indicated in the submission to be ≥96%. However,data on the purity is not provided in the physico-chemical characterization of the FINEX-50(section 3.1.4), and was also not provided in the study reference 101.
This test does not correspond to OECD Guideline 404 which indicates that rabbits should be
used for the in vivo irritation test, and gives a dermal dose of 0.5 mL per application siteusing a gauze patch.
Mucous membrane irritation
The mucosal irritation potential was determined for one ZnO nanomaterial (FINEX-50)
according to Japanese guidelines.
Study Design
Guideline/method:

Guide to marketing and manufacturing of cosmetics and quasi drugs
in Japan and according to Draize (1959) and Gullot et al. (1982) intotal comparable to OECD Guideline 405.

Species/strain:

Rabbit/Japanese WhiteGroup size:

Three males per concentration.

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Test substance:

FINEX-50 ZnO (supplied by Sakai Chemical Industry Co., primaryparticle size: 20 nm, non-coated).

Batch:

OZ52

Purity:≥96% as indicated in the submission

Vehicle:Olive oil

Dose level:

0.1 g of neat (unchanged) substance or 0.1 mL of a 25% solution inolive oil.

Dose volume:

0.1 mL

GLP:

No

Study period:

Up to seven days

Date of report:

April 2006

The potential irritant effect on the mucous membrane was investigated by instillation ofeither 0.1 g of the neat test substance or 0.1 mL of a 25% oily solution into the rightconjunctival sac of the eye of each of the three rabbits. The 25% oily solution was chosen
as the proposed consumer use concentration of the product. The eyes were not washed.
The left eyes remained untreated and served as controls. Both eyes of the animals were
examined within 1 and 4 hours after application and twice daily on days 1, 2, 3, 6 and 7
according to the method and scoring system of Draize.
Results
Neat test substance group
At 1 hour after administration, redness of the conjunctiva was observed in all animals, andslight or apparent edema in two of three animals and edema accompanying one-half
occlusion of the eyelid in one of three animals. Slight discharge was observed in two of
three animals. At 4 hours after administration, apparent or diffuse beef-like redness of the
conjunctiva was observed in all three animals, and slight or apparent edema in two of three
animals and edema accompanying more than one-half occlusion of the eyelid in one of three
animals. Slight or apparent discharge was observed in two of three animals. On day 1,
apparent redness and slight edema were observed in two of three animals, and slightdischarge in one of three animals. On day 2, apparent redness was observed in two of three
animals, and slight edema and slight discharge in one of three animals. On day 3, slight orapparent redness was observed in two of three animals. There were no adverse effects
recorded in observations made on day 6 post-administration and thereafter.

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25% test substance group
Apparent redness of the conjunctiva, slight edema, and slight discharge were observed in all
three animals at 1 hour after administration. At 4 hours after administration, there wasslight or apparent redness of the conjunctiva in two of three animals and diffuse beef-likeredness in one of three animals. In addition, slight or apparent edema was observed in twoof three animals. Slight discharge was observed in one of three animals. On day 1 postadministration, slight redness was observed in one of three animals. There were no adverse
effects recorded in observations made on day 2 post-administration and thereafter.

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The calculated eye irritation indeces based on the findings at 1 hour after administration of
the test substance and 25% test substance solution were 11.3 and 8.0, respectively.
Conclusion
The neat and the 25% solution of the test substance were concentration dependent, slightly
and transiently irritating to the eyes of three Japanese White rabbits. Therefore, both wereassessed as “mild irritant”.
(Reference: 96)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
The purity of the FINEX-50 used was indicated in the submission to be ≥96%. However,data on the purity is not provided in the physico-chemical characterization of the FINEX-50(section 3.1.4), and was also not provided in the study reference 96.
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Opinion on zinc oxide (nano form)___________________________________________________________________________________________In Reference 96, the number of animals investigated was six (three per group) and thenumbering is from 1 to 6. However, the results are presented for animal numbers 7 to 12.
3.3.3

Skin sensitization

The skin sensitization potential of one ZnO nanomaterial (FINEX-50) was evaluated using amodification of the guinea pig maximization test as a short-term adjuvant and patch test.
Study Design
Date of report:

February 2006

Guideline/method:

Short-term adjuvant and patch test method (s-APT according toReference: AR25).

Species/strain:

Guinea pig/HartleyGroup size:

Ten females in total (five per group).

Test substance:

FINEX-50 ZnO (supplied by Sakai Chemical Industry Co., primaryparticle size: 20 nm, non-coated).

Batch:

OZ52

Purity:≥96% as indicated in the submission

Vehicle:Distilled water

Concentration:

Induction:
Group A) distilled waterGroup B) 40% aqueous test substance suspension
Challenge:
Group A) 5%, 12.5%, 25%, 40% aqueous test substance suspension.
Group B) 1.25%, 2.5%, 5%, 12.5%, 25%, 40% aqueous test
substance suspension.

Route/Exposure:

Induction:
1st induction: intradermal injection with Freund’s complete adjuvant
followed by occlusive topical application of the vehicle or 40%
aqueous test concentration for 72 hours.
2nd induction: six days after 1st induction occlusive topical applicationof the vehicle or 40% aqueous test concentration for 48 hours.
Challenge: 13 days after 1st induction, open application of test
concentrations.

Skin evaluations:24, 48, 72 hours after challenge.

Study period:

16 days

GLP:

No

Published:

No

Date of report:

February 2006

The sensitizing potential of the test substance was tested in female Hartley Guinea pigs
according to the short-term adjuvant and patch test method (s-APT according to Yanagi etal. (Reference: AR25). In total ten animals were tested, divided in two groups of five37

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animals each. Prior to the 1st induction the hair was removed and the animals were shaved,
followed by an intradermal injection of Freund’s complete adjuvant at four sites. (The
Guinea pig maximization test according to OECD Guideline 406 uses three injections as
induction; one being a mixture of Freund’s complete adjuvant and water, one a mixture of
FCA and the test substance, and one of the test substance only). Thereafter, one group
received the vehicle (group A) or a 40% aqueous test substance suspension (group B) for
72 hours under an occlusive dressing. Six days after the 1st induction, the animals were
induced a 2nd time by occlusive topical application of either the vehicle or the 40% aqueous
test substance concentration.
The challenge was performed by open topical application of 5%, 12.5%, 25%, 40% aqueous
test substance suspension (group A) or 1.25%, 2.5%, 5%, 12.5%, 25%, 40% aqueous test
substance suspension (group B), and the skin was evaluated at 24, 48 and 72 hours after
the challenge.
Results
Neither the group induced with the 40% aqueous test substance suspension or with thevehicle showed any skin reaction to the challenge concentrations at the evaluation time
points of 24, 48 or 72 hours.
Conclusion
It was concluded that the test substance exhibited no potential to induce dermal
sensitization in Guinea pigs under the conditions of the study.
(Reference: 95)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on the doses expressed as surface area and number ofparticles was not provided.
The purity of the FINEX-50 used was indicated in the submission to be ≥96%. However,data on the purity is not provided in the physico-chemical characterization of the FINEX-50(section 3.1.4), and was also not provided in the study reference 95.
The sensitizing potential of the test substance was tested in female Hartley Guinea pigs
according to the short-term adjuvant and patch test method (s-APT), which is not arecognized guideline assay. In the classical Guinea pig maximization test (OECD Guideline406) the induction is performed with a mixture of the test material and FCA, and ten
animals are used in the test group. Mixing of the test substance with FCA provides optimal
conditions for inducing an immune response, i.e. induction of sensitization if allergic
substances are present in the test material.
A concurrent positive control with a well known weak sensitizer as indicated in OECD
Guideline 406 (hexylcinnamic aldehyde (CAS No. 101-86-0), mercaptobenzothiazole (CASNo. 149-30-4) and benzocaine (CAS No. 94-09-7)) was not included in this assay, so there
is no certainty as to whether the test system used was able to identify weak sensitizers.
However, in the paper of Yanagi et al. (Reference: AR25) eight contact sensitizers were
found to show similar reactions in the GPMT performed according to the OECD Guideline
406, and in this shortened test.
The SCCS considers that the validity of this (or any) test for demonstrating sensitization
potency of nanomaterials has not yet been demonstrated. The inclusion of a positive particle
control might overcome this problem. However, no positive particle control has been
identified thus far.

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Human repeat insult patch test
Study Design
Date of report:

October 2007

Guideline/method:

According to internal laboratory methodology.

Species:

HumanGroup size:

50 volunteers (six males and 44 females)

Test substance:

A) ZnO, coated (Zano 10 Plus, Umicore) (former Zano® Plus, ZnO
coated with octyl triethoxysilane).
B) ZnO uncoated (Zano 10, Umicore).

Batch:A) 22-5D
B) No data

Route:

Dermal occlusive application by patch.

Procedure:

Induction: Nine consecutive 24 hour exposures on Monday,
Wednesday and Friday for three consecutive weeks.

Rest period:

10–14 days

Challenge:

After rest period one 24 hour exposure as during the induction
period.

Reaction scoring:

24 and 48 hours after challenge application.

Concentration:

25% in corn oil (0.2 mL dispensed on occlusive, hypoallergenic
patch).,

Vehicle:Corn oil

GLP:

Yes

Published:No

The test solutions were freshly prepared prior to applications. Of 52 volunteers enrolled forthe study, 50 volunteers (six males and 44 females aged 21–66 years; Caucasian and
Hispanic) completed the study.
The oily test material dispersions were dispensed onto an occlusive, hypoallergic patch.
After 24 hours the patch was removed and the procedure was repeated until a series of nineconsecutive 24 hour exposures were made on Monday, Wednesday and Friday for threeconsecutive weeks. The volunteers were given a rest period of 10–14 days after which achallenge was applied once to a previously unexposed test site. The challenge was
equivalent to any of the original procedures. The skin reactions were scored 24 and 48
hours after application on a scale graded 0–4.
Results
Under the selected conditions of this study none of the 50 investigated volunteers showed
any skin reaction at any time. All volunteers were consequently scored with grade 0.
Conclusion

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The applicant concluded that coated and uncoated ZnO tested as 25% oily dispersion did
not produce any skin irritation or skin sensitization under the conditions of this repeat
human insult patch test.
(References: 105, 106)

Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
Two volunteers discontinued the treatment at the third application after two applications
without any clinical signs. Any follow-up and reasons for discontinuation were not presented
in the report.
3.3.4

Dermal/percutaneous absorption

3.3.4.1

In vitro studies for dermal penetration of human skin

Exploratory in vitro study for percutaneous skin penetration
Study Design
Date of publication:

1997

Guideline/method:

Exploratory percutaneous skin penetration study in vitro.

Test system:

Abdominal human skin.

Test substance:

Titanium dioxide T805 (Degussa, Germany), and Spectra veil MOTG
(Tioxide specialties, UK), a 60% dispersion of zinc oxide in mineral
oil/triglyceride.

Particle size:

Mean crystalline length 116.8 nm with a standard error of 8.5 nm.

Batch:

Not stated

Dose level:

Water in oil (W/O) emulsion similar to commercial sunscreen
formulation with ultrafine titanium dioxide (11% wt), and zinc oxide(2.5% wt), dose of formulation 1mg/cm2.

Skin preparation:

Abdominal human skin from plastic surgery.

Cells:

Not stated

Skin temperature:

Room temperature

Test chamber:

Not stated

Route:

Topical administration

Exposure time:

Not statedSampling time points: Not stated
GLP:

No

Published:

Yes

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Characterization of the mineral content of sunscreen formulations containing both TiO2 andZnO was performed. The distribution of TiO2 and ZnO at the surface of human stratum
corneum was investigated in vitro. Human abdominal skin obtained from plastic surgery was
exposed to a W/O emulsion containing 11% ultrafine titanium dioxide and 2.5% ultrafine
ZnO at 1 mg/cm2 at room temperature. Transmission electron microscopic cross-sections of
the horny layer of human epidermis showed an almost regular mineral coating of thestratum corneum but neither intercellular nor intracellular ZnO penetration was noted.
Results
This study analysed the distribution of the ZnO nanoparticles in the original preparation andin the cosmetic formulation. It showed the presence of the nanomaterial on the surface ofthe skin and a lack of nanoparticle uptake by the cells of the stratum corneum of the skin.
(Reference: 40)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
Although this study provides some evidence that there is no penetration of thenanoparticles from the formulation into the skin, the information on the study itself is rather
limited, e.g. time of incubation and surface area of treated skin were not indicated. From
the discussion it appears that a mixture of TiO2 and ZnO nanoparticles was used in theformulation. This study is of no value for the evaluation of skin penetration of ZnO
nanoparticles.
Exploratory in vitro study for percutaneous skin penetration
Study Design
Date of publication:

2009

Guideline/method:

Exploratory percutaneous skin penetration study in vitro.

Test system:

Human skin

Test substance:

Z-COTE® Max, coated with dimethoxydiphenylsilanetriethoxycaprylylsilane, cross-polymer.

Particle size:

Not stated

Batch:

Not stated

Dose level:

Water in silicone (W/Si) emulsion and water in oil (W/O) emulsion
with 1% ZnO, 2 mg/cm2 of each emulsion.

Skin preparation:

Excised human skin from female Caucasian patients from abdominal
plastic surgery, after verification of integrity and removal of
subcutaneous fat, storage at –25°C for a maximum of six months
prior to use.

Cells:

Six intact membranes

Skin temperature:

32±1°C

Test chamber:

Static diffusion cells (Franz-type)

Route:

Topical application

Exposure time:

24 hours
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Sampling time points: 1, 2, 4, 6, 8, 12 and 24 hours
GLP:

No

Published:

Yes

Two different types of emulsion; water in silicone (W/Si) and water in oil (W/O), eachcontaining 1% ZnO, were tested. Previously frozen dermatomed human Caucasian skin fromsurgery was put on the static diffusion cells and, after stabilization of the receptor fluid at atemperature of 37 ± 1°C to achieve a skin temperature of 32 ± 1°C, 2 mg/cm2 of each
formulation was applied to the skin membrane (six/formulation).
Receptor fluid was sampled at 1, 2, 4, 6, 8, 12 and 24 hours after application. Thereafter,
the skin samples were removed, rinsed to remove residual formulation, homogenized andprocessed for analysis. The content of Zn++ ions in the skin was analyzed by means ofInductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES).
Results and conclusion
The recovery rates for ZnO after application of the W/O and W/Si emulsions after
application to the skin were 76% and 86%, respectively. For the W/O emulsion, slightly
more ZnO was recovered from in the skin compared to the recovery from on the skin (about
40 and 35%, respectively). For the W/Si emulsion the amount of ZnO recovered in the skin
was about the same as the amount recovered from on the skin. No penetration through theskin could be observed with a limit of detection of 0.01 ppm and a limit of quantification of
0.1 ppm for Zn++, respectively in the W/O and the W/Si emulsions.
(Reference: 39)
Comment
The ZnO particles tested were not characterized, and data on particle size, surface area andnumber of particles per mass was not provided. Information on dose expressed as surface
area and number of particles was not provided.
Exploratory in vitro study for percutaneous skin penetration
Study Design
Date of report:

2007

Guideline/method:

Exploratory study on human skin penetration of sunscreen
nanoparticles in vitro.

Test system:

Human skin

Test substances:

A) ZnO formulation A: dispersion with 60% of siliconate coated ZnO
in caprylic capric triglyceride.
B) ZnO typical sunscreen formulation B: O/W emulsion sunscreenwith 20% of siliconate coated ZnO in caprylic capric triglyceride.
C) O/W emulsion without ZnO.

Particle size:

15–40 nm

Dose level:

10 μL/cm2.

Skin preparation:

Full thickness skin from female donors following abdominoplasty
cleaned by a heat-separation technique.

Replicates:

A and B) eight samples, C) three samples.
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Test chamber:

Static glass diffusion cell (Franz type, exposed area: 1.3 cm2,
receptor phase volume: 3.5 mL).

Route:

Topical application

Exposure time:

24 hours

Sampling time points: 12 and 24 hours
GLP:

No

Published:

Yes

Experimental design
The possible in vitro penetration of human skin was investigated using sunscreens(described as a novel micronized formulation with ZnO) containing nano-sized ZnO. The
formulations tested included: (A) ZnO dispersion made with 60% siliconate coated ZnO incaprylic capric triglyceride; (B) a typical O/W emulsion sunscreen with 20% ZnO siliconate
coated ZnO in caprylic capric triglyceride; and (C) a control (blank) O/W emulsion
sunscreen without ZnO. Particle size determination was carried out before application of thecoating using four different techniques identified as transmission electron microscopy (TEM),
Brunauer-Emmett-Teller nitrogen-gas absorption method (BET), X-ray diffraction (XRD) andphoton correlation spectroscopy (PCS). Human skin samples were from females following
abdominoplasty. Epidermal samples were prepared from full thickness tissue. These
membranes were mounted in static, horizontal Franz-type diffusion cells with an exposed
surface area of approximately 1.3 cm2. Treatment with the formulations A (n=8), B (n=8)
and C (n=3) involved application of 10 μL/cm2 and collection of receptor fluid samples at 12
and 24 hour intervals. The receptor fluid was analyzed for the presence of zinc by ICP-MSand electron microscopy (EM) was used to examine the tissue samples.
Results and conclusion
The analysis of particle size generation following MCP technology (high-energy dry milling)
revealed similar results to the detection methods; TEM (15-40 nm), BET (30 nm), XRD (26nm) and PCS (30 nm).
Results indicated penetration (over 24 hours) of zinc into the receptor fluid from untreatedand placebo treated epidermal membranes as; 0.09 ± 0.04 and 0.22 ± 0.12 μg/cm2,
respectively. The amount of zinc found in the receptor fluid following application of the twotest formulations was higher (although not statistically significant) after 24 hours exposureand the total amount found in the receptor phase was less than 0.03% of the applied dose.
Analysis with electron microscopy revealed penetration of ZnO nanoparticles limited to theouter surface of the stratum corneum (SC) and loose, desquamating cells of the upper SC
only. There was no evidence of penetration of nanoparticles in the lower SC layers or viableepidermis.

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Comment
For the ZnO used in this study, information on surface area and number of particles in dose
was not provided. Information on dose expressed as surface area and number of particles
was not provided.
In this publication, siliconate-coated ZnO was investigated as a 60% dispersion and as a
typical sunscreen preparation containing 20% ZnO. The Zn in the receptor fluid wasdetermined by elemental analysis of Zn using ICP-MS. Thus, it was not determined whether
the low amount of Zn detected in the receptor fluid was present as solubilized Zn ions or as
ZnO particles.
In the publication, the penetration of Zn++ using sunscreen formulations containing nanoparticulate ZnO is reported to be very low (40–10 times lower) compared to other cosmetic
formulations in the literature containing ionic zinc. It is stated in the publication that this
supports the idea that nanoparticle formulation decreases the absorption of ZnO across theskin which is suggested to be due to a reduction in the amount of available solubilized Zn on
the skin surface.
Based on the detection of Zn in the receptor fluid, the SCCS considers that 0.03% of theapplied dose may be absorbed after topical application of ZnO on the skin.

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Exploratory in vitro study for percutaneous skin
Study Design
Date of report:

2008

Guideline/method:

Exploratory study on human skin penetration of sunscreen
nanoparticles in vitro.

Test system:

Human skin (excised abdominal or breast human skin from plastic
surgery).

Test substance:

Commercial sunscreen (Dr. Lewinn’s private formula (19% W/W) with
26–30 nm mean size ZnO particles suspended in caprylic capric
triglycerides (preservatives: 0.3% phenoxyethanol, 0.3%hydroxybenzoate).

Dose applied:

0.3 g of commercial product (see comment below).

Route:

Topical application

Exposure time:

2–24 hours

GLP:

No

Published:Yes

The distribution of topically applied nano-sized ZnO (mean primary particle size: 26–30 nm)
in excised human skin after application of a commercial sunscreen was examined using
multiphoton microscopy (MPM) imaging with a combination of scanning electron microscopy(SEM) and an energy-dispersive X-ray (EDX) technique. Abdominal or breast skin obtained
following plastic surgery was used.
Results and conclusion
The cross-sectional imaging showed no evidence of nano-sized ZnO penetration into thecells or extracellular space.
(Reference: 120)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
It is not stated in the publication whether the ZnO particles were coated or uncoated, butthey appear to have been uncoated.
This paper also includes an in vivo experiment in humans (see 3.3.4.3). In the in vivo study
0.3g of the sunscreen formulation was applied to a skin surface of 50 cm2 and rubbed in for
5 minutes. From the study description, it is not clear if an equivalent dosing was used in thein vitro experiment.
3.3.4.2

In vitro studies for dermal penetration of non-human skin

Exploratory in vitro study for percutaneous skin penetration
Study Design
Date of publication:

2006

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Guideline/method:

OECD 428 and Guidance Document No. 28 and EU SCCNP opinion
SCCNP/0750/03.

Test system:

Porcine skin

Test substance:

ZnO (Z-COTE uncoated), mean size of 80 nm (90% of the particles
had a size below 160 nm).

Batch:

Not stated

Dose level:

Test formulation 4 mg/cm2, ZnO 400 μg/cm2 (Zn 360 μg/cm2).
Skin preparation:
Skin from the lateral abdominal region of fivemonth old domestic pigs of the Pietrain-Deutsche Landrasse-Hybrid
strain, dermatomed to a thickness of around 500 µm.

Cells:

Eight intact skin preparations, each from three pigs + two vehicle
controls + two untreated controls from each animal.

Skin temperature:

32 ± 1°C

Test chamber:

Static diffusion cells (Franz-type).

Route:

Topical application

Exposure time:

24 hours

Sampling time points: 3, 6, 12 and 24 hours
GLP:

Yes

Published:Yes

The study was performed to evaluate possible skin penetration in vitro using porcine skin.
Full skin thickness (epidermis and dermis) samples were incubated with ZnO preparations inmodified Franz static dermal penetration cells.
The zinc oxide formulation was a white viscous oil/water emulsion, containing 10.3 wt%
zinc oxide (corresponding to 8.3 wt% of zinc) as in an actual sunscreen formulation. The ZCOTE, uncoated microfine zinc oxide, had a mean primary particle size of 80 nm with 90%of the particles being <160 nm. Primary particles and loose agglomerates were present
exclusively in the water phase and primary particles were often arranged around oil
droplets.
ZnO test formulations were applied on the skin and samples of the receptor fluid wereobtained at 3, 6, 12 and 24 hours. After 24 hours incubation the skin samples were
removed and the presence of ZnO on the surface was evaluated using tape stripping. Znlevels in the samples (pooled tape strips, skin remaining after tape stripping, and allretained receptor fluid samples) were evaluated using Atomic Absorption Spectroscopy(AAS) or Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS) depending on theconcentration of the samples.
Results
The total Zn recoveries ranged from 102% to 107% with almost the total amount recoveredin the first five tape strips.
The amounts of zinc found in the skin membrane and the receptor fluid were comparable inuntreated, vehicle treated or zinc oxide treated skin preparations. In the receptor fluid, thelevels observed for Zn for the skin samples of the three pigs were 0.8%, 0.8% and 1.4% of
the applied dose, respectively. The absorbed dose was 1.5%, 1.6%, and 2.3% of theapplied dose. Background levels in the receptor fluid of vehicle treated skin were similar for
samples from three different pigs. In the skin, the Zn levels were 1.4%, 1.5% and 1.5%,

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respectively. Untreated or vehicle treated skin preparations contained 3–5 μg of zinccorresponding to about 1% of the applied dose.
(Reference: 48)
Conclusion
From the data, the authors concluded that there was no penetration of ZnO particles orsolubilized Zn through the stratum corneum of pig skin.
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
The mean size of the ZnO (Z-COTE uncoated) under investigation was 80 nm. It is unclear
whether this mean size given is mass-based or number-based. It is also not known how thematerial used in this test relates to the Z-COTE formulation presented in the dossier as thesize information given differs.
Exploratory in vitro study for percutaneous skin penetration
Study Design
Date of publication:

2011

Guideline/method:

Exploratory percutaneous skin penetration study in vitro after UVB
radiation in vivo (sunburn simulation).

Test system:

Skin of weanling Yorkshire pigs.

Test substances:

O/W sunscreen formulations.
A: Z-COTE HP1 (5% coated, CM 643; mean size 140 nm; SSA 12–24m2/g).
B: Z-COTE (5% uncoated, CM 644; mean size 140 nm; SSA 12–24m2/g).

Batch:

Not stated (source: BASF SE, Germany).

UVB exposure:Fiber optic UVB lamp (Lightningcure 200 UV-Spot light), on the day
the pig was sedated and the hair clipped. The minimal erythemic dose(MED) was determined by sequential exposure to UVB light (30–120
mJ/cm2, 6 – 24 seconds). On day 2 the pig was sedated and theexposed sites were analyzed to determine the UVB dose required to
produce a minimal erythema response. The MED was determined torange between 40 and 50 mJ/cm2.

UVB dose:

100, 110 and 120 mJ/cm2 (to obtain a +2 erythema)

Dose level:

50 μL of each formulation on 0.64 cm2 dermatomed pig skin
A 2.5 mg dose of ZnO corresponds to a dose of 0.03–0.06 m2,
surface area on 0.64 cm².

Skin preparation:

Dermatomed pig skin (400–500 μm) biopsied 24 hours after thesecond UVB exposure inducing sunburn (= consistent +2 erythema).

Cells:

Formulation A and B: four with UVB exposed skin, two with
unexposed skin.

Control:

Two with UVB exposed skin, two with unexposed skin.
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Skin temperature:

37°C

Test chamber:

Flow-through diffusion cells.

Route:

Topical application

Exposure time:

24 hours

Sampling time points: Every 2 hours for the first 12 hours, every 4 hours thereafter up to
24 hours.
Examinations:Light microscopy (LM)Transmission electron microscopy (TEM) plus X-ray microanalysis(EDS)
Scanning electron microscopy (SEM)
Time-of-flight secondary ion mass spectrometry (TOF-SIMS)

GLP:

No

Published:

Yes (abstract)

The purpose of this study was to determine whether skin damaged by UVB radiationinducing moderate sunburn with a +2 erythema reaction enhanced the penetration of TiO2
or ZnO nanoparticles present in these sunscreen formulations. The pig was sedated andmultiple sites (about 52) on the back were exposed to the UVB dose that caused a
consistent +2 erythema (a pale red in a defined area of the skin). Twenty-four hours after
UVB exposure, the pig was sedated, sites visually analyzed for consistency, and the skinprepared for in vivo or in vitro studies. For the in vitro studies, the UVB exposed and non
exposed sites were dermatomed to a thickness of approximately 400–500 μm. The
dermatomed skin was mounted in the flow-through diffusion cells with a dosing area of 0.64
cm2 and maintained at 37°C. The skin was dosed with 50 μL of each formulation (CM 643:
n=4 UVB exposed skin, n=2 unexposed skin; CM 644: n=4 UVB exposed skin, n=2
unexposed skin; and control: n=2 UVB exposed skin, n=2 unexposed skin). Uponcompletion of dosing, the perfusion was resumed and the perfusate collected every 2 hours
for the first 12 hours and every 4 hours thereafter up to 24 hours. After 24 hours, theperfusion was terminated and the skin was removed from the diffusion cells. The dose site
was removed with an 8 mm biopsy punch and cut into thirds. One third was placed in
Trump’s fixative and stored at 4°C for later processing by light microscopy (LM; flowthrough 1 and 2 only) and transmission electron microscopy (TEM). The remaining third of
the skin was cut in half and immediately frozen and stored at -20°C for later elemental
analysis. The vials containing perfusate from each timed collection were capped and thesamples immediately stored at 4°C.
Results
Light microscopy showed that UVB exposed skin showed focal intracellular epidermal
edema, sunburn cells, dermal inflammation and focal microblister. In unexposed (UV light)
skin residual sunscreen containing ZnO was limited to the stratum corneum. The
morphology of the normal and the UVB exposed skin was not affected by topical treatment
with the sunscreen formulations. The ZnO in each formulation was confirmed by TEM andelemental analysis. Energy dispersive X-ray spectroscopy (EDS) confirmed the presence of
Zn indicating ZnO (from sunscreen) and Cu (from sample grids) in CM 643 and CM 644. In
the pig skin from the flow-through studies, TEM/EDS found ZnO nanoparticles only on thesurface of the stratum corneum on both the unexposed and UVB exposed skin, and firstlayers of the stratum corneum. Elemental analysis on the SEM confirmed the presence of
ZnO within the stratum corneum and epidermis of the skin in the sunscreen treatedsamples. TOF-SIMS data indicated that ZnO did focally penetrate into the epidermis in boththe normal and photo-damaged skin treated with the sunscreen formulations. However,48

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background Zn interference was present in some of the map overlays as determined by aregion of interest analysis and the authors were not sure what caused the background insome samples.
Conclusion
Under the conditions of this study, UVB damaged skin did not enhance the penetration of Zn
nanoparticles. TOF-SIMS indicated that Zn focally penetrated into the epidermis in both theUVB exposed and unexposed skin treated with the sunscreen formulations, but theobservation remained inconclusive as background interference was also present. It wasdemonstrated by SEM and TEM that ZnO nanoparticles remained on the surface and upper
stratum corneum layers in UVB exposed and unexposed skin, while ZnO particles were
found to penetrate into the stratum corneum by TEM and Zn into the epidermis asdemonstrated by TOF-SIMS. In any case, there was no evidence from these optical methods
that the nanoparticles penetrated into the perfusate.
(References: 75, 76, AR18)
Comment
For the ZnO used in this study information on number of particles per mass was notprovided. Information on dose expressed as number of particles was not provided.
It is not clear whether the mean size given is mass-based or number-based. It is also not
known how the material used in this test relates to the Z-COTE formulation presented in thedossier as the size information given differs.
Exploratory in vitro study for percutaneous skin penetration
Study Design
Date of publication:

2009

Guideline/method:

Exploratory percutaneous skin penetration study in vitro usingchemical enhancers for skin penetration.

Test system:

Skin of ten month old nude mice.

Test substances:

Purpose made ZnO nanoparticles, size ~10 nm as determined by
TEM.

Batch:

Purpose madeChemical enhancers: Ethanol (EtOH) and oleic acid (OA) as enhancers for altering stratum
corneum lipid structure; control sample, PBS.
Dose level:

50 μL of each formulation on 0.64 cm2 dermatomed pig skin.

Skin preparation:

Removal of subcutaneous fat and mounting skin in Franz diffusion
cells.

Test chamber:Franz diffusion cells

Route:

Topical application with 400 μL of donor solution.

Exposure time:

12 hours

Examinations:

Dual channel multi photon microscopy

GLP:

No

Published:

Yes

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This study investigated mechanistical aspects of chemically enhanced penetration into thestratum corneum using skin samples of four ten month old nude mice (BALB/c). ZnOnanoparticles were prepared from commercial zinc acetate dehydrate with a primary particle
diameter of around 2 nm. However, an average particle diameter of 10 nm was measured.
The absorption and emission peaks were reported at 370 and 525 nm. The non-linear
polarization effect of second harmonic generation (SHG) was used for ZnO nanoparticles tobe distinguished from the autofluorescence (AF) of the stratum corneum (SC) using dualchannel multi-photon microscopy. The study combined the SHG of ZnO nanoparticles andthe AF of the SC to image the transdermal delivery of ZnO nanoparticles under the chemicalenhancer conditions of oleic acid (OA), ethanol (EtOH) and oleic acid–ethanol (OA–EtOH). In
addition to qualitative imaging, the microtransport properties of ZnO nanoparticles were
quantified to give the enhancements of the vehicle to skin partition coefficient (K), the SHG
intensity gradient (G) and the effective diffusion path length (L).
Results
The multilamellar lipid regions between the corneocytes were the pathways for ZnO
nanoparticle delivery. With oleic acid, the transport of ZnO nanoparticles into the stratum
corneum was facilitated by the phase-separated oleic acid domains, while the ethanol
enhancer leached a significant amount of non-covalently bound amphiphilic stratum
corneum lipids to modulate the skin barrier. As regards the oleic acid–ethanol donor
solution, the increase of stratum corneum lipid fluidity associated with the oleic acidenhancer was offset by the effect of ethanol in loosening the stratum corneum lipid
structure. Among the different chemical enhancer conditions, the oleic acid–ethanol
combination was regarded as the most effective donor solution in transdermal delivery of
ZnO nanoparticles into the stratum corneum.
Conclusion
The results showed that OA, EtOH and OA–EtOH were all capable of enhancing thetransdermal delivery of ZnO nanoparticles compared to the PBS control by increasing theintercellular lipid fluidity or extracting lipids from the stratum corneum.
(Reference: 66)
Comment
For the ZnO used in this study information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
Apparently only penetration into the stratum corneum, but not into deeper skin layers wasinvestigated.
3.3.4.3

In vivo studies for dermal absorption

Exploratory in vivo study for percutaneous skin penetration
Study Design
Guideline/method:

Comparative study according to internal laboratory methodology
considering real use conditions, recommendation of US FDA and
COLIPA SPF requirements.

Species:

HumanGroup sizes:

TiA: eight volunteers (normal skin).
TiB: nine volunteers (normal skin).
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TiHB: eight volunteers (normal skin)
TiB: ten volunteers (stripped skin and occlusive patches).
Six volunteers for basal elemental concentration in the skin.
Test substances:

Commercial products containing coated nano-sized ZnO:
TiA: contained only TiO2
TiB: contained TiO2 plus ZnO.
TiHB: contained coated rutile TiO2, size 20 nm.

Particle size:

Spherical ZnO, size 20–60 nm.

Dose applied:

Realistic use condition: 0.5–1.0 mg/cm2 on an area of 25 cm2.

SPF testing method:

2.0 mg/cm2 on an area of 25 cm2.

Skin:

Intact and tape-stripped human skin.

Skin temperature:

37°C

Exposure period:

2 hours (intact skin), 48 hours (tape-stripped skin).

GLP:

No

Date of report:

2009

Published:Yes

The localization and possible skin penetration of nanoparticles dispersed in three sunscreen
formulations under realistic use conditions was comparatively investigated in normal andaltered skin. Commercial products containing nano-sized particles of coated TiO2 and ZnO
dispersed in hydrophobic emulsions were used. One product contained only TiO2 (TiA),
another TiO2 plus ZnO (TiB) and a third material (TiHB) contained nanoparticles of thecoated rutile form of TiO2. The size and shape of nanoparticles in the three formulations
were inspected with transmission electron microscopy and X-ray microanalysis. The ZnO
nanoparticles were spherical and their size ranged from 20–60 nm. The application took into
account relevant application conditions, such as the quantity of sunscreen applied and theduration between the applications recommended by the US FDA. The protocol consisted ofan open test. The formulations were applied on the sacral region and buttocks for 2 hours
using a sunscreen application rate of approximately 0.5–1.0 mg/cm2 within an area of 25
cm2 to reflect realistic use conditions. This condition was compared to standard FDA and
COLIPA methods for the sun protection factor test (SPF), which recommend a thickness ofsunscreen application of 2.0 mg/cm2. TiB was applied to nine individuals, and TiA and TiHB
to five individuals each. Penetration was also investigated in ten individuals under
exaggerated exposure conditions, such as application to the skin after tape stripping andunder occlusive patches (IQR chamber, 48 hour application). Tape stripping consisted of aseries of strips until the tapes were free of corneocytes. A matched control group consistingof six individuals was used for the determination of basal elemental concentrations in theskin including Zn. Skin punch biopsies of 3 mm diameter were taken after application,quench-frozen and kept in containers until processing. One biopsy was taken from eachvolunteer. Sections of 14 μm thickness were cut from the frozen biopsy in a cryostat at 25°C. Biopsies were mounted in mounting medium for microscopy (OCT™ compound).
Sections were obtained from the non-immersed portion of the tissue, and sectioning
performed from inside to outside to avoid tissue contamination. Tissue integrity and theefficacy of corneocyte removal after tape stripping were checked by preparing intercalarystained sections for optical microscopy purposes. Scanning Transmission Ion Microscopy
technique and Particle Induced X-ray Emission technique were used for detection of thelocalization of the ZnO particles by analyzing the presence of elemental Zn. The minimum
detectable concentration of Zn in the skin was 0.10 μmol/g.

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Results
The coverage of the outer skin layer with the TiB sunscreen formulation was homogeneously
distributed. No differences were observed between the recommended SPF procedure (2.0mg/cm2) and the realistic use condition (0.5–1.0 mg/cm2). Sunscreen formulations
accumulated in skin wrinkles and depressions, as well as infundibulum cavities, but
exogenous Zn remained on the outer layers of the keratinized tissue that enfold the follicle,
i.e. outside the living skin. The penetration profiles of the nanoparticles obtained with thetreated skin groups (TiA, TiB and TiHB) were all similar. The high levels of Zn observed at
the outer layers of stratum corneum sharply decreased within the deeper layers to very low
levels (Zn) in both protocols (SPF and realistic use condition).
By quantitatively integrating the imaged skin areas, the Zn concentration was extremely
elevated in the stratum corneum (exogenous Zn). However, the Zn values decreased tophysiological levels in the subcorneal epidermis and finally remained within a narrowinterval of variation in this compartment with no differences between both application
procedures.
The fit estimate of the Zn concentration decrease confirmed that most of the nanoparticles
were confined to the stratum corneum outer layers and showed no significant penetration
into the living cell layers. At approximately 80% of full stratum corneum thickness, the Zn
concentration decreased to the physiological Zn level of <0.3 μmol/g. For the depth
positions where Zn nanoparticles penetration ended, an estimated error of 10% was
obtained which approximately corresponds to 0.5 μm for both application procedures.
Under non-physiological conditions using occlusive patches, there was no significant
difference in the distribution and penetration depth profiling of the ZnO nanoparticles.
Conclusion
The results of the study demonstrated that profiles and localization of Zn nanoparticles
could be accurately established in vivo. Following the 2 hour exposure period to a nanosized ZnO-containing sunscreen on intact human skin using realistic use and SPF methods,
detectable amounts of this physical UV-filter were only present at the skin surface and in
the upper most stratum corneum regions. Layers deeper than the stratum corneum were
devoid of exogenous Zn, even after 48 hours exposure to the sunscreen under occlusion.
Deposition of ZnO nanoparticles in the openings of the pilosebaceous follicles was also
observed but penetration of nanoparticles into viable skin tissue could not be detected.
(Reference: 46)
Comment
For the ZnO used in this study information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
Exploratory in vivo study for percutaneous skin penetration
Study Design
Guideline/method:

Exploratory study on human skin penetration of sunscreen
nanoparticles.

Species:

HumanGroup size:

Four volunteers (two Caucasian males, one Indian male, one Chinese
female).

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Test substances:

Commercial sunscreen (Dr. Lewinn’s private formula (19% W/W) with
26–30 nm mean size ZnO particles suspended in caprylic capric
triglycerides (preservatives: 0.3% phenoxyethanol, 0.3%hydroxybenzoate).

Dose applied:

0.3 g of commercial product.

Skin area:

50 cm2 (forearm, cheek, shoulder or feet).

Skin:

Cleaned but otherwise untreated skin.

Route:

Topical application

Exposure time:

2–24 hours

GLP:

No

Date of report:

2008

Published:Yes

The distribution of topically applied ZnO to human skin was examined by multiphoton
microscopy (MPM) imaging with a combination of scanning electron microscopy (SEM) andan energy-dispersive X-ray (EDX) technique. A commercial sunscreen product containing
nano-ZnO (mean particle size 26–30 nm) was used in this study. An area of 50 cm2 of skin
was selected on the forearm, cheek, shoulder or feet of four volunteers from different ethnic
backgrounds. The selected area of skin was cleaned with ethanol prior to the investigation.
An amount of approximately 0.3 g of the test sunscreen was applied to the selected skinareas and rubbed in for 5 minutes. Images were generated immediately, 4 hours after
application and 24 hours after topical application. Application of the commercial sunscreen
product was followed by an incubation period of 2 to 24 hours. Sections of treated skin were
either tape-stripped (10–20 times) or left untouched prior to analysis.
Results
The MPM analysis showed that nano-ZnO particles stayed on the stratum corneum (SC) andaccumulated into skin folds and/or hair follicle roots of human skin. The nano-sized ZnO
predominantly remained on the outermost surface within a several-micrometer layer at all
analysis points (at application, and after 4 and 24 hours). No penetration of nano-sized ZnO
into the cells or extracellular space was observed and the 24 hour analysis showed complete
removal of sunscreen from the skin. The test material localized in the hair follicle shaft did
not spread to the neighbouring cells and tissue. High intensity, high resolution analysis(SEM/EDX) confirmed no noticeable presence of nano-sized ZnO in the epidermis. It wasnoted that nano-sized ZnO existed predominantly in an agglomerated phase.
Conclusion
In this study, it was demonstrated that after application of a commercial sunscreen
formulation to human skin, the nano-sized ZnO remained either on the skin surface or
stayed within the stratum corneum.
(Reference: 120)
Comment
For the ZnO used in this study information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
It is not stated in the publication whether the ZnO particles were coated or uncoated.
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Exploratory in vivo study for percutaneous skin penetration
Study Design
Guideline/method:

Exploratory study on human skin penetration of sunscreen
nanoparticles.

Species:

HumanGroup size:

20 volunteers (11 in the nano-group, nine in the non-nano “bulk”
group).

Test substance:

ZnO powder enriched to >99% 68Zn. Half of the stock was used tomake nanoparticles with a final crystallite size of about 19 nm (± 8nm; minimum 3 nm, maximum 60 nm) using a proprietary method
based on high-energy attrition milling. Larger particles were also
prepared with an average crystallite size of 110 nm (± 46 nm;
minimum 25 nm, maximum 284 nm) produced by a modified versionof the same method. The uncoated particles were incorporated into an
oil-water formulation using a commercial process for preparing
sunscreens. Both sunscreens contained ~20% wt/wt 68ZnO particles.

Dose applied:

Mean dose 4.3 mg/cm2 (range 2.8–5.8 mg/cm2) twice daily for fiveconsecutive days (4.6 mg/cm2 for the males and 3.7 mg/cm2 for thefemales).

Skin area:

6 cm2 on the back

Skin:

Back skin

Route:

Topical application

Exposure time:

Sampling at 24 hours each day and at 24 hours after day 5.

Evaluation:

Blood and urine samples, before, after and during the trial.

GLP:

No

Date of report:

2010

Published:

Yes (References: 59, 60, AR10, AR27)

An exploratory dermal penetration study was performed in human volunteers using theisotope approach for tracing potential absorption of Zn from ZnO nanoparticles applied tohuman skin under conditions of normal use. In total, three trials were performed. The firsttrial involved two male subjects with two applications to their backs of a formulation
containing ZnO nanoparticles (with diameters of ~30 nm) enriched to 51% with 68Zn. The
second trial involved a female in addition, and the same formulation was applied twice daily
for five days. Blood was sampled in both trials at regular intervals for up to 126 days. These
trials formed the basis and protocol refinement for the main trial in which particles of ZnOenriched to >99% with 68Zn were incorporated into a different formulation (Reference:
AR10). Two groups, each consisting of ten people of various ages, skin classifications, andrace, participated in the study at a beach. One group of ten volunteers was tested with asunscreen containing nanoparticles of 68ZnO (about 20 nm) – the “nanoparticle” group. The
other group was tested with particles of 68ZnO (>100 nm) – the “bulk” group. Sunscreen
was applied to the backs of the volunteers twice daily for a period of five days and thesubjects experienced a minimum of 1 hour UV exposure in two episodes following sunscreen
application. Blood was sampled twice daily and urine three times daily. Blood and urine
samples were also supplied before the five day beach exposure and in a follow-up period.
Zinc was purified from blood and urine samples by ion exchange procedures. Changes in theisotopic abundance of 68Zn of the purified samples measured by the multi-collector wereused to evaluate the dermal absorption of Zn from the sunscreens.
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Results
The results from the first two trials showed changes of <0.1% in the isotope ratios in blood.
The authors estimated that this limits the dermal absorption to <0.1%. In the beach trial,changes in the 68Zn/64Zn ratio in blood samples for the nanoparticle group ranged from
0.1 to 0.8% at the end of the beach trial and all subjects showed significant increases in theabundance of 68Zn six days after the completion of the trial. The changes in blood samples
for the bulk group were similar to those for the nanoparticle group. Excluding the data for
two outliers, there was no statistically significant difference in dermal absorption for thevolunteers in the nanoparticle and the bulk groups; the mean increase was about 0.4%.
However, in females a significant difference was observed between the nanoparticle and thebulk groups; the nanoparticle group absorbing more Zn. Urine samples showed larger
increases in abundance of 68Zn over the same time intervals, but there was no simple
relationship with changes in blood for the same volunteers. For the pretreatment (zeromeasurement) samples there was a very low variation between the 68Zn content in the
blood between all volunteers. After ZnO exposure, an increase in variation was observed.
An increase in Zn levels was also found in urine. There was a delay in the detection of 68Zn
in the blood as it was detected for the first time at day 2 after the fourth application of thesunscreen in four individuals with a more rigorous follow up evaluation. The increase
continued after stopping the application until at least day 11, which was the last day the
samples were evaluated.
Conclusion
The authors concluded that these results provided evidence that Zn from ZnO particles insunscreen penetrated healthy human skin. An additional factor for the Zn uptake might be
the composition of the sunscreen formulation as it contained isopropyl-myristate, a known
chemical enhancer of skin penetration. The total amounts of Zn absorbed are rather small
when compared to the amounts of natural Zn normally present in the human body. For the
male group with the highest absorption, the amount of Zn isotope tracer originating fromthe applied sunscreen ranged from 8.6–30.8 μg. These small amounts are in contrast to theaverage amount of Zn present in whole blood under physiological conditions (about 12 mg).
It should also be noted that the blood values are very low in relation to the recommended
daily values for the dietary intake of Zn of 8 mg for females and 11 mg for males as
indicated in the paper. Overall, the amount of 68Zn tracer detected in the blood post-trial
represents less than 0.001% of the applied dose. The study did not investigate whether thetranslocating Zn was present as nanoparticles or soluble Zn ions.
Comment
For the ZnO used in this study information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
The important findings of this study are: Zn uptake via healthy human skin wasdemonstrated; the total uptake was very low compared to the normal Zn levels in the body;there was a gender difference with females absorbing slightly more than males; and there
was a delay in detection between application and detection time. The main limitation of thestudy is that it did not investigate whether the 68Zn was absorbed as ZnO nanoparticles or
as 68Zn ions. The SCCS considers that the Zn that originated from the topically applied ZnO
containin sunscreen was only a fraction of the amount of Zn present in the overall blood
zinc pool. The pilot study preceding this exploratory study was published in 2012 (AR27). In
the pilot study on three subjets a delayed absorption of Zn was observed which information
was used for the design of the final study (AR10).
(References: 59, 60, AR10, AR27)
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3.3.5

Repeated dose toxicity

3.3.5.1

Repeated dose (5 days) inhalation toxicity

Exploratory 5-day lung toxicity study
Study Design
Date of publication:

March 2010

Guideline/method:

Exploratory study with inhalation exposures according to OECD
Guideline 412.

Species/strain:

Rat/Wistar

Group size:

17 males per group.

Test substance:a) Z-COTE® HP1 coated with triethoxycaprylylsilane.
b) Pigmentary zinc oxide powder.

Batch:a) CNFC0701 (97.3 g Zn/100 g ZnO)b) S35583-206 (>99.9%, <1 μ)Route:

Inhalation

Concentrations:a) 0, 0.5, 2.5, 12.5 mg/m3, corresponding to 22,126, 87,044,
233,360 particles/cm3 (SMPS measurement during exposure).)Information on dose expressed as surface area was not provided.
b) 0, 12.5 mg/m3, corresponding to 219,031 particles/cm3 (SMPS
measurement during exposure).

Exposure period:

Five days

Frequency of
exposure:

6 hours/day

Type of exposure:Head-nose

Exposure conditions: Head-nose exposure systems: aerodynamic exposure systems (INA
60, volume V ≈ 90 L, BASF SE).
Generator systems:

Solid particle generators and glass cyclonic separators.

Generation procedure:The test substance was used unchanged. For each concentration, asolid particle feeder was used for generating the dust. The control
group was exposed to conditioned air.
Observations:

The examinations were restricted to clinical observation, body weight
data, broncho-alveolar lavage with clinico-chemical and cytologicalevaluation of lavage fluid, gross necropsy and histopathologicalexamination of the respiratory tract.

Recovery period:

About 14 days

GLP:

Yes

Published:No

The purpose of this study was to determine the pulmonary toxicity of Z-COTE® HP1 coatedwith triethoxycaprylylsilane in rats using a short-term bioassay including broncho-alveolar
lavage with clinico-chemical and cytological evaluation of lavage fluid and serum, as well as
pathological examination of the lung and pulmonary cell proliferation measurements. The
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concentration response relationship should be established as well as a potential No
Observed Adverse Effect Concentration (NOAEC). To elucidate the influence of particle size,
pigmentary ZnO power was tested at one concentration, which is comparable to the highconcentration of Z-COTE HP1. Seventeen male Wistar rats per test group and time point
were head-nose exposed to respirable dusts for 6 hours per day, on five consecutive days.
The target concentrations for Z-COTE® HP1 were: 0.5, 2.5 and 12.5 mg/m3 and for
pigmentary zinc oxide, powder < 1μ was: 12.5 mg/m3. A concurrent control group was
exposed to conditioned air. Animals were sacrificed on study days 4 and 25. On eachsacrificing day, nine animals per group were designated for histopathological examination.
Moreover, organ burdens were determined in three animals per group. On study days 7 and28, the lungs of the five animals per group were lavaged, and the broncho-alveolar lavage
fluid (BALF) was analyzed for markers indicative of injury of the broncho-alveolar region. On
exposure days clinical examination was performed before, during and after exposure.
During the post exposure period clinical findings were recorded once on each working day.
The body weight of the animals was determined. The concentrations were produced withbrush particle generators. The dust concentration was determined by gravimetricalmeasurements. Particle size was determined by gravimetrical cascade impactor
measurements, optical particle counter and a scanning mobility particle sizer.
Results
The inhalation of Z-COTE® HP1 coated with triethoxycaprylylsilane for five days resulted inlocal inflammation in the lungs of the rats, indicated by changes in several parameters in
the BALF and histological examinations.
Secondary to the effects in the lung, activation of the draining lymph nodes was noted.
Moreover, minimal to moderate necrosis of the olfactory epithelium was observed. The
effects occurred in a concentration-related manner and were reversible within the recovery
period. Only a multifocal increase in alveolar macrophages was still present at the end of
the recovery period. Similar effects were also observed in the animals exposed topigmentary ZnO powder. At the lowest concentration of 0.55 mg/m3, increased levels of afew mediators in the BALF and in serum were determined. Moreover, minimal (grade 1)
multifocal necrosis of the olfactory epithelium was noted in the nasal cavity in one of the six
animals treated with the lowest dose. Therefore, a No Observed Adverse EffectConcentration (NOAEC) could not be established in this study. The lowest concentration of
0.55 mg/m3 is considered to be the Low Observed Adverse Effect Concentration (LOAEC).
Conclusion
ZnO induced a concentration-related inflammation reaction in the lung which was associatedwith dose-dependent increases in BALF markers. In addition to the inflammation reaction,
necrosis was detected in the lung and the nose. There was no biologically relevant
difference between the nano-sized and pigmentary ZnO. As ZnO is soluble in lung fluid andzinc ions are cytotoxic at higher concentrations necrosis can be attributed to the zinc ionsdissolved from the ZnO particles. Likewise, elevated zinc levels were detected in variousorgans, most likely due to zinc ions dissolved from the ZnO particles. There was, however,no indication of systemic effects.
(References: 30, 71)Comment
For the ZnO used in this study information on surface area and number of particles permass was not provided. Information on dose expressed as surface area was not provided.
At the lowest dose investigated there were minimal alterations as indicated by the presenceof an increased number of alveolar macrophages after the recovery period at day 25.
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3.3.5.2

Repeated dose (28 days) dermal toxicity

Additional study in the open literature
Exploratory in vivo study on repeated dose dermal toxicity
Study Design
Guideline/method:

Exploratory study on repeated dose dermal toxicity of zinc oxide
nanoparticles based on OECD Guideline 410 with modifications fordose levels, biochemical parameters and measurement of collagencontent.

Species:Sprague-Dawley rats

Group size:

Ten animals (five males and five females).

Test substanceZnO nanoparticles were obtained from Nanostructured and Amorphous
Materials Inc., USA, stock no. 5810HT, surface area 50 m250m2/g.
ZnO was obtained from Sigma-Aldrich, product no. ZO385.

Particle sizes:

ZnO nanoparticles; 20 nm according to manufacturer; 63 nm by SEM
evaluation; 224.7 nm by DLS in aqueous solution.

Dose applied:ZnO nanoparticle doses, 75, 180, and 360 mg/kg bw, surface area
3.75, 9, and 18 m2/kg bw.,
ZnO 2,000 mg/kg bw.

Skin area:

10% of the total body surface

Route:

Topical application

Exposure time:

6 hours per day for five days per week for a 28 day period.

Evaluation:

Blood samples on day 29 and for satellite groups at day 42. Skin andtail for collagen content. Gross pathology and organ wet weight.
Histopathology of organs.

GLP:

No

Date of report:

2012

Published:

Yes

In this exploratory study, rats were treated for five days per week at 10% of the total bodyskin area. Animals were treated for a total of 28 days and evaluated for toxicity at day 29.
After a recovery period satellite groups were evaluated at day 42. The dosages administered
were calculated to be similar to the daily uptake of nanomaterial per kg body weight perday. The average uptake (administration) for a woman was determined to be 12 mg/kg bw
using a 2% concentration of ZnO in a sunscreen product, and a recommended use of
36,000 mg per day. Using conversion factors for the rat of 1, 2.5, and 5 and a body weightof approximately 180 g, the calculated doses were: 72 mg/kg bw (rounded to 75 mg/kg bw)
for the low dose; 180 mg/kg bw for the intermediate dose; and 360 mg/kg bw for the highdose. Toxicity was evaluated at days 28 and 42. Hydroxyproline content of the skin and tailwas estimated, and the collagen content was calculated.
Results
No significant changes were observed in the clinical chemistry parameters in both micro andnano zinc oxide treated rats. There were no statistically significant changes in thehematologic parameters compared with the control. A statistically significant increase inclotting time was observed in all treatment groups of nano zinc oxide compared to themicro-sized zinc oxide.
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No gross pathology or histopathological lesions were observed in any of the organs
investigated.
There was a significant decrease in the collagen content of the skin and the tail in all thenano ZnO treated groups of rats compared to the control, as well as with the micro-sizedzinc oxide treated groups. The loss was higher in the skin than in the tail. There was aninverse dose relationship with the higher doses inducing a lower decrease. The decreaseinduced by the micro-sized ZnO (2,000 mg/kg bw) was similar to the decrease of thehighest dose of nano-sized ZnO (360 mg/kg bw). A maximum decrease of more than 50%
was observed in three treatment groups (skin 75 mg/kg bw males, 180 mg/kg bw males,
and tail 75 mg/kg bw males).
Conclusion
The decrease in collagen content of the low dose group of nano ZnO was significant
compared to the high dose group and control group in both the tail and the skin. It wassuggested that this effect was due to potential skin penetration of ZnO nanoparticles due topartial dissolution, followed by induction of reactive oxygen species.
(Reference: AR23)
Comment
For the ZnO used in this study information on number of particles per mass was notprovided. Information on dose expressed as number of particles was not provided.
This is the only study available on repeated dose toxicity in the skin. In general, nosystemic effects were observed. The data indicate a decrease in skin collagen content.
Although statistics were not presented in the paper, the data suggest statistically significant
differences. The inverse dose response relationship for the decrease in the collagen content
cannot be explained and needs further confirmation.
3.3.5.3

Repeated dose (30 days) oral toxicity

No data available.
3.3.5.4

Sub-chronic (90 days) toxicity (oral, dermal)

No data available.
3.3.5.5

Chronic (>12 months) toxicity

No data available.
3.3.6

Mutagenicity/genotoxicity

3.3.6.1

Mutagenicity/genotoxicity in vitro

Bacterial gene mutation assays
Guideline/method:

OECD Guideline 471 (1997)

Species/strain:

Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, TA
1537.

Replicates:

Triplicate plates, two independent experiments (SPT, PIT).

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Test substance:

ZnO (Z-COTE® Max) (ZnO coated withdimethoxydiphenylsilanetriethoxy-caprylylsilane cross-polymer).

Batch:

FCGB0701

Purity:100.4 g/100 g).

Solvent:

DMSO (standard plate test) or fetal calf serum (pre-incorporation
test)

Concentrations:Standard plate test (SPT): 0, 20, 100, 500, 2,500, 5,000 μg/plateboth without and with metabolic activation (S9-mix)).
Preincubation test (PIT): 0, 20, 100, 500, 2,500, 5,000 μg/plate(with/without metabolic activation (S9-mix)).

Treatment:

direct plate incorporation with 48 - 72 h incubation without and with
S9-mixpre-incubation method was used with 20 minutes pre-incubation andat least 48 – 72 h incubation time both without and with S9-mix

GLP:

in compliance

Date of report:

March 2009

The test substance was tested for mutagenicity in the reverse mutation assay on bacteriawith and without metabolic activation (S9-mix prepared from phenobarbital/βnaphthoflavone induced male Wistar rat liver) according to the standard plate test (SPT)and the plate incorporation test (PIT). The Salmonella typhimurium strains were exposed tothe test substance dissolved in DMSO (SPT) or in fetal calf serum (PIT, added to avoid
aggregate formation, and to mimic the protein containing body fluids (e.g. blood)) at
concentrations ranging from 20–5,000 μg/plate. For control purposes, a sterility and solvent(DMSO) and positive controls (NOPD, MNNG, AAC, MIT.C, 2-AA) were also investigated. In
the standard plate test the revertant colonies were counted after incubation at 37°C for 48– 72 hours in the dark; in the preincubation test, the duration of preincubation was about
20 minutes whereas the revertant colonies were again counted after incubation at 37°C for
48 – 72 hours in the dark. Negative and positive controls were in accordance with the OECD
guidelines.
Results
No sign of bacteriotoxicity seen as reduced his- background growth, a decrease in thenumber of his+ revertants, or reduction in the titer, was noted when tested up to thehighest required concentration in the absence and presence of metabolic activation.
Precipitation of the test substance was recorded at and above 2,500 μg/plate with orwithout metabolic activation.
The test substance did not induce a biologically relevant increase in revertant colony
numbers in the bacterial strains at any concentration tested in the presence or absence of
metabolic activation compared to the background control. The sensitivity and validity of thetest system used was demonstrated by the expected induction of a significantly increased
number of revertants with the positive controls.
Conclusion
The test substance did not induce gene mutations in the bacterial strains used either in thepresence or absence of S9-mix up to precipitation concentrations. Thus, it was shown to be
non-mutagenic in this bacterial gene mutation test.
(Reference: 27)

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Comment
For the ZnO used in this study information on surface area and number of particles permass was not provided. For the exposures information on dose expressed as surface areaand number of particles was not provided.
The positive control used represents a generally accepted chemical positive control for thistest and demonstrated its performance. However it does not provide proof that a negative
response of a nanomaterial/nanoparticle is really negative. There is presently no accepted
nanoparticle positive control that demonstrates whether the assay is generally suitable forthe mutagenicity testing of insoluble/poorly soluble nanoparticles.
The behaviour of the ZnO in the test was evaluated as indicated by the observation that
there was precipitation at the two highest concentrations of ZnO investigated. Contact ofbacterial DNA (i.e. nanoparticle uptake by bacteria) with the ZnO nanomaterials was notdemonstrated. Negative findings in the Ames tests have also been shown for coated ZnO(i.e.
tetramethylammoniumhydroxide-capped
ZnO)(Reference:
AR26).
Bacterial
mutagenicity assays are considered to be less appropriate for the testing of nanoparticles
compared to mammalian cell systems (Reference: 72) due to the lack of endocytosis(Reference: AR4).
It is uncertain whether Zn ions that might be available from ZnO nanoparticles may cause
mutagenicity in bacterial assays. Reachable intra-bacterial zinc concentrations will dependon the solubility and dissolution kinetics of ZnO nanoparticles (see section 3.1.6) and theavailable zinc transporter systems. Ionic zinc (e.g. zinc acetate) has tested negative inbacterial mutagenicity tests, while it tested positive in other mutagenicity tests (Reference:
AR24).
The SCCS considers that the results of the used bacterial gene mutation assay can beconsidered negative. However, as there is no certainty on the exposure of the bacterial DNA
to the added ZnO nanoparticles this negative result is of a limited value.
Comet assay in human epidermal cells
Guideline/method:

According to published protocol

Species/strain:

Human epidermal cell line (A431).

Replicates:

Duplicate slides per tissue culture well

Test substance:

ZnO nanopowder

Batch:No dataPurity:>99%), Sigma–Aldrich, St. Louis, MO, USA.

Solvent:

DMEM

Concentrations:0.001, 0.008, 0.08, 0.8, 5 μg/mL.

Exposure:cells were exposed for 6 hours

Sampling:

immediately after the end of treatment

GLP:

Not in compliance

Date of publication:

January 2009

The DNA damaging potential of ZnO nanoparticles was investigated in a Comet assay in ahuman epidermal cell line (A431) according to a previously published protocol. At 24 hours
after seeding, cells were exposed to different concentrations of ZnO nanoparticles (0.001,
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0.008, 0.08, 0.8, 5 μg/mL) for 6 hours. Cell viability was determined with the MTT assay,
LDH relase, NR uptake and trypan blue exclusion. After exposure two slides were preparedfrom each well (one well/concentration) for analysis. These 2 slides were exposed to alkali(pH>10), followed by electrophoresis for 25 minutes at 0.7 V/cm and 300 mA, stained withethidium bromide and scored for comets. The Comet parameters used to measure DNA
damage in the cells were % tail DNA (fraction of DNA in the tail) and Olive tail moment(OTM; arbitrary units, the product of the distance of DNA migration from the body of
nuclear core and the total fraction of DNA in the tail). Images from 50 random cells (25from each replicate slide) were analyzed for each experiment.
Results
The mean hydrodynamic diameter and zeta potential of the nanoparticle suspension indeionized water, as determined by dynamic light scattering (DLS) measurement, was 165
nm and −26 mV, respectively. The average size analyzed by transmission electron
microscopy (TEM) was 30 nm. A decrease in cell viability was noted from a concentration of
0.8 μg/mL and higher at an exposure time of 24 h and 48 h. At an exposure time of 6 h, adecrease in viability occurred at 8 μg/mL and higher. The cell viability in the Comet assay
exceeded 90% for all experimental groups before and after the treatment as assessed by
Trypan blue dye exclusion assay.
A statistically significant and concentration dependent increase in DNA damage wasobserved compared to the control; in cells exposed to ZnO for 6 hours at 5 and 0.8 μg/mL.
ZnO nanoparticles were also found to induce oxidative stress in cells indicated by depletionof glutathione (59% and 51%); catalase (64% and 55%) and superoxide dismutase (72%
and 75%) at 0.8 and 0.08_g/ml respectively.

Conclusion
Under the experimental conditions used ZnO nanopowder induced DNA damage in A431
cells and, consequently, ZnO nanopowder was genotoxic (clastogenic and/or mutagenic) inthese cells. However, the authors emphasized that the observed genotoxic potential in
human epidermal cells may be mediated through lipid peroxidation and oxidative stress.
(Reference: 94)

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Comment
For the ZnO used in this study information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
S9-mix fraction was not mentioned and presumed not used. However, the usefulness of S9mix fraction in assays investigating ZnO nanoparticles may be doubtful as ZnO
nanoparticles will not be metabolised.
The use of two slides from a tissue culture well cannot be considered a replicate of two. It
consists of one single incubation for which two read out slides were used.
The applicant stated that depletion of the anti-oxidative defence systems and cellular
protection mechanisms of mitochondria in situ particularly under stress, is a major
intracellular source of oxidative stress which is obviously no longer controlled by cellularmechanisms and therefore leads directly to DNA damage. This is therefore not related tonanoparticles per se, but is probably due to the imbalance of Zn ions under artificial in vitro
conditions. The applicant thus concluded that the results are a set of cellular effects that are
not linked to ZnO nanoparticles, but to Zn solubility under in vitro test conditions.
The authors suggest that the DNA damaging effects may be attributed to lipid peroxidationand oxidative stress as one of the probable causes. This is a plausible explanation for the
induction of the observed DNA damage. However, the Zn ions were delivered by the ZnO
nanoparticles in this test situation so the damage induced is of relevance for ZnO
nanoformulations, irrespective of the mechanism.
In view of this, the SCCS concludes that the used ZnO nanoparticles were genotoxic in the
comet assay with A431 human epidermal cells.
Additional studies in the open literature
Comet assay in human nasal mucosal cells
Guideline/method:

According to published protocol

Species/strain:

Human nasal mucosal cells obtained from ten surgery patients (threefemale and seven male).

Group size:
Test substance:

ZnO nanoparticles (<100 nm, surface area 15–25 m2/g) and ZnO
powder (<5 μm) were obtained from Sigma–Aldrich (Steinheim,
Germany). Sizes: mean longitudinal diameter of 86 ± 41 nm (mean± SE) and a mean lateral diameter of 42 ± 21 nm (mean ± SE).

Batch:

Not stated

Purity:Not stated

Dose levels:

Diluted nanoparticle or powder suspension at end concentrations of
0.01, 0.1, 5, 10 and 50 μg/mL in the well, corresponding to 0.15–0.25, 1.5–-2.5, 75–-125, 150–-250, 750–1,250-1250 mm2/mL in thewell.

Route:

In vitro

Vehicle:Distilled water

Exposure:Single exposure of 24 hour.
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Sampling:

immediately after the end of treatment

GLP:

Not in compliance

Date of report:

2011

The DNA damaging potential of nano-sized and micro-sized ZnO was investigated, in humanmucosal cells. The cells were exposed to ZnO nanoparticles or ZnO powder dilutions for 24 h
at 37oC in a humidified incubator with 5% CO2. The MTT assay as well as the trypan blue
exclusion test were performed in order to measure cell viability of human nasal mucosa cells
after exposure to ZnO nanoparticles and ZnO powder. The cells were detached from thewells by trypsinization with trypsin–EDTA (0.05% ) and centrifuged at 500g for 5 min. Afterremoval of the supernatant, the cell pellet was resuspended at 105 cells/ml.
Slides were prepared and placed in the ice-cooled Plexiglas gel electrophoresis chamber(distance between electrodes: 30 cm) for 20 min. Electrophoresis was conducted foranother 20 min at 25 V and 300 mA.. The following parameters were analyzed to quantify
the induced DNA fragmentation: tail DNA (TD), tail length (TL), and Olive tail moment(OTM) as a product of the median migration distance and the percentage of DNA in the tail.
The OTM was applied for statistical analysis.
Cells were prepared for TEM for ZnO particle uptake by the cells. Airway Epithelial Cell
Growth-Medium (BEGM) served as negative control, and directly alkylating methyl methanesulphonate (MMS) at 100 μM was used as a reliable positive control of genotoxicity without
cytotoxic effects.
Results
The frequency of cells with intracytoplasmatic ZnO nanoparticles was 10%, while particle
transfer into the cell nucleus could be observed in 1.5% of the cells. While no cytotoxicity or
genotoxicity was observed for the ZnO powder (<5 μm) in the tetrazolium (MTT) assay, inthe trypan blue exclusion test, and in the Comet assay, cytotoxic effects were shown at a
ZnO nanoparticles concentration of 50 μg/mL (P-value <0.01).
In comparison to the control, a ZnO nanoparticles concentration dependent increase in the
Olive tail moment (OTM) as an indicator for genotoxic effects could be seen. The enhanced
DNA migration was statistically significant at 10 μg/mL (P-value <0.05) and 50 μg/mL (Pvalue <0.01).
Also, the proinflammatory cytokine interleukin 8 secretion into the basolateral culture
medium was increased at ZnO nanoparticles concentrations of 5 μg/mL. In contrast, thepigmentary grade ZnO did not induce cytotoxicity (MTT assay) or DNA damage (cometassay).

Conclusion
Under the experimental conditions the used ZnO nanoparticles induced DNA damage inhuman nasal mucosal cells and, consequently, ZnO nanoparticles was genotoxic (clastogenic
and/or mutagenic) in these cells.
Under the same experimental conditions, the used ZnO powder did not induce DNA damage
and, thus ZnO powder was not genotoxic (clastogenic and/or mutagenic) in these cells.
(Reference: AR11)
Comment
For the ZnO used in this study, information on number of particles per mass was notprovided. Information on dose expressed as number of particles was not provided.
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S9-mix fraction was not mentioned and presumably not used. However, the usefulness of
S9-mix fraction in assays investigating ZnO nanoparticles may be doubtful as most ZnO
nanoparticles will not be metabolised.
The SCCS agrees with the conclusion that the used ZnO nanoparticles were genotoxic in the
comet assay with primary human nasal mucosal cells.

Comet assay in human nasal mucosal cells
Guideline/method:

According to published protocol

Species/strain:

Human nasal mucosal cells.

Group size:

Ten patients (seven female and three male).

Test substance:

ZnO nanoparticles (<100 nm, surface area 15–25 m2/g) wereobtained from Sigma–Aldrich (Steinheim, Germany).

Batch:

Not reported

Purity:Not reported

Dose levels:

500 μL of diluted nanoparticle or powder suspension at end
concentrations of 0.1, and 5 μg/mL in the well, corrersponding to
1.5–-2.5, and 75–-125 mm2/mL in the well.

Route:

In vitro

Vehicle:Distilled water

Exposure:

Repeated 3 consecutive exposures of 1 h with a washing step in
betweenObservation period:
GLP:

Not in compliance

Published:

Yes

Study period:

27 hours

Date of report:

2011

To perform repetitive exposures, seven day old mini organ cultures (MOCs) (threedimensional cultures of human nasal mucosa) were incubated with 0.1 and 5 μg/mL of ZnO
NP suspension, without S9-mix, bronchial epithelium growth medium (BEGM), or methyl
methanesulfonate (MMS) for 1 hour followed by a washing step with fresh BEGM. After the
initial exposure step, the first cyto- and genotoxicity assays were performed in four MOCs
for each concentration (control, 0.1 μg/mL, 5 μg/mL, MMS). The other 48 MOCs were again
exposed to ZnO nanoparticles, BEGM, or MMS for another hour. This procedure wasrepeated one more time. Following the third exposure, the remaining 16 MOCs were further
incubated in fresh BEGM for a 24 hour regeneration period. The experiment was performedon cells after 1 h exposure, after two consecutive 1 h exposures, after three consecutive 1 h
exposures, and after three consecutive 1 h exposures followed by a regeneration period of
24 h.
Cells were prepared for TEM for ZnO particle uptake by the cells. Cell viability wasdetermined by the trypan blue exclusion test.
After a 20-min DNA “unwinding’’period, the electrophoresis was performed under standard
conditions (25 V, 300 mA, distance between electrodes 30 cm) for 20 minutes. The
following parameters were analyzed to quantify the induced DNA fragmentation: tail DNA(TD), tail length (TL), and Olive tail moment (OTM) as a product of the median65

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migration distance and the percentage of DNA in the tail. The OTM was applied for statisticalanalysis.
Results
ZnO nanoparticles had an oval shape with a mean longitudinal diameter of 76 ± 41 nm(mean ± SE) and a mean lateral diameter of 53 ± 22 nm (mean ± SE). After the exposure
period it was observed that the ZnO nanoparticles were distributed to the cytoplasm and thenucleus of the cells in some of the cultures, but this was more pronounced for the 5 μg/mL
exposure culture. There was no significant enhancement of DNA migration as determined by
the Comet assay in nasal mucosa cells from MOCs exposed to 0.1 μg/mL of ZnO
nanoparticles. At a ZnO nanoparticle concentration of 5 μg/mL, a significant increase in OTM
was observed after each of one, two, and three consecutive 1 hour exposure periods
compared to the control when measured directly after exposure. At both concentrations of
ZnO nanoparticles, DNA fragmentation increased when measured at a 24 hours
regeneration period after 3 repeated 1 hour exposures. A dose dependent increase in DNA
fragmentation was present.
Conclusion
At both highest concentrations (5 μg/mL and 10 μg/mL) of ZnO-nanoparticles, DNA
fragmentation increased after 24 hr of regeneration. In contrast, DNA damage which was
induced by the positive control, methyl methanesulfonate, was significantly reduced after
24-hr regeneration.The authors concluded that the results suggest that repetitive exposure
to low concentrations of ZnO nanoparticles results in persistent or ongoing DNA damage.
(Reference: AR12)
Comment
For the ZnO used, information on number of particles per mass was not provided.
Information on dose expressed as number of particles was not provided.
Although it was suggested that a repeated exposure was used, the exposure was actuallyeither 1, 2 or 3 hours with only a minimal non-exposed period with a washing step between
the exposures.
S9-mix fraction was not mentioned and presumably not used. However, the usefulness of
S9-mix fraction in assays investigating ZnO nanoparticles may be doubtful as most ZnO
nanoparticles will not be metabolised.
The SCCS agrees with the conclusion that in this in vitro study, the ZnO exposure resulted
in DNA damage. There fore, the ZnO was genotoxic in the used comet assay with humanmucosal cells.

3.3.6.2

Mutagenicity/genotoxicity in vivo

Mouse Micronucleus Assay
Guideline/method:

OECD Guideline 474 (1997), EC Regulation 440/2008

Species/strain:

Mouse/NMRI

Group size:

Five male mice per dose group/sacrifice time.

Test substance:

ZnO (Z-COTE® HP1) (ZnO coated with triethoxycaprylylsilane).

Batch:

CNFC0701

Dose levels:

0, 15, 30, 60 mg/kg bw
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Application volume:

10 mL/kg body weight.

Exposure:Single application

Route:

Intraperitoneal (ip) injection

Vehicle:Fetal calf serum (FCS).

Sacrifice Times:

Micronucleus test: 24 hours post-dose (vehicle, positive controls, low,
mid and high) and 48 hours post-dose (vehicle and high dose only).

Positive controls:

Cyclophosphamide (CPP): 20 mg/kg bw, vincristine sulfate (VCR):
0.15 mg/kg bw.

GLP:

Yes

Date of report:

August 2009

The potential of the test substance to cause chromosomal damage or spindle poison effects
in vivo was investigated in the mouse bone marrow micronucleus test. Based on the resultsof a dose range-finding study, where mortality was observed at and above 80 mg/kg bw
and where 60 mg/kg bw led to distinct clinical signs with no differences between sexes,
each of the five male NMRI mice per dose group received a single intraperitoneal injectionof the test substance suspension at 15, 30 or 60 mg/kg bw. Five male mice were used ascontrols receiving the vehicle (fetal calf serum), while each of the five positive control micereceived 20 mg/kg bw cyclophospamide/kg bw or 0.15 mg/kg bw vincristine sulfate. In eachcase, the application volume was 10 mL/kg bw. All animals were observed for clinical signsof intoxication at regular intervals throughout the study period.
Bone marrow cells were harvested for evaluation of micronuclei at 24 hours post-dose(vehicle, positive controls, low, mid and high) and 48 hours post-dose (vehicle and highdose only).
Bone marrow for micronuclei examination was prepared, spread on a glass slide, fixed andstained with eosin and methylene blue, rinsed followed by staining with Giemsa solution and
fixed in Corbit Balsam. Using a light microscope, the slides were evaluated. Two-thousand
polychromatic erythrocytes (PCE) per animal were scored for the presence of micronuclei(10,000 per treatment group). The number of normochromatic erythrocytes (NCE) was alsoscored. The results obtained for the test groups, the negative and positive controls werecompared to historical control data for validation purpose.
Results
The formulation analysis showed the correctness of the injected dose level and stability inthe vehicle. The single intraperitoneal injection of the test substance preparations led to adose-dependent increase in distinct clinical signs, while the animals of the negative andpositive control groups revealed no findings. There were no statistically significances or
biologically relevant differences in the number of erythrocytes containing micronuclei either
between the vehicle control groups and the three dose groups, or between the two sacrificeintervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes
containing small micronuclei or large micronuclei did not deviate from the vehicle control
values at any of the sacrifice intervals and was within the historical vehicle control data
range. Both of the positive control substances, cyclophosphamide and vincristine sulfate,induced a statistically significant increase in the number of PCEs containing small and/or
large micronuclei within the range of (or above) the historical positive control data
indicating the suitability and sensitivity of the test system.
Conclusion

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Opinion on zinc oxide (nano form)___________________________________________________________________________________________The test substance did not induce an increase in the number of polychromatic erythrocyteswith micronuclei in the bone marrow of intraperitoneally treated mice and did not lead toany impairment of chromosome distribution in the course of mitosis. Thus, there was no
indication for a clastogenic potential or any aneugenic activity in vivo under the conditionsof the study.
(Reference: 26)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
Intraperitoneal administration is proposed in OECD Guideline 474 but the text also states,“If there is evidence that the test substance, or a reactive metabolite, will not reach thetarget tissue, it is not appropriate to use this test”. For intraperitoneal ZnO, exposure ofbone marrow to the nanoparticles has not been demonstrated, although it can be assumed
that Zn ions released from the ZnO nanoparticles may have resulted in exposure of thebone marrow. The value of this test is thus limited.
The SCCS disagrees with the conclusion of the authors. This assay does not prove that ZnO
nanoparticles lack clastogenic or aneugenic activity in vivo as exposure of the target organs
was not demonstrated.

3.3.7

Carcinogenicity

No data available.

3.3.8

Reproductive toxicity

No data specific for ZnO nanoparticles have been submitted or identified from the open
literature.
3.3.9

Toxicokinetics

Additional studies in the open literature
No specific data for ZnO nanoparticles have been submitted. However, an exploratory study
was recently published indicating systemic availability of Zn after ZnO nanoparticles wereadministered orally and intraperitoneally.
ZnO nanoparticles were approximately 50 nm in size (TEM evaluation), which werecompared to ZnO microparticles showing at least one diameter >100 nm (TEM evaluation).
DLS evaluation for the ZnO microparticles showedi an average hydrodynamic diameter of
1,226 ± 120 nm, and for the ZnO nanoparticles an average hydrodynamic diameter of 93 ±14 nm was reported. Information on surface area and number of particles per mass was notprovided.
After oral and intraperitoneal administration of a single dose of 2.5 g/kg bw for both ZnO
nanoparticles and microparticles, Zn could be observed in serum indicating uptake from theGI–tract. For ZnO nanoparticles the systemic availability was somewhat higher compared tothat of ZnO microparticles as indicated by Zn measurements by ICP-MS. A higher
distribution of Zn in the liver, spleen and lung was shown after treatment with ZnO NP
compared to treatment with ZnO microparticles. Serum liver enzyme analysis (aspartate
aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH))
indicated liver toxicity due to both ZnO NP and ZnO MP treatments which was confirmed by68

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histopathology. Histopathological lesions were only observed for ZnO NP treatments in theliver.
(Reference: AR16)
Comment
The results indicate a similar uptake, tissue distribution, and toxicity for ZnO nanoparticles
and ZnO microparticles. As Zn was measured by ICP-MS, it cannot be concluded whether it
was present as particulate material or as dissolved Zn ions. Although serum levels of
aspartate aminotransferase, alanine aminotransferase, and lactyate dehydrogenase
indicated similar liver toxicity for both ZnO nanoparticles and ZnO microparticles,
histopathological lesions were only observed for ZnO nanoparticles in the liver.
3.3.10 Photo-induced toxicity

3.3.10.1

Phototoxicity/photoirritation and photosensitisation

Since submission I (2001) for ZnO, which served as a basis for the former opinion (SCCNFP
opinion dated 24-25 June 2003, SCCNFP/0649/03, Reference: 87) and submission II(2005), no further studies have been performed or provided by the sponsors.
The studies presented in opinion SCCNFP/0649/03 (Reference: 87) did not indicate aphotoirritant or photosensitizing potential of micronized ZnO (not further specified).
3.3.10.2

Phototoxicity/photomutagenicity/photoclastogenicity

Photomutagenicity in a Salmonella typhimurium Reverse Mutation Assay
Study Design
Date of report:

August 2006

Guideline/method:

Considerations on photochemical genotoxicity: Report of theInternational Workshop on Genotoxicity Test Procedures Working
Group, 2000; Japanese Ministry of Health and Welfare: Guidelines forgenotoxicity studies of drugs, 1999.

Species/strain:

Salmonella typhimurium TA1537, TA98, TA100, TA102.

Replicates:

Triplicate plates

Test substance:

FINEX-50 ZnO (supplied by Sakai Chemical Industry Co., primaryparticle size: 20 nm, non-coated).

Batch:
Concentrations:

OZ52 (purity: ≥96% as indicated in the submission)
Dose-ranging test: 5, 15, 50, 150, 500, 1,500, 5,000 μg/plate(with/without light irradiation).

Main test:

313, 625, 1,250, 2,500, 5,000 μg/plate (with/without lightirradiation).

Vehicle:DMSO

Irradiation:
Source of light:

Intensity ofirradiation:

Sunlight simulator (SOL 500) equipped with a metal halide lamp withemission of a continuous spectrum of simulated sunlight (50% at 335
nm wavelength).
1.6–1.7 mW/cm2
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Bacterial

Positive controls:

Bacterial Strains

UVA dose (mJ/cm2)

TA 1537 (8 minutes 20 seconds)

0.80–0.85

TA 98 (8 minutes 20 seconds)

0.80–0.85

TA 100 (8 minutes 20 seconds)

0.80–0.85

TA 102 (50 minutes)

4.8–5.1

Without irradiation

TA 100: AF-2, 0.01 μg/plate
TA 1537: 9AA, 80 μg/plate
TA 98: AF-2, 0.1 μg/plate
TA 102 MMC, 0.05 μg/plateWith irradiation:

TA 98, TA 100, TA 1537: CPZ, 1.0 μg/plate
TA 102: 8-MOP, 0.05 μg/plate

GLP:

No

Published:No

The test substance was investigated for its potential to induce gene mutations underirradiation with simulated sunlight according to considerations on photochemical
genotoxicity using the Salmonella typhimurium strains TA 1537, TA 98, TA 100, and TA
102. The tests were performed in the absence of S9 mix. Each concentration, including thecontrols, was tested in triplicate and at concentrations of 5, 15, 50, 150, 500, 1,500, 5,000
μg/plate in the dose range-finding part of the study and concentrations of 313, 625, 1,250,
2,500, 5,000 μg/plate in the main study in the absence of metabolic activation (S9 mix),
under the light irradiation or without light irradiation. The test substance was dissolved in
DMSO. Petri dishes containing the test substance suspension, test bacterial suspension andphosphate buffered saline were either irradiated with a sunlight simulator (SOL 500) or
remained not irradiated. The irradiation time was the treatment time. After the treatments,
aliquots of the mixtures were dispensed into test tubes, mixed with top agar, and spread on
the minimum glucose agar plates. The plates were incubated at 37°C for 48 hours andrevertant colonies on the plates were counted.
Results
The dose-range finding study was conducted at 7 dose levels, i.e., 5, 15, 50, 150, 500,
1500 and 5000μg ZnO/plate. Growth inhibition was not observed in each tester strain atany doses in both the light irradiated group and the light-unirradiated group. The plates
incubated with the test item showed normal background growth up to 5,000 μg ZnO/platein all strains used. In the main test no substantial increase in revertant colony numbers of
any of the four tester strains was observed following treatment with the test substanceunder irradiation with simulated sunlight at any dose level. Also in the absence of irradiation
no effects were found. In both the dose–range finding study and the main test, theprecipitation of FINEX-50 zinc oxide was seen on the plate at 5000 and 2500
μg/plate in both the light-irradiated group and the light-unirradiated group
The sensitivity and validity of the test system used was demonstrated by the expectedinduction of a significantly increased number of revertants with the appropriate positive
controls.
Conclusion

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Opinion on zinc oxide (nano form)___________________________________________________________________________________________The test substance did not induce gene mutations both in the presence and absence of
irradiation. It was therefore considered to be non-photomutagenic in this Salmonella
typhimurium photomutagenicity test.
(Reference: 98)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
The purity of the FINEX-50 used was indicated in the submission to be ≥96%. However,data on the purity is not provided in the physico-chemical characterization of the FINEX-50(section 3.1.4), and was also not provided in the study reference 98.
S9-mix fraction was not used. However, the usefulness of S9-mix fraction in assays
investigating ZnO nanoparticles may be doubtful as ZnO nanoparticles will not be
metabolised.
Contact of bacterial DNA (i.e. nanoparticle uptake by bacteria) with the ZnO nanomaterials
was not demonstrated. This test has been considered inappropriate for nanoparticles
because bacteria do not take up nanoparticles due to the lack of endocytosis (Reference:
AR4). However, Released Zn ions might be available as suggested for in vitro exploratory
Comet assay.
The SCCS considers that the results can be considered negative in the usedphotomutagenicity bacterial gene mutation assay. However, as there is no certainty on the
exposure of the bacterial DNA to the added ZnO nanoparticles, this negative result is of alimited value.
Photo-chromosomal aberration test with cultured mammalian cells
Study Design
Date of report:

August 2006

Guideline/method:

Considerations on photochemical genotoxicity: Report of theInternational Workshop on Genotoxicity Test Procedures Working
Group, 2000; Japanese Ministry of Health and Welfare: Guidelines forgenotoxicity studies of drugs, 1999

Species/strain:

Chinese Hamster lung fibroblasts (CHL cells).

Replicates:

Duplicate experiments.

Test substance:

FINEX-50 ZnO (supplied by Sakai Chemical Industry Co., primaryparticle size: 20 nm, non-coated).

Batch:OZ52 (purity: ≥96% as indicated in the submission)

Concentrations:

Growth inhibition test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5,
625, 1,250, 2,500, 5,000 μg/mL (with/without light irradiation).

Chromosomal
aberration test:

2.44, 4.88, 9.77, 19.53 μg/mL (with/without light irradiation).

Vehicle:0.5% aqueous carboxymethyl-cellulose solution.

Irradiation:

71

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Source of light:

Sunlight simulator (SOL 500) equipped with a metal halide lamp withemission of a continuous spectrum of simulated sunlight (50% at 335
nm wavelength).

Intensity ofirradiation:

1.6–1.7 mW/cm2

UV doses:

4.8–5.1 J/cm2

Preincubation time:

60 minutes

Irradiation time:

50 minutes

Recovery:After irradiation reaction solutions were removed and the cells were
further cultured for another 22 hours.

Negative control:

0.5% aqueous carboxymethyl-cellulose solution.

Positive controls:

With irradiation: 8-Methoxypsoralene (8-MOP), 0.01 μg/mL.
Without irradiation: MNNG, 2 μg/mL.

GLP:

No

Published:No

The cytotoxicity of the ZnO test substance was evaluated by measuring cell density. The celldensity was determined as a percentage to the untreated control value (100%) bymeasuring the degree of staining on each dish with a single-layer culture cell densimeter.
On the basis of the measured values, an approximate 50% inhibitory concentration (IC50)of the test substance was estimated. This assay was conducted in duplicate.
The test substance was investigated for its potential to induce structural chromosomal
aberrations in Chinese Hamster lung fibroblasts (CHL cells) in the absence and the presenceof simulated sunlight in two independent experiments. A sunlight simulator (SOL 500)
equipped with a metal halide lamp with emission of a continuous spectrum of simulated
sunlight (50% at 335 nm wavelength) was used as a light source. The cultures were preincubated with the test item for 60 minutes. After pre-incubation, the cultures were exposedto the solar simulator at an irradiation intensity of 1.6–1.7 mW/cm2 resulting in a UVA doseof 4.8–5.1 J/cm2. Thereafter, the cultures were washed. Corresponding cultures with thetest item were kept in the dark for the 50 minute exposure period. The chromosomes wereprepared 22 hours after washing. Two parallel cultures were investigated and at least 100
metaphase per plate were scored for structural chromosome aberrations in each culture.
Results
Cell growth inhibition test
The cell growth ratio was affected by precipitation of the test substance at 312.5 μg/mL andabove in the cultures without irradiation, while no precipitation was observed at 156.25
μg/mL and below. The cell growth ratios by test substance were reduced to 31% at 19.5
μg/mL and 77% at 9.8 μg/mL. In the irradiated cultures, the cell growth ratio was affectedby precipitation at 312.5 μg/mL and above, while no precipitation was observed at 156.25
μg/mL and below. Thus, concentrations of 19.5 μg/mL (as the maximum concentration),
followed by serial 2-fold dilution to give 2.4, 4.9, 9.8 and 19.5 μg/mL were selected for theaberration test in the presence or absence of irradiation.
Chromosomal aberration test
The non-irradiated cultures revealed structural aberrations at a concentration of 19.5 μg/mL
and a ratio of 13% between the non-irradiated and the irradiated groups was calculated,which was statistically significant positive. The ratios of numerical aberrations were not72

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statistically significant and increased at any dose level. With irradiation, the ratios ofstructural aberrations at 19.5 and 9.8 μg/mL were 17 and 14%, respectively, and attained
statistical significance. The ratios of numerical aberrations were not affected at any dose.
In order to clarify whether the test substance was photo-clastogenic or not, the difference inthe ratios of aberrations between the irradiated cultures and those which were notirradiated was analyzed, showing a significant increase at 9.8 μg/mL. The sensitivity of thesystem was demonstrated since the positive controls induced statistically significant
increases in cells showing structural chromosome aberrations.
Conclusion
Under the conditions of the study, the test substance induced structural chromosome
aberrations in the absence or presence of simulated sunlight as determined by the photochromosomal aberration test in Chinese Hamster lung fibroblasts. According to the authors,
the test substance was also shown to be photo-clastogenic when tested up to cytotoxic
concentrations. However, the study with non-, pre- or simultaneously irradiated mammaliancells (Reference: 37 below) showed that treatment with UV light resulted in an increased
susceptibility to ZnO clastogenicity and thus, is not photoclastogenic per se, according to
the applicant.
(Reference: 97)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
The purity of the FINEX-50 used was indicated in the submission to be ≥96%. However,data on the purity is not provided in the physico-chemical characterization of the FINEX-50(section 3.1.4), and was also not provided in the study reference 97.
S9-mix fraction was not mentioned and presumed not used. However, the usefulness of S9mix fraction in assays investigating ZnO nanoparticles may be doubtful as ZnO
nanoparticles will not be metabolised.
The study showed that test substance induced structural chromosome aberrations in theabsence or presence of simulated sunlight. Without irradiation, the highest dose
investigated (19.5 μg/mL) was positive in the test, while after irradiation a dose dependent
positive response was seen for the two highest doses investigated (9.8 μg/mL and 19.5
μg/mL). Even if treatment with UV light increased the sensitivity of the cells, it is thepresence of the ZnO nanoparticles that induced the chromosomal aberrations, and thiscannot be ignored.
The SCCS considers that ZnO induced chromosomal aberrations in Chinese hamster lungfibroblasts both in the absence and presence of light irradiation.
Photo-chromosomal aberration test with non-, pre- or simultaneous irradiatedmammalian cells
Study Design
Date of report:

January 2007

Guideline/method:

Requirements of the EMEA Committee for Proprietary Medicinal
Products, Note for Guidance on Photosafety Testing, 27 June 2002,
Considerations on Photochemical Genotoxicity: Report of the73SCCS/1489/12
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Species/strain:

International Workshop on Genotoxicity Test Procedures Working
Group. (Gocke et al. 2000).
Chinese Hamster ovary (CHO) cells.

Replicates:

Duplicate cultures, two flasks (test substance, positive controls), four
flasks (negative control).

Test substance:

ZnO, uncoated (Z-COTE®), size less than 200 nm according tomaterials and methods section (100 nm is mentioned in the abstract).

Batch:

EHDE0402 (purity: >99% (i.e. 100.1 g/100 g)).

Concentrations:Phototoxicity range finder: 8.201, 13.67, 22.78, 37.97, 63.28, 105.5,
175.8, 293.0, 488.3, 813.8 μg/mL (with simultaneous and preimposed UV irradiation).

Chromosomal
aberration test:

27.49, 34.36, 42.95, 53.69, 67.141, 83.89, 104.9, 131.1, 163.8,
204.8, 256.0, 320.0, 400, 500 μg/mL (non-irradiated, withsimultaneous and pre-imposed UV irradiation).

Vehicle:McCoys 5A medium

Irradiation:
Source of light:

Atlas Suntest CPS+ solar simulator (Heraeus Equipment Limited,
Brentwood, UK). The intensity of UVA and UVB was measured using a
Dr Gröbel RM 21 UV meter. The ratio of UVB:UVA was in the range ofapproximately 1:30.

UV doses:

Phototoxicity range finder: UVA: 400 and 800 mJ/cm2.

Chromosomal
aberration test:

UVA: 350 and 700 mJ/cm2.

Groups:Non-irradiated:

Without irradiation

Pre-irradiated (PI):

Irradiation 2–3 hours (350 mJ/cm2) or 1–2 hours (700 mJ/cm2) prior
to treatment with ZnO concentration.

Simultaneous
irradiated (SI):

350 mJ/cm2 and 700 mJ/cm2 cultures received simultaneous
treatment with ZnO concentration.

Incubation time:

Cells were exposed to ZnO for 3 hours, after washing, cells were
harvested after a further incubation for 17 hours in tissue culture
medium.

Negative control:

McCoys 5A medium.

Positive controls:

8-Methoxypsoralene (8-MOP): 0.5 and 1.0 μg/mL (with/without
irradiation).
4-Nitroquinoline-1-oxide (NQO): 0.25 and 0.3 μg/mL (without
irradiation).

GLP:

Yes

Published:

Yes

ZnO was tested in an in vitro cytogenetic assay using duplicate cultures of Chinese Hamster
Ovary (CHO) cells in the presence and absence of UV light to clarify whether the slightly
pronounced clastogenicity in vitro in the dark is a genuine photo-genotoxic effect.. The
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effect of ZnO exposure was investigated in the cells in the dark (D), under pre-imposed
irradiation (PI, i.e. UV irradiation of cells followed by treatment with ZnO 1-3 hours later)
and under simultaneous irradiation conditions (SI, i.e. ZnO treatment concurrent with UV
irradiation).
Concentrations used in the main experiment were selected on the basis of cytotoxicity(expressed as a decrease in population doublings relative to controls). The number ofcells/mL were measured in trypsinised samples of cell suspension using a Coulter counter.
The highest concentration of ZnO used in the range-finder was 813.8 μg/mL (equivalent to
10 mM) for zinc. After three hours of exposure to ZnO or control chemicals, cell monolayers
from all cultures were washed with sterile saline, and re-fed with fresh McCoy’s 5A medium
containing foetal calf serum and gentamycin, and incubated for 17 hours before cell
harvesting. Approximately 1.5 h prior to harvest, colchicine was added to give a final
concentration of approximately 1_g/mL to arrest dividing cells in metaphase.
At the defined sampling times, monolayers of these cultures were removed using
trypsin/EDTA and a measurement of cell counts/mL was performed on an aliquot of cellsuspension using a Coulter counter. The remaining cell suspensions from each flask were
harvested and slides prepared for chromosome aberrations (CA) analysis using standard
operating procedures.
Negative (solvent) control cultures were included in the test system under each treatment
condition. 4-Nitroquinoline-1-oxide (NQO) and 8-methoxypsoralen (8-MOP) were employed
as positive control chemicals in the absence and presence of UV light. The slides for CA
analysis were prepared 17 hours after washing. Two parallel cultures were investigated andat least 100 metaphase plates were scored for structural chromosome aberrations in each
culture.
Results
In the absence of UV light, the cytotoxicity of ZnO was concentration dependent, with 4060% cytotoxicity observed in the concentration range 256–320 μg/mL. In SI cultures, thecytotoxicity was more pronounced than in the dark and related to the UV dose: 40-60%
cytotoxicity was observed in the concentration range 131.1–256 μg/mL (350 mJ/cm2) or
83.89–13 1.1 μg/mL (700 mJ/cm2). Overall, cytotoxicity in SI cultures increased with ZnO
concentrations, although a large variability in ZnO cytotoxicity was observed at the high UVdose (700 mJ/cm2) and at high ZnO concentrations (≥204.8 μg/mL).
In PI cultures, cytotoxicity of ZnO was concentration dependent and comparable at the low
UV dose (350 mJ/cm2) to that observed in SI cultures (40-60% cytotoxicity was observed in
the concentration range 104.9–256.0 μg/mL). At the high UV dose (700 mJ/cm2) and at low
ZnO concentrations (<204.8 μg/mL), cytotoxic effects of ZnO in PI cultures wereintermediate between those observed in the dark and SI cultures, with 40-60% cytotoxicity
observed in the concentration range 131–256 μg/mL. At higher concentrations (≥204.8
μg/mL), ZnO cytotoxicity was similar in the PI and SI cultures. For each individual
irradiation dose and condition, the concentrations analyzed for CA (chromosome
aberrations) covered a range of cytotoxicity from little or none, to maximum effects.
Treatment of cultures with ZnO in the absence of UV light resulted in statistically significant
increases in the frequencies of cells with structural aberrations at 104.9 μg/mL (giving 13%
cytotoxicity, as measured by population doubling) and above, in a concentration dependent
manner. The frequencies of cells with structural aberrations (excluding gaps) exceeded thehistorical negative control (normal) range in both cultures analyzed at 163.8 μg/mL andabove, and also in single cultures analyzed at several lower concentrations. Theseobservations were considered biologically relevant and ZnO was considered clastogenic in
the dark. Under SI conditions, treatment with ZnO induced biologically relevant increases instructural aberrations at 104.9 μg/mL (23% cytotoxicity) and 53.69 μg/mL (19%cytotoxicity) following 350 and 700 mJ/cm2 UV radiation.

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Under PI conditions, treatment with ZnO resulted in biologically relevant increases in thefrequencies of cells with structural aberrations at 104.9 μg/mL and above (36%cytotoxicity) and at 53.69 μg/mL and above (0% cytotoxicity) following 350 and 700
mJ/cm2 UV radiation, respectively. When compared at similar cytotoxic concentrations, theincidence of chromosome aberrations following PI or SI was generally similar at 700
mJ/cm2.
The proportion of cells with structural aberrations treated with the vehicle (negative control)
fell within historical solvent control ranges. Both treatments with the positive controls
induced increases in the proportion of cells with structural aberrations. When added to
cultures treated in the absence of UV light, 8-MOP induced frequencies of cells withstructural aberrations that were similar to those seen in concurrent solvent control cultures(non irradiated). Thus, the validity and sensitivity of the test system was demonstrated.

ZnO induced clastogenic activity in CHO cells, both in the absence and in the presence of UV
light. The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was asfollows: SI > PI > D. The effects were more pronounced at 700 mJ/cm2. The incidence ofchromosome aberrations in SI or PI cells was generally higher than in the dark. At similar
ZnO concentrations, SI conditions generally produced the greatest incidence of chromosome
aberrations. However, at similar cytotoxic concentrations, the incidence of chromosome
aberrations following PI or SI was generally similar for 700 mJ/cm2.
Conclusion
The authors concluded that ZnO was clastogenic to CHO cells in the dark and after
irradiation of the cellular test system, either prior to (PI conditions) or concurrently with (SIconditions). The treatment with UV light resulted in an increased susceptibility of CHO cells
to ZnO clastogenicity, as indicated by clastogenic responses of higher magnitude and/or
observed at lower concentrations in both SI and PI conditions when compared to thoseobtained in the dark.

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Finally, the results provided evidence that, under conditions of in vitro photo-clastogenicity
tests, UV irradiation of the cellular test system per se may produce a slight increase in the
genotoxic potency of compounds that are clastogenic in the dark. Therefore, minor
increases in clastogenic potency under conditions of photo-genotoxicity testing do notnecessarily represent a photogenotoxic effect, but may occur due to an increased sensitivityof the test system subsequent to UV irradiation.
(References: 37, 67)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided. The uptake of the ZnO by the exposed cells was notdemonstrated.
S9-mix fraction was not mentioned and presumed not used. However, the usefulness of S9mix fraction in assays investigating ZnO nanoparticles may be doubtful as ZnO
nanoparticles will not be metabolised.
An increase in the frequency of cells with structural chromosomal aberrations was noted
after UV irradiation both in simultaneous irradiation and in pre-irradiation culture conditions.
In addition, clastogenic activity was also demonstrated without UV treatment. The observed
clastogenic effect during irradiation may not be a photo-genotoxic effect of the ZnO as also
in the pre-irradiated cultures (in which ZnO was not irradiated) a clastogenic effect wasseen.
The SCCS consideres that based on the results presented ZnO has clastogenic activity in
Chinese hamster ovary cells both in the presence and absence of UV.

Photo-micronucleus test in mice
Study Design
Date of report:

August 2008

Guideline/method:

Photo-micronucleus test in mice according to published methodology(Reference: AR13).

Species/strain:

Hairless mouse/HR-1

Group size:

Three male mice/group.

Test substance:

FINEX-50 ZnO (supplied by Sakai Chemical Industry Co., primaryparticle size: 20 nm, non-coated)

Batch:
Dose levels:

OZ52 (purity: ≥96% as indicated in the submission)
Preliminary toxicity study: 2.5%, 5%, 10%, 20% (w/v) concentrationof the test sample in Aceton Olive Oil (AOO) (with irradiation).

Micronucleus test:

5%, 10%, 20% (w/v) concentration (with/without irradiation).

Vehicle:Acetone/olive oil (4:1)

Exposure:

Once daily for two consecutive days.

Route:

Topical on the skin (open without occlusion of the test samples)

Application volume:

0.2 mlApplication area:

3 × 4 cm

Irradiation:
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Source of light:

Intensity ofirradiation:

Sunlight simulator (light emission equipment for animals (LDS-20,
ABLE) with six xenon lamps) with emission of a continuous spectrumof simulated sunlight between 300–800 nm, visible light (48,800–
67,600 lux).
UVA: 2.27–2.59 mW/cm2
UVB: 88.6–17.2 μW/cm2

Irradiation time:

2 hours, once a day for two days at an interval of 24 hours.

Negative control:

Acetone/olive oil (4:1, with/without irradiation).

Positive controls:

With irradiation:

8-Methoxypsoralene (8-MOP), 0.0015%.

Without irradiation:

Mitomycin C (MMC), 0.05%.

GLP:

No

The ability of the test substance to cause photo-chromosomal damage in vivo wasinvestigated in the photo-micronucleus test with mouse epidermal cells after topicalapplication on the skin of male hairless HR-1 mice.
After a preliminary toxicity test with light irradiation to observe skin effects and to
determine the maximum tolerated dose of the test article, the micronucleus test(with/without light irradiation) was carried out. In the preliminary toxicity test,
concentrations of 2.5, 5, 10 and 20% (w/v) of the test article suspended in acetone/olive oil(4:1) were used.
The test article suspensions were applied on the dorsal skin of mice (3–4 cm) once a day for
two days at an interval of 24 hours followed by light irradiation using a sunlight simulator(Light Emission Equipment for Animals, LDS-20: with six xenon lamps, ABLE), within 30
minutes after each application. As neither abnormal signs nor dead animals were observedin the dosing groups, concentrations of 5%, 10% and 20% were selected for the
micronucleus test.
The micronucleus test was carried out with ten experimental groups of mice, five with andfive without light irradiation: the negative control groups [vehicle (acetone/olive oil, 4:1)],
the test article groups [5, 10 and 20 % (w/v)] and the positive control groups (with lightirradiation: 8-methoxypsoralen; without light irradiation: Mitomycin C).
The dosing formulation (0.2 mL) was applied on the dorsal skin of mice once a day for twodays at an interval of 24 hours. For the light-irradiated group, the mice were exposed tolight from the sunlight simulator within 30 minutes after each application for 2 hours (UVA
intensity: 2.27–2.59 mW/cm2, UVB intensity: 88.6–117.2 μW/cm2). For the non-lightirradiated group, the mice were returned to their home cages immediately after applicationand kept in the animal room under white fluorescent light.
All animals were euthanized about 48 hours after the last treatment. The skin wasprocessed and epidermal cell suspensions were prepared. The cell suspensions were placedon glass slides, dried and stained with acridine orange solution. Each of the 2,000 epidermal
cells were examined with a fluorescence microscope and the number of micronucleatedepidermal cells was recorded.
Results
In the preliminary toxicity test, after the topical application on the skin, abnormal
behavioral signs or dead animals as indication for toxicity were not observed, so the test
sample did not induce overt toxicity in the used application.

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The fluorescence microscopic observation revealed no test substance (ZnO) related increasein the number of micronucleated epidermal cells outside the historical control data, neitherwith nor without UVA and UVB light irradiation.
In the vehicle group the incidence of micronucleated cells did not exceed the historicalcontrol group ranges, while the respective positive controls induced a significant increase in
the number of micronucleated epidermal cells with and without simulated light irradiation
indicating the suitability and sensitivity of the test system.
Conclusion
The test substance did not increase the incidence of micronucleated epidermal cells in
exposed male mice with or without simulated light irradiation. From these results, it was
concluded that FINEX-50 ZnO Batch OZ52 (purity: ≥96%) is not clastogenic or photoclastogenic in epidermal cells of mice under these experimental conditions.
(Reference: 100)Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
The purity of the FINEX-50 used was indicated in the submission to be ≥96%. However,data on the purity is not provided in the physico-chemical characterization of the FINEX-50(section 3.1.4). The submitted study report in reference 100 indicates a content of 100%.
A different product/batch is mentioned in the reference to that included in the dossier.
Product/batch 05M099 is mentioned in the results and conclusions, but not in materials andmethods. Product/batch 05M99 (lot no. 2Z51, content 100%) is mentioned in Reference:
100.
The ZnO was applied topically on the skin. Uptake by living cells in the skin was notinvestigated. As there is no information whether the substance reaches the target cells (orthe nucleus), the value of this test is very limited.
The results show that the ZnO tested did not increase the number of micronuclei in the skinafter topical application indicating that the ZnO is not a positive mutagenic substance.
However, from the negative results it cannot be concluded that ZnO is non-mutagenic.
The SCCS considers the value of this test limited.
3.3.11 Human data
Comparative human inhalation study
Study Design
Date of publication:

February 2005

Guideline/method:

According to internal laboratory methodology.

Species:

HumanGroup size:

12 volunteers (six males and six females, one male/female in eachexposure sequence).

Test substance:A) Fine ZnO particles (A) ZnO, coated (Zano 10 Plus, Umicore)(former Zano® Plus, ZnO coated with octyl triethoxysilane).
B) Ultrafine ZnO particles.

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ZnO particles were generated by an electric arc discharge system
between two consumable zinc electrodes.
Particles sizes:

For the ultrafine particle exposures, the count median diameter was
40.4 ± 2.7 nm geometric standard deviation (GSD) 1.7, whereas forthe fine particle exposures, the count median diameter was 291.2 ±20.2 nm GSD 1.7.

Batch:

Not applicable

Route:

Inhalation (by mouth piece)

Frequency:

2 hour inhalation at rest on three exposure days.

Concentration:

500 μg/m3 fine and ultrafine ZnO particles, or 4.6×10106x1010/m3 and1.9×1089x108/m3 particles, respectively, as measured duringexposure.

Examinations:

Clinical symptoms, heart rate, blood pressure, mouth temperature,oxygen saturation at rest, differential blood cell counts, expression ofactivation markers and adhesion molecules, coagulation factors,
inflammation markers, electrocardiogram (ECG) parameters, sputum
induction.

GLP:

No

Published:

Yes

A comparative exploratory inhalation study was performed in 12 human volunteers (sixmale and six female, age range 23–52 years) with ultrafine and fine ZnO particles
generated by an electric arc discharge system between two consumable zinc electrodes. The
exposure response relationships for respiratory, hematologic, and cardiovascular endpoints
between ultrafine and accumulation mode zinc oxide particles were compared. Twelve
healthy adults inhaled 500 μg/m3 of ultrafine zinc oxide, the same mass of fine zinc oxide,and filtered air while at rest for 2 hours. Pre-exposure and follow-up studies of symptoms,
leukocyte surface markers, homeostasis, and cardiac electrophysiology were conducted up
to 24 hours post-exposure. Induced sputum was sampled 24 hours after exposure.
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Frequency distribution of inhaled zinc oxide particles for the two exposures conditions (left:
ultrafine zinc oxide; right: fine or accumulation mode zinc oxide) as measured by number
concentration (number of particles per volume of air) measured in the inspiratory circuit of
the human mouthpiece exposure system during the studies is shown below.

CMD: count median diameter; dN/dlog(Dp): normalized particle number concentration,
P/cm3: particles per cubic centimeter of inspired air. Overlapping distributions at a particle
diameter of 100 nm indicate that in ultrafine exposures there was a small percentage by
number above the ultrafine size. No differences were observed after exposure to the fine or
ultrafine ZnO particles when compared to clean filtered air conditions at this level of
exposure (500 μg/m3).
Conclusion
Freshly generated zinc oxide in the fine or ultrafine fractions inhaled by healthy subjects at
rest at a concentration of 500 μg/m3 for 2 hours is below the threshold for acute systemic
effects as detected by these endpoints.
(Reference: 32)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area was not provided.
The ZnO materials given as test substances in the dossier (A and B in the table) were notused in the study.
Reference 32 only describes the effect of ZnO generated at the research facility itself.
Ultrafine and fine ZnO were generated by an electric arc discharge system (Palas generator;
Palas, Karlsruhe, Germany) between two consumable zinc electrodes. The ZnO materials
used in the reference do not relate to any of the ZnO materials submitted and included in
the dossier by the applicant.

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In view of the different type of ZnO used (spark generated) in the study, the SCCS
considers this study not relevant for the evaluation of the use of ZnO as cosmetic
ingredient.
3.3.12 Special investigations
Experimental study on toxicity after IV administration
Study Design
Date of report:

December 2009

Guideline/method:

Study according to internal laboratory methodology for investigative
purposes considering OECD Guideline 407, EC Commission Regulation
No. 440/2008, EC Commission Directive 87/302/EEC, OECD Guideline
417

Species/strain:

Rat/Wistar (Crl:WI (Han))

Group size:

Groups of five male and five female rats.

Test substances:a) ZnO nanoscale, coated with triethoxyoctylsilane (Z-COTE®HP1)b) ZnO, nanoscale uncoated (Z-COTE®)c) ZnO pigmentd) Zinc sulfate

Batches:a) CNBG1602 (ZnO >97.1%, triethoxyoctylsilane 3.4%)b) EHFA0907
c) S35583-206 (>99%)d) 02616TC-126 (100%)

Dose levels:a) 1 or 5 mg/kg bw (subsets 0, 1, 2), single administration day 28b) 1 or 5 mg/kg bw (subsets 0, 1, 2)c) 5 mg/kg bw (subsets 0, 1, 2)d) 17.6 mg/kg bw (subset 2) or 4 × 4.4 mg/kg bw on days 0, 7, 14,
21 (subsets 0, 2) as an equimolar zinc ion concentration related tothe dose level of 5 mg/kg bw of ZnO.

Exposure:I) Single application for test item groups a, b and c (subsets 0, 1, 2)
and test item group d (subset 1).
II) Four applications on study days 0, 7, 14 and 21 for test item
group d (subsets 0, 2).

Route:

Intravenous

Application volume:

1 mL/kg bw

Vehicle:a–c) Inactivated calf serumd) Physiological (0.9%) NaCl solution
Application and sampling time-points: subset 0 (five males/fivefemales per group): application on study day 0, urine sampling on
study days 24/25, blood sampling and necropsy on study day 29.
Subset 1 (five males per group): application on study day 28, blood
sampling and perfusion fixation on study day 29.
Subset 2 (five males per group): application on study day 0,
perfusion fixation on study day 29.
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GLP:

Yes

The study was divided into separate subsets according to the dosing and sampling schedule.
• Subset 0: ZnO coated and uncoated, and pigment ZnO dosing at day 0 and samplingat days 24/25 (urine) and day 29 (blood). As control Zn sulfate was used that wasadministered at days 0–7–14–21 and sampled on the same days.
• Subset 1: ZnO coated and uncoated, pigment ZnO and Zn sulfate on day 28 andsampling at day 29 (day 1 after exposure).
• Subset 2: Same as subset 0 treatment at day 0, but animals were sacrificed forhistopathology after perfusion fixation at day 29.
The total dose of Zn sulphate (17.6 mg/kg bw) was administered in four dosages of 4.4
mg/kg bw at days 0, 7, 14, and 28.
The treatment schedule is presented below.

Results
The analysis confirmed the homogenous distribution of the test items in the vehicles andthe correctness of the concentrations.
Significantly increased body weights were determined on study day 28 for female animals intest group 2 (zinc oxide [nanoscale, coated], 5 mg/kg bw) and test group 3 (zinc oxide[nanoscale, uncoated], 1 mg/kg bw), as well as on study days 21 and 28 for female animals
in test group 5 (zinc oxide [pigment], 5 mg/kg bw) compared to the control. For maleanimals in test group 26 (zinc sulfate, 4 × 4.4 mg/kg bw), a significantly lower mean bodyweight was detected on study day 21.
Significantly lower mean body weight changes were determined on study days 7, 14 and 21
for male animals in test group 14 (zinc oxide [nanoscale, uncoated], 5 mg/kg bw).
Significantly higher mean body weight change was determined on study day 28 for female
animals in test group 2 (zinc oxide [nanoscale, coated], 5 mg/kg bw).
Significant effects on body weight were noted in various groups after ZnO treatmentcompared to the vehicle (calf serum inactivated, 1 ml/kg bw) control animals.
Test group 2, females, ZnO nanoparticles, coated, 5 mg/kg bw, single dose day 0, increased
bw day 28.
Test group 3, females, ZnO nanoparticles, uncoated, 1 mg/kg bw, single dose day 0,
increased bw day 28.
Test group 5, females, ZnO-pigment, 5 mg/kg bw), single dose day 0, increased bw days 21
and 28.

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Test group 26, males, Zn-sulfate, dose 4 × 4.4 mg/kg bw, decreased bw day 21.
Test group 14, males, ZnO nanoparticles, uncoated, 5 mg/kg bw, decreased bw days 7, 14,
and 21.
The values reflect the normal range of biological variation inherent in the strain of rats used
for this study and, therefore, were assessed as incidental.
There was no mortality, no clinical findings, no effect on food consumption or body weight
gain, no impaired organ weight and no gross pathological findings in the organs of any of
the investigated in either group in the respective subsets, irrespective of whether the
animals were killed after one day or after four weeks following the single (ZnO) or four (zincsulfate) intravenous test item injections. In addition, the male and female animals of subset
0 (ZnO coated and uncoated, and pigment ZnO) showed no treatment related findings in
the urinalysis and the comprehensive clinical pathology examinations including additional
serum parameters (haptoglobulin, alpha2-macroglobulin, troponin I) at the end of theobservation period of four weeks.
However, the animals of subset 1, which were killed and investigated by clinical pathology
at day 1 after injection of the test items, revealed some minor deviations in single
parameters of differential blood count, enzymes, clinical chemistry and the additional serum
parameter haptoglobin. See table below (5.12.1.).

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Conclusion
The comparative toxicity study of ZnO (coated or uncoated nanoparticles), ZnO (inndin
pigment form) and zinc and sulfate, showed no persistent effects at the end of theobservation period of four weeks when injected as a single dose intravenously at dose levelsof 1 or 5 mg/kg bw.
Very mild, and in any case not persistent, deviations in single hematology parameters(monocyte, large unstained cells) and few clinical chemistry parameters, probably indicativeof a minimal impairment of liver function (ZnO preparations) or of kidney function in
addition (zinc sulfate) were observed one day after intravenous injection of 5 mg/kg bw
pigmented or triethoxyoctylsilane coated nanoscale ZnO or an equimolar zinc sulfate dose.
At 1 mg/kg bw no biologically relevant effects were observed. Among the known and very
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sensitive markers of acute phase inflammatory reactions towards nanoparticles, only
haptoglobulin was increased. Alpha2-macroglobulin and troponin were not affected.
Finally, the comparative screening showed only minor and in any case transient effects
without toxicological relevance. There was no biologically relevant difference in the injected
form of ZnO, and in particular, no enhancement of the observed effects due to the injectionof nanoscale material. The equimolar injection of the zinc ion as zinc sulfate led qualitatively
and quantitatively to a slight enhancement of the observed variation in homoeostasis. In
any case, four weeks after treatment there was virtually no difference in comparison to therespective control group, irrespective of the injected dose level, the usage of ZnO as coatedor uncoated nanoparticles, as ZnO pigment or as zinc sulfate.
(Reference: 28)
Comment
For the ZnO used in this study, information on surface area and number of particles permass was not provided. Information on dose expressed as surface area and number ofparticles was not provided.
In general similar minimal effects were observed for both ZnO coated and uncoatednanoparticles. Some effects on body weight were observed, but they were not consistent.
Minimal alterations were observed in blood parameters (clinical pathology) at one day after
treatment. For these effects a dose response relationship could not be established as mostalterations were only observed at the highest dose (5 mg/kg bw) administered.
For both coated and uncoated nano-ZnO indications for acute liver damage (alterations inblood parameters ALP, AST and bilirubin) were observed at the highest dose of 5 mg/kg bw
administered.
Indications for kidney damage (increase in blood urea and creatinine) were only observed
for zinc sulfate after the single high dose (17.6 mg/kg bw).
Four weeks after the administration, no alterations were observed in blood parameters
evaluated for clinical pathology and in histopathology in the major organs evaluated (brain,
lung, liver, spleen and kidney). Thus, there was no indication for persistent toxic effects.
The intravenous administration provided a 100% bioavailability of the nano-ZnO. A
limitation of the study is that only a single administration was investigated, while for zinc
sulfate the equimolar dose was administered both as a single dose or divided over four
administrations in four weeks. The comparison of coated and uncoated ZnO is limited toacute reactions as indicated by several alterations in blood parameters at day 1 after
treatment with 1 mg/kg bw or 5 mg/kg bw. A real comparison between coated anduncoated ZnO cannot be performed as toxic reactions were not observed.
In view of the indications for liver damage, a repeated dose toxicity study would haveprovided better information on potential toxicity.
In conclusion, after intravenous administration obtaining an internal dose of 5 mg/kg bw
alterations in clinical pathology were observed that were indicative of liver damage. The
liver damage appeared to be transient as similar alterations were not observed at fourweeks after the single administration.
Based on this study, the SCCS considers a NOAEL of 1 mg/kg bw for acute toxicity of ZnO inrats after intravenous administration.
Additional studies submitted
Additional information was included in the dossier on ZnO commercially obtained from
Sigma-Aldrich and not related to the ZnO presented in the dossier.
(References: 85, 94, 119)86

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In vitro cytotoxicity studies
Exploratory Comet assay in human epidermal cells
These studies demonstrated dose and time dependent cytotoxicity of ZnO nanoparticles.
LDH release started at 5, 8 and 20 μg/mL exposure. Information on doses expressed assurface area and number of particles was not provided. NRU assay showed cytotoxicity at
8–20 μg/mL exposure. Oxidative stress markers were increased at a dose of 0.008 μg/mL
exposure.
According to the applicant the cytotoxic responses in terms of disturbance of cellular
homeostasis were observed at an overdosing of ZnO nanoparticles. The effect was
attributed not to the nanoparticles per se, but to the released Zn ions as the soluble part of
the ZnO nanoparticles.
(Reference: 94)
Comment
The SCCS does not consider an effect induced at doses as low as 0.08 μg/mL or even 5
μg/mL as a result of overdosing. Therefore, the SCCS considers ZnO nanoparticles at thesedoses cytotoxic. Even if the effects were due to the release of toxic ions, these wereintroduced by the added ZnO nanoparticles so the toxicity is indirectly due to the ZnO
nanoparticles.
Comparison of in vivo and in vitro studies
For the development of a predictive in vitro assay to assess the lung hazard potential of
nanomaterials, a comparative screening study was performed. The main objectives were tocompare lung toxicity impacts of nanoscale (NZnO) versus fine zinc oxide (FZnO) particles,
assess predictability of in vitro cell culture systems, and compare the effects of instillation
versus inhalation exposures in rats. The fine and nano-sized ZnO materials were obtained
from Sigma-Aldrich, St. Louis, USA (purity: >99%). The surface area of the fine and nanoZnO was 9.6 m2/g and 12.1 m2/g, respectively. Information on the number of particles permass was not provided. The authors concluded that the comparisons of in vivo and in vitro
toxicity measurements following nano- or fine-ZnO exposures demonstrated little
convergence and few differences in potency. In addition, the implementation of the current
cell culture methodologies tested in this study did not accurately predict the pulmonary
hazards associated with in vivo exposures to ZnO.
(References: 85, 119)
Method for screening of photoactivity of nanoparticles
Undoped and doped zinc oxide was prepared by proprietary methods and characterizedchemically and physically. Besides size, no other information is presented on the ZnO used.
Information on surface area and number of particles per mass was not provided. In contrast
to a comparative study cited in the dossier, the screening method revealed photoactivity
behaviour increasing (one to twofold) with decreasing crystallite size for crystallites <100
nm. A further series of nanophase ZnO powders doped with metal ions showed that,
depending on the type and/or level of dopant, the photoactivity behaviour of nanoparticles
could be reduced by more than an order of magnitude. According to the dossier, thisenzymatic method should be considered as an initial exploratory screening study on
photoreactivity in vitro with questionable relevance for in vivo effects on the skin.
(Reference: 33)

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Comment
The paper was published in 2004 (Reference 33, submission III) and no follow up studies
were cited In view of the rather limited characterization of the ZnO preparations used, thestudy does not contribute to the evaluation of the use of ZnO as a UV-filter in sunscreen
formulations. However, scientifically the results indicate that the photoactivity of the ZnO
nanoparticles can be manipulated by doping of the particles.
The SCCS considers this study not relevant for the evaluation of the use of ZnO as acosmetic ingredient.

3.3.13 Safety evaluation (including calculation of the MoS)
The data provided, and as present in the literature, indicate that ZnO nanoparticles do not
penetrate through the skin. However, some minimal absorption of zinc was demonstrated.
Although the zinc was determined by methods which do not discriminate between
particulate and solubilized forms, considering the dissolution rate of ZnO, it is likely that thiswas in the form of solubilized zinc ion. This conclusion is based on the weight of evidence
from the available studies so far which suggest that only a small fraction of the nano-sized
ZnO is likely to be solubilized in a formulation, and possibly when in contact with the skin,and a part of this solubilized fraction may subsequently be absorbed. The maximum
solubility is estimated to be around 50 mg/L, depending on the solvent/medium under static
equilibrium conditions. However, in view of the dissolution rate of ZnO, any translocating
ZnO nanoparticles will also be completely solubilized due to the non-static conditions in the
biological environment (see section 3.1.6). Thus, until proven otherwise, it is assumed thatany transdermal penetration following application the nano-ZnO containing cosmetic
product is that of Zn ions released from the ZnO nanoparticles.

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in the EU Risk Assessment Report on Zinc Oxide (2004) (Reference 44, sub III) arerelevant:
The total database available indicates that skin-bound zinc may not become
systemically available in a way that it results in high peak levels of zinc inserum, but rather in a more gradual way. Given the efficient homeostaticmechanisms of mammals to maintain the total body zinc and thephysiologically required levels of zinc in the various tissues constant, theanticipated slow release of zinc from the skin is not expected to disturb the
homeostatic zinc balance of the body. By expert judgement, based on the
aforementioned considerations, the default for dermal absorption ofsolutions or suspensions of zinc or zinc compounds is therefore chosen to be
2%. Based on the physical appearance, for dust exposure to zinc or zinccompounds a 10-fold lower default value of 0.2% is chosen in the risk
assessment.
For sunscreen containing 10% zinc oxide the following exposure assessment
was performed:
By an application of 9 g sunscreen/event, 3 events/day during 18 days/year
the exposure will be 1,332 mg sunscreen/day, being 107 mg Zn2+/day.
Assuming a dermal absorption of 2% the uptake is estimated to be 2.14 mg
Zn2+/ day.
An NOAEL of 50 mg Zn2+/day was derived from a 10-week oral study withhuman volunteers and was used as a starting point for the risk
characterization for repeated dose toxicity. This NOAEL of 50 mg Zn2+/day
results in an internal NOAEL of 10 mg Zn2+/day by correction for oral
absorption (20%; worst case, because of the homeostasis the relative
absorption will be smaller by excess of Zn2+-intake). Given that this study
was with women (the most sensitive population in zinc supplementation
studies), and that in women clinical signs begin to appear only at a dose
three times this NOAEL, a minimal MOS of 1 is considered sufficient whencomparing
the
human
NOAEL
with
the
exposurelevelsforworkers/consumers/general population.
The MOS between this internal NOAEL and the internal exposure as a resultof exposure to sunscreen formulations is 5, which is a factor of 5 larger thanthe minimal MOS.

The SCCS agrees with the NOAEL indicated in the RAR statement. Therefore, thisinformation is used together with the data from the absorption study provided in the dossierand exposure assumptions from the SCCS Notes of Guidance for the risk assessment of zincoxide nanoparticles in sun screens as follows:89

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Calculation of the margin of safety for ZnO (nano)(See section 3.1.16 for the range of nano-ZnO covered by this opinion)

Amount of sunscreen applied*

18,000 mg

Maximum concentration of ZnO

25%

Absorption through the skin

0.03% (Reference: 36)

Amount absorbed/day(18,000 × 25%/100 × 0.03%/100)

1.35 mg

Typical body weight of human

60 kg

Systemic exposure dose
(1.35 mg/60 kg)

0.0225 mg/kg bw/d

No Observed Adverse Effect Level NOAEL(oral, human, sensitive subpopulation)**0.166 mg/kg bw/dMargin of Safety

NOAEL/SED= 7.4* Standard amount as indicated in the SCCS Notes of Guidance for the testing of cosmetic ingredients and theirsafety evaluation SCCS/1416/11** The internal NOAEL for ZnO is 10 mg Zn2+/day = 10/60= 0.166 mg /kg bw per day (Reference 44, sub III)The calculation of the exposure via sun protection products to ZnO nanoparticles assuming
Zn2+ uptake results in a MoS of 7.4. Given that the NOAEL is derived from a study on
women (the most sensitive population in zinc supplementation studies), and that in womenclinical signs begin to appear only at a dose three times this NOAEL, a minimal MOS of 1 isconsidered sufficient when comparing the human NOAEL with the exposure levels forworkers/consumers/general population (Reference 44, sub III).
Calculation of the Margin of Safety for Lip products
According to the SCCS Notes of Guidance for the testing of cosmetic ingredients and theirsafety evaluation (SCCS/1416/11) the daily exposure to lipstick/lipproducts is 0.9 mg/kg/d.
Exposure dose (0.9 x 25%)0.225 mg/kg bw/day.

No Observed Adverse Effect Level NOAEL(oral, human)*

0.83 mg/kg bw/dMargin of Safety

NOAEL/ED**= 3.7* The NOAEL for ZnO is 50 mg Zn2+/day = 50/60= 0.83 mg /kg bw per day (Reference 44, sub III)** Since route to route extrapolation is not needed, the (external) NOAEL is compared to the (external) exposure
dose

It is expected that the exposure to lipstick/lip products that contain high SPF factors (egZnO nanomaterial) is less than the exposure to ‘regular’ lipstick/lip products. Typically,
these products are used only in specific time frames, e.g. during outdoor holidays.
Therefore 0.9 mg/kg bw/d is considered a conservative value.
The calculation of the exposure via lip products to ZnO nanoparticles assuming Zn2+ uptake
results in a MoS of 3.7 (with a minimal MOS of 1 required, see above).

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3.3.14 Discussion
Several submissions on ZnO have been received during recent years. Submission I dealt
with ZnO in general, the main focus was on zinc ions as being responsible for Zn toxicity.
The ZnO used was only marginally characterized. Submission II relied mainly on the reporton ZnO of the regular risk assessment prepared in the context of Council Regulation (EEC)
No. 793/93 on the evaluation and control of existing substances. This risk assessment was
peer reviewed by the EU Commission’s Scientific Committee on Toxicity, Ecotoxicity and theEnvironment (CSTEE). Submission III, evaluated in this opinion, deals with ZnO
nanoparticles from four different sources, either coated or uncoated, with a mean size rangeof 30 nm to 55 nm as indicated by their number size distribution.
Regarding the dosimetry of nanoparticles it is not yet clear which metric is the best dose
descriptor for nanomaterials. However, it is assumed that for evaluation of toxicity
endpoints the dose metric of mass, i.e. mg/kg body weight or μg/ml in in vitro systems,
might not be the best parameter for expression of the dose response relationship. The total
surface area administered and or the number of particles have been suggested asparameters to better describe dose response relationships for nanoparticles as it is the unitof one particle that interacts with the biological system and not the number of molecules ofthat particle, in contrast to soluble chemicals in which the total number of molecules present
can initiate such interactions. However, in the present Opinion, the dose metric used for therisk assessment has only been described in mass units.
Physicochemical properties
It should be noted that it is technically possible to produce nano-dispersed ZnO withdifferent physicochemical properties. Therefore, this opinion relates to those coated anduncoated ZnO nanomaterials for which data have been submitted in this dossier. It is
required that manufacturers carefully analyze the raw materials which they obtain,
particularly if they come in a finally stabilized formulation in liquid dispersions.
It should be noted that this assessment applies to those ZnO nanoparticles that are includedin this dossier, and similar materials that have the following characteristics:• ZnO nanoparticles of purity ≥99%, with wurtzite crystalline structure, and physicalappearance as described in the dossier, i.e. clusters that are rod-like, star-like and/or
isometric shapes.
• ZnO nanoparticles with a median diameter (D50: 50% of the particles below thisdiameter) of the particle number size distribution between 30 nm and 55 nm, and D1(1% of the particles below this diameter) above 20 nm.
• ZnO nanoparticles that are either uncoated, or coated with triethoxycaprylylsilane,
dimethicone, dimethoxydiphenylsilanetriethoxycaprylylsilane cross-polymer, or octyl
triethoxy silane.
• ZnO nanoparticles that have a comparable solubility to that reported in the dossier,i.e. below 50 mg/L (approximately the maximum solubility of the ZnO nanomaterials
for which data are provided in the dossier).
In the submitted dossier on ZnO nanomaterials the purity of one of the ZnO nanomaterials
used in some safety assays was indicated to be ≥96%. However, data on the purity is notprovided in the physico-chemical characterization section of the dossier (section 3.1.4), andwas also not provided in the various study reports cited although in one report a content of
100% was indicated.
As discussed in section 3.1.6, the parameter of equilibrium solubility describes the static
dissolved fraction in a saturated solution. However, since ZnO nanoparticles and/or their
agglomerates/aggregates in the body will not be in a static equilibrium of dissolution, Zn
ions will be continuously generated and taken into the endogenous Zn pool, and the ZnO
particles will continue to dissolve until completeness. Hence the parameter of dissolution
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rate is more appropriate than the static solubility parameter when considering the fate ofZnO nanoparticles in the body. As the dermal penetration, if any, seems to be associatedwith the Zn ions released, the solubility of ZnO nanomaterials used as cosmetic ingredientsshould be below 50 mg/L because for this solubility level the data presented in this Opinion
show an acceptable Margin of Safety.
Irritation, sensitization
A skin irritation study (not according to a guideline) was performed in which male Guinea
pigs were exposed to 25% and 40% of 20 nm ZnO dispersed in ethanol. No effects wereobserved at any time during the administration and observation periods in the 25% test
substance group. Slight erythema was observed in one of three animals in the 40% test
substance group on day 3 of administration.
The ZnO nanomaterial, both as a neat dispersion and 25% solution as used in sunscreen,
was slightly and transiently irritating to the eyes when tested in rabbits.
The sensitization assay was not performed according to a recognized OECD guideline.
Furthermore, a concurrent positive control with a well known weak sensitizer was notincluded in this assay, so there is no certainty as to whether the test system used was able
to identify weak sensitizers. However, for eight contact sensitizers, similar responses were
found in the GPMT performed according to the OECD Guideline 406, and in a shortened test.
The validity of this (or any) test for demonstrating sensitization potency of nanomaterials
has not yet been demonstrated. The inclusion of a positive particle control might overcome
this problem. However, no positive particle control has been identified thus far.
Dermal absorption
The applicants have performed and provided a comprehensive review and assessment of
the available in vivo and in vitro dermal penetration studies. None of the studies or projects
yielded any evidence that nano-sized ZnO particles are able to cross the skin barrier inintact or compromised skin. The literature data and the data which were provided for thissubmission suggest that there is only minimal absorption and resulting systemic availabilityof zinc from application of ZnO nanoparticles containing sunscreens on the skin. Whether
this zinc is available as zinc oxide nanoparticles or zinc ions has not been determined. Thesestudies include numerous in vitro (using human, porcine and nude mice skin) and in vivo
human volunteer studies. In studies that analysed particles, no penetration beyond thestratum corneum was seen. In studies that analysed Zn, small amounts were detected indeeper skin layers and receptor fluid/blood. Zn could only be detected in one out of seven of
the in vitro studies evaluated, and was detected in the receptor liquid by elemental analysis
with ICP-MS indicating some passage (maximally 0.03% of the applied dose) of the skinbarrier. It was shown that some Zn may pass the skin barrier in a human volunteer study inwhich a small fraction of the blood Zn-pool was demonstrated to originate from a dermallyapplied sunscreen preparation. No major differences were seen between coated/uncoatedZnO nanomaterials, and no significant differences were observed between damaged skinversus normal healthy skin.
In view of the discussion above, it is assumed that penetration of the skin, if any, is caused
by Zn ions released from ZnO nanoparticles. Therefore the solubility of ZnO is one of thecritical parameters that should be considered in the characterization of ZnO used forsunscreen formulations.
General toxicity
In an acute oral toxicity study a dose of 2 g/kg body weight in rats induced some bodyweight decrease in one animal, although no deaths were observed. In one study all animals(mice) survived a single oral dose of 5 g/kg body weight of both nano-sized ZnO (50 nm)
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reduction in both male and female animals (Reference: AR16). After oral administration of
2.5 g/kg body weight, systemic uptake was demonstrated and indications for acute liver
toxicity (at 24-72 hours after administration) were noted in serum enzyme values andhistopathology (Reference: AR16). However, in another study a similar oral dose of 5 g/kg
body weight of ZnO nanoparticles in mice resulted in the death of one out of five animals
when ZnO of 20 nm in size was administered, whereas five out of ten animals died afteradministration of 120 nm diameter ZnO (Reference: 118). In addition, in several organs
e.g. liver and spleen, toxicity was observed for both the 20 nm and 120 nm ZnO particles.
Similar results were reported after oral administration in a recent rat study indicative ofliver toxicity following oral administration of 5 mg/kg body weight (Reference: AR20).
Overall, it can be concluded that after oral administration Zn can become systemicallyavailable resulting in liver toxicity.
A comparative (exploratory) intravenous toxicity study of ZnO as coated or uncoatednanoparticles, as a pigment form or as zinc sulfate was performed. No persistent effects
were observed at the end of the four week observation period when the various ZnO
preparations were injected intravenously as a single dose at levels of 1 or 5 mg/kg bw.
Deviations in some hematology parameters (monocyte, large unstained cells) and clinicalchemistry parameters (alterations in blood parameters ALP, AST and bilirubin) indicative ofan effect on liver function (coated and uncoated ZnO-nano preparations) and kidney
function (zinc sulfate) were observed 1 day after intravenous injection of 5 mg/kg bw
pigmented or coated nanoscale ZnO or an equimolar zinc sulfate dose. As at 1 mg/kg bw,
no effect was observed and a dose response relationship could not be established. There
was no indication for persistent toxic effects and, as at four weeks after the administration,
no alterations were observed in blood parameters evaluated for clinical pathology and forhistopathology in the major organs evaluated (brain, lung, liver, spleen and kidney). In
general there was no difference in the reactions between the various injected forms of ZnO.
There were no indications that nanoscale ZnO induced different effects compared topigmentary ZnO or zinc sulfate.
The intravenous administration provided 100% bioavailability of the nano-ZnO. A limitationof the study is that only a single administration was investigated, while for the Zn sulfate
the equimolar dose was administered both as a single dose or divided over four
administrations in four weeks. The comparison of coated and uncoated ZnO is limited toacute reactions as indicated by several alterations in blood parameters at day 1 after
treatment with 1 mg/kg bw or 5 mg/kg bw. A real comparison between coated anduncoated ZnO cannot be performed as toxic reactions were not observed. In view ofindications for liver damage, a repeated dose toxicity study would have provided better
information on potential toxicity.
In general it can be concluded that based on the observations on serum liver enzyme levels
and histopathology, the systemic availability of either ZnO nanoparticles or Zn ions has thepotential to induce liver toxicity.
No oral repeated dose toxicity study with ZnO nanoparticles is available.
Inhalation
In a 5 day exposure study in rats, ZnO induced a concentration-related inflammation
reaction in the lung which was associated with dose-dependent increases in BALF. In
addition to the inflammation reaction, necrosis was detected in the lung and the nose. There
was no difference between the nano-sized and pigmentary ZnO. Local effects in the lungwere still present 25 days after the exposure. There was no indication of systemic effects
after the 5 day inhalation exposure.
After inhalation exposure of human volunteers, no acute toxic effects were observed with adose of 500 μg/m3 air with 2 hours of exposure.

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In view of the observed effects in the respiratory tract a caution is warranted against theuse of ZnO nanoparticles in spray application despite the potentially low inhalation
exposure.
Mutagenicity
Mutagenicity has been addressed in the dossier by both in vitro and in vivo tests.
The bacterial reverse mutation assay (according to OECD Guideline 471) was performed
with dimethoxydiphenylsilanetriethoxycaprylylsilane coated ZnO nanoparticles in a panel of
Salmonella typhimurium strains, both with and without S9-mix and was negative..The
material tested negative for base pair changes and frame shift mutations, in contrast to thespecific positive controls (MNNG, MIT.C, NOPD, AAC and 2-AA). Bacterial mutagenicity
assays are judged less appropriate for testing of nanoparticles in comparison to mammaliancell systems in view of different internalisation pathways with eukaryotes and specificbactericidal effects of specific types of nanoparticles such as silver. According to the study
provided in the dossier, no signs of bacteriology were observed. However, uptake of thenanoparticles by the bacteria and the role of Zn ions were not investigated. Negative
findings in Ames tests (frame shifts, base pair substitutions) have also been reported in theliterature for tetramethylammoniumhydroxide-capped ZnO nanoparticles (Reference: AR26)
as well as ionic zinc (Reference: AR24).
A DNA damage potential of ZnO nanoparticles was revealed in A431 human epidermal cellsby an alkakine Comet assay. Lipid peroxidation and oxidative stress induction were
postulated as underlying mechanisms in the dossier. It should be emphasized however, that
this does not exclude a genotoxic potential of the treatment agent, i.e. the applied ZnOnanoparticles. Several other studies in the open literature have confirmed DNA damaging
effects with ZnO nanoparticles with the alkaline Comet assay (References: AR6, AR11,
AR12, AR14, AR22).
The mouse micronucleus assay (according to OECD Guideline 474) using intraperitoneal
administration showed an absence of clastogenic and aneugenic effects oftriethoxycaprylylsilane coated ZnO nanoparticles, whereas the controls cyclophosphamide
and vincristine sulfate tested positive. No evidence was provided that the ZnO nanoparticles(or dissolved ions) reached the bone marrow after the intraperitoneal applications, hence
the results of this study are of limited value.
The SCCS concludes that there is evidence for in vitro genotoxicity of ZnO nanoparticles as
demonstrated in the positive comet assay, but does not consider the negative results of thein vivo assay valid as exposure of the target organs was not demonstrated.
Both in vitro and in vivo data on photo-genotoxicity/mutagenicity were provided in thedossier. In Salmonella typhimurium mutation assays, ZnO revealed no gene mutations bybase pair changes or frame shifts in the presence or absence of irradiation. In otherinvestigations ZnO showed some clastogenic and photo-clastogenic potential. In CHO
fibroblasts, increased structural chromosome aberrations were found both in the absence orpresence of simulated sunlight. In CHO cells, clastogenic effects were observed after
treatment with ZnO in the presence or absence of simultaneous UV irradiation. Increased
chromosome aberrations were also observed in cells in which UV irradiation was performed
prior to ZnO treatment, suggesting that radiation increases the susceptibility of the CHO
cells to the clastogenic activity of ZnO. In vivo, ZnO was investigated in a photomicronucleus test in hairless mice after topical application and did not increase the incidenceof micronucleated epidermal cells in exposed animals with or without simulated lightirradiation.
The SCCS is of the view that ZnO nanoparticles are (photo)clastogenic in vitro. The negative
results in the in vivo micronucleus assay for epidermal cells can not be accepted as actual
exposure of the living epidermal cells was not demonstrated.

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Although the conclusion that UV-irridiation increases the sensitivity of thephotomutagenicity/clastogenicity assay may be valid, the induction of chromosomal
aberration in the absence of UV exposure should not be ignored. This should be considereda positive genotoxic activity of ZnO nanoparticles. Further studies available in the open
literature have shown that ZnO nanoparticles do not cause mutations in FE1-Muta™Mouselung epithelial cells (Reference: AR1), and induce micronuclei in A549 human lung epithelial
cells only at a concentration where marked toxicity was observed (Reference: AR2).
It has been discussed in the open literature that part of the toxicity of nano ZnO is due togeneration of reactive oxygen species, which has been associated with DNA damage(AR28). Scavenging of these oxygen radicals can be done by adding antioxidants or othercomponents to the formulations. The impact of this in relation to potential harmful effects ofnano ZnO in dermally applied cosmetic products is sofar not known.
Based on the available database and additional in depth evaluation of the studies, and inview of the uncertainties over whether or not nanoparticles reached the target cells/DNA in
the tests, there is no conclusive evidence to conclude whether or not micro-or nano-sized
ZnO particles pose a mutagenic/genotoxic, photo-toxic or photo-mutagenic/genotoxic risk
to humans. However, where ZnO nanoparticles are applied on the skin in a sunscreen
formulation, there is sufficient evidence to conclude that due to the very low if any systemic
exposure, the risk to the consumer is negligible. The evidence from in vitro and in vivo
studies presented in this dossier (section 3.3.4), and other studies on different metal/metaloxide nanoparticles (e.g. titanium dioxide – Nanoderm Project1) shows that penetration ofnano or larger particles is generally limited to the upper few layers of the stratum corneum
and there is no significant dermal penetration of the particles to systemic circulation. Whilst
this leaves the possibility for nanoparticle mediated local effects, it diminishes the possibility
for any harmful effects at the systemic level.
Toxicokinetics
After oral exposure there is some uptake of Zn in the systemic circulation. ZnOnanoparticles of approximately 50 nm in size (TEM evaluation) were compared to ZnO
microparticles showing at least one diameter >100 nm (TEM evaluation). After oral andintraperitoneal administration for both ZnO nanoparticles and microparticles, Zn could beobserved in serum indicating uptake from the GI–tract, either as particulate materials or as
dissolved Zn ions. For ZnO nanoparticles the systemic availability was somewhat higher
compared to that of ZnO microparticles as indicated by Zn measurements by ICP-MS. Zn
showed a higher distribution in the liver, spleen and lung after treatment with ZnO
nanoparticles compared to treatment with ZnO microparticles.
After inhalation exposure elevated zinc levels were detected in various organs, most likely
due to zinc ions dissolved from the ZnO particles.
There are no indications for significant if any penetration of nanoparticles through the skin,most likely only a minimal amount of Zn ions released from the nanoparticles may beavailable for systemic exposure.
Carcinogenicity
Specific data on carcinogenicity studies of ZnO nanomaterials are not available. In view ofthe occurring dissolution of the ZnO nanoparticles it can be assumed that the carcinogenic
risk is similar to the conventionally manufactured ZnO preparations. According to the EU
1

Nanoderm
Project:
nanoderm.pdf

http://ec.europa.eu/research/environment/pdf/env_health_projects/nanotechnology/n-95

SCCS/1489/12
Opinion on zinc oxide (nano form)___________________________________________________________________________________________

Risk assessment report (Reference 44, submission III), there is no clear experimental or
epidemiological evidence for a direct carcinogenic action of zinc or its compounds. .
4. CONCLUSION
Risk assessment of nanomaterials still has some issues to adress. At present, there are stillcertain gaps in the knowledge, for instance on the behaviour of nanoparticles in the test
medium or in the animals. This leads to uncertainties on whether the particles remain freeand hence able to reach the biological target sites and interact with various moieties, orthey agglomerate/ aggregate and become less biologically available. There are also
limitations in the validity of some of the tests used as they were developed for conventional(soluble) chemicals and not for nanoparticles. However, clear positive toxic responses insome of these tests clearly indicate a potential for risk to humans.

1. Does the SCCS consider zinc oxide in its nano-form safe for use as a UV-filter witha concentration up to 25% in cosmetic products taking into account the scientific
data provided?

With regard to applications resulting in dermal exposure
From the available information, there is no indication for penetration of ZnO nanoparticles
through the skin. In one study it was shown that Zn from ZnO nanoparticles in a tested
sunscreen formulation made a minor contribution to the blood Zn pool of human volunteers.
This shows that some Zn was absorbed from the sunscreen, although it was not known
whether this was absorbed in nanoparticulate form or as solubilized Zn ions. Considering thedissolution of ZnO, it is most likely that the zinc was absorbed in ionic form. The overall
weight of evidence therefore suggests that a very small proportion of Zn ions released fromthe ZnO nanoparticles may be available for systemic exposure when applied dermally.
With regard to applications resulting in other routes of exposure
Oral exposure
In one exploratory study in mice, systemic availability of Zn was indicated after a single oral
exposure. However, no differences weren observed between ZnO administered as nanoscaleor microscale particles. It is likely that absorbed Zn in the GI-tract was in the dissolved ionicform. In view of the data provided, oral exposure of nano-ZnO via applications of nano-ZnO
as a cosmetic ingredient in sunscreens should be considered to be of a similar risk to
micron-sized ZnO as previously evaluated in the RAR. The NOAEL for oral intake of ZnO is
50 mg /bw day (Reference 44, sub III). The oral exposure to ZnO nanoparticles as cosmetic
ingredient in sunscreens is limited to accidental ingestion of small fractions of lip products
and sun protection products and can be considered to be low.
Inhalation exposure
Upon inhalation of ZnO nanoparticles, serious local effects in the lung were observed. Even
if this may be due to the solubilized Zn ions, the effects are a direct result of the exposure
to the ZnO nanoparticles. Therefore, the SCCS is of the opinion that, on the basis ofavailable information, the use of ZnO nanoparticles in spray products cannot be considered
safe.

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In summary, the evidence presented in this submission and the available literature showed
that:•

There is no evidence for the absorption of ZnO nanoparticles through skin and via
the oral route. Even if there was any dermal and/or oral absorption of ZnO
nanoparticles, continuous dissolution of zinc ions would lead to complete
solubilization of the particles in the biological environment. In the MoS calculation,
the calculation of the exposure to ZnO nanoparticles assuming Zn2+ uptake resultsin acceptable MoSs for both the oral and dermal routes.

•

Nano ZnO-containing cosmetic formulations are likely to contain a small proportionof solubilized zinc, a further small proportion of which may be absorbed through skinand other routes. The rate and amount of the absorbed zinc is, however, likely to be
insignificantly small compared to the large zinc pool already present in the body.

•

Although the current evidence in relation to potenial genotoxicity of ZnO is notconclusive, the use of nano ZnO in cosmetic products should not pose a risk to theconsumer in the absence of a significant systemic exposure.

•

Based on the parameters described in the dossier, the different particle sizes, surface
modifications, and crystalline structures and morphologies investigated do notsignificantly alter the uptake, bioavailability and overall safety profile.

•

The different typical formulations as described in this submission also do not change
the overall safety profile of the tested ZnO nanoparticles.

In summary, it is concluded on the basis of available evidence that the use of ZnO
nanoparticles with the characteristics as indicated below, at a concentration up to 25% as a
UV-filter in sunscreens, can be considered not to pose a risk of adverse effects in humans
after dermal application. This does not apply to other applications that might lead toinhalation exposure to ZnO nanoparticles (such as sprayable products). Also, thisassessment only applies to ZnO nanoparticles that are included in this dossier, or are similarmaterials that have the following characteristics:• ZnO nanoparticles of purity ≥99%, with wurtzite crystalline structure and physicalappearance as described in the dossier, i.e. clusters that are rod-like, star-like and/or
isometric shapes.
• ZnO nanoparticles with a median diameter (D50: 50% of the number below thisdiameter) of the particle number size distribution between 30 nm and 55 nm, and theD1 (1% below this size) above 20 nm.
• ZnO nanoparticles that are either uncoated, or coated with triethoxycaprylylsilane,
dimethicone, dimethoxydiphenylsilanetriethoxycaprylylsilane cross-polymer, or octyl
triethoxy silane.
• ZnO nanoparticles that have a comparable solubility to that reported in the dossier,i.e. below 50 mg/L (approximately the maximum solubility of the ZnO nanomaterials
for which data are provided in the dossier).
Some tests were performed with a nano-ZnO material with a purity of ≥96% as indicated in
the submission. However, data on the purity was not provided in the physico-chemical
characterization section (3.1.4), and was also not provided in the various study reports
cited although in one report a content of 100% was indicated.
It is worth highlighting that this opinion has considered the small proportion of the absorbed
Zn following dermal application of nano ZnO to most likely be a solubilised ionic form. This
is in consideration of the solubility and dissolution aspects of ZnO described in section 3.1.6.
However, if any new evidence emerges in the future to show that the translocating species
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were in the form of insoluble and potentially persistent nanoparticles, then the SCCS may
consider revising the safety assessment of nano ZnO.
It should also be noted that the risk assessment of nanomaterials is currently evolving. In
particular, the toxicokinetics aspects have not yet been fully explored in the context ofnanoparticles (e.g. the size dependency). Also, long term stability of the coatings remains
unclear. At the moment, testing of nanomaterials and the present assessment, are both
based on the methodologies developed for substances in non-nano form, and the currently
available knowledge on properties, behavior and effects of nanomaterials. Therefore thisassessment is not intended to provide a blue-print for future assessments of othernanomaterials, where depending on the developments in methodological risk assessment
approaches and nano-specific testing requirements, additional/different data may berequired and/or requested on a case-by-case basis.
2. Does the SCCS confirm that zinc oxide in its non-nano form is safe for use as a UVfilter with a concentration up to 25% as stated in the SCCP clarification(SCCP/1215/09)?
The SCCS considers that the use of larger (non-nano) forms of ZnO as a UV-filter with aconcentration up to 25%, as stated in the SCCP clarification (SCCP/1215/09), is safe and is
not of any additional safety concern compared to the nano-forms assessed in this Opinion.
This is in view of the following:• When compared in terms of solubility, micro-sized ZnO has been shown to be lesssoluble (in water), and equally soluble (in tissue culture medium) compared to nanoZnO.
• Experimental evidence shows that both nano and non-nano particulate forms of ZnO
are not absorbed through the skin. Also on a theoretical ground, larger sized (nonnano) particles of ZnO are less likely to be absorbed through the skin than the nanoforms.
• As far as evaluated in the toxicity testing, micro-sized ZnO has been shown to induce
either similar toxic effects (in terms of general toxicity, lung toxicity after inhalation,
uptake from gastroIntestinal-tract, serum liver enzyme presence) or lower toxic
effects (in terms of genotoxicity, liver histopathology) when compared to nano-sized
ZnO. No toxic effects were observed at similar doses in a human volunteer inhalation
study for both nano-sized ZnO (40 nm) and fine ZnO (291 nm).
Thus the calculation of MoS for nano-sized ZnO for use as a cosmetic ingredient insunscreen formulations can also be used for the non-nano form of ZnO.
The SCCS would like to point out that a re-evaluation may be needed in the case of use ofother specific coatings or specific absorption enhancers in the formulation, which canpromote the dermal penetration of ZnO particles (nano or non-nano), or indications ofdermal absorption after long term use of nano ZnO containing formulations.
3.
And/or does the SCCS have any further scientific concern with regard to the use ofzinc oxide in cosmetic products?
ZnO is also used as a colorant in cosmetic products. In view of the similar toxicity of bothnano-sized and fine ZnO, it is considered that ZnO is safe when used as colorant incosmetics for dermal application. However, no information is available on whether and at
what concentrations ZnO is used in other cosmetic products that may lead to oral or
inhalation exposure. The MoS for such uses is uncertain.
In view of the lung inflammation induced by ZnO particles after inhalation exposure, the useof ZnO in cosmetic products which may result in inhalation is of concern.

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Any cosmetic products containing ZnO particles (nano or non-nano) with coatings that canpromote dermal penetration will also be of concern.
5. MINORITY OPINION

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BASF SE (2008). Composition of sun protection cream CM 643 (Z-COTE HP1®). BASF
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BASF SE (2008). Composition of sun protection cream CM 644 (Z-COTE®). BASF SE,
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BASF SE (2008). SEM analytics of sun protection solutions/crèmes (German report:
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11.

BASF SE (2008). Standard Specification, Care Materials, 3-103050, Z-COTE® HP1,
PRD 30083072. BASF SE, Ludwigshafen, Germany, 22 October 2008.

12.

BASF SE (2008). Test Method PM/00806/01e, Analysis of particle size distribution of
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13.

BASF SE (2008). Z-COTE®, Z-COTE® HP1, Z-COTE® Max, T-Lite® SF, T-Lite® SF-S,
T-Lite® max, Particle Morphology (German Report), GKP No. 96036. TEM laboratory,
BASF SE, Ludwigshafen, Germany, 4 August 2008.

14.

BASF SE (2009). Comparative table on physical-chemical properties of UV filters(ZCOTE®, Z-COTE®HP1). BASF SE, Ludwigshafen, Germany, 11 February 2009.

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BASF SE (2009). Personal Care Ingredients, Technical Information – UV Filters. BASF
SE, Ludwigshafen, Germany, 14 April 2009.

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BASF SE (2009). Sunscreen formulations: Lotions and cremes, Fa. Procter & Gamble,
all day moisture Lotion SPF 15, Olay, GKP No. 108088. SEM laboratory, BASF SE,
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BASF SE (2009). Sunscreen formulations: Lotions and cremes, Fa. Procter & Gamble,
all day moisture Lotion SPF 15, Olay, GKP No. 108088. TEM laboratory, BASF SE,
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