SCCS/1516/13
Revision of 22 April 2014

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Scientific Committee on Consumer Safety
SCCS

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OPINION ON

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Titanium Dioxide (nano form)

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COLIPA n° S75

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The SCCS adopted this opinion by written procedure on 22 July 2013

SCCS/1516/13
Revision of the opinion on Titanium Dioxide, nano form___________________________________________________________________________________________

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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.

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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).

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SCCS
The 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.).

35© European Union, 2013

Scientific Committee members
Ulrike Bernauer, Qasim Chaudhry, Pieter-Jan Coenraads, Gisela Degen, Maria Dusinska,
David Gawkrodger, Werner Lilienblum, Andreas Luch, Elsa Nielsen, Thomas Platzek, Suresh
Chandra Rastogi, Christophe Rousselle, Jan van Benthem.
Contact
European Commission
Health & Consumers
Directorate C: Public Health
Unit C2 – Health Information/ Secretariat of the Scientific Committee
Office: HTC 03/073
L-2920 Luxembourg
SANCO-C2-SCCS@ec.europa.eu

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ISSN 1831-4767

ISBN 978-92-79-30114-8

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Doi: 10.2772/70108

ND-AQ-13-007-EN-N

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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.

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http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm

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ACKNOWLEDGMENTSSCCS Members
Dr. Q. Chaudhry (chairman and rapporteur)Dr. U. Bernauer
Dr. W. Lilienblum
Dr. S. Ch. Rastogi
Prof. Th. Platzek
Dr. J. van Benthem
Dr. M. Dusinska
SCENIHR members
Prof. P. H. M. Hoet
External experts
Dr. W. Kreyling
Dr. R. Schins
Dr. W. H. de Jong
Dr. T. Jung
Prof. T. SannerDr. J. van Engelen
Dr. O. Bussolati
Prof. M-P Vinardell
Dr. Sh. DoakFor the revision
SCCS members
Dr. U. Bernauer
Dr. Q. Chaudhry
Dr. M. Dusinska
Dr. W. Lilienblum
Prof. Th. Platzek
Dr. S. Ch. Rastogi
Dr. J. van Benthem
SCENIHR members
Prof. P. H. M. Hoet
Dr. K. Rydzynski
External experts
Dr. O. Bussolati
Dr. W. H. de Jong
Dr. S. H. DoakProf. Th. Jung
Prof. T. Sanner
Dr. N. von Götz

(chairman and rapporteur)This opinion has been subject to a commenting period of six 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.
Keywords: SCCS, scientific opinion, UV filter, titanium dioxide (nano form), SCCS/1516/13,
directive 76/768/ECC, CAS number: 13463-67-7, EC: 236-675-5
Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion ontitanium dioxide (nano form), 22 July 2013, revision of 22 April 2014.
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Revision of the opinion on Titanium Dioxide, nano form___________________________________________________________________________________________1

TABLE OF CONTENTS

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1.

BACKGROUND ............................................................................................. 6
TERMS OF REFERENCE ......................................................................... 6
OPINION ............................................................................................ 7
Chemical and Physical Specifications....................................................... 7
1.3.1
Chemical identity .................................................................... 7
1.3.1.1 Primary name and/or INCI name ............................................... 7
1.3.1.2 Chemical names ...................................................................... 7
1.3.1.3 Trade names and abbreviations ................................................. 7
1.3.1.4 CAS / EC number..................................................................... 7
1.3.1.5 Structural formula ................................................................... 7
1.3.1.6 Empirical formula .................................................................... 7
1.3.2
Physical form ......................................................................... 7
1.3.3
Molecular weight .................................................................. 10
1.3.4
Purity, composition and substance codes .................................. 10
1.3.5
Impurities / accompanying contaminants ................................. 10
1.3.6
Solubility ............................................................................. 10
1.3.7
Partition coefficient (Log Pow) ................................................ 10
1.3.8
Additional physical and chemical specifications .......................... 10
1.3.9
Droplet size in formulation ..................................................... 12
1.3.10Particle size ......................................................................... 13
1.3.11
Microscopy .......................................................................... 14
1.3.12
Homogeneity and stability ...................................................... 15
1.4
Function and uses .............................................................................. 18
1.5
Toxicological Evaluation ...................................................................... 18
1.5.1
Acute toxicity ....................................................................... 18
1.5.1.1 Acute oral toxicity .................................................................. 18
1.5.1.2 Acute dermal toxicity ............................................................. 20
1.5.1.3 Acute inhalation toxicity ......................................................... 22
1.5.2
Irritation and corrosivity ........................................................ 25
1.5.2.1 Skin irritation ........................................................................ 25
1.5.2.2 Mucous membrane irritation .................................................... 29
1.5.3
Skin sensitisation .................................................................. 31
1.5.4
Dermal / percutaneous absorption........................................... 33
1.5.5
Repeated dose toxicity .......................................................... 59
1.5.5.1 Repeated Dose (30 days) oral toxicity ...................................... 59
1.5.5.2 Sub-chronic (90 days) toxicity (oral, dermal) ............................. 59
1.5.5.3 Chronic (> 12 months) toxicity ................................................ 61
1.5.6
Mutagenicity / Genotoxicity .................................................... 61
1.5.6.1 Mutagenicity / Genotoxicity in vitro .......................................... 62
1.5.6.2 Mutagenicity/Genotoxicity in vivo ............................................. 73
1.5.7
Carcinogenicity ..................................................................... 77
1.5.8
Photo-carcinogenicity ............................................................ 84
1.5.9
Reproductive toxicity ............................................................. 85
1.5.9.1 Two generation reproduction toxicity ........................................ 87
1.5.9.2 Teratogenicity ....................................................................... 87
1.5.10
Toxicokinetics ...................................................................... 87
1.5.11
Photo-induced toxicity ........................................................... 89
1.5.11.1 Phototoxicity / photoirritation and photosensitisation ................ 89
1.5.11.2 Phototoxicity / photomutagenicity / photoclastogenicity ............. 92
1.5.12
Human data ......................................................................... 93
1.5.13Special investigations ............................................................ 94
1.5.14
Human safety evaluation (including calculation of MoS) .............. 94
1.5.15
Discussion ........................................................................... 94
2.
CONCLUSIONS .......................................................................................... 99
3.
MINORITY OPINION.................................................................................. 102
4.
REFERENCES ........................................................................................... 102
1.1
1.2
1.3

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1. BACKGROUND
The first scientific opinion on the safe use of titanium dioxide as a UV-filter at a maximum
concentration of 25% in cosmetic products was adopted 24 October 2000 by the SCCNFP(SCCNFP/0005/98).
However, a review of the substance in its nanoform is deemed necessary according to theopinion on Safety of Nanomaterials in Cosmetic Products adopted on 18 December 2007
(SCCP/1147/07), where it is stated that:"The SCCNFP opinion from 2000 (SCCNFP/0005/98) is on micro-crystalline preparations ofTiO2 and preparations of coarse particles. However, since this opinion, new scientific dataon nanosized particles including, TiO2 has become available. Therefore, the SCCP considers
it necessary to review the safety of nanosized TiO2 in the light of recent information. Also, asafety assessment of nanosized TiO2, taking into account abnormal skin conditions and thepossible impact of mechanical effects on skin penetration needs to be undertaken".
Supplementary information on nanosized Titanium dioxide was submitted following ameeting with stakeholders on 1 October 2008, where data requirements were agreed.
Titanium Dioxide is currently regulated - irrespectively of its form - as a UV-filter in aconcentration up to 25% in cosmetic products in Annex VII, entry 27 of the Cosmetics
Directive.

1.1

TERMS OF REFERENCE

1.

Does SCCS consider that use of titanium dioxide in its nanoform as a UV-filterin cosmetic products in a concentration up to maximum 25.0 % is safe for the
consumers taken into account the scientific data provided?

2.

In order for the COM to differentiate in the regulation between materials in its
nanoform and its non-nano form, can the SCCS give quantitative andqualitative guidance on how this differentiation should be given based on theparticle size distribution or other parameters?

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1.2

OPINION3

1.3

Chemical and Physical Specifications

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1.3.1

Chemical identity

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Titanium Dioxide

61.3.1.1 Primary name and/or INCI name7

Titanium Dioxide

81.3.1.2 Chemical names

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Titanium Dioxide

101.3.1.3 Trade names and abbreviations

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COLIPA No. S75

121.3.1.4 CAS / EC number

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CAS number: 13463-67-7
EC: 236-675-5
Other registry numbers: 100292-32-8; 101239-53-6; 1025343-79-6; 116788-85-3; 1200059-8; 1205638-49-8; 1236143-41-1; 12701-76-7; 12767-65-6; 12789-63-8; 1309-63-3;
1344-29-2; 1377807-26-5; 1393678-13-1; 1400974-17-5; 158518-86-6; 185323-71-1;
185828-91-5; 188357-76-8; 188357-79-1; 195740-11-5; 221548-98-7; 224963-00-2;
246178-32-5; 252962-41-7; 37230-92-5; 37230-94-7; 37230-95-8; 37230-96-9; 3932058-6; 39360-64-0; 39379-02-7; 416845-43-7; 494848-07-6; 494848-23-6; 494851-77-3;
494851-98-8; 52624-13-2; 55068-84-3; 55068-85-4; 552316-51-5; 62338-64-1; 76734100-4; 859528-12-4; 861455-28-9; 861455-30-3; 866531-40-0; 97929-50-5; 98084-96-9.
[Source: ChemIdPlus]

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1.3.1.5 Structural formula

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TiO2

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1.3.1.6 Empirical formula

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Formula:

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1.3.2

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Titanium Dioxide (TiO2¸ COLIPA No. S75, CAS No. 13463-67-7) is described as a solid,
white, odourless powder. The TiO2 materials used in sunscreen products are reported to becomposed of two crystalline types: rutile and anatase or a mixture of the two. The different
materials included in the dossier have been reported to be needle, spherical, or lanceolate(longer than wide) in shape. The primary particle size of the TiO2 nanomaterials has beenreported to range from around 20 to 100 nm.

TiO2

Physical form

Nanoparticles are generally known to have a tendency to stick together to form
agglomerates and/or aggregates, and it is claimed by the Applicant that, in sunscreen
products, TiO2 is not present in the form of primary nanoparticles but as aggregates of a6

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size between 30 nm to >150 nm. These aggregates are claimed to be formed during the
manufacturing process.

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It has been stated by the Applicant that ‘[the stability of coating] is certainly less relevantfrom a human-safety aspect, especially since materials used as coating agents for TiO2 may
be present as constitutive ingredients of the same cosmetic product’. This may be true forsome materials, but it also needs to be considered that a range of materials has been used
for coating the TiO2 nanomaterials under current assessment. Some of these materials havebeen used in a substantially high coating to nanomaterial ratios (e.g. 16% alumina).
Although a few studies showing coating stability have been provided, it is important to knowwhether this, for example, could lead to the release of aluminium ions from alumina that
may be present after the coating process and which may dissolve in the final formulation.
Thus, where appropriate, safety of the coating materials should also be considered in theirown right because any significant dissolution of a coating component, such as alumina, may
require a separate safety assessment.

Fifteen (15) TiO2 nanomaterials have been presented in the submission for evaluation. They
include uncoated as well as surface-coated nanomaterials with various organic and inorganic
coating materials. A range of coating materials has been used which include hydrophilic,hydrophobic and amphiphilic materials, such as alumina/silica, methicone/silica, aluminiumhydroxide and dimethicone/methicone copolymer, trimethyloctylsilane, alumina/silicone andalumina/silica/silicone,
dimethicone,simethicone,
stearicacid,
glycerol,dimethoxydiphenylsilane, triethoxycaprylylsilane (Table 1).
The coating materials have been stated by the Applicant to be those that are common
cosmetic ingredients. The purpose of coatings has been stated to include improvement of
the dispersion of TiO2 nanomaterials within the cosmetic formulation, inhibiting orcontrolling photoactivity, and improving compatibility with other ingredients in sunscreen
formulations. The coatings applied to nanoparticle surface are also stated to be not UV
absorbers themselves.
SCCS Comment
For this opinion, the trade names of the nanomaterials under assessment have been codedby the SCCS and are referred to by the relevant codes.

Three studies have been provided (submission II – Ref 62 and 63, and Submission III – Ref68) to indicate that the coatings (e.g. silica/alumina) are stable in formulation, as well as
under different conditions of pH, temperature, shear force, etc. However, from the otherphysicochemical data provided, it is less clear how stable the coatings are in final
formulations. The photocatalytic activity data, measured in formulations, indicate that eithersome of the materials were not completely coated, or some of the coatings were not stablein the formulations.
Despite the fact that the materials used as coatings to TiO2 nanomaterials have a wide
diversity, and some of them have been used in substantially high proportions (e.g. 16%
alumina), putative exposure to the coating materials has not been considered in theassessment. Although a few studies showing coating stability have been provided, it isimportant to know the concentration of any dissolved coating materials, e.g. aluminium
ions, in the final formulation. For example, in a recent study, Virkutyte et al (2012) foundthat chlorine in swimming pools could potentially strip the coating from titanium dioxide
nanoparticles in sunscreens. The study, however, relates to a specific use scenario – i.e.
where TiO2 nanomaterials are coated with aluminium hydroxide (Al(OH)3), and the productis used in chlorinated water (e.g. in a swimming pool). It is also likely that the coatingstripped nanoparticles will be washed off the skin during swimming or bathing afterswimming. Although this specific type of coating/use scenario relates more to risk
management than risk assessment, any significant dissolution of some coating materials(e.g. alumina) may require a separate safety assessment for the uncoated nanomaterial aswell as the coating material.
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In view of this, the SCCS has only recommended the types of coatings covered in this2
opinion. Other cosmetic ingredients applied as stable coatings on TiO2 nanomaterials can
3also be used, provided that they can be demonstrated to the SCCS to be safe and the4coatings do not affect the particle properties related to behaviour and/or effects, compared5
to the nanomaterials covered in this opinion.
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Table-1: Form and composition TiO2 nanomaterials *8Material TiO2
Coating material
Doping
Form
Bulk
VSSA
code
crystalinematerial
density(m2 cm-3)
form(g/cm3)
S75-A> 99.5%6% silica, 16% alumina None
Oil
0.35
460
Rutiledispersion
S75-B> 99.5%6% silica, 16% alumina None
Aqueous0.35
460
Rutiledispersion
S75-C> 99.5%
7.5% alumina, 9,5%
None
Oil
0.31
220
Rutilealuminium stearate
dispersion
S75-D> 99.5%10% alumina, 13.5%
None
Oil
0.58
300
Rutilestearate
dispersion
S75-E> 99.5%10% alumina, 13.5%
None
Aqueous0.58
300
Rutilestearate
dispersion
S75-F
Anatase
7.5%
None
Hydrophobic 0.2
19285%, Rutile
trimethoxycaprylylsila
powder15%
ne
S75-G
AnataseNone
None
Hydrophilic
0.13
21385%, Rutile
powder15%
S75-H> 99,5%6% alumina, 1%
None
Hydrophilic
0.31
260
Rutileglycerin
powder
S75-I> 99,5%7% alumina 10%
None
Hydrophobic 0.28
300
Rutilestearic acidpowder
S75-J> 99,5%6% alumina 1%
None
Hydrophobic 0.31
260
Rutiledimethicone
powder
S75-K> 94% Rutile 6-8% aluminium
None
Hydrophobic 0.12-0.28 426
hydroxide, 3.5-4.5%
powderdimethicone/methicone copolymer
S75-L> 94% Rutile 6.5-8.5% hydrated
None
Hydrophobic 0.07-0.2426
silica, 2.5-4.5%
powderaluminium hydroxide,
4.5-6.5%
dimethicone/methicone copolymer
S75-M> 98% Rutile, 17% silica
None
Hydrophilic
0.09
260<2% anatasepowder
S75-N> 95% Rutile, Alumina 10%1000 ppm
Amphiphilic
0.16
400<5% anatase simethicone 2%Fepowder
S75-O
100%
Simethicone 5%
None
Hydrophobic 0.75
400
Anatasepowder910* Regarding purity/impurity all materials are claimed by the applicant to conform with USP
1135 requirements: TiO2 (99.0-100.5%), Loss on Ignition (≤ 13%), Water-soluble substances12(≤ 0.25%), Acid-soluble substances (≤ 0.5%), Arsenic (≤ 1 ppm), Residual Solvents (No13solvents used),14
and FDA requirements: Lead (HCl-soluble) (≤ 10 ppm), Antimony (HCl-soluble) (≤ 2 ppm),
15
Mercury (≤ 1 ppm).
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For purity/impurity, all materials were tested as uncoated and untreated material.

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1.3.3

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Molecular weight of TiO2: 79.9 g/mol.

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1.3.4

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According to the Applicant, the TiO2 nanomaterials have been produced according to USP
31 specifications, in high purity, with concentration of the active material ≥99.0 %. It isalso stated that the materials do not contain heavy metals (e.g., Hg, Cd, Pb, As or Sb)
beyond the generally accepted limits.

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Purity/impurity has been referred to USP-35 in the additional information provided by theapplicant. USP-31 is an earlier edition of USP-35.

SCCS comment
Analytical data on purity and impurities were not submitted, purity was only referred to USP
and FDA requirements. Analytical data on purity and impurities of each nanomaterial should
be provided.
Molecular weight

Purity, composition and substance codes

SCCS Comments
The nanomaterials included in the submission have been stated to be manufactured
according to USP-31 specifications, with no heavy metals beyond the ‘generally accepted
limits’. The Applicant should provide the contents of heavy metals, such as Hg, Cd, Pb, As
and Sb, which are considered ‘acceptable’ under USP-31, as they may or may not be
considered acceptable under the EU regulations. In addition, impurities of well-known
metallic contact allergens, such as Cr, Co, Ni, should also be reported.

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1.3.5

Impurities / accompanying contaminants

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See SCCS comment under 1.3.2

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1.3.6

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TiO2 is insoluble in water and organic solvents. It also has a very low dissociation constantin water and aqueous systems, and thus can in practice be considered as insoluble also
under the physiological conditions.
(Numerous references in open literature)

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1.3.7

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Log Pow: Not applicable for uncoated TiO2.

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1.3.8

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Melting point:

Solubility

Partition coefficient (Log Pow)
(Reference: 137)

SCCS Comment
A method to determine partition coefficient of nano particles coated with organic materials
is not yet available. However, distribution of TiO2 nanomaterials coated with organic
substances between polar and non polar phases should be described.

Additional physical and chemical specifications
Not provided
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Boiling point:
Not applicable2
Flash point:
Not applicable
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Vapour pressure:
Not applicable4
Density:
The Tap Density of the titanium dioxide powders was
5measured according to DIN ISO 787/11 (Table 1)
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Viscosity:
Not provided
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pKa:
Not applicable for uncoated TiO2
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Refractive index:
Not provided
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UV_Vis spectrum (200-800 nm): UV data only (see Table 3)
1011SCCS Comment
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The dissociation kinetics of the materials in acidic media can be potentially modified by
13certain coatings. However, considering the physicochemical properties of TiO2, it is agreed
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that, for TiO2 nanomaterials, coatings are unlikely by definition to change the dissociation
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constant of TiO2 in water.
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Table-2: Physicochemical properties of TiO2 nanomaterials
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Material
Crystal Aspect UV Absorption
Zeta
Photo-catalytic
PhotoCoating
code
size
ratio
(Extinction coefficient) potential activity*
stability
stability(XRD)(L /W)
E308
E360
E400
(IEP)
∆E
% to
Reference
S75-A
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3.8
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11
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3
9
PhotoStablestable
S75-B

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3.8

51

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N/A

3

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S75-C

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3.7

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N/A

7.8

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S75-D

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4.5

48

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3

N/A

7.2

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S75-E

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4.5

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10

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N/A

7.2

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S75-F

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1.2

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N/A

11.8

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S75-G

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1.2

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16

9

7

25.1

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S75-H

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1.7

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17

9

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0.3

1

S75-I

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3.2

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14

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N/A

0.8

2

S75-J

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1.5

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16

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N/A

0.6

2

S75-K

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3.9

60

12

1

N/A

2.3

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S75-L

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4.3

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2

N/A

0.8

2

S75-M

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2.6

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2

0.6

2

S75-N

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4.1

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0.7

2

S75-O

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1.2

20

8

5

N/A

15.7

46

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21

Photostable
Photostable
Photostable
Photostable
Photostable
Photostable
Photostable
Photostable
Photostable
Photostable
Photostable
Photostable
Photostable
Photostable

StableStableStableStableStableNA
StableStableStableStableStableStableStableStable* Photocatalytic activity 5% TiO2 formulation irradiated in a Suntest CPS+ solar simulator
for 30 minutes at 300 W/m^2. Sample measured before and after using a colourimeter.
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Calculation ∆E = [(∆L*)^2+(∆a*)^2 + (∆b*)^2]^1/2; Reference uncoated TiO2 ∆E = 34.
See Egerton et al. (2007) for more details on the method.

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1.3.9

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SCCS Comment
The photoreactivity of a chemical is generally determined in terms of degradation of anorganic substance (e.g. iso-propanol, propanone, salicylic acid, or an organic dye such as
methylene blue) on exposure to UV irradiation. Regarding measurement of photocatalytic
activity of nanomaterials, the OECD guidance (2010) provides further information and also
cites the methods described in ISO TC 206/WG37 (Fine ceramics – Test methods forphotocatalytic material).
In regard to the TiO2 nanomaterials under evaluation, the SCCS accepted the applicant’s
provided data from a different method used for measuring photocatalytic activity. The
method, which is described by Egerton et al. (2007), is based on photogreying of the TiO2
material on exposure to UV irradiation. Although the test is based on a non-standard
method, the SCCS accepted the data in view of the published work by Egerton et al. (2007),
which indicates measurable photogreying of TiO2 nanomaterials upon UV irradiation. As
such, the method will not be applicable to other nanomaterials because they may not turngrey on exposure to UV irradiation, and/or may already have a colour.
Nanomaterials used in cosmetic products should ideally be non photocatalytic. However, inview of measurement uncertainties, the SCCS has considered acceptable an arbitrary levelof up to 10% photocatalytic activity of a coated or doped nanomaterial, measured in terms
of % to a reference standard (which is uncoated/undoped form of the same nanomaterial).

Droplet size in formulation

According to the information provided by the Applicant, sunscreen spray products containing
nano-sized TiO2 are available on the EU market. These spray products are formulated with
non-volatile ingredients in pump sprays (without propellant gas) to generate minimal
aerosol cloud. It is stated that these products comply with current standards andrequirements in terms of droplet size, Mass Median Aerodynamic Diameter (MMAD) of atleast 30 μm, with no more than 1% of the droplets having an aerodynamic diameter of 10
μm or less. The Applicant has quoted the Technical Guidance Document on Risk Assessmentof the European Chemical Bureau (2003), which considers aerosols with an MMAD >10-15
μm as not respirable for humans because of deposition mainly in the upper regions of thelungs (Reference 148). It is also quoted that the U.S. Silicones Environmental, Health and
Safety Council (2001) suggests that a consumer aerosol application for any silicone-basedmaterial, regardless of the method of aerosol generation, should have particle size MMAD atleast 30 μm, with no more than 1% of the particles having an aerodynamic diameter of 10
μm or less (Reference 203). The Applicant has provided droplet size distribution
measurements for a few sprayable products. The technique used for droplet size
measurement was based on Laser Diffraction by Malvern method.
SCCS Comments- The trade name of one sprayable product suggests that it may be for use by children.

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The droplet size of an aerosolised formulation would affect the entry and uptake ofnanomaterial in the lung. It is therefore noteworthy that whilst droplet size would
depend on nebulizer/ matrix, it may change due to evaporation/sublimation of the fluid
used in the emulsion. Thus, the characteristic dimension of a nanomaterial contained in
the formulation would have little relevance to the droplet size, which is typically muchlarger (tens of micron).

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Although the measurement results indicate that droplet sizes were largely above therespirable range (>10 µm), and only 0.24 to 0.37% of the droplets were in the size
range below 20 µm, it should be noted that even a low fraction based on droplet weightis still relevant because it will contain a large number of nanoparticles. The possibility ofdroplets drying and becoming smaller in size following spraying, and the possible lung11

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exposure to dried residual particles after inhalation also needs to be taken into account.
The measurement of the droplet size distribution therefore needs to be complemented
by measurements of the size distribution of the dried residual aerosol particles as well, if
they can dry on the timescale in a practical use scenario.

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1.3.10 Particle size

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The size distribution of the droplets and dried droplets/ particles should be presented as
number size distribution.

Table 3: Particle size of TiO2 nanomaterials
Material
Particle Size Distribution*
code
Lower Cut Off level (nm)Volume weighted median,X50,3 (nm)LUMisizer
DLS
71
111

Average**
78

Number weighted median, X50,0(nm)
LUMiAverag
CPS sizer
DLS e**
37
48
79
55

S75-A

CPS
20

LUMisizer
33

DLS
35

Averag
e**
29

CPS
53

S75-B

28

34

47

36

68

76

145

96

47

56

105

69

S75-C

20

25

26

24

52

49

78

60

39

48

59

49

S75-D

17

23

15

18

35

44

56

45

28

34

34

32

S75-E

21

27

41

30

45

51

104

67

37

42

81

53

S75-F

35

49

63

49

75

92

139

102

55

70

115

80

S75-G

25

58

54

46

77

99

129

102

45

79

102

75

S75-H

29

63

41

44

71

120

112

101

50

79

82

70

S75-I

22

58

41

40

73

107

140

107

40

76

103

73

S75-J

33

52

35

40

71

103

125

100

48

69

85

67

S75-K

26

34

30

30

48

52

75

58

41

44

58

48

S75-L

33

37

41

37

56

64

103

74

46

53

80

60

S75-M

42

75

73

63

119

124

173

139

75

99

133

102

S75-N

21

37

26

28

51

61

91

68

41

51

65

52

S75-O

24

71

47

47

354

653

146

384

33

87

85

68

* The particle size distribution was measured by three different methods - Differential
Sedimentation Analysis (CPS disc centrifuge); Integral Sedimentation Analysis (LUMiSizer
centrifuge); and Dynamic Light Scattering (Malvern HPPS). In addition, Electron microscopy(SEM and TEM) images of representative nanomaterials have been provided.
** average of median values from the three measurement methods.
According to the applicant, all samples were measured in a standardized fashion according
to specific standard operating procedures as follows:
1. Hydrophilic Powder: 1) Add 30 ml SHMP-solution (0.02 g sodium hexametaphosphate to
30 ml deionised water) to 0.2 g titanium dioxide powder in the glass beaker and agitate thesample gently with an overhead or magnetic stirrer for 15 minutes to ensure homogeneity;
2) Disperse the probe using an ultrasonic probe (power 50 Watts) for 10 minutes. The
ultrasonic horn should not touch the side of the glass beaker or the bottom. The
suspensions should be cooled during the dispersion.
2. Hydrophobic Powder: 1) Add 1 ml isopropyl alcohol to 0.2 g titanium dioxide powder inthe glass beaker. To wet the powder slew the beaker carefully; 2) Add 1 drop Disperbyk
190 (BYK Chemie, Germany) after adding isopropyl alcohol; 3) Add 30 ml SHMP-solution
into the beaker and agitate the sample gently with an overhead or magnetic stirrer for 15
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minutes to ensure homogeneity; 4) Disperse the probe using an ultrasonic horn (50 Watts)
for 10 minutes. The ultrasonic horn should not touch the side of the glass beaker or thebottom. The suspensions should be cooled during the dispersion.

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1.3.11 Microscopy

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An example transmission electron microscopy (TEM) image of TiO2 nanomaterial is shown
below:

3. Oil based Dispersion: Dilute the dispersion to 1% solids by cyclohexane (solids content ofdispersion must be supplied by company.
4. Water based dispersion: Dilute the dispersion to 1% solids by deionised water (solids
content of dispersion must be supplied by company). Agitate every sample gently with thestirrer for 1 hour for equilibration before measurement.
SCCS Comment
The different materials included in the dossier have different particle sizes. These range
from ~45 nm to 384 nm on volume weighted median basis (average of 3 measurement
methods), and ~32 nm to ~102 nm on the basis of number weighted median (average of 3
measurement methods). The lower size cut offs (average of 3 measurement methods)
range between 18 nm and 63 nm. Note that different methods are typically characterised bysystematic, or partially systematic, different measurement uncertainties depending on thesize range measured. Therefore the average of different measurement methods on thesame nanomaterial does not necessarily provide a more reliable value than measured by anindividual method, but has been adopted as a practical approach to size determination.

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An example Cryo-TEM image of TiO2 nanomaterial in formulation is shown below:

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SCCS Comment
The different nanomaterials included in the dossier have primary particles that have either
spherical, needle, or lanceolate (longer than wide) shapes, and appear to be present inaggregated clusters.

1.3.12 Homogeneity and stability
According to the Applicant, the term “dispersion” has been used in relation to the dispersionof TiO2 clusters/ aggregates in the cosmetic product, whereas aggregates bound by strong
forces could not be dissociated. They also claim that coating materials on the TiO2 particle
are stable under various conditions of pH, temperature and shear forces, and that thematerials used as coating agents for TiO2 may also be present as constitutive ingredients ofthe same cosmetic product.
SCCS Comments on Physicochemical Characterisation
The physicochemical characterisation data provided in the dossier relates to fifteen (15)
TiO2 nanomaterials. The data are reasonably extensive, which show that:

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1. Ten out of the 15 materials (S75-A, S75-B, S75-C, S75-D, S75-E, S75-H, S75-I, S75J, S75-K, S75-L) are rutile. Two other materials (S75-M, S75-N) are mainly rutile witha small proportion (2-5%) of anatase.

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2. One material (S75-O) is anatase. Two other materials (S75-F and S75-G) are mainly
anatase (85%) with rutile (15%).

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3. The primary crystal size of the materials range between 9 and 21 nm. The average
particle sizes in dispersions (measured by 3 different methods) range from ~45 nm to384 nm on volume weighted median basis (average of 3 measurement methods), and~32 nm to ~102 nm on the basis of number weighted median (average of 3

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measurement methods). The lower size cut offs (average of 3 measurement methods)
range between 18 nm and 63 nm.

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4. One material (S75-G) is uncoated, all other materials are surface coated with different
coating materials (silica, alumina, organo-silanes).

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5. All coatings are reported to be stable at least in the short-term in vitro test systems.
In view of the diversity of the coating materials and some high coating tonanomaterial ratios, it is important to know the concentration of dissolved coating
materials, e.g. alumina that could release aluminium ions, in the final formulation. A
significant dissolution of a coating material (e.g. alumina) may require a separate
safety assessment for the coating material.

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6. One material (S75-N) is doped with 1000 ppm iron. All other materials are not doped.

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7. The apparent bulk density of the materials ranges between 0.09 to 0.75 g/cm3. The
SCCS notes that the lowest density reported for some materials does not fit in thenormal range. As all materials have core particles of TiO2, with sizes in the nanoscale, it is not clear why there is such a large variation in their bulk densities. The
Applicant needs to clarify whether the materials with low bulk densities have a porous
structure, as in such a case they may have different physicochemical properties fromthe other TiO2 materials.

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8. One material (S75-E) is in aqueous dispersion. All other materials are eitherhydrophilic or hydrophobic powders, or are in oil dispersions.

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9. The VSSAs of the materials range between 192 to 460 m2/cm3 for the different
materials, indicating that they are indeed nanomaterials (i.e. VSSA ≥60 m2/cm3).

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10. Aspect ratios of the different materials range between 1.2 and 4.5, indicating that thehigh aspect ratio materials have needle or lanceolate shaped particle structures.

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11. All materials are stated to be photostable.

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12. UV absorption data for the materials have been provided.

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13. Zeta potential measurements have been provided for some materials, and not forothers due to difficulties in measuring zeta potential for hydrophobic nanomaterials.

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14. Photocatalytic activity data have been provided for all materials (see Table 2, andcorresponding SCCS comments). The data show that the materials have differinglevels of photocatalytic activity, which ranges from insignificant to weak (S75-A, S75B, S75-H, S75-I, S75-J, S75-K, S75-L, S75-M, S75-N), to moderate (S75-C, S75-D,
S75-E), and strong (S75-F, S75-G; S75-O). All 3 nanomaterials with strong
photocatalytic activity are also either anatase form of TiO2, or mainly anatase withsome rutile.

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From the physicochemical characterisation data provided, the materials could be broadlygrouped as shown below for the purpose of this assessment. This grouping is based on the
differences between physicochemical properties and the potential effects of anatase/rutile,
coated/uncoated, and photocatalytic/non-photocatalytic forms of TiO2 nanomaterials. It is
known that uncoated and non-doped TiO2 nanoparticles are photocatalytic when exposed to
UV light. The anatase form has been shown to be more photoreactive than rutile oranatase-rutile mixtures (e.g. Sayes et al., 2006). Another indicator of catalytic activity ofnanomaterials is the increased generation of reactive oxygen species (ROS) in biologicalsystems and the resulting toxicological effects, such as cytotoxicity. Jiang et al. (2008)
noted that the generation of ROS (per unit surface area) was the highest in amorphous
nano-TiO2, followed by anatase, anatase/rutile mixture, and rutile. Anatase form of nanoTiO2 has also been reported to be 100 times more cytotoxic under UV than rutile of asimilar size (e.g. Sayes et al., 2006). These aspects have already been highlighted in the
SCCP opinion on Safety of Nanomaterials in Cosmetic Products (SCCP/1147/07) in thephototoxicity part (page 33):

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‘When coupled with UV irradiation, anatase TiO2 (hydrophilic, circa 20 nm) was clearly more
photogenotoxic than TiO2 (anatase and rutile, both 255 nm) in mouse lymphoma L5178Y
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cells, as measured by the comet assay (Nakagawa et al. 1997). Rutile of larger particle size(420 nm) was not photogenotoxic. The nanosized anatase TiO2 was also photogenotoxic in
Chinese hamster lung CHL/IU cells, when assessed by chromosome aberration induction,
but not in Salmonella typhimurium or in mouse lymphoma L5178Y tk+/- cells, when studied
for mutation induction (Nakagawa et al. 1997). Furthermore, this nanosized TiO2
(hydrophilic surface) only induced DNA damage, chromosome aberrations and mutations
with UV radiation.’

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* S75-K and S75L have stated purity of >94% with no impurity profile provided.

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On the basis of above physicochemical considerations, the SCCS has considered the TiO2
nanomaterials in the following 3 groups of for the purpose of this assessment:

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9 materials (S75-A, S75-B, S75-H, S75-I, S75-J, S75-K, S75-L, S75-M, S75-N) on thebasis that they are (mainly) rutile with a relatively low photocatalytic activity. However,two of these materials (S75-K and S75-L) have a stated purity of >94%, with noimpurity profile provided. These two materials (S75-K and S75-L) were considered by
the SCCS to be not sufficiently pure to include in this opinion.

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3 materials on the basis that they are rutile with a moderate photocatalytic activity(S75-C; S75-D; S75-E);

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3 materials on the basis that they are (mainly) anatase, and also that they have astrong photocatalytic activity (S75-F, S75-G, S75-O).

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In view of the foregoing, it is important to note that this opinion applies to all fifteen (15)nanomaterials presented in this submission. The opinion may, however, be also applicableto other TiO2 nanomaterials that have similar characteristics to the 15 nanomaterials in thissubmission in terms of the physicochemical parameters listed in Tables 1-3, and otherspecific provisions laid out in Section 2 below.

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1.4

Function and uses

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Titanium dioxide is used as an UV-filter in a concentration up to 25% in cosmetic products.
It is regulated in Annex VII, entry 27 of the Cosmetics Directive

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1.5

Toxicological Evaluation

1.5.1

Acute toxicity

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1.5.1.1 Acute oral toxicity
Acute toxicity, single oral administration, rat
Guideline:
OECD Guidelines 401 and EEC Guidelines 92/32/EEC
Species/strain:
8 week old rats/Hsd-Win: WUGroup size:
5 male/ 5 female
Test substance:
TiO2 T805, hydrophobic fluffy white powder, CAS 100209-12-9
Batch:
27073
Purity:TiO2 96.5%, SiO2 3%, carbon approx 4%.
Vehicle:suspension in peanut oil
Dose levels:
2150 mg/kg
Dose volume:
21.5 ml/kg of 100 mg/ml
Route:
Oral
Administration:
single dose
GLP:
yes
Study period:
August 1993
References
Submission I - Evonik (Degussa) 1993 (5) and DHS Evonik (Degussa) 1993 (1)Results
No signs of toxicity recorded during the observation period, no deaths recorded, necroscopy
showed no alterations, LD50 for male and female rats >2150 mg/kg.
SCCS Comment
The study relates to S75-F material included in the dossier, which is anatase/rutile material,with organic coating of trimethoxy-caprylylsilane, in oily suspension. This study is relevantto the nanomaterial group (85% anatase, 15% rutile).
Acute toxicity, multiple oral administration, rat
Guideline:
OECD Guidelines 401 and EEC Guidelines 92/32/EEC
Species/strain:
7 week old male rats, 8 week old female rats /Hsd-Cpb: WUGroup size:
5 male/ 5 female
Test substance:
TiO2 T817, hydrophobic fluffy white powder, CAS 100209-12-9
Batch:
04095
Purity:TiO2 >97%, Fe2O3 2±1%, carbon 3.5-4.5%.
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Vehicle:suspension in olive oil
Dose levels:
Total dose of 2150 mg/kg (dosed twice in equal amount)
Dose volume:
twice dose of 21.5 ml/kg of 50 mg/ml
Route:
Oral
Administration:
single dose
GLP: yes
Study period:
DHS Evonik (Degussa), 1993 (2)Results
No signs of toxicity were recorded during the observation period, no deaths recorded, only
signs of diarrhoea in 2 male and 1 female rats from day 1 until day 2 after administration.
Necroscopy showed no alterations, LD50 for male and female rats were >2150 mg/kg.
SCCS Comment
The study relates to S75-F material included in the dossier, which is a coated, anatase/rutile
material, with organic coating of trimethoxy-n-octyl-silane, in oily suspension. This study is
relevant to the nanomaterial group (85% anatase, 15% rutile).
Approximate Lethal Dose study, Intragastric intubation, Rats
Guideline:
OECD Guidelines 401 and EEC Guidelines 92/32/EEC
Species/strain:
7 week old Male rats/Crl-CD®BR
Group size:
not mentioned
Test substance:
TiO2 T805, white powder, CAS number 13463-67-7
Batch:
H-20762
Purity:TiO2 100%.
Vehicle:suspension in deionised water
Dose levels:
2,300 to 11,000 mg/kg
Dose volume:
not described
Route:
Oral
Administration:
single dose
GLP:
No (not mentioned)
Study period:
August-October 1994
Reference:
Submission I - DuPont, 1994 (1)Results
No signs of toxicity were recorded during the observation period, no deaths recorded,
pathological examination not performed, weight loss (up to 6%) in some animals after 1
day of dosing, ALD >11000 mg/kg, considered as very low toxicity.
SCCS Comment
The study relates to S75-F material included in the dossier, which is anatase/rutile material,with organic coating of trimethoxy-caprylylsilane, in oily suspension. This study is relevantto the nanomaterial group (85% anatase, 15% rutile).
Exploratory study, acute toxicity, oral, mice (Wang et al., 2007)
Guideline:
OECD Guidelines, No. 420
Species/strain:
mice/ CD-1 (ICR)
Group size:
80 (40 female, 40 male)
Test substance:
TiO2 nanoparticles (25, 80 and 155 nm) - not mentioned whether
rutile or anatase
Batch:
not mentioned
Purity:not mentioned
Vehicle:0.5% hydroxypropylmethylcellulose K4M used as a suspending agent.
Dose levels:
5 gram/kg bw
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Dose volume:
not mentioned
Route:
single oral gavage
Administration:
single high dose 5 g/kg bw gavage.
GLP:
Study period:
Reference 213: (Wang, J., Zhou, G., Chen, C., Yu, H., Wang, T., Ma, Y., Jia, G., Gao, Y., Li,
B., Sun, J., Li, Y., Jiao, F., Zhao, Y. and Chai, Z. 2007. Acute toxicity and biodistribution ofdifferent sized titanium dioxide particles in mice after oral administration. Toxicol Lett 168(2): 176-85).

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In addition, the following two articles have been provided on acute toxicity, but they are of
no value to this assessment:

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An article by Ferch, Habersang, 1982 (SI-3) is in fact an old review article (up to 1982)
which focuses mainly on the possible health effects of amorphous and crystalline silica. It
also includes literature review on possible effects of Degussa P25 TiO2 on the formation andinduction of granulomatous changes in the lungs or the peritoneum. Since these were notfound, the authors claim that P25 TiO2 is not toxic. As such the article does not provide
experimental data, but is solely a review of the literature, with the main emphasis on SiO2
and only a few remarks on P25 TiO2.

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An article by Warheit et al., 2007 (SI-II-215) is a review of different studies on ultrafine
TiO2 particles to develop a base set of toxicity tests. As such it does not provide any details
on the studies or any experimental data that could be used for this assessment.

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From the limited data available, the acute oral toxicity of nano-TiO2 (anatase and rutile
mixtures) appears to be very low.

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1.5.1.2 Acute dermal toxicity

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Results
Retention of a small percentage of titanium (measured by ICP-MS) showed predominantly inthe liver and spleen. Kidney, liver and heart pathology was observed with all sizes, withmore pronounced effects for 80 and 155 nm particles. Changes in serum biochemicalparameters (increased lactate dehydrogenase (LDH) and alphahydroxybutyrate
dehydrogenase (alpha-HBDH) levels) were most pronounced for 80 nm particles.
SCCS Comment
The study has a number of flaws, and is therefore of little value to this assessment.
Sufficient characterisation of the nanomaterials used was not carried out, the administereddose (5 g/kg/bw) was very high, frequent oesophageal ruptures were reported that led toanimal deaths, translocation of TiO2 from GI tract was measured as titanium with noevidence that it was in nanoparticulate form. It is not clear whether any of the effects
observed were due to TiO2 toxicity, or simply overloading the gut at high dose of theparticulate material.
SCCS Comment on Acute Oral Toxicity
The TiO2 nanomaterials tested for this endpoint are mainly anatase/rutile mixtures, coatedwith trimethoxy-n-octyl-silane. The derived LD50 in rat is >2150 mg/kg. One study has
determined the approximate lethal dose at >11000 mg/kg.

Exploratory study, Acute toxicity and Skin and Eye irritation tests, Mouse andRabbitGuideline:
Species/strain:
Group size:

OECD Guidelines 401 and EEC Guidelines 92/32/EEC
Male albino mice (acute toxicity tests), and male albino rabbits (skinirritation tests), male albino rabbits (eye irritation tests)
10 mice for toxicity tests, 4 rabbits for skin irritation tests, 3 rabbits
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Test substance:
Batch:
Purity:Vehicle:Dose levels:
Dose volume:
Route:
Administration:
GLP:
Study period:for eye irritation tests
TiO2 (referred to as natural colour)
not statedsuspension in water
up to 10 g/kg for toxicity study, 100mg/square inch for skin patch
tests, 100 mg for eye irritation tests
Oral intubation for toxicity tests, skin patch for irritation test,
instillation in lower conjunctival sac of eye,
7 days for toxicity tests, 48 hours for skin irritation tests, eye washedafter 5 minutes of instillation.
No

Reference 2(Roy, D. and Saha, J. (1981) Acute toxicity of dyes used in drugs and cosmetics, The
Eastern Pharmacist, May 1981, pages 125-126)
Results
No mortality recorded in mice, even at 10 g/kg. No sign of skin irritation or eye irritation.
LD50 >10,000 mg/kg, TiO2 regarded as non-toxic, non-irritant to both skin and eye.
SCCS Comment
The study is of little value in relation to the current assessment for nano-forms of TiO2 as
characterisation data (particle size distribution) have not been provided to show that thetested materials were nanomaterials.
Acute dermal toxicity, limit test, rat
Guideline:
OECD Guidelines 401 and EEC Guidelines 92/32/EEC
Species/strain:
8 week old rats/Sprague DawleyGroup size:
5 male/ 5 female
Test substance:
TiO2 NP88/296 (ultrafine), fluffy white powder, CAS
100209-12-9
Batch:
control No. 27073; July 27th, 93.
Purity:TiO2 96.5%, SiO2 3%, carbon approx 4%.
Vehicle:suspension in peanut oil
Dose levels:
2000 mg/kg
Dose volume:
Route:
DermalAdministration:
single application under occlusion
GLP:
yes
Study period:
February 1989
Submission I
Croda (Tioxide UK), 1989 (Reference 6)
Results
No deaths recorded after 24 hour dermal administration, under occlusion, of NP 88/296 at
2000 mg/kg. Clinical signs noted only after day 1 of dosing, and included hypokinesia,
ataxia, chromodacryorrhoea (eyes and nose), animals hot to the touch. All animals werenormal 2 days after dosing. Median Dermal lethal dose (LD50) of NO 88/296 in rats is>2000 mg/kg. No significant abnormalities noted after post-mortem.
SCCS Comment
The study used ultrafine TiO2, and lacks data on characterisation (particle size distribution)of the tested material. According to the Applicant, the material used in this study relates to21

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rutile material coated with alumina/silica (i.e. S75-A, S75-B, S75-C, S75-L). It is however
not clear how the test material relates to those included in the dossier and what proportionof the micronized material was in the nano-scale.

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1.5.1.3 Acute inhalation toxicity

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SCCS Comment on Acute Dermal Toxicity
The TiO2 material tested in one study is described as ‘natural colour’. The other study has
used ultrafine TiO2, and it is not clear what proportion of the micronized material (coatedwith alumina/silica) was in the nano-scale. Another reference provided in relation to acutetoxicity (Submission I – ref 4, Trochimowicz et al., 1988) is in fact a secondary citation of
the oral lethal dose cited in another article which relates to chronic inhalation toxicity.
From the provided test data, acute dermal LD50 of TiO2 has been derived at >2000 mg/kg(ultrafine material), and >10,000 mg/kg (natural colour material). However, the provided
studies are of no value to the current assessment of nano forms of TiO2.

No study has been provided on acute inhalation toxicity. The SCCS has therefore considered
relevant studies in the open literature:
Respiratory deposition of particles
Inhaled particulate materials may deposit in the lung depending on size (and shape) of theparticles, structure of the lung, and breathing pattern (Sarangapani & Wexler, 2000). The
mammalian respiratory tract is often divided into three regions - the extrathoracic (mouth
or nose and throat), the trachea-bronchial and the alveolar regions with each having atypical structure and function. In general, particles >10 µm deposit in the extrathoracic
region. Nanoparticles also mainly deposit in the extrathoracic region, but alveolar deposition
has been noted for particles with a size of 300-200 nm down to 3-2 nm (ICRP 1994 –Oberdorster 2005, Cassee et al. 2002).

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Particulate materials getting into the lung are generally cleared from the respiratory system.
Large insoluble particles are cleared mechanically, whereas those that dissolve in the lungare removed via adsorption. Particles in the extrathoracic region are generally removed bycoughing or swallowed into the gastrointestinal tract. Particles deposited into the tracheabronchial region are in contact with the mucus layer covering the ciliated cells, and are
generally cleared via the ’mucociliary escalator‘, which moves the mucus (and the particles)
toward the epiglottis where they are subsequently swallowed and cleared via the GI-tract.
Clearing of particles from the alveolar region is much slower and may take weeks to years.
The most important pathway here involves alveolar macrophages. These phagocytic cells
reside on the alveolar epithelium, and phagocytize the particles. The particle-laden
macrophages can be removed via the mucociliary escalator, or can translocate to theinterstitial tissue – together with free particles. These clearance mechanisms are similar inhumans and most mammals, although clearance rates can significantly differ between
species.

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Some particles may be retained in the alveoli for long periods (months) before being
cleared. A small fraction of the inhaled particles can reach the systemic circulation bypassing the pulmonary epithelial barrier; another small fraction can probably reach thebrain via olfactory nerve route. It has been shown that ultrafine (including nano) particles
have a longer retention time in the alveoli compared to larger particles (Oberdorster, 1994).
During chronic and/or cumulative exposure nanoparticles in the alveoli potentiallyaccumulate in the tissue of the entire lungs.

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Exposure to ultrafine particles has been linked to inflammatory and neurodegenerativechanges in the olfactory mucosa, olfactory bulb, and cortical and subcortical brain structures(Oberdorster, 2005). So far there are no toxicological studies available which show
extrapulmonary effects when the exposure was performed under relevant occupational orenvironmental conditions. Yet there exists a vast epidemiological literature which clearly
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indicates exposures to urban ambient aerosols containing nano-sized particles at highnumber concentrations are associated with cardiovascular morbidity and mortality (Pope etal., 2009).
4-Hour Acute Inhalation Toxicity Study in Rats
Authors:
Dekker, U.
Reference:
RCC-Report B25007, internal report
Guideline:
The following guidelines were considered:European Communities, Directive 92/69/EEC, Part B.2 "Acute Toxicity(Inhalation)", published December 29, 1992 and European Communities
Directive 93/21/EEC, April 27, 1993 amending the aforementioned
Directive.
OECD Guidelines for Testing of Chemicals, Section 4, No. 403: "AcuteInhalation Toxicity", adopted May 12, 1981.
U.S. Environmental Protection Agency, Health Effects Test Guidelines
OPPTS 870.1300, Acute Inhalation Toxicity, August 1998.
Species/strain: 15 males and 15 females HanRcc:WIST(SPF) rats; 9-10 weeks oldGroup size:
15 rats per group, one TiO2 exposed group, one placebo exposed group
Test substance: TiO2;
Batch:/CAS No.
/Purity:unknown
Dose levels:
A mean TiO2 aerosol concentration of 4.877 mg/L was inhaled by the rats.
TiO2 particles were resuspended in water and jet nebulized. Medianaerodynamic diameters (MMADs) and geometric standard deviations (GSD)
were 1.4 μm (GSD 2.10)
Route:
Acute 4-hour nose-only inhalation. After a 4-hour inhalation BAL was
performed in satellite groups of 5 rats at 14 hours and 2 days after
inhalation. The rats were studied at day 15 after exposure.
GLP:
No
Study period:
Results
In BALF collected at 14 hours post end of exposure, total cell count (neutrophil numbers)
and total protein were significantly elevated in both sexes of the exposed group comparedto the control group. The changes in BALF were consistent with the histopathology findingsof diffuse alveolar histiocytosis and alveolar lining cell activation seen in all animals of the
exposed group. Significant increases of the absolute and relative lung weights andhistopathology findings of diffuse alveolar histiocytosis and alveolar lining cell activation
were found in the exposed group on day 2. These findings were consistent with TNFα andIL-6 levels in BALF higher in females of the exposed group than in control group on day 2.
SCCS Comments
It is not clear which of the three noted guidelines were followed. The distribution was notinvestigated. The deposited TiO2 particle dose was not determined. The exposed group
showed signs of inflammation based on the methodology applied. The study was poorly
performed and important control parameters are missing. This is by no means acomprehensive study and is of questionable value to this assessment.
Chronic inhalation Exposure of rats to titanium dioxide dust
Authors:
Trochimowicz, H.J. et al. (1988)
Reference:
Chronic inhalation study ref. No. 4
Guideline:
not specified
Species/strain: 3-6 months ChR-CD rats at the begin of the study
Group size:
11 males + 11 females
Test substance: TiO2 not specified
Batch:
not specified.
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Purity:Dose levels:

not specified
250 mg/m³, 50 mg/m³, 10 mg/m³,0 mg/m³, 6h/day, 5 days/week, 104
weeks
Route:
chronic inhalation for 104 weeks;
Administration: whole body exposure
GLP:
not specified
Study period:
/

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-

Rats inhaled a TiO2 aerosol of 22 mg/m³ concentration consisting either ofnanostructured or pigmentary TiO2 particles for 6h/d 5d/wk for 12 consecutive weeks
and were followed up for 1 year (Ferin et al., 1992).

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-

Rats, mice and hamsters inhaled a nanostructured TiO2 aerosol at concentrations of 10,
50 or 250 mg/m³ for 6h/d 5d/wk for 13 consecutive weeks and were followed up for 1
year (Bermudez et al., 2002; Everitt et al., 2000).

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-

Rats, mice and hamsters inhaled a nanostructured TiO2 aerosol at concentrations of 0.5
or 2 or 10 mg/m³ for 6h/d 5d/wk for 13 consecutive weeks and were followed up for 1
year (Bermudez et al., 2004)

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Common findings of these sub-chronic studies were: substantial responses of inflammationand overload associated with diminishing particle clearance in a dose dependent manner,and histologically clear indications of epithelial hypertrophy and hyperplasia. Most
pathophysiological responses disappeared after 1 year of recovery and only the very highdoses led to persistent adverse effects. Rats always responded more sensitively than mice;
hamsters had the least responce. When nanostructured or pigmentary TiO2 particles werecompared, stronger effects were observed for the nanostructured particles.

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Two 5-day inhalation-exposure studies in rats with a follow-up of 28 days as a substitute of
sub-chronic 90-days studies with a follow-up of 1 year have been conducted:

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-

TiO2 nanoparticles at a concentration of 100 mg/m³, and pigmentary TiO2 particles at aconcentration of 250 mg/m³ - with a positive control exposure to quartz particles at 100
mg/m³ (van Ravenzwaay et al., 2009) were investigated. Mild inflammation wasreported in lung histology and BAL with subsequent reversibility. All responses weretransient but the quartz effects persisted. The authors suggested that the effects seen inthese short term studies would be similar to those after 90-day exposure studies. It is
however not clear to the SCCS how the major differences seen in these and the otherstudies can be equated.

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-

Nanostructured TiO2 particles at concentration of 2, 10 and 50 mg/m³ were
investigated. Transient inflammatory responses were observed in lung histology and
BAL. (Ma-Hock et al., 2009).

Results
After 3 months: alveolar cell hyperplasia at doses of 250 mg/m³, 50 mg/m³,
After 6 months: alveolar cell hyperplasia at all dose levels
After 12 months: additionally minute areas of collagen fiber deposition at 250 mg/m³ dose
After 24 months: massive alveolar hyperplasia, focal patches of pneumonia, areas ofcollagenized fibrosis; only at 250 mg/m³ dose; occurrence of lung tumours
The authors conclude significant patho-physiological alterations at doses of 250 mg/m³, 50
mg/m³ but not at 10 mg/m³
SCCS Comment
This study is one of the early chronic inhalation studies on titanium dioxide which triggered
later chronic inhalation studies in the 1980s and 1990s and later investigations into
biokinetics and more toxicological endpoints.
Studies in open literature
Several sub-chronic (90 days) TiO2 inhalation exposure studies have been reported:

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Another intratracheal instillation study used nanostructured anatase TiO2 particles of 5, 23
and 154 nm (actual hydrodynamic diameters of 19, 28 and 176 nm) at a concentration of 5
mg/kg bw administered to the rats and studied until three months after instillation. The
results showed that the smaller the particles, the larger the inflammatory response andhypertrophy. However the effects were transient, (Kobayashi et al., 2009). Several otherinstillation studies have been published that used nano- and submicron-sized TiO2 particles
but they have not been considered here because the particles had already formed larger
sized agglomerates.

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SCCS Comment on acute inhalation toxicity
No study on acute inhalation toxicity was provided. Studies (including open literature) on
acute and sub-chronic inhalation exposure to TiO2 nanomaterials have indicated substantial
inflammatory responses, and histologically clear indications of epithelial hypertrophy andhyperplasia at high exposure dose. In view of this, the SCCS does not recommend the useof nano TiO2 in applications that would lead to any significant inhalation exposure (e.g.
powder or sprayable products).

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1.5.2

Irritation and corrosivity

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1.5.2.1 Skin irritation
Skin irritation/corrosion, Patch test, Rabbit
Guideline:
OECD Guidelines 404 and EEC Guidelines 92/32/EEC
Species/strain:
11 month old Rabbit/white RussianGroup size:
3 maleTest substance:
TiO2 T805, hydrophobic fluffy white powder, CAS 100209-12-9
Batch:
27073.
Purity:TiO2 96.5%, SiO2 3%, carbon approx 4%.
Vehicle:Paraffin
Dose levels:
0.5 g in 0.64 ml paraffin to dorsal skin area patch 6.25 cm2.
Dose volume:
Route:
skin patch
Administration:
single application, observation over 3 days
GLP:
yes
Study period:
August 1993
Submission I
Evonik (Degussa), 1993 (13)DHS Evonik (Degussa), 1993 (5)Results
Very slight erythema (grade 1 in 2 animals), very slight edema (one animal) after one dayof exposure. Primary Irritation Index is 0.3, TiO2 was regarded non-irritant on rabbit skin.
SCCS Comment
The study relates to S75-F material included in the dossier, which is anatase/rutile material,with organic coating of trimethoxy-caprylylsilane, in oily suspension. This study is relevantto the nanomaterial group (85% anatase, 15% rutile).
Skin irritation/corrosion, Patch test, Rabbit
Guideline:
OECD Guidelines 404 and EEC Guidelines 92/69/EEC
Species/strain:
48 month old male, 43 month old female Rabbit/white RussianGroup size:
3 (1 male, 2 female)
Test substance:
TiO2 T817, hydrophobic fluffy white powder, CAS number
100209-12-9
Batch:
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Purity:Vehicle:Dose levels:
Dose volume:
Route:
Administration:
GLP:
Study period:
Reference:

TiO2 > 97%, Fe2O3 2±1%, carbon approx 3.5-4.5%.
0.5 g in peanut oil to dorsal skin area patch 6.25 cm2.
skin patch
single application, observation over 3 days
Yes
February 1998
DHS Evonik (Degussa), 1998 (6)Results
No changes observed, neither erythema nor edema observed. Primary Irritation Index was
0.0, TiO2 regarded non-irritant on rabbit skin. No systemic effects observed.
SCCS Comment
The study relates to S75-F material included in the dossier, which is anatase/rutile material,with organic coating of trimethoxy-n-octyl-silane, in oily suspension. The study is relevantto the nanomaterial group (85% anatase, 15% rutile).
Skin irritation/corrosion, Patch test, Rabbit
Guideline:
OECD Guidelines 404 and EEC Guidelines 92/32/EEC
Species/strain:
11 month old Rabbit/ New Zealand whiteGroup size:
3 male, 3 female
Test substance:
TiO2 H20762, CAS number 13463-67-7
Batch:
Purity:TiO2 100%.
Vehicle:Dose levels:
0.5 g in pre-moistened patch (2 inch square gauze)
Dose volume:
Route:
skin patch
Administration:
single application, observation over 3 days
GLP:
No (not mentioned)
Study period:
August-September 1994
Reference:
Submission I - DuPont, 1994 (10)Results
Three rabbits showed no dermal irritation during the study, no to mild erythema by 1 hour
after patch removal. By 24, 48 and 72 hours, no to slight erythema observed, no edema
observed during the study. H-20762 is regarded a mild skin irritant.
SCCS Comment
The study is of little value in relation to assessment for nano-form of TiO2 as there is a lackof data on characterisation (particle size distribution) of the tested materials to show thatthey were nanomaterials.
Skin irritation/corrosion, Patch test, Rabbit
Guideline:
not mentioned
Species/strain:
Rabbit/ albino
Group size:
6 maleTest substance:
TiO2 - referred to as Haskell Nos. (H 12684, H 12685, H 12686)
Batch:
Purity:not mentioned
Vehicle:Dose levels:
0.5 g pre-moistened with physiological saline (1½ inch squaregauze)
Dose volume:
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Route:
Administration:
GLP:
Study period:
Reference:skin patch
single application, observation over 2 days
No (not mentioned)

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Results

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No skin irritation observed on intact guinea pig skin.

Submission I DuPont, 1978 (11)Results
No skin irritation observed on intact rabbit skin.
SCCS Comment
The study is of little value in relation to assessment for nano-form of TiO2 as there is a lackof data on characterisation (particle size distribution) of the tested materials to show thatthey were nanomaterials.
Skin irritation/corrosion, Patch test, guinea pig
Guideline:
not mentioned
Species/strain:guinea pig/ albino
Group size:
12 maleTest substance:
TiO2 - referred to as 99.5% active ingredientBatch:
Purity:Vehicle:Dose levels:
0.5 g powder and 0.1 g 28% paste were slightly rubbed into
shaved back skin, covered with impervious film and wrapped.
Dose volume:
Route:
skin patch
Administration:
single application, 24 hours, then rinsed in water, observation
over 2 days
GLP:
No (not mentioned)
Study period:
Reference:
Submission I - DuPont, 1969 (12)

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SCCS Comment
The study is of little value in relation to assessment for nano-form of TiO2 as there is a lackof data on characterisation (particle size distribution) of the tested materials to show thatthey were nanomaterials.

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Skin irritation/corrosion, 5 day repeat application study, Rabbit
Guideline:
not mentioned
Species/strain:
RabbitGroup size:
2 male, 1 female
Test substance:
TiO2 utrafine dispersion - referred to as NP 89/97, NP 89/98.
Batch:
Purity:not mentioned
Vehicle:Dose levels:
around 0.5 g (2.5 cm2 patch)
Dose volume:around 0.5 ml
Route:
skin patch

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Administration:
GLP:
Study period:
Reference:

4x repeated (application, removal, skin observation)
No
Submission I - Croda (Tioxide UK), 1989 (14)27

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Results
One animal died on day 4 (unrelated to the test), 5 day repeat applications produced mean
irritation scores of 1.58 and 1.92 for 89/97, NP 89/98 respectively. NP 89/98 consideredslightly more irritant than NP 89/97.

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SCCS Comment
The study used ultrafine TiO2, however, data on characterisation (particle size distribution)of the tested material has not been reported. It is therefore not clear whether the material
had a nano-sized fraction, and if so, in what proportion.

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Skin irritation/corrosion, 5 day repeat application study, Rabbit
Guideline:
not mentioned
Species/strain:
Rabbit/ New Zealand whiteGroup size:
3 (2 male, 1 female)
Test substance:
TiO2 utrafine dispersion - referred to as NP 88/296.
Batch:
Purity:not mentioned
Vehicle:Dose levels:
2 dispersions tested (40% A.I. and 10% A.I. which was diluted
with carrier oil NP88/310)
Dose volume:around 0.5 ml
Route:
skin patch

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Administration:
GLP:
Study period:
Reference:

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Two studies provided in the submission are relevant to the TiO2 nanomaterials. They relate
to anatase/rutile mixture, coated with trimethoxy-n-octyl-silane. In one of the studies, thetest animals showed signs of very slight erythema and oedema. The primary irritation index
was estimated to be zero and 0.3, and the materials regarded as non-irritant on rabbit skin.

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Two other studies used ultrafine grade materials and showed the mean irritation scores of
0.3 and 1.58-1.92 during 5 day repeat applications on rabbit skin, but the proportion of
nano-scale fraction in the materials used has not been reported.

4x repeated (application, removal, skin observation)
No (not mentioned)
Submission I - Croda (Tioxide UK), 1989 (15)Results
5 day repeat applications produced mean irritation scores of 0.13 for both dispersions
tested (i.e. no dose response). Neither the undiluted or diluted test material NP 88/296
produced significant reactions. One rabbit did not react, and the other 2 rabbits showed only
slight to non persistent erythema.
SCCS Comment
The study used ultrafine TiO2. However, there is a lack of data on characterisation (particle
size distribution) of the tested material. According to Applicant, the material used in thisstudy relates to rutile material coated with alumina/silica (i.e. S75-A, S75-B, S75-C, S75-L).
However it is not clear how the test material relates to the nanomaterials included in thedossier and what proportion of the micronized material was in the nano-scale.
SCCS Comment on Skin irritation
The study by Warheit et al., 2007 (SI-II-215) is of no use to this assessment because it is adetailed literature review on the possible effects of different TiO2 ultrafine particles. As suchit does not provide details on the studies, or any experimental data, that could be used forthis assessment.

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The remaining 3 studies showing the tested materials as either mild irritant or non irritant
to rabbit and guinea pig skin are of little value to this assessment because there is a lack ofdata on characterisation (particle size distribution) of the tested materials, and it is not clear
whether they were in fact nanomaterials.

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From the limited useful data presented in the dossier, it appears that the TiO2
nanomaterials are either mild or non-irritant to skin.

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1.5.2.2 Mucous membrane irritation
Eye irritation, single application, rabbit
Guideline:
OECD Guidelines 405 (1) and EEC Guidelines 92/32/EEC
Species/strain:
10-11 month old Rabbits/ white Russian (albino)
Group size:
3 (males)Test substance:
TiO2 T805, hydrophobic fluffy white powder, CAS 100209-12-9
Batch:
27073
Purity:TiO2 96.5%, SiO2 3%, carbon approx 4%.
Vehicle:Dose levels:
22.8 to 24.3 mg
Dose volume:
0.1 mlRoute: eye instillation
Administration:
single application, 3 days observation period
GLP:
Yes
Study period:
August 1993
Reference:
Submission I - Evonik (Degussa), 1993 (9); DHS Evonik(Degussa), 1993 (3)Results
No alterations detected in cornea, iris and conjunctiva, primary irritation index is zero, TiO2(805) regarded as non-irritant on rabbit eye. No systemic toxic effects detected.
SCCS Comment
The study relates to S75-F material included in the dossier, which is anatase/rutile material,with organic coating of trimethoxy-caprylylsilane, in oily suspension. This study is relevantto the nanomaterial group (85% anatase, 15% rutile).
Eye irritation, single application, rabbit
Guideline:
OECD Guidelines 405 (1) and EEC Guidelines 92/69/EEC
Species/strain:
35 month old Rabbits/ white Russian (albino)
Group size:
3 (females)
Test substance:
TiO2 T817, hydrophobic fluffy white powder, CAS number
100209-12-9
Batch:
04095
Purity:TiO2 >97%, Fe2O3 2±1%, carbon 3.5-4.5%.
Vehicle:Dose levels:
11.5 to 16.8 mg
Dose volume:
0.1 mlRoute:
eye instillation
Administration:
single application, 3 days observation period
GLP:
Yes
Study period:
February 1998
Reference:
DHS Evonik (Degussa), 1993 (4)Results

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Some blood vessels definitely hyperaemic in two animals after one hours of application.
Primary irritation index is 0.3, TiO2 regarded as non-irritant on rabbit eye. No systemic
toxic effects detected.
SCCS Comment
The study relates to S75-F material included in the dossier, which is anatase/rutile material,coated with organic coating of trimethoxy-n-octyl-silane, in oily suspension. This study is
relevant to the nanomaterial group (85% anatase, 15% rutile).
Eye irritation, single application, rabbit
Guideline:
OECD Guidelines 405 (1) and EEC Guidelines 92/69/EEC
Species/strain:
Rabbits/ New Zealand whiteGroup size:
2 (females)
Test substance:
TiO2 H-20762, CAS number 13463-67-7
Batch:
Purity:TiO2 100%.
Vehicle:Dose levels:
approx. 10 mg
Dose volume:
Route:
eye instillation
Administration:
single application, eye washed after 20 seconds of application. 3
days observation period
GLP: yes
Study period:
September 1994
Reference:
Submission I - DuPont, 1994 (7)Results
Moderate redness and slight chemosis observed in both treated and untreated washed eyes
(normal after 1 and 3 days respectively). No clinical signs of toxicity obeserved, TiO2
(H20762) regarded as moderate eye irritant but could be classified as non-irritant under the
EEC Directive 93/21, Annex VI.
SCCS Comment
The study is of little value in relation to assessment for nano-form of TiO2 as there is a lackof data on characterisation (particle size distribution) of the tested materials to show thatthey were nanomaterials.
Eye irritation, single application, rabbit
Guideline:
OECD Guidelines 405 (1) and EEC Guidelines 92/69/EEC
Species/strain:
Rabbits/ New Zealand whiteGroup size:
3 (2 male, 1 female)
Test substance:
TiO2 NP 88/296 (ultrafine)
Batch:
not mentioned
Purity:not mentioned
Vehicle:Dose levels:
not mentioned
Dose volume:
0.1 mlRoute:
eye instillation
Administration:
single application, eye washed after 20 seconds of application. 3
days observation period
GLP:
Yes
Study period:
Reference:
Submission I - Croda (Tioxide UK), 1989 (8)Results
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No corneal or iridial reactions, slight conjunctival redness (score 1) which disappeared after
72 hours of treatment. TiO2 (NP88/296) is regarded slightly irritant to rabbbit eyes.

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2. An article by Warheit et al., 2007 (SI-II-215) is a review of different studies on
ultrafine TiO2 particles to develop a base set of toxicity tests. As such it does notprovide any details on the studies or any experimental data that could be used forthis assessment.

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Two other studies provided used TiO2 anatase/rutile mixtures, coated with trimethoxy-noctyl-silane. From these studies, primary irritation index was between zero and 0.3. Anotherstudy has regarded the tested material (TiO2-NP88/296) as slightly irritant to rabbit eye. In
this study, the material used has been described as ultrafine rutile material coated withalumina/silica (relating to S75-A, S75-B, S75-C, S75-L) but information on characterisation(particle size distribution) has not been reported to indicate what proportion was in the
nano-scale. Similarly, another study has regarded the tested material (TiO2-H20762)
moderately irritant to rabbbit eye, but it is not clear whether the tested material was ananomaterial.

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From the limited useful data provided, eye irritation potential of nano-TiO2 appears to be
low.

SCCS Comment
The study relates to ultrafine TiO2. However, information on the characterisation (particle
size distribution) of the tested material has not been reported. According to the Applicant,the material used in this study relates to rutile material coated with alumina/silica (i.e. S75A, S75-B, S75-C, S75-L). It is however not clear how the test material relates to thenanomaterials included in the dossier and what proportion of the micronized material was in
the nano-scale.
SCCS Comments on Eye Irritation
The following two articles provided with the submission on acute toxicity are of no value tothis assessment:
1. An article by Frosch and Kligman (Reference 16 - S75 irritation skin) refers mainly tothe development of a scarification chamber test for irritancy of materials. It doesrefer irritancy of titanium dioxide as low, but it is not clear whether the tested TiO2
was a nanomaterial.

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1.5.3

Skin sensitisation

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Skin sensitisation, Guinea Pig, maximisation test
Guideline:
OECD Guidelines 406 and EEC Guidelines 84/449/EEC
Species/strain:
8 week old 12 male, 10 female guinea pigs/Pirbright whiteGroup size:
3 (1 male, 2 female)
Test substance:
TiO2 T805, hydrophobic fluffy white powder, CAS 100209-12-9
Batch:
030492
Purity:TiO2 96.5%, SiO2 3%, carbon approx 4%.
Vehicle:paraffin oil, Freunds Complete Adjuvant for immunisation
Dose levels:
0.5 g in paraffin oil to dorsal skin area 5 cm2 patch.
Dose volume:
0.1 ml of 0.5% dispersion, 0.2 ml of 5% dispersion forchallenge
Route:
Induction application intradermal and epidermal, challenge
application epidermal
Administration:
single application, 48 hours, challenge on day 22 for 24 hours,
observation over 48 hours
GLP:
yes
Study period:
June 1992
Reference:
Submission I Evonik (Degussa), 1992 (19); DHS Evonik(Degussa), 1992 (7)31SCCS/1516/13
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Results
Following epidermal challenge neither treated nor control animals showed any changes atthe skin. TiO2 regarded as non-sensitiser in maximisation test on guinea pig skin. No
systemic effects observed.
SCCS Comment
The study relates to S75-F material included in the dossier, which is anatase/rutile material,with organic coating of trimethoxy-caprylylsilane, in oily suspension. This study is relevantto the nanomaterial group (85% anatase, 15% rutile).
Skin sensitisation, Guinea Pig, Buehler test
Guideline:
OECD Guidelines 406 and EC Guidelines 96/54/EC
Species/strain:
8 week old guinea pigs/PsdPCC: DHGroup size:
20 male, 20 female (2 vehicle control groups of 10, and 1 test
group of 20)
Test substance:
TiO2 T817, hydrophobic fluffy white powder, CAS 100209-12-9
Batch:
04095
Purity:TiO2 > 97%, Fe2O3 2±1%, carbon approx 3.5-4.5%.
Vehicle: paraffin oil
Dose levels:
0.5 g applied, 3 applications on day 1,8,15.
Dose volume:
Route:
Induction phase duration 15 days, epidermal, challenge
application epidermal (occlusive patch)
Administration:
epidermal, 48 hours, challenge on day 29 for 6 hours,
observation over 48 hours.
GLP:
yes
Study period:
November-December 1997
Reference:
DHS Evonik (Degussa), 1992 (8)Results
Following first challenge, 3 out of 10 animals reacted with an erthyema and 1 in 10 animals
showed edema. Following epidermal challenge neither treated nor control animals showed
any changes at the skin. TiO2 regarded as non-sensitiser in Buehler test on guinea pig skin.
No systemic effects observed.
SCCS Comment
The study relates to S75-F material included in the dossier, which is anatase/rutile material,coated with organic coating of trimethoxy-n-octyl-silane, in oily suspension. This study is
relevant to the nanomaterial group (85% anatase, 15% rutile). Due to the absence of skinpenetration of TiO2 as demonstrated by many studies included in this dossier, theusefulness of the Buehler test for assessing sensitisation potency of nanomaterials is
doubtful as it is based on exposure to intact skin.
Skin sensitisation, Guinea Pig, Magnusson-Kligman maximisation test
Guideline:
Species/strain:guinea pigs/Dunkin Hatley strain
Group size:
20 test group, 16 control group
Test substance:
TiO2 NP89/145
Batch:
Purity:TiO2 96.5%, SiO2 3%, carbon approx 4%.
Vehicle:Freunds Complete Adjuvant for immunisation
Dose levels:
2 cm x 4 cm patch, 2cm x 2 cm patch for challenge
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Route:

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From two of the other studies, TiO2 nanomaterials (anatase/ rutile mixture, coated withtrimethoxy-caprylylsilane or trimethoxy-n-octyl-silane) have been regarded non-sensitiser.
Another material (rutile, coated with alumina/silica) is classified as a weak sensitiser
according to the Magnusson-Kligman classification (that considers 0 to 8% response a weaksensitizer category). The material used in this study is described as ultrafine rutile material
coated with alumina/silica (relating to S75-A, S75-B, S75-C, S75-L) but information on
characterisation (particle size distribution) of the tested materials has not been reported toindicate what proportion was in the nano-scale.

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Due to the absence of skin penetration of TiO2 as demonstrated by many studies includedin this dossier, the usefulness of the Buehler test for assessing sensitisation potency ofnanomaterials is doubtful as it is based on exposure to intact skin.

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From the limited useful data, TiO2 nanomaterials appear to be weak or non- skin sensitiser.

Administration:
GLP:
Study period:
Reference:

Induction with NP 89/145 at 10% v/v in NP 88/310 (injection)and 100% (topical), challenge application at 100% and 50% v/v
in NP88/310.
Patch, 48 hours (induction patch), 24 hour (challenge patch),observation period 24 and 48 hours
Yes
April-May 1989
Submission I - Croda (Tioxide, UK), 1989 (20)Results
At challenge, none of the test or control group animals treated with NP 89/145 at 100% or
50% v/v (in NP 88/310) showed a positive response. No evidence that NP 89/145 is asensitiser in guinea pigs. Classified as a weak sensitiser according to the MagnussonKligman classification. No clinical signs were noted, body weight gains were acceptable.
SCCS Comment
The study used ultrafine TiO2, however, there is a lack of information on thecharacterisation (particle size distribution) of the tested material. According to Applicant,the material used in this study relates to rutile material coated with alumina/silica (i.e. S75A, S75-B, S75-C, S75-L). It is however not clear how the test material relates to thenanomaterials included in the dossier because the proportion of the nano fraction in the
micronized material has not been provided.
SCCS Comment on Skin Sensitisation
The article by Warheit et al., 2007 (SI-II-215) is a review of different studies on ultrafine
TiO2 particles to develop a base set of toxicity tests. As such it does not provide any details
on the studies or any experimental data that could be used for this assessment.

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1.5.4

Dermal / percutaneous absorption

In vitro studies:
Guideline/method:
Species:
Test substances:
Particle size:
Group sizes:
Dose applied:

human abdominal epidermis
Titanium dioxide T805, comprising 5% micronized titanium
dioxide; not radiolabelled.
not given2 female donors in experiment 1, 1 male and 1 female donor inexperiment 2
3.6g/cm2 of cream with a content of 5% micronized titanium
dioxide (actual dose 3.55 mg/cm2)
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Skin area:
Skin temperature:
Test chamber:
Receptor fluid:
Exposure period:
GLP:
Published:
Study period:
Reference:

0.32 cm2
30-32°Cflow through diffusion cells
0.9% saline
6 hoursyesno1995
Reference 24 submission 1

MethodThe amount applied to each cell was 3.55 mg/cm2. Skin integrity was checked. The
penetration through the skin membranes was determined over a period of 6 hours under
non-occluded conditions. The receptor fluid was delivered at a flow rate of about 1.5 mL/hduring the testing period. The perfusate from each cell was collected separately at ambient
temperature for 0-8h post application. Eight hours post application the perfusate sampling
was terminated. All skin membrane rinse fractions were combined according to theindividual cells and added to the 0-8h perfusate.
Results
The perfusate samples were analysed by IPCMS, the TiO2 content ranged from 2.6 to 4.8
ng/ml. These concentrations were reported to be in the same range as the ‘blind’ solutions(2.-2.9 ng/ml). Transmission electronic microscopy of titanium dioxide in the skin samples
showed presence only in the outer skin layers and not in the deeper layers of the epidermis.
Thus TiO2 nanoparticles did not penetrate through human skin under the experimental
conditions described above.
SCCS Comments
The study shows lack of detectable skin penetration of the test nanomaterial which relates
to S75-F included in the dossier (anatase/rutile material, with organic coating oftrimethoxy-caprylylsilane, in oily suspension). This study is relevant to the nanomaterialgroup (85% anatase, 15% rutile).
The particle size of the tested nano-material was not determined in this study. It is assumed
that the particle size is similar to the data shown in Table 1.3. However most likely theparticles were present as agglomerates as the test item was used in a cream formulation.
Study Design:
Guideline/method:
Species:
Test substances:micronized TiO2:
vehicleParticle size:
Group sizes:
Dose applied:
Skin area:
Skin temperature:
Test chamber:
Receptor fluid:
Exposure period:
GLP:
Published:
Study period:
Reference:

human abdominal epidermis
micronized TiO2: Eusolex TA (5% O/W lotion),
Eusolex TC (5% W/O cream)(O/W lotion and W/O cream)
particle sizes not provided, Eusolex TA: BET= 84.2 m2/g Eusolex TC:
BET= 58.8 m2/g
4 cells per donor; 4 donors
between 3.19 and 4.28 mg/cm2
0.32 cm2
30-32°Cflow through diffusion cells
0.9% saline
6 hoursyesno1995
Reference 25 submission 1

Method34

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The amount applied to each cell was 3.19-3.31 mg/cm2 (Eusolex TC and TA, respectively;
applied amount of vehicle only was slightly higher). Skin integrity was checked. The
penetration through the skin membranes was determined over a period of 6 hours under
non-occluded conditions. The receptor fluid was delivered at a flow rate of about 1.5 mL/hduring the testing period. The perfusate from each cell was collected separately at ambient
temperature for 0-8h post application.
Eight hours post application the perfusate sampling was terminated. All skin membranerinse fractions were combined according to the individual cells and added to the 0-8h
perfusate.
Results
The perfusate samples were analysed by ICP-OES, the TiO2 content were below
0.05ug/sample. No, or only slight traces of TiO2 particles were detectable on the skinsamples treated with Eusolex® TA under the light microscope. The refracting colourless
TiO2 particles were localized on the outer surface of the stratum corneum. One skin sample
revealed two particles sited intracellularly at one location at the stratum granulosum.
Whether these were refracting particles of TiO2 could not be resolved unequivocally under
the optical microscope. Multiple foci of TiO2 particles were observed on most of the skinsamples that had been treated with Eusolex® TC. The refracting particles were localized on
the outer surface of the stratum corneum. It was concluded that titanium dioxide
nanoparticles did not penetrate through human skin under the experimental conditions
described above.
SCCS Comments
The study shows lack of detectable dermal penetration of TiO2 nanoparticles. The test
material possibly (as it is not clear from the different code) relates to S75-M, S75-N, and/or
S75-O. The particle size of the tested nano-material was not determined in this study.
Test for penetration of micronized TiO2 through the egg membrane or the chorioallantoic membrane (CAM).
Guideline:
Species/strain:
White Leghorn chicken eggs, freshly fertilizedGroup size:
3 eggs per group (control group: 2 eggs)
Test substance:
micronized Eusolex TC (TC);
Batch:TO 118279
Purity:not reported
Particle size:
not reported
GLP:
Reference:
Reference 26 submission I
MethodThe testing material was prepared on the day of exposure. The concentration was 5 g/100
ml carrier. The carrier used was water for injection to which 0.01 % of the cationic tenside
UCARE 10 had been added. To enable the test material to be applied to the egg membrane,
the eggshell was opened with the aid of a dentist's drill and the material was introducedwith the aid of a needle. The volume introduced was 0.06 ml per egg. To enable thematerial to be applied to the CAM (chorio-allantoic membrane), the eggshell was taken off,the egg membrane removed and the material introduced onto the exposed CAM. The
volume introduced was 0.3 ml. After the prescribed period of exposure, the treated surface
was fixed for 24h with approximately 10% formaldehyde solution. The fixed CAM or egg
membrane with CAM was removed, embedded in paraffin, sliced, and then stained with
nuclear fast red and H. E. The sections were evaluated under an optical microscope.
Results

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No signs of penetration by TiO2 through the egg membrane or the chorio-allantoic
membrane were seen under an optical microscope. The introduction of TiO2 was fully
tolerated in this sensitive model.
SCCS Comments
The test report is very concise. No positive control was used in this test. This test istherefore of very limited use for this assessment.
Study Design:
Guideline/method:
Species:
Test substance:
Particle size:
Group sizes:
Dose applied:
Skin area:
Skin temperature:
Test chamber:
Receptor fluid:
Exposure period:
GLP:
Published:
Study period:
Reference:

human abdominal skin
J&J Baby Sunblock SPF 30 (2723L) containing microfine titaniumoxide (Hombifine 535) (conc unknown)
not reported.
not reported (1 donor?)
400 um formulation
not reported
not reported
flow through diffusion cells
0.9% saline
24h hours
no
no
1990
Reference 28 submission 1

MethodA layer of about 400 um of formulation was applied on each human cadaver skin sample
and left to dry for 15 minutes. The treated skin samples with the epidermis side facing up
were then mounted on each of the modified diffusion cells. The receptor compartment wasfilled with 0.9% NaCl adjusted to pH 7.4 and 5 respectively. The permeation was conducted
for 24 hrs and the receptor solutions were collected at the end of the experiment. The
amount of cream left on the skin surface was then recovered using wipes and rinsed withmethanol (methanol washings).
Results
In these diffusion cell based tests, samples of stripped human cadaver skin and mouse skinwere used. The stripped skin does not have a stratum corneum and can thus be regarded tosimulate injured skin. The study showed that only a negligible amount of titanium
permeated through either whole skin or the simulated "damaged skin". About 15% of
titanium oxide was found in the skin tissue and most of the titanium (ca. 85%) wasrecovered from the skin surface for both whole skin and stripped skin when the receptor pH
was adjusted at pH 7. 4. It appears that titanium has little tendency to permeate throughthe skin. The amount of titanium oxide recovered in the skin tissue may include the physicaladsorption of titanium oxide to the skin surface, which was difficult to be rinsed off bymethanol.
The effect of pH in the receptor fluid may play an important role towards the penetration oftitanium oxide. The point of zero charge (pzc) of microfine titanium oxide (Hombifine S35)
is 5.6. Therefore, the receptor fluid will provide better "sink" conditions if its pH is adjusted
further away from 5. 6. Less titanium was found in the skin when the receptor pH wascontrolled at pH 5. This can be explained by the fact that pH 5 (0.6 pH unit away frompzc) is providing less "sink condition" compared to pH 7.4 (1.8 pH unit from zpc). It was
concluded that the chance for titanium oxide to penetrate across human cadaver skin is
slim.
SCCS Comments
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This is a special and limited study to investigate the influence of different pH conditions.
Reporting is very concise. Therefore this study provides some additional but limited
information for the risk assessment.
Guideline/method:
Species:
Test substances:
Particle size:
Group sizes:

Dose applied:
Skin area:
Skin temperature:
Test chamber:
Receptor fluid:
Exposure period:
GLP:
Published:
Study period:
Reference:

human abdominal skin
Sunscreen cream with 5% UV-Titan M160 formulation containing 5%titanium dioxide Sunscreen cream without UV-Titan (ca 50 ml).
not given
3 donors, 1 male and 2 females; 17 samples from 3 donors weretreated with sunscreen cream with UV -titan M 160 formulation. A
total of 4 samples of epidermis taken from the 3 different donors
were treated with the control formulation
2.06 mg/cm2 of cream with a content of 5% micronized titanium
dioxide0.32 cm2
30-32°Cflow through diffusion cells
0.9% saline
8 hoursyesno1996
Reference 30 submission 1

MethodThe amount applied to each cell was 2.06 mg/cm2. Skin integrity was checked. The
penetration through the skin membranes was determined over a period of 6 hours under
non-occluded conditions. The receptor fluid was delivered at a flow rate of about 1.5 mL/hduring the testing period. The perfusate from each cell was collected separately at an
ambient temperature for 0-8h post application. Eight hours post application the perfusate
sampling was terminated.
Results
The absorbed amount of Titanium Dioxide was below the detection limit of 5 ng (1ug/l in
ICP-MS) in all samples. The analyses of the samples did not indicate significant penetrationof Titanium Dioxide UV-TITAN within the detection limit of the method.
SCCS Comments
The study shows lack of detectable dermal penetration of TiO2 nanoparticles. The tested
material is S75-I (>99.5% Rutile, coated with 7% alumina 10% stearic acid).
Guideline/method:
Species:
Test substances:
C- Oil/Water lotion:
Particle size:
Group sizes:
Dose applied:
Skin:
Skin temperature:
Exposure period:
GLP:

Human (4 females, mean age 26), upper arm
A- Oil/Water lotion: 5% w/w Ti02 from 12.5% Tioveil AQG
B- Water/Oil cream: 7.5% w/w Ti02 from 18.75% Tioveil TG
7.5% w/w Ti02 from 18.75% Tioveil OP
not reported
4 volunteers, 3 different locations of the upper arm (for application A,
B and C)
2.0 ul/cm2 : 8 ul spread over 4 cm2 area of skin.
Intact human skin
37 °C
8h under occlusion
No
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Study period:
Reference:

1993
Reference 29 submission 1

MethodThe three test products were randomly allocated to three of the four test sites on theforearm. After the 8 hour occlusion, the dressings were removed. The sites were not wipedprior to removal of stratum corneum by the skin surface biopsy (SSB) procedure.
Successive SSBs were taken from the same site such that a profile across the stratum
corneum was obtained. Four SSBs were taken from each of the treated sites.
The migration of titanium dioxide from sunscreen formulas into the skin was investigatedusing a range of sunscreen formulas (A-C) and four subjects. Consecutive 4cm2 skinsamples (biopsies) were taken from test areas. A maximum of 4 biopsies were taken fromany one skin area, providing 16-20 skin layers in total. Selected skin biopsies were thenanalysed using X-ray microanalysis to determine the concentration of titanium dioxide in thebiopsy and to show the migration of the titanium dioxide through the skin.
Results
Emulsion A did not appear to have migrated past the first biopsies from subjects 1, 2 and 4
but had migrated to the second biopsy from subject 3.
Comparing the emulsions tested on subject 1, emulsions A and B showed little differencewith titanium only present in the first biopsy, but the titanium from emulsion C had
migrated to the second biopsy. These results have been confirmed by transmission electron
microscopy examination of these samples where titanium dioxide crystals were shown to bepresent through the first biopsy and in the second skin biopsy of the area treated withemulsion C but not in the second biopsies of the areas treated with any of the otheremulsions. Repeat analyses on selected samples showed that there was an error of ±0.2%
in the measurement of titanium in these samples. This indicates that there is some variation
across the samples possibly due to uneven migration of the sunscreen or uneven thicknessof the biopsies. The detection limit of the analyser was -0.1% and though comparative
results were obtained by this method it is not as accurate as observations made bytransmission electron microscopy. Any measurements less than 0.3% were confirmed byrepeat analyses. It was concluded that in all cases no Ti02 was detected beyond the top two(out of four) skin surface biopsies. No evidence of penetration to the viable epidermis wasfound.
SCCS Comments
The study shows some penetration of TiO2 nanoparticles to the outer layers of skin, but not
to the viable epidermis. The tested material relates to S75-B (>99.5% Rutile, coated with
6% silica, 16% alumina).
Study Design:
Guideline/method:
Species:
Test substances:titanium dioxide.

Particle size:
Group sizes:

Comparative study according to an internal laboratory methodology
considering real use conditions and recommendation of US FDA and
COLIPA SPF requirements
Human (25- to 65-year-old adults)
Commercial products containing coated (Al2O3 and SiO2) nano-sized
No information on size except for Eusolex T-2000
TiA: contained only TiO2
TiB: contained TiO2 plus ZnO
TiHB: (Eusolex T-2000) contained coated rutile TiO2 (average size of
20 nm)
Nanoparticles of TiO2 needle-shaped; dimension not given
TiA, TiHB: 8 volunteers (intact skin)
TiB: 9 volunteers (intact skin)
TiA, TiB, TiHB: 10 volunteers (stripped skin)
TiA: 4 psoriatic patients38

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Controls:
Dose applied:
Skin:
Skin temperature:
Exposure period:
GLP:
Published:
Study period:
Reference:

6 volunteers for basal elemental concentration in the skin
0.5 – 1.0 mg/cm² on an area of 25 cm²
Intact and tape stripped human skin
37 °C
2 h (intact) or 48 h (stripped skin and psoriatic patients)
No
Yes
Before 2009
Filipe et al., 2009 (54, 155)

MethodThe localization and possible skin penetration of TiO2 nanoparticles dispersed in threesunscreen formulations, in use under certain conditions were 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 coatedrutile form TiO2.
The nanoparticles were dispersed in hydrophobic basis gel composed by high pressurepolyethylene and viscous paraffin with Al2O3 (8-11%) and SiO2 (1-3%). The coatedpreparations contained 76 – 82% TiO2. The size and shape of nanoparticles in the three
formulations were inspected with transmission electron microscopy and X-ray microanalysis.
Nanoparticles were needle-shaped and similar in both commercial and test formulation. The
application protocol consisted of an open test. The formulation was applied on the sacral
region and buttocks for 2 h, using a sunscreen application of approximately 0.5-1.0 mg/cm²
within an area of 25 cm².
The 3 formulations used in the study were tested in normal skin: TiB was applied to 9
individuals and both TiA and TiHB to 8 individuals. Nanoparticle penetration (TiA, TiB, TiHB)
was also evaluated in normal skin in an independent group of 10 individuals under nonphysiological conditions induced by tape stripping and occlusive patches (48-hourapplication). Tape stripping consisted of series of strips until the tapes were free of
corneocytes. A TiA-containing commercial sunscreen was further tested in involved skinareas of 4 psoriatic patients. A matched control group constituted by 6 individuals was used
for the determination of basal elemental concentrations in skin including Ti.
Skin punch biopsies of 3 mm diameter were taken after application, quench-frozen and kept
in containers until processing. One biopsy was taken from each volunteer. 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. Sections were obtained from the nonimmersed portion of the tissue, and sectioning performed from inside to outside to avoid
tissue contamination. Tissue integrity and the efficacy of corneocyte removal after tape
stripping were checked by preparing intercalary stained sections for optical microscopy
purposes. Scanning Transmission Ion Microscopy technique and Particle Induced X-ray
Emission technique were used for detection. The minimum detectable concentration of TiO2
in the skin was 0.31 μmol/g (24.8 μg/g tissue or 25 ppm).
Results
For imaging and localizing TiO2 and ZnO nanoparticles in intact skin, the coverage of theouter skin layer with the TiA and TiB sunscreen formulations was homogeneously
distributed. The TiHB formulation showed a patchy distribution. Sunscreen formulations
accumulated in skin wrinkles and depressions as well as infundibulum cavities. Exogenous Ti
and Zn remained at the outer layers of the keratinized tissue that enfold the follicle i.e.
outside the living skin.
The nanoparticles penetration profiles obtained with the treated skin groups (TiA, TiB and
TiHB) were all similar. The high levels of TiO2 observed at the outer layers of stratum
corneum sharply decreased within deeper layers to become undetectable (as Ti by x-ray
emission technique). High Ti concentrations levels were only determined in the stratum
corneum of skin treated with the three formulations. In the subcorneal regions Ti
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concentration was below the minimum detectable concentration estimated for the analyticaltechnique. In non-treated skin Ti was below the minimum detection limit in all strata
inspected. For the depth positions, where TiO2 nanoparticle penetration ended an estimatederror of 10% was obtained, which approximately corresponds to 0.5 μm. In occluded skin,
there was no significant difference in TiO2 nanoparticles distribution and penetration depth
profiling.
Nanoparticle localization in damaged skin
Parts of the outer layers of the stratum corneum were removed by tape stripping (at least
15 strips) before sunscreen application. Removal of the stratum corneum was confirmed byhistological examination and ultimately by nuclear microprobe examination. Under thiscondition there was negligible adhesion of the formulation tested (TiA). The TiO2 contents
determined on the skin outer layers were unimportant suggesting that, in normal skin, theouter layers of stratum corneum trapped nanoparticles inside the desquamating corneocytes
network.
Results
In psoriatic skin, where the horny layer is thicker and less compacted than in normal skinshowed that the sunscreen formulation remained only, in the first layers of the stratum
corneum. The Ti distribution was often non-uniform and in some "hot-spots" sunscreen was
deposited at the outer layers of stratum corneum partly even in the hair follicle
infundibulum region.
Conclusion
The authors concluded that following 2 h exposure period of normal human skin to nanosized TiO2-containing sunscreens, detectable amounts of these physical UV filters were only
present at the skin surface and in the upper most stratum corneum regions. Layers deeper
than the stratum corneum were devoid of TiO2, even after 48 h exposure to the sunscreen
under occlusion. Deposition of TiO2 and ZnO nanoparticles in the openings of thepilosebaceous follicles was also observed. Penetration of nanoparticles into viable skin tissue
could not be detected.
SCCS Comments
The study is of good quality. Although for the TiO2 nanomaterial used in this study
information on surface area, number of particles per mass was not provided, the results
showed penetration of the nanoparticles only to the outer layers of Stratum corneum, but
not to the viable epidermis. The tested material relates to S75-N (>95% Rutile, <5%anatase, coated with alumina 10% simethicone 2%, doped with 1000 ppm Fe).
Study Design:
Guideline/method:
Species:
Test substances:

Formulations:

Dose applied:

Porcine and human skinTiO2 uncoated nanoparticles, mixture of rutile and anatase,average primary particle size 21 nm, uncoated, approximately
spherical platelets (Degussa-P25)
TiO2 coated nanoparticles, rutile, composition 76-82% TiO2, 811% Al2O3 and 1-3% SiO2, primary particle size about 20-100
nm, needle shaped (Eusolex T-2000, Merck KGaA)
All formulations contained 5% TiO2 nanoparticles.
1. TiO2 uncoated: carbomergel, 20% propylenglycol, 0.5%
carbomer 500,000, 0.3% trometamol, and 79.2% purified
water.
2. TiO2 coated: hydrophobic basisgel, 5% high pressurepolyethylene and 95% viscous paraffin
3. TiO2 coated: polyacrylategel, 20% propylenglycol, 0.5%
carbopol 980, 0.3% trometamol, and 79.2% purified water.
2 mg/cm²
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Skin:

Skin temperature:
Exposure period:
GLP:
Published:
Study period:
Reference

Porcine skin. The porcine skin specimens (n=12) were obtained
from domestic pigs. Specimens were sampled from the innerparts of thighs in the form of punch biopsies.
Human skin. The human skin was obtained from the dorsal
region and buttocks of healthy adult volunteers (n=8).
Human grafted skin samples were produced from normal humanforeskins obtained from circumcision and grafted on a severe
combined immunodeficient (SCID) mouse model (n=4).
Not stated
Porcine and human skin 2 h under semi-occlusive conditions,human grafted skin 1 h, 24 h, and 48 h under occlusive
conditions.
No
Yes
Before 2008
Gontier et al., 2008 (158)

MethodAll three formulations were topically applied at 2 mg/cm2 and for 2 h to porcine and humanskin under semi-occlusive conditions, i.e., a breathable plaster protected the area. In aprevious pilot study with exposure times between 8 and 48 h no significant differences were
found for different exposure times. The sunscreen was applied to human skin grafted on
SCID mice for 1 h, 24 h, and 48 h under occlusive conditions. Untreated control samples
were also prepared for each analysis.
The skin biopsies (3 mm in diameter) were studied by Transmission Electron Microscopy(HRTEM) and Scanning Transmission Ion Microscopy (STIM) combined with Rutherford
Backscattering Spectrometry (RBS) and Particle Induced X-Ray Emission (PIXE) on ultrathin and thin cross-sections, respectively.
Results
Porcine skinTiO2 uncoated. By superimposing the titanium distribution obtained by the PIXE map
on to the STIM map, it was possible to unambiguously determine the distribution of TiO2
particles via their chemical fingerprint with a close correlation to the epidermal layers. TiO2
particles were exclusively localized on the surface of the outermost SC layer. No titanium
could be found in the layers containing vital cells. The porcine skin after application ofhydrophobic basisgel exhibited a similar titanium distribution. To quantify the penetration
depth of TiO2 particles, a region of interest was chosen to extract the titanium depth profile
displayed. The extent of the profile was about 30 µm. A clear titanium peak is visible at theskin surface, the titanium being strictly limited to the SC. The nuclear microprobe
observations were cross-checked by the results obtained on the same type of samples
studied by HRTEM. Apart from corneocyte layers, nanoparticles and agglomerates on and in
between the corneocytes are clearly visible. Electron X-ray microanalysis on individual
nanoparticles proved that they contain Ti. In addition, morphological features of the TiO2
particles were examined. The TiO2 particles sometimes appear as individual particles, butmore frequently agglomerated to clusters of different sizes.
TiO2 coated. An average size of 12 nm in width and of 60 nm for the length was estimatedfor the primary needle-shaped particles. The large amount of the titanium particles forboth test emulsions, carbomergel and hydrophobic basisgel, was strictly located at thesurface of the last corneocyte layer with the possible exception of agglomerates below the
first and third corneocyte layer.
Human skin
The STIM map exhibits a thick SC and a well delineated SS containing keratinocyte cellbodies. The Ti PIXE-maps are superimposed onto the STIM image, demonstrating that the41

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particles were exclusively located on the outermost layers of the SC. This observation iscorroborated by the superimposition of the same titanium PIXE-map onto the RBS carbon
map. The depth profile of titanium extracted from the region of interest demonstrates thatthe presence of this element is limited to a layer with a thickness of about 20 µm.
On the STIM map obtained for the commercial formulation, the SC is easily observable due
to its high density despite its unusually low thickness. In the titanium PIXE-map issuperimposed onto the STIM image. Ti is exclusively localized on the surface of the horny
layer. From the titanium depth profile, extracted from the region of interest, titanium wasfound to penetrate into a 10 µm thickness layer of the SC only, but no titanium was
detected in the SS.
In the HRTEM micrograph, TiO2 particles were identified by the presence of large
homogeneous electron dense objects on the surface of the horny layer. At low magnification
the particles appear to be spread in a very homogeneous thin layer. With a highmagnification, the particles occasionally appeared as needle-shaped individual particles, but
most frequently aggregated in clusters of different sizes. The primary particles have a widthof 12 nm and an average length of 60 nm. Some particles were seen four to five layers
deeper, apparently only when a passage exists due to the looseness of corneocytes.
Human skin grafted to SCID-mice
The murine SCID model allows human skin to be grafted without any rejection. The
commercial product was applied for 2 h under occlusive conditions. Here, the STIM image
enables to delineate the SC from the large SS by its high density. In addition it shows thepapillary dermal-epidermal junction and the dermis. When the PIXE-titanium maps weresuperimposed onto the STIM images obtained from the two different areas of interest a
microlesion, i.e., a partly detached horny layer, with Ti in the cleft was seen. The result
seemed to indicate that in some areas of the SC titanium penetrated more deeply comparedto other skin samples. The HRTEM micrographs revealed a thinner SC constituted by two or
three layers of corneocytes only. In fact, this sample was taken from the border between
mouse and human skin. The corneocytes are separated by larger spaces which have allowed
the product to penetrate down to the innermost corneocyte layer. The TiO2 particles seemto be attached to the corneocyte layers. Nevertheless no TiO2 particles were observed in
the very close SG.
Conclusion
The authors concluded that whereas the HRTEM and STIM/PIXE images reveal clear
differences – mainly related to the different thickness of the cross-sections – they
unambiguously show that penetration of TiO2 nanoparticles is restricted to the topmost 3–5
corneocyte layers of the stratum corneum.
SCCS Comments
The study is of good quality. Although for the TiO2 nanomaterial used in this study
information on surface area, number of particles per mass was not provided, the results
showed penetration of the nanoparticles only to the outer layers of Stratum corneum, but
not to the viable epidermis. The tested material relates to S75-G (uncoated, anatase 85%,
rutile 15%), and S75-N (>95% Rutile, <5% anatase, coated with alumina 10% simethicone
2%, doped with 1000 ppm Fe).
Compromised skin
Study Design:
Guideline/method:
Test system:
Test substances:

Exploratory comparative percutaneous skin penetration study in vitro
after UVB radiation in vivo (sunburn simulation)
Skin of weanling Yorkshire pigs (approximately 20–30 kg)
O/W and W/O sunscreen formulations
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Batch:UVB exposure:
Reference:

A: T-Lite SF (coated, 10% O/W formulation, CM 630)
B: T-Lite SF (coated, 10% W/O formulation, CM 634)
CM 630 and CM 634 consist of TiO2 (rutile, crystallite of 14–16 nm)
coated with hydrated silica, dimethicone/methicone copolymer, andaluminium hydroxide for a primary particle size of 10 x 50 nm andspecific surface area of 100 m2/g. The mean size of the agglomerates
was 200 nm with a range of ca. 90–460 nm
Not stated (source: BASF SE, Germany)
A Fiber optic UVB lamp (Lightning cure 200 UV-Spot light) was used.
Monteiro-Riviere et al., (2011) (181, 182).

MethodOn day 1 a pig was sedated and the hair clipped. The minimal erythemic dose (MED) was
determined by sequential exposure to UVB light (30 – 110 mJ/cm², - 22 sec.). On day 2 theexposed sites were analyzed to determine the UVB dose required to produce 1 MED. The pig
was subsequently sedated and multiple sites (52 sites) on the back were exposed to theUVB dose that caused a consistent +2 erythema, a pale red in a defined area of the skin.
Twenty-four hours after UVB exposure (Day 3), the pig was sedated, sites visually analyzed
for consistency, and the pig euthanatized. The UVB-exposed sites were dermatomed to a
thickness of approximately 400-500 μm and placed dermis side down on paper towels
saturated with physiological saline.
The skin prepared for the in vitro or in vivo studies.
UVB dose:
100, 110 and 120 mJ/cm² (pig 1, 2, 3 for MED of about 2.5)
In vitro part:
Dose level:
Skin preparation:
Cells:
Control:
Skin temperature:
Test chamber:
Route:
Exposure time:
Sampling time
points:Examinations:

In vivo part:
Dose level:
Controls:
UV-B exposed:
Sunscreen in a Hill
Top chamber:
Route:
Exposure time:
Sampling:
Examinations:
GLP:
Published:

50 μl of each formulation on 0.64 cm² dermatomed pig skin
Exposed and unexposed skin sites were dermatomed to 400 µm.
Dermatomed skin, placed dermis side down on towels saturated withphysiological saline, was cut into with a 19 mm circular punch.
Formulation A and B: 4 with UVB exposed skin, 2 with unexposedskin
2 with UVB exposed skin, 2 with unexposed skin
37 °C
Flow-through diffusion cells
Topical application
24 hours
Every 2 h for the first 12 h, every 4 h thereafter up to 24 h
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)
250 μl of each formulation on exposed sites (n = 3 per formulation)
on 2 pigs on 1.0 cm² pad Hill Top chamber
Normal pig skin (no UVB, no sunscreen, no Hill Top chamber (n = 2
per pig)
No sunscreen, dry chamber (n = 2 per pig)
No UVB (n = 2 per formulation)
Topical application
2x 24 h and termination after 48 hSkin was removed by 8-mm biopsy punch
As in vitro part
No
Yes

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MethodThe purpose of the 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 (see Opinion on ZnO (nanoform)) present in sunscreen formulations.
Weanling Yorkshire pigs (approximately 20–30 kg) were sedated and multiple sites (about52) on the back were exposed to the UVB dose that caused a consistent +2 erythema (apale red in a defined area of the skin).
Twenty-four hours after UVB exposure, the pig was sedated, sites visually analyzed forconsistency, and the skin prepared 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 flowthrough diffusion cells with a dosing area of 0.64 cm2 and maintained at 37°C. The skin was
equilibrated in perfusate and a flow rate of 2 ml/h for 30 min prior to dosing. The skin was
subsequently dosed with 50 μL of each formulation (CM 630: (n=4 UVB exposed skin, n=2
unexposed skin; CM 634: n=4 UVB exposed skin, n=2 unexposed skin; and control: n=2
UVB exposed skin, n=2 unexposed skin). After completion 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, the perfusion was terminated and the skin wasremoved from the diffusion cells.
The dose site was removed with an 8 mm biopsy punch and cut into thirds. One third wasplaced in Trump’s fixative and stored at 4°C for later processing by light microscopy (LM;flow-through 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 the samples immediately stored at 4°C.
For in vivo treatment exposed sites (n = 3 per formulation) on two pigs were treated with
250 μl of each formulation; 200 μl was loaded onto the pad of the Hill Top chamber (1.0
cm² area) and 50 μl was placed directly on the skin within a template. Controls included
normal pig skin (no UVB, no sunscreen, no Hill Top chamber; n = 2 per pig), UVB-exposed(no sunscreen, dry chamber; n = 2 per pig), and sunscreen in a Hill Top chamber (no UVB;
n = 2 per pig per formulation). Sites were redosed with new Hill Top chambers after 24 h,and the treatment was terminated after 48 h. Erythema was scored for each site, and thepigs were euthanatized as above. Skin from all of these sites was removed with an 8-mm
biopsy punch for microscopy studies as stated above.
Results
For the in vitro studies, light microscopy showed that UVB exposed skin showed focal
intracellular epidermal oedema, sunburn cells, dermal inflammation and focal microblisterand residual sunscreen containing TiO2 limited to the stratum corneum. The morphology ofthe normal and the UVB-exposed skin was not affected by topical treatment with thesunscreen formulations. The TiO2 in each formulation was confirmed by TEM and elemental
analysis. EDS found the presence of Ti and Cu (copper grid)) in CM 630 and CM 634. Si forthe coating, Pb for lead citrate and U for uranyl acetate staining.
In the in vitro flow-through studies, TEM/EDS found penetration of Ti to a depth of 9 layers
in the stratum corneum of normal skin and 17 layers in the stratum corneum of UVBexposed skin. TEM/energy dispersive x-ray spectroscopy or inductively coupled plasma
mass spectrometry detected no Ti or Zn, indicating minimal transdermal absorption.
For in vivo tests, skin was dosed at 24 h occluded with formulations and at 48 h. TiO2 NP ino/w formulation penetrated 13 layers into UVB-damaged SC, whereas only 7 layers innormal skin; TiO2 in w/o penetrated deeper in UVB-damaged SC. Coated and uncoated ZnO
NP in o/w were localized to the upper one to two SC layers in all skin. TOF-SIMS showed Ti
within epidermis and superficial dermis, whereas Zn was limited to SC and upper epidermis
in both treatments. In summary, UVB-damaged skin slightly enhanced TiO2 NP or ZnO NP
penetration in sunscreen formulations but no transdermal absorption was detected.

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Conclusion
In summary, UVB-sunburned skin slightly enhanced the in vitro or in vivo penetration of the
TiO2 or ZnO NPs present in the sunscreen formulations into the stratum corneam (SC).
Although penetration of the two NPs into the SC was shown by TEM, and into the epidermis
and dermis by TOF-SIMS, there was no definitive evidence that they penetrated the skin in
vitro into the perfusate. In most cases, TiO2 penetration into the SC was greater than ZnO.
These results viewed together suggest minimal penetration of TiO2 and ZnO NPs into theupper epidermal layers when applied topically in sunscreen formulations to normal and
UVB-sunburned skin, with no evidence of systemic absorption.
SCCS Comments
The study is of a good quality. The test material relates to S75-K (>94% rutile, coated with
6-8% aluminium hydroxide, 3.5-4.5% dimethicone/ methicone copolymer). The results oftransmission electron microscopy indicated penetration of TiO2 nanoparticles into stratum
corneum, whereas TOF-SIMS analysis indicated penetration into the epidermis and dermis.
However, analysis of perfusate by TEM/Energy Dispersive Analysis or ICP-MS did not detect
Ti or Zn indicating nanoparticles did not penetrate the skin in vitro.
In Vitro study (Senzui et al., 2010 - Ref 204)
Study Design:
Guideline/method:
Species:
Test substances:

Formulations:
Dose applied:
Skin:

Skin temperature:
Exposure period:
GLP:
Published:
Study period:
Reference:

Yucatan micropig skin
All TiO2 are rutile-type
T-35. size 35 nm, uncoatedTC-35, size 35 nm, coated alumina + silica + silicone
T-disp, size 10 x 100 nm, mixture of alumina coated and silicone
coated
T-250, size 250 nm, uncoatedAll
formulations
contained
10%
TiO2
nanoparticles.
Cyclopentasiloxane (silicone, KF-995) used as dispersing medium)2 µl/cm²
Yucatan micropig skin removed the subdermal tissue and fat wasused as full-thickness skin (intact skin). The SC was removed from
intact skin with adhesive tape (Scotch 313, 3M) (stripped skin). Hair
was removed from intact skin using tweezers (hair removed skin)
Not stated
24 h
No
Yes
Before 2010
Senzui et al., 2010 (204)

MethodThe TiO2 was suspended in a volatile silicone fluid used for cosmetics, cyclopentasiloxane,
at a concentration of 10%. The suspension was applied at a dose of 2 mg/cm2 for 24 h.
The skin penetration was investigated in vitro with intact skin and with stripped skin (the SC
removed from intact skin with adhesive tape) as a model of injured skin. In addition hairremoved skin (hair was removed from intact skin using tweezers) was used to represent
skin damaged by hair-removal treatment.
Two µl of suspension were applied to an area of skin of approximately 1 cm2. Then the skin
was placed on a modified Franz-type diffusion cell. After 24 h, the receptor phase (pH 7.1
isotonic phosphate buffer solution) was collected, the skin was removed from the diffusion
cell and cut off at the rim for mounting the cell. Residues on the skin surface were removedby two cyanoacrylate stripping and Ti in the skin was determined. For some samples, theepidermis and dermis were separated by heating after cyanoacrylate stripping.
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Skin conditions after application of TiO2 was observed using two methods. After applicationand drying, the skin surface was observed by digital fine scope microscopy. The epidermisof the skin prepared by a heat separation method was mounted on a scanning electron
microscope (SEM) stage with adhesive tape.

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Results
The particle size distribution of TiO2 in silicone was determined. The mean particle size of T35 was 1700 nm, which was larger than that of T-250, 1200 nm. In contrast, suspensions of
the coated TC-35 and T-disp contained nanoparticles with mean diameter of 80 and 130
nm, respectively.
Ti concentration in the receptor phase was similar in all skin conditions and formulation
applied. For intact and stripped skin, no significant difference in Ti concentration was foundbetween the control and suspension applied, which indicates TiO2 did not penetrate into theskin regardless of particles size and even when the SC was removed. For hair-removed skin,
Ti concentration in skin after application of TC-35 suspension was significantly higher thanthat of the control, and after application of T-disp suspension, tended to be high. The Ti
concentration in the dermis was not different from the control.
Ti concentration in the epidermis after application of TiO2 nanoparticles tended to begreater than that of the control, but the difference was not significant. The epidermis
consists of SC, viable epidermis and hair follicles. Ti was detected in the hair follicle pocketsof hair-removed skin, but not in the surrounding viable skin. The radius of a hair follicle is0.05 – 0.2 mm which allow solvent to enter the hair shaft and sebum did not fill the follicle
space. When fluid enters a small space by capillary action, small particles of Ti in fluid may
be able to enter the follicle. Large particles cannot be moved by such small force, but TC-35
well dispersed in solvent might enter a follicle more easily than other types of TiO2. For Tdisp, the dispersing agent had some effect, resulting in particles left in the skin after dryingof the suspension
Conclusion of the Applicant
The authors concluded that TiO2 does not penetrate into viable skin, even if the particle size
is less than 100 nm and the SC is damaged. However, immediately after hair removal theconcentration of Ti in skin was higher when TC-35 was applied, which was most probably
caused by dispersion. SEM-EDS observation showed that Ti penetrated into vacant hairfollicles but in any case did not penetrate into dermis or viable epidermis. It was noted thatsince this was an in vitro study, inflammation could affect the results and further in vivo
studies on viable skin with hair removal are needed.
SCCS Comments
The quality of the study is difficult to evaluate. Moreover, the study was performed with skinfrom Yucatan micropigs and experience with this skin type in skin absorbance studies islimited.
In vitro exploratory study - percutaneous skin penetration - pig skin (Ref 70)
Study design.
Guideline/method:
Species:
Test substances:
Particle size:
Group sizes:
Dose applied:
Skin:
Skin area
Skin temperature:
Test chamber:
Receptor fluid:
Exposure period:
GLP:
Published:
Study period:

exploratory study
pigs
T805 (Degussa), hydrophobically coated with trimethyloctylsilane
about 20 nm
n=2 skin samples
0.8 mg total (20 mg with 4% TiO2), 0.16 mg TiO2 per cm2
fresh skin obtained from pigs used within 3 h after collection
4.9 cm2
32oC
custom-made Franz-type diffusion cells
0.9% w/v NaCl, 0.1% w/v gentamycin sulfate, 1% w/v bovine serum
albumin in bi-distilled water
24 h
no
yes
1999
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Reference:

Reference 70 submission III+IV Pflücker et al., 1999

MethodFresh pig skin was obtained from the butcher, and used within 3 hours after collection. Skinsamples were punched (5 cm in diameter). The dermal absorption study was performed
with custom-made Franz-type diffusion cells. The lower cell was placed on a magnetic stirrer(Variomag, Germany) and connected by tygon tubes to a thermostat (Type CS-C6, Lauda,
Germany) set at a temperature of 32°C (in vivo skin temperature). Magnetic stirring bars
were placed in the lower cells, which were filled with the receptor fluid (0.9% w/v NaCl,
0.1% w/v gentamycin sulfate, 1% w/v bovine serum albumin in bidistilled water). 20 mg of
the test emulsion, which contained 4% titanium dioxide, were topically applied with agloved finger to two excised pig skin discs (area 4.9 cm2, 2.5 cm in diameter, giving a
concentration of 4 mg cm2). After 24 h incubation 2 mm punch biopsies were obtained forhistological evaluation (TEM and SEM). SEM micrographs were recorded to evaluate themorphology of the freeze-dried skin samples and the stripped stratum corneum sheets.
Freeze dried skin samples were investigated before and after 10–fold tape stripping.
Results
TiO2 was found exclusively on the outermost SC layer. No titanium dioxide could be found in
the living cell layers of the stratum granulosum. The surface deposit, as displayed by TEM,
featured clearly distinguishable agglomerates as well as single particles with a characteristic
cubic shape and a primary particle size of about 20–50 nm. Concurrently, SEM/EDXA
micrographs first showed an even distribution of TiO2 on the skin surface. After 10-fold
stripping, however, TiO2 was found to be localized only in the furrows and not on thepartially removed ridges of the skin surface. In the upper part of the hair follicle TiO2 wasdemonstrated.
SCCS Comments
The actual TiO2 dose was 0.16 mg, and not 20 mg as mentioned in the paper. The study
does not show quantitative results but demonstrates by electron microscopy that the TiO2
nanoparticles are present on the skin mainly as aggregates. The study is of limited value
with number of samples investigated was only 2, but can be considered as supporting
evidence that TiO2 nanoparticles do not penetrate to the viable cell layers of the dermis.
In vitro exploratory study - percutaneous skin penetration and in vivo - humanskin (Ref 78)
Study design.
Guideline/method:
Species:
Test substances:

Particle size:
Group sizes:
Dose applied:
Skin:
Skin area

exploratory study
human healthy volunteers (female)
Mixture of broad spectrum UV water-in –oil emulsions containing
water, glycerin, dimethicone, ethylhexyl methoxycinnamate,isododecane, cyclomethicone, C12-15 alkyl benzoate, PEG-30
dipolyhydroxystearate, decyl glucoside, dodecyl glycol copolymer,
magnesium aluminium silicate, preservatives, zinc oxide, tocopheryl
acetate, o -cymen-5-ol, fragrance, xanthan gum and 3% ultrafine
TiO2 (T805, Degussa, Germany) and 8% methylene bisbenzotriazoyl tetramethylbutylphenol (MBBT) in a dispersion of decyl
glucoside.
TiO2 was coated with trimethyloctylsilane.
TiO2 20 nm
n=3
2 mg/cm2 of formulation, 60 μg TiO2 / cm2
in vitro abdominal and face skin frozen until use,in vivo skin of upper arm
in vivo 10 cm2 (2x5 cm)
Teflon static diffusion cell 10 cm2 (2x5 cm)
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Test chamber:
Receptor fluid:
Skin temperature:
Exposure period:
GLP:
Published:
Study period:
Reference:Franz diffusion cell 1.13 cm2
Teflon® homemade static diffusion cell with a 10 cm2 (5x2 cm)
surface and a receptor volume of 8 ml.
Franz diffusion cell with a 1.13-cm2 surface and 5 ml of receptor fluid.
0.9% NaCl water solution with 3% bovine serum albumin
32oC
5h
no
yes
2007
Reference 78 Submission VII Mavon et al., 2007.

MethodSamples of the mixture of broad spectrum UV water-in –oil emulsions were applied on skinof volunteers (10 cm2, 2x5 cm) and on two types of diffusion chambers, one Teflon®
homemade static diffusion cell with a 10 cm2 surface allowing tape stripping of the test
system, and a Franz diffusion cell with a 1.13-cm2 surface. The applied dose for the in vitro
study was 60.6 ± 3.1 μg/cm2, and for the in vivo study 58.4 ± 1.9 μg/cm2.
The distribution of the sunscreens in the skin was directly assessed by the tape stripping
method, using adhesive tape (Scotch TM No. 6204, 3M Corp.). A total of 15 tape strippings
were applied onto the surface of the skin, and each was pressed on the skin 10 times with aroller. Each strip was removed with 1 quick movement. No washing procedure was used.
The titanium analysis in the tape strippings and skin samples (epidermis, dermis andreceptor fluid) was based on a microwave assisted treatment, which digested the organic
components in the presence of sulphuric and nitric acid. The samples were then analyzed bycolorimetric assay, using diantipyrylmethane (0.5 g in 20 ml HCl 1 N). One ml of the coloredsolution and 1 ml of the solution to be tested were mixed. The absorbance was read at 390
nm with a spectrophotometer (Anthelie Advanced, France) 30 min later. Using thistechnique, a LOD of 0.2 μg/ml was obtained.
Transmission electron microscopy and particle-induced X-ray emission (PIXE) techniques
were used to localize the TiO 2 in skin sections. Punch biopsies of 6 mm in diameter weremade on skin samples, consecutively after 1, 8 and 15 tape strippings and were fixed with
2% glutaraldehyde in a Sorensen buffer for TEM analysis.
Results
For the in vitro experiments with n=3 >94.2% of the recovered TiO2 was found in the 15
tape strippings and in the stratum corneum. In the epidermis 5.6% was found, and <0.1%
was found in the dermal compartment. No TiO2 was found in the receptor fluid (below LOD).
The amount recovered accounted for 88.8% of the applied dose of TiO2. In the in vivo study(n=3) the recovery was 93% of the TiO2 dose. Most of the recovered dose was in the first
three tape strippings. After 15 tape strippings a few grains could be distinguished in the
TEM samples (amplification x 15,000), attributed to TiO 2 nanoparticles, but they were very
few and isolated in the stratum corneum (SC) layer. Deeper in the SC, no particles could beobserved, which suggested an absence of penetration into the viable skin tissue.
The 2-dimensional mapping of titanium using Micro-PIXE analysis of the skin showed thatmost of the Ti applied at the skin surface remained there or penetrated only into the opened
infundibulum. Quantitative analysis revealed a concentration of Ti at the LOD, in theunderlying layer of the epidermis, the dermis, the follicle and the sebaceous glands.
It was concluded by the authors that the study confirms that TiO2 accumulates in theuppermost layers of the SC and in the opened infundibulum only. No TiO2 was detected in
the viable skin layers through either transcorneal or transfollicular pathways. From thesedata the authors concluded that the amount of TiO 2 found in the in vitro ‘epidermal’compartment is located mainly in the furrows or the opened infundibulum and does not
represent actual transcorneal penetration.

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SCCS Comments
Both TiO2 and MBBT were present in the broad spectrum UV water-in–oil emulsions. Lack ofpenetration of TiO2 was supported by both in vitro and in vivo studies. Whether the detectedparticles were attributed to TiO2 or not has not been identified by the study.
In vitro study - Percutaneous skin penetration pig skin (Ref 56)
Study design.
Guideline/method:
Species:
Test substances:
Particle size:

Group sizes:
Dose applied:
Skin:
Skin area
Skin temperature:
Test chamber:
Receptor fluid:
Exposure period:
GLP:
Published:
Study period:
Reference:yes (OECD 428, SCCNFP/0750/03) skin absorption in vitro methodpig
T-Lite SF-S coated with silica (2%-5% wt%) and methicone (4.5%6.5%)
T-Lite SF coated with methicone (3.5%-5.5%)
T-Lite SF-S, needle like with a size of 30-60x10 nm
T-Lite SF, needle like with a size of 30-60x10 nm
Both TiO2 materials were present including aggregates up to 200 nm
and higher (1 μm)
skin from 3 pigs, and per sample 3 skin preparations (n=9)
4mg/cm2 corresponding to nominal doses of about 400 μg/cm2 oftitanium dioxide or to nominal doses of
240 μg/cm2 of titanium,
full thickness skin samples from lateral abdominal region
about 1 cm232 ± 1oC
modified Franz static dermal penetration cells
physiological saline containing 5% bovine serum albumin
24 h, sampling at various time intervals (3, 6, 12, and 24 h)
yes
yes
2007
Reference 56 Submission VII Gamer et al., 2007

MethodAfter removal of the receptor fluid the skin was removed from the diffusion cell and put onto
parafilm. Titanium was removed from the skin preparations by washings with sponge pieces
dipped into soap solution, and subsequent tape stripping was used to remove titanium
together with the superficial layers of the stratum corneum. Ti was determined byinductively coupled plasma-atomic emission spectrometry (ICP-AES) or ICP-mass
spectrometry (ICP-MS).
Results
For the titanium dioxide formulations T-Lite SF-S and Tlite SF, mean total recoveries of Ti
ranged from 98% to 100% and 86% to 93% of the total Ti applied, respectively. Virtually
the total amount of applied Ti could be removed from the skin surface by washing. The
amounts of titanium found in the tape strips and skin preparations were in the order of theanalytical determination limit. No Ti was found in the receptor fluid at any sampling time.
SCCS Comments
This is a GLP study with three independent measurements indicating lack of TiO2
penetration in an in vitro assay using pig skin. Although the number of measurements(n=3) per skin is limited, it was repeated in skin samples of three different pigs.

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In vitro exploratory study percutaneous skin penetration, and in vivo study onhuman skin (Ref 130)
Study design.
Guideline/method:
Species:
Test substances:
Particle size:
Group sizes:
Dose applied:
Skin:

Skin area
Skin temperature:
Test chamber:
Receptor fluid:
Exposure period:
GLP:
Published:
Study period:
Reference:

exploratory study
human (male and female)commercial microfine TiO2 dispersion either in octyl palmitate or inwater (Tioxide Specialities Ltd)
not reported
n=3
in vitro 150 μl/cm2 of commercial preparation (5% TiO2, 7.5mg/cm2)
in aqueous or oily dispersion
In vivo 2 μl/cm2 (5% TiO2, 0.1 mg/cm2) in aqueous or oily dispersion
in vitro human skin from abdominal area (samples stirred at -20oC),
and skin equivalents with cultivated human keratinocytes andfibroblasts
In vivo ventral side of forearm of male and female volunteers
not reported
32oC
penetration cells identified with figure.
phosphate buffer pH 7.4
24 h for in vitro studies
45 minutes for the in vivo studies
no
yes
2000
Reference 130 Submission VII Bennat and Müller-Goymann 2000

MethodA penetration cell was used for both skin samples and the human skin equivalent studies.
For in vitro test the amount added was 150 μl per skin sample, for the in vivo tests 2 μl perskin area. This results in TiO2 administrations of 7.5 mg/cm2 and 0.1 mg/cm2, respectively.
All dispersions were removed after the exposure period (in vitro 24 h, in vivo 45 minutes)
with a paper towel. Both in vivo and in vitro Tesa® were used for collection of cell layers of
the skin treated with the TiO2 formulations. Atomic absorption spectrometry (AAS) was used
for determination of the Ti content. Tests were performed in triplicate. The formulations
investigated were: an oil/water emulsion with carboxymethylcellulose (CMC), anddimethicon and silicon oil; a liposomal formulation with phospholipid and water.
Results
The amounts of Ti observed after the in vitro and in vivo exposure of skin was in the μg
range. In the sequential tape strips starting at about 25-35 μg/cm2 in the first tape strip
and declining just above the limit of detection (0.1 μg/cm2) level at tape strip #6-#12. For
the oily dispersion having the highest Ti levels were measured in the first tape strips. For
the in vivo exposure the Ti recovery started at about 7.5 μg/cm2 and declined in thefollowing tape strips. Microfine TiO2 was found to penetrate deeper in the human skin froman oily dispersion than from an aqueous one.
SCCS Comments
No information was provided on the actual size of the used microfine TiO2, so the study can
only be considered as supporting evidence.

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In vitro exploratory study percutaneous skin penetration - human skin (Ref 142)
Study design.
Guideline/method:
Species:
Test substances:
Particle size:
Group sizes:
Dose applied:
Skin:
Skin area
Skin temperature:
Test chamber:
Receptor fluid:
Exposure period:
GLP:
Published:
Study period:
Reference:

exploratory study
human (female Caucasian)Solaveil CT10W 3% W/Si emulsion, 3% W/O emulsion, both used assprayable product (Uniqema, UK)
not reported in M&M section. Mentioned in Discussion to be between
20-70 nmn=6 (experiments)
2 mg/cm2
abdominal skin from plastic surgery stored at -25oC for maximally 6months5.31 cm2
32 ± 1 oC
static Franz-type diffusion cell,
PBS with 4% bovine serum albumin
1, 2, 4, 6, 8, 12 and 24 h
no
yes
2009
Reference 142 submission VII Durand et al., 2009

MethodThe incubation in the diffusion cells was performed in wells covered with Parafilm paper toavoid drying, and the whole system was protected from sunlight by opaque paper. Receptor
fluid was removed at several time points and replaced immediately with fresh solution. Ti
level was quantified and determined by Inductively Coupled Plasma-Optical Emission
Spectroscopy (ICP-OES). Samples were digested and dissolved before Ti determination.
At the end of the experiment, the skin samples were removed from the cell and rinsed with
PBS solution and tetrahydrofuran/ acetonitrile (THF/CAN, 80 : 20, v : v) until no productwas left on the skin. The skin was then ground and mixed with a THF/ACN (80 : 20, v : v)
solution and placed in an ultrasonic bath for 30 min. Each solution was then divided in two
parts: one part was kept at -25oC for the further analysis of the TiO2 by spectrometricmethods. Three types of sample were taken and analysed:
1 The receptor fluid (5 mL).
2 A solution of the recovered product remaining on the skin (after evaporation of all liquid).
3 The mixture of ground skin (after evaporation of all the liquid).
The samples were heated at 450oC in a muffle furnace for 10–12 h. They were then fusedwith 5 g of K2S2O7 in a flame. The resulting substance was dissolved in 10 mL hot H2SO4
solution (1 : 1 v/v) and diluted with ultra-pure water to 100 mL.
The solutions obtained were then injected into the ICP-OES apparatus.
Results
The recovery of the TiO2 from the emulsions and spiked PBS solution with 4% BSA was
92.5% (W/O emulsion), 92.4% (W/Si emulsion, and 96.8% for the BSA-PBS solution ,respectively, demonstrating the validity of the method for determination of Ti. In each partof the skin and in the receptor fluid for W/O and W/Si, respectively. the levels of recovery
were between 76% and 86% for Ti present in the skin and/or on the skin. The limits ofdetection and of quantification are respectively 0.01 ppm (0.01 μg/g) and 0.1 ppm (0.1μg/g) for the W/O and W/Si emulsion. Presence on skin after washing was about 40% and
50% for the W/O and W/Si emulsion, respectively. Approximately 20% (W/O) to 40%(W/Si) of the TiO2 was observed in the skin. After 24 h of experiment titanium levels were
below the limit of detection. So it was considered that no TiO2 passed into the receptor fluid.
There was a loss in the recovery up to 25% of the administered dose.

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SCCS Comment
No characterization data for TiO2 is presented - only size has been indicated in discussionsection of the paper. Presence of coating indicated in Table 4.2 of supplicant but notmentioned in the paper. Data on receptor fluid indicated in text but not shown in paper.
Level of Ti at 24 h mentioned to be below limit of detection but data on
recovery/determinations at various time points are not presented in the paper. Results areof limited value for the evaluation of skin penetration of TiO2 as no data on the receptor
fluid were presented. It was demonstrated that approximately 20% to 40% of the TiO2 wasobserved in the skin. No further evaluation of localization was done.
In vitro exploratory study percutaneous skin penetration - human skin (Ref 143)
Study design.
Guideline/method:
Species:
Test substances:
Particle size:
Group sizes:
Dose applied:
Skin:
Skin area
Skin temperature:
Test chamber:
Receptor fluid:
Exposure period:
GLP:
Published:
Study period:
Reference:

exploratory study
human
Titanium dioxide T805, and Spectra veil MOTG, a 60% dispersion ofzinc oxide in mineral oil/triglyceride.
not reported
not reported
1 mg/cm2 in vitro
abdominal skin recovered from plastic surgery
not reported
room temperature
not reported
not reported
not reported
no
yes
1997
Reference 143 submission VII Dussert et al., 1997.

MethodThe presence of TiO2 in the skin was evaluated by TEM. At TEM characterization the TiO2
was identified as a mixture of rutile and anatase crystal forms. The sunscreen formulation
investigated was a mixture of both TiO2 and ZnO. The test formulation was a w/o emulsion
formulated with ultrafine titanium dioxide (11% wt), and zinc oxide (2.5% wt). The
formulation was used as topical administration in vitro with a dose of 1 mg/cm2 Skin
penetration was evaluated by TEM.
Results
Cross-sections of the horny layer of human epidermis, after topical application of thesunscreen emulsion, show an almost regular mineral-coating of the stratum corneum. The
crystals appear to surround the desquamating corneocytes. However, neither intercellular
nor intracellular penetration of crystallites is evident in transmission electron microscopy.
The TEM evaluation shows the presence of particles above the stratum corneum andbetween desquamating stratum corneum cells.
SCCS Comments
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. A
mixture of TiO2 and ZnO nanoparticles was used in the formulation. In the TEM evaluations
the TiO2 and ZnO could not be identified separately. This study is of no value for theevaluation of skin penetration of TiO2 nanoparticles. Presence of coating is indicated in Table
4.2 of supplicant but not mentioned in the paper.

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2

Kertesz et al. 2004, Ion-microscopic evaluation of porcine or human skin after
treatment with TiO2 samples (Ref 66)

3

Samples investigated by ion microscopy are 14-16 µm thick porcine and human skin.

4
5

Quantitative elemental concentrations and distributions a new measurement setup and data
evaluation system has been developed.

6
7
8

The penetration studies using different formulations were started on domestic pig skin,which resembles human skin closest. In a next step, human skin xenografts transplanted
into SCID mice were used.

922 pig skin, 11 transplanted human skin and 13 human skin samples were investigated.

10
11
12
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16
17

Results
The results obtained by ion microscopy or electron microscopy show that in the case ofhealthy skin the nanoparticles penetrate into the deepest corneocyte layer of the skin, butnever reach the vital layers.

18

Nanoderm - Quality of skin as a barrier to ultra-fine particles (ref 67)

19
20

Penetration of TiO2-nanoparticles through the epidermis of human foreskin grafts
transplanted into SCID (Severe Combined Immune Deficiency) mice.

21
22

The skin grafts were treated with a hydrophobic emulsion (Antheil’s XL F60) containing
micronized TiO2-nanoparticles in occlusion, for 1, 24 and 48 h.

23
24
25
26

Quantitative elemental concentrations and distributions have been determined in 14-16 µm
thick freeze-dried sections obtained from quick frozen punch biopsies using PIXE (Particle
Induced X-ray Emission), STIM (Scanning Transmission Ion Microscopy) and RBS(Rutherford Backscattering) analytical methods.

27
28
29
30
31
32
33
34
35

ResultIn most cases it was found that the remnant of the liposome crème together with theoutermost stratum corneum was removed during the sample preparation. When the crème
remained on the skin the Ti was quasi homogeneously distributed in the outermost layers,
and the penetration seemed to be limited to the outermost part of the stratum corneum.
However, in two cases, both after 48 h exposure, penetration through the stratum corneum
to the limit of the vital stratum granulosum was observed. The sample originates from theentry of a sweat gland.

36
37
38
39

Conclusions
No penetration to the viable skin was reported except for some limited observations ofmaterial entering sweat glands.

40
41

Adachi et al., 2010, In vivo effect of industrial titanium dioxide nanoparticles
experimentally exposed to hairless rat skin (Ref 126)

42

Guideline/method:

No specific guidelines followed

43
44

Test system:

Hairless Rat (Male Westar Yogi Rats) 8 weeks old, weighing 202–267
g, (Japan SLC, Hamamatsu)

45
46

Test items:

Uncoated anatase TiO2 nanoparticles (ST-01) from Ishihara Sangyo,
Ltd, Japan.

47
48
49

Formulation

White water/oil (W/O) emulsion containing 10 wt% TiO2, 4 wt%
Nikko Nikkomulese WO (cyclopentasiloxane, PEG-10 dimethicone,dosteardionium hectrite), 50.0 wt% decamethylcyslopentasiloxane

Conclusion
No penetration of the test material into viable porcine or human skin54

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2

KF-995 and 0.55 wt% acetic acid, and purified water was added to a
final volume of 100 wt%

3
4
5

Concentrations:

Four mg/cm2 emulsion (0.4 mg/cm2 TiO2) was applied to a 15 cm2
area on the rat dorsal skin in the absence of ultraviolet (UV)radiation.

6
7
8
9
10

Exposure:

Skin samples at 4 h after exposure were observed using light,
electron, and confocal laser scanning microscopy over 48 hrs. Timecourse study for light microscopic evaluation in the other groups ofrats (10 TiO2-treated and five control rats) was carried out at 24, 72
and 168 h after exposure.

11
12
13
14
15
16
17

Results
After 24 h, no particles were observed in keratinized layers of the follicular infundibulum,
but a small amount of particles remained in the superficial part of the stratum disjunctum.
After 72 h, the particles were still observed in upper keratinized layers of the infundibulum
but were not found in the interfollicular horny cell layer (Figure 3d). After 168 h, small cropsof particles were found in the uppermost keratinized layer of only a few follicular openings.

18
19
20
21

Conclusion
The study shows no penetration of TiO2 in water / oil emulsion into viable skin througheither the transcorneal or transfollicular pathway.

22
23
24

Gopee et al., 2009, Lack of dermal penetration following topical application ofcoated and uncoated nano- and micron-sized titanium dioxide to intact anddermabraded skin in mice (Ref 162 - poster presentation)

25

Guideline/method:

No

26

Test system:

Mice (hairless)

27
28
29
30
31
32
33

Test item:

TiO2 (Unreported batch) roughly spherical uncoated particles,with 25.1 ± 8.2 nm diameter (minimum particle size was 13 nm
and maximum particle size was 71 nm). Formulation consistedof titanium dioxide suspended in polyglyceryl-3 distearate,cetearyl alcohol, light mineral oil, propylene glycol, k-phosphatebuffer, methyl paraben, propyl paraben, and propylene
glycol:water (1:4, v:v).

34
35
36
37
38
39

Treatment:

Mice (hairless) were treated with 5 uL of 5% uncoated anatase
TiO2 (intact or dermabraded skin). At 6 and 24 hr postapplication, mice were sacrificed and skin, right regional lymph
nodes, blood, liver, kidney and spleen were collected andanalyzed for titanium (Ti) by ICP-MS. Tissues of one mouse wasanalyzed microscopically.

40
41
42
43
44
45
46

ResultNo significant elevations in Ti levels were observed in any of the organs analyzed for Ti.

47
48
49

Kiss et al. 2008, Investigation of micronized titanium dioxide penetration inhuman skin xenografts and its effect on cellular functions of human skin-derived
cells (Ref 167)

50

Guideline/method:

Conclusion
The results suggest that both intact and compromised skin of hairless mice may be aneffective barrier for nano-sized TiO2.

No

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2

Test system:

In vivo SCID mice, grafts area, 6-mm diameter human foreskinpunch biopsies were taken.

3
4
5

In vitro:

human immortalized HaCaT keratinocyte cells, human dermal
fibroblasts (HDFs) & human immortalized sebaceous gland cellline SZ95.

6
7

Test items:

TiO2, 9 nm Anatase (gift from Prof. Z. Stachura, Krakow,
Poland)89Vehicle:hydrophobic emulsion (‘TiO2-emulsion’) was used (Anthelios XL
SPF 60, La Roche Posay, La Roche Posay, France)

10

Concentrations:

2 mg / cm³

11

Exposure:

24 h

12
13
14
15
16
17
18
19
20

ResultTiO2 particles did not penetrate through the stratum corneum of human skin transplants.
TiO2 nanoparticles are internalized by in vitro cultured fibroblasts and melanocytes but notby keratinocytes and sebocytes.

21
22

Pinheiro et al. 2007, The influence of corneocyte structure on the interpretation of
permeation profiles of nanoparticles across skin (Ref 191)

23

Guideline/method:

24
25

Test system:
Healthy and psoriatic human skin was collected by .punch
biopsy (3 mm diameter) at lumbar-sacral region,

26
27

Test material:

Commercial
sunscreencontaining nano TiO2.

28

Concentrations:Unknown

29

Exposure:

2h

30
31
32
33
34

Results
The TiO2 permeation in psoriatic skin reached deeper regions of the stratum corneum thanin healthy skin. However, for both cases TiO2 nanoparticles did not reach the living layers of
the granulosa or spinosum strata.

35
36
37
38
39

Conclusion
Psoriasis seems to have only a limited effect on the permeation profile of TiO2
nanoparticles. It has to be mentioned that the source and concentration of the particles is
not specified in this study.

40

Popov et al. 2005, 2005, 2010 (Ref 192, 193, 194)

41
42
43
44
45
46

Test system:

human skin (volunteers). Sunscreen including rutile TiO2
particles (100 nm) was administered five times over a period of
4 days onto the surface area of flexor forearm skin. The tapestripping procedure started on the fourth day, 1 h after last
application. The surface density of TiO2 particles on the tape
strips was analyzed by x-ray fluorescent measurements.

47
48

Test material:

Sunscreen including rutile TiO2 particles (100 nm), this was notfurther specified.

Conclusions
This type of TiO2 (custom made, anatase) does not penetrate human foreskin grafts. In
vitro uptake is cell type dependent.

No

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formulation(unknown

source),SCCS/1516/13
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2

Concentration:

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5
6
7
8
9
10
11

Results
Approximately 14 µg/cm2 of TiO2 was found on the first tape strip and almost zero on thosetaken at the depth of 15 µm. The particles were mainly located at a depth range of 0 to 3
µm.

12
13
14

Sadrieh et al. 2010, Lack of significant dermal penetration of titanium dioxide(TiO2) from sunscreen formulations containing nano- and sub-micron-size TiO2
particles (Ref 199)

15
16

Test system:

Female Yucatan minipigs (~4 months of age; n ¼ 12) from
Sinclair Research Center (Auxvasse, MO, USA).

17
18

Test items:

Uncoated nano titanium dioxide (Degussa Aeroxide P25, a
mixture of anatase and rutile and known to be photocatalytic;

19
20
21
22
23
24
25
26
27

2 mg/cm² sunscreen. skin area of 10 X 8 cm (160 mg
sunscreen).

Conclusions
No penetration into living layer of skin. The source and nature of TiO2 is not well reported.
Three different papers all presenting the same experiment as an original study.

Vehicle1. coated (aluminum hydroxide/dimethicone copolymer) nanotitanium dioxide (BASF T-Lite SF obtained from BASF,
Shreveport, LA; rutile; ‘‘coated nano’’)2. uncoated submicron titanium dioxide (treated withaluminum hydroxide, Ishihara Tipaque CR-50 obtained from
Ishihara
Corporation,
San
Francisco,CA;rutile;‘‘submicron’’)All used particles were added to the same sunscreen
preparation, preparation without particles was used as control.

28

Concentrations:

Approximately 5% preparations were achieved.

29
30
31
32

Exposure:

Topical application four times daily, 5 days a week, for a total of
22 days. Dose of 2 mg/cm², each animal received a total of 176
mg/cm² cream resulting in a average of ~1.32 l of cream per
animal33

Negative control:

cream without TiO2

34
35
36
37
38
39
40
41
42

Result
The epidermis from minipigs treated with sunscreens containing TiO2 showed elevatedtitanium levels. Increased titanium was detected in abdominal and neck dermis of minipigs
treated with uncoated and coated nano TiO2. EM-energy dispersive x-ray analysis showedthat TiO2 particles were found in the stratum corneum and upper follicular lumens in alltreated skin samples. Isolated titanium particles were present at various locations in thedermis of animals treated with any of the three types of TiO2 sunscreens; however, there
was no pattern of distribution or pathology.

43
44
45
46
47
48
49
50
51

Conclusion
These findings indicate that there is some, though probably not significant, penetration of
TiO2 nanoparticles through the intact normal epidermis in minipigs. The quantification of theconcentration in the dermis is difficult since the removal of the epidermis is almost never
perfect (resulting in possible false positive results).
Exploratory study, dermal penetration and toxicity, hairless mice and porcine skin,subchronic dermal exposure (Wu et al., 2009)57

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8

The paper has its focus on the penetration of TiO2 nanoparticles through the skin after
dermal exposure.

No penetration in in vitro porcine skin model of TiO2 (4, 10, 25, 60 and 90nm). The
amount of TiO2 was below detection limit, but materials and methods stated that TiO2
was not removed. Not clear whether the TiO2 was removed before tape stripping.
Results indicate that tape stripping most probably was done after removal of TiO2,
hence there was a low levels in the tape strip pools.

9
10
11



Pig skin in vivo: TiO2 present in stratum corneum, stratum granulosum, prickle cell layerand stratum basale of the epidermis but not in dermis. Only 4nm TiO2 in basal cell
layer. Figure 2 does NOT clearly show presence of TiO2 nanoparticles in epidermis.

12
13
14



Hairless mice: Effect of TiO2 on body weight observed. Decreased growth compared tocontrol mice and mice treated with normal sized TiO2. 10-25- and 21 (P25) nm TO2
induced growth retardation.

15
16
17



Biochemical parameters for skin and liver malondialdehyde (MDA) increase (10-2521nm), superoxide dismutase (SOD) skin and liver decrease (10-21nm), skinhydroxyproline (HYP) decrease (10-25-21-60nm)

18
19
20



Organ distribution after 60 days skin exposure showed 10, 25, 21, 60nm TiO2 in skin,
sub muscles, heart, liver, spleen, 21, 60nm TiO2 in lung, 21nm TiO2 in brain, whereas
TiO2 in kidney was similar to control. However the differences were not significant.

21
22
23
24
25
26
27
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30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54

Conclusion
Local effects on skin are demonstrated by biochemical parameters SOD, MDA, and HYP, andhistopathology (keratinization). Systemic effects are not clearly identified because of
possible alternative route of exposure by oral uptake. Also the lesions shown in variousorgans may be due to background lesions present in animal strain. This is not excluded byscoring of lesions in control versus treated animals. However, the treatment resulted in
growth retardation of the animals.
Studies with limited information
In vivo study (Gottbrath et al., 2003; FitzGeral, 2005)
Penetration of nano-sized titanium dioxide (Tioveil AQ N; (rutile, coated with alumina/silica)
into human stratum corneum after in vivo application of two formulations was studied.
Penetration was measured by tape stripping of skin (10 strips). Tape strips from thetitanium dioxide-treated skin sites were assayed for titanium by atomic absorption
spectrometry. Tape strips from the vehicle control treated sites were viewed with aninverted microscope to estimate the amount of corneocyte aggregates. Titanium dioxide
nanoparticles in the formulations and tape strips were visualized by transmission electron
microscopy (TEM). The authors concluded that, after application of the liposomal
formulation, a fraction of the TiO2 nanoparticles penetrated into the stratum corneum anddid not remain in shallow valleys formed by the corneocytes, explaining the water resistanceof the liposomal formulation, i.e. the deposition of TiO2 nanoparticles depends on theformulation used.
In vivo study (Tan et al., 1996; FitzGeral, 2005)
Review of recent literature on safety of nanomaterials in cosmetics with special references
to skin absorption and resorption of ultrafine titanium dioxide and zinc oxide, prepared forPhysical Sunscreens Manufacturers Association (PSMA), European Cosmetic, Toiletry and
Perfumery Association and BASF AG, 28 September 2005.
A study with 10-50 nm TiO2 particles was performed in order to evaluate if the particles
could penetrate the stratum corneum to the dermis following repeated application involunteers (13 patients with compromised skin scheduled to have surgery for skin lesions).
58

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55
56

The patients received repeated application (twice a day for 2-6 weeks) of a sunscreen lotion
containing 8% microfine TiO2. Chemical analysis (ICPMS) were performed on skin biopsies.
The authors concluded that non-statistically significantly higher Ti levels in the dermis oftreated subject vs. controls (cadaver skin) were found.
In vivo study (Lademann et al., 1999)
The dermal penetration of 20 nm TiO2 nanoparticles (Titan M 160, coated, rutile) (assumed
particle size, based on description of product used) in a sunscreen formulation (o/wemulsion) was studied. The sunscreen was applied repeatedly (11 times) over 4 days to theforearm skin (2 mg/cm2) of human volunteers. UV/Vis spectroscopic evaluation, X-ray
fluorescence measurements LIFM, SRLSM and Raman spectroscopy of skin tape strips andhistological evaluation of skin biopsies were performed. The only significant finding
concerning a potential penetration of TiO2 beyond the upper skin layers was their depositionin single hair follicle openings, although there was no evidence that these residues werelocated within the living skin. The concentration of Ti in the hair follicle openings was twoorders of magnitude lower than that in the upper skin layers. The authors concluded thatthat there was no penetration of TiO2 particles in living skin and that the TiO2 particles
were mainly located in the outer layers of the SC.
In vivo study (Schulz et al., 2002)
The influence of particle size on the dermal absorption of three TiO2 preparations wasinvestigated (T805 [20 nm, cubic, Ti/Si coating, rutile/anatase], Eusolex T-2000 [rutile, 1015 nm NPs in 100 nm aggregates, needles, Ti/ Al2O3/SiO2 coated] Tioveil AQ-1 0P [100nm, needles, Ti/Al/Si coated]). Each had a different primary particle size (10-15 nm, 20 nm
and 100 nm), shape (cubic or needles) and hydrophobic/hydrophilic characteristics. The
preparations were topically applied (4 mg/cm2) in an oil-in water emulsion containing 4%
TiO2 to the forearm skin of human volunteers for 6 hours. Skin biopsies were examined byscanning electron microscopy to visualize the distribution of particles within the skin layers.
TiO2 particles were only deposited on the outermost surface of the SC, and were notdetected in deeper SC layers, the human epidermis and dermis. The authors concluded thatnone of the particles penetrated beyond the outer layer of the stratum corneum.
Another study provided under dermal penetration (Reference 10, submission 1) seems to be
an irritation study and has therefore not been reviewed.
SCCS Comments on Dermal/ Percutaneous Absorption
The studies presented in the submission cover a range of nanomaterials of which somerelate to the materials under assessment. The studies range from in vitro to ex vivo and invivo experimental conditions, and intact and UV damaged skin. The results from thesestudies suggest that TiO2 nanoparticles, when applied to skin in a sunscreen formulation,
are likely to stay largely on the skin, whilst a small proportion of the particles may
penetrate to the outer layers of stratum corneum. A few reports have suggested thepossibility that TiO2 nanoparticles may penetrate deeper to reach stratum granulosum –e.g. in human foreskin grafts transplanted onto SCID mice (Kertész et al., 2005) - or todermis of minipigs treated with nano TiO2 (Sadrieh et al., 2010 (Ref 199)). There is,however, a consistent and large body of evidence from the submitted studies, and otherstudies published in open literature (e.g. NANODERM, 2007; Nohynek et al., 2007), whichshows that nanoparticles do not penetrate deep enough to reach the viable epidermis ordermis cells of healthy skin. In psoriatic skin, Pinheiro et al. (2007) showed that nano-TiO2
in a sunscreen formulation penetrated into deeper areas of the stratum corneum than in
healthy skin, but did not reach living cells in either psoriatic or healthy skin. Some in vitro
test systems, however, lack a stratum corneum layer, which can block penetration of TiO2
nanoparticles. Toxicological effects from such tests therefore need a careful consideration
since they may be difficult to extrapolate to the effects in vivo (Nohynek et al., 2007).

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29

A recent study by Bennett et al. (2012) investigated the penetration of TiO2 particles
through isolated pig skin sections and found a small fraction of the total dose in the skinsections. The study found nanoparticles, or small clusters, in the interstitial spaces of the
porcine dermis after irradiation up to 500 µm depth, in comparison to the control skinsamples (tested under dark) where TiO2 was only found on the surface of the stratum
corneum. This study does raise questions over the possible disagglomeration of nanoparticle
clusters and enhanced penetration of TiO2 nanoparticles into skin under use conditions. The
study used TiO2 (anatase, non-coated) material, the type which is not recommended in thisopinion. Further studies will be needed on different crystalline forms and coated materials to
draw any conclusions on other TiO2 nanomaterials.

30

1.5.5

Contrary to the strong evidence suggesting a lack of penetration of TiO2 nanoparticles toviable epidermis or dermis cells, there are a number of studies (in this submission andpublished elsewhere), which indicate that nanoparticles can enter hair follicles. According to
SCCP opinion (2007) and NANODERM report (2007), adverse effects are not expected fromdermal exposure of healthy unflexed skin to photostable nano-TiO2 in sunscreens. However,
if photocatalytic nano-TiO2 is present in a sunscreen, it can potentially lead to generation ofreactive oxygen species (ROS) on exposure to UV light.
Most, if not all, studies provided in the submission were performed with nano TiO2 as
present in sunscreen formulations depicting consumer use. The studies were not directed
towards hazard identification using either a dose response approach or a worst casescenario (overdosing situation). It is also of note that currently there are certain knowledge
gaps in relation to the possible dermal penetration of nano TiO2 on repeated or long term
use of cosmetic products, which may not only be used on flexed healthy skin but also on
skin that may have lesions or cuts. Studies provided in support of this submission haveshown that TiO2 nanoparticles do not penetrate the (simulated) sunburnt skin, whereas
such information on flexed or damaged skin is currently not available.

Repeated dose toxicity

31
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50
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52

1.5.5.1 Repeated Dose (30 days) oral toxicity
Exploratory subchronic oral study – Mice 30 day oral (gavage)
Guideline:
Species/strain:
Group size:
Test substance:
Batch:
Purity:Vehicle:Dose levels:
Dose volume:
Route:
Administration:
GLP:
Study period:
Reference:

No guideline
Mice/CD-1
20 females per group
TiO2 (Anatase, prepared from hydrolysis of Ti-tetrabutoxide, Primaryparticle size 5 nm)

0, 62.5, 125 and 250 mg/kg bw/day
Oral
Intragastric administration every other day for 30 days
No
2009
SI-II-Duan et al., 2010, (140)

Results

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7

Mice treated with doses ≥125 mg/kg bw/d showed body weight reduction, an increase incoefficients of the liver and increased coefficients of the liver, kidney, spleen and thymus
and serious damage to liver function as shown by: A decrease in interleukin-2 activity, white blood cells, red blood cells, haemoglobin,mean corpuscular haemoglobin concentration, thrombocytes, reticulocytes, Tlymphocytes (CD3+, CD4+, CD8+), NK lymphocytes, B lymphocytes, and the ratio of
CD4 to CD8 of mice.

89

An increase in NO level, mean corpuscular volume, mean corpuscular haemoglobin, red(cell) distribution width, platelets, hematocrit, mean platelet volume of mice.

10
11
12
13



Disruption of the liver function in terms of enhanced activities of alanine
aminotransferase,
alkaline
phosphatase,aspartateaminotransferase,lactatedehydrogenase and cholinesterase, increase of total protein, and reduction of albumin to
globulin ratio, total bilirubin, triglycerides, and the total cholesterol levels.

14
15
16
17

No such effects were seen at low dose, and the NOAEL appears to be 62.5 mg/kg bw/d.

18

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

19
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34
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38
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54

SCCS Comment
NOAEL derived from this study is 62.5 mg/kg bw/d.

Subchronic oral toxicity – Rat 90 day oral (diet)
Guideline:
Species/strain:
Group size:
Test substance:
Batch:
Purity:Vehicle:Dose levels:
Dose volume:
Route:
Administration:
GLP:
Study period:
Reference:

No guideline
Rat/F344
10 m, 10 f per group
TiO2 (uncoated, Unitane®, Anatase), CAS No. 13463-67-7
402110C46
98%6250, 12500, 25000, 50000, 100000 ppm
Oral
Diet
No
1978
I-NCI, 1979 (22); DHS-NCI, 1979 (9)Results
No deaths, no differences in body weight gains, no substance-related gross or microscopic
pathological finding, NOAEL: 100000 ppm.
SCCS Comment
No information has been provided on the particle size profile of the material tested in thisstudy. The study is therefore of little value in relation to the current assessment for nanoforms of TiO2.
Note
The two references provided (I-NCI, 1979 (22) and DHS-NCI, 1979 (9)) are in fact thesame.
Subchronic oral toxicity – Mouse 90 day oral (diet)
Guideline:
Species/strain:

No guideline
Mouse/B6C3Fi
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Group size:
Test substance:
Batch:
Purity:Vehicle:Dose levels:
Dose volume:
Route:
Administration:
GLP:
Study period:
Reference:

10 m, 10 f per group
TiO2 (uncoated, Unitane®, Anatase), CAS No. 13463-67-7
402110C46
98%

49
50



Inhibition of the activities of Na+/K+-ATPase, Ca2+-ATPase, Ca2+/Mg2+-ATPase,
acetylcholine esterase, and nitric oxide synthase;

51
52
53
54



Disturbed function of the central cholinergic system – significantly decreased levels of
monoamines neurotransmitters such as norepinephrine, dopamine and its metabolite 3,
4- dihydroxyphenylacetic acid, 5-hydroxytryptamine and its metabolite 5hydroxyindoleacetic acid,55Increased levels of acetylcholine, glutamate, and nitric oxide.

6250, 12500, 25000, 50000, 100000 ppm
Oral
Diet
No
1978
I-NCI, 1979 (22); DHS-NCI, 1979 (9)Results
No deaths, no differences in body weight gains, no substance-related gross or microscopic
pathological finding, NOAEL: 100000 ppm.
SCCS Comment
No information has been provided on the particle size profile of the material tested in thisstudy. The study is therefore of little value in relation to the current assessment for nanoforms of TiO2.
Note: Two references provided (I-NCI, 1979 (22); DHS-NCI, 1979 (9)) are in fact the same.
Exploratory subchronic oral study – Mice 60 day oral (gavage)
Guideline:
Species/strain:
Group size:
Test substance:
Batch:
Purity:Vehicle:Dose levels:
Dose volume:
Route:
Administration:
GLP:
Study period:
Reference:

No guideline
Mice/CD-1
20 females per group
TiO2 (Anatase, prepared from hydrolysis of Ti-tetrabutoxide, Primaryparticle size 5 nm)

0, 5, 10, 50 mg/kg bw/d
Oral
Intragastric administration every day for 60 days
No
2010
SI-II- Hu et al., 2010 (163)

Results
Potential effects on nervous system function, significant impairment of the behaviours ofspatial recognition memory. Indications for impaired neurofunction and behaviour at alldose levels, indicated by: Significantly altered levels of Ca, Mg, Na, K, Fe and Zn in brain

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SCCS Comment
From the 60 day oral (gavage) study in mice, a LOAEL of 5 mg/kg bw/d may be derived.

5

1.5.5.3 Chronic (> 12 months) toxicity

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SCCS Comment on Repeated Dose Toxicity:
Two out of the 4 subchronic studies provided are of little value to the assessment of nanoforms of TiO2 because particle size distribution of the tested materials is not provided. The
other two studies used anatase nanomaterials. From the 60 day oral (gavage) study inmice, a LOAEL of 5 mg/kg bw/d may be derived.

1.5.6

Mutagenicity / Genotoxicity

There are a number of issues in regard to in vitro testing of nanomaterials for mutagenicity.
Bacterial mutagenicity assays are considered to be less appropriate for the testing of
nanoparticles compared to mammalian cell systems due to the lack of endocytosis bybacterial cells (EFSA, 2011). Therefore, for a negative outcome of such tests to be
acceptable, it is essential that contact of the test materials with bacterial DNA (i.e.
nanoparticle uptake by bacteria) is demonstrated. Furthermore, for testing of (conventional)
chemical substances, generally accepted positive controls are used for the various
Salmonella strains. The use of such chemical positive controls in testing nanomaterials
would not provide a proof for a negative response of the nanomaterial. Currently, there isno accepted nanoparticle positive control that can demonstrate whether the assay issuitable for the mutagenicity testing of insoluble/poorly soluble nanoparticles.
It is of note that the following studies have not been reviewed as part of this assessment
because they relate to test materials that are either not nanomaterials, or lack data on
material characterisation to establish whether they were relevant nanomaterials for thisassessment.
SI-Dunkel et al., 1985 (32); SI-Tennant et al. 1987 (33 (i, ii)); SI-Ivett et al., 1989 (35);
SIII-Lu et al., 1998 (56c), Nohynek, 1999 (56), PSMA statement, 1999 (66); SI-II Warheit
et al., 2007 (215); SIII-Lu et al., 1998 (56c), SI-Myhr, Caspary, 1991 (34); SI-Poole et al.
1986, (36); SI-Lemaire et al., 1982 (37); SI-II Msiska et al., 2010 (183); SI-Casto et al.,1979 (38); SI-Mikalsen et al., 1988 (39); SI-DiPole, Casto, 1979 (40); SI-Tripathy et al.,1990 (44); SI-Kitchin, Brown, 1989 (43); SI-II Pan et al., 2010 (189), SI-II DiVirglilio etal., 2010 (139), Osman et at 2012 (188).

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1.5.6.1 Mutagenicity / Genotoxicity in vitro

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Bacterial gene mutation test
Guideline/method: OECD 471 (1997)
Test system:
Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 andEscherichia coli WP2uvrA. Tests were performed in absence or presenceof S9-mix
Replicates:
Triplicate cultures in 2 independent experiments
Test items:
T805 (coated, A/R, PSMA 1 type)
Batch:/
Solvent:
Ethanol
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Concentrations:
Exposure:
Negative control:
Positive control:
GLP:
Date of report:
Reference:

8, 40, 200, 1000 and 5000 μg/plate in 1st experiment (range findings
experiment); 312.5, 625, 1250, 2500 and 5000 μg/plate in 2nd
experiment48 h using the direct plate incorporation method
yes (vehicle)
ENNG for WP2uvrA, TA100 and TA1535; 9AA for TA1537 and 4NQO for
TA98; 2AA in all strains in experiments with S9-mix.
in compliance
19 June 1994 – 25 August 1994
Submission DHS (11), II(67)The test substance was tested for mutagenicity in bacterial gene mutation assays with andwithout metabolic activation (S9-mix prepared from Arochlor 1254 induced male Sprague
Dawley rat livers) using the direct plate incorporation method. Test concentrations werebased on the results of a preliminary toxicity study. The S. typhimurium strains TA98,
TA100, TA1535 and TA1537, and the E. coli strain WP2uvrA- were exposed for 48 h to thetest substance (suspended in ethanol) in concentrations ranging from 8 - 5000 μg/plate (1stexperiment) and 312.5 - 5000 μg/plate (2nd experiment).
Results
The test substance caused no visible growth reductions. Precipitation was observed atconcentrations of 625 μg/plate and above. All positive controls showed marked effects onrevertant colony numbers and the ethanol vehicle tested negative. Exposure to the test
substance did not result in biologically relevant increases in revertant colony numbers.
Conclusion
Under the experimental conditions used T805 was not mutagenic in this gene mutation tests
in bacteria.
SCCS Comment
See comments under 3.3.6 on the issues relating to the suitability of bacterial mutagenicity
assays for nanomaterials.
Bacterial gene mutation test
Guideline/method: OECD 471 (1983)
Test system:
Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537.
Tests were performed in absence or presence of S9-mix
Replicates:
Triplicate cultures in 2 independent experiments
Test items:
T817 (coated, A/R, PSMA 1 type)
Batch:
04095
Solvent:
Ethanol
Concentrations:33.3, 100, 333.3, 1000, 2500 and 5000 μg/plate
Exposure:
48 h using the direct plate incorporation method
Negative control: vehicle
Positive control:
NaN3 for TA100 and TA1535; 4-NOPD for TA1537 and TA98; 2AA in all
strains in experiments with S9-mix.
GLP:
in compliance
Date of report:
1997
Reference:
Submission DHS (12), II(67)The test substance was tested for mutagenicity in a bacterial gene mutation test with andwithout metabolic activation (S9-mix was prepared from phenobarbital/-naphtoflavone
induced male Wistar Rat livers). The S. typhimurium strains TA98, TA100, TA1535 andTA1537 were exposed for 48 h to the test substance (suspended in ethanol) atconcentrations ranging from 33.3 to 5000 μg/plate.

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Normal background growth was observed up to 5000 μg/plate. All positive controls showeddistinct increases in revertant colony numbers. Exposure to the test substance did not resultin biologically relevant increases in revertant colony numbers.

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The test substances were tested for mutagenicity in the reverse mutation assay in bacteriawith and without metabolic activation. The S9 fraction was prepared from phenobarbital/βnaphthoflavone induced male Wistar rat liver. Both the standard plate test (SPT) and theplate incorporation test (PIT) were used. The S/ typhimurium strains TA98, TA100, TA102,
TA1535 and TA1537 were exposed to the test substance (dissolved in DMSO (SPT) or fetal
calf serum (PIT)) at concentrations ranging from 20–5000 μg/plate. For control purposes,
DMSO) as negative control and the positive controls (NOPD, MNNG, AAC, MIT.C, 2-AA) werealso investigated.

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Results
With the T-LiteTM SF a weak bacteriotoxicity was occasionally observed from 2500 μg/plate
onward in the presence of S9-mix only. With T-LiteTM MAX no bacteriotoxicity was noted.
Precipitation of the test substance was recorded from 100 μg/plate onward for T-LiteTM SF
and from 2500 μg/plate with T-LiteTM MAX.
The test substances 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.

Conclusion
Under the experimental conditions used T817 was not mutagenic in this gene mutation tests
in bacteria.
SCCS Comment
The study is on T817 (coated, A/R, 95%, PSMA 1 type) which relates to Eusolex T in thedossier. This study is relevant to the nanomaterial group (anatase).
See comments under 3.3.6 on the issues relating to the suitability of bacterial mutagenicity
assays for nanomaterials.
Bacterial Gene Mutation Test
Guideline/method: OECD 471 (1997)
Test system:
Salmonella typhimurium strains T 98, T 100, T 102, T 1535 and TA1537,
in presence or absence of S9-mix
Replicates:
Triplicate plates
Test items:
T-LiteTM SF, pure rutile, primary particle size 10 x 50 nm, mean
agglomerates approximately 200 nm (d10: 90 nm, d90: 460 nm);coating consisting of aluminium hydroxide and dimethicone/methicone
copolymer
T-LiteTM MAX, pure rutile, primary particle size 10 x 50 nm, mean
agglomerates approximately 200 nm (d10: 90 nm, d90: 460 nm);coating consisting of dimethoxydiphenylsilane, triethoxycaprylylsilane
crosspolymer, hydrated silica and aluminium hydroxide
Batch:/
Solvent:
DMSO (SPT), FCS (PIT)
Concentrations:0, 20, 100, 500, 2500, 5000 μg/plate
Exposure:Standard plate test and or preincubation test
GLP:
in compliance
Reference:
Landsiedel et al., 2010

Conclusion
Under the experimental conditions used T-LiteTM SFand T-LiteTM MAX were not mutagenic inthis gene mutation tests in bacteria.

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SCCS Comment
The tested materials relate to S75-K (94% rutile, coated with aluminium hydroxide,
dimethicone/methicone copolymer). See comments under 3.3.6 on the issues relating to thesuitability of bacterial mutagenicity assays for nanomaterials.
Chromosome aberration test in mammalian cells
Guideline/method: OECD 473 (1997)
Test system:
CHO cells. Tests were performed in absence or presence of S9-mix
Replicates:
Duplicate cultures in 2 independent experiments
Test items:
T805 (coated A/R, PSMA 1 type)
Batch:
0510067
Solvent:
Ethanol
Concentrations:
Experiment 1: 86.72, 209.7 and 800 μg/ml without S9 mix
167.8, 640 and 800 μg/ml with S9-mix
Experiment 2: 167.8, 512 and 800 μg/ml
Exposure:
Experiment 1: 20 h treatment without S9 mix
3 h treatment and 17 h recovery with S9-mix
Experiment 2: 3 h treatment and 17 h recovery with S9-mix
Negative control: Vehicle
Positive control:
NQO (without S9), CPA (with S9)
GLP:
yes
Date of report:
17 November 1998 – 11 January 1999
Reference:
Submission DHS (13), II(67)The test substance was evaluated for potential cytogenetic effects in Chinese hamster ovary(CHO) cells in the absence or presence of S9-mix. The S9 fraction was prepared from liversof rats treated with Arochlor 1254 (experiment 1) or phenobarbital/-naphtoflavone(experimen2t). Cytotoxicity was measured as a reduction in cell number compared to thesolvent control. In the absence of S9-mix only one experiment was performed. 4nitroquilonine 1-oxide and cyclophosphamide were used as positive controls in theexperiments without and with S9-mix respectively. For each culture cells with structuralaberrations excluding gaps, and polyploidy, endoreduplication or hyperdiploidy werecategorized.
Results
The number of cells with structural aberrations in the negative control cultures were within
normal range. A biologically relevant increase in the number of cells with chromosome
aberrations was not observed due to exposure to T805 both without and with S9-mix. The
positive controls NQO and CPA induced statistically significant increases in the number ofcells with structural aberrations in the absence or presence of S9 mix respectively.
Conclusion
Under the experimental conditions used T805 was not genotoxic (clastogenic) in thischromosome aberration test in mammalian cells.
SCCS Comment
The experiment in the absence of S9-mix was performed only once.
Chromosome aberration test in mammalian cells
Guideline/method: OECD 473 (1997)
Test system:
CHO cells. Tests were performed in absence or presence of S9-mix
Replicates:
Duplicate cultures in 2 independent experiments
Test items:
T817 (coated A/R, PSMA 1 type)
Batch:
04095
Solvent:
Ethanol
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Concentrations:

Experiment 1:

85.9, 640 and 800 μg/ml without S9-mix
167.8, 512 and 800 μg/ml with S9-mix
209.7, 512 and 800 μg/ml with S9-mix
20 h treatment without S9 mix
3 h treatment and 17 h recovery with S9-mix
3 h treatment and 17 h recovery with S9-mix

Exposure:

Experiment 2:Experiment 1:

Negative control:
Positive control:
GLP:
Date of report:
Reference:

Experiment 2:
VehicleNQO (without S9), CPA (with S9)
yes
June 1999
Submission DHS (14), II(67)The test substance was evaluated for potential cytogenetic effects in Chinese hamster ovary(CHO) cells in the absence or presence of S9-mix. The S9-mix was prepared from livers ofrats treated with Arochlor 1254 (experiment 1) or phenobarbital/-naphtoflavone(experiment 2). In the absence of S9-mix only one experiment was performed. Cytotoxicity
was measured as a reduction in cell number compared to the solvent control. 4nitroquilonine 1-oxide and cyclophosphamide were used as positive controls in theexperiments without and with S9-mix respectively. For each culture cells with structuralaberrations excluding gaps, and polyploidy, endoreduplication or hyperdiploidy werecategorized.
Results
The number of cells with structural aberrations in the negative control cultures was within
normal range. In the experiment without S9-mix, a slight but not statistically significant
increase in the number of cells with chromosomal aberrations was observed. In theexperiments with S9-mix no biologically relevant increase in the number of cells withchromosomal aberrations was observed. The positive controls NQO and CPA induced
statistically significant increases in the number of cells with structural aberrations in theabsence or presence of S9-mix, respectively.
Conclusion
Under the experimental conditions used T805 was not genotoxic (clastogenic) in thischromosome aberration test in mammalian cells.
SCCS Comment
The experiment in the absence of S9 mix was performed only once. A tendency of anincreasing number of cells with structural aberrations was noted in the experiment without
S9-mix.
In vitro micronucleus test in human epidermal cells
Guideline/method: According to an generally accepted published protocol
Test system:
Human epidermal cell line, A431
Replicates:
3 independent experiments
Test item:
TiO2 NP (Anatase, 99.7%), commercial
Batch:/
Solvent:
DMEM with 10% FBS
Concentrations:
0.008, 0.08, 0.8, 8, 80 µg/ml
Exposure:
6 h treatment without S9-mix, harvest time 24 h after the start oftreatment
Negative control: Vehicle
Positive control:
Ethyl methanesulfonate (6 mM)
GLP:
Not in compliance
Published:
Shukla et al., 2011
Reference:
Submission SI-II, (205)67

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The cytokinesis-block micronucleus (CBMN) assay was carried out to determine thepotential genotoxicity of TiO2 NP in the human epidermal cell line A431. The cells weretreated for 6 h with different concentrations of TiO2 NP (0, 0.008, 0.08, 0.8, 8, and 80µg/ml). Ethyl methanesulfonate was used as positive control. After the 6 h exposure, theNPs were removed by washing with medium and cells were grown for additional 18 h in
fresh DMEM medium containing Cytochalasin-B (3 µg/ml medium). Cytospin preparations
were examined for the presence of micronuclei in binucleate cells. From each concentration
2000 binucleate cells were scored; the cytokinesis block proliferation index (CBPI) was
calculated from 500 cells/concentration as recommended in OECD Guideline 487.
Transmission electron microscopy (TEM) was used to evaluate uptake of the TiO2 NP into
the cells.

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Results
The TiO2 NP caused a significant concentration-dependent induction of DNA damage. Effects
were statistically significant at the two highest testing concentrations. These concentrations
were not cytotoxic after 6 or 24 h treatment in the MTT or NRU assay. Significant
cytotoxicity for both concentrations was found in these assays after 48 h treatment. Uptakeof NP into the A431 cells was shown by TEM analysis. Particles were observed mostly in thecytoplasm, but occasionally also in the nucleus. Oxidative stress in the cells was indicatedResults
CBPI was not significantly different from the control treatments. TEM analysis showed thatNPs were taken up by the cells. The NPs were found to be distributed mostly in cytoplasm,
some NP were also localised in the nucleus. A statistically significant induction in thenumber of cells with micronuclei was observed after 6 h exposure to TiO2 NP.
The particles were also found to induce oxidative stress in the cells indicated by a significant
depletion of glutathione, induction of lipid peroxidation and reactive oxygen species
generation.
Conclusion
Under the experimental conditions used TiO2 NPs induced an increase in the number of cellswith micronuclei and, consequently, TiO2 NPs is genotoxic (clastogenic and/or aneugenic) in
the human epidermal cell line A431.
Fpg modified Comet assay in human epidermal cells
Guideline/method: According to an generally accepted published protocol
Test system:
Human epidermal cell line A431
Replicates:
2 cultures
Test item:
TiO2 NP (Anatase, 99.7%), commercial
Batch:/
Solvent:
DMEM with 10% FBS
Concentrations:
0.008, 0.08, 0.8, 8, 80 µg/ml
Exposure:
6 h treatment
Negative control: Vehicle
Positive control:
25 µM hydrogen peroxide
GLP:
Not in compliance
Published:
Shukla et al., 2011
Reference:
Submission SI-II, (205)TiO2 NP was assayed for DNA damage in the human epidermal cell line A431 with the Comet
assay. The cells were treated for 6 hours with TiO2 NP in a concentration range up to 80µg/ml. DNA damage was evaluated by formamidopyrimidine DNA glycosylase (fpg) modified
Comet assay. The fpg allows for detection of oxidative DNA base damage lesions, inparticular, 8-OH guanine. Hydrogen peroxide was included as a positive control andcytotoxicity was evaluated by MTT and NRU assay.

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from the significant depletion of glutathione, induction of lipid peroxidation and reactive
oxygen species generation.

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Guideline/method: According to an generally accepted published protocol for the alkaline
Comet assay
Test system:
Human lymphocytes
Replicates:
triplicate culture in 2 independent experiments
Test items:
TiO2 NP commercial, declared size of 100 nm and surface area of 14.0
m2/g
Batch:/
Solvent:
RPMI-1640
Concentrations:0, 0.25, 0.50, 0.75, 1, 1.25, 1.50, 1.75, 2 mM
Exposure:
3 hour treatment
GLP:
Not in compliance
Published:
Ghosh et al., 2010
Reference:
Submission SI-II, (157)

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The DNA damaging potential of TiO2 NP was evaluated using the Comet assay in human
lymphocytes obtained by venipuncture from peripheral blood of healthy volunteers. Cells
were isolated by gradient centrifugation using Histopaque and resuspended in RPMI-1640
culture medium. Cells were treated for 3 hours with the TiO2 NP at a concentration range of
0 to 2mM. The Comet assay was performed according to published methods. DNA damage
was reported as % tail DNA in treated lymphocytes. Slides were prepared in triplicates perconcentration and each experiment was repeated twice. Viability was determined by trypan
blue dye exclusion, MTT assay and WST-1 assay in the same concentration range as used
for the Comet assay.

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Results
Trypan blue indicated viability above 80% at the highest treatment concentrations. MTT and
WST-1 assay showed increased toxicity, with an LC50 in the range of 1.0 to 1.25 mM. A
statistically significant increase in DNA damage was observed in lymphocytes treated with
the TiO2 NP at a concentration of 0.25 mM. No concentration dependent effect and nostatistically significant effects were found at any of the other testing concentrations.

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SCCS Comment
The authors of the paper conclude that TiO2 NP were genotoxic to human lymphocytes.
They propose that the absence of a dose-dependent effect on DNA damage may be due tothe agglomeration behaviour of the nanoparticles.
SCCS concludes that in view of the absence of a dose-dependent effect, this study does notprovide evidence for the genotoxicity of TiO2 in human lymphocytes.

Conclusion
Under the experimental conditions used the results of the study indicate that TiO2 NPs
possess DNA damaging potential in human epidermal cells.
Comet assay in human lymphocytes

Conclusion:
Under the experimental conditions used, the results of the study indicate that TiO2 NPs
possess DNA damaging potential in human epidermal cells.

In vitro mammalian cell gene mutation test
Guideline/method:
Test system:/
gpt delta transgenic mouse primary embryo fibroblasts
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Replicates:
Test items:

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Mutant frequencies in red/gam loci by Spi- detection by two nano-sized and one fine TiO2
materials were evaluated in gpt delta transgenic mouse primary embryo fibroblasts. The
samples were suspended in distilled water, subsequently sonicated for 30 min, sonicated onice, and diluted in medium before addition to the cells. S9-mix was not included in theassay.

Batch:
Solvent:
Concentrations:
Exposure:
Solvent:
GLP:
Reference:/
TiO2 NP anatase (5 nm, 114m2/g), Sigma Aldrich,
TiO2 NP anatase (40 nm, 38 m2/g), Inframat Advanced Materials LLC
Fine TiO2 (325 mesh, 8.9m2/g), Sigma-Aldrich//0, 0.1, 1, 10 and 30 µg/ml
24 h treatment
Distilled water (sonicated and then further diluted in culture medium)not in compliance
Xu et al., 2009

Results
Concurrent cytotoxicity of the samples was evaluated by the MTT assay and uptake of the
NP in the cells was assessed by flow-cytometry. Increased mutants frequencies wereobserved with both the nanosize TiO2 samples, but not with the fine TiO2, demonstratingthat these nanomaterials can cause kilo-base pair deletion mutations. These effects could be
abrogated by co-treatment of the endocytosis inhibitor (lipid raft/caveolae disrupting agent)
Nystatin, the nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMMA) and thecyclooxygenase-2 activity inhibitor NS-398.
Conclusions
TiO2 NP were taken up by the cells and induced kilo-base pair deletion mutations in atransgenic mouse mutation system.
It was suggested that induction of [ONOO]-, triggered by the signalling events associatedwith the transporting of nanoparticles into the cells, rather than the chemicalcomposition/surface area combination of the nanoparticles may be a critical event for theobserved genotoxicity.
SCCS Comment
Translocation and contact of the test items with the fibroblast DNA has not been
demonstrated in the tests. The effects of the applied inhibitors suggest an indirect effectmediated by TiO2 triggered formation of reactive oxidants. Uptake cannot be verified byflow-cytometry on the basis of side-scattering, as particles may merely be adhered to thecell membranes.
In vitro micronucleus test in mammalian cells
Guideline/method: Draft OECD 487
Test system:
V79 cells
Replicates:
Quadruplicate cultures
Test item:
T-LiteTM SF, pure rutile, primary particle size 10 x 50 nm, mean
agglomerates approximately 200 nm (d10: 90nm, d90: 460 nm);coating consisting of aluminium hydroxide and dimethicone/methicone
copolymere
Batch:/
Solvent:
FCS
Concentrations:75, 150 and 300 µg/ml with 4 h exposure
18.8, 37.5 and 75 µg/ml with 24 h exposure
Exposure:
4 h treatment and harvest 24 h after start of the treatment
24 h treatment and harvest immediately after the end of treatment
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Positive control:
GLP:
Reference:

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DNA damage of five different types of TiO2 particles (which included AEROXIDE-P25) was
evaluated by alkaline Comet assay in A549 cells. No S9-mix was added to the test system.
Cells were treated with one concentration (100 µg/ml) for 4 h, 24 h and 48 h. Cytotoxicity
was evaluated by the MTT-assay. Electron microscopy was performed to evaluate uptake ofthe test samples into the A549 cells after 4 h.

Micronucleus formation was evaluated in V79 cells after treatment with TiO2 rutile NP coatedwith aluminium hydroxide and dimethicone/methicone copolymere (T-LiteTM SF rutile). The
study was performed without S9 mix which was considered scientifically justified because of
the nanoparticulate nature of the material. Cells were either treated for 4 hours with 75,
150 and 300 µg/ml, followed by 24 h recovery, or for 24 h at concentrations of 18.8, 37.5
and 75 µg/ml without recovery. The concentrations were selected on the basis of pilot
experiment on cytotoxicity. Ethyl methanesulfonate was used as positive control.
Results
The occurrence of precipitation at higher concentrations influenced the toxicity assessment.
Concurrent evaluation of cytotoxicity by analysis of proliferation index (PI) demonstrated
the absence of cytotoxicity up to highest scorable concentrations. A biologically relevant
increase in the number of cells with micronuclei was not observed after exposure to T-LiteTM
SF.
Conclusions
Under the experimental conditions used T-LiteTM SF did not induce an increase in thenumber of cells with micronuclei and, consequently, T-LiteTM SF is not genotoxic (clastogenic
and/or aneugenic) in V79 cells.
SCCS Comment
The test material relates to S75-K (94% rutile, coated with aluminium hydroxide anddimethicone/methicone copolymer). Translocation and contact of the test material with the
V79 cells and its possible translocation into the nucleus and interaction with DNA has notbeen demonstrated.
Alkaline Comet assay in mammalian lung cells
Test system:
A549 human lung carcinoma cells
Replicates:
Triplicate cultures
Test items:
TiO2 synthesized by laser pyrolysis (spherical, 12 nm, 92 m2/g, 95%
anatase, PZC (point of zero charge) = 6.4)
TiO2 synthesized by laser pyrolysis (spherical, 21 nm, 73 m2/g, 90%
rutile)TiO2-A25 AEROXIDE-P25 (spherical, 24 nm, 46 m2/g, 86% anatase, PZC= 7.0) uncoatedTiO2 ref. 637262 from Sigma-Aldrich (Elongated, L: 68 nm, d: 9nm, 118
m2/g, 100% rutile)
TiO2 ref. T8141 from Sigma-Aldrich (spherical, 142 nm, 10 m2/g, 100%
anatase, PZC = 5.2)
Batch:/
Solvent:
Ultrapure sterile water (pH5.5), suspended at 10 mg/ml, further dilutedin cell culture medium
Concentrations:
0 and 100 µg/ml
Exposure:
4 h, 24h and 48 h after start of the exposure
GLP:
not in compliance
Reference:
Jugan et al., 201171

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Results
Electron microscopic evaluation demonstrated a rapid uptake of the various test materials
into the cytoplasm of the A549 cells. Samples were tested only at one concentration.
Cytotoxicity, evaluated by MTT-test, revealed that cell death was less than 25% for all
samples after 48 h of exposure.
DNA damage was significantly increased with all samples at 4 h, with three out of the fivesamples at 24 h. After 48 h no significant increase was detected with the exception of one
sample (i.e. laser pyrolysis synthesized rutile, 21 nm). The uncoated sample (AEROXIDEP25) caused a significant increase in DNA single strand breakage at treatment times of 4
and 24 h. For all smallest, including the uncoated sample cellular internalization andaccumulation into cytoplasm was reported. For one sample (i.e. 12 nm laser pyrolysis
synthesized), nanoparticles were found located in the nucleus.
It was concluded that several types of TiO2 can cause DNA single strand breaks. In parallelinvestigations, they also showed capacity of TiO2 to cause formation of the oxidative DNA
damage lesion 8-OHdG as well as an inhibition of DNA (base excision) repair activity. In
contrast, they did not detect double strand breaks evaluated by H2AX
immunohistochemistry or clastogenic/aneugenic effects evaluated by micronucleus assay inthe same cells.
Conclusions
Under the experimental conditions used it was concluded that TiO2 nanoparticles have agenotoxic potential in this alkaline Comet assay in mammalian lung cells.
Alkaline Comet assay in mammalian liver cells
Guideline/method: According to generally accepted and published protocols
Test system:
Human hepatoblastoma cell line C3A
Replicates:
Triplicate cultures
Test items:
NM101 Anatase 9 nm (XRD), 4-8/50-100 nm; two different particle
types (TEM), 322 m2/g
NRCWE001 Rutile 10 nm (XRD), 80-400 (TEM), 99 m2/g
NRCWE002 Rutile 10 nm (XRD), 80-400 (TEM), 84 m2/g, negative
charged
NRCWE003 Rutile 10 nm (XRD), 80-400 (TEM), 84 m2/g, positive
charged
NRCWE004 Rutile approx. 100 nm (XRD), 1-4/10-100/100-200/10002000; five different types of particles (TEM)
Batch:/
Solvent:
Distilled water with FCS
Concentrations:Three concentrations, i.e. LC20, ½ of LC20 and 2x LC20
Exposure:
4 h treatment
Positive controls: H2O2
GLP:
not in compliance
Reference:
Kermanizadeh et al., 2012
DNA damage in human hepatoblastoma C3A cell line was evaluated by the alkaline Comet
assay (evaluated as % tail DNA), with inclusion of fpg enzyme to detect oxidative DNA
damage. A total of five different types of TiO2 were tested at a concentration that caused
20% viability loss (LC20), as well as twice or half of this concentration. The toxicity wasevaluated by WST-1 assay (24 h treatment), the treatment time for the Comet assay was 4hours. S9 mix was not included in the assays.
Results
Biologically relevant and small but statistically significant increases in DNA damage were
found with several of the samples. The most pronounced effects were seen with NM101 and
RWCE001. No biologically relevant increase in DNA damage was observed with the72

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negatively charged RCWE003. In view of the observed effects in the presence of fpg (as wellas based on further analysis of oxidative stress markers in the study), the authors suggestthat the DNA damage effects are mediated by reactive oxygen species (ROS).

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The in vitro mutagenicity genotoxicity studies on TiO2 nanomaterials have been recently
reviewed by Magdolenova et al. (2013). In many of these studies, particle size (andchemistry) is not, or poorly specified in the publications. As such, these studies do not allow
for evaluation of the potential effects of the nanosize aspect of the potential genotoxicity of
TiO2 (Le Boeuf et al., 1996; Endo-Capron et al., 1993; Pelin et al., 1995; Miller et al., 1995;
Lu et al., 1998; Kamp et al., 1995; Dunford et al., 1997; Wamer et al., 1997). In several
studies only fine TiO2 was used (e.g. Driscoll et al., 1997; Van Maanen et al., 1999, in boththese studies TiO2 anatase 180nm with a BET value of 8.8 m2/g was used; Notably
however, one may argue that this sample contains a particle distribution “tail” in thenanosize range).

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Nagakawa et al. (1997) tested four TiO2 samples, i.e. 21 nm and 255 nm anatase and 255
nm and 420 nm rutile for DNA strand breaks by alkaline Comet assay in the mouse
lymphoma cell clone L5178Y/tk+/-. In the presence of UV/light all samples showed enhanced
DNA strand breaks at concentrations which also elicited cell death. Without irradiation only
the 255 nm anatase showed enhanced strand breakage. The 21 nm anatase sample wasalso evaluated for the induction of chromosomal aberrations in the Chinese Hamster cell line
CHL/IU, for mutagenicity in the Salmonella typhimurium strains TA100, TA98 and TA102,and colony formation in the L5178Y/tk+/- cells. Chromosomal aberrations (mainly polyploidy,
chromatid breaks and chromatid exchanges) were found only in the presence of UV/visible
light, and occurred at cyotoxic concentrations. In the absence of light the 21 nm anatase did
not elicit chromosomal aberrations in contrast to the positive control (ofloxacin).
Irrespective of UV/light irradiation, the 21 nm anatase failed to enhance the frequencies ofrevertant Salmonella colonies or mutant L5178Y colonies, in contrast to the positive control
methyl methanesulfonate (MMS).

Conclusions
Under the experimental conditions used, it was concluded that short term exposure of liver
cells to some TiO2 particles caused small but significant increases in DNA damage.
SCCS Comments
Translocation and contact of the test material with the hepatoblastoma cells and its' possible
translocation into the nucleus and interaction with DNA have not been demonstrated. Someof the effects are minor but are concentration dependent, this might become significant at a
certain exposure level.
Further mutagenicity/genotoxicity in vitro studies (open literature):

Linnainmaa et al. (1997) investigated micronucleus formation in rat liver epithelial cells
after treatment with various TiO2 samples in the presence or absence of UV light. Mitomycin
C was used as positive control. TiO2 samples were a 170 nm and a 20 nm anatase sample,
and a 20 nm coated rutile sample. The coated sample was prepared with aluminiumhydroxide and stearic acid. The sample was ethanol washed to remove the stearic acidbefore treatment of the cells. In contrast to the positive control, none of the samples
induced an increase in cells with micronuclei.
Rahman et al. (2002) studied micronucleus formation in SHE fibroblasts after treatment
with fine TiO2 (>200nm) and nanosize TiO2 (20nm). Apart from size, no further details of
the samples were provided. Increased micronuclei were found only with the ultrafine TiO2.
The authors reported (but did not show in the manuscript) that further kinetochore-staining
experiments revealed indications for chromosomal non-disjunction during mitosis. The
nanosize TiO2 also elicited apoptosis shown by DNA fragmentation analysis and theappearance of apoptotic bodies (transmission electron microscopy evaluation).

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Gurr et al. (2005) tested a variety of TiO2 samples for micronucleus formation as well as theinduction of oxidative DNA damage using the Fpg-modified Comet assay in BEAS-2B humanbronchial epithelial cells. The samples used were four different anatase samples, withrespective sizes of 10, 20, 200 and >200 nm, and one rutile sample with the size of 200
nm. Micronucleus induction was found with the 10 and 200 nm anatase sample, but notwith the >200 nm anatase and the 200 nm rutile samples. For the 20 nm anatase sample
no data were provided. Enhanced oxidative DNA damage (fpg-Comet assay) was observedwith the 10 and 20 nm anatase samples and with the 200 nm rutile. All other samples were
negative. Finally, the authors showed that a 1:1 mixture of 200 nm anatase and 200 nm
rutile caused stronger oxidative DNA damage than the 200 nm anatase or 200 nm rutile
alone.

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Bhattacharya et al. (2009) investigated the genotoxicity of anatase TiO2 in BEAS-2B humanbronchial epithelial cells and IMR-90 human lung fibroblasts. The TiO2 nanoparticles caused
induction of the oxidative DNA adduct 8-OHdG in IMR-90 cells (measured by an ELISA
method), but did not cause increased strand breaks (measured by Comet assay) in the IMR90 and BEAS-2B cells. Electron microscopy demonstrated that both particles translocatednear to nucleus, but were not found inside the nucleus, mitochondria or ribosomes.
Falck et al. (2009) investigated the genotoxicity of three TiO2 samples in BEAS-2B humanbronchial epithelial cells by the alkaline Comet assay and the micronucleus test. The
samples were a nanosize rutile sample coated with <5 SiO2 (10x40nm needle shaped, BET
132 m2/g), a fine rutile sample (<5 µm, 2 m2/g), and a nanosize anatase sample (<25 nm,
222 m2/g). Hydrogen peroxide and mitomycin-C were used as respective positive controls.
All samples showed mild but significant DNA damaging effects. The effects of the nanosize
rutile were much weaker than those of the nanosize anatase and fine rutile sample. The
nanosize anatase, in contrast to both other samples, also caused increased micronuclei. For
the observed DNA damaging and micronucleus effects mostly no clear dose-dependency
could be observed. It was also reported that the micronucleus scoring was difficult due tothe presence of the particles during microscopy.
Magdolenova et al. (2012a) showed in human TK6, EUE and Cos-1 cells that genotoxicity of
TiO2 (DNA damage and oxidised DNA lesions) measured by the Comet assay (with andwithout fpg) depends on the stock dispersion protocol. The same TiO2 (Aeroxide P25,
primary particle size 21 nm, mixture of anatase /rutile), but prepared with different stock
dispersion protocol, following further with the same media and exposure conditions resulted
in differed state of agglomeration and gave different results. Larger agglomerates gave
positive results. Thus differences in stock dispersion preparation could explain contradictory
results published on the same nanoparticles. Magdolenova et al. (2012b) studied thepossible interference of TiO2 and other nanoparticles with the fpg enzyme in the Comet
assay but did not find this to cause any artefacts.

Open literature studies
Micronuclei in peripheral blood erythrocytes after oral uptake
Guideline/method: /Species/strain:
C57Bl/6Jpun/pun mice.
Group size:
5 mice/treatment group
Test substance:
Aeroxide P25, Degussa/Evonik, primary particle size 21 nm, BET surface
area 50 m2/g, DLS in water: 21-1446 nm)
Batch:/
Vehicle:water
Dose levels:
0, 50, 100, 250, and 500 mg/kg bw (estimated dose)
Treatment:/
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GLP:
Reference:

not in compliance
Trouiller et al., 2009

Methods
C57Bl/6Jpun/pun mice, containing naturally occurring 70-kb internal duplication in the pinkeyed dilution (p) gene, were exposed via drinking water to the TiO2 NP. The suspensions
were ultrasonicated for 15 min before providing to animals. Water (with/without the NP)
was provided ad libitum during 5 days. Peripheral blood was collected and erythrocytes
were evaluated for the presence of micronuclei. The estimated exposures were 0, 50, 100,
250 and 500 mg/kg bw. The doses were estimated on the basis of estimated drinking water
consumption (set at 5 ml) and the average weight of the animals. The authors also
evaluated DNA damage, measured as 8-hydroxy-2’-deoxyguanosine in liver tissue by
HPLC/ECD analysis, and alkaline Comet assay in blood cells, but these were tested only at
one concentration (500 mg/kg bw). Moreover, DNA deletions were evaluated in theoffspring of pregnant C57Bl/6Jpun/pun mice treated for 10 days at 500 mg/kg bw/day, toevaluate in utero effects.
Results
A biologically relevant increase in the number of peripheral blood erythrocytes after oral
administration of TiO2 NP was found in mice at the highest treatment dose only (500mg/kg). This concentration also caused increased DNA strand breakage in white blood cells(Comet assay), γ-H2AX foci in bone marrow cells, and 8-hydroxy-2′-deoxyguanosine
formation in liver cells. A 10-day exposure in pregnant mice also led to DNA deletions inoffspring. The TiO2 NP exposure also caused a mild but statistically significant increase insystemic inflammation, as shown by qRT-PCR analysis of the mRNA expression ofproinflammatory genes (TNFalpha, IFNgamma, KC/IL-8) in peripheral blood. It was
concluded that oral TiO2 NP exposure causes genotoxicity in mice, possibly caused by asecondary genotoxic mechanism associated with inflammation and/or oxidative stress.
Conclusions:
Under the experimental conditions used, Aeroxide P25 was genotoxic (clastogenic and/or
aneugenic) in human lymphocytes in vitro.
SCCS Comments
The test material relates to S75-G (anatase/rutile, not coated). However, the description of
the test material given in the paper suggests a different proportion of anatase and rutile(75%:25%) than the proportion specified for S75-G. Data indicate genotoxic effects of TiO2
NP after oral exposure in mice in organs/tissues other than those that are in direct contact
via the exposure route (i.e. effects in blood, bone marrow, liver and foetuses). Insufficientdetails have been provided in the article regarding methodology. This makes the findings ofthe study of limited value to this risk assessment.
Further limitations of the study are:
- The work does not contain biokinetics, i.e. dosimetry cannot be accurately
determined. Actual intake of the NP is not measured, only indirect by calculation of
the amount of drinking water. Translocation of particles and accumulation in different
organs was also not determined.
- Potential local effects (histopathology, genotoxicity assays) in gastrointestinal tract
target cells are not provided, and thus do not allow for assessment of potential
effects on epithelial barrier integrity, inflammation and local mutagenicity.
- The effects were observed at a rather high dose (calculated cumulative oral dose of
500 mg/kg). The authors do not report whether these concentrations affect intestinal
physiology. The high surface burden of TiO2 NP in the G.I. tract may have significant
impact on the adsorption and transport of nutrients.
DNA double strand breakage in bone marrow cells after oral uptake
Guideline/method: According to published protocols
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Species/strain:
Group size:
Test substance:
Batch:
Vehicle:Dose levels:
Treatment:
GLP:
Reference:

C57Bl/6Jpun/pun mice.
5 / treatment group
Aeroxide P25, Degussa/Evonik, primary particle size 21nm, BET surface
area 50m2/g, DLS in water: 21-1446nm)/
water
0, 50, 100, 250, and 500 mg/kg bw (estimated dose)/
not in compliance
Trouiller et al., 2009

Methods
DNA double strand breaks were analysed by immunohistochemical detection of γ-H2AX fociin C57Bl/6Jpun/pun mice exposed to TiO2 NP via drinking water. Bone marrow smears wereanalysed after 5 exposure days for γ-H2AX foci, at estimated exposure of the mice to 0, 50,
100, 250, and 500 mg/kg bw TiO2 NP.
Results
Oral TiO2 NP caused increased γ-H2AX foci in a clear dose dependent manner, being
significant from the lowest dose (50 mg/kg bw) onwards. DNA double-strand break wasconsidered the most sensitive parameter among a variety of genotoxicity endpoints. It wastherefore concluded that oral TiO2 NP exposure causes DNA double strand breaks in bonemarrow of the mice and suggest that this may be caused by a secondary genotoxic
mechanism associated with inflammation and/or oxidative stress.
The TiO2 NP exposure also caused mild but significantly increased systemic inflammation, as
shown by qRT-PCR analysis of the mRNA expression of proinflammatory genes (TNFalpha ,IFNgamma, KC/IL-8) in peripheral blood.
Conclusions:
Under the experimental conditions used, Aeroxide P25 was genotoxic rats causing DNA
double strand breaks in bone marrow cells.
SCCS Comments
The test material relates to S75-G (anatase/rutile, not coated). Marked dose dependent
effects are observed, suggesting that the bone marrow may be a sensitive target for TiO2
nanoparticle (after oral uptake). Whether the nanoparticles actually reached this target is
not shown in the study. The effects were observed at high concentrations. Other limitationsof the study are:
- The work does not contain biokinetics, i.e. dosimetry cannot be accurately
determined. Actual intake of the NP is not measured, only indirect by calculation of
the amount of drinking water. Translocation of particles and accumulation in different
organs was also not determined.
- Potential local effects (histopathology, genotoxicity assays) in gastrointestinal tract
target cells are not provided, and thus do not allow for assessment potential effects
on epithelial barrier integrity, inflammation and local mutagenicity.
- The effects were observed at a rather high dose (calculated cumulative oral dose of
500 mg/kg bw). The authors have not reported whether these concentrations affectintestinal physiology. The high surface burden of TiO2 NP in the G.I. tract may havesignificant impact on the adsorption and transport of nutrients.
Comet assay in vivo in rat lungs (five day inhalation study)
Guideline/method: According to generally accepted and published protocols
Species/strain:
Male Wistar Crl:W1 Han rats
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Test substance:

Batch:
Vehicle:Dose levels:
Treatment:
GLP:
Reference:

T-LiteTM SF, pure rutile, primary particle size 10 x 50 nm, mean
agglomerates approximately 200 nm (d10: 90 nm, d90: 460 nm);coating consisting of aluminium hydroxide and dimethicone/methicone
copolymere//
0 and 10 mg/m3/treatment/day
6 h/day for 5 consecutive days
not in compliance
Landsiedel et al., 2010

Rats were exposed by inhalation (head-nose exposure) for 6 hours on five consecutive days
to 0 or 10 mg/m3/treatment/day. DNA damage was evaluated by alkaline Comet assay inthe rat lung cells (isolated by in situ perfusion) from three animals per group. Viability of
the isolated cells was determined by trypan blue dye exclusion. Further parameters
evaluated included body weight, and bronchoalveolar lavage levels of LDH and ALP.
Results
The treated animals showed significantly increased LDH and ALP concentrations in BAL.
Average viability of the cells isolated for the Comet assay were 95% and 88.7% respectively
for air and TiO2 exposed animals. A biologically relevant increase in DNA damage was notdetected by the Comet assay.
Conclusion
Under the experimental conditions used it was concluded that T-LiteTM SF has a genotoxic
potential in this alkaline Comet assay in lung cells.
SCCS Comment
The test material relates to S75-K (94% rutile, coated with aluminium hydroxide anddimethicone/methicone copolymere). The applied method is not yet validated, but
represents tissue that at least in part is directly exposed to the testing material. The
isolation procedure may have affected the background damage in the cells from theanimals.
Further mutagenicity/genotoxicity studies in vivo (open literature)
In specific animal studies no information is provided on the size of the particles used, or
only non-ultrafine samples were used for effects of nano-sized TiO2 (Shelby, 1993; Driscoll
et al., 1997).
Rehn et al. (2003) investigated oxidative DNA damage induction by two samples of TiO2 in
rat lungs after intratracheal instillation at the dosages of 0, 0.15, 0.3, 0.6 and 1.2 mg/kg
bw/day. The samples used were an untreated TiO2 and a trimethoxyoctylsilane-treated TiO2
sample, both approximately 20 nm. DQ12 crystalline silica was used as a positive control at
0.6 mg/kg. Oxidative damage induction was determined after 90 days byimmunohistochemical analysis of lung sections using an 8-oxoguanine antibody. Enhanced
oxidative DNA damage was not observed with the untreated or silanised TiO2 nanoparticles,in contrast to the DQ12 crystalline silica. Analysis of markers of pulmonary inflammationand toxicity at 3, 21, and 90 days indicated a strong progressing inflammation with DQ12
crystalline silica, whereas for both TiO2 samples only mild inflammatory effects were
noticed. Proliferation in lung tissue, as determined using Ki-67 staining, showed only minor
differences between control and TiO2 treated rats in contrast to DQ12 treated rats whichshowed strong increase in % Ki-67 positive cells after 90 days. The contrasting observations
with regard to oxidative DNA damage induction and proliferation were considered to be dueto the marked contrasts in severity and persistence of pulmonary inflammation.

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Similar to these observations, Driscoll et al. (1997) have demonstrated the likely role ofpulmonary inflammation in driving mutagenesis in rat lungs after in vivo instillation ofdifferent particles. These included a fine crystalline silica sample, a nano-sized carbon black
sample and a fine anatase TiO2 sample (180 nm median diameter, 8.8 m2/g). Mutagenicity
was studied by hprt-analysis of lung epithelial cells isolated from the lungs of female SPF
F334 Fischer rats, 15 months after intratracheal instillation of each of the particles at 10
mg/kg or 100 mg/kg. For the fine TiO2 sample, enhanced hprt-mutagenesis was observedwith 100 mg/kg, the dose which also elicited persistent lung inflammation, but not with the
10 mg/kg dose. Similar for the other particles used (carbon black, silica) in vivo
mutagenicity was only observed at doses that also caused persistent inflammation. The
inflammatory cells obtained by bronchoalveolar lavage from the particle-treated animals
were found to induce hprt-mutagenesis in a rat lung epithelia cell line in vitro.

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From the studies discussed above, the potential to cause DNA damage has been clearly
demonstrated for some TiO2 nanomaterials. However, it is not clear how this relates to theother nanomaterials presented in the submission.

SCCS Comments on Mutagenicity/Genotoxicity

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1.5.7

Carcinogenicity

Two stage skin painting carcinogenicity studies
Study Design:

Two stage mouse skin carcinogenicity (Initiator: DMBA)

Date of publication:
Guideline/method:

Available online 30 November 2010.
Two stage muse skin carcinogenicity test. Coated and uncoatedtitanium dioxide nanoparticles were used as promoter with 7,12dimethylbenz[a]anthracene (DMBA) as initiator.
CD1 (ICR) female mice.
Industrial material-grades of coated (alumina and stearic acid)titanium dioxide nanoparticles (CTDN, titanium dioxide content:
79.2%, spindle shape, long axis of 50–100 nm, short axis of 10–20nm) and uncoated titanium dioxide nanoparticles (UCTDN, titanium
dioxide content: 96.0%, spindle shape, long axis of 50–100 nm, shortaxis of 10–20 nm) from Ishihara Sangyo Kaisha, Ltd., Osaka, Japan.
No data
CTDN and UCTDN dispersed in Pentalan 408 (pentaerythrityl
tetraethylhexanoate) at concentrations of 5 mg/0.1 g, 10 mg/0.1 g
and 20 mg/0.1 g on ultra sonic cleaner.
Twice weekly for 19 weeks
Pentalan 408 (pentaerythrityl tetraethylhexanoate)
Solvent
12-o-tetradecanoylphorbol 13-acetate (TPA)
No
Furukawa et al., 2011

Test system:
Test substance:

Batch:
Concentrations:
Exposure:
Solvent:
Negative control:
Positive Controls:
GLP:
Reference:

This study was conducted to examine the promoter potential of coated and uncoatedtitanium dioxide nanoparticles (CTDN and UCTDN) in a two-stage mouse skin carcinogenesis
model using 7 week old CD1 (ICR) female mice. Initiation treatment: 0.1 ml (0.1 mg) DMBA
or vehicle alone was applied to furclipped back skin one time, using a micropipetter withdisposable tips. Starting 1 week after the initiation treatment, aliquots of 5, 10 and 20 mg
of CTDN or UCTDN in 0.1–0.09 ml of Pentalan were applied using a disposable syringe andglass spreader daily, or 0.2 ml (4 µg) of TPA were applied using a micropipetter twice
weekly for 19 weeks to the animals as post-initiation treatments. TPA was used as a
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positive control promoter. Pentalan 408 served as a vehicle control as well as negative
control.

Study Design:

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Date of publication:
Guideline/method:

Published 2012
Two stage mouse skin carcinogenicity test. Coated and uncoatedtitanium dioxide nanoparticles were used as promoter with 7,12dimethylbenz[a]anthracene (DMBA) as initiator.
Female rasH2 mice and their wild-type counterparts CB6F1 mice andCD1 mice.
sTiO2 particles (rutile type, silicone coated, mean particulate diameter
35 nm) and ncTiO2 rutile type mean particulate diameter 20 nm)
were provided by Japan Cosmetics Association, Tokyo.
No data
0, 50 and 100 mg/ml
sTiO2 rasH2 mice 5 times a week for 8 weeks, CB6F1 mice 5 times aweek for 40 weeks.
ncTiO2 CD1 mice 2 times a week for 52 weeks
sTiO2 silicon oil, ncTiO2 Pentalan 408 (pentaerythrityl
tetraethylhexanoate)
Solvent
sTiO2 no positive control, ncTiO2, 12-o-tetradecanoylphorbol 13acetate (TPA)
No
Sagawa et al., 2012

No changes in survival rate, general condition and body weight related to the test materials
were observed. On macroscopic observation, 1–2 nodules/group on the skin were observedin each group applied CTDN and UCTDN as well as the control group after DMBA initiation.
The nodules were histopathologically diagnosed as squamous cell hyperplasia, sebaceous
gland hyperplasia, squamous cell papilloma and keratoacanthoma. While in CTDN andUCTDN experiments enlargement of the mandibular, pancreatic, lumbar region andinguinofemoral lymph nodes, spleen and thymus was observed in mice given 5 and 10 mg
but not 20 mg, the lack of dose-dependence suggests no biological significance.
The study authors concluded that CTDN and UCTDN applied as promoter at doses of up to
20 mg/mouse did not increase the development of nodules. There were no significant
differences between the number of nodules in the negative control (no initiator) and theexperiments with TiO2 as promoter. In the positive control, DMBA as initiator and TPA as
promoter, 100% of the animals developed nodules. The authors concluded that titanium
dioxide nanoparticles do not possess promoter activity for mouse skin carcinogenesis.
SCCS Comment
The test material used in this study might be comparable to one type of materials includedin this dossier. It was a good experiment with a procedure that is generally accepted forstudying initiation and promoter activity. SCCS agree that under the experimental
conditions uncoated and alumina- and stearic acid- coated nano TiO2 do not show any
carcinogenic promoter activity.

Test system:
Test substance:
Batch:
Concentrations:
Exposure:
Solvent:
Negative control:
Positive Controls:
GLP:
Reference:

TEM analysis showed that the shape of sTiO2 particles was generally round to oval while
ncTiO2 particles were more club shaped. The mean length of sTiO2 particles suspended insilicone was 0.28±0.22 µm. The mean length of ncTiO2 particles suspended in Pentalan 408
was 4.97±0.50 µm.
sTiO2 nano particles
The skin on the backs of 7-week old female rasH2 mice and wild type CB6F1 mice wasshaved and the animals received a single topical application of 0.1 ml DMBA (0.2 mg). Two
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weeks later the animals were divided into 3 groups. Group 1 (control, only initiation thanvehicle) (15 mice of each strain) were painted with 0.2 ml silicone oil. Group 2 (15 mice ofeach strain) were painted with 0.2 ml of 50 mg/ml sTiO2 suspended in silicone oil. Group 3(15 mice of each strain) were painted with 0.2 ml of 100 mg/ml sTiO2 suspended in siliconeoil. Group 4 (control, no initiation)(15 mice of each strain) were painted with 0.2 ml of 100
mg/ml sTiO2 suspended in silicone oil without prior DMBA treatment. The mice were painted
5 times a week. The rasH2 mice were killed after 8 weeks and the wild-type CB6F1 mice
after 40 weeks.

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Study Design

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Date of publication:
Guideline/method:rasH2 mice
The incidence of squamous cell papillomas was 100% in all groups (Group 1 – 3) of rasH2
mice treated with DMBA. No skin tumours were found in the group (Group 4) which was
only treated with sTiO2. The incidence of squamous cell carcinomas was 33% in Group 1(only DMBA and silicone oil), 60% in Group 2 (DMBA + 10 mg TiO2), and 53% in Group 3(DMBA + 20 mg TiO2). The difference in carcinomas was not significant. No difference wasfound in the multiplicity of tumours.
CB6F1 mice
The incidence of squamous cell papillomas was 7% (1 mouse) in Group 1 (only DMBA andsilicone oil) and 13% (2 mice) in Group 2 and 3 (DMBA + 10 and 20 mg TiO2). No skintumours were found in the group (Group 4) which was only treated with sTiO2. The
incidence of squamous cell carcinomas was 7% (1 mouse in Group 1 (only DMBA andsilicone oil). No squamous cell carcinomas were found in any of the other groups.
ncTiO2 nano particles
The skin on the backs of 10-week old female CD1 mice was shaved and the animals
received a single topical application of 0.1 ml DMBA (0.2 mg). Two weeks later the animals
were divided into 4 groups. Group 1 (control, only initiation than vehicle) (16 mice) werepainted with 0.2 ml Pentalan 408. Group 2 (16 mice) were painted with 0.2 ml of 50 mg/ml
ncTiO2 suspended in Pentalan 408. Group 3 (15 mice) were painted with 0.2 ml of 100
mg/ml ncTiO2 suspended in Pentalan 408. Group 4 (positive control)(15 mice) were painted
with 0.2 ml of TPA 200 nmol/ml in acetone. Groups 1 – 3 were painted 2 times a week andkilled after 52 weeks. Group 4 was painted 4 times a week and killed after 40 weeks.
CD1 mice
The incidence of squamous cell papillomas was 19% (3 mice) in Group 1 (only DMBA andsilicone oil), 6% (1 mice) in Group 2 (DMBA + 10 mg TiO2) and 13% (2 mice) in Group 3(DMBA + 20 mg TiO2). None of the mice in Groups 1 – 3 had developed squamous cell
carcinomas. In the positive control (DMBA + TPA), 87% (13 mice) had developed squamouscell papillomas and 13% (2 mice) had squamous cell carcinomas.
SCCS Comment
The results indicate that ncTiO2 does not promote skin tumours in mice. With sTiO2 anincrease in the number of tumours was found among mice initiated with DMBA. The
increase was not significant and no conclusion can be drawn.
Two stage rat skin carcinogenicity

Test system:
Test substance:

Published 2012
Two stage rat skin carcinogenicity test. Uncoated titanium dioxide
nanoparticles (ncTiO2) was used as promoter with 7,12dimethylbenz[a]anthracene (DMBA) as initiator.
Male Hras128 rats and their wild-type counterparts SD rats.
ncTiO2 rutile type mean particulate diameter 20 nm) were provided
by Japan Cosmetics Association, Tokyo.
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Concentrations:
Exposure:

Study Design:

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Date of publication:
Guideline/method:

2011
Exploratory Dermal UV-B initiated skin carcinogenesis promotion
study.
Rat/Sprague-Dawley (wild-type and transgenic Hras128). 10 weeksold
5 – 8 male and 5 – 8 female per group.
TiO2 NP (uncoated, rutile type, R, PPS: 20 nm, Ishihara Sangyo
Kaisha, Japan)
No data
0, 100 mg/ml per rat (0.5 ml on 9 cm²)
Topical application
42 weeks with/without pre-irradiation with UV-B for 10 weeks
UV-B radiation unit, Dermaray 100, Eisai-Toshiba, Tokyo, Japan
800 mJ/cm²P, 2x/week for 10 weeks
Pentalan 408 (pentaerythrityl tetraethylhexanoate)

Solvent:
Negative control:
Positive Controls:
GLP:
Reference:

No data
0, 100 and 200 mg/ml
ncTiO2 Hras128 rats 2 times a week for 28 weeks and SD rats 2 times
a week for 40 weeks.
Pentalan 408 (pentaerythrityl tetraethylhexanoate)
Solvent
None
No
Sagawa et al., 2012

TEM analysis showed that the shape ncTiO2 particles were clubbed shaped. The mean lengthof the ncTiO2 particles suspended in Pentalan 408 was 4.97±0.50 µm.
ncTiO2 nano particles
The skin on the backs of 10-week old male Hras128 rats and wild type SD rats was shaved
and the animals received a single topical application of 0.5 ml DMBA (2.5 mg). Two weeks
later the animals were divided into 3 groups. Group 1 (control, only initiation than vehicle)(17 Hras128 rats and 12 SD rats) was painted with 0.5 ml Pentalan 408. Group 2 (16Hras128 rats and 12 SD rats) was painted with 0.5 ml (50 mg) ncTiO2 suspended inPentalan 408. Group 3 (17 Hras128 rats and 12 SD rats) was painted with 0.5 ml (100 mg)
ncTiO2 suspended in Pentalan 408. The rats were painted twice a week. The Hras128 rats
were killed after 28 weeks and the SD rats after painting for 40 weeks.
Hras128 rats
The incidence of squamous cell papillomas was 94% (16 rats) in Group 1 (only DMBA andPentalan 408), 88% (14 rats) in Group 2 (DMBA + 50 mg TiO2) and 94% (16 rats) in Group
3 (DMBA + 100 mg TiO2). None of the rats Groups 1 had developed squamous cell
carcinomas, while 13% (2 rats) in both Group 2 and Group 3 had developed squamous cell
carcinomas.
SD rats
The incidence of squamous cell papillomas was 25% (3 rats) in Group 1 (only DMBA andPentalan 408), 17% (2 rats) in Group 2 (DMBA + 50 mg TiO2) and 8% (1 rat) in Group 3(DMBA + 100 mg TiO2). None of the rats in Groups 1 and 3 had developed squamous cell
carcinomas, while 17% (2 rats) in both Group 2 had developed squamous cell carcinomas.
SCCS Comment
This rat model is less developed than the mouse two-stage carcinogenicity model. Since
94% of the Hras rats treated with DMBA only developed tumours, the model is not adequate
and no conclusion can be drawn from the study.

Test system:
Group size:
Test substance:
Batch:
Concentrations:Route:
Exposure:
Source of UV-light:
Irradiation: UV-B:
Solvent:

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Negative control:
Positive Controls:
GLP:
Reference:

Solvent
None
No
Xu et al., 2011

Study Design:

Intra-pulmonary spraying

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Date of publication:
Guideline/method:

Advance Access publication February 25, 2010.
Two stage rat skin carcinogenicity test. Uncoated titanium dioxide
nanoparticles (ncTiO2) were used as promoter with DHPN as initiator.

The potential of TiO2 NPs (uncoated, R, PPS: 20 nm) to promote skin tumours after dermal
application after UV-B irradiation was studied in transgenic rats carrying the human c-Haras proto-oncogene (Hras128 rats), known to be sensitive to chemically induced skincarcinogenesis in males and mammary carcinogenesis in females, and their wild-type
counterparts. A total of 80 Hras128 rats and their wild-type siblings were investigated.
The size of TiO2 particles suspended in Pentalan 408 ranged from 10 nm to 300 μm (meansize of 5.0 μm, median size of 4.6 μm) indicating that a large majority of the particles
formed aggregates in the Pentalan 408 suspension.
Group 1 (initiation and promotion) received ultraviolet B (UV-B) radiation (UV-B radiation
unit, Dermaray 100, Eisai-Toshiba, Tokyo, Japan) 2 times per week for 10 weeks at 800
mJ/cm2, on the shaved target skin, followed by painting with 0.5 ml of TiO2 suspended inPentalan 408 at 100 mg/ml on the shaved (9 cm²) area twice a week until sacrifice. Group2 (negative control, initiation + vehicle) received UV-B radiation and painting with thevehicle Pentalan 408 on the shaved area twice a week until sacrifice, and Group 3 (noinitiation, only TiO2 as promoter) received painting with 0.5 ml of TiO2 suspension as inGroup1 but without prior UV-B radiation.
Any grossly visible papilloma lesions were carefully examined every day. All the animals
were sacrificed at week 52 (after 42 weeks painting) except for the female Hras128 rats,
which were terminated at week 16 (after 6 weeks painting) due to early mammary tumour
development. The skin, brain, lung, liver, mammary gland, mesenterial lymph nodes, spleen
and kidney, were excised, fixed and processed for light microscopic examination.
In male Hras128 rats, papillomas on the back skin developed from week 32 and theincidence of skin papillomas was 12.5% (1/8) in Groups 1 and Group 3. No skin tumours
were observed on the targeted back skin in female Hras128 rats or wild-type rats of either
sex. Eye lid squamous cell papillomas were found in wild type female rats exposed to UVB(Groups 1 and 2) with incidences of 12.5% (1/8) and 14.3% (1/7). No statisticallysignificant inter-group differences in incidence, multiplicity or weight were found. Mammary
tumours (adenocarcinomas) were induced with high incidence in Hras128 rats of both
sexes. Wild-type female rats also had an increased incidence of mammary tumours but nostatistically significant inter-group differences in incidence, multiplicity or weight wereobserved.
Conclusions by the authors
TiO2 particles were detected in the upper stratum corneum but not in the underlying skintissue layers. TiO2 did not induce or promote skin carcinogenesis in transgenic (Hras128)
and wild-type Sprague-Dawley rats under the conditions of this study. The data suggestthat TiO2 does not cause skin carcinogenesis, probably due to its inability to penetrate
through the epidermis and reach underlying skin structures.
SCCS Comment
This is not a generally accepted model for studying initiation and promotion of skin tumours.
Since no positive control was included it is not possible to make any conclusion with regard
to potential carcinogenic properties of TiO2 from the study.

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Test system:
Test substance:
Batch:
Concentrations:
Exposure:

Solvent:
Negative control:
Positive Controls:
GLP:
Reference:

Female transgenic rats carrying the human c-Ha-ras gen (Hras128
rats) and female wild-type SD rats were obtained from CLEA Japan
Co., Ltd (Tokyo, Japan)
ncTiO2 rutile type mean particulate diameter 20 nm) were provided
by Japan Cosmetics Association, Tokyo.
No data
TiO2 particles were suspended in saline at 250 µg/ml or 500 µg/ml.
Initiation: 0.2% DHPN (N-nitrosobis(2-hydroxypropyl)amine), (Wako
Chemicals Co., Ltd Osaka, Japan) in the drinking water for 2 weeks.
Promotion: Two weeks after DHPN treatment, the rats were exposed
intratracheally every second week to TiO2 suspensions underisoflurane anesthesia for a total of 7 times. The rats were killed 3days after the last exposure.
SalineOnly DHPN in drinking water
None
No
Xu et al., 2011

Female transgenic Hras128 rats and female wild-type SD rats were used in the study. TiO2
particles were suspended in saline at 250 µg/ml or 500 µg/ml. The TiO2 suspension was
intratracheally administered to animals under isoflurane anesthesia using a Microsprayer(Series IA-1B Intratracheal Aerosolizer, Penn-Century, Philadelphia, PA) connected to a 1 ml
syringe; the nozzle of the sprayer was inserted into the trachea through the larynx and a
total of 0.5 ml suspension was sprayed into the lungs synchronizing with spontaneous
respiratory inhalation (IPS).
IPS-initiation–promotion protocol
Female Hras128 rats aged 6 weeks were given 0.2% DHPN, in the drinking water for 2
weeks. Two weeks later, the rats were divided into four groups. Group 1 (9 rats). DHPN
alone. Group 2 (10 rats). DHPN followed by 250 µg/ml TiO2. Group 3 (11 rats). DHPN
followed by 500 µg/ml TiO2. Group 4 (9 rats). 500 µg/ml TiO2 without DHPN initiation.
The TiO2 particle preparations were administered by IPS once every 2 weeks from the endof week 4 to week 16 (a total of seven exposures). The total amount of TiO2 administered toGroups 1, 2, 3 and 4 were 0, 0.875, 1.75 and 1.75 mg per rat, respectively. Three days
after the last treatment, animals were killed and the organs (brain, lung, liver, spleen,kidney, mammary gland, ovaries, uterus and neck lymph nodes) were excised
TiO2 was distributed primarily to the lung, but minor amounts of TiO2 were also found inother organs. Various sizes of TiO2 aggregates were observed in alveolar macrophages. The
TiO2-laden macrophages were evenly scattered throughout the lung alveoli. Of 452 particle
aggregates examined, 362 (80.1%) were nanosized, i.e.100 nm. Overall, the average size
was 84.9 nm and the median size was 44.4 nm.
The author concluded that TiO2 treatment significantly increased the multiplicity of DHPNinduced alveolar cell hyperplasias and adenomas in the lung. In the rats, which received
TiO2 treatment without prior DHPN treatment, alveolar proliferative lesions were notobserved although slight inflammatory lesions were observed. TiO2 aggregates werelocalized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm.
In the mammary gland, TiO2 treatment significantly increased the multiplicity of
adenocarcinomas from about 3 tumours per rat in Group 1 to about 6 tumours per rat inGroup 2 and 3. The treatment did also tend to increase the weight of the mammary tumors
from about 6 g per tumour in Group 1 to about 12 – 15 g per tumour in Group 2 and 3 (onlyshown in Figure with no Table).

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IPS 9 day protocol
Twenty female SD rats (wild-type counterpart of Hras128) aged 10 weeks were treated by
IPS with 0.5 ml suspension of 500 µg/ml TiO2 particles in saline five times over a 9 day
period. The total amount of TiO2 administered was 1.25 mg per rat. Six hours after the lastdose, animals were killed and the lungs and inguinal mammary glands were excised. Fatty
tissue surrounding the mammary gland was removed as much as possible. The left lungs
and inguinal mammary glands were used for biochemical analysis, and the right lungs were
fixed in 4% paraformaldehyde solution in PBS adjusted at pH 7.3 and processed forhistopathological examination and immunohistochemistry.
Morphologically, TiO2 particles were observed as yellowish, polygonal bodies in thecytoplasm of cells. These cells are morphologically distinct from neutrophils and strongly
positive for CD68, indicating that the TiO2 engulfing cells were macrophages. TiO2
aggregates of various sizes were found in macrophages, and aggregates larger than a single
macrophage were surrounded by multiple macrophages. Of 2571 particle aggregates
examined, 1970 (76.6%) were <100 nm and five particles were >4000 nm in size. Overall,
the average size was 107.4 nm and the median size was 48.1 nm.
TiO2 treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide
dismutase activity and macrophage inflammatory protein 1a (MIP1a) expression in the lung
Comment by the authors
TiO2 treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide
dismutase activity, and macrophage inflammatory protein 1a (MIP1a) expression in thelung. MIP1a, detected in the cytoplasm of TiO2-laden alveolar macrophages in vivo and in
the media of rat primary alveolar macrophages treated with TiO2 in vitro, enhanced
proliferation of human lung cancer cells. Furthermore, MIP1a, also detected in the sera andmammary adenocarcinomas of TiO2-treated Hras128 rats, enhanced proliferation of rat
mammary carcinoma cells. These data indicate that secreted MIP1a from TiO2-ladenalveolar macrophages can cause cell proliferation in the alveoli and mammary gland andsuggest that TiO2 tumor promotion is mediated by MIP1a acting locally in the alveoli anddistantly in the mammary gland after transport via the circulation.
SCCS Comment
TiO2 treatment significantly increased the multiplicity of DHPN-induced alveolar cell
hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas.
Thus, non-coated TiO2 administered intratracheally had tumour promoter activity.
Oral carcinogenicity studies in non-nano TiO2
Oral study with F344 rats. Each groups consisted of 60 male and 60 female rats. The control
diet contained 1% corn oil, while experimental diets contained 1.0, 2.0, or 5.0% titanium
dioxide-coated mica and 1% corn oil.
The article states: “TiO2-coated mica is a nonfibrous, naturally occurring silicate, which,when coated with TiO2 is used as a pearlescent pigment in plastics, industrial coatings,
simulated leather, and cosmetic preparations. Annual worldwide production of TiO2-coatedmica exceeds 1 million pounds and the potential for human exposure is great.”
The test material consisted of a 1:1 blend of two samples of titanium dioxide-coated mica.
The material was in the form of flat platelet with the longest dimension ranging from 10 to
35 µm. The final blend of test material contained 28% TiO2 and 72% mica. A purity of
100% was assumed for purposes of diet formulations.

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The rats (6 week old) received the TiO2 containing for up to 130 weeks. The study authors
stated that “there was no evidence that TiO2-coated mica produced either toxicologic or
carcinogenic effects at dietary concentrations as high as 5.0%.
Ref.: Bernard et al., 1990

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Groups of 50 male and 50 female B6C3F1 mice, 5 weeks of age, were fed diets containing
0, 2.5 or 5% titanium dioxide (size unspecified; anatase; purity, ≥98%) daily for 103
weeks. Mice were killed at 109 weeks of age, at which time no significant difference insurvival was observed between treated and control males. In females, a dose-related trend
in decreased survival was noted. No significant differences in body weights or incidence oftumours were observed between treated and control groups.
Groups of 50 male and 50 female Fischer rats, 9 weeks of age, were fed diets containing 0,
2.5 or 5% titanium dioxide (size unspecified; anatase; purity, ≥98%) daily for 103 weeks.
The rats were killed at 113 weeks of age, at which time no significant difference in survival
was observed between treated and control groups of either sex. No significant differences inbody weights or incidence of tumours were observed between treated and control groups.
Ref.: National Cancer Institute, 1979
SCCS Comment
From the studies, exposure to non-nano titanium dioxide via the oral route does not appear
to lead to carcinogenic effects.

Photo-carcinogenicity

Photo-carcinogenicity studies in non-nano TiO2
The ability of MTD (titanium dioxide, not further specified) and 2-EHMC (2-ethylhexyl-pmethoxycinnamate) to protect mice from the “promotion phase” of tumorigenesis wasstudied.
The dorsal trunks of inbred female C3H/HeJ mice (10 – 12 weeks old) were shaved and therelevant groups (15 mice) initiated with 10 nmol DMBA. Five days later UV-irradiation
and/or sunscreen treatment was commenced and this was continued for 32 weeks. The
mice were monitored for a further 14 weeks after cessation of irradiation.
The sunscreens were in oil-in-water emulsion and contained MTD (7.2%) or 2-EHMC (8%).
The MTD was a broad-spectrum-reflecting physical sunscreen with an SPF of 7, while the 2EHMC was shown to be a UVB-absorbing sunscreen with an SPF of 4. The sunscreens orbase lotion (BL) were applied at least 10 min prior to UV exposure at approximately 2
mg/cm2. The integrated irradiance was 1.7 W/m2 for UVB and 34 W/m2 for UVA.
The mice were irradiated 5 days per week for 32 weeks, i.e. until 50% of the DMBA plus UV
irradiated groups had tumors. The average cumulative dose was 571 kJ/m2 for UVB and
11.4 mJ/m2.
The DMBA-initiation alone and DMBA-initiated sunscreen-treated groups did not develop
tumours. UV alone induced tumours in 46% of the mice at week 48. Initiation with DMBA
prior to UV irradiation enhanced tumour formation such that 87% had tumours at week 48.
Both MTD and 2-EHMC completely protected the mice from UV-induced tumour formation.
Ref.: Bestak and Haliday, 1996.
Groups of female inbred mice (hr/hr, strain Skh:HR-1) treated with an SPF 15 sunscreen
formulated with MT100T microfine titanium dioxide coated with aluminium stearate (notfurther specified) were exposed daily to minimally skin reddening UV radiation over 1285

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weeks. Throughout a 200 day observation period substantial protection was afforded fromthe induction of skin cancer compared to unprotected controls.

-

According to the evaluation of titanium dioxide by IARC (2010), induction of lungtumours was observed in two inhalation studies with rats while two inhalation studies inrats and one in female mice gave negative results.

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Intratracheally instilled female rats showed an increased incidence of lung tumours
following treatment with two types of titanium dioxide. Tumour incidence was notincreased in intratracheally instilled hamsters and female mice.

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Oral, subcutaneous and intraperitoneal administration did not produce a significant
increase in the frequency of any type of tumour in mice or rats.

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IARC concluded that there is inadequate evidence in humans for the carcinogenicity oftitanium dioxide but sufficient evidence in experimental animals for the carcinogenicity
of titanium dioxide. Titanium dioxide was classified as a Group 2B carcinogen (Possibly
carcinogenic to humans).

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In their recent evaluation of TiO2 NIOSH has determined that ultrafine TiO2 with equal
nano-sized TiO2 is a potential occupational carcinogen and, that there is insufficient data
to classify fine TiO2 as potential occupational carcinogen after inhalation (NIOSH 2011).

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Nano titanium dioxide has been studied in 2 two-stage skin carcinogenicity studies withmice, 2 two-stage skin carcinogenicity studies with rats, and one two-stage lung study
with rats.

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Both non-coated (ncTiO2) and coated titanium dioxide have been studied in the twostage mouse skin carcinogenicity studies with CD1 mice and a transgenic mouse strain(rasH2). In one well performed study with non-coated and alumina and stearic acidcoated titanium dioxide, no promoter activity was found (Furukawa et al., 2011).
Promoter activity was also not found for ncTiO2 in the other study (Sagawa et al., 2012).
However, it is difficult to draw a firm conclusion from this study with silica coatedtitanium dioxide due to lack of positive controls and very high tumour activity in the“initiated” mice.

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Non-coated titanium dioxide was studied in 2 two-stage rat skin carcinogenicity studies.
Although, no tumour promoter activity was observed, it is difficult to draw any

Two groups of sunscreen protected mice were treated immediately following the radiation
regime with the tumour promoter croton oil. UV + croton oil induced tumours in 100% of
the mice. The mice protected by a sunscreen showed only 3.7% with tumours, which wasless than with treatment with croton oil alone. However, where sunscreen protected mice
were exposed to croton oil about 25% proved to have been initiated.
The authors concluded that the superfine titanium dioxide sunscreen provided a high levelof protection similar to that by conventional sunscreen formulations.
Ref.: Greenoak et al., 1993.
SCCS Comment
The studies above are of little value because size and specifications of the titanium dioxide
particles are unknown.
SCCS Comments on Carcinogenicity
Pigmentary and ultrafine titanium dioxide has been tested for carcinogenicity by oral
administration in mice and rats, by inhalation exposure in rats and female mice, byintratracheal administration in hamsters and female rats and mice, by subcutaneous
injection in rats, and by intraperitoneal administration in male mice and female rats.

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conclusion since little experience with the model used is available and no positive
controls have been used in the studies.

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One two-stage rat lung carcinogenicity studyhas been carried out with non-coatedtitanium dioxide. The rats were “initiated” by DHPN in the drinking water prior to intrapulmonary spraying with ncTiO2. The experiment demonstrated promoter activity ofncTiO2 (Xu et al., 2011).

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Since TiO2 particles have shown carcinogenic activity and since nano ncTiO2 also showedpromoter activity after intra-pulmonary spraying, the use of nano TiO2 in sprayable
applications needs specific considerations.

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1.5.9

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In the submission, no studies have been provided with reproductive toxicity data relevant to
the nanomaterials under assessment. A review of reproductive and developmental toxicity
studies of manufactured nanomaterials (including TiO2) has been provided (Ema et al.,2010 - Reference 146). The two TiO2 materials referred to include a TiO2 material withparticle size <10µm (no further information), and a TiO2 nanomaterial with primary particle
size 25-70 nm (20–25m2/g surface area, anatase). Relevant studies in the review by Ema
et al. (2010) showed that:

Reproductive toxicity

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-

Pregnant BALB/c mice administered on gestational day 14 with <10 µm TiO2
suspended in phosphate-buffered saline at 50 µg/mouse by a single intranasal
insufflation had higher serum levels of cytokines, including interleukin-1β, tumor
necrosis factor-α, interleukin-6 and chemokine, 48 h after exposure compared with
nonpregnant mice. The offspring of the dams exposed to TiO2 showed increased
airway hyperresponsiveness, increased percentage of eosinophils, and pulmonary
inflammation. These findings showed that TiO2 caused acute cellular inflammation inpregnant mice and increased allergic susceptibility in their pups.

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Pregnant Slc:ICR mice administered on gestational days 6, 9, 12 and 15 with TiO2
nanomaterial suspended in saline with 0.05% Tween 80 via subcutaneous injection
at 100µg/mouse/day caused changes in the expression of genes associated withbrain development, cell death, response to oxidative stress, and mitochondria in thebrain during the prenatal period, and genes associated with inflammation andneurotransmitters in the later stages of the offsprings.

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In vitro exposure of testis-constituent cells (mouse Leydig cell line TM3) to nanoTiO2 showed uptake of the nanoparticles after incubation of cells at 30µg/mL for
48h, and a remarkable inhibition of viability and transient reduction in proliferation of
the cells at 100µg/mL after 24 h.

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The article is, however, a review of exploratory studies, and as such is of a limited
usefulness to this assessment.

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Other studies in open literature, including some of those reviewed by Ema et al. (2010)
have demonstrated the possibility of placental transport of different manufactured
nanomaterials in pregnant animals into the fetus, or found effects in the offspring.
Yamashita et al. (2011) reported on the presence of nano-TiO2 in fetuses after the
intravenous administration of nano-TiO2 in pregnant mice. Nano-TiO2 was detected by TEM
in the placenta, fetal liver and fetal brain, and induced a decrease in uterine weight andhigher fetal absorption. A limitation of the study was that relatively high doses (about 32
mg/kg body weight on gestation days 16 and 17) were used. In addition, the chemical
nature of the nanomaterials observed in the organs was not confirmed. For the silica
nanoparticles investigated in the same paper a size dependency of transplacental migration
was demonstrated as 70 nm nanoparticles did show placental transport while 300 nm and1000 nm silica nanoparticles did not (Yamashita et al., 2011).

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After subcutaneous administration to dams (Slc:ICR mice) on gestation days 3, 7, 10 and
14 at 100µg/mouse/day, Takeda et al. (2009) observed TiO2 particle aggregates (identified
by Energy Dispersive X-ray Spectroscopy, EDS) in the testis of male offsprings at day 4 andweek 6 after birth. Also histopathological alterations were observed in the testis. In
addition, nano-TiO2 particles were demonstrated in the brain of offspring mice (Takeda etal., 2009), suggesting that nano-TiO2 might have passed through undeveloped ordeveloping Blood Brain Barrier (BBB) in embryos of the young mice. However, since mice
were tested at 4 days or 6 weeks of age, it is not clear whether exposure to nano-TiO2
occurs in utero via the placenta or through milk. A previous study of the same researchgroup observed alterations in gene expression in the brain (Shimizu et al., 2009). The gene
expression alterations were already observed in 16 days old embryos. As only the motheranimals were exposed to nano-TiO2 it seems likely that the offspring received the Ti via themother either during pregnancy or in the weaning period via the milk (Takeda et al., 2009).
For some effects, like reduced pup weight and gene alterations, indirect mechanisms due toeffects on the pregnant animals themselves could not be excluded.

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After inhalation exposure to nano-TiO2 during gestation days 8-18 moderate behaviouraleffects were observed in the offspring (Hougaard et al., 2010). Time to first litter was
prolonged after mating the exposed male offspring to unexposed mice but did not reachstatistical significance. For females there was no difference. After inhalation of a surface
coated nano-TiO2 by pregnant mice, no effects were seen on DNA damage inbronchoalveolar lavage fluid (BALF) cells and liver cells (Jackson et al., 2011), nor in
offspring that had been prenatally exposed. Some changes were noted in liver gene
expression profiles of female offspring. However, as in general the exposure of the fetuses
would be rather low, the observed alterations might have been caused as a secondary
response to the maternal inflammation in the lungs.

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Shimuzu et al. (2009), from the same research group as Takeda et al. (2009) performed asimilar study in which pregnant mice were injected subcutaneously (100 µl of 1 mg/ml TiO2
solution) with nano-TiO2 (25-70 nm, anatase) on gestational days 6, 9, 12, and 15. This
study also investigated the effects of maternal exposure to nano-TiO2 on gene expression inbrain during the developmental period using cDNA analysis. Expression levels of the genes
associated with apoptosis were altered in the brain of newborn pups, whereas genes
associated with brain development were altered in early age. The genes associated withresponse to oxidative stress were changed in the brains of 2 and 3 weeks old mice. Using
Medical Subject Headings (MeSH) terms information, the changes of the expression ofgenes was found to be associated with neurotransmitters and psychiatric diseases.

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In conclusion, although after inhalation or subcutaneous exposure of pregnant mice theexposure of offspring in the uterus has been reported, exposure through this route is likely
to be low and some of the effects might be secondary to maternal toxicity induced by thenanomaterials. The reported fetal effects were observed after high doses of intravenously
administered nano-TiO2, which are unlikely to occur in real life with the use of sunscreen
products.

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SCCS Comment
No relevant study on reproductive toxicity is provided. One review article covering
exploratory studies has been provided (SI-II, Ema et al., 2010 (146)). Overall informationon this endpoint is as yet patchy and inconclusive.

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1.5.9.1 Two generation reproduction toxicity

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SCCS Comment
No data on two-generation reproductive toxicity is provided

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SCCS Comment
No data on teratogenicity is provided

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1.5.10 Toxicokinetics

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The following studies on toxicokinetics and metabolism have been provided:

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Exploratory distribution, excretion study in rat
Reference:
Fabian et al., Arch Toxicol. 2007 ref. No. 28 + 53; andFabian E. + Landsiedel R. ref. No. 28)
Guideline:
Study considered a number of guidelines: EC Commission Directive
87/302/EEC (EC Commission Directive 1988), OECD Guidelines for
Testing of Chemicals (Method No. 417) (OECD Guidelines 1984), U.S.
EPA, Health EVects Guidelines, OPPTS 870.7485 (U.S. EPA 1998), and theJapan/MAFF: Guidelines on the Compiling of Test Results on Toxicity(Japan/MAFF2001).

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SCCS Comments
It is not clear which of the numerous noted guidelines were followed. Ti contents of theorgans were not corrected for background levels but untreated rats were analysed as well.
This means only 3 rats per group were analysed. Questions arise where the rest of theadministered TiO2 particles went, since an estimated dose of about 1.25 mg per rat were
injected and liver, spleen, lungs and kidneys amounted only to 600-700 µg per rat providing
no information on the remainder 500 µg.

Species/strain:

male Wistar rat, 7–12 weeks old and weighed 200– 300g

Group size:
Test substance:

Batch:CAS No.
Purity:12 rats; 3 rats per group
TiO2; 06/0489; P25 consisted of both anatase and rutile forms (70/30),
had no surface coating, the TiO2 primary particles were in the size range
20–30 nm; approximately 10 wt.% of the particle
agglomerates/aggregates are found in the nano-size range. BET specificsurface area of 48.6 m2/g.
4165012298 (FI);13463-67-7
unknown

Dose levels:
Route:

5 mg/kg body weight, TiO2 particles suspended in serum
A single intravenous injection followed by biokinetics study

GLP:
Study period:

not applied

Results
Analysis was performed on ICP-AES. According to the analytical method there were no
detectable levels of TiO2 in blood cells, plasma, brain, or lymph nodes. There were nochanges in the cytokines and enzymes measured in blood samples. Highest Ti retention wasobserved in the liver at about 100-150 µg/g of organ with a limited clearance during thenext four weeks. Ti concentrations in spleen were only slightly lower than in the liver, but Ti
concentrations in kidneys and in lungs were about one order of magnitude lower with rather
remarkable clearance of about 66% during the next 14 days.

Exploratory distribution, excretion study in rat
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Reference:

Sugibayashi K., Todo H., Kimura E Safety evaluation of titanium dioxide
nanoparticles by their absorption and elimination profiles. Journal of
Toxicological Sciences 33(3), 293-8 (2008).

Guideline:
Species/strain:
Group size:
Test substance:

not specified
mouse of unspecified strain
not clearly identified, probably 3-5 mice at each time pointrod-shaped TiO2 rutile surface-coated with silica; (primary particle
diameter: 15 nm; agglomerated particle size: 220 nm);
HD-AW-150 from a Japanese company
rutile analysis by XRD, 27.5% silica content from surface modification, nofurther analysis on impurities

Batch:
Purity:Dose levels:
Route:
Administration:
GLP:
Study period:

no dose levels specified
intravenous injection followed by biokinetics study in mice;intravenous injection of titanium dioxide nanoparticles, single intravenous
injection; biokinetics after 5 min, 72 h and 30 d
not specified

Results
Distribution of TiO2 (measured as Ti) was in blood and several tissues (primarily liver) but
not in brain. A slow decrease of TiO2 in liver was observed over time (~30% decrease in
one month). Observation of substantial amounts of Ti found in untreated mice prior to any
treatment due to significant natural food contamination; this led to an estimated dose of 90µg/ Ti per day. After i.v. injection the Ti level was significantly increased in blood andtissues. Ti concentrations per organs are provided but it is not clear whether or not thesewere corrected for background Ti in all organs nor is the administered dose given.
SCCS Comment
Neither the strain nor the number of mice is clearly identified. The i.v. injected dose of TiO2
NP is also not specified. This study is therefore of no use to the current assessment.

Open literature
There are other toxicokinetic data of inhaled agglomerated TiO2 nanoparticles (Ma Hock et
al. 2008, 2009) showing oxidative stress and inflammatory reactions similar to previously
described 90-days exposure investigations. As far as toxicokinetic parameters were
evaluated, due to the detection limits, extrapulmonary TiO2 particles were not detected.
There are also toxicokinetic studies in which TiO2 NP were intravenously injected into thevein of rodents (Fabian et al., 2007, and other papers). Retention was highest in the liver
followed by spleen, lungs, kidneys and it was highest at the first day compared to days 14
and 28. Cytokine levels remained unchanged indicating no detectable toxicity.
There are no new toxicokinetic data on the absorption of TiO2 NP after administration to thegastrointestinal-tract (GIT). The most recent study from Wang et al. 2008 usedunrealistically high doses of 5 g/kg BW in rats such that their findings are not useful andmay even be modulated by uncontrolled other forms of intake like inhalation of aspiration.
Their biodistribution data showed the highest retention in the liver followed by spleen,kidneys and lungs. Thus toxicokinetics data after GIT administration still rely on the studiesof the group of Alexander Florence, in the 1990ies. These suggest that about 5-7% of theadministered 500nm TiO2 particles were absorbed and retained in the body, mainly in theliver.

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Applicant’s conclusions
Intravenous administration of large doses of nano TiO2 did not result in adverse effects orsigns of toxicity in rodents. A non-specific and expected tissue distribution of TiO2 wasobserved. No TiO2 was detected in brain, and the levels in other organs decreased over
time.

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1.5.11 Photo-induced toxicity

SCCS Comment
The limited available evidence suggests that if TiO2 nanoparticles become systemicallyavailable, they may accumulate mainly in liver with a very slow clearance.

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1.5.11.1 Phototoxicity / photoirritation and photosensitisation

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Photo- irritation

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Guidelines:

OECD good laboratory principles

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Product tested:

TiO2 T805 (1992 batch 030492)

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Species:SPF NZ white rabbits (Ch. River), Female

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Groups:3 animals/group

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Dosing:3, 10, 30% in ethanol 96% during 100 min

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Exposure area:

15 – 7.5 cm total, each exposure side spot approximately 2 cm
diameter24

UVA-light:

310-420 nm peak 365nm total dose 10J/cm² (approx. 50 min dosing)25

Readings:

30 min, 24h, 48h, 72 h after UV-exposure

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Observations:No irritation found, neither non-irradiated as irradiated TiO2 treatedanimals.

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Reference:

15

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Conclusion:

TiO2 (T805) is not photo-irritating for rabbit skin under the assay
conditions after UVA irradiation up to 10 J/cm².

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Guidelines:

OECD good laboratory principles

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Product tested:

TiO2 T805 (1992 batch 030492)

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Species:

SPF albino guinea pigs (Ch. River)

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Sex:

Males & Female

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Experimental protocol: Following Ichikawa, Armstrong & Harber 1981, Induction treatment
followed by challenge 12 days later

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Groups:Test groups 5 animals of each sex

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Dosing:30% TiO2 in ethanol (96%) at induction treatment & challenge
treatment (day 12)

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Induction protocol:

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6-8 cm area cleared from fur
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Challenge protocol:

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Observations:No irritation found, neither during induction phase or challenge
phase, in both non-irradiated as irradiated TiO2 treated animals.

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Conclusions:TiO2 (T805) is not photo-sensitizer for guinea pigs under the assay
conditions after UVA irradiation up to 10 J/cm².

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Reference:

17-

Area is subcutaneously pre-treated with Freund adjuvant and exposed to 0.2 ml ofsuspension followed by UVA-light: 310-420 nm (peak 365 nm) total dose 10 J/cm²
In total 5 treatment over 2 weeks (only first time Freund adjuvant was used).
Skin was not cleared after treatment
Reading after each treatment
12 days after last induction
5-10 cm area cleared from fur
Exposed to 0.5 ml of 30% TiO2 (T805) suspension direct followed by light: 310-420 nm(peak 365 nm) total dose 10 J/cm² - 37 min

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Human data:

18

Product tested:

0685115 (No other info in the document)

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Species:

60 volunteers (19-77 y) of which 50 completed the study

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Sex:

Males & Female

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Protocol:

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26

-

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Result:No effects observed, in any of the volunteers

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Conclusion:

Product 0685115 is not a sensitizer for humans under the assay
conditions

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Reference:

27-

Induction: 3 patches per week (Mon, Wed, Fri) during 3 weeks (0.2 ml TiO2 suspension
per patch – no concentration reported). Patches remain at place 24 h (removal byvolunteers). If reaction, next patch was moved to adjacent area (testing was
discontinued if severe reaction was noted)
Challenge: 2 weeks after last induction at different spot

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SCCS Comment
The study is not a photosensitisation study but is only sensitization study.

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Product tested:

0685115 (No other information in the document)

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Species:

29 human volunteers (18-60 y) of which 25 finished the whole study(drop-out were not related to the test)

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Sex:

Males & Female

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Pre-testing:

MED (Minimal Erythemal Dose) of unprotected skin of each volunteer
was assessed. [MED = time interval or dose of UV sufficient toproduce minimal perceptive erythema]

43

Light source:

UV A (320-400 nm), 3 min(approximately 10.08 Joules)

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Result:No effects observed, in any of the volunteers

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Conclusion:

Product 0685115 is not a photo-sensitizer for humans under the assay
conditions

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Reference:

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Induction: 2 spot prepared for exposure to compound 0685115, of which one isirradiated while the other is not be irradiated.
The areas cleared from hair of 1 inch/ 1 inch, and 0.2 ml (no concentration of TiO2
suspension reported) of test material is placed on the spots. Exposed side is kept underpatch during 24 h.
2 applications applied per week for 3 weeks (total 6 applications).
After removal of patch spots irradiated with a dose of 2x MED of the volunteer
Challenge: 2 weeks after last induction at different spots on the back.
Spots are under patch for 24 h, then irradiated for 3 min (non erythemogenic dose).
Reading after 24, 48 & 72 h

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SCCS Comments
Ref 16 and 18 could not be found. The given references are not correct, as they do notreport photo-irritation (Sonnenschutzformulierungen: Lotions und Cremes)

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1.5.11.2 Phototoxicity / photomutagenicity / photoclastogenicity

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A number of studies has not been reviewed as part of this assessment, because theexperiments were performed with bacterial cells. As discussed in section 3.3.6, bacterial
mutagenicity assays are not considered to be appropriate for the testing of nanoparticles
compared to mammalian cell systems. Other studies were not reviewed because they are
related to test materials that are either not nanomaterials, or they lack data on material
characterisation to establish whether they were relevant nanomaterials to this assessment.
Phototoxicity test in vitro

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Guideline/method: OECD TG432
Test system:
Balb/c 3T3 fibroblasts, neutral red uptake (NRU)
Replicates:
no replicates
Test item:
T805 (coated, A/R, PSMA 1 type), T817 (coated, A/R, PSMA 1 type),
TiO2 P25 (non coated)
Batch:
05 10067 (T805), 04095 (T817), P1S-3087 (p25)
Vehicle:EBSS wit 1% ethanol
Concentrations:0.78 t0 1—mg/L
UV-A: 5.0 J/cm²
Exposure:
0, 0.79, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L
Negative control: vehicle
Positive control:
not included
GLP:
noDate of report:
1999
Reference:
Submission DHS, 24 and 25

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Balb/c 3T3 cells were pre-incubated with eight different concentrations (0.79, 1.56, 3.13,
6.25, 12.5, 25, 50 and 100) of the nanoparticles in two 96-well plates, one plate wassubsequently exposed to 5 J/cm2 UVA while the other plate was kept in the dark. Medium
was then replaced and after 24 h cell viability was determined by spectrophotometricalevaluation of neutral red dye uptake (3 h incubation of neutral red). The phototoxic
potential was determined by calculation of the ratio of the nanoparticle concentration that
reduced viability by 50% (NR50) in presence versus absence of UV irradiation.

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Results
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T805 and T817 showed neither cytotoxicity nor phototoxicity up to a concentration of
100mg/L. The p25 (non coated NP) sample also was not cytotoxic up to the highest
concentrations, but in the presence of irradiation a viability reduction of 82 % (at 50 mg/L)
and 44% (at 100 mg/ml) was observed.

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Conclusion
p25 sample is phototoxic towards Balb/c 3T3 cells, while T805 is not phototoxic.

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Guideline/method: Chromosomal aberration test in presence or absence of UV treatment
Test system:
CHO-WBL cells
Replicates:
Duplicate
Test item:
See table
Batch:
Vehicle:Ethanol (sample A), PBS (samples B and C), DMSO (D, E,F,G and H)
Concentrations:Three concentrations for each sample with as higest concentration either
5000 µg/ml or a dose that resulted in less than 50% cytotoxicity
Exposure:
3 h followed by 17 h recovery
UV dose:
750 mJ/cm2 (provided 15 min after NP treatment initiation)
Negative control: Vehicle
Positive control:
8-methoxypsoralen (8-MOP), 4-nitroquinoline-1-oxide (NQO)
GLP:
Published:
yes
Reference:
Theogaraj et al., 2007

SCCS Comment
This study is indicative of the importance of coating on the phototoxic properties of TiO2
nanoparticles.
Photoclastogenicity test in vitro

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Test items used:

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The photoclastogenicity of TiO2 was determined in CHO cells. S9 mix was not included in
the protocol. Cells were treated in the dark for 15 min and then UV radiated. After
irradiation the cultures were incubated in the dark, after which the medium was removed.
Cultures were washed and fresh medium was added for a further 17 h. Cells were thenharvested and stained slides were then evaluated for the presence of chromosomal
aberrations.

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Results
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No increases in chromosomal aberration frequencies were found either in the presence orabsence of UV up to the highest treatment concentrations.

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Conclusion
No photogenotoxicity was observed under the applied testing conditions.

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1.5.12 Human data

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A number of human studies have been quoted on different versions of skin patch test. Someof the studies have used TiO2 materials for which no information on material
characterisation has been provided, whist others have been reviewed in relevant sections.

17

1.5.13 Special investigations

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A number of studies have been provided, relating to cytotoxicity, coating stability andphotostability of TiO2 materials. Many of these studies have used TiO2 materials for which
information on material characterisation has not been provided.

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1.5.14 Human safety evaluation (including calculation of MoS)

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SCCS Comment
Uptake of the NP into the cells was not evaluated. The UV treatment was performed shortly
after initial exposure to the particles (15min). At this time uptake may have been limited.

Given the very low, if any, dermal penetration of nano-TiO2 when applied on skin, and inconsideration of the low toxicity observed, the calculation of a margin of safety (MoS) is not
relevant for this assessment.
Any exposure to nano-TiO2 via oral route from a dermally applied product is also likely to
be insignificantly low. Again in consideration of the low toxicity observed, the calculation ofa margin of safety (MoS) for the oral route is not relevant.
In view of the concerns over safety of nano-TiO2 via inhalation route, its use in applications
that might lead to inhalation exposure (such as powders or sprayable products) is notrecommended and therefore has not been considered in the calculation of MoS.

1.5.15 Discussion
General considerations:
The submission consists of fifteen (15) TiO2 nanomaterials that vary in terms of various
physicochemical parameters. The studies provided in support of the submission range fromold to recent ones. A major proportion of the (old) studies are on materials for which little
or no information on characterisation has been provided, which makes it difficult to relate
many of them to the nanomaterials under current assessment.
The evaluation by the SCCS of these and other studies provided in this submission has
shown that many of them are not relevant to the nanomaterials in the submission.
Therefore the relevance and usefulness of the data provided for this evaluation is poor andpatchy. It is difficult (in some cases impossible) to relate the studies to the types ofnanomaterials under evaluation. It would have been more productive if a complete set of
supporting data was provided on one (or a few) rather than several different TiO2
nanomaterials in a single submission.
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Physicochemical properties:- The studies provided in the submission relate to a range of TiO2 materials that comprisemicronized, ultrafine, or nano-sized particles. The physicochemical characterisation data
include coated and non-coated materials, composed of rutile and/or anatase forms of
TiO2. On the basis of the physicochemical data provided, the SCCS has considered thematerials in three broad groups on the basis of crystalline form and photocatalytic
activity.

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The SCCS agrees that TiO2 nanoparticles, due to agglomerative behaviour, are likely tobe present in the final sunscreen products mainly in the form of agglomerates, which
can also be in the nanoscale. It can therefore be assumed that the consumer is likely to
be exposed mainly to TiO2 agglomerates. However, it is also possible for the
agglomerates to de-agglomerate under certain conditions of formulation/use. Therefore,
the SCCS has considered the size of the primary particles more important than the sizeof agglomerates for the purposes of risk assessment.

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-

As nanoparticles may have different properties and biokinetic behaviour than theirsoluble equivalents, it is important to know the exact purity/impurity profile of ananomaterial intended for use in a cosmetic product (SCCS Guidance, SCCS/1484/12).
This opinion therefore does not cover TiO2 nanomaterials that have TiO2 purity less than
99%, and for which an acceptable impurity profile has not been provided. The opinion
may, however, be also applicable to other TiO2 nanomaterials that are similar to thenanomaterials in this opinion in terms of the physicochemical parameters listed in Tables
1-3, and other specific provisions laid out in Section 2.

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-

None of the materials evaluated in the submission is comprised of completely sphericalparticles because their reported aspect ratios are >1.0. However, the SCCS has
accepted an aspect ratio range between 1.0 and 4.5 on the basis that a lower aspectratio particle is less likely to be of a concern compared to higher aspect ratio ones.

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Zeta potential measurements have been provided for some materials, and not for others
due to difficulties in measuring zeta potential for hydrophobic nanomaterials.

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Among the nanomaterials assessed, the SCCS has noted a potential concern in relation
to photocatalytic activity, and stability of the coating, of some of the materials. It is
stated by the Applicant that all coatings on the materials included in the submission arestable. Three (3) studies have been provided, which show that coatings are stable.
However, from the other physicochemical data provided, it is less clear how stable thecoatings are in final formulations. The photocatalytic activity data, which is measured informulations, clearly indicate that either some of the materials were not completely
coated, or some of the coatings (e.g. organic, organosilanes) were not so stable in theformulations. This is an important aspect to ascertain because application of aformulation containing a nanomaterial that has a significant photocatalytic activity may
lead to local effects on sun-exposed skin. Such effects may or may not manifest during
the immediate use, and it is important to investigate the possibility of latent effects
following the use of a skin product that contained photocatalytic nanoparticles. This isbecause, whilst most studies on dermal absorption indicate that TiO2 nanoparticles are
not able to penetrate the skin deep enough to reach live cells of the epidermis/dermis,
they do show that nanoparticles can penetrate into stratum corneum, and can also enter
hair follicles and sweat glands. It is therefore possible that a trace amount of
nanoparticles may remain embedded in stratum corneum, in hair follicles, and/or sweat
glands, potentially over several days after skin application of a product and washing off.
If the nanoparticles have a significant photocatalytic activity, there is a possibility thatthey may cause generation of reactive radical species on exposure to sunlight, long after
the skin formulation had been applied and washed off. This, in a close proximity of living
cells, raises a concern over the possibility of harmful effects. Generally metal(oxide)nanomaterials which exhibit a high photocatalytic activity are those that are either
uncoated, partially coated, or have not been quenched by other means (e.g. doping) toadequately reduce photoreactivity. The TiO2 nanomaterials in the current submission
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that have a high photocatalytic activity include anatase materials in uncoated (S75-G)
and coated forms (S75-F, S75-O). Three (3) other rutile coated nanomaterials also havecomparatively lower but still significant levels of photocatalytic activity (S75-C, S75-D,
S75-E).

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The SCCS considers up to 10% photocatalytic activity compared to corresponding noncoated or non-doped reference as acceptable.

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-

In view of this, the SCCS does not recommend the use of nanomaterials that have ahigh photocatalytic activity (S75-F, S75-G, S75-O) in dermal formulations. Thesematerials can only be recommended after appropriate coating/doping has been applied
to quench their photocatalytic activity down to acceptable levels.

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Three rutile materials (S75-C, S75-D, S75-E) with relatively lower but still significant
levels of photocatalytic activity may be used in dermal formulations, but further
investigations over longer post-application periods may be necessary to ascertain thatthey do not pose a risk due to photocatalytic activity.

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Acute toxicity:- The studies provided on acute oral toxicity in the submission mainly relate to TiO2
nanomaterials that are anatase/rutile mixtures, coated with trimethoxy-n-octyl-silane.
From the limited relevant information provided, and considering that oral intake is not
likely to be the major route of exposure to TiO2 nanomaterials from dermal applicationof formulations, the acute oral toxicity of TiO2 is unlikely to be of a concern.

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The studies provided on acute dermal toxicity relate to an ultrafine TiO2 material and amaterial described as ‘natural colour’, and are therefore of no relevance to theassessment of nanomaterials.

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No study has been provided on acute inhalation toxicity. Sub-chronic (inhalation) andchronic (instillation) studies have indicated substantial inflammatory responses andoverload associated with diminishing particle clearance in a dose dependent manner,and histological indications of epithelial hypertrophy and hyperplasia.

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-

The limited relevant information provided in the submission, and other information in
the open literature, indicates that TiO2 nanomaterials are likely to be non-toxic via oral
or dermal application routes. However, inhalation exposure to TiO2 nanoparticles islikely to cause substantial inflammatory effects in the lung.

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Skin irritation:- Only two of the studies provided are relevant to the TiO2 nanomaterials. They relate toanatase/rutile mixtures, coated with trimethoxy-n-octyl-silane. The results showedprimary irritation index between zero and 0.3. Two studies using ultrafine grade
materials showed the mean irritation scores of 0.3 and 1.58-1.92 during 5 day repeat
applications on rabbit skin. Other studies also showed the tested materials to be either
mild- or non- irritant to rabbit and guinea pig skin, but it is not clear whether the tested
materials were nanomaterials.
-

From the limited relevant information, it can be considered that TiO2 nanomaterials arelikely to mild- or non- irritant to skin.

Eye irritation:- Two studies tested TiO2 anatase/rutile mixtures, coated with trimethoxy-n-octyl-silane.
From the studies, the derived primary irritation index was between zero and 0.3. A
different study used ultrafine rutile material coated with alumina/silica and regarded thetested material as slightly irritant to rabbit eye. Another study found the tested TiO2
materials to be moderately irritant to rabbbit eye, but it is not clear whether thematerial was a nanomaterial.
-

From the limited relevant data provided, eye irritation potential of nano-TiO2 appears to
be low.
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Skin sensitisation:- Two of the provided studies have regarded TiO2 nanomaterials (anatase/ rutile mixture,coated with trimethoxy-caprylylsilane or trimethoxy-n-octyl-silane) as non-sensitiser.
Another ultrafine material (rutile, coated with alumina/silica) is classified as a weaksensitiser, but characterisation data (particle size distribution) has not been reported toindicate what proportion of the particles was in the nano-scale.

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-

Due to the absence of skin penetration of TiO2 as demonstrated by many studies
included in this dossier, the usefulness of the Buehler test for assessing sensitisation
potency of nanomaterials is doubtful as it is based on exposure to intact skin.

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12

-

From the limited relevant data provided, TiO2 nanomaterials appear to be non- or weakskin sensitisers.

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Dermal absorption:- A number of in vitro and in vivo dermal penetration studies have been provided with thesubmission. In addition, there is a body of open literature on this subject. The evidence
from these studies supports the conclusion that TiO2 nanoparticles are unlikely topenetrate across the skin to reach viable cells of the epidermis. In these studies, TiO2
nanoparticles have been shown to penetrate only to the outer layers of the stratum
corneum, and there is as yet no conclusive evidence to show that they do reach living
cells of the epidermis/dermis. Studies have also shown that TiO2 nanoparticles do not
penetrate the (simulated) sunburnt skin.

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26

-

Despite the extensive database showing a general lack of TiO2 nanoparticle absorption
via the dermal route, there are a few gaps in the knowledge. For example, it is not clear
whether TiO2 nanoparticles will be able to penetrate through cuts and bruises, or overrepeated or long term applications of a sunscreen formulation.

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-

A number of studies have indicated that TiO2 nanoparticle can enter the hair follicles
and sweat glands, and that they may remain there for a number of days. This is a
scenario in which TiO2 nanoparticles are likely to get and remain in a close proximity to
the living cells for a length of time. A photocatalytic nanoparticle in such a situation may
cause generation of reactive oxyradical species (ROS) and potential harmful effects
when exposed to sunlight. As mentioned before, more data would be needed to justify
the use of those TiO2 nanoparticles in skin applications that have a considerable level ofphotocatalytic activity.

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Repeated dose toxicity:
Only two of the four provided subchronic studies on repeated dose toxicity are relevantto the TiO2 nanomaterials under evaluation. However, these studies relate to oral
exposure only, from which a LOAEL of 5 mg/kg bw/d has been derived.

-

No chronic toxicity study (>12 months) is provided, although a chronic inhalation study
has been provided (Section 3.3.1.3).

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Inhalation toxicity:- Studies in open literature indicate that subacute repeated dose respiratory toxicity
studies with nano size TiO2 induce an acute inflammation in the lungs that may bereversible depending on the dose and the time evaluated after exposure. In view of this,acute inflammation (spray) applications, which may result in inhalation exposure is notrecommended by the SCCS.

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Mutagenicity/ Genotoxicity:
Although an extensive range of studies on mutagenicity has been provided in thesubmission, most of them have not been conducted in any special consideration of thenano-related properties of the test materials.
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Several studies have been performed mainly to investigate mechanistic effects relating
to DNA damage and genotoxic properties. These studies are usually not performed
according to specific genotoxicity guidelines (e.g. OECD). Many of the studies have notevaluated the effects in a dose- and/or time- dependent manner. Those that haveaddressed this, often reveal no clear dose- or time- dependent effects.

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7-

From the provided studies, and open literature, TiO2 particles have also beenreported, or suggested, to interfere with the assays, because:

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10

-

Micronucleus scoring is difficult in the presence of TiO2 particles. This effectwas suggested to explain for the occasionally observed decreases in MN counts
after TiO2 treatment (Falck et al., 2009).

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It has been suggested (although not shown) that artefacts may be caused inrelation to the use of cytochalasin B for micronucleus testing. On one hand, it issuggested that nanoparticles may interfere with cytochalasin B (binding), andon the other, that the cytochalasin B may act as an inhibitor of the uptake ofnanoparticles in cells potentially leading to false negatives (Landsiedel et al.,2010).

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-

Due to the current lack of information on the possible cellular uptake andsubsequent translocation of TiO2 nanoparticles to nucleus, it is not possible todraw a conclusion on whether or not exposure to TiO2 nanomaterials can leadto mutagenic effects.

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-

Overall in a number of assays, TiO2 nano particles were observed to induce DNA
damage, so TiO2 nano particles have to be considered genotoxic.

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25

-

It is also of note that appropriate coating of nanomaterial to quench surface
photocatalytic activity will also reduce the likelihood of generation of reactive oxygen
species (ROS), which may in turn reduce the chances of genotoxicity.

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Carcinogenicity:- Pigmentary and ultrafine TIO2 materials have been tested for carcinogenicity by oral
administration in mice and rats, by inhalation exposure in rats and female mice, byintratracheal administration in hamsters and female rats and mice, and by subcutaneous
injection in rats and by intraperitoneal administration in male mice and female rats.

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-

According to the evaluation of TiO2 by IARC (2010), induction of lung tumours wasobserved in two inhalation studies with rats. Two other inhalation studies in rats, andone in female mice gave negative results. Intratracheally instilled female rats showed
an increased incidence of lung tumours following treatment with two types of titanium
dioxide. Tumour incidence was not increased in intratracheally instilled hamsters andfemale mice. Oral, subcutaneous and intraperitoneal administration did not produce asignificant increase in the frequency of any type of tumour in mice or rats. IARC
concluded that there is inadequate evidence in humans for the carcinogenicity oftitanium dioxide but sufficient evidence in experimental animals for the carcinogenicity
of titanium dioxide. Both nano and non nano size Titanium dioxide was classified as aGroup 2B carcinogen (Possibly carcinogenic to humans).

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-

In their recent evaluation of TiO2 NIOSH has determined that ultrafine TiO2 whichcontains nano-sized TiO2 is a potential occupational carcinogen and, that there isinsufficient data to classify fine TiO2 as potential occupational carcinogen after inhalation(NIOSH 2011).

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-

Nano titanium dioxide has been studied in 2 two-stage skin carcinogenicity studies withmice, 2 two-stage skin carcinogenicity studies with rats, and one two-stage lung study
with rats. Both noncoated (ncTiO2) and coated titanium dioxide have been studied in the
two-stage mouse skin carcinogenicity studies with CD1 mice and a transgenic mousestrain (rasH2). In one well performed study with non-coated and alumina and stearicacid coated TiO2, no promoter activity was found (Furukawa et al., 2011). Promoter
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activity was also not found for ncTiO2 in the other study (Sagawa et al., 2012).
However, it is difficult to draw a firm conclusion from this study with silica coatedtitanium dioxide due to lack of positive controls and very high tumour incidence in the‘initiated’ mice.

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8

-

Non-coated titanium dioxide was studied in 2 two-stage rat skin carcinogenicity studies.
Although, no tumour promoter activity was observed, it is difficult to draw any
conclusion since little experience with the model used is available and no positive
controls have been used in the studies.

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-

One, two-stage rat lung carcinogenicity study has been carried out with non coatedtitanium dioxide. The rats were ‘initiated’ by DHPN in the drinking water prior to intrapulmonary spraying with ncTiO2. The experiment demonstrated promoter activity ofncTiO2 (Xu et al., 2011).

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-

Since TiO2 particles have shown carcinogenic activity (after inhalation) and since nanoncTiO2 showed promoter activity after intra-pulmonary spraying, the use of nano TiO2
in sprayable applications is not recommended by the SCCS.

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Reproductive toxicity- No study has been provided on reproductive toxicity that is relevant to thenanomaterials under assessment. A review article covering exploratory studies in micehas been provided, which relates to the use of a TiO2 material which is <10µm (with nofurther information), and a TiO2 nanomaterial with primary particle size 25-70 nm (nofurther information).

-

Other studies in open literature have indicated the possibility of placental transport inpregnant animals into the foetus, or found effects in the offspring for various
manufactured nanomaterials including nano-TiO2. However, the information relating tothis endpoint is patchy and therefore inconclusive.

Photo-induced toxicity- Only a few studies have been provided that are relevant to the nanomaterials underassessment.

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-

These indicate that TiO2 materials may not be photo-sensitisers. However, concerns
regarding the use of photocatalytic nanomaterials in dermal formulations discussed
above need to be taken into consideration.

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-

Several studies have specifically addressed photo-sensitization effects TiO2. However,
the outcomes of these studies need to differentiate between photo-sensitization andother local effects on skin (taking into account the aspect of penetration), versus
potential effects at other target sites.

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Toxicokinetics:- Two studies have been provided in the submission on toxicokinetics of TiO2 following
intravenous injection in rats and mice. In addition, there are few other relevant studies
in the open literature relating to inhalation and intravenous, as well as limited(questionable) information on oral administration routes.
-

The available evidence suggests that, if TiO2 particles become systemically available by
the oral and inhalation uptake pathway, they are likely to accumulate mainly in the liver,followed by a very slow rate of clearance.

Special investigations:
No relevant specific studies have been provided apart from those already discussed above
under relevant endpoints.

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2. CONCLUSIONS

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This opinion is based on the risk assessment of nano-sized titanium dioxide (TiO2) for use
as a UV filter in sunscreen formulations. It is important to note that risk assessment of
nanomaterials in general still has certain gaps in the knowledge - for instance in relation to
the behaviour of nanoparticles in a test medium, or in the animals. This has led to
uncertainties over whether the nanoparticles are able to reach and interact with various
moieties and biological target sites, and whether, on dermal application, they may penetrate
through damaged skin, or during repeated or long term applications. There are also
uncertainties over the validity of the currently available tests used for nanomaterials.
However, a positive toxic response in these tests is still considered valid for risk assessment
as it would indicate a hazard potential.

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As discussed above, the safety data provided in support of the fifteen (15) nanomaterials isquite patchy, and is only partially useful for any of the given nanomaterials. However, the
SCCS took the view that this submission could be considered for evaluation as an exception.
This is because some additional information on TiO2 nanomaterials is available in open
literature which is relevant for this evaluation. Also, for example, although the safety data
provided in the submission on rutile nanomaterials is insufficient, the studies on anataseform (or rutile/anatase mixtures) could be considered as a surrogate because publishedstudies in open literature have regarded anatase a greater safety concern than the rutile
form. However, as the evaluation is still based on limited information which could be related
to specific nanomaterial types in the submission, this opinion is limited to the nanomaterials
indicated below:-

On the basis of physicochemical considerations discussed above, this opinion applies to
the TiO2 nanomaterials presented in this submission. In addition, the opinion may also
be applicable to other TiO2 nanomaterials that are similar to the nanomaterials coveredin this opinion in terms of physicochemical parameters listed in Tables 1-3, and thespecific provisions laid out in the overall conclusions below.

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36

-

It needs to be stressed that the main consideration in the current assessment is theapparent lack of penetration of TiO2 nanoparticles through skin, which is supported by abody of evidence both in the form of studies provided by the Applicant and other studies
reported in open literature. In the absence of a systemic exposure, a margin of safety(MoS) could not be calculated for TiO2 nanomaterials in this assessment. From thelimited relevant information provided in the submission, and the information from open
literature, the SCCS considers that TiO2 nanomaterials in a sunscreen formulation areunlikely to lead to:

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o

systemic exposure to nanoparticles through human skin to reach viable cells of
the epidermis, dermis, or other organs;

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o

acute toxicity via dermal application or incidental oral ingestion. This, however,does not apply to sprayable applications that may lead to inhalation exposure of
TiO2 nanomaterials, which may result in lung inflammation;

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o

skin irritation, eye irritation, or skin sensitisation when (repeatedly) applied onhealthy skin (except possible photoxicity of insufficiently coated nanomaterials);44oreproductive effects when applied on healthy skin.

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-

Some TiO2 nanoparticles have been shown to be able to damage DNA and should beconsidered genotoxic. However as negative results have also been reported, the current
evidence in relation to potential genotoxicity of TiO2 nanomaterials is not conclusive.
TiO2 particles have also shown to lead to carcinogenic effects after inhalation. These
manifestations are a major hazard concern. However, no penetration was found throughthe stratum corneum of reconstructed human full thickness skin models and no DNA
damage was detected by the Comet assay in these cells in contrast to epidermal cellline. Considering the absence of a systemic exposure, the SCCS considers that the use

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of nano TiO2 in dermally applied cosmetic products should not pose any significant risk
to the consumer.

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7
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Evidence on acute and sub-chronic inhalation toxicity does not support the overall safetyof use of TiO2 nanomaterial formulations for spray applications. In addition, tumour
promoter activity of nano (non-coated) TiO2 has been shown after intra-pulmonary
spraying. Therefore the SCCS does not recommend the use of nano TiO2 in sprayable
applications. This may be reconsidered if further evidence is provided to rule out thepossibility that the nanoparticles can reach the lower respiratory tract during spray
applications.

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-

Although there is no conclusive evidence at present to indicate penetration of TiO2
nanoparticles through the skin to viable cells of the epidermis, a number of studies haveshown that they can penetrate into the outer layers of the stratum corneum, and canalso enter hair follicles and sweat glands. It is therefore recommended not to use TiO2
with substantially high photocatalytic activity (e.g. S75-F, S75-G, S75-O) in sunscreen
formulations. Other TiO2 nanomaterials that have a relatively lower but still significant
level of photocatalytic activity (e.g. S75-C, S75-D, S75-E) may be used, but further
investigations over longer post-application periods taking into account the potential
photocatalytic activity post-application, whilst allowing for appropriate lag-time andusing realistic application scenarios may be necessary to ascertain that they do not posea risk due to photocatalytic activity.

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Overall conclusion

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1. Does SCCS consider that use of titanium dioxide in its nanoform as an UV-filter incosmetic products in a concentration up to maximum 25.0 % is safe for the consumers
taken into account the scientific data provided?

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 have TiO2 purity of ≥99%, or in case of a lesser purity, the impurities must be
demonstrated to be safe for use in cosmetic formulations;

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 are composed of mainly the rutile form, or rutile with up to 5% anatase, withcrystalline structure and physical appearance as described in the current submission,i.e. clusters of spherical, needle, or lanceolate shapes;

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 have a median particle size based on number size distribution of 30 to 100 nm(measured by different methods) as submitted in the dossier, or larger. Thus whilst
primary particle size may be smaller (around 10 nm), the median particle size of TiO2
nanomaterials in a cosmetic formulation must not be smaller than 30 nm in terms ofnumber based size distribution;45
46

 have an aspect ratio from 1.0 and up to 4.5, and volume specific surface area up to
460 m2/cm3;

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 are coated with one of the coating materials described in Table 1, and the coatings arestable in the final formulation and during use. Other cosmetic ingredients applied asstable coatings on TiO2 nanomaterials can also be used, provided that they can bedemonstrated to the SCCS to be safe and the coatings do not affect the particle
properties related to behaviour and/or effects, compared to the nanomaterials coveredin this opinion.

On the basis of the available evidence, the SCCS has concluded that the use of TiO2
nanomaterials with the characteristics as indicated below, at a concentration up to 25% as a
UV-filter in sunscreens, can be considered to not pose any risk of adverse effects in humans
after application on healthy, intact or sunburnt skin. This, however, does not apply toapplications that might lead to inhalation exposure to TiO2 nanoparticles (such as powders
or sprayable products). Furthermore, this assessment applies to the TiO2 nanoparticles
presented in the submission, but may also be applicable to other TiO2 nanomaterials thatare similar to the parameters in Tables 1-3, i.e. TiO2 nanomaterials that:103

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 are photostable in the final formulation;

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 do not have photocatalytic activity. However, the SCCS considers up to 10%
photocatalytic activity compared to corresponding non-coated or non-doped reference
as acceptable.

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9

It is also worth highlighting again that this opinion is based on the currently available
scientific evidence which shows an overall lack of dermal absorption of TiO2 nanoparticles.
If any new evidence emerges in the future to show that the TiO2 nanoparticles used in asunscreen formulation can penetrate skin (healthy, compromised, or damaged skin) toreach viable cells, then the SCCS may consider revising this assessment.

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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, behaviour and effects of nanomaterials. This assessment
is, therefore, 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.

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It is also important to note that the potential ecotoxicological impacts of nano TiO2 whenreleased into the environment have not been considered in this opinion.

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2. In order for the COM to differentiate in the regulation between materials in its nanoform
and its non-nano form, can the SCCS give quantitative and qualitative guidance on how thisdifferentiation should be given based on the particle size distribution or other parameters?

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A detailed SCCS guidance on risk assessment of nanomaterials in cosmetics has recently
been published (SCCS/1484/12). The guidance provides a detailed account of the importantnano-related parameters that should be considered in relation to physicochemical
characterisation, hazard identification, exposure assessment and risk assessment of
nanomaterials.

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3. MINORITY OPINION/

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4. REFERENCES

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Bellmann, B., Muhle, H. and Heinrich, U. (1983). Lung clearance after long time exposure ofrats to airborne pollutants. J. Aerosol Sci. 14(3), 194-196.

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6

Bennat C, Müller-Goumann CC. Skin penetration and stabilization of formulations containing
microfine titanium dioxide as physical UV filter. Int J Cosmetic Sc 22, 271-283, 2000.

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8
9Bennett, SW, Zhou, D, Mielke, R., Keller, AA (2012) Photoinduced Disaggregation of TiO2
Nanoparticles Enables Transdermal Penetration, PLoS ONE 7(11): e48719.
doi:10.1371/journal.pone.0048719.

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Xu J, Futakuchi M, Iigo M, Fukamachi K, Alexander DB, Shimizu H, Sakai Y, Tamano S,
Furukawa F, Uchino T, Tokunaga H, Nishimura T, Hirose A, Kanno J, Tsuda H (2010).
Involvement of macrophage inflammatory protein 1a (MIP1a) in promotion of rat lungand mammary carcinogenic activity of nanoscale titanium dioxide particles
administered by intra-pulmonary spraying. Carcinogen31: 927–935.

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Xu J, Sagawa Y, Futakuchi M, Fukamachi K, Alexander DB, Furukawa F, Ikarashi Y, Uchino
T, Nishimura T, Morita A, Suzui M, Tsuda H (2011). Lack of promoting effect oftitanium dioxicde particles on ultraviolet B-initiated skin carcinogenesis in rats, Food
Chem Toxicol. 49: 1298-1302.

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ABBREVIATIONS AND GLOSSARY OF TERMS
BETBrunauer-Emmett-Teller method based on nitrogen gas absorption

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CAS

A chemical registry system established by the Chemical Abstracts
Service (CAS)

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ECVAM

European Centre for the Validation of Alternative Methods

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EDX

Energy Dispersive X-ray

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HPLC

High performance liquid chromatography

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ICP-MS

Inductively coupled plasma mass spectrometry

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12
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In vitro test method

Biological method that uses organs, tissue sections and tissue
cultures, isolated cells and their cultures, cell lines and subcellular
fractions, or non-biological method that uses chemical interaction
studies, receptor binding studies, etc [Rogiers and Beken 2000]

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ISO

International Organization for Standardization

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IARC

International Agency for Research against Cancer

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IUPAC

A system of chemical nomenclature established by the International
Union of Pure and Applied Chemistry (IUPAC)

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Local effects

A Local effect refers to an adverse health effect that takes place atthe point or area of contact. The site may be skin, mucous
membranes, the respiratory tract, gastrointestinal system, eyes, etc.
Absorption does not necessarily occur.

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Nanomaterial

An insoluble or biopersistent and intentionally manufactured material
with one or more external dimensions, or an internal structure, on
the scale from 1 to 100 nm [Regulation (EC) No 1223/2009]

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Nanoparticle

A nano-object with all three external dimensions in the nanoscale[ISO/TS 27687:2008, Nanotechnologies -- Terminology anddefinitions for nano]. For the purpose of this assessment the term
‘nanoparticle’ is used to also include other forms of nano-object, suchas nano-rods, nano-tubes, etc.

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NPs

Nanoparticles

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NanoscaleSize range from approximately 1 nm to 100 nm [ISO/TS 800041:2010, Nanotechnologies -- Vocabulary]

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OECD

Organisation for Economic Co-operation and Development

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PBS

Phosphate buffered saline

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ROS

Reactive Oxygen Species

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SCCNFP

Scientific Committee on Cosmetic products and Non-Food Products
intended for consumers

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SCCP

Scientific Committee on Consumer Products

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SCCS

Scientific Committee on Consumer Safety

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SED

Systemic Exposure Dosage

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SEM

Scanning electron microscopy

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Solubility

The terms ‘solubility’ and ‘persistence’ are often used to describe therate of “degradation”. As such there are a number of definitions ofsolubility (see SCENIHR Opinion ‘Scientific Basis for the Definition of
the Term “Nanomaterial”, 8 December 2010). In the context of thisassessment, solubility means disintegration of a nanomaterial in an
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aqueous medium or biological environment
components with the loss of nano features.

intomolecular

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Systemic effects

Systemic effect refers to an adverse health effect that takes place at
a location distant from the body's initial point of contact andpresupposes absorption has taken place.

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TEM

Transmission electron microscopy

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TiO2:

Titanium Dioxide

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UV-VisUltraviolet-visible spectrophotometry

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Validated method

A standard method for which the relevance and reliability have been
established for a particular purpose, usually through an inter-lab
comparison, which found uncertainties in the measurements
acceptable..

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VSSA

Volume specific surface area (see Kreyling et al., 2010)

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XRD:

X-ray diffraction

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