Nanotoxicology, December 2010; 4(4): 364–381

Gene toxicity studies on titanium dioxide and zinc oxide nanomaterials

used for UV-protection in cosmetic formulations

ROBERT LANDSIEDEL1, LAN MA-HOCK1, BEN VAN RAVENZWAAY1,

MARKUS SCHULZ1, KARIN WIENCH2, SAMANTHA CHAMP3, STEFAN SCHULTE3,
WENDEL WOHLLEBEN4, & FRANZ OESCH5
1
Experimental Toxicology and Ecology, 2Product Safety Department, 3Care Chemicals Division, 4Polymer Physics
Department, BASF SE, Ludwigshafen, and 5Institute of Toxicology, University of Mainz, Mainz, Germany
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(Received 5 February 2010; accepted 2 July 2010)

Abstract
Titanium dioxide and zinc oxide nanomaterials, used as UV protecting agents in sunscreens, were investigated for their
potential genotoxicity in in vitro and in vivo test systems. Since standard OECD test methods are designed for soluble materials
and genotoxicity testing for nanomaterials is still under revision, a battery of standard tests was used, covering different
endpoints. Additionally, a procedure to disperse the nanomaterials in the test media and careful characterization of the
dispersed test item was added to the testing methods. No genotoxicity was observed in vitro (Ames’ Salmonella gene mutation
test and V79 micronucleus chromosome mutation test) or in vivo (mouse bone marrow micronucleus test and Comet DNA
damage assay in lung cells from rats exposed by inhalation). These results add to the still limited data base on genotoxicity test
results with nanomaterials and provide congruent results of a battery of standard OECD test methods applied to
nanomaterials.
For personal use only.

Keywords: Cosmetics, genotoxicity, test methods, titanium dioxide, zinc oxide

Introduction (coated zinc oxide nanomaterial) were tested for their

Extensive exposure to sunlight is responsible for
harmful effects in the skin. The high energy content
in the UV portion of the sunlight leads to sunburn,
accelerated ageing of the skin giving rise to wrinkles
and, with frequent intensive exposure to sun light, an
increased risk of skin cancer. Titanium dioxide and
zinc oxide have broad UV spectrum attenuation prop-
erties and are therefore used in sunscreen applications
to protect the human skin against UV radiation. The
optical properties of these inorganic oxides of various
particle sizes can be calculated by the use of Mie
theory, showing that a particle size of less than
100 nm is necessary to achieve cosmetic and efficacy
benefits by reducing the ‘scattering’ of visible light
providing ‘transparent’ products which retained UV
absorption characteristics (Maier and Korting 2005).
Four inorganic UV-filter products, T-LiteTM SF
and T-LiteTM MAX (coated titanium dioxide nano-
material) and Z-COTE
HP1 and Z-COTE
MAX
potential genotoxic potential. The materials were
investigated in standard OECD guideline tests such
as the Salmonella Reverse Mutation Assay (OECD
TG 471) and the in vitro Mammalian Cell Micronu-
cleus Test (MNvit) (OECD Draft TG 487) only
modified in regard to dispersing the material in the
test systems, giving a special focus on the particle
size distribution of the metal oxides in the media.
In addition, two in vivo tests were performed: The
Mammalian Erythrocyte Micronucleus Test (OECD
TG 474) after i.p. injection of Z-COTE
HP1 and a
Comet DNA damage assay in broncho-alveolar lavage
cells from rats exposed by inhalation to T-LiteTM SF.
Materials and methods
Since methods for genotoxicity testing of nanoparti-
cles are currently refined, materials and methods are
given in detail, especially the preparation of the

Correspondence: Dr Robert Landsiedel, Experimental Toxicology and Ecology, BASF SE, GV/TB-Z 470, Ludwigshafen, 67056, Germany.
E-mail: robert.landsiedel@basf.com

ISSN 1743-5390 print/ISSN 1743-5404 online  2010 Informa UK, Ltd.

DOI: 10.3109/17435390.2010.506694

nanomaterial dispersions and the characterization of
the test dispersions.
Test substances
Physico-chemical characterization was performed
according to the ‘characterization matters’ initiative
(MinChar Initiative 2008). Primary particle size
was determined by monolayer transmission electron
microscopy (TEM) (Supplementary Figure S1, avail-
able online). Samples were wetted in ethanol, then
gently spread on a sample holder and transferred into
vacuum. The electron microscope from FEI, Type
Strata 400 DB was equipped with a field emission
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cathode. Crystallinity was determined by X-ray diffrac-
tion (XRD). The intensity of the diffracted X-ray beam
was recorded by a D8 Advance (Fa. Bruker/AXS) as a
function of the diffraction angle (2 < 2q < 150 ).
Quantitative phase analysis was performed using Riet-
veld refinement. Impurities and surface modification
were determined by X-ray photoelectron spectroscopy
(XPS) with a Phi XPS 5500 system with 300 W
monochromatic Al- K alpha radiation, pass energy
for surveys 117 eV (measurement time of 45 min),
detailed spectra at 23.5 eV (measurement time of
6 min). Evaluation was performed by CasaXPS
For personal use only.
2.3.15, based on the Phi standard-sensitivity factors,
with Shirley background subtraction and peak shape
fits as sum of 90% Gaussian and 10% Lorentzian.
Information depth is limited to the surface 10 nm of the
material. We performed two measurements per sam-
ple, each integrating over 0.5 mm2. The results in at %
are derived from relative concentration of elements and
their chemical bonds from line shape analyses.
The results of the physico-chemical characteriza-
tion are summarized in Table I. T-LiteTM SF and
T-LiteTM MAX are nano-particular (primary particle
size 10  50 nm, mean agglomerates about 200 nm,
range 90 [d10]–460 nm [d90]) coated titanium diox-
ide (CAS No. 13463-67-7). The titanium dioxide
had a purity of ‡99%. The coating consists of
aluminium hydroxide and dimethicone/methicone
copolymer (T-LiteTM SF) and dimethoxydiphenylsi-
lane, triethoxycaprylylsilane crosspolymer, hydrated
silica and aluminium hydroxide (T-LiteTM MAX).
The T-LiteTM primary particles are pure rutile (from
XRD) and have an acicular-shaped morphology with
a width/length around 10 nm/50 nm (Supplementary
Figure S1, available online). The low aspect ratio
around 5 classifies them still as particles. The two
variants originate from the same titanium dioxide
core, which is then coated with a thin AlOH layer
and polymers in order to adjust the hydrophobicity.
Typically, hydrophobic surfaces are generated such
Genotoxicity studies on sunscreen nanomaterials 365
that in a sunscreen formulation the particles accumu-
late in the oily phase.
Z-COTE
MAX and Z-COTE
HP1 are nano-
particular (primary particle size range 30–200 nm)
zinc oxides and their inorganic core has a purity
of ‡99%. The crystalline structure of the core is
pure zinc oxide in hexagonal zincite formation (from
XRD). The material has a mass-specific surface of
9 m2/g (measured by BET adsorption). The two var-
iants originate form the same zinc oxide core, which is
then coated non-covalently with different polymers.
Both Z-COTE
types share a relatively broad particle
size range from 25–900 nm diameter with both iso-
metric and slightly elongated morphologies.
All test materials were stable throughout the study
periods. The analyses of the test substances were
carried out at Competence Center Characterization,
BASF SE, Ludwigshafen, Germany.
In addition to the commonly used vehicle dimethyl-
sulfoxide (DMSO) (obtained from Mallinckrodt
Baker, Deventer, The Netherlands) fetal bovine
serum (FCS) (obtained from Biochrom, Berlin,
Germany) was used to mimic the protein containing
body fluids (e.g., blood) in the bacterial reverse muta-
tion assays. It was shown in previous particle size
distribution measurements (Schulze et al. 2008)
that the vehicle FCS is appropriate for the dispersion
of particles, leading to reduced numbers of test sub-
stance agglomerates. Therefore, FCS was used as sole
vehicle in the micronucleus test in vitro and in the
micronucleus test in vivo. The test substance stock
dispersion was stirred in a closed vessel for 24 h at
1,000–1,200 rpm at room temperature. The further
concentrations were diluted from the stock dispersion
and were stirred for about 1 h prior to use.
Analytics on the state of agglomeration in aerosol and test
dispersion
The state of agglomeration changes when the particle
powder is nebulized in air for inhalation testing or
dispersed in a medium for in vitro genotoxicity assays
(Landsiedel et al. 2010). We monitored closely these
changes. The aerosol characterization is described in
details below, under the section Comet assay in vivo in the
rat lungs. For the in vitro assays and except where stated
otherwisetheparticlesizedistributionandagglomeration
state was determined by analytical ultracentrifugation
which is an adequate method for determination of
particle size distribution (Schulze et al. 2008,
Landsiedel et al. 2010). At the acceleration of up to
300,000 g used in analytical ultracentrifugation, solutes
and nanoparticles sediment into fractions that are sep-
aratedaccordingtotheirsizeintherange0.5–10,000nm.
 366 R. Landsiedel et al.

Table I. Identification and properties of the titanium dioxide and zinc oxide products applied to genotoxicity tests.

T-Lite SF T-Lite Max

INCIa Titanium dioxide (and) aluminum Titanium dioxide (and) dimethoxydiphenylsilane/

hydroxide (and) dimethicone/methicone triethoxycaprylylsilane crosspolymer (and) hydrated silica
copolymer (and) aluminum hydroxide
CAS-no.b 13463-67-7, 21645-51-2, 68037-59-2 13463-67-7, 827036-50-0, 1343-98-2, 21645-51-2
Surface coating Yes Yes
2
TiO -content [%] 79–89 69–73
Purity of TiO2 core ‡99% ‡99%
Lattice structure Rutile Rutile
Primary particle size [nm]
Length: 50 50
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Width: 10 10
Morphology Acicular-shaped Acicular-shaped
Mean agglomerates [nm] About 200 About 200
Range [nm] 90 (d10)–460 (d90) 90 (d10)–460 (d90)
Specific surface area [m2/g]c 100 100
Low aspect ratio Around 5 Around 5


Z-COTE HP 1 Z-COTE
MAX

INCIa Zinc oxide (and) Zinc oxide (and) dimethoxy- diphenylsilane/triethoxy

triethoxycaprylylsilane caprylylsilane crosspolymer
CAS-no.b 1314-13-2, 2943-75-1 1314-13-2, 827036-50-0
Surface coating Yes Yes
For personal use only.

ZnO-content [%] 96–99 96–99

Purity of ZnO core ‡99% ‡99%
Crystalline structure Hexagonal zincite Hexagonal zincite
of core:
Primary particle size [nm] 30–200 30–200
Specific surface area 12–24 12–24
[m2/g]c

INCI = International Nomenclature of Cosmetic Ingredients; bCAS = Chemical Abstract Service; cAccording to BET.
a

Agglomerates larger than ~10 mm diameter sediment
before data acquisition starts and cannot be quantified.
Simultaneous detection by synchronized optics quanti-
fied the amount and the diameter of each fraction inde-
pendently. A Beckman model XL ultracentrifuge
modified for the online recording of sedimentation
with turbidity, interference, and Schlieren or ultraviolet
(UV) detection allowed the use of interference and
turbidity optics with a ramped rpm-profile to quantify
the amounts of ultrafine (diameter <100 nm), fine
(100–1000 nm), and larger-scale material (>1 mm).
Bacterial reverse mutation assay
The Ames tests for the detection of gene muta-
tions in bacteria were performed following the recom-
mendations of the OECD test guideline No. 471 with
Salmonella strains TA 98, TA 100, TA 102, TA 1535
and TA 1537. Although for nano-particular com-
pounds the omission of metabolic activation by exo-
geneous rat liver enzymes may be scientifically
justified, in accordance with the general OECD guide-
lines the five strains were tested in the absence
and presence of exogenous metabolic activation
(phenobarbital/ß-naphthoflavone-induced rat liver
S9) using standard plate and preincubation method.
Standard plate method (Ames et al. 1975; Maron and
Ames 1983). For testing in the absence of S9 mix,
100 ml test substance preparation (dispersed in
dimethylsulfoxide) and 100 ml bacterial suspension
were mixed with 500 ml phosphate buffer (0.015 M,
pH 7.4) and 2 ml overlay agar. In case of metabolic
 Genotoxicity studies on sunscreen nanomaterials 367

activation instead of phosphate buffer 500 ml S9 mix
was used. 500 ml S9 mix equates to 50 ml of S9 fraction
(ratio 1:9). The samples were poured onto minimal
agar plates. After incubation of the plates for 48 h at
37 C the bacterial colonies were counted.
Preincubation method (Yahagi et al. 1977; Matsushima
et al. 1980). For testing in the absence of S9 mix,
100 ml test substance preparation (dispersed in fetal
bovine serum) and 100 ml bacterial suspension were
mixed with 500 ml phosphate buffer (0.015 M,
pH 7.4) and were incubated at 37 C for 20 min on
a shaker. In case of metabolic activation instead of
phosphate buffer 500 ml S9 mix (ratio 1:9; see above)
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Quadriperm dish. About 6 h after seeding, the medium
was removed and culture medium containing 10% test
substance preparation in FCS was added. Concurrent
vehicle controls (MEM with 10% FCS) and appropri-
ate positive controls (500 mg/ml ethylmethanesulfonate
[EMS] dissolved in MEM without FCS) were treated
in parallel. The cultures were incubated for the expo-
sure period of 4 or 24 h at 37 C, 5% CO2 and ‡90%
humidity. In the case of 4 h exposure, the medium was
removed after end of exposure period and the cultures
were rinsed twice with Hanks Balanced Salt Solution
(HBSS). Then complete culture medium was added
and the cultures were incubated for the respective
recovery time. In the case of continuous treatment,

the cell preparation was started directly at the end of

was used. Subsequently, 2 ml of overlay agar was
exposure. At the harvest time, 24 h after start of
added and, after mixing, the samples were poured
exposure, the medium was removed. After hypotonic
onto minimal agar plates. After incubation of the
treatment with 1.5% sodium citrate solution the cells
plates at 37 C for 48 h the bacterial colonies were
were fixed by adding a mixture of cold ethanol/
counted.
glacial acetic acid/formaldehyde. Finally, the cells
Five doses of test substance together with concur-
were prepared by passing through a flame. After dry-
rent vehicle controls and appropriate positive controls
ing, the slides were stained with an aqueous solution of
were tested in triplicate on each tester strain without
Acridine Orange (50 mg/ml) (Hayashi et al. 1983). The
and with exogenous metabolic activation by induced
slides were stored in the dark, then wet mounted with
rat liver S9 preparation with both methods. The dose
cover slips using purified water just prior to scoring
levels in the study were 20, 100, 500, 1000, 2500 and
using a fluorescence microscope (ZEISS Axioplan,
5000 mg per plate.
Jena, Germany) at 400-fold magnification and a filter
For personal use only.

The test chemical is considered positive if a dose-

setting providing blue excitation at 440–490 nm and
related and reproducible increase in the mean number
emission at approximately 520 nm. Quadruplicate
of revertants per plate was obtained, leading at least to
cultures were prepared and 2,000 cells were analyzed
a doubling of the spontaneous mutation rate in at least
for micronuclei for each test group.
one tester strain either without or with exogenous
metabolic activation.
Evaluation. The analysis of micronuclei followed the
criteria of Countryman and Heddle (1976): (i) The
Micronucleus test in vitro in V79 cells diameter of the micronucleus is less than 1/3 of the
main nucleus; (ii) the micronucleus and main nucleus
This method for the detection of aneugenic and/or
retain the same colour; and (iii) the micronucleus is
clastogenic effects in mammalian cells in vitro was
not linked to the main nucleus and is located within
conducted according to the draft OECD Guideline
the cytoplasm of cells clearly surrounded by a nuclear
No. 487. The test substance T-LiteTM SF is a nano-
membrane.
particular compound; therefore, the omittance of
For the determination of cytotoxicity, additional
metabolic activation by exogeneous rat liver enzymes
cell cultures in flasks (2.5  105 cells per 25 cm2
was scientifically justified. The V79 cells (lung fibro-
flask) were treated in parallel. At the harvest time cell
blasts from Chinese Hamster) were grown in MEM
suspensions were prepared by trypsination and cell
(minimal essential medium with Earle’s salts) con-
counts were measured with a cell counter (CASY,
taining a L-glutamine source supplemented with 10%
Innovatis, Germany) in all test groups, except the
FCS, 1% penicillin/streptomycin (10,000 IU/10,000
positive controls. In addition, at the end of the treat-
mg/ml) and 1% amphotericin B (250 mg/ml).
ment period, the test cultures of the test groups
Cell incubation occurred with 5% CO2 at 37 C
except the positive controls, were examined micro-
and ‡90% humidity.
scopically with regard to cell morphology (200
magnification) as an indication of attachment of the
Test performance (Kalweit et al. 1999). A single cell cells to the slides. For dose selection for scoring the
suspension was prepared and about 5  104 cells were quality and the number of analyzable cells was
seeded on sterile glass slides in each chamber of a determined.
 368 R. Landsiedel et al.
For the assessment of test substance induced cyto-
toxicity the proliferation index (PI) as measure of the
proliferative activity of the cells was determined in
2,000 cells per test group. The number of clones
(packs) containing 1, 2, 3–4 or 5–8 cells was recorded
and the PI was calculated using the following formula:
(n cl1) × 1 + (n cl2) × 2 + (n cl4) × 3 + (n cl8) × 4
PI =
( n cl1) + ( n cl2) + ( n cl4) + ( n cl8)
where, cl1 = cell clone with 1 cell; cl2 = cell clone with
2 cells; cl4 = cell clone with 3 or 4 cells; cl8 = cell clone
with 5, 6, 7 or 8 cells.
Additional parameters like pH value, osmolarity
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and test substance precipitation were checked. The
test chemical is considered positive if a statistically
significant (Fisher’s exact test), dose-related and
reproducible increase in the number of micronu-
cleated cells was obtained, with values exceeding
both the value of the concurrent vehicle control
and the range of the historical negative control data
(0.3–1.8% micronucleated cells).
Micronucleus test in vivo in mouse bone marrow cells
The Mammalian Erythrocyte Micronucleus Test for
For personal use only.
determination of aneugenic and/or clastogenic effects
in vivo was conducted according to the OECD Guide-
line No. 474.
Animals. In this study 5- to 8-week-old healthy male
Crl:NMRI mice from Charles River Laboratories,
Sulzfeld, Germany were acclimatized for at least
five days and kept at 20–24 C at a relative humidity
of 30–70% at a day/night rhythm of 12 h light
from 06:00–18:00 h in Makrolon cages using
single housing. They were fed standardized pelleted
feed (Provimi Kliba, Kaiseraugst, Switzerland). Tap
water was available ad libitum. Five animals were
assigned to each test group according to a random-
ization plan prepared with an appropriate computer
program.
Dose and application of the test and control materials. The
test substance Z-COTE
HP1 was disperged in FCS
(see Test substances) and was administered i.p. each in
10 ml/kg body weight. In pretests for the determina-
tion of the acute toxicity of Z-COTE
HP1, deaths
occurred down to 80 mg/kg body weight. At
60 mg/kg, all animals survived but piloerection,
hunched posture and reduced general condition
were observed. The vehicle (single i.p. administration
of pure FCS in a volume of 10 ml/kg body weight) was
tolerated well by all animals. Due to lacking distinct
differences in the symptoms between males and
females only male animals were used for the main
experiment in which 0 (vehicle control), 15 mg, 30 mg
and 60 mg Z-COTE
HP1 per kg body weight were
administered to the animals which were sacrificed
24 h later, while only the dose of 60 mg/kg body
weight and the vehicle control were used for the
animals which were sacrificed 48 h after treatment.
20 mg cyclophosphamide (Baxter Oncology, Unter-
schleißheim, Germany) (clastogen) and 0.15 mg vin-
cristine sulfate (Sigma, Deisenhofen, Germany)
(aneugen) per kg body weight (both dissolved in
purified water) were used as positive controls in
animals sacrificed 24 h after treatment. Five animals
were used per dose and exposure time. After treat-
ment up to the time of sacrifice, the animals were
examined for clinically evident signs of toxicity several
times.
Preparation of the bone marrow, staining of the slides and
microscopic evaluation. The bone marrow was pre-
pared based on the method by Schmid (1976,
1977) and Salamone et al. (1980) as follows: The
two femora of the animals sacrificed by cervical
dislocation were prepared by dissection and remov-
ing soft tissues. After cutting off the epiphyses,
the bone marrow was flushed out of the diaphysis
into a centrifuge tube using a cannula filled with
preheated FCS. The suspension was mixed and
centrifuged at 300 g for 5 min. The pellet was
resuspended in about 50 ml FCS. One drop of
this suspension was given onto a microscopic slide
with ground edges, using a Pasteur pipette. The
preparations were dried in the air and stained with
modified May-Grünwald/Giemsa solution. Then the
slides were mounted in Corbit-Balsam. 10,000 poly-
chromatic erythrocytes (PCE) were scored per test
group. The following parameters were recorded:
. Number of PCE and of PCE containing micro-
nuclei the increase in the number of micronuclei in
PCE providing an index of a chromosome-
breaking (clastogenic) effect or damage of the
mitotic apparatus (aneugenic activity)
. Number of normochromatic erythrocytes (NCE)
and of NCE containing micronuclei (the number
of micronuclei in NCE at the early sacrifice interval
indicating the situation before test substance
administration while a test substance induced
increase in the number of micronuclei in normo-
cytes may be found with an increase in the duration
of the sacrifice interval)

. Ratio of PCE to NCE (an alteration of this ratio
indicating that the test substance reached the bone
marrow)
. Number of small micronuclei (d < D/4) and of
large micronuclei (d ‡ D/4) [d = diameter of
micronucleus, D = cell diameter] (d < D/4 indicat-
ing a clastogenic effect, d ‡ D/4 indicating a spindle
poison effect (Yamamoto and Kikuchi 1980).
The test chemical is considered positive if a statis-
tically significant (one-sided Wilcoxon test), dose-
related increase in the number of PCE containing
micronuclei was obtained, with values exceeding
both the value of the concurrent vehicle control
and the range of the historical negative control data
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(0.3–3.3‰ PCE containing micronuclei).
Comet assay in vivo in the rat lung
The Comet assay for detection of DNA single and
double-strand breaks and alkaline labile DNA sites
was integrated in a subacute five-day lung toxicity
study with inhalation using a five-day inhalation test
method for nanomaterials (Ma-Hock et al. 2007,
2009).
For personal use only.
Animals. Seven-week-old male Wistar Crl:WI Han
rats from Charles River Laboratories, Sulzfeld, Ger-
many, were used. They were acclimatized for at least
five days and kept at 20–24 C at a relative humidity of
30–70% at a day/night rhythm of 12 h light from
06:00– 18:00 h in H-Temp (PSU) cages from TEC-
NIPLAST, Hohenpeißenberg, Germany. Standard-
ized pelleted feed (Provimi Kliba, Kaiseraugst,
Switzerland) was fed and tap water was available
ad libitum.
Generation of the inhalation atmospheres. The test sub-
stance was stirred in its container before a sample for
dust aerosol generation was taken. For each concen-
tration the dust aerosol was generated with a solid
particle generator (brush generator) and compressed
air inside a mixing tube (glass), mixed with condi-
tioned dilution air and passed via a cyclonic separator
(glass) into the inhalation system.
Analytical determination of concentrations. Sampling for
gravimetric analyses was performed using the sam-
pling equipment (Millipore, Schwalbach, Germany)
with an internal probe diameter of 7 mm, a MN 85/90
BF filter (d = 4.7 cm) and a Millipore vacuum pump,
sampling velocity 1.25 m/s, flow rate 3 l/min, sam-
pling site immediately adjacent to the animals’ noses,
Genotoxicity studies on sunscreen nanomaterials 369
sampling frequency as a rule two samples per expo-
sure and concentration group. By means of a vacuum
compressed air pump a defined volume of the dust
aerosol was drawn through the filter. The dust con-
centration was calculated from the difference between
the weight of the filter before and after sampling, with
reference to the sample volume of the inhalation
atmosphere. Scattered light photometers (VisGuard
from Sigrist-Photometer, Unterpleichfeld, Germany)
were used to monitor the constancy of concentrations
of test substance aerosols in the inhalation systems.
Particle size analysis. This was carried out with a
cascade impactor (stack sampler Marple 298 [Sierra],
vacuum compressed air pump [Millipore], limiting
orifice 3 l/min [Millipore], sampling probe internal
diameter 7 mm). Metal collecting discs and a back-
up particle filter were placed into the cascade impactor
and two samples were taken in each concentration at a
sampling velocity of 1.25 m/sec from the breathing
zones of the animals. The amount of dust deposited by
each stage was calculated from the difference between
the weight of the metal collecting disc and back-
up filter before and after sampling. The deposits in
the probe and the wall losses in the impactor were
determined as difference of the total mass increase of
the impactor and the sum of masses on the collecting
discs and backup filter. The particle size distribution
was calculated by the mathematical methods for eval-
uating particle measurements DIN 66141 and DIN
66161. Measurements with a light-scattering spec-
trometer WELAS
2100 (Palas, Karlsruhe, Germany)
were performed to determine the size distribution of
particles with diameters larger than 250 nm. The
measuring range of the sensor was 0.25–10 mm and
the sampling flow rate 5 l/min. For all test groups
10 repeats were measured. To determine the particle
size distribution in the sub-micrometer range, each test
atmosphere was measured with the Scanning Mobility
Particle Sizer (SMPS, Grimm Aerosol Technik, Ain-
ring, Germany) comprising an Electrostatic Classifier
(Model Vienna U-DMA) which separates the particles
into known size fractions and a Condensation Particle
Counter which measures particle count concentra-
tions. The DMA was equipped with Am-241 neutral-
izer. The instrument measures particles in the size
range from 0.011–1.083 mm. Using a conductive sam-
ple hose, the SMPS sampled at 0.3 l/min with a sheath
flow of 3 l/min. At this setting the single-stage, inertial
impactor incorporated into the inlet to remove larger
particles had a 50% cut size of 1.082 mm according to
the software calculation. The sampling duration was
about 7 min. As a rule, nine repeats were measured for
each exposure concentration.
 370 R. Landsiedel et al.
Head-nose exposure of the animals. The inhalation atmo-
sphere was maintained inside aerodynamic exposure
systems (INA 60, volume V » 90 l, BASF SE) con-
sisting of a cylindrical inhalation chamber made of
stainless steel sheeting and cone-shaped outlets and
inlets. The rats were restrained in glass exposure tubes.
Their snouts projected into the inhalation chamber to
inhale the aerosol. A positive pressure was maintained
inside the exposure systems by adjusting the air flow of
the exhaust air system. This ensured that the aerosol in
the breathing zones was not diluted by laboratory air.
To accustom the animals to exposure they were treated
with air under conditions comparable to exposure on
two days before start of exposure (preflow period).
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Then they were exposed for 6 h on five consecutive
days without access to water or feed during the expo-
sure. Recording of exposure parameters was performed
as follows: Supply air and exhaust air by orifice plate
with differential pressure measurement, chamber
humidity by dielectric probe (Testo), chamber tem-
perature by thermosensor, substance flow by reading
from display once per exposure, real time concentra-
tion surveillance by scattered light photometers. The
measurements were transferred to instrumentation
consoles where the values were displayed in an anal-
ogous way (where this is provided for) and some were
used for regulating the specific parameter. The values
For personal use only.
were scanned every 10 sec, converted from analog to
digital, transferred to a personal computer, displayed
on its screen, and saved on hard disk. The computer
checked the arriving values against preset threshold
values, displayed warnings if violations of thresholds
occurred and recorded the start and the end of thresh-
old violations. After the end of the exposure all data
were backed up on optical media.
Clinical examinations. The animals were examined for
evident signs of toxicity twice a day from Mondays to
Fridays and once a day on Saturdays, Sundays and
public holidays. The clinical condition of the test
animals was recorded once during the preflow period
and on post-exposure observation days and before,
during and after exposure on exposure days. The
body weight was determined at the start of the pre-
flow, at the start of the exposure and then, as a rule,
once a week as well as prior to gross necropsy or
lavage. Body weight change was calculated as the
difference between body weight on the respective
day and on the day of the first exposure.
Clinical pathology. The animals were killed by exsan-
guination from aorta abdominalis and vena cava under
Narcoren
anesthesia. The lung was lavaged by two
instillations of physiologic saline. Total cell counts
were determined using a haematology analyzer (Advia
120 Diagnostics; Bayer, Fernwald, Germany). The
following parameters were determined: By cytochem-
istry coupled with flow cytometry (Operator’s Guide
for Advia 120 System): Total cell count; by staining
followed by microscopic evaluation (Warheit and
Hartsky 1993): Macrophages, polymorphonuclear
neutrophils, lymphocytes, eosinophils, monocytes
and atypical cells. An automatic analyzer (Hitachi
917; Roche, Mannheim, Germany) was used to
examine the humoral parameters in the bronchoal-
veolar fluid (BALF) according to Roche working
instructions: By the benzethoniumchloride method:
protein; by kinetic UV test, 340 nm, 37 C: Lactate
dehydrogenase (LDH) (L-Lactate-NAD oxidoreduc-
tase; EC 1.1.1.27) and alkaline phosphatase (ALP)
(ortho-phosphoric acid monoester phosphohydrolase
EC 3.1.3.1.); by kinetic color test, 415 nm, 37 C: g-
glutamyltransferase (GGT) (g-glutamyl) peptide:
aminoacid-g-glutamyltransferase; EC 2.3.2.2.); by
color test, 580 nm, 37 C: N-acetyl-b-D-glucosami-
nidase (NAG) (2- acetamido-2-deoxy-b-D-glucoside
acetamidodeoxyglucohydrolase; EC 3.2.1.30).
Statistics. Means and standard deviations were calcu-
lated. Additionally body weight and body weight
change were evaluated by comparison of each group
with the control group using Dunnett’s test (two-
sided) for the hypothesis of equal means (Dunnett
1955, 1964).
Toxicity pretest. Three rats per test group were head-
nose exposed for five days (6 h/day) to aerosol of
0 (negative controls: conditioned air only), 0.5 mg,
2 mg or 10 mg of T-LiteTM SF per m3. On day 5 from
the start of exposure the animals were sacrificed
and bronchoalveolar lavage was performed. In the
lavage fluid the enzymatic activities of LDH and
ALP were determined. For an additional group of
three rats exposed to the highest concentration (10 mg
of T-LiteTM SF per m3) also by inhalation for five days
(6 h/day) a recovery period of 23 days was allowed
and subsequently the same clinico-pathological
parameters (LDH, ALP) were determined.
Comet assay. The Comet assay was performed with
lung cells after alveolar lavage of these two recovery
groups: 10 mg of T-LiteTM SF per m3 and control
group treated in the same way by conditioned air only.
The lung cells were isolated by means of in situ
perfusion and lavage following the method described
by Brendler-Schwaab et al. (1994). Under anaesthesia

the rib cage of the animals was opened and the lungs
were perfused through the right ventricle. After
complete whitening of the tissue the lungs were
lavaged three times with saline at room temperature
followed by instillation with Williams medium con-
taining collagenase IV (400 U/ml) and trypsin
(0.125%). After removal of the lungs with closed
trachea the lungs were rinsed in washing buffer and
cut in small pieces, which were incubated in Williams
medium containing collagenase IV (400 U/ml) and
trypsin (0.125%) at 37 C for 20 min on a shaker (60–
100 rpm). Then Hanks Balanced Salt Solution
(HBSS) containing 5% FCS was added. The tissue
pieces were gently forced through 60 mesh (230 mm)
Nanotoxicology Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/12/13
and 200 mesh (73.7 mm), consecutively. Then the
cells were centrifuged at 250 g for 5 min (4 C). The
supernatant was carefully removed and the pellet was
re-suspended in washing buffer. The cell suspension
was transferred to 1,040 g/ml Percoll gradient and was
centrifuged a second time at 250 g for 20 min (4 C).
The supernatant was removed and the cells were re-
suspended in washing buffer. The cell density and
viability were determined by trypan blue vital dye-
exclusion (Burlinson et al. 2007) using a Neubauer
cell counter.
For personal use only.
Procedure of the Comet assay. The cell preparation
was carried out according to Tice et al. (2000)
and Singh et al. (1988) under alkaline conditions.
10 ml of the single cell suspension (1–3  104 viable
lung cells per slide) were carefully mixed with 90 ml
low melting agarose and layered on an agarose-
coated slide. After solidification on ice the slides
were transferred to lysis solution (pH 10) for at least
1 h at about 4 C in the dark. The following steps until
neutralization of the slides were carried out sheltered
from daylight to prevent UV light-induced DNA
damage. After lysis the slides were placed randomly
in the electrophoresis chamber (cooled on ice) and
were covered with electrophoresis buffer (pH > 13) for
45 min to allow DNA unwinding. Afterwards the
electrophoresis was conducted (25 V; 300 mA;
0.85 V/cm) for 30 min. Then the slides were trans-
ferred into neutralization buffer for about 10 min.
Subsequently, they were exposed for 1 min to abso-
lute ethanol. Finally, the slides were air-dried and
stored at room temperature until scoring.
Microscopic evaluation. The DNA on the coded slides
(stained with 40 ml ethidium bromide per slide) was
evaluated by fluorescence microscopy (ZEISS Axio-
plan 2, Jena, Germany; 40 objective). The scoring
and evaluation of Comet images (DNA migration)
Genotoxicity studies on sunscreen nanomaterials 371
was carried out via a CCD with an Image Analysis
System (Comet Assay IV, Perceptive Instruments
Ltd, Suffolk, UK). The relative tail intensity was
determined. This parameter is a measure of the rel-
ative fluorescence intensity in the head and the tail.
The relative tail intensity covers a wide range of DNA
damage (from 0–100%) and is independent of the
threshold settings of the Image Analysis program used
(Hartmann et al. 2003; Collins et al. 2008). At least
two slides per test group and 50 images per slide were
analyzed to achieve a total number of at least
100 images per animal. The images were selected
randomly. Each slide scored for DNA damage was
also examined for possible indications of cytotoxicity,
i.e., ‘hedgehog’ images (indication of severe genotoxi-
city, necrosis or apoptosis). A test substance is con-
sidered positive if the mean relative tail intensity of
one dose group clearly exceeds the concurrent neg-
ative control value.
Results
Bacterial reverse mutation assays
T-LiteTM SF, T-LiteTM MAX and Z-COTE
MAX
were tested for their mutagenic potential based on the
ability to induce point mutations in a reverse mutation
assay in Salmonella typhimurium TA 1535, TA 100,
TA 1537, TA 98 and TA 102 at 20–5,000 mg/plate
both with and without metabolic activation (induced
rat liver S9 mix) (see Supplementary Tables S1–S11,
available online).
Since the choice of the vehicle is one of the most
critical steps and has a great impact on the particle size
distribution and the bioavailability of a test com-
pound, different vehicles were used in the standard
plate incorporation test (DMSO) and in the preincu-
bation test (FCS). The test substance preparations
were dispersions in the respective vehicles.
The particle size distribution determined by ana-
lytical ultracentrifugation of the test substance pre-
parations showed in the sample prepared in DMSO
two fractions of particles in the range below 10 mm,
whereas in the sample in FCS only one. We assume
that additionally, some agglomerates with diameters
larger than 10 mm are present, but not recorded.
A good dispersion in the ultrafine fraction is obtained
for the organic solvent DMSO as carrier, because
hydrophobic interaction is avoided. Using FCS the
dispersion contained still a significant amount of
ultrafine particles (diameter <100 nm) including
some non-aggregated primary particles. This is in
strong contrast to the (attempted) dispersion in water
without the protein components, where T-LiteTM SF,
 372 R. Landsiedel et al.
T-LiteTM MAX and Z-COTE
MAX agglom-
erate strongly. The effect is attributed to interface-
active serum proteins that act as wetting and dispers-
ing agents (Schulze et al. 2008). 50 mg material/ml
showed in DMSO a diameter of 60 nm (T-LiteTM
SF), 92 nm (T-LiteTM MAX) and 31 nm (Z-COTE

MAX) (fraction 1) and of 240 nm (T-LiteTM SF),
378 nm (T-LiteTM MAX) and 173 nm (Z-COTE

MAX) (fraction 2); in FCS a diameter of 239 nm
(T-LiteTM SF), 744 nm (T-LiteTM MAX) and
519 nm (Z-COTE
MAX) as determined by analyt-
ical ultracentrifugation.
In the standard plate incorporation assay and in the
preincubation assay in the absence and presence of
Nanotoxicology Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/12/13
S9 mix T-LiteTM SF precipitated clearly from 100 mg
per plate onward, T-LiteTM MAX and Z-COTE

MAX from 2,500 mg per plate onward (see Supple-
mentary Tables S1–S11, available online).
In the tests with T-LiteTM SF a weak bacteriotoxic
effect (slight decrease in the number of his+ rever-
tants) was occasionally observed in the standard plate
test and in the preincubation assay from 2,500 mg
onward in the presence of metabolic activation only
(Supplementary Tables S1b and S3 versus Table S1a
and S2). In the tests with T-LiteTM MAX and
Z-COTE
MAX no bacteriotoxic effect (decrease
in the number of his+ revertants [Supplementary
For personal use only.
Tables S4–S11, available online], reduced his- back-
ground growth, reduction in the titer [data not
shown]) was observed in the standard plate test nor
in the preincubation assay with the vehicles DMSO
and FCS up to the highest concentration tested in the
absence and presence of metabolic activation.
No increase in the number of his+ revertants was
observed with T-LiteTM SF (Supplementary Tables
S1–S3, available online), T-LiteTM MAX (Supplemen-
tary Tables S4–S7) or Z-COTE
MAX (Supplemen-
tary Tables S8–S11, available online) in the standard
plate test (Supplementary Tables S1, S4–S5, S8–S9,
online) nor in the preincubation test (Supplementary
Tables S2–S3, S6–S7, S10–S11, online) using different
vehicles (in the standard plate incorporation test
DMSO, in the preincubation test fetal bovine serum)
with (Supplementary Tables S1, S3, S5, and S7,
online) or without (Supplementary Tables S1, S2,
S4, and S6) the addition of an S9 mix as an exogenous
xenobiotic metabolizing system while the positive con-
trols used led to the expected increases in the mutation
rates (Supplementary Tables S1–S11, online).
T-LiteTM SF, T-LiteTM MAX and Z-COTE

MAX did not show mutagenic activity in the stan-
dard plate incorporation nor in the preincubation
S. typhimurium reverse mutation assay with and with-
out exogenous metabolic activation tested up to
5,000 mg per plate.
Micronucleus formation in vitro
T-LiteTM SF was assessed in V79 cells in vitro for
possible clastogenic or aneugenic activity leading
to the induction of micronuclei either after short-
term exposure for 4 h or continuous exposure for 24 h.
In an initial range-finding cytotoxicity test per-
formed following the method described under Mate-
rials and methods for the main experiment, the cells
were prepared at a harvest time of 24 h (about 2 cell
cycles) after 4 and 24 h exposure time (as was the case
in the main experiment). The pH value and osmo-
larity were not relevantly influenced by the addition of
the test substance preparation to the culture medium.
No cytotoxicity indicated by reduced cell numbers of
below 50% of control was observed up to the highest
concentration recommended by the current guide-
line, i.e., 5 mg/ml. However, clear toxicity indicated
by strongly reduced quality of the cells was observed
from 625 mg/ml onward after 4 h treatment and from
156.3 mg/ml onward after 24 h treatment. In addition,
the occurrence of precipitation on the slides strongly
influenced the toxicity assessment from 625 mg/ml
onward (in culture medium test substance particles
were observed already at 2.5 mg/ml and above at the
end of the exposure period).
Based on these results of the dose range-finding
test, concentrations from 75 up to 300 mg/ml were
evaluated for micronucleus formation in the main
experiment after 4 h exposure to the test substance
and 18.8 up to 75 mg/ml after 24 h exposure time
(Table II).
The particle size distribution determined by ana-
lytical ultracentrifugation showed in the sample pre-
pared in FCS a single fraction of particles. The
dispersion contained still a significant amount of
ultrafine particles (diameter <100 nm) including
some non-aggregated primary particles. This is in
strong contrast to the (attempted) dispersion in water
without the protein components, where T-LiteTM SF
agglomerates strongly. T-LiteTM SF in the vehicle
FCS (50 mg/ml) showed a diameter of 239 nm, in
FCS/MEM (0.6 mg/ml) a diameter of 562 nm as
determined by analytical ultracentrifugation.
No increase in the number of micronucleated cells
was observed in the main experiment after 4 h and
after 24 h exposure to the test substance (Table II).
The values after treatment with the test substance
(0.2–0.7% micronucleated cells) were close to the
concurrent negative control values (0.4% and 0.7%
micronucleated cells for 4 and 24 hours exposure,
respectively). The frequencies of micronucleated
cells in the vehicle controls were clearly within our
historical negative control data range for the V79 cell
line (0.3–1.8% micronucleated cells). The positive

Table II. Micronuclei formation in vitro in V79 cells with T-Lite SF.
Dose mg/mL culture Exposure time Cells in test
0 (only FCS) 4 hours 2000
75 T-Lite SF 4 hours 2000
150 T-Lite SF 4 hours 2000
300 T-Lite SF 4 hours 2000
500 EMS 4 hours 2000
0 (only FCS) 24 hours 2000
18.8 Lite SF 24 hours 2000
37.5 Lite SF 24 hours 2000
75.0 Lite SF 24 hours 2000
500 EMS 24 hours 2000
Nanotoxicology Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/12/13
No S-9 Mix was used. PI = proliferation index; EMS = ethyl methanesulfonate; **p < = 0.01 (one-sided Fisher’s exact test, pairwise comparison
of each dose group with the vehicle control group).
control substance ethyl methanesulfonate (500 mg/ml)
led to the expected increase in the number of cells
containing micronuclei (2.8–5% after 24 and 4 h
exposure, respectively; Table II, compared with
2.6–8.3% in historical controls after these two expo-
sure times).
Also, as expected from the results of the initial dose
range-finding cytotoxicity test, after 4 and 24 h expo-
For personal use only.
sure no cytotoxicity indicated by reduced PI values was
observed up to the highest concentrations scorable for
micronuclei induction (Table II). After both expo-
sure times two-fold higher concentrations applied
(600 mg/ml after 4 h exposure and 150 mg/ml after
24 h exposure) were neither scorable for PI values nor
for micronuclei induction due to strong test substance
precipitation on the slides although an extensive wash-
ing step of the slides at the end of treatment was
included. No growth inhibition indicated by reduced
cell counts in cell culture flasks was observed through-
out the investigated test substance dose range which
exceeded the concentration range which was scorable
for micronuclei formation and for PI value determi-
nation by a factor of two (Table III). The attachment of
the cells to the slides was complete as judged from the
cell morphology (rounded, fibroblast-like cell mor-
phology) up to the highest applied concentrations
albeit the assessment was influenced by the precipita-
tion of the test substance (Table III). The pH value and
the osmolarity were not significantly changed at any
concentration of the test substance.
T-LiteTM SF did not increase the number of cells
containing micronuclei up to the highest scorable
concentrations of the test substance. Therefore
T-LiteTM SF is considered not to be a chromo-
some-damaging or spindle-apparatus-disturbing sub-
stance under the experimental conditions of this study.
Genotoxicity studies on sunscreen nanomaterials 373
Cells with micronuclei % Micronuclei PI
8 0.4 2.75
4 0.2 2.66
14 0.7 2.69
9 0.5 2.69
99 5.0** 2.46
13 0.7 2.87
13 0.7 2.99
9 0.5 2.86
11 0.6 2.78
55 2.8** 1.84
Micronucleus formation in vivo
Z-COTE
HP1 was assessed in NMRI mice in vivo for
possible clastogenic or aneugenic activity. 15 mg,
30 mg or 60 mg Z-COTE
HP1 per kg body weight,
dispersed in pure FCS were administered once i.p. to
five male NMRI mice per dose and exposure time. The
animals were sacrificed and the bone marrow of the
two femora was prepared 24 and 48 h after adminis-
tration in the dose group of 60 mg/kg body weight and
in the vehicle controls. In the test groups of 30 and
15 mg/kg body weight and in the positive control
groups, the 24-h sacrifice interval was investigated
only. After staining of the preparations, 2,000 PCE
per animal (10,000 PCE per group) were investigated
for micronuclei. The NCE with and without micro-
nuclei occurring per 2,000 PCE were also recorded.
After the single administration of 60 mg Z-COTE

HP1 per kg body weight, 1.3‰ PCE containing micro-
nuclei were found after 24 h and 1.0‰ after 48 h. In the
two lower dose groups, rates of micronuclei of 0.9‰
(30 mg/kg group) and 1.2‰ (15 mg/kg group) were
detected after a sacrifice interval of 24 h (Table IV).
These values were not significantly different from
those obtained in the concurrent vehicle controls:
The single i.p. administration of the vehicle FCS led
to 0.9‰ PCE containing micronuclei after the 24-h
sacrifice interval and to 1.2‰ after the 48-h sacrifice
interval (Table IV). Historical controls with the same
vehicle (FCS) were not available, but vehicle control
values using other vehicles (water, DMSO, olive oil,
corn oil) led to rates of micronuclei formation up to
3.3‰ in PCE (mean 1.5‰, standard deviation 0.5‰;
data not shown) well above those obtained in the
present experiment with Z-COTE
HP1. Both posi-
tive control substances, cyclophosphamide for
 374 R. Landsiedel et al.

Table III. Cell count, cell attachment and cell quality upon treatment of V79 cells with T-Lite SF.

Cell count
Dose mg/mL culture Exposure time Number 106/ml % Cell morphology/attachment to slides Quality of cells

0 (only FCS) 4 hours 3.59 100.0 1 E1

9.4 T-Lite SF 4 hours 3.92 109.2 1 E4
18.8 T-Lite SF 4 hours 3.94 109.8 1 E4
37.5 T-Lite SF 4 hours 3.87 107.5 1 R3
75 T-Lite SF 4 hours 3.76 104.7 1 R3
150 T-Lite SF 4 hours 3.91 108.9 1 R3
300 T-Lite SF 4 hours 3.91 108.9 1 R3
600 T-Lite SF 4 hours 3.86 107.5 n.s. N4
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0 (only FCS) 24 hours 4.31 100.0 1 E1

2.4 T-Lite SF 24 hours 3.77 87.5 1 E4
4.7 T-Lite SF 24 hours 3.90 90.5 1 E4
9.4 T-Lite SF 24 hours 3.79 87.9 1 E4
18.8 T-Lite SF 24 hours 3.86 89.6 1 R3
37.5 T-Lite SF 24 hours 4.05 94.0 1 R3
75.0 T-Lite SF 24 hours 4.15 96.3 1 R3
150 T-Lite SF 24 hours 4.17 96.8 1 N4

Harvest time 24 h after start of treatment.

Cell morphology/attachment to slides:
1 = complete attachment, i.e., fibroblast-like cells;
2 = slightly reduced attachment, i.e., few cells rounded;
For personal use only.

3 = reduced attachment, i.e., most cells rounded;

4 = complete detachment, i.e., all cells rounded;
E = evaluation possible;
E1 = sufficient cells of good quality;
E2 = reduced number of analyzable cells;
E3 = reduced number of analyzable cells, some with fragmentations;
E4 = evaluation of the cells is influenced by the precipitation of the test substance;
R = evaluation possible but with reservations;
R1 = considerably reduced number of analyzable cells;
R2 = considerably reduced number of analyzable cells and with fragmentations;
R3 = evaluation is influenced by the precipitation of the test substance;
N = evaluation not possible;
N1 = no or only a few cells for evaluation;
N2 = no or only a few cells for evaluation and with fragmentations;
N3 = complete cell death (lysed cells);
N4 = evaluation of the cells is strongly influenced by the precipitation of the test substance;
n.s. = not scorable due to strong test substance precipitation on the slides.

clastogenicity and vincristine sulfate for spindle poison
effects, led to the expected biologically relevant and
statistically significant increase in the rate of
PCE containing small or large micronuclei (cyclophos-
phamide 14.6‰ containing mainly small micronuclei;
vincristine sulfate 66.2‰, a significant portion
[20.1‰] attributable to large micronuclei) (Table
IV). A slight inhibition of erythropoiesis determined
from the ratio of PCE to NCE was observed at the
highest administered dose of 60 mg Z-COTE

HP1 per kg body weight at the 48-h sacrifice interval
(Table IV). The alteration of this ratio indicates that
the test substance actually reached the bone marrow,
means the target determined for genotoxic effects.
Z-COTE
HP1 did not increase the number of PCE
containing small or large micronuclei. The rate of
micronuclei was close to the range of the concurrent
vehicle control in all dose groups and at all time
intervals and within the range of historical vehicle
control data. Therefore, Z-COTE
HP1 did not
induce the formation of micronuclei in bone marrow
cells of NMRI mice in vivo under the experimental
conditions of this study.
Comet assay in vivo in the rat lung
A standard five-day inhalation study for nanomater-
ials (Ma-Hock et al. 2009) was performed with
 Genotoxicity studies on sunscreen nanomaterials 375

Table IV. Micronuclei formation in vivo in NMRI mouse bone marrow with Z-Cote HP1.

Polychromatic erythrocytes Normochromatic erythrocytes

‰ with MN per animal ‰
Doses mg/kg bw Exposure time [h] n Small Large Total Mean SD With MN

0 (only FCS) 24 10,000 0.8 0.1 0.9 1156 280 0.3

15 Z-Cote HP1 24 10,000 1.2 0.0 1.2 1013 178 1.0
30 Z-Cote HP1 24 10,000 0.9 0.0 0.9 972 235 0.8
60 Z-Cote HP1 24 10,000 1.2 0.1 1.3 1094 132 0.7
20 CPP 24 10,000 14.1** 0.5 14.6** 897 320 1.6
0.15 VCR 24 10,000 46.1** 20.1** 66.2** 1231 182 1.5
0 (only FCS) 48 10,000 1.2 0.0 1.2 754 220 0.0
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60 Z-Cote HP1 48 10,000 0.9 0.1 1.0 1165 707 0.9

SD = standard deviation; bw = body weight; n = total number; MN = micronuclei, Small = MN diameter < 1/4 of cell diameter; Large = MN
diameter ‡ 1/4 of cell diameter. FCS = 10% fetal calf serum; CPP = cyclophosphamide; VCR = vincristine sulphate. **p £ 0.01 (one-sided
Wilcoxon test, pairwise comparison of each dose group with the vehicle control group)

T-LiteTM SF. Wistar rats were exposed by head-nose-
only inhalation for six hours per day on five conse-
cutive days to aerosol of 0 (negative control), 0.5, 2 or
10 mg of T-LiteTM SF per m3. Clinical signs, mean
body weights and mean body weight changes of all test
groups were not statistically significantly different
For personal use only.
The animals were anesthetized and the lung cells
were harvested by in situ perfusion after lavage. After
enzymatic digestion and density gradient centrifuga-
tion the obtained single cells were embedded in
agarose gel, lysed to liberate the DNA and DNA
unwinding single cell gel electrophoresis under alka-

from the control groups. Effects in the lung were
most pronounced two days after the last exposure:
Slight to moderate increases of neutrophils and
monocytes, of total protein and of activities of
LDH, GGT, ALP and NAG in the broncheo-
alveolar lavage fluid were observed at aerosol concen-
trations of 2 and 10 mg/m3. No changes were
seen after exposure to the lowest concentration of
0.5 mg/m3 which therefore is considered the
NOAEC. The effects were partly reversible within
the post-exposure observation period of three weeks:
LDH and ALP activities were still significantly chan-
ged in the animals exposed to the highest concentra-
tion (10 mg/m3) (Table V). Therefore, the lungs of
those recovery animals and the respective control
animals were used for the investigation of DNA single
and double strand breaks and alkali-labile DNA sites
in the alkaline Comet assay (Single Cell Gel
Electrophoresis).
line conditions (pH 13) was performed. The DNA
was stained with ethidium bromide and 100 cell
images per animal were scored for DNA migration
(relative tail intensity) using a fluorescence micro-
scope. Indicators of organ specific toxicity like cell
viability after lung cell preparation and the number of
highly damaged cells (hedgehog images) occurring
per 100 images were also recorded.
For the time being, there are only a small number of
published studies available that describe the in vivo
Comet assay in rat lung cells. The test method is a
newly established method in our laboratory, there-
fore, currently only a small data base for negative
controls of male Wistar rats exists. For the negative
control group low values (mean tail moment range:
0.89–2.13; mean tail length range: 27.0–40.2 mm;
mean tail intensity range: 5.15–8.97%) were obtained
from the three animals that were in the expected range
based on our current experience with the Comet assay

Table V. Body weight and clinical pathology findings at time point chosen for Comet assay in alveolar lavage cells from the lungs of Wistar rats
exposed in vivo to T-lite SF by inhalation.

Concentration, mg/m3 Body weight, g BW change, g LDH, mkat/1 ALP, mkat/1

0 336.6 ± 15.0 81.7 ± 8.3 0.28 ± 0.04 0.47 ± 0.11

10 335.7 ± 50.8ns 80.1 ± 26.6ns 0.47 ± 0.18* 1.09 ± 0.27**
ns
not significant, p > 0.05, Dunnett’s test, two-sided; *p < 0.05, Wilcoxon test, two-sided; **p < 0.01, Wilcoxon test, two-sided.
 376 R. Landsiedel et al.

in Wistar rats in vivo in several tissues. The quality of
the preparation of the lung cells was demonstrated by
high cell viabilities (93% and above) in the negative
control animals (Table VI).
All parameters determined for DNA damage – mean
tail moment (0.6), mean tail length (28.3 mm) and
mean tail intensity (3.04%) – after test substance
inhalation exposure were below the values of the
respective negative control group (mean tail moment:
1.82; mean tail length: 35.2 mm; mean tail intensity:
6.50%). Additionally, the numbers of hedgehog
images when scoring 100 Comet images per animal
were at 15–33% in the negative control group and from
14–23% in the exposed test group indicating that this
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medications used in dental practice, chromosome
aberrations were induced in D824 cells (human den-
tal pulp cells) treated with zinc oxide. All studies used
either non-nano-sized zinc oxide or the size was not
reported. Dufour et al. (2006) reported that uncoated
zinc oxide particles of a mean diameter of 100 nm
induced chromosome aberrations in vitro (slightly
increased by UV light; called ‘pseudo-photo-
clastogenicity’).
Up to now, there is only a limited number of data
published on the genotoxicity of nano-scale zinc
oxide. Numerous studies with titanium dioxide
(nano- and pigmentary/micron-scale materials) have
been published in the scientific literature. The

parameter was not influenced by test substance
administration.
As shown in Table VI, the exposure by inhalation to
10 mg T-LiteTM SF per m3 compared with the
negative control did not lead to an increase in the
tail intensity, nor in the tail length, nor in the tail
moment, nor in the hedgehogs formed indicating that
there is no DNA damage in the rat lung in vivo upon
inhalation of T-LiteTM SF three weeks after the last
exposure.
For personal use only.
majority of the test results found no genotoxic or
photo-genotoxic effects using standard test protocols
(Theogaraj et al. 2007; see for review Nohynek et al.
2008). No major differences in the hazard profile was
observed for micro- and nano-structured particles,
and no evidence was found suggesting that nano-
structured particles pose greater or qualitatively
new mutagenic/genotoxic hazards. Other authors,
however, reported somewhat contradictory results
for uncoated titanium dioxide.
With ordinary titanium dioxide, no induction of
chromosomal aberrations, sister chromatid exchanges
(SCEs), or micronuclei was observed in Chinese

Discussion
The Scientific Committee on Cosmetic Products and
Non-Food Products Intended for Consumers opinion
concerning zinc oxide (SCCNFP 2003) reviewed the
genotoxicity data of zinc oxide and concluded that
‘micronized zinc oxide has been demonstrated to be
photoclastogenic, possibly photo-aneugenic, and a
photo-DNA damaging agent in mammalian cells cul-
tured in vitro. The relevance of these findings needs to
be clarified by appropriate investigations in vivo.’ In a
more recent study by Someya et al. (2008) on
hamster ovary (CHO) cells in vitro (Ivett et al.
1989; Miller et al. 1995). However, Lu et al.
(1998) obtained a dose-dependent increase in both
micronuclei and SCEs in Chinese hamster ovary
CHO-K1 cells.
Genotoxicity studies on titanium dioxide nanoma-
terial available in the literature (presented here
below) show a variety of positive and negative results
highlighting the necessity: (a) To ascertain the
genotoxicity of the individual titanium dioxide nano-
material, and (b) to document precisely both the exact

Table VI. Comet assay in alveolar lavage cells from the lung of Wistar rats exposed in vivo to T-lite SF by inhalation.

Tail intensity Tail length Tail moment

Concentration, mg/m 3
Viability, % Hedge hogs, % Mean, % Median, % Mean, mm Median, mm Mean Median

0 93 15 5.15 1.39 38.28 34.94 0.89 0.19

0 95 24 8.97 2.49 40.22 32.64 2.13 0.26
0 98 33 5.39 1.21 27.00 25.94 0.83 0.11
Average 95 24 6.50 1.70 35.17 31.17 1.82 0.19
10 92 16 4.42 0.69 26.28 23.85 0.76 0.07
10 78 23 2.99 0.34 34.20 30.54 0.88 0.06
10 96 14 1.70 0.26 24.48 24.27 0.16 0.02
Average 88.7 18 3.04 0.43 28.32 26.22 0.60 0.05

Data from each of the three animals per exposure and per control group are given. Data represent at least 100 images per animal.

nature of this material as well as the exact procedure
of the genotoxicity investigation. Ultrafine titanium
dioxide (diameter £20 nm) was reported to induce
micronuclei in Syrian hamster embryo cells in vitro
when fine titanium dioxide (diameter ‡200 nm) did
not (Rahman et al. 2002). Nanosized (10 and 20 nm)
anatase induced DNA damage (alkaline Comet assay
positive with and without FPG), micronuclei (micro-
nucleus assay with cytochalasin B), lipid peroxidation,
and the formation of hydrogen peroxide and nitric
oxide in human bronchial epithelial BEAS 2B cells,
while anatase of larger particle size (200 nm
and >200 nm) did not; however, also 200 nm-sized
rutile produced oxidative DNA damage and H2O2
Nanotoxicology Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/12/13
(Gurr et al. 2005). Ultrafine titanium dioxide (particle
size distribution by volume 6.57 nm:100%, and by
intensity 8.2 nm: 80.4% and 196.5 nm:19.4%) also
increased micronuclei (determined by the micronu-
cleus assay with cytochalasin B), nucleoplasmic
bridges, DNA damage (alkaline Comet assay without
FPG), and HPRT mutations in human B-cell lym-
phobalastoid WIL2-NS cells in vitro (Wang et al.
2007). In contrast, a recent study by Bhattacharya
et al. (2009) reports that titanium dioxide (anatase,
diameter <100 nm) did not induce DNA strand
breaks and/or alkali-labile sites as determined by
the alkaline Comet assay (without FPG) in human
For personal use only.
bronchial epithelial BEAS 2B cells or in IMR-90
human lung fibroblasts, but did induce the formation
of oxidative DNA damage (8-oxo-guanine) in the
IMR-90 cells (in BEAS 2B cells not determined)
which may suggest that where greater genotoxicity
of nano-sized material is observed as compared with
larger scale material, this may be secondary to oxida-
tive stress and inflammation (see also Oberdörster
et al. 1994a, 1994b). Indeed, in RAW 264.7 macro-
phages ultrafine titanium dioxide (Aeroxide TiO2
P-25, primary particle diameter 21 nm) enhanced
intracellular generation of reactive oxygen species to
a greater extent than fine titanium dioxide (primary
particle size 1 mm); ultrafine titanium dioxide induced
ERK1/2 activation, while the fine titanium dioxide-
induced activation was minimal; the potency of ultra-
fine titanium dioxide to enhance tumor necrosis
factor-alpha and macrophage inflammatory protein-
2 secretion was higher than that of fine titanium
dioxide; all of this suggesting greater biological activ-
ity of ultrafine titanium dioxide as measured by these
endpoints (Kang et al. 2008). Also, intranasal expo-
sure of mice to ovalbumin in combination with fine
(250 and 260 nm) or ultrafine (29 and 14 nm) tita-
nium dioxide showed that exposure to ultrafine tita-
nium dioxide induced airway inflammation, and
had immune adjuvant activity as demonstrated by
increasing the peribronchial lymph nodes cell
Genotoxicity studies on sunscreen nanomaterials 377
numbers and the production of ovalbumin-specific
T-helper type 2 cytokines (IL-4, IL-5, IL-10 and
IL-13). Also ovalbumin-specific IgE and IgG1 levels
in serum were only increased in animals exposed to
the ultrafine titanium dioxide (de Haar et al. 2006).
In contrast, a study by Veranth et al. (2007) which
measured cytotoxicity and the release of the proin-
flammatory cytokines IL-6 and IL-8 by human
bronchial epithelial cells treated with manufactured
nano- and micron-sized particles of titanium dioxide
(5–6 nm anatase versus 1–2 mm rutile) (as well as
Al2O3, CeO2, Fe2O3, NiO and SiO2) (and also soil-
derived particles from dust) observed that the nano-
sized particles were not consistently more potent than
micron-sized particles of the same nominal composi-
tion for the induction of IL-6 and IL-8 secretion in the
in vitro models used, albeit for titanium dioxide the
nanosized material was more active as compared with
the microsized material (interestingly the manufac-
tured oxides were much less potent than natural
PM2.5 particles derived from soil dust). The nano-
sized particles caused artifacts in the measurement of
IL-6 due to adsorption of the cytokine on the high-
surface-area particles. The metal oxide particles had
low potency to induce IL-6 secretion in BEAS-2B
cells suggesting that manufactured metal oxide nano-
particles are not highly toxic to lung cells compared to
environmental particles. Moreover there are also
reports that ultrafine titanium dioxide (P25, uncoated
anatase as well as UV-TITAN MI60 [rutile coated by
aluminium hydroxide and stearic acid, before expo-
sure washed with ethanol to remove the stearic acid in
order to make the particles hydrophobic and suspen-
sable], both with an average crystal size of 20 nm and
both with and without UV treatment) did not increase
micronuclei (as determined by the micronucleus
assay with cytochalasin B in rat liver epithelial cells)
(Linnainmaa et al. (1997). Moreover, ultrafine tita-
nium dioxide (79% rutile, 21% anatase, median par-
ticle size 140 nm in water, neutralization of acidic
chloride groups on the particle surface) was negative
in the chromosomal aberration test in CHO cells
(Warheit et al. 2007). Thus beside variations in the
test systems and conditions also simple considerations
of properties such as particle size or form of titanium
dioxide (e.g., anatase versus rutile) appear not to
allow predictions on whether a titanium dioxide nano-
material will be genotoxic in vitro or not, therefore it is
essential that the properties of the tested material are
exactly documented as was done in this study.
Instillation of titanium dioxide P25 nanomaterial
(hydrophilic surface) and trimethoxyoctylsilane-
coated titanium dioxide T805 (hydrophobic), both
with primary particle diameter of approximately
20 nm, did not induce DNA damage in lung cell
 378 R. Landsiedel et al.
DNA of rats in vivo (Rehn et al. 2003). DNA damage
was assessed by immunohistochemical detection of
8-oxoguanine in lung sections of the rats; in quartz-
exposed animals a significant increase in the amount
of 8-oxoguanine in DNA of lung cells was detected.
When coupled with UV irradiation, ultrafine tita-
nium dioxide (P-25 anatase, diameter 21 nm) was
clearly more photogenotoxic than coarse titanium
dioxide (anatase and rutile, both 255 nm by diameter)
in mouse lymphoma L5178Y cells, as measured by
the alkaline Comet assay (Nakagawa et al. 1997).
Rutile of larger particle size (420 nm) was not photo-
genotoxic. The P-25 anatase nanomaterial was also
photogenotoxic in Chinese hamster lung CHL/IU
Nanotoxicology Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/12/13
cells, when assessed by chromosome aberration
induction, but not in Salmonella typhimurium or in
mouse lymphoma L5178Y tk+/- cells, when studied
for mutation induction, and it did not induce DNA
damage (alkaline Comet assay), chromosome aberra-
tions or mutations without UV radiation (Nakagawa
et al. 1997). Titanium dioxide samples (0.0125% w/v
titanium dioxide diameter 20–50 nm) extracted from
over-the-counter sunscreens showed positive results
in MRC-5 human fibroblasts in presence of UV-
light as determined by the alkaline Comet assay (tita-
nium dioxide without illumination was negative).
Also illumination of 0.025% w/v titanium dioxide
For personal use only.
mixed with supercoiled plasmids DNA with simu-
lated sunlight showed in agarose gel electrophoresis
that plasmids were converted to the relaxed form and
to the linear form, demonstrating strand breakage.
Sunlight alone had very little effect, 100% anatase
standard was more active than 100% rutile standard
(Dunford et al. 1997). Chen et al. (2007) reported
that irradiation by UV light of titanium dioxide nano-
particles (actual size not specified) led to oxidative
DNA damage determined by an electrochemical
method. Hidaka et al. (1997) reported on damage
to DNA and RNA visualized by scanning micro-
graphs after UV irradiation of commercial P25 tita-
nium dioxide. However, Theogaraj et al. (2007)
reported that none of eight different forms of titanium
dioxide induced increases in the frequency of chro-
mosome aberrations in CHO-WBL cells treated with
or without UV irradiation from a solar simulator with
a temperature of the irradiated area of 35 C main-
tained by a SunCool
unit and under removal of
wavelengths <290 nm by a glass filter (the tested
materials: A, B and C: crystal type anatase 80%, rutile
20%, primary particle 21 nm; A: coating trimethoxy
caprylylsilane; B: no coating, doped with 2% di-iron
trioxide; C: no coating; D: 100% rutile, primary
particle 14 nm, coating 8–11% alumina, 1–3%
simethicone; E: 100% anatase, aggregate size
60 nm, coating 37% alumina, 12–18% silica; F–H:
100% rutile, F: primary particle 20 nm, coating
5–6.5% alumina, 1–4% dimethicone; G: primary
particle 15 nm, coating 3–8% alumina, 5–11% stearic
acid; H: primary particle 20–22, coating 10.5–12.5%
alumina, 3.5–5% silica; the primary particles in dis-
persions typically forming aggregates of 30–150 nm).
To the best of knowledge of the present authors,
titanium dioxide (whether nano-sized or larger)
was always negative in bacterial mutation assays
(Landsiedel et al. 2009).
The Japanese risk assessment of titanium dioxide
nanomaterials remarked that most of the positive
responses in genotoxicity tests of titanium dioxide
have been reported in in vitro DNA damage assays
and in the in vitro micronucleus test. Since different
experimental conditions have always been used, it is
difficult to comprehensively examine the relationship
between the test results and experimental parameters
such as, e.g., particle size or irradiation. Therefore
they propose that for clarification of the genotoxicity
of titanium dioxide it is necessary to perform in vivo
genotoxicity tests and to evaluate genotoxicity by a
standard genotoxicity testing battery covering a wide
range of mechanisms (New Energy and Industrial
Technology Development Organization [NEDO]
2009).
Following this recommendation, we used of a bat-
tery of standard genotoxicity testing methods, includ-
ing in vivo methods, covering a wide range of
mechanisms. Application of these standard methods
to nanomaterials demands, however, several adapta-
tions in substance dispersion for application and
characterization of the test items. Early genotoxicity
studies with nanomaterials were often deficient in
sufficient preparation and characterization of the
test items. And many genotoxicity investigations pub-
lished in the open literature employed only one gen-
otoxicity method; in some cases without proper
control for cytotoxic effects. And for most in vitro
studies correlating in vivo data are missing.
In the present paper, four inorganic nanomaterials
were investigated under GLP conditions for potential
genotoxicity in vitro (generally more sensitive) and
in vivo (generally reflecting more closely the risk to
human) by tests recognizing DNA damage, gene
mutations and chromosome mutations.
No increase in genotoxicity induced by any of the
investigated nanomaterials was observed in a battery
of OECD guideline tests as well as in a modified
in vivo Comet assay, determining possible DNA
damage in the lungs after inhalation exposure.
The lack of an increase of revertant colonies in the
Salmonella reverse mutation assay is in agreement with
and also adds to the limited experience gained in this
test with nanomaterials so far. A recent review

(Landsiedel et al. 2009) reported that of the only six
Ames test studies found in the open literature at the
time of writing the review almost all were negative
(only one Ames test was positive, only weakly
positive and in a single strain; investigated material:
Water-soluble FePt nanoparticles capped with
(CH3)4NH4OH [Maenosono et al. 2007], while the
other five Ames tests were negative). Also, in a very
recent bacterial reverse mutation test on water-soluble
zinc oxide nanoparticles capped with tetramethylam-
monium hydroxide using Salmonella typhimurium
TA98, TA100, TA1535 and TA1537, and Escherichia
coli WP2uvrA(-), with and without metabolic activa-
tion by S9 mix in the pre-incubation method, no
Nanotoxicology Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/12/13
mutagenicity was observed (Yoshida et al. 2009).
The genotoxicity tests which so far have most
frequently and successfully been used for genotoxicity
testing of nanomaterials were the Comet assay
(19 studies available in the open literature at the
time of writing the Landsiedel et al. 2009 review)
and the micronucleus test (14 studies available at the
time of writing the Landsiedel et al. 2009 review). The
nanomaterials tested in the present study were clearly
negative in both of these tests which provides well-
founded confidence on their lack of genotoxicity,
since most of the genotoxicity tests on nanomaterials
reported in the literature using any of these two
For personal use only.
methods were positive: Comet assay: Of the reported
19 studies 14 with positive outcome; micronucleus
test: of the reported 14 studies 12 with positive out-
come (Landsiedel et al. 2009). The confidence is still
increased by the fact that in the present study one
micronucleus test was performed in vitro (titanium
dioxide T-LiteTM SF in V79 Chinese hamster cells),
the other micronucleus test was performed in vivo
(zinc oxide Z-COTE
HP1 in bone marrow cells of
NMRI mice) and the Comet assay was performed
in vivo in another species (broncho-alveolar fluid of
Wistar rats exposed by inhalation to aerosol of tita-
nium dioxide T-LiteTM SF under well-controlled
conditions assuring arrival of most of the T-LiteTM
SF in the deep lung alveolar region).
Our results are in contrast to the just recently
published study of Trouiller et al. (2009), which
reports DNA damage as well as genetic instability
in mice after application of nanoscale titanium diox-
ide in the drinking water. The authors discussed that
the genotoxic effect might be associated with inflam-
mation and/or oxidative stress. In this study P25, a
material with a different crystal structure (75% ana-
tase) was tested, known to have a catalytic reactivity.
Another critical aspect in this study is the application
in drinking water, knowing that the material used in
the study sediments very fast (Wiench et al. 2009),
therefore, the tested doses might be much higher than
Genotoxicity studies on sunscreen nanomaterials 379
those described in the publication calculated via the
drinking water consumption. Trouiller et al. dis-
cussed the genotoxic effects as secondary genotoxic
mechanism associated with inflammation and/or oxi-
dative stress, whereas our inhalation study with a
coated non-catalytic material examined DNA damage
after a recovery period when most of the inflammatory
response subsided. This recent example again empha-
sizes the necessity of carefully describing the exact test
conditions and of looking at the genotoxic effect of
the individual nanomaterials under the specific test
conditions.
Thus, the results of this study: (i) Indicate that the
investigated nanomaterials are devoid of genotoxicity
in a range of standard testing methods covering var-
ious endpoints and mechanisms, and (ii) provide
congruent results obtained in a battery of OECD
standard genotoxicity test methods applied to nano-
materials complemented by appropriate procedure
for the preparation and characterization of the
nanomaterials.
Acknowledgements
The authors thank ECNIS (Environmental Cancer,
Nutrition and Individual Susceptibility), a network of
excellence operating within the European Union 6th
Framework Program, Priority 5: Food Quality and
Safety (Contract no. 513943) for support.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper. How-
ever, Robert Landsiedel, Lan Ma-Hock, Ben van
Ravenzwaay, Stefan Schulte, Markus Schulz,
Samantha Champ, Karin Wiench and Wendel Wohl-
leben are employees of BASF SE, a company mar-
keting nano-scale titanium dioxide and zinc oxide.
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