TOXICOLOGICAL SCIENCES 123(1), 264280 (2011)

doi:10.1093/toxsci/kfr148
Advance Access publication June 3, 2011

Safety Evaluation of Sunscreen Formulations Containing Titanium

Dioxide and Zinc Oxide Nanoparticles in UVB Sunburned Skin: An
In Vitro and In Vivo Study
N. A. Monteiro-Riviere,*,1 K. Wiench, R. Landsiedel, S. Schulte, A. O. Inman,* and J. E. Riviere*
*Center for Chemical Toxicology Research and Pharmacokinetics, North Carolina State University, Raleigh, North Carolina 27606; BASF SE, GUP/PB-Z470,
67056 Ludwigshafen, Germany; and BASF SE, Care Chemicals, 67056 Ludwigshafen, Germany

1
To whom correspondence should be addressed at Center for Chemical Toxicology Research and Pharmacokinetics, North Carolina State University,
4700 Hillsborough Street, Raleigh, NC 27606. Fax: (919) 513-6358. E-mail: nancy_monteiro@ncsu.edu.

Received April 22, 2011; accepted May 22, 2011

Downloaded from http://toxsci.oxfordjournals.org/ by guest on February 9, 2016

Sunscreens containing titanium dioxide (TiO2) and zinc oxide
(ZnO) nanoparticles (NP) are effective barriers against ultraviolet
B (UVB) damage to skin, although little is known about their
disposition in UVB-damaged skin. Pigs were exposed to UVB that
resulted in moderate sunburn. For in vitro studies, skin in flow-
through diffusion cells were treated 24 h with four sunscreen
formulations as follows: 10% coated TiO2 in oil/water (o/w), 10%
coated TiO2 in water/oil (w/o), 5% coated ZnO in o/w, and 5%
uncoated ZnO in o/w. TiO2 (rutile, crystallite) primary particle
size was 10 3 50 nm with mean agglomerates of 200 nm (range ca.
90nm460nm); mean for ZnO was 140 nm (range ca. 60200nm).
Skin was processed for light microscopy, scanning (SEM) and
transmission electron microscopy (TEM), and time-of-flight
secondary ion mass spectrometry (TOF-SIMS). UVB-exposed
skin had typical sunburn histology. TEM showed TiO2 NP 17
layers into stratum corneum (SC), whereas ZnO remained on the
surface. TOF-SIMS showed TiO2 and ZnO epidermal penetration
in both treatments. Perfusate analyzed by TEM/energy dispersive
x-ray spectroscopy or inductively coupled plasma mass spectrom-
etry detected no Ti or Zn, indicating minimal transdermal
absorption. In vivo, skin was dosed at 24 h occluded with
formulations and at 48 h. TiO2 NP in o/w formulation penetrated
13 layers into UVB-damaged SC, whereas only 7 layers in normal
skin; TiO2 in w/o penetrated deeper in UVB-damaged SC. Coated
and uncoated Zn NP in o/w were localized to the upper one to two
SC layers in all skin. By SEM, NP were localized as agglomerates
in formulation on the skin surface and base of hair. 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.
Key Words: sunscreens; sunburn skin; titanium dioxide (TiO2);
zinc oxide (ZnO); nanoparticles; skin penetration.
Skin cancer is the most common form of cancer in the United
States with more than 1 million cases diagnosed annually in the
The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For permissions, please email: journals.permissions@oup.com
form of basal cell and squamous cell carcinomas associated with
solar ultraviolet (UV) radiation (American Cancer Society, 2009;
Armstrong and Kricker, 1993). The incidence of melanoma skin
cancer is rising significantly and is the most common cancer
among 25- to 29-year-olds (National Cancer Institute, 2006).
Sunscreen use is recommended to prevent skin cancer, sunburn,
photoaging, and skin wrinkles. Traditional sunblocks incorpo-
rate chemicals that require constant reapplication and may cause
skin irritation. Zinc oxide (ZnO) lotions provide a longer lasting
nonirritating broad spectrum physical sunblock. However, large
ZnO particles result in a white barrier that is not cosmetically
appealing. Currently, nanoscale titanium dioxide (TiO2) and
micronized ZnO nanoparticles (NP) less than 100 nm have been
incorporated in sunscreens to provide effective broad spectrum
physical sunblock.
Borm et al. (2006) estimated that global production of TiO2
and ZnO for use in skincare products was 1000 tons in 2003
and 2004. A 2007 survey by iVillage found that 11% of
respondents used some type of sunscreen every day, whereas
59% used a sunscreen occasionally (Skin Cancer Foundation,
2007). Extrapolation of this data suggests that 33 million
people in the United States use a sunscreen product daily and
177 million apply sunscreen occasionally. With this wide-
spread use and the potential for TiO2 or ZnO NP exposure in
sunscreens, concerns have focused on their possible penetration
through the stratum corneum (SC) skin barrier to contact viable
keratinocytes. Often sunscreens are applied on water-soaked
skin or severely burned skin in which the integrity of the SC
may be impaired. The European Union (EU) Scientific
Committee on Cosmetics and Non-Food Products (SCCNFP,
2000) rendered an opinion on the safety of TiO2, finding no
evidence of in vitro or in vivo skin penetration of NP. However,
the safety of nanomaterials in cosmetic products issued by the
EU Scientific Committee on Consumer Products in 2007 stated
that a safety assessment of nanosized TiO2 should take into
account abnormal skin conditions and the possible impact of
 SAFETY EVALUATION OF SUNSCREENS IN SUNBURN SKIN
mechanical effects on skin penetration (SCCP, 2007). An
earlier opinion on ZnO found a lack of reliable data on the
percutaneous absorption of micronized ZnO (SCCNFP, 2003).
A number of in vitro and in vivo studies have investigated
the penetration of TiO2 and ZnO NP found in sunscreen
formulations through normal skin. Dussert and Gooris (1997)
studied water/oil (w/o) emulsions of TiO2 and ZnO on excised
human skin and found that the emulsion remained on the
surface of the SC; however, TiO2/ZnO formulations (80200
nm, up to 1 lm) applied to in vitro porcine skin for 24 h
showed no penetration (Gamer et al., 2006). Absorption studies
with in vitro and in vivo human skin (20 nm TiO2 in a w/o
emulsion for 5 h) showed no penetration into the viable
epidermal layers (Mavon et al., 2007). TiO2 oil/water (o/w)
emulsions applied to in vitro human organotypic cultures for
24 h and to in vivo human forearms for 6 h (Bennat and
Muller-Goymann, 2000) showed that penetration was greater
in vitro than in vivo, but TiO2 tape stripping indicated that
particles remained within SC layers. Similar studies with
hydrophobic (100 nm), amphiphilic (1015 nm), and hydro-
philic (20 nm) micronized TiO2 in o/w emulsions applied
nonocclusive to human forearms for 6 h showed formation of
a thin film on the SC (Schulz et al., 2002), and neither particle
shape, formulation, nor exposure had a significant impact on
penetration. Human foreskin grafted onto severe combined
immunodeficiency (SCID) mice treated with hydrophobic
emulsions of occluded micronized TiO2 and analyzed by
particle induced x-ray emission (PIXE) and scanning trans-
mission ion microscopy tracked Ti particles to the stratum
granulosum (SG) layer of the epidermis (Kertesz et al., 2005).
Alternatively, Kiss et al. (2007) showed that no TiO2 NP
penetrated intact skin of SCID models. Lekki et al. (2007)
applied TiO2 to porcine and human skin by gentle rubbing and
found Ti in the upper three to five layers of the SC and to
a depth of 400 lm in hair follicles by PIXE, concluding that
mechanical movement (tensile and compressive) played a role
in penetration. Studies by Lademann et al. (1999) reported
TiO2 within some follicular orifices, but most remained on the
skin surface. In contrast, studies conducted 824 h in vivo
porcine skin by Menzel et al. (2004) suggested that hair
follicles were not important but detected four different
formulations of TiO2 on the SC and in the SG using ion beam
analysis. Tan et al. (1996) applied micronized TiO2 in older
patients for 931 days and detected insignificant levels of Ti in
the dermis.
In vitro static diffusion cell (Cross et al., 2007; Zvyagin
et al., 2008) and in vivo (Zvyagin et al., 2008) studies with
human skin treated with ZnO (2630 nm) formulations found
that NP remained with the SC by multiphoton microscopy.
Normal and damaged (tape stripped) skin from pigs exposed to
four different types of TiO2 (35250 nm) in vitro for 24 h in
Franz cells showed no Ti penetration in the receptor fluid
(Senzui et al., 2010). Three different TiO2 formulations applied
daily for 22 days to minipigs showed localization of TiO2 in
265
the upper SC and follicular lumen, with isolated TiO2 within
the dermis with all three formulations (Sadrieh et al., 2010).
Finally, a 5-day study with 68ZnO particles (19 nm, average
agglomeration size of 110 nm) in o/w formulation applied to
the backs of human skin depicted small increases of 68Zn in the
blood and urine samples. This stable enriched isotope found in
the samples suggested that Zn from the sunscreen formulations
was absorbed; however, it is not known if 68Zn was absorbed
as ZnO particles or soluble Zn (Gulson et al., 2010).
Currently, only a few studies have been conducted with TiO2
or ZnO NP applied to compromised skin as would occur when
sunscreen was reapplied to sunburned skin. If the integrity of
the skin barrier is breached either by mechanical, physical, or
chemical damage, the rate-limiting barrier is disrupted, and the
potential for many chemicals as well as very small particles to
cross the SC lipid barrier increases (Monteiro-Riviere and
Baroli, 2010). In addition, solvents, hydration, and surfactants
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can influence the barrier function (Monteiro-Riviere, 2008).
Studies in healthy and psoriatic skin showed that Ti deeply
penetrated the disorganized SC of psoriatic skin relative to
normal skin but did not reach the viable layers of the epidermis
(Pinheiro et al., 2007). However, little data are available
regarding the penetration of TiO2 and ZnO NP found in
sunscreen formulations in skin damaged by UV radiation.
The objective of this study was to assess the absorption and
penetration of commercially available TiO2 and ZnO in
sunscreen formulations in ultraviolet B (UVB)-damaged skin
in both in vitro flow-through diffusion cells and in vivo porcine
skin using multiple detection modalities.
MATERIAL AND METHODS
Ultrastructure and composition of sunscreen formulations. Four hydro-
phobic and hydrophilic sunscreen formulations containing commercially avail-
able TiO2 and ZnO products (BASF, Ludwigshafen, Germany) were used in this
study: CM 630 (10% TiO2 [T-Lite SF] in o/w formulation), CM 634 (10% TiO2
[T-Lite SF] in w/o formulation), CM 643 (5% ZnO [Z-COTE HP1] in o/w
formulation), and CM 644 (5% ZnO [Z-COTE] in o/w formulation). CM 630 and
CM 634 consist of TiO2 (rutile, crystallite of 1416 nm) coated with hydrated
silica, dimethicone/methicone copolymer, and aluminum hydroxide for a primary
particle size of 10 3 50 nm (estimated by scanning electron microscopy [SEM])
and specific surface area of 100 m2/g. The mean size of the agglomerates was
200 nm with a range ca. 90460 nm. CM 643 consists of ZnO coated with
triethoxycaprylylsilane for a mean size of 140 nm (weight based) with a range
between ca. 60200 nm and specific surface area of 1224 m2/g, whereas CM
644 consists of uncoated zinc oxide with a mean size of 140 nm (weight based,
range ca. 60200 nm) and a specific surface area of 1224 m2/g. Particle size
distributions of ZnO were determined by laser diffraction.
For NP sizing in the formulations, each viscous sunscreen was diluted in
ultrapure water (ca. 1:200) and vortexed to provide a suspension. In addition, CM
634 was sonicated for 40 min to facilitate suspension. A suspension of 8 ll was
placed onto formvar-coated carbon grids and allowed to air-dry. Samples were
imaged on an FEI/Philips EM208S TEM operating at an accelerating voltage of
80 KV. In addition, samples were analyzed by energy-dispersive X-ray
spectroscopy (EDS) on a Hitachi HF2000 FE transmission electron microscope
equipped with an Oxford Instruments INCA EDS operating at an accelerating
voltage of 200 KV.
266 MONTEIRO-RIVIERE ET AL.
UV exposure to in vivo pig skin. Experiments were conducted on
weanling white Yorkshire pigs approximately 2030 kg in accordance with the
guidelines prepared by the Institute of Laboratory Animal Resources (1996).
Animals, acclimated for 5 days prior to UVB exposure, were housed in an
Association for Assessment and Accreditation of Laboratory Animal Care
accredited facility on an elevated floor and provided water ad libitum and 15%
protein pellets. The pigs were sedated by an im injection of a telazol-ketamine-
xylazine (TKX) cocktail and the hair on the back was clipped with electric
clippers. Skin was exposed to UVB as previously reported (Lin et al., 2003,
2005). UV radiation source was a 1000 W lamp solar stimulator (Lightning
Cure 200; Hamamatsu, Japan) combined with a dichroic mirror assembly to
reflect the visible and infrared emission and fitted with a 1-mm WG295 Schott
selective UVB band-pass filter to eliminate wavelengths less than 295 nm. This
delivers UV to the skins surface through a 1 cm diameter liquid light guide at
an intensity of 5 mW/cm2 of UVB and 40 mW/cm2 UVA as measured by an
IL1400 radiometer (International Light, Newburyport, MA). UVB was the
dominant wave band, and to simplify terminology, the UV radiation will be
referred to as UVB in the manuscript.
Determination of minimal erythemic dose. To determine a minimal
erythemic dose (MED) for each pig, the skin along the midline of the back was
sequentially exposed within a template to the following UVB doses (mJ/cm2):
30 (6 s), 40 (8 s), 50 (10 s), 60 (12 s), 70 (14 s), 80 (16 s), 90 (18 s), 100 (20 s),
110 (22 s), and 120 (24 s). The MED was determined to range between 40 and
50 mJ/cm2, which was consistent with historical studies in our laboratory.
Exposed sites were analyzed after 24 h to determine the UVB dose required to
produce a 2 erythema (ranging from 100 to 120 mJ/cm2) on the skin of each
pig (n 4 pigs), which is considered a moderate erythemic dose. Pigs were
sedated with TKX, and multiple sites on the back were exposed to the UVB
dose that caused a consistent 2 erythema, a pale red irritation within a defined
area of skin (Draize et al., 1944). Then 24 h later, the pig was sedated with
TKX, the sites visually scored for 2 erythema, and the skin prepared for the
in vitro or in vivo studies.
In vitro flow-through diffusion cells. Pigs were euthanatized via iv
injection into the ear vein with Euthasol (390 mg/ml of pentobarbital sodium
and 50 mg of phenytoin sodium), and exposed and unexposed skin sites were
dermatomed to 400 lm. Dermatomed skin, placed dermis side down on towels
saturated with physiological saline, was cut into with a 19 mm circular punch.
Skin was mounted in flow-through diffusion cells maintained at 37C with
a dosing area of 0.64 cm2 and equilibrated in perfusate (1.2mM KH2PO4,
32.7mM NaHCO3, 2.5mM CaCl2, 4.8mM KCl, 1.2mM MgSO4, 118mM NaCl,
1200 mg/l D-glucose, 4.5% bovine serum albumin, 5 U/ml heparin, 30 lg/ml
amikacin, and 12.5 U/ml penicillin G; pH 7.37.5) at a flow rate of 2 ml/h for
30 min prior to dosing. Perfusion was halted and skin was (n 16 with UVB-
exposed skin sites; n 8 with unexposed skin sites) dosed with 50 ll of the
four sunscreen formulations (n 4 replicates of diffusion cells per formulation)
and control sites (n 4 of UVB-exposed and normal skin without sunscreen).
Upon completion of dosing, perfusion was resumed, and the perfusate was
collected every 2 h for the first 12 h, then every 4 h up to 24 h. After 24 h,
perfusion was terminated, the skin was removed from the diffusion cells, and
the dose site was removed with an 8-mm biopsy punch and cut into thirds. Skin
was placed in Trumps fixative for light microscopy (LM) and for transmission
electron microscopy (TEM) and stored at 4C. The remaining skin was
immediately frozen in liquid nitrogen and stored at
20C for time-of-flight
secondary ion mass spectrometry (TOF-SIMS). Perfusate samples from each
timed collection were capped and stored at 4C for TiO2 and ZnO analysis by
inductively coupled plasma mass spectrometry (ICP-MS).
In vivo treatment. Exposed sites (n 3 per formulation) on two pigs were
treated with 250 ll of each formulation; 200 ll was loaded onto the pad of the
Hill Top chamber (1.0 cm2 area) and 50 ll 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 the pigs were euthanatized as above. Skin from all of these sites were
removed with an 8-mm biopsy punch for microscopy studies as stated above.
Light microscopy. Fixed skin was rinsed in 70% ethanol, processed
through graded ethanol series, cleared in Clear-Rite 3, and infiltrated and
embedded in Paraplast tissue embedding medium. Sections of 5 lm were
mounted on positive-charged slides and stained with hematoxylin and eosin.
Transmission electron microscopy. Fixed skin was rinsed in 0.1M
phosphate buffer, postfixed in phosphate-buffered osmium tetroxide, dehy-
drated through graded ethanols, cleared in acetone, and infiltrated and
embedded in Spurrs resin (Ted Pella, Inc., Redding, CA). Thin sections
(8001000 A) were mounted on formvar carboncoated copper grids and
examined on an FEI/Philips EM 208S TEM. Some sections were stained with
lead citrate and uranyl acetate and others were left unstained. Stained sections
provided better contrast and thus resolution of the tissue, whereas unstained
sections allowed better visualization of the Ti and Zn NP and ensured the
absence of stain artifacts that may have resulted from lead citrate or uranyl
acetate precipitation. In addition, stained and unstained samples were analyzed
by EDS to determine element distribution with a Hitachi HF2000 FE TEM
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equipped with an Oxford Instruments INCA EDS.
Scanning electron microscopy. Frozen skin from the in vivo study was
fixed in Trumps fixative, rinsed in 0.1M phosphate buffer, and dehydrated
through graded ethanols to absolute ethanol. The tissue was critical point dried,
mounted on aluminum stubs, and sputter coated with a gold/palladium alloy.
The samples were imaged on a high-resolution JEOL 6400F field emission
SEM at 20 KV, and the presence of Ti and Zn was confirmed by EDS.
Time-of-flight secondary ion mass spectrometry. Frozen skin was aligned
with the epidermis perpendicular to the knife, sectioned at 15 lm in a cryostat,
and mounted onto silicon chips. Samples were air-dried for 3 h prior to being
introduced into the chamber of the microscope. An Ion TOF SIMS5 (Physical
ION TOF, Munster, Germany) employing a Bi pulsed primary ion beam was
used to sputter the surface of the skin sample. Ionized species eroded from the
sample surface were extracted into a TOF mass spectrometer and analyzed
according to their mass to charge ratio by measuring TOF from the sample
surface to the detector, obtaining high spatial and mass resolution data. The
pulsed Bi beam was rastered across the sample (256 3 256 pixels), and a mass
spectrum was acquired at each pixel. Ion images were obtained retrospectively
from the acquired data for selected Ti and Zn isotopes. The images from the Ti
and Zn isotopes were summed to improve signal to noise. Ion images were
aligned with optical images of the tissue to determine the upper surface of the
SC and the lower surface of the dermis and to localize the NP in the skin.
Inductively coupled plasma mass spectrometry. Perfusate samples were
analyzed for Ti and Zn by ICP-MS. The samples (0.2 g) were weighed and
mixed with 0.2 ml of nitric acid up to 10 ml with deionized water. The Ti or Zn
content (isotope: 66Zn) of the solutions was analyzed by ICP-MS with an
Agilent 7500a with Meinhardt atomizer (generator: 1300 W and analysis time:
1.5 s) using 45Sc as an internal standard. Additionally, perfusate samples were
concentrated by analytical ultracentrifugation and the sediment was analyzed
by TEM/energy dispersive x-ray spectroscopy (TEM/EDS) for Ti and Zn.
Spherical particles were identified by EDS as Si/O and by field emission gun
TEM and high-angle annular dark fieldscanning TEM. Small angle x-ray
scattering (SAXS) was used to confirm the Ti and Zn used in the sunscreen
formulations.
RESULTS
Particle Ultrastructural Characterization and Composition in
Diluted Sunscreen Formulations
TiO2 in CM 630 (Fig. 1a) and CM 634 (Fig. 1b) appear
similar in length (up to 100 nm) and fusiform/spindle in shape.
 SAFETY EVALUATION OF SUNSCREENS IN SUNBURN SKIN
ZnO found in CM 643 (Fig. 1c) and CM 644 (Fig. 1d) was
heterogeneous, with a mean size of 140 nm and elongated to
cuboidal shape up to 200 nm. EDS spectra (insets within
micrographs) confirm that the NP in CM 630 and CM 634 are
Ti and the NP in CM 643 and CM 644 are Zn. In addition to
the Ti in CM 630 and CM 634, EDS also identified the
presence of Si in the Ti spectra and Cu in all samples. Si is
a coating for the Ti, and the samples are mounted on copper
grids.
Characterization of UVB-Exposed Sunburned Pig Skin
Pigs were exposed to UVB that resulted in a 2 sunburn
erythema for the in vitro flow-through experiments and the
in vivo experiments. Typical sunburn cells (SBC) with
a pyknotic nucleus and eosinophilic cytoplasm are indicative
of UVB skin exposure and can be identified in Figures 2b2d
(in vitro) and Figures 4b4d (in vivo). SBC have a distinct
morphology, consisting of a pyknotic nucleus (apoptotic) and
eosinophilic cytoplasm, and their presence indicates that the
skin had received UVB damage. For the in vivo studies,
termination at 48 h resulted in most of the UVB-exposed sites
reaching a 3 erythema, which exhibits a definite red in
a well-defined area.
In Vitro Flow-Through Diffusion Cells
At the end of the experiments (24-h postdose), skin was
removed from the diffusion cells. Three sunscreen formulations
were dry on the skin surface with the exception of CM 634 that
was moist.
LM of In Vitro
Control skin (no UVB and no sunscreen) exhibited
intracellular epidermal edema (Fig. 2a), which is typical of
perfused skin after 24 h. UVB control skin (UVB and no
sunscreen) and all UVB-treated skin with the four formulations
had intracellular epidermal edema and showed typical
expression of SBC in the lower layers of the epidermis
(Fig. 2b). All UVB-exposed areas showed a normal SC and
inflammatory infiltrates in the dermis. Residual CM 630 (10%
TiO2 in o/w vehicle) was prominently visible as a dark
precipitant on the surface and in the invaginations or furrows of
the SC in unexposed skin. UVB-exposed skin treated with CM
634 (10% TiO2 in w/o vehicle) depicted focal parakeratosis and
intracellular epidermal edema with SBC (Fig. 2c). Unexposed
skin treated with CM 643 (5% ZnO; Z-COTE HP1 in o/w
vehicle) was typical of normal perfused skin, with only small
residues of sunscreen ZnO remaining on the SC. Treatment
with CM 643 was similar to CM 630. Unexposed skin treated
with CM 644 (5% ZnO; Z-COTE in o/w vehicle) was similar to
normal perfused skin. UVB-exposed skin with CM 644 was
typical of UVB-damaged skin as described above and
consisted of focal areas of discrete sunscreen on the surface
of the SC (Fig. 2d).
267
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FIG. 1. Transmission electron micrographs of sunscreen NP diluted 1:200
in ultrapure water. (a) CM 630, 10% TiO2 (T-Lite SF); (b) CM 634, 10% TiO2
(T-Lite SF); (c) CM 643, 5% ZnO (Z-COTE HP1); (d) CM 644, 5% ZnO
(Z-COTE). Note size homogeneity of the TiO2 NP and size heterogeneity of the
ZnO NP. X-ray microanalysis spectrum (inset) confirms the presence of Ti or
Zn (arrows); arrowheads denote Si peaks in (a) and (b). Bar 100 nm.
TEM of In Vitro
TEM was used to assess TiO2 and ZnO particle penetration
through the SC skin layers. The SC layers of the control (no
UVB, no sunscreen) appeared normal, with numerous compact
layers of SC cells interconnected by desmosomes. Stratum
basale (SB) layer had a few intracytoplasmic lipid droplets,
swollen mitochondria, focal intercellular edema, and attached
to the basement membrane by hemidesmosomes. Nonmem-
brane-bound lipid droplets were seen and typical of degenerat-
ing cells. EDS of the stained tissue found spectra specific for
the presence of Cu (copper grid), Os (osmium tetroxide
postfix), Pb (lead citrate stain), and U (uranyl acetate stain). SC
of the UVB control skin (no sunscreen) appeared similar to
normal skin with no UVB exposure. The SG consisted of
numerous degenerating cells containing vacuoles and cellular
remnants. At times, isolated necrotic cells were found in the SG
and SB. The SB layer contained many cytoplasmic vacuoles,
swollen and altered mitochondria, lipid droplets, condensed
and fragmented nuclei, and remnants of disintegrating cells. At
times, focal microvesication and SBC were noted throughout
the epidermis.
The presence of TiO2 or ZnO in the treated skin was
confirmed by the ultrastructure of the NP as well as by EDS.
Skin without UVB exposure and treated with CM 630 (10%
TiO2; T-Lite SF in o/w) or CM 634 (10% TiO2; T-Lite SF in
w/o) appeared similar to the unexposed controls. TiO2 NP were
268 MONTEIRO-RIVIERE ET AL.

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FIG. 2. Light micrographs of in vitro porcine skin after 24 h of perfusion. (a) Control skin (no UVB and no sunscreen). Note the presence of intracellular
epidermal edema (arrows). (b) UVB control skin (UVB and no sunscreen). Arrows denote intracellular epidermal edema and arrowheads increased numbers of
SBC in the SB layer of the epidermis. (c) UVB-exposed skin treated with CM 634 exhibiting SBC (arrowheads) in the stratum spinosum layer of the epidermis.
Arrows denote residual sunscreen containing TiO2 on the upper SC. (d) UVB-exposed skin treated with CM 644. Sunscreen containing ZnO was located above the
SC (inset, upper right). Arrowheads denote SBC in the upper stratum spinosum layers. E, epidermis; D, dermis. Hematoxylin and eosin. Bar 50 lm.

occasionally found as deep as nine layers in the compacted SC,
with large aggregates often present on the surface and within
the upper two to three layers of the SC. UVB-exposed skin
treated with CM 630 showed the typical aggregates of TiO2 on
the surface and up to 17 SC layers deep (Fig. 3a). Occasionally,
several TiO2 NP were found in the intercellular lipid moiety
space at the lateral overlapping edges between adjacent SC
cells. The epidermal morphology was typical of UVB-damaged
skin, including the condensed pyknotic nucleus of SBC. EDS
of the stained tissue confirmed that the NP in the SC were Ti,
with minor peaks specific for Si (coating), Cu (grid), and Pb
and U (staining). The distribution of TiO2 in unexposed skin
treated with CM 634 was comparable to the unexposed skin
treated with CM 630. The epidermal morphology was normal.
TiO2 NP were never found within any Langerhans cell or cell
process. TiO2 agglomerates were seen on the surface and in the
upper SC layers of the UVB skin treated with CM 634
(Fig. 3b). In addition, separation of the epidermal-dermal
junction was noted. EDS of unstained CM630 and CM634
tissue confirmed TiO2 NP in the SC.
Unexposed skin treated with CM 643 (5% ZnO; Z-COTE
HP1 in o/w) or CM 644 (5% ZnO; Z-COTE in o/w) was also
normal with focal areas of ZnO NP, as confirmed by EDS, on
the surface of the SC and superficial stratum disjunctum. In
UVB-exposed skin treated with CM643 (Fig. 3c) or CM 644
(Fig. 3d), focal ZnO NP, as confirmed by EDS, were noted
above the SC. NP were adjacent to an electron-translucent area
that probably represents residual sunscreen vehicle. ZnO NP
were not found in the upper SS layer, the SG layer, or the lower
SC layers. TEM of the epidermis of UVB-exposed skin treated
with CM 643 and CM 644 depicted typical UVB damage with
intracellular and intercellular epidermal edema, SBC, vacuoles,
necrotic cells, and Langerhans cells that did not contain ZnO
NP.
In Vivo Treatment
All skin exposed to UVB had a 2 erythema score based on
the Draize test at 24 h. Pads in the used chambers as well as
the underlying skin were relatively dry, indicating that the
formulation vehicles had been absorbed into the skin. At the
termination of the experiment (48 h), most UVB-exposed sites
had an increase in erythema to 3, exhibiting a definite red in
a well-defined area. Normal pig skin and unexposed sunscreen
controls showed no erythema.
LM of In Vivo
All sunscreens remained on the pig for 48 h following UVB
exposure. Control skin (no UVB, no sunscreen) exhibited
normal epidermis and dermis with a compact SC (Fig. 4a).
UVB control skin (UVB, no sunscreen) had focal intracellular
epidermal edema, slight dermal inflammation, and typical SBC
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FIG. 3. Transmission electron micrographs of in vitro porcine skin. (a) UVB-exposed skin treated with CM 630, with TiO2 NP found 17 layers into the SC
(arrows) Bar 4 lm. (b) Higher magnification of UVB-exposed skin treated with CM 634 depicting TiO2. Arrows denote residual sunscreen containing TiO2 (as
confirmed by x-ray microanalysis) on the surface and in between the layers of the SC. Bar 1 lm. (c) UVB-exposed skin treated with CM 643. Residual vehicle
containing ZnO (arrows) as confirmed by x-ray microanalysis above the surface of SC. Bar 250 nm. (d) UVB-exposed skin treated with CM 644. Residual
vehicle containing Zn located above the surface of SC. Bar 4 lm. Unstained.

in the lower layers of the epidermis (Fig. 4b), with early
parakeratosis similar to our other published studies with UVB
exposure in porcine skin (Lin et al., 2003, 2004, 2005; Tournas
et al., 2006). At times, focal microblisters were present with
epidermal-dermal separation. UVB-exposed skin treated with
all four sunscreen formulations had typical UVB damage as
mentioned above but with more severe lesions than the in vitro
studies due to the 48 h time following exposure. UVB-exposed
sites treated with CM 630 (Fig. 4c) and CM 634 typically had
a thin layer of the sunscreen formulation on the surface of the
skin and some early parakeratosis. Sites treated with CM 643
(Fig. 4d) and CM 644 were similar with little residual Zn
sunscreen on the SC, slight dermal inflammation, SBC, and
superficial epidermal necrosis with hypereosinophilia consis-
tent with UVB
exposure.
TEM of In Vivo
To assess the skin penetration of all the formulations of
sunscreens, TEM was used to confirm the localization and
depth of penetration in skin. The SC and SB of normal pig skin
(no UVB, no sunscreen, and without a chamber) were normal.
Langerhans cells were identified within the SS layers. The SG
contained cellular remnants, with Langerhans cell processes
throughout the SS. Skin without UVB exposure and treated
with CM 630 appeared normal. TiO2 NP were found only up to
seven layers deep in areas of the compact SC (Fig. 5a), with
large aggregates usually present on the surface and within the
upper two to three layers of the SC. The epidermis was normal,
with occasional Langerhans cells in the viable layers. UVB-
exposed skin treated with CM 630 in an o/w formulation
depicted typical aggregates of TiO2 on the surface and
intercellular space of the SC and was found deeper to 13 cell
layers (Fig. 5b). In one focal area of UVB-damaged skin, TiO2
crystals were found 29 layers deep into the SC. Degenerating
cells containing vacuoles and cellular remnants were present
within the SG. The SB layer contained numerous cytoplasmic
vacuoles, swollen and altered mitochondria, lipid droplets,
pyknotic cells with condensed nuclei, and remnants of
disintegrating cells. Most of the distribution of TiO2 in normal
skin with CM 634 was found in the upper nine layers of the SC
(Fig. 5c); however, in one instance, TiO2 was found 20 layers
deep in this w/o formulation. The epidermis was normal. In the
UVB skin treated with CM 634, TiO2 accumulated on the
surface and between adjoining SC cells. Ti was continuously
seen in the SC usually up to 12 cell layers in depth.
A degenerating cell that contained Ti within the cytoplasm
and within vacuoles was noted in the SC (Fig. 5d). TiO2
agglomerates were noted between the cell and adjacent SC
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FIG. 4. Light micrographs of in vivo porcine skin after 48-h exposure. (a) Control skin (no UVB, no sunscreen). (b) UVB control skin (UVB, no sunscreen).
Note the presence of focal intracellular epidermal edema (arrow) and SBC in the SB layer of the epidermis and early parakeratosis. (c) UVB-exposed skin treated
with CM 630 exhibiting SBC (arrowheads) in the SB layer of the epidermis. Arrows denote residual sunscreen containing TiO2 on the surface of the SC. (d) UVB-
exposed skin treated with CM 643. Sunscreen containing ZnO was located above the stratum corneum (arrow). Arrowheads denote SBC in the SB and stratum
spinosum layers with superficial epidermal necrosis with hypereosinophilia. E, epidermis; D, dermis. Hematoxylin and eosin. Bar 50 lm.

cells and within an invagination of the cell membrane of the
degenerative cell (Fig. 5d, inset).
Pig skin not exposed to UVB and treated with CM 643 or
CM 644 was normal, with focal areas of ZnO NP on and
immediately above the superficial layers of the SC. Langerhans
cells were present in the SS and SB and ZnO NP were not
observed within them. ZnO NP were found up to 10 cell layers
deep in the SC (Fig. 5e) of UVB-exposed skin treated with CM
643, and in one instance up to 16 layers deep. In the UVB-
exposed skin treated with CM 644, the ZnO NP were localized
solely to the upper SC (Fig. 5f). In general, TEM showed that
TiO2 NP penetrated deeper into the SC in normal and UVB-
damaged skin than ZnO but still remained localized to the
intercellular space of the SC layers. We noticed that NP in the
w/o formulation penetrated deeper into the SC.
SEM of In Vivo
SEM/EDS was performed on the in vivo normal porcine
skin exposed only to UVB to verify that no contaminants of
Ti or Zn were present on the skin. In the UVB-exposed skin
treated with the CM 630 formulation, TiO2 NP were primarily
localized as large agglomerates on the surface of the skin.
Discrete areas of Ti were found on the outer periphery of
these agglomerates. Near the base of the hair, just as it
emerges from the skin (Fig. 6a), in the area referred to as the
infundibulum, EDS confirmed that TiO2 NP were present
(Fig. 6b, inset). Large agglomerates of TiO2 from the CM 634
formulation were also present on the hair (Fig. 6c) and
localized in the crevices between the overlapping cuticle
scales (Fig. 6d) as confirmed by EDS (Fig. 6d, inset). Skin
adjacent to these hairs also contained agglomerates of TiO2.
The heterogeneous ZnO NP in CM 643 were distributed on
the surface of the UVB skin, and ZnO agglomerates were also
noted near the base of the hair (Fig. 7a) and on the hair
(Fig. 7b) as confirmed by EDS (Fig. 7b, inset). The CM 644
sunscreen was distributed evenly across the UVB-exposed
skin and was present near the base of the hair (Fig. 7c) and on
the hair (Fig. 7d) and confirmed by EDS (Fig. 7d, inset). In
one focal area of the skin near the base of a hair, large ZnO
crystals (confirmed by EDS) measuring up to 15 lm in
diameter were noted within a large agglomerate of Zn NP and
were not seen in any other samples.
Time-of-Flight Secondary Ion Mass Spectrometry
TOF-SIMS provides a sensitive analytical method to detect
the NP in the skin. TOF-SIMS images of mapping overlays
localized Ti and Zn in the skin samples. Homogeneous
background for Ti and Zn were present in some of the maps
as individual pixels. Clustering of pixels indicated the presence
of Ti or Zn above background.
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FIG. 5. Transmission electron micrographs of in vivo porcine. (a) Unexposed (no UVB) skin treated with CM 630. Upper SC, with Ti (arrows) seven layers
deep. Unstained bar 1.2 lm. (b) UVB-exposed skin treated with CM 630. Ti (arrows) 13 layers deep within the SC. Unstained. Bar 1 lm. (c) Unexposed skin
treated with CM 634. Ti (arrows) between layers of the upper SC. Stained with lead citrate and uranyl acetate. Bar 600 nm. (d) UVB-exposed skin treated with
CM 634. Degenerating cell embedded in the upper SC containing Ti (arrows). Arrowheads denote Ti on SC cells. Bar 3 lm. Inset: Ti within an invagination
(arrow) of the degenerating cell. High magnification of area in (d). (e) UVB-exposed skin treated with CM 643. Zn localized 910 layers deep (arrows) in the SC.
Unstained. Bar 1 lm. (f) UVB-exposed skin treated with CM 644. Zn NP on the surface of SC (arrows). Stained with lead citrate and uranyl acetate. Bar 2 lm.

In Vitro Flow-Through of TOF-SIMS
Mapping showed no Ti or Zn above background levels were
present in the normal and UVB-exposed control skin (no
sunscreen). In the UVB-exposed control skin (UVB, no
sunscreen), neither Ti nor Zn was detected above background.
Normal unexposed skin treated with the CM 630 formulation
revealed that the concentration of Ti (and isotopes 46Ti, 47Ti,
48
Ti, and/or 48TiH) was the greatest in the epidermis, with
some penetration into the superficial papillary dermis (Fig. 8a).
In the UVB-exposed skin treated with CM 630, Ti was the
greatest in the epidermis and similar penetration in the dermis
to the nonUVB-exposed skin (Fig. 8b). In the overlay panel of
Figure 8b, which provides an orientation of the tissue and
localization of the NP, a focal area of Ti was noted and appears
to be adjacent to a hair follicle. In unexposed skin treated with
CM 634, Ti also was localized primarily to the upper epidermis
(Fig. 8c). In the UVB-exposed skin, focal areas of Ti were
present in the SC, epidermis, and dermis (Fig. 8d). In normal
skin treated with CM 643, Zn (64Zn and/or 66Zn) was typically
localized to a narrow band in the SC (Fig. 9a). In one sample,
Zn was present within the focal clusters in the epidermis and
superficial dermis, sometimes aligning with structures such as
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FIG. 6. Scanning electron micrographs of in vivo porcine skin. (a) Low magnification of CM 630 sunscreen on a hair at the surface of UVB-exposed skin.
Note Ti agglomeration (arrow) near the base of the hair (H). Bar 20 lm. (b) Higher magnification of area denoted by arrow. X-ray microanalysis (EDS) (inset)
confirmed the NP as Ti (arrow). Bar 300 nm. (c) Low magnification of CM 634 sunscreen on a hair and adjacent skin of UVB-exposed skin. Bar 50lm.
(d) Higher magnification area denoted by box. Ti present on surface of hair (arrows) and between overlapping cuticle scales (*). Bar 300 nm. EDS (inset)
confirmed Ti NP (arrow).

hair follicles and sebaceous glands. In the UVB-exposed skin
treated with CM 643, Zn localization had a small but thicker
band in the epidermis compared with the unexposed skin (Fig.
9b). In both the unexposed (Fig. 9c) and exposed (Fig. 9d) skin
treated with CM 644, Zn was primarily concentrated in the
upper epidermis within a much thicker band than CM 643. The
UVB-exposed skin also had higher background levels in the
epidermis
(Fig. 9d).
In Vivo Study of TOF-SIMS
Mapping of the normal and UVB-exposed control skin (no
sunscreen) showed no Ti or Zn above background levels.
Normal unexposed skin treated with CM 630 revealed only
slight Ti above background in the epidermis and superficial
papillary dermis (Fig. 10a). In the UVB-exposed skin treated
with CM 630, the greatest concentration of Ti was found in the
upper epidermis (Fig. 10b). Homogeneous distribution of Ti
was found in the SC and upper epidermis, with some focal
areas of Ti in the superficial dermis in unexposed skin treated
with CM 634 (Fig. 10c). At times, focal areas of Ti were noted
in the dermis and based on the overlay, could not be correlated
to any morphological structures such as hair follicles or
sebaceous glands. Ti mapping in UVB-exposed skin treated
with CM 634 was similar to that of the unexposed UVB skin
(Fig. 10d). Normal unexposed skin treated with CM 643
revealed focal Zn slightly above background level in the
epidermis (Fig. 11a). In the UVB-exposed skin treated with
CM 643, focal areas of Zn were present in the SC and in the
upper epidermis (Fig. 11b). In the unexposed skin treated with
CM 644, Zn was present primarily on the surface of the SC
(Fig. 11c). In the UVB-exposed skin, Zn was slightly above
background in the SC (Fig. 11d).
Figure 12 is a schematic illustrating the penetration path that
NP follow in the skin and subsequent interpretation by TEM. In
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FIG. 7. Scanning electron micrographs of in vivo porcine. (a) Low magnification of CM 643 sunscreen on a hair at the surface of UVB-exposed skin. Note
agglomerated Zn (rectangle) near the base of the hair (H). Bar 50 lm. (b) Higher magnification of heterogeneous Zn (arrows) within area denoted by rectangle.
X-ray microanalysis (EDS) (inset) confirmed the NP as Zn (arrow). Bar 600 nm. (c) Low magnification of CM 644 sunscreen on a hair and adjacent skin of
UVB-exposed skin. Bar 50 lm. (d) High magnification of Zn within the area (rectangle) on the surface of the hair (arrows). Bar 600 nm. EDS (inset)
confirmed the Zn NP (arrows).

some micrographs, heterogeneous NP were not found in each
sequential layer of the SC. In many cases, NP were observed
on the surface of the SC and again in deeper layers of the SC
intercellular space, thus appearing to miss a few layers. This
may be explained by the plane of section of the skin that was
examined by TEM. When viewing by TEM, a very small
sample size is examined. When a tissue section a is viewed,
the NP appeared only on the surface and in layer 6. When
section b is viewed, the NP appeared on the surface, in the
next layer down, and in layers 5 and 6; when section c is
viewed, the NP appeared in all SC cell layers.
concentrations between 0.3 and 0.5 lg/ml were background
levels. Neither of the NP species was found to have penetrated
the pig skin into the perfusate. This indicates that although the
TiO2 and ZnO NP were shown to penetrate the SC by TEM
and into the skin by TOF-SIMS as depicted by the presence of
NP in the epidermis and into the dermis, there was not
a significant amount of NP penetration into the dermis for
sufficient systemic absorption to occur. Additionally, the
perfusate samples concentrated by analytical ultracentrifuga-
tion and analyzed by TEM/EDS also confirmed that TiO2 and
ZnO NP did not penetrate through the skin into the perfusate.

ICP-MS of In Vitro Perfusate Samples

ICP-MS analysis of the 6, 12, 20, and 24 h perfusate samples
from the UVB-exposed skin in the in vitro cell diffusion studies
is summarized in Table 1. Ti was below detectable limits
(< 0.1% of applied dose) in all samples tested while Zn
DISCUSSION
The use of sunscreens containing TiO2 and ZnO NP has
increased worldwide in recent years. The Australian Thera-
peutic Goods Administration estimated that in 2005, 70% of all
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FIG. 8. TOF-SIMS of in vitro pig skin. First panel of each figure is an NP overlay on the skin and the second panel is NP alone. (a) Unexposed (no UVB)
skin with the sunscreen formulation CM 630. Mapping shows the highest Ti concentrations in the epidermis. (b) UVB-exposed pig skin treated with CM 630
shows the highest Ti concentrations in the epidermis and superficial dermis. (c) Unexposed skin with the sunscreen CM 634. Mapping shows the highest Ti
concentrations in the upper epidermis. (d) UVB-exposed pig skin treated with CM 634. Mapping shows the highest Ti concentrations in the epidermis.
(e) Intensity scale for Ti, from highest to lowest. Each vertical bar segment equals 100 lm. Narrow lines in the overlay panel are within the target area analyzed
by the TOF-SIMS.

sunscreens containing TiO2 and 30% containing ZnO were
formulated with NP (Faunce et al., 2008). Sunscreens
containing micronized TiO2 and ZnO NP appeal to consumers
because they are transparent and provide effective UV
protection by reducing the scattering effect of visible light.
No genotoxicity was noted when TiO2 and ZnO in cosmetic
formulations were tested with the Ames Salmonella gene
mutation test, the V79 micronucleus chromosome mutation
test, the in vivo mouse bone marrow micronucleus test, and the
Comet assay (Landsiedel et al., 2010). The toxicological
profiles of TiO2 and ZnO are very comprehensive; however,
little is known about the effects of these NP in UVB sunburned
skin. TiO2 and ZnO have been used as UV filters in cosmetics
and in sunscreens because they do not cause irritation or act as
sensitizers. They are considered safe and thought to be similar
to their bulk counterparts.
As discussed in the Introduction, ZnO and TiO2 NP do not
appear to penetrate through intact healthy skin. In the present
study, we extend these findings with ZnO and TiO2 NP to skin
damaged by sunburn, a common application scenario to
anyone exposed to UV radiation. Using in vitro and in vivo
porcine skin, an accepted animal model for human skin
(Bronaugh et al., 1982; Monteiro-Riviere, 1986, 2001;
Monteiro-Riviere et al., 1994, 2008; Monteiro-Riviere and
Riviere, 1996, 2005; Monteiro-Riviere and Stromberg, 1985;
Wester and Maibach, 1977) minimal penetration of both ZnO
and TiO2 NP to epidermal cellular elements occurred. Any
material that did penetrate resided primarily in the upper layers
of the SC. This superficial penetration was modulated by the
nature of the topical formulations, a finding we have seen
previously with topical exposure to carbon NP (Xia et al.,
2010). Significantly, NP could not be detected in the perfusate
from in vitro exposures using multiple sensitive analytical
approaches, suggesting systemic absorption did not occur
following topical application to UVB-damaged skin. The
strength of this study includes the use of multiple and very
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FIG. 9. TOF-SIMS of in vitro pig skin. First panel of each figure is an NP overlay on the skin and the second panel is NP alone. (a) Unexposed skin treated with
the sunscreen formulation CM 643. Mapping shows a slight band of Zn in the SC. (b) UVB-exposed skin treated with the formulation CM 643. Mapping shows
a narrow band of Zn in the upper epidermis. (c) Unexposed skin treated with the sunscreen formulation CM 644. Mapping shows a thicker band of Zn in the
epidermis. (d) UVB-exposed skin treated with the sunscreen formulation CM 644. Mapping shows a wider band of Zn in the epidermis. (e) Intensity scale for Zn,
from highest to lowest. Each vertical bar segment equals 100 lm. Narrow lines in the overlay panel are within the target area analyzed by the TOF-SIMS.

precise analytical techniques to detect Zn and Ti in two well-
accepted and sensitive model systems.
To date, absorption studies have been restricted primarily to
normal skin. Both TiO2 and ZnO used in skin care products
have been reported to penetrate the SC of rabbit skin
(Lansdown and Taylor, 1997), with the highest absorption
with water and oil vehicles. Other studies with different surface
coatings of TiO2 in o/w emulsions applied to human skin for
6 h only detected Ti in the uppermost SC (Pflucker et al.,
2001). Lademann et al. (1999) found TiO2 in an emulsion
localized to the upper SC with distribution to the pilosebaceous
orifices, and Cross et al. (2007) found that ZnO was limited to
the outer surface of the SC. Other studies showed that TiO2 in
sunscreens applied to minipigs four times per day for 5 days per
week for 4 weeks observed high levels of Ti in the epidermis by
TEM/EDS, primarily in the SC layers and near hair follicle
openings but also observed a few particles of TiO2 in the dermis,
which were regarded as contamination (Sadrieh et al., 2010).
A few investigators have suggested that when the skin
barrier is damaged, penetration may occur. Sunscreens
containing ZnO NP exposed to humans for 5 days under
typical outdoor exposures found small increases in the Zn
tracer in the blood and urine by very sensitive methods, but it is
not known whether 68Zn was absorbed as ZnO or soluble Zn
(Gulson et al., 2010). Although the authors state that their
study shows evidence of absorption in healthy skin exposed to
sunlight, they are omitting the fact that skin exposed to UV
irradiation is damaged and that the barrier properties are
different. Mortensen et al. (2008) investigated quantum dot
(QD) penetration through UVB-exposed mouse skin and found
silver-enhanced QD penetrated through the SC into the
epidermis and dermis. However, they did not determine the
MED of each exposed animal to standardize UVB exposure
and did not use a relevant animal skin model. The size, coating,
and charge of these QD do not provide a good model for TiO2
and ZnO skin penetration. Also, mouse skin is known to have
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FIG. 10. TOF-SIMS of in vivo pig skin. First panel of each figure is an NP overlay on the skin and the second panel is NP alone. (a) Unexposed skin treated
with the sunscreen formulation CM 630. Mapping shows Ti in the epidermis. (b) UVB-exposed skin treated with the sunscreen formulation CM 630. Mapping
shows Ti concentrated in the upper epidermis. (c) Unexposed skin treated with the sunscreen formulation CM 634. Mapping shows Ti concentrated in the SC, with
slight diffusion into the epidermis and superficial dermis. (d) UVB-exposed skin treated with the sunscreen formulation CM 634. Mapping shows Ti concentrated
in the SC, with slight diffusion into the epidermis. (e) Intensity scale for Ti, from highest to lowest. Each vertical bar segment equals 100 lm. Narrow lines in the
overlay panel are within the target area analyzed by the TOF-SIMS.

minimal barrier properties to prevent topical absorption and is
therefore not similar to human skin.
The purpose of the current study was to determine whether
skin damaged by moderate UVB radiation (sunburn, as scored
by a 2 Draize score) enhanced the penetration of TiO2 or
ZnO NP present in commercial sunscreen formulations. TiO2
and ZnO in the sunscreen formulations were confirmed by
x-ray microanalysis. EDS found the presence of the ZnO NP in
CM 643 and CM 644, and TiO2 NP and Si (Ti coating) in CM
630 and CM 634. Because Si is present in the TiO2 samples
and not the ZnO sample, it is formulation specific; the rutile
TiO2 has a coating of hydrated silica, dimethicone/methicone
copolymer, and aluminum hydroxide. Sunscreen formulations
that contained TiO2 (CM 630 and CM 634) were much more
persistent on the surface of the skin than formulations that
contained ZnO (CM 643 and CM 644). Ti aggregates were
readily found in or on the upper layers of the SC, whereas ZnO
aggregates were smaller, more focal, and typically found just
above the SC layer. Sunscreens containing TiO2 were
apparently more persistent on the surface of the SC, as seen
by LM and TEM. TOF-SIMS also detected less ZnO on the
surface, which was also influenced by the poor secondary ion
yield of Zn. The macroscopic anatomy of normal and
UVB-exposed skin was not affected by topical treatment with
any sunscreen formulation.
In the flow-through studies, TEM found penetration of TiO2
to a depth of 9 layers in the SC of normal skin and 17 layers in
the SC of UVB-exposed skin. ZnO was detected only on the
surface of the SC on both the unexposed and UVB-exposed
skin. In addition to Ti and Zn, Si and Os were detected within
the upper SC by EDS. Although the Os originated from the
osmium tetroxide postfixation during tissue processing, the
source of the Si in skin treated with the ZnO formulations is not
as obvious. Si is probably a contaminant from the perfusate or,
more likely, an environmental contaminate on the pig. Si is
ubiquitous in the environment as well as in many products.
Pigs were housed on open elevated floors and unavoidably still
come in contact with their own feces. ICP-MS analysis of the
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FIG. 11. TOF-SIMS of in vivo pig skin. First panel of each figure is an NP overlay on the skin and the second panel is NP alone. (a) Unexposed skin treated
with the sunscreen formulation CM 643. Mapping shows Zn in the epidermis. (b) UVB-exposed skin treated with the sunscreen formulation CM 643. Mapping
shows Zn in the SC, with slight diffusion into the upper epidermis. (c) Unexposed skin treated with the sunscreen formulation CM 644. Mapping shows Zn on the
surface of the SC. (d) UVB-exposed skin treated with the sunscreen formulation CM 644. Mapping shows Zn in the SC, with slight diffusion into the upper
epidermis. (e) Intensity scale for Zn, from highest to lowest. Each vertical bar segment equals 100 lm. Narrow lines in the overlay panel are within the target area
analyzed by the TOF-SIMS.

perfusate found that Ti was below detectable limits, whereas
Zn was present in all samples. Cross et al. (2007) also reported
background levels of Zn in the receptor fluid from flow-
through studies with human skin after 24 h. This is not
surprising because Zn is the most abundant essential trace
metal after iron (St. Croix et al., 2005) and is ubiquitous in the
body, especially in the skin and associated appendages and
commonly found in powered gloves, glass, and stainless steel
(Rostan et al., 2002).
Overall, the in vivo studies showed that TiO2 penetrated
deeper into the SC in both normal and UVB-exposed skin
compared with ZnO. The depth of the NP in the SC of the
UVB-exposed skin may have been underestimated in our study
because some of the SC was sloughed from the damaged skin.
Formulations with TiO2 in nonUVB-exposed skin depicted
TiO2 as deep as 20 cell layers, whereas in UVB-exposed skin
TiO2 penetrated 1213 SC cell layers. TiO2 in the w/o
formulation was detected down to 1820 cell layers in normal
skin. ZnO was localized primarily to the upper one to two
layers of the SC in nonUVB- and UVB-exposed skin and in
one instance to a depth of 16 layers in the UVB-exposed skin
treated with CM 643. Retention of ZnO NP was much greater
in the in vivo compared with the in vitro study, probably due to
dose occlusion and the increase in time the formulations were
in contact with the skin. SEM/EDS localized the Ti and Zn
primarily in agglomerates on the surface of the UVB-exposed
skin and at the base of the hair in vivo. Although the NP
agglomerates at the openings of the hair, there are no data to
suggest that hair plays a role in NP penetration into the
epidermis or dermis. TiO2 NP reported in the hair follicle
remained outside the viable epidermis and dermis and were
shown to be eliminated by sebum flow (Lademann et al., 1999;
Nohynek et al., 2007). Hair follicles have been suggested to be
weak points in the skin barrier (Patzelt et al., 2010) and may
provide an area for potential absorption, thereby serving as
a reservoir for drugs and nanoparticles. Many authors have
278 MONTEIRO-RIVIERE ET AL.

normal and UVB-damaged skin treated with the sunscreen

FIG. 12. Schematic illustrating NP penetration into the SC layers of the
skin. (a), (b), and (c) represent different sections through the skin as imaged by
TEM. (a) NP found only in deeper layers of the SC, (b) NP in the upper and
lower layers of the SC, and (c) NP in all layers of the SC. Arrows denote
presence of nanoparticles in the intercellular spaces between the SC cells.
formulations. Slight Ti and Zn background interferences were
present in some of the map overlays as determined by a region
of interest analysis. We are not sure what caused this
interference in some samples, although the counts may be
due to molecular fragments from molecules of higher mass that
did not survive the trip from the sample to the detector. The
concentrations of Ti and Zn in the skin as determined by TOF-
SIMS were less in vivo than in vitro. In vitro, neither Ti nor Zn
was present in the untreated control skin (no sunscreen), with
the exception of one sample showing Ti slightly elevated above
background. Ideally, the absorption of Ti or Zn through porcine
skin is determined by analyzing the perfusate. In our studies,
no absorption was detected. However, depending on the
methodology, the detection limit may be too high to detect very
low levels of NP absorption.
In summary, UVB-sunburned skin slightly enhanced the

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reported that follicular density effects penetration (Feldman in vitro or in vivo SC penetration of the TiO2 or ZnO NP
and Maibach, 1967; Maibach et al., 1971; Monteiro-Riviere, present in the sunscreen formulations. Although TiO2 and ZnO
2004, 2008). In addition, the lower region of the hair NP were found to penetrate into the SC by TEM and into the
infundibulum has a weak barrier where corneocytes are smaller epidermis and dermis by TOF-SIMS, we found no definitive
and crumbly, which can increase the permeability for drugs evidence that the NP penetrated the skin in vitro into the
(Lademann et al., 2006; Patzelt et al., 2010). perfusate. In most cases, TiO2 penetration into the SC was
Much of the sunscreen vehicle containing the TiO2 and ZnO greater than ZnO. These results viewed together suggest
NP was probably removed from the skin during tissue minimal penetration of TiO2 and ZnO NP into the upper
processing for LM and TEM. To circumvent this problem, epidermal layers when applied topically in sunscreen for-
we imaged the metallic oxides stabilized in the tissue by mulations to normal and UVB-sunburned skin, with no
freezing, therefore leaving the NP undisturbed on the skin. evidence of systemic absorption.
TOF-SIMS allowed us to investigate the distribution of Ti and
Zn on skin tissue sections mounted on silicon wafers. Although
the spatial resolution of the microscope is low, TOF-SIMS FUNDING
provided a very sensitive tool to map the Ti and Zn distribution
Portions of this research were funded by BASF.
within the tissue sections. TOF-SIMS data indicate that both
the Ti and Zn did focally penetrate into the epidermis in both
ACKNOWLEDGMENTS
TABLE 1
ICP-MS Analysis of Perfusate from In Vitro UVB-Exposed Skin Portions of this research were selected for presentation at the
platform session on NanotoxicologyMetals and Metal Oxide
Time Titanium Time Zinc
Formulation (h) (lg/ml)a Formulation (h) (lg/ml)b at the 49th Annual Meeting of the National Society of
Toxicology Meeting in Salt Lake City, Utah, on 10 March
None 6 <0.1 None 6 0.5 2010 and at the 12th International Perspectives in Percutaneous
12 <0.1 12 0.5 Penetration Conference in La Grande Motte, Paris, on 7 April
20 <0.1 20 0.4
2010. The authors would like to thank Dr Dieter Griffis,
24 <0.1 24 0.3
CM 630 6 <0.1 CM 643 6 0.5 Director of the Analytical Instrumentation Facility at North
12 <0.1 12 0.4 Carolina State University, for his help with TOF-SIMS
20 <0.1 20 0.5 analysis. No conflict of interest for N.A.M.-R., J.E.R., and
24 <0.1 24 0.3 A.O.I. K.W., R.L., and S.S. are employees of BASF.
CM 634 6 <0.1 CM 644 6 0.5
12 <0.1 12 0.5
20 <0.1 20 0.4
24 <0.1 24 0.3 REFERENCES

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