Concern has been expressed in the published literature that nanosized particles have unknown toxicological profiles differing from those of larger sized particles, and because of their small size, they may penetrate the skin and pose safety concerns. This matter has gained more attention due to the large number of sunscreens available on the market claiming to contain nanosize TiO2
. In order to evaluate whether nanosize TiO2
can penetrate intact skin, several studies have been conducted and published. Tan et al. (1996)
excised skin from human volunteers following 2- to 6-week applications of a sunscreen containing 8% microfine (i.e., nanosized) TiO2
for 2–6 weeks. TiO2
was determined in the lower epidermis and dermis by ICP-MS. The authors found elevated but not significant (p
= 0.14) levels of TiO2
in the skin of the test patients when compared to skin from cadavers; however, if one extreme value is removed from the cadaver group, a significant difference was detected (p
= 0.006) with an ~50% increase in skin TiO2
levels. This work was one of the first reports to suggest TiO2
might penetrate the skin.
Lansdown and Taylor (1997)
applied suspensions of 20% “microfine” TiO2
and ZnO to the shaved backs of New Zealand white rabbits. Surface coated TiO2
or ZnO was removed by tape stripping and the skin removed after 24 or 48 h posttreatment. TiO2
applied as a caster oil suspension penetrated the skin; TiO2
applied as an aqueous suspension did not penetrate the skin. These results suggested that dermal penetration by micronized ZnO or TiO2
Bennat and Müller-Goymann (2000)
examined the penetration of microfine TiO2
into human skin in vitro
and in vivo
. The TiO2
was suspended either as an aqueous or as an oil/water emulsion and applied at a dose of 2 mg/cm2
. The stratum corneum of the epidermis was removed 24 h later using tape strips and the TiO2
content on the strip was determined. The authors concluded that TiO2
penetrated the upper layers of the epidermis when applied as a 40% suspension in oil, with less penetration from a water suspension. This penetration was enhanced ~10-fold when the material was applied on hairy skin, suggesting that either the hair follicles could be a route of penetration or in this study, tape stripping was inadequate in removing the TiO2
that penetrated into the follicle. Application of the TiO2
as a 5% liposomal suspension resulted in consistent presence of TiO2
in the epidermis, while application as an oil/water emulsion did not achieve the same level of penetration.
Lademann et al. (1999)
applied a coated micronized TiO2
sunscreen formulation to the skin and found that epidermal penetration could not be detected in intrafollicular areas of the skin; however, TiO2
was detected in follicular-containing areas following tape stripping. Histological examination of the skin indicated TiO2
in the open part of the follicle.
Schulz et al. (2002)
used nanosize TiO2
from several sources to determine the penetration of TiO2
into human skin. TiO2
that was hydrophobic (20 nm; coated with trimethyloctylsilane), amphilic (10–15 nm; coated with Al2
, aggregating to 100-nm needles), and hydrophilic (100-nm needles) were applied as 4% emulsions in a complex cream to human skin and the skin biopsied after 36 h. The authors examined the skin for TiO2
particles using TEM and did not find evidence of skin penetration. Agglomerations of TiO2
were detected on the surface of the skin.
While none of these studies were conclusive, they did raise concerns about the possibility that nanosize TiO2
can penetrate skin. The ambiguity in the literature, combined with the public perception of a health concern (demonstrated in a citizen petition submitted to FDA), led us to conduct the present study. Because the pig and minipig have skin that more closely resembles human skin, as compared to other species, in regards to epidermal cellular thickness, overall physiology, and hair follicle density, we chose this animal model to conduct our study (Prunieras, 1993
; Sekkat and Guy, 2001
; Tiemessen, 1993
; Wester and Maibach, 1999
In order to have our study provide the highest relevance to the sunscreen-using public, the doses and vehicle used in this study were chosen to closely resemble the material currently used in marketed products. The formulations used in this study were very similar to those used in a “typical” sunscreen. TiO2 may be included in sunscreens as an ingredient as long as it does not exceed 25% by weight (21 CFR 352.10). We chose to include TiO2 at ~5%. In addition, instead of monitoring TiO2 levels following a single administration of sunscreen, we chose to mimic the continuous use of sunscreen over the course of 22 days.
To optimize assessment of penetration by TiO2
through intact pigskin, we used both ICP-MS and EM-EDX. Using the lymphatics and liver as sentinel organs for TiO2
penetration, we did not detect by ICP-MS consistent increases in these organs following 22 days of sunscreen application. The sentinel organ approach has been previously demonstrated to detect the distribution of PEG-coated quantum dots following intradermal administration (Gopee et al., 2007
) and dermal penetration following disruption of the skin (Gopee et al., 2009
). In this study, we show that titanium levels in lymph nodes and liver are not elevated as would be expected if TiO2
had penetrated the epidermis.
The use of EM was important to demonstrate whether the titanium found in dermis was due to contamination from epidermis or actual penetration. However, to put our findings into context, the following calculations were done, as a means to estimate the dose of TiO2 detected in the skin based on EM results.
The levels of titanium found in the epidermis from control, submicron, uncoated nanosized, and coated nanosized TiO2 was 0.035, 2.11, 2.57, 5.05 mg/g tissue, respectively, whereas the levels of titanium found in the dermis were 1.003, 7.34, 5.17, 10.92 μg/g. These results indicate that the amount of titanium found in the epidermis was 35-, 287-, 498-, and 463-fold higher than the levels detected in the dermis. Although the dry heat method for separating the epidermis from dermis was very good, it is possible that, as shown in , a small piece of epidermis could occasionally remain on the dermis, following separation. If the epidermis left behind on the dermis was ~0.2–0.3% of the original epidermis, this could account for the level of titanium that was detected in the dermis. To address this concern, we used TEM-EDX, which could resolve whether the titanium detected in the dermis was due to contamination from residual epidermis.
TEM images of the skin exposed to submicron TiO2 show electron-dense spots of the appropriate morphology to be TiO2 particles primarily in the upper stratum corneum layers of all three skin samples. These particles appear slightly aggregated and are smaller than expected (the average particle diameter is ~210 nm). EDX analysis confirmed that these particles were TiO2. Only a few particles (< 50) were observed in the lower epidermis layer or viable dermis layer, suggesting that minimal nanoparticle penetration occurs through intact epidermis. Scattered electron-dense individual particles were also detected in the upper follicular lumens and in the lower follicular lumens. These particles have similar sizes and shapes as those observed in the stratum corneum at nonfollicular areas.
We also found TiO2 particles primarily in the stratum corneum and upper follicular lumens in skin samples exposed to uncoated nano-TiO2. These particles display a similar degree of agglomeration and similar morphology to those in the samples treated with submicron TiO2 but are even smaller in size (around 30 nm in diameter). The TEM micrographs of the skin samples exposed to the coated nanosize TiO2 cream showed that these samples contain significantly more TiO2 nanoparticles than the skin samples treated with the creams containing submicron TiO2 or uncoated nanosize TiO2, though this qualitative finding does not necessarily reflect a difference in the total amount of TiO2 in the skin. Thousands of these particles were detected in the upper stratum corneum layers and appear highly aggregated (the aggregations reaching several microns in size), forming fibril-like structures with the individual particles being, on average, 57 nm in length and 15 nm in width (as seen in ).
All three types of TiO2 particles were most concentrated in the stratum corneum layer, and these particles were highly aggregated in between the layers of keratin, which compose the stratum corneum. In lower skin layers, only a very few isolated TiO2 particles were observed. These few particles appeared isolated and randomly distributed. There was no evidence of penetration via expected routes such as follicular lumens or evidence that the particle concentration decreased with depth in skin. The presence of these particles in the lower skin layers may have been the result of cross-contamination during tissue sectioning.
Although a very small number of scattered isolated TiO2 particles were seen in the dermis of pigs treated with sunscreen formulations containing nanoparticles, these are possibly the result of contamination of samples during sample handling. Even if these particles did reach the dermis via penetration, they represent a very small fraction of the applied TiO2. The electron microscopy results allowed us to consider a quantitative approach to determine the level of TiO2 that were confirmed to be present in the electron micrographs. If one considers the area covered by the electron micrographs that were examined, the corresponding surface area equates to 8 × 10−8 cm2. Since 176 mg/cm2 of total cream was applied containing 5% by weight TiO2, 7.0 × 10−7 mg TiO2 was applied to the skin above the EM viewing area.
If we assume that the P25 nano-TiO2 had a nominal primary particle size of 30 nm, the volume of that sphere would be 1.4 × 10−17 cm3, and if the specific density is 4 g/cm3, the weight of each uncoated nano-TiO2 of primary particle size would be 5.6 × 10−14 mg. Therefore, each particle detected by electron microscopy would equate to 8 × 10−6 percent of the total dose that was applied. As a result, and assuming the TiO2 particles of uncoated nano-TiO2 that were detected were primary particles, the detection of 10 confirmed particles of uncoated nano-TiO2 would equate to 8 × 10−5 percent of the total applied dose. Using a similar analysis as described above for the coated nanoscale TiO2 (15 × 57–nm rods), the detection of one particle in the dermis would equate to 5.7 × 10−6 percent of the total dose applied. Similarly, the detection of one submicron TiO2 (50 × 207–nm rods) in the dermis would equate to 2.3 × 10−4 percent of the applied dose. This evaluation of the doses that were detected is based on several assumptions; however, it provides a reasonable measure of the order of magnitude of dose that is detected by electron microscopy. This quantitative analysis of the skin penetration of nanoscale uncoated TiO2 showed that our sensitive electron microscopic methods detected a measurable but insignificant amount of the TiO2 applied to the skin of pigs, regardless of the type or particle size used in the study.
Recently Wu et al. (2009)
have shown that dermal application of TiO2
(4 or 60 nm) to pig ears for a period of 30 days did not result in penetration of nanoparticles beyond the deep epidermal layers. The results of Wu et al. (2009)
are in agreement with the results of the present study, where we did not find any significant dermal penetration of nanoparticles of TiO2
in pigs treated for 1 month with sunscreens formulated with various forms of TiO2
, including coated nano, uncoated nano, and submicron-sized particles of TiO2
; however, the study by Wu et al. (2009)
did report dermal penetration of TiO2
nanoparticles in hairless mice, with subsequent appearance of lesions in multiple organs. The relevance of the finding in mice to our present study in minipigs and to human exposure is unclear. Hairless mouse skin does contain abnormal hair follicles, and mouse stratum corneum has a higher lipid content than human stratum corneum, which in part probably explains why some drugs penetrate mouse skin differently than human skin.
Overall, our study demonstrates that nanosized or submicron-sized TiO2
suspended in the specific formulation used in this study do not penetrate the intact epidermis to any significant extent. While this study does not address any issues within the epidermis where concentrated TiO2
could have an effect on the immune system (e.g., contact dermatitis) or cause toxicity following irradiation with ultraviolet light (e.g., photoactivation, phototoxicity), the study does show that the dermis and other organs may not be at risk from topically applied sunscreens containing nanosize TiO2
. Furthermore, no obvious structural abnormalities were observed in the skin cells, which can be attributed to the presence or absence of TiO2
particles. This study supports the conclusion that nanosized TiO2
included in a formulation similar to currently marketed sunscreens does not significantly penetrate intact normal pigskin to any significant degree and therefore is unlikely to significantly penetrate human skin. We believe that the inclusion of nanosized titanium dioxide in OTC sunscreens does not pose a significant health threat because nanosized particles do not appear to significantly penetrate intact skin; however, since some studies have shown that compromised skin allows particle penetration through the skin (Gopee et al., 2009
, and references therein), we cannot at this time rule out the possibility that damaged skin could be a risk factor for TiO2 penetration.