- Top of page
- UV filters in sunscreens
- Organic UV filters toxicity
- Inorganic UV filter toxicity
- Inorganic UV filter association with organic UV filters
Sunscreens provide broad-spectrum UV skin protection and contain more often UV filter combinations. Their efficacy reducing skin photo carcinogenesis and photo ageing is widely documented. However, there are many concerns about UV filter safety. Organic UV filters were the first targeted by scientist concerns, as they were showed to trigger skin allergic reactions. Inorganic UV filters were then at the heart of scientist debate especially because of their nanometric size. Over the last years, many studies have been published tending to highlight that organic as well as inorganic UV filters could lead to variable side effects after sunscreen application. However, these studies are still very controversial due to different experimental conditions and models. This review reveals that complementary studies using standardized methods are mandatory before ascertaining that UV filters threaten human health.
Les produits solaires destinés à protéger la peau contre les irradiations UV contiennent le plus souvent des combinaisons de filtres UV. Ces filtres permettent de réduire le vieillissement prématuré de la peau ainsi que la survenue de cancer cutané. Cependant, la nocivité des filtres UV est toujours un sujet de controverse. Ainsi, des réactions allergiques liées à l'utilisation de produits solaires contenant des filtres UV organiques ont été rapportées dans la littérature. Récemment, l'utilisation de filtres UV minéraux à l'échelle nanométrique, dispersés dans les produits destinés à la protection solaire a fait l'objet de travaux scientifiques visant à établir le niveau de pénétration cutanée. Ces dernières années, de nombreuses études ont été publiées tendant à montrer que les filtres UV organiques et inorganiques peuvent entrainer divers effets indésirables. Mais, ces diverses études restent très controversées notamment à cause des différences dans les conditions expérimentales et les modèles utilisés. Cette revue révèle que des études complémentaires utilisant des méthodes standardisées sont nécessaires pour confirmer ou infirmer que les filtres UV présentent un risque sanitaire.
Organic UV filters toxicity
- Top of page
- UV filters in sunscreens
- Organic UV filters toxicity
- Inorganic UV filter toxicity
- Inorganic UV filter association with organic UV filters
Organic UV filters are commonly used in sunscreens because of their ability to filter UV rays without leaving unpleasant white marks onto the skin. They permit lighter and more pleasant sunscreen formulations than those containing inorganic UV filters. More often, organic UV filter combinations are used to achieve a sufficient SPF. However, some of these chemical compounds are known to be photounstable, decomposing under UV light, generating ROS and toxic derivative compounds and losing their UV filter properties . Organic UV filters are known to be allergenic triggering photoallergic contact dermatitis [4, 9]. Photoallergic contact dermatitis is a skin allergy reaction to light mediated by contact with some chemical compounds . A correlation between photoallergic contact dermatitis case reports and organic UV filter use into sunscreen formulations (concentration and first appearance on the market) has been described by Kerr et al. . In 2010, a study reported that sunscreen chemical compounds were the main photoallergen agents responsible for photoallergic contact dermatitis [17, 19]. Organic UV filter compounds absorb UV radiations and convert them through chemical reactions that can lead to their breakdown and thus to photounstable reactive intermediate generation in direct contact with the skin [10, 20]. These reactive intermediates can behave as photo oxidants promoting phototoxic or photoallergic reactions and no longer provide protection against UV ray damages . Moreover, organic UV filters can be inactivated by UV radiations  losing their photoprotective effect . Another concern about UV filter photoinstability is their ability to generate ROS with or without UV light stimulation. However, ROS transfer through the SC is still the object of controversy. There are many organic UV filters used in sunscreen formulations. This review focuses on para-aminobenzoic acid, avobenzone, octocrylene, benzophenone compounds, organic camphor derivatives, octyl-methoxycinnamate, octyl-triazone and salicylate compounds summarized in Table 1.
Table 1. CAS numbers, chemical formula, molecular weights, log P, maximum authorized concentrations in European Union and in United States, solubility and absorption spectra of the different organic UV filters referred in this review
|Compounds||CAS number ||Chemical formula ||Molecular weight (gmol−1) ||Log P ||Maximum authorized concentrations in European Union (%) ||Maximum authorized concentrations in United-States FDA (%) ||Solubility ||Absorption spectrum |
|Benzophenone 3||131-57-7||C14-H12-O3||228.2||3.790||10||6||Lipophilic||UVB, UVAII|
Para-aminobenzoic acid (PABA) and its derivatives (e.g. octyl-dimethyl-PABA (OD-PABA)) are known to trigger photoallergies . A recent published report documented the development of photocontact allergies to PABA, highlighting the need of continued vigilance to these organic UV filters . An in vitro experiment focusing on PABA showed that it induces ROS and DNA thymine dimer formation in aqueous solution under sunlight irradiation [26, 27]. DNA thymine dimers consist of double liaison formation between two consecutive DNA thymine bases after nonionizing irradiation like UV rays. These dimers prevent DNA polymerase to make cell replication and, if they are not repaired, lead to cell death or cancer formation. Moreover, it was showed that PABA produced ROS in phosphate-buffered saline (PBS) solution without sunlight irradiation [10, 28]. This is quite worrying because PBS mimics human organism extracellular fluid and thus this mean that this organic UV filter could be cytotoxic reaching extracellular fluid. Another study assessing oestrogenic activity of OD-PABA revealed that it presents oestrogenic activity on MCF-7 cells in vitro leading to their proliferation .
Avobenzone (AVB) was revealed to be one of the most photoallergenic UV filters [17, 24]. Actually, AVB is highly photolabile photodegrading to benzyls and arylglyoxals [10, 20]. The latter compound is a strong skin sensitizer, and thus photocontact allergy to this UV filter may be due to the formation of arylglyoxals in contact with the skin . AVB photoinstability lead to a 60% loose of its photoprotective properties after sun exposure [10, 30]. Moreover, AVB can affect the stability of others organic UV filters it is in presence with . However, this chemical compound can be stabilized notably with octocrylene, another organic UV filter, to avoid its photoinstability [21, 32].
Octocrylene (OC) was also shown to be one of the most photoallergenic organic UV filters . However, OC seems to maintain high UV filter abilities after being irradiated compared to others organic UV filters . In 2006, Hanson et al. demonstrated the ability of OC to generate ROS in human living epidermis cell layers after UV exposure . Other organic UV filters such as benzophenone compounds, organic camphor derivatives, octyl methoxycinnamate and salicylate, have already been reported to penetrate through human SC [[34-39]] due to their relative low molecular weight and their lipophilic character permitting them to pass through intercellular spaces of SC cells . Given the structural similarity of these organic UV filters, OC is also susceptible to penetrate human SC. Consequently, OC could generate ROS in human epidermis nucleated keratinocytes cytoplasm after UV exposure, as it was observed using two-photon fluorescence microscopy . However, in this experiment, OC was incorporated into oil in water emulsions that are susceptible to enhance its penetration into the skin. Actual sunscreen formulations are elaborated without seriously considering their ability to enhance organic UV filter penetration into the skin. Given that sunscreen formulations are applied on a large skin area for a long period of time, a constant and high input of organic UV filters into skin viable cell layers and into systemic circulation might be assured. Moreover, it was showed that OC produces ROS in phosphate-buffered saline (PBS) solution without sunlight irradiation [10, 28].
Different chemical types of benzophenone compounds from benzophenone-1 (Bp-1) to benzophenone-12 (Bp-12) are used as organic UV filters. Bp-3, also known as oxybenzone, is largely incorporated into sunscreens even if it was showed to have the highest incidence of photoallergic contact dermatitis [17, 41]. In 1997 and then in 2002, studies revealed that Bp-3 can be detected in urine after sunscreen topical application on human volunteers [34, 35, 39]. It appeared that between 1 and 2% of Bp-3 total amount applied onto the skin was recovered into human volunteer urines. Bp-3 has also been detected in human plasma after 4 days of whole-body topical application . Concentrations used in this study were 10%, the maximum allowed in the European Union, whereas maximum Bp-3 concentration approved in the United States is 6% . Bp-3 has also been detected in human breast milk after topical application [39, 42]. Tape stripping experiments also suggested that Bp-3 penetrates into human epidermis [34, 38, 39]. This ability could be due to its low molecular weight and lipophilic character permitting it to pass through intercellular spaces of SC cells . In this experiment, Bp-3 was incorporated in oil in water emulsions susceptible to enhance organic UV filter penetration into the skin. In 2006, a study demonstrated Bp-3 ability to generate ROS in human living epidermis keratinocytes cytoplasm using two-photon fluorescence microscopy .
Given that sunscreen formulations are applied on a large skin area for a long period of time, a constant and high input of organic UV filters into skin viable cell layers and into systemic circulation are permitted. In 2007, Kasichayanula et al. demonstrated that Bp-3 penetrates porcine skin reaching blood circulation and being excreted in urine . According to data observed in these different studies, it is relevant to wonder if benzophenone compounds have the possibility, reaching systemic blood circulation, to act on others organism tissues and cells. Another study assessed oestrogenic activity of three commonly used benzophenone derivative organic UV filters: Bp-1, Bp-2 and Bp-3. It appeared that all of them presented estrogenic activity on MCF-7 cells (human tumour mammary cells) in vitro leading to their proliferation . Oestrogenic activity of these organic UV filters was also evaluated in vivo using uterotrophic assays on rat. Data revealed that Bp-1, Bp-2 and Bp-3 significantly increase uterine weight in immature rat demonstrating their strong oestrogenic activity in vivo. Another study reporting in vitro Bp-3 increase effect on MCF-7 cells proliferation also noted a dose-dependent increase in immature Long-Evans rat uterine weight when fed with Bp-3 . In 2005, Morohoshi et al. published ERs competitive binding assays results carried out using 37 commonly used organic UV filters . Data confirmed benzophenone derivatives oestrogenic activity. A study revealed that Bp-4 seems to be more photoallergenic, as it is combined with others organic compounds, including Bp-3 .
Organic camphor derivatives, such as 3-benzilidene camphor (3-BC) or 4-methylbenzilidene camphor (4-MBC), are largely used in cosmetic industries for their ability to protect the skin against UV rays. A recent study showed an important skin permeation concerning both of these chemicals using porcine skin model . Skin permeation of these compounds permits them to reach blood vessels localized in deeper skin cell layers and thus systemic circulation. After in vitro identification of 4-MBC oestrogenic activity in rat , this activity was also revealed in vivo in mammals [47, 48]. 4-MBC binds competitively to oestrogen receptors (ERs) and initiates their transactivation [48, 49]. In 2005, a study carried out in vivo in rat model revealed that 4-MBC ingestion changes oestrogen-regulated genes expression in uterus . Moreover, it showed that 4-MBC acts as a partial estrogenic receptor agonist in vitro on MCF-7 cells and in vivo on immature rat uterus. Other studies assessing oestrogenic activity of 3-BC and 4-MBC revealed that both presented oestrogenic activity on MCF-7 cells in vitro leading to their proliferation [29, 44]. Globally, these data demonstrated that 4-MBC exposure at the minimal dose of 0.7 mg kg−1, during early development and postnatal life can lead to significant changes in the expression of ERs nuclear receptor coregulators and oestrogen target genes in rat uterus at mRNA and protein expression level. In this study, 4-MBC was ingested by rat, but this can happen to human sunscreen consumers notably using lipstick sunscreens, and even with classical sunscreens. Oestrogenic activity of these organic UV filters was also evaluated in vivo using uterotrophic assays on rat. Data revealed that 4-MBC significantly increase uterine weight in immature rat demonstrating their strong oestrogenic activity in vivo. Moreover, data showed that 4-MBC pre and postnatal exposure affects hypothalamo-pituitary-gonadal system development in rat male and female.
Octyl methoxycinnamate (OMC), also known as octinoxate, produces ROS in phosphate-buffered saline (PBS) solution without sunlight irradiation [10, 28]. Another in vitro study, carried out on porcine skin in 2006 showed that about 9% from the applied dose of OMC penetrates the skin with a flux of 27 μg cm−2 h−1 when hydroethanolic solution is used as receiver medium . Tape stripping experiments also suggested that OMC penetrates into human epidermis [38, 39].
In 2006, Hanson et al. demonstrated the ability of OMC to generate ROS in human living epidermis cell layers after UV exposure . OMC has previously been reported to penetrate through human SC [34, 38] due to its relative low molecular weight and its lipophilic character . Consequently, OMC could generate ROS in the cytoplasm of epidermis-nucleated keratinocytes, as it was observed using two-photon fluorescence microscopy. OMC has been detected in human plasma and urine after 4 days of whole-body topical application . OMC concentration used in this study was 10%, the maximum allowed in European Union, whereas the maximum approved concentration of this organic UV filter in United States is 7.5% . OMC has also been demonstrated to trigger MCF-7 cell proliferation in vitro [29, 44]. This study also noted a dose-dependent increase in immature Long-Evans rat uterine weight when fed with OMC. However, in 2005, Morohoshi et al. published controversial data. ERs competitive binding assays were carried out on 37 commonly used organic UV filters notably OMC. In vitro and in vivo assays showed that OMC has no agonistic effect on oestrogenic receptors . However, other cinnamate derivative UV filters have been reported to be poorly photoallergenic [51, 52]. Another study revealed that OMC is not photostable loosing 60% of its photoprotective properties after sun exposure [10, 30]. In 2003, Yener et al. studied OMC entrapment into solid lipid microspheres (SLM) . It appeared that OMC does not penetrate rat skin SC when entrapped into SLM whereas, when it is spread into oleaginous cream, it cans freely penetrate in it. Moreover, when OMC is entrapped into SLM, it does not decompose into derivative compounds contrary to when it is dispersed into oleaginous cream, oil in water emulsion or carbopol gel. More important, entrapped into SLM, OMC keeps his UV filter effectiveness. Thus, organic UV filter vehicles in sunscreens are very important to prevent their UV dependant decomposition leading to ROS formation and to the lost of their effectiveness.
Octyl-triazone organic UV filter has been reported as a strong sensitizer causing allergic contact dermatitis in children and photoallergic contact dermatitis in adults [54, 55].
Salicylate compounds are rarely associated with allergic and photoallergic contact dermatitis or with any oestrogenic activity and are thus often incorporated into sunscreens [45, 51, 52]. However, a recent study reported the development of allergic contact dermatitis to octyl-salicylate . Tape stripping experiments also suggested that it penetrates into human epidermis [38, 39]. Another study assessing homosalate oestrogenic activity revealed that it enhances MCF-7 cell proliferation in vitro .
Previous in vitro experiments concerning 2-phenyl-benzimidazle-5-sulfonic acid (PBSA) organic UV filters showed that it induces ROS and DNA thymine dimer formation in aqueous solution under sunlight irradiation [26, 27].
Some organic UV filter combinations are not suitable to elaborate sunscreens because they can react with each other leading to derivative chemical compound formation and also to their UV filter abilities decrease when they are subjected to sunlight or artificial UV radiations. AVB can affect the stability of other organic UV filters it is in presence with . However, this chemical compound can be stabilized notably with OC to avoid its photoinstability [21, 32]. In 2006, Gaspar et al. studied photostability of some organic UV filter combinations . The most photostable combination revealed was OMC, Bp-3 and OC that do not lead to chemical derivative formation. However, it appeared that OMC in presence of AVB lead to photoproduct formation, as they react each other. OC seems to be more effective than 4-MBC to avoid this chemical reaction occurring between OMC and AVB. Thus, combining OMC, BP-3 and OC or OC, OMC and AVB could be less deleterious than combining 4-MBC, OMC and AVB in sunscreen formulations. Given that some organic UV filter combinations are not suitable to elaborate sunscreens, a list of permitted combinations of approved organic UV filters has been established. This list also gives maximal concentrations that can be used for each chemical compound .
Organic UV filter vehicles in sunscreens are very important to prevent their UV dependant decomposition leading to ROS formation and the lost of their effectiveness, as shown by Yener et al. study focusing on OMC entrapment into SLM . In 2006, a study showed that grafting UV filter chemical compounds on a jojoba wax backbone permit to avoid compound absorption into nude mice skin . Moreover, this strategy tends to enhance UV filter ability to protect the skin against UV damages.
Organic UV filter toxicity studies are numerous. However, considerable variations concerning allergen testing methods, result interpretations and data comparison difficulties have to be taken into account. This lack of standardization in methodology was summarized in a study highlighting differences in irradiation wavelengths, UVA dosage, duration of test allergen application and post irradiation reading times . Controversial data observed concerning organic UV filter oestrogenic activity assessment could be due to differences in organic UV filter concentrations used for the assays and also to experiment conditions that were not similar. Thus, other studies on organic UV filter oestrogenic activity are needed as well as standardized study methods. In the same way, studies concerning organic UV filter penetration into the skin are very controversial. However, these studies were not carried out in same conditions using different experiments, and models.
However, nowadays, formulators start looking for more elaborated sunscreen formulations preventing UV filter penetration across skin SC.
Inorganic UV filter toxicity
- Top of page
- UV filters in sunscreens
- Organic UV filters toxicity
- Inorganic UV filter toxicity
- Inorganic UV filter association with organic UV filters
The two most common inorganic UV filters used in sunscreen formulations are zinc oxide (ZnO) and titanium dioxide (TiO2) particles. They are traditionally used in sunscreens because of their ability to strongly filter UV rays. TiO2 and ZnO bulk form leads to thick unaesthetic formulations leaving unpleasant white marks onto the skin after application. To avoid this nuisance, sunscreens formulators use nanosized forms of these compounds. Moreover, these forms reflect more efficiently UV light. Actually, ZnO and TiO2 nanoparticles permit to develop transparent, fluid and easy-spreading sunscreen formulations. By definition, nanoparticles are particles sizing less than 100 nm. Despite of poor knowledge about biological effects of nanoparticles, we can find them in most of sunscreens. For example, in Australia, it has been estimated that 70% of sunscreens contain TiO2 nanoparticles and 30% of them contain ZnO nanoparticles [58, 59].
Potential toxicity concern about nanoparticles in sunscreens is due to their small size, their ability to evade immunologic defence mechanisms, to form complexes with proteins and to induce ROS formation. Actually, particle toxicity is determined by its surface reactivity. Given their structure, nanoparticles exhibit more important reactivity surface area than larger particles and thus are more likely to generate ROS under UV light catalysing reaction.
Concerns about ZnO and TiO2 nanoparticle toxicity focus mainly on ROS generation because both of these compounds are well-known photocatalysts used notably to generate electricity in photovoltaic cells. When these compounds are exposed to UV light, they emit electrons inducing free radicals, peroxides and others ROS generation . Particle ability to penetrate SC is defined by its molecular size. Intercellular spaces between SC cells measure around 100 nm. These spaces can be modified and notably widened by various topical products [61, 62] or after a stress like UV ray exposition.
Several studies have already reported ZnO nanoparticle cytotoxic effects on different mammalian cell lines, such as on human mesothelioma and rodent fibroblasts , neuroblastoma cells , vascular endothelial cells , human neural cells , human alveolar epithelial cells , and also neural stem cells . Skin barrier represents the first level of exposure to ZnO nanoparticle toxicity after sunscreen use. Toxicity of these particles can occur at two different levels: on cells constituting skin barrier and in a second time on tissues reached from systemic circulation. In the last years, some studies concerning ZnO nanoparticle toxicity on human dermal fibroblasts have been published. It appeared that ZnO nanoparticles (Ø ≈ 20 nm) could trigger human dermal fibroblast apoptosis, through p53-p38, pathway even at the very low concentration of 10 μg mL−1 (Fig. 1) . P38 MAP kinases are a protein family that is activated by genotoxic stresses. When activated, they enhance p53 activity by phosphorylation and leads to cell cycle arrest or apoptosis. It has also recently been tested bulk (Ø ≈ 550 nm) and nanoparticulate ZnO (Ø ≈ 60 nm) phototoxicity in Caenorhabditis elegans nematode . Results showed that nanosized and bulk ZnO phototoxicity are significantly enhanced under natural sunlight and also that ZnO nanoparticles seem to be more likely to induce phototoxicity than bulk ZnO particles.
Figure 1. Schematic representation of a possible toxicity model of ZnO nanoparticles on human epidermal cells according to Meyer et al. study . ZnO nanoparticles are macropinocytosed by skin cell. They induce ROS formation in cell cytoplasm. Oxidative stress leads to MAPK p38 and p53 expression increase. P38 MAPK phosphorylates p53 triggering its activation. p53 MAPK activated trigger mitochondrial pathway apoptosis of skin cell.
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There is a way to reduce undesirable ZnO particle photoreactivity in sunscreens. These particles can be modified notably coating them with organic (such as alkoxy titanates, silanes and methyl polysiloxanes) or inorganic (such as alumina, silica and zirconia) elements and doping them with manganese ions [71, 72]. Particle coat forms hydrated oxides, which can capture hydroxyl radicals and thus reduce phototoxicity minimizing or eliminating potential reactivity of photoactivated ZnO particles by quenching and/or reducing ROS generated before they interact with skin components.
In 2011, Cao et al. demonstrated that capping ZnO nanoparticles using organically modified silica permits to avoid their photocatalytic activity .
The extent ZnO cutaneous toxicity is related to nanoparticle transport within the stratum corneum then into basal cells.
Nowadays, studies concerning ZnO nanoparticle ability to cross normal or altered skin barrier tend to demonstrate that this kind of particles cannot reach living skin cells [6, 74-77].
In 2009, Filipe et al. studied ZnO nanoparticle penetration into normal and psoriatic human skin in vivo . Data revealed that following a 2-h application period of ZnO nanoparticles contained in three marketed sunscreens on intact human skin, these particles were only detectable in SC. There were no ZnO nanoparticles in deeper skin layers even after a 48-h exposure of sunscreens under occlusion. Thus, this study suggests that ZnO nanoparticles cannot reach viable skin tissues. It was also observed that hair follicles were preferential accumulation sites for these ZnO nanoparticles. However, this study was carried out using only three different hydrophobic marketed sunscreen formulations containing coated ZnO nanoparticles and thus results cannot guarantee the same observations using others marketed sunscreens. Some studies reported the lack of nanoparticle penetration in nonfollicular porcine skin  and even on human reconstructed epidermis model [78-81].
Another studies confirmed ZnO nanoparticle harmlessness towards epidermal cells [76, 82].
However, UV radiations can weaken skin barrier by disorganizing intercellular lipid lamellae [83, 84]. Early study demonstrated that tight junction protein expression was disrupted following UVB exposure thus facilitating nanoparticle penetration [85, 86].
It was showed in hairless mice that skin UV-ray exposure led to prostaglandin synthesis [87, 88]. Prostaglandins target E-cadherin receptors and thus prostaglandin synthesis increase led to E-cadherin receptors significant decrease . Cadherin proteins are expressed at cell surface to ensure cell liaisons notably skin cell tight junctions. In vitro studies carried out on hairless mouse epidermis and keratinocytes demonstrated that after UV ray exposure, there was an increase in cyclo-oxygenase 2 (COX-2) protein expression. COX-2 protein expression is induced in the epidermis in response to injuries, such as those triggered by UV radiations. This protein is responsible for arachidonic acid conversion into prostaglandin. Thus, there was an increase in prostaglandin proteins expression and notably of Prostaglandin E2 (PGE2) protein. This protein activated growth stimulatory prostaglandin receptors (EP2) that led to reaction cascade in cell triggering E-cadherin protein expression decrease on cell surface. Consequently, there was a disorganization of cell tight junctions and thus of skin cell adhesions [85, 87, 88]. Skin cell adhesion disorganization after UV ray exposure could permit ZnO nanoparticles to penetrate skin layers and reach living cells. UV ray energy applied in these experiments is not higher than the one from sun rays. Data showed that ZnO nanoparticles were able to disturb skin cell functions through direct contact when skin barrier was disrupted or through hair follicles reaching viable epidermis [89-91].
There is no evidence about ZnO nanoparticle permeation across the skin. Until now, studies tend to demonstrate that this kind of particles cannot cross SC skin layer. Thus, even if it was showed that ZnO nanoparticles trigger deleterious damages on different cell lines notably on human skin fibroblasts, it is not really worrying given that it has not been proved that they can reach living cells.
Skin barrier represent the first level of exposure to TiO2 nanoparticle toxicity after sunscreen use. In actual commercial sunscreens, TiO2 nanoparticle concentrations are between 3% and 15%. Recommended quantity of sunscreen that must be applied to protect the skin is 2 mg cm−2. Thus, an adult should apply 31.25 mg of sunscreen to protect his skin and would be exposed up to 938–4688 mg of TiO2 nanoparticles. A recent study showed cytotoxic and genotoxic effects of TiO2 nanoparticles sizing (50 < Ø <125 nm) on human skin cells in vitro . Maximal TiO2 concentration tested was of 0.080 mg mL−1, which is far less than concentrations used in sunscreens. TiO2 nanoparticle mechanism of toxicity resided in their ability to induce ROS generation due to their photounstable character leading to DNA epidermal cell damages (Fig. 2). Wamer et al. have already reported cytotoxicity of 450 nm TiO2 particles on human skin fibroblasts after their photoactivation through UVA irradiation . Human skin fibroblast treatment with microcrystalline TiO2 particles followed by irradiation with UVA resulted in cytotoxicity with human skin fibroblasts significant reduction. Data also revealed that RNA isolated from treated fibroblasts contained high levels of photo oxidation products namely hydroxylated guanine bases. In 2007, Rampaul et al. assessed UVA dependant cytotoxicity of various TiO2 nanoparticles (11 < Ø < 30 nm) directly extracted from marketed sunscreens on a human skin cell line . Some of these TiO2 nanoparticles induced real phototoxic activity on human skin cells after UVA exposure. This study clearly demonstrated the ability of TiO2 nanoparticles found in commercialized sunscreens to cause skin cell damages. This could lead to DNA skin cell mutation apparition that will be passed to daughter cells and thus to tumour development.
Figure 2. Schematic representation of a possible toxicity model of TiO2 nanoparticles on human epidermal cells according to Shukla et al., Rampaul et al. and Tiano et al. studies [93, 95, 96].TiO2 nanoparticles are macropinocytosed by skin cell. They induce ROS formation in cell cytoplasm. Oxidative stress leads to DNA damages and membrane lipid peroxidation that enhance cell apoptosis.
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But, it seems that TiO2 particle cytotoxicity depends on particle size and crystal form. Anatase TiO2 particle form sizing between 15 and 120 nm gave more OH radicals in vitro than rutile and amorphous forms sizing between 17 and 400 nm after UVA irradiation . Considering that photocatalytic activity of TiO2 anatase form is approximately 1.5 times higher than the rutile one, these results are logical . This is quite reassuring considering that TiO2 crystalline rutile form is used in sunscreens .
Another study showed that TiO2 particles themselves were cytotoxic on human bronchial epithelial cells without being activated by UVA radiations . Data demonstrated that cell treatment with 10 μg mL−1 of anatase TiO2 nanoparticles (10 < Ø < 20 nm) led to oxidative stress and thus to DNA damages without UVA irradiation, whereas anatase and rutile TiO2 particles (≥200 nm) did not lead to such cytotoxicity. In conclusion, the smaller TiO2 nanoparticles, the higher potency of oxidative stress in absence of photoactivation. ROS formed by UV-exposed TiO2 particles are able to damage proteins, lipids and DNA they are in contact with. Applying sunscreen formulation containing TiO2 nanoparticles, skin barrier and notably nucleated cell constituting it, will be the first line where deleterious damages will happen.
To reduce their photoreactivity, TiO2 particles can be coated with organic or inorganic elements doped with manganese ions [71, 72]. A recent study with coated TiO2 particles photocatalytically inactive, but still providing protection against UV damaging sunlight radiations showed minimal DNA damages on yield cultures under UV illumination . Despite this process, Dunford et al. showed that some TiO2 particle coatings did not permit to prevent phototoxicity leading to plasmid DNA and human skin fibroblast damages . It has also been demonstrated that some TiO2 particle coatings such as TiO2/Al2O3 or TiO2/SiO2 enhanced photoreactivity . These results were confirmed by Rampaul et al. in 2007 showing that some coated inorganic UV filters incorporated into actual sunscreens, such as a mixed anatase and rutile crystal form of TiO2 with a dimethicone or organosilane coating, was still destructive leading to photocatalytic human skin and others, animal epithelium DNA cell damages triggering their apoptosis, whereas others particle types, such as manganese doped or alumina-coated rutile TiO2 particles protected the skin without being phototoxic . More recently, Tiano et al. demonstrated that TiO2 particle anatase form was cytotoxic on human skin fibroblasts either coated or uncoated . Moreover, this cytotoxicity was more aggressive after fibroblast exposition to UVA radiations. However, several previous studies demonstrated that TiO2 particle anatase form was also cytotoxic without fibroblast exposition to UVA radiations [98, 102]. Tiano et al. concluded that coating treatments, including organic compounds, were improper for sunscreen elaboration. Actually, this kind of coating enhanced TiO2 particle phototoxicity. However, it appeared that aluminia/dimethicone, aluminia/manganese and aluminia hydroxide coatings were well adapted to sunscreens preventing TiO2 particle phototoxicity. Importantly, it appeared that some modified TiO2 particles specially developed and marketed for sun care and skin care still exhibited photocatalytic activity .
All these studies concerning TiO2 nanoparticle toxicity on human skin cells would be relevant only if these particles could cross SC barrier. It is then interesting to focus on studies about TiO2 nanoparticle absorption through skin barrier. In 2009, an in vivo study made on porcine skin demonstrated that TiO2 nanoparticles (4 < Ø < 60 nm) could penetrate skin SC . This study revealed that TiO2 nanoparticle penetration was size and time dependant. Further investigations carried out on hairless mice revealed that after 60 consecutive days of daily TiO2 nanoparticle application at 8 mg cm−2 under dorsal skin, particles crossed the skin to be entrapped notably in liver, spleen and lung using systemic circulation. However, it is important to precise that mice skin is a half thinner than human skin. For percutaneous penetration studies, it is judicious to choose pig skin that show many parallels to human skin . In 2004, Menzel et al. tended to show ultrafine TiO2 nanoparticle ability to cross skin barrier . In this study, penetration tests were carried out on pig skin model using TiO2 nanoparticles (45–150 nm long and 17–35 nm wide) contained into four different marketed sunscreens. Results revealed that TiO2 nanoparticles mainly concentrated at skin surface and in skin SC. TiO2 particles were also found in skin stratum granulosum, but not in stratum spinosum. Thus, this study showed that TiO2 particles present in some marketed sunscreen formulations were able to penetrate SC intercellular spaces reaching stratum granulosum living skin tissue layers.
In 2008, Mortensen et al. studied quantum dots (Ø = 30 nm) penetration across mouse skin beforehand exposed to UV radiations . Results showed quantum dots penetration into viable epidermis and dermis when skin is injured notably by UV rays. This study concludes saying that TiO2 nanoparticles could also penetrate skin barrier. However, they did not use TiO2 quantum dots in these experiments. Thus, it is not possible to extend results obtained to TiO2 nanoparticles.
Some studies tend to demonstrate TiO2 nanoparticle ability to cross skin barrier and reach systemic blood circulation. It is then relevant to wonder if these particles exhibit any toxicity against erythrocytes that are blood dominant cells (99%) and on others blood circulating cells. In 2008, Li et al. assessed TiO2 nanoparticle toxicity on rabbit erythrocytes . It appeared that TiO2 nanoparticles significantly increased erythrocyte sedimentation and agglutination damaging their membranes in a dose-dependent way. Moreover, photonic microscopy analysis of erythrocytes treated with TiO2 microparticles and nanoparticles revealed erythrocyte abnormal shape. Results also exhibited that in presence of TiO2 nanoparticles, lipid peroxidation increased. Erythrocytes are incapable to produce SOD and catalase that are cell enzymes taking charge of generated ROS. Thus, this kind of cells is particularly vulnerable to extraneous toxicants and their cell membrane could be easily damaged during lipid peroxidation occurring in presence of TiO2 particles.
It also showed genotoxicity and cytotoxicity of TiO2 nano (90 < Ø < 110 nm) and bulk particles due to their capacity to generate ROS on human blood circulating lymphocytes leading to deleterious damages in their DNA .
These results are very controversial. Actually, some studies demonstrated that this kind of insoluble nanoparticles cannot cross normal or even altered human skin [6, 74-77].
Nohynek et al. tended to demonstrate that TiO2 nanoparticles do not penetrate healthy or altered skin. Moreover, their results tended to show that this kind of nanoparticles incorporated into sunscreens do not present any risk to human health [6, 74].
In 2007, Pinheiro et al. in vivo study reported that TiO2 nanoparticle (contained in a commercial sunscreen formulation) permeation profile was similar in human normal skin and in human psoriatic skin . However, in human normal skin, TiO2 nanoparticles seemed to be retained at SC outermost corneocyte layers, whereas in human psoriatic skin, SC fragility seemed to facilitate TiO2 nanoparticle penetration inside SC, although they did not reach living cell layers. Thus, results presented in this study tended to demonstrate that TiO2 nanoparticles do not penetrate deeper into altered human skin.
In 2009, Filipe et al. also studied TiO2 nanoparticle penetration into human normal skin and human psoriatic skin in vivo . It was observed the same results like for ZnO nanoparticles. There was not TiO2 nanoparticles in deeper skin layers even after exposure to sunscreen under occlusion. Hair follicles were also showed to be preferential accumulation sites for these particles. Some studies reported the lack of nanoparticle penetration in nonfollicular porcine skin  and even on human reconstructed epidermis model [79-81, 90].
Gopee et al. showed that TiO2 nanoparticle subcutaneous administration did not produce toxicity or unexpected body distribution . Theogaraj et al. showed that TiO2 nano and micro particles were safety, no causing cytotoxicity, genotoxicity or phototoxicity on Chinese Ovarian Cell (CHO) cultures .
However, UV radiation effects on skin barrier [83, 84] organization have to be taking into account considering that tight junctions can be altered permitting TiO2 nanoparticles to penetrate easily into living skin cell layers [80, 84, 85, 91, 92].
Mixed ZnO/TiO2 powders were showed to be destructive on skin cells. This is not very surprising given that ZnO is a photocatalyst  able to enhance TiO2 photocatalytic activity , but it is quite worrying given that many marketed sunscreens contain both ZnO and TiO2 nanoparticles.
As it was observed for ZnO nanoparticles, there is no evidence about TiO2 nanoparticle permeation across the skin.
Considering all these studies, ZnO and TiO2 nanoparticles seem to be susceptible to enhance deleterious damages on living cells. Crossing this biological barrier, these particles could reach systemic blood circulation with cytotoxic effects on blood circulating cells before to be entrapped into vital organs. ZnO and TiO2 nanoparticle damage cell mechanisms rely on their photoinstability leading to ROS generations that are highly cytotoxic. However, most of studies concerning these nanoparticles tend to demonstrate that they cannot cross skin barrier. Thus, sunscreens containing TiO2 and ZnO nanoparticles seem to pose no risk to human health.
The lack of standardized methods to study ZnO and TiO2 nanoparticle cytotoxicity made these studies controversial. In the present time, there are no definite agreements in the literature on whether these nanoparticles, regardless their size, present in sunscreens penetrate skin barrier entering in the epidermis or not. It is then not possible to certify ZnO and TiO2 toxicity in sunscreens.