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Safety of titanium dioxide and zinc oxide nanoparticles in sunscreens

Introduction

    This scientific report reviewed the safety concerns surrounding zinc oxide (ZnO) and titanium dioxide (TiO2) nanoparticles (NPs) present in sunscreens. It is the second update of the report published by the Therapeutic Goods Administration (TGA) in 2006 and already updated in May 2013. The two main issues considered in this review were the evidence for the ability of these NPs to penetrate the skin and to reach viable cells as well as their potential toxicity.

    The Australian Therapeutic Goods Administration (TGA) has been continuously monitoring the emerging scientific literature in this area and working cooperatively with international regulatory agencies to ensure that appropriate regulatory action is undertaken if any unacceptable risk of harm/toxicity is identified.

    Why are titanium dioxide and zinc oxide used in sunscreens?

      Titanium dioxide (TiO2) has strong UV light absorbing capabilities, which, together with the fact that it keeps its colour and its UV-absorbing properties when exposed to UV light , are the reasons why it is found with zinc oxide (ZnO) in the majority of physical sunscreens for over three decades to protect the skin from ultraviolet light. It is a physical blocker both for UV-A (ultraviolet light with wavelength of 315–400 nm) and UV-B (ultraviolet light with wavelength of 280–315 nm) radiations.

      Why are titanium dioxide and zinc oxide used in sunscreens in the form of nanoparticles?

        One apparent disadvantage of zinc oxide (ZnO) and titanium dioxide (TiO2) is that, in their macro particulate (bulk) form in sunscreens, they are visible on the skin as an opaque layer resulting in reluctance of consumers to use the products. This undesirable visual effect has been addressed by decreasing the particle size of these metal oxides to nanoparticles (NP) form.

        When used in this NP form, these oxides cannot be seen on the skin but retain or even augment their UV-sunscreening properties. These NPs maintain their intrinsic ability to filter ultraviolet light (UV-A as well as UV-B) with a broader protection than any other sunscreening agent. Patents on TiO2 and ZnO NPs were filed in the USA in the 1980s.

        What are the characteristics of sunscreen nanoparticles?

          For the purpose of this report, the definition of TiO2 and ZnO NPs includes materials within the nanosize range of 1 to 100 nm. Nano-sized TiO2 and ZnO exist in three separate states: primary particles (5-20 nm), aggregates (30-150 nm) and agglomerates (1-100 microns).

          Primary particles cluster together to form aggregates and are the smallest units present in a final sunscreen formulation. Larger agglomerates form when aggregates bind loosely during the manufacturing process but these are not efficient UV absorbers so they need to be broken down into the more efficient aggregates, which are chemically bound.

          This review includes assessments of NP preparations that contain aggregates and agglomerates. The latter have been included although not normally found in sunscreen formulations because their yet unclear potential to disaggregate and disagglomerate when applied on the skin in a sunscreen formulation.

          Agglomerates may form on the skin surface after application of sunscreens, suggesting fewer primary NPs would be available for skin penetration. A preliminary experimental study suggested that exposure to sunlight can lead to disaggregation of TiO2 NPs which facilitate their penetration. Thus, if the NP particles aggregate and form structures above the nanoscale, this reduces their ability to penetrate the skin and, likely, any potential hazard linked with nano-sized particles.

          TiO2 NPs (but not ZnO NPs) exist under different crystal forms: the anatase form, which is substantially more photocatalytic and adheres more strongly to skin and the rutile form. A mixture of the rutile and anatase forms are generally used in sunscreens.

          Do titanium and zinc oxide nanoparticles have skin irritation or sensitisation effects?

            In 2007, in a review of photo-protection, neither TiO2 nor ZnO NPs were found to possess notable skin irritation or sensitisation properties when used in sunscreens on humans. Since this safety review, a number of new studies published have further assessed the potential of ZnO NPs or TiO2 NPs to cause skin irritation (reversible skin damage), corrosion (irreversible necrotic damage extending into the dermis) or sensitisation in the absence of UV or non-UV light.

            Zn Oxide

            Experiments demonstrated that ZnO NPs could potentially penetrate the dermis, exert an anti-inflammatory effect on allergic skin but enhance IgE generation. Despite this potential for increasing an allergic response, the authors concluded that the benefits of using sunscreens to combat the development of skin malignancies outweigh the possible risks of exacerbating allergic symptoms, and in other studies it was reported that ZnO NPs failed to cause irritation or sensitisation. In particular, ZnO NPs did not cause acute dermal toxicity or dermal irritation in rabbits at the doses applied and failed to induce skin sensitization in the guinea pigs after an induction phase that involved weekly application of NPs for 3 weeks.

            Based on the results of two studies on topical application of ZnO to intact skin of human volunteers, the European Commission’s Scientific Committee on Consumer Safety (SCCS)1 also concluded that there is still no evidence of any positive findings suggesting photo-irritation and photosensitisation.

            Titanium dioxide

            A dose-dependent increase in ear swelling (used as an indicator of skin irritancy) was observed in female mice after dermal administration of TiO2 NPs but skin sensitisation was not observed. In the same mice strain, dermal pre-treatment with TiO2 NPs exacerbated subsequent sensitisation induced by dinitro-chloro-benzene. However, exposure to TiO2 NPs of a commercially available human 3D skin model composed of human-derived epidermal keratinocytes did not induce signs of dermal irritation.

            Titanium dioxide and Zn oxide combined

            When applied to another 3D human skin model or to intact rabbit skin, TiO2 NPs, ZnO NPs or a mixture of the two (at a final concentration of 25% (w/v)) did not cause irritation or corrosion.

            1 2014 Sunscreens with titanium dioxide as nanoparticles. Health risks?
            The SCCS has published detailed guidance documents on the evaluation of risks of nanomaterials in cosmetics (SCCS/1484/12 and SCCS/1524/13).

            Are titanium and zinc oxide nanoparticles penetrating the skin?

              The findings of the great majority of a large set of in vitro and in vivo studies indicated an apparent inability of TiO2 and ZnO NPs to reach viable cells in the dermis. Given the majority of studies, including with human volunteers, found no evidence of skin penetration of NPs when applied on the skin, it is highly unlikely that the high systemic NP concentrations attained in the experimental animals would be achieved in people, even if accidental intake occurred via these non-dermal routes.

              The SCCS Committee of the European Commission also concluded in 2015 that TiO2 nanomaterials in a sunscreen formulation were unlikely to lead to systemic exposure to nanoparticles through human skin and to reach viable cells of the epidermis, dermis, or other organs2.

              The few in vitro studies that reported findings suggesting that TiO2 NPs could penetrate beyond the stratum corneum suffered from important methodological limitations that put in doubt the validity and extrapolation of these findings to humans. Experimental approaches biologically relevant for nanoparticle-induced hazard and inter-species variability in skin penetration demand also caution when extrapolating positive findings from animal studies to potential for hazard in humans.

              Globally, the currently available evidence from in vitro studies suggested that the likelihood of penetration of TiO2 or ZnO NPs beyond the surface layers into viable cells of the dermis is extremely low, with penetration largely limited to the stratum corneum.

              Observations from in vivo studies

              in vivo and under real-life conditions, there were also concerns relating to the methodology and conduct of a study of sunscreen used by human volunteers over a period of five days through the determination of Zn ion concentration in the blood as evidence of systemic or dermal exposure to ZnO NPs. This was because such blood concentration does not necessarily prove the dermal penetration of, or systemic exposure to ZnO NPs.

              Similarly a review of the issue of altered penetration of nanosized ingredients through damaged skin (i.e. diseased or sunburnt) concluded that penetration through compromised skin was likely to be similar to normal skin and a study suggested that the risk of TiO2 NP penetration into the dermis via hair follicles was very low.

              2 https://ec.europa.eu/health/sites/health/files/scientific_committees/consumer_safety/docs/sccs_o_206.pdf  
              See the summary: Sunscreens with titanium dioxide as nanoparticles. Health risks?

              Can titanium and zinc oxide as nanoparticles induce toxic effects?

                Both ZnO and TiO2 NPs can generate, via UV-induced photocatalysis, reactive oxygen species (ROS) such as superoxide anions or hydroxyl radicals. ROS can damage cellular components and macromolecules such as lipids, proteins and nucleic acids, and ultimately cause cell death if produced in excess or if they are not neutralised by innate antioxidant defences.

                6.1 Via skin exposure

                There is conclusive evidence from in vitro experiments that in the presence of UV light, ZnO and TiO2 nanoparticles can induce reactive oxygen species (ROS), which have the capacity in a wide range of cell types to damage cellular components and mediate cytotoxicity and genotoxicity.

                However, of paramount importance is the finding that the vast majority of studies did not demonstrate NP skin penetration; the current weight of evidence suggested that TiO2 and ZnO NPs do not reach viable skin cells (even in compromised skin) or the general circulation, but rather remain on the skin surface and in the outer layer of the stratum corneum, a surface layer of non-viable, keratinized cells.

                It is therefore highly likely that, if sunscreens are used as is intended, NPs from these sunscreens applied dermally will not achieve significant concentrations in the systemic circulation and suggested thus that the likelihood of NPs causing cytotoxicity or pathology in internal organs or tissues is very low since systemic absorption is highly unlikely.

                6.2. Via other routes of exposure (inhalation or oral absorption)

                There are numerous studies that demonstrated a diversity of potential pathological sequelae upon administration of ZnO and TiO2 nanoparticles to experimental animals via a range of administration routes.

                Orally or intravenously administered, ZnO NPs failed to cross the blood brain barrier in a 28 day repeat dose study conducted in rats; but in a mouse study where NPs were administered orally for 21 days both ZnO and TiO2, NPs were detected within neurons with evidence of oxidative stress in the liver and brain and increased levels of some neurotransmitters. In a study conducted in human volunteers TiO2 NPs were not absorbed systemically after oral administration because nanoparticles agglomerated in the gastrointestinal tract.

                When administered ZnO or TiO2 NPs via oral, intravenous or intraperitoneal routes, evidence of cardiovascular pathology, mild perturbations in some haematological and biochemical parameters, and mild pathological features in the stomach and pancreas, as well as retinal atrophy was observed at the highest dose in rats orally administered. Significant kidney pathology attributed to NP-induced oxidative stress was observed in mice.

                In inhalation studies conducted in rodents, ZnO NPs caused mild acute pulmonary and systemic inflammation, pulmonary cell damage and a reduction in the antioxidant capacity of the lungs while other studies have shown that ZnO NPs cause only minor changes in the lungs including in a chronic inhalational study conducted in mice.

                Meanwhile, endogenous protective mechanisms, such as antioxidant activity mediated by a range of intracellular enzymes and factors, will likely protect against the damaging effects of oxidative stress generated by any exposure to nanoparticles. The minimal dermal penetration of NPs is likely to be adequately counteracted by these natural cellular defences.

                Do zinc oxide and titanium dioxide have genotoxic and/or carcinogenic properties?

                  The genotoxicity and/or mutagenicity of ZnO and TiO2 NPs have been addressed by several studies and reviews. Reactive oxygen species generated by TiO2 or ZnO NP have been documented to induce DNA damage and cytotoxicity in variety of cells types. When applied in vitro to HaCaT cells, TiO2 NPs reduced cell viability and induced membrane damage at concentrations at or greater than 0.7µg/cm2 regardless of the exposure time. Induction of ROS and reduction in cell viability, which were both enhanced by UVB irradiation, were also observed and ZnO NPs (100 nm in diameter) were internalised and induced dose- and time-dependent cytotoxicity with ZnO NPs inducing cell death by apoptosis. Other data suggested that TiO2 NPs could induce apoptosis but could not induce tumorigenic changes in HaCaT cells.

                  DNA damage and clastogenic changes (disruption or breakages of chromosomes) in hepatocytes and other cell types were also observed with ZnO NP administered intraperitoneally to mice. These studies are reported in details in the Literature review3, which constitutes the basis of these highlights

                  In 2003 already, the Scientific Committee Cosmetic Non-Food Products (SCCNFP) to the European Commission already concluded from three unpublished studies that “micronised material (ZnO) has been found to be clastogenic, possibly aneugenic and inducing DNA damage in cultured mammalian cells in vitro, under the influence of UV light”. In the same review, the SCCNFP noted that micronized ZnO was non-photo-mutagenic in the bacterial Ames test but this result has been questioned because as NPs may not penetrate the bacterial cell wall, and this makes that their intrinsic genotoxicity evaluation with bacterial test could be considered as largely not relevant. Nevertheless, the OECD guidance manual on “nanoparticles genotoxicity testing” still includes the Ames assay.

                  In 2012, a comprehensive review by the E.U. Scientific Committee on Consumer Safety (SCCS), which assessed both in vitro and in vivo studies on photo-mutagenicity/genotoxicity of ZnO NPs concluded that there is no conclusive evidence to ascertain whether or not ZnO NPs pose a mutagenic/genotoxic, photo-toxic or photo-mutagenic/genotoxic risk to humans. A similar position was upheld in their review of TiO2 NPs.

                  The authors of the present report noted that this view was confirmed in a review conducted by industry representatives4, which stated that ”in vitro genotoxic and photogenotoxic profiles of these nano-structured oxides are of no consequence to human health.”

                  Meanwhile, in parallel to potential genotoxic effects, ZnO NPs were also shown to affect metabolic parameters, such as glycogenolysis and gluconeogenesis, in hepatocyte cell lines and interfered with the cell cycle in human intestinal epithelial cells. In human gastric epithelial cells, TiO2 NPs induced a pre-malignant phenotype characterised by enhanced DNA damage and proliferation, apoptosis resistance and increased invasiveness. Treatment with ROS scavenger or anti-oxidant was shown to prevent NP-induced cytotoxicity, which highlighted the central role oxidative stress plays in NP-induced cell death pathways.

                  3See chapter 4.3 Genotoxicity, pg 12
                  4 Schilling K, Bradford B, Castelli D, Dufour E, Nash JF, Pape W, Schulte S, Tooley I, van den Bosch J and Schellauf F. (2010) Human safety review of "nano" titanium dioxide and zinc oxide. Photochem Photobiol Sci; 9(4): 495-509.

                  How can the potential toxic effects of titanium dioxide and zinc oxide nanoparticles be reduced in sunscreen products?

                    The potential of ZnO NP to generate ROS can be effectively reduced by coating them or by adding anti-oxidant compounds to the sunscreen formulation. Indeed, the inherent cytotoxic properties of ZnO NPs were markedly reduced by curtailing the release of Zn2+ ions and decreasing the contact area of the ZnO NP by the TiO2 NPs shell.

                    • Coating materials include aluminium hydroxide (Al(OH)3), polymers and inert oxides of silica, while anti-oxidant compounds include vitamins (e.g. A, E, C). It was demonstrated that coatings based on silicon dioxide were also highly effective in ameliorating UV-induced TiO2 NP derived ROS. ZnO particles dispersed in medium-chain triglycerides were shown also experimentally to reduce ROS formation;  
                    • It has been shown, however, that, under certain circumstances, coating of NPs can be disrupted or stripped by alteration of the integrity of the Al (OH)3 surface layer, principally by Ca2+ and OCl ions, which are present for example in swimming pool water. ). 

                    What are the main conclusions of this updated Literature Review?

                      The global conclusion of the present Literature review was that there is conclusive in vitro evidence that in the presence of UV light and when coming into contact with water rich in Ca2+ and OCl- ions, such as swimming pool water, ZnO and TiO2 NPs can induce reactive oxygen species (ROS), which have the capacity to damage cellular components. ZnO NP- and TiO2 NP-mediated cytotoxicity and genotoxicity have indeed been demonstrated in a wide range of cell types. In addition, there were numerous studies that demonstrated a diversity of potential pathological effects upon administration of ZnO and TiO2 NPs to experimental animals via a range of administration routes.

                      However, when coated with silica or alumina, the potential of these NPs to produce ROS was largely supressed, making their use as sunscreens via such coating very safe.

                      In the meantime, the majority of studies (both in vitro studies using animal and human skin, and in vivo studies) have shown that both ZnO and TiO2 nanoparticles either did not penetrate or minimally penetrate the stratum corneum and underlying layers of skin, and suggested that systemic (whole body) absorption, hence toxicity, was highly unlikely.

                      Based on this current evidence, the conclusion of the report was that neither ZnO nor TiO2 NPs were likely to cause harm when used as ingredients in sunscreens. On the contrary, the current state of knowledge strongly indicated that the minor risks potentially associated with NPs in sunscreens were vastly outweighed by the benefits that NPs-containing sunscreens afford against skin damage and, importantly, skin cancer.

                      The report also highlights that in 2012, the Scientific Committee on Consumer Safety (SCCS) of the European Commission concluded “in sunscreens, ZnO nanoparticles can be considered to not pose any risk of adverse effects in humans after application on healthy, intact or sunburnt skin”. A similar position was upheld in their review of TiO2 NPs.


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