5. Do ZnO nanoparticles pass through the skin?
Dermal/percutaneous absorption
In vitro studies for dermal penetration of human skin
Exploratory in vitro study for percutaneous skin penetration
Study Design
Date of publication: 1997
Guideline/method: Exploratory percutaneous skin penetration study in vitro.
Test system: Abdominal human skin.
Test substance: Titanium dioxide T805 (Degussa, Germany), and Spectra veil MOTG (Tioxide specialties, UK), a 60% dispersion of zinc oxide in mineral oil/triglyceride.
Particle size: Mean crystalline length 116.8 nm with a standard error of 8.5 nm.
Batch: Not stated
Dose level: Water in oil (W/O) emulsion similar to commercial sunscreen formulation with ultrafine titanium dioxide (11% wt), and zinc oxide (2.5% wt), dose of formulation 1mg/cm2.
Skin preparation: Abdominal human skin from plastic surgery.
Cells: Not stated
Skin temperature: Room temperature
Test chamber: Not stated
Route: Topical administration
Exposure time: Not stated
Sampling time points: Not stated
GLP: No
Published: Yes
Characterization of the mineral content of sunscreen formulations containing both TiO2 and ZnO was performed. The distribution of TiO2 and ZnO at the surface of human stratum corneum was investigated in vitro. Human abdominal skin obtained from plastic surgery was exposed to a W/O emulsion containing 11% ultrafine titanium dioxide and 2.5% ultrafine ZnO at 1 mg/cm2 at room temperature. Transmission electron microscopic cross-sections of the horny layer of human epidermis showed an almost regular mineral coating of the stratum corneum but neither intercellular nor intracellular ZnO penetration was noted.
Results
This study analysed the distribution of the ZnO nanoparticles in the original preparation and in the cosmetic formulation. It showed the presence of the nanomaterial on the surface of the skin and a lack of nanoparticle uptake by the cells of the stratum corneum of the skin.
(Reference: 40)
Comment
For the ZnO used in this study, information on surface area and number of particles per mass was not provided. Information on dose expressed as surface area and number of particles was not provided.
Although this study provides some evidence that there is no penetration of the nanoparticles from the formulation into the skin, the information on the study itself is rather limited, e.g. time of incubation and surface area of treated skin were not indicated. From the discussion it appears that a mixture of TiO2 and ZnO nanoparticles was used in the formulation. This study is of no value for the evaluation of skin penetration of ZnO nanoparticles.
Exploratory in vitro study for percutaneous skin penetration
| Study Design | |
| Date of publication: | 2009 |
| Guideline/method: | Exploratory percutaneous skin penetration study in vitro. |
| Test system: | Human skin |
| Test substance: | Z-COTE® Max, coated with dimethoxydiphenylsilanetriethoxy-caprylylsilane, cross-polymer. |
| Particle size: | Not stated |
| Batch: | Not stated |
| Dose level: | Water in silicone (W/Si) emulsion and water in oil (W/O) emulsion with 1% ZnO, 2 mg/cm2 of each emulsion. |
| Skin preparation: | Excised human skin from female Caucasian patients from abdominal plastic surgery, after verification of integrity and removal of subcutaneous fat, storage at –25°C for a maximum of six months prior to use. |
| Cells: | Six intact membranes |
| Skin temperature: | 32±1°C |
| Test chamber: | Static diffusion cells (Franz-type) |
| Route: | Topical application |
| Exposure time: | 24 hours |
Sampling time points: 1, 2, 4, 6, 8, 12 and 24 hours
GLP: No
Published: Yes
Two different types of emulsion; water in silicone (W/Si) and water in oil (W/O), each containing 1% ZnO, were tested. Previously frozen dermatomed human Caucasian skin from surgery was put on the static diffusion cells and, after stabilization of the receptor fluid at a temperature of 37 ± 1°C to achieve a skin temperature of 32 ± 1°C, 2 mg/cm2 of each formulation was applied to the skin membrane (six/formulation).
Receptor fluid was sampled at 1, 2, 4, 6, 8, 12 and 24 hours after application. Thereafter, the skin samples were removed, rinsed to remove residual formulation, homogenized and processed for analysis. The content of Zn++ ions in the skin was analyzed by means of Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES).
Results and conclusion
The recovery rates for ZnO after application of the W/O and W/Si emulsions after application to the skin were 76% and 86%, respectively. For the W/O emulsion, slightly more ZnO was recovered from in the skin compared to the recovery from on the skin (about 40 and 35%, respectively). For the W/Si emulsion the amount of ZnO recovered in the skin was about the same as the amount recovered from on the skin. No penetration through the skin could be observed with a limit of detection of 0.01 ppm and a limit of quantification of 0.1 ppm for Zn++, respectively in the W/O and the W/Si emulsions.
(Reference: 39)
Comment
The ZnO particles tested were not characterized, and data on particle size, surface area and number of particles per mass was not provided. Information on dose expressed as surface area and number of particles was not provided.
Exploratory in vitro study for percutaneous skin penetration
Study Design
| Date of report: | 2007 |
| Guideline/method: | Exploratory study on human skin penetration of sunscreen nanoparticles in vitro. |
| Test system: | Human skin |
| Test substances: | A) ZnO formulation A: dispersion with 60% of siliconate coated ZnO in caprylic capric triglyceride. |
| B) ZnO typical sunscreen formulation B: O/W emulsion sunscreen with 20% of siliconate coated ZnO in caprylic capric triglyceride. | |
| C) O/W emulsion without ZnO. | |
| Particle size: | 15–40 nm |
| Dose level: | 10 μL/cm2. |
| Skin preparation: | Full thickness skin from female donors following abdominoplasty cleaned by a heat-separation technique. |
| Replicates: | A and B) eight samples, C) three samples. |
Test chamber: Static glass diffusion cell (Franz type, exposed area: 1.3 cm2, receptor phase volume: 3.5 mL).
Route: Topical application
Exposure time: 24 hours
Sampling time points: 12 and 24 hours
GLP: No
Published: Yes
Experimental design
The possible in vitro penetration of human skin was investigated using sunscreens (described as a novel micronized formulation with ZnO) containing nano-sized ZnO. The formulations tested included: (A) ZnO dispersion made with 60% siliconate coated ZnO in caprylic capric triglyceride; (B) a typical O/W emulsion sunscreen with 20% ZnO siliconate coated ZnO in caprylic capric triglyceride; and (C) a control (blank) O/W emulsion sunscreen without ZnO. Particle size determination was carried out before application of the coating using four different techniques identified as transmission electron microscopy (TEM), Brunauer-Emmett-Teller nitrogen-gas absorption method (BET), X-ray diffraction (XRD) and photon correlation spectroscopy (PCS). Human skin samples were from females following abdominoplasty. Epidermal samples were prepared from full thickness tissue. These membranes were mounted in static, horizontal Franz-type diffusion cells with an exposed surface area of approximately 1.3 cm2 . Treatment with the formulations A (n=8), B (n=8) and C (n=3) involved application of 10 μL/cm2 and collection of receptor fluid samples at 12 and 24 hour intervals. The receptor fluid was analyzed for the presence of zinc by ICP-MS and electron microscopy (EM) was used to examine the tissue samples.
Results and conclusion
The analysis of particle size generation following MCP technology (high-energy dry milling) revealed similar results to the detection methods; TEM (15-40 nm), BET (30 nm), XRD (26 nm) and PCS (30 nm).
Results indicated penetration (over 24 hours) of zinc into the receptor fluid from untreated and placebo treated epidermal membranes as; 0.09 ± 0.04 and 0.22 ± 0.12 μg/cm2, respectively. The amount of zinc found in the receptor fluid following application of the two test formulations was higher (although not statistically significant) after 24 hours exposure and the total amount found in the receptor phase was less than 0.03% of the applied dose. Analysis with electron microscopy revealed penetration of ZnO nanoparticles limited to the outer surface of the stratum corneum (SC) and loose, desquamating cells of the upper SC only. There was no evidence of penetration of nanoparticles in the lower SC layers or viable epidermis.
Comment
For the ZnO used in this study, information on surface area and number of particles in dose was not provided. Information on dose expressed as surface area and number of particles was not provided.
In this publication, siliconate-coated ZnO was investigated as a 60% dispersion and as a typical sunscreen preparation containing 20% ZnO. The Zn in the receptor fluid was determined by elemental analysis of Zn using ICP-MS. Thus, it was not determined whether the low amount of Zn detected in the receptor fluid was present as solubilized Zn ions or as ZnO particles.
In the publication, the penetration of Zn++ using sunscreen formulations containing nano-particulate ZnO is reported to be very low (40–10 times lower) compared to other cosmetic formulations in the literature containing ionic zinc. It is stated in the publication that this supports the idea that nanoparticle formulation decreases the absorption of ZnO across the skin which is suggested to be due to a reduction in the amount of available solubilized Zn on the skin surface.
Based on the detection of Zn in the receptor fluid, the SCCS considers that 0.03% of the applied dose may be absorbed after topical application of ZnO on the skin.
Exploratory in vitro study for percutaneous skin
Study Design
Date of report: 2008
Guideline/method: Exploratory study on human skin penetration of sunscreen nanoparticles in vitro.
Test system: Human skin (excised abdominal or breast human skin from plastic surgery).
Test substance: Commercial sunscreen (Dr. Lewinn’s private formula (19% W/W) with 26–30 nm mean size ZnO particles suspended in caprylic capric triglycerides (preservatives: 0.3% phenoxyethanol, 0.3% hydroxybenzoate).
Dose applied: 0.3 g of commercial product (see comment below).
Route: Topical application
Exposure time: 2–24 hours
GLP: No
Published: Yes
The distribution of topically applied nano-sized ZnO (mean primary particle size: 26–30 nm) in excised human skin after application of a commercial sunscreen was examined using multiphoton microscopy (MPM) imaging with a combination of scanning electron microscopy (SEM) and an energy-dispersive X-ray (EDX) technique. Abdominal or breast skin obtained following plastic surgery was used.
Results and conclusion
The cross-sectional imaging showed no evidence of nano-sized ZnO penetration into the cells or extracellular space.
(Reference: 120)
Comment
For the ZnO used in this study, information on surface area and number of particles per mass was not provided. Information on dose expressed as surface area and number of particles was not provided.
It is not stated in the publication whether the ZnO particles were coated or uncoated, but they appear to have been uncoated.
This paper also includes an in vivo experiment in humans (see 3.3.4.3). In the in vivo study 0.3g of the sunscreen formulation was applied to a skin surface of 50 cm2 and rubbed in for 5 minutes. From the study description, it is not clear if an equivalent dosing was used in the in vitro experiment.
In vitro studies for dermal penetration of non-human skin
Exploratory in vitro study for percutaneous skin penetration
Study Design
Date of publication: 2006
| Guideline/method: | OECD 428 and Guidance Document No. 28 and EU SCCNP opinion SCCNP/0750/03. |
| Test system: | Porcine skin |
| Test substance: | ZnO (Z-COTE uncoated), mean size of 80 nm (90% of the particles had a size below 160 nm). |
| Batch: | Not stated |
| Dose level: | Test formulation 4 mg/cm2, ZnO 400 μg/cm2 (Zn 360 μg/cm2). |
| Skin preparation: | Skin from the lateral abdominal region of five month old domestic pigs of the Pietrain-Deutsche Landrasse-Hybrid strain, dermatomed to a thickness of around 500 µm. |
| Cells: | Eight intact skin preparations, each from three pigs + two vehicle controls + two untreated controls from each animal. |
| Skin temperature: | 32 ± 1°C |
| Test chamber: | Static diffusion cells (Franz-type). |
| Route: | Topical application |
| Exposure time: | 24 hours |
Sampling time points: 3, 6, 12 and 24 hours
GLP: Yes
Published: Yes
The study was performed to evaluate possible skin penetration in vitro using porcine skin. Full skin thickness (epidermis and dermis) samples were incubated with ZnO preparations in modified Franz static dermal penetration cells.
The zinc oxide formulation was a white viscous oil/water emulsion, containing 10.3 wt% zinc oxide (corresponding to 8.3 wt% of zinc) as in an actual sunscreen formulation. The Z-COTE, uncoated microfine zinc oxide, had a mean primary particle size of 80 nm with 90% of the particles being <160 nm. Primary particles and loose agglomerates were present exclusively in the water phase and primary particles were often arranged around oil droplets.
ZnO test formulations were applied on the skin and samples of the receptor fluid were obtained at 3, 6, 12 and 24 hours. After 24 hours incubation the skin samples were removed and the presence of ZnO on the surface was evaluated using tape stripping. Zn levels in the samples (pooled tape strips, skin remaining after tape stripping, and all retained receptor fluid samples) were evaluated using Atomic Absorption Spectroscopy (AAS) or Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS) depending on the concentration of the samples.
Results
The total Zn recoveries ranged from 102% to 107% with almost the total amount recovered in the first five tape strips.
The amounts of zinc found in the skin membrane and the receptor fluid were comparable in untreated, vehicle treated or zinc oxide treated skin preparations. In the receptor fluid, the levels observed for Zn for the skin samples of the three pigs were 0.8%, 0.8% and 1.4% of the applied dose, respectively. The absorbed dose was 1.5%, 1.6%, and 2.3% of the applied dose. Background levels in the receptor fluid of vehicle treated skin were similar for samples from three different pigs. In the skin, the Zn levels were 1.4%, 1.5% and 1.5%, respectively. Untreated or vehicle treated skin preparations contained 3–5 μg of zinc corresponding to about 1% of the applied dose.
(Reference: 48)
Conclusion
From the data, the authors concluded that there was no penetration of ZnO particles or solubilized Zn through the stratum corneum of pig skin.
Comment
For the ZnO used in this study, information on surface area and number of particles per mass was not provided. Information on dose expressed as surface area and number of particles was not provided.
The mean size of the ZnO (Z-COTE uncoated) under investigation was 80 nm. It is unclear whether this mean size given is mass-based or number-based. It is also not known how the material used in this test relates to the Z-COTE formulation presented in the dossier as the size information given differs.
Exploratory in vitro study for percutaneous skin penetration
| Study Design | |
| Date of publication: | 2011 |
| Guideline/method: | Exploratory percutaneous skin penetration study in vitro after UVB radiation in vivo (sunburn simulation). |
| Test system: | Skin of weanling Yorkshire pigs. |
| Test substances: | O/W sunscreen formulations. |
| A: Z-COTE HP1 (5% coated, CM 643; mean size 140 nm; SSA 12–24 m2/g). | |
| B: Z-COTE (5% uncoated, CM 644; mean size 140 nm; SSA 12–24 m2/g). | |
| Batch: | Not stated (source: BASF SE, Germany). |
| UVB exposure: | Fiber optic UVB lamp (Lightningcure 200 UV-Spot light), on the day the pig was sedated and the hair clipped. The minimal erythemic dose (MED) was determined by sequential exposure to UVB light (30–120 mJ/cm2, 6 – 24 seconds). On day 2 the pig was sedated and the exposed sites were analyzed to determine the UVB dose required to produce a minimal erythema response. The MED was determined to range between 40 and 50 mJ/cm2. |
| UVB dose: | 100, 110 and 120 mJ/cm2 (to obtain a +2 erythema) |
| Dose level: | 50 μL of each formulation on 0.64 cm2 dermatomed pig skin A 2.5 mg dose of ZnO corresponds to a dose of 0.03–0.06 m2, surface area on 0.64 cm². |
| Skin preparation: | Dermatomed pig skin (400–500 μm) biopsied 24 hours after the second UVB exposure inducing sunburn (= consistent +2 erythema). |
| Cells: | Formulation A and B: four with UVB exposed skin, two with unexposed skin. |
| Control: | Two with UVB exposed skin, two with unexposed skin. |
Skin temperature: 37°C
Test chamber: Flow-through diffusion cells.
Route: Topical application
Exposure time: 24 hours
Sampling time points: Every 2 hours for the first 12 hours, every 4 hours thereafter up to 24 hours.
Examinations: Light microscopy (LM)
Transmission electron microscopy (TEM) plus X-ray microanalysis (EDS)
Scanning electron microscopy (SEM)
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) GLP: No
Published: Yes (abstract)
The purpose of this study was to determine whether skin damaged by UVB radiation inducing moderate sunburn with a +2 erythema reaction enhanced the penetration of TiO2 or ZnO nanoparticles present in these sunscreen formulations. The pig was sedated and multiple sites (about 52) on the back were exposed to the UVB dose that caused a consistent +2 erythema (a pale red in a defined area of the skin). Twenty-four hours after UVB exposure, the pig was sedated, sites visually analyzed for consistency, and the skin prepared for in vivo or in vitro studies. For the in vitro studies, the UVB exposed and non exposed sites were dermatomed to a thickness of approximately 400–500 μm. The dermatomed skin was mounted in the flow-through diffusion cells with a dosing area of 0.64 cm2 and maintained at 37°C. The skin was dosed with 50 μ L of each formulation (CM 643: n=4 UVB exposed skin, n=2 unexposed skin; CM 644: n=4 UVB exposed skin, n=2 unexposed skin; and control: n=2 UVB exposed skin, n=2 unexposed skin). Upon completion of dosing, the perfusion was resumed and the perfusate collected every 2 hours for the first 12 hours and every 4 hours thereafter up to 24 hours. After 24 hours, the perfusion was terminated and the skin was removed from the diffusion cells. The dose site was removed with an 8 mm biopsy punch and cut into thirds. One third was placed in Trump’s fixative and stored at 4°C for later processing by light microscopy (LM; flow-through 1 and 2 only) and transmission electron microscopy (TEM). The remaining third of the skin was cut in half and immediately frozen and stored at -20°C for later elemental analysis. The vials containing perfusate from each timed collection were capped and the samples immediately stored at 4°C.
Results
Light microscopy showed that UVB exposed skin showed focal intracellular epidermal edema, sunburn cells, dermal inflammation and focal microblister. In unexposed (UV light) skin residual sunscreen containing ZnO was limited to the stratum corneum. The morphology of the normal and the UVB exposed skin was not affected by topical treatment with the sunscreen formulations. The ZnO in each formulation was confirmed by TEM and elemental analysis. Energy dispersive X-ray spectroscopy (EDS) confirmed the presence of Zn indicating ZnO (from sunscreen) and Cu (from sample grids) in CM 643 and CM 644. In the pig skin from the flow-through studies, TEM/EDS found ZnO nanoparticles only on the surface of the stratum corneum on both the unexposed and UVB exposed skin, and first layers of the stratum corneum. Elemental analysis on the SEM confirmed the presence of ZnO within the stratum corneum and epidermis of the skin in the sunscreen treated samples. TOF-SIMS data indicated that ZnO did focally penetrate into the epidermis in both the normal and photo-damaged skin treated with the sunscreen formulations. However, background Zn interference was present in some of the map overlays as determined by a region of interest analysis and the authors were not sure what caused the background in some samples.
Conclusion
Under the conditions of this study, UVB damaged skin did not enhance the penetration of Zn nanoparticles. TOF- SIMS indicated that Zn focally penetrated into the epidermis in both the UVB exposed and unexposed skin treated with the sunscreen formulations, but the observation remained inconclusive as background interference was also present. It was demonstrated by SEM and TEM that ZnO nanoparticles remained on the surface and upper stratum corneum layers in UVB exposed and unexposed skin, while ZnO particles were found to penetrate into the stratum corneum by TEM and Zn into the epidermis as demonstrated by TOF-SIMS. In any case, there was no evidence from these optical methods that the nanoparticles penetrated into the perfusate.
(References: 75, 76, AR18)
Comment
For the ZnO used in this study information on number of particles per mass was not provided. Information on dose expressed as number of particles was not provided.
It is not clear whether the mean size given is mass-based or number-based. It is also not known how the material used in this test relates to the Z-COTE formulation presented in the dossier as the size information given differs.
Exploratory in vitro study for percutaneous skin penetration
Study Design
Date of publication: 2009
Guideline/method: Exploratory percutaneous skin penetration study in vitro using chemical enhancers for skin penetration.
Test system: Skin of ten month old nude mice.
Test substances: Purpose made ZnO nanoparticles, size ~10 nm as determined by TEM.
Batch: Purpose made
Chemical enhancers: Ethanol (EtOH) and oleic acid (OA) as enhancers for altering stratum corneum lipid structure; control sample, PBS.
Dose level: 50 μL of each formulation on 0.64 cm2 dermatomed pig skin.
Skin preparation: Removal of subcutaneous fat and mounting skin in Franz diffusion cells.
Test chamber: Franz diffusion cells
Route: Topical application with 400 μL of donor solution.
Exposure time: 12 hours
Examinations: Dual channel multi photon microscopy
GLP: No
Published: Yes
This study investigated mechanistical aspects of chemically enhanced penetration into the stratum corneum using skin samples of four ten month old nude mice (BALB/c). ZnO nanoparticles were prepared from commercial zinc acetate dehydrate with a primary particle diameter of around 2 nm. However, an average particle diameter of 10 nm was measured. The absorption and emission peaks were reported at 370 and 525 nm. The non-linear polarization effect of second harmonic generation (SHG) was used for ZnO nanoparticles to be distinguished from the autofluorescence (AF) of the stratum corneum (SC) using dual-channel multi-photon microscopy. The study combined the SHG of ZnO nanoparticles and the AF of the SC to image the transdermal delivery of ZnO nanoparticles under the chemical enhancer conditions of oleic acid (OA), ethanol (EtOH) and oleic acid–ethanol (OA–EtOH). In addition to qualitative imaging, the microtransport properties of ZnO nanoparticles were quantified to give the enhancements of the vehicle to skin partition coefficient (K), the SHG intensity gradient (G) and the effective diffusion path length (L).
Results
The multilamellar lipid regions between the corneocytes were the pathways for ZnO nanoparticle delivery. With oleic acid, the transport of ZnO nanoparticles into the stratum corneum was facilitated by the phase-separated oleic acid domains, while the ethanol enhancer leached a significant amount of non- covalently bound amphiphilic stratum corneum lipids to modulate the skin barrier. As regards the oleic acid–ethanol donor solution, the increase of stratum corneum lipid fluidity associated with the oleic acid enhancer was offset by the effect of ethanol in loosening the stratum corneum lipid structure. Among the different chemical enhancer conditions, the oleic acid–ethanol combination was regarded as the most effective donor solution in transdermal delivery of ZnO nanoparticles into the stratum corneum.
Conclusion
The results showed that OA, EtOH and OA–EtOH were all capable of enhancing the transdermal delivery of ZnO nanoparticles compared to the PBS control by increasing the intercellular lipid fluidity or extracting lipids from the stratum corneum.
(Reference: 66)
Comment
For the ZnO used in this study information on surface area and number of particles per mass was not provided. Information on dose expressed as surface area and number of particles was not provided.
Apparently only penetration into the stratum corneum, but not into deeper skin layers was investigated.
In vivo studies for dermal absorption
Exploratory in vivo study for percutaneous skin penetration
| Study Design | |
| Guideline/method: | Comparative study according to internal laboratory methodology considering real use conditions, recommendation of US FDA and COLIPA SPF requirements. |
| Species: | Human |
| Group sizes: | TiA: eight volunteers (normal skin). |
| TiB: nine volunteers (normal skin). |
TiHB: eight volunteers (normal skin)
TiB: ten volunteers (stripped skin and occlusive patches).
Six volunteers for basal elemental concentration in the skin.
Test substances: Commercial products containing coated nano-sized ZnO:
TiA: contained only TiO2
TiB: contained TiO2 plus ZnO.
TiHB: contained coated rutile TiO2, size 20 nm.
Particle size: Spherical ZnO, size 20–60 nm.
Dose applied: Realistic use condition: 0.5–1.0 mg/cm2 on an area of 25 cm2.
SPF testing method: 2.0 mg/cm2 on an area of 25 cm2.
Skin: Intact and tape-stripped human skin.
Skin temperature: 37°C
Exposure period: 2 hours (intact skin), 48 hours (tape-stripped skin).
GLP: No
Date of report: 2009
Published: Yes
The localization and possible skin penetration of nanoparticles dispersed in three sunscreen formulations under realistic use conditions was comparatively investigated in normal and altered skin. Commercial products containing nano-sized particles of coated TiO2 and ZnO dispersed in hydrophobic emulsions were used. One product contained only TiO2 (TiA), another TiO2 plus ZnO (TiB) and a third material (TiHB) contained nanoparticles of the coated rutile form of TiO2. The size and shape of nanoparticles in the three formulations were inspected with transmission electron microscopy and X-ray microanalysis. The ZnO nanoparticles were spherical and their size ranged from 20–60 nm. The application took into account relevant application conditions, such as the quantity of sunscreen applied and the duration between the applications recommended by the US FDA. The protocol consisted of an open test. The formulations were applied on the sacral region and buttocks for 2 hours using a sunscreen application rate of approximately 0.5–1.0 mg/cm2 within an area of 25 cm2 to reflect realistic use conditions. This condition was compared to standard FDA and COLIPA methods for the sun protection factor test (SPF), which recommend a thickness of sunscreen application of 2.0 mg/cm2. TiB was applied to nine individuals, and TiA and TiHB to five individuals each. Penetration was also investigated in ten individuals under exaggerated exposure conditions, such as application to the skin after tape stripping and under occlusive patches (IQR chamber, 48 hour application). Tape stripping consisted of a series of strips until the tapes were free of corneocytes. A matched control group consisting of six individuals was used for the determination of basal elemental concentrations in the skin including Zn. Skin punch biopsies of 3 mm diameter were taken after application, quench-frozen and kept in containers until processing. One biopsy was taken from each volunteer. Sections of 14 μm thickness were cut from the frozen biopsy in a cryostat at - 25°C. Biopsies were mounted in mounting medium for microscopy (OCT™ compound). Sections were obtained from the non-immersed portion of the tissue, and sectioning performed from inside to outside to avoid tissue contamination. Tissue integrity and the efficacy of corneocyte removal after tape stripping were checked by preparing intercalary stained sections for optical microscopy purposes. Scanning Transmission Ion Microscopy technique and Particle Induced X-ray Emission technique were used for detection of the localization of the ZnO particles by analyzing the presence of elemental Zn. The minimum detectable concentration of Zn in the skin was 0.10 μmol/g.
Results
The coverage of the outer skin layer with the TiB sunscreen formulation was homogeneously distributed. No differences were observed between the recommended SPF procedure (2.0 mg/cm2) and the realistic use condition (0.5–1.0 mg/cm2). Sunscreen formulations accumulated in skin wrinkles and depressions, as well as infundibulum cavities, but exogenous Zn remained on the outer layers of the keratinized tissue that enfold the follicle, i.e. outside the living skin. The penetration profiles of the nanoparticles obtained with the treated skin groups (TiA, TiB and TiHB) were all similar. The high levels of Zn observed at the outer layers of stratum corneum sharply decreased within the deeper layers to very low levels (Zn) in both protocols (SPF and realistic use condition).
By quantitatively integrating the imaged skin areas, the Zn concentration was extremely elevated in the stratum corneum (exogenous Zn). However, the Zn values decreased to physiological levels in the subcorneal epidermis and finally remained within a narrow interval of variation in this compartment with no differences between both application procedures.
The fit estimate of the Zn concentration decrease confirmed that most of the nanoparticles were confined to the stratum corneum outer layers and showed no significant penetration into the living cell layers. At approximately 80% of full stratum corneum thickness, the Zn concentration decreased to the physiological Zn level of <0.3 μmol/g. For the depth positions where Zn nanoparticles penetration ended, an estimated error of 10% was obtained which approximately corresponds to 0.5 μm for both application procedures.
Under non-physiological conditions using occlusive patches, there was no significant difference in the distribution and penetration depth profiling of the ZnO nanoparticles.
Conclusion
The results of the study demonstrated that profiles and localization of Zn nanoparticles could be accurately established in vivo. Following the 2 hour exposure period to a nano-sized ZnO-containing sunscreen on intact human skin using realistic use and SPF methods, detectable amounts of this physical UV-filter were only present at the skin surface and in the upper most stratum corneum regions. Layers deeper than the stratum corneum were devoid of exogenous Zn, even after 48 hours exposure to the sunscreen under occlusion. Deposition of ZnO nanoparticles in the openings of the pilosebaceous follicles was also observed but penetration of nanoparticles into viable skin tissue could not be detected.
(Reference: 46)
Comment
For the ZnO used in this study information on surface area and number of particles per mass was not provided. Information on dose expressed as surface area and number of particles was not provided.
Exploratory in vivo study for percutaneous skin penetration
Study Design
Guideline/method: Exploratory study on human skin penetration of sunscreen nanoparticles.
Species: Human
Group size: Four volunteers (two Caucasian males, one Indian male, one Chinese female).
Test substances: Commercial sunscreen (Dr. Lewinn’s private formula (19% W/W) with 26–30 nm mean size ZnO particles suspended in caprylic capric triglycerides (preservatives: 0.3% phenoxyethanol, 0.3% hydroxybenzoate).
Dose applied: 0.3 g of commercial product.
Skin area: 50 cm2 (forearm, cheek, shoulder or feet).
Skin: Cleaned but otherwise untreated skin.
Route: Topical application
Exposure time: 2–24 hours
GLP: No
Date of report: 2008
Published: Yes
The distribution of topically applied ZnO to human skin was examined by multiphoton microscopy (MPM) imaging with a combination of scanning electron microscopy (SEM) and an energy-dispersive X-ray (EDX) technique. A commercial sunscreen product containing nano-ZnO (mean particle size 26–30 nm) was used in this study. An area of 50 cm2 of skin was selected on the forearm, cheek, shoulder or feet of four volunteers from different ethnic backgrounds. The selected area of skin was cleaned with ethanol prior to the investigation. An amount of approximately 0.3 g of the test sunscreen was applied to the selected skin areas and rubbed in for 5 minutes. Images were generated immediately, 4 hours after application and 24 hours after topical application. Application of the commercial sunscreen product was followed by an incubation period of 2 to 24 hours. Sections of treated skin were either tape-stripped (10–20 times) or left untouched prior to analysis.
Results
The MPM analysis showed that nano-ZnO particles stayed on the stratum corneum (SC) and accumulated into skin folds and/or hair follicle roots of human skin. The nano-sized ZnO predominantly remained on the outermost surface within a several-micrometer layer at all analysis points (at application, and after 4 and 24 hours). No penetration of nano-sized ZnO into the cells or extracellular space was observed and the 24 hour analysis showed complete removal of sunscreen from the skin. The test material localized in the hair follicle shaft did not spread to the neighbouring cells and tissue. High intensity, high resolution analysis (SEM/EDX) confirmed no noticeable presence of nano-sized ZnO in the epidermis. It was noted that nano-sized ZnO existed predominantly in an agglomerated phase.
Conclusion
In this study, it was demonstrated that after application of a commercial sunscreen formulation to human skin, the nano-sized ZnO remained either on the skin surface or stayed within the stratum corneum.
(Reference: 120)
Comment
For the ZnO used in this study information on surface area and number of particles per mass was not provided. Information on dose expressed as surface area and number of particles was not provided.
It is not stated in the publication whether the ZnO particles were coated or uncoated.
Exploratory in vivo study for percutaneous skin penetration
Study Design
Guideline/method: Exploratory study on human skin penetration of sunscreen nanoparticles.
Species: Human
Group size: 20 volunteers (11 in the nano-group, nine in the non-nano "bulk” group).
Test substance: ZnO powder enriched to >99% 68Zn. Half of the stock was used to make nanoparticles with a final crystallite size of about 19 nm (± 8 nm; minimum 3 nm, maximum 60 nm) using a proprietary method based on high-energy attrition milling. Larger particles were also prepared with an average crystallite size of 110 nm (± 46 nm; minimum 25 nm, maximum 284 nm) produced by a modified version of the same method. The uncoated particles were incorporated into an oil-water formulation using a commercial process for preparing sunscreens. Both sunscreens contained ~20% wt/wt 68ZnO particles.
Dose applied: Mean dose 4.3 mg/cm2 (range 2.8–5.8 mg/cm2) twice daily for five consecutive days (4.6 mg/cm2 for the males and 3.7 mg/cm2 for the females).
Skin area: 6 cm2 on the back
Skin: Back skin
Route: Topical application
Exposure time: Sampling at 24 hours each day and at 24 hours after day 5.
Evaluation: Blood and urine samples, before, after and during the trial.
GLP: No
Date of report: 2010
Published: Yes (References: 59, 60, AR10, AR27)
An exploratory dermal penetration study was performed in human volunteers using the isotope approach for tracing potential absorption of Zn from ZnO nanoparticles applied to human skin under conditions of normal use. In total, three trials were performed. The first trial involved two male subjects with two applications to their backs of a formulation containing ZnO nanoparticles (with diameters of ~30 nm) enriched to 51% with 68Zn. The second trial involved a female in addition, and the same formulation was applied twice daily for five days. Blood was sampled in both trials at regular intervals for up to 126 days. These trials formed the basis and protocol refinement for the main trial in which particles of ZnO enriched to >99% with 68Zn were incorporated into a different formulation (Reference: AR10). Two groups, each consisting of ten people of various ages, skin classifications, and race, participated in the study at a beach. One group of ten volunteers was tested with a sunscreen containing nanoparticles of 68ZnO (about 20 nm) – the "nanoparticle” group. The other group was tested with particles of 68ZnO (>100 nm) – the "bulk” group. Sunscreen was applied to the backs of the volunteers twice daily for a period of five days and the subjects experienced a minimum of 1 hour UV exposure in two episodes following sunscreen application. Blood was sampled twice daily and urine three times daily. Blood and urine samples were also supplied before the five day beach exposure and in a follow-up period. Zinc was purified from blood and urine samples by ion exchange procedures. Changes in the isotopic abundance of 68Zn of the purified samples measured by the multi-collector were used to evaluate the dermal absorption of Zn from the sunscreens.
Results
The results from the first two trials showed changes of <0.1% in the isotope ratios in blood. The authors estimated that this limits the dermal absorption to <0.1%. In the beach trial, changes in the 68Zn/64Zn ratio in blood samples for the nanoparticle group ranged from 0.1 to 0.8% at the end of the beach trial and all subjects showed significant increases in the abundance of 68Zn six days after the completion of the trial. The changes in blood samples for the bulk group were similar to those for the nanoparticle group. Excluding the data for two outliers, there was no statistically significant difference in dermal absorption for the volunteers in the nanoparticle and the bulk groups; the mean increase was about 0.4%. However, in females a significant difference was observed between the nanoparticle and the bulk groups; the nanoparticle group absorbing more Zn. Urine samples showed larger increases in abundance of 68Zn over the same time intervals, but there was no simple relationship with changes in blood for the same volunteers. For the pretreatment (zero measurement) samples there was a very low variation between the 68Zn content in the blood between all volunteers. After ZnO exposure, an increase in variation was observed. An increase in Zn levels was also found in urine. There was a delay in the detection of 68Zn in the blood as it was detected for the first time at day 2 after the fourth application of the sunscreen in four individuals with a more rigorous follow up evaluation. The increase continued after stopping the application until at least day 11, which was the last day the samples were evaluated.
Conclusion
The authors concluded that these results provided evidence that Zn from ZnO particles in sunscreen penetrated healthy human skin. An additional factor for the Zn uptake might be the composition of the sunscreen formulation as it contained isopropyl-myristate, a known chemical enhancer of skin penetration. The total amounts of Zn absorbed are rather small when compared to the amounts of natural Zn normally present in the human body. For the male group with the highest absorption, the amount of Zn isotope tracer originating from the applied sunscreen ranged from 8.6–30.8 μg. These small amounts are in contrast to the average amount of Zn present in whole blood under physiological conditions (about 12 mg). It should also be noted that the blood values are very low in relation to the recommended daily values for the dietary intake of Zn of 8 mg for females and 11 mg for males as indicated in the paper. Overall, the amount of 68Zn tracer detected in the blood post-trial represents less than 0.001% of the applied dose. The study did not investigate whether the translocating Zn was present as nanoparticles or soluble Zn ions.
Comment
For the ZnO used in this study information on surface area and number of particles per mass was not provided. Information on dose expressed as surface area and number of particles was not provided.
The important findings of this study are: Zn uptake via healthy human skin was demonstrated; the total uptake was very low compared to the normal Zn levels in the body; there was a gender difference with females absorbing slightly more than males; and there was a delay in detection between application and detection time. The main limitation of the study is that it did not investigate whether the 68Zn was absorbed as ZnO nanoparticles or as 68Zn ions. The SCCS considers that the Zn that originated from the topically applied ZnO containin sunscreen was only a fraction of the amount of Zn present in the overall blood zinc pool. The pilot study preceding this exploratory study was published in 2012 (AR27). In the pilot study on three subjets a delayed absorption of Zn was observed which information was used for the design of the final study (AR10).
(References: 59, 60, AR10, AR27)
Discussion
Dermal absorption
The applicants have performed and provided a comprehensive review and assessment of the available in vivo and in vitro dermal penetration studies. None of the studies or projects yielded any evidence that nano-sized ZnO particles are able to cross the skin barrier in intact or compromised skin. The literature data and the data which were provided for this submission suggest that there is only minimal absorption and resulting systemic availability of zinc from application of ZnO nanoparticles containing sunscreens on the skin. Whether this zinc is available as zinc oxide nanoparticles or zinc ions has not been determined. These studies include numerous in vitro (using human, porcine and nude mice skin) and in vivo human volunteer studies. In studies that analysed particles, no penetration beyond the stratum corneum was seen. In studies that analysed Zn, small amounts were detected in deeper skin layers and receptor fluid/blood. Zn could only be detected in one out of seven of the in vitro studies evaluated, and was detected in the receptor liquid by elemental analysis with ICP-MS indicating some passage (maximally 0.03% of the applied dose) of the skin barrier. It was shown that some Zn may pass the skin barrier in a human volunteer study in which a small fraction of the blood Zn-pool was demonstrated to originate from a dermally applied sunscreen preparation. No major differences were seen between coated/uncoated ZnO nanomaterials, and no significant differences were observed between damaged skin versus normal healthy skin.
In view of the discussion above, it is assumed that penetration of the skin, if any, is caused by Zn ions released from ZnO nanoparticles. Therefore the solubility of ZnO is one of the critical parameters that should be considered in the characterization of ZnO used for sunscreen formulations.
The Three-Level Structure used to communicate this Scientific Committee on Consumer Safety (SCCS) opinion is copyrighted by Cogeneris SPRL.