Evaluation of Silver Nanoparticle Toxicity in Skin in Vivo and Keratinocytes in Vitro

Introduction Products using the antimicrobial properties of silver nanoparticles (Ag-nps) may be found in health and consumer products that routinely contact skin. Objectives This study was designed to assess the potential cytotoxicity of Ag-nps in human epidermal keratinocytes (HEKs) and their inflammatory and penetrating potential into porcine skin in vivo. Materials and Methods We used eight different Ag-nps in this study [unwashed/uncoated (20, 50, and 80 nm particle diameter), washed/uncoated (20, 50, and 80 nm), and carbon-coated (25 and 35 nm)]. Skin was dosed topically for 14 consecutive days. HEK viability was assessed by MTT, alamarBlue (aB), and CellTiter 96 AQueous One (96AQ). Release of the proinflammatory mediators interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) were measured. Results The effect of the unwashed Ag-nps on HEK viability after a 24-hr exposure indicated a significant dose-dependent decrease (p < 0.05) at 0.34 μg/mL with aB and 96AQ and at 1.7 μg/mL with MTT. However, both the washed Ag-nps and carbon-coated Ag-nps showed no significant decrease in viability at any concentration assessed by any of the three assays. For each of the unwashed Ag-nps, we noted a significant increase (p < 0.05) in IL-1β, IL-6, IL-8, and TNF-α concentrations. We observed localization of all Ag-nps in cytoplasmic vacuoles of HEKs. Macroscopic observations showed no gross irritation in porcine skin, whereas microscopic and ultrastructural observations showed areas of focal inflammation and localization of Ag-nps on the surface and in the upper stratum corneum layers of the skin. Conclusion This study provides a better understanding Ag-nps safety in vitro as well as in vivo and a basis for occupational and risk assessment. Ag-nps are nontoxic when dosed in washed Ag-nps solutions or carbon coated.

Historically, silver (Ag) compounds have been used in numerous fields to prevent microbial growth. Like many non essential heavy met als, Ag is a natural biocide, but compared with titanium, zinc, and copper, Ag nano particles (Agnps) show the highest anti microbial efficacy against bacteria, viruses, and other eukaryotic micro organisms (Gong et al. 2007). The Phoenicians coated milk bottles with Ag to inhibit bacterial growth; doctors have adminis tered drops of Ag nitrate solutions to newborn babies to prevent neo natal conjunctivitis (Crede 1881); and Ag sulfa diazine creams have long been consid ered the standard of care for the preven tion of widespread bacterial growth on burn patients' denuded skin (Moyer et al. 1965). Both dietary supplements and homemade varieties of Ag colloids have been sold for decades as a "cureall" for such diseases as tuberculosis, syphilis, scarlet fever, shingles, herpes, pneumonia, and arthritis (National Center for Complementary and Alternative Medicine 2009). Furthermore, advances in nano technology have facilitated the increase of Agcontaining merchandise available to the public, making Ag the most used nano material of all manufactureridentified products in the world (Project on Emerging Nanotechnologies 2009). Products such as room deodorizing sprays, acne creams, cloth ing that prevents body odor, baby wipes, and pacifiers all exploit the natural anti microbial activity of Ag (Project on Emerging Nanotechnologies 2009). In a study inves tigating the release of Agnps from com mercially available sock fabric, Benn and Westerhoff (2008) showed that socks could contain up to 1,360 µg Ag/g per sock and could release as much as 1.3 µg/mL of Ag into distilled water.
Ingestion of Ag can cause argyria, the benign condition characterized by the bluish graying of the skin that occurs through the preferential deposition of Ag in the basal lam ina of soft tissues such as the skin, liver, and spleen (Fung and Bowen 1996) and blood vessels, gastro intestinal tract, liver, and kidney (Danscher 1980). Although argyria is most commonly reported clinically after exces sive Ag ingestion, Ag deposition has been seen after treatment of burned skin with Ag sulfa diazine (Lee and Lee 1994;Marshall and Schneider 1977;Temple and Farooqi 1985). In response to argyria, but not to Ag toxic ity, the National Institute for Occupational Safety and Health (2003) set a daily exposure limit for all forms of Ag at 0.01 mg/m 3 , and the U.S. Environmental Protection Agency (2003) established the oral reference dose at 0.005 mg/kg/day. Studies indicating Ag toxicity exist from as early as 1983, when Rungby and Danscher (1983) showed that Ag salts intra peritoneally adminis tered to rats can accumulate in neurons and in protoplasmic glial cells of the brain and spinal cord. In vitro cell line studies have shown decreased mitochondrial function after expo sure to Agnps in murine neuro blastoma cells (Schrand et al. 2008), hepatic cells , germ line stem cells (Braydich Stolle et al. 2005), human skin carcinoma cells (Arora et al. 2008), and human epidermal kera tinocytes (HEKs) and fibroblasts (Burd et al. 2007). Although in vivo studies have not been performed with Agnps, poly vinyl pyrrolidone stabilized Agnps with a mean size of 25 nm were shown to penetrate into the upper lay ers of the epidermis in excised human skin in static diffusion cells (Larese et al. 2009). Other nano materials, such as quantum dots (QDs) and fullerenes (Rouse et al. 2006;Ryman Rasmussen et al. 2006;, as well as zinc oxide (Cross et al. 2007;Gamer et al. 2006), are able to penetrate into the stratum corneum; thus, examination of the ability of Agnps to penetrate the skin is warranted.
Our objectives in this study were to determine the optimal viability assay for use with Agnps in order to assess their toxicity

Evaluation of Silver Nanoparticle Toxicity in Skin in Vivo and Keratinocytes in Vitro
IntroductIon: Products using the antimicrobial properties of silver nanoparticles (Ag-nps) may be found in health and consumer products that routinely contact skin. objectIves: This study was designed to assess the potential cytotoxicity of Ag-nps in human epidermal keratinocytes (HEKs) and their inflammatory and penetrating potential into porcine skin in vivo. MaterIals and Methods: We used eight different Ag-nps in this study [unwashed/uncoated (20, 50, and 80 nm particle diameter), washed/uncoated (20, 50, and 80 nm), and carbon-coated (25 and 35 nm)]. Skin was dosed topically for 14 consecutive days. HEK viability was assessed by MTT, alamarBlue (aB), and CellTiter 96 AQueous One (96AQ). Release of the pro inflammatory mediators interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) were meas ured. results: The effect of the unwashed Ag-nps on HEK viability after a 24-hr exposure indicated a significant dose-dependent decrease (p < 0.05) at 0.34 µg/mL with aB and 96AQ and at 1.7 µg/mL with MTT. However, both the washed Ag-nps and carbon-coated Ag-nps showed no significant decrease in viability at any concentration assessed by any of the three assays. For each of the unwashed Ag-nps, we noted a significant increase (p < 0.05) in IL-1β, IL-6, IL-8, and TNF-α concentrations. We observed localization of all Ag-nps in cyto plasmic vacuoles of HEKs. Macroscopic observations showed no gross irritation in porcine skin, whereas microscopic and ultrastructural observations showed areas of focal inflammation and localization of Ag-nps on the surface and in the upper stratum corneum layers of the skin. conclusIon: This study provides a better understanding Ag-nps safety in vitro as well as in vivo and a basis for occupational and risk assessment. Ag-nps are non toxic when dosed in washed Ag-nps solutions or carbon coated.

Ag-nps.
To encompass the variety of manufac tured Agnps on the market, we used eight dif ferent forms of Agnps with different sizes and surface conditions (Table 1). All Agnps used in this study were supplied by nanoComposix (San Diego, CA): commercially used unwashed and uncoated Agnps suspended in deionized water with diameters of 20, 50, and 80 nm ("unwashed"); washed and uncoated Agnps suspended in deionized water with diameters of 20, 50, and 80 nm ("washed"); and commer cially used dried carboncoated Agnps with diameters of 25 and 35 nm ("carboncoated"). The sizes of each type of Agnps were deter mined by the manufacturer and confirmed in this study by dynamic light scattering (DLS) and transmission electron microscopy (TEM).
Both the unwashed and washed Agnps were synthesized by ammonium hydroxidecatalyzed growth of Ag onto 5nm gold seed particles. Concentration of the particles was achieved via tangential flow filtration. The unwashed Agnps solution contained approxi mately 5.55 mg/mL formaldehyde solvent and methanol byproduct from their forma tion. These unwashed Agnps were then ultra centrifuged to obtain the solution super natant for toxicity testing ("as synthesized" super natant). The Agnps were then serially washed with 20 vol equivalents of 2 mM phosphate buffer (pH 7.5) and the 5, 10, 15, and 20 washing permeates were collected. The wash ing permeates contained 20-50 ppb dissolved Ag. The colloidal Agnps were stored at 4°C in the dark. The carboncoated Agnps, synthe sized by pulsed plasma reactor and coated with poly aromatic graphitic carbon, were supplied as a powder and stored at room temperature.
Cell culture. Cryopreserved primary neo natal HEKs (Lonza, Walkersville, MD) were grown in Keratinocyte Growth Medium2 (KGM2; Lonza) in cell culture flasks (75 cm 2 ; 1,000,000 cells) to approximately 80% confluency in a 37°C humidified 5% CO 2 incubator. The cells were passed into clear or black 96well microplates (12,500 cells/well; 200 µL) in which the peripheral wells con tained only KGM2 to prevent the evapora tion of treatment medium. Between 18 and 24 hr later, after reaching approximately 80% confluency, the HEKs were exposed to either KGM2 (control) or serial dilutions of each Agnps type for the following experiments.
Evaluation of best viability assay. A nano particle (nocell) control (nanoparticles in collagencoated wells with no HEKs) and a nanoparticle/cell control (nanoparticles exposed to HEKmetabolized assay dye) were run in parallel with each viability assay as described by MonteiroRiviere et al. (2009) to assess the interactions between the viability assays and the Agnps [see Supplemental Material avail able online (doi:10.1289/ehp.0901398.S1 via http://dx.doi.org/)].
Ag-nps treatment of HEKs. We conducted an initial dose-response study to assess the concentrations of Agnps that could affect HEK after 24hr exposure. Most of the col loidal Agnps tested were supplied in both low volume and concentration, which limited the highest HEK dosing concentration to 1.7 µg/mL. Combined with KGM2 medium, a 1.7 µg/mL solution of the Agnps was serially diluted (1:5) to provide concentrations rang ing from 1.7 to 0.000544 µg/mL. The effect of the Agnps on HEK viability was assessed by three different toxicity assays: 3(4,5dimethyl thiazol2yl)2,5diphenyltetrazolium bro mide ( We tested dosing concentrations of the washed and carboncoated Agnps up to 42.5 µg/mL but saw no response. Additionally, to differentiate the potential cyto toxicity between the particles and the contaminants present in the colloidal solution, HEKs were treated with the "as synthesized" supernatant and the 5, 10, 15, and 20 washing perme ate for 24 hr at concentrations ranging from 1.7 µg/mL to 0.068 µg/mL. Cytokine release. For the concentrations of Agnps that showed toxicity, we conducted cytokine analysis to determine their pro inflammatory potential by assessing the release of interleukin (IL)8, IL6, tumor necrosis factorα (TNFα), IL10, and IL1β; the procedure is described in the Supplemental Material (doi:10.1289/ehp.0901398.S1).
In vivo porcine skin exposure. We com pared the effects of unwashed Agnps with those of washed Agnps in vivo. Assuming the two smallest Agnps could penetrate the skin, the comparison was limited to the 20 and 50nm washed and unwashed samples. Pigs were dosed on the back skin with Agnps solutions ranging from 34.0 to 0.34 µg/mL [for details, see Supplemental Material (doi:10.1289/ehp.0901398.S1)]. Skin was evaluated for erythema and edema according to the Draize system (Draize et al. 1944): for erythema: 0, no change; 1, very slight change; 2, pale red in defined area; 3, definite red in welldefined area; 4, crimson red; and for edema: 0, no change; 1, very slight change; 2, slight change with edges barely defined; 3, moderate change, with area raised 1 mm; 4, severe change, with area raised > 1 mm and extending beyond the exposure area. All  -, No change in absorbance or fluorescence value was found, indicating a lack of interaction between assay and Ag-nps. a No cell control value at which a significant increase in absorbance or fluorescence was observed (p < 0.05). b Cell control value at which a significant increase in absorbance was observed before and after Ag-nps (p < 0.05). c A significant decrease was observed for this data set.
animals were treated humanely and with regard for alleviation of suffering. Microscopic observations. To assess mor phologic alterations during the in vivo study, we harvested tissue samples after the pigs were euthanized and processed them rou tinely for light microscopy [see Supplemental Material (doi:10.1289/ehp.0901398.S1)]. Approximately 1cm sections were evaluated for inter cellular and intra cellular epidermal edema, dermal edema, and inflammation using the following scoring system: 0, no change; slight, inflammation on less than half the sample; moderate, inflammation on half the sample; severe, inflammation on more than half the sample.
Ultrastructural observations. Particle size analysis was conducted with both DLS and TEM to confirm the manufacturer identified diameters and surface characteriza tion [for details, see Supplemental Material (doi:10.1289/ehp.0901398.S1)]. To localize Agnps uptake in vitro, HEKs were grown to approximately 70% confluency in cell cul ture flasks (25 cm 2 ) and treated for 24 hr with each Ag particle at 1.7 µg/mL in KGM2. The cells and skin samples were processed rou tinely for TEM (for details, see Supplemental Material). We viewed all TEM samples on an FEI/Philips EM 208S TEM (FEI, Hillsboro, OR), operating at an accelerating voltage of 80 kV. Additionally, unstained samples were analyzed by Xray micro analysis [energy dispersive Xray spectroscopy (EDS)] with a Hitachi HF2000 FE TEM (Hitachi High Technologies America Inc., Lexington, KY) equipped with an Oxford Instruments INCA EDS (Oxford Instruments, Oxfordshire, UK).
Statistical analysis. We calculated the mean values for HEK percent viability (nor malized by viability) and cytokine concen tration for each treatment and determined the significant differences (p < 0.05) using the PROC GLM procedure (SAS, version 9.1 for Windows; SAS Institute Inc., Cary, NC). When significant differences were found, we performed multiple comparisons using the Tukey's studentized range highest significant difference test, with p < 0.05 as the level of significance. Dunnett's ttest was performed to determine the significance at p < 0.5 of differ ences between control and treatment groups. Data are expressed as the mean ± SEM of two plates (n = 6/plate). Table 2 summa rizes the control studies for the three viability assays, listing the absorbance or fluorescence value for each Agnps type that shows a signifi cant inter action. For MTT, at 1.7 µg/mL both the 25nm ( Figure 1A) and 35nm ( Figure 1B) carboncoated Agnps nano particle/cell con trols show a statistically significant increase in absorbance after exposure of Agnps to cellreacted assay dye. The nano particle (no cell) controls showed a statistically significant increase in absorbance at 1.7 µg/mL with the 20nm unwashed Agnps for 96AQ and MTT but not for aB ( Figure 1C).

Evaluation of viability assays.
All other nanoparticle control data are available in the Supplemental Material (doi:10.1289/ehp.0901398.S1). We saw no significant changes in the fluorescence values for aB for the 20, 50, or 80nm unwashed Agnps (see Supplemental Material, Figure 1a). The unwashed 50 and 80nm Agnps caused a significant increase in absor bance values for MTT and 96AQ but not for aB (see Supplemental Material, Figure 1b,c). The fluorescence value for aB increased sig nificantly at 0.034 µg/mL for both 20 and 50nm washed Agnps and at 1.7 for 80nm washed Agnps (see Supplemental Material, Figure 2a). The absorbance value for 96AQ changed significantly at 1.7 µg/mL for each of the 20, 50, and 80nm washed Agnps (see Supplemental Material, Figure 2b). The 80nm washed Agnps caused a signifi cant increase in absorbance value for MTT at 1.7 µg/mL (see Supplemental Material, Figure 2c). The fluorescence value for aB increased significantly at 1.7 µg/mL for 35nm carboncoated Agnps (see Supplemental Material, Figure 3a). The absorbance value increased significantly for 96AQ at 1.7 µg/mL for 25nm carboncoated Agnps and at 0.034 µg/mL for 35nm carboncoated Agnps (see Supplemental Material, Figure 3b). We observed a significant increase in the absorbance value for MTT at 1.7 µg/mL for 25 and 35nm carboncoated Agnps (see Supplemental Material, Figure 3c).
Ag-nps treatment of HEK. Generally, for concentrations ranging from 0.000544 to 1.7 µg/mL and exposures of 24 hr, treatment with unwashed Agnps resulted in a dose dependent decrease in viability with all three *p < 0.05; nanoparticle/cell controls were compared for each concentration before and after Ag-nps, nanoparticle controls were run with multiple comparisons between concentrations, and each Ag-nps type was assessed independently. In D, different letters denote significant differences for each nanoparticle at each concentration (p < 0.05); each Ag-nps type was assessed independently.   Figure 1D, the 20 and 50nm unwashed Agnps caused a significant decrease in viability at 0.34 µg/mL for both aB and 96AQ and at 1.7 µg/mL for MTT. The 80nm unwashed Agnps caused a sig nificant decrease in viability at 0.34 µg/mL for all three assays. The "as synthesized" supernatant showed a significant decrease in viability at 0.34 µg/mL for the MTT and aB assays and at 1.7 µg/mL for 96AQ. Toxicity was not present for any of the supernatants obtained from the 5, 10, 15, or 20 wash ing permeates. HEKs exposed for 24 hr to washed 20, 50, or 80nm Agnps or the carboncoated Agnps ranging in concentra tion from 0.000544 to 1.7 µg/mL showed no significant decrease in viability with any assay [see Supplemental Material, Figures 4-6 (doi:10.1289/ehp.0901398.S1)].
Macroscopic observations of topically applied Ag-nps on porcine skin. We noted no gross erythema or edema based on the Draize system in any of the treated sites during the entire 14day in vivo study. Treated sites were gray in appearance, representing residual Agnps on the surface of the skin.
Microscopic observations. Morphologic observations of untreated porcine skin exhib ited normal epidermis and dermis ( Figure 3A). Skin dosed daily with Agnps for 14 days exhibited a concentrationdependent response, regardless of particle size or if washed or unwashed. Skin treated with the lowest dosing concentration of 0.34 µg/mL 20nm washed Agnps typically showed a slight intra cellular and inter cellular epidermal edema ( Figure 3B); with 3.4 µg/mL of 20nm washed Agnps there was moderate focal intra cellular and inter cellular epidermal edema and focal epi dermal and dermal inflammation ( Figure 3C). Skin treated with the highest concentration of 20nm washed Agnps (34 µg/mL) showed severe intra cellular and inter cellular epidermal edema with severe focal dermal inflammation (spongiosis), epidermal hyperplasia, and para keratosis. Also, the extension of the rete pegs increased into the superficial papillary layer of the dermis ( Figure 3D).
Results for the 20nm unwashed Agnps [see Supplemental Material, Figure 7 (doi:10.1289/ehp.0901398.S1)] were similar to those for the 20nm washed Agnps. In skin treated with 0.34 µg/mL of 20nm unwashed Agnps, we observed slight intra cellular epider mal edema; with 3.4 µg/mL, moderate intra cellular and inter cellular epidermal edema; and with 34 µg/mL, severe intracellular and inter cellular epidermal edema with focal areas of intra epidermal infiltrates and superficial papillary dermal inflammation.  Ultrastructural observations of HEKs exposed to Ag-nps. Ultrastructural observations of the 20nm washed and 25nm carbon coated Agnps are shown in Figure 4A and B [for all other Agnps, see Supplemental Material, Figure 8 (doi:10.1289/ehp.0901398. S1)]. The HEK controls appeared normal, with prominent nucleus, nucleolus, and mito chondria ( Figure 4C). In HEKs dosed with Agnps of all sizes and surface conditions, Agnps were localized within membrane bound cytoplasmic vacuoles; Figure 4D shows a representative image of internalized Agnps (for all other Agnps electron micro graphs, see Supplemental Material, Figure 9). EDS analysis of HEKs dosed with the 20nm washed Agnps confirmed the presence of Ag in the vacuoles; copper from the grid and gold from the particle seed were present (see Supplemental Material, Figure 10).
The control skin consisted of a normal compact stratum corneum with approxi mately 20-30 layers attached by desmo somes ( Figure 4E). For skin dosed daily for 14 days, all Agnps localization was within or on top of the stratum corneum. TEM images show the presence of Agnps within the superficial layers of the stratum corneum of skin dosed with 34 µg/mL 50nm washed Agnps ( Figure 4F), and on top of the stratum corneum of skin dosed with 20nm washed Agnps [see Supplemental Material, Figure 9H (doi:10.1289/ehp.0901398.S1)]. EDS analy sis conducted in these areas detected Ag, osmium from the post fixation process, and copper from the grid (see Supplemental Material, Figure 10).

Discussion
Agnps have been integrated into hundreds of products that affect the daily lives of mil lions of people in many countries. Their main use is for disinfection in wound care and in products such as odorreducing clothing, acne creams, and face masks. Most of these prod ucts come into direct contact with skin, the largest organ of the human body, and could serve as a potential route for nano particle penetra tion. Therefore, the relationship of Agnps in skin needs to be investigated with particular focus on their irritation potential, toxicity, and penetration into skin and skin cells. In this study we evaluated the cyto toxic potential of Agnps of varying sizes and sur face conditions in HEK cells, their penetrat ing capacity into porcine skin after topical repetitive daily dosing for 2 weeks, and the localization of the Agnps within HEKs and porcine skin.
The use of several viability assays is impor tant to determine the optimal assay to assess Agnps toxicity; therefore, mortality of HEKs after Agnps exposure was evaluated with three different assays that use colorimetric or fluorescent dyes as markers to determine cell viability by assessing cell metabolism.
Nanomaterials, such as singlewalled carbon nanotubes (Zhang et al. 2007), carbon black (MonteiroRiviere and Inman 2006), fuller enes, and QDs (MonteiroRiviere et al. 2009), are capable of interfering with dye and dye products in viability assays through the adsorp tion of cell medium constituents and cyto kines. We assessed the potential interactions between assays and Agnps using nanoparticle and nanoparticle/cell controls, which showed an increase in absorbance and fluorescence val ues at the highest concentration. The increase in absorbance and fluorescence values could cause the toxicity of the Agnps in HEKs to be underestimated. Additionally, the nano particle/ cell control showed that both the 25 and 35nm carboncoated Agnps interfered with the MTT assay at the 1.7 µg/mL concentration  Figure 1A,B). Overall, all assays were affected by Agnps; based on its fluorescence values, aB may be the best viabil ity assay to use when conducting experiments with Agnps.
MTT, aB, and 96AQ viability assays did not show toxicity for the 25 and 35nm carboncoated Agnps or for the 20, 50, or 80nm washed Agnps. All three assays also showed that the 20, 50, and 80nm unwashed Agnps contributed to a decrease in HEK viability 24 hr after exposure to the 0.34-1.7 µg/mL concentrations, but there was no size dependent decrease in viability. However, the difference in toxicity between the unwashed and washed Agnps is inferred to be due to the presence of contaminants in the unwashed solu tion such as formaldehyde, which has shown to have cyto toxic effects on cell culture (Ku and Billings 1984). In the present study, these residual contaminants were removed by the fifth washing step, as indicated by the lack of cell death after exposure to the 5, 10, 15, and 20 washing super natants. We also found that Agnps of different sizes, surface conditions, and synthesis methods are all internalized into membranebound vacuoles in HEKs, without a decrease in viability, after 24 hr.
In vitro cell line studies have shown that 25 µg/mL of 25nm Agnps in murine neuro blastoma cells decreases mitochondrial func tion and causes the production of reactive oxygen species that could potentially contrib ute to neuro degenerative diseases (Schrand et al. 2008). A significant decrease in mito chondrial function has been shown in hepatic cells after single exposures to 15 and 100nm Agnps at concentrations ranging from 5 to 50 µg/mL , in germline stem cells exposed to 10 µg/mL of 15nm Agnps (BraydichStolle et al. 2005), and in HEKs and fibroblasts after exposure to approxi mately 15 µg/mL Agnps extracted from commercially available Agbased wound dressings with Ag content ranging from 13 to 934 µg/cm 2 (Burd et al. 2007). Interactions between Agnps ranging in size from 7 to 20 nm and human skin carcinoma cells showed a decrease in mitochondrial function and the onset of apoptosis at concentrations of 0.78 µg/mL and 1.56 µg/mL, respectively (Arora et al. 2008). The toxic concentrations of the 20, 50, and 80nm unwashed Agnps (0.34-1.7 µg/mL) are slightly more sensitive in HEKs compared with in vitro toxicity stud ies conducted by others in different cell lines, although it is important to consider such fac tors as agglomeration, surface conditions, size, cell lines, and interactions with the assay dye products when comparing across studies.
Keratinocytes produce proinflamma tory cytokines, such as IL8, IL6, TNFα, and IL1β, that serve as mediators for inflam matory and immunologic reactions in skin exposed to irritants (Allen et al. 2000(Allen et al. , 2001a(Allen et al. , 2001bBarker et al. 1991;Corsini and Galli 2000;Grone 2002;MonteiroRiviere et al. 2003;Nickoloff et al. 1991). Although differ ent toxicants may elicit different responses in HEKs, studies in our laboratory have shown cytokine release by HEKs in response to jet fuel exposure (Allen et al. 2000(Allen et al. , 2001a(Allen et al. , 2001bChou et al. 2002Chou et al. , 2003MonteiroRiviere et al. 2003MonteiroRiviere et al. , 2004, multi walled carbon nano tubes (MonteiroRiviere et al. 2005), 6amino hexanoic acidfunctionalized singlewalled carbon nanotubes (Zhang et al. 2007), fuller enes (Rouse et al. 2007), and QDs (Ryman Rasmussen et al. 2006;. The inflammatory potential of Agnps was con firmed by the increases in IL1β, IL6, IL8, and TNFα detected in the media from HEK cell cultures exposed to 0.34 µg/mL of each of the unwashed Agnps. Nanomaterials are also capable of being internalized into cells and penetrating through skin; QD621 have the ability to penetrate into the inter cellular lipid layers of the stra tum corneum of porcine skin ); QD565 and QD655, with diverse physiochemical properties, have been shown to penetrate into the dermis of abraded skin (Zhang and MonteiroRiviere 2008); and derivatized fullerenes are localized within the inter cellular space of the stratum granulosum layer of flexed excised porcine skin (Rouse et al. 2006). Topical application of 26-30 nm zinc oxide in a sunscreen formulation on in vitro human skin localized nano particles in the upper stratum corneum with minimal penetration (Cross et al. 2007), and microfine zinc oxide, with a mean size of 80 nm, and agglomerates of titanium dioxide < 160 nm did not penetrate the porcine stratum cor neum layer of in vitro static diffusion cells (Gamer et al. 2006).
Porcine skin is an excellent model for studying penetration of human skin because its thickness and absorption rates are compa rable to those of human skin (Bronaugh et al. 1982;MonteiroRiviere and Riviere 1996;Reifenrath et al. 1984). In the present study, we were surprised that after 14 consecutive days of topical dosing, the Agnps did not cause any macro scopic irritation, although the gray appearance of the skin due to the deposi tion of Agnps may have masked any subtle signs of erythema. When viewed microscopi cally, focal inflammation and edema increased with an increase in Agnps concentration. The highest concentration consistently caused epidermal hyperplasia with elongated exten sion of rete pegs down into the dermis, which is typical of a chronic irritation reaction as reported with exposure to jet fuels (Monteiro Riviere et al. 2001;Muhammad et al. 2005).
TEM depicted the localization of Agnps only in the super ficial layers of the stratum cor neum, which was similar to results found in a static cell diffusion study (Larese et al. 2009), and suggests that ionic flux into the epidermis could attribute to focal inflammation. Many Agnps that were not bound to the skin were washed away during both the light and elec tron microscopy processing techniques, yet their location is confirmed with other nano particles that were not shown to penetrate into the deeper epidermis (Cross et al. 2007;Zhang and MonteiroRiviere 2008).

Conclusion
This study indicates that toxicity of Agnps in HEKs is influenced by the residual con taminants in the Agnps solutions, and that the Agnps themselves may not be responsible for an increase in cell mortality. Complete characterization of not only the nano particles but also the vehicle is important in order to distinguish between Agnps and contaminant toxicity. Additionally, this study shows that 20, 50, and 80nm washed and unwashed Agnps, as well as 25 and 35nm carbon coated Agnps, interfered and/or reacted with MTT, 96AQ, and aB viability assays and that aB may be the best viability assay because of its lower interference with these Agnps. Because Agnps of several types have been shown to enter cells and remain on the skin, the possibility of Agnps entry into the body through damaged or abraded skin is important to consider, particularly because many Agcontaining products are specialized for wound care. With the ability for Agnps to enter HEKs, their degradation within the cell may create reactive oxygen species that would be damaging to cell machinery and DNA (Arora et al. 2008). We observed focal inflammation, specifically intra cellular and inter cellular epidermal edema, after 14 days of topical application of Agnps to skin. Studies that use longer exposures (several weeks) and Agnps in different vehicles should be car ried out, possibly also investigating effects on compromised skin. Overall, the present study provides knowledge onAgnps toxicity and penetration in vitro and in vivo over 14 days and provides a basis for occupational and risk assessment.