Nowadays, various industries using nanomaterials are growing rapidly, and in particular, as the commercialization and use of nanomaterials increase in the cosmetic field, the possibility of exposure of nanomaterials to the skin of product producers and consumers is increasing. Due to the unique properties of nanomaterials with a very small size, they can act as hapten and induce immune responses and skin sensitization, so accurate identification of toxicity is required. Therefore, we selected silica nanomaterials used in various fields such as cosmetics and biomaterials and evaluated the skin sensitization potential step-by-step according to in-vitro and in-vivo alternative test methods. KeratinoSens^TM cells of modified keratinocyte and THP-1 cells mimicking dendritic-cells were treated with silica nanoparticles, and their potential for skin sensitization and cytotoxicity were evaluated, respectively. We also confirmed the sensitizing ability of silica nanoparticles in the auricle-lymph nodes of BALB/C mice by in-vivo analysis. As a result, silica nanoparticles showed high protein binding and reactive oxygen species (ROS) mediated cytotoxicity, but no significant observation of skin sensitization indicators was observed. Although more studies are needed to elucidate the mechanism of skin sensitization by nanomaterials, the results of this study showed that silica nanoparticles did not induce skin sensitization.

Carbon nanotubes (CNTs) are one of the major types of nanomaterials that have various industrial and biomedical applications. However, there is a risk of accidental exposure to CNTs in individuals involved in their large-scale production and in individuals who use products containing CNTs. This study aimed to evaluate the skin sensitization induced by CNTs using two alternative tests. We selected single-wall carbon nanotubes and multi-walled carbon nanotubes for this study. First, the physiochemical properties of the CNTs were measured, including the morphology, size, and zeta potential, under various conditions. Thereafter, we assessed the sensitization potential of the CNTs using the ARE-Nrf2 Luciferase KeratinoSens™ assay, an in vitro alternative test method. In addition, the CNTs were evaluated for their skin sensitization potential using the LLNA: BrdU-FCM in vivo alternative test method. In this study, we report for the first time the sensitization results of CNTs using the KeratinoSens™ and LLNA: BrdU-FCM test methods in this study. This study found that both CNTs do not induce skin sensitization. These results suggest that the KeratinoSens™ and LLNA: BrdU-FCM assay may be useful as alternative assays for evaluating the potential of some nanomaterials that can induce skin sensitization.

Graphene nanoplatelets (GNPs) are one of the major types of carbon based nanomaterials that have different industrial and biomedical applications. There is a risk of exposure to GNP material in individuals involved in their large-scale production and in individuals who use products containing GNPs. Determining the exact toxicity of GNP nanomaterials is a very important agenda. This research aimed to evaluate the skin sensitization potentials induced by GNPs using two types of alternative to animal testing. We analyzed the physicochemical characteristics of the test material by selecting a graphene nanomaterial with a nano-size on one side. Thereafter, we evaluated the skin sensitization effect using an in vitro and an in vivo alternative test method, respectively. As a result, we found that GNPs do not induce skin sensitization. In addition, it was observed that the administration of GNPs did not induce cytotoxicity and skin toxicity. This is the first report of skin sensitization as a result of GNPs obtained using alternative test methods. These results suggest that GNP materials do not cause skin sensitization, and these assays may be useful in evaluating the skin sensitization of some nanomaterials.

The nano-market has grown rapidly over the past decades and a wide variety of products are now being manufactured, including those for biomedical applications. Despite the widespread use of nanomaterials in various industries, safety and health effects on humans are still controversial, and testing methods for nanotoxicity have not yet been clearly established. Nanomaterials have been reported to interfere with conventional cytotoxicity tests due to their unique properties, such as light absorption or light scattering. In this regard, the colony-forming efficacy (CFE) assay has been suggested as a suitable test method for testing some nanomaterials without these color-interferences. In this study, we selected two types of GNPs (Graphene nanoplatelets) as test nanomaterials and evaluated CFE assay to assess the cytotoxicity of GNPs. Moreover, for further investigation, including expansion into other cell types, GNPs were evaluated by the conventional cytotoxicity tests including the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), Cell Counting Kit-8 (CCK-8), and Neutral red uptake (NRU) assay using MDCK, A549 and HepG2 cells. The results of CFE assay suggest that this test method for three cell lines can be applied for GNPs. In addition, the CFE assay was able to evaluate cytotoxicity regardless more accurately of color interference caused by residual nanomaterials.

Zinc oxide nanoparticles (ZnO NPs) are used in various industries such as food additives, cosmetics, and biomedical applications. In this study, we evaluated lung damage over time by three types of ZnO NPs (L-serine, citrate, and pristine) following the regulation of functional groups after a single intratracheal instillation to rats. The three types of ZnO NPs showed an acute inflammatory reaction with increased LDH and inflammatory cell infiltration in the alveoli 24 h after administration. Especially in treatment with L-serine, citrate ZnO NPs showed higher acute granulocytic inflammation and total protein induction than the pristine ZnO NPs at 24 h. The acute inflammatory reaction of the lungs recovered on day 30 with bronchoalveolar fibrosis. The concentrations of IL-4, 6, TNF-α, and eotaxin in the bronchoalveolar lavage fluid (BALF) decreased over time, and the levels of these inflammation indicators are consistent with the following inflammatory cell data and acute lung inflammation by ZnO NP. This study suggests that single inhalation exposure to functionalized ZnO NPs may cause acute lung injury with granulocytic inflammation. Although it can recover 30 days after exposure, acute pulmonary inflammation in surface functionalization means that additional studies of exposure limits are needed to protect the workers that produce it.

Choi JH, et al. Cancer Res Treat. 2008 Mar;40(1):22-26. http://dx.doi.org/10.4143/crt.2008.40.1.22