Arthropods can transmit pathogens and infect other organisms, contributing to endemics worldwide (Benelli, 2015; Benelli and Mehlhorn, 2016). Aedes aegypti acts as a vector for Zika, Dengue, Chikungunya, and Yellow Fever and becomes a point of convergence for all four diseases, critically spreading these arthropod-borne viruses. This mosquito is very well adapted to urban spaces due to its heterogeneous and long-lived population (Brady and Hay, 2020). In addition to adapting to urban environments, Aedes aegypti is a versatile vector capable of hosting and transmitting Dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) viruses simultaneously in a single inoculation (Rückert et al., 2017; Caron et al., 2012; Dupont-Rouzeyrol et al., 2015; Estofolete et al., 2019; Carrillo-Hernández et al., 2018). Co-infection with different DENV strains can cause more severe clinical manifestations than those observed with mono-infection (Dhanoa et al., 2016). For example, co-infection could increase viremia and trigger immune system syndromes such as Guillain-Barr (Donalisio et al., 2017).

Repellent compounds are important in the prevention of insect-borne diseases. Among a variety of compounds validated as repellents, the most widely used are N,N-diethyl-meta-toluamide (DEET), IR3535 (Ethyl butyl-acetylamino propionate) and Icaridin (Picaridin), the latter being the most effective against Aedes aegypti (Abdel-Ghaffar et al., 2015). Additionally, natural repellents have been widely used for indoor repellency; for example, Eugenol (4-Allyl-2-Methoxyphenol), a volatile compound found in clove and cinnamon oil, and Geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol), present in Citronella essential oil (Chen and Viljoen, 2010). Repellents are commonly used in high concentrations to ensure long-lasting, low-requirements reapplications, such as Icaridin used in concentrations of 25% to 50% (Stefani et al., 2009). For DEET, concentration variations ranging from 4.7% to 95% of the active ingredient are reported in formulations registered by the U.S. Environmental Protection Agency (Patel et al., 2016). However, user exposure to high concentrations of these active ingredients may increase the likelihood of toxic effects.

Many strategies have been used to improve the pharmaceutical properties of repellent compounds, such as drug delivery systems using micro- and nanoparticles (Tavares et al., 2018). Some of these delivery systems have already been explored in drug solutions (Wang et al., 2013) and agriculture (Fraceto et al., 2016). Zein, a botanical protein extracted from maize, demonstrates noteworthy potential in the development of nanoparticles due to its intrinsic attributes, which encompass reproducibility, biodegradability, biocompatibility, and its capacity for encapsulating bioactive substances along with both hydrophobic and hydrophilic compounds onto its surface (Pascoli et al., 2018). In this context, nanomaterials hold the potential for successful optimization in topical formulations with active ingredients.

Orthogonal and multiparametric approaches provide a richer scenario for determining toxicity profiles. Validated methods rely on classic cytotoxicity assessment techniques such as OECD Guideline 432, which uses the Neutral Red assay (OECD, 2019), and OECD Guideline 491, Short-Time Exposure Test (STE) (OECD, 2020a), which uses the MTT reduction assay (Borenfreund and Puerner, 1985; Mosmann, 1983). In addition to the technical limitations of these methods, there are still interferences caused by nanomaterials in their underlying mechanisms, which can lead to misinterpretations (Azhdarzadeh et al., 2015; Tournebize et al., 2013). Multiparametric assays account for more than one response in a single assay, allowing simultaneous responses to the same stimulus observed. For example, the Cell Painting Assay is based on cellular phenotypic profiling using fluorescent markers with organelle-specific affinities. Data are acquired using fluorescence microscopy, and with CellProfiler and CellProfiler Analyst software, it is possible to segment the images and perform single-cell analysis, extracting up to 1500 features per cell (size, shape, texture, fluorescence intensity) (Bray et al., 2016). These features can be used to generate a phenotypic profile capable of discriminating subtle changes in treated cells. The improvement and development of in vitro methods to assess nanomaterials are crucial, as are the concepts of more comprehensive and complex strategies to more accurately predict the fate and realistic risks that nanomaterials pose to our organisms and environment.

Although geraniol and icaridin have established uses and zein nanoparticles are considered safe to ingest, combining these components in a new formulation has received a limited evaluation. In this work, we combined different biological models, concentrations, exposures, and methods through an oriented hazard and risk assessment approach to investigate the contribution of zein nanoparticles to the cytotoxicity of the formulation. For the hazard determination, we evaluated the intrinsic toxicity potential of each component of our formulation. For the risk assessment, we considered different exposure scenarios depicting the use of repellents: isolated (single, 1 day), consistent (repeated, 6 h daily up to 5 days), and accidental (eye contact).