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Walter Brand,1,* Cornelle W Noorlander,1,* Christina Giannakou,2,3 Wim H De Jong,2 Myrna W Kooi,1 Margriet VDZ Park,2 Rob J Vandebriel,2 Irene EM Bosselaers,4 Joep HG Scholl,5 Robert E Geertsma2 1Centre for Safety of Substances and Products, 2Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, 3Department of Toxicogenomics, Maastricht University, Maastricht, 4Section Pharmacology, Toxicology and Pharmacokinetics, Medicines Evaluation Board (CBG-MEB), Utrecht, 5Research & Analysis Department, Netherlands Pharmacovigilance Centre Lareb, ‘s-Hertogenbosch, the Netherlands *These authors contributed equally to this work Abstract: Due to their specific properties and pharmacokinetics, nanomedicinal products (NMPs) may present different toxicity and side effects compared to non-nanoformulated, conventional medicines. To facilitate the safety assessment of NMPs, we aimed to gain insight into toxic effects specific for NMPs by systematically analyzing the available toxicity data on approved NMPs in the European Union. In addition, by comparing five sets of products with the same active pharmaceutical ingredient (API) in a conventional formulation versus a nanoformulation, we aimed to identify any side effects specific for the nano aspect of NMPs. The objective was to investigate whether specific toxicity could be related to certain structural types of NMPs and whether a nanoformulation of an API altered the nature of side effects of the product in humans compared to a conventional formulation. The survey of toxicity data did not reveal nanospecific toxicity that could be related to certain types of structures of NMPs, other than those reported previously in relation to accumulation of iron nanoparticles (NPs). However, given the limited data for some of the product groups or toxicological end points in the analysis, conclusions with regard to (a lack of) potential nanomedicine-specific effects need to be considered carefully. Results from the comparison of side effects of five sets of drugs (mainly liposomes and/or cytostatics) confirmed the induction of pseudo-allergic responses associated with specific NMPs in the literature, in addition to the side effects common to both nanoformulations and regular formulations, eg, with liposomal doxorubicin, and possibly liposomal daunorubicin. Based on the available data, immunotoxicological effects of certain NMPs cannot be excluded, and we conclude that this end point requires further attention. Keywords: adverse effects, drug safety, immunotoxicity, nanomedicine, nanotoxicology, pharmacovigilance

Christina Giannakou,1,2 Margriet VDZ Park,1 Wim H de Jong,1 Henk van Loveren,1,2 Rob J Vandebriel,1 Robert E Geertsma1 1Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, 2Department of Toxicogenomics, Maastricht University, Maastricht, the Netherlands Abstract: Nanomaterials (NMs) are attractive for biomedical and pharmaceutical applications because of their unique physicochemical and biological properties. A major application area of NMs is drug delivery. Many nanomedicinal products (NMPs) currently on the market or in clinical trials are most often based on liposomal products or polymer conjugates. NMPs can be designed to target specific tissues, eg, tumors. In virtually all cases, NMPs will eventually reach the immune system. It has been shown that most NMs end up in organs of the mononuclear phagocytic system, notably liver and spleen. Adverse immune effects, including allergy, hypersensitivity, and immunosuppression, have been reported after NMP administration. Interactions of NMPs with the immune system may therefore constitute important side effects. Currently, no regulatory documents are specifically dedicated to evaluate the immunotoxicity of NMs or NMPs. Their immunotoxicity assessment is performed based on existing guidelines for conventional substances or medicinal products. Due to the unique properties of NMPs when compared with conventional medicinal products, it is uncertain whether the currently prescribed set of tests provides sufficient information for an adequate evaluation of potential immunotoxicity of NMPs. The aim of this study was therefore, to compare the current regulatory immunotoxicity testing requirements with the accumulating knowledge on immunotoxic effects of NMPs in order to identify potential gaps in the safety assessment. This comparison showed that immunotoxic effects, such as complement activation-related pseudoallergy, myelosuppression, inflammasome activation, and hypersensitivity, are not readily detected by using current testing guidelines. Immunotoxicity of NMPs would be more accurately evaluated by an expanded testing strategy that is equipped to stratify applicable testing for the various types of NMPs. Keywords: nanomedicine, regulatory requirements, safety, testing methods, immunotoxicity

Various nanomedicinal products (NMPs) have been reported to induce an adverse immune response, which may be related to their tendency to accumulate in or target cells of the immune system. Therefore, before their market authorization, NMPs should be thoroughly evaluated for their immunotoxic potential. Nonclinical regulatory immunotoxicity testing of nonbiological medicinal products, including NMPs, is currently performed by following the guideline S8 "Immunotoxicity Studies for Human Pharmaceuticals" of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). However, this guideline does not cover all the immunotoxicity endpoints reported for NMPs in the literature, such as complement activation related pseudo allergy, hypersensitivity and immunosuppression. In addition, ICH-S8 does not provide any nanospecific testing considerations, which is important given their tendency to interfere with many commonly used toxicity assays. We therefore propose a nonclinical regulatory immunotoxicity assessment strategy, which considers the immunotoxicity endpoints currently missing in the ICH-S8. We also list the known pitfalls related to the testing of NMPs and how to tackle them. Next to defining the relevant physicochemical and pharmacokinetic properties of the NMP and its intended use, the proposed strategy includes an in vitro assay battery addressing various relevant immunotoxicity endpoints. A weight of evidence evaluation of this information can be used to shape the type and design of further in vivo investigations. The final outcome of the immunotoxicity assessment can be included in the overall risk assessment of the NMP and provide alerts for relevant endpoints to address during clinical investigation. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials., (© 2020 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals, Inc.)

Aim: Nanomaterials and nanomedicinal products tend to interfere with various commonly used assays, including regulatory required endotoxin detection methods for medicines. We developed a method to quantify endotoxin levels that is compatible with nanomaterials and nanomedicinal products. Materials & methods: The method is based on measuring endotoxin indirectly via 3-hydroxylated fatty acids of lipid-A, using Ultra High Performance Liquid Chromatography coupled with mass spectrometry. The outcome was related to results of the commonly used Limulus Amebocyte Lysate method. Results: The ultra high performance liquid chromatography coupled with mass spectrometry method has clear advantages compared with other endotoxin determination assays; particularly the absence of nanospecific interference. Conclusion: The method is sensitive, straightforward and accurate in determining and quantifying endotoxin in nanomedicinal product samples.

To study the effects of nanomaterials after inhalation, a large number of in vitro lung models have been reported in literature. Although the in vitro models contribute to the reduction of animal studies, insufficient data exists to determine the predictive value of these in vitro models for the in vivo situation. The aim of this study was to determine the correlation between in vitro and in vivo data by comparing the dose metrics of silver nanoparticles in an in vitro lung model of increasing complexity to our previously published in vivo inhalation study. In vivo, the previously published study showed that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation. The results of the present study show that particle surface area is a suitable dose metric to describe the effects of silver nanoparticles when using a simple monolayer of lung epithelial cells. The dose metric shifted from particle surface area to particle mass when adding an increasing number of macrophages. In addition, a co-culture of endothelial cells, epithelial cells and macrophages on a Transwell® insert correlated less well to the in vivo results compared to the epithelial monolayer. We conclude that for studying the acute pulmonary toxicity of nanoparticles simple in vitro models using an epithelial monolayer better predict the in vivo response compared to complex co-culture models.

The increasing number of nanotechnology products on the market poses increasing human health risks by particle exposures. Adverse effects of silver nanoparticles (AgNPs) in various cell lines have been measured based on exposure dose after a fixed time point, but NP uptake kinetics and the time-dependent internal cellular concentration are often not considered. Even though knowledge about relevant timescales for NP uptake is essential, e.g. for time- and cost-effective risk assessment through modelling, insufficient data are available. Therefore, the authors examined uptake rates for three different AgNP sizes (20, 50 and 75 nm) and two tissue culture medium compositions (with and without foetal calf serum, FCS) under realistic exposure concentrations in pulmonary epithelial 16HBE14o-cells. The quantification of Ag in cells was carried out by high-resolution inductively coupled plasma mass spectrometry. We show for the first time that uptake kinetics of AgNPs into 16HBE14o-cells was highly influenced by medium composition. Uptake into cells was higher in medium without FCS, reaching approximately twice the concentration after 24 h than in medium supplemented with FCS, showing highest uptake for 50-nm AgNPs when expressed on a mass basis. This optimum shifts to 20 nm on a number basis, stressing the importance of the measurand in which results are presented. The importance of our research identifies that not just the uptake after a certain time point should be considered as dose but also the process of uptake (timing) might need to be considered when studying the mechanism of toxicity of nanoparticles.

Human health risks by silver nanoparticle (AgNP) exposure are likely to increase due to the increasing number of NP-containing products and demonstrated adverse effects in various cell lines. Unfortunately, results from (toxicity) studies are often based on exposure dose and are often measured only at a fixed time point. NP uptake kinetics and the time-dependent internal cellular concentration are often not considered. Macrophages are the first line of defense against invading foreign agents including NPs. How macrophages deal with the particles is essential for potential toxicity of the NPs. However, there is a considerable lack of uptake studies of particles in the nanometer range and macrophage-like cells. Therefore, uptake rates were determined over 24 h for three different AgNPs sizes (20, 50 and 75 nm) in medium with and without fetal calf serum. Non-toxic concentrations of 10 ng Ag/mL for monocytic THP-1 cells, representing realistic exposure concentration for short-term exposures, were chosen. The uptake of Ag was higher in medium without fetal calf serum and showed increasing uptake for decreasing NP sizes, both on NP mass and on number basis. Internal cellular concentrations reached roughly 32/10 %, 25/18 % and 21/15 % of the nominal concentration in the absence of fetal calf serum/with fetal calf serum for 20-, 50- and 75-nm NPs, respectively. Our research shows that uptake kinetics in macrophages differ for various NP sizes. To increase the understanding of the mechanism of NP toxicity in cells, the process of uptake (timing) should be considered., Competing Interests: The authors report no conflicts of interest.