Options for imaging cellular therapeutics in vivo: a multi-stakeholder perspective.

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics.

Bridging communities in the field of nanomedicine.

An early dialogue between nanomedicine developers and regulatory authorities are of utmost importance to anticipate quality and safety requirements for these innovative health products. In order to stimulate interactions between the various communities involved in a translation of nanomedicines to clinical applications, the European Commission's Joint Research Centre hosted a workshop titled "Bridging communities in the field of Nanomedicine" in Ispra/Italy on the 27th -28th September 2017. Experts from regulatory bodies, research institutions and industry came together to discuss the next generation of nanomedicines and their needs to obtain regulatory approval. The workshop participants came up with recommendations highlighting methodological gaps that should be addressed in ongoing projects addressing the regulatory science of nanomedicines. In addition, individual opinions of experts relevant to progress of the regulatory science in the field of nanomedicine were summarised in the format of a survey.

Progress and future of in vitro models to study translocation of nanoparticles.

The increasing use of nanoparticles in products likely results in increased exposure of both workers and consumers. Because of their small size, there are concerns that nanoparticles unintentionally cross the barriers of the human body. Several in vivo rodent studies show that, dependent on the exposure route, time, and concentration, and their characteristics, nanoparticles can cross the lung, gut, skin, and placental barrier. This review aims to evaluate the performance of in vitro models that mimic the barriers of the human body, with a focus on the lung, gut, skin, and placental barrier. For these barriers, in vitro models of varying complexity are available, ranging from single-cell-type monolayer to multi-cell (3D) models. Only a few studies are available that allow comparison of the in vitro translocation to in vivo data. This situation could change since the availability of analytical detection techniques is no longer a limiting factor for this comparison. We conclude that to further develop in vitro models to be used in risk assessment, the current strategy to improve the models to more closely mimic the human situation by using co-cultures of different cell types and microfluidic approaches to better control the tissue microenvironments are essential. At the current state of the art, the in vitro models do not yet allow prediction of absolute transfer rates but they do support the definition of relative transfer rates and can thus help to reduce animal testing by setting priorities for subsequent in vivo testing.

Physicochemical characteristics of nanomaterials that affect pulmonary inflammation.

The increasing manufacture and use of products based on nanotechnology raises concerns for both workers and consumers. Various studies report induction of pulmonary inflammation after inhalation exposure to nanoparticles, which can vary in aspects such as size, shape, charge, crystallinity, chemical composition, and dissolution rate. Each of these aspects can affect their toxicity, although it is largely unknown to what extent. The aim of the current review is to analyse published data on inhalation of nanoparticles to identify and evaluate the contribution of their physicochemical characteristics to the onset and development of pulmonary inflammation. Many physicochemical characteristics of nanoparticles affect their lung deposition, clearance, and pulmonary response that, in combination, ultimately determine whether pulmonary inflammation will occur and to what extent. Lung deposition is mainly determined by the physical properties of the aerosol (size, density, shape, hygroscopicity) in relation to airflow and the anatomy of the respiratory system, whereas clearance and translocation of nanoparticles are mainly determined by their geometry and surface characteristics. Besides size and chemical composition, other physicochemical characteristics influence the induction of pulmonary inflammation after inhalation. As some nanoparticles dissolve, they can release toxic ions that can damage the lung tissue, making dissolution rate an important characteristic that affects lung inflammation. Fibre-shaped materials are more toxic to the lungs compared to spherical shaped nanoparticles of the same chemical composition. In general, cationic nanoparticles are more cytotoxic than neutral or anionic nanoparticles. Finally, surface reactivity correlates well with observed pulmonary inflammation. With all these characteristics affecting different stages of the events leading to pulmonary inflammation, no unifying dose metric could be identified to describe pulmonary inflammation for all nanomaterials, although surface reactivity might be a useful measure. To determine the extent to which the various characteristics influence the induction of pulmonary inflammation, the effect of these characteristics on lung deposition, clearance, and pulmonary response should be systematically evaluated. The results can then be used to facilitate risk assessment by categorizing nanoparticles according to their characteristics.

Particle size dependent deposition and pulmonary inflammation after short-term inhalation of silver nanoparticles.

BACKGROUND: Although silver nanoparticles are currently used in more than 400 consumer products, it is not clear to what extent they induce adverse effects after inhalation during production and use. In this study, we determined the lung burden, tissue distribution, and the induction and recovery of adverse effects after short-term inhalation exposure to 15 nm and 410 nm silver nanoparticles. METHODS: Rats were nose-only exposed to clean air, 15 nm silver nanoparticles (179 µg/m³) or 410 nm silver particles (167 µg/m³) 6 hours per day, for four consecutive days. Tissue distribution and the induction of pulmonary toxicity were determined at 24 hours and 7 days after exposure and compared with the internal alveolar dose. Presence of silver nanoparticles in lung cells was visualized by transmission electron microscopy (TEM). RESULTS: Exposure to 15 nm silver nanoparticles induced moderate pulmonary toxicity compared to the controls, indicated by a 175-fold increased influx of neutrophils in the lungs, a doubling of cellular damage markers in the lungs, a 5-fold increase in pro-inflammatory cytokines, and a 1.5-fold increase in total glutathione at 24 hours after exposure. All the observed effects disappeared at 7 days after exposure. No effects were observed after exposure to 410 nm silver particles. The internal alveolar mass dose of the 15 nm nanoparticles was 3.5 times higher compared to the 410 nm particles, which equals to a 66,000 times higher particle number. TEM analysis revealed 15 nm nanoparticles in vesicles and nuclei of lung cells, which were decreased in size to <5 nm at 24 hours after exposure. This demonstrates substantial dissolution of the silver nanoparticles. CONCLUSION: The results show a clear size-dependent effect after inhalation of similar mass concentrations of 15 nm and 410 nm silver (nano)particles. This can be partially explained by the difference in the internal alveolar dose between the 15 nm and 410 nm silver (nano)particles as well as by a difference in the release rate of silver ions.

The value of organs-on-chip for regulatory safety assessment.

Organs-on-chip (OC) have gained much interest as animal-free toxicity testing methods due to their closer resemblance to human tissues and longer culture viability than conventional in vitro methods. The current paper discusses where and how OCs may take a role in the transition to a more predictive, animal-free safety assessment for regulatory purposes. From a preliminary analysis of a repeated dose toxicity database, ten organs of priority for OC development for regu­latory use have been identified. For a number of these organs (lung, skin, liver, kidney, heart, and intestine), OCs are already at rather advanced stages of development, such that involvement of regulators becomes of value in the optimi­zation towards fitness-for-purpose of these methods. For organs such as testis, spleen, brain, and stomach, OCs are much more premature, if existing at all. Therefore, developmental work on OCs for these latter organs is expected to stay in the academic arena for the coming time. A number of technical recommendations and some challenges to reaching final implementation are discussed. We recommend that the development of OCs goes forward together with the development of adverse outcome pathways (AOP) and that they are combined with other methods into integrated testing strategies. Overall, opportunities exist, but much still needs to be done. In our view, regular interactions in multi-stakeholder work­shops on the application of animal-free innovations such as OCs will be beneficial.

Nonclinical regulatory immunotoxicity testing of nanomedicinal products: Proposed strategy and possible pitfalls.

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.

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles.

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes. Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering. Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 microg/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested. Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.

Sensitive method for endotoxin determination in nanomedicinal product samples.

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.

Assessment of oxidative damage induced by iron oxide nanoparticles on different nervous system cells.

Iron oxide nanoparticles (ION) have received much attention for their utility in biomedical applications, such as magnetic resonance imaging, drug delivery and hyperthermia, but concerns regarding their potential harmful effects are also growing. Even though ION may induce different toxic effects in a wide variety of cell types and animal systems, there is a notable lack of toxicological data on the human nervous system, particularly important given the increasing number of applications on this specific system. An important mechanism of nanotoxicity is reactive oxygen species (ROS) generation and oxidative stress. On this basis, the main objective of this work was to assess the oxidative potential of silica-coated (S-ION) and oleic acid-coated (O-ION) ION on human SH-SY5Y neuronal and A172 glial cells. To this aim, ability of ION to generate ROS (both in the absence and presence of cells) was determined, and consequences of oxidative potential were assessed (i) on DNA by means of the 8-oxo-7,8-dihydroguanine DNA glycosylase (OGG1)-modified comet assay, and (ii) on antioxidant reserves by analyzing ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG). Conditions tested included a range of concentrations, two exposure times (3 and 24 h), and absence and presence of serum in the cell culture media. Results confirmed that, even though ION were not able to produce ROS in acellular environments, ROS formation was increased in the neuronal and glial cells by ION exposure, and was parallel to induction of oxidative DNA damage and, only in the case of neuronal cells treated with S-ION, to decreases in the GSH/GSSG ratio. Present findings suggest the production of oxidative stress as a potential action mechanism leading to the previously reported cellular effects, and indicate that ION may pose a health risk to human nervous system cells by generating oxidative stress, and thus should be used with caution.

Development of a systematic method to assess similarity between nanomaterials for human hazard evaluation purposes - lessons learnt.

Within the EU FP-7 GUIDEnano project, a methodology was developed to systematically quantify the similarity between a nanomaterial (NM) that has been tested in toxicity studies and the NM for which risk needs to be evaluated, for the purpose of extrapolating toxicity data between the two materials. The methodology is a first attempt to use current knowledge on NM property-hazard relationships to develop a series of pragmatic and systematic rules for assessing NM similarity. Moreover, the methodology takes into account the practical feasibility, in that it is based on generally available NM characterization information. In addition to presenting this methodology, the lessons learnt and the challenges faced during its development are reported here. We conclude that there is a large gap between the information that is ideally needed and its application to real cases. The current database on property-hazard relationships is still very limited, which hinders the agreement on the key NM properties constituting the basis of the similarity assessment and the development of associated science-based and unequivocal rules. Currently, one of the most challenging NM properties to systematically assess in terms of similarity between two NMs is surface coating and functionalization, which lacks standardized parameters for description and characterization methodology. Standardization of characterization methods that lead to quantitative, unambiguous, and measurable parameters describing NM properties are necessary in order to build a sufficiently robust property-hazard database that allows for evidence-based refinement of our methodology, or any other attempt to systematically assess the similarity of NMs.

Nanomedicinal products: a survey on specific toxicity and side effects.

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.

Considerations for Safe Innovation: The Case of Graphene.

The terms "Safe innovation" and "Safe(r)-by-design" are currently popular in the field of nanotechnology. These terms are used to describe approaches that advocate the consideration of safety aspects already at an early stage of the innovation process of (nano)materials and nanoenabled products. Here, we investigate the possibilities of considering safety aspects during various stages of the innovation process of graphene, outlining what information is already available for assessing potential hazard, exposure, and risks. In addition, we recommend further steps to be taken by various stakeholders to promote the safe production and safe use of graphene.

Simple in vitro models can predict pulmonary toxicity of silver nanoparticles.

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.

A comparison of immunotoxic effects of nanomedicinal products with regulatory immunotoxicity testing requirements.

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.

Identification of the appropriate dose metric for pulmonary inflammation of silver nanoparticles in an inhalation toxicity study.

A number of studies have shown that induction of pulmonary toxicity by nanoparticles of the same chemical composition depends on particle size, which is likely in part due to differences in lung deposition. Particle size mostly determines whether nanoparticles reach the alveoli, and where they might induce toxicity. For the risk assessment of nanomaterials, there is need for a suitable dose metric that accounts for differences in effects between different sized nanoparticles of the same chemical composition. The aim of the present study is to determine the most suitable dose metric to describe the effects of silver nanoparticles after short-term inhalation. Rats were exposed to different concentrations (ranging from 41 to 1105 µg silver/m(3) air) of 18, 34, 60 and 160 nm silver particles for four consecutive days and sacrificed at 24 h and 7 days after exposure. We observed a concentration-dependent increase in pulmonary toxicity parameters like cell counts and pro-inflammatory cytokines in the bronchoalveolar lavage fluid. All results were analysed using the measured exposure concentrations in air, the measured internal dose in the lung and the estimated alveolar dose. In addition, we analysed the results based on mass, particle number and particle surface area. Our study indicates that using the particle surface area as a dose metric in the alveoli, the dose-response effects of the different silver particle sizes overlap for most pulmonary toxicity parameters. We conclude that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation.

Horizon scan of nanomedicinal products.

AIM: A horizon scan of nanomedicinal product on the market or undergoing clinical investigation by analyzing the current nanomedicinal landscape. MATERIALS & METHODS: The horizon scan includes a search of literature, clinical trial registries and the internet. RESULTS: This horizon scan yielded 175 nanomedicinal products. Most products were intended for cancer treatment, followed by infectious diseases. Polymer conjugates, liposomes and protein nanoparticles were the most used structures for nanomedicinal products. CONCLUSIONS: This paper provides an overview of nanomedicinal products on the market or undergoing clinical investigation, their application areas and specific properties.

A practical approach to determine dose metrics for nanomaterials.

Traditionally, administered mass is used to describe doses of conventional chemical substances in toxicity studies. For deriving toxic doses of nanomaterials, mass and chemical composition alone may not adequately describe the dose, because particles with the same chemical composition can have completely different toxic mass doses depending on properties such as particle size. Other dose metrics such as particle number, volume, or surface area have been suggested, but consensus is lacking. The discussion regarding the most adequate dose metric for nanomaterials clearly needs a systematic, unbiased approach to determine the most appropriate dose metric for nanomaterials. In the present study, the authors propose such an approach and apply it to results from in vitro and in vivo experiments with silver and silica nanomaterials. The proposed approach is shown to provide a convenient tool to systematically investigate and interpret dose metrics of nanomaterials. Recommendations for study designs aimed at investigating dose metrics are provided.

A perspective on the developmental toxicity of inhaled nanoparticles.

This paper aimed to clarify whether maternal inhalation of engineered nanoparticles (NP) may constitute a hazard to pregnancy and fetal development, primarily based on experimental animal studies of NP and air pollution particles. Overall, it is plausible that NP may translocate from the respiratory tract to the placenta and fetus, but also that adverse effects may occur secondarily to maternal inflammatory responses. The limited database describes several organ systems in the offspring to be potentially sensitive to maternal inhalation of particles, but large uncertainties exist about the implications for embryo-fetal development and health later in life. Clearly, the potential for hazard remains to be characterized. Considering the increased production and application of nanomaterials and related consumer products a testing strategy for NP should be established. Due to large gaps in data, significant amounts of groundwork are warranted for a testing strategy to be established on a sound scientific basis.

Systemic and immunotoxicity of silver nanoparticles in an intravenous 28 days repeated dose toxicity study in rats.

Because of its antibacterial activity nanosilver is one of the most commonly used nanomaterials. It is increasingly used in a variety of both medical and consumer products resulting in an increase in human exposure. However, the knowledge on the systemic toxicity of nanosilver is relatively limited. To determine the potential systemic toxicity of silver nanoparticles (Ag-NP) with different sizes (20 nm and 100 nm) a 28-days repeated dose toxicity study was performed in rats using intravenous administration. The toxic effect of the 20 nm Ag-NP was performed using the bench mark dose (BMD) approach. Treatment with a maximum dose of 6 mg/kg body weight was well tolerated by the animals. However, both for 20 nm and 100 nm Ag-NP growth retardation was observed during the treatment. A severe increase in spleen size and weight was present which was due to an increased cell number. Both T and B cell populations showed an increase in absolute cell number, whereas the relative cell numbers remained constant. At histopathological evaluation brown and black pigment indicating Ag-NP accumulation was noted in spleen, liver, and lymph nodes. Ag-NP was also detected incidentally in other organs. Clinical chemistry indicated liver damage (increased alkaline phosphatase, alanine transaminase, and aspartate transaminase) that could not be confirmed by histopathology. Hematology showed a decrease in several red blood cell parameters. The most striking toxic effect was the almost complete suppression of the natural killer (NK) cell activity in the spleen at high doses. Other immune parameters affected were: decreased interferon-γ and interleukin (IL)-10 production by concanavalin-A stimulated spleen cells, increased IL-1ß and decreased IL-6, IL-10 and TNF-α production by lipopolysaccharide stimulated spleen cells, increase in serum IgM and IgE, and increase in blood neutrophilic granulocytes. For the spleen weight a critical effect dose of 0.37 mg/kg body weight (b.w.) could be established. The lowest critical effect dose (CED) for a 5% change compared to control animals was observed for thymus weight (CED05 0.01 mg/kg b.w.) and for functional immune parameters, i.e. decrease in NK cell activity (CED05 0.06 mg/kg b.w.) and LPS stimulation of spleen cells (CED05 0.04 mg/kg b.w.). These results show that for nanosilver the most sensitive parameters for potential adverse responses were effects on the immune system.