Ongoing Laboratory Performance Study on Chemical Analysis of Hydrophobic and Hydrophilic Compounds in Three Aquatic Passive Samplers.

The quality of chemical analysis is an important aspect of passive sampling-based environmental assessments. The present study reports on a proficiency testing program for the chemical analysis of hydrophobic organic compounds in silicone and low-density polyethylene (LDPE) passive samplers and hydrophilic compounds in polar organic chemical integrative samplers. The median between-laboratory coefficients of variation (CVs) of hydrophobic compound concentrations in the polymer phase were 33% (silicone) and 38% (LDPE), similar to the CVs obtained in four earlier rounds of this program. The median CV over all rounds was 32%. Much higher variabilities were observed for hydrophilic compound concentrations in the sorbent: 50% for the untransformed data and a factor of 1.6 after log transformation. Limiting the data to the best performing laboratories did not result in less variability. Data quality for hydrophilic compounds was only weakly related to the use of structurally identical internal standards and was unrelated to the choice of extraction solvent and extraction time. Standard deviations of the aqueous concentration estimates for hydrophobic compound sampling by the best performing laboratories were 0.21 log units for silicone and 0.27 log units for LDPE (factors of 1.6 to 1.9). The implications are that proficiency testing programs may give more realistic estimates of uncertainties in chemical analysis than within-laboratory quality control programs and that these high uncertainties should be taken into account in environmental assessments.

New insights into the association of air pollution and kidney diseases by tracing gold nanoparticles with inductively coupled plasma mass spectrometry.

Exposure to particles from air pollution has been associated with kidney disease; however, the underlying biological mechanisms are incompletely understood. Inhaled particles can gain access to the circulation and, depending on their size, pass into urine, raising the possibility that particles may also sequester in the kidney and directly alter renal function. This study optimised an inductively coupled plasma mass spectrometry (ICP-MS) method to investigate the size dependency of particle accumulation in the kidneys of mice following pulmonary instillation (0.8 mg in total over 4 weeks) to gold nanoparticles (2, 3-4, 7-8, 14 or 40 nm or saline control). Due to the smallest particle sizes being below the limit of detection in single particle mode, ICP-MS was operated in total quantification mode. Gold was detected in all matrices of interest (blood, urine and kidney) from animals treated with all sizes of gold nanoparticles, at orders of magnitude higher than the methodological limit of detection in biological matrices (0.013 ng/mL). A size-dependent effect was observed, with smaller particles leading to greater levels of accumulation in tissues. This study highlights the value of a robust and reliable method by ICP-MS to detect extremely low levels of gold in biological samples for indirect particle tracing. The finding that nano-sized particles translocate from the lung to the kidney may provide a biological explanation for the associations between air pollution and kidney disease.

Human Health Risk Assessments and Characterization of Nanomaterials: Are We Ready for the Next (Active) Generations?

Driven by the concept of the 'four generations of nanomaterials', the current state of the knowledge on risk assessment of future generation is explored for active nanomaterials. Through case studies, we identify challenges and evaluate the preparedness of characterization methods, available risk assessment modeling tools, and analytical instrumentation for such future generation active nanomaterials with dynamic hybrid structures of biotic-abiotic and organic-inorganic combinations. Currently available risk assessment tools and analytical instrumentation were found to be lacking the risk preparedness and characterization readiness for active nanomaterials, respectively. Potential future developments in risk assessment modeling tools and analytical techniques can be based upon this work which shall ensure long-term safety of the next generation of nanomaterials.

Analytical human biomonitoring method for the identification and quantification of the metabolite BDCPP originated from the organophosphate flame retardant TDCPP in urine.

Tris(1,3-dichloropropyl) phosphate (TDCPP, CAS 13674-87-8) is one of the most commonly used organophosphate flame retardants (OPFRs) in cars, residential furniture and other products containing polyurethane foam to meet the required flammability standards. For the tasks of the working group Analyses in Biological Material from the German Research Foundation (DFG), a human biomonitoring process for TDCPP is developed. The metabolism of TDCPP is described in different in vivo studies and it is already shown that Bis (1,3-dichloropropyl) phosphate (BDCPP, CAS 72236-72-7) is the primary compound specific metabolite of TDCPP which is often detectable in urine samples. BDCPP is also the most appropriate metabolite because it is unique to TDCPP since no other OPFR known today is transformed or hydrolyzed to BDCPP. A combined method by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is implemented by optimizing atmospheric pressure chemical ionization (APCI) and Electron Spray Ionization (ESI) operating in negative ionization mode. Simultaneous, multiple reaction monitoring is studied to achieve the best performance with respect to selectivity, detectability and robustness of BDCPP. During an expanded validation assessment, the methodological performance characteristics are determined in details and the method is applied in a specific human biomonitoring study among non-occupationally exposed humans of randomly chosen volunteers from the Netherlands.

Measurement of the enriched stable isotope 58Fe in iron related disorders- comparison of INAA and MC-ICP-MS.

As a safer alternative for the use of radioactive tracers, the enriched stable 58Fe isotope has been introduced in studies of iron metabolism. In this study this isotope is measured with instrumental neutron activation analysis (INAA) in blood samples of patients with iron related disorders and controls after oral ingestion of a 58Fe containing pharmaceutical. Results were compared with those derived from MC-ICP-MS, applied on the same samples, and analytical and practical aspects of the two techniques were compared. Both techniques showed an increased absorption and incorporation in red blood cells of the 58Fe isotope in iron deficient patients in contrast to the controls. In all individuals results of INAA measurements were in good agreement with those of MC-ICP-MS (|zeta| < 2). Uncertainties in INAA are substantially higher than those achievable by MC-ICP-MS but the INAA technique offers a high specificity and selectivity for iron close to 100%. In contrast to INAA, sample preparation before measurement is very critical in MC-ICP-MS and interferences with 58Ni and 54Cr may hamper the measurement of 58Fe and 54Fe respectively. Since it takes at least five days after irradiation to reduce the activity of interfering radionuclides (mainly 24Na), INAA is a more time consuming procedure; the need of a nuclear reactor facility makes it also less accessible than MC-ICP-MS. Costs are comparable. Both INAA and MC-ICP-MS are able to adequately measure changes in iron isotope composition in blood when an enriched stable iron isotope is applied in clinical research. Although MC-ICP-MS is more sensitive, is faster and has easier access, in INAA preparative steps before measurement are simpler and there are hardly demands on the kind and size of the samples. This may be relevant working with biomaterials in a clinical setting.

Sampling and pre-treatment effects on the quantification of (nano)silver and selected trace elements in surface water - Application in a Dutch case study.

Detection and quantification of trace elements in aqueous samples is crucial in terms of environmental monitoring and risk assessment for (heavy) metals in the environment. Silver (Ag) in its nanoparticulate form is commonly used as antimicrobial additive in consumer products and pharmaceuticals. Since released dissolved Ag species act as the actual antimicrobial agent, Ag nanomaterials are supposed to pose risks to the environment by a release of dissolved species. Unfortunately, no standard protocols exist yet to gain reliable information about the presence and distribution of nanomaterials in the environment. Therefore, we present an interlaboratory collaboration involving three laboratories to quantify silver, silver based nanoparticles (Ag-b-NPs) and a wide range of relevant trace elements after different sample pre-treatments for profiling surface water of a Dutch channel. Besides quantification of the elements, different sample pretreatments like acidification, with or without filtration, and their effect on the measurable elemental content were studied. Total Ag and Ag-b-NPs were quantified at lower ng L-1 range in the channel water whereas reasonable differences depending on the pre-treatment were identified; Ba, As, Pb, Co, Cr, Cu, Ni and Zn were detected at µg L-1 range and Na, K, Mg, Ca and Fe at mg L-1 range. Significant sample pre-treatment effects were observed for the elements Cr, Cu, Fe, Pb and Zn, which is very likely due to the existence of particulate species. Measured concentrations were well comparable among the three laboratories underpinning method validity and correctness allowing for a comprehensive, reliable risk assessment for nanomaterials in the environment.

Inhaled Nanoparticles Accumulate at Sites of Vascular Disease.

The development of engineered nanomaterials is growing exponentially, despite concerns over their potential similarities to environmental nanoparticles that are associated with significant cardiorespiratory morbidity and mortality. The mechanisms through which inhalation of nanoparticles could trigger acute cardiovascular events are emerging, but a fundamental unanswered question remains: Do inhaled nanoparticles translocate from the lung in man and directly contribute to the pathogenesis of cardiovascular disease? In complementary clinical and experimental studies, we used gold nanoparticles to evaluate particle translocation, permitting detection by high-resolution inductively coupled mass spectrometry and Raman microscopy. Healthy volunteers were exposed to nanoparticles by acute inhalation, followed by repeated sampling of blood and urine. Gold was detected in the blood and urine within 15 min to 24 h after exposure, and was still present 3 months after exposure. Levels were greater following inhalation of 5 nm (primary diameter) particles compared to 30 nm particles. Studies in mice demonstrated the accumulation in the blood and liver following pulmonary exposure to a broader size range of gold nanoparticles (2-200 nm primary diameter), with translocation markedly greater for particles <10 nm diameter. Gold nanoparticles preferentially accumulated in inflammation-rich vascular lesions of fat-fed apolipoproteinE-deficient mice. Furthermore, following inhalation, gold particles could be detected in surgical specimens of carotid artery disease from patients at risk of stroke. Translocation of inhaled nanoparticles into the systemic circulation and accumulation at sites of vascular inflammation provides a direct mechanism that can explain the link between environmental nanoparticles and cardiovascular disease and has major implications for risk management in the use of engineered nanomaterials.

Uptake of silver nanoparticles by monocytic THP-1 cells depends on particle size and presence of serum proteins.

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.

Exploring the effect of silver nanoparticle size and medium composition on uptake into pulmonary epithelial 16HBE14o-cells.

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.

Exploring methods for compositional and particle size analysis of noble metal nanoparticles in Daphnia magna.

The identification and quantification of the bioaccumulation of noble metal engineered nanoparticles (ENPs) by aquatic organisms is of great relevance to understand the exposure and potential toxicity mechanisms of nanoscale materials. Four analytical scenarios were investigated in relation to various sized and composed noble metal (gold (Au), platinum (Pt) and silver (Ag)) ENPs during acute, short-term exposure of Daphnia (D.) magna. Next to the total elemental quantification of absorbed ENPs by D. magna, especially information on the size and particle distribution of ENPs in D. magna is of relevance. Dissolution of the exposed biological material prior to measurement by asymmetric flow field flow fractionation coupled to inductively coupled plasma mass spectrometry (AF4-ICPMS) is challenging because the ENPs must stay stable regarding to particle size and composition. Next to dissolution of exposed D. magna by tetra methyl ammonium hydroxide (TMAH), a new enzymatic dissolution approach was explored by using trypsin. The presence of various sized and composed ENPs has been confirmed by AF4-ICPMS but the chosen dissolution medium was crucial for the results. TMAH and trypsin led to comparable results for medium-sized (50nm) noble metals ENPs in exposed D. magna. But it was also shown that the dissolution of biological materials with smaller (<5nm) ENPs led to different results in particle size and elemental concentration depending on the selected dissolution medium. A significant uptake of Au and Pt ENPs by D. magna or adsorption to particles occurred because only 1-5% of the exposed ENPs remained in the exposure medium.

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.

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.

Comparative hazard identification by a single dose lung exposure of zinc oxide and silver nanomaterials in mice.

Comparative hazard identification of nanomaterials (NMs) can aid in the prioritisation for further toxicity testing. Here, we assessed the acute lung, systemic and liver responses in C57BL/6N mice for three NMs to provide a hazard ranking. A silver (Ag), non-functionalised zinc oxide (ZnO) and a triethoxycaprylylsilane functionalised ZnO NM suspended in water with 2% mouse serum were examined 24 hours following a single intratracheal instillation (I.T.). An acute pulmonary inflammation was noted (marked by a polymorphonuclear neutrophil influx) with cell damage (LDH and total protein) in broncho-alveolar lavage fluid (BALF) after administration of both non-functionalised and functionalised ZnO. The latter also induced systemic inflammation measured as an increase in blood neutrophils and a decrease in blood lymphocytes. Exposure to Ag NM was not accompanied by pulmonary inflammation or cytotoxicity, or by systemic inflammation. A decrease in glutathione levels was demonstrated in the liver following exposure to high doses of all three nanomaterials irrespective of any noticeable inflammatory or cytotoxic effects in the lung. By applying benchmark dose (BMD) modeling statistics to compare potencies of the NMs, we rank functionalised ZnO ranked the highest based on the largest number of affected endpoints, as well as the strongest responses observed after 24 hours. The non-functionalised ZnO NM gave an almost similar response, whereas Ag NM did not cause an acute response at similar doses.

Percutaneous penetration of silver from a silver containing garment in healthy volunteers and patients with atopic dermatitis.

Human data on dermal absorption of silver under "in use" scenario are scarce which hampers health risk assessment. The main objective of the present study was to determine percutaneous penetration of silver after dermal exposure to silver containing garment in healthy individuals and atopic dermatitis (AD) patients. Next to assess pro-inflammatory effect of silver in the skin. Healthy subjects (n=15) and patients with AD (n=15) wore a sleeve containing 3.6% (w/w) silver on their lower arms for 8h during 5 consecutive days. The percutaneous penetration parameters were deduced from the silver concentration-depth profiles in the stratum corneum (SC) collected by adhesive tapes. Furthermore, silver was measured in urine samples collected before and after exposure. Inflammatory response was assessed by measuring IL-1α and IL-1RA in the exposed and non-exposed skin sites. Dermal flux of silver in healthy subjects and AD patients was respectively 0.23 and 0.20 ng/cm(2)/h. The urine silver concentrations showed no increase after exposure. Furthermore, exposure to silver did not lead to the changes in the profiles of IL-1α and IL-1RA. Dermal absorption of silver under "real life scenario" was lower than the current reference dose. Furthermore, dermal exposure did not lead to altered expression of inflammatory IL-1 cytokines in the skin.

Pilot study on the identification of silver in skin layers and urine after dermal exposure to a functionalized textile.

Silver (Ag) is increasingly used in consumer products like functionalized textiles and medical devices owing to its strong antimicrobial activity which is largely assigned to Ag ions released after oxidation of metallic Ag. To increase generation of Ag ions, in various products Ag is often present as nanoparticles. Ideally, Ag ions would remain on the surface of the skin to combat the bacteria and the uptake of Ag into the body should be limited. However, the Ag ions might penetrate across the skin into the body leading to adverse health effects. Data on in vivo uptake of Ag due to dermal exposure are scarce partly caused by the lack of suitable analytical approaches for the determination of Ag in biological matrices, but strongly needed to enable risk assessment of skin exposure to (nano) Ag containing products. With the developed approach, the presence of Ag in a functionalized textile is confirmed by using scanning electron microscopy (SEM). After in vivo dermal exposure to Ag containing textile material under ׳׳in use׳׳ exposure scenarios, the outermost layers of the skin (Stratum Corneum, SC) were sampled by using adhesive tapes with a size of 3.8cm(2). Different leaching and dissolution procedures of Ag from biological samples prior analysis by inductively coupled plasma mass spectrometry (ICPMS) have been evaluated. The developed method results in a limit of detection (LOD) of 2ng Ag per removed SC layer. The method allows the measurement of the Ag concentrations at different depths of the SC enabling the deduction of the percutaneous penetration kinetics. Due to the possible bio distribution within the whole body, an indirect exposure matrix (urine) was studied too. The detection power of the method permits measuring the ultra-trace concentrations of Ag in urine before and after dermal exposure; LOD is 0.010µg Ag/L urine.

Analytical assessment about the simultaneous quantification of releasable pharmaceutical relevant inorganic nanoparticles in tap water and domestic waste water.

For pharmaceutical applications, the use of inorganic engineered nanoparticles is of growing interest while silver (Ag) and gold (Au) are the most relevant elements. A few methods were developed recently but the validation and the application testing were quite limited. Therefore, a routinely suitable multi element method for the identification of nanoparticles of different sizes below 100 nm and elemental composition by applying asymmetric flow field flow fraction (AF4) - inductively coupled plasma mass spectrometry (ICPMS) is developed. A complete validation model of the quantification of releasable pharmaceutical relevant inorganic nanoparticles based on Ag and Au is presented for the most relevant aqueous matrices of tap water and domestic waste water. The samples are originated from locations in the Netherlands and it is of great interest to study the unwanted presence of Ag and Au as nanoparticle residues due to possible health and environmental risks. During method development, instability effects are observed for 60 nm and 70 nm Ag ENPs with different capping agents. These effects are studied more closely in relation to matrix effects. Besides the methodological aspects, the obtained analytical results and relevant performance characteristics (e.g. measuring range, limit of detection, repeatability, reproducibility, trueness, and expanded uncertainty of measurement) are determined and discussed. For the chosen aqueous matrices, the results of the performance characteristics are significantly better for Au ENPs in comparison to Ag ENPs; e.g. repeatability and reproducibility are below 10% for all Au ENPs respectively maximal 27% repeatability for larger Ag ENPs. The method is a promising tool for the simultaneous determination of releasable pharmaceutical relevant inorganic nanoparticles.

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.

Tissue distribution and elimination after oral and intravenous administration of different titanium dioxide nanoparticles in rats.

OBJECTIVE: The aim of this study was to obtain kinetic data that can be used in human risk assessment of titanium dioxide nanomaterials. METHODS: Tissue distribution and blood kinetics of various titanium dioxide nanoparticles (NM-100, NM-101, NM-102, NM-103, and NM-104), which differ with respect to primary particle size, crystalline form and hydrophobicity, were investigated in rats up to 90 days post-exposure after oral and intravenous administration of a single or five repeated doses. RESULTS: For the oral study, liver, spleen and mesenteric lymph nodes were selected as target tissues for titanium (Ti) analysis. Ti-levels in liver and spleen were above the detection limit only in some rats. Titanium could be detected at low levels in mesenteric lymph nodes. These results indicate that some minor absorption occurs in the gastrointestinal tract, but to a very limited extent.Both after single and repeated intravenous (IV) exposure, titanium rapidly distributed from the systemic circulation to all tissues evaluated (i.e. liver, spleen, kidney, lung, heart, brain, thymus, reproductive organs). Liver was identified as the main target tissue, followed by spleen and lung. Total recovery (expressed as % of nominal dose) for all four tested nanomaterials measured 24 h after single or repeated exposure ranged from 64-95% or 59-108% for male or female animals, respectively. During the 90 days post-exposure period, some decrease in Ti-levels was observed (mainly for NM-100 and NM-102) with a maximum relative decrease of 26%. This was also confirmed by the results of the kinetic analysis which revealed that for each of the investigated tissues the half-lifes were considerable (range 28-650 days, depending on the TiO(2)-particle and tissue investigated). Minor differences in kinetic profile were observed between the various particles, though these could not be clearly related to differences in primary particle size or hydrophobicity. Some indications were observed for an effect of crystalline form (anatase vs. rutile) on total Ti recovery. CONCLUSION: Overall, the results of the present oral and IV study indicates very low oral bioavailability and slow tissue elimination. Limited uptake in combination with slow elimination might result in the long run in potential tissue accumulation.