Airborne particulate matter and diesel engine exhaust on infrastructure construction sites in the Copenhagen metropolitan area.

Diesel engine exhaust (DEE) is carcinogenic and potentially hazardous for those working in close proximity to diesel-powered machines. This study characterizes workplace exposure to DEE and its associated particulate matter (PM) during outdoor construction activities. We sampled at 4 construction sites in the Copenhagen metropolitan area. We used portable constant-flow pumps and quartz-fiber filters to quantify personal exposure to elemental carbon (EC), and used real-time instruments to collect activity-based information about particle number and size distribution, as well as black carbon (BC) concentration. Full-shift measurements of EC concentration ranged from < 0.3 to 6.4 µg/m3. Geometric mean (GM) EC exposure was highest for ground workers (3.4 µg/m3 EC; geometric standard deviation, GSD = 1.3), followed by drilling rig operators (2.6 µg/m3 EC; GSD = 1.4). Exposure for non-drilling-rig machine operators (1.2 µg/m3 EC; GSD = 2.9) did not differ significantly from background (0.9 µg/m3 EC; GSD = 1.7). The maximum 15-min moving average concentration of BC was 17 µg/m3, and the highest recorded peak concentration was 44 µg/m3. In numbers, the particle size distributions were dominated by ultrafine particles ascribed to DEE and occasional welding activities at the sites. The average total particle number concentrations (PNCs) measured in near-field and far-field positions across all worksites were 10,600 (GSD = 3.0) and 6,000 (GSD = 2.8)/cm3, respectively. Sites with active drilling rigs saw significantly higher average total PNCs at their near-field stations (13,600, 32,000, and 9,700/cm3; GSD = 2.4, 3.4, and 2.4) than sites without (4,700/cm3; GSD = 1.6). Overall, the DEE exposures at these outdoor construction sites were below current occupational exposure limits for EC (10 µg/m3 in Denmark; 50 µg/m3 in the European Union), but extended durations of exposure to the observed DEE levels may still be a health risk.

The Underlying Mechanism of Poisoning after the Accidental Inhalation of Aerosolised Waterproofing Spray.

Waterproofing sprays can cause acute respiratory symptoms after inhalation, including coughing and dyspnoea shortly after use. Here, we describe two cases where persons used the same brand of waterproofing spray product. In both cases the persons followed the instructions on the product and maximized the ventilation by opening windows and doors; however, they still became affected during the application of the product. Products with the same batch number as that used in one case were tested for their effect on respiration patterns of mice in whole-body plethysmographs and lung surfactant function inhibition in vitro. The product was used in spraying experiments to determine the particle size distribution of the aerosol, both using a can from one case and a can with an identical batch number. In addition, the aerosols in the mouse exposure chamber were measured. Aerosol data from a small-scale exposure chamber and data on the physical and temporal dimensions of the spraying during one case were used to estimate the deposited dose during the spraying events. All collected data point to the spraying of the waterproofing product being the reason that two people became ill, and that the inhibition of lung surfactant function was a key component of this illness.

Use of the dustiness index in combination with the handling energy factor for exposure modelling of nanomaterials.

The use of modelling tools in the occupational hygiene community has increased in the last years to comply with the different existing regulations. However, limitations still exist mainly due to the difficulty to obtain certain key parameters such as the emission rate, which in the case of powder handling can be estimated using the dustiness index (DI). The goal of this work is to explore the applicability and usability of the DI for emission source characterization and occupational exposure prediction to particles during nanomaterial powder handling. Modelling of occupational exposure concentrations of 13 case scenarios was performed using a two-box model as well as three nano-specific tools (Stoffenmanager nano, NanoSafer and GUIDEnano). The improvement of modelling performance by using a derived handling energy factor (H) was explored. Results show the usability of the DI for emission source characterization and respirable mass exposure modelling of powder handling scenarios of nanomaterials. A clear improvement in modelling outcome was obtained when using derived quartile-3 H factors with, 1) Pearson correlations of 0.88 vs. 0.52 (not using H), and 2) ratio of modelled/measured concentrations ranging from 0.9 to 10 in 75% cases vs. 16.7% of the cases when not using H. Particle number concentrations were generally underpredicted. Using the most conservative H values, predictions with ratios modelled/measured concentrations of 0.4-3.6 were obtained.

Toxicity dose descriptors from animal inhalation studies of 13 nanomaterials and their bulk and ionic counterparts and variation with primary particle characteristics.

This study collects toxicity data from animal inhalation studies of some nanomaterials and their bulk and ionic counterparts. To allow potential grouping and interpretations, we retrieved the primary physicochemical and exposure data to the extent possible for each of the materials. Reviewed materials are compounds (mainly elements, oxides and salts) of carbon (carbon black, carbon nanotubes, and graphene), silver, cerium, cobalt, copper, iron, nickel, silicium (amorphous silica and quartz), titanium (titanium dioxide), and zinc (chemical symbols: Ag, C, Ce, Co, Cu, Fe, Ni, Si, Ti, TiO2, and Zn). Collected endpoints are: a) pulmonary inflammation, measured as neutrophils in bronchoalveolar lavage (BAL) fluid at 0-24 hours after last exposure; and b) genotoxicity/carcinogenicity. We present the dose descriptors no-observed-adverse-effect concentrations (NOAECs) and lowest-observed-adverse-effect concentrations (LOAECs) for 88 nanomaterial investigations in data-library and graph formats. We also calculate 'the value where 25% of exposed animals develop tumors' (T25) for carcinogenicity studies. We describe how the data may be used for hazard assessment of the materials using carbon black as an example. The collected data also enable hazard comparison between different materials. An important observation for poorly soluble particles is that the NOAEC for neutrophil numbers in general lies around 1 to 2 mg/m3. We further discuss why some materials' dose descriptors deviate from this level, likely reflecting the effects of the ionic form and effects of the fiber-shape. Finally, we discuss that long-term studies, in general, provide the lowest dose descriptors, and dose descriptors are positively correlated with particle size for near-spherical materials.

Dissolution Rate of Nanomaterials Determined by Ions and Particle Size under Lysosomal Conditions: Contributions to Standardization of Simulant Fluids and Analytical Methods.

Dissolution of inhaled engineered nanomaterials (ENM) under physiological conditions is essential to predict the clearance of the ENM from the lungs and to assess their biodurability and the potential effects of released ions. Alveolar macrophage (AM) lysosomes contain a pH 4.5 saline brine with enzymes and other components. Different types of artificial phagolysosomal simulant fluids (PSFs) have been developed for dissolution testing, but the consequence of using different media is not known. In this study, we tested to which extent six fundamentally different PSFs affected the ENM dissolution kinetics and particle size as determined by a validated transmission electron microscopy (TEM) image analysis. Three lysosomal simulant media were consistent with each other and with in vivo clearance. These media predict the quick dissolution of ZnO, the partial dissolution of SiO2, and the very slow dissolution of TiO2. The valid media use either a mix of organic acids (with the total concentration below 0.5 g/L, thereof citric acid below 0.15 g/L) or another organic acid (KH phthalate). For several ENM, including ZnO, BaSO4, and CeO2, all these differences induce only minor modulation of the dissolution rates. Only for TiO2 and SiO2, the interaction with specific organic acids is highly sensitive, probably due to sequestration of the ions, and can lead to wrong predictions when compared to the in vivo behavior. The media that fail on TiO2 and SiO2 dissolution use citric acid at concentrations above 5 g/L (up to 28 g/L). In the present selection of ENM, fluids, and methods, the different lysosomal simulant fluids did not induce changes of particle morphology, except for small changes in SiO2 and BaSO4 particles most likely due to ion dissolution, reprecipitation, and coalescence between neighboring particles. Based on the current evidence, the particle size by TEM analysis is not a sufficiently sensitive analytical method to deduce the rate of ENM dissolution in physiological media. In summary, we recommend the standardization of ENM dissolution testing by one of the three valid lysosomal simulant fluids with determination of the dissolution rate and halftime by the quantification of ions. This recommendation was established for a continuous flow system but may be relevant as well for static (batch) solubility testing.

Response Letter to Koivisto et al. 'Evaluating the Theoretical Background of STOFFENMANAGER® and the Advanced REACH Tool'.

In this article, we have responded to the key statements in the article by Koivisto et al. (2022) that were incorrect and considered to be a biased critique on a subset of the exposure models used in Europe (i.e. ART and Stoffenmanager®) used for regulatory exposure assessment. We welcome scientific discussions on exposure modelling (as was done during the ISES Europe workshop) and criticism based on scientific evidence to contribute to the advancement of occupational exposure estimation tools. The tiered approach to risk assessment allows various exposure assessment models from screening tools (control/hazard banding) through to higher-tiered approaches. There is a place for every type of model, but we do need to recognize the cost and data requirements of highly bespoke assessments. That is why model developers have taken pragmatic approaches to develop tools for exposure assessments based on imperfect data. We encourage Koivisto et al. to focus on further scientifically robust work to develop mass-balance models and by independent external validations studies, compare these models with alternative model tools such as ART and Stoffenmanager®.

Pulmonary toxicity of synthetic amorphous silica - effects of porosity and copper oxide doping.

Materials can be modified for improved functionality. Our aim was to test whether pulmonary toxicity of silica nanomaterials is increased by the introduction of: a) porosity; and b) surface doping with CuO; and whether c) these modifications act synergistically. Mice were exposed by intratracheal instillation and for some doses also oropharyngeal aspiration to: 1) solid silica 100 nm; 2) porous silica 100 nm; 3) porous silica 100 nm with CuO doping; 4) solid silica 300 nm; 5) porous silica 300 nm; 6) solid silica 300 nm with CuO doping; 7) porous silica 300 nm with CuO doping; 8) CuO nanoparticles 9.8 nm; or 9) carbon black Printex 90 as benchmark. Based on a pilot study, dose levels were between 0.5 and 162 µg/mouse (0.2 and 8.1 mg/kg bw). Endpoints included pulmonary inflammation (neutrophil numbers in bronchoalveolar fluid), acute phase response, histopathology, and genotoxicity assessed by the comet assay, micronucleus test, and the gamma-H2AX assay. The porous silica materials induced greater pulmonary inflammation than their solid counterparts. A similar pattern was seen for acute phase response induction and histologic changes. This could be explained by a higher specific surface area per mass unit for the most toxic particles. CuO doping further increased the acute phase response normalized according to the deposited surface area. We identified no consistent evidence of synergism between surface area and CuO doping. In conclusion, porosity and CuO doping each increased the toxicity of silica nanomaterials and there was no indication of synergy when the modifications co-occurred.

Nanoparticle Exposure and Workplace Measurements During Processes Related to 3D Printing of a Metal Object.

Metal 3D printing has many potential uses within prototyping and manufacturing. Selective laser melting (SLM) is a process that uses metal powders in the micrometer range as printing material. The particle release from the entire SLM printing process is not well-studied. While the 3D printing itself often occurs in a sealed chamber, activities related to the process can potentially release harmful metal particles to the indoor working environment through resuspension of the printing powder or via incident nanoparticles generated during printing. The objective of this study was to improve the understanding of particle exposure in work processes associated with 3D printing and potential needs for interventions by a case study conducted in a 3D printing facility. In this setting, direct release and dispersion of particles throughout the workspace from processes related to metal 3D printing was investigated. The release from five activities were studied in detail. The activities included post-printing cleaning, object annealing, and preparation of new base substrate for the next printing was. Three of the five measured activities caused particles number concentrations in the working environment to increase above background levels which were found to be 8·102 cm-3. Concentrations during chamber emptying and the open powder removal system (PRS) cleaning processes increased to 104 and 5·103 cm-3, respectively, whereas grinding activity increased number concentrations to 2.5·105 cm-3. Size distributions showed that particles were mainly smaller than 200 nm. Respirable mass concentrations were 50.4 µg m-3, collected on filters. This was corroborated by respirable mass measured with a DustTrak of 58.4 µg m-3. Respirable mass concentrations were below the occupational exposure limits in Denmark for an 8 h time-weighted average.

Short-term oral administration of non-porous and mesoporous silica did not induce local or systemic toxicity in mice.

In this study, two sets of methyl-coated non-porous and mesoporous amorphous silica materials of two target sizes (100 and 300 nm; 10-844 m2/g) were used to investigate the potential role of specific surface area (SSA) and porosity on the oral toxicity in mice. Female Swiss mice were administered by oral gavage for 5 consecutive days. Two silica dose levels (100 and 1000 mg/kg b.w.) were tested for all four materials. All dispersions were characterized by transmission electron microscopy (TEM) and Nanoparticle tracking analysis (NTA). Batch dispersions of porous silica were rather unstable due to agglomeration. Animals were sacrificed one day after the last administration or after a three-week recovery period. No relevant toxicological effects were induced by any of the silica materials tested, as evaluated by body weight, gross pathology, relative organ weights (liver, spleen, kidneys), hematology, blood biochemistry, genotoxicity (Comet assay in jejunum cells and micronucleus test in peripheral blood erythrocytes), liver and small intestine histopathology, and intestinal inflammation. The presence of silica particles in the intestine was evaluated by a hyperspectral imaging microscopy system (CytoViva) using histological samples of jejunum tissue. Silica spectral signatures were found in jejunum samples with all the treatments, but only statistically significant in one of the treatment groups.

Pulmonary toxicity of Fe2O3, ZnFe2O4, NiFe2O4 and NiZnFe4O8 nanomaterials: Inflammation and DNA strand breaks.

Exposure to metal oxide nanomaterials potentially occurs at the workplace. We investigated the toxicity of two Fe-oxides: Fe2O3 nanoparticles and nanorods; and three MFe2O4 spinels: NiZnFe4O8, ZnFe2O4, and NiFe2O4 nanoparticles. Mice were dosed 14, 43 or 128 µg by intratracheal instillation. Recovery periods were 1, 3, or 28 days. Inflammation - neutrophil influx into bronchoalveolar lavage (BAL) fluid - occurred for Fe2O3 rods (1 day), ZnFe2O4 (1, 3 days), NiFe2O4 (1, 3, 28 days), Fe2O3 (28 days) and NiZnFe4O8 (28 days). Conversion of mass-dose into specific surface-area-dose showed that inflammation correlated with deposited surface area and consequently, all these nanomaterials belong to the so-called low-solubility, low-toxicity class. Increased levels of DNA strand breaks were observed for both Fe2O3 particles and rods, in BAL cells three days post-exposure. To our knowledge, this is, besides magnetite (Fe3O4), the first study of the pulmonary toxicity of MFe2O4 spinel nanomaterials.

Data supporting the investigation of interaction of biologically relevant proteins with ZnO nanomaterials: A confounding factor for in vitro toxicity endpoints.

Test materials, like manufactured nanomaterials (MN), may interact with serum proteins, interleukins (IL) and lactate dehydrogenase (LDH) and cause measurement artefacts as a result of e.g., physical adsorption and electrostatic forces, and/or interaction with dissolved species or conditional chemical changes during testing. In this article, data are given on the zeta-potentials of two manufactured ZnO nanomaterials (NM-110 and NM-111) dispersed in 0.05% w/v Bovine Serum Albumin (BSA) water batch dispersions and in Ham's F12 nutrient mixture added Fetal Bovine Serum (FBS), penicillin, and streptomycin and particle free mediums (cHam's F12). Data on the Zeta-potential and the iso-electrical point of lactate hydrogenase in pure Ham's F12 nutrient mixture is also provided. The percentage of added IL-6, IL-8 and LDH remaining after 24-h incubation in cHam's F12 are given as function of MN concentrations. Finally data from thermodynamic chemical reaction modeling of changes in pH and Zn-speciation during dissolution of ZnO or dissolved ZnCl2 additions to Ham's F12 using Geochemist Workbench® are given. For further information, data interpretation and discussion please refer to the research article "Interaction of biologically relevant proteins with ZnO nanomaterials: a confounding factor for in vitro toxicity endpoints" (E. Da Silva et al. 2019).

Effects of maternal inhalation of carbon black nanoparticles on reproductive and fertility parameters in a four-generation study of male mice.

BACKGROUND: Previous findings indicate that in utero exposure to nanoparticles may affect the reproductive system in male offspring. Effects such as decreased sperm counts and testicular structural changes in F1 males have been reported following maternal airway exposure to carbon black during gestation. In addition, a previous study in our laboratory suggested that the effects of in utero exposure of nanoparticles may span further than the first generation, as sperm content per gram of testis was significantly lowered in F2 males. In the present study we assessed male fertility parameters following in utero inhalation exposure to carbon black in four generations of mice. RESULTS: Filter measurements demonstrated that the time-mated females were exposed to a mean total suspended particle mass concentration of 4.79 ± 1.86 or 33.87 ± 14.77 mg/m3 for the low and high exposure, respectively. The control exposure was below the detection limit (LOD 0.08 mg/m3). Exposure did not affect gestation and litter parameters in any generation. No significant changes were observed in body and reproductive organ weights, epididymal sperm parameters, daily sperm production, plasma testosterone or fertility. CONCLUSION: In utero exposure to carbon black nanoparticles, at occupationally relevant exposure levels, via maternal whole body inhalation did not affect male-specific reproductive, fertility and litter parameters in four generations of mice.

Source specific exposure and risk assessment for indoor aerosols.

Poor air quality is a leading contributor to the global disease burden and total number of deaths worldwide. Humans spend most of their time in built environments where the majority of the inhalation exposure occurs. Indoor Air Quality (IAQ) is challenged by outdoor air pollution entering indoors through ventilation and infiltration and by indoor emission sources. The aim of this study was to understand the current knowledge level and gaps regarding effective approaches to improve IAQ. Emission regulations currently focus on outdoor emissions, whereas quantitative understanding of emissions from indoor sources is generally lacking. Therefore, specific indoor sources need to be identified, characterized, and quantified according to their environmental and human health impact. The emission sources should be stored in terms of relevant metrics and statistics in an easily accessible format that is applicable for source specific exposure assessment by using mathematical mass balance modelings. This forms a foundation for comprehensive risk assessment and efficient interventions. For such a general exposure assessment model we need 1) systematic methods for indoor aerosol emission source assessment, 2) source emission documentation in terms of relevant a) aerosol metrics and b) biological metrics, 3) default model parameterization for predictive exposure modeling, 4) other needs related to aerosol characterization techniques and modeling methods. Such a general exposure assessment model can be applicable for private, public, and occupational indoor exposure assessment, making it a valuable tool for public health professionals, product safety designers, industrial hygienists, building scientists, and environmental consultants working in the field of IAQ and health.

Interaction of biologically relevant proteins with ZnO nanomaterials: A confounding factor for in vitro toxicity endpoints.

The results of in vitro toxicological studies for manufactured nanomaterials (MNs) are often contradictory and not reproducible. Interference of the MNs with assays has been suggested. However, understanding for which materials and how these artefacts occur remains a major challenge. This study investigated interactions between two well-characterized ZnO MNs (NM-110 and NM-111) and lactate dehydrogenase (LDH), and two interleukins (IL-6 and IL-8). Particles (10 to 640 µg/mL) and proteins were incubated for up to 24 h in routine in vitro assays test conditions. LDH activity (ODLDH), but not interleukins concentrations, decreased sharply in a dose-dependent manner within an hour after exposure (ODLDH < 60% of ODref for both MNs at 10 µg/mL). A Freundlich adsorption isotherm was successfully applied, indicating multilayer adsorption of LDH. ZnO MNs and LDH had neutral to slightly negative surface charges in dispersion, precluding electrostatic attachment. Particle sedimentation was not a limiting factor. Fast dissolution of ZnO MNs was shown and Zn2+ could play a role in the ODLDH drop. To summarize, ZnO MNs quickly reduced ODLDH due to concentration-dependent adsorption and LDH inhibition by interaction with dissolved Zn. The control of particle interference in toxicological in vitro assays should become mandatory to avoid misleading interpretation of results.

Maternal inhalation of carbon black nanoparticles induces neurodevelopmental changes in mouse offspring.

BACKGROUND: Engineered nanoparticles are smaller than 100 nm and designed to improve or creating even new physico-chemical properties. Consequently, toxicological properties of materials may change as size reaches the nm size-range. We examined outcomes related to the central nervous system in the offspring following maternal inhalation exposure to nanosized carbon black particles (Printex 90). METHODS: Time-mated mice (NMRI) were exposed by inhalation, for 45 min/day to 0, 4.6 or 37 mg/m3 aerosolized carbon black on gestation days 4-18, i.e. for a total of 15 days. Outcomes included maternal lung inflammation (differential cell count in bronchoalveolar lavage fluid and Saa3 mRNA expression in lung tissue), offspring neurohistopathology and behaviour in the open field test. RESULTS: Carbon black exposure did not cause lung inflammation in the exposed females, measured 11 or 28-29 days post-exposure. Glial fibrillary acidic protein (GFAP) expression levels were dose-dependently increased in astrocytes around blood vessels in the cerebral cortex and hippocampus in six weeks old offspring, indicative of reactive astrogliosis. Also enlarged lysosomal granules were observed in brain perivascular macrophages (PVMs) in the prenatally exposed offspring. The number of parvalbumin-positive interneurons and the expression levels of parvalbumin were decreased in the motor and prefrontal cortices at weaning and 120 days of age in the prenatally exposed offspring. In the open field test, behaviour was dose-dependently altered following maternal exposure to Printex 90, at 90 days of age. Prenatally exposed female offspring moved a longer total distance, and especially males spent significantly longer time in the central zone of the maze. In the offspring, the described effects were long-lasting as they were present at all time points investigated. CONCLUSION: The present study reports for the first time that maternal inhalation exposure to Printex 90 carbon black induced dose-dependent denaturation of PVM and reactive astrocytes, similarly to the findings observed following maternal exposure to Printex 90 by airway instillation. Of note, some of the observed effects have striking similarities with those observed in mouse models of neurodevelopmental disorders.

Particle release and control of worker exposure during laboratory-scale synthesis, handling and simulated spills of manufactured nanomaterials in fume hoods.

Fume hoods are one of the most common types of equipment applied to reduce the potential of particle exposure in laboratory environments. A number of previous studies have shown particle release during work with nanomaterials under fume hoods. Here, we assessed laboratory workers' inhalation exposure during synthesis and handling of CuO, TiO2 and ZnO in a fume hood. In addition, we tested the capacity of a fume hood to prevent particle release to laboratory air during simulated spillage of different powders (silica fume, zirconia TZ-3Y and TiO2). Airborne particle concentrations were measured in near field, far field, and in the breathing zone of the worker. Handling CuO nanoparticles increased the concentration of small particles (< 58 nm) inside the fume hood (up to 1 × 105 cm-3). Synthesis, handling and packaging of ZnO and TiO2 nanoparticles did not result in detectable particle release to the laboratory air. Simulated powder spills showed a systematic increase in the particle concentrations inside the fume hood with increasing amount of material and drop height. Despite powder spills were sometimes observed to eject into the laboratory room, the spill events were rarely associated with notable release of particles from the fume hood. Overall, this study shows that a fume hood generally offers sufficient exposure control during synthesis and handling of nanomaterials. An appropriate fume hood with adequate sash height and face velocity prevents 98.3% of particles release into the surrounding environment. Care should still be made to consider spills and high cleanliness to prevent exposure via resuspension and inadvertent exposure by secondary routes.

Particle emission rates during electrostatic spray deposition of TiO2 nanoparticle-based photoactive coating.

Here, we studied the particle release rate during Electrostatic spray deposition of anatase-(TiO2)-based photoactive coating onto tiles and wallpaper using a commercially available electrostatic spray device. Spraying was performed in a 20.3m3 test chamber while measuring concentrations of 5.6nm to 31µm-size particles and volatile organic compounds (VOC), as well as particle deposition onto room surfaces and on the spray gun user hand. The particle emission and deposition rates were quantified using aerosol mass balance modelling. The geometric mean particle number emission rate was 1.9×1010s-1 and the mean mass emission rate was 381µgs-1. The respirable mass emission-rate was 65% lower than observed for the entire measured size-range. The mass emission rates were linearly scalable (±ca. 20%) to the process duration. The particle deposition rates were up to 15h-1 for <1µm-size and the deposited particles consisted of mainly TiO2, TiO2 mixed with Cl and/or Ag, TiO2 particles coated with carbon, and Ag particles with size ranging from 60nm to ca. 5µm. As expected, no significant VOC emissions were observed as a result of spraying. Finally, we provide recommendations for exposure model parameterization.

Influence of dispersion medium on nanomaterial-induced pulmonary inflammation and DNA strand breaks: investigation of carbon black, carbon nanotubes and three titanium dioxide nanoparticles.

Intratracheal instillation serves as a model for inhalation exposure. However, for this, materials are dispersed in appropriate media that may influence toxicity. We tested whether different intratracheal instillation dispersion media influence the pulmonary toxicity of different nanomaterials. Rodents were intratracheally instilled with 162 µg/mouse/1620 µg/rat carbon black (CB), 67 µg/mouse titanium dioxide nanoparticles (TiO2) or 54 µg/mouse carbon nanotubes (CNT). The dispersion media were as follows: water (CB, TiO2); 2% serum in water (CB, CNT, TiO2); 0.05% serum albumin in water (CB, CNT, TiO2); 10% bronchoalveolar lavage fluid in 0.9% NaCl (CB), 10% bronchoalveolar lavage (BAL) fluid in water (CB) or 0.1% Tween-80 in water (CB). Inflammation was measured as pulmonary influx of neutrophils into bronchoalveolar fluid, and DNA damage as DNA strand breaks in BAL cells by comet assay. Inflammation was observed for all nanomaterials (except 38-nm TiO2) in all dispersion media. For CB, inflammation was dispersion medium dependent. Increased levels of DNA strand breaks for CB were observed only in water, 2% serum and 10% BAL fluid in 0.9% NaCl. No dispersion medium-dependent effects on genotoxicity were observed for TiO2, whereas CNT in 2% serum induced higher DNA strand break levels than in 0.05% serum albumin. In conclusion, the dispersion medium was a determinant of CB-induced inflammation and genotoxicity. Water seemed to be the best dispersion medium to mimic CB inhalation, exhibiting DNA strand breaks with only limited inflammation. The influence of dispersion media on nanomaterial toxicity should be considered in the planning of intratracheal investigations.

Impact of serum as a dispersion agent for in vitro and in vivo toxicological assessments of TiO2 nanoparticles.

Nanoparticles (NP) have a tendency to agglomerate after dispersion in physiological media, which can be prevented by the addition of serum. This may however result in modification of the toxic potential of particles due to the formation of protein corona. Our study aimed to analyze the role of serum that is added to improve the dispersion of 10 nm TiO2 NPs on in vitro and in vivo effects following the exposure via the respiratory route. We characterized NP size, surface charge, sedimentation rate, the presence of protein corona and the oxidant-generating capacity after NP dispersion in the presence/absence of serum. The effect of serum on NP internalization, cytotoxicity and pro-inflammatory responses was assessed in a human pulmonary cell line, NCI-H292. Serum in the dispersion medium led to a slower sedimentation, but an enhanced cellular uptake of TiO2 NPs. Despite this greater uptake, the pro-inflammatory response in NCI-H292 cells was lower after serum supplementation (used either as a dispersant or as a cell culture additive), which may be due to a reduced intrinsic oxidative potential of TiO2 NPs. Interestingly, serum could be added 2 h after the NP treatment without affecting the pro-inflammatory response. We also determined the acute pulmonary and hepatic toxicity in vivo 24 h after intratracheal instillation of TiO2 NPs in C57BL/6N mice. The use of serum resulted in an underestimation of the local acute inflammatory response in the lung, while a systemic response on glutathione reduction remained unaffected. In conclusion, serum as a dispersion agent for TiO2 NPs can lead to an underestimation of the acute pro-inflammatory response in vitro and in vivo. To avoid potential unwanted effects of dispersants and medium components, we recommend that the protocol of NM preparation should be thoroughly tested, and reflect as close as possible realistic exposure conditions.