Lan Ma-Hock

Testing Metal‐Oxide Nanomaterials for Human Safety

Nanomaterials can display distinct biological effects compared with bulk materials of the same chemical composition. The physico-chemical characterization of nanomaterials and their interaction with biological media are essential for reliable studies and are reviewed here with a focus on widely used metal oxide and carbon nanomaterials. Available rat inhalation and cell culture studies compared to original results suggest that hazard potential is not determined by a single physico-chemical property but instead depends on a combination of material properties. Reactive oxygen species generation, fiber shape, size, solubility and crystalline phase are known indicators of nanomaterials biological impact. According to these properties the summarized hazard potential decreases in the order multi-walled carbon nanotubes >> CeO(2), ZnO > TiO(2) > functionalized SiO(2) > SiO(2), ZrO(2), carbon black. Enhanced understanding of biophysical properties and cellular effects results in improved testing strategies and enables the selection and production of safe materials.

Development of a Short-Term Inhalation Test in the Rat Using Nano-Titanium Dioxide as a Model Substance

Evidence suggests that short-term inhalation studies may provide comparable prediction of respiratory tract toxicity to 90-day studies, presenting the opportunity to save time and resources in screening inhalation toxicity of test substances. The aim of this study was to develop a short-term inhalation test that could be employed to provide early evidence on respiratory tract effects which might occur from long-term exposure to aerosols of nano-materials. Male Wistar rats were exposed to aerosols of 0 (control), 2, 10 and 50 mg/m3 nano-titanium dioxide (TiO2) by inhalation for 6 h/day for 5 days. Necropsies were performed either immediately after the last exposure or after 3 and 16 days post exposure (study days 5, 8 and 21, respectively). Treatment with nano-TiO2 resulted in morphological changes in the lung, with 50 mg/m3 nano-TiO2 producing an increase in lung weight. Lung inflammation was associated with dose-dependent increases in bronchoalveolar lavage fluid (BALF) total cell and neutrophil counts, total protein content, enzyme activities and levels of a number of cell mediators. No indications of systemic effects could be found by measurement of appropriate clinical pathology parameters. Cell replication (determined by incorporation of 5-bromo-2′-deoxyuridine) was increased at all nano-TiO2 dose levels in large/medium bronchi and terminal bronchioles. The effects on the parameters measured were most prominent either on study day 5 or 8, with some endpoints returning to control levels by day 21. Overall, the pulmonary effects of nano-TiO2 observed in this short-term study were comparable to those previously reported in subchronic inhalation studies.

Comparing fate and effects of three particles of different surface properties: Nano-TiO2, pigmentary TiO2 and quartz

The fate of nano-TiO(2) particles in the body was investigated after inhalation exposure or intravenous (i.v.) injection, and compared with pigmentary TiO(2) and quartz. For this purpose, a 5-day inhalation study (6h/day, head/nose exposure) was carried out in male Wistar rats using nano-TiO(2) (100mg/m(3)), pigmentary TiO(2) (250mg/m(3)) and quartz dust DQ 12 (100mg/m(3)). Deposition in the lung and tissue distribution was evaluated, and histological examination of the respiratory tract was performed upon termination of exposure, and 2 weeks after the last exposure. Broncho-alveolar lavage (BAL) was carried out 3 and 14 days after the last exposure. Rats were also injected with a single intravenous dose of a suspension of TiO(2) in serum (5mg/kg body weight), and tissue content of TiO(2) was determined 1, 14 and 28 days later. The majority of the inhaled nano-TiO(2) was deposited in the lung. Translocation to the mediastinal lymph nodes was also noted, although to smaller amounts than following inhalation of pigmentary TiO(2), but much higher amounts than after exposure to quartz. Systemically available nano-TiO(2), as simulated by the i.v. injection, was trapped mainly in the liver and spleen. The (agglomerate) particle size of lung deposited nano-TiO(2) was virtually the same as in the test atmosphere. Changes in BAL fluid composition and histological examination indicated mild neutrophilic inflammation and activation of macrophages in the lung. The effects were reversible for nano- and pigmentary TiO(2), but progressive for quartz. The effects observed after 5-day inhalation exposure to nano-TiO(2) were qualitatively similar to those reported in sub-chronic studies.

Application of short-term inhalation studies to assess the inhalation toxicity of nanomaterials

BackgroundA standard short-term inhalation study (STIS) was applied for hazard assessment of 13 metal oxide nanomaterials and micron-scale zinc oxide.MethodsRats were exposed to test material aerosols (ranging from 0.5 to 50 mg/m3) for five consecutive days with 14- or 21-day post-exposure observation. Bronchoalveolar lavage fluid (BALF) and histopathological sections of the entire respiratory tract were examined. Pulmonary deposition and clearance and test material translocation into extra-pulmonary organs were assessed.ResultsInhaled nanomaterials were found in the lung, in alveolar macrophages, and in the draining lymph nodes. Polyacrylate-coated silica was also found in the spleen, and both zinc oxides elicited olfactory epithelium necrosis. None of the other nanomaterials was recorded in extra-pulmonary organs. Eight nanomaterials did not elicit pulmonary effects, and their no observed adverse effect concentrations (NOAECs) were at least 10 mg/m3. Five materials (coated nano-TiO2, both ZnO, both CeO2) evoked concentration-dependent transient pulmonary inflammation. Most effects were at least partially reversible during the post-exposure period.Based on the NOAECs that were derived from quantitative parameters, with BALF polymorphonuclear (PMN) neutrophil counts and total protein concentration being most sensitive, or from the severity of histopathological findings, the materials were ranked by increasing toxic potency into 3 grades: lower toxic potency: BaSO4; SiO2.acrylate (by local NOAEC); SiO2.PEG; SiO2.phosphate; SiO2.amino; nano-ZrO2; ZrO2.TODA; ZrO2.acrylate; medium toxic potency: SiO2.naked; higher toxic potency: coated nano-TiO2; nano-CeO2; Al-doped nano-CeO2; micron-scale ZnO; coated nano-ZnO (and SiO2.acrylate by systemic no observed effect concentration (NOEC)).ConclusionThe STIS revealed the type of effects of 13 nanomaterials, and micron-scale ZnO, information on their toxic potency, and the location and reversibility of effects. Assessment of lung burden and material translocation provided preliminary biokinetic information. Based upon the study results, the STIS protocol was re-assessed and preliminary suggestions regarding the grouping of nanomaterials for safety assessment were spelled out.

Generation and Characterization of Test Atmospheres with Nanomaterials

To ensure the product safety of nanomaterials, BASF has initiated an extensive program to study the potential inhalation toxicity of nanosize particles. As preparation work for upcoming inhalation studies, the following manufactured nanomaterials have been evaluated for their behavior in an exposure system designed for inhalation toxicity studies: titanium dioxide, carbon black, Aerosil R104, Aerosil R106, aluminum oxide, copper(II) oxide, amorphous silicon dioxide, zinc oxide, and zirconium(IV) oxide. As the physicochemical properties and the complex nature of ultrafine aerosols may substantially influence the toxic potential, the particle size, specific surface area, zeta potential, and morphology of each of the materials were determined. Aerosols of each material were generated using a dry powder aerosol generator and by nebulization of particle suspensions. The mass concentration of the particles in the inhalation atmosphere was determined gravimetrically and the particle size was determined using a cascade impactor, an optical particle counter, and a scanning mobility particle sizer. The dispersion techniques used generated fine aerosols with particle size distributions in the respiratory range. However, as a result of the significant agglomeration of nanoparticles in the test materials evaluated, no more than a few mass percent of the materials were present as single nanoparticles (i.e., < 100 nm). Considering the number, a greater percentage of nanoparticles was present. Based on the obtained results and experience with the equipment, a technical setup for inhalation studies with nanomaterials is proposed. Furthermore, a stepwise testing approach is recommended that also could reduce the number of animals used in testing.

Hazard identification of inhaled nanomaterials: making use of short-term inhalation studies

A major health concern for nanomaterials is their potential toxic effect after inhalation of dusts. Correspondingly, the core element of tier 1 in the currently proposed integrated testing strategy (ITS) is a short-term rat inhalation study (STIS) for this route of exposure. STIS comprises a comprehensive scheme of biological effects and marker determination in order to generate appropriate information on early key elements of pathogenesis, such as inflammatory reactions in the lung and indications of effects in other organs. Within the STIS information on the persistence, progression and/or regression of effects is obtained. The STIS also addresses organ burden in the lung and potential translocation to other tissues. Up to now, STIS was performed in research projects and routine testing of nanomaterials. Meanwhile, rat STIS results for more than 20 nanomaterials are available including the representative nanomaterials listed by the Organization for Economic Cooperation and Development (OECD) working party on manufactured nanomaterials (WPMN), which has endorsed a list of representative manufactured nanomaterials (MN) as well as a set of relevant endpoints to be addressed. Here, results of STIS carried out with different nanomaterials are discussed as case studies. The ranking of different nanomaterials potential to induce adverse effects and the ranking of the respective NOAEC are the same among the STIS and the corresponding subchronic and chronic studies. In another case study, a translocation of a coated silica nanomaterial was judged critical for its safety assessment. Thus, STIS enables application of the proposed ITS, as long as reliable and relevant in vitro methods for the tier 1 testing are still missing. Compared to traditional subacute and subchronic inhalation testing (according to OECD test guidelines 412 and 413), STIS uses less animals and resources and offers additional information on organ burden and progression or regression of potential effects.

Pulmonary toxicity of nanomaterials: a critical comparison of published in vitro assays and in vivo inhalation or instillation studies

To date, guidance on how to incorporate in vitro assays into integrated approaches for testing and assessment of nanomaterials is unavailable. In addressing this shortage, this review compares data from in vitro studies to results from in vivo inhalation or intratracheal instillation studies. Globular nanomaterials (ion-shedding silver and zinc oxide, poorly soluble titanium dioxide and cerium dioxide, and partly soluble amorphous silicon dioxide) and nanomaterials with higher aspect ratios (multiwalled carbon nanotubes) were assessed focusing on the Organisation for Economic Co-Operation and Development (OECD) reference nanomaterials for these substances. If in vitro assays are performed with dosages that reflect effective in vivo dosages, the mechanisms of nanomaterial toxicity can be assessed. In early tiers of integrated approaches for testing and assessment, knowledge on mechanisms of toxicity serves to group nanomaterials thereby reducing the need for animal testing.

In vivo-in vitro comparison of acute respiratory tract toxicity using human 3D airway epithelial models and human A549 and murine 3T3 monolayer cell systems.

The usefulness of in vitro systems to predict acute inhalation toxicity was investigated. Nineteen substances were tested in three-dimensional human airway epithelial models, EpiAirway™ and MucilAir™, and in A549 and 3T3 monolayer cell cultures. IC(50) values were compared to rat four-hour LC(50) values classified according to EPA and GHS hazard categories. Best results were achieved with a prediction model distinguishing toxic from non-toxic substances, with satisfactory specificities and sensitivities. Using a self-made four-level prediction model to classify substances into four in vitro hazard categories, in vivo-in vitro concordance was mediocre, but could be improved by excluding substances causing pulmonary edema and emphysema in vivo. None of the test systems was outstanding, and there was no evidence that tissue or monolayer systems using respiratory tract cells provide an added value. However, the test systems only reflected bronchiole epithelia and alveolar cells and investigated cytotoxicity. Effects occurring in other cells by other mechanisms could not be recognised. Further work should optimise test protocols and expand the set of substances tested to define applicability domains. In vivo respiratory toxicity data for in vitro comparisons should distinguish different modes of action, and their relevance for human health effects should be ensured.

Inhalation of formaldehyde does not induce systemic genotoxic effects in rats.

Male Fischer-344 rats were exposed to formaldehyde (FA) by inhalation for 4 weeks (6 h/day, 5 days/week). Groups of six rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15 ppm. Potential systemic genotoxic effects were investigated as part of a comprehensive study on local and systemic toxic and genotoxic effects. For this purpose, peripheral blood samples were obtained by puncturing the retro-orbital venous plexus at the end of the exposure period. Blood sampling was carried out in a randomized sequence and samples were coded by sequence number to ensure blind evaluation. Blood samples were used for the comet assay, the sister chromatid exchange test (SCE test) and the micronucleus test (MNT). DNA migration in the comet assay was measured both directly and after irradiation of the blood samples with 2 Gy gamma-radiation. The latter modification of the comet assay was included to increase its sensitivity for the detection of DNA-protein cross-links (DPX). The following positive control groups were included: one group (six animals) was treated with 50mg/kg methyl methanesulfonate (MMS) once by gavage 4h before blood sampling. Another group (six animals) was treated twice orally with 10mg/kg cyclophosphamide (CP) with an interval of 24 h. The last application of CP was 24h before blood sampling. For the comet assay, four slides were analysed from each blood sample, two without and two with irradiation. From each slide, 50 randomly selected cells were measured by image analysis, and tail intensity (% tail DNA) and tail moment were evaluated. For the SCE test, blood was cultured for 56 h in the presence of BrdU (10 microg/ml for the last 35 h) and SCE were counted in 30 second-division metaphases per sample. The MNT with peripheral blood was performed according to the instructions for the micronucleus analysis kit MICROFLOW (Litron Laboratories). Approximately 20,000 cells per sample were analysed by flow cytometry and the percentage of reticulocytes with micronuclei (MN) was determined. The positive control substances induced a significant effect in the genotoxicity tests and thus demonstrated the sensitivity of the test systems. FA did not induce any significant effect in any of the genotoxicity tests performed. It can be concluded that inhalation of FA in a 28-day study with FA concentrations up to 15 ppm does not lead to systemic genotoxic effects in the blood of rats.

Applicability of rat precision-cut lung slices in evaluating nanomaterial cytotoxicity, apoptosis, oxidative stress, and inflammation

The applicability of rat precision-cut lung slices (PCLuS) in detecting nanomaterial (NM) toxicity to the respiratory tract was investigated evaluating sixteen OECD reference NMs (TiO₂, ZnO, CeO₂, SiO₂, Ag, multi-walled carbon nanotubes (MWCNTs)). Upon 24-hour test substance exposure, the PCLuS system was able to detect early events of NM toxicity: total protein, reduction in mitochondrial activity, caspase-3/-7 activation, glutathione depletion/increase, cytokine induction, and histopathological evaluation. Ion shedding NMS (ZnO and Ag) induced severe tissue destruction detected by the loss of total protein. Two anatase TiO₂ NMs, CeO₂ NMs, and two MWCNT caused significant (determined by trend analysis) cytotoxicity in the WST-1 assay. At non-cytotoxic concentrations, different TiO₂ NMs and one MWCNT increased GSH levels, presumably a defense response to reactive oxygen species, and these substances further induced a variety of cytokines. One of the SiO₂ NMs increased caspase-3/-7 activities at non-cytotoxic levels, and one rutile TiO₂ only induced cytokines. Investigating these effects is, however, not sufficient to predict apical effects found in vivo. Reproducibility of test substance measurements was not fully satisfactory, especially in the GSH and cytokine assays. Effects were frequently observed in negative controls pointing to tissue slice vulnerability even though prepared and handled with utmost care. Comparisons of the effects observed in the PCLuS to in vivo effects reveal some concordances for the metal oxide NMs, but less so for the MWCNT. The highest effective dosages, however, exceeded those reported for rat short-term inhalation studies. To become applicable for NM testing, the PCLuS system requires test protocol optimization.