Silke Treumann

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.

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.

Short term inhalation toxicity of a liquid aerosol of CdS/Cd(OH)2 core shell quantum dots in male Wistar rats

Colloidal quantum dots (QD) show great promise as fluorescent markers. The QD used in this study were obtained in aqueous medium rather than the widely used colloidal QD. Both methodologies used for the production of QD are associated with the presence of heavy metals such as cadmium (Cd). Here we investigate the short-term inhalation toxicity of water-soluble core-shell CdS/Cd(OH)₂ QD. Male Wistar rats were head-nose exposed for 6 h/day on 5 days at the technically maximum concentration (0.52 mg Cd/m³). Histological examination was performed directly after the last exposure. Additional rats were used for Cd organ burden determinations. Clinical parameters in blood, bronchoalveolar lavage fluid and lung tissue were determined 3 days after the last exposure. To analyze the reversibility or progression of effects, the examinations were performed again after a recovery period of 3 weeks. The results of the study indicate that CdS/Cd(OH)₂ QD caused local neutrophil inflammation in the lungs that partially regressed after the 3-week recovery period. There was no evidence that QD were translocated to the central nervous system nor that a systemic acute phase response occurred.

Deposition behavior of inhaled nanostructured TiO2 in rats: fractions of particle diameter below 100 nm (nanoscale) and the slicing bias of transmission electron microscopy.

Context: In experimental studies with nanomaterials where translocation to secondary organs was observed, the particle sizes were smaller than 20 nm and were mostly produced by spark generators. Engineered nanostructured materials form microsize aggregates/agglomerates. Thus, it is unclear whether primary nanoparticles or their small aggregates/agglomerates occur in non-negligible concentrations after exposure to real-world materials in the lung. Objective: We dedicated an inhalation study with nanostructured TiO2 to the following research question: Does the particle size distribution in the lung contain a relevant subdistribution of nanoparticles? Methods: Six rats were exposed to 88 mg/m3 TiO2 over 5 days with 20% (count fraction) and <0.5% (mass fraction) of nanoscaled objects. Three animals were sacrificed after cessation of exposure (5 days), others after a recovery period of 14 days. Particle sizes were determined morphometrically by transmission electron microscopy (TEM) of ultra-thin lung slices. Since the particles visible are two-dimensional surrogates of three-dimensional structures we developed a model to estimate expected numbers of particle diameters below 100 nm due to the TEM slicing bias. Observed and expected numbers were contrasted in 2 × 2 tables by odds ratios. Results: Comparisons of observed and expected numbers did not present evidence in favor of the presence of nanoparticles in the rat lungs. In simultaneously exposed satellite animals agglomerates of nanostructured TiO2 were observed in the mediastinal lymph nodes but not in secondary organs. Conclusions: For nanostructured TiO2, the deposition of nanoscaled particles in the lung seem to play a negligible role.

Additional Histopathologic Examination of the Lungs from a 3-Month Inhalation Toxicity Study with Multiwall Carbon Nanotubes in Rats

For hazard assessment of multiwalled carbon nanotubes (MWCNTs), a 90-day inhalation toxicity study has been performed with Nanocyl NC 7000 in accordance with OECD 413 test guideline. MWCNTs produced no systemic toxicity. However, increased lung weights, multifocal granulomatous inflammation, diffuse histiocytic and neutrophilic infiltrates, and intra-alveolar lipoproteinosis were observed in lung and lung-associated lymph nodes at 0.5 and 2.5mg/m(3). Additional investigations of the lungs were performed, including special stains for examination of connective tissue, and electron microscopy was performed to determine the location of the MWCNTs. The alveolar walls revealed no increase of collagen fibers, whereas within the microgranulomas a slight increase of collagen fibers was observed. The pleura did not reveal any increase in collagen fibers. Only a slight increase in reticulin fibers in the alveolar walls in animals of the 0.5 and 2.5mg/m(3) concentration group was noted. In the 0.1mg/m(3) group, the only animal revealing minimal granulomas exhibited a minimal increase in collagen within the granuloma. No increase in reticulin was observed. Electron microscopy demonstrated entangled MWCNTs within alveolar macrophages. Occasionally electron dense particles/detritus were observed within membrane-bound vesicles (interpreted as phagosomes), which could represent degraded MWCNTs. If so, MWCNTs were degradable by alveolar macrophages and not persistent within the lung. Inhalation of MWCNTs caused granulomatous inflammation within the lung parenchyma but not the pleura in any of the concentration groups. Thus, there are some similarities to effects caused by inhaled asbestos, but the hallmark effects, namely pleural inflammation and/or fibrosis leading to mesotheliomas, are absent.

Short term inhalation toxicity of a liquid aerosol of glutaraldehyde-coated CdS/Cd(OH)2 core shell quantum dots in rats

Quantum dots exhibit extraordinary optical and mechanical properties, and the number of their applications is increasing. In order to investigate a possible effect of coating on the inhalation toxicity of previously tested non-coated CdS/Cd(OH)2 quantum dots and translocation of these very small particles from the lungs, rats were exposed to coated quantum dots or CdCl2 aerosol (since Cd(2+) was present as impurity), 6h/d for 5 consecutive days. Cd content was determined in organs and excreta after the end of exposure and three weeks thereafter. Toxicity was determined by examination of broncho-alveolar lavage fluid and microscopic evaluation of the entire respiratory tract. There was no evidence for translocation of particles from the respiratory tract. Evidence of a minimal inflammatory process was observed by examination of broncho-alveolar lavage fluid. Microscopically, minimal to mild epithelial alteration was seen in the larynx. The effects observed with coated quantum dots, non-coated quantum dots and CdCl2 were comparable, indicating that quantum dots elicited no significant effects beyond the toxicity of the Cd(2+) ion itself. Compared to other compounds with larger particle size tested at similarly low concentrations, quantum dots caused much less pronounced toxicological effects. Therefore, the present data show that small particle sizes with corresponding high surfaces are not the only factor triggering the toxic response or translocation.

Comparative short-term inhalation toxicity of five organic diketopyrrolopyrrole pigments and two inorganic iron-oxide-based pigments.

Abstract Diketopyrrolopyrroles (DPP) are a relatively new class of organic high-performance pigments. The present inhalation and particle characterization studies were performed to compare the effects of five DPP-based pigments (coarse and fine Pigment Red 254, coarse and fine meta-chloro DPP isomer and one form of mixed chlorinated DPP isomers) and compare it to coarse and fine inorganic Pigment Red 101. Wistar rats were exposed head-nose to atmospheres of the respective materials for 6 h/day on 5 consecutive days. Target concentrations were 30 mg/m3 as high dose for all compounds and selected based occupational exposure limits for respirable nuisance dust. Toxicity was determined after end of exposure and after 3-week recovery using broncho-alveolar lavage fluid (BALF) and microscopic examinations of the entire respiratory tract. Mixed chlorinated DPP isomers and coarse meta-chloro DPP isomer caused marginal changes in BALF, consisting of slight increases of polymorphonuclear neutrophils, and in case of coarse meta-chloro DPP increased MCP-1 and osteopontin levels. Mixed chlorinated DPP isomers, Pigment Red 254, and meta-chloro DPP caused pigment deposits and phagocytosis by alveolar macrophages, slight hypertrophy/hyperplasia of the bronchioles and alveolar ducts, but without evidence of inflammation. In contrast, only pigment deposition and pigment phagocytosis were observed after exposure to Pigment Red 101. All pigments were tolerated well and caused only marginal effects in BALF or no effects at all. Only minor effects were seen on the lung by microscopic examination. There was no evidence of systemic inflammation based on acute-phase protein levels in blood.

Spontaneous Occurrence of Dissecting Aneurysms in the Region of the Ductus Arteriosus in Four-Day-Old Wistar Rat Pups

Dissecting aortic aneurysms, generally involving the thoracic aorta, have been shown to be caused by specific aliphatic amines in developing rats. Whether such lesions might occur spontaneously in control rats is not known. Therefore, in this study, 1,016 four-day-old, untreated rats culled from ongoing scheduled breeding studies were subjected to gross and histopathological examination in order to create a background control data base on the incidence of spontaneous aortic dissecting aneurysms. Two animals (0.2%) were found to have small dissecting aortic aneurysms, and an additional 2 (0.2%) had only hemorrhagic lesions. All of these lesions were limited to the region of the ductus arteriosus. An additional 18 findings were judged to be artifacts. These findings suggest that small vascular dissections may rarely occur in the aortic arch adjacent to the ductus arteriosus. Special attention should be paid in experimental studies to avoid confusing these small spontaneous lesions with treatment-induced lesions or artifacts.

Occurrence of Pineal Gland Tumors in Combined Chronic Toxicity/Carcinogenicity Studies in Wistar Rats

Pineal gland tumors are very rare brain lesions in rats as well as in other species including humans. A total of 8 (out of 1,360 examined) Wistar rats from 3 different combined chronic toxicity/carcinogenicity or mere carcinogenicity studies revealed pineal gland tumors. The tumors were regarded to be spontaneous and unrelated to treatment. The morphology and immunohistochemical evaluation led to the diagnosis malignant pinealoma. The main characteristics that were variably developed within the tumors were the following: cellular atypia, high mitotic index, giant cells, necrosis, Homer Wright rosettes, Flexner-Wintersteiner rosettes and pseudorosettes, positive immunohistochemical reaction for synaptophysin, and neuron-specific enolase. The pineal gland is not a protocol organ for histopathological examination in carcinogenicity studies. Nevertheless, the pineal gland can occasionally be encountered on the routine brain section or if it is the origin of a tumor protruding into the brain, the finding will be recorded. Therefore, although known to be a rare tumor in rats, pineal neoplasms should be included in the list of possible differential diagnoses for brain tumors, especially when the tumor is located in the region of the pineal body.

Particle diameter estimates of Creutzenberg et al. (2012) are distorted due to the slicing bias of transmission electron microscopy

Dear Editor, We read with interest the study by Creutzenberg et al. (2012) and Schaudien et al. (2012) on the presence of nanoparticles in vivo following lung exposure in rats. Particle sizes were determined morphometrically by transmission electron microscopy (TEM) of ultrathin lung slices. A particular methodological challenge is the two dimensional presentation of larger particles in the ultrathin TEM section planes and the analytical approach to extract informative data thereof. The typical thickness of an ultrathin section is about 80 nm or smaller (Hayat, 1986). Therefore, a larger particle (e.g. with a diameter of 300 nm) is cut into a slice at a random section plane. The profiles of this slice visible and measured in the TEM sections predictably underestimate the true particle diameter: this is the TEM slicing bias. This slicing bias is amplified by the systematic oversampling of large particles (Boyce et al., 2010; Cruz-Orive, 1987; Hsia et al., 2010). We developed a model to estimate expected numbers of particle diameters below 100 nm due to the TEM slicing bias and contrasted observed and expected numbers in 2 × 2 tables by odds ratios (Morfeld et al., 2012). Our results showed pronounced overestimates of the prevalence of small particles if the bias is not taken into account. Creutzenberg et al. (2012) and Schaudien et al. (2012) did not take care of the TEM slicing bias in their analyses. The results presented in Table 3, Figure 2 and Figure 3 in Creutzenberg et al. (2012) are distorted suggesting erroneously the presence of too many small particles. This may irritate readers who are interested in the question whether nano-objects are present in the lung after exposure to nanostructured materials. As an example for such an irritation we note that the MAK committee appears to take such an observation for granted: “Ultrafine primary particles are measured according to their mobility-equivalent diameter (DM) <100 nm (corresponds to a diffusionequivalent diameter (Dae) <100 nm). They can occur as single particles in the workplace air or more often as basic units of aggregates and agglomerates. In these forms they can be seen under an electron microscope”. (Deutsche Forschungsgemeinschaft, 2012). We would suggest correcting the particle size distributions given in Creutzenberg et al. (2012) for the immanent TEM slicing bias to avoid confusion.