Toxicological inhalation studies in rats to substantiate grouping of zinc oxide nanoforms.

BACKGROUND: Significant variations exist in the forms of ZnO, making it impossible to test all forms in in vivo inhalation studies. Hence, grouping and read-across is a common approach under REACH to evaluate the toxicological profile of familiar substances. The objective of this paper is to investigate the potential role of dissolution, size, or coating in grouping ZnO (nano)forms for the purpose of hazard assessment. We performed a 90-day inhalation study (OECD test guideline no. (TG) 413) in rats combined with a reproduction/developmental (neuro)toxicity screening test (TG 421/424/426) with coated and uncoated ZnO nanoforms in comparison with microscale ZnO particles and soluble zinc sulfate. In addition, genotoxicity in the nasal cavity, lungs, liver, and bone marrow was examined via comet assay (TG 489) after 14-day inhalation exposure. RESULTS: ZnO nanoparticles caused local toxicity in the respiratory tract. Systemic effects that were not related to the local irritation were not observed. There was no indication of impaired fertility, developmental toxicity, or developmental neurotoxicity. No indication for genotoxicity of any of the test substances was observed. Local effects were similar across the different ZnO test substances and were reversible after the end of the exposure. CONCLUSION: With exception of local toxicity, this study could not confirm the occasional findings in some of the previous studies regarding the above-mentioned toxicological endpoints. The two representative ZnO nanoforms and the microscale particles showed similar local effects. The ZnO nanoforms most likely exhibit their effects by zinc ions as no particles could be detected after the end of the exposure, and exposure to rapidly soluble zinc sulfate had similar effects. Obviously, material differences between the ZnO particles do not substantially alter their toxicokinetics and toxicodynamics. The grouping of ZnO nanoforms into a set of similar nanoforms is justified by these observations.

Proceedings of the 2020 Classic Examples in Toxicologic Pathology XXVII.

The histopathology slide seminar "Classic Examples in Toxicologic Pathology XXVII" was held on February 21 and 22, 2020, at the Department of Pathology at the University of Veterinary Medicine in Hannover, Germany, with joint organization by the European Society of Toxicologic Pathology. The goal of this annual seminar is to present and discuss classical and actual cases of toxicologic pathology. This article summarizes the presentations given during the seminar, including images of representative lesions. Ten actual and classical cases of toxicologic pathology, mostly induced by a test article, were presented. These included small intestine pathology and transcriptomics induced by a γ-secretase modulator, liver findings in nonhuman primates induced by gene therapy, drug-induced neutropenia in dogs, device-induced growth plate lesions, polycystic lesions in CAR/PXR double knockout mice, inner ear lesions in transgenic mice, findings in Beagle dogs induced by an inhibitor of the myeloid leukemia cell differentiation protein MCL1, findings induced by a monovalent fibroblast growth factor receptor 1 antagonist, kidney lesions induced by a mammalian target of rapamycin inhibitor in combination therapy, and findings in mutation-specific drugs.

Metazachlor: Mode of action analysis for rat liver tumour formation and human relevance.

In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000 ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000 ppm metazachlor for 3, 7, 14 and 28 days. The treatment of male rats with 200 and 8000 ppm metazachlor and female rats with 8000 ppm metazachlor resulted in significant increases in relative liver weight, which was associated with a centrilobular hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 8000 ppm metazachlor for 3 and 7 days and in female rats given 200 ppm metazachlor for 7-28 days and 8000 ppm metazachlor for 3-28 days. Significant increases in relative liver weight, centrilobular hepatocyte hypertrophy and hepatocyte RDS were also observed in male and female Wistar rats given and 500 ppm sodium phenobarbital (NaPB) for 3-28 days. The treatment of female Wistar rats with either 8000 ppm metazachlor for 7 days or with 500 ppm NaPB for 3 and 7 days resulted in the nuclear translocation of the hepatic constitutive androstane receptor (CAR). Treatment of male and female Wistar rats with 8000 ppm metazachlor for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content, CYP2B subfamily-dependent enzyme activities and mRNA levels, together with some increases in CYP3A enzyme activity and mRNA levels. The treatment of male Wistar rat hepatocytes with metazachlor (concentration range 0.5-50 µM) and NaPB (500 µM) for 4 days resulted in increased CYP2B enzyme activities and mRNA levels; with metazachlor and NaPB also producing significant increases in hepatocyte RDS levels. Studies were also performed with hepatocytes from male Sprague-Dawley wild type (WT) rats and CAR knockout (CAR KO) rats. While both treatment with metazachlor and NaPB for 4 days increased CYP2B enzyme activities and mRNA levels in WT rat hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both metazachlor and NaPB only increased RDS in WT but not in CAR KO rat hepatocytes. The treatment of hepatocytes from two male human donors with 0.5-25 µM metazachlor or 500 µM NaPB for 4 days resulted in increases in CYP2B6 and CYP3A4 mRNA levels but had no effect on hepatocyte RDS. EGF as concurrently used positive control demonstrated the expected RDS response in all rat and human hepatocyte cultures. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that metazachlor is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for metazachlor-induced female rat liver tumour formation has been established. Based on the lack of effect of metazachlor on RDS in human hepatocytes, it is considered that the MOA for metazachlor-induced rat liver tumour formation is qualitatively not plausible for humans.

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

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/m(3) 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.

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.

Dissecting aortic aneurysm induced by N-(2-aminoethyl) ethanolamine in rat: Role of defective collagen during development.

BACKGROUND: Dissecting aortic aneurysm (DAA) is a tear in the wall of the aorta that causes blood to flow, or "dissect," between the medial layers of the media. METHODS: Pregnant rats (dams) were treated with the industrial chemical n-(2-aminoethyl) ethanolamine (AEEA) by intraperitoneal injection or gavage. The histology and pathology of aorta in the thorax from newborn pups were examined. Aortas of fetuses of gestational day 20 from dams exposed to AEEA were harvested for immunohistochemical staining and native Western blot to study the changes of collagen type 1 and type 3 in aorta. RESULTS: Dissecting aortic aneurysm of newborn rats was induced by treating with AEEA through intraperitoneal injection or gavage. The incidence of DAA reached 100% in live pups at the high dose by means of gavage of AEEA, but without lethality compared with intraperitoneal injection. A grading system for the dose-response of DAA lesions associated with AEEA by gavage was established. Gestational day 20 fetuses from treated dams showed a decreased content and altered distribution of medial and adventitial collagen type 1 and 3 in aorta by immunohistochemistry; this decrease was confirmed by native Western blot. CONCLUSION: This in vivo model of spontaneous aortic dissection bears striking similarities histologically to human aortic dissection. As such, the model conceivably could contribute to elucidating the mechanisms of DAA formation and to exploring diagnostic and therapeutic strategies. The pathogenesis of AEEA-induced DAA may be related to defects in the normal developmental progression of collagen types 1 and 3 in the vascular wall.

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

BACKGROUND: A standard short-term inhalation study (STIS) was applied for hazard assessment of 13 metal oxide nanomaterials and micron-scale zinc oxide. METHODS: Rats 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. RESULTS: Inhaled 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)). CONCLUSION: The 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.

Comparative inhalation toxicity of multi-wall carbon nanotubes, graphene, graphite nanoplatelets and low surface carbon black.

BACKGROUND: Carbon nanotubes, graphene, graphite nanoplatelets and carbon black are seemingly chemically identical carbon-based nano-materials with broad technological applications. Carbon nanotubes and carbon black possess different inhalation toxicities, whereas little is known about graphene and graphite nanoplatelets. METHODS: In order to compare the inhalation toxicity of the mentioned carbon-based nanomaterials, male Wistar rats were exposed head-nose to atmospheres of the respective materials for 6 hours per day on 5 consecutive days. Target concentrations were 0.1, 0.5, or 2.5 mg/m3 for multi-wall carbon nanotubes and 0.5, 2.5, or 10 mg/m3 for graphene, graphite nanoplatelets and low-surface carbon black. Toxicity was determined after end of exposure and after three-week recovery using broncho-alveolar lavage fluid and microscopic examinations of the entire respiratory tract. RESULTS: No adverse effects were observed after inhalation exposure to 10 mg/m3 graphite nanoplatelets or relatively low specific surface area carbon black. Increases of lavage markers indicative for inflammatory processes started at exposure concentration of 0.5 mg/m3 for multi-wall carbon nanotubes and 10 mg/m3 for graphene. Consistent with the changes in lavage fluid, microgranulomas were observed at 2.5 mg/m3 multi-wall carbon nanotubes and 10 mg/m3 graphene. In order to evaluate volumetric loading of the lung as the key parameter driving the toxicity, deposited particle volume was calculated, taking into account different methods to determine the agglomerate density. However, the calculated volumetric load did not correlate to the toxicity, nor did the particle surface burden of the lung. CONCLUSIONS: The inhalation toxicity of the investigated carbon-based materials is likely to be a complex interaction of several parameters. Until the properties which govern the toxicity are identified, testing by short-term inhalation is the best option to identify hazardous properties in order to avoid unsafe applications or select safer alternatives for a given application.

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.

Proliferative and non-proliferative lesions of the rat and mouse integument.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) project is a joint initiative of the societies of toxicological pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP). Its aim is to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory rodents. A widely accepted international harmonization of nomenclature in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and will provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. The purpose of this publication is to provide a standardized nomenclature for classifying microscopical lesions observed in the integument of laboratory rats and mice. Example colour images are provided for most lesions. The standardized nomenclature presented in this document and additional colour images are also available electronically at http://www.goreni.org. The nomenclature presented herein is based on histopathology databases from government, academia, and industrial laboratories throughout the world, and covers lesions that develop spontaneously as well as those induced by exposure to various test materials. (DOI: 10.1293/tox.26.27S; J Toxicol Pathol 2013; 26: 27S-57S).

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 TiO(2) 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/m(3) TiO(2) 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 TiO(2) were observed in the mediastinal lymph nodes but not in secondary organs. CONCLUSIONS: For nanostructured TiO(2), the deposition of nanoscaled particles in the lung seem to play a negligible role.

Proliferative and nonproliferative lesions of the rat and mouse mammary, Zymbal's, preputial, and clitoral glands.

The mammary gland of laboratory rodents is an important organ for the evaluation of effects of xenobiotics, especially those that perturb hormonal homeostasis or are potentially carcinogenic. Mammary gland cancer is a leading cause of human mortality and morbidity worldwide and is a subject of major research efforts utilizing rodent models. Zymbal's, preputial, and clitoral glands are standard tissues that are evaluated in animal models that enable human risk assessment of xenobiotics. A widely accepted and utilized international harmonization of nomenclature for mammary, Zymbal's, preputial, and clitoral gland lesions in laboratory animals will improve diagnostic alignment among regulatory and scientific research organizations and enrich international exchanges of information among toxicologists and pathologists.

Nanostructured calcium silicate hydrate seeds accelerate concrete hardening: a combined assessment of benefits and risks.

Nanotechnology creates new possibilities to control and improve material properties for civil infrastructure. Special focus in this area is put on Portland cement and gypsum. Together their annual production is by far larger than for any other material worldwide. Nanomodification of these materials can be done during the few hours between dissolution and hardening, especially by nucleation of the re-crystallization with suitable colloids. Here we report first results in homogeneous seeding of the precipitation of calcium silicate hydrates within a real Portland cement composition. The occupational safety during the production phase and during mixing of concrete paste is addressed in detail by in vivo testing. We perform 5-day inhalation with 21-day recovery in rats and analyze organ-specific toxicity and 71 endpoints from bronchoalveolar lavage (BALF) and blood. In BALF parameters, no test-related changes were observed, indicating the generally low toxicity of the test material. Some mild lesions were observed in larynx level. In the lungs, all animals of the 50 mg/m³ concentration group revealed a minimal to mild increase in alveolar macrophages, which recovered back to control level.

Malformations of the great vessels in the neonatal rat induced by N-(2-aminoethyl)ethanolamine.

The reproductive and developmental toxicity of aminoethylethanolamine was evaluated in a standard screening study (OECD, 1995: Organisation for economic co-operation and development. Paris, France), in which groups of Wistar rats (10/sex/group) were administered the test substance by gavage at dosage levels of 50, 250, or 1000 mg/kg/day (groups 2-4, respectively). A control group received the vehicle, doubly distilled water. No live pups were delivered in group 4, and there was a higher incidence of stillborn offspring and reduced postnatal survival in group 3. Macroscopic changes in groups 2 and 3 were primarily related to the great vessels and characterized by dilations, aneurysms, and altered course of the aorta, pulmonary trunk, carotids, and the ductus arteriosus. A follow-up study was conducted to characterize the low dose-response, using dosage levels of 0, 0.2, 1, 5, or 50 mg/kg/day (groups 1-5, respectively). Given the expected scarcity of the lesions in control offspring, each group consisted of 25 animals of each sex. Macroscopic examination revealed a high incidence (18.5%) of aneurysm-bearing offspring in group 5 litters, and single offspring (0.3-0.4%) with aneurysms in groups 3 and 4. Microscopic examination revealed dissecting aneurysms in offspring from all aminoethylethanolamine treatment groups, without a clear dose-response between groups 2 and 4 (0.6%, 1.2%, and 0.3%, respectively), and focal hemorrhages in all groups including the control. In comparison, the background incidence of aneurysms in untreated 4-day old offspring was 0.2% (Treumann et al., 2011: Toxicol Pathol 39:969-974). Consequently, the findings in groups 2-4 cannot be conclusively attributed to treatment.

Investigation on the genotoxicity of different sizes of gold nanoparticles administered to the lungs of rats.

Nanomaterials are already used today and offer even greater use and benefits in the future. The progress of nanotechnology must be accompanied by investigations of their potential harmful effects. For airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. While standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. We have investigated two genotoxic endpoints - alkaline Comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow - in a combined study 72 h after a single instillation of 18 µg gold nanoparticles (NP) into the trachea of male adult Wistar rats. The administration of three test materials differing only in their primary particle size (2, 20 and 200 nm) did not lead to relevant DNA damage in the mentioned tests. The measurement of clinical pathology parameters in bronchoalveolar lavage fluid (BALF) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. Minor histopathology findings occurred in the lung of the animals exposed to 20 nm and 200 nm sized nanomaterials. In conclusion, under the conditions of this study the different sized gold NP tested were non-genotoxic and showed no systemic and local adverse effects at the given dose.

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.

On the lifecycle of nanocomposites: comparing released fragments and their in-vivo hazards from three release mechanisms and four nanocomposites.

Nanocomposites are the dominating class of nanomaterials to come into consumer contact, and were in general assumed to pose low risk. The first data is now emerging on the exposure from nanocomposites, but little is yet known about their hypothetical nanospecific physiological effects, giving ample room for speculation. For the first time, this comprehensive study addresses these aspects in a systematic series of thermoplastic and cementitious nanocomposite materials. Earlier reports that 'chalking', the release of pigments from weathered paints, also occurs for nanocomposites, are confirmed. In contrast, mechanical forces by normal consumer use or do-it-yourself sanding do not disrupt nanofillers (nanoparticles or nanofibers) from the matrix. Detailed evidence is provided for the nature of the degradation products: no free nanofillers are detected up to the detection threshold of 100 ppm. Sanding powders measuring 1 to 80 µm in diameter are identified with the original material, still containing the nanofillers. The potential hazard from aerosols generated by sanding nanocomposites up to the nuisance dust limit is also investigated. In-vivo instillation in rats is used to quantify physiological effects on degradation products from abraded nanocomposites, in comparison to the abraded matrix without nanofiller and to the pure nanofiller. In this pioneering and preliminary evaluation, the hazards cannot be distinguished with or without nanofiller.

Inhalation toxicity of multiwall carbon nanotubes in rats exposed for 3 months.

Carbon nanotubes (CNT) are of great commercial interest. Theoretically, during processing and handling of CNT and in abrasion processes on composites containing CNT, inhalable CNT particles might be set free. For hazard assessment, we performed a 90-day inhalation toxicity study with a multiwall CNT (MWCNT) material (Nanocyl NC 7000) according to Organisation for Economic Co-operation and Development test guideline 413. Wistar rats were head-nose exposed for 6 h/day, 5 days/week, 13 weeks, total 65 exposures, to MWCNT concentrations of 0 (control), 0.1, 0.5, or 2.5 mg/m(3). Highly respirable dust aerosols were produced with a proprietary brush generator which neither damaged the tube structure nor increased reactive oxygen species on the surface. Inhalation exposure to MWCNT produced no systemic toxicity. However, increased lung weights, pronounced multifocal granulomatous inflammation, diffuse histiocytic and neutrophilic inflammation, and intra-alveolar lipoproteinosis were observed in lung and lung-associated lymph nodes at 0.5 and 2.5 mg/m(3). These effects were accompanied by slight blood neutrophilia at 2.5 mg/m(3). Incidence and severity of the effects were concentration related. At 0.1 mg/m(3), there was still minimal granulomatous inflammation in the lung and in lung-associated lymph nodes; a no observed effect concentration was therefore not established in this study. The test substance has low dust-forming potential, as demonstrated by dustiness measurements, but nonetheless strict industrial hygiene measures must be taken during handling and processing. Toxicity and dustiness data such as these can be used to compare different MWCNT materials and to select the material with the lowest risk potential for a given application.