Toxicokinetic study following intratracheal instillation or oral gavage of two [7Be]-tagged carbon black samples.

BACKGROUND: The toxicokinetic behaviour of nanostructured particles following pulmonary or oral deposition is of great scientific interest. In this toxicokinetic study, following the general principles of OECD TG 417, the systemic availability of carbon black, a nanostructured material consisting of agglomerated aggregates was characterised. METHODS: Each of two grades of beryllium-7 labelled carbon black (Monarch® 1000, oxidized and Printex® 90; untreated) was administered either intratracheally or orally to adult rats. Independent of route, rats received a single dose of approximately 0.3 mg radiolabelled carbon black. A total of 12 rats were treated per grade and per exposure route: 4 females each for feces/urine/organs and serial blood kinetics; 4 males for organs. At necropsy, the complete suite of organs was analysed for females, but only the lungs, liver, kidney, reproductive organs for males. RESULTS: In the pulmonarily exposed animals, 7Be-Monarch® 1000 and 7Be-Printex® 90 was detected in feces in the first 3 days after treatment at significant levels, i.e. 17.6% and 8.2%, respectively. In urine, small percentages of 6.7% and 0.4% were observed, respectively. In blood, radioactivity, representative of carbon black was within the background noise of the measurement method. At necropsy, 20 days post-instillation, both test items were practically exclusively found in lungs (75.1% and 91.0%, respectively) and in very small amounts (approximately 0.5%) in the lung-associated lymph nodes (LALN). In the other organs/tissues the test item was not detectable. BAL analyses indicated that carbon black particles were completely engulfed by alveolar macrophages. In orally exposed animals, 98% (7Be-Monarch® 1000) and 99% (7Be-Printex® 90) of the measured radioactivity was detected in feces. Excretion was complete within the first 3 days following treatment. 1.3% and 0.5% of measured activity was attributable to urine in animals that received 7Be-Monarch® 1000 and 7Be-Printex® 90, respectively. Radioactivity was absent in blood and other organs and tissues. CONCLUSION: Radioactivity, representative of carbon black, was not detected beyond the experimentally defined limit of quantitation systemically after deposition in lungs or stomach in rats. Under these experimental conditions, the two CB samples were not shown to translocate beyond the lung or the GI tract into the blood compartment.

A tiered approach to investigate the inhalation toxicity of cobalt substances. Tier 4: Effects from a 28-day inhalation toxicity study with tricobalt tetraoxide in rats.

Lung cancer following inhalation in rodents is a major concern regarding exposure to cobalt substances. However, little information is available on adverse effects and toxicity following long-term inhalation exposure to poorly soluble cobalt substances with low bioavailability. Thus, the present study focused on pulmonary effects of the poorly soluble tricobalt tetraoxide (5, 20, 80 mg/m³) in a 28-day inhalation exposure study. Lung weights increased with increasing exposures. Bronchoalveolar lavage fluid analysis and histopathology revealed lung tissue inflammation at the mid-dose with increasing severity in the high-dose group and post-exposure persistency. Markers for cellular damage and cell proliferation were statistically significantly increased. No increase in 8-OH-dG lesions was observed, indicating an absence of oxidative DNA lesions. The primary effect of inhaled Co3O4 particles is inflammation of the respiratory tract strongly resembling responses of inhaled "inert dust" substances, with a NOAEC of 5 mg/m³ under the conditions of this test.

Femtosecond pulsed laser microscopy: a new tool to assess the in vitro delivered dose of carbon nanotubes in cell culture experiments.

BACKGROUND: In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs). METHODS AND RESULTS: We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1ß by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1ß responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples. CONCLUSION: The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.

Pyrrolizidine alkaloid-induced transcriptomic changes in rat lungs in a 28-day subacute feeding study.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites synthesized by a wide range of plants as protection against herbivores. These toxins are found worldwide and pose a threat to human health. PAs induce acute effects like hepatic sinusoidal obstruction syndrome and pulmonary arterial hypertension. Moreover, chronic exposure to low doses can induce cancer and liver cirrhosis in laboratory animals. The mechanisms causing hepatotoxicity have been investigated previously. However, toxic effects in the lung are less well understood, and especially data on the correlation effects with individual chemical structures of different PAs are lacking. The present study focuses on the identification of gene expression changes in vivo in rat lungs after exposure to six structurally different PAs (echimidine, heliotrine, lasiocarpine, senecionine, senkirkine, and platyphylline). Rats were treated by gavage with daily doses of 3.3 mg PA/kg bodyweight for 28 days and transcriptional changes in the lung and kidney were investigated by whole-genome microarray analysis. The results were compared with recently published data on gene regulation in the liver. Using bioinformatics data mining, we identified inflammatory responses as a predominant feature in rat lungs. By comparison, in liver, early molecular consequences to PAs were characterized by alterations in cell-cycle regulation and DNA damage response. Our results provide, for the first time, information about early molecular effects in lung tissue after subacute exposure to PAs, and demonstrates tissue-specificity of PA-induced molecular effects.

The pulmonary toxicity of carboxylated or aminated multi-walled carbon nanotubes in mice is determined by the prior purification method.

BACKGROUND: Inhalation of multi-walled carbon nanotubes (MWCNTs) poses a potential risk to human health. In order to safeguard workers and consumers, the toxic properties of MWCNTs need to be identified. Functionalization has been shown to either decrease or increase MWCNT-related pulmonary injury, depending on the type of modification. We, therefore, investigated both acute and chronic pulmonary toxicity of a library of MWCNTs derived from a common pristine parent compound (NC7000). METHODS: MWCNTs were thermally or chemically purified and subsequently surface functionalized by carboxylation or amination. To evaluate pulmonary toxicity, male C57BL6 mice were dosed via oropharyngeal aspiration with either 1.6 or 4 mg/kg of each MWCNT type. Mitsui-7 MWCNT was used as a positive control. Necropsy was performed at days 3 and 60 post-exposure to collect bronchoalveolar lavage fluid (BALF) and lungs. RESULTS: At day 3 all MWCNTs increased the number of neutrophils in BALF. Chemical purification had a greater effect on pro-inflammatory cytokines (IL-1ß, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-ß1). At day 60, thermally purified, carboxylated MWCNTs had the strongest effect on lymphocyte numbers in BALF. Thermally purified MWCNTs caused the greatest increase in LDH and total protein in BALF. Furthermore, the thermally purified and carboxyl- or amine-functionalized MWCNTs caused the greatest number of granulomatous lesions in the lungs. The physicochemical characteristics mainly associated with increased toxicity of the thermally purified derivatives were decreased surface defects and decreased amorphous content as indicated by Raman spectroscopy. CONCLUSIONS: These data demonstrate that the purification method is an important determinant of lung toxicity induced by carboxyl- and amine-functionalized MWCNTs.

Hepatotoxic pyrrolizidine alkaloids induce DNA damage response in rat liver in a 28-day feeding study.

Pyrrolizidine alkaloids (PA) are secondary plant metabolites that occur as food and feed contaminants. Acute and subacute PA poisoning can lead to severe liver damage in humans and animals, comprising liver pain, hepatomegaly and the development of ascites due to occlusion of the hepatic sinusoids (veno-occlusive disease). Chronic exposure to low levels of PA can induce liver cirrhosis and liver cancer. However, it is not well understood which transcriptional changes are induced by PA and whether all hepatotoxic PA, regardless of their structure, induce similar responses. Therefore, a 28-day subacute rat feeding study was performed with six structurally different PA heliotrine, echimidine, lasiocarpine, senecionine, senkirkine, and platyphylline, administered at not acutely toxic doses from 0.1 to 3.3 mg/kg body weight. This dose range is relevant for humans, since consumption of contaminated tea may result in doses of ~ 8 µg/kg in adults and cases of PA ingestion by contaminated food was reported for infants with doses up to 3 mg/kg body weight. ALT and AST were not increased in all treatment groups. Whole-genome microarray analyses revealed pronounced effects on gene expression in the high-dose treatment groups resulting in a set of 36 commonly regulated genes. However, platyphylline, the only 1,2-saturated and, therefore, presumably non-hepatotoxic PA, did not induce significant expression changes. Biological functions identified to be affected by high-dose treatments (3.3 mg/kg body weight) comprise cell-cycle regulation associated with DNA damage response. These functions were found to be affected by all analyzed 1,2-unsaturated PA.In conclusion, 1,2-unsaturated hepatotoxic PA induced cell cycle regulation processes associated with DNA damage response. Similar effects were observed for all hepatotoxic PA. Effects were observed in a dose range inducing no histopathological alterations and no increase in liver enzymes. Therefore, transcriptomics studies identified changes in expression of genes known to be involved in response to genotoxic compounds at PA doses relevant to humans under worst case exposure scenarios.

Cerium oxide and barium sulfate nanoparticle inhalation affects gene expression in alveolar epithelial cells type II.

BACKGROUND: Understanding the molecular mechanisms of nanomaterial interacting with cellular systems is important for appropriate risk assessment. The identification of early biomarkers for potential (sub-)chronic effects of nanoparticles provides a promising approach towards cost-intensive and animal consuming long-term studies. As part of a 90-day inhalation toxicity study with CeO2 NM-212 and BaSO4 NM-220 the present investigations on gene expression and immunohistochemistry should reveal details on underlying mechanisms of pulmonary effects. The role of alveolar epithelial cells type II (AEII cells) is focused since its contribution to defense against inhaled particles and potentially resulting adverse effects is assumed. Low dose levels should help to specify particle-related events, including inflammation and oxidative stress. RESULTS: Rats were exposed to clean air, 0.1, 0.3, 1.0, and 3.0 mg/m3 CeO2 NM-212 or 50.0 mg/m3 BaSO4 NM-220 and the expression of 391 genes was analyzed in AEII cells after one, 28 and 90 days exposure. A total number of 34 genes was regulated, most of them related to inflammatory mediators. Marked changes in gene expression were measured for Ccl2, Ccl7, Ccl17, Ccl22, Ccl3, Ccl4, Il-1α, Il-1ß, and Il-1rn (inflammation), Lpo and Noxo1 (oxidative stress), and Mmp12 (inflammation/lung cancer). Genes related to genotoxicity and apoptosis did not display marked regulation. Although gene expression was less affected by BaSO4 compared to CeO2 the gene pattern showed great overlap. Gene expression was further analyzed in liver and kidney tissue showing inflammatory responses in both organs and marked downregulation of oxidative stress related genes in the kidney. Increases in the amount of Ce were measured in liver but not in kidney tissue. Investigation of selected genes on protein level revealed increased Ccl2 in bronchoalveolar lavage of exposed animals and increased Lpo and Mmp12 in the alveolar epithelia. CONCLUSION: AEII cells contribute to CeO2 nanoparticle caused inflammatory and oxidative stress reactions in the respiratory tract by the release of related mediators. Effects of BaSO4 exposure are low. However, overlap between both substances were detected and support identification of potential early biomarkers for nanoparticle effects on the respiratory system. Signs for long-term effects need to be further evaluated by comparison to a respective exposure setting.

Nanomaterials: certain aspects of application, risk assessment and risk communication.

Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public.

Effects from a 90-day inhalation toxicity study with cerium oxide and barium sulfate nanoparticles in rats.

BACKGROUND: Nanomaterials like cerium oxide and barium sulfate are frequently processed in industrial and consumer products and exposure of humans and other organisms is likely. Generally less information is given on health effects and toxicity, especially regarding long-term exposure to low nanoparticle doses. Since inhalation is still the major route of uptake the present study focused on pulmonary effects of CeO2NM-212 (0.1, 0.3, 1.0, 3.0 mg/m3) and BaSO4NM-220 nanoparticles (50.0 mg/m3) in a 90-day exposure setup. To define particle-related effects and potential mechanisms of action, observations in histopathology, bronchoalveolar lavage and immunohistochemistry were linked to pulmonary deposition and clearance rates. This further allows evaluation of potential overload related effects. RESULTS: Lung burden values increased with increasing nanoparticle dose levels and ongoing exposure. At higher doses, cerium clearance was impaired, suggesting lung overload. Barium elimination was extremely rapid and without any signs of overload. Bronchoalveolar lavage fluid analysis and histopathology revealed lung tissue inflammation with increasing severity and post-exposure persistency for CeO2. Also, marker levels for genotoxicity and cell proliferation were significantly increased. BaSO4 showed less inflammation or persistency of effects and particularly affected the nasal cavity. CONCLUSION: CeO2 nanoparticles penetrate the alveolar space and affect the respiratory tract after inhalation mainly in terms of inflammation. Effects at low dose levels and post-exposure persistency suggest potential long-term effects and a notable relevance for human health. The generated data might be useful to improve nanoparticle risk assessment and threshold value generation. Mechanistic investigations at conditions of non-overload and absent inflammation should be further investigated in future studies.

Biological effects of carbon black nanoparticles are changed by surface coating with polycyclic aromatic hydrocarbons.

BACKGROUND: Carbon black nanoparticles (CBNP) are mainly composed of carbon, with a small amount of other elements (including hydrogen and oxygen). The toxicity of CBNP has been attributed to their large surface area, and through adsorbing intrinsically toxic substances, such as polycyclic aromatic hydrocarbons (PAH). It is not clear whether a PAH surface coating changes the toxicological properties of CBNP by influencing their physicochemical properties, through the specific toxicity of the surface-bound PAH, or by a combination of both. METHODS: Printex®90 (P90) was used as CBNP; the comparators were P90 coated with either benzo[a]pyrene (BaP) or 9-nitroanthracene (9NA), and soot from acetylene combustion that bears various PAHs on the surface (AS-PAH). Oxidative stress and IL-8/KC mRNA expression were determined in A549 and bronchial epithelial cells (16HBE14o-, Calu-3), mouse intrapulmonary airways and tracheal epithelial cells. Overall toxicity was tested in a rat inhalation study according to Organization for Economic Co-operation and Development (OECD) criteria. Effects on cytochrome monooxygenase (Cyp) mRNA expression, cell viability and mucociliary clearance were determined in acute exposure models using explanted murine trachea. RESULTS: All particles had similar primary particle size, shape, hydrodynamic diameter and ζ-potential. All PAH-containing particles had a comparable specific surface area that was approximately one third that of P90. AS-PAH contained a mixture of PAH with expected higher toxicity than BaP or 9NA. PAH-coating reduced some effects of P90 such as IL-8 mRNA expression and oxidative stress in A549 cells, granulocyte influx in the in vivo OECD experiment, and agglomeration of P90 and mucus release in the murine trachea ex vivo. Furthermore, P90-BaP decreased particle transport speed compared to P90 at 10 µg/ml. In contrast, PAH-coating induced IL-8 mRNA expression in bronchial epithelial cell lines, and Cyp mRNA expression and apoptosis in tracheal epithelial cells. In line with the higher toxicity compared to P90-BaP and P90-9NA, AS-PAH had the strongest biological effects both ex vivo and in vivo. CONCLUSIONS: Our results demonstrate that the biological effect of CBNP is determined by a combination of specific surface area and surface-bound PAH, and varies in different target cells.

Translocation and biokinetic behavior of nanoscaled europium oxide particles within 5 days following an acute inhalation in rats.

Nanoscaled europium oxide (Eu2O3) particles were inhaled by rats after acute exposure and the potential translocation of particles followed by chemical analysis and transmission electron microscopy (TEM) was investigated. An aqueous dispersion (phosphate buffer/bovine serum albumin) of a commercially available Eu2O3 particle fraction consisting partially of nanoscaled particles was aerosolized with pressurized air. After rapid evaporation, rats inhaled the dry aerosol for 6 h in a single exposure resulting in an alveolar calculated dose of approximately 39.5 µg Eu2O3. Using chemical analysis, 36.8 µg Eu2O3 was detected 1 h after lung inhalation. The amount declined slightly to 34.5 µg after 1 day and 35.0 µg after 5 days. The liver showed an increase of Eu2O3 from 32.3 ng 1 h up to 294 ng 5 days after inhalation. Additionally, lung-associated lymph nodes, thymus, kidneys, heart and testis exhibited an increase of europium over the period investigated. In the blood, the highest amount of europium was found 1 h after treatment whereas feces, urine and mesenteric lymph nodes revealed the highest amount 1 day after treatment. Using TEM analysis, particles could be detected only in lungs, and in the liver, no particles were detectable. In conclusion, the translocation of Eu2O3 within 5 days following inhalation could be determined very precisely by chemical analysis. A translocation of Eu2O3 particulate matter to liver was not detectable by TEM analysis; thus, the overproportional level of 0.8% of the lung load observed in the liver after 5 days suggests a filtering effect of dissolved europium with accumulation.

The carcinogenic effect of various multi-walled carbon nanotubes (MWCNTs) after intraperitoneal injection in rats.

BACKGROUND: Biological effects of tailor-made multi-walled carbon nanotubes (MWCNTs) without functionalization were investigated in vivo in a two-year carcinogenicity study. In the past, intraperitoneal carcinogenicity studies in rats using biopersistent granular dusts had always been negative, whereas a number of such studies with different asbestos fibers had shown tumor induction. The aim of this study was to identify possible carcinogenic effects of MWCNTs. We compared induced tumors with asbestos-induced mesotheliomas and evaluated their relevance for humans by immunohistochemical methods. METHODS: A total of 500 male Wistar rats (50 per group) were treated once by intraperitoneal injection with 109 or 5 × 109 WHO carbon nanotubes of one of four different MWCNTs suspended in artificial lung medium, which was also used as negative control. Amosite asbestos (108 WHO fibers) served as positive control. Morbid rats were sacrificed and necropsy comprising all organs was performed. Histopathological classification of tumors and, additionally, immunohistochemistry were conducted for podoplanin, pan-cytokeratin, and vimentin to compare induced tumors with malignant mesotheliomas occurring in humans. RESULTS: Treatments induced tumors in all dose groups, but incidences and times to tumor differed between groups. Most tumors were histologically and immunohistochemically classified as malignant mesotheliomas, revealing a predominantly superficial spread on the serosal surface of the abdominal cavity. Furthermore, most tumors showed invasion of peritoneal organs, especially the diaphragm. All tested MWCNT types caused mesotheliomas. We observed highest frequencies and earliest appearances after treatment with the rather straight MWCNT types A and B. In the MWCNT C groups, first appearances of morbid mesothelioma-bearing rats were only slightly later. Later during the two-year study, we found mesotheliomas also in rats treated with MWCNT D - the most curved type of nanotubes. Malignant mesotheliomas induced by intraperitoneal injection of different MWCNTs and of asbestos were histopathologically and immunohistochemically similar, also compared with mesotheliomas in man, suggesting similar pathogenesis. CONCLUSION: We showed a carcinogenic effect for all tested MWCNTs. Besides aspect ratio, curvature seems to be an important parameter influencing the carcinogenicity of MWCNTs.

Particle aerosol generation and potential altering in airflow used for acute/repeated inhalation toxicity testing.

Reproducible aerosol generation in combination with stable aerosol properties are essential prerequisites for compliant performance of acute or repeated inhalation toxicity tests of particulate materials according to OECD TG 403, 436, 412, or 413. A frequent problem of powder aerosol generation is the formation of coarse agglomerates with low shear resistance, which are beyond the tolerable size range but not detected by the prescribed aerodynamic measurement techniques by cascade impactor as the measurement conditions cause a disintegration into smaller fragments. But such agglomerates are observed during the transport to the inhalation chambers. These effects particularly apply to high mass concentrations and low-density powders, i.e., pyrogenic oxides. This study describes the transport influence in the airflow on the change of powder aerosols and on their respirability. A simplified short tube set-up was developed for the aerosol transport pre-tests, which allows the determination of the optimal aerosol formation conditions for the inhalation tests. The particles were measured with low shear using laser diffraction measurement or optical particle counters. The calculation of the aerodynamic particle sizes prescribed in the guidelines requires knowledge of the effective particle density of the porous aerosol particles. A practicable method for determining these is presented and described. In the outlook, first low concentration measurements show that clear agglomeration effects can also occur at particle concentrations around 20 mg/m³.

Enhanced study design for acute inhalation studies with hydrophobic surface-treated particles to determine toxicological effects including suffocation.

High concentrations of low-density particles may cause effects in acute inhalation toxicity studies which can be easily underestimated or misinterpreted following strictly the OECD TG 436, i.e., limited parameters as mortality and gross lesions will be evaluated only. Seven particle types (synthetic amorphous silica (SAS) HMDZ-SAS, silica gel, pyrogenic SAS, and precipitated SAS, calcium carbonate, aluminum oxide pyrogenic alumina, organic red pigment) were chosen at the highest technically feasible concentration of approximately 500 mg/m3 for acute inhalation studies with an expanded endpoint setup. Therefore additional parameters and a thorough histopathological evaluation of an extensive set of organs, including the respiratory tract emphasizing the nasal cavities were added. Six Crl:WI rats per study were exposed for four hours from which three animals were sacrificed after 24 hours and three animals after 14 days. HMDZ-SAS caused early death in all animals due to blockage of the nasal passages caused by its hydrophobicity. For all other Si-containing compounds, histology revealed minor inflammatory and reactive lesions in lungs after 24 hours that were still present after 14 days, except in silica gel-treated animals. After 14 days, for pyrogenic SAS, precipitated SAS, and pyrogenic alumina, granulomas formed in the BALT and lung-associated lymph nodes. In contrast, the calcium carbonate induced almost no findings, and the red pigment (also tested for the additional dose of 1000 mg/m3) stuck partially to the nasal mucosa without causing pathological damage and partly entered the lungs without showing any adverse effects. The results of the present study highlight the advantage of improving the rather simple study design of acute inhalation studies by implementing an extended study design.

Relative oral bioavailability of glycidol from glycidyl fatty acid esters in rats.

In order to quantify the relative bioavailability of glycidol from glycidyl fatty acid esters in vivo, glycidyl palmitoyl ester and glycidol were orally applied to rats in equimolar doses. The time courses of the amounts of glycidol binding to hemoglobin as well as the excretion of 2,3-dihydroxypropyl mercapturic acids were determined. The results indicate that glycidol is released from the glycidyl ester by hydrolysis and rapidly distributed in the organism. In relation to glycidol, there was only a small timely delay in the binding to hemoglobin for the glycidol moiety released from the ester which may be certainly attributed to enzymatic hydrolysis. In both cases, however, an analogous plateau was observed representing similar amounts of hemoglobin binding. With regard to the urinary excretion of mercapturic acids, also similar amounts of dihydroxypropyl mercapturic acids could be detected. In an ADME test using a virtual double tag (³H, ¹4C) of glycidyl palmitoyl ester, a diverging isotope distribution was detected. The kinetics of the ¹4C-activity reflected the kinetics of free glycidol released after hydrolysis of the palmitoyl ester. In view of this experimental data obtained in rats, it is at present justified for the purpose of risk assessment to assume complete hydrolysis of the glycidyl ester in the gastrointestinal tract. Therefore, assessment of human exposure to glycidyl fatty acid ester should be regarded as an exposure to the same molar quantity of glycidol.

Relative oral bioavailability of 3-MCPD from 3-MCPD fatty acid esters in rats.

In order to quantify the relative oral bioavailability of 3-chloropropane-1,2-diol (3-MCPD) from 3-MCPD fatty acid diesters in vivo, 1,2-dipalmitoyl-3-chloropropane-1,2-diol (3-MCPD diester) and 3-MCPD were orally applied to rats in equimolar doses. In both cases, the time courses of 3-MCPD concentrations were measured in blood, various organs, tissues and intestinal luminal contents. The results show that 3-MCPD is released by enzymatic hydrolysis from the 3-MCPD diester in the gastrointestinal tract and distributed to blood, organs and tissues. Based on the measurements in blood, the areas under the curve (AUC) for 3-MCPD were calculated. By comparing both AUC, the relative amount of 3-MCPD bioavailable from the 3-MCPD diester was calculated to be 86 % on average of the amount bioavailable following administration of 3-MCPD. In view of limited experimental data, it is justified for the purpose of risk assessment to assume complete hydrolysis of the diesters in the gastro-intestinal tract. Therefore, assessment of the extent of exposure to 3-MCPD released from its fatty acid esters should be performed in the same way as exposure to the same molar quantity of 3-MCPD.

Pulmonary toxicity of printer toner following inhalation and intratracheal instillation.

The pulmonary effects of a finished toner were evaluated in intratracheal instillation and inhalation studies, using toners with external additives (titanium dioxide nanoparticles and amorphous silica nanoparticles). Rats received an intratracheal dose of 1 mg or 2 mg of toner and were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months. The toner induced pulmonary inflammation, as evidenced by a transient neutrophil response in the low-dose groups and persistent neutrophil infiltration in the high-dose groups. There were increased concentrations of heme oxygenase-1 (HO-1) as a marker of oxidative stress in the bronchoalveolar lavage fluid (BALF) and the lung. In a 90-day inhalation study, rats were exposed to well-dispersed toner (mean of MMAD: 3.76 µm). The three mass concentrations of toner were 1, 4 and 16 mg/m(3) for 13 weeks, and the rats were sacrificed at 6 days and 91 days after the end of the exposure period. The low and medium concentrations did not induce neutrophil infiltration in the lung of statistical significance, but the high concentration did, and, in addition, upon histopathological examination not only showed findings of inflammation but also of fibrosis in the lung. Taken together, the results of our studies suggest that toners with external additives lead to pulmonary inflammation and fibrosis at lung burdens suggest beyond the overload. The changes observed in the pulmonary responses in this inhalation study indicate that the high concentration (16 mg/m(3)) is an LOAEL and that the medium concentration (4 mg/m(3)) is an NOAEL.

Regenerative and progressing lesions in lungs and lung-associated lymph nodes from fourteen 90-day inhalation studies with chemically different particulate materials.

Rat lungs and lung-associated lymph nodes from 14 inhalation studies with chemically different particulate materials were histopathologically re-evaluated, and the bronchoalveolar lavage fluid (BALF) data and lung burden analyses were compared. All investigated substances caused similar lesions. For most substances, 1 mg/m3 of respirable particulate matter was established as the borderline for adverse morphological changes after the 90-day exposure period, confirmed by the increase in polymorphonuclear neutrophils in BALF. Possible reversibility was demonstrated when recovery groups are included in the study especially allowing the differentiation between regeneration or progressing of inflammatory changes during the recovery period. It was concluded, that the major driver of toxicity is not an intrinsic chemical property of the particle but a particle effect. Concerning classification for specific target organ toxicant (STOT) repeated exposure (RE), this paper highlights that merely comparing the lowest concentration, at which adverse effects were observed, with the Classification Labelling and Packaging (CLP) regulation (EC) no. 1272/2008 guidance values is inappropriate and might lead to a STOT classification under CLP for a large part of the substances discussed in this paper, on the basis of typically mild to moderate findings in rat lung and lung-associated lymph nodes on day 1 after exposure. An in-depth evaluation of the pathologic findings is required and an expert judgement has to be included in the decision on classification and labeling, evaluating the type and severity of effects and comparing these with the classification criteria.

Issues in the inhalation toxicity testing and hazard assessment for low density particulate materials such as synthetic amorphous silica (SAS).

Inhalation toxicity testing of particulate materials is mandated for classification. According to CLP, particulate materials should be tested as marketed and many particulate materials are marketed as non-respirable particles. However, OECD TG 413 requires exposure to particle sizes that are respirable and reach the alveoli. The requirement for exposure of rats to respirable particles is thus in contrast to CLP and requires the application of high shear forces. The exposure to artificially small particles causes a number of issues that hamper the interpretation of the results of the testing. These issues are aerosol altering in the exposure system, assessment of the adversity of the inflammatory lung responses, inclusion of recovery groups, and extrapolation of the results to humans exposed under occupational condition. In addition, effects of many particulate materials after testing according to OECD 413 are not intrinsic properties, but a general reaction of the lung to the deposited material, show very similar NOAECs for chemical diverse materials, and often are completely reversible.

Biological interactions and toxicity of nanomaterials in the respiratory tract and various approaches of aerosol generation for toxicity testing.

After deposition in the respiratory tract, nanoparticles exhibit acute, neutrophil-driven inflammatory and oxidative reactions, fibrotic responses and in chronic studies under overload conditions carcinogenic effects, more severely than the microscaled materials of the same chemistry. Besides these effects also known to be induced by microsized particles, nanoparticles principally can translocate from the site of exposure to circulation and become systemically available. This may either increase the toxic outcome (e.g. cardio-vascular effects and potential responses in remote organs) or facilitate an elimination of nanomaterials. For example, in combination with partial dissolution, a strong lung response after a short-term inhalative exposure may be followed by a rapid recovery effect. Mechanistically, in vitro and in vivo tests demonstrated that nanoparticles induce inflammation and oxidative stress after interaction with macrophages and lung epithelial cells; consequently, a cytotoxic and genotoxic potential may exist. The deposition, retention and clearance behaviour of inhaled nanomaterials and the toxic effects observed are decisively dependent on the particle agglomeration status of the aerosol. Two principally different experimental approaches are used for inhalative exposure to nanoparticles: either (1) a basic research-oriented approach using very small aerosol mass concentrations or particle formulations that result in at least partially nanoscaled aerosols; in this way, the potential hazard and the translocation potential for individual nanoparticles can be followed effectively; or (2) exposure scenarios mimicking the occupational situation (risk-oriented) with mostly agglomerated nanoparticles; consequently, the probable risk deriving from incidental/accidental exposure can be assessed more adequately.