Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

Molecular signatures of angiogenesis inhibitors: a single-embryo untargeted metabolomics approach in zebrafish.

Angiogenesis is a key process in embryonic development, a disruption of this process can lead to severe developmental defects, such as limb malformations. The identification of molecular perturbations representative of antiangiogenesis in zebrafish embryo (ZFE) may guide the assessment of developmental toxicity from an endpoint- to a mechanism-based approach, thereby improving the extrapolation of findings to humans. Thus, the aim of the study was to discover molecular changes characteristic of antiangiogenesis and developmental toxicity. We exposed ZFEs to two antiangiogenic drugs (SU4312, sorafenib) and two developmental toxicants (methotrexate, rotenone) with putative antiangiogenic action. Molecular changes were measured by performing untargeted metabolomics in single embryos. The metabolome response was accompanied by the occurrence of morphological alterations. Two distinct metabolic effect patterns were observed. The first pattern comprised common effects of two specific angiogenesis inhibitors and the known teratogen methotrexate, strongly suggesting a shared mode of action of antiangiogenesis and developmental toxicity. The second pattern involved joint effects of methotrexate and rotenone, likely related to disturbances in energy metabolism. The metabolites of the first pattern, such as phosphatidylserines, pterines, retinol, or coenzyme Q precursors, represented potential links to antiangiogenesis and related developmental toxicity. The metabolic effect pattern can contribute to biomarker identification for a mechanism-based toxicological testing.

Application of high throughput in vitro metabolomics for hepatotoxicity mode of action characterization and mechanistic-anchored point of departure derivation: a case study with nitrofurantoin.

Omics techniques have been increasingly recognized as promising tools for Next Generation Risk Assessment. Targeted metabolomics offer the advantage of providing readily interpretable mechanistic information about perturbed biological pathways. In this study, a high-throughput LC-MS/MS-based broad targeted metabolomics system was applied to study nitrofurantoin metabolic dynamics over time and concentration and to provide a mechanistic-anchored approach for point of departure (PoD) derivation. Upon nitrofurantoin exposure at five concentrations (7.5 µM, 15 µM, 20 µM, 30 µM and 120 µM) and four time points (3, 6, 24 and 48 h), the intracellular metabolome of HepG2 cells was evaluated. In total, 256 uniquely identified metabolites were measured, annotated, and allocated in 13 different metabolite classes. Principal component analysis (PCA) and univariate statistical analysis showed clear metabolome-based time and concentration effects. Mechanistic information evidenced the differential activation of cellular pathways indicative of early adaptive and hepatotoxic response. At low concentrations, effects were seen mainly in the energy and lipid metabolism, in the mid concentration range, the activation of the antioxidant cellular response was evidenced by increased levels of glutathione (GSH) and metabolites from the de novo GSH synthesis pathway. At the highest concentrations, the depletion of GSH, together with alternations reflective of mitochondrial impairments, were indicative of a hepatotoxic response. Finally, a metabolomics-based PoD was derived by multivariate PCA using the whole set of measured metabolites. This approach allows using the entire dataset and derive PoD that can be mechanistically anchored to established key events. Our results show the suitability of high throughput targeted metabolomics to investigate mechanisms of hepatoxicity and derive point of departures that can be linked to existing adverse outcome pathways and contribute to the development of new ones.

Genotoxicity testing of nanomaterials.

Nanomaterials have outstanding and unprecedented advantageous material properties but may also cause adverse effects in humans upon exposure. Testing nanomaterials for genotoxic properties is challenging because traditional testing methods were designed for small, soluble molecules and may not be easily applicable without modifications. This review critically examines available genotoxicity tests for use with nanomaterials, including DNA damage tests such as the comet assay, gene mutation tests such as the mouse lymphoma and hprt assay, and chromosome mutation tests such as the micronucleus test and the chromosome aberration test. It presents arguments for the relative usefulness of various tests, such as preferring the micronucleus test over the chromosome aberration test for scoring chromosome mutations and preferring mammalian cell gene mutation tests because the Ames test has limited utility. Finally, it points out the open questions and further needs in adapting genotoxicity tests for nanomaterials, such as validation, reference nanomaterials, and the selection of top test concentrations, as well as the relevance and applicability of test systems and the need to define testing strategies. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Enigmatic mechanism of the N-vinylpyrrolidone hepatocarcinogenicity in the rat.

N-vinyl pyrrolidone (NVP) is produced up to several thousand tons per year as starting material for the production of polymers to be used in pharmaceutics, cosmetics and food technology. Upon inhalation NVP was carcinogenic in the rat, liver tumor formation is starting already at the rather low concentration of 5 ppm. Hence, differentiation whether NVP is a genotoxic carcinogen (presumed to generally have no dose threshold for the carcinogenic activity) or a non-genotoxic carcinogen (with a potentially definable threshold) is highly important. In the present study, therefore, the existing genotoxicity investigations on NVP (all showing consistently negative results) were extended and complemented with investigations on possible alternative mechanisms, which also all proved negative. All tests were performed in the same species (rat) using the same route of exposure (inhalation) and the same doses of NVP (5, 10 and 20 ppm) as had been used in the positive carcinogenicity test. Specifically, the tests included an ex vivo Comet assay (so far not available) and an ex vivo micronucleus test (in contrast to the already available micronucleus test in mice here in the same species and by the same route of application as in the bioassay which had shown the carcinogenicity), tests on oxidative stress (non-protein-bound sulfhydryls and glutathione recycling test), mechanisms mediated by hepatic receptors, the activation of which had been shown earlier to lead to carcinogenicity in some instances (Ah receptor, CAR, PXR, PPARα). No indications were obtained for any of the investigated mechanisms to be responsible for or to contribute to the observed carcinogenicity of NVP. The most important of these exclusions is genotoxicity. Thus, NVP can rightfully be regarded and treated as a non-genotoxic carcinogen and threshold approaches to the assessment of this chemical are supported. However, the mechanism underlying the carcinogenicity of NVP in rats remains unclear.

N-vinyl compounds: studies on metabolism, genotoxicity, carcinogenicity.

Several N-vinyl compounds are produced in high volumes and are widely employed in the production of copolymers and polymers used in chemical, pharmaceutical, cosmetic and food industry. Hence, information on their genotoxicity and carcinogenicity is requisite. This review presents hitherto available information on the carcinogenicity and genotoxicity of N-vinyl compounds as well as their metabolism potentially generating genotoxic and carcinogenic derivatives. The genotoxicity and carcinogenicity of the investigated N-vinyl compounds vary widely from no observed carcinogenicity tested in lifetime bioassays in two rodent species (up to very high doses) to carcinogenicity in rats at very low doses in the absence of apparent genotoxicity. Despite of the presence of the vinyl group potentially metabolized to an epoxide followed by covalent binding to DNA, genotoxicity was observed for only one of the considered N-vinyl compounds, N-vinyl carbazole. Carcinogenicity was investigated only for two, of which one, N-vinyl pyrrolidone was carcinogenic (but not genotoxic) and ranitidine was neither carcinogenic nor genotoxic. As far as investigated, neither a metabolically formed epoxide nor a therefrom derived diol has been reported for any of the considered N-vinyl compounds. It is concluded that the information collected in this review will further the understanding of the carcinogenic potentials of N-vinyl compounds and may eventually allow approaching their prediction and prevention. A suggestion how to prevent genotoxicity in designing of N-vinyl compounds is presented. However, the available information is scarce and further research especially on the metabolism of N-vinyl compounds is highly desirable.

Aerogels are not regulated as nanomaterials, but can be assessed by tiered testing and grouping strategies for nanomaterials.

Aerogels contribute to an increasing number of novel applications due to many unique properties, such as high porosity and low density. They outperform most other insulation materials, and some are also useful as carriers in food or pharma applications. Aerogels are not nanomaterials by the REACH definition but retain properties of nanoscale structures. Here we applied a testing strategy in three tiers. In Tier 1, we examined a panel of 19 aerogels (functionalized chitosan, alginate, pyrolyzed carbon, silicate, cellulose, polyurethane) for their biosolubility, and oxidative potential. Biosolubility was very limited except for some alginate and silicate aerogels. Oxidative potential, as by the ferric reduction ability of human serum (FRAS), was very low except for one chitosan and pyrolyzed carbon, both of which were <10% of the positive control Mn2O3. Five aerogels were further subjected to the Tier 2 alveolar macrophage assay, which revealed no in vitro cytotoxicity, except for silicate and polyurethane that induced increases in tumor necrosis factor α. Insufficiently similar aerogels were excluded from a candidate group, and a worst case identified. In the Tier 3 in vivo instillation, polyurethane (0.3 to 2.4 mg) elicited dose-dependent but reversible enzyme changes in lung lavage fluid on day 3, but no significant inflammatory effects. Overall, the results show a very low inherent toxicity of aerogels and support a categorization based on similarities in Tier 1 and Tier 2. This exemplifies how nanosafety concepts and methods developed on particles can be applied to specific concerns on advanced materials that contain or release nanostructures.

In Silico Models to Predict the Perturbation of Molecular Initiating Events Related to Thyroid Hormone Homeostasis.

Disturbance of the thyroid hormone homeostasis has been associated with adverse health effects such as goiters and impaired mental development in humans and thyroid tumors in rats. In vitro and in silico methods for predicting the effects of small molecules on thyroid hormone homeostasis are currently being explored as alternatives to animal experiments, but are still in an early stage of development. The aim of this work was the development of a battery of in silico models for a set of targets involved in molecular initiating events of thyroid hormone homeostasis: deiodinases 1, 2, and 3, thyroid peroxidase (TPO), thyroid hormone receptor (TR), sodium/iodide symporter, thyrotropin-releasing hormone receptor, and thyroid-stimulating hormone receptor. The training data sets were compiled from the ToxCast database and related scientific literature. Classical statistical approaches as well as several machine learning methods (including random forest, support vector machine, and neural networks) were explored in combination with three data balancing techniques. The models were trained on molecular descriptors and fingerprints and evaluated on holdout data. Furthermore, multi-task neural networks combining several end points were investigated as a possible way to improve the performance of models for which the experimental data available for model training are limited. Classifiers for TPO and TR performed particularly well, with F1 scores of 0.83 and 0.81 on the holdout data set, respectively. Models for the other studied targets yielded F1 scores of up to 0.77. An in-depth analysis of the reliability of predictions was performed for the most relevant models. All data sets used in this work for model development and validation are available in the Supporting Information.

Predictivity of the kinetic direct peptide reactivity assay (kDPRA) for sensitizer potency assessment and GHS subclassification

Several in vitro OECD test guidelines address key events 1-3 of the adverse outcome pathway for skin sensitization, but none are validated for sensitizer potency assessment. The reaction of sensitizing molecules with skin proteins is the molecular initiating event and appears to be rate-limiting, as chemical reactivity strongly correlates with sensitizer potency. The kinetic direct peptide reactivity assay (kDPRA), a modification of the DPRA (OECD TG 442C), allows derivation of rate constants of the depletion of the cysteine-containing model peptide upon reaction with the test item. Its reproducibility was demonstrated in an inter-laboratory study. Here, we present a database of rate constants, expressed as log kmax, for 180 chemicals to define the prediction threshold to identify strong sensitizers (classified as GHS 1A). A threshold of log kmax -2 offers a balanced accuracy of 85% for predicting GHS 1A sensitizers according to the local lymph node assay. The kDPRA is proposed as a stand-alone assay for identification of GHS 1A sensitizers among chemicals identified as sensitizers by other tests or defined approaches. It may also be used for the prediction of sensitizer potency on a continuous scale, ideally in combination with continuous parameters from other in vitro assays. We show how the rate constant could be combined with read-outs of other in vitro assays in a defined approach. A decision model based on log kmax alone has, however, a high predictivity and can be used as stand-alone model for identification of GHS 1A sensitizers among chemicals predicted as sensitizers.

The kinetic direct peptide reactivity assay (kDPRA): Intra- and inter-laboratory reproducibility in a seven-laboratory ring trial

While the skin sensitization hazard of substances can be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide are evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times. The reaction is stopped by addition of monobromobimane, which forms a fluorescent complex with the free cysteine of the model peptide. The relative remaining non-depleted amount of peptide is determined. Kinetic rate constants are derived from the depletion vs concentration and time matrix and used to distinguish between UN GHS sub-category 1A sensitizers and test substances in sub-category 1B/not classified test substances. In this study, we present a ring trial of the kDPRA with 24 blind-coded test substances in seven laboratories. The intra- and inter-laboratory reproducibility were 96% and 88%, respectively (both for differentiating GHS Cat 1A sensitizers from GHS Cat 1B/not classified). Following an independent peer review, the kDPRA was considered to be acceptable for the identification of GHS Cat 1A skin sensitizers. Besides GHS Cat 1A identification, the kDPRA can be used as part of a defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment. For this aim, next to reproducibility of classification, the quantitative reproducibility and variability of the rate constants were quantified in this study.

A novel 3D intestine barrier model to study the immune response upon exposure to microplastics.

The plausibility of human exposure to microplastics has increased within the last years. Microplastics have been found in different food types including seafood, salt, sugar and beverages. So far, human health effects of microplastics after ingestion are unknown. Herein, we designed a novel, three-dimensional in vitro intestinal model consisting of the human intestinal epithelial cell lines Caco-2 and HT29-MTX-E12 as well as human blood monocyte-derived macrophages and dendritic cells that is suitable to assess the possible effects of ingested microplastics. Relevant microplastic particles (in the order of 50-500 µm), including polymers representing tire wear and polyolefins, which represent major sources of microplastic in the EU, were compared to other polymer classes and an inorganic microparticle, healing earth, which is intended for human consumption. Microplastic particles were exposed at concentrations of 823.5-1380.0 µg/cm2 to the model using a dry powder insufflator system to aerosolize the particles directly on the intestinal model's surface. Cytotoxicity was investigated after 6, 24 and 48 h of exposure via measuring the release of lactate dehydrogenase. Inflammatory end points including the cytokines IL-8, TNFα and IL-1ß as well as changes of the barrier integrity after exposure were additionally monitored. We demonstrated that all of the microplastics and the healing earth particles did not cause any significant cytotoxicity or release of (pro-)inflammatory cytokines and did not change the barrier integrity of the co-culture at any of the time points investigated.

In vitro-to-in vivo extrapolation (IVIVE) by PBTK modeling for animal-free risk assessment approaches of potential endocrine-disrupting compounds.

While in vitro testing is used to identify hazards of chemicals, nominal in vitro assay concentrations may misrepresent potential in vivo effects and do not provide dose-response data which can be used for a risk assessment. We used reverse dosimetry to compare in vitro effect concentrations-to-in vivo doses causing toxic effects related to endocrine disruption. Ten compounds (acetaminophen, bisphenol A, caffeine, 17α-ethinylestradiol, fenarimol, flutamide, genistein, ketoconazole, methyltestosterone, and trenbolone) have been tested in the yeast estrogen screening (YES) or yeast androgen-screening (YAS) assays for estrogen and androgen receptor binding, as well as the H295R assay (OECD test guideline no. 456) for potential interaction with steroidogenesis. With the assumption of comparable concentration-response ratios of these effects in the applied in vitro systems and the in vivo environment, the lowest observed effect concentrations from these assays were extrapolated to oral doses (LOELs) by reverse dosimetry. For extrapolation, an eight-compartment Physiologically Based Toxicokinetic (PBTK) rat model based on in vitro and in silico input data was used. The predicted LOEL was then compared to the LOEL actually observed in corresponding in vivo studies (YES/YAS assay versus uterotrophic or Hershberger assay and steroidogenesis assay versus pubertal assay or generation studies). This evaluation resulted in 6 out of 10 compounds for which the predicted LOELs were in the same order of magnitude as the actual in vivo LOELs. For four compounds, the predicted LOELs differed by more than tenfold from the actual in vivo LOELs. In conclusion, these data demonstrate the applicability of reverse dosimetry using a simple PBTK model to serve in vitro-in silico-based risk assessment, but also identified cases and test substance were the applied methods are insufficient.

In Vitro and In Vivo Short-Term Pulmonary Toxicity of Differently Sized Colloidal Amorphous SiO2.

In vitro prediction of inflammatory lung effects of well-dispersed nanomaterials is challenging. Here, the in vitro effects of four colloidal amorphous SiO2 nanomaterials that differed only by their primary particle size (9, 15, 30, and 55 nm) were analyzed using the rat NR8383 alveolar macrophage (AM) assay. Data were compared to effects of single doses of 15 nm and 55 nm SiO2 intratracheally instilled in rat lungs. In vitro, all four elicited the release of concentration-dependent lactate dehydrogenase, ß-glucuronidase, and tumor necrosis factor alpha, and the two smaller materials also released H2O2. All effects were size-dependent. Since the colloidal SiO2 remained well-dispersed in serum-free in vitro conditions, effective particle concentrations reaching the cells were estimated using different models. Evaluating the effective concentration-based in vitro effects using the Decision-making framework for the grouping and testing of nanomaterials, all four nanomaterials were assigned as "active." This assignment and the size dependency of effects were consistent with the outcomes of intratracheal instillation studies and available short-term rat inhalation data for 15 nm SiO2. The study confirms the applicability of the NR8383 AM assay to assessing colloidal SiO2 but underlines the need to estimate and consider the effective concentration of such well-dispersed test materials.

Genotoxicity testing of different surface-functionalized SiO2, ZrO2 and silver nanomaterials in 3D human bronchial models.

Inhalation is considered a critical uptake route for NMs, demanding for sound toxicity testing using relevant test systems. This study investigates cytotoxicity and genotoxicity in EpiAirway™ 3D human bronchial models using 16 well-characterized NMs, including surface-functionalized 15 nm SiO2 (4 variants), 10 nm ZrO2 (4), and nanosilver (3), ZnO NM-110, TiO2 NM-105, BaSO4 NM-220, and two AlOOH NMs. Cytotoxicity was assessed by LDH and ATP assays and genotoxicity by the alkaline comet assay. For 9 NMs, uptake was investigated using inductively coupled plasma-mass spectrometry (ICP-MS). Most NMs were neither cytotoxic nor genotoxic in vitro. ZnO displayed a dose-dependent genotoxicity between 10 and 25 µg/cm2. Ag.50.citrate was genotoxic at 50 µg/cm2. A marginal but still significant genotoxic response was observed for SiO2.unmodified, SiO2.phosphate and ZrO2.TODS at 50 µg/cm2. For all NMs for which uptake in the 3D models could be assessed, the amount taken up was below 5% of the applied mass doses and was furthermore dose dependent. For in vivo comparison, published in vivo genotoxicity data were used and in addition, at the beginning of this study, two NMs were randomly selected for short-term (5-day) rat inhalation studies with subsequent comet and micronucleus assays in lung and bone marrow cells, respectively, i.e., ZrO2.acrylate and SiO2.amino. Both substances were not genotoxic neither in vivo nor in vitro. EpiAirway™ 3D models appear useful for NM in vitro testing. Using 16 different NMs, this study confirms that genotoxicity is mainly determined by chemical composition of the core material.

No genotoxicity in rat blood cells upon 3- or 6-month inhalation exposure to CeO2 or BaSO4 nanomaterials.

In the course of a 2-year combined chronic toxicity-carcinogenicity study performed according to Organisation for Economic Co-operation and Development (OECD) Test Guideline 453, systemic (blood cell) genotoxicity of two OECD representative nanomaterials, CeO2 NM-212 and BaSO4 upon 3- or 6-month inhalation exposure to rats was assessed. DNA effects were analysed in leukocytes using the alkaline Comet assay, gene mutations and chromosome aberrations were measured in erythrocytes using the flow cytometric Pig-a gene mutation assay and the micronucleus test (applying both microscopic and flow cytometric evaluation), respectively. Since nano-sized CeO2 elicited lung effects at concentrations of 5mg/m3 (burdens of 0.5mg/lung) in the preceding range-finding study, whereas nano-sized BaSO4 did not induce any effect, female rats were exposed to aerosol concentrations of 0.1 up to 3mg/m3 CeO2 or 50mg/m3 BaSO4 nanomaterials (6h/day; 5 days/week; whole-body exposure). The blood of animals treated with clean air served as negative control, whereas blood samples from rats treated orally with three doses of 20mg/kg body weight ethylnitrosourea at 24h intervals were used as positive controls. As expected, ethylnitrosourea elicited significant genotoxicity in the alkaline Comet and Pig-a gene mutation assays and in the micronucleus test. By contrast, 3- and 6-month CeO2 or BaSO4 nanomaterial inhalation exposure did not elicit significant findings in any of the genotoxicity tests. The results demonstrate that subchronic inhalation exposure to different low doses of CeO2 or to a high dose of BaSO4 nanomaterials does not induce genotoxicity on the rat hematopoietic system at the DNA, gene or chromosome levels.

Assessment of the oxidative potential of nanoparticles by the cytochrome c assay: assay improvement and development of a high-throughput method to predict the toxicity of nanoparticles.

Oxidative stress has increasingly been demonstrated as playing a key role in the biological response induced by nanoparticles (NPs). The acellular cytochrome c oxidation assay has been proposed to determine the intrinsic oxidant-generating capacity of NPs. Yet, there is a need to improve this method to allow a rapid screening to classify NPs in terms of toxicity. We adapted the cytochrome c assay to take into account NP interference, to improve its sensitivity and to develop a high-throughput method. The intrinsic oxidative ability of a panel of NPs (carbon black, Mn2O3, Cu, Ag, BaSO4, CeO2, TiO2 and ZnO) was measured with this enhanced test and compared to other acellular redox assays. To assess whether their oxidative potential correlates with cellular responses, we studied the effect of insoluble NPs on the human bronchial epithelial cell line NCI-H292 by measuring the cytotoxicity (WST-1 assay), pro-inflammatory response (IL-8 cytokine production and expression) and antioxidant defense induction (SOD2 and HO-1 expression). The adapted cytochrome c assay had a greatly increased sensitivity allowing the ranking of NPs in terms of their oxidative potential by using the developed high-throughput technique. Besides, a high oxidative potential revealed to be predictive for toxic effects as Mn2O3 NPs induced a strong oxidation of cytochrome c and a dose-dependent cytotoxicity, pro-inflammatory response and antioxidant enzyme expression. BaSO4, which presented no intrinsic oxidative potential, had no cellular effects. Nevertheless, CeO2 and TiO2 NPs demonstrated no acellular oxidant-generating capacity but induced moderate cellular responses. In conclusion, the novel cytochrome c oxidation assay could be used for high-throughput screening of the intrinsic oxidative potential of NPs. However, acellular redox assays may not be sufficient to discriminate among low-toxicity NPs, and additional tests are thus needed.

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.

Assessment of Pre- and Pro-haptens Using Nonanimal Test Methods for Skin Sensitization.

Because of ethical and regulatory reasons, several nonanimal test methods to assess the skin sensitization potential of chemicals have been developed and validated. In contrast to in vivo methods, they lack or provide limited metabolic capacity. For this reason, identification of pro-haptens but also pre-haptens, which require molecular transformations to gain peptide reactivity, is a challenge for these methods. In this study, 27 pre- and pro-haptens were tested using nonanimal test methods. Of these, 18 provided true positive results in the direct peptide reactivity assay (DPRA; sensitivity of 67%), although lacking structural alerts for direct peptide reactivity. The reaction mechanisms leading to peptide depletion in the DPRA were therefore elucidated using mass spectrometry. Hapten-peptide adducts were identified for 13 of the 18 chemicals indicating that these pre-haptens were activated and that peptide binding occurred. Positive results for five of the 18 chemicals can be explained by dipeptide formations or the oxidation of the sulfhydryl group of the peptide. Nine of the 27 chemicals were tested negative in the DPRA. Of these, four yielded true positive results in the keratinocyte and dendritic cell based assays. Likewise, 16 of the 18 chemicals tested positive in the DPRA were also positive in either one or both of the cell-based assays. A combination of DPRA, KeratinoSens, and h-CLAT used in a 2 out of 3 weight of evidence (WoE) approach identified 22 of the 27 pre- and pro-haptens correctly (sensitivity of 81%), exhibiting a similar sensitivity as for directly acting haptens. This analysis shows that the combination of in chemico and in vitro test methods is suitable to identify pre-haptens and the majority of pro-haptens.

An in vitro alveolar macrophage assay for predicting the short-term inhalation toxicity of nanomaterials.

BACKGROUND: Most in vitro studies investigating nanomaterial pulmonary toxicity poorly correlate to in vivo inhalation studies. Alveolar macrophages (AMs) play an outstanding role during inhalation exposure since they effectively clear the alveoli from particles. This study addresses the applicability of an in vitro alveolar macrophage assay to distinguish biologically active from passive nanomaterials. METHODS: Rat NR8383 alveolar macrophages were exposed to 18 inorganic nanomaterials, covering AlOOH, BaSO4, CeO2, Fe2O3, TiO2, ZrO2, and ZnO NMs, amorphous SiO2 and graphite nanoplatelets, and two nanosized organic pigments. ZrO2 and amorphous SiO2 were tested without and with surface functionalization. Non-nanosized quartz DQ12 and corundum were used as positive and negative controls, respectively. The test materials were incubated with the cells in protein-free culture medium. Lactate dehydrogenase, glucuronidase, and tumour necrosis factor alpha were assessed after 16 h. In parallel, H2O2 was assessed after 1.5 h. Using the no-observed-adverse-effect concentrations (NOAECs) from available rat short-term inhalation studies (STIS), the test materials were categorized as active (NOAEC < 10 mg/m(3)) or passive. RESULTS: In vitro data reflected the STIS categorization if a particle surface area-based threshold of <6000 mm(2)/mL was used to determine the biological relevance of the lowest observed significant in vitro effects. Significant effects that were recorded above this threshold were assessed as resulting from test material-unspecific cellular 'overload'. Test materials were assessed as active if ≥2 of the 4 in vitro parameters undercut this threshold. They were assessed as passive if 0 or 1 parameter was altered. An overall assay accuracy of 95 % was achieved. CONCLUSIONS: The in vitro NR8383 alveolar macrophage assay allows distinguishing active from passive nanomaterials. Thereby, it allows determining whether in vivo short-term inhalation testing is necessary for hazard assessment. Results may also be used to group nanomaterials by biological activity. Further work should aim at validating the assay.

In vitro and in vivo genotoxicity investigations of differently sized amorphous SiO2 nanomaterials.

In vitro and in vivo genotoxic effects of differently sized amorphous SiO2 nanomaterials were investigated. In the alkaline Comet assay (with V79 cells), non-cytotoxic concentrations of 300 and 100-300µg/mL 15nm-SiO2 and 55nm-SiO2, respectively, relevant (at least 2-fold relative to the negative control) DNA damage. In the Alkaline unwinding assay (with V79 cells), only 15nm-SiO2 significantly increased DNA strand breaks (and only at 100µg/mL), whereas neither nanomaterial (up to 300µg/mL) increased Fpg (Formamidopyrimidine DNA glycosylase)-sensitive sites reflecting oxidative DNA base modifications. In the Comet assay using rat precision-cut lung slices, 15nm-SiO2 and 55nm-SiO2 induced significant DNA damage at ≥100µg/mL. In the Alkaline unwinding assay (with A549 cells), 30nm-SiO2 and 55nm-SiO2 (with larger primary particle size (PPS)) induced significant increases in DNA strand breaks at ≥50µg/mL, whereas 9nm-SiO2 and 15nm-SiO2 (with smaller PPS) induced significant DNA damage at higher concentrations. These two amorphous SiO2 also increased Fpg-sensitive sites (significant at 100µg/mL). In vivo, within 3 days after single intratracheal instillation of 360µg, neither 15nm-SiO2 nor 55nm-SiO2 caused genotoxic effects in the rat lung or in the bone marrow. However, pulmonary inflammation was observed in both test groups with findings being more pronounced upon treatment with 15nm-SiO2 than with 55nm-SiO2. Taken together, the study shows that colloidal amorphous SiO2 with different particle sizes may induce genotoxic effects in lung cells in vitro at comparatively high concentrations. However, the same materials elicited no genotoxic effects in the rat lung even though pronounced pulmonary inflammation evolved. This may be explained by the fact that a considerably lower dose reached the target cells in vivo than in vitro. Additionally, the different time points of investigation may provide more time for DNA damage repair after instillation.