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.

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.

Thyroid Hormone Metabolites Quantified in Pup and Adult Rat Cerebellum, Cortex and Whole-Brain Samples Using an Automated Online SPE-LC-MS/MS Method.

Changes in thyroid hormone (TH) levels in rat brain at early developmental stages are correlated with adverse effects on offspring development. To characterize the ability of substances to interfere with the TH concentrations in, e.g., rat brain, it is essential to know the mean TH concentrations in this tissue under control conditions. In this publication, an online solid-phase extraction (SPE) liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was validated and used to measure TH metabolites (T4, T3, rT3, T2 and T1) in the brains of untreated rats. Data on TH concentrations in the whole brain and separate data from the cerebellum and the cortex are shown. The corresponding samples were gathered from young rats at postnatal days (PND) 4 and 21/22 and from adult rats. The results show inter alia the high accuracy and precision of the method, and LOQs of 0.02 ng/mL were determined for T1, T2 and rT3 and of 0.15 ng/mL for T3 and T4. Technical variability is low, as shown by the relative standard deviations of 7.5-20%. For our rat model, we found that T4, T3 and T2 concentrations rise from PND4 to PND21, whereas the rT3 concentration decreases; as well as there is no statistical difference between TH concentrations in the male and female rat brain. This method is suitable to analyze TH metabolites in the brain and build up a database of historical TH concentrations in control rats. Together, this yields a robust diagnostic tool to detect potentially adverse disturbances of TH homeostasis in the most vulnerable anatomic structure.

A Novel and Fast Online-SPE-LC-MS/MS Method to Quantify Thyroid Hormone Metabolites in Rat Plasma.

Since the focus in regulatory toxicology has drifted toward the identification of endocrine disruptors, the improvement in determination of alterations in the thyroid hormone system has become more important. THs are involved in several molecular processes important for a proper pre- and postnatal development so that disturbances can inter alia lead to incorrect brain maturation and/or disturbed metabolic processes (thermogenesis or lipolysis). In this publication, a new automated online solid-phase extraction (SPE)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS, xLC-MS/MS) is introduced which simultaneously analyzes total T4, T3, rT3, T2, and T1. Method validation parameters are presented, and the method was positively verified by analyzing control and PTU-treated rat plasma samples (time points day 7, 14, and 28) for their total TH content. The obtained results were compared to published results by using a radioimmunoassay method. The automated SPE system ensures a consistent unified sample preparation, and this method overall showed sufficient specificity and accuracy to detect the given analytes in rat plasma. For the preparation of 50 µL of rat plasma, the following LOQs were established: 0.020 nM for T1, 0.029 nM for T2, 0.023 nM for rT3 and T3, and 3.22 nM for T4. This method is suitable to assess the identification of mechanisms leading to adverse effects, such as disturbed TH metabolism and regulation.

Analysis of health concerns not addressed by REACH for low tonnage chemicals and opportunities for new approach methodology.

In Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) the criterion for deciding the studies that must be performed is the annual tonnage of the chemical manufactured or imported into the EU. The annual tonnage may be considered as a surrogate for levels of human exposure but this does not take into account the physico-chemical properties and use patterns that determine exposure. Chemicals are classified using data from REACH under areas of health concern covering effects on the skin and eye; sensitisation; acute, repeated and prolonged systemic exposure; effects on genetic material; carcinogenicity; and reproduction and development. We analysed the mandated study lists under REACH for each annual tonnage band in terms of the information they provide on each of the areas of health concern. Using the European Chemicals Agency (ECHA) REACH Registration data base of over 20,000 registered substances, we found that only 19% of registered substances have datasets on all areas of health concern. Information limited to acute exposure, sensitisation and genotoxicity was found for 62%. The analysis highlighted the shortfall of information mandated for substances in the lower tonnage bands. Deploying New Approach Methodologies (NAMs) at this lower tonnage band to assess health concerns which are currently not covered by REACH, such as repeat and extended exposure and carcinogenicity, would provide additional information and would be a way for registrants and regulators to gain experience in the use of NAMs. There are currently projects in Europe aiming to develop NAM-based assessment frameworks and they could find their first use in assessing low tonnage chemicals once confidence has been gained by their evaluation with data rich chemicals.

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.

A metabolomics approach to reveal the mechanism of developmental toxicity in zebrafish embryos exposed to 6-propyl-2-thiouracil.

A crucial component of a substance registration and regulation is the evaluation of human prenatal developmental toxicity. Current toxicological tests are based on mammalian models, but these are costly, time consuming and may pose ethical concerns. The zebrafish embryo has evolved as a promising alternative model to study developmental toxicity. However, the implementation of the zebrafish embryotoxicity test is challenged by lacking information on the relevance of observed morphological alterations in fish for human developmental toxicity. Elucidating the mechanism of toxicity could help to overcome this limitation. Through LC-MS/MS and GC-MS metabolomics, we investigated whether changes to the endogenous metabolites can indicate pathways associated with developmental toxicity. To this aim, zebrafish embryos were exposed to different concentrations of 6-propyl-2-thiouracil (PTU), a compound known to induce developmental toxicity. The reproducibility and the concentration-dependence of the metabolome response and its association with morphological alterations were studied. Major morphological findings were reduced eye size, and other craniofacial anomalies; major metabolic changes included increased tyrosine, pipecolic acid and lysophosphatidylcholine levels, decreased methionine levels, and disturbance of the 'Phenylalanine, tyrosine and tryptophan biosynthesis' pathway. This pathway, and the changes in tyrosine and pipecolic acid levels could be linked to the mode of action of PTU, i.e., inhibition of thyroid peroxidase (TPO). The other findings suggested neurodevelopmental impairments. This proof-of-concept study demonstrated that metabolite changes in zebrafish embryos are robust and provide mechanistic information associated with the mode of action of PTU.

A high-throughput metabolomics in vitro platform for the characterization of hepatotoxicity.

Cell-based metabolomics provides multiparametric physiologically relevant readouts that can be highly advantageous for improved, biologically based decision making in early stages of compound development. Here, we present the development of a 96-well plate LC-MS/MS-based targeted metabolomics screening platform for the classification of liver toxicity modes of action (MoAs) in HepG2 cells. Different parameters of the workflow (cell seeding density, passage number, cytotoxicity testing, sample preparation, metabolite extraction, analytical method, and data processing) were optimized and standardized to increase the efficiency of the testing platform. The applicability of the system was tested with seven substances known to be representative of three different liver toxicity MoAs (peroxisome proliferation, liver enzyme induction, and liver enzyme inhibition). Five concentrations per substance, aimed at covering the complete dose-response curve, were analyzed and 221 uniquely identified metabolites were measured, annotated, and allocated in 12 different metabolite classes such as amino acids, carbohydrates, energy metabolism, nucleobases, vitamins and cofactors, and diverse lipid classes. Multivariate and univariate analyses showed a dose response of the metabolic effects, a clear differentiation between liver toxicity MoAs and resulted in the identification of metabolite patterns specific for each MoA. Key metabolites indicative of both general and mechanistic specific hepatotoxicity were identified. The method presented here offers a multiparametric, mechanistic-based, and cost-effective hepatotoxicity screening that provides MoA classification and sheds light into the pathways involved in the toxicological mechanism. This assay can be implemented as a reliable compound screening platform for improved safety assessment in early compound development pipelines.

Connecting Gut Microbial Diversity with Plasma Metabolome and Fecal Bile Acid Changes Induced by the Antibiotics Tobramycin and Colistin Sulfate.

The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics.

Rapid equilibrium dialysis, ultrafiltration or ultracentrifugation? Evaluation of methods to quantify the unbound fraction of substances in plasma.

In pharmacokinetics plasma protein binding (PPB) is a well-established parameter impacting drug disposition. The unbound fraction (fu) is arguably regarded the effective concentration at the target site. Pharmacology and toxicology, increasingly use in vitro models. The translation of in vitro concentrations to in vivo doses can be supported by toxicokinetic modelling, e.g. physiologically based toxicokinetic models (PBTK). PPB of a test substance is an input parameter for PBTK. We compared three methods to quantify fu: rapid equilibrium dialysis (RED), ultrafiltration (UF) and ultracentrifugation (UC) using twelve substances covering a wide range of Log Pow (-0.1 to 6.8) and molecular weights (151 and 531 g/mol): Acetaminophen, Bisphenol A, Caffeine, Colchicine, Fenarimol, Flutamide, Genistein, Ketoconazole, α-Methyltestosterone, Tamoxifen, Trenbolone and Warfarin. After RED and UF separation, three polar substances (Log Pow < 2) were largely unbound (fu > 70%), while more lipophilic substances were largely bound (fu < 33%). Compared to RED or UF, UC resulted in a generally higher fu of lipophilic substances. fu obtained after RED and UF were more consistent with published data. For half of the substances, UC resulted in fu higher than the reference data. UF, RED and both UF and UC, resulted in lower fu of Flutamide, Ketoconazole and Colchicine, respectively. For fu quantifications, the separation method should be selected according to the test substance's properties. Based on our data, RED is suitable for a broader range of substances while UC and UF are suitable for polar substances.

Plant extracts, polymers and new approach methods: Practical experience with skin sensitization assessment.

Over the last decade, research into methodologies to identify skin sensitization hazards has led to the adoption of several non-animal methods as OECD test guidelines. However, predictive accuracy beyond the chemical domains of the individual validation studies remains largely untested. In the present study, skin sensitization test results from in vitro and in chemico methods for 12 plant extracts and 15 polymeric materials are reported and compared to available in vivo skin sensitization data. Eight plant extracts were tested in the DPRA and h-CLAT, with the 2 out of 3 approach resulting in a balanced accuracy of 50%. The balanced accuracy for the 11 plant extracts assessed in the SENS-IS was 88%. Excluding 5 polymers inconclusive in vitro, the remainder, assessed using the 2 out of 3 approach, resulted in 63% balanced accuracy. The SENS-IS method, excluding one polymeric material due to technical inapplicability, showed 68% balanced accuracy. Although based on limited numbers, the results presented here indicate that some substance subgroups may not be in the applicability domains of the method used and careful analysis is required before positive or negative results can be accepted.

Toward Realistic Dosimetry In Vitro: Determining Effective Concentrations of Test Substances in Cell Culture and Their Prediction by an In Silico Mass Balance Model.

Nominal concentrations (CNom) in cell culture media are routinely used to define concentration-effect relationships in the in vitro toxicology. The actual concentration in the medium (CMedium) can be affected by adsorption processes, evaporation, or degradation of chemicals. Therefore, we measured the total and free concentration of 12 chemicals, covering a wide range of lipophilicity (log KOW -0.07-6.84), in the culture medium (CMedium) and cells (CCell) after incubation with Balb/c 3T3 cells for up to 48 h. Measured values were compared to predictions using an as yet unpublished in silico mass balance model that combined relevant equations from similar models published by others. The total CMedium for all chemicals except tamoxifen (TAM) were similar to the CNom. This was attributed to the cellular uptake of TAM and accumulation into lysosomes. The free (i.e., unbound) CMedium for the low/no protein binding chemicals were similar to the CNom, whereas values of all moderately to highly protein-bound chemicals were less than 30% of the CNom. Of the 12 chemicals, the two most hydrophilic chemicals, acetaminophen (APAP) and caffeine (CAF), were the only ones for which the CCell was the same as the CNom. The CCell for all other chemicals tended to increase over time and were all 2- to 274-fold higher than CNom. Measurements of CCytosol, using a digitonin method to release cytosol, compared well with CCell (using a freeze-thaw method) for four chemicals (CAF, APAP, FLU, and KET), indicating that both methods could be used. The mass balance model predicted the total CMedium within 30% of the measured values for 11 chemicals. The free CMedium of all 12 chemicals were predicted within 3-fold of the measured values. There was a poorer prediction of CCell values, with a median overprediction of 3- to 4-fold. In conclusion, while the number of chemicals in the study is limited, it demonstrates the large differences between CNom and total and free CMedium and CCell, which were also relatively well predicted by the mass balance model.

The Evolution of Regulatory Toxicology: Where is the Gardener?

There is a need for paradigm change in the methodology employed for toxicological testing and assessment. It could be said that this change is well on its way, through an evolutionary progress analogous to that of natural selection. Darwin's Theory of Evolution has defined the idea of evolution and descendancy since the last third of the 19th century. Increasingly, this concept of 'evolution' is being applied beyond the field of biology. This Comment article discusses the progress of toxicological testing in the context of 'evolutionary pressure' and deliberates how this process can help foster the development, implementation and acceptance of mechanistic and human-relevant methods in this field. By comparing the current regulatory landscape in toxicity testing and assessment to specific elements in Charles Darwin's evolutionary theory, we aim to better understand the needs and requirements for the future.

The thyroid hormone converting enzyme human deiodinase 1 is inhibited by gold ions from inorganic salts, organic substances, and by small-size nanoparticles.

The selenocysteine-containing enzyme class deiodinases (DIO) consists of three isoforms. DIOs play a role in regulation of thyroid hormone (TH) signaling through the removal of iodide from TH leading to TH that interact with the hypothalamic-pituitary-thyroid (HPT) axis with differing potency. Some gold-containing organic substances are known to inhibit many selenoenzymes, including DIOs. It is, however, unclear whether the Au-containing substances or the Au ions are causing the inhibition. In this study, five organic and inorganic gold substances as well as three gold nanoparticles (AuNPs) were tested for their potential to inhibit DIO1. The enzyme activity was tested using human liver microsomes as an enzyme source and reverse T3 as a substrate; iodide release was measured by the Sandell-Kolthoff method. The three organic gold substances aurothioglucose, auranofin and sodium aurothiomalate inhibited DIO1 with IC50 between 0.38 and 0.75 µM while their structural analogues lacking the gold ion did not. Likewise, the two tested gold salts, Au(I) and Au(III) chloride, showed a concentration-dependent inhibition of the DIO1 with IC50 values of 0.95 and 0.57 µM. Further, AuNPs of different sizes (100, 30 and 5 nm diameter) were tested with only the 5 nm AuNPs leading to inhibition with an IC50 of 8 × 1014 AuNP/L. This inhibition was not caused by the Au ions released by the AuNP into the incubation media. The exact mechanism of inhibition of DIO1 by 5 nm AuNPs should be further examined. In conclusion, the microsomal DIO1 assay demonstrated the inhibition of DIO1 by gold ions originating from different gold-containing substances, but not by Au released from AuNPs; rather DIO1 is inhibited by 5 nm, but not larger, AuNPs.

Investigation of ruminant xenobiotic metabolism in a modified rumen simulation system (RUSITEC).

The approving agencies for plant protection agents request xenobiotic metabolism and residue studies in rats, farm animals and plants (e.g. EU regulation 1107/2009) according to OECD guidelines. The specific intestinal physiology of ruminants might lead to specific residues, which should be investigated very carefully. Specific aspects of xenobiotic metabolism in ruminants may arise, which are investigated by performing additional in vivo studies. The aim of the present work is to asses a modified rumen simulation system (RUSITEC) for such studies in vitro. Rumen constituents from sheep were incubated over 8 days. Physiological parameters followed by monitoring the pH (6.70 ± 0.07), the redox potential (301 mV ± 30 mV), the microbial composition and determination of ß-glucosidase activity. After anequilibration period of three to four days the fermenters were probed with 14C-labelled triazolederivatives, i.e.common metabolites of azole fungicides. Only triazole-alanine was cleaved to 1,2,4-triazole, while triazole-acetic acid and triazole-lactic acid remained stable up to 96 h. Moreover, glycosides are often determined as the main residues in the plants. This analysis showed, that the two glucosides octyl-ß-D-glucopyranoside and polydatin were both rapidly cleaved in this rumen in vitro system. These data showed that the modified RUSITEC system is stable, viable and maintained metabolic capacity over a longer period (at least 8 days). This makes many animal experiments obsolete and lead to a significant contribution of the 3R (refine, reduce, replace). The modification of the RUSITEC system enables safe routine use for unlabeled but also for radioactive labelled compounds.