Human IPSC 3D brain model as a tool to study chemical-induced dopaminergic neuronal toxicity.

Oxidative stress is caused by an imbalance between the generation and detoxification of reactive oxygen and nitrogen species (ROS/RNS). This imbalance plays an important role in brain aging and age-related neurodegenerative diseases. In the context of Parkinson's disease (PD), the sensitivity of dopaminergic neurons in the substantia nigra pars compacta to oxidative stress is considered a key factor of PD pathogenesis. Here we study the effect of different oxidative stress-inducing compounds (6-OHDA, MPTP or MPP+) on the population of dopaminergic neurons in an iPSC-derived human brain 3D model (aka BrainSpheres). Treatment with 6-OHDA, MPTP or MPP+ at 4 weeks of differentiation disrupted the dopaminergic neuronal phenotype in BrainSpheres at (50, 5000, 1000 µM respectively). 6-OHDA increased ROS production and decreased mitochondrial function most efficiently. It further induced the greatest changes in gene expression and metabolites related to oxidative stress and mitochondrial dysfunction. Co-culturing BrainSpheres with an endothelial barrier using a transwell system allowed the assessment of differential penetration capacities of the tested compounds and the damage they caused in the dopaminergic neurons within the BrainSpheres In conclusion, treatment with compounds known to induce PD-like phenotypes in vivo caused molecular deficits and loss of dopaminergic neurons in the BrainSphere model. This approach therefore recapitulates common animal models of neurodegenerative processes in PD at similarly high doses. The relevance as tool for drug discovery is discussed.

Description of neurological mimics presented to the neurology service of a small animal referral hospital.

BACKGROUND: Clinicians observe that cats and dogs referred to neurology services often do not have an underlying neurological disorder. There has been no analysis of the frequency or categorisation of these neurological mimics. METHODS: Retrospective study of 520 cases was carried out. Data on signalment, presenting clinical signs, neurological examination findings and final diagnosis were collected. Final diagnoses were classified as primary neurological, non-neurological in origin but with neurological clinical manifestation, completely non-neurological (neurological mimics) or undiagnosed. Presenting clinical signs and neurological examination results were compared between neurological mimics and primary neurological cases using Chi-square or Fischer exact test. Relative risk (RR) was calculated for significant associations. RESULTS: A total of 74% were primary neurological conditions, 8% neurological mimics, 3% non-neurological with neurological manifestation and 15% undiagnosed. An animal referred for lameness was approximately five times more likely to be diagnosed as a neurological mimic than as a primary neurological disorder (RR = 5.42, p < 0.001). Cases with a normal neurological examination were approximately 15 times more likely to be a neurological mimic (RR = 14.97, p < 0.001). CONCLUSION: Thorough examination with consideration of alternative diagnoses is important when a neurological condition is suspected in an animal that presents with lameness or normal neurological examination.

Primary orthostatic tremor and orthostatic tremor-plus in dogs: 60 cases (2003-2020).

BACKGROUND: Orthostatic tremor (OT) is a rare movement disorder characterized by high-frequency (>12 Hz) involuntary, rhythmic, sinusoidal movements affecting predominantly the limbs while standing. OBJECTIVE: To describe the signalment, presenting complaints, phenotype, diagnostic findings, treatment, and outcome of a large sample of dogs with OT. ANIMALS: Sixty dogs diagnosed with OT based on conscious electromyography. METHODS: Multicenter retrospective case series study. Dogs were included if they had a conscious electromyography consistent with muscle discharge frequency >12 Hz while standing. RESULTS: Fifty-three cases were diagnosed with primary OT (POT). Giant breed dogs represented most cases (83%; 44/53). Most dogs (79%; 42/53) were younger than 2 years of age at onset of signs, except for Retrievers which were all older than 3.5 years of age. The most common presenting complaints were pelvic limb tremors while standing (85%; 45/53) and difficulty when rising or sitting down (45%; 24/53). Improvement of clinical signs occurred in most dogs (85%; 45/53) treated medically with phenobarbital, primidone, gabapentin, pregabalin or clonazepam, but it was mostly partial rather than complete. Orthostatic tremor-plus was seen in 7 dogs that had concurrent neurological diseases. CONCLUSIONS AND CLINICAL IMPORTANCE: Primary OT is a progressive disease of young, purebred, giant/large-breed dogs, which appears to begin later in life in Retrievers. Primary OT apparently responds partially to medications. Orthostatic tremor-plus exists in dogs and can be concomitant or associated with other neurological diseases.

One Hundred Cases of Localized Laryngeal Amyloidosis - Evidence for Future Management.

OBJECTIVE/HYPOTHESIS: To update the current understanding of localized laryngeal amyloidosis by analyzing the NHS National Amyloidosis Database and to further clarify the important ongoing management issues. STUDY DESIGN: Retrospective review, case series. METHODS: Patients with laryngeal amyloid were identified from the database of the NHS National Amyloidosis Center, UCL, Royal Free Hospital, London between 2000 and 2017. Patient demographics and disease profile were collated, including the exact location of amyloid deposit, treatments if any, and progression of disease. RESULTS: One hundred and three patients with localized laryngeal amyloid where identified from the database, with a mean age of 54 at diagnosis and female to male ratio of 54:49. Three patients were excluded from further analysis due to limited database information. The majority of amyloid was found in either the supraglottis (44) or glottis (53) but all the laryngeal subsites were involved. One-third of the patients (34) had amyloid in more than one laryngeal subsite. No patients were found to progress to systemic amyloid, but many progressed locally to other subsites or further down the LTB tree (29%). Three patients were successfully treated with radiotherapy after other modalities had failed. CONCLUSIONS: This is the largest case series reported to date of localized laryngeal amyloidosis. It highlights the high incidence of multifocal disease and the significant proportion of patients who progressed, not to systemic amyloidosis but to more extensive localized amyloid. We recommend that in all cases of laryngeal amyloid, patients should undergo a thorough assessment of the upper and lower airways and have ongoing surveillance for at least 15 years. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E1912-E1917, 2021.

Performance Analysis of Cooperative and Non-Cooperative Relaying over VLC Channels.

The line-of-sight (LoS) channel is one of the requirements for efficient data transmission in visible-light communications (VLC), but this cannot always be guaranteed in indoor applications for a variety of reasons, such as moving objects and the layout of rooms. The relay-assisted VLC system is one of the techniques that can be used to address this issue and ensures seamless connectivity. This paper investigates the performance of half-duplex (HD) conventional DF relay system and cooperative systems (i.e., selective DF (SDF) and incremental DF (IDF)) over VLC channels in terms of outage probability and energy consumption. Analytical expressions for both outage probability and the minimum energy-per-bit performance of the aforementioned relaying systems are derived. Furthermore, Monte Carlo simulations are provided throughout the paper to validate the derived expressions. The results show that exploiting SDF and IDF relaying schemes can achieve approximately 25% and 15% outage probability enhancement compared to single-hop and DF protocols, respectively. The results also demonstrate that the performance of the single-hop VLC system deteriorates when the end-to-end distances become larger. For example, when the vertical distance is 3.5m, the single-hop approach consumes 20%, 40% and 45% more energy in comparison to the DF, SDF, and IDF approaches, respectively.

Antidepressant Paroxetine Exerts Developmental Neurotoxicity in an iPSC-Derived 3D Human Brain Model.

Selective serotonin reuptake inhibitors (SSRIs) are frequently used to treat depression during pregnancy. Various concerns have been raised about the possible effects of these drugs on fetal development. Current developmental neurotoxicity (DNT) testing conducted in rodents is expensive, time-consuming, and does not necessarily represent human pathophysiology. A human, in vitro testing battery to cover key events of brain development, could potentially overcome these challenges. In this study, we assess the DNT of paroxetine-a widely used SSRI which has shown contradictory evidence regarding effects on human brain development using a versatile, organotypic human induced pluripotent stem cell (iPSC)-derived brain model (BrainSpheres). At therapeutic blood concentrations, which lie between 20 and 60 ng/ml, Paroxetine led to an 80% decrease in the expression of synaptic markers, a 60% decrease in neurite outgrowth and a 40-75% decrease in the overall oligodendrocyte cell population, compared to controls. These results were consistently shown in two different iPSC lines and indicate that relevant therapeutic concentrations of Paroxetine induce brain cell development abnormalities which could lead to adverse effects.

Introduction of Disc Material into the Vertebral Canal by Fenestration of Thoracolumbar Discs Following Decompressive Surgery.

OBJECTIVE: Intervertebral disc extrusion is a common disease affecting chondrodystrophic dogs. It has been reported that fenestration of thoracolumbar intervertebral discs reduces recurrence of disc extrusion and is associated with a low complication rate. One complication reported is iatrogenic introduction of disc material into the canal directly following fenestration. This study aimed to ascertain if, and at what frequency, additional disc material may be introduced into the vertebral canal by fenestration of the affected disc following decompressive surgery. STUDY DESIGN: Twenty-one dogs that underwent hemilaminectomy and disc fenestration for the treatment of thoracolumbar intervertebral disc extrusion had intraoperative assessment of the vertebral canal before and after fenestration. The spinal cord was first decompressed by hemilaminectomy and removal of all visible extruded disc material within the vertebral canal. Once no further material was visible, manual fenestration of the affected disc was performed. The vertebral canal was re-inspected, and the presence or absence of additional material was noted and included in the surgery report. RESULTS: Seven dogs showed the presence of new disc material in the vertebral canal post fenestration.This preliminary study shows that additional disc material can be forced into the vertebral canal by fenestration following decompressive surgery, with a frequency of 7/21. CONCLUSION: This illustrates the importance of checking the vertebral canal after fenestration of an extruded intervertebral disc.

In vitro and in silico Models to Study Mosquito-Borne Flavivirus Neuropathogenesis, Prevention, and Treatment.

Mosquito-borne flaviviruses can cause disease in the nervous system, resulting in a significant burden of morbidity and mortality. Disease models are necessary to understand neuropathogenesis and identify potential therapeutics and vaccines. Non-human primates have been used extensively but present major challenges. Advances have also been made toward the development of humanized mouse models, but these models still do not fully represent human pathophysiology. Recent developments in stem cell technology and cell culture techniques have allowed the development of more physiologically relevant human cell-based models. In silico modeling has also allowed researchers to identify and predict transmission patterns and discover potential vaccine and therapeutic candidates. This review summarizes the research on in vitro and in silico models used to study three mosquito-borne flaviviruses that cause neurological disease in humans: West Nile, Dengue, and Zika. We also propose a roadmap for 21st century research on mosquito-borne flavivirus neuropathogenesis, prevention, and treatment.

Suitability of 3D human brain spheroid models to distinguish toxic effects of gold and poly-lactic acid nanoparticles to assess biocompatibility for brain drug delivery.

BACKGROUND: The blood brain barrier (BBB) is the bottleneck of brain-targeted drug development. Due to their physico-chemical properties, nanoparticles (NP) can cross the BBB and accumulate in different areas of the central nervous system (CNS), thus are potential tools to carry drugs and treat brain disorders. In vitro systems and animal models have demonstrated that some NP types promote neurotoxic effects such as neuroinflammation and neurodegeneration in the CNS. Thus, risk assessment of the NP is required, but current 2D cell cultures fail to mimic complex in vivo cellular interactions, while animal models do not necessarily reflect human effects due to physiological and species differences. RESULTS: We evaluated the suitability of in vitro models that mimic the human CNS physiology, studying the effects of metallic gold NP (AuNP) functionalized with sodium citrate (Au-SC), or polyethylene glycol (Au-PEG), and polymeric polylactic acid NP (PLA-NP). Two different 3D neural models were used (i) human dopaminergic neurons differentiated from the LUHMES cell line (3D LUHMES) and (ii) human iPSC-derived brain spheroids (BrainSpheres). We evaluated NP uptake, mitochondrial membrane potential, viability, morphology, secretion of cytokines, chemokines and growth factors, and expression of genes related to ROS regulation after 24 and 72 h exposures. NP were efficiently taken up by spheroids, especially when PEGylated and in presence of glia. AuNP, especially PEGylated AuNP, effected mitochondria and anti-oxidative defense. PLA-NP were slightly cytotoxic to 3D LUHMES with no effects to BrainSpheres. CONCLUSIONS: 3D brain models, both monocellular and multicellular are useful in studying NP neurotoxicity and can help identify how specific cell types of CNS are affected by NP.

Lumbosacral transitional vertebrae in cats and its relationship to lumbosacral vertebral canal stenosis.

OBJECTIVES: Although a clear relationship has been demonstrated between the presence of lumbosacral transitional vertebrae and the development of lumbosacral stenosis in dogs, this relationship has not been evaluated in cats. The aim of this study was to investigate the relationship between the presence of lumbosacral transitional vertebrae and lumbosacral stenosis in cats. METHODS: The medical records and diagnostic imaging studies of 13 cats diagnosed with lumbosacral stenosis were retrospectively reviewed for lumbosacral abnormalities and compared with findings of 405 cats that underwent CT for reasons unrelated to spinal disease. RESULTS: Clinical signs associated with lumbosacral stenosis included lumbosacral pain, low tail carriage, difficulty jumping, and urinary or faecal incontinence. Neurological signs included proprioceptive deficits, ambulatory paraparesis, pelvic limb ataxia, reduced spinal reflexes and reduced perianal reflex. Duration of clinical signs ranged from 1 day to 10 months (mean 3 months). Of the 13 cats with lumbosacral stenosis, seven (53.8%) were diagnosed with lumbosacral transitional vertebrae. In the control population of 405 cats, 24 (5.9%) were diagnosed with lumbosacral transitional vertebrae. Results indicated that lumbosacral transitional vertebrae were significantly ( P <0.0001) more prevalent in cats with lumbosacral stenosis compared with the control feline population (odds ratio 18.52, 95% confidence interval 6.1-62.1). Development of clinical signs of lumbosacral stenosis in cats with lumbosacral transitional vertebrae (mean 10.8 years) was not significantly different from that of cats without lumbosacral transitional vertebrae (mean 12.7 years). Likewise, there was no significant influence of breed ( P >0.99) or sex ( P = 0.29) on the occurrence of lumbosacral transitional vertebrae. CONCLUSIONS AND RELEVANCE: Despite lumbosacral stenosis being a rare spinal condition in cats, lumbosacral transitional vertebrae can be considered a risk factor for its development.

Toxicity, recovery, and resilience in a 3D dopaminergic neuronal in vitro model exposed to rotenone.

To date, most in vitro toxicity testing has focused on acute effects of compounds at high concentrations. This testing strategy does not reflect real-life exposures, which might contribute to long-term disease outcome. We used a 3D-human dopaminergic in vitro LUHMES cell line model to determine whether effects of short-term rotenone exposure (100 nM, 24 h) are permanent or reversible. A decrease in complex I activity, ATP, mitochondrial diameter, and neurite outgrowth were observed acutely. After compound removal, complex I activity was still inhibited; however, ATP levels were increased, cells were electrically active and aggregates restored neurite outgrowth integrity and mitochondrial morphology. We identified significant transcriptomic changes after 24 h which were not present 7 days after wash-out. Our results suggest that testing short-term exposures in vitro may capture many acute effects which cells can overcome, missing adaptive processes, and long-term mechanisms. In addition, to study cellular resilience, cells were re-exposed to rotenone after wash-out and recovery period. Pre-exposed cells maintained higher metabolic activity than controls and presented a different expression pattern in genes previously shown to be altered by rotenone. NEF2L2, ATF4, and EAAC1 were downregulated upon single hit on day 14, but unchanged in pre-exposed aggregates. DAT and CASP3 were only altered after re-exposure to rotenone, while TYMS and MLF1IP were downregulated in both single-exposed and pre-exposed aggregates. In summary, our study shows that a human cell-based 3D model can be used to assess cellular adaptation, resilience, and long-term mechanisms relevant to neurodegenerative research.

Epidemiology, clinical management, and outcomes of dogs involved in road traffic accidents in the United Kingdom (2009-2014).

OBJECTIVE: To estimate the prevalence and risk factors for road traffic accidents (RTA) in dogs and describe the management and outcome of these dogs attending primary-care veterinary practices in the United Kingdom. DESIGN: Retrospective cross-sectional study. SETTING: Primary-care veterinary practices in the United Kingdom. ANIMALS: The study population included 199,464 dogs attending 115 primary-care clinics across the United Kingdom. MEASUREMENTS AND MAIN RESULTS: Electronic patient records of dogs attending practices participating in the VetCompass Programme were assessed against selection criteria used to define RTA cases. Cases identified as RTAs were identified and manually verified to calculate prevalence. Univariable and multivariable logistic regression methods were used to evaluate associations between risk factors and RTA. The prevalence of RTA was 0.41%. Of the RTA cases, 615 (74.9%) were purebred, 322 (39.2%) were female, and 285 (54.8%) were insured. The median age at RTA was 2.5 years. After accounting for the effects of other factors, younger dogs had increased odds of an RTA event: dogs aged under 3 years showed 2.9 times the odds and dogs aged between 6-9 years showed 1.8 times the odds of an RTA event compared with dogs aged over 14 years. Males had 1.4 times the odds of an RTA event compared with females. Overall, 22.9% of cases died from a cause associated with RTA. Of dogs with information available, 34.0% underwent diagnostic imaging, 29.4% received intravenous fluid-therapy, 71.1% received pain relief, 46.0% were hospitalized, and 15.6% had surgery performed under general anesthetic. CONCLUSIONS: This study identified important demographic factors associated with RTA in dogs, notably being young and male.

Rotenone exerts developmental neurotoxicity in a human brain spheroid model.

Growing concern suggests that some chemicals exert (developmental) neurotoxicity (DNT and NT) and are linked to the increase in incidence of autism, attention deficit and hyperactivity disorders. The high cost of routine tests for DNT and NT assessment make it difficult to test the high numbers of existing chemicals. Thus, more cost effective neurodevelopmental models are needed. The use of induced pluripotent stem cells (iPSC) in combination with the emerging human 3D tissue culture platforms, present a novel tool to predict and study human toxicity. By combining these technologies, we generated multicellular brain spheroids (BrainSpheres) from human iPSC. The model has previously shown to be reproducible and recapitulates several neurodevelopmental features. Our results indicate, rotenone's toxic potency varies depending on the differentiation status of the cells, showing higher reactive oxygen species (ROS) and higher mitochondrial dysfunction during early than later differentiation stages. Immuno-fluorescence morphology analysis after rotenone exposure indicated dopaminergic-neuron selective toxicity at non-cytotoxic concentrations (1 µM), while astrocytes and other neuronal cell types were affected at (general) cytotoxic concentrations (25 µM). Omics analysis showed changes in key pathways necessary for brain development, indicating rotenone as a developmental neurotoxicant and show a possible link between previously shown effects on neurite outgrowth and presently observed effects on Ca2+ reabsorption, synaptogenesis and PPAR pathway disruption. In conclusion, our BrainSpheres model has shown to be a reproducible and novel tool to study neurotoxicity and developmental neurotoxicity. Results presented here support the idea that rotenone can potentially be a developmental neurotoxicant.

3D Differentiation of LUHMES Cell Line to Study Recovery and Delayed Neurotoxic Effects.

Current neurotoxicity testing and the study of molecular mechanisms in neurodegeneration in vitro usually focuses on acute exposures to compounds. 3D Lund human mesencephalic (LUHMES) cells allow long-term treatment or pulse exposure in combination with compound washout to study delayed neurotoxic effects as well as recovery and neurodegeneration pathways. In this unit we describe 3D LUHMES culture and characterization. Characterization of the model involves immunocytochemistry, flow cytometry, and qPCR measurements. Studying the delayed effects of compounds is more relevant to human exposures and neurodegenerative diseases with a strong genetic or environmental component. Most assays for molecular endpoints have been developed for monolayer cell culture and therefore need to be adapted for 3D models. In this unit, we further describe toxicological assays for molecular endpoints such as ATP levels, mitochondrial viability, and neurite outgrowth, which have been adapted for use in 3D LUHMES cultures. © 2017 by John Wiley & Sons, Inc.

A human brain microphysiological system derived from induced pluripotent stem cells to study neurological diseases and toxicity.

Human in vitro models of brain neurophysiology are needed to investigate molecular and cellular mechanisms associated with neurological disorders and neurotoxicity. We have developed a reproducible iPSC-derived human 3D brain microphysiological system (BMPS), comprised of differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over eight weeks and show the critical elements of neuronal function: synaptogenesis and neuron-to-neuron (e.g., spontaneous electric field potentials) and neuronal-glial interactions (e.g., myelination), which mimic the microenvironment of the central nervous system, rarely seen in vitro before. The BMPS shows 40% overall myelination after 8 weeks of differentiation. Myelin was observed by immunohistochemistry and confirmed by confocal microscopy 3D reconstruction and electron microscopy. These findings are of particular relevance since myelin is crucial for proper neuronal function and development. The ability to assess oligodendroglial function and mechanisms associated with myelination in this BMPS model provide an excellent tool for future studies of neurological disorders such as multiple sclerosis and other demyelinating diseases. The BMPS provides a suitable and reliable model to investigate neuron-neuroglia function as well as pathogenic mechanisms in neurotoxicology.

Cellular resilience.

Cellular resilience describes the ability of a cell to cope with environmental changes such as toxicant exposure. If cellular metabolism does not collapse directly after the hit or end in programmed cell death, the ensuing stress responses promote a new homeostasis under stress. The processes of reverting "back to normal" and reversal of apoptosis ("anastasis") have been studied little at the cellular level. Cell types show astonishingly similar vulnerability to most toxicants, except for those that require a very specific target, metabolism or mechanism present only in specific cell types. The majority of chemicals triggers "general cytotoxicity" in any cell at similar concentrations. We hypothesize that cells differ less in their vulnerability to a given toxicant than in their resilience (coping with the "hit"). In many cases, cells do not return to the naive state after a toxic insult. The phenomena of "pre-conditioning", "tolerance" and "hormesis" describe this for low-dose exposures to toxicants that render the cell more resistant to subsequent hits. The defense and resilience programs include epigenetic changes that leave a "memory/scar" - an alteration as a consequence of the stress the cell has experienced. These memories might have long-term consequences, both positive (resistance) and negative, that contribute to chronic and delayed manifestations of hazard and, ultimately, disease. This article calls for more systematic analyses of how cells cope with toxic perturbations in the long-term after stressor withdrawal. A technical prerequisite for these are stable (organotypic) cultures and a characterization of stress response molecular networks.

Quality assurance of metabolomics.

Metabolomics promises a holistic phenotypic characterization of biological responses to toxicants. This technology is based on advanced chemical analytical tools with reasonable throughput, including mass-spectroscopy and NMR. Quality assurance, however - from experimental design, sample preparation, metabolite identification, to bioinformatics data-mining - is urgently needed to assure both quality of metabolomics data and reproducibility of biological models. In contrast to microarray-based transcriptomics, where consensus on quality assurance and reporting standards has been fostered over the last two decades, quality assurance of metabolomics is only now emerging. Regulatory use in safety sciences, and even proper scientific use of these technologies, demand quality assurance. In an effort to promote this discussion, an expert workshop discussed the quality assurance needs of metabolomics. The goals for this workshop were 1) to consider the challenges associated with metabolomics as an emerging science, with an emphasis on its application in toxicology and 2) to identify the key issues to be addressed in order to establish and implement quality assurance procedures in metabolomics-based toxicology. Consensus has still to be achieved regarding best practices to make sure sound, useful, and relevant information is derived from these new tools.

Sex differences in liver toxicity-do female and male human primary hepatocytes react differently to toxicants in vitro?

There is increasing amount of evidence for sex variation in drug efficiency and toxicity profiles. Women are more susceptible than men to acute liver injury from xenobiotics. In general, this is attributed to sex differences at a physiological level as well as differences in pharmacokinetics and pharmacodynamics, but neither of these can give a sufficient explanation for the diverse responses to xenobiotics. Existing data are mainly based on animal models and limited data exist on in vitro sex differences relevant to humans. To date, male and female human hepatocytes have not yet been compared in terms of their responses to hepatotoxic drugs. We investigated whether sex-specific differences in acute hepatotoxicity can be observed in vitro by comparing hepatotoxic drug effects in male and female primary human hepatocytes. Significant sex-related differences were found for certain parameters and individual drugs, showing an overall higher sensitivity of female primary hepatocytes to hepatotoxicants. Moreover, our work demonstrated that high content screening is feasible with pooled primary human hepatocytes in suspension.

Iron oxide nanoparticle toxicity testing using high-throughput analysis and high-content imaging.

Applying validated in vitro assays to the study of nanoparticle toxicity is a growing trend in nanomaterial risk assessment. Precise characterisation of reference nanomaterials and a well-regulated in vitro testing system are required to determine the physicochemical descriptors which dictate the toxic potential of nanoparticles. The use of automated, high-throughput technologies to facilitate the identification and prioritisation of nanomaterials which could pose a risk is desirable and developments are underway. In this study, two mammalian fibroblast lines (Balb/c 3T3 and COS-1 cells) were treated with a range of concentrations of iron oxide nanomaterials manufactured for use in medical diagnostics, using an automated platform and high-content-imaging endpoints for cell viability, oxidative stress and DNA damage (double-strand breaks). At the same time, the high-throughput comet assay was employed to measure DNA strand breaks and oxidised bases. Our results show that these methods provide a fast way to determine the toxicity of coated and uncoated iron oxide nanoparticles and, furthermore, to predict the mechanism of toxicity in vitro.

Consensus report on the future of animal-free systemic toxicity testing.

Since March 2013, animal use for cosmetics testing for the European market has been banned. This requires a renewed view on risk assessment in this field. However, in other fields as well, traditional animal experimentation does not always satisfy requirements in safety testing, as the need for human-relevant information is ever increasing. A general strategy for animal-free test approaches was outlined by the US National Research Council`s vision document for Toxicity Testing in the 21st Century in 2007. It is now possible to provide a more defined roadmap on how to implement this vision for the four principal areas of systemic toxicity evaluation: repeat dose organ toxicity, carcinogenicity, reproductive toxicity and allergy induction (skin sensitization), as well as for the evaluation of toxicant metabolism (toxicokinetics) (Fig. 1). CAAT-Europe assembled experts from Europe, America and Asia to design a scientific roadmap for future risk assessment approaches and the outcome was then further discussed and refined in two consensus meetings with over 200 stakeholders. The key recommendations include: focusing on improving existing methods rather than favoring de novo design; combining hazard testing with toxicokinetics predictions; developing integrated test strategies; incorporating new high content endpoints to classical assays; evolving test validation procedures; promoting collaboration and data-sharing of different industrial sectors; integrating new disciplines, such as systems biology and high throughput screening; and involving regulators early on in the test development process. A focus on data quality, combined with increased attention to the scientific background of a test method, will be important drivers. Information from each test system should be mapped along adverse outcome pathways. Finally, quantitative information on all factors and key events will be fed into systems biology models that allow a probabilistic risk assessment with flexible adaptation to exposure scenarios and individual risk factors.