Towards an alternative testing strategy for nanomaterials used in nanomedicine: Lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

Integrated testing strategy (ITS) for bioaccumulation assessment under REACH

REACH (registration, evaluation, authorisation and restriction of chemicals) regulation requires that all the chemicals produced or imported in Europe above 1 tonne/year are registered. To register a chemical, physicochemical, toxicological and ecotoxicological information needs to be reported in a dossier. REACH promotes the use of alternative methods to replace, refine and reduce the use of animal (eco)toxicity testing. Within the EU OSIRIS project, integrated testing strategies (ITSs) have been developed for the rational use of non-animal testing approaches in chemical hazard assessment. Here we present an ITS for evaluating the bioaccumulation potential of organic chemicals. The scheme includes the use of all available data (also the non-optimal ones), waiving schemes, analysis of physicochemical properties related to the end point and alternative methods (both in silico and in vitro). In vivo methods are used only as last resort. Using the ITS, in vivo testing could be waived for about 67% of the examined compounds, but bioaccumulation potential could be estimated on the basis of non-animal methods. The presented ITS is freely available through a web tool.

Optimizing the aquatic toxicity assessment under REACH through an integrated testing strategy (ITS).

To satisfy REACH requirements a high number of data on chemical of interest should be supplied to the European Chemicals Agency. To organize the various kinds of information and help the registrants to choose the best strategy to obtain the needed information limiting at the minimum the use of animal testing, integrated testing strategies (ITSs) schemes can be used. The present work deals with regulatory data requirements for assessing the hazards of chemicals to the aquatic pelagic environment. We present an ITS scheme for organizing and using the complex existing data available for aquatic toxicity assessment. An ITS to optimize the choice of the correct prediction strategy for aquatic pelagic toxicity is described. All existing information (like physico-chemical information), and all the alternative methods (like in silico, in vitro or the acute-to-chronic ratio) are considered. Moreover the weight of evidence approach to combine the available data is included.

Novel Machine-Learning Based IC Testing Strategy

The final goal of the thesis should be a real-world application in the production test data environment. This includes the pre-processing of the data, building models and visualizing the results. To do this, different machine learning models, outlier prediction oriented, should be investigated using a real dataset. Finally, the different outlier prediction algorithms should be compared, and their performance discussed.

Testing I(1) against I(d) alternatives in the presence of deteministic components

This paper discusses the role of deterministic components in the DGP and in the auxiliary regression model which underlies the implementation of the Fractional Dickey-Fuller (FDF) test for I(1) against I(d) processes with d ∈ [0, 1). This is an important test in many economic applications because I(d) processess with d & 1 are mean-reverting although, when 0.5 ≤ d & 1,, like I(1) processes, they are nonstationary. We show how simple is the implementation of the FDF in these situations, and argue that it has better properties than LM tests. A simple testing strategy entailing only asymptotically normally distributed tests is also proposed. Finally, an empirical application is provided where the FDF test allowing for deterministic components is used to test for long-memory in the per capita GDP of several OECD countries, an issue that has important consequences to discriminate between growth theories, and on which there is some controversy.

Testing I(1) against I(d) alternatives in the presence of deteministic components

This paper discusses the role of deterministic components in the DGP and in the auxiliaryregression model which underlies the implementation of the Fractional Dickey-Fuller (FDF) test for I(1) against I(d) processes with d [0, 1). This is an important test in many economic applications because I(d) processess with d < 1 are mean-reverting although, when 0.5 = d < 1, like I(1) processes, they are nonstationary. We show how simple is the implementation of the FDF in these situations, and argue that it has better properties than LM tests. A simple testing strategy entailing only asymptotically normally distributedtests is also proposed. Finally, an empirical application is provided where the FDF test allowing for deterministic components is used to test for long-memory in the per capita GDP of several OECD countries, an issue that has important consequences to discriminate between growth theories, and on which there is some controversy.

Viewpoint-based testing of concurrent components

The use of multiple partial viewpoints is recommended for specification. We believe they also can be useful for devising strategies for testing. In this paper, we use Object-Z to formally specify concurrent Java components from viewpoints based on the separation of application and synchronisation concerns inherent in Java monitors. We then use the Test-Template Framework on the Object-Z viewpoints to devise a strategy for testing the components. When combining the test templates for the different viewpoints we focus on the observable behaviour of the application to systematically derive a practical testing strategy. The Producer-Consumer and Readers-Writers problems are considered as case studies.

Elevated Risk for HIV-1 Infection in Adolescents and Young Adults in Sao Paulo, Brazil

Background. Recent studies have sought to describe HIV infection and transmission characteristics around the world. Identification of early HIV-1 infection is essential to proper surveillance and description of regional transmission trends. In this study we compare people recently infected (RI) with HIV-1, as defined by Serologic Testing Algorithm for Recent HIV Seroconversion (STARHS), to those with chronic infection. Methodology/Principal Findings. Subjects were identified from 2002-2004 at four testing sites in Sao Paulo. Of 485 HIV-1-positive subjects, 57 (12%) were defined as RI. Of the participants, 165 (34.0%) were aware of their serostatus at the time of HIV-1 testing. This proportion was statistically larger (p<0.001) among the individuals without recent infection (n = 158, 95.8%) compared to 7 individuals (4.2%) with recently acquired HIV-1 infection. In the univariate analysis, RI was more frequent in,25 and >59 years-old age strata (p < 0.001). The majority of study participants were male (78.4%), 25 to 45 years-old (65.8%), white (63.2%), single (61.7%), with family income of four or more times the minimum wage (41.0%), but with an equally distributed educational level. Of those individuals infected with HIV-1, the predominant route of infection was sexual contact (89.4%), with both hetero (47.5%) and homosexual (34.5%) exposure. Regarding sexual activity in these individuals, 43.9% reported possible HIV-1 exposure through a seropositive partner, and 49.4% reported multiple partners, with 47% having 2 to 10 partners and 37.4% 11 or more; 53.4% of infected individuals reported condom use sometimes; 34.2% reported non-injecting, recreational drug use and 23.6% were reactive for syphilis by VDRL. Subjects younger than 25 years of age were most vulnerable according to the multivariate analysis. Conclusions/Significance. In this study, we evaluated RI individuals and discovered that HIV-1 has been spreading among younger individuals in Sao Paulo and preventive approaches should, therefore, target this age stratum.

Powder forging of a sintered Al-3.8Cu-1Mg-0.8Si-O.1Sn alloy

The forging characteristics of an Al-Cu-Mg-Si-Sn alloy are examined using it new testing strategy which incorporates a double truncated cone specimen and finite element modelling. This sample geometry produces controlled strain distributions within a single specimen and can readily identify the specific strain required to achieve a specific microstructural event by matching the metallographic data with the strain profiles calculated from finite element software, The friction conditions were determined using the conventional friction ring test, which was evaluated using finite element software. The rheological properties of the alloy, evaluated from compression testing of right cylinders, are similar to the properties of conventional aluminium forgings. A hoop strain develops at the outer diameter of the truncated cones and this leads to pore opening at the outer few millimetres. The porosity is effectively removed when the total strain equals the net compressive strain. The strain profiles that develop in the truncated cones are largely independent of the processing temperature and the strain rate although the strain required for pore closure increases as the forging temperature is reduced. This suggests that the microstructure and the strain rate sensitivity may also be important factors controlling pore behaviour. (C) 2002 Elsevier Science B.V. All rights reserved.

Establiment de bases científiques per al desenvolupament d'una estratègia integrada per avaluar toxicitat en reproducció

Report for the scientific sojourn at the National Institute for Public Health and the Environment (RIVM), Netherlands, from 2006 to 2008. This project aimed at creating scientific elements that could help deriving an integrated testing strategy for reproductive toxicity. Part of the project focused on the use of alternative tests for regulatory purposes. An in vitro-in vivo extrapolation method for embryotoxicity was proposed and evaluated. In vitro and in vivo dose descriptors were correlated; however, the scatter in the correlation was too large to allow an accurate estimation of an in vivo dose from an in vitro dose. The in vitro-in vivo extrapolation method together with toxicokinetic data was also applied to a category of substances (phthalates). Although based on a limited number of substances, the results suggested that in vitro-in vivo extrapolation for embryotoxicity is possible within a category of compounds if adequate toxicokinetic data is available. Because of the limitations that still remain in the use of alternative tests for reproductive toxicicity, other approaches to reduce animal testing were explored. Thus, a database of reproductive toxicity studies was created to retrospectively evaluate the comparative value of some studies or elements in a particular study. When compared to the subchronic toxicity study, the rat two-generation reproductive toxicity study had a considerable impact on the identification of hazard for reproductive toxicity, but not on the overall NOAEL. Among the two-generation studies included in our database, the second generation affected neither the overall NOAEL nor the critical effect. The rat and the rabbit developmental toxicity studies were, on average, similarly sensitive. However, for certain substances the developmental study in either one of the two species appeared to be more sensitive than in the other species.

The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European Project ACuteTox.

ACuteTox is a project within the 6th European Framework Programme which had as one of its goals to develop, optimise and prevalidate a non-animal testing strategy for predicting human acute oral toxicity. In its last 6 months, a challenging exercise was conducted to assess the predictive capacity of the developed testing strategies and final identification of the most promising ones. Thirty-two chemicals were tested blind in the battery of in vitro and in silico methods selected during the first phase of the project. This paper describes the classification approaches studied: single step procedures and two step tiered testing strategies. In summary, four in vitro testing strategies were proposed as best performing in terms of predictive capacity with respect to the European acute oral toxicity classification. In addition, a heuristic testing strategy is suggested that combines the prediction results gained from the neutral red uptake assay performed in 3T3 cells, with information on neurotoxicity alerts identified by the primary rat brain aggregates test method. Octanol-water partition coefficients and in silico prediction of intestinal absorption and blood-brain barrier passage are also considered. This approach allows to reduce the number of chemicals wrongly predicted as not classified (LD50>2000 mg/kg b.w.).

Predictive models for nanotoxicology: current challenges and future opportunities.

Characterizing the risks posed by nanomaterials is extraordinarily complex because these materials can have a wide range of sizes, shapes, chemical compositions and surface modifications, all of which may affect toxicity. There is an urgent need for a testing strategy that can rapidly and efficiently provide a screening approach for evaluating the potential hazard of nanomaterials and inform the prioritization of additional toxicological testing where necessary. Predictive toxicity models could form an integral component of such an approach by predicting which nanomaterials, as a result of their physico-chemical characteristics, have potentially hazardous properties. Strategies for directing research towards predictive models and the ancillary benefits of such research are presented here.

Evaluation of drug-induced neurotoxicity based on metabolomics, proteomics and electrical activity measurements in complementary CNS in vitro models.

The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation.

Amiodarone biokinetics, the formation of its major oxidative metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures.

The difficulty in mimicking nervous system complexity and cell-cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure.

An Investigation of the Role of Attention in the Cross-Race Effect: An Ecological Approach

The current set of studies was conducted to examine the cross-race effect (CRE), a phenomenon commonly found in the face perception literature. The CRE is evident when participants display better own-race face recognition accuracy than other-race recognition accuracy (e.g. Ackerman et al., 2006). Typically the cross-race effect is attributed to perceptual expertise, (i.e., other-race faces are processed less holistically; Michel, Rossion, Han, Chung & Caldara, 2006), and the social cognitive model (i.e., other-race faces are processed at the categorical level by virtue of being an out-group member; Hugenberg, Young, Bernstein, & Sacco, 2010). These effects may be mediated by differential attention. I investigated whether other-race faces are disregarded and, consequently, not remembered as accurately as own-race (in-group) faces. In Experiment 1, I examined how the magnitude of the CRE differed when participants learned individual faces sequentially versus when they learned multiple faces simultaneously in arrays comprising faces and objects. I also examined how the CRE differed when participants recognized individual faces presented sequentially versus in arrays of eight faces. Participants’ recognition accuracy was better for own-race faces than other-race faces regardless of familiarization method. However, the difference between own- and other-race accuracy was larger when faces were familiarized sequentially in comparison to familiarization with arrays. Participants’ response patterns during testing differed depending on the combination of familiarization and testing method. Participants had more false alarms for other-race faces than own-race faces if they learned faces sequentially (regardless of testing strategy); if participants learned faces in arrays, they had more false alarms for other-race faces than own-races faces if ii i they were tested with sequentially presented faces. These results are consistent with the perceptual expertise model in that participants were better able to use the full two seconds in the sequential task for own-race faces, but not for other-race faces. The purpose of Experiment 2 was to examine participants’ attentional allocation in complex scenes. Participants were shown scenes comprising people in real places, but the head stimuli used in Experiment 1 were superimposed onto the bodies in each scene. Using a Tobii eyetracker, participants’ looking time for both own- and other-race faces was evaluated to determine whether participants looked longer at own-race faces and whether individual differences in looking time correlated with individual differences in recognition accuracy. The results of this experiment demonstrated that although own-race faces were preferentially attended to in comparison to other-race faces, individual differences in looking time biases towards own-race faces did not correlate with individual differences in own-race recognition advantages. These results are also consistent with perceptual expertise, as it seems that the role of attentional biases towards own-race faces is independent of the cognitive processing that occurs for own-race faces. All together, these results have implications for face perception tasks that are performed in the lab, how accurate people may be when remembering faces in the real world, and the accuracy and patterns of errors in eyewitness testimony.