Estimation of acute oral toxicity using the No Observed Adverse Effect Level (NOAEL) from the 28 day repeated dose toxicity studies in rats

Acute systemic toxicity is one of the areas of particular concern due to the 2009 deadline set by the 7th Amendment of the Cosmetics Directive (76/768/EEC), which introduces a testing and marketing ban of cosmetic products with ingredients tested on animals. The scientific community is putting considerable effort into developing and validating non-animal alternatives in this area. However, it is unlikely that validated and regulatory accepted alternative methods and/or strategies will be available in March 2009. Following the initiatives undertaken in the pharmaceutical industry to waive the acute oral toxicity testing before going to clinical studies by using information from other in vivo studies, we proposed an approach to identify non-toxic compounds (LD50>2000mg/kg) using information from 28 days repeated dose toxicity studies. Taking into account the high prevalence of non-toxic substances (87%) in the New Chemicals Database, it was possible to set a NOAEL threshold of 200mg/kg that allowed the correct identification of 63% of non-toxic compounds, while <1% of harmful compounds were misclassified as non-toxic. Since repeated dose toxicity studies can be performed in vivo until 2013, the proposed approach could have an immediate impact for the testing of cosmetic ingredients.     

Generation and Evaluation of a Homology Model of PfGSK‐3

Plasmodial GSK‐3 is a potential new target for malaria therapy. For a structure‐based design project, the three‐dimensional information of the designated target is needed. Unfortunately, experimental structure data for plasmodial GSK‐3 is not yet available. Homology building can be used to generate such three‐dimensional structure data using structure information of a homologous protein. GSK‐3 possesses a very flexible ATP‐binding site, a fact reflected in the variety of X‐ray structures of the human GSK‐3β which are deposited in the protein data base and are crystallized with different ligands. We used ten different HsGSK‐3β templates for the model building of plasmodial GSK‐3 and generated 200 models for each template with different modeling protocols. The quality of the models was evaluated with different tools. The results of these evaluations were used to calculate a rank‐by‐rank consensus score. The top models of this were used to compile an ensemble of PfGSK‐3 models that reflect the flexibility of the ATP‐binding site and that will be used for the structure‐based design of potential ATP‐binding site inhibitors of PfGSK‐3.     

“ToxRTool”, a new tool to assess the reliability of toxicological data

Evaluation of the reliability of toxicological data is of key importance for regulatory decision-making. In particular, the new EU Regulations concerning the registration, evaluation, authorisation and restriction of chemicals (REACH) and classification, labelling and packaging (CLP) according to the new globally harmonised system (GHS) rely on the integration of all available toxicological information. The so-called Klimisch categories, although well established and widely used, lack detailed criteria for assigning data quality to categories. A software-based tool (ToxRTool) was developed within the context of a project funded by the European Commission to provide comprehensive criteria and guidance for reliability evaluations of toxicological data. It is applicable to various types of experimental data, endpoints and studies (study reports, peer-reviewed publications) and leads to the assignment to Klimisch categories 1, 2 or 3. The tool aims to increase transparency and to harmonise approaches of reliability assessment. The tool consists of two parts, one to evaluate in vivo and one to evaluate in vitro data. The prototypes of the tool were tested in two independent inter-rater experiments. This approach allowed the analysis of the performance of the tool in practice and the identification and minimisation of sources of heterogeneity in evaluation results. The final version, ToxRTool, is publicly available for testing and use.     

Monitoring direct FXa-inhibitors and fondaparinux by Prothrombinase-induced Clotting Time (PiCT): relation to FXa-activity and influence of assay modifications

Introduction

FXa-activity can be measured by the Prothrombinase induced Clotting time (PiCT). The manufactured assay uses bovine FXa as component and employs a incubation period before re-calcification. Its use with new direct FXa-inhibitors is challenged by reports on decreased sensitivity.

Methods

Blood was incubated with 3 investigational, structurally related (oxazolidinones) direct FXa-inhibitors including the recently approved agent rivaroxaban (0 - 2.0 µM), with the structurally distinct direct FXa-inhibitor DX 9065a (0 - 18 µM) and with the indirect inhibitor fondaparinux (0 - 0.6 µM). We tested modifications of PiCT regarding the source of FXa (bovine or human) and the incubation step (incubation before re-calcification = 2-step, no incubation =1-step), and compared results with inhibition of human or bovine FXa-activity.

Results

The bovine 2-step PiCT showed a paradoxical decrease with all direct FXa-inhibitors, this effect is surmounted only at high concentrations and is not seen with the bovine 1-step PiCT. The decrease in PiCT is not observed in antithrombin-depleted plasma. The humanized PiCT (1 or 2 step) showed a consistent prolongation under all direct inhibitors. Fondaparinux prolonged PiCT with either assay. The correlation between PiCT and corresponding FXa-activity was significant both for humanized 2-step PiCT or bovine 1 step PiCT (r2 = 0.80), but the 95% prediction interval was large and covered a span of 40% FXa-activity between one agent and another.

Conclusions

The customary bovine PiCT should only be used to monitor direct FXa-inhibitors when modified as 1-step procedure. PiCT is not suitable to assess similarity of FXa-inhibition when different agents are interchanged.     

Clinical correlates of word recognition memory in obsessive-compulsive disorder: an event-related potential study

Memory disturbances found in obsessive-compulsive disorder (OCD) may partially be related to dysfunction of cortico-subcortical circuits. However, it is still unknown how OCD symptomatology is related to memory processing. To explore this question, event-related potentials (ERPs) were recorded in a continuous word-recognition paradigm in OCD patients with either severe or moderate scores on the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) (group S and group M, n=8 each) and in normal healthy controls (n=16). Typically ERPs to repeated items are characterized by more positive waveforms beginning approximately 250 ms post-stimulus. This "old/new effect" has been shown to be relevant for memory processing. The early old/new effect (ca. 300-500 ms) with a frontal distribution is proposed to be a neural correlate of familiarity-based recognition. The late old/new effect (post-500 ms) is assumed to reflect conscious memory retrieval processes. The OCD group S showed a normal early old/new effect and a reduced late old/new effect compared with group M and the control group, but no difference was found between group M and the control group. Source analyses for the late old/new effect showed statistically reduced cerebral activation in the anterior cingulate for OCD group S in contrast to the control group. Additionally, the early old/new effect in OCD group S was negatively correlated with the Y-BOCS total scores, and the late old/new effect was negatively correlated with obsession sub-scores. The severely, not moderately, ill OCD patients showed an impaired conscious recollection of the word-to-be-remembered, which suggested an impairment of working memory capacity in these patients due to a dysfunction in the frontal and cingulate cortex.     

Differential amygdala activation to negative and positive emotional pictures during an indirect task

The role of the amygdala for the processing of valence and arousal is a matter of debate. Using event-related functional magnetic resonance imaging, we tested valence-specific amygdala effects during attentional distraction. Subjects attended to a matching task in the foreground of neutral pictures, and of negative and positive pictures matched for arousal. Negative pictures elicited stronger amygdala activation than neutral or positive pictures, suggesting valence-specific amygdala responses under attentional load.