Date: 2010-04-14
Source: BDS

A) Final conclusions of projects in which BDS CALUX technologies have been applied

Fast Advanced Cellular and Ecosystems Information Technologies (Face-It)

This project aimed to develop rapid methods to predict the toxicologcal and environmental impact of oil spills. To measure the impact to vertebrates rapid fish embryo and human cell bioassays have been established; a molecular DarT test and two CALUX reporter gene assays. These tests closely match with regulatory requirements and in their original form they are DIN accredited (DarT), accepted in international legislation (DR CALUX), and can be run under ISO17025 and related international quality standards. 

The cyp1A (zebrafish)/1A1(human) gene was identified as key responsive element in the toxicity chain of oil exposed higher vertebrates. This gene is responsive to activation of the dioxin receptor and our results suggest that its activation is mainly due to the carcinogenic polycyclic aromatic hydrocarbon fraction in oil. To measure this fraction a simple PAH CALUX test was designed. In addition, a more broad CALUX screening method for oil-derived carcinogens (the p53 CALUX assay) was designed and is currently being validated. We were able to measure the major biologically active fraction of PAHs after a simple extraction procedure in experimental samples including a series from an experimental oil spill at full sea.

In human cells and particularly in zebrafish additional target genes were identified that may be related to additional effects of oil exposure such as teratogenicity. Our results indicate that some of these genes represent markers of activation of pathways independent of  dioxin receptor activation, and could form the basis for additional assay development.


B) Newly approved projects applying BDS CALUX technologies

Chemical Substance In Vitro/In Silico Screening System To Predict Human And Ecotoxicological Effects (ChemScreen)

The current system of risk assessment of chemicals is complex, very resource-intensive and extremely time-consuming. Because of this, there is a great need to modernize this process. However, this is not feasible without alternative, integrated testing strategies in which chemical characteristics are used to more advantage and where costly and time consuming animal tests are replaced to a large extent by more rapid, cheap and ethically less controversial methods. This is particularly needed for reproductive toxicity testing of chemicals. Reproductive toxicity is important to assess both human and environmental toxicity and uses the most animals in toxicity testing. Unfortunately, there are very few alternative methods. The EU project ChemScreen is a partnership between 9 European institutes and companies from 5 different countries. The project is part of a transatlantic collaboration with the US EPA. BDS will be the coordinator of the FP7 project. It aims to generate alternative methods and place the tests in a more general innovative animal-free testing strategy. For this, we will generate a simple rapid screening system, which aims at widespread implementation within the tight time schedule of the REACH program. It will be a flexible tool that can be adapted and used for applications beyond the scope of REACH and in the post-REACH period. It will use in silico methods for prescreening chemicals for all relevant toxic effects. When found positive, this will be followed by further in silico and in vitro tests (including a panel of CALUX assays), most of which are available already. To fill the gap of suitable alternative methods for reproductive toxicity testing we will use a novel high throughput approach combining in silico/in vitro methods. In this approach we will combine knowledge of critical processes affected by reproductive toxicants with knowledge on the mechanistic basis of such effects. Straightforward data interpretation and decision trees will be developed in which all information on the potential toxicity of a chemical is considered. In this way we will provide a cost-effective means to generate a basic set of data on toxicological properties of chemicals and a decision tool to assess if further testing of chemicals is required.


C) Dutch project alternative non-animal genotoxicity testing “Dierproeven begrensd”

Validation and implementation of rapid and cost-effective human in vitro screening assay to predict genotoxicity and carcinogenicity of chemicals and pharmaceuticals

A paradigm shift is needed to improve toxicity testing of chemicals and pharmaceuticals. Key to this change is the use of more defined, mechanism-based in vitro methods that have a great potential to reduce and replace animal experiments. The current system of risk assessment of chemicals is complex, resource-intensive and time-consuming, and besides extensive animal experimentation the attrition rate of novel pharmaceuticals is still very high due to toxicity that is discovered in late development. Furthermore, the REACH legislation aims to evaluate the safety of at least 30.000 chemicals. If toxicity testing is not quickly adapted this will lead to the use of a large number of animals. This provides a strong incentive to replace animal studies by more rapid, cheap and ethically less controversial methods. At the laboratories of BDS and Schering Plough  recently promising human cell-based assays for high throughput toxicity testing of compounds were developed, with promising perspectives to classify genotoxic and non-genotoxic carcinogens. A lot of animals are used to evaluate the carcinogenic potential of compounds. Furthermore the current regulatory in vitro mutagenicity assays induces many ´false positive´ results leading to unnecessary in vivo testing. The proposed high throughput reporter gene assays already produced very promising results in comparison with more classical genotoxicity assays.

This project aims to (pre)validate and implement these predictive tests through the following steps:

1.Optimization and standardization of the procedure and materials to start prevalidation of the novel promising HT tests. Robustness and transferability of the methods will be demonstrated. The reporter gene assays will be transferred to an independent laboratory at Research Centre of Utrecht University of Applied Sciences (Hogeschool Utrecht)

2. Selection of compounds, based on ToxCast database and completion of the pre-evaluation of a range of human high throughput tests for genotoxicity/carcinogenicity testing. Compounds include: non-carcinogens, true genotoxic carcinogens, false positive genotoxins and non-genotoxic carcinogens.

3. Determine strengths/weaknesses, in terms of sensitivity, specificity and predictivity, of various tests and select a minimal panel for a comprehensive high throughput approach.

4. Introduction of the individual tests and/or a panel into the regulatory circuit. The project team is in close contact with regulatory bodies and furthermore TNO is involved as advisor and will implement these assays to industry.

5. Set up a structure for global implementation using existing training and dissemination expertise. This will be done in parallel with a recent FP7 initiative ChemScreen, using global distribution and training networks.