Work Programme

WP1: Project management

 

The WP1 is devoted to the management of the project. The objectives are a) ensuring a high standard of supervision, quality control and overall coordination of research tasks described in WPs 2-9; b) constant supervision of all partner-related issues, anticipating any major problems in advance; c) providing sound administrative and financial management coordination based on accepted rules and FP7 guidelines; d) setting up a framework for communication and managing communication flow among partners and with PHARMAS project; e) interacting with the PHARMAS project selected from the same call (FP7-ENV-2010) within the framework of the "PharmaCluster" cluster to ensure good coordination and synergy of the activities of the two projects

 

 

WP2: Quantitative chemical analysis of cytostatic pharmaceutical residues in aqueous samples

The objectives of WP2 are to develop and validate analytical method for determination of cytostatic compounds in aqueous samples, and to assess the occurrence of cytostatic pharmaceuticals in wastewaters and in aquatic environment.

 

Within this WP analytical procedures for the determination of cytostatics will be developed, validated and applied to environmental samples . The key compounds to be studied within the frame of the project will be determined in WP8 .

 

 

WP3: Transformation of cytostatic pharmaceuticals in water treatment and in the environment

 

The objectives of WP3 are to identify transformation products (TPs) of cytostatic pharmaceuticals formed during water treatment and in the environment; to study the cycling of cytostatic compounds including stable TP formation in the waste water treatment and in the environment, and to chemicaly and toxicologicaly characterize the selected environmental samples.

 

The degradation processes of key compounds (selected in WP8) will be simulated on laboratory level and the formation of stable transformation products will be followed. After the identification  and adaptation of analytical methods for TP determination, their presence will be assessed in the environment. Finally, toxicological characterisation will be performed.

 

 

WP4: Effects of cytostatics in plant and invertebrate experimental models

 

The main objective of WP4 is to provide ecotoxicity data by testing the most relevant cytostatics (selected within WP8) for acute and chronic toxicity in standard ecotoxicity tests that cover organisms that represent primary producers (algae and plants) and primary consumers invertebrate (water flea Daphnia sp and mussel Unio sp).

 

The toxicity for algae  will be determined my growth inhibition according to the OECD Guideline 201assay using Pseudokirchneriella subcapitata and cyanobacteria indicator species. The multiple adverse effects in higher plants  will be determined by measuring growth retardation, and micronuclei induction in Tradescantia pollen and root tips of Allium cepa, and by pollen abortion assay in wildelife plants. The acute and long-term effects in crustacea will be determined in two species D. magnaand C. dubia according to established standards and guidelines.  To assess whether reduced growth rate in long-term exposure is associated with the disturbance of DNA integrity the effect of exposure to cytostatics on DNA integrity will be measured by means comet assay. The genotoxic effects of cytostatics in mussel Unio sp.  will be performed to explore the sensitivity mussel to cytostatics and to evaluate their potential for the use as the biosensors for exposure to genotoxic contaminants including cytostatics.

 

From the data of these experiments the LC50, EC50, LOEC and NOEC values for the tested  cytostatics will be calculated. The values of these parameters will then be used for the risk assessment in WP8. In addition these patrameters be will used to compare the effects of degradation products and real samples (obtained in WP3), and mixtures (obtained in WP8). The obtained data will be shared also with PHARMAS project to include them into their risk assessment modelling.

 

 

WP5: Long-term effects of cytostatics in a vertebrate experimental model

 

The aim of WP5 is to explore the short- and long-term effects of cytostatics (selected in WP8;) in a vertebrate, the zebrafish (Danio rerio), to generate data (LC50, EC50, NOEC) required for environmental risk assessment (required in WP8) and to determine molecular biomarkers of exposure to cytostatics (in association with WP7). The acute toxicity of cytostatics will be initially determined in adult zebrafish according to the OECD guideline 203 and in zebrafish embryos (FET test) according to the OECD draft guideline (May 30, 2006). The responses in the two tests will be compared and if the results enable, later on the fish embryo toxicity (FET) will be used for establishing acute toxicity cytostatics, their TPs, complex samples and mixtures, because it is considered as an in vitro assay and is in better agreement with the directives of experimental animal protection.

 

The long-term toxic effects of cytostatics will be studied using the standardized Fish Early-life Stage Toxicity Test (OECD 210) and the Two Generation Toxicity Test, which has been currently not yet standardized for fish. The latter test is particularly relevant as it will provide information concerning the effects of a test substance on the integrity and performance of the male and female reproductive systems, information about the effects of the test substance on neonatal morbidity and mortality, and preliminary data on prenatal and postnatal developmental toxicity.

 

In the FET test and the two generation toxicity test lethal and sublethal effects, genotoxic effects (comet assay in liver and MN in peripheral blood; performed by IMI), as well as changes in gene expression in liver  will be studied at different developmental stages during the exposure.

 

The study design will enable the collection of both mechanism-specific toxicity data and information concerning fecundity and viability. The former is useful for the diagnosis and/or extrapolation across species (WP6 and 7), while the latter is essential for population modelling in risk assessment scenarios (WP8). The obtained data will shared with PHARMAS to include them into risk assessment modelling.

 

 

WP6: Comparative studies of the responses to exposure to cytostics in in vitro fish and human models

 

The objectives of WP6 are to evaluate the sensitivity of an in vitro fish cell model (fish liver cell line ZFL) to predict in vivo long-term sublethal effects of cytostatic drugs and to evaluate differences in responses of fish liver cell models, human hepatoma HepG2 cells and human peripheral lymphocytes (HPL) to selected cytostatics in order to evaluate the predictive potential of fish experimental models for human exposure. The effects of cytostatics on different model cell lines will be assessed at the level of cyto-and genotoxic responses  and at the level of alterations of gene expression. Based on predictive potential of zebra fish liver (ZFL) cells for long-term in vivoeffects and the differences in responses between ZFL cells and human cell lines, attempts will be made to evaluate the predictive potential of zebrafish experimental models for long-term effects of  human exposure.

 

 

WP7: Relationship between gene expression patterns and toxic effects

 

The objective of WP7 is to investigate the relationship between transcriptome alterations and observed toxiological effects (i.e., reproductive effects and genotoxicity biomarker responses) in order to:  a) identify predictive transcript signatures caused by exposure to low concentrations of cytostatic drugs in zebrafish; b) identify predictive markers for reproductive and genotoxic effects on embryo stage of zebrafish; and c) determine the differences in transcript signatures induced by exposure to low doses of cytostatic pharmaceuticals in human vs. fish in vitro experimental models.

Ecotoxicogenomic tools will be used to assay molecular responses in reaction to selected  cytostatics that give indications  on a) toxic effects experienced by the organism; and b) compensatory mechanisms the organism has mobilized in its defence.

 

The particular pattern of responses can give indications  on  the mechanism of action (MoA) of the pollutant and attempts will be made to correlate the effects observed on the transcriptional level with those observed on the phenotype level.

 

In the first step predictive transcriptomic signatures of the exposure to cytostatic drugs will be determined at four life stages: embryo, larvae, juvenile fish, and adult fish. The phenotypic effect that will be determined in WP5 will allow phenotypic anchoring of transcription signatures. A database of the predictive signatures will be set up for data sharing between partners and for investigation of further pollutants.

 

In the next step transcriptomic markers will be identified and validated that will allow for faster and more sensitive detection of potential long-term effects of cytostatics. A few gene expression markers for qPCR detection will be selected from the pool of differentially expressed genes and by predictive analysis and data mining approaches. Preferably, markers that predict genotoxic and reproductive effects on the embryo stage will be selected. Selected markers will then be validated using exposure experiments of zebrafish embryos to other cytostatics with same or different MoA.  Attempts will be made to define a threshold concentration at which no change in marker genes are detected – NOTEL a parameter that will be included in the risk assessment.

 

To find out if transcript changes observed in vitro in ZFL cells can predict damage on the organ level, the transcription patterns obtained in ZFL cells will be compared to those obtained in in vivo experiments with zebrafish. Data analysis will focus on comparisons of transcript signatures in the organism (fish liver) and cell cultures.

 

Finally, the differences at the transcription level in responses to cytostatic drugs between different cell lines: fish ZFL and human HepG2  and HPL  will be evaluated by comparing the expression of markers, identified in Task 7.2. The suitability of selected gene expression markers to predict genotoxic effects in human cells will be evaluated by correlation analysis between DNA damaging effects and transcription patterns

 

 

WP8: Complex mixtures and risk assessment

 

The aims of WP8 are to: a) analyse cytostatics consumption data and select model compounds to be included in the studies ; b) explore possible synergistic and antagonistic effects of cytostatic drugs in mixtures and their relative contribution to observed toxicity of real environmental samples ; and c)  utilize the data generated by and available to the project to perform environmental risk assessment for representative cytostatic drugs and mixtures.

 

The cytostatic that will be included in chemical-analytical  and toxicological studies will be selected according to (i) consumption data of cytostatics on European level including the type and amount of compounds used, (ii)  following a literature study of potential chemical analysis that can be performed given current instrumental capabilities and (iii) taking into account different MoAs.

 

Based on the data on the most commonly used cytostatic cocktails used in clinical applications and based on their consumption and prediction or measurements of most common combinations occurring in the hospital wastewater (data from WP2 and WP3), defined mixtures of cytostatics will be prepared. The mixtures will be tested for toxicity, genotoxicity and changes in gene expression profiles that will be compared to the changes in profiles induced by single compounds to explore the possibility to predict the synergistic or antagonistic effect of the mixtures based on gene expression profiles.

 

The same approach will be applied to explore potential  synergistic and antagonistic effects of cytostatic drugs in combination with disinfectants (i.e., quarternary ammonium compounds) that are commonly found in hospital wastewaters together with cytostatics.The results  will contribute to better prediction of the environmental risks of complex waste containing cytostatics..

 

The environmental risk assessment  will be performed based on predicted environmental concentration (PEC) of the selected cytostatics according to the procedures described in the EMEA guideline  on the environmental risk assessment of medicinal products for human use (http://www.emea.eu.int) and compared to the measured environmental concentrations (MEC) (data from WP2 and WP3), to evaluate to what extent and into which direction (over- or under-estimation) predicted and measured values differ.

Based on the principles of the environmental risk assessment for single pharmaceuticals, models for risk the assessment of mixtures will be developed in which data from the project will be used to establish predicted no-effect concentrations (PNECs) for mixtures .

 

This workpackage will be linked also to the research performed within PHARMAS. PHARMAS will address the difficult issue of mixtures in WP3 (Prediction and assessment of mixture toxicology and ecotoxicology of antibiotics and anticancer drug) and plans to study the combined effect predominantly in complex periphyton community, while CYTOTHREAT  intends to study combined effects in ecotoxicological experimental models including genotoxicity endpoints. PHARMAS also intends to optimize and validate modelling approaches for prediction of hazards of mixtures for which enough experimental data is crucial. This clearly indicates complemetaraity of the two projects within this area.

 

 

WP9: Dissemination

 

The goals of WP9 devoted to dissemination of the project results are to a)  create the critical mass of interest and   raise awareness among the relevant stakeholders and policy makers necessary for the exploitation  of the results of the present project related to the effect of cytostatic drugs released into environment and the potential threat that they present to aquatic biota and human health; b)  share the results of the project with the academic and research community interested in the topics addressed by the project and  stimulate further research in the field covered by the present project; and c)  disseminate information on the planned and ongoing activities in the project through various media. The dissemination actions are planned in line with the dissemination actions of PHARMAS project in order to address wider spectrum of stakeholders and to disseminate the findings and outcomes of both projects that are highly complementary. The goals will be achieved through preliminary dissemination actions  that include the selection of common and individual project logos; the setup of a common webpage with the sections of individual projects; press releases; the development of promotional and informational materials such as brochures, leaflets, articles in mass media; scientific dissemination by means of  scientific papers, conference presentations; dissemination of relevant results to specific public and stakeholders such as policy makers; dissemination events by means of conferences and workshops  as well as through continuous public relation actions .

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