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CHEMICAL SUBSTANCE IN VITRO/IN SILICO SCREENING SYSTEM TO PREDICT HUMAN- AND ECOTOXICOLOGICAL EFFECTS

Final Report Summary - CHEMSCREEN (CHEMICAL SUBSTANCE IN VITRO/IN SILICO SCREENING SYSTEM TO PREDICT HUMAN- AND ECOTOXICOLOGICAL EFFECTS)



Executive Summary:

The complexity of the system of risk assessment of chemicals has led to huge delays. Because of this, the toxicological properties of most industrial chemicals that are in common use are largely unknown. New legislation such as REACH aims to end this unacceptable situation by modernizing and streamlining chemical risk assessment. However, this approach is unlikely to be successful without incorporating 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 rapid and cost-effective alternative testing 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 ChemScreen project aimed to fill this gap and identify and when needed generate to be used in an innovative animal free testing strategy. In our approach we combined knowledge on critical processes affected by reproductive toxicants with knowledge on the mechanistic basis of such effects. We now have generated a simple rapid screening system, aiming at widespread implementation within the tight time schedule of the REACH program. It is a flexible tool that can be adapted and used for applications beyond the scope of REACH and in the post-REACH period. It uses in silico methods for prescreening chemicals for all relevant toxic effects. These test results determine the route of further in silico and in vitro testing. For instance, when genotoxic or carcinogenic properties are found, testing will concentrate on these routes of toxicity. For those chemicals that need testing for reproductive toxicity we have set up a high throughput screening CALUX assay panel that can efficiently identify endocrine active compounds (with 100% accuracy), and compounds leading to sex organ deformities (with 87% accuracy). In combination with EST test results more than 90% accuracy was reached to detect reproductive toxicants. Alternatively, in combination with existing repeated dose study data potential reproductive and developmental toxicants could be identified ahead of in vivo studies, allowing prioritizations of animal testing.

Exposure assessment is critical in risk assessment of chemicals. Several modules for rapid exposure predictions via diverse exposure routes have been set up by TNO and P&G. The resulting in vivo concentration estimates were shown to greatly improve predictivity of the in vitro tests. As a further step, we have developed methods for integrated testing of chemicals using all available data, complemented with in vitro screening data, and reference data for so-called read-across purposes using a range of databases.

We are actively engaged in promoting regulatory acceptance of the tools developed since that is an essential step towards practical application of the test battery developed. With this, a significant saving in animal use and associated costs seems very feasible.

In conclusion, all elements of the ChemScreen integrated high throughput testing strategy have been set up and are in an advanced state of integration and effective dissemination routes, including training and support facilities are available through the coordinator’s office.

Project Context and Objectives:

The complexity of the system of risk assessment of chemicals has led to huge delays. Because of this, the toxicological properties of most industrial chemicals that are in common use are largely unknown. New legislation such as REACH aims to end this unacceptable situation by modernizing and streamlining chemical risk assessment. However, this approach is unlikely to be successful without incorporating 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 rapid and cost-effective alternative testing 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. We aimed to fill this gap and select suitable tests and place them in a more general innovative animal free testing strategy. For this, we have been generating a simple rapid screening system, aiming at widespread implementation within the tight time schedule of the REACH program. It is a flexible tool that can be adapted and used for applications beyond the scope of REACH and in the post-REACH period. It uses in silico methods for prescreening chemicals for all relevant toxic effects. When found positive, this is followed by further in silico and in vitro tests. To fill the gap of suitable alternative methods for reproductive toxicity testing we use a novel high throughput approach combining in silico/in vitro methods. In this approach we aimed to combine knowledge of critical processes affected by reproductive toxicants with knowledge on the mechanistic basis of such effects. Data interpretation and decision trees were developed in which all information on the potential toxicity of a chemical is considered. In this way we 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 or can be waived.

ChemScreen focuses on delivering a practical approach how to use molecular screening tools for reproductive toxicants in the context of chemical risk assessment in REACH and related programs. The approach includes integrated testing strategies, molecular screening tools combined with more apical tests and various in silico methods, including QSAR. To this end the following objectives were formulated, corresponding to the ChemScreen work packages (WPs):

Objectives (see Fig 1):

1. Establish in silico prescreening methods prioritizing in vitro toxicity testing; WP1, leading partner: Danish Technical University Food Institute (DTU; see fig 1)
2. Establish a database and an in silico prescreen to identify potential reproductive toxicants; WP2, leading partner: Fraunhofer Institute for Toxicology and Experimental Medicine (FhG)
3. Establishment of sensitive parameters and a medium throughput ‘minimal essential’ in vitro assay panel; WP3, leading partner: National Institute for Public Health and the Environment (RIVM)
4. Establish a high throughput mechanistic pathway screen, ReproScreen HTP, for reproductive toxicants; WP4, leading partner: Eberhard Karls University of Tübingen (EKUT)
5. Integrative methods to predict in vivo reprotoxicity for both human- and environmental toxicity allowing informed decisions on eventual further testing; WP5, Leading partner: Netherlands Organization for Applied Scientific Research (TNO)
6. Integration into one user-friendly tool, including uncertainty assessment; WP6, Procter & Gamble Eurocor (P&GEN)
7. Efficient dissemination to facilitate widespread implementation; WP7, leading partner: BioDetection Systems (BDS)

The ChemScreen project started at January 1, 2010. The program aimed to collaboratively generate an innovative testing strategy combining unique expertise of the participants. To attain this level of interaction, frequent meetings, and workshop have been planned, as well as a high profile Scientific Advisory Board (see table 2) to help guide this process. In the international advisory board major stakeholders (JRC/ECVAM, OECD, US EPA, Industry, ECETOC) are represented. Beginning 2010 the ChemScreen project has entered an important transatlantic collaboration with the US EPA National Center for Computational Toxicology (NCCT), and the Texas Indiana Virtual Star Centre (TIVSC). Agreements on scientific collaboration, data- and chemical sharing have been established

Project Results:

WP1 concentrated on the establishment and selection of in silico prescreening methods to categorize chemicals, mainly using (quantitative) structure-activity relationships (Q)SARs. This was done for major classes of toxicity prioritized in REACH (carcinogenesis, mutagenesis and reproductive (CMR) toxicity and persistent, (very) bioaccumulative toxic (PBT/vPvB) compounds). As a starting point DTU scientists have been using a unique database which comprises abbreviated predictions from more than 70 (Q)SAR models on endpoints for physico-chemical properties, fate, eco-toxicity, absorption, metabolism and toxicity. Positive predictions for carcinogenicity, mutagenicity and reproductive toxicity can be applied to avoid unnecessary animal testing of compounds as replacement for animal tests for these endpoints. As no testing for reproductive effects should be performed in REACH on known genotoxic carcinogens or germ cell mutagens with appropriate risk management measures implemented, as stated in the REACH annexes VIII-X, predictions for genotoxic carcinogens and germ cell mutagens can furthermore be applied to avoid testing for reproductive toxicity. A developmental toxicity screen was carried out as well, but predictivity of this analysis expectedly is rather low. To make the (Q)SAR screening as comprehensive as possible it should be based on as many as possible of the 143,835 chemicals, which were pre-registered (PRS) between 1 June and 1 December 2008. Therefore the work was carried out in collaboration with the Computational Toxicology Group within the Joint Research Centre (JRC), that has generated structure information for 80,413 PRS chemicals, of which 70,983 chemicals were suitable for generation of QSAR predictions for all relevant endpoints As part of the in vitro pre-screen, it was envisaged to develop an exposure module to identify chemicals that will have no or a negligible systemic availability (and anticipating that gonads and embryos will not be exposed) after exposure via different routes. To that effect, literature searches have been performed to investigate on one hand whether specific chemical properties can be identified to predict internal exposure after exposure via the oral, respiratory and dermal routes, and on the other hand which (publicly available) in silico tools exist leading to reliable prediction of negligible internal exposure. Based on the current knowledge, no cut-offs based on physicochemical properties could be identified for the three main exposure routes below or above which internal exposure is zero or negligible. REACH does provide physicochemical criteria to waive toxicity testing via the respiratory route, but these criteria cannot waive testing via other routes of exposure. Therefore, it was concluded that inclusion of these criteria in a module to predict internal exposure aimed at excluding reproductive toxicity testing based on physicochemical criteria is not a viable option. Instead, the PK models will now be used exclusively in WP5.

Main conclusions:

• A priori exposure based waiving was found not to be a viable option
• In silico pre-screening (Q)SAR tools have been developed for human health effects including predictions for genotoxic carcinogenicity, in vivo mutagenicity, limited developmental toxicity and in vitro estrogenic and antiandrogenic activity.
• These tools have been applied to over 70,000 REACH pre-registration chemicals and were summarized in a searchable spreadsheet form.

WP2 focused on expansion of databases which contain information relevant to reproductive toxicity; RepDose (repeated dose) and FeDTex (fertility and developmental toxicity). First reproductive toxicity studies were identified from peer reviewed publications, as well as other databases provided by other partners. Available studies on reprotoxicity were selected for data entry. In particular, the amount of chemicals in FeDTex were more than doubled in the course of ChemScreen, mounting up to over 300. The expanded databases were used to identify redundant data coming from animal studies and thus possibilities to reduce testing. Evaluating the responsiveness of the different generations, a comparison of the dose levels of NOELs was performed. These data suggest a tendency of F0 and F1 being mostly equal sensitive in one- and multi-generational studies, whereas F2 seems to be less indicative for NOEL derivation.

A significant overlap with RepDose was created to allow comparison of in vivo data with the same chemical and evaluate possible predictivity of repeated dose toxicity for reproductive toxicity. Target organs and effects of repeated dose toxicity are suitable to inform on possibly occurring effects in reprotoxicity of chemicals. Whether and how minimal effect concentrations of repeated dose toxicity can be extrapolated to reprotoxicity on the chemical class of the compounds studied. For example with chlorinated phenols, including phenol as parental substance, in none of the chlorinated compounds, repeated dose studies displayed a direct indication for repro-toxic effects. The major effects observed after repeated exposure are body weight loss related to reduced availability of energy and increased catabolism as well as changes in liver weight and pathology. The reproductive toxicity observed might also be related to insufficient energy supply due to uncoupled oxidative phosphorylation resulting in growth retardation or even increased prenatal mortality. The mechanism of toxicity is thus likely to be the same in repeated dose as well as reproductive toxicity but comparison of in vivo study results without further knowledge on the mode of action of the substances will not reveal any predictivity of the repeated dose toxicity study. To enhance this predictivity further knowledge on the specific mechanism causing the adverse effects after repeated exposure and its implication in gestation and offspring development is needed. For Alcoholic solvents, phthalates and glycol ethers comparable conclusions can be drawn.

The correlation of estrogen and androgen receptor activity, the interplay of conversion enzymes of sex steroids and pathways of their disturbance are correlated to structural categories and correlations between in vitro and in vivo results of the on-going research are drawn. In a further approach, all sex organ related effects, retrieved from the Fraunhofer ITEM database FeDTex on reproductive toxicity, were correlated to the chemical structures causing them and structural alerts are addressed. Structural alerts were identified and summarized, resulting in 32 structural alert structures. Additional sub-analyses were performed for 3 sub-categories, and explained certain inconsistencies in the structure-activity relations.

Main conclusions:

• Evaluating the NOELs of the different generations, suggest a tendency of F0 and F1 being mostly equal sensitive in one- and multi-generational studies, whereas F2 seems to be less indicative for NOEL derivation.
• Target organs and effects of repeated dose toxicity are suitable to inform on possibly occurring effects in reprotoxicity of chemicals. Chemical class formation and additional information on the mode of action of the chemical, allow a better extrapolation of observed effects.
• The correlation of estrogen and androgen receptor activity, the interplay of conversion enzymes of sex steroids and pathways of their disturbance were correlated to structural categories. This resulted in 32 structural alert structures.
• Additional sub-analyses were performed for 3 sub-categories, and explained certain inconsistencies in the structure-activity relations.

The aim of WP3 is the design of a ‘minimal essential’ reproductive toxicity screen, consisting of a number of in vitro assays that are representative of those parameters in reproductive toxicity that are crucial for reproductive hazard assessment. One way to simplify testing schemes is the approach of identifying critical endpoints for reproductive toxicity using literature and database searches, and focusing on development of tests for these endpoints. So far most endpoints identified so far are quite generic and non-specific in nature, which may mean that screening panels should also include quite simple, relatively non-specific tests (cytotoxicity) in addition to more mechanistic assays.

Meanwhile, progress was obtained at RIVM with the optimization and amendment to higher throughput of two tests that are very likely to be included in the final battery, namely a genomics-based improved embryonal stem cell test, and steroidogenesis assays, in particular four CYP17/19 assays. Two Ph.D students are working on enhancing the predictive value of these assays. In the embryonic stem cell test, transcriptomics readouts were employed to improve the mechanistic readout of the system and improve its predictive value. The CYP17/19 assays were being optimized and microsomes versus whole cell assays were being compared for their predictive value. The embryonic stem cell assay was applied to the category of phthalates and predictivity within this class of compounds on a comparative basis with existing transcriptomics patterns of other phthalates was shown. A ring trial among ChemScreen partners employing the battery was carried out, analysed as to predictability and kinetic extrapolation, and the publication on this work was published.

Main conclusions:

• A pragmatic medium/low-throughput ChemScreen battery of alternative developmental toxicity assays was defined.
• A ring trial among ChemScreen partners employing the battery was carried out, analysed as to predictability and kinetic extrapolation, and a second one is underway.
• Novel CYP17 and CYP19 assays were developed and a preliminary assessment of their performance was published.
• The transcriptomics-enhanced embryonic stem cell assay was applied to the category of phthalates and shown to be suitable for read-across purposes within this class of compounds.
• The embryonic stem cell assay was applied to the category of phthalates and predictivity within this class of compounds on a comparative basis with existing transcriptomics patterns of other phthalates was shown.

WP4 focused on the establishment of high throughput screening methods (ReproScreen HTP) predictive of reprotoxic potential, based on insight in molecular mechanisms that are relevant to reproductive toxicity. These assays comprise a panel of highly specific human CALUX® reporter gene assays at BDS, which includes about 50 different assays. A systematic approach has been taken developing the screening panel using very sensitive and selective reporter genes, covering a wide range of receptors and signaling pathways that potentially are involved in reproductive toxicity. The approach taken aims to provide several advantages. First of all, by providing a mechanistic base, regulatory acceptance is strongly facilitated. Secondly, this mechanistic base will provide input for decisions on further test requirements and risk assessment. Thirdly, cost, speed, robustness, and quantification is superior in these types of assays. In the fourth place, this facilitates predictions on both human- and ecotoxicological properties of chemicals, when different prediction models are used. One approach taken to develop additional reporter gene assays for ReproScreen is to use central intracellular pathways that are involved in regulating transcription in a mammalian cell. These pathways include besides nuclear hormone receptors also reporter gene assays for quite generic responses to toxicants, including cytotoxicity, apoptosis and genotoxicity. These responses can also be relevant in reproductive toxicity, however. A control cell line, called cytotox CALUX has been generated which constitutively expresses the luciferase gene.

In addition to these highly selective CALUX HTS reporter gene assays, have been established in more complex, differentiated cells such as murine ES cells by introducing reporter systems for signalling pathways controlling key differentiation pathways in embryonic development (so-called ReproGlo assays). Despite several attempts only the original ReProGlo (Wnt-D3) assay turned out to be successful. All CALUX and ReProGlo assays have now been automated in 384 or 96 wells format, respectively, and a series of reference compounds has been used to deselect less informative assays and identify missing endpoints. Data storage and analysis has been set up in collaboration with WP6 partners. More than 250 compounds have been screened in the CALUX panel and a subset in the ReProGlo assay.

The panel contains (pre)validated assays that are highly predictive to assess endocrine disruptive activity of compounds acting through the androgen- or estrogen- receptor.

It was shown that a single selective and extensively validated CALUX assay (ERalpha CALUX) can replace a battery of tests measuring the same endpoint.

Preliminary assessment of the application domain of the screening panel shows that it is very suitable to identify not only estrogenic and androgenic endocrine disrupting compounds (100% accuracy), but also can predict chemically induced sex organ deformations with 83% accuracy. This screening panel can be used with in combination with more apical tests, such as the EST test (i.e. components of the minimal essential screen identified in D3.3) efficiently predicting reproductive toxicity of chemicals

Main conclusions:

• A significant expansion of the CALUX battery of highly selective human cell based assays has been attained, with an almost doubling of the battery relative to ChemScreen’s start.
• Efficient automation of a CALUX assay panel has been attained allowing rapid screening of reproductive toxicants. A large amount of chemicals has been screened in all CALUX assays and the ReProGlo assay
• A selection of the most relevant assays has been made to be included in a smaller ReProScreen HTP screening panel for reproductive toxicants.
• The panel contains (pre)validated assays that are highly predictive to assess endocrine disruptive activity of compounds acting through the androgen- or estrogen- receptor. It was shown that a single selective and extensively validated CALUX assay (ERalpha CALUX) can replace a battery of tests measuring the same endpoint.
• Preliminary assessment of the application domain of the screening panel shows that it is very suitable to identify not only estrogenic and androgenic endocrine disrupting compounds (100% accuracy), but also can predict chemically induced sex organ deformations with 83% accuracy.
• Currently this screening panel can be used with in combination with more apical tests, such as the EST test, efficiently predicting reproductive toxicity of chemicals.

WP5 has been establishing integrative methods to predict in vivo reprotoxicity for both human- and environmental toxicity using in vitro benchmark (threshold) concentrations as a starting point. Prediction of the correct in vivo dose level at which adverse effects can be expected is one of the key issues. High throughput pharmacokinetic models are being set up describing the bioavailability via relevant routes (oral, dermal, inhalation) and the pharmacokinetics of the most relevant reference chemical classes.

A rapid PBPK modeling strategy has been developed to translate in vitro concentrations to in vivo dose levels. Instead of building custom models for each chemical (class) as planned, it was chosen to develop one generic PBPK model framework describing all relevant compartments, physiological processes and exposure routes. The generic model with its (chemical-independent) anatomical and physiological parameter values in principle applies to all chemicals. The PK of specific compounds has been addressed by providing chemical-specific parameters (relating to permeability, partitioning and clearance) that can be obtained by either in silico predictions or routine in vitro measurements, as input to the generic model. To this effect, an algorithm has been implemented to predict the binding and partitioning behaviour of compounds in water, protein and lipid phases of blood (plasma and red blood cells) and tissues (interstitial fluid and cells). This algorithm takes as input readily obtainable chemical properties. It can also be used to predict binding and partitioning behaviour in culture medium if its pH and protein and lipid fractions are known, and thus to correct for differences in unbound concentrations between in vitro and in vivo. This generic framework is more compatible with the rapid screening character of the ChemScreen tool as a whole, and allows PK prediction of a much larger number of compounds in a relatively short time. Till now, the collection of chemical-specific parameter values and the comparison of model simulations with in vivo PK data has been performed for the first list of test compounds. In addition, in vitro-in vivo extrapolation of the available data so far, determined by other partners of the ChemScreen project, has been performed.

In risk assessment, methods determining defined conserved mechanisms of toxicity have a great advantage over more classical black-box approaches, such as morphological endpoints. This is because interpretation of data is much more straightforward. By focusing on molecular mechanisms conserved in various species (e.g. various receptor-mediated processes) possibilities are explored to predict not only for humans but also for aquatic organisms (particularly fish) and thus for ecotoxicological effects. For this extrapolation methods have been designed to establish if there are conserved molecular mechanisms in aquatic organisms responsive to the action of different chemicals. As one of the first steps an expansion and restructuring of the large EDUKON ecotox database (at UKON) has been carried out as well as an inventory of possible chemo-biological interactions that can be related to distinct molecular mechanisms. All relevant technical prerequisites to screen and to use the EDUKON DB as intended within the project description were fulfilled, and the integration of approximately 1,400 new data sets of peer-reviewed studies into the DB was accomplished in accordance with the intention to integrate the newest scientific findings into the DB and to set it to the current state of science. The heterogeneity of the data involving many species and effects together with the complexities of reproductive toxicity, however, hampered the generation of firm conclusions on the applicability of mechanistic insights in environmental risk assessment.

Recently, this workpackage has designed a state-of-the-art draft REACH Integrated Testing Strategy (ITS) for reproductive toxicity including the novel ChemScreen modules. This represents a major step towards the final aim of the ChemScreen project.

Main conclusions:

• A high throughput PBPK modelling strategy to translate in vitro concentrations to in vivo dose levels. A workflow is established that allows comparison in vitro effect data with effective in vivo effective dose levels, such as those in contained in RepDose and FeDTex databases.
• First explorations of the developed in vitro / HTS battery appear promising: both for substances that have structural analogues, and for those that have not. ChemScreen has created a framework which shows success on a case-by-case basis.
• The battery approach provides mechanistic insights greater than the sum of the individual assays.
• The current database and assay battery provides an excellent foundation for further refinements and for exploring the suitability of broader chemical domains.
• Methods to assess the environmental bioavailability of the ChemScreen compounds in biota have been established.
• All relevant technical prerequisites to screen and to use the EDUKON DB as intended within the project description were fulfilled, and the integration of approximately 1,400 new data sets of peer-reviewed studies into the DB was accomplished in accordance with the intention to integrate the newest scientific findings into the DB and to set it to the current state of science. The heterogeneity of the data involving many species and effects together with the complexities of reproductive toxicity, however, hampered the generation of firm conclusions on the applicability of mechanistic insights in environmental risk assessment.
• A model structure for the calculation or prediction of compounds effect on individuals and populations was created.

WP6 was devoted towards the setup of software tools that integrate the methods, modules and databases generated. It has produced a document with a complete description of the external interfaces of the tool with other software components and databases. Because of the need of a practical tool to store and process the large amount of high throughput data this part of the tool was completed first by Simpple. The goal of the tool is to provide the features required to store, manage and quickly analyze assay data generated in high throughput screening (HTP) bioassays allowing access to primary data. This tool was successfully constructed and installed and all screening data have been uploaded at BDS. Next steps will include coupling to reference datasets facilitating its use in read across and integrated testing strategies.

In addition, a methodology for in vitro - dermal in vivo extrapolation was developed. The method enables the calculation of an external exposure dose leading to systemic toxicity by combining in vitro experimental data relevant to the toxicity affected pathways with in silico predictions of skin permeation and whole-body ADME. The method is in line with the current development of mechanistic methods used to predict in vivo toxicity from sets of reference data, which may combine data from in vivo experiments, in vitro cell-based assays and/or computational results. Due to incomplete understanding of the AOPs and data limitations determination of the representative concentration in vitro will need to most likely rely on statistical approach of choosing the lowest value of EC50 distribution. The use of the internal systemic concentration external to estimate a safe dose has been compared to an approach based on in vivo dose (LOEL). Results suggest that LOELs may be too conservative for use in realistic in prediction of toxicity and risk assessment.

Main conclusions:

• The dependency of dermal safe dose on exposure scenario and application conditions has been shown.
• The method is in line with the current development of mechanistic methods used to predict in vivo toxicity from sets of reference data, which may combine data from in vivo experiments, in vitro cell-based assays and/or computational results.
• Due to incomplete understanding of the AOPs and data limitations determination of the representative concentration in vitro will rely on a statistical approach of choosing the lowest value of EC50 distribution.
• The final version of the software tool to support analysis and storage of HTP generated data has been developed. The goal of the tool is to provide the features required to store, manage and quickly analyze assay data generated in HTP bioassays.

Final ChemScreen Integrated Testing Battery

The combined efforts of the ChemScreen consortium as described in the workpackage descriptions have led to a final ChemScreen Integrated Testing Battery. A flexible in vitro/in silico screening technology that forms the heart of the ChemScreen technology which has been incorporated in flexible modules that can be used in integrated testing strategies:

Module 1. In silico prescreening module

This module includes in silico prescreening methods run at DTU to categorize chemicals into major classes of toxicity prioritized in REACH. Positive predictions for carcinogenicity, mutagenicity and reproductive toxicity can be applied to avoid unnecessary animal testing of compounds as replacement for animal tests for these endpoints, in particularly in integrated testing and read across.

Module 2. In vitro screening module

a. Integrated low/medium throughput models

Because of the complexity of the reproductive system and developmental processes the ChemScreen minimal essential screening panel contains zebrafish early life stage test or the EST test as integrative lower throughput models. These models have been extensively validated in the past and detailed OECD/ECVAM protocols are available.

b. High throughput models

BDS has set up a high throughput screening CALUX assay panel that can efficiently identify endocrine active compounds (with 100% accuracy), and compounds leading to sex organ deformities (with 87% accuracy). In addition, in combination with existing repeated dose study data potential reproductive and developmental toxicants could be identified ahead of in vivo studies, allowing prioritizations of animal testing.

These CALUX models have been extensively validated in the past and detailed OECD/ECVAM protocols have been set up and were submitted. EKUT has established the ReProGlo assay with partially complementary application domains. Both types of assays are running at BDS. Ample experience is available at BDS in training of external parties and world-wide dissemination of these assays. Simpple in collaboration with BDS has generated a ChemScreen software tool for bioanalytical data storage and analysis. This module is running at BDS when required Simpple can adapt this tool to the needs of individual clients.

Module 3. Exposure assessment

Several modules for exposure predictions via diverse exposure routes have been set up by TNO and P&G, and are available as a third party service via TNO.

Module 4. Integrative models and risk assessment

a. Integrated testing strategies and innovative risk assessment

TNO has been involved in setting up methods for integrated testing of chemicals using all available data, complemented with in vitro screening data. BDS is setting up methods for hazard- and risk assessment linked to high throughput bioanalytical data, based on OECD guidelines and linkage to adverse outcome pathways. FhG showed that a number of potential reproductive and developmental toxicants could be identified ahead of in vivo studies by using existing repeated dose study data, in vitro tests or structural alerts thus supporting prioritizations of animal testing.

b. databases

UKON, RIVM and FhG have been completing databases with reference (in vivo) data for reproductive toxicants. These databases are not readily accessible for outside users but processed data will be included in international databases, while remaining data remain available upon request directly to those partners. A more accessible version of these databases, allowing direct linkage to bioanalytical data, and integrated testing approaches is in preparation.

Potential Impact:

1. Potential Impact

The ChemScreen program particularly targeted Theme 6, Environment and Climate Change, Sub-activity 6.3.3. Technology assessment, verification and testing. More specifically, the proposal very clearly and precisely addressed the expected impact of ENV.2009.3.3.1.1 “Screening methods for assessing the toxicological and ecotoxicological properties of chemicals”, with the expected impact: “Possible use of results for regulatory purposes in the screening of industrial chemicals”. Not only was the main aim of our project the generation of a simple, rapid screening system for prioritized effects of concern, it also aimed at widespread implementation, preferably within the tight time schedule of the REACH program. It aimed to provide a cost-effective means to generate a basic set of data suitable for regulatory purposes on toxicological properties of chemicals and a decision tool to assess if further testing of chemicals is required or can be waived. Of course such a method will also be used outside the scope of REACH and in the post-REACH period. However, since the call text referred to REACH as one of the regulatory frameworks, we have specifically focused on the use within REACH, since this legislation now is implemented and expectedly will pose the tightest time schedule compared to any of the international regulatory frameworks. We argued that if we were successful in REACH, this will be a major incentive for an even broader and prolonged use of our novel tools. Because of our strong commitment to deliver widely applied tools tools that go far beyond “scientific methods”, we involved all major stakeholders including ECHA, JRC, OECD, CEFIC, ECETOC, Individual industries, CRO’s, etc. With this our proposal fitted almost exactly to the expected impacts of the work program and aimed to have a major impact in the use of alternative testing strategies in REACH and beyond.

In the 4 years that this project has been running it has reached much of its S&T aims and was able to keep in pace and actively collaborate with major international activities such as the NCCT ToxCast program. In addition, the ChemScreen project paid ample attention to reaching practical goals and dissemination activities specifically addressed European and global regulatory bodies and related institutes, including EURL-ECVAM, OECD (Molecular Screening Group, Validation Management Group on Non Animal Testing and National Coordinators of the Test Guidelines Program) and ECHA. The first two organisations have been approached successfully and ChemScreen scientists have actively worked together with EURL-ECVAM and OECD to promote regulatory acceptance of individual assays from the ChemScreen battery (as reported in the non-contractual deliverable D7.14) and have explored avenues how to validate the entire battery.

From the beginning of the ChemScreen program onwards, however, it has been difficult to obtain high quality contacts with regulatory toxicologists and other scientists at ECHA. In a way this could be expected since in general the work of regulatory toxicologists is quite strictly dictated by existing guidelines, i.e. the animal studies that ChemScreen wishes to reduce. The route via EURL-ECVAM/OECD to new guidelines unfortunately cannot be completed within the time frame of a EU project. This poses a generic problem to all project in this area. Because of the intention of the REACH legislation to use animal experiments only as a last resource, a more pro-active attitude of ECHA was expected. Fortunately, these contact now have been established, but only in the course of the program.

A more general problem with the ChemScreen approach of using molecular screening assays to predict toxicity in animals is related to the different types of results obtained. Trying to couple assay results to (morphological) endpoints in experimental animals cannot be readily done. In the first place, there may be multiple mechanisms leading to the same endpoint, while the same mechanism may be involved in different endpoint toxicities when time and tissue distribution is different. In addition, a single test only gives part of the puzzle and only batteries such as the ChemScreen battery can be effective in replacing animal studies. The validation of such a battery is complex and unprecedented. Because of the strong interest in the ChemScreen and related approaches from ToxCast, currently there are significant activities ongoing to improve these linkages of mechanistic in vitro tools to results in animal experiments, through the design of so-called Adverse Outcome Pathways. ChemScreen’s scientists have embarked on these new and important activities thought to be essential missing links in application of molecular screening tools.

In conclusion, we are actively engaged in promoting regulatory acceptance of the tools developed, and in due time we hope to reach a significant saving in animal use and associated costs.

2. Main dissemination activities

WP7 concentrated on the dissemination of the project’s results through various channels, including publications, folders, news items (see project’s website: www.chemscreen.eu) participation to and organisation of meetings with scientists and various stakeholders, etc. As one of the project’s first deliverables ChemScreen’s strategy has been further defined and published in the form of a position paper (Van der Burg et al., 2011). The second collaborative paper is being drafted and planned and will be included in the special issue of Reproductive Toxicology on the ChemScreen project and the EPA sister project of the Texas-Indiana Virtual STAR Centre (TIVSC).

In addition, ample attention is paid to validation and regulatory acceptance of the test methods developed in ChemScreen. To this end close contacts with relevant ECVAM and OECD working groups, scientists and regulators are being established. Novel science- and performance criteria-based methods will be employed to validate the ReproScreen system, and allow rapid acceptance. Methods aim to be robust and simple allowing widespread dissemination using ample expertise available at BDS to disseminate bioassays worldwide. Methods to screen for endocrine disrupting compounds, such as the ER- and AR CALUX assay already were prevalidated in the context of the ReProTect FP6 program, and subsequently have been submitted to ECVAM and OECD to allow formal validation, and regulatory acceptance. Since our battery of tests of reporter gene assays is very comparable to these tests, they may be used as “validation anchors” in a screening battery. Through close collaboration, synergy will be attained with ongoing framework projects, and the NCCT molecular screening programs. In order to prevalidate our screening battery and select relevant tests a feasibility study has been initiated early in the project using a set of reproductive toxicants that are run in all available tests.

As an important integrative effort to test the feasibility of using the ChemScreen approach to screen for reproductive toxicants, a ring trail has been initiated. RIVM has been involved in the selection of the first set of chemicals for the ChemScreen ring trial, based on available information about the toxicity profiles of chemicals. A selection of twelve chemicals was made with varying toxicity profiles including reproductive and developmental toxicants as well as endocrine active substances. The outcome of the ring trial, after data collection by BDS, has been published (Piersma et al., 2013). In addition, a follow-up ring trial is being analysed among ChemScreen partners as well as with the related US STAR project partners. The results will be published in a special issue of Reproductive Toxicology.

ChemScreen aimed to collaboratively generate an efficient innovative testing strategy combining unique expertise of the participants. To attain this level of interaction, frequent meetings, and workshops have been organized with the partners and the Scientific Advisory Board. Two very successful open symposia were organized aimed on molecular screening tools for reproductive toxicants. At this meeting results of ChemScreen, ToxCast and related molecular screening programs were presented by leading scientists and the possibilities to use such data in chemical risk assessment were discussed with a broad audience.

Main conclusions:

• A range of topical workshop have been organized (on planning of review papers, chemical selection, test selection, in silico tools, data interpretation, adverse outcome pathway construction).
• An international ChemScreen symposium has been organized twice, including and interactive workshop/panel discussion with stakeholders.
• Six general project meetings have been organized
• Twenty meetings of the executive board were held
• Two collaborative review papers have been published, one is in preparation.
• More than 25 publications have been produced, many of them already accepted in leading journals.
• A special issue on special issue of Reproductive Toxicology on the ChemScreen project and the EPA sister project of the Texas-Indiana Virtual STAR Centre (TIVSC) has been planned with approximately 7 ChemScreen papers and 3 TIVSC papers.
• A homepage including a news item section has been established and maintained and a flyer has been made.
• Annual reports have been disseminated to the general public through the AxlR8 project
• Significant progress has been made in promoting the project’s objectives within and outside the scientific-, industrial-, and regulatory community
• Significant steps towards validation of molecular screening batteries have been made and regulators were actively approached; the ERalpha- and AR-CALUX assays are well underway in the process of formal EUR-ECVAM/OECD validation, aiming at inclusion in OECD test guidelines.
• All methods developed in ChemScreen are available at the partner’s laboratories and commercial services are available or are being set-up. Coordinated access is being arranged through the ChemScreen coordinator.
• All elements of the ChemScreen integrated high throughput testing strategy are in an advanced state of integration and for all relevant elements training capacity has been set up or will be set up when the module is finalized.
• A maintenance-, service and support system for the screening system and its components developed is available.
• The work on novel test systems as well as on the battery approach was disseminated extensively in the scientific literature as well as in scientific conferences. This work generated significant attention in view of its innovative nature and relevance for other ongoing work on AOPs (e.g. OECD) and battery approaches (e.g. US EPA).

3. Exploitation of results

Final ChemScreen Integrated Testing Battery

The combined efforts of the ChemScreen consortium as described in the workpackage descriptions have led to a final ChemScreen Integrated Testing Battery. A flexible in vitro/in silico screening technology that forms the heart of the ChemScreen technology which has been incorporated in flexible modules that can be used in integrated testing strategies:

Module 1. In silico prescreening module

This module includes in silico prescreening methods to categorize chemicals into major classes of toxicity prioritized in REACH. Positive predictions for carcinogenicity, mutagenicity and reproductive toxicity can be applied to avoid unnecessary animal testing of compounds as replacement for animal tests for these endpoints, in particularly in integrated testing and read across.

Module 2. In vitro screening module

a. Integrated low/medium throughput models

Because of the complexity of the reproductive system and developmental processes the ChemScreen minimal essential screening panel contains zebrafish early life stage test or the EST test as integrative lower throughput models. These models have been extensively validated in the past and detailed OECD/ECVAM protocols are available.

b. High throughput models

BDS has set up a high throughput screening CALUX assay panel that can efficiently identify endocrine active compounds (with 100% accuracy), and compounds leading to sex organ deformities (with 83% accuracy). In combination with EST test results more than 90% accuracy was reached to detect reproductive toxicants. Alternatively, in combination with existing repeated dose study data potential reproductive and developmental toxicants could be identified ahead of in vivo studies, allowing prioritizations of animal testing.

These CALUX models have been extensively validated in the past and detailed OECD/ECVAM protocols have been set up and were submitted. EKUT has established the ReProGlo assay with partially complementary application domains. Both types of assays are running at BDS. Ample experience is available at BDS in training of external parties and world-wide dissemination of these assays. Simpple in collaboration with BDS has generated a ChemScreen software tool for bioanalytical data storage and analysis. This module is running at BDS when required Simpple can adapt this tool to the needs of individual clients.

Module 3. Exposure assessment

Exposure assessment is critical in risk assessment of chemicals. Several modules for rapid exposure predictions via diverse exposure routes have been set up by TNO and P&G. The resulting in vivo concentration estimates were shown to greatly improve predictivity of the in vitro tests. High throughput PBPK modeling is available as a third party service via TNO.

Module 4. Integrative models and risk assessment

a. Integrated testing strategies and innovative risk assessment

TNO has been involved in setting up methods for integrated testing of chemicals using all available data, complemented with in vitro screening data. BDS is setting up methods for hazard- and risk assessment linked to high throughput bioanalytical data, based on OECD guidelines and linkage to adverse outcome pathways. FhG showed that a number of potential reproductive and developmental toxicants could be identified ahead of in vivo studies by using existing repeated dose study data, in vitro tests or structural alerts thus supporting prioritizations of animal testing.

b. databases

UKON, RIVM and FhG have been completing databases with reference (in vivo) data for reproductive toxicants. These databases are not readily accessible for outside users but processed data will be included in international databases, while remaining data remain available upon request directly to those partners. A more accessible version of these databases, allowing direct linkage to bioanalytical data, and integrated testing approaches is in preparation.

In conclusion, all elements of the ChemScreen integrated high throughput testing strategy have been set up and are in an advanced state of integration and effective dissemination routes, including training and support facilities are available through the coordinators office.

List of Websites:

Dr. Bart van der Burg
Coordinator ChemScreen
BioDetection Systems BV
1098XH Amsterdam
The Netherlands
Tel: +31 20 4350750/62
Email: bart.van.der.burg@bds.nl
Web: www.chemscreen.eu