- Project no.: LSHB-CT-2004-503257
Instrument: Integrated Project (IP)
Project title: Development of a novel approach in hazard and risk assessment or reproductive toxicity by a combination and application of in vitro, tissue and sensor technologies
Thematic Priority: Development of new in vitro tests to replace animal experimentation
Date of preparation: the 25th of March 2003
Start date of project: the 1st of July 2004
Duration: 5 years
- Project coordinator:
Prof. Michael Schwarz
Department of Toxicology
Institute of Pharmacology und Toxicology
University of Tuebingen
- Project manager:
Dr. Cristian Pellizzer
ECVAM, IHCP, Joint Research Centre, EU
21020 Ispra (VA), Italy TP 580
Tel: +39-0332 786475
Fax: +39-0332 785336
e-mail: firstname.lastname@example.org, email@example.com
- The development of reliable testing strategies for industrial chemicals as well as for cosmetic ingredients (Cosmetic Directive 7th amendment and drugs development) is not only relevant to reduce testing costs and animal experimentation but it also serves as a sound basis for adequate risk assessment of chemicals.
It has been estimated that the detection of reproductive/developmental toxicants under the Registration, Evaluation and Authorisation of CHemicals (REACH) will have the highest impact on the animal use for regulatory safety testing. A report prepared by the Joint Research Centre of the European Commission (Pederson et al., 2003) is estimating an average testing of 2893 chemicals for reproductive
and 2135 chemicals for developmental toxicity testing. By following the guidelines for regulatory safety testing, millions of animals will be required only for the detection of reproductive/developmental toxicants. In addition several estimations of cost have led to the assumption that up to 60% of testing cost could be to 2 generation studies and developmental toxicity studies.
Thus, both ethical and feasibility consideration prompt to a reduction of the number of animals and testing costs, whilst ensuring the accurate identification of chemical hazards. Intelligent testing strategies are needed which make use of existing information as well as in vitro tests, QSARs and read across approaches.
In certain areas of reproductive toxicity testing, a number of useful and promising in vitro models are already available but they need to be converted into tests with a predictive power for toxicological safety testing. ReProTect is aiming to optimize these tests in order to prepare them for formal validation studies. Targeting a limited set of effects/mechanisms is a potential shortcoming of individual in vitro/in silico assays for complex events such as reproductive and developmental
toxicity. Thus, ReProTect aims at optimizing an integrated set of tests as a basis for a reproductive/developmental battery. In vitro test batteries will provide more detailed understanding of the main chemical target tissues or targeted biological mechanism in the reproductive cycle such as gametogenesis, steroidogenesis, embryogenesis etc. and will so support regulatory decisions.
Pedersen et al. (2003). Assessment of additional testing needs under REACH. http://ihcp.jrc.cec.eu.int/
- The complementary expertise of the partners will allow the sharing of methodology developed in different centres. All partners have been working in different fields of reproductive toxicology such as germ cell toxicity, embryotoxicity, endocrine disrupters, sterodiogenesis etc. and have collaborated with industrial partners. This ensures the understanding of the specific needs for industrial application of in vitro test systems. The strong involvement of the European Centre
for the Validation of Alternative Methods (ECVAM) in the scientific management will ensure a focused test development with the aim of proofing the relevance and reliability of developed tests in future validation studies. In close collaboration with the European Chemical Bureau (ECB) testing strategies will be established that allow a smooth transition of successful validated in
vitro tests into strategies that are accepted for appropriate regulation of a substance.
The ReProTect Consortium composition
- The ReProTect project, assembling 35 different European partners from Academia, Small Medium Enterprises, Governmental Institutions and Industries intends to explore a strategy for reproductive/developmental toxicity testing.
Structure of the project
- Due to the complexity of the mammalian reproductive cycle it is not possible to model the whole cycle in one in vitro system in order to detect chemical effects on mammalian reproduction.Therefore, the reproductive cycle has been broken down into 3 major research areas:
3. Prenatal development
Furthermore, a research area for
4. Cross-cutting technologies
The research areas consist of different numbers of workpackages reflecting various aspects of reproductive toxicology. The workpackages include the development and prevalidation of in vitro tests that are at various stages of test development. In some of the workpackages no or only insufficiently developed in vitro systems are available.
The development of in vitro models will be supported by cross-cutting technologies, which can be applied in the different research elements.
Complex tiered test strategies shall be composed and integrated in a conceptual framework as it is under discussion at OECD level (draft guidance 43). This entirely new approach and instrumentarium for hazard assessment will have to be fully assessed as to its relevance and reliability. This conceptual framework shall be deduced and elaborated in a series of consensus workshops.
Research area 1:
Fertility (key area leader Giovanna Lazzari, partner 7)
Infertility is a medical problem that can be related to the man or the woman. Approximately 35 per cent of cases are due to a female problem, 35 per cent due to a male problem and in the rest of the cases the causes are unexplained. Different toxicological mechanisms involving various target organs/tissues/cells that can lead to impaired fertility have been identified. The major cellular targets covering male and female fertility have been combined in the research area “Fertility”:
Mature spermatozoa/Spermatogenesis (W.P. I.1.)
(partners involved: 26, 29, 30)
Milestones: Test system for detecting germ cell mutagenicity on mature sperm cells; prevalidated test system for sperm functionality after chemical exposure.
Leydig cells (W.P. I.2.)
(partners involved: 2, 13)
Milestones: genetically engineered Leydig cell line that is showing the biosynthesis of testosterone; prevalidation of model.
Sertoli cells (W.P. I.3.)
(partners involved: 11)
Milestones: alternative test which is detecting chemical effects on the blood testis barrier.
Meiotically competent oocytes (W.P. I.4.)
(partners involved: 7, 28)
Milestones: three in vitro models based on bovine and mouse oocytes that can be further developed to predictive tests that can be used for analyzing adverse effects on oocyte maturation, fertilisation and preimplantation of embryos.
Granulosa/Thecal cells (W.P. I.5.) and Folliculogenesis (W.P. I.6.)
(partners involved: 5, 10, 28)
Milestones: two test systems that are able to detect adverse effects on the female steroidogenesis and one test system for detecting adverse effects on the folliculogenesis.
Research area 2:
Implantation (key area leader Lennart Denker, partner 21a)
This research area will commence its work at month 24 (July 2006) after start of ReProTect. Within the first 18 month reporting period promising in vitro models were selected within a symposium (Copenhagen, 13-14 January 2005). In addition, a workshop was held in order to develop a test strategy that is defining the most sensitive target tissues/organs or biological mechanisms of this research area (Copenhagen, 23 May 2005). Further partners will be recruited after the 18 months conference.
The research outlined within ReProTect has the aim of exploring available cell systems with definite outputs related to hazard identification for human reproduction. WP II will promote the set up of experimental tests to be developed and evaluated by the ECVAM principles on test validity.
The research area of implantantion has been divided into two workpackages in order to cover the regular preparation of the uterus for implantation as target for chemical insult as well as placental functions.
Endometrium/implantation and oviduct studies (W.P. II.1.)
(partners involved: 21b, 21c, 39)
Milestones: the main aim of this project is to study the endometrial angiogenesis, and to study how different agents affect endothelial cells. The second aspect of female reproductive toxicity is covered by additional research on the uterine function, which should lead to a successful implantation of the embryo. In vitro systems mirroring vessel formation and/or endometrial function, or placental function, would fill an important gap of test protocols mirroring the complete reproductive cycle. An in vitro model using human trophoblast and endometrium has been developed in order to study the biology of attachment and invasion of the early embryo and the influence of hormonal therapy and xenobiotics.
Placental toxicity (W.P. II.2.)
(partners involved: 21a, 32, 40, 41)
Milestones: identification of important cells types in the process of placentation by study of human tissues at early stages of pregnancy. The aim is to conduct feasibility studies of the assessment of functions of the selected cell types (trophoblasts and/or pericytes) in successful implantation and functioning of the placenta. Placenta is also considered a valuable organ for studies of fetal exposure by the placenta perfusion set up and this system will be described and tested according to the modular approach. Data from placental perfusion studies will feed into the QSAR calculations.
Research area 3:
Prenatal Development (key area leader Horst Spielmann, partner 12)
The third major effect on the female reproductive system consists in an alteration of the viability and morbidity of the offspring. The research area "Prenatal Development" is taking this aspect into account. Developmental toxicity - taken in its widest sense - includes any effect interfering with normal development. It encompasses effects induced or manifested prenatally as well as those manifested postnatally. This includes embryotoxic/foetotoxic effects such as reduced body weight, growth and developmental retardation, organ toxicity, death, abortion, and structural or functional defects. However, due to the complexity of the development of the unborn child only a battery of tests will provide information that allows a mechanism based identification of embryotoxic chemicals.
Early prenatal development (W.P. III.1.)
(partners involved: 6, 12, 14, 17, 35)
The consortium will focus on the further development of the validated murine Embryonic Stem Cell Tests (EST), in order to detect adverse effects on cardiaomyocytes differentiation and effects on neural and skeletal tissues. Related toxicological endpoints based on histochemical and molecular biological parameter (e.g. flow cytometry and/or TaqMan PCR) will be developed. In addition, another objective is the enlargement of
the database for the validated endpoints in the EST. A metabolic system will be evaluated by testing selected compounds. Adaptation of the murine system to human embryonic stem cells will provide a more precise forecast of developmental toxic effects in humans and avoid the need for inter-species extrapolation. Ultra-sensitive, differential protein expression analysis of differentiating ES cell cultures exposed to embryotoxic drugs and other chemicals will be
used to identify toxicant-specific protein expression patterns.
Milestones: SOPs for induction of embryonic stem cell differentiation into neural cell types and chondrocytes and culturing human embryonic stem cells; list of test chemicals with reliable in vivo animal and human data; start of identification of embryotoxic-signatures on protein basis.
Late prenatal development (W.P. III.2.)
(partners involved: 4)
This workpackage aims at the development and evaluation of an in vitro metabolizing system as a pre-incubation step in the existing whole embryo culture (WEC) technique.
Milestones: Protocol for WEC including biotransformation system.
Research area 4:
Cross-cutting Technologies (key area leader Alberto Mantovani, partner 8)
WP 4 represents a cross-cutting research area involving i) critical biological mechanisms shared by the research areas identified by WP 1-3, such as receptor interaction or biotransformation and ii) the development and assessment for possible validation of high-tech methodologies such as toxicogenomics, sensor technologies or QSARs. These approaches should be extended with proteomics and protein arrays to relate gene expression profiles to protein markers for confirmation of elucidated mechanisms. The relevance of Cross-cutting in testing strategies has been discussed in a ReProTect workshop (Vienna, 5-6 December 2005).
Sensor technologies (W.P. IV.1.)
(partners involved: 35)
This Workpackage will develop microelectrode arrays to monitor ion channel activity in murine embryonic stem cells. This approach, coupled with an optimized system for signal processing and data acquisition, will identify chemicals interacting with cardiac cell development. An assessment of the perspectives of sensor technologies in reproductive toxicity testing has been carried in a ReProTect Workshop (Ispra, 3-4th May 2005).
Quantitative Structure/Activity Relationships (QSARs) (W.P. VI.2.)
(partners involved: 23,27)
This Workpackage intends to exploit the QSAR potential as a high-throuput tool to support toxicological risk assessment, e.g., to support priority setting of chemicals or to provide mechanistic information; in the meanwhile, issues relevant to model development will be dealt, also interfacing with the OECD work on QSAR validation. Main activities will QSARs for a) steroid binding/transactivation, b) passage of blood-testis barrier; further step will be QSARs for placental transfer.
Biotransformation (W.P. IV.3.)
(partners involved: 3,5,17)
This Workpackage will optimize biotransformation systems in order to support the predictive power of in vitro assays. Activities will target the integration of biotransformation systems into the validated embryonic stem cell test (WP III.1) and the assays for receptor binding/transactivation (WP. IV.5). An assessment of biotransformation systems in reproductive toxicity testing has been carried in a ReProTect Workshop (Ispra, 30 November 2004).
Array technology (W.P. IV.4.)
(partners involved: 8, 31)
This Workpackage will target the characterization of gene expression profile changes and arrays as possible high-throughput tool for the identification of endocrine disrupters, with special attention to androgens/antiandrogens. Issues relevant to the development of array technologies will be dealt with, including the partnering of gene profiling with relevant toxicological endpoints, as well as quality assurance and efficiency problems.
Receptor interaction (W.P. IV.5.)
(partners involved: 3, 13, 25, 31)
This Workpackage will integrate as main EU partner the OECD validation work on cell- based assays ERalpha and AR binding/transactivation. Prediction models, optimized protocols and transferred SOPs shall be developed for the relevant systems (eg., PALM, MVLN, ER-CALUX, AR-CALUX). Moreover, a yeast-based assay will be developed in order to identify heterodimerization events among nuclear receptors (ERalph and -beta, AhR).
- Workpackage 5: Management activities
(partners involved: 1, 7, 8, 12, 21, 35)
The scientific and technical management is supported by a Project Office (PO) installed by Partner 35. The PO ensures a communication flow between the partners via the intranet and e-mail contacts. The PO is coordinating the scientific and technical activities of the project. Furthermore, the PO initiates and organizes general assemblies as well as workshops in agreement with the supervising board and the research area leaders. The project office ensures that timelines will be hold and will alert responsible research area leader if reports etc are not delivered in time. The project office also informs the supervising board about delays and eventually delivery problems of results. The management support group prepares activity reports twice a year. This will include information about the status of the project, responsible persons, dates when deliverables are expected and problems that are occurring during the project. The PO reports on a regular basis to the co-ordinator and to the members of the supervising board.
- Workpackage 6: Webpage
(partners involved: 5)
The principal aim of the workpackage is to facilitate the dissemination of the results, in order to ensure a very broad basis of the acquired know how all across Europe. The ReProTect webpage and a Newsletter will allow sharing scientific knowledge, which will include publications / workshop reports, organization of project meetings, public relations, SME support and industry integration.
- Workpackage 7: Technology and information scout
(partners involved: 35, 36)
The establishment of an "information and technology scout" has two major functions in ReProTect:
1. The scout ensures that all existing, non-confidential or shared toxicological data as information sources for reproductive/developmental toxicants are identified which allows the establishment of an appropriate database for each compound based on existing information. The review of of this database will allow an assessment on whether the existing information is already sufficient for hazard identification in a weight-of-evidence analysis before additional testing will be performed.
The existing data review will then be used as a justification why certain (animal) test data that relate to information requirements of REACH do not have to be generated, using one or several of the arguments indicated in Annex IX of the REACH proposal.
In case the data are insufficient but alerts have been identified, the existing data will be the basis for the development of a tailored testing scheme. Depending of the nature of the alerts, test batteries of specific in vitro tests will be triggered in order to confirm or refute concerns. This approach is seeking for a targeted testing that provides sufficient toxicological data for hazard identification but it also keeps testing to a minimum which is essential for complex toxicological endpoints
such as reproductive toxicity.
2. In addition, the scout is responsible for monitoring international and national projects that are aiming to develop new technologies and in vitro tests that can be used to identify reproductive toxicants. This function ensures an early adaptation of testing strategies to new technologies and testing methods as soon as their relevance and reliability has been proven. This can either enhance the throughput of chemicals in a screening process or it will provide more detailed information of the toxicological mechanism of the reviewed chemicals.