The ReProTect Feasibility Study, a novel comprehensive in vitro approach to detect reproductive toxicants.

ReProTect is a project within the 6th European Framework Program which has developed alternative methods aimed to reduce or replace animal experimentation in the field of reproductive toxicology. In its final year, a ring trial, named the "Feasibility Study", was conducted, in which 10 blinded chemicals with toxicologically well-documented profiles were analyzed by employing a test battery of 14 in vitro assays. EC(50) (half maximal effective concentration) or equivalent endpoints were determined and the test compounds were ranked relative to chemicals previously assayed in the tests of the battery. This comparative analysis together with a weight of evidence approach allowed a robust prediction of adverse effects on fertility and embryonic development of the 10 test chemicals in vivo. In summary, the vast majority of the predictions made based on the in vitro results turned out to be correct when compared to the whole animal data. The procedure used here, a nearest neighbor analysis coupled with a weight of evidence approach, may guide future activities in the field of alternative toxicity testing.

The test chemical selection procedure of the European Centre for the Validation of Alternative Methods for the EU Project ReProTect.

The selection of reference compounds is crucial for a successful in vitro test development in order to proof the relevance of the test system. This publication describes the criteria and the selection strategy leading to a list of more than 130 chemicals suitable for test development within the ReProTect project. The presented chemical inventory aimed to support the development and optimization of in vitro tests that seek to fulfill ECVAM's criteria for entering into the prevalidation. In order to select appropriate substances, a primary database was established compiling information from existing databases. In a second step, predefined selection criteria have been applied to obtain a comprehensive list ready to undergo a peer review process from independent experts with industrial, academic and regulatory background. Finally, a peer reviewed chemical list containing 13 substances challenging endocrine disrupter tests, additional 50 substances serving as reference chemicals for various tests evaluating effects on male and female fertility, and finally 61 substances were identified as known to provoke effects on the early development of mammalian offspring. The final list aims to cover relevant and specific mode/site of actions as they are known to be relevant for various substance classes. However, the recommended list should not be interpreted as a list of reproductive toxicants, because such a description requires proven associations with adverse effects of mammalian reproduction, which are subject of regulatory decisions done by involved competent authorities.

Transferability and inter-laboratory variability assessment of the in vitro bovine oocyte maturation (IVM) test within ReProTect.

The new European chemicals policy for the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) will most probably impose a dramatic increase in the number of animals required for reproductive toxicity testing. For this purpose, the development and validation of alternative methods is urgently needed in order to reduce the use of laboratory animals. The present study describes the inter-laboratory variability and the transferability assessment of an in vitro test able to identify chemical effects during the process of oocyte maturation in a bovine model. The test was developed/optimised within ReProTect, an integrated research project funded by the European Union, joining together 35 partners with complementary expertise in reproductive toxicology. Eight chemicals with well-known toxic properties were tested (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486 and DMSO as solvent) on the in vitro maturation (IVM) assay in two well-trained laboratories using the established Standard Operating Procedures. The statistical analysis demonstrated the concordance of results across the laboratories and the reproducibility of the test. We therefore conclude that the IVM test could advance toward the process of validation as alternative in vitro method that, in combination with additional in vitro tests, can become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.

Development of an in vitro test battery for assessing chemical effects on bovine germ cells under the ReProTect umbrella.

Current European legislation for the registration and authorization of chemicals (REACH) will require a dramatic increase in the use of animals for reproductive toxicity testing. Since one objective of REACH is to use vertebrates only as last resort, the development and validation of alternative methods is urgently needed. For this purpose ReProTect, an integrated research project funded by the European Union, joining together 33 partners with complementary expertise in reproductive toxicology, was designed. The study presented here describes a battery of two tests developed within ReProTect. The objective of these tests is the detection of chemical effects during the processes of oocyte maturation and fertilisation in a bovine model. The corresponding toxicological endpoints are the reaching of metaphase II and the formation of the pronuclei respectively. Fifteen chemicals have been tested (Benzo[a]pyrene, Busulfan, Butylparaben, Cadmium Chloride, Carbendazim, Cycloheximide, Diethylstilbestrol, Genistein, Ionomycin, Ketoconazole, Lindane, Methylacetoacetate, Mifepristone, Nocodazole and DMSO as solvent) demonstrating high intra-laboratory reproducibility of the tests. Furthermore, the responses obtained in both tests, for several substances, had a good correlation with the available in vivo and in vitro data. These tests therefore, could predictably become part of an integrated testing strategy that combines the bovine models with additional in vitro tests, in order to predict chemical hazards on mammalian fertility.

First steps in establishing a developmental toxicity test method based on human embryonic stem cells.

The use of embryonic stem cells is currently the most promising approach to assess developmental toxicity in vitro. In addition, the possibility of using human embryonic stem (hES) cells will increase safety of consumers and patients as false classification of substances due to inter-species variations can be avoided. One validated test based on murine embryonic stem cells, the embryonic stem cell test (EST), consists of following endpoints: IC(50) values of fibroblasts and embryonic stem cells as well as the inhibition of differentiation of mES cells into cardiomyocytes. As a follow up of its successful validation study we established a cytotoxicity assay based on hES cells and human fibroblasts employing two developmental toxicants: 5-fluorouracil (5-FU) and all-trans retinoic acid (RA). The results were compared to historical data from the EST. For 5-FU, no significant differences were obtained between the different cell lines. However, for RA, both test systems produced higher IC(50) values for the fibroblasts than for the stem cells, which is a well-known effect of developmental toxicants. Moreover, the reliability and relevance of several marker genes as possible toxicological endpoints were tested. During early differentiation Oct-4, hTert and Dusp6 showed the most reliable results. Brachyury and GATA-4 were found to be best suited to monitor cardiac differentiation. The late cardiac marker gene TNNT2 demonstrated significant results until day 18. Therefore, these marker genes have the highest potential to serve as endpoints for a developmental toxicity test.

Modulation of different stress pathways after styrene and styrene-7,8-oxide exposure in HepG2 cell line and normal human hepatocytes.

Styrene is one of the most important monomers produced worldwide. IARC classified styrene as a possible carcinogen to humans (group 2B). Styrene-7,8-oxide (SO) is the main reactive metabolite of styrene, and it is found to be genotoxic in several in vitro test systems. Styrene and styrene-7,8-oxide (SO) toxicity to HepG2 cells was investigated by evaluating end-points such as heat shock proteins (Hsps), metallothioneins (MT), apoptosis-related proteins, accumulation of styrene within the cells and expression of two isoforms of cytochrome P450. The potential activity of styrene and styrene-7,8-oxide in modulating gene expression was also investigated. The results showed induction of Hsp70, metallothioneins, BclX(S/L) and c-myc expression and a decrease in Bax expression in HepG2 after treatments, confirming that these compounds activated protective mechanisms. Moreover, up-regulation of TGFbeta2 and TGFbetaRIII in HepG2 cells was found after exposure to styrene, while in human primary hepatocytes these genes were down-regulated after both treatments. Finally, it was found that styrene and SO treatments did not induce CYP1A2 and CYP2E1 protein expression. In conclusion, both compounds caused toxic stress in HepG2 cells, with SO being more toxic; in the meantime, a different effect of the two compounds in HepG2 cells and primary human hepatocytes was observed regarding their activity in gene modulation.

The value of alternative testing for neurotoxicity in the context of regulatory needs.

Detection and characterisation of chemical-induced toxic effects in the central and peripheral nervous system represent a major challenge for employing newly developed technologies in the field of neurotoxicology. Precise cellular predictive test batteries for chemical-induced neurotoxicity are increasingly important for regulatory decision making, but also the most efficient way to keep costs and time of testing within a reasonable margin. Current in vivo test methods are based on behavioural and sensory perturbations coupled with routine histopathological investigations. In spite of the empirical usefulness of these tests, they are not always sensitive enough and often, they do not provide information that facilitates a detailed understanding of potential mechanisms of toxicity, thus enabling predictions. In general, such in vivo tests are unsuitable for screening large number of agents. One way to meet the need for more powerful and comprehensive tests via an extended scientific basis is to study neurotoxicity in specific cell types of the brain and to derive generalised mechanisms of action of the toxicants from such series of experiments. Additionally, toxicokinetic models are to be developed in order to give a rough account for the whole absorption, distribution, metabolism, excretion (ADME) process including the blood-brain barrier (BBB). Therefore, an intensive search for the development of alternative methods using animal and human-based in vitro and in silico models for neurotoxic hazard assessment is appropriate. In particular, neurotoxicology represents one of the major challenges to the development of in vitro systems, as it has to account also for heterogeneous cell interactions of the brain which require new biochemical, biotechnological and electrophysiological profiling methods for reliable alternative ways with a high throughput.

The detection of differentiation-inducing chemicals by using green fluorescent protein expression in genetically engineered teratocarcinoma cells.

The murine embryonal teratocarcinoma cell line, P19, was genetically manipulated in order to provide preliminary information on compounds that induce differentiation. Without chemical induction, P19 cells remain in an undifferentiated state, but can be induced to differentiate into specific cell types. For example, dimethyl sulphoxide (DMSO) induces cardiac and skeletal muscle differentiation, whereas retinoic acid stimulates neuronal differentiation. P19 cells were transfected with a construct containing a segment of the murineTert (mTert) promoter sequence combined with the green fluorescent protein (GFP) gene, which acts as a reporter gene. mTert expression, the reverse transcriptase component of murine telomerase, is closely linked to telomerase activity and is down-regulated during differentiation. Three retinoids and DMSO induced the differentiation of P19 cells, which was determined by a reduction in mTert_GFP expression, detected by flow cytometry and confocal microscopy as independent methods of detection. A test substance, ethanol, and a control substance, saccharin, did not cause a decrease in mTert_GFP expression. In addition, it could be demonstrated that the mTert_GFP test detects developmentally relevant effects at non-cytotoxic concentrations. The ID50 values derived for the reduction of mTert_GFP expression were lower than the IC50 values detected with the MTT test, by a factor of 21.4 for all-trans retinoic acid, 12.7 for 9-cis retinoic acid, 29.6 for 13-cis retinoic acid, and 8.7 for DMSO. In comparison to the IC50 value for the P19 cell line, a similar IC50 value was obtained with 3T3 cells for ethanol, but there was a 2-fold increase for DMSO. The retinoids were not cytotoxic to 3T3 cells at the concentrations tested. This newly developed test is capable of detecting differentiation-inducing compounds at non-cytotoxic concentrations within 4 days. It offers a method for detecting chemicals with specific toxicological mechanisms, such as the retinoids, which could provide additional information in embryotoxicity testing as different promoters could be employed. Here, we report the use of this novel test system for the successful analysis of DMSO and three retinoids with different in vivo teratogenic potentials.

Developmental toxicity testing from animal towards embryonic stem cells.

Developmental toxicology is the study of undesirable effects on the development of the organism, which may result from exposure before conception, from the period of prenatal development, or postnatally during the time of sexual maturation. The principal manifestations of developmental toxicity include: embryolethality, malformations, growth retardation, and functional impairment. In 2001, the European Commission published the future chemicals policy entitled "White Paper: Strategy for a Future Chemicals Policy". The new regulation requires a toxicological evaluation with strong emphasis on reproductive toxicity, by using in vitro methods, especially for those chemicals marketed at more than 1 ton per year. For this reason, the establishment of in vitro models capable of detecting major undesirable manifestations in the fetus, are urgently required. The aim of the present review is to explore the capacity of existing in vitro systems, based on embryonic stem (ES) cells, to identify embryotoxicity with a focus on specific effects such as teratogenicity and growth retardation. In addition, we discuss the possibility to adapt the mouse ES cells based tests to human ES cells, avoiding inter-species variations in developmental toxicity studies and address related ethical issues. Considering the different manifestations of developmental toxicity, only a battery of in vitro tests will provide the necessary information for regulatory developmental toxicity assessment.

The integrated project ReProTect: a novel approach in reproductive toxicity hazard assessment.

Validated alternative test methods are urgently needed for safety testing of drugs, chemicals and cosmetics. Whereas some animal tests for topical toxicity have been successfully replaced by alternative methods, systemic toxicity testing requires new test strategies in order to achieve an adequate safety level for the consumer. Substantial numbers of animals are required for the current in vivo assays for drugs, chemicals and cosmetics and a broad range of pioneering alternative methods were already developed. These prerequisites motivate the development of a tiered testing strategy based on alternative tests for reproductive toxicity hazard. In the Integrated Project ReProTect, a consortium set up by the European Centre for the Validation of Alternative Methods (ECVAM) takes the lead to manage the development of a testing strategy in the area of reproductive toxicity. The reproductive cycle can be broken down into well-defined sub-elements, namely male and female fertility, implantation and pre/postnatal development. In this project, in vitro, in silico and sensor technologies will be developed, leading to testing strategies, that shall be implemented and disseminated.

Detection of tissue-specific effects by methotrexate on differentiating mouse embryonic stem cells.

BACKGROUND: Pluripotent embryonic stem (ES) cells offer a unique possibility to monitor the differentiation of several cell types in vitro. This study attempts to identify marker genes during in vitro cell differentiation of murine ES cells and allow a prediction of chemical effects on cell differentiation of specific target tissues. The study focused on the expression pattern of key genes involved in cardiomyocyte and osteoblast differentiation: Oct-4, Brachyury, Nkx2.5, alpha myosin heavy chain, Cbfa1, and Osteocalcin. METHODS: Methotrexate was selected due to its well-characterized teratogenic effects. Several in vivo studies have demonstrated the specific interactions of methotrexate with bone formation whereas the cardiovascular system is not specifically affected after exposure to low concentration. The capability of murine ES cells to differentiate in vitro into cardiomyocytes as well as into osteoblasts have been used to demonstrate the target cell specificity in vitro, at non-cytotoxic concentration. RESULTS: Exposure of differentiating ES cells did not result in any gene profile modification of the selected cardiomyocyte specific genes, whereas the expression of osteoblast specific key genes, Cbfa1 and Osteocalcin, decreased. At the latter stages of skeletal differentiation we observed a 30% decrease in gene expression for Cbfa1 and a 60% decrease for Osteocalcin, with reference to the control. Early marker genes for undifferentiated cells and mesodermal cells were not modified after methotrexate treatment. CONCLUSIONS: These results show the possibility to integrate specific in vitro tests for teratogenicity in a test strategy for developmental toxicity.

Development of a testing strategy for detecting embryotoxic hazards of chemicals in vitro by using embryonic stem cell models.

The importance of developing in vitro tests for embryotoxicity is discussed, and ECVAM's work with its collaborators is summarised. Studies are in progress to find new endpoints for use in the scientifically validated embryonic stem (ES) cell test, so that the potential for chemical effects on endodermal, mesodermal and/or ectodermal differentiation can be identified. This involves, inter alia, the use of genetically modified ES cells.