EURL ECVAM GLP Test Facility
Standardization, validation and regulatory applicabilty assessment (under GLP conditions) of in vitro assay protocols
The EURL ECVAM GLP Test Facility is responsible for the execution of validation studies that establish the reliability and relevance of in vitro methods suitable for regulatory safety assessment.
The group hosts the EURL ECVAM GLP Test facility and acts as a lead laboratory for studies conducted within the EU Network of Validation Laboratories (EU-NETVAL).
Its activities include:
- Theoretical and experimental assessment of in vitro assay protocols after submission to EURL ECVAM with the main objective to assess the applicability of the assay protocol in a regulatory environment.
- Optimise test definition and test description of the in vitro assay protocols where needed based on the factorial design approach including aspects related to procedures for the control and management of the test system, test items (and reference and controls items) and the endpoint detection system(s).
- Establish, coordinate and sustain the Network of Validation laboratories (EU-NETVAL) based on a specific set of eligibility criteria and work in close collaboration with DG ENV and DG ENTR to assure interactions with the national contact points for the Directive 2010/63/EU on the protection of animals used for scientific purposes and the European GLP compliant test facilities.
- GLP regulatory applicability assessment of the in vitro assay protocols to obtain within laboratory variability data and initiate the preparation of the training material for the EU-NETVAL laboratories.
On 9 May 2012 the EURL ECVAM Test Facility received the OECD Good Laboratory Practice (GLP) Certificate of GLP Conformity, as the first GLP test facility in the world with an OECD compliance statement for “Validation of in vitro toxicological methods”.
The EURL ECVAM Test Facility implemented GLP for its specific mandate to validate in vitro methods for regulatory purposes and to carry out the duties as laid down in the European Directive 2010/63/EU of 22 September 2010 on the protection of animals used for scientific purposes that established EURL ECVAM according to the highest quality standards.
Reference: OECD Series on principles of Good Laboratory Practice and compliance monitoring:
- After a successful GLP regulatory applicability assessment of the in vitro assay protocols in the EURL ECVAM GLP Test Facility, transfer protocols to selected EU-NETVAL laboratories complemented by the necessary training and guidance and use the appropriate communication platforms for managing EURL ECVAM coordinated Validation projects carried out by EU-NETVAL Test Facilities.
- Prepare e-learning modules on in vitro assay protocols as complementary resource to the in vitro assay EURL ECVAM GLP SOPs and forms.
- Provide purchase, characterization, coding and distribution services for supplying selected test items (chemicals) to the EURL ECVAM GLP Test Facility and the EU-NETVAL test facilities participating in EURL ECVAM coordinated Validation projects.
- Appoint an Assay Validation group liaison to manage the EU-NETVAL validation and establish an EURL ECVAM expert panel to assist in the overall Validation Management during the EU-NETVAL phase.
- Provide support during the transfer of in vitro assay protocols to an automated version, when required.
- Provide final EURL ECVAM GLP SOP for public distribution via the EURL ECVAM DB-ALM.
The GLP test facility Study Directors and Study Personnel continuously assess the reliability and relevance of in vitro test methods and has an extensive experience in in vitro method development, optimisation, pre-validation and validation, related to different (human) cell and tissue-based test systems and to endpoint detection methods such as flow cytometry (FCM), spectroscopy, high performance liquid chromotography (HPLC) and mass spectrometry (MS).
Their skills and experience enable the team to assess the transferability, the reliability and regulatory applicability (under GLP) of standard operating procedures provided by test developers, as initial step before formal validation starts.
The thorough quality control of assay protocols by the EURL-ECVAM GLP test facility ensures that:
- The amount of issues arising during the validation ring trials carried out by the EU-NETVAL laboratories is reduced.
- Assay protocols are harmonised as much as possible between validation projects.
- The time needed for the complete validation of the assay protocol will be reduced.
Work relates to high priority areas such as in vitro test method validation for Skin Sensitisation, Toxicokinetics and Metabolism and Endocrine disruption based in vitro assay protocols.
The following in vitro alternatives methods for dynamic assessments are, have been or will be running in the EURL ECVAM GLP test facility:
- Skin sensitisation test methods
- Human Cell Line Activation Test (h-CLAT) for skin sensitisation. THP-1 cells (an acute monocytic leukaemia cell line) are treated with the test item, followed by the flow cytometric detection of two cell surface molecules CD86 and CD54.
- Direct Peptide Reactivity Assay (DPRA) for skin sensitisation. This test method aims to model protein haptenation in chemico by measuring the depletion of two synthetic peptides (containing either a single cysteine or lysine side-chain as a reaction target) using HPLC.
- Estrogen receptor method for endocrine disruption. Endpoint detection method is luminescence.
- Lumicell (BG1Luc ER transcriptional activation assay). The BG1Luc4E2 cell line derived from BG-1 ovarian cell line immortalized adenocarcinoma cells that endogenously express both human ER forms, ERα and ERβ , stably transfected with the plasmid pGudLuc7.ERE. This plasmid contains 4 copies of a synthetic oligonucleotide containing ER response element upstream of the mouse mammary tumor viral promoter and the firefly luciferase gene. BG-1Luc4E2 cells express luciferase activity in response to estrogen and estrogen-like substances measure by a luminometer.
- MELN (MCF-7 Estrogen receptor-Luciferase-N Assay). Human MCF-7 cells, which express the oestrogen receptor alpha (ER alpha) were transfected with a plasmid containing the luciferase gene downstream detecting luciferase activity in response to estrogen and estrogen-like substances measure by a luminometer.
- Androgen receptor methods for endocrine disruption. Endpoint detection method is luminescence.
- PALM (PC-3-Androgen receptor-Luciferase-MMTV) human stable prostatic cell line expressing the human androgen receptor (AR) and the AR-responsive reporter gene to generate a tool for investigating androgen action and for rapid screening of agonists and antagonists
- AR (androgen receptor) and ERα (estrogen receptor alpha) CALUX® (Chemically Activated LUciferase eXpression), human U2-OS osteosarcoma cell line, all using highly selective reporter constructs based with a basal promoter element linked to multimerized response elements, allowing efficient and specific measurement of compounds interfering with androgen receptor
Toxicokinetics is the endpoint that informs about the penetration into and fate within the body of a toxic substance, including the possible emergence of metabolites, and their absorption, distribution, metabolism and excretion (ADME). When there is an increasing reliance on non-animal testing approaches, toxicokinetics has been identified as a key element to integrate the results from in silico, in vitro and in vivo studies.
In the EURL GLP Test Facility a validation project is running to develop a kinetic methods (Absorption, Distribution, Metabolism, Excretion, ADME methods ) that can cover the need for introduction of metabolic activity in any in vitro kinetic/dynamic test strategy, using cryopreserved human hepatocytes and cryopreserved HepaRG® cells as the metabolic competent test systems.
Metabolism is a great bottleneck in any type of our work so the search for a relevant metabolic competent test system is addressed in the validation project contributing to both the kinetic and dynamic aspects of metabolism-mediated aspects toxicology.
CYP enzymes, such as CYP1A2, CYP2B6, 2C9 and CYP3A4 which are susceptible to induction, are monitored as sensitive indicator of de novo protein synthesis, the critical metabolism enzymes in a metabolic competent system.
The in vitro CYP induction assays are using therefore, plateable cryohepatocytes and HepaRG® cells to identify compounds that may induce CYP activities and as a qualitative measure a s a reliable test system for metabolic competence. The endpoint detection method is LC-MS.
The two in vitro assay protocols which are being validated to provide metabolically competent test systems for kinetic and dynamic applications are:
- in vitro cryoHepaRG CYP induction assay
- in vitro cryoHepatocytes CYP induction assay
Pulmonary absorption was identified as a high priority area since no validated methods are available (Adler et al., 2011). In this context, inhalation barrier test systems that are commercially available have been identified to assess their potential for kinetic applications.See also: GLP test facility photo gallery
Photos: a) The EURL ECVAM GLP Test Facility team. Copyright EU 2013; b) and c) Scientists at work in the EURL ECVAM GLP Test Facility. Copyright EU 2012.