Periodic Report Summary - TESTMETEDECO (Development of test methods for the detection and characteristation of endorcrine disrupting chemicals in environmental species)
The overall objective of the TESTMETEDECO project was to improve international coordination of research and test method development in the area of endocrine disruption in wildlife species, within Europe, Japan and the United States.
The TESTMETEDECO project contributed to this objective by the following specific actions:
- Collect existing information on test methods identified as priority within the OECD Conceptual Framework for Endocrine Disrupters Testing and Assessment (EDTA) for the detection and characterisation of endocrine active substances, and produce Detailed Review Papers.
- Organise and coordinate the different phases of the validation of protocols for testing endocrine active substances, in accordance with the OECD validation principles, and assist laboratories in preparing the work and reporting the deliverables.
- Produce statistical analysis and validation reports for each phase of the validation of a test method protocol.
- Publish the validation reports and communicate on the outcome of the validation to the regulatory community, as one of the bases for member countries to decide on the development of OECD Test Guidelines for the testing of chemicals.
The objective for the second and final reporting period of the project was to get all statistical analysis and validation reports prepared by consultants submitted within the timelines. All deliverables planned in the description of work were submitted.
The project contained five work packages:
- Work package 01 (Fish Partial Life Cycle Test),
- work package 02 (Fish Life Cycle Testing),
- Work package 03 (Amphibian Metamorphosis Assay),
- Work package 04 (Invertebrate Development and Reproduction Tests),
- Work package 05 (Coordination and Dissemination).
During the course of the project, all planned deliverables were produced for the various work packages.
Work package 01 (WP01): Fish Partial Life-Cycle Test
This test was developed in Denmark and Sweden, and originally adapted from the OECD Test Guideline 210 (fish early life stage test). The enhanced version of the test measured Vitellogenin, a protein used as a biomarker endpoint of estrogenic exposure and sex ratio of juvenile fish exposed to a test chemical. Two test designs were considered and evaluated in the validation study, the choice of one or the other being dependent on the regulatory purpose and on the nature of the data.
Overall objective at the OECD level: develop and validate a protocol for the fish partial life-cycle test (fish sexual development) that could be used by regulators for the detection of endocrine active substances.
Objectives under TESTMETEDECO:
- conduct two successive phases of the validation to demonstrate the sensitivity, relevance and reproducibility of the test method,
- analyse test results and produce validation reports,
- get sub-contractors to undertake the statistics (including power analysis) and the drafting of the report,
- circulate validation reports for comments, revise, finalise and publish as time allows.
Work package 02 (WP02): Fish Full life-Cycle/2-Generation Tests
This test is definitive and confirmatory: the endpoints measured are direct indicators of growth and reproduction, and usable for setting Predicted No Effect Concentration (PNEC). From the various versions and fish species used, there was no rigorous comparison/evaluation of endpoints sensitivity and reproducibility.
Overall objective at the OECD level:
- compare the various test designs used in OECD countries, their advantages and limitations;
- analyse the sensitivity and variability of the measured endpoints;
- if possible give guidance on the usefulness of these endpoints for regulatory use, and gauge countries' interest to agree on a common approach for the fish long-term testing;
- produce the necessary review document(s) to support a harmonised fish long-term test.
Objectives under TESTMETEDECO:
- conduct the initial pre-validation work (Phase 1) to assess sensitivity and reproducibility of tremarkable endpoints;
- proceed to a detailed statistical analysis and produce the report of Phase 1 of the validation;
- finalise the Detailed Review Paper which provides the background of fish definitive testing for the characterisation of adverse effects of endocrine disrupters and other chemicals;
- circulate documents for comments, revise, finalise and publish as time allows.
Work package 03 (WP03): Amphibian Metamorphosis Assay
The amphibian metamorphosis assay was intended to detect substances interfering with the thyroid system in vertebrates, more specifically aquatic vertebrates. Frog metamorphosis was used as a model as it represented a well-characterised transformation process triggered by the thyroid hormone; importantly there exists an analogy of thyroid regulating genes among vertebrates, which allows to a certain extent extrapolations between an amphibian model and higher vertebrates. In the assay, tadpoles were exposed in the water to a range of concentrations of the test substance during a defined period of the metamorphosis. Endpoints measured were development stage, body length, hind-limb length and thyroid histology. Endpoints were measured either at termination of exposure (day 21) or at a sensitive window of the assay (day 7) for hind limb length.
Overall objective at the OECD level: develop and validate a protocol for the amphibian metamorphosis assay that can be used by regulators for the detection of thyroid active substances.
Objectives under TESTMETEDECO:
- finalise Phase 1 of the validation (TESTMETEDECO funding not used for that purpose);
- organise Phase 2 of the validation of the amphibian metamorphosis assay, in the form of a multi-chemical / multi-laboratory study; and
- coordinate the statistical analysis and the preparation of the report;
- organise Phase 3 of the validation for the testing of weakly active and negative substances, and coordinate the statistical analysis and preparation of the report;
- circulate documents for comments, revise, finalise and publish as time allows;
- organise the preparation of a review paper on the comparison of the amphibian metamorphosis assays versus the rodent model.
Work package 04 (WP04): Invertebrates Testing
The invertebrate tests comprised in this work package were the copepod development and reproduction test and the enhanced Daphnia reproduction test. Both tests aimed at detecting chemicals having an adverse effect on growth or reproduction of aquatic arthropods in life-cycle tests in marine water (Copepod) or freshwater (Daphnia), with the enhanced version of the Daphnia test being more specific for the detection of substances active on the hormonal system of arthropods (e.g. juvenile hormone agonists). These tests would allow determining the effect level of substances tested, including potential hormone disrupters of aquatic arthropods, and more generally reproductive and growth toxicants.
Overall objective at the OECD level: develop Test Guideline(s) on aquatic invertebrate(s), including development and reproduction endpoints, to be used for the testing of substances suspected to have an effect on growth and reproduction (whether through endocrine related pathways or not) in aquatic arthropods.
Objective under TESTMETEDECO:
- conduct validation studies for copepods and daphnia using one or two test substances; substances are selected by the Invertebrate Expert Group and the Validation Management Group for Ecotoxicity Testing, based on the relevance of the substance, the minimum number of substance(s) to achieve validation, and the financial means available in the laboratories;
- assess the reproducibility of the tests through a statistical analysis and produce validation reports;
- circulate documents for comments, revise, finalise and publish as time allows.
Work package 05 (WP05): Coordination and Dissemination
Overall objective at the OECD level:
- provide the necessary coordination at all stages of test development and validation for test methods for endocrine disrupters in wildlife species, report on progress with the validation exercises to EDTA Task Force and VMG-eco;
- ensure in the long-run the mutual acceptance of data through the regulatory acceptance of Test Guidelines.
Objective under TESTMETEDECO:
- provide the necessary coordination at all stages of test development and validation (as described in WP01 to WP04),
- ensure that information is circulated rapidly,
- post information on the website on time,
- ensure dissemination of project outcomes on appropriate forum (conferences, workshops, expert meetings, OECD meetings),
- report on progress with the validation exercises to EDTA Task Force and VMG-eco.
The TESTMETEDECO project contributed to this objective by the following specific actions:
- Collect existing information on test methods identified as priority within the OECD Conceptual Framework for Endocrine Disrupters Testing and Assessment (EDTA) for the detection and characterisation of endocrine active substances, and produce Detailed Review Papers.
- Organise and coordinate the different phases of the validation of protocols for testing endocrine active substances, in accordance with the OECD validation principles, and assist laboratories in preparing the work and reporting the deliverables.
- Produce statistical analysis and validation reports for each phase of the validation of a test method protocol.
- Publish the validation reports and communicate on the outcome of the validation to the regulatory community, as one of the bases for member countries to decide on the development of OECD Test Guidelines for the testing of chemicals.
The objective for the second and final reporting period of the project was to get all statistical analysis and validation reports prepared by consultants submitted within the timelines. All deliverables planned in the description of work were submitted.
The project contained five work packages:
- Work package 01 (Fish Partial Life Cycle Test),
- work package 02 (Fish Life Cycle Testing),
- Work package 03 (Amphibian Metamorphosis Assay),
- Work package 04 (Invertebrate Development and Reproduction Tests),
- Work package 05 (Coordination and Dissemination).
During the course of the project, all planned deliverables were produced for the various work packages.
Work package 01 (WP01): Fish Partial Life-Cycle Test
This test was developed in Denmark and Sweden, and originally adapted from the OECD Test Guideline 210 (fish early life stage test). The enhanced version of the test measured Vitellogenin, a protein used as a biomarker endpoint of estrogenic exposure and sex ratio of juvenile fish exposed to a test chemical. Two test designs were considered and evaluated in the validation study, the choice of one or the other being dependent on the regulatory purpose and on the nature of the data.
Overall objective at the OECD level: develop and validate a protocol for the fish partial life-cycle test (fish sexual development) that could be used by regulators for the detection of endocrine active substances.
Objectives under TESTMETEDECO:
- conduct two successive phases of the validation to demonstrate the sensitivity, relevance and reproducibility of the test method,
- analyse test results and produce validation reports,
- get sub-contractors to undertake the statistics (including power analysis) and the drafting of the report,
- circulate validation reports for comments, revise, finalise and publish as time allows.
Work package 02 (WP02): Fish Full life-Cycle/2-Generation Tests
This test is definitive and confirmatory: the endpoints measured are direct indicators of growth and reproduction, and usable for setting Predicted No Effect Concentration (PNEC). From the various versions and fish species used, there was no rigorous comparison/evaluation of endpoints sensitivity and reproducibility.
Overall objective at the OECD level:
- compare the various test designs used in OECD countries, their advantages and limitations;
- analyse the sensitivity and variability of the measured endpoints;
- if possible give guidance on the usefulness of these endpoints for regulatory use, and gauge countries' interest to agree on a common approach for the fish long-term testing;
- produce the necessary review document(s) to support a harmonised fish long-term test.
Objectives under TESTMETEDECO:
- conduct the initial pre-validation work (Phase 1) to assess sensitivity and reproducibility of tremarkable endpoints;
- proceed to a detailed statistical analysis and produce the report of Phase 1 of the validation;
- finalise the Detailed Review Paper which provides the background of fish definitive testing for the characterisation of adverse effects of endocrine disrupters and other chemicals;
- circulate documents for comments, revise, finalise and publish as time allows.
Work package 03 (WP03): Amphibian Metamorphosis Assay
The amphibian metamorphosis assay was intended to detect substances interfering with the thyroid system in vertebrates, more specifically aquatic vertebrates. Frog metamorphosis was used as a model as it represented a well-characterised transformation process triggered by the thyroid hormone; importantly there exists an analogy of thyroid regulating genes among vertebrates, which allows to a certain extent extrapolations between an amphibian model and higher vertebrates. In the assay, tadpoles were exposed in the water to a range of concentrations of the test substance during a defined period of the metamorphosis. Endpoints measured were development stage, body length, hind-limb length and thyroid histology. Endpoints were measured either at termination of exposure (day 21) or at a sensitive window of the assay (day 7) for hind limb length.
Overall objective at the OECD level: develop and validate a protocol for the amphibian metamorphosis assay that can be used by regulators for the detection of thyroid active substances.
Objectives under TESTMETEDECO:
- finalise Phase 1 of the validation (TESTMETEDECO funding not used for that purpose);
- organise Phase 2 of the validation of the amphibian metamorphosis assay, in the form of a multi-chemical / multi-laboratory study; and
- coordinate the statistical analysis and the preparation of the report;
- organise Phase 3 of the validation for the testing of weakly active and negative substances, and coordinate the statistical analysis and preparation of the report;
- circulate documents for comments, revise, finalise and publish as time allows;
- organise the preparation of a review paper on the comparison of the amphibian metamorphosis assays versus the rodent model.
Work package 04 (WP04): Invertebrates Testing
The invertebrate tests comprised in this work package were the copepod development and reproduction test and the enhanced Daphnia reproduction test. Both tests aimed at detecting chemicals having an adverse effect on growth or reproduction of aquatic arthropods in life-cycle tests in marine water (Copepod) or freshwater (Daphnia), with the enhanced version of the Daphnia test being more specific for the detection of substances active on the hormonal system of arthropods (e.g. juvenile hormone agonists). These tests would allow determining the effect level of substances tested, including potential hormone disrupters of aquatic arthropods, and more generally reproductive and growth toxicants.
Overall objective at the OECD level: develop Test Guideline(s) on aquatic invertebrate(s), including development and reproduction endpoints, to be used for the testing of substances suspected to have an effect on growth and reproduction (whether through endocrine related pathways or not) in aquatic arthropods.
Objective under TESTMETEDECO:
- conduct validation studies for copepods and daphnia using one or two test substances; substances are selected by the Invertebrate Expert Group and the Validation Management Group for Ecotoxicity Testing, based on the relevance of the substance, the minimum number of substance(s) to achieve validation, and the financial means available in the laboratories;
- assess the reproducibility of the tests through a statistical analysis and produce validation reports;
- circulate documents for comments, revise, finalise and publish as time allows.
Work package 05 (WP05): Coordination and Dissemination
Overall objective at the OECD level:
- provide the necessary coordination at all stages of test development and validation for test methods for endocrine disrupters in wildlife species, report on progress with the validation exercises to EDTA Task Force and VMG-eco;
- ensure in the long-run the mutual acceptance of data through the regulatory acceptance of Test Guidelines.
Objective under TESTMETEDECO:
- provide the necessary coordination at all stages of test development and validation (as described in WP01 to WP04),
- ensure that information is circulated rapidly,
- post information on the website on time,
- ensure dissemination of project outcomes on appropriate forum (conferences, workshops, expert meetings, OECD meetings),
- report on progress with the validation exercises to EDTA Task Force and VMG-eco.