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CHARGE TO PANEL

Introduction

The Food Quality Protection Act (FQPA) of 1996 requires that EPA

“develop a screening program, using appropriate validated test systems and other 

scientifically relevant information, to determine whether certain substances may have an effect 

in humans that is similar to an effect produced by a naturally occurring estrogen, or other such 

endocrine effect as the Administrator may designate.”

Subsequent to passage of the FQPA the EPA formed a federal advisory committee, the 

Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), to provide 

recommendations on how to implement its Endocrine Disruptor Screening Program (EDSP). The 

EDSP is described in detail at the following website: http://www.epa.gov/scipoly/oscpendo/. 

Based on the recommendations from the EDSTAC (EDSTAC 1998), the EPA, in addition to the 

estrogen pathway, included the androgen and thyroid pathways of the endocrine system in 

humans as well as in wildlife. The EDSTAC also recommended the Agency adopt a two-tiered 

screening and testing program. 

Tier 1 is an integrated battery of relatively short-term in vitro and in vivo assays designed 

to detect the potential of a chemical to interact with endocrine system, principally the estrogen, 

androgen, and thyroid hormonal pathways. Test chemicals determined to have the potential to 

interact with the endocrine system, based on a weight-of-evidence analysis of the results of Tier 

1 screening and inclusive of other scientifically relevant information, would be considered for 

Tier 2 testing. 

Tier 2 has the purpose to “characterize the nature, likelihood, and dose-response 

relationship of endocrine disruption of estrogen, androgen, and thyroid in humans and wildlife.” 

Tier 2 tests consist of more comprehensive, long-term tests during various life stages and 

multiple generations enhanced with endocrine-specific endpoints across multiple taxonomic 

groups, including mammals, birds, fish, amphibians, and invertebrates. The purpose of Tier 2 

testing is to identify any potential adverse outcome and provide quantitative concentrationresponse information that may be used for risk assessment. The following ecotoxicity tests, as 

generally recommended by EDSTAC, are being considered as part of the EDSP Tier 2:

• Japanese Quail Two-generation Test (JQTT)

• Medaka Multi-generation Test (MMT)

• Larval Amphibian Growth and Development Assay (LAGDA)

• Mysid Two-generation Toxicity Test (MTTT)

The Integrated Summary Reports (ISRs) and accompanying appendices for each assay provide a 

detailed account of the historical development and validation of a standardized protocol for each 

Tier 2 ecotoxicity assay. The EPA is requesting the SAP to provide independent peer review of 

each Tier 2 ecotoxicity test method. 

(The criteria used to guide the validation process for each test method were based on the 

principles of validation developed by the U.S. Interagency Coordinating Committee for the 

Validation of Alternative Methods (ICCVAM) and the Organisation for Economic and 

Cooperative Development (OECD). These questions follow a format consistent with those 

international validation standards and are similar to the questions provided for the EDSP Tier 

I validation).
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Validation Charge Questions:

Japanese Quail Two-Generation Test (JQTT)

1. A rationale for the test method should be available, including a clear statement of scientific 

basis and the regulatory purpose and need for the test method. The EDSTAC described the 

Tier 2 tests as having the purpose to “characterize the nature, likelihood, and dose-response 

relationship of endocrine disruption of estrogen, androgen, and thyroid in humans and 

wildlife.” Tier 2 tests were designed to be definitive tests that generate sufficient data to 

characterize the specific hazard of the substance and provide sufficient information on doseresponse and adverse effects to permit risk decisions. 

Please comment on the rationale and purpose of the assay as part of the Tier 2 testing in

the EDSP, as described in Sections 2.5 and 2.6 of the JQTT ISR. 

2. Test methods and their associated endpoint(s) should be scientifically relevant to the 

biological processes of interest and should be demonstrated to be responsive to the specific 

type of effect/toxicity of interest. Each species presents unique characteristics from a 

biological perspective and allows for specialized endpoints to address a specific toxicological 

mode of action. 

Please comment on the biological and toxicological relevance of the assay in regards to 

the stated purpose of characterizing endocrine disruptors, as described in Sections 3 and 

4 of the JQTT ISR. 

3. The test protocol should be sufficiently detailed and should include a description of what is 

measured and how it is measured. The selection of endpoints within the assay should be 

reflective of the biological processes of interest and the endpoints should be intrinsically 

relevant and have established sensitivity. The test protocol should demonstrate the ability to 

measure the endpoints and provide adequate performance criteria for evaluation. 

3.1 Please comment on the selection, optimization and demonstration of the assay 

endpoints, as outlined in Sections 4 and 5 of the JQTT ISR. 

Pathological evaluation of histological tissue preparations is an established, sensitive and 

integral endpoint in the assessment of effects in long term in vivo assays. The tissues targeted 

for histopathological evaluation should be shown to be sensitive to exposure and relevant to a 

mode of action or pathway determination. Tissue samples from several organs or glands (e.g., 

kidney, liver, thyroid, gonads) underwent histopathological examination in the JQTT interlaboratory validation and a discussion of histopathology as an endpoint is provided in Section 

4.1.13 of the JQTT ISR. 

3.2 Please comment on the value of histopathological analyses in the JQTT assay for each 

of the tissue types examined and what (if any) critical information is gained from their 

inclusion or would be lost if histology were not examined. 
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4. Demonstration of the test method performance should be based on the testing of reference 

chemicals representative of the types of substances for which the test method will be used. 

Test substances should adequately represent an appropriate range of responses and 

physical/chemical properties for which the test method is proposed to be appropriate. The 

selection of the most appropriate statistical approaches depends in part on the nature of the data 

and also on the design of the validation study. Statistical and non-statistical methods used to 

analyze should be described. 

Please comment on the selection of test substances and methods (analytical and statistical 

where appropriate) chosen for the demonstration and validation of the JQTT assay. 

5. Considering the variability inherent in biological and chemical test methods, a test method 

needs to be repeatable and reproducible. A test is robust and reliable if the results are 

repeatable and reproducible within a laboratory and between different laboratories, respectively. 

A test protocol should provide sufficient guidance to ensure proper and consistent performance 

across labs and chemicals. 

Please comment on the test method robustness and reliability and the repeatability and 

reproducibility of the results obtained with the JQTT assay. 

6. The test protocol should be descriptive enough to be fully transferable to a competent

laboratory. The protocol should describe the methodology of the assay in a clear and concise 

manner so that a laboratory could comprehend the objective, conduct the assay, observe and 

measure prescribed endpoints, compile and prepare data for statistical analyses, and report the 

results. Section 6 of the JQTT ISR outlines the process of and challenges experienced in the 

inter-laboratory studies. 

Please comment on the transferability across labs and provide any suggestions or 

recommendations for improvement of the JQTT assay. 

7. The purpose of the validation process is to determine the readiness of a test for inclusion in a 

testing program. A component of readiness of a test is the evaluation of the usefulness and 

limitations of the test, including the classes and types of test substances that can and cannot be 

tested. 

Please comment on the strengths and/or limitations of the JQTT assay, as described in 

Section 7 of the JQTT ISR. 

8. There is sufficient evidence to indicate that EDCs can disrupt normal development and 

reproductive success, however the sensitivity of the F2 generation compared to the P0 or F1 is 

less clearly defined. In the JQTT, there is an increase in endpoint CVs with each subsequent 

generation (JQTT ISR Table 6-4), which indicates decreased power of discrimination. 
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Please comment from a scientific and risk assessment perspective on the value added of 

multiple generations in the JQTT assay. 
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Medaka Multigeneration Test (MMT)

Section 5 of the MMT ISR outlines the EPA proposed Medaka Reproduction Test (MRT) assay 

which utilizes the culmination of knowledge and experience gained from many successive U.S. 

and Japanese studies over multiple years. 

1. A rationale for the test method should be available, including a clear statement of scientific 

basis and the regulatory purpose and need for the test method. The EDSTAC introduced the 

Tier 2 tests as having the purpose to “characterize the nature, likelihood, and dose-response 

relationship of endocrine disruption of estrogen, androgen, and thyroid in humans and 

wildlife.” Tier 2 tests were designed to be definitive tests which generate sufficient data to 

characterize the specific hazard of the substance and provide sufficient information on doseresponse and adverse effects to permit risk decisions. 

Please comment on the rationale and purpose of the assay as part of the Tier 2 testing in

the EDSP, as described in Sections 2.1 and 2.2 of the MMT ISR. 

2. Test methods and their associated endpoint(s) should be scientifically relevant to the 

biological processes of interest and should be demonstrated to be responsive to the specific 

type of effect/toxicity of interest. Each species presents unique characteristics from a 

biological perspective and allows for specialized endpoints to address a specific toxicological 

mode of action. 

Please comment on the biological and toxicological relevance of the assay in regards to 

the stated purpose of characterizing endocrine disruptors, as described in Section 3 of the 

MMT. 

3. The test protocol should be sufficiently detailed and should include a description of what is 

measured and how it is measured. The selection of endpoints within the assay should be 

reflective of the biological processes of interest and the endpoints should be intrinsically 

relevant and have established sensitivity. The test protocol should demonstrate the ability to 

measure the endpoints and provide adequate performance criteria for evaluation. 

Please comment on the selection, optimization and demonstration of the assay endpoints, 

as outlined in Section 3 of the MMT ISR. 

4. Demonstration of the test method performance should be based on the testing of reference 

chemicals representative of the types of substances for which the test method will be used. 

Test substances should adequately represent an appropriate range of responses and 

physical/chemical properties for which the test method is proposed to be appropriate. The 

selection of the most appropriate statistical approaches depends in part on the nature of the data 

and also on the design of the validation study. Statistical and non-statistical methods used to 

analyze should be described. 
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Please comment on the selection of test substances and methods (analytical and statistical 

where appropriate) chosen for the demonstration and validation of the MMT and MRT 

assays. 

5. Considering the variability inherent in biological and chemical test methods, a test method 

needs to be repeatable and reproducible. A test is robust and reliable if the results are 

repeatable and reproducible within a laboratory and between different laboratories, respectively. 

A test protocol should provide sufficient guidance to ensure proper and consistent performance 

across labs and chemicals. 

Please comment on the test method robustness and reliability and the repeatability and 

reproducibility of the results obtained with the MTT and MRT assays.

6. The test protocol should be descriptive enough to be fully transferable to a functional 

laboratory. The protocol should describe the methodology of the assay in a clear and concise 

manner so that a laboratory could comprehend the objective, conduct the assay, observe and 

measure prescribed endpoints, compile and prepare data for statistical analyses, and report the 

results. Section 4 of the MMT ISR outlines the process of and challenges experienced in the 

inter-laboratory studies and Section 5 presents the optimization and proposed Medaka 

Reproduction Test (MRT) protocol. 

Please comment on the transferability across labs and provide any suggestions or 

recommendations for improvement of the.MMT and MRT assays.

7. The purpose of the validation process is to determine the readiness of a test for inclusion in a 

testing program. A component of readiness of a test is the evaluation of the usefulness and 

limitations of the test, including the classes and types of test substances that can and cannot be 

tested. 

Please comment on the strengths and/or limitations of the MMT and MRT assays. 

8. There is sufficient evidence to indicate that EDCs can disrupt normal development and 

reproductive success, however the value added of the F2 generation compared to the P0 or F1 is 

less clearly defined. The EPA is proposing the Medaka Reproduction Test (MRT) as an EDSP 

Tier 2 assay. The MRT test terminates after the F2 embryo hatch, and a rationale for the 

proposed protocol is provided in Section 5 of the MMT ISR. 

Please comment on the Agency’s rationale that the value added by the F2 generation is 

not sufficient to warrant its inclusion in the Tier 2 fish test protocol. 

9. It is the Agency’s opinion that the outcomes of the various MMT trials have provided enough 

information to recommend a medaka reproduction test (MRT) for use as the fish test in Tier 2 

of the EDSP. Two major changes from the MMT are proposed, i.e., an increase in the number 

of replicates per treatment for evaluating effects on reproduction, and terminating the test after 
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the embryos hatch in F2. Other proposed changes include minimizing the collection of endpoint 

data from F0, and evaluating pathology in only the F1 adults sampled after the assessment of 

reproduction.

Overall, the authors conclude that both the MMT and the MRT are transferable methods and 

are capable of adequately characterizing potential disruption of the endocrine system by 

putative endocrine disrupting chemicals. However, the MRT is recommended as the preferred 

EDSP Tier 2 test method for fish because it is less resource intensive with improved statistical 

power, appears to be as sensitive, and is better able to ensure consistent findings when 

performed routinely by testing laboratories. 

Please comment on the scientific rationale of the Agency’s proposed Tier 2 fish test, the 

MRT, with respect to statistical power, sensitivity, and consistency in performance across 

laboratories. In addition, please comment on the adequacy of the MRT to characterize 

potential endocrine disruption, a requirement of Tier 2 of the EDSP.
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Larval Amphibian Growth and Development Assay (LAGDA)

1. A rationale for the test method should be available, including a clear statement of scientific 

basis and the regulatory purpose and need for the test method. The EDSTAC introduced the 

Tier 2 tests as having the purpose to “characterize the nature, likelihood, and dose-response 

relationship of endocrine disruption of estrogen, androgen, and thyroid in humans and 

wildlife.” Tier 2 tests were designed to be definitive tests which generate sufficient data to 

characterize the specific hazard of the substance and provide sufficient information on doseresponse and adverse effects to permit risk decisions. 

Please comment on the rationale and purpose of the assay as part of the Tier 2 testing in

the EDSP, as described in Section 3 of the LAGDA ISR. 

2. Test methods and their associated endpoint(s) should be scientifically relevant to the 

biological processes of interest and should be demonstrated to be responsive to the specific 

type of effect/toxicity of interest. Each species presents unique characteristics from a 

biological perspective and allows for specialized endpoints to address a specific toxicological 

mode of action. 

Please comment on the biological and toxicological relevance of the assay in regards to 

the stated purpose of characterizing endocrine disruptors, as described in Sections 3 and 

4 of the LAGDA ISR. 

3. The test protocol should be sufficiently detailed and should include a description of what is

measured and how it is measured. The selection of endpoints within the assay should be 

reflective of the biological processes of interest and the endpoints should be intrinsically 

relevant and have established sensitivity. The test protocol should demonstrate the ability to 

measure the endpoints and provide adequate performance criteria for evaluation. 

3.1 Please comment on the selection, optimization and demonstration of the assay 

endpoints, as outlined in Sections 3 and 4 of the LAGDA ISR. 

Three of the four labs in the inter-laboratory validation of the LAGDA were unsuccessful in 

properly performing the thyroxine (T4) analyses using commercially available enzyme-linked 

Immunosorbent assay (ELISA) kits with antibodies specific for human or canine T4. 

Subsequently, the EPA developed an extraction method (Section 5.3.2) and presents a revised 

ELISA method for measuring T4 in amphibian samples in Appendix 7 of the LAGDA ISR. 

3.2 Please comment on the technical feasibility, reproducibility, and accuracy of the 

revised ELISA T4 measurement method. Provide any recommendations regarding 

additional guidance to ensure the reproducibility of T4 measurements. 
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Vitellogenin (VTG) is an established biomarker of estrogenic exposure and is used as a key

endpoint in endocrine disruptor testing. There is currently not a standardized commercial 

source for Xenopus laevis VTG antibodies for an ELISA. 

3.3 Please comment on the protocol for measuring and reporting VTG levels. Provide 

any recommendations regarding additional guidance to ensure the consistency, 

repeatability and reproducibility of VTG measurements. 

4. Demonstration of the test method performance should be based on the testing of reference 

chemicals representative of the types of substances for which the test method will be used. 

Test substances should adequately represent an appropriate range of responses and 

physical/chemical properties for which the test method is proposed to be appropriate. The 

selection of the most appropriate statistical approaches depends in part on the nature of the data 

and also on the design of the validation study. Statistical and non-statistical methods used to 

analyze should be described. 

Please comment on the selection of test substances and methods (analytical and statistical 

where appropriate) chosen for the demonstration and validation of the LAGDA assay. 

5. Considering the variability inherent in biological and chemical test methods, a test method 

needs to be repeatable and reproducible. A test is robust and reliable if the results are 

repeatable and reproducible within a laboratory and between different laboratories, respectively. 

A test protocol should provide sufficient guidance to ensure proper and consistent performance 

across labs and chemicals. 

Please comment on the test method robustness and reliability and the repeatability and 

reproducibility of the results obtained with the LAGDA assay. 

6. The test protocol should be descriptive enough to be fully transferable to a functional 

laboratory. The protocol should describe the methodology of the assay in a clear and concise 

manner so that a laboratory could comprehend the objective, conduct the assay, observe and 

measure prescribed endpoints, compile and prepare data for statistical analyses, and report the 

results. Sections 5 and 6 of the LAGDA ISR outline the process of and challenges experienced 

in the inter-laboratory studies. 

Please comment on the transferability across labs and provide any suggestions or 

recommendations for improvement of the LAGDA assay.

7. The purpose of the validation process is to determine the readiness of a test for inclusion in a 

testing program. A component of readiness of a test is the evaluation of the usefulness and 

limitations of the test, including the classes and types of test substances that can and cannot be 

tested. 

Please comment on the strengths and/or limitations of the LAGDA assay. 
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Mysid Two-Generation Toxicity Test (MTTT)

1. A rationale for the test method should be available, including a clear statement of scientific 

basis and the regulatory purpose and need for the test method. The EDSTAC introduced the 

Tier 2 tests as having the purpose to “characterize the nature, likelihood, and dose-response 

relationship of endocrine disruption of estrogen, androgen, and thyroid in humans and 

wildlife.” Although the hormones produced and used by invertebrates are not directly 

analogous to those of vertebrates (e.g., estrogen, androgen, and thyroid), growth, reproduction, 

development, and other aspects of invertebrate physiology and life cycle are known to be under 

endocrine control. EDSTAC went on to note that “chemicals that affect these vertebrate 

hormones may also affect invertebrate hormones resulting in altered reproduction, 

development, and growth.” Tier 2 tests were designed to be definitive tests which generate 

sufficient data to characterize the specific hazard of the substance and provide sufficient 

information on dose-response and adverse effects to permit risk decisions. 

Please comment on the rationale and purpose of the assay as part of the Tier 2 testing in

the EDSP, as described in Sections 2.1 and 2.2 of the MTTT ISR. 

2. Test methods and their associated endpoint(s) should be scientifically relevant to the 

biological processes of interest and should be demonstrated to be responsive to the specific 

type of effect/toxicity of interest. Each species presents unique characteristics from a 

biological perspective and allows for specialized endpoints to address a specific toxicological 

mode of action. 

Please comment on the biological and toxicological relevance of the assay in regards to 

the stated purpose of characterizing endocrine disruptors, as described in Section 2 of the 

MTTT ISR. 

3. The test protocol should be sufficiently detailed and should include a description of what is 

measured and how it is measured. The selection of endpoints within the assay should be 

reflective of the biological processes of interest and the endpoints should be intrinsically 

relevant and have established sensitivity. The test protocol should demonstrate the ability to 

measure the endpoints and provide adequate performance criteria for evaluation. 

Please comment on the selection, optimization and demonstration of the assay endpoints, 

as outlined in Sections 3 and 4 of the MTTT ISR. 

4. Demonstration of the test method performance should be based on the testing of reference 

chemicals representative of the types of substances for which the test method will be used. 

Test substances should adequately represent an appropriate range of responses and 

physical/chemical properties for which the test method is proposed to be appropriate. The 

selection of the most appropriate statistical approaches depends in part on the nature of the data 

and also on the design of the validation study. Statistical and non-statistical methods used to 

analyze should be described. 
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4.1 Please comment on the selection of test substances and methods (analytical and 

statistical where appropriate) chosen for the demonstration and validation of the MTTT 

assay. 

The proposed MTTT study design uses three replicates per control and treatment group and 

additional replicates require greater resources. 

4.2 Please comment on the proposed level of replication and the subsequent statistical 

analysis of the data. 

5. Considering the variability inherent in biological and chemical test methods, a test method 

needs to be repeatable and reproducible. A test is robust and reliable if the results are 

repeatable and reproducible within a laboratory and between different laboratories, respectively. 

A test protocol should provide sufficient guidance to ensure proper and consistent performance 

across labs and chemicals. 

Please comment on the test method robustness and reliability and the repeatability and 

reproducibility of the results obtained with the MTTT assay.

6. The test protocol should be descriptive enough to be fully transferable to a functional 

laboratory. The protocol should describe the methodology of the assay in a clear and concise 

manner so that a laboratory could comprehend the objective, conduct the assay, observe and 

measure prescribed endpoints, compile and prepare data for statistical analyses, and report the 

results. Section 5 of the MTTT ISR outlines the process of and challenges experienced in the 

inter-laboratory studies. 

6.1 Please comment on the transferability across labs and provide any suggestions or 

recommendations for improvement of the MTTT assay.

Based on the validation results using the two different invertebrate species, mysid (MTTT) and 

copepod (HCDRT), the EPA is proposing the mysid protocol as the preferred Tier 2 assay. 

6.2 Please comment on the rationale to recommend the mysid protocol as the preferred 

Tier 2 invertebrate assay, as described in Section 6.4 of the MTTT ISR.

7. The purpose of the validation process is to determine the readiness of a test for inclusion in a 

testing program. A component of readiness of a test is the evaluation of the usefulness and 

limitations of the test, including the classes and types of test substances that can and cannot be 

tested. 

Please comment on the strengths and/or limitations of the assay, as described in Section 6 

of the MTTT ISR. 
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8. There is sufficient evidence to indicate that EDCs can disrupt normal development and 

reproductive success, however the sensitivity of the F1 generation compared to the F0 is less 

clearly defined. 

Please comment from a scientific and risk assessment perspective on the value added of 

multiple generations in the MTTT assay