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 concentration-response 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. 

Charge Questions

Slide 1 of 38
Question 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 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 2.5 and 2.6 of 

the JQTT ISR. 

Japanese Quail Two-Generation Test (JQTT)

Charge Questions

Slide 2 of 38
Question 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. 

Japanese Quail Two-Generation Test (cont.) 

Slide 3 of 38
Question 3.1

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 4 

and 5 of the JQTT ISR. 

Japanese Quail Two-Generation Test (cont.) 

Slide 4 of 38
Question 3.2

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 

inter-laboratory validation and a discussion of histopathology as an 

endpoint is provided in Section 4.1.13 of the JQTT ISR. 

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. 

Japanese Quail Two-Generation Test (cont.) 

Slide 5 of 38
Question 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. 

Japanese Quail Two-Generation Test (cont.) 

Slide 6 of 38
Question 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. 

Japanese Quail Two-Generation Test (cont.) 

Slide 7 of 38
Question 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. 

Japanese Quail Two-Generation Test (cont.) 

Slide 8 of 38
Question 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. 

Japanese Quail Two-Generation Test (cont.) 

Slide 9 of 38
Question 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. 

Please comment from a scientific and risk assessment 

perspective on the value added of multiple generations in the 

JQTT assay. 

Japanese Quail Two-Generation Test (cont.) 

Slide 10 of 38
Question 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 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 MMT ISR. 

Medaka Multigeneration Test (MMT)

Charge Questions

Slide 11 of 38
Question 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. 

Medaka Multigeneration Test (cont.)

Slide 12 of 38
Question 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.

Medaka Multigeneration Test (cont.)

Slide 13 of 38
Question 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 MMT and MRT assays. 

Medaka Multigeneration Test (cont.)

Slide 14 of 38
Question 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 

MMT and MRT assays.

Medaka Multigeneration Test (cont.)

Slide 15 of 38
Question 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.

Medaka Multigeneration Test (cont.)

Slide 16 of 38
Question 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. 

Medaka Multigeneration Test (cont.)

Slide 17 of 38
Question 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. 

Medaka Multigeneration Test (cont.)

Slide 18 of 38
Question 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 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.

Medaka Multigeneration Test (cont.)

Slide 19 of 38
Question 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 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 Section 3 of the LAGDA 

ISR. 

Larval Amphibian Growth and Development Assay (LAGDA)

Charge Questions

Slide 20 of 38
Question 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. 

Larval Amphibian Growth and Development Assay (cont.)

Slide 21 of 38
Question 3.1

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 LAGDA 

ISR. 

Larval Amphibian Growth and Development Assay (cont.)

Slide 22 of 38
Question 3.2

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. 

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. 

Larval Amphibian Growth and Development Assay (cont.)

Slide 23 of 38
Question 3.3

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. 

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. 

Larval Amphibian Growth and Development Assay (cont.)

Slide 24 of 38
Question 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. 

Larval Amphibian Growth and Development Assay (cont.)

Slide 25 of 38
Question 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. 

Larval Amphibian Growth and Development Assay (cont.)

Slide 26 of 38
Question 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.

Larval Amphibian Growth and Development Assay (cont.)

Slide 27 of 38
Question 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.

Larval Amphibian Growth and Development Assay (cont.)

Slide 28 of 38
Question 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. 

Mysid Two-Generation Toxicity Test (MTTT)

Charge Questions

Slide 29 of 38
Question 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. 

Mysid Two-Generation Toxicity Test (cont.)

Slide 30 of 38
Question 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. 

Mysid Two-Generation Toxicity Test (cont.)

Slide 31 of 38
Question 4.1

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 MTTT assay. 

Mysid Two-Generation Toxicity Test (cont.)

Slide 32 of 38
Question 4.2

The proposed MTTT study design uses three replicates per control and 

treatment group and additional replicates require greater resources. 

Please comment on the proposed level of replication and the 

subsequent statistical analysis of the data. 

Mysid Two-Generation Toxicity Test (cont.)

Slide 33 of 38
Question 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.

Mysid Two-Generation Toxicity Test (cont.)

Slide 34 of 38
Question 6.1

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. 

Please comment on the transferability across labs and provide any 

suggestions or recommendations for improvement of the MTTT 

assay.

Mysid Two-Generation Toxicity Test (cont.)

Slide 35 of 38
Question 6.2

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. 

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.

Mysid Two-Generation Toxicity Test (cont.)

Slide 36 of 38
Question 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. 

Mysid Two-Generation Toxicity Test (cont.)

Slide 37 of 38
Question 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.

Mysid Two-Generation Toxicity Test (cont.)

Slide 38 of 38