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 38Question 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 38Question 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 F 2 

### generation compared to the P 0 

### or F 1 

### 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 8

There is sufficient evidence to indicate that EDCs can disrupt normal development and reproductive success, however the value added of

the F 2 generation compared to the P 0 or F 1 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 F 2 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 38Question 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

F 2 . Other proposed changes include minimizing the collection of endpoint data from F 0 , and evaluating pathology in only the F 1 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 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 38Question 8

There is sufficient evidence to indicate that EDCs can disrupt normal

development and reproductive success, however the sensitivity of the

F 1 generation compared to the F 0 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