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FIFRA Scientific Advisory Panel Meeting May 21-23, 2013

Endocrine Disruptor Screening Program (EDSP) Tier 1

Screening Assays and Battery Performance

CHARGE QUESTIONS

As recommended by a Federal Advisory Committee, (Endocrine Disruptor Screening 

and Testing Advisory Committee; EDSTAC, 1998), the EPA established a two-tiered 

Endocrine Disruptor Screening Program (EDSP) to address the potential of chemicals 

to pose risks to humans and wildlife by interacting with the estrogen, androgen or 

thyroid (E, A or T) hormonal pathways. To address whether a chemical interacts with E, 

A, or T, the EPA has designed the Tier 1 screening battery of 11 assays that have been 

validated and subsequently published as Office of Chemical Safety and Pollution 

Prevention (OCSPP) harmonized test guidelines (Series 890).

In 2008, the FIFRA SAP submitted their review of the EDSP Tier 1 screening battery 

and concluded that “[t]he ability to identify endocrine active substances is enhanced in 

the Tier 1 battery because the tests provide adequate replication and redundancy.” The 

FIFRA SAP recommended that the agency “continue to develop, refine, and review the 

battery.” (SAP, 2008). Moreover, a joint committee of the SAB/SAP reviewed the initial 

framework for an EDSP (USEPA, 1999) and recommended the EPA “...should convene 

a panel of independent scientists to review all the screening data for 50-100 

compounds, with an eye towards revising the process and eliminating those methods 

that don’t work.”

The purpose of this FIFRA SAP review is to evaluate the performance of EDSP Tier 1 

screening assays and battery. To this end, the agency’s review is presented in two 

parts: 1. individual assay performance, and 2. battery performance. The SAP charge 

questions below are presented in this sequence.

Individual EDSP Tier 1 Assay Performance

The results of the EPA’s evaluation to determine how well each Tier 1 assay was 

executed relative to defined performance criteria in the respective test guideline are 

presented in Section III of the white paper. This evaluation was based on a subset of 

pesticide active ingredients that represent a broad range of physical-chemical properties 

(e.g., water solubility, octanol-water partition coefficients, volatility), as well as a range of 

chemical classes and pesticidal activities. This preliminary analysis generally 

demonstrates that the laboraotories executing the assays can meet the performance 
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critieria defined in the test guidelines and the results provide useful information to 

determine whether a chemical has the potential interact with the endocrine system. 

During this initial analysis of individual study data, however, EPA identified a few issues 

with some assays. The following questions seek comment from the FIFRA SAP on 

assay (laboratory) performance and advice on how to address technical deviations and

other issues encountered with specific assays.

Charge Question 1. Based on the analysis of the data presented in Section III, please 

comment on the proficiency of the contributing laboratories to execute each assay in 

accordance with the test guidelines and achieve the performance criteria.

Charge Question 2. The performance criteria for each in vitro assay are clearly stated 

in the test guidelines for the ER binding (OCSPP 890.1250), AR binding (OCSPP 

890.1150), ERα Transcriptional Activation (OCSPP 890.1300, OECD 455), H295R

steroidogenesis (OCSPP 890.1550) and aromatase human recombinant (OCSPP 

890.1200) assays. Although contributing laboratories did not always demonstrate that 

results were within the specified boundaries of the performance criteria, the majority of 

the deviations were still close to the performance criteria. In this regard, the EPA

concluded that the data were still adequate for use. Please comment on the EPA’s 

conclusion. Please comment on when a deviation from the recommended performance 

criteria would render the study unreliable.

Charge Question 3. Unlike the Hershberger and Uterotrophic assays, a positive control 

is not required in the male (OCSPP 890.1500) and female (OCSPP 890.1450) pubertal

assays. For these in vivo assays with rats, coefficient of variation (CV) limits are 

specified in the test guidelines for most endpoints. Submissions from different 

laboratories sometimes fell short of meeting all the test guideline-recommended CV 

limits for the endpoints evaluated. However, in most cases these shortcomings were 

considered of minor importance to the overall results, and EPA concluded that the data 

are still adequate for endocrine screening. Please comment on the EPA’s conclusion. 

Please comment on when a deviation from the recommended CV limits would render 

the study unreliable.

Charge Question 4. The test guidelines for the six in vivo assays (Hershberger assay -

OCSPP 890.1400, OECD 441; Uterotrophic assay- OCSPP 890.1600, OECD 440; Male 

Pubertal assay- OCSPP 890.1500; Female Pubertal assay - OCSPP 890.1450; FSTRA 

- OCSPP 890.1350, OECD 229 and AMA - OCSPP 890.1100) offer some guidance on 

setting the dose/concentration range when testing for specific effects on the E, A, or T 

signaling pathways. In some of the in vivo assays, overt toxicity was noted based on 

effects on growth, other sublethal effects, and even mortality at the highest 
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dose/concentration tested. Positive Tier 1 findings indicating the potential for endocrine 

activity can be difficult to interpret in the presence of overt toxicity.

Please comment on the nature and severity of overt toxicity that would render study 

results unreliable for the purpose of Tier 1 screening of a chemical for endocrine 

activity. Please comment on what, if any, additional guidance is needed to minimize 

overt toxicity while ensuring adequate dose selection for these screening assays. Also, 

specifically comment on the validity of including treatment concentrations with apparent 

body weight effects in the analysis for potential endocrine interaction for FSTRA?

Charge Question 5. Spinal curvature, usually manifesting as “bent tail” in X. laevis

tadpoles, was reported in 15 of 18 AMA studies reviewed thus far. The anomaly 

appears to be first observed several days after study initiation, and prevalence 

increases with time. Overall, the prevalence of spinal curvature in these studies ranged 

from “a few per replicate” to 92% of a given treatment group by test termination. 

Experimental work by the EPA Office of Research and Development suggests that 

overfeeding can be a primary cause of spinal curvature in their Xenopus test

populations; however, spinal curvature remained prevalent (range: 16-92%) in the five 

industry AMA studies in which feed was reduced by 50% compared to guideline 

recommendations.

Overall, the incidence of spinal curvature appears to be highly variable. From a 

qualitative review of the data, there appear to be no consistent differences in the 

incidence or variability of spinal curvature when studies using guideline versus reduced 

feeding regimes are compared. Please comment on whether the presence or 

prevalence of spinal curvature in test specimens, including controls, compromises the 

utility or validity of an AMA submission. If so, when does the prevalence of spinal 

curvature render the study unreliable? What technical guidance may be useful for 

laboratories in reducing the occurrence of spinal curvature and determining if, or at what 

point within the study, a study may be compromised because of this phenomenon?

Charge Question 6. With the exception of thyroid gross pathology findings (thyroid 

gland atrophy and hypertrophy) in the AMA, severity grades are generally assigned 

based on comparison to “normal” X. laevis thyroid findings depicted in the guidance or 

based on the professional opinion of the pathologist conducting the assessment; they 

are not assigned in comparison to concurrent control findings from a given study. 

(Please refer to Section III.2.f in the document entitled “Interpreting Amphibian Thyroid 

Histopathology Diagnoses” and supporting documents, OECD Guidance Document on

Amphibian Thyroid Histology No. 82, 2007 and Grim et al., 2009).
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a. In one study, the pathologist’s report identified a lower incidence and 

severity of follicular cell hypertrophy when compared to the incidence and 

severity of this trait in control specimens. Similar trends have been observed in 

other studies. In this case, the pathologist concluded that the finding was 

potentially consistent with treatment-related delay of metamorphosis because 

thyroid follicular cells normally increase in height during tadpole development. 

Please comment on the validity of this conclusion.

b. What guidance may be given to better distinguish between histological 

changes in the thyroid associated with the normal progression of metamorphosis 

and treatment-related effects? Are there certain lesions or diagnoses which may, 

by their absence or lessened severity as compared to controls, be indicative of 

treatment-related HPT effects such as delayed metamorphosis?

EDSP Tier 1 Screening Battery Performance

The Tier 1 Battery of 11 validated assays is intended to function as a suite of assays to 

screen for the “potential” of a chemical to interact with the E, A, or T hormonal pathways 

of humans and wildlife. Section IV of the white paper describes the performance of the 

Tier 1 Battery using assay data from a subset of chemicals that were issued Test 

Orders in 2009 and 2010. The purpose of this initial analysis is to evaluate 

“complementarity” relationships or concordance among endpoints within an assay and 

“redundancy” or the concordance of endpoints across assays that are intended to 

evaluate an interaction with a particular hormonal pathway (E, A, or T). Section IV 

discusses the anticipated complementarity and redundancy in the Tier 1 battery and 

whether or not a modified battery of assays could reduce animal usage and lower costs

while adequately ensuring the identification of potential interactions with the E, A or T 

hormonal pathways.

Charge Question 7. In 2008, the SAP acknowledged that the in vivo assays included in 

the Tier 1 battery provide both redundancy and complementarity for evaluating 

interactions with the E, A, or T signaling pathways. The panel also noted that all of the 

Tier 1 assays and the broad range of endpoints appeared to be necessary to 

“discriminate positive and negative results”.

a. Please comment on the battery performance with respect to the 

anticipated complementary nature of the more complex, multi-parameter in vivo

assays in the context of the observed responses with the case studies. Please 

comment separately on the E-, A-, and T-related assays and endpoints.
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b. Please comment on the battery performance with respect to the 

anticipated redundancy across the 11 assays in the context of the observed 

responses with the case studies. Please comment separately on the E-, A-, and 

T-related assays and endpoints.

c. The EPA concluded that the battery has performed as anticipated by the 

2008 SAP. Please comment on this conclusion.

Charge Question 8. The EPA is committed to minimizing animal usage in the 

screening battery while maintaining the effectiveness of the battery to answer the 

question of whether a chemical has the “potential” to interact with the endocrine system.

a. In 1998, the EDSTAC described the conceptual framework for Tier 1 

assays and recommended the strategy to “require the minimal number of 

screens and tests necessary to make sound decisions, thereby reducing the time 

needed to make these decisions”, and that the screens should be conducted at a 

minimal cost necessary to make decisions. Based on the preliminary battery 

performance evaluation, to what extent can the current Tier 1 battery of 11 

assays be modified to reduce animal usage and/or lower cost while adequately 

ensuring the EPA’s ability to answer the question of “whether a chemical has the 

potential to interact with the endocrine system?” More specifically, please 

comment on whether the Uterotrophic and Hershberger assays provide 

necessary redundancies in the Tier 1 battery based on this preliminary analysis. 

Please include in your comments what information may be lost and what 

uncertainties may be introduced by absence of either or both of these assays.

b. Please comment on the scientific criteria the Agency should consider in 

evaluating necessary redundancies and eliminating assays from the current 

battery.