FIFRA SAP Review of Tier 1 

Screening Assays and 

Battery Performance

Charge Questions

Office of Chemical Safety and Pollution Prevention

U.S. Environmental Protection Agency
Individual EDSP 

Tier 1 Assay Performance

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Overview

Laboratory performance of individual assays were 

generally conducted in accordance with test guidelines 

but initial analysis indicated specific issues with some 

assays.

The following charge questions seek comment from the 

SAP on laboratory performance of individual assays 

and advice on how to address technical deviations and 

other assay-specific issues.

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Individual EDSP Tier 1 Assay Performance

Charge Question 1: 

Based on analyses 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.

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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.

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Individual EDSP Tier 1 Assay Performance

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Individual EDSP Tier 1 Assay Performance

Charge Question 2 (continued): 

•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.

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Individual EDSP Tier 1 Assay Performance
Charge Question 3 (continued):

•Please comment on the EPA’s conclusion.

•Please comment on when a deviation from the 

recommended CV limits would render the study unreliable.

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Individual EDSP Tier 1 Assay Performance
Charge Question 4: 

The test guidelines for the six in vivo assays (Hershberger 

assay - OCSPP 890.1400, OECD 441; Uterotrophic assayOCSPP 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 

dose/concentration tested. Positive Tier 1 findings indicating 

the potential for endocrine activity can be difficult to interpret 

in the presence of overt toxicity.

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Individual EDSP Tier 1 Assay Performance

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Charge Question 4 (continued): 

•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.

Specifically comment on the validity of including 

treatment concentrations with apparent body weight 

effects in the analysis for potential endocrine interaction

for FSTRA.

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Individual EDSP Tier 1 Assay Performance
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 

certain feeding regimes are associated with 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.

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Individual EDSP Tier 1 Assay Performance
Charge Question 5 (continued): 

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?

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Individual EDSP Tier 1 Assay Performance

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Charge Question 5 (continued): 

•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?

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Individual EDSP Tier 1 Assay Performance
Charge Question 6 (a & b):

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.

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Individual EDSP Tier 1 Assay Performance
Charge Question 6a:

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.

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Individual EDSP Tier 1 Assay Performance
Charge Question 6b:

•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?

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Individual EDSP Tier 1 Assay Performance
EDSP Tier 1 Screening 

Battery Performance
Based on evaluation of “complementary” relationships or 

concordance among endpoints within an assay and 

“redundancy” or the concordance of endpoints across assays 

that are intended to detect an interaction with a particular E, 

A, or T hormonal pathway, the following charge questions 

seek comment from SAP on 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 E, A or T.

Overview

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EDSP Tier 1 Screening Battery Performance

Charge Question 7 (a, b & c):

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.”

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Charge Question 7a:

•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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EDSP Tier 1 Screening Battery Performance
Charge Question 7b:

•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.

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EDSP Tier 1 Screening Battery Performance
Charge Question 7c:

The EPA concluded that the battery has performed as 

anticipated by the 2008 SAP.

•Please comment on this conclusion.

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EDSP Tier 1 Screening Battery Performance
Charge Question 8 (a & b):

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.

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EDSP Tier 1 Screening Battery Performance
Charge Question 8a:

•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?”

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.

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EDSP Tier 1 Screening Battery Performance

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Charge Question 8a:

•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?”

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.

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EDSP Tier 1 Screening Battery Performance

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Charge Question 8b:

•Please comment on the scientific criteria the Agency should 

consider in evaluating necessary redundancies and 

eliminating assays from the current battery.

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EDSP Tier 1 Screening Battery Performance
This Concludes the 

Reading of the Charge Questions