## 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

2

# 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 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 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

th at 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 PerformanceBased 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