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FIFRA Scientific Advisory Panel Meeting May 21

- 23, 20 13

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 scree ning 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

c ompounds, 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. T o 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 ea ch 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 p hysical

- 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
demonstrate s that the laboraotories executing the assays can meet the performance2 | Page

critieria defined in the test guidelines and the results provide useful information to determine whether a chemical has the potential inter act 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 a nd ach ieve 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), H295 R

stero i dogenesis (OCSPP 890.1550) and aromatase human recombinant (OCSPP 890.1200) assays. Although contributing laboratories did no t 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 890.150 0) 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 wou ld render the study unreliable.

Charge Question 4

. T he 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/conce ntration 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 highest3 | Page

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 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 sc reening 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 th ese 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
lab oratories 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, O ECD Guidance Document on Amphibian Thyroid Histology No. 82, 2007 and Grim et al.

, 2009 ).4 | Page

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 ta dpole development.
Please comment on th e 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 w ithin 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 t he anticipated complementarity and redundancy in the Tier 1 battery and
whether or n ot a modified battery of assays could redu ce 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 include d 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 app eared 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 res ponses with the case studies. Please comment separately on the E

- , A

- , and T

- related assays and endpoints.5 | Page

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 . Plea se 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 inter act with the endocrine system.

a. In 1998, the EDSTAC described the c onceptual f ramework for Tier 1 assays and recommended the strategy to “ require the minimal number of
screens and te sts 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 exte nt can the current Tier 1 battery of 11 assays be modi fied 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 sp ecifically, p lease

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 ass ays.

b

. Please comment on the scientific criteria the A gency should consider in evaluating necessary redundancies and eliminating assays from the current battery

.