USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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# U.S. Environmental Protection Agency Endocrine Disruptor Screening Program 

# SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance

May 21

- 23, 2013

This document was developed by the EPA to provide general background and technical information to support and facilitate SAP review of the performance of EDSP Tier 1 screening assays and battery

.
This document does not create or confer legal rights or impose any legally binding requirements on the EPA or any other party. This document is distributed solely for the purpose of sharing this information with the public, consistent with the EPA transparency objectives. It is not intended to serve any other purpose, and should not be construed to represent formal dissemination of any agency determination or policy. As such, the information correction process under the agency's Information Quality Guidelines does not apply to this document.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 2 of 160 Table of Contents

SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance

List of Acronyms ............................................................................................................................ 6
Executive Summary ........................................................................................................................ 9

I. Introduction ........................................................................................................................... 13

II. Methods ................................................................................................................................. 20

III. Evaluation of Results from Individual Assays ...................................................................... 24

1. In vitro ................................................................................................................................ 24

a. ER Binding .................................................................................................................................. 24

b. ERα Transcriptional Activation .................................................................................................. 32

c. AR Binding ................................................................................................................................. 37

d. Steroidogenesis ........................................................................................................................... 45

e. Aromatase (Human Recombinant) Assay ................................................................................... 53

2. In vivo

................................................................................................ ................................. 59

a. Uterotrophic ................................................................................................................................ 59

b. Hershberger ................................................................................................................................. 64

c. Pubertal Female .......................................................................................................................... 70

d. Pubertal Male .............................................................................................................................. 78

e. Fish short- te rm reproduction ....................................................................................................... 86

f. Amphibian Metamorphosis Assay ............................................................................................ 110

IV. Performance of Battery ....................................................................................................... 130

1. Interaction with the HPT Axis ......................................................................................... 131

2. Interaction with the Androgen Pathway .......................................................................... 141

3. Interaction with the Estrogen Hormonal Pathway ........................................................... 147

V. Overall Summary and Conclusions ..................................................................................... 152

VI. References ........................................................................................................................... 156USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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### List of Tables

Table 1. Current EDSP Tier 1 Screening Assays. ...................................................................................... 16
Table 2. Number of Chemicals Included in the Assay and Battery Performance Analysis ....................... 22 Table 3. Overview of Case Study Chemical Product Types and Chemical Classes. ................................. 23 Table 4. ER Binding Classification Results for the 18 Chemicals Evaluated. ........................................... 27 Table 5. Performance Criteria for Saturation Binding ............................................................................... 27 Table 6. Cytosolic Preparations Used for More than One Chemical. ........................................................ 28 Table 7. Upper and Lower Limits for Parameters in Competitive Binding Assay Curves for Positive
Control. ..................................................................................................................................... 29
Table 8. Classification of the 19 Chemicals Evaluated in the ERTA ........................................................ 35 Table 9. AR Binding Classification Results for the 17 Chemicals Evaluated ........................................... 39
Table 10. Performance Criteria for Satura tion Binding ............................................................................. 40 Table 11. Prostate Cytosol Preparations Used for More Than One Chemical. .......................................... 41 Table 12. Upper and Lower Limits for Parameters in Competitive Binding Assay Curves for Strong and
Weak Positive Controls. ........................................................................................................... 42 Table 13. Performance Criteria to be Met on Each QC Plate .................................................................... 49 Table 14. Summary of Fold- Change in Estradiol and Testosterone Levels ............................................... 51
Table 15. Results from the 18 Steroidogenesis Studies. ............................................................................ 52 Table 16. Summary of the High Test Concentration for the 18 Aromatase Assays. ................................. 55 Table 17. Classification of the 18 Chemicals Evaluated in the Aromatase Assay. .................................... 56 Table 18. Performance Criteria for Aromatase Assay ............................................................................... 56 Table 19. Summary of Overt Toxicity Observed in the Uterotrophic Assay ............................................. 60 Table 20. Summary of Overt Toxicity Observed in the Hershberger Assay. ............................................ 66 Table 21. Maximum recommended % CVs for the Mandatory Sex Accessory Tissues. .......................... 68 Table 22. Summary of Overt Toxicity Observed in the Pubertal Female Assay ....................................... 73 Table 23. Performance Criteria for Controls (Sprague

- Dawley Strain). .................................................... 77 Table 24. Summary of Overt Toxicity Observed in the Pubertal Male Assay. .......................................... 81 Table 25. Performance Criteria for Controls (Sprague

- Dawley and Wistar Strains). ................................ 85 Table 26. Test Validity Elements for the FSTRA ...................................................................................... 91 Table 27. Identification of the Only Test Validity Criteria Not Achieved in 11 Studies ........................... 92 Table 28. Summary of the Types of Overt Toxicity Observed in the 20 Assays ....................................... 98 Table 29: Validity Elements for the Amphibian Metamorphosis Assay. ................................................ 115 Table 30: General Performance Criteria for the Amphibian Metamorphosis Assay, when Conducted

Un der Flow-Through or Static-Renewal Conditions .............................................................. 115 Table 31: Additional Performance Criteria for the Amphibian Metamorphosis Assay, when Conducted
Under Static-Renewal Conditions .......................................................................................... 116 Table 32: Prevalence of Spinal Curvature in the Amphibian Metamorphosis Assay. ............................. 120

T able 33: Developmental and Thyroid Gross Pathology/Histopathology Endpoints , in the AMA with
Chemical F. ............................................................................................................................. 123 Table 34: Growth Endpoints a,b in the AMA with Chemical F. ................................................................ 123 Table 35. Endpoints for Detecting Potential Effects on the Thyroid Hormonal Pathway According to the
2008 FIFRA SAP. ................................................................................................................... 132 Table 36. Preliminary Results of Tier 1 Assays Relevant to the HPT Axis or Thyroid Hormonal Pathway, by Case Study Chemical. ........................................................................................................ 134USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 4 of 160 Table 37. Associations between Endpoint Responses in Tier 1 Assays Relevant to the HPT and Thyroid Hormonal Pathway, by Case Study Chemical (Preliminary). ................................................. 136 Table 38. Endpoints for Detecting Potential Effects on the Androgen Hormonal Pathway. ................... 142 Table 39. Summary of Results in Tier 1 Assays Relevant to the Androgen Hormonal Pathway by
Chemical. ................................................................................................................................ 145 Table 40. Endpoints for Detecting Potential Effects on the Estrogen Hormonal System. ....................... 148
Table 41. S ummary of Results in Tier 1 Assays Relevant to the Estrogen Hormonal Pathway, by
Chemical. ................................................................................................................................ 151USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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### List of Figures

Figure 1. Saturation Binding Curves. ..................................................................................................... 28 Figure 2. Performance Criteria Results for the Saturation Binding for Cytosolic Preparations. ............ 29 Figure 3. ER Competitive Binding Curves ............................................................................................. 30 Figure 4. Performance Crit eria Results for the Strong Positive Control. ............................................... 31 Figure 5. Performance Criteria Results for the Saturation Binding for Cytosolic Pr eparations. ............ 41 Figure 6. Saturation Binding Curves. ..................................................................................................... 42 Figure 7. Competitive Binding Curves ................................................................................................... 43 Figure 8. Performance Criteria Results for Strong (R1881) Positive Control. ....................................... 44 Figure 9. Performance Criteria Results for Weak (Dexamethasone) Positive Control. .......................... 44 Figure 10. Steroidogenic Pathway in H295R cells a . ................................................................................. 46 Figure 11. Quality Control Plate Performance for the 18 Steroidogenesis Studies. ................................. 50 Figure 12. Concentration Response Curve for Positive Control 4

- OH ASDN. ........................................ 54 Figure 13. Aromatase Performance Criteria Results. ............................................................................... 58 Figure 14. Summary of Number of Studies Achieving Each Individual Test Validity Criteria ............... 91 Figure 15. Summary of the Number of Individual Test Validity Criteria Achieved by Each Study ........ 92 Figure 16. Summary of the Range of Reported Coefficient of Variation (CV) for the Studies .................. 96 Figure 17. Male VTG data ...................................................................................................................... 106 Figure 18. The majority of AMA study submissions reviewed to date have met the validity elements and performance criteria outlined in the test guideline. ......................................... 116 Figure 19. Suggested D ecision Logic for the Conduct and Interpretation of the AMA (OCSPP 890.1100, OECD 2009). ......................................................................................................................... 133USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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### List of Acronyms

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

AMA Amphibian Metamorphosis Assay

ANOVA Analysis of Variance

AR Androgen Receptor

ATCC American Type Culture Collection

B max Binding at maximum
CG Cowper’s Gland

ChE Cholinesterase
CV Coefficient of Variation

DER Data Evaluation Record

DMEM Dulbecco's Modified Eagle Medium

DMF Dimethyl

- Formamide

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid
DO Dissolved Oxygen

DP Dorsolateral Prostrate

DRP Detailed Review Paper

EACPD Exposure Assessment Coordination and Policy

ECVAM European Centre for the Validation of Alternative Methods

EDMVAC Endocrine Disrupt o r Methods Validation Advisory Committee

EDMVS Endocrine Disrupt o r Methods Validation Sub

- committee

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol
ELISA Enzyme Linked Immunosorb e nt Assay

E Estrogen (hormonal pathway)

ER Estrogen Receptor

ERTA Estrogen Receptor Transcr iptional Activation

EtOH Ethanol

FACA Federal Advisory Committee Act

FIFRA Federal Insecticide, Fungicide, Rodenticide Act

FFDCA Federal Food, Drug, and Cosmetic Act FQPA Food Quality Protection Act

FRN Federal Register Notice

FSHR Follicle

- Stimulating Hormone Receptor

FSTRA Fish Short

- Term Reproduction Assay

FT Flutamide

GLP Good Laboratory Practices GP Glans Penis
GSI Gonado

- Somatic Index

HBD Hormone Binding Domain

HLL Hind Limb Length

HPG Hypothalamic

- Pituitary

- Gonadal AxisUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 7 of 160 Abbreviation Terminology

HPLC/MS/MS High Pressure Liquid Chromatography/ tandem Mass Spectroscopy

HPT Hypothalamic

- Pituitary

- Thyroidal Axis

IC 50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods

ISR Integrated Summary Report

Kd Equilibrium Dissociation Constant
LABC Levator Ani

- Bulbocave rn osus LC 50 Lethal Concentration in 50% of test organisms

LHR Luteinizing Hormone Receptor

MDL Minimum Detection Level MoA Mode of Action
MRID Master Record Identifier
MTC Maximum Tolerated Concentration

MTD Maximum Tolerated Dose

NADPH Nicotinamide Adenine Dinucleotide Phosphate
NF stage Nieukoop and Faber’s Staging Atlas

NIEHS National Institute of Environmental Health Sciences

NOAEC No Observed Adverse Effect Concentration

NSB Non

- Specific Binding
OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organis ation for Economic Co

- Operation and Development
ORD Office of Research and Development

OSCP Office of Science Coordination and Policy OSRI Other Scientifically Relevant Information
PC Positive Control

PC10 Positive Control at 10% of response

PC50 Positive Control at 50% of response PND Post

- Natal Day QC Quality Control
RBA Relative Binding Affinity

RBC Red Blood Cells

RPC
max Relative to Positive Control at maximum

SAB Science Advisory Board SAP Scientific Advisory Panel
SC Solvent Control

s.c Subcutaneous

SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague

- Dawley
SVL Snout

- to

- Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T 3 3,3’,5 triiodothyronine

T 4 Thyroxine (tetraiodothyronine)

TP Testosterone Propionate
TR Thyroid ReceptorUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 8 of 160 Abbreviation Terminology

TSH Thyroid Stimulating Hormone

VC Vehicle Control

VO Vaginal Opening
VP Ventral Prostate
VTG Vitellogenin
WoE Weight

- of

- EvidenceUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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### Executive Summary

The EPA developed the Endocrine Disruptor Screening Program in response to the statutory mandate in the Federal Food, Drug, and Cosmetic Act (FFDCA) to “develop a screening program...to determine whether certain substances may have an effect in humans that i s similar to an effect produced by a naturally occurring estrogen, or such other endocrine effects as the Administrator may designate.” In addition to FFDCA, the Safe Drinking Water Act (SDWA) provides the EPA with authority to provide for testing “of any other substances that may be found in sources of drinking water if the Administrator determines that a substantial population may be exposed to such substance.” To begin meeting this statutory mandate, the EPA in 1996 chartered a Federal Advisory Committ ee to address endocrine disruption, the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC). One of the key recommendations made by the EDSTAC in 1998 was to develop a two

- tiered screening and testing strategy, now known as the EDSP. Th e two

- tiered screening and testing process is intended to ensure that only those chemicals that were screened to have potential endocrine activity would be advanced for further Tier 2 testing to determine whether the chemical interacts
with the endocrine s ystem. The purpose of the Tier 1 screening is to identify chemicals that have
the potential to interact with the estrogen, androgen or thyroid hormone systems.

EPA issued the first test orders of the Endocrine Disruptor Screening Program (EDSP) Tier 1 screening on 67 chemicals (List 1) between October 29, 2009 and February 26, 2010 ( http://www.epa.gov/endo/ ). Of these, there were eight voluntary cancellations and seven inert ingredient manufacturers that chose to "opt out" of the pesticide market. Consequently, data were submitted on 50 pesticide active ingredients and 2 inert ingredients. For some test orders, EPA accepted “other scientifically relevant information” (OSRI) in lieu of specific study data ( http://www.epa.gov/endo/ ).

A subsample of up to 21 of the 52 chemicals on List 1 provided case studies to evaluate the performance of the EDSP Tier 1 individual assays and the entire battery . The 21 chemicals were comprised of 20 compounds representing a range of chemical classes ( e.g ., azoles, imides, organophosphates) and pesticidal activities ( e.g ., insecticides, herbicide and fungicides) and one inert ingredient. The case studies provide a robust chemical and biological domain to evaluate each of the Tier 1 assays and overall battery performance given the breadth of physico

- chemical properties and given that all but one of these chemicals are designed to be biologically ac tive.

The EDSP Tier 1 screening con sists of a battery of 11 assays: five in vitro (E strogen Receptor binding , ER transcriptional activation assay, Androgen Receptor binding, Aromatase, and Steroidogenesis) and six in vivo ( Uterotrophic, Hershberger, Female Pubertal, Male Pubertal,
Fish Short- term Reproduction, and Amphibian Metamorphosis ). The assays were to identify whether a chemical has the potential to interact with estrogen, androgen or thyroid (E, A and T) hormone systems. Understanding that not all in vitro and in vivo data are equal, the in vitro data may be quite informative to the in vivo assay results. Tier 1 in vitro assays were intended toUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 10 of 160 provide some mechanistic data for single known pathways, whereas the in vivo assays would
capture mu ltiple modes of action including those not yet known mechanisms and effects in an intact organism , accounting for both toxicokinetic and toxicodynamics parameters

. The purpose of this review of the Tier 1 assays was to: (i) evaluate how well each Tier 1 a ssay test guideline can be consistently executed based on defined performance criteria and to highlight any issues associated with interpretation of responses within each assay ( discussed in Section III), (ii) evaluate the complementary relationships among responses within the assays (discussed in Section IV), and to (iii) evaluate the redundancy of responses across the Tier 1 battery ( discussed in Section IV).

Additionally, this review considered 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 ( as discussed in Section IV). No determinations concerning chemical interactions with the E, A, or T signaling pathways are made in the current effort. A more detailed analysis of chemical endocrine interaction will be presented in a future SAP on the Agency’s proposed weight -of- evidence (WoE) approach during July 30 through August 2, 2013.

E ach of the Tier 1 assays was pre viously validated with a known set of reference compounds (FR Notice, October 21, 2009) for endocrine screening specific to E, A, and T signaling pathways through an extensive process of test method development, prevalidation and interlaboratory
validation that culminated in an evaluation by a 2008 FIFRA SAP 1 , which supported using the 11 assays to initiate Tier 1 screening. Thus, one aspect the focus of current assay performance analysis i s on the proficiency of the contributing laboratories to execute each Tier 1 assay in accordance with the test guidelines

, including the proper execution of the performance criteria and to obtain the expected results for those assays utilizing positive controls. The laboratory performance of each Tier 1 assay was typic ally consistent across the 21 chemicals evaluated. Although there were situations where performance criteria were not met, those deviations were considered minor and did not impact the interpretation or reliability of the data. Overall, laboratory performance for each Tier 1 assay met expectations, and the submitted Tier 1 assay data provide the expected useful information to evaluate potential interactions of test chemicals with the E, A and T signaling pathways.

Another purpose of the current review of assay performance was to identify any issues associated with the interpretation of Tier 1 assays. The most noteworthy issue encountered was associated
with dose selection and the presence of overt toxicity ( e.g ., body weight reductions >10%, clinical signs , abnormal behavior, etc .) in the execution of some submitted Hershberger, R at
P ubertal s and the Fish Short- Term Reproduction Assays

. When positive Tier 1 findings are observed in the presence of overt toxicity there is uncertainty as to whether or not the responses are mediated through an endocrine pathway. A more detailed analysis of overt toxicity and the

1 March 25

- 28, 2008 Session of the FIFRA Scientific Advisory Panel entitled “Review of the Endocrine Disruptor Screening Program (EDSP) Proposed Tier 1 Screening Battery”.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 11 of 160 potential for chemical interactions with endocrine systems will be presented in at the WoE SAP during July 30 through August 2, 2013.

In 2008, the SAP was also asked to comment on the extent of redundancies in the battery and to recommend ways in which the battery could be made more efficient. In addressing these questions, the 2008 SAP considered each Tier 1 assay in the context of a conceptual construct that used different “modalities” that span multiple levels of biological organization including molecular interactions, and cellular, organ and organism responses for each hormonal class ( i.e

., E, A, and T hormonal systems) the battery was expected to assess. The 2008 SAP defined these biological levels of organization as consisting of “receptor/target binding, cell responses, enzyme activity, organ response, males and female system integrity, development (thyroid) and comparative system integrity”. The 2008 SAP examined the “complementarity” and “redundancy” in the Tier 1 battery

, i.e ., “complementarity” describes the concordance of endpoints within an assay and “redundancy” describes the concordance of endpoints across the assays that a re intended to evaluate an interaction with a particular pathway . The extent to which different endpoints within and across the assays reflected the test che micals interaction with E, A, and T hormonal system was e valuated in terms of the extent to which t he endpoints and assays were complementary and redundant . From its analysis, the 2008 SAP concluded that the Tier 1 battery is enhanced because the assays provide adequate “replication” ( i.e

., compl e mentary responses within assays) and redundancy across assays.

The current analysis used the 2008 FIFRA SAP concept as a guide in evaluating battery performance by qualitatively describing the degree to which responses within multi

- parameter

assays are “ c omplementary ” and the degree to which responses across assays are “ redundant”. The findings are discussed with consideration to whether there appear to be any unnecessary redundancies in assessing chemical interactions with E, A, or T signaling pathways based on the diversity of the representative compounds evaluated to date. Because the purpose of the current review is to evaluate battery and assay performance in the broader context, this review presents qualitative information a s to the general presence or absence of responses within assays ( i.e., compl e mentary responses) and across assays ( i.e., redundancy). The directionality or magnitude of Tier 1 responses and the process for determining a specific chemical’s potential to interact with E, A, or T are not discussed in this evaluation. A s discussed previously, the WoE analysis of the Tier 1 battery results, with OSRI, will be examined at the WoE SAP during July 30 through August 2, 2013.

T he range of responses evaluated in the Tier 1 battery using a subset of List 1 chemicals generally demonstrate the battery contains the necessary complementarity within and redundancy across assays to effectively screen for interactions with the E, A, or T hormonal systems. For the
E and A pathways, there were compl ementary responses with two or more endpoints responding in the pubertal assays and in the Fish Short-T erm R eproduction A ssay

. In some case studies, the male and female pubertal assays and/or the FSTRA identified endpoint responses along with endpoint responses in the relevant in vitro assays for E and A. The 2008 SAP pointed out that theUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 12 of 160 in vitro assays represent “ specificity and sensitivity....and do not provide complementary or redundant assessments of androgenic activities”, and in the current evaluation t here were also
cases in which potential specific modes of action were not detected in the in vitro assays; however, in vivo responses were obtained in the Hershberger, Pubertals and the Fish Short

- Term Reproduction Assays

. The later observations emphasize the importance of these multi

parameter in vivo assays as a component of the battery to evaluate hypothalamic pituitary gonadal alterations, as well as potential differences between taxa. In some cases, the results of
the Estrogen R eceptor binding assay and the Uterotrophic assay demonstrated concordance in that there was a lack of response in both assays which suggests a lack of estrogenicity among the case studies. There was also general agreement among the Hershberger a ssay, the M ale P ubertal
and/or F ish Short-T erm R eproduction A ssay as all three generally showed responses in androgen dependent tissues. With respect to the thyroid hormone pathway, less complementarity and redundancy were established in the design of the battery compared to the E - and A- related assays/endpoints, which is consistent with the results in the current analysis. Thus, this current analysis reinforces the need of both the A mphibian M etamorphosis A ssay and the Rat Pubertal assays as essential components of the battery for evaluating the thyroid pathway.

In general, the current analysis illustrated both complementarity within assays and redundancy across assays and demonstrated the battery d esign provided complement arity and redundancy for evaluating potential interactions with the E, A, or T signaling pathways across taxa. However, chemicals that elicited a response in the Hershberger also elicited a response in the

M ale P ubertal assay and/or F ish Short- Term R epro duction A ssay . Additionally, the Uterotrophic assay never showed a response with the subset of chemicals used to evaluate battery performance while the multi

- parameter female pubertal assay and/or FSTRA did . The agency will seek guidance from the 2013 Ma y 21 -23 SAP on whether the EDSP Tier 1 battery of eleven assays can be optimized and reduced, and more specifically, whether the Hershberger and Uterotrophic assays are necessary redundancies in the current battery.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

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### I. I ntroduction

a. Historical background

In 1996, Congress passed t he Food Quality Protection Act (FQPA) and amendments to the Safe Drinking Water Act (SDWA) . Passage of the FQPA provided for amendments to the Federal

F ood, Drug, and Cosmetic Act [ FFDCA , Section 408(p) ] which required the US Environmental Protection (EPA) to “ develop a screening program, using appropriate validated test systems and other scientifically relevant information, to determine whether certain substances may have an effect in humans that is similar to an effec t produced by a naturally occurring estrogen, or other such endocrine effect as the Administrator may designate. ” 21 U.S.C. 346a(p)(1). In addition, amendment to the SDWA (Section 1457) indicated that “ the Administrator may provide for testing under the screening program ... any other substance that may be found in sources of drinking water if the Administrator determines that a substantial population may be exposed to
such substance .” 42 U.S.C. 300j-17.

• Expand the number of hormonal pathways by including test systems to detect interactions along the androgen and thyroid hormonal pathways, in addition to the estrogen pathway,

• Expand the number of organisms to include wildlife ( i.e ., amphibian, fish, reptiles, birds, and invertebrates), in addition to humans, and,

• I ncorporate a two

- tiered screening and testing approach.

In gener al, Tier 1 screening sh ould consist of a battery of less complex in vitro and in

vivo assays designed to effectively and efficiently detect the potential of a chemical t o interact with estrogen, androgen, or thyroid (E, A, or T) hormonal pathways and, based on a weight - of

- evidence analysis (WoE), determine whether a chemical is a candidate for Tier 2 testing. Tier 2 testing would involve more complex in vivo studies to clarify the potential interaction with the endocrine or non-endocrine systems and establish dose- response relationships that may be
needed for risk assessment.

Based on EDSTAC’s recommendations and pursuant to the Administrator’s discretionary authority, the Agency expanded the program to include the androgen and thyroid hormonal pathways of the endocrine system and ecological effects within a two

- tiered screening and

te sting strategy (EDSTAC, 1998). This framework was generally supported by a joint commit tee of the SAB/SAP (SAB/SAP, 1999) who recommended to the Agency, that after review of the initial set of Tier 1 data, the results be subject to external scientific peer review “...with an eye towards revising the process and eliminating those methods that don’t work.”USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 14 of 160 b. Tier 1 Screening Assay and Validation Process

In general, Tier 1 screening inclusive of other scientifically relevant information (OSRI) was envisioned to provide data that would be subject to a WoE analysis to determine whether or

not a chemical has the potential to interact with the E, A, or T hormonal pathways and assess the
need for Tier 2 testing. Under FFDCA, the EDSP is mandated to use “validated” assays to screen for endocrine disrupting chemicals. Validation principles establis hed by the Interagency Coordinating Committee Validation of Alternative Methods (ICCVAM, 2000) and Organization
for Economic Co- Operation and Development ( OECD, 2005) were adopted and followed by EPA (USEPA, 2007) to develop, standardize, and validate the current EDSP Tier 1 screening

assays as shown in Table 1.

Generally, validation has been defined as “the process by which the relevance and reliability of a test method is evaluated for a particular use” (OECD, 1996; NIEHS, 1997):

• Relevance describes whether or not a test is meaningful and useful for a particular purpose (OECD, 1996). For EDSP Tier 1 screening assays, relevance is generally described as the ability of an assay to detect chemicals with the potential to interact with the E, A, or T hormonal pathways.

• Reliability is a measure of the variability or reproducibility of assay results within and between or among laboratories.

The validation process of individual EDSP Tier 1 screening assays has largely been a
collaborative effort c oordinated by and funded through the Agency’s EDSP, with other offices within EPA, especially the Office of Research and Development (ORD), as well as industry and academic institutions providing necessary scientific and technical support. In addition, tes t development, prevalidation, and inter -laboratory validation work for some of the Tier 1 screening assays were conducted internationally through coordination with the OECD.

In addition, the EDSP sought guidance under the FACA to provide independent techni cal expertise and recommendations throughout the validation process. Federal advisory committees such as the Endocrine Disrupter Methods Validation Sub

- committee (EDMVS) and the Endocrine Disrupter Methods Validation Advisory Committee (EDMVAC) provided gu idance from 2001 through 2006. Thereafter, the FIFRA SAP has primarily served the advisory role for EDSP. Generally, for each Tier 1 screening assay, the EPA has followed a five

- stage validation

process:

• Test Development , an applied research function using reference chemicals known
to interact with the endocrine- dependent endpoints culminating in an initial assay protocol and establishing relevanceUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 15 of 160 • Prevalidation consisting of the refinement, optimization, and standardizat ion of the protocols. In addition, assessing the transferability and performance of the protocols being considered as part of the Tier 1 EDSP assay battery.

• Inter -laboratory Validation in which independent laboratories conducted studies with optimized, standardized protocols used to primarily determine inter

laboratory variability ( i.e ., repeatability) and to set or cross

- check performance
criteria.

• Peer Review , an independent scientific review by qualified experts. Peer review of individual screening as says was conducted in compliance with the EPA’s Peer Review Handbook (EPA, 2006).

• Regulatory Acceptance , adoption for regulatory use by an agency. The inclusion of an assay in the proposed Tier 1 battery, review of the proposed battery by the SAP (SAP, 2008), and final adoption of the battery by EPA (Federal Register Notice issued October 21, 2009, 74 FR 54416) based on SAP recommendations constitutes the regulatory acceptance stage of the process.

c. Tier 1 Screening Battery

Considering the results of assay validat ion, comments from scientific peer review of individual assays, and recommendations from various FACAs, the EPA compiled the current EDSP Tier 1 screening battery (Table 1). In accord with EDSTAC’s recommendation (EDSTAC, 1998), the current co mbination of assays within the battery was designed to maximize sensitivity and reliability for determining the potential of a chemical to interact with the E, A, or T hormonal pathways. T he basis for selecting individual assays to include in the battery involved various factors that generally included the capability of the assays to detect :

• E, A, and T hormonal signaling in different taxa,

• estrogen - and androgen

- mediated receptor binding,

• estrogen

- mediated gene transactivation,

• enzyme inhibition involving the reproductive steroidogenesis pathway,

• interactions with gonadal estrogen and androgen production that may alter feedback mechanisms involving the hypothalamic

- pituitary

- gonadal (HPG) axis,

• androgen and estrogen endpoints within and complementary among assays, and

• interactions with thyroid hormone production that may alter feedback mechanisms involving the hypothalamic

- pituitary

- thyroid (HPT) axis.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 16 of 160 Table 1

. Current EDSP Tier 1 Screening A ssays. The current EDSP Tier 1 screening assays encompass key endpoints within a mode of action ( e.g., receptor binding) as well as along endocrine pathways ( e.g., steroidogenesis, effects on hypothalamic

- pituitary -gonadal and – thyroid axes) through which a chemical has the potential to interact with the estrogen, androgen, or thyroid (E, A, or T) hormonal pathways.*

Screening Assay Test

Guideline Receptor Binding Steroidogenesis HPG

Axis HPT

Axis E Anti

- E A Anti

- A E A

In vitro
ER Binding (Rat uterine cytosol) OCSPP 890.1250 ■ ■

ERα Transcriptional Activation (Human cell line
HeLa -9903) OCSPP 890.1300 OECD 455 ■

AR Binding (Rat prostate cytosol) OCSPP 890.1150 ■ ■

Steroidogenesis (Human Cell Line H295R) OCSPP 890.1550 ■ ■

Aromatase (Human target tissue or cell line microsomes) OCSPP 890.1200 ■

In vivo Uterotrophic (Rat) OCSPP 890.1600 OECD 440 ■

Hershberger (Rat) OCSPP 890.1400 OECD 441 ■ ■ ■ 1

Pubertal Male (Rat) OCSPP 890.1500 ■ ■ ■ ■ ■

Pubertal Female (Rat) OCSPP 890.1450 ■ ■ ■ ■ ■

Fish Short

- term Reproduction OCSPP 890.1350 OECD 229 ■ ■ ■ ■ ■ ■ ■

Amphibian Metamorphosis (Frog) OCSPP 890.1100 OECD 231 ■

* Complementary endpoints across assays are indicated (solid black box) within each column.

1 5α

- reductase inhibition only.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 17 of 160 (i) Assays for Detecting the Effect of Chemicals on the Estrogen Hormonal Pathway

Seven Tier 1 assays are capable of detecting che micals with estrogenic and
anti estrogenic- like activity that may disrupt estrogen hormonal signaling as shown in Table 1.

Details of individual assays are described in respective assays sections within the
docume nt as well as test guidelines. An introduction and overview of the assays are as follows:

• ER binding assay, uses rat uterine cytosol and has the potential to detect estrogen receptor binding ( i.e., ER α or ER β ) of a chemical. Note, cannot distinguish between estrogen agonist or antagonist activity.

• ER transcriptional activation assay, uses a human cervical tumor cell line and has the potential to detect estrogen agonists through receptor binding ( i.e., ER α ) and gene transcription.

• Aromatase assay, uses human microsomes and has the potential to detect the inhibition of aromatase activity ( i.e ., conversion of androgen to estrogen)

• Steroidogenesis assay, uses a human cell line an d has the potential to detect a change in production (increase or decrea se) of testosterone and estradiol.

• Uterotrophic assay, uses adult ovariectomized rat or mouse, or sexually immature intact female rats and has the potential to d etect estrogen

- like activity tha t may involve receptor binding and effect a change (increase ) in uterine weight . Note, oral or subcutaneous routes of

exposure are acceptable where the latter bypasses first

- pass liver metabolism

.

• Pubertal female assay

, uses intact rats post

- weaning and has the potential to detect both estrogen and antiestrogen

- like activity tha t may involve receptor binding or inhibition of steriodogenesis that may alter hormonal signaling along the HPG axis and a ffect a change in multiple endpoints generally associated with female sexual development ( e.g ., vaginal opening), and gross and histological changes of reproductive organs.

• Fish short -term reproduction assay (FSTRA) , uses mature male and female fathead minnows and has the potential to detect both estrogen and antiestrogen

- like activity that may
involve rece ptor binding or inhibition of steroidogenesis that may alter hormonal signaling along the HPG axis and a ffect a change in multiple endpoints generally associated with survival, and changes in growth, secondary sex characteristics, histology of gonads, and plasma concentrations of vitellogenin.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 18 of 160 ( ii ) Assays for Detecting the Effect of Chemicals on the Androgen Hormonal Pathway

Six Tier 1 assays are capable of det ecting chemicals with androgenic and antiandrogenic- like activity that may disrupt androgen hormonal signaling as shown in Table 1.

Details of individual assays are described in respective assays sections within the document. An introduction and overview of the assays are as follows:

• AR binding assay , uses rat prostate cytosol and has the potential to detect androgen receptor binding; cannot distinguish between androgen agonist or antagonist activity.

• Aromatase assay , similar to that previously described

.

• Steroidogenesis assay , similar to that previ ously described

.

• Hershberger assay

, uses castrated male peripubertal rats and has the potential to primarily detect androgen and antiandrogen- like activity tha t may involve receptor binding or inhibition of steriodogenesis that could effect a change (increase or decrease) on multiple endpoints associated with gross changes in organ weights of ventral prostate, seminal vesicles, levator ani plus bulbocavernous muscle complex, Cowper’s gland, and glans penis.

• Pubertal male assay, uses intact rats post

- weani ng and has the potential to detect both androgen and antiandrogen

- like activity tha t may involve receptor binding or inhibition of steriodogenesis that may alter hormonal signaling along the HPG axis and affect a change in multiple endpoints generally associated with male sexual development ( e.g ., preputial separation) and gross and histological changes of reproductive organs.

• The fish short -term reproduction assay , similar to that previously described expect there is the potential to detect both andro gen and antiandrogen

- like activity

.

( iii ) Assays for Detecting the Effect of Chemicals on the Thyroid Hormonal Pathway

Three Tier 1 assays are capable of detecting chemicals that may disrupt the thyroid hormonal signaling pathway as shown in Table 1.

Details of individual assays are described in respective assays sections within the
document. An introduction and overview of the assays are as follows:

• Pubertal female and males assays , similar to that previously describe except there is the potential to detect effects on the thyroid hormonal signaling pathway ( i.e ., HPT axis) through an effect on thyroid gland weight, histology, and serum total thyroxine concentrations.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 19 of 160 • Amphibian metamorphosis assay , uses tadpoles and has the potential to detect effects on the thyroid hormonal signaling pathway ( i.e ., HPT axis) through effects on metamorphosis and thyroid gland histology.

In accord with EDSTAC, “...the value of each individual assay cannot be considered in isolation from other assays in the battery, as they have been combined in a manner such that limitations of one assay are complemented by the strengths of another” (EDSTAC, 1998). Thus, the combination of Tier 1 screening assays and potential for concordance or corroboration of results among assays maximi zes the sensitivity and reliability for determining the potential of a chemical to interact with the E, A, or T hormonal pathways.

d. FIFRA SAP Review of the Tier 1 Screening Battery in 2008

In 2008, the FIFRA SAP reviewed the combination of EDSP Tier 1 screening assays of the current battery based on several charge questions (USEPA, 2008). A couple ke y questions
were whether the battery could :

• M inimize the potential for "false negatives" and "false positives" and

• did not result in unnecessary redundanci es for mode s of action across the battery.

In addressing these questions, the SAP considered the different modalities within the battery for E, A, and T ( e.g ., receptor binding, cell signaling, organ response) and evaluated the degree of redundancy and compl e mentarity of the assays and endpoints across the battery (Table 1). In response, the SAP indicated the battery provided for both redundancy and compl e mentarity, especially in regard to the in vivo assays and recommended to EPA that “... the proposed set of Tier 1 assays are appropriate to begin screening for disruptors of the EAT

axes

. ” ( USEPA , 1998)

.

The EPA subsequently published a Federal R egister Notice (FRN) in 2009 (USEPA, 2009c) notifying the public of the current EDSP T ier 1 screening battery (Table 1 ) and availability of harmonized test guidelines (OCSPP Series 890).

e. Test Orders for Tier 1 Screening

The first list of test chemicals for EDSP Tier 1 screening consisted of 67 chemicals, 58 pesticide active ingredients belonging to several chemical classes and 9 High Production Volume (HPV) chemicals used as pesticide inert ingredients (USEPA, 2009d). The criteria for sel ecting
chemicals for List 1 were based on the finding of a chemical in at least three of four exposure pathways (food, water, residential, or occupational exposures). In October 2009, EPA began issuing test orders for Tier 1 screening (USEPA, 2009d). Recipients complied by submitting the
results of all 11 assays of the battery or, t o avoid duplicity in test data according to the FFDCAUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 20 of 160 statute that directs the EPA to use “ ... appropriate validated test systems and other scientifically relevant information...” , O SRI was submitted and accepted in some instances by the Agency in
fulfillment of test order requirement s for one or more of the Tier 1 assays. In general, information that is submitted voluntarily and is applicable to support or clarify an EPA action is commonly referred to as “other” s cientifically relevant information ( USEPA, 2002b ). Sources of OSRI may in clude results from EPA or OECD equivalent test guideline studies ( e.g ., EPA Part 158 studies) and information from published or publically available peer -reviewed studies.

f. F IFRA SAP Review of the Tier 1 Screening Assay and Battery Performance in 2013

It is emphasized that the current 2013 SAP performance review of EDSP Tier 1 screening assays and battery is primarily based on the 2008 SAP review of the current battery of assays as
shown in Table 1. The fundamental difference between then and now is the 2008 review was
based on individual assay validation results with selected reference chemicals known to interact with the E, A and T hormonal pathways, whereas the current review is based the application of the entire battery of 11 assays with chemicals n ot necessarily known to interact with the endocrine system. Moreover, the current SAP is c onsistent with the joint SAB/SAP panel in
1999 who recommended the EPA “ 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 ” ( SAB/SAP, 1999) and SAP panel in 2008 who recommended

“... that the EPA will continue to develop, refine, and review the battery.”



### II. Methods

Section III provides a preliminary analysis of the performance of each EDSP Tier 1 assay using an initial subset of chemicals which were issued Test Orders (List 1) in October 2009. The purpose of Section III is to:

• Evaluate how well each Tier 1 assay test guideline can be consistently executed based on defined performance criteria

• Highlight any issues associated with interpretation of responses within each assay

It is important to note that all 11 Tier 1 assays are validated methods. This analysis is not
a “ re validat ion” o f the Tier 1 assays . Each of the Tier 1 assays was validated with a known set of reference compounds (FR Notice, October 21, 2009) for endocrine screening specific to the estrogen, androgen, and thyroid (EAT) pathways through an extensive process of test m ethod development, prevalidation and interlaboratory validation which culminated in evaluation by a
FIFRA S AP in 2008 (USEPA, 2008).

This review is intended to transparently communicate observations on performance of the assays with a set of test chemical s drawn from data submitted in response to the first set of Tier 1USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 21 of 160 test orders issued for 67 chemicals by EPA on October 29, 2009 2

. Fifteen chemicals were voluntarily cancelled or the manufacturer/importer agreed to cease all sales and distribution of
the chemical in the pesticide market. Tier 1 assay d ata were submitted on a total of 52 chemicals. To facilitate timely feedback, a sub sample consisting of 21 test chemicals that adequately represented the chemical space of the 52 pesticide actives were avai lable for consideration. These chemicals represent a broad range of physical

- chemical properties including water solubility, octanol

- water partition coefficient (K ow ), and volatility. The chemicals also represent a range of chemical classes ( e.g ., organophosphates, imides, azoles) and pesticidal activities ( e.g ., fungicides, herbicides, insecticides). The review of Tier 1 submitted data is an ongoing process. Thus, for the purposes of this review the chemicals were selected to be illustrative and

r epresent ative of the 52 chemicals included in the test orders. Given the bread th of physico

chemical properties and that many of these chemical are designed to be biologically active ( i.e

., pesticidal modes of action), this group of chemicals represents a robust domain over which to evaluate assay and battery performance.

As with other data requirements intended to support regulatory decisions, EPA has developed a set of standard evaluation procedures (SEPs) which provide guidance for the review
and evaluat ion of environmental and human health effects data. Similar guidance documents have been developed for the review of the data submitted in response to the test orders and these
are outlined in the 890 Guideline Series 3 for the EDSP Tier 1 battery. The SE Ps are used in conjunction with the appropriate EDSP Series 890 Guideline. Using the guidance within the relevant SEP, Agency reviewers develop Data Evaluation Records (DERs) for each of the submitted assays using study profile templates 4 that have been e stablished for th is purpose. These DERs capture data to determine how well the assay conforms to the Series 890 EDSP Test Guidelines and document basic information on the materials, methods and results of the study. The DER provides both registrant conclusions and EPA reviewers’ conclusion.

The Agency currently follows a multi- tiered approach to reviewing EDSP data submissions. Primary reviews originate with an Agency staff scientist or contract scientist who is responsible for transcribing the materials, methods and results into the appropriate study profile template. These reviews generally consist of a reanalysis of study raw data for independent verification of study results and the Agency’s interpretation of the study is recorded in the DER. Secondary review of the DER is conducted by an Agency scientist who is trained to review the test guideline study and is experienced in reviewing data for the specific test
chemical. A division

- level consistency check may then be performed befor e the DER is elevated to an interdisciplinary committee of scientists with specialized knowledge of the Tier 1 assays. The committee reviews several data submissions for a given guideline concurrently to ensure

2 Federal R egister. 2009.Endocrine Disruptor Screening Program: Tier 1 Screening Order Issuing Announcement. http://www.gpo.gov/fdsys/pkg/FR

- 2009

- 10

- 21/html/E9

- 25352.htm

3 USEPA 2009. OCSPP Harmonized Test Guidelines Series 890. Endocrine Disruptor Screening Program Test Guidelines. http://www.epa.gov/ocspp/pubs/frs/publications/Test _Guidelines/series890.htm

4 USEPA. 2011. Study Profile Templates. Endocrine: Series 890
– Endocrine Disruptor Screening Program Test Guidelines. http://www.epa.gov/pesticides/regulating/studyprofile_templates/studyprofile_templatelist.htm#series

- 890USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 22 of 160 consistency in interpretation. Any changes recommended by the committee or at any previous point in the review process are reflected in the draft DER. The Agency anticipates that each review will remain pr eliminary until all Tier 1 data submissions for a given chemical are reviewed as a battery al ong with other scientifically relevant information consistent with the approach outlined in the WoE guidance document 5

. Given the preliminary nature of the reviews,
actual chemical names are omitted from this document.

Up to 21 chemicals were evaluated in this analysis. The number of case study chemicals evaluated for each assay type and the pathways evaluated for battery performance differ since some of the assays were previously satisfied by OSRI. Table 2 summarizes the number of case study chemicals for the assay and battery analysis. This assay/battery performance review
focuses only Tier 1 test guideline data. All available OSRI will be considered in the future WoE
determinations for each chemical.

Table 2. Number of Chemicals Included in the Assay and Battery Performance Analysis

Assay Performance Assay Type Number of Case Studies Evaluated
In Vitro Estrogen Receptor Binding 18 Estrogen Receptor Transcriptional Activation 19 Androgen Receptor 17 Steroidogenesis 18

Aromatase 1 8

In Vivo Uterotrophic 17 Hershberger 17
Pubertal Female 17
Pubertal Male 16
Fish Short

- Term Reproduction 21 Amphibian Metamorphosis 18 Battery Performance Interaction with the Estrogen Hormonal Pathway 17 Interaction with the Androgen Pathway 17
Interaction with the HPT Axis 15
Table 3 provides an overview of the product types and chemical classes represented by the case study chemicals.

5 USEPA. 2011. Weight

- of

- evidence: Evaluating results of EDSP Tier 1 screening to identify the need for Tier 2 testing. 14 September 2011. Office of Chemical Safety and Pollution Prevention, Washington, DC. Regulations.gov Document ID: EPA

HQ

- OPPT

- 2010

- 0877

- 0021USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 23 of 160 Table 3 . Ove rview of Case Study Chemical Product Types and Chemical C lasses.

Number of Case Study Chemical s Product Type Chemical Class

1 Herbicide Phenoxy

2 Herbicide Dinitroaniline

1 Herbicide Chlorinated benzoic

1 Herbicide Benzonitrile

1 Herbicide Chloroacetanilide

1 Herbicide Triazinon

1 Herbicide Triazine

1 Herbicide Fluorinated pyridazinone

1 Insecticide, nematicide N

- methyl carbamate

4 Insecticide, nematicide, acaracide Organophosphate

1 Insecticide Pyrethroid

1 Acaricide Organotin

1 Acaricide Organosulphite

1 Fungicide Imide

2 Fungicide Azole

1 Pesticide Inert N/A

In some cases, the Agency’s preliminary review of data submissions identified procedural inadequacies or other confounding factors, such as excess overt toxicity or cytotoxicity, which may compromise the interpretation of results. The recommendations for successful performa nce of a Tier 1 assays and for gauging overt toxicity are guideline

- specific and are addressed in more detail in the preceding chapters for each assay

Whether a particular measurement endpoint within a study was deemed affected depended on its statistical significance at a particular level of confidence, its biological relevance
in terms of the nature and magnitude of the effect, and the extent to which the response appeared to be concentration dependent. In some cases, the biological relevance in terms of magnitude of the effect was used to indicate an effect even in the absence of statistical significance.

Within each assay, a particular pathway (E, A, or T) was scored based on the guidance provided by the SAP (USEPA, 2008).USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 24 of 160 

### III. Evaluation of Results from Individual Assays

This s ection describes the technical conduct of individual assays and interpretation of
results in accordance with respective test guidelines and SEPs

. Up to 21 chemicals were included for this analysis.

1. In vitro

a. ER Binding

(i) Background

The purpose of the Estrogen Receptor Binding (Rat Uterine Cytosol) Assay (OCSPP 980.1250) 6 is to provide information on the ability of a compound to interact with estrogen receptors (ERs) isolated from the rat uterus. The assay measures the ability of a test chemical to interact with ERs as reflected by the displacement of radiolabeled [ 3 H]

- 17β -estradiol. As conducted, this assay utilizes all cytosolic estrogen receptor subtypes that are expressed in this tissue including ERα and ERβ. The ER binding assay on ly has the ability to determine if a chemical can interact and displace the endogenous hormone; it provides no information on whether a chemical will act as an agonist or antagonist at the receptor(s) to either activate or inactivate an estrogen

- dependent response.

The Estrogen Receptor Binding (Rat Uterine Cytosol) Assay consists of a saturation binding experiment and a competitive binding experiment. The purpose of the saturation binding experiment is to demonstrate that each batch of rat uterine cytos ol contains an adequate number of ER with a binding affinity for the native ligand, 17β

- estradiol that is consistent with historical
values. ER saturation binding experiments measure total and non

- specific binding of increasing concentrations of [ 3 H]

- 17β

- estradiol under conditions of equilibrium. From these measurements, specific binding at each concentration can be calculated. A graph of specific [ 3 H]

- 17β

- estradiol binding versus radioligand concentration should reach a plateau indicative of saturation o f the ER with the radioligand. In addition, analysis of the data should document the binding of the [ 3 H]

- 17β

- estradiol to a single, high

- affinity binding site ( i.e ., K d = 0.03 to 1.5 nM). The

c ompetitive binding assay measures the binding of a single concentration of [ 3 H]

- 17β

- estradiol in the presence of increasing concentrations (10 - 10 to 10 -3 M) of a test substance. Each test concentration is evaluated in triplicate (three concurrent replicates) within one run. Three independent (non

- concurrent) runs are performed for each chemical tested.

6 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1250: Estrogen Receptor Binding (Rat Uterine Cytosol). Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 74 0- C- 09

- 005. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0576

- 0005

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 25 of 160 Each run of the competitive binding assay includes concurrent solvent (vehic le), a 17β

estradiol reference curve, a weak positive control, and a negative. Dose response curves for the negative, weakly positive and reference estrogen are evaluated to demonstrate that the assay is conducted properly. The solvent control indicates the maximum specific binding that is possible
under the conditions of that run. The negative control (octyltriethoxysilane) provides assurance that the assay as conducted does not report binding for chemicals that do not bind to the ER. A weak positive sub stance (norethynodrel) is included to demonstrate the sensitivity of each experiment and to allow an assessment of variability in the conduct of the assay across time. The reference standard (17β -estradiol) is also included to ensure that the run has been properly performed, and to allow an assessment of variability in the conduct of the assay across time.

Results from e ach run of the competitive binding assay are used to classify the test
chemical as “interacting”, “not interacting”, “equivocal”, or “equivocal up to the limit of the
concentrations tested”. The classification criteria are summarized below.

Interactive:
A chemical is classified as “interactive” with the ER if the lowest point on the fitted response curve within the range of the data is less than 50% ( i.e

. , more than 50% of the radiolabeled estradiol has been displaced from the receptor ).

Not Interactive:
A chemical is classified as “not interactive” if the lowest data point on the fitted response curve is above 75%. ( i.e.

, less than 25% of the radiolabeled estradiol has been displaced) or if a binding curve cannot be fitted and the lowest average percent binding among the concentration groups in
the data is above 75%.

Equivocal up to the limit of concentrations tested:

A chemical is classified as “equivocal up to the limit of concentrations tested” if there are no data points at or above a test chemical concentration of 10 -6 M

, and (A) a binding curve can be fit
but 50% or less of t he radiolabeled estradiol is displaced by concentration 10 -6 M, or (B) a binding curve cannot be fit and the lowest average percent binding among the concentration groups in the data is above 50%.

Equivocal:

A chemical is classified as “equivocal” if it d oes not meet the criteria of the categories above.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 26 of 160 Chemical Classification:
After each chemical is classified from each run , the chemical is classified by assigning the following values to each run and averaging across runs:
interactive: 2 equivocal: 1
not interactive: 0

Chemical classification, based on the average of all the runs performed for a chemical: interactive: average ≥ 1.5 equivocal: 0.5 ≤ average < 1.5 not interactive: average < 0.5

(ii) Strengths and Limitations of the Assay

The hormone binding domain (HBD) of the ER is highly conserved across species. Environmental chemicals that compete with the endogenous ER have the potential to either
induce hormone- dependent processes (agonist) or block normal hormone function by preventing endogen ous hormone from binding to the receptor (antagonist). The assay cannot distinguish between ER agonism and ER antagonism.

(iii) Observations of Test Chemicals

There were 18 chemicals available at the time of analysis to evaluate laboratory performance of this assay. Seven different laboratories were involved in conducting the ER Binding Assays, with the majority of the assays being performed by two laboratories (Laboratory 1, 7 chemicals; Laboratory 2, 5 chemicals). Uterine tissue from Sprague

- Dawley rats was used as the source tissue for cytosolic ER for all compounds. The cytosols were prepared and stored as specified in the Guideline. The protein concentrations of the cytosol preparations ranged from 1.1 to 5.1 mg/mL, and the final protein concentrations used in the assays (where reported) were 0.341 to 1.191 mg/mL or 40 to 320 μg/assay tube. The purity of the test chemicals used in the assays was >89%. Dimethyl sulfoxide (DMSO) or ethanol (EtOH) was used as the solvent
vehicle for the test chemicals in all assays. Solvent concentration limits were met for all compounds. Test chemicals were initially evaluated at serial log concentrations from 1 mM to 100 pM (10 − 10 to 10 − 3 M) in the assay as recommended in the test guideline, with the exceptions of two chemicals (Chemicals Q and S) which were tested to a maximum of 10 -4 M and 10 -5 M, respectively, based on preliminary solubility findings. For the remaining 16 compounds, 10 were soluble up to the limit concentration of 10 − 3 M and six were only soluble u p 10 -4 M. If a
chemical is not soluble at 10 − 3 M, the guideline recommends testing the chemical at a concentration that is 1⁄2 log lower ( e.g. 10 - 3.5 M). All but one laboratory (Laboratory 1) followed this guidance when insolubility was encountered. In some instances when precipitation wasUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 27 of 160 observed, Laboratory 1 added additional concentrations at the lower end of the concentration range (10 − 11 or 10 − 12 M), rather than utilizing mid

- log concentrations at the upper end of the concentration range (10 − 4.5 M or 10 − 5.5 M) which would better characterize the response curve and provide more confidence in the interpretation of the results.

Based on the results of the 18 assays, 16 of the test compounds were classified as non

interactive, and two compounds were classified as equivocal based on the classification criteria presented above. None of the 18 chemicals was classified as interactive (T able 4).

Table 4. ER Binding Classification Results for the 1 8 Chemicals Evaluated.
Binding Classification Number of Chemicals
Interactive 16
Not interactive 0 Equivocal 2

(iv) Performance Criteria

ER Guideline performance criteria for the saturation binding experiment are provided in
Table 5 . The performance criteria are provided : (i) to ensure that there are adequate numbers of

ERs (B max ) in the uterine cytosolic preparations ; (ii) to ensure that the ERs have the appropriate affinity (K d ) for [ 3 H]

- 17β

- estradiol and (iii) to serve as guidance in identifying competitive runs that provide parameters in the preferred ranges

.

Table 5 . Performance Criteria for Saturation Binding

Parameter Value Range

B max 10

- 150 fmol/100 μg protein

K d 0.03

– 1.5 nM

Non

- Specific Binding (NSB) <50% of specific binding at maximum concentration

Saturation Binding: The saturation binding data were included within the ER binding study reports from five laboratories (representing six chemicals)

. However, s aturation binding data were not included with the original ER binding study repor ts that were submitted by two of the testing laboratories (Laboratories 1 and 2). Upon request by the Agency, summary saturation binding information was subsequently submitted to support the chemicals. The available data from the saturation binding assays indicate that binding reached a plateau and that the Scatchard plots for each run were generally linear, although several of the runs showed curving of the plots. Some laboratories used a single cytosol preparation to test multiple chemicals. In those ca ses, a single saturation binding experiment was conducted for the cytosol batch and the saturation binding data (or a summary of the data) for that batch were submitted for more than one
chemical.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 28 of 160 As shown in Table 6, all three binding runs for Chemicals A and K and one binding run for Chemical G were performed using the same cytosol preparation (Cytosol A). Three runs for Chemicals B and 2 runs for Chemical G were tested with Cytosol B; Chemicals V and L were tested with Cytosol C; Chemicals E, F, I, J and N were tested with Cytosol D; and Chemicals D and M were tested using Cytosol E. The remaining five studies submitted saturation binding data that were used for only one chemical.

Table 6

. Cytosolic Preparations Used for More than One Chemical.

La boratory Cytosol ID Chemicals Tested
1 A A, K (all 3 runs); 1 run for G
1 B B (all 3 runs); 2 runs for G
1 C V, L (all runs)
2 D E, F, I, J, N (all runs)
3 E D, M

Figure 1 below represents a typical saturation binding curve. The measured values for total binding and non- specific binding as well as the calculated values for specific binding (total binding minus non- specific binding) are shown for three independent r uns.

Figure 1. Saturation Binding Curves.

TB= Total binding; NSB= Non- specific binding; SB= Specific binding; E2= EstradiolUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 29 of 160 The saturation binding performance criteria for B max and K d were met by all seven testing laboratories for the cytosol preparations used for the 18 chemicals. Non

- specific binding (NSB), when reported as a value or depicted in graphical form, was less than 24% of specific binding at the highest concentration. A summary of the performance criteria for the saturation binding experiments is presented in Figure 2.

Figure 2. Performance Criteria Results for the Saturation Binding for Cytosolic Preparations.

Competitive Binding:

Performance Criteria for Competitive Binding

Guideline performance criteria for positive controls for the competitive binding experiment are provided in Table 7. The performance criteria are provided to serve as guidance in identifying competitive runs that provide parameters in the preferred ranges and to ensure that assay is responding properly.

Table 7. Upper and Lower Limits for Parameters in Competitive Binding Assay Curves for Positive Control.

Parameter Unit Estradiol

Test Guideline Criteria

Range of N on
- performance

Lower
Limit Upper Limit

Log e (S yx) ( i.e. Loge(Residual Std. Dev.) - NA 2.35 Values were not reported for 3 chemicals.
All others met the criteria

Bottom plateau level % binding

- 4 1

- 5 to 1.1 Top plateau

level % binding 94 111 93 - 111

Hill Slope Log 10 (M) -1

- 1.1

- 0.7

- 2.0 to

- 0.63

Note: 19

- norethindrone was used as a positive control. None of the testing laboratories used Norethynodrel

. NA= not applicable 18 18

Bmax Kd (nM) 

## ER Saturation Binding
Criteria

MetUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 30 of 160 Figure 3 below shows typical competitive binding curves for the positive control (19

- norethindrone), strong positive control (17β

- estradiol ), negative control (octyltriethoxysilane) and a test chemical that was classified as not interactive in this assay.

Figure 3 . ER Competitive Binding Curves

All competitive binding performance criteria for the positive control, 17β

- estradiol, were met for 11 of the 18 chemicals. For the remaining chemicals, the laboratory did not meet one or more of the 17β

- estradiol positive control performance criteria; however, in most cases the
values were o nly slightly outside the expected range and did not impact the interpretation of the study. The Log e (S yx ), with an upper maximum performance criteria of 2.35, was met for 16 of the 19 compounds, but was not reported for three studies (Chemicals D, M, P). The top % binding (94 to 111% ) was generally met for all reviewed studies ; the exceptions included only a single run for three different chemicals (D, L, V), each only slightly below (93%) the lower recommended limit of 94%. The bottom % binding (

− 4 to 1% ) was also slightly below range for a single run in two studies (L, V; −5%) and above range for single runs with for two other chemicals (Chemicals C and K; 2.1 and 5%, respectively). The Hill slope was slightly below the recommended range (−1.1 to −0.7) in one of the five runs in one study (Chemical P; −1.126), and the Hill slope performance criteria were not met in any run for two studies from the same laboratory (Chemicals M, D; −2 to −1.7) The Hill slope was greater than -1.1 in one or two runs
in two other studies: 2 of 3 runs in the study in Chemical K (−1.4 and −1.7), and in a single run in the study for Chemical A (−1.6). A summary of the performance for the 17β

- estradiol positive control is presented in Figure 4.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 31 of 160 Figure 4. Performance Criteria Results for the Strong Positive Control.

Although the Guideline recommends the use of norethynodrel as the weak positive control ligand, it allows the use of 19

- norethindrone as a replacement if norethynodrel is
unavailable. All 18 of the reviewed studies used 19- norethindrone in place of norethynodrel. The Guideline does not contain performance criteria for 19 -norethindrone.

The single performance criterion for the negative control, octyltriethoxysilane, requires that the displacement of radioligand from ER be <25%. This criterion was met in all but three cases (Chemicals L and V, which shared control data; and Chemical Q) that were all conducted by the same laboratory (Laboratory 1). For these three chemicals, the negative contro l criterion
was met in one run and was not met in the two remaining runs, with displacement values ranging from 51.8 to 58.5%. NSB (no criteria provided for NSB in the Guideline) was generally less
than 10%.

(v) Issues or Problems with Laboratory Performance

One of the issues identified in the laboratory performance of the ER Binding Assay was
where two sets of negative control runs by Laboratory 1 did not m eet the negative control performance criterion in two out of three assay runs (data were used for three separate study compounds). Two of the compounds, Chemical L and T, shared the same control data, and Chemical Q had different control data. The assays with Chemicals L and T used the same cytosol preparation and the Chemical Q used a cytosol prepared on a different date; however, the other compounds assayed with either of these cytosol preparations did not show a similar issue with the negative control. Although the study report for Chemical T stated that precipitation was observed at an octyltriethoxysi lane concentration of 10 − 3 M in all three competitive binding runs (resulting in exclusion of this concentration), the study reports for Chemical L (which shares control data with Chemical T) and Chemical Q stated that precipit at e was not observed at any 14

16 15

16

2 3 4

3 3

Hill Slope Top plateau level Bottom plateau level Loge(Syx) (i.e. Loge(Residual Std. Dev.) 

## 17 β

- Estradiol Performance Criteria

Met (all runs) Did not meet in one run

Did not meet in >1 run Not reportedUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 32 of 160 concentration of octyltriethoxysilane. Although an explanation for the >25% displacement is proposed in the two latter compound study reports, a positive response with the negative control and/or precipitation at a concentration that has been validated by the EPA may indicate an issue with the laboratory performance for those assay runs. Otherwise, only minor deficiencies were noted in the 18 studies, and few of these impacted study interpretation. In general, the data were
considered reliable.

(vi) Overall conclusion

The assay performance was generally consistent across all 18 test compounds. Although a number of the evaluated assays did not meet all of the performance criteria, overall the laboratories performance of the ER binding assays was generally acc eptable.

b. ERα Transcriptional Activation

(i) Background

The Estrogen Receptor Transcriptional Activation Assay (OSCPP 890.1300) 7 is used to determine if a chemical interacts with the endocrine system by functioning as an ERα ligand and activating transcription of an estrogen

- dependent reporter gene. This protocol is validated for the detection of an agonistic response. The assay e xclusively uses the stably

- transfected hERα

HeLa

- 9903 cell line derived from human cervical tumor cells (Japanese Collection of Research Bioresources [JCRB] Cell Bank). The cells were transfected with an expression construct encoding human ERα and a reporter construct containing an estrogen response element and encoding firefly luciferase; the bioluminescence product is evaluated to determine the agonist response. The cells are required to be grown for more than one passage from frozen stock, but

not more than 40 passages. The stability of the cells is monitored with four reference chemicals: 17β

- estradiol (strong agonist ), 17α

- estradiol (weak agonist ), 17α

- methyltestosterone (very weak agonist), and corticosterone (negative control). A group of ten comp ounds should be used to evaluate the proficiency of the cells prior to initiation of the assay runs. The proficiency compounds include positive [diethylstilbestrol, 17

- ethynyl estradiol, hexestrol, genistein, estrone, butyl paraben, and 1,3,5

- tris (4

- hydro xyphenyl)benzene] and negative [dibutyl phthalate, atrazine, and corticosterone] chemicals. The proficiency test should be performed in duplicate on two different days (and include the three positive reference chemicals).

The assay is performed under cell culture conditions in 96- well plates as described in Guideline 890.1300. After allowing the cells to attach for 3 hours, cells are exposed to multiple concentrations of the test chemical (in triplicate wells) for 20 -24 hours. Medium is then
removed ; luciferin substrate is added; and the luciferase activity is measured using a

7 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1300: Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa

- 9903). Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 740 -C- 09

- 006. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

OPPT

- 2009

- 0576

- 0006USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 33 of 160 luminometer. The amount of light emitted is proportional to the potency and/or concentration of the estrogen (or estrogenic compound). A test chemic al is determined to be posi tive if the maximum response induced by the test chemical (RPC Max ) is equal to or exceeds 10% of the response of the positive control in at least two of two or two of three runs.

(ii) Strengths and Limitations of the Assay

The use of an immortalized cell line in this assay eliminates the use of live animals for this purpose. The assay can detect chemicals that bind ERα and activate transcription of an ERα

dependent gene. The activity of the test compound can be quan tified and compared to the activity
of the well characterized ER α ligand

, 17 β

- estradiol.

This assay was validated for ER agonist activity only (does not identify ER antagonists). The construct used in this cell line identifies only chemicals that activate ER α (does not detect
activation of ER β or me mbrane- associated ERs). Also, l uminescence signals in excess of signals produced by the known endogenous ligands have been reported at phytoestrogen and synthetic estrogen concentrations in the micromolar range. The guid eline includes an additional test cotreating cells with test chemical and a known ERα antagonist, to be used when a false positive is suspected. Finally, t he HeLa

- 9903 cell line exhibits limited metabolism so the assay may not adequately evaluate metabolites f or potential estrogenic activity.

(iii) Observations of Test Chemicals
Nineteen compounds were used to evaluate the performance of the estrogen receptor transcriptional activation assay (ERTA). Test chemical purity was >89%. All of the test facilities
used the recommended hER α

- HeLa

- 9903 cell line from JCRB, and the cell passages used for the
test runs were maintained between 1 < n < 40. All test materials were dissolved in either DMSO

or EtOH and final vehicle concentrations in the assays did not exceed 0.1% m eeting Guideline requirements. Only a single chemical (Chemical N) was tested up to the test limits of 10 -3 M; all other chemicals were limited by solubility and/or cytotoxicity. Selected test concentrations ranged from 10 - 3.3 and 10 - 3.5 to 10 - 13 M. Most test facilities spaced the test chemical concentrations by orders of whole logs; however, one chemical was tested with half log separations (Chemical L) after solubility and cytotoxicity issues were observed in the first assay

run.
All test facilities, wi th the exception of Laboratory 1 included the appropriate figures
and/or tables for calculation of the positive control (PC, 1 nM 17β

- estradiol) values (PC 10 ) relative to the vehicle control (VC) . Laboratory 1 did not report if PC 10 values were greater than 1 plus 2 standard deviations (SD) of the concurrent VC ; however, PC 10 and 1 plus2 SD values
could be determined from the reported data and did conform to the Guideline

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 34 of 160 Proficiency Test

The Guideline- required proficiency testing of the cell passage(s) to be used for assay runs was conducted, but reported differently, by each testing facility. A few laboratories (3/8) provided proficiency data an d classification per guideline requirements. However several
lab oratories (4/8) provided some data or assessment that did not completely me e t g uideline requirements. Also, L aboratory #1 did not provide proficiency testing information initially (see below).

• None of the reports issued by Laboratory 1 ( C hemicals: A, B, G, K, L, P, Q, S and V) contain any information regarding proficiency testing, other than a statement claiming that all study personnel had demonstrated proficiency in performing the assay. Subsequently, on request, a summary proficiency data report was submitted to the Agency. This report included the methodology and qualitative results, but no data or figures, from replicate tests for the ten proficiency chemicals (over three assay runs with three different passages of cells). Sever al criteria for the reference chemicals included with each proficiency run were not met.

• Reports issued by Laboratory # 2 (C hemicals : E, F, I, J, N and O ) stated that proficiency testing was performed and that the results demonstrated proficiency; however, the data and results were included in a separate, unreferenced report.

• Reports issued by Laboratory # 3 (Chemicals D and M) included summary and classification data from two proficiency test runs, but did not include the individual data
While nine of the ten proficiency chemicals were classified accurately, corticosterone was positive in the first run and negative (correct) in the second run.

• Laboratory #4 (Chemical C) did not include any proficiency data or a summary, but stated that a separate study w as conducted and the results met guideline requirements.

• Laboratory #5 (Chemical H) provided curve fit, RPC max data and classifications for proficiency chemicals (2 to 4 runs), as an appendix to the main study report.

Transcriptional Activation Test

Of the 19 chemicals tested, the RPC max values were <10% for 14 chemicals that were classified as negative (except for one, Chemical Q, that was considered inconclusive). Of the
five chemicals that had RPC max values >10% in at least two runs , Chemical E (19. 1%),), Chemical F (18.0%), Chemical I (25.4%) and Chemical L (29.7%), and Chemical F (18.0%) were considered positive agonists. Chemical B (RPC max = 115%) was suspected to be a false positive because the ER α antagonist ICI 182,780 did not inhibit the respo nse . A PC 50 value was determined only for Chemical B, and PC 10 values were reported for three of the chemicals (Chemicals E, F, and I) with a positive response. Neither PC 50 nor PC 10 were rep orted for
Chemical L. L ogEC 50 and/or Hill Slope estimates were determined for all positive chemicals except Chemical L, as well as for Chemical M (negative response). A summary of the results is
shown in Table 8.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 35 of 160 Table 8. Classification of the 19 Chemicals E valuated in the ERTA

.

As say Classification Number of Chemicals
Positive 5* Negative 13

Inconclusive 1

*Positive includes one suspected false positive (Chemical B).

(iv) Performance Criteria

The performance criteria for the four reference compounds were determined in conjunction with each test chemical. The ranges for each reference compound met the Guideline required ranges; Laboratory 1 included an additional lower concentration (beyond the lowest
Guideline- recommended concentration) for each reference compound. This additional low concentration had no impact on the study.

Few issues were observed for any testing facility regarding the negative reference compound, corticosterone, namely, cytotoxicity at the high concentration (10 -4 M) observed by Laboratory # 1 during five test chemical runs (Chemicals: B, G, K, and L) . Corticosterone
showed a slight positive response in 1 of 3 runs for Chemical C by Laboratory #4

.

All response curve parameters for the strong estrogen agonist (17 β

- estradiol) were met
for 10 of the 19 chemicals. Nevertheless, out -of- range values were very close to the test guideline ranges except the LogEC 50 for Chemical O and Chemical F as noted below :

• LogEC 50 (-9.4 versus the criteria range of -11.3 to -10.1) and Hill Slope (0.3 versus the
criteria range of 0.7 to 1.5) estimates were out of range for two of four runs for Chemical O and one of the four runs for Chemical F tested by Laboratory # 2 that shared the same reference compound data.

• Hill Slope estimates were out of range (0.4 versus the criteria range of 0.7 to 1.5) for one out of three runs for three chemicals (Chemicals I, J, N) tested by Laboratory # 1 that shared the same reference compound data.

• LogEC 50 (-9.7) , Hill Slope (0.66) , and logPC 10 (-10.9 versus the criteria range of -11.4 to -10.1) estimates were out of range for one out of three runs for Chemical C tested by Laboratory # 4

• The LogPC 50 estimate (-11.5) was out of range for one out of two run s for Chemical A tested by Laboratory # 1.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 36 of 160 • LogEC 50 (-11.5 and -11.4) and LogPC 50 (-11.7 and -11.5) estimates were out of range for
Chemicals G and L in two out of two valid runs tested by Laboratory # 1 that shared the same reference compound data ; Hill Slope (1.6) was out of range for one run only.

R esponse c ur ve par am eters for the weak estrogen agonist (17 α

- estradiol) were met for only 5 of the 19 chemicals. Nevertheless, out-of- range values were very close to the g uideline

ranges except for Ch emicals M, D and Q as noted below:

• LogPC 50 (-9.9 versus validated range of -9.6 to -8.1) , LogPC 10 (-11.0 versus criteria range of -10.7 to -9.3) , and LogEC 50 (-10.0 versus criteria range of -9.6 to -8.4) estimates were
out of range for Chemicals G and L , in one out of two valid runs tested by Laboratory # 1 that shared the same reference compound data.

• LogPC 50 (-10.1) , LogPC 10 (-10.9) , and Hill Slope (0.5 versus criteria range of 0.9 to 2.0) estimates were out of range for three out of four runs, for C hemicals O and F tested by Laboratory #2 that shared the same reference compound data.

• LogPC 10 estimate (-11.9) was out of range for one run and Hill Slope estimates (0.3 and 0.8 versus 0.9 to 2.0) were out of range for both two runs, for Chemicals D and M that were tested by Laboratory # 1 that shared the same reference compound data

.

• Hill Slope estimates (0.6) were out of range for one out of three runs for Chemicals I, J
and N tested by Laboratory #2 that shared the same reference compound data, as well as
one out of three runs (0.7) for Chemical E (Laboratory #2), and one out of two runs (2.5) for Chemicals Q and P (Laboratory 1).

• LogEC 50 estimates (-8.2) were out of range for two of three runs fo r Chemical C that was tested by Laboratory # 4.

All of the test facilities had difficulties meeting the Guideline ranges for the very weak estrogen agonist, 17 α

- methyltestosterone. Often , the logPC 10 and logPC 50 estimates were less than the lower limits described in the Guideline, indicating increased sensitivity of the assay
system to weak agonists during tests for 11 of the 19 chemi cals. Alternatively, eight test (from a
total of three laboratories) had logPC 10 and /or logPC 50 values for methyltestosterone that were above the Guideline range upper limit (Chemicals P and S, Laboratory 1; Chemical C, Laboratory 4; Chemical H, Laboratory 5) or were not calculable due to cytotoxicity (Chemicals B, K, Q and V; Laboratory #1)

.

(v) Issues or Problems with Laboratory Performance

Majority of the deviations are considered minor. However, cytotoxicity observed with corticosterone in Laboratory 1, and cytotoxicity and high logPC 10 and/or logPC 50 estimates
observed with 17 α

- methyltestosterone in Laboratories #1, 4 and 5 impact interpretations for eight
chemicals.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 37 of 160 (vi) Overall conclusion

While performance criteria were generally not met for the majority of the chemical, out

of- range values were often close to the Guideline ranges. All but one chemical were classified as either negative or positive in the assay, however inability to fulfill the performance criteria may affect the interpretation of at least 8 of 19 chemicals.

c. AR Binding

(i) Background

The Androgen Receptor Binding (Ra t Prostate Cytosol) (OSCPP 890.1150) 8 Assay is used to determine if a chemical competes with the reference [ 3 H] androgen ( [ 3 H] -R1881) for binding of the cytosolic androgen receptor (AR) from rat ventral prostate tissue homogenate. The assay measures the ability of radiolabeled [ 3 H] -R1881 to interact with the AR in the presence of increasing concentrations of a test chemical. Rat prostate cytosol containing AR is incubated in test tubes with increasing concentrations of the test substance and an aliquot o f
radiolab e led [ 3 H] -R1881. If the test substance interacts with the hormone binding domain of the AR, less radiolab e led [ 3 H] -R1881 can bind to the AR producing a descending concentration

response curve.

The Androgen Receptor Binding Assay consists of a saturation binding experiment and a competitive binding experiment. The purpose of the saturation binding experiment is to ensure that the AR in the cytosolic preparation is present in reasonable numbers and is functioning with the appropriate affinity for the radiolabeled reference ligand. AR saturation binding experiments
measure total and non- specific binding of increasing concentrations of [ 3 H] -R1881 under conditions of equilibrium. From these measurements, specific binding at each concentration can
be calculated. A graph of specific [ 3 H] -R1881 binding versus radioligand concentration should reach a plateau indicative of saturation of the AR with the radioligand. In addition, analysis of the data should document the binding of the [ 3 H]

- R1881 to a single, high

- affinity binding site ( i.e ., K d = 0.685 to 1.57 nM and a linear Scatchard plot). The competitive binding assay measures the binding of a single concentration of [ 3 H]

- R1881 in the presence of increasing concentrations (10 - 10 to 10 -3 M) of a test substance. Each test concentration is evaluated in triplicate (three concurrent replicates) within one run. Three independent (non -concurrent) runs are performed for each chemical tested.

Each run also includes concurrent solvent (vehicle) , and weak and strong positive
controls. The solvent control indicates the total binding that is possible under the conditions of
that run. A weak positive substance (dexamethasone) is included to demonstrate the sensitivity

8 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1150: Androgen Receptor Binding (Rat Prostate Cytosol). Office of Saf ety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 640 -C- 09

- 003. October 2009. http://www.regulations.gov/#!d ocumentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0576

- 0003

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 38 of 160 of each experiment and to all ow an assessment of variability in the conduct of the assay across time. The reference standard (unlabeled R1881) is also included to ensure that the run has been properly performed, and to allow an assessment of variability in the conduct of the assay ac ross time.

Although the classification terminology used in the AR binding Test Guideline differs from the ER binding assay

, t he term “binder” in the AR assay and “interacting” in the ER assay are considered equivalent for the purposes of chemical classific ation.

Binder Classification

Results from e ach run of the competitive binding assay are used to classify the test
chemical as a “binder”, “no n-binder ”, “equivocal”, or “ untestable”. The classification criteria
are summarized below.

If the data fit a 4- parameter nonlinear regression model, the test chemical would typically be
classified as:

Binder: A chemical is classified as a “binder” if t he average curve for t he test chemical across runs displaces 50% or more of the radioligand.

Equivocal: A chemi cal is classified as “equivocal” if the average lowest portion of the curves across runs is between 50% and 75% activity, or if the slope of the curve falls outside the range for the weak positive control ( -0.6 to -1.4).

Non-Binder: A chemical is classifie d as a “non -binder” if t he average lowest portion of the curves across runs is greater than 75% activity, or the data do not fit the model.

Untestable: A chemical is classified as “untestable” if the test compound is not soluble above 10 -6 M and the binding curve does not cross 50%.

(ii) Strengths and Limitations of the Assay
Environmental chemicals that compete with the endogenous AR have the potential to
either induce hormone- dependent processes (agonist) or block normal hormone function by preventing endogenous hormone from binding to the receptor (antagonist). There is a h igh degree of DNA sequence conservation in the AR across mammalian phylogenetic lines (Kelce et al , 1998; Betney and McEwan, 2003). Therefore substances that bind the AR from rats are presumed to be capable of binding the AR in humans and other species. The assay cannot distinguish between AR agonism and AR antagonism.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 39 of 160 (iii) Observations of Test Chemicals

Seventeen compounds were evaluated in the AR Binding Assay. Six different
laboratories were involved in conducting the 17 AR Binding assays, with the major ity of the assays being conducted by Laboratory 1 (7 tests) or Laboratory 2 (4 tests). Each assay utilized ventral prostate tissue from castrated, male Sprague

- Dawley rats as the source of cytosolic AR. The cytosol was prepared and stored as specified in the Guideline. The protein concentrations of the cytosolic preparations ranged from 4.3 to 8.8 mg protein/mL, and the final protein concentrations used in the assays were 1.6 - 6.57 mg/mL, or 0.48- 1.97 mg protein/ assay tube. The purity of the test chemicals used in the assays was >89%. DMSO and EtOH were used as the solvent vehicles for the test chemicals in all assays. Solvent concentration limits were met for all compounds (max: 3%). Test chemicals were initially tested at serial log concentrations
from 10 - 10 to 10 -3 M (as recommended in the Test Guideline), with the exception of one chemical (Chemical S) which was tested at a maximum of 10 -5 M based on preliminary solubility findings. No solubility issues were noted for nine of the compounds tested at up to 10 -3 M. However, seven of the test chemicals were insoluble in the assay at a concentration of 10 -3 M, and two of these compounds were also not soluble at 10 -4 or 10 -5 M (Chemical G and C, respectively). Similar to what was observed in the ER Binding Assay, when precipitation was observed, Laboratory 1 added additional concentrations at the lower end of the concentration range (10 − 11

or 10 − 12 M), rather than utilizing mid

- log concentrations at the upper end of the concentration range (10 − 4.5 M or 10 − 5.5 M) which would better characterize the response curve and provide more confidence in the interpretation of the results those runs. Based on the results of the 17 assays, 10 of the test compounds were classified as non

- binders, and three compounds (Chemicals A, H and K) were classified as “binders”, with Log IC 50 values of -5.0 to -3.8 M. Another three compounds were classified as equivocal, based on minimum 3 H-ligand binding between 50 and 75% (Chemicals I and P) or because of an excessively steep Hill Slope (Chemical J). One compound (Chemical C) was classified as untestable as it was not soluble
>10 -6 M and binding was >75% at all lower concentrations. A summary of the classification of
the chemicals is shown in Table 9.

Table 9

. AR Binding Classification Results for the 1 7 Chemicals Evaluated
Binding Classification Number of Chemicals
Binder 3

Non

- binder 10 Equivocal 3

Untestable 1USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 40 of 160 (iv) Performance Criteria:

Saturation Binding

The A R Binding Guideline performance criteria for the saturation binding experiment are provided in Table 10 . The performance criteria are provided to serve as guidance in identifying competitive runs that provide parameters in the preferred ranges and to ensure that there are adequate numbers of ARs in the prostate cytosolic preparations and that the A Rs have the appropriate affinity for [ 3 H] -R1881.

Table 10. Performance Criteria for Saturation Binding

Parameter Value Range

K d 0.685 – 1.57 nM

Non

- Specific Binding (NSB) <20% of specific binding at maximum concentration

Saturation binding data were not always included with the original AR binding study

repor ts ; however, saturation binding information was subsequently submitted to the Agency upon request. Saturation binding data were often submitted as summary data in graph and
tabular form without the accompanying raw data. Some laboratories used a single prostate cytosol preparation to test multiple chemicals. In those cases, a single saturation binding experiment was conducted for the cytosol batch and the saturation binding data (or summary data) f or that batch were submitted for more than one chemical. As shown in Table 11, Chemicals E, I, J, and N were tested using the same cytosol preparation (Cytosol A); Chemicals B, G, K, U and V were tested with Cytosol B; and Chemicals D and, M, were tested using Cytosol C. For Chemicals A and L, one competitive binding run was conducted with Cytosol B, with Cytosol D and a third run was conducted using a different cytosol prep (Cytosol E). The remaining four studies (Chemicals C, H, P and S) submitted satu ration binding data used for only
one chemical.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 41 of 160 Table 11. Prostate Cytosol Preparations U sed for M ore T han One Chemical.
Laboratory Cytosol ID Chemicals Tested
2 Cytosol A E, I, J, N (all 3 runs)

1 Cytosol B B, G, K, U, V (all 3 runs)
3 Cytosol C D, M (all 3 runs)

1 Cytosols B, D and E A, L (1 run per cytosol)

The available data from the saturation binding experiments indicated that binding reached a plateau. The saturation binding performance criteria for K d (0.685 to 1.57 nM) was not met for
th e cytosol prepared by Laboratory 2 (Cytosol A) (K d : 0.464 nM), which was utilized in assays
for 4 chemicals. The K d saturation binding performance criteria were also not met for two cytosols prepared by Laboratory 1, Cytosol preparation D (0.614 nM) and Cytosol E (0.613 nM). A third batch of cytosol prepared by Laboratory 1, Cytosol B, met the criteria for K d (1.080 nM). Additionally, the saturation binding performance criteria for K d was not met by Laboratory 5 for the cytosol preparation used for Chemical H (0.65 nM). The saturation binding performance criteria were met for the rat prostate cytosol prepared by Laboratory 3 (2 studies), and the individual studies conducted by the two remaining laboratories. A summary of the performance
for the saturati on binding experiments is presented in Figure 5. This figure represents the saturation binding performance for 6 different testing laboratories and 8 different cytosol preparations. The non

- specific binding criterion was met for all cytosol preparations.

Figure 5. Performance Criteria Results for the Saturation Binding for Cytosolic Preparations.

18 9

0 9

0% 20% 40% 60% 80% 100% Non

- Specific Binding (NSB) Kd (nM) 

## AR Saturation Bindng

Met Did not meetUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 42 of 160 Figure 6 below represents an AR saturation binding curve where the performance criteria were met. The measured values for total binding and non- specific binding as well as the calculated values for specific binding (total binding minus non

- specific bindin g) are shown for one independent run.

Figure 6. Saturation Binding Curves.

TB= Total binding; NSB= Non

- specific binding; SB= Specific binding

(v) Performance Criteria:

Competitive Binding

Guideline performance criteria for positive controls for the competitive binding experiment are provided in Table 12. The performance criteria are provided to serve as guidance in identifying competitive runs that provide parameters in the preferred ranges and to ensure that assay is responding properly.

Table 12. Upper and Lower Limits for Parameters in Competitive Binding Assay Curves for Strong and Weak Positive Controls.

Parameter Unit R1881 Dexamethasone

Lower Limit Upper Limit Lower Limit Upper Limit Bottom plateau level % binding

- 2.0 2.0

- 12 12 Top plateau level % binding 82 114 87 106 Hill Slope Log 10 (M) - 1

- 1.2

- 0.8

- 1.4

- 0.6USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 43 of 160 Figure 7 below shows representative competitive binding curves for the positive control (dexamethasone), strong positive control (R1881) and a test chemical that was classified as a binder in this assay.

Figure 7. Competitive Binding Curves

The performance criteria for the Competitive Binding Assays were generally met with only a few exceptions. For the positive control, R1881, the top % binding (82 to 114 %) was met for all reviewed studies, and bottom % binding (

- 2.0 to 2.0%) was met except in Laboratory 2 studies (Chemical E and I) and the Laboratory 5 study (Chemical H), which had values slightly below the bottom range (-2.6 to - 2.1%). Except for one run ( -1.588) in the study conducted for Chemical P, the Hill Slope performance criteria for R1881 ( -1.2 to 0.8) were met. The Log IC 50 for the reference androgen ranged from -10.1 to - 8.6 Log M in runs across all the assays, but was typically -9.9 to -9.0 log M.

For the weak positive control, dexamethasone, the performance criteria for top % binding (8 7 to 106 %) and bottom % binding (-12 to 1 2%) were met in all 17 studies, and the Hill Slopes for dexamethasone (

- 1.2 to

- 0.6) also met the performance criteria ( -1.4 to 0.6). The Log IC 50 for
the weak positive control were relative consistent across all the assays , r ang ing from -4.6 to -4.0 log M . The % relative binding affinity (%RBA) for dexamethasone compared to R1881 was
0.0013-0.0071% , with the range typically being 0.0021-0.0036%.

A summary of how well the positive controls met the performance criteria is shown in Figure 8 and Figure 9.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 44 of 160 Figure 8 . Performance Criteria Results for Strong ( R1881 ) Positive Control.

Figure 9 . Performance Criteria Results for Weak (Dexamethasone ) P ositive Control.

(vi) Issues or Problems with Laboratory Performance

The performance criteria for the saturation binding experiments were not consistently met by two of testing laboratories. In addition, for compounds with limited solubility at 10 -4

to 10 -3 M, the addition of 1⁄2 log concentrations at the upper end of the test concentration range would provide more useful information than including testing concentrations at the lower end (<10 - 10 M). Otherwise, only minor deficiencies were noted in the 17 studies, and none of these had an impact on study interpretation. 17

18 15

1 3

Hill Slope Top plateau level (%) Bottom plateau level (%) 

## Competive Binding - R1881

Met Did not meet

18 18 18

Hill Slope Top plateau level (%) Bottom plateau level (%) 

## Competitive Binding Dexamethasone

Met Did not meetUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 45 of 160 (vii) Overall conclusion

The laboratory performance of the assay was generally consistent across all 17 test compounds. Although several laboratories generated rat prostate cytosolic preparations that did not meet the performance criteria for the saturation binding experiment, when these same preparations were used in the competitive binding assays, the preparations were able to identify test chemicals that bound to the AR (b inders) and the control chemicals responded appropriately as indicated by meeting (or only slightly exceeding) the competitive binding performance criteria for R1881 and dexamethasone. Overall, the laboratories performance of the AR binding assays

was gen erally acceptable and the data were considered reliable.

d. Steroidogenesis

(i) Background

The Steroidogenesis Assay (OCSPP Guideline 890.1550 9 ) is an in vitro screen intended to identify xenobiotics that affect the steroidogenic pathway beginning with the sequence of
reactions involved in the production of testosterone and estradiol/estrone occurring after gonad otropin hormone receptors [follicle

- stimulating hormone receptor ( FSHR ) and luteinizing hormone receptor ( LHR) ] are activated. The steroidogen esis assay is not intended to identify substances that affect steroidogenesis due to effects on the hypothalamus or pituitary gland ( i.e. HPG axis)

. The assay utilizes a human H295R adreno- carcinoma cell line (NCI -H295R) which expresses genes that encode all t he key enzymes needed for steroidogenesis as shown in Figure
10. The assay is intended to identify chemicals that have the potential to induce or inhibit the production of testosterone and/or estradiol.

9 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1550: Steroidogenesis (Human Cell Line - H295R). Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and To xic Substances (7101). EPA 740 -C- 09

- 004. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0576

- 0004

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 46 of 160 Figure 10 . Steroidogenic P athway in H295R cells a

.

a Taken from OECD (2011), Test No. 456: H295R Steroidogenesis Assay, OECD Guidelines for the Testing of Chemicals, Section 4, O ECD Publishing. Gray background indicates corticosteroid pathways/products. Sex steroid pathway/products are circled. CYP= cytochrome P450; HSD = hydroxysteroid hydrogenase; DHEA= dehydroepiandrosterone

. The assay is typically conducted using 24

- well culture plates. H295R cells are plated
under standard culture conditions and allowed to acclimate for 24 hours. The cells are then exposed for 48 hour to seven concentrations (plus solvent control) of the test chemical (10 -4 - 10 -

10 M) in triplicate. A separate 24

- well plate containing solvent control and positive controls for steroid hormone inhibition (prochloraz) and induction (forskolin) are included as a quality control (QC) plate for each run. A minimum of three runs is recommended. At the end of the exposure period, the culture medium is removed, cell viability is evaluated and the
concentrations of estradiol and testoste rone in the medium are measured. Laboratories may use the hormone measurement system of their choice to analyze production of testosterone and estradiol. Data are expressed as fold change relative to the control. The Guideline defines a chemical to be p ositive in this assay if the fold induction is statistically significantly different from the solvent control at doses that fall within the increasing or decreasing portion of the

concentration

- response curve. Statistically significant (p ≤ 0.05) differenc es at concentrations
that do not follow a concentration- response curve may be due to random effects; such results are considered equivocal. Concentrations exceeding the limits of solubility or at cytotoxic (<80% cell viability) concentrations are not inclu ded in the data analyses.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 47 of 160 (ii) Strengths and Limitations of the Assay

The assay uses a cell line available from the American Type Culture Collection (ATCC). It can respond with both increases and decreases in the production of both testosterone and estradiol and allows direct assessment of cell viability within the assay to distinguish effects on steroidogenesis due to chemical interaction with hormone pathways from effects due to cytotoxicity. (OECD Guideline). The inclusion of the H295R Steroidogenesis Assay in the battery provides some redundancy with the aromatase assay and it also addresses multiple targets in the steroidogenic pathway (SAP, 2008). The total metabolic capacity of the cell line is unknown. H295R cells possess enzymes that are capable of catalyzing the production of glucocorticoids and mineralocorticoids so chemicals that alter production of these hormones could influence the levels of testosterone and estradiol production.

(iii) Observations of Test Chemicals

Eighteen compounds were used to evaluate the test laboratories performance of the Steroidogenesis Assay. The purity of the test chemicals used in the assays was > 89%. Five
different laboratories were involved in conducting the 18 steroidogenesis assays, with the majority of the assays being conducted by Laboratory 1 (eight chemicals) or Laboratory 2 (six chemicals)

. All assays were performed with H295R cells (ATCC CLR -2128) grown in DMEMHam’s F -12 medium in a 24- well plate setup as specified in the Guid eline . Due to changes in hormone production in H295R cells with increasing cell passages, specific guidance is provided in the test guideline to ensure that the cells are responsive in this assay.

In the 18 studies evaluated, the cells were all frozen at passage 5 and cultured at least an additional four passages before use in the assays. The total number of passages was ≤10 in all but two assays. Passage 11 cells were used in two assays (Chemicals E, F), but it was stated that since the QC plate met the criteria for hormone production, induction, and inhibition, the assay results were acceptable. All assays used forskolin as a positive control for hormone induction (1 and 10 μM) and prochloraz as a positive control for hormone inhibition (0.1 and 1 μM ). DMSO was used as the solvent in all but one assay. EtOH was used as the solvent for the remaining assay (Chemical S). The concentration of solvents was acceptable for all studies.

One laboratory (Laboratory 3) provided details of the laboratory profic iency test for two chemicals and Laboratory 4 provided details of an abbreviated proficiency test (single high concentrations of forskolin and prochloraz) for Chemical C; the results indicated that the ED 50 values were within ranges specified by the guidelines. All other performing laboratories either only stated that a laboratory proficiency test was conducted or made no reference to one. All assays provided dosing schematics for the test chemicals, and QC plates indicated that the cells

were seeded at a n appropriate concentration (2 -3 × 10 5 cells/mL), and stated that the cells were cultured under the appropriate conditions (5% CO 2 at 37°C for 24 h) prior to exposure. Cytotoxicity was determined by one of three assays: the MTT assay; the modified MTT as say;USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 48 of 160 or the XTT assay. Cytotoxicity was observed in 4 of the 18 steroidogenesis assays, at the highest test compound concentrations (1

- 100 μM).

Hormone concentrations were determined by high pressure liquid chromatography coupled with tandem mass spectroscopy (HPLC/MS/MS) in 14 assays; enzyme linked immunosorbent assays (ELISAs) were used in the remaining four assays (Chemicals C, D, H, M). The limit of quantitation for testosterone (Guideline requirement: 100 pg/mL) ranged from

25- 100 pg/mL for HPLC/MS/M S and from 5-95.7 pg/mL for ELISA (the single exception was a MDL [minimum detection level] of 200 pg/mL for Chemical C). The limit of quantitation for estrogen/estradiol (Guideline requirement: 10 pg/mL) was 10 pg/mL for HPLC/MS/MS and 5.7

- 8.7 pg/mL for ELISA (the single exception was a MDL of 25 pg/mL for Chemical C). Acceptable recovery of testosterone and estrogen from spiked samples was demonstrated in all but three assays. Hormone cross-reactivity in the ELISAs was checked in two of the four studie s that used ELISA for determining hormone concentrations. Hormones were extracted from the culture medium with either methylene chloride or methyl tert

- butyl ether in the HPLC/MS/MS
studies.

Test chemicals were tested at concentrations from 10 0 to 0.0001 μM, with the exceptions
of Chemicals C and S. Chemical C was tested at concentrations from 37.5 to 0.01 μM in Run 2 and 20 to 0.01 μM in Run 3, based on results from the previous runs. Chemical S was tested at concentrations of 0.1 to 0.00001 μM, as cytot oxicity was observed at 1 μ M. No solubility issues
were noted for 16 of the compounds tested at up to 10 0 μ M; h owever, precipitation was noted at 100 μM in one run for Chemical A, and at 10 μM in one run and at 100 μM in all three runs for
Chemical B.

All eight steroidogenesis assays that were conducted by Laboratory 1 included 10 μM

22R- hydroxycholesterol (Guideline recommendation is 20 to 40 μM) to increase the basal production of estradiol. Background wells (that did not include 22R -hydroxycholesterol) were included on the QC plates; the background values were subtracted from the forskolin values to correct for the influence of 22R-hydroxycholesterol on forskolin induction.

(iv) Performance Criteria

Guideline performance criteria for QC plates in the steroidogenesis assay are provided in Table 13. The performance criteria are provided to serve as guidance in identifying runs that provide parameters in the preferred ranges and to ensure that the cell line is responding properly
to known inducers and inhibitors.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 49 of 160 Table 13 . Performance Criteria to be Met on Each QC Plate

Parameter Testosterone Estradiol

Minimum Basal Production 500 pg/mL 40 pg/mL

Basal Production ≥5

- times MDL ≥2.5

- times MDL

Induction (10 μM forskolin) ≥1.5

- times SC a ≥7.5

- times SC

Inhibition (1 μM prochloraz) ≤ 0.5- times SC ≤ 0.5- times SC

a T his value was presented as ≥2

- times SC in the Test Guideline (OCSPP 890.1550)

. Abbreviations: MDL= minimum detectable limit; SC= solvent control

QC Plate Results

All of the QC plates were considered to have had acceptable hormone production for basal levels, as well as for induction (forskolin) and inhibition (prochloraz). Basal testosterone production met or exceeded the minimal basal level of 500 pg/mL for 13 of the 18 assays (range

719- 10897 pg/mL). For the five plates that did not (Chemicals E, K, N, J, F; all from the same laboratory), the lowest concentration range was 433

- 470 pg/mL. All plates met the guideline requirement of having a basal testosterone production ≥5 times the MDL. Two assays by Laboratory 1 had initial induction levels with 10 μM forskolin that were below the minimum (1.5 times the solvent control [SC]), but all values were ≥2 after subtracting the background values. The remaining 16 chemicals had induction level s with 10 μM forskolin that exceeded 1.5 times the SC. Two assays had inhibition levels with 1 μM prochloraz that were above the minimum (≤0.5 times the SC), ranging from 0.52 times to 0.53 times the SC. Basal estradiol production met or exceeded the min imal basal level of 40 pg/mL for 13 of the 18 assays (range

41- 370 pg/mL). For the five plates that did not, the lowest concentration range was 26.7 -35.5 pg/mL. All plates met the guideline requirement of having a basal estrogen production ≥2.5
times the minimum detection level (MDL). All assays had induction levels with 10 μM forskolin that were ≥7.5 times the solvent control. Seven assays had inhibition levels with 1 μM prochloraz that were above the minimum (0.5 times the SC), ranging from 0.53 times to 0.60 times the SC. All intraplate coefficients of variation (CVs) were within acceptable limits (≤30%), but interplate CVs for the A, D, M, N, Q and SQC plates were ≥30%. Overall, when the performance criteria were not met, the values only slightly exceeded the expected values and the data do not indicate that the assay was not responding appropriately. A summary of the QC plate performance data is shown in Figure 11.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 50 of 160 Figure 11. Quality Control Plate Performance for the 18 Steroidogenesis Studies.

Test Compound Results

Table 14 summarizes the results of changes in estrogen and testosterone levels. Six test compounds increased estrogen levels, with no effect on testosterone production. Estrogen levels were significantly (p<0.05) increased by Chemicals, P, D, and F (10 μM and 100 μM). Chemical B increased estrogen levels (p<0.05) in one run at 1 μM and in two runs at 10 μM (precipitation was noted in one run at 10 μM). Estrogen levels were significantly (p<0.05)
increased a t 100 μM by Chemicals V and O. Testosterone levels were decreased by Chemical A (p<0.05), with no effect on estradiol production. Two test compounds showed decreased testosterone levels and increased estrogen levels

. Chemical I decreased (p<0.05) testos terone levels at 10 and 100 μM and increased (p<0.05) estrogen levels at 10 and 100 μM . Chemical C decreased (p<0.05) testosterone levels at 5 (2 runs), 10 (3 runs), and 20 μM (3 runs) and increased (p<0.05) estrogen levels at 5 (2 runs), 10 (3 runs), and 20 μM ( 3 runs). Chemical J decreased (p<0.05) both testosterone (100 μM) and estrogen levels (0.1 μM, 2 runs; 1 μM, 3 runs; 10 μM, 3 runs; and 100 μM, 3 runs). Two test compounds (Chemicals M and H) were considered to have equivocal effects on testoste rone and estrogen levels due to variable findings between runs, and the remaining six test compounds (Chemicals E, G, K, N, S, U) were considered to have negative results for effects on testosterone and estrogen concentration. A summary of the results is shown in Table 15. 11 18 18 13 16 18 13 18

7 5 2 5

Estradiol Inhibition (1 μM prochloraz) Estradiol Induction (10 μM forskolin) Basal Production Estradiol Minimum Basal Production Estradiol Testosterone Inhibition (1 μM prochloraz) Testosterone Induction (10 μM forskolin) Basal Production Testosterone Minimum Basal Production Testosterone 

## Performance Criteria for QC Plates

Met Did not meetUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 51 of 160 Table 14. Summary of Fold- Change in Estradiol and Testosterone Levels

Chemical Concentration (μM) Significant Change in Estradiol a Significant Change in
Testosterone a

P 10 ↑

-- b

100 ↑

--

A 10

-- ↓

100

-- NA c

D 10 ↑ d

-- 100 ↑ d

--

F 10 ↑

-- 100 ↑

--

B 1 ↑ (1 run)

-- 10 ↑ (2 runs) e

--
V 100 ↑

--
O 100 ↑

--

I 10 ↑ ↓

100 ↑ ↓

C g 5 ↑ (2 runs) ↓ (2 runs)

10 ↑ ↓

20 ↑ ↓

J 0.1 ↓ (2 runs)

-- 1 ↓

-- 10 ↓

-- 100 ↓ ↓

M 0.0001

- 100 equivocal equivocal

H 0.0001

- 100 equivocal equivocal

a = statistically significant fold

- change relative to solvent (p<0.05) in all 3 runs, unless otherwise noted.

b No significant changes noted.

c NA = not analyzed due to precipitation. Precipitation noted in all 3 runs

d Statistical significant only for the overall mean of 3 runs per concentration.

e 1 run not analyzed due to precipitation. Precipitation noted in all 3 runs at 100 μM.

f not test ed.

g Extra concentrations beyond protocol requirements tested. Precipitation or cytotoxicity noted at ≥ 37.5 μM.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 52 of 160 Table 15 . Results from the 18 Steroidogenesis Studies.
Assay Results Number of Chemicals Negative 6
Induced Estradiol 6

Inhibited Testosterone 1

Induced Estradiol and Inhibited Testosterone 2

Inhibited Estradiol and Testosterone 1 Equivocal 2

All intraplate CVs were within acceptable limits (≤30%), but interplate CVs for the Chemicals A, H, M and S test plates were slightly ≥30%. MRIDs submitted by Laboratories 2 and 3 reported if CV criteria were met or not met, but did not include individual values.

(v) Issues or Problems with Laboratory Performance

Four of the five performing laboratories (16 of 18 compounds) did not provide data and results for the laboratory proficiency test which is recommended to demonstrate laboratory proficiency with running the assay.

(vi) Overall conclusion

The laboratories performance of the assay was generally consistent across all 18 test compounds, and the performance criteria were generally met for all compounds. In most cases where the performance criteria were not met, the values only slightly exceeded the expected values and did not impact the interpretation or reliability of the study. The results demonstrate that the Steroidogenesis Assay as performed by the testing laboratories can distinguish between
chemicals that alter or do not alter testoster one and/or estrogen levels in vitro

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 53 of 160 e. A romatase (Human Recombinant) Assay

(i) Background

The Aromatase Assay (OCSPP Guideline 890.1200 10 ) is used to determine if a chemical could affect the endocrine system by inhibiting the catalytic activity of the aromata se enzyme, which is responsible for the conversion of androgens to estrogens. The assay utilizes recombinant microsomes containing human aromatase (CYP19) and cytochrome P450 reductase,
and measures the release of 3 H 2 O during the conversion of 3 H-androstenedione to estrone. Competitive inhibition of aromatase activity by a test chemical is detected by serial reaction tubes containing increasing concentrations of the chemical of interest. A depiction of the reaction is presented below:

Follo wing a 15 minute incubation, the reaction is terminated using methylene chloride. The aqueous fraction is extracted and the amount of 3 H 2 O is quantified for each assay tube by liquid scintillation spectrometry. Each mole of 3 H 2 O generated represents the conversion of one mole of substrate to estrone. Each run is an independent experiment and contains replicate tubes of full activity control, background activity control, positive control and test chemical. The known aromatase inhibitor 4-hydroxyandrostendione (4- OH ASDN) is used as the positive control. Eight concentrations of the positive control (10 - 10 to 10 -5 M) and eight concentrations of test chemical (10 - 10 to 10 -3 M) are evaluated in each run. A minimum of three runs is
recommended. After the co mpletion of the first run, the Test Guideline recommends adjusting the concentration of the test chemical in the second and third runs if evidence of insolubility is observed at the highest concentration (10 -3 M). When insolubility is observed, it is

reco mmended that mid -log concentrations ( i.e . 10 - 3.5 M) be tested to define the lower portion of the concentration response curve.

10 Endocrine Disruptor Screening Progr am Test Guidelines OPPTS 890.1200: Aromatase Assay (Human Recombinant). Office of Chemical Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 740 -C- 09

- 004. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0576

- 0004

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 54 of 160 The data are analyzed by first converting raw data to aromatase activity (mmol/mg protein/min) and calculating percent of control values. The percent of control values are plotted versus logarithm of the test chemical concentration. Chemicals that reduce enzyme activity levels by 50% or more and fit a four parameter, one

- site competitive binding inhibition model
are considered t o be inhibitors of aromatase. Chemicals that fit the inhibition curve, but allow

50- 75% activity, i.e . reduce activity by 25

- 50%, are considered equivocal. Chemicals that do not fit the model or that fit the model, but do not reduce activity by 25%, are considered to be noninhibitors of aromatase. Typically, an inhibition curve will fall from 90% to 10% over 2 log units with a slope near

- 1. Unusually steep curves may indicate solubility issues or protein denaturation. If the slope of the curve is ste eper than

- 2.0, the classification is considered equivocal. A typical positive control response curve is shown in Figure 12 below:

Figure 12. Concentration R esponse C urve for Positive Control 4- OH ASDN.

(ii) Strengths and Limitations of the Assay

The assay utilizes commercially available recombinant microsomes and does not require culturing cell lines or animal testing. The assay i s considered an important assay in the battery since it might indirectly inform on the potential developmental effects mediated by disruption of
testosterone to estradiol conversion. The assay detects chemicals that inhibit aromatase activity, and cannot detect chemicals that may induce aromatase activity through an upregulation of aromatase gene expression. The assay provides li mited/no ability to metabolize xenobiotics.

(iii) Observations from Initial Screening of Test Chemicals

Eight een compounds were used to evaluate the performance of the Aromatase Assay.
Seven different laboratories we re involved in conducting the Aromatase Assa ys, with the majority of the assays (13) being conducted by two labs. Although any source of human recombinant microsomes containing CYP19 + reductase may be used in the assay, a ll laboratories utilized recombinant microsomes purchased from one source (BD Biosciences). The

a ssays were generally conducted in accordance with the recommendations in Guideline
890. 1200. The purity of the test chemicals used in the assays was > 89%. Dimethyl sulfoxideUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 55 of 160 (DMSO) or ethanol w as used as the solvent vehicle in all studies, each at ≤1% of assay volume. Each assay included data from three successful runs, although a total four or seven test runs were
conducted in five studies.

In general, the t est chemicals were initially tested at serial log concentrations from 10 - 10

to 10 -3 M as recommended in the Test Guideline. There was one exception (Chemical S) where a solubility test was performed prior to conducting the study using DMSO and ethanol as
solvents. The test chemical was soluble at 10 -5 M in ethanol and that concentration was used as the high test concentration in the initial run. Additional mid -log concentrations of 10 - 5.5 and
10 - 7.5 M were included in the runs for that chemical. Of the remaining 17 chemicals, no solubility issues were noted for eight of the compounds tested at up to 10 -3 M. However, 10 of the test chemicals were not soluble in the assay at a concentration of 10 -3 M, and f ive of these compounds were also not soluble at 10 - 3.5 M. When solubility issues were noted, additional mid

log concentrations (10 - 3.5 , 10 - 4.5 or 10 - 4.5 M) were added at the upper end of the concentration

range for six compounds, in order to better define the lower portion of the inhibition curve. However, for three chemicals with solubility issues, additional mid -log concentrations were not added to the assay. These values are summarized in Table 16.

Table 16. Summary of the High T est Concentration for the 18 Aromatase Assays.

Number of Test C hemicals Hig hest Soluble Concentration
T ested Mid

- log Concentrations
T ested Comments

8 10 - 3 M N/A Soluble up to 10 - 3 M
3 10 - 4 M Yes Insoluble at 10 - 3.5 M
3 10 - 4 M No Insoluble at 10 - 3 M
2 10 - 4.5 M Yes Insoluble at 10 - 4 M
1 10 - 5 M Yes Insoluble at 10 - 4.5 M
1 10 - 5 M Yes Solubility pre

- test performed. N/A= not applicable

Two of the studies included data demonstrating the laboratory’s proficiency in running the assay by testing either the four Guideline

- recommended proficiency chemicals (econazole, fenarimol, nitrofen and atrazine) or a variation of these chemicals (prochloraz, fenarimol, nitrofen and ronidazole), and another five study reports stated that proficiency testing was successfully conducted, but results were not included in the study reports. The remai ning 12 studies provided no information regarding proficiency testing.

Results

Of the 1 8 chemicals tested , 1 1 were classified as non -inhibitors (Table 17). However, two of these compounds had precipitation at 10 -3 M and did not include mid- log concentrations at the higher concentrations to better define the inhibition curve

, consequently there is less
confidence in their classification

. Five of the chemicals were classified as equivocal due toUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 56 of 160 having either steep H ill slopes (Chemicals M, P) or minimum inhibition between 50

- 75% at the highest tested concen tration (Chemicals A, D, and S). However, there is less confidence in the classification for one (Chemical M) of these compounds as it did not include mid

- log concentrations in the upper range. The remaining 2 compounds were classified as inhibitors (Chemicals J and K).

Table 17. Classification of the 1 8 C hemicals E valuated in the Aromatase Assay

. Assay Results Number of Chemicals
Inhibitor 2
Non

- inhibitor 11 Equivocal 5

(iv) Performance criteria

Guideline performance criteria for the aromatase assay are provided in Table 18. The performance criteria are provided to serve as guidance in identifying runs that provide parameters in the preferred ranges.

Table 18. Performance Criteria for Aromatase Assay

Parameter Recommended Values Minimum Aromatase Activity 0.1 nmol/mg

- protein/min
Mean Background Control Activity ≤ 10% of Full Activity

Coefficient of Variation (CV) for replicates within each sample type and concentration of 4

- OH ASDN <15%

Criteria for Positive Control Response Curves

Parameter Lower Limit Upper Limit Slope -1.2 -0.8 Top (%) 90 110
Bottom (%) -5 +6

Log IC 50 -7.0 -7.3

Background and Full Activity Controls

Full enzyme activity control samples contain substrate, NADPH, propylene glycol, vehicle and microsomes. Aromatase activity in the full enzyme activity controls was above the recommended minimum activity level (0.1 nmol/mg protein/min ) , except in two studies, which were slightly below the recommend ed levels with minimum activities of 0.090 and 0.098 nmol/mg protein/min in selected runs. In all the assays, the average enzyme activity across all three runs ranged from 0.114 to 0.616 nmol/mg protein/min. Background activity controlsUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 57 of 160 (samples that contain all components of the full aromatase activity controls minus NADPH) w ere ≤11.5% of the full activity in all assays and was typically ≤3.5% of full activity . The recommended mean background control activity is ≤ 15% of full activity control

. The range of values for the full activity controls (as % of average full activity) were within the recommended 90-110% range in 8 of the 18 studies, and the remaining 1 0 studies had only a few full activity values slightly out side the range (84 -115%). Quality con trol

- assay drift is monitored by measuring the aromatase activity of the full enzyme activity controls and background activity controls at the beginning and end of each assay run. No assay drift was noted in 10 of the 18 studies, and slight assay drift wa s occasionally noted in the other eight studies. When drift was noted, the changes were minor and did not adversely impact the study results.

Positive Control

All studies utilized 4

- hydroxy -androstenedione (4- OH ASDN) as the positive control compound for inhibition of aromatase activity. The performance criteria for the 4

- OH ASDN were generally met with a few exceptions. The top of the mean inhibition curve for the positive control was within the recommended range (90-110%) in 12 of the studies and w as out of range in the remaining 6 studies (77.5- 117%). The bottom of the mean inhibition curve for the positive control was also within the recommended range (

- 5 to 6%) in 17 studies, and was only slightly out of range in the remaining study (-5.2%). Th e Hill Slope values for 4

- OH ASDN were acceptable ( -1.2 to - 0.8), with the exception of one run in one study (- 0.76). The Log IC 50 values
for 4 -OH ASDN were within the recommended range (-7.3 to -7.0) in all runs for 10 of the 18 studies. Of eight studie s with Log IC 50 values outside the recommended range, six studies had
runs that were lower values ( -7.79 to -7.35 log M) and two tests had slightly higher values (-6.90 to -6.74 log M). However, the a verage log IC 50 values (across runs for a chemical) wer e generally within range, with the exception of the two studies, which had average log IC 50 values
of -7.54 and - 7.38 log M, respectively. The variability (%CV) within % inhibition values for 4

- OH ASDN at each concentration tested were generally acceptabl e (<15%), although 1 0 of the studies had %CV in excess of 15% at one or more concentrations. The higher %CVs typically occurred at the higher concentrations of 4- OH ASDN, which had high levels of inhibition (>95%). At these low activity levels, small cha nges in the standard deviations resulted in large changes in the %CV. A summary of how well the positive controls met the performance criteria is shown in Figure 13.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 58 of 160 Figure 13. Aromatase Performance Criteria Results.

(v) Issues or Problems with Laboratory Performance

The issues were noted in the conduct of the Aromatase Assays relating to laboratory proficiency testing and the selection of test chemical concentrations. Although the assay generally performed as expected in each study, actual laboratory pr oficiency data utilizing four reference compounds was provided in only two study reports. The other challenge, which was noted in 4 of the 18 studies, was that the activity of the test compounds was not adequately characterized at higher concentrations. For three chemicals with solubility issues at 10 -3 M

, additional mid -log concentrations , such as 10 - 3.5 or 10 - 4.5 M, were not included in the assay to better characterize the response of the test chemical and provide a complete inhibition curve.

(vi) Overall co nclusion

The assay performance was generally consistent across all 18 test compounds, and the performance criteria for the assay were generally met in each study. Aside from the lack of mid

log concentrations for some studies, the deviations noted in the performance criteria were often minor, slightly exceeding the ranges for the performance criteria and did not adversely impact study interpretation. Overall the assay as performed by the testing laboratories is able to distinguish between inhibitors and non- inhibitors of aromatase activity. 17 10 17 12 17 8 18 16

1 8 1 6 1 10 2

Mean Positive Control (mean of all runs) - Log
IC50 Positive Control (all runs) - Log IC50 Positive Control Response Curve - Bottom (%) Postive Control Response Curve - Top (%) Positive Control Slope Coefficient of Variation (CV) for replicates of 4

- OH ASDN Mean Background Control Activity Minimum Aromatase Activity



## Aromatase Performance Criteria

Met Did not meetUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 59 of 160 2. In vivo

a. Uterotrophic

(i) Background

The Uterotrophic Assay (OCSPP 890.1600) 11 is intended as an in vivo screening assay, providing data about a single endocrine mechanism ( i.e ., the estrogenicity of a test compound).
The Uterotrophic assay employs an animal test system in which there are very low levels of or no endogenous estrogen and concomitant low baseline uterine weights with minimal variation. This enhances the ability to evaluate the uterine response to exogenous 1 7 β -estradiol and potential estrogen agonists. Two animal models have been validated for this assay: young adult females (mouse or rat) after ovariectomy with adequate time for uterine tissue regression; or immature female rats after weaning and prior to puberty. The G uideline states a preference for the adult ovariectomized rat. The Uterotrophic Assay evaluates the ability of test chemical(s) to elicit agonism of estrogenicity as determined by a statistically significant increase in uterine weight.

(ii) Stre ngths and Limitations of the Assay

The Uterotrophic Assay is a relatively rapid screen which incorporates metabolism. The test guideline states a preference for subcutaneous injection as the route of administration to provide absorption, distribution, metabolism, and excretion ( ADME ) insight and contrast with the rat pubertal assays with gavage administration. However, oral administration is acceptable and is the most frequent route of administration in uterotrophic studies re viewed to date. For humans, oral administration will model a primary exposure route and incorporates ADME of the
test chemical. A positive finding in this assay can be corroborated by information such as ER binding or transcriptional activation.

(iii) Observations of Test Chemicals

Of the 17 chemicals evaluated, 10 assays utilized mature, ovariectomized female rats at ages of 56- 75 days, and the other seven assays (Chemicals D, E, F, J, K, M, Q) utilized sexually immature females at an age of 19 days. All compounds were evaluated for estrogenic activity, and none of the tests included the optional testing for anti -estrogenic activity. The route of exposure for the test compounds was via oral gavage, with the exception of one chemical (N)
which was administered subcutaneously (s.c.). The dietary concentrations of phytoestrogens (total isoflavones or genistein equivalents) were reported for all but one assay (Chemical C). Dietary phytoestrogen concentrations were reported within two distinct ranges: 6.2

- 43 μg/g diet, reflecting the actual measured concentrations, or <300

- 350 μg/g diet, meeting Guideline specified limits. The Guideline recommends that the highest dose level be at or just below the

11 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1600: Uterotrophic Assay. O ffice of Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 740 -C- 09

- 0010. October 2009.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 60 of 160 MTD but need not exceed the Limit Dose (1000 mg/kg/day); however, typically, the a gency also considers the toxicity profile of the chemical ( i.e ., ≥10% decrease in body weight, cholinesterase inhibition, target organ toxicity, etc .) in dose selection. As a screen, large intervals, e.g. , one half log units (corresponding to a dose progression of 3.2) or even one log units, between dosages

were acceptable.

As shown in Table 19, of the 17 chemicals tested in this assay, dose levels were selected
based on the resu lts of range -finding studies (11 chemicals), Part 158 standard toxicology studies (3 chemicals), or the Limit Dose (3 chemicals). Seven chemicals had two dose levels (A, B, D, G, K, Q, and S) tested

, and 10 chemicals had three doses tested. No overt toxicity was seen in 16
of 17 chemicals; overt toxicity was seen for Chemical A as describe below

. The doses tested were determined to be adequate for all chemicals.

Table 19. Summary of Overt Toxicity Observed in the Uterotrophic Assay

Chemical Code Highest Dose Tested (mg/kg/day) Body weight ( > 10%) Clinical Signs Methods Used to Select Dose Levels
A 250 X X Part 158 studies
B 1000 Limit Dose
C 1000 Limit Dose
D 120 Range finding study
E 200 Range finding study
F 225 Range finding study
G 400 Range finding study
H 180 Range finding study
I 4.0 Range finding study
J 4 50 Range finding study
K 20 Part 158 studies
M 20 Range finding study
N 50 Range finding study
P 1000 Limit Dose Q 400 Range finding study
S 75 Range finding study
V 0.3 Part 158 studies X= Toxicity which was observed only at the highest dose tested

Chemical - A : Doses were selected based on the results of Part 158 standard toxicology studies
where diarrhea, rust colored fur, and tremors were observed in female rats within 9 hrs following a single oral dose at 300 mg/kg. Based on these studies, the highest dose selected for the main study w as 250 mg/kg/day. In the main study, r ats a t the highest do se tested ( HDT ) exhibited the following adverse clinical effects: 2 rats displayed nasal discharge, thinness, and fur discoloration on Day 2; on Day 3

, those same 2 rats plus 3 additional rats (total of five) displayed head and scapula fur discoloration, lethargy and hunched posture; these same 5 rats displayed head and scapula fur discoloration prior to euthanasia. There was a significant decrease (15%) in mean body weight at HDT.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 61 of 160 Chemical - D : In the range finding study, when tested at doses of at doses of 0, 5, 20, 50, 80 or 150 mg/kg/day, clinical signs and body weight changes were seen at 150 mg/kg/day. Based on
these results

, the highest dose selected for the main study was 1 20 mg/kg/day.

Chemical - E : In the range finding study, when tested at 250, 500, 750 and 1000 mg/kg/day b y D ay 15, females weighed 12.2%, 9.0%, 13.5% and 12.9% less than controls, respectively. Based on these results, the highest dose selected for the main study was 200 mg/kg/day.

Chemical - F : In the range finding study, when tested at doses of 0, 50, 200, 400, or 600 mg/kg/day for 15 days, absolute and relative liver weights were increased at doses ≥ 200 mg/kg/day

. Based on these results, the highest dose selected for the main study w as 225 mg/kg/day.

Chemical - G : Two range finding studies were performed

. In the first study , no clinical signs of toxicity or change in body weights were observed in doses up to 150 mg/kg/day. In the second study m ortality was observed in ≥600 mg/kg dose groups

. Based on these results

, the highest dose selected for the main study was 4 00 mg/kg/day.

Chemical - H : In the range finding study, when tested at doses of 0, 20, 60, or 180 mg/kg/day for 3 consecutive days , n o adverse effects were seen at any dose including the high dose. The highest dose tested in the main study was 180 mg/kg/day where mean body weights were decreased (p<0. 05) by 8 and 9% on Days 3 and 4, respectively, at this dose

.

Chemical - I : In the range finding study, when tested at doses of 0, 1, 2, 4, or 8 mg/kg/day for 15 days, decreased bo dy weight gains were observed in the 4 and 8 mg/kg/day dose groups. Red
blood cell cholinesterase (ChE) activity was inhibited by 75

- 81% at 1 mg/kg/day and ≥ 95% at doses greater than 2 mg/kg/day ; brain ChE was inhibited at doses ≥2 mg/kg/day. Based on these results, the highest dose selected for the main study was 4 mg/kg/day

.

Chemical - J : In the range finding study, when tested at doses of 0, 25, 100, 300, or 450 mg/kg/day for 15 days, no adverse effects were seen at any dose. Based on these results, the highest dose se lected for the main study w as 450 mg/kg/day

.

Chemical - K : Dose levels were selected based on data available from the standard Part 158
studies. No mortality was observed in the repeated

- dose dietary studies conducted at doses of up to 27 mg/kg/day, or in repeated

- dose gavage studies with doses up to 15 mg/kg/day. Based on
the se data , the highest dose selected for the main study was 20 mg/kg/day

.

Chemical - M : In the range finding study, when tested at doses of 20 or 40 mg/kg/day for 10 days, mortality, severe clinical signs and reduced body weight gain were seen at 40 mg/kg/day. Based on these results, the highest dose selected for the main study was 20 mg/kg/day.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 62 of 160 Chemical - N : In a range

- finding study, rats were given subcutaneous (s.c) injections at 0, 25, 75 or 250 mg/kg/day. Rats given 75 mg/kg/day, struggled and vocalized during dosing, suggesting
irritation due to s.c administration; therefore, the 250 mg/kg/day dose was not administered

. Based on body weight loss and the presence of a small focus of hemorrhage at the injection site,
the dose of 75 mg/kg/day was determined to exceed MTD. Based on the se results

, the highest
dose selected for the main study w as 50 mg/kg/day

.

Chemical - Q : In the range finding study, when tested at dose levels of 0, 100, 300, 500, 750 and 1000 mg/kg for 14 consecutive days, mortality occurred at doses > 500 mg/kg/day. At 300 mg/kg /day, transient uncoordinated movement was seen in all rats. Based on these results, the highest dose selected for the main study was 400 mg/kg/day.

Chemical - S : In the range finding study, when tested at doses of 0, 10, 50, or 100 mg/kg/day for
28 da ys, mortality, clinical signs, and decreases in body weight were seen at 100 mg/kg/day. Based on these results, the highest dose selected for the main study was 75 mg/kg/day.

Chemical - V : In Part 158 standard toxicology studies, brain ChE activity was significantly decreased by 50

- 59% in adult rats at 0.3 mg/kg/day. Based on these results, the highest dose selected for the main study was 0.3 mg/kg/day. In the main study, t he cholinesterase phase of the study was conducted separately ; s ig nificant inhibition of red blood cell ( RBC ) and whole
brain ChE activity was seen at the high dose ( 0.3 mg/kg/day ) on the first and last day of dose
administration . All animals survived until scheduled termination and no clinical signs of toxicity

were observed.

(vi) Results

None of the 17 test compounds evaluated showed a response of estrogenic activity under the conditions of this assay. Except for Chemical A, d oses tested in all studies were deemed adequate based on the absence of overt systemic toxicity at the doses te sted , attainment of an adequate dose and/or inclusion of the Limit Dose.

Positive Control: 17 α

- ethynyl estradiol (EE) is recommended as the positive control for this assay. In the examined studies, EE was administered by subcutaneous ( s.c. ) injection at doses ranging from 0.27 to 0.5 μg/kg/day in three studies, at 3 μg/kg/day in two studies, and at 300 μg/kg/day in two studies. Additionally, EE was administered by oral gavage at doses of 1.0 μg/kg/day in three studies, 10 μg/kg/day in four studies, and 100 μg/kg/day in three studies.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 63 of 160 The expected effect of the positive control, EE, in the agonist assay is increased uterine weight. Following s.c. administration , EE increased absolute we t uterine weights by 1.9- to 3.9-
fold at doses of 0.27- 0.5 μg/kg/day, 8.7 - to 9.3- fold at doses of 3 μg/kg/day, and 6.6 - to 10.4-fold at doses of 300 μg/kg/day. The concomitant increases in blotted uterine weights were less varied, with increases ranging from 1.9 2 - to 2.8-fold at 0.27 - 0.5 μg/kg/d ay , 3.3 - 3.5 at 3 μg/kg/day and 3.3- 6.0 at 300 μg/kg/day

. Following administration by oral gavage, EE increased absolute wet uterine weights by 2.1- to 2.9 - fold at 1 μg/kg/day, 6.3 - to 9.9- fold at 10μg/kg/day,
and 2.6- to 5.1- fold at 100 μg/kg/day. As with the s.c. dosing, the increases in blotted uterine weights following oral dosing were less varied, ranging from 2.1 - to 3.0- fold at 1 μg/kg/day, 5.0 - to 6.2- fold at 10 μg/kg/day, and 2.3 - to 3.3- fold at 100 μg/kg/day. EE dose s were high enough for the positive control to show the appropriate response in terms of statistically (p≤ 0.05) increased uterine weights in all 17 assays. However, for three assay s , the EE doses used were above the maximum dose (10 μg) validated by OECD for this assay (Kanno et al ., 2001); such high doses are often beyond the maximal response plateau for EE and may lead to nonmonotonic positive control results.

(iv) Performance Criteria : The performance criteria for the Uterotrophic assay are:

• Blotted uterine weights after dosing of the vehicle control are expected to be 0.09% or 0.04% of final body weight in immature or ovariectomized female rats, respectively.

• A baseline positive control study with ≥ 4 doses of EE to show dose response vs. uterine weight is expected to have been conducted, and the dose of EE administered in the assay should be 70-80% of the effective dose determined from the baseline study.

The first criterion related to relative uterine weight was met in all studies; however, the second criterion was not met consistently. I ssues regarding the second performance criterion are:

• Baseline control studies were not reported for 2 test chemicals.

• One laboratory (8) conducted a baseline control study, but selected the reference EE dose based on other study results.

• Data were not routinely reported to assess the dose response of the positive control (EE)

vs. effective dose from the control baseline study.

(v) Issues or Problems with Assay

No technical deficiencies sufficient to compromise the conduct and/or interpretation of the assay were identified

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 64 of 160 (vi) Overall C onclusion

The assay performance was consistent across all 17 test compounds. Vehicle control uterine weights were ≤ 0.09% or 0.04% of final body weight in immature or ovariectomized female rats, respectively, and positive control animals showed the appropriate response of statistically (p≤0.05) increased uterine weights. Although a wide range of doses were utilized for the EE positive control, the resulting maximal increases in wet and blotted uterine weights consistent ly reached at dose s ≥10μg and demonstrated that the assay was performing as expected.

b. Hershberger

(i) Background

The Tier 1 Hershberger Bioassay (OCSPP 890.1400) 12 is a short- term in vivo screening test that is designed to detect chemicals that act as androgen agonists (androgenic) or androgen antagonists (anti- androgenic) or chemicals that inhibit 5α -reductase. Androge ns are essential for the initial development of the reproductive system in utero, the maturation of the reproductive system and accessory sex organs, development of secondary and tertiary sex characteristics at puberty, and maintenance of the reproductive system and accessory sex organs structures and functions until death. Androgens are needed for feedback regulation of the hypothalamus

pituitary

- gonadal (HPG) axis and spermatogenesis.

In this assay, castrated pubertal rats are dosed with the test chemical by oral gavage or subcutaneous (s.c) injection for 10 consecutive days. Critical measurements include the weights of five androgen- dependent sex accessory tissues (ventral prostate [VP], seminal vesicle [SV] with fluids and coagulating glands, levator ani

- bulbocavernosus [LABC] muscle, paired Cowper’s glands [CG] and glans penis [GP]) . These five tissues all respond to androgens with an increase in absolute weight. When testing for anti- androgenicity, t hese tissues are stimulated to increase in weight by s.c. administration of a potent reference androgen (testosterone propionate [TP]) and the five tissues measured respond to anti

- androgens with a decrease in absolute weight. Other endpoints include clinical observations and body weight changes. For and rogen agonism, a statistically significant (p ≤ 0.05) increase in two or more of the five androgen responsive tissues is considered a positive result. For androgen antagonism, a statistically significant (p ≤ 0.05) reduction in two or more of the five androge n responsive tissues is considered a positive result.

12 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1400: Hershberger Assay. Office of Sa fety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 740 -C- 09

- 008. October 2009.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 65 of 160 (ii) Strengths and Limitations of the Assay

Th e Hershberger A ssay is a relatively rapid screen that is quite specific to androgenic effects. The assay can effectively detect (and distinguish) androgen agonists, antagonists, and
inhibitors of 5α

- reductase. The assay incorporates metabolism and can detect chemicals that

requ ire metabolic activation. Oral administration models a primary exposure route and incorporates ADME. Compounds other than androgenic agonist s can alter the weight of certain
tissues ; h owever, the growth response of several tissues concomitantly substanti ates a more androgen

- specific mechanism.

(iii) Observations of Test Chemicals

Of the 17 available Hershberger Bioassays, 16 used Sprague

- Dawley rats as the test species, and one (Chemical S) utilized Wistar rats. All of the assays utilized male rats, surgically castrated on post

- natal day (PND) 42

- 48. The rats were at PND 51-60 and weighed 194- 314 g at
the initiation of the 10

- day treatment period. All test compounds were evaluated for both androgenic and anti- androgenic activity. The route of exposure for all of the test compounds was oral gavage. The Guideline recommends that the highest dose level be at or just below the MTD but need not exceed the Limit Dose (1000 mg/kg/day); however, typically, the a gency also considers the toxicity profile of the chemical ( i.e

., ≥ 10% decrease in body weight

, cholinesterase inhibition, target organ toxicity, etc.) in dose selection. In addition to the guideline recommended endpoints, blood and br ain cholinesterase activity were measured in 2 studies, and
serum testosterone levels were measured in three studies.

(iv) Results

As shown in Table 20, of the 17 chemicals tested in this assay, dose levels were selected based on the results of range- finding studies (11 chemicals), Part 158 standard toxicology studies (5 chemicals), or the Limit Dose (1 chemical). For the androgen phase (A) of the assay, 9
chemicals had two dose levels tested and 7 chemicals had three doses (E, F, I, J, N, P and V)
tested. For the anti- androgen phase (aA), three dose levels were used for all chemicals

. No overt toxicity was seen for 14 of 17chemicals and the dose levels tested were determined to be adequate for these chemicals. Overt toxicity was seen for the remaining 3 chemicals, (B, E and K). No overal l androgenic response was seen for any chemical. In contrast,
anti

- androgenic response was seen for 7 chemicals (A, B, E, G, H, K and S) . For 3 of the 7 (B, E and K), effects were only seen in the presence of overt toxicity ( e.g

. , ≥ 10 % decreases in body weight and/or clinical signs) . Anti- androgenic responses were seen in the absence of overt toxicity for the remaining 4 Chemicals (Table 20).USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 66 of 160 Table 20. Summary of Overt Toxicity Observed in the Hershberger Assay

.

Chemical C ode Highest Doses
Tested (mg/kg/day) Body Weight ( > 10%) Food Consumption Clinical Signs Methods Used to Select
Dose Levels
A 150 Part 158 studies
B 1000 x x Range finding study
C 1000 Limit dose
D 80 Range finding study
E 750 x x Range finding study
F 450 Range finding study
G 150 Part 158 studies
H 180 Range finding study
I 12 Range finding study
J 500 Range finding study
K 25 x Part 158 studies
L 100 Range finding study
M 20 Range finding study
N 800 Range finding study
P 500 Part 158 studies study
S 75 x Range finding study
V 0.3 Part 158 studies

X = Toxicity seen only at the highest dose tested Shaded rows indicate a response in the assay

Chemical - B : Based on the results of a range finding study, the highest dose tested (HDT) was
1000 mg/kg/day for both the androgen agonist and anti

- androgen phase of the assay. In androgen phase of the Hershberger assay, at 1000 mg/kg/day, mean body weights were
decreased (p≤0.05) on Days 3 ( ↓13%), 4 (↓ 15%)

, and 5 thru 9 ( ↓ 11%). Food consumption was affected. In the anti

- androgen phase, at the HDT, mean body weights were (p≤0.05) decreased on Days 3 (12%), 4 (18%), 5 and 6 (↓21%), 7 (↓22%), 8 (↓24%), 9 (↓23%), 10 (↓ 22%) and 11 ( ↓22%), and food consumption was decreased (p<0.05) by 31% on Days 1 -11. Effects on androgen sensitive tissues w ere seen only at the HDT, but not at the lower doses [300 m g/kg/ day
and 100 mg/kg/ day] where no overt toxicity was seen.

Chemical - E : Based on the results of a range finding study, the high dose selected was 750 mg/kg/day. In the androgen phase of the Hershberger assay, at 750 mg/kg/day, mean body weights were decreased (p≤0.05) on Days 4 ( ↓11%), 7 (↓ 7%) and 11 ( ↓7%, not significant).
Food cons umption was decreased (p≤0.05) in the mid (250 mg/kg/day; ↓ 12%) and high (750 mg/kg/day; ↓44%) dose groups during Days 1

- 4. There were no effe cts on food consumption during D ays 4 -7 or 7- 11 in any of the dose groups. In the anti- androgen phase at the HDT, mean body weights were signif icantly (p<0.05) decreased on Days 4 (↓14%), 7 (↓ 10%) and 11 ( ↓ 12%).

F ood consumption was decreased (p≤0.05) in the mid (250 mg/kg/day; ↓ 23%) and high (750 mg/kg/day; ↓ 50%) dose gro ups during Days 1

- 4, and only in the high dose (750 mg/kg/day;USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 67 of 160 ↓ 13%) group during D ays 4 through 11. Effects on androgen sensitive tissues were seen only at the HDT but not at the lower doses, 250 mg/kg/day and 50 mg/kg/day.

Chemical - K : Based on the results of a range finding study, the highest dose selected was 25 mg/kg/day. In the androgen phase of the Hershberger assay at the HDT

, tremors ( Days 3 -9) and uncoordinated movement (Days 4 and 5 ) were seen in one rat, and tremors were noted in six
other rats on Days 3 -9. There were no effects on body weight at any dose level. In the anti

androgen assay , tremors (Days 3 and 7) and uncoordinated movement ( Day 3 ) were seen in one
rat at the HDT. Another rat at the HDT exhibited tremors on D ay s 3

- 9 and uncoordinated movement on Day 5; six other rats at this dose also exhibited tremors between Days 4 and 8. At 24 hours after dosing, no clinical signs were observed in any dose group. Mean body weights were decreased ( ↓12%; p≤0.05) on Day 11 only at the high dose. Effects on androgen sensitive tissues were seen only at the HDT but not at the lower doses, 15 mg/kg/day and 5 mg/kg/day.

Chemical – S : Based on the results of a range finding study, the highest dose selected was 75 mg/kg/day. In the Hershberger assay, clinical signs of toxicity were limited to salivation in 3/6 and 6/6 rats at the 10 and 75 mg/kg/day groups, respectively, in the androgen agonist phase, and in 4/6 and 6/6 rats in the 40 and 75 mg/kg/day groups in the anti

- androgen study.

Positive Controls: The Guideline recommended reference androgen agonist is TP administered at 0.2 or 0.4 mg/kg/day by s.c. injection. In the examined studies, TP was administered by s.c. injection at 0.2 mg/kg/day in four studies and at 0.4 mg/kg/day in 13 studies. The Guideline recommended reference androgen antagonist is flutamide (FT) administered at a dose of 3 mg/kg/day by oral gavage, and co

- administered with TP. FT was administered at the recommended dose and by the reco mmended route in all 17 assays

.

The expected effect of the reference androgen agonist, TP, in the androgenic assay is increased weight of the androgen- dependent sex accessory tissues. Below are the ranges of reference androgen responses across the 17 assa ys evaluated. Following administration by s.c. injection at 0.2 mg/kg/day, TP increased tissue weights in all 5 sex accessory organs examined compared to solvent control as follows: ( i) SV by 327-381%;( ii) VP by 381 -676%; ( iii) LABC by 132-188%; (iv) CG by 322-579%; and (v) GP by 48-72%. F ollowing administration by s.c. injection at 0.4 mg/kg/day, TP increased sex accessory tissue weights compared to the control as follows: i) SV by 530-1926%; ii) VP by 493-2200%; iii) LABC by 96-287%; iv) CG by 285-794%; and v) GP by 49-112%.

The expected effect of the reference androgen antagonist, FT, when co

- administered with
TP in the anti- androgenic assay is decreased weight of the androgen

- dependent sex accessory tissues compared to the TP controls. Below are the ranges of reference androgen agonist responses across the 17 assays evaluated F ollowing administration by oral gavage at 3 mg/kg/day with TP co

- administered by s.c. injection at 0.2 mg/kg/day, FT decreased sexUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 68 of 160 accessory tissue weights compared to the solvent control as follows: ( i) SV by 70 -74%; ( ii) VP by 73

- 75%; ( iii) LABC by 48

- 58%; (iv) CG by 67-75%; and (v) GP by 18 -33%. Following administration by oral gavage at 3 mg/kg/day with TP co -administered by s.c. injection at 0.4 mg/kg/day for the 17 assays , FT decreased sex accessory tissue weights compared to the control
as follows: ( i) SV by 62 -86%; ( ii) VP by 59

- 81%; ( iii) LABC by 36 -58%; (iv) CG by 51- 69%; and (v) GP by 14 -35%.

In the androgenic assay, the reference androgen agonist (TP) showed the appropriate

re sponse in terms of significantly (p≤0.05) increased sex accessory tissue weights in all 17 assays. In the anti- androgenic assay, the reference androgen antagonist (FT), co

- administered with the reference androgen agonist, showed the appropriate response in terms of s ignificantly (p≤0.05) decreased sex accessory tissue weights in all 17 assays.

(v) Performance Criteria: The performance criteria for the Hershberger A ssay are presented in Table 21.

Table 21. Maximum recommended % CVs for the Mandatory Sex Accessory Tissues.

Tissue Anti

- androgenic effects Androgenic effects

Seminal vesicles 40% 40% Ventral prostate 40% 45%

LABC 20% 30% Cowper's glands 35% 55% Glans penis 17% 22%

In the androgenic assays , the CVs [ expressed as a percent (%CVs) ] for the sex accessory tissues from the control and high- dose animals were less than the specified maxima for all five tissues in 10 of the 17 assays. For the control animals, the maximum recommended %CVs were exceeded in one assay for the SV (49%, Chemical D), four assays for the VP (48 -94%; Chemicals D, E, F, and G), one assay for the CG (71%; Chemical G), and one assay for the GP (38%; Chemical G). For the high- dose animals, the maximum recommended %CVs were exceeded in one assay for th e SV (49%; Chemical D), three assays for the VP (49 -78%; Chemicals D, N, P), one assay for the CG (83%; Chemical G), and three assays for the GP (25- 36%; Chemicals C, D, P). Although %CVs above the recommended maxima were noted in 7 of the assays, only 2 (Chemicals D and G) of the 17 assays had %CVs that exceeded maximum performance criteria for multiple tissues from both the control and high -dose animals. For the positive control (TP), the %CVs were less than the recommended maxima for all five androgen sensitive tissues in all 17 evaluated assays.

In the anti -androgenic assays , the %CVs for the sex accessory tissues from the control and high- dose animals were less than the specified maximums for all five tissues in 13 of the 17

assays.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 69 of 160 • For the control animals, the %CVs were less than the recommended maxima for the SV, VP, LABC, and CG tissues in all 17 assays, and the maximum %CV for the GP was exceeded only slightly in one assay (22%; Chemical B).

• For the high- dose animals, the %CVs were less than the recommended maxima for the SV, GC, and GP in all 17 assays, and the %CV maxima were exceeded in one assay for the VP (49%; Chemical J) and three assays for the LABC (24- 33%; Chemicals A, B, K). Only one of the anti- androgenic assays ( Chemical B ) had %CVs above the recommended maxima in multiple tissues from both the control and high

- dose animals.

• For the positive control (FT+TP) animals, the %CVs were less than the recommended maxima for all five androgen sensitive tissues in 8 of the 17 evaluated assays , and another 7 of the assays had %CVs above the recommended maxima in only one tissue. The maximum recommend %CVs for the positive control were exceeded in one assay for the SV (44%), two assays for the VP (41 % and 57%), four assays for the LABC (22 %- 30%), one assay for the CG (37%), and two assays for the GP (19 % and 20%). The maximum %CV was exceeded by ≥10% in only one tissue (SV or VP) in two different assays.

(vi) Issues or Problems with Assay

No technical deficiencies sufficient to compromise the conduct and/or interpretation of the assay were identified. The %CVs in excess of the performance criteria were noted in 14 of the 17 studies; none of these had an impact on study interpretation. Two of the studies (Chemicals D and G) had %CVs in excess of the recommended maxima in at least three separate tissues in the androgenic assay for the control and/or high

- dose animals, and both these studies were negative for androgenic activity. One C hemical (B) had high %CVs in the anti-androge nic assay in at least three separate tissues for the control and/or high

- dose animals; Chemical B showed a response for androgen antagonist activity in this assay

.
(vii) Overall C onclusion

Overall, the assays were conducted in accordance with the test guidelines, including proper dose selection, methodology, study design, and statistical analyses. The assay performance was relatively consistent across all 17 test compounds. With regards to changes in sex accessory tissue weights, the reference androgen agonist and antagonist both performed as expected. Although the maximum %CVs were occasionally exceeded for all tissues across all 17 assays in the anti

- androgen assay, the overall responses were relatively consistent and demonstrated that the screening assay can detect the androgenic and anti

- androgenic potential of
chemicals.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 70 of 160 c. Pubertal F emale

(i) Background

The purpose of the female pubertal assay (OCSPP 890.1450) 13 is to identify chemicals that have the potential to interact with the endocrine system, by identifying effects on pubertal development and thyroid function in the intact juvenile/peripubertal female rat. This assay is also capable of detecting not only anti

- thyroid, estrogenic, or anti

- estrogenic chemicals, but also agents that alter pubertal development through mechanisms that induce changes in luteinizing hormone, follicle stimulating hormone, prolactin, or growth hormone levels or via alterations in hypothalamic function.

Endpoints assessed in the Female Pubertal Assay include body weights, age and weight at vaginal opening, organ weights (thyroid, liver, kidneys, pituitary, and adrenals), histology (uterus, ovary, thyroid, and kidney), hormones [thyroxine ( T 4 ) and thyroid- stimulating hormone ( TSH ) ], estrous cyclicity (age at first vaginal estrus after vaginal opening, length of cycle, percent cycling, and percent cycling regularly), and standard blood panel (including creatinine and blood urea nitrogen).

(ii) Strengths and Limitations of the Assay

The Female Pubertal A ssay is an apical assay performed in the intact animal that has redundant confirmatory endpoints, involves the pubertal period of development, and has a well

established base of knowledge of the relationship of the endpoints to endocrine activity

. Collectively, all of these facto rs contribute to the strength of this assay to detect potential interactions with the E or T pathways. Moreover, they also contribute to an evaluation of a chemical’s potential to affect the HPG axis. Having redundant confirmatory endpoints is helpful
in a n assay such as this where variability in proficiency in measuring specific endpoints can be a factor in the ability of the assay to detect a response.

Metabolism takes place in this in vivo assay, which is advantageous over in vitro assays. For chemical s whose metabolites are the most active agents, in vitro systems may not identify the parent compound as having the potential to interact with the endocrine system. Thus, the use
of an in vivo system reduces the likelihood of false negative or false positive results. In addition, the integrated nature of the endocrine system in the developing organism and the relationship of the endocrine toxicity to other systemic effects cannot be simulated in vitro . Also, the use of a

13 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1450: Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female Rats. Office of Safety and Pollution Prevention formerly the Office of Prevention, Pesti cides and Toxic Substances (7101). EPA 740 -C- 09

- 009. October 2009.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 71 of 160 mammalian model usually gives rise to greater confidence that results are relevant to humans than if a phylogenetically more removed model is used.

The Female Pubertal A ssay focuses on a period of development when the endpoints selected for the assay are particularly sensitive to endocrine modulation. T herefore , s ensitivity is greater than for other life stages which are practically feasible to include in a screen for endocrine activity. One of the strengths particular to the fe male pubertal protocol lies in the fact
that the measures included for the identification of endocrine effects are based on a solid knowledge of how the reproductive and thyroid axis mature in the rat. Thus, the extensive basic literature in this area, r eviewed in the detailed review pap er , provides the background for the underlying assumptions in the assay endpoints and assists in the interpretation of results. The assay offers certain advantages over the proposed in vitro tests for determining alterati ons in AR and ER binding or steroidogenesis in that the in vitro assays cannot account for metabolic
activation of xenobiotics which would result in “false negative” responses.

The F emale P ubertal A ssay does have limitations. One limitation is the cyclical variability of the female reproductive organ weights after sexual maturity has been reached . The female pubertal assay utilizes ovary

- intact animals . Th us, once puberty is attained, the fluctuations associated with the normal ovarian cycle could b e viewed as problematic, as evidenced by the high coefficients of variation. Although identification of mechanism of action is not necessary for identification of interaction with the endocrine system, the inability of this assay to identify mechanisms of action is a limitation. However, the pubertal assays are not
stand alone assays , and the results are to be interpreted within the context of the Tier 1 battery. Thus, effects observed with the pubertal assay are used to compl e ment any data in the other battery assays that may be more informative as far as mechanisms are concerned.

(iii) Observations of Test Chemicals

Of the 17 Female Pubertal A ssays, Sprague

- Dawley rats were used as the test strain for all but one chemical (Chemical S), which utilized Wistar rats. Dose groups in each assay consisted of 15-16 juvenile female rats. Six different laboratories were involved in testing. The route of exposure for all of the test compounds was oral gavage, using corn oil, aqueous 0.4

- 1.0% methylcellulose, or 1% gu m tragacanth as the dosing vehicle. The Guideline recommends that the highest dose level be at or just below the MTD but need not exceed the Limit Dose (1000 mg/kg/day); however, typically, the Agency also considers the toxicity profile of the
chemical (i.e ., ≥10% decrease in body weight, cholinesterase inhibition, target organ toxicity, etc.) in dose selection. The two or three dose levels used for the test compounds were considered to be adequate in all assays

. In all 17 tests, juvenile female rats were dosed daily from PND 22
to PND 42

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 72 of 160 The dietary concentrations of phytoestrogens (total isoflavones or genistein -equivalents; aglycone forms of genistein plus daidzein) in the study diet were reported for all assays. The most common diets provided wer e manufactured by Teklad (14

- 18% protein), except for two studies conducted by Laboratory 2, which used A04C

- 10 diet. Reported dietary phytoestrogen concentrations fell into two distinct ranges; either 6.2 to 29 μg/g diet, reflecting the actual
measured c oncentrations, or ≤350 μg/g diet, indicating the concentrations were within the Guideline specified limits. Two or three dose groups of the test compounds plus a vehicle control group were included in the studies. The dose levels used were considere d to be adequate in all assays.

The Guideline recommends that females be sacrificed on PND 42 beginning 2 hours after the final dose, and that sacrifices are to be completed by 1300 hours due to normal diurnal fluctuation in thyroid hormone levels. The Guidel ine recommendation is sacrifice by injectable anesthetic, followed immediately by decapitation. Decapitation without anesthesia is an acceptable, although less preferred method. Only one laboratory (for Chemical S) used an injectable anesthetic (sodium t hiopentone); eight laboratories used decapitation without anesthetic, and eight laboratories used decapitation after isoflurane inhalation.

In all 17 assays , the animals were observed for clinical signs throughout the assay. At sacrifice, the appropriate reproductive organs were collected, along with the liver, pituitary, adrenals, kidney and thyroid. All samples were weighed and selected organs were examin ed histologically. Blood samples were also collected for analyses of clinical chemistry parameters (including creatinine and blood urea nitrogen ) and serum T 4 and TSH levels

. Of the four cholinesterase inhibiting compounds that were tested, two of the tes ts included analysis of RBC and brain cholinesterase (ChE) activity. The statistical analyses were deemed appropriate in all 17 assays.

(iv) Results

As shown in Table 21, o f the 17 chemicals evaluated in this assay, dose levels were selected based on the results of range -finding studies (11 chemicals), Part 15 8 standard toxicology studies (5 chemicals), or the Limit Dose (1 chemical). Eleven chemicals had two
doses tested and 6 chemicals had three dos es (E, F, I, J, N, and V) tested.

No overt toxicity was seen for 12 of 17 chemicals

, and the dose levels tested were determined to be adequate for these chemicals. For the remaining 5 chemicals (D, G, K, M and S), effects on estrogen

- dependent and/or thyro id

- related endpoints were seen only at doses that elicited overt toxicity (e.g., ≥ 10 % decreases in body weight, food consumption, and clinical signs ) as shown in Table 22.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 73 of 160 Table 22. Summary of O vert Toxicity Observed in the Pubertal Female Assay

Chemical Code Highest
Dose Tested (mg/kg/day) Mortality Body weight ( > 10%) Food Consumption Clinical Signs Methods Used to Select Dose
Levels
A 100 Part 158 studies

B 500 Range finding study

C 1000 Limit Dose

D 120 x Range finding study

E 300 Range finding study

F 250 Part 158 studies

G 125 x x x Range finding study

H 60 Range finding study

I 2.0 Range finding study

J 400 Range finding study

K 20 x x x Part 158 studies

M 20 x Range finding study

N 800 Range finding study

P 600 Part 158 studies Q 300 Range finding study

S 75 x x x Range finding study

V 0.3 Part 158 studies

X = Toxicity seen only at the highest dose tested XX = Toxicity seen at both doses tested

Chemical - D : Based on the results of a range finding study, the highest dose selected for the main study was 120 mg/kg/day. In the female pubertal assay, increased incidence s of salivation

w ere seen in 6/15 rats at the low dose (60 mg/kg/day) and in 7/15 rats at the high dose (120 mg/kg/day) when compared to controls

. No treatment

- related effects were seen in estrogen

dependent endpoints at any dose l evel. Treatment

- related change s in thyroid

- related end points

w ere seen only at the HDT

, but not at the lower dose, 60 mg/kg/day.

Chemical - G : Based on the results of a range finding study, the highest dose selected for the main study was 125 mg/kg/day. In the pubertal assay a t the HDT, piloerection, diarrhea thinness, hunched posture and/ or a distended abdomen was seen in one rat on Days 10 to 15 which was sacrificed on Day 15 (not attributed to dosing error). Another rat died on Day 19. Also, at the HDT, there was a significant (p< 0.01) decrease ( ↓ 14%) in final body weights.

Treatment

- related effect on estrogen

- dependent and thyroid- related endpoints were seen only at the HDT, but not at the lower dose (62.5 mg/kg/day).

Chemical - K : Based on the data available from the standard Part 158 toxicology studies, the highest dose selected for the main study was 20 mg/kg/day. In the pubertal assay, t remors wereUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 74 of 160 seen at various times post

- dose in up to 5 of 16 rats at the low dose (10 mg/kg/day) on 10 days, and up to 16 rats at the high dose (20 mg/kg/day) on 18 days. At the high dose, mean body weights were significantly (p<0.01) decreased on Days 27 (↓8%), 19 (↓ 10%) and 36 ( ↓ 9%).

Treatment

- related effects were seen in estrogen

- dependent e ndpoints only at HDT whereas thyroid

- related endpoints were seen both at the HDT and at the lower dose (10 mg/kg/day)

. Overt toxicity was seen at both dose levels.

Chemical - M : Based on the results of a range finding study, the highest dose selected for the main study was 20 mg/kg/day. In the pubertal assay, i ncreased incidence of salivation was seen
in 5 of 15 rats at HDT

. Treatment

- related effect s on estrogen- dependent endpo ints w ere seen only at the HDT, but not at the lower dose (10 mg/kg/day). No treatment

- related effects were seen in thyroid- related endpoints at any dose level.

Chemical - S : Based on the results of a range finding study, the highest dose levels selected for the main study was 75 mg/kg/day. In the pubertal assay, t he following findings were noted at
HDT : (i) decreases (p<0.01) in mean body weights on Days 24 ( ↓8%), 28 ( ↓ 14%) , 36 ( ↓ 20%) and 42 ( ↓ 13%); (ii) decreases (p<0.01) in food consumption during PND 22 -29 ( ↓ 37%) and during PND 29- 36 ( ↓ 27%); and (iii) increased incidences of slight to moderate salivation ( post

dose) in 15/15 rats compare d to controls (0 /15). Treatment

- related effects were seen in estrogen endpoints at both low and high dose groups; however, overt toxicity was seen only at the HDT

. Effects on t hyroid

- related endpoint s w ere seen only at the HDT

, but not at the lower dose (10 mg/kg/day)

.

Of the 17 chemicals evaluated

, 6 showed responses in at least one estrogen -d ependent endpoint (E, G, K, M, Q, and S). The remaining 11 chemicals did not show a ny estrogen

dependent responses . E strogen

- dependent response s were defined as : (i) an advance or delay in VO ( > 2 day s); (ii) advance or delayed first estrous ; (iii) estrous cyclicity disruption; and/or (iv)
uterine and ovarian weight changes

.

For 4 of 6 chemicals that showed estrogen

- dependent responses , effects occurred at doses that elicited overt toxicity (Chemicals G, K, M and S).

• For Chemical G, estrogen

- depende nt effects seen at the HDT in the presence of overt toxicity (mortality, decreased body weight and clinical signs ) included delayed VO and changes in ovarian and uterine weighs.

• For Chemical K

, estrogen

- depende nt effects seen at the HDT in the presence of overt toxicity ( decreased body weight and tremors) w ere changes in ovarian histopathology

.

• For Chemical M

, estrogen

- depende nt effects seen at the HDT in the presence of overt toxicity (salivation) were delayed VO and first estrus.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 75 of 160 • For Chemical S

, estrogen

- depended effects seen at the HDT in the presence of overt toxicity (decreases in body weight and food consumption, salivation ) included delayed
VO and changes in ovarian and uterine weights

.

For the remaining two (Chemicals E and Q), estrogen

- dependent responses were seen in the absence of overt toxicity.

Of the 17 chemicals evaluated , 11 showed an alteration for at least one thyroid

- related endpoint (Chemicals A, B, C, D, E, F, G, K, P, Q and S). The remaining six chemicals (H, I, J,

M , N and V) did not show a response in any thyroid related endpoint s. Thyroid

- related

responses were defined as: (i) changes in serum thyroxine (T 4 ); (ii) changes in serum TSH; (iii)
alterations in thyroid weights, and/ or (iv) thyroid histopathology.

For 4 of 11 chemicals that showed a thyroid

- related response, effects occurred at doses
that elicited overt toxicity (Chemicals D, G, K and S).

• For Chemical D, changes in thyroid weight alterations in serum T 4 levels were seen at the
HDT in the presence of increased incidence of salivation.

• For Chemical G at the HDT, alterations in serum T 4 levels and thyroid histopathological changes were seen in the presence of mortality, decreased body weight and clinical signs

.

• For Chemical K

, alterations in serum T 4 level an d histopathological changes (increase in the follicular cell height) were seen at the HDT

.

• For Chemical S

, changes in thyroid weight were the sole finding at HDT, which was seen in the presence of decreases in body weight and food consumption and salivation.

• For the remaining 7 chemicals, thyroid

- related responses were seen in the absenc e of overt toxicity.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 76 of 160 (v) Performance Criteria

The Guideline includes performance criteria for the vehicle

- treated contr ol animals (Spr a gue

- Dawley for the Female Pubertal assay (OCSP P Guideline 890.1500, Section Q [ reproduced as Table 23)] . The table lists eleven performance criteria, including the average value and coefficient of variation (CV) in percentage for critical organ weights; age and body weight at vaginal opening (VO); final body weight; and serum concentration of T 4 . The “mean”, “2 SDs”, “CV”, and “1.5 CV” columns in Table 19 provide the mean, two standard deviations, coefficient of variation, and 1.5 times the coefficient of variation for that endpoint in historical controls. Mean values and CVs for the vehicle control group must fall within the acceptable range of each to be consid ered fully acceptable. The Guideline states that similar results have been produced with Wistar and Long

- Evans strains.

The performance criteria for blotted uterus, ovaries, thyroid, T 4 , and pituitary weights
were met for all 16 chemicals tested in SD ra ts. The most common exception to the performance criteria not fully met was mean adrenal weight, which was below the acceptable range (38.34

- 48.84 mg) for 11 chemicals (range was 31.6-37.4 mg). Although adrenal weight is a relevant secondary endpoint, it is not the most sensitive endpoint for this assay. The performance criteria for the remaining endpoints were met in all 16 assays performed in SD rats, with the following exceptions.

• For age at VO, the mean was above the acceptable range for two chemicals (Chemical C,

PND 36.1 ; and Chemical F, PND 36.4), and the CV was slightly above the acceptable range for four chemicals (Chemical A, 8.6%; Chemical P, 9.39%; Chemical D, 7.5%; Chemical G, 11.7%).

• For body weight at VO, the mean was above the acceptable range for t wo chemicals (Chemical C, 137.1 g; Chemical A, 135.7 g) and the CV was above the acceptable range for three chemicals (Chemical A, 14.2%; Chemical G, 18.3%; Chemical F 15.58%).

• For final body weight, the CV was above the acceptable range for three chemicals (Chemical E, 11.91%; Chemical B, 9.57%; Chemical G, 10.1%).

• For kidney weight, the CV was above the acceptable range for three chemicals (Chemical B, 12.75%; Chemical K, 11.4%; Chemical G, 12.7%).

• For liver weight, the CV was above the acceptable range for five chemicals (Chemical E, 14.71%; Chemical N, 13.29%; Chemical P, 13.36%; Chemical B, 15.26%; Chemical G, 14.76%).

For the one test chemical that utilized the Wistar rat, the CV exceeded the acceptable range for kidney weight and total serum T 4 concentration

, based on performance criteria for the
SD strain.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 77 of 160 Table 23. Performance Criteria for Controls (Sprague -Dawley Strain).

Endpoint Mean 2 SDs Acceptable range CV 1.5 CV Top of Acceptable

R ange a Uterus, blotted (milligrams)
298.89 111.49 187.40 to 410.38 28.26 9.48 37.73 Ovaries (milligrams)

75.65 39.11 36.54 to 114.77 17.37 5.83 23.20

T 4 (total, μg/dl)

4.03 1.34 2.69 to 5.38 21.38 8.01 29.39 Thyroid weight (milligrams)
14.20 8.00 6.20 to 22.20 23.97 14.61 38.58 Age at VO (postnatal day, where day of birth = PND 0)
33.15 2.47 30.67 to 35.62 4.69 1.82 6.52 Weight at VO (grams)

116.58 14.86 101.71 to 131.44 9.81 4.16 13.97 Final body weight (grams)

154.70 49.85 104.86 to 204.55 6.69 2.25 8.93 Adrenals (milligrams)
43.59 5.25 38.34 to 48.84 16.02 6.95 22.97 Kidneys (grams)
1.57 0.63 0.95 to 2.20 8.42 2.34 10.76 Liver (grams)
8.05 3.73 4.32 to 11.78 9.58 3.55 13.13 Pituitary (milligrams)

8.97 3.11 5.86 to 12.08 15.76 11.22 26.97

a= Bottom of the acceptable range for coefficient of variation is zero.

(vi) Issues or Problems with Assay

No technical deficiencies sufficient to compromise t he conduct and/or interpretation of the assay were identified. Although not a technical deficiency in the conduct of the assay, prolonged delays in VO precluded an evaluation of cyclicity for some assays. Minor deficiencies, including mean body or organ weights or %CV outside the limits of the performance criteria, were noted in 16 of the 17 studies (Chemical Q had no deficiencies), and none of these minor deficiencies had an adverse impact on study interpretation. One study (Chemical G) had six exceptions to the performance criteria with one exception, % CV for age at VO that was higher than acceptable criterion (11.7% treated vs. 6.52% control), indicating variability of the data. All other studies had three or fewer exceptions. Eleven of the 16 studies used the body weight on PND 21 instead of PND 22 as the covariate for statistical analyses, but
thi s was not considered to have any meaningful effect on study interpretation.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 78 of 160 Several assays utilized radioimmunoassay for T 4 and TSH measurements. Details on the methodology were not provided in the study report for three assays.

(vii) Overall Conclusion

Overall, the assays were conducted in accordance with the test guidelines, including proper dose selection, methodology, study design, and statistical analyses. The assay performance was generally consistent across all 17 test compounds. Although the %CV and mean body or organ weights were occasionally outside the limits of the performance criteria, the overall responses were relatively consistent and demonstrated that the screening assay can detect potential effects of chemicals on pubertal development an d thyroid function in the intact juvenile/peripubertal female rat.

d. Pubertal M ale

(i) Background

The Male Pubertal Assay (OCSPP 890.1500) 14 is designed to determine if a chemical interacts with the endocrine system by identifying effects on pubertal development and thyroid function in the intact juvenile/peripubertal male rat. This assay is capable of detecting chemicals
with anti

- thyroid, androgenic, or anti

- androgenic androgen receptor (AR) or steroid

- enzyme

mediated activity. This assay may also detect agents which alter pubertal development via changes in gonadotropins, prolactin, or hypothalamic function. Therefore, this screening assay can be useful in detecting chemicals that act on androgen s or through the hypothalamus

pituitary

- gonadal (HPG) axis that controls the estrogen and androgen hormone systems, as well as chemicals that may interfere with the thyroid system.

Endpoints assessed in the Male Pubertal Assay include age and weight at preputial separation, body weights, organ weights (seminal vesicles with coagulating glands, ventral and dorsolateral prostate, levator ani/bulbocarvernosus (LABC) muscle complex, epididymides,
testes, thyroid, liver, kidneys, pituitary, and adrenals), histology (epididymis, testis, thyroid, and kidney), hormones (testosterone, thyroxine [T 4 ], and thyroid stimulating hormone [TSH]), and standard blood panel including creatinine and blood urea nitrogen.

14 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1500: Pubertal Development and Thyroid Function in

Intact Juvenile/Peripubertal Male Rats. Office of Safety and Pollution Prevention formerly the Office of Prevention, Pestici des and Toxic Substances (7101). EPA 740 -C- 09

- 012. October 2009.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 79 of 160 (ii) Strengths and Limitations of the Assay

The M ale Pubertal Ass ay is an apical assay performed in the intact animal that has redundant confirmatory endpoints, involves the pubertal period of development, and has a well

established base of knowledge of the relationship of the endpoints to endocrine activity. Collectively, all of these factors contribute to the strength of this assay to detect potential interactions with the A or T pathways. Moreover, they also contribute to an evaluation of a chemical’s po tential to affect the HPG axis. Having redundant, confirmatory endpoints is helpful in an assay such as this where variability in proficiency in measuring specific endpoints can be a factor in the ability of the assay to detect a response.

Metabolism takes place in this in vivo assay, which is advantageous over in vitro assays. For chemicals whose metabolites are the most active agents, in vitro systems may not identify the parent compound as having the potential to interact with the endocrine system. In addition, the integrated nature of the endocrine system in the developing organism and the relationship of the endocrine toxicity to other systemic effects cannot be simulated in vitro . Also, the use of a mammalian model usually gives rise to greater confi dence that results are relevant to humans than if a phylogenetically more removed model is used.

As is the case with any study design, the male pubertal assay also has limitations. One potential limitation is the variability of the testosterone measurements. The hormone assays themselves are not difficult, as they are available commercially and incl ude their own quality control samples. However, sample collection methodology and the changing levels of testosterone over time in the juvenile male rat present possible sources of increased variability in the assay. Collecting the sample in a non

- stress ed animal will reduce the variability. However, the pubertal as say are not stand alone assays nor is testosterone measurement the only endpoint that might be associated with endocrine disruption in the m a le, and the results are to be interpreted within the context of the Tier 1 battery. Thus, effects observed with the p ubertal assay are used to comple ment any data in the other battery assays that may be more definitive as
far as mechanisms are concerned.

(iii) Ob servations of Test Chemicals

Of the 16 Pubertal Male Assays , Sprague

- Dawley rats were used as the test strain for 15 assays, and the remaining assay utilized Wistar rats. Dose groups in each assay consisted of 15 or 16 juvenile male rats. Six different laboratories were involved in the testing. For each test compound, the route of exposure was oral gavage, using either corn oil, aqueous 0.5 -1.0% carboxymethyl cellulose, or 1% gum tragacanth as the dosing vehicle. The Guideline recommends that the highest dose level be at or just below the MTD but need not exceed the Limit Dose (1000 mg/kg/day); however, typically, the Agency also considers the toxicity profile of the chemical ( i.e ., ≥ 10% decrease in body weight, cholinesterase inhibition, target organUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 80 of 160 t oxicity, etc.) in dose selection. The two or three dose levels used for the test compounds were considered to be adequate in all assays . In all 16 assays , juvenile male rats were dosed daily
from PND 23 to PND 53 or 54.

The dietary concentration of phytoestrogens (total isoflavones or genistein -equivalents; aglycone forms of genistein plus daidzein) in the study diet is recommended to be ≤ 300 μg/g diet. The most common diets were Teklad diets (14- 18% protein), except for studies conducted by Labora tory 3 (Diet A04C- 10). Reported dietary phytoestrogen concentrations fell into two distinct ranges, either 6.2 to 29 μg/g diet or ≤300

- 350 μg/g diet.

In each assay, animals were sacrificed by 1300 hours on the final day of dosing, 2 hours
after the last dose. With regards to anesthesia, the Guideline recommends sacrifice by decapitation without anesthesia in order to minimize any effect on testosterone. The use of CO 2 inhalation is not considered acceptable as an anesthetic, but if used must be administered for ≤60 seconds. In the 16 tests, six laboratories used decapitation without anesthetic, eight laboratories used decapitation after isoflurane inhalation, and one laboratory used decapitation after brief CO 2 inhalation (<60 sec).

In all 16 assays , t he animals were observed for clinical signs throughout the assay. At sacrifice, the appropriate reproductive organs were collected, along with the liver, pituitary, adrenals, and thyroid. All samples were weighed and selected organs were examined
histolo gically. Blood samples were also collected for clinical chemistry analyses and analysis of

serum T 4 , TSH, and testosterone; none of the assays tested for serum levels of 3,3’5

- triiodothyronine (T 3 ). Of the four cholinesterase (ChE) inhibiting compounds that were tested, two of the assays included analysis of red blood cell ( RBC ) and brain ChE activity. The statistical analyses were deemed appropriate in all 16 assays.

(vi) Results

As shown in Table 24, o f the 16 chemicals evaluated in this assay, dose levels were selected based on the results of range- finding studies (12 chemicals), Part 158 standard toxicology studies (3 chemicals), or at the Limit Dose (1 chemical). Ten chemicals had two dose levels tested and 6
chemicals had three doses (E, F, I, J, N, and V) tested.

No overt toxicity was seen for 13 of 16 chemicals and the dose levels tested were determined to be adequate for these chemicals. For the three chemicals (D, G and S) effects on androgen

- dependent and/or thyroid

- related endpoints were seen only at doses that elicited overt toxicity (e.g., as < 10% decreases in body weight , food consumption, and clinical signs) a s shown in Table 24.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 81 of 160 Table 24. Summary of Overt Toxicity Observed in the Pubertal Male Assay

. C hemical Code Highest Dose Tested (mg/kg/day) Body weight (> 10%) Food Consumption Clinical Signs Methods Used to Select
Dose Levels

A 100 Part 158 studies

B 375 Range finding study
C 1000 Limit Dose
D 120 x x Range finding study
E 400 Range finding study
F 250 Range finding study
G 125 x Range finding study
H 60 Range finding study
I 2.0 Range finding study
J 400 Range finding study
K 20 Part 158 studies
M 20 Range finding study
N 800 Range finding study
P 800 Range finding study
S 75 x x x Range finding study
V 0.3 Part 158 studies X = Toxicity seen only at the highest dose tested XX = Toxicity was seen at both doses tested

Chemical - D : Based on the results of a range finding study, the highest dose selected for the main study was 120 mg/kg/day. In the male pubertal assay, i ncreased incidence s of salivation
were seen in 5/15 rats at the low dose (60 mg/kg/day) and in all rats at the high dose (120 mg/kg/day) groups. No treatment

- related effects were seen in androgen

- dependent endpoints at any dose level. Treatment

- related changes in thyroid

- related end points were seen at both dose
levels.

Chemical - G : Based on the results of a range finding study, the highest dose selected for the main study was 125 mg/kg/day. In the male pubertal assay at the HDT, final body weights were decreased (23%; p < 0.01). Treatment

- related effects in androgen

- dependent and thy roid

- related endpoints were seen in the absence of any toxicity at the low dose (62.5 mg/kg/day) but in the presence of overt toxicity at HDT.

Chemical - S : Based on the results of a range finding study, the highest dose selected for the main study w as 75 mg/kg/day. At the HDT, mean body weights were significantly (p<0.01)
decreased on PND 27 ( ↓10%), PND 41 (↓ 22%) and PND 53 ( ↓ 20% ). Food consumption was significantly (p<0.01) decreased (10 -28%) during the measured intervals from PND 23-53. Increased incidence of slight to moderate salivation was seen in 15/15 rats compared to 0/15 in
the controls. Treatment

- related effects in androgen

- dependent endpoints were seen only at HDT.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 82 of 160 Of the 1 6 chemicals evaluated

, 9 showed responses in at least o ne andro gen -d ependent endpoint (B, C, E, F, G, J, N, P and S ). The remaining 7 chemicals did not show a nyandrogendependent responses. Androgen- dependent response s were defined as : (i) a delay preputial separation (ii) changes in testosterone levels ; and/or (iii) androgen -sensitive organ weights changes

.

For 2 of 9 chemicals that showed androgen- mediated responses, effects occurred at doses that elicited overt toxicity (Chemicals G and S)

.

• For Chemical G, at the HDT

, androgen

- dependent effects, seen in the presence of decreased body weight, included increased age at PPS ; alterations in testosterone levels;
and changes in androgen -sensitive organ weights.

• For Chemical S, at the HDT high dose, androgen

- dependent effects were increased age of PPS, alterations in testosterone levels, and changes in androgen -sensitive organ weights. These changes were seen in the presence of increased salivation and decreases in body weight and food consumption

.

For the remaining 7 chemicals, androgen

- dependent responses were seen in the absence of overt toxicity

. Of the 16 chemicals evaluated

, 7 showed a response for at least one thyroid

- related endpoint (Chemicals C, D, E, F, G, J, and P). T he remaining 9 chemicals did not show a response in any thyroid

- related endpoints. Thyroid

- related responses were defined as: (i) changes in serum thyroxine (T 4 ); (ii) changes in serum TSH; (iii) alterations in thyroid weights,
and/ or (iv) thyroid histopathology.

For 2 of 7 chemicals that showed thyro id

- related responses, effects occurred at doses that elicited overt toxicity (Chemicals D and G)

.

• For Chemical D, alterations in serum T 4 levels

, changes in thyroid weight , and enlarged thyroid were seen at the HDT in the presence of overt toxicity (salivation)

.

• For Chemical G

, alterations in serum T 4 levels, changes in thyroid weight and thyroid histopathological changes were seen in the presence of decreased body weight.

(v) Performance Criteria

The Guideline includes the performance criteria for the vehicle

- treated control animals (Sprague

- Dawley and Wistar strains) for the Male Pubertal assay (OCSPP Guideline 890.1500 [ reproduced as Table 2 5] ). The table lists the performance criteria (average value and CV inUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 83 of 160 percentage) for critical organ weights; age at PPS; and serum concentrations of TSH, testosterone, and total T 4 . The “mean”, “2 SDs”, “CV”, and “1.5 CV” columns in Table 20 provide the mean, two standard deviations, coefficient of variation, and 1.5 times the coefficient
of variation f or that endpoint in historical controls. Mean values and CVs for the vehicle control group must fall within the acceptable range of each criterion to be considered fully acceptable. Performance criteria are not included for LABC and thyroid weight and TSH concentration for Wistar rats, for the testis weight for Sprague

- Dawley r ats, or for the dorsolateral prostate (DP) of
either strain. For Sprague

- Dawley r ats, there are currently 16 parameters having performance criteria for the average value (weight, age, or concentration) and the %CV, totaling 32 performance criteria values. As stated in the Guideline, more emphasis is placed on meeting performance criteria for the CV than for the endpoint control means since the CVs relate directly to sensitivity of t he endpoints.

The performance criteria were generally met for each assay, although all tests had 2 or more values that were outside the acceptable range. For the 15 assays that utilized Sprgue

Dawley rats, each assay had 2 to 11 values out of range. However, the majority of these out -ofrange values were only marginally outside the performance criteria. If these marginal values are excluded, then five of the assays met all the performance criteria; nine of the assays had only 1-3 values out of range; and one assay had only 4-6 values out of range. For the one assay that utilized Wistar rats, 16 of the 28 performance criteria values exceeded the normal range.

The individual performance criteria for each parameter (organ weight, body weights, age,

or hormone concentration) across the 15 assays that utilize S prague -D awley rats are as follows.

• For the weight of the seminal vesicle plus coagulating gland (with fluid), only 1 assay had an average weight above the performance criteria (Chemical C), and 8 assays had %CVs in excess of the performance criteria maximum value (Chemicals B, C, E, F, I, J, K, and N)

.

• For the ventral prostate weight, the average weight met the performance criteria in all 15 assays, and 4 assays (Chemicals A, B, K, and P) had %CVs in excess of the recommended maximum . T he %CVs were substantially out of range in only 2

assays (Chemicals A and B).

• For the LABC weight, only 1 assay (Chemical N) had an average weight below the performance criteria ; otherwise

, the performance criteria for LABC were met in all 15 assays.

• For the weight of the epididymis (combined left and right), the average weight met the performance criter ia in all 15 assays, and only 3 assays (Chemicals D , K, and S) had %CVs in excess of the recommended maximum. Th e %CV was substantially out of range in only 1 assay (Chemical K).

• The T 4 concentration was low in 1 assay (Chemical M); otherwise, the performance criteria for T 4 were met in all 15 assays.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 84 of 160 • For TSH concentration, 5 of the assays (Chemicals A, D, G, K, and M) had concentrations below the performance criteria, and 7 assays (Chemicals D, F, G, K, M, N, and S) had %CVs above the maximum. T he %CVs were substantially out of range i n only 3 assays (Chemicals F, M, and S).

• The performance criteria for testost erone concentration were met in all 15 assays.

• For thyroid weight, the average weights were below the performance criteria in 7

assays (Chemicals E, G, H, I, J, N, and P), and 3 assays (Chemicals B, K, and N) had %CV above the recommended maximum. T he %CV was substantially out of range in only 1 assay (Chemical B).

• For adrenal weight, the maximum %CV was slightly exceeded in only 1 assay (Chemical P); otherwise, the performance criteria for adrenal weight were met in all 15 assays.

• For kidneys, average weights were below the performance criteria in 12 of the 15 assays, but the maximum %CV was only slightly exceeded in 1 assay (Chemical B).

• For liver, the average weight met the performance criter ion in all 15 assays, and the maximum %CV was exceeded in only 2 assays (Chemicals B and I).

• For the pituitary gland, the average weight met the performance criteria in all 15 assays, and recommended maximum %CV were exceeded in 5 assays (Chemicals G, N, P, S, and V). T he %CVs were substantially out of range i n only 3 assay s (Chemicals N, P, and S).

• For the initial body weight, the average weight was above the performance criteria in 6 assays (Chemicals A, C, D, G, K, and P), but the maximum %CVs was only slightly exceeded in 3 assays (Chemicals B, F, and P).

• F or the final body weight, the average weight met the performance criteria in all 15

assays . T he maximum %CVs was exceeded in 12 assays; however, the values were substantially out of range i n only 3 assays (Chemicals B, I, and P).

• For the body weight at P PS, the average weight was slightly high in 1 assay (Chemical D) of the 15 assays . T he maximum %CVs was exceeded in 12 assays, and the values were substantially out of range i n 6 assays (Chemicals A, B, G, J, K, and I)

.

• The average age at PPS met the perf ormance criteria in all 15 assays, but the maximum %CV was slightly exceeded in 5 of the assays (Chemicals B, C, D, M, and P).

Although the Guideline for this a ssay does not currently specify performance criteria (weight ranges and CVs) for the testes or the dorsolateral prostate (DP) of Sprague

- Dawley rats, the data from the 15 assays that utilized S prague Dawley rats indicate that testis weights (and variances) for control animals were consistent across the assays. For each assay, the left testis weight s averaged 1.343-1.502 g with CVs of 5.2- 27.7% (typically 6.7

- 9.2%). Right testis weights averaged 1.352-1.543 g with CVs of 5.7- 22.2% (typically 6.7 -9.2%). Across the 15 assays, the overall average weigh ts %( CV) were 1.428 g (3.0%) for the left testis a nd 1.449 g (3.3%) for the right testis. For the dorsolateral prostate (DP), average study weights ranged from 90.4 to 194.6 mg with CVs of 13.9- 31.1% (typically 18.4

- 27.6%). Across the 15 assays, the overall average weights (%CV) for the DP were 140.0 mg (19.0%).USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 85 of 160 Table 25. Performance Criteria for Controls (Sprague-Dawley and Wistar Strains).

Endpoint Rat Strain Mean 2 SD s Acceptable Range CV 1.5 CV Top of Acceptable

Range a
Ventral prostate wt (grams) Wistar 0.223 0.072 0.151 to 0.295 16.67 5.65 22.32 S pr a gue -D awley 0.246 0.086 0.160 to 0.332

LABC wt (grams) Wistar NA NA NA 15.77 11.33 27.100 S pr a gue

- D awley 0.651 0.204 0.447 to 0.855 Epididymis wt (g) Wistar 0.474 0.124 0.350 to 0.598 10.94 5.45 16.39 S pr a gue

- D awley 0.446 0.082 0.364 to 0.528 Seminal vesicle w/ coagulating gland wt. (g) Wistar 0.576 0.234 0.342 to 0.810

20.61 0.45 21.06 S pr a gue -D awley 0.507 0.212 0.295 to 0.719

Testis wt (grams) Wistar 1.341 0.250 1.091 to 1.591 9.27 8.35 17.62 S pr a gue

- D awley NA NA NA Thyroid wt (milligrams) Wistar NA NA NA 15.39 8.24 23.63 S pr a gue

- D awley 20 6 14 to 26 Adrenals wt (mg) Wistar 54.597 13.768 40.829 to 68.365 15.42 7.34 22.77 S prague

- D awley 46.478 14.636 31.842 to 61.114 Kidneys wt (g) Wistar 2.516 0.550 1.966 to 3.066 9.56 5.20 14.76 S prague

- D awley 2.646 0.404 2.242 to 3.050 Liver wt (g) Wistar 14.070 2.874 11.196 to 16.944 10.24 4.69 14.93 S prague

- D awley 12.670 2.680 9.990 to 15.350 Pituitary wt (mg) Wistar 8.051 1.934 6.117 to 9.985 12.14 3.83 15.98 S prague

- D awley 10.354 2.544 7.810 to 12.898
T4 concen. (μg/dl) Wistar 5.478 2.164 3.314 to 7.642 18.27 9.20 27.46 S prague

- D awley 5.716 1.660 4.056 to 7.376
TSH concen. (ng/mL) Wistar NA NA NA 34.04 24.26 58.29 S prague

- D awley 14.162 9.950 4.212 to 24.112
Testosterone

concen. (ng/mL) Wistar 2.118 2.540 0 to 4.658 58.82 30.88 89.70 S prague -D awley 2.110 1.850 0.260 to 3.960

Weaning wt (g) Wistar 58.238 11.058 47.180 to 69.296 8.04 2.21 10.25 S prague

- D awley 52.642 7.170 45.472 to 59.812 Age at PPS (PND, PND 0 = day of birth) Wistar 43.124 2.948 40.176 to 46.072 3.64 2.03 5.67 S prague -D awley 43.147 3.366 39.781 to 46.513 Weight at PPS (grams) Wistar 209.142 31.850 177.292 to 240.992 7.54 0.03 7.57 S prague

- Dawley 222.223 33.946 188.277 to 256.169 Final body weight (grams) Wistar 291.818 41.578 250.24 to 333.396 6.62 0.85 7.47 Sprague

- Dawley 295.647 36.412 259.235 to 332.059

a Bottom of the acceptable range for coefficient of variation is zeroUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 86 of 160 (vi) Issues or Problems with Assay

No technical deficiencies sufficient to compromise the conduct and/or interpretation of the assay were identified

. Five of the 16 studies used the body weight on PND 23 (instead of PND 21) as the covariate for statistical analyses, but this was not considered to have any meaningful effect on study interpretation.

(viii) Overall Conclusion

Overall, the assays were conducted in accordance with the OPPTS guidelines, including proper dose selection, methodology, study design, and statistical a nalyses. Assay performance was generally consistent across all 1 6 test compounds. Although the average values and %CVs were occasionally outside the limits of the performance criteria, the overall responses were relatively consistent and demonstrated tha t the screening assay can detect potential effects of chemicals on pubertal development and thyroid function in the intact juvenile/peripubertal male

rat.

e. F ish short

- term reproduction

(i) Background

The fish short- term reproduction assay (FSTRA; OCSPP Guideline 890.1350 15 ) is intended to evaluate reproductive fitness as an integrated measure of toxicant effects. The test includes a suite of biochemical and histological endpoints that reflect effects associated with interactions to the hypothalamic

- pitui tary -gonadal (HPG) endocrine axis and its downstream hormonal pathways. The assay uses sexually mature male and female fathead minnow fish ( Pimephales promelas ). During a 21- day chemical exposure, survival, reproductive behavior, secondary sex characteri stics, fecundity, and fertility are monitored. At the end of the test, additional measurements that are meant to be directly reflective of the status of the reproductive endocrine system such as secondary sex characteristics, gonadal histopathology, gonado

- somatic index (GSI), and plasma concentrations of vitellogenin (VTG) are collected. The FSTRA is intended to serve in a screening capacity to provide an indication of potential endocrine interactions, not necessarily to confirm any specific mechanism or mode of action. Specifically, the FSTRA is intended to provide information on potential estrogen agonists or antagonists, androgen agonists or antagonists, and/or chemicals that act on steriodogenesis or at a higher level along the HPG axis. The specifics on the test methodology are presented in the OCSPP 890.1350
Fish Short- term Reproduction Assay guideline (USEPA 2009). A summary of the assay is presented below.

15 USEPA 2009. Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1350: Fish Short

- term Reproduction Assay. Office of Chemical Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 740 -C- 09

- 007. October 2009. http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0576

- 0007USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 87 of 160 Prior to initiating the chemical exposure, breeding groups (consisting of two males and
four females) of mature fish, typically 4

- 6 months old, are monitored for a minimum of 14 days for survival, behavior, fecundity and fertility. This is to ensure adequate spawning performance and to allow for a systematic (typically a randomized block) distribution of the breeding groups to individual treatment group replicates. Each treatment group (including the dilution water -only negative control) has four replicate tanks. There are typically three chemical treatment concentrations and a negative control group. If a solvent carrier is required to facilitate dosing, then a solvent control group is also included.

The exposure route for the test chemical is by way of tank water using an appropriate
flow

- through delivery system. Breeding groups are exposed for 21

- days during which mortality, reproductive behavior, secondary sex characteristics, fecundity and fertility are monitored daily. At test termination, additional measurements are collected, which include nuptial tubercle score, gonadal histopathology, GSI, and plasma concentrations of VTG. Additionally, plasma sex steroids (17β

- estradiol and testosterone) may be measured at test termination. Collectively, these measurement endpoints are meant to be indicative of the status of the reproductive endocr ine
system.

According to the test guideline, “ statistically significant effect in one or more of the core endpoints (fecundity, secondary sex characteristics, VTG, GSI, and histopathology) may be indicative of the test material to potentially disturb the HPG axis of fishes .” (pg 20). Effects on fertility should also be considered when evaluating potential disruptions. Throughout this chapter, unless otherwise specified, “HPG axis” is used generically and should be considered inclusive of its downstream p athways. As the statistical methods used to analyze study data tend to favor detection of monotonic response, any statistically significant effect on one of these endpoints may be considered indicative of a “positive” response. Also, the test guideline

su ggests that results which are indicative of a trend but which are not necessarily statistically significant at the 95% confidence level may be considered biologically relevant. However, if a given treatment results in substantial mortality (> 2 mortalitie s in any replicate per the guideline) or other overt signs of toxicity, responses in the core endpoints may be due to general toxicity and not necessarily mediated primarily via interaction with the HPG axis. In such a case, the guideline recommends that t he lower treatment level(s) be examined for potentially HPG

- related effects outside of the range of general toxicity.

It is recommended that the suite of responses across the FSTRA and the overall weight of evidence across the EDSP Tier I battery be examined collectively to provide a comprehensive assessment of any potential to interact with the HPG axis in fish. Furthermore, fish are intended to serve as surrogates for other species as HPG pathways are conserved across vertebrate species. Data obtained through the FSTRA are considered applicable as a line of evidence in evaluatingUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 88 of 160 the potential for a chemical to impact endocrine- mediated processes beyond fish, although the degree of applicability for other vertebrates is partially dependent upon other factors such as anticipated route of exposure and the pharmacodynamics of a given chemical.

Evaluation of the assay performance was conducted using data submitted in response to Tier 1 EDSP test orders. Strengths and previously identified limitations of the assay are acknowledged. However, as acknowledged by the 2008 FIFRA SAP, the previously identified
limitations do no t affect the general appropriateness of the experimental design to screen for potential endocrine interacting chemicals. The performance of the assay is evaluated using the test validity criteria in the guideline as a benchmark for comparison between studies. Finally, issues identified during the preliminary review of the submitted studies are discussed. These issues do not suggest critical flaws of the FSTRA experimental design, but they may indicate that additional, targeted guidance may be warranted to improve the laboratory execution of the assay

.
(ii) Strengths and Limitations of the Assay

The FSTRA is intended for detection of effects potentially associated with disturbance to the HPG axis using the suite of measurement endpoints identified in the guideline. The assay is conducted using whole fish reflecting an intact HPG axis and its complex feedback mechanisms, which may be relevant to other taxa. Since the assay is conducted on mature fish, it reflects relevant pharmacokinetics and pharmacodynamics that may impact the potential exposure in terms of absorption, distribution, metabolism, and excretion of the test chemical and alterations at various levels of biological organization, i.e ., biochemical (VTG, plasma steroids), tissue (gonad histology), organ (gonado

- somatic index), and the whole animal (phenotype, survival). The test species , fathead minnow is a routinely- used species for which husbandry conditions are relatively well defined thus enabling environmental conditions in the laboratory to be optimized to reduce confounding effects on reproduction that might be attributed to test conditions. The test design utilizes standard aquatic toxicology methodology where measurement endpoints are well defined and accepted internationally as measures of reproductive fitness (OECD 2012). The results of this assay have also been reproduced ac ross several laboratories and have yielded relatively consistent results for reference chemicals (USEPA 2007a).

In 2008, a FIFRA SAP charged with evaluating the proposed Tier 1 EDSP battery (USEPA 2008) emphasized the importance of the FSTRA because of its inclusion of apical endpoints. The inclusion of the apical endpoints may allow for detection of potential endocrine active chemicals that may operate by mechanisms not yet specified. The assay also has utility as a screen for perturbations of the VTG and secondary sex characteristic endpoints in intact
animals.

While there are many strengths of the FSTRA, some limitations have been previously identified in other summary reports for the FSTRA. The technical document for the FIFRAUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 89 of 160 Scientific Advisory Pa nel in 2008 on the EDSP Tier 1 screening battery (USEPA 2008 16 ) identified the inherent difficulties of testing poorly soluble test materials and the specialized technical expertise that would be required for analysis of the sex steroids. However, difficul ties in testing poorly soluble materials are generally addressed in Organization for Economic Cooperation and Development (OECD) Guidance Document 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000) as well a s the OCSPP Guideline 850.1000 (draft) (USEPA 1996 17 ).

In an earlier Integrated Summary Report (USEPA 2007), other limitations were discussed
and included:

1) VTG analysis requires additional calibration in the individual laboratory; 2) fecundity can be influenced by both chemical and non

- chemical factors [potentially non

endocrine specific toxicity] and requires a pre

- exposure period to establish adequate spawning; 3) histology requires additional time and resources; and

, 4) the sensitivit y and specificity of secondary sex characteristics are not applicable to all modes of action; Secondary sex characteristics are not as amenable to quantitative analysis as other endpoints; and the test duration may not be adequate to observe some physiolog ical alterations of secondary sex characteristics.

Upon reviewing submitted Tier 1 test order data, it is recognized that for the limitations discussed above that involve additional time and/or resources (VTG, plasma sex steroids, gonadal histology), the inclusions of these endpoints when blood/tissue samples are available are extremely useful measurement endpoints when considered collectively in determining whether a chemical is potentially interacting with the endocrine system. The pre

- exposure phase to evaluate fecundity is critical for this assay to establish relatively uniform spawning performance and allow for a systematic distribution of breeding groups to replicate treatment tanks. With respect to the potential lack of endocrine

- mediated specificit y of the fecundity endpoint, the 2008 SAP noted that the fecundity observations were nonetheless “ an essential part of apical analysis of HPG activity ” (USEPA 2008, pg 13). Similarly, although secondary sex characteristics are generally evaluated qualitat ively or only semi- quantitatively and may vary in their response to different modes of action, the 2008 SAP concluded that the presence of the male secondary sex characteristic of nuptial tubercles in females was a particularly “ robust endpoint to identify androgenic compounds ” (USEPA 2008, pg 15). Additionally, although the 2008 SAP and prior supporting documents noted certain limitations and potential challenges in the performance and

16 USEPA. 2008. Transmittal of Meeting Minutes of the FIFRA Scien tific Advisory Panel Meeting Held on March 25

- 26, 2008 to review and consider the Endocrine Disrupter Screening Program (EDSP) Proposed Tier 1 Screening Battery. FIFRA Scientific Advisory Panel. Office of Science Coordination and Policy. June 11, 2008.

17 USEPA. 1996. Ecological Effects Test Guidelines OPPTS 850.1000 Special Considerations for Conducting Aquatic Laboratory Studies. Office of Chemical Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Tox ic Substances (7101). EPA 7120C096

- 113. April 1996. http://www.epa.gov/opptsmnt/pubs/frs/publications/OPPTS_Harmonized/850_Ecologica l_Effects_Test_Guidelines/Drafts/850

- 1000.pdfUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 90 of 160 conduct of the FSTRA (as with any assay), these limitations did not de tract from the overall SAP conclusion that the FSTRA was a critical component of the proposed battery (USEPA 2008).

(iii) Observations from Initial Screening of Chemicals

Currently, of the FSTRAs that tested chemicals of unknown potential for endocrine
inter action and which were submitted by Test Order Recipients, twenty

- one were selected by the Agency as case studies to evaluate assay performance, consistency in experimental conduct, and interpretation of the results (see Section II).

Generally, the Agency documents its evaluation of pesticide ecotoxicity or mammalian toxicity data in a Data Evaluation Record (DER). Each DER typically reflects a single data submission (study); the DER outlines the study conditions, provides a comparison of the reported

test methods to those outlined in the test guideline ( e.g. , OCSPP 890.1350), and summarizes and discusses the Agency’s interpretation of results based on its own independent analysis of the data. In the case of the 21 case study chemicals, the Agency’s review s of these studies remain preliminary. The current review of FSTRA performance is based on these preliminary reviews as captured in the associated draft DERs.

Quality Control

In the OCSPP 890.1350 guideline, there are six separate test validity elements identified ( Table 26 ). These validity criteria provide an indication of the general performance of the
FSTRA under test conditions. When met, the validity criteria are used to support the scientific soundness of a given study and the degree of confidenc e in its results. An additional test performance criterion is that the water quality characteristics described in the test guideline should remain within the reported tolerance limits. These characteristics include temperature and dissolved oxygen as wel l as limits on residual chlorine, unionized ammonia, total organic carbon and alkalinity. This additional performance criterion is meant to provide information on general water characteristics that are typically adequate to promote reproduction and genera l health of the fish. The validity criteria for temperature, control survival, fecundity and fertility are also referred to as performance criteria in the guideline (pg 19). In this evaluation, the validity criteria were used to evaluate the scientific s oundness and level of confidence in the results. However, a deviation from a defined test validity criterion does not necessarily indicate that the study cannot provide useful information. Rather, as with any of the identified criterion, it is anticipate d that slight deviations from a criterion will occur and that decisions regarding acceptability of a study will be influenced by factors such as the observed variability in the endpoint and the magnitude and duration of departure from a criterion. Overall, the conduct and results of each study are evaluated as a whole to determine the degree of confidence in the ability to discern the potential for endocrine interactions. Each of the identified criteria in Table 2 6 is
discussed in more detail below.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 91 of 160 Tab le 2 6 . Test Validity Elements for the FSTRA

Criteria Reference Value(s)

Measured Test Concentration Coefficient of Variance (CV) ≤20% for all replicates

Control Fertility ≥95% (of eggs produced) Control Fecundity ≥15 eggs/female/day/replicate or spawning every 4 days (per replicate) Control Survival ≥90% Dissolved Oxygen ≥60% saturation (at 25°C) Mean Water Temperature 25 ± 1°C

Evaluations of these six criteria were conducted using the information provided in the 21 submitted studies. The number of studies that met each of the identified test validity criteria is presented in Figure 1 4.

Figure 1 4. Summary of Number of Studies Achieving Each Individual Test Validity Criteria

Four of the 21 studies met all of the ide ntified test validity criteria (Figure 15) . Eleven studies met all but one of the listed criteria ( Table 2 7 ), indicating that 71% of the evaluated studies either met all or only missed one of the identified test validity criteria. The six other studies had two or more deficiencies. A CV of greater than 20% for test concentration was the most common deficiency noted and was observed in multiple laboratories and in both studies
that did and did not utilize solvent carriers. The increase in CV above the recommended value was observed in three studies that also reported decreased dissolved oxygen levels or control fecundity rates. 21 15 20 15 17 12

6 1 6 4 9

0 5 10 15 20 Mean Temperature

Dissolved Oxygen

Control Survival Control Fecundity

Control Fertility Test Concentration CV

# of Studies met

didn't meetUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 92 of 160 Figure 15. Summary of the Number of Individual Test Val idity Criteria Achieved by Each Study

Table 2 7. Identification of the Only Test Validity Criteria Not Achieved in 11 Studies
Test Validity Criteria Number of Studies Measured Test Concentration Coefficient of Variance (CV) 4 Control Fecundity 3 Dissolved Oxygen 2

Control Survival 1 Control Fertility 1

Water Quality Parameters

Environmental test conditions are described in OCSPP 890.1350 guideline (Table 1 in guideline, pg 3) and include dilution water characteristics, appropriate water temperature ranges, and minimum dissolved oxygen levels. The guideline test validity criteria for dissolved oxygen and water temperature are dissolved oxygen ≥ 60% of saturation (4.9 mg/L at 25°C), and that the mean water temperature 25 ± 1°C. Additional water quality characteristics specified in the guideline (Table 1 in guideline) include limits on total alkalinity, total organic carbon, unionized
ammonia and residual chlorine.

Based on an analysis of the data, the performing laborator ies were able to maintain the specified mean water temperature of 25 ± 1°C throughout the duration of the test. As depicted in Figure 1 4 , all 21 studies reported an overall mean temperature of 25 ± 1°C. However, three of the studies were not able to keep the temperature within 1°C between tanks at any one time, and in seven other studies there was insufficient temperature data reported to evaluate this. all 5 of 6 4 of 6 3 of 6 2 of 6 0 2 4 6 8 10 12

# of Met Criteria# of StudiesUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 93 of 160 Additionally, in two studies, the minimum temperature was reported to have dropped to 23°C. However, these types of deviations were not of sufficient duration and/or frequency to affect the overall mean temperature and its standard devia tion. Short durations of this magnitude of temperature fluctuations did not appear to negatively impact the performance of the fish as reproductive performance in the affected tanks was within the test validity criteria.

Dissolved oxygen (DO) levels wer e also generally maintained above the required minimum of 60% saturation. In 15 of the study reports, the individual DO measurements were equal to or greater than 60% of saturation. In the other six study reports, at least one
measurement of DO was below 60% saturation (minimums of 45- 58% reported). As DO was not measured daily nor in every replicate when measured, the duration of the decline for all the studies is unknown. However, in these six studies, once a decrease in DO was measured, efforts

to ra ise the DO was initiated, and aeration was generally added to the system sometimes as early as at test initiation to Day 2 of the test. In one instance, a drop in DO was measured after aeration was initiated; however based on the data provided it appears that this may have occurred in two other instances as well. In one case, the temporary drop in DO was reported not to have adversely affected the interpretation of the study; the other five studies did not speculate on the potential impact.

In addition to the specified test validity criteria for temperature and DO in Table 1 of the guideline, measured limits on several dilution water characteristics were also listed. These included total alkalinity, total organic carbon (TOC), unionized ammonia and resi dual chlorine. All of the study reports measured total alkalinity from the control and treatment group test solutions, and the reported values were greater than the minimum level specified in Table 1 of the guideline. Seventeen of the 21 studies reported measuring TOC, but values were only reported for 10 of the studies which were all below the maximum level specified in the guideline. Measurements of residual chlorine levels were reported in eight of the studies. However, only
two of the studies had re ported residual chlorine levels below the guideline maximum level whereas for the other six, the limit of detection was greater than the maximum recommended level. Unionized ammonia levels were recorded in one study which was below the guideline
maximum l evel; six other studies reported the nitrogen levels (as either ammonia (as N) or ammonium (as N)) in the dilution source water. In these 21 studies, the reported levels of total organic carbon, unionized ammonia and residual chlorine were from periodic sa mples collected from the dilution water house source and not from the dilution water collected directly from the study chambers. The laboratories that performed these 21 FSTRAs routinely perform other ecotoxicological studies in support of pesticide registrations. As such, historical data are available on the quality of the dilution water source used in these studies. In general and based on the historical data from specific laboratories, the quality of dilution water sources used in
these studies has be en adequate to promote good health and reproduction in fathead minnow.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 94 of 160 Control Survival

The test validity criterion in the guideline for control survival is 90%. This criterion of
90% survival applies to the negative control and, if used, the solvent cont rol as well. In all but one study, overall control survival was equal to or greater than 90%. In the one study, the control survival was 83%. While not used as test validity criteria, the survival of each gender between the controls and treatment groups was evaluated to determine if mortality may have been gender specific. While no evidence of gender

- specific mortality was observed, except for in one study, tracking the gender

- specific mortality is useful when trying to interpret the results
for other e ndpoints. This is especially true for the gender

- specific endpoints as a slight decrease in the sample size for males or females may have an impact on the ability to reliably interpret or discern differences in sublethal endpoints. In regards to mortality, the guideline specifies that if all the treatment groups exhibit mortality

, then the assay should be repeated at lower test concentrations to be better enable reliable inferences about possible endocrine interactions.
Observations of non- lethal effects such as morbidity and other clinical signs of toxicity ( e.g., loss of equilibrium and decreased feeding) should also be considered when evaluating whether a test should be repeated and also when evaluating for potential endocrine interactions. Overt toxi city is described in the guideline as “>2 mortalities in any replicate that can only be explained by toxicity rather than technical error” (pg 18). Other signs of overt toxicity identified in the guideline include “hemorrhage, abnormal behaviors, abnormal swimming patterns, anorexia, and any other clinical signs of disease”.

Control Fecundity

The guideline indicates that for a successful study, the fish in the control(s) should be actively spawning. This can be indicated by the fish spawning at least ev ery 4 days or reproducing at a rate of 15 eggs/female/reproductive day/replicate (abbreviated: E/F/D/R) during the exposure phase of the assay. While not a test validity criteria, the guideline states that in the pre

- exposure phase, the fish should spawn at least two times in the 7 days immediately preceding the exposure phase and that egg production should exceed 15 eggs per female per reproductive day per replicate. In terms of the spawning groups in the pre

- exposure phase that
were carried over into th e actual exposure, the majority (20/21) of the evaluated studies met the

pre

- exposure recommended spawning frequency and/or fecundity rate. In the one remaining study, the pre

- exposure production rate was <15 E/F/D/R for all but two of the tanks used in t he exposure phase (range approximately 6

- 16 E/F/D/R); there were insufficient data reported to determine whether fish in these two tanks had spawned two times in the 7 days prior to exposure.

Spawning performance of controls during the exposure phase fo r each of the 21 studies was evaluated. In 17 of the 21 studies, the overall control (includes negative and solvent) mean fecundity was >15 E/F/D/R and generally was >20 E/F/D/R. The negative control mean fecundity for the other four studies ranged from 8.8- 12 E/F/D/R; two of the studies also had a solvent control with fecundity values of 6.5 and 13 E/F/D/R. Based on the wording in theUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 95 of 160 guideline, adequate spawning is to be demonstrated in all replicates of the control. Therefore,
the stated criteria wer e evaluated against each replicate in the negative control, and solvent control if present. On an individual replicate basis, 15 of the studies met the fecundity performance criterion. In the other six studies, for at least one replicate in the negative control, and solvent control if present, the fecundity was <15 E/F/D and the spawning interval was greater than 4 days. It should be noted that in 10 studies for at least one control replicate the reported fecundity was >15 E/F/D, but spawning s till did not occur every 4 days; however

, the number of eggs produced allowed for demonstration of adequate spawning. It has been reported that on average, spawn ing will occur every 3 - 4 days; however

, longer intervals of as much as 15 days have also been observed (Jensen et al ., 2001 in USEPA 2007(ISR report ).

In reviewing the submitted data, it was observed that laboratories are calculating fecundity in two slightly different ways. Some of the labs are calculating a daily fecundity rate in each replicate expressed as ‘number of eggs/female’ and then taking the mean of the 21 daily fecundity estimates for an overall mean ‘number of eggs/female/reproductive day’. Other labs are summing the total number of eggs produced and the total number of female reproduction days over the 21 days and dividing the two which is then expressed as ‘number of eggs/female reproduction day’. Both methods take into account female mortality that occurs during the test. If there is no female mortality in the tank, then the overall mean value is the same regardless of
which method is u sed. However, if there is mortality, then there may be differences in the overall fecundity value for that replicate. Both methods of calculating fecundity have been presented in previously published documents on the development of this assay. As long a s the method is consistent within a study, either methodology should be acceptable.

Control Fertility

The guideline reported test validity criterion for fertility is ≥95% for eggs produced in the control(s). As depicted in Figure 14 , in 17 of the studies, the overall mean fertility was ≥95% in the controls. In the other four studies, the overall mean fertility ranged from 90 -94%.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 96 of 160 Variation in Measured Test Concentrations

The validity criterion for the CV in a measured test concentration over the duration of the exposure period is reported as 20% in the guideline where the reported CVs are based on the 21

- day treatment mean or time -weighted mean test concentration and its associated standard deviation. There was a wide range of test chemical solubility and/or stability across the test chemicals evaluated. In some cases, a chemical’s low solubility and/or instability influenced its overall ability to be maintained at a consistent concentration in the water column. As depicted in Figure 1 4 , 12 of the 21 studies maintained CV’s within 20% during the exposure period. In the other nine studies, CVs ranged from slightly greater than 20% to up to 80% ( Figure 16 ). A solvent carrier was used in eight of the 21 studies, whereas a saturator column was used to

ge nerate test solution in three other studies. CV values higher than 20% were generally attributed to poor water solubility or the adsorptive properties of the test chemical ( e.g. sorbed to aquaria glass). Generally, laboratories took steps to reduce the v ariation in the overall measured
concentrations. These steps included increasing the delivery flow rate of the test solutions and cleaning the tanks more frequently. Some laboratories included additional analytical
measurements to better characterize the actual test concentrations over time. However, CV values that exceed the stated performance criterion will likely continue to be associated with chemicals with limited water solubility, adsorption and/ or degradation properties. CV values greater than 20 % may be considered acceptable on a case

- by

- case basis if the time

- weighted average can be estimated and demonstrated not to overlap between test levels.

Figure 16

. Summary of the Range of Reported Coefficient of Variation (CV) for the Studies

0 2 4 6 8 10 12 14

≤ 20% up to 30% up to 40% up to 50% up to 80%# of Studies

Range of Reported CVsUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 97 of 160 (iv) Adequate Dosing and Overt Toxicity

The guideline states that the highest concentration tested should be one that does not result in increased mortality or signs of morbidity compared to the control. The use of 1⁄3 of a
derived 96- hr LC 50, using fish of similar age and size as those used in the FSTRA, is suggested as an option for establishing the high test concentration, with a 10

- fold decrease between subsequent test concentrations. It is important to demonstrate though that the study was conducted at a high enoug h concentration to allow for the possible observation of effects. The guideline alternatively suggests that, in the absence of observed toxicity, the highest concentration may be set at the solubility limit of the chemical in water or at the historical limit test concentration of 100 mg/L (part per million; ppm). Twelve of the 21 evaluated studies used, at least in part, 1⁄3 of an LC 50 value when setting the highest test concentration ( Table 28 ). Other studies used a variation of a reported maximum tolerated concentration, solubility limit or the maximum test concentration of 100 mg/L to set the high concentration. When evaluating the results, the use of a 1⁄3 LC 50 value sometimes resulted in substantial mortality as well as effects on physical appearance, behavior and/or changes in body weight. The LC 50 value used in dose selection was sometimes generated using fish of similar age and size, but other times was from studies with juvenile fish and in some cases even a different species. In 17 of the 21 studi es, effects on survival, decreased body weight and/or observation of clinical toxicity were reported for at least one test concentration ( Table 28). For some sublethal effects ( e.g., body weight) it is not always clear if the observed effect is due to overt toxicity, but was included in the table to illustrate the types of lethal and sublethal effects observed.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 98 of 160 Table 28. Summary of the Types of Overt Toxicity Observed in the 20 Assays

Chemical Code Type of Effect Observed (test levels affected 1 ) Method Used to Set High Test Concentration Survival 2 Sublethal clinical signs of toxicity 3 Body Weight 2,4

O

− − − Max. necessary conc. (100 mg/L)
E High

− − 1⁄3 96h LC50

I

− High (discoloration, loss of equilibrium) High 14-d MTC

C

− − High Solubility
A

− High (abnormal swimming) Mid, High Not reported
H

− High (discoloration)

− Not reported Q

− 5

− − 1⁄3 96h NOAEC
J

− − High 1⁄3 96h LC50

K High 6 Mid, High (loss of equilibrium, lethargy , deformed) − 1⁄3 96h LC50

G

− − Low, High 8 1⁄3 LC50 (unknown duration)

L

− − − ELS

T

− − Low, Mid, High 1⁄3 96h LC50

M High

− − 1⁄3 96h LC50

U

− − Low, Mid, High Max. necessary conc.

S High Not well reported

− 1⁄3 LC50 (duration unknown)
N

− High (decreased feeding, emaciated) High 1⁄3 96h LC50 & ELS

P High High (decreased feeding, abnormal swimming) − 1⁄3 96h LC50

D

− 7

− − 1⁄3 96h LC50 & NOAEC
B

− High (decreased feeding) Mid, High Solubility

F

− High (loss of equilibrium, discolored, paralysis) High 8 1⁄3 96h LC50

T

− Not well reported

− Not well reported Total # of Studies 7 8 10

-- 13 (reported effects on survival or clinical signs of toxicity) -- --

17 (reported effects on survival, clinical signs of toxicity, or decreased body weight) --USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 99 of 160 ‘

— ‘ = no effects observed; MTC = maximum tolerated concentration; ELS = fish early life

- stage test

1 The three test concentrations in the assays referred to as low, mid, and high

2 Indicates a significant (p≤0.05) overall effect at the listed test concentration

3 Clinical signs of toxicity (other than survival and growth) were visually evaluated.

4 Changes in body wt. may or may not be due to overt toxicity. Other lines of evidence from the study should be used to distinguish between overt toxicity and endocrine mediated changes

5 While overall survival was not significantly (p>0.05) decreased, one replicate at high concentration had >2 mortalities

6 Additionally, one replicate at lo w concentration had >2 mortalities

7 While overall survival was not significantly (p>0.05) decreased, where was a significant decrease in female survival at the h igh
concentration

8 Significant increase in body weight compared to control (p≤0.05), so likel y not indicative of overt toxicity.

Overt toxicity is described in the guideline (pg 18) as “>2 mortalities in any replicate that can only be explained by toxicity rather than technical error” (pg 18). Other signs of overt toxicity identified in the guide line include “hemorrhage, abnormal behaviors, abnormal swimming patterns, anorexia, and any other clinical signs of disease”. According to the guideline, if such signs of overt toxicity are observed then the replicate or possibly the entire test

concentra tion should be removed from statistical analysis. This definition of ‘overt toxicity’ is broad and may be interpreted differently among laboratory investigators or reviewers. Additionally, if overt toxicity is observed, and a 10

- fold decrease in test con centration is used, the large differences between concentrations may limit the ability to observe potential chemical interactions with the endocrine system as the next lower concentration may result in responses that are statistically indistinguishable fro m control(s). It is recognized that changes in some endpoints ( e.g ., fecundity) in this assay may be due to non

- endocrine mediated pathways, and the observance of clinical signs of toxicity should be used to help distinguish those pathways. However, for some sublethal endpoints ( e.g ., changes in body weight), it is not definitively
known if the observed effect was due to a non- endocrine mode of action or possibly the result of a downstream effect from a modification to the endocrine system.

Survival

In 21 of the evaluated studies, there were reported effects on survival in seven studies. Five studies (29%) reported statistically significant effects (p≤0.05) on overall survival (males and females combined) at the highest concentration tested. In four of the five studies, the percent mortality in the highest treatment group at test termination ranged from 29 to 92%, and in the fifth study, the high est treatment group was terminated during the study due to >30% of the fish showing signs of overt toxicit y ( i.e., mortality, loss of equilibrium). In the sixth study, there was a significant decrease in female mortality at the high concentration compared to the control. Lastly, in the seventh study, at the highest concentration tested, there was one replica te with more than 2 mortalities and per the guideline, this replicate may be considered for exclusion in the data analysis; it is expected that inclusion or exclusion of replicates/treatments will be analyst dependent. In addition to the seven studies wit h effects on survival at the highest concentration tested, six other studies reported sublethal clinical signs of toxicity at the highest concentrationUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 100 of 160 tested. The reported sublethal effects ranged in severity and included decreased feeding and lethargy, abnormal swimming and loss of equilibrium to complete paralysis.

Body Weight

Body wet weight and length are measured in the surviving fish at test termination. In only one study were length and weight significantly decreased (p≤0.05) at the highest

con centration compared to the control. In 5 of the 21 studies, significant (p≤0.05) changes in male or female body weight were observed at concentrations where other sublethal and/or lethal effects were also reported. However, in four other studies, significant (p≤0.05) decreases on body weight were reported at concentrations where there were no other sublethal or lethal effects reported. Another study reported a significant increase in body weight compared to the control with no reported overt toxicity; wher eas there was one study that reported a significant increase in weight in the presence of observed overt toxicity. The magnitude difference in body weight in the studies that reported a significant difference relative to controls varied but was generally
between 10 and 30%.

For females, the timing of the last spawning event relative to test termination may influence overall body weight for an individual. Evaluation of body weight was not based on changes in body weight over the course of the study but rather was based on the absolute body weights at test termination. The test is not designed for evaluating changes of an individual fish’s body weight throughout the exposure period; although, tracking body weight by gender in a replicate is possible, it would require more effort. This could be accomplished, for example, by recording not only the female body weight but also which replicate tank it was placed into during the pre

- exposure phase and then using the test termination body weights to calculate a c hange in body weight for females for that replicate. However, this value would represent both the pre

exposure as well as the treatment group exposure which could confound the interpretation. Prior to distribution to the pre

- exposure tanks, each fish is weighed and the goal is to keep the wet weight with ±20% of the arithmetic mean wet weight for the same gender. This mean wet weight is determined from a sub

- set of the population of fish to be used in the study. While most of the studies reported that the initial wet weight was within 20%, there were four studies that reported a range of approximately 30%. As there is allowed variability in the initial wet weight of the fish and weights of individual fish are not tracked throughout the exposure, anticip ating whether a given magnitude of change will result in a statistically significant difference may not be feasible. In terms of whether a change in body weight, compared to the control, is the result of generalized overt toxicity or an alteration in an e ndocrine mediated pathway is not always clear. The collective evaluation of the other measured endpoints, especially the endocrine specific endpoints, as well as reported feeding behavior, if available, is key toward determining the potential cause for th e change in body weight.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 101 of 160 As lethal and/or sublethal effects (not including changes in body weight) were reported in at least the highest test concentration in 13 of the 21 evaluated studies, this could lead to the
exclusion of at least one treatment conc entration from the analysis. Furthermore, in an additional four studies, fish exhibited decreases in body weight which may or may not be due to endocrine interactions. Given that there were generally three test concentrations, the interpretation of
poten tial endocrine interactions is then limited to two concentrations which are generally 10 and 100 times lower than where the lethal and/or sublethal effects were observed. Additionally, given that there would only be two test concentrations for data analys is, the ability to observe potential increasing or decreasing trends compared to the control for an endpoint is more limited.

The Use of Solvent Carriers in the FSTRA

The use of a solvent carrier (co -solvent) to facilitate dosing is discouraged in the FSTR A test guideline. Co

- solvents are discouraged as they have the potential to interfere with the interpretation of the results by potentially modifying the fish’s response to a chemical or increasing the microbial community in a tank which may influence wat er quality, food availability, and/or potential degradation of the test chemical. Co -solvents were used in seven of the 21 evaluated studies. Acetone and dimethyl- formamide (DMF) were the reported solvents,
with all solvent concentrations at or below the maximum recommended concentration of 0.1 mL/L in regulatory fish chronic toxicity guidelines (OCSPP 850.1400 18 ; OECD 210 19 ). In five
of the seven studies where co

- solvent was used, the solvent concentration was consistent ( i.e ., at the same concentration) between the solvent control and all the treatment groups. The other two studies utilized proportional diluters and only the highest test chemical concentration had the
same solvent concentration as the solvent control.

If a co

- solvent was used, an evaluation of potential solvent interferences is conducted by considering results from a concurrent solvent control group. If there are statistically significant differences detected between the negative (dilution water only) cont rol and the solvent control for a given endpoint(s), then best professional judgment may be used by Agency reviewers to evaluate if the study is valid. In one of the seven studies which utilized a co- solvent, there was a significant (p≤0.05) reduction of 34% in fecundity between the solvent (DMF 0.04mL/L) and negative control; female body weight was also significantly (p≤0.05) increased by 11% relative to the negative control. Reduced dissolved oxygen (<60% saturation) was reported in four of the

seven st udies which utilized solvents (3 used DMF and one acetone), an observation perhaps reflective of enhanced microbial growth. However, the decreases in DO appeared to be relatively short in duration and the laboratories did take measures to rectify the situa tion. Overall, it

18 USEPA. 1996. Ecological Effects Test Guidelines OPPTS 850.1400 Fish Early

- Life Stage Toxicity Test. Office of Chemical Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Toxic Substances (7101). EPA 7120C096

- 113. April 1996. http://www.epa.gov/opptsmnt/pubs/frs/publications/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Drafts/850

- 1400.pdf

19 OECD 1992. Test No. 210: Fish, Early

- life stage toxicity test. Organisat ion of Economic Co

- operation and Development. http://www.oecd

- ilibrary.org/docserver/download/9721001e.pdf?expires=1361831113&id=id&accname=guest&checksum =8F099EABD91311E2B51F27DA93614DE4USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 102 of 160 appears that in one of these studies that utilized a co

- solvent, that there was a potential solvent
interference.

Incorrectly Sexed Fish in the FSTRA

In this test, fish are distributed into the test chambers in a pre

- determined sex ratio of two males and four females per replicate. The guideline recognizes the potential for incorrectly identifying the sex of a fish (referred to as a mis -sexed fish) and recommends that additional tanks be established in the pre

- exposure phase to ensure adequate sample size at test initiation. Upon evaluation of the 21 FSTRAs, six of the 21 studies had at least one male fish mistaken for a female but the error was not caught until test termination. Three of these studies contained one misidentified fish, with the other three studies resulting in 2, 3, and 5 replicates with a misidentified fish. In all of these studies, calculation of fecundity in these replicates with the
mis

- sexed fish was adjusted to account for the additional male. However, for the ot her gender

specific endpoints, some laboratories included measurements from the mis -sexed fish in the analyses while other labs excluded the fish. As these fish were most likely not expressing male characteristics ( i.e., may have been immature males) prior to test chemical exposure, the Agency considered it appropriate to exclude them from data analyses.

Reasons for mis- sexed fish vary but may include technician inexperience and/or the use of insufficiently mature fish. The guideline states that the fish should be within the range of 4.5 to 6 months old and reproductively mature; this assumes the fish were cultured at 25±2°C with adequate feed. The OECD 229 FSTRA also provides guidance on fish selection, including recommended mean wet weights 20 . Given th at there may be slight differences in time to maturation between batches of fish in a laboratory and among laboratories, fish between 4.5 to 6 months old for a given cohort may not always be in appropriate reproductive status necessary for this assay. How ever, as there are well

- defined culturing protocols for fathead minnows (ASTM

2005 21 , Benoit 1982 22 , Brauhn, and Schoettger 1975 23 , and USEPA 1987 24 ), the likelihood of this occurring is expected to be low . In 18 of the 21 evaluated assays, the reported age of the fish at the beginning of pre

- exposure was within the 4.5 to 6 months age range, with all but one ranging from 5 to 6 months. The age for two other studies was between 6 – 7 months, and the final study indicated that the fish were at least 120 days old. For the assays with the mis- sexed fish, the reported age ranged from 4.5 to 7 months with no discernible pattern. To increase the

20 OECD. 2009. Fish Short Term Reproduction Assay. Test No. 229. O rganisation of Economic Co

- operation and Development . http://miranda.sourceoecd.org/vl=68881146/cl=11/nw=1/rpsv/cgi

- bin/fulltextew.pl?prpsv=/ij/oecdjournals/1607310x/v1n2/s30/p1.idx (accessed February 6, 2013).

21 American Society for Testing and Materials. ASTM E 1241

- 05, Standard Guide for Conducting Early Life

- Stage Toxicity Tests with Fishes. In Annual Book of ASTM Standards, Vol. 11.06, ASTM, West Conshohocken, PA. Current edition approved

2005.

22 Benoit, D.A., 1982. User’s guide for conducting life

- cycle chronic toxicity tests with fathead minnows ( Pim ephales promelas ). Environ. Res. Lab - Duluth, Duluth, MN. EPA 600/8

- 81

- 011.

23 Brauhn, J.L. and Schoettger, R.A., 1975. Acquisition and Culture of Research Fish: Rainbow trout, Fathead minnows, Channel catfish and Bluegills. p. 54, Ecological Research Series, EPA

- 660/3

- 75

- 011, Duluth, MN

24 Denny, J.S..1988 Guidelines for the culture of fathead minnows, Pimephales promelas for use in toxicity tests U.S Environmental Protection Agency, Environmental Research Laboratory. Duluth, MN. EPA/ 600/ S 3

- 87/001USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 103 of 160 likelihood of success, it is considered important to have a well

- established healthy culture of fathead minnows ( i.e., pr oper density, adequate feed and environmental conditions) from which to draw upon for each test. Having a culture that is sufficient in numbers as well as old enough to establish fully mature females ( i.e., the presence of a fully

- developed ovipositor) should help
reduce the incidence of mis

- sexed fish. As investigators gain experience with this assay, the
incidence of mis

- sexing fish may diminish; however, it still may occur from time to time. While there is potential for incorrect sexing, the presumpti on was that either the mis -sexed fish would be identified during the pre

- exposure period and excluded from further chemical exposure or, if they were not identified in the pre

- exposure period, that the occurrence would be minimal and
not have a substantive impact on the study conclusions. However, if the presence of mis

- sexed fish occurs, it will influence the analysis of the data for endpoints such as fecundity and gender

- specific endpoints ( i.e. VTG and GSI) as well as potentially increase the variabili ty for a measured endpoint.

(v) Measured Endpoints in the FSTRA

The FSTRA is intended to provide insight on whether a chemical may interact with the HPG axis. The FSTRA has been validated using chemicals with known modes of action (MOA, for discussion see Ankley et al. 2010) and is expected to provide information on a chemical’s potential to disrupt any of several endocrine pathways ( e.g ., estrogen, androgen, steriodogenesis) related to the HPG axis ( USEPA 2007a). Reviewing the results from a chemical with a known mode of action may be useful when evaluating the resul ts a chemical with an ‘unknown’ potential for endocrine interaction. This review included chemicals with previously unknown potential for endocrine interaction. This section will discuss each of the endpoints in the assay and the preliminary results of these endpoints for each of the 21 available assays. In some cases, significant differences (p≤0.05) were reported for endpoints at concentrations where effects on survival or other clinical signs of toxicity were reported. Additionally, effects on endpoi nts were reported at concentrations that also reported effects on body weight.

Fecundity and Fertility : As pointed out previously, the 2008 SAP noted that the fecundity
observations were “ an essential part of apical analysis of HPG activity ” (USEPA 2008, pg 13). Significant (p≤0.05) reductions in fecundity were reported in 16 of the 21 assays in at least one test concentration. In eight of the studies, there were statistically significant (p ≤ 0.05) effects on fecundity only at the same concentration as reported effects on survival, female body weight or measures of clinical toxicity. However, in four of the 16 studies, there was a significant decrease in fecundity without a reported lethal or sublethal effect in the study. Lastly, in the four other

stu dies, overt toxicity was observed in some, but not all, of the concentrations that reported a significant decrease in fecundity.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 104 of 160 Fertility was reported to be significantly decreased in 9 of the 21 assays for at least one
test concentration. In seven of these studies, effects on fertility were only reported at concentrations that had effects on survival, male body weight or other clinical signs of toxicity.

Secondary Sex Characteristics: Evaluation of secondary sex characteristics in the FSTRA are evaluated both qualitatively ( e.g ., presence of vertical banding, fat pad (dorsal nape pad), ovipositor, body coloration) and semi

- quantitatively ( i.e ., nuptial tubercles and sometimes fat pad) in males and females. Nuptial tubercles are located on the head (dorsal pad) of reproductively active male fathead minnows, and are not exhibited by control females and juvenile males and females (Jensen et al . 2001). However, exposure to certain types of endocrine disrupting chemicals may cause the appearance of nuptial tubercles in females (androgen) or diminish them in males (estrogens). In this assay, if present on a fish, the nuptial tubercles are enumerated and graded based on prominence, and the overall scores between the control and treatment groups are compared. In the 21 assays evaluated, no nuptial tubercles were observed in either control females or in females exposed to the test chemicals. Control male fish did exhibit nuptial tubercles, expect for the four fish that were mistakenly identified as femal es (mis

sexed) during the distribution of the fish to the pre

- exposure tanks. Eight of the 21 assays reported a statistically significant decrease (p≤0.05) in male nuptial tubercle scores compared to the control, and these effects were only reported at te st concentrations with overt toxicity for six of these studies. In an additional study, a decrease in tubercles was reported at test concentrations both with and without reported overt toxicity. In the final study, all reported
effects were in the absenc e of overt toxicity.

Gonado-somatic Index (GSI): The gonado

- somatic index (GSI) is calculated for each fish at test termination. As with the other endpoints identified as core endpoints in the test guideline, GSI is evaluated in concert with other endpoints to determine potential chemical -induced effects on the reproductive system of a fish. The guideline states that typical GSI values for reproductively active fathead minnows range from 8 to 13% for females and from 1 to 2% for males. For the 21

assays, the range of male GSI in the controls was 0.98 – 1.75% with a median value of 1.3% and
was 11.1 – 16.4% with a median value of 13% for control females. Therefore, the reported GSIs appear to be generally within typical values. For males, there were thre e studies out of the 21 which reported a statistically significant (p< 0.05) decrease in GSI for at least one test concentration compared to the control with another four studies reporting a statistically significant (p < 0.05) increase in GSI. In three of t hese seven studies, the change was reported only at concentrations where there was an effect on survival, male body weight or other clinical signs of toxicity were reported; however, this was not the case for other concentrations in four of
the studies. F or females, a statistically significant (p≤0.05) decrease in GSI was reported for three studies as well as a significant increase in three other studies. In two of these studies, the
effect was not dose- dependent, and signs of potential overt toxicity were reported in corresponding concentrations in thr ee of the studies.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 105 of 160 Vitellogenin (VTG) : Vitellogenin, an egg yolk precursor glycolipoprotein, is an important endpoint in the FSTRA for evaluating if a chemical is potentially interacting with the endocrine system and has been characterized as the most v aluable biochemical endpoint in this assay ( USEPA 2007a). VTG levels are normally very low in healthy male fathead minnows , but VTG production can be induced in both male and female fathead minnows when exposed to estrogenic chemicals, whereas other types of endocrine disrupting chemicals ( e.g., androgens) can cause a decrease in female VTG ( USEPA 2007a). Upon review of the 21 assays, a significant (p≤0.05) increase in VTG in a treatment group compared to the control was observed in males for two
studies whereas a significant decrease (p≤0.05) in VTG was reported in another study. In two of these three studies, effects on survival or other clinical signs of toxicity were reported at the same concentration as the significant change in VTG. For females, a significant decrease (p≤0.05) in VTG was reported in four of the 21 studies, and a significant increase (p≤0.05) was reported in another study. In three of these studies, effects on female VTG were only reported in
the same concentrations that had surviva l effects or other clinical signs of toxicity.

When reviewing the VTG results, occasionally the mean VTG value for a test concentration visually appeared to be substantially greater or lower than the control mean VTG (treatment mean based on the average of the four replicate means). This was observed in nine of
the studies for male VTG and in two of the studies for female VTG. However, none of these

cases was not statistically significantly different, and may not have been dose

- responsive. As
male VTG is normally very low and the measured concentration can be near the limit of detection in an assay, considerable variation in plasma VTG can occur. Examination of the CVs for these treatments also provided insight on the variability within a treatment group. Additionally, plotting the data is an effective means of determining whether an observed increase or decrease in mean VTG in a treatment group was due to a marked change in a limited number of fish or if it represented an overall change in response in the exposed fish. Data from one of the 21 studies demonstrating where male VTG was statistically significant in a treatment group compared to the control as well for another study in which it was not significant is shown in Figure 1 7. As d epicted in the F igure 17B for Chemical A , all of the male VTG values are similar except for two values in the mid concentration (L2), illustrating the magnitude of variability sometimes observed in the 21 evaluated assays. For Chemical L (depicted in Figure 17 A), the
h ighest treatment group was significantly increased (p≤0.05) compared to the control, l ikely due to two outliers, with greater variability observed in this treatment compared to the control and lower treatment groups . G enerating a simple plot of the data and looking for general trends is not limited to VTG and may be beneficial for other endpoints as well.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 106 of 160 Figure 1 7. Male VTG data

.

Figure 17.A demonstrates male VTG data (ng/mL) from Chemical L where the high treatment group was statistically different (p≤0.05) than the control. A (Chemical L).USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 107 of 160 Figure 17.B is presenting data from Chemical A where there were no significant effects (p>0.05). The median of the individual data values is represented by the solid line, with the box representing the 25 th and 75 th percentile and the whiskers are the data range, with suspected outliers denoted by a small open circle next to the data point.

B (Chemical A)

Sex Steroids

Currently, analysis of plasma sex steroids (17β

- estradiol and testosterone) is an optional measurement endpoint in the test guideline. Due to the small blood volumes available from fathead minnow, an individual sample of adequate volume is not always available after removing a sample for VTG analysis. While routine in many mammalian toxicology studies, sex steroid measurements have not historically been required in most regulatory ecotoxicity studies and labs performing the FSTRA may have limited experience with performing the immunoassays and reporting the relevant data. In the 21 studies evaluated, only three analyzed sex steroids with effects reported for females in one study and for males in another study. In these cases, availability of the sex steroid data improved the Agency’s ability to interpret any effects on the other endpoints. 11, 700USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 108 of 160 Gonadal Histopathology

Histopathology is another important endpoint in the FSTRA because it provides a direct
evaluation o f the gonads, and changes observed can encompass the integration of several molecular, cellular and physiologic processes. In doing so, histopathology can provide insight on the potential reproductive impacts of chemical disruption, and it is useful to co nfirm changes in other assay endpoints ( USEPA 2007). Histopathological evaluation of the gonads of surviving male and female fish is conducted on fixed specimens collected at test termination. Histopathology guidelines for the fathead minnow ( Pimephales promelas ) are provided in Appendix E of the guideline. The guideline is intended to provide general direction on evaluating gonadal tissue sections, promote awareness of potential pathological findings, and to foster consistency in diagnostic terminology.

Gonadal staging as well as individual pathological findings were reported in the studies. Increase in oocyte atresia and changes in sperm maturation were the two main histopathological observations reported in the evaluated data set. Ten studies reporte d an increase in oocyte atresia and 8 studies reported changes in sperm maturation. In eight of these studies, effects on survival and/or other clinical signs of toxicity were recorded in the same treatments as the reported histopathological observations. In a few other studies, the observed findings did not appear to be

treatment

- related.

(vi) Statistical Approach for the FSTRA

Statistical analysis of the data for 20 assays generally followed the statistical path outlined in Figure 1 in the guideline and its supporting text (USEPA 2009, pg 17); one other assay used slightly different statistical tests than outlined in the guideline. All statistical tests were performed at the 0.05 significance level (α = 0.05). The guideline states that “for all
continuo us quantitative endpoints ( i.e., fecundity, VTG, GSI) consistent with a monotonic dose

response, the Jonckheere

- Terpstra test should be applied in step -down manner to establish a significant treatment effect” (USEPA 2009, pg 15). For the endpoints in the evaluated assays, monotonicity was visually assessed using professional judgment by examining the treatment and/or replicate means or medians. Generally, a trend test was used by reviewers when the mean or median values, as applicable, for each test conce ntration followed a clear increasing or decreasing pattern. Per the guideline, the non

- parametric Jonckheere

- Terpstra test was used for monotonic data sets regardless of whether the data met the assumptions for normality and variance homogeneity. The test was run under a one- sided hypothesis, with the direction based on whether the data was monotonically increasing or decreasing.

For all endpoints, if the data were not judged to be consistent with a monotonic dose

response, the data were assessed for norm ality and variance homogeneity. If the data met these assumptions for parametric data, Dunnett’s test was used to analyze the data; if not, then the non

parametric Mann -Whitney Wilcoxon U test was used. Both tests were run under a two- sidedUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 109 of 160 hypothesis in order to detect both increases and decreases in the data. Per current Agency practice, when data do not meet normality or homogeneity assumptions for parametric tests, transformations are not applied and analysis proceeds directly to non

- parametric metho ds. The statistical approach for the FSTRA and the potential impact of alternative statistical methods are discussed in more detail in Appendix 1.

(vii) Overall Conclusions for the FSTRA

In terms of the test validity criteria depicted in Figure 14 , the FSTRA is generally being performed successfully. Four of the 21 reviewed assays met all the validity criteria, whereas another 11 assays deviated from only one of the six identified criteria. In the cases of DO, if decreases were observed, the laboratories appeared to be responsive to rectifying the situation.
This was also the case for measured concentrations of some chemicals which are considered more difficult to test due to their physio

- chemical properties, as investigators generally took
measures to try to regulate exposure. Maintaining consistent test concentrations is expected to be challenging for difficult to test substances and as laboratories gain experience with non

solvent delivery methods, this observed variability should decrease, but is not expec ted to be
eliminated. It is re

- iterated, that a deviation from a defined test validity criterion does not necessarily indicate that the study cannot provide useful information, but rather, the conduct and results of each study are evaluated as a whole to determine the degree of confidence in the ability to discern the potential for endocrine interactions.

Statistically significant (p≤0.05) effects on survival and/or the observation of overt toxicity were observed in the highest concentration tested in 13 o f the submitted studies with another four studies reporting a decrease in body weight. Evaluation of potential endocrine interaction needs to be considered in the context of whether overt toxicity was present in the study and the degree to which is was ob served.

Thirty percent of the studies contained at least one replicate with incorrectly sexed fish. If a misidentified fish is observed, while adjustments were made to endpoints such as fecundity, it may also be appropriate to exclude the misidentified fi sh from the data analysis for the gender specific endpoints. If there are numerous misidentified fish in a study, the potential implications in the interpretation of the results should be addressed. Ultimately, as laboratories gain experience with this assay, the occurrence of mis

- sexed fish is expected to decrease.

Although the measurement of plasma sex steroids is an optional endpoint, the endpoints are endocrine specific and are anticipated to provide a very useful line of evidence on whether a chemical is potentially impacting the HPG axis. Therefore, the Agency emphasizes the importance of measuring sex steroids when the blood sample volume is sufficient.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 110 of 160 f. Amphibian Metamorphosis Assay

(i) Background

The Amphibian Metamorphosis Assay (AMA) is a s creening assay intended to empirically identify substances which may interfere with the normal function of the hypothalamic

- pituitary

- thyroid (HPT) axis (Fort et al . 2007). The AMA represents a generalized
vertebrate model to the extent that it is based o n the conserved structures and functions of the HPT axis. It is an important assay because amphibian metamorphosis provides a well

- studied, thyroid

- dependent process which responds to substances known to be active along the HPT axis, and it is the only as say in the EDSP Tier 1 battery that detects thyroid activity in an animal undergoing metamorphosis. As described in the test guideline (OCSPP Guideline 890.1100) and the Standard Evaluation Procedure (SEP) for the AMA (USEPA 2011a ), each of the Tier 1

EDS P assays, including the AMA, is intended to be interpreted within a battery of in vitro and in

vivo tests to identify substances with potential to interact with the endocrine system.

The general experimental design entails exposing Nieuwkoop

- Faber (NF; Ni euwkoop
and Faber 1956 25 ) stage 51 African clawed frog ( Xenopus laevis ) tadpoles, in separate treatment groups, to a minimum of three concentrations of a test chemical or a negative (clean water) control for 21 days. There are four replicates tanks within each test treatment. Larval density at test initiation is 20 tadpoles per test tank (replicate) for all treatment groups (80 larvae per treatment). The observational endpoints are hind limb length (HLL), snout

- to -vent length (SVL), developmental stage, body weight, thyroid histopathology, and daily observations of mortality and clinical signs. (Fort et al . 2007, OCSPP Guideline 890.1100).

This chapter uses data submitted in response to Tier 1 EDSP test orders to explore
whether Test Order Recipients and their contracted laboratories are able to successfully execute
the AMA . Evaluation criteria presented in the test guideline are perhaps the most straightforward benchmarks for comparison. Previously acknowledged limitations and uncertainties of the assay are summarized. These uncertainties are not considered “fatal” flaws
of the AMA ; rather, they underscore the utility of the holistic, battery

- type approach that has been implemented in Tier 1 for evaluating the potential for endocrine interaction. Consi deration is given to observations and recommendations from the previous peer reviews of the AMA, which culminated in the 2008 SAP review of the EDSP Tier 1 battery. Finally, issues identified during the preliminary review of the submitted studies are disc ussed. Again, t hese issues do not suggest critical flaws of the AMA experimental design, but they may indicate that additional, targeted guidance could improve study conduct and interpretation.

25 Nieuwkoop, P.D., and J. Faber. 1956. Normal table of Xenopus laevis. Amsterdam: North Holland Publishing.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 111 of 160 (ii) Strengths and Limitations

The AMA provides direct evidence of the potential for effects of chemical exposure on aquatic

- phase amphibians. More specifically to the purposes of Tier 1 EDSP, it allows inferences to be drawn about potential chemical -induced endocrine interactions which may have consequences for devel opment and general fitness in vertebrate taxa. The developmental and histological endpoints in the AMA may reveal effects which are reflective of perturbations in physiological pathways which are known to be conserved across amphibians and mammals: the
be st understood of these is likely the negative feedback loop through which thyroid hormone (TH) levels are regulated via the controlled release of TSH (see Brown and Cai 2007).

Since the assay is conducted on intact larvae, it includes toxicokinetics and

t oxicodynamics that may impact the potential exposure of a compound in terms of absorption, distribution, metabolism and excretion of the test chemical and the effects of the chemical throughout various levels of biological organization, i.e ., biochemical ( thyroxine ; T 4 ), tissue (thyroid histology), and the whole animal (phenotype, survival). The study is conducted under
flow

- through conditions and measured concentrations are reported to document and ensure exposure over the 21

- day test period. Exposure concentrations typically span a broad range with
a factor of 3

- to

- 10 between test concentrations. Study conditions are optimized to ensure that environmental conditions promote development and do not obscure potential effects from the test
chemical.

The degree of homology among the amphibian and mammalian HPT axes is somewhat uncertain. Many aspects, including specific enzymes, synthetic pathways, regulatory feedback mechanisms, cell types, and transporter proteins are known to be conserved (Fort et al

. 2007, Shi 2000), but some degree of divergence is expected in downstream phenotypic responses to HPT perturbations. For example, goitrogens (iodine uptake inhibitors) are known to be active in both mammals (Brucker

- Davis 1998, Green 1996) and amphibians ( Kaltenback 1996); whereas in mammals, the primary biomarkers of goitrogenic effects are circulating thyroid hormone levels and neurological endpoints, the AMA is potentially more diagnostic of this mechanism in that exposed tadpoles exhibit inhibited de vel opment (OECD No. 77, 2007). Such differences in potential HPT

- related responses between amphibians and mammals are not determined but rather underscore the value of the AMA as a unique screening tool.

The AMA is expected to be sensitive to HPT perturbation s which may not be detected in the mammalian assays with pubertal rats or non

- EDSP guideline studies with maternal exposure
to the test substance. For example, a recent study by Tietge et al . (2013) showed that 2

- mercaptobenzothiazole (MBT) was a potent i nhibitor of thyroid peroxidase ( TPO ) activity in the

AMA

, whereas MBT had not been identified as a thyroid toxicant in mammals in studies conducted by the National Toxicology Program. The reasons for such potential differences inUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 112 of 160 sensitivity across taxa a re varied but may include differences in route of exposure and metabolism which result in different doses in a given tissue. Additionally, the peripheral tissue changes associated with metamorphosis provide a suite of bioindicators in the AMA that allow
f or observation at multiple scales of biological organization, from organismal to histological, and the test substance exposure in the AMA occurs independent of any maternal signal. Therefore,
the AMA is a useful counterpart to the thyroid endpoints in the Tier 1 assays with male and female pubertal rats.

Based on validation studies, the AMA has demonstrated sensitivity to various MOAs. Reference chemicals have included T4 (thyroid agonism; OECD No. 76 and No. 77, 2007), sodium perchlorate (inhibition o f the sodium- iodide symporter (NIS); OECD No. 77, 2007), iopanoic acid (Type I, II, and III deiodinase inhibition; OECD No. 77, 2007), 2

- mercaptobenzothiazole (thyroid peroxidase inhibition; Tietge et al. 2013), propylthiouracil (inhibition of iodine organ ification; OECD No. 76, 2007), and to the putatively weakly anti

thyroid substance benzophenone

- 2 (OECD No. 91, 2008). The native estrogen 17 β

- estradiol (E2) has also been tested in the AMA and was determined not to be a thyroid disruptor; a single exposure at high concentrations relative to background levels resulted in slight developmental delay, based on NF stage, with only a slight reduction in thyroid follicular cell height (OECD No. 91., 2008). Results from these studies are also published in the traditionally peer

- reviewed literature (Degitz et al. 2005, Tietge et al. 2005).

As with any assay, limitations of the AMA were identified through the research and development and the validation of the test design (OECD, 2004; 2007a). These limitations were discussed by the 2008 FIFRA SAP, by prior Federal Advisory Committee Act (FACA ) Committees, and in supporting documents from Agency and independent peer reviews of the

AMA . The conclusions regarding these potential limitations are r eiterated in the test guideline
and in various other documented in publicly available records. The y are highlighted here for transparency and because they are relevant when considering the performance and interpretation
of the AMA. G enerally , the findings include technical performance issues and uncertainties in interpretation due to an incomplete understanding of amphibian physiology and endocrinology; broadly speaking, similar types of caveats are common to most ecotoxicology assays. Importantly, the acknowledged limitations did not detract from the overall SAP conclusion that the AMA was a critical comp onent of the proposed Tier 1 screening battery (USEPA 2008).

Relevance to Other Taxa

Amphibian metamorphosis, as a life stage, has been compared to the postembryonic (pre and postnatal) stage in mammals, when thyroid hormone levels first become elevate d and the organs are rapidly developing (Shi 2009, Tata 1993, Hetzel 1989). Nonetheless, our understanding of the relevance of the amphibian metamorphosis model for understanding mechanisms of HPT disruption in other taxa, including fish and mammals, cont inues to evolve. Genomic and transcriptomic approaches are revealing multiple functions of the thyroid hormoneUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 113 of 160 receptor (TR) in amphibians: Shi (2009) and others have proposed that unliganded and liganded TH, respectively, impact the timing of metamorphos is in X. laevis by recruiting protein complexes which act either as co

- repressors or co

- activators of TH

- dependent genes. It is
unknown whether mamma lian TR function similarly in post

- embryonic development. There is an even greater paucity of information with respect to how peripheral [ i.e. , other than thyroid hormone receptor (TR)

- mediated ) ] mechanisms of effect may compare across taxa. Examples of
such effects include tissue

- level TH regulation, TH transport in the circulatory system, and hepatic metab olism. Certain differences have been identified: with respect to binding of thyroid hormones to transport proteins, T 3 has a higher binding affinity for the transthyretin (TTR) in amphibians, whereas T 4 has the higher TTR affinity in mammals. Thus, test compounds which affect TTR binding may elicit a different response in amphibians than in mammals.

Differences in Sensitivities Based on Life -Phase and Developmental Stage
In amphibians and other tetrapods, the thyroid axis of a developing animal is regulated and responds differently than in the adult

- life phase. The regulatory relationships between and amongst the different juvenile organs and hormone action on tissues var y with the stage of the life cycle. Since the morphological and molecular signatures of thyroid axis perturbation can differ markedly from larval phase to adult, extrapolating effects of a substance on larval phase amphibians to effects of exposure on juvenile and adult life phases is difficult. This is particularly true since for many amphibians, the juvenile and adult phases are adapted to the terrestrial environment as opposed to the aquatic environment. Given the purely aquatic nature
of X. laevis , concerns have been expressed regarding its relevancy to amphibian species that have a distinct terrestrial phase (USEPA 2007 26 ). The amphibian life stage recommended for testing in the AMA was selected to maximize sensitivity to potential HPT interaction an d not necessarily to predict adverse consequences of exposure in adult organisms.

Even within the larval phase, the dynamic regulatory environment of tissue formation, growth, and remodeling (morphogenesis), accompanied by programmed cell death (apoptosis), is a moving target for endocrine stressors. The potential consequences of disruption along this spectrum of development range from subtle to profound and from reversible to fatal. The developmental trajectory of the recommended test species, X. laevis

, is extremely well

characterized; its use in the AMA allows investigators to test specific hypotheses about whether the test substance may behave agonistically or antagonistically vis -a- vis particular HPT

dependent tissues or processes at a specific developmental stage. While a response at one time point or in one tissue may be suggestive or even diagnostic of an endocrine interaction, it is not necessarily informative of whether exposure at a different developmental or life stage would
result in the same type of interaction ( e.g. , apparent TH agonism) or a similar functional response ( e.g. , advanced development).

26 USEPA. 2007, Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting Held October 9
– 11, 2007 on the Potential for Atraz ine to Affect Amphibian Gonadal Development. http://www.epa.gov/scipoly/sap/meetings/2007/october/finalminutes.pdfUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 114 of 160 Logistical Considerations for Aquatic Exposure Methods
The suitability of the aqueous exposure method recommended in the AMA, as with other aquatic toxicity test methods, may be limited by the physico

- chemical characteristics of the test substance. Aquatic testing of substances with low solubility, high octanol:water partition coefficients (K ow ), high volatility, rapid hydrolysis, and photoly sis under ambient laboratory conditions is challenging and sometimes uninformative. Methods for delivering such materials have evolved and are discussed in the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000) and OCSPP Guideline 850.1000 (USEPA
1996 27 ). The AMA test guideline directs the investigator to this OECD (2000) guidance document, but the concepts therein were not directly incorporated into validation work with the AMA. Delivery of test substances via food for difficult test materials has been proposed by OECD, and may be considered an appropriate alternative approach in the future if adequately
demonstrated.

Non -Thyroidal Toxicity

Non

- thyroidal toxicity of test materials has the potential to affect some of the endpoints included in the AMA. For example, changes in body weight and organism length can be altered by processes independent of the thyroid axis. The use of multiple endpoints is intended to provide a WoE to identify cases where HPT

- mediated effects may be occurring while accounting for other forms of toxicity ( e.g. , mortality, malformation, other clinical signs).

(iii) Observations from Initial Screening of Case Study Chemicals

Results from 18 industry- submitte d studies with chemicals previously untested using the AMA method and with unknown potential for endocrine interaction are reviewed and discussed within the context of experimental performance, consistency of approach, and interpretation of
results. For t he purposes of this review, the chemicals were selected to be illustrative, but not necessarily all

- encompassing, of a range of pesticide product types, physico

- chemical characteristics, technical performance issues, and study outcomes. Given the prelimin ary nature of these reviews, actual chemical names are omitted.

Quality Control

The AMA test guideline presents a series of validity elements ( Table 29 ) based upon the recommended experimental design. These validity elements provide an indication of the g eneral performance of the AMA under test conditions, e.g. , control mortality that exceeds the reference value may be an indication of underlying problems associated with husbandry, environmental conditions, or the particular strain of test organisms used in the study. Alternatively, high

27 USEPA. 1996. Ecological Effects Test Guidelines OPPT S 850.1000 Special Considerations for Conducting Aquatic Laboratory Studies. Office of Chemical Safety and Pollution Prevention formerly the Office of Prevention, Pesticides and Tox ic Substances (7101). EPA 7120C096

- 113. April 1996. http://www.epa.gov/opptsmnt/pubs/frs/publications/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Drafts/850

- 1000.pdfUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 115 of 160 treatment mortality may limit or preclude the ability to detect potential endocrine

- mediated effects in the remaining dataset. When met, the validity elements are used to support the scientific soundness of a given study and the degree of confidence in its results.

Similarly, performance criteria ( Tables 30 and 31 ) are identified in the AMA test guideline to promote consistent and biologically appropriate environmental conditions and to support the interpretation of result s. Performance criteria are considered when evaluating each study but may be especially useful when deviations from the guideline are reported, as satisfied criteria may demonstrate that the deviations had a minor impact on study outcome. That said, a

st udy that did not meet one or more performance criteria may still provide useful information and is not necessarily rejected. In some cases, performance criteria overlap with validity elements; for example, a threshold for control mortality is presented in both categories in the AMA test guideline.

Table 29 : Validity Elements for the Amphibian Metamorphosis Assay.

Element Reference Value(s) Mortality (each treatment, including controls) < 10%
Mortality (each replicate) If positive for thyroid interaction If negative for thyroid interaction < 2 tadpoles/replicate (< 10%)

< 3 tadpoles/replicate (< 15%) Uncompromised treatment levels (all 4 replicates available for analysis) > 2, in addition to controls

Treatment levels without overt toxicity (mortality) > 2, in addition to controls

Table 30 : General Performance Criteria for the Amphibian Metamorphosis Assay, when Conducted Under Flow -Through or Static- Renewal Conditions

Criteria Reference Value(s)

Measured test concentrations CV < 20% over 21 days Control mortality < 2 tadpoles/each replicate ( < 10%) Median NF stage in controls at test termination > 57

Difference between 10 th and 90 th percentile of NF stage distribution in controls at test
termination < 4 stages

Dissolved oxygen > 40% air saturation

pH 6.5-8.5 ( < 0.5 difference between replicates, treatments)

Water temperature 22±1°C ( < 0.5°C difference between replicates, treatments)USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 116 of 160 Table 31 : Additional Performance Criteria for the Amphibian Metamorphosis Assay, when Conducted Under Static

- Renewal Conditions

Criteria Guideline Recommendation(s)

Measured test concentrations Analysis immediately prior to and immediately following renewal General water quality parameters, including
ammonia Analysis immediately prior to renewal

Renewal period < 72 hours ( < 24 hours preferred)

Feeding schedule Adjusted as necessary ( e.g. , 50%) based on
renewal scheme

When a study is reviewed by the Agency, the Data Evaluation Record (DER) notes whether each validity element and performance criterion was met. The DER includes a discussion of any rationale provided in the study report for any deviations or deficiencies. This includes identification of whether, to what extent, and in what way an inability to meet the criteria has an impact on the quality or utility of the study. Figure 1 8 illustrates the proportion of preliminarily reviewed AMA studies which met each of the major validity elements and performance criteria outlined in the test guideline. No studies using a static

- renewal exposure method were submitted; therefore, those cri teria are not evaluated.

Figure 18. The majority of AMA study submissions reviewed to date have met the validity elements and performance criteria outlined in the test guideline.

0 3 6 9 12 15 18 Control and treatment mortality ≤ 10% Replicate mortality ≤ 10% Replicate mortality ≤ 15% Uncompromised treatments ≥ 2 Treatments without overt toxicity ≥ 2 Test concentration CV ≤ 20% over 21 days

Median Day 21 NF stage in controls ≥ 57 Control Day 21 NF stage (10th

- 90th

... Dissolved oxygen ≥ 40% pH 6.5

- 8.5 (≤ 0.5 difference) Water temperature 22 1 C (≤0.5 C

...

Met

Not Met
Not reported

# StudiesUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 117 of 160 Consistency in exposure levels, demonstrated by a test concentration CV < 20%, has been the most difficult performance criteria for laboratories to meet; only 11 of 18 (61%)
submissions satisfied this criterion across the 21

- day exposure period. It is unc ertain to what extent the sample of existing study submissions is biased toward higher performing studies since time extensions have been granted in some cases due to technical challenges. “Not reported” indicates that the study report did not directly address the validity element or performance criterion or did not provide enough information ( e.g., individual data points) for the Agency
reviewer to determine if it was met.

Uncertain Association b etween Alternative Feeding Approaches, Developmental Progression, and Spinal Curvature in X. laevis

The AMA test guideline recommends the use of Sera Micron  feed for tadpoles during
the in

- life portion of the test. A specific feeding regime is recommended, with the quantity of feed increasing as tadpoles develop. The guideline recommendations to use Sera Micron  and at these particular rates are based upon experience gained during the development and validation of the AMA, and because the iodide content in this product has been documented. Other diets and feeding regimes may be equally or more appropriate for X. laevis husbandry within the context of the AMA, but publicly available documentation of the performance of such alternatives is
minimal. Theref ore, the Agency recommends that test submitters provide information supporting the performance of the feeding regime, if the feed or amount and frequency of feeding differ from the guideline recommendations. In addition to general nutritional information, analyses of iodide content and for potential contaminants are particularly important.

In the initial data submissions, some labs have reported challenges regarding tadpole development in controls that advances beyond the recommended stage at test terminat ion by Day 21 in the AMA when using the guideline recommended feeding regime. For example, the guideline recommends that the minimum median stage in controls at test termination be NF Stage 57, when X. laevis tadpoles are undergoing toe differentiation an d hind limb growth during prometamorphosis, and endogenous TH levels are rising (Shi 2000, Nieuwkoop and Faber 1956). Tadpoles which have reached NF Stage 61 or beyond are routinely excluded from analyses other than the analysis of NF stage; these tadpoles have entered metamorphic climax and TH levels have peaked and drastic tissue remodeling is occurring (Shi 2000, Nieuwkoop and Faber 1956). Although a precise threshold has not been calculated, the test guideline concludes that the development of more than 20% of control tadpoles beyond this stage may compromise the ability
to detect treatment

- related effects in the study.

The cause of the more rapid control development observed in some AMA study submissions, as compared to the validation studies, is not well understood. Similarly, there appears to be high variance in the incidence of spinal curvature observed in X. laevis tadpoles across studies and laboratories ( Table 32 ). It is uncertain whether these phenomena are independent of one another or if, and to what extent, they are both dependent upon the sameUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 118 of 160 causal factors. Such factors may include differences in the strain or even individual parentage of test specimens, environmental conditions such as temperature and photoperiod, iodide availability in the water and feed, nutritional variables other than iodide (Crawshaw 2003, Whitaker 2001) , or a combination of these elements. Experimental work by the USEPA Office
of Researc h and Development demonstrated that excessive feed can be a particularly strong factor in both outcomes ( Olmstead et al ., unpublished manuscript ). Generally, the Agency
recommends examin ing whether any patterns of spinal curvature are consistent between
treatments and controls or whether there is evidence of a potential treatment -related induction of spinal curvature (USEPA 2011b)

.

As shown in Table 32 , the initial data submissions reflect that some labs are exploring the use of different feeding regimes i n an attempt to synchronize the developmental stage of control tadpoles with that which is expected for the 21

- day timeframe of the guideline AMA. Specifically, certain studies are being conducted with feeding rates that are approximately 50%
of the rates recommended in the test guideline. While this appears to slow the developmental rate of control tadpoles in these studies so that guideline performance criteria for developmental stage are met, without elevating mortality, it is unclear whether such a dr astic reduction may have other adverse effects on the health or endocrine status of the test specimens which may limit the sensitivity of the AMA.

Based on the initial data submissions, musculo

- skeletal deformities ( e.g. , spinal curvature or “bent tail, ” a s it is typically confined to the tail region ) have been reported in fifteen of the eighteen studies reviewed by the Agency, but the incidence varies widely across studies. The remaining three studies did not indicate whether spinal curvature was observed

. The deformities have been reported both in studies which observe the guideline

- recommended feeding regime
and in studies which deviate from the guideline by reducing the amount of feed by approximately one

- half. Therefore, a hypothesis of excessive feed as the primary factor in the development of spinal curvature does not appear to be supported in these cases. However, t hese observations are anecdotal; the Agency is unaware of any intra- lab comparisons from private laboratory facilities regarding fe eding rates and musculo

- skeletal deformities within a single population of X. laevis . There is no clear pattern in the metadata for the initial data submissions that reveals a single, predictive factor for the prevalence of spinal curvature ( Table 32 ).

The submitted studies vary with respect to the amount of detail provided regarding observations of spinal curvature. In ten cases, observations were reported as the number of surviving tadpoles with spinal curvature at test termination. Three studies rep orted the number observed and the first onset of noticeable spinal curvature, e.g. , beginning day 8. Three studies reported only an approximation (“a few”) or a range (“one

- to

- five per replicate”). In one study (Chemical U ) that reported the number of observations at test termination, investigators assigned a ranked severity score (0, 1, 2, or 3) for spinal curvature to each test specimen at test termination and provided these individual

- level data as an appendix to the study report. Absence of spinal curvature was graded as 0, and 3 represented the most severe form of spinal curvatureUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 119 of 160 observed. The criteria used to assign the numerical grades were not further described. The investigators then reported incidence of spinal curvature of severity grade 2 and 3, together, on a percentage basis per treatment level. Using this approach, the incidence of spinal curvature (falling within either grade 2 or 3) was 40%, 27%, 21%, and 7% in the negative control and low, medium, and high treatment levels, respective ly. When grade 1 observations are included with grades 2 and 3, the incidence rises to 74%, 52%, 59%, and 10% in the negative control and low, medium, and high treatment levels, respectively.

In this example, Sera Micron  feed was supplied during the exposure period at rates approximately half of the guideline recommended rate; no additional iodide supplementation was provided. The report contained insufficient information to determine whether test specimens were selected from the “best single spawn.” Th ere was no correlation between NF stage at test termination and incidence of spinal curvature. With the exception of tadpole mortality in one replicate, all validity elements and performance criteria were met. Similar and apparently
inverse concentration

- related trends in the occurrence of spinal curvature were observed in some other studies; h owever, the significance and reproducibility of these findings is unclear.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 120 of 160 Table 32 : Prevalence of Spinal Curvature in the Amphibian Metamorphosis Assay.

Lab Test Chemical Animals Feeding Add’l Sources
of Iodide Prevalence of Spinal Curvature (%)

ID ID Product Type Suppplier h

Location Product Reduced Iodide (ug/g) Water i
(ug/L) Supplem

. Negative
Control Solvent
Control Trtmt.
1 Trtmt.
2 Trtmt.
3

1 0 A Insecticide Massachusetts Xenopus Express ® No . a 7 No 12 NA 17 29 12

Q Fungicide Massachusetts Xenopus Express ® No . a 7.4 No 41 NA 30 37 20

K Insecticide Wisconsin Xenopus Express ® Yes . a 7.8 No 41 NA 49 46 30

G b Acaricide Wisconsin Xenopus Express ® No . a 7 No

. NA

. . .

9 V c Insecticide

. Sera Micron ® No

. 13 No

. NA

. . . S d Acaricide

. Sera Micron ® No

. 13 No

. . . . . 3 M Insecticide Wisconsin Nasco Frog
Brittle ® Yes 48 e 1.2 No 29 29 21 16 26

6 C Herbicide

. Sera Micron ® Yes

. 4.5 No 43 53 39 40 36

U Insecticide

. Sera Micron ® Yes

. 4.5 No 74 NA 52 59 10

P Herbicide Michigan Sera Micron ® No

. 4.5 No 66 NA 85 98 48

B Herbicide Michigan Sera Micron ® Yes

. 4.5 No 87 74 92 87 60

5 H Herbicide

. f Sera Micron ® No 40 3.6 Yes 22 28 23 23 13

2 O d Herbicide Florida Sera Micron ® No 54 <50 No

. NA

. . . E d Herbicide Florida Sera Micron ® No 40 <10 No

. . . . . I g Insecticide Florida Sera Micron ® No 40 <10 No

. . . . .

Color Key : Prevalence 0 to 25% 26 to 50% 50 to 100%

a Iodide content was not addressed in the study report. The Agency has requested information from the manufacturer of Xenopus Express ® Tadpole Diet and is awaiting a

response. b The study report described “several” tadpoles exhibiting spinal curvature beginning on Day 10 in controls and the lowest trea tment, Day 15 in the intermediate treatment, and Day 19 in the highest treatment level.

c The incidence of spinal curvature was reported as one to five tadpoles per replicate between Day 8 and test termination.

d The study report did not provide information on observations of spinal curvature.

e Reported on manufacturer’s website as 48 mg iodine/kg. http://www.enasco.com/page/xen_brittle

f X. laevis strain: Daudin. Supplier not reported. No in

- house parental stock; embryos were purchased for the selection of test specimens.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 121 of 160 Lab Test Chemical Animals Feeding Add’l Sources
of Iodide Prevalence of Spinal Curvature (%)

ID ID Product Type Suppplier h

Location Product Reduced Iodide (ug/g) Water i
(ug/L) Supplem

. Negative
Control Solvent
Control Trtmt.
1 Trtmt.
2 Trtmt.
3

N Inert

. Sera Micron ® Yes 54 <50

. 17 NA 20 29 17

J Funcigide Michigan
Florida Sera Micron ® No 40 7 No 27 NA 8 11 19

F Herbicide Florida Sera Micron ® No 50 <50 No 2 5 2 13 2

g Spinal curvature was observed in a “few” tadpoles but incidence values were not reported.

h Original source of parental stock.

i Background iodide in laboratory water.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 122 of 160 Interpreting Growth Effects within an Endocrine Context

Morphogenesis (development) and somatic growth in amphibians are mediated through only partially overlapping hormonal pathways (Denver 1997); although both events are directed through the hypothalamic

- pituitary axis, the cascades of chemical messengers, their proximate ligands, and the phenotypic responses can differ markedly. Moreover, there is some indication that morphogenesis, and specifically amphibian metamorphosis, may be altered (either promoted or inhibited) based on body condition, effects on growth at critical phases of development, and ecological factors such as intr aspecific competition and hydroperiod ( e.g. , Wissinger et al. 2010,
Downie et al. 2004). Various explanations for these sometimes contradictory phenomena have been postulated and tested across scales of biological organization, but our understanding of the interplay between the molecular and, specifically, the hormonal pathways of growth and development remain incomplete.

Based on preliminary analysis, 12 of the 18 studies demonstrated some effect on growth endpoints, such as body weight or SVL, in at least one treatment level. In 10 of these studies, the effects on growth accompanied effects on at least one developmental endpoint (NF stage, normalized HLL) or thyroid (histo)pathology, although these effects were not always observed at the same
treatment l evel, and the direction of the effect ( i.e ., increasing or decreasing) was occasionally different. In two cases, a promotion of SVL was observed at one treatment level, but an inhibition of
SVL was observed at another treatment level. Both of these studi es also showed evidence of a solvent effect based on comparison (Student’s t -test) of the solvent control to the negative control. As an example, results from one of these studies, Chemical F , are shown in Tables 33 and 34 below (solvent: DMF, at a concentration of up to 0.1 mL/L). The study report did not state whether proportional or serial diluters were used; therefore, it is unknown whether all treatments used equal
concentrations of solvent.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 123 of 160 Table 33 : Developmental and Thyroid Gross Pathology/Histopathology Endpoints 35 , 36 in the AMA with Chemical F.

Table 34 : Growth Endpoints a,b in the AMA with Chemical F.

Color Key Promotion Inhibition

Abbreviations: Diff. Difference. NA Not applicable.

35 Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board

- certified pathologist.

36 Unless otherwise specified, effects are considered statistically significant at p < 0.05.

37 H ind

- limb length is normalized to snout

- vent length (SVL).

38 p values for solvent control represent the p value from a two

- sided t

- test assuming unequal variance. Treatment (mg a.i./L)

[TWA

measured] NF Developmental Stage Hind Limb Length 37 Thyroid Gross

and Histopathology

Day 7 Day 21 Day 7 Day 21 Day 21

Median p Median p %
Diff. p 38 %
Diff. p d Treatment

Related Effects? (Yes/No)

Negative
Control 54

-- 58

-- N/A

-- N/A

-- --

Solvent Control 54

-- 59

-- 0.80 0.84 16.1 0.05 No

0.003 54 >0.99 59 0.10 3.05 0.77 25.4 0.01 No

0.03 54 > 0.99 59 0.10

- 0.76 0.99 17.9 0.04 No

0.23 54 0.48 57 0.10

- 1.53 0.96

- 19.6 0.02 No
Statistical Test Mann

- Whitney Mann

- Whitney Dunnett’s Dunnett’s N/A

Treatment (mg a.i../L) [TWA

- measured] Snout

- Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

% Diff. p d % Diff. p d % Diff. p d %
Diff. p d

Negative Control 0 N/A 0 N/A N/A

-- N/A

--

Solvent Control 6.90 0.04 1.00 0.35 18.6 0.08 10.0 0.05

0.003 5.63 0.22 1.80 0.24 14.8 0.31 12.6 0.18

0.03 8.79 0.04

- 0.17 >0.99 23.3 0.07

- 5.23 0.77

0.23

- 16.9 <0.01

- 5.76 0.08

- 38.5 <0.01 14.4 0.11

Statistical method Dunnett’s Mann

- Whitney Dunnett’s Dunnett’sUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 124 of 160 In the case of Chemical F , the presence of a solvent effect similar in direction and magnitude to effects in the lower treatment levels, without any corresponding effect on thyroid (histo)pathology, suggests that the effects observed are driven by the solvent and not the test substance. Conversely, the statistically significant reductions in Day 21 normalized HLL, Day 7 SVL, and Day 7 body weight at the highest treatment level (p<0.05), without any corresponding effects on the thyroid gland, are consistent with generalized toxicity of the test substance. Ov erall, the growth effects observed in this study suggest that the range of concentrations tested was adequate to challenge the organism, but the presence of solvent effects makes it difficult to interpret the findings.

Apparent Absence of Effect with Difficult

- to

- Test Substances

Of the 18 studies in this preliminary analysis, seven did not meet the performance criterion for maintaining consistent measured test concentrations over the entire exposure period, as indicated by the percent coefficient of va riability (%CV) ( i.e. , the %CV was > 20%). Across studies, the CVs tend to vary either directly or inversely with increasing test concentration. For example, in cases where the variability is driven by difficulties testing or quantifying very low concent rations of a test substance, the CVs are greater at lower treatment levels. Conversely, where the variability is driven more by difficulties testing near the limit of solubility of the test substance, the CVs tend to increase with increasing test concentr ation. Measured test concentrations are used to place the biological results of the study in context of aquatic exposure to the test substance. When recoveries are
variable but treatment

- related effects are observed within a given study, there may still be enough information within that study to determine whether exposure has the potential to elicit an effect. However, when test substance recoveries are poor and no effects are observed within the study, it may be questionable whether the assay was succes sfully performed. A different exposure method may be better suited for highly unstable compounds. Two examples follow from the initial screening of pesticides with the AMA.

One study (Chemical M ) reported an error in stock solution preparation that incre ased the variability in test substance recovery: the initial stock solution concentration was inadvertently doubled prior to study initiation. As a result, percent recovery in individual samples at Day 0 ranged
from 79% of the intended nominal concentrati on in a low treatment replicate to 161% in an intermediate treatment replicate. By Day 7, recovery ranged from 29% in a high treatment replicate to 44% in a low treatment replicate. Recovery at Day 14 was similar, ranging from 31% in a high
treatment rep licate to 57% in a low treatment replicate. At test termination on Day 21, recovery was only 20% in the high treatment and up to 37% in the low treatment. (Recovery percentages in this example are all based on comparison to the intended nominal concentra tions.) Given the low and declining recoveries throughout the exposure period, it is unlikely that the study would have met the performance criterion for %CV even if the stock solution had been correctly prepared at test initiation. The biological result s of this study included the advancement of median NF stage by one stage in the intermediate and high treatment levels; an initial increase and subsequent decrease of SVL at the intermediate and high treatment levels, respectively; and the increased body w eight inUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 125 of 160 both the intermediate and high treatment levels, all based on comparison to the negative control. The biological results are difficult to interpret within the context of actual exposure to the test substance, which is known to be a low solubility, highly adsorptive compound. The use of a solvent (DMF, up to 0.1 mL/L) and the presence of effects on NF stage and growth parameters in solvent controls further complicates interpretation.

In the study with Chemical S , mean %CVs for test substance concentrations were 69% in the lowest treatment level, 33% in the intermediate treatment level, and 49% in the high treatment level. Test substance recovery was only 9% of nominal in the high treatment level at test termination. Other than %CV, all valid ity elements and performance criteria were met. No biological effects
were observed. The treatment concentrations were based on three 24

- to -96 hour range-finding
studies with X. laevis tadpoles (NF stage not reported). However, given the high %CVs and p oor recovery in the definitive test, it is questionable whether the investigators achieved and maintained a high enough exposure level to elicit a potential response in the 21

- day AMA. A dietary exposure approach, which is mentioned in the test guideline as a potential alternative test method, may have
been more useful in this case.

Interpreting Amphibian Thyroid Histopathology Diagnoses

Thyroid gross pathology and histopathology observations are critical components of the AMA, particularly in cases when other biological effects are absent or difficult to interpret. To assist investigators in consistently preparing, analyzing, and reporting an assessment of thyroid histopathology diagnoses, the Agency served as a lead in the preparation of a Guidance Document on Thyroid Histopathology (OECD No. 82, 2007), which was informed by international expertise in pathology and experience gained during the validation of the AMA (Grim et al. 2009). The guidance document includes micrographs of “normal” X. laevis thyr oid glands and recommends a four- point (0, 1, 2, or 3) severity grading scale for assessment, with 0 representing findings that are “not remarkable,” and 3 generally representing findings that are “severe.”

With the exception of gross pathology findings (thyroid gland atrophy and hypertrophy), severity grades are generally assigned based on comparison to “normal” thyroid findings depicted in the guidance or based on the professional opinion of the pathologist conducting the assessment, and not in comparison to control findings from a given study. The severity grades are to be assigned based on a reading of at least two

- to

- three slides per specimen. Pathologists are expected to have experience or have received training in amphibian and specifically X. laevi s thyroid histopathology; otherwise, the guidance document suggests additional oversight or peer review of the findings. The study reports reviewed thus far by the Agency indicate that histopathology analyses were performed by a certified histopathologist, but the level of individual experience with amphibian histopathology is generally not described. Finally, consistent with practice in the discipline, the guidance document recommends that samples be evaluated initially in a non

- blinded manner, with poss ible confirmation of results by a subsequent, blinded reading (Crissman 2004). The degree of compliance with thisUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 126 of 160 recommendation varies based on the AMA histopathology reports submitted to date; when a non

blinded reading has been used, a subsequent blinded reading is rarely reported.

The AMA test guideline recommends that five of the fifteen tadpoles remaining per replicate (assuming no mortality) at test termination (Day 21) be selected for thyroid histopathology. To the extent possible, the guideline states that these specimens should be stage

- matched to the median NF stage in controls. If precise stage

- matching is not possible because of an apparent treatment

- related effect on NF stage development, the investigator is encouraged select specimens as c losely matched as possible to the median NF stage in controls, but consistent with the directionality of any apparent

treatment

- related effect. For example, if there are less than fives tadpoles in a treatment replicate
which are matched to the median NF stage in controls, and the data suggest a possible treatment

related developmental delay, then the investigators would select tadpoles of the next lower NF stage when compared to controls. The Agency considers stage

- matching of histopathology specimens to be important to the interpretation of histopathology findings because tadpoles undergoing metamorphosis ( i.e. , at different NF stages) are expected to have different histological presentations (Grim et al. 2009). In the absence of significant effects on developmental biomarkers (NF stage or normalized HLL), the inability to stage

- match thyroid specimens may reduce the likelihood of detecting a treatment

- related lesion caused by a less potent thyroid inhibitor

. Thyroid histopathology was performed in all e ighteen of the studies reviewed here. Seven of these eighteen studies reported that tadpoles selected for histopathology were stage

- matched to controls. Two studies reported that specimens were not stage

- matched but instead were impartially
selected. Th e remaining nine study reports did not provide specific information on specimen selection, although some inferences may be made by comparing information in the pathology report to the raw data from the study, when available.

Other Guideline Recommendations

Use of the Best Single Spawn

With respect to embryo selection for the AMA, the test guideline recommends that two

- to

three of the best individual spawns from in -house adults be retained to evaluate the quality of the spawns. From these two

- to

- three spawns, it is recommended that the test organisms originate from the best single spawn, based upon embryo viability/appearance and the presence of an adequate number (>1,500) of embryos. The guideline recommendation to concurrently evaluate multiple spawns was intended to increase the likelihood of identifying a single spawn with an adequate number of viable embryos in a time- efficient manner ( i.e. , instead of evaluating spawns serially) (pers. comm., J. Tietge, March 2013). The guideline recommends against co

- mixing spawns because this has been demonstrated to increase variability and decrease the statistical power of the test (OECD 2007).USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 127 of 160 In its review of the data, the Agency considers and documents in the DER how many spawns
were evaluated. The DER also incl udes details of the evaluation method ( e.g. , timing of evaluation and number of eggs evaluated per sample) and whether the organisms selected for the definitive test originated from the best single spawn, when this information is provided in the study report. Of the 18 studies in the initial analysis, only seven reported using the best single spawn consistent with test guideline recommendations. Seven studies reported using another method, either co

- mixing embryos from different spawns based on NF sta ge or using embryos from a single (but not necessarily the best of two

- to

- three) spawn. Four study reports did not contain enough information to determine the method of embryo selection.

Optional Size Selection Method

The exposure is initiated when a sufficient number of tadpoles in the pre

- exposure stock population, which are less than or equal to 17 days of age post

- fertilization, have reached NF stage 51. The test guideline includes additional guidance on optional size selection, based on total length (not snout- vent length), that complements the stage selection. The optional addition of size selection further reduces variability by ensuring that the NF stage 51 tadpoles fall within a specified size range. None of the 18 studies reviewed thus far have implemented the optional size selection
method.

Iodide Measurement and Supplementation

Sufficient iodine intake, generally in the form of iodide, is necessary for normal functioning of the HPT axis because iodine serves as a substrate for the synthesis of thyroid hormone (Shi 2000). Iodination of thyroglobulin produces T 4 , and partial deiodination of T 4 produces the more biologically active T 3 (Dodd and Dodd 1976). The AMA was successfully performed in validation studies when test water iodide (I - ) conce ntrations ranged between 0.5 and 10μg/L. This is in addition to dietary iodide exposure at levels present in the guideline recommended feeding regime using Sera Micron  . Therefore, the test guideline recommends that the iodide concentration in test

water be determined. Iodide supplementation is recommended if the concentration is naturally below 0.5 μg/L in the test water or if test water is reconstituted from deionized water. It is recommended that iodide supplementation not exceed 2 μg/L. The Agency considers both dietary and test water sources of iodide that are reported for a given study when reviewing the data. For the eighteen studies reviewed thus far, these values are summarized previously in Table 32 . Iodide supplementation was reported in on ly one study (Chemical H).

(iv) Statistical Approach and Considerations

The Agency routinely verifies the statistical conclusions of industry data submissions in response to AMA test orders. Agency reviewers typically re -analyze individual- level data using

sta tistical methods recommended in the AMA test guideline, as appropriate. Some differences between the AMA test guideline recommendations for statistical analyses and current Agency practice have emerged. Numerous reasons exist for these deviations, such a s differences inUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 128 of 160 analytical software capability, characteristics of a given dataset, historical practice, uncertainty regarding the ability of a particular statistical test to detect an effect given the guideline
recommended

- experimental design, and occasionally perceived ambiguity in the guideline
recommendations.

Exclusion of Late Stage (>NF Stage 60) Tadpoles

Sixteen of the eighteen AMA studies which have undergone preliminary review reported that some proportion of tadpoles had moved beyond NF stage 60 by test termination (Day 21). At this stage of development, X. laevis tadpoles are shifting from prometamorphosis to metamorphic climax; the regression of tissues ( e.g. , tail resorption) and overall changes in size and shape during metamorphic climax are reflected in changing morphometric ratios. For this reason, the test guideline recommends that measurements from individual tadpoles which have advanced beyond NF stage 60 (termed “late stage”) be censored from the analysis of continuous endpoints ( i.e. , SVL, HLL, body weight). When more than 20% of tadpoles are classified as “late stage” at test termination, t he Agency currently recommends conducting two analyses for each continuous endpoint: one on the “non

- late stage” tadpoles and a second analysis on the “late stage” tadpoles. Note that in this case the guideline recommends a two

- factor analysis of variance (ANOVA) with a nested variance structure be undertaken on all tadpoles to assess growth effects due to chemical treatments while taking into account the effect of late stage development on growth. However, the Agency’s attempt at this approach was unsucc essful due to the greater variance between individuals than between replicates. Four of the eighteen studies reviewed reported “late stage” in more than 20% of tadpoles in at least one treatment group. All data points are considered together when analyzi ng non- continuous endpoints ( i.e. , NF stage).

Normalization of Morphometric Measurements

The test guideline recommends that each individual hind limb length (HLL) measurement be normalized to the snout-vent length (SVL) of the same individual ( i.e. , HLL:SVL) prior to analysis
of the dataset for an HPT

- mediated effect. The resulting, calculated value is referred to simply as “normalized hind limb length.” The normalization is intended to control for effects on growth which are analyzed separately and may be due to non- specific toxicity. Before evaluating the statistical conclusions of a given AMA study submission, the Agency confirms whether the HLL data are normalized consistent with guideline recommendations.

In some cases, the investigators are ap plying a different transformation to HLL data. Rather than reporting and analyzing normalized HLL as the ratio of HLL

- to

- SVL, some investigators are calculating the ratios and then multiplying the original HLL values by these ratios prior to statistical

a nalysis ( i.e. , (HLL/SVL)*HLL) . While there is nothing inherently flawed in the alternate approach, for consistency, the Agency currently abides by the test guideline recommendations and uses only the HLL:SVL ratios when analyzing normalized HLL.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 129 of 160 (viii) Overall Conclusions for the AMA

The preliminary analysis of 18 AMA studies submitted by industry test order recipients suggests that laboratory performance of the AMA generally meets expectations . Previous peer review conclusions during the development and valida tion of the AMA test design are reinforced by the initial data submissions. Briefly, these conclusions were that (1) the AMA provides useful, complementary, and sometimes unique information about potential perturbations of the HPT axis in amphibians, (2) conclusions regarding the potential for a test substance to interact with the HPT axis in amphibians and, more broadly, in vertebrate taxa need to be based on a holistic examination of all endpoints in the AMA and within the context of other available data ( i.e. , the Tier 1 EDSP battery), (3) in some cases, it may be difficult to discern HPT

- specific effects in the AMA from other types of toxicity, e.g. , when growth is inhibited or with only weakly active compounds, and (4) certain test substances may not b e amenable to the 21

- day aquatic exposure design of the AMA. The previously acknowledged limitations and uncertainties do not suggest critical flaws of the AMA experimental design as presented in the test guideline, but they may indicate that additional, targeted guidance could improve study conduct and interpretation.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 130 of 160 

### IV. Performance of Battery

In 1998, when developing the Tier 1 battery, the EDSTAC considered screening endpoints for their utility in screening chemicals substances or mixtures for their potential to interact with the endocrine system. The goal of Tier 1 assays is to detect chemical substances or mixtures capable of have the “potential” of interacting with estrogen, androgen, or thyroid hormone systems. Assess ing these activities is impor tant as changes may adversely affect the development, reproductive function,
or chronic health status of humans or wildlife. The ob jective of the Tier 1 assays is not to determine
dose- response relationships, confirm the mechanisms of action, or determine the adverse outcome(s). The screening assays , however, must be sensitive enough to detect all known xenobiotics that act via the mechanism of action each assay is designed to detect. The Tier 1 screening battery as a whole should maximize sensitivity to minimize false negatives , while recognizing some acceptable level of false positives. Based on EDSTAC conclusions in 1998, when the Tier 1 screening battery is validated, it should include a combination of in vitro and in vivo studies that are not weight ed equally, but when combined together, should detect all known modes of action for the endocrine endpoints of concern, include sufficient range of taxonomic groups among the test organisms, incorporate sufficient diversity among endpoints and assays and overall, it should minimize miss ing potential endocrine active chemicals. (EDSTAC, 1998)

A decade after the publication of the EDSTAC report, the SAP in 2008 reviewed the Tier 1 proposed battery of 11 assays and concluded that “ the proposed set of Tier 1 assays are appropriate to begin screening for disruptors of the EAT axes .” The 2008 SAP panel also 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 .” and should “ provide safeguards against false positive or false negatives .” The 2008 SAP did recommend that the agency “ will continue to develop, refine, and review the battery .”

Introduction

This c hapter describes the performance of the Tier 1 Battery using data from a subset of chemicals issued Test Orders (List 1) beginning in October 2009. This analysis used the 2008 FIFRA SAP report 39 as a guide, borrowing the concepts of “complementarity” and “redund ancy” as defined by the SAP in 2008; terms such as “complementarity” describes the concordance of endpoints within an assay and “redundancy” describes the concordance of endpoints across the assays that are intended to evaluate an interaction with in a particular pathway, E, A, or T. Complementarity and redundancy will be evaluated in the context of different “modalities” that span multiple levels of biological organization, including molecular interactions, and cellular, organ and

39 March 25

- 28, 2008 Session of the FIFRA Scientific Advisory Panel entitled “Review of the Endocrine Disruptor Screening Program (EDSP) Proposed Tier 1 Screening Battery”.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 131 of 160 organism responses for each hormonal class ( i.e ., E, A, and T hormonal systems) that the battery was expected to assess. The 2008 SAP defined these different biological levels of organization as consisting of “receptor/target binding, cell responses, enzyme activity, organ response, males and female system i ntegrity, development (thyroid) and comparative system integrity”. Thus, the purpose of this preliminary battery review is to: 1) e valuate the complementary relationships among responses within the assays in the Tier 1 battery, and 2) e valuate the redundancy of responses across the Tier 1 battery

. Subsequently, this review will also consider whether a different combination o f assays, reduced in number, can adequately ensure the identification of potential interactions with the E, A, or T hormonal p athways.

For this systematic preliminary assessment, a subset of List 1 chemicals with submitted Tier 1 data were selected as a representative group of chemicals to provide a broad range of responses across 11 Tier 1 assays. This subset of Tier 1 battery results allowed the agency to assess the performance of the battery in terms of the complementary relationships within an assay (namely the
multi

- parameter in vivo assays) and redundancies across a ssays. The battery performance analysis is
also separated i nto the three hormone classes ( i.e., E, A, or T) to evaluate the extent of complementary relationships within assays and redundancy across assays for each of the hormonal pathways using the subset of List 1 chemicals whose identity has been coded.

Indivi dual Tier 1 Assay Assumption: Observations of Overt Toxicity

The review of the battery performance is based on the assumption that individual assays are performing adequately. As noted in Section III, overt toxicity ( e.g ., mortality, reduction in body weight, behavior) was reported in some assays. Effects on endocrine- related endpoints observed in the Hershberger, male and female pubertal rat assays, and FSTRA were in some cases coincident with signs of overt systemic toxicity at some doses; however, at this time such effects were not
excluded from the battery performance analysis. When positive Tier 1 findings are observed in the presence of overt toxicity, the agency is aware that there is uncertainty as to whether the responses are mediated through a n endocrine pathway. The agency, however, will evaluate the degree of confidence for endocrine responses that co

- occur with systemic toxicity in subsequent weight of evidence determinations, on a case by case basis. To ensure full transparency, cases whe re overt toxicity was observed are clearly identified and are noted in all tables included in the battery review
section.

1. Interaction with the HPT Axis

Three assays in the Tier 1 battery are intended to detect potential interaction with the HPT axis or its downstream thyroid hormonal pathways . These assays include the Female and M aleUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 132 of 160 P ubertal rat assay s , and the AMA

. Table 35 lists the endpoints from these guideline assays which
the 2008 FIFRA SAP 40 identified as biomarkers of potential thyroid interaction.

Table 35. Endpoints for Detecting Potential Effects on the Thyroid Hormonal Pathway According to the 2008 FIFRA SAP.
Assay Endpoint(s) Male Pubertal • Thyroid stimulating hormone (TSH)

• Thyroxine (T 4 )

• Thyroid gland histology Female Pubertal • Thyroid stimulating hormone (TSH)

• Thyroxine (T 4 )

• Thyroid gland histology
AMA • Amphibian (tadpole) development

• Thyroid gland histology

In its preliminary analysis of Tier 1 battery performance, the Agency considers a treatment

related effect on thyroid stimulating hormone (TSH), thyroxine (T 4 ), or thyroid weight or histology in the male or female pubertal rat to indicate a potential interaction with the thyroid hormonal pathway; however, any observations of overt to xicity are noted and will be considered in the future
WoE determinations. The 2008 FIFRA SAP recommended, “[a] significant effect of test substance on serum T4 must be considered a positive finding, whether or not serum TSH is affected, and
whether or not the AMA is positive ” (USEPA 2008a, p. 31). For the AMA, the Agency is following the decision logic outlined in the AMA test guideline (OCSPP 890.1100) and Standard Evaluation Procedure (SEP), which was informed by the results of earlier validation studie s using chemicals with known thyroidal MOAs (USEPA 2008b). This decision logic is illustrated in Figure 1 9 . Using this approach, the direction of effect is considered when evaluating whether treatment

- related effects on amphibian development are indicative of thyroid activity: Developmental delay is considered a thyroid finding only if corroborated by treatment- related effects on thyroid gland histology, whereas advanced development, asynchronous development, or effects on thyroid gland histology may each be considered to be diagnostic findings independently.

40 USEPA. 2008a. Transmittal of meeting minutes of the FIFRA Scientific Advisory Pa nel meeting held on March 25

- 26, 2008, to review and consider the Endocrine Disruptor Screening Program (EDSP) proposed Tier 1 screening battery. SAP Minutes No. 2008

- 03. 11 June 2008. Washington, DC.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 133 of 160 Figure 19. Suggested D ecision Logic for the Conduct and I nterpretation of the AMA (OCSPP 890.1100, OECD 2009).

Sixteen case study chemicals were used in the preliminary analysis of performance of the battery for HPT/thyroid hormonal pathway screening. In 13 of these cases, Tier 1 guideline data showed indications of potential thyroid activity in one or a combination of assays ( Table 36 ). Responses in HPT

- relevant endpoints were identified in s even of 16 guideline Male Pubertal studies, in 9 of 16 guideline Female Pubertal studies, and in seven of 16 submitted AMA studies , and in 4 instances where all 3 assays had effects noted in the studies that have been reviewed. Of these, one
Male Pubertal study and three Female Pubertal studies only showed effects at the same dose level where overt toxicity was recorded. Potential solvent interference was identified in two of the three AMA studies for chemicals which only showed responses in this particular assay

. Overall, all 3 assays for a given chemical reported effects in three cases. Submissions of Other Scientifically Relevant Information (OSRI) which were used to potentially satisfy guideline requirements are
noted in the table.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 134 of 160 Table 36. Preliminary Results of Tier 1 Assays R elevant to the HPT A xis or Thyroid
Hormonal P athway, by Case Study C hemical.

Chemical Code Male Pubertal Female Pubertal AMA
A x B x x C x x x E x x F x x x G x x a x
H x b

I
J x K x a x a

M x b

N

P x x S x a c

T d

V x

x : =E ffects were seen on thyroid

- related endpoint(s); Blank cell = no thyroid

- relevant effects observed

a = Effects on thyroid

- related endpoints only observed at doses where overt toxicity was observed

b = Potential solvent interference made results difficult to interpret

c = Did not achieve adequate exposures

d = P ubertal rat assay Test Orders were satisfied by OSRI; effect only on T3 (endpoint not included in the guideline) in the male rat

.

(i) Complementarity of Results Within Each Tier 1 Assay

When considering the possible complementarity of responses among endpoints within an assay, the following general patterns were noted in the initial data submissions ( Table 3 6 ). A more
detailed examination of the directio n and magnitude of each effect will be performed in the future when evaluating complementarity within the chemical

- specific WoE analyses.

Male Pubertal Rat

• A response in T 4 was accompanied by a response in TSH in only one case (Chemical E). An effect on thyroid weight, but not histology, was also observed in this case.

• A response in T 4 occurred without a response in TSH in three cases (Chemicals C, F, and G).

• A response in TSH occurred without a response in T4 in two cases (Chemicals J and P).USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 135 of 160 • The response in TSH was accompanied by a difference in thyroid weight in only one case (P).

• The response in TSH was accompanied by an effect on thyroid histology in only one case (Chemical J).

• In one case, an effect on thyroid histology occurred without responses in any other diagnostic endpoints (Chemical K). However, overt toxicity was observed at the same dose levels as histological effects.

• Effects on thyroid histology were not observed in association with effects on T 4 in the male

rat.

Fem ale Pubertal Rat

• A response in T 4 was accompanied by a response in TSH in two case s (Chemical s E, and F).

• In six cases, T 4 was affected without a concomitant response in TSH (Chemicals A, B, C, G, K, P).

• In four of the cases where an effect on T 4 was observed, a n effect on thyroid histology was observed (Chemicals B, G, K, and P). Effects on thyroid histology only occurred
concomitant with alterations in T 4 in the female pubertal rat.

• In two of these cases, the effects on T 4 and thyroid histology occurred only in the presence of overt toxicity (Chemicals G and K).

• In one case, TSH was altered in the absence of any other effects (Chemical Q).

• In one case, thyroid gland weight was affected in the presence of overt toxicity (Che mical S).
No other effects were observed.

Amphibian Metamorphosis

• Observations of advanced development (based on developmental stage and/or normalized hind limb length) were complemented by thyroid histopathology diagnoses in three cases (Chemicals C, H, and V). In one of these cases, potential solvent interference was identified (Chemical H).

• Advanced development was observed in the absence of effects on thyroid histopathology in two cases (Chemicals F and M). In one of these cases, potential solvent interference was identified (Chemical M).

• Observations of developmental delay (based on developmental stage and/or normalized hind limb length) were complemented by effects on thyroid histopathology in only one case (Chemical G).

• Developmental delay occurred in the absence of effects on thyroid histopathology in five cases (Chemicals I, J, K, P and T). According to the AMA test guideline deci sion logic outlined in Figure 19 , these cases are considered “likely thyroid inactive.”USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 136 of 160 • Notably, one of these putati vely inactive cases (Chemical T) represents a special data submission based on examination of preserved specimens at a later developmental stage (NF stage 66). This special submission lacks observations at the interim time point (Day 7 of the AMA) which a re expected to be more sensitive to potential thyroid agonism ( e.g. , advanced development)

.

The variety of responses observed among the HPT

- relevant assays illustrates two main points: 1) Less complementarity is built into the individual Tier 1 assays for detecting potential thyroid effects, as compared to the estrogen and androgen hormonal pathways. Therefore, all three thyroid

- relevant assays and each of their endpoints need to be considered when screening for potential interaction. 2) The AMA decision logic ( Figure 19) is a simple but useful and important tool for interpreting the results of this assay. Although developmental delay was observed in six cases (Chemicals G, I, J, K, P, and T), only one of these cases (Chemical G) is characterized as
pote ntially thyroid

- active in the AMA when the decision logic is implemented.

Table 37 summarizes the preliminary diagnostic endpoint responses in each assay. Effects determined to be indicative of potential thyroid interaction are shown with an “x ”. Effec ts on amphibian development are identified consistent with the AMA decision logic ( i.e. , they are not marked in the table if the effect was a delay and there were no corroborating histological effects). The responses will be considered using a WoE approach to determine potential for interaction along the thyroid pathway; therefore, the binary responses shown in this table (effect: “x”, no effect: blank cell) are only preliminary indicators and do not reflect any overall chemical conclusions.

Table 37 . As sociations b etween Endpoint R e sponses in Tier 1 Assays R elevant to the HPT and Thyroid Hormonal Pathway, by Case Study C hemica l (P reliminary)

.

Chemical

Code Assay Thyroid Weight Thyroid Histology TSH T 4 Amphibian Development

A Male Pubertal

Female Pubertal x AMA

B Male Pubertal

Female Pubertal x x
AMA x

C Male Pubertal x
Female Pubertal x AMA x x

E Male Pubertal x x x
Female Pubertal x x AMA

F Male Pubertal x
Female Pubertal x x AMA xUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 137 of 160 Chemical

Code Assay Thyroid Weight Thyroid Histology TSH T 4 Amphibian Development

G Male Pubertal x
Female Pubertal x a x a

AMA x x

H Male Pubertal

Female Pubertal

AMA x x b

I Male Pubertal

Female Pubertal

AMA

J Male Pubertal x x
Female Pubertal

AMA

K Male Pubertal x a

Female Pubertal x a x a

AMA

M Male Pubertal

Female Pubertal

AMA x b

N Male Pubertal

Female Pubertal

AMA

P Male Pubertal x x Female Pubertal x x AMA

S Male Pubertal

Female Pubertal x a

AMA c

T Male Pubertal d

Female Pubertal e

AMA f

V Male Pubertal
Female Pubertal
AMA x x

x = treatment

- related effect; B lank cell = no thyroid

- relevant effect; shaded cell: not measured in the assay

a = Effects were observed only at dose levels with overt toxicity
b = Potential solvent interference

c = Did not achieve adequate exposure
d = T est order requirement satisfied by OSRI (Stoker et al. 2000); dose

- dependent effects were observed on T3 but no effects were observed on T4, TSH, or thyroid histopa t hology

e = T est order requirement satisfied by OSRI (Laws et al. 2000); no effects on thyroid hormones or thyroid gland morphology
f = S pecial da ta submission: observations were based on preserved NF stage 66 specimensUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 138 of 160 (ii) Redundancy Across Tier 1 Assays

As shown in Table 3 6 and Table 3 7 , there was general consistency in the overall responses
between the M ale and Female Pubertal assays, but not necessarily complete agreement between the individual endpoints that were affected. The AMA was sometimes responsive when the pubertal assays were not, and vice versa

. Comparisons of Male and Female Pubertal Rat

• B oth the male and female pubertal rat assays were responsive in six cases (Chemicals C, E, F, G, K, and P). However, Chemical K caused overt toxicity in both the male and female rat.

• Six chemicals showed no response on guideline endpoints in either the male or female pubertal rat (Chemicals H, I, M, N, T, and V)

. However, an OSRI submission showed that Chemical V caused an effect on T3 in the male pubertal rat. T 3 is not typically included as an endpoint in the guideline studies.

• Chemical J caused an effect in the male rat but n ot the female rat

.

• Three chemicals caused an effect in the female rat but not the male rat (Chemicals A, B, and S) . Chemical S was considered overtly toxic in the female rat

.
Comparisons of Pubertal Rats and Amphibian Metamorphosis

• For chemicals having a ll three guideline studies available, a relevant effect in the AMA was accompanied by a relevant effect in at least one pubertal rat study in four of seven cases ( Chemicals B, C, F, and G ).

• In three of these cases, HPT

- relevant responses were observed in each of the male pubertal,
Female Pubertal , and Amphibian Metamorphosis Assay s (Chemicals C, F, and G)

.

• Chemical S, which was overtly toxic in the female rat and caused no effect in the male rat,

was not successfully tested in the AMA.

• Chemical B , which caused an effect in the female rat, also affected thyroid histopathology in
the AMA.

• In two cases, a test chemical did not cause a significant effect on a ny diagnostic endpoint in any of the three guideline assays (Chemicals I and N)

.

• The special data submission for Chem ic al T showed no significant effects in the frog at NF stage 66. The OSRI for this chemical showed an effect on a non- guideline endpoint (T 3 ) in the male rat only.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 139 of 160 (iii) Discussion

Visually, the summary results illustrated i n Table 36 show a degree of consistency between the overall results in the Male and Female Pubertal rat studies. At a high level, the assays both showed either some effect or no effect on guideline endpoints in 12 of 16 cases where both Male and
Female Pu bertal rat studies were available for review. Similarly, the specific endpoint responses in male and female rat mirror one another in many, but not all, cases ( Table 37 ). This observation underscores the intended redundancy of these assays with respect t o mechanisms and downstream effects which may be conserved across sexes. However, sex

- specific differences in the preliminary overall results for guideline endpoints were observed in the remaining four of 16 cases. In its review of the proposed Tier 1 battery, the 2008 SAP advised, “ Replacement of the pubertal male or female assays with an alternative assay is not considered a reasonable alternative because it is possible to miss gender specific effects from certain chemicals ” (USEPA 2008a, p. 23). The p reliminary results from Tier 1 studies provide support for the assertion that the Male and Female Pubertal rat assays are both useful: in some cases, they show redundancy in responses which increases confidence in the overall findings, and in other cases, they provide information on possible sex

- specific effects which may be missed if one of the assays were excluded.

This preliminary review shows that thyroid

- relevant results in the rat are not necessarily predictive of results in the amphibian, and vice ve rsa . The 2008 SAP observed that the AMA was the only assay in the Tier 1 battery that provided an evaluation of an early developmental timeframe. The AMA may provide information on the downstream effects of potential thyroid pathway interaction, as captured by peripheral tissue endpoints associated with amphibian metamorphosis, whereas the Male and Female Pubertal rat assays provide information on circulating TSH and T 4 levels. The three assays all include examinations of thyroid gland histology; however , different effects across assays may be observed due to differences in exposure route, metabolism, life stage, and taxonomic differences in downstream responses to certain types of perturbations. Such differences will need to be considered when interpreting the nature, magnitude, and relevance of various endpoint responses when the chemical

- specific WoE analyses are conducted in the future. At a high level, the preliminary results reinforce the importance of including both taxa in the screening battery t o reduce the potential for false negatives.

• In one case (Chemical A) , an effect on T 4 was observed in the female rat, but no effects were observed in the male rat, and no effects were observed in the amphibian. In the absence of other information, the screen would have missed a potential interaction if the Female Pubertal assay were dropped from the battery

.

• In the second case (Chemical B) , effects were observed on T 4 in the female rat and on thyroid gland histology in both the female rat and the amphibian, but no effects were observed in the
male rat.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 140 of 160 • In the third case (Chemical S) , thyroid gland weight was affected in the female rat, but only at overtly toxic doses. No effects were seen in the male rat, and the chemical was not amenable to aquatic exp osure testing in the AMA. The latter case illustrates the importance of considering overt toxicity and general experimental performance when interpreting results, especially when results across assays do not appear to be concordant.

• The male rat showed greater sensitivity in only one case (Chemical J) wherein both thyroid gland histology and TSH were affected; no significant diagnostic effects of this chemical were observed in the female rat or in the amphibian.

• Using the test guideline decision logic depi cted in Figure 19 , t he AMA showed meaningful

responses to three chemicals which caused no relevant effects in either male or Female
Pubertal rat assays (Chemicals H, M, and V). However, potential co

- solvent interference
was identified in the AMA in two of these three cases (Chemicals H and M)

.

• In the third case (Chemical V) , effects on amphibian thyroid histology and developmental biomarkers were observed in the absence of any potential confounding effects, while no
effects were seen in the rat. Such resu lts may support the 2008 SAP prediction that “... weakly acting chemicals that affect thyroid hormone receptor or deiodinase activity in peripheral tissues would score positive only in the AMA ” (USEPA 2008a, p. 23).

Therefore, i n the evaluation of battery performance, it is useful to consider the inherent differences between each assay, its biological domain of applicability, and the physiology and life
stage of its test species, in addition to any confounding factors in individual studies. A thorough
unde rstanding of these issues will be helpful in preparing for the future WoE determinations for individual chemicals subjected to Tier 1 EDSP screening.

Across the domain of chemicals evaluated, less complementarity and redundancy in results was observed for endpoints within and across Tier 1 assays with respect to the HPT/thyroid hormonal pathway than was observed with the estrogen (E) and androgen (A) hormonal pathways (Sections b and c below). This observation is likely reflective of the design of the battery; as compared to the E and A pathways, fewer assays and biomarkers are available in the battery for the detection of thyroidal MOAs. Accordingly, this preliminary review agrees with the recommendations of the 2008 SAP that all three thyroid- relevant assays, and each of their diagnostic endpoints, need to be considered when screening for potential thyroid activity.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 141 of 160 2. Interaction with the Androgen Pathway

Six assays in the Tier 1 battery are designed to , either specifically or at least in part, screen for potential androgen interaction . These assays include three in vitro assays: the androgen receptor binding cytosol assay; the aromatase assay; and, the steroidogenesis assay. Additionally, three in

vivo assays are included in evaluating interaction with the androgen pathway: the Hershberger assay; the male pubertal assay; and the fish short

- term reproduction assay (FSTRA) . The 2008 FIFRA
SAP 41 did not specifically identify the aromatase and steroidogenesis assays when discussing the androgen pathway; however, they noted that “ [ a ] ndrogens could also influence enzyme activity in the aromatase and H295R cell line [steroi dogenesis] assays ”. Theref ore, all six assays were considered when evaluating the complementary and redundant nature of the assays relative to potential androgen interactions. Table 38 lists the endpoints from these guideline assays which the

2008 FIFRA SAP 42 identified as indicators of potential androgen interaction. In some cases, the endpoints are indicative of a potential androgen agonist ( e.g., development of male secondary sex characteristics, such as tubercles, in female fish). However, for other endpoints, the observed effec t may be due to a potential androgen antagonism or an estrogen agonist ( e.g., attenuated tubercle expression in male fish). This is consistent with the 2008 SAP which stated, “ The comparative fish short-term reproduction assay would also be sensitive to the effects of an androgen. In this assay, androgen would affect organ responses, male and female system integrity with the apical endpoints being manifest on development and comparative reproductive system integrity .” The panel also stated that “[a] ndrogens will affect organ responses and male system integrity in the male pubertal

assay ,” and that the effects are mediated through sub

- organ activities. The Panel also pointed out that apical responses such as those in the FSTRA are critical components to the battery ’s consistency and redundancy, i.e ., “ the role of fecundity . . . is paramount for evaluations of the HPG axis ” recognizing that “ EPA should be alert to possible non-endocrine mechanisms ” (pg . 27).

41 USEPA. 2008. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held on March 25

- 26, 2008, to review and consider the Endocrine Disruptor Screening Program (EDSP) proposed Tier 1 screening battery. SAP Minutes No. 2008

- 03. 11 June 2008. Washington, DC.

42 USEPA. 2008. Tran smittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held on March 25

- 26, 2008, to review and consider the Endocrine Disruptor Screening Program (EDSP) proposed Tier 1 screening battery. SAP Minutes No. 2008

- 03. 11 June 2008. Washin gton, DC.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 142 of 160 Table 38 . Endpoints for D etecting Potential E ffects on the A ndrogen Hormonal Pathway.

Assay Endpoint(s) AR Binding • Receptor binding
Aromatase • Enzyme activity (stero i dogenesis)
Stero i dogenesis • Enzyme activity (stero i dogenesis)

Hershberger • Changes (increase or decrease) in weight of androgen

- dependent tissues

Male Pubertal HPG • Sexual developmental characteristic (age at preputial separation (PPS) )

• Weight and/or histology of androgen

- dependent reproductive organs or tissues

• Total serum concentrations of testosterone

Fish Reproduction HPG • Fecundity

• Fertilization success

• Secondary sex characteristics

• Gonadal size (gonado

- somatic index [GSI] ) and histopathology

• Plasma concentrations of vitellogenin [VTG] in females and males

• Plasma concentrations of testosterone in males (optional)

In the current analysis of Tier 1 battery performance, for the androgen receptor binding and

aromatase in vitro assays, the Agency considers the extent to which a chemical elicits a response, based on the chemical’s percent activity under assay conditions surpassing the threshold established in the guideline. A statistically significant (p≤0.05) difference in a chemical’s response compared to the control was used in the steroidogenesis assay to evaluate responsiveness. Similarly for the Hershberger assay, response was defined when any two of the five measured androgen

- dependent tissue weights in the treated animals were statistically different (p≤0.05) from the control animals. In the male pubertal assays, a change in any of the three endpoint categories listed in Table 38 could be considered as indicative of a response with the androgen pathway. Finally, for the FSTRA assay, a statistically significant differences (p≤0.05) from the control was found for on fecundity, fertilization success, tubercles, GSI, VTG or sex stero ids and thus could be considered indicative of a response in androgen dependent tissues/processes.

(i) Complementarity of Results Within Each Tier 1 Assay

Results for each of the six identified assays are presented in Table 39 . It is noted that for the

in vivo studies summarized in Table 39 , the determination of a treatment

- related effect reflects a range of responses over what may be multiple endpoints for a chemical. There are chemicals that exhibited multiple treatment

- related effects across several different modalities as described by the 2008 SAP within and across assays. While for other chemicals, a single significant effect was observed within a multiple endpoint assay. Additionally, effects may have been detected in the presence of overt toxicity; however, at this time such effects were not excluded from the analysis. To help in evaluating if the Tier 1 screening assays provide redundancy and compl e mentarily for androgenic responses, examples of the types and number of responses observed are discuss ed below.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 143 of 160 Male Pubertal Assay :

• S ix chemicals (E, F, J, N, G, and S) resulted in alterations in two or more androgen

responsive endpoints ( e.g. , testosterone and multiple organ weights); whereas,

• Three other chemicals (B, C, and P) only reported a single type of androgen

- responsive effect ( e.g ., multiple organ weights or testosterone);

• Chemicals G and S, an alteration on PPS as well as effects in testosterone and androgen

depend e nt organ weights was observed but in the presence of overt toxicity (salivation and/or decreased body weight).

Hershberger A ssay

• Three chemicals (Chemical A, G, and H ) exhibited significant alterations in two or more of the measured androgen responsive tissues within the assay; whereas,

• Chemicals E , K and S elicited a response by alterations in androgen responsive tissues, but in the presence of overt toxicity.

• Chemical B also reported effects on both androgen

- dependent tissues and on testosterone, but in the presence of overt toxicity.

FSTRA

• With the F STRA, for Chemical J, there were complementary responses in the endpoints which included alterations in fecundity, female GSI, female VTG and E2, and female testosterone with alterations in female gonads; additionally there was a change in male GSI
and alt erations in male gonads.

• Chemical V reported an effect on tubercles in males alon g with alterations in fertility.

E ffects on fecundity were also observed, however, an effect on female body weight ( i.e

., overt toxicity) was reported at the same test conce ntrations.

• For Chemical A, while there were responses in two or more endpoints which include fecundity, fertilization success, male tubercles, male and female GSI, female and male gonadal histopathology, and female VTG, overt toxicity was observed at some, but not all of the concentrations where the responses were observed.

• Additionally, Chemicals B and Q also reported alterations in fecundity, gonadal histopathology, and/or fertility and male tubercles, but also at test concentrations both with and without overt toxicity.

• For Chemical H, a reported effect in male tubercles, as well as on fecundity and female histopathology, was reported at test concentrations both with and without overt toxicity

.

• While the responses were of a complementary nature (f ertility, male tubercles and male gonadal histopathology or male GSI and male gonadal histopathology) among endpoints for Chemicals K and M, these responses were observed in the presence of overt toxicity.

• Additionally, effects on fertility or alterations in male gonadal histopathology were also observed in the presence of overt toxicity for Chemicals C, D, N, and P.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 144 of 160 (ii) Redundancy Across Tier 1 Assays

An examination among the three in vivo assays and three in vitro assays was conducted to look for potential redundant responses across the nine different modalities for the assays, with some examples presented in Table 39 and discussed below. These example s though should not be construed as an interpretation of whether the androgen pathway has been perturbed since such a determination would require a WoE analysis.

Based on the evaluation of redundancy in identifying potential interactions across the assays using the available dataset and consistent with input from the SAP it appears that the in vitro assays do not consistently exhibit significant effects when effects were observed in the in vivo assays

. For
the in vitro assays , given the specificity and different mechanisms of action represented in the three

in vitro assays, the mode of action by which the interaction may be occurring may not be consistent

or necessarily redundant across the in vitro assays. The AR binding assay evaluates binding potential whereas the aromatase and steriodogenesis assays examine enzy matic activity. This is
consistent with the Panel comments that the in vitro assays represent “ specificity and sensitivity...and do not provide complementary or redundant assessments of androgenic activities ”.

• Chemical J reported responses in s ex steroids a nd organ responses (androgen

- dependent tissue weight and female VTG levels) in both the male pubertal and fish assays as well as responses in the in vitro aromatase and steroidogenesis assays and an equivocal response in the androgen binding assay.

• For Ch emical A, organ responses were observed in both the Hershberger and FSTRA; however, the organ response in the FSTRA ( effects on female VTG ) was observed in the presence of overt toxicity. Additionally, effects on cell response, male and female systems
and fertility were also observed in the fish assay, but again some of those responses were observed in the presence of overt toxicity. For Chemical A, there were also responses in the androgen binding and steroidogenesis assays and an equivocal response in the aromatase

assay.

• For Chemical E, effects on androgen

- responsive organ weights were observed in the Hershberger and male pubertal assays with effects on testosterone observed in the pubertal

assay ; overt toxicity was observed in the Hershberger.

• In Chemical B, effects on androgen

- dependent organs weights were observed in the Hershberger and male pubertal, but overt toxicity was observed in the Hershberger. Additional effects including male tubercles and alterations in male gonads were affected in the FSTRA assay for Chemical B, but there may be potential solvent interferences with this

assay.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 145 of 160 Table 39. Summary of R esults in Tier 1 A ssays R elevant to the Androgen Hormonal Pathway by C hemical.

Chemical

C ode ARB Aromatase Stero i dogenesis Hershberger Male Pubertal FSTRA

A x equivocal x x x

B x a x x c

C U x x x b

D equivocal x a

E x a x

F NA x x
G x x a x d

H x equivocal x x

I equivocal x x a

J equivocal x x x x

K x x x a x a

M equivocal equivocal x

N x x

P equivocal equivocal x x a

S equivocal x a x a x a

T NA NA NA x I

V x
x = T reatment

- related effect(s) on measured endpoint(s) indicative of potential androgen interaction reported Blank cells represent the absence of a statistically significant effect). U = C hemical could not be tested under assay conditions
I = I nconclusive NA = D ata submission not yet reviewed (OSRI)

a = E ffects only observed at test levels with overt toxicity ( e.g ., substantial decrease in body weight in rats; for fish - reduced survival, abnormal swimming, reduced feeding)
b = C hanges in fish body weight reported at concentrations were an effect was also reported

c = P otential solvent interferences
d = S tudy conduct questionable, results possibly not reliable

(iii) Discussion

Th e six assays discussed are intended to provide insight on a chemical’s potential to interact with the androgen pathway as measured through different levels of biological organization. The identified assays are in t en ded to provide complementary and/or redundant assessments of androgenic activity. This is reflected in the 2008 SAP comment that “ the in vivo assays included in the Tier I battery provide for both redundancy and complementarily for androgenic responses ”, and
that the aromatase and stero i dogenesis assay “ complete the complement of in vitro assays in the Tier
I b attery ”. However, the in vitro assays may not provide the same level of redundancy, nor are they expected to provide the same level of data compared to the in vivo assays

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 146 of 160 As discussed above and as summarized in Table 39 , some concordance was observed across
the in vitro and in vivo assays with the chemicals evaluated in support of this white paper.

In terms of responses across the in vitro assays, t here were three ( Chemicals A, J, and K ) out
of the 17 cases that had effects in two in vitro assays which were considered to have potential activity where two of those cases (Chemicals A and K ) included the AR binding assay (Table 39). For Chemicals M and P, there were tw o of the three in vitro assays that had equivocal results. For Chemicals A, H, I, and J there an equivocal result for one in vitro assay along with an effect in at
least one other in vitro assay.

Based on the evaluation of responses within the more complex assays ( i.e ., FSTRA and the male pubertal ) , two or more endpoints were altered indicating the in vivo assays provide concordance with which to evaluate androgenic responses.

For the three in vivo assays (Hershberger, Male Pubertal, and FSTRA), ther e were alterations in an endpoint (s) among them providing sufficient information to evaluate potential androgenic responses. However, evaluation of responses among the three in vivo assays is complicated by the observation of overt toxicity in some of th e assays. Overt toxicity was observed for at least one study in each of the three in vivo assays for chemicals where effects were reported , with six of the FSTRAs reporting significant effects only where overt toxicity was also observed. Additionally,

an other FSTRA study had potential co

- solvent interferences and in another, the results of the study were technically deficient and could not be readily evaluated . A discussion on the observation of overt toxicity in the FSTRA is presented in a previous s ection ( Section 2e)

. Although the in vitro assays did not exhibit effects as frequently as the in vivo , there are a number of plausible biological reasons why this may occur and does not necessarily refute the intended redundancy of the assays within the battery. The importance of the intentional redundancy
is illustrated where of seven chemicals ( Chemicals A, B, E, G, H, K, and S ) that elicited significant
results in the Hershberger assay

, four ( B, E, G, and S) also produced a significant response in th e male pubertal assay ; for Chemicals A, J and K effects were observed in the in vitro assays as well

. For the three chemicals that did elicit a response in the Hershberger but not the male pubertal assay (Chemicals A, H, and K), a response was observed in the FSTRA . H owever, overt toxicity was
observed in some of the in vivo assays

. Therefore, the range of endpoints evaluated in the Tier 1 battery provides complementary response within the in vivo assays and redundancies across the
relevant in vitro and i n vivo assays to evaluate androgenic responses.

To what extent is this level of redundancy among the in vivo studies necessary for the Tier 1 assay is a question posed to the SAP panel. The agency will seek guidance from the 2013 SAP on whether the Hershberger assay is a necessary redundancy in the current battery given that the Hershberger typically showed responses in androgen sensitive tissues when the male rat pubertal
and/or FSTRA also exhibited effects (Table 39)

.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 147 of 160 3. Interaction with the Estrogen Hormonal Pathway

The EDSP Tier 1 battery covers the Estrogen hormonal pathway more extensively ( i.e., has more responsive endpoint s) than the Androgen or T hyroid pathways. However, battery performance for the estrogen pathway is difficult to fully assess because of the apparent absence of case studies with unequivocal estrogen receptor agonist activity (as indicated by positive ER binding and ERTA, and/or Uterotrophic assay results) in this initial review of 18 chemicals.

Seven assays in the Tier 1 battery are designed to identify potential interactions on the estrogen hormonal system and with different sexes and taxa. These include four in

- vitro assays: the

ER binding assay; ER α transcriptional activation (ERTA) assay; the aromatas e assay; and the
steroi dogenesis assay. Additionally, three in vivo assays are useful in evaluating interaction with the estrogen pathway: the uterotrophic assay; the female rat p ubertal assay; and the FSTRA. With regard to the female pubertal assay, the 2008 FIFRA SAP identified vaginal opening and onset of first estrus as suitable endpoints for estrogen

- dependent responses, but the panel noted that ovarian cyclicity may not be a useful endpoint because of high variability caused by other factors . The 2008 FIFRA SAP considered these seven assays as a means of evaluating the complementary and redundant nature of the assays relative to potential estrogen interactions. E ndpoints fr om these guideline assays which the 2008 FIFRA SAP 43 identified as biological indcators of potential estrogen interaction are presented in Table 40

.

In this preliminary analysis of Tier 1 battery performance, for the estrogen receptor binding
and aromatase in

- vitro assays, the Agency considers a chemical to have the potential to interact with the estrogen pathway based on the chemical’s percent activity under assay conditions surpassing the threshold established in the guideline. A statistically significant (p≤0.05) difference in a chemical’s response (alteration in estradiol levels) compared to the control was used to determine a potential estrogen interactio n in the steroidogenesis assay. In the female pubertal assay, a change in any endpoint listed in Table 40 could be considered and indication of a response in estrogen dependent tissues/ processes. Finally, for the FSTRA assay, a statistically significant differences (p≤0.05) fr om the control was found for fecundity, fertilization success, tubercles, GSI, VTG or sex steroids and thus could be considered indicative of a response in estrogen dependent tissues/processes.

43 USEPA. 2008. Transmittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held on March 25

- 26, 2008, to review and consider the Endocrine Disruptor Screening Program (EDSP) proposed Tier 1 screening battery. SAP Minutes No. 2008

- 03. 1 1 June 2008. Washington, DC.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 148 of 160 The inclusion of the FSTRA assay was reported by the 2008 SAP Panel as “ an important component of the estrogenicity screen ”, with a specific mention of the measurement of plasma VTG. Ultimately, the deg ree to which the responses across a pathway are complementary and redundant
are considered in the context of any observations of overt toxicity and procedu ral adequacy to support the WoE determination for each chemical , which will be the subject of a future SAP

.

Table 40. Endpoints for Detecting Potential Effects on the Estrogen Hormonal Sys tem.

Assay Endpoint(s)

ER Binding • Receptor binding

ERTA • Cell Signalling

Aromatase • Enzyme activity (steroidogenesis)

Steroidogenesis • Enzyme activity (steroidogenesis)

Uterotrophic • Increased uterine weight

Female Pubertal

HPG • Sexual developmental characteristics (vaginal opening and onset of first estrus)

• Weight and/or histology of estrogen

- dependent reproductive organs or tissues Fish Reproduction

HPG • Fecundity

• Fertilization success

• Secondary sex characteristics

• Gonadal size (gonado- somatic index) and histopathology

• Plasma concentrations of vitellogenin in females and males

• Plasma concentrations of sex steroids (optional)

(i) Complementarity of Results Within Each Tier 1 Assay

Results for each of the seven identified assays ar e presented in Table 41. It is noted that for
the in vivo studies summarized in Table 41 , the determination of a treatment

- related effect reflects a range of responses over what may be multiple endpoints for a chemical. There are chemicals that
exhibited multiple treatment

- related effects across several different modalities as described by the 2008 SAP within and across assays. For other chemicals, a single significant effect was observed within a multiple endpoint assay. Additionally, effects may have been detected in the presence of overt toxicity; however, at this time such effects were not excluded from the analysis. To help in evaluating if the Tier 1 screening assays provide redundancy and compl e mentari t y for estrogenic responses, examples of the types and number of responses observed are discussed below.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 149 of 160 Female Pubertal Assay:

• Chemical E resulted in changes in VO and first estrus as well as effects on estrogen dependent tissues;

• Chemical Q had effects on VO and first estrus;

• Chemical G, effects were reported in the presence of overt toxicity (mortality, decreased body weight and clinical signs) and included alterations in VO and effects on ovarian and uterus (wet, and blotted ) weights;

• For Chemical K, effects observed in the presence of over t toxicity (tremors) were ovarian follicle changes;

• Chemical M, effects reported in the presence of overt toxicity (salivation) were alterations in
VO and first estrus ; and

• For Chemical S, effects reported in the presence of overt toxicity (decreases in body weight and food consumption) included changes in VO and in ovarian and uterus (wet and blotted) weights.

FSTRA Assay:

• Chemical V reported effects on fecundity, fertility and male tubercles; however, there was an effect on female body weight r eported.

• Chemical H had alterations in fecundity, male tubercles and female gonadal histopathology, although overt toxicity was observed at some, but not all, concentrations with the reported
effects.

• Chemical M reported alterations in fecundity, male and female GSI and gonads; however, the histopathology and male GSI effects corresponded with observed overt toxicity along with some of the effect concentrations for fecundity.

• Che mical F reported effects on fecundity, fertility, male tubercles, GSI, estra diol levels and male and female gonads; however, except for fertility and male GSI, all the remaining effects were only observed in the presence of overt toxicity.

• Similarly, for Chemical S, alterations in fertility, male tubercles, male GSI, and male VTG were observed only at levels with observed overt toxicity.

• In Chemical N, alterations in fecundity, fertility, female GSI and gonadal histopathology were only observed in the presence of overt toxicity, except for female VTG.

• Chemicals C, D, I, K, and P also reported alterations in fecundity and/or gonadal histopathology, fertility, male tubercles, GSI or VTG only in the presence of overt toxicity.

Uterotrophic Assay:

• None of t he chemicals were active in the in vivo uterotr ophic assay. However, the results in the ER binding and the uterotrophic assay demonstrated concordance in that there was a lack of response in both assays which suggests a lack of estrogenicity among the case studies.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 150 of 160 (ii) Redundancy Across Tier 1 Assay

An examination among the four in vivo assays and three in vitro assays was conducted to
determine potential redundant responses across the nine different modalities for the assays, with some examples presented in Table 41 and discussed below. These example s should not be construed as an interpretation of whether the estro gen pathway has been perturbed since such a determination would require a WoE analysis.

In general, concordance of receptor

- mediated in vitro responses (ER binding and ERTA) and

in vivo assays including HPG effects (female pubertal rat and FSTRA) results was limited, suggesting that estrogen pathway effects observed in the evaluation of battery performance are likely mediated through mechanisms other than receptor binding. Potent ial effects of enzymatic conversion of androgens to estrogens are evaluated directly by the aromatase assay (inhibition only) and indirectly by the steroidogenesis assay (induction or inhibition relative to controls of
testosterone and estradiol from chole sterol). Results of these two assays were biologically consistent
for the chemical s included in this evaluation; C hemical J significantly inhibited aromatase activity and estradiol production in the steroidogenesis assay and all other chemicals with stati stically significant (p ≤ 0.05) steroidogenesis assay results (Chemicals B, C, D, F, I, O, P, and V) appeared to induce estradiol production and had no significant effect on aromatase activity.

• Chemical Q reported responses in female estrogen

- dependent systems ( i.e ., alterations in vaginal opening/estrus or fecundity) in the female pubertal and FSTRA; however, an effect on fecundity was only reported in the presence of overt toxicity.

• Chemical M reported responses in female estrogen

- dependent systems ( i. e ., alterations in vaginal opening/estrus or fecundity) in the female pubertal and FSTRA; however for the pubertal assay, these effects were only observed in the presence of overt toxicity. Equivocal results were reported for the aromatase and steroidogenesis assays.

• In C hemical S, organ responses were observed in both the pubertal and FSTRA . But only in the presence of toxicity. An equivocal result was reported for the aromatase assay.

• Similarly, for Chemical K, alterations in cell response (gonadal histopathology) and female
estrogen

- dependent systems (vaginal opening and fecundity) were observed in the female pubertal and FSTRA in the presence of overt toxicity. An effect in the aromatase assay was reported as well

.

• In Chemical T, there were effects reported on female estrogen

- dependent systems in the pubertal but were equivocal in the FSTRA.

It may be worth noting that if studies with overt toxicity are excluded from consideration, statistically significant (p ≤ 0.05) estrogen responses ob serv ed in the female pubertal rat (C hemicalsUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 151 of 160 E, T, and Q) and FSTRA (Chemicals A, F, H, J, N, Q, and V) possibly represent two di stinct chemical sets and may indicate different sensitivities of these two test species or different chemical
actions due to or al (rat) versus aquatic (fish) exposure routes. Though significant (p ≤ 0.05) estrogen pathway effects were observed in in vivo assays without accompanying in vitro effects, for no chemical were significant in vitro effects observed without accompanying i n vivo effects.

Table 41. Summary of Results in Tier 1 Assays Relevant to the Estrogen Hormonal Pathway, by Chemical.

Chemical Code ERB ERT A Aromatase Steroidogenesis Utero

trophic Female Pubertal FSTRA

A equivocal equivocal x
B x X x c

C equivocal X x b

D equivocal X x a

E x x
F x X x G x a x d

H equivocal x
I x X x a

J x X x
K x x a x a

M equivocal equivocal x a x
N x
P equivocal X x a Q I x x
S equivocal x a x a

T NA NA x I

V X x
x = T reatment

- related effect(s) on measured endpoint(s) indicative of potential estrogen interaction reported
I = I nconclusive NA = D ata submission not yet reviewed (OSRI)

a = E ffects observed only in the presence of overt toxicity ( e.g ., substantial decrease in body weight in rats; for fish - reduced survival, abnormal swimming, reduced feeding)
b = C hanges in fish body weight reported at concentrations were an effect was also reported

c = P otential co

- solvent interferences
d = Study conduct questionable, results possibly not reliableUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 152 of 160 (iii) Discussion

The seven Tier 1 screening assays discussed above are meant to provide insight on a chemical’s potential to interact with the estrogen hormonal system as measured through different
levels of biological organization. The identified assays are intended to provide complementary or redundant assessments of estrogenic activity. The 2008 SAP stated that the “ in vivo assays are well represented with a breadth of complementary endpoints. In vitro assays on the other hand have few or no complements such as indicated for receptor binding or transactivation assay .” However, the
Panel did comment that the estrogenic pathway for each modality did appear to retain at least some redundancy within the battery.

In conclusion, the range of endpoints evaluated in the Tier I battery is consider to provide the necessary redundancies to evaluate estrogenic responses. The different mechanisms of action and
exposure routes among assays relevant to the estrogen hormonal pathway were designed to minimize false negatives and detect effects of chemicals at multiple levels of biological organization. It should be noted that the set of chemicals on which this initial evaluation of battery p erformance does not include estrogen receptor agonists, thus making it difficult to assess the performance of the battery for this mode of action. Nonetheless, in 2008, the battery of assays was validated for estrogen agonism using known reference chemica ls

.

Based on the results of this subset of chemicals as reported in Table 41, the uterotrophic assay did n ot appear responsive for this subset of chemicals. While acknowledging that there does not appear to be estrogen agonists in this subset of chemicals, it is important to note that there were no cases where the uterotrophic assay was positive and pubertal and/or FSTRA was negative. To what extent is this level of redundancy across the in vivo assays necessary for the Tier 1 screen will be posed to the SAP panel. T he agency seeks advice on whether the uterotrophic assay provide s a necessary redundancy in the battery for the estrogen signaling pathway given the mechanistic specificity of the in vitro assays and the multi- parameter female pubertal assay an d FSTRA.



### V. Overall Summary and C onclusion s

In 1999, following the EDSTAC recommendations to develop a two

- tiered screening and testing program, a joint subcommittee of the Agency’s Science Advisory Board and FIFRA Scientific Advisory Panel recommended to the Agency, after review of the initial set of Tier 1 data, to subject that data to external scientific peer review for consideration to further optimize the Tier 1 screening
batter y . The scientific analyses included in this white paper are intended to eva luate the performance of the individual Tier 1 assays and the overall battery.

Individual Assay Performance Conclusions:

EDSP Tier 1 assay data were reviewed on a subset (up to 21 tes t chemicals) of the List 1 chemicals ( consisting of 50 pesticides and 2 i nerts). The subset represents a broad range of physical

-USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 153 of 160 chemical properties ( e.g ., water solubility, octanol

- water partition coefficients, volatility), as well as a range of chemical classes and pesticidal biological activities. The purpose of the analys is was to
evaluate:

• How well each Tier 1 assay test guideline can be consistently executed based on defined performance criteria;

• Issues associated with interpretation of responses within each assay;

• Complementary relationships within the multi

- parameter in vivo assays in the Tier 1 battery which may be indicative of potential interactions with the estrogen, androgen or thyroid hormonal pathways; and,

• Redundancy across the in vitro and in vivo assays that comprise the Tier 1 battery.

Additionally , this r eview considered whether 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

.
Based on the case studies evaluated, the Tier 1 assays provide useful information to evaluate potential interactions with the E, A, and T signaling pathways. There were no major problems identified with the Tier 1 assays; in general, contributing laboratories were able to execute each assay protocol in accordance with the respective test guidelines and achieve the specified performance criteria. Although there were some minor deviations from the performance criteria, these differences were not substantial so as to render the study unreliable in the va st majority of cases. Although recommended CV values for test concentrations were not always achieved with the

in vivo assays using aquatic organisms, it is not unexpected for those test substances that are poorly soluble or prone to absorb to surface mat rices within these experimental test systems.

Battery Performance Conclusions:

In evaluating the battery performance with the current subset of test order chemicals, there appears to be reasonable representation of endpoint alterations intended to evaluate potential interactions on the androgen hormonal pathways, the HPG axis and HPT axis. However, based on this initial evaluation, the subset of List 1 chemicals may be limited with respect to estrogen ic modes of action (particularly, agonism). It should be noted, that the Tier 1 assays were validated against compounds that act directly on the estrogen receptor. The discussion below will present the analysis for the battery performance by each hormonal pathway.

Thyroid Pathway:

There are three Tier 1 assay s ( i.e ., the male and female pubertal assays, and AMA) relevant to evaluating the HPT pathway. There is less complementarity built into the design for the T pathway compared to the E - and A- pathways which is reflected in the results of the current analysis

. In the rat pubertal assays, thyroid hormone changes were the most common observation which wasUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 154 of 160 accompanied by thyroid histology only in a few cases. In the AMA, histology was generally the most sensitive endpoint. There is also less redundancy built into the battery for the thyroid pathway. Thyroid

- related results in the AMA were not necessarily predictive of the rat findings and vice versa

. This initial analysis supports the view of the 2008 SAP that the AMA and the rat pubertal studies can provide different types of information and it reinforces the importance of including both taxa in the screening battery to reduce the potential for false negatives.

Androgen Pathway:

There are six Tier 1 assays relevant to evaluating the potential of a chemical to interact with
the A pathway ( i.e ., the androgen receptor binding cytosol assay, aromatase assay, steroidogenesis assay, Hershberger assay, male pubertal assay, and the fish short

- term reproduction assay. The degree of complementary findings relevant to the androgen hormonal pathway was evaluated for the more complex multi

- parameter in vivo assays. In the male pubertal assay, responses were generally observed for two or more different types of endpoints or for two or more of the androgen

- dependant

organs. With the FSTRA, effects were observed for all the chemicals and complementarity was generally found with two or more endpoints affected. It should be noted that about a third of the time the findings in the FSTR A were at exposure concentrations that also elicited signs of overt toxicity, and thus, distinguishing effects due to generalized toxicity versus potential interaction with the androgen (or estrogen) pathway for each chemical will be an important consideration in future weight of evidence determin ations. The male pubertal also showed a number of responses but overt toxicity did not appear to be a major issue.

Consistent with the 2008 SAP, the in vitro assays do not consistently exhibit significant
effects when effects are observed in the in vivo assays. But this analysis did find redundancy across
the in vivo assays (Hershberger, male pubertal, and FSTRA) with this subset of chemicals, albeit

some in vivo responses were complicated by the observation of overt toxicity. A number of the
chemicals that elicited responses in the Hershberger assay also produced a response in the male pubertal assay. For the chemicals that elicited a response in the Hershberger but not in the male pubertal assay, a response was observed in the FSTRA indicating that th e in vivo redundancy for the androgen signaling pathway enables the battery to serve as an effective means of minimizing false negatives. The agency will seek guidance from the 2013 SAP on whether the Hershberger assay is a necessary redundancy in the cu rrent battery given that the Hershberger typically showed responses in androgen sensitive tissues when the male rat pubertal and/or FSTRA also exhibited effects.

Estrogen Pathway:

There are seven assays in the Tier 1 battery de signed to screen for potenti al interaction with the E pathway ( i.e ., the estrogen receptor (ER) binding assay, ERa transcriptional activation (ERTA) assay, aromatase assay, steriodogenesis assay, uterotrophic assay, the female rat pubertal assay, and the FSTRA). As indicated earlier, the current case studies may be limited in potential modes of
action relevant to the E pathway. Again, the FSTRA showed a number of responses complicated byUSEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 155 of 160 overt toxicity about one thi rd of the time. Effects found in the female rat pubertal assay were generally associated with responses in the fish, but not always. Based on this data set, the female rat pubertal and fish assays are important in evaluating the E pathway across taxa, an d therefore are important components to the battery. The uterotrophic assay was consistently negative for all the chemicals examined, an effect which would be viewed as consistent with the lack of responses found
for the in vitro estrogen receptor binding assay. T he lack of response in the uterotrophic assay, ER binding assay and ERTA, and the discordant results with ER binding and ERTA, suggests that there are no ER agonists in this subset of chemicals. There are also some examples of responses in the in

vitro steroidogenesis assay that were associated with the female rat pubertal and/or fish assays. The agency seeks advice on whether the uterotrophic assay represents an unnecessary redundancy in the battery for estrogen signaling pathway given the mechanistic specificity of in vitro assays and the
multi

- parameter femal pubertal assay and FSTRA, noting that the results of the uterotrophic assay

were consistently negative and that there were no cases when the uterotrophic was positive but not in the puberta l or FSTRA.USEPA — SAP Review of EDSP Tier 1 Screening: Assay and Battery Performance — May 2013

Page 156 of 160 

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